Herman T.B. van Moerkerk

14CO2 production is no adequate measure of [14C] fatty acid oxidation

Palmitate oxidation was comparatively assayed in various cell-free and cellular systems by 14CO2 production and by the sum of 14CO2 and 14C-labeled acid-soluble products. The 14CO2 production rate was dependent on incubation time and amount of tissue in contrast to the total oxidation rate. The 14CO2 contribution to the oxidation rate of [1-14C]palmitate varied with homogenates from 1% with rat liver to 28% with rat kidney and amounted to only 2-4% with human muscles. With cellular systems the 14CO2 contribution varied between 20% in human fibroblasts and 70% in rat muscles and myocytes. Addition of cofactors increased the oxidation rate, but decreased the 14CO2 contribution. Various conditions appeared also to influence to a different extent the 14CO2 production and the total oxidation rate with rat tissue homogenates and with rat muscle mitochondria. Incorporation of radioactivity from [1-14C]palmitate into protein was not detectable in cell-free systems and only 2-3% of the sum of 14CO2 and 14C-labeled acid-soluble products in cellular systems. Assay of 14CO2 and 14C-labeled acid-soluble products is a much more accurate and sensitive estimation of fatty acid oxidation than assay of only 14CO2.

Ligand specificity and conformational stability of human fatty acid-binding proteins

Fatty acid binding proteins (FABPs) are small cytosolic proteins with virtually identical backbone structures that facilitate the solubility and intracellular transport of fatty acids. At least eight different types of FABP occur, each with a specific tissue distribution and possibly with a distinct function. To define the functional characteristics of all eight human FABPs, viz. heart (H), brain (B), myelin (M), adipocyte (A), epidermal (E), intestinal (I), liver (L) and ileal lipid-binding protein (I-LBP), we studied their ligand specificity, their conformational stability and their immunological crossreactivity. Additionally, binding of bile acids to I-LBP was studied. The FABP types showed differences in fatty acid binding affinity. Generally, the affinity for palmitic acid was lower than for oleic and arachidonic acid. All FABP types, except E-FABP, I-FABP and I-LBP interacted with 1-anilinonaphtalene-8-sulphonic acid (ANS). Only L-FABP, I-FABP and M-FABP showed binding of 11-((5-dimethylaminonaphtalene-1-sulfonyl)amino)undecanoic acid (DAUDA). I-LBP showed increasing binding of bile acids in the order taurine-conjugated>glycine-conjugated>unconjugated bile acids. A hydroxylgroup of bile acids at position 7 decreased and at position 12 increased the binding affinity to I-LBP. The fatty acid-binding affinity and the conformation of FABP types were differentially affected in the presence of urea. Our results demonstrate significant differences in ligand binding, conformational stability and surface properties between different FABP types which may point to a specific function in certain cells and tissues. The preference of I-LBP (but not L-FABP) for conjugated bile acids is in accordance with a specific role in bile acid reabsorption in the ileum.

Immunochemical quantitation of fatty acid-binding proteins. Tissue distribution of liver and heart FABP types in human and porcine tissues

1. Antisera against heart and liver fatty acid-binding proteins (FABPs) were used in enzyme-linked immunosorbent assay to study the cross-reactivity between these FABP types of man, pig and rat, and to assess their tissue distribution in man and pig. 2. No cross-reactivities were found of heart FABPs with anti-liver FABP sera and vice versa. With the liver FABPs, marked species differences were found, but the three proteins are clearly related. Human and pig heart FABP are immunochemically closer related to each other than to this protein from rat heart. 3. The tissue distribution of the heart and liver FABP types is similar in man, pig and rat. Liver FABP is only found in liver and intestine, and heart FABP is present in heart, skeletal muscle, kidney, lung, brain and placenta. 4. Cardiac FABP is also found in cultured human and rat endothelial cells. 5. The FABP content of human and pig liver is comparable to that of rat liver, but the tissue concentrations of heart FABP are lower in man and pig than in rat. When the latter values are expressed relative to the FABP content in heart, analogous distribution patterns are observed in man, pig and rat.

