1.

Comparison of plasma and serum levels of H₂S determined by several commonly used techniques: relevance to the cardiovascular system

Species	Model	Method of H2S detection	Fluid	H2S level reported	Comment	References
Male Sprague-Dawley rats	Myocardial injury induced by homo-cysteine treatment	Commercial sulfide-sensitive electrode	Plasma	Control rats, ∼30 μM* Homocysteine treatment, ∼50 μM*	Homocysteine is a substrate for CSE. H2S prevented myocardial injury. CSE expression and activity were lowered by high concentrations of H2O2 (0.1–10mM).	[34]
Rats –species not specified	Isoproterenol- induced myocardial injury	Zinc acetate / N,N-dimethyl-p-phenylenedi- amine (DMPD)	Plasma	Control rats, ∼65 μM*Isoprotenerol treated rats, ∼18 μM*	CSE involved in mediating cardiac contraction. Treatment of rats with isoproterenol and NaHS significantly lowered CPK and LDH release from myocardial tissue, lowered the levels of lipid peroxidation products and preserved left ventricular function. NaHS increased survival, induced capillary dilatation and reduced leucocyte infiltration into myocardial tissue. Part of the mechanism for these observations may include H2S-mediated ‘scavenging’ of H2O2 and O2.	[25]
Male Sprague- Dawley rats	Endotoxemia induced by bacterial lipopolysaccharide from E. Coli	Zinc acetate /DMPD	Plasma	Control rats, ∼26 μM* LPS treated rats, ∼45 μM*	LPS increase plasma PGE2 levels; an effect decreased by the H2S-releasing drug S-diclofenac LPS increased plasma NO2- levels; decreased by S-diclofenac suggesting H2S–nitric oxide cross-talk	[113]
Male Sprague- Dawley rats, WKY rats, Spontaneously hypertensive rats (SHR)	Development of a novel slow releasing H2S donor (GYY4137); Hypertension: induced by L-NAME induced hypertension, nor-motensive rats and SHR	Zinc acetate /DMPD	Plasma	Baseline levels of ∼35 μM* rose to ∼80 μM* 90 min after i.v. or i.p. administration of H2S donor.	First demonstration of the biphasic effects of H2S on cardiac function. Rapid release of H2S by NaHS reduced cardiac contractility (left ventricular diastolic pressure) by 42% and heart rate by 53%. In contrast, the slow and sustained release of H2S via GYY4137 had no effect on cardiac contractility or heart rate. NaHS increased mean arterial blood pressure in L-NAME treated rats whereas GYY4137 prevented L-NAME-induced hypertension. Furthermore GYY4137 markedly reduced systolic pressure in SHR but not WKY rats.	[79]
Male Sprague- Dawley rats	Type I diabetes v	Zinc acetate /DMPD	Plasma	Control rats, ∼40 μM STZ treated rats, ∼38 μM	Insulin treatment significantly elevated plasma H2S levels. STZ elevated liver, kidney and pancreas CSE and CBS expression and activity. STZ is a nitric oxide donating molecule and the decreased plasma levels of H2S after STZ treatment could reflect consumption of H2S by nitric oxide and / or derived intermediates	[15]
Male Sprague-Dawley rats	High blood-flow induced pulmonary hypertension induced by abdominal aorta-inferior cava vein shunt	Zinc acetate / DMPD	Plasma	Control rats, 50.9 ± 3.9 μM Pulmonary hypertensive rats, 36.4 ± 2.6 μM	Shunt decreased CSE mRNA expression in lung CSE mRNA localized in smooth muscle cells on small pulmonary muscular arteries with minimal expression in endothelial cells	[110]
Male Sprague-Dawley rats	High blood flow induced pulmonary hypertension induced by abdominal aorta-inferior cava vein shunt	Zinc acetate / DMPD	Plasma	Control rats, 50.83 ± 4.01 μM Shut, 36.42 ± 3.12 μM	Shunting decreased CSE mRNA expression in medial and small pulmonary arteries and rate of H2S synthesis in lung tissue	[111]
Male WKY and spontaneous hypertensive (SHR) rats	Hypertension	Zinc acetate /DMPD	Plasma	WKY rats 48 ± 13 μM SHR rats 20 ± 9 μM	PAG treatment decreased H2S levels in WKY (to 21 ± 7 μM) and SHR (to 12 ± 10 μM) and also increased the expression of CSE in aorta in WKY and SHR suggesting H2S involvement in regulation of blood pressure	[61]
