ApoE and apoC-III-defined HDL subtypes: a descriptive study of their lecithin cholesterol acyl transferase and cholesteryl ester transfer protein content and activity.

BACKGROUND: The functionality of high-density lipoproteins (HDL) is a better cardiovascular risk predictor than HDL concentrations. One of the key elements of HDL functionality is its apolipoprotein composition. Lecithin-cholesterol acyl transferase (LCAT) and cholesterol-ester transfer protein (CETP) are enzymes involved in HDL-mediated reverse cholesterol transport. This study assessed the concentration and activity of LCAT and CETP in HDL subspecies defined by their content of apolipoproteins E (apoE) and C-III (apoC-III) in humans. METHODS: Eighteen adults (ten women and eight men, mean age 55.6, BMI 26.9 Kg/m2, HbA1c 5.4%) were studied. HDL from each participant were isolated and divided into four subspecies containing respectively: No apoE and no apoC-III (E-C-), apoE but not apoC-III (E + C-), apoC-III but no apoE (E-C+) and both apoE and apoC-III (E + C+). The concentration and enzymatic activity of LCAT and CETP were measured within each HDL subspecies using immunoenzymatic and fluorometric methods. Additionally, the size distribution of HDL in each apolipoprotein-defined fraction was determined using non-denaturing electrophoresis and anti-apoA-I western blotting. RESULTS: HDL without apoE or apoC-III was the predominant HDL subtype. The size distribution of HDL was very similar in all the four apolipoprotein-defined subtypes. LCAT was most abundant in E-C- HDL (3.58 mg/mL, 59.6% of plasma LCAT mass), while HDL with apoE or apoC-III had much less LCAT (19.8, 12.2 and 8.37% of plasma LCAT respectively for E + C-, E-C+ and E + C+). LCAT mass was lower in E + C- HDL relative to E-C- HDL, but LCAT activity was similar in both fractions, signaling a greater activity-to-mass ratio associated with the presence of apoE. Both CETP mass and CETP activity showed only slight variations across HDL subspecies. There was an inverse correlation between plasma LCAT activity and concentrations of both E-C+ pre-beta HDL (r = - 0.55, P = 0.017) and E-C- alpha 1 HDL (r = - 0.49, P = 0.041). Conversely, there was a direct correlation between plasma CETP activity and concentrations of E-C+ alpha 1 HDL (r = 0.52, P = 0.025). CONCLUSIONS: The presence of apoE in small HDL is correlated with increased LCAT activity and esterification of plasma cholesterol. These results favor an interpretation that LCAT and apoE interact to enhance anti-atherogenic pathways of HDL.

Time Course of Decrease in Skeletal Muscle Mitochondrial Biogenesis after Discontinuation of High-Fat Diet.

It is known that a high-fat diet induces an increase in mitochondrial biogenesis in skeletal muscle. To examine the time course of decrease in mitochondrial biogenesis in skeletal muscle after discontinuing a high-fat diet feeding, C57BL/6 mice were fed a high-fat diet for 4 wk and then switched to the control diet for another 3 or 7 d. During the high-fat diet withdrawal period, the protein content of the mitochondrial respiratory chain decreased faster than the fatty acid oxidation enzymes. The mitochondrial DNA copy number remained high for at least 1 wk after withdrawing the high-fat diet. These results suggested that after switching to the control diet following a period of high-fat diet, the increased mitochondrial biogenesis levels are maintained for a few days, and the rate of decline is divergent between the different mitochondrial components.

Isoform analysis of LC-MS/MS data from multidimensional fractionation of the serum proteome.

We developed a visualization approach for the identification of protein isoforms, precursor/mature protein combinations, and fragments from LC-MS/MS analysis of multidimensional fractionation of serum and plasma proteins. We also describe a pattern recognition algorithm to automatically detect and flag potentially heterogeneous species of proteins in proteomic experiments that involve extensive fractionation and result in a large number of identified serum or plasma proteins in an experiment. Examples are given of proteins with known isoforms that validate our approach and present a subset of precursor/mature protein pairs that were detected with this approach. Potential applications include identification of differentially expressed isoforms in disease states.

