Fragment-based QSAR study to explore the structural requirements of DPP-4 inhibitors: a stepping stone towards better type 2 diabetes mellitus management.

Dipeptidyl peptidase-4 (DPP-4) inhibitors belong to a prominent group of pharmaceutical agents that are used in the governance of type 2 diabetes mellitus (T2DM). They exert their antidiabetic effects by inhibiting the incretin hormones like glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide which, play a pivotal role in the regulation of blood glucose homoeostasis in our body. DPP-4 inhibitors have emerged as an important class of oral antidiabetic drugs for the treatment of T2DM. Surprisingly, only a few 2D-QSAR studies have been reported on DPP-4 inhibitors. Here, fragment-based QSAR (Laplacian-modified Bayesian modelling and Recursive partitioning (RP) approaches have been utilized on a dataset of 108 DPP-4 inhibitors to achieve a deeper understanding of the association among their molecular structures. The Bayesian analysis demonstrated satisfactory ROC values for the training as well as the test sets. Meanwhile, the RP analysis resulted in decision tree 3 with 2 leaves (Tree 3: 2 leaves). This present study is an effort to get an insight into the pivotal fragments modulating DPP-4 inhibition.

The biochemistry and toxicology of benzoic acid metabolism and its relationship to the elimination of waste nitrogen.

Detoxification of sodium benzoate by elimination as a conjugate with glycine, a nonessential amino acid, provides a pathway for the disposal of waste nitrogen. Since 1979, sodium benzoate has been widely used in the therapeutic regimen to combat ammonia toxicity in patients born with genetic defects in the urea cycle. Although the clinical use of benzoate is associated with improved outcome, the search for biochemical evidence in support of the rationale for benzoate therapy has produced conflicting results. This review begins with an historical account leading to elucidation of the biochemistry of benzoate detoxification and early work indicating the potential utility of the pathway for elimination of waste nitrogen. An introduction to contemporary efforts at employing benzoate to treat hyperammonemia is followed by a detailed review of studies on benzoate metabolism and resultant toxic interactions with other major metabolic pathways. With this background, the several metabolic routes by which benzoate is thought to promote the disposal of waste nitrogen are then examined, followed by a consideration of alternative mechanisms by which benzoate might combat ammonia toxicity.

Reduction in the MK-801 binding sites of the NMDA sub-type of glutamate receptor in a mouse model of congenital hyperammonemia: prevention by acetyl-L-carnitine.

Our earlier studies on the pharmacotherapeutic effects of acetyl-L-carnitine (ALCAR), in sparse-fur (spf) mutant mice with X linked ornithine transcarbamylase deficiency, have shown a restoration of cerebral ATP, depleted by congenital hyperammonemia and hyperglutaminemia. The reduced cortical glutamate and increased quinolinate may cause a down-regulation of the N-methyl-D-aspartate (NMDA) receptors, observed by us in adult spf mice. We have now studied the kinetics of [3H]-MK-801 binding to NMDA receptors in spf mice of different ages to see the effect of chronic hyperammonemia on the glutamate neurotransmission. We have also studied the Ca2+-dependent and independent (4-aminopyridine (AP) and veratridine-mediated) release of glutamate and the uptake of [3H]-glutamate in synaptosomes isolated from mutant spf mice and normal CD-1 controls. All these studies were done with and without ALCAR treatment (4 mmol/kg wt i.p. daily for 2 weeks), to see if its effect on ATP repletion could correct the glutamate neurotransmitter abnormalities. Our results indicate a normal MK-801 binding in 12-day-old spf mice but a significant reduction immediately after weaning (21 day), continuing into the adult stage. The Ca2+-independent release of endogenous glutamate from synaptosomes was significantly elevated at 35 days, while the uptake of glutamate into synaptosomes was significantly reduced in spf mice. ALCAR treatment significantly enhanced the MK-801 binding, neutralized the increased glutamate release and restored the glutamate uptake into synaptosomes of spf mice. These studies point out that: (a) the developmental abnormalities of the NMDA sub-type of glutamate receptor in spf mice could be due to the effect of sustained hyperammonemia, causing a persistent release of excess glutamate and inhibition of the ATP-dependent glutamate transport, (b) the modulatory effects of ALCAR on the NMDA binding sites could be through a repletion of ATP, required by the transporters to efficiently remove extracellular glutamate.

Insect repellent, N,N-diethyl-m-toluamide, effect on ammonia metabolism.

