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PubMed1987

Rhodamine 123 uptake and mitochondrial DNA content in rabbit articular chondrocytes evolve differently upon transfer from cartilage to culture conditions.

Mitochondrial DNA (mtDNA) represents 0.15% of the total cell DNA (at least an order of magnitude less than in liver or heart) of rabbit articular chondrocytes. Besides the already well-documented low respiratory activity, chondrocyte differentiation thus involves a specific control of mitochondrial biogenesis. When transferred to in vitro conditions, chondrocytes increase their stock of mtDNA at the same time they resume growth, even more efficiently (8 times) than they do for cell volume (4.4 times). On the contrary, overall mitochondrial activity, estimated as the uptake of rhodamine 123, does not follow the same trend (2.5 times increase). Chondrocytes apparently keep these functional characteristics for some generations in culture.

Animals↗

PubMed1996

Mitochondrial DNA is required for regulation of glucose-stimulated insulin secretion in a mouse pancreatic beta cell line, MIN6.

To determine whether mtDNA and mitochondrial respiratory function in pancreatic beta cells are necessary for the phenotypic expression of glucose-stimulated insulin secretion, we used a cultured mouse pancreatic beta cell line, MIN6, and two derivative lines, mtDNA knockout MIN6 (rho0 MIN6) and mtDNA repopulated cybrid MIN6. The MIN6 cells retain the property of glucose-stimulated insulin secretion, but their mtDNA knockout induced the loss of mitochondrial transcription, translation, and respiration activity, without inhibition of transcription of the insulin gene or loss of succinate dehydrogenase activity, indicating that the observed mitochondrial dysfunction in rho0 MIN6 cells was not due to a cytotoxic side effect derived from the mtDNA knockout. Moreover, the mtDNA depletion also inhibited both the glucose-stimulated increase in the intracellular free Ca2+ content and the elevation of insulin secretion. The possibility of the involvement of nuclear genome-encoded factors in this process was excluded by the observation that the missing sensitivity to extracellular glucose stimulation in rho0 MIN6 cells was restored reversibly by repopulation with foreign mtDNA and isolating cybrid MIN6 clones. Therefore, these findings provide unambiguous evidence for the involvement of the mitochondrial dysfunction induced by mtDNA impairment in developing pathogeneses of some forms of diabetes mellitus.

Animals↗

PubMed2001

Effects of contractile activity on mitochondrial transcription factor A expression in skeletal muscle.

Mitochondrial transcription factor A (Tfam) is a nuclear-encoded gene product that is imported into mitochondria and is required for the transcription of mitochondrial DNA (mtDNA). We hypothesized that conditions known to produce mitochondrial biogenesis in skeletal muscle would be preceded by an increase in Tfam expression. Therefore, rat muscle was stimulated (10 Hz, 3 h/day). Tfam mRNA levels were significantly elevated (by 55%) at 4 days and returned to control levels at 14 days. Tfam import into intermyofibrillar (IMF) mitochondria was increased by 52 and 61% (P < 0.05) at 5 and 7 days, respectively. This corresponded to an increase in the level of import machinery components. Immunoblotting data indicated that IMF Tfam protein content was increased by 63% (P < 0.05) at 7 days of stimulation. This was associated with a 49% (P < 0.05) increase in complex formation at the mtDNA promoter and a 65% (P < 0.05) increase in the levels of a mitochondrial transcript, cytochrome-c oxidase (COX) subunit III. Similarly, COX enzyme activity was elevated by 71% (P < 0.05) after 7 days of contractile activity. These results indicate that early events in mitochondrial biogenesis include increases in Tfam mRNA, followed by accelerations in mitochondrial import and increased Tfam content, which correspond with increased binding to the mtDNA promoter region. This was accompanied by increased mitochondrial transcript levels and elevated COX activity. These data support the role of Tfam as a regulatory protein involved in contractile activity-induced mitochondrial biogenesis.

