Green tea upregulates the low-density lipoprotein receptor through the sterol-regulated element binding Protein in HepG2 liver cells.

Green tea from Camellia sinensis lowers plasma cholesterol in animal models of hypercholesterolemia. The aim of this study was to determine the effects of green tea on the expression of the hepatic low-density lipoprotein (LDL) receptor, a cell surface protein involved in the control of plasma cholesterol. Incubating human HepG2 liver cells in culture with green tea increased both LDL receptor binding activity and protein. An ethyl acetate extract of green tea, containing 70% (w/w) catechins, also increased the LDL receptor binding activity, protein, and mRNA, indicating that (1) the effect was at the level of gene transcription and that (2) the catechins were the active constituents. The mechanism by which green tea up-regulated the LDL receptor was then investigated. Green tea decreased the cell cholesterol concentration (-30%) and increased the conversion of the sterol-regulated element binding protein (SREBP-1) from the inactive precursor form to the active transcription-factor form. Consistent with this, the mRNA of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis, was also increased by green tea. In conclusion, green tea up-regulated the LDL receptor in HepG2 cells. The effect was most likely mediated through SREBP-1 in response to a decrease in the intracellular cholesterol concentration. The LDL receptor may therefore play a role in the hypocholesterolemic effect of green tea in vivo.

Binding and uptake of chylomicron remnants by primary and THP-1 human monocyte-derived macrophages: determination of binding proteins.

The binding and uptake of chylomicron remnants by human macrophages was studied in order to resolve paradoxical observations that have described the putative mechanisms by which postprandial lipoproteins induce foam cell formation. Chylomicron remnants bound to human monocyte-derived macrophages (HMMs) and to the transformed monocytic cell line THP-1 with high affinity (Kd of approx. 5.5 microg of chylomicron remnant protein/ml). Binding was found to be saturable for both cell types, and was strongly inhibited in the presence of unlabelled chylomicron remnants. Ligand blot studies with colloidal-gold-labelled chylomicron remnants identified two cell surface binding sites on both HMMs and THP-1 cells, with molecular masses of approx. 128 kDa and 43 kDa. The high-molecular-mass binding site was found to be the low-density lipoprotein (LDL) receptor, based on the strong inhibition of chylomicron remnant binding in the presence of unlabelled LDL, Fab2 antibody fragments to the LDL receptor or calcium chelators. Competition studies suggested that, in HMMs, the LDL receptor appeared to facilitate approximately half of the total chylomicron remnant uptake. In contrast, the LDL receptor was not significantly involved in macrophage uptake of chylomicron remnants by THP-1 cells. The identity of the 43 kDa binding site is presently unknown, but, importantly, expression was not inhibited as a consequence of sterol loading, which was induced by incubating HMMs and THP-1 cells with 25-hydroxycholesterol. In contrast, the expression of the LDL receptor was substantially attenuated following lipid loading. Collectively, our data suggest that, while the macrophage LDL receptor can bind chylomicron remnants and facilitate uptake in non-lipid-loaded HMMs, other sterol-insensitive sites are responsible for the unabated uptake of chylomicron remnants by macrophages. We propose that the 43 kDa macrophage chylomicron remnant binding protein may be a candidate for the sterol loading of macrophages.

Effects of simvastatin on hepatic cholesterol metabolism, bile lithogenicity and bile acid hydrophobicity in patients with gallstones.

BACKGROUND AND AIMS: There is limited information available on the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on hepatic and biliary cholesterol metabolism in patients with gallstones. The aims of this study were to determine the effect of simvastatin on the regulatory elements of cholesterol metabolism that determine the concentrations of cholesterol in plasma and bile. METHODS: Thirty-one gallstone patients were enrolled in the study; 17 were treated with 20 mg simvastatin daily for 3 weeks prior to cholecystectomy and 14 served as controls. Samples of blood, liver, gall-bladder bile and bile from the common bile duct (CBD) were collected and analysed. RESULTS: The plasma cholesterol (-30%), triacylglycerol (-23%) and low-density lipoprotein (LDL) cholesterol (-42%) concentrations were significantly lowered by simvastatin treatment, as was the plasma lathosterol: cholesterol (-70%), which reflects whole-body cholesterol synthesis. Despite these changes, the hepatic LDL receptor protein and LDL receptor activity in circulating mononuclear cells were similar in both groups. There were no differences in the plasma phytosterol: cholesterol, which reflects the intestinal cholesterol absorption capacity or in the activity of hepatic acyl-coenzyme A: cholesterol acyltransferase. There were however, lower cholesterol concentrations in CBD (-68%) and gall bladder (-41%) bile, and decreased lithogenic (-47%) and bile acid hydrophobicity (-22%) indices of CBD bile in the simvastatin group. CONCLUSIONS: These data indicate that simvastatin reduced plasma and biliary cholesterol levels primarily by reducing cholesterol synthesis. The reduction in CBD bile lithogenicity and bile acid hydrophobicity by simvastatin suggests that this agent may be useful for people who have early stages of cholesterol gallstone development and in whom a choleretic effect is required.

