Regulation of ascorbic acid and of xylulose synthesis in rat-liver extracts. The effect of alloxan-diabetes on the glucuronic acid pathway.

1. The synthesis of l-ascorbic acid and of l-xylulose from d-glucuronolactone, d-glucuronate, l-gulonate and l-gulonolactone has been studied with liver extracts from normal and alloxan-diabetic rats. 2. In diabetic animals the synthesis of ascorbic acid is impaired, and more from glucuronolactone and glucuronate than from gulonate and gulonolactone, whereas the formation of xylulose from gulonate and gulonolactone is enhanced. These changes are reversed by insulin therapy. 3. The activity of the NAD-linked gulonate dehydrogenase is enhanced during diabetes.     

The influence of dietary protein on ascorbic acid metabolism in rats

1. d-Glucuronolactone reductase, l-gulonolactone oxidase, uronolactonase, dehydroascorbatase, l-gulonate dehydrogenase and l-gulonate decarboxylase have been measured in the tissues of rats fed on diets containing variable amounts of protein. Rats fed on a protein-free or a 2% casein diet for 15 days showed a marked decline in the activities of d-glucuronolactone reductase, l-gulonolactone oxidase, uronolactonase and dehydroascorbatase in the liver, and no change in l-gulonate dehydrogenase and l-gulonate decarboxylase activities in the kidney when compared with rats fed on diets containing 9%, 18% or 25% casein. Giving diets containing 60% or 88% casein to rats did not appreciably alter the activities of uronolactonase, dehydroascorbatase, l-gulonate dehydrogenase and l-gulonate decarboxylase, but inhibited considerably the activities of d-glucuronolactone reductase and l-gulonolactone oxidase in the liver, resulting in decreased synthesis of ascorbic acid. 2. Rats fed on a 25% casein diet showed maximal weight gain, higher tissue reserve of ascorbic acid and higher urinary excretion of both ascorbic acid and glucuronic acid when compared with rats fed on diets containing lower or higher amounts of protein.     

The effect of molybdenum on ascorbic acid metabolism in rats

1. The effect of dietary molybdenum on the growth rate and also on ascorbic acid metabolism in rats was studied. An excess of dietary molybdenum resulted in growth retardation and loss of weight. Tolerance to molybdenum was affected by the nature of the molybdenum salt administered. 2. Molybdenum ingestion altered certain aspects of ascorbic acid metabolism in rats. The conversion of d-glucuronolactone into l-ascorbic acid in vitro and the oxidative breakdown of l-ascorbic acid by liver enzymes decreased with high molybdenum intakes. The activity of liver uronolactonase was slightly inhibited. The activities of l-gulonate dehydrogenase and l-gulonate decarboxylase were not affected appreciably. 3. Molybdenum supplementation of the control diet resulted in an increase in ascorbic acid content of spleen and adrenal gland, and in a marked decrease in the urinary excretion of ascorbic acid and glucuronic acid. The implications of these findings are discussed.     

Sequestration of ascorbic acid by the larval labial gland and haemolymph of the tobacco hornworm,Manduca sexta L. (Lepidoptera: Sphingidae)

Tissues from Manduca sexta were examined for the presence of l-ascorbic acid and l-gulonolactone oxidase. l-Ascorbic acid was found in eggs, larval labial gland, haemolymph, gut, muscle, cuticle, adult nervous tissue and gonads at concentrations ranging from < 10 to > 150 mg per 100 g wet tissue. No ascorbate was detected in larval fat body and Malphigian tubule or adult salivary gland. Concentrations in labial gland and haemolymph increased 80- and 10-fold, respectively, during the fifth larval instar such that the labial gland surpassed all other tissues in ascorbate concentration. Since tissues from insects reared on an l-ascorbate-deficient diet contained no detectable vitamin C and l-gulonolactone oxidase was absent from tissue extracts, the hornworm apparently acquired l-ascorbate solely from the diet.     

