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Background & Aims: To investigate the acute effects of intravenous vs enteral meal administration on circulating bile acid and gut hormone responses., Methods: In a randomized crossover design, we compared the effects of duodenal (via a nasoduodenal tube) vs parenteral (intravenous) administration over 180 min of identical mixed meals on circulating bile acid and gut hormone concentrations in eight healthy lean men. We analysed the bile acid and gut hormone responses in two periods: the intraprandial period from time point (T) 0 until T180 during meal administration and the postprandial period from T180 until T360, after discontinuation of meal administration., Results: Intravenous meal administration decreased the intraprandial (AUC (μmol/L∗min) duodenal 1469 ± 284 vs intravenous 240 ± 39, p < 0.01) and postprandial bile acid response (985 ± 240 vs 223 ± 5, p < 0.05) and was accompanied by decreased gut hormone responses including glucose-dependent insulinotropic polypeptide, glucagon-like peptide 1, glucagon-like peptide 2 and fibroblast growth factor 19. Furthermore, intravenous meal administration elicited greater glucose concentrations, but similar insulin concentrations compared to enteral administration., Conclusions: Compared to enteral administration, parenteral nutrition results in lower postprandial bile acid and gut hormone responses in healthy lean men. This was accompanied by higher glucose concentrations in the face of similar insulin concentrations exposing a clear incretin effect of enteral mixed meal administration. The alterations in bile acid homeostasis were apparent after only one intravenous meal., Competing Interests: Conflict of interest None., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Patients with chronic intestinal failure (CIF) often develop cholestatic liver injury, which may lead to liver failure and need for organ transplantation., Objectives: The aim of this study was to investigate whether citrulline (CIT) and the enterokine fibroblast growth factor 19 (FGF19) are associated with chronic cholestasis and survival in adult CIF patients, and to develop a risk score to predict their survival., Methods: We studied 135 adult CIF patients on intravenous supplementation (>3 mo). Associations of plasma CIT and FGF19 with chronic cholestasis and survival were estimated by logistic and Cox regression models. A predictive risk score was developed and validated internally., Results: Patients with chronic cholestasis (17%) had a reduced 5-y survival rate compared with patients without chronic cholestasis (38% and 62%, respectively). In multivariable analysis, low FGF19, low CIT, and female sex were associated with chronic cholestasis. Patients with low rather than high CIT or FGF19 also had reduced 5-y survival rates (29% compared with 69%; 54% compared with 66%, respectively). Risk factors identified in multivariable analysis of survival were low FGF19 (HR: 3.4), low CIT (HR: 3.3), and number of intravenous infusions per week (HR: 1.4). These 3 predictors were incorporated in a risk model of survival termed Model for End-Stage Intestinal Failure (MESIF) (C-statistic 0.78). The 5-y survival rates for patients with MESIF scores of 0 to <20 (n = 47), 20-40 (n = 75), and >40 (n = 13) were 80%, 58%, and 14%, respectively., Conclusions: CIT and FGF19 predict chronic cholestasis and survival in this cohort of adult CIF patients, and the derived MESIF score is associated with their survival. Pending external validation, the MESIF score may help to identify patients for closer clinical monitoring or earlier referral to intestinal transplantation centers., (Copyright © American Society for Nutrition 2019.)

