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Biomedical subjects

J M Land

Publications and source records attributed to J M Land.

At least 55 records · Page 3Linked to original sources
PubMed1997

Adaptation of the nitrate reductase and Griess reaction methods for the measurement of serum nitrate plus nitrite levels.

Nitrite and nitrate determinations in biological fluids are increasingly being used as markers of nitric oxide production. We have modified a nitrate reductase and Griess reaction method for the measurement of serum nitrate and nitrite in ultrafiltrated samples using a microtitre plate. The recoveries of nitrate and nitrite were 95% (range = 86-113%) and 100% (range = 92-109%), respectively. The intra and inter assay coefficients of variation for nitrate plus nitrite in the concentration range 40-50 microM were 9.1% and 7.8%, and in the concentration range of 2.5-10 microM 23.4% and 25.5%, respectively. At its lower limit the assay is able to detect 125 pmoles of nitrate plus nitrite in 50 microL of sample (2.5 mumol/L). A mean serum nitrate plus nitrite level of 32.8 mumol/L (SD 12.3) was measured in 24 healthy adult volunteers (12 men and 12 women), no age or sex differences were noted.

Adult↗
PubMed1996

Impairment of the nitric oxide/cyclic GMP pathway in cerebellar slices prepared from the hph-1 mouse.

In this study, the effect of tetrahydrobiopterin deficiency on the nitric oxide/cGMP pathway has been investigated in cerebellar slices derived from the hph-1 mouse. This animal displays a partial deficiency of tetrahydrobiopterin. Basal levels of cGMP were significantly reduced (-29.5%) in the hph-1 mouse cerebellum compared to controls. Following kainate stimulation (500 microM) cGMP levels increased in both control and hph-1 preparations but were again significantly lower (-29.1%) in the hph-1 mouse. Exposure of slices to the nitric oxide donors, S-nitroso-N-acetylpenicillamine and S-nitroso-glutathione, revealed no difference in cGMP accumulation between the two groups. These findings suggest that the cerebellar nitric oxide/cGMP pathway may be impaired in partial tetrahydrobiopterin deficiency states due to diminished nitric oxide formation.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1996

Depletion of brain glutathione results in a decrease of glutathione reductase activity; an enzyme susceptible to oxidative damage.

Loss of the intracellular antioxidant glutathione (GSH) from the substantia nigra is considered to be an early event in the pathogenesis of Parkinson's disease (PD). While the cause of the loss is unclear, an imbalance in the enzymes associated with the synthesis, utilisation, degradation and translocation of GSH has been implicated. The enzyme glutathione reductase is also important in GSH homeostasis: it regenerates GSH from the oxidised from (GSSG). However, to date the activity and regulation of glutathione reductase in conditions such as PD have not been explored. In view of this we have measured the effects of GSH depletion on glutathione reductase activity of the rat brain. Other glutathione related enzymes were also measured. Using pre-weanling rats, brain GSH was depleted by up to 60% by subcutaneous administration of L-buthionine sulfoximine. The only enzyme affected by GSH depletion was glutathione reductase; its activity being reduced by approximately 40%. As GSH inactivates a number of oxidising species including peroxynitrite (ONOO-), we additionally investigated the susceptibility of glutathione reductase to ONOO- in vitro, using purified enzyme. ONOO- decreased glutathione reductase activity in a concentration dependent manner with an apparent 50% inhibition occurring at an initial concentration of 0.09 mM. These data suggest that GSH is important in the maintenance glutathione reductase activity. This may arise in part from its ability to inactivate oxidising agents such as ONOO-.

Animals↗
PubMed1996

Nitric oxide and antioxidant status in glucose and oxygen deprived neonatal and adult rat brain synaptosomes.

Nitric oxide (NO.) has been implicated in the process of cerebral ischemia/reperfusion injury. We have examined the production of NO., as reflected by nitrite (NO2-) + nitrate (NO3-) accumulation, from synaptosomes isolated from neonatal or adult rat brain and subjected to a period of glucose and oxygen deprivation. There was a significant increase in the amount of NO2- + NO3- production from adult synaptosomes under these conditions, whereas there was no difference compared to control in the production of NO2- + NO3- from the neonatal synaptosomes. The total antioxidant status of the synaptosomes at these different stages of brain development was found to be the same. These data suggest that the vulnerability of the adult brain to ischemia/reperfusion injury may be associated with the production of NO. from nerve terminals. The ratios of antioxidant capacity to NO. production under such conditions have been shown here to be different between the neonatal and adult nerve terminals. Thus the well documented resistance of neonatal brain to ischemia/reperfusion injury may involve the neonatal nerve terminal being under less oxidative stress than the adult.

