Prospective clinical trial examining the impact of genetic variation in FADS1 on the metabolism of linoleic acid- and ɣ-linolenic acid-containing botanical oils.

BACKGROUND: Unexplained heterogeneity in clinical trials has resulted in questions regarding the effectiveness of É£-linolenic acid (GLA)-containing botanical oil supplements. This heterogeneity may be explained by genetic variation within the fatty acid desaturase (FADS) gene cluster that is associated with circulating and tissue concentrations of arachidonic acid (ARA) and dihomo-É£-linolenic acid (DGLA), both of which may be synthesized from GLA and result in proinflammatory and anti-inflammatory metabolites, respectively. OBJECTIVES: The objective of this study was to prospectively compare the capacity of a non-Hispanic white cohort, stratified by FADS genotype at the key single-nucleotide polymorphism (SNP) rs174537, to metabolize 18-carbon omega-6 (n-6) PUFAs in borage oil (BO) and soybean oil (SO) to GLA, DGLA, and ARA. METHODS: Healthy adults (n = 64) participated in a randomized, double-blind, crossover intervention. Individuals received encapsulated BO (Borago officinalis L.; 37% LA and 23% GLA) or SO [Glycine max (L.) Merr.; 50% LA and 0% GLA] for 4 wk, followed by an 8-wk washout period, before consuming the opposite oil for 4 wk. Serum lipids and markers of inflammation (C-reactive protein) were assessed for both oil types at baseline and during weeks 2 and 4 of the intervention. RESULTS: SO supplementation failed to alter circulating concentrations of any n-6 long-chain PUFAs. In contrast, a modest daily dose of BO elevated serum concentrations of GLA and DGLA in an rs174537 genotype-dependent manner. In particular, DGLA increased by 57% (95% CI: 0.38, 0.79) in GG genotype individuals, but by 141% (95% CI: 1.03, 2.85) in TT individuals. For ARA, baseline concentrations varied substantially by genotype and increased modestly with BO supplementation, suggesting a key role for FADS variation in the balance of DGLA and ARA. CONCLUSIONS: The results of this study clearly suggest that personalized and population-based approaches considering FADS genetic variation may be necessary to optimize the design of future clinical studies with GLA-containing oils. This trial was registered at clinicaltrials.gov as NCT02337231.

Impact of methods used to express levels of circulating fatty acids on the degree and direction of associations with blood lipids in humans.

Numerous studies have examined relationships between disease biomarkers (such as blood lipids) and levels of circulating or cellular fatty acids. In such association studies, fatty acids have typically been expressed as the percentage of a particular fatty acid relative to the total fatty acids in a sample. Using two human cohorts, this study examined relationships between blood lipids (TAG, and LDL, HDL or total cholesterol) and circulating fatty acids expressed either as a percentage of total or as concentration in serum. The direction of the correlation between stearic acid, linoleic acid, dihomo-γ-linolenic acid, arachidonic acid and DHA and circulating TAG reversed when fatty acids were expressed as concentrations v. a percentage of total. Similar reversals were observed for these fatty acids when examining their associations with the ratio of total cholesterol:HDL-cholesterol. This reversal pattern was replicated in serum samples from both human cohorts. The correlations between blood lipids and fatty acids expressed as a percentage of total could be mathematically modelled from the concentration data. These data reveal that the different methods of expressing fatty acids lead to dissimilar correlations between blood lipids and certain fatty acids. This study raises important questions about how such reversals in association patterns impact the interpretation of numerous association studies evaluating fatty acids and their relationships with disease biomarkers or risk.

LTC4 synthase polymorphism modifies efficacy of botanical seed oil combination in asthma.