There appear to be at least three mechanisms for systemic reactions to diethyltoluamide. As with most substances, allergy is possible. The ingestion of large doses can produce seizures and coma by a direct action on the CNS. This occurs in experimental animals in which seizures and coma followed by death can be produced rapidly if sufficiently large doses are given. Finally, with smaller systemic doses as may occur with absorption during heavy topical use, there is also the possibility of a perturbation of ammonia metabolism. Diethyltoluamide may then pose a substantial hazard to individuals with defects in ammonia metabolism.

Effect of sodium benzoate on cerebral and hepatic energy metabolites in spf mice with congenital hyperammonemia.

The sparse-fur (spf) mutant mouse has an X-linked deficiency of ornithine transcarbamylase and develops congenital hyperammonemia similar to that seen in human patients. We studied the effect of sodium benzoate (2.5, 5 and 10 mmol/kg body wt) on ammonia, glutamine and glutamate, as well as various intermediates of energy metabolism in brain and liver of normal CD-1/Y and hyperammonemic spf/Y mice. The ammonia concentration of brain was decreased with 2.5 mmol sodium benzoate in spf/Y mice, whereas higher doses resulted in a significant increase in both liver and brain. Cerebral glutamine content decreased generally in a dose-dependent manner, both in normal and affected mice, following treatment with various doses of sodium benzoate. Cerebral glutamate concentrations were increased only in spf mice treated with sodium benzoate, whereas ATP and acetyl CoA were decreased (P < 0.001), in both normal and affected mice, indicating that glutamine synthesis may be affected by ATP availability. Free CoA levels were decreased (P < 0.05) only in liver in both groups of treated mice, whereas pyruvate concentrations were elevated (P < 0.05) in affected mice following sodium benzoate administration. The results demonstrate that a dose of 2.5 mmol sodium benzoate/kg body wt has a beneficial effect in reducing cerebral ammonia with a concomitant decrease in glutamine. However, the results suggest that many of the metabolite changes observed following higher doses of benzoate could be due to depletion of ATP, free CoA and acetyl CoA levels, possibly secondary to benzoyl CoA accumulation. The response of the spf/Y mouse to sodium benzoate was different from that of the control CD-1/Y mouse, which could be due to its urea cycle dysfunction and a chronic hyperammonemic state. Hence, the spf/Y mouse may be the ideal animal model for studying the pharmacology of sodium benzoate in hyperammonemic disorders at both the cerebral and hepatic levels.

Restoration of hepatic cytochrome c oxidase activity and expression with acetyl-L-carnitine treatment in spf mice with an ornithine transcarbamylase deficiency.

The sparse fur (spf) mutant mouse, with an X-linked ornithine transcarbamylase deficiency, is a model of congenital hyperammonemia in children. Our earlier studies indicated a deficiency of hepatic carnitine, CoA-SH, acetyl CoA, and ATP in spf mice. We have now studied the effects of a 7-day treatment with acetyl-L-carnitine (ALCAR) in the spf/Y mice on the activity and expression of the respiratory chain enzyme cytochrome c oxidase (COX; EC 1.9.3.1). We found decreased hepatic activity and expression of COX in the untreated hyperammonemic spf/Y mice, which was restored upon ALCAR treatment. Because COX is a mitochondrial membrane protein, we also carried out studies to explain the mechanism of ALCAR through its effect on membrane stability. Our results indicate a decrease of the mitochondrial membrane cholesterol/phospholipid molar ratio (CHOL/PL ratio) with the activity and expression of COX in untreated spf/Y mice. While ALCAR treatment normalized the ratios, it also restored the hepatic ATP production to normal. To study further if there was any effect of ALCAR on the mitochondrial matrix urea cycle enzymes, we measured the activity and expression of mutant ornithine transcarbamylase (OTC; EC 2.1.3.3) and normal carbamyl phosphate synthase-I (CPS-I; EC 6.3.4.16) in spf/Y mice. There was no general effect on the specific activities of the matrix enzymes upon ALCAR treatment, although their mRNA levels were enhanced. Our studies point towards the feasibility of an ALCAR treatment in conjunction with other treatment modalities, e.g. sodium benzoate and/or arginine, to improve the availability of cellular ATP and to counteract the effects of hereditary hyperammonemic syndromes in children.

Effect of L-carnitine on cerebral and hepatic energy metabolites in congenitally hyperammonemic sparse-fur mice and its role during benzoate therapy.

