Simultaneous GC-MS/MS measurement of malondialdehyde and 4-hydroxy-2-nonenal in human plasma: Effects of long-term L-arginine administration.

Here, we report the simultaneous derivatization and quantification of malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) in human plasma by GC-MS/MS using [1,3-2H2]-MDA (d2-MDA) and [9,9,9-2H3]-HNE (d3-HNE) as the internal standards, respectively. MDA, d2-MDA, HNE and d3-HNE were converted to their pentafluorobenzyl oximes (PFBOX) by pentafluorobenzyl hydroxylamine. Subsequently, the hydroxyl groups of the PFBOX of HNE and d3-HNE were trimethylsilylated with N,O-bis(trimethylsilyl)trifluoroacetamide/1% trimethylchlorosilane. GC-MS/MS analyses were performed in the electron-capture negative-ion chemical ionization mode. Quantification was performed by selected-reaction monitoring the mass transitions m/z 442 to m/z 243 for MDA, m/z 444 to m/z 244 for d2-MDA, m/z 403 → m/z 283 for HNE and m/z 406 → m/z 286 for d3-HNE. The method was applied to measure MDA and HNE in plasma of patients suffering from coronary artery disease (CAD) or peripheral artery occlusive disease (PAOD) before and after oral supplementation of L-arginine (3 g/day) or placebo for 3 (CAD and PAOD) and 6 months (PAOD). All plasma samples were analyzed after completion of the studies. Our results revealed that storage of plasma samples (at -80 °C) leads to lower MDA and HNE plasma concentrations in the plasma samples that were collected at the end of the studies as compared to those collected at the begin of the studies. Based on MDA and HNE measurements in plasma, L-arginine did not influence lipid peroxidation in CAD and PAOD patients. Long-term studies on lipid peroxidation are best performed by measuring oxidative stress biomarkers such as MDA and/or HNE in plasma samples immediately after their collection. Long-term storage of plasma samples even at -80 °C is not recommended.

Effects of chronic oral L-arginine administration on the L-arginine/NO pathway in patients with peripheral arterial occlusive disease or coronary artery disease: L-Arginine prevents renal loss of nitrite, the major NO reservoir.

Despite saturation of nitric oxide (NO) synthase (NOS) by its substrate L-arginine (Arg), oral and intravenous supplementation of Arg may enhance NO synthesis, a phenomenon known as "The L-arginine paradox". Yet, Arg is not only a source of NO, but is also a source for guanidine-methylated (N (G)) arginine derivatives which are all inhibitors of NOS activity. Therefore, Arg supplementation may not always result in enhanced NO synthesis. Concomitant synthesis of N (G)-monomethyl arginine (MMA), N (G),N (G)-dimethylarginine (asymmetric dimethylarginine, ADMA) and N (G),N (G´)-dimethylarginine (symmetric dimethylarginine, SDMA) from supplemented Arg may outweigh and even outbalance the positive effects of Arg on NO. Another possible, yet little investigated effect of Arg supplementation may be alteration of renal function, notably the influence on the excretion of nitrite in the urine. Nitrite is the autoxidation product of NO and the major reservoir of NO in the circulation. Nitrite and Arg are reabsorbed in the proximal tubule of the nephron and this reabsorption is coupled, at least in part, to the renal carbonic anhydrase (CA) activity. In the present placebo-controlled studies, we investigated the effect of chronic oral Arg supplementation of 10 g/day for 3 or 6 months in patients suffering from peripheral arterial occlusive disease (PAOD) or coronary artery disease (CAD) on the urinary excretion of nitrite relative to nitrate. We determined the urinary nitrate-to-nitrite molar ratio (UNOxR), which is a measure of nitrite-dependent renal CA activity before and after oral intake of Arg or placebo by the patients. The UNOxR was also determined in 6 children who underwent the Arg test, i.e., intravenous infusion of Arg (0.5 g Arg/kg bodyweight) for 30 min. Arg was well tolerated by the patients of the three studies. Oral Arg supplementation increased Arg (plasma and urine) and ADMA (urine) concentrations. No appreciable changes were seen in NO (in PAOD and CAD) and prostacyclin and thromboxane synthesis (in PAOD). In the PAOD study, UNOxR did not change in the Arginine group (480 ± 51 vs 486 ± 50), but fell in the Placebo group (422 ± 67 vs 332 ± 42, P = 0.025). In the CAD study, UNOxR did not change significantly in the Arginine group (518 ± 77 at start vs 422 ± 40 after 3 months vs 399 ± 66 after 6 months), but fell in the Placebo group (524 ± 69 vs 302 ± 36 vs 285 ± 31; P = 0.025 for 0 vs 3 months). Infusion of Arg tended to decrease the UNOxR in the children (317 ± 41 vs 208 ± 16, P = 0.06). We propose that oral long-term Arg supplementation prevents loss of NO bioactivity by saving nitrite. The optimum Arg dose needs to be elaborated and is likely to be less than 10 g per day in adults. Orally and intravenously administered arginine was well tolerated by the elderly patients and young children, respectively.

