Intestinal mycoplasma in Crohn's disease.

Intestinal diversion with reconnection in active Crohn's disease (CD) indicates that luminal contents or bacteria contribute to the formation of CD lesions. Fluorescent staining for mycoplasma in freshly resected Crohn's tissue and electron microscopy reveal intracellular organisms akin to mycoplasma. Historically, tissue culture of CD has shown mycoplasma described as contaminants. Mycoplasma are surface epithelial parasites requiring exogenous cholesterol for membrane stability and cell entry. PCR of intestinal tissue has shown Mycoplasma pneumoniae to be detectable more significantly in CD. Oral M. iowae in experimental poultry localizes to the distal small bowel and colon. Hypothetically, lipopeptides of mycoplasmal membranes are proposed to cause chronicity and stronger immune responses than by other bacteria. 'Intestinal' mycoplasmas, from a number of observations, deserve consideration as organisms mediating inflammation of acute and chronic CD.

Thiol methyltransferase activity in colonocytes and erythrocyte membranes.

AIMS: To verify the improved thiol methyltransferase (TMT) assay and measure activity in isolated colonocytes and erythrocyte membranes of the same subjects. METHODS: High performance liquid chromatography with radioactivity detection was used to measure 14C-methylmercaptoethanol formation, the reaction product of cell extracts incubated with mercaptoethanol and 14C-S-adenosylmethionine. RESULTS: Verification of radiolabelled 14C-methylmercaptoethanol was by exogenous addition of methylmercaptoethanol and simultaneous ultraviolet detection at 214 nm. Using a substrate concentration of 10 mM mercaptoethanol, the Km for S-adenosylmethionine was 25 microM. The sensitivity of the radioactive method was 2 pmol, with coefficients of variation of 7% within assay and 6.4% between assay. TMT activities (mean +/- SE; n = 17) were 471 +/- 64 pmol/hour/mg protein for colonocytes and 73 +/- 7 pmol/hour/mg protein for erythrocyte membranes. CONCLUSIONS: The direct assay of TMT activity is sensitive, specific and eliminates concern over non-enzymatic methylation of thiol compounds. High activities in colonic epithelial cells deserve evaluation in disease states.

Estimation of phenolic conjugation by colonic mucosa.

Conjugation of phenol by the colonic mucosa was assessed in vivo using dialysis tubing containing 1.5 ml of 1 mmol/l acetaminophen (paracetamol) and 10 mmol/l butyrate. These were allowed to equilibrate in the rectum for one hour. The glucuronidated and sulphated conjugates of acetaminophen were measured by high pressure liquid chromatography and bicarbonate concentrations by gas analysis. In 21 subjects without colonic disease sulphate conjugation was observed in all cases, with a mean (SE) of 3.86 (0.66) nmol/hour, while glucuronide conjugation was found in seven of 21 cases. Mean (SE) bicarbonate output of 42.9 (3.9) mumol/hour (n = 21) indicated healthy colonic mucosal metabolism and phenolic sulphation in dialysate and agreed with published sulphation rates obtained with cultured cells of colonic epithelium. Acetaminophen sulphation suggests that the colonic mucosa has an important role in the conjugation of phenols, and the method reported here would be useful in assessing the detoxification capacity of the colonic mucosa in diseases of the rectal mucosa.

Luminal ions and short chain fatty acids as markers of functional activity of the mucosa in ulcerative colitis.

Luminal concentrations of short chain fatty acids (SCFA), ammonia, sodium and potassium were measured in colonic dialysate of 16 control subjects and in 65 cases with ulcerative colitis (UC), which were graded according to mucosal changes into mild (1), moderate (2), or severe (3) inflammatory activity. Sodium concentrations were mildly but not significantly increased in severe ulcerative colitis while luminal potassium concentrations were markedly decreased in severe ulcerative colitis (p less than 0.025). Concentrations of SCFA were increased in severe ulcerative colitis. Butyrate concentrations were significantly raised in all stages of active ulcerative colitis even when other fatty acids were not raised. Of all the parameters a lowered pH and raised butyrate concentration most strikingly correlate with the severity of mucosal change. Results indirectly suggest that control of luminal pH, potassium secretion and utilisation of butyrate by the colonic mucosa are impaired with progressive mucosal inflammation.

Colonic bicarbonate output as a test of disease activity in ulcerative colitis.

