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.

Antioxidant enzyme levels in the erythrocytes of riboflavin-deficient and Trichinella spiralis-infected rats.

The erythrocyte antioxidant enzyme levels of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of riboflavin-deficient and Trichinella spiralis-infected rats were investigated. The rats were deprived of riboflavin at the 8th week of the experiment. At that time, the erythrocyte glutathione reductase activity coefficient (EGR AC), as an indicator of riboflavin status, was > or = 1.30 in rats fed a riboflavin-deficient diet and T. spiralis-infected rats fed a riboflavin-deficient diet showed no biochemical sign of riboflavin deficiency. At the 12th week of the experiment, the levels of catalase, SOD and GSH-Px were significantly lower in the riboflavin-deficient, T. spiralis-infected, and combined riboflavin-deficient and T. spiralis-infected, rats, compared to the control group. This may have been due to an increase in free oxygen radicals caused by riboflavin deficiency and parasitic infection.

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.

Glutathione peroxidase (EC 1.11.1.9) and superoxide dismutase (EC 1.15.1.1) activities in riboflavin-deficient rats infected with Plasmodium berghei malaria.

Riboflavin deficiency interferes with the growth and multiplication of malaria parasites as well as the host response to malaria. The objective of the present work was to determine the effects of riboflavin deficiency on erythrocyte glutathione peroxidase (EC 1.11.1.9; GPx) and superoxide dismutase (EC 1.15.1.1; SOD) in rats infected with Plasmodium berghei malaria. Riboflavin in its co-enzyme form, FAD, is required by glutathione reductase (EC 1.6.4.1) to regenerate GSH and GSH is an important cellular antioxidant both in its own right and also as a substrate for the enzyme GPx. Weanling rats were deprived of riboflavin for 8 weeks before intraperitoneal injection of 1 x 10(6) P. berghei parasites. Control animals were weight-matched to the respective riboflavin-deficient group. At 10 d post-infection, parasite counts were higher in the weight-matched control group than the riboflavin-deficient group (P = 0.004). GPx activity was higher in erythrocytes of rats parasitized with P. berghei than comparable non-infected rats regardless of riboflavin status (P < 0.05). As mature erythrocytes do not synthesize new protein, the higher GPx activities were probably due to the presence of the parasite protein. In erythrocytes from riboflavin-deficient rats, GPx activity tended to be lower than in those rats fed on diets adequate in riboflavin (weight-matched controls) whether parasitized or not, but the difference was not significant. Neither riboflavin deficiency nor malaria had any effect on erythrocyte SOD activity. It was concluded that riboflavin deficiency has no marked effect on erythrocyte GPx or SOD activity in the rat.

Glutathione reductase activity, riboflavin status, and disease activity in rheumatoid arthritis.

OBJECTIVE: To measure erythrocyte glutathione reductase (EGR) activity and riboflavin status, and their relations to disease activity, in rheumatoid arthritis patients compared to healthy controls. METHODS: Patients with rheumatoid arthritis were classified as active if there were features of articular inflammation which required initiation or change of disease modifying therapy, and as inactive if they had little evidence of articular inflammation. EGR was measured in patients and healthy controls by a functional assay with or without the addition of flavin adenine dinucleotide (FAD). The ratio of stimulated EGR (with FAD added) to basal EGR (no added FAD), which measures riboflavin status, is known as the EGR activation coefficient (EGRAC). An EGRAC > or = 1.3 represents biochemical riboflavin deficiency. RESULTS: 91 patients with rheumatoid arthritis, including 57 with active disease, and 220 healthy controls were studied. Both basal and stimulated EGR were significantly higher in patients with rheumatoid arthritis (P = 0.0001) than in controls. Biochemical riboflavin deficiency was identified in 41% controls and 33% patients with active rheumatoid arthritis but was significantly less frequent (9%) in patients with inactive compared to active disease (P = 0.02) or healthy controls (P = 0.0006). Pain score, articular index, C reactive protein, and erythrocyte sedimentation rate were increased in patients with riboflavin deficiency (all P < 0.02). CONCLUSIONS: Higher basal and stimulated EGR might be expected in patients with rheumatoid arthritis in response to chronic oxidative stress due to synovial inflammation. The association of riboflavin deficiency with increased disease activity suggests that impaired EGR activity could facilitate continuing inflammation in these patients.

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.

