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The interaction of a dietary excess of vitamin A (retinoid) and deficiency of methyl-donor compounds was examined in murine early-organogenesis embryonic development. Female mice were fed one of six diets from the time of vaginal plug detection until gestational d 8.0, when embryos were removed and grown in whole embryo culture for 46 h, using serum from rats fed the same diet for 36 d as the culture medium. The six diets were either methyl-donor deficient (designated -FCM: devoid of folic acid, choline and supplemental L-methionine, but having methionine as a component of the protein portion of the diet) or methyl-donor sufficient (designated +FCM: containing folic acid, choline and L-methionine supplementation), in combination with one of three concentrations of retinyl palmitate (0.016, 0.416 or 4.016 g/kg diet). The high dose of retinyl palmitate induced a failure of anterior neuropore closure and hypoplasia of the visceral arches, both of which were significantly ameliorated by simultaneous administration of the methyl-donor-deficient diet. The primary acidic retinoid detected in the rat serum was 9,13-di-cis-retinoic acid, although we hypothesize that teratogenic retinoids were formed by embryonic biotransformation of the retinyl esters to toxic metabolites. Biochemical measurements of metabolites in relevant pathways were performed. We propose that the amelioration of these malformations may be used to determine biochemical pathways critical for retinoid teratogenesis.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The chronically uremic rat has been used as a model to study amino acid metabolism in uremia. Uremic rats fed low protein diets (6% casein) survived longer than uremic rats receiving either higher levels of dietary protein or a low protein diet supplemented with a mixture of nonessential amino acids. Alterations in plasma amino acid levels were observed in the uremic rats and were similar to those found in patients with renal failure. Plasma concentrations of citrulline, free tryptophan, glycine and the methylhistidines were increased and levels of serine, ornithine, lysine, total tryptophan, tyrosine, and the tyrosine-phenylalanine ratio were reduced. The metabolic basis of the altered tyrosine-phenylalanine ratio in plasma was studied. Tyrosine aminotransferase (TAT) and phenylalanine hydroxylase (PHL) activity were normal in the liver, but renal PHL activity of was decreased as compared to control rats. Tissue concentrations of citrulline were also found to be raised in liver and muscle of uremic rats. The activity of ornithine transcarbamoylase, was reduced in the liver and arginine synthetase activity was decreased in the kidneys of uremic rats. Thus elevated citrulline levels in uremic tissue appear to be caused by a decrease conversion of citrulline to arginine in the kidney. Preliminary studies of tryptophan metabolism in uremic rats have shown elevated brain levels of 5-hydroxyindoleacetic acid and increased hepatic tryptophan oxygenase activity. Increased plasma amine levels were associated with altered activities of monoamine oxidase and diamine oxidase in kidney and other tissues.

The enzyme, diamine oxidase, is present in many tissues and plays a role in the metabolism of certain amines, some of which may be toxic. In renal failure, plasma diamine oxidase activity was found to be increased in chronically uremic patients and before and after dialysis therapy in patients undergoing maintenance hemodialysis. Diamine oxidase activity was decreased in urine of the chronically uremic patients as compared to normal subjects. In chronically uremic rats, diamine oxidase activity was observed to be increased in plasma and reduced in urine as compared to sham-operated, pair-fed control rats. In the uremic rats diamine oxidase activity was also decreased in kidney and unchanged in liver and muscle. Total amine levels were elevated in plasma and reduced in urine of patients and rats with chronic renal failure. Although the clinical significance of abnormal diamine oxidase activity in renal failure is not clear, it is possible that this enzyme may have a pathophysiologic role in uremia.

Diamine oxidase activity was measured in plasma or urine in 12 normal men, 4 men with chronic liver or heart disease, 13 men with chronic renal failure, and 12 men undergoing maintenance hemodialysis. Also in five studies in 4 patients, plasma diamine oxidase activity and total amine levels were measured at hourly intervals during a hemodialysis treatment. Plasma diamine oxidase activity was normal in patients with liver or heart disease and was at least three times normal in chronically uremic patients and in patients undergoing maintenance hemodialysis. Plasma diamine oxidase activities before and after a hemodialysis therapy were similar and did not change during dialysis until the 4th hour when they fell transiently; plasma total amine levels, which were elevated initially, tended to rise during the 4th hour of dialysis. Urinary diamine oxidase activity was reduced in the chronically uremic patients as compared to normal subjects. These observations are consistent with three alterations in diamine oxidase in patients with renal failure: activity (a) is increased in plasma of chronically uremic patients and those undergoing maintenance hemodialysis, (b) does not increase normally in response to heparin administration during dialysis therapy, and (c) is reduced in urine of chronically uremic patients.