Introduction: Endotoxins, in the form of lipopolysaccharides (LPS), are potent inducers of biliary injury. However the mechanism by which injury develops remains unclear. We hypothesized that hepatic macrophages are pivotal in the development of endotoxin-induced biliary injury and that no injury would occur in their absence., Material and Methods: Clodronate liposomes were used to deplete macrophages from the liver. Forty-eight rats were equally divided across six study groups: sham operation (sham), liposome treatment and sham operation (liposomes+sham), 1mg/kg LPS i.p. (LPS), liposome treatment and LPS administration (liposomes+LPS), hepatic ischaemia-reperfusion injury with LPS administration (IRI+LPS) and liposome treatment followed by IRI+LPS (liposomes+IRI+LPS). Following 6h of reperfusion, blood, bile, and liver tissue was collected for further analysis. Small bile duct injury was assessed, serum liver tests were performed and bile composition was evaluated. The permeability of the blood-biliary barrier (BBB) was assessed using intravenously administered horseradish peroxidase (HRP)., Results: The presence of hepatic macrophages was reduced by 90% in LPS and IRI+LPS groups pre-treated with clodronate liposomes (P<0.001). Severe small bile duct injury was not affected by macrophage depletion, and persisted in the liposomes+IRI+LPS group (50% of animals) and liposomes+LPS group (75% of animals). Likewise, BBB impairment persisted following macrophage depletion. LPS-induced elevation of the chemokine Mcp-1 in bile was not affected by macrophage depletion., Conclusions: Depletion of hepatic macrophages did not prevent development of biliary injury following LPS or LPS-enhanced IRI. Cholangiocyte activation rather than macrophage activation may underlie this injury. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background & Aims: Bile acids (BA) are pleiotropic hormones affecting glucose and lipid metabolism. The physiochemical properties of different BA species affect their enterohepatic dynamics and their affinity for bile acid receptors. The BA pool composition is altered in patients with type 2 diabetes and obesity. In this study we used a 2-week very-low-calorie diet (VLCD) to investigate the effects of weight loss on BA pool composition and postprandial dynamics., Methods: We performed mixed meal tests in obese, insulin resistant subjects before and after the VLCD. We measured postprandial plasma levels of glucose, insulin, BA and the BA-induced enterokine fibroblast growth factor 19 (FGF19)., Results: The VLCD decreased weight by 4.5 ± 2.3 kg (p < 0.0001) within 14 days. Weight loss increased peak postprandial deoxycholate (DCA) levels (median [IQR]: 0.90 [0.90] vs. 1.25 [1.35] μmol/L; p = 0.045*). Other BA species, glucose, insulin and FGF19 levels and prandial excursions were not significantly affected. The VLCD decreased resting and postprandial energy expenditure by 7 and 11% respectively., Conclusions: VLCD induced weight loss increased postprandial DCA peak levels and decreased resting energy expenditure in obese insulin resistant subjects., (Copyright © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Background & Aims: Parenteral nutrition (PN), a lifesaving therapy in patients with intestinal failure, has been associated with hepatobiliary complications including steatosis, cholestasis and fibrosis, collectively known as parenteral nutrition-associated liver disease (PNALD). To date, the pathogenesis of PNALD is poorly understood and therapeutic options are limited. Impaired bile salt homeostasis has been proposed to contribute PNALD. The objective of this study was to establish a PNALD model in rats and to evaluate the effects of continuous parenteral nutrition (PN) on bile salt homeostasis., Methods: Rats received either PN via the jugular vein or received normal diet for 3, 7 or 14 days. Serum biochemistry, hepatic triglycerides, circulating bile salts and C4, IL-6 and TNF-alpha, and lipogenic and bile salt homeostatic gene expression in liver and ileum were assessed., Results: PN increased hepatic triglycerides already after 3 days of administration, and resulted in conjugated bilirubin elevation after 7 or more days. This indicates PN-induced steatosis and impaired canalicular secretion of bilirubin, the latter which is in line with reduced hepatic expression of Mrp2 mRNA. There was no histological evidence for liver inflammation after PN administration, and circulating levels of pro-inflammatory cytokines IL-6 and TNF-α, were comparable in all groups. Hepatic expression of Fxr mRNA was decreased after 7 days of PN, without apparent effect on expression of Fxr targets Bsep and Shp. Nonetheless, Cyp7a1 expression was reduced after 7 days of PN, indicative for lowered bile salt synthesis. Circulating levels of C4 (marker of bile salt synthesis) were also decreased after 3, 7 and 14 days of PN. Levels of circulating bile salts were not affected by PN., Conclusions: This study showed that PN