Trans unsaturated fatty acids inhibit lecithin: cholesterol acyltransferase and alter its positional specificity

Although dietary trans unsaturated fatty acids (TUFA) are known to decrease plasma HDL, the underlying mechanisms for this effect are unclear. We tested the hypothesis that the decreased HDL is due to an inhibition of lecithin:cholesterol acyltransferase (LCAT), the enzyme essential for the formation of HDL, by determining the activity of purified LCAT in the presence of synthetic phosphatidylcholine (PC) substrates containing TUFA. Both human and rat LCATs exhibited significantly lower activity (-37% to -50%) with PCs containing 18:1t or 18:2t, when compared with the PCs containing corresponding cis isomers. TUFA-containing PCs also inhibited the enzyme activity competitively, when added to egg PC substrate. The inhibition of LCAT activity was not due to changes in the fluidity of the substrate particle. However, the inhibition depended on the position occupied by TUFA in the PC, as well as on the paired fatty acid. Thus, for human LCAT, 18:1t was more inhibitory when present at sn-2 position of PC, than at sn-1, when paired with 16:0. In contrast, when paired with 20:4, 18:1t was more inhibitory at sn-1 position of PC. Both human and rat LCATs, which are normally specific for the sn-2 acyl group of PC, exhibited an alteration in their positional specificity when 16:0-18:1t PC or 16:1t-20:4 PC was used as substrate, deriving 26-86% of the total acyl groups for cholesterol esterification from the sn-1 position. These results show that the trans fatty acids decrease high density lipoprotein through their inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, and also alter LCAT's positional specificity, inducing the formation of more saturated cholesteryl esters, which are more atherogenic.     

Minimally oxidized LDL is a potent inhibitor of lecithin:cholesterol acyltransferase activity

The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. As a variety of highly reactive lipid peroxidation products can transfer from oxidized LDL to HDL, we evaluated the potential deleterious effects of LDL oxidation on HDL-cholesterol metabolism. To address this issue, we exposed the HDL-containing d > 1.063 g/ml fraction of human plasma to copperoxidized LDL and assessed lecithin:cholesterol acyltransferase (LCAT) activity and apolipoproteinA-I (apoA-I) structure. To determine whether LCAT was directly affected by oxidized LDL, independent of crosslinking of apoA-I, we used an exogenous, [14C]cholesterol-labeled proteoliposome substrate to measure plasma LCAT activity. We observed an inhibition of LCAT activity where copper-oxidized LDL possessing only 2.3 +/- 0.1 and 7.3 +/- 1.4 TBARS produced 24 +/- 3% and 47 +/- 10% reductions in [14C]cholesterol esterification by 1 h, respectively. Copper-oxidized LDL that had been passed through a GF-5 desalting column, while retaining only one-third of its original TBARS, possessed nearly all of its LCAT inhibitory capacity suggesting that the LCAT inhibitory factor(s) was a lipophilic oxidation product. Analysis of polarlipids isolated from copper-oxidized LDL indicated that phospholipid and sterol fractions effectively inhibited LCAT. Copper-oxidized LDL, with as little as 6.3 TBARS, also produced intermolecular crosslinking of apoA-I molecules. Taken together, these data suggest that products of LDL oxidation may adversely affect HDL-cholesterol metabolism by two separate mechanisms: 1) a direct inhibitory effect on LCAT activity and 2) through crosslinking of apoA-I. If occurring in vivo, minimally oxidized LDL may impair cholesteryl ester formation on HDL thereby limiting the ability of HDL to function efficiently in the putative antiatherogenic reverse cholesterol transport pathway.     

On the interpretation and potential diagnostic value of the measurements related to lecithin:cholesterol acyltransferase activity

At least one reliable method is available for measuring LCAT activity in human subjects(25), and this assay may help to probe even subtle changes in plasma lipids and lipoproteins. Since plasma lipids and lipoproteins are known to be present in abnormal amounts in a large number of metabolic diseases, the method of measuring the rate of cholesterol esterification in plasma could assume a substantial role in the diagnosis and (or) monitoring of treatment of pathologic states.     

