Intracellular S-adenosylhomocysteine concentrations predict global DNA hypomethylation in tissues of methyl-deficient cystathionine beta-synthase heterozygous mice.

Because S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are the substrate and product of essential methyltransferase reactions; the ratio of SAM:SAH is frequently used as an indicator of cellular methylation potential. However, it is not clear from the ratio whether substrate insufficiency, product inhibition or both are required to negatively affect cellular methylation capacity. A combined genetic and dietary approach was used to modulate intracellular concentrations of SAM and SAH. Wild-type (WT) or heterozygous cystathionine beta-synthase (CBS +/-) mice consumed a control or methyl-deficient diet for 24 wk. The independent and combined effect of genotype and diet on SAM, SAH and the SAM:SAH ratio were assessed in liver, kidney, brain and testes and were correlated with relative changes in tissue-specific global DNA methylation. The combined results from the different tissues indicated that a decrease in SAM alone was not sufficient to affect DNA methylation in this model, whereas an increase in SAH, either alone or associated with a decrease in SAM, was most consistently associated with DNA hypomethylation. A decrease in SAM:SAH ratio was predictive of reduced methylation capacity only when associated with an increase in SAH; a decrease in the SAM:SAH ratio due to SAM depletion alone was not sufficient to affect DNA methylation in this model. Plasma homocysteine levels were positively correlated with intracellular SAH levels in all tissues except kidney. These results support the possibility that plasma SAH concentrations may provide a sensitive biomarker for cellular methylation status.

Urinary excretion of folate catabolites responds to changes in folate intake more slowly than plasma folate and homocysteine concentrations and lymphocyte DNA methylation in postmenopausal women.

Folate turnover involves urinary excretion, fecal excretion, and catabolism that involves cleavage of the C9-N10 bond to yield pterins and para-aminobenzoylglutamate (pABG). Little is known about the relationship between the function of folate pools and their rates of catabolism. We report here an investigation of excretion of urinary pABG and its primary excretory form, para-acetamidobenzoylglutamate (ApABG) in samples collected during a previously published study of postmenopausal women. Ten women (49-63 y) were fed a low folate diet (56 microg/d) supplemented with folic acid to yield total folate intakes of 195 microg/d (d 1-5), 56 microg/d (d 6-41), 111 microg/d (d 42-69), 286 microg/d (d 70-80) and 516 microg/d (d 81-91). This caused changes in plasma folate, plasma homocysteine and global methylation of lymphocyte DNA. For each subject, a 7-d pooled urine sample was collected over d 1-7, 36-42, 64-70 and 85-91. ApABG constituted >85% of total catabolite excretion, and folate intake did not significantly influence ApABG or pABG excretion. The molar ratio of total catabolite excretion/folate intake varied significantly, with ratios of 1.0 +/- 0.17 (d 1-7), 3.0 +/- 0.55 (d 36-42), 1.1 +/- 0.18 (d 64-70) and 0. 33 +/- 0.054 (d 85-91). These observations indicate that the rate of folate catabolite excretion is related mainly to masses of slow-turnover folate pools governed by long-term folate intake. Folate pools functioning in some forms of methyl group metabolism respond to dietary changes in folate intake much more rapidly.

Folate nutriture alters choline status of women and men fed low choline diets.