UNLABELLED: Botanical seed oils reduce the generation of leukotrienes in patients with asthma. Our objective was to determine the efficacy of a botanical seed oil combination against airflow obstruction in asthma, and to determine the pharmacogenomic effect of the leukotriene C4 synthase (LTC4S) polymorphism A-444C. We conducted a randomized, double-blind, placebo-controlled, cross-over clinical trial in mild to moderate asthmatics to determine the change in FEV1 after 6 weeks of therapy with borage and echium seed oils versus corn oil placebo. We also examined the effect of the variant LTC4S -444C allele on the change in lung function. We did not identify a difference in FEV1 in the study cohort as a whole (n = 28), nor in the group of A homozygotes. In the C allele carriers (n = 9), FEV1 improved by 3% after treatment with borage and echium seed oils and declined by 4% after placebo corn oil (p = 0.02). All 9 C allele carriers demonstrated an improvement in their FEV1 on active treatment compared to placebo as compared to only 7 out of 19 A allele homozygotes (p = 0.007). We observed transient differences in ex vivo leukotriene generation from circulating basophils and granulocytes. We did not observe significant differences in urinary LTE4 levels. We conclude that compared to corn oil, a combination of borage and echium seed oils improves airflow obstruction in mild to moderate asthmatics who carry the variant allele in the LTC4S gene (A-444C). Botanical oil supplementation may have therapeutic potential in asthma if used in a personalized manner. TRIAL REGISTRATION: This trial was registered at http://www.clinicaltrials.gov as NCT00806442.

The impact of polyunsaturated fatty acid-based dietary supplements on disease biomarkers in a metabolic syndrome/diabetes population.

BACKGROUND: Ingestion of polyunsaturated fatty acids (PUFAs) has been proposed to influence several chronic diseases including coronary heart disease (CHD) and type-2 diabetes (T2D).There is strong evidence that omega-3 (n-3) PUFAs provide protection against CHD and biomarkers of atherosclerosis. In contrast, there is more limited and inconsistent data for T2D. Few studies have examined the impact of n-3 PUFA-containing botanical oils on T2D. METHODS: Fifty-nine subjects with early-stageT2D or metabolic syndrome participated in an 8-week, randomized, single-blind, parallel intervention study and were provided PUFA-containing oils. Individuals received either corn oil (CO), a botanical oil (BO) combination (borage [Borago officinalis L.]/echium oil [Echium plantagineum L.]) or fish oil (FO). The BO combination was enriched in alpha-linolenic, gamma-linolenic, and stearidonic acids and the FO in eicosapentaenoic and docosahexaenoic acids. Serum fatty acids and other serum lipids(triglycerides and total, HDL and LDL cholesterol), as well as markers of inflammation (leptin, and C-reactive protein) and glucose regulation (glucose and hemoglobin A1c) were assessed from fasting participants at baseline and after the intervention. RESULTS: Compliance was verified by expected increases in specific PUFAs in each of the three oil arms. Participants in the CO group showed no differences in serum lipids, markers of inflammation or glucose regulation between pre- and post-treatment measures. Supplementation with BO significantly lowered total and LDL cholesterol levels and FO reduced serum triglycerides, hemoglobin A1c and increased HDL-cholesterol. CONCLUSION: Short-term dietary supplementation with BO and FO improved biomarkers associated with T2D/metabolic syndrome. TRIAL REGISTRATION: Clinicaltrial.gov NCT01145066.

Relationship between a common variant in the fatty acid desaturase (FADS) cluster and eicosanoid generation in humans.

Dramatic shifts in the Western diet have led to a marked increase in the dietary intake of the n-6 polyunsaturated fatty acid (PUFA), linoleic acid (LA). Dietary LA can then be converted to arachidonic acid (ARA) utilizing three enzymatic steps. Two of these steps are encoded for by the fatty acid desaturase (FADS) cluster (chromosome 11, 11q12.2-q13) and certain genetic variants within the cluster are highly associated with ARA levels. However, no study to date has examined whether these variants further influence pro-inflammatory, cyclooxygenase and lipoxygenase eicosanoid products. This study examined the impact of a highly influential FADS SNP, rs174537 on leukotriene, HETE, prostaglandin, and thromboxane biosynthesis in stimulated whole blood. Thirty subjects were genotyped at rs174537 (GG, n = 11; GT, n = 13; TT, n = 6), a panel of fatty acids from whole serum was analyzed, and precursor-to-product PUFA ratios were calculated as a marker of the capacity of tissues (particularly the liver) to synthesize long chain PUFAs. Eicosanoids produced by stimulated human blood were measured by LC-MS/MS. We observed an association between rs174537 and the ratio of ARA/LA, leukotriene B4, and 5-HETE but no effect on levels of cyclooxygenase products. Our results suggest that variation at rs174537 not only impacts the synthesis of ARA but the overall capacity of whole blood to synthesize 5-lipoxygenase products; these genotype-related changes in eicosanoid levels could have important implications in a variety of inflammatory diseases.

Impact of botanical oils on polyunsaturated fatty acid metabolism and leukotriene generation in mild asthmatics.

