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.

Creatine homeostasis and the kidney: comparison between kidney transplant recipients and healthy controls.

Creatine is a natural nitrogenous organic acid that is integral to energy metabolism and crucial for proper cell functioning. The kidneys are involved in the first step of creatine production. With kidney transplantation being the gold-standard treatment for end-stage kidney disease, kidney transplant recipients (KTR) may be at risk of impaired creatine synthesis. We aimed to compare creatine homeostasis between KTR and controls. Plasma and urine concentrations of arginine, glycine, guanidinoacetate, creatine and creatinine were measured in 553 KTR and 168 healthy controls. Creatine intake was assessed using food frequency questionnaires. Iothalamate-measured GFR data were available in subsets of 157 KTR and 167 controls. KTR and controls had comparable body weight, height and creatine intake (all P > 0.05). However, the total creatine pool was 14% lower in KTR as compared to controls (651 ± 178 vs. 753 ± 239 mmol, P < 0.001). The endogenous creatine synthesis rate was 22% lower in KTR as compared to controls (7.8 ± 3.0 vs. 10.0 ± 4.1 mmol per day, P < 0.001). Despite lower GFR, the plasma guanidinoacetate and creatine concentrations were 21% and 41% lower in KTR as compared to controls (both P < 0.001). Urinary excretion of guanidinoacetate and creatine were 66% and 59% lower in KTR as compared to controls (both P < 0.001). In KTR, but not in controls, a higher measured GFR was associated with a higher endogenous creatine synthesis rate (std. beta: 0.21, 95% CI: 0.08; 0.33; P = 0.002), as well as a higher total creatine pool (std. beta: 0.22, 95% CI: 0.11; 0.33; P < 0.001). These associations were fully mediated (93% and 95%; P < 0.001) by urinary guanidinoacetate excretion which is consistent with production of the creatine precursor guanidinoacetate as rate-limiting factor. Our findings highlight that KTR have a disturbed creatine homeostasis as compared to controls. Given the direct relationship of measured GFR with endogenous creatine synthesis rate and the total creatine pool, creatine supplementation might be beneficial in KTR with low kidney function.Trial registration ID: NCT02811835.Trial registration URL: https://clinicaltrials.gov/ct2/show/NCT02811835 .

Acetazolamide and human carbonic anhydrases: retrospect, review and discussion of an intimate relationship.

Acetazolamide (AZM) is a strong pharmacological sulphonamide-type (R-SO2-NH2, pKa 7.2) inhibitor of the activity of several carbonic anhydrase (CA) isoforms, notably of renal CA II (Ki, 12 nM) and CA IV (Ki, 74 nM). AZM is clinically used for about eighty years in various diseases including epilepsy and glaucoma. Pharmacological AZM increases temporarily the urinary excretion of bicarbonate (HCO3-) and sodium ions (Na+) and sustainably the urinary pH. AZM is excreted almost unchanged over several hours at high rates in the urine. Closely parallel concentrations of circulating and excretory AZM are observed upon administration of therapeutical doses of AZM. In a proof-of-principle study, we investigated the effects of the ingestion of a 250-mg AZM-containing tablet by a healthy volunteer on the urinary excretion of organic and inorganic substances over 5 h (range, 0, 0.5, 1, 1.5, 2, 3, 4, 5 h). Measured analytes included: AZM, amino acids and their metabolites such as guanidinoacetate, i.e. the precursor of creatine, of asymmetrically (ADMA) and symmetrically (SDMA) dimethylated arginine, nitrite (O = N-O-, pKa 3.4) and nitrate (O2N-O-, pKa -1.37), the major metabolites of nitric oxide (NO), the C-H acidic malondialdehyde (MDA; (CHO)2CH2, pKa 4.5), and creatinine for correction of analytes excretion. All analytes were measured by validated isotopologues using gas chromatography-mass spectrometry (GC-MS) methods. AZM excretion in the urine reached its maximum value after 2 h and was fairly stable for the next 3 h. Time series analysis by the ARIMA method was performed. AZM ingestion increased temporarily the urinary excretion of the amino acids Leu + Ile, nitrite and nitrate, decreased temporarily the urinary excretion of other amino acids. AZM decreased sustainably the urinary excretion of MDA, a biomarker of oxidative stress (i.e. lipid peroxidation). Whether this decrease is due to inhibition of the excretion of MDA or attenuation of oxidative stress by AZM is unknown. The acute and chronic effects of AZM on the urinary excretion of electrolytes and physiological substances reported in the literature are discussed in depth in the light of its extraordinary pharmacokinetics and pharmacodynamics. Tolerance development/drug resistance to AZM in chronic use and potential mechanisms are also addressed.

