Ground Up Strength Nutrition

The Effects of Alcohol on Athletic Performance and Recovery

Sat, 31 Jul 2010 19:48:25 +0000

by Luke D. Vella and David Cameron-Smith

Molecular Nutrition Unit, School of Exercise and Nutrition Sciences, Deakin University, Burwood,

Nutrients 2010

Alcohol consumption within elite sport has been continually reported both anecdotally within the media and quantitatively in the literature. The detrimental effects of alcohol on human physiology have been well documented, adversely influencing neural function, metabolism, cardiovascular physiology, thermoregulation and skeletal muscle myopathy. Remarkably, the downstream effects of alcohol consumption on exercise performance and recovery, has received less attention and as such is not well understood. The focus of this review is to identify the acute effects of alcohol on exercise performance and give a brief insight into explanatory factors.

Introduction

Athletes, like the rest of the population, consume alcohol. Sporting clubs and associations are frequently reported in the media to place bans or restrictions on the availability and consumption of alcohol by contracted athletes. Yet the same media organizations also report on alcohol-fuelled violence or misdemeanors perpetrated by these same athletes, suggesting anecdotally that athletes consume alcohol, occasionally to excess. This is quantitatively supported by dietary surveys of athletic populations that demonstrate self-reported alcohol intake constitutes up to 5% of the total daily energy intake in elite athletes [1]. However this is far from universal, as survey data reports either greater [2,3], or reduced [4,5] alcohol ingestion in athletic populations than the general community. This high variability in reported alcohol intake within athletic groups may in part be due to the characteristics of each sporting discipline. Alcohol intake appears to be positively associated with team sports where alcohol consumption is often encouraged as a component of team/group bonding and can be related to stress relief [6].

The detrimental effects of alcohol on human physiology have been well documented with acute alcohol ingestion affecting many aspects of metabolism, neural function, cardiovascular physiology, thermoregulation and skeletal muscle myopathy [7-9]. Yet the impact that alcohol ingestion has on exercise performance and more critically recovery has received less detailed scrutiny. This review aims to provide insights into the current knowledge around how alcohol acts to impair both exercise performance and the critical mechanisms by which alcohol acts at the cellular level to retard recovery following strenuous activity.

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Effect of Alcohol on Human Physiology

Alcohol consumption has a deleterious effect on a multitude of systems within the body and an in-depth analysis of each is beyond the scope of this review. However a brief insight into common symptoms linked to acute alcohol misuse will guide the following discussion on how alcohol influences human performance and recovery.

Effect of Alcohol in Skeletal Muscle

Multiple detrimental actions of lcohol within skeletal muscle are likely. Firstly, alcohol inhibits Ca2+ transients into the myocyte by inhibiting sarcolemmal Ca2+ channel actions. This action is reported in isolated human myotubes and rodent muscle tubes in-vitro [10-12]. Consequently this will impair excitation-contraction coupling, decreasing strength output. Yet human clinical data fails to support this in-vitro evidence [13]. Secondly, alcohol consumption may compromise sarcolemmal integrity, with evidence of greater plasma rises in the intracellular enzyme creatine kinase (CK), following alcohol ingestion and exercise [14]. Indeed, in rodents a supraphysiological dose of alcohol markedly increased plasma CK. Furthermore, in both electrically stimulated rodent muscles [15] and in human subjected to eccentric loading this not evident [13,16-18]. Thus clear mechanisms remain elusive, with a need for supporting clinical data. It is well understood that muscle cramps, pain and a loss of proprioception are common symptoms of alcohol misuse [19]; however the underlying mechanisms remain speculative.

Effect of Alchohol on Thermoregulation and Hydration

The effects of alcohol on hydration and its diuretic function are historically well recognised. The identification of alcohol as a potent diuretic date back to 1948, where a 10 mL excess urine production was evident following each gram of ethanol consumed [5]. The mechanism subsequently identified is the inhibition of anti-diuretic hormone (ADH) by ethanol [21], although this relationship is evident only in beverages containing greater than 4% (w/v) ethanol [21].

Alcohol has further been shown to act as a peripheral vasodilator. This presents several complications. Primarily this increases fluid loss through evaporation which further exacerbates the dehydration that is potentially already present. There is further an interference of central thermoregulatory mechanisms consequently resulting in a reduction in core body temperature [5,22]. Thus, not surprisingly, alcohol consumption has been repeatedly shown to decrease work tolerance in both high and low ambient temperatures [22-24].

Effect of Alcohol on Metabolism

In additional to being a readily accessible source of energy (29kJ per gram) [5], alcohol has a number of effects that bear ramifications for human metabolism. Alcohol-induced hypoglycemia has been proposed as a possible cause of symptoms common to alcohol misuse. The intake of high alcohol doses has been demonstrated to impair hepatic gluconeogenesis and subsequent glucose output [25,26], decrease the uptake of gluconeogenic precursors lactate and glycerol [27], and reduce muscle glycogen uptake and storage [27]. Alcohol has also been shown to induce a reactive hypoglycemia by exacerbating insulin secretion in the presence of a high carbohydrate meal [28]. While the detrimental effect of alcohol on glucose metabolism is strongly documented, some authors maintain that this can be negated if glycogen stores are maintained at homeostatic levels [25,29]. Specifically to exercise, acute alcohol intoxication inhibited the exercise induced rise in serum glucose concentration and caused a mild decrease in serum glucose during recovery from anaerobic exercise [25]. Further acute alcohol intoxication has been implicated in attenuating post exercise increases in serum fatty acid concentration [25]. These findings bear considerable ramifications for exercise performance and recovery. It is well documented in the literature that glucose availability plays a pivotal role in endurance performance [30,31] and further readily available stores of energy are necessary to fuel protein synthesis during muscle recovery from exercise [32].

Neurological Effects of Alchohol

Alcohol is a well-known depressant and thus acts to reduce central nervous system (CNS) excitability and cerebral activity [33], as demonstrated by a slowed encephalographic rhythm [34]. Functionally alcohol has been repeatedly shown to exhibit a dose-dependent impairment of balance, reaction time, visual search, recognition, memory and accuracy of fine motor skills [1,9]. Variances in neurological activity have also been intricately linked to a disturbance in sleep length and quality with some authors observing a loss in sleep depth with a shorter time of rapid eye movement (REM) sleep and an increase in sleep at stage 1 [35,36].

The effect of alcohol on neurological function is likely to be caused by a myriad of factors. The aforementioned effect of alcohol on glucose metabolism could affect cerebral functioning leading to symptoms of alcohol intoxication [25]. Alternatively, the accumulation of acetaldehyde, a bi-product of alcohol metabolism, has been theorized as a potential cause of the aversive neurological symptoms associated with alcohol misuse [37]; however this prospect remains speculative [19]. Further the toxic effect of a group of substances collectively termed congeners often produced during the fermentation of alcohol, are likely to contribute to the reduction in CNS activity. Methanol, histamine and polyphenols are amongst the congeners best studied [38]. Serotonin regulation provides a further prospective mechanism as this hormone has both been shown to be increased in the presence of alcohol [38] and performs various cognitive functions including memory and learning.

Alcohol and Exercise Performance

Given the numerous and complex mechanisms by which ethanol impacts on physiological systems it can be strongly hypothesized that elevated blood alcohol concentrations at the time of exercise will impair performance. Remarkably there are relatively few clinical trials that address this question.

Aerobic Performance

Earlier studies found no significant consequence of alcohol on a sub-maximal endurance performance and a 5-mile treadmill time trial respectively [39,40]. Contrastingly and not surprisingly, there is also literature that demonstrates that alcohol is detrimental to endurance performance [41-45]. What is apparent is that a threshold exists at which point alcohol becomes detriment to aerobic performance. Cofan and colleagues describe an alcohol intoxication threshold of 20mmol/L of ethanol in both animal [12] and human [10] studies, beyond which did performance decrements become significant. Further research has elaborated that this cause-effect relationship may exist in a dose dependent manner [43].

Anaerobic Performance

Despite the long list of skeletal muscle and neurological symptomatology associated with alcohol consumption, the majority of literature has been unable to establish a significant cause-effect relationship between alcohol and anaerobic performance. To the reviewers knowledge McNaughton and Pierce [43] have conducted the only research that has identified an effect of alcohol on sprint performance. This research examined five sprinters using sprint time as a measure of performance and established a detrimental, albeit inconsistent, association between alcohol dosage and sprint performance. Alcohol was ingested immediately prior to exercise testing so this data is limited to the acute effects of alcohol intoxication and does not apply to more chronic hangover symptoms. Recent research has been unable to validate these findings, and have consistently seen no change in strength or power characteristics following acute alcohol ingestion [13,16,42]. Contrasting to McNaughton and Pierce, these studies have examined force output using an isokinetic dynamometer as their outcome measure of anaerobic performance. Comparatively time trial sprint performance incorporates a high degree of motor control and coordination and may provide insight as to why these findings cannot be replicated.

Alcohol and Exercise Recovery

Most of the studies examining alcohol and athlete recovery have focused predominately on functional measures of muscle performance and blood borne markers of cellular tissue damage. To date, these studies have produced inconclusive results that fail to demonstrate a dose-dependency or critical threshold above which muscular recovery is compromised. Creatine kinase (CK) is an intra-muscular enzyme which when present in the peripheral circulation is widely used as a measure of muscle damage. Despite the clinical association between chronic alcohol abuse and skeletal muscle myopathy, acute ingestion appears to have little impact on exercise-mediated muscular damage [13,16,18].

The lack of results may be attributable to the parameters measured within these above mentioned trials. CK is highly variable and may not provide the best measure of muscle damage [45,48]. More recently, circulating levels of pro-inflammatory cytokines, released from the musculature may provide alternative measures of muscular stress and damage [47]. Inflammatory processes appear to be variably modulated by chronic and acute alcohol use. Prolonged alcoholism is associated with high circulating levels of pro-inflammatory mediators [49], whilst conversely acute consumption has been shown to decrease production of TNF-a and (IL-1) in rodent studies [50-52] It has yet to be established if cytokine concentrations are altered by acute alcohol ingestion during or immediately following intense exercise.

Similarly to the analysis of markers of muscle damage, the intra-muscular consequences of acute alcohol ingestion on aspects metabolic pathways of recovery are also ambiguous in humans. Alcohol ingestion immediately following prolonged cycling exercise has a modest impact to impair glycogen re-synthesis [48]. This action is dependent in part on alcohol replacing carbohydrates in energy-matched meals. Although acute suppression of glycogen synthesis may have been evident, examination of glycogen repletion over 24 hours demonstrated no long term detrimental impact of alcohol ingestion on muscle glycogen stores. Of particular relevance to the recovery of strength athletes is the enhanced protein synthesis that occurs post-exercise to facilitate repair and adaptive hypertrophy [53].

Acute alcohol ingestion decreases muscle protein synthesis in a dose- and time-dependent manner, in the absence of an exercise stimulus. Alcohol facilitates this firstly by suppressing the phosphorylation and activation of the mTOR pathways, the critical kinase cascade regulating translation initiation [54]. Complementing the decreased activation of the protein synthetic pathway, alcohol increases the expression of muscle specific E3 ligases; atrogin-1 and Muscle-specific RING finger 1 (MuRF1) [55]. These proteins are up regulated by conditions that promote skeletal muscle atrophy. Interestingly this was not associated with increased proteolysis, suggesting alcohol primarily impairs protein synthesis. It remains to be confirmed in rodents subjected either to muscle loading or resistance exercise that alcohol impairs protein synthesis. Subsequent clinical data is also lacking and this remains a critical absence in the scientific literature.

Functionally, the consumption of moderate amounts of alcohol augments the loss of force associated strenuous eccentric exercise [18,56]. To the researchers knowledge Barnes, Mündel and Stannard have produced the only research that has used functional measures of muscle performance to identify an interaction between post-exercise muscle damage and alcohol [18,56]. This research established a significant decrease in average peak isometric, concentric and eccentric torques at 36 hours post-exercise. This decrement appeared to be exacerbated across all three variables in the group that consumed 1g per kg of body weight immediately post-exercise. Whilst this research provides new insights into the effect of alcohol consumption on post-exercise muscle recovery, further research is required to ascertain how this relationship exists and establish the physiological mechanisms governing this response.

Conclusion

Both the affects of alcohol on human physiology and the parameters that determine athletic performance are multi-factorial and extremely complicated. A significant body of literature has established an array of adverse symptoms caused by acute alcohol ingestion. However the notion that alcohol consumption effects performance has not received enough consistent validation to advance beyond being anecdotal. Nevertheless, just because alcohol is not yet comprehensively shown to have a negative influence on performance, does not imply this review advocates its use prior to, or following competition. Indeed, the data demonstrates a severe lack of analysis on the possible detrimental action of alcohol in the recovering athlete. However, based on the available experimental evidence in cellular and rodent-models, athletes should remain wary of ingesting alcohol following intense exercise, focusing instead on effective dietary strategies proven to enhance recovery.

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© 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an Open Access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

by EricT

Dietary Protein and Kidney Function

Thu, 29 Jul 2010 19:11:53 +0000

by William F Martin¹, Lawrence E Armstrong² and Nancy R Rodriguez³

Nutrition & Metabolism, 2005

Recent trends in weight loss diets have led to a substantial increase in protein intake by individuals. As a result, the safety of habitually consuming dietary protein in excess of recommended intakes has been questioned. In particular, there is concern that high protein intake may promote renal damage by chronically increasing glomerular pressure and hyperfiltration. There is, however, a serious question as to whether there is significant evidence to support this relationship in healthy individuals. In fact, some studies suggest that hyperfiltration, the purported mechanism for renal damage, is a normal adaptative mechanism that occurs in response to several physiological conditions. This paper reviews the available evidence that increased dietary protein intake is a health concern in terms of the potential to initiate or promote renal disease. While protein restriction may be appropriate for treatment of existing kidney disease, we find no significant evidence for a detrimental effect of high protein intakes on kidney function in healthy persons after centuries of a high protein Western diet.

Dietary protein Intake and Kidney Function

Dietary protein intake can modulate renal function [1] and its role in renal disease has spawned an ongoing debate in the literature. At the center of the controversy is the concern that habitual consumption of dietary protein in excess of recommended amounts promotes chronic renal disease through increased glomerular pressure and hyperfiltration [2,3]. Media releases often conclude that, "too much protein stresses the kidney" [4]. The real question, however, is whether research in healthy individuals supports this notion. In fact, studies suggest that hyperfiltration in response to various physiological stimuli is a normal adaptative mechanism [5-10].

The purpose of this paper is to review the available evidence regarding the effects of protein intake on renal function with particular emphasis on renal disease. This review will consider research regarding the role of dietary protein in chronic kidney disease, normal renal function and kidney stone formation and evaluate the collective body of literature to ascertain whether habitual consumption of dietary protein in excess of what is recommended warrants a health concern in terms of the initiation and promotion of renal disease. In the following review, high protein (HP) diets will be defined as a daily consumption of greater than or equal to 1.5 g/kg/day, which is almost twice the current Recommended Dietary Allowance but within the range of current Dietary Reference Intakes (DRIs) for protein [11]. The Institute of Medicine DRI report concluded that there was insufficient scientific evidence for recommendations of an upper limit of protein intake but suggested an acceptable macronutrient distribution range of 10–35% of total energy for protein intake [11].

While the optimal ratio of macronutrient intake for adults has typically focused on fat and carbohydrate [12], contemporary discussions include the role of dietary protein [13-15]. This is particularly true given the recent popularity of high protein diets in weight management [16]. Although the efficacy of these diets with regard to weight loss is still subject to debate, several studies have demonstrated favorable physiological effects [12,16-24]. This has led to a substantial increase in protein intake by individuals adhering to contemporary weight loss plans. As a result, the safety of habitually consuming dietary protein in excess of the Recommended Daily Allowance (RDA) has been questioned.

An Overview of Chronic Kidney Disease

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Chronic Kidney Disease (CKD) is defined as either kidney damage or a decline in renal function as determined by decreased glomerular filtration rate (GFR) for three or more months [25]. It is estimated that 1 in 9 adults in the United States meet this criteria, while an additional 1 in 9 adults are at increased risk for CKD [26]. In the general population, a decline in renal function is considered an independent risk factor for both cardiovascular disease and all-cause mortality [27]. However, the extent to which a mild diminution in renal function influences this risk is not known [28].

According to the National Kidney Foundation guidelines, CKD is classified into five stages, each of which directly correlates with the severity of the disease [25]. As one progresses from stage 1 to 5 there is a concomitant decline in GFR and thus renal function. The final stage, known as end stage renal disease, represents the most severe manifestation of CKD [29]. This classification system provides a universal standard for application of clinical treatment guidelines.

Hypertension is the second leading cause of CKD and accounts for approximately 30% of all cases in the U.S. [30,31]. In one study, hypertension was associated with a premature decline in renal function in men with normal kidney function [32]. Although, initial estimates of CKD prevalence in hypertensive individuals were about 2%, recent evidence suggests that prevalence rates may be significantly higher [33]. Blood pressure control is of particular importance in hypertensive individuals with CKD. This point has been demonstrated in several trials in which antihypertensive therapy slowed the progression of CKD [34-36].

Race, gender, age and family history are four risk factors for CKD [37-40]. Recent findings suggest that modifiable lifestyle risk factors (i.e., physical inactivity, smoking, obesity) are also associated with CKD. Limited data exist regarding the role of dietary protein intake as an independent risk factor for either the initiation or progression of renal disease but population studies have consistently demonstrated an inverse relationship between dietary protein intake and systemic blood pressure [41,42]. In a randomized control trial [43], dietary protein and fiber had additive effects in lowering 24-hour and awake systolic blood pressure in a group of 36 hypertensives. While these findings suggest that high protein diets may be beneficial to hypertensive individuals, additional research is warranted since increased protein intakes often result in increased consumption of certain micronutrients known to impact blood pressure (e.g., potassium, magnesium, calcium) [44].
Dietary protein and renal function

The relationship between dietary protein and renal function has been studied for over half a century [1]. In 1923, Addis and Drury [45] were among the first to observe a relationship between level of dietary protein and rates of urea excretion. Soon after, it was established that increased protein intake elevated rates of creatinine and urea excretion in the dog model [46]. The common mechanism underlying increased excretion rates was eventually attributed to changes in GFR [47,48] and Van Slyke et al. [49] demonstrated that renal blood flow was the basis for GFR mediated changes in clearance rates in response to increased protein intake. Clearly dietary protein effects GFR [50], with both acute and chronic increases in protein consumption elevating GFR [50,51].

Dietary Protein and the Progression of renal disease

Observational data from epidemiological studies provide evidence that dietary protein intake may be related to the progression of renal disease [52]. In the Nurses' Health Study, protein intake, assessed with a semi-quantitative food frequency questionnaire, was compared to the change in estimated GFR over an 11-year span in individuals with pre-existing renal disease [53]. Regression analysis showed an association between increased consumption of animal protein and a decline in kidney function suggesting that high total protein intake may accelerate renal disease leading to a progressive loss of renal capacity. However, no association between protein intake and change in GFR was found in a different cohort of 1,135 women with normal renal function (Figure 1.). The latter finding led the authors to conclude that there were no adverse effects of high protein intakes on kidney function in healthy women with normal renal status.

Figure 1: This figure is a plot of multivariate linear regression for change in estimated GFR according to quintile of total protein intake* in participants with normal renal function (n = 1135). Data are taken from Knight et al., Ann Intern Med 2003 Mar 18;138(6):460-7 [53].

Research by Johnson et al. [54], showed protein intake as a possible risk factor for progressive loss of remaining renal function in dialysis patients. Indeed, dietary protein restriction is a common treatment modality for patients with renal disease [55,56] and practice guidelines exist regarding reduced dietary protein intakes for individuals with chronic renal disease in which proteinuria is present [57]. The National Kidney Foundation (NKF) has extensive recommendations with regard to protein intake, which are a byproduct of the Dialysis Outcome Quality Initiative [58]. Again, it is important to note that these recommendations are not indicated for individuals with normal renal function nor are they intended to serve as a prevention strategy to avoid developing CKD. Despite the clarity of these guidelines, their mere existence has resulted in concern regarding the role of dietary protein in the onset or progression of renal disease in the general population [59].
Dietary protein and renal disease

Allen and Cope's observation that increased dietary protein induced renal hypertrophy in dogs [60] led to speculation that dietary protein intake may have deleterious effects on the kidney. Later research in the rat model produced evidence supporting earlier observations from canine research [61-63]. Recently, Hammond and Janes [64] demonstrated an independent effect of increased protein intake on renal hypertrophy in mice. In this study, changes in renal function (i.e., increased glomerular filtration rate and renal hypertrophy) were observed.

Currently, a combination of hormonal interactions and renal processes are thought to explain protein-induced hyperfiltration [65]. Increased glucagon secretion in response to protein administration induced hyperfiltration [66] subsequent to a cascade of events referred to as the"pancreato-hepatorenal cascade" [67]. It has been hypothesized that cAMP works in concert with glucagon to mediate GFR [68]. To date, however, this hypothesis has not been tested and other competing hypotheses suggest other novel mechanisms of protein-induced hyperfiltration [69].

While the effect of hyperfiltration on renal function in those individuals with pre-existing renal disease is well documented [52], the application of these observations to healthy persons with normal renal function is not appropriate. To date, scientific data linking protein-induced renal hypertrophy or hyperfiltration to the initiation or progression of renal disease in healthy individuals is lacking. The possibility that protein-induced changes in renal function are a normal physiological adaptation to nitrogen load and increased demands for renal clearance is supported by changes noted in renal structure and function during pregnancy [70]. GFR increases by as much as 65% in healthy women [8] during pregnancy, typically returning to nonpregnant levels by three months postpartum [7]. Despite these changes in renal function, pregnancy is not a risk factor for developing CKD [6].

The renal hypertrophy and accompanying improvements in renal function in the contralateral kidney that occur subsequent to unilateral nephrectomy also suggest these processes are an adaptive, and possibly beneficial, response [5]. Studies show, despite prolonged hyperfiltration, remnant kidney function remained normal and did not deteriorate during long-term (> 20 yrs) follow-up in nephrectomized patients [9,10]. Thus, compensatory hyperfiltration appears to be a biological adaptation to a variety of renal challenges that is not associated with increased risk of chronic kidney disease in healthy individuals.
The Brenner Hypothesis

Perhaps the most consistently cited reference with regard to the potentially harmful effects of dietary protein intake on renal function is that of Brenner et al. [3]. In brief, the Brenner Hypothesis states that situations associated with increased glomerular filtration and glomerular pressure cause renal injury, ultimately compromise renal function, and potentially increase the risk for or progression of renal disease. Brenner proposed that habitual consumption of excessive dietary protein negatively impacted kidney function by a sustained increased in glomerular pressure and renal hyperfiltration [3]. Since the majority of scientific evidence cited by the authors was generated from animal models and patients with co-existing renal disease, extension of this relationship to healthy individuals with normal renal function is inappropriate. Indeed, a relationship between increased glomerular pressure or hyperfiltration and the onset or progression of renal disease in healthy individuals has not been clearly documented in the scientific literature. Rather, findings from individuals with compensatory hyperfiltration during pregnancy and following unilateral nephrectomy suggest otherwise [9].

The Modification of Diet in Renal Disease (MDRD) study was the largest randomized multicenter, controlled trial undertaken to evaluate the effect of dietary protein restriction on the progression of renal disease [71]. Several variables, including GFR, were measured in patients with chronic renal disease at baseline and throughout the approximately 2 year follow-up period. Patients with renal disease randomized to the very low-protein diet group had slightly slower decline in GFR decline compared with patients randomized to the low-protein diet group. Further data analyses showed patients with lower total protein intake would have a longer time to renal failure and suggested that a lower protein intake postponed the progression of advanced renal disease. Using meta-analysis to assess the efficacy of dietary protein restriction in previously published studies of diabetic and nondiabetic renal diseases, including the MDRD Study, Pedrini et al. concluded that the progression of both nondiabetic and diabetic renal disease could be effectively delayed with restriction of dietary protein [56]. Indeed, current clinical guidelines for the management of patients with renal disease continue to be based on the premise that protein intake greater than that recommended or which results in a renal solute load in excess of the kidney's excretory capabilities will contribute to progressive renal failure in persons with compromised renal function. However, of significance to this review, is the fact that imposing these guidelines on healthy individuals with normal renal function is overzealous given the current status of the scientific literature in this area.
Dietary protein and renal strain

Concerns about level of dietary protein and renal function are often presented in public health guidelines [59]. In addition to the claims that high protein intake causes renal disease, some studies have suggested that renal function may be negatively affected by routine consumption of high protein diets [72-75]. Although high protein diets cause changes in renal function (i.e., increased GFR) and several related endocrine factors [1,76,77] that may be harmful to individuals with renal disease [52,53], there is not sufficient research to extend these findings to healthy individuals with normal renal function at this time.

The lay public is often told that high protein diets "overwork" the kidney and may negatively impact renal function over time [78]. In addition, a number of highly regarded organizations appear to support this line of reasoning [79] given the physiological processes required for excretion of protein-related metabolic waste products to maintain homeostasis following consumption of protein at levels in excess of recommended amounts. Increased consumption of dietary protein is linearly related to the production of urea [80] and urea excretion is controlled by the kidney. These processes are of significant energetic cost to the kidney and represent the physiological "strain" associated with increased protein intake [81].

The word "strain" is misleading given its negative connotation. In a press release [82], one group asserted that increased dietary protein "strains" the kidney via increased urea production, and causes dehydration and accumulation of blood urea nitrogen. This press release also suggested that these events synergistically overwork the kidney and predispose humans to CKD. Scientific research is often misrepresented in this context. Research from our laboratory [83] which is cited in the press release, does not support these contentions. Rather, we found that habitual consumption of a high protein diet minimally affected hydration indices. Changes in total body water and renal function were not measured.

