Kinetic Studies of Mouse Brain Transketolase

Abstract: The activity of transketolase in mouse brain was 5.7 nmol/min/mg protein measured by an enzyme-coupled spectrophotometric assay. The apparent K_m for ribose-5-phosphate was 330 μ M , for d -xylulose-5-phosphate was 120 μ M , and for thiamine pyrophosphate was 7 μ M . However, thiamine pyrophosphate remained tightly bound to transketolase in homogenates in which it dissociated completely from another thiamine pyrophosphate- dependent enzyme, the pyruvate dehydrogenase complex. These data suggest that loss of transketolase activity is likely to be a later consequence of thiamine deficiency in mammalian brain than is decreased activity of pyruvate dehydrogenase complex.     

Pyruvate Dehydrogenase Activity in Osmotically Shocked Rat Brain Mitochondria: Stimulation by Oxaloacetate

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.     

Activities of thiamine-dependent enzymes in two experimental models of thiamine-deficiency encephalopathy:

Chronic thiamine deprivation in the rat leads to selective neuropathological damage in brainstem structures whereas treatment with the central thiamine antagonist, pyrithiamine, results in more widespread damage. In order to further elucidate the neurochemical mechanisms responsible for this selective damage, the thiamine-dependent enzyme complex pyruvate dehydrogenase (PDHC) was measured in 10 brain structures in the rat during progression of thiamine deficiency produced by chronic deprivation or by pyrithiamine treatment. Feeding of a thiamine-deficient diet to adult rats resulted in 5–7 weeks in ataxia and loss of righting reflex accompanied by decreased blood transketolase activities. PDHC activities were selectively decreased by 15–30% in midbrain and pons (lateral vestibular nucleus). Thiamine treatment of symptomatic rats led to reversal of neurological signs and to concomitant reductions of the cerebral PDHC abnormalities. Daily pyrithiamine treatment led within 3 weeks to loss of righting reflex and convulsions and to decreased blood transketolase of a comparable magnitude to that observed in chronic thiamine-deprived rats. No significant regional alterations of PDHC, however, were observed in pyrithiamine-treated rats.     

Studies on the Pyruvate Dehydrogenase Complex in Brain with the Arylamine Acetyltransferase-Coupled Assay

Abstract: A spectrophotometric assay for the brain pyruvate dehydrogenase complex (PDHC) with arylamine acetyltransferase (ArAT; EC 2.3.1.5) to follow the production of acetyl-CoA has been standardized. Activity was proportional to time and protein. It depended completely on added pyruvate, CoA, NAD, and MgCI₂, and partially on thiamine pyrophosphate, Triton X-100, and a sulfhydryl compound. The activities are the highest in the literature for brain PDHC (50 nmol/min/mg protein) and equal the maximum recorded rates of pyruvate flux for brain in vivo . Activities as low as 0.6 nmol/min could be measured. Use of ArAT of different purities (1–2-fold and 11–%-fold) allowed convenient measurement of total PDHC (ArAT-I) and of the active form of PDHC (ArAT-II). The proportion of PDHC in the active form was 50% in mouse brain, 30% in rat brain, and 10% in mouse liver. Total PDHC activity was unchanged postmortem during storage of mouse brain in situ at +4°C or at -20°C for 3 days or at +20°C for 24 h. The relative specific activity of PDHC in cytoplasmic or synaptoplasmic fractions was less than that of two other mitochondrial enzymes, fumarase (EC 4.2.1.2) and monoamine oxidase (EC 1.4.3.4), which argues strongly against the hypothesis of a cytoplasmic PDHC in cholinergic nerve endings.     

Determination of pyruvate dehydrogenase and acetyl-CoA synthetase activities using citrate synthase

Methods are described for the assay of pyruvate dehydrogenase and acetyl-CoA synthetase activities in rat brain subcellular fractions. Citrate synthase and oxaloacetate serve as a trapping system in these assays. The methods permit the determination of a large number of samples of different turbidity with satisfactory precision. Highest activities of pyruvate dehydrogenase and acetyl-CoA synthetase (117.7 and 7.29 nmol/min/mg of protein, respectively) were found in rat brain mitochondria. A three times lower activity of acetyl-CoA synthetase and negligible of pyruvate dehydrogenase was found in brain cytosol.     

A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver.

