GC-MS analysis of 4-hydroxyproline: elevated proline hydroxylation in metformin-associated lactic acidosis and metformin-treated Becker muscular dystrophy patients.

Metformin (N,N-dimethylbiguanide), an inhibitor of gluconeogenesis and insulin sensitizer, is widely used for the treatment of type 2 diabetes. In some patients with renal insufficiency, metformin can accumulate and cause lactic acidosis, known as metformin-associated lactic acidosis (MALA, defined as lactate ≥ 5 mM, pH < 7.35, and metformin concentration > 38.7 µM). Here, we report on the post-translational modification (PTM) of proline (Pro) to 4-hydroxyproline (OH-Pro) in metformin-associated lactic acidosis and in metformin-treated patients with Becker muscular dystrophy (BMD). Pro and OH-Pro were measured simultaneously by gas chromatography-mass spectrometry before, during, and after renal replacement therapy in a patient admitted to the intensive care unit (ICU) because of MALA. At admission to the ICU, plasma metformin concentration was 175 µM, with a corresponding lactate concentration of 20 mM and a blood pH of 7.1. Throughout ICU admission, the Pro concentration was lower compared to healthy controls. Renal excretion of OH-Pro was initially high and decreased over time. Moreover, during the first 12 h of ICU admission, OH-Pro seems to be renally secreted while thereafter, it was reabsorbed. Our results suggest that MALA is associated with hyper-hydroxyprolinuria due to elevated PTM of Pro to OH-Pro by prolyl-hydroxylase and/or inhibition of OH-Pro metabolism in the kidneys. In BMD patients, metformin, at the therapeutic dose of 3 × 500 mg per day for 6 weeks, increased the urinary excretion of OH-Pro suggesting elevation of Pro hydroxylation to OH-Pro. Our study suggests that metformin induces specifically the expression/activity of prolyl-hydroxylase in metformin intoxication and BMD.

GC-MS Studies on the Conversion and Derivatization of γ-Glutamyl Peptides to Pyroglutamate (5-Oxo-Proline) Methyl Ester Pentafluoropropione Amide Derivatives.

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, γ-Glu-Cys-Gly) is the most abundant intra-cellular dicarboxylic tripeptide with multiple physiological roles. In biological samples, glutathione exists in its reduced form GSH and in two stable oxidized forms, i.e., in its symmetric disulfide form GSSG and as S-glutathionyl residue in proteins. S-Glutathionylation is a post-translational modification, which is involved in several pathophysiological processes, including oxidative stress. The GSH-to-GSSG molar ratio is widely used as a measure of oxidative stress. γ-Glutamyl is the most characteristic structural moiety of GSH. We performed gas chromatography-mass spectrometry (GC-MS) studies for the development of a highly specific qualitative and quantitative method for γ-glutamyl peptides. We discovered intra-molecular conversion of GSH, GSSG, γ-Glu-Cys and of ophthalmic acid (OPH; γ-glutamyl-α-amino-n-butyryl-glycine) to pyroglutamate (pGlu; 5-oxo-proline, also known as pidolic acid) during their derivatization with 2 M HCl/CH3OH (60 min, 80 °C). For GC-MS analysis, the methyl esters (Me) were further derivatized with pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v; 30 min, 65 °C) to their PFP derivatives. At longer reaction times, pGlu is hydrolyzed to Glu. Internal standards were prepared by derivatizing GSH, GSSG, γ-Glu-Cys and OPH in 2 M HCl/CD3OD. Quantification of the Me-PFP derivative of pGlu was performed in the electron-capture negative-ion chemical ionization (ECNICI) mode by selected-ion monitoring (SIM) of the mass-to-charge (m/z) ions 269 for unlabeled pGlu (d0Me-PFP-pGlu) and m/z 272 for the in situ prepared deuterium-labeled pGlu (d3Me-PFP-pGlu). Although not inherent to the analysis of small peptides, the present GC-MS method is useful to study several biochemical aspects of GSH. Using pentafluorobenzyl bromide (PFB-Br) as the derivatization reagent, we found that synthetic pGlu is converted in aqueous acetone (60 min, 50 °C) into its pentafluorobenzyl (PFB) ester (PFB-pGlu). This derivatization procedure is useful for the GC-MS analysis of free pGlu in the ECNICI mode. Quantitative analysis of PFB-pGlu by GC-MS requires the use of stable-isotope labeled analogs of pGlu as an internal standard.

Unusual Derivatization of Methylmalonic Acid with Pentafluorobenzyl Bromide to a Tripentafluorobenzyl Derivative and Its Stable-Isotope Dilution GC-MS Measurement in Human Urine.

