Identification of Arsenobetaine,a Water Soluble Organo-arsenic Compound in Muscle and Liver of a Shark,Prionace glaucus

After male rats of the Sprague Dawley strain, 5 weeks old, were fed a 20% casein diet for 12 days, 70 mg of streptozotocin/kg body weight (STZ group) or 70 mg of streptozotocin and 500 mg of nicotinamide/kg body weight (STZ-Nam group) was injected intraperitoneally into the rats. The rats were kept for 21 more days on the 20% casein diet and killed by decapitation. Urine was collected for the last 2 days. The level of blood glucose was 2-fold higher in the STZ group than in the STZ-Nam group. Urinary excretion of large amounts of glucose was observed only in the STZ group. Extremely reduction of urinary excretion of nicotinamide was observed in the STZ group, but, urinary excretion of N¹-methylnicotinamide (MNA) and N-¹-methyl-2-pyridone-5-carboxamide (2-py) was about the same in the two groups and that of N¹-methyl-4-pyridone-3-carboxamide (4-py) was higher in the STZ group than in the STZ-Nam group. The sum of urinary excretion of nicotinamide, MNA, 2-py, and 4-py was higher in the STZ group than in the STZ-Nam group. The levels of NAD in liver, pancreas, and blood in the STZ group tended to be higher, or rather not to decrease compared to the STZ-Nam group. For enzyme activities concerned with the tryptophan-NAD metabolism, a marked increase was observed in the activities of aminocarboxymuconate-semialdehyde decarboxylase, 3-hydroxyanthranilic acid oxygenase, and nicotinamide methyltransferase, on the other hand, the activity of NAD⁺ synthetase decreased in the STZ group compared to the STZ-Nam group. The activities of tryptophan oxygenase, kynureninase, NMN adenylyltransferase, and MNA oxidase were about the same in the two groups. These changes in the above enzyme activities mean that the conversion ratio from tryptophan to NAD is lower in the streptozotocin diabetic rats than normal rats, but the tryptophan metabolites such as NAD and 4-py were higher in the STZ group than in the STZ-Nam group. This might be due to the higher food intake and the lower body weight gain in the STZ group than in the STZ-Nam group. Similar phenomena have reported in alloxan diabetic rats.     

Evidence for an Ester Linkage between Lignin and Glucuronic Acid in Lignin–Carbohydrate Complexes by DDQ-Oxidation

The effects of ethanol feeding on the tryptophan-niacin metabolism were investigated in rats. Male rats of the Wistar strain (3 weeks old) were fed with a 20% casein diet and 15% ethanol ad libitum for 56 days. Urine samples were collected every week during the experimental period. Urinary excretion of N¹-methylnicotinamide (MNA) was always higher in the ethanol-fed group than in the control group, but urinary excretion of its oxidized metabolites N¹-methyl-2-pyridone-5-carboxamide (2-Py) and N¹-methyl-4-pyridone-3-carboxamide (4-Py) was always lower. Therefore, the ratio of (2-Py + 4-Py)/MNA excretion was lower in the ethanol-fed group than in the control group. The rats were killed on day 57 and liver enzyme activities involved in the tryptophan-niacin metabolism were also measured. Tryptophan oxygenase, kynureninase, nicotinamide mononucleotide adenylyltransferase, NAD⁺ synthetase, and nicotinamide methyltransferase activities were similar in both groups, but, 2-Py-forming MNA oxidase and 4-Py-forming MNA oxidase activities in the ethanol-fed group were 43% and 18% of the control, respectively. Therefore, the increase in MNA excretion and the decrease in the ratio of (2-Py + 4-Py)/MNA excretion might be attributed to the inhibition of the two MNA oxidase activities by ethanol feeding. Furthermore, it happened to be found that this excretion ratio also increased with growth up to nine weeks and this change was attributed to the increased reaction MNA → 4-Py with growth.     

