Norbert Maassen

Human plasma concentrations of malondialdehyde (MDA) and the F2-isoprostane 15(S)-8-iso-PGF2α may be markedly compromised by hemolysis: Evidence by GC-MS/MS and potential analytical and biological ramifications

OBJECTIVES Malondialdehyde (MDA) and the F(2)-isoprostane 15(S)-8-iso-prostaglandin F(2alpha) (15(S)-8-iso-PGF(2alpha)) belong to the most frequently analyzed biomarkers of oxidative stress in basic and clinical research. The objective of the present study was to examine the effect of hemolysis on free MDA and total (free+esterified) 15(S)-8-iso-PGF(2alpha) concentrations in human plasma. DESIGN AND METHODS MDA and 15(S)-8-iso-PGF(2alpha) were determined by GC-MS/MS in plasma samples from venous heparinized blood drawn under resting conditions (n=22) as well as under physical exercise (n=158) in 22 healthy young subjects. In vitro, we prepared plasma samples with hemolysis degrees up to 0.8% using artificially hemolyzed, freshly obtained heparinized blood. RESULTS In some plasma samples of the exercise study both under resting and exercise conditions, clinically significant hemolysis was macroscopically visible. Both in vivo (r=0.74) and in vitro (r=0.87), we found a significant positive correlation between hemolysis degree (0-0.2%) and MDA plasma concentrations (50-250 nmol/L). Unlike in vitro (r=0.84), in vivo, 15(S)-8-iso-PGF(2alpha) and MDA plasma concentrations correlated weakly (r=0.50). CONCLUSIONS We hypothesize that free hemoglobin catalyzes the formation of MDA and 15(S)-8-iso-PGF(2alpha) from free and esterified arachidonic acid. Plasma concentrations of MDA and total 15(S)-8-iso-PGF(2alpha) may be markedly compromised by hemolysis. Measurements of MDA and 15(S)-8-iso-PGF(2alpha) should be treated with caution regarding involvement of oxidative stress in disease as well as in health both under resting conditions and under exercise.

Positive correlation between plasma nitrite and performance during high-intensive exercise but not oxidative stress in healthy men

Several studies suggest that exercise is associated with elevated oxidative stress which diminishes NO bioavailability. The aim of the present study was to investigate a potential link between NO synthesis and bioavailability and oxidative stress in the circulation of subjects performing high-intensive endurance exercise. Twenty-two male healthy subjects cycled at 80% of their maximal workload. Cubital venous blood was taken before, during and after exercise, and heparinized plasma was generated. Plasma concentrations of nitrite and nitrate were quantified by GC-MS and of the oxidative stress biomarker 15(S)-8-iso-PGF(2alpha) by GC-MS/MS. pH and pCO(2) fell and HbO(2) increased upon exercise. The duration of the 80% phase (d80) was 740+/-210s. Subjects cycled at 89.2+/-3.3% of their peak oxygen uptake. Plasma concentration of nitrite (P<0.01) and 15(S)-8-iso-PGF(2alpha) (P<0.05) decreased significantly during exercise. At the end of exercise, plasma nitrite concentration correlated positively with d80 and performed work (w80) (each P<0.05). Changes in nitrate concentration also correlated positively with d80 (P<0.05) and w80/kg (P<0.01). These findings provide evidence of a favorable effect of nitrite on high-intensive endurance exercise. The lack of association between 15(S)-8-iso-PGF(2alpha) and NO bioavailability (nitrite concentration) and NO biosynthesis (nitrate concentration) suggest that oxidative stress, notably lipid peroxidation, is not linked to the l-arginine/NO pathway in healthy male subjects being on endurance exercise.

Point:Counterpoint: Lactic acid is/is not the only physicochemical contributor to the acidosis of exercise

to the editor: During intense exercise, pH markedly decreases in working muscle fibers, interstitial fluid, and blood. Here we consider not the effects of the rising Pco2 in muscles and venous blood but the nonrespiratory acidosis caused, according to traditional views, by lactic acid (HLa).

Development, validation and biomedical applications of stable-isotope dilution GC-MS and GC-MS/MS techniques for circulating malondialdehyde (MDA) after pentafluorobenzyl bromide derivatization: MDA as a biomarker of oxidative stress and its relation to 15(S)-8-iso-prostaglandin F2α and nitric oxide (NO).

