Free Radical Biology and Medicine 60 (2013) 195–200

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Free Radical Biology and Medicine

0891-58http://d

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Jon.lund

journal homepage: www.elsevier.com/locate/freeradbiomed

Original Contribution

Microbial regulation of host hydrogen sulfide bioavailability and metabolism

Xinggui Shen a, Mattias Carlströmb, Sara Borniquel b, Cecilia Jädert b, Christopher G. Kevil a,n,Jon O. Lundberg b

a Department of Pathology, Department of Molecular and Cellular Physiology, and Department of Cellular Biology and Anatomy, Louisiana State University Health Shreveport,Shreveport, LA 71130, USAb Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden

a r t i c l e i n f o

Article history:Received 10 January 2013Accepted 22 February 2013Available online 1 March 2013

Keywords:SulfideBacteriaCysteineMicrofloraMetabolismFree radicals

49/$ - see front matter & 2013 Elsevier Inc. Ax.doi.org/10.1016/j.freeradbiomed.2013.02.024

esponding author. Fax: þ1 318 675 7662.ail addresses: ckevil@lsuhsc.edu (C.G. Kevil),berg@ki.se (J. Lundberg).

a b s t r a c t

Hydrogen sulfide (H2S), generated through various endogenous enzymatic and nonenzymatic pathways,is emerging as a regulator of physiological and pathological events throughout the body. Bacteria in thegastrointestinal tract also produce significant amounts of H2S that regulates microflora growth andvirulence responses. However, the impact of the microbiota on host global H2S bioavailability andmetabolism remains unknown. To address this question, we examined H2S bioavailability in its variousforms (free, acid labile, or bound sulfane sulfur), cystathionine γ-lyase (CSE) activity, and cysteine levelsin tissues from germ-free versus conventionally housed mice. Free H2S levels were significantly reducedin plasma and gastrointestinal tissues of germ-free mice. Bound sulfane sulfur levels were decreased by50–80% in germ-free mouse plasma and adipose and lung tissues. Tissue CSE activity was significantlyreduced in many organs from germ-free mice, whereas tissue cysteine levels were significantly elevatedcompared to conventional mice. These data reveal that the microbiota profoundly regulates systemicbioavailability and metabolism of H2S.

& 2013 Elsevier Inc. All rights reserved.

Hydrogen sulfide has emerged as an important endogenousgasotransmitter in vivo that contributes to numerous physiologicaland pathological responses of various organs through its ability tomodulate oxidative stress, signal transduction, and nitric oxidebioavailability [32]. Endogenous generation of hydrogen sulfide iscomplex, with enzymatic synthesis occurring through three pro-teins comprising cystathionine γ-lyase (CSE) and cystathionine β-synthase, which use cysteine as a substrate, and 3-mercaptosul-furtransferase, using 3-mercaptopyruvate as a substrate [25,32].Once formed, hydrogen sulfide is very reactive, resulting in rapidmetabolism into one of three major pools comprising free, acid-labile, and bound sulfane sulfur forms [28,29]. In this way,hydrogen sulfide bioequivalents may be regulated allowing forconversion and use for various cellular biochemical processes.

Hydrogen sulfide production has long been studied in prokaryoticcells, with its generation being important for antioxidant defense,energy production, and cell cycle regulation [1,11,17,20,21]. Variousspecies of sulfate-reducing bacteria typically use thiosulfate togenerate hydrogen sulfide, although disproportionation of S2O3

− tohydrogen sulfide and SO4

2− and decay of S-containing amino acids arealternative generation pathways [3,17]. Studies suggest that gastro-intestinal hydrogen sulfide generation plays a critical role in

ll rights reserved.

regulating physiological responses such as motility, epithelial cellhealth, and inflammation [4,16,30]. Conversely, other reports suggesta pathological role of gastrointestinal hydrogen sulfide generationpresumably due to differential microbial colonization contributing tovarious conditions such as inflammatory bowel disease, colonicnociception, and colorectal cancer [18,22]. However, the importanceof microflora on host hydrogen sulfide formation, bioavailability, andmetabolism remains unknown, as examination of tissue H2S synth-esis has been performed using only conventional mice [13,29]. Herewe report that the normal microflora profoundly alters H2S bioavail-ability along with alterations in synthesis enzyme activity andsubstrate availability.

