The synthesis of radioactive 3-methylthiopropionate and other alkylthio fatty acids.

Aprocedure is described for the synthesis of radioactive 3-methylthiopropionate, a recently isolated metabolite of mammalian methionine metabolism. The method is a two-step synthesis whereby correspondingly labeled methionine is degraded by ninhydrin to 3-methylthiopropionaldehyde and then specifically oxidized to 3-methylthiopropionate without oxidation of the sulfur atom by the yeast enzyme, aldehyde dehydrogenase. Radiochemical purity of the isolated product was established by paper, thin-layer, and gas-liquid chromatography. This procedure is economical and readily applicable to the synthesis of other alkylthio fatty acids for the study of S-methylcysteine and ethionine metabolism and probably for the synthesis of radioactive intermediates of branched chain amino acids.     

Involvement of glycoconjugates in insulin-receptor interactions Studies in liver plasma membranes of control and diabetic mice

The involvement of glycoconjugates in the insulin-receptor interactions in mouse liver is tested by digestions of membranes with various enzymes. Trypsin decreased the binding of [¹²⁵I]insulin to liver membranes. After digestion with β-galactosidase no “high affinity” receptor sites could be detected. The effects observed with plant lectins confirm the involvement of galactoconjugates in the insulin binding process. Sophora japonica and Ricinus communis lectins (with galactose specificity) and concanavalin A largely inhibit the binding process of insulin and those effects concern the “high affinity” receptor sites. Other lectins (wheat germ agglutinin, Dolichos) and enzymes (α-l-fucosidase, $\text{β-N-}\text{acetyl-hexosaminidase and neuraminidase}$) are without effect on insulin binding.Comparative studies performed on diabetic mouse liver membrane (KK mice), previously characterized by decreased number of insulin receptors, are in good agreement with qualitatively similar receptor sites in both non-diabetic (control) and diabetic mice. Effects of enzymes and lectins yielded same results as compared to control membranes. Plasma membrane proteins and glycoproteins in both types of mouse are indistinguishable with respect to enzymic and chemical analysis. Sodium dodecyl sulphate acrylamide gel electrophoresis shows identical patterns. Moreover, the decrease in the number of insulin receptors is easily reversed with diet restriction. These data are consistent with the similarity of receptor sites in control and diabetic liver membrane.     

Inhibition of hepatic gluconeogenesis and lipogenesis by benzoic acid,p-tert.-butylbenzoic acid,and a structurally related hypolipidemic agent SC-33459

Benzoic acid, p-tert.-butylbenzoic acid, and a structurally related hypolipidemic agent SC-33459 were found to inhibit glucose synthesis by hepatocytes isolated from 48-h fasted rats as well as fatty acid synthesis by hepatocytes isolated from meal-fed rats. Glucose synthesis was less sensitive than fatty acid synthesis. Benzoic acid was the least effective inhibitor of both processes; SC-33459 and p-tert.-butylbenzoic acid were very potent inhibitors with similar efficacy. Glycine prevented the inhibition of fatty acid synthesis caused by benzoic acid, but had no effect on that caused by p-tert.-butylbenzoic acid. Octanoate opposed the inhibitory effects of both benzoic acid and p-tert.-butylbenzoic acid. Oxidation of [1-¹⁴C]oleate to ketone bodies and acid-soluble radioactive products was inhibited by both p-tert.-butylbenzoic acid and SC-33459. Preincubation of hepatocytes with SC-33459 was required for the latter effect, suggesting catabolism of this compound may be involved. SC-33459 is a p-tert.-butylphenyl derivative which should be readily converted to p-tert.-butylbenzoic acid by β oxidation. Both p-tert.-butylbenzoic acid and SC-33459 decreased citrate levels dramatically. All three compounds reduced CoA and acetyl-CoA levels and increased medium-chain acyl-CoA ester levels. p-tert.-Butylbenzoic acid and SC-33459 also increased long-chain acyl-CoA ester levels. The increase in medium-chain acyl-CoA levels presumably reflects benzoyl-CoA formation from benzoic acid and p-tert.-butylbenzoyl-CoA formation from p-tert.-butylbenzoic acid and SC-33459. Inhibition of glucose and fatty acid synthesis by these compounds may be due to effects on specific enzymes or to CoA sequestration.     

Sequential changes in ornithine decarboxylase thymidine kinase, and other enzyme activities in regenerating liver in rats on controlled feeding schedules.