Nutrition and lysosomal activity. The influence of the vitamin A status on the proteolytic activity of extracts from the livers and kidneys of rats

1. Young rats were kept for several weeks on a diet deficient in vitamin A. Some were undosed, others were given marginal (25i.u. weekly), adequate (1000i.u. weekly) or excessive (50000i.u. daily) doses of vitamin A acetate. The undosed rats developed signs of vitamin A deficiency, and the overdosed animals had skeletal fractures indicative of hypervitaminosis A. 2. The rats were decapitated. Their livers, and sometimes their kidneys, were homogenized and processed by centrifugal methods to sediment most of the lysosome fractions. Proteolytic activity was measured, after treatment with a detergent, in the whole homogenate (;total' activity), in the pellet obtained after 20min. at 15000g (;bound' activity) and, without treatment with detergent, in the supernatant (;free' activity). 3. In rats suffering from hypervitaminosis A the free activity and to a smaller extent the total activity were increased. Free activity was also raised in most rats suffering from avitaminosis A, but less than in those suffering from hypervitaminosis. 4. The vitamin A status appeared to have little effect on the proteolytic activity of the kidneys. Results for total and free activities, but not for bound activities, were higher than for corresponding liver preparations. 5. Control experiments were done on starved rats and on rats which were pair-fed with hypervitaminotic animals. Short periods of starvation caused an increase in free activity in young rats, but not in adults. The increases caused by starvation were much less than those caused by hypervitaminosis A. 6. For studies of the distribution of vitamin A more complete separation of the subcellular fractions was carried out on the combined livers from several hypervitaminotic rats. The concentration of vitamin A in the lysosome fraction was less than in the liver as a whole. 7. Our finding that the free proteolytic activity of the liver is increased by toxic oral dosing with vitamin A can be considered an extension of the previous observation that proteolytic enzymes are liberated when lysosomes are treated in vitro with vitamin A.     

Up-regulation of the intestinal 1,25-dihydroxyvitamin D receptor during hypervitaminosis D: a comparison between vitamin D2 and vitamin D3

Concentrations of intestinal 1,25-dihydroxyvitamin D receptor were measured in rats receiving pharmacological amounts (25,000 IU/rat daily for 6 days) of either vitamin D2 or vitamin D3. The data showed that both hypervitaminosis D2 and hypervitaminosis D3 resulted in significant up-regulation of intestinal 1,25-dihydroxyvitamin D receptor (fmol/mg protein) relative to controls (409 +/- 24, vitamin D2-treated; 525 +/- 41, vitamin D3-treated; and 249 +/- 19, control). The 1,25-dihydroxyvitamin D receptor enhancement also was accompanied by elevated plasma 25-hydroxyvitamin D and hypercalcemia. These data suggest that increased target-tissue 1,25-dihydroxyvitamin D receptor may play a role in enhancing target-tissue responsiveness and, thus, have a significant role in mediating the toxic effects of hypervitaminosis D.     

Role of adrenals in the vitamin A-mediated increase in the activities of gluconeogenic enzymes of rat liver.

Oral administration of large doses of vitamin A to rats even for two days was found to cause marked increase in the activities of PEP-carboxykinase, fructose-1, 6-diphosphatase, glucose-6-phosphatase and alanine aminotransferase in liver. However, overdosage of this vitamin failed to enhance the activities of these enzymes in the livers of bilaterally adrenalectomized rats. The adrenalectomy was also found to abolish the vitamin A-mediated increase in the levels of glucose and lactic acid in the blood. Thus, it is concluded that stimulation of gluconeogenesis in hypervitaminosis is, perhaps, caused by the increase in the activities of the key gluconeogenic enzymes of the liver, and that adrenal hormones are directly or indirectly involved in this process.     

Occurrence in yeast of L-galactonolactone oxidase which is similar to a key enzyme for ascorbic acid biosynthesis in animals, L-gulonolactone oxidase.

l-Galactonolactone oxidase is believed to catalyze the last step of l-ascorbic acid biosynthesis in yeast. A highly purified preparation of this enzyme from baker's yeast was obtained by a seven-step procedure. The molecular weight of the purified enzyme was estimated to be 290,000 by gel filtration, while the dissociated enzyme possessed a molecular weight of 56,000, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzes the reaction, l-galactono-γ-lactone + O₂ → l-ascorbic acid + H₂O₂. l-Gulono- and d-altrono-γ-lactone also serve as substrates. The enzyme was found to contain a flavin which is covalently bound to the enzyme protein. By comparing the properties of this enzyme with those of isofunctional enzymes of higher plants and animals, it became evident that the yeast enzyme is more like the l-gulonolactone oxidase (EC 1.1.3.8) of animals than the l-galactonolactone dehydrogenase (EC 1.3.2.3) of higher plants. Since phylogenetically lower animals are reported to lack l-gulonolactone oxidase, the finding of a similar enzyme in yeast is of great interest.     

