mitochondrial dysfunction brief: reference abstracts

Research Advisory Committee on Gulf War Veterans’ Illnesses (RAC-GWVI)Mitochondrial Dysfunction Brief: Reference Abstracts

References with abstractsAbstracts not available or not entered for all citations

1. Barbiroli, B., et al., Coenzyme Q10 improves mitochondrial respiration in patients with mitochondrial cytopathies. An in vivo study on brain and skeletal muscle by phosphorous magnetic resonance spectroscopy. Cellular and Molecular Biology, 1997. 43(5): p. 741-9.

2. Barbiroli, B., S. Iotti, and R. Lodi, Improved brain and muscle mitochondrial respiration with CoQ. An in vivo study by 31P-MR spectroscopy in patients with mitochondrial cytopathies. Biofactors, 1999. 9(2-4): p. 253-60. We used in vivo phosphorus magnetic resonance spectroscopy (31P-MRS) to study the effect of CoQ10 on the efficiency of brain and skeletal muscle mitochondrial respiration in ten patients with mitochondrial cytopathies. Before CoQ, brain [PCr] was remarkably lower in patients than in controls, while [Pi] and [ADP] were higher. Brain cytosolic free [Mg2+] and delta G of ATP hydrolysis were also abnormal in all patients. MRS also revealed abnormal mitochondrial function in the skeletal muscles of all patients, as shown by a decreased rate of PCr recovery from exercise. After six-months of treatment with CoQ (150 mg/day), all brain MRS-measurable variables as well as the rate of muscle mitochondrial respiration were remarkably improved in all patients. These in vivo findings show that treatment with CoQ in patients with mitochondrial cytopathies improves mitochondrial respiration in both brain and skeletal muscles, and are consistent with Lenaz's view that increased CoQ concentration in the mitochondrial membrane increases the efficiency of oxidative phosphorylation independently of enzyme deficit.

3. Boitier, E., et al., A case of mitochondrial encephalomyopathy associated with a muscle coenzyme Q10 deficiency. Journal of the Neurological Sciences, 1998. 156(1): p. 41-6.

4. Chen, R.S., C.C. Huang, and N.S. Chu, Coenzyme Q10 treatment in mitochondrial encephalomyopathies. Short-term double-blind, crossover study. European Neurology, 1997. 37(4): p. 212-8.

5. Darley-Usmar, V.M., et al., Deficiency in ubiquinone cytochrome c reductase in a patient with mitochondrial myopathy and lactic acidosis. Proc Natl Acad Sci U S A, 1983. 80(16): p. 5103-6. The skeletal muscle of a patient with a mitochondrial myopathy was examined. A defect in the electron transport chain was identified at the position of complex III by activity measurements and the low levels of reducible cytochrome b. The polypeptide composition of complex III in the patient's mitochondria was determined by antibody binding experiments. The method allowed detection of individual polypeptides at a lower limit of 10-40 ng of protein. Characterization of protein composition is thus possible by using a biopsy sample of 1 g of tissue. The level of core proteins, FeS protein, and subunit VI was greatly diminished in the patient's mitochondria. Cytochrome c1 polypeptide was found at normal levels but was sensitive to proteolysis by trypsin. These results show that complex III is not assembled in the patient's mitochondria. The possible role of cytochrome b as the site of the primary lesion is discussed.

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6. Di Giovanni, S., et al., Coenzyme Q10 reverses pathological phenotype and reduces apoptosis in familial CoQ10 deficiency. Neurology, 2001. 57(3): p. 515-8. Two brothers with myopathic coenzyme Q10 (CoQ10) deficiency responded dramatically to CoQ10 supplementation. Muscle biopsies before therapy showed ragged-red fibers, lipid storage, and complex I + III and II + III deficiency. Approximately 30% of myofibers had multiple features of apoptosis. After 8 months of treatment, excessive lipid storage resolved, CoQ10 level normalized, mitochondrial enzymes increased, and proportion of fibers with TUNEL-positive nuclei decreased to 10%. The authors conclude that muscle CoQ10 deficiency can be corrected by supplementation of CoQ10, which appears to stimulate mitochondrial proliferation and to prevent apoptosis.

7. England, J.D., et al., Mitochondrial myopathy developing on treatment with the HMG CoA reductase inhibitors--simvastatin and pravastatin [letter]. Australian and New Zealand Journal of Medicine, 1995. 25(4): p. 374-5.

8. Fosslien, E., Mitochondrial medicine--molecular pathology of defective oxidative phosphorylation. Ann Clin Lab Sci, 2001. 31(1): p. 25-67. Different tissues display distinct sensitivities to defective mitochondrial oxidative phosphorylation (OXPHOS). Tissues highly dependent on oxygen such as the cardiac muscle, skeletal and smooth muscle, the central and peripheral nervous system, the kidney, and the insulin-producing pancreatic beta-cell are especially susceptible to defective OXPHOS. There is evidence that defective OXPHOS plays an important role in atherogenesis, in the pathogenesis of Alzheimer's disease, Parkinson's disease, diabetes, and aging. Defective OXPHOS may be caused by abnormal mitochondrial biosynthesis due to inherited or acquired mutations in the nuclear (n) or mitochondrial (mt) deoxyribonucleic acid (DNA). For instance, the presence of a mutation of the mtDNA in the pancreatic beta-cell impairs adenosine triphosphate (ATP) generation and insulin synthesis. The nuclear genome controls mitochondrial biosynthesis, but mtDNA has a much higher mutation rate than nDNA because it lacks histones and is exposed to the radical oxygen species (ROS) generated by the electron transport chain, and the mtDNA repair system is limited. Defective OXPHOS may be caused by insufficient fuel supply, by defective electron transport chain enzymes (Complexes I - IV), lack of the electron carrier coenzyme Q10, lack of oxygen due to ischemia or anemia, or excessive membrane leakage, resulting in insufficient mitochondrial inner membrane potential for ATP synthesis by the F0F1-ATPase. Human tissues can counteract OXPHOS defects by stimulating mitochondrial biosynthesis; however, above a certain threshold the lack of ATP causes cell death. Many agents affect OXPHOS. Several nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit or uncouple OXPHOS and induce the 'topical' phase of gastrointestinal ulcer formation. Uncoupled mitochondria reduce cell viability. The Helicobacter pylori induces uncoupling. The uncoupling that opens the membrane pores can activate apoptosis. Cholic acid in experimental atherogenic diets inhibits Complex IV, cocaine inhibits Complex I, the poliovirus inhibits Complex II, ceramide inhibits Complex III, azide, cyanide, chloroform, and methamphetamine inhibit Complex IV. Ethanol abuse and antiviral nucleoside analogue therapy inhibit mtDNA replication. By contrast, melatonin stimulates Complexes I and IV and Gingko biloba stimulates Complexes I and III. Oral

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Q10 supplementation is effective in treating cardiomyopathies and in restoring plasma levels reduced by the statin type of cholesterol-lowering drugs.

9. Ogasahara, S., et al., Muscle coenzyme Q deficiency in familial mitochondrial encephalomyopathy. Proc Natl Acad Sci U S A, 1989. 86(7): p. 2379-82. The electron transport system of muscle mitochondria was examined in a familial syndrome of lactacidemia, mitochondrial myopathy, and encephalopathy. The propositus, a 14-year-old female, and her 12-year-old sister had suffered from progressive muscle weakness, abnormal fatigability, and central nervous system dysfunction since early childhood. In the propositus, the state 3 respiratory rate of muscle mitochondria with NADH-linked substrates and with succinate was markedly reduced. The levels of cytochromes a + a3, b, and c + c1 were normal. The activities of complexes I, II, III, and IV of the electron transport chain were normal or increased. By contrast, the activities of complex I-III and of complex II-III, both of which need coenzyme Q10 (CoQ10), were abnormally low. On direct measurement, the mitochondrial CoQ10 content was 3.7% of the mean value observed in 10 controls. Serum and cultured fibroblasts of the propositus had normal CoQ10 contents. In the younger sister, the respiratory activities and CoQ10 level of muscle mitochondria were similar to those observed in the propositus. The findings establish CoQ10 deficiency as a cause of a familial mitochondrial cytopathy and suggest that the disease results from a tissue-specific defect of CoQ10 biosynthesis.

10. Takusa, Y. and S. Yamaguchi, [Myopathies with miscellaneous disorders related to mitochondrial fatty acid oxidation: defective synthesis of ketone body, long-chain fatty acid transport defect, and muscular coenzyme Q10 deficiency]. Ryoikibetsu Shokogun Shirizu, 2001(36): p. 90-4.

11. Tedeschi, D., et al., Potential involvement of ubiquinone in myotonic dystrophy pathophysiology: new diagnostic approaches for new rationale therapeutics. Neurol Sci, 2000. 21(5 Suppl): p. S979-80. An impairment of mitochondrial function may contribute to the pathophysiology of myotonic dystrophy (MyD). Coenzyme Q10 (CoQ10) deficiency has been previously observed, even if in a restricted sample of patients. The aim of this investigation was to obtain more information about coenzyme Q10 and its relationships to the aerobic metabolism in a group of MyD patients. Serum CoQ10 appeared significantly reduced with respect to normal controls: 0.93 +/- 0.22 vs. 1.58 +/- 0.28 micrograms/ml (p < 0.05). Moreover, the results demonstrated an inverse tendency between CoQ10 levels and the CTG expansion degree. Basal blood lactate levels were significantly higher than controls (p < 0.05). A borderline inverse correlation between CoQ10 and lactate, corresponding to lactate threshold, was found. These data suggest a possible role of CoQ10 in the pathogenesis of MyD, which may be mediated by mechanisms of cellular damage common to the oxidative pathway. Therapeutic strategies may be devised by virtue of this rationale.

12. Fukuda, K., et al., Chronic multisymptom illness affecting Air Force veterans of the Gulf War. JAMA, 1998. 280: p. 981-988.

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13. Bowman, P.D., et al., Myopathic changes in diaphragm of rats fed pyridostigmine bromide subchronically. Fundam Appl Toxicol, 1989. 13(1): p. 110-7. To determine if alterations in muscle morphology occur after subchronic oral administration of pyridostigmine bromide, rats were fed 90 mg/kg continuously in meal and examined at 1, 2, 4, 7, and 15 days. Within the first day, cholinesterase activity was reduced by 87% and remained inhibited by 74-91% for the entire course of the feeding. Light microscopy demonstrated that by the first day approximately 1 in 100 myofibers was shrunken and contained centralized nuclei. Electron microscopic examination showed that while presynaptic areas of neuromuscular junctions were relatively unaffected by this dose, postsynaptic areas invariably showed maximal changes. Ultrastructural alterations included disruption of myofilaments, mitochondrial changes consistent with accumulation of calcium, and nuclear alterations. These effects appeared not to be cumulative and were greatly diminished by 15 days even under constant drug administration and inhibition of cholinesterase activity. We conclude that subchronic feeding of pyridostigmine bromide induces primarily myopathic rather than neurogenic changes in the diaphragm and that some mechanism of accommodation may be activated that minimizes continued muscle injury.

14. Glass-Marmor, L., M. Chen-Zion, and R. Beitner, Effects of carbamylcholine and pyridostigmine on cytoskeleton-bound and cytosolic phosphofructokinase and ATP levels in different rat tissues. Gen Pharmacol, 1996. 27(7): p. 1241-6. 1. The effects of carbamylcholine (CaCh) (acetylcholine agonist) and pyridostigmine (Pyr) (acetylcholinesterase inhibitor), on the activity of cytoskeleton-bound and cytosolic phosphofructokinase (PFK), the rate-limiting enzyme in glycolysis, and ATP levels, were studied in rat tibialis anterior (TA) muscle, heart, and brain. 2. In the TA muscle, a marked (about three-fold) increase in the allosteric activity of cytosolic (soluble) PFK was found, 3-5 min following the injection of CaCh or Pyr. The intracellular distribution of the enzyme was not affected by both drugs. Stimulation of glycolysis in this muscle was also expressed by a significant increase in the concentrations of glycolytic intermediates and lactate. Glucose 1,6-bisphosphate (Glc-1,6-P2) levels were unchanged, whereas fructose-2,6-bisphosphate (Fru-2,6-P2) was increased. Glycogenolysis was also stimulated, as deduced from the decrease in glycogen content. The stimulation of glycolysis, induced by both drugs, was accompanied by an increase in ATP level in the TA muscle. 3. In contrast to the stimulatory action of CaCh or Pyr on glycolysis in the TA muscle, both drugs had no effect on cytosolic and cytoskeletal PFK in heart and brain. However, ATP content in both heart and brain was markedly reduced by these drugs, most probably due to their reported harmful effects on mitochondrial function, leading to tissue damage. 4. Electron microscopic studies of TA muscle and heart from rats treated with CaCh or Pyr, revealed severe damage of heart but no harmful effects on TA muscle, which is a muscle with high glycolytic and low oxidative capacity. The present experiments suggest that the accelerated glycolysis in this muscle induced by both drugs, supplies ATP, thus preventing muscle damage.

15. Glass-Marmor, L. and R. Beitner, Effects of carbamylcholine and pyridostigmine on mitochondrial-bound hexokinase in skeletal muscle and heart. Biochem Mol Med, 1996. 57(1): p. 67-70. We show here that carbamylcholine (acetylcholine agonist) or

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pyridostigmine (acetylcholinesterase inhibitor), drugs which are widely used in medical treatments, exerted a rapid reduction in mitochondrial-bound hexokinase. This reduction was inversely proportional to the changes in glucose-6-phosphate levels in skeletal and heart muscle. Increased concentration of acetylcholine, occurring in various diseases or induced by acetylcholinesterase inhibitors, was reported to cause deterioration of mitochondrial function, resulting in heart and muscle damage. The present experiments suggest that the reduction in mitochondrial-bound hexokinase, which is closely linked to intramitochondrial oxidative metabolism, may play an important role in the mechanism which leads to tissue damage.

