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Published on: June 21, 2018
Pyruvate dehydrogenase complex activity in normal and deficient fibroblasts
The Journal of Clinical Investigation
|May 1, 1981
Summary
Pyruvate dehydrogenase complex (PDC) activity in human skin fibroblasts is regulated by phosphorylation. Dichloroacetate (DCA) activates PDC by inhibiting pyruvate dehydrogenase kinase, revealing deficiency in patients and enabling detection of heterozygous carriers.
Area of Science:
- Biochemistry
- Cell Biology
- Human Genetics
Background:
- Pyruvate dehydrogenase complex (PDC) activity is crucial for cellular energy metabolism.
- PDC activity is regulated by phosphorylation and dephosphorylation in animal cells.
- Human skin fibroblasts provide a model for studying PDC regulation and deficiency.
Purpose of the Study:
- To investigate the regulation of pyruvate dehydrogenase complex (PDC) activity in human skin fibroblasts.
- To establish a reliable assay for measuring PDC activity and detecting deficiencies.
- To explore the potential of PDC assays in identifying heterozygous carriers of PDC deficiency.
Main Methods:
- Fibroblast cultures were treated with dichloroacetate (DCA) to activate PDC by inhibiting pyruvate dehydrogenase kinase.
- PDC activity was measured by quantifying [1-(14)C]pyruvate decarboxylation.
- Assays were validated by assessing cofactor dependence and product ratios.
- PDC activity was also modulated using purified pyruvate dehydrogenase phosphatase and metal ions.
- NaF was used to inhibit dephosphorylation and assess its effect on PDC activity.
- PDC activity was measured in fibroblasts from patients with known deficiencies and their families.
Main Results:
- Dichloroacetate (DCA) treatment activated PDC 5-20 fold in normal fibroblasts, reaching 5-6 nmol/min per mg protein in infant cells.
- The assay accurately reflected PDC activity, dependent on thiamin pyrophosphate, CoA, Mg(++), and NAD(+).
- Fibroblasts from patients with PDC deficiency showed significantly reduced activity (0.1-0.3 nmol/min per mg protein) even after DCA activation.
- Familial studies indicated an autosomal inheritance pattern for PDC deficiency, with carriers exhibiting approximately half the normal activated PDC activity.
- The assay successfully identified heterozygous carriers.
Conclusions:
- PDC activity in human skin fibroblasts is regulated by a phosphorylation-dephosphorylation mechanism.
- Dichloroacetate (DCA) is effective in activating PDC and revealing deficiencies.
- The developed assay is sufficiently sensitive to detect pyruvate dehydrogenase complex deficiency and identify heterozygous carriers.
- This assay has significant implications for diagnosing and understanding inherited metabolic disorders related to PDC.
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