Short-chain fatty acid production from mono- and disaccharides in a fecal incubation system: implications for colonic fermentation of dietary fiber in humans.

An in vitro fecal incubation system was used to demonstrate how lactose, lactulose and monosaccharides (mainly constituents of dietary fiber) influence short-chain fatty acid production in colon. Short-chain fatty acids were formed from all mono- and disaccharides tested (except L-glucose): D-glucose, D-galactose, D-fructose, D-mannose, L-rhamnose, D-sorbitol, D-arabinose, D-xylose, D-ribose, D-galacturonate, D-glucuronate, lactose and lactulose. All saccharides increased acetate formation; propionate production was increased from rhamnose, arabinose, xylose, ribose, galacturonic and glucuronic acid, whereas the synthesis of butyrate was elevated in assays incubated with sorbitol, galacturonic and glucuronic acid, and to a lesser degree ribose. Isobutyrate, valerate, isovalerate and hexanoate were produced in increased amounts in assays incubated with albumin, but in fact decreased in many incubations with saccharides. It is speculated that saccharide fermentation always results in formation of acetate, and that the relative production of acetate, propionate and butyrate is related to the monosaccharide composition of dietary fiber available for colonic bacteria. However, the production of isobutyrate, valerate, isovalerate and hexanoate is probably not due to saccharide fermentation, but is rather of polypeptide origin.

[Influence of fasting and the dietary lipid content on ketogenesis in isolated rabbit hepatocytes]. / Influence du jeûne et de la teneur en lipides du régime sur la cétogenèse dans les hépatocytes isolés de lapin.

Ketogenesis was measured in isolated liver cells from fed or 48 h starved rabbits given either a low fat diet (3%) or a high fat diet (18%). In the fed rabbits, ketogenesis with butyrate, octanoate and oleate was greatly enhanced by the high fat diet. In the starved animals the increase in ketogenesis was moderate and only observed with oleate and butyrate. Results are discussed in relation to in vivo observations.

Biochemical changes during the aerobic-anaerobic transition in Ascaris suum larvae.

Ascaris suum L3 larvae isolated from rabbit lungs undergo the third ecdysis to L4 larvae after 3 days in culture under a gas phase of 85% N2/10% CO2/5% O2. The L3 larvae contain substantial malic enzyme activity and are capable of producing small amounts of the reduced organic acids characteristic of the fermentative pathways which operate in the adult. However, only a small portion of the total carbon utilized is accounted for by these reduced acids and their motility is cyanide-sensitive, suggesting that their energy-generating pathways are predominantly aerobic. In contrast, after ecdysis, the L4 larvae begin to utilize glucose at a greater rate and the proportion of total carbon utilized which is accounted for as propionate, 2-methylbutyrate and 2-methylvalerate also increases. In addition, motility becomes increasingly cyanide-insensitive, suggesting that these L4 larvae are able to utilize the anaerobic energy-generating pathways of the adult. Surprisingly, on day 10 in culture, these L4 larvae, although capable of producing reduced volatile acids, still retain substantial cyanide-sensitive cytochrome oxidase activity.

Characterization of Flavobacterium species by analysis of volatile fatty acid production.

Seventy-four Flavobacterium strains were characterized by gas-liquid chromatographic analysis of volatile fatty acids produced in the culture medium. Principal components analysis permitted the graphic representation of the relative positions of the different strains, and aggregation according to the variance enabled a hierarchical classification to be established. The study revealed three subgroups each for F. meningosepticum and F. odoratum. Our F. breve, Flavobacterium sp. group IIb and F. multivorum strains appeared to be homogeneous. These results tallied with those of previous studies on DNA base composition and reassociation, electrophoretic protein profiles and cellular fatty acid composition.

Lipid composition in the classification of the butyric acid-producing clostridia.

An examination of 20 strains of butyric acid-producing Clostridium species for phospholipid class compositions, plasmalogen content, and acyl and alk-l-enyl chains showed that the deoxyribonucleic acid homology groups I (Clostridium butyricum) and II (Clostridium beijerinckii) could be distinguished by their lipid compositions. The phospholipids of C. butyricum strains had ethanolamine as the major nitrogenous lipid polar head-group moiety, more octadecenoate plus C19-cyclopropane than hexadecenoate plus C17-cyclopropane acyl chains, and the predominant alk-l-enyl chain was C18-monounsaturated. Clostridium beijerinckii strains had N-methylethanolamine plus ethanolamine in phospholipid head-groups, more hexadecenoate plus C17-cyclopropane than octadecenoate plus C19-cyclopropane acyl chains, and the major alk-l-enyl chain was C16-saturated. Three species falling outside the two homology groups Clostridium fallax, Clostridium pseudofallax and Clostridium acetobutylicum had ethanolamine as the major phospholipid base, but these species could be distinguished from C. butyricum by their acyl and alk-l-enyl chain compositions. The lipid composition of Clostridium pasteurianum is even more distinct.

Organic acids and anaerobic microorganisms in the contents of the cholesteatoma sac.