Aging↗
PubMed1996

Nitric oxide-mediated mitochondrial damage: a potential neuroprotective role for glutathione.

In this study we have investigated the mechanisms leading to mitochondrial damage in cultured neurons following sustained exposure to nitric oxide. Thus, the effects upon neuronal mitochondrial respiratory chain complex activity and reduced glutathione concentration following exposure to either the nitric oxide donor, S-nitroso-N-acetylpenicillamine, or to nitric oxide releasing astrocytes were assessed. Incubation with S-nitroso-N-acetylpenicillamine (1 mM) for 24 h decreased neuronal glutathione concentration by 57%, and this effect was accompanied by a marked decrease of complex I (43%), complex II-III (63%), and complex IV (41%) activities. Incubation of neurons with the glutathione synthesis inhibitor, L-buthionine-[S,R]-sulfoximine caused a major depletion of neuronal glutathione (93%), an effect that was accompanied by a marked loss of complex II-III (60%) and complex IV (41%) activities, although complex I activity was only mildly decreased (34%). In an attempt to approach a more physiological situation, we studied the effects upon glutathione status and mitochondrial respiratory chain activity of neurons incubated in coculture with nitric oxide releasing astrocytes. Astrocytes were activated by incubation with lipopolysaccharide/interferon-gamma for 18 h, thereby inducing nitric oxide synthase and, hence, a continuous release of nitric oxide. Coincubation for 24 h of activated astrocytes with neurons caused a limited loss of complex IV activity and had no effect on the activities of complexes I or II-III. However, neurons exposed to astrocytes had a 1.7-fold fold increase in glutathione concentration compared to neurons cultured alone. Under these coculture conditions, the neuronal ATP concentration was modestly reduced (14%). This loss of ATP was prevented by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. These results suggest that the neuronal mitochondrial respiratory chain is damaged by sustained exposure to nitric oxide and that reduced glutathione may be an important defence against such damage.

Animals↗
PubMed1996

Comparison of two cotside methods for the detection of hypoglycaemia among neonates in Nepal.

AIMS: To compare two cotside methods of blood glucose measurement (HemoCue and Reflolux II) against a standard laboratory method for the detection of neonatal hypoglycaemia in a developing country maternity hospital where hypoglycaemia is common. METHODS: 94 newborn infants and 75 of their mothers had blood glucose assessed on the same venous sample using three different methods in the Special Care Baby Unit and postnatal wards, Prasuti Griha Maternity Hospital, Kathmandu, Nepal: HemoCue and Reflolux II at the cotside; Roche Ultimate glucose oxidase method (GOM) in the laboratory. RESULTS: The mean (SD) values for blood glucose in newborn infants were GOM 2.5 (1.1) mmol/l; Reflolux II 2.1 (0.9); and HemoCue 4.2 (1.2). For mothers the values were GOM 5.3 (1.2) mmol/l; Reflolux II 3.6 (1.2); and HemoCue 5.6 (1.0). Bland-Altman plots showed that Reflolux II consistently underreads GOM blood glucose in neonates by 0.5 mmol/l (SD 0.7) and that HemoCue overreads glucose by 1.7 mmol/l (SD 0.8). For the detection of hypoglycaemia (< 2.0 mmol/l), Reflolux II achieved a sensitivity of 83%, a specificity of 62%, and a likelihood ratio of 2.2. HemoCue produced a sensitivity of 0% and a specificity of 100% using measured values. If 2.0 mmol were subtracted from all Hemocue values this rose to 81% and 68% and a likelihood ratio of 2.5. CONCLUSION: Although more accurate than Reflolux II for the measurement of blood glucose in mothers, HemoCue overreads glucose concentrations in neonates and is therefore potentially dangerous as a screening method for neonatal hypoglycaemia. Reflolux II is useful as a screening method for high risk infants (low birthweight, post-term) and could achieve a post-test probability of detecting hypoglycaemia in a high risk setting like Nepal of 50-60%.

Adult↗
PubMed1996

Glutathione protects astrocytes from peroxynitrite-mediated mitochondrial damage: implications for neuronal/astrocytic trafficking and neurodegeneration.

In this study we have examined the susceptibility of the mitochondrial respiratory chain of astrocytes and astrocytes depleted of glutathione to peroxynitrite exposure. Astrocytes, as reported previously by us, appeared resistant to the actions of peroxynitrite. In contrast, depletion (-94%) of astrocytic glutathione rendered the cells susceptible with mitochondrial complexes I and II/III being decreased in activity by 80 and 64%, respectively, after peroxynitrite exposure. Furthermore, cell death, as judged by lactate dehydrogenase release, was significantly increased (+81%) in the glutathione-depleted astrocytes exposed to peroxynitrite. Glutathione depletion alone had no effect on any of the measured parameters. It is concluded that glutathione is an important intracellular defence against peroxynitrite and that when glutathione levels are compromised the mitochondrial respiratory chain is a vulnerable target and cell death ensues. In view of the relative paucity of neuronal glutathione, it is possible that astrocyte-derived peroxynitrite may, in certain pathological conditions, be released and diffuse into neighboring neurones where mitochondrial damage may occur.

