Determination of NG,NG-dimethylarginine in human plasma by high-performance liquid chromatography

N^G,N^G-Dimethylarginine (asymmetric dimethylarginine, ADMA) can be directly separated and measured from deproteinized human plasma using o-phthaldialdehyde-mercaptoethanol (OPA reagent) as a fluorogenic reagent by reversed-phase high-performance liquid chromatography. The mean recovery of ADMA was over 96% and the inter- and intra-assay coefficients of variation of amounts were lower than 3.80% and those of retention time were below 0.37% for five runs. The detection limit of the assay is 1 pmol when the signal-to-noise is 3:1. It was observed that the concentration of ADMA was significantly elevated in plasma of patients with pregnancy induced hypertension (PIH) in contrast to healthy pregnant women.     

Development of an Enzyme-Linked Immunosorbent Assay System for the Determination of Asymmetric Dimethylarginine Using a Specific Monoclonal Antibody

We produced a monoclonal antibody (mAb) against N ^G,N ^G-dimethyl-L-arginine (asymmetric dimethylarginine: ADMA), an endogenous competitive inhibitor of nitric oxide synthase (NOS), and developed an enzyme-linked immunosorbent assay (ELISA). The competitive ELISA method using the mAb determined 5 nM–100 nM ADMA, and ADMA levels in human plasma and urine were found to be 0.78 μM and 51.3 μmol/g of creatinine respectively.     

Asymmetric Dimethylarginine,an Endogenous NOS Inhibitor,Is Actively Metabolized in Rat Erythrocytes

N ^G,N ^G-Dimethyl-L-arginine (asymmetric dimethylarginine: ADMA) is an endogenous competitive inhibitor of nitric oxide synthase (NOS). Plasma ADMA concentrations have been reported to increase in connection with diseases associated with an impaired endothelial L-arginine/NO pathway. In this study, we investigated the metabolism of ADMA in circulating blood cell populations to elucidate the regulatory mechanism of elevation of plasma ADMA, a novel risk factor for cardiovascular disease. We found by RT-PCR and Western blot analyses that protein arginine methyltransferase (PRMT)1 and dimethylarginine dimethylaminohydrolase (DDAH)-1, responsible for the biosynthesis and degradation of ADMA respectively, are expressed in erythrocytes (ECs), leukocytes, and platelets. We also identified a major ADMA-containing protein in ECs as catalase, confirmed by GST-pull down assay to bind to PRMT1 in vitro. This is the first report that the ADMA-metabolizing system, including the arginine methylation of proteins and the breakdown of free ADMA, occurs in circulating blood cell-populations, and that catalase in ECs might be a potential protein targeted by PRMT1.     

Synthesis of N-monomethylagmatine, a decarboxylation product of N-monomethyl- -arginine

N^G-Monomethylagmatine, a decarboxylation product of N^G-monomethyl- -arginine, has been synthesized by reacting putrescine with N,S-dimethylthiopseudouronium iodide. The structural identity of the product was confirmed by proton NMR and mass spectroscopy, and its properties were determined on thin-layer and electrophoretic chromatography.     

Detection and quantification of α-keto-δ-(N,N-dimethylguanidino)valeric acid: A metabolite of asymmetric dimethylarginine

Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate l-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N^G,N^G-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of DMGV. D₆-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 μmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 μmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2.     

Biosynthesis of homoarginine (hArg) and asymmetric dimethylarginine (ADMA) from acutely and chronically administered free <Emphasis Type="SmallCaps">l</Emphasis>-arginine in humans