The total activity of pyruvate dehydrogenase in mitochondria isolated from rat brain and liver was 53.5 and 14.2nmol/min per mg of protein respectively. Pyruvate dehydrogenase in liver mitochondria incubated for 4 min at 37 degrees C with no additions was 30% in the active form and this activity increased with longer incubations until it was completely in the active form after 20 min. Brain mitochondrial pyruvate dehydrogenase activity was initially high and did not increase with addition of Mg2+ plus Ca2+ or partially purified pyruvate dehydrogenase phosphatase or with longer incubations. The proportion of pyruvate dehydrogenase in the active form in both brain and liver mitochondria changed inversely with changes in mitochondrial energy charge, whereas total pyruvate dehydrogenase did not change. The chelators citrate, isocitrate, EDTA, ethanedioxybis(ethylamine)tetra-acetic acid and Ruthenium Red each lowered pyruvate dehydrogenase activity in brain mitochondria, but only citrate and isocitrate did so in liver mitochondria. These chelators did not affect the energy charge of the mitochondria. Mg2+ plus Ca2+ reversed the pyruvate dehydrogenase inactivation in liver, but not brain, mitochondria. The regulation of the activation-inactivation of pyruvate dehydrogenase in mitochondria from rat brain and liver with respect to energy charge is similar and may be at least partially regulated by this parameter, and the effects of chelators differ in the two types of mitochondria.     

Fatty acid activation in the brains of neurologically mutant mice

1. Palmitoyl-CoA synthetase activity was assayed in subfractions of control and Quaking, Jimpy, Shiverer and Trembler mouse brain. 2. Mouse brain palmitoyl-CoA synthetase activity is not altered during myelination. 3. Mouse brain enzyme activity (homogenate 1.5 +/- 0.3 nmol palmitoyl carnitine/min/per mg protein crude mitochondria 0.6 +/- 0.1 nmol/min/per mg protein and microsomes 1.9 +/- 0.3 nmol/min/per mg protein) does not differ markedly from rat and rabbit brain activity. 4. The lesions of the above mutants which affect myelination and lipid synthesis do not include the enzyme palmitoyl-CoA synthetase.     

Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide.

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.     

THE CONTROL OF PYRUVATE DEHYDROGENASE IN ISOLATED BRAIN MITOCHONDRIA

Abstract— The activity and control of the pyruvate dehydrogenase complex in isolated rat brain mitochondria has been studied. The activity of this complex in mitochondria as isolated from normal fed rats was 78 ± 10nmol.min⁻¹ mg mitochondrial protein⁻¹ (n = 18) which represented 70% of the total pyruvate dehydrogenase activity. The pyruvate dehydrogenase in isolated brain mitochondria could be inactivated by incubation in the presence of ATP, oligomycin and NaF. The rate of inactivation was dependent upon the added ATP concentration but inactivation below approx 30% of the total pyruvate dehydrogenase activity could not be achieved. The inactivation of pyruvate dehydrogenase in brain mitochondria was inhibited by pre-incubation with pyruvate. Reactivation of inactivated pyruvate dehydrogenase in rat brain mitochondria was incomplete in the incubation medium unless 10mM-Mg²⁺+ 1 mM-Ca²⁺ were added; NaF, however, prevented any reactivation (Fig. 4). It is concluded that the pyruvate dehydrogenase complex in rat brain mitochondria is controlled in a manner similar to that in other tissues, and that pyruvate protection of pyruvate dehydrogenase activity may be important in maintaining brain energy metabolism.     

Measurement of alpha-ketoglutarate dehydrogenase activity in tissue extracts and human platelets using reversed-phase high-performance liquid chromatography

A new method for the determination of the activity of alpha-ketoglutarate dehydrogenase complex (KGDHC) in mouse brain and liver mitochondria and in human platelets using reversed-phase high-performance liquid chromatography is described. This method is based on the quantification of succinyl-CoA formed in the reaction catalyzed by KGDHC. Succinyl-CoA was separated using a YMC-Pack C8 column employing isocratic elution and detected spectrophotometrically at 254 nm. The detection limit of succinyl-CoA was 0.05 nmol. Succinyl-CoA in the supernatant of the assay mixture was stable for several hours at 4 degrees C and for a week when stored at -20 degrees C. The KGDHC assay showed good linearity with time and added protein, and all tissues demonstrated an absolute requirement for added alpha-ketoglutarate, nicotinamide dinucleotide, and coenzyme A and partial or no requirement for thiamine pyrophosphate, magnesium chloride, and dithiothreitol. The specific activities in liver and brain mitochondria and platelet homogenates determined by the present method were 19.2 +/- 0.9, 18.1 +/- 2.8, and 2.6 +/- 0.3 nmol/min/mg protein, respectively. In human platelets, the present method gives higher specific activity and lower blank values than a prior method using 14CO2 and may be useful in the diagnosis of KGDHC deficiency. This method is simple, rapid, and can be readily employed for the determination of KGDHC activity in various animal tissues and human platelets.     

