INTRODUCTION

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THE SYNTHESIS AND ASSEMBLY of mitochondrial components are remarkable in several respects. The number or mass of mitochondria per cell varies, depending on physiological as well as pharmacological factors, even in cells that are terminally differentiated (9, 11, 20). Mitochondrial mass can, therefore, be regulated independently from cell division. Mitochondrial DNA (mtDNA) is also distinct biochemically from nuclear DNA (nDNA). The mitochondrial genome in animals consists of 16- to 20-kb circular DNA molecules. The mitochondrial genome is strikingly compact in its organization, being almost completely saturated with coding regions (i.e., lacking introns) that are joined to each other directly or separated by only a few nucleotides (1, 5, 6). Mitochondria also contain a distinct DNA polymerase (DNA polymerase-) that is of nuclear origin (5, 6, 12).

Although the great majority of mitochondrial respiratory enzymes are imported from the cytosol and coded by nDNA, gene products from mtDNA are essential for mitochondrial function and normal tissue health (1, 5, 6). Several key enzymes of oxidative phosphorylation are coded by mtDNA (11). Genetic and acquired disorders of mitochondria have been identified (18, 27). Assessment of mtDNA replication rate in vivo has been problematic, however (10, 13). Static measurements (cellular DNA content or expression of cell cycle antigens), which have limitations even as markers of nDNA replication and cell proliferation (14), have no relation to or utility for assessing mitochondrial biogenesis.

Our laboratory recently described a nonradioactive, stable isotope-mass spectrometric method for measuring DNA replication (14, 22, 24, 25). Purine deoxyribonucleotides of replicating DNA are labeled through the de novo nucleotide synthesis pathway from deuterated glucose or ²H₂O. In contrast, previously described techniques for labeling DNA utilized the nucleoside salvage pathway from labeled pyrimidine nucleosides (³H-dT or bromodeoxyuridine). The stable isotope, de novo nucleotide synthesis pathway labeling technique has technical advantages, as discussed previously (22, 24, 25), in addition to being without toxicity and, therefore, applicable to humans (22, 24). Here we apply the ²H₂O incorporation method to the measurement of mtDNA synthesis in rodents and humans. Portions of this work have been presented previously in abstract form (7, 8).

	METHODS

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Animal Studies

Labeling protocol. Sprague-Dawley rats (weight, 210-350 g) from Simonsen were housed in wire cages, three per cage, with a 12:12-h light-dark cycle. All procedures were approved by the University of California Berkeley Office of Laboratory Animal Care. Purina rat chow was provided ad libitum. There were two groups of rats: a young male growing group (2-4 mo of age at the beginning of studies), and an older, weight-stable female group (8-10 mo of age). Initial average rat weight from the young rat group was ~210 g, whereas the initial average body weight from the older, weight-stable group was ~225 g.

Delabeling protocol. Female Sprague-Dawley rats (weight, 210-250 g) from Simonsen were used. Food was provided ad libitum. All rats were labeled for 8 wk with 4% ²H₂O as described above. Rats were then delabeled by replacement of 4% ²H₂O by natural abundance water for 1-5 wk.

Isolation of bone marrow and cardiac and hindlimb muscle mitochondria. Bone marrow and cardiac (0.5 g) and hindlimb muscle (0.3 g) samples from individual animals were removed immediately after death. Muscle samples were homogenized as previously described (16, 26). Mitochondria from the homogenate were then isolated by density gradient centrifugation (26). nDNA contamination is removed enzymatically by treatment with DNase (Sigma Chemical). Absence of nDNA contamination in muscle samples was confirmed by PCR followed by gel electrophoresis (see below). More than sufficient mtDNA is obtained from 0.3-0.5 g of muscle tissue for measurement of mtDNA kinetics in individual animals.

PCR. PCR was performed according to manufacturer's instructions (2) on mtDNA preparations and compared with genomic DNA standards from brain. Comparison of band intensities from mtDNA preparations to serial dilutions from genomic DNA standards revealed 0.00045% contamination (see below).