Giardiasis in ringed seals from the western arctic.

Sixteen beluga whales (Delphinapterus leucas) and fifteen ringed seals (Phoca hispida) from the western arctic region of Canada were examined for giardiasis and cryptosporidiosis. Intestinal contents from the rectum and colon were collected from animals slaughtered by Inuit hunters. A fluorescent monoclonal antibody identified Giardia sp. cysts in three of 15 (20%) seals. Thus, ringed seals are implicated as a potential reservoir for this zoonosis in the arctic.

Circadian rhythm in hepatic low-density-lipoprotein (LDL)-receptor expression and plasma LDL levels.

On the basis of studies in vivo and in vitro that involved the use of pharmacological amounts of drugs and hormones or excess cholesterol supplementation, the expression of the low-density lipoprotein (LDL) receptor appears to be tightly coupled to the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity and to extracellular levels of LDL. The present study was undertaken to see how these three entities are regulated under normal physiological conditions over a 24 h period. The results show that, in the rat, hepatic LDL-receptor expression and plasma LDL levels exhibit diurnal periodicity, with a 2-3-fold difference between the peak and trough of each rhythm. Both rhythms showed high inverse correlation (r = -0.86, P < 0.01), plasma LDL levels being lowest at the onset of darkness when LDL-receptor expression was at its peak. The results also showed that the LDL-receptor protein in rat liver has a shorter half-life than that reported for cultured fibroblasts or HepG2 cells. The maximal expression of the LDL receptor occurred several hours before the peak activity of HMG-CoA reductase and appeared not to be influenced by cellular or membrane cholesterol levels during the 24 h cycle. Treatment with dexamethasone increased the LDL-receptor activity significantly at both the lowest and highest points of the rhythm, but the receptor rhythm was still maintained, suggesting that the signal for the circadian variation of the receptor expression is not mediated by adrenal hormones.

The effects of hypercholesterolaemia, simvastatin and dietary fat on the low density lipoprotein receptor of unstimulated mononuclear cells.

The in vivo expression and regulation of the LDL receptor of circulating mononuclear cells was studied using a sensitive spectrophotometric assay with low density lipoproteins conjugated to colloidal gold (LDL-gold). The high plasma cholesterol of familial hypercholesterolemic subjects was shown to be related to a low in vivo LDL receptor activity; cells from a homozygote had virtually no activity and those from 24 heterozygotes expressed 45% of the activity of cells from 35 normals. The average receptor activity of cells from 18 polygenic hypercholesterolemic (PH) subjects was not significantly different from normal but a low expression may have been a factor in six of these subjects. Simvastatin increased the LDL receptor activity of cells from the PH subjects by 70% while lowering their plasma cholesterol by 26%, but reducing the fat intake from 38% to 20% of energy and cholesterol from 239 to 96 mg/day had no effect on the receptor despite a 10% reduction in plasma cholesterol. Upregulation of the LDL receptor may therefore have been involved in the lowering of plasma cholesterol by simvastatin but not by the reduction in dietary fat and cholesterol.

The low-density lipoprotein receptor and cholesterol synthesis are affected differently by dietary cholesterol in the rat.