16. Hudson, C.S., R.E. Foster, and M.W. Kahng, Neuromuscular toxicity of pyridostigmine bromide in the diaphragm, extensor digitorum longus, and soleus muscles of the rat. Fundam Appl Toxicol, 1985. 5(6 Pt 2): p. S260-9. The neuromuscular junctions from diaphragm, soleus, and extensor digitorum longus (EDL) muscles of male albino rats were assessed for morphological alterations following acute (30-min) and subacute (2-day) exposure to pyridostigmine bromide in Mestinon-equivalent buffer. These muscles were selected to compare the effects of the drug on muscles of different fiber type composition. The diaphragm has approximately equal numbers of type I and type II fibers while the soleus and EDL possess primarily type I and type II fibers, respectively. Pyridostigmine was administered to each acute-exposure animal by a single subcutaneous injection of 0.36 mg/kg pyridostigmine and to each subacute-exposure animal by a subcutaneously implanted osmotic minipump containing 10 mg/ml pyridostigmine. Both treatments resulted in whole blood cholinesterase (ChE) depression of approximately 60-70% as determined by radiometric assay. Control animals received only Mestinon-equivalent buffer. Both acute and subacute exposures resulted in morphological alteration of the neuromuscular junctions (NMJs) of all three muscles, although considerable variation in the extent of damage occurred even within individual NMJs. The most frequently observed presynaptic alterations were mitochondrial damage and partial withdrawal of nerve terminal branches (partial denervation). Post-synaptic changes included occasional rarefaction of mitochondrial matrices and disruption of the myofibrillar organization in small numbers of subjunctional sarcomeres. The data indicate that acute or subacute exposure to pyridostigmine bromide at a whole blood ChE depression of 60-70% results in similar alterations to the NMJs of three muscles with substantially different fiber type compositions. Although the severity of the damage varies from fiber to fiber, the variability appears random and not related to a specific fiber type or dosage regimen.

17. Kato, T., et al., Role of acetylcholine in pyridostigmine-induced myocardial injury: possible involvement of parasympathetic nervous system in the genesis of cardiomyopathy. Arch Toxicol, 1989. 63(2): p. 137-43. Although acetylcholine is known to be involved in the genesis of skeletal muscle disturbance, its effect on cardiac muscle has been scarcely studied. In the present paper, using pyridostigmine, a cholinesterase inhibitor, the possible role of acetylcholine in the genesis of cardiomyopathy was investigated. In a mortality study, it was shown that pyridostigmine (100 mg/kg) caused death of 9/10 rats within 8 h, and that the lethality of such a dose could be significantly diminished by the subsequent administration of a total dose of 4 mg/kg atropine. In all

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other experiments, rats were divided into three groups; the control, untreated group; the pyridostigmine + atropine group in which atropine (2 mg/kg) was administered 5 min after pyridostigmine (60 mg/kg) administration; and the pyridostigmine group in which pyridostigmine (60 mg/kg) was administered orally. Rats were killed 3 h after pyridostigmine administration, and hearts were isolated. Heart mitochondrial electron transport activity (NADH-cytochrome c reductase, succinate-cytochrome c reductase, and cytochrome c oxidase) were measured enzymatically, and mitochondrial respiratory rates and control indices were measured polarographically. Structural changes in cardiac muscles of each group were observed by electron microscopy of cardiac sections. Acetylcholine levels of left ventricle were measured by high performance liquid chromatography. Activities of NADH-cytochrome c reductase and succinate-cytochrome c reductase were not affected by pyridostigmine administration; however, cytochrome c oxidase activity was significantly reduced in the pyridostigmine group. Atropine markedly lessened this reduction in activity. A protective effect of atropine was also observed morphologically. A protective effect of atropine was also observed morphologically. In the pyridostigmine group and the pyridostigmine + atropine group, left ventricular acetylcholine levels were increased significantly compared with the control.(ABSTRACT TRUNCATED AT 250 WORDS).

18. Schuschereba, S.T., et al., Myopathic alterations in extraocular muscle of rats subchronically fed pyridostigmine bromide. Toxicol Pathol, 1990. 18(3): p. 387-95. To determine if alterations in extraocular muscle morphology occur after subchronic oral administration of pyridostigmine bromide, rats were continuously fed 90 mg/kg in meal and examined at 1, 2, 4, 7, and 15 days. Within the first day, blood acetylcholinesterase activity was reduced by 87% and remained inhibited by 74-91% during the study. Light microscopy demonstrated that by day 1 approximately 3% of the extraocular myofibers were shrunken and invaded by inflammatory cells. The most severe degenerative changes consisting of vacuoles and inflammatory cell infiltration occurred at day 1 with progressively less severe changes at days 2 and 4. At days 7 and 15, 1.3-4.5% of the myofibers still exhibited damage. Ultrastructurally, all presynaptic areas were normal but the postsynaptic areas of affected myofibers at days 1, 2, and 4 showed myofilament and Z-band dissolution, mitochondrial inclusions, subneural fold and T-tubule/sarcoplasmic reticulum vacuolization and subneural fold depth reduction. By days 7 and 15, these changes were diminished in some cases and in others alterations appeared similar to day 1. We conclude that subchronic feeding of pyridostigmine bromide induces myopathic rather than neurogenic changes in rat extraocular muscle and that the myopathy is different in these muscles than in the diaphragm from the the same rats.

19. Kawabuchi, M., Neostigmine myopathy is a calcium ion-mediated myopathy initially affecting the motor end-plate. J Neuropathol Exp Neurol, 1982. 41(3): p. 298-314. Morphological techniques were used to determine the acute and chronic effects of neostigmine on rat muscles. Transient calcium deposits, eliminated by prior treatment of sections with ethyleneglycol bis (aminoethylether) tetracetate (EGTA), were independent of fiber type and found at sites corresponding to neostigmine-induced focal lesions. The dimension and number of focal lesions and calcium deposits gradually decreased with chronic drug treatment. Size, shape, and density of the calcium deposits varied.

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Alterations in the motor nerve terminal, synaptic space, and junctional fold persisted even when banding patterns at the motor end plates were intact. Characteristic intermediate findings consisted of rod bodies and ribosomal clusters. Such clusters were frequently mingled and clumped sarcoplasmic reticulums, T-systems, or mitochondria. Despite continued administration of neostigmine, focal myopathic changes, other than in the synaptic region of the end plates, were reversible.

20. Babu, S.R., et al., Effect of physostigmine on plasma lactate and pyruvate in untrained/trained rats. Pharmacol Biochem Behav, 1992. 42(1): p. 67-73. Physostigmine (Phy) is metabolized to eseroline, a phenolic compound that appears to alter mitochondrial functions. The effect of Phy on recovery from exercise and on time course of plasma lactate and pyruvate levels following an acute bout of exercise (AE) was examined in untrained and trained (ET) rats. Phy alone elicited significantly higher plasma lactate and pyruvate levels than sedentary control. AE + Phy had a significantly higher plasma lactate and pyruvate levels compared to AE 2 min postadministration. From 5-30 min postexercise, lactate and pyruvate levels did not differ between these two acutely exercised groups. ET + Phy exhibited significantly lower levels of plasma lactate and pyruvate from 5-60 min postexercise compared to ET. The data show that the "additive" effect of Phy on postexercise plasma lactate and pyruvate levels can be attenuated by an enhanced fitness level in these rats.

21. De Pinieux, G., et al., Lipid-lowering drugs and mitochondrial function: effects of HMG-CoA reductase inhibitors on serum ubiquinone and blood lactate/pyruvate ratio. British Journal of Clinical Pharmacology, 1996. 42(3): p. 333-7. 1. Statins inhibit synthesis of mevalonate, a precursor of ubiquinone that is a central compound of the mitochondrial respiratory chain. The main adverse effect of statins is a toxic myopathy possibly related to mitochondrial dysfunction. 2. This study was designed to evaluate the effect of lipid-lowering drugs on ubiquinone (coenzyme Q10) serum level and on mitochondrial function assessed by blood lactate/pyruvate ratio. 3. Eighty hypercholesterolaemic patients (40 treated by statins, 20 treated by fibrates, and 20 untreated patients, all 80 having total cholesterol levels > 6.0 mmol l-1) and 20 healthy controls were included. Ubiquinone serum level and blood lactate/pyruvate ratio used as a test for mitochondrial dysfunction were evaluated in all subjects. 4. Lactate/pyruvate ratios were significantly higher in patients treated by statins than in untreated hypercholesterolaemic patients or in healthy controls (P < 0.05 and P < 0.001). The difference was not significant between fibratetreated patients and untreated patients. 5. Ubiquinone serum levels were lower in statin-treated patients (0.75 mg l-1 +/- 0.04) than in untreated hypercholesterolaemic patients (0.95 mg l-1 +/- 0.09; P < 0.05). 6. We conclude that statin therapy can be associated with high blood lactate/ pyruvate ratio suggestive of mitochondrial dysfunction. It is uncertain to what extent low serum levels of ubiquinone could explain the mitochondrial dysfunction.

22. King, B.F. and S.M. Somani, Distribution of physostigmine and metabolites in brain subcellular fractions of the rat. Life Sci, 1987. 41(17): p. 2007-15. The distribution of 3H-physostigmine (Phy) has been studied in the rat brain subcellular fractions at various time intervals following i.v. injection. 3H-Phy or its metabolites rapidly

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accumulate into the cytoplasm of cells and penetrates the intracellular compartments. Kinetic studies of the subcellular distribution of radioactivity (RA) per gm of rat brain following i.v. injection of 3H-Phy show peak concentrations at 30 min in all subcellular fractions with the exception of mitochondria. In the mitochondrial fraction the RA levels continue to rise from 4682 +/- 875 DPM/gm at 5 min to 27474 +/- 2825 DPM/gm at 60 min (P less than .05). The cytosol contains the highest RA: 223341 +/- 21044 DPM/gm at 30 min which declined to 53475 +/- 3756 DPM/gm at 60 min. RA in synaptosome, microsomes and myelin increases from 5 to 30 min, and declines at 60 min. In vitro studies did not show a greater uptake of RA by the mitochondrial or synaptosomal fractions. The finding of relatively high concentrations of RA in the mitochondrial fraction at 60 min increases the likelihood that Phy or its metabolites could interfere with the physiological function of this organelle.

23. Li, L., et al., Pyridostigmine-induced acute and delayed neuronal apoptosis [abstract]. Soc Toxicol Abstract, 1999.

24. Li, L., et al., Muscarinic receptor-mediated pyridostigmine-induced neuronal apoptosis. Neurotoxicology, 2000. 21(4): p. 541-52. Pyridostigmine is a reversible cholinesterase (ChE) inhibitor that is associated with neurologic dysfunction involving both central and peripheral nervous systems. To determine the neurotoxic potential of pyridostigmine, rats were sacrificed at intervals after drug administration (0.5-1.85 mg/kg, i.p., twice daily for 4 days) and brains examined histologically. ChE inhibition was used as a biomarker of pyridostigmine activity. Using the in situ terminal deoxynucleotidyl transferase nick-end labeling of DNA fragments (TUNEL) method and electron microscopy, apoptotic brain cell death was noted in cerebral cortex over a dose range of 0.5-1.85 mg/kg and at the higher dose (1.85 mg/kg), apoptosis was also noted in striatum and hippocampus. These responses were blocked by pretreatment with atropine. Rat cortical cells in culture also underwent apoptosis when exposed to pyridostigmine (250 microM for 24 hr), indicating that the pyridostigmine can initiate apoptosis, independent of peripheral mechanisms. Pretreatment of cells with atropine (10 microM) inhibited pyridostigmine-induced apoptosis, confirming the response was mediated by muscarinic receptors. Short term treatment of rats with pyridostigmine (1.85 mg/kg twice daily for 4 days) induced a prolonged apoptotic response, which was evident in rat cortex up to 30 days after the last dose. Active apoptosis persisted, despite recovery of serum ChE activity. These in vivo and in vitro observations indicate that pyridostigmine can initiate a prolonged neurodegeneration.

25. Isom, G.E., Pyridostigmine-induced neurodegeneration: role of neuronal apoptosis, in Annual Report to Congress: Federally Sponsored Research on Gulf War Veterans' Illnesses for 1998. Appendices, The Research Working Group of the Persian Gulf Veterans Coordinating Board, Editor. 1999. p. 100.