Expression of human liver fatty acid-binding protein in Escherichia coli and comparative analysis of its binding characteristics with muscle fatty acid-binding protein

Human liver fatty acid-binding protein (L-FABP) has been efficiently expressed in Escherichia coli. The cDNA encoding human liver FABP was under the control of T7 RNA polymerase promoter in the expression vector pET-3b. Expression required overnight induction with isopropyl beta-D-thiogalactopyranoside in the presence of the bacterial RNA polymerase inhibitor, rifampicin. The protein could be purified by (NH4)2SO4 fractionation, anion-exchange and gel filtration chromatography, and was recognized by anti-(human L-FABP) antiserum. The binding characteristics of delipidated recombinant human L-FABP and muscle FABP (M-FABP) for fatty acids of different chain length and saturation grade, and for various hydrophobic ligands, were determined by radiochemical analysis and also by fluorescence for L-FABP. The apparent binding affinity of the ligands was calculated by using displacement curves of oleic acid and dansylamino-undecanoic acid (DAUDA). L-FABP showed a preference for the binding of long-chain saturated and unsaturated fatty acids up to C24:1, whereas the M-FABP has a preference for unsaturated fatty acids, especially those with 18 C atoms. L-FABP also binds palmitoyl derivatives and many other hydrophobic ligands--however, generally with a lower affinity than fatty acids. M-FABP binds--besides with fatty acids--only with oestradiol and testosterone with high affinity. Fatty acids with fluorescent reporter groups are also more tightly bound by L-FABP. A direct assay and displacement study of oleic acid gave the same Kd value of DAUDA for L-FABP. Fluorescence enhancement and displacement studies indicate that the binding of fluorescent fatty acids is determined by both the fluorescent reporter group and the acyl carbon chain.

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.

Metabolic capacity, fibre type area and capillarization of rat plantaris muscle. Effects of age, overload and training and relationship with fatigue resistance

1. The influences of age (5, 13 and 25-month-old rats), overload as obtained by denervation of synergists, and training on the metabolic capacity, relative muscle cross-sectional area occupied by each fibre type, capillarization and fatigue resistance of the rat m. plantaris were investigated. 2. Creatine kinase, phosphorylase and citrate synthase activities were lower in muscles of 25 than in those of 13-month-old rats (P < 0.001). 3. Overload resulted in an increased relative area of type I and IIa fibres at all ages (P = 0.001). 4. Capillary density decreased with overload and increasing age (P < 0.001). 5. Fatigue resistance was higher in muscles of 13 than in those of 5-month-old rats (P < 0.05), and increased with overload (P < 0.05) at all ages. 6. Fatigue resistance of the whole muscle was not closely related to its oxidative capacity in contrast to what is generally found for single fibres or motor units.

Oxidative metabolism of cultured human skeletal muscle cells in comparison with biopsy material.

Human muscle cell cultures were examined for capacities to oxidize several substrates, and for activities of some enzymes related to intermediate metabolism. The results indicate that mitochondrial activities attained appreciable degrees of maturity. The specific activity of creatine kinase increased during myoblast fusion. In contrast, parameters of oxidative metabolism (palmitate and pyruvate oxidation, and cytochrome c oxidase and citrate synthase) did not significantly change throughout myogenesis and thereafter. In differentiated cells (myotubes) the oxidation capacities were pyruvate greater than 2-oxoglutarate greater than malate (+ acetylcarnitine) greater than malate (+ pyruvate), as in muscle biopsies. With regard to protein the cultured human muscle cells showed higher activities than the original biopsies (= 100%) with respect to citrate synthase (179%), but lower values for cytochrome c oxidase (50%) and creatine kinase (7%). Palmitate oxidation capacities were the same in both systems. The presence of antimycin and rotenon inhibited to a comparable extent the palmitate oxidation in cultured muscle and biopsies.

