Increased cholesterol efflux from cultured fibroblasts to plasma from hypertriglyceridemic type 2 diabetic patients: roles of pre beta-HDL, phospholipid transfer protein and cholesterol esterification.

We tested whether hypertriglyceridemia associated with type 2 diabetes mellitus is accompanied by alterations in pre beta-HDL, which are considered to be initial acceptors of cell-derived cholesterol, and by changes in the ability of plasma to promote cellular cholesterol efflux. In 28 hypertriglyceridemic and 56 normotriglyceridemic type 2 diabetic patients, and in 56 control subjects, we determined plasma lipids, HDL cholesterol and phospholipids, plasma pre beta-HDL and pre beta-HDL formation, phospholipid transfer protein (PLTP) activity, plasma cholesterol esterification (EST) and cholesteryl ester transfer (CET) and the ability of plasma to stimulate cholesterol efflux out of cultured human fibroblasts. HDL cholesterol and HDL phospholipids were lower, whereas plasma PLTP activity, EST and CET were higher in hypertriglyceridemic diabetic patients than in the other groups. Pre beta-HDL levels and pre beta-HDL formation were unaltered, although the relative amount of pre beta-HDL (expressed as % of total plasma apo A-I) was increased in hypertriglyeridemic diabetic patients. Cellular cholesterol efflux to plasma from hypertriglyceridemic diabetic patients was increased compared to efflux to normotriglyceridemic diabetic and control plasma, but efflux to normotriglyceridemic diabetic and control plasma did not differ. Multiple linear regression analysis demonstrated that cellular cholesterol efflux to plasma was positively and independently related to pre beta-HDL formation, PLTP activity and EST (multiple r=0.48), but not to the diabetic state. In conclusion, cholesterol efflux from fibroblasts to normotriglyceridemic diabetic plasma is unchanged. Efflux to hypertriglyceridemic diabetic plasma is enhanced, in association with increased plasma PLTP activity and cholesterol esterification. Unaltered pre beta-HDL formation in diabetic hypertriglyceridemia, despite low apo A-I, could contribute to maintenance of cholesterol efflux.

Biliary anionic peptide fraction/calcium binding protein inhibits apolipoprotein A-I-mediated cholesterol efflux from cultured cells.

The ABC transporter ABCA1 has been implicated to control cholesterol efflux in a variety of cell types including macrophages, fibroblasts, and intestinal epithelial cells. In this study we have investigated whether the 6-kD protein anionic peptide fraction/calcium binding protein (APF/CBP) which has homology to apolipoprotein AI may regulate efflux mediated by lipoproteins. APF/CBP was purified from T-tube bile by ultracentrifugation and preparative reversed phase HPLC. Cholesterol efflux to a variety of acceptors was determined using cultured fibroblasts from controls and patients with Tangiers disease. APF/CBP (0.1 to 2.4 microg/ml) inhibited ApoA-1 (2 microg/ml) mediated cholesterol efflux from normal fibroblasts in a dose dependent manner but had no effect on aspecific efflux to methyl-beta-cyclodextrin or phosphatidylcholine liposomes. In ABCA1 deficient fibroblasts no effect of APF/CBP on efflux was seen. We conclude that APF/CBP specifically interferes with ApoA-I mediated cholesterol trafficking. We hypothesize that competitive binding to ABCA1 may explain the decreased ApoA-I mediated efflux from fibroblasts.

Common genetic variation in ABCA1 is associated with altered lipoprotein levels and a modified risk for coronary artery disease.

BACKGROUND: Low plasma HDL cholesterol (HDL-C) is associated with an increased risk of coronary artery disease (CAD). We recently identified the ATP-binding cassette transporter 1 (ABCA1) as the major gene underlying the HDL deficiency associated with reduced cholesterol efflux. Mutations within the ABCA1 gene are associated with decreased HDL-C, increased triglycerides, and an increased risk of CAD. However, the extent to which common variation within this gene influences plasma lipid levels and CAD in the general population is unknown. METHODS AND RESULTS: We examined the phenotypic effects of single nucleotide polymorphisms in the coding region of ABCA1. The R219K variant has a carrier frequency of 46% in Europeans. Carriers have a reduced severity of CAD, decreased focal (minimum obstruction diameter 1.81+/-0.35 versus 1.73+/-0.35 mm in noncarriers, P:=0.001) and diffuse atherosclerosis (mean segment diameter 2.77+/-0.37 versus 2.70+/-0.37 mm, P:=0.005), and fewer coronary events (50% versus 59%, P:=0.02). Atherosclerosis progresses more slowly in carriers of R219K than in noncarriers. Carriers have decreased triglyceride levels (1.42+/-0.49 versus 1.84+/-0.77 mmol/L, P:=0.001) and a trend toward increased HDL-C (0.91+/-0.22 versus 0.88+/-0.20 mmol/L, P:=0.12). Other single nucleotide polymorphisms in the coding region had milder effects on plasma lipids and atherosclerosis. CONCLUSIONS: These data suggest that common variation in ABCA1 significantly influences plasma lipid levels and the severity of CAD.