Fine structure and cytochemistry of lysosomes in the Ito cells of the rat liver

Summary Ultracytochemical studies of the performic acid-phosphotungstic acid (PFP) reaction and acid phosphatase (ACPase) activity in the Ito cells (fat-storing cells) of the rat liver revealed two kinds of lipid droplets: one surrounded by a structure giving PFP- and ACPase-positive reactions, recognized as a lysosome, the other without such a reactive structure displaying a limiting membrane.To elucidate the function of the lysosomes surrounding lipid droplets, experiments were carried out on the following groups of animals: (1) Vitamin A-deficient rats were fed a normal diet containing vitamin A, and (2) hypervitaminosis A was experimentally induced in previously untreated rats. Lipid droplets were studied in both groups.No lipid droplets reappearing in an early stage after restoration of the regular diet were either membrane-bounded or surrounded by lysosomes. Lipid droplets surrounded by lysosomes could be seen in rats fully restored from vitamin-A deficiency and more frequently in animals suffering from hypervitaminosis A. It seems likely that as a result of the lysosomal activity in the immediate vicinity of the lipid droplets a degradation of the vitamin A-containing lipid droplets takes place in the Ito cells. Therefore, the lysosome-surrounded lipid droplets can be regarded as a sort of autophagolysosome; these lysosomes may play a role in preventing an unrestricted increase in the number and volume of lipid droplets.This work was supported by Grants-in-Aid for Co-operative Research (Nos. 437001 and 57370001) from the Ministry of Education, Science and Culture, Japanese Government     

Early effects of feeding excess vitamin A: mechanism of fatty liver production in rats

Oral administration of vitamin A (30,000 IU daily for 2 days) to young rats caused a marked increase in hepatic glycogen, cholesterol, and glycerides, while hepatic phospholipid content remained almost unaltered. In an examination of the pathogenesis of the lipid accumulation, it was found that more glucose-(14)C was incorporated into liver lipids in vitamin A-fed rats, whereas incorporation of glucose-(14)C and dl-glycine-(14)C into liver protein remained unaltered. The increase in glucose-(14)C incorporation was confined to the glyceride-glycerol portion of the lipids; incorporation into liver fatty acids was inhibited. Plasma free fatty acid concentrations were elevated. It is postulated that in the vitamin A-fed rats, increased accumulation of lipids in the liver is caused by a stimulation of fatty acid mobilization from adipose tissue and enhanced formation of glycerophosphate through glycolysis, with consequent increase in the glyceride synthesis in the liver. The weight of the adrenals was increased, whereas cholesterol concentration in the gland was decreased, after administration of vitamin A to rats. This indicates adrenocortical stimulation. Interestingly enough, vitamin A feeding did not affect either the level of liver lipids or of plasma FFA in adrenalectomized rats.     

Alterations in the lipids of bone caused by hypervitaminosis A and D

1. The total lipid, phospholipid, total and free fatty acid, free and esterified cholesterol contents of the long bones of normal, hypervitaminotic A, D and A plus D rats were determined. 2. Toxic amounts of vitamin A decreased the total fatty content, whereas toxic amounts of vitamin D increased triglycerides, esterified cholesterol and in particular the phospholipids of bone. 3. An interaction occurred between toxic amounts of vitamins A and D, which prevented, to a large extent, the alterations in bone lipids that occur in hypervitaminosis D. 4. The studies suggest an involvement of vitamin D in lipid metabolism and tend to support the idea that lipids are involved in ossification.     

