High plasma guanidinoacetate-to-homoarginine ratio is associated with high all-cause and cardiovascular mortality rate in adult renal transplant recipients

l-Arginine:glycine amidinotransferase (AGAT) is the main producer of the creatine precursor, guanidinoacetate (GAA), and l-homoarginine (hArg). We and others previously reported lower levels of circulating and urinary hArg in renal transplant recipients (RTR) compared to healthy subjects. In adults, hArg emerged as a novel risk factor for renal and cardiovascular adverse outcome. Urinary GAA was found to be lower in children and adolescents with kidney transplants compared to healthy controls. Whether GAA is also a risk factor in the renal and cardiovascular systems of adults, is not yet known. In the present study, we aimed to investigate the significance of circulating GAA and the GAA-to-hArg molar ratio (GAA/hArg) in adult RTR. We hypothesized that GAA/hArg represents a measure of the balanced state of the AGAT activity in the kidneys, and would prospectively allow assessing a potential association between GAA/hArg and long-term outcome in RTR. The median follow-up period was 5.4 years. Confounders and potential mediators of GAA/hArg associations were evaluated with multivariate linear regression analyses, and the association with all-cause and cardiovascular mortality or death-censored graft loss was studied with Cox regression analyses. The study cohort consisted of 686 stable RTR and 140 healthy kidney donors. Median plasma GAA concentration was significantly lower in the RTR compared to the kidney donors before kidney donation: 2.19 [1.77-2.70] mu M vs. 2.78 [2.89-3.35] mu M (P <0.001). In cross-sectional multivariable analyses in RTR, HDL cholesterol showed the strongest association with GAA/hArg. In prospective analyses in RTR, GAA/hArg was associated with a higher risk for all-cause mortality (hazard ratio (HR): 1.35 [95% CI 1.19-1.53]) and cardiovascular mortality (HR: 1.46 [95% CI 1.24-1.73]), independent of potential confounders. GAA but not GAA/hArg was associated with death-censored graft loss in crude survival and Cox regression analyses. The association of GAA and death-censored graft loss was lost after adjustment for eGFR. Our study suggests that in the kidneys of RTR, the AGAT-catalyzed biosynthesis of GAA is decreased. That high GAA/hArg is associated with a higher risk for all-cause and cardiovascular mortality may suggest that low plasma hArg is a stronger contributor to these adverse outcomes in RTR than GAA

Asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and homoarginine (hArg): the ADMA, SDMA and hArg paradoxes

Abstract NG-Methylation of l-arginine (Arg) residues in certain proteins by protein arginine methyltransferases and subsequent proteolysis yields NG-monomethyl-l-arginine (MMA), NG,NG-dimethyl-l-arginine (asymmetric dimethylarginine, ADMA) and NG,N′G-dimethyl-l-arginine (symmetric dimethylarginine, SDMA). Biological MMA, ADMA and SDMA occur as free acids in the nM-range and as residues of proteins of largely unknown quantity. Arginine:glycine amidinotransferase (AGAT) catalyzes the synthesis of L-homoarginine (hArg) from free Arg and l-lysine. Biological hArg is considered to occur exclusively as free acid in the lower µM-range. Nitric oxide synthase (NOS) catalyzes the conversion of Arg (high affinity) and hArg (low affinity) to nitric oxide (NO) which is a pleiotropic signaling molecule. MMA, ADMA and SDMA are inhibitors (MMA > ADMA ≫ SDMA) of NOS activity. Slightly elevated ADMA and SDMA concentrations and slightly reduced hArg concentrations in the circulation are associated with many diseases including diabetes mellitus. Yet, this is paradox: (1) free ADMA and SDMA are weak inhibitors of endothelial NOS (eNOS) which is primarily responsible for NO-related effects in the cardiovascular system, with free hArg being a poor substrate for eNOS; (2) free ADMA, SDMA and hArg are not associated with oxidative stress which is considered to induce NO-related endothelial dysfunction. This ADMA/SDMA/hArg paradox may be solved by the assumption that not the free acids but their precursor proteins exert biological effects in the vasculature, with hArg antagonizing the effects of NG-methylated proteins

GC-MS and GC-MS/MS measurement of ibuprofen in 10-μL aliquots of human plasma and mice serum using [α-methylo-(2)H3]ibuprofen after ethyl acetate extraction and pentafluorobenzyl bromide derivatization: Discovery of a collision energy-dependent H/D isotope effect and pharmacokinetic application to inhaled ibuprofen-arginine in mice.