Choline and folate share methylation pathways and, in studies of rats, were shown to be metabolically inter-related. To determine whether choline status is related to folate intake in humans, we measured the effect of controlled folate depletion and repletion on the plasma choline and phosphatidylcholine concentrations of 11 healthy men (33-46 y) and 10 healthy women (49-63 y) fed low-choline diets in two separate metabolic unit studies. Total folate intake was varied by supplementing low folate (25 and 56 microg/d for men and women, respectively) and low choline (238 and 147 mg/d for men and women, respectively) diets with pteroylglutamic acid for 2-6 wk following folate-depletion periods of 4-5 wk. The low folate/choline intakes resulted in subclinical folate deficiencies; mean plasma choline decreases of 28 and 25% in the men and women, respectively; and a plasma phosphatidylcholine decrease of 26% in the men (P < 0. 05). No functional choline deficiency occurred, as measured by serum transaminase and lipid concentrations. The decreases in choline status measures returned to baseline or higher upon moderate folate repletion and were more responsive to folate repletion than plasma folate and homocysteine. Feeding methionine supplements to the men did not prevent plasma choline depletion, indicating that folate is a more limiting nutrient for these methylation pathways. The results indicate that 1) choline is utilized as a methyl donor when folate intake is low, 2) the de novo synthesis of phosphatidylcholine is insufficient to maintain choline status when intakes of folate and choline are low, and 3) dietary choline is required by adults in an amount > 250 mg/d to maintain plasma choline and phosphatidylcholine when folate intake is low.

Moderate folate depletion increases plasma homocysteine and decreases lymphocyte DNA methylation in postmenopausal women.

To determine the human folate requirement on the basis of changes in biochemical pathways, we studied the effect of controlled folate intakes on plasma homocysteine and lymphocyte DNA methylation and deoxynucleotide content in healthy postmenopausal women. Eight women (49-63 y of age) were housed in a metabolic unit and fed a low folate diet containing 56 microg/d of folate for 91 d. Folate intake was varied by supplementing 55-460 microg/d of folic acid (pteroylglutamic acid) to the diet to provide total folate intake periods of 5 wk at 56 microg/d, 4 wk at 111 microg/d and 3 wk at 286-516 microg/d. A subclinical folate deficiency with decreased plasma folate was created during the first two periods. This resulted in significantly elevated plasma homocysteine and urinary malondialdehyde, and lymphocyte DNA hypomethylation. The folate depletion also resulted in an increased ratio of dUTP/dTTP in mitogen-stimulated lymphocyte DNA and decreased lymphocyte NAD, changes suggesting misincorporation of uracil into DNA and increased DNA repair activity. The DNA hypomethylation was reversed with 286-516 microg/d of folate repletion, whereas the elevated homocysteine decreased with 516 but not 286 microg/d of folate. The results indicate that marginal folate deficiency may alter DNA composition and that the current RDA of 180 microg/d may not be sufficient to maintain low plasma homocysteine concentrations of some postmenopausal women.

Plasma carotenoids and the prevalence of adenomatous polyps of the distal colon and rectum.

In a case-control study, the authors investigated relations between plasma carotenoid concentrations and the prevalence of colorectal adenomatous polyps (precursors to colorectal cancer) in residents of Los Angeles County and Orange County, California, from 1991 through 1993. Plasma concentrations of six carotenoids were compared in 472 asymptomatic cases with a first-time diagnosis of at least one adenomatous polyp of the distal colon or rectum and 502 matched controls. Odds ratios adjusted for age, sex, smoking, alcohol intake, and energy, saturated fat, and fruit and vegetable intake revealed no associations between any of the individual carotenoids and polyp prevalence or between total carotenoids and polyp prevalence.

Male rats fed methyl- and folate-deficient diets with or without niacin develop hepatic carcinomas associated with decreased tissue NAD concentrations and altered poly(ADP-ribose) polymerase activity.

Folate is an essential cofactor in the generation of endogenous methionine, and there is evidence that folate deficiency exacerbates the effects of a diet low in choline and methionine, including alterations in poly(ADP-ribose) polymerase (PARP) activity, an enzyme associated with DNA replication and repair. Because PARP requires NAD as its substrate, we postulated that a deficiency of both folate and niacin would enhance the development of liver cancer in rats fed a diet deficient in methionine and choline. In two experiments, rats were fed choline- and folate-deficient, low methionine diets containing either 12 or 8% casein (12% MCFD, 8% MCFD) or 6% casein and 6% gelatin with niacin (MCFD) or without niacin (MCFND) and were compared with folate-supplemented controls. Liver NAD concentrations were lower in all methyl-deficient rats after 2-17 mo. At 17 mo, NAD concentrations in other tissues of rats fed these diets were also lower than in controls. Compared with control values, liver PARP activity was enhanced in rats fed the 12% MCFD diet but was lower in MCFND-fed rats following a further reduction in liver NAD concentration. These changes in PARP activity associated with lower NAD concentrations may slow DNA repair and enhance DNA damage. Only rats fed the MCFD and MCFND diets developed hepatocarcinomas after 12-17 mo. In Experiment 2, hepatocarcinomas were found in 100% of rats fed the MCFD and MCFND diets. These preliminary results indicate that folic acid deficiency enhances tumor development. Because tumors developed in 100% of the MCFD-fed rats and because tissue concentrations of NAD in these animals were also low, further studies are needed to clearly define the role of niacin in methyl-deficient rats.