Measurement of ²H₂O enrichments of body water. Enrichment of ²H₂O in body water (blood) was measured by a new GC-MS technique, after chemical conversion to tetrabromoethane (25). Briefly, the hydrogen atoms in H₂O were first transferred to acetylene by addition of 1-10 µl water via syringe to a chip of calcium carbide in a sealed vial, equipped with a 3-ml syringe inserted into the septum. The resulting acetylene gas was drawn into the 3-ml syringe and expelled into another sealed vial containing 0.5 ml Br₂ (0.1 mM) dissolved in CCl₄. After 2 h of incubation at room temperature, the remaining Br₂ is reacted with cyclohexene dissolved in CCl₄ (10% solution). This solution is injected into the GC-MS for analysis. GC-MS anaylsis was with a DB-225, 30-m column at 220°C, with the use of methane chemical ionization with selected ion monitoring. The C₂H₂Br₃+ fragment [mass-to-charge ratio (m/z) 265 and 266, representing the M₀ and the M₊₁ ion, respectively, of the ⁷⁹Br⁷⁹Br⁸¹Br isotopologue] was used for calculating ²H enrichment, by comparison to standard curves generated by mixing 100% ²H₂O with natural abundance H₂O in known proportions (25).

Human Studies

²H₂O administration. We administered ²H₂O to human subjects for the first 24 h under observation in a metabolic ward setting, at the General Clinical Research Center of the San Francisco General Hospital. This was done to avoid the possibility of transient vertigo or dizziness, which has been reported as a rare, adverse effect of rapid changes in body water enrichment (24). The ²H₂O administration protocol consisted of 50-ml doses of 70% ²H₂O given every 3-4 h for 18-24 h in the metabolic ward. No subject experienced any adverse symptoms using this protocol. The study volunteers were subsequently maintained on 50- to 70-ml daily intake of ²H₂O, with a goal of maintaining ~1.5-2% body water enrichment (assuming total body water turnover of roughly 3.5 l/day in healthy, ambulatory subjects) (24). Subjects underwent a blood draw at week 5 or 6 for collection of platelets and monocytes.

Isolation of mtDNA from human platelets. Whole blood (10 ml) from patients was collected in two separate anticoagulated (heparinized) tubes (one for platelets and a second 10-ml tube for monocyte isolation; see below). Blood from the first tube was centrifuged, and the white blood cell upper layer was removed, leaving behind the red blood cells. This platelet-rich layer was then centrifuged, resulting in a white blood cell upper layer and a platelet pellet. The platelet pellet was removed and incubated with DNase. The cell membrane was disrupted with a lysis buffer AL (Qiagen) before incubation, to allow DNase access to any nDNA present. Absence of nDNA contamination in platelet mtDNA samples was evaluated quantatively by use of real-time PCR (28). Electron microscopy of the platelet fraction confirmed pure platelets, uncontaminated by red cells or other white blood cells (not shown).

PCR amplification. Real-time quantitative PCR analysis, the principle of which has previously been described (28), was performed with an Applied Biosystems 5700 sequence detector. The Alu TaqMan system consisted of the amplification primers, AluF 5'-GGAGGCTGAGGCAGGAGAA-3' and AluR 5'-ATCTCGGCTCACTGCAACCT-3', and a fluorescent probe, 5'-(FAM)CGCCTCCCGGGTTCAAGCG-3'.

Isolation of DNA from blood monocytes. Blood monocytes were isolated from Ficoll-Hypaque gradient isolated peripheral blood mononuclear cells by use of anti-CD14 magnetic beads (Miltenyi Biotec, Auburn, CA). The monocyte fraction was resuspended in 200 µl PBS, and nDNA was isolated as described previously (24). Monocyte nDNA is used as a nearly completely turned over tissue for calculation of fractional DNA synthesis in comparison tissues, as described previously (24).

DNA Measurements and Calculations

Isolation of DNA and deoxyadenosine. Bone marrow nDNA was isolated by use of a Qiamp column (Qiagen), as described previously (15, 25, 22). mtDNA was also isolated from cardiac muscle, hindlimb muscle, and platelets by using the Qiagen kit (Qiagen) after isolation of the mitochondrial fraction from the tissue (3, 4). mtDNA and nDNA were hydrolyzed enzymatically to free deoxyribonucleosides, as described previously (24, 25). In brief, an LC18 solid-phase extraction column (Supelco, Bellefone, PA) was used to separate deoxyadenosine (dA) from the other deoxyribonucleosides. The column was washed with 100% methanol (2 ml) and water (2 ml). The hydrolyzed DNA sample was then added to the column, and nucleosides other than dA were eluted with an H₂O wash (5 ml). The dA was then eluted with 50% methanol (1 ml), as previously described (25).