Vitamin B6 deficiency was evaluated in 37 patients with chronic renal failure and in 71 patients undergoing maintenance hemodialysis (HD) or intermittent peritoneal dialysis (PD). Vitamin B6 deficiency was assessed by the in vitro activity of erythrocyte glutamic pyruvic transaminase (EGPT), without (basal) and with (stimulated) the addition of pyridoxal-5-phosphate to the assay, and the EGPT index (stimulated activity ./. basal activity). Basal and stimulated EGPT activities were below normal in the HD patients, and the EGPT index was increased in each group of patients, indicating vitamin B6 deficiency. Supplemental pyridoxine hydrochloride was given to 30 HD patients who received 1.25 to 50 mg/day (37 studies), 6 PD patients who were given 1.25 or 2.5 mg/day (7 studies), and 8 nondialyzed patients with mild to severe renal failure who received 2.5 mg/ day. In all HD patients, 10 or 50 mg/day of pyridoxine hydrochloride rapidly corrected the abnormal EGPT index and maintained normal values; with supplements of 5.0 mg/day or less, the index was often abnormal, particularly in those who were septic or taking pyridoxine antagonists. In PD patients and nondialyzed patients with renal failure, 2.5 mg/day of pyridoxine hydrochloride was inadequate to correct rapidly the abnormal index in all patients. These findings suggest that HD patients should receive 10 mg/day of supplemental pyridoxine hydrochloride (8.2 mg/day pyridoxine). PD patients and patients with chronic renal failure should receive about 5.0 mg/day of supplemental pyridoxine hydrochloride (4.1 mg/day pyridoxine). When sepsis intervenes or vitamin B6 antagonists are taken, 10 mg/day of pyridoxine hydrochloride may be a safer supplement for all patients.

There is evidence that the kidney has a major role in the formation of the arginine used for extrahepatic protein synthesis. The effects of arginine-free diets were studied, therefore, in female Sprague-Dawley rats made uremic by partial left-renal artery ligation and contralateral nephrectomy. Uremic and sham-operated control rats were fed diets with amino acids proportioned as in casein or similar isonitrogenous diets in which the arginine was replaced by glutamic acid and alanine. Weight gain and the food efficiency ratio were determined, and 6 weeks after nephrectomy, a pulse dose of 14C-guanido arginine was administered. The rats were killed 2 hours later, and the radioactivity of proteins in various tissues was determined. Free arginine levels in tissues were also measured. Control rats fed diets devoid of arginine had reduced growth and a low food efficiency ratio. Free arginine levels in tissues and 14C-arginine incorporation into tissue protein in these rats were not different from controls receiving arginine except that 14C-incorporation into brain protein was decreased. Uremic rats fed an arginine-containing diet had a reduced growth rate as compared to control rats, and 14C-incorporation into brain protein was less. In uremic rats, when arginine was removed from the diet, there was no further effect on weight gain but the plasma arginine level was decreased and the incorporation of 14C-guanido arginine into protein of muscle and of kidney was reduced. Hence, the effects of an arginine-free diet appears to be different in chronically uremic as compared to control rats.

The effects of soy or collagen protein, 1.3 g/kg desirable body weight per day, on fasting and postprandial plasma free amino acid concentrations were evaluated in eight obese men during a 40-day very-low-calorie reducing regimen. The interrelationships among individual plasma amino acids were also examined. In both protein-fed groups, fasting plasma histidine, phenylalanine, tyrosine, threonine and alanine levels decreased by day 40 whereas glycine increased. The decrease in plasma threonine and increase in plasma glycine were more pronounced in the collagen-fed group (n = 4) than in the soy-fed group (n = 4). Serine increased only in the collagen-fed group. The postprandial increases of all essential amino acids, with the exception of valine and phenylalanine, were less on day 26 than on day zero. Except for threonine levels, plasma amino acid profiles were similar during very-low-calorie dieting and during prolonged fasting. However, essential amino acid levels were better maintained by soy than by collagen protein diets.

The requirement for dietary histidine was investigated in four normal and three chronically uremic men. Subjects lived in a metabolic unit where they were fed three isonitrogenous diets in the following order: a 40-g protein diet (28 plus or minus SD 8 days), a semi-synthetic amino acid diet deficient in histidine (35 plus or minus 2 days), and an amino acid diet which contained histidine (31 plus or minus 5 days). With ingestion of the histidine-deficient diet, nitrogen balance gradually became negative, and serum albumin decreased in six subjects. Plasma histidine fell by 82 plus or minus 6 per cent; muscle histidine decreased by 62 plus or minus 19 per cent; the hematocrit fell by 25 plus or minus 9 per cent; and serum iron rose. Subjects felt unwell, and in five cases a skin lesion consisting of fine scales, dry skin, and mild erythema developed. After administration of the histidine-repletion diet, nitrogen balance became positive in six subjects; serum albumin increased in five cases; plasma and muscle histidine rose; serum iron fell abruptly; a reticulocytosis ensued; and the hematocrit rose. The clinical symptoms and skin lesions disappeared. These observations indicate that histidine is an essential amino acid in normal and chronically uremic man. The absence of dietary histidine is associated with failure of normal erythropoiesis.