The changes in activity of five enzymes including ornithine decarboxylase (ODC), tyrosine aminotransferase (TAT), thymidine kinase (TK), ornithine aminotransferase (OAT) and serine dehydratase (SDH) in the early stage of the regenerating rat liver have been studied under a controlled feeding and lighting schedule. The first three enzyme activities were stimulated sequentially by partial hepatectomy. The earliest response was observed in ODC activity. A significant increase in this enzyme activity was observed at 2 hrs and the maximal level was at 4 hrs after the operation. TAT began to increase at 4 hrs and the maximal level was at 8 hrs. The TK activity was induced at about 24 hrs and the highest value was at 48 hrs after partial hepatectomy.A significant decrease in OAT activity was observed at 24 hrs after the operation and subsequently. Although a decrease in SDH activity was also observed this decrease did not seem to correlate directly with the regeneration process, since a lowered level of the enzyme activity was also found in the sham operated group.     

A nonspecific inhibitor of leukocyte migration in serum from rats bearing large fibrosarcomata.

A 3 M KCl crude extract of the syngeneic benzpyrene-induced fibrosarcoma termed BP 238 specifically inhibits migration out of glass capillary tubes of immune spleen cells from tumor amputee and small tumor-bearing rats, as does supernatant medium from tumor cells grown in culture. Serum from rats bearing small (< 2 cm³) tumors does not inhibit migration of immune spleen cells, while serum from rats with larger tumors (>4 cm³) nonspecifically inhibits migration of both immune and nonimmune spleen cells, thoracic duct lymphocytes, and thymocytes. This nonspecific inhibition increases with increasing tumor size, does not correlate with the presence of bacterial infection, and is presumably due to a circulating factor produced in vivo during tumor growth. Production of macrophage inhibitory-like factor (MILF) by neoplasms in vivo may offer a mechanism for tumors to escape immunosurveillance by systemic immobilization of cytotoxic lymphocytes. From Sephadex and ultrafiltration fractionation experiments, the molecular weight of MILF in serum is polydisperse (30,000–100,000 daltons), and is heat and chymotrypsin resistant, in contrast with the properties reported for LIF (leukocyte inhibitory factor) and MIF (macrophage migration inhibitory factor) produced in vitro.     

Uptake and metabolism of purine nucleosides and nucleotides in isolated frog skeletal muscle.

When isolated frog skeletal muscles were incubated with ¹⁴C-labeled adenosine, the nucleoside was rapidly taken up by the cells and was either immediately incorporated into adenine nucleotides or deaminated to inosine. Incorporation was predominant at low (micromolar) concentrations whereas, deamination was the major route of metabolism at high (millimolar) concentrations. When muscles were incubated with ¹⁴C-labeled inosine the nucleoside, after entry into the cells, was metabolized to a lesser extent than adenosine. ATP and hypoxanthine were the major products of its metabolism. Intracellular concentrations were calculated using ³H-labeled sorbitol to measure the extracellular space.Because of its lower rate of intracellular metabolism inosine was used to investigate the characteristics of the nucleoside transport system. The uptake of inosine was saturable at high concentrations and was specifically inhibited by the presence of adenosine or uridine in the incubation media. Persantin, a well known specific inhibitor of nucleoside transport, also competitively inhibited inosine uptake, as did theophylline [1, Woo et al. Can J. Physiol. Pharmacol. $\text{52}$, 1063, 1974]. These data, along with the knowledge that in a well-oxygenated muscle, inosine entry follows a downhill chemical potential gradient, strongly support the view that the transport mechanism is facilitated diffusion.The muscle cell membrane does not appear to be permeable to ¹⁴C-labeled ATP under the conditions studied. Investigations of the permeability to the major extracellular degradation products of ATP suggest that AMP was the compound most likely to cross the cell membrane.     

Diurnal variations in activity of four pyridoxal enzymes in rat liver during metabolic transition from high carbohydrate to high protein diet.