Animals↗

PubMed2005

Flutamide restores cardiac function after trauma-hemorrhage via an estrogen-dependent pathway through upregulation of PGC-1.

Although previous studies have shown that flutamide improves cardiovascular function after trauma-hemorrhage, the mechanisms responsible for the salutary effect remain unknown. We hypothesized that flutamide mediates its beneficial effects via an estrogen-dependent pathway through upregulation of peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC-1). PGC-1, a key regulator of cardiac mitochondrial ATP production, induces mitochondrial DNA (mtDNA)-encoded genes such as cytochrome-c oxidase (COX) subunit I, II, and III (COX I, COX II, and COX III), which regulates mitochondrial oxidative phosphorylation. To test this hypothesis, male rats underwent trauma-hemorrhage (mean arterial pressure of 35-40 mmHg for approximately 90 min) followed by resuscitation. At the onset of resuscitation, rats received vehicle, flutamide (25 mg/kg body wt), flutamide in combination with estrogen receptor (ER) antagonist ICI-182,780 (3 mg/kg body wt), or ICI-182,780 alone. Flutamide administration after trauma-hemorrhage restored the depressed cardiac function and increased cardiac testosterone, estrogen levels, and aromatase activity. These increases were accompanied by normalized cardiac ER-alpha and ER-beta protein levels, PGC-1, and COX I mRNA expression, mitochondrial COX activity, and ATP contents. However, cardiac dihydrotestosterone, 5alpha-reductase II, androgen receptor protein levels, and mtDNA-encoded genes COX II and COX III were unaffected by flutamide treatment. The flutamide-mediated restoration of cardiac function, the increases in aromatase activity and estrogen levels, ER-alpha, ER-beta, PGC-1, COX I, COX activity, and ATP contents were, however, abolished when ER antagonist ICI-182,780 was administrated along with flutamide. These findings suggest that the salutary effect of flutamide on cardiac function after trauma-hemorrhage is mediated via an estrogen-dependent pathway through upregulation of PGC-1.

Abdominal Injuries↗

PubMed1985

The expression of human glycerol-3-phosphate dehydrogenase in human/rodent somatic-cell hybrids.

Our previous studies using rodent/human somatic-cell hybrids suggested that the expression of human mitochondrial glycerol-3-phosphate dehydrogenase (GPDM) is dependent on the presence of human mitochondria. This has now been tested directly by analysis of GPDM activity in a series of nine hybrid-cell lines, four segregating human chromosomes and five losing rodent chromosomes (reverse segregants). The chromosome composition of the hybrids was deduced from analysis of biochemical markers and examination of G- and G11-banded metaphase spreads and the mitochondrial content was determined by Southern blot analysis, using cloned mouse and human mtDNA sequences as probes. We found that the mtDNA species present in these hybrids correlated exactly with the pattern of chromosome segregation such that the conventional hybrids contained rodent mtDNA and the reverse segregants human mtDNA. However, the pattern of GPDM expression was not directly correlated with the species of chromosomes or mitochondria present: all the hybrids showed strong rodent GPDM activity and two from each class of hybrid also showed human GPDM activity but the other hybrids were negative for human GPDM. We conclude that rodent GPDM readily integrates into human mitochondria, that the expression of rodent GPDM is not dependent on the presence of rodent mitochondria, and that GPDM is not coded by mtDNA. Human GPDM either is not capable of being inserted into the rodent mitochondrial membrane or is regulated in some way in the hybrid cells by an unidentified rodent factor.

Animals↗

PubMed1984

The sequence of the gene for cytochrome c oxidase subunit I, a frameshift containing gene for cytochrome c oxidase subunit II and seven unassigned reading frames in Trypanosoma brucei mitochondrial maxi-circle DNA.