in rats caused early mild steatosis and cholestasis, while hepatic and systemic inflammation were not present. The onset of these abnormalities was associated with alterations in bile salt synthesis and transport. This animal model serves as an experimental model to further investigate the pathogenesis of PNALD inflicted by steatosis and cholestasis., (Copyright © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Background & Aims: Prognostic biomarkers are lacking in primary sclerosing cholangitis, hampering patient care and the development of therapy. We aimed to identify novel protein biomarkers of disease severity and prognosis in primary sclerosing cholangitis (PSC)., Methods: Using a bead-based array targeting 63 proteins, we profiled a derivation panel of Norwegian endoscopic retrograde cholangiography bile samples (55 PSC, 20 disease controls) and a Finnish validation panel (34 PSC, 10 disease controls). Selected identified proteins were measured in serum from two Norwegian PSC cohorts (n=167 [1992-2006] and n=138 [2008-2012]), inflammatory bowel disease (n=96) and healthy controls (n=100)., Results: In the bile derivation panel, the levels of 14 proteins were different between PSC patients and controls (p<0.05); all were confirmed in the validation panel. Twenty-four proteins in the bile derivation panel were significantly (p<0.05) different between PSC patients with mild compared to severe cholangiographic changes (modified Amsterdam criteria); this was replicated for 18 proteins in the validation panel. Interleukin (IL)-8, matrix metallopeptidase (MMP)9/lipocalin (LCN)2-complex, S100A8/9, S100A12 and tryptophan hydroxylase (TPH)2 in the bile were associated with both a PSC diagnosis and grade of cholangiographic changes. Stratifying PSC patients according to tertiles of serum IL-8, but not MMP9/LCN2 and S100A12, provided excellent discrimination for transplant-free survival both in the serum derivation and validation cohort. Furthermore, IL-8 was associated with transplant-free survival in multivariable analyses in both serum panels independently of age and disease duration, indicating an independent influence on PSC progression. However, the Enhanced Liver Fibrosis (ELF®) test and Mayo risk score proved to be stronger predictors of transplant-free survival., Conclusions: Based on assaying of biliary proteins, we have identified novel biliary and serum biomarkers as indicators of severity and prognosis in PSC., Lay Summary: Prognostic biomarkers are lacking in primary sclerosing cholangitis, hampering patient care and the development of therapy. We have identified inflammatory proteins including calprotectin and IL-8 as important indicators of disease severity and prognosis in bile and serum from patients with primary sclerosing cholangitis., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Hepatic failure is a feared complication that accounts for up to 75% of mortality after extensive liver resection. Despite improved perioperative care, the increasing complexity and extensiveness of surgical interventions, in combination with an expanding number of resections in patients with compromised liver function, still results in an incidence of postresectional liver failure (PLF) of 1-9%. Preventive measures aim to enhance future remnant liver size and function. Numerous non-invasive techniques to assess liver function and predict remnant liver volume are being developed, along with introduction of novel surgical strategies that augment growth of the future remnant liver. Detection of PLF is often too late and treatment is primarily symptomatic. Current therapeutic research focuses on ([bio]artificial) liver function support and regenerative medicine. In this review we discuss the current state and new developments in prediction, prevention and management of PLF, in light of novel insights into the aetiology of this complex syndrome., Lay Summary: Liver failure is the main cause of death after partial liver resection for cancer, and is presumably caused by an insufficient quantity and function of the liver remnant. Detection of liver failure is often too late, and current treatment focuses on relieve of symptoms. New research initiatives explore artificial support of liver function and stimulation of regrowth of the remnant liver., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Introduction: Postoperative liver failure (PLF) is a dreaded complication after partial hepatectomy. The peak bilirubin criterion (>7.0 mg/dL or ≥120 μmol/L) is used to define PLF. This study aimed to validate the peak bilirubin criterion as postoperative risk indicator for 90-day liver-related mortality., Methods: Characteristics of 956 consecutive patients who underwent partial hepatectomy at the Maastricht University Medical Centre or Royal Free London between 2005 and 2012 were analyzed by uni- and multivariable analyses with odds ratios (OR) and 95% confidence intervals (95%CI)., Results: Thirty-five patients (3.7%) met the