Determination of N-Acetyl-L-cysteine Ethyl Ester (NACET) by Sequential Injection Analysis.

New sequential injection analysis (SIA) methods with optical sensing for the determination of N-acetyl-L-cysteine ethyl ester (NACET) have been developed and optimized. NACET is a potential drug and antioxidant with advantageous pharmacokinetics. The methods involve the reduction of Cu(II) in its complexes with neocuproine (NCN), bicinchoninic acid (BCA), and bathocuproine disulfonic acid (BCS) to the corresponding chromophoric Cu(I) complexes by the analyte. The absorbance of the Cu(I) complexes with NCN, BCA, and BCS was measured at their maximum absorbance wavelengths of 458, 562, and 483 nm, respectively. The sensing manifold parameters and experimental conditions were optimized for each of the Cu(II) complexes used. Under optimal conditions, the corresponding linear calibration ranges, limits of detection, and sampling rates were 8.0 × 10-6-2.0 × 10-4 mol L-1, 5.5 × 10-6 mol L-1, and 60 h-1 for NCN; 6.0 × 10-6-1.0 × 10-4 mol L-1, 5.2 × 10-6 mol L-1, and 60 h-1 for BCA; and 4.0 × 10-6-1.0 × 10-4 mol L-1, 2.6 × 10-6 mol L-1, and 78 h-1 for BCS. The Cu(II)-BCS complex was found to be best performing in terms of sensitivity and sampling rate. Usual excipients in pharmaceutical preparations did not interfere with NACET analysis.

Application of the Bland-Altman and Receiver Operating Characteristic (ROC) Approaches to Study Isotope Effects in Gas Chromatography-Mass Spectrometry Analysis of Human Plasma, Serum and Urine Samples.

The Bland-Altman approach is one of the most widely used mathematical approaches for method comparison and analytical agreement. This work describes, for the first time, the application of Bland-Altman to study 14N/15N and 1H/2H (D) chromatographic isotope effects of endogenous analytes of the L-arginine/nitric oxide pathway in human plasma, serum and urine samples in GC-MS. The investigated analytes included arginine, asymmetric dimethylarginine, dimethylamine, nitrite, nitrate and creatinine. There was a close correlation between the percentage difference of the retention times of the isotopologs of the Bland-Altman approach and the area under the curve (AUC) values of the receiver operating characteristic (ROC) approach (r = 0.8619, p = 0.0047). The results of the study suggest that the chromatographic isotope effects in GC-MS result from differences in the interaction strengths of H/D isotopes in the derivatives with the hydrophobic stationary phase of the GC column. D atoms attenuate the interaction of the skeleton of the molecules with the lipophilic GC stationary phase. Differences in isotope effects in plasma or serum and urine in GC-MS are suggested to be due to a kind of matrix effect, and this remains to be investigated in forthcoming studies using Bland-Altman and ROC approaches.

Malondialdehyde-Induced Post-Translational Modification of Human Hemoglobin.

Lysine residues in proteins undergo multiple enzymatic and nonenzymatic post-translational modifications (PTMs). The terminal ε amine group of lysine residues in proteins is carbonylated chemically by carbonyl species such as glyoxal (GO; OCH-CHO, C2H2O2; MW 58) and methylglyoxal (MGO; OCH-C(=O)-CH3, C3H4O2; MW 72) that are derived from the metabolism of endogenous substances including glucose. The dicarbonyl species malondialdehyde (MDA, OCH-CH2-CHO, C3H4O2; MW 72) is generated by enzymatic and nonenzymatic peroxidation of polyunsaturated fatty acids (PUFAs). GO, MGO, and MDA occur in biological systems in their free forms and in their conjugated forms adducted to free amino acids and amino acid residues in proteins, notably to lysine. MDA is a C-H-acidic acid (pKa, 4.45). Biological MDA is widely used as a biomarker of lipid peroxidation. The most frequently analyzed biological samples for MDA are plasma and serum. Reportedly, MDA concentrations in plasma and serum samples of healthy and ill humans range by several orders of magnitude. The most severe preanalytical contributor is artificial formation of MDA in lipid-rich samples such as plasma and serum. In very few publications, plasma MDA concentrations were reported to lie in the lower mM-range.

