Julie K. Furne

Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values

Hydrogen sulfide is gaining acceptance as an endogenously produced modulator of tissue function. The present paradigm of H(2)S (diprotonated, gaseous form of hydrogen sulfide) as a tissue messenger consists of H(2)S being released from the desulfhydration of l-cysteine at a rate sufficient to maintain whole tissue hydrogen sulfide concentrations of 30 microM to >100 microM, and these tissue concentrations serve a messenger function. Utilizing physiological concentrations of l-cysteine and aerobic conditions, we found that catabolism of hydrogen sulfide by mouse liver and brain homogenates exceeded the rate of enzymatic release of this compound such that measureable hydrogen sulfide release was less with tissue-containing vs. tissue-free buffers. Analyses of the gas space over rapidly homogenized mouse brain and liver indicated that in situ tissue hydrogen sulfide concentrations were only about 15 nM. Human alveolar air measurements indicated negligible free H(2)S concentrations in blood. We conclude rapid tissue catabolism of hydrogen sulfide maintains whole tissue brain and liver concentrations of free hydrogen sulfide that are three orders of magnitude less than conventionally accepted values and only 1/5,000 of the hydrogen sulfide concentration (100 microM) required to alter cellular function in vitro. For hydrogen sulfide to serve as an endogenously produced messenger, tissue production and catabolism must result in intracellular microenvironments with a sufficiently high hydrogen sulfide concentration to activate a local signaling mechanism, while whole tissue concentrations remain very low.

Detoxification of hydrogen sulfide and methanethiol in the cecal mucosa

Colonic bacteria liberate large quantities of the highly toxic gases hydrogen sulfide (H(2)S) and methanethiol (CH(3)SH). The colonic mucosa presumably has an efficient means of detoxifying these compounds, which is thought to occur through methylation of H(2)S to CH(3)SH and CH(3)SH to dimethylsulfide (CH(3)SCH(3)). We investigated this detoxification pathway by incubating rat cecal mucosal homogenates with gas containing H(2)S, CH(3)SH, or CH(3)SCH(3). Neither CH(3)SH nor CH(3)SCH(3) was produced during H(2)S catabolism, whereas catabolism of CH(3)SH liberated H(2)S but not CH(3)SCH(3). Thus, H(2)S and CH(3)SH are not detoxified by methylation to CH(3)SCH(3). Rather, CH(3)SH is demethylated to H(2)S, and H(2)S is converted to nonvolatile metabolites. HPLC analysis of the homogenate showed the metabolite to be primarily thiosulfate. Analysis of cecal venous blood obtained after intracecal instillation of H(2)(35)S revealed that virtually all absorbed H(2)S had been oxidized to thiosulfate. The oxidation rate of H(2)S by colonic mucosa was 10,000 times greater than the reported methylation rate. Conversion to thiosulfate appears to be the mechanism whereby the cecal mucosa protects itself from the injurious effects of H(2)S and CH(3)SH, and defects in this detoxification possibly could play a role in colonic diseases such as ulcerative colitis.

Fecal Hydrogen Sulfide Production in Ulcerative Colitis

Objective:Sulfide, a product of sulfate-reducing bacteria, has been proposed to play an etiologic role in ulcerative colitis. Ulcerative colitis feces have increased numbers and activity of sulfate-reducing bacteria, but only modestly increased sulfide. However, fecal sulfide exists largely in the volatile, highly toxic H2S form that moves rapidly from feces to surrounding gas. Our aim was to quantify the fecal release of H2S and other volatiles (CO2, H2, CH2, methanethiol, and dimethylsulfide).Methods:Fecal samples from 25 subjects with ulcerative colitis and 17 controls were incubated in 4-L containers, and gas release was assessed at intervals over 24 h.Results:H2S release by ulcerative colitis feces was elevated 3–4-fold at every measurement point compared with normal feces (p < 0.003 at 24 h). The only other significant difference was increased CO2 release by ulcerative colitis feces at 1 h. Supplementation of fecal homogenates with sulfur-containing substrates showed that organic compounds (mucin, cysteine, taurocholate) provided more readily utilizable substrate for H2S production than did sulfate.Conclusions:Increased H2S release is a relatively localized metabolic aberration of ulcerative colitis feces. This increased H2S may reflect abnormalities of the fecal bacteria and/or substrate availability.