The concept that increased dietary protein leads to dehydration may have originated from an unsubstantiated extension of a 1954 review of the nitrogen balance literature [84]. This review focused on the design of survival rations for military operations in the desert or at sea, when water supply and energy intake are limited. Since the excretion of 1 gram of urea nitrogen requires 40 – 60 mL of additional water, increased protein intakes in the study translated into an increased water requirement (i.e., +250 mL water per 6 grams of dietary nitrogen in a 500 Kcal diet) for excretion of urea nitrogen. This increased fluid requirement is situation specific and is not necessarily applicable to individuals whose calorie and water intakes are adequate. Presently, we know of no studies executed in healthy individuals with normal renal function which demonstrate a clear relation between increased dietary protein intake and dehydration or a detrimental "strain" on the kidney. Therefore, claims that a high protein diet promotes dehydration or adversely "strains" the kidney remain speculative.

Evidence in Healthy Individuals

Although the efficacy of high protein diets for weight loss has been evaluated, there have been no reports of protein-induced diminutions in renal function despite subject populations that are generally at risk for kidney disease (e.g., dyslipidemia, obesity, hypertension) [14,15,22,85-87]. A randomized comparison of the effects of high and low protein diets on renal function in obese individuals suggested that high protein diets did not present a health concern with regard to renal function their study population [65]. In this study, 65 overweight, but otherwise healthy, subjects adhered to a low or high protein diet for six months. In the high protein group, both kidney size and GFR were significantly increased from that measured at baseline. No changes in albumin excretion were noted for either group and the authors concluded that, despite acute changes in renal function and size, high protein intake did not have detrimental effects on renal function in healthy individuals. Similar findings were recently reported by Boden et al. [88] in a study of 10 subjects who consumed their typical diet for 7 days followed by strict adherence to a high protein diet for 14 days. No significant changes were noted in serum or urinary creatinine and albumin excretion, suggesting no ill-effects of a high protein diet on renal function.

Athletes, particularly in sports requiring strength and power, consume high levels of dietary protein [89,90]. In fact, many athletes habitually consume protein in excess of 2.0 g/kg/day [91]. Supplementation with amino acids will further increase dietary protein levels in these individuals [92]. Yet there is no evidence that this population is at greater risk for kidney disease or losses in renal function [90]. Poortsmans and Dellalieux [93] found that protein intakes in the range of ~1.4–1.9 g/kg/day or 170–243% of the recommended dietary allowance did not impair renal function in a group of 37 athletes. We found no data in the scientific literature to link high protein intakes to increased risk for impaired kidney function in healthy, physically active men and women.

Dietary Protein and Kidney function in Animal Models

Although there is limited research regarding the long-term effects of high protein intakes on renal function in humans, animal models have provided insight into this quandary. Mammals fed acute and chronic high protein diets exhibit increases in GFR and renal blood flow [94]. These changes, which are comparable to those observed in humans, led to the hypothesis that high protein intakes are associated with progressive glomerulosclerosis in the rat. Recently, Lacroix et al. [95] studied the effects of a diet containing 50% protein on renal function in Wistar rats and noted no abnormalities in renal function or pathology. Collins et al. [96] also reported no adverse effects of long-term consumption of high protein diets on renal function when two years of a diet containing 60% protein failed to evoke changes in the percentage of sclerotic glomeruli in rats. Robertson et al., [97] studied the effect of increased protein intake on hyperperfusion and the progression of glomerulosclerosis in dogs that were 75% nephrectomized. After four years of feeding diets that were either 56, 27 or 19% protein, no association between diet and structural changes in the kidney were observed.

To the best of our knowledge, there has been only one report of a potentially toxic effect of excessive protein intake on renal function in the rat. Stonard et al. [98] found a diet containing 33% protein produced tubular damage in a specific strain of female rats. However, findings from this study are limited by the fact that damage was induced by a bacterial single-cell protein (Pruteen).

In summary, studies documenting high protein intake as a cause of renal disease in any animal model have not been done. Rather, studies have typically focused on the interaction between protein intake and renal function in the diseased state. As a result, findings from these investigations should not be used as a basis for dietary recommendations for humans. Studies designed to characterize the effects of dietary protein intake on renal function in healthy subjects are warranted.
Dietary protein and kidney stones

The role of high protein diets in kidney stone formation has received considerable attention. Excessive protein intake increases excretion of potentially lithogenic substances such as calcium and uric acid [99,100]. Reddy et al. [101] noted that consumption of a high protein diet for six weeks was associated aciduria and urinary calcium and claimed that this constituted increased risk of stone formation in ten healthy subjects although none of the ten subjects developed renal stones. The severe carbohydrate restriction imposed in this study may have increased keto-acid production thereby contributing acid formation. Since consumption of fruits and vegetables usually produces a marked base load [102], restriction of these foods subsequent to the diet intervention may have also contributed to the net acid load.

Studies that claim an increased propensity for stone formation as a result of increased protein intake should be taken at face value because propensity is a surrogate marker and does not represent actual stone formation. Further, randomized control trials have not been done to test whether an increased tendency for stone formation is enhanced with consumption of a high protein diet.

Epidemiological studies provide conflicting evidence with regard to the association between protein intake and the predisposition for kidney stone formation. In a prospective study of over 45,000 men, researchers found a direct correlation between animal protein intake and risk of stone formation [103]. However, findings in women are difficult to interpret due to conflicting reports in the literature. While some studies have shown a direct relationship between animal protein intake and risk of stone formation in women [104,105], other work suggests an inverse relationship exists [106].

Conflicting findings regarding the role of dietary protein in kidney stone formation limit the development of universal guidelines with regard to a recommended protein intake for individuals at increased risk for stone formation [107]. It is not likely that diet alone causes kidney stone formation [108]. Rather, metabolic abnormalities are typically the underlying cause [109]. For example, Nguyen et al. [110] found that high intakes of animal protein adversely affected markers of stone formation in those afflicted with a stone causing disorder, while no changes were observed in healthy individuals. It has been suggested that one must have a preexisting metabolic dysfunction before dietary protein can exert an effect relative to stone formation [108]. This notion has been coined the "powderkeg and tinderbox" theory of renal stone disease by Jaeger [111]. This theory asserts that dietary excesses, such as high protein intake, serve as a tinderbox which, only in tandem with a metabolic abnormality (the powderkeg), can bring about stone formation. At the present time, however, evidence showing that a high protein intake is an inherent cause of this renal abnormality or is consistently associated with increased kidney stone formation does not exist.

Conclusion

Although excessive protein intake remains a health concern in individuals with pre-existing kidney disease, the literature lacks significant research demonstrating a link between protein intake and the initiation or progression of kidney disease in healthy individuals. More importantly, evidence suggests that protein-induced changes in renal function are likely a normal adaptative mechanism well within the functional limits of a healthy kidney. Without question, long-term studies are needed to clarify the scant evidence currently available regarding this relationship. At present, there is not sufficient proof to warrant public health directives aimed at restricting dietary protein intake in healthy adults for the purpose of preserving renal function.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

WFM conducted literature search, prepared the manuscript and assisted in presentation of final draft, LEA and NRR conceived the idea, organized contents and participated in preparation of final manuscript.

© 2005 Martin et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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by EricT

The Other Side Of Dogma - Alternative a Euphemism for Untested?

Wed, 21 Jul 2010 17:21:26 +0000

My last post about strength training and nutrition dogma dealt with the downside of the popular and untested beliefs that we cling to in the face of little to no evidence. Even so I pointed out that not all beliefs which appear to be dogmatic are "bad". Well, it just so happens that I think there are worse things than dogma.

The thing is that just because there are beliefs that people in the strength training and nutrition industries believe are not to be disputed does not mean they are wrong! Shouting DOGMA, DOGMA, is not a valid argument. Just because your opponent aligns himself with the popular doctrines does not mean you are correct in your beliefs by default.

Being able to recognize when you or someone else is clinging to dogma is a good thing. But recognizing when dogma has taken the place of critical thought is only the first step.

Alternative Sanctimony

One attitude that irritates me more than most is a "sanctimonious" attitude. Recently on another forum I frequent a new member posted on a very old thread and tried to dismiss the popular opinion expressed in that thread by assigning emotion to those who expressed it. Basically, everything he said was "Vulcan Logic" and everything everyone else said was because he had "hit a nerve". So my reaction was to begin pointing out every logical fallacy this fellow committed. Not because I think that knowing a persons argumentative fallacies or recognizing their cognitive biases wins an argument - it doesn't - but because I wanted to fight fire with fire. You see, those who think themselves to be immune to fallacy are setting themselves up to be taken down by their own logic! Because nobody is immune to fallacy and when a discussion is complex enough you can always find them on both sides!

The claim of dogma is of that vein. The sanctimonious cry of dogma in response to every 'mainstream' belief is not more relevant or justified than to cry crank or zealot. Popular beliefs are not always true. Alternative beliefs, however, are even more unlikely to be mistaken. When it comes to alternative medicine, for instance, alternative is a euphemism for untested and without credible evidence. Some of the most popular and influential "thinkers" are those who always express "alternative" opinions. They lead people to believe that they are right just by virtue of not being swayed by mainstream beliefs. It is amazing how powerful this is. "Dr" Mercola is considered to have more authority by many than their own doctor.

Even a token effort to investigate the information sources of people like Mercola should persuade most from being a blind follower of the alternative. The problem is most don't bother to check up on their heroes.

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Nutritionists a Frequent Target

Let's look at nutrition. Some of the most popular "strength coaches" around fancy themselves as nutrition experts and yet seem to have little to no knowledge of the subject. For every Jamie Hale or Alan Aragon there are one hundred others who would profess the same level of expertise. However, what these would-be experts actually express is a contrariness. There is a certain arrogance at work here. Nutritionists are wrong. I know more than nutritionists. Therefore I am a nutrition expert.

Look at the terminology. The word "nutritionist" is the nutrition equivalent of mainstream. All things mainstream are "dogmatic" and therefore wrong. Nutritionists are therefore wrong and by opposing them I am therefore right. So I am a nutrition expert! I'm not making this up. I've seen these very opinions expressed by the likes of Mike Boyle, for instance, who basically declared that all nutritionists are idiots and that he knew more about nutrition than any nutritionist. Boyle even went so far as to claim that Michael Pollen would be a better source of nutrition information than a nutritionist. Which would mean that "The Omnivore's Dilemma" would be a better source than a nutrition textbook since such books are, of course, written by nutritionists. Yet Pollen's book is without the slightest scientific merit as far as nutrition goes (the next few posts will discuss this).

But what's a nutritionist? Well nutritionist is nothing but a word. Look around and you will find that more people declare nutritionists to be dogmatists than "dietitians" for example. That is because nutritionist, lacking a standardized or "official" definition is a better target. You can have a PhD in biology, for instance, but devote your career to nutrition. You can lack a university degree of any kind and be a great and influential nutritionist. If we were going to pull a definition out of this then I think that one characteristic of a nutritionist would be that he or she spent most of their time studying and consulting about nutrition! So, would you rather take your nutrition advice from someone who specializes in nutrition or someone who arrogantly asserts that he is smarter than these people even though he spends very little of his time actually studying nutrition?

The kind of arrogance that leads one to believe that they are superior to nutritionists because of a sterotyped image of nutritionists being dogmatic is the same type of arrogance that leads one to believe that their beliefs are superior owing to the simple fact that they are not popular. Being different does not make us superior or correct and when it comes to science. The strength of a theory does not rest on how different it is from other theories.!

As you can see from this forum thread on trans fats the high horse attitude that many in the fitness industry bring to health and wellness is not an attitude I appreciate. As I was writing my responses in that thread I began to wonder if the phrase "that's easy for you to say" wouldn't be a good test for "alternative" opinions. Just how 'invested' is the person in the subject being discussed? Having read one book expressing alternative ideas about nutrition does not give one the stake in nutrition that someone has who has spent their career studying the subject. Likewise, having read a couple of studies related to the subject at hand does not give one the means to cure someone's high blood pressure.

This kind of arrogance leads people to never question their own conclusions no matter how quick they have jumped to them. Even the suggestion that omega-3 fatty acids EPA and DHA have an anti-inflammatory role has lead "experts" to advise arthritis sufferers to ditch their medication and pop fish oil capsules instead. Whereas nobody who was actually invested in these patients would discontinue all medication all at once due to the addition of a dietary supplement.

But it strikes deeper than that. As I point out in the thread, many opinions about nutrition and health coincide with value judgements. I know that "that's easy for you to say" is not a logical argument but when you consider the things that the fitness industry professes to have the answers to you can see how I would ask the question. Many of them are telling you they have the answers that your doctor doesn't. Yet, despite all the criticisms of the medical community, a doctor finds his patient's life in his hands quite often. So when some guy on an internet blog claims to know more than the M.D. maybe you should consider that his investment is likely not much more than a few strokes on a keyboard and some internet browsing! This is not an appeal to authority, although it may seem so at first glance.

I want you to notice that the term I am using, investment has nothing to do with degrees or other authority badges. Time, dedication, passion, experience, research, etc…is what I am talking about. What does it cost the individual to make the statements he or she is making? How does it affect those that seek their expertise? And in this industry there is one thing I value more than the ability to be right or simply to win arguments: genuine caring about whether what you do helps other people.

One Sound Argument

It should be clear that the sheer number of people who believe a certain thing does not make it true. But neither does "mass appeal" make it false. It simply means that the majority accepts a certain view. Only through our own sound thinking and weighing of evidence can we decide for ourselves what to believe while keeping in mind that the facts may change the next time you turn around. But remember that it only takes ONE sound argument to derail any view. There is an anecdote about Albert Einstein which says he once made jibes about a book condemning his theory of relativity. Supposedly one hundred Nazi professors contributed to the condemnation in said book. Einstein is reported to have said, "Were I wrong, one professor would have been enough." I think that about sums it up.¹

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by EricT

Nutrition is Not a True or False Proposition

Tue, 29 Jun 2010 20:18:26 +0000

By Eric Troy

I just came across a "nutrition quiz".

I got eight out of ten correct. Only eight? ME? You have got to be kidding me. I am a nutrition stud. Well one of them involved brown rice. Go figure. I knew they wanted to hear that brown rice was healthier than white rice so that is what I answered but really I refuse to capitulate to the nonsense about brown rice being magical health pellets and white rice being evil little starch monsters.

More of the native nutrition of the grain is retained in brown rice, sure but it's the glycemic index and "insulin spike that has 97 percent of bodybuilders thinking that white rice will blow you up like Harry Potter did his aunt. Which is all a bunch of baloney. White rice as part of a varied diet that consists of mixed food meals is quite fine. Remember it is the glycemic load as the meal as whole that counts, anyway. However, although glycemic load seems to have influence on various health parameters, it is not a very good fat loss tool. If you like white rice, eat white rice. Just don't eat TOO much whi…anything.

Here's a quiz/poll of my own. Only one question.

But here are the questions I got wrong.

What's the best food for improving eyesight?

Pomegranate
Collard greens
Carrots

Ok so you know how your mom told you that carrots will make you see better? Well no, they won't. Even though they contain cartenoids and especially beta carotene. The rub is, then, the word "improve". I thought that they wanted me to say collard greens because of the whole leafy green thing and lutein. But lutein has not been "shown" to make you see better either. The hype on lutein has to do with slowing down age related macular degeneration. If "improve" means not to go blind as fast then I guess lutein does improve eyesight, if you believe the studies. But I don't think slowing down the loss of eyesight through age related degeneration is quite the same as improving eyesight, do you? so out of shear stubborness I said carrots. Because they still have just as much claim to "eye health" as leafy greens. Unlikely though, that any one food will save your eyes.

The next one I got wrong is the one that really got under my skin and motivated this post. It was a true/false question. True/False questions are pretty much worthless for nutrtion and in this case, fat loss, folks. Because there are very few "trues" and "untrues" which equate to black and white.

True or False: Eating bananas (the "Monkey Diet") is a great way to stay slim?

True
False

I said false. It is false. The "Monkey Diet" is NOT a great way to stay slim. I got it wrong though. Because according to the quiz makers bananas are not fattening. Of course bananas are not fattening. But in what universe does that mean that bananas are "slimming" and the Monkey Diet is a good way to stay slim or lose weight? Sure you could lose weight eating mostly bananas because you'd probably take in much fewer calories and that will result in a net loss especially at first. But it's a terrible diet and it's doomed to failure. Just because the statement "bananas are fattening" is false does not make the inverse true! C'mon.

The Monkey Diet. Cuz monkeys don't get fat.
image by Chris May via flickr

Nutrition is not a true or false proposition. There are very few concrete answers. There are no "evil" foods. There are no superfoods. Every time someone takes a quiz such as this there is the potential for perpetuating the kind of myths the quiz hopes to discourage. Don't start thinking that 'entertainment' is the same as 'research'.

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by EricT

Health Benefits Of Nut Consumption

Sat, 26 Jun 2010 03:14:36 +0000

by Emilio Ros

Lipid Clinic, Endocrinology and Nutrition Service, Institut d’Investigacions Biomèdiques August Pii Sunyer, Hospital Clínic, Barcelona and Ciber Fisiopatología de la Obesidady Nutrición (CIBERobn), Instituto de Salud Carlos III (ISCIII), Spain

Nutrients 2010

Introduction

Nuts (tree nuts and peanuts) are nutrient dense foods with complex matrices rich in unsaturated fatty and other bioactive compounds: high-quality vegetable protein, fiber, minerals, tocopherols, phytosterols, and phenolic compounds. By virtue of their unique composition, nuts are likely to beneficially impact health outcomes. Epidemiologic studies have associated nut consumption with a reduced incidence of coronary heart disease and gallstones in both genders and diabetes in women. Limited evidence also suggests beneficial effects on hypertension, cancer, and inflammation. Interventional studies consistently show that nut intake has a cholesterol-lowering effect, even in the context of healthy diets, and there is emerging evidence of beneficial effects on oxidative stress, inflammation, and vascular reactivity. Blood pressure, visceral adiposity and the metabolic syndrome also appear to be positively influenced by nut consumption. Thus it is clear that nuts have a beneficial impact on many cardiovascular risk factors. Contrary to expectations, epidemiologic studies and clinical trials suggest that regular nut consumption is unlikely to contribute to obesity and may even help in weight loss. Safety concerns are limited to the infrequent occurrence of nut allergy in children. In conclusion, nuts are nutrient rich foods with wide-ranging cardiovascular and metabolic benefits, which can be readily incorporated into healthy diets.

Extensive research has been carried out on nuts and health outcomes during the last two decades since publication of a report from the pioneering Adventist Health Study showing an association of nut consumption with a lower risk of coronary heart disease (CHD) in 1992 [1], shortly followed by the seminal clinical trial of Sabaté et al. [2] demonstrating that a diet enriched with walnuts reduced serum cholesterol levels compared to a standard healthy diet. The interested reader will find complete information regarding research published up to 2005 on nuts and health outcomes in a recent monograph [3] and up to 2007 in the proceedings of a Symposium on Nuts and Health held at the U.S. Department of Agriculture Research Laboratory in the University of California at Davis [4].

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By definition, tree nuts are dry fruits with one seed in which the ovary wall becomes hard at maturity. The most popular edible tree nuts are almonds (Prunus amigdalis), hazelnuts (Corylus avellana), walnuts (Juglans regia), and pistachios (Pistachia vera). Other common edible nuts are pine nuts (Pinus pinea), cashews (Anacardium occidentale), pecans (Carya llinoiensis), macadamias (Macadamia integrifolia), and Brazil nuts (Bertholletia excelsa). The consumer definition also includes peanuts (Arachis hypogea), which botanically are groundnuts or legumes but are widely identified as part of the nuts food group. In addition, peanuts have a similar nutrient profile to tree nuts [5,6]. Although chestnuts (Castanea sativa) are tree nuts as well, they are different from all other common nuts because of being starchier and having a different nutrient profile. For the purpose of this review, the term “nuts” includes all common tree nuts [with the exception of chestnuts] plus peanuts.

Nuts, seeds and pulses are all nutrient dense foods and have been a regular constituent of mankind’s diet since pre-agricultural times [7]. In Western countries nuts are consumed as snacks, desserts or part of a meal, and are eaten whole (fresh or roasted), in spreads (peanut butter, almond paste), as oils or hidden in commercial products, mixed dishes, sauces, pastries, ice creams and baked goods. In the last century, nut consumption in most industrialized nations followed a downward trend to become only a marginal source of energy in the daily diet, except for vegetarians and other health-conscious populations, such as Seventh Day Adventists [8,9].

However, nut consumption has increased in recent times in Western countries following both the inclusion of this food group in many guidelines for healthy eating and wide media coverage of recent evidence connecting nut consumption to a wide range of health benefits. Thus, nuts have been proposed as an important component of optimal diets for the prevention of CHD by leading experts in the field [10] and, in the summer of 2003, the US Food and Drug Administration issued a health claim for nuts because of the link between nut consumption and a reduced risk of both CHD and intermediate biomarkers, such as blood cholesterol [11]. Since then, nuts have become an indispensable component of healthy diets [12,13] and are included in the American Heart Association dietary metrics for defining ideal cardiovascular health in their recent report on setting goals for health promotion and disease reduction for 2020 [14].

The scientific evidence behind the proposal of nuts as cardio-protective foods stem from both epidemiological observations suggesting a consistent inverse association between the frequency of nut intake and development of CHD [reviewed in 13,15,16] and numerous short-term clinical trials showing beneficial effects of nut intake on the lipid profile [reviewed in 13,17-20] and other intermediate markers of CHD [reviewed in 13,20-22]. The mechanism for these salutary effects probably lies in the synergistic interaction of the many bioactive constituents of nuts, which may allfavorably influence human physiology. Thus, nuts contain high amounts of vegetable protein [5] and fat, mostly unsaturated fatty acids [6]. They are also dense in a variety of other nutrients and provide dietary fiber [23], vitamins (e.g., folic acid, niacin, tocopherols, and vitamin B6), minerals (e.g., calcium, magnesium, potassium) and many other bioactive constituents such as phytosterols [24] and phenolic compounds [25].

Contrary to expectations due to the high energy density of nuts, evidence from both epidemiological studies and clinical trials suggests that their regular consumption neither contributes to obesity nor increases the risk of developing diabetes, as reviewed [13,16,26,27]. The present review summarizes current knowledge on the expanding topic of nuts and health outcomes. First an outline of the unique nutrient content of nuts is necessary in order to better understand their health effects.

Nutrient Content of Nuts

Nuts are clearly nutrient dense foods. With the exception of chestnuts, which contain little fat, nuts have a high total fat content, ranging from 46% in cashews and pistachios to 76% in macadamia nuts, and they provide 20 to 30 kJ/g (Table 1). Thus, nuts are one of the natural plant foods richest in fat after vegetable oils. However, the fatty acid composition of nuts is beneficial because the saturated fatty acid (SFA) content is low (4–16%) and nearly half of the total fat content is made up of unsaturated fat, monounsaturated fatty acids MUFA (oleic acid) in most nuts, similar proportions of MUFA and polyunsaturated fatty acids (PUFA), mostly linoleic acid, in Brazil nuts, a predominance of PUFA over MUFA in pine nuts, and mostly PUFA, both linoleic acid and a-linolenic acid (ALA), the plant omega-3 fatty acid, in walnuts [6]. With regard to walnuts, it must be underlined that they are a whole food with the highest content in ALA of all edible plants [28]. As discussed below, the particular lipid profile of nuts in general and walnuts in particular is likely to be an important contributor to the beneficial health effects of frequent nut consumption.

Nuts are also rich sources of other bioactive macronutrients that have the potential to beneficially affect metabolic and cardiovascular outcomes. They are an excellent source of protein (approximately 25% of energy) and often have a high content of L-arginine [5]. As this amino acid is the precursor of the endogenous vasodilator, nitric oxide (NO) [29], nut intake might help improve vascular reactivity, as discussed below. Nuts also are a good source of dietary fiber, which ranges from 4 to 11 g per 100 g (Table 1), and in standard servings provide 5–10% of daily fiber requirements [23].

Table 1. Average nutrient composition of nuts (per 100 g).

Nuts	Energy (kJ)	Fat (g)	SFA(g)	MUFA (g)	PUFA (g)	LA (g)	ALA (g)	Protein (g)	Fiber (g)	Folate (μg)	PS (mg)
Almonds	2418	50.6	3.9	32.2	12.2	12.2	0.00	21.3	8.8	29	120
Brazil nuts (dried)	2743	66.4	15.1	24.5	20.6	20.5	0.05	14.3	8.5	22	NR
Cashews	2314	46.4	9.2	27.3	7.8	7.7	0.15	18.2	5.9	25	158
Hazelnuts	2629	60.8	4.5	45.7	7.9	7.8	0.09	15.0	10.4	113	96
Macadamia nuts	3004	75.8	12.1	58.9	1.5	1.3	0.21	7.9	6.0	11	116
Peanuts	2220	49.2	6.8	24.4	15.6	15.6	0.00	25.8	8.5	145	220
Pecans	2889	72.0	6.2	40.8	21.6	20.6	1.00	9.2	8.4	22	102
Pine nuts (dried)	2816	68.4	4.9	18.8	34.1	33.2	0.16	13.7	3.7	34	141
Pistachios	2332	44.4	5.4	23.3	13.5	13.2	0.25	20.6	9.0	51	214
Walnuts	2738	65.2	6.1	8.9	47.2	38.1	9.08	15.2	6.4	98	72

Data for raw nuts, except where specified. SFA, saturated fatty acids; MUFA, monounsaturated
fatty acids; PUFA, polyunsaturated fatty acids; LA, linoleic acid; ALA, a-linolenic acid; PS, plant
sterols; NR, not reported.
Source: US Department of Agriculture Nutrient Data Base at: http://www.nal.usda.gov/fnic/
cgi-bin/nut_search.pl (Accessed on 26 April 2010).