Homoarginine in health and disease.

PURPOSE OF REVIEW: Homoarginine (hArg) is an endogenous, nonproteinogenic amino acid. It is enzymatically synthesized from L-arginine and L-lysine. Low hArg concentrations appear to be a risk factor in the renal and cardiovascular systems. This review discusses advances in-vitro and in-vivo experimental and clinical research on hArg in health and disease. RECENT FINDINGS: Recent studies indicate that low circulating and low urinary concentrations of hArg are associated with morbidity and worse outcome. Although the biological activities of hArg remain still unexplored, hArg supplementation is intensely investigated as a strategy to increase hArg concentration to reach normal levels in cases of low hArg concentrations. The greatest changes in circulating hArg concentrations are observed during pregnancy and after delivery. In healthy adults, a daily dose of 125 mg hArg seems to be optimum to normalize circulating levels. Short-term supplementation of inorganic nitrate enhances hArg biosynthesis in healthy young men. Apart from hArg supplementation, dietary L-arginine and L-citrulline appear to be a promising alternative. SUMMARY: Considerable progress has been made in recent years, but hArg remains still enigmatic. Further research is required to explore the biological activities of hArg. Supplementation of hArg or its precursors L-citrulline/L-arginine seem to be promising strategies to prevent and overcome altered hArg synthesis.

Determination of equilibria constants of arginine:glycine amidinotransferase (AGAT)-catalyzed reactions using concentrations of circulating amino acids.

Arginine:glycine amidinotransferase (AGAT) catalyzes mainly two reactions that generate 1) L-homoarginine (hArg) from L-arginine and L-lysine (Kharg) and 2) guanidinoacetate (GAA) and L-ornithine from L-arginine and glycine (Kgaa). Previously, we found that pharmacological treatment of Becker muscular dystrophy (BMD) patients with metformin or L-citrulline resulted in antidromic effects on serum hArg and GAA concentrations, seemingly acting as an inhibitor and effector of AGAT activity, respectively. Here, we used data of this study as a model to determine Kharg and Kgaa values by using the concentrations of the participating amino acids measured in serum samples of the BMD patients. The study aimed to prove the general utility of this approach to investigate effects of amino acids and drugs on AGAT-catalyzed reactions in vivo in humans.

Amino acids, post-translational modifications, nitric oxide, and oxidative stress in serum and urine of long COVID and ex COVID human subjects.

In this study, we investigated the status of amino acids, their post-translational modifications (PTM), major nitric oxide (NO) metabolites and of malondialdehyde (MDA) as a biomarker of oxidative stress in serum and urine samples of long COVID (LoCo, n = 124) and ex COVID (ExCo, n = 24) human subjects collected in 2022. Amino acids and metabolites were measured by gas chromatography-mass spectrometry (GC-MS) methods using stable-isotope labelled analogs as internal standards. There were no differences with respect to circulating and excretory arginine and asymmetric dimethylarginine (ADMA). LoCo participants excreted higher amounts of guanidino acetate than ExCo participants (17.8 ± 10.4 µM/mM vs. 12.6 ± 8.86 µM/mM, P = 0.005). By contrast, LoCo participants excreted lower amounts of the advanced glycation end-product (AGE) NG-carboxyethylarginine (CEA) than ExCo participants did (0.675 ± 0.781 µM/mM vs. 1.16 ± 2.04 µM/mM, P = 0.0326). The serum concentrations of MDA did not differ between the groups, indicating no elevated oxidative stress in LoCo or ExCo. The serum concentration of nitrite was lower in LoCo compared to ExCo (1.96 ± 0.92 µM vs. 2.56 ± 1.08 µM; AUC, 0.718), suggesting altered NO synthesis in the endothelium. The serum concentration of nitrite correlated inversely with the symptom anxiety (r = - 0.293, P = 0.0003). The creatinine-corrected urinary excretion of Lys and its metabolite L-5-hydroxy-Lys correlated positively with COVID toes (r = 0.306, P = 0.00027) and sore throat (r = 0.302, P = 0.0003). Our results suggest that amino acid metabolism, PTM and oxidative stress are not severely affected in long COVID. LoCo participants may have a lower circulating NO reservoir than ExCo.

l-Arginine/nitric oxide pathway and oxidative stress in adults with ADHD: Effects of methylphenidate treatment.