Methylmalonic acid (MMA) is a very short dicarboxylic acid (methylpropanedioic acid; CH3CH(COOH)2; pKa1, 3.07; pKa2, 5.76) associated with vitamin B12 deficiency and many other patho-physiological conditions. In this work, we investigated several carboxylic groups-specific derivatization reactions and tested their utility for the quantitative analysis of MMA in human urine and plasma by gas chromatography-mass spectrometry (GC-MS). The most useful derivatization procedure was the reaction of unlabeled MMA (d0-MMA) and trideutero-methyl malonic acid (d3-MMA) with 2,3,4,5,6-pentafluorobenzyl bromide (PFB-Br) in acetone. By heating at 80 °C for 60 min, we observed the formation of the dipentafluorobenzyl (PFB) ester of MMA (CH3CH(COOPFB)2). In the presence of N,N-diisopropylamine, heating at 80 °C for 60 min resulted in the formation of a tripentafluorobenzyl derivative of MMA, i.e., CH3CPFB(COOPFB)2). The retention time was 5.6 min for CH3CH(COOPFB)2 and 7.3 min for CH3CPFB(COOPFB)2). The most intense ions in the negative-ion chemical ionization (NICI) GC-MS spectra of CH3CH(COOPFB)2 were mass-to-charge (m/z) 233 for d0-MMA and m/z 236 for d3-MMA. The most intense ions in the NICI GC-MS spectra of CH3CPFB(COOPFB)2 were mass-to-charge (m/z) 349 for d0-MMA and m/z 352 for d3-MMA. These results indicate that the H at C atom at position 2 is C-H acidic and is alkylated by PFB-Br only in the presence of the base N,N-diisopropylamine. Method validation and quantitative analyses in human urine and plasma were performed by selected ion monitoring (SIM) of m/z 349 for d0-MMA and m/z 352 for the internal standard d3-MMA in the NICI mode. We used the method to measure the urinary excretion rates of MMA in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). The creatinine-corrected excretion rates of MMA were 1.50 [0.85-2.52] µmol/mmol in the black boys and 1.34 [1.02-2.18] µmol/mmol in the white boys (P = 0.85; Mann-Whitney). The derivatization procedure is highly specific and sensitive for MMA and allows its accurate and precise measurement in 10-µl of human urine by GC-MS.

Stable-Isotope Dilution GC-MS Measurement of Metformin in Human Serum and Urine after Derivatization with Pentafluoropropionic Anhydride and Its Application in Becker Muscular Dystrophy Patients Administered with Metformin, l-Citrulline, or Their Combination.

Metformin (N,N-dimethylguanylguanidine) is one of the most prescribed drugs with pleiotropic, exerted in part by not fully elucidated mechanisms of action. We developed and validated a gas chromatography-mass spectrometry (GC-MS) method for the quantitative analysis of metformin (metformin-d0) in 10-µL aliquots of human serum and urine using N,N-[dimethylo-2H6]guanylguanidine (metformin-d6) as the internal standard. The method involves evaporation of the samples to dryness, derivatization with pentafluoropropionic (PFP) anhydride in ethyl acetate (30 min, 65 °C), and extraction into toluene. The negative-ion chemical ionization GC-MS spectra of the PFP derivatives contain a single intense ion with mass-to-charge (m/z) ratios of m/z 383 for metformin-d0 and m/z 389 for metformin-d6. Our results suggest that all amine/imine groups of metformin-d0 and metformin-d6 are converted to their N,N,N-tripentafluoropropionyl derivatives, which cyclize to form a symmetric triazine derivative, of which the non-ring amine group is amidated. Quantification was performed by selected-ion monitoring (SIM) of m/z 383 and m/z 389. Upon validation, the method was applied to determine serum and urine metformin concentrations in 19 patients with Becker muscular dystrophy (BMD). Serum and urine samples were collected at baseline (Visit I), after six weeks of supplementation (Visit II) with metformin (3 × 500 mg/d; metformin group; n = 10) or l-citrulline (3 × 1500 mg/d; citrulline group; n = 9) followed by a six-week supplementation with 3 × 500 mg/d of metformin plus 3 × 1500 mg/d l-citrulline. At Visit I, the metformin concentration in the serum and urine was very low in both groups. The metformin concentrations in the serum and urine of the patients who first took metformin (MET group) were higher at Visit II and Visit III. The metformin concentration in the serum and urine samples of the patients who first took l-citrulline (CITR group) were higher at Visit III. The serum and urine concentrations of metformin were insignificantly lower in the CITR group at Visit III. The mean fractional excretion (FE) rate of metformin was 307% (Visit II) and 322% (Visit III) in the MET group, and 290% in the CITR group (Visit III). This observation suggests the accumulation of metformin in the kidney and its secretion in the urine. The GC-MS is suitable to measure reliably circulating and excretory metformin in clinical settings.

