Search

The measurement of the level of mitochondrial DNA (mtDNA) in the blood is a difficult problem due to high variability of mitochondrial genes, deletions in the mitochondrial genome in some pathological conditions, different sources of mtDNA into the bloodstream (mtDNA from tissues, from blood cells, etc.). We designed primers and TaqMan probes for highly conserved regions of the ND1 and ND2 genes outside the mitochondrial deletions "hot zones". For standardizing the technique, the true concentration of low-molecular-weight mtDNA was determined by real-time PCR for two targets: a fragment of the ND2 gene (122 bp) and the ND1 and ND2 genes (1198 bp). The sensitivity and specificity of the developed approach were verified on a DNA pool isolated from the blood plasma of healthy donors of various nationalities. The concentration of low-molecular-weight mtDNA in the blood plasma of two patients with COVID-19 was monitored over two weeks of inpatient treatment. A significant increase in the content of low-molecular-weight mtDNA was observed during the first 5 days after hospitalization, followed by a drop to the level of healthy donors. The developed technique makes it possible to assess the blood level of low-molecular-weight mtDNA regardless of the quality of sampling and makes it possible to standardize this biological marker in a wide range of infectious and non-infectious pathologies., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Liquid biopsy represents a diagnostic and monitoring tool and the circulating cell-free mitochondrial DNA (mtDNA) plays a vital role in tumor diagnosis and dynamic assessment. Colorectal cancer (CRC) is one of the most common fatal cancers worldwide. Mitochondrially encoded NADH dehydrogenase subunit 1 (MT-ND1) encodes the biggest subunit of respiratory complex I of mtDNA, and mutations in the MT-ND1 are common in CRC. We sought to determine if mutations in circulating MT-ND1 could be a potential biomarker for colorectal cancer. In this study, twenty-two CRC patients at Zhujiang Hospital were included. We mainly used droplet digital PCR to determine the mutation status of MT-ND1, combined with clinical data. In the experiment in vivo, cell-free mtDNA generally presented high concordance with tumor tissues. By quantitative PCR, the MT-ND1 content of plasma in CRC patients was significantly higher than that in healthy individuals (58.01 vs. 0.64, p=0.027). The detection of circulating MT-ND1 content and variants (m.3606 A>G, m.3970 C>T, m.4071 C>T, m.4086 C>T) in cfDNA showed a good correlation with predicted tumor response and progression to chemotherapy. In conclusion, the content and variants of circulating MT-ND1 may become a versatile tool for the diagnosis and monitoring of colorectal cancer.

Parkinson's disease (PD) is a neurodegenerative disease with multifactorial aetiology that influences the quality of life. However, the association of possible factors with PD is need to be investigated in Indian population, hence we aimed to determine the association of lifestyle, environmental factors, biochemical parameters and genetic insights of MT-ND1 gene in PD patients. Using a standardised questionnaire, PD patients and control group of about 146 subjects were interviewed on demographic, lifestyle and environmental factors. The subjects includes n = 73 Parkinson's patients [juvenile (n = 4); early-onset (n = 8); late-onset (n = 61)] with equal number of age and sex matched controls, further we had obtained institutional ethical clearance and informed consent from study participants. Biomarker investigations and MT-ND1 alterations were investigated by appropriate molecular techniques. During the average follow-up years of 5.1, significant association was observed among smoking, alcohol, caffeinated drinks, surgery, pesticide exposure at p < 0.05 in varied PD age groups. Occupational exposure to agriculture and industry showed an increased risk among the late-onset group. The biomarkers uric acid (UA) and dopamine (DA) were significant at p < 0.05 in all the three PD age groups. The MT-ND1 alteration with A3843 G variant was significant at p < 0.05 for AG allele in all the three PD groups but the highest prevalence was observed in late-onset group. From our study, smoking, alcohol, caffeinated drinks, occupational influence of agriculture and industry and pesticide exposure had more association with PD occurrence. Hence, to the best of our knowledge, this is the first kind of study in Tamil Nadu population, India to validate the various factors with PD. Therefore we suggest that further research is mandatory to detect other possible associations among PD, using comprehensive larger sample size., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: There is a lack of mechanism-driven, clinically relevant biomarkers in chronic obstructive pulmonary disease (COPD). Mitochondrial dysfunction, a proposed disease mechanism in COPD, is associated with the release of mitochondrial DNA (mtDNA), but plasma cell-free mtDNA has not been previously examined prospectively for associations with clinical COPD measures., Methods: P-mtDNA, defined as copy number of mitochondrially-encoded NADH dehydrogenase-1 (MT-ND1) gene, was measured by real-time quantitative PCR in 700 plasma samples from participants enrolled in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) cohort. Associations between p-mtDNA and clinical disease parameters were examined, adjusting for age, sex, smoking status, and for informative loss to follow-up., Results: P-mtDNA levels were higher in participants with mild or moderate COPD, compared to smokers without airflow obstruction, and to participants with severe COPD. Baseline increased p-mtDNA levels were associated with better CAT scores in female smokers without airflow obstruction and female participants with mild or moderate COPD on 1-year follow-up, but worse 6MWD in females with severe COPD. Higher p-mtDNA levels were associated with better 6MWD in male participants with severe COPD. These associations were no longer significant after adjusting for informative loss to follow-up., Conclusion: In this study, p-mtDNA levels associated with baseline COPD status but not future changes in clinical COPD measures after accounting for informative loss to follow-up. To better characterize mitochondrial dysfunction as a potential COPD endotype, these results should be confirmed and validated in future studies., Trial Registration:  ClinicalTrials.gov NCT01969344 (SPIROMICS).