INTRODUCTION: Attention deficit hyperactivity disorder (ADHD) is a mental disorder that was once thought to occur only in children. Meanwhile, it is known that adults can also be affected. The first-line drug in children and adults to treat symptoms of inattention, impulsivity, lack of self-regulation, and hyperactivity is methylphenidate (MPH). Known adverse effects of MPH include cardiovascular problems, such as elevated blood pressure and heart rate. Therefore, biomarkers to monitor potential cardiovascular side effects of MPH are needed. The l-Arginine/Nitric oxide (Arg/NO) pathway is involved in noradrenaline and dopamine release as well as in normal cardiovascular functioning and is therefore a prime candidate for the search of biomarkers. The aim of the present study was to investigate the Arg/NO pathway as well as oxidative stress in adult ADHD patients in plasma and urine and the potential influence of MPH medication. METHODS: In plasma and urine samples of 29 adults with ADHD (39.2 ± 10.9 years) and 32 healthy adults serving as controls (CO) (38.0 ± 11.6 years) the major NO metabolites nitrite and nitrate, Arg, the NO synthesis inhibitor asymmetric dimethylarginine (ADMA) and its major urinary metabolite dimethylamine (DMA) as well as malondialdehyde (MDA) were measured by gas chromatography-mass spectrometry. RESULTS: Of the 29 patients with ADHD 14 were currently without MPH treatment (-MPH) and 15 were treated with MPH (+MPH). Plasma nitrate concentrations were significantly higher in patients not treated with MPH vs. CO (-MPH 60.3 µM [46.2-76.0] vs. CO 44.4 µM [35.0-52.7]; p = 0.002), while plasma nitrite tended to be higher in -MPH patients (2.77 µM [2.26-3.27]) vs. CO (2.13 µM [1.50-2.93]; p = 0.053). Additionally, plasma creatinine concentrations were significantly different, with -MPH showing significantly higher concentrations than the other two groups (-MPH 141 µM [128-159]; +MPH 96.2 µM [70.2-140]; Co 75.9 µM [62.0-94.7]; p < 0.001). Urinary creatinine excretion tended to be lowest in -MPH group vs. +MPH and CO (-MPH 11.4 ± 8.88 mM; +MPH 20.7 ± 9.82 mM; 16.6 ± 7.82 mM; p = 0.076). None of the other metabolites, including MDA, a marker of oxidative stress, showed a difference between the groups. CONCLUSION: Adult patients with ADHD, who are not treated with MPH (-MPH), showed varied Arg/NO pathway, but Arg bioavailability seemed to be consistent over the groups. Our findings imply that urinary reabsorption may be increase and/or excretion of nitrite and nitrate may be decreased in ADHD, resulting in an increase in the plasma concentration of nitrite. MPH seems to partially reverse these effects by not yet known mechanisms, and does not affect oxidative stress.

Endocannabinergic modulation of central serotonergic activity in healthy human volunteers.

BACKGROUND: The serotonergic and the endocannabinoid system are involved in the etiology of depression. Depressive patients exhibit low serotonergic activity and decreased level of the endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2AG). Since the cannabinoid (CB) 1 receptor is activated by endogenous ligands such as AEA and 2AG, whose concentration are controlled by the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, respectively, we investigated the effects on serotonergic utilization. In this study, we investigated the impact of the rs1049353 single-nucleotide polymorphism (SNP) of the cannabinoid receptor 1 (CNR1) gene, which codes the endocannabinoid CB1 receptor, and the rs324420 SNP of the FAAH gene on the serotonergic and endocannabinoid system in 59 healthy volunteers. METHODS: Serotonergic activity was measured by loudness dependence of auditory-evoked potentials (LDAEP). Plasma concentrations of AEA, 2AG and its inactive isomer 1AG were determined by mass spectrometry. Genotyping of two SNPs (rs1049353, rs344420) was conducted by polymerase chain reaction (PCR) and differential enzymatic analysis with the PCR restriction fragment length polymorphism method. RESULTS: Genotype distributions by serotonergic activity or endocannabinoid concentration showed no differences. However, after detailed consideration of the CNR1-A-allele-carriers, a reduced AEA (A-allele-carrier M = 0.66, SD = 0.24; GG genotype M = 0.72, SD = 0.24) and 2AG (A-allele-carriers M = 0.70, SD = 0.33; GG genotype M = 1.03, SD = 0.83) plasma concentration and an association between the serotonergic activity and the concentrations of AEA and 2AG has been observed. CONCLUSIONS: Our results suggest that carriers of the CNR1-A allele may be more susceptible to developing depression.