Advanced Glycation End-Products (AGEs) of Lysine and Effects of Anti-TCR/Anti-TNF-α Antibody-Based Therapy in the LEW.1AR1-iddm Rat, an Animal Model of Human Type 1 Diabetes.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D). Previously, we have shown that combination with anti-TCR/anti-TNF-α antibody-based therapy re-established normoglycemia and increased proteinic arginine-dimethylation in the spleen, yet not in the pancreas. High blood glucose is often associated with elevated formation of advanced glycation end-products (AGEs) which act via their receptor (RAGE). Both anti-TCR and anti-TNF-α are inhibitors of RAGE. The aim of the present work was to investigate potential biochemical changes of anti-TCR/anti-TNF-α therapy in the LEW.1AR1-iddm rat. We determined by stable-isotope dilution gas chromatography-mass spectrometry (GC-MS) the content of free and proteinic AGEs and the Nε-monomethylation of lysine (Lys) residues in proteins of pancreas, kidney, liver, spleen and lymph nodes of normoglycemic control (ngCo, n = 6), acute diabetic (acT1D, n = 6), chronic diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Analyzed biomarkers included Lys and its metabolites Nε-carboxymethyl lysine (CML), furosine and Nε-monomethyl lysine (MML). Other amino acids were also determined. Statistical methods including ANOVA, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to evaluate the effects. Most statistical differences between the study groups were observed for spleen, pancreas and kidney, with liver and lymph nodes showing no such differences. In the pancreas, the groups differed with respect to proteinic furosine (p = 0.0289) and free CML (p = 0.0023). In the kidneys, the groups differed with respect to proteinic furosine (p = 0.0076) and CML (p = 0.0270). In the spleen, group differences were found for proteinic furosine (p = 0.0114) and free furosine (p = 0.0368), as well as for proteinic CML (p = 0.0502) and proteinic MML (p = 0.0191). The acT1D rats had lower furosine, CML and MML levels in the spleen than the rats in all other groups. This observation corresponds to the lower citrullination levels previously measured in these rats. PCA revealed diametric associations between PC1 and PC2 for spleen (r = -0.8271, p < 0.0001) compared to pancreas (r = 0.5805, p = 0.0073) and kidney (r = 0.8692, p < 0.0001). These findings underscore the importance of the spleen in this animal model of human T1D. OPLS-DA showed that in total sixteen amino acids differed in the experimental groups.

Measurement of the tripeptides glutathione and ophthalmic acid by gas chromatography-mass spectrometry.

Glutathione (GSH, γ-glutamyl-cysteinyl-glycine) is the most abundant intracellular dicarboxylic tripeptide with multiple physiological roles. GSH and its symmetric disulfide GSSG co-exist in biological samples. The GSH-to-GSSG molar ratio is high within cells and low in extracellular compartments. It is widely used as a biomarker of oxidative stress. Here, we report on a novel two-step derivatization procedure which allows for the specific quantitative measurement of GSH by gas chromatography-mass spectrometry (GC-MS) in the electron-capture negative-ion chemical ionization mode. The method is based on the derivatization of GSH first with pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C) and subsequently with 2 M HCl/CH3OH (30 min, 80 °C). Esterification in 2 M HCl/CD3OD is used for the in situ synthesis of deuterium-labelled GSH for use as internal standard. Quantification is performed by selected-ion monitoring of m/z 557 for GSH and m/z 560 for the internal standard (1 mM). Linear regression analysis between the peak area ratio of m/z 557 to m/z 560 (y) and the GSH concentration (x; range, 0-1 mM) resulted in the regression equations y = 0.02 + 0.62x (r2 = 0.976) in the absence and y = 0.02 + 0.63x (r2 = 0.997) in the presence of GSSG (range, 0-1 mM), underlying the linearity of the method for GSH and suggesting lack of interference by GSSG. γ-Glu-Cys and Cys-Gly also did not interfere. The GC-MS method demonstrated that in human hemolysate, nitrite at toxicological concentrations (range, 0-15 mM) depletes erythrocytic GSH. For the GC-MS analysis of the GSH tripeptide analogue ophthalmic acid, which lacks a Cys moiety, the derivatization order was HCl/CH3OH followed by pentafluoropropionic anhydride in ethyl acetate.

Development, validation of a GC-MS method for the simultaneous measurement of amino acids, their PTM metabolites and AGEs in human urine, and application to the bi-ethnic ASOS study with special emphasis to lysine.

A gas chromatography-mass spectrometry (GC-MS) method was developed and validated in relevant concentration ranges for the simultaneous measurement of L-lysine (Lys, L) and its Nε- and Nα-methylated (M), Nε- and Nα-acetylated (Ac), Nε-carboxymethylated (CM) and Nε-carboxyethylated (CE) metabolites in human urine. Analyzed Lys metabolites were the post-translational modification (PTM) products Nε-mono-, di- and trimethyllsine, Nε-MML, Nε-DML, Nε-TML, respectively, Nα-ML, Nε-AcL, Nα-AcL, and its advanced glycation end-products (AGEs) Nε-CML, Nε-CM-[2,4,4-2H3]Lys (d3-CML), Nε-CEL and furosine. AGEs of arginine (Arg) and cysteine (Cys) were also analyzed. De novo synthesized trideutero-methyl esters (R-COOCD3) from unlabelled amino acids and derivatives were used as internal standards. Native urine samples (10 µL aliquots) were evaporated to dryness under a stream of nitrogen. Analytes were esterified using 2 M HCl in methanol (60 min, 80 °C) and subsequently amidated by pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C). The generated methyl ester-pentafluoropropionyl (Me-PFP) derivatives were reconstituted in borate buffer and extracted immediately with toluene. GC-MS analyses were performed by split-less injection of 1-µL aliquots, oven-programmed separation and negative-ion chemical ionization (NICI). Mass spectra were generated in the scan mode (range, m/z 50-1000). Quantification was performed in the selected-ion monitoring (SIM) mode using a dwell time of 50 or 100 ms for each ion. The GC-MS method was suitable for the measurement of Lys and all of its metabolites, except for the quaternary ammonium cation Nε-TML. The Me-PFP derivatives of Lys, Arg and Cys and its metabolites eluted in the retention time window of 9 to 14 min. The derivatization of Nε-CML, d3-CML and Nε-CEL was accompanied by partial Nε-decarboxylation and formation of the Me-PFP Lys derivative. The lowest derivatization yield was observed for Nε-DML, indicating a major role of the Nε-DML group in Lys derivatization. The GC-MS method enables precise (relative standard deviation, RSD < 20%) and accurate (bias, < ± 20%) simultaneous measurement of 33 analytes in human urine in relevant concentration ranges. We used the method to measure the urinary excretion rates of Lys and its PTM metabolites and AGEs in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). No remarkable differences were found indicating no ethnic-related differences in PTM metabolites and AGEs except for Nε-monomethyllysine and S-(2-carboxymethylcysteine).

