HPLC of nucleic acid components with volatile mobile phases part. 2: Separations on polymeric supports

Krauss, Gerhard; Pissarek, Margit; Blasig, Ingolf E.

doi:10.1002/jhrc.1240201214

Cited by 15 publications

(5 citation statements)

References 11 publications

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“…Adenine nucleotides were determination by high performance liquid chromatography (HPLC) with a ProPac PA1 column (4 × 250 mm: Dionex Corp., Sunnyvale, CA, USA) and a binary gradient of 0.3 m ammonium carbonate and water [46]. Purified mitochondria were rapidly resuspended in ice‐cold 0.5 m perchloric acid, mixed during 120 s in vortex (to break the mitochondrial membranes) and centrifuged at 25,000 g for 15 min at 2°C to precipitate proteins.…”

Section: Methodsmentioning

confidence: 99%

“…After stabilizing the column with the mobile phase, 20 μ L of each sample were injected onto the HPLC system. The mobile phase consisted in water (phase A) and 0.3 M ammonium carbonate pH 8.9 (phase B), and the following time schedule for the binary gradient (flow rate 1 mL/min) was used: 5 min, 50% A and 50% B; 5 min 50–100% B and then maintained 100% B during 25 min; 5 min 100–50% B and then another 5 min with 50% B [46]. For calibration, water was used as blank and 3.125, 6.250, 12.5 and 25 μ g/mL of each nucleotide (AMP, ADP and ATP) was used for constructing the standard curves.…”

Section: Methodsmentioning

confidence: 99%

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Long‐term melatonin administration protects brain mitochondria from aging

Carretero

Escames

López

et al. 2009

Journal of Pineal Research

126

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We tested whether chronic melatonin administration in the drinking water would reduce the brain mitochondrial impairment that accompanies aging. Brain mitochondria from male and female senescent prone (SAMP8) mice at 5 and 10 months of age were studied. Mitochondrial oxidative stress was determined by measuring the levels of lipid peroxidation and nitrite, glutathione/glutathione disulfide ratio, and glutathione peroxidase and glutathione reductase activities. Electron transport chain activity and oxidative phosphorylation capability of mitochondria were also determined by measuring the activity of the respiratory chain complexes and the ATP content. The results support a significant age-dependent mitochondrial dysfunction with a diminished efficiency of the electron transport chain and reduced ATP production, accompanied by an increased oxidative/nitrosative stress. Melatonin administration between 1 and 10 months of age completely prevented the mitochondrial impairment, maintaining or even increasing ATP production. There were no major age-dependent differences between males in females, although female mice seemed to be somewhat more sensitive to melatonin treatment than males. Thus, melatonin administration as a single therapy maintained fully functioning brain mitochondria during aging, a finding with important consequences in the pathophysiology of brain aging.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Long‐term melatonin administration protects brain mitochondria from aging

Carretero

Escames

López

et al. 2009

Journal of Pineal Research

126

View full text Add to dashboard Cite

show abstract

“…After stabilization of the column with the mobile phase, samples (20 µL) were injected onto the HPLC system. The mobile phase consisted of water (phase A) and 0.3 m ammonium carbonate (pH 8.9) (phase B), and the following time schedule for the binary gradient (flow rate 1 mL·min −1 ) was used: 5 min, 50% A and 50% B; 5 min, 50% to 100% B, and then 100% B for 25 min; 5 min, 100% to 50% B, and then another 5 min with 50% B [71]. Water was used for calibration purposes.…”

Section: Methodsmentioning

confidence: 99%

Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice

et al. 2007

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Sepsis-induced multiple organ failure is the major cause of mortality in critically ill patients, and its incidence is rising [1]. The heart and cardiovascular systems are seriously affected during sepsis [2]. Although myocardial impairment in sepsis has been extensively studied, its etiology remains unclear [3]. Some reports The existence of an inducible mitochondrial nitric oxide synthase has been recently related to the nitrosative ⁄ oxidative damage and mitochondrial dysfunction that occurs during endotoxemia. Melatonin inhibits both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase activities, a finding related to the antiseptic properties of the indoleamine. Hence, we examined the changes in inducible nitric oxide synthase ⁄ inducible mitochondrial nitric oxide synthase expression and activity, bioenergetics and oxidative stress in heart mitochondria following cecal ligation and puncture-induced sepsis in wild-type (iNOS + ⁄ + ) and inducible nitric oxide synthase-deficient (iNOS -⁄ -) mice. We also evaluated whether melatonin reduces the expression of inducible nitric oxide synthase ⁄ inducible mitochondrial nitric oxide synthase, and whether this inhibition improves mitochondrial function in this experimental paradigm. The results show that cecal ligation and puncture induced an increase of inducible mitochondrial nitric oxide synthase in iNOS + ⁄ + mice that was accompanied by oxidative stress, respiratory chain impairment, and reduced ATP production, although the ATPase activity remained unchanged. Real-time PCR analysis showed that induction of inducible nitric oxide synthase during sepsis was related to the increase of inducible mitochondrial nitric oxide synthase activity, as both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase were absent in iNOS -⁄ -mice. The induction of inducible mitochondrial nitric oxide synthase was associated with mitochondrial dysfunction, because heart mitochondria from iNOS -⁄ -mice were unaffected during sepsis. Melatonin treatment blunted sepsis-induced inducible nitric oxide synthase ⁄ inducible mitochondrial nitric oxide synthase isoforms, prevented the impairment of mitochondrial homeostasis under sepsis, and restored ATP production. These properties of melatonin should be considered in clinical sepsis.