Plasma homoarginine, arginine, asymmetric dimethylarginine and total homocysteine interrelationships in rheumatoid arthritis, coronary artery disease and peripheral artery occlusion disease.

Elevated circulating concentrations of total L-homocysteine (thCys) and free asymmetric dimethylarginine (ADMA) are long-established cardiovascular risk factors. Low circulating L-homoarginine (hArg) concentrations were recently found to be associated with increased cardiovascular morbidity and mortality. The biochemical pathways of these amino acids overlap and share the same cofactor S-adenosylmethionine (SAM). In the present study, we investigated potential associations between hArg, L-arginine (Arg), ADMA and thCys in plasma of patients suffering from rheumatoid arthritis (RA), coronary artery disease (CAD) or peripheral artery occlusive disease (PAOD). In RA, we did not find any correlation between ADMA or hArg and thCys at baseline (n = 100) and after (n = 83) combined add-on supplementation of omega-3 fatty acids, vitamin E, vitamin A, copper, and selenium, or placebo (soy oil). ADMA correlated with Arg at baseline (r = 0.446, P < 0.001) and after treatment (r = 0.246, P = 0.03). hArg did not correlate with ADMA, but correlated with Arg before (r = 0.240, P = 0.02) and after treatment (r = 0.233, P = 0.03). These results suggest that hArg, ADMA and Arg are biochemically familiar with each other, but unrelated to hCys in RA. In PAOD and CAD, ADMA and thCys did not correlate.

Biosynthesis of homoarginine (hArg) and asymmetric dimethylarginine (ADMA) from acutely and chronically administered free L-arginine in humans.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis, whereas L-arginine (Arg) and L-homoarginine (hArg) serve as substrates for NO synthesis. ADMA and other methylated arginines are generally believed to exclusively derive from guanidine (N (G))-methylated arginine residues in proteins by protein arginine methyltransferases (PRMTs) that use S-adenosylmethionine (SAM) as the methyl donor. L-Lysine is known for decades as a precursor for hArg, but only recent studies indicate that arginine:glycine amidinotransferase (AGAT) is responsible for the synthesis of hArg. AGAT catalyzes the formation of guanidinoacetate (GAA) that is methylated to creatine by guanidinoacetate methyltransferase (GAMT) which also uses SAM. The aim of the present study was to learn more about the mechanisms of ADMA and hArg formation in humans. Especially, we hypothesized that ADMA is produced by N (G)-methylation of free Arg in addition to the known PRMTs-involving mechanism. In knockout mouse models of AGAT- and GAMT-deficiency, we investigated the contribution of these enzymes to hArg synthesis. Arg infusion (0.5 g/kg, 30 min) in children (n = 11) and ingestion of high-fat protein meals by overweight men (n = 10) were used to study acute effects on ADMA and hArg synthesis. Daily Arg ingestion (10 g) or placebo for 3 or 6 months by patients suffering from peripheral arterial occlusive disease (PAOD, n = 20) or coronary artery disease (CAD, n = 30) was used to study chronic effects of Arg on ADMA synthesis. Mass spectrometric methods were used to measure all biochemical parameters in plasma and urine samples. In mice, AGAT but not GAMT was found to contribute to plasma hArg, while ADMA synthesis was independent of AGAT and GAMT. Arg infusion acutely increased plasma Arg, hArg and ADMA concentrations, but decreased the plasma hArg/ADMA ratio. High-fat protein meals acutely increased plasma Arg, hArg, ADMA concentrations, as well as the plasma hArg/ADMA ratio. In the PAOD and CAD studies, plasma Arg concentration increased in the verum compared to the placebo groups. Plasma ADMA concentration increased only in the PAOD patients who received Arg. Our study suggests that in humans a minor fraction of free Arg is rapidly metabolized to ADMA and hArg. In mice, GAMT and N (G)-methyltransferases contribute to ADMA and hArg synthesis from Arg, whereas AGAT is involved in the synthesis of hArg but not of ADMA. The underlying biochemical mechanisms remain still elusive.

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).