Materials and methods

Animals and tissue collection

All animal experiments were approved by the ethics committeein Stockholm, Sweden. Eleven- to 12-week-old male germ-freeC57BL/6 J mice (n¼10) and specific-pathogen-free (conventional)C57BL/6 J mice (n¼10) were used. All mice were maintained onautoclaved standard chow (R36; Lactamin, Stockholm, Sweden)and water ad libitum and kept under a controlled 12-h light–darkcycle. The germ-free status was checked weekly by culturing fecal

Figbiochanfidan

X. Shen et al. / Free Radical Biology and Medicine 60 (2013) 195–200196

samples, both aerobically and anaerobically, at þ20 and þ37 1Cfor up to 4 weeks [10].

On the day of the experiment, animals were anesthetized byinhalation of 2.2% isoflurane (Forane; Abbot Scandinavia AB, Kista,Sweden) in air. After blood sampling (inferior vena cava) theanimals were sacrificed and tissues rapidly collected. Plasma andtissue samples were immediately homogenized in a stabilizationbuffer (degassed 100 mM Tris–HCl buffer, pH 9.5, 0.1 mM diethy-lenetriaminepentaacetic acid (DTPA)) to preserve the bioavailablepools of H2S and metabolic proteins. Samples were snap-frozenand stored in liquid nitrogen until analyzed.

Detection of free sulfide, acid-labile sulfide, bound sulfide, andcysteine

Concentrations of sulfide and cysteine were measured by RP-HPLC after derivatization with excess monobromobimane (MBB)as stable sulfide-dibimane and cysteine-S-bimane as we havepreviously reported [23]. Briefly, 30 μl of samples was added to70 μl of 100 mM Tris–HCl buffer (pH 9.5, 0.1 mM DTPA), followedby addition of 50 μl of 10 mM MBB and incubated for 30 min. Thereaction was terminated with 50 μl of 200 mM 5-sulfosalicylic acidand the mixture centrifuged. The resulting supernatant wasanalyzed by RP-HPLC equipped with a fluorescence detector (λex390 nm and λem 475 nm) and an Eclipse XDB-C18 column (4.6 �250 mm). Typical retention times for bimane adducts of hydrogensulfide and cysteine were 15.75 and 10.12 min, respectively.

Hydrogen sulfide can exist in many biochemical forms asillustrated in Fig. 1. Acid-labile sulfide and bound sulfane sulfurwere measured as we have previously reported [24]. Acid-labilesulfide was released by incubating samples in an acidic solution(pH 2.6, 100 mM phosphate buffer, 0.1 mM DTPA), in an enclosedsystem to contain volatilized hydrogen sulfide. Volatilized hydro-gen sulfide was then trapped in 100 mM Tris–HCl buffer (pH 9.5,0.1 mM DTPA). The bound sulfane sulfur pool was measured byincubating the sample with 1 mM Tris (2-carboxyethyl) phosphine(TCEP) in 100 mM phosphate buffer at pH 2.6 with 0.1 mM DTPA,and sulfide measurement was performed in a manner analogousto that described above. The acid-labile pool was determined bysubtracting the free hydrogen sulfide value from the valueobtained by the acid-liberation protocol. The bound sulfane sulfurpool was determined by subtracting the hydrogen sulfide mea-surement from the acid-liberation protocol alone from that of theTCEP plus acidic conditions.

. 1. Various biochemical forms of H2S bioavailability. H2S exists in variouschemical forms within biological systems that can be classified based onemical properties and/or structure. Freely available H2S represents gaseous H2Sd its HS− anion, acid-labile sulfide represents iron–sulfur clusters and persul-es, and bound sulfane sulfur represents thiol sulfides, polysulfides, sulfate/sulfite,d bound elemental sulfur.