Effect of heparin on lecithin: cholesterol acyltransferase and lipids in cholestasis

Injury to the hippocamp disturbs learning processes and short-term memory in 20-, 50- and 110-day rats. In 50-day rats, hippocampectomy results in lesser changes in learning and memory than in 20- and 110-day animals. Anatomo-physiological characteristics of the hippocamp in 20-day rats presumably indicate a particular importance of this structure at early stages of ontogenesis, when the brain cortex is not yet sufficiently mature and its connections with other structures are not completely formed. Non-linear dependence of learning disturbance in rats of varying age after hippocampectomy suggests that in rat hippocampal function undergoes changes during individual development of animals.     

Molecular diagnosis of lecithin: cholesterol acyltransferase deficiency in a presymptomatic proband

OBJECTIVE: The study compared two commercial chlorhexidine mouthrinses (Chlorhexamed 0.1% and Corsodyl 0.2%) for their effects on dental plaque and gingival inflammation, their side effects (eg, tooth staining and mucosal irritation), and patient acceptance. METHOD AND MATERIALS: One hundred thirty healthy volunteers were randomly distributed into two groups of 65 each. Each volunteer had gingivitis or chronic marginal periodontitis and used the rinse two times a day for 4 weeks. The sulcular bleeding index, approximal plaque index, gingival index, and a discoloration index were taken at baseline and once a week thereafter. The patients were questioned about taste disturbances, mucosal irritation, and their perception of the taste of the mouthrinse. RESULTS: In both groups, after 4 weeks, the mean sulcular bleeding index, approximal plaque index, and gingival index scores had decreased significantly. The discoloration index had increased significantly in both groups. There were no statistically significant differences between the two mouthrinses in any of these measurements. There were no significant differences in side effects reported by the two groups. CONCLUSION: The increase in concentration of chlorhexidine provided no clinical advantages or disadvantages.     

Biochemical and biophysical characterization of human recombinant lecithin: cholesterol acyltransferase

We established a Chinese hamster ovary cell line that constitutively expresses up to 5 mg/L of human recombinant lecithin: cholesterol acyltransferase (rLCAT). We purified the rLCAT to > 96% purity, and characterized it along with plasma LCAT (pLCAT) biochemically and biophysically. The recombinant enzyme is more heavily glycosylated and more heterogeneous in its carbohydrate content than the plasma enzyme, as revealed by differences in molecular weight and pI isoforms, determined by mass spectrometry and isoelectric focusing. Recombinant LCAT is half as active enzymatically as pLCAT. The difference in activity is due to differences in the catalytic rates rather than in the apparent K(m) values, suggesting that the binding of the rLCAT to interfaces is not altered by its different glycosylation pattern. Despite these differences, rLCAT has essentially the same intrinsic tryptophan fluorescence emission spectrum and far-UV CD spectrum as pLCAT, indicating that the tertiary and secondary structures of both enzyme forms are very similar. Both enzyme forms have a propensity to self-associate, and their multimers appear resistant to dissociation by SDS and dilution. The free energies of unfolding (delta G(H2O)) of rLCAT and pLCAT are 3.4 +/- 0.2 and 3.2 +/- 0.2 kcal/mol, respectively, as determined by guanidine hydrochloride denaturation monitored by fluorescence. These relatively low delta G(H2O) values support the notion that LCAT is capable of undergoing major conformational changes upon interaction with interfacial substrates.     

