An NADH-linked spectrophotometric assay for pyruvate dehydrogenase complex in crude tissue homogenates.

Hinman, Lois M.; Blass, John P.

doi:10.1016/s0021-9258(19)69029-0

Cited by 127 publications

(16 citation statements)

References 18 publications

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“…Pyruvate dehydrogenase activity was measured using a coupled reaction with iodonitrotetrazolium, as described in [108], with several modifications, including omission of dithiothreitol from the reaction medium, suggested in [109]. The final assay medium comprised of 50 mM potassium phosphate buffer, 5 mM L-carnitine, 2.5 mM NAD + , 0.2 mM ThDP, 0.1 mM coenzyme A, 1 mM MgCl 2 , 1 mg/mL bovine serum albumin, 0.6 mM iodonitrotetrazolium chloride, 6.5 µM phenazine methosulfate, and 5 mM pyruvate, pH = 7.5.…”

Section: Metabolite and Enzyme Activity Assaysmentioning

confidence: 99%

Increasing Inhibition of the Rat Brain 2-Oxoglutarate Dehydrogenase Decreases Glutathione Redox State, Elevating Anxiety and Perturbing Stress Adaptation

et al. 2022

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Specific inhibitors of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) are administered to animals to model the downregulation of the enzyme as observed in neurodegenerative diseases. Comparison of the effects of succinyl phosphonate (SP, 0.02 mmol/kg) and its uncharged precursor, triethyl succinyl phosphonate (TESP, 0.02 and 0.1 mmol/kg) reveals a biphasic response of the rat brain metabolism and physiology to increasing perturbation of OGDH function. At the low (TE)SP dose, glutamate, NAD+, and the activities of dehydrogenases of 2-oxoglutarate and malate increase, followed by their decreases at the high TESP dose. The complementary changes, i.e., an initial decrease followed by growth, are demonstrated by activities of pyruvate dehydrogenase and glutamine synthetase, and levels of oxidized glutathione and citrulline. While most of these indicators return to control levels at the high TESP dose, OGDH activity decreases and oxidized glutathione increases, compared to their control values. The first phase of metabolic perturbations does not cause significant physiological changes, but in the second phase, the ECG parameters and behavior reveal decreased adaptability and increased anxiety. Thus, lower levels of OGDH inhibition are compensated by the rearranged metabolic network, while the increased levels induce a metabolic switch to a lower redox state of the brain, associated with elevated stress of the animals.

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Section: Metabolite and Enzyme Activity Assaysmentioning

confidence: 99%

Increasing Inhibition of the Rat Brain 2-Oxoglutarate Dehydrogenase Decreases Glutathione Redox State, Elevating Anxiety and Perturbing Stress Adaptation

et al. 2022

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“…Activities of OGDHC, glutamate dehydrogenase, malate dehydrogenase, and malic enzyme were measured via spectrophotometric assay, detecting NADH or NADPH absorbance change at 340 nm. Pyruvate dehydrogenase complex was assayed spectrophotometrically using a coupled reaction between the reaction product, NADH, and iodonitrotetrazolium, detecting absorbance change at 500 nm [ 75 , 76 ]. Glutamine synthase was assayed by endpoint method, measuring the absorbance of its side reaction product, γ-gutamyl hydroxamate-Fe 3+ complex in acidic condition at 540 nm [ 77 ].…”

Section: Methodsmentioning

confidence: 99%

Acute Prenatal Hypoxia in Rats Affects Physiology and Brain Metabolism in the Offspring, Dependent on Sex and Gestational Age

Граф

Maslova

Artiukhov

et al. 2022

IJMS

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Hypoxia is damaging to the fetus, but the developmental impact may vary, with underlying molecular mechanisms unclear. We demonstrate the dependence of physiological and biochemical effects of acute prenatal hypoxia (APH) on sex and gestational age. Compared to control rats, APH on the 10th day of pregnancy (APH-10) increases locomotion in both the male and female offspring, additionally increasing exploratory activity and decreasing anxiety in the males. Compared to APH-10, APH on the 20th day of pregnancy (APH-20) induces less behavioral perturbations. ECG is changed similarly in all offspring only by APH-10. Sexual dimorphism in the APH outcome on behavior is also observed in the brain acetylation system and 2-oxoglutarate dehydrogenase reaction, essential for neurotransmitter metabolism. In view of the perturbed behavior, more biochemical parameters in the brains are assessed after APH-20. Of the six enzymes, APH-20 significantly decreases the malic enzyme activity in both sexes. Among 24 amino acids and dipeptides, APH-20 increases the levels of only three amino acids (Phe, Thr, and Trp) in male offspring, and of seven amino acids (Glu, Gly, Phe, Trp, Ser, Thr, Asn) and carnosine in the female offspring. Thus, a higher reactivity of the brain metabolism to APH stabilizes the behavior. The behavior and brain biochemistry demonstrate sexually dimorphic responses to APH at both gestational stages, whereas the APH effects on ECG depend on gestational age rather than sex.

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“…PGDHC activity was measured spectrophotometrically using a modified method of Hinman and Blass [50] by following the formation of NADH at 340nm at 37 °C. Isolated mitochondria were resuspended in the assay buffer (50mM phosphate buffer, pH 7.4).…”

Section: Assays Of Mitochondrial Enzymes Pyruvate Dehydrogenase Complex (Pgdhc)mentioning

confidence: 99%

Potential Role of Mitochondrial Dysfunction in Diabetic Hypertriglyceridemia

Parthasarathy¹

2017

JETR

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Type 2 diabetes patients have increased oxidative stress and hypertriglyceridemia. We tested the hypothesis that these two are related and the latter could be the result of peroxide-mediated mitochondrial dysfunction and increased acetate production. Treatment of isolated liver mitochondria or primary hepatocytes with oxidized linoleic acid (LOOH) or hydrogen peroxide (H 2 O 2 ) resulted in a drastic decrease in activities of pyruvate dehydrogenase (PDHC), aconitase and α-ketoglutarate dehydrogenase (KDHC). In contrast, the incorporation of 14C-acetate into lipids was not affected by peroxides suggesting that fatty acid synthesis was not affected. The livers of Db/db diabetic mice showed reduced enzyme activities as compared to control non-diabetic mice. In vitro reaction of pyruvate in the presence of LOOH or H 2 O 2 showed that pyruvate was non-enzymatically converted to acetate together with the release of carbon dioxide (CO 2 ). These results show that diabetic mice may convert more pyruvate non-enzymatically into acetate in the cytoplasm in the presence of peroxides. In addition, mitochondria in diabetic state may have poor capacity to utilize acetate by TCA cycle to generate energy. Combined with the findings that peroxides did not affect acetate incorporation of acetate into fatty acids, one could expect a net increase in fatty acid triacylglycerol (TG) production.

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An NADH-linked spectrophotometric assay for pyruvate dehydrogenase complex in crude tissue homogenates.

Cited by 127 publications

References 18 publications

Increasing Inhibition of the Rat Brain 2-Oxoglutarate Dehydrogenase Decreases Glutathione Redox State, Elevating Anxiety and Perturbing Stress Adaptation

Increasing Inhibition of the Rat Brain 2-Oxoglutarate Dehydrogenase Decreases Glutathione Redox State, Elevating Anxiety and Perturbing Stress Adaptation

Acute Prenatal Hypoxia in Rats Affects Physiology and Brain Metabolism in the Offspring, Dependent on Sex and Gestational Age

Potential Role of Mitochondrial Dysfunction in Diabetic Hypertriglyceridemia

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