Comparison of the Activity of the Tryptophan-NAD Pathway between Rats Fed a Fat-free Diet and a Fat Diet

The effect of dietary fat on tryptophan-NAD metabolism was investigated. Weanling male rats of the Sprague Dawley strain were fed a 40% casein diet (nicotinic acid-free) with or without 20% fat for 13 days. Although the food intake in 13 days was significantly higher in the fat-free group than in the fat group, the gains in body weight in the two groups were almost the same, because of the same energy intakes. The urinary excretion of tryptophan metabolites such as quinolinic acid, niacin and N¹-methylnicotinamide was greatly increased in the fat group in comparison with that in the fat-free group. The urinary excretion of xanthurenic acid was almost the same in the two groups. The blood NAD level of the fat group was significantly increased. The activities of liver amino-carboxymuconate-semialdehyde decarboxylase and liver nicotinamide methyltransferase in the fat group were significantly reduced, and that of liver NMN adenylyltransferase was significantly increased. The changes of these three enzymes could be advantageous for the increased formation of NAD from tryptophan. As a result, the feeding of a high fat diet to rats increased the formation of niacin and niacin-related compounds.     

Effects of Peroxidation Products of Linoleic Acid on Tryptophan–nicotinamide Metabolism in Rats

The flavin and pyridine nucleotide coenzymes are involved in the detoxication of autoxidation products of lipids. In tryptophan-nicotinamide metabolism, kynurenine 3-hydroxylase and N¹-methylnicotinamide (MNA) oxidase contain FAD as a coenzyme. So, the effects of dietary autoxidation products of linoleic acid on the metabolism of tryptophan-nicotinamide were investigated using rats. The administration of linoleic acid hydroperoxides or secondary products reduced the urinary excretion of xanthurenic acid, nicotinamide and its metabolites such as MNA, N¹-methyl-2-pyridone-5-carboxamide (2-Py), and N¹-methyl-4-pyridone-3-carboxamide (4-Py) as compared with the group administered saline or linoleic acid. Among the enzyme activities involved in the tryptophan-nicotinamide metabolism, the activity of NAD⁺ synthetase was decreased by the administration of linoleic acid hydroperoxides or secondary products. The activities of tryptophan oxygenase and 4-Py-forming MNA oxidase were also decreased by the administration of secondary products. These results indicate that the conversion of tryptophan to nicotinamide would be lower in the groups administered the hydroperoxides and secondary products than in saline and linoleic acid groups.     

Enzyme activities along the tryptophan-nicotinic acid pathway in alloxan diabetic rabbits

Recent data from our laboratory have indicated that the rabbit is a suitable animal model for the study of enzyme activities of the tryptophan-nicotinic acid pathway. We report here the pattern of tryptophan metabolism in rabbits made diabetic with alloxan treatment, and hypercholesterolemic with a high-cholesterol diet. A group of rabbits with only hypercholesterolemia was also considered. The enzymes assayed were: liver tryptophan 2,3-dioxygenase (TDO), intestine indoleamine 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase.TDO showed a reduction of specific activity in liver of diabetic-hyperlipidemic and hyperlipidemic rabbits compared to controls. Intestine IDO activities and liver and kidney kynurenine monooxygenase were unchanged with respect to controls.Kynurenine-oxoglutarate transaminase and kynureninase activities were reduced in the kidneys, but not in the liver, of diabetic-hyperlipidemic rabbits.The main finding was the reduction of 3-hydroxyanthranilate 3,4-dioxygenase activity (expressed as activity per g of fresh tissue) in the liver and kidneys of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared to controls. Conversely, aminocarboxymuconate-semialdehyde decarboxylase activity was significantly higher in diabetic hypercholesterolemic rabbits in comparison with control and hypercholesterolemic rabbits.These data demonstrate that also in diabetic rabbits there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid-->nicotinic acid step. Also dyslipidemia seems to be involved in enzyme activity variations of the tryptophan metabolism along the kynurenine pathway.     

A Novel Sesquiterpene,Tuberonone, from Solanum tuberosum L.