Correction of hypoalphalipoproteinemia in LDL receptor-deficient rabbits by lecithin:cholesterol acyltransferase

Familial hypercholesterolemia (FH), a disease caused by a variety of mutations in the low density lipoprotein receptor (LDLr) gene, leads not only to elevated LDL-cholesterol (C) concentrations but to reduced high density lipoprotein (HDL)-C and apolipoprotein (apo) A-I concentrations as well. The reductions in HDL-C and apoA-I are the consequence of the combined metabolic defects of increased apoA-I catabolism and decreased apoA-I synthesis. The present studies were designed to test the hypothesis that overexpression of human lecithin:cholesterol acyltransferase (hLCAT), a pivotal enzyme involved in HDL metabolism, in LDLr defective rabbits would increase HDL-C and apoA-I concentrations. Two groups of hLCAT transgenic rabbits were established: 1) hLCAT+/LDLr heterozygotes (LDLr+/-) and 2) hLCAT+/LDLr homozygotes (LDLr-/-). Data for hLCAT+ rabbits were compared to those of nontransgenic (hLCAT-) rabbits of the same LDLr status. In LDLr+/- rabbits, HDL-C and apoA-I concentrations (mg/dl), respectively, were significantly greater in hLCAT+ (62 +/- 8, 59 +/- 4) relative to hLCAT- rabbits (21 +/- 1, 26 +/- 2). This was, likewise, the case when hLCAT+/ LDLr-/- (27 +/- 2, 19 +/- 6) and hLCAT-/LDLr-/- (5 +/- 1, 6 +/- 2) rabbits were compared. Kinetic experiments demonstrated that the fractional catabolic rate (FCR, d(-1)) of apoA-I was substantially delayed in hLCAT+ (0.376 +/- 0.025) versus hLCAT- (0.588) LDLr+/- rabbits, as well as in hLCAT+ (0.666 +/- 0.033) versus hLCAT- (1.194 +/- 0.138) LDLr-/- rabbits. ApoA-I production rate (PR, mg x kg x d(-1)) was greater in both hLCAT+/LDLr+/- (10 +/- 2 vs. 6) and hLCAT+/LDLr-/- (9 +/- 1 vs. 4 +/- 1) rabbits. Significant correlations (P < 0.02) were observed between plasma LCAT activity and HDL-C (r = 0.857), apoA-I FCR (r = -0.774), and apoA-I PR (r = 0.771), while HDL-C correlated with both apoA-I FCR (-0.812) and PR (0.751). In summary, these data indicate that hLCAT overexpression in LDLr defective rabbits increases HDL-C and apoA-I concentrations by both decreasing apoA-I catabolism and increasing apoA-I synthesis, thus correcting the metabolic defects responsible for the hypoalphalipoproteinemia observed in LDLr deficiency.     

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)     

Amino acid residue 149 of lecithin:cholesterol acyltransferase determines phospholipase A2 and transacylase fatty acyl specificity

Human LCAT prefers phosphatidylcholine (PC) with sn-1-palmitoyl-2-oleoyl PC (POPC) as substrate for cholesteryl ester synthesis, whereas rat LCAT (which is 92% similar in amino acid sequence) prefers sn-1-palmitoyl-2-arachidonoyl PC (PAPC). Six recombinant human LCAT cDNA clones were constructed with unique clusters of rat sequence substitutions in the human background spanning the region encoding amino acids 121-296. Media from transfected COS cells expressing each of the constructs were assayed for LCAT cholesterol esterification (CE) or phospholipase A2 (PLA2) activity using substrate particles containing POPC or PAPC. The PAPC/POPC CE activity ratio of the cluster 1 construct (amino acids 149-158) was 1.3, resembling rat LCAT, whereas cluster 2-5 clones produced CE activity ratios <0.3, unchanged from human LCAT. The cluster 6 clone (Y292H/W294F) had an intermediate ratio (0.6). Similar results were observed for LCAT PLA2 activity. In additional studies, position 149 of human LCAT was changed to the rat sequence (hE149A) and compared to a triple mutation containing the remainder of the cluster 1 changes (G151R/E154D/R158Q). CE and PLA2 activity ratio for the hE149A construct was >1.7, similar to rat LCAT, whereas the triple mutation construct retained a ratio similar to human LCAT (<0.6). Thus, a single amino acid substitution (E149A) was sufficient to alter the fatty acyl specificity of human LCAT to that of rat LCAT, with an increase in activity toward PAPC. This is the first example of a point mutation in an enzyme with PLA2 activity that results in an increase in activity toward arachidonic acid.     