BACKGROUND: Dietary supplementation with botanical oils that contain n-6 and n-3 eighteen carbon chain (18C)-PUFA such as γ linolenic acid (GLA, 18:3n-6), stearidonic acid (SDA, 18:4n-3) and α linolenic acid (ALA, 18:3n-3) have been shown to impact PUFA metabolism, alter inflammatory processes including arachidonic acid (AA) metabolism and improve inflammatory disorders. METHODS: The diet of mild asthmatics patients was supplemented for three weeks with varying doses of two botanical seed oils (borage oil [Borago officinalis, BO] and echium seed oil [Echium plantagineum; EO]) that contain SDA, ALA and GLA. A three week wash out period followed. The impact of these dietary manipulations was evaluated for several biochemical endpoints, including in vivo PUFA metabolism and ex vivo leukotriene generation from stimulated leukocytes. RESULTS: Supplementation with several EO/BO combinations increased circulating 20-22 carbon (20-22C) PUFAs, including eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and dihommo-gammalinolenic acid (DGLA), which have been shown to inhibit AA metabolism and inflammation without impacting circulating AA levels. BO/EO combinations also inhibited ex vivo leukotriene generation with some combinations attenuating cysteinyl leukotriene generation in stimulated basophils by >50% and in stimulated neutrophils by >35%. CONCLUSIONS: This study shows that dietary supplementation with BO/EO alters 20-22C PUFA levels and attenuates leukotriene production in a manner consistent with a reduction in inflammation.

Differences in arachidonic acid levels and fatty acid desaturase (FADS) gene variants in African Americans and European Americans with diabetes or the metabolic syndrome.

Over the past 50 years, increases in dietary n-6 PUFA, such as linoleic acid, have been hypothesised to cause or exacerbate chronic inflammatory diseases. The present study examines an individual's innate capacity to synthesise n-6 long-chain PUFA (LC-PUFA) with respect to the fatty acid desaturase (FADS) locus in Americans of African and European descent with diabetes or the metabolic syndrome. Compared with European Americans (EAm), African Americans (AfAm) exhibited markedly higher serum levels of arachidonic acid (AA) (EAm 7·9 (sd 2·1), AfAm 9·8 (sd 1·9) % of total fatty acids; P < 2·29 × 10⁻9) and the AA:n-6-precursor fatty acid ratio, which estimates FADS1 activity (EAm 5·4 (sd 2·2), AfAm 6·9 (sd 2·2); P = 1·44 × 10⁻5). In all, seven SNP mapping to the FADS locus revealed strong association with AA, EPA and dihomo-γ-linolenic acid (DGLA) in the EAm. Importantly, EAm homozygous for the minor allele (T) had significantly lower AA levels (TT 6·3 (sd 1·0); GG 8·5 (sd 2·1); P = 3·0 × 10⁻5) and AA:DGLA ratios (TT 3·4 (sd 0·8), GG 6·5 (sd 2·3); P = 2·2 × 10⁻7) but higher DGLA levels (TT 1·9 (sd 0·4), GG 1·4 (sd 0·4); P = 3·3 × 10⁻7) compared with those homozygous for the major allele (GG). Allele frequency patterns suggest that the GG genotype at rs174537 (associated with higher circulating levels of AA) is much higher in AfAm (0·81) compared with EAm (0·46). Similarly, marked differences in rs174537 genotypic frequencies were observed in HapMap populations. These data suggest that there are probably important differences in the capacity of different populations to synthesise LC-PUFA. These differences may provide a genetic mechanism contributing to health disparities between populations of African and European descent.

The impact of FADS genetic variants on ω6 polyunsaturated fatty acid metabolism in African Americans.

