Abstract SNACC-16

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Dipping Our Toes in the Hot Springs: Protocol Development For Capturing the Endogenous Gasotransmitter Hydrogen Sulfide in Human Serum

Collins N
UVM/FAHC, Burlington, VT, USA

INTRODUCTION
Of the known gasotransmitters hydrogen sulfide(H2S) is of particular interest to the clinical researcher. Its function as a localized marker of ischemic response make this gas significant both for purposes of monitoring physiologic response and as a target for therapeutic intervention. Despite the increasingly robust biochemical work in the field of gasotransmitters there is little published that addresses the difficulties of translating primarily laboratory based animal studies into the human subject. To this end a reliable and safe methodology suitable to the clinical medical environment is needed. The fluorescent moiety dansyl azide was selected as a model agent for protocol development based on ease of synthesis, compound stability, and demonstrated selectivity for reduction by H2S over other reductive biologic agents.

MATERIALS AND METHODS
Protocol development with the fluorescent H2S trapping agent dansyl azide in buffer systems, whole blood, and human serum took place prior to initiation of an IRB approved healthy volunteer pilot study. During the development stage of the project solubility and stability of both the fluorescent reagent and the H2S equivalent internal standard were investigated in relation to human blood components, exposure sequence, and equipment tolerance. Correlation coefficients derived from internal standard curves were utilized for inclusion of all buffer (R2≥0.95) and serum samples (R2≥0.90).

A pilot project consisting of ten healthy male volunteers who ventilated room air, hypoxic (15% oxygen, 85% nitrogen), and hyperoxic (100%) gas mixtures was undertaken. Venous whole blood samples were collected at internal jugular and antecubital sites following seven minutes of exposure to each oxygen environment. Resultant serum aliquots were loaded with a 200μmol solution of dansyl azide and submitted to fluorescence reading (excitation 340nm, emission 517nm).

RESULTS
The dansyl azide exhibited minimal solubility in many commonly used solvents when applied to whole blood and buffer. This lack of solubility was ultimately ameliorated through dilution techniques with ethanol that saw return of acceptable internal standard trendlines in both buffer and serum samples. Repeat application of the dansyl azide for maximal gas capture following separation of the serum was noted to be a necessary step for reliable application of the protocol.

Compiled mean data from volunteer serum samples demonstrated statistically significant findings (p<0.05) in measurement of fluorescent intensity between hypoxic and normoxic/hyperoxic samples obtained from the antecubital collection site.

CONCLUSION
Dansyl azide was successfully demonstrated as a viable fluorescent trapping agent for measurement of change to the endogenous production of H2S under mild hypoxic conditions. Equipment substitution and sample collection was successfully integrated into this protocol and accommodated hospital laboratory, clinical setting, and human subject needs without undue loss of sample integrity. It is anticipated that future development and testing of more hydrophilic fluorescent trapping agents using the protocol described will aid in further translational work on this gasotransmitter.


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