A 9.2 kb segment of the maxi-circle of Trypanosoma brucei mitochondrial DNA contains the genes for cytochrome c oxidase subunits I and II (coxI and coxII) and seven Unassigned Reading Frames ("URFs"). The genes for coxI and coxII display considerable homology at the aminoacid level (38 and 25%, respectively) to the corresponding genes in fungal and mammalian mtDNA, the only striking point of divergence being an unusually high cysteine content (about 4.5%). The reading frame coding for cytochrome c oxidase subunit II is discontinuous: the C-terminal portion of about 40 aminoacids, is present in the DNA-sequence in a -1 reading frame with respect to the N-terminal moiety. URF5, 8 and 10, show a low but distinct homology (about 20%) to mammalian mitochondrial URF-1, 4 and 5, respectively. In URF5, the first AUG is found at codon 145, whereas extensive homology to mammalian URF-1 sequences occurs upstream of this position. The possibility exists that UUG can serve as an initiator codon. URF7 and URF9 have a highly unusual aminoacid composition and do not possess AUG or UUG initiator codons. These URFs probably do not have a protein-coding function. The segment does not contain conventional tRNA genes.

Amino Acid Sequence↗

PubMed2001

Complete sequence of the mitochondrial genome of the tapeworm Hymenolepis diminuta: gene arrangements indicate that Platyhelminths are Eutrochozoans.

Using "long-PCR," we amplified in overlapping fragments the complete mitochondrial genome of the tapeworm Hymenolepis diminuta (Platyhelminthes: Cestoda) and determined its 13,900-nt sequence. The gene content is the same as that typically found for animal mitochondrial DNA (mtDNA) except that atp8 appears to be lacking, a condition found previously for several other animals. Despite the small size of this mtDNA, there are two large noncoding regions, one of which contains 13 repeats of a 31-nt sequence and a potential stem-loop structure of 25 bp with an 11-member loop. Large potential secondary structures were identified also for the noncoding regions of two other cestode mtDNAS: Comparison of the mitochondrial gene arrangement of H. diminuta with those previously published supports a phylogenetic position of flatworms as members of the Eutrochozoa, rather than placing them basal to either a clade of protostomes or a clade of coelomates.

Animals↗

PubMed1994

Age-dependent increase of mitochondrial DNA deletions together with lipid peroxides and superoxide dismutase in human liver mitochondria.

We previously reported an age-dependent deterioration of mitochondrial respiration as well as two age-associated mitochondrial DNA (mtDNA) deletions in the human liver. In this study, we further determined the relative quantities of the deleted mtDNAs in liver biopsies from 64 subjects of different ages. The results showed that both mtDNA deletions increase in frequency and quantity with age. Moreover, we measured hepatic lipid peroxides (malondialdehyde; MDA) of isolated mitochondria and manganese superoxide dismutase (Mn-SOD) activity of submitochondrial particles. We found a significant age-dependent increase in both MDA and Mn-SOD levels in liver mitochondria. These results confirm the previous contention that enhanced generation of lipid peroxides in the mitochondria during the aging process may damage mtDNA, and mtDNA deletions may be one of the important factors contributing to aging in humans.

Adult↗

PubMed1995

The mitochondrial DNA of the amoeboid protozoon, Acanthamoeba castellanii: complete sequence, gene content and genome organization.

