Charles C. Schwartz

An in vivo evaluation in man of the transfer of esterified cholesterol between lipoproteins and into the liver and bile

The metabolism of the esterified cholesterol fractions of HDL and LDL has been studied in vivo in man with regard to their ability to serve as precursors (after intrahepatic hydrolysis) for bile acid synthesis and biliary cholesterol secretion. Information was also obtained on the exchange of cholesterol esters between the lipoprotein classes. Fasting subjects were intravenously administered autologous HDL (or LDL) labeled with esterified [3H]cholesterol and free [3H]- and [14C]cholesterol. Following the administration of the labeled lipoproteins, bile and blood were collected at frequent intervals. In each experiment the observed 3H/14C ratios in bile acids, biliary cholesterol, lipoprotein free cholesterol and red blood cell cholesterol were similar and markedly divergent from the lipoprotein esterified cholesterol 3H/14C ratios. Following the administration of labeled HDL, the 3H/14C ratios observed in the esterified cholesterol fractions of VLDL and LDL closely resembled the ratios in HDL indicating that VLDL and LDL received esterified cholesterol by direct transfer from HDL. Following the administration of labeled LDL, the 3H/14C ratios in HDL esterified cholesterol were midway between the ratio in LDL esterified cholesterol and plasma free cholesterol, indicating that HDL esterified cholesterol is derived from more than one source. These sources could be LDL esterified cholesterol and esters formed de novo from plasma free cholesterol. A precursor-product relationship was found between the specific activities of lipoprotein free cholesterol and the bile steroids. Assuming direct entry of lipoprotein free and esterified cholesterol (after hydrolysis) into the bile acid and biliary cholesterol precursor pools, it was calculated that less than 20% of these biliary steroids could be derived from HDL esterified cholesterol. The results support the view that lipoprotein free cholesterol is the major source of bile acids in man. Also, the results suggest that in vivo esterified cholesterol fractions of VLDL and LDL originate from HDL, that some LDL ester is transferred back to HDL, and that the cholesterol liberated form hydrolyzed esters undergoes recirculation into the free cholesterol pool rather than excretion as biliary cholesterol or bile acids.

Gallbladder mucin, arachidonic acid, and bile lipids in patients who develop gallstones during weight reduction

BACKGROUND Arachidonic acid (AA) and hydrophobic bile salts (BS) stimulate gallbladder mucin (GBM) secretion, which is thought to be an essential step in gallstone pathogenesis. The present study was performed to evaluate the relationship between AA, BS, and GBM in patients who develop gallstones following weight reduction. METHODS Eleven patients who underwent gastric bypass, developed symptomatic gallstones, and then underwent cholecystectomy were evaluated. Gallbladder bile was obtained for analysis during each procedure. Matched patients who did not develop gallstones following gastric bypass served as controls. RESULTS GBM increased in every patient who developed stones (mean increase: 5000%). The largest increase was observed soon after gastric bypass, and this declined curvilinearly with time. Gallbladder bile cholesterol was initially elevated but then rapidly declined before increasing back to pregastric bypass levels after weight loss was complete. No significant changes in phosphatidylcholine molecular species (including AA) or BS composition were observed following weight reduction. Concentrations of cholesterol, phospholipids, and changes in [AA] over time were each a linear function of [BS]. No relationship between GBM and any of these bile constituents was apparent. CONCLUSIONS These observations strongly suggest that increases in GBM, which occur with gallstone formation in humans, are not the result of alterations in biliary AA or BS composition.

Preferential binding of chlordecone to the protein and high density lipoprotein fractions of plasma from humans and other species