CSE activity measurement

CSE activity was measured as previously reported [12,36].Tissue lysates were incubated with 2 mM cystathionine, 0.25 mMpyridoxal 5′-phosphate in 100 mM Tris–HCl buffer (pH 8.3) for60 min at 37 1C. Trichloroacetic acid (10%) was added into thereaction mixture. After centrifugation, the supernatant was mixedcompletely with 1% ninhydrin reagent and incubated for 5 min in aboiling-water bath. After heating, the solution was cooled on icefor 2 min and the color reaction development measured 20 min at455 nm using a SmartSpect Plus spectrophotometer (Bio-Rad). CSEactivity was assessed by cystathionine consumption and enzymeactivity expressed as nanomoles of cystathionine consumed permilligram of total protein per hour of incubation.

Statistical analysis

Resulting hydrogen sulfide species measurements and cysteineand CSE activity levels were statistically compared with Prismsoftware (GraphPad, Inc.) using an unpaired Student t test betweenconventional and germ-free mice per organ examined. Distribu-tion of tissue H2S metabolite levels and CSE enzyme activity wasalso compared within each subject group using one-way ANOVAwith the Newman–Keuls multiple comparison test to identifytissues with the most significant differences. A minimumpo0.05 was necessary for significance.

Results

Plasma H2S bioavailability in conventional versus germ-free mice

Bioavailable H2S can be compartmentalized in various bio-chemical forms as illustrated in Fig. 1 [28]. Therefore, we employedspecific analytical methods that we have developed to measurethese various H2S pools comparing conventional versus germ-freemice. Fig. 2 illustrates the amount of plasma free H2S, acid-labilesulfide, and bound sulfane sulfur in conventional and germ-freemice (Fig. 2A–C, respectively). Germ-free mice had significantlyreduced plasma free H2S and bound sulfane sulfur levels comparedto conventional mice.

Tissue free H2S levels in conventional versus germ-free mice

Fig. 3 shows distinct differences regarding the amount of freelyavailable H2S in various organs. In conventional mice, the kidney,stomach, and heart showed the highest levels of free H2S, whereaslung and fat tissues were found to have the lowest levels.Germ-free mice had significantly less free H2S in cecum and coloncompared to conventional mice. These data indicate that thepresence of an intestinal microflora contributes significantly toplasma and gastrointestinal organ free H2S levels.

Tissue acid-labile sulfide and bound sulfane sulfur levels inconventional versus germ-free mice

Experiments were performed to measure tissue levels of acid-labile sulfide and bound sulfane sulfur. Fig. 4A reports varioustissue levels of acid-labile sulfide in conventional and germ-freemice. The absence of a microflora did not significantly affect acid-labile H2S pools in any of the organs examined, although compar-isons of tissue acid-labile H2S levels in conventional and germ-freemice revealed significantly higher amounts in fat and aorta. Fig. 4Bshows the various tissue levels of bound sulfane sulfur levels (i.e.,polysulfides) in conventional and germ-free mice. Interestingly, fatand aorta tissues of conventional mice contained the greatest

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Fig. 2. Plasma H2S bioavailable pools in conventional and germ-free mice. (A–C) Plasma free H2S, acid-labile sulfide, and bound sulfane sulfur levels, respectively, inconventional versus germ-free mice. The amount of bioavailable H2S is normalized to milligrams of total protein. nnpo0.01, n¼10.

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Fig. 3. Organ tissue free H2S levels in conventional and germ-free mice. Multiple organs were collected from conventional and germ-free mice, and free H2S was measuredand normalized to milligrams of total protein. ^po0.05, tissues with significantly higher amounts of free H2S in conventional mice. ∨po0.05, tissues with significantlyhigher amounts of free H2S in germ-free mice. nnnpo0.001 conventional versus germ-free tissue levels. n¼10.

