The effect of desferal on rat heart mitochondrial function, iron content, and xanthine dehydrogenase/oxidase conversion during ischemia-reperfusion.

Cardiac mitochondrial function as measured by oxidative phosphorylation is impaired by ischemia; and, this deteriorates even further on reperfusion of the heart. Free oxygen radicals, especially the formation of hydroxyl radicals via the iron-catalyzed Haber-Weiss and Fenton reactions have been implicated in the reperfusion injury. In this study, the effect of desferrioxamine (desferal) in the perfusate on mitochondrial function of isolated rat hearts during different periods of normothermic ischemic cardiac arrest (NICA), and subsequent reperfusion was investigated. Mitochondrial functions measured were the QO2 (state 3); ADP/O ratio and oxidative phosphorylation; the mitochondrial, loosely bound (chelateable) iron (LB-iron); the xanthine dehydrogenase and xanthine oxidase activities. Inclusion of desferal in the perfusion solution significantly improved mitochondrial function during the different NICA periods, and prevented the deterioration of mitochondrial function resulting from reperfusion. Desferal did not significantly affect the LB-iron content of the mitochondria or the ratio of xanthine dehydrogenase/xanthine oxidase activities in the mitochondria during NICA or reperfusion. Our experiments suggest that iron, which is free to be chelated by desferal, plays a role in this injury to the rat myocardium.

Effect of desferrioxamine on reperfusion damage of rat heart mitochondria.

Ischaemia of the myocardium leads to necrosis unless oxygen supply is restored but it has only recently been realised that reperfusion is not without danger. The greatest rate of myocardial damage, as measured by mitochondrial function, occurred during the first 5 minutes of reperfusion in rat hearts subjected to normothermic ischaemic cardiac arrest. Addition of desferrioxamine to the perfusate after 5 minutes of reperfusion did not reverse the mitochondrial damage. It is therefore concluded that desferrioxamine prevents mitochondrial damage caused by ischaemia-reperfusion but does not reverse the damage already present.

Vitamin B-6 nutrition status and cigarette smoking.

We investigated the vitamin B-6 status in smokers, nonsmokers, and exsmokers by measuring both B-6 aldehyde vitamers, pyridoxal-5'-phosphate (PLP) and pyridoxal (PL), in the plasma as well as in the erythrocyte compartment. Two hundred eighty-six healthy, sedentary male workers from a middle-income group were investigated. There were 159 smokers, 59 exsmokers, and 68 nonsmokers. Plasma PLP and PL concentrations were significantly lower in smokers than in the nonsmokers and exsmokers whereas erythrocyte PLP and PL did not differ significantly between groups. Because PLP mainly functions as an intracellular coenzyme, the clinical significance of a depressed plasma PLP concentration alone is uncertain. It is concluded that circulating plasma PLP is labile and not necessarily indicative of intracellular PLP concentrations. The measurement of erythrocyte PLP and/or PL may be more informative about vitamin B-6 status than is plasma PLP alone.

Purine and oxypurine production in mitochondria of ischemic and reperfused myocardium.

The present study was undertaken to determine whether significant breakdown of adenine nucleotides to purine bases and oxypurines occurred in mitochondria following myocardial ischemia and ischemia followed by reperfusion, and whether allopurinol prevented this effect. The adenine nucleotides adenosine, hypoxanthine, xanthine and uric acid were measured in the mitochondria and the results suggest that breakdown did occur. Malondialdehyde concentration was determined to gauge lipid peroxidation. This substance did not increase during ischemia or reperfusion, but did so in the presence of allopurinol. Xanthine dehydrogenase was converted to xanthine oxidase during reperfusion and the activity of both enzymes were inhibited by allopurinol. The results also suggested the presence of a mitochondrial 5'-nucleotidase. We conclude that significant breakdown of adenine nucleotide took place in myocardial mitochondria during ischemia and ischemia followed by reperfusion and that allopurinol may have a protective effect.

The effect of allopurinol and deferrioxamine on rat heart mitochondrial oxidative phosphorylation after normothermic ischemic cardiac arrest and of reperfusion.

The electron transport chain of the mitochondria is highly sensitive to myocardial ischemia. As free oxygen radicals take part in the damage that occurred during ischemia, this study was undertaken to determine if allopurinol and deferrioxamine had any beneficial effect on mitochondrial function. Our results showed that perfusion with allopurinol did not improve the mitochondrial function, but that reperfusion with allopurinol and deferrioxamine had a beneficial effect. We came to the conclusion that xanthine oxidase, as a generator of superoxide anions, is of minor importance in comparison with the hydroxyl radicals, which are probably formed in the presence of iron in the cell.

Towards standardisation of pre- and post-analytical variables in the assessment of lipid risk factors for ischaemic heart disease.

