Myocardial S-adenosylhomocysteine hydrolase is important for adenosine production during normoxia

(1) The coronary vasodilator adenosine can be formed in the heart by breakdown of AMP or S-adenosylhomocysteine (SAdoHcy). The purpose of this study was to get insight into the relative importance of these routes of adenosine formation in both the normoxic and the ischemic heart. (2) A novel HPLC method was used to determine myocardial adenosine and SAdoHcy. Accumulation of SAdoHcy was induced in isolated rat hearts by perfusion with L-homocysteine thiolactone or L-homocysteine. The release of adenosine, inosine, hypoxanthine, xanthine and uric acid was determined. Additional in vitro experiments were performed to determine the kinteic parameters of S-adenosylhomocysteine hydrolase. (3) During normoxia the thiolactone caused a concentration-dependent increase in SAdoHcy. At 2000 μM of the thiolactone an SAdoHcy accumulation of 0.49 nmol/min per g wet weight was found during normoxia. L-Homocysteine (200 μM) caused an increased of 0.37 and 4.17 nmol SAdony/soc per g wet weight during normaxia and ischemia, respectively. (4) The adenosine concentration in ischemic hearts was significantly lower when homocysteine was infused (6.2 vs. 115 nmol/g; P < 0.05). Purine release was increased 4-fold during ischemia. (5) The K_m for hydrolysis of SAdoHcy was about 12 μM. At in vitro conditions favoring near-maximal SAdoHcy synthesis (72 μM adenosine, 1.8 mM homocysteine), the synthesis rate in homogenates was 10 nmol/min per g wet weight. (6) From the combined in vitro and perfusion studies, we comclude that S-adenosylhomocysteine hydrolase can contribute significantly to adenosine production in normoxic rat heart, but not during ischemia.     

Boron deprivation decreases liver S-adenosylmethionine and spermidine and increases plasma homocysteine and cysteine in rats

Two experiments were conducted with weanling Sprague–Dawley rats to determine whether changes in S-adenosylmethionine utilization or metabolism contribute to the diverse responses to boron deprivation. In both experiments, four treatment groups of 15 male rats were fed ground corn-casein based diets that contained an average of 0.05 mg (experiment 1) or 0.15 mg (experiment 2) boron/kg. In experiment 2, some ground corn was replaced by sucrose and fructose to increase oxidative stress. The dietary variables were supplemental 0 (boron-deprived) or 3 (boron-adequate) mg boron/kg and different fat sources (can affect the response to boron) of 75 g corn oil/kg or 65 g fish (menhaden) oil/kg plus 10 linoleic acid/kg. When euthanized at age 20 (experiment 1) and 18 (experiment 2) weeks, rats fed the low-boron diet were considered boron-deprived because they had decreased boron concentrations in femur and kidney. Boron deprivation regardless of dietary oil increased plasma cysteine and homocysteine and decreased liver S-adenosylmethionine, S-adenosylhomocysteine, and spermidine. Plasma concentration of 8-iso-prostaglandin F_2α (indicator of oxidative stress) was not affected by boron, but was decreased by feeding fish oil instead of corn oil. Fish oil instead of corn oil decreased S-adenosylmethionine, increased spermidine, and did not affect S-adenosylhomocysteine concentrations in liver. Additionally, fish oil versus corn oil did not affect plasma homocysteine in experiment 1, and slightly increased it in experiment 2. The findings suggest that boron is bioactive through affecting the formation or utilization of S-adenosylmethionine. Dietary fatty acid composition also affects S-adenosylmethionine formation or utilization, but apparently through a mechanism different from that of boron.     

Inhibition of phospholipid methylation in isolated rat hepatocytes by analogues of adenosine and S-adenosylhomocysteine

Phospholipid methylation in isolated hepatocytes was inhibited in the presence of 3-deazaadenosine (ID₅₀ = 1.7 μM) 9-β-d-arabinofuranosyladenine (ID₅₀ = 6.0 μM), S-tubercidinylhomocysteine (ID₅₀ = 30 μM), and 5′-deoxy-5′-isobutylthioadenosine (ID₅₀ = 177 μM). A transient inhibitory effect was observed with adenosine, whereas S-adenosyl-l-homocysteine and Sinefungin were essentially without effect. The inhibition of phospholipid methylation by S-tubercidinylhomocysteine and 9-β-d-arabinofuranosyladenine showed a lag-phase, whereas the effect of the other inhibitors was apparent within a few minutes. Cells exposed to 9-β-d-arabinofuranosyladenine or 3-deazaadenosine accumulated large amounts of AdoHcy, and adenosine induced a transient increase in the AdoHcy level. In addition, 3-deazaadenosine served as a precursor for the formation of S-3-deazaadenosylhomocysteine, which accumulated rapidly in cells exposed to this agent. The inhibitory effects of 3-deazaadenosine, 9-β-d-arabinofuranosyladenine and adenosine could be explained by the increase in total nucleosidylhomocysteine induced by these agents. In contrast, only a slight (less than 2-fold) increase in S-adenosyl-l-homocysteine content was observed in hepatocytes treated with 5′-deoxy-5′-isobutylthioadenosine, and this metabolic effect could not explain the inhibition of phospholipid methylation induced by this agent. None of the compounds tested reduced the amount nor the specific radioactivity of S-adenosylmethionine. Biological processes determining the inhibitory effects of adenosine, S-adenosyl-l-homocysteine and their analogues on phospholipid methylation in intact cells are discussed.     