Heterozygosity for ABCA1 gene mutations: effects on enzymes, apolipoproteins and lipoprotein particle size.

A cohort of 13 female and 14 male heterozygotes for ATP binding cassette A1 (ABCA1) gene defects was directly compared with 13 and 14 unaffected female and male family members of almost exact same age. The activities of three proteins that play key roles in HDL metabolism were measured in addition to extensive lipid and (apo) lipoprotein subfraction analysis. Compared to controls, LCAT activity was reduced by 15% in affected subjects (P < 0.001) while PLTP activity was unaffected. Interestingly, CETP activity was elevated by 50% in the heterozygote siblings of one kindred but was unaffected in heterozygotes of the three other families. With respect to lipids, the heterozygotes had normal total cholesterol (TC), and LDL-cholesterol concentrations but presented with a trend towards increased triglyceride levels (13%; P = 0.08). HDL metabolism, by contrast, was severely affected as illustrated by 40% reductions in HDL-cholesterol (P < 0.001) with concomitant reductions in apoAI (25%; P < 0.001) levels and in lipoprotein subfraction LpAI (28%; P < 0.001), LpAI:AII (24%; P=0.014), and LpCIII:nonB (34%; P < 0.001) concentrations. We furthermore observed reduced average HDL particle size (5%; P = 0.004; 16% in female and 3.6% in male) and reduced plasma apoCIII concentration (15%; P = 0.006) while apoAII, apoAIV, apoE and apoB levels were unchanged. In conclusion, heterozygosity for ABCA1 defects was associated with reduced LCAT activity in absence of effects on PLTP activity. Of special interest was our finding that the effects of compromised ABCA1 function on HDL were more pronounced in women than in men.

Analysis of apolipoprotein A-I, lecithin:cholesterol acyltransferase and glucocerebrosidase genes in hypoalphalipoproteinemia.

Hypoalphalipoproteinemia (HALP) is a dyslipidemia characterized by low HDL-cholesterol (HDL-C) levels with important genetic contribution. However, no common genetic mutations have been found to be associated with this disorder. We screened the promoter and coding sequence of apolipoprotein (apo) A-I and lecithin:cholesterol acyltransferase (LCAT) genes and the 5' apo C-III region by SSCP and heteroduplex analysis, and DNA sequencing in 66 unrelated subjects with recurrent low HDL-C levels. We also analyzed the N370S and L444P variants, in the glucocerebrosidase (GBA) gene by restriction fragment analysis. Three mutations in the apo A-I gene (L144R, W108R, g.1833C>T) and 3 mutations in the LCAT gene (S208T, I178T, IVS3-23C>A) were detected, in six heterozygous subjects. In addition, a novel polymorphic site in LCAT gene (g.4886C>T) has been identified. Allelic frequencies of polymorphisms g.(-636)C>A, g.(-625)G>A, g.(-620)T>del, g.(-479C>T and g.(-452)T>C, located upstream of the apo C-III gene, were in normal range, and no other mutation was found in this region. Two HALP subjects were found to carry the N370S mutation at GBA locus. In conclusion, 12% of HALP subjects were found to carry mutations in apo A-I, LCAT, or GBA genes, which could explain this phenotype. Our results confirm the molecular, genetic and phenotypic heterogeneity of HALP.

Impaired serum lipids and lipoproteins in fetal macrosomia related to maternal obesity.