Thyroid cancer is the most common endocrine malignancy, and its incidence continues to rise. For clinicians with cancer patients, choosing and interpreting diagnostic laboratory studies has become increasingly important. Previously, changes in plasma free mitochondrial DNA levels have been found in colorectal, breast, lung, and urinary cancers, and have demonstrated diagnostic value. In this study, we investigated whether the occurrence and development of thyroid cancer might be predicted using mtDNA copy number (ND1), mtDNA integrity (ND4/ND1) and levels of cell-free nDNA (GAPDH). We analyzed ND1, ND4, and GAPDH levels in plasma and blood cells from 75 patients with thyroid cancer, 40 patients with nodular goiter, and 107 normal controls using real-time PCR. Although both the thyroid nodule and thyroid cancer patients had significantly increased ND1 levels, the ND4/ND1 ratio in the thyroid cancer group was higher than the thyroid nodule group (P < 0.05), and significantly higher than the normal control group (P < 0.01). Plasma levels of nuclear DNA (GAPDH) in the thyroid cancer group were also higher compared to normal (P < 0.05). These results indicate that increased intactness of plasma free mtDNA is associated with increased levels of plasma cell-free nDNA, and that the ND4/ND1 ratio has the potential to be a new detection indicator in thyroid cancer. Furthermore, we classified thyroid cancer patients according to clinical data including age, tumor size, and metastasis. We found significantly higher levels of GAPDH in malignant tissues. Because ND4/ND1 correlated with plasma GAPDH in the plasma studies, this also suggests a potential relationship between ND4 intactness and thyroid tumor tissue size. Taken together, our findings suggest a tumor-specific process involving increased release of intact mtDNA, detectable in the plasma, which differentiates normal patients from patients with thyroid cancer., (Copyright © 2020 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

The systemic cytokine response during surgery has been reported to be stimulated by the molecules released from damaged cells, called damage-associated molecular patterns (DAMPs). The relationship between DAMPs and liver transplantation has not been reported. We aimed to clarify the relationship between the plasma levels of DAMPs and the short-term post-transplant outcomes, including mortality and postoperative multi-organ dysfunction syndrome (MODS). This retrospective cohort study enrolled 61 patients who underwent liver transplantation. Mitochondrial DNA fragments, as mitochondria-derived DAMPs (mtDAMPs), were isolated from frozen plasma obtained at the start and the end of transplantation and were quantified by polymerase chain reaction. The short-term post-transplant outcomes were compared among the groups categorized based on the median value of the intraoperative fluctuation of mtDAMPs levels. The mtDAMPs levels were increased from the start to the end of transplantation in 52 recipients (85.2%, n = 61). Regarding mortality, no significant differences were noted between the high group (n = 30) and the low group (n = 31). The higher plasma levels of mtDAMPs were correlated with the longer duration of postoperative vasopressor support (P < 0.05). Importantly, the rate of MODS on post-operative day 1 was significantly higher in the high group (high vs. low group: 21 patients [70%] vs. 11 patients [35.1%], P < 0.01). In conclusion, mtDAMPs were increased in plasma during liver transplantation in most recipients. This elevation was not associated with mortality, but associated with the post-transplant recovery. Measuring plasma mtDAMPs may be helpful for predicting posttransplant recovery among liver-transplant recipients.