Among the constituents of nuts there are significant amounts of essential micronutrients that are associated with an improved health status when consumed at doses beyond those necessary to prevent deficiency states. Nuts contain sizeable amounts of folate (Table 1) [24], a B-vitamin necessary for normal cellular function that plays an important role in detoxifying homocysteine, a sulfur-containing amino acid with atherothrombotic properties that accumulates in plasma when folate status is subnormal [30].

Nuts are also rich sources of antioxidant vitamins (e.g., tocopherols) and phenolic compounds, necessary to protect the germ from oxidative stress and preserve the reproductive potential of the seed, but also bioavailable after consumption and capable of providing a significant antioxidant load [25]. Almonds in particular are especially rich in a-tocopherol, while walnuts contain significant amounts of its isomer g-tocopherol, which has been investigated much less than atocopherol, but is increasingly recognized as a relevant antiatherogenic molecule [31]. Remarkably, in all nuts most of the antioxidants are located in the pellicle or outer soft shell, as shown for almonds [32,33] and peanuts [34], and 50% or more of them are lost when the skin is removed [25]. Bleaching of nuts when the hard shells are cracked, as it occurs naturally in pistachios, also destroys most of the antioxidants [35]. Interestingly, roasting of almonds preserves the phenolic compounds better than blanching [36]. These facts, rarely taken into consideration in prior studies with nuts, should not be overlooked when giving advice on nut intake in healthy diets. Walnuts are an exception because they are almost always consumed as a raw, unpeeled product.

Nuts are cholesterol-free, but their fatty fraction contains sizeable amounts of chemically related noncholesterol sterols belonging to a heterogeneous group of compounds known as plant sterols or phytosterols (Table 1) [24]. They are non-nutritive components of all plants that play an important structural role in membranes, where they serve to stabilize phospholipid bilayers just as cholesterol does in animal cell membranes [37]. Phytosterols interfere with cholesterol absorption and thus help lower blood cholesterol when present in sufficient amounts in the intestinal lumen. The mechanism of action of phytosterols has been linked to their hydrophobicity, which is higher than cholesterol because of a bulkier hydrocarbon molecule and entails a higher affinity for micelles than has cholesterol. Consequently, cholesterol is displaced from micelles and the amount available for absorption is limited [36]. In all probability the phytosterol content of nuts contributes to their cholesterol-lowering effect (see below).

Compared to other common foods, nuts have an optimal nutritional density with respect to healthy minerals, such as calcium, magnesium, and potassium. Like that of most vegetables, the sodium content of raw or roasted but otherwise unprocessed nuts is very low, ranging from undetectable in hazelnuts to 18 mg/100 g in peanuts (Table 2) [24]. A high intake of calcium, magnesium and potassium, together with a low sodium intake, is associated with protection against bone demineralization, arterial hypertension, insulin resistance, and overall cardiovascular risk [39]. Obviously, the advantage of the low sodium content of nuts is lost if they are consumed as a salted product.

In summary, the macronutrient, micronutrient and non-nutrient components of nuts shown in Table 1 and Table 2 have all been documented to contribute to a reduced risk of CHD and related metabolic disturbances. For these reasons, whole raw, unpeeled and otherwise unprocessed nuts may be considered as natural health capsules, where the whole is always better than the parts [40].

Table 2. Calcium, magnesium, sodium and potassium content of nuts and other foods in
mg/100 g of edible portion.

Nuts	Calcium	Magnesium	Sodium	Potassium
Almonds	248	275	1	728
Brazil nuts	160	376	3	659
Cashew nuts	37	292	12	660
Hazelnuts	114	163	0	680
Macadamia nuts	85	130	5	368
Peanuts	92	168	18	705
Pecans	70	121	0	410
Pine nuts	16	251	2	597
Pistachios	107	121	1	1025
Walnuts	98	158	2	441
Other foods	Calcium	Magnesium	Sodium	Potassium
Apples (with skin)	6	5	1	107
Bananas	5	27	1	358
Beans (white, cooked)	90	63	6	561
Broccoli (cooked)	40	21	41	293
Cheese (cheddar)	721	28	621	98
Chickpeas (cooked)	49	48	7	291
Ham	24	22	1304	287
Lettuce (romaine)	33	14	8	247
Milk	113	10	40	143
Rice (white, cooked)	10	38	1	35
Sardines (canned in oil)	382	39	505	397
Spinach (cooked)	136	87	70	466
Tomato	10	11	5	237
Veal (cooked)	22	26	87	325
Wheat bread	72	86	527	252

Epidemiological Studies of Nut Consumption and Health Outcomes

Nut Consumption and Coronary Heart Disease Risk

Four prospective studies conducted in the US have reported a beneficial effect of nut consumption on CHD incidence after follow-up ranging from six to 18 years of large cohorts of previously healthy subjects [1,41-43]. A pooled analysis of these studies shows that subjects in the highest intake group for nut consumption had a 37% reduction in multivariable-adjusted risk of fatal CHD [15]. The combined relative risk for total CHD mortality derived from the comparison of rates between the highest and lowest frequency of nut intake in all four studies was 0.63 [95% confidence interval [CI], 0.51 to 0.83]. Importantly, a dose-response relationship between nut consumption and reduced CHD mortality rates was reported for all four studies, strengthening the causal link (Figure 1). Of particular note are the results of the Physicians’ Health Study [43], where the inverse association between nut consumption and total CHD mortality was primarily due to a reduction in sudden cardiac death. Compared with men who rarely or never consumed nuts, those who consumed nuts two or more times per week had a 47% reduced risk of sudden cardiac death (relative risk, 0.53; CI, 0.30 to 0.92).

Figure 1. Results of prospective studies of nut consumption and risk of death from
coronary heart disease.

In the Nurses’ Health Study [41], nut intake could be subdivided between all tree nuts and peanuts and peanut butter. Consumption of peanut products was also associated with a decreased relative risk of CHD. Subjects who consumed peanuts two or more times per week had a relative risk of CHD of 0.66 (CI, 0.46 to 0.94), while for other nuts the relative risk for consumption of two or more times per week was 0.79 (CI, 0.50 to 1.25). A recent report of the same study concerning a subgroup of 6309 women with type-2 diabetes at baseline shows similar findings, as consumption of at least five servings per week of nuts or peanut butter (serving size, 28 g [1 ounce] for nuts and 16 g [1 tablespoon] for peanut butter) was significantly associated with a lower risk of cardiovascular disease, with a relative risk of 0.56 (CI, 0.36 to 0.89) [44]. It must be underlined that in all these studies [1,41-44] the protective effect of nut consumption on total CHD or sudden cardiac death was independent of gender, age, body mass index, alcohol use, other nutritional characteristics or presence of cardiovascular risk factors. The dose-relationship between nut intake and incident CHD translates into an average 8.3 reduction for each weekly serving of nuts [15]. The consistency of findings in all studies points to a causal association between nut consumption and reduced CHD, indicating that nuts possibly are one of the most cardioprotective foods in the habitual diet.

Since inflammation is a key process in atherogenesis, one mechanism by which nut consumption may decrease CHD risk is by improving inflammatory status, which can be ascertained from levels of circulating inflammatory markers. Three cross-sectional studies have investigated nut consumption in relation to circulating inflammatory biomarkers [45-47].

In an analysis of data from nearly 6000 participants in the Multi-Ethnic Study of Atherosclerosis (MESA) [45], consumption of nuts and seeds was inversely associated with levels of inflammatory markers, C-reactive protein (CRP), interleukin-6 (IL-6) and fibrinogen. Another study of 987 diabetic women from the prospective Nurses’ Health Study [46] showed a direct association between nut consumption and increased plasma levels of adiponectin, an adipose tissue-secreted cytokine with antiinflammatory and antiatherosclerotic properties. The third study was carried out in 772 older subjects at high risk for CHD living in Spain for the purpose of assessing adherence to the Mediterranean dietary pattern and its food components in relation to levels of soluble inflammatory markers. Adjusted mean serum levels of intercellular adhesion molecule-1 (ICAM-1), but not those of CRP or IL-6, decreased across increasing tertiles of nut consumption [47]. Thus, increasing epidemiologic evidence links frequent consumption of nuts to a reduced inflammatory status, which might help explain their cardio protective properties.

Nut Consumption and Risk of Type-2 Diabetes

In addition to cardiac outcomes, the Nurses’ Health Study also ascertained the incidence of type-2 diabetes, a major risk factor for CHD, by frequency of nut and peanut butter intake during a 16-year follow-up [48]. Nut consumption was inversely associated with risk of type-2 diabetes after multivariate adjustment for traditional risk factors, with relative risks across categories of nut consumption (never/almost never, 4 times/week) for a 28 g serving of 1.0, 0.92 (CI, 0.85 to 1.00), 0.84 (CI, 0.76 to 0.93), and 0.73 (CI, 0.60 to 0.89). Considering only lean women (BMI< 25 kg/m2), a 45% risk reduction was observed in those consuming nuts five times or more per week. Consumption of peanut butter was also inversely associated with type-2 diabetes with an adjusted relative risk of 0.79 (CI, 0.68 to 0.91) in women consuming peanut butter more than four times a week (equivalent to15 ounces of peanuts per week) compared with those who never or almost never ate peanut butter.

However, in the Iowa Women’s Health Study [49] the association between nut consumption and diabetes risk was less clear. In the 11 years of follow up, the postmenopausal women who ate nuts often had no reduced risk of diabetes compared to those who ate nuts occasionally after adjusting for multiple confounders. These negative findings might have been due to over adjustment for nutrients that mediate in part the protective effect of nuts, such as fiber and unsaturated fatty acids, because when adjusting for age only a significant 18% reduction in relative risk was observed between the highest and lowest categories of peanut butter consumption.

To ascertain whether menopausal status influenced the association between nut consumption and diabetes risk, in response to the analysis of the Iowa Women’s Health Study authors of the Nurse’s Health Study report performed additional analysis stratifying by menopausal status, and the inverse association of nut consumption and diabetes risk changed little. Among premenopausal women the multivariate relative risk was 0.67 (CI, 0.46 to 0.97) comparing those who ate nuts five times or more times a week with those who never or almost never ate nuts. Among postmenopausal women the corresponding relative risk was similar at 0.73 (CI, 0.57 to 0.95) [49]. A subsequent report from a Chinese cohort of nearly 64,000 women followed up for 4.6 years also suggests a protective effect of nuts on diabetes risk [50]. This study showed an adjusted 20% risk reduction between the lowest quintile (0.1 g) and upper quintile (3.1 g) of daily peanut consumption.

At odds with the findings in women, a recent report from the Physicians’ Health Study [51] suggests no protective effect of nut consumption on diabetes risk in men. In this study 20,224 male participants were followed for an average of 19 years. Adjusted hazard ratios for development of diabetes ranged from 1.06 (CI, 0.93 to 1.20) for men consuming less than one serving of nuts per week to 0.87 (CI, 0.61 to 1.24) for those eating at least a daily serving of nuts, and the results were similar in lean or overweight/obese participants. Figure 2 (ommitted) illustrates the findings of the main prospective studies relating nut consumption to the risk of developing type-2 diabetes. In summary, regular consumption of nuts is clearly beneficial for CHD risk, but confirmation of any protective role on diabetes risk must await further studies.

Nut Consumption and other Health Outcomes

Two prospective studies have assessed the frequency of nut consumption in relation to incident hypertension, with discordant results [52,53]. In a cohort of 15,966 participants in the Physicians’ Health Study [52] who were free of hypertension at baseline and had 237,585 person-years of follow up, adjusted hazard ratios for hypertension ranged from 0.97 (CI, 0.91 to 1.03) for nut consumption 1-2 times per month to 0.82 (CI, 0.71 to 0.94) for nut consumption of seven or more times per week. In a secondary analysis stratified by BMI, there was an inverse relationship between nut intake and hypertension in lean subjects but not in those who were overweight or obese at baseline. These results must be taken with caution, however, because salt intake and changes in weight, two major factors that influence the risk of hypertension, were not accounted for in this study. The second study, which involved 9919 Spanish university graduates followed-up for a median of 4.3 years in the SUN cohort [53], found no association between nut consumption and incidence of hypertension after adjusting for several confounders, including exposure to salt and weight changes during follow-up.

The hazard ratio for the highest versus lowest nut consumption category was 0.77 (CI, 0.46 to 1.30) in this relatively young sample of well educated adults at little baseline risk for hypertension, thus a larger sample and longer duration of follow-up might have provided a better level of evidence. In summary, limited epidemiologic data provide only circumstantial evidence for a protective effect of nut consumption on development of hypertension. The fact that nuts are often eaten with salt from snack packs is an added source of confusion in the relationship between nut consumption and hypertension.

The incidence of two major complications of hypertension, stroke and heart failure, was unrelated to the frequency of nut consumption in recent reports from the prospective Physicians’ Health Study [54,55]. Regarding stroke, while no association with total or ischemic stroke was observed, there was a suggestive non-linear relation between nut intake and hemorrhagic stroke: compared to subjects who did not consume nuts, adjusted hazard ratios for hemorrhagic stroke for subjects consuming nuts <1, 1, 2–4, 5–6, and 7 or more times per week were 1.13 (CI, 0.78 to 1.62), 1.05 (CI, 0.70 to 1.58), 0.49 (CI, 0.27 to 0.89), 1.50 (CI, 0.79 to 2.84), and 1.84 (CI, 0.95 to 3.57), respectively (p for quadratic trend 0.12) [50]. However, there were a limited number of hemorrhagic strokes in the highest categories of nut consumption, thus further studies are clearly warranted to confirm or discard this improbable adverse effect of nuts.

Some bioactive constituents of nuts, such as tocopherols, phytosterols, folic acid, selenium, and magnesium, are purported to have antioxidant, antiinflammatory or anticarcinogenetic properties [24], a reason why a protective effect of nut consumption on cancer risk might be hypothesized [56]. Old epidemiological evidence of the role of nut consumption on cancer incidence was inconclusive [57-59]. More recent reports support a preventive role, although limited to women [56-59]. A small case-control study in Greek women [60] suggests that a diet rich in nuts, seeds and pulses reduces the risk of endometrial cancer by 27% compared to infrequent consumption of such foods.

Results from the large EPIC study [61] showed no relation between higher intake of nuts and seeds and risk of colorectal cancers in the whole cohort or in men alone, but an inverse association was detected in women between the highest quintile of nut consumption (>6.2 g/day) and the lowest quintile (non-consumers), with an adjusted odds ratio of 0.69 (CI, 0.50 to 0.95). A gender discrepancy in the risk of colorectal cancer associated with peanut consumption was also reported from a population-based cohort study of approximately 24,000 people in Taiwan [62]. This study showed that women consuming peanuts had a remarkable risk reduction of 58% compared to non-consumers. However, the protective effect was not observed in men. A small clinical study in men at risk for prostate cancer showed increased serum -tocopherol and a trend towards an increase in the ratio of free prostate specific antigen (PSA): total PSA after eight weeks of a diet supplemented with 75 g walnuts per day compared with a control diet [63]. Two recent experimental studies using human cancer cell lines [64] and a mice model of human breast cancer [65] suggest an antiproliferative effect of walnuts. Clearly, more research is necessary on the important topic of nuts and cancer.

Again because of the richness of nuts in bioactive components, particularly unsaturated fatty acids, fiber, and minerals, a protective effect of nut intake on gallstone disease is biologically plausible. Two separate studies by the same authors, each on different populations, examined the relationship between frequency of nut intake and gallstone disease risk. After following 80,718 women for 20 years, the Nurses’ Health Study [66] showed that frequent nut consumers (5/week) had a 25% reduced risk of cholecystectomy compared to non-consumers. Similar findings were observed among nearly 43,000 men in the Health Professional’s Follow-up study [67]. During 457,305 person-years of follow-up, men who consumed 5 or more servings of nuts per week showed a risk of developing clinical gallstone disease that was 30% lower compared to those who rarely or never ate nuts. The results of the two studies suggest that frequent nut consumption is equally protective of gallstone disease in men and women.

Finally, patients with diverticular disease of the colon are frequently advised to avoid eating nuts and seeds to reduce the risk of complications but there is little evidence to support this recommendation, a reason why investigators from the Health Professionals Follow-up Study [68] evaluated the frequency of nut consumption in relation to new diagnoses of diverticular disease and its complications during 18 years of follow-up in 47,228 men. The results showed an inverse association between nut consumption and the risk of diverticulitis, with a multivariate hazard ratio for men with the highest intake compared with those with the lowest intake of 0.80 (CI, 0.63 to 1.01). No associations were seen between nut consumption and diverticular bleeding or uncomplicated diverticulosis. Clearly there are no reasons to recommend avoiding nuts to prevent diverticular complications.

Nut Feeding Trials with Outcomes on Cardiovascular Risk Factors

The epidemiologic evidence reporting benefits of nut consumption on CHD risk was the impetus for clinical studies designed to assess the effects on cardiovascular risk factors and begin to understand the underlying mechanisms that explained the observational data. Most clinical studies with nuts have been short-term and have compared diets supplemented with nuts with control diets for outcomes on blood lipid changes in healthy subjects or patients with moderate hypercholesterolemia. There have been fewer studies with nuts in patients with obesity, the metabolic syndrome, or type-2 diabetes investigating insulin sensitivity or glycemic control besides the lipid profile. More recent clinical trials have dealt with intermediate risk markers, such as blood pressure, oxidation biomarkers, antioxidant defenses and oxidative modification of lipids or DNA, and inflammation status. Some studies have focused on the relevant question of whether unrestricted nut intake leads to weight changes. Long-term studies targeting effects of nut consumption on metabolic syndrome, diabetes, CHD events and risk for chronic degenerative diseases are underway.

Effects of Nuts on the Lipid Profile

The first clinical trial using nuts was the Loma Linda University walnut study, published in 1993 [2]. In this landmark study, a cholesterol-lowering diet that provided 20% of energy from walnuts and 31% of energy from fat, of which 6% came from SFA and 16% from PUFA, was compared to a standard Step-I diet that provided 30% of energy from fat, of which 10% was from SFA and 10% from PUFA. Total cholesterol and LDL cholesterol decreased significantly by 12% and 18%, respectively in the healthy subjects studied. Since then, over 40 clinical studies have been conducted assessing the effects of nut-enriched diets versus isoenergetic, usually healthy comparator diets, on serum lipids and lipoproteins, as reviewed up to December 2004 in a pooled analysis of 25 intervention trials using different nut types [69] and through May 2008 in a meta-analysis of 13 feeding studies with walnuts [70].

The nuts most frequently studied have been almonds and walnuts. Some feeding trials used peanuts, pecans, macadamia nuts, hazelnuts, pistachios, cashews, and Brazil nuts. To date there have been no clinical studies with pine nuts. In the feeding trials the nut-supplemented diets were compared to various control diets: low in total fat and high in carbohydrate; high in SFA; a Mediterranean diet; the Japanese diet; or subjects’ usual diet. Although the degree of dietary control was variable, ranging from being tightly controlled (i.e., all foods provided by the investigators) to simply providing dietary advice to free-living participants eating on their own, the results have been consistent in showing a cholesterol-lowering effect of regular nut intake, usually without any significant effect on triglycerides or HDL cholesterol [2,13,17-20,69,70].

Recently the findings of a pooled analysis of 1,284 observations contributed by 583 unique participants from 25 clinical studies performed with different nuts, including peanuts, and conducted in seven different countries have been reported [69]. The results show a dose-response cholesterol-lowering effect and indicate that, for an average daily intake of 67 g of nuts (roughly equivalent to 20% of energy), the mean estimated reductions of total cholesterol and LDL-cholesterol were 11 mg/dL (5%) and 10 mg/dL (7%), respectively.

Nuts had no significant effect on HDL-cholesterol or triglycerides, except in participants with serum triglycerides >150 mg/dL, in whom a significant 10.2 mg/dL reduction was observed. Importantly, the lipid effects of nuts were dose-related, similar by gender and across all age groups, and independent of the type of nut tested. The statistical power of this pooled analysis also allowed detection of differential responses by baseline LDL-cholesterol level and BMI. The estimated cholesterol lowering effect of nuts was greater for participants with higher initial values of LDL-cholesterol and, noticeably, for those with lower baseline BMI (Figure 3). A recent meta-analysis [70] examined 13 clinical trials involving 365 participants who received diets supplemented with walnuts accounting for 5% to 25% of total energy and lasting 4–24 weeks. When compared with control diets, walnut-rich diets resulted in a significantly greater decrease in total and LDL-cholesterol concentrations, with weighted mean decreases of 10.3 and 9.2 mg/dL, respectively. The overall result indicated that the walnut diets compared with the control diets were associated with a 6.7% greater decrease in LDL-cholesterol concentration, which concurs with the mean 7% decrease reported with various nut types in the pooled analysis [69]. HDL-cholesterol and triglycerides were not significantly affected by walnut diets more than by control diets.

Recent well controlled intervention studies with walnuts [71-74], almonds [75], hazelnuts [76], pistachios [77], macadamias [78], and peanuts [79] showed LDL-cholesterol reductions ranging from 4% to 11% versus comparator diets, confirming the cholesterol-lowering efficacy of various nut types. A Mediterranean diet supplemented with 30 g of mixed nuts (walnuts, almonds and hazelnuts) per day also showed beneficial effects on the lipid profile compared with advice on a low-fat diet in diabetic and non diabetic participants in the PREDIMED study, a randomized trial of dietary intervention for the primary prevention of cardiovascular disease [80]. Of note, two randomized trials that used cashews or walnuts [81] and mixed nuts [82] compared to control diets in obese patients with the metabolic syndrome failed to show the predictable cholesterol lowering-effect, which supports the findings of the pooled analysis [69] (Figure 3) regarding the inverse association between cholesterol responses to nut feeding and BMI.

There may be a mechanistic explanation for decreased lipid responsiveness to dietary intervention in patients with the metabolic syndrome. Studies have shown that the LDL cholesterol response to diets low in SFA [83] or to egg feeding as dietary cholesterol challenge [84] are blunted in obese, insulin-resistant subjects compared with lean, insulin-sensitive individuals. Prior studies had shown that higher BMI is associated with decreased LDL-cholesterol responses to hypolipidemic diets [85-87]. High cholesterol synthesis and reduced intestinal cholesterol absorption in insulin-resistant states [88,89] might explain these findings, as an enhanced cholesterol flux through the liver will down-regulate LDL receptors and make them refractory to additional regulation by dietary fatty acid changes, while a decreased cholesterol flux though enterocytes would lessen both the cholesterol-raising response to dietary cholesterol and the cholesterol-lowering effect of plant sterols. Nuts are rich in plant sterols, which are likely to contribute to their cholesterol lowering effect [24], but this would be less operative when cholesterol absorption is low.

Figure 3. LDL-cholesterol response to nut feeding by baseline LDL-cholesterol level and
BMI. Data from a pooled study of 25 nut feeding trials (adapted from ref. 69).

Nut consumption decreases total and LDL-cholesterol, but the response does not completely agree with that expected on the basis of the dietary fatty acid and cholesterol exchange between nut diets and control diets [90]. As discussed [19], the decrease in LDL-cholesterol by nut diets is greater than predicted in most studies. This suggests that nut constituents other than fatty acids, such as fiber and/or phytosterols [23,24] are also bioactive in lowering blood cholesterol. Recently suggestive evidence has been provided that phytosterols in nuts relate to the LDL-cholesterol response observed after their consumption [91].

Nuts, Insulin resistance and Glycemic Control

Some interventional studies have examined the effects of nut-enriched diets on glycemic control in diabetic patients and insulin sensitivity in insulin-resistant states. Nuts had no discernible effect on fasting or postprandial glucose and hemoglobin A1C in patients with diabetes [73,92-94]. Changes in insulin sensitivity in response to nut diets have been inconsistent. No effects were seen in feeding studies of healthy subjects [92], hyperlipidemic patients [95], or patients with insulin-resistant states, such as obesity [96] or the metabolic syndrome [97,98]. Two recent small studies, however, found reduced insulin levels in patients with metabolic syndrome [82] and diabetes [73] after nut feeding. The three-month report of the larger PREDIMED study [80] also showed that the Mediterranean diet enriched with nuts was associated with improved insulin sensitivity and fasting glucose levels in non diabetic and diabetic participants, respectively. Finally, two studies from the same group [99,100] reported reduced postprandial glucose and insulin excursions after almond meals compared with those elicited after meals containing carbohydrates with a high glycemic index. Thus, in spite of their high energy and fat load, nuts do not worsen and may even improve metabolic control or insulin sensitivity in insulin-resistant states, but more evidence is necessary.

Effects of Nuts on Emerging Cardiovascular Risk Factors

By virtue of their unique fat and non-fat composition, nuts are likely to affect markers of atherogenesis other than the lipid profile or carbohydrate metabolism. More recently, the effects of nuts on novel CHD risk factors have been evaluated, including oxidative stress, inflammation and vascular reactivity, as reviewed [13,20-22]. The emerging picture is that frequent nut consumption has beneficial effects on cardiovascular risk factors beyond well-established cholesterol lowering.