In phylogenetic trees based on comparison of nuclear small subunit rRNA sequences, Acanthamoeba castellanii (an amoeboid protozoon) is positioned near the base of the radiation leading to the animals, fungi and plants. However, the specific affiliation of this protist with the major multicellular lineages of eukaryotes is currently uncertain. To further explore the evolutionary position of A. castellanii, we have determined the complete primary sequence of its mitochondrial genome. We find that the circular mtDNA (41,591 bp; 70.6% A+T) encodes two rRNAs (small subunit and large subunit), 16 tRNAs and 33 proteins (17 subunits of the respiratory chain and 16 ribosomal proteins). As well, this genome contains eight open reading frames (ORFs) larger than 60 codons and of undefined function. Two of these ORFs (orf124 and orf142) have homologs in other mtDNAs ("orf25" and "orfB", respectively), three are unique to A. castellanii mtDNA (orf83, orf115 and orf349), and three are intronic ORFs. Among notable features of A. castellanii mtDNA are the following: (1) Genes and ORFs are all encoded on the same strand and are tightly packed, with only 6.8% of the total sequence not having an evident coding function and intergenic spacer sequences ranging from only 1 to 616 bp (average 64 bp). Ten pairs of protein-coding genes overlap by up to 38 bp and two subunits of cytochrome oxidase (COX1 and COX2) are specified by a single continuous ORF. (2) Only three introns, all group I and each containing a free-standing ORF, are present; these are localized in the 3'-half of the large subunit rRNA gene. (3) The genome encodes fewer than the minimal number of tRNA species required to support mitochondrial protein synthesis, suggesting that additional tRNAs are imported from the cytosol into A. castellanii mitochondria. Of the 16 tRNAs specified by A. castellanii mtDNA (one with an 8-nucleotide anticodon loop), 13 have been shown or are predicted to undergo a novel form of RNA editing within the acceptor stem. (4) A modified genetic code is used in which UGA specifies tryptophan. (5) Repeated sequences and obvious small sequence motifs that might represent regulatory elements are absent. In overall size, gene content and organizational pattern, A. castellanii mtDNA most closely resembles the mtDNA of the chlorophycean alga Prototheca wickerhamii (55,326 bp; 74.2% A+T), but is quite different in these respects from the mtDNA of Chlamydomonas reinhardtii (15,758 bp; 54.8% A+T), another chlorophycean alga, as well from characterized animal and fungal mitochondrial genomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acanthamoeba↗

PubMed1997

Mitochondrial membrane fluidity and oxidative damage to mitochondrial DNA in aged and AD human brain.

Oxidative damage on biological molecules has been proposed as a major cause of alterations observed in aging brain as well as in neurodegenerative diseases. In this study, we measured membrane fluidity in mitochondria extracted from three cerebral regions and cerebellum of Alzheimer disease (AD) patients and age-matched controls by means of fluorescence polarization technique. A significant reduction of mitochondrial membrane fluidity was found in AD, except in cerebellum. In controls, a decrease of membrane fluidity was observed along with age, and it was also related to the content of the oxidized nucleoside 8-hydroxy-2'-deoxyguanosine (OH8dG) in mitochondrial DNA (mtDNA). Alteration in membrane fluidity seems to be a result of lipid peroxidation, since it dramatically decreased when mitochondria were exposed to FeCl2 and H2O2. The parallel increase of viscosity in mitochondrial membrane and the amount of OH8dG in mtDNA is suggestive of a relationship between these biological markers of oxidative stress. These results provide further evidence that oxidative stress may play a role in the pathogenesis of AD.

Aged↗

PubMed1995

Oxidative damage to mitochondrial DNA and its relationship to ageing.

Mitochondria are the most important intracellular source of reactive oxygen species and are protected against them by enzymatic and nonenzymatic antioxidants. Nevertheless, mitochondrial DNA (mtDNA) is subject to severe oxidative damage, and much more so than nuclear DNA (nDNA). Damage is indicated by the detection of various base modifications, particularly 8-hydroxydeoxyguanosine (8OHdG), which can lead to point mutations because of mispairing. MtDNA is also fragmented to some extent. Conceivably, such fragmentation relates to the deletions found in mtDNA. Several hypotheses suggest that defective mitochondria contribute to, or are responsible for, ageing. Recent observations indicate that mitochondria in an old organism differ in many respects from those in a young organism. Thus, with ageing there is an increased production of reactive oxygen species, a decrease in certain antioxidants, a decreased transcription, translation, and cytochrome oxidase content, and an increase in the extent of DNA modifications. Major unresolved questions concerning the role of mtDNA changes in ageing are addressed: is there a causal relationship; what is the true extent of DNA damage; what are significance and functional consequences of mtDNA oxidation; are reactive oxygen species the cause of the DNA modifications found in vivo; what is the relationship between DNA damage and alterations of RNAs and proteins? Future studies promise to clarify the possible causal relationship between mitochondrial dysfunction, reactive oxygen species production, mtDNA modifications, and ageing.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2001

Doxorubicin-induced persistent oxidative stress to cardiac myocytes.

