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.

Biochemical markers for assessment of niacin status in young men: levels of erythrocyte niacin coenzymes and plasma tryptophan.

Seven male subjects housed in a controlled metabolic unit for 80 d were fed diets containing amounts of niacin and tryptophan ranging from 6.1 to 32 niacin equivalents (NE) per day. Erythrocyte nicotinamide adenine dinucleotide (NAD) and nicotinamide nucleotide phosphate (NADP), activity of nicotinic acid mononucleotide phosphoribosyltransferase (NMNPRT), plasma tryptophan levels and the urinary excretion of organic acids were measured during dietary periods of low (6.1 or 10.1), adequate (19) and high (25 or 32) NE intake. With both low NE diets, NAD levels in erythrocytes decreased by approximately 70% and increased during repletion with an adequate NE diet. NADP levels remained relatively unchanged. Plasma tryptophan levels decreased by 40% and 10% in subjects ingesting diets of 6.1 and 10.1 NE/d, respectively. A daily 7.8-g leucine supplement during repletion was not associated with changes in plasma tryptophan levels or erythrocyte NAD and NADP levels at the end of the period. No changes in NMNPRT activity or organic acid excretion were found during the study. The results indicate that the erythrocyte NAD level may serve as a sensitive indicator for the assessment of niacin status. Also, a niacin index, the ratio of erythrocyte NAD to NADP, below 1.0 may identify subjects at risk of developing a niacin deficiency.

Hepatic content of S-adenosylmethionine, S-adenosylhomocysteine and glutathione in rats receiving treatments modulating methyl donor availability.

Because of evidence linking methyl group deficiency and increased tumor formation in experimental animals, we explored other possible methods of producing a methyl group deficiency. Rats fed a low methionine diet lacking choline (MCD) were injected intraperitoneally daily for 3 wk with large doses of nicotinamide. Hepatic levels of lipids were elevated, S-adenosylmethionine (SAM) levels and the SAM:S-adenosylhomocysteine (SAH) ratio were decreased, and SAH level was not consistently changed. In livers of rats fed the MCD diet without folate (MCFD), lipids were also elevated and SAM reduced as compared to MCD-fed rats. In rats fed the MCD diet plus a methionine (Met) supplement (MCD + Met), hepatic SAM levels and the SAM:SAH ratio were higher and lipid levels lower than in MCD-fed rats, indicating that the MCD diet is marginally deficient in methyl donor groups. The injection of nicotinamide or the removal of folate from the MCD diet increased the severity of methyl donor deficiency, as shown by lower hepatic SAM levels and higher hepatic lipid levels. Hepatic glutathione levels were similar in MCD- and MCFD-fed rats and were lower than in rats fed the methionine-supplemented MCD diet or injected with nicotinamide.

Hepatic poly(ADP ribose) polymerase activity in methyl donor-deficient rats.

Hepatic poly(ADP ribose) polymerase (EC 2.4.2.30) activity as an indicator of DNA damage was measured in rats fed a low methionine, choline-devoid diet (MCD) for a 3-wk period. Additional groups of rats were either injected intraperitoneally (i.p.) with large doses of nicotinamide (NAM) or saline or fed the MCD diet without folic acid (MCFD). As a positive control, some rats were fed the MCD diet supplemented with methionine and choline (MCD + Met). In all groups of methyl donor-deficient rats and associated with increases in hepatic lipid levels, hepatic malondialdehyde concentrations were found to be increased. This observation is evidence for the occurrence of lipid peroxidation in methyl donor deficiency. Methyl donor deficiency was also associated with a significantly elevated hepatic poly(ADP ribose) polymerase activity in all groups of rats as compared to the positive control, suggesting a stimulation of DNA repair processes. The highest enzyme activity was observed in the MCD-NAM i.p. group.

Biochemical markers for assessment of niacin status in young men: urinary and blood levels of niacin metabolites.

Biochemical markers of niacin status were studied in healthy young men fed 6.1 to 32 niacin equivalents (NE) per day over an 11-wk period while residing in a metabolic unit. Methylated metabolites of niacin, N1-methylnicotinamide (NMN) and N1-methyl-2-pyridone-5-carboxamide (2-pyr), in urine and plasma were determined during periods of low (6.1 or 10.1 NE per day), adequate (19 NE per day = 1 RDA) and high (25 or 32 NE per day) niacin intakes and after small test doses of nicotinamide. Urine excretion of less than 1.2 mg/d of either NMN or 2-pyr was a reliable indicator of subjects receiving the lowest intake of 6.1 NE/d, but the NMN metabolite was a better marker of subjects ingesting 10.1 NE/d. The ratio of 2-pyr/NMN in urine was not as good a measure of the 6.1 NE/d intake as the individual metabolite excretions and was not responsive to the 10.1 NE/d intake. Plasma niacin metabolites were generally not as reliable as urinary metabolites for identifying subjects receiving low niacin intakes, however, values for plasma 2-pyr dropped quickly and were eventually nondetectable. After a 1 RDA oral dose of nicotinamide, increases in urine and plasma 2-pyr levels above pre-dose baseline values were significantly decreased in subjects receiving low, as compared to adequate, niacin intake. A leucine supplement had no effect on the rate of repletion of niacin-deficient subjects nor on the level of methylated niacin metabolites in urine or plasma.

Methylated niacin derivatives in plasma and urine after an oral dose of nicotinamide given to subjects fed a low-methionine diet.

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.

Metabolic effects of nicotinamide administration in rats.

Male rats fed a 12% casein diet without choline were injected i.p. daily for 2 or 5 weeks with either saline or 6, 20 or 60 mg of nicotinamide (NAM) per 100 g body weight. Weight gain, food intake and gain/food were lower for the NAM-treated groups compared to the controls. Urinary excretion of the major metabolite, N1-methylnicotinamide (NMN) increased with increasing dose of NAM. NAM administration did not alter the activity of hepatic nicotinamide methyltransferase. Excretion of another metabolite, N1-methyl-2-pyridone-5-carboxamide (2-PYR) was low and showed minimal changes in response to NAM administration. NAM administration did not affect urinary creatinine excretion. Livers of the NAM-injected groups were hypertrophied, and the total lipid content was increased. Kidney hypertrophy was also noted. Plasma and liver choline levels were decreased in response to NAM administration. We conclude that chronic NAM administration resulted in a methyl-group deficiency state due to the greatly increased need for methylation of NAM.

Determination of nicotinamide and metabolic products in urine by high-performance liquid chromatography.

A high-performance liquid chromatographic procedure has been developed for the quantitation of nicotinamide, nicotinic acid, nicotinuric acid, 1-methylnicotinamide and 1-methyl-2-pyridone-5-carboxamide in rat and human urines. The procedure utilizes a Varian Model 5020 liquid chromatograph with a UV detector, and an Altex 15 cm X 4.6 mm Ultrasphere-ODS column, employing a linear ion-pair mobile phase gradient. Solvent A contains 10 mM concentrations of pentanesulfonic acid (PSA), tetramethylammonium chloride (TMA) and KH2PO4, and solvent B contains PSA, TMA and acetonitrile. Different pH values for solvent A vary the retention times and thus the separation of the five compounds. Temperature of the system is critical. The conditions found most satisfactory were pH 3.30 and 24.5 degrees C.