Stability of the human sperm DNA methylome to folic acid fortification and short-term supplementation.

STUDY QUESTION: Do short-term and long-term exposures to low-dose folic acid supplementation alter DNA methylation in sperm? SUMMARY ANSWER: No alterations in sperm DNA methylation patterns were found following the administration of low-dose folic acid supplements of 400 µg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. WHAT IS KNOWN ALREADY: Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate's role in one-carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high-dose (5 mg/day) folic acid supplementation. STUDY DESIGN, SIZE, DURATION: This is a control versus treatment study where genome-wide sperm DNA methylation patterns were examined prior to fortification of food (1996-1997) in men with no history of infertility at baseline and following 90-day exposure to placebo (n = 9) or supplement containing 400 µg folic acid/day (n = 10). Additionally, pre-fortification sperm DNA methylation profiles (n = 19) were compared with those of a group of post-fortification (post-2004) men (n = 8) who had been exposed for several years to dietary folic acid fortification. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole-genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. MAIN RESULTS AND THE ROLE OF CHANCE: Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first-generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays, no significant changes were observed in individual probes following low-level supplementation; when compared with those of the post-fortification cohort, there were also few differences in methylation despite exposure to years of fortified foods. LARGE SCALE DATA: Illumina HumanMethylation450 BeadChip data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE89781. LIMITATIONS, REASONS FOR CAUTION: This study was limited to the number of participants available in each cohort, in particular those who were not exposed to early (pre-1998) fortification of food with folic acid. While genome-wide DNA methylation was assessed with several techniques that targeted genic and CpG-rich regions, intergenic regions were less well interrogated. WIDER IMPLICATIONS OF THE FINDINGS: Overall, our findings provide evidence that short-term exposure to low-dose folic acid supplements of 400 µg/day, over a period of 3 months, a duration of time that might occur during infertility treatments, has no major impact on the sperm DNA methylome. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR: MOP-89944). The authors have no conflicts of interest to declare.

The prevalence of cardiovascular risk factors among elderly Chinese Americans.

In 1981 to 1983, the nutrition and health status of 346 Chinese immigrants in Boston, Mass, aged 60 to 96 years was surveyed and analyzed for cardiovascular risk factors. These elderly Chinese were physically active and seldom obese and consumed a high-carbohydrate (57% of total energy intake), low-fat (24% of total energy intake), low-ascorbic acid (0.62 mmol/d) diet. Current cigarette smoking was common (39%) only in men, while alcoholism was rare in both sexes. Compared with elderly whites, they had lower mean blood pressure and blood levels of total, low-density lipoprotein, and high-density lipoprotein cholesterol, apolipoproteins A-I and B, and ascorbic acid. These characteristics resemble those of the urban population in mainland China, where hemorrhagic stroke is the major cause of cardiovascular mortality.

Vitamin A analogs as tests for liver vitamin A status in the rat.

Two chlorinated retinol analogs (Ro 11-0503 and Ro 11-8284) were assayed in rat serum and correlated to retinol in liver. Rats were fed a retinol-free diet to deplete their liver stores, then repleted with 0.5, 1.0, 3.0, and 10.0 mg retinyl palmitate/kg diet. Rats were given intraperitoneal injections of the analog then killed after an additional week on the diets. Analogs were measured by HPLC. The relative abundance of both analogs in serum was inversely correlated with the amount of retinyl palmitate in the liver. Serum analog concentrations may be useful as indications of liver retinol stores.

Oxidative damage and defense.

Increased production of reactive oxygen species is a feature of most, if not all, human disease, including cardiovascular disease and cancer. Dietary antioxidants may be especially important in protecting against human diseases associated with free radical damage to cellular DNA, lipids, and proteins. Ascorbic acid is an effective water-soluble antioxidant, and epidemiologic studies suggest that increased ascorbate nutriture is associated with reduced risk of some degenerative diseases, especially cancer and eye cataracts. Population studies have also shown that high vitamin E intakes are associated with decreased risk of coronary heart disease, possibly as a result of inhibition of atherogenic forms of oxidized low-density lipoprotein. Recent data suggest that beta-carotene provides protection against lipid peroxidation in humans, as well as provitamin A activity. Yet, present data are not sufficient to quantitate micronutrient requirements needed to protect against oxidative damage. The antioxidant roles of many food constituents, such as polyphenols, have not been clarified. Most antioxidants can act as prooxidants under certain conditions, and more research is needed to determine the occurrence and importance of this in vivo. The few controlled intervention trials carried out so far have shown mixed results as to the potential of antioxidant supplements for reducing the incidence of chronic diseases. Definitive recommendations on antioxidant intakes for disease prevention must await evidence from controlled studies and intervention trials, some currently in progress. Overall, the present data suggest that protection against oxidative damage and related disease is best served by the variety of antioxidant substances found in fruit and vegetables.

