Analysis of NO and its metabolites by mass spectrometry. Comment on 'Detection of nitric oxide in tissue samples by ESI-MS' by Z. Shen, A. Webster, K. J. Welham, C. E. Dyer, J. Greenman and S. J. Haswell.

Recently, Shen et al. (Analyst, 2010, 135, 302) reported on a flow injection analysis (FIA) ESI-MS/MS approach for the determination of the short-lived gaseous nitric oxide (NO) in biological samples. This method is based on the reaction of NO, and presumably of other NO-derived oxides such as N(2)O(3), with the vicinal amino groups of methylpiperazinobenzendiamine to form a benzotriazole derivative. Under MS/MS conditions, the protonated derivative loses molecular nitrogen (N(2)) from the triazole ring and the product ion formed is utilized for quantitative analyses. This seems to be the first ESI-MS/MS method for authentic NO detection and quantification. However, the ESI-MS/MS method reported by Shen et al. deserves some critical discussion.

Evaluation of vardenafil for the treatment of subjective tinnitus: a controlled pilot study.

BACKGROUND: Vardenafil (Levitra(R)) represents a potent and highly selective phosphodiesterase type 5 (PDE5) inhibitor, which is established for treatment of various diseases. There are several unpublished reports from patients stating that vardenafil has a considerable therapeutic effect on their concomitant tinnitus. This pilot study was conducted to specifically assess the effect of vardenafil in patients with chronic tinnitus. METHODS: This trial was based on a prospective, randomized, double-blind, placebo-controlled, parallel group design. Fourty-two consecutive subjects with mon- or binaural chronic tinnitus received 10 mg vardenafil (N = 21) or matching placebo tablets (N = 21) administered orally twice a day over a period of 12 weeks. Clinical examination and data acquisition took place at each visit: at baseline, after 4 weeks, after 12 weeks (end of treatment with study medication), and at non-medicated follow-up after 16 weeks. Assessment of clinical effectiveness was based on a standardized tinnitus questionnaire (TQ), the Short Form 36 health survey (SF-36), audiometric measurements (mode, pitch and loudness of tinnitus; auditory thresholds) and biomarkers of oxidative stress in patients' blood (malondialdehyde, protein carbonyl, homocysteine and total antioxidative status). Therapeutic efficacy was evaluated by comparison of subjective and objective parameters with baseline data between both treatment groups (ANCOVA). RESULTS: Vardenafil had no superior efficacy over placebo in the treatment of chronic tinnitus during this study. The primary efficacy criterion 'TQ total score' failed to demonstrate significant improvement compared to placebo. Subjective reports of TQ subscales and general quality of life areas (SF-36), objective audiometric examinations as well as investigated biomarkers for oxidative stress did not reveal any significant treatment effects. The safety profile was favorable and consistent with that in other vardenafil studies. CONCLUSION: Although hypoxia and ischemia play a special role in the pathogenesis of tinnitus, the PDE5-inhibitor-induced increase of nitric oxide-mediated vasodilatation exerted no specific influence on tinnitus symptomatology. Considering the unclear risk of rarely associated hearing impairment, systemic application of vardenafil or other PDE5 inhibitors prove to be not appropriate for therapy of chronic tinnitus.

Application of a stable-isotope dilution technique to study the pharmacokinetics of human 15N-labelled S-nitrosoalbumin in the rat: possible mechanistic and biological implications.