The aim of this work was to determine lipoprotein metabolism alterations in macrosomic newborns and to see whether these lipoprotein abnormalities are parallel or not to those found in their obese or nonobese mothers. Serum lipids, apo A-I, apo B100, lipoproteins (VLDL, LDL, HDL2, and HDL3), and LCAT activity were investigated in obese and nonobese mothers and cord blood of their macrosomic or appropriate-for-gestational-age (AGA) newborns. Serum and VLDL triglyceride concentrations were higher in obese mothers of AGA newborns than in nonobese mothers. Serum triglyceride, VLDL, and apo B100 levels were higher, while serum apo A-I and HDL2 cholesterol concentrations were lower in obese mothers of macrosomic newborns than in the other groups. In their macrosomic newborns, serum lipid, lipoprotein, apo B100, and apo A-I levels were higher as compared with those of other newborns. Macrosomic newborns of nonobese mothers had lipoprotein profiles similar to those in AGA newborns. LCAT activity was similar in both mother groups and in both newborn groups. In conclusion, maternal obesity and fetal macrosomia were associated with lipoprotein abnormalities consistent with high atherogenic risk.

Dietary safflower phospholipid reduces liver lipids in laying hens.

This experiment was conducted to determine the effects of dietary safflower phospholipids (crude safflower phospholipid and purified safflower phospholipid) on performance and lipid metabolism of laying hens. Sixty-week-old Single Comb White Leghorn laying hens were divided into four groups of seven birds each, and were given one of four experimental diets containing 5% beef tallow (served as a control, tallow), a mixture of safflower oil and palm oil (SP-oil), crude safflower phospholipid (Saf-PLcrude), or purified safflower phospholipid (Saf-PL) for 7 wk. Egg production ratio and daily egg mass were significantly higher in hens fed Saf-PLcrude diets than in hens of the other diet groups. There were no significant differences in egg weight among groups. Liver cholesterol and triglyceride contents were significantly decreased in all treated groups as compared with the control. The activity of hepatic 3-hydroxy-3 methylglutaryl coenzyme A reductase was the highest in hens fed the Saf-PLcrude diet. Serum esterified cholesterol concentration was decreased by feeding of SP-oil, Saf-PLcrude, or Saf-PL diets. Serum lecithin-cholesterol acyltransferase activity was highest in hens fed the tallow diet. Excreta neutral steroid excretion was significantly increased in the Saf-PLcrude or Saf-PL diet groups, although acidic steroid excretion was not affected by dietary treatments. Total cholesterol, triglyceride, and phospholipid contents in egg yolks were not different for any dietary treatments. The fatty acid compositions of egg yolks from hens fed Saf-PLcrude diets were not different with those fed the SP-oil diet, although eggs of hens fed the Saf-PL diet showed lower total polyunsaturated fatty acids. These results suggest that dietary safflower phospholipids may be a valuable ingredient to layers for reducing liver triglycerides and serum cholesterol without any adverse effects.

Determinants of plasma HDL-cholesterol in hypertriglyceridemic patients. Role of cholesterol-ester transfer protein and lecithin cholesteryl acyl transferase.

Hypertriglyceridemic patients commonly have low levels of HDL cholesterol. Elevated triglycerides per se may be one cause of low HDL levels, but other factors also may be involved. The current study was designed to define the role of cholesterol-ester transfer protein (CETP) in causation of a low HDL cholesterol in hypertriglyceridemic patients; in addition other factors-lecithin cholesterol acyl transferase (LCAT), hepatic triglyceride lipase (HTGL), and lipoprotein lipase (LPL)-were examined. Plasma activities of CETP and LCAT were measured in 137 male patients with moderate hypertriglyceridemia (plasma triglycerides [TGs] 200 to 500 mg/dL and LDL cholesterol < 160 mg/dL). Results were compared with those from 50 normolipidemic men of similar age and body habitus. In addition, lipase activities in postheparin plasma were measured in 118 of the subjects with hypertriglyceridemia. The activities of CETP and LCAT were 17% (P < .01) and 7% (P < .05), respectively, higher in the hypertriglyceridemic group than in control subjects. By stepwise regression analysis CETP appeared to contribute 15.2% and LCAT 9.8% to variation in HDL-cholesterol levels. Activities of LPL and HTGL together contributed an additional 14.1% to HDL-cholesterol variation. In contrast, levels of plasma TG accounted for only 5.4% of the variation. There were no differences in relative contributions of these parameters in patients with and those without coronary heart disease. This study indicates that several factors contribute to the variation in HDL-cholesterol levels in hypertriglyceridemic patients, and five factors-CETP, LCAT, HTGL, LPL, and triglyceride levels-account for almost half of this variation.