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amounts of bound sulfane sulfur pools, similar to the acid-labileH2S measurements. However, fat and lung tissue bound sulfanesulfur levels were significantly decreased in germ-free mice. Thesedata highlight that the microflora significantly influences boundsulfane sulfur bioavailability in discrete tissue compartments.

CSE activity and cysteine levels in conventional versus germ-free mice

Fig. 5A reports the amount of CSE activity among varioustissues in conventional and germ-free mice. Comparison of tissueCSE activity in conventional mice revealed that aorta, fat, andmuscle tissue contain the most abundant enzyme activity. Absenceof a microflora elicited a significant decrease in tissue CSE activityin multiple organs including the cecum, colon, small intestine,kidney, liver, aorta, heart, and brain, although the aorta, fat, andmuscle tissues still displayed the greatest CSE activity. Fig. 5Billustrates the amount of cysteine measured in various tissues fromconventional and germ-free mice. In germ-free mice, cysteinelevels were significantly increased in the plasma, cecum, colon,small intestine, kidney, liver, fat, and muscle compared withconventional mice. Interestingly, only aorta tissue from germ-free mice showed a significant decrease in cysteine levels. Thesedata reveal that the presence of a microflora significantly influ-ences CSE activity and cysteine bioavailability.

Discussion

It is increasingly apparent that the gut microbiota plays a keyrole in modulating health and disease of its host [2]. Numerousstudies demonstrate that gut commensal bacteria participate in

regulating gastrointestinal neurophysiology, mucosal immunity,epithelial health, and survival as well as overall metabolic activity[5,26]. These responses are most probably due to the copiousamount of bacterial products that are produced and releasedwithin the host that influence various physiological and patholo-gical responses involving chronic inflammation and metabolicdysfunction [8,19,31,33]. Of these metabolites, sulfur/sulfate-redu-cing bacteria generate significant amounts of H2S luminally thathas been posited to be involved in gastrointestinal pathophysiol-ogy [18,22]. However, other findings suggest that the role ofmicrobial H2S production may not be completely deleterious, withpossible benefits conveyed to the host [16,30].

The contribution of the microbiota to host H2S metabolism andregulation of its bioavailability in various biochemical forms ispoorly understood. A recent study by Flannigan and colleagues [6]found no differences in colonic tissue H2S synthesis rates betweengerm-free mice and mice colonized with altered Schaedler flora.However, in our study we found significant differences in H2Sbiochemical pool bioavailability coupled with altered synthesisenzyme activity and substrate levels using sensitive analyticalHPLC-based methods that allow for accurate measurement ofH2S metabolism compared to the methylene blue detectionmethod that we and others have reported to be subject toexperimental artifact [9,23,24]. Our findings highlight the impor-tance of the microbiota in contributing to the regulation of H2Smetabolism and synthesis that was hereto unknown. Intriguingly,our results demonstrate that the gut microbiota regulates H2Shomeostasis not only locally in the gut but also systemically invarious tissues.

The physiological and pathological importance of the variousH2S biochemical pools (free, acid labile, and bound sulfane sulfur)

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Fig. 4. Organ tissue acid-labile and bound sulfane sulfur levels in conventional and germ-free mice. (A) Tissue concentrations of acid-labile sulfide levels reported asnanomoles per milligram of total protein in conventional and germ-free mice. ^po0.05, tissues with significantly higher amounts of acid-labile sulfide in conventional mice.∨po0.05, tissues with significantly higher amounts of acid-labile sulfide in germ-free mice. (B) Tissue concentrations of bound sulfane sulfur levels reported as nanomolesper milligram of total protein in conventional and germ-free mice. ^po0.05, tissues with significantly higher amounts of bound sulfane sulfur in conventional mice.∨po0.05, tissues with significantly higher amounts of bound sulfane sulfur in germ-free mice. npo0.05, conventional versus germ-free bound sulfane sulfur levels. n¼10.