Mass Spectrometry-Based Evaluation of the Bland-Altman Approach: Review, Discussion, and Proposal.

Reliable quantification in biological systems of endogenous low- and high-molecular substances, drugs and their metabolites, is of particular importance in diagnosis and therapy, and in basic and clinical research. The analytical characteristics of analytical approaches have many differences, including in core features such as accuracy, precision, specificity, and limits of detection (LOD) and quantitation (LOQ). Several different mathematic approaches were developed and used for the comparison of two analytical methods applied to the same chemical compound in the same biological sample. Generally, comparisons of results obtained by two analytical methods yields different quantitative results. Yet, which mathematical approach gives the most reliable results? Which mathematical approach is best suited to demonstrate agreement between the methods, or the superiority of an analytical method A over analytical method B? The simplest and most frequently used method of comparison is the linear regression analysis of data observed by method A (y) and the data observed by method B (x): y = α + ßx. In 1986, Bland and Altman indicated that linear regression analysis, notably the use of the correlation coefficient, is inappropriate for method-comparison. Instead, Bland and Altman have suggested an alternative approach, which is generally known as the Bland-Altman approach. Originally, this method of comparison was applied in medicine, for instance, to measure blood pressure by two devices. The Bland-Altman approach was rapidly adapted in analytical chemistry and in clinical chemistry. To date, the approach suggested by Bland-Altman approach is one of the most widely used mathematical approaches for method-comparison. With about 37,000 citations, the original paper published in the journal The Lancet in 1986 is among the most frequently cited scientific papers in this area to date. Nevertheless, the Bland-Altman approach has not been really set on a quantitative basis. No criteria have been proposed thus far, in which the Bland-Altman approach can form the basis on which analytical agreement or the better analytical method can be demonstrated. In this article, the Bland-Altman approach is re-valuated from a quantitative bioanalytical perspective, and an attempt is made to propose acceptance criteria. For this purpose, different analytical methods were compared with Gold Standard analytical methods based on mass spectrometry (MS) and tandem mass spectrometry (MS/MS), i.e., GC-MS, GC-MS/MS, LC-MS and LC-MS/MS. Other chromatographic and non-chromatographic methods were also considered. The results for several different endogenous substances, including nitrate, anandamide, homoarginine, creatinine and malondialdehyde in human plasma, serum and urine are discussed. In addition to the Bland-Altman approach, linear regression analysis and the Oldham-Eksborg method-comparison approaches were used and compared. Special emphasis was given to the relation of difference and mean in the Bland-Altman approach. Currently available guidelines for method validation were also considered. Acceptance criteria for method agreement were proposed, including the slope and correlation coefficient in linear regression, and the coefficient of variation for the percentage difference in the Bland-Altman and Oldham-Eksborg approaches.

GC-MS Studies on Nitric Oxide Autoxidation and S-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using 15N and 18O Isotopes.