Characterization and potential uses of rabbit polyclonal antibodies against human plasma lecithin-cholesterol acyltransferase

Familial and secondary deficiency of plasma lecithin-cholesterol acyltransferase (LCAT) produce circulating lipoprotein particles with gross structural and compositional abnormalities; these have adverse effects on a variety of cellular functions. Factors affecting hepatic synthesis and secretion of this plasma enzyme are largely unknown but, potentially, some of them can be investigated with monospecific antibodies. In the present study, enzymically active LCAT was purified 40,000-fold from human plasma and then used to raise polyclonal antibodies in New Zealand White rabbits. Addition of this antiserum (1 mul) to human plasma (25 mul) completely inhibited LCAT activity, although it was less effective against plasma from other species. The antibodies appeared to be monospecific to plasma LCAT. They gave a single precipitin arc by crossed immunoelectrophoresis, while immunodiffusion established that there was no cross-reactivity with several apolipoproteins or with serum albumin. Moreover, the antiserum was successfully used to detect LCAT in normal human plasma by Laurell rocket immunoelectrophoresis. By contrast, Western blotting of plasma proteins using whole LCAT antiserum was largely unsuccessful because of high background staining, although this could be substantially reduced by use of an IgG fraction. However, the whole antiserum readily immunoprecipitated LCAT secreted into the culture medium of HepG2 cells, a human hepatoblastoma cell line, pre-labelled with [35S]methionine, the [(35)S]-labelled LCAT appearing as a narrow 65-kDa protein band by electrophoresis and fluorography. We conclude that polyclonal antibodies may be an important tool to investigate the characteristics and underlying mechanisms of secondary LCAT deficiencies, including those associated with hepatic cirrhosis and schistosomiasis.

Apolipoprotein AIV of human interstitial fluid is associated with apolipoprotein AI-containing but not with AII-containing particles.

Apolipoproteins and lipoprotein particles from human interstitial fluid and plasma were analyzed. The interstitial fluid was enriched in apolipoproteins AI, AII, and AIV compared with apo B, apo CIII, and apo E, LpAI was found to contain apo AIV which was absent from LpAI: AII. Moreover, the bulk of lecithin-cholesterol acyl-transferase was present in LpAI. The concentration range of these particles was in agreement with those required in vitro for cholesterol efflux. Thus the interstitial fluid contains particles in which two agonists but no antagonists of cholesterol efflux are associated with lecithin-cholesterol acyltransferase activity. This supports apolipoprotein AI- and/or AIV-containing particles playing a critical role in the first step of reverse cholesterol transport.

Two different allelic mutations in a Finnish family with lecithin:cholesterol acyltransferase deficiency.

Lecithin:cholesterol acyltransferase (LCAT) deficiency is a genetic disorder associated with low levels of serum HDL cholesterol. The proband of the Finnish LCAT-deficient family had corneal opacities, proteinuria, anemia with stomatocytosis, low serum HDL cholesterol (0.27 mmol/L), and low LCAT activity. Sequence analysis of his LCAT gene revealed compound heterozygosity for two different mutations: a C insertion in exon 1 between nucleotides 932 and 937 and a C-to-T point mutation in exon 6 at position 4976. The C insertion in exon 1 is predicted to result in premature termination and a truncated polypeptide containing only 16 amino acids. The C-to-T point mutation in exon 6 substitutes cysteine for arginine at residue 399. The functional significance of the Arg399-->Cys mutation was examined by expressing the mutated and wild-type LCAT cDNAs in COS cells. COS cells transfected with mutated and wild-type cDNAs showed comparable levels of mature LCAT mRNA. However, LCAT activity in the cell media of COS cells transfected with the mutant LCAT cDNA was significantly lower than that of COS cells transfected with the wild-type cDNA (1.4% versus 12.0% cholesterol esterified, respectively). A polymerase chain reaction-based duplex assay, in which both mutations can be detected simultaneously, was used for preliminary screening of Finnish subjects with serum HDL levels below 0.9 mmol/L; two additional individuals heterozygous for the Arg399-->Cys mutation were identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein composition in insulin-dependent diabetes mellitus with chronic renal failure: effect of kidney and pancreas transplantation.