Urinary excretion of amino acids and their advanced glycation end-products (AGEs) in adult kidney transplant recipients with emphasis on lysine: furosine excretion is associated with cardiovascular and all-cause mortality.

Arginine (Arg) and lysine (Lys) moieties of proteins undergo various post-translational modifications (PTM) including enzymatic NG- and Nε-methylation and non-enzymatic NG- and Nε-glycation. In a large cohort of stable kidney transplant recipients (KTR, n = 686), high plasma and low urinary concentrations of asymmetric dimethylarginine (ADMA), an abundant PTM metabolite of Arg, were associated with cardiovascular and all-cause mortality. Thus, the prediction of the same biomarker regarding mortality may depend on the biological sample. In another large cohort of stable KTR (n = 555), higher plasma concentrations of Nε-carboxymethyl-lysine (CML) and Nε-carboxyethyl-lysine (CEL), two advanced glycation end-products (AGEs) of Lys, were associated with higher cardiovascular mortality. Yet, the associations of urinary AGEs with mortality are unknown. In the present study, we measured 24 h urinary excretion of Lys, CML, and furosine in 630 KTR and 41 healthy kidney donors before and after donation. Our result indicate that lower urinary CML and lower furosine excretion rates are associated with higher mortality in KTR, thus resembling the associations of ADMA. Lower furosine excretion rates were also associated with higher cardiovascular mortality. The 24 h urinary excretion rate of amino acids and their metabolites decreased post-donation (varying as little as - 24% for CEL, and as much as - 62% for ADMA). For most amino acids, the excretion rate was lower in KTR than in donors pre-donation [except for S-(1-carboxyethyl)-L-cysteine (CEC) and NG-carboxyethylarginine (CEA)]. Simultaneous GC-MS measurement of free amino acids, their PTM metabolites and AGEs in urine is a non-invasive approach in kidney transplantation.

Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC-MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene Extracts.

Analysis of amino acids by gas chromatography-mass spectrometry (GC-MS) requires at least one derivatization step to enable solubility in GC-MS-compatible water-immiscible organic solvents such as toluene, to make them volatile to introduce into the gas chromatograph and thermally stable enough for separation in the GC column and introduction into the ion-source, and finally to increase their ionization by increasing their electronegativity using F-rich reagents. In this work we investigated the long-term stability of the methyl esters pentafluoropropionic (Me-PFP) derivatives of 21 urinary amino acids prepared by a two-step derivatization procedure and extraction by toluene. In situ prepared trideuteromethyl ester pentafluoropropionic derivatives were used as internal standards. GC-MS analysis (injection of 1 µL aliquots and quantification by selected-ion monitoring of specific mass fragments) was performed on days 1, 2, 8, and 15. Measured peak areas and calculated peak area ratios were used to evaluate the stability of the derivatives of endogenous amino acids and their internal standards, as well as the precision and the accuracy of the method. All analyses were performed under routine conditions. Me-PFP derivatives of endogenous amino acids and their stable-isotope labelled analogs were stable in toluene for 14 days. The peak area values of the derivatives of most amino acids and their internal standards were slightly higher on days 8 and 15 compared to days 1 and 2, yet the peak area ratio values of endogenous amino acids to their internal standards did not change. Our study indicates that Me-PFP derivatives of amino acids from human urine samples can easily be prepared, are stable at least for 14 days in the extraction solvent toluene, and allow for precise and accurate quantitative measurements by GC-MS using in situ prepared deuterium-labelled methyl ester as internal standard.

Diminished Systemic Amino Acids Metabolome and Lipid Peroxidation in Ureteropelvic Junction Obstruction (UPJO) Infants Requiring Surgery.