Nitrite (O=N-O-, NO2-) and nitrate (O=N(O)-O-, NO3-) are ubiquitous in nature. In aerated aqueous solutions, nitrite is considered the major autoxidation product of nitric oxide (●NO). ●NO is an environmental gas but is also endogenously produced from the amino acid L-arginine by the catalytic action of ●NO synthases. It is considered that the autoxidation of ●NO in aqueous solutions and in O2-containing gas phase proceeds via different neutral (e.g., O=N-O-N=O) and radical (e.g., ONOO●) intermediates. In aqueous buffers, endogenous S-nitrosothiols (thionitrites, RSNO) from thiols (RSH) such as L-cysteine (i.e., S-nitroso-L-cysteine, CysSNO) and cysteine-containing peptides such as glutathione (GSH) (i.e., S-nitrosoglutathione, GSNO) may be formed during the autoxidation of ●NO in the presence of thiols and dioxygen (e.g., GSH + O=N-O-N=O → GSNO + O=N-O- + H+; pKaHONO, 3.24). The reaction products of thionitrites in aerated aqueous solutions may be different from those of ●NO. This work describes in vitro GC-MS studies on the reactions of unlabeled (14NO2-) and labeled nitrite (15NO2-) and RSNO (RS15NO, RS15N18O) performed in pH-neutral aqueous buffers of phosphate or tris(hydroxyethylamine) prepared in unlabeled (H216O) or labeled H2O (H218O). Unlabeled and stable-isotope-labeled nitrite and nitrate species were measured by gas chromatography-mass spectrometry (GC-MS) after derivatization with pentafluorobenzyl bromide and negative-ion chemical ionization. The study provides strong indication for the formation of O=N-O-N=O as an intermediate of ●NO autoxidation in pH-neutral aqueous buffers. In high molar excess, HgCl2 accelerates and increases RSNO hydrolysis to nitrite, thereby incorporating 18O from H218O into the SNO group. In aqueous buffers prepared in H218O, synthetic peroxynitrite (ONOO-) decomposes to nitrite without 18O incorporation, indicating water-independent decomposition of peroxynitrite to nitrite. Use of RS15NO and H218O in combination with GC-MS allows generation of definite results and elucidation of reaction mechanisms of oxidation of ●NO and hydrolysis of RSNO.

Pentafluoropropionic Anhydride Derivatization and GC-MS Analysis of Histamine, Agmatine, Putrescine, and Spermidine: Effects of Solvents and Starting Column Temperature.

Gas chromatography-mass spectrometry (GC-MS) is useful for the quantitative determination of the polyamines spermidine (SPD) and putrescine (PUT) and of the biogenic amine agmatine (AGM) in biological samples after derivatization. This GC-MS method involves a two-step extraction with n-butanol and hydrochloric acid, derivatization with pentafluoropropionic anhydride (PFPA) in ethyl acetate, and extraction of the pentafluoropropionic (PFP) derivatives by toluene of SPD, PUT, and AGM. We wanted to extend this GC-MS method for the biogenic amine histamine (HA), but we faced serious problems that did not allow reliable quantitative analysis of HA. In the present work, we addressed this issue and investigated the derivatization of HA and the effects of toluene and ethyl acetate, two commonly used water-insoluble organic solvents in GC-MS, and oven temperature program. Derivatization of unlabelled HA (d0-HA) and deuterium-labelled HA (d4-HA) with PFPA in ethyl acetate (PFPA-EA, 1:4, v/v; 30 min, 65 °C) resulted in the formation of d0-HA-(PFP)2 and d4-HA-(PFP)2 derivatives. d4-HA and 13C4-SPD were used as internal standards for the amines after standardization. Considerable quantitative effects of toluene and ethyl acetate were observed. The starting GC column temperature was also found to influence considerably the GC-MS analysis of HA. Our study shows the simultaneous quantitative analysis of HA as HA-(PFP)2, AGM as AGM-(PFP)3, PUT as PUT-(PFP)2, and SPD as SPD-(PFP)3 derivatives requires the use of ethyl acetate for their extraction and injection into the GC-MS apparatus and a starting GC column temperature of 40 °C instead of 70 °C. The PFP derivatives of HA, AGM, PUT, and SPD were found to be stable in ethyl acetate for several hours at room temperature. Analytically satisfactory linearity, precision, and accuracy were observed for HA, AGM, PUT, and SPD in biologically relevant ranges (0 to 700 pmol). The limits of detection of AGM, PUT, and SPD were about two times lower in ethyl acetate compared to toluene (range, 1-22 fmol). The limits of detection were 1670 fmol for d0-HA and 557 fmol for d4-HA. Despite the improvements achieved in the study for HA, its analysis by GC-MS as a PFP derivative is challenging and less efficient than that of PUT, AGM, and SPD.