Chronic renal failure (CRF) in nondiabetics is associated with a number of lipoprotein abnormalities that place these patients at high risk for atherosclerosis. This study compared the lipoprotein composition of nondiabetic controls (n = 68) with that of patients with insulin-dependent diabetes mellitus ([IDDM] n = 13) and of patients with IDDM and CRF ([IDDM + CRF] n = 74). Six lipoprotein subfractions (very-low-density lipoprotein [VLDL], intermediate-density lipoprotein [IDL], low-density lipoprotein [LDL], high-density lipoprotein-light [HDL-L], HDL-medium [HDL-M], and HDL-dense [HDL-D]) were isolated by rapid gradient ultracentrifugation using a fixed-angle rotor. The apolipoprotein (by reverse-phase high-performance liquid chromatography [HPLC]) and lipid (by enzymatic assays) composition of each subfraction was determined. The only abnormalities found in IDDM patients were increases in IDL and HDL-L triglyceride (TG) levels and an increase in the HDL-L free cholesterol (FC) level. The IDDM + CRF group had multiple abnormalities including (1) elevated TG, apolipoprotein (apo) C-II, and apo C-III levels in all lipid subfractions; (2) elevated VLDL and IDL apo B, TG, FC, cholesterol ester (CE), and phospholipid (PL) levels (with an increased CE/TG ratio in VLDL only); (3) decreased HDL-M apo A-I, apo A-II, CE, and PL levels, but an increased HDL-D apo A-I level; and (4) decreased lecithin:cholesterol acyltransferase (LCAT) activity. Twenty-five of the IDDM + CRF patients underwent combined pancreas and kidney (P + K) transplantation, and 12 patients received only a kidney transplant. Lipoprotein composition was determined at 3, 6, and 12 months posttransplant. Both types of transplantation resulted in similar alterations in lipoprotein composition, even though there was essential normalization of blood glucose levels in most of the patients who received a pancreas transplant (hemoglobin A1C [HbA1C], 9.1% +/- 1.1% v 5.7% +/- 0.3% at 12 months, P < .01). These posttransplant changes included (1) no improvement in the elevated TG level in any lipid subfraction even though there was some reduction in apo C-III levels in VLDL; (2) reductions in levels of VLDL and IDL apo B but increases in LDL apo B; (3) increases in HDL apo C-III and FC concentrations despite an increase in LCAT activity; and (4) increases in apo A-I levels in HDL-L and HDL-M. The addition of a pancreas to a kidney transplant had no obvious impact on the lipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipoproteins containing apolipoprotein A-IV: composition and relation to cholesterol esterification.

In order to investigate the relationship of lipid and apolipoprotein composition to cholesterol esterification in lipoproteins containing apolipoprotein (apo) A-IV, apo A-containing lipoprotein particles were isolated from fresh human plasma using a system of sequential immunoaffinity chromatography. Plasma was first depleted of apo B- and apo E-containing lipoproteins. Four major subpopulations of apo A-containing lipoprotein particles were separated: Lp A-I, Lp A-I: A-II, Lp A-IV and Lp A-I: A-IV: A-II. Lp A-IV and Lp A-I: A-IV: A-II contained less total lipid, less cholesterol and more triacylglycerol than Lp A-I and Lp A-I: A-II. Lp A-IV and Lp A-I: A-IV: A-II contained more sphingomyelin and less phosphatidylcholine than Lp A-I and Lp A-I: A-II and were richer in (16:0 + 18:0) saturated fatty acids. Among these isolated lipoprotein particles, Lp A-IV contained the highest lecithin: cholesterol acyltransferase (LCAT) activity per micrograms of protein. Cholesterol esterification rates were 2.6 +/- 0.5, 5.3 +/- 0.4 and 0.8 +/- 0.2 mumol of cholesterol per hour per mg of lipoproteins for Lp A-IV, Lp A-I and Lp A-I: A-II, respectively. The apolipoprotein and lipid composition and LCAT activity of Lp A-IV suggest that this lipoprotein may be a source of cholesterol esterification in plasma.