Derivatization of dA and GC-MS analysis. The deoxyribose (dR) moiety of dA was analyzed by GC-MS, after conversion to its pentane-tetraacetate derivative, as described elsewhere (25). The isotopic enrichment of dR was determined by GC-MS analysis (m/z 245 and 246, representing M₀ and M₁ masses, respectively). There is no exchange between solvent water or other sample matrix protons and hydrogen atoms in C-H bonds of dR in DNA or free deoxyribonucleosides (24, 25). The derivative that we analyzed contained only the dR moiety, not the base portion, of purine deoxyribonucleosides (15), so label incorporation into the base moiety via base salvage pathways is not a confounding factor (22, 24, 25).

Calculations

Fractional synthesis (f) rates of cells were calculated by use of the precursor-product relationship. The isotopic enrichment of a completely (or nearly completely) turned over tissue can be used as a measure of the true precursor enrichment for the cells of interest. Under conditions of steady state in the pool size, the f rate also represents the fractional replacement rate (i.e., assuming that every cell or molecule produced must be balanced by a cell or molecule destroyed). The replacement constant (k) and half-life (t_1/2) of mtDNA after ²H₂O labeling were calculated, as described previously (14, 22, 24, 29). The central principle behind the mathematics of the precursor-product relationship is that the isotopic enrichment of a product derived exclusively from a precursor pool will approach the isotopic enrichment of the precursor pool, with the shape of an exponential curve (15, 29)

(1)

where M₁ is the fractional abundance of the M₊₁ mass isotopomer and EM₁ represents the excess molar fraction of the M₊₁ mass isotopomer

(2)

where t is time in days

(3)

	RESULTS

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Animal Studies

Purity of mtDNA. PCR data, using genomic DNA as a control against mtDNA, confirmed minimal contamination of genomic DNA in mtDNA isolated from rodent tissues. Comparison of band intensities from mtDNA preparations to serial dilutions from genomic DNA standard revealed 0.00045% contamination (data not shown).

Body water enrichments. Body water ²H₂O enrichments were measured serially. The steady-state body ²H₂O enrichments attained in rats maintained on 4% ²H₂O in drinking water were ~3.0% and were stable over time in individual animals (Fig. 1A). The delabeling kinetics of body water in rats after discontinuing ²H₂O in drinking water was also measured (Fig. 1B). By weeks 2-3 of delabeling, the body water enrichments had fallen to essentially zero from the plateau values of ~3%.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Body water enrichments in female Sprague-Dawley rats. A: during intake of 4% 2H2O in drinking water for 7 wk. B: after discontinuing 2H2O in drinking water (delabeling) after 8 wk of 2H2O intake. EM1, excess molar fraction of M+1 mass isotopomer. Values are means ± SD.

mtDNA labeling in tissues of rodents. Young male rats were studied first, as a model of mitochondrial biogenesis that included somatic growth. Body weights from young male rats increased by >50% over the duration of ²H₂O labeling (Fig. 2A). Enrichments of bone marrow nDNA remained stable at 9.5-10.0% through 11 wk of labeling (Fig. 2B and Ref. 14). Enrichments of cardiac and hindlimb mtDNA increased from 0.0% to 3.5% over time (Fig. 2, C and D). The f of mtDNA from hindlimb and cardiac muscle also increased over time (Table 1). Approximately 40-50% of mtDNA were newly synthesized after 11 wk of labeling with ²H₂O in both tissues, with the value in hindlimb muscle appearing to plateau at ~45%. New mtDNA synthesized was not greater in magnitude than whole body somatic growth (~50% for each), so we cannot determine from these results whether true turnover (i.e., replacement of mtDNA in the absence of growth) occurs.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Results on growing (2- to 4-mo-old) male rats. A: body weights during period of labeling study. B: enrichments of deoxyadenosine (dA) from nuclear DNA (nDNA) of bone marrow. C: enrichments of dA from mitochondrial DNA (mtDNA) of cardiac muscle. Each point represents the average of 2 consecutive week's values. D: enrichments of dA from mtDNA of hindlimb muscle. Each point represents the average of 2 consecutive week's values. Values are means ± SD.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Results in older, weight-stable female rats (8-10 mo old). A: body weights during period of labeling study. B: enrichments of dA from nDNA of bone marrow. C: enrichments of dA from mtDNA of cardiac muscle. Each point represents the average of 2 consecutive week's values. D: enrichments of dA from mtDNA of hindlimb muscle. Each point represents the average of 2 consecutive week's values. Values are means ± SD.