Congenital anomalies of the urinary tract, and particularly of obstructive nephropathy such as ureteropelvic junction obstruction (UPJO) in infants, can later lead to chronic kidney disease and hypertension. Fundamental questions regarding underlying mechanisms remain unanswered. The aim of the present study was to quantitate the systemic amino acids metabolome in 21 UPJO infants requiring surgery (Group A) and 21 UPJO infants under conservative treatment (Group B). Nineteen healthy age-matched infants served as controls (Group C). Serum amino acids involved in several pathways and representative metabolites, including the L-arginine-derived nitric oxide (NO) metabolites nitrite and nitrate and the lipid peroxidation biomarker malondialdehyde (MDA) were measured by gas chromatography-mass spectrometry (GC-MS) methods using their stable-isotope labeled analogs as internal standards after derivatization to their methyl esters N-pentafluoropropionic amides (amino acids) and to their pentafluorobenzyl derivatives (nitrite, nitrate, MDA). The concentrations of the majority of the biomarkers were found to be lower in Group A compared to Group B. Statistical analysis revealed clear differentiation between the examined study groups. Univariate statistical analysis highlighted serum homoarginine (q = 0.006), asymmetric dimethylarginine (q = 0.05) and malondialdehyde (q = 0.022) as potential biomarkers for UPJO infants requiring surgery. Group A also differed from Group B with respect to the diameter of the preoperative anterior-posterior renal pelvis (AP) as well as regarding the number and extent of inverse correlations between AP and the serum concentrations of the biomarkers. In Group A, but not in Group B, the AP diameter strongly correlated with hydroxy-proline (r = -0.746, p = 0.0002) and MDA (r = -0.754, p = 0.002). Our results indicate a diminished amino acids metabolome in the serum of UPJO infants requiring surgery comparing to a conservative group.

GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N5-Carboxy-ornithine and N6-Carboxy-lysine.

Derivatization of amino acids by 2 M HCl/CH3OH (60 min, 80 °C) followed by derivatization of the intermediate methyl esters with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) is a useful two-step derivatization procedure (procedure A) for their quantitative measurement in biological samples by gas chromatography-mass spectrometry (GC-MS) as methyl ester pentafluoropropionic (PFP) derivatives, (Me)m-(PFP)n. This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (d3Me)m-(PFP)n, from synthetic amino acids and 2 M HCl/CD3OD for use as internal standards. However, procedure A converts citrulline (Cit) to ornithine (Orn) and homocitrulline (hCit) to lysine (Lys) due to the instability of their carbamide groups under the acidic conditions of the esterification step. In the present study, we investigated whether reversing the order of the two-step derivatization may allow discrimination and simultaneous analysis of these amino acids. Pentafluoropropionylation (30 min, 65 °C) and subsequent methyl esterification (30 min, 80 °C), i.e., procedure B, of Cit resulted in the formation of six open and cyclic reaction products. The most abundant product is likely to be N5-Carboxy-Orn. The second most abundant product was confirmed to be Orn. The most abundant reaction product of hCit was confirmed to be Lys, with the minor reaction product likely being N6-Carboxy-Lys. Mechanisms are proposed for the formation of the reaction products of Cit and hCit via procedure B. It is assumed that at the first derivatization step, amino acids form (N,O)-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP)4 and hCit-(PFP)4 are esterified on their C1-Carboxylic groups and on their activated Nureido groups. Procedure B also allows in situ preparation of (d3Me)m-(PFP)n from synthetic amino acids for use as internal standards. It is demonstrated that the derivatization procedure B enables discrimination between Cit and Orn, and between hCit and Lys. The utility of procedure B to measure simultaneously these amino acids in biological samples such as plasma and urine remains to be demonstrated. Further work is required to optimize the derivatization conditions of procedure B for biological amino acids.

Whole-body arginine dimethylation is associated with all-cause mortality in adult renal transplant recipients.

Arginine residues in proteins can be singly or doubly methylated post-translationally. Proteolysis of arginine-methylated proteins provides monomethyl arginine, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). ADMA and SDMA are considered cardiovascular risk factors, with the underlying mechanisms being not yet fully understood. SDMA lacks appreciable metabolism and is almost completely eliminated by the kidney, whereas ADMA is extensively metabolized to dimethylamine (DMA), with a minor ADMA fraction of about 10% being excreted unchanged in the urine. Urinary DMA and ADMA are useful measures of whole-body asymmetric arginine-dimethylation, while urinary SDMA serves as a whole-body measure of symmetric arginine-dimethylation. In renal transplant recipients (RTR), we previously found that higher plasma ADMA concentrations and lower urinary ADMA and SDMA concentrations were associated with a higher risk of all-cause mortality. Yet, in this RTR collective, no data were available for urinary DMA. For the present study, we additionally measured the excretion rate of DMA in 24-h collected urine samples of the RTR and of healthy kidney donors in the cohort, with the aim to quantitate whole-body asymmetric (ADMA, DMA) and symmetric (SDMA) arginine-dimethylation. We found that lower DMA excretion rates were associated with higher all-cause mortality, yet not with cardiovascular mortality. In the healthy donors, kidney donation was associated with considerable decreases in ADMA (by - 39%, P < 0.0001) and SDMA (by - 21%, P < 0.0001) excretion rates, yet there was no significant change in DMA (by - 9%, P = 0.226) excretion rate. Our results suggest that protein-arginine dimethylation is altered in RTR compared to healthy kidney donors and that it is pronouncedly shifted from symmetric to asymmetric arginine-dimethylation, with whole-body protein-arginine dimethylation being almost unaffected.

Urinary albumin-to-creatinine ratio is inversely related to nitric oxide synthesis in young black adults: the African-PREDICT study.