Isoalloxazine ring of FAD is required for the formation of the core in the Hsp60-assisted folding of medium chain acyl-CoA dehydrogenase subunit into the assembly competent conformation in mitochondria.

We studied the role of FAD in the intramitochondrial folding and assembly of medium-chain acyl-CoA dehydrogenase (MCAD), a homotetrameric mitochondrial enzyme containing a molecule of non-covalently bound FAD/monomer. In the MCAD molecule, FAD is buried in a crevice containing the active center. We have previously shown that upon import into mitochondria, newly processed MCAD is first incorporated into a high molecular weight (hMr) complex and that the hMr complex mainly consisted of MCAD-heat-shock protein 60 (hsp60) complex (Saijo, T., Welch, W.J., and Tanaka, K (1994) J. Biol. Chem. 269, 4401-4408). In the present study, we incubated in vitro synthesized precursor MCAD with mitochondria isolated from normal and riboflavin-deficient rat liver for 10-60 min and fractionated the solubilized mitochondria using gel filtration. The amount of MCAD in the hMr complex was larger and that of tetramer was smaller in riboflavin-deficient mitochondria than in control at any time point. In addition, riboflavin-deficient mitochondria were solubilized after 10-min import in a buffer containing ATP and were chased in the presence of FAD, FMN, or NAD+ or without any addition. The mitochondrial proteins were analyzed using gel filtration or immunoprecipitated with anti-hsp60 antibody. After 60-min chase in the presence of FAD, the majority of MCAD in the complex with hsp60 was transferred to tetramer, whereas no such transfer occurred after the chase in the absence of FAD. When chase was done in the presence of FMN, a significant amount of MCAD was transferred from the complex with hsp60 to tetramer, but the transfer was not as efficient as in the presence of FAD. The chase in the presence of NAD+ resulted in no transfer. These data suggest that isoalloxazine ring of FAD plays a critical role, exerting nucleating effect, in the hsp60-assisted folding of MCAD subunit into an assembly competent conformation, probably assisting the formation of the core.

Acute ethanol exposure alters hepatic glutathione metabolism in riboflavin deficiency.

Since acute ethanol consumption and riboflavin deficiency each induces oxidative stress within tissues, we examined whether their combined effects compromise the major antioxidative system in liver, namely, reduced glutathione (GSH) metabolism. Four hours before sacrifice, half the riboflavin-deficient (RD) and riboflavin-sufficient (RS) rats were treated with ethanol (3 g/kg). Livers were excised and analyzed for GSH and enzymes that control its metabolism. In RD rats, GSH increased while glucose-6-phosphate dehydrogenase (G6PD) activity decreased. Ethanol had no effect on these measurements in RS rats. In RD rats, ethanol administration decreased GSH along with the activities of GSH peroxidase, glutathione reductase, and G6PD. These data suggest that riboflavin deficiency alone does not compromise hepatic GSH metabolism. By contrast, ethanol consumption together with riboflavin deficiency depletes hepatic GSH, blunts enzyme activities controlling GSH metabolism and may enhance alcohol-induced liver injury.

Is the flavin-deficient red blood cell common in Maremma, Italy, an important defense against malaria in this area?

There is a high prevalence of a familial flavin-deficient red blood cell in Ferrara province in the Po delta in northern Italy, believed to have been selected for by malaria which was endemic from the 12th century. In the present study, activities of FAD-dependent red-cell glutathione reductase (EGR) in the Grosseto area of Maremma on the west coast of Italy where malaria was endemic from 300 B.C. are compared both with activities in the Ferrara area and with activities where there was no history of endemic malaria--in the Florence area and in London in people of Anglo-Saxon origin. EGR activities were similar in Grosseto and Ferrara and were significantly lower than in Florence and London. As previously found in Ferrara, low EGR activity in Grosseto was shown to be unrelated to low dietary riboflavin intake. These findings in Grosseto, suggesting selection by malaria, are particularly interesting because, unlike the situation in Ferrara and most other malarial areas, the prevalence of thalassemia and glucose-6-phosphate dehydrogenase deficiency is very low, and they do not appear to have been selected for in Maremma. It is possible that a flavin-deficient red cell, known to inhibit growth of the malaria parasite, was an important protecting factor in the population of this area over the centuries.

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.

Low red blood cell glutathione reductase and pyridoxine phosphate oxidase activities not related to dietary riboflavin: selection by malaria?

