Mette Rye Clausen

Colonic Fermentation to Short-Chain Fatty Acids Is Decreased in Antibiotic-Associated Diarrhea

To elucidate the pathogenesis of antibiotic-associated diarrhea, colonic carbohydrate fermentation was investigated in three groups of subjects--a group of controls, a group of patients with antibiotic-associated diarrhea, and a group of patients receiving antibiotic therapy without diarrhea. Compared with controls, the colonic fermentation was markedly impaired in patients with antibiotic-associated diarrhea reflected by both very low concentrations (22.1 vs. 59.5 mmol/L; P less than 0.01) and production rates of short-chain fatty acids. In the group of patients without diarrhea, the effect on the colonic fermentation was dependent on the antibiotic administered. Penicillin and pivampicillin PO did not reduce the concentrations (69.9 and 66.7 mmol/L, respectively) or production rates. Dicloxacillin, erythromycin, and combined IV treatment with ampicillin, netilmicin, and metronidazole reduced both concentrations (27.1, 38.2, and 18.8 mmol/L; P less than 0.01) and production rates of short-chain fatty acids to levels seen in patients with diarrhea. L-Lactate and D-lactate concentrations were normal in all patients (less than 5 mmol/L), but lactate production was reduced in the patients who had reduced production of short-chain fatty acids, including patients with diarrhea. Thus, antibiotic-associated diarrhea was always related to reduced fecal concentrations and production rates of short-chain fatty acids and production rates of lactate. These results suggest that the antibiotic-associated diarrhea might be secondary to impaired colonic fermentation in otherwise disposed subjects, resulting in accumulation of luminal carbohydrate and/or decreased short-chain fatty acid-stimulated sodium and water absorption.

Short-Chain Fatty Acids in Pouch Contents From Patients With and Without Pouchitis After Ileal Pouch-Anal Anastomosis

Fecal concentrations of short-chain fatty acids were markedly reduced in 6 patients with pouchitis (mean +/- SE, 56.2 +/- 13.3 mmol/L) compared with 28 patients without pouchitis (139.0 +/- 8.5 mmol/L; P less than 10(-3)). The ratios of acetate to propionate to butyrate were not changed (pouchitis, 75:12:11%; normal pouches, 76:12:11%), i.e., all acids were equally reduced. The 24-hour production of total short-chain fatty acids in 16.6% fecal homogenates from patients with pouchitis was decreased (17.5 +/- 5.3 mmol/L) compared with patients without pouchitis (33.3 +/- 3.4 mmol/L; P less than 0.05), which could be overcome by the addition of saccharides to the homogenates. Pouch excretions of saccharides were similar in the two groups, but dilution occurred during pouchitis because of the increased outputs. Concentrations and productions of short-chain fatty acids correlated with pouch concentrations and excretions of sodium and saccharides. L-Lactate was elevated in pouchitis outputs, but differences in stool culture counts, mucosal histology, fecal concentration, assimilation or production of ammonia, nitrogen excretion, pH, and osmolality were not found. Pouchitis is characterized by decreased fecal concentrations and productions of short-chain fatty acids possibly caused by low pouch concentrations of fermentable saccharides.

Kinetic studies on the metabolism of short-chain fatty acids and glucose by isolated rat colonocytes