Malondialdehyde (MDA, CH2(CHO)2) is one of the best investigated and most frequently measured biomarkers of lipid peroxidation in biological fluids, a constituent of the so called thiobarbituric acid reactive substances (TBARS). The reaction of thiobarbituric acid with MDA and other carbonyl compounds is the basis for the batch TBARS assay, one of the most commonly and widely used assays of oxidative stress. Yet, the TBARS assay lacks specificity even if combined with HPLC separation prior to visible absorbance or fluorescence detection. In this article, we report highly specific and sensitive stable-isotope dilution GC-MS and GC-MS/MS methods for the quantitative determination of MDA in human plasma (0.1 mL). These methods utilize the acidity (pKa, 4.46) of the two methylene H protons of MDA in aqueous solution, which are as acidic as acetic acid. Endogenous MDA in native plasma and the externally added internal standard [1,3-(2)H2]-MDA (d2-MDA, CH2(CDO)2) are derivatized in aqueous acetone (400 μL) with pentafluorobenzyl (PFB) bromide (10 μL). The reaction products were identified as C(PFB)2(CHO)2 (molecular weight, 432) and C(PFB)2(CDO)2) (molecular weight, 434), respectively. After solvent extraction with toluene (1 mL) quantification is performed by selected-ion monitoring (SIM) in GC-MS and by selected-reaction monitoring (SRM) in GC-MS/MS in the electron-capture negative-ion chemical ionization (ECNICI) mode. In the SIM mode, the anions [M-PFB](-) at m/z 251 for MDA and m/z 253 for d2-MDA are detected. In the SRM mode, the mass transitions m/z 251 to m/z 175 for MDA and m/z 253 to m/z 177 for d2-MDA are monitored. The method was thoroughly validated in human plasma. Potential interfering substances including anticoagulants and commercially available monovettes commonly used for blood sampling were tested. The lowest MDA concentrations were measured in serum followed by heparinized and EDTA plasma. The GC-MS and GC-MS/MS methods were found to be specific, precise, accurate and sensitive. Thus, the LOD of the GC-MS/MS method was determined to be 2 amol (2 × 10(-18)mol) MDA. The GC-MS/MS method is exceedingly useful in clinical settings. We report several biomedical applications and discuss the utility of circulating MDA as a biomarker of lipid peroxidation, especially in long-term clinical studies, and its relation to the F2-isoprostane 15(S)-8-iso-prostaglandin F2α and nitric oxide (NO).

The effects of creatine pyruvate and creatine citrate on performance during high intensity exercise

BackgroundA double-blind, placebo-controlled, randomized study was performed to evaluate the effect of oral creatine pyruvate (Cr-Pyr) and creatine citrate (Cr-Cit) supplementation on exercise performance in healthy young athletes.MethodsPerformance during intermittent handgrip exercise of maximal intensity was evaluated before (pretest) and after (posttest) 28 days of Cr-Pyr (5 g/d, n = 16), Cr-Cit (5 g/d, n = 16) or placebo (pla, 5 g/d, n = 17) intake. Subjects performed ten 15-sec exercise intervals, each followed by 45 sec rest periods.ResultsCr-Pyr (p < 0.001) and Cr-Cit (p < 0.01) significantly increased mean power over all intervals. Cr-Cit increased force during the first and second interval (p < 0.01) compared to placebo. The effect of Cr-Cit on force decreased over time and the improvement was not significant at the sixth and ninth interval, whereas Cr-Pyr significantly increased force during all intervals (p < 0.001). Cr-Pyr (p < 0.001) and Cr-Cit (p < 0.01) resulted in an increase in contraction velocity, whereas only Cr-Pyr intake significantly (p < 0.01) increased relaxation velocity. Oxygen consumption measured during rest periods significantly increased with Cr-Pyr (p < 0.05), whereas Cr-Cit and placebo intake did not result in significant improvements.ConclusionIt is concluded that four weeks of Cr-Pyr and Cr-Cit intake significantly improves performance during intermittent handgrip exercise of maximal intensity and that Cr-Pyr might benefit endurance, due to enhanced activity of the aerobic metabolism.