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis, whereas l-arginine (Arg) and l-homoarginine (hArg) serve as substrates for NO synthesis. ADMA and other methylated arginines are generally believed to exclusively derive from guanidine (N ^G)-methylated arginine residues in proteins by protein arginine methyltransferases (PRMTs) that use S-adenosylmethionine (SAM) as the methyl donor. l-Lysine is known for decades as a precursor for hArg, but only recent studies indicate that arginine:glycine amidinotransferase (AGAT) is responsible for the synthesis of hArg. AGAT catalyzes the formation of guanidinoacetate (GAA) that is methylated to creatine by guanidinoacetate methyltransferase (GAMT) which also uses SAM. The aim of the present study was to learn more about the mechanisms of ADMA and hArg formation in humans. Especially, we hypothesized that ADMA is produced by N ^G-methylation of free Arg in addition to the known PRMTs-involving mechanism. In knockout mouse models of AGAT- and GAMT-deficiency, we investigated the contribution of these enzymes to hArg synthesis. Arg infusion (0.5 g/kg, 30 min) in children (n = 11) and ingestion of high-fat protein meals by overweight men (n = 10) were used to study acute effects on ADMA and hArg synthesis. Daily Arg ingestion (10 g) or placebo for 3 or 6 months by patients suffering from peripheral arterial occlusive disease (PAOD, n = 20) or coronary artery disease (CAD, n = 30) was used to study chronic effects of Arg on ADMA synthesis. Mass spectrometric methods were used to measure all biochemical parameters in plasma and urine samples. In mice, AGAT but not GAMT was found to contribute to plasma hArg, while ADMA synthesis was independent of AGAT and GAMT. Arg infusion acutely increased plasma Arg, hArg and ADMA concentrations, but decreased the plasma hArg/ADMA ratio. High-fat protein meals acutely increased plasma Arg, hArg, ADMA concentrations, as well as the plasma hArg/ADMA ratio. In the PAOD and CAD studies, plasma Arg concentration increased in the verum compared to the placebo groups. Plasma ADMA concentration increased only in the PAOD patients who received Arg. Our study suggests that in humans a minor fraction of free Arg is rapidly metabolized to ADMA and hArg. In mice, GAMT and N ^G-methyltransferases contribute to ADMA and hArg synthesis from Arg, whereas AGAT is involved in the synthesis of hArg but not of ADMA. The underlying biochemical mechanisms remain still elusive.     

First detection and quantification of N-monomethylarginine,a structural isomer of N-monomethylarginine,in humans using MS

The l-arginine metabolites methylated at the guanidino moiety, such as N^G-monomethyl-l-arginine (LNMMA), asymmetric N^G,N^G-dimethyl-l-arginine (ADMA), and symmetric N^G,N^G'-dimethyl-l-arginine (SDMA), are long known to be present in human plasma. Far less is known about the structural isomer of LNMMA, N^δ-monomethyl-l-arginine (δ-MMA). In prior work, it has been detected in yeast proteins, but it has not been investigated in mammalian plasma or cells. In this work, we present a method for the simultaneous and unambiguous quantification of LNMMA and δ-MMA in human plasma that is capable of detecting δ-MMA separately from LNMMA. The method comprises a simple protein precipitation sample preparation, hydrophilic interaction liquid chromatography (HILIC) gradient elution on an unmodified silica column, and triple stage mass spectrometric detection. Stable isotope-labeled D₆-SDMA was used as internal standard. The calibration ranges were 25–1000 nmol/L for LNMMA and 5–350 nmol/L for δ-MMA. The intra- and inter-batch precision determinations resulted in relative standard deviations of less than 12% for both compounds with accuracies of less than 6% deviation from the expected values. In a pilot study enrolling 10 healthy volunteers, mean concentrations of 48.0 ± 7.4 nmol/L for LNMMA and 27.4 ± 7.7 nmol/L for δ-MMA were found.     

Decomposition of N-nitrosamines, and concomitant release of nitric oxide by Fenton reagent under physiological conditions

N-Nitrosodimethylamine (NDMA) in phosphate buffer was rapidly decomposed by Fenton reagent composed of H₂O₂, and Fe(II) ion. Electron spin resonance (ESR) studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that characteristic four line 1:2:2:1 ESR signals due to the DMPO-OH adduct formed on treatment of DMPO with Fenton reagent disappeared in the presence of NDMA, and N-nitrosodiethylamine (NDEA), suggesting the interaction of the N-nitrosamines with Fenton reagent. Treatment of the N-nitrosamines with Fenton reagent generated nitric oxide (NO) as estimated by ESR technique using cysteine–Fe(II), and N-methyl- -glucaminedithiocarbamate (MGD)–Fe(II) complexes. Characteristic 3, and single line signals due to 2 cysteine–Fe(II)–NO, and 2 cysteine–Fe(II)–2 NO complexes, respectively, and three line signals due to MGD–Fe(II)–NO were observed. Considerable amount of NO were liberated as determined by NO₂⁻, the final oxidation product of NO formed by reaction with dissolved oxygen in the aqueous medium. Spontaneous release of a small amount of NO from the N-nitrosamines was observed only on incubation in neutral buffers. Above results indicate that the N-nitrosamines were decomposed accompanying concomitant release of NO on contact with reactive oxygen species.     

Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.     