Midkine and cyclooxygenase-2 promoters are promising for adenoviral vector gene delivery of pancreatic carcinoma.

Midkine (MK), a heparin binding growth factor, and cyclooxygenase-2 (COX-2), a key enzyme in the conversion of arachidonic acid to prostaglandin, are both up-regulated at the mRNA or protein level in many human malignant tumors. Here, we investigated the tumor specificity of both MK and COX-2 promoters in human pancreatic cancer, with the aim to improve the selectivity of therapeutic gene expression. We constructed recombinant adenoviral (Ad) vectors containing either the luciferase (Luc) reporter gene under the control of the COX-2 or MK promoter or the herpes simplex virus thymidine kinase (HSV Tk) gene under the control of the COX-2 promoter and compared the expression with the cytomegalovirus (CMV) promoter. AdMKLuc achieved moderate to relatively high activity upon infection to both primary and established pancreatic carcinoma cells. Of the two COX-2 promoter regions (COX-2M and COX-2L), both revealed a high activity in primary pancreatic carcinoma cells, whereas in the established pancreatic carcinoma cell lines, COX-2L has an approximately equal high activity compared to CMV. In addition, both AdCOX-2M Tk and AdCOX-2L Tk induced marked cell death in response to ganciclovir (GCV) in three of four established pancreatic carcinoma cell lines. From these results, and because it has been reported that AdMKTk and AdCOX-2L Tk in combination with GCV did not reveal significant liver toxicity, we conclude that the MK as well as the COX-2 promoters are promising tumor-specific promoters for Ad vector-based gene therapy of pancreatic cancer.

Regulation of mdr2 P-glycoprotein expression by bile salts.

The phosphatidyl translocating activity of the mdr2 P-glycoprotein (Pgp) in the canalicular membrane of the mouse hepatocyte is a rate-controlling step in the biliary secretion of phospholipid. Since bile salts also regulate the secretion of biliary lipids, we investigated the influence of the type of bile salt in the circulation on mdr2 Pgp expression and activity. Male mice were led a purified diet to which either 0.1% (w/w) cholate or 0.5% (w/w) ursodeoxycholate was added. This led to a near-complete replacement of the endogenous bile salt pool (mainly tauromuricholate) by taurocholate or tauroursodeoxycholate respectively. The phospholipid secretion capacity was then determined by infusion of increasing amounts of tauroursodeoxycholate. Cholate feeding resulted in a 55% increase in maximal phospholipid secretion compared with that in mice on the control diet. Northern blotting revealed that cholate feeding increased mdr2 Pgp mRNA levels by 42%. Feeding with ursodeoxycholate did not influence the maximum rate of phospholipid output or the mdr2 mRNA content. Female mice had a higher basal mdr2 Pgp mRNA level than male mice, and this was also correlated with a higher phospholipid secretion capacity. This could be explained by the 4-fold higher basal cholate content in the bile of female compared with male mice. Our results suggest that the type of bile salts in the circulation influences the expression of the mdr2 gene.

The role of bile salt composition in liver pathology of mdr2 (-/-) mice: differences between males and females.