THE REGULATION OF PYRUVATE DEHYDROGENASE IN BRAIN IN VIVO

—The activity of pyruvate dehydrogenase in the brains of mice frozen in liquid nitrogen was 14·0 nmol/min per mg protein. It rose to 23·8 nmol/min per mg protein after incubation of the brain homogenate with 10mm -MgCl₂ to activate (dephosphorylate) the enzyme, indicating that approx 60% of the enzyme was originally in the active form. Treatment with amobarbital or pentobarbital halved the proportion of pyruvate dehydrogenase in the active form. The proportion of pyruvate dehydrogenase in the active form increased during ischemia, activation being complete within one min. Anesthesia with amobarbital slowed the activation during ischemia but did not alter the total amount of pyruvate dehydrogenase activity. The concentration of ATP, the ATP/ADP ratio and the adenylate energy charge increased as the proportion of pyruvate dehydrogenase in the active form decreased during barbiturate anesthesia, and they decreased as the proportion of pyruvate dehydrogenase in the active form increased during ischemia. After treatment with insulin, the proportion of pyruvate dehydrogenase in the active form increased by 30%. but the energy charge did not change. Treatment of mice with ether, morphine, ethanol, or diazepam did not change the proportion of pyruvate dehydrogenase in the active form although these treatments have been reported to alter pyruvate oxidation in brain in vivo. Treatments which altered pyruvate oxidation in the brain did not consistently alter the proportion of pyruvate dehydrogenase in the active form, unless they also altered energy charge.     

Development of a quantitative assay method for 3 beta-hydroxy-delta 5-steroid dehydrogenase in the rat testis

We investigated the first step of the sex steroid hormone biosynthesis pathway by assaying the activities of 3 beta-hydroxy-delta 5-steroid dehydrogenase, the rate-limiting enzyme in this pathway. We have developed a simple and rapid colorimetric assay for 3 beta-hydroxy-delta 5-steroid dehydrogenase in rat testis. The supernatant from rat testis tissue homogenates were used for the enzyme assay. The enzyme activity was determined by measuring the absorbance at 570nm which indicates the rate of conversion of pregnenolone into progesterone in the presence of NAD, using phenazine methosulfate and nitro blue tetrazolium as the color reagent. The activity of this enzyme ranged from 4.57+/-1.34 to 10.56+/-2.13 nmol/mg protein/min with a mean activity of 8.96+/-1.27 nmol/mg protein/min. The K(m) of the enzyme at an optimum pH of 7.25 was about 4.7+/-0.12 nM.     

Effect of Pyrithiamine Treatment and Subsequent Thiamine Rehabilitation on Regional Cerebral Amino Acids and Thiamine-Dependent Enzymes

Pyrithiamine-induced thiamine-deficiency encephalopathy in the rat shows many neuropathological and biochemical similarities to Wernicke's encephalopathy in humans. Treatment of rats with pyrithiamine resulted in moderate reductions of glutamate in thalamus and pons and in generalized severe reductions of aspartate in pons (by 89%, p less than 0.01), thalamus (by 83%, p less than 0.01), cerebellum (by 53%, p less than 0.01), and cerebral cortex (by 33%, p less than 0.05). Alanine concentrations were concomitantly increased. Activities of the thiamine-dependent enzyme alpha-ketoglutarate dehydrogenase (alpha KGDH) were decreased in parallel with the aspartate decreases; pyruvate dehydrogenase complex activities were unchanged in all brain regions. Following thiamine administration to symptomatic pyrithiamine-treated rats, neurological symptoms were reversed and concentrations of glutamate, aspartate, and alanine, as well as alpha KGDH activities, were restored to normal in cerebral cortex and pons. Aspartate levels and alpha KGDH activities remained below normal values, however, in thalamus. Thus, pyrithiamine treatment leads to reductions of cerebral alpha KGDH and (1) decreased glucose (pyruvate) oxidation resulting in accumulation of alanine and (2) decreased brain content of glutamate and aspartate. Such changes may be of key significance in the pathophysiology of the reversible and irreversible signs of Wernicke's encephalopathy in humans.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of the matrix calcium level in the enhancement of mitochondrial pyruvate carboxylation by glucagon pretreatment.

