Effects of dietary riboflavin levels on antioxidant defense of the juvenile grouper Epinephelus coioides.

This study was conducted to evaluate the effects of dietary riboflavin on antioxidant defense in the juvenile grouper Epinephelus coioides. Graded levels of riboflavin (0.9, 1.6, 4.4, 6.7, 12.9 and 19.4 mg kg(-1) dry diet) were fed to grouper juveniles (mean weight: 14.90 +/- 0.46 g) for 12 weeks. Higher levels of liver thiobarbituric acid reactive substances (TBARS) content were observed in grouper fed low doses (0.9 and 1.6 mg kg(-1) diet) of riboflavin. Both liver glutathione reductase (GR) activity and its activation coefficient (GR-AC) poorly responded to riboflavin deficiency. In addition, other indices of the glutathione-dependent defense system, including the activities of glutathione peroxidase (GSH-PX) and glutathione-S-transferase (GST), and the content of glutathione (GSH), were also non-significantly affected by dietary riboflavin levels. However, the activities of liver superoxide dismutase (SOD) and catalase (CAT) were significantly lower in fish fed 0.9 mg kg(-1) diet, with a positive correlation between the different groups. In conclusion, the present study indicated that the juvenile grouper fed the riboflavin-unsupplemented diet was susceptible to lipid peroxidation (LPO), with lower SOD and CAT activities in the liver. However, the glutathione-dependent defense system of grouper was not affected by dietary riboflavin levels.

Glutathione reductase deficiency in Saudi Arabia.

Glutathione reductase (GR) is a ubiquitous enzyme required for the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH) concomitantly oxidizing reduced nicotinamide adenine dinucleotide phosphate (NADPH) in a reaction essential for the stability and integrity of red cells. Mutations in the GR gene and nutritional deficiency of riboflavin, a co-factor required for the normal functioning of GR, can cause GR deficiency. We conducted a study on 1691 Saudi individuals to determine the overall frequency of GR deficiency and to identify whether the deficiency results from genetic or acquired causes or both. The activity of GR was measured in freshly prepared red cell haemolysate in the presence and absence of flavin adenine dinucleotide (FAD) and the activity coefficient (AC) was determined. Samples with low GR activity (> 2.0 IU/g haemoglobin) both in the presence and absence of FAD and an AC between 0.9 and 1.2 were considered GR-deficient. Samples with AC > or = 1.3 were considered riboflavin-deficient. The overall frequency of partial GR deficiency was 24.5% and 20.3% in males and females respectively. In addition, 17.8% of males and 22.4% of females suffered from GR deficiency due to riboflavin deficiency. This could be easily corrected by dietary supplementation with riboflavin. No cases of severe GR deficiency were identified.

Modulation of carcinogen-induced DNA damage and repair enzyme activity by dietary riboflavin.

Formation of single strand breaks in nuclear DNA induced by hepatocarcinogens aflatoxin B1 and N-nitrosodimethylamine was observed to be more pronounced in rats maintained on a riboflavin-deficient diet compared to that on a normal diet. This increased damage was reversed on riboflavin supplementation. The induction of repair enzymes poly(ADP-ribose) polymerase, DNA polymerase beta and DNA ligase was significantly higher in riboflavin-deficient rats following DNA damage caused by the administration of carcinogens. Riboflavin supplementation brought down the induction to the levels found in rats maintained on normal diet. Since damage to DNA and its altered repair may relate to carcinogenesis, modulation of these parameters by riboflavin suggests a potential chemopreventive role of this vitamin.

Optimal and stable conditions for the determination of erythrocyte glutathione reductase activation coefficient to evaluate riboflavin status.

To measure the activation coefficient (AC) of erythrocyte glutathione reductase (GR), suspended whole blood was lysed in a preincubation solution, with or without 100 microM flavin adenine dinucleotide (FAD). Upon addition of the reaction mixture, FAD concentration decreased about 10-fold. No AC values < 1 were obtained in any of the subjects. The range of unstimulated activity per g hemoglobin (Hb) was 5 to 12 U. AC values of healthy subjects (1.3) decreased to about 1.15 after vitamin supplementation of 1 RDA for 3 wk. In healthy young subjects consumption of dietary riboflavin at levels as low as 0.5 mg/d resulted in an AC of 1.6.

Transcriptional regulation of carnitine palmitoyltransferase synthesis in riboflavin deficiency in rats.

