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Clinical Chemistry 51: 151-160, 2005. First published November 18, 2004; 10.1373/clinchem.2004.033852
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(Clinical Chemistry. 2005;51:151-160.)
© 2005 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Optimized Spectrophotometric Assay for the Completely Activated Pyruvate Dehydrogenase Complex in Fibroblasts

Marina A. Schwab1,2,a, Stefan Kölker1,2, Lambert P. van den Heuvel2,4, Sven Sauer1, Nicole I. Wolf3, Dietz Rating3, Georg F. Hoffmann1, Jan A.M. Smeitink2,4 and Jürgen G. Okun1

1 Division of Metabolic and Endocrine Diseases and 3 Department of Neuropediatrics, University Children’s Hospital Heidelberg, Heidelberg, Germany.
2 Department of Pediatrics, University Medical Center Nijmegen, Nijmegen, The Netherlands.
4 Nijmegen Center for Mitochondrial Disorders (NCMD), Nijmegen, The Netherlands.

aAddress correspondence to this author at: Division of Metabolic and Endocrine Diseases, University Children’s Hospital Heidelberg, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany. Fax 49-6221-565565; e-mail marina_schwab{at}med.uni-heidelberg.de.

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 14CO2, an endproduct of the E1 component of PDHc, from [1-14C]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).

Conclusion: The simple, optimized spectrophotometric assay for PDHc analysis allows reliable investigation of the enzyme complex in human skin fibroblasts.




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