A wide disparity exists between statistical and optimal reference ranges for total cholesterol (TC) levels in South Africa. The haphazard downward adjustments by some laboratories over the years have resulted in highly variable and confusing 'normal cholesterol' values. Using essentially the same enzymatic method and equipment the TC upper limits varied by almost 50% (5.7-8.5 mmol/l) in 24 private laboratories, while it ranged from 5.7 mmol/l to 6.7 mmol/l in 6 academic hospital laboratories. The greatest disparity occurred in the lower limits of 'normal' for high-density lipoprotein cholesterol (HDLC), which ranged from 0.45 mmol/l to 1.43 mmol/l. Although triglycerides may not be an independent risk factor for ischaemic heart disease, the upper limit ranged from 1.69 mmol/l to 2.5 mmol/l despite comparable methods and equipment used. Lipid data obtained on approximately 5,000 healthy adults from the Transvaal, Natal and the Orange Free State revealed remarkable similarities to those obtained in the CORIS study. It therefore confirms the applicability of the CORIS epidemiological findings to other westernized groups in different regions of South Africa. This congruency in blood lipid values and methodologies used in different laboratories should also enable all state and private laboratories to use the same age-specific limits for desirable TC and HDLC levels.

Compartmentalization of pyridoxal-5'-phosphate during the acute phase of myocardial infarction.

Vitamin B6 nutritional status is assessed by measuring the plasma concentration of one of its vitamers, pyridoxal-phosphate (PLP). Several conditions, e.g., myocardial infarction (MI), can disturb the dynamic equilibrium between the different vitamers resulting in transiently low plasma PLP levels. An important question is whether these low plasma PLP levels observed during MI represent a transient state of deficiency and what the possible clinical consequences of such a fall in plasma PLP could be. Since the main metabolic function of PLP is to act as an intracellular coenzyme, it was decided to monitor the changes not only of PLP but also of PL (transport form of vitamin B6) in both the plasma and red blood cells (RBCs) in patients with myocardial infarctions (MI): 16 patients with proven MI were investigated measuring the aforementioned parameters at regular intervals. It was found that the approximately 40% fall in plasma PLP levels was accompanied by an equivalent increase in RBC PLP levels. Subsequently plasma PLP concentrations returned to normal but RBC PLP values were maintained at the newly elevated steady state (without any vitamin supplementation). Since membranes are impermeable to PLP, the only way in which PLP could have been redistributed to the intracellular compartment was through hydrolysis to PL and rephosphorylation once inside the RBCs. This compartmentalization could be an important adaptive response since it has been shown that PLP reduces O2 affinity of deoxygenated hemoglobin, thereby facilitating O2 delivery to the tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of normothermic ischemic cardiac arrest and of reperfusion on the free oxygen radical scavenger enzymes and xanthine oxidase (a generator of superoxide anions).

Recent evidence suggests that free oxygen radicals are produced by ischaemic tissues, accounting for at least part of the damage that results. These free oxygen radicals are produced by xanthine oxidase, amongst others, and removed by scavenger enzymes (catalase, superoxide dismutase and glutathione peroxidase) and anti-oxidants. As mitochondria are oxygen-utilising organelles, they are capable of producing free oxygen radicals. Our results indicate that the removal of free oxygen radicals are not diminished during ischaemia, but the activity of the free oxygen radical generator, xanthine oxidase, is increased. This could lead to an increased superoxide anion concentration.

Enzymatic and structural modifications of mitochondrial NADH-ubiquinone reductase with autolysis as experimental model.

Complex I (nicotinamide adenine dinucleotide-ubiquinone reductase) is a complex enzyme system located in the inner mitochondrial membrane. It has the ability to catalyze several different enzymatic reactions in electron transport, and is known to be one of the respiratory chain components most sensitive to ischaemia. Mitochondria and two complexes I (complex IA and complex IB) were isolated from normal and ischaemic myocardial tissue. Enzymatic activities, polypeptide composition, as well as other components such as non-haem iron, acid-labile sulphur and ubiquinone, were determined. The results indicated that complex IB reflected the enzymatic changes in the mitochondria during myocardial ischaemia, but complex IA did not. The lesion that resulted from ischaemia was localised as altered enzymatic activities due to a different polypeptide composition, as well as loss of ubiquinone and non-haem iron from complex IB.

A new perspective in the assessment of vitamin B-6 nutritional status during pregnancy in humans.