BACKGROUND: Arachidonic acid (AA) is a long-chain omega-6 polyunsaturated fatty acid (PUFA) synthesized from the precursor dihomo-gamma-linolenic acid (DGLA) that plays a vital role in immunity and inflammation. Variants in the Fatty Acid Desaturase (FADS) family of genes on chromosome 11q have been shown to play a role in PUFA metabolism in populations of European and Asian ancestry; no work has been done in populations of African ancestry to date. RESULTS: In this study, we report that African Americans have significantly higher circulating levels of plasma AA (p = 1.35 × 10(-48)) and lower DGLA levels (p = 9.80 × 10(-11)) than European Americans. Tests for association in N = 329 individuals across 80 nucleotide polymorphisms (SNPs) in the Fatty Acid Desaturase (FADS) locus revealed significant association with AA, DGLA and the AA/DGLA ratio, a measure of enzymatic efficiency, in both racial groups (peak signal p = 2.85 × 10(-16) in African Americans, 2.68 × 10(-23) in European Americans). Ancestry-related differences were observed at an upstream marker previously associated with AA levels (rs174537), wherein, 79-82% of African Americans carry two copies of the G allele compared to only 42-45% of European Americans. Importantly, the allelic effect of the G allele, which is associated with enhanced conversion of DGLA to AA, on enzymatic efficiency was similar in both groups. CONCLUSIONS: We conclude that the impact of FADS genetic variants on PUFA metabolism, specifically AA levels, is likely more pronounced in African Americans due to the larger proportion of individuals carrying the genotype associated with increased FADS1 enzymatic conversion of DGLA to AA.

Depression and altered serum lipids in cynomolgus monkeys consuming a Western diet.

Research over the past 15 years has suggested a high comorbidity of depression and coronary heart disease (CHD). However the mechanisms responsible for this relationship are poorly understood. This study was designed to examine the relationships between depressive behaviors and concentrations of circulating lipids and lipid signaling molecules that may be common to both CHD and depression in a cohort of cynomolgus monkeys (Macaca fascicularis) consuming a 'Western' diet, enriched with saturated fat and cholesterol. Socially-housed adult female cynomolgus monkeys (n=36) were fed the Western diet for 27 months and depressive behavior was recorded weekly. Body weight, body mass index and circulating cholesterol profiles were measured in all animals, and fatty acids (FA) and FA-based signaling molecules were measured in the 6 least and 6 most depressed monkeys. Monkeys consuming the Western diet exhibited a broad range of percent time spent in depressive behavior. The percent time spent depressed was positively correlated with total plasma and LDL cholesterol and negatively correlated with HDL cholesterol. Despite being leaner, depressed monkeys had higher concentrations of monounsaturated fats (C16:1 and C17:1), a higher ω6/ω3 polyunsaturated fatty acid (PUFA) ratio and higher concentrations of omega-6 (ω6) PUFAs, particularly C18:2ω6 and C20:3ω6. FA ratios suggest that stearoyl CoA desaturase 1 activity was increased in depressed monkeys. Depressed female cynomolgus monkeys had elevated concentrations of serum lipids and lipid signaling molecules that are typically associated with obesity, insulin resistance and cardiovascular disease, which may account in part for the comorbidity of depression and CHD.

FADS genetic variants and omega-6 polyunsaturated fatty acid metabolism in a homogeneous island population.

Long-chain polyunsaturated fatty acids (PUFA) orchestrate immunity and inflammation through their capacity to be converted to potent inflammatory mediators. We assessed associations of FADS gene cluster polymorphisms and fasting serum PUFA concentrations in a fully ascertained, geographically isolated founder population of European descent. Concentrations of 22 PUFAs were determined by gas chromatography, of which ten fatty acids and five ratios defining FADS1 and FADS2 activity were tested for genetic association against 16 single nucleotide polymorphisms (SNP) in 224 individuals. A cluster of SNPs in tight linkage disequilibrium in the FADS1 gene (rs174537, rs174545, rs174546, rs174553, rs174556, rs174561, rs174568, and rs99780) were strongly associated with arachidonic acid (AA) (P = 5.8 x 10(-7) - 1.7 x 10(-8)) among other PUFAs, but the strongest associations were with the ratio measuring FADS1 activity in the omega-6 series (P = 2.11 x 10(-13) - 1.8 x 10(-20)). The minor allele across all SNPs was consistently associated with decreased omega-6 PUFAs, with the exception of dihomo-gamma-linoleic acid (DHGLA), where the minor allele was consistently associated with increased levels. Our findings in a geographically isolated population with a homogenous dietary environment suggest that variants in the Delta-5 desaturase enzymatic step likely regulate the efficiency of conversion of medium-chain PUFAs to potentially inflammatory PUFAs, such as AA.

Effect of a multifaceted, church-based wellness program on metabolic syndrome in 41 overweight or obese congregants.