The aim of this research was to detect potential serum biomarkers of melamine diet-induced bladder stones in C57BL/6 mice. Magnetic bead-based weak cationexchange chromatography (MB-WCX) and matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) were employed to detect serum biomarkers in 10 mice fed a melamine diet and 10 control mice. Seventeen peaks (fold change>1.5) with a mass to charge (m/z) value of 1000-10,000 Da were detected in the two groups. Among the significant peaks, five were upregulated and the other 12 were downregulated in the model group. Among the upregulated peaks, 2954.49 and 1710.49 were found to correspond to the peptide regions of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8(Ndufα8) and basigin, respectively, by liquid chromatography with electrospray ionization and tandem triple quadrupole mass spectrometry(LC-ESI-MS/MS). Western blot analysis was used to detect the expression of Ndufα8 and basigin in another 10 model mice and 10 control mice. The western blot results confirmed the LC-ESI-MS/MS data. The expression of serum basigin and Ndufα8 was partly dependent the concentration of melamine, but no time dependence. In conclusion, Ndufα8 and basigin may be potential serum biomarkers for the detection of melamine diet-induced bladder stones in C57BL/6 mice., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: Obesity is an independent risk factor for chronic kidney disease. Recently, urinary mitochondrial DNA (mtDNA) has been used as a surrogate marker of mitochondrial damage in various kidney diseases. However, there are no data regarding its use in patients with obesity or the change in urinary mtDNA copy number after surgery., Design: We prospectively recruited age- and sex-matched healthy volunteers and patients with obesity (n = 22 in each group: nine men and 13 women). The copy number of urinary and serum mtDNA nicotinamide adenine dinucleotide dehydrogenase subunit-1 (mtND-1) and cytochrome-c oxidase 3 (mtCOX-3) was measured using quantitative PCR. We measured urinary mtDNA and body weight and carried out laboratory tests, 6 months after surgery., Results: Urinary mtND-1 copy number was significantly higher in the obese group than in healthy volunteers. However, urinary mtCOX-3 and serum ND-1 copy numbers in the obese group did not differ from that in the healthy volunteers. When patients with obesity were divided into two groups, according to their baseline mtND-1 copy number, bariatric surgery reduced the mtND-1 copy number (P = 0.006) in the high baseline mtDNA copy-number group. The change in urinary mtND-1 copy number was correlated with a change in urinary albumin (r = 0.478, P = 0.025)., Conclusions: Obesity is associated with elevated urinary mtND-1 copy number. Bariatric surgery reduces the elevated urinary mtND-1 copy number in patients with obesity. This suggests that bariatric surgery could attenuate mitochondrial damage in the kidney cells of patients with obesity., (Copyright © 2019 Endocrine Society.)

This study aimed to identify the potential target genes for the treatment of ankylosing spondylitis (AS).Dataset GSE25101 was downloaded from Gene Expression Omnibus, including 16 AS and 16 normal control blood samples. Differentially expressed genes (DEGs) were identified using unmatched t-test in limma package with adjusted P < .05. Gene ontology-biological process (GO-BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using multifaceted analysis tool for human transcriptome. Protein-protein interaction (PPI) network was constructed using STRING and Cytoscape, and module analysis was performed using MCODE plug-in. Webgestal was utilized to predict transcriptional factor (TF)-microRNA-target network and Comparative Toxicogenomics Database (CTD) was applied to predict chemical-target network.A total of 334 DEGs were identified, including 136 upregulated genes and 198 downregulated genes. According to STRING, a PPI network was constructed and 1 significant clustered module was screen out with score = 6.33. MAPK7 (degree = 11) and NDUFS4 (degree = 10) were 2 important nodes in PPI network, and both of them were significantly enriched in cAMP mediated signaling pathway (P = 2.02E-02). MAPK7 could be regulated by NFY. Both MAPK7 and NDUFS4 were 2 potential targets for Indomethacin.MAPK7 and NDUFS4 played important roles in the pathogenesis of AS via cAMP mediated signaling pathway. Both of them could be targeted by Indomethacin.

The objective of the present work was to study the population composition and functional activity of lymphocytes in the spleen and peripheral blood of the rats exposed experimentally to the toxic effect of household gas. The study included the morphofunctional examination of the state of the immune organs and the immunological investigation of the population composition and functional activity of lymphocytes from the peripheral blood of the experimental animals. We also evaluated the activity of nucleic acids, NADH2-dehydrogenase, and 5'-nucleotidase. The study revealed the relationship between the pathological and histochemical changes and the shifts in the population composition and functional activity of lymphocytes in the spleen and peripheral blood of the rats. Specifically, the action of household gas induced by the profound inhibition of the proliferative activity of the lymphocytes, enhanced the suppressive activity of the immunoregulatory cells (T-suppressors), and altered the population composition of the effector cells in the spleen and peripheral blood. It is concluded that the impairment of the functional activity of T-lymphocytes under the influence of household gas should be attributed not only to its direct toxic action but also to the increased activity of T-suppressors.

Peripheral blood mononuclear cells (PBMC) represent an easily available population of cells for the studies on remote effects of lung cancer. NADH dehydrogenase (ubiquinone) Fe-S protein-1 (Ndufs1), a marker of mitochondrial complex I, and mitochondrially encoded cytochrome c oxidase 1 (MTCO1), a marker of complex IV, may participate in cognitive decline during the course of lung cancer. In this study, Ndufs1 and MTCO1 expression in PBMC was evaluated by means of ELISA in 80 lung cancer patients. Mini-Mental State Examination (MMSE) were conducted Trail Making Tests (TMT-A and TMT-B) at baseline and after the 6 months' follow-up. Autoantibodies were identified by means of indirect immunofluorescence and line blot. We found that enhanced levels of Ndufs1 in PBMC were related to impaired cognitive performance; TMT-A of 13.6 ± 3.1 s and TMT-B of 162.5 ± 46.4 s compared with 8.6 ± 4.5 s (p = 0.003) and 124.8 ± 51.8 s (p < 0.05), respectively, in the case of low Ndufs-1 levels. The Ndufs1 expression at baseline was associated with MMSE - τb (Kendall's tau-b) = -0.31; p = 0.024; TMT-A - τb = 0.30; p = 0.001), and TMT-B - τb = 0.199; p = 0.012) after the 6 months' follow-up. Higher MTCO1 expression was accompanied by worse TMT-A results than in case of inhibited MTCO1; 11.1 ± 5.8 s vs. 8.5 ± 4.1 s; respectively; p = 0.048. MTCO1 expression was correlated with TMT-A results (τb = 0.17; p = 0.034) at baseline. We conclude that stimulation of PBMC mitochondrial function in lung cancer patients is associated with cognitive impairment. Mitochondrial dysfunction in PBMC may reflect cytotoxicity responsible for neurological deficits.