The effect of Ca2+ on the rate of pyruvate carboxylation was studied in liver mitochondria from control and glucagon-treated rats, prepared under conditions that maintain low Ca2+ levels (1-3 nmol/mg of protein). When the matrix-free [Ca2+] was low (less than 100 nM), the rate of pyruvate carboxylation was not significantly different in mitochondria from control and glucagon-treated rats. Accumulation of 5-8 nmol of Ca2+/mg, which increased the matrix [Ca2+] to 2-5 microM in both preparations, significantly enhanced pyruvate carboxylase flux by 20-30% in the mitochondria from glucagon-treated rats, but had little effect in control preparations. Higher levels of Ca2+ (up to 75 nmol/mg) inhibited pyruvate carboxylation in both preparations, but the difference between the mitochondria from control and glucagon-treated animals was maintained. The enhancement of pyruvate dehydrogenase flux by mitochondrial Ca2+ uptake was also significantly greater in mitochondria from glucagon-treated rats. These differential effects of Ca2+ uptake on enzyme fluxes did not correlate with changes in the mitochondrial ATP/ADP ratio, the pyrophosphate level, or the matrix volume. Arsenite completely prevented 14CO2 incorporation when pyruvate was the only substrate, but caused only partial inhibition when succinate and acetyl carnitine were present as alternative sources of energy and acetyl-CoA. Under these conditions, mitochondria from glucagon-treated rats were less sensitive to arsenite than the control preparations, even at low Ca2+ levels. We conclude that the Ca(2+)-dependent enhancement of pyruvate carboxylation in mitochondria from glucagon-treated rats is a secondary consequence of pyruvate dehydrogenase activation; glucagon treatment is suggested to affect the conditions in the mitochondria that change the sensitivity of the pyruvate dehydrogenase complex to dephosphorylation by the Ca(2+)-sensitive pyruvate dehydrogenase phosphatase.     

Simultaneous measurement of D-serine dehydratase and d-amino acid oxidase activities by the detection of 2-oxo-acid formation with reverse-phase high-performance liquid chromatography

N-methyl-D-aspartate receptors (NMDARs) play critical roles in excitatory synaptic transmission in the vertebrate central nervous system. NMDARs need D-serine for their channel activities in various brain regions. In mammalian brains, D-serine is produced from L-serine by serine racemase and degraded by D-amino acid oxidase (DAO) to 3-hydroxypyruvate. In avian organs, such as the kidney, in addition to DAO, D-serine is also degraded to pyruvate by D-serine dehydratase (DSD). To examine the roles of these two enzymes in avian brains, we developed a method to simultaneously measure DAO and DSD activities. First, the keto acids produced from D-serine were derivatized with 3-methyl-2-benzothiazolinone hydrazone to stable azines. Second, the azine derivatives were quantified by means of reverse-phase high-performance liquid chromatography using 2-oxoglutarate as an internal standard. This method allowed the simultaneous detection of DAO and DSD activities as low as 100 pmol/min/mg protein. Chicken brain showed only DSD activities (0.4+/-0.2 nmol/min/mg protein) whereas rat brain exhibited only DAO activities (0.7+/-0.1 nmol/min/mg protein). This result strongly suggests that DSD plays the same role in avian brains, as DAO plays in mammalian brains. The present method is applicable to other keto acids producing enzymes with minor modifications.     

Activities of thiamine-dependent enzymes in two experimental models of thiamine-deficiency encephalopathy 2. α-ketoglutarate dehydrogenase

Chronic thiamine deprivation in the rat leads to selective neuropathological damage to pontine structures. Onset of neurological symptoms of thiamine deprivation (ataxia, loss of righting reflex) was accompanied by selective decreases (of the order of 30%) in the activity of -ketoglutarate dehydrogenase (KGDH) in lateral vestibular nucleus and hypothalamus. Enzyme activities were decreased to a lesser extent in medulla oblongata, striatum and hippocampus and were unchanged in other brain structures. No changes in KGDH occurred prior to the onset of neurological signs of thiamine deprivation. Administration of the central thiamine antagonist, pyrithiamine, results within 3 weeks in loss of righting reflex and convulsions and in more widespread neuropathological changes than those observed following thiamine deprivation. KGDH activities were found to be substantially diminished in all brain regions studied following pyrithiamine treatment with most severe changes occurring in brain regions found to be vulnerable to pyrithiamine (lateral vestibular nucleus, hypothalamus, midbrain, medullapons). In some cases, KGDH changes preceded the appearance of neurological symptoms of pyrithiamine treatment. Such decreases in KGDH may explain previous findings of region-selective changes in energy metabolism and of decreased synthesis of glucose-derived neurotransmitters (acetylcholine, GABA, glutamate) in pyrithiamine-treated rat brain. Thiamine administration to symptomatic pyrithiamine treated rats resulted in reversal of neurological signs of encephalopathy and in normalisation of defective KGDH activity in all brain regions. These findings suggest that the reversible neurological symptoms associated with Wernicke's Encephalopathy in man likely result from region-selective impairment of KGDH.     