The effects of the ovarian hormones progesterone and estrone on the conversion of tryptophan to nicotinamide in rats were investigated. Female rats were fed for 35 days with a 20% casein diet, or with a 20% casein diet containing 0.1% progesterone, or 0.001% estrone, or 0.1% progestrone and 0.001% estrone. The conversion ratio of tryptophan to nicotinamide on the last day of the experiment was 2% in the groups fed with the 20% casein diet and the diet containing 0.1% progesterone, but around 1.2% in the group fed with 0.001% estrone, and 0.7% in the group fed with 0.1% progesterone and 0.001% estrone. These results demonstrated that administration of ovarian hormones significantly decreased the conversion of tryptophan to nicotinamide.     

Effects of Dietary Pyrazinamide on the Metabolism of Tryptophan to Niacin in Streptozotocin-diabetic Rats

We investigated the effects of feeding with a diet containing pyrazinamide (PYR) on the metabolism of l-tryptophan (Trp) to nicotinamide in streptozotocin (STZ)-diabetic rats and whether the diabetic action of STZ is prevented by feeding with the PYR diet, which is known as an inhibitor of aminocarboxy-muconate-semialdehyde decarboxylase and poly(ADP)ribose synthetase and therefore, significantly increases the formation of nicotinamide from Trp in normal rats. As was expected, feeding with the PYR diet to the STZ-injected rats caused a significantly increased excretion of nicotinamide and its metabolites like that in normal rats. The body weight increased in the STZ-injected rats fed with the PYR diet, while it was lost in the STZ-injected rats fed with the non-PYR diet. However, the blood glucose level and the urinary excretion of glucose were not improved even when the rats were fed with the PYR diet. Therefore, it was suggested that chronically increasing the formation of nicotinamide from Trp could not completely prevent the STZ-diabetic action.     

Induction of rat liver glycine methyltransferase by high methionine diet

Dietary experiments were carried out to evaluate the physiological role of glycine methyltransferase. When rats received a 18% casein diet containing excess methionine, the activity of the enzyme in liver extracts increased with increasing methionine content in the diet. Adenosylmethionine synthetase and adenosylhomocysteinase activities were also elevated, while guanidoacetate methyltransferase activity showed no significant change. The glycine methyltransferase activity reached a maximal level after 4–6 days on the 3% methionine diet. Immunological titration showed that the increase in activity was associated with the increase in amount of the enzyme.     

Tryptophan catabolism in Bacillus megaterium.

Bacillus megaterium grows in a medium containing L-tryptophan as the sole carbon, nitrogen, and energy source. Kynurenine, anthranilic acid, and catechol are metabolic intermediates, suggesting that this organism used the anthranilic acid pathway for tryptophan degradation. Cells that grow on L-tryptophan oxidize kynurenine, alanine, and anthranilic acid and the presence of tryptophan oxygenase (EC 1.13.1.12), kynureninase (EC 3.7.1.3), and catechol oxygenase (EC 1.13.1.1) in cell extracts provide additional evidence for the degradative pathway in B. megaterium. Tryptophan oxygenase is inhibited by sodium azide, potassium cyanide, and hydroxylamine, indicating that the enzyme has a functional heme group. D-Tryptophan is not a substrate for tryptophan oxygenase, and the D-isomer does not inhibit this enzyme. Formamidase (EC 3.5.1.9) and anthranilate hydroxylase are not detectable in extracts. Tryptophan catabolism is inducible in B megaterium and is subject to catabolite repression by glucose and glutamate. Arginine does not cause repression, and kynurenine induces both tryptophan oxygenase and kynureninase.     

Intrauterine Malnutrition and the Brain: Effects on Enzymes and Free Amino Acids Related to Glutamate Metabolism

Abstract: The effect of feeding pregnant rats with wheat and Bengal gram (black chick pea) diets during the later part of pregnancy on brain growth, enzymes, and free amino acids of glutamate metabolism in 1-day-old rats was investigated. These diets did not induce growth dissociation, and the body and brain weights were equally affected. The concentrations of DNA, RNA, protein, and free α-amino nitrogen in brain decreased significantly and the activities of glutamine synthetase, glutamine transferase, glutaminase 1, glutaminase 11, and glutamate decarboxylase and the concentrations of free amino acids, glutamic acid, glutamine, alanine, and GABA were also decreased. The concentration of aspartic acid, however, was increased. Wheat and Bengal gram diets fortified with lysine and with methionine, cystine, and tryptophan respectively showed various beneficial effects on the changes observed in the brain. A 20% casein diet induced higher body and brain weights and better brain protein and free α-amino nitrogen concentrations than those observed on a 10% casein diet.     