Oxidation of hydrogen sulfide and methanethiol to thiosulfate by rat tissues: a specialized function of the colonic mucosa

Colonic bacteria release large quantities of the highly toxic thiols hydrogen sulfide (H(2)S) and methanethiol (CH(3)SH). These gases rapidly permeate the colonic mucosa, and tissue damage would be expected if the mucosa could not detoxify these compounds rapidly. We previously showed that rat cecal mucosa metabolizes these thiols via conversion to thiosulfate. The purpose of the present study in rats was to determine if this conversion of thiols to thiosulfate is (a) a generalized function of many tissues, or (b) a specialized function of the colonic mucosa. The tissues studied were mucosa from the cecum, right colon, mid-colon, ileum, and stomach; liver; muscle; erythrocytes; and plasma. The metabolic rate was determined by incubating homogenates of the various tissues with H(2)(35)S and CH(3)(35)SH and measuring the rate of incorporation of (35)S into thiosulfate and sulfate. The detoxification activity of H(2)S (expressed as nmol/mg per min) that resulted in thiosulfate production was at least eight times greater for cecal and right colonic mucosa than for the non-colonic tissues. Thiosulfate production from CH(3)SH was at least five times more rapid for cecal and right colonic mucosa than for the non-colonic tissues. We conclude that colonic mucosa possesses a specialized detoxification system that allows this tissue to rapidly metabolize H(2)S and CH(3)SH to thiosulfate. Presumably, this highly developed system protects the colon from what otherwise might be injurious concentrations of H(2)S and CH(3)SH. Defects in this detoxification pathway possibly could play a role in the pathogenesis of various forms of colitis.

Production of the gaseous signal molecule hydrogen sulfide in mouse tissues

The gaseous molecule hydrogen sulfide (H2S) has been proposed as an endogenous signal molecule and neuromodulator in mammals. Using a newly developed method, we report here for the first time the ability of intact and living brain and colonic tissue in the mouse to generate and release H2S. This production occurs through the activity of two enzymes, cystathionine‐γ‐lyase and cystathionine‐β‐synthase. The quantitative expression of messenger RNA and protein localization for both enzymes are described in the liver, brain, and colon. Expression levels of the enzymes vary between tissues and are differentially distributed. The observation that, tissues that respond to exogenously applied H2S can endogenously generate the gas, strongly supports its role as an endogenous signal molecule.

Free and Acid-Labile Hydrogen Sulfide Concentrations in Mouse Tissues: Anomalously High Free Hydrogen Sulfide in Aortic Tissue

Endogenously produced hydrogen sulfide is thought to function as an intracellular messenger. There is, however, little information on tissue concentrations of free hydrogen sulfide, the putative messenger form of this molecule, versus that of the bound (acid-labile) form. The present report describes the application of a novel technique to measure free and acid-labile hydrogen sulfide in mouse tissues. Very low free hydrogen sulfide concentrations (<0.050 μmol/kg) were observed in brain, liver, blood, heart, kidney, striated muscle, and esophagus. Aortic concentrations of free hydrogen sulfide were 20 to 100 times greater than that of the other tissues. Acid-labile hydrogen sulfide concentrations were multiple orders of magnitude greater than that of the free form in every tissue other than aorta. Previous reports of tissue hydrogen sulfide concentrations of 30 to >100 μmol/kg measured bound rather than free hydrogen sulfide, the observation that aorta contains anomalously high free hydrogen sulfide concentrations lends support for a vasodilator function for this molecule, and the very low free hydrogen sulfide concentrations in most tissues seemingly requires intermediation of a yet to be described receptor-like mechanism if this molecule is to serve as a gasotransmitter.

Differentiation of mouth versus gut as site of origin of odoriferous breath gases after garlic ingestion

Utilizing the sulfur-containing gases of garlic as probes, we investigated the gut versus mouth origin of odoriferous breath gases. Five individuals ingested 6 g of garlic, and sulfur gases in mouth, alveolar air, and urine samples were measured. The mouth normally contained low concentrations of hydrogen sulfide, methanethiol, and dimethyl sulfide. Immediately after garlic ingestion, transient high concentrations of methanethiol and allyl mercaptan and lesser concentrations of allyl methyl sulfide (AMS), allyl methyl disulfide, and allyl disulfide were observed. With the exception of AMS, all gases were present in far greater concentrations in mouth than alveolar air, indicating an oral origin. Only AMS was of gut origin as evidenced by similar partial pressures in mouth, alveolar air, and urine. After 3 h, AMS was the predominant breath sulfur gas. The unique derivation of AMS from the gut is attributable to the lack of gut and liver metabolism of this gas versus the rapid metabolism of the other gases. Breath odor after garlic ingestion initially originates from the mouth and subsequently from the gut.Utilizing the sulfur-containing gases of garlic as probes, we investigated the gut versus mouth origin of odoriferous breath gases. Five individuals ingested 6 g of garlic, and sulfur gases in mouth, alveolar air, and urine samples were measured. The mouth normally contained low concentrations of hydrogen sulfide, methanethiol, and dimethyl sulfide. Immediately after garlic ingestion, transient high concentrations of methanethiol and allyl mercaptan and lesser concentrations of allyl methyl sulfide (AMS), allyl methyl disulfide, and allyl disulfide were observed. With the exception of AMS, all gases were present in far greater concentrations in mouth than alveolar air, indicating an oral origin. Only AMS was of gut origin as evidenced by similar partial pressures in mouth, alveolar air, and urine. After 3 h, AMS was the predominant breath sulfur gas. The unique derivation of AMS from the gut is attributable to the lack of gut and liver metabolism of this gas versus the rapid metabolism of the other gases. Breath odor after garlic ingestion initially originates from the mouth and subsequently from the gut.