In the hamster and the rabbit, the low-density lipoprotein (LDL) receptor and cholesterol synthesis are coordinately downregulated by dietary cholesterol. In the rat, cholesterol synthesis is downregulated but LDL kinetic studies suggest that the LDL receptor is not. The aim of this study was to determine the effect of dietary cholesterol on the expression of the hepatic LDL receptor in the rat. Young (2 months) hooded and albino Wistar rats and older (9 months) Sprague-Dawley rats were used because of their reported different propensities to develop hypercholesterolaemia when fed cholesterol. Hepatic LDL receptor activity was measured using a dot blot assay with LDL-gold and LDL receptor mass was measured using an electroblot assay with a polyclonal antibody. Dietary cholesterol had no effect on the plasma cholesterol concentration in both strains of young Wistar rats but increased it in the older Sprague-Dawley rats. Cholesterol synthesis as measured with 3H2O or as indicated by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity or the ratio of plasma lathosterol to cholesterol was effectively downregulated by dietary cholesterol (1% w/w) in all three strains. In contrast, dietary cholesterol increased both hepatic LDL receptor activity and mass in the young Wistar rats and had no effect on either receptor activity or mass in the older Sprague-Dawley rats. Increases in receptor activity occurred despite increases in hepatic cholesterol especially when cholic acid was added to the cholesterol diet. The effect was systemic because CL 277082, an inhibitor of intestinal cholesterol absorption, prevented the increase in LDL receptor activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordinate changes in the low density lipoprotein receptor activity of liver and mononuclear cells in the rabbit.

In the rabbit, dietary cholesterol downregulates the hepatic LDL receptor and concomitant treatment with 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors partly restores its expression. The aim of this study was to determine whether the LDL receptor activity of circulating mononuclear cells would reflect the changes seen in liver. New Zealand White rabbits were fed for 3 weeks either a normal diet or diets containing 0.25% (w/w) cholesterol, 0.25% cholesterol plus 22 mg/kg per day pravastatin or 0.25% cholesterol plus 6 mg/kg per day simvastatin. Dietary cholesterol increased plasma cholesterol 8.9-fold, liver membrane cholesterol 1.8-fold and bile cholesterol saturation 2.3-fold, and decreased the LDL receptor activities of liver and mononuclear cells by 69% and 58%, respectively. In the cholesterol-fed rabbit, pravastatin decreased plasma cholesterol by 55%, liver membrane cholesterol by 29% and bile cholesterol saturation by 23%, and increased liver and mononuclear cell LDL receptor activities by 120% and 77%, respectively. Similarly, simvastatin decreased plasma cholesterol by 74%, liver membrane cholesterol by 24% and bile cholesterol saturation by 38%, and increased liver and mononuclear cell LDL receptor activities by 80% and 62%, respectively. Liver and mononuclear cell LDL receptor activities were directly correlated (r = 0.73, P < 0.005) and both activities were inversely correlated with plasma cholesterol concentration in a log-linear fashion (r = -0.70, P < 0.005 and r = -0.69, P < 0.01, respectively). The LDL receptor activity of mononuclear cells therefore reflected the hepatic LDL receptor activity in these rabbits.

Waterborne Giardia cysts and Cryptosporidium oocysts in the Yukon, Canada.

Several outbreaks of waterborne giardiasis have occurred in southern Canada, but nothing has been reported from the Canadian North. The objective of this study was to collect information relevant to waterborne giardiasis and cryptosporidiosis in the Yukon including epidemiological data and analyses of water, sewage, and animal fecal samples. Remote, pristine water samples were found to be contaminated with Giardia cysts (7 of 22 or 32%) but not with Cryptosporidium oocysts. Giardia cysts were found in 21% (13 of 61) of animal scats, but no Cryptosporidium oocysts were observed (small sample size). Whitehorse's drinking water was episodically contaminated with Giardia cysts (7 of 42 or 17%) and Cryptosporidium oocysts (2 of 42 or 5%). Neither were found in Dawson City's water supply. The only water treatment in the Yukon is chlorination, but contact times and free chlorine residuals are often too low to provide adequate protection by disinfection. Raw sewage samples from the five largest population centers in the Yukon contained 26 to 3,022 Giardia cysts and 0 to 74 Cryptosporidium oocysts per liter. Treated sewage from Whitehorse contained fewer Giardia cysts but more Cryptosporidium oocysts on average. Both were detected in Lake Laberge, downstream of Whitehorse, which has a history of fecal coliform contamination. Daily monitoring of raw sewage from the suburbs of Whitehorse showed a summertime peak of Giardia cysts and occasional Cryptosporidium oocysts after springtime contamination of drinking water. Despite this evidence, epidemiological data for the Yukon showed an endemic infection rate of only 0.1% for giardiasis (cryptosporidiosis is not notifiable).(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the low-density-lipoprotein-receptor-related protein and its relevance to chylomicron-remnant metabolism.