Organic acids in the contents of the cholesteatoma sac from 28 cases were studied by gas chromatographic technique. Five volatile fatty acids (acetate, propionate, isobutyrate, butyrate and isovalerate) and lactate were detected in large amounts, which may lower the pH of the cholesteatoma content. These acids were considered to be derived from products of anaerobic microorganisms. Therefore, the contents from 12 cases were cultured anaerobically in a glove box. Obligate microorganisms were identified in 92% of the cases and Peptococcus, Bacteroides, and Clostridium species were frequently isolated. In vitro, such obligate anaerobes produced various organic acids from the cholesteatoma content. Facultatives such as Staphylococcus aureus and Proteus mirabilis produced acetate in the content under aerobic and anaerobic conditions, whereas no organic acid was produced by Pseudomonas aeruginosa. Organic acids in the cholesteatoma content could be fermentative products made by the microorganisms, anaerobes and facultatives, which use the content as a substrate for acid production.

Pathway of formation of branched-chain volatile fatty acids in Ascaris mitochondria.

Disrupted Ascaris mitochondria formed 2-methylbutyrate (2-MB) and 2-methylvalerate (2-MV) when incubated anaerobically with acetyl CoA, propionyl CoA and NADH. However, when mitochondrial membranes were removed by high speed centrifugation and the mitochondrial soluble fraction was incubated with the same substrates, 2-methylcrotonate (tiglate) and a compound tentatively identified as 2-methyl-2- pentenoate accumulated rather than 2-MB or 2-MV. These data suggest that the terminal reduction of the unsaturated intermediates to the saturated 2-MB and 2-MV was catalyzed by an enzyme system at least partially bound to membranes. This supposition was further supported by the findings that disrupted Ascaris mitochondria also formed 2-MB and lesser amounts of 2-MV when incubated with tiglyl CoA plus NADH, and both soluble and membrane-bound components appear to be involved in this reduction. The possibility that electron transport associated ATP synthesis may be coupled to these reductions remains to be examined.

A role for threonine deaminase in the regulation of alpha-acetolactate biosynthesis in Escherichia coli K12.

The flow of carbon to alpha-acetolactate is Escherichia coli K12 is shown to involve the endogenous pool of alpha-ketobutyrate (alpha-KB). In vivo, the acetohydroxy acid synthase (AHAS) isoenzymes have an affinity for alpha-KB sufficiently high that alpha-acetolactate production is severely limited when alpha K-B is supplied exogenously. The ability of threonine deaminase to make alpha-KB is correlated with the synthesis of the AHAS isoenzymes. Mutations in ilvA that alter the catalytic and allosteric properties of threonine deaminase affect alpha-KB production and the expression of the AHAS isoenzymes in a direct way. The ilv A538 mutation results in a feedback-hypersensitive threonine deaminase ans slow alpha-KB and AHAS production. A spontaneous revertant of an ilvA538 strain expressing a feedback-resistant threonine deaminase produces alpha-KB and AHAS more quickly. A physiological role for the activator (valine) site on threonine deaminase is proposed and valine is shown to increase alpha-KB production in vivo. Valine can thus regulate its own biosynthetic pathway without jeopardizing the production of isoleucine. The physiological implications of the role of alpha-KB in the biosynthesis of acetolactate are discussed.

Presence of diaminopimelic acid in propionate-negative Bacteroides species and in some butyric acid-producing strains.

AThe presence of diaminopimelic acid (m-DAP) in strains of Bacteroides melaninogenicus, B. bivius and other species as well as in unidentified strains of Bacteroides was investigated by thin-layer chromatography. Strains of B. bivius and B. disiens all contained m-DAP as did the subspecies intermedius and melaninogenicus of B. melaninogenicus. Strains of B. asaccharolyticus and similar black pigment-producing butyrate-positive isolates showed heterogeneity. Asaccharolytic strains were DAP negative, whereas two strains fermenting glucose were positive. Some of the non-pigmented propionate-negative and butyrate-negative unidentified strains also contained DAP. The consistent finding of m-DAP in strains of B. bivius, B. disiens, and B. melaninogenicus indicates that DAP detection might be of value in the identification of these species.

Formation of methionine from alpha-amino-n-butyric acid and 5'-methylthioadenosine in the rat.

Adult rats may utilize two metabolites of methionine for the biosynthesis of this essential amino acid. In separate experiments methionine, labeled with 14C or with 35S was observed in plasma and urine following the administration of [2-14C]-alpha-amino-n-butyric acid or [35S]-5'-methylthioadenosine by stomach tube. Although alpha-amino-n-butyric acid (ABA) or homoserine, alone or with dietary sodium sulfate, choline, and/or S-methylcysteine, was not utilized for growth, weight loss in weanling rats was decreased by dietary cysteine when fed as an additive to a basal methionine-free, cysteine-free, labile methyl-free, sulfur-free diet. Following the addition of 10 mg ABA and 28 mg 5'-methylthioadenosine/day to the basal diet, growth response was equivalent to that occurring in rats receiving 27 mg of methionine/day with the basal diet. The implications of these findings for adaptation to protein restriction and a discussion of equilibrium and steady state conditions related to the increase in methionine content in the blood are presented.