26. Li, L., et al., Reactive oxygen species mediate pyridostigmine-induced neuronal apoptosis: involvement of muscarinic and NMDA receptors. Toxicol Appl Pharmacol, 2001. 177(1): p. 17-25. Pyridostigmine bromide (PB) is a reversible cholinesterase inhibitor used for treatment of myasthenia gravis and for prophylactic protection against

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organophosphate nerve agent. We previously showed PB can induce apoptotic death in rat brain following systemic treatment. To study mechanisms by which PB induces brain cell death, cultured rat cerebellar granule cells were used. Cytotoxicity was determined after exposure to PB (10-1000 microM) for 24 h; a high concentration of PB (>500 microM) significantly increased lactate dehydrogenase release, which was reduced by pretreatment with the antioxidant, N-t-butyl-alpha-phenyl-nitrone (PBN). Apoptosis, as determined by TUNEL staining, was concentration dependent (10-250 microM) after a 24-h exposure and cytotoxicity was confirmed by gel electrophoresis of DNA, release of cytochrome c from mitochondria, elevation of caspase activity, and electron microscopy. The oxidant-sensitive fluorescent dye 2',7'-dichlorofluorescin diacetate was used to detect reactive oxidative species (ROS) generation. Pretreatment with PBN, superoxide dismutase, catalase, or the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) blocked PB-induced ROS generation and apoptotic cell death. Pretreatment with atropine or MK-801 blocked ROS generation and the subsequent neurotoxicity, showing that both muscarinic and NMDA receptors mediate the response. DNA extracted from PB-treated cells revealed oligonucleosomal fragmentation on gel electrophoresis and antioxidants attenuated the DNA fragmentation, providing further evidence for a link of ROS generation and apoptosis. These results indicate that muscarinic receptor-mediated ROS generation is an initiating factor in PB-induced apoptotic cell death and activation of the NMDA glutamate receptor is directly linked to the response.

27. Quillet-Mary, A., et al., Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis. J Biol Chem, 1997. 272(34): p. 21388-95. The key events implicated in ceramide-triggered apoptosis remain unknown. In this study we show that 25 &mgr;M C6-ceramide induced significant H2O2 production within 60 min, which increased up to 180 min in human myeloid leukemia U937 cells. Inactive analogue dihydro-C6-ceramide had no effect. Furthermore, no H2O2 production was observed in C6-ceramide-treated U937 rho degrees cells, which are mitochondrial respiration-deficient. We also present evidence that ceramide-induced activation of the transcription factors NF-kappaB and AP-1 is mediated by mitochondrial derived reactive oxygen species. Both H2O2 production, transcription factor activation as well as apoptosis could be inhibited by rotenone and thenoyltrifluoroacetone (specific mitochondrial complexes I and II inhibitors) and antioxidants, N-acetylcysteine and pyrrolidine dithiocarbamate. These effects could be potentiated by antimycin A (specific complex III mitochondrial inhibitor). H2O2 production was also inhibitable by ruthenium red, suggesting a role of mitochondrial calcium homeostasis alterations in ceramide-induced oxidative stress. Finally, C6-ceramide had no influence on mitochondrial membrane potential within the first 6 h. Altogether, our study points to reactive oxygen species, generated at the ubiquinone site of the mitochondrial respiratory chain, as an early major mediator in ceramide-induced apoptosis.

28. Haley, R.W., et al., Brain abnormalities in Gulf War syndrome: Evaluation with 1H MR spectroscopy. Radiology, 2000. 215: p. 807-817. PURPOSE: To test for neuronal brain damage in the basal ganglia and brainstem in Gulf War veterans by using magnetic resonance (MR) spectroscopy. MATERIALS AND METHODS: Twenty-two Gulf War

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veterans with one of three factor analysis-derived syndromes (case patients); 18 well veterans matched for age, sex, and education level (control subjects); and six Gulf War veterans with syndrome 2 from a different population (replication sample) underwent long echo time (272 msec) proton (hydrogen 1) MR spectroscopy on a 4 x 2 x 2-cm voxel in the basal ganglia bilaterally and a 2 x 2 x 2-cm voxel in the pons. Syndromes 1-3 are described as "impaired cognition," "confusion-ataxia," and "central pain," respectively. RESULTS: The N-acetylaspartate-to-creatine (NAA/Cr) ratio, which reflects functional neuronal mass, was significantly lower in the basal ganglia and brainstem of Gulf War veterans with the three syndromes than in those structures of the control subjects (P =.007). The finding was corroborated in the replication sample (P =.002). Veterans with syndrome 2 (the most severe clinically) had evidence of decreased NAA/Cr in both the basal ganglia and the brainstem; those with syndrome 1, in the basal ganglia only; and those with syndrome 3, in the brainstem only. CONCLUSION: Veterans with different Gulf War syndromes have biochemical evidence of neuronal damage in different distributions in the basal ganglia and brainstem.

29. Asa, P.B., Y. Cao, and R.F. Garry, Antibodies to squalene in Gulf War syndrome. Exp Mol Pathol, 2000. 68(1): p. 55-64. Gulf War Syndrome (GWS) is a multisystemic illness afflicting many Gulf War-era veterans. The molecular pathological basis for GWS has not been established. We sought to determine whether the presence of antibodies to squalene correlates with the presence of signs and symptoms of GWS. Participants in this blinded cohort study were individuals immunized for service in Desert Shield/Desert Storm during 1990-1991. They included 144 Gulf War-era veterans or military employees (58 in the blinded study), 48 blood donors, 40 systemic lupus erythematosus patients, 34 silicone breast implant recipients, and 30 chronic fatigue syndrome patients. Serum antibodies to squalene were measured. In our small cohort, the substantial majority (95%) of overtly ill deployed GWS patients had antibodies to squalene. All (100%) GWS patients immunized for service in Desert Shield/Desert Storm who did not deploy, but had the same signs and symptoms as those who did deploy, had antibodies to squalene. In contrast, none (0%) of the deployed Persian Gulf veterans not showing signs and symptoms of GWS have antibodies to squalene. Neither patients with idiopathic autoimmune disease nor healthy controls had detectable serum antibodies to squalene. The majority of symptomatic GWS patients had serum antibodies to squalene.

30. Powell, M.F. and M.J. Newman, eds. Chapter 8. Adjuvant properties of aluminum and calcium compounds. Vaccine Design. The Subunit and Adjuvant Approach. 1995, Plenum Press: New York.

31. Melinda Plaisier, FDA Associate Commissioner of Legislation, Letter to Congressmand Jack Metcalf, 20 March 2000. Cited in: [Committee on Government Reform Hearings for the United States House of Representatives, 2000 #1320]. 2000. Cited in: Accountability of DoD, FDA adn BioPort Officials for the Anthrax Vaccine Immunization Program (AVIP_. Committee on Government Reform Hearings for th United States House of Representatives. Oct 3 and 11, 2000.

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32. Ivins, B., et al., Experimental anthrax vaccines: efficacy of adjuvants combined with protective antigen against an aerosol Bacillus anthracis spore challenge in guinea pigs. Vaccine, 1995. 13(18): p. 1779-84. The efficacy of several human anthrax vaccine candidates comprised of different adjuvants together with Bacillus anthracis protective antigen (PA) was evaluated in guinea pigs challenged by an aerosol of virulent B. anthracis spores. The most efficacious vaccines tested were formulated with PA plus monophosphoryl lipid A (MPL) in a squalene/lecithin/Tween 80 emulsion (SLT) and PA plus the saponin QS-21. The PA+MPL in SLT vaccine, which was lyophilized and then reconstituted before use, demonstrated strong protective immunogenicity, even after storage for 2 years at 4 degrees C. The MPL component was required for maximum efficacy of the vaccine. Eliminating lyophilization of the vaccine did not diminish its protective efficacy. No significant alteration in efficacy was observed when PA was dialyzed against different buffers before preparation of vaccine. PA+MPL in SLT proved superior in efficacy to the licensed United States human anthrax vaccine in the guinea pig model.

33. Ivins, B. and et al, Adjuvant efficacy in experimental anthrax vaccines: Protection studies in guinea pigs. Abstracts of the 91st General Meeting of the American Society for Microbiology, 1991: p. 121.

34. Matsumoto, G., The Pentagon's toxic secret, in Vanity Fair. 1999. p. 83-96.

35. Tomich, N., Anthrax vaccine program scrutiny intensifies. U.S. Medicine, 2000. 36(11): p. 1,32,33,36.

36. Carlson, B.C., et al., The endogenous adjuvant squalene can induce a chronic T-cell-mediated arthritis in rats. Am J Pathol, 2000. 156(6): p. 2057-65. Squalene is a cholesterol precursor, which stimulates the immune system nonspecifically. We demonstrate that one intradermal injection of this adjuvant lipid can induce joint-specific inflammation in arthritis-prone DA rats. Histopathological and immunohistochemical analyses revealed erosion of bone and cartilage, and that development of polyarthritis coincided with infiltration of alphabeta(+) T cells. Depletion of these cells with anti-alphabeta TcR monoclonal antibody (R73) resulted in complete recovery, whereas anti-CD8 and anti-gammadelta TcR injections were ineffective. The apparent dependence on CD4(+) T cells suggested a role for genes within the major histocompatibility complex (MHC), and this was concluded from comparative studies of MHC congenic rat strains, in which DA.1H rats were less susceptible than DA rats. Furthermore, LEW.1AV1 and PVG.1AV1 rats with MHC identical to DA rats were arthritis-resistant, demonstrating that non-MHC genes also determine susceptibility. Some of these genetic influences could be linked to previously described arthritis susceptibility loci in an F2 intercross between DA and LEW.1AV1 rats (ie, Cia3, Oia2 and Cia5). Interestingly, some F2 hybrid rats developed chronic arthritis, a phenotype not apparent in the parental inbred strains. Our demonstration that an autoadjuvant can trigger chronic, immune-mediated joint-specific inflammation may give clues to the pathogenesis of rheumatoid arthritis, and it raises new questions concerning the role of endogenous molecules with adjuvant properties in chronic inflammatory diseases.

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37. Gajkowska, B., et al., The experimental squalene encephaloneuropathy in the rat. Exp Toxicol Pathol, 1999. 51: p. 75-80.

38. Lorentzen, J.C., Identification of arthritogenic adjuvants of self and foreign origin. Scand J Immunol, 1999. 49(1): p. 45-50.

39. Sverdrup, B., L. Klareskog, and S. Kleinau, Common commercial cosmetic products induce arthritis in the DA rat. Environ Health Perspect, 1998. 106(1): p. 27-32. squalene arthritis.

40. Dijkstra, J., et al., Interaction of anti-cholesterol antibodies with human lipoproteins. J Immunol, 1996. 157(5): p. 2006-13. Inoculation of mice with cholesterol-rich liposomes containing the adjuvant monophosphoryl lipid A results in the production of antiserum containing IgM Ab to cholesterol. The specificity of the Ab was to cholesterol and structurally similar sterols containing a 3 beta-hydroxyl group. Anti-cholesterol binding activity was significantly diminished if the 3 beta-hydroxyl was altered by either epimerization, substitution, oxidation, or esterification. A similar specificity for 3 beta-hydroxy-sterols was observed for an anti-cholesterol IgM mAb. Both hyperimmune serum and the mAb reacted with intact human very-low-/intermediate-density lipoprotein (VLDL/IDL) and low-density lipoproteins (LDL), but not high-density lipoproteins (HDL), in an ELISA, but could react with total lipid extracts containing cholesterol that were prepared from all three lipoprotein classes. Functionally, immune serum or the mAb aggregated and induced a fusion-like reaction with VLDL/IDL and LDL at low temperatures: these aggregates result in spherical structures visible with light microscopy. Similarly, binding of anti-cholesterol A to small cholesterol-rich liposomes resulted in the appearance of vesicular structures with approximately 20- to 200-fold increased diameters. These data demonstrate that the anti-cholesterol Ab recognize unesterified cholesterol in VLDL/IDL and LDL; high-density lipoprotein cholesterol in the intact lipoprotein, however, appears to be protected from reaction with these Ab.

41. Ordovas, J.M., Anticholesterol antibodies and plaque formation. Nutr Rev, 1996. 54(4 Pt 1): p. 124-7. Immunization of rabbits with a protein-free formulation consisting of liposomes containing 71% cholesterol and lipid A induced cholesterol antibodies in rabbits fed a diet containing 0.5% and 1.0% cholesterol. Elevation in plasma cholesterol levels was significantly less in immunized than in nonimmunized rabbits. Immunization also resulted in a marked decrease in the risk of developing atherosclerosis as determined by analysis of aortic atherosclerosis by quantitative histological examination and fatty streaks by automated morphometric probability-of-occurrence mapping.

42. Alving, C.R., et al., Immunization with cholesterol-rich liposomes induces anti-cholesterol antibodies and reduces diet-induced hypercholesterolemia and plaque formation. J Lab Clin Med, 1996. 127(1): p. 40-9. Immunization of rabbits with a protein-free formulation consisting of liposomes containing 71% cholesterol and lipid A as an adjuvant induced anticholesterol antibodies that caused complement-dependent

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lysis of liposomes lacking lipid A. The antibodies, immunoglobulin G (IgG) and immunoglobulin M (IgM), also recognized nonoxidized crystalline cholesterol as an antigen by enzyme-linked immunosorbent assay (ELISA). The effects of immunization against cholesterol on elevations in serum cholesterol and development of atherosclerosis were examined in rabbits fed a diet containing 0.5% to 1.0% cholesterol. Although the mean serum cholesterol level, mainly in the form of very-low-density lipoprotein cholesterol, rose as much as 60-fold in the nonimmunized rabbits, the elevation was significantly less--as much as 35% lower--in the immunized rabbits. Elevation of serum cholesterol was accompanied by an apparent drop in the level of antibodies on initiating the diet, followed by a rebound on stopping the diet, thus suggesting that the antibodies were adsorbed to cholesterol that was present in circulating lipoproteins. When lipoprotein fractions--composed of either very-low-density and intermediate-density lipoproteins derived from cholesterol-fed nonimmunized rabbits or human low-density lipoproteins--were tested as capture antigens by solid-phase ELISA, reactivity was observed with IgG and IgM antibodies present in the serum of immunized rabbits. Immunization also resulted in a marked decrease in the risk of developing atherosclerosis. Analysis of aortic atherosclerosis by quantitative histologic examination and fatty streaks by automated morphometric probability-of-occurrence mapping showed diminished atherosclerosis in most areas of the aorta in vaccine recipients. It is proposed that immunization with liposomes containing 71% cholesterol and lipid A can reduce diet-induced hypercholesterolemia and atherosclerosis.