Riboflavin deficiency leads to depressed mitochondrial fatty acid oxidation rates but increased activity of carnitine palmitoyltransferase (CPT). Starvation leads to increased CPT activity in ad libitum-fed, riboflavin-supplemented rats. The present studies examined the mechanism of the increase in CPT activity in riboflavin deficiency and whether it was additive to that seen in starvation. Rats were divided into three groups initially: riboflavin-sufficient, ad libitum-fed; riboflavin-deficient, ad libitum-fed; and pair-fed. These groups were subdivided after 5 wk into fed and 24- and 48-h starved groups. When riboflavin-deficient rats were starved for 24 or 48 h, there was only a 30-40% increase in hepatic CPT activity, in contrast to the ad libitum-fed, riboflavin-supplemented rats, in which activity increased twofold. CPT activity of pair-fed rats was similar to that of controls in the fed state and did not increase significantly with starvation. CPT translation, mRNA levels and transcription rates correlated with CPT activity, as did immunoreactive CPT. Concurrently, hepatic ketone production and plasma beta-hydroxybutyrate concentration increased during starvation in the control and pair-fed but not in the riboflavin-deficient rats. The results indicate that increased CPT activity in riboflavin deficiency and starvation results at least in part from increased synthesis. Furthermore, the data support previous work suggesting that the block in fatty acid oxidation occurs in the beta-oxidation pathway at the level of acyl-CoA dehydrogenases.

A simple fluorimetric assay for pyridoxamine phosphate oxidase in erythrocyte haemolysates: effects of riboflavin supplementation and of glucose 6-phosphate dehydrogenase deficiency.

The formation of pyridoxal and its phosphate from pyridoxamine phosphate by red cell haemolysates was measured in a centrifugal analyser by the formation of the fluorescent adduct with semicarbazide. Pyridoxal phosphate was found to react more rapidly than pyridoxal, thus permitting a distinction between the two products, and hence the measurement of phosphatase activity. Activity of the enzyme, pyridoxamine phosphate:oxygen oxidoreductase (deaminating) EC 1.4.3.5 (PPO) was measured in haemolysates from 72 Gambian women with evidence of riboflavin deficiency, and was repeated after 6 weeks of placebo or riboflavin supplementation. Those who received the riboflavin supplement responded with a marked increase in PPO activity, which was matched by a decrease in the activation coefficient (AC) of erythrocyte NAD(P)H2:glutathione oxidoreductase, EC 1.6.4.2 (glutathione reductase, EGR). No difference between the supplemented and unsupplemented groups was observed in the capacity of haemolysates to hydrolyse pyridoxal 5-phosphate, nor in the extent of activation of erythrocyte L-aspartate:2-oxoglutarate aminotransferase EC 2.6.1.1. by pyridoxal phosphate. Although the three subjects with low levels of D-glucose 6-phosphate: NADP 1-oxidoreductase EC 1.1.1.49 (G6P-D) had, as expected, correspondingly low AC's of EGR, their unsupplemented activities of PPO were in the same low range as those of the G6P-D-normal subjects, and they responded as G6P-D-normal subjects did to riboflavin supplementation. PPO thus does not appear to resemble EGR in retaining its flavin coenzyme during riboflavin depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of a food supplement in correcting riboflavin deficiency in pregnant Gambian women.

Pregnant women living in rural Gambian villages, whose natural riboflavin intake is about 0.5 mg/d, have abnormal biochemical riboflavin status and signs of clinical deficiency. A vitamin-fortified food supplement given in one village which increased the riboflavin intake to about 1.3 mg/d was followed by a substantial improvement in biochemical status, although seasonally-related variations in status somewhat complicated the picture. It was calculated that the amount of riboflavin needed to satisfy the requirement, for normal biochemical status, of the majority of pregnant women throughout pregnancy and throughout the year, is about 2.6 mg/d. Clinical signs associated with riboflavin deficiency, especially atrophic lingual papillae, showed significantly reduced incidence in the supplemented, compared with an unsupplemented, village. Cord blood values of the activation coefficient of erythrocyte glutathione reductase were in the abnormal range for 84 per cent of infants before introduction of the supplement, but were abnormal for only one of 12 infants after its introduction. Thus even a suboptimal maternal riboflavin intake of about 1.3 mg/d appears to be sufficient to prevent biochemical deficiency in cord blood, and to reduce considerably the incidence of clinical deficiency signs during pregnancy.

Effects of riboflavin on the dimethylnitrosamine demethylase system in the rat liver.

