Hydrogen sulfide (H 2 S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors including pharmaco-therapeutic manipulation. Amongst the challenges facing the field is the accurate measurement of biologically active H 2 S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bonafide H 2 S. The complexity of analytical H 2 S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in different forms, including a free form, an acid labile pool and as bound sulfane sulfur. Here we describe a new protocol to discretely measure specific H 2 S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping and derivatization of H 2 S. Acid-labile H 2 S is released by incubating the sample in an acidic solution (pH 2.6) of 100 mM phosphate buffer with 0.1 mM DTPA, in an enclosed system to contain volatilized H 2 S. Volatilized H 2 S is then trapped in 100 mM Tris-HCl (pH 9.5, 0.1 mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of bound sulfane sulfur pool was measured by incubating the sample with 1 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent to reduce disulfide bonds, in 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA), and H 2 S measurement performed in an analogous manner to the one described above. The acid labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H 2 S measurement from the acid liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method protocol allows very sensitive and accurate measurement of the three primary biological pools of H 2 S including free, acid labile, and bound sulfane sulfur in various biological specimens.
BackgroundHydrogen sulfide (H2S) has been implicated in regulating cardiovascular pathophysiology in experimental models. However, there is a paucity of information regarding the levels of H2S in health and cardiovascular disease. In this study we examine the levels of H2S in patients with cardiovascular disease as well as bioavailability of nitric oxide and inflammatory indicators.Methods and ResultsPatients over the age of 40 undergoing coronary or peripheral angiography were enrolled in the study. Ankle brachial index (ABI) measurement, measurement of plasma‐free H2S and total nitric oxide (NO), thrombospondin‐1 (TSP‐1), Interleukin‐6 (IL‐6), and soluble intercellular adhesion molecule‐1 (sICAM‐1) levels were performed. Patients with either coronary artery disease alone (n=66), peripheral arterial disease (PAD) alone (n=13), or any vascular disease (n=140) had higher plasma‐free H2S levels compared to patients without vascular disease (n=53). Plasma‐free H2S did not distinguish between disease in different vascular beds; however, total NO levels were significantly reduced in PAD patients and the ratio of plasma free H2S to NO was significantly greater in patients with PAD. Lastly, plasma IL‐6, ICAM‐1, and TSP‐1 levels did not correlate with H2S or NO bioavailability in either vascular disease condition.ConclusionsFindings reported in this study reveal that plasma‐free H2S levels are significantly elevated in vascular disease and identify a novel inverse relationship with NO bioavailability in patients with peripheral arterial disease.Clinical Trial RegistrationURL: http://www.clinicaltrials.gov. Unique identifier: NCT01407172.
We report a case of invasive pulmonary filamentous fungal infection in a patient with chronic obstructive pulmonary disease who was treated with a conventional dose of inhaled fluticasone in the absence of other causes of immunosuppression. This case demonstrates the potential risk for opportunistic fungal infections in patients treated with high-potency lipophilic inhaled corticosteroids.
Nebivolol is a novel beta1-selective beta-blocker with vasodilator properties mediated through activation of the l-arginine-nitric oxide pathway. There is no published report of coronary artery spasm associated with nebivolol. We describe a 64-year-old female patient who developed unstable angina secondary to nebivolol-induced vasospastic angina which was also visible during coronary angiography.
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