The diurnal variations in enzyme activities including tyrosine aminotransferase (TAT), ornithine decarboxylase (ODC), ornithine aminotransferase (OAT) and serine dehydratase (SDH) have been studied in rats trained to a 2 hour meal feeding schedule (″2+22″) during metabolic transition from 12.5 to 60% protein diets over a period of 21 days. Although the maximal TAT activity on the first day was slightly lower compared with other days, both TAT and ODC activities adapted rapidly to the increased dietary protein from the first day. The responses of TAT and ODC to the food were so rapid that the maximal value was observed only 4 hrs after the onset of feeding. After each feeding ODC activity decreased rapidly after 4 hours, while TAT activity declined only after 6 hours had elapsed. No clear diurnal rhythm was observed in either OAT or SDH, though OAT activity tended to decrease from the beginning of the dark period and to resume a slow adaptation after about four hours. In contrast to ODC and TAT both OAT and SDH required about 7 days to fully adapt to the high protein diet. The activities of the four enzymes were also compared after 4 groups of rats had been adapted to the ″2+22″ feeding of 12.5, 30 and 60% protein diets and to 60% diet, $\text{ad}$$\text{libitum}$, respectively. The enzyme activities were not directly proportional to the protein content of the diets although higher activity was observed on the high protein diets. The diurnal variations in both TAT and ODC were observed in all ″2+22″ groups although the timing of the peak values were slightly different from each other. The maximal activities of TAT were found at earlier times in 12.5 and 30% protein groups than in the 60% protein group. The peak time for ODC activity was found at a later time in the 12.5% protein group than in rats fed 30% and 60% protein. $\text{Ad}$$\text{libitum}$ rats fed 60% protein maintained relatively high levels of TAT activity compared to the rats on the schedule. However, the maximal activity of ODC on the 60% ″2+22″ protein diet $\text{ad}$$\text{libitum}$ was so low that a diurnal rhythm was not clearly evident.     

Ornithine decarboxylase activity and DNA synthesis in Morris hepatomas 5123-C and 7800.

The activities of ornithine decarboxylase (ODC) and thymidine kinase (TK) and the rates of DNA synthesis were determined in hepatomas and livers of rats bearing Morris hepatoma 5123-C or 7800 and entrained to a schedule of 12 hours of light followed by 12 hours of darkness, with food (60% protein) available only during the first 2 hours of the dark period. ODC activity in hepatoma 5123-C displayed a diurnal oscillation, increasing 2-fold during the feeding period and then rapidly decaying to 20% of the peak level. The livers of rats bearing hepatoma 5123-C exhibited a similar oscillation of ODC activity, with peak values lower than in the hepatomas but higher than in the livers of control (non-tumor bearing) animals. TK activity and the rate of DNA synthesis in hepatoma 5123-C were low during most of the dark period but increased rapidly towards the end of the dark period. DNA synthesis reached a plateau at the dark-light interface and then rapidly declined, but TK activity remained high during the light period. Similar studies on hepatoma 7800 established that ODC activity in this hepatoma did not oscillate but remained at low levels throughout the day. Similarly, host livers of rats bearing hepatoma 7800 did not exhibit the diurnal oscillation of ODC activity characteristic of liver from control rats, but showed a slow increase in activity followed by a plateau and a slow decline to base-line levels. DNA synthesis in hepatoma 7800 was constant throughout the day, whereas TK activity may have increased during the dark period. In the livers of control rats and animals bearing hepatoma 5123-C or 7800, TK activity and rate of DNA synthesis were at low levels at all times studied and appeared not to oscillate.     

Subunit interactions in liver alcohol dehydrogenase: A partial alkylation study.