Animals↗
PubMed1995

Tetrahydrobiopterin deficiency and brain nitric oxide synthase in the hph1 mouse.

Tetrahydrobiopterin (BH4) is the cofactor for the aromatic amino acid monoxygenase group of enzymes and for all known isoforms of nitric oxide synthase (NOS). Inborn errors of BH4 metabolism lead to hyperphenylalaninaemia and impaired catecholamine and serotonin turnover. The effects of BH4 deficiency on brain nitric oxide (NO) metabolism are not known. In this study we have used the hph-1 mouse, which displays GTP cyclohydrolase deficiency, to study the effects of BH4 deficiency on brain NOS. In the presence of exogenous BH4, NOS specific activity was virtually identical in the control and hph-1 preparations. However, omission of BH4 from the reaction buffer led to a significant 20% loss of activity in the hph-1 preparations only. The Km for arginine was virtually identical for the control and hph-1 NOS when BH4 was present in the reaction buffer. In the absence of cofactor, the Km for arginine was 3-fold greater for control and 5-fold greater for hph-1 preparations. It is concluded that (a) BH4 does not regulate the intracellular concentration of brain NOS; (b) less binding of BH4 to NOS occurs in BH4 deficiency states; (c) BH4 has a potent effect on the affinity of NOS for arginine; and (d) the availability of arginine for NOS activity may become severely limiting in BH4 deficiency states. Since, in the presence of suboptimal concentrations of BH4 or arginine, NOS may additionally form oxygen free-radicals, it is postulated that in severe BH4 deficiency states NO formation is impaired and the central nervous system is subjected to increased oxidative stress.

Amino Acid Oxidoreductases↗
PubMed1995

Neonatal carnitine palmitoyltransferase-2 deficiency: a case presenting with myopathy.

Mitochondria were isolated from liver, heart and skeletal muscle of a 34-day-old female infant who died from a myopathic illness. Muscle biopsy showed lipid accumulation and no obvious pathology in any other organ. Enzymatic analysis of skeletal muscle extracts revealed normal activities of the markers pyruvate dehydrogenase and citrate synthase. Malonyl-CoA-sensitive carnitine palmitoyltransferase (CPT1) was detected but malonyl-CoA-insensitive carnitine palmitoyltransferase (CPT2) appeared to be absent. Quantitative immunoblotting revealed the presence of a normal abundance of CPT2 protein in the patient's muscle. It is concluded that enzymically inactive CPT2 protein was present.

Carnitine O-Palmitoyltransferase↗
PubMed1995

Effect of peroxynitrite on the mitochondrial respiratory chain: differential susceptibility of neurones and astrocytes in primary culture.

The effect of the neurotoxic nitric oxide derivative, the peroxynitrite anion (ONOO-), on the activity of the mitochondrial respiratory chain complexes in cultured neurones and astrocytes was studied. A single exposure of the neurones to ONOO- (initial concentrations of 0.01-2.0 mM) caused, after a subsequent 24-h incubation, a dose-dependent decrease in succinate-cytochrome c reductase (60% at 0.5 mM) and in cytochrome c oxidase (52% at 0.5 mM) activities. NADH-ubiquinone-1 reductase was unaffected. In astrocytes, the activity of the mitochondrial complexes was not affected up to 2 mM ONOO-. Citrate synthase was unaffected in both cell types under all conditions studied. However, lactate dehydrogenase activity released to the culture medium was increased by ONOO- in a dose-dependent manner (40% at 0.5 mM ONOO-) from the neurones but not from the astrocytes. Neuronal glutathione concentration decreased by 39% at 0.1 mM ONOO-, but astrocytic glutathione was not affected up to 2 mM ONOO-. In isolated brain mitochondria, only succinate-cytochrome c reductase activity was affected (22% decrease at 1 mM ONOO-). We conclude that the acute exposure of ONOO- selectively damages neurones, whereas astrocytes remain unaffected. Intracellular glutathione appears to be an important factor for ameliorating ONOO(-)-mediated mitochondrial damage. This study supports the hypothesis that the neurotoxicity of nitric oxide is mediated through mitochondrial dysfunction.

Animals↗
PubMed1994

Trolox protects mitochondrial complex IV from nitric oxide-mediated damage in astrocytes.