INTRODUCTION: A rise in obesity, poor-quality diets, and low physical activity has led to a dramatic increase in the number of Americans with metabolic syndrome and diabetes. Our objective was to determine the effect of a short-term, multifaceted wellness program carried out in a church setting on weight, metabolic syndrome, and self-reported wellness. METHODS: Forty-one overweight or obese adults in a church congregation provided fasting blood samples and answered a wellness questionnaire before and after completing an 8-week diet and exercise program. We also measured weight, body fat, body mass index, and waist and hip circumference. RESULTS: The intervention decreased weight, body fat, and central adiposity; improved indexes of metabolic syndrome; and increased self-reported wellness. CONCLUSION: A multifaceted wellness intervention that emphasizes diet and exercise can rapidly influence weight, insulin resistance, metabolic syndrome, and self-reported wellness.

Effect of dietary fatty acids on inflammatory gene expression in healthy humans.

Over the past 100 years, changes in the food supply in Western nations have resulted in alterations in dietary fatty acid consumption, leading to a dramatic increase in the ratio of omega-6 (omega6) to omega3 polyunsaturated fatty acids (PUFA) in circulation and in tissues. Increased omega6/omega3 ratios are hypothesized to increase inflammatory mediator production, leading to higher incidence of inflammatory diseases, and may impact inflammatory gene expression. To determine the effect of reducing the omega6/omega3 ratio on expression of inflammatory pathway genes in mononuclear cells, healthy humans were placed on a controlled diet for 1 week, then given fish oil and borage oil for an additional 4 weeks. Serum and neutrophil fatty acid composition and ex vivo leukotriene B(4) production from stimulated neutrophils were measured at the start and end of the supplementation period and after a 2-week washout. RNA was isolated from mononuclear cells and expression of PI3K, Akt, NFkappaB, and inflammatory cytokines was measured by real-time PCR. A marked increase was seen in serum and neutrophil levels of long-chain omega3 PUFA concomitant with a reduction in the omega6/omega3 PUFA ratio (40%). The ex vivo capacity of stimulated neutrophils to produce leukotriene B(4) was decreased by 31%. Expression of PI3Kalpha and PI3Kgamma and the quantity of PI3Kalpha protein in mononuclear cells was reduced after supplementation, as was the expression of several proinflammatory cytokines. These data reveal that PUFA may exert their clinical effects via their capacity to regulate the expression of signal transduction genes and genes for proinflammatory cytokines.

The content of favorable and unfavorable polyunsaturated fatty acids found in commonly eaten fish.

Changes in diet during the past century have caused a marked increase in consumption of saturated fatty acids and n-6 polyunsaturated fatty acids (PUFAs) with a concomitant decrease in the intake of n-3 PUFAs. Increased fish consumption has been shown to be the only realistic way to increase dietary quantities of beneficial long-chain n-3 PUFAs such as eicosapentaenoic acid and docosahexaenoic acid and re-establish more balanced n-6:n-3 ratios in the diets of human beings. Our objective in this research was to characterize some of the relevant fatty acid chemistry of commonly consumed fish, with a particular focus on the four most commonly consumed farmed fish. To do this, 30 commonly consumed farmed and wild fish were collected from supermarkets and wholesalers throughout the United States. Fatty acid composition of samples from these fish was determined using gas chromatography. The 30 samples studied contained n-3 PUFAs ranging from fish having almost undetectable levels to fish having nearly 4.0 g n-3 PUFA per 100 g fish. The four most commonly farmed fish, Atlantic salmon, trout, tilapia, and catfish, were more closely examined. This analysis revealed that trout and Atlantic salmon contained relatively high concentrations of n-3 PUFA, low n-6:n-3 ratios, and favorable saturated fatty acid plus monounsaturated fatty acid to PUFA ratios. In contrast, tilapia (the fastest growing and most widely farmed fish) and catfish have much lower concentrations of n-3 PUFA, very high ratios of long chain n-6 to long chain n-3 PUFAs, and high saturated fatty acid plus monounsaturated fatty acid to PUFA ratios. Taken together, these data reveal that marked changes in the fishing industry during the past decade have produced widely eaten fish that have fatty acid characteristics that are generally accepted to be inflammatory by the health care community.

Ethanol self-administration and alterations in the livers of the cynomolgus monkey, Macaca fascicularis.

