Intracellular trafficking of the free cholesterol derived from LDL cholesteryl ester is defective in vivo in Niemann-Pick C disease: insights on normal metabolism of HDL and LDL gained from the NP-C mutation.

Niemann-Pick C disease (NP-C) is a rare inborn error of metabolism with hepatic involvement and neurological sequelae that usually manifest in childhood. Although in vitro studies have shown that the lysosomal distribution of LDL-derived cholesterol is defective in cultured cells of NP-C subjects, no unusual characteristics mark the plasma lipoprotein profiles. We set out to determine whether anomalies exist in vivo in the cellular distribution of newly synthesized, HDL-derived or LDL-derived cholesterol under physiologic conditions in NP-C subjects. Three affected and three normal male subjects were administered [14C]mevalonate as a tracer of newly synthesized cholesterol and [3H]cholesteryl linoleate in either HDL or LDL to trace the distribution of lipoprotein-derived free cholesterol. The rate of appearance of free [14C]- and free [3H]cholesterol in the plasma membrane was detected indirectly by monitoring their appearance in plasma and bile. The plasma disappearance of [3H]cholesteryl linoleate was slightly faster in NP-C subjects regardless of its lipoprotein origin. Appearance of free [14C] cholesterol ill the plasma (and in bile) was essentially identical in normal and affected individuals as was the initial appearance of free [3H]cholesterol derived from HDL, observed before extensive exchange occurred of the [3H]cholesteryl linoleate among lipoproteins. In contrast, the rate of appearance of LDL-derived free [3H]cholesterol in the plasma membrane of NP-C subjects, as detected in plasma and bile, was retarded to a similar extent that LDL cholesterol metabolism was defective in cultured fibroblasts of these affected subjects. These findings show that intracellular distribution of both newly synthesized and HDL-derived cholesterol are essentially unperturbed by the NP-C mutation, and therefore occur by lysosomal-independent paths. In contrast, in NP-C there is defective trafficking of LDL-derived cholesterol to the plasma membrane in vivo as well as in vitro. The in vivo assay of intracellular cholesterol distribution developed herein should prove useful to quickly evaluate therapeutic interventions for NP-C.

Disappearance of two major phosphatidylcholines from plasma is predominantly via LCAT and hepatic lipase.

Metabolism of 1-stearoyl-2-arachidonyl-phosphatidyl-choline (SAPC), a major phosphatidylcholine (PC) species in rat plasma, was compared with 1-palmitoyl-2-linoleoyl-PC (PLPC) metabolism. High-density lipoproteins containing SAPC and PLPC tracers labeled in the sn-2 fatty acid with 3H and 14C isotopes, respectively, were administered. The rats were depleted of endogenous bile acids and infused via the ileum with individual bile acids that ranged widely in hydrophobicity. The half-lives for SAPC and PLPC in plasma were 48 and 57 min, respectively. Most of the 3H activity that disappeared from plasma at 1 h was found in the liver in 1-palmitoyl-2-arachidonyl-PC, SAPC, and 1-oleoyl-2-arachidonyl-PC, indicating phospholipase A1 hydrolysis of plasma SAPC forming 2-arachidonyl-lysophosphatidylcholine, which was reacylated in the liver. Plasma PLPC also underwent phospholipase A1 hydrolysis, as reported previously. The fraction of 3H dose that accumulated in plasma cholesteryl arachidonate was two- to threefold higher than the fraction of 14C dose in cholesteryl linoleate. Multicompartmental models for SAPC and PLPC were developed that included lysophosphatidylcholines and cholesteryl esters. Bile acids did not influence plasma PC metabolism. Lecithin-cholesterol acyltransferase and phospholipase A1 (hepatic lipase) hydrolysis accounted for > or = 90% of the SAPC and PLPC that disappeared from plasma; SAPC and PLPC are comparable as substrates for hepatic lipase, but SAPC is preferred by lecithin-cholesterol acyltransferase.

Cholesterol kinetics in subjects with bile fistula. Positive relationship between size of the bile acid precursor pool and bile acid synthetic rate.

Our aim was to identify and quantitate cholesterol pools and transport pathways in blood and liver. By studying bile fistula subjects, using several types of isotopic preparations, simultaneous labeling of separate cholesterol pools and sampling all components of blood and bile at frequent intervals, we developed a comprehensive multicompartmental model for cholesterol within the rapidly miscible pool. Data in six components (bile acids, esterified cholesterol in whole plasma, and free cholesterol in blood cells, bile, alpha lipoproteins, and beta lipoproteins) were modeled simultaneously with the SAAM program. The analysis revealed extensive exchange of free cholesterol between HDL and liver, blood cells, and other tissues. There was net free cholesterol transport from HDL to the liver in most subjects. The major organ that removed esterified cholesterol from blood was the liver. A large portion (4,211 mumol) of total hepatic cholesterol comprised a pool that turned over rapidly (t1/2 of 72 min) by exchanging mainly with plasma HDL and was the major source of bile acids and biliary cholesterol. Only 6% of hepatic newly synthesized cholesterol was used directly for bile acid synthesis: the analysis showed that 94% of newly synthesized cholesterol was partitioned into the large hepatic pool (putative plasma membrane free cholesterol) which exchanged rapidly with plasma lipoproteins. Bile acid synthetic rate correlated directly with the size of the large hepatic pool. In conclusion, hepatic and blood cholesterol pools and transports have been quantitated. HDL plays a central role in free cholesterol exchange/transport between all tissues and plasma. In humans, the metabolically active pool comprises a large portion of total hepatic cholesterol that, in part, regulates bile acid synthesis.

Plasma 1-palmitoyl-2-linoleoyl phosphatidylcholine. Evidence for extensive phospholipase A1 hydrolysis and hepatic metabolism of the products.

1-Palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) labeled in either the choline, glycerol, palmitate, or linoleate component in reconstituted rat high density lipoprotein (rHDL), was administered by vein to rats with bile fistula and taurocholate infusion. PLPC disappeared from plasma in a monoexponential fashion with a half-life of 50 min. A small fraction, about 14%, of PLPC disappearance was due to removal of linoleate from the sn-2 ester bond to form plasma cholesterol esters, presumably by lecithin-cholesterol acyltransferase. Otherwise, nearly all of the PLPC components that disappeared from blood in 1 h were recovered in the liver. The choline, glycerol, and linoleate components appeared predominantly in hepatic phosphatidylcholine (PC). These three components remained together in the liver with similar fractions of each in individual PC molecular species, most notably 1-stearoyl-2-linoleoyl-PC and dilinoleoyl-PC as well as PLPC. However, the palmitate component was spread among hepatic triglyceride, free fatty acid, other phospholipids, and all palmitate-containing molecular species of PC. Less than 2% of any administered PLPC component appeared in 1-stearoyl-2-arachidonyl-PC, the major species by mass in the liver. The palmitate component from plasma PLPC appeared in biliary PC at a more rapid rate than glycerol and linoleate components; the latter components appeared in bile in identical fashion. The results show that about two-thirds of plasma PLPC disappearance is due to phospholipase A1 hydrolysis, probably hepatic lipase. The putative produce, 2-linoleoyl-lysoPC, is efficiently reacylated with a saturated fatty acid in the liver, conserving PC.