Effects of riboflavin deficiency upon prostaglandin biosynthesis in rat kidney.

The effects of riboflavin deficiency on the activity in vitro of prostaglandin synthetase were determined in rat kidney homogenates. For a period of two to five months, weaning rats were fed either a diet deficient in riboflavin or equal amounts of a diet identical in composition except for the addition of riboflavin at four times the RDA for this vitamin. In further experiments, each group of rats was treated for 10 days with either an inhibitor of cyclooxygenase (flurbiprofen) or buffer. Following sacrifice, prostaglandin biosynthesis in vitro was measured both in the absence and presence of reduced glutathione, and subsequently in the presence of reduced glutathione with and without flurbiprofen. Reaction products were extracted from supernatant solutions with diethylether, and the PGE2 and PGF2 alpha formed were measured by radioimmunoassay. Dietary riboflavin deficiency increased biosynthesis rates in vitro of both PGE2 and PGF2 alpha in rat renal medulla and papilla. When both control and riboflavin deficient rats were treated with flurbiprofen for a 10 day period, PGE2 biosynthesis in vitro was markedly inhibited. This inhibition of PGE2 biosynthesis was partially overcome by the addition of reduced glutathione in vitro. The addition of flurbiprofen in vitro to samples containing reduced glutathione prevented the restoration of PGE2 biosynthesis by the latter. The rate of prostaglandin biosynthesis in kidney homogenates from riboflavin deficient rats remained higher than that of controls with each experimental manipulation. These data in their entirety suggest a possible role for riboflavin in the regulation of renal prostaglandin biosynthesis in the rat.

Accelerated development of riboflavin deficiency by treatment with chlorpromazine.

The present study was undertaken to determine whether treatment with chlorpromazine accelerates the depletion of tissue stores of flavin adenine dinucleotide during dietary riboflavin deficiency. These investigations derived their impetus from earlier findings that low doses of chlorpromazine in rats fed abundant riboflavin increase urinary riboflavin excretion and reduce hepatic flavin stores. From 6 to 10 days after beginning to feed on a riboflavin-deficient diet, rats treated with chlorpromazine, 2 mg/kg body weight twice daily, had approximately twice the urinary riboflavin excretion of that of pair-fed saline-treated controls. When the riboflavin-deficient diets and chlorpromazine treatments were extended for 3 weeks and the animals killed, FAD levels in liver, kidney, and heart were markedly lower in drug-treated than in saline-treated animals. When studies were extended for 7 weeks, tissue FAD levels in saline-treated animals declined further and were equal to those of chlorpromazine-treated rats after only 3 weeks of dietary deficiency. Thus, chlorpromazine treatment accelerated urinary riboflavin loss and accelerated tissue depletion of FAD levels during dietary riboflavin deficiency. Brain levels of FAD by contrast were relatively resistant to both dietary riboflavin withdrawal and treatment with chlorpromazine. Subsequent studies showed that urinary riboflavin excretion began to increase within 6 hr of treatment with chlorpromazine. It is concluded that significant riboflavin depletion occurs following treatment with low doses of chlorpromazine, both in animals fed a normal diet and in animals fed a riboflavin-deficient diet, particularly during the early stages of deficiency.

Cardiac sensitivity to the inhibitory effects of chlorpromazine, imipramine and amitriptyline upon formation of flavins.

Chlorpromazine, imipramine and amitriptyline, drugs structurally related to riboflavin, each inhibited the formation in vivo of flavin adenine dinucleotide (FAD) from riboflavin in rat heart at 2-5 mg/kg body weight, doses comparable on a weight basis to those used clinically. All three drugs inhibited FAD formation in heart within 5 hr after a single dose of 25 mg/kg. Chlorpromazine under these conditions also inhibited FAD formation in liver, cerebrum and cerebellum. A series of psychoactive agents structurally unrelated to riboflavin did not inhibit flavin formation in the organs tested. These findings indicate that the inhibitory effects of the drugs studied have organ specificity with respect to FAD formation.

A deficiency of citrate synthase results in constitutive expression of the glyoxylate pathway in Arthrobacter pyridinolis.

Previous studies of Arthrobacter pyridinolis indicated that during the first half of the growth cycle on D-fructose, the organism utilizes a respiration-coupled transport system and exhibits glyoxylate pathway activity; during the second half of the growth cycle, a phosphoenolypyruvate:D-fructose phosphotransferase system is used for transport and no glyoxylate pathway activity is found [Pelliccione et al. (1979) Eur. J. Biochem. 95, 69--75]. A citrate-synthase-deficient mutant had the following properties: (a) high constitutive levels of glyoxylate pathway enzymes on various substrates, while such levels were only found in the wild type when it was grown on acetate; (b) acetyl-CoA levels much higher than in the wild type grown on several different substrates, whereas other metabolite levels were similar in the two strains; and (c) under conditions for induction of the phosphotransferase system, the wild type exhibited at least twice as much phosphotransferase activity as the mutant strain. A mutant lacking acetyl-CoA synthetase exhibited no induction of the glyoxylate pathway in the presence of acetate, although acetate uptake was normal. The results indicate a role for acetyl-CoA as inducer of the glyoxylate pathway. They further suggest a possible role, perhaps indirect, in repression of the phosphotransferase system.

Induction of the phosphoenolpyruvate: hexose phosphotransferase system associated with relative anaerobiosis in an obligate aerobe.

Arthrobacter pyridinolis possesses alternative transport systems for D-fructose: a respiration-coupled transport system whereby D-fructose transport occurs with concomitant oxidation of L-malate, and a phosphoenolpyruvate: D-fructose phosphotransferase system. Studies of D-fructose uptake by whole cells in the presence and absence of cyanide demonstrate that respiration-coupled transport is used almost exclusively during the first half of logarithmic growth, after which it accounts for only 15-20% of D-fructose uptake. Phosphotransferase levels are low during log phase, peak during late log, and then slowly decline. In a mutant of A. pyridinolis which requires delta-aminolevulinic acid for growth, the growth rate, cell cytochrome content, and activity of the respiration-coupled transport system increased with increasing concentrations of delta-aminolevulinic acid up to 50 microgram/ml. By contrast, phosphotransferase activity was highest in cells grown on limiting delta-aminolevulinic acid. L-Malate, which stimulates respiration-coupled transport, repressed the phosphotransferase system. The respiratory activity and the ability to release CO2 from internalized d-fructose was consistently low in D-fructose-grown cells. A cyanide-resistant cytochrome, tentatively identified as cytochrome d, appeared in the late exponential phase of growth. Isocitrate lyase activity, required for aerobic growth of this organism, declined markedly during the late exponential phase. Thus the phosphotransferase system is maximally induced, in this obligate aerobe, under conditions of relative anaerobiosis during which metabolism is primarily fermentative.