Delabeling results. The delabeling experiment was performed to determine whether mtDNA die-away curves generate similar kinetic results as label incorporation curves (i.e., as an internal validation). Body water ²H₂O enrichments fell off to near-zero levels by 2 wk after ²H₂O was discontinued (Fig. 1B). Bone marrow enrichments from aged female rats decreased rapidly and reached near zero values by week 3 (Fig. 4A), consistent with rapid turnover of these cells. In contrast, delabeling of DNA from hindlimb and cardiac muscle mtDNA fell modestly in female rats (Fig. 4B), consistent with the slow turnover rates calculated from the isotope incorporation studies. Although the dilution of labeled mtDNA during the die-away period was too small quantitatively to allow precise calculations of kinetic parameters, calculated fractional decay rates were 0.3 and 0.8%/day (average values for hindlimb and cardiac muscle mitochondria, respectively).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Delabeling results in weight-stable female rats after prior 8 wk of 2H2O administration. A: delabeling kinetics of dA from bone marrow nDNA. B: delabeling kinetics of dA from cardiac and skeletal muscle mtDNA. Values are means ± SD.

Comparison of mtDNA to nDNA synthesis in muscle tissue. Another way to correct for somatic growth in evaluating mtDNA turnover is to compare nDNA synthesis in the tissues of interest. The f of nDNA and mtDNA in cardiac and hindlimb muscle was compared in weight-stable female rats. Higher synthesis rates of mtDNA compared with genomic DNA, by about twofold after 6-9 wk, were observed in both cardiac and hindlimb muscle tissues (Fig. 5, A and B, respectively). Of note, skeletal muscle nDNA synthesis was of lower magnitude (~5% after 9 wk) than whole body somatic growth (~10%) in these animals, whereas cardiac muscle nDNA synthesis (~10%) was of similar magnitude as somatic growth. The observation that mtDNA synthesis is greater than nDNA synthesis in these tissues is important because these results are consistent with mtDNA replication, independent of cell division. If mtDNA synthesis is corrected for new myocyte proliferation (nDNA synthesis), the half-life of mtDNA in nongrowing tissue can be estimated, as was done based on somatic growth (see above). Calculated half-life in cardiac muscle was ~350 days (replacement rate constant 0.2%/day), and in skeletal muscle was ~700 days (replacement rate constant 0.1%/day).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Comparison of mtDNA to nDNA synthesis in cardiac and hindlimb muscle of weight-stable female rats A: fractional synthesis (f) in cardiac muscle. B: f in hindlimb muscle. Values are means ± SD.

Human Studies

Purity of mtDNA. Purity of mtDNA isolated from human platelets was confirmed by quantitative analysis by using real time PCR (Table 3). Because mtDNA do not contain Alu 1 sequences, as opposed to genomic DNA (5, 6), the absence of amplification from mtDNA samples is indicative of no or minimal genomic DNA contamination.

Platelet mtDNA labeling during ²H₂O intake. The mtDNA isolated from human platelets exhibited label incorporation during 6 wk of ²H₂O administration (Fig. 6). Comparison to monocyte nDNA enrichments in the same subjects (used as a marker of a fully turned over tissue, or maximal labeling) indicated that platelet mtDNA was close to 100%, replaced by week 5 samples (platelet mtDNA = 4.98 ± 0.01% vs. monocyte nDNA = 5.00 ± 0.23%, week 5 subjects; platelet mtDNA = 7.12 ± 1.02% vs. monocyte nDNA = 7.58 ± 1.26%, week 6 subjects).