BACKGROUND/AIMS: The mouse mdr2 gene encodes a P-glycoprotein expressed in the canalicular membrane of the hepatocyte. Mice in which this gene has been inactivated (mdr2 -/-) show a defect in biliary phospholipid and cholesterol secretion and develop non-suppurative cholangitis. We hypothesized that secretion of bile salts without lipids initiates this liver disease. METHODS: To delineate the pathologic process, mdr2 (-/-) mice were fed different bile salt-supplemented diets for 22 weeks after weaning. Aspects of liver pathology including eosinophilic bodies, portal inflammation, ductular proliferation, mitotic activity and fibrosis were semi-quantitatively scored. RESULTS: It was observed that liver pathology was more severe in female than in male mice when fed a purified control diet. This correlated with a more hydrophobic bile salt composition of female vs. male bile. When increasing amounts of cholate were added to the diet (0.01% and 0.1%), the secretion of taurocholate increased and this was accompanied by a more severe liver pathology. At the high dose of cholate (0.1%), the bile salt compositions of male and female mice became similar, as did the severity of the histological score. Addition of cholate to the diet did not induce liver pathology in (+/+) mice. Addition of ursodeoxycholate to the diet (0.5%) led to a near complete replacement of biliary bile salts by tauroursodeoxycholate and this reduced pathology and dissipated the difference between males and females. CONCLUSIONS: These observations support our hypothesis that liver pathology in the mdr2 (-/-) mouse is caused by bile salts and depends on the hydrophobicity c.q. cytotoxicity of biliary bile salts.

Distinct immuno-localization of mucin and other biliary proteins in human cholesterol gallstones.

BACKGROUND/AIMS: Cholesterol gallstones consist of cholesterol crystals and smaller amounts of pigments and calcium salts, arrayed on a mucin plus protein matrix. The localization of the various biliary proteins in the stones has not been characterized. We aimed to localize several biliary proteins in gallstones in order to determine their possible role in stone formation and growth. METHODS: The distribution of several matrix proteins and their relationships to the minerals were determined using immunostaining and EDAX microanalysis on hemisected cholesterol gallstones. RESULTS: Pigment areas were rich in calcium and contained Cu, P and S. These elements were absent in cholesterol regions. Mucin was identified in a three-dimensional network intercalated between cholesterol crystals and as septa between deposits of pigments and cholesterol; APF/CBP and ApN coated only the pigment deposits. No specific topographical localization was found for albumin or IgA. CONCLUSIONS: This suggests a role for mucin, APF/ CBP and ApN in the formation of cholesterol gallstones. We propose that cholesterol crystals bind directly to mucin, whereas calcium salts and pigments deposit on APF/CBP and ApN bind to the mucin.

Uncoupling of biliary phospholipid and cholesterol secretion in mice with reduced expression of mdr2 P-glycoprotein.

Mice in which the gene for mdr2 P-glycoprotein has been disrupted have a severe deficiency in biliary phospholipid and cholesterol secretion. We studied the relation between mdr2 gene expression and biliary lipid secretion with emphasis on the role of bile salt hydrophobicity. Control mice (+/+), and mice with a homozygous (-/-) or heterozygous (+/-) disruption of the mdr2 gene, were infused with taurodeoxycholate (TDC) or tauroursodeoxycholate (TUDC). In mdr2 (-/-) mice, virtually no phospholipids were secreted into bile, irrespective of the type of bile salt infused. In contrast, cholesterol secretion in (-/-) mice increased upon TDC infusion from less than 0.1 to more than 2 nmol/min . 100 g, which was similar to controls under the same conditions. After infusion of TUDC in (-/-) mice. cholesterol secretion also rose (to 1.8 nmol/min . 100 g) but remained much lower than in controls (8 nmol/min x 100 g). In (+/-) mice, cholesterol secretion was equal to (+/+) mice during secretion of endogenous bile salts and during TDC infusion, but was 50% of control levels during maximal TUDC infusion. We conclude that biliary phospholipid secretion completely depends on mdr2 gene expression but cholesterol can, at least partially, be secreted in an mdr2 Pgp-independent mechanism. The extent to which cholesterol is secreted via this mechanism may depend on the hydrophobicity (i.e., cholesterol-solubilizing capacity) of the secreted bile salt.

Influence of bile salts on hepatic mdr2 P-glycoprotein expression.

Mdr2 P-glycoprotein is expressed in the canalicular membrane of the mouse hepatocyte and is responsible for phospholipid secretion into bile. It is our hypothesis that it functions as a flippase in the translocation of phosphatidylcholine from the inner leaflet to the outer leaflet of the canalicular membrane. We have investigated the influence of different types of bile salts on the expression levels of mdr2 Pgp. Feeding mice a cholate-supplemented diet results in an increased mdr2 mRNA level, and this is accompanied by an increased biliary phospholipid secretion capacity. Cholate is a more hydrophobic bile salt than the main endogenous bile salt, muricholate. The induction of mdr2 gene expression and concomitant increase in phospholipid secretion are in line with the function of biliary phospholipids to inactivate the detergent action of hydrophobic bile salts.

