Hydrogen Sulfide Metabolizing Enzymes in the Intestinal Mucosa in Pediatric and Adult Inflammatory Bowel Disease

Abstract

Hydrogen sulfide (H₂S) is a toxic gas that has important regulatory functions. In the colon, H₂S can be produced and detoxified endogenously. Both too little and too much H₂S exposure are associated with inflammatory bowel disease (IBD), a chronic intestinal disease mainly classified as Crohn's disease (CD) and ulcerative colitis (UC). As the pathogenesis of IBD remains elusive, this study's aim was to investigate potential differences in the expression of H₂S-metabolizing enzymes in normal aging and IBD. Intestinal mucosal biopsies of 25 adults and 22 children with IBD along with those of 26 healthy controls were stained immunohistochemically for cystathionine-γ-lyase (CSE), 3-mercapto-sulfurtransferase (3-MST), ethylmalonic encephalopathy 1 protein (ETHE1), sulfide:quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST). Expression levels were calculated by multiplication of the staining intensity and percentage of positively stained cells. Healthy adults showed an overall trend towards lower expression of H₂S-metabolizing enzymes than healthy children. Adults with IBD also tended to have lower expression compared to controls. A similar trend was seen in the enzyme expression of children with IBD compared to controls. These results indicate an age-related decrease in the expression of H₂S-metabolizing enzymes and a dysfunctional H₂S metabolism in IBD, which was less pronounced in children.

Keywords: H2S metabolism; IBD pathogenesis; enzymatic expression; hydrogen sulfide; inflammatory bowel disease.

Figure A1

Expression levels of cystathionine-γ-lyase (CSE), 3-mercapto-sulfurtransferase (3-MST), ethylmalonic encephalopathy 1 protein (ETHE1), Sulfide:quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST) of biopsies from the terminal ileum compared to the histologic severity score (HSS) of each sample, separated into Crohn’s disease (CD) and controls (C).

Figure A2

Expression levels of cystathionine-γ-lyase (CSE), 3-mercapto-sulfurtransferase (3-MST), ethylmalonic encephalopathy 1 protein (ETHE1), Sulfide:quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST) of biopsies from the rectum compared to the histologic severity score (HSS) of each sample, separated into ulcerative colitis (UC) and controls (C).

Figure A3

Expression levels of cystathionine-γ-lyase (CSE), 3-mercapto-sulfurtransferase (3-MST), ethylmalonic encephalopathy 1 protein (ETHE1), Sulfide:quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST) of biopsies from the ascending colon compared to the histologic severity score (HSS) of each sample, separated into Crohn’s disease, ulcerative colitis (UC) and controls (C).

Figure 1

Endogenous hydrogen sulfide (H₂S) production and detoxification. H₂S is produced from L-cysteine with or without homocysteine by cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) in the cytosol and from 3-mercaptopyruvate (3MP) by 3-mercapto-sulfurtransferase (3-MST) in mitochondria. The detoxification is catalyzed by sulfide:quinone oxidoreductase (SQOR) and subsequently by thiosulfate sulfurtransferase (TST) or ethylmalonic encephalopathy 1 protein (ETHE1). This process takes place in the mitochondria. Cysteine aminotransferase (CAT); D-amino acid oxidase (DAO); electron (e⁻), coenzyme Q (CoQ); glutathione persulfide (GSSH); thiosulfate (SSO₃²⁻); sulfite (SO₃²⁻); glutathione (GSH); ammonia (NH₃).

Figure 2

Assessment of staining intensity. (A) 0 = no staining (case number 36677: adult with Crohn’s disease; biopsy of the ascending colon stained with cystathionine-γ-lyase (CSE) antibody), (B) 1 = weak staining (case number 21480: child with Crohn’s disease; biopsy of the ascending colon stained with cystathionine-γ-lyase (CSE) antibody), (C) 2 = moderate staining (case number 10145: adult from control group; biopsy of the ascending colon stained with 3-mercapto-sulfurtransferase (3-MST) antibody), (D) 3 = strong staining (case number 13366: child from control group; biopsy of the ascending colon stained with ethylmalonic encephalopathy 1 protein (ETHE1) antibody). Scale bar: 50 μm.