View larger version (8K): [in this window] [in a new window]   Fig. 6.   Label incorporation into mtDNA from human blood platelets during 6 wk of daily 2H2O intake. The f results in platelets during 5 and 6 wk of 2H2O intake are shown. Values are means ± SD.

	DISCUSSION

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We describe here a method for measuring mtDNA synthesis in vivo in rodents and humans. This method may be useful for the evaluation of aerobic training regimens or other factors that alter mitochondrial biogenesis. This method may also prove useful in testing hypothesized mitochondrial toxicities of certain drugs (11, 15, 20, 29).

The results confirm relatively slow synthesis rates of hindlimb and cardiac muscle mtDNA in normal rats. True turnover (i.e., replacement in the absence of somatic growth) does seem to occur, based on mtDNA synthesis in excess of somatic growth in cardiac muscle in relatively weight-stable rats (Table 2) and, perhaps more convincingly, based on comparison of mtDNA to nDNA synthesis in both cardiac and skeletal muscle (Fig. 5). By both approaches, somatic growth of tissue accounted for ~50% of mtDNA synthesis. The apparent half-life of mtDNA in weight-stable rats ranged between 12 and 24 mo for cardiac and hinblimb muscle, respectively, after correction for new mitochondria that was added during growth of tissues.

The human results were promising, although preliminary. The protocol was well tolerated, and the mtDNA isolated was free of genomic DNA. Because blood platelets are highly accessible, abundant, rich in mitochondria, and free of nuclei, they may be an ideal tissue for human testing of drugs or genes with putative effects on mtDNA synthesis. Measurements of mtDNA kinetics in human tissues of greater physiological interest, such as skeletal muscle or adipose tissue, are not shown here but are in progress.

Some technical issues deserve comment. Body ²H₂O enrichments are very stable in rodents (Fig. 1A) and humans (24) during long-term administration of oral ²H₂O. The classic form of the precursor-product relationship, wherein the isotopic enrichment of the precursor pool is held constant and the enrichment of the product pool approaches this value over time (29), is thereby made applicable even for very slow turnover end products, such as mtDNA in muscle tissue. This feature represents a great operational advantage of the ²H₂O labeling technique. Application of this approach requires knowledge of the enrichment of the true precursor pool [deoxyribonucleotide-triphosphates (dNTP)] for mtDNA synthesis, however, a value that is most easily estimated from plateau isotope enrichment nDNA of fully turned over tissues (24). Previous studies have shown that plateau isotope enrichments in nDNA from a variety of tissues (e.g., liver, cardiac muscle, skin, brain, bone marrow) are essentially identical after ²H₂O administration in utero and in early postnatal life (24), when all tissues have 100% new cells; thus dNTP pools in different tissues appear typically to exhibit similar labeling during long-term ²H₂O administration. The assumption that mtDNA and nDNA isotope enrichments approach the same value (i.e., derive from a common dNTP precursor pool) was supported by our studies in humans showing identical plateau enrichments in platelet mtDNA and monocyte nDNA (Fig. 6). The assumption of a common dNTP precursor pool for mtDNA and nDNA replication is also reasonable biochemically, because dNTP are synthesized in the cytosolic compartment and imported into both nuclei and mitochondria (5, 6).

In summary, mtDNA synthesis and turnover are now routinely measurable by use of a stable isotope-mass spectrometric technique. The technique is simple and inexpensive, can be applied to experimental animals as well as humans, is applicable to DNA that turns over slowly (as appears to be the case for hindlimb and cardiac muscle mitochondria), and can be compared with concurrently measured nDNA in a tissue. We report here that mtDNA does turn over independent of somatic growth in hindlimb and cardiac muscle, that human platelets represent a highly accessible tissue, and that the method is well tolerated in people as well as rodents. Many possible research questions can be addressed by this approach, including inborn errors of mtDNA or mitochondrial biogenesis, assessment of aerobic fitness or aerobic demand on tissues, and toxicities of antiretroviral drugs (9, 11, 17, 20, 21). Future applications include isolation of mtDNA from other tissues, such as adipose or human skeletal muscle.