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Enhanced fatty acid uptake may lead to the accumulation of lipid intermediates. This is related to insulin resistance and type 2 diabetes mellitus. Rodent studies suggest that fatty acid transporters are acutely regulated by insulin. We investigated differences in fatty acid transporter content before and at the end of a hyperinsulinemic euglycemic clamp in skeletal muscle (m. vastus lateralis) of obese, glucose-intolerant men (IGT) and obese normal glucose tolerant controls (NGT). The fatty acid transporter FAT/CD36 protein content increased 1.5-fold (P < 0.05) after 3-hrs of insulin stimulation with no difference between IGT and control subjects. No change was seen in cytosolic fatty acid binding protein (FABPc) protein content. The increase in FAT/CD36 protein content was positively related to insulin resistance as measured during the clamp (r = 0.56, P < 0.05). An increase in FAT/CD36 protein content in skeletal muscle may result in a higher fractional extraction of fatty acids (larger relative uptake) after a meal, enhancing triglyceride accumulation in the muscle. We conclude that also in obese humans the FAT/CD36 protein content in skeletal muscle is dynamically regulated by insulin in vivo on the short term.

The increased use of dietary plant oil supplementation combined with high dietary lipid loads challenges the lipid transport systems of cultivated fish species. Fatty acid binding proteins (FABPs) have been thoroughly studied as intracellular fatty acid transporters in vertebrates, but no data have been reported in Atlantic salmon. In the present study, comparative characterizations were performed, and dietary influence of plant oil supplementation on FABP3 and FABP10 expression was studied for several tissues in two separate dietary trials. In trial I, groups (6 fish each) were fed diets for 42 weeks (body mass 142+/-1 to 1463+/-83 g) (mean+/-S.D.), containing graded levels of rapeseed oil substituting for fish oil using a linear regression design. In trial II, groups (3 fish each) were fed 100% fish oil or 100% plant oil for 22 months (0.160+/-0.052 to 2523+/-590 g) (mean+/-S.D.) and sampled at regular intervals. Liver and muscle tissues appeared to express several FABPs possibly linked to different metabolic functions. FABPs mRNA expression did not change with dietary inclusion of 75% rapeseed oil, whereas FABP3 protein expression seemed to be affected by dietary rapeseed oil inclusion. Significant changes in red muscle FABP3 mRNA expression correlate to significant changes in total beta-oxidation capacity during the energy consuming process of smoltification.

Human heart-type fatty acid-binding protein (FABP) is suggested as an early plasma marker of acute myocardial infarction (AMI), and several studies have proved that, for early diagnosis of AMI, FABP performs better than myoglobin, which is a more often used early marker protein. Because serial measurement of biochemical markers in plasma is now universally accepted as an important determinant in AMI diagnosis, a rapid and continuous measuring method for FABP would be desirable. The aim of the present study was to develop an immunoassay based on the principle of displacement and using a column for rapid and continuous measurement of FABP in plasma. Glass columns filled with Sepharose-bound FABP were loaded with a horseradish peroxidase (HRP)-labeled antibody (Ab) and equilibrated with human plasma. After reaching a stable baseline, human plasma spiked with FABP or plasma from AMI patients was added. The Ab-HRP complex dissociated due to the presence of FABP in the plasma and was subsequently quantified. For plasma from AMI patients (n=5), the Ab-HRP level thus measured correlated with the corresponding plasma FABP concentration (R=0.96). The results of this study show the feasibility of a sensor for continuous monitoring of FABP in plasma.

Comparison of reperfusion injury in kidneys transplanted from LD, HBD or NHBD donors is presented in the paper. Central venous blood samples (taken during perioperative period) was assessed for free radicals, total antioxidant activity and various markers of tissue injury. There was demonstrable ischemia reperfusion injury occurring at the time of revascularization, which was particularly notable in kidneys transplanted from NHBD donors.