We recently reported a cardioselective and cumulative oxidation of cardiac mitochondrial DNA (mtDNA) following subchronic administration of doxorubicin to rats. The mtDNA adducts persist for up to 5 weeks after cessation of doxorubicin treatment. Since the evidence suggests that this persistence of mtDNA adducts cannot be attributed to a lack of repair and replication, we investigated whether it might reflect a long-lasting stimulation of free radical-mediated adduct formation. Male Sprague-Dawley rats received weekly s.c. injections of either doxorubicin (2 mg/kg) or an equivalent volume of saline. Cardiac myocytes isolated from rats following 6 weekly injections of doxorubicin expressed a much higher rate of reactive oxygen species (ROS) formation compared to saline controls. This higher rate of ROS formation persisted for 5 weeks following the last injection. Associated with this was a persistent depression of GSH in heart tissue, while protein-thiol content was not markedly altered. These data suggest that the accumulation and persistence of oxidized mtDNA may be due, not to the stability of the adducts, but to some as yet undefined toxic lesion that causes long-lasting stimulation of ROS generation by doxorubicin. This persistent generation of ROS may contribute to the cumulative and irreversible cardiotoxicity observed clinically with the drug.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1995

Defective brain and muscle energy metabolism shown by in vivo 31P magnetic resonance spectroscopy in nonaffected carriers of 11778 mtDNA mutation.

In vivo phosphorus magnetic resonance spectroscopy (31P-MRS) showed defective brain and muscle energy metabolism in three affected siblings in a family with Leber's hereditary optic neuropathy (LHON) with the 11778 mtDNA mutation. We studied 14 nonaffected members of the same pedigree by 31P-MRS and molecular genetics. Nine of 14 individuals studied had the 11778 mtDNA mutation, with various degrees of heteroplasmy. A decreased brain energy reserve, as shown by low phosphocreatine content and phosphorylation potential and high [ADP], was present in eight of these nine subjects with the 11778 mutation. A low rate of postexercise phosphocreatine recovery in muscle was present in six of the nine mutated individuals. Normal MRS findings in the brain of one and the muscle of three carriers were accompanied by a low percentage of mutated mtDNA. All subjects without mutation had normal brain and muscle MRS. 31P-MRS disclosed defective bioenergetics in the brain or muscle or both of all asymptomatic carriers studied from our pedigree.

Adenosine Diphosphate↗

PubMedDate not supplied

PCR-select subtraction for characterization of messages differentially expressed in brown compared with white adipose tissue.

To understand the energy metabolism occurring in brown adipose tissue (BAT), we subtracted the messages expressed in white adipose tissue (WAT) from those in BAT. Thereby we succeeded in identifying 37 cDNA clones as being significantly expressed in BAT but not in WAT. Of these, 24 clones were found to code for mitochondrial proteins. Since BAT is well known to have a higher mitochondrial content than WAT, these results would seem to reflect simply the differences in mitochondrial content between BAT and WAT. To examine this possibility, we next measured the amount of mitochondrial DNA (mtDNA) in various rat tissues. As a result, the mtDNA copy number per cell was found to be markedly different among the tissues analyzed, and the highest value of about 5.3x10(4) copies per cell was observed with the rat brain. BAT showed a value similar to that of brain, but this value was only about 3.5-fold higher than that for WAT. Since observed differences in mitochondrial content between BAT and WAT was smaller than those observed with transcript levels of proteins, we conclude that the observed differences in the transcript levels of certain proteins between BAT and WAT reflect the functional differences between BAT and WAT, and do not reflect the differences in mitochondrial content between BAT and WAT.