Physiological Measurements of Luminal Stirring in the Dog and Human Small Bowel

The resistance to absorption resulting from poor stirring of luminal contents (RLum) is considered to be equivalent to an unstirred layer of greater than 600 microns in the human small intestine. We measured RLum in the jejunum of conscious dogs by assessing the absorption rate of two rapidly absorbed probes, glucose, and [14C]warfarin. When RLum was expressed as an unstirred layer, the maximal thickness of the unstirred layer (assuming negligible epithelial cell resistance) was only approximately 35 and 50 microns for perfusion rates of 26 and 5 ml/min, respectively. Maximal unstirred layer thickness for the human jejunum, calculated from previous studies of glucose absorption, yielded a mean value of only 40 microns (range: 23 to 65 microns). Since epithelial resistance appears to be negligible during absorption of low concentrations of glucose, the maximal unstirred layer of 40 microns should be close to the true value for glucose in the human small intestine. We conclude that the unstirred layer for rapidly absorbed compounds in dogs and man are less than one-tenth of previously reported values, but this layer still may remain the rate limiting step in absorption of rapidly transported compounds.

Paracellular intestinal transport of six-carbon sugars is negligible in the rat

BACKGROUND & AIMS Active D-glucose absorption has been theorized to increase convective flow and enhance tight junction permeability such that paracellular transport becomes the major mechanism of D-glucose absorption. This concept was tested in rats by measuring the absorption of four gavaged, nonmetabolizable six-carbon sugars (L-glucose, L-galactose, L-mannose, and D-mannitol) thought to be absorbed solely by the paracellular route. METHODS Uptake of gavaged probes was measured by recovery in 24-hour urine specimen collections. RESULTS L-glucose (71.2% +/- 2.4%) absorption exceeded that of the other probes (1.4%-9%). Coadministration of 3.0 mol/L D-glucose, 0.22 mol/L D-glucose, or chow significantly reduced the absorption of L-glucose to 38.1% +/- 7.2%, 61% +/- 3.3%, and 53.6% +/- 3.5%, respectively, but did not influence the absorption of the other six-carbon probes. CONCLUSIONS (1) L-glucose seems to have a weak affinity for a D-glucose carrier and is not a marker of paracellular transport, and (2) paracellular transport accounts for a minimal fraction of D-glucose uptake; this fraction is not enhanced by ingestion of D-glucose or chow.

Production and elimination of sulfur-containing gases in the rat colon

Highly toxic sulfur-containing gases have been pathogenetically implicated in ulcerative colitis. Utilizing a rat model, we studied the production and elimination of sulfur-containing gases within the unperturbed colon. The major sulfur-containing gases were hydrogen sulfide (H2S), methanethiol, and dimethyl sulfide with cecal accumulation rates of 2.6, 0.096, and 0.046 μl/min, respectively. The dependence of H2S production on dietary components was demonstrated via a sixfold reduction with fasting and a fivefold increase with carrageenan (a nonabsorbable, sulfur compound) feeding. Zinc acetate reduced cecal H2S by fivefold, indicating the importance of H2S binding by divalent cations. During passage from the cecum to the rectum, >90% of the sulfur gases were absorbed or metabolized. An H235S turnover of 97%/min was observed in the isolated cecum. Thus mucosal exposure is >10 times the measured accumulation rate. Cecal mucosal tissue very rapidly metabolized H2S and methanethiol via a nonmethylating reaction.Highly toxic sulfur-containing gases have been pathogenetically implicated in ulcerative colitis. Utilizing a rat model, we studied the production and elimination of sulfur-containing gases within the unperturbed colon. The major sulfur-containing gases were hydrogen sulfide (H2S), methanethiol, and dimethyl sulfide with cecal accumulation rates of 2.6, 0.096, and 0.046 microliter/min, respectively. The dependence of H2S production on dietary components was demonstrated via a sixfold reduction with fasting and a fivefold increase with carrageenan (a nonabsorbable, sulfur compound) feeding. Zinc acetate reduced cecal H2S by fivefold, indicating the importance of H2S binding by divalent cations. During passage from the cecum to the rectum, > 90% of the sulfur gases were absorbed or metabolized. An H2 35S turnover of 97%/min was observed in the isolated cecum. Thus mucosal exposure is > 10 times the measured accumulation rate. Cecal mucosal tissue very rapidly metabolized H2S and methanethiol via a nonmethylating reaction.