Optimized spectrophotometric assay for the completely activated pyruvate dehydrogenase complex in fibroblasts.
BACKGROUND
Analysis of the pyruvate dehydrogenase complex (PDHc) activity in human skin fibroblasts is hampered by low enzyme activity in the cells. The most commonly used radiochemical method detects the formation of (14)CO(2), an endproduct of the E1 component of PDHc, from [1-(14)C]pyruvate.


METHODS
We report a spectrophotometric method for the analysis of PDHc activity in fibroblasts based on detection of NADH formation via a p-iodonitrotetrazolium violet (INT)-coupled system. We investigated in detail the specific requirements of this assay, such as cofactor requirements and the effects of suggested stimulatory compounds and different cell disruption procedures. The reliability of the optimized assay was studied by investigation of patients previously diagnosed with PDHc deficiency and by comparison with results from the radiochemical method.


RESULTS
Mean (SD) total PDHc activities were 136 (31) and 58 (21) mU/U of citrate synthase in fibroblast homogenates from 10 healthy volunteers and 7 PDHc-deficient patients, respectively, by the spectrophotometric assay. Similar results were obtained in a mitochondrial fraction. Dithiothreitol (DTT) increased the nonspecific inhibitor-insensitive rate with less pronounced effect on the specific rate of PDHc activity. Administration of DTT increased PDHc activity to 193 (3)% of control activity (without DTT), but decreased the inhibitor-sensitive rate from 99 (0.3)% (without DTT) to 69 (2)% (with 0.3 mmol/L DTT).


CONCLUSIONS
The simple, optimized spectrophotometric assay for PDHc analysis allows reliable investigation of the enzyme complex in human skin fibroblasts.
Schwab
Marina A
MA
Division of Metabolic and Endocrine Diseases, University Children's Hospital Heidelberg, 69120 Heidelberg, Germany. marina_schwab@med.uni-heidelberg.de
Kölker
Stefan
S
van den Heuvel
Lambert P
LP
Sauer
Sven
S
Wolf
Nicole I
NI
Rating
Dietz
D
Hoffmann
Georg F
GF
Smeitink
Jan A M
JA
Okun
Jürgen G
JG
D002478:Cells, Cultured; D004229:Dithiothreitol / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology* / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015325:Pyruvate Dehydrogenase Complex Deficiency Disease / Q000201:enzymology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012867:Skin / Q000166:cytology; D013053:Spectrophotometry / Q000379:methods
D011768:Pyruvate Dehydrogenase Complex; D004229:Dithiothreitol