GC-MS and GC-MS/MS methods were developed and validated for the quantitative determination of ibuprofen (d0-ibuprofen), a non-steroidal anti-inflammatory drug (NSAID), in human plasma using α-methyl-(2)H3-4-(isobutyl)phenylacetic acid (d3-ibuprofen) as internal standard. Plasma (10μL) was diluted with acetate buffer (80μL, 1M, pH 4.9) and d0- and d3-ibuprofen were extracted with ethyl acetate (2×500μL). After solvent evaporation d0- and d3-ibuprofen were derivatized in anhydrous acetonitrile by using pentafluorobenzyl (PFB) bromide and N,N-diisopropylethylamine as the base catalyst. Under electron-capture negative-ion chemical ionization (ECNICI), the PFB esters of d0- and d3-ibuprofen readily ionize to form their carboxylate anions [M-PFB](-) at m/z 205 and m/z 208, respectively. Collision-induced dissociation (CID) of m/z 205 and m/z 208 resulted in the formation of the anions at m/z 161 and m/z 164, respectively, due to neutral loss of CO2 (44 Da). A collision energy-dependent H/D isotope effect was observed, which involves abstraction/elimination of H(-) from d0-ibuprofen and D(-) from d3-ibuprofen and is minimum at a CE value of 5eV. Quantitative GC-MS determination was performed by selected-ion monitoring of m/z 205 and m/z 208. Quantitative GC-MS/MS determination was performed by selected-reaction monitoring of the mass transitions m/z 205 to m/z 161 for d0-ibuprofen and m/z 208 to m/z 164 for d3-ibuprofen. In a therapeutically relevant concentration range (0-1000μM) d0-ibuprofen added to human plasma was determined with accuracy (recovery, %) and imprecision (relative standard deviation, %) ranging between 93.7 and 110%, and between 0.8 and 4.9%, respectively. GC-MS (y) and GC-MS/MS (x) yielded almost identical results (y=4.00+0.988x, r(2)=0.9991). In incubation mixtures of arachidonic acid (10μM), d3-ibuprofen (10μM) or d0-ibuprofen (10μM) with ovine cyclooxygenase (COX) isoforms 1 and 2, the concentration of d3-ibuprofen and d0-ibuprofen did not change upon incubation at 37°C up to 60min. The trough pharmacokinetics of an inhaled arginine-containing ibuprofen preparation in mice was studied after once-daily treatment (0.0, 0.07, 0.4 and 2.5mg/kg body weight) for three days. A linear relationship between ibuprofen concentration in serum (10μL) and administered dose 24h after the last drug administration was observed

Low plasma homoarginine concentration is associated with high rates of all-cause mortality in renal transplant recipients

In renal transplant recipients (RTR), we recently found that low urinary excretion of homoarginine (hArg) is associated with mortality and graft failure. However, it is not known whether such prospective associations also hold true for plasma concentrations of hArg. In the present study, we therefore determined plasma concentrations of hArg in the same cohort, i.e. in 687 RTR (functioning graft ≥1 year), and in 140 healthy donors, before and after kidney donation. Plasma hArg concentrations were significantly lower in RTR compared to healthy controls [1.24 (0.95-1.63) µM vs. 1.58 (1.31-2.03) µM, P &lt; 0.001], and kidney donation resulted in a decrease in plasma hArg concentration to 1.41 (1.10-1.81) µM (P &lt; 0.001). In RTR, multivariable linear regression analysis revealed BMI (β = 0.124), heart rate (β = -0.091), pre-emptive transplantation (β = 0.078), antidiabetic medication (β = -0.091), eGFR (β = 0.272), plasma PTH (β = -0.098), uric acid (β = 0.137), alkaline phosphatase (β = -0.100), HDL (β = -0.111), NT-pro-BNP (β = -0.166), and urinary urea excretion (β = 0.139) as main determinants of plasma hArg (all P &lt; 0.05). In RTR, plasma hArg concentration was inversely associated with all-cause [hazard ratio (HR) 0.59 (95% CI 0.50-0.70), P &lt; 0.001] and cardiovascular mortality [HR 0.50 (0.39-0.66), P &lt; 0.001], both expressed per standard deviation change in log-transformed hArg, independent of potential confounders. To conclude, our results suggest that the kidney is a major hArg production site and an important modulator of hArg homeostasis in the renal and cardiovascular systems. Moreover, low plasma hArg is independently associated with increased risk of cardiovascular mortality in RTR, which corroborates the cardiovascular importance of preserving kidney function after transplantation.</p