Analysis of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine, the mercapturic acid derived from N,N-dimethylformamide

Human biotransformation of the industrial solvent N,N-dimethylformamide gives raise to N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) which has the longest half-life (about 23 h) among urinary metabolites of N,N-dimethylformamide. It could be used for monitoring industrial exposure over several workdays, by measuring it in urine samples collected at the end of the working week. This is consistent with the suggestions of the American Conference of Governmental Industrial Hygienists, which established a limit of 40 mg/l for the year 2000. An easy, cheap and user-friendly method has been developed for determination of urinary AMCC. Unlike currently available methods, it requires neither a time-consuming preparation phase nor gas chromatographic analysis with a nitrogen-phosphorus or mass detector. The method uses high-performance liquid chromatography (HPLC), with an UV detector at 436 nm. A 10-μl volume of urine is added to a carbonate–hydrogen carbonate buffer and mixed with a dabsyl chloride solution in acetonitrile. The reaction between AMCC and the reagent is performed at 70°C for 10 min. The ‘dabsylated’ product is stable for at least 12 h. After brief centrifugation, the solution is ready for HPLC analysis using a C₁₈ column (250×4.6 mm, 5 μm). The method is sensitive (detection limit 1.8 mg/l) and specific. It identified urinary AMCC in urine of 40 subjects not exposed to N,N-dimethylformamide with a median concentration of 3.9 mg/l. In urine samples from 20 workers exposed to N,N-dimethylformamide (5–40.8 mg/m³), AMCC concentrations ranged from 16 to 170 mg/l. Industrial toxicology laboratories with limited instrumentation will be able to use it in the biological monitoring of workers exposed to N,N-dimethylformamide.     

Analysis of cysteine and N-acetylcysteine in human plasma by high-performance liquid chromatography at the basal state and after oral administration of N-acetylcysteine

A high-performance liquid chromatographic method for the determination of free reduced cysteine and N-acetylcysteine in human plasma at the basal state and after oral administration of N-acetylcysteine is described. The method is based on acid-catalysed conversion of plasma thiols to the corresponding S-nitroso derivatives by excess of nitrite and their subsequent cation-pairing RP-HPLC with detection at 333 nm. Recovery rates of cysteine and N-acetylcysteine added to human plasma were 94.6 and 99.6%, respectively. Inter- and intra-day precision were below 6%. In healthy humans (n=5), free reduced cysteine was determined to be (mean±S.E.) 10.0±0.96 μM. No N-acetylcysteine was detected in plasma of these subjects above the limit of detection (e.g. 170 nM). The method was successfully applied to a pharmacokinetic study on orally administered N-acetylcysteine to healthy volunteers.     

DNA sequence dependence of guanine-O alkylation by the N-nitroso carcinogens N-methyl- and N-ethyl-N-nitrosourea

After intracellular in vitro exposure to the mutagenic and carcinogenic N-nitroso compounds N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), respectively, the average relative amounts of the premutational lesion O⁶-alkylguanine represent about 6% and 8% of all alkylation products formed in genomic DNA. At the level of individual DNA molecules gunine-O⁶ alkylation does nor occur at random; rather, the probability of a substitution reaction at the nucleophilic O⁶ atom is influenced by nucleotide sequence, DNA conformation, and chromatin structure. In the present study, 5 different double-stranded polydeoxynucleotides and 15 double-stranded oligodeoxynucleotides (24-mers) were reacted with MeNU or EtNU in vitro under standardized conditions. Using a competitive radioimmunoassay in conjunction with an anti-(O⁶-2′-deoxyguanosine) monoclonal antibody, the frequency of guanine-O⁶ alkylation was found to be strongly dependent on the nature of the nucleotides flanking guanine on the 5t́ and 3′ sides. Thus, a 5′ neighboring guanine, followed by 5t́ adenine and 5′ cytosine, provided an up to 10-fold more ‘permissive’ condition for O⁶-alkylation of the central guanine than a 5′ thymine (with a 5-methylcytocine in the 5′ position being only slightly less inhibitory). Thymine and cytosine were more ‘permissive’ when placed 3′ in comparison with their affects in the 5′ flanking position.     

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C₁₈ Sep-Pak^® cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150–200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4±10.6, 71.1±11.0, 82.3±6.7, 60.4±11.8, 63.6±10.1, 69.3±8.5, 68.3±12.0, 82.6±8.1, and from urine 55.9±9.8, 60.3±7.4, 77.9±9.1, 61.7±13.5, 68.6±8.9, 62.0±9.5, 72.9±9.1, and 72.1±8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.     