Circulating tumor cells (CTCs) and/or their relating molecules are promising determinants during the course of cancer treatment, especially for post-therapeutic monitoring. We recently reported the clinical relevance of detecting circulating tumor-associated mutant mitochondrial DNAs (mut-mtDNAs) at three different regions including the displacement loop, 12S-rRNA and 16S-rRNA in oral squamous cell carcinomas (OSCCs). In the present study, to further investigate if the other mut-mtDNAs have novel efficiency for detecting potential tumoral micrometastasis, mut-mtDNAs on the ND2 and ND3 regions of the genome in 240 clinical samples from patients with OSCC were assessed in vitro and in vivo by quantitative real-time PCR combined with high-resolution melting curve analysis. Furthermore, the clinical relevance was evaluated by the area under the receiver operating characteristic curve (AUC) analysis. Three discrete sequence variations were identified in OSCC derived cell lines at the regions of ND2 (T:A to C:G at position 5108) and ND3 (A:T to G:C at position 10397 and C:G to T:A at position 10400), whereas no mutation was observed in normal control human normal oral keratinocytes. In OSCC patients examined, the presence of mut-mtDNAs in serum during the postoperative period accurately predicted poor prognoses (ND2 AUC, 0.761; ND3 AUC, 0.704). The data presented here provide a novel approach for detecting the circulating mut-mtDNAs that are promising molecular markers for evaluating tumoral micrometastasis in OSCCs.

Gene expression in peripheral blood has the potential to inform on pathophysiological mechanisms and has emerged as a viable avenue for the identification of biomarkers. Here, we aimed to identify gene expression candidate genes and to explore the potential for a composite gene expression measure as a diagnostic and state biomarker in bipolar disorder. First, messenger RNA levels of 19 candidate genes were assessed in peripheral blood mononuclear cells of 37 rapid cycling bipolar disorder patients in different affective states (depression, mania and euthymia) during a 6-12-month period and in 40 age- and gender-matched healthy control subjects. Second, a composite gene expression measure was constructed in the first half study sample and independently validated in the second half of the sample. We found downregulation of POLG and OGG1 expression in bipolar disorder patients compared with healthy control subjects. In patients with bipolar disorder, upregulation of NDUFV2 was observed in a depressed state compared with a euthymic state. The composite gene expression measure for discrimination between patients and healthy control subjects on the basis of 19 genes generated an area under the receiver-operating characteristic curve of 0.81 (P < 0.0001) in sample 1, which was replicated with a value of 0.73 (P < 0.0001) in sample 2, corresponding with a moderately accurate test. The present findings of altered POLG, OGG1 and NDUFV2 expression point to disturbances within mitochondrial function and DNA repair mechanisms in bipolar disorder. Further, a composite gene expression measure could hold promise as a potential diagnostic biomarker.

The study was carried out to analyze dynamics of diaphorase and esterase activity ofneutrophils of blood in patients with chronic viral hepatitis C of lower degree of activity depending on gender characteristics in dynamics of treatment. The examination and treatment were organized concerning sampling of 113 patients with chronic viral hepatitis C of lower degree of activity. The diaphorase and esterase activity of neutrophils in dynamics of treatment was detected The analysis of diaphorases was carried out according R.P. Nartsissov technique. The content of esterase was estimated by V.M. Wachstein-FG. Wolf technique. The count of results was implemented using Kaplow semiquantitative technique. In patients with chronic viral hepatitis C of lower degree of activity prior to treatment the activity of NAD-diaphorase was lowered both in males and females. The activity of NADF-diaphorase prior to treatment significantly exceeded standard in males and matched standard in females. The application of basic therapy resulted in qualitative redistribution of cellular composition of reacting cells. All of them reacted following medium degree of activity (b). At that, average cytochemical indicator of reaction was normal in males and in females increasing of activity was observed. The activity og both diaphorases after application of complex therapy (basic therapy and cycloferon) totally returned to normality both in males and females. The esterase activity prior to treatment was decreased in males and increased in females (alpha-naphthylacetate esterase) and vice versa (alpha-naphthylbutyrate esterase) was increased in males and decreased infemales. After application of basic therapy in males increasing of esterase activity was registered and total redistribution of qualitative composition of reacting neutrophils (from degree "a" to degree "b"). In females after treatment the activity of alpha-naphthylacetate esterase was decreased and alpha-naphthylbutyrate esterase was increased. The redistribution of qualitative composition of cells was absent. The application of cycloferon brings to normality the activity of alpha-naphthylbutyrate esterase in males and activity of diaforaselpha- naphthylacetate esterase in females. In patients with chronic viral hepatitis C of lower degree of activity the differences in diaphorase and esterase activity ofneutrophils depending on gender characteristics in dynamics of treatment are observed.

