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J Appl Physiol (January 31, 2003). doi:10.1152/japplphysiol.00691.2002
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Submitted on July 29, 2002
Accepted on January 21, 2003

Measurement of Mitochondrial DNA Synthesis In Vivo in Rodents and Humans Using a Stable Isotope-Mass Spectrometric Technique

Michelle L Collins1*, Shannon Eng1, Hoh Rebeccah1, and Marc K Hellerstein1

1 Molecular and Biochemcial Nutrition, University of California at Berkeley, Berkeley, CA, USA

* To whom correspondence should be addressed. E-mail: mcollins{at}nature.berkeley.edu.

We describe here a new stable isotope/ mass spectrometric technique for measuring mitochondrial (mt) DNA synthesis. Growing (2-4 month old) and weight stable (8-10 month old) Sprague-Dawley rats were primed with 2H2O (deuterated water) to 2.0 -2.5% body water enrichment, via intra peritoneal injection, then given 4% 2H2O in drinking water for 3-11 weeks. Mitochondria were isolated from cardiac and hind-limb muscle and mtDNA was isolated and enzymatically hydrolyzed to deoxyribonucleosides. PCR confirmed absence of nuclear DNA contamination. The isotopic enrichment of the deoxyribose moiety of deoxyadenosine (dA) was determined by gas chromatographic-mass spectrometric analysis and percent new mtDNA was calculated by comparison to genomic DNA enrichments in a tissue with nearly complete turnover (bone marrow). Initial label incorporation into dA of mtDNA was linear and turnover of mtDNA was observed in non-growing adult female rats (1.1-1.3 % new mtDNA per day in cardiac and skeletal muscle). Die-away curves of mtDNA after discontinuing 2H2O administration gave a similar turnover rate constant. Human subjects were also given 2H2O for up to 6 weeks and mitochondria from platelets were isolated. Incubation with DNAse removed any contaminating genomic DNA; platelet mtDNA exhibited linear incorporation from 2H2O and reached plateau values identical to those in genomic DNA from fully turned-over cells (circulating monocytes and granulocytes). In conclusion, replication of mtDNA can be directly measured in vivo in rodents and humans without the use of radioactivity. Use of this technique may allow improved understanding of the regulation of mitochondrial biogenesis in health and disease.




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