The necessity of niacin in rats fed on a high protein diet

It is known that niacin itself is not necessary in rats when tryptophan is given in adequate amounts, because rats can biosynthesize niacin from tryptophan. In our experiment, young rats were fed on a 20%, 40%, 60%, or 70% casein diet with or without niacin. The rats fed on the 20%, 40%, and 60% casein diets did not require niacin for growth, but the rats fed on the 70% casein diet needed it. This phenomenon was attributed to the supposition that liver aminocarboxymuconate-semialdehyde decarboxylase activities increased according with the dietary casein levels. The conversion ratio of tryptophan-niacin in rats fed on the 70% casein diet became extremely low, and then the rats needed niacin.     

EFFECTS OF DIETARY PROTEINS ON FETAL BRAIN PROTEIN AND GLUTAMIC ACID METABOLISM IN RAT

Abstract— The effects of feeding dietary wheat and Bengal gram proteins to pregnant rats on brain protein and glutamic acid metabolism in 15-, 17- and 19-day fetuses were investigated. Wheat and Bengal gram diets resulted in loss of brain weight with decreased DNA, RNA, protein, free x amino N and deficits in the activities of brain glutamine synthetase, glutaminase I. glutaminase II and glutamate decarboxylase at all the gestational ages studied without any change in glutamine transferase activity. The concentrations of the amino acids alanine, glutamic acid, glutamine and GABA were found to be significantly lower on wheat and Bengal gram diets than the control on a 10% casein diet. The wheat with lysine and Bengal gram with methionine, cystine and tryptophan resulted in similar mean values of all the characteristics studied to the mean values observed in rats on the control diet. However, glutaminase I activity remained significantly low on lysine fortified wheat diet, and aspartic acid content was found to increase on both fortified and unfortified wheat and Bengal gram diets. A 20% casein diet showed increased brain weight, DNA. RNA. protein and free x amino N concentrations as compared with the 10% casein diet, while the other parameters remained unchanged.     

Properties and Structure of a Novel Peptide Antibiotic No. 1907

There are three NAD biosynthetic pathways: the nicotinic acid-NAD, nicotinamide-NAD, and quinolinic acid (derived from tryptophan)-NAD pathways. To discover the main pathways of NAD biosyntheses in various tissues of the rat, the tissue distribution of nicotinamidase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase, nicotinamide phosphoribosyl-transferase, nicotinamide mononucleotide adenylyltransferase, and NAD⁺ synthetase were investigated. All of the tissues could synthesize NAD from nicotinamide, judging from that the activities of nicotinamide phosphoribosyltransferase and NMN adenylyltransferase detected in all of the tissues. From nicotinic acid, only liver, kidneys, and heart could. Liver and kidney can also synthesize NAD de novo from quinolinic acid.     

Effects of fatty liver induced by niacin-free diet with orotic acid on the metabolism of tryptophan to niacin in rats

The effects of dietary orotic acid on the metabolism of tryptophan to niacin in weaning rats was investigated. The rats were fed with a niacin-free, 20% casein diet containing 0% (control diet) or 1% orotic acid diet (test diet) for 29 d. Retardation of growth, development of fatty liver, and enlargement of liver were observed in the test group in comparison with the control group. The concentrations of NAD and NADP in liver significantly decreased, while these in blood did not decrease compared to the control group. The formation of the upper metabolites of tryptophan to niacin such as anthranilic acid, kynurenic acid, and 3-hydroxyanthranilic acid were not affected, but the quinolinic acid and beyond, such as nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, were significantly reduced by the administration of orotic acid. Therefore, the conversion ratio of tryptophan to niacin significantly decreased in the test group in comparison with the control group.     