Oxidation

Nuts are important sources of tocopherols and phenolic compounds with potent antioxidant effects, as shown by reduction of lipid peroxidation or oxidative DNA damage with nut extracts in studies in vitro and the beneficial effects of nut intake on lipid oxidation, antioxidant enzyme activity, and formation of cholesterol oxidation products in both acute and chronic experimental animal studies [22,25]. Recently, walnuts were shown to contain substantial amounts of melatonin, which contributed a significant antioxidant effect in an experimental rat model [101]. In addition, because an important fraction of the fat contained in most nuts is made of MUFA, which is not a substrate for oxidation, enrichment of lipoprotein lipids with these fatty acids after nut consumption might decrease their susceptibility to oxidation. Nuts, especially walnuts, are also good sources of PUFA, and double bonds in the molecular structures of these fatty acids are preferred initiation sites for oxidation reactions [102]. Consequently, detrimental changes of lipoprotein oxidation might be expected to occur after walnut consumption unless counteracted by endogenous antioxidants in these nuts.

Oxidative markers after feeding of MUFA-rich nuts, predominantly almonds, but also hazelnuts, peanuts, pistachios, macadamia nuts, cashews, pecans, and Brazil nuts, have been examined in several randomized feeding studies, usually of small size and lasting from three to eight weeks, as comprehensively reviewed up to 2008 [22]. Biomarkers of oxidation were secondary outcomes in most of these studies, which showed inconsistent results, with either reduced or unchanged oxidation, but in no case worse oxidative status, compared with various control diets. Several feeding studies of similar characteristics have assessed oxidative biomarkers after consumption of diets supplemented with PUFA-rich walnuts versus other healthy diets [reviewed in 21,22]. In general, there were no between-diet differences in oxidative status, probably because as discussed antioxidants present in walnuts likely prevented the potentially adverse effects of increasing the PUFA content of biological membranes.

Four recent studies have assessed the acute effects of meals enriched with nuts on postprandial oxidation in comparison with nut-free meals [99,103-105]. Results have again been mixed, as two studies using walnuts [103] and almonds [104] had no discernible effect on oxidation biomarkers, while one study with almonds [99] and another study examining both almond and walnut meals [105] showed beneficial effects on postprandial oxidative stress.

In a recent parallel feeding trial with higher statistical power than usual clinical studies with nuts, the PREDIMED study [106], a Mediterranean diet enriched with 30 g mixed nuts (half of it walnuts, the rest almonds and hazelnuts) given daily for 12 weeks to subjects at high cardiovascular risk resulted in a lower oxidized LDL level compared with the control diet. Conversely, a smaller study using the same mixed-nut diet against a similar healthy diet without nuts for 12 weeks in patients with metabolic syndrome failed to show any between-diet differences in oxidized LDL and other oxidation biomarkers, except for reduced DNA damage with the nut diet [107].

In summary, available evidence from clinical studies suggests that MUFA-rich nuts may
moderately improve oxidative status, while PUFA-rich nuts (walnuts) have a neutral or slightly beneficial effect, but no studies have shown that frequent nut consumption reduces antioxidant defenses.

Inflammation

The high content of phenolic compounds in nuts, particularly in the pellicle, might anticipate an antiinflammatory effect of frequent nut consumption [108], as suggested in cross-sectional studies [43-45]. Walnuts could be predicted to be more antiinflammatory than other nuts for two reasons. First, as discussed, walnuts are the only nuts that contain substantial amounts of ALA, which is described as one of the more anti-inflammatory fatty acids [109,110]. And second, walnuts are also particularly rich in the phenolic compound ellagic acid, which has shown potent anti-inflammatory properties in experimental studies [111,112]. Nevertheless, plasma levels of CRP, a standard measure of systemic low-grade inflammation, were usually unaffected in controlled feeding trials with almonds, walnuts, or mixed nuts, as reviewed up to 2008 [21].

On the other hand, other inflammatory mediators such as plasma levels of ICAM-1, vascular cell adhesion molecule [VCAM]-1, or IL-6 decreased after nut diets in two studies [80,113]. It must be noted that inflammatory biomarkers were always secondary outcomes of nut-feeding trials, thus problems of statistical power to detect significant changes are a problem. The same can be said of a recent small interventional study using diets enriched with two doses of almonds versus a healthy, nut-free diet [114]. However, in this study almond diets were superior to the control diet to reduce circulating CRP and also E-selectin, another potent inflammatory cytokine, but not IL-6.

Two recent studies have examined the acute effects of walnut-rich meals on postprandial inflammation [103,115]. The study of Cortés et al. [103] compared high-SFA meals supplemented with either walnuts or olive oil on postprandial events in healthy and hypercholesterolemic subjects and found that postprandial rises of inflammatory markers were similarly blunted after the two meals, except for soluble E-selectin, which was lower after the walnut meal than after the olive oil meal. Jiménez-Gómez et al. [115] used meals enriched with walnuts, olive oil and butter in healthy subjects and found similar reductions in postprandial levels of circulating inflammatory biomarkers with the walnut and olive oil meals compared with the SFA-meal. However, these authors also examined mRNA expression of some inflammatory cytokines in circulating blood mononuclear cells and reported that the walnut meal elicited a reduced expression of IL-6 compared to the other two meals [115]. A further sub-study of the PREDIMED trial analyzed both three-month changes in circulating inflammatory biomarkers and in the expression of ligands for inflammatory molecules in circulating monocytes after the study diets, one of which was supplemented with 1-oz (30 g) mixed nuts per day [116]. The findings indicate reductions in both soluble ICAM-1 and IL-6 and, importantly, reduced monocyte expression of pro-inflammatory ligands after the walnut-rich mixed nut diet compared with the low-fat diet.

In conclusion, nut consumption appears to have little effect on CRP but it evokes a reduction in concentrations of other inflammatory biomarkers. The gaps in our knowledge of the anti-inflammatory effects of nuts from clinical studies using enriched diets probably stem from the fact that most of them were not designed to evaluate this specific outcome. More recent studies of the effects of nut diets and nut meals on postprandial events and expression of pro-inflammatory molecules in circulating mononuclear cells support a stronger beneficial effect for walnuts while beginning to unravel the molecular bases for the anti-inflammatory effects of nut consumption. Nevertheless, more studies are warranted to conclusively resolve the important question of the antiinflammatory effects of nuts.

Vascular reactivity

Endothelial dysfunction is a critical event in atherogenesis that is implicated both in early disease and in advanced atherosclerosis, where it relates to perfusion abnormalities and the causation of ischemic events [117]. It is characterized by a decreased bioavailability of the endogenous vasodilator NO, synthesized from L-arginine as discussed earlier [29], and increased expression of proinflammatory cytokines and cellular adhesion molecules. Endothelial injury caused by cardiovascular risk factors or atherosclerotic vascular disease reduces NO production and this is followed by arterial wall abnormalities, both functional [inhibition of vasodilatation or paradoxical vasoconstriction] and structural [smooth muscle cell growth and blood cell adhesion] that are responsible for the initiation, development, and progression of atherosclerosis [117].

It has been known for some time that food intake affects vascular reactivity. Short-term feeding studies have consistently shown that diets rich in SFA impair endothelial function [118-120]. In addition, a single fatty meal rich in SFA usually is followed by transient endothelial dysfunction in association with elevated triglyceride-rich lipoproteins [121]. Whether acute or chronic, these detrimental effects can be counteracted by the administration of healthy nutrients, such as n-3 PUFA [120], antioxidant vitamins and phenolic compounds [122,123], and L-arginine [124], all of them nut constituents. Thus, it could be predicted that nut consumption could beneficially influence endothelial function.

Walnut diets have been examined versus control diets in three randomized crossover
studies [74,103,113] by using the standard method for noninvasive assessment of conduit artery endothelial function, flow-mediated dilatation (FMD) in the brachial artery [117]. The first study showed that, by comparison with an isoenergetic Mediterranean diet with similar SFA content, a four-week walnut diet attenuated the endothelial dysfunction associated with hypercholesterolemia [113]. In a follow-up of this trial, it was shown that adding walnuts to a high-fat, high-SFA meal counteracted ensuing postprandial endothelial dysfunction compared to the same meal with added olive oil [103]. The third study, conducted in diabetic patients, compared a walnut diet with an isoenergetic ad libitum diet with similar SFA content but without walnuts, each lasting eight weeks, and confirmed that walnuts improve FMD [74]. By analogy with the improvement of endothelial function observed after supplementation of marine n-3 PUFA [118-120], this beneficial effect of walnuts may be ascribed in part to their high ALA content. Antioxidants and L-arginine also might have played a role. Of note, supplementing a high-fat meal with ALA from canola oil was also associated with improved postprandial endothelial function in patients with diabetes [125].

Two further studies have assessed the effects of nuts on vascular reactivity [107,126]. A recent study from Turkey reported improved FMD after a diet supplemented with pistachios compared with a healthy diet, but the sequential design of the interventions precludes firm conclusions [126]. Finally, a 12-week parallel design study in patients with metabolic syndrome comparing healthy diets with or without supplementation with 30 g of mixed nuts per day found no between-diet differences in vascular reactivity, as assessed by digital pulse amplitude tonometry [107]. However, it must be noted that this is a non-standard technique to measure endothelial function with much less clinical trial experience than FMD of the brachial artery [117].

In summary, there is consisting evidence from two studies that walnut diets improve FMD and from another study that this effect can be observed after a single walnut meal. In support of these observations, Davis et al. [127] showed that walnut feeding also reduced the expression of endothelin-1, a potent endothelial activator, in an animal model of accelerated atherosclerosis, and this effect was attributable to the fat component of walnuts. Although there is a paucity of vascular reactivity studies after consumption of diets enriched with nuts other than walnuts, they might be expected to show similar beneficial effects because, with the exception of ALA, particular to walnuts, all nuts contain substantial quantities of bioactive compounds that can favorably influence vascular reactivity. This is a key area for future research.

When endothelial function improves, a lower blood pressure could be predicted. This has not been observed in the usually small-sized clinical studies performed to date, but the larger PREDIMED trial did show significant reductions in both systolic and diastolic blood pressure after the nut-supplemented Mediterranean diet compared with the control diet [80]. A recent report of the PREDIMED trial provides an insight into the possible mechanism of this antihypertensive effect by showing that the nut diet was associated with reduced cholesterol: phospholipid ratios of erythrocyte membranes, which would translate into an increase of membrane fluidity [128]. Possibly future adequately powered studies might uncover a true antihypertensive effect of nut intake. As discussed, there is also insufficient evidence from prospective studies of the relationship between nut consumption and hypertension [50,51]. Further epidemiological and clinical studies on this important topic are clearly needed.

Safety of Nut Consumption

There are two main concerns regarding the safety of increasing nut consumption: possible weight gain (and worsening metabolic complications of increased adiposity, such as the metabolic syndrome and diabetes) and allergic reactions. While the first can reasonably be dispelled, the second merits attention in particular situations. An additional concern is potential toxicity through contamination of nuts with mycotoxins, particularly aflatoxins, which is a problem that affects agricultural economies and is beyond the scope of this review [reviewed in 129,130].

Body Weight

The common perception that fatty foods provide excess energy and thus promote obesity has had a negative effect on the image of nuts. The question of whether increasing the intake of nuts and therefore calories could lead to unwanted weight gain and related health problems is a critical one. However, as thoroughly reviewed [13,16,26,27], there is considerable scientific evidence indicating that there are no adverse effects of frequent nut consumption on energy balance or body weight. Some studies suggest that nut consumption might even help lose weight.

First, the epidemiological studies that related the frequency of nut consumption with a reduction of incident CHD [1,39-41] or diabetes [46] showed a neutral or even inverse association between nut intake and BMI. Recent reports from two large prospective cohorts [131,132] and a cross-sectional study [133] support these findings. In a 28-month prospective study of the SUN cohort conducted in Spain in 8865 university graduates, a significant inverse association between nut consumption and weight gain was reported.

Compared with those who never or almost never ate nuts, participants who ate nuts ³2 times/wk had a 31% lower risk of gaining ³5 kg during follow-up, while participants who frequently consumed nuts had an average 0.42 kg less weight gain than did those who rarely consumed nuts after multivariate adjustment [131]. The Nurses’ Health Study followed 51,188 women for eight years and showed that women who reported eating nuts ³2 times/wk had 0.4 kg less mean weight gain than did women who rarely ate nuts, a small but significant difference. The results were similar for tree nuts and peanuts in normal-weight, overweight, and obese participants. In multivariate analyses, greater nut consumption (³2 times/wk compared with never/almost never) was associated with a slightly lower risk of obesity, with a hazard ratio of 0.77 (CI, 0.57 to 1.02) [132]. In a cross-sectional study of a sample of 847 subjects recruited into the PREDIMED study, nut consumption was inversely associated with adiposity measures (BMI and waist circumference) independently of other lifestyle variables. From regression coefficients of nut intake versus adiposity variables, it was predicted that BMI and waist circumference decreased by 0.78 kg/m2 and 2.1 cm, respectively, for each serving of 30 g of nuts [133].

Second, the nut intervention trials with outcomes on lipid changes that were carried out in free-living individuals showed no weight gain or a tendency to lose weight in those assigned to nut diets compared with control diets, as reviewed [26,27]. A similar lack of weight gain was documented in the mostly overweight or obese participants in the PREDIMED study who consumed 30 g of mixed nuts per day during three months [80] and in overweight diabetics consuming 30 g of walnuts for six months in a study from Australia [94]. Recent evidence from the PREDIMED study shows a decreased prevalence of the metabolic syndrome, mainly due to reduced visceral adiposity, after intervention for 12 months in participants following a Mediterranean diet supplemented with 30 g of nuts per day [134].

Third, four clinical studies specifically investigated the effects on body weight of supplementing the customary diets of free-living subjects with nuts without constraints on energy balance [135-138]. In these studies, sizeable quantities of peanuts, almonds or walnuts were provided for daily consumption during periods ranging from eight weeks to six months, without advice on how to include them in their
diet. Compared with the corresponding control diet periods, there were insignificant increases or no changes in body weight after the nut diets in all these studies.

Finally, two clinical trials have assessed the efficacy of low-calorie diets with added nuts versus conventional low-fat diets for weight loss, and the results showed the nut diets resulting in superior long-term participation and adherence, with consequent improvements in weight loss [139,140].

There are potential mechanisms for the lack of a weight-promoting effect or even a tendency to reduce adiposity of nut consumption in spite of increased energy acquisition. Probably because nuts are fatty foods containing substantial amounts of fiber, their chronic consumption in a free feeding situation causes satiation and elicits a strong dietary compensation, whereby intake of other energy-dense foods is curtailed, which accounts for roughly two thirds of the energy derived from nuts [135,141]. Increased satiation subsequent to nut consumption has been more difficult to detect in acute studies [98,142]. An enhanced thermogenic effect of nut intake was also postulated based on findings from a 19-week feeding trial with peanuts (88 g/day) in healthy subjects, who showed an 11% increase in resting energy expenditure [136]. These findings could not be reproduced in acute studies with walnuts [98,142]. Fat malabsorption, documented during nut diets as increased fecal fat excretion, also can contribute to the lack of weight gain. Increased stool fat losses may be due in part to the high fiber content of nuts [23] or to incomplete digestion of their matrices because the fat of nuts is enclosed within cell membranes, which are not readily available to digestive enzymes [143], an effect that can be compounded by incomplete mastication [144]. At any rate, the satiating effect of nuts with subsequent food compensation appears to be the main reason for their lack of weight-promoting effect.

Allergic Reactions to Nuts

Nuts are a well known cause of food allergy, with estimated prevalence rates of approximately 1% in the general population [reviewed in 145]. A recent systematic review of population-based studies points to a prevalence of 4.3% when diagnosis is based on food challenge tests and <1% when sensitization is assessed by skin prick test [146]. Allergic reactions to nuts are due to allergenic seed storage proteins that elicit specific IgE antibodies. They affect principally young children and may be particularly severe, even life-threatening. Indeed, fatal anaphylactic reactions following nut ingestion have been documented. Severity of coexisting atopic diseases [asthma, rhinitis, and eczema] predicts which patients are most likely to develop life-threatening allergic reactions to tree nuts and peanuts [147]. A minority of children with peanut allergy developed tolerance with time. Once nut allergy is firmly established, prevention of subsequent episodes, which tend to be clinically worse, includes patient and family education to avoid all types of nuts and be careful of hidden nut products in processed foods. Patients and relatives must be instructed on how to recognize early symptoms of an allergic reaction and how to treat promptly an anaphylactic episode.

Conclusion

Nuts are energy dense foods rich in bioactive macronutrients, micronutrients and phytochemicals. The unique composition of nuts is critical for their health effects. Indeed, there are consistent evidences from epidemiologic and clinical studies of the beneficial effects of nut consumption on risk of CHD, including sudden cardiac death, as well as on diabetes in women, and on major and emerging cardiovascular risk factors, as summarized in Table 3 (ommitted).

The evidence to date is convincing that including nuts in a healthy dietary pattern will extend the cardioprotective effects beyond those attributable to the components of any healthy diet exclusive of nuts. Importantly, these effects take place without undue weight gain, or even with reduced adiposity, and target multiple cardiovascular risk factors and mechanisms, which help explain why nuts so potently reduce the risk for CHD. There is also emerging evidence from acute studies that single meals enriched with nuts can have a beneficial impact on postprandial events related to atherogenesis, such as glucose and triglyceride raises, inflammation, and endothelial activation. Understanding the underlying biological mechanisms of the effects of nuts on mediators of CHD, obesity, metabolic syndrome, diabetes, and cancer should help in the design of diets that include nuts to maximally reduce chronic disease risk. The vegetarian, Mediterranean, and many Asian diets are traditional plant-based dietary patterns that include nuts and are reputed for their beneficial effects on health.

A healthy dietary pattern is high in vegetables, fruits, legumes, nuts, whole grains, and lean protein sources and low-fat dairy products [10]. Nuts are a popular and important source of unsaturated fat and high-quality vegetable protein in vegetarian diets, where they rank high on the list of foods most frequently consumed, above meat substitutes [9,148]. The optimal nutrient composition of nuts and the impressive evidence gained from epidemiologic and clinical studies on their health benefits indicates that they are an indispensable contribution to a well-balanced vegetarian diet. Also, knowledge has accumulated that dietary patterns close to the Mediterranean diet, in which nuts are a key food item, are associated with many beneficial health outcomes [16,149]. Indeed both exposure to the Mediterranean diet and frequency of nut consumption are among the dietary factors with stronger evidences for a causal link with CHD prevention [150]. Ongoing research like the large randomized PREDIMED trial, wherein one daily serving of mixed nuts within the context of the Mediterranean
diet is provided to participants at high cardiovascular risk in one arm of this six-year study [80], might eventually settle the critical issues of whether, in comparison with a healthy control diet without nuts, a healthy diet supplemented with one daily serving of nuts prevents cardiovascular events and development of other prevalent chronic disorders, including diabetes, cancer and neurodegenerative diseases.

Conflict of Interest

The author has received research funding from the California Walnut Commission, Sacramento, CA and is a non paid member of its Scientific Advisory Committee.

Acknowledgements

Work supported in part by grants from the Spanish Health Ministry (FIS Thematic Research Networks C03/01 and G03/140) and the California Walnut Commission, Sacramento, CA. CIBERobn is an initiative of ISCIII, Spain.

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by EricT

Omega 3 Fatty Acids And Inflammation

Sun, 16 May 2010 03:17:53 +0000

Omega-3 Fatty Acids and Inflammatory Processes

by Philip C. Calder

Institute of Human Nutrition, School of Medicine, University of Southampton, MP887 Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK

Nutrients 2010

Long chain fatty acids influence inflammation through a variety of mechanisms; many of these are mediated by, or at least associated with, changes in fatty acid composition of cell membranes. Changes in these compositions can modify membrane fluidity, cell signaling leading to altered gene expression, and the pattern of lipid mediator production. Cell involved in the inflammatory response are typically rich in the n-6 fatty acid arachidonic acid, but the contents of arachidonic acid and of the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can be altered through oral administration of EPA and DHA. Eicosanoids produced from arachidonic acid have roles in inflammation. EPA also gives rise to eicosanoids and these often have differing properties from those of arachidonic acid-derived eicosanoids. EPA and DHA give rise to newly discovered resolvins which are anti-inflammatory and inflammation resolving. Increased membrane content of EPA and DHA (and decreased arachidonic acid content) results in a changed pattern of production of eicosanoids and resolvins. Changing the fatty acid composition of cells involved in the inflammatory response also affects production of peptide mediators of inflammation (adhesion molecules, cytokines etc.). Thus, the fatty acid composition of cells involved in the inflammatory response influences their function; the contents of arachidonic acid, EPA and DHA appear to be especially important. The anti-inflammatory effects of marine Omega 3 PUFAs suggest that they may be useful as therapeutic agents in disorders with an inflammatory component.

Introduction

Inflammation is a normal defense mechanism that protects the host from infection and other insults; it initiates pathogen killing as well as tissue repair processes and helps to restore homeostasis at infected or damaged sites. It is typified by redness, swelling, heat, pain and loss of function, and involves interactions amongst many cell types and the production of, and responses to, a number of chemical mediators. Where an inflammatory response does occur, it is normally well regulated in order that it does not cause excessive damage to the host, is self-limiting and resolves rapidly. This self-regulation involves the activation of negative feedback mechanisms such as the secretion of anti-inflammatory mediators, inhibition of pro-inflammatory signaling cascades, shedding of receptors for inflammatory mediators, and activation of regulatory cells. As such, when controlled properly, regulated inflammatory responses are essential to remain healthy and maintain homeostasis. Pathological inflammation involves a loss of tolerance and/or of regulatory processes [1]. Where this becomes excessive, irreparable damage to host tissues and disease can occur. Irrespective of the cause of the inflammation, the response involves four major events:

An increased blood supply to the site of inflammation;

Increased capillary permeability caused by retraction of endothelial cells. This permits larger molecules, not normally capable of traversing the endothelium, to do so and thus delivers soluble mediators to the site of inflammation;

Leukocyte migration from the capillaries into the surrounding tissue. This is promoted by release of chemoattractants from the site of inflammation and by the upregulation of adhesion molecules on the endothelium. Once in the tissue the leukocytes move to the site of inflammation;

Release of mediators from leukocytes at the site of inflammation. These may include lipid

mediators (e.g., prostaglandins (PGs), leukotrienes (LTs)), peptide mediators (e.g., cytokines), reactive oxygen species (e.g., superoxide), amino acid derivatives (e.g., histamine), and enzymes (e.g., matrix proteases) depending upon the cell type involved, the nature of the inflammatory stimulus, the anatomical site involved, and the stage during the inflammatory response. These mediators normally would play a role in host defense, but when produced inappropriately or in an unregulated fashion they can cause damage to host tissues, leading to disease. Several of these mediators may act to amplify the inflammatory process acting, for example, as chemoattractants. Some of the inflammatory mediators may escape the inflammatory site into the circulation and from there they can exert systemic effects. For example, the cytokine interleukin (IL)-6 induces hepatic synthesis of the acute phase protein C-reactive protein, while the cytokine tumour necrosis factor (TNF)-. elicits metabolic effects within skeletal muscle, adipose tissue and bone.

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Fatty Acid Composition of Cells Involved in Inflammation and its Modification by Marine Omega 3 Fatty Acids

Polyunsaturated fatty acids (PUFAs) are important constituents of the phospholipids of all cell membranes. Laboratory animals that have been maintained on standard chow have a high content of arachidonic acid (20:4n-6) and low contents of eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) in the bulk phospholipids of tissue lymphocytes [2,3], peritoneal macrophages [4-8], alveolar macrophages [9,10], Kupffer cells [10], and alveolar neutrophils [11-13]. Feeding laboratory animals a diet containing fish oil, which provides EPA and DHA, results in a higher content of these fatty acids in lymphocytes [3], macrophages [4-7,9,10], Kupffer cells [10] and neutrophils [11-13]; typically enrichment in marine n-3 PUFAs is accompanied by a decrease in content of arachidonic acid. The bulk phospholipids of blood cells representing those that become involved in inflammatory responses (e.g., neutrophils, lymphocytes, monocytes) and collected from humans consuming typical Western diets contain about 10 to 20% of fatty acids as arachidonic acid, with about 0.5 to 1% EPA and about 2 to 4% DHA [14-25], although there are differences between the different phospholipid classes in terms of the content of these fatty acids [16]. The fatty acid composition of these cells can be modified by increasing intake of marine n-3 fatty acids [14-21,23-25]. This occurs in a dose response fashion [25] and over a period of days to weeks, with a new steady-state composition reached within about 4 weeks (Figure 1). Typically the increase in content of n-3 PUFAs occurs at the expense of n-6 PUFAs, especially arachidonic acid.

Figure 1. Time course of incorporation of EPA and DHA into human blood mononuclear
cells. Healthy subjects supplemented their diet with fish oil capsules providing 2.1 g EPA
plus 1.1 g DHA per day for a period of 12 weeks. Blood mononuclear cell phospholipids
were isolated at 0, 4, 8 and 12 weeks and their fatty acid composition determined by gas
chromatography. Data are mean ± SEM from 8 subjects and are from Yaqoob et al. [19].