No available test objectively measures impairment of function of the inflamed colonic mucosa in ulcerative colitis. To study function we assessed rectal bicarbonate output by rectal dialysis in the presence of water and bacterial fatty acid (n-butyrate) in 21 controls, 18 patients with acute ulcerative colitis, 12 patients with ulcerative colitis in remission, and 12 patients with other forms of colitis. In acute ulcerative colitis, compared with controls, bicarbonate output and pH was reduced (p less than 0.001); stimulated bicarbonate output with bacterial fatty acid (incremental bicarbonate output) was reduced by 80% in acute ulcerative colitis (p less than 0.01). Results indicate that bicarbonate output is a useful and selective test of mucosal function in acute ulcerative colitis. A diminished incremental bicarbonate output with n-butyrate supports the view of inadequate oxidation of bacterial fatty acids in vivo by the mucosa in ulcerative colitis. Whether the test will prove to be an index of prognosis or will aid choice between medical or surgical therapy requires further study.

Endocrine and cytokine changes during elective surgery.

Elective surgery was used as a model of severe non-thyroidal illness (SNTI) to study the inter-relation between changes in serum thyroid hormones, thyroid stimulating hormone (TSH), cortisol, and interleukin 6 concentrations. The study was designed to determine whether the expected interleukin 6 increases after surgery are the cause of decreased serum tri-iodothyronine (T3) concentration normally observed following severe trauma. Blood was sampled for 24 hours before, during, and for 48 hours after abdominal surgery under general anaesthesia in 11 patients. Total T3 decreased 30 minutes after induction and continued to decrease at 24 hours. After a transient increase at 30 minutes, free T3 also decreased, and free thyroxine (T4) concentrations, other than a similar transient increase, did not change. TSH concentrations were increased at four hours and the nocturnal surge was suppressed. The increase in the serum interleukin 6 concentration was not observed until four hours. Cortisol concentrations were increased at 30 minutes and peaked at four hours. Therefore, the early changes in thyroid hormones and TSH accompanying surgery do not seem to be caused by changes in interleukin 6 concentrations.

Diverticular colitis - therapeutic and aetiological considerations.

OBJECTIVE: Treatment of diverticulitis may change when associated with mucosal inflammation of either ulcerative-like or Crohns-like colitis. To determine effective treatment of diverticular colitis, four cases were analysed. Mechanisms to account for the colitis associated with diverticulitis are put forward. PATIENTS AND TREATMENT: Four cases had colitis and active diverticulitis established by clinical, colonoscopic or CT evidence of active inflammation. Biopsies confirmed mucosal inflammation: two with ulcerative colitis-like and two with granulomata suggestive of Crohns-like colitis. Treatment for colitis in all cases included sulphasalazine and steroids in two cases. RESULTS: Colitis subsided in three cases but one had continuing inflammatory polyps and one case did not resolve. Segmental resection was performed in two cases one with continuing colitis and one with inflammatory polyps. No further attacks of colitis have occurred since the initial observations were made or operations performed. CONCLUSION: An association of colitis with diverticulitis has been confirmed by present observations and case reports of others. Colitis requires medical treatment and if inflammation fails to resolve, segmental resection is indicated. Diverticular colitis, either ulcerative colitis-like or Crohns-like, is part of the spectrum of acute diverticulitis.

Famine, fiber, fatty acids, and failed colonic absorption: does fiber fermentation ameliorate diarrhea?

The salvage function of the colon for absorption of unabsorbed sodium and water from the jejunum and ileum depends upon the metabolic integrity of colonic epithelial cells maintained by luminal short-chain fatty acids. With the depletion of luminal short-chain fatty acids under conditions of starvation, metabolic compensation from vascular substrates is incomplete. Loss of luminal short-chain fatty acids diminishes cell membrane integrity and causes secretion by colonic epithelial cells, leading to starvation diarrhea. Because sodium absorption is dependent upon CO2 production from n-butyrate, no compensatory absorption occurs during starvation. Under conditions in which luminal short-chain fatty acids are depleted, dietary fiber is useful as a low osmolality food constituent and for renewal of short-chain fatty acid levels by bacterial fermentation. The "antisecretory" effect of dietary fiber depends on the degree of the preexisting depletion of short-chain fatty acids and the methodology used to assess absorptive function. Dietary fiber has not been found harmful in refeeding starvation victims for whom it is an essential food constituent.

Selective reduction of fatty acid oxidation in colonocytes: correlation with ulcerative colitis.

Attempts were made to define which fatty acid (2:0 to 18:1) was optimally oxidized by isolated colonocytes (colonic epithelial cells) and to select inhibitors of fatty acid oxidation which would be analogous in their action to the inhibition of fatty acid oxidation observed in colonocytes involved with ulcerative colitis. Isolated colonic epithelial cells of Sprague-Dawley rats were used with 2-mercaptoacetate, dichloroacetate, 3-mercaptopropionate, 4-mercaptobutyrate, 4-sulfatebutyrate, 2-bromobutyrate, sulfite ions and nitrite ions. n-Butyrate (4:0) was maximally oxidized to CO2 and ketone bodies (mean value 5.46 mumols/min/g dry wt). Oxidation of butyrate to CO2 was diminished by 2-bromobutyrate, sulfite ions and all mercapto fatty acids. Both fatty acid oxidation and glucose oxidation were significantly inhibited by 2-bromobutyrate, while mercapto fatty acids and sulfite inhibited fatty acid oxidation (p less than 0.01) without significantly changing glucose oxidation. Observation with 2-mercaptoacetate and sulfite correlate with early changes of fatty acid oxidation observed in cases of ulcerative colitis, and warrant further study with isolated colonocytes of man.