The effects of riboflavin on the dimethylnitrosamine [(DMN) CAS: 62-75-9; N-nitrosodimethylamine] demethylase system in inbred F344 rat liver were investigated. Dietary riboflavin deficiency markedly stimulated the activity of DMN demethylase I operating at a low substrate concentration (4 mM), but it caused no change in the activity of DMN demethylase II operating at a high substrate concentration (200 mM). This effect could be reversed by short-term supplementation of the vitamin by a series of three ip injections (0.5 mg/dose/day) to the deficient animals. This dosage regimen of riboflavin did not change the activity of DMN demethylase I in the normal animals. The study in vitro demonstrated that flavins were inhibitory to DMN demethylase I. The enzyme activity in the microsomes of both control and deficient animals was inhibited by preincubation with flavins, suggesting that the interaction between flavins and the enzyme rendered the enzyme inactive. Flavin adenine dinucleotide (FAD) appeared to have a more pronounced effect than riboflavin. In agreement with the earlier observation, DMN demethylase II was unaffected by FAD or riboflavin.

FAD-induced in vitro activation of glutathione reductase in the lens of B2 deficient rats.

We studied the FAD-induced in vitro stimulation of lenticular glutathione reductase in riboflavin-deficient rats. The stimulatory effect of FAD on lenticular glutathione reductase in rats fed a B2-deficient diet for 4 weeks was remarkably higher than in paired control rats fed a B2-supplemented basal diet and control rats had ad libitum access to a B2-supplemented basal diet. The in vitro FAD stimulation effect on rat lenticular glutathione reductase represents a sensitive indicator of the B2 deficient status.

The effect of reduced dietary protein level on the activity of transketolase and glutathione reductase in pig erythrocytes.

The effect was investigated of decreased protein level in a food ration on covering the requirements for vitamins B1 and B2. Two feeding experiments were conducted, each on 32 fattening pigs of Polish Landrace breed. The activity of transketolase and glutathione reductase in erythrocytes and vitamin B1 and B2 level in blood and liver were determined. It was shown that a reduction of the diatery protein level by 25% caused a decrease of thiamine utilization by pigs. It indicates the necessity of a dietary supplementation with a synthetic vitamin B1 preparation in such a condition. A reduced activity of glutathione reductase, indicating a riboflavin deficiency, was observed only when the low protein diet was additionally deprived of a vitamin-mineral premix containing a fodder B2 preparation. Reduction of the dietary protein level by 25% was not associated with the risk of riboflavin deficiency, provided that the ration was supplemented with a standard dose of synthetic vitamin B2.

A biochemical evaluation of the erythrocyte glutathione reductase (EC 1.6.4.2) test for riboflavin status. 2. Dose-response relationships in chronic marginal deficiency.

1. Chronic marginal riboflavin deficiency was induced in groups of weanling rats by feeding a deficient diet supplemented with 0, 0.5, 1.0 and 1.5 mg riboflavin/kg diet. Ad lib.- and pair-fed controls received 3.0 and 15 mg riboflavin/kg diet respectively. 2. Serial measurement of erythrocyte NAD(P)H2 glutathione oxidoreductase (glutathione reductase; EC 1.6.4.2) and its activation coefficient revealed that after 12 weeks a steady-state of deficiency had been reached following initial fluctuations in status; the animals were then killed, and their tissues analysed. 3. Food intake, growth rate and the appearance of pathological signs were directly proportional to riboflavin content; however relative liver weight was increased above control levels only in the most-severely-deficient group, and anaemia was not detected in any group. 4. The activation coefficient of glutathione reductase in erythrocytes and liver was closely related to dietary riboflavin content; that of skin responded maximally even in the least-severely-depleted animals. 5. Hepatic and renal flavin contents were directly proportional to dietary riboflavin, FAD being conserved at the expense of riboflavin and FMN. ATP:riboflavin 5-phosphotransferase (flavokinase; EC 2.7.1.26) activity was reduced, even in the least-severely-deficient animals; ATP:FMN adenylyltransferase(FAD pyrophosphorylase; EC 2.7.7.2) was increased in liver, but only in the most-severely-deficient animals. 6. Hepatic succinate:(acceptor) oxidoreductase (succinate dehydrogenase; EC 1.3.99.1) activity fell sharply between 1.5 and 0.5 mg riboflavin/kg diet, producing an S-shaped dose-response curve; it showed smaller or less specific changes in other tissues such as brain, skin and intestine. NADH:(acceptor) oxidoreductase (NADH dehydrogenase; EC 1.6.99.3) activity declined in liver and intestine, but not in skin or brain. 7. The activation coefficient of glutathione reductase was correlated strongly with nearly all the riboflavin-sensitive variables measured, once equilibrium had been reached in this chronic deficiency model, and it was particularly strongly correlated with hepatic and renal FAD levels. Under equilibrium conditions, therefore, it appears to represent a good index of the extent of riboflavin deficiency, and significant changes in flavin levels and enzymes in the internal organs were detected even under conditions of marginal deficiency, associated with relatively small increases in the activation coefficient.