Hepatic levels of the low-density-lipoprotein (LDL)-receptor-related protein (LRP) and the LDL receptor were measured in rats subjected to treatments known to affect the expression of the LDL receptor. Propylthiouracil decreased both hepatic LRP and LDL receptor expression by 30-40%. Thyroxine treatment increased LDL receptor levels by 3-fold without altering LRP levels. In contrast, 17 alpha-ethinyloestradiol decreased LRP by 50%, whereas the LDL receptor was increased 5-fold. Plasma chylomicrons and their remnants were decreased to insignificant levels with this treatment. In rats fed with cholesterol there was a significant increase in these particles in plasma (1.21 +/- 0.4 versus 5.71 +/- 0.4 mg/dl), whereas the expression of LRP was unaltered. In Watanabe heritable hyperlipidaemic and cholesterol-fed rabbits, in which the LDL receptor expression is absent or decreased, the expression of LRP was not significantly different from that in normal rabbits. These results suggest that the expression of hepatic LRP can be modulated by changes in the hormonal status of the rat and that this modulation is not tightly co-ordinated with that of the LDL receptor. Moreover, LRP does not appear to have a significant role in chylomicron-remnant clearance, whereas the LDL receptor is actively involved in this process.

Fish oil and oat bran in combination effectively lower plasma cholesterol in the rat.

Male rats were fed a semi-purified diet containing oat bran or wheat bran with or without a marine fish oil to investigate the effects of such combinations on lipid metabolism. Oat bran alone and wheat bran plus fish oil gave lower plasma cholesterol concentrations than wheat bran alone while oat bran plus fish oil gave the lowest. Oat bran increased plasma triacylglycerols compared with wheat bran but oat bran plus fish oil gave concentrations similar to those seen with wheat bran plus fish oil. Oat bran gave higher hepatic cholesterol synthesis rates and a higher activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase compared to wheat bran. The addition of fish oil to either bran diet decreased cholesterol synthesis but HMG CoA reductase activity was not reduced. Oat bran increased hepatic acyl coenzyme A:cholesterol acyl transferase (ACAT) activity and increased the ratio of esterified to unesterified cholesterol in hepatic microsomal membranes compared with wheat bran. Fish oil decreased hepatic LDL receptor activity and increased HDL binding activity when added to the wheat bran diet but these effects were not seen with oat bran. Oat bran also had no effect on hepatic lipoprotein receptor activity compared with wheat bran. These results show that fish oil and oat bran have complementary cholesterol lowering effects in the rat.

The disappearance rate of human versus rat intermediate density lipoproteins from rat liver perfusion.

The aim of this work was to compare the disappearance rate of human and rat intermediate density lipoproteins (IDL) using the rat liver perfusion system. Human and rat IDL were produced in vitro by incubating human or rat very low density lipoproteins (VLDL) with either rat post-heparin plasma (method I) or a resolubilized isopropanol precipitate of rat post-heparin plasma (method II). With both methods, the degree of triacylglycerol lipolysis was approximately 55%. The different preparations of IDL were labelled with 125I and added to perfusates of rat livers. The disappearance rates of 125I-labelled IDL were monitored by measuring the radioactivity associated with apolipoprotein (apo) B in the perfusate during a 15-min period. Both human and rat IDL prepared with method I had an increased apoE to apoC ratio as compared with their native counterparts. Furthermore, human IDL had a significantly higher apoE to apoC ratio than rat IDL. However, when IDL were produced in the absence of exchangeable apolipoproteins (method II), no change in the apoE to apoC ratios was observed for the transformation of VLDL to IDL and the ratios were similar for human and rat IDL. Despite these differences, human IDL were always removed at a lower rate than rat IDL. The only striking difference between the two types of IDL made by method II was that the apoB100 to apoB48 ratio was considerably higher in human than in rat IDL. These results suggest that the apoB100 to apoB48 ratio is likely to be responsible for the observed differences in liver uptake between rat and human IDL.

Retroendocytosis of high density lipoproteins by the human hepatoma cell line, HepG2.