To risk-stratify patients with chest pain who are admitted to emergency rooms and for whom initial evaluation is not conclusive, the use of cardiac markers has become a standard procedure. A recently introduced early plasma marker for acute myocardial infarction (AMI) is the 14.5-kDa cytoplasmic heart-type fatty acid-binding protein (FABP). To fully exploit its early release from injured myocardium, a rapid method for repeated measurements or continuous monitoring of FABP in plasma is desirable. Such an on-line method could be an immunosensor based on displacement. The aim of the present study was to further investigate the principles underlying the displacement assay of FABP, both in buffer and in plasma. Batches of sepharose-bound FABP were loaded with an antibody-horseradish peroxidase (HRP) conjugate (anti-FABP). Continuous measurement of FABP was mimicked by repeated addition of FABP containing solutions followed by several washing steps. In the presence of free FABP the antibody-HRP complex dissociated and was subsequently quantified. Significant displacement in the presence of free FABP was observed in both buffer and human plasma. Anti-FABP could be intermittently displaced in the same batch, for at least 9 h, and the displacement was concentration-dependent. These results show the feasibility of a sensor based on the displacement principle to be used for the diagnosis of AMI in emergency medicine.

Background: With a universal shortage of donor organs, screening and selection of marginal kidneys from non-heart-beating donors (NHBDs) provides a valuable source for transplantation. Pre-transplant viability assessment is based on a combination of flow characteristics and assessment of ischaemic tissue injury during NHBD kidney machine perfusion by measurement of total glutathione S-transferase (GST) activity. Successful viability screening has facilitated 69 renal transplants from 60 NHBDs within our transplant centre since 1998, with a first-year graft survival of 90.5%., Methods: We have investigated alanine aminopeptidase (Ala-AP) and fatty acid binding protein (FABP) as alternative biochemical markers to GST for pre-transplantation viability assessment. They were measured, together with GST, in tissue perfusate samples from machine-perfused kidneys from controlled and uncontrolled NHBDs., Results: During machine perfusion, a parallel response was seen for each of the three markers in the perfusates of controlled and uncontrolled NHBD kidneys over the 4-h perfusion. A highly significant correlation was obtained between GST and Ala-AP activities (P<0.0001) and between GST activity and FABP concentration (P<0.0001) in corresponding samples., Conclusion: In this study, GST, Ala-AP and FABP represent equivalent biochemical markers in terms of their ability to quantitate renal tissue injury in human NHBD kidneys. However, there may be some advantage in using all three analytes to provide complementary information on kidney allograft viability.

Giant vesicles were used to study the rates of uptake of long-chain fatty acids by heart, skeletal muscle, and adipose tissue of obese and lean Zucker rats. With obesity there was an increase in vesicular fatty acid uptake of 1.8-fold in heart, muscle and adipose tissue. In some tissues only fatty acid translocase (FAT) mRNA (heart, +37%; adipose, +80%) and fatty acid-binding protein (FABPpm) mRNA (heart, +148%; adipose, +196%) were increased. At the protein level FABPpm expression was not changed in any tissues except muscle (+14%), and FAT/CD36 protein content was altered slightly in adipose tissue (+26%). In marked contrast, the plasma membrane FAT/CD36 protein was increased in heart (+60%), muscle (+80%), and adipose tissue (+50%). The plasma membrane FABPpm was altered only in heart (+50%) and adipose tissues (+70%). Thus, in obesity, alterations in fatty acid transport in metabolically important tissues are not associated with changes in fatty acid transporter mRNAs or altered fatty acid transport protein expression but with their increased abundance at the plasma membrane. We speculate that in obesity fatty acid transporters are relocated from an intracellular pool to the plasma membrane in heart, muscle, and adipose tissues.

Ligation of the main left coronary artery in mice serves as a model for myocardial infarction (MI). We tested whether plasma concentrations of heart-type fatty acid-binding protein (H-FABP) and/or cardiac troponin T (cTnT) discriminate between infarcted and sham-operated mice and allow estimation of infarct size. Mice were subjected to coronary artery ligation or sham surgery and release curves of H-FABP and cTnT were determined. At 4 h after surgery the mean (+/-SD) H-FABP plasma concentration was 461+/-134 microg/l (n=10) in MI and 185+/-51 microg/l (n=6; P<0.001) in sham-operated mice. By 24 h after surgery H-FABP levels had returned to normal in both groups. cTnT plasma concentrations increased up to 48 h after MI to 13.5+/-6.2 microg/l (n=6; P<0.001) compared with 0.031+/-0.063 microg/l (n=7) in sham-operated mice. Linear regression analysis revealed a significant correlation between plasma H-FABP at 4 h and infarct size assessed 7 days after surgery. Plasma cTnT did not correlate significantly with infarct size. In conclusion, plasma cTnT concentration at 48 h after infarction can be used to distinguish MI from sham mice, whereas H-FABP concentration at 4 h can be used for stratification of animals according to infarct size.