The transient kinetics of aldehyde reduction by NADH catalyzed by liver alcohol dehydrogenase consist of two kinetic processes. This biphasic rate behavior is consistent with a model in which one of the two identical subunits in the enzyme is inactive during the reaction at the adjacent protomer. Alternatively, enzyme heterogeneity could result in such biphasic behavior. We have prepared liver alcohol dehydrogenase containing a single major isozyme; and the transient kinetics of this purified enzyme are biphasic.Addition of two [¹⁴C]carboxymethyl groups per dimer to the two “reactive” sulfhydryl groups (Cys46) yields enzyme which is catalytically inactive toward alcohol oxidation. Alkylated enzyme, as initially isolated by gel filtration chromatography at pH 7·5, forms an NAD⁺-pyrazole complex. However, the ability to bind NAD⁺-pyrazole is rapidly lost in pH 8·75 buffer; therefore, our alkylated preparations, as isolated by chromatography at pH 8·75, are inactive toward NAD⁺-pyrazole complex formation. We have prepared partially inactivated enzyme by allowing iodoacetic acid to react with liver alcohol dehydrogenase until 50% of the NAD⁺-pyrazole binding capacity remains; under these reaction conditions one [¹⁴C]carboxymethyl group is added per dimer. This partially alkylated enzyme preparation is isolated by gel filtration and has been aged sufficiently to lose NAD⁺-pyrazole binding ability at alkylated subunits. When solutions of native liver alcohol dehydrogenase and partially alkylated liver alcohol dehydrogenase containing the same number of unmodified active sites are allowed to react with substrate under single turnover conditions, partially alkylated enzyme is only half as reactive as native enzyme. This indicates that some molecular species in partially alkylated liver alcohol dehydrogenase that react with pyrazole and NAD⁺ during the active site titration do not react with substrate. These data are consistent with a model in which a subunit adjacent to an alkylated protomer in the dimeric enzyme is inactive toward substrate. In addition, NAD⁺-pyrazole binding at the protomers adjacent to alkylated subunits is slowly lost so that 75% of the enzyme-NAD⁺-pyrazole binding capacity is lost in 50% alkylated enzyme. These data supply strong evidence for subunit interactions in liver alcohol dehydrogenase.Binding experiments performed on partially alkylated liver alcohol dehydrogenase indicate that coenzyme binding is normal at a subunit adjacent to an alkylated protomer even though active ternary complexes cannot be formed. One hypothesis consistent with these results is the unavailability of zinc for substrate binding at the active site in subunits adjacent to alkylated protomers in monoalkylated dimer.     

A comparative study of mouse liver and mouse blastocyst DNA-dependent RNA polymerases.

DNA-dependent RNA polymerase has been studied in adult mouse liver and mouse blastocysts. The enzyme from mouse liver was resolved into three enzyme forms by DEAE-Sephadex chromatography. Two of the forms, IA and IB, are insensitive to α-amanitin, have low $\text{Mn}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ activity ratios, and are optimally active at low ionic strength. Form II is inhibited by α-amanitin, has a higher $\text{Mn}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ activity ratio, and is most active at high ionic strength. An optimal reaction temperature of 37 ° C was found for all enzyme forms. All of the isolated enzyme forms are inhibited by the exotoxin from Bacillus thuringiensis and the inhibition can be partially reversed by increased ATP levels. Forms IA and IB are most active with native template while form II prefers denatured DNA.The blastocyst RNA polymerase activity exhibits similar requirements for divalent metal ions and ionic strength to the purified liver enzymes. The maximum inhibition of blastocyst RNA polymerase obtained with α-amanitin and exotoxin differs from that observed for purified liver enzymes but is similar to the inhibition of liver homogenate. However, the concentrations of inhibitor required for maximum inhibition by α-amanitin and exotoxin is different for the blastocyst and liver homogenate enzymes.     

A new class of model glycolipids: synthesis, characterization, and interaction with lectins.

A method is described for preparing model glycolipids by linking aldobionic acids to an alkylamine through an amide bond. These compounds may be rapidly prepared in large quantities. The glycolipids precipitate specifically with lectins. Precipitation occurs at glycolipid concentrations just above their critical micelle concentration.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. III. Cholera enterotoxin induces lymph node cells to release soluble factor(s) which enhance(s) antibody synthesis by antigen-treated lymph node cells.

Unprimed or KLH-primed rabbit lymph node cells were pulsed with cholera enterotoxin or KLH for 2 hr and washed. KLH-treated LNC were mixed with equal numbers of CT-treated LNC or boiled CT-treated LNC. Cocultivation of CT-treated LNC with KLH-treated cells resulted in at least a 100% increase in antibody synthesis compared to control cultures. Delaying cocultivation for 24 hr reduced enhancement to 25%. Thus it appears that an early event—before 24 hr—is involved in CT enhancement. Using ¹²⁵I-CT, it was shown that these effects were not due to CT carry-over. When KLH- and CT-pulsed LNC were cultured in chambers separated by polycarbonate membranes (0.2- to 0.4-μm pore size) antibody production was enhanced 50–80%. Supernates of CT-treated LNC also enhanced antibody production by KLH-treated LNC. These results suggest that CT triggers the release of soluble factor(s) which enhance(s) antibody synthesis by antigen-primed and antigen-challenged LNC.     