NADPH-cytochrome P-450 reductase, cytochrome P-450 2C11 and P-450 1A1, and the aryl hydrocarbon receptor in livers of rats fed methyl-folate-deficient diets.

We investigated three hepatic cytochrome P-450 isozymes and the aryl hydrocarbon (Ah) receptor in rats fed one of the following three diets for 15 months: a diet containing the AIN vitamin mixture (control), the control diet devoid of choline and folate (CFD), or the CFD diet devoid of niacin (CFND). Hepatic tumors developed in all CFD- and CFND-fed rats. Western blot analyses of nontumor hepatic tissue showed that NADPH-cytochrome P-450 reductase (P-450 reductase) increased significantly in the CFD and CFND groups compared with the control group. Hepatic cytochrome P-450 2C11 (CYP2C11) was not detectable in the CFD and CFND groups compared with the control group. Ah receptor and cytochrome P-450 1A1 (CYP1A1) were detected in higher amounts in livers of both deficient groups. CYP1A1 is an enzyme associated with bioactivation of exogenous genotoxins. To our knowledge, this is the first time it has been shown that CYP1A1 and the Ah receptor are induced by dietary deficiencies.

Vitamins C, E and A and heme oxygenase in rats fed methyl/folate-deficient diets.

There is evidence that the development of hepatocarcinoma in rats fed a methyl-deficient diet is associated with oxidative stress. We investigated, therefore, whether the tissue concentrations of the antioxidant vitamins ascorbic acid (AA) and alpha- and gamma-tocopherol (T) are altered in methyl/folate deficiency. We also measured retinol concentrations in tissues and hepatic mRNA expression of heme oxygenase (HO1). A 6% gelatin, 6% casein diet, devoid of choline and folate (CFD) was selected based on the high rate of tumor development in rats fed this diet. Spectrophotometric measurement of AA and HPLC determination of tissue T and retinol showed decreased concentrations of AA in blood; alpha- and gamma-T in lung, heart and plasma, alpha-T and retinol in liver; retinol in lung; and increased expression of hepatic HO1 mRNA. Similar alterations in tissue vitamin concentrations were found when the CFD diet devoid of niacin (CFND) was fed. Reducing alpha-T in the CFND diet (CFNED) further decreased hepatic alpha-T concentrations. These results show that chronic methyl/folate deficiency is associated with a compromised antioxidant defense system.

Blood antioxidants changes in young women following beta-carotene depletion and repletion.

OBJECTIVE: This study was undertaken to investigate the relationship between beta-carotene intake and biochemical indices of antioxidant status in the blood of nine premenopausal women ages 18 to 42. METHODS: Nine healthy adult women were fed a low beta-carotene diet for 68 days. They were repleted with the same diet supplemented with beta-carotene (15 mg beta-carotene) for 28 days. During the last week of the study, they received an additional mixed carotenoid supplement. Indices of blood antioxidant status were measured on days 1, 29, 36, 43, 50, 64, 71, 92, and 99. RESULTS: We found significant increases of erythrocyte conjugated dienes between the 71st and 99th day of the study; increases of glutathione (GSH) peroxidase (GP) on day 43 and day 92 compared to a decrease on day 29; and decreases of GSH reductase throughout the treatment period. Erythrocyte catalase activities seemed to parallel GP activities. Erythrocyte oxidized glutathione (GSSG) levels were depressed both after beta-carotene depletion and repletion. beta-Carotene depletion/repletion had no effect on plasma vitamin E or GSH levels. Platelet GSH levels were depressed after beta-carotene depletion followed by elevated GSH levels after beta-carotene repletion. CONCLUSION: A diet low in beta-carotene and adequate in all other nutrients, including vitamin A, resulted in altered erythrocyte and platelet antioxidant indices; however, it had little impact on plasma GSH or vitamin E levels in young healthy women. Our results are consistent with the suggestion that carotenes may be important in the prevention of oxidative damage.