43. Alving, C.R., N.M. Wassef, and M. Potter, Antibodies to cholesterol: biological implications of antibodies to lipids. Curr Top Microbiol Immunol, 1996. 210: p. 181-6. Injection of silicone gel or silicone oil intraperitoneally into BALB/c mice induced the formation of antibodies that reacted by ELISA with highly purified crystalline cholesterol and, to a much lesser extent, antibodies that reacted with a phospholipid (dimyristoyl phosphatidylglycerol). Although IgM and IgG antibodies to cholesterol were detected, the titers of IgG antibodies were low when compared with IgM. The titers of IgM antibodies to cholesterol in certain sera exhibited activities that reached baseline values at dilutions as high as 1:5000, thus making them equivalent to titers that have been previously published for ascites fluid containing murine monoclonal antibodies to cholesterol. The antibodies to cholesterol induced by silicone compounds are indistinguishable in their binding to crystalline cholesterol from naturally-occurring antibodies to cholesterol in normal human serum. They are also indistinguishable from antibodies induced by a proposed vaccine to cholesterol that is currently in late preclinical development for prevention of hypercholesterolemia in humans. The anti-cholesterol vaccine, which consists of liposomes heavily laden with cholesterol as an antigen and lipid A as an adjuvant, induces antibodies that react with low density lipoproteins (LDL) and opsonize them for removal by liver macrophages. It appears that silicone gel or silicone oil causes recruitment and adsorption of cholesterol at the injection site, and also serves as an adjuvant that may have immunostimulant properties similar to lipid A for inducing antibodies to lipids. Antibodies to lipids such as cholesterol or phospholipids are not harmful to intact cell membranes because of steric hindrance from surrounding lipids and larger macromolecules that block binding of the antibodies.

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44. Lahita, R.G., et al., Low levels of total cholesterol, high-density lipoprotein, and apolipoprotein A1 in association with anticardiolipin antibodies in patients with systemic lupus erythematosus. Arthritis Rheum, 1993. 36(11): p. 1566-74. OBJECTIVE. To determine if there is an association between low levels of high-density lipoprotein cholesterol (HDL), apolipoprotein A1 (Apo A1), total cholesterol, and anticardiolipin antibody (aCL) in patients with systemic lupus erythematosus (SLE) who are not taking corticosteroids. METHODS. We studied 75 outpatients with documented SLE who were attending our hospital clinics: 57 were aCL positive and 18 were aCL negative. Both IgG and IgM aCL levels were determined by enzyme-linked immunosorbent assay. Lipid fractions (total cholesterol, HDL, low-density lipoprotein, very-low-density lipoprotein, and triglycerides) were determined by standard enzymatic techniques. Apo A1 and Apo B levels were determined by nephelometry. RESULTS. Patients with SLE who were IgG aCL+ had low levels of serum cholesterol (mean +/- SD 173.6 +/- 34.6 mg/dl) and HDL (43.9 +/- 16.3 mg/dl) compared with aCL- SLE patients, normal donors, and patients with other diseases. Apo A1 levels were also low in the aCL+ group (95.5 +/- 50.9 mg/dl) compared with the aCL- group (152.7 +/- 32.6 mg/dl). There was no association of total cholesterol level or aCL titer with clinical activity. CONCLUSION. These data indicate that in SLE patients, there is an association between antibody against the phospholipid cardiolipin and low levels of cholesterol, HDL, and Apo A1.

45. Alving, C.R. and G.M. Swartz, Jr., Antibodies to cholesterol, cholesterol conjugates and liposomes: implications for atherosclerosis and autoimmunity. Crit Rev Immunol, 1991. 10(5): p. 441-53. Polyclonal and monoclonal antibodies to cholesterol are readily induced by injecting cholesterol-loaded liposomes containing lipid A as an adjuvant. Analysis of the literature reveals that conjugates of cholesterol, and conjugates of analogues of cholesterol, with heterologous proteins or lipids have been used as antigens in various studies since 1925, and this has led to successful development of immunoassays for steroid hormones. It is concluded that cholesterol is a highly immunogenic molecule. The ability of monoclonal antibodies to cholesterol to react with liposomes containing cholesterol to cause complement-dependent immune damage to the liposomes is strongly influenced by the lipid composition of the liposomes, the amount of cholesterol in the liposomes, and the reaction temperature. The antibodies also react with crystalline cholesterol in a solid-phase ELISA and, depending on the particular monoclonal antibody, immune reactivity may or may not be observed with cholesterol esters, cholesterol analogues, or steroid hormones. Analysis by ELISA has revealed that virtually all normal human sera contain varying levels of naturally occurring IgG and IgM autoantibodies to cholesterol. Naturally occurring autoantibodies to cholesterol are also observed in pigs, but not in guinea pigs. Possible implications of these investigations for theories of immune mechanisms that may have beneficial or detrimental roles in processes of aging, atherosclerosis, and vascular diseases are discussed.

46. Alving, C.R., Antibodies to liposomes, phospholipids, and cholesterol: implications for autoimmunity, atherosclerosis, and aging. Prog Clin Biol Res, 1990. 343: p. 41-51.

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47. Swartz, G.M., Jr., et al., Antibodies to cholesterol. Proc Natl Acad Sci U S A, 1988. 85(6): p. 1902-6. Cholesterol-dependent complement activation has been proposed as a factor that might influence the pathogenesis of atherosclerosis. Although antibodies to cholesterol conjugates have been reported, cholesterol is widely regarded as a poorly immunogenic substance. Monoclonal IgM complement-fixing antibodies to cholesterol were obtained in the present study after immunizing mice with liposomes containing high amounts of cholesterol (71 mol % relative to phosphatidylcholine) and lipid A as an adjuvant. Clones were selected for the ability of secreted antibodies to react with liposomes containing 71% cholesterol but not with liposomes containing 43% cholesterol. The antibodies also reacted with crystalline cholesterol in a solid-phase enzyme-linked immunosorbent assay. Binding of monoclonal antibodies to the surface of crystalline cholesterol was demonstrated by electron microscopy by utilizing a second antibody (anti-IgM) labeled with colloidal gold. The immunization period required to induce monoclonal antibodies was very short (3 days) and a high fraction of the hybrid cells (at least 70%) were secreting detectable antibodies to cholesterol. The results demonstrate that cholesterol can be a highly immunogenic molecule and that complement-fixing antibodies to cholesterol can be readily obtained.

48. Folkers, K. and J.L. Nilsson, [Coenzyme Q regarded as a vitamin]. Nord Med, 1970. 83(16): p. 489-93.

49. Estornell, E., et al., Effects of vitamin A deficiency on mitochondrial function in rat liver and heart. Br J Nutr, 2000. 84(6): p. 927-34. The aim of this study was to investigate comparative effects of vitamin A deficiency on respiratory activity and structural integrity in liver and heart mitochondria. Male rats were fed a liquid control diet (control rats) or a liquid vitamin A-deficient diet (vitamin A-deficient rats) for 50 days. One group of vitamin-A deficient rats was refed a control diet for 15 days (vitamin A-recovered rats). To assess the respiratory function of mitochondria the contents of coenzyme Q (ubiquinone, CoQ), cytochrome c and the activities of the whole electron transport chain and of each of its respiratory complexes were evaluated. Chronic vitamin A deficiency promoted a significant increase in the endogenous coenzyme Q content in liver and heart mitochondria when compared with control values. Vitamin A deficiency induced a decrease in the activity of complex I (NADH-CoQ reductase) and complex II (succinate-CoQ reductase) and in the levels of complex I and cytochrome c in heart mitochondria. However, NADH and succinate oxidation rates were maintained at the control levels due to an increase in the CoQ content in accordance with the kinetic behaviour of CoQ as an homogeneous pool. On the contrary, the high CoQ content did not affect the electron-transfer rate in liver mitochondria, whose integrity was preserved from the deleterious effects of the vitamin A deficiency. Ultrastructural assessment of liver and heart showed that vitamin A deficiency did not induce appreciable alterations in the morphology of their mitochondria. After refeeding the control diet, serum retinol, liver and heart CoQ content and the activity of complex I and complex II in heart mitochondria returned to normality. However, the activities of both whole electron transfer chain and complex I in liver were increased over the control values. The interrelationships between physiological antioxidants in biological membranes and the beneficial effects of their administration in mitochondrial diseases are discussed.

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50. Anderson, S.S., I.G. Lyle, and R. Paterson, Electron transfer across membranes using vitamin K1 and coenzyme Q10 as carrier molecules. Nature, 1976. 259(5539): p. 147-8.

51. Sobreira, C., et al., Mitochondrial encephalomyopathy with coenzyme Q10 deficiency. Neurology, 1997. 48(5): p. 1238-43.

52. DiMauro, S., et al., Mitochondria in neuromuscular disorders. Biochimica et Biophysica Acta, 1998. 1366(1-2): p. 199-210.

53. Suzuki, Y., et al., A case of diabetic amyotrophy associated with 3243 mitochondrial tRNA(leu; UUR) mutation and successful therapy with coenzyme Q10. Endocrine Journal, 1995. 42(2): p. 141-5.

54. Hansen, I.L., et al., Bioenergetics in clinical medicine. IX. Gingival and leucocytic deficiencies of coenzyme Q10 in patients with periodontal disease. Res Commun Chem Pathol Pharmacol, 1976. 14(4): p. 729-38. The specific activities of the succinate dehydrogenase-coenzyme Q10 reductase in mitochondria were determined for patients from a normal periodontal practice. The criteria for selection were patients having a bone score of 1.0-4.0 and a pocket depth of 2.5-5.2 mm. All 29 patients showed a deficiency of 20-63% of CoQ10-enzyme activity in gingival biopsies. The mean value was elevated (P less than 0.001) over that of controls. For corresponding blood samples, 24/28 (86%) showed deficiencies of 20-66% and a higher (P less than 0.001) mean value than that of controls. Periodontal patients frequently have significant gingival and leucocytic deficiencies of CoQ10. The leucocytic deficiency indicates a systemic nutritional imbalance and is not likely caused by neglected oral hygiene. A gingival deficiency could predispose this tissue to periodontitis and this disease could even augment the deficiency. These results support previously suggested adjunctive use of CoQ10 with oral hygiene for improved treatment presumably through bioenergetics.

55. Wilkinson, E.G., et al., Bioenergetics in clinical medicine. II. Adjunctive treatment with coenzyme Q in periodontal therapy. Res Commun Chem Pathol Pharmacol, 1975. 12(1): p. 111-23. Eight patients under routine care for periodontitis received oral treatment with a form of coenzyme Q (7 / CoQ10 and 1 / hexahydrocoenzyme Q4). An unchanged plaque score showed the patients cooperated and were under plaque control. The periodontal score decreased (p less than 0.01) on CoQ treatment. Unexpectedly, the periodontal pocket depth decreased (P less than 0.05) on CoQ treatment since all patients were considered candidates for surgical intervention. Healing was so excellent 5-7 days post-biopsy that the biopsy sites were difficult to locate. The healing was viewed as extraordinarily effective. The mean value of the specific activities of the succinate dehydrogenase-coenzyme Q10 reductase of gingival biopsies increased (P less than 0.05) during treatment which could correlate with the extraordinarily healing. Treatment of periodontitis with coenzyme Q should be considered as adjunctive treatment with current dental practice.

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56. Folkers, K. and R. Simonsen, Two successful double-blind trials with coenzyme Q10 (vitamin Q10) on muscular dystrophies and neurogenic atrophies. Biochimica et Biophysica Acta, 1995. 1271(1): p. 281-6.

57. Kuz'menko, I.V., et al., [Efficiency of ubiquinone and p-oxybenzoic acid in prevention of E-hypovitaminosis-induced development of muscular dystrophy]. Ukr Biokhim Zh, 1981. 53(5): p. 73-9. It is shown that E-hypovitaminosis-induced muscular dystrophy in rabbits is accompanied by a sharp decrease in the body mass, an increase in the urine creatine-index, a decrease in the vitamin E and ubiquinone contents in the liver and skeletal muscle tissues. In the myocardium mitochondria a decrease in the vitamin E content and an increase in the ubiquinone content are observed. The activity of NADH-cytochrome c-, NADH-ubiquinone- and succinate-ubiquinone-reductase also varies in mitochondria of the studied tissues. In myocardium organellas a direct dependence is found between the content of ubiquinone, NADH- and succinate-ubiquinone-reductase activity and an inverse one-between its content and the activity of the NADH-cytochrome c-reductase system. It is established that p-oxybenzoic acid as well as vitamin E prevents development of muscular dystrophy and causes changes analogous in direction in the activity of the ubiquinone-dependent enzymic systems of mitochondria. Ubiquinone-9 is less efficient in preventing the development of muscular dystrophy.

58. Shimomura, Y., et al., Protective effect of coenzyme Q10 on exercise-induced muscular injury. Biochem Biophys Res Commun, 1991. 176(1): p. 349-55. The effect of coenzyme Q10 administration on exercise-induced muscular injury was examined in rats. Coenzyme Q10-treated and control rats were exercised by 90 min of downhill treadmill running. A part of the animals in both groups were killed immediately after exercise and the others were 40 h postexercise. After the exercise bout, serum creatine kinase and lactate dehydrogenase activities were elevated in the control rats, but not in the coenzyme Q10-treated rats. These enzyme activities in the latter increased to the similar level of the former 40 h postexercise. The muscle coenzyme Q10 content increased by the coenzyme Q10 treatment. These results suggest that the coenzyme Q10 treatment protected skeletal muscles against injury caused during exercise, but not against damage related with inflammatory processes after exercise.