Mechanisms by which Polyunsaturated Fatty Acids can Influence Inflammatory Cell Function

Poly Unsaturated Fatty Acids can influence inflammatory cell function, and so inflammatory processes, by a variety of mechanisms as follows:

PUFA intake can influence complex lipid, lipoprotein, metabolite and hormone concentrations that in turn influence inflammation;

Non-esterified PUFAs can act directly on inflammatory cells via surface or intracellular “fatty acid receptors” – the latter may include transcription factors like peroxisome proliferator activated receptors (PPARs);

PUFAs can be oxidized (enzymatically or non-enzymatically) and the oxidized derivatives can act directly on inflammatory cells via surface or intracellular receptors – oxidation can occur to the non-esterified form of the PUFA or to PUFAs esterified into more complex lipids including circulating or cell membrane phospholipids and intact lipoproteins such as low density lipoprotein (LDL);

PUFAs can be incorporated into the phospholipids of inflammatory cell membranes (as

described above). Here they play important roles assuring the correct environment for
membrane protein function, maintaining membrane order (“fluidity”) and influencing lipid raft formation [26]. Membrane phospholipids are substrates for the generation of second
messengers like diacylglycerol and it has been demonstrated that the fatty acid composition of such second messengers, which is determined by that of the precursor phospholipid, can
influence their activity [27]. In addition, membrane phospholipids are substrates for the release of (non-esterfied) PUFAs intracellularly – the released PUFAs can act as signaling molecules, ligands (or precursors of ligands) for transcription factors, or precursors for biosynthesis of lipid mediators which are involved in regulation of many cell and tissue responses, including aspects of inflammation and immunity (see below). Thus, changes in membrane phospholipid fatty acid composition, as described above, can influence the function of cells involved in inflammation via:

o alterations in the physical properties of the membrane such as membrane order and raft
structure;

o effects on cell signaling pathways, either through modifying the expression, activity or
avidity of membrane receptors or modifying intracellular signal transduction mechanisms that lead to altered transcription factor activity and changes in gene expression;

o alterations in the pattern of lipid mediators produced, with the different mediators having different biological activities and potencies (see below).

The multitude of potential mechanisms involved and their complexity has made it difficult to fully understand the actions of PUFAs within inflammatory processes. This difficulty has been further compounded by the variety of experimental approaches that have been used, including the method of presentation of PUFAs of interest to inflammatory cells in order to study their effects. For example, many in vitro studies have exposed cells to non-esterified fatty acids, often at concentrations that might not be achieved physiologically. Thus, effects of non-esterified PUFAs on responses of lymphocytes [2], monocytes [28], macrophages [8,29-33], neutrophils [34-36] and endothelial cells [37-39] have been demonstrated.

These effects may involve a direct effect of the non-esterified PUFA or of an oxidized derivative of the PUFA [40-42] or they may be secondary to incorporation of the PUFA into cell membrane phospholipids. Physiologically, the concentration of non-esterified n-3 PUFAs (and also arachidonic acid) is quite low. These fatty acids are carried in the bloodstream at much higher concentrations in more complex lipids (triglycerides, phospholipds, cholesteryl esters) within lipoproteins. Many of the cell types involved in inflammatory responses express lipoproteinreceptors (e.g., LDL receptor, very low density lipoprotein receptor, scavenger receptors) and so are able to take up intact lipoproteins, subsequently utilising the fatty acid components. Thus, lipoproteins may affect inflammatory cell function [43,44], perhaps due to their component fatty acids.

Inflammatory cells may also access fatty acids from lipoproteins by hydrolysing them extracellularly as has been demonstrated for macrophages [45] and lymphocytes [46]. Thus, cells involved in inflammatory processes are exposed to fatty acids, including PUFAs, in many different forms, and they may access fatty acids from their environment by a variety of mechanisms. The effect of the form of presentation of PUFAs to inflammatory cells can be examined in the cell culture setting and studies to date indicate that non-esterified fatty acids [28-39], complex lipids like triglycerides [46], intact lipoproteins [44], and oxidized forms of fatty acids and other lipids [40-42] all influence inflammatory cell responses, frequently with different effects or different potencies of n-6 and n-3 PUFAs.

Following increased dietary intake of marine n-3 PUFAs their concentrations increase in complex lipids within the bloodstream (triglycerides, phospholipids, cholesteryl esters), as well as within the membrane phospholipids of cells and tissues including those involved in inflammatory responses (see above), and there is a small increase in their concentration within the circulating non-esterfied fatty acid pool; the latter increase is small because circulating non-esterfied fatty acids derive principally from adipose tissue triglyceride breakdown and adipose tissue triglycerides contain very little EPA and DHA. Thus, following increased intake of EPA and DHA, both the cells involved in inflammation and their extracellular environment (e.g., blood plasma) are enriched in those fatty acids, so that the n-3 PUFA enriched inflammatory cells will be in contact with n-3 PUFA-rich complex lipids and lipoproteins. Many studies have examined the effect of increased intake of marine n-3 PUFAs on the function of cells typically involved in inflammation taken from the bloodstream (neutrophils, eosinophils, monocytes, lymphocytes) or, in the case of animal studies tissues and subsequently cultured. In many, probably most, cases the in vivo situation is not maintained during the ex vivo culture period, in that the n-3 PUFA enriched cells are maintained in an environment that is different from that to which they were exposed in vivo i.e., to an n-3 PUFA poor environment. Thus, the in vivo situation is not replicated in the ex vivo setting. This hampers the full interpretation of the findings of
such research.

Lipid Mediators: Biosynthesis, Roles in Inflammation, and the Impact of Marine Omega 3 Fatty Acids

Eicosanoids Generated from Arachidonic Acid

Eicosanoids are key mediators and regulators of inflammation and immunity and are generated from 20 carbon PUFAs. Eicosanoids, which include PGs, thromboxanes, LTs and other oxidised derivatives, are generated from arachidonic acid by the metabolic processes summarized in Figure 2. Eicosanoids are involved in modulating the intensity and duration of inflammatory responses [47,48], have cell- and stimulus-specific sources and frequently have opposing effects. Thus, the overall physiological (or pathophysiological) outcome will depend upon the cells present, the nature of the stimulus, the timing of eicosanoid generation, the concentrations of different eicosanoids generated and the sensitivity of target cells and tissues to the eicosanoids generated. Because of the relatively high amount of arachidonic acid in membrane phospholipids of cells involved in inflammation, this fatty acid is typically the major precursor for eicosanoid mediators, which are produced in greatly increased amounts upon cellular stimulation. Thus, amongst the mix of eicosanoids produced, those synthesized from arachidonic acid (e.g., PGE2 and LTB4) predominate although the exact eicosanoid profile depends upon the cell type concerned (e.g., neutrophils and mast cells produce a lot of PGD2 whereas monocytes produce a lot of PGE2) and the nature of the stimulus; the profile will also change over time as the nature of the response to the stimulus alters. In general arachidonic acid-derived eicosanoids act in a pro-inflammatory way, although this is an over-simplification since it is now recognised that PGE2, for example, has both pro- and anti-inflammatory effects, and that another eicosanoid derived from arachidonic acid, lipoxin A4, is anti-inflammatory [49-52].

Figure 2. Outline of the pathway of eicosanoid biosynthesis from arachidonic acid. COX,
cyclooxygenase; HETE, hydroxyeicosatetraenoic acid; HpETE, hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; LT, leukotriene; LX, lipoxin; oxoETE, oxoeicosatetraenoic acid; PG, prostaglandin; TX, thromboxane.

Fatty Acid Modification of Eicosanoid Profiles

Animal studies have shown a direct relationship between arachidonic acid content of inflammatory cell phospholipids and ability of those cells to produce PGE2 [53], such that PGE2 production is increased by arachidonic acid feeding [53] and decreased by EPA or DHA feeding [53-55]. It is well documented that PGE2 and 4 series-LT production by human inflammatory cells can be significantly decreased by fish oil supplementation of the diet for a period of weeks to months [14-16,18,56,57].

EPA is also a substrate for the cyclooxygenase and lipoxygenase enzymes that produce eicosanoids, but the mediators produced have a different structure from the arachidonic acid-derived mediators, and this influences their potency. Increased generation of 5-series LTs has been demonstrated using macrophages from fish oil-fed mice [55] and neutrophils from humans supplemented with oral fish oil for several weeks [14-16]. The functional significance of the generation of eicosanoids from EPA is that EPA-derived mediators are often much less biologically active than those produced from arachidonic acid (Figure 3). For example EPA-derived LTB5 is 10- to 100-fold less potent as a neutrophil chemoattractant compared with LTB4 [58,59]. Furthermore, EPA-derived eicosanoids may antagonise the action of those produced from arachidonic acid, as was recently demonstrated for PGD3 vs. PGD2 [60]. However, in some cases arachidonic acid-derived and EPA-derived eicosanoids appear to behave with similar potency (e.g., inhibition of TNF-. production by monocytes [61,62]).

Figure 3. General overview of synthesis and actions of lipid mediators produced from arachidonic acid, EPA and DHA. COX, cyclooxygenase; LOX, lipoxygenase; LT, leukotriene; PG, prostaglandin.

Resolvins: Novel Anti-Inflammatory and Inflammation Resolving Mediators Produced from EPA

and DHA

EPA and DHA also give rise to resolvins and related compounds (e.g., protectins) through pathways involving cyclooxygenase and lipoxygenase enzymes (Figure 4) [63-65]. These mediators have been demonstrated in cell culture and animal feeding studies to be anti-inflammatory and inflammation resolving (Figure 3). For example, resolvin E1, resolvin D1 and protectin D1 all inhibit transendothelial migration of neutrophils, so preventing neutrophilic infiltration at sites of inflammation, resolvin D1 inhibits IL-1. production, and protectin D1 inhibits TNF and IL-1. production (see [65] for references). The role of resolvins and related compounds may be very important because resolution of inflammation is important in shutting off the ongoing inflammatory process and in limiting tissue damage.

Influence of Marine n-3 Fatty Acids on Leukocyte Chemotaxis

A number of dietary supplementation studies with fish oil have demonstrated a time-dependent
decrease in chemotaxis of human neutrophils and monocytes towards various chemoattractants
including LTB4, bacterial peptides and human serum [14-16,66-68]. Both the distance of cell migration and the number of cells migrating were decreased. Despite the high dose of marine n-3 PUFAs used in many of these studies (3.1 to 14.4 g EPA+DHA/day), a dose response study by Schmidt et al. [69] suggests that near-maximum inhibition of chemotaxis occurs at an intake of 1.3 g EPA+DHA/day.

Some studies report no effect of n-3 PUFAs on neutrophil chemotaxis [20,70,71]. An explanation for this lack of effect with regard to one of these studies [70] may be that the dose of EPA+DHA used was too low to be active (0.55 g EPA+DHA/day). However the other two studies [20,71] used higher doses of EPA+DHA, but in both cases this was a DHA-rich preparation with little EPA being provided. If this is the explanation for the lack of effect, then this suggests that the anti-chemotactic effects of fish oil might be due to EPA rather than DHA. There have been no studies attempting to discriminate the effects of EPA and DHA on chemotaxis. The mechanism by which n-3 PUFAs inhibit chemotaxis is not clear but may relate to reduced expression or antagonism of receptors for chemoattractants.

Figure 4. Outline of the pathway of synthesis of resolvins and related mediators from EPA and DHA. COX, cyclooxygenase; HpDHA, hydroperoxydocosahexaenoic acid; HpEPE, hydroperoxyeicosapentaenoic acid; LOX, lipoxygenase; LT, leukotriene; PG, prostaglandin; Rv, resolvin; TX, thromboxane

Influence of Marine n-3 Fatty Acids on Adhesion Molecules and Adhesive Interactions

Cell culture [28,37-39] and animal feeding studies [72,73] report decreased expression of some adhesion molecules on the surface of monocytes [28], macrophages [72], lymphocytes [73] or endothelial cells [37-39] following exposure to marine n-3 PUFAs. In some cases this was shown to result in decreased adhesion between leukocytes and endothelial cells. Supplementing the diet of healthy humans with fish oil providing about 1.5 g EPA+DHA/day resulted in a lower level of expression of ICAM-1 on the surface of blood monocytes stimulated ex vivo with interferon-. [74]. Dietary fish oil providing 1.1 g EPA+DHA/day was found to decrease circulating levels of soluble VCAM-1 in elderly subjects [75], but it is not clear if this represents decreased surface expression of VCAM-1.

Influence of Marine n-3 Fatty Acids on Inflammatory Cytokines

Transcription Factors Involved in Regulating Inflammatory Gene Expression

In addition to effects on inflammation mediated by changes in the pattern of eicosanoids and other lipid mediators produced, marine n-3 PUFAs have also been shown to alter the production of inflammatory proteins including chemokines, cytokines, growth factors and matrix proteases. This effect may be mediated by altered activation of key transcription factors involved in regulating inflammatory gene expression. Two transcription factors that are likely to play a role in inflammation are nuclear factor . B (NF.B) and PPAR-.. NF.B is the principal transcription factor involved in upregulation of inflammatory cytokine, adhesion molecule and cyclooxygenase-2 genes [76,77]. NF.B is activated as a result of a signalling cascade triggered by extracellular inflammatory stimuli and involving phosphorylation of an inhibitory subunit (inhibitory subunit of NF.B (I.B)) which then allows translocation of the remaining NF.B dimer to the nucleus [78]. The second transcription factor, PPAR-., is believed to act in an anti-inflammatory manner. While PPAR-. directly regulates inflammatory gene expression, it also interferes with the activation of NF.B creating an intriguing interaction between these two transcription factors [79]. Both NF.B and PPAR-. may be regulated by n-3 PUFAs.

Fatty Acid Modulation of Inflammatory Cytokine Production and of Transcription Factor Activation

EPA and DHA inhibited endotoxin-stimulated production of IL-6 and IL-8 by cultured human endothelial cells [38,80] and EPA or fish oil inhibited endotoxin-induced TNF-. production by cultured monocytes [29,30]. EPA or fish oil decreased endotoxin-induced activation of NF.B in human monocytes and this was associated with decreased I.B phosphorylation [31], perhaps due to decreased activation of mitogen-activated protein kinases [32]. These observations suggest effects of marine n-3 PUFAs on inflammatory gene expression via inhibition of activation of the transcription factor NF.B.

Animal feeding studies with fish oil support the observations made in cell culture with respect to the effects of marine n-3 PUFAs on NF.B activation and inflammatory cytokine production. Compared with feeding corn oil, fish oil lowered NF.B activation in endotoxin-activated murine spleen lymphocytes [81]. Feeding fish oil to mice decreased ex vivo production of TNF-., IL-1. and IL-6 by endotoxin-stimulated macrophages [54,82,83]. Several studies in healthy human volunteers involving supplementation of the diet with fish oil have demonstrated decreased production of TNF-., IL-1., IL-6 and various growth factors by endotoxin-stimulated monocytes or mononuclear cells (a mixture of lymphocytes and monocytes) [15,18,56,84-86], although not all studies confirm this effect. Some of the studies that fail to show an effect of n-3 PUFAs on cytokine production have provided < 2 g EPA+DHA/day [70,87-90], which may be an insufficient dose, although others have provided higher doses [19,24,91-94]. It is not clear what the reason for these discrepancies in the literature is, but technical factors are likely to be contributing factors, as discussed in detail elsewhere [95]. The relative contributions of EPA and DHA might also be important in determining the effect of fish oil. One other factor that has recently been identified is polymorphisms in genes affecting cytokine production [96].In this study it was found that the effect of dietary fish oil upon cytokine production by human mononuclear cells was dependent upon the nature of the -308 TNF-. and the +252 TNF-. polymorphisms.

Anti-Inflammatory Effects of Marine n-3 Fatty Acids Suggest a Therapeutic Value

Inflammation is an overt or covert component of numerous human conditions and diseases
(Table 1) [1]. Although the inflammation may afflict different body compartments, one common
characteristic of these conditions and diseases is excessive or inappropriate production of
inflammatory mediators including eicosanoids and cytokines [1]. The foregoing discussion has
highlighted that marine n-3 PUFAs can act in a number of ways to reduce inflammation. They:

decrease production of eicosanoid mediators from arachidonic acid, many of which have pro-

inflammatory roles;

increase production of weakly inflammatory or anti-inflammatory eicosanoids from EPA;

increase production of anti-inflammatory and inflammation resolving resolvins from EPA and

DHA;

decrease chemotactic responses of leukocytes;

decrease adhesion molecule expression on leukocytes and on endothelial cells and decrease

intercellular adhesive interactions;

decrease production of pro-inflammatory cytokines and other pro-inflammatory proteins induced via the NF.B system.

The roles marine n-3 PUFAs in shaping and regulating inflammatory processes and responses suggest that the level of exposure to these fatty acids might be important in determining the development and severity of inflammatory diseases. The recognition that marine n-3 PUFAs have anti-inflammatory actions has lead to the idea that supplementation of the diet of patients with inflammatory diseases may be of clinical benefit. Each of the diseases or conditions listed in Table 1 is a possible therapeutic target for marine n-3 PUFAs. Perhaps unsurprisingly, supplementation trials have been conducted in most of these diseases. Those conducted in patients with rheumatoid arthritis appear to be the most successful with most trials reporting several clinical benefits [97]; these benefits are supported by meta-analyses of the available data [98,99]. Studies in patients with inflammatory bowel diseases (Crohn’s disease and ulcerative colitis) provide equivocal findings with some showing some benefits and others not [100,101].

Likewise studies conducted in patients with asthma do not provide a clear picture; most studies conducted in adults do not show a clinical benefit, while there are indications of benefits of marine n-3 PUFAs in children and adolescents, although there are few studies in those groups [102]. An extension of the latter studies is recent work in pregnancy which shows an impact of marine n-3 PUFAs on the maternal and foetal immune system [103-105], that may reduce risk of development of allergic type diseases in infancy [104] and childhood [106]. Although this is an emerging area with few published studies at present, the possibility of an early effect of marine n-3 PUFAs on immune maturation hints at an important, novel role for these fatty acids in early development [102,107,108]. In most other inflammatory diseases and conditions there are too few studies to draw a clear conclusion of the possible efficacy of marine n-3 PUFAs as a treatment. One exception to this may be related to cardiovascular disease morbidity and mortality. There is evidence that marine n-3 PUFAs slow the progress of atherosclerosis [109,110], which has an inflammatory component [111,112]. Further, marine n-3 PUFAs decrease mortality due to cardiovascular disease [113,114]; this may be, in part, due to stabilization of atherosclerotic plaques against rupture [115], which again has an inflammatory component [112,116]. Thus, the anti-inflammatory effects of marine n-3 PUFAs may contribute to their protective actions towards atherosclerosis, plaque rupture and cardiovascular mortality.

Table 1. List of diseases and conditions with an inflammatory component in which marine
n-3 fatty acids might be of benefit. Note: this list is not exhaustive.

Disease/condition
Rheumatoid arthritis
Crohn’s disease
Ulcerative colitis
Lupus
Type-1 diabetes
Cystic fibrosis
Childhood asthma
Adult asthma
Allergic disease
Chronic obstructive pulmonary disease
Psoriasis
Multiple sclerosis
Atherosclerosis
Acute cardiovascular events
Obesity
Neurodegenerative diseases of ageing
Systemic inflammatory response to surgery, trauma and critical illness

The dose of marine n-3 PUFAs required to prevent or to treat different inflammatory conditions is not clear, although it is evident that the anti-inflammatory effects of these fatty acids are dose-dependent [25]. As alluded to above, studies in healthy human volunteers suggest that an intake of >2 g EPA+DHA/day is required to affect inflammatory processes. There are few dose response studies investigating the effect of marine n-3 PUFAs in patients with inflammatory conditions. Studies in rheumatoid arthritis have used 1.5 to 7 g EPA+DHA/day (average about 3.5 g/day) and have been of long duration (3 to 12 months), with effects becoming apparent after some months [97]. One study that used two doses of n-3 PUFAS [117] reported that both doses induced benefit, but that the effect was seen earlier with the higher dose. If doses of 2 g, or even more, of EPA+DHA per day are required before an anti-inflammatory effect is seen, it is possible that some studies in patients fail to show a benefit because they have used an insufficient dose, or been of insufficient duration. It is not known whether different doses of marine n-3 PUFAs are required to treat different inflammatory conditions, but this is a possibility because the precise nature of the inflammation (i.e., the cells, mediators and signaling systems involved) will differ from condition to condition [1] and it may be that these different components of inflammation show different sensitivities to n-3 PUFAs.

Conclusions

Fatty acids influence inflammation through a variety of mechanisms; many of these are mediated by, or at least associated with, changes in fatty acid composition of cell membranes. Changes in these compositions can modify membrane fluidity, cell signaling leading to altered gene expression, and the pattern of lipid mediator production. Cells involved in the inflammatory response are typically rich in the n-6 fatty acid arachidonic acid, but the contents of arachidonic acid and of the n-3 fatty acids EPA and DHA can be altered through oral administration of EPA and DHA. Eicosanoids produced from arachidonic acid have roles in inflammation. EPA also gives rise to eicosanoids and these may have differing properties from those of arachidonic acid-derived eicosanoids. EPA and DHA give rise to newly discovered resolvins which are anti-inflammatory and inflammation resolving. Increased membrane content of EPA and DHA (and decreased arachidonic acid content) results in a changed pattern of production of eicosanoids and probably also of resolvins. Changing the fatty acid composition of inflammatory cells also affects production of peptide mediators of inflammation (adhesion molecules, cytokines etc.). Thus, the fatty acid composition of human inflammatory cells influences their function; the contents of arachidonic acid, EPA and DHA appear to be especially important. As a result of their anti-inflammatory actions marine n-3 PUFAs have therapeutic efficacy in rheumatoid arthritis, although benefits in other inflammatory diseases and conditions have not been unequivocally demonstrated. The anti-inflammatory effects of marine n-3 PUFAs may contribute to their protective actions towards atherosclerosis, plaque rupture and cardiovascular mortality.

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by EricT

Beneficial Effects of Probiotic and Food Borne Yeasts on Human Health

Sat, 15 May 2010 19:13:21 +0000

by Saloomeh Moslehi-Jenabian, Line Lindegaard Pedersen and Lene Jespersen

Department of Food Science, Food Microbiology, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark;

Nutrients 2010

Besides being important in the fermentation of foods and beverages, yeasts have shown numerous beneficial effects on human health. Among these, probiotic effects are the most well known health effects including prevention and treatment of intestinal diseases and immunomodulatory effects. Other beneficial functions of yeasts are improvement of bioavailability of minerals through the hydrolysis of phytate, folate biofortification and detoxification of mycotoxins due to surface binding to the yeast cell wall.

Introduction

Fermentation is one of the oldest forms of food processing and preservation in the world. Since very early times, humans have been exploiting yeasts and their metabolic products, mainly for baking and brewing. Nowadays, the products of modern yeast biotechnology form the backbone of many commercially important sectors, including foods, beverages, pharmaceuticals, industrial enzymes and others. Saccharomyces cerevisiae, which according to EFSA (The European Food Safety Authority) has a QPS (Qualified Presumption of Safety) status [1], is the most common yeast used in food fermentation where it has shown various technological properties. Yeasts do also play a significant role in the spontaneous fermentation of many indigenous food products.

A review on S. cerevisiae in African fermented foods has been provided by Jespersen [2]. Several beneficial effects on human health and well-being have been reported and there seems to be a need to understand the positive effects of yeasts, their mechanisms and employment of them. The present article reviews the major beneficial effects of yeasts, i.e., probiotic effects, biodegradation of phytate, folate biofortification and detoxification of mycotoxins, which has been summarized in Table 1. However, there are other reported effects such as enrichment of foods with prebiotics as fructooligosaccharides [3], lowering of serum cholesterol [4,5], antioxidative properties, antimutagenic and antitumor activities [6] etc. These topics will meanwhile not be the focus of the present review. Additional information on health significance and food safety of yeasts in foods and beverages can be obtained from Fleet and Balia [7].

Table 1. Overview of the major beneficial effects of yeasts.

Activity	Yeast species	Heath effects	Ref.
Probiotic effect	• Saccharomyces cerevisiae var. boulardii	Effect on enteric bacterial pathogen Maintenance of epithelial barrier integrity Anti-inflammatory effects Effects on immune response Trophic effects on intestinal mucosa Clinical effects on diarrheal diseases	[16-35] [21,22,31,36] [21,22,31-35,37, 39-41] [42-45] [46-49,52-53] [62-63,65-75]
Biodegradation of phytate	• Saccharomyces cerevisiae, Saccharomyces kluyveri, Schwanniomyces castellii, Debaryomyces castellii, Arxula adeninivorans, Pichia anomala, Pichia rhodanensis, Pichia spartinae, Cryptococcus laurentii, Rhodotorula gracilis, Torulaspora delbrueckii, Kluyveromyces lactis Candida krusei (Issatchenkia orientalis) and Candida spp.	Nutritional importance, i.e., bioavailability of divalent minerals such as iron, zink, calcium and magnesium	[87-108]
Folate biofortification	• S. cerevisiae • Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces pastorianus, Metschnikowia lochheadii, Debaryomyces melissophilus, Debaryomyces vanrijiae var. vanrijiae, Debaryomyces hansenii, Pichia philogaea, Kodamaea anthophila, Wickerhamiella lipophilia, Candida cleridarum and Candida drosophilae • Candida milleri and T. delbrueckii • Saccharomyces exiguous and Candida lambica • P. anomala and Candida glabrata • Kluyveromyces marxianus and C. krusei (I. orientalis	Prevention of neural tube defects in the foetus, megaloblastic anaemia and reduction of the risk for cardiovascular disease, cancer and Alzheimer's disease	[119-126,130] [121] [126] [128-129] [130] [128,130]
Degradation of mycotoxins	• S. cerevisiae • Phaffia rhodozyma and Xanthophyllomyces dendrorhous	Antitoxic in some degree	[138-141] [142]
Absorption of mycotoxins	• S. cerevisiae	Antitoxic	[143-155]

Beneficial Effects of Yeast as Probiotics

Taxonomic Characterization of Probiotic Yeasts

Probiotics are defined as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’ [8]. Probiotics may be consumed either as food components or as non-food preparations. There is a great interest in finding yeast strains with probiotic potential. Different yeast species such as Debaryomyces hansenii, Torulaspora delbrueckii [9], Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces lodderae [10] have shown tolerance to passage through the gastrointestinal tract or inhibition of enteropathogens. However, Saccharomyces boulardii is the only yeast with clinical effects and the only yeast preparation with proven probiotic efficiency in double-blind studies [11].

