Effect of a Deficiency of Thiamine on Brain Pyruvate Dehydrogenase: Enzyme Assay by Three Different Methods

Abstract

A simple and rapid method based on the NADH-linked reduction of a tetrazolium dye was described for the determination of pyruvate dehydrogenase activity in rat brain homogenates. The method (method 3) gave a value of 36.06 +/- 1.24 nmol of pyruvate utilised/min/mg of whole brain protein. This value was higher than that obtained by measurement of the rate of decarboxylation of [1-14C]pyruvate (15.10 +/- 0.88 nmol/min/mg of protein; method 1) and was comparable with the rate of transfer of acetyl groups to an arylamine (39.04 +/- 1.32 nmol/min/mg of protein; method 2). A critique of the values reported by others by different methods was given. The pyruvate dehydrogenase activity in the mitochondria isolated from rat brain was in the "active" (nonphosphorylated) form. A deficiency of thiamine in rats was produced by treatment with pyrithiamine, an antagonist of thiamine. This treatment resulted in abnormal neurological signs, such as ataxia and convulsions. The measurement of the total activity of pyruvate dehydrogenase in the brain by all three methods showed no significant change in the enzymic activity in thiamine-deficient rats after treatment with pyrithiamine. The activities of the enzyme in the brains of pair-fed animals were similar to those in the controls.

Selective Reductions in the Activity of the Pyruvate Dehydrogenase Complex in Mitochondria Isolated from Brain Subregions following Forebrain Ischemia in Rats

“…Consistent with previous investigations (Sims, 1990), the specific activities of mitochondrial en zymes measured in the present study were two-to threefold greater than values reported previously (Lai and Cooper, 1986;Mizuno et aI., 1987;Elnageh and Gaitonde, 1988) for brain mitochondria isolated using alternative preparative procedures. As the isolated mitochondrial population is likely to in clude organelles from both neuronal and glial cells, no conclusions can be reached as to the cellular location of the observed changes in the dorsolateral striatum.…”

“…PDHC activity was determined spectrophotometrically by the method of Hinman and Blass (1981) with minor modifications as described by Elnageh and Gaitonde (1988). For measurements of the total activity of the PDHC, mitochondria were suspended in 250 ILl buffer containing 1 mM 2-mercaptoethanol, 1 mM EDT A, 10 mM MgCI2, 0.…”

“…For spectrophotometric determination of PDHC activ ity, the assay mixture in a final volume of 1 ml contained 50 mM Tris-HCI (pH 7.8), 0.5 mM EDTA, 0.2% (wt/vol) Triton X-IOO, 2.5 mM NAD+, 0.1 mM coenzyme A, 1 mM MgCI2, 0.1 mM oxalate, 1 mg/ml bovine serum albu min, 0.6 mM INT, 7 U of lipoamide dehydrogenase, 0.2 mM thiamine pyrophosphate, 5 mM pyruvate (Elnageh and Gaitonde, 1988), and 150 ILl stopped mitochondrial suspension containing -0.03 mg of mitochondrial pro tein. The reference cuvette was identical except that no pyruvate was added.…”

“…Assays were performed at 3rC and the production of reduced INT was followed at 500 nm. The activity of PDHC was linear between 2 and 10 min of incubation and for mitochondrial protein values up to at least 0.06 mg. An extinction coefficient of 15,400 was used to calculate enzyme activity (Elnageh and Gaitonde, 1988).…”

“…PDHC activity was measured using a dye coupled spectrophotometric assay that monitors production of NADH (Hinman and Blass, 1981;EI nageh and Gaitonde, 1988). This procedure was chosen as NADH production requires all three en zymes that contribute to the overall reaction of PDHC (Wieland, 1983;Randle, 1986), whereas al ternative procedures based on monitoring CO2 pro duction (Elnageh and Gaitonde, 1988) principally measure the initial decarboxylation step (catalyzed by the El component of PDHC) and may underes timate the effects of changes in the other compo nents.…”