N-(4-hydroxyphenyl)-retinamide increases lecithin:retinol acyltransferase activity in rat liver

N-(4-Hydroxyphenyl)-retinamide (4-HPR; Fenretinide) is a synthetic retinoid which is undergoing investigation as a cancer chemopreventive agent. However, 4-HPR alters vitamin A kinetics and reduces the concentration of plasma retinol. We have conducted studies to examine the effects of 4-HPR on the activity of the enzyme lecithin:retinol acyltransferase (LRAT). This enzyme is implicated in the absorption and storage of vitamin A and is regulated, in liver, by vitamin A nutritional status. To determine whether 4-HPR, like retinoic acid, is able to induce liver LRAT activity, vitamin A-deficient rats having negligible liver LRAT activity were treated with single doses of 4-HPR (0.02-2.5 mg) and liver homogenates were assayed for LRAT activity using 3H-retinol bound to the cellular-retinol binding protein, CRBP, as substrate. Treatment with 4-HPR resulted in a dose- and time-dependent increase in liver LRAT activity which reached a maximum at 24 h. The activity of LRAT assayed in vitro and of hepatic 3H-retinyl ester content determined after an in vivo pulse of 3H-retinol were highly correlated (r = 0.802, P < 0.0002). When vitamin A-sufficient rats were fed a 4-HPR-supplemented diet for 30 d, LRAT activity differed significantly from control values in the liver (P < 0.0001) but not the small intestines. Changes in hepatic retinol metabolism which favor the esterification of vitamin A may be related to the mechanism by which 4-HPR alters vitamin A kinetics in vivo.     

Interaction of N-methyl-anthraniloyl-labelled porcine apolipoprotein A-I with porcine lecithin: cholesterol acyltransferase: an energy transfer study

In an 8-month strictly controlled dietary study of 16 healthy young men, the long-term effect of a low-fat (26% of energy) high-fiber (4.5 g/MJ) diet on cardiovascular risk markers of the hemostatic system was assessed. Fasting blood sampling was performed during a 4-week baseline period and then monthly during the intervention. A matched control group of 16 men on habitual diets was also monitored. Median fibrinolytic activity of tissue-type plasminogen activator (t-PA) in plasma was significantly elevated (twofold to fourfold) by the experimental diet. A significant increase in the systemic fibrinolytic activity of the euglobulin fraction of plasma was also observed. Median plasma factor VII coagulant activity (F VIIc) was depressed by 5-10% during the first 2 months and the last month of the study period. The dietary change did not significantly affect plasma levels of fibrinogen, t-PA antigen, or plasminogen activator inhibitor type I antigen. In conclusion, young men who were switched from a typical Danish diet high in saturated fat to a low-fat/high-fiber diet showed a permanent increase in plasma fibrinolytic activity and a biphasic decrease in F VIIc. The dietary change thus had a favorable effect on cardiovascular risk markers of the hemostatic system.     

Regulation of hepatic lecithin:retinol acyltransferase activity by retinoic acid receptor-selective retinoids