59. Muller, D.P., J.K. Lloyd, and O.H. Wolff, Vitamin E and neurological function: abetalipoproteinaemia and other disorders of fat absorption. Ciba Found Symp, 1983. 101: p. 106-21. Evidence that vitamin E is important for normal neurological function in humans is presented. First, in abetalipoproteinaemia early therapy with vitamin E delays and may prevent the development of the neurological complications, and in patients with established lesions treatment can arrest or reverse the neuropathy. Second, in other chronic disorders of fat absorption with severe vitamin E deficiency, neurological manifestations which are very similar to those described in untreated abetalipoproteinaemia can be improved by vitamin E. Vitamin E supplementation is therefore advisable for all patients with chronic fat malabsorption who have low serum vitamin E concentration. Serum vitamin E concentrations should also be measured in patients with spinocerebellar disorders, whatever the aetiology.

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60. Muller, D.P.R., J.K. Lloyd, and A. Bird, Long-term management of abetalipoproteinemia. Possible role for vitamin E. Arch Dis Child, 1977. 52: p. 209-14.

61. Azizi, E., et al., Abetalipoproteinemia treated with parenteral and oral vitamin A and E and with medium chain triglycerides. Acta Pediatr Scand, 1978. 67: p. 797-801.

62. Hegele, R.A. and A. Angel, Arrest of neuropathy and myopathy in abetalipoproteinemia with high-dose vitamin E therapy. Can Med Assoc J, 1985. 132(1): p. 41-4. A 16-year-old girl, one of dizygotic twins, presented in 1976 complaining of a 1-year history of a lack of coordination and an inability to run. The results of biochemical tests confirmed the diagnosis of classic abetalipoproteinemia. In addition to the recognized neurologic features of this disorder, she had a reduced evoked motor unit potential and markedly elevated serum levels of muscle enzymes, which suggested myositis. The serum vitamin E level was markedly decreased. Oral therapy with vitamin E, 800 mg daily, was begun, and in 1981 the dosage was increased to 3200 mg daily. Over the 7 years of follow-up she improved clinically, there was an increase in the evoked motor unit potential, the serum levels of some of the muscle enzymes decreased to normal, and the serum and tissue vitamin E levels increased significantly. It was concluded that treatment with high doses of vitamin E was responsible for the arrest of the usually progressive neuropathy and myopathy.

63. Iannaccone, S.T. and R.J. Sokol, Vitamin E deficiency in neuropathy of abetalipoproteinemia. Neurology, 1986. 36(7): p. 1009.

64. Alleva, R., et al., Coenzyme Q blocks biochemical but not receptor-mediated apoptosis by increasing mitochondrial antioxidant protection. FEBS Lett, 2001. 503(1): p. 46-50. Generation of free radicals is often associated with the induction and progression of apoptosis. Therefore, antioxidants can prove anti-apoptotic, and can help to elucidate specific apoptotic pathways. Here we studied whether coenzyme Q, present in membranes in reduced (ubiquinol) or oxidised (ubiquinone) forms, can affect apoptosis induced by various stimuli. Exposure of Jurkat cells to alpha-tocopheryl succinate (alpha-TOS), hydrogen peroxide, anti-Fas IgM or TRAIL led to induction of apoptosis. Cell death due to the chemical agents was suppressed in cells enriched with the reduced form of coenzyme Q. However, coenzyme Q did not block cell death induced by the immunological agents. Ubiquinol-10 inhibited reactive oxygen species (ROS) generation in cells exposed to alpha-TOS, and a mitochondrially targeted coenzyme Q analogue also blocked apoptosis triggered by alpha-TOS or hydrogen peroxide. Therefore, it is plausible that ubiquinol-10 protects cells from chemically-induced apoptosis by acting as an antioxidant in mitochondria. Our results also indicate that generation of free radicals may not be a critical step in induction of apoptosis by immunological agents.

65. Fernandez-Ayala, D.J., et al., Coenzyme Q protects cells against serum withdrawal-induced apoptosis by inhibition of ceramide release and caspase-3 activation. Antioxid Redox Signal, 2000. 2(2): p. 263-75. Coenzyme Q10 (CoQ10) is a component of the antioxidant machinery that protects cell membranes from oxidative damage and decreases apoptosis in leukemic cells cultured in serum-depleted media.

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Serum deprivation induced apoptosis in CEM-C7H2 (CEM) and to a lesser extent in CEM-9F3, a subline overexpressing Bcl-2. Addition of CoQ10 to serum-free media decreased apoptosis in both cell lines. Serum withdrawal induced an early increase of neutral-sphingomyelinase activity, release of ceramide, and activation of caspase-3 in both cell lines, but this effect was more pronounced in CEM cells. CoQ10 prevented activation of this cascade of events. Lipids extracted from serum-depleted cultures activated caspase-3 independently of the presence of mitochondria in cell-free in vitro assays. Activation of caspase-3 by lipid extracts or ceramide was prevented by okadaic acid, indicating the implication of a phosphatase in this process. Our results support the hypothesis that plasma membrane CoQ10 regulate the initiation phase of serum withdrawal-induced apoptosis by preventing oxidative damage and thus avoiding activation of downstream effectors as neutral-sphingomyelinase and subsequent ceramide release and caspase activation pathways.

66. Kagan, T., et al., Coenzyme Q10 can in some circumstances block apoptosis, and this effect is mediated through mitochondria. Ann N Y Acad Sci, 1999. 887: p. 31-47. The mitochondrial component coenzyme Q10 (CoQ10) has been used for many years as a dietary supplement intended to promote good health by trapping free radicals, thus preventing lipid peroxidation and DNA damage. We have tested its use as a generic anti-apoptotic compound and have found that its ability to protect against apoptosis varies depending on both cell type and mode of cell death induction. We have further established that this protection may be mediated by its effect on mitochondrial function and viability. We provide additional evidence that CoQ10's protective effect on mitochondrial membrane potential does not always result in altered mitochondrial enzyme activity and neither does it guarantee survival. These observations open the way for further investigations into the mechanisms involved in mitochondrial control of apoptosis.

67. Mosca, L., et al., Modulation of apoptosis and improved redox metabolism with the use of a new antioxidant formula. Biochem Pharmacol, 2002. 63(7): p. 1305-14. Oxidative stress is involved in the pathogenesis of a wide spectrum of diseases, implicating that strategies directed at counterbalancing oxidative processes could have a role in clinical medicine. There is also an evidence that oxidative stress acts as a major determinant of apoptotic cell death. Many studies have reported favourable effects of antioxidant formulas on several parameters of the oxidant-antioxidant balance, but none of them has focused whether antioxidant formulas could modulate apoptosis. We investigated in 20 healthy individuals the effect of supplementation with a formula containing alpha-tocopherol, alpha-lipoic acid, coenzyme Q(10), carnitines, and selenomethionine, on plasma oxidant status and peroxide levels, erythrocyte antioxidant enzymes, lymphocyte apoptosis, and generation of ROS at the mitochondrial level. Control subjects received only carnitines or an incomplete formula with alpha-tocopherol, alpha-lipoic acid, coenzyme Q(10), and selenomethionine. Supplementation with the complete formula resulted in a significant increase in the plasma antioxidant status that was mirrored by a decrease in blood peroxide levels and a reduced generation of ROS at the mitochondrial level. This was associated with a significant decrease in the frequency of peripheral blood lymphocytes, with either CD4 or CD8 phenotype,

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undergoing apoptosis. Less consistent results were found when either incomplete formula was used. Our study suggests that supplementation with antioxidant formulas can modulate the process of apoptosis under in vivo conditions. The clinical potential of this strategy in the treatment of diseases with an elevated commitment to apoptosis should be explored.

68. Buckenmeyer, P.J., et al., Effect of concurrent exercise and physostigmine on lactate and pyruvate in plasma, muscle, and brain tissue of rats. Pharmacol Biochem Behav, 1994. 47(4): p. 779-88. The purpose of this investigation was to determine the effect of physostigmine (Phy) and/or concurrent exercise on lactate, pyruvate, and L/P ratio in plasma, skeletal muscle, and brain tissue in male Sprague-Dawley rats. The Phy-dosed (Phy-D) and Phy-dosed + concurrent acute exercise (Phy-D + CAE) groups elicited significantly higher L/P ratios in plasma compared to the acutely exercised (AE) group at 30 min postexercise. Physostigmine dosing, with or without exercise, resulted in significantly lower muscle pyruvate levels, from 30 to 50 min postdrug administration, in Phy-D and Phy-D + CAE groups compared to the AE group. In the brain, lactate values were significantly elevated in the acutely exercised groups at 5 min postexercise with or without Phy dosing. However, at 15 to 30 min postexercise, lactate values were significantly elevated in the Phy-D + CAE compared to the AE group. These data suggest that when Phy is administered prior to a 20-min moderately intensive exercise bout, there is an accumulation of lactate for a prolonged period of time in recovery.

69. Phillips, P. and et al, Title Not Yet on Current Contents. Annals of Internal Medicine, 2002. Oct 1, 2002: p. Pages to be secured.

70. Ackrell, B.A., et al., Effect of iron deficiency on succinate- and NADH-ubiquinone oxidoreductases in skeletal muscle mitochondria. J Biol Chem, 1984. 259(16): p. 10053-9. The effects of iron deficiency on the NADH- and succinate-oxidizing complexes of rat skeletal muscle mitochondria have been investigated. Both systems were similarly affected: activities were about 30% of normal in dehydrogenase, ubiquinone reductase, and oxidase assays, and similar reductions in the concentration of their respective flavin prosthetic groups were also evident in the iron-deficient membranes. Thus, the turnover numbers of the two enzymes were unchanged in iron deficiency. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed similarly reduced levels of those peptide components of Complexes I and II that could be unequivocally distinguished. Soluble beef heart succinate dehydrogenase added to alkaline-treated rat skeletal muscle mitochondrial membranes attached to binding sites exposed by the treatment, forming a hybrid complex indistinguishable from the original skeletal muscle complex, with restoration of succinoxidase and succinate-ubiquinone reductase activities to the levels observed in the original rat membranes. Iron-deficient particles behaved like the normal in these tests. No unfilled binding sites for the enzyme could be detected prior to alkaline treatment. The data are interpreted as indicating that the lower activities of these two respiratory complexes in iron deficiency are due to lower content of the enzymes rather than to the presence of impaired enzymes in the membrane, that only fully competent complexes are present in these membranes, and that iron-deficient complexes are either not assembled or are lost after assembly.

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71. Shingwekar, A.G., M. Mohanram, and V. Reddy, Effect of zinc supplementation on plasma levels of vitamin A and retinol-binding protein in malnourished children. Clin Chim Acta, 1979. 93(1): p. 97-100. The relationship between plasma levels of vitamin A and zinc was studied in 45 children suffering from vitamin A deficiency and 20 children with protein-energy malnutrition. Thirty apparently normal children of the same age group were also studied for comparison. The mean levels of plasma vitamin A, retinol-binding protein and zinc were significantly lower in vitamin A-deficient children and in children with PEM, as compared to controls. Supplementation with 40 mg zinc daily for 5--10 days resulted in a significant increase in plasma vitamin A and RBP levels in children with PEM but not in the vitamin A-deficient group. There was, however, no correlation between plasma levels of vitamin A and zinc. The data suggest that in children with PEM, apart from deficiencies of protein and vitamin A, zinc deficiency may also contribute to the lowering of plasma vitamin A levels. They also suggest that in vitamin A-deficient children, without protein-energy malnutrition, zinc deficiency does not seem to have a role.

72. Vadhanavikit, S. and H.E. Ganther, Decreased ubiquinone levels in tissues of rats deficient in selenium. Biochem Biophys Res Commun, 1993. 190(3): p. 921-6. The effect of selenium deficiency (-Se) on the levels of ubiquinones in liver, heart, kidney, and leg muscle was studied in the rat. Levels of ubiquinone 9 and ubiquinone 10 in the liver of -Se rats were about 50% of the levels in selenium adequate animals. Both ubiquinones in the heart were about 15% lower in -Se rats. Only ubiquinone 9 was significantly lower in the kidney of -Se rats. There was no difference in ubiquinone levels in leg muscle. Glutathione peroxidase activity in the tissues of -Se rats was > 95% lower. It is concluded that Se, as an integral part of the enzyme glutathione peroxidase, may protect tissues from oxidative damage, thereby preserving the ability of the cells to synthesize ubiquinone and preventing ubiquinone from oxidative degradation.

73. Pedersen, H.S., et al., High serum coenzyme Q10, positively correlated with age, selenium and cholesterol, in Inuit of Greenland. A pilot study. Biofactors, 1999. 9(2-4): p. 319-23.

74. Pelton, R., et al., Drug-Induced Nutrient Depletion Handbook 1999-2000. 1999, Hudson, Ohio: Lexi-Comp, Inc. 485.

75. Pfizer reports 38% increase, in San Diego Union Tribune, January 24. 2002: San Diego.

76. Sinzinger, H., Does vitamin E beneficially affect muscle pains during HMG-Co-A-reductase inhibitors without CK-elevation. Atherosclerosis, 2000. 149: p. 225. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10799017.