Studies on the bioavailability of zinc in humans: effects of heme and nonheme iron on the absorption of zinc.

The change in the concentration of plasma zinc after oral administration of zinc was used as the index of zinc absorption in the study of the interaction of zinc and iron in the human intestine. With zinc sulfate as the source of inorganic zinc, and ferrous sulfate as the source of nonheme iron, a ratio of Fe/Zn of 1:1 slightly inhibited zinc absorption while Fe/Zn ratio of 2:1 and 3:1 substantially inhibited zinc uptake. No effect on zinc absorption was observed, however, when heme iron, as heme chloride, was ingested in a 3:1 Fe/Zn ratio with inorganic zinc. Atlantic oysters providing about 54 mg of zinc were consumed with or without 100 mg of ferrous iron; Fe/Zn ratio was 2:1. With this "organic" form of zinc, iron did not significantly affect zinc absorption. The evidence for competitive interaction of zinc and iron was strongest with nonheme iron and inorganic zinc. Thus, the possibility that intrinsic iron in formulas for feeding infants and in vitamin-mineral supplement might inhibit the absorption of zinc justifies concern about the Fe/Zn ratio in the formulation of these products.

Urinary hydroxyproline excretion and vitamin C status in healthy young men.

The relationship between ascorbic acid status and the urinary excretion of hydroxyproline was examined in 11 healthy male subjects fed an ascorbic acid-deficient diet for 14 wk while in a metabolic unit. The diet provided 5 mg ascorbic acid/d and was supplemented with ascorbic acid to give intakes of 65 mg/d (2 wk), 5 mg/d (4 wk), 605 mg/d (3 wk), 5 mg/d (4 wk), and an average 375 mg/d (1 wk). The urinary excretion of hydroxyproline increased by an average of 16% and 30% after the first and second depletion periods, respectively, and decreased to baseline values after supplementation with normal or high doses of vitamin C. Significant (p less than 0.05) inverse correlations were found between urinary hydroxyproline and plasma, red cell, and leukocyte ascorbic acid. These results show that urinary hydroxyproline excretion increases during human vitamin C deficiency but that this effect is not strong enough to provide a reliable marker of mild vitamin C deficiency.

Effect of fiber from fruits and vegetables on metabolic responses of human subjects. III. Zinc, copper, and phosphorus balances.

Intakes and excretions of zinc, copper, and phosphorus were determined for 12 men when they consumed a diet low in fiber and when they consumed a diet containing fiber from fruits and vegetables (high-fiber diet). All subjects consumed both diets for 26 days each. The low-fiber diet was supplemented with copper so that it was equivalent to the high-fiber diet in this respect. Mean daily intakes on the low- and high-fiber diets, respectively, were: zinc, 13.2 and 12.6 mg; copper, 1.8 and 1.6 mg; phosphorus, 1.639 and 1.690 g. Mean daily balances on the low- and high-fiber diets, respectively, were: zinc, +3.5 and -0.9 mg; copper, +0.2 and -0.4 mg; phosphorus, +0.361 and +0.292 g. Zinc and copper balances differed significantly on the two diets (P less than 0.001 and P less than 0.005, respectively, but phosphorus balances did not. The fiber supplied by the fruits and vegetables contained more lignin and cellulose and less hemicellulose than would an equivalent amount of fiber from bran. The bran would supply more magnesium, zinc, and copper than this particular diet containing fruits and vegetables.

Biochemical indices of human vitamin C status.