Regulation of protein secretion into bile: studies in mice with a disrupted mdr2 p-glycoprotein gene.

BACKGROUND & AIMS: Protein is secreted into bile via several independent pathways. The aim of this study was to investigate whether these pathways are influenced by secretion of biliary lipid. METHODS: Protein secretion and biliary lipid output were studied in wild-type mice (+/+), heterozygotes (+/-), and homozygotes (-/-) for mdr2 gene disruption. Biliary lipid and protein output were varied by infusion with taurocholate (TC) and tauroursodeoxycholate (TUDC). RESULTS: Exocytosis and transcytosis were unaltered in (-/-) mice. Infusion with TC strongly induced secretion of alkaline phosphatase in (-/-) mice but had little effect in (+/-) and (+/+) mice. Infusion with TUDC had little effect on alkaline phosphatase output. In contrast, both TUDC and TC strongly stimulated secretion of aminopeptidase N and lysosomal enzymes in (+/+) mice but had no effect in (-/-) animals. Aminopeptidase N secretion correlated with phospholipid output, but only at high flux. At low flux, aminopeptidase N was secreted independently from both phospholipid and bile salts. CONCLUSIONS: The canalicular membrane enzymes alkaline phosphatase and aminopeptidase N are secreted via separate pathways. Part of alkaline phosphatase output is controlled by bile salt hydrophobicity, whereas at high lipid flux, aminopeptidase N secretion seems to be coupled to phospholipid output. Lysosomal enzymes follow the latter pathway.

Quantification of mucin in human gallbladder bile: a fast, specific, and reproducible method.

It is generally accepted that gallbladder mucin (GBM) plays an important role in the pathogenesis of cholesterol gallstone (ChG) disease. However, it remains unclear whether ChG patients have higher GBM concentrations than controls. Discrepant findings regarding biliary mucin concentrations may be due to methodological problems with the assays commonly used. The methods currently used to quantitate mucin in bile have not been systematically evaluated. To establish a reliable method for mucin quantification in bile, we evaluated three mucin assays: the classic Pearson-PAS (periodic acid Schiff) assay, a direct fluorometric assay, and a new PAS or fluorometric assay with the following modifications of the Pearson assay: preincubation of bile samples with TBS containing KSCN and sodium taurocholate and micellation of biliary lipids during gel chromatographic fractionation using 25 mM sodium taurocholate in the elution buffer. SDS-PAGE and monoclonal anti-human-GBM (GBM59) were used to identify mucin. Highly specific and reproducible mucin isolation was achieved with the modified method. We found considerable loss of mucin during the different purification steps in the Pearson method. The direct fluorometric assay showed unspecific fluorometric signal with low molecular constituents of bile. Our experiments showed that human-GBM can be accurately measured after a simple modified chromatographic fractionation followed by a PAS or fluorometric assay.

Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse.

Disruption of the mdr2 gene in mice leads to a complete absence of phospholipid from bile (Smit, J. J. M., et al. 1993. Cell. 75:451-462). We have investigated the control of both mdr2 P-glycoprotein (Pgp) expression and bile salt secretion on biliary lipid secretion in the mouse. Lipid secretion was monitored at various bile salt output rates in wild-type mice (+/+), heterozygotes (+/-), and homozygotes (-/-) for mdr2 gene disruption. In (-/-) mice, phospholipid secretion was negligible at all bile salt output rates. In (+/-) mice, a curvilinear relation between bile salt and phospholipid secretion was observed similar to that in (+/+) mice; however, at all bile salt secretion rates phospholipid secretion was reduced compared to (+/+) mice, indicating that mdr2 Pgp exerts a strong control over secretion. Infusion of increasing amounts of taurocholate up to maximal secretory rate led to a decline in the phospholipid and cholesterol secretion in both (+/+) and (+/-) mice in accordance to what has been observed in other species. In contrast, in (-/-) mice cholesterol secretion increased under these conditions while phospholipid output remained extremely low. The increased cholesterol secretion may represent extraction of cholesterol from the canalicular plasma membrane by taurocholate micelles as opposed to the concomitant secretion of both phospholipid and cholesterol in the presence of a functional mdr2 Pgp. Increased bile flow in (-/-) mice could be attributed completely to an increase in the bile salt-independent fraction and may therefore be caused by the bile duct proliferation in these mice.

Heterogeneity of human gallbladder mucin in bile.

