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.

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).

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.

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.

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.

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.

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 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.

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.

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.

Asymmetric dimethylation and citrullination in the LEW.1AR1-iddm rat, an animal model of human type 1 diabetes, and effects of anti-TCR/anti-TNF-α antibody-based therapy.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D). We determined by GC-MS the extent of asymmetric dimethylation (prADMA) and citrullination (prCit) of L-arginine residues in organ proteins (pr) of normoglycaemic control (ngCo, n = 6), acutely diabetic (acT1D, n = 6), chronically diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Pancreatic prCit and prADMA did not differ between the groups but were correlated (r = 0.728, P = 0.0003, n = 20). acT1D rats had lower prCit levels in spleen and kidney than ngCo rats. cuT1D rats had higher prADMA levels than chT1D rats only in the spleen. Combination therapy re-established normoglycaemia and increased prADMA in the spleen without altering pancreatic prADMA and prCit. Western blotting demonstrated the presence of different prADMA pattern, especially an ≈ 50-kDa prADMA in spleen and pancreas, and an ≈ 25-kDa prADMA in the pancreas only, with the kidney showing only a very faint and small prADMA. Besides the changes in the pancreas during different metabolic states, the spleen may play a stronger role for the recognition of metabolic changes in T1D than thought thus far.

Synthesis of homoagmatine and GC-MS analysis of tissue homoagmatine and agmatine: evidence that homoagmatine but not agmatine is a metabolite of pharmacological L-homoarginine in the anesthetized rat.

Low L-homoarginine (hArg) concentrations in human blood and urine are associated with renal and cardiovascular morbidity and mortality, yet the underlying mechanisms and the biological activities of hArg are elusive. In humans and rats, hArg is metabolized to L-lysine. The aim of the present work was to study hArg metabolism to agmatine (Agm) and homoagmatine (hAgm) in the anesthetized rat. Using a newly developed and validated GC-MS method and a newly synthesized and structurally characterized hAgm we investigated the metabolism of i.p. administered hArg (0, 20, 220, 440 mg/kg) to hAgm and Agm in lung, kidney, liver and heart in anesthetized rats. Our study provides unequivocal evidence that hArg is metabolized to hAgm but not to Agm. Whether hAgm derived from hArg's metabolism may contribute to the pathophysiological significance of endogenous hArg and for the favoured effects of pharmacological hArg remains to be demonstrated. The biology of hArg warrants further investigations.

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.

Evidence by GC-MS that lysine is an arginase-catalyzed metabolite of homoarginine in vitro and in vivo in humans.

l-Homoarginine (hArg) is biosynthesized from l-arginine (Arg) and l-lysine (Lys) by arginine:glycine amidinotransferase (AGAT). AGAT also catalyzes the formation of guanidinoacetate (GAA) from Arg and glycine (Gly). GAA is converted to creatine (N-methyl guanidinoacetate) by guanidinoacetate N-methyl-transferase (GAMT). Low circulating and excretory concentrations of hArg are associated with worse cardiovascular outcome and mortality. hArg is a poor substrate of nitric oxide synthase (NOS) and a weak inhibitor of arginase. The metabolism of hArg in humans is little investigated. Previously, we found that orally administered hArg (125 mg/day) increased the plasma concentration of hArg, but not of Arg, the substrate of NOS, in healthy subjects. We newly analyzed the plasma samples collected in that study for Lys and other amino acids. Repeated measures ANOVA revealed statistically significant differences between the groups (P = 0.008) with respect to plasma Lys concentration which increased by about 8% after a 4-week hArg supplementation. In vitro, recombinant human arginase and bovine liver arginase I were demonstrated by a specific and sensitive stable-isotope GC-MS assay to hydrolyze hArg to Lys. Our results suggest that Lys is a metabolite of hArg produced by the hydrolytic activity of arginase. Arginase may play a key role in hArg homeostasis in humans.

Pharmacological activation of dimethylarginine dimethylaminohydrolase (DDAH) activity by inorganic nitrate and DDAH inhibition by NG-hydroxy-L-arginine, Nω,Nω-dimethyl-L-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-L-citrulline: results and overview.

Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of nitric oxide (NO) synthase. SDMA is excreted in the urine without major metabolization. About 10% of daily produced ADMA are excreted unchanged in the urine. The major elimination route of ADMA (about 90%) involves its hydrolysis to dimethylamine (DMA) and L-citrulline by dimethylarginine dimethylaminohydrolase (DDAH) and excretion of DMA in the urine. High circulating and low excretory concentrations of ADMA are considered risk factors. Experimentally, DDAH activity can be inhibited by SH-specific agents such as inorganic and organic mercury compounds, and by S-nitrosothiols which block the SH group of a particular cysteine moiety of DDAH that is essential for its hydrolytic activity. Alternatively, DDAH activity can be inhibited by organic compounds that compete with the substrate ADMA for DDAH. Arginine analogs that contain substituents on guanidine nitrogen atom(s) (NG) represent a class of DDAH inhibitors. In the present study, we investigated the effects of physiological and natural amino acid derivatives of L-arginine and L-citrulline as well as of nitrate and nitrite, the major circulating and excretory metabolites of NO and NO donating drugs. Here, we report for the first time that the physiological NG-hydroxy-L-arginine, an isolable intermediate in NO synthesis, inhibits recombinant DDAH-1 activity (IC50 ≈ 100 µM). Two plant L-citrulline derivatives, i.e., Nω,Nω-dimethyl-L-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-L-citrulline (connatin), were found to inhibit almost completely hepatic DDAH activity in vitro in rat homogenate at a concentration of 100 µM each. At pharmacological concentrations (i.e., 1 mM), inorganic nitrate, but not inorganic nitrite, was found to increase rat liver DDAH activity. In urine of 18 patients with Becker's muscular dystrophy, nitrate was found to correlate closely with DMA (Spearman, r = 0.73, p = 0.002). In summary, NG-hydroxy-L-arginine, Nω,Nω-dimethyl-L-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-L-citrulline are novel inhibitors of DDAH activity. This article provides an overview of amino acid-based DDAH inhibitors and discusses potential underlying inhibition mechanisms.

Asymmetric dimethylation and citrullination of proteinic arginine and homoarginine synthesis in human Helicobacter pylori infection.

The importance of L-arginine (Arg) and relatives, including L-homoarginine (hArg) and asymmetric dimethylarginine (ADMA), in humans infected with Helicobacter pylori (Hp) is little understood. ADMA is produced by asymmetric dimethylation of the guanidine group of Arg residues in certain proteins and is released by proteolysis. High concentrations of circulating free ADMA are considered a risk factor for morbidity and mortality in adults. This risk is considered to arise from the inhibition of the synthesis of nitric oxide (NO), which is a potent vasodilator and inhibitor of platelet aggregation. In the present study, we quantified by stable isotope dilution gas chromatography-mass spectrometry (GC-MS) the concentration of free (f) and total (t) ADMA, Arg, hArg, lysine (Lys) and the sum of citrulline (Cit) and ornithine (Orn) (6 M HCl, 20 h, 110 °C) in serum samples of apparently healthy elderly subjects (n = 27; age, 31-105 years) who were tested for Hp infection. Nine subjects (5 males, 4 females) were found to be Hp seropositive (Hp+) and 18 subjects (8 males, 9 females) were found to be Hp seronegative (Hp‒). Proteinic (p) concentrations were determined by difference. fADMA (0.493 ± 0.068 vs 0.466 ± 0.081 µM, P = 0.382), pADMA (113 ± 73 vs 76 ± 59 nM, P = 0.169) and tADMA (0.606 ± 0.126 vs 0.543 ± 0.121 µM, P = 0.280) serum concentrations were found not to differ between the Hp+ and Hp- subjects. Serum concentrations of fArg (162 ± 30 vs 177 ± 36 µM, P = 0.471), fhArg (1.600 ± 0.638 vs 1.831 ± 0.742 µM, P = 0.554), and fLys (388 ± 170 vs 395 ± 149 µM, P = 0.700) also did not differ statistically between Hp+ and Hp- subjects. tArg (12.4 ± 1.49 vs 13.0 ± 1.33 mM, P = 0.190), tLys (23.0 ± 2.65 vs. 23.9 ± 2.66 mM, P = 0.456) and tCit + Orn (2.53 ± 0.76 vs 2.63 ± 0.85 mM, P = 0.817) did not differ between Hp+and Hp‒ subjects as well. phArg concentration was close to the limit of quantitation of the method (Hp+: 30 ± 210 nM; Hp-: 42 ± 205 nM), suggesting that hArg is virtually absent in serum proteins of the investigated subjects. pCit + Orn did not differ between infected and non-infected subjects. Our study suggests that Hp infection is not associated with elevated asymmetric dimethylation and citrullination of Arg proteins present in the serum or with the hArg synthesis from free Arg in elderly subjects. However, asymmetric Arg dimethylation was found to correlate inversely with Arg citrullination in Hp- (r2 = 0.408, P = 0.004) but not in Hp+ (r2 = 0.065, P = 0.506), with Arg citrullination decreasing and Arg asymmetric dimethylation increasing with subjects' age.