To generate antibodies to defined cell-surface antigens, we used a large phage antibody fragment library to select on cell transfectants expressing one of three chosen receptors. First, in vitro panning procedures and phage antibody screening ELISAs were developed using whole live cells stably expressing the antigen of interest. When these methodologies were applied to Chinese hamster ovary (CHO) cells expressing one of the receptors for a neuropeptide, somatostatin, using either direct cell panning or a strategy of depletion or ligand-directed elution, many different pan-CHO-cell binders were selected, but none was receptor specific. However, when using direct panning on CHO-cells expressing the human membrane protein CD36, an extraordinary high frequency of antigen-specific phage antibodies was found. Panning on myoblasts expressing the rat homologue of CD36 revealed a similar selection dominance for anti-(CD36). Binding of all selected 20 different anti-(CD36) phage was surprisingly inhibited by one anti-(CD36) mAb CLB-IVC7, which recognizes a functional epitope that is also immunodominant in vivo. Similar inhibition was found for seven anti-(rat) CD36 that cross-reacted with human CD36. Our results show that, although cells can be used as antigen carriers to select and screen phage antibodies, the nature of the antigen target has a profound effect on the outcome of the selection.

The long-distance migratory flights of birds are predominantly fueled by the oxidation of fatty acids, which are sourced primarily from extracellular adipose stores. These fatty acids have to be transported, via the circulatory system, to the mitochondria of the active muscles. An important facilitator of fatty acid transport within the cytoplasm of muscle cells is fatty acid binding protein (FABP), which serves as an intracellular carrier of long-chain fatty acids. In mammals, the muscular FABP content is related to the fatty acid oxidation capacity of the tissue. The aim of this study was to measure FABP in samples taken from the cardiac, pectoralis, and semimembranosus muscles of a long-distance avian migrant, the barnacle goose (Branta leucopsis), at various stages of development. Western blot analysis identified a single goose muscle protein of 15 kDa that was able to bind fatty acids and showed a 66% cross-reactivity with antibodies against human heart-type FABP. Captive goslings showed no significant changes in FABP content of either the heart (62.6 +/- 10.6 microgram/g wet wt) or the semimembranosus muscle (8.4 +/- 1.9 microgram/g wet wt) during development. However, in both peripheral and deep sites within the pectoralis muscle, FABP content of samples taken from captive goslings were approximately 10-fold higher throughout development and reached values of 30-40 microgram/g wet wt in fledging goslings at 7 wk of age. A further twofold higher value was seen in wild but not in captive goslings immediately before migration (12 wk of age). Similarly, FABP content was significantly higher in pectoralis samples taken from wild adults (94.3 +/- 3.6 microgram/g wet wt) compared with those from captive adults (60.5 +/- 3.6 micro/g wet wt). These results suggest that the experience of flight activity may be of critical importance in achieving maximal expression of FABP in the pectoralis muscles of postfledging and mature geese immediately before migration.

The rat membrane protein fatty acid translocase (FAT), which shows sequence similarity to human CD36 (a membrane protein supposedly involved in a variety of membrane processes), is implicated in the transport of long-chain fatty acids across cellular membranes. To set up an immunoassay for quantification of FAT in different tissues, we isolated a series of anti-FAT antibodies by panning a large naive phage antibody library on FAT-transfected H9c2 cells. All seven different phage antibody fragments isolated reacted specifically with FAT, and most likely recognize the same or closely located immunodominant sites on FAT, as a competitive monoclonal antibody (mAb) (CLB-IV7) completely blocked the binding of all these phage antibodies to cells. A sandwich ELISA was set up using mAb 131. 4 (directed against purified CD36 from human platelets) as capture antibody and phage antibodies and anti-phage sera as detector. With this ELISA (sensitivity 0.05 microgram/ml), the FAT content in isolated cardiomyocytes was found to be comparable with that of total heart ( approximately 3 mg/g of protein), while liver tissue and endothelial cells were below the detection limit (<0.1 mg of FAT/g of protein). During rat heart development, protein levels of FAT rose from 1.7+/-0.7 mg/g of protein on the day before birth to 3.6+/-0.4 mg/g of protein on day 70. Comparing control with streptozotocin-induced diabetic rats, a statistically significant (P<0.05) 2-4-fold increase of FAT was seen in heart (from 4.2+/-2.3 to 11.0+/-5.7 mg/g of protein), soleus (from 0.6+/-0.1 to 1.4+/-0.5 mg/g of protein) and extensor digitorum longus (EDL) muscle (from 0.3+/-0.1 to 1. 2+/-0.8 mg/g of protein). In addition, the FAT contents of each of these muscles were found to be of similar magnitude to the contents of cytoplasmic heart-type fatty-acid-binding protein in both diabetic rats and controls, supporting the suggested roles of these two proteins in cellular fatty acid metabolism. This is the first time phage display technology has been succesfully applied for direct selection, from a large naive antibody library, of antibodies that recognize selected membrane proteins in their natural context.