High urinary homoarginine excretion is associated with low rates of all-cause mortality and graft failure in renal transplant recipients

Renal transplant recipients (RTR) have an increased cardiovascular risk profile. Low levels of circulating homoarginine (hArg) are a novel risk factor for mortality and the progression of atherosclerosis. The kidney is known as a major source of hArg, suggesting that urinary excretion of hArg (UhArg) might be associated with mortality and graft failure in RTR. hArg was quantified by mass spectrometry in 24-h urine samples of 704 RTR (functioning graft a parts per thousand yen1 year) and 103 healthy subjects. UhArg determinants were identified with multivariable linear regression models. Associations of UhArg with all-cause mortality and graft failure were assessed using multivariable Cox regression analyses. UhArg excretion was significantly lower in RTR compared to healthy controls [1.62 (1.09-2.61) vs. 2.46 (1.65-4.06) A mu mol/24 h, P <0.001]. In multivariable linear regression models, body surface area, diastolic blood pressure, eGFR, pre-emptive transplantation, serum albumin, albuminuria, urinary excretion of urea and uric acid and use of sirolimus were positively associated with UhArg, while donor age and serum phosphate were inversely associated (model R (2) = 0.43). During follow-up for 3.1 (2.7-3.9) years, 83 (12 %) patients died and 45 (7 %) developed graft failure. UhArg was inversely associated with all-cause mortality [hazard risk (HR) 0.52 (95 % CI 0.40-0.66), P <0.001] and graft failure [HR 0.58 (0.42-0.81), P = 0.001]. These associations remained independent of potential confounders. High UhArg levels are associated with reduced all-cause mortality and graft failure in RTR. Kidney-derived hArg is likely to be of particular importance for proper maintenance of cardiovascular and renal systems

Effect of renal function on homeostasis of asymmetric dimethylarginine (ADMA): studies in donors and recipients of renal transplants

Asymmetric dimethylarginine (ADMA) is a methylated form of arginine and an endogenous nitric oxide synthase inhibitor. Renal function decline is associated with increase of plasma ADMA in chronic kidney disease populations. It is yet unknown how isolated renal function impairment affects ADMA homeostasis in healthy humans. Here, we measured plasma concentrations and urinary excretion of ADMA using GC-MS/MS in 130 living kidney donors before and at 1.6 (1.6-1.9) months after donation. We additionally analyzed 201 stable renal transplant recipients (RTR) that were included > 1 year after transplantation, as a model for kidney disease in the context of single kidney state. We measured true glomerular filtration rate (mGFR) using 125I-iothalamate. To study enzymatic metabolism of ADMA, we also measured L-citrulline as primary metabolite. Mean age was 52 ± 10 years in donors and 54 ± 12 years in RTR. Renal function was significantly reduced from pre- to post-donation (mGFR: 104 ± 17 vs. 66 ± 10 ml/min per 1.73 m2 BSA, - 36 ± 7%, P < 0.001). Urinary ADMA excretion strongly and significantly decreased from pre- to post-donation (60.6 ± 16.0 vs. 40.5 ± 11.5 µmol/24 h, - 31.5 ± 21.5%, P < 0.001), while plasma ADMA increased only slightly (0.53 ± 0.08 vs. 0.58 ± 0.09 µM, 11.1 ± 20.1%, P < 0.001). Compared to donors post-donation, RTR had significantly worse renal function (mGFR: 49 ± 18 ml/min/1.73 m2, - 25 ± 2%, P < 0.001) and lower urinary ADMA excretion (30.9 ± 12.4 µmol/24 h, - 23.9 ± 3.4%, P < 0.001). Plasma ADMA in RTR (0.60 ± 0.11 µM) did not significantly differ from donors post-donation (2.9 ± 1.9%, P = 0.13). Plasma citrulline was inversely associated with mGFR (st. β: - 0.23, P < 0.001), consistent with increased ADMA metabolism to citrulline with lower GFR. In both groups, the response of urinary ADMA excretion to renal function loss was much larger than that of plasma ADMA. As citrulline was associated with GFR, our data indicate that with renal function impairment, a decrease in urinary ADMA excretion does not lead to a corresponding increase in plasma ADMA, likely due to enhanced metabolism, thus allowing for lower renal excretion of ADMA