Plasma ferritin, iron intake, and the risk of colorectal polyps.

High iron exposure has been associated with colorectal neoplasia in several studies. The authors investigated plasma ferritin, an indicator of iron stores, and iron intake as risk factors for adenomatous polyps, intermediate markers for colorectal cancer. During 1991-1993, they collected fasting blood samples from and administered questionnaires to men and women 50-75 years old who visited free sigmoidoscopy clinics at a health maintenance organization. Data from 965 subjects (467 cases, 498 controls) were analyzed. Compared with those who had low-normal plasma ferritin concentrations (73-141 micrograms/liter), those with elevated concentrations ( > 289 micrograms/liter) had a multivariate-adjusted odds ratio of 1.5 (95% confidence interval (CI) 1.0-2.3) after excluding subjects with possible non-iron-related elevations in ferritin. Compared with subjects consuming an adequate amount of iron (11.6-13.6 mg/day), multivariate-adjusted odds ratios were 1.6 (95% CI 1.1-2.4) for < 11.6 mg/day and 1.4 (95% CI 0.9-2.0) for > 27.3 mg/day. These results provide further support for a weak positive association between iron exposure and colorectal polyps.

Red cell and plasma folate, folate consumption, and the risk of colorectal adenomatous polyps.

Epidemiological and experimental evidence suggests that dietary folate may protect against colorectal carcinogenesis. The epidemiological relationship between a biochemical measure of folate status and colorectal neoplasia in a sizeable and generally healthy population does not yet appear to have been reported. We conducted a case-control study of the relationships among red cell folate, plasma folate, folate intake, and adenomatous polyps, intermediate markers for colorectal cancer. During 1991-1993, fasting blood samples were assayed and dietary and nondietary risk factor questionnaires were administered to men and women ages 50-75 years who had a free sigmoidoscopy at a health maintenance organization. We analyzed data from 682 subjects (332 cases and 350 controls), controlling for potential confounding by sex, age, sigmoidoscopy date, and clinic. For red cell folate levels 160 ng/ml (363 nmol/liter) or more, compared to lower levels, the odds ratio was 0.76 [95% confidence interval (CI) = 0.53-1.08]. For men, the corresponding odds ratio was 0.53 (CI = 0.32-0.87); for women, it was 1.16 (CI = 0.67-2.00). Results were essentially unchanged when adjusted for levels of blood nutrients and other potential confounding variables. Plasma folate and folate intake results were similar to red cell folate results, but the associations with polyps were weaker. Results are consistent with a protective effect of red cell folate concentration against the development of colorectal polyps, at least in men. A folate effect may depend on sex-specific interactions with other nutritional or physiological factors.

In vivo methylation capacity is not impaired in healthy men during short-term dietary folate and methyl group restriction.