A simple electrochemical biosensor was developed for the detection of the mitochondrial NADH dehydrogenase 6 gene (MT-ND6) and its enzymatic digestion by BamHI enzyme. This biosensor was fabricated by modification of a glassy carbon electrode with gold nanoparticles (AuNPs/GCE) and a probe oligonucleotide (ssDNA/AuNPs/GCE). The probe, which is a thiolated segment of the MT-ND6 gene, was deposited by self-assembling immobilization on AuNPs/GCE. Two indicators including methylene blue (MB) and neutral red (NR) were used as the electroactive indicators and the electrochemical response of the modified electrode was measured by differential pulse voltammetry. The proposed biosensor can detect the complementary sequences of the MT-ND6 gene. Also the modified electrode was used for the detection of an enzymatic digestion process by BamHI enzyme. The electrochemical biosensor can detect the MT-ND6 gene and its enzymatic digestion in polymerase chain reaction (PCR)-amplified DNA extracted from human blood. Also the biosensor was used directly for detection of the MT-ND6 gene in all of the human genome., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: Our objective was to execute a prospective cohort study to determine relationships between plasma mtDNA DAMP levels and the occurrence of systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), and mortality., Background: Mitochondrial DNA damage-associated molecular patterns (DAMPs) accumulate in the circulation after severe injury. Observations in animal models demonstrate that mtDNA DAMPs contribute to organ dysfunction; however, the link between plasma mtDNA DAMPs and outcome in severely injured human subjects has not been established., Methods: DNA was isolated from plasma samples taken from severely injured patients at hospital days 0, 1, and 2. Real-time PCR was used to quantify selected ≈200 base pair sequences of mtDNA within the COX1, ND1, and ND6 genes, as well as from the D-Loop transcriptional regulatory region. MODS was defined as a Denver Multiple Organ Failure score of 4 or greater., Results: MtDNA DAMPs were quantified as PCR threshold cycle number. Lower threshold cycles indicate increased mtDNA DAMP content. Patients with SIRS had significantly increased mtDNA DAMP levels in all 4 sequences examined (32.14 ± 0.90 vs 29.00 ± 1.15 for COX1, 31.90 ± 0.47 vs 30.16 ± 1.42 for ND1, 32.40 ± 0.61 vs 28.94 ± 1.13 for ND6, and 33.12 ± 0.83 vs 28.30 ± 1.14 for D-Loop). Patients who developed MODS also had elevated mtDNA DAMP levels compared with those who did not (32.57 ± 0.74 vs 27.12 ± 0.66 for COX1, 32.45 ± 0.65 vs 28.20 ± 0.73 for ND1, 32.52 ± 0.56 vs 27.60 ± 0.79 for ND6, and 32.85 ± 0.75 vs 27.86 ± 1.27 for D-Loop). Patients with above-median mtDNA DAMP levels had a significantly elevated relative risk for mortality. Four patients died secondary to severe MODS., Conclusions: These findings comprise the first observational evidence that plasma mtDNA DAMPs is associated with the evolution of SIRS, MODS, and mortality in severely injured human subjects.

The response of rainbow trout (Oncorhynchus mykiss, Walbaum) towards probiotics present in the feed was investigated by examining the proteome of serum as a measure of the acute phase response (APR). Proteomic analysis by two-dimensional electrophoresis (2D) concurrently with mass spectrometry was used to detect APR related proteins in rainbow trout serum following feeding with probiotics Aeromonas sobria GC2 and Bacillus sp. JB-1. Three candidate proteins increased following use of GC2, and were putatively identified as NADH dehydrogenase, dystrophin and mKIAA0350. Conversely, one of the proteins, which were induced following use of JB-1 was identified as transferrin.

Diabetes mellitus is associated with increased ROS generation, oxidative injury and obesity. To elucidate the relationship between nutrition and ROS generation, we have investigated the effect of glucose challenge on ROS generation by leucocytes, p47phox protein, a key protein in the enzyme NADPH oxidase and alpha-tocopherol levels. Blood samples were drawn from 14 normal subjects prior to, at 1, 2 and 3 h following ingestion of 75 g glucose. ROS generation by polymorphonuclear leucocytes (PMNL) and mononuclear cells (MNC) increased to a peak of 244 +/- 42% and 233 +/- 34% of the basal respectively at 2h. The levels of p47phox in MNC homogenates increased significantly at 2 h and 3 h after glucose intake. alpha-Tocopherol levels decreased significantly at 1 h, 2 h and 3 h. We conclude that glucose intake stimulates ROS generation and p417phox of NADPH oxidase; increases oxidative load and causes a fall in alpha-tocopherol concentration.