Hypertension is common in black populations and is known to be associated with low nitric oxide (NO) bioavailability. We compared plasma and urinary NO-related markers and plasma creatine kinase (CK) levels between young healthy black and white adults along with the associations of these markers with the urinary albumin-to-creatinine ratio (uACR), which is a surrogate marker of endothelial and kidney function. We included 1105 participants (20-30 years). We measured the uACR, plasma CK, plasma and urinary arginine, homoarginine, asymmetric (ADMA) and symmetric dimethylarginine (SDMA), urinary ornithine/citrulline, nitrate and nitrite, and malondialdehyde (MDA). In addition, the urinary nitrate-to-nitrite ratio (UNOxR) was calculated and used as a measure of circulating NO bioavailability. The uACR was comparable between the groups, yet the black group had lower urinary nitrate (by -15%) and UNOxR values (by -18%) (both p ≤ 0.001), higher plasma (by +9.6%) and urinary (by +5.9%) arginine (both p ≤ 0.004), higher plasma (by +13%) and urinary (by +3.7%) ADMA (both p ≤ 0.033), and higher CK (by +9.5%) and MDA (by +19%) (both p < 0.001) compared with white adults. Plasma and urinary homoarginine were similar between the groups. In the multiple regression analysis, we confirmed the inverse associations of the uACR with both plasma (adj. R2 = 0.066; ß = -0.209; p = 0.005) and urinary (adj. R2 = 0.066; ß = -0.149; p = 0.010) homoarginine and with the UNOxR (adj. R2 = 0.060; ß = -0.122; p = 0.031) in the black group only. The overall less favorable NO profile and higher CK and MDA levels in the black cohort along with the adverse associations with the uACR may reflect the vulnerability of this cohort to the early development of hypertension.

Local and Systemic Alterations of the L-Arginine/Nitric Oxide Pathway in Sputum, Blood, and Urine of Pediatric Cystic Fibrosis Patients and Effects of Antibiotic Treatment.

Alterations in the L-arginine (Arg)/nitric oxide (NO) pathway have been reported in cystic fibrosis (CF; OMIM 219700) as the result of various factors including systemic and local inflammatory activity in the airways. The aim of the present study was to evaluate the Arg/NO metabolism in pediatric CF patients with special emphasis on lung impairment and antibiotic treatment. Seventy CF patients and 78 healthy controls were included in the study. CF patients (43% male, median age 11.8 years) showed moderately impaired lung functions (FEV1 90.5 ± 19.1% (mean ± SD); 21 (30%) had a chronic Pseudomonas aeruginosa (PSA) infection, and 24 (33%) had an acute exacerbation). Plasma, urinary, and sputum concentrations of the main Arg/NO metabolites, nitrate, nitrite, Arg, homoarginine (hArg), and asymmetric dimethylarginine (ADMA) were determined in pediatric CF patients and in healthy age-matched controls. Clinical parameters in CF patients included lung function and infection with PSA. Additionally, the Arg/NO pathway in sputum samples of five CF patients was analyzed before and after routine antibiotic therapy. CF patients with low fractionally exhaled NO (FENO) showed lower plasma Arg and nitrate concentrations. During acute exacerbation, sputum Arg and hArg levels were high and dropped after antibiotic treatment: Arg: pre-antibiotics: 4.14 nmol/25 mg sputum vs. post-antibiotics: 2.33 nmol/25 mg sputum, p = 0.008; hArg: pre-antibiotics: 0.042 nmol/25 mg sputum vs. post-antibiotics: 0.029 nmol/25 mg sputum, p = 0.035. The activated Arg/NO metabolism in stable CF patients may be a result of chronic inflammation. PSA infection did not play a major role regarding these differences. Exacerbation increased and antibiotic therapy decreased sputum Arg concentrations.

Upregulation of the TRPA1 Ion Channel in the Gastric Mucosa after Iodoacetamide-Induced Gastritis in Rats: A Potential New Therapeutic Target.

Acute gastritis is often untreatable by acid secretion-inhibiting drugs. Understanding the protective mechanisms including the role of Transient Receptor Potential Ankyrin1 (TRPA1) and Vanilloid1 (TRPV1) channels localized on capsaicin-sensitive afferents and non-neuronal structures might identify novel therapeutic approaches. Therefore, we characterized a translational gastritis model using iodoacetamide (IAA) and investigated TRPA1/V1 expressions. Wistar rats and CD1, C57Bl/6J mice were exposed to IAA-containing (0.05, 0.1, 0.2, 0.3, 0.5%) drinking water for 7 or 14 days. Body weight and water consumption were recorded daily. Macroscopic lesions were scored, qualitative histopathologic investigation was performed, TRPA1/V1 immunopositivity and mRNA expressions were measured. IAA induced a concentration-dependent weight loss and reduced water intake in both species. Hyperemia, submucosal edema, inflammatory infiltration and hemorrhagic erosions developed after 7 days, while ulcers after 14 days in rats. Trpa1 mRNA/protein expressions were upregulated at both timepoints. Meanwhile, TRPV1 immunopositivity was upregulated in the gastric corpus after 0.05% IAA ingestion, but downregulated after 0.2%, whereas Trpv1 mRNA did not change. Interestingly, no macroscopic/microscopic changes were observed in mice. These are the first data for the concentration- and duration-dependent changes in the IAA-induced gastritis in rats accompanied by TRPA1 upregulation, therefore, its therapeutic potential in gastritis should further be investigated.