Radioenzymatic determination of CMP-N-acetylsialic acid and free N-acetylsialic acid in biological material

Free N-acetylsialic acid (NeuNAc) and CMP-N-acetylsialic acids (CMP-NeuNAc) are extracted from freeze-clamped or liquid nitrogen-frozen biological material by sequential extraction with cold acetone and acetone/water. [¹⁴C]NeuNAc and [¹⁴C]CMP-NeuNAc (20,000 dpm each) are added to the frozen material to correct for small losses occurring during the subsequent steps. NeuNAc and CMP-NeuNAc are separated by anion-exchange chromatography. CMP-NeuNAc is hydrolyzed with formic acid and again chromatographed on an ion-exchange column. The NeuNAc-containing fractions (representing free NeuNAc and CMP-NeuNAc) are converted to [¹⁴C]CMP-NeuNAc in the presence of [¹⁴C]CTP and CMP-NeuNAc synthetase. [¹⁴C]CMP-NeuNAc is separated by paper chromatography and the radioactivity measured by liquid scintillation counting. The amount of NeuNAc is calculated from a calibration curve obtained with NeuNAc standards. The small amounts of [¹⁴C]NeuNAc and [¹⁴C]CMP-NeuNAc added initially do not interfere with the final assay. The method gives reliable values down to 50 pmol/assay, but the sensitivity can be easily increased by a factor of 10. Recoveries, with NeuNAc and CMP-NeuNAc added to biological extracts, were 98.3 and 98.5% for NeuNAc and CMP-NeuNAc, respectively. With this method values of 61.2 ± 12.8 and 24.4 ± 5.2 nmol/g wet wt were found in rat liver for free NeuNAc and CMP-NeuNAc, respectively. Values for free NeuNAc found in human blood plasma were 600 ± 476 and 373 ± 180 pmol/g plasma for healthy persons and patients with breast cancer, respectively. Free CMP-NeuNAc could not be found in plasma.     

N-acetylglucosaminyltransferase V-deficiency increases susceptibility to murine malaria

It is considered that several glycoproteins on erythrocytes in mammalian species are involved in malaria parasite infection. To elucidate the role of N-glycans on malaria parasite infection, we induced experimental murine malaria infection (using Plasmodium berghei ANKA) in mice deficient in N-acetylglucosaminyltransferase V (Mgat5), which is one of the enzymes involved in β1,6-GlcNAc N-glycan biosynthesis. After infection, Mgat5^-/- mice showed severe body weight loss and parasitemia compared with wild-type mice. The Mgat5^-/- mice, but not wild-type mice, also showed severe pathology accompanied by marked infiltration of plasma cells into the lungs and liver. These results suggest that β1,6-GlcNAc N-glycans on/in host erythrocytes may interfere with invasion of the parasites and progression to severe malaria.     

Simultaneous bioanalysis of L-arginine, L-citrulline, and dimethylarginines by LC-MS/MS

Purposel-Arginine (ARG) is converted to nitric oxide (NO) and l-citrulline (CIT) by endothelial nitric oxide synthase which is competitively inhibited by asymmetric dimethylarginine (ADMA). We have developed a liquid chromatography–mass spectrometric method for the simultaneous determination of endogenous ARG, labeled ARG (¹⁵N₄-ARG), CIT, ADMA, and its inactive isomer, symmetric dimethylarginine (SDMA) in biological samples.MethodsConcentrations of unlabeled ARG, ¹⁵N₄-ARG, CIT, ADMA, and SDMA in EA.hy926 human endothelial cell lysate, cell incubation media, rat plasma or rat urine were measured by hydrophilic-interaction liquid chromatography electrospray tandem mass spectrometry. ¹³C₆-ARG, D₄-CIT and D₇-ADMA were used as internal standards for ARG and ¹⁵N₄-ARG, CIT, and dimethylarginines, respectively.ResultsThe calibration curves of ARG, ¹⁵N₄-ARG, CIT, ADMA, and SDMA were linear and independent of several sample matrices. Intra- and inter-day variabilities for the quantification of all the compounds were below 15% in quality control samples. Application of this method to determine the uptake as well as efflux of these compounds was illustrated through in vitro cell study by exposing human endothelial cells to ¹⁵N₄-ARG, which allowed the observation of generation of ¹⁵N₃-CIT and ¹⁵N₃-ARG in the cell lyate. Use of these isotopes adds insights into the cellular handling of endogenous vs. exogenous ARG. Application of this method for rat plasma and rat urine assays was demonstrated after ARG oral supplementation in rats.ConclusionAn LC–MS/MS method was developed to quantify 6 ARG-related compounds simultaneously, utilizing 3 separate internal standards. This assay allows concurrent monitoring of uptake, efflux and metabolic processes when isotope-labeled ARG and CIT are measured, and can be applied for determination of these compounds in rat plasma and rat urine.     