Follicular fluid lipoproteins in the mare: evaluation of HDL transfer from plasma to follicular fluid.

Using a density gradient ultracentrifugal procedure, we have separated equine plasma and follicular fluid high-density lipoproteins (HDL). The density distribution of the follicular fluid HDL was clearly displaced towards the highest densities in comparison with that of plasma HDL. Similarly, an analysis of size distributions showed a decrease in follicular fluid HDL diameters (4.2 to 9.2 nm) compared to plasma HDL (5.5 to 9.5 nm). HDL were isolated into three subfractions on the basis of the disposition of the Sudan Black stained bands in the centrifuge tubes. Concentrations of each subfraction were clearly lower in the follicular fluid, and the relative percentages with regard to the plasma equivalents were inversely proportional to the molecular weights (23.8% for HDL-1, 49.9% for HDL-2 and 63.7% for HDL-3). The cholesterol/phospholipid molar ratio and the esterified/free cholesterol molar ratio were clearly increased in the follicular HDL-2 and HDL-3 subfractions. The apolipoprotein distribution in follicular fluid HDL was very close to that in plasma HDL. LCAT activity measured in human as well as equine samples was weaker in follicular fluid compared to plasma in both species (4.0 nmol of free cholesterol esterified per h per ml vs. 24 nmol per h per ml). Theoretical concentrations of follicular fluid HDL were calculated assuming that the HDL particles would be merely a filtration product undergoing no detectable metabolic modifications. Biochemical measurements showed that the lightest particles (HDL-1) were less numerous than suggested by the theoretical calculation. Thus, although follicular fluid HDL appear to be a filtration product of plasma HDL, they undergo metabolic transformations that we suggest may be linked to hormonal synthesis and reverse cholesterol transport.

Tangier disease: isolation and characterization of LpA-I, LpA-II, LpA-I: A-II and LpA-IV particles from plasma.

Tangier disease (TD) is characterized by extremely low plasma levels of HDL, apoA-I and apoA-II due to very rapid catabolism. However, the risk of premature coronary heart disease (CHD) is not markedly increased in TD. In order to gain insight into reverse cholesterol transport in TD, we isolated LpA-I, LpA-I:A-II, LpA-II and LpA-IV particles from fasting plasma of 5 TD patients. LpA-I composition was similar to control LpA-I, but TD LpA-I had more LCAT and CETP activity (respectively, 0.35 +/- 0.14 and 0.14 +/- 0.04 mumol of cholesterol esterified/h/micrograms of protein, and 7 +/- 2.5 and 1.4 +/- 0.3 mumol of cholesteryl ester transferred/h/micrograms of protein). In contrast, TD LpA-I:A-II had abnormal composition, with a low molar ratio of apoA-I to apoA-II (0.2-1.33). In addition, LpA-I:A-II in TD contained a substantial amount of apoA-IV compared with control, making this particle an LpA-I:A-II:A-IV complex. LpA-I:A-II from normal plasma do not promote cholesterol efflux from adipocytes cells, whereas TD LpA-I:A-II:A-IV complexes promoted cholesterol efflux from these cells. Moreover LpA-I:A-II:A-IV complexes have more LCAT and CETP activity than control (respectively 1.2 +/- 0.16 and 0.05 +/- 0.01 mumol of cholesterol esterified/h/micrograms of protein and, 41 +/- 3.7 and 1 +/- 0.4 mumol of cholesteryl ester transferred/h/micrograms of protein). The LpA-II particle in TD represented in fact an LpA-II:A-IV complex (75% mol apoA-II and 22% mol apoA-IV).(ABSTRACT TRUNCATED AT 250 WORDS)

Short-term changes in lipid and protein metabolism in liver transplants from living-related donors.