Alteration of Tryptophan-mediated Regulation in Neurospora crassa by Indoleglycerol Phosphate

The accumulation of imidazoleglycerol phosphate during growth of Neurospora crassa in the presence of 3-amino-1,2,4-triazole was found to cause derepression of tryptophan synthetase and to inhibit the induction of kynureninase. Accumulation of indoleglycerol phosphate in response to growth in the presence of indole acrylic acid or anthranilic acid was also accompanied by derepressed synthesis of tryptophan synthetase. Enzyme synthesis in mutants (his-7 and trp-4) unable to form these intermediates was not altered under similar conditions. The rate of formation of tryptophan synthetase and kynureninase was found to differ in the presence of tryptophan and indole.     

The effect of pyrazines on the metabolism of tryptophan and nicotinamide adenine dinucleotide in the rat Evidence of the formation of a potent inhibitor of aminocarboxy-muconate-semialdehyde decarboxylase from pyrazinamide

The intraperitoneal or oral administration of pyrazinamide and pyrazinoic acid (pyrazine 2-carboxylic acid) resulted in a marked increase of the NAD content in rat liver. The injections of pyrazine and pyrazine 2,3-dicarboxylic acid exhibited no significant effect on the hepatic NAD content. The boiled extract obtained from liver and kidney of rat injected with either pyrazinamide or pyrazinoic acid exhibited a potent inhibitory effect on the aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) activity in either liver or kidney, although pyrazinamide or pyrazinoic acid per se did not inhibit the enzyme activity. The unknown inhibitor of aminocarboxymuconate-semialdehyde decarboxylase was dialysable and heat-stable, and mostly excreted in urine by 6 and 12 h after injection of pyrazinoic acid and pyrazinamide, respectively. Pyrazine 2,3-dicarboxylic acid, pyrazine, nicotinamide, nicotinic acid, tryptophan, anthranilic acid, 5-hydroxyanthranilic acid and quinolinic acid exhibited no significant effect on the aminocarboxymuconate-semialdehyde decarboxylase activity in liver and kidney at the concentration of 1 mM in the reaction mixture. The expired ¹⁴CO₂ from l-[benzen ring-U-¹⁴C]tryptophan was markedly decreased by the pyrazinamide injection, while the urinary excretion of ¹⁴C-labeled metabolites from l-tryptophan, mainly quinolinic acid, was markedly increased. These results suggest that the glutarate pathway of l-tryptophan was strongly inhibited by the inhibitor produced after the administration of pyrazinoic acid and pyrazinamide. Pyrazinamide but not pyrazinoic acid also exhibited a significant inhibition of the nuclear enzyme poly(ADP-ribose) synthetase in rat liver.     

Cloricromene effect on the enzyme activities of the tryptophan-nicotinic acid pathway in diabetic/hyperlipidemic rabbits