BACKGROUND/AIMSnAlthough the interest in colonic mucosal metabolism of short-chain fatty acids is steadily increasing, the kinetic parameters Vmax (maximum velocity) and Km (Michaelis constant) of the complete oxidation of these acids into CO2 by colonic epithelial cells have not previously been determined.nnnMETHODSnIsolated rat colonocytes were incubated in the presence of a concentration range of 14C-labeled acetate, propionate, butyrate, and glucose. Oxidation rates were obtained by quantifying the production of 14CO2. Vmax and Km were calculated by computer-fitting of the data to a Michaelis-Menten plot.nnnRESULTSnThe apparent Vmax values were similar comparing acetate, propionate, and butyrate (1.114 +/- 0.061, 0.991 +/- 0.072, and 1.007 +/- 0.070 mumol/min.g, respectively), but significantly lower for glucose (0.339 +/- 0.022 mumol/min.g). The corresponding Km values were all different and in the order of butyrate (0.184 +/- 0.017 mmol/L) < propionate (0.339 +/- 0.025 mmol/L) < acetate (0.487 +/- 0.019 mmol/L) < glucose (0.777 +/- 0.051 mmol/L). In substrate competition experiments, butyrate caused a strong noncompetitive inhibition of acetate oxidation and a mixed type of inhibition of propionate oxidation. Propionate inhibited the oxidation of acetate noncompetitively and that of butyrate competitively. Acetate only slightly inhibited the oxidation of propionate and butyrate.nnnCONCLUSIONSnColonic epithelial cells seem to utilize short-chain fatty acids in a preferential order of butyrate > propionate > acetate. Oxidation of propionate or acetate, however, may provide the energy needed for cellular functions if the metabolism of butyrate is impaired or the luminal supply is limited.

The degradation of amino acids, proteins, and blood to short-chain fatty acids in colon is prevented by lactulose.

Short-chain (C2-C5) fatty acids account for 60%-70% of the anions in the colon. Acetate (C2) is nontoxic in contrast to C(3)4-C5 fatty acids (propionate, butyrate, isobutyrate, valerate, and isovalerate), which induce coma in animals and may be important in the pathogenesis of hepatic coma in humans. An in-vitro fecal incubation system was used to map out short-chain fatty acid production in the presence of lactulose, amino acids, albumin, or blood. Albumin and blood increased production of all C2-C5 fatty acids. In contrast, lactulose was converted to acetate only and increased fecal acidity. The degradation of amino acids, albumin, and blood to short-chain fatty acids was completely inhibited by 10-25 mM lactulose. This was caused mainly by the acidifying effect of lactulose. pH-independent inhibition of blood and amino acid degradation to short-chain fatty acids required concentrations of lactulose exceeding 50-100 mM. Thus, the effect of lactulose in the treatment of hepatic coma may be related to its rapid fermentation into organic acids at rates exceeding colonic buffering capacity. This probably reduces formation of toxic fatty acids and ammonia from amino acids, polypeptides, and blood in the colon.

Lactulose, disaccharides and colonic flora. Clinical consequences.

SummaryLactulose is one of the most frequently utilised agents in the treatment of constipation and hepatic encephalopathy because of its efficacy and good safety profile. The key to understanding the possible modes of action by which lactulose achieves its therapeutic effects in these disorders lies in certain pharmacological phenomena: (a) lactulose is a synthetic disaccharide that does not occur naturally; (b) there is no disaccharidase on the microvillus membrane of enterocytes in the human small intestine that hydrolyses lactulose; and (c) lactulose is not absorbed from the small intestine. Thus, the primary site of action is the colon in which lactulose is readily fermented by the colonic bacterial flora with the production of short-chain fatty acids and various gases. The purpose of this review is to focus on some pertinent basic aspects of the clinical pharmacology of lactulose and to discuss the possible mechanisms by which lactulose benefits patients with constipation and hepatic encephalopathy.

Comparison of diarrhea induced by ingestion of fructooligosaccharide Idolax and disaccharide lactulose: role of osmolarity versus fermentation of malabsorbed carbohydrate.