postoperative peak bilirubin criterion at median day 19 with a median bilirubin level of 183 [121-588] μmol/L. Sensitivity and specificity for liver-related mortality after major hepatectomy were 41.2% and 94.6%, respectively. The positive predictive value was 22.6%. Predictors of liver-related mortality were the peak bilirubin criterion (p < 0.001, OR = 15.9 [95%CI 5.2-48.7]), moderate-severe steatosis and fibrosis (p = 0.013, OR = 8.5 [95%CI 1.6-46.6]), ASA 3-4 (p = 0.047, OR = 3.0 [95%CI 1.0-8.8]) and age (p = 0.044, OR = 1.1 [95%CI 1.0-1.1])., Conclusion: The peak bilirubin criterion has a low sensitivity and positive predictive value for 90-day liver-related mortality after major hepatectomy., (Copyright © 2016 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Background & Aims: Ursodeoxycholic acid (UDCA) exerts anticholestatic, antifibrotic and antiproliferative effects in primary biliary cirrhosis (PBC) via mechanisms not yet fully understood. Its adequate biliary enrichment is considered mandatory for therapeutic efficacy. However, precise determination of biliary enrichment of UDCA is not possible in clinical practice. Therefore, we investigated (i) the relationship between biliary enrichment and plasma pharmacokinetics of UDCA, (ii) the effect of UDCA on plasma and biliary bile acid composition and conjugation patterns, and (iii) on the intestinal detoxification machinery in patients with PBC and healthy controls., Methods: In 11 PBC patients and 11 matched healthy subjects, cystic bile and duodenal tissue were collected before and after 3 weeks of administration of UDCA (15 mg/kg/day). Extensive pharmacokinetic profiling of bile acids was performed. The effect of UDCA on the intestinal detoxification machinery was studied by quantitative PCR and Western blotting., Results: The relative fraction of UDCA and its conjugates in plasma at trough level[x] correlated with their biliary enrichment[y] (r=0.73, p=0.0001, y=3.65+0.49x). Taurine conjugates of the major hydrophobic bile acid, chenodeoxycholic acid, were more prominent in bile of PBC patients than in that of healthy controls. Biliary bile acid conjugation patterns normalized after treatment with UDCA. UDCA induced duodenal expression of key export pumps, BCRP and P-glycoprotein., Conclusions: Biliary and trough plasma enrichment of UDCA are closely correlated in PBC and health. Taurine conjugation may represent an adaptive mechanism in PBC against chenodeoxycholic acid-mediated bile duct damage. UDCA may stabilize small intestinal detoxification by upregulation of efflux pumps., (Copyright © 2012. Published by Elsevier B.V.)

Apolipoprotein AV (apoAV) overexpression causes a decrease in plasma triglyceride (TG) levels, while deficiency of apoAV causes hypertriglyceridemia in both men and mice. However, contrary to what would be expected, plasma apoAV and TG levels in humans are positively correlated. To address this apparent paradox, we determined plasma apoAV levels in various mouse models with median TG levels ranging from 30 mg/dl in wild-type mice to 2089 mg/dl in glycosylphosphatidylinositol-anchored HDL binding protein 1-deficient mice. The data show that apoAV and TG levels are positively correlated in mice (r = +0.798, P < 0.001). In addition, we show that LPL gene transfer caused a simultaneous decrease in TG and apoAV in LPL-deficient mice. The combined data suggest that apoAV levels follow TG levels due to an intimate link between the apoAV molecule and TG-rich lipoproteins, comprising both secretion and removal of these lipoproteins. Taken together, the data suggest that higher plasma apoAV levels reflect an increased demand for plasma TG hydrolysis under normal physiological conditions.

Variation in the apolipoprotein A5 (APOA5) gene has consistently been associated with increased plasma triglyceride (TG) levels in epidemiological studies. In vivo functionality of these variations, however, has thus far not been tested. Using adenoviral over-expression, we evaluated plasma expression levels and TG-lowering efficacies of wild-type human apoAV, two human apoAV variants associated with increased TG (S19W, G185C) and one variant (Q341H) that is predicted to have altered protein function. Injection of mice with adenovirus encoding wild-type or mutant apoAV resulted in an identical dose-dependent elevation of human apoAV levels in plasma. The increase in apoAV levels resulted in pronounced lowering of plasma TG levels at two viral dosages. Unexpectedly, the TG-lowering efficacy of all three apoAV variants was similar to wild-type apoAV. In addition, no effect on TG-hydrolysis-related plasma parameters (free fatty acids, glycerol and post-heparin lipoprotein lipase activity) was apparent upon expression of all apoAV variants. In conclusion, our data indicate that despite their association with hypertriglyceridemia and/or predicted protein dysfunction, the 19W, 185C and 341H apoAV variants are equally effective in reducing plasma TG levels in mice.