A new structural class of S-adenosylhomocysteine hydrolase inhibitors

Effective inhibitors of S-adenosylhomocysteine hydrolase hold promise towards becoming useful therapeutic agents. Since most efforts have focused on the development of nucleoside analog inhibitors, issues regarding bioavailability and selectivity have been major challenges. Considering the marine sponge metabolite ilimaquinone was found to be a competitive inhibitor of S-adenosylhomocysteine hydrolase, new opportunities for developing selective new inhibitors of this enzyme have become available. Based on the activities of various hybrid analogs, SAR studies, pharmacophore modeling, and computer docking studies have lead to a predictive understanding of ilimaquinone’s S-adenosylhomocysteine hydrolase inhibitory activities. These studies have allowed for the design and preparation of simplified structural variants possessing new furanoside bioisosteres with 100-fold greater inhibitory activities than that of the natural product.     

A Novel Family of S-Nitrosothiols: Chemical Synthesis and Biological Actions

S-Nitrosothiols are a class of chemical compounds that decompose to release nitric oxide and show promise in the treatment of a variety of cardiovascular diseases. Some of these are present in vivo and others have been synthesized in vitro. However, those discovered or synthesized to date have very little tissue selectivity or specificity. We synthesized a number of novel S-nitrosated dipeptides of high purity and examined their effects on vasorelaxation using rat mesenteric arteries and on inhibition of platelet aggregation using platelets from healthyhuman subjects. For comparison, we also tested the effects of S-nitroso- -glutathione (GSNO, an S-nitrosothiol present in vivo) and S-nitroso-N-acetyl- -β,β-dimethylcysteine (SNAP(D), the -isomer of SNAP, a commonly used S-nitrosothiol previously synthesized in vitro) in these biological systems. Satisfactory elemental analyses were obtained for all compounds synthesized (less than ± 0.3%), and all accurate mass measurements were within 1–5 ppm of the expected mass. The novel S-nitrosated dipeptides all elicited vasorelaxation with significantly higher potency, of the order of one log molar unit, than either GSNO or SNAP(D). However, all compounds inhibited U46619-induced platelet aggregation with similar potency to GSNO and SNAP(D). These findings indicate a degree of tissue selectivity which may prove to be of therapeutic usefulness.     

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.     

Application of capillary gas chromatography to the study of hydrolysis of the nerve agent VX in rat plasma

We present here a gas chromatography technique allowing the detection and quantification of VX [O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate] a as well as its P---S bond hydrolysis product diisopropylaminoethanethiol directly from spiked rat plasma. This technique was applied to study VX hydrolysis in rat plasma. We observed that 53±4% of 374 μM VX disappeared from spiked plasma after 2 h. VX disappearance was mainly related to enzymatic cleavage of the P---S bond (K_m=2.5 mM and V_max=13.3 nmol min⁻¹ of rat plasma). The activity was totally inhibited by 1 mM Hg²⁺ and was also inhibited by metal chelators.     

Analysis of the 5′ non-coding region of rat liver S-adenosylmethionine synthetase mRNA and comparison of the Mr deduced from the cDNA sequence and the purified enzyme

A 3 kb cDNA coding for rat liver S-adenosylmethionine (AdoMet) synthetase has been isolated. The M_r of the protein has been unequivocally determined by cDNA sequencing and enzyme purification on a thiopropyl-Sepharose column. The length of the mRNA 5′ non-coding region has been defined by primer-extension analysis. The rat liver cloned cDNA has been also used to detect S-adenosylmethionine synthetase mRNA in human liver.     