Activated L-Arginine/Nitric Oxide Pathway in Pediatric Cystic Fibrosis and Its Association with Pancreatic Insufficiency, Liver Involvement and Nourishment: An Overview and New Results.

Cystic fibrosis (CF; OMIM 219700) is a rare genetic disorder caused by a chloride channel defect, resulting in lung disease, pancreas insufficiency and liver impairment. Altered L-arginine (Arg)/nitric oxide (NO) metabolism has been observed in CF patients' lungs and in connection with malnutrition. The aim of the present study was to investigate markers of the Arg/NO pathway in the plasma and urine of CF patients and to identify possible risk factors, especially associated with malnutrition. We measured the major NO metabolites nitrite and nitrate, Arg, a semi-essential amino acid and NO precursor, the NO synthesis inhibitor asymmetric dimethylarginine (ADMA) and its major urinary metabolite dimethylamine (DMA) in plasma and urine samples of 70 pediatric CF patients and 78 age-matched healthy controls. Biomarkers were determined by gas chromatography-mass spectrometry and high-performance liquid chromatography. We observed higher plasma Arg (90.3 vs. 75.6 µM, p < 0.0001), ADMA (0.62 vs. 0.57 µM, p = 0.03), Arg/ADMA ratio (148 vs. 135, p = 0.01), nitrite (2.07 vs. 1.95 µM, p = 0.03) and nitrate (43.3 vs. 33.1 µM, p < 0.001) concentrations, as well as higher urinary DMA (57.9 vs. 40.7 µM/mM creatinine, p < 0.001) and nitrate (159 vs. 115 µM/mM creatinine, p = 0.001) excretion rates in the CF patients compared to healthy controls. CF patients with pancreatic sufficiency showed plasma concentrations of the biomarkers comparable to those of healthy controls. Malnourished CF patients had lower Arg/ADMA ratios (p = 0.02), indicating a higher NO synthesis capacity in sufficiently nourished CF patients. We conclude that NO production, protein-arginine dimethylation, and ADMA metabolism is increased in pediatric CF patients. Pancreas and liver function influence Arg/NO metabolism. Good nutritional status is associated with higher NO synthesis capacity and lower protein-arginine dimethylation.

An Ethnic Comparison of Arginine Dimethylation and Cardiometabolic Factors in Healthy Black and White Youth: The ASOS and African-PREDICT Studies.

Proteinic arginine dimethylation (PADiMe) is a major post-translational modification. Proteolysis of asymmetric and symmetric PADiMe products releases asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), respectively, two endogenous atherogenic substances. SDMA, ADMA, and its major metabolite dimethylamine (DMA) are eliminated by the kidney. The urinary concentrations of DMA+ADMA, SDMA, and DMA+ADMA+SDMA are useful measures of the whole-body asymmetric and symmetric PADiMe, respectively. Urinary (DMA+ADMA)/SDMA is an index of the asymmetric to symmetric PADiMe balance. In two bi-ethnic studies, the ASOS (39 black boys, 41 white boys) and the African-PREDICT (292 black young men, 281 white young men) studies, we investigated whether ethnicity is a major determinant of PADiMe, and whether PADiMe is associated with blood pressure and ethnicity-dependent growth and inflammatory factors, including HDL. DMA, ADMA, and SDMA were measured in spot urine samples by gas chromatography-mass spectrometry, and their excretion was corrected for creatinine excretion. In black boys, creatinine-corrected DMA, DMA+ADMA, and DMA+ADMA+SDMA concentrations were lower by 11.7%, 9.5%, and 7.6% (all p < 0.05), respectively, compared to the white boys, and 3.4%, 2.0%, and 1.8% lower (all p < 0.05), respectively, in black compared to white men. (DMA+ADMA)/SDMA did not differ between black boys and black men, but was higher in white boys compared to white men. ADMA did not differ between black and white boys, or between black and white men. Creatinine-corrected SDMA excretion was lower in black boys compared to white boys (by 8%) and to white men (by 3.1%). None of the PADiMe indices were associated with blood pressure in either study. IGF-binding protein 3 correlated inversely with all PADiMe indices in the black men only. Our study showed that asymmetric proteinic arginine dimethylation is higher in white boys than in black boys, and that this difference disappears in adulthood. ADMA metabolism and SDMA excretion were lower in the black subjects compared to the white subjects, suggesting ethnicity-dependent hepatic and renal elimination of ADMA and SDMA in the childhood. The results of our study may have clinical relevance beyond atherosclerosis, such as in growth and inflammation, which have not been sufficiently addressed thus far.

GC-MS and LC-MS/MS pilot studies on the guanidine (NG)-dimethylation in native, asymmetrically and symmetrically NG-dimethylated arginine-vasopressin peptides and proteins in human red blood cells.

