We observed earlier that phytanic acid activated subtype alpha of the peroxisome proliferator-activated receptor (PPAR) via the cytosolic liver-type fatty acid-binding protein (L-FABP). In a further search for minor lipid nutrients that interact with genes, we explored here the potential of branched-chain fatty acids to serve as agonists for the PPAR subtypes alpha, beta and gamma in rodent and human molecular test systems. Beyond chlorophyll-derived pristanic and phytanic acids, bacteria-derived iso- and anteiso-fatty acids and avian-derived 'uropygial' fatty acids were tested by transactivation assay. In addition, we studied binding of these fatty acids to recombinantly expressed PPAR ligand binding domains (LBDs) and to L-FABP by competition with fluorescent parinaric acid. In contrast to single methyl-branched agonists, multi methyl-branched fatty acids had high transactivation potentials in either test system; in addition, some agonists of the latter were highly subtype selective. Multi methyl-branched chain fatty acids were superior activators of human PPAR alpha, a preference not seen in the murine test system. High-affinity binding of isoprenoid-derived pristanic and phytanic acids to PPAR gamma-LBD and to L-FABP was observed, and also of pristanic acid to PPAR alpha-LBD. Polyketidic uropygial fatty acids bound to PPAR gamma-LBD only, though weakly. As both isoprenoid and polyketidic fatty acids showed high activation potentials, it became clear that binding data determined in vitro cannot predict biological activity as determined by transactivation assay. For pristanic acid, however, a signalling path similar to that found for phytanic acid can be concluded. Taken together, multi methyl-branched fatty acids of the human food chain can affect cellular metabolism through regulating gene expression.

To investigate the potentials of cis-9,trans-11 and trans-10,cis-12 conjugated linoleic acid (9-CLA and 10-CLA, respectively) isomers and their precursors trans-11 vaccenic (VA), linoleic (LA) and oleic (OA) acids for interactions with genes, we determined their binding affinities to the ligand binding domains of the human peroxisome proliferator-activated receptor (PPAR) alpha, beta and gamma subtypes by a fluorescent binding assay. Then, we elaborated a transactivation-chemiluminescent assay using HepG2 cells transfected with full-length cDNAs encoding human PPARs and retinoic acid (RA) receptors (RXRs) and with the ideal PPAR response element (iPPRE)-luciferase reporter. Binding affinity of 9-CLA was two times higher (but weaker than of precursor VA and OA) than that of 10-CLA for PPAR alpha, the opposite was observed with PPAR beta; binding affinities of CLAs and precursor fatty acids with PPAR gamma were comparable. Unlike in binding studies, transactivation potentials of 9- and 10-CLAs were comparable in the human system. Comparing the transactivation potentials of CLAs and their precursors in toto, those obtained for PPARa (two- to fourfold) in both human and murine systems (the latter was used in this study as reference) were comparable, but for PPAR and 7, fold inductions obtained in the human system were unique. Inclusion of the coactivator RXR and its natural ligand RA in the system was found unnecessary and would lead to false positive values. Taken together, the human transactivation-based test system - which was found to be superior to the murine system - comprises only iPPRE and human PPARs for rapid screening of potential CLA and other PPAR agonists.

This study aimed at an analysis of the release of Braintype and Heart-type Fatty Acid- Binding Proteins (B-FABP and HFABP) in acute ischaemic stroke and their potential value as neurobiochemical markers of brain damage.We investigated 42 consecutive patients admitted within 6 hours after ischaemic stroke. Serial venous blood samples were taken hourly between 1 to 6 hours, and at 12, 18, 24, 48, 72, 96, and 120 hours after stroke onset. In all patients lesion topography was assessed and infarct volume was calculated. The neurological deficit was quantified by the National Institutes of Health stroke scale score, and functional outcome was assessed with the modified Rankin Scale 3 months after stroke.H-FABP and B-FABP concentrations showed peak values already 2 to 3 hours after stroke onset and remained elevated up to last measurements at 120 hours.Unlike BFABP, early H-FABP concentrations were significantly associated with the severity of the neurological deficit and the functional outcome. High H-FABP release was associated with large infarction on CT.Our study shows for the first time quantitative data of serum BFABP and H-FABP being elevated early in acute ischaemic stroke indicating that especially H-FABP might have the potential to be a rapid marker of brain damage and clinical severity. As both FABPs indicate damage to neuronal and glial tissue but are not specific for cerebral infarction, further investigations are needed to better understand the prolonged release of both in ischaemic stroke which is in contrast to the transient increase after myocardial infarction and can not be explained by their renal extraction.