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remains poorly understood [24,28]. Our detailed examination ofthese different pools in the major organs reveals new informationregarding basal and microbial modulation of tissue H2S bioavail-ability and synthesis rates. Interestingly, we found that aorta andadipose tissue contained the highest amount of H2S bioavailableequivalents primarily due to greater amounts of acid-labile andbound sulfane sulfur forms. Our observation of abundant aorticH2S bioavailability affirms recent findings from Levitt et al. [13]that the aorta contained the largest amount of H2S (free and acid-labile forms) as measured using gas chromatography–chemilumi-nescence techniques. With the use of our selective H2S poolliberation methods coupled with HPLC analysis, we further iden-tified adipose tissue as an equally important biological reservoirfor H2S bioequivalents that was not previously examined [13].These observations are consistent with numerous studies docu-menting the importance of H2S for cardiovascular health andendocrine and metabolic disease such as diabetes [32].

It is particularly interesting that the presence of a microbiotasignificantly altered adipose tissue bound sulfane sulfur, CSEenzyme activity, and cysteine levels, given the facts that germ-free mice have less fat mass compared to conventional mice andthat the type of enteric flora significantly influences fat pad mass[7,27]. It is possible that the presence of a microflora differentiallyaffects distal H2S metabolism in the adipocyte that modulatessubsequent adipose metabolism responses. This notion is sup-ported by two reports demonstrating (1) alterations in plasma H2Slevels in obese patients and (2) that the H2S donor diallyl trisulfidecan suppress 3T3-L1 adipogenesis stimulation [15,34]. Additionalstudies are clearly needed to determine whether the microbiota

may contribute to adipocyte function and obesity due to altera-tions in H2S metabolism.

To our surprise, the absence of a microflora was associated witha significantly reduced CSE activity in numerous tissues coincidentwith an increase in tissue cysteine levels. These observationssuggest an interesting hypothesis that bacterial products couldpossibly influence CSE activity or expression. Alternatively, cal-cium/calmodulin has been reported to modulate CSE activity, andthe enzyme may also be targeted for sumoylation, both of whichmight be altered in germ-free animals [25,35]. Tissue cysteinelevels were significantly elevated in many of the same tissues withblunted CSE activity. This may simply reflect less utilization ofsubstrate because of decreased enzyme activity or it could indicateenhanced cellular cysteine uptake and shunting of H2S synthesisthrough cystathionine β-synthase activity or altered regulation ofredox status, as many of these tissues did not manifest abundantdeficiency of H2S bioavailability. Although a clear explanation forthese differences is not readily apparent, future studies willaddress these questions in greater detail. It will also be necessaryto better understand exact mechanistic and signaling pathwaysinvolved in the induction of systemic H2S synthesis by the gutmicrobiota. One possibility is that bacterial products leak into thebloodstream and induce H2S-generating enzymes systemically.Indeed, bacterial lipopolysaccharide has been shown to upregulateCSE in certain cell types [14]. It is also possible that a portion of theH2S bioequivalents found in blood and tissues of conventionalmice comes from sulfur-metabolizing bacteria in the gut lumen.

In conclusion, we have shown that the host microbiota servesan important role in controlling host tissue H2S bioavailability and

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Fig. 5. Organ tissue CSE activity and cysteine levels in conventional and germ-free mice. (A) Differences in tissue CSE enzyme activity levels reported as nanomoles ofsubstrate consumed per milligram of total protein. ^po0.05, tissues with significantly higher amounts of CSE enzyme activity in conventional mice. ∨po0.05, tissues withsignificantly higher amounts of CSE enzyme activity in germ-free mice. npo0.05 and nnpo0.01, conventional versus germ-free tissue CSE enzyme activity. (B) Tissue cysteinelevels as nanomoles per milligram of total protein in conventional and germ-free mice. npo0.05, nnpo0.01, and nnnpo0.001, conventional versus germ-free tissue cysteinelevels. n¼10.

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metabolism. These data highlight the possibility that severaleffects of the microflora on physiological or pathological responsescould involve its modulation of H2S bioavailability.

Acknowledgment

This work was funded in part by NIH Grant HL113303 to C.G.K.

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