Effect of dichloroacetate and glucagon on the incorporation of labeled substrates into glucose and on pyruvate dehydrogenase in hepatocytes from fed and starved rats

Dichloroacetate (2 mm) stimulated the conversion of [1-¹⁴C]lactate to glucose in hepatocytes from fed rats. In hepatocytes from rats starved for 24 h, where the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio is elevated, dichloroacetate inhibited the conversion of [1-¹⁴C]lactate to glucose. Dichloroacetate stimulated ¹⁴CO₂ production from [1-¹⁴C]lactate in both cases. It also completely activated pyruvate dehydrogenase and increased flux through the enzyme. The addition of β-hydroxybutyrate, which elevates the intramitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio, changed the metabolism of [1-¹⁴C]lactate in hepatocytes from fed rats to a pattern similar to that seen in hepatocytes from starved rats. Thus, the effect of dichloroacetate on labeled glucose synthesis from lactate appears to depend on the mitochondrial oxidation-reduction state of the hepatocytes. Glucagon (10 nm) stimulated labeled glucose synthesis from lactate or alanine in hepatocytes from both fed and starved rats and in the absence or presence of dichloroacetate. The hormone had no effect on pyruvate dehydrogenase activity whether or not the enzyme had been activated by dichloroacetate. Thus, it appears that pyruvate dehydrogenase is not involved in the hormonal regulation of gluconeogenesis. Glucagon inhibited the incorporation of 10 mm [1-¹⁴C]pyruvate into glucose in hepatocytes from starved rats. This inhibition has been attributed to an inhibition of pyruvate dehydrogenase by the hormone (Zahlten et al., 1973, Proc. Nat. Acad. Sci. USA70, 3213–3218). However, dichloroacetate did not prevent the inhibition of glucose synthesis. Nor did glucagon alter the activity of pyruvate dehydrogenase in homogenates of cells that had been incubated with 10 mm pyruvate in the absence or presence of dichloroacetate. Thus, the inhibition by glucagon of pyruvate gluconeogenesis does not appear to be due to an inhibition of pyruvate dehydrogenase.     

The relation between the unit thread of chromosomes and isolated nucleohistone

Changes in the physical properties and molecular structure of isolated nucleohistone, induced by binding magnesium or other ions, have been studied. Nucleohistone has a net negative charge. Added magnesium binds tightly to the DNA phosphate groups that are not already complexed with histone. This results in neutralization of the net negative charge, a reduction in intermolecular repulsion, aggregation and compaction of the nucleohistone gel. The “unit thread” of nucleohistone observed in the electron microscope changes diameter from 110 Å to 230 Å on addition of magnesium before drying. However, X-ray diffraction studies fail to detect any changes in regular tertiary structure on adding divalent ions. The methods for dehydration commonly used in specimen preparation for electron microscopy appear to cause a complete loss of regular tertiary structure.We conclude that the DNA in hydrated nucleohistone is supercoiled, that the degree of regular supercoiling is independent of the presence of ions and that both the 110 Å and 230 Å threads observed in the electron microscope probably contain the distorted remnant of a single supercoil.     

Regulation of collagen synthesis by ascorbic acid. Ascorbic acid increases type I procollagen mRNA

Ascorbic acid specifically stimulates collagen production in cultured human skin fibroblasts, an effect that appears to be independent of its cofactor role in prolyl and lysyl hydroxylation. In order to investigate the level of regulation of ascorbic acid on collagen synthesis, we have translated mRNA in a cell-free system derived from rabbit reticulocytes. Total RNA was prepared from normal human skin fibroblasts and similar fibroblasts which had been exposed to 100 uM ascorbic acid for four days. Ascorbic acid treatment resulted in a twofold stimulation of procollagen mRNA whereas non-collagenous mRNA was unchanged. These results reveal that ascorbic acid has a preferential stimulating effect on type I procollagen mRNA.     

Tumor-mediated immunosuppression: Prevention by inhibitors of prostaglandin synthesis

The addition of MC16 tumor cells (a prostaglandin E₂-producing cell line induced in C57B1/6J mice by methylcholanthrene) to cultures of normal syngeneic spleen cells inhibits the antibody response of these cells to sheep red blood cells. This inhibition can be blocked by adding to the cultures prostaglandin synthetase inhibitors, such as indomethacin, flufenamic acid and aspirin. These MC16 tumor cells are also immunosuppressive . Mice bearing the syngeneic MC16 tumor become unresponsive to sheep red blood cells as the tumor grows. As in the test system, inhibitors of prostaglandin synthetases seem to block the immunosuppressive activity of MC16 cells since tumor-bearing mice, treated therapeutically with indomethacin, responded normally in their production of antibody to sheep red blood cells.     