When human HepG2 hepatoma cells were pulsed with 125I-labeled high density lipoproteins (HDL) and chased in fresh medium, up to 65% of the radioactivity released was precipitable with trichloroacetic acid. Cell-internalized 125I-HDL contributed to the release of acid-precipitable material; when cells were treated with trypsin before the chase to remove 125I-HDL bound to the outer cell membrane, 50% of the released material was still acid-precipitable. Characterization of the radioactive material resecreted by trypsinized cells revealed the presence of particles that were similar in size and density to mature HDL and contained intact apolipoproteins (apo) A-I and A-II. The release of internalized label occurred at 37 degrees C but not at 4 degrees C. Monensin, which inhibits endosomal recycling of receptors, decreased the binding of 125I-HDL to cells by 75%, inhibited the release of internalized radioactivity as acid-precipitable material by 80%, and increased the release of acid-soluble material by 90%. In contrast, the lysosomal inhibitor chloroquine increased the association of 125I-HDL to cells by 25%, inhibited the release of precipitable material by 10%, and inhibited the release of acid-soluble radioactivity by 80%. Pre-incubation with cholesterol caused a 50% increase in the specific binding, internalization, and resecretion of HDL label. Cholesterol affected the release of acid-precipitable label much more (+90%) than that of acid-soluble material (+20%). Taken together, these findings suggest that HepG2 cells can bind, internalize, and resecrete HDL by a retroendocytotic process. Furthermore, the results with cholesterol and monensin indicate that a regulated, recycling, receptor-like molecule is involved in the binding and intracellular routing of HDL.

On the mechanism by which an ACAT inhibitor (CL 277,082) influences plasma lipoproteins in the rat.

CL 277,082 is an inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT). The effects of this drug on lipoprotein metabolism have been examined in cholesterol-fed rats. An optimal dose of drug incorporated into the diet (0.1% w/w) for 7 days reduced plasma cholesterol by 48% and plasma triglycerides by 72%. The decrease in plasma cholesterol was due to a reduction in triglyceride-rich lipoproteins and in HDL cholesterol. There was a significant 72% reduction in intestinal ACAT activity, accompanied by a 41% reduction in hepatic cholesterol content. There was a smaller 21% reduction in hepatic ACAT activity. Hepatic HMG-CoA reductase activity increased 3-fold. HDL binding activity by liver membranes was not altered significantly. The decrease in plasma cholesterol with this ACAT inhibitor is most likely due to decreased absorption of dietary cholesterol resulting from inhibition of intestinal ACAT.

Modulation of the hypolipidemic effect of fish oils by dietary fiber in rats: studies with rice and wheat bran.

Adult male rats were fed a purified diet containing rice bran or wheat bran with or without a marine fish oil to investigate the possible effects of such dietary combinations on lipid metabolism. Plasma and hepatic triacylglycerols and hepatic lipogenesis were lowered significantly by feeding fish oil with rice bran but not with wheat bran. Plasma cholesterol and hepatic cholesterol synthesis were significantly lower in animals fed fish oil with either bran. Liver microsomal free cholesterol was significantly lower in rats fed rice bran alone than in all other groups. Hepatic low density lipoprotein (LDL) receptor activity was significantly higher in the two groups fed rice bran than in the two groups fed wheat bran. Fish oil significantly decreased hepatic LDL receptor activity and increased hepatic high density lipoprotein (HDL) binding activity with wheat bran but had no significant effects on these parameters when added to the rice bran diet. However, when the data for all groups were pooled, there was a significant negative correlation between hepatic HDL binding activity and LDL receptor activity. Cecal volatile fatty acids were significantly higher in rats fed rice bran, were unaffected by adding fish oil to either bran diet and did not appear to mediate any of the effects of the brans and fish oil on plasma lipids and hepatic lipid metabolism. The combination of rice bran plus fish oil therefore appears to have more beneficial effects on lipid metabolism than wheat bran plus fish oil.

The use of metronidazole, tinidazole and dimetridazole in eliminating trichomonads from laboratory mice.

Metronidazole, tinidazole and dimetridazole were administered in the drinking water for 5 days to mice experimentally infected with Tritrichomonas muris and Tetratrichomonas microta. Mice were successfully infected with T. muris and T. microta recovered from infected gerbils. The trichomonas infection was successfully eliminated in mice given a 1% sucrose solution containing 2.5 mg/ml metronidazole or tinidazole. Mice receiving 1.0 mg/ml metronidazole, 1.0 mg/ml tinidazole and 1.2, 5.0 and 10.0 mg/ml dimetridazole failed to eliminate the trichomonas organism. A reduction in water intake was only noted with mice receiving 10 mg/ml dimetridazole. In mice receiving only 1% sucrose the infection was not eliminated.