NADH dehydrogenase in the plasma membrane transfers electrons from NADH to external oxidants like ferricyanide, through pathways which are linked to metabolic processes in the cell. Hormone binding to specific sites (receptors) can modify the enzyme activity, suggesting a direct or indirect coupling between the redox system and the hormone receptors. Reduction of external ferricyanide to ferrocyanide by human erythrocytes was stimulated by beta-adrenergic agonists (adrenaline, ritodrine and isoxsuprine), this effect being dependent upon concentration and pH. The agonist-stimulatory effect was attenuated in the presence of metoprolol (10(-4) M), a beta-adrenergic antagonist, and was not modified in the presence of prazosin, an alpha-adrenergic antagonist, suggesting that modification of the redox activity is mediated by binding of the agonists to beta-adrenergic receptors present in the human erythrocytes. Basal and agonist-dependent activities were inhibited in the presence of sulfhydryl reagents p-chloromercuribenzoate (PCMB, 10(-5) M) and N-ethylmaleimide (NEM, 10(-3) M), indicating the involvement of -SH groups. Inactivation by NEM was reversed by washing the cells with GTP (10(-3) M) and GTP gamma S (10(-4) M), suggesting that the specific alkylated -SH group(s) is located on a G protein in the hormone-receptor-G-protein complex. The human erythrocytes contain G proteins, displaying both guanine-nucleotide-binding properties and GTPase activity. Fluoride (10(-2) M) and fluoroaluminate (AlF4- (F-, 10(-2) M + Al3+, 10(-5) M), G protein activators, enhanced the basal and agonist-dependent activities, suggesting the involvement of G proteins in this system. The overall results indicated that one of the coupling components between the hormonal receptors and the redox system is probably a G protein, and the mechanism of enzyme activation after hormone binding to the receptor is based on the redox state of cysteine residues probably within the receptor-G-protein complex.

Human peripheral blood polymorphonuclear leukocytes were preincubated with lanthionine, S-(2-aminoethyl)-L-cysteine, and some of their derivatives found in normal human urine and bovine brain. Among these compounds, lanthionine ketimine and to a lesser extent S-(2-aminoethyl)- L-cysteine ketimine enhanced the N-formyl-methionyl-leucyl-phenylalanine-induced superoxide generation. These ketimines induced tyrosyl phosphorylation of 45 kDa protein of cells. The tyrosyl phosphorylation was markedly increased with time, and the phosphorylation process was dependent on the concentration of both ketimines. However, lanthionine, 1,4-thiomorpholine-3,5-dicarboxylic acid, S-(2-aminoethyl)-L-cysteine and 1,4-thiomorpholine-3-carboxylic acid were without effect both on superoxide generation and on tyrosyl phosphorylation of 45 kDa protein. Lanthionine ketimine and S-(2-aminoethyl)-L-cysteine ketimine also enhanced superoxide generation induced by opsonized zymosan but not the one induced by arachidonic acid and phorbol 12-myristate 13-acetate. Ketimine-primed superoxide generation and tyrosyl phoshorylation of 45 kDa protein were inhibited by genistein, an inhibitor of protein tyrosine kinase, but not by 1-(5-isoquinoline sulfonyl)-2- methylpiperazine, an inhibitor of protein kinase C.

Aims: To evaluate the mitochondrial respiratory enzyme activities in blood cells of Leber's hereditary optic neuropathy (LHON) with 11778 point mutation of mitochondrial DNA., Methods: Assays for the activities of NADH-cytochrome c reductase (complex I+complex III), succinate-cytochrome c reductase (complex II+complex III), and cytochrome c oxidase (complex IV) on blood cell mitochondria of seven LHON patients and 15 normal controls., Results: There was no statistically significant difference in NADH-cytochrome c reductase and cytochrome c oxidase activities between LHON patients and controls, but activities of succinate-cytochrome c reductase in LHON patients was significantly elevated compared with normal controls., Conclusion: The observations that the activity of NADH-cytochrome c reductase is normal but that of succinate-cytochrome c reductase is increased in LHON patients with 11778 point mutation of mitochondrial DNA indicate an elevation of complex II activity, which may be due to a nuclear compensatory effect for defects of the respiratory function of mitochondria.

To investigate energy metabolism in migraine, we determined platelet mitochondrial enzyme activities in 40 patients with migraine with aura and in 40 patients with migraine without aura during attack-free intervals and in 24 healthy control subjects. NADH-dehydrogenase, citrate synthase and cytochrome-c-oxidase activities in both patient groups were significantly lower than in controls (p < 0.01), while NADH-cytochrome-c-reductase activity was reduced only in migraine with aura (p < 0.01). No alteration in succinate-dehydrogenase was observed. Monoamine-oxidase activity differed between sexes (p < 0.05) but within each sex group no difference was observed between patients and controls. We hypothesize that the defect in mitochondrial enzymes observed indicates a systemic impairment of mitochondrial function in migraine patients.

