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.

Aortic thrombus and pulmonary embolism in a patient with hyperhomocysteinemia.

BACKGROUND: A 32-year-old man presented at hospital with persistent pain, hypothermia and paraesthesia in his right leg, caused by embolic occlusion of all three large arteries as a result of massive thrombi in the abdominal aorta. Previously, the patient had been diagnosed with pulmonary embolism and admitted at least a 6-month history of alcohol abuse. Laboratory assessment of the patient's lipid levels, platelet function and coagulation factors yielded normal results. Duplex ultrasound revealed substantial media thickening of the carotid and femoral arteries, without evidence of calcification. Further laboratory tests revealed elevated plasma levels of homocysteine, asymmetric dimethylarginine, symmetric dimethylarginine and 8-isoprostaglandin F2alpha. INVESTIGATIONS: Physical examination, laboratory analyses, bronchoscopy, duplex ultrasonography, CT scan and CT angiography. DIAGNOSIS: Severe hyperhomocysteinemia associated with acute aortic thrombi and peripheral emboli. MANAGEMENT: Diet supplementation with folic acid, vitamin B6 and vitamin B12, low-molecular-weight heparin and L-arginine.

Oral L-arginine improves endothelial function in healthy individuals older than 70 years.

Ageing is associated with progressive endothelial dysfunction in normal humans. Flow-mediated dilation (FMD) of the brachial artery is impaired in elderly individuals with cardiovascular disease and vascular nitric oxide (NO) bioavailability is reduced. We investigated whether oral L-arginine, the substrate for NO synthesis, can improve impaired FMD in healthy very old people. In a prospective, double-blind, randomized crossover trial, 12 healthy old subjects (age 73.8 +/- 2.7 years) took L-arginine (8 g p.o. two times daily) or placebo for 14 days each, separated by a wash-out period of 14 days. FMD was determined by high-resolution ultrasound in the brachial artery during reactive hyperaemia. Baseline artery diameter was 3.88 +/- 0.18 mm. L-Arginine significantly improved FMD (to 5.7 +/- 1.2%, p < 0.0001), whereas placebo had no effect (-0.25 +/- 0.7%; n.s.). After L-arginine, plasma levels of L-arginine increased significantly (114.9 +/- 11.6 versus 57.4 +/- 5.0 micromol/l), but placebo had no effect. As NO synthesis can be antagonized by its endogenous inhibitor asymmetric dimethyl L-arginine (ADMA), we determined ADMA plasma concentrations, which were elevated at baseline in comparison to healthy middle-aged individuals (3.9 +/- 0.2 versus 1.0 +/- 0.1 micromol/l; p < 0.0001). ADMA remained unchanged during treatment, but L-arginine supplementation normalized the L-arginine/ADMA ratio (p < 0.05). We conclude that in healthy very old age endothelial function is impaired and may be improved by oral L-arginine supplementation, probably due to normalization of the L-arginine/ADMA ratio.

ADMA and oxidative stress are responsible for endothelial dysfunction in hyperhomocyst(e)inemia: effects of L-arginine and B vitamins.

OBJECTIVES: Hyperhomocyst(e)inemia is a risk factor for atherosclerotic vascular disease, and it is associated with endothelial dysfunction. Mechanisms responsible for endothelial dysfunction in hyperhomocyst(e)inemia may involve impaired bioavailability of NO, possibly secondary to accumulation of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) and increased oxidative stress. We investigated whether oral treatment with B vitamins or L-arginine normalizes endothelium-dependent, flow-dependent vasodilation (FDD) in patients with peripheral arterial occlusive disease (PAOD) and hyperhomocyst(e)inemia. METHODS: 27 patients with PAOD and hyperhomocyst(e)inemia were assigned to oral treatment with combined B vitamins (folate, 10 mg; vitamin B-12, 200 microg; vitamin B-6, 20 mg/day), L-arginine (24 g/day) or placebo, for 8 weeks in a double-blind fashion. FDD was determined by high-resolution ultrasound in the radial artery. RESULTS: Vitamin B supplementation significantly lowered plasma homocyst(e)ine concentration from 15.8+/-1.8 to 8.7+/-1.1 micromol/l (P<0.01). However, B vitamins had no significant effect on FDD (baseline, 7.8+/-0.7%, B vitamins, 8.3+/-0.9%, placebo 8.9+/-0.7%; P=n.s.). In contrast, L-arginine treatment did not affect homocyst(e)ine levels, but significantly improved FDD (10.2+/-0.2%), probably by antagonizing the impact of elevated ADMA concentration (3.8+/-0.3 micromol/l) and reducing the oxidative stress by lowering urinary 8-iso-prostaglandin F(2alpha) (baseline, 76.3+/-7.1 vs. 62.7+/-8.3 pmol/mmol creatinine after 8 weeks). CONCLUSIONS: Oral supplementation with combined B vitamins during 8 weeks does not improve endothelium-dependent vasodilation in PAOD patients with hyperhomocyst(e)inemia, whereas L-arginine significantly improved endothelial function in these patients. Thus, accumulation of ADMA and increased oxidative stress may underlie endothelial dysfunction under hyperhomocyst(e)inemic conditions. These findings may have importance for evaluation of homocyst(e)ine-lowering therapy.