The effects of liver transplantation involving living-related donors were investigated in 20 pediatric cases in terms of protein and lipid metabolism using the extent of cholesterol esterification and the levels of total cholesterol, lecithine-cholesterol acyltransferase, apolipoprotein A-I, cholinesterase, and rapid turnover proteins as parameters. Cholesterol esterification increased from preoperative values of 39% +/- 4% to 67% +/- 1% (mean +/- SEM, n = 17) at 3 weeks after liver transplantation in successful cases but decreased from the preoperative value of 45% +/- 10% to 26% +/- 6% (n = 3) at 3 weeks in unsuccessful cases. Cholinesterase, transferrin, and prealbumin levels remained low after 3 weeks even in successful cases. Patients who had partial liver transplantations from living-related donors showed rapid recovery of cholesterol esterification. However, patients with graft livers required an extensive period before normalization of protein metabolism occurred, indicating the necessity for long-term follow-up of recipient development.

Expression and characterization of recombinant human lecithin:cholesterol acyltransferase.

We have established a baby hamster kidney (BHK) cell line that constitutively expresses significant quantities of human recombinant lecithin:cholesterol acyltransferase (rLCAT). LCAT cDNA was cloned into a mammalian expression vector containing the metallothionein promoter and the dihydrofolate reductase gene. After transfection, the BHK cells were treated with 500 microM methotrexate for 2 weeks to select the successfully transfected cells. Surviving colonies were subcloned and high level secretors were identified by measurement of LCAT activity and mass in the culture medium. The attachment of transfected cells to microcarrier beads enabled the efficient production of large quantities of rLCAT in a serum-free medium. After a single-step chromatography procedure, the rLCAT was purified to homogeneity with yields exceeding 1 mg of rLCAT per 100 ml of culture medium. The molecular weight of rLCAT (approximately 66,000) was identical to that of purified human plasma LCAT on SDS polyacrylamide electrophoresis. The rLCAT was activated by apolipoprotein A-I and had an average specific activity that was similar to purified plasma LCAT. After selective deglycosylation with either neuraminidase or N-glycanase, rLCAT and plasma LCAT had identical molecular weights. The simplification of the production and purification of rLCAT reported here will enable a more in depth analysis of the structure and function of this enzyme.

Continuous fluorescence assay for lecithin:cholesterol acyltransferase using a water-soluble phosphatidylcholine.

A water-soluble fluorescent phosphatidylcholine, 1,2-bis[4-(1-pyreno-butanoyl]-sn-glycero-3-phosphocholine (DPybPC) has been used to develop a sensitive, continuous assay for pure lecithin:cholesterol acyltransferase (LCAT) in solution. The monomeric substrate allowed us to examine the reaction of LCAT in the absence of a lipid/water interface in terms of the sensitivity of the enzymatic reaction to anions, ionic strength, apolipoproteins A-I and A-II, and a series of lysophosphatidylcholines and fatty acids. In contrast to the reaction of LCAT with aggregated phosphatidylcholines, the reaction of DPybPC with LCAT was not significantly affected by anions, ionic strength, nor apolipoproteins, indicating that these are only effectors of the interfacial reaction. Lysophosphatidylcholines and fatty acids inhibited LCAT in a chain-length-dependent manner below the critical micellar concentrations of these amphiphiles, indicating that the products of the LCAT reaction can bind to the enzyme and affect its kinetics even in the absence of an interface.

Biochemical assessment of cholelithiasis.

Bile and serum were analysed in 45 cases of cholelithiasis and 25 control subjects for cholesterol, phospholipids, bilirubin, alkaline phosphatase and LCAT activity. Serum phospholipids were found to be elevated in sixty percent of cases, whereas phospholipids in bile were found to be decreased. Serum alkaline phosphatase and alanine aminotransferase were normal. Serum and bile LCAT activity was found to be significantly depressed.