Male Sprague-Dawley rats	Haemorrhagic shock (blood with-drawl, 9–10ml over an hour in 2 min periods)	Zinc acetate / DMPD	Plasma	Prior to blood withdrawl, 28.9 ± 1.4 μM 60 min after blood withdrawl, 37.5 ± 1.3 μM	PAG or β-cyanoalanine injected either pro-phylactically or therapeutically, inhibited the increase in plasma H2S and drop in mean arterial blood pressure. This response was not inhibited by gliben-clamide (KAT P channel antagonist). Blood withdrawl increased liver CSE mRNA expression	[62]
Wistar rats	Hypoxic pulmonary hypertension (HPH)	Zinc acetate / DMPD	Plasma	Control rats, 301.6 ± 32.41 μM HPH, 92.2 ± 22.1 μM	NaHS increased lung CSE expression; lev- els of control rats approximately 10 fold higher than the reported findings of others with the same species of rat (see below). Nevertheless, HPH induced a significant decrease in plasma H2S.	[43]
Male Wistar rats	Myocardial infarc-tion; ligation of left anterior descend- ing artery from its origin between the pulmonary artery conus and the left atrium	Zinc acetate / DMPD	Plasma	Control rats, 38.2 ± 2.07 μM 48 hrs after MI induction, 59.2 ± 7.16 μM	PAG treatment reduced plasma H2S levels to 39.2 ± 5.02 μM whereas treatment with the H2S donor, NaHS, significantly increased plasma H2S levels to 92.2 ± 12.40 μM. MI and PAG treatment decreased CSE mRNA expression. PAG treatment reduced myocardial oedema and inflammatory cell infiltrate. CSE immunoreactivity detected in infarct area as well as in the endothelium of small vessels of area at-risk. In contrast, CSE was not detected in cardiomyocytes under these experimental conditions.	[70]
Male Wistar rats	Hypoxic pulmonary hypertension	Zinc acetate / DMPD	Plasma	Control group, 299.6 ± 12.4 μM Hypoxia, 187.2 ± 13.1 μM	NaHS increased lung CSE expression; levels of control rats approximately 10 fold higher than the reported findings of others with the same species of rat . Administration of NaHS to hypoxia treated rats increased plasma H2S levels to 309.2 ± 13.6 μM Potential antioxidant action of H2S: Hypoxia decreased plasma antioxidant enzyme superoxide dismutase (SOD), increased levels of oxidized glutathione (GSSG) and lipid peroxidation markers (malondialdehyde); these effects were reversed by NaHS administration.	[114]
Male Sprague- Dawley rats	Cardiac ischemia reperfusion; occlusion of left anterior descending coronary artery	Zinc acetate /DMPD	Plasma	Control group, 58.28 ± 7.86 μM Ischemia-reperfusion group 30.32 ± 5.26 μM	NaHS decreased infarct size and improved haemodynamics (increased left ventricular diastolic pressure) Suggested mechanism for cardioprotection via down-regulation of c-fos expression in myocardium.	[115]
Male Sprague- Dawley rats		Commercial sulfide-sensitive	Plasma	Healthy rats, 45.6 ± 10.59 μM		[109]
Lobund-Wistar (LW) rats Harlan- Sprague-Dawley (HSD) rats C57 Black/6 (C57) mice	Method comparison study	electrode Polarographic H2S sensor and Ion-selective electrode Zinc acetate / DMPD	Plasma	H2S was not detected in LW or HSD rats or C57 mice. H2S was not detected in LW rats but 4.3 ± 0.5 μM detected in HSD rat plasma.	This study also shows the rapid removal, metabolism or sequestration of H2S added to vertebrate plasma; half time of decay of 10 μM Na2S added to plasma at 37°C, 13.0 ± 0.2 sec.	[38]
Male Swiss albino mice	Septic shock (induced by LPS)	Zinc acetate /DMPD	Plasma	Untreated animals, ∼32μM* LPS 4 hrs, ∼40μM LPS 20 hrs, ∼65 μM	LPS induced liver and kidney CSE expression and activity was inhibited by PAG. H2S levels correlated with myeloperoxidase expression and activity.	[14]
Male Balb/C mice	Acute pancreatitis induced by caerulein	Zinc acetate / DMPD	Plasma	Control mice, 22.5 ± 1.9 μM Caerulein treatment, 31.1 ± 3.3 μM	Pancreatitis-induced CSE expression elevated plasma H2S levels. PAG treatment lowered plasma H2S levels, levels pancreatic amylase and myeloperoxidase activity, inhibited acinar cell death and lung injury.	[116]
		Plasma				Control mice, ∼30 μM* Caerulein treatment, ∼35 μM*						[117]
Male Swiss albino mice	Cecal ligation and puncture-induced sepsis (CLP)	Zinc acetate /DMPD	Plasma	Normal, ∼10 μM* Sham, ∼13 μM* CLP, 20 μM*	Sepsis-induced CSE expression and elevated plasma H2S levels. Plasma H2S levels were significantly lowered after prophylactic or therapeutic treatment with PAG.	[118]