In the year 1992, S-nitrosoalbumin (SNALB) has been proposed to be the most abundant physiological carrier and pool of nitric oxide (NO) activity in human circulation, by which NO-dependent biological functions are regulated. The concentration, the metabolism and the mechanisms of the biological actions of SNALB are controversial and still incompletely understood. Moreover, the suitability of SNALB as a biomarker of diseases associated with altered NO bioactivity in human circulation has not been demonstrated convincingly so far. In the present study, we report on the development and application of a stable-isotope technique to study the pharmacokinetics of (15)N-labelled SNALB (S(15)NALB) in anesthetized rats. S(15)NALB was synthesized from albumin isolated by affinity chromatography from freshly prepared human plasma. This technique was also applied to study and quantify the formation of S(15)NALB from endogenous rat plasma albumin and intravenously applied S-[(15)N]nitrosoglutathione (GS(15)NO) or S-[(15)N]nitrosocysteine (S(15)NC) in anesthetized rats. In these investigations the mean arterial pressure (MAP) was monitored continuously. The elimination half-life (t(1/2)) of S(15)NALB from rat plasma was determined to be 4.1 min (t(1/2)alpha) and 9.4 min (t(1/2)beta). S(15)NALB (125 nmol) produced long-lasting decreases in MAP (by 49% for 18 min). Thirty minutes after intravenous (i.v.) injection of S(15)NALB (125 nmol), repeated i.v. injection of L-cysteine or D-cysteine (10 micromol each) produced repeatedly potent (by 44-55%) but short-lasting (about 4 min) MAP falls. Intravenously administered GS(15)NO and S(15)NC (each 500 nmol) could not be isolated from rat blood. (15)N-Labelled nitrite and nitrate were identified as the major metabolites of all investigated S-nitrosothiols in rat plasma. The results of this study suggest that in the rat S(15)NALB is a potent S-transnitrosylating agent and that the blood pressure-lowering effect of S(15)NALB and other S-nitrosothiols are mediated largely by L-cysteine via S-transnitrosylation to form S(15)NC that subsequently releases (15)NO. Our results also suggest that S-transnitrosylation of the single reduced cysteine moiety of albumin by endogenous GSNO or SNC in blood is possible but does not represent an effective mechanism to produce SNALB in vivo. This stable-isotope dilution GC-MS technique is suitable to perform in vivo studies on SNALB using physiologically and pharmacologically relevant doses.

Specific transport of S-nitrosocysteine in human red blood cells: Implications for formation of S-nitrosothiols and transport of NO bioactivity within the vasculature.

The transport of various S-nitrosothiols, NO and NO donors in human red blood cells (RBC) and the formation of erythrocytic S-nitrosoglutathione were investigated. Of the NO species tested only S-nitrosocysteine was found to form S-nitrosoglutathione in the RBC cytosol. L-Serine, L-cysteine and L-lysine inhibited formation of S-nitrosoglutathione. Incubation of RBC pre-incubated with S-[15N]nitroso-L-cysteine with native plasma or platelet-rich plasma led to formation of S-[15N]nitrosoalbumin and inhibited platelet aggregation, respectively. The specific transporter system of S-nitroso-L-cysteine in the RBC membrane may have implications for formation of S-nitrosoalbumin and S-nitrosohemoglobin and for transport of NO bioactivity within the vasculature.

Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative.

Asymmetric dimethylarginine (ADMA; N(G),N(G)-dimethyl-L-arginine) is the most important endogenous inhibitor of nitric oxide synthase and a potential risk factor for cardiovascular diseases. This article describes a gas chromatographic-tandem mass spectrometric (GC-tandem MS) method for the accurate quantification of ADMA in human plasma or serum and urine using de novo synthesized [2H(3)]-methyl ester ADMA (d(3)Me-ADMA) as the internal standard. Aliquots (100 microl) of plasma/serum ultrafiltrate or native urine and of aqueous solutions of synthetic ADMA (1 microM for plasma and serum; 20 microM for urine) are evaporated to dryness. The residue from plasma/serum ultrafiltrate or urine is treated with a 100 microl aliquot of 2M HCl in methanol, whereas the residue of the ADMA solution is treated with a 100 microl aliquot of 2M HCl in tetradeuterated methanol. Methyl esters are prepared by heating for 60 min at 80 degrees C. After cooling to room temperature, the plasma or urine sample is combined with the d(3)Me-ADMA sample, the mixture is evaporated to dryness, the residue treated with a solution of pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v) and the sample is incubated for 30 min at 65 degrees C. Solvent and reagents are evaporated under a stream of nitrogen gas, the residue is treated with a 200 microl aliquot of 0.4M borate buffer, pH 8.5, and toluene (0.2 ml for plasma, 1 ml for urine). Reaction products are extracted by vortexing for 1 min, the toluene phase is decanted, and a 1 microl aliquot is injected into the GC-tandem MS instrument. Quantitation is performed by selected reaction monitoring (SRM) of the common product ion at m/z 378 which is produced by collision-induced dissociation of the ions at m/z 634 for endogenous ADMA and m/z 637 for d(3)Me-ADMA. In plasma and urine of healthy humans ADMA was measured at concentrations of 0.39+/-0.06 microM (n=12) and 3.4+/-1.1 micromol/mmol creatinine (n=9), respectively. The limits of detection and quantitation of the method are approximately 10 amol and 320 pM of d(3)Me-ADMA, respectively.

Health disturbances of German battle tank officers: results of an interview with 64 commanding officers of a tank battalion.