Sparse-fur (spf) mutant mice with X-linked ornithine transcarbamylase deficiency were used to study the effect of L-carnitine on energy metabolites in congenital hyperammonemia. L-Carnitine was used at doses of 2, 4, 8, or 16 mmol/kg body weight (BW), and levels of ammonia, glutamine, glutamate, and some intermediates of energy metabolism were measured in brain and liver of spf/Y mice. Cerebral and hepatic levels of ammonia were decreased with 4 mmol L-carnitine (P < .001), whereas other doses did not seem to have any effect on this metabolite. Cerebral levels of glutamine were decreased following administration of L-carnitine at doses of up to 4 mmol/kg BW, whereas hepatic glutamine levels remained unaltered at all doses of L-carnitine. Both cerebral and hepatic levels of pyruvate, lactate, and alpha-ketoglutarate were decreased at doses of up to 8 mmol L-carnitine/kg BW. L-Carnitine treatment elevated adenosine triphosphate (ATP), free coenzyme A (CoA), and acetyl CoA levels in both brain and liver of spf/Y mice. Cytosolic and mitochondrial redox ratios of spf/Y mice, which were altered by congenital chronic hyperammonemia, were partially corrected by L-carnitine administration. L-Carnitine supplementation to spf/Y mice during sodium benzoate therapy also restored the availability of free CoA and ATP, thus counteracting the adverse effects of higher doses of sodium benzoate. These changes in free CoA and acetyl CoA levels could be due to the deinhibition of pantothenate kinase and stimulation of fatty acid oxidation by L-carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)

Guanidino compounds in serum, urine, liver, kidney, and brain of man and some ureotelic animals.

Guanidino compound levels were quantitatively determined in serum, urine, liver, kidney, and brain of man and of some ureotelic animals. The guanidino compounds were separated over a cation exchange resin, using sodium citrate buffers, and detected with the fluorescence ninhydrin method. Species-specific differences in the levels of some guanidino compounds in the studied ureotelic animals are shown. alpha-Keto-delta-guanidinovaleric acid is a naturally occurring guanidino compound in ureotelic animals, and is not restricted to the pathobiochemistry of hyperargininemic patients. The fasting serum levels observed in beagles are the same as those found in hyperargininemic patients. In serum, liver, and kidney, the homoarginine, beta-guanidinopropionic acid, and gamma-guanidinobutyric acid levels are the highest in rats. The last two compounds have the highest levels of the studied guanidino compounds, with the exception of creatinine, in kidney. Specific high levels of gamma-guanidinobutyric acid and argininic acid are found in brain of rabbits.

The pathobiochemistry of uremia and hyperargininemia further demonstrates a metabolic relationship between urea and guanidinosuccinic acid.

To better understand the biosynthesis of guanidinosuccinic acid, we determined urea, arginine, and guanidinosuccinic acid levels in nondialyzed uremic and hyperargininemic patients. These substances were also determined during several years of therapy in one hyperarginiemic patient. Interrelationships of guanidinosuccinic acid levels with their corresponding urea and arginine levels were assessed by linear correlation studies. In uremic patients, a significant positive linear correlation (r = .821, p less than .001) was found between serum urea and guanidinosuccinic acid levels A significant positive linear correlation was also found between serum urea levels and urinary guanidinosuccinic acid levels (r = .828, P less than .001), but not between serum arginine levels and urinary guanidinosuccinic acid levels in hyperargininemic patients. In the intrahyperargininemic patient study, a similar significant positive correlation was found between serum urea levels and the corresponding urinary guanidinosuccinic acid levels (r = .866, P less than .001); the correlation between serum arginine levels and the corresponding urinary guanidinosuccinic acid levels was smaller. The presented analytical findings in uremic and hyperargininemic patients clearly demonstrate a metabolic relationship between urea and guanidinosuccinic acid.

Epidermal growth factor in acute promyelocytic leukemia treated with retinoic acid.

We studied 18 patients with acute promyelocytic leukaemia and 13 with relapsed APL. We found a significantly elevated EGF in acute leukaemia, especially in APL, being 418.59 +/- 19.2 micrograms in the 24-h urine that was much higher than that of the normal controls. When eight APL patients achieved complete remission by RA treatment, the EGF value decreased to 149.9 +/- 27.3 micrograms in the 24-h urine near to normal. In 13 patients with relapsed APL, EGF rose to 446.9 +/- 82.6 micrograms in the 24-h urine. Most interestingly, this elevated EGF could be detected before the relapse by 5 +/- 0.84 months in seven out of eight APL with relapse. We suggest that the unaccountably elevated EGF during remission period may be an indicator of the occurrence of relapse.

