Differential in vivo and in vitro intestinal permeability to lactulose and mannitol in animals and humans: a hypothesis.

BACKGROUND/AIMS: Clinical interpretation of urinary recovery ratios of lactulose and mannitol is hampered by incomplete understanding of the mechanisms of transmucosal passage. The aim of this study was to compare in vivo and in vitro probe permeability. METHODS: Stripped sheets of small intestine from rodents and human biopsy specimens were mounted in Ussing chambers, and mucosa-to-serosa fluxes of lactulose and mannitol were determined. Urinary recovery of orally applied probes was measured in rodents, cats, and humans. RESULTS: In vitro lactulose/mannitol flux ratios were close to 0.8 in all species. Urinary recovery ratios differed between rodents and cats or humans; low ratios in cats and humans were due to high mannitol recovery. CONCLUSIONS: Interspecies variation in urinary recovery of mannitol is caused by differences specific for the intact small intestines in vivo. Because hyperosmolality of villus tips in vivo varies, being highest in humans and cats as a result of vascular countercurrent multiplication, it is hypothesized that the high urinary recovery of mannitol in these species is caused by solvent drag through pores that allow the passage of mannitol but not of lactulose. Therefore, the lactulose/mannitol ratio is primarily a standard for the normal functioning of villus epithelial cells in metabolite absorption and for normal villus blood flow.

Expression in Escherichia coli and characterization of the fatty-acid-binding protein from human muscle.

The coding part of the cDNA encoding human muscle fatty-acid-binding protein (FABP) was ligated in the pET8c vector and expressed under the control of the lacUV5 promoter. After induction with isopropyl beta-D-thiogalactopyranoside, almost 12% of the cytoplasmic proteins consisted of FABP. The protein could be isolated after sonication of the bacterial pellet followed by (NH4)2SO4 precipitations, anion-exchange chromatography and gel filtration. The muscle FABP produced in Escherichia coli has an isoelectric point of 5.3 and is recognized by anti-(human muscle FABP) antiserum after Western blotting. The purified FABP has a preference for binding to palmitic acid and C18-C22 (poly)unsaturated fatty acids, and no affinity to palmitoyl-CoA or other hydrophobic ligands tested. The dissociation constant for oleic acid is 0.58 microM, with a binding stoichiometry of 0.72 mol of fatty acid/mol of protein. The physicochemical and binding characteristics of the protein were in complete agreement with those of FABP isolated from human skeletal muscle.

Cloning of the cDNA encoding human skeletal-muscle fatty-acid-binding protein, its peptide sequence and chromosomal localization.

A cDNA clone for the human skeletal-muscle fatty-acid-binding protein (FABP) was isolated by screening of a human adult muscle lambda gt11 expression library with an anti-(muscle FABP) serum. The identify of the clone was confirmed by transcription/translation in vitro in plasmid pSP6.5, followed by immunoprecipitation. The nucleotide sequence of the 551 bp cDNA insert showed an open reading frame of 399 nucleotides, coding for a protein of 133 amino acid residues with a calculated molecular mass of 14858 Da and a pI of 4.94. Only one cysteine residue was found, at position 125. Peptide sequence analyses of human skeletal-muscle and heart FABP, after carbamoylmethylation and lysyl endopeptidase digestion followed by automatic Edman degradation, showed that both proteins are identical. No evidence was found for the existence of isoproteins in muscle. The chromosomal localization of the human muscle FABP gene was determined by analysing 31 human x rodent somatic-cell hybrid lines. The human muscle FABP gene could be assigned to chromosome 1pter-q31.

Detection, tissue distribution and (sub)cellular localization of fatty acid-binding protein types.

This overview of recent work on FABP types is focussed on their detection and expression in various tissues, their cellular and subcellular distribution and their binding properties. Besides the 3 well-known liver, heart and intestinal types, new types as the adipose tissue, myelin and (rat) renal FABPs have been described. Recent observations suggest the occurrence of more tissue-specific types, e.g. in placenta and adrenals. Heart FABP is widely distributed and present in skeletal muscles, kidney, lung, brain and endothelial cells. The cellular distribution of FABP types appears to be related to the function of the cells in liver, muscle and kidney. The presence of FABP in cellular organelles requires more evidence. The functional significance of the occurrence of more FABP types is unclear, in spite of the observed differences in their ligand-protein interaction.

The fatty acid-binding protein from human skeletal muscle.