Rat liver glutathione S-transferase-catalyzed conjugation of glutathione to the endogenous epoxides of oleic acid and cholesterol.

cis-9,10-Epoxy-octadecanoic acid (oleic acid epoxide, OAE) and 5α,6α-epoxy-cholesterol (ChE) are endogenous epoxides. Unlike other epoxides, the oxirane groups of OAE and ChE are relatively stable against nucleophiles. OAE lacks toxicity and mutagenicity, while ChE is considered harmful, mutagenic and cancerogenic to animals. In humans, ChE is associated with cancer. The metabolism of OAE and ChE includes hydrolysis by cytosolic and microsomal hydrolases to their diols and glutathione (GSH) conjugation by GSH S-transferases (GST) to form the GSH conjugates (R-SG; R, residue). The GST-catalyzed GSH conjugation of OAE and ChE is poorly investigated. This article reports on the GSH conjugation of OAE, its methyl ester (OAEMe) and of ChE by rat liver homogenate GST. The GSH conjugates of OAE, OAEMe and ChE, i.e., OAE-SG, OAEMe-SG and ChE-SG, respectively, were determined by pre-column derivatization with o-phthaldialdehyde (OPA)/2-mercaptoethanol, high-performance liquid chromatography (HPLC) and fluorescence detection. Complex biphasic kinetics were observed with substrate inhibition of GST activity by OAE, OAEMe and ChE, an optimum pH of about 8.3 for OAE, and no measurable chemical GSH conjugation, underlying the importance of GST for the biotransformation of these epoxides. The results confirm the substrate concentration-dependent kinetic mechanism of GST isoforms first reported by William B. Jakoby (J. Biol. Chem. 1974) for exogenous electrophiles including the epoxide 1,2-epoxy-3-(p-nitrophenoxy)propane and the organic nitrates. This mechanism allows for maximal GST activity that can be achieved under given concentrations of GSH, epoxides and other electrophiles.

Biotransformation of organic nitrates by glutathione S-transferases and other enzymes: An appraisal of the pioneering work by William B. Jakoby.

Organic nitrates (R-ONO2; R, organic residue) such as nitroglycerin are used as drugs in part for more than a century. Their pharmacological use is associated with clinically relevant tolerance which is reportedly known since 1888. The underlying mechanisms of both, the mechanisms of action and the main pharmacological effect, which is vasodilatation and reduction of blood pressure, and the development of tolerance, which means increasing need of drug amount in sustained long-term therapy, are still incompletely understood. William B. Jakoby and associates were the first to report the biotransformation of organic nitrates, notably including nitroglycerin (i.e., glycerol trinitrate; GTN), by glutathione S-transferase (GST)-catalyzed conjugation of glutathione (GSH) to the nitrogen atom of one of the three nitrate groups of GTN to generate glutathione sulfenyl nitrite (glutathione thionitrate, S-nitroglutathione; GSNO2). Jakoby's group was also the first to suggest that GSNO2 reacts with a second GSH molecule to produce inorganic nitrite (ONO-) and glutathione disulfide (GSSG) without the catalytic involvement of GST. This mechanism has been adopted by others to the biotransformation of GTN by mitochondrial aldehyde dehydrogenase (mtALDH-(CysSH)2) which does not require GSH as a substrate. The main difference between these reactions is that mtALDH forms an internal thionitrate (mtALDH-(CysSH)-CysSNO2) which releases inorganic nitrite upon intra-molecular reaction to form mtALDH disulfide (mtALDH-(CysS)2). Subsequently, ONO- and GSNO2 are reduced by several proteins and enzymes to nitric oxide (NO) which is a very potent activator of soluble guanylyl cyclase to finally relax the smooth muscles thus dilating the vasculature. GSNO2 is considered to rearrange to GSONO which undergoes further reactions including GSNO and GSSG formation. The present article is an appraisal of the pioneering work of William B. Jakoby in the area of the biotransformation of organic nitrates by GST. The two above mentioned enzymatic reactions are discussed in the context of tolerance development to organic nitrates, still a clinically relevant pharmacological concern.

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.

N-Acetyl-L-cysteine in human rheumatoid arthritis and its effects on nitric oxide (NO) and malondialdehyde (MDA): analytical and clinical considerations.