Figure 3

Expression of cystathionine-γ-lyase (CSE), 3-mercapto-sulfurtransferase (3-MST), ethylmalonic encephalopathy 1 protein (ETHE1), sulfide:quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST) in pediatric (P) and adult (A) samples in the terminal ileum of pediatric (P) and adult (A) healthy controls. Range of expression levels from a minimum of 0 to a maximum of 300. ** = p-value < 0.01.

Figure 4

Expression of cystathionine-γ-lyase (CSE), 3-mercapto-sulfurtransferase (3-MST), ethylmalonic encephalopathy 1 protein (ETHE1), sulfide:quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST) in pediatric (P) and adult (A) samples in the rectum of pediatric (P) and adult (A) healthy controls. Range of expression levels from a minimum of 0 to a maximum of 300.

Figure 5

Expression of cystathionine-γ-lyase (CSE), 3-mercapto-sulfurtransferase (3-MST), ethylmalonic encephalopathy 1 protein (ETHE1), sulfide:quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST) in pediatric (P) and adult (A) samples in the ascending colon of pediatric (P) and adult (A) healthy controls. Range of expression levels from a minimum of 0 to a maximum of 300. * = p-value < 0.05.

Figure 6

Immunohistochemical staining of 3-mercapto-sulfurtransferase (3-MST) (a,f,k), cystathionine-γ-lyase (CSE) (b,g,l), ethylmalonic encephalopathy 1 protein (ETHE1) (c,h,m), sulfide:quinone oxidoreductase (SQOR) (d,i,n) and thiosulfate sulfurtransferase (TST) (e,j,o) in the ascending colon of an pediatric Crohn’s disease (CD) patient (case number: 21480; a–e), ulcerative colitis (UC) patient (case number: 17921; f–j) and a healthy control (case number: 41952; k–o). Scale bar: 50 μm.

Figure 7

Immunohistochemical staining of 3-mercapto-sulfurtransferase (3-MST) (a,f,k), cystathionine-γ-lyase (CSE) (b,g,l), ethylmalonic encephalopathy 1 protein (ETHE1) (c,h,m), sulfide:quinone oxidoreductase (SQOR) (d,i,n) and thiosulfate sulfurtransferase (TST) (e,j,o) in the ascending colon of an adult Crohn’s disease (CD) patient (case number: 1593; a–e), ulcerative colitis (UC) patient (case number: 118884; f–j) and a healthy control (case number: 14411; k–o). Scale bar: 50 μm.

Figure 8

Expression of cystathionine-γ-lyase (CSE), 3-mercapto-sulfurtransferase (3-MST), ethylmalonic encephalopathy 1 protein (ETHE1), sulfide:quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST) in pediatric (P) and adult (A) samples in the terminal ileum in Crohn’s disease (CD) compared to healthy controls (C). Range of expression levels from a minimum of 0 to a maximum of 300. * = p-value < 0.05.

Figure 9

Expression of cystathionine-γ-lyase (CSE), 3-mercapto-sulfurtransferase (3-MST), ethylmalonic encephalopathy 1 protein (ETHE1), sulfide:quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST) in pediatric (P) and adult (A) samples in the rectum of ulcerative colitis (UC) compared to healthy controls (C). Range of expression levels from a minimum of 0 to a maximum of 300. * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001.

Figure 10

Expression of cystathionine-γ-lyase (CSE), 3-mercapto-sulfurtransferase (3-MST), ethylmalonic encephalopathy 1 protein (ETHE1), sulfide:quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST) in pediatric (P) and adult (A) samples in the ascending colon in Crohn’s disease (CD), ulcerative colitis (UC) and healthy controls (C). Range of expression levels from a minimum of 0 to a maximum of 300. * = p-value < 0.05, ** = p-value < 0.01, *** = p < 0.001.