Integrating microdensitometry was used to study changes in the intracellular activity of 4 enzymes during macrophage development. Suspension cultures of blood monocytes from 19 healthy human subjects were examined at 0, 2, 4 and 6 d. Mononuclear phagocytes were harvested by glass adherence and standard methods were used for cytochemical staining for NADH dehydrogenase, succinate dehydrogenase, acid phosphatase and alpha-naphthyl butyrate esterase. All specimens from all subjects were stained at the same time and staining intensities in individual cells were measured at appropriate wavelengths. A highly significant increase in enzyme activity with culture time was found for all 4 enzymes. These increases in mitochondrial, lysosomal and ectoenzyme activities during development indicate the increasing functional capabilities of the macrophages.

Vitamin E, the major lipid chain-breaking antioxidant in erythrocyte membranes, is present in low concentration, suggesting that mechanisms should exist to protect against its loss. Enzymatic pathways for the recycling of vitamin E from its tocopheroxyl radical have been observed previously in inner membranes of mitochondria and microsomes. These pathways use electron transport enzymes and their substrates to regenerate vitamin E. Erythrocyte membranes also contain significant NADH-cytochrome c reductase activity, as well as cytochrome b5, the function of which is not yet known. Using an enzymatic oxidation system composed of lipoxygenase and arachidonic acid, free radicals were produced in human erythrocyte membranes, and their reaction with chromanols was followed by ESR and high performance liquid chromatography (HPLC). Since the endogenous vitamin E content of the membranes is very low, we used a vitamin E homologue lacking the hydrocarbon chain (2,2,5,7,8-pentamethyl-6-hydroxychromane) as a probe molecule for ESR measurements. However, parallel HPLC determinations of lipid hydroperoxides and of endogenous vitamin E confirmed the results obtained by ESR. It was found that protection against the loss of vitamin E can be provided either by NADH-cytochrome b5-dependent enzymatic recycling or by a nonenzymatic pathway involving ascorbate and dihydrolipoic acid.

Respiratory chain enzyme activities were studied in lymphocytes from patients with Parkinson disease (PD) (n = 16) and age-matched control subjects (n = 15). The patients had received no therapy before the study was conducted. Complex I, III, and IV activities were significantly lower (P < 0.05) in patients than in control subjects. A complex I defect was found in one patient, whereas complex IV was defective in another. Two patients had combined defects of both complexes. The use of lymphocytes for investigating the respiratory chain enzymes provides an easy, noninvasive method to assess mitochondrial function in patients with PD. Furthermore, our study supports the hypothesis that a biochemical defect in the respiratory chain may be involved in the pathogenesis of PD.

The NADPH-dependent superoxide-generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell-free system. The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat-germ-agglutinin-agarose column chromatography. The chromatography resulted in loss of the O2--generating activity in the cell-free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N-acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre-mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent-free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 +/- 0.53 nmol/mg protein (mean +/- SD, n = 5). The potency of fraction B in the reconstitution of the O2--generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O2--generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O2--generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p-chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0-7.5. The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 +/- 3.5 mumol O2-.min-1.mg-1 membrane-derived protein (mean +/- SD, n = 5) which shows that the membrane component was purified about 100-fold. These findings indicate that cytochrome b558 is probably a membrane component of the O2--generating NADPH oxidase and its activation in the cell-free system requires the reconstitution with phospholipids.

Ca2+-dependent K+ transport and plasma membrane NADH dehydrogenase activities have been studied in several 'high-K+' (human, rabbit and guinea pig) and 'low-K+' (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca2+-dependent K+ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome c reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca2+-dependent K+ channel activities in the species studied. Nor were any of the above activities correlated with (Na+ + K+)-ATPase activity.

An NADH cytochrome c reductase has been identified in plasma membrane fractions from neutrophils in addition to the superoxide producing NADPH oxidase which has been extensively studied by other investigators. Activation of neutrophils resulted in increased enzyme activities but to different degrees; the NADH cytochrome c reductase increased 2 fold in specific activity and the NADPH oxidase 30 fold. Treatment of the plasma membrane fraction with sonication and differential centrifugation yielded a particulate fraction (R2) with a 2 fold increase in specific activities of both enzymes and concentrations of cytochrome b and FAD. The cytochrome b in the preparation was not reduced under anaerobic conditions by either NADH or NADPH. Treatment of preparations of R2 with deoxycholate or potassium thiocyanate separated the two enzymes yielding particulate preparations with only NADPH oxidase or NADH cytochrome c reductase activity, respectively.