Apolipoprotein E2 (apoE2)-associated hyperlipidemia is characterized by a disturbed clearance of apoE2-enriched VLDL remnants. Because excess apoE2 inhibits LPL-mediated triglyceride (TG) hydrolysis in vitro, we investigated whether direct or indirect stimulation of LPL activity in vivo reduces the apoE2-associated hypertriglyceridemia. Here, we studied the role of LPL and two potent modifiers, the LPL inhibitor apoC-III and the LPL activator apoA-V, in APOE2-knockin (APOE2) mice. Injection of heparin in APOE2 mice reduced plasma TG by 53% and plasma total cholesterol (TC) by 18%. Adenovirus-mediated overexpression of LPL reduced plasma TG by 85% and TC by 40%. Both experiments indicate that the TG in apoE2-enriched particles is a suitable substrate for LPL. Indirect activation of LPL activity via deletion of Apoc3 in APOE2 mice did not affect plasma TG levels, whereas overexpression of Apoa5 in APOE2 mice did reduce plasma TG by 81% and plasma TC by 41%. In conclusion, the hypertriglyceridemia in APOE2 mice can be ameliorated by the direct activation of LPL activity. Indirect activation of LPL via overexpression of apoA-V does, whereas deletion of apoC-III does not, affect the plasma TGs in APOE2 mice. These data indicate that changes in apoA-V levels have a dominant effect over changes in apoC-III levels in the improvement of APOE2-associated hypertriglyceridemia.

Objective: The recently discovered apoAV is hypothesized to affect triglyceride metabolism by stimulating the lipolysis of triglycerides in VLDL and chylomicrons. We set out to determine the association between increased serum TG levels, plasma apoAV levels, and polymorphism of the APOA5 gene, with specific emphasis on the APOA5 S19W variation. This mutation alters the endoplasmic reticulum signal peptide and is hypothesized to impair apoAV secretion into the circulation., Methods and Results: Two haplotype-tagging APOA5 polymorphisms, APOA5 S19W and APOA5 -1131T>C and plasma apoAV levels were determined in a population of patients with severe hypertriglyceridemia (HTG). As compared to a random control population, the allele frequencies of the APOA5 S19W and -1131T>C rare variants were significantly increased in HTG patients. Furthermore, the HTG population exhibited markedly elevated plasma apoAV levels that were positively correlated with serum TG levels. Plasma apoAV levels were positively correlated with occurrence of the APOA5 S19W rare variant., Conclusions: The increased allele frequencies of the APOA5 S19W and -1131T>C rare variants in the HTG population are in agreement with previous reports. Our data show a positive correlation between apoAV and TG levels. Moreover the finding of a positive association between apoAV levels and the APOA5 S19W rare variant is in disagreement with the hypothesis that this variant is poorly secreted.

The relevance of apolipoprotein A-V (apoA-V) for human lipid homeostasis is underscored by genetic association studies and the identification of truncation-causing mutations in the APOA5 gene as a cause of type V hyperlipidemia, compatible with an LPL-activating role of apoA-V. An inverse correlation between plasma apoA-V and triglyceride (TG) levels has been surmised from animal data. Recent studies in human subjects using (semi)quantitative immunoassays, however, do not provide unambiguous support for such a relationship. Here, we used a novel, validated ELISA to measure plasma apoA-V levels in patients (n = 28) with hypertriglyceridemia (HTG; 1.8-78.7 mmol TG/l) and normolipidemic controls (n = 42). Unexpectedly, plasma apoA-V levels were markedly increased in the HTG subjects compared with controls (1,987 vs. 258 ng/ml; P < 0.001). In the HTG group, apoA-V and TG were positively correlated (r = +0.44, P = 0.02). In addition, we noted an increased level of the LPL-inhibitory protein apoC-III in the HTG group (45.8 vs. 10.6 mg/dl in controls; P < 0.001). The correlation between apoA-V and TG levels in the HTG group disappeared (partial r = +0.09, P = 0.65) when controlling for apoC-III levels. In contrast, apoC-III and TG remained positively correlated in this group when controlling for apoA-V (partial r = +0.43, P = 0.025). Our findings suggest that in HTG patients, increased TG levels are accompanied by high plasma levels of apoA-V and apoC-III, apolipoproteins with opposite modes of action. This study provides evidence for a complex interaction between apoA-V and apoC-III in patients with severe HTG.