An experiment was conducted with rats to determine the effects on the tissue concentrations of Zn, Fe, Cu, and Mg of feeding various amounts of zinc. The rats were pair-fed one of the following diets for four weeks: Diet A, a zinc-deficient diet; a diet containing the recommended amoun of zinc (diet A plus 55 microgram of zinc per gram of diet), or diet A plus 550 microgram zinc per gram of diet. Concentrations of these elements in various tissues were determined by atomic absorption spectrometry after wet digestion. Feeding the rats zinc-supplemented diets resulted in increased zinc in blood, heart, kidney, and liver, and a marked decrease of iron in kidney and liver. Concentrations of the other two elements were unchanged in all tissues. Thus, the effect of zinc in decreasing iron concentrations in liver, observed by several investigators when rats were fed toxic amounts of zinc, also occurs when zinc is administered in normal or subtoxic amounts.

The effects of zinc supplementation on levels of various blood constituents and the outcome of pregnancy in 213 Hispanic women attending a prenatal clinic in Los Angeles was assessed in this double-blind study. The women were randomized into either a control (C) or a zinc-supplemented (Z) group and received similar vitamin and mineral supplements except that 20 mg zinc was added to the Z group's capsules. At the final interview, women (C + Z) with low serum Zn levels (less than or equal to 53 micrograms/dl) had higher (p less than 0.01) mean ribonuclease activity and lower (p less than 0.01) mean delta-aminolevulinic acid dehydratase activity than women with acceptable serum zinc levels. The incidence of pregnancy-induced hypertension was higher (p less than 0.003) in the C than in the Z group, but pregnancy-induced hypertension was not associated with low serum zinc levels at either the initial or final interview. The expected increase in serum copper levels was greater (less than 0.001) in women with pregnancy-induced hypertension (C + Z) than in normotensives. Except for pregnancy-induced hypertension, there was a higher incidence of abnormal outcomes of pregnancy in the noncompliers than in the compliers (C + Z).

A woman appeared to clinically benefit from high doses of amphetamine taken for a period of 27 years. During amphetamine administration, elevated urinary 3-methoxy-4-hydroxy-phenylglycol (MHPCG) excretion values returned to normal accompanied by reductions in plasma phenylalanine.

Biochemical measurements and 24-h dietary recalls were conducted early (18.9 +/- 5.9 wk) and late (35.1 +/- 2.0 wk) in pregnancy in women attending clinics in Montemorelos, Mexico. Mean weight gain per week (0.4 +/- 0.2 kg) and birth weight (3381 +/- 456 g) were normal. Intakes tended to decline during pregnancy and declined significantly for zinc (p less than 0.05) and vitamin B-6 (p less than 0.03). Mean Zn intake late in pregnancy was low (7.8 +/- 3.3 mg/d). Various supplements were taken but none contained Zn. During pregnancy mean plasma Zn levels fell (p less than 0.001) and late in pregnancy 57% of the women had values suggestive of poor Zn status (less than or equal to 8.1 mumol/L). These data indicate that Zn intakes of approximately 8 mg/d will not maintain plasma Zn levels in late pregnancy. Erythrocyte glutamic-pyruvic transaminase (EGPT) index and the index of diamine oxidase (DAO), a vitamin B-6-requiring enzyme of placental origin, were correlated suggesting that DAO index may be useful in evaluating vitamin B-6 status in pregnancy.

Five healthy males, age 25-32 y, were fed in sequence a diet of ordinary foods (10 d, PI), a low-methionine diet (285 mg/d, 14 d, PII), and an adequate-methionine diet (725 mg/d, 7 d, PIII). Diets contained 9 g nitrogen (N) per day with soy protein and synthetic L-amino acids as the N sources in PII and PIII. In PII, subjects were in negative N balance whereas, in PIII, four subjects were in positive N balance. On the last day of each period, fasting subjects ingested a dose of nicotinamide (NAM, 102 mumol/kg body wt). Plasma and urine samples were analyzed for methylated derivatives of NAM by high-performance liquid chromatography (HPLC) methods. Mean values of methylated metabolites in urine from the three diet periods (for four subjects in N balance during PIII) were not different (59.8, 56.7, and 59.9 mumol/(kg body wt X 24 h) for PI, PII, and PIII, respectively). Plasma values of these metabolites also were similar. Results suggest that during a 2-wk period of negative N balance due to a low-methionine intake hepatic methylation processes are not impaired. These processes appear to have a higher metabolic priority than maintenance of the net protein synthesis rate.

The activity of S-adenosyl methionine decarboxylase (SAM decarboxylase), an enzyme mediating polyamine biosynthesis, was measured in liver and kidney of chronically uremic rats and their pair-fed controls. Effects of protein intake were assessed in groups of rats fed either 8 or 18% casein or switched from 18 to 70% casein for 14 hours. In control rats, both hepatic and renal SAM decarboxylase activities increased with the higher levels of dietary casein. In uremic rats, SAM decarboxylase activity was not responsive to alterations in protein intake. Hepatic and renal SAM decarboxylase activity was significantly higher in control rats than in uremic rats, but this was due to increases in activity that occurred with diets containing higher amounts of protein (18 and 70% casein versus 8% casein).