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Brief Communications |
1 Department of Chemistry, the University of Michigan, Ann Arbor, MI; 2 Department of Internal Medicine, University of Cincinnati, Cincinnati, OH; 3 College of Pharmacy, University of Michigan, Ann Arbor, MI;
aaddress correspondence to this author at: Department of Chemistry, the University of Michigan, Ann Arbor, MI 48109-1055. Fax 734-647-4865; e-mail: mmeyerho{at}umich.edu
Abstract
Background: The determination of reference intervals for the concentration of total S-nitrosothiols (RSNOs) in blood is a highly controversial topic, likely because of the inherent instability of these species. Most currently available techniques to quantify RSNOs in blood require considerable sample handling and multiple pretreatment steps during which light exposure is difficult to completely eliminate. We investigated the effect of brief light exposure on the stability of RSNO species in blood during the initial sampling process.
Methods: A novel amperometric RSNO sensor, based on an immobilized organoselenium catalyst at the distal tip of an electrochemical nitric oxide detector, was used to determine RSNO species in diluted whole blood without centrifugation or pretreatment. Porcine blood was collected into aluminum foil–wrapped syringes via a 12-inch butterfly needle tube assembly. Two blood samples were collected from the same animal—one with the butterfly needle tubing wrapped in aluminum foil and one with the tubing exposed to ambient room light. The RSNO concentrations in these sequential blood samples were determined by a standard addition procedure.
Results: Eight sets of measurements were made in 6 animals. Samples exposed to light yielded RSNO concentrations only 23.6% (7.2%) [mean (SD)] of the RSNO concentrations determined in samples that were shielded from light and obtained from the same animals.
Conclusions: These results suggest significant photoinstablity of RSNOs in whole blood and indicate the critical importance of proper light protection during sampling and processing of blood samples for the accurate determinations of endogenous RSNO concentrations.
S-Nitrosothiols (RSNOs), including nitrosoglutathione (GSNO), nitrosocysteine, and nitroso-albumin, exist in fresh blood and are formed from the reaction of the corresponding thiols with oxidation intermediates of nitric oxide (NO) produced by endothelial (EC) and other cells(1). In recent years, there has been growing interest in measuring concentrations of endogenous RSNO species in plasma because they are thought to serve as stable carriers of NO in the bloodstream(2) and may be a useful biomarker for assessing endothelial function(3). Despite considerable research efforts to quantify RSNOs in biological fluids, the reference intervals for plasma concentration of RSNOs reported in the literature vary substantially, with reported mean values ranging from 20 nmol/L to approximately 10 µmol/L(4).
This variation in concentration reference intervals may result from the inherent instability of RSNOs, which decompose readily when exposed to light, heat, and trace metal ions (e.g., Cu+)(5). At present, most techniques to measure blood RSNOs require the preparation of plasma by centrifugation and subsequent treatment of the plasma samples with various reagents to liberate NO(6). During these processes, exposure to ambient light is difficult to completely eliminate, and most methods reported to date do not take any precautions in this regard. Furthermore, because whole blood must first be centrifuged to remove red cells, even minimal hemolysis during this process can create free oxyhemoglobin in the plasma that can readily scavenge NO liberated from the RSNO species via added reagents (e.g., N-ethylmaleimide) before NO detection(1)(7). For these reasons, although measurement of total RSNO concentrations may be a good biomarker for endothelial function testing (as well as thrombotic risk assessment due to the inherent antiplatelet activity of RSNOs(8)), methods must be devised to determine total RSNO concentrations in the plasma phase in whole blood samples freshly drawn from the patient without time delay or excessive pretreatment steps.
We recently developed a novel amperometric RSNO sensor that is based on the use of an immobilized organoselenium catalytic species at the surface of an electrochemical NO detector(9). This sensor was shown to respond nearly equally to all low molecular weight (LMW) RSNO species present in blood. Unlike conventional methods (reduction of RSNOs and measurement of NO by fluorescence or chemiluminescence), the RSNO sensor measurement approach does not require blood separation or sample pretreatment, making it potentially useful for simple and rapid testing of blood RSNO concentrations for diagnostic purposes.
In the process of developing a practical analytical method that uses this new sensor technology to monitor total RSNOs in whole blood samples (initially employing animal models), we found that even normal sampling of the venous blood via a conventional butterfly type needle/tubing/syringe assembly can dramatically alter the observed response of the sensor toward the endogenous RSNO species that are present (unpublished data). We initially thought that the inherent absorbance of blood would be high enough to protect the endogenous RSNO species from photodecomposition (via 550–600 nm light, which causes homolytic cleavage of the S-N bond(5)), but we have discovered that this is not the case.