Versatile wedge-based system for the construction of unidirectional collagen scaffolds by directional freezing: practical and theoretical considerations

Aligned unidirectional collagen scaffolds may aid regeneration of those tissues where alignment of cells and extracellular matrix is essential, as for instance in cartilage, nerve bundles, and skeletal muscle. Pores can be introduced by ice crystal formation followed by freeze-drying, the pore architecture reflecting the ice crystal morphology. In this study we developed a wedge-based system allowing the production of a wide range of collagen scaffolds with unidirectional pores by directional freezing. Insoluble type I collagen suspensions were frozen using a custom-made wedge system, facilitating the formation of a horizontal as well as a vertical temperature gradient and providing a controlled solidification area for ice dendrites. The system permitted the growth of aligned unidirectional ice crystals over a large distance (>2.5 cm), an insulator prolonging the freezing process and facilitating the construction of crack-free scaffolds. Unidirectional collagen scaffolds with tunable pore sizes and pore morphologies were constructed by varying freezing rates and suspension media. The versatility of the system was indicated by the construction of unidirectional scaffolds from albumin, poly(vinyl alcohol) (a synthetic polymer), and collagen-polymer blends producing hybrid scaffolds. Macroscopic observations, temperature measurements, and scanning electron microscopy indicated that directed horizontal ice dendrite formation, vertical ice crystal nucleation, and evolutionary selection were the basis of the aligned unidirectional ice crystal growth and, hence, the aligned unidirectional pore structure. In conclusion, a simple, highly adjustable freezing system has been developed allowing the construction of large (hybrid) bioscaffolds with tunable unidirectional pore architecture.

Effect of clofibrate feeding on palmitate and branched-chain 2-oxo acid oxidation in rat liver and muscle.

Oxidation rates of palmitate (total and antimycin-insensitive), pyruvate, leucine, 4-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate and activities of two mitochondrial marker enzymes (citrate synthase and cytochrome c oxidase) were assayed in liver and muscle homogenates of fed, clofibrate-treated and 18 hr-starved rats. Significant alterations in the clofibrate-treated and the starved rats were predominantly observed in the liver. Clofibrate feeding increased antimycin-insensitive (peroxisomal) and antimycin-sensitive (mitochondrial) palmitate oxidation and 4-methyl-2-oxopentanoate and pyruvate oxidation in liver. In muscle, only the activities of citrate synthase and cytochrome c oxidase were slightly decreased. Short starvation increased antimycin-sensitive palmitate and 4-methyl-2-oxopentanoate oxidation in liver. The rates of pyruvate and 3-methyl-2-oxobutanoate oxidation were decreased in muscle homogenates. Results suggest that myopathic phenomena observed after chronic clofibrate administration are not related to changes in the capacity of oxidative metabolism of muscle.

Fetal bladder wall regeneration with a collagen biomatrix and histological evaluation of bladder exstrophy in a fetal sheep model.

Objectives: To evaluate histological changes in an animal model for bladder exstrophy and fetal repair of the bladder defect with a molecular-defined dual-layer collagen biomatrix to induce fetal bladder wall regeneration. Methods: In 12 fetal lambs the abdominal wall and bladder were opened by a midline incision at 79 days’ gestation. In 6 of these lambs an uncorrected bladder exstrophy was created by suturing the edges of the opened bladder to the abdominal wall (group 1). The other 6 lambs served as a repair group, where a dual-layer collagen biomatrix was sutured into the bladder wall and the abdominal wall was closed (group 2). A caesarean section was performed at 140 days’ gestation, followed by macroscopic and histological examination. Results: Group 1 showed inflammatory and maturational changes in the mucosa, submucosa and detrusor muscle of all the bladders. In group 2, bladder regeneration was observed, with urothelial coverage, ingrowth of fibroblasts and smooth muscle cells, deposition of collagen, neovascularization and nerve fibre formation. This tissue replaced the collagen biomatrix. No structural changes of the bladder were seen in group 2. Conclusions: The animal model, as in group 1, for bladder exstrophy shows remarkable histological resemblance with the naturally occurring anomaly in humans. This model can be used to develop new methods to salvage or regenerate bladder tissue in bladder exstrophy patients. Fetal bladder wall regeneration with a collagen biomatrix is feasible in this model, resulting in renewed formation of urothelium, blood vessels, nerve fibres, ingrowth of smooth muscle cells and salvage of the native bladder.