Klavs Holtug

Degradation of Amino Acids to Short-Chain Fatty Acids in Humans: An in Vitro Study

Short-chain fatty acids (SCFA) originate mainly in the colon through bacterial fermentation of polysaccharides. To test the hypothesis that SCFA may originate from polypeptides as well, the production of these acids from albumin and specific amino acids was examined in a faecal incubation system. Albumin was converted to all C2-C5-fatty acids, whereas amino acids generally were converted to specific SCFA, most often through the combination of a deamination and decarboxylation of the amino acids, although more complex processes also took place. This study indicates that a part of the intestinal SCFA may originate from polypeptides, which apparently are the major source of those SCFA (isobutyrate, valerate, and isovalerate) only found in small amounts in the healthy colon. Moreover, gastrointestinal disease resulting in increased proteinous material in the colon (exudation, mucosal desquamation, bleeding, and so forth) may hypothetically influence SCFA production.

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.

The colon in carbohydrate malabsorption : short-chain fatty acids, pH, and osmotic diarrhoea

Short-chain (C2-C6) fatty acids (SCFA) are the major anions in colonic contents and the result of anaerobic fermentation of mainly saccharides. The effects and regulation of saccharide fermentation were studied in vitro and in vivo. In vitro faecal incubation was used to study the effects of lactose, glucose, and galactose and of pH on SCFA formation. Changing the pH to below 5 or above 11 abolished SCFA formation in the faecal incubates; in the pH 5-9 interval SCFA production was high, with only minor pH dependence. Adding glucose, galactose, or lactose to the incubation system increased SCFA production, but at high saccharide concentrations (100-300 mmol/l) SCFA formation was inhibited by the pH change. In vivo disaccharide malabsorption with increasing doses of lactulose caused a decrease in faecal pH to less than 5, values inhibitory to fermentation, before the appearance of carbohydrate in faeces. In 6 of 12 volunteers diarrhoea occurred suddenly and was caused by malabsorbed non-fermented carbohydrate. The six other volunteers had a gradual increase in faecal output with lactulose dose and developed diarrhoea before the appearance of saccharide in faeces. The intake of lactulose tolerated before diarrhoea ensued varied between individuals, with the majority having diarrhoea of more than 11/day at 160 g lactulose per day. At this dose SCFA absorption was estimated to be in the range 550 to 1150 mmol/day.

Fermentation to short-chain fatty acids and lactate in human faecal batch cultures : intra- and inter-individual variations versus variations caused by changes in fermented saccharides

The fermentation to short-chain fatty acids, lactate, and ammonia from several non-starch polysaccharides, glucose, and albumin was investigated in 16.6% faecal homogenates. Increasing concentrations (0-30 mg/ml) of glucose, wheat bran, pectin, ispaghula, cellulose, or albumin incubated for 24 h in homogenates pooled from three individuals increased short-chain fatty acid production linearly. Amounts and ratios of short-chain fatty acids formed were highly dependent on the type of substrate fermented. Fermentable saccharides increased ammonia assimilation, in contrast to the metabolic inert cellulose. Nine faecal homogenates sampled from three individuals at three occasions were incubated for 6 and 24 h. The production of total short-chain fatty acids, acetate, propionate, and butyrate and the accumulation of D- and L-lactate changed considerably in relation to the type of substrate added (cellulose, ispaghula, wheat bran, pectin, gum arabic, and glucose; p less than 10(-4)-10(-7). In contrast, there were no significant (p greater than 0.05) differences in organic acid formation between the nine homogenates, and the intra- and inter-individual variations were of the same magnitude. Variations in fermentation, when measured as organic acid formation, were therefore related to the type of substrate fermented rather than the individual tested.

An in vitro study of short-chain fatty acid concentrations, production and absorption in pig (Sus scrofa) colon

1. Short-chain fatty acid concentration was 180 mmol/l in the proximal colon and decreased to 108 mmol/l in the rectum. 2. Fermentation in chymus from different regions of the colon, showed the pattern of end products to reflect the substrate and not the site of the colon. 3. Isolated mucosa from proximal and distal colon had electroneutral sodium absorption of 4.8 +/- 0.2 and 2.9 +/- 0.8 mueq/cm2 hr in bicarbonate free media, which was abolished in the absence of chloride. 4. Electroneutral sodium absorption was enhanced by short-chain fatty acids in the proximal colon and could be described by Michaelis-Menten kinetics with Km 2.0-11 mmol/l and Jm 1.6-3.6 mueq/cm2 hr. In the distal colon the stimulation was smaller and propionate even inhibited sodium absorption. 5. Butyrate was absorbed in the proximal colon, whereas acetate and propionate, and butyrate in the distal colon had a flux ratio of one. 6. Amiloride (5 mmol/l) inhibited sodium absorption and net butyrate absorption.