N-Acetyl-L-cysteine (NAC) is an endogenous cysteine metabolite. The drug is widely used in chronic obstructive pulmonary disease (COPD) and as antidote in acetaminophen (paracetamol) intoxication. Currently, the utility of NAC is investigated in rheumatoid arthritis (RA), which is generally considered associated with inflammation and oxidative stress. Besides clinical laboratory parameters, the effects of NAC are evaluated by measuring in plasma or serum nitrite, nitrate or their sum (NOx) as measures of nitric oxide (NO) synthesis. Malondialdehyde (MDA) and relatives such as 4-hydroxy-nonenal and 15(S)-8-iso-prostaglandin F2α serve as measures of oxidative stress, notably lipid peroxidation. In this work, we review recent clinico-pharmacological studies on NAC in rheumatoid arthritis. We discuss analytical, pre-analytical and clinical issues and their potential impact on the studies outcome. Major issues include analytical inaccuracy due to interfering endogenous substances and artefactual formation of MDA and relatives during storage in long-term studies. Differences in the placebo and NAC groups at baseline with respect to these biomarkers are also a serious concern. Modern applied sciences are based on data generated using commercially available instrumental physico-chemical and immunological technologies and assays. The publication process of scientific work rarely undergoes rigorous peer review of the analytical approaches used in the study in terms of accuracy/trueness. There is pressing need of considering previously reported reference concentration ranges and intervals as well as specific critical issues such as artefactual formation of particular biomarkers during sample storage. The latter especially applies to surrogate biomarkers of oxidative stress, notably MDA and relatives. Reported data on NO, MDA and clinical parameters, including C-reactive protein, interleukins and tumour necrosis factor α, are contradictory in the literature. Furthermore, reported studies do not allow any valid conclusion about utility of NAC in RA. Administration of NAC patients with rheumatoid arthritis is not recommended in current European and American guidelines.

Specific and sensitive GC-MS analysis of hypusine, Nε-(4-amino-2-hydroxybutyl)lysine, a biomarker of hypusinated eukaryotic initiation factor eIF5A, and its application to the bi-ethnic ASOS study.

Hypusination is a unique two-step enzymatic post-translational modification of the Nε-amino group of lysine-50 of the eukaryotic initiation factor 5A (eIF5A). We developed a specific and sensitive gas chromatography-mass spectrometry (GC-MS) method for the measurement of biological hypusine (Hyp), i.e., Nε-(4-amino-2-hydroxybutyl)lysine. The method includes a two-step derivatization of Hyp: first esterification with 2 M HCl in CH3OH (60 min, 80 °C) to the methyl ester (Me) and then acylation with penta-fluoro-propionic (PFP) anhydride in ethyl acetate (30 min, 65 °C). Esterification with 2 M HCl in CD3OD was used to prepare the internal standard. The major derivatization product was identified as the un-labelled (d0Me) and the deuterium-labelled methyl esters (d3Me) derivatives: d0Me-Hyp-(PFP)5 and d3Me-Hyp-(PFP)5, respectively. Negative-ion chemical ionization generated the most intense ions with m/z 811 for d0Me-Hyp-(PFP)5 and m/z 814 for the internal standard d3Me-Hyp-(PFP)5. Selected-ion monitoring of m/z 811 and m/z 814 was used in quantitative analyses. Free Hyp was found in spot urine samples (10 µL) of two healthy subjects at 0.60 µM (0.29 µmol Hyp/mmol creatinine) in the female and 1.80 µM (0.19 µmol Hyp/mmol creatinine) in the male subject. The mean accuracy of the method in these urine samples spiked with 1-5 µM Hyp was 91-94%. The limit of detection (LOD) of the method is 1.4 fmol Hyp. The method was applied to measure the urinary excretion rates of Hyp in healthy black (n = 38, age 7.8 ± 0.7 years) and white (n = 41, age 7.7 ± 1.0 years) boys of the Arterial Stiffness in Offspring Study (ASOS). The Hyp concentrations were 3.55 [2.68-5.31] µM (range 0.54-9.84 µM) in the black boys and 3.87 [2.95-5.06] µM (range 1.0-11.7 µM) in the white boys (P = 0.64). The creatinine-corrected excretion rates were 0.25 [0.20-0.29] µmol/mmol (range 0.11-0.36 µmol/mmol) in the black boys and 0.26 [0.21-0.30] µmol/mmol (range 0.10-0.45 µmol/mmol) in the white boys (P = 0.82). These results suggest that there is no ethnic-related difference in the ASOS population in the eIF5A modification. Remarkable differences were found between black and white boys with respect to correlations of urinary Hyp with amino acids and advanced glycation end-products of Lys, Arg and Cys. Deoxyhypusine, formally the direct precursor of Hyp, seems not to be excreted in the urine by healthy subjects.

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).