Adipose Tissue↗

PubMed2001

Mitochondrial abnormalities in tumor necrosis factor-alpha-induced heart failure are associated with impaired DNA repair activity.

BACKGROUND: Recent studies suggest that mutations in cardiac mitochondrial DNA (mtDNA) may contribute to the development of dilated cardiomyopathy. The mechanisms that regulate those mutations, however, remain undefined. Thus, we studied cardiac mtDNA repair mechanisms, mtDNA damage, and mitochondrial structure and function in mice with heart failure secondary to overexpression of TNF-alpha (TNF1.6 mice). METHODS AND RESULTS: We studied mtDNA repair by measuring the uracil DNA glycosylase (mtUDG) and base excision repair activities. mtDNA damage was assessed by Southern blot of Fpg protein-digested mtDNA. Mitochondrial ultrastructural changes were examined by electron microscopy, and function by cytochrome c oxidase and succinate dehydrogenase activity assays. The results showed that both mtUDG and base excision repair activities were significantly reduced in TNF1.6 mouse heart. Fpg-sensitive sites were markedly increased in TNF1.6 mouse cardiac mtDNA, suggesting increased mtDNA damage. Mitochondrial function as demonstrated by cardiac cytochrome c oxidase activity was also markedly reduced. Cardiac ATP content was not changed, however, suggesting a shift from oxidative phosphorylation to glycolysis, as shown by increased LDH and ALT activities and lactate/pyruvate ratio. Ultrastructurally, the TNF1.6 mouse cardiac mitochondria became irregular in shape and smaller, and the cristae were decreased and appeared disorganized, with breaks. CONCLUSIONS: These results suggest that mtDNA mutations and mitochondrial structural and functional alterations in TNF-alpha-induced heart failure may be associated with reduced mtDNA repair activity, and the pathophysiological effects of TNF-alpha on the heart may be mediated, at least in part, through these changes in mitochondria.

Animals↗

PubMed1987

Structural elements highly preserved during the evolution of the D-loop-containing region in vertebrate mitochondrial DNA.

A detailed comparative study of the regions surrounding the origin of replication in vertebrate mitochondrial DNA (mtDNA) has revealed a number of interesting properties. This region, called the D-loop-containing region, can be divided into three domains. The left (L) and right (R) domains, which have a low G content and contain the 5' and the 3' D-loop ends, respectively, are highly variable for both base sequence and length. They, however, contain thermodynamically stable secondary structures which include the conserved sequence blocks called CSB-1 and TAS which are associated with the start and stop sites, respectively, for D-loop strand synthesis. We have found that a "mirror symmetry" exists between the CSB-1 and TAS elements, which suggests that they can act as specific recognition sites for regulatory, probably dimeric, proteins. Long, statistically significant repeats are found in the L and R domains. Between the L and R domains we observed in all mtDNA sequences a region with a higher G content which was apparently free of complex secondary structure. This central domain, well preserved in mammals, contains an open reading frame of variable length in the organisms considered. The identification of common features well preserved in evolution despite the high primary structural divergence of the D-loop-containing region of vertebrate mtDNA suggests that these properties are of prime importance for the mitochondrial processes that occur in this region and may be useful for singling out the sites on which one should operate experimentally in order to discover functionally important elements.

Amino Acid Sequence↗

PubMed1997

An ancestral mitochondrial DNA resembling a eubacterial genome in miniature.