Plasma pyridoxal-5'-phosphate (PLP) concentration has been suggested as a valid indicator to assess vitamin B-6 nutritional status. Animal and human studies have shown that plasma PLP concentrations decrease progressively during pregnancy and large doses of vitamin B-6 supplementation are required to maintain plasma PLP at early or prepregnant levels. PLP is known to be hydrolyzed to pyridoxal (PL) by alkaline phosphatase (ALP), resulting in an inverse relationship between PLP and ALP. The object of this study was to compare the PLP-PL equilibrium in a group of healthy pregnant females with that of an age-matched nonpregnant control group from a similar socioeconomic background. The mean plasma PLP level was 37% lower, (P less than 0.0001), whereas the mean PL level was almost 90% higher (P less than 0.001) in the pregnant group than in the nonpregnant control group. The total amount of plasma PLP and PL levels, however, did not differ significantly (P greater than 0.24) between the two groups. Because the PL vitamer is regarded as the ultimate transport form of vitamin B-6, it may serve as a readily available source of vitamin B-6 to meet possible increased metabolic demands. Therefore, the estimation of plasma PLP alone does not permit an accurate assessment or understanding of the nutritional status and the physiology of vitamin B-6 in conditions associated with altered vitamin B-6 homeostases.

Vitamin B6 and coronary artery disease. Epidemiological observations and case studies.

The finding of low plasma pyridoxal-5'-phosphate levels in patients suffering from myocardial infarction has been construed as possible evidence for the pathogenetic role that vitamin B6 deficiency may play in causing premature ischaemic heart disease. However, the presence of normal plasma pyridoxal-5'-phosphate levels in patients with angiographic evidence of coronary artery narrowing but with no previous infarctions prompted the investigation of possible short-term alterations in plasma pyridoxal-5'-phosphate levels during the acute phase of myocardial infarction. In the follow-up of 30 patients with acute myocardial infarction, all of them showed a continuous decrease of approximately 45% in plasma pyridoxal-5'-phosphate levels during the acute phase. These levels subsequently returned back to normal before discharge from hospital. A large number of volunteers from an ethnic group known to have a very low incidence of ischaemic heart disease were found to have both significantly lower total cholesterol and plasma pyridoxal-5'-phosphate levels than a Caucasian group in the same geographic area which is known to have a high incidence of ischaemic heart disease. These findings therefore do not support the contention that vitamin B6 deficiency may be a risk index for ischaemic heart disease.

Cholinesterase variants in a South African population.

Results of cholinesterase studies in 56 white and 22 black patients in Bloemfontein hospitals who developed suxamethonium apnoea between March 1974 and April 1985 are reported. Genotypes of patients and families are compared with figures for the UK and Denmark. A higher incidence of the silent cholinesterase allele is confirmed; the incidence of other variants is similar. Black patients were found to be only of the silent genotype.

Plasma pyridoxal-5'-phosphate levels in myocardial infarction.

In 34 patients with acute myocardial infarction (MI) plasma pyridoxal-5'-phosphate (PLP) levels were significantly lower (5.22 +/- 1.88 ng/ml) than those in an age- and sex-matched control group (11.5 +/- 2.03 ng/ml). In another group of patients who had clinical and angiographic evidence of ischaemic heart disease but had not had an MI plasma PLP levels were not significantly different from those in the control group (10.07 +/- 2.98 ng/ml). However, plasma high-density lipoprotein cholesterol levels in this group (0.75 +/- 0.28 mmol/l) as well as in the MI group (0.76 +/- 0.28 mmol/l) were significantly lower than those in the control group (1.26 +/- 0.23 mmol/l). On follow-up, all of 15 patients who had had an acute MI showed a continuous decrease in plasma PLP levels of approximately 50% during the first 48 hours after admission. Sixteen healthy volunteers subjected to a period of prolonged fasting (+/- 30 hours) displayed a decrease of approximately 43% over this period. We conclude that an acute reduction in plasma PLP levels occurred during the acute phase of MI. Other factors, for example prolonged acute starvation, may also produce a rapid decrease in plasma PLP levels.

Isolation of a physiologically active and a physiologically inactive mitochondrial NADH-ubiquinone reductase (complex I) from donkey hearts.

The method described for the isolation of mitochondrial complex I (NADH-ubiquinone reductase) from bovine hearts could not be applied to donkey hearts as unacceptably large losses in enzyme activity occurred. This method was modified for the isolation of complex I using donkey hearts and two complexes were obtained: complex IA which was physiologically inactive and complex IB which was physiologically active as it catalyzed the reaction from NADH to ubiquinone. Both complexes had relatively low enzyme activity with artificial electron acceptors, except with potassium ferricyanide, and had more or less the same amount of acid-labile sulfur and nonheme iron although the polypeptide composition differed to a great extent.

Changes in NADH-ubiquinone reductase (complex I) with autolysis in the rat heart as experimental model.

Complex I (NADH-ubiquinone reductase) is a complex system located in the inner mitochondrial membrane and has the ability to catalyse several different enzymatic reactions concerned in electron transport. It is known to be one of the first components of the respiratory chain to be damaged by ischemia. Our results, using autolysis in the rat heart as experimental model, indicate that the NADH dehydrogenase system was impaired relatively early during ischemia while transhydrogenation and NADPH dehydrogenation appeared to be relatively resistant.

