Platelet mitochondrial function in Leber's hereditary optic neuropathy.

We report the effect of the 11,778 and 3460 base pair mitochondrial DNA mutations, found in Leber's hereditary optic neuropathy (LHON), on platelet mitochondrial respiratory chain enzyme activity. We measured respiratory chain enzyme activities in platelets from 4 patients with the 3460 mutation, 17 patients with the 11,778 mutation and compared them with those of 41 healthy age-matched controls. We observed a 67% (P < 0.001) reduction in the mean NADH CoQ1 reductase (complex I) activity of the 3460 group compared to the control group. It has been shown previously that platelet mitochondrial biochemistry is affected by cigarette smoking. A significant reduction (25%, P < 0.03) in the mean complex I activity of the 11,778 group was only observed when the non-smokers within that group were compared to the non-smoking controls. The effect of smoking observed in this study may explain why previous workers have not observed a decrease in complex I activity associated with the 11,778 mutation. There was no significant change in the activity of complexes II/III or IV with either of these mutations. There was a significant increase (26%, P < 0.008) in citrate synthase (CS) activity with the non-smoking 11,778 group compared to the non-smoking control group, rising to 40% (P < 0.002) in those with this mutation who smoked. This reflects an increase in mitochondrial mass with the 11,778 mutation. This effect was not observed with the 3460 mutation even though the complex deficiency was much more severe.

Smoking and mitochondrial function: a model for environmental toxins.

Defects of the human mitochondrial respiratory chain have been associated with several diseases including, most recently, certain neurodegenerative disorders. Several studies have used platelet mitochondrial function as a means to determine the potential contribution of respiratory chain defects to the pathogenesis of Parkinson's disease. Platelet biochemistry is subject to modulation by numerous factors that may circulate in the blood, including environmental agents, some of which may be relevant to mitochondrial dysfunction and neuronal toxicity. We measured mitochondrial respiratory chain enzyme activities in platelets from 18 normal healthy non-smoking controls and compared them with those from 23 similarly healthy cigarette smoking individuals. A 24% decrease (p < 0.02) was observed in the mean NADH CoQ1 reductase (complex I) activity of the smoking group compared with that of the non-smoking group. There was no significant change in the activity of any of the other respiratory chain enzymes. This is the first demonstration in vivo of mitochondrial inhibition by a common environmental agent. The results offer a novel mechanism of action for the cellular toxicity, or even mutagenicity, associated with cigarette smoking. In addition, these data have important implications for the interpretation of platelet mitochondrial complex I activities in disease states. They are particularly relevant to our interpretation and understanding of the complex I deficiency in Parkinson's disease platelets.

Isolation and partial characterization of the plasma membrane of purified bovine neutrophils.

1. Large amounts of granulocytes can be isolated from bovine blood by differential centrifugation and hypotonic lysis of erythrocytes, followed by separation of neutrophils and eosinophils by centrifugation through a gradient of colloidal silica. 2. Careful homogenization of the purified neutrophils and subfractionation of the postnuclear supernatant by centrifugation through a discontinuous sucrose gradient provides a membrane fraction (at the 20/32%, w/w, sucrose interface), which collects about 20--35% of the activity of plasma membrane marker enzymes. 3. Treatment of the plasma membrane fraction with 0.5 M KCl removes some protein and activity of granule enzymes, leading to an about 20--35-fold enrichment in specific activity of plasma membrane marker enzymes. In particular, there is a 25-fold enrichment in a Ca2+-dependent ATPase, whose half-maximal reaction velocity is reached at 2.2 X 10(-7) M Ca2+. 4. High-resolution sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals a complex composition of the neutrophil plasma membrane, with about 40 polypeptides stained by Coomassie blue. Ten of these peptides are more intensely stained by the dye; their apparent molecular weight ranges from 81 000 to 34 000. All these ten polypeptides but one (which is likely to be actin) are markedly enriched in the plasma membrane fraction over the original homogenate. 5. Since bovine blood can be obtained in unlimited amounts, the procedures here described can be applied to a large-scale purification of the neutrophil plasma membrane, suitable for biochemical studies.

Blood mononuclear cell coenzyme Q10 concentration and mitochondrial respiratory chain succinate cytochrome-c reductase activity in phenylketonuric patients.

Coenzyme Q10 (CoQ10) serves as an electron carrier within the mitochondrial respiratory chain (MRC), where it is integrally involved in oxidative phosphorylation and consequently ATP production. It has recently been suggested that phenylketonuria (PKU) patients may be susceptible to a CoQ10 deficiency as a consequence of their phenylalanine-restricted diet, which avoids foods rich in CoQ10 and its precursors. Furthermore, the high phenylalanine level in PKU patients not on dietary restriction may also result in impaired endogenous CoQ10 production, as previous studies have suggested an inhibitory effect of phenylalanine on HMG-CoA reductase, the rate-controlling enzyme in CoQ10 biosynthesis. We investigated the effect of both dietary restriction and elevated plasma phenylalanine concentration on blood mononuclear cell CoQ10 concentration and the activity of MRC complex II + III (succinate:cytochrome-c reductase; an enzyme that relies on endogenous CoQ10) in a PKU patient population. The concentrations of CoQ10 and MRC complex II + III activity were not found to be significantly different between the PKU patients on dietary restriction, PKU patients off dietary restriction and the control group, although plasma phenylalanine levels were markedly different. The results from this investigation suggest that dietary restriction and the elevated plasma phenylalanine levels of PKU patients do not effect mononuclear cell CoQ10 concentration and consequently the activity of complex II + III of the MRC.