In the present study the electrophysiological characteristics and the proarrhythmic potential of cisapride and a structurally related drug, mosapride, were compared. In the anesthetized guinea pig, cisapride and d-sotalol (0.01-10 micromol/kg i.v., n = 6) dose-dependently prolonged the duration of the monophasic action potential recorded from the left ventricle. The maximal lengthening was 18 +/- 3.2% at 1.0 micromol/kg (mean +/- S.E.M., P < .01 vs. base line) and 19 +/- 2.5% at 10 micromol/kg (P < .001) for cisapride and d-sotalol, respectively. In contrast, mosapride did not increase this variable. In a rabbit model of the acquired long QT syndrome, infusion of cisapride (0.3 micromol/kg/min for 10 min maximum, n = 6), but not mosapride or vehicle, was associated with a significant lengthening of the QTU interval (43 +/- 3.8 ms, P < .01). Furthermore, torsades de pointes appeared in two of the six rabbits given cisapride. In isolated rabbit Purkinje fibers (PF), cisapride increased the action potential duration (48 +/- 5.6% at 0.1 micromol/l, P < .01 vs. control, n = 4). Mosapride did not significantly influence the action potential duration (3 +/- 2.0% increase at 1.0 micromol/l, n = 6). However, after mosapride was washed out, the addition of cisapride (0.1 micromol/l) caused a 46 +/- 3.2% lengthening of the action potential duration (P < .01 vs. 1.0 micromol/l mosapride). Early afterdepolarizations and triggered activity appeared in four of eight cisapride-superfused PF stimulated at a very low frequency (0.1 Hz). In isolated rabbit cardiomyocytes, cisapride concentration-dependently blocked (IC50 = 9 nmol/l) the rapid component of the delayed rectifying K+ current (I(Kr)). Mosapride was approximately 1000-fold less potent in blocking I(Kr) (IC50 = 4 micromol/l). It is concluded that the electrophysiological characteristics of cisapride may explain the recently reported propensity to prolong the QT interval and to induce torsades de pointes in susceptible patients, although a structurally related benzamide, mosapride, did not appear to have electrophysiological features of relevance for induction of torsades de pointes in common with cisapride.     

The hydrophobic face orientation of apolipoprotein A-I amphipathic helix domain 143-164 regulates lecithin:cholesterol acyltransferase activation

Apolipoprotein A-I (apoA-I) activates the plasma enzyme lecithin:cholesterol acyltransferase (LCAT), catalyzing the rapid conversion of lipoprotein cholesterol to cholesterol ester. Structural mutants of apoA-I have been used to study the details of apoA-I-LCAT-catalyzed cholesterol ester formation. Several studies have shown that the alpha-helical segments corresponding to amino acids 143-164 and 165-186 (repeats 6 and 7) are essential for LCAT activation. In the present studies, we examined how the orientation of the hydrophobic face, independent of an increase in overall hydrophobicity, affects LCAT activation. We designed, expressed, and characterized a mutant, reverse of 6 apoA-I (RO6 apoA-I), in which the primary amino acid sequence of repeat 6 (amino acids 143-164) was reversed from its normal orientation. This mutation rotates the hydrophobic face of repeat 6 approximately 80 degrees. Lipid-free RO6 apoA-I showed a marked stabilization when denatured by guanidine hydrochloride, but showed significant destabilization to guanidine hydrochloride denaturation in the lipid-bound state compared with wild-type apoA-I. Recombinant high density lipoprotein discs (rHDL) formed from RO6 apoA-I, sn-1-palmitoyl-sn-2-oleoyl phosphati-dylcholine, and cholesterol were approximately 12 A smaller than wild-type apoA-I rHDL. The reduced size suggests that one of the repeats did not effectively participate in phospholipid binding and organization. The sn-1-palmitoyl-sn-2-oleoyl phosphatidylcholine RO6 rHDL were a less effective substrate for LCAT. Mapping the entire lipid-free and lipid-bound RO6 apoA-I with a series of monoclonal antibodies revealed that both the lipid-free and lipid-bound RO6 apoA-I displayed altered or absent epitopes in domains within and adjacent to repeat 6. Together, these results suggest that the proper alignment and orientation of the hydrophobic face of repeat 6 is an important determinant for maintaining and stabilizing helix-bilayer and helix-helix interactions.     

Fatty acids modulate lecithin:cholesterol acyltransferase secretion independently of effects on triglyceride secretion in primary rat hepatocytes