Comparison of the metabolic effects of chylomicrons and their remnants on isolated hepatocytes

A comparison was made between the effects of chylomicrons and chylomicron remnants on metabolic processes of isolated hepatocytes. Since isolated triacylglycerol-rich lipoproteins are contaminated with nonesterified fatty acids, control incubations were conducted with an amount of fatty acid equivalent to the contaminating fatty acids present in the chylomicrons and the remnant preparations, respectively. Chylomicron remnants, produced in vitro by incubation of chylomicrons in postheparin rat plasma, caused marked inhibition of glycolysis, fatty acid synthesis, and cholesterol synthesis, along with marked stimulation of ketogenesis. These effects were traced to the release of nonesterified fatty acids from these remnant particles as a consequence of contamination with lipoprotein lipase, picked up by the particles during the incubation with rat plasma. Fatty acids inhibit glycolysis, cholesterol, and fatty acid synthesis, but enhance ketone body formation by isolated hepatocytes. Chylomicrons and remnants prepared in vivo by the injection of chylomicrons into functionally hepatectomized rats were not contaminated with lipoprotein lipase and did not inhibit glycolysis and cholesterol synthesis nor increase ketone body formation. These lipoprotein particles did, however, cause significant inhibition of fatty acid synthesis, with the chylomicrons being more effective on a protein basis than the remnants produced in vivo. The mechanism responsible for the inhibition of fatty acid synthesis by chylomicrons and remnants prepared in vivo remains to be resolved.     

Effects of tunicamycin upon glycoprotein synthesis and development of early mouse embryos

Tunicamycin, an antimetabolite which inhibits the N-glycosylation of proteins, does not block the initial cleavages of mouse embryos, even at relatively high concentrations. However, it can interfere with compaction and blastocyst formation. Although tunicamycin treatment from the two-cell or eight-cell stage can cause developmental arrest prior to hatching from the zona pellucida, much higher (sublethal) concentrations of the antimetabolite added at the morula or blastocyst stage do not specifically affect hatching of blastocysts, their attachment to the substratum, or outgrowth of trophoblast cells. The consequence of continuous exposure of embryos to moderate amounts (0.05 to 0.1 μg/ml) of tunicamycin through peri-implantation stages is death of trophoblast cells with little effect upon the cells of the inner cell mass (ICM). The latter give rise to apparently normal early endoderm cells in the presence of the antimetabolite. The incorporation of leucine, mannose, and fucose into acid-insoluble material by ICM cells is only minimally inhibited by tunicamycin. On the other hand, the antimetabolite causes a severe inhibition of incorporation of not only mannose, but also leucine, into acid-insoluble material in trophoblast cells. Thus, trophoblast cells resemble transformed cells by their extreme sensitivity to tunicamycin.     

Influence of valproic acid on hepatic carbohydrate and lipid metabolism

Valproic acid (dipropylacetic acid), an antiepileptic agent known to be hepatotoxic in some patients, caused inhibition of lactate gluconeogenesis, fatty acid oxidation, and fatty acid synthesis by isolated hepatocytes. The latter process was the most sensitive to valproic acid, 50% inhibition occurring at ca. 125 microM with cells from meal-fed female rats. The medium-chain acyl-CoA ester fraction was increased whereas coenzyme A (CoA), acetyl-CoA, and the long chain acyl-CoA fractions were decreased by valproic acid. The increase in the medium chain acyl-CoA fraction was found by high-pressure liquid chromatography to be due to the accumulation of valproyl-CoA plus an apparent CoAester metabolite of valproyl-CoA. Salicylate inhibited valproyl-CoA formation and partially protected against valproic acid inhibition of hepatic metabolic processes. Octanoate had a similar protective effect, suggesting that activation of valproic acid in the mitosol is required for its inhibitory effects. It is proposed that either valproyl-CoA itself or the sequestration of CoA causes inhibition of metabolic processes. Valproyl-CoA formation also appears to explain valproic acid inhibition of gluconeogenesis by isolated kidney tubules. No evidence was found for the accumulation of valproyl-CoA in brain tissue, suggesting that the effects of valproic acid in the central nervous system are independent of the formation of this metabolite.