Whether carbohydrate malabsorption causesdiarrhea probably depends on the balance between theosmotic force of the carbohydrate and the compensatorycapacity of the colon to dispose of the carbohydrate by bacterial fermentation. The present studyevaluated the specific role of the osmolarity bycomparing the severity of diarrhea after ingestion oftwo nonabsorbable carbohydrates, thefructooligosaccharide Idolax and the disaccharide lactulose. Bothcarbohydrates are readily fermented by the colonic florabut differ in osmolarity, the osmotic force being twiceas high for lactulose as for Idolax. Twelve subjects were given increasing doses (0, 20, 40, 80, 160g/d) of Idolax and lactulose in a crossover design.Every dose level was administered for three days withintervals of one week. Stools were collected on the third day to determine 24-hr volume,concentrations of short-chain fatty acids, L- andD-lactate, residues of Idolax or lactulose, sodium,potassium, pH, osmolarity, and in vitro productions oforganic acids. Measured by short-chain fatty acid andlactate formation in a fecal incubation system, thefermentation of Idolax and lactulose was identical andvery rapid compared with a range of referencecarbohydrates. A laxative effect of both Idolax and lactulosewas demonstrated. The increment in fecal volume as afunction of the dose administered was twice as high forlactulose (slope of the regression line = 7.3, r = 0.64, P < 10-5) as for Idolax(slope = 3.7, r = 0.51, P < 10-3), i.e.,isosmolar doses of lactulose and Idolax had the sameeffect on fecal volume. The variation in fecal volumewas substantial (lactulose 80 g/day: 110-1360 g/day; Idolax 160g/day: 130-1440 g/day). High responders had earlier andlarger fecal excretions of the saccharide compared withlow-responders. Fecal volume in carbohydrate-induced diarrhea is proportional to the osmotic forceof the malabsorbed saccharide, even though all or themajority of the saccharide is degraded by colonicbacteria. The capacity to modify the diarrhea varies considerably from person to person and isassociated with colonic saccharide disposal, whereas thevariation in response to isosmolar amounts of differentsaccharides is small within the sameindividual.

Colonic Fermentation of Ispaghula, Wheat Bran, Glucose, and Albumin to Short-Chain Fatty Acids and Ammonia Evaluated in Vitro in 50 Subjects

The production of short-chain fatty acids and ammonia was measured in 16.6% fecal homogenates from 50 subjects incubated at 37 degrees C for 6 and 24 hours. All 50 homogenates produced ammonia and short-chain fatty acids of any chain length (C2-C5). Incubation for 24 hours with dietary fiber (ispaghula husk or wheat bran), albumin, or glucose (10 mg/mL) increased the short-chain fatty acid production (43.6 +/- 2.8, 45.4 +/- 2.0, 60.3 +/- 3.2, and 65.8 +/- 3.1 mmol/L, respectively) compared with controls (21.4 +/- 1.3 mmol/L). The degradation of different substrates was associated with the production of different amounts of ammonia and short-chain fatty acids. Ispaghula, wheat bran, albumin, and glucose were fermented to acetate (> 2 mmol/L; 24-hour incubations) in 86%, 96%, 98%, and 98% of the homogenates, to propionate in 80%, 76%, 100%, and 68%, and to butyrate in 32%, 94%, 88%, and 54% of the homogenates, respectively. Isobutyrate, valerate, and isovalerate were produced from albumin in all (100%) of the homogenates, but only in 2 to 4% of the homogenates incubated with ispaghula or glucose. Ammonia was always (100%) produced after the addition of albumin and always (98%) consumed (assimilated) when glucose was fermented. Surgery (sigmoid or right- or left-sided colonic resection) did not change the pattern of ammonia and short-chain fatty acid production from these substrates. This study suggests that the different colonic flora from a large number of subjects share general biochemical characteristics, which metabolize different substrates to specific patterns of ammonia and short-chain fatty acids.

The bifurcation of the cyanogenic glucoside and glucosinolate biosynthetic pathways