The effect of riboflavin deficiency on white cell glutathione reductase in rats.

1. The measurement of glutathione reductase in white cells from peripheral rat blood is described; the contribution from red cell glutathione reductase was virtually eliminated. 2. The activation of the white cell enzyme by flavin adenine dinucleotide (FAD) in vitro was measured during an eight week period of dietary riboflavin deficiency in weanling rats. 3. The activation increased progressively during the deficiency reaching about fifty per cent after eight weeks, at which time liver FAD levels had declined to 30% of the control levels and clinical symptoms of deficiency were beginning to appear. No increase in activation of white cell glutathione reductase was observed in pair-fed controls, over this time period. 4. These observations suggest that the measurement of the activation coefficient of white cell glutathione reductase could usefully supplement that of the red cell enzyme, in defining riboflavin status.

The effect of certain vitamin deficiencies on hepatic drug metabolism.

There is increasing evidence that the liver microsomal drug metabolizing system is affected by various vitamins such as ascorbic acid, riboflavin, and alpha-tocopherol. In regard to ascorbic acid deficiency there is a decrease in the quantity of hepatic microsomal electron transport components such as cytochrome P-450 and NADPH-cytochrome P-450 reductase, as well as decreases in a variety of drug enzyme reactions such as N-demethylation, O-demethylation, and steroid hydroxylation. In addition, young animals given high supplements of vitamin C have increased quantities of electron transport components and overall drug metabolism activities. Kinetic studies indicate no change in the apparent Km of N-demethylase, O-demethylase or hydroxylase for drug substrates in animals depleted or given high amounts of the vitamin. However, there are qualitative changes in both type I and II substrate-cytochrome P-450 binding. Ascorbic acid is not involved in microsomal lipid peroxidation or in any qualitative or quantitative change in phosphatidylcholine. Replenishing vitamin C-deficient animals with ascorbic acid required 3 to 7 days for the electron transport components and drug metabolism activities to return to normal levels. Induction with phenobarbital and 3-methylcholanthrene is not impaired in the deficient animal since drug metabolism activities are induced to the same extent as normal controls; however, the administration of delta-aminolevulinic acid, a precursor of heme synthesis, to deficient animals caused an increase in the quantity of cytochrome P-450. The effects of riboflavin deficiency on electron transport components and drug metabolism activities have been noted only in adult animals after prolonged periods of deficiency. Decreases in drug metabolism activities occur with both type I (aminopyrine and ethylmorphine) and type II (aniline) substrates. As was found with ascorbic acid deficiency, drug enzyme induction occurred to the same extent with phenobarbital in deficient and normal animals. In addition, it required from 10 to 15 days for the drug metabolism activities to return to normal levels when deficient animals were replenished with riboflavin. The effect of vitamin E on drug metabolism is specific in N-demethylase activities decrease while O-demethylase activities are not affected in the deficient state. This vitamin differs from ascorbic acid and riboflavin in that several laboratories have reported no quantitative decrease in cytochrome P-450, although there are some reports that it and delta-aminolevulinic acid dehydratase are lowered quantity of cytochrome in E-deficient animals. The effect of vitamin E, if any, on the P-450 is unresolved; an important question that requires further clarification. As with ascorbic acid there is no difference in the apparent Km of N-demethylase enzymes for varous substrates and the protective effect of vitamin E does not appear to be one of an antioxidant inhibiting microsomal lipid peroxidation.

Evaluation of methods of coenzyme activation of erythrocyte enzymes for detection of deficiency of vitamins B1, B2, and B6.

We describe optimized, ultraviolet spectrophotometric procedures for determination of erythrocyte transketolase, glutathione reductase, and aspartate aminotransferase activity, and their activation by their respective coenzymes--thiamine pyrophosphate, flavin-adenine dinucleotide, and pyridoxal-5-phosphate--as tests for vitamin B1, B2, and B6 deficiency. With these procedures we have investigated healthy subjects on normal and vitamin-supplemented diets, and a series of (mainly) alcoholic hospital in-patients. The enzyme procedures described have good precision and can be readily carried out in the routine laboratory. Abnormal transketolase activation correlated well with clinical evidence of vitamin B1 deficiency.