The efficacy of cystine, ascorbate and trolox, a vitamin E analogue, at protecting against nitric oxide-mediated mitochondrial complex IV damage has been investigated in cultured astrocytes. Of these compounds, only trolox afforded protection. It is suggested that lipid peroxidation is responsible for nitric oxide-mediated mitochondrial damage and that inhibitors of this process may be of therapeutic benefit in conditions where excessive nitric oxide production is implicated.

Amino Acid Oxidoreductases↗
PubMed1994

Nitric oxide-mediated inhibition of the mitochondrial respiratory chain in cultured astrocytes.

The Ca(2+)-independent form of nitric oxide synthase was induced in rat neonatal astrocytes in primary culture by incubation with lipopolysaccharide (1 microgram/ml) plus interferon-gamma (100 U/ml), and the activities of the mitochondrial respiratory chain components were assessed. Incubation for 18 h produced 25% inhibition of cytochrome c oxidase activity. NADH-ubiquinone-1 reductase (complex I) and succinate-cytochrome c reductase (complex II-III) activities were not affected. Prolonged incubation for 36 h gave rise to a 56% reduction of cytochrome c oxidase activity and a 35% reduction in succinate-cytochrome c reductase activity, but NADH-ubiquinone-1 reductase activity was unchanged. Citrate synthase activity was not affected by any of these conditions. The inhibition of the activities of these mitochondrial respiratory chain complexes was prevented by incubation in the presence of the specific nitric oxide synthase inhibitor NG-monomethyl-L-arginine. The lipopolysaccharide/interferon-gamma treatment of the astrocytes produced an increase in glycolysis and lactate formation. These results suggest that inhibition of the mitochondrial respiratory chain after induction of astrocytic nitric oxide synthase may represent a mechanism for nitric oxide-mediated neurotoxicity.

Amino Acid Oxidoreductases↗
PubMed1994

Dilated cardiomyopathy due to type II X-linked 3-methylglutaconic aciduria: successful treatment with pantothenic acid.

A case of dilated cardiomyopathy in a young boy secondary to type II 3-methylglutaconic aciduria is described. A metabolic cause for his dilated cardiomyopathy was suspected because of the development on the electrocardiogram of an unusual "camel's hump" shape of the T waves, and of progressive thickening with increasing echogenicity of the left ventricular wall. He initially improved on digoxin treatment, but did not maintain the response with conventional dietary treatment for this condition. Supplementation with L-carnitine was associated with rapid deterioration in cardiac state, and may be contraindicated in this condition. At a point when the patient was moribund, large doses of pantothenic acid, a precursor of coenzyme A, produced a dramatic and sustained improvement in myocardial function and in growth, neutrophil cell count, hypocholesterolaemia, and hyperuricaemia, which suggests that limitation of availability of coenzyme A is a fundamental pathological process in this condition. The clinical improvement has been maintained for 13 months, and myocardial function is now nearly normal. Oral pantothenol, unlike pantothenic acid, is not efficacious.

Amino Acid Metabolism, Inborn Errors↗
PubMed1994

Hypoglycaemia in the neonate: how and when is it important?

Cerebral metabolism is known to be highly dependent upon glucose utilisation. In the neonate, severe hypoglycaemia is now recognized to be associated with subsequent adverse neurodevelopment and in extremis glial and neuronal cell death. However, the level at which hypoglycaemia becomes clinically important is not well understood. Clinical methods for assessing effects of hypoglycaemia are at best crude. Noninvasive assessment of neuronal function suggests that cerebral dysfunction occurs at blood glucose levels well above clinical action levels. Thus, we appear to ignore apparently asymptomatic hypoglycaemia at our and indeed the patient's peril. Furthermore, glycaemic status invariably is interpreted with scant regard to alternative catabolic substrates. In the neonate, recent evidence suggests this approach is unfounded. This presentation will review evidence that glycaemic status in neonates should no longer be considered as an entity in itself, but rather in the light of compensatory metabolic processes and treated accordingly.

Humans↗
PubMed1993

Oral phosphate supplements reverse skeletal muscle abnormalities in a case of chronic fatigue with idiopathic renal hypophosphatemia.

A 57-yr-old man presented with a long history of undiagnosed fatigue but no evidence of bone disease. He was noted to have hypophosphatemia due to an idiopathic phosphaturia. Marked abnormalities of exercising skeletal muscle detected by phosphorus magnetic resonance spectroscopy and by plasma metabolite measurements were consistent with mitochondrial dysfunction. Oral phosphate supplements restored plasma phosphate concentration and muscle biochemistry to normal and produced considerable improvement in symptoms and exercise tolerance, although the phosphate concentration in muscle was only marginally low and increased little by treatment. We conclude that hypophosphatemia should be excluded in unexplained fatigue.

Adenosine Diphosphate↗