This study was designed to confirm that low dietary riboflavin does not contribute to the flavin-deficient red blood cells commonly found in subjects in Ferrara Province, northern Italy. In this area it is primarily an inherited characteristic believed to have been selected for by malaria, which was endemic from the 12th century. In parallel with assessment of daily riboflavin intake (DRI), flavin adenine dinucleotide-dependent glutathione reductase (EGR) and flavin mononucleotide-dependent pyridoxine phosphate oxidase (PPO) were measured in beta-thalassemic heterozygotes, their normal relatives, and normal spouses (representative of the normal population). In all of these groups there is a high incidence of deficiency of these flavin enzymes. We found that the majority had an adequate riboflavin intake and there was no significant correlation of EGR and PPO activities with DRI. Thus, interpretation of low EGR activity is discussed with reference to studies of EGR done to detect nutritional riboflavin deficiency in countries where there is malnutrition and endemic malaria.

FAD-dependent regulation of transcription, translation, post-translational processing, and post-processing stability of various mitochondrial acyl-CoA dehydrogenases and of electron transfer flavoprotein and the site of holoenzyme formation.

The most prominent biochemical consequence of riboflavin deficiency in rats is a drastic decrease in various acyl-CoA dehydrogenase activities, especially that of short chain and isovaleryl-CoA dehydrogenase (IVD). As a result, oxidation of fatty acids and leucine is severely inhibited. We studied the effects of FAD at various stages of acyl-CoA dehydrogenase biogenesis. Immunoblot revealed severe losses of various acyl-CoA dehydrogenases and electron transfer flavoprotein in riboflavin-deficient rat liver mitochondria. The decreases in IVD and short chain acyl-CoA dehydrogenase were particularly severe, reaching values of 17 and 34% of controls, respectively. With the exception of IVD, the rate of in vitro transcription of the respective genes and the amounts of mRNAs of these flavoproteins in tissues increased 3-8.5-fold over controls. The amount of IVD mRNA and its transcription rate remained unchanged, suggesting that IVD expression is regulated separately from other acyl-CoA dehydrogenases. When riboflavin was depleted, in vitro translation of acyl-CoA dehydrogenase and electron transfer flavoprotein alpha-subunit mRNAs was moderately inhibited. Translation of non-flavoproteins was also inhibited. The stability of precursor acyl-CoA dehydrogenases and their mitochondrial import/processing were unaffected. However, mature acyl-CoA dehydrogenases degraded markedly faster in deficient mitochondria than in controls. Regardless of whether precursors were translated under riboflavin-depleted or riboflavin replete conditions, mature acyl-CoA dehydrogenases survived well when imported into normal mitochondria but degraded faster when imported into deficient mitochondria. These findings indicate that FAD ligand binds to mature acyl-CoA dehydrogenase inside the mitochondria.

Riboflavin deficiency is associated with selective preservation of critical flavoenzyme-dependent metabolic pathways.

Riboflavin is a water soluble vitamin that serves as a precursor of flavin mononucleotide and flavin adenine dinucleotide. These two compounds are coenzymes in a variety of electron transfer reactions that occur in energy producing, biosynthetic, detoxifying and electron scavenging pathways. When an organism is confronted with inadequate dietary riboflavin, characteristic changes occur in the cellular distribution of the various flavin fractions as well as in the activities of flavin-dependent enzymes. These changes suggest a specific hierarchic response to riboflavin deficiency, e.g. the core electron transfer chain required for ATP synthesis is preserved while the enzymes required for the first step of fatty acid beta-oxidation are diminished. The mechanisms by which the specific changes in enzyme activity are mediated have not been completely identified, but appear to result from a combination of diminished access of normal or near normal levels of apoenzyme to coenzyme and diminished abundance of apoenzyme. The changes in apoenzyme content potentially result from alterations in either protein stability or gene expression. The response to riboflavin deficiency of several key enzyme systems and the pathways affected will be discussed and a hierarchic order by which specific enzyme activities are preserved while others are decreased will be proposed. The current understanding of the molecular mechanisms by which these changes are mediated will be discussed.

Effect of vitamin B2 deficiency on rat liver dihydropyrimidine dehydrogenase activity.

