The measurement of coenzyme A and a coenzyme A-dependent enzyme. In microdissected epidermal material using coupled enzyme and bioluminescent reactions.
A sensitive micromethod for the determination of Coenzyme A and its esters down to about 0.2 pmol in a volume of 10 microliters and of the activity of citrate synthase is outlined. Epidermal material from healthy and psoriatic skin was utilized in microgram quantity as tissue source. The assay utilizes the ketoglutarate dehydrogenase reaction to yield NADH on addition of free Coenzyme A and the subsequent measurement of NADH by a bioluminescent reaction with Acromobacter fischerii. The total Coenzyme A content in six healthy subjects measured in stratum Malpighii was 1.58 +/- 0.19 mmol per kg dry weight. In six psoriatic patients non-involved and involved epidermis contained 1.51 +/- 0.27 and 1.50 +/- 0.25 mmol/kg, respectively. Long-chain acyl-Coenzyme A comprised about 20% in lesion-free skin and 60% of total content in the involved psoriatic epidermis. The activity of citrate synthase in basal layers of healthy epidermis was 0.30 +/- 0.04 mkat/kg dry weight.
D000818:Animals; D002950:Citrate (si)-Synthase / Q000032:analysis; D003065:Coenzyme A / Q000032:analysis*; D006168:Guinea Pigs; D006801:Humans; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008722:Methods; D011565:Psoriasis / Q000201:enzymology; D012867:Skin / Q000201:enzymology*
D007655:Ketoglutarate Dehydrogenase Complex; D002950:Citrate (si)-Synthase; D003065:Coenzyme A