Biochemical indicators of ascorbic acid (AA) status were studied in eleven young adult males fed the same AA deficient diet for 14 wk in a live-in metabolic unit. Supplements of AA were added to the diet to give AA-intake periods of 65 mg/d (2 wk), 5 mg/d (4 wk), 605 mg/d (3 wk), 5 mg/d (4 wk), 605 mg/d (4 d), and 65 mg/d (3 d). Blood plasma, erythrocyte, and leukocyte AA levels all reflected AA intake, however, plasma AA showed less variability than red cell AA levels and was considerably easier to determine than leukocyte AA. Plasma AA values less than 0.40 mg/dL (23 mumol/L) reflected marginal AA status. The daily AA intake calculated to maintain plasma AA levels of at least 0.4 mg/dL (23 mumol/L) in healthy young men was 41 mg. The average AA intake estimated to maximize the total body pool was 138 mg/d. Urine and salivary AA levels were not useful indicators of AA status because urinary AA levels did not discriminate well between adequate and deficient AA intakes and salivary AA levels did not consistently reflect AA intake.

Plasma ascorbic acid in adult males: effects of depletion and supplementation.

We investigated effects of moderately elevated oral doses of ascorbic acid (AS) on plasma AS turnover in healthy men after abrupt alterations in AS intake. Subjects ate a basal diet of conventional food in which only AS intake was changed. Blood specimens were collected from fasted subjects twice each week during depletion periods. Deproteinized plasma was analyzed after derivatization with 2,4-dinitrophenylhydrazine. Subjects remained healthy and never became frankly scorbutic. The kinetics of log-converted plasma AS values for the depletion periods demonstrated that the slopes calculated by least squares were up to 56% higher for six of nine subjects after 600 mg AS/day. The difference between the mean slopes (M) for loss of plasma AS was significantly different (p less than 0.05, n = 6) after 60 mg AS (M = -0.0222 +/- 0.0145) than after 600 mg AS (M = -0.0246 +/- 0.0003). Leukocyte AS concentration decreased 44.6% in the first depletion period and 77.1% in the second.

Studies on the bioavailability of zinc in man. III. Effects of ascorbic acid on zinc absorption.

As ascorbic acid is known to enhance the absorption of dietary iron and to inhibit the absorption of dietary copper, studies were undertaken to examine the effect of ascorbic acid on the bioavailability of zinc in human subjects. The index of absorption was the change in plasma zinc concentration after a 110-mg aqueous dose of ZnSO4.7H2O (containing 25 mg of elemental zinc). Doses of 0.5, 1.0, and 2.0 g of ascorbic acid, representing a spectrum of Zn:ascorbic acid molar ratios from the dietary to the pharmacological range, failed to produce any significant change in the pattern of zinc absorption. Moreover, 2.0 g of ascorbic acid, equivalent to a Zn:ascorbic acid ratio of 0.145 failed to improve the absorption of 108 mg of elemental zinc incorporated into 120 g of black bean gruel. Ascorbic acid over a range of dosages commonly consumed by man had no demonstrable effect on the absorption of inorganic zinc.

Hair as a biopsy material. V. Hair metal as an index of hepatic metal in rats: copper and zinc.

Hair copper in 500 day-old adult rats was found to correlate directly with copper in whole liver (P less than 0.001) and the subcellular liver nuclear (P less than 0.022), and cytosol fractions (P less than 0.001). An inverse correlation was found between copper in liver microsomes and plasma cholesterol (P less than 0.044). Similar data analysis for zinc showed no significant correlations (P greater than 0.05). The determination of copper in hair may be useful for assessing total liver copper content in human beings. Copper in liver microsomes may participate in the control of plasma cholesterol.

Zinc deficiency in pregnant rhesus monkeys: effects on behavior of infants.

Zinc deprivation from day 110 to 150 of gestation in rhesus monkeys resulted in rash, alopecia, anorexia, decreased feed efficiency, and low plasma zinc in the mothers. Infants of the dams that had been deprived of zinc during gestation displayed a more rapid postnatal growth rate than infants of the control mothers. Infants of the zinc-deprived dams played and explored less than the control infants. They also associated with their mothers a greater percentage of the time and were less active. This study has shown that third trimester maternal zinc deprivation in nonhuman primates can impair behavioral development of offspring.

Abnormal cellular immune responses during acquired zinc deficiency.