Impaired cognitive performance in ornithine transcarbamylase-deficient mice on arginine-free diet.

Sparse-fur (spf) mice are a model for the congenital deficiency of ornithine transcarbamylase (OTC), the most common inborn error of urea synthesis in man. In this study, performance of clinically stable spf and control mice (8-10-weeks-old) on two learning tests was assessed under normal Arg(+) or arginine-free Arg(-) diet conditions. Used as an indicator of the metabolic status of the animals, plasma ammonia concentrations were significantly higher in spf than in controls on normal diet, and increased even more during the Arg(-) diet episode. Behaviourally, we found no difference in passive avoidance learning between control and spf mice on Arg(+) diet, whereas in spf mice receiving Arg(-) diet during training, retention performance was significantly reduced. In the hidden-platform water maze, spf mice on Arg(+) diet only showed decreased swimming velocity compared to controls. In mice on Arg(-) diet during the first week of acquisition training, performance on acquisition and retention (probe) trials showed that spf mice experienced more difficulties in actually locating the platform. Visible-platform control experiments only showed a reduction in swimming velocity in spf mice on either diet. We conclude that cognitive performance is impaired in spf mice as a consequence of Arg(-) diet-induced neurochemical alterations.

Evidence for cholinergic neuronal loss in brain in congenital ornithine transcarbamylase deficiency.

Congenital ornithine transcarbamylase (OTC) deficiency in humans is associated with seizures and mental retardation. As part of a series of studies to delineate the neurochemical features of OTC deficiency, activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), respectively, were measured in brain regions of the congenitally hyperammonemic sparse-fur (spf) mouse, a mutant with an X-linked inherited defect of OTC. ChAT activities were reduced by 63% (P < 0.01) in cerebral cortex of spf mice compared with CD-1/Y controls. Activities of the GABA nerve terminal marker enzyme, glutamic acid decarboxylase, on the other hand, were within normal limits. Using an immunohistochemical technique with a monoclonal antibody to ChAT, a significant loss of ChAT-positive neurons was observed throughout the cerebral cortex, septal area and diagonal band of spf mice. These results suggest that a loss of forebrain cholinergic neurons is a feature of congenital OTC deficiency in these mutants. Possible pathogenetic mechanisms responsible for the cholinergic neuronal loss in congenital OTC deficiency include neurotoxic effects of ammonia and accumulation of quinolinic acid.

Activities of monoamine oxidase-A and -B are altered in the brains of congenitally hyperammonemic sparse-fur (spf) mice.

Activities of monoamine oxidases, MAOA and MAOB, were measured using radiometric assays in different brain regions of the sparse-fur (spf/Y) mouse, a model of congenital hyperammonemia resulting from an X-chromosomal defect of ornithine transcarbamylase. MAOA activities were decreased in cerebellum (by 23%, P < 0.05) and brainstem (by 16%, P < 0.05) of spf mice; activities of MAOB were concomitantly increased in cerebellum (by 22%, P < 0.05), brainstem (by 20%, P < 0.05) and cerebral cortex (by 22%, P < 0.05). These findings offer a rational explanation for previous findings of increased acidic metabolites of monoamines in the brain of spf mice. Altered monoaminergic function could be a key factor in the pathogenesis of neurological dysfunction in congenital hyperammonemias.

Na+,K(+)-ATPase activities are increased in brain in both congenital and acquired hyperammonemic syndromes.

Activities of Na+,K(+)-ATPase were measured in brain regions of experimental animals with either congenital or acquired hyperammonemia. In the sparse-fur (spf) mutant mouse, with a genetic X-linked deficiency of ornithine transcarbamylase, an animal model of congenital hyperammonemia, Na+,K(+)-ATPase was increased in frontal cortex (by 57%, P < 0.001), cerebellum (by 61%, P < 0.001), brainstem (by 71%, P < 0.001) and striatum (by 48%, P < 0.01). Four weeks following portacaval anastomosis in the rat, Na+,K(+)-ATPase activities were increased in cerebellum and striatum (by 19%, P < 0.01) and in brainstem (by 28%, P < 0.01). Stimulation of Na+,K(+)-ATPase and the subsequent alteration of neuronal excitability could contribute to the CNS dysfunction characteristic of chronic hyperammonemic syndromes.