Evidence is presented for a transmembranous NADH-dehydrogenase in human erythrocyte plasma membrane. We suggest that this enzyme is responsible for the ferricyanide reduction by intact cells. This NADH-dehydrogenase is distinctly different from the NADH-cytochrome b5 reductase on the cytoplasmic side of the membrane. Pretreatment of erythrocytes with the nonpenetrating inhibitor diazobenzene sulfonate (DABS) results in a 35% loss of NADH-ferricyanide reductase activity in the isolated plasma membrane. Since NADH and ferricyanide are both impermeable, the transmembrane enzyme can only be assayed in open membrane sheets with both surfaces exposed, and not in closed vesicles. The transmembrane dehydrogenase has affinity constants of 90 microM for NADH and 125 microM for ferricyanide. It is inhibited by p-chloromercuribenzoate, bathophenanthroline sulfonate, and chlorpromazine.

A study of NADH ferricyanide reductase activity in oriented vesicles or open ghosts of human and porcine erythrocytes shows that the dehydrogenase activity can have three types of orientation in the membrane. There is activity which responds only to acceptors and NADH exclusively on the inside face, or exclusively on the outer surface. There is also activity which requires exposure of both sides of the membrane and thus is transmembranous. The transmembrane activity is inhibited by insulin, whereas the internal and external enzymes do not respond to insulin. The transmembrane dehydrogenase can be a basis for proton transport in the plasma membrane.

Previous studies by Roth & Majerus [J. Clin. Invest. (1975) 56, 624-632] showed that exposure of platelets to [acetyl-14C]aspirin resulted in the radioactive labelling of three polypeptides, two of which were in the cytosol and not saturable, whilst the third was located in particulate material, and was saturated at 30 microM-aspirin. By using high voltage free flow electrophoresis to separate a platelet mixed membrane fraction into highly purified surface and intracellular membrane subfractions, we have confirmed that the major polypeptide acetylated after exposing whole platelets to [acetyl-14C]aspirin is almost exclusively associated with intracellular membrane structures. We have shown previously that these intracellular membranes are the major site for prostanoid biosynthesis [Carey, Menashi & Crawford (1982) Biochem. J. 204, 847-851] and in the present study the extent of the radioactive labelling correlated well with inhibition of the cyclo-oxygenase activity in these intracellular membranes. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the [14C]acetylated component, which appears to be a dimer, migrates with a mobility corresponding to 72kDa. Although cyclo-oxygenase is inhibited, there is no discernible radioactive labelling when the platelets are exposed to aromatic-ring-labelled [14C]aspirin. We suggest that the site or sites for aspirin acetylation and cyclo-oxygenase activity are structurally associated in the platelet's intracellular membranes referred to by electron microscopists as the dense tubular membrane system.

Previous evidence suggests that the increase in platelet membrane fluidity associated with a subgroup of patients with Alzheimer's disease results from the accumulation of internal membrane. The specific activities of enzyme markers for selective cell membrane compartments were compared in platelets from subgroups of demented patients with normal or increased fluidity as well as from normal control subjects. A statistically significant change in enzyme activity was observed only for antimycin A-insensitive NADH-cytochrome reductase, a selective marker for smooth endoplasmic reticulum (SER) in platelets. This reduction was limited to the subgroup of demented patients who had increased platelet membrane fluidity, and therefore is not a nonspecific concomitant of neurodegeneration, medication exposure, or chronic illness in general. Since the platelet membrane alteration associated with Alzheimer's disease results from the inheritance of a single major locus, these results suggest that a defect in SER function may exist in brain cells as well as peripheral cells that express this genotype.

Reduction of extracellular ferricyanide by intact erythrocytes proceeds by a membrane bound, NADH-dependent reaction. It is depressed by a glycolysis inhibitor and a non penetrable sulfhydryl reagent, and activated by dehydroascorbate. Dehydroascorbate activation cannot be accounted for by release of reducing equivalents from the cells. It is concluded that the observed reaction is brought about by transmembrane NADH-acceptor oxidoreductase with donor binding at the inner and acceptor binding at the outer cell surface.

High voltage free flow electrophoresis has been applied to the separation of human platelet membranes. After short treatment with neuraminidase at the whole cell level, three membrane vesicle subpopulations have been isolated. Using a surface label (125I-labeled Lens culinaris lectin), the marker enzyme NADH-cytochrome c reductase, and lipid analysis, two of the fractions have been identified as of surface origin and the other consists of intracellular membrane elements. The distribution of adenylate cyclase, leucyl aminopeptidase, 5'-nucleotidase and Ca2+-ATPase has also been investigated, and their usefulness as markers for the different membrane fractions has been evaluated. All three fractions are vesicular but differ in size and character. Their phospholipid and cholesterol contents have been determined, and the cholesterol/phospholipid ratios of the two surface fractions are over twice that of the intracellular membrane, which also has a significantly lower microviscosity as determined by fluorescence polarization using diphenyl hexatriene. The polypeptide profiles from sodium dodecyl sulfate-polyacrylamide gel electrophoresis are particularly distinctive, with actin present in the two surface membrane fractions and absent from the intracellular membranes. Myosin, confirmed by its ATPase characteristics, is almost exclusively localized in one of the surface membrane fractions, and actin-binding protein is a prominent feature of the other.