Effect of vitamin B2 deficiency on rat liver dihydropyrimidine dehydrogenase was investigated. It was found that the purified enzyme contains 2 mol flavin per molecule, which consists of equal proportions of flavin-adenine dinucleotide (FAD) and riboflavin 5'-phosphate (FMN). When rats were fed on a vitamin B2-deficient diet for 5 weeks, dihydropyrimidine dehydrogenase activity in the liver was diminished, followed by a decrease in enzyme concentration. Moreover, the addition of exogenous FAD or FMN did not restore the activity. Thus endogenous flavin may regulate the enzyme half-life or synthesis. Lowering of dihydropyrimidine dehydrogenase activity in the livers of rats fed on a vitamin B2-deficient diet did not affect the uridine, uracil and sigma UMP (the sum of acid soluble uracil 5'-nucleotides) pool in liver.

Enhanced depletion of lens reduced glutathione Adriamycin in riboflavin-deficient rats.

The anticancer drug Adriamycin has photosensitizing properties which potentially may be detrimental to lens tissue. Since reduced glutathione (GSH) serves to protect lens from photo-oxidative stress and dietary riboflavin is required by glutathione reductase to regenerate GSH, we investigated whether Adriamycin intensifies the depletion of GSH levels in rat lens during dietary riboflavin deficiency. Three-week-old rats were divided into two groups. One group was fed a diet deficient in riboflavin (less than 1 ppm) and the other group was pair-fed a control diet containing adequate riboflavin (8.5 ppm). After 6-12 weeks of dietary treatment, half the animals in each dietary group received Adriamycin (8 mg/kg/day) intraperitoneally for 3 days. After killing the rats, lenses were removed, and GSH content and glutathione reductase activity were measured in freshly prepared homogenates. To determine the extent of systemic oxidative stress and the degree of riboflavin deficiency, glucose-6-phosphate dehydrogenase and glutathione reductase activities, respectively, were measured in erythrocytes. In lens of rats fed the riboflavin-sufficient diet, treatment with Adriamycin did not diminish GSH content or alter glutathione reductase activity. In confirmation of reports by others, lenses of animals fed the riboflavin-deficient diet had diminished GSH levels, lower basal glutathione reductase activity, and elevated glutathione reductase activity coefficients compared to those of animals pair-fed the control diet. The present study shows that in riboflavin-deficient rats, Adriamycin exacerbated the depletion of GSH but did not reduce further glutathione reductase activity. The implications of these findings are that nutritional deficiencies, in particular riboflavin deprivation, may pose a potential risk to lenticular tissue following Adriamycin treatment.

Riboflavin deficiency in cultured rat hepatoma cells: a model for studying the hepatic effects of riboflavin deficiency.

The acyl-CoA dehydrogenases are a family of mitochondrial flavoenzymes required for fatty acid beta-oxidation and branched-chain amino acid degradation. The hepatic activity of these enzymes, particularly the short-chain acyl-coenzyme A (CoA) dehydrogenase, is markedly decreased in riboflavin deficient rats. We now report that the in vivo effects of riboflavin deficiency on the beta-oxidation enzymes of this group are reproduced in FAO rat hepatoma cells cultured in riboflavin-deficient medium. Although it has been long known that hepatic short-chain acyl-CoA dehydrogenase activity is the most severely affected of the straight-chain specific enzymes in riboflavin deficiency, the mechanism by which its activity is decreased has not been reported. We have used this new cell culture system to characterize further this mechanism. Whole cell extracts from riboflavin-deficient and control cells were subjected to analysis by denaturing polyacrylamide gel electrophoresis. The contents of the gels were then electroblotted onto nitrocellulose filters and probed with short-chain acyl-CoA dehydrogenase-specific antiserum. The relative abundance of enzyme antigen was estimated autoradiographically. Our findings indicate that short-chain acyl-CoA dehydrogenase activity changes in parallel with its antigen, suggesting that riboflavin deprivation does not affect the activity of individual enzyme molecules. Further, no evidence of extramitochondrial enzyme precursor was found on the blots, making unlikely a significant block in the mitochondrial uptake process. These findings suggest that changes in short-chain acyl-CoA dehydrogenase activity in riboflavin deficiency result from either increased synthesis or decreased degradation of the enzyme.

Altered acyl-CoA metabolism in riboflavin deficiency.