Progressive decrease of cerebral cytochrome C oxidase activity in sparse-fur mice: role of acetyl-L-carnitine in restoring the ammonia-induced cerebral energy depletion.

Sparse-fur (spf) mice with a deficiency of hepatic ornithine transcarbamylase (OTC) are congenitally hyperammonemic, showing elevated cerebral ammonia and glutamine and depleted levels of energy metabolites. This mouse disorder is akin to the human OTC deficiency, in which neuronal loss and Alzheimer's type II astrocytosis is reported. Reduced cytochrome C oxidase (COX) activity is characteristic of neurodegeneration in Alzheimer's type disorders. We have studied the causal relationship between cerebral COX activity and energy depletion in spf mice. Our results indicate a progressive decrease in the COX activity in various brain regions in spf mice, up to 40 weeks of age, which severely effected the cerebral levels of various energy metabolites. A quantitative estimation of cerebral COX subunit I mRNA also showed a tendency to decrease in spf mice. Short-term acetyl L-carnitine (ALCAR) treatment restored these abnormalities. Our study points out that: (a) ammonia-induced alterations in the cerebral reducing equivalents could cause a decrease in COX activity and its mRNA expression, and (b) ALCAR administration could normalize the cerebral energy metabolism and induce COX mRNA expression and activity.

Arginine-related guanidino compounds and nitric oxide synthase in the brain of ornithine transcarbamylase deficient spf mutant mouse: effect of metabolic arginine deficiency.

The sparse-fur (spf) mouse, with an X-linked hepatic ornithine transcarbamylase (OTC, E.C.2.1.3.3) deficiency, exhibits significantly lower levels of arginine in the brain as compared to normal controls. In the present study, the effect of a sustained lower metabolic arginine was studied by measuring the levels of several arginine-related guanidino compounds in brain. The concentrations of gamma-guanidinobutyric acid (gamma-GBA), N-alpha-acetylarginine (N-alpha-AA), argininic acid (Arg-A), guanidinoacetic acid (GAA), and creatine were significantly lower in spf mice as compared to controls. Since arginine is the precursor for nitric oxide, we also measured the activity of nitric oxide synthase which was significantly reduced in cerebellum, striatum, hippocampus and cerebral cortex of spf mice. The changes seen in cerebral guanidino compound and nitric oxide metabolism of spf mice could be due to a sustained deficiency of arginine, caused by a metabolic block in the area cycle.

Guanidino compounds in plasma, urine and cerebrospinal fluid of hyperargininemic patients during therapy.

The concentrations of guanidino compounds were determined in urine, plasma and cerebrospinal fluid of two patients with hyperargininemia during dietary therapy. alpha-Keto-delta-guanidinovaleric acid, N-alpha-acetylarginine, argininic acid and gamma-guanidinobutyric acid were increased in urine. In plasma, these compounds together with creatine, guanidinoacetic acid, arginine and homoarginine were also increased. In cerebrospinal fluid, only arginine, homoarginine and argininic acid were increased. Trace amounts of alpha-keto-delta-guanidinovaleric acid were found in cerebrospinal fluid of the patient treated with only a low-arginine diet. The concentrations of guanidinosuccinic acid are decreased in urine, plasma and cerebrospinal fluid. During a low-arginine diet, together with sodium benzoate therapy, the plasma and cerebrospinal fluid arginine values returned to normal. There was also a normalization of plasma guanidinoacetic acid and a marked decrease in plasma N-alpha-acetylarginine and argininic acid.

Intraocular pressure and pregnancy: a comparison between normal and ocular hypertensive subjects.

Decrease in intraocular pressure (IOP) during pregnancy has been reported by previous studies, but these studies have concentrated on the last trimester of pregnancy or one reading per trimester. Moreover, IOP changes during pregnancy in ocular hypertensive subjects have never been described. Therefore, the present study was planned to determine IOP throughout the pregnancy, in both normal and ocular hypertensive subjects. Intraocular pressure was measured at six-week intervals throughout the pregnancy in 44 normal and 32 ocular hypertensive women. Intraocular pressure was also measured in 44 normal and 32 ocular hypertensive non-pregnant controls of the same age group. IOP measurements were taken with the Goldmann applanation tonometer. In normal subjects, IOP decreased significantly at the 18th week (p < 0.05). The IOP differences between first and second (p < 0.05) and second and third (p < 0.01) trimesters of pregnancy were significant. In these subjects, pregnancy decreased IOP by 19.6%. About 35% of total decrease occurred between 12th and 18th weeks of pregnancy. In ocular hypertensive subjects, IOP decreased significantly at the 24th week (p < 0.05). The IOP differences between second and third (p < 0.001) trimesters of pregnancy were significant. In these subjects, pregnancy decreased IOP by 24.4%. About 61% of total decrease occurred between 24th and 30th weeks of pregnancy. In both groups, decreases in IOP were independent of systolic and diastolic blood pressures, body weight, height, and number of previous pregnancies. With advancing pregnancy, intraocular pressure decreases. The higher decrease in ocular hypertensive subjects may be due to their higher level of ocular pressure. In ocular hypertensive subjects, pregnancy can decrease intraocular pressure up to a level of normal limit.