GC-MS measurement of biological NG-hydroxy-L-arginine, a stepmotherly investigated endogenous nitric oxide synthase substrate and arginase inhibitor.

L-Arginine is converted by nitric oxide synthase (NOS) to L-citrulline and nitric oxide (NO). NG-Hydroxy-L-arginine (NOHA) is the isolable intermediate of this reaction. NOHA has been identified in biological samples by gas chromatography-mass spectrometry (GC-MS) and quantified by high-performance liquid chromatography (HPLC). Reportedly, NOHA concentrations in human plasma and serum range over four orders of magnitude (e.g., 2 nM-34 µM). The natural occurrence of NOHA in urine has not been reported thus far. Here, we report a validated stable-isotope dilution GC-MS method for the quantitative determination of NOHA in 10-µL aliquots of human serum and urine samples. The method is based on a two-step derivatization of NOHA to the methyl ester pentafluoropropionyl (PFP) derivatives using newly synthesized trideuteromethyl ester NOHA (d3Me-NOHA) as the internal standard and GC-MS quantification. NOHA was found to form a methyl ester-NG,Nδ,Nα-pentafluoropropionyl derivative, i.e., Me-(PFP)3 (M, 642) with the NG-hydroxy group remaining non-derivatized. Selected-ion monitoring of mass-to-charge (m/z) ratio of 458 for endogenous NOHA and m/z 461 for d3Me-NOHA in the negative-ion chemical ionization mode revealed NOHA concentrations of the order of 0.2 µM in human serum and 3 µM in urine samples. Accuracy (recovery, %) was 91.6 ± 1.6% in serum and 39.9 ± 4.5% in urine. Inorganic nitrate was found to decrease NOHA recovery from urine presumably through the reaction of the OH group of NOHA with nitric acid. Imprecision (RSD,  %) ranged between 1.4 and 14.8% in serum, and between 5.3 and 18.4% in urine in the investigated concentration range (0-15 µM NOHA). Ten healthy kidney donors excreted in the urine (mean ± SEM) 13.9 ± 1.81 µmol NOHA per day before and 10.9 ± 1.4 µmol NOHA per day after kidney donation (P = 0.24). Similar results were observed for dimethylamine (DMA), the major urinary metabolite of asymmetric dimethylarginine (ADMA). Changes in NOHA and DMA correlated positively (r = 0.718, P = 0.019). This is the first report on the occurrence and measurement of NOHA in human urine and on the effect of human unilateral nephrectomy on urinary NOHA and DMA. Healthy kidney donation may be useful as a model for kidney disease.

Development and validation of GC-MS methods for the comprehensive analysis of amino acids in plasma and urine and applications to the HELLP syndrome and pediatric kidney transplantation: evidence of altered methylation, transamidination, and arginase activity.

We developed and validated gas chromatography-mass spectrometry (GC-MS) methods for the simultaneous measurement of amino acids and their metabolites in 10-µL aliquots of human plasma and urine. De novo synthesized trideutero-methyl esters were used as internal standards. Plasma proteins were precipitated by acidified methanol and removed by centrifugation. Supernatants and native urine were evaporated to dryness. Amino acids were first esterified using 2 M HCl in methanol and then amidated using pentafluoropropionic anhydride for electron-capture negative-ion chemical ionization. Time programmes were used for the gas chromatograph oven and the selected-ion monitoring of specific anions. The GC-MS methods were applied in clinical studies on the HELLP syndrome and pediatric kidney transplantation (KTx) focusing on L-arginine-related pathways. We found lower sarcosine (N-methylglycine) and higher asymmetric dimethylarginine (ADMA) plasma concentrations in HELLP syndrome women (n = 7) compared to healthy pregnant women (n = 5) indicating altered methylation. In plasma of pediatric KTx patients, lower guanidinoacetate and homoarginine concentrations were found in plasma but not in urine samples of patients treated with standard mycophenolate mofetil-based immunosuppression (MMF; n = 22) in comparison to matched patients treated with MMF-free immunosuppression (n = 22). On average, the global arginine bioavailability ratio was by about 40% lower in the MMF group compared to the EVR group (P = 0.004). Mycophenolate, the major pharmacologically active metabolite of MMF, is likely to inhibit the arginine:glycine amidinotransferase (AGAT), and to enhance arginase activity in leukocytes and other types of cell of MMF-treated children.