77. Sinzinger, H., F. Chehne, and G. Lupattelli, Oxidation Injury in Patients Receiving HMG-CoA Reductase Inhibitors: Occurrence in Patients Without Enzyme

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Elevation or Myopathy. Drug Saf, 2002. 25(12): p. 877-83. BACKGROUND: Myopathy in its severe forms including rhabdomyolysis is a very rare adverse effect occurring during monotherapy with the HMG-CoA reductase inhibitors ('statins') and is associated with pronounced signs of oxidation injury. This has been found at a local (muscle) as well as at a systemic level (blood). Several lines of evidence indicate that even mild forms of myopathy during statin treatment may be associated with in vivo oxidation injury. In contrast, statin therapy has been shown to be associated with a decrease in oxidation injury. OBJECTIVE: The aim of this study was to investigate whether patients with heterozygous familial hypercholesterolaemia who did not exhibit any symptoms or abnormalities in safety parameters during 6 months of treatment with various statins (atorvastatin, fluvastatin, lovastatin, pravastatin, simvastatin) did exhibit a change in oxidation injury as assessed by the isoprostane levels. METHODS: Blood (plasma and serum) as well as urine was tested before and 1, 3 and 6 months after starting statin therapy. Results: Out of 111 treated patients (63 males, 48 females; aged 19 to 58 years) who did not experience any adverse effects during statin treatment, 11 (seven males, four females; aged 24 to 51 years) showed a pronounced increase in 8-epi-prostaglandin (PG) F(2alpha) in all the compartments examined. In the remaining 100 patients (56 males, 44 females; aged 19 to 58 years) there was either no change in or even an apparent decrease in 8-epi-PGF(2alpha). This increase was monitored with all the statins administered. If elevated, the increase in 8-epi-PGF(2alpha) remained without change throughout the entire follow-up period. No sex difference or differential response between smokers and nonsmokers was observed. DISCUSSION: These findings indicate that in the absence of other clinically observable adverse effects, in some of the patients, for an as yet unknown reason, statin therapy may be associated with increased oxidation injury. The fact that changing to another statin is apparently not necessarily associated with an identical response raises the question of a specific predisposition for certain compounds in a given patient. These data add a further piece of evidence that mild adverse effects of statins that are difficult to assess might be much more prevalent than widely considered. The clinical relevance and consequence of these findings still remains to be assessed.

78. Sinzinger, H., P. Schmid, and J. O'Grady, Two different types of exercise-induced muscle pain without myopathy and CK -elevation during HMG-Co-enzyme-A-reductase inhibitor treatment. Atherosclerosis, 1999. 143(11): p. 459-460.

79. Orsi, A., O. Sherman, and Z. Woldeselassie, Simvastatin-associated memory loss. Pharmacotherapy, 2001. 21(6): p. 767-9. The statins are widely used to treat dyslipidemias. They are generally associated with mild adverse effects, but rarely, more serious reactions may occur. A 51-year-old man experienced delayed-onset, progressive memory loss while receiving simvastatin for hypercholesterolemia. His therapy was switched to pravastatin, and memory loss resolved gradually over the next month, with no recurrence of the adverse effect.

80. Muldoon, M.F., et al., Effects of lovastatin on cognitive function and psychological well-being. Am J Med, 2000. 108(7): p. 538-46.

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81. Bruckert, E., C. Simonetta, and P. Giral, Compliance with fluvastatin treatment characterization of the noncompliant population within a population of 3845 patients with hyperlipidemia. CREOLE Study Team. J Clin Epidemiol, 1999. 52(6): p. 589-94. The objectives of the study were to analyze 1) compliance with an HMG-CoA reductase inhibitor, 2) the relationship between treatment compliance and sociodemographic, clinical, and psychological criteria, and 3) the effect of raising patients' awareness through distribution of an information notice. The results analyzed in this article compare the noncompliant and compliant population independently of awareness. This open-label study was conducted in two randomized parallel groups: a control group that received the information normally given by the practitioners, and an awareness group that received specific informational brochures on diet and cardiovascular risk factors and the reasons for the treatment. Male and female patients (n = 3845) aged 18 to 75 years with primary hypercholesterolemia (type IIa and IIb), not taking a cholesterol-lowering drug or for whom an ongoing treatment was poorly tolerated or ineffective, were to be included. Cholesterol levels had to be greater than 250 mg/dL (or 200 mg/dL if previous coronary history) and triglyceride levels less than 350 mg/dL. A total of 2888 subjects (75%) were defined as compliant (taking more than 90% of the prescription) and 957 (25%) noncompliant. Both populations are identical for age, sex ratio, and different risk factors, with the exception of diabetes. The adverse effects in noncompliant subjects were very clearly different, with an overrepresentation of gastrointestinal and neurologic effects and the noncompliant patients more frequently having more than one adverse effect. Noncompliant patients had an identical duration of follow-up, and the number of patients claiming to have a symptom related to hypercholesterolemia, self-evaluation of cardiovascular risk level, and source of knowledge about cholesterol and diet was similar in both groups. In contrast, in the noncompliant group, there were a larger number of symptomatic patients who thought the drug did not improve the symptoms. In practice, these results show that physicians should systematically evaluate compliance by looking for and analyzing adverse effects and by reassuring the patient when these effects are minor or probably unrelated to the treatment. Diabetics and polymedicated patients deserve special attention in this regard.

82. Gaist, D., et al., Statins and risk of polyneuropathy: A case-control study. Neurology, 2002. 58: p. 1333-1337. Background: Several case reports and a single epidemiologic study indicate that use of statins occasionally may have a deleterious effect on the peripheral nervous system. The authors therefore performed a population-based study to estimate the relative risk of idiopathic polyneuropathy in users of statins. Method: The authors used a population-based patient registry to identify first-time-ever cases of idiopathic polyneuropathy registered in the 5-year period 1994 to 1998. For each case, validated according to predefined criteria, 25 control subjects were randomly selected among subjects from the background population matched for age, sex, and calendar time. The authors used a prescription register to assess exposure to drugs and estimated the odds ratio of use of statins (ever and current use) in cases of idiopathic polyneuropathy compared with control subjects. Results: The authors verified a diagnosis of idiopathic polyneuropathy in 166 cases. The cases were classified as definite (35), probable (54), or possible (77). The odds ratio linking idiopathic polyneuropathy with statin use was 3.7 (95% CI 1.8 to 7.6) for all cases and 14.2 (5.3 to 38.0) for definite

23


cases. The corresponding odds ratios in current users were 4.6 (2.1 to 10.0) for all cases and 16.1 (5.7 to 45.4) for definite cases. For patients treated with statins for 2 or more years the odds ratio of definite idiopathic polyneuropathy was 26.4 (7.8 to 45.4). Conclusions: Long-term exposure to statins may substantially increase the risk of polyneuropathy.

83. Blasiak, J. and Z. Walter, Protective action of cholesterol against changes in membrane fluidity induced by malathion. Acta Biochimica Polonica, 1992. 39(1): p. 49-52. no abstract.

84. Nevin, D.N., et al., Paraoxonase genotypes, lipoprotein lipase activity, and HDL. Arterioscler Thromb Vasc Biol, 1996. 16(10): p. 1243-9. Paraxonase, an enzyme associated with the high density lipoprotein (HDL) particle, hydrolyzes paraoxon, the active metabolite of the insecticide parathion. Several studies have shown that paraxonase levels in humans have a distribution characteristic of two alleles, one with low activity and the other with high activity. Paraoxonase also has arylesterase activity, which does not exhibit activity polymorphism and can therefore serve as an estimate of enzyme protein. Although the ability of paraoxon to irreversibly inhibit lipoprotein lipase (LPL) has been exploited experimentally for many years, the role of plasma paraoxonase in lipoprotein metabolism is unknown. Seventy-two normal individuals were examined for paraoxonase genotypes, plasma paraoxonase and arylesterase activities, postheparin LPL and hepatic lipase (HL) activities, and lipoprotein levels to determine whether (1) paraoxonase activity or genotype determines lipoprotein levels via an effect on LPL or HL activity or (2) variation in LPL and HL activities determines HDL levels and indirectly affects paraoxonase activity and protein levels in plasma. In the entire group, paraoxonase activity was related to arylesterase activity and genotype. Whereas arylesterase activity was correlated with HDL cholesterol (HDL-C) and apolipoproteinA-I (apoA-I) levels, neither arylesterase nor paraoxonase was correlated with LPL or HL activity. Furthermore, LPL activity was positively correlated and HL inversely correlated with HDL cholesterol and apoA-I levels, whereas LPL was inversely correlated with triglyceride levels. The paraoxonase genotypes of the study group were 30 individuals homozygous for the low-activity allele, 38 heterozygotes, and 4 individuals homozygous for the high-activity allele. Paraoxonase genotype accounted for approximately .75 of the variation in paraoxonase activity. Paraoxonase activity was linearly related to arylesterase activity within each subgroup. No difference in either LPL or HL activity was seen as a function of paraoxonase genotype, nor were differences seen in plasma triglyceride or HDL-C by genotype by ANOVA. The relation between LPL and HL and components of HDL in the paraoxonase genotypic subgroups in general reflected the associations seen in the group as a whole. Multivariate analysis showed that LPL, HL, and arylesterase, a measure of paraoxonase mass, were independent predictors of HDL cholesterol, while paraoxonase genotype or activity was not. Thus, variation in LPL and HL appears to be significantly related to HDL cholesterol and apoA-I levels. The levels of HDL are a major correlate of paraoxonase protein levels, while paraoxonase genotype is the major predictor of plasma paraoxonase activity.

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85. Kishi, H., T. Kishi, and K. Folkers, Bioenergetics in clinical medicine. III. Inhibition of coenzyme Q10-enzymes by clinically used anti-hypertensive drugs. Res Commun Chem Pathol Pharmacol, 1975. 12(3): p. 533-40. Background data revealed that some American and Japanese patients with essential hypertension, including many who were not being treated with any anti-hypertensive drug, had a deficiency of coenzyme Q10. Eight clinically used anti-hypertensive drugs have now been tested for inhibition of two mitochondrial coenzyme Q10-enzymes of heart tissue, succinoxidase and NADH-oxidase. Diazoxide and propranolol significantly inhibited the CoQ10-succinoxidase and CoQ10-NADH-oxidase, respectively. Metoprolol did not inhibit succinoxidase, and was one-fourth as active as propranolol for inhibition of NADH-oxidase. Hydrochlorothiazide, hydralazine, ans clonidine also inhibited CoQ10-NADH-oxidase. Reserpine did not inhibit either CoQ10-enzyme, and methyldopa was a very eak inhibitor of succinoxidase. The internationally recognized clinical side-effects of propranolol may be due, in part, to inhibition of CoQ10-enzymes which are indispensable in the bioenergetics of cardiac function. A pre-existing deficiency of coenzyme Q10 in the myocardium of hypertensive patients could be augmented by subsequent treatment with propranolol, possibly to the "life-threatening" state described by others.

86. Kishi, T., T. Watanabe, and K. Folkers, Bioenergetics in clinical medicine XV. Inhibition of coenzyme Q10-enzymes by clinically used adrenergic blockers of beta-receptors. Res Commun Chem Pathol Pharmacol, 1977. 17(1): p. 157-64. Adrenergic blockers for beta-receptors were studied for inhibition of mitochrondrial CoQ10-enzymes. These enzymes are indispensable for the bioenegetics of the myocardium. Propranolol is frequently used to treat hypertension; in some patients, it depresses myocardial function as an adverse reaction. This side effect may be related to the inhibition by propranolol of CoQ10-enzymes of the myocardium. Timolol showed negligible inhibition of the CoQ10-enzyme, NADH-oxidase. Metoprolol was less inhibitory than propranolol. Five alprenolols showed inhibition which approached that of propranolol. The 1-isomer of alprenolol showed weak inhibition of another CoQ10-enzyme, succinoxidase, but the other beta-blockers were essentially non-inhibitory to this enzyme. The drug of choice is timolol, based on negligible inhibition of these bioenergetic enzymes of the heart, which correlates with its pharmacologically low cardiac depressant effects.

87. Reyes, A.J., et al., Diuretics and zinc. S Afr Med J, 1982. 62(11): p. 373-5. Distal tubule diuretics (DTDs), including chlorothiazide, hydrochlorothiazide, bendroflumethiazide, chlorthalidone and xipamide, have been found to increase urinary zinc output through a poorly understood mechanism which could involve both direct and hormone-mediated processes. Significant zinc depletion may occur during long-term administration of DTDs, principally in conditions associated with diminished total body zinc levels such as hepatic cirrhosis, diabetes mellitus, gastro-intestinal disorders and several renal diseases. Attention to the early symptoms of zinc deficiency such as hypogeusia, hyposmia, abnormal dark adaptation and impotence and the monitoring of serum zinc levels are advisable during long-term treatment with common DTDs.

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88. Cohanim, M. and E.R. Yendt, The effects of thiazides on serum and urinary zinc in patients with renal calculi. Johns Hopkins Med J, 1975. 136(3): p. 137-41. Urine zinc (Zn) excretion was significantly higher in 338 male patients with renal calculi than in 21 normal male subjects, but there was no significant difference between 102 female patients with renal calculi and 15 normal female subjects. In 33 patients with renal calculi urinary Zn was less than 50% of the mean normal value; in these patients there is a possibility that lower urine Zn excretion contributes to the formation of renal calculi. Hydrochlorothiazide produced a striking and sustained increase in urinary Zn excretion, which might contribute to the efficacy of this drug in the prevention of calcific renal stones. Serum Zn levels remained normal in the majority of subjects on long term thiazide therapy, but the occasional finding of subnormal levels suggests that long term thiazide therapy carries with it the hazard of Zn deficiency.