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“…Nucleotides were separated with a modified procedure according to Krauss et al (1997) in a gradient System Gold (Beckman, Munich, Germany) using a Propac-PA1 column (Dionex, Idstein, Germany) for anion-exchange chromatography. The eluent was ammonium carbonate 0.3 M (pH 8.9).…”

Section: Biochemical Experimentsmentioning

confidence: 99%

Changes by short-term hypoxia in the membrane properties of pyramidal cells and the levels of purine and pyrimidine nucleotides in slices of rat neocortex; effects of agonists and antagonists of ATP-dependent potassium channels

Pissarek

Arriba

Schäfer

et al. 1998

Naunyn-Schmiedeberg's Arch Pharmacol

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In a first series of experiments, intracellular recordings were made from pyramidal cells in layers II-III of the rat primary somatosensory cortex. Superfusion of the brain slice preparations with hypoxic medium (replacement of 95%O2-5%CO2 with 95%N2-5%CO2) for up to 30 min led to a time-dependent depolarization (HD) without a major change in input resistance. Short periods of hypoxia (5 min) induced reproducible depolarizations which were concentration-dependently depressed by an agonist of ATP-dependent potassium (K(ATP)) channels, diazoxide (3-300 microM). The effect of 30 but not 300 microM diazoxide was reversed by washout. Tolbutamide (300 microM), an antagonist of K(ATP) channels, did not alter the HD when given alone. It did, however, abolish the inhibitory effect of diazoxide (30 microM) on the HD. Neither diazoxide (3-300 microM) nor tolbutamide (300 microM) influenced the membrane potential or the apparent input resistance of the neocortical pyramidal cells. Current-voltage (I-V) curves constructed at a membrane potential of -90 mV by injecting both de- and hyperpolarizing current pulses were not altered by diazoxide (30 microM) or tolbutamide (300 microM). Moreover, normoxic and hypoxic I-V curves did not cross each other, excluding a reversal of the HD at any membrane potential between -130 and -50 mV. The hypoxia-induced change of the I-V relation was the same both in the absence and presence of tolbutamide (300 microM). In a second series of experiments, nucleoside di- and triphosphates separated with anion exchange HPLC were measured in the neocortical slices. After 5 min of hypoxia, levels of nucleoside triphosphates declined by 29% (GTP), 34% (ATP), 44% (UTP) and 58% (CTP). By contrast, the levels of nucleoside diphosphates either did not change (UDP) or increased by 13% (GDP) and 40% (ADP). In slices subjected to 30 min of hypoxia the triphosphate levels continued to decrease, while the levels of GDP and ADP returned to control values. The tri- to diphosphate ratios progressively declined for ATP/ADP and GTP/GDP, but not for UTP/UDP when the duration of hypoxia was increased from 5 to 30 min. Hence, the rapid fall in the ratios of nucleoside tri- to diphosphates without the induction of a potassium current failed to indicate an allosteric regulation of a plasmalemmal K(ATP) channel by purine and pyrimidine nucleotides. Diazoxide had no effect on neocortical pyramidal neurons and was effective only in combination with a hypoxic stimulus; it is suggested that both plasmalemmal and mitochondrial K(ATP) channels are involved under these conditions. The hypoxic depolarization may be due to blockade of K+,Na+-ATPase by limitation of energy supplying substrate.

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HPLC of nucleic acid components with volatile mobile phases part. 2: Separations on polymeric supports

Cited by 15 publications

References 11 publications

Long‐term melatonin administration protects brain mitochondria from aging

Long‐term melatonin administration protects brain mitochondria from aging

Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice

Changes by short-term hypoxia in the membrane properties of pyramidal cells and the levels of purine and pyrimidine nucleotides in slices of rat neocortex; effects of agonists and antagonists of ATP-dependent potassium channels

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