Turnover of N-acetylglutamate in isolated rat hepatocytes

Isolated hepatocytes from starved rats were loaded with N-[¹⁴C]acetylglutamate by preincubating them with [¹⁴C]bicarbonate, oleate, NH₃, ornithine and lactate. Turnover of N-acetylglutamate in these cells was subsequently measured in an unlabelled medium under conditions of minimal flux (oleate alone present) and maximal flux (oleate, NH₃, ornithine and lactate present) through the urea cycle. 1. Direct measurement of the distribution of N-[¹⁴C]acetylglutamate across the mitochondrial membrane in the hepatocytes showed that, under the conditions studied, the rate of degradation of total intracellular N-[¹⁴C]acetylglutamate was about equal to the rate of efflux of N-acetylglutamate from the mitochondria. 2. In the presence of oleate alone, intramitochondrial N-acetylglutamate decreased because mitochondrial N-acetylglutamate efflux predominated over the synthesis of N-acetylglutamate in the mitochondria. 3. In the presence of oleate, NH₃, ornithine and lactate both the rate of synthesis of N-acetylglutamate and the rate of its transport out of the mitochondria were increased when compared with the condition with oleate alone. However, the intramitochondrial concentration of N-acetylglutamate increased because initially the rate of its synthesis exceeded that of its efflux from the mitochondria. Finally, a steady state was reached in which both rates were equal. 4. The data indicate that in hepatocytes from starved rats N-acetylglutamate transport out of the mitochondria takes place at a rate proportional to its intramitochondrial concentration. It is concluded that transport of N-acetylglutamate either occurs by diffusion or is mediated by a transport system with a high K_m for intramitochondrial N-acetylglutamate.     

Intracellular localization of N-formyl chemotactic receptor and Mg dependent ATPase in human granulocytes

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83 000 × g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40–60% were achieved with a 20–70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g·cm⁻³. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimanted with specific granules (g9 = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg²⁺ -ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg²⁺ -ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.     

Gas chromatographic–mass spectrometric determination of α-ketoisocaproic acid and [H7]α-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

A method for determination of α-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-²H₇]α-ketoisocaproic acid ([²H₇]KIC) in rat plasma was developed using gas chromatography–mass spectrometry-selected ion monitoring (GC–MS-SIM). [5,5,5-²H₃]α-Ketoisocaproic acid ([²H₃]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [²H₃]KIC and [²H₇]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [²H₇]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [²H₇]KIC spiked to rat plasma in the range of 0.1 to 10 μM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [²H₇]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [²H₇]KIC after an intravenous administration of [²H₇]KIC in rat.     

Hydrazides of N-blocked amino acids as sole substrates with unique difunctional behavior under papain catalysis

Hydrazides of five N-acylamino acids have been used alone as substrates for papain catalysis to yield N¹,N²-diacylhydrazines. With the exception of N-(benzyloxycarbonyl)(Z)- -alanine hydrazide, they were very effective as both acylating agents of the enzyme and nucleophiles in attacking the enzyme-substrate intermediate. Although Z- -alanine hydrazide was a minimal acylating agent, it was a satisfactory nucleophile. The most favorable reaction involved Z- -alanine hydrazide in producing N¹,N²-bis(Z- -alanyl)hydrazine. When Z- -alanine hydrazide was the substrate, this same chiral diacylhydrazine was formed along with meso N¹-(Z- -alanyl)-N²-(Z- -alanyl)hydrazine. For the acylation step, the enzyme displayed powerful, essentially stereospecific, bias toward the enantiomer. Once the thioester intermediate was formed, little preference was detected for attack by the enantiomers as nucleophiles. The most direct procedure for synthesis of substrates was conversion of Z-amino acids to their esters by means of dry HCl in an absolute alcohol. Treatment with hydrazine produced the hydrazides in excellent yield.