Since alterations of tryptophan metabolism have been reported in diabetes and atherosclerosis, it was thought of interest to investigate any role of cloricromene through the influence on the oxidative metabolism of the amino acid by using diabetic/hyperlipidemic rabbits.Male 4-month-old New Zealand white rabbits, fed a diet enriched with 1% cholesterol and 10% corn oil, were made diabetic with alloxan. During the hyperlipidemic diet, a group of rabbits was treated with cloricromene (10 mg/kg/day subcutaneously plus 1.5 mg/kg/day intravenously, for 5 weeks). The other group received saline. Normometabolic New Zealand rabbits fed standard diet, treated or not with cloricromene, were used as control.The specific activities of liver tryptophan 2,3-dioxygenase and small intestine indole 2,3-dioxygenase were not significantly changed by the drug treatment. Also the specific activities of other enzymes of the kynurenine pathway in the liver and kidneys, specifically kynurenine 3-monooxygenase, kynureninase and kynurenine-oxoglutarate transaminase, did not show any significant difference in both tissues between the two groups of rabbits. On the contrary, 3-hydroxyanthranilate 3,4-dioxygenase activity in the liver of diabetic/hyperlipidemic rabbits and control rabbits treated with cloricromene showed a slight increase in comparison with untreated animals. Conversely, the specific activity of the enzyme in kidneys was not affected by the drug treatment in diabetic/hyperlipidemic animals but was reduced in controls. Aminocarboxymuconate-semialdehyde decarboxylase specific activity remained unchanged in the liver following cloricromene treatment, instead the specific activity of the enzyme in the kidneys of the diabetic/hyperlipidemic rabbits was significantly increased by the drug, with a value more than double in comparison to untreated animals. The activity of the scavenger enzyme Cu/Zn superoxide dismutase (Cu/Zn SOD) in the small intestine was also determined and found significantly increased of about twice as much in the group of diabetic/hyperlipidemic rabbits treated with cloricromene.In conclusion, in diabetic/hyperlipidemic rabbits, cloricromene appeared to influence the enzymes involved in the last steps of tryptophan oxidative metabolism through the kynurenine pathway. This, together with the antioxidant action through the activation of Cu/Zn SOD, might deserve further investigation for evaluating any link between the observed experimental findings at the level of the kynurenine pathway and the clinical effect of the drug.     

THE EFFECT OF A LOW PROTEIN DIET AND EXOGENOUS INSULIN ON BRAIN TRYPTOPHAN AND ITS METABOLITES IN THE WEANLING RAT

—Male Wistar rats aged 24 days were divided into three groups. Two groups were given a high protein (250 g/kg casein) and a low protein (30 g/kg casein) diet respectively. The third group was given an amount of the high protein diet containing the same amount of energy as that consumed by the low protein diet rats. The plasma of the animals on low protein contained 20% of the concentration of tryptophan of animals on the other two diets. In these animals the concentration of tryptophan was reduced in the forebrain, cerebellum and brain stem, and the concentrations of 5-HT and 5-hydroxyindoleacetic acid were reduced in the forebrain and brain stem. The low protein diet decreased the total uptake of l -[G-³H]tryptophan into the brain and its incorporation into brain protein. Plasma insulin concentrations were reduced in the low protein and ‘restricted high protein’ animals and the plasma corticosterone concentration was raised in the low protein animals. Exogenous insulin did not raise the plasma tryptophan concentration in the low protein animals but it increased the uptake of l -[G-³H]tryptophan into the brain and its incorporation into protein. Rehabilitation for 7 days restored the plasma and brain tryptophan concentrations and those of brain 5-HT and 5-hydroxyindoleacetic acid to control values.     

Enzyme activities involved in tryptophan metabolism along the kynurenine pathway in rabbits

The following enzyme activities of the tryptophan-nicotinic acid pathway were studied in male New Zealand rabbits: liver tryptophan 2,3-dioxygenase, intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase. Intestine superoxide dismutase and serum tryptophan were also determined. Liver tryptophan 2,3-dioxygenase exists only as holoenzyme, but intestine indole 2,3-dioxygenase is very active and can be considered the key enzyme which determines how much tryptophan enters the kynurenine pathway also under physiological conditions. The elevated activity of indole 2,3-dioxygenase in the rabbit intestine could be related to the low activity of superoxide dismutase found in intestine. Kynurenine 3-monooxygenase appeared more active than kynurenine-oxoglutarate transaminase and kynureninase, suggesting that perhaps a major portion of kynurenine available from tryptophan may be metabolized to give 3-hydroxyanthranilic acid, the precursor of nicotinic acid. In fact, 3-hydroxyanthranilate 3,4-dioxygenase is much more active than the other previous enzymes of the kynurenine pathway. In the rabbit liver 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase show similar activities, but in the kidney 3-hydroxyanthranilate 3,4-dioxygenase activity is almost double. These data suggest that in rabbit tryptophan is mainly metabolized along the kynurenine pathway. Therefore, the rabbit can also be a suitable model for studying tryptophan metabolism in pathological conditions.     

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.