1. Human gallbladder mucin has been implicated to play a role in gallstone disease. In spite of this fact relatively little is known about the structure of human gallbladder mucin. In this study we have investigated the possible heterogeneity of mucin. For this purpose polyclonal and monoclonal antibodies against gallbladder mucin were raised. All antibodies reacted primarily with carbohydrate antigenic determinants. With these antibodies the immunoreactivity of gallbladder mucin from 60 patients with cholesterol gallstones and 20 subjects without stones was screened. In addition, reactivity with several lectins was studied. 2. Considerable heterogeneity was found with both antibody and lectin typing, but there was no significant difference in heterogeneity between mucin from patients with gallstones and control subjects. Immunoblotting revealed that there was similarity between the reaction of the polyclonal antibody and the Helix pomatia agglutinin. All mucin preparations reacting with the polyclonal antibody also bound to Helix pomatia agglutinin. Nineteen of the 21 reacting mucins (90%) were from patients with blood group A (18 patients) or AB (one patient) and expression of A antigen could be demonstrated on the mucin of these patients. The resulting two reacting mucins were from patients with type O. However, expression of the blood group antigen could not account for the lack of reactivity of the mucin of other patients. The Helix pomatia agglutinin partially blocked the reactivity of the polyclonal antibody, whereas anti-A antibody did not show inhibition, indicating that more than only blood group A epitopes were recognized by this antibody. 3. We conclude that considerable patient to patient heterogeneity of human gallbladder mucin exists.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of human biliary mucin: functional implications.

1. Human gallbladder mucin has been implicated as playing a role in the pathogenesis of gallstones. In previous studies no differences have been found in the content or composition of mucins derived from control bile or cholesterol gallstone bile. Until now, no differences were also found between these two groups of mucins with regard to their ability to cause cholesterol nucleation. In the accompanying paper we have reported that there is a strong heterogeneity of gallbladder mucins derived from individual patients (M. J. A. van Wijland, J. H. Klinkspoor, L. Th. de Wit, R. P. J. Oude Elferink, G. N. J. Tytgat and A. K. Groen, Clin Sci 1994; 86: 67-74). In the present study we further investigated a possible patient to patient heterogeneity of mucin by means of immunological and functional characterization of mucins isolated from hepatic bile of six different patients with gallstones. 2. Considerable heterogeneity was found. Two of the mucins barely reacted with a polyclonal anti-mucin antibody, whereas the other four mucins reacted very strongly. Lectin-binding studies indicated that the glycans of these two mucins expressed less D-N-acetylgalactosamine residues than the other four mucins. This was confirmed by analyses of the glycan compositions. These studies furthermore indicated that the glycans were of the O-linked type, contained alpha-D-N-acetylglucosamine and were fucosylated, sialylated and sulphated to different extents. Except for a strong heterogeneity in the sugar composition of the mucins, heterogeneity was also found in the biological activity of the mucins.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct demonstration that the deficient oxidation of very long chain fatty acids in X-linked adrenoleukodystrophy is due to an impaired ability of peroxisomes to activate very long chain fatty acids.

A method was developed to prepare peroxisome-enriched fractions depleted of microsomes and mitochondria from cultured skin fibroblasts. The method consists of differential centrifugation of a postnuclear supernatant followed by density gradient centrifugation on a discontinuous Metrizamide gradient. The activity of hexacosanoyl-CoA synthetase was subsequently measured in postnuclear supernatants and peroxisome-enriched fractions prepared from cultured skin fibroblasts from control subjects and patients with X-linked adrenoleukodystrophy. Whereas the hexacosanoyl-CoA synthetase activity in postnuclear supernatants of X-linked adrenoleukodystrophy fibroblasts was only slightly decreased (77.8 +/- 4.4% of control (n = 15], enzyme activity was found to be much more markedly reduced in peroxisomal fractions isolated from the mutant fibroblasts (19.6 +/- 6.7% of control (n = 5]. This is a direct demonstration that the defect in X-linked adrenoleukodystrophy is at the level of a deficient ability of peroxisomes to activate very long chain fatty acids, as first suggested by Hashmi et al. [Hashmi, M., Stanley, W. and Singh, I. (1986) FEBS Lett. 86, 247-250].

A new peroxisomal disorder with enlarged peroxisomes and a specific deficiency of acyl-CoA oxidase (pseudo-neonatal adrenoleukodystrophy).