The preferential distribution of the relatively nonpolar pesticide chlordecone (CD) to liver rather than to fat tissues in humans suggests that it may be transported in plasma differently from other organochlorine pesticides. The plasma binding of [14C] CD was investigated in vitro in human, rat, and pig plasma and in vivo in rat plasma. Protein and lipoprotein fractions were separated by serial ultracentrifugation. Heparin-manganese precipitation and agarose gel electrophoresis were also carried out to determine whether separation techniques altered CD binding to plasma components. In human plasma, the distribution of [14C] CD among proteins and high density, low density, and very low density lipoproteins (HDL, LDL, and VLDL) was 46, 30, 20, and 6%, respectively. The distribution of cholesterol in the same plasma fractions was 4, 20, 63, and 7%, respectively. In the pig and rat the order of binding was similar to that in humans, with protein greater than or equal to HDL greater than LDL greater than or equal to VLDL. Separation by heparin-Mn precipitation confirmed the results obtained by ultracentrifugation. The distribution of [14C] CD in rat lipoprotein was similar whether the CD was administered in vivo or incubated with plasma in vitro, with approximately 80% bound to HDL, 11% to LDL, and 9% to VLDL in either case. Agarose gel electrophoresis of plasma-bound [14C] CD indicated that albumin was the major component of the protein fraction responsible for CD binding. Preferential binding of CD by albumin and HDL may explain its unusual tissue distribution compared to other organochlorine pesticides such as aldrin and dieldrin, which bind preferentially to VLDL and LDL and distribute preferentially to fat tissues.

α-Tocopherol modulates lipoprotein cytotoxicity in obstructive nephropathy

Abstract Oxidative stress in unilateral ureteral obstruction (UUO) contributes to the development of glomerular and tubulointerstitial lesions. The present study investigated whether oxidized low-density lipoprotein (oLDL) contributes to the pathogenesis of kidney injury in UUO, and whether α-tocopherol modulates such cytotoxicity and promotes repair. Male Sprague-Dawley rats weighing 100–125 g were assigned to three groups of 6 animals each: (1) sham, regular chow; (2) UUO, regular chow; and (3) UUO, α-tocopherol supplementation. We found a significant increase in the level of oxidative stress in the UUO group as measured by malondialdehyde (MDA) content in both plasma and kidneys. The LDL isolated from this group was cytotoxic to rat mesangial cells. The level of oxidation and cytotoxicity was significantly reduced when animals were treated with α-tocopherol. Plasma cholesterol concentration, kidney MDA, and transforming growth factor β1 mRNA expression were all significantly increased in the UUO animals, and partially reduced in α-tocopherol-treated animals. Our data suggest that oxidative modification of LDL is associated with the renal injury in UUO. Taken together, our data support the concept that α-tocopherol can modulate LDL oxidation and its cytotoxic effects on rat mesangial cells in vitro.

A quantitative evaluation of the conversion of 25-hydroxycholesterol to bile acids in man

The present study was directed toward providing additional information in man on the nature of a potential alternative pathway to cholic acid not involving an initial 7 alpha-hydroxylation of cholesterol. Two bile fistula patients and one normal subject each received 25-hydroxy[G-3H]cholesterol; [14C]cholic and [14C]chenodeoxycholic acids were also simultaneously administered to one bile fistula patient and normal subject. The labeled 25-hydroxycholesterol was found to be poorly converted to primary bile acids by all three patients; the range of conversion was 9.7 to 18.9%. Cholic acid was favored over chenodeoxycholic acid by a margin of about 1.4/1. It is concluded that a pathway to primary bile acid via the 25-hydroxylation of cholesterol is of minor importance under conditions of normal or accelerated synthesis in man.

Chlordecone metabolism in the pig.

The biotransformation of chlordecone (CD) to chlordecone alcohol (CDOH) occurs in man and gerbils but not in rats, guinea pigs and hamsters [1, 2]. Because of the species differences in CDOH formation and the need for a suitable animal model, pigs were administered CD by intraperitoneal (i.p.) injection. Plasma, gallbladder bile, hepatic bile, liver and feces were collected and analyzed by gas chromatography for CD metabolites. CDOH was present in bile and feces with up to 85% conjugated in the bile but only 15% was conjugated in the feces. Up to 20% of the CD in plasma and bile and less than 3% in feces was in the conjugated form. Both reduction and conjugation of CD in the pig are similar to those in man.