The rate of transmethylation in mouse liver as measured by trapping S-adenosylhomocysteine

Periodate-oxidized adenosine has previously been shown to be a potent inhibitor in vitro of S-adenosylhomocysteine hydrolase (E.C. 3.3.1.1). This paper describes the inhibition of this enzyme in liver following injection of mice with periodate-oxidized adenosine. A maximally effective dose of 100 nmol/g of this compound causes liver S-adenosylhomocysteine to increase from 12 to 600 nmol/g within 30 min. This accumulation of S-adenosylhomocysteine provides an estimate of the rates of transmethylation, as well as adenosine and homocysteine production, as being at least 20 nmol/min/g liver. A doubling of S-adenosylmethionine in the liver of mice treated with periodate-oxidized adenosine suggests that the high levels of S-adenosylhomocysteine inhibit some transmethylation reactions.     

Comparison of anion-exchange and ion-modified reversed-phase liquid chromatography for the determination of S-sulfocysteine

A dual Hg–Au amalgam electrode is used to detect S-sulfocysteine (SSC) in this study. There exist two main components in the acetonitrile (ACN) rat brain extracts, namely, Cl⁻ and GSSG (oxidized glutathione), that are active in our detection system (GSH is not extracted in ACN). Two strong anion-exchange columns from different companies were used to separate the samples under different conditions, but SSC and Cl⁻ were not separated at the optimum detection pH of 5.2. The signal from Cl⁻ was greatly decreased by lowering the potential at the downstream electrode, though it cannot be completely eliminated. While a silver cartridge removed Cl⁻ from micromoles to several millimoles without any negative effect on the SSC signal in aqueous standards, a large negative peak which interferes with SSC detection was unfortunately introduced when a silver cartridge was applied to brain tissue samples. However, SSC and Cl⁻ in the samples are successfully separated by ion-modified reversed-phase LC in acetate buffer at the optimum detection pH (5.2). The separation conditions are 20 mM acetic acid, 2% methanol, 0.5 mM cetyltrimethylammonium p-toluene sulfonate (CTMA) (pH 5.2). Most importantly, the sensitivity of SSC under the optimum separation conditions is not sacrificed. The detection limit is 8 nM (20 μl injected).     

Gas chromatographic-mass spectrometric determination of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol for study on human urinary excretion of ginsenosides after ingestion of ginseng preparations

An improved gas chromatographic-mass spectrometric method (GC-MS) with a fast solid-phase extraction on a newly introduced C₁₈ microcolumn, was applied to study the urinary excretion of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol glycosides in man after oral administration of ginseng preparations. Using panaxatriol as internal standard, 20(S)-protopanaxadiol and 20(S)-protopanaxatriol (the aglucones of ginesenosides) could be determined at a detection level of a few ng per ml urine by GC-MS with selected-ion monitoring after their release from glycosides which occur in urine. The extraction recovery of ginsenosides from urine was more than 80% and the intra-assay coefficient of variation was less than 5.0%. The results after intake of single doses of ginseng preparations demonstrated a linear relation between the amounts of ginsenosides consumed and the 20(S)-protopanaxatriol glycosides excreted in urine. About 1.2% of the dose was recovered in five days.     

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.     

Modulation by the ratio of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio can modulate phosphorylation of a specific protein.     

High-performance liquid chromatography of the renal blood flow marker p-aminohippuric acid (PAH) and its metabolite N-acetyl PAH improves PAH clearance measurements

PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart^® (125 mm×4 mm I.D.) Nucleosil 100-5 μm C₁₈ AB cartridge column, using a gradient elution of MeOH–buffer pH 3.9 1:99→15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate–citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H₃PO₄ 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2±2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (Cl_R) and systemic (Cl_S) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney Cl_R/Cl_S calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73±0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87±0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.     

Determination of 5-S-cysteinyldopa in plasma and urine using a fully automated solid-phase extraction–high-performance liquid chromatographic method for an improvement of specificity and sensitivity of this prognostic marker of malignant melanoma

5-S-Cysteinyldopa (5-SCD) in plasma and urine was determined by means of a newly developed method. This method incorporates optimized conditions for blood collection and storage, as well as a new extraction and separation technique, required for the strong oxidation and light sensitive 5-SCD. The new aspects of the method are the following: immediate centrifugation and freezing of the samples after blood collection, fully automatical solid-phase extraction (SPE) with phenylboronic acid (PBA) cartridges and immediate HPLC injection of the eluate, nearly complete exclusion of light and air–oxygen during extraction, constant sample cooling, use of the more suitable internal standard 5-S- -cysteinyldopa and easy, sensitive and selective HPLC conditions (RP18-column with isocratic separation and electrochemical detection). The method has a linear range from 0.25 to 50 μg l⁻¹ and 25 to 5000 μg l⁻¹ for plasma and urine samples, respectively, a limit of detection of 0.17 μg l⁻¹, intra-assay variabilities from 1.7 to 3.6%, inter-assay variabilities from 4.0 to 18.3% and an average relative recovery of 103.5% for plasma and 105.4% for urine samples. In our study the measured 5-SCD concentrations of patients with melanomas at various stages correlated better with their clinical pictures than described in literature up to date. The results were obtained in comparison to patients with other skin tumors and in comparison to healthy control persons.     