In mouse models, apolipoprotein A-V (apoA-V) exhibits triglyceride (TG)-lowering effects. We investigated the apoA-V/TG relationship and the association of apoA-V with coronary artery disease (CAD) risk by determining serum apoA-V levels and genotypes in a nested case-control (n = 1,034/2,031) study. Both univariate and multivariate apoA-V levels showed no association with future CAD (P = 0.4 and 0.5, respectively). Unexpectedly, there was a significant positive correlation between serum apoA-V and TG in men and women (r = 0.36 and 0.28, respectively, P < 0.001 each) but a negative correlation between apoA-V and LPL mass (r = -0.14 and -0.12 for men and women respectively, P < 0.001 each). The frequency of the c.56C>G polymorphism did not differ between cases and controls despite significant positive association of c.56G with both apoA-V and TG levels. For -1131T>C, the minor allele was significantly associated with lower apoA-V yet higher TG levels and was overrepresented in cases (P = 0.047). The association of -1131T>C with CAD risk, however, was independent of apoA-V levels and likely acts through linkage disequilibrium with APOC3 variants. The positive correlation of apoA-V levels with TG levels, negative correlation with LPL levels, and lack of association with CAD risk highlight the need for further human studies to clarify the role of apoA-V.

Background/aim: Mutations in genes encoding the ATP-binding cassette (ABC)-transporters ABCG5 and ABCG8 underlie sitosterolemia, which is characterized by elevated plasma levels of phytosterols due to increased intestinal absorption and impaired biliary secretion of sterols. The aim of our study was to correlate the expression levels of Abcg5 and Abcg8 to biliary cholesterol secretion in various (genetically-modified) mouse models., Methods: Bile was collected from genetically-modified mice fed a chow diet, or from mice fed either a chow diet, or chow supplemented with either 1% diosgenin, 0.1% simvastatin, or a synthetic liver X receptor agonist, for determination of biliary lipids. Livers and small intestines were harvested and expression levels of Abcg5, Abcg8 and Abcb4 were determined by real-time polymerase chain reaction., Results: Intestinal expression of Abcg5 and Abcg8 did not show much variation between the various models. In contrast, a linear correlation between hepatic expression levels of Abcg5 and Abcg8 and biliary cholesterol secretion rates was found. This relation was independent of Abcb4-mediated phospholipid secretion. However, in diosgenin-fed mice showing cholesterol hypersecretion, hepatic Abcg5 and Abcg8 expression levels remained unchanged., Conclusions: Our results strongly support a role for Abcg5 and Abcg8 in regulation of biliary cholesterol secretion, but also indicate the existence of a largely independent route of cholesterol secretion.

Mice lacking ApoA-V, a novel HDL-associated apolipoprotein identified by our group and independently by Pennacchio et al. [Science 294 (2001) 169], were recently shown to be hypertriglyceridemic. To study the role of ApoA-V in triglyceride homeostasis, we compared lipid profiles in mice expressing normal and highly elevated levels of ApoA-V. For this purpose, adenoviral vectors expressing sense or antisense ApoA-V cDNA were constructed. Treatment of mice with sense adenoviral constructs resulted in circa 20-fold higher serum ApoA-V levels compared with mice injected with either PBS or antisense adenoviral constructs. ApoA-V overexpressing mice had markedly decreased (-70%) serum triglyceride levels caused primarily by lowered triglyceride content of the VLDL fraction. Furthermore, in these mice cholesterol levels were found to be lowered in all lipoprotein fractions with the largest mass decrease in the HDL fraction. This resulted in a 40% drop of serum cholesterol content. These findings suggest a role of ApoA-V in regulating levels of circulating triglycerides and cholesterol.

Heart-type fatty acid-binding protein (H-FABP) is a member of a family of 14-15 kDa lipid binding proteins which are believed to enhance intracellular transport of lipids by facilitating their cytoplasmic diffusion. To obtain sufficient amounts of protein for in vitro studies, we expressed rat H-FABP in Escherichia coli and compared its biochemical properties with the protein isolated from rat heart. An effective method was developed to purify recombinant rat H-FABP from cell lysates in a single step using anion-exchange chromatography. This method also proved to be applicable for purifying heterologously expressed human H-FABP. Recombinant rat H-FABP, which made up approximately 25% of the soluble proteins in E. coli, was obtained in a yield of 30-40 mg/l culture. Characterization showed that recombinant rat H-FABP was indistinguishable from the protein isolated from rat heart regarding molecular mass and oleic acid binding. Some heterogeneity upon isoelectric focusing was observed, presumably due to differences in N-terminal processing of the proteins. In conclusion, a method is presented for efficient high-yield production of recombinant rat H-FABP.