The biosynthetic pathway for the cyanogenic glucoside dhurrin in sorghum has previously been shown to involve the sequential production of (E)- and (Z)-p-hydroxyphenylacetaldoxime. In this study we used microsomes prepared from wild-type and mutant sorghum or transiently transformed Nicotianaxa0benthamiana to demonstrate that CYP79A1 catalyzes conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime whereas CYP71E1 catalyzes conversion of (E)-p-hydroxyphenylacetaldoxime into the corresponding geometrical Z-isomer as required for its dehydration into a nitrile, the next intermediate in cyanogenic glucoside synthesis. Glucosinolate biosynthesis is also initiated by the action of a CYP79 family enzyme, but the next enzyme involved belongs to the CYP83 family. We demonstrate that CYP83B1 from Arabidopsisxa0thaliana cannot convert the (E)-p-hydroxyphenylacetaldoxime to the (Z)-isomer, which blocks the route towards cyanogenic glucoside synthesis. Instead CYP83B1 catalyzes the conversion of the (E)-p-hydroxyphenylacetaldoxime into an S-alkyl-thiohydroximate with retention of the configuration of the E-oxime intermediate in the final glucosinolate core structure. Numerous microbial plant pathogens are able to detoxify Z-oximes but not E-oximes. The CYP79-derived E-oximes may play an important role in plant defense.

Short-chain fatty acids, lactate, and ammonia in ileorectal and ileal pouch contents: a model of cecal fermentation.

Fecal concentrations of total short-chain fatty acids were normal in 16 patients with ileorectal anastomoses (mean +/- SEM, 99.7 +/- 10.3 mmol/L) and 28 patients with ileal pouch-anal anastomoses (138.8 +/- 8.5 mmol/L) and did not differ from those in 14 healthy noncolectomized controls (130.7 +/- 12.6 mmol/L). Acetate:propionate:butyrate:isobutyrate+valerate+isovalerate ratios were similar in the ileorectum (71:12:12:5%) and in the colorectum (66:14:13:7%) of healthy noncolectomized controls, whereas the concentration of acetate was increased at the expense of the polypeptide-derived isobutyrate, valerate, and isovalerate in the ileal pouch (77:12:11:1%). Ammonia was accordingly significantly diminished in ileal pouch contents (28.8 +/- 3.2 mmol/L vs 45.2 +/- 4.1 mmol/L in controls) in contrast to concentrations in ileorectal contents (36.2 +/- 5.3 mmol/L). Concentrations of lactate were normal and low. Twenty-four-hour productions of total short-chain fatty acids in 16.6% fecal homogenates from both groups of patients were normal. Addition of saccharides (eg, glucose, starch, pectin, ispaghula husk) increased the production of acetate, propionate, and butyrate and decreased the production of ammonia and isobutyrate, valerate, and isovalerate, which was increased in homogenates with albumin added. This pattern of substrate fermentation was similar in homogenates from ileal pouch, ileorectum, and control colorectum. In conclusion, the concentrations of short-chain fatty acids, lactate, and ammonia indicate that ileorectal fermentation resembles normal colorectal fermentation in noncolectomized healthy individuals, whereas the fermentation in ileal pouch contents seems to be more carbohydrate predominated.(ABSTRACT TRUNCATED AT 250 WORDS)

Fecal ammonia in patients with adenomatous polyps and cancer of the colon.

The correlation between high intakes of protein and high incidence of human colonic cancer is unexplained. Appreciable amounts of ammonia are generated in the large bowel through bacterial degradation of proteins and peptides, and experimental studies indicate that ammonia may select for neoplastic growth. Fecal concentrations of ammonia did not differ among 17 patients with former colonic adenomas [40.6 +/- 4.5 (SE) mM], 17 patients with former colonic cancer (51.4 +/- 3.9 mM), and 16 healthy controls (46.4 +/- 6.1 mM). By use of an in vitro fecal incubation system, possible alterations in bacterial fermentation and formation of ammonia were investigated. Fecal suspensions were incubated for 6 and 24 hours with and without addition of fermentable substrates (ispaghula husk, wheat bran, albumin, and glucose; 10 mg/ml). The in vitro production of ammonia in unsupplemented fecal homogenates from both groups of patients was comparable with the production found in homogenates from healthy controls, and the response to fermentable substrates was similar in all three groups. Addition of albumin caused a marked increase in the production of ammonia, and addition of glucose increased bacterial assimilation of ammonia considerably. These well-known characteristics of bacterial metabolism of ammonia apparently did not differ between healthy individuals and patients investigated more than three months after colonoscopic polypectomy or colonic cancer resection.