l-NAME has Opposite Effects on the Productions of S-adenosylhomocysteine and S-adenosylmethionine in V12-H-Ras and M-CR3B-Ras Pheochromocytoma Cells

Homocysteine is a sulfur-containing, nonproteinogenic, neurotoxic amino acid biosynthesized during methyl cycles after demethylation of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) and subsequent hydrolysis of SAH into homocysteine and adenosine. Formed homocysteine is either catabolized into cystathionine (transsulfuration pathway) by cystathionine β-synthase, or remethylated into methionine (remethylation pathway) by methionine synthase. To demonstrate the specificity of Ras-elicited effects on the activity of methyl cycles, wild-type pheochromocytoma PC12, mutant oncogenic rasH gene (MVR) expressing PC12 pheochromocytoma and normal c-rasH stably transfected M-CR3B cells were incubated with the N^ω-nitro-l-arginine methyl ester (l-NAME), and manumycin, (inhibitors of nitric oxide synthase and farnesyltransferase, respectively). We have found that l-NAME significantly changes the SAM/SAH ratio in both MCR and MVR cells. Moreover, these alterations have reciprocal character; in the MCR cells, the SAM/SAH ratio was raised, whereas in the MVR cells this ratio was decreased. We conclude that depletion of endogenous NO with l-NAME increased the production of SAH only in cells with mutated oncogenic RasH, possibly through enhancement of production of reactive oxygen species (ROS). Oxidative stress can increase cystathionine β-synthase activity that switches methyl cycles from remethylation into transsulfuration pathway to maintain the intracellular glutathione pool (essential for the redox-regulating capacity of cells) via an adaptive process.     

Properties of γ-aminobutyraldehyde dehydrogenase from Escherichia coli

γ-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an M_r of 195 000±10 000 in its dimeric form with an M_r of 95 000±1000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. K_m values are 31.3±6.8 μM and 53.8±7.4 μM for Δ-1-pyrroline and NAD⁺, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme.     

Fast liquid chromatographic determination of urinary trans,trans-muconic acid

trans,trans-Muconic acid (1,3-butadiene-1,4-dicarboxylic acid, MA), a minor urinary metabolite of benzene exposure, was determined, after clean-up by solid-phase anion-exchange chromatography, by reversed-phase HPLC on a C₁₈ column (5 × 0.46 cm I.D., 3 μm particle size), using formic acid-tetrahydrofuran-water (14:17:969) as mobile phase and UV detection at 263 nm. The recovery of MA from spiked urine was > 95% in the 50–500 μg/l range; the quantification limit was 6 μg/l; day-to-day precision, at 300 μg/l, C.V. = 9.2%; the run time was less than 10 min. Urinary MA excretion was measured in two spot urine samples of 131 benzene environmentally exposed subjects: midday values obtained in non-smokers (mean±S.D.=77±54 μg/l, N = 82) were statistically different from those of smoerks (169±85 μg/l, N = 30) (P<0.0001); each group showed a statistically significant increase between MA excretion in midday over morning samples. Moreover, in subjects grouped according to tobacco-smoke exposure level, median values of MA were positively associated with and increased with daily smoking habits.     

The S-n-propyl analogue of S-adenosylmethionine

A special strain of Saccharomyces cerevisiae responded to a supplement of S-n-propyl-l-homocysteine in the culture medium by synthesizing S-adenosyl-(S-n-propyl)l-homycysteine, the S-n-propyl analogue of S-adenosylmethionine. S-n-Butyl-l-homocysteine reacted sparingly with this strain, but S-isopropyl-l-homocysteine failed to form detectable quantities of the corresponding S-adenosylsulfonium were compound. The S-n-propyl compound was isolated by extraction of the cells, followed by ion-exchange chromatography, which separated it from endogenous S-adenosylmethionine. The structure was determined by hydrolytic procedures leading to overlapping fragments of known structure, 5′-n-propylthioadenosine and S-n-propyl-l-homocysteine. The new sulfonium compound was examined for its activity as n-propyl donor by substituting it for S-adenosylmethionine in methyltransferase systems. Enzymatic transpropylation was observed with S-adenosylmethionine: l-homocysteine S-methyltransferase (EC 2.1.1.10). Its rate was low in the S-adenosylmethionine: N-acetylserotonin O-methyltransferase system (EC 2.1.1.4), and below recognition with S-adenosylmethionine: guanidonoacetate methyltransferase (EC 21.1.2) and S-adnosylmethionine: histame N-methyltransferase (EC 2.1.1.8).