In a former presentation, we reported the failure of commanding officers after a 320-km-long chain march. In this study, 64 commanding officers of a tank battalion were questioned regarding subjective health disturbances similar to the disturbances we have seen before. The soldiers indicated subjective troubles and frequency in dependence of the load duration. After 36 hours, one-half of the soldiers indicated feeling some pain, which consisted primarily of knee and back troubles. In addition to the marked disturbances after 24 hours, 50% of the soldiers indicated suffering from at least one health disturbance, and after 36 hours, 80% of the soldiers suffered at least one health disturbance. The disturbances appear faster and more frequently the longer the soldiers are on the battle tank as commanding officer. There is also a subjective increase in troubles with the years of service. Our results show that the vibration load on the battle tank can cause health disturbances. These disturbances seem to be a relevant problem of the commanding officers with no single case report.

Clinical and endocrine follow-up of patients after testicular sperm extraction.

OBJECTIVE: To evaluate the risk of testicular damage from testicular biopsies that are carried out for testicular sperm extraction (TESE) in infertile men. DESIGN: Prospective controlled clinical study. SETTING: Academic hospital. PATIENT(S): Forty infertile males with azoospermia. Examination of the clinical, endocrine, biochemical, and sonographic data in average after 18 months after TESE was performed. MAIN OUTCOME MEASURE(S): Measurements before and after TESE: hormone values, testicular size, morphologic characteristics, and power Doppler after scrotal sonography. RESULT(S): Comparison of preoperative and postoperative values of basal testosterone, FSH, LH, and estradiol levels did not reveal any differences. Twelve of 26 patients had subnormal testosterone values before TESE; 14 of 39 patients had subnormal levels afterward. Postoperative sonographic measurements showed no significant difference of the testicular volume as compared with the preoperative values. Results of power Doppler sonography revealed pathological conditions (n = 5) in patients with former iliacal or testicular operations. CONCLUSION(S): Endocrine testicular function and testicular size were not impaired after testicular biopsy when compared with preoperative data. However, patients with nonobstructive azoospermia seem to be at risk for androgen deficiency due to primary testicular failure after repeated testicular biopsies.

Measurement of S-nitrosoalbumin by gas chromatography--mass spectrometry. III. Quantitative determination in human plasma after specific conversion of the S-nitroso group to nitrite by cysteine and Cu(2+) via intermediate formation of S-nitrosocysteine and nitric oxide.

Highly contradictory data exist on the normal plasma basal levels in humans of S-nitrosoproteins, in particular of S-nitrosoalbumin (SNALB), the most abundant nitric oxide (.NO) transport form in the human circulation with a range of three orders of magnitude (i.e., 10 nM-10 microM). In previous work we reported on a GC-MS method for the quantitative determination of SNALB in human plasma. This method is based on selective extraction of SNALB and its 15N-labeled SNALB analog (S(15)NALB) used as internal standard on HiTrapBlue Sepharose affinity columns, HgCl(2)-catalysed conversion of the S-nitroso groups to nitrite and [15N]nitrite, respectively, their derivatization to the pentafluorobenzyl derivatives and quantification by GC-MS. By this method we had measured SNALB basal plasma levels of 181 nM in healthy humans. It is generally accepted that HgCl(2)-catalysed conversion of S-nitroso groups into nitrite is specific. In consideration of the highly divergent SNALB plasma levels in humans reported so far, we were interested in an additional method that would allow specific conversion of S-nitroso groups into nitrite. We found that treatment with cysteine plus CuSO(4) is as effective and specific as treatment with HgCl(2). The principle of the cysteine/CuSO(4) procedure is based on the transfer of the S-nitroso group from SNALB to cysteine yielding S-nitrosocysteine, and its subsequent highly Cu(2+)-sensitive conversion into nitrite via intermediate.NO formation. Similar SNALB concentrations in the plasma of 10 healthy humans were measured by GC-MS using HgCl(2) (156+/-64 nM) and cysteine/CuSO(4) (205+/-96 nM). Our results strongly suggest that SNALB is an endogenous constituent in human plasma and that its concentration is of the order of 150-200 nM under physiological conditions.

The use of cryopreserved mature and immature testicular spermatozoa for intracytoplasmic sperm injection: risks and limitations.

Treatment of severe male subfertility has become available since the intracytoplasmic injection of a single sperm into an oocyte was successfully applied for the first time in 1992. Moreover, with the use of fresh and cryopreserved epididymal and testicular spermatozoa for this procedure, fertilization and pregnancies could be accomplished. This review addresses the development and performance of these techniques and discusses achievements and problems as well as future aspects of the feasibility of early spermatid injection. Furthermore, limitations of these procedures and concerns with regard to genetic and epigenetic risks of using immature gametes are stressed.