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 Influence of Ispaghula Husk and Lactulose on the In Viva and the In Vitro Production Capacity of Short-Chain Fatty Acids in Humans

To evaluate factors influencing the short-chain fatty acid (SCFA) concentrations in stools, three different experiments were performed: faecal concentrations of SCFA at defecation were determined by gas liquid chromatography in nine healthy volunteers on a free diet. SCFAs were 114 +/- 15.0 mmol/l (means +/- SD). The coefficient of variation (CV) of the assay was 4-15%, the intraindividual CV 12-33%, and the interindividual CV 11-29%. On incubation of faeces at 37 degrees C concentrations of SCFA doubled in 6 h and rose fourfold in 72 h. In three volunteers the experiments were extended by adding ispaghula husk or lactulose to the diet for two 14-day periods each; no change in faecal SCFA concentrations was seen, either at defecation or after incubation. When ispaghula husk or lactulose was added to faeces in an in vitro incubation system, the concentrations of SCFA were five times higher than those of controls. We conclude that instant handling of faeces is essential for determinations of SCFA concentrations to obtain interpretable and comparable results; that determination of total SCFA output is of limited value; that addition of fibre to the diet does not influence faecal SCFA concentrations; and that the capacity for SCFA production in faeces is large provided a sufficient amount of substrate is available.

Stimulation of butyrate absorption in the human rectum in vivo.

BACKGROUND Models of short-chain fatty acid absorption have focused on the stimulation of sodium absorption, an effect mainly located in the proximal colon of man. With the present efforts to utilize butyrate enemas as a treatment of ulcerative colitis, it seemed important to assess the transport in the rectum. METHODS Non-equilibrium dialysis of the rectum was applied by placing dialysis bags containing various electrolyte solutions in the rectum of volunteers for 30 min. In this period changes in ion concentrations were linear with time. Net absorption and secretion rates were calculated from the change in fluid composition. RESULTS Sodium absorption was highest (24 +/- 8 mumol/cm2 h) in the presence of chloride and lowest (16 +/- 2 mumol/cm2 h) in the presence of bicarbonate and butyrate. Butyrate (70 mmol/l) was absorbed at a high rate of 7.1 +/- 2.2 mumol/cm2 h, independent on the presence of chloride, and was accompanied by increased bicarbonate secretion. Butyrate absorption increased to 9.6 +/- 1.8 mumol/cm2 h in sodium-free high-potassium media containing bicarbonate. CONCLUSION The results show that it is possible to increase butyrate uptake by manipulation of the electrolyte composition in the rectal lumen. Maximal uptake occurred with an electrolyte composition that was similar to the natural rectal content. The information gathered could be useful in designing enemas for trial in ulcerative colitis, provided the findings can be confirmed in these patients.

Localization of sodium absorption and chloride secretion in an intestinal epithelium.

SummaryHen coprodeum absorbs sodium electrogenically and, when stimulated by theophylline, secretes chloride. In this study the vibrating microprobe technique was used to localize the transport of these ions to intestinal villi/folds and crypts. With the isolated, stretched epithelium, controlled by light microscopy and scanning electron microscopy, in open circuit, currents were inward, 40±7 μA/cm2, 50 μm vertically above villi, and outward, 36±7 μA/cm2 above crypts. The currents decayed exponentially to near zero at 300 μm with the same length constant. A physical model simulating the observed loci of current sources and sinks predicts potential profiles consistent with our data. Extrapolation of the currents gives a surface potential of 45 μV, negative on villi and positive above crypts. Short circuiting increased villus current to 86±27 μA/cm2 at 50 μm, and amiloride treatment reduced it to −8 μA/cm2; in both cases crypt currents were abolished. The inward currents are compatible with sodium absorption. Induction of chloride secretion after amiloride treatment, resulted in current circuits similar to those induced by sodium absorption, with villus currents of 23±7 μA/cm2. This is in accord with chloride secretion at the villi. Quantitative estimates of crypt number (860/cm2) and opening diameter (15 μm), in conjunction with isotopic measurements of active and electrical potential-driven ion fluxes demonstrate, however, that only 4% of the potential-driven co-ion transport occurs through the crypts. This indicates that nearly all chloride secretion comes from the sodium-absorbing villar area. Were the chloride secretion to occur solely from the crypts, the current should have been in the opposite direction and 10,000-fold larger.

Short-chain fatty acids in bowel contents after intestinal surgery

Short-chain fatty acids are produced in the human colon by bacterial fermentation of dietary fibers and other saccharides escaping absorption in the small bowel. Short-chain fatty acid concentrations were determined together with production rates in 6- and 24-h incubations of intestinal outputs from 56 patients with various types of intestinal resections. Concentrations and 6- and 24-h production rates in feces from 9 healthy persons (controls; median +/- SD) were 98.9 +/- 21.4 mmol/L and 17.2 +/- 5.1 and 9.3 +/- 1.5 mmol/L.h, respectively. Colectomized patients with short bowel syndrome had extremely low concentrations (0.8 mmol/L) compared with controls (p less than 10-5), patients with ileostomy (p = 0.003), and ileal reservoirs (p less than 10-5), and showed low 6- and 24-h production rates (1.5 and 0.9 mmol/L.h, respectively; p less than 10-5 vs. controls). Short-chain fatty acids in ileostomic digesta (11.1 mmol/L) were decreased (p = 0.011) compared with outputs from ileal reservoirs (51.5 mmol/L), although production rates were in the same order of magnitude--all below control values (p less than 0.001). Patients partially colectomized and patients with small bowel bypass or short bowel syndrome with preserved colon had normal fecal concentrations with decreased production rates of short-chain fatty acids vs. controls (p less than 0.01). Only minor changes in ratios between individual acids were found. Reciprocal values of short-chain fatty acid concentrations correlated to volumes of outputs from both small intestine (r = 0.86, p less than 10-6) and colon (r = 0.79, p less than 10-6) when results were cumulated. It is concluded that partial resections of colon and the small bowel do not influence the fecal concentration level of short-chain fatty acids as long as colon is not totally resected.