Simultaneous extraction and reverse-phase high-performance liquid chromatographic determination of adenine and pyridine nucleotides in human red blood cells

Stocchi, Vilberto; Cucchiarini, Luigi; Magnani, Mauro; Chiarantini, Laura; Palma, Pierangela; Crescentini, G.

doi:10.1016/0003-2697(85)90405-1

Cited by 339 publications

(164 citation statements)

References 20 publications

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“…After neutralization with 3 M KOH/1.5 M Tris, samples were centrifuged at 20,800 g for 5 min at 4°C. The resulting supernatants were assayed for ATP determination by separation in a reverse-phase high-performance liquid chromatography (HPLC), with detection at 254 nm, as described previously (Stocchi et al, 1985). ADP and AMP levels were undetectable in our samples.…”

Section: Measurement Of Atp and Phosphocreatinementioning

confidence: 99%

Bioenergetic dysfunction in Huntington's disease human cybrids

Ferreira¹,

Cunha‐Oliveira²,

Nascimento³

et al. 2011

Experimental Neurology

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In this work we studied the mitochondrial-associated metabolic pathways in Huntington's disease (HD) versus control (CTR) cybrids, a cell model in which the contribution of mitochondrial defects from patients is isolated. HD cybrids exhibited an interesting increase in ATP levels, when compared to CTR cybrids. Concomitantly, we observed increased glycolytic rate in HD cybrids, as revealed by increased lactate/pyruvate ratio, which was reverted after inhibition of glycolysis. A decrease in glucose-6-phosphate dehydrogenase activity in HD cybrids further indicated decreased rate of the pentose-phosphate pathway. ATP levels of HD cybrids were significantly decreased under glycolysis inhibition, which was accompanied by a decrease in phosphocreatine. Nevertheless, pyruvate supplementation could not recover HD cybrids' ATP or phosphocreatine levels, suggesting a dysfunction in mitochondrial use of that substrate. Oligomycin also caused a decrease in ATP levels, suggesting a partial support of ATP generation by the mitochondria. Nevertheless, mitochondrial NADH/NAD t levels were decreased in HD cybrids, which was correlated with a decrease in pyruvate dehydrogenase activity and protein expression, suggesting decreased tricarboxylic acid cycle (TCA) input from glycolysis. Interestingly, the activity of alpha-ketoglutarate dehydrogenase, a critical enzyme complex that links the TCA to amino acid synthesis and degradation, was increased in HD cybrids. In accordance, mitochondrial levels of glutamate were increased and alanine was decreased, whereas aspartate and glutamine levels were unchanged in HD cybrids. Conversely, malate dehydrogenase activity from total cell extracts was unchanged in HD cybrids. Our results suggest that inherent dysfunction of mitochondria from HD patients affects cellular bioenergetics in an otherwise functional nuclear background.

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Section: Measurement Of Atp and Phosphocreatinementioning

confidence: 99%

Bioenergetic dysfunction in Huntington's disease human cybrids

Ferreira¹,

Cunha‐Oliveira²,

Nascimento³

et al. 2011

Experimental Neurology

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“…The supernatants were neutralized with 10 M KOH in 5 M Tris and further centrifuged at 15,800 Â g, for 10 min at 4 8C to remove the potassium perchlorate salt. The resulting supernatants were assayed for adenosine triphosphate (ATP) using a reverse-phase HPLC methodology, as described by Stocchi et al (1985) The chromatographic apparatus was a Beckman System Gold, consisting of a 126 binary Pump Model and a 166 Variable UV detector controlled by a computer. The column was a Lichrospher 100 RP-18 (5 mM) from Merck (Germany).…”

Section: Determination Of Atp Levelsmentioning

confidence: 99%

3,4-Dihydroxyphenylacetic acid (DOPAC) modulates the toxicity induced by nitric oxide in PC-12 cells via mitochondrial dysfunctioning

2008

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“…The pellets were used to determine the protein content by the Sedmak method [20], whereas the supernatants were neutralized with 10 M KOH/5 M Tris and centrifuged at 15,800 ϫ g for 10 min. The resulting supernatants were analyzed by separation in a reverse-phase HPLC, as described by Stocchi et al [21]. The chromatographic apparatus used was a Beckman-System Gold, consisting of a 126 Binary Pump Model, a 166 Variable UV detector and a Lichrospher 100 RP-18 (5 m) column, from Merck (Darmstadt, Germany).…”

Section: Determination Of Cellular Energy Chargementioning

confidence: 99%

Influence of the antioxidants vitamin E and idebenone on retinal cell injury mediated by chemical ischemia, hypoglycemia, or oxidative stress

Rego

Santos

Oliveira

1999

Free Radical Biology and Medicine

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Abstract-A role for the antioxidants vitamin E and idebenone in decreasing retinal cell injury, after metabolic inhibition induced by chemical ischemia and hypoglycemia, was investigated and compared with oxidative stress conditions. Preincubation of the antioxidants, vitamin E (20 M) and idebenone (10 M), effectively protected from retinal cell injury after oxidative stress or hypoglycemia, whereas the protection afforded after postincubation of both antioxidants was decreased. Delayed retinal cell damage, mediated by chemical ischemia, was attenuated at 10 or 12 h postischemia, only after exposure to the antioxidants during all the experimental procedure. An antagonist of the N-methyl-D-aspartate (NMDA) receptors, an inhibitor of nitric oxide synthase (NOS) or a blocker of L-type Ca 2ϩ channels were ineffective in reducing cell injury induced by chemical ischemia, hypoglycemia or oxidative stress. Oxidative stress and hypoglycemia increased (about 1.2-fold) significantly the fluorescence of the probe DCFH 2 -DA, that is indicative of intracellular ROS formation. Free radical generation detected with the probe dihydrorhodamine 123 (DHR 123) was enhanced after oxidative stress, chemical ischemia or hypoglycemia (about 2-fold). Nevertheless, the antioxidants vitamin E or idebenone were ineffective against intracellular ROS generation. Cellular energy charge decreased greatly after chemical ischemia, was moderately affected after hypoglycemia, but no significant changes were observed after oxidative stress. Preincubation with vitamin E prevented the changes in energy charge upon 6 h posthypoglycemia. We can conclude that irreversible changes occurring during chemical ischemia mainly reflect the alterations taking place at the ischemic core, whereas hypoglycemia situations may reflect changes occurring at the penumbra area, whereby vitamin E or idebenone may help to increase cell survival, exerting a beneficial neuroprotective effect.

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Simultaneous extraction and reverse-phase high-performance liquid chromatographic determination of adenine and pyridine nucleotides in human red blood cells

Cited by 339 publications

References 20 publications

Bioenergetic dysfunction in Huntington's disease human cybrids

Bioenergetic dysfunction in Huntington's disease human cybrids

3,4-Dihydroxyphenylacetic acid (DOPAC) modulates the toxicity induced by nitric oxide in PC-12 cells via mitochondrial dysfunctioning

Influence of the antioxidants vitamin E and idebenone on retinal cell injury mediated by chemical ischemia, hypoglycemia, or oxidative stress

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