The regulation of plasma lecithin:cholesterol acyltransferase (LCAT) expression is not well understood. Although oleic acid increases both the secretion of triglycerides and LCAT by primary rat hepatocytes, the effect of other fatty acids (FA) on LCAT secretion is not known. This study was designed to examine the effect of FA on the hepatic secretion of LCAT, triglyceride and apolipoprotein A-1 (apoA-1). Primary rat hepatocytes were incubated with serum-free medium, supplemented with individual FA (0-1 mmol/L) for 22-24 h. Preliminary studies indicated a linear secretion of LCAT up to 24 h in both control and FA-treated cells. When hepatocytes were incubated with 1 mmol/L FA, the LCAT secretion increased 50-100% (P < 0.01) in the presence of the 18-carbon FA (stearic, oleic, elaidic and linoleic acids), whereas the presence of butyric, lauric and palmitic acids had no significant effect. LCAT secretion decreased (P < 0.01) in the presence of docosahexaenoic acid (DHA). All FA (except DHA) significantly enhanced triglyceride secretion; however, only the 18 carbon FA significantly stimulated the synthesis and secretion of apoA-1 and secretion of LCAT. The secretion of LCAT correlated with apoA-1 secretion (r = 0.88, P = 0.004) but not with triglyceride secretion (r = 0.55, P = 0.12). Treatment with oleic acid resulted in a 1.5-fold increase in hepatocyte LCAT mRNA accumulation, whereas butyrate and palmitate had no effect. These data indicate that FA that promote the apparent synthesis and secretion of apoA-1 also stimulate the secretion of LCAT in vitro, suggesting a coordinate regulatory mechanism for apoA-1 and LCAT expression.     

Diagnosis of fatty liver in dairy cows

From 27 dairy cows with a mean milk yield of 6900 kg FCM (4% milk fat) per 305 day lactation period liver bioptates 2 weeks post partum (p.p.), milk samples 2, 4 weeks p.p., blood samples 0 (partus), 2, 4 week p.p., measurement of backfat thickness 2 weeks prior to calving, 0, 6, 17 weeks p.p. were taken and body weight and milk yield were determined. Fertility results and disorders appearance were recorded too. Total lipid and triglyceride content were analysed in liver tissue. Acetone concentration was determined in milk and 15 clinical-chemical parameters were elucidated in blood samples. Liver fat concentration shows a great variability from 3.9% to 24%. There is no strong reference value for the distinction between physiological and pathological liver fat concentration. Assessment as to whether increased liver fat levels in dairy cow are indicative of liver damage due to a pathological process should include detection of liver cell damage on the basis of plasma enzymes with closest possible specificity of liver. Glutamate-dehydrogenase (GLDH) is recommended. Liver fat content clearly could be defined exclusively from investigation of liver tissue rather than from analysis of blood or milk parameters. Measurement of backfat thickness provided useful information on the post partum lipolysis rate with a good correlation to liver fat (r to -0.72).     

Transfer of cholesterol and lecithin between erythrocytes and serum in vitro

1. In human blood incubated in vitro, the transfer of free cholesterol and lecithin from erythrocytes to serum was not related to glycolytic activity in erythrocytes or esterification of cholesterol in serum. 2. The stability of free cholesterol concentration in serum was dependent on the activity of lecithin:cholesterol acyltransferase (EC 2.3.1.43), and the stability of lecithin concentration in erythrocytes, on glycolytic activity.     

Comparative studies on the substrate specificity of lecithin:cholesterol acyltransferase towards the molecular species of phosphatidylcholine in the plasma of 14 vertebrates