To prove that photodecomposition of the RSNO species in blood can occur during normal blood collection, we measured RSNOs in whole blood by drawing animal blood through a butterfly needle/tubing either with or without protection from light (via an aluminum foil covering of the plastic tubing that connects the needle with the syringe). Blood samples from pigs were collected by venipuncture in the abdomen via a butterfly needle (23G, Abbott) into a 5 mL heparinized syringe (5 U/mL final heparin) wrapped in aluminum foil. To evaluate the effect of light exposure on the stability of RSNO species in blood, another blood sample was drawn from the same animal with the butterfly tubing also wrapped with aluminum foil. During typical blood collection, it normally takes <15 s for blood to pass through the 12-inch length of plastic tubing. The blood RSNO concentrations were determined immediately after collection via a dual RSNO/NO sensor setup(9), using a standard addition technique. Within 1 min of blood draw, 5 mL of fresh animal blood was injected into an amber chamber thermostatted at 34 °C, containing 10 mL PBS (10 mol/L phosphate, pH 7.4), 100 µmol/L glutathione, and 0.5 mmol/L EDTA (to chelate trace metal ions), under an N2 atomosphere in which the RSNO and NO sensors were placed. The signal from the NO sensor (without catalyst) was used to correct for any low concentrations of other components in blood (e.g., ammonia) that may elicit a slight electroactive interference with both the RSNO and NO sensors (typically a very minor correction). After the amperometric responses from blood samples reached steady state, an aliquot of GSNO calibrator was added to calibrate the sensor response in situ.
Fig. 1
shows typical sensor response patterns to blood collected using the covered and exposed sampling devices from the same animal, followed by the standard addition of 3 µmol/L GSNO to the diluted whole blood sample. It can be seen that exposure to light, though brief, substantially decreases the detected RSNO concentration from 1.45 µmol/L in the covered blood sample (whole blood concentration, not plasma phase concentration, which can be reported if values are corrected for hematocrit) to 0.40 µmol/L in the exposed sample (based on converting the steady-state response in current units to RSNO concentration units). The sensor response to the subsequent addition of the GSNO standard is reproducible between the 2 runs. Further, we confirmed the reproducibility of the sensor’s response to endogenous RSNOs in a separate experiment by consecutively measuring the RSNO concentration during a 90 min period in 4 fully covered/protected blood samples from another animal. The measured RSNO concentrations were consistent, with a mean (SD) of 3.36 (0.32) µmol/L. Thus, the only plausible explanation for the large decrease in initial response obtained immediately after injecting blood that was not protected from ambient light is the photodecomposition of a significant fraction of the RSNO species present. A total of 8 experiments of this type were performed using 6 animals. When the RSNO concentrations determined in samples collected with the protected butterfly tubing were normalized to 100%, the RSNO concentrations measured using the exposed tubing were only 23.6% (7.2%) [mean (SD)] of the values obtained when the protected tubing device was employed for collection. These findings clearly suggest that exposure to light, even if it is brief, has a dramatic impact on the concentration of total LMW RSNOs in blood samples. Using the new sensor technology we have observed similar results in preliminary testing for RSNO concentrations in human whole blood samples (data not shown).
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S-Nitrosothiols exhibit ultraviolet/visible absorbance maxima at 330–350 nm and 550–600 nm(5). Despite a nearly 100-fold higher molar absorptivity at the lower wavelength, results of previous studies suggest that photodecomposition of RSNOs is primarily the result of light absorption in the 550–600 nm region(10). The light effect can be expected to be much greater for plasma samples, given the lack of any shielding from light offered by the presence of red blood cells. Hence, this light effect may explain the wide discrepancy of mean reference range values reported in the literature, because additional photoinduced losses can occur during the extra sample preparation steps (e.g., preparing plasma) that are required for all other RSNO detection methods. Indeed, some researchers have found LMW RSNO species to be "undetectable" in plasma samples(11).
Our data highlight the critical importance of even the initial sample-handling step in RSNO determinations. Furthermore, the findings described here regarding RSNO instability under normal blood collection conditions may also have profound consequence for the interpretation of results of various in vitro blood clotting and other coagulation tests that rely on inherent platelet functionality in the blood sample. Because it is known that RSNOs are potent inhibitors of platelet aggregation(8), if a significant fraction of endogenous RSNO species is lost during collection and handling of blood samples drawn for such in vitro testing, one may conclude that results obtained will not necessarily be a true indictor of in vivo platelet functionality in the presence of the in vivo concentrations of RSNO species. It would therefore be interesting to assess whether shielding blood samples from light during all collection and handling steps significantly alters results obtained for such in vitro coagulation tests.
Acknowledgments
Grant/Funding Support: The National Institutes of Health (grant no. EB-000783 and EB 004527).
Financial Disclosures: None declared.
Acknowledgment: We would like to thank Terry C. Major in the Department of Surgery at the University of Michigan Medical School for assistance in some of the animal studies.
References
The following articles in journals at HighWire Press have cited this article:
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  D. T. Hess, M. W. Foster, and J. S. Stamler Assays for S-Nitrosothiols and S-Nitrosylated Proteins and Mechanistic Insights Into Cardioprotection Circulation, July 21, 2009; 120(3): 190 - 193. [Full Text] [PDF]
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 R. Rossi and D. Tsikas S-Nitrosothiols in Blood: Does Photosensitivity Explain a 4-Order-of-Magnitude Concentration Range? Clin. Chem., May 1, 2009; 55(5): 1036 - 1038. [Full Text] [PDF]
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Y. Wu, W. Cha, F. Zhang, and M. E. Meyerhoff In Reply Clin. Chem., May 1, 2009; 55(5): 1038 - 1040. [Full Text] [PDF]
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