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S. boulardii, isolated from litchi fruit in Indochina by Henri Boulard in the 1920s, is commonly used as a probiotic yeast especially in the pharmaceutical industry and in a lyophilized form for prevention and treatment of diarrhoea. In a study conducted by van der Aa Kühle and Jespersen [12] on commercial strains of S. boulardii, it was found that the S. boulardii strains morphologically and physiologically could be characterized as S. cerevisiae. Sequences of the D1/D2 domain of the 26S rRNA gene were identical for all isolates examined and had 100 % similarity with the sequences of the type strain of S. cerevisiae (CBS 1171T) and the sequenced S. cerevisiae strain S288c. All S. boulardii isolates were found to have the same ITS1-5.8S rRNA-ITS2 sequence, which displayed a close resemblance with the sequences published for S288c (99.9%), CBS 1171T (99.3%) and other S. cerevisiae strains. Sequence analysis of the mitochondrial cytochrome-c oxidase II gene (COX2) also resulted in identical sequences for the S. boulardii strains and comparisons with available nucleotide sequences revealed close relatedness to strains of S. cerevisiae including S288c (99.5%) and CBS 1171T (96.6%).

The electrophoretic karyotypes of the S. boulardii strains appeared quite uniform and although very typical of S. cerevisiae, they formed a cluster separate from other strains within this species. The results of the study strongly indicated a close relatedness of S. boulardii to S. cerevisiae and thereby support the recognition of S. boulardii as a member of S. cerevisiae and not as a separate species. The fact that strains of S. boulardii should be seen as a separate cluster within the S. cerevisiae species is further supported by the fact that strains of S. boulardii previously have been reported to differ from strains of S. cerevisiae due to a specific microsatellite allele [13] as well as trisomy of the chromosome IX and altered copy numbers of specific genes [14]. Others have reported S. boulardii strains to tolerate acidic stress better and grow faster at 37 °C than S. cerevisiae [15]. Due to the fact that S. boulardii from a taxonomic point of view should not be recognized as a separate species, S. boulardii will in the following be referred to as S. cerevisiae var. boulardii. It is worth to notice that contrary to e.g., probiotic strains of lactic acid bacteria, apparently there seems not to be different strains within S. cerevisiae var. boulardii. Based on the similarity in different molecular analyses, all isolates appear to originate from the one isolated from litchi fruit in Indochina by Henri Boulard [12].

Experimental Effects of S. cerevisiae var. boulardii

Effects on enteric bacterial pathogens

Several studies have shown that S. cerevisiae var. boulardii confer beneficial effects against various enteric pathogens, involving different mechanisms as: (i) prevention of bacterial adherence and translocation in the intestinal epithelial cells, (ii) production of factors that neutralize bacterial toxins and (iii) modulation of the host cell signalling pathway associated with pro-inflammatory response during bacterial infection.

Prevention of bacterial adherence and translocation in the intestinal epithelial cells is due to the fact that the cell wall of S. cerevisiae var. boulardii has the ability to bind enteropathogens. S. cerevisiae var. boulardii cell wall has shown binding capacity to enterohaemorrhagic Escherichia coli and Salmonella enterica serovar Typhimurium [16]. Additionally, the yeast inhibits adherence of Clostridium difficile to Vero cells (derived from kidney epithelial cells). Pre-treatment of C. difficile or the Vero cells with S. cerevisiae var. boulardii or its cell wall particles results in lowering the adherence of bacteria to the Vero cells. Yeast cells or cell wall particles are able to modify the surface receptors involved in adhesion of C. difficile through a proteolytic activity and by steric hindrance [17].

Administration of S. cerevisiae var. boulardii reduces adherence of enterotoxigenic E. coli to mesenteric lymph node in pigs intestine [18]. S. cerevisiae var. boulardii has also beneficial effect on Citrobacter rodentium-induced colitis in mice, which is due to attenuating the adherence of C. rodentium to host epithelial cells, through reduction in EspB and Tir protein secretions, respectively a translocator and an effector protein implicated in the type III secretion system (TTSS) [19]. In a study on rats, ingestion of S. cerevisiae var. boulardii decreased the incidence of antibiotic-induced bacterial translocation. The total bacteria count of fecal flora and especially the number of Gram-negative bacteria were significantly lower after intake of the yeast in addition to antibiotic [20]. However, in other studies on enteropathogenic E. coli-or Shigella-infected T84 cells (human colonic adenocarcinoma cell line) and on mice infected with S. enterica serovar Typhimurium or Shigella flexneri, in which S. cerevisiae var. boulardii demonstrated beneficial effects, no effect on modifying the bacterial adherence was observed [21-23].

S. cerevisiae var. boulardii produces two proteins of 54 and 120 kDa being responsible for degradation or neutralisation of bacterial toxins. The 54 kDa protein is a serine protease that decrease the enterotoxic and cytotoxic activities of C. difficile by proteolysis of the toxin A and inhibition of binding of the toxin to its brush border membrane receptor. In vivo studies have shown that oral administration of S. cerevisiae var. boulardii or its supernatant decreases toxin A-induced intestinal secretion and permeability due to activity of this enzyme [24-26]. The 120 kDa protein has a nonproteolytic activity, competes specifically with the chloride secretion stimulated by the toxins of Vibrio cholera by reducing the cyclic adenosine monophosphate (cAMP) in the intestinal cells [27,28]. Both S. cerevisiae var. boulardii and S. cerevisiae W303 have the ability to protect Fisher rats against cholera toxin [29]. S. cerevisiae var. boulardii also synthesizes a protein phosphatase that dephosphorylates endotoxins such as lipopolysaccharide of E. coli 055B5 and inactivates its cytotoxic effects [30].

In vitro studies using mammalian cell cultures have shown that S. cerevisiae var. boulardii modifies host cell signalling pathways associated with pro-inflammatory response during bacterial infection. The mechanism is based on blocking activation of nuclear factor-kappa B (NF-.B) and mitogenactivated protein kinase (MAPK) which decreases the expression of inflammation-associated cytokines such as interleukin 8 (IL-8), tumor necrosis factor alpha (TNF-a) and interferon gamma (IFN-.) [22,31,32]. The exposure of mammalian cells to S. cerevisiae var. boulardii before addition of enteropathogenic and enterohaemorrhagic E. coli reduces activation of NF-.B and MAPK, diminish production of TNF-a and secretion of IL-8 [21,31], delay enterohaemorrhagic E. coli -induced apoptosis (due to the reduction of TNF-a) and decline pro-inflammatory cytokine synthesis [32].

S. cerevisiae var. boulardii produces a 10 kDa protein that exerts anti-inflammatory effects after stimulation with C. difficile-toxin A due to decrease in secretion of IL-8 in human colonocytes and activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in both human colonocytes and murine ileal loops [33]. Sougioultzis et al. [34] has shown that S. cerevisiae var. boulardii produces a low molecular weight soluble factor (< 1 kDa) which blocks NF-.B activation and NF-.B-mediated IL-8 gene expression in intestinal epithelial cells and monocytes. Expression of the pro-inflammatory cytokine IL-1a also decreased in IPEC-J2 cells (porcine intestinal epithelial cell lines) exposed to enterohaemorrhagic E. coli, when cells were pre- and co-incubated with S. cerevisiae var. boulardii [35].

Maintenance of epithelial barrier integrity

Klingberg et al. [36] have shown that exposure of different strains of S. cerevisiae var. boulardii and S. cerevisiae to Caco-2 cells (human epithelial colorectal adenocarcinoma cell lines) increased the transepithelial electrical resistance (TER) across polarized monolayers of cells. In another study, infection of T84 cells with enteropathogenic E. coli reduced the monolayer transepithelial resistance and distribution of tight-junction-associated protein Zonula occludens (ZO-1) was altered, which caused disruption of epithelial barrier structure [21]. Presence of S. cerevisiae var. boulardii in the infection showed no alteration in the transepithelial resistance and ZO-1 protein distribution, suggesting a protective effect of S. cerevisiae var. boulardii on the tight-junctions structure of T84infected cells. During bacterial infection, the myosin light chain protein (MLC) is phosphorilated and the tight-junctions are disrupted. Dahan et al. [31] have shown that S. cerevisiae var. boulardii abolished phosphorylation of MLC and thereby eliminated the reduction of TER after infection of cells with enterohaemorrhagic E. coli and in that way preserved the barrier function. In Shigella-infected T84 cells, the yeast positively affected tight-junctions proteins (claudin-1 and ZO-2) and significantly protected the barrier function [22]. Shigella-infected cellular monolayer had a dramatic decrease in claudin-1 and ZO-2 levels. In the presence of yeast, cellular monolayer exhibited larger amounts of these proteins. These results demonstrate that S. cerevisiae var. boulardii enhances the ability of intestinal epithelial cells to restore the tight-junction structure and the barrier permeability.

Anti-inflammatory effects

Besides reducing inflammation during bacterial infection by interfering with the host cell signalling pathways, S. cerevisiae var. boulardii also stimulates the peroxisome proliferator-activated receptor-gamma (PPAR-.) expression in human colonocytes and reduces the response of human colon cells to pro-inflammatory cytokines [37]. PPAR-. is a nuclear receptor expressed by several cell types including intestinal epithelial cells, dendritic cells, T and B cells, and can act as a regulator of the inflammation [38]. S. cerevisiae var. boulardii has been reported to modify the migratory behaviour of lymphocytes. This was observed in a mice model of inflammatory bowel disease (IBD), where inhibition of inflammation in the colon was detected in animals treated with S. cerevisiae var. boulardii.

The inhibition was due to decrease in the production of IFN-. and a modification of T cell distribution. There was a decrease in IFN-.-producing CD4+ T cells within the colonic mucosa and an increase in IFN-.-producing T cells in the mesenteric lymph nodes. In addition, S. cerevisiae var. boulardii supernatant modifies the capacity of endothelial cells to adhere to leucocytes, allowing better cell rolling and adhesion [39]. In inflammatory bowel disease (IBD), production of high levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) activity is associated with inflammatory effects [40]. The inhibitory effect of S. cerevisiae var. boulardii on iNOS activity has been investigated by Girard et al. [41] in rats with castor oil-induced diarrhoea. Administration of yeast blocked the production of the citrulline (a marker of NO production). The iNOS inhibition by S. cerevisiae var. boulardii may be beneficial in the treatment of diarrhoea and/or IBD associated with overproduction of NO.

Effects on immune response

There are several studies indicating the stimulation of the host cell immunity, both innate and adaptive immunity, by S. cerevisiae var. boulardii in response to pathogen infections. Oral administration of S. cerevisiae var. boulardii in healthy volunteers revealed several cellular and humoral changes in peripheral blood. This contributes to the activation of the reticuloendothelial and complement system, demonstrating the stimulation of the innate immune system by the yeast [42]. Oral ingestion of S. cerevisiae var. boulardii stimulated secretion of immune factors, i.e., adaptive immunity. In a study by Buts et al. [43], the level of secretory immunoglobulin A (sIgA) increased 57% in the duodenal fluid and the secretory component of immunoglobulins enhanced 69% in villus cells and 80% in crypt cells of rats treated with the high dose of yeast. Application of S. cerevisiae var. boulardii to mice treated with C. difficile toxin A caused a 1.8-fold increase in total sIgA levels and a 4.4-fold increase in specific antitoxin A sIgA levels [44]. In another study, after intravenous administration of E. coli, germ-free mice mono-associated with S. cerevisiae var. boulardii showed higher clearance of the pathogen from the bloodstream compared to germ-free mice, which was correlated with earlier production of IFN-. and IL-12 in the serum [45].

Trophic effects on intestinal mucosa

Several studies have shown that S. cerevisiae var. boulardii exerts trophic effects restoring the intestinal homeostasis. Oral administration of yeast by human volunteers or rats enhanced the activity of brush border membrane enzymes, e.g., sucrase-isomaltase, lactase, maltase-glucoamylase, aglucosidase and alkaline phosphatase, which have a positive influence on nutrient degradation and absorption [46,47]. Oral administration of yeast after partial resection of the small bowel, increased disaccharidase activities and improved the absorption of D-glucose as well as the expression of the sodium/glucose cotransporter-1 (SGLT-1) in the brush border of the remaining intestinal segments [48]. Improvement of expression of SGLT-1 by S. cerevisiae var. boulardii, which is implicated in water and electrolyte re-absorption, could be beneficial in the treatment of diarrhoea and congenital sucrase-isomaltase deficiency. S. cerevisiae var. boulardii cells contain high level of polyamines and it has been suggested that endoluminal release of polyamines (mainly spermine and spermidin) by S. cerevisiae var. boulardii, may contribute to rise in expression of intestinal enzymes, i.e., increase in sucrase and maltase activity [49].

Modification of luminal short-chain fatty acids (SCFAs) concentration is another trophic effect of the yeast. SCFAs are among the most important metabolites produced by anaerobic bacteria in the colon and are involved in water and electrolyte absorption by the colonic mucosa [50]. Patients on long-term total enteral nutrition have a decrease in number of fecal anaerobic bacteria and in the level of fecal SCFAs [51]. Schneider et al. [52] have shown that administration of S. cerevisiae var. boulardii in these patients increased the level of total fecal SCFAs up to 9 days after termination of the treatment. However, yeast did not modify the fecal flora. This increase in fecal SCFAs concentration may explain the preventive effects of the yeast in enteral nutrition-induced diarrhoea.

S. cerevisiae var. boulardii further has the ability to prevent reactions to food antigens. In neonates and young infants, the quality of endoluminal proteolysis is very important in the absorption of completely or incompletely degraded proteins and antigens by the mucosal barrier with increased permeability. This is one of the fundamental mechanisms involved in food protein intolerance. Buts et al. [53] have shown the endoluminal release of a leucine aminopeptidase by S. cerevisiae var. boulardii in rats and thereby enhancement of N-terminal hydrolysis of oligopeptides in both endoluminal fluid and intestinal mucosa. Thus, they proposed that this function of S. cerevisiae var. boulardii could be important in preventing reactions to food antigens when mucosal permeability is increased.

Application of S. cerevisiae var. boulardii in Clinical Trails

S. cerevisiae var. boulardii has been used in different clinical trails against different diarrhoeal diseases and has shown promising results. Treatment with S. cerevisiae var. boulardii is well tolerated, except for sporadic reports of fungemia, in immune-compromised patients or patients with severe general or intestinal diseases in most cases infected through an indwelling central venous catheter [54-56]. One of the benefits of using S. cerevisiae var. boulardii as a probiotic is the natural resistance of that to antibacterial antibiotics, thus it can be prescribed to patients receiving antibacterial antibiotic therapy.

Antibiotic-associated diarrhoea (AAD) is a common complication of treatment with antibiotics
caused by disruption of normal gut microbiota and colonization of pathogenic bacteria which results in an acute inflammation of the intestinal mucosa. The most common opportunistic pathogen related to AAD is C. difficile [57-59]. Among other infectious organisms Staphylococcus aureus, Clostridium perfringens, Klebsiella oxytoca, Candida species, E. coli and Salmonella species can be mentioned [60,61]. S. cerevisiae var. boulardii has been comprehensively evaluated for the prevention of AAD and the potential effect of the yeast in decreasing the ADD in adults and children has been proven [62,63].

Traveller’s diarrhoea is a common health complaint among persons travelling from low risk regions to developing countries where enteric infection is hyper-endemic. Enterotoxigenic E. coli, Shigella and Salmonella account for about 80% of the cases with an identified pathogen [64]. In a meta-analysis study performed by McFarland [65], it has been concluded that S. cerevisiae var. boulardii has a significant efficacy on the prevention of Traveller’s diarrhoea.

Several randomized placebo-controlled studies have proven the efficacy of S. cerevisiae var.
boulardii in the treatment and prevention of acute infectious [66,67]. Intestinal disorder and diarrhoea are also common complications in critically ill patients with enteral nutrition which is caused by alteration in the colonic microbiota [51]. The effect of S. cerevisiae var. boulardii to prevent and reduce the incidence of diarrhoea and to decrease the length of this disease has been demostrated [68]. In the patient with AIDS-associated diarrhoea, the efficacy of S. cerevisiae var. boulardii has been proven by a randomized, double-blind trail [69,70].

S. cerevisiae var. boulardii has also shown positive results in patients with irritable bowel syndrome (IBS). In a double-blind, placebo-controlled study, performed on patients with diarrhoea-predominant IBS, administration of S. cerevisiae var. boulardii decreased the daily number of stools and improved the consistency of the stools [71]. A double-blind study on the patients with Crohn's disease with moderate activity showed that the addition of S. cerevisiae var. boulardii to conventional therapy considerably reduced bowel movements [72]. In patients with Crohn's disease of the ileum or colon who had been in remission for more than 3 months, treatment with S. cerevisiae var. boulardii together with the conventional therapy was more efficient in preventing relapse, compared to conventional therapy alone [73]. In patients with mild-to-moderate ulcerative colitis, addition of yeast to the conventional therapy resulted in clinical remission for 68% of patients [74]. In a randomized-placebo study on the patients with Crohn’s disease in remission, addition of S. cerevisiae var. boulardii to the baseline medications improved intestinal permeability with a decrease in the lactulose/mannitol ratio [75].

Beneficial Effects of Yeasts on Bioavailability of Nutrients

Biodegradation of Phytate by Yeasts

Antinutritional effects of phytate

Phytic acid or phytate (myo-inositol hexakisphosphate, IP6) is the primary storage form of phosphorus in mature seeds of plants and it is particularly abundant in many cereal grains, oilseeds, legumes, flours and brans. Phytate has a strong chelating capacity and forms insoluble complexes with divalent minerals of nutritional importance such as iron, zink, calcium and magnesium [76-78]. Human as well as monogastric animals like poultry and pigs, lack the required enzymes in the gastrointestinal tract for degradation and dephosphorylation of the phytate complex. Besides, lowering the bioavailability of divalent ions, phytate may have negative influence on the functional and nutritional properties of proteins such as digesting enzymes [79]. In addition, lower inositol phosphates attained from degradation of phytate have a positive role in cancer prevention and treatment [80,81].

Dephosphorylation of phytate is catalyzed by phytases (myo-inositol-hexakisphosphate 6-phosphohydrolases). Characterized phytases are nonspecific phosphatase enzymes, which release free inorganic phosphate (Pi) and inositol phosphate esters with a lower number of phosphate groups. Organisms such as plants and microorganisms extensively produce phytase enzymes and make the minerals and phosphorus present in the phytates available through a stepwise phytate hydrolysis [82]. In food processing, degradation of phytate can be catalyzed either by endogenous enzymes, naturally present in cereals, or by microbial enzymes produced by e.g., yeasts or/and lactic acid bacteria naturally present in flour or added as starter cultures [83]. Accordingly, improved adsorption of iron, zinc, magnesium and phosphorus can be achieved by degradation of phytate during food processing [84,85] or by degradation of phytate in the intestine [86].

Phytase activity by yeasts

Phytases are widespread in various microorganisms including filamentous fungi, Gram-positive and Gram-negative bacteria and yeasts [87]. Among yeasts, Candida krusei (Issatchenkia orientalis) [88], Schwanniomyces castellii [89], Debaryomyces castellii [90], Arxula adeninivorans [91,92], Pichia anomala [92,93], Pichia rhodanensis, Pichia spartinae [94], Cryptococcus laurentii [95], Rhodotorula gracilis [96], S. cerevisiae [97-100], Saccharomyces kluyveri, Torulaspora delbrueckii, Candida spp. and Kluyveromyces lactis [94] have been identified as phytase producers. In a study by Olstorpe et al. [92] on the ability of different yeast strains (122 strains from 61 species) to utilize phytic acid as sole phosphorus source, strains of A. adeninivorans and P. anomala showed the highest volumetric phytase activities.

Production of phytase by S. cerevisiae has been investigated in different studies [83,99]. The phytase activity of S. cerevisiae is partly due to the activity of the secretory acid phosphatases (SAPs), which are secreted by the cells to the growth media and are repressed by inorganic phosphate (Pi) [99]. However, the phytase activity of yeasts, e.g., during bread leavening, is relatively low [83,101,102]. This could be due to the repression of the SAPs by Pi [99]. Besides, repression of phytate-degrading enzymes is dependent on the pH and the medium composition. Andlid et al. [99] have shown that repression of phytate-degrading enzymes is weak in complex medium with pH 6.0 and high amount of phosphate. Regardless of Pi addition, the capacity to degrade phytase is highest at the pH far from the optimum pH for the SAPs, suggesting that pH has more effect on the expression of the enzyme that on
the enzyme activity.

S. cerevisiae as a phytase carrier in the gastrointestinal tract and hydrolysis of phytate after digestion has also been investigated. In a study using a high-phytase producing recombinant yeast strain at simulated digestive conditions, a strong reduction of phytate (up to 60%) in the early gastric phase was observed as compared to no degradation by wild-type strains. The phytase activity during digestion was influenced by the type of yeast strain, cell density, and phytate concentration. However, degradation in the late gastric and early intestinal phases was insignificant, in spite of high phytate solubility, high resistance against proteolysis by pepsin, and high cell survival [103]. This study also showed the importance of pH as a limiting factor for phytase expression and/or activity, as observed by Andlid et al. [99].

Application of yeast phytases in foods

Yeasts or yeast phytases can be applied for pre-treatment of foods to reduce the phytate contents or they can be utilized as food supplement in order to hydrolysis the phytate after digestion. The phytase activity of yeast during bread making for reduction of phytate content of bread have been examined. However, it seems to be too low to significantly influence the iron absorption [99]. Nevertheless, as explained earlier, during bread making, the content of phytic acid decreases. This is due to the action of phytases in the dough (cereal) and the activity of starter culture [83,104-106]. Chaoui et al. [106] have shown that phytase activity in sourdough bread is highest using combinations of yeasts and lactic acid bacteria as starter culture. The same result was found by Lopez et al. [105]. They found that phytate contents in yeast and sourdough bread were lower than in reconstituted whole-wheat flour and that mineral bioavailability could be improved by bread making especially using both yeast and lactic acid bacteria. Therefore a high-phytase S. cerevisiae strain, may be suitable for the production of food-grade phytase and for direct use in food production [98]. Increasing the bioavailability of minerals is especially of importance in low-income countries. Therefore it is important to notice that apart from bread, reduction of phytates by yeast phytases have been observed in other plant-derived foods such as in ‘Icacina mannii paste’, a traditional food in Senegal, during fermentation with S. cerevisiae [107] and in ‘Tarhana’, a traditional Turkish fermented food, using baker's yeast as a phytase source [108].

Folate Biofortification by Yeasts

Importance of folate in the human diet

Folates (vitamin B9) are essential cofactors in the biosynthesis of nucleotides and therefore crucial for cellular replication and growth [109,110]. Plants, yeast and some bacterial species contain the folate biosynthesis pathway and produce natural folates, but mammals lack the ability to synthesize folate and they are therefore dependent on sufficient intake from the diet [111]. During the last years, folates have drawn much attention due to the various beneficial health effects following an increased intake. The role of folate in the prevention of neural tube defects in the foetus has been established [112,113] and sufficient folate intake may reduce the risk of cardiovascular disease [112,114], cancer [112,115] and even Alzheimer's disease [116]. The recommended dietary intake (RDI) for the adult population is between 200–300 µg/day for males and between 170–300 µg/day for females according to the FAO/WHO in the USA and several European countries [117]. Insufficient folate levels result in prolonged cell division, which leads to megaloblastic anaemia [118].

Folate production by yeasts

S. cerevisiae is a rich dietary source of native folate and produces high levels of folate per weight [119]. Besides the role as a biofortificant in fermented foods, high producing strains may be used as biocatalysts for biotechnological production of natural folates. The folate level can be considerably augmented in fermented foods using an appropriate yeast strain and by optimizing the growth phase and cultivation conditions for the selected strain. Hjortmo et al. [120] have found that the growth medium and physiological state of cells are important factors in folate production. In synthetic growth medium, high growth rate subsequent to respiro-fermentative growth resulted in the highest specific folate content (folate per unit biomass). In complex media, the level of folate was much lower and less related to growth phase. The specific content of folate in yeast is not only species-specific but also dependents on the yeast strain. In another study, Hjortmo et al. [121] investigated the folate content and composition and the dominating forms of folate found in 44 different strains of yeasts belonging to 13 different yeast species cultivated in a synthetic medium at standard conditions. There was a large diversity in relative amounts of folate content among the studied yeasts. Tetrahydrofolate (H4folate) and -methyl-tetrahydrofolate (5-CH3-H4folate) were the dominating forms, which were varying extensively in relative amounts between different strains. Several strains showed a 2-fold or higher folate content as compared to the control strain, i.e., a commercial strain of Baker's yeast. This indicates that by choosing an appropriate strain, the folate content in yeast-fermented foods may be enhanced more than 2-fold. These scientists have shown that using a specific strain of S. cerevisiae cultured in defined medium and harvested in the respiro-fermentative phase of growth prior to dough preparation the folate content increased 3 to 5-fold (135–139 µg/100 g dry matter) in white wheat bread, compared to white wheat bread industrially processed with commercial S. cerevisiae (27–43 µg/100 g dry matter) 122].