Demonstration of a high density lipoprotein (HDL)-binding protein in Hep G2 cells using colloidal gold-HDL conjugates.

Solubilized membrane proteins of Hep G2 cells were electrophoretically separated on polyacrylamide gels and electrotransferred onto nitrocellulose paper. Overlaying the nitrocellulose with human high density lipoproteins conjugated to colloidal gold revealed the presence of a single protein band with an apparent molecular mass of 80 kDa. Binding of the conjugates to this protein was specific for high density lipoproteins in as much as it was effectively displaced by an excess of unlabelled high density lipoproteins but not by a similar excess of unlabelled low density lipoproteins. Binding was not dependent on Ca2+ as 10 mM EDTA had no effect. The binding activity of the solubilized membranes was increased by incubating the cells with non-lipoprotein cholesterol. This was detected on electroblots and quantified with a new dot blot assay using the colloidal gold-high density lipoprotein conjugates.

Detection of the low density lipoprotein (LDL) receptor on nitrocellulose paper with colloidal gold-LDL conjugates.

Gold-low density lipoprotein (LDL) conjugates were used to detect the LDL receptor on nitrocellulose paper. Solubilized rat liver membrane proteins were subjected to electrophoresis and electroblotted onto nitrocellulose paper. The receptor was then detected as a red band (within 10 min) by overlaying with the LDL conjugates. The coloration was prevented by unlabeled LDL, EDTA, and suramin but not by unlabeled HDL3. In the dot blot assay, detection with the colloidal gold-LDL conjugates was as sensitive as both the autoradiographic method with 125I-labeled LDL and the biotinylated LDL method; the estimated limit of detection by scanning densitometry was 1.6 femtomoles of receptor protein. When the coloration obtained with the colloidal gold-LDL conjugates was intensified by photochemical silver staining, down to 200 attomoles of the LDL receptor could be detected. In this assay, the EDTA-sensitive binding of colloidal gold-LDL to solubilized hepatic membrane proteins was 12 times higher for rats treated with 17 alpha-EE than for normal rats. The use of colloidal gold-LDL conjugates is therefore a very easy, safe, inexpensive, fast and sensitive method for the detection of the LDL receptor on nitrocellulose paper. Furthermore, with silver staining and scanning densitometry, the colloidal gold-LDL conjugates could be used in a dot blot assay to quantify tissue and cell LDL receptors down to attomolar levels.

Biotinylation of low density lipoproteins via free amino groups without loss of receptor binding activity.

Low density lipoproteins were biotinylated via free amino groups using carbodiimide-activated biotin or D-biotin-N-hydroxysuccinimide ester. The receptor binding activity of the biotinylated LDL was determined by their ability to displace 125I-labeled LDL from the rat hepatic LDL receptor in the liposome filtration assay. LDL biotinylated with either of the two reagents was able to compete effectively with 125I-labeled LDL providing less than twenty biotin moieties were incorporated per lipoprotein particle. When more than twenty biotins were linked there was a marked loss of activity. The following conditions were adopted as standard for the biotinylation of LDL via free amino groups: 0.3 mumol of D-biotin-N-hydroxysuccinimide ester was incubated with 2 mg of LDL for 1 hr at room temperature. These conditions reproducibly yielded 11.3 +/- 0.6 biotins per LDL particle. With the biotinylated LDL and a performed streptavidin-biotinylated peroxidase complex, the hepatic LDL receptor from rats treated with 17 alpha-ethinyl estradiol was visualized as a single band on electroblots. Finally, the biotinylated LDL was used in an enzyme-linked sorbent assay for the LDL receptor. When solubilized liver membrane proteins from rats treated with 17 alpha-ethinyl estradiol were fixed to the wells with 1.6% paraformaldehyde, a specific binding greater than 0.4 absorbance units was observed which was about ninefold higher than with solubilized proteins from normal rats. We therefore suggest that D-biotin-N-hydroxysuccinimide ester can be used to biotinylate LDL reliably without destroying the lipoprotein's ability to bind specifically to its high affinity receptor.