Cytoplasmic heart-type fatty acid-binding protein has recently gained much attention in clinical diagnosis as a very early marker of acute myocardial infarction. Immunoassays have been developed for determination of this protein in plasma and urine samples. In the present study it is shown that those types of fatty acid-binding proteins which are abundant in tissues other than heart and muscle do not interfere with immunochemical determination of heart-type fatty acid-binding protein. To provide sufficient protein of consistent quality as standard in these immunoassays, human heart-type fatty acid-binding protein was cloned, expressed in Escherichia coli and purified to homogeneity. For quantitation of the recombinant protein its extinction coefficient was determined. Comparison of the recombinant and tissue-derived proteins by a variety of methods revealed both proteins to show similar kinetic as well as equilibrium constants with respect to two monoclonal antibodies currently applied in immunochemical detection of heart-type fatty acid-binding protein. Both preparations were indistiguishable in sandwich-ELISA and immunosensor measurements. A high stability of the recombinant protein was proven by ELISA measurements during storage and several freeze and thaw cycles. Thus, recombinant and tissue-derived heart-type fatty acid-binding proteins are immunochemically equivalent. The recombinant human heart-type fatty acid-binding protein is now available as standard for immunoassays.

To allow a more rapid determination of heart-type fatty acid-binding protein (FABP) concentration in plasma a direct non-competitive (sandwich-type) ELISA was developed which uses high-affinity monoclonal antibodies to FABP. Total performance time of the one-step immunoassay is 45 min. The standard curve was linear between 0.2-6 micrograms/L, and the within-run and between-run coefficients of variations were below 6 and 11%, respectively. The serum FABP concentration measured in 79 healthy individuals was 1.6 (0.8) [mean (SD), range 0.3-5.0] micrograms/L. The assay can be used for rapid plasma or serum FABP measurement in the early diagnosis of acute myocardial infarction.

beta 2-Glycoprotein I (beta 2GPI) is an essential cofactor for the binding to lipids of anticardiolipin antibodies (ACA), isolated from patients with anti-phospholipid syndrome. We used ellipsometry to study the binding of beta 2GPI and the beta 2GPI-mediated binding of ACA to planar membranes composed of phosphatidylcholine (PC) and 5-20 mol % phosphatidylserine (PS). No binding of beta 2GPI was observed to neutral (PC) membranes. Maximal binding of beta 2GPI was 3.2-3.6 pmol.cm-2. Affinity decreased strongly with decreasing PS content; increasing the NaCl and CaCl2 concentrations also led to a decrease in affinity. At physiologic conditions (10 mol % PS, 120 mM NaCl, and 3 mM CaCl2), a Kd of 14 microM was observed. Binding constants were insensitive to the chemical composition of the negatively charged phospholipid headgroup. ACA (1.25-10 micrograms.mL-1) caused a 30-40-fold enhancement of beta 2GPI binding to PS/PC membranes (20 mol % PS), resulting in the binding of about 2 pmol.cm-2 divalent ACA-(beta 2GPI)2 complexes at 100 nM beta 2GPI. In the absence of beta 2GPI, binding of ACA was negligible. Ad- and desorption kinetics of ACA-beta 2GPI complexes indicate that the initial monovalent association of ACA to membrane-bound beta 2GPI is rapidly followed by formation of divalent ACA-(beta 2GPI)2 complexes. Experiments with monovalent Fab1 fragments of ACA showed no appreciable effect on the beta 2GPI binding to lipid, substantiating the notion that divalent interactions are essential for the high-affinity binding of ACA-beta 2GPI. The anticoagulant effect of ACA is rationalized by the observation that binding of ACA-beta 2GPI complexes to the PSPC membrane severely restricts the adsorption of blood coagulation factor Xa.