Effect of yeasts on folate biofortification of food

Cereals, especially whole grain products, are the main supplier of folate in the diet. Yeast has crucial effects on the folate contents of breads. Breads prepared with baking powder have the lowest folate contents, while addition of yeast results in higher folate content in bread [123]. The variety of sourdoughs and baking processes obviously lead to great variation in folate content of breads. Total folate content increases considerably during sourdough fermentation due to the growth of yeasts [123,124]. However, there would be some losses (about 25%) in the amount of folate following the baking [123]. Final folate content is dependent on the microflora and amylolytic activity of flour, starter cultures and baking conditions [125]. Other microorganisms present in the sourdough like lactic acid bacteria may also influence the folate content. In a study, Kariluoto et al. [126] investigated the ability of typical sourdough yeasts (S. cerevisiae, Candida milleri, and T. delbrueckii) and lactic acid bacteria to produce or consume folates during sourdough fermentation. Yeasts increased the folate contents of sterilised rye flour-water mixtures to about 3-fold after 19 h, whereas lactobacilli not only did not produce folates but also decreased it to ultimately half amount. Although the lactobacilli consumed folates, their effect on folate contents in co-cultivations with yeasts was minimal.

In beer, the amount of folate enhances due to synthesis by the yeast during the initial period of the fermentation. However, since yeast folate is intracellular, after cropping the yeast, folate will be eliminated from the beer and this is regardless the type of yeast. Some beer brands, which have a secondary fermentation step (often in the bottle), contain higher level of folate [125].

Production of folate in kefir has also been investigated. Kefir is a fermented milk beverage that originated in Eastern Europe and regarded as a natural probiotic product, i.e., a health promoting product [127]. It is produced by the fermentation of milk with kefir granules (grains) and contains different vitamins and minerals. Kefir granules have a varying and complex microbial composition including species of lactic acid bacteria (as the largest portion of microorganism), acetic acid bacteria, yeasts and mycelial fungi. Yeasts isolated from Kefir grains include Kluyveromyces marxianus, Saccharomyces exiguus, Candida lambica and C. krusei (I. orientalis) [128]. Kefir contains high folate content, which is produced by the yeast and not the lactic acid bacteria [129]. In a study, Patring et al. [129] investigated the folate content of different yeast strains isolated from Russian kefir granules, belonging to different Saccharomyces and Candida species. Kefir yeast strains showed high folate-producing capacity. The most abundant folate forms were 5-CH3-H4folate (43–59%) and 5formyltetrahydrofolate (5-HCO-H4folate, 23–38%), whereas H4folate occurred in a minor proportion (19–23%). By choosing yeast strains that produce a higher proportion of the most stable folate forms such as 5-HCO-H4folate and 5-CH3-H4folate, it is possible to improve the stability of folates during fermentation and storage, and thus to increase the folate content in kefir products.

Recently, the folate content of a traditionally fermented maize-based porridge, called togwa, consumed in rural areas in Tanzania has been investigated by Hjortmo et al. [130]. The yeasts strains belonged to C. krusei (I. orientalis), P. anomala, S. cerevisiae, K. marxianus and Candida glabrata. The major folate forms found during the fermentations were 5-CH3-H4folate and H4folate. The content of H4folate, per unit togwa, remained quite stable at a low level throughout the experiment for all strains, while the concentration of 5-CH3-H4folate was highly strain- and time-dependent. The highest folate concentration was found after 46 h of fermentation with C. glabrata, corresponding to a 23-fold increase compared with unfermented togwa. As for degradation of phytate, selection of appropriate yeast strains as starter cultures in indigenous fermented foods appears to have high potential in especially developing countries where the vitamin intake generally is lower. Compared to e.g., lactic acid bacteria yeast are much more robust and may therefore more easily be distributed as starter cultures.

Beneficial Effects of Yeasts on Detoxification of Mycotoxins

Prevention of Toxic Effects of Mycotoxins

Mycotoxins are econdary metabolites produced by fungi belonging mainly to the Aspergillus, Penicillium and Fusarium genera. Agricultural products, food and animal feeds can be contaminated by these toxins and lead to various diseases in humans and livestocks [131]. Contamination of agricultural products by mycotoxins is a worldwide dilemma, however it is rigorous in tropical and subtropical regions [132]. The most important mycotoxins are the aflatoxins, ochratoxins, fumonisins, deoxynivalenol (DON), zearalenone (ZEA) and trichothecenes [133,134]. There are three general strategies in order to prevent the toxic effects of mycotoxins in foods: (i) prevention of mycotoxin contamination (ii) decontamination/detoxification of foods contaminated with mycotoxins and (iii) inhibition of absorption of consumed mycotoxin in the gastrointestinal tract [135]. The ideal solution to reduce the health risk of mycotoxins is to prevent contamination of foods with them. Unfortunately, this can not be completely avoided and sporadically mycotoxin contamination is reported in food products, especially in the developing world [136].

Therefore, there is an increased focus on effective methods for detoxification of mycotoxins present in foods and also on the inhibition of mycotoxin absorption in the gastrointestinal tract. Various physical and chemical methods are available for the detoxification of food products contaminated with mycotoxins. However, due to disadvantages of these methods, such as possible losses in the nutritional quality of treated commodities, limited efficacy, reduction of sensory quality and high cost of equipment, their application has been restricted [135]. An alternative strategy could be utilization of microorganisms capable of detoxifying mycotoxins in contaminated foods and feeds.

Biodegradation of Mycotoxins by Yeasts

Interests in biodegradation of mycotoxins have been increased significantly, since it is specific and environmentally friendly to reduce or eliminate the possible contaminations of mycotoxins in foods. Various microorganisms such as soil or water bacteria, fungi, and protozoa as well as specific enzymes isolated from microbial systems are able to some extent and with varied efficiency to degrade mycotoxins to less- or non-toxic products [137]. Degradation of mycotoxins subsequent to yeast fermentation has been reported in different studies. Degradation of patulin during fermentation of apple juice by S. cerevisiae with E-ascladiol and Z-ascladiol as major metabolites [138] and degredation of zearalenone by several yeast strains has been observed [139]. However, degradation of zearalenone leads to conversion of that to a- and ß-zearalenol, which are still toxic. Degradation of ochratoxin A, fumonisins B1 and B2 [140], deoxynivalenol and T-2 toxin [141] by S. cerevisiae has been reported. Two yeast strains, Phaffia rhodozyma and Xanthophyllomyces dendrorhous, have also been shown to have ochratoxin A (OTA) degrading activity by converting OTA to ochratoxin a possibly mediated by an enzyme related to carboxypeptidases [142].

Mycotoxin Absorption by Yeasts

Inhibition of mycotoxin absorption in the gastrointestinal tract is another way to prevent the toxic effects of mycotoxins. There has been increased interest in the use of mycotoxin binding agents, e.g., yeasts and yeast-derived products, which can be added to the diet to bind mycotoxins. S. cerevisiae has the ability to bind mycotoxins as reviewed by Shetty and Jespersen [143]. The mechanism of detoxification by yeast is due to the adhesion of mycotoxins to cell-wall components. As for binding of pathogenic bacteria [16], mannan components of the cell wall play a major role in mycotoxin binding [144]. In vitro efficacy of esterified glucomannan to bind aflatoxin B1, ochratoxin A and T-2 toxin, when present alone or in combination, was assessed in toxin-contaminated feed. Esterified glucomannan showed significantly higher binding ability to aflatoxin B1 than to ochratoxin A and T-2 toxin in a dose dependent manner [145].

In a study by Aravind et al. [146] performed on broiler chicks to determine the efficacy of esterified glucomannan in counteracting the toxic effects of mycotoxins in naturally contaminated diet (aflatoxin, ochratoxin, zearalenone and T-2 toxin), it was observed that esterified glucomannan effectively improved the growth depression caused by mycotoxins. Strains of S. cerevisiae have been shown to bind ochratoxin A [147] and zearalenone as well [148]. Ochratoxin A and T-2 toxins also bind to glucomannan component of cell wall [145,149]. However, zearalenone bind to ß-d-glucans of yeast cell wall [148]. It has been shown that yeast cell wall derived products efficiently adsorbed zearalenone (>70%) in an in vitro model that resembled the different pH conditions in the pig gastrointestinal tract, but they were not able to bind deoxynivalenol in a considerable percentage [150]. In study on mice, when dried yeast and yeast cell walls were added to the diet along with aflatoxin B1, a significant reduction in the toxicity was observed [151]. Similarly, Madrigal-Santillán et al. [152] described the potential of S. cerevisiae to improve weight gain and reduce genotoxicity of aflatoxin B1 in mice fed with contaminated corn.

Even though several trials have been made for decontamination of animal feeds by yeast, very little have so far been conducted on decontamination of foods and beverages. Binding of mycotoxins to yeast has especially been investigated during winemaking. It has been shown that yeasts can bind to ochratoxin A and remove it from the white and red wine. Ochratoxin A removal from grape must was due to binding of the toxin to the yeast cell wall, and mannoproteins were involved in the mycotoxin absorption during winemaking. The implication of this finding could be very important in the winemaking of must contaminated with ochratoxin A [153,154]. Oenological strains of Saccharomyces yeasts can also be used for the decontamination of ochratoxin A in synthetic and natural grape juice. Heat-treated cells showed higher absorption (90% w/w) compared to viable cells (35% w/w) showing the involvement of physical binding, and cell density played an important role in absorption efficiency. Dead yeasts do not pose any quality or safety problems and can be potentially used for detoxification of the grape juice [147].

S. cerevisiae is one of the most important microorganisms involved in food fermentations in tropical countries with high level of mycotoxin contamination in the foods. Shetty et al. [155] have investigated Aflatoxin B1 binding abilities of S. cerevisiae strains isolated from fermented maize dough (Kenkey) and sorghum beer (Pito), indigenous fermented foods from Ghana, West Africa. They showed that aflatoxin binding was strain specific and strains were found to bind 10–40% and some of them more than 40% of the added aflatoxin B1 at standard condition. Highest binding capacity was observed at the exponential growth phase with 53% binding of the total toxin and the binding reduced towards the stationary phase. Aflatoxin B1 binding increased in a dose dependent manner after addition of aflatoxin, regardless to the temperatures ranging from 20 to 37 °C, but was significantly reduced at 15 °C. Heat and acid treated cells showed higher binding capacity, i.e., up to 78% binding of the total added toxin [155]. Following this study unpublished results by Jespersen et al. have shown the yeast-aflatoxin B1 complex to be stable during the passage of an in vitro gastrointestinal tract model indicating that the aflatoxin will not be absorbed in the gastrointestinal tract but excreted together with the yeast cells in the human feces. Additionally, strains of S. cerevisiae isolated from indigenous fermented foods which are effective aflatoxin binders have been proven to be usable as starter cultures with additional capacities to decontaminate mycotoxins in fermented maize products.

Conclusions

Yeasts are used in preparation of human foods and beverages, where they besides having technological functions, confer different beneficial effects on human health and well-being. Among these, the most well known is the probiotic effect, which has been proven for S. cerevisiae var. boulardii. This is the only yeast produced and used as a pharmaceutical product offering numerous valuable effects such as prevention and treatment of intestinal diseases and immunomodulatory effects. Since S. cerevisiae var. boulardii is recognised as a member of the species S. cerevisiae, it is most likely that also other strains within S. cerevisiae might show probiotics properties. Even though to date, most efforts have been focused on analysing the probiotic effects of S. cerevisiae var. boulardii isolates. Similarly, other yeast species might have probiotic effects and confer positive effects on human health. Therefore, it is recommended that additional attempts are placed on discovering the probiotic properties among other yeast species and strains.

Besides the role as probiotics, yeasts have other beneficial functions such as dephosphorylation of phytate and folate biofortification of foods. By choosing appropriate yeast strains as starter cultures and using optimized food processing techniques, it is possible to improve the nutritional value of foods in general. In low-income areas such as the developing countries, application of yeast strains with high phytase and/or folate producing capacity in indigenous food fermentation will increase the bioavailability of minerals and folate content in the foods and will reduce the risk of different diseases such as neural tube defects and anaemia. Therefore, investigation of potential yeast strains isolated from indigenous native foods with high phytase activity and/or high folate content is encouraged.

Contamination of agricultural products by mycotoxins causes severe threat to both livestock productivity and human health and brings huge worldwide economic losses each year. This is especially a rigorous problem in tropical and subtropical regions. For this reason, isolation and application of yeast strains with high mycotoxin binding capacity from indigenous fermented foods would be a valuable alternative for decontamination of mycotoxins in food. Strains of yeast, e.g., S. cerevisiae with high mycotoxin binding abilities can be used as part of the starter cultures in the fermentation of food and beverages, and heat treated cell walls or purified components can be applied as additives in small quantities without compromising the characteristics of the final product. The application of S. cerevisiae as mycotoxin binders in human foods highly depends on stability of the yeast-mycotoxin complex through the passage of the gastrointestinal tract. Additional research efforts are required in this area to explore the great potential of using S. cerevisiae as a detoxifying agent in contaminated foods.

So far, tremendous efforts have been placed on utilising the probiotic effects of especially lactic acid bacteria, whereas rather limited emphasis has been placed on the beneficial effects offered by yeast. Yeasts do meanwhile offer several advantages compared to lactic acid bacteria. They do have a more diverse enzymatic profile, they appear to have a more versatile effect on the immune system, they do provide protection against pathogenic bacteria and toxic compounds by surface binding and appear to be better suited for nutritional enrichment and delivery of bio-active molecules. Besides yeast are much more robust than lactic acid bacteria which make them easier to produce and to distribute, especially in less developed areas. It is therefore encouraged that additional efforts are placed on exploring the health beneficial effects of yeasts, especially those properties that can not be replaced by lactic acid bacteria.

Acknowledgements

This work was supported by Faculty of Life Sciences, University of Copenhagen, Denmark.

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by EricT

Nonclassical Vitamin D Actions

Sat, 15 May 2010 17:04:01 +0000

Armin Zittermann and Jan F. Gummert

Clinic for Thoracic and Cardiovascular Surgery, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum, Georgstrasse 11, 32545 Bad Oeynhausen, Germany

Nutrients 2010

It is becoming increasingly clear that vitamin D has a broad range of actions in the human body. Besides its well-known effects on calcium/phosphate homeostasis, vitamin D influences muscle function, cardiovascular homeostasis, nervous function, and the immune response. Vitamin D deficiency/insufficiency has been associated with muscle weakness and a high incidence of various chronic diseases such as cardiovascular disease, cancer, multiple sclerosis, and type 1 and 2 diabetes. Most importantly, low vitamin D status has been found to be an independent predictor of all-cause mortality. Several recent randomized controlled trials support the assumption that vitamin D can improve muscle strength, glucose homeostasis, and cardiovascular risk markers. In addition, vitamin D may reduce cancer incidence and elevated blood pressure. Since the prevalence of vitamin D
deficiency/insufficiency is high throughout the world, there is a need to improve vitamin D status in the general adult population. However, the currently recommended daily vitamin D intake of 5–15 µg is too low to achieve an adequate vitamin D status in individuals with only modest skin synthesis. Thus, there is a need to recommend a vitamin D intake that is effective for achieving adequate circulating 25-hydroxyvitamin D concentrations (>75 nmol/L).

Introduction

Vitamin D has long been known for its effects on calcium and bone metabolism. Severe vitamin D deficiency causes a lack of bone mineralization, which manifests as rickets in children and osteomalacia in adults. There is also accumulating evidence that insufficient vitamin D status contributes to the bone disease osteoporosis. Adequate vitamin D supplementation can reduce the risk of osteoporotic fractures by approximately 20% [1]. However, it is now becoming increasingly clear that vitamin D has a much broader range of actions in the human body than believed before. Its physiological effects are not only limited to bone. Various other chronic diseases that are frequently observed in modern societies are probably at least in part caused by inadequate vitamin D supply. The present article describes the potential clinical relevance of nonclassical vitamin D actions. It refers to randomized, controlled clinical trials (RCTs) or meta-analyses of RCTs whenever it is possible. Results from non-RCTs are also presented in fields where no RCTs are available yet. Although the article primarily refers to the literature of the last four years, some useful older data are also included. Note that this article should provide evidence for nonclassical vitamin D actions. It is not a systemic review of the available literature.

Vitamin D Metabolism

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Vitamin D is unique among vitamins in that humans can produce it themselves in their skin provided they have sufficient exposure to ultraviolet radiation B (290–315 nm). Vitamin D is also found naturally in small amounts in milk and eggs, and in relatively large amounts in fatty fish such as herring and mackerel. Nevertheless, skin synthesis of vitamin D usually contributes 80% to 90% to vitamin D supply in free-living persons. This assumption is based on the fact that in healthy young adults circulating 25(OH)D concentrations usually lie between 30–80 nmol/L [2], dietary vitamin D intake is usually below 5 µg daily [3], and 1 µg vitamin D increases circulating 25(OH)D concentrations by approximately 1–3 nmol/L [4,5]. The exact amount of vitamin D production in human skin depends on the geographic latitude, season, time of day, as well as on the weather conditions (cloudiness), amount of air pollution and surface reflection. In addition, clothing habits, lifestyle, and workplace (e.g., indoor versus outdoor), sunscreen use, and sun avoidance practices have a strong impact on vitamin D synthesis. It is also noteworthy that skin type determines a person’s effectiveness in producing vitamin D. The darker the skin is pigmented, the more ultraviolet radiation is absorbed by melanin and the less vitamin D is produced [6,7]. Migrant populations and their descendants often have skin types that do not fit to the ambient ultraviolet environment. To achieve a similar effect on vitamin D production compared to a fair-skinned person, the exposure time to ultraviolet radiation in a dark-skinned person living in Europe or North America must be up to six times longer [8].

Vitamin D can be produced very effectively by humans when ultraviolet radiation B (UVB) from sunlight or artificial sources reaches skin cells. A whole body exposure to UVB radiation of 15 to 20 minutes daily is able to produce up to 250 µg vitamin D (10,000 IU) [9,10]. Once in the circulation, vitamin D is converted by a hepatic hydroxylase into 25-hyroxyvitamin D (25(OH)D). The circulating 25(OH)D level is an indicator of vitamin D status. This level reflects both, ultraviolet exposure and dietary vitamin D intake. As needed, 25(OH)D is converted in the kidney to its active hormonal form ,25-dihydroxyvitamin D3 (calcitriol) in a process which is usually tightly controlled by parathyroid hormone. In spite of this, inadequate vitamin D supply lowers the circulating level of this important hormone [11]. Circulating calcitriol is also adversely affected by a reduced number of viable nephrons, high serum concentrations of fibroblast growth factor-23, and high levels of inflammatory cytokines [12,13].

If vitamin D production or intake is low, vitamin D insufficiency or even deficiency is the result. Parathyroid hormone levels start rising at 25(OH)D cutoff levels of 75 nmol/l or lower (Table 1). The following cut-offs are used for different stages of vitamin D inadequacy: <25 nmol/L for deficiency (divide by 2.496 to convert into ng/ml), 25-49.9 nmol/L for insufficiency, 50-74.9 nmol/L for hypovitaminosis/suboptimal supply. Although there is still some debate on how to classify vitamin D status, the vast majority of vitamin D researchers agree that 25(OH)D levels below 50 nmol/l are insufficient.

Cellular vitamin D actions are mediated by a membrane-bound and a cytosolic vitamin D receptor (VDR). The VDR is nearly ubiquitously expressed, and almost all cells respond to vitamin D exposure; about 3% of the human genome is regulated, directly and/or indirectly, by the vitamin D endocrine system [14]. Calcitriol is also produced by local 1a-hydroxylases from its precursor 25(OH)D in various extra-renal cells, among them vascular smooth muscle cells, colonocytes, and immune cells such as monocytes, dendritic cells (DCs), and B-lymphocytes [15,16]. Here, calcitriol plays an important paracrine and autocrine role. Uptake of 25(OH)D into extra-renal tissues is reduced in case of low circulating calcitriol levels, e.g., in patients with renal insufficiency [17].

Table 1. Vitamin D status classified according to circulating 25-hydroxyvitamin D
concentrations [according to reference 18, with modifications according to reference 6].

Stage	25-hydroxyvitamin D (nmol/l)	Clinical/biochemical alterations
Deficiency	<25	Rickets, osteomalacia, myopathy, calcium malabsorption, severe hyperparathyroidism, low calcitriol concentrations, impaired immune and cardiac function?, death
Insufficiency	25 to 49.9	Reduced bone mineral density, impaired muscle function, low intestinal calcium absorption rates, elevated PTH levels, slightly reduced calcitriol levels
Hypovitaminosis D /suboptimal supply	50 to 74.9	Low bodily stores of vitamin D, slightly elevated PTH levels
Adequacy	75 to 372	No disturbances of vitamin D-dependent functions
Intoxication		Intestinal calcium hyperabsorption, hypercalcemia, soft tissue calcification, death

Abbreviation: PTH, parathyroid hormone

Worldwide Vitamin D Status

A recent review [19] summarized human vitamin D status according to region of the world. Six regions of the world were reviewed - Asia, Europe, Middle East and Africa, Latin America, North America, and Oceania–through a survey of published literature. Based on the articles referred to in this review, it was concluded that insufficient vitamin D status is prevalent in every of the six regions studied. Depending on the region, between 50% and more than 90% of people had 25(OH)D concentrations below 50 mol/L.

Low vitamin D status is most common in regions such as South Asia and the Middle East. Data demonstrate that insufficient vitamin D status is widespread and is reemerging as a major health problem globally. Urbanization in combination with modern and also traditional lifestyles such as indoor working, indoor leisure time activities, and traditional Islamic clothing, and in combination with the aging process (institutionalization) is an important risk factor for vitamin D insufficiency/deficiency in large parts of the adult population. In highly urbanized areas, individual daily sun exposure is usually too low to achieve a 25(OH)D level of 75 nmol/L. Due to the fact that the vast majority of foods naturally contain no or only modest amounts of vitamin D, diet is not able to close the gap in vitamin D supply. It is noteworthy that urbanization and industrialization has long been known as a major cause of childhood rickets in western countries [7]. Rickets is now on the increase in many developing countries, and is also re-emerging as an important health problem in countries with strong sun avoidance policies and cultures requiring modest dress.

Diseases Associated with Nonclassical Vitamin D Actions

Figure 1 illustrates that vitamin D deficiency/insufficiency can result in impaired musculo-skeletal function, impaired immune function, cardiac and vascular impairment and impaired nervous function. As outlined in Figure 1, the development of various chronic diseases may be the consequence.

Figure 1. Suggested association of vitamin D deficiency/insufficiency with chronic
diseases.

Vitamin D Deficiency/Insufficiency

Musculoskeletal Sytem → Falls and Fractures

Immune System → Infections, Allergies, Tumors

Endocrine System → Type 1 and II Diabetes

Circulatory System → Hypertension, Stroke, Heart Attack, Heart Failure

Nervous System → Multiple Sclerosis

Vitamin D and Muscle Strengthening

Vitamin D deficiency causes reduced aktomyosin content of myofibrils, low calcium content of mitochondria, reduced calcium uptake into the sarcoplasmic reticulum, and low serum levels of muscle enzymes [3]. The importance of vitamin D-repletion for adequate muscle function was underscored in a recent study in institutionalized people =60 years of age with insufficient vitamin D status [20]: This RCT demonstrated that six-month supplementation (December to May) of oral vitamin D (3,750 µg once a month during the first two months, followed by 2,250 µg once a month for the last four months) was able to improve lower limb muscle strength by 16–24%.

Data support results of a recently performed meta-analysis of randomized controlled trials (RCTs), indicating that daily doses of 17.5 to 20 µg supplemental vitamin D are able to prevent falls in elderly adults [21]. The relative risk of falls was reduced by approximately 20% if the achieved serum 25(OH)D concentrations is 60 nmol/l or more. In contrast to “high dose” supplemental vitamin D, low dose daily supplemental vitamin D (5 to 15 µg) is not able to prevent falls. Thus, doses of supplemental vitamin D of less than 17.5 µg or serum 25-hydroxyvitamin D concentrations of less than 60 nmol/L may not reduce the risk of falling among older individuals. It is noteworthy that in elderly people the risk of falling predicts the risk of developing osteoporotic fractures. Therefore, the effects of vitamin D on muscle strength may contribute to the preventive effect of vitamin D on osteoporotic fractures. There is also evidence that adequate vitamin D supply is important for muscle function in children. Already more than 50 years ago, Ronge [22] has demonstrated that children who have hands and face exposed to UVB radiation in their classroom at school for 3–5 hours during wintertime show better endurance performance compared to a control group without UVB exposure. Endurance performance was assessed by bicycle ergometry. In that study, a similar positive effect on endurance performance was seen in children who received a single vitamin D bolus of 6.25 mg vitamin D in February.

Infections

There is mounting evidence for a pivotal role of vitamin D in the immune system. Calcitriol is able to induce the differentiation of monocytes into macrophages. In addition, calcitriol increases the activity of macrophages and facilitates their cytotoxic activity. Macrophages represent the first unspecific defence line of the immune system. It is well known that the prevalence of infections such as pneumonia is high in infants with rickets [3]. The use of vitamin D (or cod liver oil) as a treatment of infections have been practised for over 150 years. As early as 1903, Niels Finsen was awarded the Nobel Prize for Medicine and Physiology for his theory to cure Lupus vulgaris (skin-tuberculosis) using phototherapy. In 2007, Schauber et al. [23] published data demonstrating that vitamin D is able to stimulate synthesis of the anti-microbial peptide cathelicidin in human skin cells to enhance innate immunity. A meta-analysis of observational studies has demonstrated that patients with tuberculosis have lower circulating 25(OH)D concentrations compared to healthy controls [24].