The relationships between plasma potassium, muscle excitability and fatigue during voluntary exercise in humans

The relationships between extracellular potassium elevation and EMG variables in relation to muscle fatigue were investigated during handgrip exercise in humans. Acid–base state, lactate, potassium ([K+]v) and sodium in venous plasma, as well as variables of surface voluntary and evoked (M‐wave) EMG were determined during repeated dynamic (DE) and static (SE) exercise (1 min exercise, 4 min rest). The different rises of [K+]v were induced by randomly varied workloads. After 15 min of warming up, the M‐wave area increased to 124.9 ± 19.6% (P < 0.001) in comparison with the control value. Simultaneously, the [K+]v decreased from 4.1 ± 0.3 to 3.6 ± 0.3 mmol l−1 (P < 0.01). During both SE and DE, there were marked intensity‐dependent signs of fatigue. The [K+]v correlated with changes of the integrated EMG (r= 0.87, P < 0.001 for both DE and SE). Changes in the M‐wave area during the exercise bouts correlated inversely with the [K+]v (r=−0.73, P < 0.001). The M‐wave area did not decrease below the control value at any intensity. The median frequency of the EMG decreased during exercise, depending on the exercise intensity (r=−0.73 for SE, r=−0.47 for DE, P < 0.001) with a maximal decrease to about 80% after SE with the maximal workload. The muscle action potential propagation velocity changed in the range of about ±2%. For the first time, a negative relationship between venous potassium and M‐wave area was shown during voluntary exercise. However, there was no evidence that the decrease in muscle performance was mainly caused by a decrease in sarcolemmal excitability resulting from a high extracellular [K+].

Simultaneous GC-ECNICI-MS measurement of nitrite, nitrate and creatinine in human urine and plasma in clinical settings.

Creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous substances. Nitrite (ONO-) and nitrate (ONO2-) are metabolites of nitric oxide (NO), a signalling molecule with multiple biological functions. Under certain and standardized conditions, the concentration of nitrate in the urine is a suitable measure of whole body NO synthesis. The urinary nitrate-to-nitrite molar ratio (UNOxR) may indicate nitrite-dependent renal carbonic anhydrase (CA) activity. In clinical studies, urine is commonly collected by spontaneous micturition. In those cases the nitrate and nitrite excretion must be corrected for creatinine excretion. Pentafluorobenzyl (PFB) bromide (PFB-Br) is a useful derivatization reagent of numerous inorganic and organic compounds, including urinary nitrite, nitrate and creatinine, for highly sensitive and specific quantitation by GC-MS. Here, we report on the simultaneous PFB-Br derivatization (60min, 50°C) of ONO-, O15NO-, ONO2-, O15NO2-, creatinine (do-Crea) and [methylo-2H3]creatinine (d3-Crea) in acetonic dilutions of native human urine and plasma samples (4:1, v/v) and their simultaneous quantification by GC-MS as PFBNO2, PFB15NO2, PFBONO2, PFBO15NO2, do-Crea-PFB and d3-Crea-PFB, respectively. Electron capture negative-ion chemical ionization (ECNICI) of these derivatives generates anions due to [M-PFB]-, i.e., the starting analytes. Quantification is performed by selected-ion monitoring (SIM) of m/z 46 (ONO-), m/z 47 (O15NO-), m/z 62 (ONO2-), m/z 63 (O15NO2-), m/z 112 (do-Crea), and m/z 115 (d3-Crea). Retention times were 2.97min for PFB-ONO2/PFB-O15NO2, 3.1min for PFB-NO2/PFB-15NO2, and 6.7min for do-Crea-PFB/d3-Crea-PFB. We used this method to investigate the effects of long-term oral NaNO3 or NaCl (serving as placebo) supplementation (each 0.1mmol/kg body weight per day for 3 weeks) on creatinine excretion and UNOxR in 17 healthy young men. Compared to NaCl (n=8), NaNO3 (n=9) supplementation increased UNOxR (1709±355 vs. 369±77, P<0.05). Creatinine excretion did not differ between the groups (6.67±1.34mM vs. 5.72±1.27mM, P=0.57). The method is also applicable to human plasma. In 78 adults patients newly diagnosed for cerebrovascular disease (CVD), there was a close correlation (r=0.9833) between the creatinine concentrations measured in plasma by GC-ECNICI-MS and those measured in serum by an enzymatic assay. Creatinine-corrected plasma nitrate and nitrite concentrations (P=0.035 and P=0.004, respectively) but not their concentrations (P=0.68 and P=0.40, respectively) differ between male (n=54) and female (n=24) CVD patients. No such differences were found between preterm newborn boys (n=25) and girls (n=22). Like in urine, circulating creatinine may be useful to correct for gender-specific differences in plasma nitrite and nitrate in adults. Chronic NaNO3 supplementation to healthy young men does not affect renal CA-dependent nitrite excretion or creatinine synthesis and excretion.

Interrelationship between pH, plasma potassium concentration and ventilation during intense continuous exercise in man

SummaryDuring resting conditions plasma hydrogen ion concentration ([H+]P) is known to influence ventilation

Even and carbon dioxide independent distribution of nitrite between plasma and erythrocytes of healthy humans at rest

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