Fatty acid-binding protein (FABP) was isolated from human skeletal muscle by gel filtration and anion- and cation-exchange chromatography. The isolation procedure, however, with rat and pig skeletal muscle gave mostly inactive preparations. Rat muscle FABP preparations contained parvalbumin as a contaminant. FABP from human muscle had a Mr of about 15 kDa, a pI value of 5.2, and a Kd value with oleic acid of 0.50 microM. Skeletal muscle and heart FABPs and their antisera showed a strong cross-reactivity on Western blots and in enzyme-linked immunosorbent assays (ELISA). No cross-reactivity was observed with liver FABP and its antiserum. On the basis of amino acid composition, electrophoretic behavior, fatty acid binding, and immunochemical properties, human skeletal muscle FABP must be similar or closely related to human heart FABP. The FABP content determined by ELISA was comparable in various human muscles and cultured muscle cells, but lower than that in rat muscles.

Does fatty acid-binding protein play a role in fatty acid transport?

The possible property of fatty acid-binding proteins (FABPs) to transport fatty acid was investigated in various model systems with FABP preparations from liver and heart. An effect of FABP, however, was not detectable with a combination of oleic acid-loaded mitochondria and vesicles or liposomes due to the rapid spontaneous transfer. Therefore, the mitochondria were separated from the vesicles in an equilibrium dialysis cell. The spontaneous fatty acid transfer was much lower and addition of FABP resulted in an increase of fatty acid transport. Oleic acid was withdrawn from different types of monolayers by FABP with rates up to 10%/min. When two separate monolayers were used, FABP increased fatty acid transfer between these monolayers and an equilibrium was reached.

Are fatty acid-binding proteins involved in fatty acid transfer?

The possible function of fatty acid-binding protein (FABP) to act as a fatty acid carrier protein was investigated in model systems with regard to three aspects. (1) does FABP release fatty acids from membranes? (2) does it facilitate fatty acid transport in an aqueous environment? (3) are FABP-bound fatty acids released for use by mitochondria? FABPs could bind oleic acid from liposomes and mitochondrial membranes with a ratio of 1 mol per mol protein. Oleic acid was withdrawn from negative, neutral or cholesterol-containing monolayers by FABP with rates up to 10%/min. Only about 5% of FABP penetrated into the monolayer. Spontaneous transfer of oleic acid between mitochondria and vesicles or liposomes occurred so rapidly that an effect of FABP was not detectable. When the mitochondria were separated from the vesicles in an equilibrium dialysis cell, a stimulating effect of FABP on fatty acid transfer could be demonstrated. Injected FABP increased also transfer of oleic acid between two separate monolayers. FABP-bound fatty acid was well oxidized by rat liver mitochondria. The results indicate that the FABP-fatty acid complex may function as an intermediate in the transfer of fatty acids between membranes. No functional differences were detected between heart and liver FABPs in this respect.

The binding affinity of fatty acid-binding proteins from human, pig and rat liver for different fluorescent fatty acids and other ligands.

1. Two forms of fatty acid-binding proteins (FABPs) were isolated from human, pig and rat liver cytosols by gelfiltration and anion-exchange chromatography. 2. Both forms did not show physicochemical or chemical differences. They had an Mr of about 14.5 kDa for all species. pI Values were 5.8 for both forms of human and pig liver FABP and 6.4 for both forms of rat liver FABP. In contrast to heart FABPs no tryptophan was present in liver FABPs. 3. Liver FABPs show a much higher enhancement of fluorescence at binding of 11-dansylaminoundecanoic acid, 16-anthroyloxy-palmitic acid and 1-pyrene-dodecanoic acid than heart FABPs and additionally a blue shift in excitation and emission wavelengths with the first fatty acid. 4. The bulky side-chain did not affect fatty acid binding since binding constants of liver FABPs were comparable for these fluorescent fatty acids and oleic acid (0.3-0.7 microM). 5. A 1:1 binding stoichiometry was obtained for oleic acid binding with heart and liver FABPs. 6. Liver FABPs have a high binding affinity for C16-C22 saturated and unsaturated fatty acids, palmitoyl-CoA, bromo-substituted fatty acids, POCA, tetradecylglycidic acid and flavaspidic acid. 7. Fatty acid binding could be reduced to less than 50% by arginine modification with 2,3-butadione or by enzymatic degradation of FABPs with trypsin or pronase.