Ecological studies also support a preventive role of vitamin D in influenza: the seasonal and latitudinal distribution of outbreaks of influenza A in the world in 1967–1975, and weekly consultation rates for illnesses diagnosed clinically as influenza or influenza-like in England 1968-1970 were inversely associated with solar UVB radiation [25]. Very recently, it has been demonstrated in an RCT that supplementation with 30 µg vitamin D daily reduces the risk of wintertime influenza A in Japanese nursery school children [26]. Some epidemiological data support the assumption that vitamin D may reduce the usceptibility to respiratory tract infections [27,28]. In addition, vitamin D users of the RECORD trial [29], an RCT with approximately 3,500 participants who received 20 µg vitamin D or placebo, reported a lower tendency for infections and antibiotic use in March compared to vitamin D nonusers. In another RCT in individuals with baseline circulating concentrations below 50 nmol/L, supplementation with 20 µg or 50 µg vitamin D daily for three years significantly reduced upper respiratory tract infections compared to placebo [30].

In contrast, a daily vitamin D supplement of 50 µg for 12 weeks did not prevent upper respiratory tract infections in individuals with baseline circulating 25(OH)D concentrations above 50 nmol/L [31]. Consequently, there is currently insufficient data to conclusively state that vitamin D supplementation could result in lowered infection [32]. One factor that has to be considered in future studies is baseline 25(OH)D concentration. In addition, the relation between vitamin D supplementation, local calcitriol, and local cathelicidin production has to be investigated more detailed. Interestingly, oral intake of activated vitamin D in rickets patients for four weeks significantly increased human cathelicidin expression in neutrophils compared to age-matched healthy controls without administration of activated vitamin D [33], indicating a critical role of adequate calcitriol availability for regulation of the innate immune response.

Allergies

Activation of the adaptive immune system is complex. Generally, it is of importance that specific pathways of the specific immune system are adequately suppressed in order to avoid autoimmune diseases or allergic reactions. Regulatory T cells are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells. A strong Th2 predominance leads to pathologic conditions such as overproduction of IgE and allergic diseases, whereas a strong Th1 predominance leads to autoimmunity and severe allograft rejection. Of clinical importance is the fact that DCs may induce naïve T cells in an immunogenetic direction but also in a tolerogenic direction, depending on the state of their maturation and their cell surface receptor. Tolerogenic DCs generally are semimature.
There is accumulating evidence that vitamin D modulates the adaptive immune system [16]. Calcitriol appears to generate tolerogenic DCs in vivo, as demonstrated in models of transplantation and autoimmune disease. DCs appear to be key targets of calcitriol. Calcitriol arrest the differentiation and maturation of DCs, maintaining them in an immature state. Calcitriol is able to enhance the secretion by DCs of the anti-inflammatory and anti-allergic cytokine IL-10.

At present, the vitamin D hypothesis of allergies takes two forms: Some argue that vitamin D deficiency may cause allergic reactions whereas others argue that vitamin D excess leads to an increased allergy risk. Wjst is a representative of the latter hypothesis. He argues that the increase in allergies in Bavaria after 1960 coincided with vitamin D supplementation intervention programs to prevent rickets in childhood. Moreover, both, adherence to these programs and prevalence of allergies in children seem to be lower in farming communities in Bavaria [34]. The farm protection is observed mainly during the first year of life [35], when vitamin D supplementation is also recommended. Wjst’s hypothesis is based on the assumption that vitamin D may lead to Th2 predominance and increased IgE production. Generally, his hypothesis is supported by findings that children whose mothers' concentration of 25(OH)-vitamin D in late pregnancy was >75 nmol/l had an increased risk of eczema on examination at nine months and asthma at age nine years compared to children whose mothers' concentration was <30 nmol/L [36]. In addition, vitamin D supplementation during infancy was associated with a higher allergy risk [37,38], and the prevalence of allergic rhinitis increased across quartile groups of 25(OH)D serum levels in adults of NHANES III [39].

It is, however, noteworthy that several other epidemiological studies support the vitamin D deficiency hypothesis of allergic reactions [40-44]. Moreover, administration of calcitriol to blood cells of healthy persons and steroid-resistant asthmatic patients enhanced subsequent responsiveness to dexamethasone for induction of IL-10 [43]. Very few intervention trials are available so far. In a small, randomized, double-blind, placebo-controlled trial, vitamin D2 supplementation (25 µg/day) significantly improved skin symptoms in children with winter-related atopic dermatitis [45]. In a study in heart failure patients, vitamin D3 supplementation (50 µg/day) was able to increase blood levels of the anti-allergic cytokine IL-10 [46]. However, the effect on allergic reactions has not been elucidated in that earlier investigation.

In total, it cannot be ruled out that vitamin D deficiency as well as vitamin D excess may increase the risk of allergic reactions. This assumption is supported by recent findings. Hyppönen et al. [47] observed a biphasic effect of vitamin D with both low and high 25(OH)D levels associated with elevated IgE concentrations in participants of the 1958 British birth cohort. Compared with the reference group with the lowest IgE concentrations [25(OH)D 100–125 nmol/L], adjusted IgE concentrations were 29% higher for participants with the 25(OH)D < 25 nmol/L, and 56% higher for participants with 25(OH)D > 135 nmol/L.

Cancer

Since vitamin D is a key regulator of various cellular metabolic pathways, it is important for cellular maturation, differentiation, and apoptosis [3]. In 2008, the WHO published a report from the International Agency for Research on cancer [48] that came to the conclusion that there is (i) consistent epidemiological evidence for an inverse association between 25(OH)D and colorectal cancer and colorectal adenomas, (ii) suggested epidemiological evidence for an inverse association between 25(OH)D and breast cancer, (iii) insufficient evidence for an inverse association between 25(OH)D and other types of cancer, and (iv) the need for new randomized controlled trials (RCTs). One such RCT has already been published [49]: In a four-year, population-based study, where the primary outcome was fracture incidence, and the principal secondary outcome was cancer incidence, 1179 community-dwelling women were randomly assigned to receive 1500 mg supplemental calcium/d alone (Ca-only), supplemental calcium plus 27.5 µg vitamin D/d (Ca + D), or placebo.

Cancer incidence was 60–77% lower in the Ca + D women and 43% lower in the Ca-only group than in the placebo control subjects (P < 0.03). Gorham et al. [50] have estimated that in North America, Europe, and East Asia approximately 32% of colon cancer and approximately 26% of breast cancer can be prevented with 50 µg vitamin D daily and 3–10 min daily of noon sunlight seasonality, when weather permits. Garland et al. [51] estimated that raising the minimum year-around serum 25(OH)D level to 100–150 nmol/L would prevent approximately 58,000 new cases of breast cancer and 49,000 new cases of colorectal cancer each year, and three fourths of deaths from these diseases in the United States and Canada. Such intakes also are expected to reduce case-fatality rates of patients who have breast, colorectal, or prostate cancer by half. Nevertheless, there is also some concern that cancer risk is not only enhanced in individuals with deficient/insufficient vitamin D status, but also if 25(OH)D concentrations rise above 80 nmol/L [52], a concentration several vitamin D researchers consider adequate. However, this increase in cancer risk has only been observed in observational studies after multivariable adjustments have been made for confounding factors. This kind of exploratory data analysis has been criticized by some researchers [53].

Diabetes Mellitus

In vitro and in vivo studies suggest that vitamin D can prevent pancreatic beta-cell destruction and reduces the incidence of autoimmune diabetes. This may at least in part be due to a suppression of proinflammatory cytokines such as tumor necrosis factor (TNF)-a. Recently, the relationship between UVB irradiance, the primary source of circulating vitamin D in humans, and age-standardized incidence rates of type 1 diabetes mellitus in children aged <14 years, was analyzed according to 51 regions of the world [54]. Incidence rates were generally higher at higher latitudes and were inversely associated with UVB irradiance.

As early as 2001, Hyppönen et al. [55] has demonstrated in a birth cohort study that vitamin D supplementation was associated with a decreased frequency of type 1 diabetes. In contrast, children suspected of having rickets during the first year of life had a three times higher relative risk compared with those without such a suspicion. Meanwhile, a meta-analysis of four case-control studies has shown that the risk of type 1 diabetes is reduced by 29% in infants who are supplemented with vitamin D compared to those who are not supplemented [56]. There is also some evidence of a dose-response effect, with those using higher amounts of vitamin D being at lower risk of developing type 1 diabetes. Finally, timing of supplementation might also be important for the subsequent development of type 1 diabetes. In a recent RCT [57], the majority of patients with latent autoimmune diabetes in adults increased their concentrations of plasma C–peptide levels in fasting state after 1 year of treatment with activated vitamin D, whereas only a minority of patients treated with insulin alone maintained stable fasting C-peptide levels.

In 2007, Pittas et al. [58] conducted a systemic review and meta-analysis for observational studies and clinical trials in adults with outcomes related to glucose homeostasis in type 2 diabetes mellitus. Observational studies show a relatively consistent association between low vitamin D status and prevalent type 2 diabetes, with an odds ratio of 0.36 among non-Blacks for highest versus lowest 25-hydroxyvitamin D. Evidence from RCTs with vitamin D and/or calcium supplementation suggests that combined vitamin D and calcium supplementation may have a role in the prevention of type 2 diabetes only in populations at high risk (i.e,. glucose intolerance). Whereas vitamin D supplementation did not improve glycemic control in diabetic subjects with normal serum 25(OH)D levels [59], administration of 100 µg vitamin D3 improved insulin sensitivity in vitamin D deficient and insulin resistant South Asian women [60]. Insulin resistance was most improved when endpoint serum 25(OH)D reached = 80 nmol/L. Optimal vitamin D concentrations for reducing insulin resistance were shown to be 80–119 nmol/L.

Cardiovascular Disease

Globally, cardiovascular disease (CVD) is the number one cause of death. In 2005, CVD was responsible for approximately 30% of deaths worldwide. CVD includes various illnesses such as coronary heart disease (CHD), peripheral arterial disease, cerebrovascular disease such as stroke, and congestive heart failure. There is accumulating evidence that the vitamin D hormone calcitriol exerts important physiological effects in cardiomyocytes, vascular smooth muscle cells, and the vascular endothelium. The mechanisms have been reviewed in detail elsewhere [61]. Hypertension is a key risk factor for CVD. A recently published systematic review and meta-analysis came to the conclusion that vitamin D produces a fall in systolic blood pressure of -6.18 mm Hg and a nonsignificant fall in diastolic blood pressure of -2.56 mm Hg in hypertensive patients. No reduction in blood pressure is seen in studies examining patients who are normotensive at baseline [62]. Since these studies had small sample sizes, future studies should investigate their generalizability.

Several large prospective observational or cohort studies have demonstrated that a higher vitamin D status is associated with approximately 50% lower cardiovascular morbidity and mortality risk compared with low vitamin D status (Table 2).

Table 2. Evidence for association of circulating 25-hydroxyvitam in D level with
cardiovascular morbidity and mortality.

Study	Design	Number of individuals	Comparator	Odds/hazard ratio or Relative risk (95% CI)
Fatal stroke Pilz et al. 2009 [63]	Prospective cohort study with coronary angiograp	3258	Per z value of 25(OH)D	OR 0.58 (0.43 to 0.78)
Cardiovascular morbidity Wang et al. 2008 [64]	Prospective observational study	1739	25(OH)D > 37.5 nmol/L versus < 25 nmol/L	HR 0.55 (0.32 to 0.97)
Cardiovascular mortality Dobnig et al. 2008 [66] Pilz et al. 2009 [67] Ginde et al. 2009 [68]	Prospective cohort study with coronary angiography Prospective observational study in individuals 50-75 years Prospective observational study in individuals > 65 years.	3258 614 3408	Median 25(OH)D 70 nmol/L versus 19 nmol/L Three highest versus lowest 25 (OH)D quartile 25(OH)D > 100 nmol/L versus < 25 nmol/L	HR 0.45 (0.32 to 0.64) HR 0.19 (0.07 to 0.50) HR 0.42 (0.21 to 0.86)

The Women’s Health Initiative (WHI) calcium/vitamin D (CaD) trial could however not demonstrate a reduction in cardiovascular mortality by daily supplementation of 1,000 mg calcium and 10 µg vitamin D [69]. Meanwhile it is clear that an amount of 10 µg vitamin D is far too low to result in a meaningful increase in serum 25(OH)D levels (see before) and that a daily calcium supplement of 1,000 mg increases the risk for cardiovascular events in healthy older women. Both, the supplemental calcium in the vitamin D arm of the WHI study and the low amount of vitamin D might have countermanded its cardiovascular benefits. In line with this assumption, a recent meta-analysis of seven randomized trials showed a slight but statistically nonsignificant reduction in CVD risk (relative risk: 0,90; 95% CI: 0.77 to 1.05) with vitamin D supplementation at moderate to high doses (approximately 25µg/d) but not with calcium supplementation (relative risk: 1,14; 95% CI: 0.92 to 1.41) or a combination of vitamin D and calcium supplementation (relative risk: 1.04; 95% CI: 0.92 to 1.18) [70].

In line with a potential beneficial effect of vitamin D on CVD risk, a daily vitamin D supplement of 83 µg could improve some traditional and nontraditional cardiovascular risk markers in healthy overweight and obese subjects with mean 25(OH)D concentrations of 30 nmol/L who attended a weight-reduction program [71].

Multiple Sclerosis

Multiple sclerosis (MS) is a demyellinating disease of the central nervous system that is debilitating and can be fatal. Manifestation of the disease is typically between the age of 20 and 40. In Europe and North America, regions with higher UVB radiation have low rates of MS and vice versa [3]. In Israel, MS prevalence depends on the country of origin. The prevalence is high in people who were born in a country with low UVB irradiance [72], indicating that vitamin D status during the period of early life is of importance for MS susceptibility. MS disease activity shows inverse fluctuations according to season and vitamin D status [73]. In a prospective, nested case-control study among more than seven million US military personnel [74], MS prevalence was lower in those people who had circulating 25-hydroxyvitamin D concentrations between 100 and 150 nmol/L compared with those who had 25-hydroxyvitamin D concentrations below 63 nmol/L. However, this association was only seen in Whites and not in Blacks, indicating that genetic factors play an important role in the pathogenesis of MS. Therefore, the recent finding is of importance that expression of the MS-associated MHC class II allele HLA-DRB1*1501 is regulated by Vitamin D [75].

Mortality

As mentioned before, vitamin D status is an important independent predictor of CVD and specific types of cancer. In addition, vitamin D status predicts CVD and cancer mortality. Both, CVD and cancer are the most important causes of mortality in developed countries. In 2007, Autier and Gandini [76] published a meta-analysis of randomized controlled trials (RCTs) on vitamin D and mortality that were not primarily designed to assess mortality. The authors found out that in middle-aged and elderly patients with low serum concentrations of 25-hydroxyvitamin D (25(OH)D) vitamin D supplementation was linked to lower all-cause mortality compared to no vitamin D supplementation.

Daily dose of vitamin D ranged between 10 µg and 50 µg. Risk reduction was 7% during a mean follow-up of 5.7 years. Based on the aforementioned meta-analysis, several large prospective cohort studies were recently published on all-cause mortality and vitamin D status (Table 3). They demonstrate a consistent increase in mortality risk in patients with insufficient or deficient 25(OH)D concentrations. However, low 25(OH)D was not an independent predictor for mortality in patients with advanced disease [77,78]. One may speculate that in this case, vitamin D supplementation is unable to reverse the already existing severe pathophysiologic derangements.

Table 3. Evidence for association of circulating 25-hydroxyvitamin D level or vitamin D supplementation with all-cause mortality.

Study	Design	Number of individuals	Comparator	Hazard ratio or relative risk (95% CI)
Autier and Gandini, 2007 [76]	Meta-analysis of 18 vitamin D supplementation studies	57,311	Supplemented versus unsupplemented	RR 0.93 (0.87 to 0.99)
Dobnig et al. 2008 [66]	Prospective cohort study with coronary angiography	3,258	Median 25(OH)D 70 nmol/L versus 19 nmol/L	HR 0.48 (0.37 to 0.63)
Kuroda et al. 2009 [77]	Prospective observational study in postmenopausal women	1,232	≥ 50 nmol/L versus < 50 nmol/L	HR 0.46 (0.27 to 0.79)
Ng et al. 2008 [78]	Prospective cohort study in patients with colorectal cancer	304	Mean 41 nmol/L versus 100 nmol/L	HR 0.52 (0.29 to 0.94)
Ginde et al. 2009 [68]	Prospective observational study in individuals > 65 years.	3,408	25(OH)D > 100 nmol/L versus < 25 nmol/L	HR 0.55 (0.34to 0.88)
Pilz et al. 2009 [67]	Prospective observational study In individuals 50-75 years	614	Three highest quartiles versus lowest quartile	HR 0.51 (0.28 to 0.93)

Conclusions

In 2003, a review article had summarized the association of insufficient vitamin D status with various diseases such as myopathy, CVD, cancer, diabetes mellitus, MS, and infections [8]. Meanwhile, evidence has accumulated that vitamin D may indeed play an important role in the etiology of many of these diseases. Meta-analyses of RCTs demonstrate that vitamin D improves muscle function and seems to reduce blood pressure in hypertensive patients. In addition, some RCTs demonstrate that vitamin D reduces cancer incidence, and improves glucose homeostasis in patients with type 2 diabetes and cardiovascular risk markers in overweight people [49,60,71]. The most exiting result is however the fact that vitamin D may reduce mortality rate. This latter finding fits well together with the fact that severe deficiency of several other vitamins such as retinol, thiamine, niacin, and ascorbic acid is also associated with enhanced mortality. Nevertheless, additional large RCTs are needed to confirm whether or not vitamin D is able prolong survival in individuals with inadequate vitamin D status. In this context, the effect of vitamin D in deficient and insufficient individuals should be investigated separately.

Some aforementioned beneficial data on glucose homeostasis and cardiovascular risk markers were not confirmed by recent RCTs [59,81]. All these RCTs performed so far were relative small in sample size [59,60,71,81]. In addition, individual medication and baseline circulating 25(OH)D concentrations may have influenced study results. Therefore, additional research is necessary to clarify whether or not vitamin D supplementation is indeed effective in secondary prevention and also in tertiary prevention of chronic diseases. But we should be aware of the fact that many chronic diseases are of multi-factorial origin. Vitamin D is certainly only one factor among others. In addition, there may be individual differences with respect to the metabolic pathways that are disturbed in vitamin D deficient persons. Therefore, we should not be too enthusiastic that future RCTs will show clear beneficial vitamin D effects. For example, the meta-analysis by Autier and Gandini was based on more than 55,000 individuals. None of the single studies included in this analysis showed a significant vitamin D effect on mortality, indicating that huge sample seizes are probably needed to demonstrate a clear vitamin D effect. Even so, the consequences on a population scale may be important because of the large number of people who are affected.

The effect of vitamin D on MS, type 1 diabetes, infections, and allergies is less clear at present. Although newborns usually receive vitamin D supplements for preventing rickets, possible adverse effects of deficient vitamin D concentrations during fetal development such as increased susceptibility for type I diabetes and MS have to be considered as well. It is noteworthy that many women of childbearing age worldwide are vitamin D insufficient or even deficient [19,82]. With respect to MS, type 1 diabetes, and allergies, more birth cohort studies are needed.

Despite some uncertainties with respect to vitamin D and health, there is general agreement that currently a high percentage of people worldwide have low vitamin D status [19,83]. The recommended daily vitamin D intake of 5–15 µg is too low to achieve an adequate vitamin D status in people with only modest UVB exposure. Generally, treating vitamin D deficiency is easy to perform, safe, and inexpensive. Sources of vitamin D could include a combination of food fortification, supplements, and natural and artificial UV-B irradiation, if properly acquired. It has been calculated that 1 µg vitamin D increases circulating 25(OH)D levels by approximately 1 nmol/L [4]. Thus, a daily intake of approximately 50 µg vitamin D would be necessary for increasing the circulating 25(OH)D level from 25 nmol/L to 75 nmol/L. In order to achieve a 25(OH)D concentration above 75 nmol/L in almost all individuals of a group with mean baseline 25(OH)D concentrations of 38 nmol/L, daily supplementation with up to 100 µg vitamin D is necessary [5]. In otherwise healthy adults, the risk of vitamin D intoxication is extremely rare [3,84]. Vitamin D intoxications such as hypercalcemia do not occur until oral vitamin D intake and serum 25(OH)D concentrations exceed 250 µg/day (approximately 3–5 µg/kg body weight) [84] and 372 nmol/L [6], respectively. A daily amount of up to 250 µg vitamin D is similar to the amount that is produced by daily whole body exposure to UVB radiation [10].

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© 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

by EricT

Strength Training and Nutrition Dogma

Tue, 11 May 2010 16:36:45 +0000

By Eric Troy

Modern strength training has, in recent times, aligned itself with science more than ever in the past. Unfortunately the majority of the industry has no clear knowledge of the scientific process and in fact, doesn't really know what science is. Most strength trainers who use science tend to point to science as if it is a thing. However, although we use the word as if it means a concrete thing it is rather a practice or system of acquiring knowledge. When we ask "what's the science on this?" what we really should be asking is "what is the state of knowledge on this?".

Viewing science as a static entity gives rise to the greatest strength training enemy of all: Dogma.

Many of the unquestionable beliefs that are repeated as "absolute truth" borrow scientific sounding terms and claim scientific foundations. We call these beliefs "dogma" or "dogmatic". Disparagingly.

One example of such a widespread dogmatic belief is that exercise enhances your immunity and protects you from colds and flu. People repeat this as if it is fact while simply assuming that there is scientific evidence behind it.

Another area of dogma is the subject of "neural fatigue". Many statements circulate about the effects of strength training on the CNS as if they are absolute scientific fact when in fact this is one of the least understood areas of strength. You may hear statements like "too much maximal strength training will burn out your CNS" and most will take this as gospel. Yet there is no standard definition for what actually constitutes "neural fatigue" (which is probably not even an accurate term) let alone just what it means to "burn out your CNS". There is nothing in this area that can be stated with absolute certainty and little that can be stated even with a degree of assurance.

Here is a common belief I'll bet you didn't know was dogma. The hook lift is stronger than the alternating "over-under" grip. Dogma. There is scientific reasoning to suggest that the hook lift has any special efficacy as compared to the alternating grip. This belief probably comes from the use of the hook grip by Olympic lifters, who are unable to use an alternating grip and would be unwise to resort to lifting straps. However, besides "safety for safety's sake" there is not even a reason to think that the use of the hook grip results in "more kilos lifted" for the average O lifter of average grip strength. The simple fact is that deadlifters have much more need of special grips than Olympic lifters for a variety of reasons but there is no reason for a deadlifter to think that the hook grip has an advantage over the alternated grip.¹

It would be improper of me not to mention nutrition and fatloss dogma. After all proper nutrition is essential to your strength training success and nutrition is subject to as many, if not more dogmatic beliefs as training. Anything you eat after 7 or 8 PM turns to fat. Dogma. You must eat multiple meals throughout the day and many small meals every few hours is better than three big ones. Dogma. Limit your salt intake because salt will give you high blood pressure (and if you're a bodybuilder "bloat" you). Dogma.

Here's a big one. Don't eat too many egg yolks because it will raise your cholesterol. This one is so big that Jamie Hale chose the subject to put in the title of his new book: "Should I Eat the Yolk? Separating Facts from Myths to Get You Lean, Fit, and Healthy"*

How about bananas cure muscle cramps? That's right. Dogma. The only way to get big is to "bulk" and then "cut". Dogma. You shouldn't deadlift more 1x5 reps a week. Dogma!!!!

You might make the point that dogma isn't necessarily wrong. And you'd be right. Many of the established doctrines of any discipline, although they seem to be based on a stubborn belief system rather than any real science, may still have solid underpinnings and be valid as far as our understanding of the world at this time.

The dogma we are talking about, however, is part of a system of belief and attitude. Any stubborn clinging to "absolutes" is, at it's heart, unscientific. The concept of absolute truth itself, as a matter of fact, is unscientific. Proof only exists in mathematics.

So when you hear the phrase, "it's been proven that", your dogma radar should ping.

My mission in this blog, if I were to call it a mission, is not only to provide strength training ideas and instruction but to help you learn to recognize false science. And also to help you spot what I would call "tricks of the trade". These are methods of persuasion that are used to help lend a sense of authority not to the information, but to the person providing it.

Persuasion itself is not a bad thing. We all must persuade others to except our ideas. I am trying to persuade you right now as you are reading this.

I think the problem may be in thinking that you must always persuade yourself to one side or another. There actually exists an attitude that says you cannot criticize a concept unless you have an alternative. That, to me, is like saying we must accept vinegar unless we can come up with a bottle of wine. There isn't always an acceptable answer and the argument that says we must either take a side or provide an alternative is a false one. It is better to say "I don't know" sometimes.

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by EricT

Footnotes

1. I am not saying to not use a hook grip to do Olympic lifts. The choice is yours. My assertions are based on the added inertia that a lifter with "some" experience may add to the weight by acceleration. With elite lifters where inertia may increase up to 50% the hook grip may certainly be necessary. For instance, consider a 120 kilo lift by a VERY accomplished lifter who manages to add that 50% inertia. So the initial pull becomes 180 kilos which is well within the range of grip challenge for a male of average grip strength. However, when comparing this to the deadlift, a "slow" lift, being conservative, the deadlifter must support the bar for AT LEAST twice as long if not longer. A dead lift of the same weight would simply require more supporting grip strength than the Snatch or Clean. For those who "dabble" in the O lifts, it is unlikely that they will encounter grip as a serious issue since they are simply not able to accelerate the bar to a degrees that would challenge the grip. The use of the hook grip for most lifters is a precaution rather than a necessity and the reason you have never heard of an Olympic lifter having a grip problem or using extra grip training is simply because it rarely comes up.