Congenital thyroid cyst of ultimobranchial gland origin.

The ultimobranchial gland (UBG) is an endocrine gland which contributes C cells to the thyroid gland in mammals. Dysembryonic tissues derived from the UBG have been described as cystic bodies. The following case illustrates the criteria which characterize dysembryogenesis of the UBG.

Review article: nitric oxide from dysbiotic bacterial respiration of nitrate in the pathogenesis and as a target for therapy of ulcerative colitis.

BACKGROUND: Factors initiating human ulcerative colitis (UC) are unknown. Dysbiosis of bacteria has been hypothesized to initiate UC but, to date, neither the nature of the dysbiosis nor mucosal breakdown has been explained. AIM: To assess whether a dysbiosis of anaerobic nitrate respiration could explain the microscopic, biochemical and functional changes observed in colonocytes of UC. METHODS: Published results in the gastroenterological, biochemical and microbiological literature were reviewed concerning colonocytes, nitrate respiration and nitric oxide in the colon in health and UC. A best-fit explanation of results was made regarding the pathogenesis and new treatments of UC. RESULTS: Anaerobic nitrate respiration yields nitrite, nitric oxide (NO) and nitrous oxide. Colonic bacteria produce NO and UC in remission has a higher lumenal NO level than control cases. NO with sulphide, but not NO alone, impairs beta-oxidation, lipid and protein synthesis explaining the membrane, tight junctional and ion channel changes observed in colonocytes of UC. The observations complement therapeutic mechanisms of those probiotics, prebiotics and antibiotics useful in treating UC. CONCLUSIONS: The prolonged production of bacterial NO with sulphide can explain the initiation and barrier breakdown, which is central to the pathogenesis of UC. Therapies to alter bacterial nitrate respiration and NO production need to evolve. The production of NO by colonic bacteria and that of the mucosa need to be separated to pinpoint the sequential nature of NO damage in UC.

Bacterial short-chain fatty acids and mucosal diseases of the colon.

This review examines the potential association between short-chain fatty acids and mucosal diseases of the colon, and discusses the implication of these relationships and the surgical management of large bowel disorders.

What sequence of pathogenetic events leads to acute ulcerative colitis?

The etiology of ulcerative colitis remains unresolved despite new immunologic, biochemical, and microbiologic observations made in this disease. A sequence of pathogenetic events has been adduced from abnormalities reported from human and experimental colitis with the express purpose of establishing priority of factors that may lead to an attack of acute ulcerative colitis. The presence of undefined bacterial metabolites in the colonic lumen causing specific breakdown of fatty acid oxidation in colonic epithelial cells is proposed to be the initiating event of the disease process that leads to an immune response and eicosanoid response perpetuating epithelial cell damage. The proposals embody the thesis that primary metabolic damage to colonocytes determines the clinical and pathologic manifestations of ulcerative colitis.

Short chain fatty acids as metabolic regulators of ion absorption in the colon.

Between 50%-80% of sodium absorption in the colonic mucosa is dependent upon CO2 generation from SCFAs derived from the colonic lumen. Lumenal starvation leading to diminished SCFAs levels in the colon greatly reduce the absorptive capacity of the colonic mucosa. Mechanisms whereby SCFAs regulate sodium absorption in the colon include 1) an enzyme induced adaptive regulation of SCFA-oxidation in colonocytes, 2) a flexible CO2 supply from SCFA depending upon stimulation or suppression of fatty acid oxidation., 3) the variable sidedness of Na(+)-H+ and Cl(-)-HCO3- exchange pumps in colonic epithelial cells, and 4) colonic epithelial cell membrane synthesis from SCFAs. Precise details of these regulatory mechanisms need to be elucidated by further experimental investigation.

New directions in the aetiology of ulcerative colitis.

The causative factors of ulcerative colitis remain unknown. Cellular biochemistry has revealed altered oxidative metabolism, membrane function and synthesis of mucus in colonic epithelial cells in the early stages of ulcerative colitis. Immune studies have highlighted more precise changes in neutrophil and macrophage function in cells of the lamina propria while microbiological evidence has shown a dysbiosis of colonic bacteria related to entero-adhesion, release of bacterial peptides and formation of secondary bacterial metabolites (mercapto fatty acids). A precise sequence of pathological events still needs to be established to account for an acute attack of ulcerative colitis.