[Studies on biosynthesis of short-chain fatty acids (author's transl)].

Studies were reported here on biosynthesis of short-chain fatty acids in lactating rabbit mammary glands. The maximal incorporation from [1-14C] acetate into total fatty acids were observed in microsomes and supernatant fractions of mammary glands but the synthetic rate either in the microsomes alone or in the supernatant alone was rather low. However approximately 80% of the maximal rate was restored in the supernatant for n-butyric acid synthesis. The incorporation from [1-14C] acetate into-n-butyric acid was markedly stimulated in the presence of NADH, compared to NADPH while total fatty acids synthesis was more dependent on NADPH. Long-chain fatty acids synthesis from [1-14C] acetate was decreased markedly by the addition of avidin although n-butyric acid formation was restored to 80%. Then by the addition of malonyl CoA or biotin the avidin system, middle- and long-chain fatty acids were recovered again. [1-14C] propionate was incorporated into even-numbered chain fatty acids as well as odd chain fatty acids. The synthesis of total fatty acids from [1-14C] propionate was more dependent on NADPH-generating system than either NADH or NADPH. [1-14C] bicarbonate was also incorporated slightly into fatty acids, such as decanoic or dodecanoic acids. In the reduction from either acetoacetyl CoA or crotonyl CoA, the tritium of NADP3H was stereospecifically incorporated into the beta-position of n-butyric acid. The reaction of acetoacetyl CoA reduction was much more dependent on NADH while crotonyl CoA was more reduced with NADPH. There was no difference between the dependencies on NADH and NADPH in the reduction of 2-hexenyl CoA.

[Investigations on the structure of the sphingolipids of the genus Bacteroides (author's transl)]. / Untersuchungen zur Struktur der Sphingolipoide von Bacteroides-Species

In 1972 Fritsche and Thelen have described the difference between the structure of the komplex lipids of the genus Bacteroides and the genus Sphaerophorus. Further investigations of Fritsche demonstrated the possibility of grouping gramnegative anaerobes into the genus Bacteroides in spite of the fact, that one of the final products of metabolism of these strains is butyric acid. These strains are the so-called butyric acid producing Bacteroides. This paper describes the structure of the still unknown fatty acids of the komplex lipids of Bacteroides strains and confirms the heterogenity of the sphingosine bases of Bacteroides as a principle. Fife strains of Bacteroides - with and without production of butyric acid - were used for purification of their long chain bases, which were characterized by degradation. The unknown fatty acids were isolated from B. thetaiotaomicron and analyzed by Dr. Rosenfelder with the aid of mass spectrometry, O-methylation and dehydratisation. The experiments of Rosenfelder demonstrate, that the unknown fatty acids have the behaviour of 3-hydroxy fatty acids, the two main peaks are a hexadecanoic and a heptade-behaviour of 3-hydroxy fatty acids, the two main peaks are a hexadecanoic and a heptadecanoic acid. They have an identical behaviour with the 3-hydroxy-15-methyl-palmitic acid of Myxococcus fulvus. Therefore the genus Bacteroides differs from the genus Sphaerophorus by synthesis of 3-hydroxy fatty acids. The production of sphingolipids is a common characteristic of the genus Bacteroides, each of the five strains demonstrated a heterogeneous pattern of bases with sphingosines with 16 to 20, perhaps also 12 to 14 carbon atoms, sometimes predominantly the branched and n-heptadeca- and the octadeca-sphinganine can be identified. The possibility of the production of phyto-sphingosines is discussed.

Studies on stereospecific reduction of acetoacetyl CoA and crotonyl CoA in lactating rabbit mammary glands.

Stereospecificity as well as the dependency on reduced pyridine nucleotides of the enzymatic reduction of acetoacetyl CoA and crotonyl CoA in lactating rabbit mammary glands are reported. In the reduction of both acetoacetyl CoA and crotonyl CoA as substrates the tritium from NADP3H was stereospecifically incorporated into the beta-position of n-butyric acid. The reaction of acetoacetyl CoA reduction was much more dependent on NADH while the reduction of crotonyl CoA was rather more dependent on NADPH. There was no difference between the dependencies on NADH and NADPH in the reduction of 2-hexenyl CoA.

Studies on lankacidin-group (T-2636) antibiotics. X. Microbial conversion of lankacidin-group antibiotics.

Lankacidin C, a component of lankacidin-group (T-2636) antibiotics, was esterified to lankacidin C 8-butyrate in the presence of methyl butyrate by culture broth and by cell-free extract of Bacillus megaterium IFO 12108. In addition, methyl isobutyrate, methyl valerate and methyl isovalerate served as acyl donors for the esterification, and lankacidin C 8-isobutyrate, lankacidin C 8-valerate and lankacidin C 8-isovalerate were formed respectively. Lankacidin C 8, 14-dibutyrate was hydrolyzed to lankacidin C 14-butyrate by the same organism.