Isolation of chlordecone binding proteins from pig liver cytosol

Cytosolic proteins may play an important role in the transport of water insoluble substances through the cytosol to various subcellular locations. The binding of chlordecone (CD) to pig liver cytosolic proteins was studied after the simultaneous administration of [14C]CD and [3H]cholesterol via the portal vein. The isolation of chlordecone binding proteins (CDBPs), from liver cytosol consisted of repeated ultracentrifugation, ammonium sulfate fractionation, and chromatography on Bio-Gel A 0.5m, carboxymethyl cellulose (CMC), and Sephadex G-100. Three proteins retained [14C]CD after elution from the CMC column. CDBP I and CDBP II also retained [3H]cholesterol through this stage of purification, while CDBP III did not. The molecular weights, estimated by Sephadex G-100 gel filtration, were 33,500 and 67,000 for CDBP IA and CDBP IB, 49,000 for CDBP II, and 44,000 for CDBP III. Since dissociation of bound cholesterol could not be avoided during the purification procedures, binding of cholesterol to the CDBPs eluted from Sephadex G-100 was investigated. Incubation of CDBPs with [3H]cholesterol resulted in a 2000-fold increase of 3H associated with CDBP II, an 800-fold increase with CDBP IA, a 100-fold increase for CDBP IB, and a 300-fold increase for CDBP III. The isolation characteristics, molecular weights, and cholesterol binding properties of the CDBPs are compared with cytosolic cholesterol binding proteins previously isolated. The high specific activity binding of both CD and cholesterol by CDBP I and CDBP II suggests that CD and cholesterol share a common transport pathway in the liver cytosol.

Evidence for a common hepatic cholesterol precursor site for cholic and chenodeoxycholic acid synthesis in man.

Summary Bile fistula patients were administered [14C]cholesterol and labeled mevalonic acid by constant infusion (for 5 hr) or by intravenous pulse. Bile was collected at frequent time intervals. The specific activities of newly synthesized cholic and chenodeoxycholic acids were virtually identical at all time periods (up to 24 hr). These observations provide evidence for the presence of a common cholesterol substrate pool for primary bile acid synthesis in man. Both cholic and chenodeoxycholic acids appear to be synthesized via the 7α-hydroxy-cholesterol pathway.

High-density lipoproteins decrease the biliary concentration of chlordecone in isolated perfused pig liver

Chlordecone (CD) is an organochlorine pesticide associated with albumin and high-density lipoproteins (HDL) in the plasma. It is found in higher concentrations in the liver than in other tissues and is excreted in the bile. The influence of plasma HDL on the biliary excretion of CD was studied using isolated pig liver perfused with a Krebs-Ringer bicarbonate solution containing albumin, dextrose, and pig red blood cells. Within 5 min after administration into the perfusion medium of [14C]CD bound to albumin or to HDL, only 13% of the [14C]CD dose remained in the perfusate, showing that CD is rapidly taken up by the liver. After 60 min the plasma concentration was constant at 0.008% dose/ml when [14C]CD was administered bound to albumin in the absence of HDL and at 0.004% dose/ml when administered bound to HDL. The mean concentration of CD in the bile was higher when CD was administered bound to albumin in the absence of HDL (0.039% dose/ml) than when it was administered bound to HDL (0.010% dose/ml). The elimination rate constant of CD from the liver into the bile was 0.007/min whether CD was administered bound to albumin or to HDL. The addition of HDL to the perfusion system after the administration of albumin-bound CD resulted in lower biliary CD concentrations. The results suggest that HDL affects the distribution of CD between the perfusate and liver and between liver and bile. In both cases, distribution toward the liver is favored.

Modeling Cholesterol in Humans: Update and Dealing with the Problem of Exchange in Vivo Using the Blood Cell-Lipoprotein Paradigm

The cholesterol molecule, like some other lipids, is very weakly soluble in water. It is present in all cells of humans, mainly in membranes and especially in specialized areas of the plasma membrane such as caveolae and rafts. These areas are rich in sphingomye-lin and carry out important functions like signaling. Cholesterol is also present in plasma and bile, but it is nearly absent from normal urine and spinal fluid. In plasma, cholesterol is localized in lipoprotein particles; in bile, it is in vesicles and micelles. The common denominators at these locations are phosphatidylcholine and sphingomyelin. Cholesterol is very mobile in this phospholipid environment; it flip-flops across the bilayer with a t1/2 of seconds, diffuses laterally with similar speed, and exchanges/transfers between membranes in proximity by aqueous diffusion or with the facilitation of soluble lipid transfer proteins. The reasons why lipoproteins, bile vesicles, and areas of the plasma membrane are cholesterol enriched are active areas of investigation.