Role of anaerobic bacteria in the metabolic welfare of the colonic mucosa in man.

Suspensions of isolated epithelial cells (colonocytes) from the human colon were used to assess utilisation of respiratory fuels which are normally available to the colonic mucosa in vivo. Cells were prepared from operative specimens of the ascending colon (seven) and descending colon (seven). The fuels that were used were the short chain fatty acid n-butyrate, produced only by anaerobic bacteria in the colonic lumen, together with glucose and glutamine, normally present in the circulation. The percentage oxygen consumption attributable to n-butyrate, when this was the only substrate, was 73% in the ascending colon and 75% in the descending colon. In the presence of 10 mM glucose these proportions changed to 59% and 72%. Aerobic glycolysis was observed in both the ascending and descending colon. Glucose oxidation accounted for 85% of the oxygen consumption in the ascending colon and 30% in the descending colon. In the presence of 10 mM n-butyrate these proportions decreased to 41% in the ascending colon and 16% in the descending colon. Based on the assumption that events in the isolated colonocytes reflect utilization of fuels in vivo, the hypothesis is put forward that fatty acids of anaerobic bacteria are a major source of energy for the colonic mucosa, particularly of the distal colon.

The aldehyde fuchsin and colloidal iron staining reactions in the canine thyroid C cell.

The cytochemically reactive groups which are responsible for Aldehyde Fuchsin (AF) and colloidal iron (CI) staining of C cells were investigated in the canine thyroid gland. To this end, stains for proteoglycans and peptide groups were utilized in conjuction with hydrolysis of glycosidic and amide bonds. In addition, the following procedures were used: acetylation, benzoylation, nitrozation, aldehyde blockade, sulphydryl blockade, methylation and mild acid hydrolysis. No acidic proteoglycan, sialic acid, polyphosphate or polysaccharide ester sulphate were detected in C cells; the results suggest that AF staining, after an oxidation step, and CI staining are due to polypeptides. Sulphydryl and carboxyl groups together are necessary for mediating the attachment of AF in C cells and it is adduced that this attachment is due to the combined charges of sulphonic and carboxylic acids. Methylation and acetylation inhibit CI staining and those staining reactions that depend upon carboxylic acid (TB) and hydroxyl groups (PAS) for their dye attachment in C cells. Acid hydrolysis, which increases the demonstration of carboxylic acid in C cells, decreases the attachment of hydroxyferric ions. I speculate that this inhibition is due to extraction of iron sites in the C cell and conclude that it is not solely carboxylic acids in C cells that are responsible for CI staining.

Utilization of nutrients by isolated epithelial cells of the rat colon.

Isolated suspensions of colonocytes from the rat were used to assess utilization, interaction, and fate of metabolic substrates normally obtained from colonic bacteria (acetate, propionate, butyrate) or derived from the blood circulation to the colonic mucosa (D-glucose, acetoacetate, L-glutamine). The short-chain fatty acid n-butyrate (10 mM), on its own, accounted for 86% of the total oxygen consumption and suppressed oxidation of endogenous fuel by 82%. Ths value was not altered by the addition of acetoacetate (5 mM), of L-glutamine (5 mM), or of D-glucose (10 mM). Activation of short-chain fatty acids by colonocytes proceeded in the order of butyrate greater than acetate greater than propionate. D-Glucose on its own accounted for 30% of the oxygen consumption by colonocytes and hardly suppressed utilization of endogenous fuels. Colonocytes utilized ketone bodies (acetoacetate) and produced them (acetoacetate and beta-hydroxybutyrate) from short-chain fatty acids. Considering the interaction of substrates, isolated colonic epithelial cells utilized respiratory fuels in the preferential order of butyrate greater than acetoacetate greater than glutamine greater than glucose. The high rate of CO2 production from butyrate should be a worthwhile means of examining the functional activity of the colonic mucosa clinically and in vivo.

The starved colon--diminished mucosal nutrition, diminished absorption, and colitis.

Nutrition of colonic epithelial cells is mainly from short chain fatty acids (SCFAs) produced by bacterial fermentation in the colonic lumen. n-Butyrate contributes more carbon of oxidation to epithelial cells than glucose or glutamine from the vasculature. Incomplete starvation of colonic epithelial cells through lack of luminal SCFAs leads, in the short term, to mucosal hypoplasia with either diminished absorption or diarrhea. A chronic lack of SCFAs or complete organ starvation in conjunction with other factors leads to nutritional colitis, either "diversion colitis" or "starvation colitis." Whether predominantly diarrhea or colitis develops in mucosal malnutrition appears to depend upon the severity and duration of starvation. Ulcerative colitis may be classified as a nutritional colitis in that colonic epithelial cells are unable to utilize SCFAs reflecting epithelial starvation despite abundant SCFAs.