BACKGROUND: Most of the studies of alcoholic liver disease use models in which animals undergo involuntary administration of high amounts of ethanol and consume diets that are often high in polyunsaturated fatty acids. The objectives of this study were (1) to evaluate whether cynomolgus monkeys (Macaca fascicularis) drinking ethanol voluntarily and consuming a diet with moderate amounts of lipid would demonstrate any indices of alcoholic liver disease past the fatty liver stage and (2) to determine whether these alterations were accompanied by oxidative stress. METHODS: Six adult male and 6 adult female cynomolgus monkeys were allowed to consume ethanol voluntarily for 18 to 19 months. Additional monkeys were maintained on the same consumption protocol, but were not provided with ethanol. During the course of the study, liver biopsy samples were monitored for lipid deposition and inflammation, serum for levels of liver enzymes, and urine for concentrations of the isoprostane (IsoP) metabolite, 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP, a biomarker for oxidative stress. Liver mitochondria were monitored for respiratory control and liver for concentrations of neutral lipids, adenine nucleotides, esterified F(2) isoprostanes, oxidized proteins, 4-hydroxynonenal (HNE)-protein adducts, and protein levels of cytochrome P-450 2E1 and 3A4. RESULTS: Ethanol consumption ranged from 0.9 to 4.05 g/kg/d over the period of the study. Serum levels of aspartate amino transferase were elevated in heavy-consuming animals compared with those in ethanol-naïve or moderate drinkers. Many of the ethanol consumers developed fatty liver and most showed loci of inflammation. Both hepatic energy charge and phosphorylation potential were decreased and NADH-linked respiration was slightly, but significantly depressed in coupled mitochondria as a result of heavy ethanol consumption. The urinary concentrations of 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP increased as high as 33-fold over that observed in ethanol-abstinent animals. Liver cytochrome P-450 2E1 concentrations increased in ethanol consumers, but there were no ethanol-elicited increases in hepatic concentrations of the esterified F(2) isoprostanes, oxidized proteins, or HNE-protein adducts. CONCLUSION: Our studies show that cynomolgus monkeys undergoing voluntary ethanol consumption for 1.5 years exhibit many of the features observed in the early stages of human alcoholic liver disease. Ethanol-elicited fatty liver, inflammation, and elevated serum aspartate amino transferase were evident with a diet that contained modest amounts of polyunsaturated lipids. The dramatic increases in urinary IsoP demonstrated that the animals were being subjected to significant oxidative stress that correlated with their level of ethanol consumption.

The effects of moderate ethanol consumption on the liver of the monkey, Macaca fascicularis.

BACKGROUND: Although evidence has accumulated for the cardioprotective effects of moderate ethanol consumption, little is known about the effects on the liver of consuming the equivalent of two drinks per day. The objective of this study was to determine the effects of moderate ethanol administration on the hepatic content of enzymes involved in ethanol oxidation, on hepatic lipid accumulation, and on serum markers of liver function/damage in the monkey, Macaca fascicularis. METHODS: Ovariectomized, adult monkeys were maintained for 34 months on an atherogenic diet containing cholesterol 1.21 mg/kJ. They were trained to drink ethanol plus vehicle at a dose of 0.5 g/kg body weight, which was administered 5 days a week for 2 years. Blood was collected for ethanol concentrations (1 hr after ethanol administration) and was also assayed for gamma-glutamyltransferase, alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities. Liver obtained at necropsy was analyzed for triglyceride and cholesterol contents and for alcohol dehydrogenase, cytochrome P450 2E1, and cytochrome P450 3A4 by Western blots. RESULTS: The blood ethanol concentrations measured 1 hr after ethanol administration were relatively constant over the 2-year dosing period. Hepatic levels of alcohol dehydrogenase and the cytochrome P450s were not significantly different between ethanol-consuming animals and control animals. Ethanol-associated increases in liver triglyceride were not significant due to high variability in hepatic lipid content in both the controls and ethanol consumers. However, covariance analyses using pretreatment concentrations of plasma cholesterol and apolipoprotein A-I suggested that the ethanol-related increase in hepatic free cholesterol was significant. Relative to controls, alcohol consumers had higher levels of serum ALT and a transient increase in ALP at 5 months. CONCLUSIONS: The observations made in this study on primates administered an atherogenic diet suggest that moderate ethanol ingestion has modest effects on the liver, including slightly increased ALT and ALP values. However, additional studies will be required to verify that this level of consumption is hepatotoxic when ingested over extended periods. This is still a concern because some human studies suggest that levels of ethanol considered to be cardioprotective cause liver injury when consumed over a lifetime.