Dose of 3-methylcholanthrene enhances vitamin C accumulation and mRNA expression of its transporter in the liver of ODS rats and in HepG2 cells

Polycyclic aromatic hydrocarbon (PAH) compounds including 3-methylcholanthrene induce harmful reactive intermediates and reactive oxygen species. This study reports the effect of 3-methylcholanthrene on the accumulation of vitamin C and the expression of vitamin C transporters. ODS rats were given l-ascorbic acid daily and intraperitoneal injections of 10 mg 3-methylcholanthrene in total. On day 10, vitamin C concentrations and the expression of vitamin C transporter in the tissues were measured. As a result, the levels of sodium-dependent vitamin C transporter (SVCTs) 1 and the l-ascorbic acid concentration in 3-methylcholanthrene-treated livers and hepatocytes have increased significantly. However, the content of vitamin C in the urine and TBARS in the liver have not changed. These results suggest that the administration of 3-methylcholanthrene elevates the requirement for vitamin C via (SVCTs) 1 due to xenobitics-metabolizing, such as the induction of cytochrome P450 family.     

Transfer ribonucleic acid methylases of bone. Studies on vitamin A and D deficiency

Methods were devised for the assay of tRNA methylases of rat bone. The activities of bone tRNA methylases are similar to those from other mammalian tissues. However, unlike reports on liver methylases, no inhibitors were found in the supernatant fraction from pH5 precipitate of bone extracts. The effects of vitamins A and D on the methylation of tRNA by cell-free extracts of rat bone were studied. Deficiency of either vitamin resulted in a decrease in the rate and extent of tRNA methylation, whereas the administration of vitamin A to hypovitaminotic-A rats and vitamin D to hypovitaminotic-D rats increased the rate and extent of tRNA methylation. These effects appear to be apart from changes in ribonuclease activity or in concentrations of calcium or magnesium. No evidence of inhibitors of tRNA methylases was found in bone extracts from vitamin-deficient rats nor of activators in bone extracts from deficient rats given vitamin A or D. The pattern of tRNA methylation under conditions of vitamin A or D deficiency was not changed, suggesting a generalized cellular deficiency. It was of significance to find that the specificity for methylation of specific bases in tRNA was different after the administration of vitamin A as contrasted with the effects of vitamin D. The possible significance of tRNA methylation to the biochemical action of the vitamins on bone is discussed.     

Studies on the effect of vitamin D on calcium absorption and transport

1. Mucosal cells of the small intestine obtained from rats deprived of vitamin D or given excessive amounts of the vitamin accumulated significantly more calcium than did cells from control animals. 2. Mucosal cells from vitamin D-deficient rats released less calcium than did cells from normal or hypervitaminotic D animals. 3. Studies in vivo showed that the transfer of (45)Ca from the intestine to the blood was delayed in vitamin D deficiency, but was accelerated in hypervitaminosis D. 4. The findings support the thesis that vitamin D is involved in the release of calcium rather than in its uptake by mucosal cells. 5. Further evidence is presented suggesting that uptake of calcium by intestinal mucosal cells at 0 degrees is primarily passive, whereas at 38 degrees uptake and release are effected by an active process that depends on energy derived from glycolytic activity.     

Absorption, storage and distribution of 3-dehydrovitamin A in the rat

1. The metabolism of 3-dehydroretinal was found to be similar to that of retinal. It alleviated all the symptoms of vitamin A deficiency, and promoted the growth of vitamin A-deficient rats. 2. When administered orally, 3-dehydroretinal was reduced in the intestine of the rat and subsequently esterified and transported to the liver, where it was stored mainly as the higher fatty acid ester. 3. Intraperitoneal administration of the compound led to the accumulation of 3-dehydrovitamin A in liver and other tissues. Subcutaneous administration of the compound showed a good growth response in the rat. 4. The ratio of 3-dehydroretinyl higher fatty acid ester to 3-dehydroretinol in liver, in the post-absorptive state, was nearly 93:7. 5. There was a linear relationship between the 3-dehydroretinol concentrations of blood and liver of rats. 6. Administration of 3-dehydroretinal at a dosage of 7.5mg./day for 3 days brought about hypervitaminosis A in the rat. 7. The maximal retention of 3-dehydrovitamin A by the kidneys was at an optimum dosage of 4.5mg./day for 3 days.     