Ten healthy adult men were fed a diet low in folate and exogenous methyl groups to study the effects on in vivo methylation capability. The men were housed in a metabolic unit for the entire 108 d of the study. After a 9-d baseline period (Period 1), the men were fed a soy-product-amino acid defined diet for 45 d, which provided 25 micrograms/d of folate for 30 d (Period 2) and, with a folate supplement, 99 micrograms/d for 15 d (Period 3). During Period 2 and Period 3, the low methionine and choline diet was supplemented with methionine for half the subjects to vary the dietary methyl group intake. The periods were then repeated over the next 54 d (Periods 4-6), with a crossover of methionine intakes in Period 5 and Period 6. A 1-g oral dose of nicotinamide was given at the end of each period and methylated urine metabolites determined. Other measures related to in vivo methylation capability included urine creatinine, and plasma and urine carnitine. Even with moderate folate depletion, none of these measures was decreased by low methionine and choline intakes. Plasma methionine concentrations were unchanged throughout. Limiting exogenous methyl group intake by restricting dietary methionine and choline did not impair in vivo methylation capabilities for the variables tested, even at low folate intake.

Homocysteine increases as folate decreases in plasma of healthy men during short-term dietary folate and methyl group restriction.

Ten healthy adult men were fed a diet low in folate and exogenous methyl groups to study the effects on folate requirement and status. The men were housed in a metabolic unit for the entire 108-d study. After a 9-d base-line period (P1), the men were fed an amino acid-defined soybean product diet for 45 d, which provided 25 micrograms/d of folate for 30 d (P2) and (with a folate supplement) 99 micrograms/d for 15 d (P3). During P2 and P3, the low methionine and choline diet was supplemented with methionine for half the subjects to vary the dietary methyl group intake. The periods were then repeated over the next 54 d (P4-P6), with a cross-over of methionine intakes in P5 and P6. Restricting dietary methyl group intake did not increase the dietary folate requirement. Plasma total homocysteine rose during folate depletion and correlated inversely with plasma folate; however, the response of homocysteine to changes in folate intake varied among individuals from very strong to absent. The results support previous suggestions that increased plasma homocysteine concentrations provide a marker of functional folate deficiency, and further indicate that individuals may differ greatly in their susceptibility to hyperhomocysteinemia due to low folate intakes. Judged by the lack of normalization of high homocysteine concentrations during folate repletion, the current folate RDA for adult men may not provide the expected margin of protection.

Erythrocyte folate levels, oral contraceptive use and abnormal cervical cytology.

The initial hypothesis of this study was that folate depletion is a risk factor for human papillomavirus infection and cervical epithelial cell abnormalities, including dysplasia. The prevalences of low erythrocyte folate levels (defined as < 140 ng/mL erythrocytes and determined by the growth of Lactobacillus) were measured in 250 University of California at Los Angeles students. Among oral contraceptive users, low erythrocyte folate was a risk factor for an abnormal cytologic smear in both benign atypia and squamous intraepithelial lesions. Odds ratios were statistically significant for biopsied women who did not have condyloma and for those who did not have squamous intraepithelial lesions but not for those with histologically confirmed intraepithelial lesions. Low erythrocyte folate was a risk factor for a positive Virapap result in oral contraceptive users. If the folate effects are causal, the findings suggest that erythrocyte folate levels should be higher in oral contraceptive users than in nonusers to protect against an abnormal cytologic smear.

Poly(ADP-ribose) polymerase activity and DNA strand breaks are affected in tissues of niacin-deficient rats.

The niacin cofactor, NAD, is the substrate for poly(ADP-ribose) polymerase, an enzyme associated with DNA repair. We investigated, therefore, whether hepatic poly(ADP-ribose) polymerase activity was altered and DNA strand breaks in lymphocytes and liver were greater in niacin-deficient rats. A niacin deficiency was established in weanling rats with diets containing 1.5 mg/kg of niacin. Based on lower growth rates and NAD concentrations in blood, liver and skeletal muscle, this diet maintained rats in a deficient state for 1 mo, and, when the dietary niacin was reduced to 0.5 mg/kg, rats remained deficient for an additional month. The hepatic poly(ADP-ribose) polymerase activity was decreased in one experiment when mean hepatic NAD concentrations were 0.60 and 0.51 mumol/g at d 34 and d 60, respectively, compared with 0.77 and 0.80 mumol/g in pair-fed controls. Enzyme activity, however, was greater than in controls when hepatic NAD concentrations were < 0.30 mumol/g. Strand breaks in DNA did not accumulate except after tissues were exposed to hypoxanthine-xanthine oxidase, a free radical-generating system. Exposure to this system caused more DNA strand breaks in lymphocytes and hepatic nuclei from niacin-deficient rats compared with the same tissues from controls. The results suggest that, in rats, although hepatic poly(ADP-ribose) polymerase activity can be elevated, a severe niacin deficiency may increase the susceptibility of DNA to oxidative damage, likely due to a lower availability of NAD.