We have studied, in a prospective blinded fashion, the effects of regular and extended-release gemfibrozil on plasma lipoprotein and apolipoprotein (apo) levels in hypercholesterolemic subjects with decreased high density lipoprotein (HDL) cholesterol (C) levels. Study participants were men and women 19 to 80 years of age with baseline plasma low density lipoprotein (LDL) C levels > or = 4.5 mmol/l (175 mg/dl), HDL-C levels < or = 1.2 mmol/l (45 mg/dl), and triglyceride levels < or = 3.4 mmol/L (300 mg/dl). All subjects were stabilized on a diet for eight weeks prior to entry into two different protocols. In the first protocol 229 subjects were randomized to placebo or extended-release gemfibrozil (1200 mg/day) for 3 months (placebo trial). In the second protocol 655 subjects were randomized to regular or extended-release gemfibrozil (1200 mg/day) for 6 months (equivalency trial). Changes in lipids and apos were stratified by baseline HDL-C levels (< 0.9 mmol/l, and 0.9-12.2 mmol/l). In both studies, treatment with gemfibrozil, either regular or extended-release, was associated with significant (P < 0.05) decreases in plasma very low density lipoprotein (VLDL) C and triglyceride levels of 42-45% and 33-37%, respectively, in subjects with HDL-C level < 0.9 mmol/l, and of 38-47% and 32-39%, respectively, in patients with HDL-C levels of 0.9-1.2 mmol/l. Modest reductions from baseline in directly measured LDL-C levels were observed in both groups (3-6% and 8-9%, respectively). These reductions were less than those observed for calculated LDL-C (7-10% and 11%, respectively). For apo B, reductions were 11-14% and 16-17% in the two groups. HDL-C, apo A-I, and apo A-II levels increased by 15-16%, 5-6%, and 21-25%, respectively, in patients with HDL-C < 0.9 mmol/l, and by 6-7%, 2-3%, and 19-22%, respectively, in patients with HDL-C of 0.9-1.2 mmol/l. These differences in HDL-C levels reached statistical significance in the equivalency trial (P < 0.0001) and were independent of baseline triglyceride levels. Our data indicate that gemfibrozil, either regular or extended-release, is highly effective in lowering plasma triglyceride levels and increases HDL-C levels by approximately 15% in hypercholesterolemic patients with low HDL-C levels (< 0.9 mmol/l). Moreover, this agent lowers VLDL-C somewhat more than triglyceride, resulting in an underestimation of calculated VLDL-C reductions and in an overestimation of calculated LDL-C reductions. This agent also raises apo A-II levels much more than apo A-I levels.     

Reduction of acyloxyacyl hydrolase activity in circulating neutrophils from cows after parturition

Bovine neutrophils contain the enzyme acyloxyacyl hydrolase, which hydrolyzes the acyloxyacyl linkage of the two nonhydroxylated fatty acyl chains to two 3-hydroxy fatty acids in the highly conserved lipid A part of endotoxins with high specificity. This hydrolysis decreases the toxicity of lipid A, but the immunostimulatory capacity of endotoxins is largely maintained. In two trials, we studied the activity of acyloxyacyl hydrolase in neutrophils that had been isolated from the blood of 18 dairy cows around parturition. Between 10 and 26 d after parturition, the activity of acyloxyacyl hydrolase in neutrophils decreased approximately 20% below prepartum activity. At about 2 mo after parturition, acyloxyacyl hydrolase activity returned to prepartum values. Changes in acyloxyacyl hydrolase activity could not be attributed to changes in binding of lipopolysaccharides by the CD14 molecules on neutrophils or monocytes. We hypothesize that decreased acyloxyacyl hydrolase activity in neutrophils shortly after parturition is a factor that increases the susceptibility of dairy cows to coliform mastitis during early lactation.     

Transmission of two novel mutations in a pedigree with familial lecithin:cholesterol acyltransferase deficiency: structure-function relationships and studies in a compound heterozygous proband

Two novel mutations were identified in a compound heterozygous male with lecithin:cholesterol acyltransferase (LCAT) deficiency. Exon sequence determination of the LCAT gene of the proband revealed two novel heterozygous mutations in exons one (C110T) and six (C991T) that predict non-conservative amino acid substitutions (Thr13Met and Pro307Ser, respectively). To assess the distinct functional impact of the separate mutant alleles, studies were conducted in the proband's 3-generation pedigree. The compound heterozygous proband had negligible HDL and severely reduced apolipoprotein A-I, LCAT mass, LCAT activity, and cholesterol esterification rate (CER). The proband's mother and two sisters were heterozygous for the Pro307Ser mutation and had low HDL, markedly reduced LCAT activity and CER, and the propensity for significant reductions in LCAT protein mass. The proband's father and two daughters were heterozygous for the Thr13Met mutation and also displayed low HDL, reduced LCAT activity and CER, and more modest decrements in LCAT mass. Mean LCAT specific activity was severely impaired in the compound heterozygous proband and was reduced by 50% in individuals heterozygous for either mutation, compared to wild type family members. It is also shown that the two mutations impair both catalytic activity and expression of the circulating protein.