Energy and glucose pathways in thiamine deficient primary rat brain microvascular endothelial cells

Thiamine deficiency (TD) results in lactate acidosis, which is associated with neurodegeneration. The aim of this study was to investigate this alteration in primary rat brain endothelia. Spectrophotometric analysis of culture media revealed that only a higher concentration of pyrithiamine, which accelerates the intracellular blocking of thiamine, significantly elevated the lactate level and lactate dehydrogenase activity within 7 days. The medium without pyrithiamine and with a thiamine concentration comparable to pathophysiological plasma levels mildly reduced only the activity of transketolase. This suggests that significant metabolic changes may not occur at the early phase of TD in cerebral capillary cells, while anaerobic glycolysis in capillaries may be mediated during late stage/chronic TD.     

Acute starvation affects rat adrenal steroidogenesis

To determine how starvation affects adrenal steroidogenesis we measured the activities of 3 adrenal enzymes involved in corticosterone biosynthesis in a group of adult female rats. The animals were either starved for 7 days or fed ad libitum for the same period. Relative adrenal weight and plasma corticosterone levels were increased in the experimental group of animals compared to the control group (40 +/- 2 vs 27 +/- 1 mg/100 g body weight, P less than 0.001, and 45 +/- 4 vs 30 +/- 5 ng/dl, P less than 0.05 respectively). There were no differences in plasma ACTH levels between the groups (34 +/- 5 vs 26 +/- 4 pg/ml). 11-Hydroxylase activity was increased in the starved group of animals (18 +/- 3 vs 8 +/- 2 nmol/mg protein/min, P less than 0.01). 3 beta-Hydroxysteroid dehydrogenase and 21-hydroxylase activities were not different between the groups (19 +/- 2 vs 16 +/- 1 nmol/mg protein/min, and 100 +/- 10 vs 110 +/- 10 pmol/mg protein/min respectively). These results suggest that acute starvation in rats produces an increase in adrenal 11-hydroxylase activity.     

The effects of a thiamine antagonist, pyrithiamine, on levels of selected metabolic intermediates and on activities of thiamine-dependent enzymes in brain and liver

(1) The effects of thiamine deficiency as produced by pyrithiamine injections have been studied in the weanling mouse. Selected metabolites were measured in extracts from brain and liver of quick-frozen animals. Pyruvate and α-oxoglutarate dehydrogenases and transketolase were also measured. (2) In deficient brain, pyruvate and α-oxoglutarate levels were greatly increased. Xylulose-5-P and 6-P-gluconate were more than doubled. Lactate, glucose-6-P, glucose and P-creatine were moderately elevated, and ATP was increased a little. Glutamate was depressed. (3) In deficient liver, α-oxoglutarate was much increased and ATP was twice normal. Glycogen, glucose, glucose-6-P, 6-P-gluconate, pyruvate, and glutamate were not different from the controls. Lactate was depressed. (4) Pyruvate dehydrogenase activity was reduced to 25 per cent or less in brain and liver. Transketolase and α-oxoglutarate dehydrogenase activities were reduced to 50 per cent in both organs. (5) Thiamine treatment, within 5 hr, largely reversed the metabolite changes brought on by pyrithiamine in brain. At the same time pyruvate and α-oxoglutarate dehydrogenase activities were increased 60 per cent or more in both brain and liver. Transketolase activity in liver was only increased 20 per cent at this time, however, and in brain was unchanged. (6) The results are interpreted to indicate that inhibition of pyruvate and α-oxoglutarate dehydrogenases in brain is sufficient to depress in vivo function. The same seems true for the inhibition of α-oxoglutarate dehydrogenase in liver. However, the changes seen in brain 6-P-gluconate and xyluIose-5-P probably depend on factors other than, or in addition to, the decrease in transketolase activity. It seems worthy of emphasis that in spite of the partial metabolic blocks high-energy phosphate stores were actually increased.