The cellular immune response of a 17-year-old decerebrate male with acquired zinc deficiency was studied. He had been fed a commercial formula which contained 7.6 mg zinc per kilogram. His caloric intake had been inadequate as judged by his cachexia. A detailed pretreatment nutritional assessment (five separate observations) which included total serum protein and globulins, albumin, folate, vitamins A, B2, C, ceruloplasmin, and plasma zinc, copper, iron, and total iron binding capacity revealed that the patient was deficient only in zinc and calories. His plasma zinc was 41 +/- 5 microgram/d1 compared with our laboratory norm of 89 +/- 9 microgram/d1 for young adult males. Cellular immunity was assessed by delayed skin reactivity to dinitrochlorobenzene and by in vitro lymphocyte transformation studies. Before zinc therapy the patient rendered a negative skin reaction to dinitrochlorobenzene, and the ability of his lymphocytes to undergo blast transformation in response to mitogen stimulation was significantly depressed with a stimulation index of 4.7 +/- 0.8 as compared with 139.1 +/- 77.3 for controls. Within 3 weeks after zinc therapy (22.7 mg zinc per day) he demonstrated a positive delayed skin reaction to dinitrochlorobenzene and a normal lymphocyte response stimulation index = 205.5 +/- 42.6 versus 199.3 +/- 58.2 for control). In addition, a pretreatment facial seborrhea and a decubitus ulcer rapidly healed.

Ascorbate is depleted by smoking and repleted by moderate supplementation: a study in male smokers and nonsmokers with matched dietary antioxidant intakes.

BACKGROUND: Lack of reliable dietary data has hampered the ability to effectively distinguish between effects of smoking and diet on plasma antioxidant status. As confirmed by analyses of comprehensive food-frequency questionnaires, the total dietary intakes of fruit and vegetables and of dietary antioxidants were not significantly different between the study groups in the present study, thereby enabling isolation of the effect of smoking. OBJECTIVE: Our objective was to investigate the effect of smoking on plasma antioxidant status by measuring ascorbic acid, alpha-tocopherol, gamma-tocopherol, beta-carotene, and lycopene, and subsequently, to test the effect of a 3-mo dietary supplementation with a moderate-dose vitamin cocktail. DESIGN: In a double-blind, placebo-controlled design, the effect of a vitamin cocktail containing 272 mg vitamin C, 31 mg all-rac-alpha-tocopheryl acetate, and 400 microg folic acid on plasma antioxidants was determined in a population of smokers (n = 37) and nonsmokers (n = 38). The population was selected for a low intake of fruit and vegetables and recruited from the San Francisco Bay area. RESULTS: Only ascorbic acid was significantly depleted by smoking per se (P < 0.01). After the 3-mo supplementation period, ascorbic acid was efficiently repleted in smokers (P < 0.001). Plasma alpha-tocopherol and the ratio of alpha- to gamma-tocopherol increased significantly in both supplemented groups (P < 0.05). CONCLUSIONS: Our data suggest that previous reports of lower concentrations of plasma vitamin E and carotenoids in smokers than in nonsmokers may primarily have been caused by differences in dietary habits between study groups. Plasma ascorbic acid was depleted by smoking and repleted by moderate supplementation.

A mathematical model of creatine metabolism in normal males--comparison between theory and experiment.

Based upon knowledge that the body creatine pool in normal males undergoes a constant fractional conversion rate to creatinine and that 24-hr urinary creatinine excretion is dependent upon creatine and protein intakes, we developed a mathematical model with feedback which describes the creatine pool size and, consequently, the 24-hr urinary creatinine excretion as a function of time after change in diet. Validity of the model was tested by comparing calculated with experimental changes in 24-hr urinary creatinine excretion rates of male volunteers who were participating in studies of effects of "high" and "low" protein diets on mineral requirements. The model was further verified by comparing published changes in creatinine excretion rates with changes predicted by the model. It is concluded that the effects of changes in creatine and protein intakes upon the body creatine pool size in healthy males can be described mathematically by a model with a feedback component. It is also cautioned that 24-hr urinary creatinine excretion may not be a good reference for quantifying other urinary substances under circumstances where the creatine or protein intakes are not constant.

Whole body surface loss of trace metals in normal males.

Whole body surface losses of zinc, copper, and iron were measured in 13 male volunteers who lived in a controlled environment for 4 to 9 months. For 88 daily samples, the mean losses for zinc, copper, and iron were 0.50, 0.34, and 0.33 mg/day (3.9, 26, and 2.1% of the mean dietary intakes, respectively). There was a large variance in metal losses with no significant differences in variance within-versus-between subjects or on the same-versus-different diets. The surface losses of zinc and iron increased the apparent dietary requirements determined by balance measurements only 5 and 3%, respectively, while the increase in copper requirement due to surface loss was 25 to 30%.

