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In Reply: Drs Tsikas and Rossi question the validity of the S-nitrosoglutathione assay and the clinical relevance of S-nitrosoglutathione for the blood pressure reduction described in our article. Our study reported only data from methods that have been validated according to established medical, analytical, or regulatory guidelines. To facilitate assessment of data quality for the methods used to measure main outcomes—including the S-nitrosoglutathione concentrations—essential validation parameters were presented. Requiring preceding publication of the complete validation process before allowing publication of clinical data would hamper clinically relevant "real-world" research without improving quality and clarity of the studies.

Regarding use of an internal standard, isotope-labeled standards are preferred for the liquid chromatography tandem mass spectrometry quantification of stable analytes. However, S-nitrosoglutathione undergoes transnitrosylation reactions. Thus, the addition of labeled S-nitrosoglutathione (such as S-[¹⁵N]nitrosoglutathione)¹ to the plasma samples leads to a reequilibration of nitric oxide (NO) and ¹⁵NO among the different thiol pools, causing changes in the S-nitrosoglutathione and S-[¹⁵N]nitrosoglutathione concentrations and thereby producing invalid analytical results.² Thiamazole is an ideal internal standard. We found that its chromatographic properties (retention time) and the mass spectrometric features (collision-induced fragmentation energy) are similar to S-nitrosoglutathione. In particular, thiamazole does not react with nitric oxide or S-nitrosothiols because its thiol function lacks nucleophilic properties due to mesomeric delocalization of the free sulfur electron pair.³

Previous studies have demonstrated that cocoa inhibits platelet aggregation, which has been attributed to the cocoa phenol-induced production of bioactive nitric oxide.⁴ Thus, the notion raised by Tsikas and Rossi that S-nitrosoglutathione inhibits platelet aggregation supports our hypothesis for the mechanism of the vascular activity of cocoa.

Biological plausibility as well as evidence from in vitro and in vivo studies indicate that S-nitrosothiols (and especially S-nitrosoglutathione) may be involved in the regulation of blood pressure.^{2, 5} In contrast to Tsikas and Rossi, in more recent studies S-nitrosoglutathione injections have been found to decrease blood pressure and elicit vasodilation with an efficacy similar to nitric oxide.⁶ Moreover, results from studies with exogenous administration of high concentrations of nitric oxide compounds cannot be simply translated into hemodynamic efficacy of the low endogenous concentrations of bioactive nitric oxide. Although maximum plasma concentrations of free nitric oxide and low-molecular weight S-nitrosothiols (including S-nitrosoglutathione) are found to fall in the low nanomolar range,^{2, 7} the administration of inhibitors of the nitric oxide synthases causes an increase in blood pressure accompanied by a significant decrease of the plasma levels of S-nitrosothiols.²

The strong correlation between changes in plasma S-nitrosoglutathione levels and blood pressure in our study adds to the existing evidence that bioactive nitric oxide species such as S-nitrosoglutathione are involved in blood pressure regulation and provides the basis for targeting research to the stimulation of endogenous nitric oxide synthesis as a novel blood pressure–lowering principle.

Financial Disclosures: None reported.

Dirk Taubert, MD, PhD
dirk.taubert{at}medizin.uni-koeln.de
Department of Pharmacology

Norma Jung, MD
Department of Internal Medicine

Renate Roesen, PhD
Department of Pharmacology
University Hospital of Cologne
Cologne, Germany

1. Tsikas D, Raida M, Sandmann J, Rossa S, Forssmann WG, Frolich JC. Electrospray ionization mass spectrometry of low-molecular-mass S-nitroso compounds and their thiols. J Chromatogr B Biomed Sci Appl. 2000;742(1):99-108. FULL TEXT | PUBMED 2. Stamler JS, Jaraki O, Osborne J, et al. Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc Natl Acad Sci U S A. 1992;89(16):7674-7677. FREE FULL TEXT 3. Jocelyn PC. Biochemistry of the SH Group: The Occurrence, Chemical Properties, Metabolism and Biological Function of Thiols and Disulphides. New York, NY: Academic Press; 1972:1-46.4. Murphy KJ, Chronopoulos AK, Singh I, et al. Dietary flavanols and procyanidin oligomers from cocoa (Theobroma cacao) inhibit platelet function. Am J Clin Nutr. 2003;77(6):1466-1473. FREE FULL TEXT 5. Liu L, Yan Y, Zeng M, et al. Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock. Cell. 2004;116(4):617-628. FULL TEXT | ISI | PUBMED 6. Rassaf T, Kleinbongard P, Preik M, et al. Plasma nitrosothiols contribute to the systemic vasodilator effects of intravenously applied NO: experimental and clinical study on the fate of NO in human blood. Circ Res. 2002;91(6):470-477. FREE FULL TEXT 7. Neishi Y, Mochizuki S, Miyasaka T, et al. Evaluation of bioavailability of nitric oxide in coronary circulation by direct measurement of plasma nitric oxide concentration. Proc Natl Acad Sci U S A. 2005;102(32):11456-11461. FREE FULL TEXT

Letters Section Editor: Robert M. Golub, MD, Senior Editor.

JAMA. 2007;298:1863-1864.

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RELATED LETTER

Cocoa Intake and Blood Pressure
Dimitrios Tsikas and Ranieri Rossi
JAMA. 2007;298(16):1862-1863.
EXTRACT | FULL TEXT