Correlation between pyridoxal-5'-phosphate levels and the percentage activation of aspartate aminotransferase enzyme in haemolysate and plasma during in vitro incubation studies with different B6 vitamers.

Conflicting results using erythrocyte aminotransferase (eAST) stimulation to assess vitamin B6 nutritional status in patients with less severe B6 deficiencies are common. It has been claimed that the presence of different B6 vitamers may modify the activation of eAST by pyridoxal-5'-phosphate (PLP) leading to stimulatory or even inhibitory effects. To investigate the possible role of this phenomenon in producing inconsistent AST stimulations, aliquots of whole blood were incubated with equivalent amounts of different B6 vitamers, and the AST stimulation was correlated with the concentrations of PLP, measured by high-performance liquid chromatography. At the end of the incubation period the erythrocytes and plasma were separately analyzed. The conversion of non-PLP B6 vitamers to PLP, by the erythrocytes, was similar (approximately 70%) for all B6 vitamers used in the incubation experiments. The newly formed PLP accumulated in the erythrocytes, but the percentage activation of AST did not change significantly from the basal levels, in spite of the presence of increased levels of PLP and other B6 vitamers used for incubation. When PLP was used in the incubation studies, all of it was retained by the plasma and was associated with a marked suppression of plasma AST stimulation. To determine the degree to which plasma and erythrocyte AST was dose-dependent, plasma and haemolysates were incubated with increasing concentrations of PLP. A very significant inverse relationship was obtained in plasma between AST stimulation and PLP even at modest PLP levels, while haemolysates required incubation with much higher PLP concentrations to demonstrate the same effect. Since plasma PLP is considered to be the most reliable indicator of B6 nutritional status in man, our findings suggest that plasma percentage AST stimulation more closely reflects the B6 nutritional status than erythrocyte AST stimulation test which may reflect B6 status only in severe, longstanding B6 deficiencies. Conflicting results using erythrocyte AST stimulations may be attributed to the insensitivity of red cell AST to changes in PLP content. It is unlikely that the presence of non-PLP B6 vitamers in haemolysate may affect the percentage stimulation of aminotransferase enzymes by PLP.

An apparatus for collecting blood samples by radiotelemetry from horses during exercise.

An apparatus was designed to collect four consecutive blood samples from exercising horses. The collection of each sample was controlled by valves activated by radiotelemetry signals transmitted by an observer. Using the device, venous blood samples were collected from ten thoroughbred racehorses before, during and after a 400 m training gallop. Blood glucose increased markedly post-exercise. Both phosphorus and potassium concentrations increased during exercise, decreased post-exercise and recovered to pre-exercise levels within 120 minutes. The system was modified to collect anaerobic samples of arterial and venous blood, and the efficiency of the modified system was investigated in a standing conscious horse. Blood gas values of samples collected by means of the apparatus were compared with those collected manually and simultaneously, directly from the neck of the intravascular catheter. For eight pairs of arterial and venous samples, the coefficients "r" were 0.998 and 0.997 for PO2 and PCO2 respectively. It was concluded that the system worked efficiently and that the anaerobic sealing of the modified version was adequate.

The effect of acute prolonged starvation on the concentrations of vitamin B6 aldehyde derivatives in whole blood.

Since plasma pyridoxal-x-5'o-phosphate (PLP) levels are used to assess vitamin B6 status low levels are frequently interpreted to indicate B6 deficiency. However plasma PLP is in a dynamic equilibrium with pyridoxal (PL) through the action of non specific alkaline phosphatases (ALP). The object of this study was to monitor possible disturbances of this equilibrium in whole blood during acute prolonged fasting (40 hrs) and the subsequent repletion period in 16 healthy male dogs. Mean plasma PLP decreased by 15 percent (p less than 0.025) and PL increased by 20 percent (p less than 0.05) at the end of the starvation period and returned to baseline values after 48 hrs of refeeding. However total plasma aldehyde (PLP and PL) B6 vitamer concentrations remained unchanged throughout the investigation period. A 35 percent increase in haemolysate PL was the only significant change (p less than 0.0005) in PLP and PL levels observed in erythrocytes during fasting. It is concluded that the use of plasma PLP alone to assess vitamin B6 status may be misleading in conditions with a disturbed plasma PLP/PL equilibrium.

The effect of seminal plasma on gonadotrophin secretion by sheep pituitary cells in culture.

We tested the effect of seminal plasma fractions of pooled normozoospermic and azoospermic seminal plasma on secretion of LH by sheep pituitary cells in culture. From these experiments we conclude that there is activity in seminal plasma (molecular mass 10.000-80.000) which inhibits LH secretion. This activity is not steroid-mediated and is absent in azoospermic seminal plasma. These data support our thesis that LH secretion is not only controlled by testosterone but also by spermatogenesis per se.