An evaluation of trace metals, vitamins, and taste function in anorexia nervosa.

Trace metals, vitamins, and other biochemical parameters were measured in 30 female patients hospitalized for anorexia nervosa with the aim of relating them to taste function, biochemical changes, and clinical signs found in this illness. Plasma zinc (71.9 +/- 14 microgram/100 ml; P < 0.01), urinary zinc (129.5 +/- 121 microgram/24 hr), and copper (84 +/- 17 microgram/100 ml; P < 0.001), were depressed, whereas zinc and copper content of hair was normal. Anorexia nervosa patients showed hypogeusia, with the bitter and sour taste most severely affected, however plasma zinc levels did not correlate with taste recognition scores. Patients showed hypercarotenemia (214 +/- 129 microgram/100 ml; P < 0.01) with normal plasma vitamin A and retinol-binding protein levels. Total iron binding capacity was depressed (261 +/- 62 microgram/100 ml; P < 0.001) in contrast to plasma iron, ceruloplasmin and folic acid, which were normal. In nine patients, who were retested before discharge, taste function improved; plasma zinc, copper, and total iron binding capacity levels increased whereas plasma carotene and cholesterol decreased to normal levels. It is concluded that the observed zinc, copper, and iron binding protein deficiencies, and hypogeusia, reflect the self-imposed nutritional restriction of anorexia nervosa patients. Zinc and other micronutrients released from catabolized tissue along with vitamin intake may mitigate against more severe deficiency states in anorexia nervosa.

Zinc status and vitamin A transport in cystic fibrosis.

Zinc status and the retinol transport system were examined in 18 retinol supplemented cystic fibrosis (CF) patients and 40 age-matched controls. Plasma vitamin A was significantly lower in the CF group as compared to the controls and correlated positively with plasma retinol-binding protein (RBP) in both the CF and control groups. Plasma zinc of the CF group was not significantly lower than controls whereas hair zinc was. Plasma zinc was positively correlated with plasma RBP, vitamin A, and albumin in the CF group but not in the controls. Plasma concentrations of vitamin A, RBP, albumin, and zinc decreased with age in the CF group but not in the controls. The data support previous suggestions that low plasma vitamin A levels in CF are due to defects in the retinol transport system. The zinc status of the CF groups as a whole was judged to be low-normal however a subgroup of CF patients were in the marginal to deficient category. This subgroup also had lower levels of plasma vitamin A and RBP. The data suggest that zinc may be a contributing factor in the low plasma vitamin A/RBP levels of CF patients with marginal or deficient zinc status.

Vitamin C status and nutrient interactions in a healthy elderly population.

Vitamin C status and interactions with other nutrients were studied in 677 healthy, noninstitutionalized elderly people aged 60-98 y. Although 6% of the males and 3% of the females showed marginal vitamin C status (plasma ascorbic acid 11 to less than 23 mumol/L), only one person had a plasma ascorbic acid (AA) level less than 11 mumol/L. At all levels of total vitamin C intake, mean plasma AA levels were higher in females than males. Vitamin C supplement use was associated with generally higher blood levels of vitamins B-6, B-12, and E and folate in both sexes and with higher levels of retinol in females. However, after both age and the total dietary intake of the specific nutrient being examined were controlled for, plasma AA levels were significantly correlated only with plasma levels of vitamin E and folate in females.

Dietary intakes and biochemical indicators of nutritional status in an elderly, institutionalized population.

A dietary and biochemical assessment of the nutritional status of 260 elderly men and women, 60-101 y (average 80.5 y), was conducted in 15 long-term-care facilities in the Boston area. Subjects were free of clinically apparent terminal or wasting illness. Nutrient intakes were comparable to those in a simultaneously studied free-living population as were most biochemical markers of nutrient status. Although no specific nutrient deficiencies were identified, blood levels of vitamin A and retinol-binding protein in males and of zinc in both sexes were lower in this institutionalized group than in the free-living subjects. Hematologic indices, albumin, prealbumin, and transferrin levels were also lower than in noninstitutionalized elderly populations. These differences may reflect the greater prevalence of chronic diseases and medication use in a long-term-care population. However, there is no evidence that institutionalization in itself leads to impairment of nutritional status.