Vitamin C. Biosynthesis, recycling and degradation in mammals

Vitamin C, a reducing agent and antioxidant, is a cofactor in reactions catalyzed by Cu(+)-dependent monooxygenases and Fe(2+)-dependent dioxygenases. It is synthesized, in vertebrates having this capacity, from d-glucuronate. The latter is formed through direct hydrolysis of uridine diphosphate (UDP)-glucuronate by enzyme(s) bound to the endoplasmic reticulum membrane, sharing many properties with, and most likely identical to, UDP-glucuronosyltransferases. Non-glucuronidable xenobiotics (aminopyrine, metyrapone, chloretone and others) stimulate the enzymatic hydrolysis of UDP-glucuronate, accounting for their effect to increase vitamin C formation in vivo. Glucuronate is converted to l-gulonate by aldehyde reductase, an enzyme of the aldo-keto reductase superfamily. l-Gulonate is converted to l-gulonolactone by a lactonase identified as SMP30 or regucalcin, whose absence in mice leads to vitamin C deficiency. The last step in the pathway of vitamin C synthesis is the oxidation of l-gulonolactone to l-ascorbic acid by l-gulonolactone oxidase, an enzyme associated with the endoplasmic reticulum membrane and deficient in man, guinea pig and other species due to mutations in its gene. Another fate of glucuronate is its conversion to d-xylulose in a five-step pathway, the pentose pathway, involving identified oxidoreductases and an unknown decarboxylase. Semidehydroascorbate, a major oxidation product of vitamin C, is reconverted to ascorbate in the cytosol by cytochrome b(5) reductase and thioredoxin reductase in reactions involving NADH and NADPH, respectively. Transmembrane electron transfer systems using ascorbate or NADH as electron donors serve to reduce semidehydroascorbate present in neuroendocrine secretory vesicles and in the extracellular medium. Dehydroascorbate, the fully oxidized form of vitamin C, is reduced spontaneously by glutathione, as well as enzymatically in reactions using glutathione or NADPH. The degradation of vitamin C in mammals is initiated by the hydrolysis of dehydroascorbate to 2,3-diketo-l-gulonate, which is spontaneously degraded to oxalate, CO(2) and l-erythrulose. This is at variance with bacteria such as Escherichia coli, which have enzymatic degradation pathways for ascorbate and probably also dehydroascorbate.     

24-Dehydrovitamin D is potent inhibitor of rat liver microsomal vitamin D-25-hydroxylase

A pathway has been described in the skin for the synthesis of 24-dehydrovitamin D3 (delta 24D3) from 24-dehydroprovitamin D3. The physiologic function of delta 24D3 is unknown, but has been proposed as a potential inhibitor of hepatic vitamin D-25-hydroxylase. We validated an assay for vitamin D-25-hydroxylase in rat hepatic microsomes, using nanomolar amounts of [3H]D3 as substrate, and found that delta 24D3 competitively inhibits vitamin D-25-hydroxylase activity. The apparent Ki was approximately 17 nM, indistinguishable from the Km of approximately 15 nM, suggesting that both delta 24D3 and cholecalciferol have similar affinity for the enzyme. We found no [3H]delta 24D3 in serum or liver extracts after repletion of vitamin D-depleted rats with [3H]vitamin D3 for 4 h or 6 days. A dose of 1 microgram delta 24D3 to vitamin D- and calcium-depleted rats was unable to promote any elevation in the 45Ca transport by everted duodenal sacs or to increase levels of plasma calcium: thus no evidence for biological conversion of delta 24D3 to vitamin D3 was observed. Further studies are needed to determine whether delta 24D3 is released from the skin to the circulation and is taken up by the liver, before physiological relevance can be attributed to this inhibitor.     

Investigations on vitamin D esters synthesized in rats. Detection and identification

1. Vitamin D-deficient rachitic rats were given [1-(3)H]cholecalciferol by gastric intubation. After 24hr., diethyl ether extracts of liver and kidney contained 5-11% and 4.5-20% respectively of total vitamin D apparently esterified with long-chain fatty acids. 2. A two-dimensional thin-layer chromatographic technique was devised that completely separated seven synthetic vitamin D esters according to the chain length and number of double bonds in the fatty acid component. When the ;vitamin D ester' fraction from liver or kidney was co-chromatographed with the standard esters, radioactivity appeared mainly in vitamin D palmitate, stearate, oleate and linoleate regions. The proportion of radioactivity in the saturated fatty acid esters was higher in kidney than in liver. 3. The same percentage of tissue vitamin D in the esterified form was found at each of two dosages of vitamin D. 4. The possible specificity of a vitamin D esterification mechanism is discussed.