Plasma beta-carotene response in humans after meals supplemented with dietary pectin.

The purpose of this study was to determine the effect of pectin on plasma response to beta-carotene in humans. Using a crossover design, we evaluated the effect on plasma beta-carotene in seven subjects when 12 g citrus pectin was added to a 2092 kJ (500 kcal) controlled meal with 25 mg beta-carotene. Plasma samples were collected at 0, 8, 30, 48, and 192 h after the meals. Plasma beta-carotene was quantified with the use of HPLC. The increase in plasma beta-carotene concentration was significantly reduced by pectin at 30 and 192 h (paired t test; P less than 0.005 and less than 0.05, respectively). Mean percent increase in plasma beta-carotene concentration at 30 h after the meal with beta-carotene was reduced by more than one-half when pectin was added to the meal. These results indicate that the inhibitory effect of pectin may provide one explanation for observations of reduced plasma beta-carotene response in humans after the ingestion of carotenoid-rich foods when compared with equivalent doses of beta-carotene supplements.

Plasma carotenoid levels in human subjects fed a low carotenoid diet.

The purpose of this study was to investigate the effect of a low carotenoid diet on plasma carotenoid levels in humans. Twelve healthy male subjects were fed a low carotenoid diet under controlled conditions for 13 wk in a live-in metabolic unit, as part of a study of vitamin C requirement. Plasma carotenoids (zeaxanthin/lutein, cryptoxanthin, lycopene, alpha-carotene, beta-carotene) were measured with HPLC on study days 2-3, 14-15, 35-36 and 63-64. The rate of decline was rapid between d 2-3 and d 14-15, when the concentration of each carotenoid decreased significantly (P less than 0.05). Although accurate figures for half-life are not possible without more frequent sampling points, mean plasma depletion half-life seemed to be less than 12 d for beta-carotene, alpha-carotene and cryptoxanthin, between 12 and 33 d for lycopene and between 33 and 61 d for zeaxanthin/lutein. Because the decline was not linear over the study period, these data suggest the possibility of at least two body pools of these compounds, with one pool having a more rapid turnover rate. Because there is a significant decline in plasma carotenoid levels within the first 2 wk of a low carotenoid diet, determination of levels of these compounds may be useful only in the assessment of short-term intake.

Glutathione blood levels and other oxidant defense indices in men fed diets low in vitamin C.

Because ascorbic acid is an important contributor to the oxidant defense system in body tissues, we studied the effects of a low dietary intake of ascorbic acid on various indicators of oxidant defense and oxidant damage. During a 13-wk study eight healthy men (25-43 y), residing in a live-in metabolic unit, were fed controlled diets containing different amounts of ascorbic acid for four consecutive periods: period 1 = 250 mg/d for 4 d; period 2 = 5 mg/d for 32 d; period 3 = 10 or 20 mg/d for 28 d and period 4 = 60 or 250 mg/d for 28 d. Measurements were made at several time intervals of the activities of glutathione peroxidase and superoxide dismutase in RBC, DNA strand breaks in mononuclear leucocytes, glutathione concentrations in plasma and RBC and NAD and NADP in RBC. After 60 d of low ascorbic acid intakes and associated with plasma ascorbic acid levels less than 6 mumol/L, the total glutathione concentration and the reduced glutathione:oxidized glutathione ratio were decreased in plasma. At the same time NAD and NADP levels in RBC were elevated. It seems that chronic marginal vitamin C deficiency states may be associated with selected biochemical changes in oxidant defense indices.