In the present paper two siblings are presented with clinical manifestations very similar to those of patients affected by neonatal adrenoleukodystrophy. In contrast to neonatal adrenoleukodystrophy patients, hepatic peroxisomes in these siblings were enlarged in size and not decreased in number. Accumulation of very-long-chain fatty acids (VLCFA) was associated with an isolated deficiency of the fatty acyl-CoA oxidase, the enzyme that catalyzes the first step of the peroxisomal beta-oxidation. Plasma levels of di- and trihydroxy-coprostanoic acid, phytanic acid, and pipecolic acid were normal; furthermore, acyl-CoA:dihydroxyacetone phosphate acyltransferase activity in cultured fibroblasts was also found to be normal. The clinical, biochemical, and cytochemical features found in these two siblings are compared with those seen in two other disorders characterized by the absence of a decreased number of hepatic peroxisomes and the presence of VLCFA: (1) pseudo-Zellweger syndrome (deficiency of peroxisomal thiolase activity) and (2) X-linked childhood adrenoleukodystrophy (deficiency of activation of lignoceric acid). Review of the different biochemical defects possible in very-long-chain fatty-acid oxidation reveals different clinical pictures of varying severity, depending on the level at which the biochemical defect occurs.

Peroxisomal fatty acid beta-oxidation in relation to the accumulation of very long chain fatty acids in cultured skin fibroblasts from patients with Zellweger syndrome and other peroxisomal disorders.

The peroxisomal oxidation of the long chain fatty acid palmitate (C16:0) and the very long chain fatty acids lignocerate (C24:0) and cerotate (C26:0) was studied in freshly prepared homogenates of cultured skin fibroblasts from control individuals and patients with peroxisomal disorders. The peroxisomal oxidation of the fatty acids is almost completely dependent on the addition of ATP, coenzyme A (CoA), Mg2+ and NAD+. However, the dependency of the oxidation of palmitate on the concentration of the cofactors differs markedly from that of the oxidation of lignocerate and cerotate. The peroxisomal oxidation of all three fatty acid substrates is markedly deficient in fibroblasts from patients with the Zellweger syndrome, the neonatal form of adrenoleukodystrophy and the infantile form of Refsum disease, in accordance with the deficiency of peroxisomes in these patients. In fibroblasts from patients with X-linked adrenoleukodystrophy the peroxisomal oxidation of lignocerate and cerotate is impaired, but not that of palmitate. Competition experiments indicate that in fibroblasts, as in rat liver, distinct enzyme systems are responsible for the oxidation of palmitate on the one hand and lignocerate and cerotate on the other hand. Fractionation studies indicate that in rat liver activation of cerotate and lignocerate to cerotoyl-CoA and lignoceroyl-CoA, respectively, occurs in two subcellular fractions, the endoplasmic reticulum and the peroxisomes but not in the mitochondria. In homogenates of fibroblasts from patients lacking peroxisomes there is a small (25%) but significant deficiency of the ability to activate very long chain fatty acids. This deficient activity of very long chain fatty acyl-CoA synthetase is also observed in fibroblast homogenates from patients with X-linked adrenoleukodystrophy. We conclude that X-linked adrenoleukodystrophy is caused by a deficiency of peroxisomal very long chain fatty acyl-CoA synthetase.

Peroxisomal very long-chain fatty acid beta-oxidation in human skin fibroblasts: activity in Zellweger syndrome and other peroxisomal disorders.

Since very long-chain fatty acids with a chain length of 24 carbons or more are known to accumulate in tissues and body fluids from patients with the cerebrohepato-renal (Zellweger) syndrome, infantile Refsum disease, neonatal adrenoleukodystrophy and X-linked adrenoleukodystrophy, we studied very long-chain fatty acid oxidation in cultured skin fibroblasts from these patients. In this paper, we report that in accordance with earlier results the first step in the beta-oxidation of the very long-chain fatty acid lignoceric acid (C24:0) primarily occurs in peroxisomes in control human skin fibroblasts. Furthermore, it was found that peroxisomal lignoceric acid beta-oxidation was strongly deficient in fibroblasts from patients with Zellweger syndrome, infantile Refsum disease, neonatal and X-linked adrenoleukodystrophy, which explains for the accumulation of very long-chain fatty acids in all four disease entities. In Zellweger syndrome, infantile Refsum disease and neonatal adrenoleukodystrophy the impairment in peroxisomal very long-chain fatty acid beta-oxidation is probably caused by a strong deficiency of all peroxisomal beta-oxidation enzyme proteins due to a deficiency of peroxisomes.