Protein-arginine methyltransferases catalyze the methylation of the guanidine (NG) group of proteinic L-arginine (Arg) to produce monomethyl and dimethylarginine proteins. Their proteolysis releases the free amino acids monomethylarginine (MMA), symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA), respectively. MMA, SDMA and ADMA are inhibitors of the nitric oxide synthase (NOS) activity. High circulating and low urinary concentrations of ADMA and SDMA are considered risk factors in the cardiovascular and renal systems, mainly due to their inhibitory action on NOS activity. Identity, biological activity and concentration of NG-methylated proteins are largely unknown. The present study addressed these issues by using GC-MS and LC-MS/MS approaches. GC-MS was used to quantify free ADMA released by classical HCl-catalyzed hydrolysis of three synthetic Arg-vasopressin (V) peptides and of unknown endogenous NG-dimethylated proteins. The cyclic (c) disulfide forms of Arg-vasopressin analogs, i.e., Arg-vasopressin (cV-Arg-Gly-NH2), asymmetrically NG-dimethylated vasopressin (cV-ADMA-Gly-NH2) and symmetrically NG-dimethylated vasopressin (cV-SDMA-Gly-NH2) were used as model peptides in quantitative GC-MS analyses of ADMA, SDMA and other expected amino acids from the hydrolyzed Arg-vasopressin analogs. cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 were discriminated from cV-Arg-Gly-NH2 by LC-MS and LC-MS/MS, yet they were indistinguishable from each other. The same applies to the respective open (o) reduced and di-S-acetamide forms of oV-ADMA-Gly-NH2, oV-SDMA-Gly-NH2 and oV-Arg-Gly-NH2. Our LC-MS and LC-MS/MS studies suggest that the Arg-vasopressin analogs form [(M-H)]+ and [(M-H)+H]+ in the positive ESI mode and undergo in part conversion of their terminal Gly-NH2 (NH2, 16 Da) group to Gly-OH (OH, 17 Da). The product ion mass spectra of the di-S-acetamide forms are complex and contain several intense mass fragments differing by 1 Da. cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 induced platelet aggregation in platelet-rich human plasma with moderately different initial velocity and maximal aggregation rates compared to cV-Arg-Gly-NH2. Previous studies showed that human red blood cells are rich in large (>50 kDa) ADMA-containing proteins of unknown identity. Our LC-MS/MS proteomic study identified several membrane and cytosolic erythrocytic NG-dimethylated proteins, including spectrin-α (280 kDa), spectrin-ß (247 kDa) and protein 4.1 (80 kDa). Being responsible for the stability of the erythrocyte membrane, the newly identified main targets for NG-dimethylation in human erythrocytes should be given a closer look in erythrocytic diseases like hereditary spherocytosis.

Enhanced Nitric Oxide (NO) and Decreased ADMA Synthesis in Pediatric ADHD and Selective Potentiation of NO Synthesis by Methylphenidate.

Attention deficit hyperactivity disorder (ADHD) is a common pediatric psychiatric disorder, frequently treated with methylphenidate (MPH). Recently, MPH's cardiovascular safety has been questioned by observational studies describing an increased cardiovascular risk in adults and blood pressure alterations in children. We considered members of the L-arginine (Arg)/nitric oxide (NO) pathway as possible early cardiovascular risk factors in pediatric ADHD children. They include the NO metabolites, nitrite and nitrate, the NO precursor Arg, and asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor and a cardiovascular risk factor in adults. We conducted a prospective clinical trial with 42 ADHD children (aged 6-16 years) with (n = 19) and without (n = 23) MPH treatment. Age-matched children without ADHD (n = 43) served as controls. All plasma and urine metabolites were determined by gas chromatography-mass spectrometry. We observed higher plasma nitrite and lower plasma ADMA concentrations in the ADHD children. MPH-treated ADHD children had higher plasma nitrite concentrations than MPH-untreated ADHD children. As NOS activity is basally inhibited by ADMA, MPH treatment seems to have decreased the inhibitory potency of ADMA. Percentiles of systolic blood pressure were higher in MPH-treated ADHD children. The underlying mechanisms and their implications in the MPH therapy of pediatric ADHD with MPH remain to be elucidated in larger cohorts.

GC-MS measurement of spermidine and putrescine in serum of elderly subjects: intriguing association between spermidine and homoarginine.

Gas chromatography-mass spectrometry (GC-MS) methods were developed, validated and used to measure serum spermidine (SPD) and putrescine (PUT) in 9 seropositive Helicobacter pylori (Hp +) and 18 seronegative Helicobacter pylori (Hp -) subjects (31-105 years). Homoarginine (hArg) was also measured by GC-MS. There were no statistical differences (unpaired t test) between the Hp + and Hp - subjects with respect to the serum concentrations of SPD (67.6 ± 40.3 vs. 93.7 ± 37.7 nM, P = 0.109), PUT (220 ± 139 vs. 236 ± 85 nM, P = 0.708) and hArg (1.60 ± 0.64 µM vs. 1.83 ± 0.74 µM, P = 0.554). Serum SPD and hArg concentrations correlated with each other (r = 0.426, P = 0.026, n = 27). The PUT/SPD molar ratio correlated inversely with the hArg concentration (r = - 0.406, P = 0.034, n = 27) and proteinic citrulline (r = - 0.487, P = 0.01, n = 27). These results suggest that SPD and PUT synthesis is associated with hArg formation and protein citrullination in healthy elderly subjects. The mechanisms underlying these associations and their significance remain to be elucidated.