BACKGROUNDDetection of brain injury by serum markers is not a standard procedure in clinical practice, although several proteins, such as S100B, neuron-specific enolase (NSE), myelin basic protein, and glial fibrillary acidic protein, show promising results. We investigated the tissue distribution of brain- and heart-type fatty acid-binding proteins (B-FABP and H-FABP) in segments of the human brain and the potential of either protein to serve as plasma marker for diagnosis of brain injury.METHODSB-FABP and H-FABP were measured immunochemically in autopsy samples of the brain (n = 6) and in serum samples from (a) patients with mild traumatic brain injury (MTBI; n = 130) and (b) depressed patients undergoing bilateral electroconvulsive therapy (ECT; n = 14). The protein markers S100B and NSE were measured for comparison. Reference values of B-FABP and H-FABP were established in healthy individuals (n = 92).RESULTSThe frontal, temporal, and occipital lobes, the striatum, the pons, and the cerebellum had different tissue concentrations of B-FABP and of H-FABP. B-FABP ranged from 0.8 microg/g wet weight in striatum tissue to 3.1 microg/g in frontal lobe. H-FABP was markedly higher, ranging from 16.2 microg/g wet weight in cerebellum tissue to 39.5 microg/g in pons. No B-FABP was detected in serum from healthy donors. H-FABP serum reference value was 6 microg/L. In the MTBI study, serum B-FABP was increased in 68% and H-FABP in 70% of patients compared with S100B (increased in 45%) and NSE (increased in 51% of patients). In ECT, serum B-FABP was increased in 6% of all samples (2 of 14 patients), whereas H-FABP was above its upper reference limit (6 microg/L) in 17% of all samples (8 of 14 patients), and S100B was above its upper reference limit (0.3 microg/L) in 0.4% of all samples.CONCLUSIONSB-FABP and H-FABP patterns differ among brain tissues, with the highest concentrations in the frontal lobe and pons, respectively. However, in each part of the brain, the H-FABP concentration was at least 10 times higher than that of B-FABP. Patient studies indicate that B-FABP and H-FABP are more sensitive markers for minor brain injury than the currently used markers S100B and NSE.

Human epidermal-type fatty acid-binding protein (E-FABP) belongs to a family of intracellular 14-15 kDa lipid-binding proteins, whose functions have been associated with fatty acid signalling, cell growth, regulation and differentiation. As a contribution to understanding the structure-function relationship, we report in the present study features of its solution structure and backbone dynamics determined by NMR spectroscopy. Applying multi-dimensional high-resolution NMR techniques on unlabelled and 15N-enriched recombinant human E-FABP, the 1H and 15N resonance assignments were completed. On the basis of 2008 distance restraints, the three-dimensional solution structure of human E-FABP was subsequently obtained (backbone atom root-mean-square deviation of 0.92+/-0.11 A; where 1 A=0.1 nm), consisting mainly of 10 anti-parallel beta-strands that form a beta-barrel structure. 15N relaxation experiments (T1, T2 and heteronuclear nuclear Overhauser effects) at 500, 600 and 800 MHz provided information on the internal dynamics of the protein backbone. Nearly all non-terminal backbone amide groups showed order parameters S(2)>0.8, with an average value of 0.88+/-0.04, suggesting a uniformly low backbone mobility in the nanosecond-to-picosecond time range. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the beta-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics elaborated in the present study differ markedly from those of the phylogenetically closely related heart-type FABP and the more distantly related ileal lipid-binding protein, implying a strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family.

Members of the phylogenetically related intracellular lipid binding protein (iLBP) are characterized by a highly conserved tertiary structure, but reveal distinct binding preferences with regard to ligand structure and conformation, when binding is assessed by the Lipidex method ( removal of unbound ligand by hydrophobic polymer) or by isothermal titration calorimetry, a true equilibrium method. Subfamily proteins bind retinoids, subfamily II proteins bind bulky ligands, examples are intestinal bile acid binding protein (I-BABP) and liver fatty acid binding protein (L-FABP) which binds 2 ligand molecules, preferably monounsaturated and n-3 fatty acids. Subfamily III intestinal fatty acid binding protein (I-FABP) binds fatty acid in a bent conformation. The fatty acid bound by subfamily IV FABPs has a U-shaped conformation; here heart (H-)FABP preferably binds n-6, brain (B-)FABP n-3 fatty acids. The ADIFAB-method is a fluorescent test for fatty acid in equilibrium with iLBP and reveals some correlation of binding affinity to fatty acid solubility in the aqueous phase; these data are often at variance with those obtained by the other methods. Thus, in this review published binding data are critically discussed, taking into account on the one hand binding increments calculated for fatty acid double bonds on the basis of the solubility hypothesis, on the other hand the interpretation of calorimetric data on the basis of crystallographic and solution structures of iLBPs.

Isoprenoid phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is degraded in peroxisomes by alpha -oxidation to pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) and then via beta -oxidation. Branched-chain phytanic add is an activator of the peroxisome proliferator activated receptor alpha (PPAR alpha) which in liver cells regulates expression of genes encoding peroxisomal and mitochondrial beta -oxidative enzymes as well as cytosolic/nuclear liver-type fatty acid binding protein (L-FABP). In this report we address the question whether pristanic acid also acts as activator of PPARa and thus mediates the expression of its catabolizing enzymes. In a first in vivo approach we fed pristanic acid for 14 days to wildtype mice and to mice lacking sterol carrier protein 2/sterol carrier protein x which leads to a phenotype having high concentrations of branched-chain fatty acids. In either genotype, feeding pristanic acid was associated with a strong induction of peroxisomal beta -oxidation enzymes tested (acyl-CoA oxidase, bifunctional enzyme, thiolase) as well as of L-FABP. The link between pristanic acid and protein expression observed was established by carrying out assays for transactivation of PPAR alpha(. in transfected HepG2 cells. In comparison to hypolipidemic drugs and to straight-chain fatty acids known to be PPAR alpha. agonists, branched-chain phytanic and pristanic acids were substantially stronger activators, pristanic acid being even superior to phytanic acid.