89. Mountokalakis, T., et al., Zinc deficiency in mild hypertensive patients treated with diuretics. J Hypertens Suppl, 1984. 2(3): p. S571-2. Mean hair zinc was found to be significantly lower in 20 mild hypertensive patients treated with thiazides for 6-36 months than in 19 mild hypertensive patients who had not been given diuretics for at least six months before study. Mean serum zinc did not differ significantly between the two groups. Chronic diuretic treatment can result in zinc deficiency through enhanced urinary excretion of zinc.

90. Lockett, C.J., et al., Zinc, angiotensin I-converting enzyme and hypertension. S Afr Med J, 1983. 64(26): p. 1022-4. Angiotensin-converting enzyme, or kininase II, is a zinc metallo-enzyme; the cation forms part of its active site. The enzyme transforms angiotensin I into angiotensin II and hydrolyses bradykinin into inactive peptides. Antihypertensive diuretics may decrease the activity of the angiotensin-converting enzyme by increasing urinary zinc excretion. Captopril inhibits the angiotensin-converting enzyme, but also affects electrolyte excretion and should be reassessed as a possible first-choice antihypertensive agent in the treatment of essential hypertension.

91. Bunning, P. and J.F. Riordan, The functional role of zinc in angiotensin converting enzyme: implications for the enzyme mechanism. J Inorg Biochem, 1985. 24(3): p. 183-98. Zinc is essential to the catalytic activity of angiotensin converting enzyme. The enzyme contains one g-atom of zinc per mole of protein. Chelating agents abolish activity by removing the metal ion to yield the inactive, metal-free apoenzyme. Zinc does not stabilize protein structure since the native and apoenzymes are equally susceptible to heat denaturation. Addition of either Zn2+, Co2+, or Mn2+ to the apoenzyme generates an active metalloenzyme; Fe2+, Ni2+, Cu2+, Cd2+, and Hg2+ fail to restore activity. The activities of the metalloenzymes follow the order Zn greater than Co greater than Mn. The protein binds Zn2+ more firmly than it does Co2+ or Mn2+. Hydrolysis of the chromophoric substrate, furanacryloyl-Phe-Gly-Gly, by the active metalloenzymes is subject to chloride activation; the activation constant is not metal dependent. Metal replacement mainly affects Kcat with very little change in Km, indicating that the role of zinc is to catalyze peptide hydrolysis.

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92. Reeves, P.G. and B.L. O'Dell, An experimental study of the effect of zinc on the activity of angiotensin converting enzyme in serum. Clin Chem, 1985. 31(4): p. 581-4. The activity in serum of zinc-dependent angiotensin converting enzyme (ACE), is measured to aid in diagnosis and monitor treatment of certain diseases. This report shows the effect of dietary zinc deprivation on ACE activity in the serum of rats. The mean (and SE) of the zinc concentration (mumol/L) in serum was 3.5 (0.3) in rats deprived of dietary zinc for four days, 16.3 (0.2) in control rats, and 19.8 (0.9) in rats deprived of zinc for four days, then repleted with zinc for 12 h. The respective mean (and SE) of ACE activities (nmol/mL per min) in serum were 390 (15), 543 (13), and 545 (20). Serum ACE activity was restored also by adding zinc to the assay mixture in vitro. The Vmax for ACE was 1.4 times greater when serum was diluted 40-fold as compared with twofold dilution. There was a small effect on the Km for the substrate, but the Km for zinc was decreased by 22-fold when serum was diluted 40-fold. The Vmax under these conditions was decreased by only 9%.

93. Reeves, P.G. and B.L. O'Dell, Effects of dietary zinc deprivation on the activity of angiotensin-converting enzyme in serum of rats and guinea pigs. J Nutr, 1986. 116(1): p. 128-34. Angiotensin-converting enzyme (ACE) is a zinc-dependent peptidyldipeptide hydrolase found in blood and in the endothelium of many tissues. This study was designed to determine the effects of dietary zinc deprivation in rats and guinea pigs on ACE activity in serum. Rats were fed zinc-deficient (less than 1 mg/kg) or zinc-adequate (25 mg/kg) diets for 3 d. Guinea pigs were fed zinc-deficient (less than 1 mg/kg) or zinc-adequate (100 mg/kg) diets for 3 wk. The plasma zinc concentration in the zinc-deprived rats was 26% and that of the deprived guinea pigs 35% of the control values. When serum was diluted by one-half in the assay medium, the respective ACE activities were 42 and 59% of controls. In spite of decreased ACE activity there were no differences in plasma angiotensin II levels in either species or in bradykinin levels in the rat. To determine the effect of zinc on the kinetics of serum ACE, rat serum was treated to remove low-molecular-weight components and diluted 30-fold in an assay medium that contained a final zinc concentration of 0.68 microM. Supplemental zinc (total, 24 microM) increased both Vmax and Km. EDTA inhibited the enzyme activity, and the inhibition was totally reversible by zinc. Copper at 30 microM had no effect on ACE activity. The results of this study show that ACE activity in serum of rats and guinea pigs is highly sensitive to the dietary intake of zinc and suggest that metabolism of the vasoactive hormones, angiotensin II and bradykinin, might be affected in vivo.

94. White, C.L., et al., The effect of zinc in vivo and in vitro on the activities of angiotensin converting enzyme and kininase-I in the plasma of rats. Biochem Pharmacol, 1986. 35(15): p. 2489-93. Two groups of rats were pair fed, for 18 days, diets containing either 2.6 (Zn deficient) or 100 mg Zn/kg (control diet). Plasma was assayed spectrophotometrically for the activity of kininase-I and angiotensin converting enzyme (ACE) in the presence of varying concentrations of added Zn2+. Zinc deficient rats had only 76% of the activity of both kininase-I and ACE compared with zinc supplemented control rats. There was a significant linear relationship between enzyme activity and concentration of zinc in plasma for both enzymes. When zinc was added to the enzyme incubation mixture for zinc deficient rats, the activity of ACE increased by 73% and that

27


of kininase-I by 33%. This Zn2+-stimulated increase in enzyme activity was negatively correlated with the in vivo concentration of zinc in plasma, and a plateau in enzyme activity was seen at concentrations of plasma zinc that were commensurate with normal zinc status (over 14 mumol/1). The results demonstrate that the activities of both kininase-I and ACE are dependent on the concentration of zinc in vivo and in vitro, and suggest that information concerning the concentration of zinc in plasma and assay solutions be a prerequisite solutions be a prerequisite for the use of these enzymes in the clinical diagnosis of disease states. The results also showed that the activity of ACE and kininase-I in plasma could be used for the biochemical diagnosis of a suboptimal zinc status.

95. Schullek, J.R. and I.B. Wilson, The binding of zinc to angiotensin-converting enzyme. Arch Biochem Biophys, 1988. 265(2): p. 346-50. The equilibrium constant for the dissociation of zinc ion from angiotensin-converting enzyme (ACE) was measured using zinc ion buffers of zinc chloride and nitrilotriacetic acid (NTA). The dissociation constant is 6.4 X 10(-10) M. The fraction of active enzyme at equilibrium is independent of the presence of substrate which indicates that hippuryl-histidylleucine binds equally well to the holoenzyme and apoenzyme. The rate constant for the dissociation of zinc from ACE was measured as 0.68 min-1 for the free enzyme; the rate constant for the enzyme substrate complex was roughly 0.18 min-1. The association of zinc ion and ACE is very fast; the rate constant is 1.06 X 10(9) M-1 min-1. Ethylenediaminetetraacetic acid (EDTA) and NTA rapidly remove zinc from ACE with rate constants of 1.27 X 10(3) and 2.2 X 10(3) M-1 min-1. The equilibrium constant for the reaction of NTA with ACE was measured as 4.6 X 10(-2) and was calculated for EDTA as 3.8 X 10(3).

96. Dahlheim, H., et al., Effect of zinc depletion on angiotensin I-converting enzyme in arterial walls and plasma of the rat. Miner Electrolyte Metab, 1989. 15(3): p. 125-9. The effect of zinc depletion and of additional angiotensin I-converting enzyme (ACE) inhibitor treatment was studied on ACE in aortic and other tissues, in plasma and on systolic blood pressure of the rat. Zinc deprivation significantly reduced plasma zinc concentration, plasma and testicular ACE activities and blood pressure, but stimulated aortic ACE while lung values remained constant. Zinc deficiency combined with ACE inhibition further suppressed plasma ACE and stimulated the aortic enzyme earlier. Zinc repletion experiments (in vitro) suggest the existence of a feedback mechanism controlling ACE synthesis depending on plasma ACE activity.

97. Kagan, B.L., et al., Elevated lipid levels in Vietnam veterans with chronic posttraumatic stress disorder. Biological Psychiatry, 1999. 45(3): p. 374-377. Elevated cholesterol levels have been reported in panic disorder and anger attacks, but not major depression. No data have been reported in posttraumatic stress disorder (PTSD). In this study, 73 male Vietnam veterans (mean age 43.8 yrs) with chronic PTSD had serum lipid screening upon entry to a 90-day inpatient program. Results showed that elevated cholesterol, low-density lipoprotein, triglycerides, and reduced high-density lipoprotein were frequent in Vietnam veterans with chronic PTSD and are significant risk factors for coronary artery disease. It is concluded that routine lipid screening may be warranted in

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this at-risk population. Altered lipid levels may result from activation of the noradrenergic system.

98. Peter, H., S. Tabrizian, and I. Hand, Serum cholesterol in patients with obsessive compulsive disorder during treatment with behavior therapy and SSRI or placebo. International Journal of Psychiatry in Medicine, 2000. 30(1): p. 27-39. 33 patients with obsessive compulsive disorder (OCD) participated in the study. Serum cholesterol was measured as pretreatment and at the end of a 10-wk treatment-period. All patients received behavior therapy and, in a double-blind fashion, fluvoxamine or placebo. Severity of OCD was assessed by the Yale-Brown Obsessive Compulsive Scale (Y-BOCS). Pretreatment cholesterol values of OCD patients were compared with cholesterol levels of 30 panic disorder patients and 30 normal controls. OCD patients had elevated cholesterol levels comparable with those of panic disorder patients. Cholesterol levels decreased significantly from pre- to posttreatment. OCD patients with high cholesterol levels could make best use of the treatment whereas patients with desirable cholesterol levels did not change their cholesterol during treatment. Data support the assumption that not only panic disorder but also other anxiety disorders, e.g., obsessive compulsive disorders, may be associated with serum cholesterol elevations.

99. Kuczmierczyk, A.R., et al., Serum cholesterol levels in patients with generalized anxiety disorder (GAD) and with GAD and comorbid major depression. Canadian Journal of Psychiatry, 1996. 41(7): p. 465-8. Investigated risk for cardiovascular disease in 38 patients (mean age 40.2 yrs) with general anxiety disorder (GAD), as well as the effects of comorbid major depression (MD). Predrug-trial serum cholesterol and triglyceride levels were assessed in Subjects with pure GAD and compared with those of 21 patients (mean age 36.7 yrs) with mixed GAD and comorbid MD. Significantly higher cholesterol and triglyceride levels were found in the GAD group. It is concluded that increased noradrenergic activity may be responsible for elevations in lipid levels in patients with pure GAD.

100. Bajwa, W.K., et al., High cholesterol levels in patients with panic disorder. American Journal of Psychiatry, 1992. 149(3): p. 376-378. 30 outpatients (mean age 37.7 yrs) with panic disorder were found to have higher serum cholesterol levels than 30 outpatients with major depression and 30 matched controls. Current or past histories of anxiety disorders in Subjects with major depression were associated with higher cholesterol levels. It is hypothesized that higher levels of cholesterol in panic disorder are a result of increased noradrenergic activity, which may be the underlying mechanism for symptoms of panic disorder.

OBJECTIVE: This study was undertaken to help clarify whether the higher cholesterol levels found in patients with panic disorder are a complication of panic disorder only or are associated with any psychiatric disorder. METHOD: The subjects of the study were 30 patients with panic disorder and 30 patients with major depression, diagnosed according to the Structured Interview for DSM-III-R, and 30 normal control subjects. The three groups were matched for sex and age, and none of the subjects had alcohol/drug abuse, abnormal ECGs, or unstable medical conditions. Blood samples were drawn at

29


random times, and serum cholesterol levels were determined. RESULTS: The patients with panic disorder had significantly higher serum cholesterol levels than did the patients with major depression and the normal control subjects. Among the patients with major depression, histories (current or past) of anxiety disorders were associated with significant elevation of serum cholesterol levels. The presence of stable medical conditions was not associated with higher cholesterol levels in any of the three groups of subjects. CONCLUSIONS: Higher cholesterol levels were particularly associated with panic disorder in comparison with major depression. Higher levels of cholesterol in panic disorder are hypothesized to be a result of increased noradrenergic activity, which may be the underlying biological/neurochemical mechanism for symptoms of panic disorder, including anticipatory anxiety.

101. Bajwa, W.K., et al., High cholesterol levels in patients with panic disorder. Am J Psychiatry, 1992. 149(3): p. 376-8. OBJECTIVE: This study was undertaken to help clarify whether the higher cholesterol levels found in patients with panic disorder are a complication of panic disorder only or are associated with any psychiatric disorder. METHOD: The subjects of the study were 30 patients with panic disorder and 30 patients with major depression, diagnosed according to the Structured Interview for DSM-III-R, and 30 normal control subjects. The three groups were matched for sex and age, and none of the subjects had alcohol/drug abuse, abnormal ECGs, or unstable medical conditions. Blood samples were drawn at random times, and serum cholesterol levels were determined. RESULTS: The patients with panic disorder had significantly higher serum cholesterol levels than did the patients with major depression and the normal control subjects. Among the patients with major depression, histories (current or past) of anxiety disorders were associated with significant elevation of serum cholesterol levels. The presence of stable medical conditions was not associated with higher cholesterol levels in any of the three groups of subjects. CONCLUSIONS: Higher cholesterol levels were particularly associated with panic disorder in comparison with major depression. Higher levels of cholesterol in panic disorder are hypothesized to be a result of increased noradrenergic activity, which may be the underlying biological/neurochemical mechanism for symptoms of panic disorder, including anticipatory anxiety.