We have recently described the effects of riboflavin deficiency on the metabolism of dicarboxylic acids (Draye et al. (1988) Eur. J. Biochem. 178, 183-189). As both mitochondria and peroxisomes are thought to be involved, we have examined the activities of various enzymes in these organelles in the livers of riboflavin-deficient rats. Mitochondrial beta-oxidation of fatty acids was severely depressed due to loss of activity of the three fatty acyl-CoA dehydrogenases, whereas there was an enhancement of peroxisomal beta-oxidation due to an increased activity of the FAD-dependent fatty acyl-CoA oxidase, although the activities of other peroxisomal flavoproteins, D-amino acid oxidase and glycolate oxidase, were lowered. Hepatocyte morphometry revealed an increase in the numbers of peroxisomes, indicating a proliferation induced by the deficiency. The mitochondrial acyl-CoA dehydrogenases involved in branched-chain amino acid metabolism were also severely decreased leading to characteristic organic acidurias. There was some loss of activity of the flavin-dependent sections of the electron transport chain (complexes I and II), but these were probably not sufficient to affect normal function in vivo. The specificity of these effects allows the use of the riboflavin-deficient rat as a model for the study of dicarboxylate metabolism.

[Mechanism of riboflavin deficiency facilitating carcinogenesis of N-nitrosamine--effect on carcinogen-metabolizing enzymes].

Previous studies have demonstrated the facilitating effect of riboflavin deficiency on the carcinogenesis of the liver of rats induced by N-nitrosamine. However, the mechanism was still not clear. In the present investigation, the alterations of microsomal carcinogen-metabolizing enzymes of rat liver during riboflavin-deficiency with simultaneous administration of nitrosodimethylamine (NDMA) were studied. The results showed that the enzyme activities of hepatic microsomal cytochrome P-450 and NDMA demethylase of riboflavin deficient rats and riboflavin deficient rats receiving NDMA were increased and significantly different from the control rats (P less than 0.05). The enzyme activities of hepatic microsomal NADPH-cytochrome P-450 reductase of riboflavin deficient rats and riboflavin deficient rats receiving NDMA were significantly decreased (P less than 0.01). All the alterations disappeared after supplying riboflavin to the deficient rats. This result indicates that the effect of riboflavin deficiency on carcinogen-metabolizing enzymes of rat liver is reversible.

Riboflavin status in Saudi Arabia--a comparative study in different regions.

Riboflavin (vitamin B2) status was investigated in male and female Saudis of different age groups in different regions of Saudi Arabia using the assay of glutathione reductase (GR) in presence and absence of flavin adenine dinucleotide (FAD) and determination of the activity coefficient (AC) for GR. The overall prevalence of riboflavin deficiency as determined by an AC value of 1.3 or above, in the male population of Al-Hafouf, Jaizan and Riyadh was found to be 32.6%, 17.8% and 17.1%, respectively, while in the female population the prevalence was 41.5%, 22.3% and 37.9%, respectively. Within each region a marked variation was found in the different age groups. In this paper the riboflavin status and the prevalence of riboflavin deficiency in three regions of Saudi Arabia is presented and the possible causes for a high prevalence of riboflavin deficiency in the Saudi population are discussed.

Effects of riboflavin deficiency and clofibrate treatment on the five acyl-CoA dehydrogenases in rat liver mitochondria.

Rats were maintained on a riboflavin-deficient diet or on a diet containing clofibrate (0.5%, w/w). The activities of the mitochondrial FAD-dependent straight-chain acyl-CoA dehydrogenases (butyryl-CoA, octanoyl-CoA and palmitoyl-CoA) and the branched-chain acyl-CoA dehydrogenases (isovaleryl-CoA and isobutyryl-CoA) involved in the degradation of branched-chain acyl-CoA esters derived from branched-chain amino acids were assayed in liver mitochondrial extracts prepared in the absence and presence of exogenous FAD. These activities were low in livers from riboflavin-deficient rats (11, 28, 16, 6 and less than 2% of controls respectively) when prepared in the absence of exogenous FAD, and were not restored to control values when prepared in 25 microM-FAD (29, 47, 28, 7 and 17%). Clofibrate feeding increased the activities of butyryl-CoA, octanoyl-CoA and palmitoyl-CoA dehydrogenases (by 48, 116 and 98% of controls respectively), but not, by contrast, the activities of isovaleryl-CoA and isobutyryl-CoA dehydrogenases (62 and 102% of controls respectively). The mitochondrial fractions from riboflavin-deficient and from clofibrate-fed rats oxidized palmitoylcarnitine in State 3 at rates of 32 and 163% respectively of those from control rats.