Tissue acylcarnitine and acyl-coenzyme A profiles in chronically hyperammonemic mice treated with sodium benzoate and supplementary L-carnitine.

The aim of the present study, was to establish the hepatic profile of acyl-coenzyme A (acyl-CoA) in relation to the hepatic profile of acylcarnitines in chronically hyperammonemic spf mice (hereditary deficiency in ornithine transcarbamylase) treated with sodium benzoate alone or in combination with L-carnitine. The muscular profile of the acylcarnitines and the stability of sarcolemma were also assessed in the same mice. Following administration of sodium benzoate, we observed decreases in hepatic total and free coenzyme A and in acetyl-CoA, which was accompanied by an increase in hepatic acyl-CoA. This treatment also resulted in increased free carnitine, decreased total carnitine, and decreased short and medium chain acylcarnitines in the liver. Increases in plasma creatine kinase levels, muscular free, total, and in short and medium chain acylcarnitines were also observed in this treatment group. In mice receiving a combination of sodium benzoate and L-carnitine, increases in free and total coenzyme A, acetyl-CoA and in free, total and esterified hepatic carnitines were observed. In this treatment group, the plasma level of creatine kinase was found to be reduced, while the free muscular carnitine was increased. Our results indicate that sodium benzoate is implicated in the decrease of total hepatic coenzyme A, through either an inhibition of CoA synthesis or activation of its degradation. The distribution of hepatic coenzyme-A and of hepatic and muscular carnitine (free or esterified) is altered following administration of sodium benzoate which results in a further destabilization of the sarcolemma induced by hyperammonemia. Supplemental treatment with L-carnitine was shown to have a positive effect by increasing hepatic coenzyme A and carnitine levels and restoring the stability of the sarcolemma caused by the treatment of sodium benzoate alone.

The effects of various inhibitors on the regulation of orotic acid excretion in sparse-fur mutant mice (spf/Y) deficient in ornithine transcarbamylase.

Experiments were conducted to determine whether the excessive orotic aciduria, induced in sparse-fur male mice (spf/Y) deficient in ornithine transcarbamylase (OTC), may be regulated by some inhibitors, such as acivicin (0.014 mmol/100 g body weight, i.p.), N-(phosphonoacetyl)-L-aspartate (PALA, 2.5 mg/100 g body weight, i.p.), adenine (3 g/kg diet) and cycloheximide (0.35 mmol/kg body weight, i.p.). We also administered ornithine (1 mmol/100 g body weight, i.p.), a substrate of the urea cycle, to alleviate the metabolic deficiency of arginine in spf/Y mice which may also be responsible for excessive orotic aciduria. The orotic aciduria remained insensitive to acivicin, indicating mitochondria as the source of carbamyl phosphate. However, orotate excretion was significantly decreased by PALA (P < 0.01), due to its effect on the aspartate transcarbamylase activity. The ingestion of adenine resulted in an increase (P < 0.05) of urinary orotate, suggesting the blockage of the utilization of orotate for nucleotide biosynthesis. Ornithine administration led to a reduction (P < 0.01) of the excretion of orotate induced by the OTC deficiency in these mice, indicating that one of the regulatory steps in its synthesis may be the availability of ornithine. There were no changes in urinary orotate excretion in spf/Y mice when treated with cycloheximide. On the other hand, pretreatment with cycloheximide in an artificial model of OTC deficiency (Swiss-ICR normal mice on an arginine-deficient diet treated thereafter with norvaline, an inhibitor of OTC), caused a significant decrease in urinary orotate. These results suggest that spf/Y mice are unique in that the increased synthesis of orotate is not sensitive to cycloheximide. Perhaps this may reflect an adaptive phenomenon developed by the mutant mice to handle excess mitochondrial carbamyl phosphate and orotic acid.