Mitochondria, organelles specialized in energy conservation reactions in eukaryotic cells, have evolved from eubacteria-like endosymbionts whose closest known relatives are the rickettsial group of alpha-proteobacteria. Because characterized mitochondrial genomes vary markedly in structure, it has been impossible to infer from them the initial form of the proto-mitochondrial genome. This would require the identification of minimally derived mitochondrial DNAs that better reflect the ancestral state. Here we describe such a primitive mitochondrial genome, in the freshwater protozoon Reclinomonas americana. This protist displays ultrastructural characteristics that ally it with the retortamonads, a protozoan group that lacks mitochondria. R. americana mtDNA (69,034 base pairs) contains the largest collection of genes (97) so far identified in any mtDNA, including genes for 5S ribosomal RNA, the RNA component of RNase P, and at least 18 proteins not previously known to be encoded in mitochondria. Most surprising are four genes specifying a multisubunit, eubacterial-type RNA polymerase. Features of gene content together with eubacterial characteristics of genome organization and expression not found before in mitochondrial genomes indicate that R. americana mtDNA more closely resembles the ancestral proto-mitochondrial genome than any other mtDNA investigated to date.

Animals↗

PubMed2004

Platelets, a typical source of error in real-time PCR quantification of mitochondrial DNA content in human peripheral blood cells.

Nucleoside analogues can induce toxic effects on mitochondria by inhibiting the human DNA polymerase-gamma. The clinically observed toxicities can range from slightly increased serum lactate levels to potentially severe and fatal lactic acidosis. A growing interest exists for detection of changes in mitochondrial (mt) DNA content in patients receiving antiretroviral therapy (HAART). Most studies use peripheral blood mononuclear cell (PBMC) fractions to investigate mt DNA content via Real-Time PCR in patients, not accounting platelets falsifying the mitochondrial (mt)DNA:nuclear (n)DNA-ratio. In this study we suggest a procedure to eliminate disturbing platelets totally. 8 healthy controls (G1), 6 therapy-naive HIV-infected patients (G2) and 9 HIV-infected patients under HAART (G3) were examined for mtDNA:nDNA-ratio using Real-Time PCR technology. Different blood collection and/or PBMC isolation strategies were analysed for variances of outcome at examinations of the same blood donor. Using DNA prepared of whole blood specimens, mtDNA:nDNA-ratios showed no differences in all investigated groups (G1, G2, G3). Comparing mtDNA:nDNA-ratios of platelet-depleted PBMC fractions of G1 with G2 revealed a reduction of 22% (p = 0.128) and a steeper reduction of 40% (p = 0.0036) comparing specimens of G1 with G3. Scrutinising differently processed specimens within the groups themselves, in G2 whole blood versus platelet-containing PBMC specimens showed a difference in mtDNA:nDNA-ratios of 26% (p = 0.0406), whereas a comparison of whole blood versus platelet-free PBMC specimens led to a comparatively more distinct reduction of 35% (p = 0.0089). The same effect was seen in G3, where whole blood versus platelet-containing PBMC specimens revealed a reduction of 32% (p = 0.01) and whole blood versus platelet-free PBMC specimens showed a 42% (p = 0.0011) decrease. Furthermore analysing each single patient in relation to the different methods, a minor fluctuation margin could be found using platelet-free PBMC specimens for Real-Time PCR. Using platelet-free PBMCs for mt DNA content detection, a correlation of low mtDNA:nDNA-ratios to clinical signs, like elevated lactate levels or lipodystrophy, could be observed. Light-microscopic evaluation for platelets, comparing platelet-containing PBMC fractions versus platelet-depleted PBMC fractions reinforced the Real-Time PCR results. Our data demonstrate that the first step of the blood sample collection/preparation is critical for valid illustration of mt DNA content in HIV-infected patients using ultra-sensitive Real-Time PCR technology. The use of serum tubes for blood collection is an easy and low-cost alternative to expensive cell sorting for elimination of disturbing platelets. Using platelet-free PBMC fractions for measurement mt DNA content could be a surrogate marker for clinical signs mediated by HAART.

Acquired Immunodeficiency Syndrome↗