102. Hayward, C., et al., Plasma lipid levels in patients with panic disorder or agoraphobia. Am J Psychiatry, 1989. 146(7): p. 917-9.

103. Patterson, S., et al., Effects of acute mental stress on serum lipids: mediating effects of plasma volume. Psychosomatic Med, 1993. 55: p. 525-32.

104. Peterson, J., R. Keith, and A. Wilcox, Hourly changes in serum cholesterol concentration: Effects of the anticipation of stress. Circulation, 1962. 25: p. 798-803. Over 100mg/dl variability in cholesterol over a period of hours in some people -- in response to stress or concern about stress, putatively -- though not well demo'd.

105. Thomas, C. and E. Murphy, Further studies on cholesterol levels in the Johns Hopkins medical students: the effect of stress at examinations. J Chron Dis, 1958. 8(6): p. 661-8. chol high at anatomy exam, falls thereafter;

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No good preexam unstressed measure: done on adm to med school, within 3 wks, also high.

106. Wertlake, P., et al., Relationship of mental and emotional stress to serum cholesterol levels. Proc Soc Exper Biol & Med, 1958. 97: p. 163.

107. Muldoon, M., et al., Effects of acute psychological stress on serum lipid levels, hemoconcentration, and blood viscosity. Arch Intern Med, 1995. 155: p. 615-20.

108. Muldoon, M., et al., Acute cholesterol responses to mental stress and change in posture. Arch Intern Med, 1992. 152: p. 775-80.

109. McCabe, W., et al., Elevations of serum cholesterol in man in association with life stress and independent of diet and exercise. Abstract, J. Lab & Clin Med, 1959. 54: p. 922.

110. Dreyfuss, F. and J. Czaczkes, Blood cholesterol and uric acid of healthy medical students under the stress of an examination. Ann Intern Med, 1959. 103: p. 708-11.

111. Muldoon, M., et al., Effects of acute psychological stress on serum lipid levels, hemoconcentration, and blood viscosity. Arch Intern Med, 1995. 155: p. 615-20.

112. Patterson, S., et al., Effects of acute mental stress on serum lipids: mediating effects of plasma volume. Psychosomatic Med, 1993. 55: p. 525-32.

113. Hata, S., H. Kunida, and Y. Oyama, [Antihypertensive effects of coenzyme Q10 in essential hypertension--in relation to the renin-aldosterone system]. Horumon To Rinsho, 1977. 25(9): p. 1019-23.

114. Hamdy, B.H., et al., Effectiveness of periodic oral vitamin A dosage on hypovitaminosis in Egypt. Int J Vitam Nutr Res, 1982. 52(3): p. 235-40. Seventy boys, aged 6-13 years were subjected to periodic oral vitamin A program. Non-parasitized boys (n = 63) responded to the oral treatment with significant increase in plasma retinol level, even five weeks after the end of oral treatment. On the other hand, boys infested with parasites, none of whom had originally acceptable plasma-retinol level, did not respond significantly to the oral treatment. Vitamin A was found also to have an effect on growth, and there was highly significant correlation between retinol level and percentile weight for age.

115. Marinho, H.A., et al., Influence of enteral parasites on the blood vitamin A levels in preschool children orally supplemented with retinol and/or zinc. Eur J Clin Nutr, 1991. 45(11): p. 539-44. A sample of 471 pre-school children who frequented schools and creches in a poor district of Manaus (Amazonas), Brazil, were randomly submitted to faecal parasitological tests. Two-hundred-and-forty children from both sexes between the ages of 3 and 7 years with Ascaris lumbricoides and/or Giardia lamblia were selected.

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The objective of the study was to determine the possible influence of these two intestinal parasites and vitamin A and/or zinc supplementation on the serum retinol levels of primary school children. The children were submitted to clinical and anthropometric examinations, dietary interviews and biochemical examinations of retinol and carotene in the serum and of zinc in the hair. The parasitic incidence was 85.0% and about 54% of the children were polyparasitic. During the pretreatment phase, the retinol and carotene serum levels were 36% and 57%, respectively, below the normal levels. Using the Waterlow classification, the anthropometric analyses revealed that 88% of the children showed normal growth. A significant effect was observed of the anti-parasitic medicine on the serum retinol levels.

116. Morris, M.C., Archives of Neurology, 2002. 59: p. 1125-32.

117. Vitamin E slows cognitive decline in the elderly. Internal Medicine World Report, 2002: p. 4.

118. Sato, Y., et al., Hypovitaminosis D and decreased bone mineral density in amyotrophic lateral sclerosis. Eur Neurol, 1997. 37(4): p. 225-9. To assess the bone health of patients with amyotrophic lateral sclerosis (ALS), we evaluated the bone density and serum biochemical indices of bone metabolism in 11 ALS patients. The serum concentration of 25-hydroxyvitamin D (25-OHD) was significantly lower in patients (14.0 +/- 3.7 ng/ml) than in controls (25.2 +/- 4.0 ng/ml), at deficient levels (< 10 ng/ml) in 2, and at insufficient levels (10-20 ng/ml) in 9 patients. Serum levels of parathyroid hormone (PTH) and ionized calcium were elevated in 8 and 6 patients, respectively. Dietary intake of vitamin D was below the recommended level (100 IU) in 10 patients, and 10 patients were in a sunlight-deprived state. The metacarpal bone density (MBD) and the metacarpal index (MCI) of the second metacarpal bone were measured by computed X-ray densitometry. Z scores of the MBD and the MCI were negative in 7 and 6 patients, respectively. The serum concentration of 25-OHD was positively correlated with the Z score of the MBD (p < 0.05, r = 0.727) and negatively with the PTH level (p < 0.05, r = -0.410). The degree of dysfunction of hand grip also correlated with the Z score of the MBD (p < 0.05, r = 0.749). These data underscore the importance of hypovitaminosis D and compensatory hyperparathyroidism in the development of osteopenia in patients with ALS.

119. Prabhala, A., R. Garg, and P. Dandona, Severe myopathy associated with vitamin D deficiency in western New York. Arch Intern Med, 2000. 160(8): p. 1199-203. Five cases of severe myopathy associated with vitamin D deficiency are described. Each patient was confined to a wheelchair because of weakness and immobility. Two were elderly, 1 was a 37-year-old African American with type 1 diabetes mellitus, 1 was being treated for carcinoid syndrome, and 1 was severely malnourished due to poor oral intake. In each, weakness had previously been attributed to other causes, including old age, concomitant diabetic neuropathy, or general debility. Correct diagnosis was made initially by a high index of suspicion, following the demonstration of clinical proximal myopathy; confirmation was made by the demonstration of low 25-hydroxyvitamin D and elevated parathyroid hormone concentrations. Treatment with vitamin D caused a

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resolution of body aches and pains and a restoration of normal muscle strength in 4 to 6 weeks. Four patients became fully mobile and had normal 25-hydroxyvitamin D concentrations, and the fifth also became mobile. In the 4 fully recovered cases, parathyroid hormone levels on follow-up were lower but still elevated. This finding suggests a degree of autonomy of parathyroid secretion known to occur in cases of long-standing vitamin D deficiency. Myopathy, due to chronic vitamin D deficiency, probably contributes to immobility and ill health in a significant number of patients in the northern United States. An awareness of this condition may significantly improve mobility and quality of life in patient populations vulnerable to vitamin D deficiency.

120. Gennari, A., et al., Organotins induce apoptosis by disturbance of [Ca(2+)](i) and mitochondrial activity, causing oxidative stress and activation of caspases in rat thymocytes. Toxicol Appl Pharmacol, 2000. 169(2): p. 185-90. Di-n-butyltin dichloride (DBTC) and tri-n-butyltin chloride (TBTC) cause thymus atrophy in rodents. At low doses, antiproliferative modes of action have been shown to be involved, whereas at higher doses apoptosis seems to be the mechanism of thymotoxicity by these chemicals. In vitro, a similar concentration-dependency has been observed. The purpose of the present research was to investigate the mechanisms underlying DNA fragmentation induced by these organotin compounds in freshly isolated rat thymocytes. As previously shown for TBTC, DBTC is also able to significantly increase intracellular Ca(2+) level ([Ca(2+)](i)). The rise in [Ca(2+)](i), already evident 5 min after treatment, was followed by a dose- and time-dependent generation of reactive oxygen species (ROS) at the mitochondrial level. Simultaneously, organotins induced the release of cytochrome c from the mitochondrial membrane into the cytosol. ROS production and the release of cytochrome c were reduced by BAPTA, an intracellular Ca(2+) chelator, or rotenone, an inhibitor of the electron entry from complex I to ubiquinone, indicating the important role of Ca(2+) and mitochondria during these early intracellular events. Furthermore, we demonstrated that rotenone prevents apoptosis induced by 3 microM DBTC or TBTC and, in addition, that both BAPTA and Z-DEVD FMK (mainly a caspase-3 inhibitor) decreased apoptosis by DBTC (already shown for TBTC). Taken together these data show the apoptotic pathway followed by organotin compounds starts with an increase of [Ca(2+)](i), then continues with release of ROS and cytochrome c from mitochondria, activation of caspases, and finally results in DNA fragmentation.

121. Ravikumar, A., et al., Isoprenoid pathway and free radical generation and damage in neuropsychiatric disorders. Indian J Exp Biol, 2000. 38(5): p. 438-46. Two substances which are products of the isoprenoid pathway, can participate in lipid peroxidation. One is digoxin, which by inhibiting membrane Na(+)-K+ ATPase, causes increase in intracellular Ca2+ and depletion of intracellular Mg2+, both effects contributing to increase in lipid peroxidation. Ubiquinone, another products of the pathway is a powerful membrane antioxidant and its deficiency can also result in defective electron transport and generation of reactive oxygen species. In view of this and also in the light of some preliminary reports on alteration in lipid peroxidation in neuropsychiatric disorders, a study was undertaken on the following aspects in some of these disorders (primary generalised epilepsy, schizophrenia, multiple sclerosis, Parkinson's disease and CNS glioma)--1) concentration of digoxin, ubiquinone, activity

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of HMG CoA reductase and RBC membrane Na(+)-K+ ATPase 2) activity of enzymes involved in free radical scavenging 3) parameters of lipid peroxidation and 4) antioxidant status. The result obtained indicates an increase in the concentration of digoxin and activity of HMG CoA reductase, decrease in ubiquinone levels and in the activity of membrane Na(+)-K+ ATPase. There is increased lipid peroxidation as evidenced from the increase in the concentration of MDA, conjugated dienes, hydroperoxides and NO with decreased antioxidant protection as indicated by decrease in ubiquinone, vit E and reduced glutathione in schizophrenia, Parkinson's disease and CNS glioma. The activity of enzymes involved in free radical scavenging like SOD, catalase, glutathione peroxidase and glutathione reductase is decreased in the above diseases. However, there is no evidence of any increase in lipid peroxidation in epilepsy or MS. The role of increased operation of the isoprenoid pathway as evidenced by alteration in the concentration of digoxin and ubiquinone in the generation of free radicals and protection against them in these disorders is discussed.

122. Schults, C.W., et al., Effects of coenzyme Q10 in early Parkinson disease. Evidence of slowing of the functional decline. Archives of Neurology, 2002. 59(10): p. 1541-50. Background: Parkinson disease (PD) is a degenerative neurological disorder for which no treatment has been shown to slow the progression. Objective: To determine whether a range of dosages of coenzyme Q10 is safe and well tolerated and could slow the functional decline in PD. Design Multicenter, randomized, parallel-group, placebo-controlled, double-blind, dosage-ranging trial. Setting: Academic movement disorders clinics. Patients: Eighty subjects with early PD who did not require treatment for their disability. Interventions: Random assignment to placebo or coenzyme Q10 at dosages of 300, 600, or 1200 mg/d.Main Outcome Measure: The subjects underwent evaluation with the Unified Parkinson Disease Rating Scale (UPDRS) at the screening, baseline, and 1-, 4-, 8-, 12-, and 16-month visits. They were followed up for 16 months or until disability requiring treatment with levodopa had developed. The primary response variable was the change in the total score on the UPDRS from baseline to the last visit. Results: The adjusted mean total UPDRS changes were +11.99 for the placebo group, +8.81 for the 300-mg/d group, +10.82 for the 600-mg/d group, and +6.69 for the 1200-mg/d group. The P value for the primary analysis, a test for a linear trend between the dosage and the mean change in the total UPDRS score, was .09, which met our prespecified criteria for a positive trend for the trial. A prespecified, secondary analysis was the comparison of each treatment group with the placebo group, and the difference between the 1200-mg/d and placebo groups was significant (P = .04). Conclusions: Coenzyme Q10 was safe and well tolerated at dosages of up to 1200 mg/d. Less disability developed in subjects assigned to coenzyme Q10 than in those assigned to placebo, and the benefit was greatest in subjects receiving the highest dosage. Coenzyme Q10 appears to slow the progressive deterioration of function in PD, but these results need to be confirmed in a larger study.

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