Female NOR/Ltj and female NOD/Ljt mice	Non-obese diabetic mouse model and controls	Zinc acetate / DMPD	Plsama	Control mice (NOR/Ljt), ∼60 μM* NOD mice stage I, ∼60 μM* stage II, ∼33 μM* stage III, ∼27 μM*	Plasma levels of H2S decreased markedly as disease progressed. L-cysteine stimulated synthesis of H2S and vasorelaxant effect in aortic tissue were significantly decreased with increasing disease progression.	[119]
Male C57BL/6J mice	Genetic knock-out studies; Wild-type (CSE+/+), CSE+/− and CSE−/−	Commerical sulfide ion-selective electrode	Serum	CSE+/+ mice ∼40 μM* CSE+/∼ mice ∼31 μM* CSE_/∼ mice ∼18 μM*	CSE / mice had higher systolic blood pressure than CSE+/∼ mice; CSE+/∼ mice had higher systolic blood pressure than CSE+/+ mice. CSE_/_ more sensitive to H2S-induced vasodilatation than CSE+/+.	[55]
Human	Chronic obstructive pulmonary disease (COPD); 27 patients with acute exacerbation of COPD (AECOPD), 37 patients with stable COPD	Commercial sulfide-sensitive electrode	Serum:	healthy volunteers; aged 71–80yrs, 35.7 ± 1.2 μM aged 61–70yrs, 34.0 ± 0.9 μM aged 50–60yrs, 36.1 ± 1.1 μM No significant difference between healthy and AECOPD (∼35 μM*) but elevated to in stable COPD patients (non-smokers, 51.1 ± 3.0 μM; smokers 49.8 ± 3.8 μM)	Smoking significantly lowered plasma levels of H2S in healthy controls and AECOPD patients. High levels of nitric oxide (measured as total nitrite/nitrate) correlated to higher H2S levels. H2S levels also correlated with stage of lung obstruction with COPD* (Stage I, ∼72 μM; Stage II, ∼50 μM; Stage III, ∼40 μM; Stage IV, ∼48 μM) H2S levels negatively correlated with sputal neutrophil count and positively with lung function (predicted FEV1).	[33]
Human	Chronic obstructive pulmonary disease (COPD); 18 patients before and after theophylline treatment	Commercial sulfide-sensitive electrode	Serum	COPD 30–100 μM* Serum levels unaffected by theophylline treat- ment.	H2S levels positively correlated with percentage of predicted FEV1, sputum macrophage levels but negatively correlated with sputum neutrophil count. Sputum levels of H2S were equivalent to levels of NO2.	[120]
Human	40 patients with coronary heart dis-ease (CHD), 17 angiographically normal patients	Commercial sulfide-sensitive electrode	Plasma:	Normal controls, 51.7 ± 11.9 μM CHD, 26.1 ± 14.2 μM Single vessel CHD, 33.0 ± 15.0 μM Double vessel CHD, 16.9 ± 7.9 μM Multi-vessel CHD, 18.4 ± 7.8 μM Unstable angina, 23.6 ± 14.4 μM Acute myocar- dial infarction, 19.9 ± 7.5 μM Stable angina, 38.4 ±14.5 μM CHD with coronary artery occlusion,	Patients with CHD had significantly lower H2S levels compared to angiographically normal controls. The number of affected vessels correlated with a decrease in H2S levels suggesting decreased H2S levels correlate with disease severity but could also reflect either H2S consumption by vascular oxidants. Plasma levels were also significantly negatively correlated to blood glucose levels and significantly lower in smokers compared to non-smokers.	[60]
Human	Essential hypertension in children	Ionic conductance meter/sulfide specific detector	Plasma	Control group 65.7 ± 5.5 μM (mean age 10.5 ± 0.73) Hypertensive children (mean age 10.48 ± 3.2yrs), 51.9 ± 6.0 μM	High plasma levels of H2S correlated to low homocysteine concentrations. Hypertensive children had a lower H2S: homocysteine ratio compared to the control group (5.8 ± 2.9 cf. 11.6 ± 3.3). Higher systolic pressure was associated with a lower plasma H2S:homocysteine ratio	[95]
Human	Healthy controls	Gas chromatography-mass spectrometry with pentafluorobenzyl bromide dramatization	Whole frozen blood	35–80 μM	Method evaluation paper.	[35]
Human	Healthy volunteers	Microdistillation and ion chromatography	Plasma	Levels differed with varying dietary meat intake Meat free, 42 ± 15 μM 240 g meat, 47 ± 12 μM 420 g meat, 25 ± 0.8 μM	This study also directly compared the methylene blue assay with this chromatographic technique and found the levels obtained in both systems to be comparable	[36]
Human	Healthy volunteers and septic shock patients	Zinc acetate / DMPD	Plasma	Age and sex matched healthy controls, 43.8 ± 5.1 μM Septic shock, 150.5 ± 43.7 μM		[14]

^*Estimated by the authors from published figures.