Radioimmunoassay of human plasma lecithin-cholesterol acyltransferase.

A sensitive and precise competitive-displacement double-antibody radioimmunoassay was developed for the human plasma enzyme lecithin-cholesterol acyltransferase (LCAT; Ec 2.3 1.43). The ability of plasma from various animal species to displace labeled human LCAT from goat anti-human LCAT could be ranked in the following order: man and sheep > nonhuman primates > cat or dog > pig > rabbit or guinea pig > mouse > rat. Normolipidemic subjects had levels of LCAT of 6.14 +/- 0.98 micrograms/ml (mean +/- SD, n = 66). Subjects with dysbeta-lipoproteinemia had the highest plasma LCAT levels (7.88 +/- 0.39 micrograms/ml, n = 7, P < 0.05), followed by hypercholesterolemic subjects (7.00 +/- 1.30, n = 41) and hypertriglyceridemic subjects (6.96 +/- 1.3, n = 10). LCAT-deficient subjects had the lowest enzyme levels (0.89, 0.83, and 0.05 micrograms/ml, respectively, and two subjects with no detectable enzyme). Males had lower LCAT levels (6.42 +/- 1.05 micrograms/ml, n = 90, for all subjects; 5.99 +/- 1.03, n = 44, for normolipidemics) than females (7.01 +/- 1.14, n = 34, for all subjects P < 0.01; 6.44 +/- 0.79, n = 22, for normolipidemics, P < 0.01). LCAT levels correlated significantly with total cholesterol (males, r = 0.384, P < 0.001; females, r = 0.519, P < 0.002); and total triglyceride (only in females, r = 0.512, P < 0.002). LCAT levels in females correlated inversely with HDL cholesterol (r = 0.341, P < 0.05) and apoprotein D (r = 0.443, P < 0.02), but no such relationship existed in males.

Defective enzyme causes lecithin-cholesterol acyltransferase deficiency in a Japanese kindred.

Lecithin-cholesterol acyltransferase mass levels and activity and apolipoproteins A-I, A-II, B and D were measured in a Japanese family who have a familial lecithin-cholesterol acyltransferase deficiency. This analysis was performed to gain insight into the molecular basis of the enzyme deficiency and to compare findings in this family with other families with familial lecithin-cholesterol acyltransferase deficiency. The mass of the enzyme in plasma was determined by a sensitive double antibody radioimmunoassay, and enzyme activity was measured by using a common synthetic substrate comprised of phosphatidylcholine, cholesterol and apolipoprotein A-I liposomes prepared by a cholate dialysis procedure. The lecithin-cholesterol acyltransferase-deficient subject had an enzyme mass level that was 35% of normal (2.04 micrograms/ml, as compared with an average normal level of 5.76 +/- 0.95 micrograms/ml in 19 Japanese subjects) and an enzyme activity of less than 0.1% of normal (0.07 nmol/h per ml, as compared with normal levels of 100 nmol/h per ml). This subject also had lower levels of apolipoproteins: apolipoprotein A-I was 53 mg/dl (42% of normal), apolipoprotein A-II was 10.6 mg/dl (31% of normal), apolipoprotein B was 68 mg/dl (68% of normal), and apolipoprotein D was 3.6 mg/dl (60% of normal). The three obligate heterozygotes had enzyme mass levels ranging from 65% to 100% of normal and enzyme activity levels ranging from 23% to 65% of normal (23.4, 56.8, and 64.7 nmol/h per ml, respectively). The proband's sister had an enzyme mass level of 6.55 micrograms/ml (114% of normal) and an enzyme activity of only 64.8 nmol/h per ml (65% of normal), suggesting that she was also a heterozygote for lecithin-cholesterol acyltransferase deficiency. The obligate heterozygotes and the sister had normal apolipoprotein levels. We conclude that the lecithin-cholesterol acyltransferase deficiency in this family is due to the production of a defective enzyme that is expressed in the homozygote as well as in the heterozygotes, and, further, that this family's mutation differs from that reported earlier for other Japanese lecithin-cholesterol acyltransferase-deficient families.

Population-based reference values for lecithin-cholesterol acyltransferase (LCAT).

Plasma unesterified cholesterol is converted to cholesteryl ester by the enzyme lecithin-cholesterol acyltransferase (LCAT). Plasma levels of LCAT were measured by a sensitive double antibody radioimmunoassay in a sample from an adult employee population, ages 20-59 years, in the Pacific Northwest. After adjusting for differences in relative body mass, women had significantly higher LCAT levels (5.90 +/- 1.06, n = 154) than men (5.49 +/- 0.89, n = 83). For ages 20-59 years, LCAT levels showed a slight association with age: r = 0.13 for men and 0.29 for women. LCAT was positively correlated with relative body mass, total cholesterol, and LDL cholesterol. Men who smoked cigarettes had significantly lower LCAT mass than men who did not smoke cigarettes. No statistical differences in mean LCAT values were found between drinkers and nondrinkers. The 5th percentile LCAT value was 4.3 micrograms/ml for both men and women not using hormones. The 95th percentile value was 7.3 micrograms/ml for men and 7.8 micrograms/ml for women regardless of hormone use. Subjects phenotypically LCAT-deficient by clinical criteria and by the absence or near absence of LCAT activity had levels of LCAT mass well below the reference values: 0.73 +/- 0.70, range 0.10 micrograms/ml to 2.65 micrograms/ml, n = 20. Parents or children of LCAT-deficient subjects, i.e., obligate heterozygotes for familial LCAT deficiency, had reduced levels: 3.59 +/- 0.69, range 2.59-4.61 micrograms/ml, n = 19.

Studies of lipoproteins and fatty acids in maternal and cord blood of two racial groups in Trinidad.

The high mortality rate from coronary heart disease (CHD) among Indians compared to Negroes in Trinidad led us to test plasma lipid profiles to see whether dietary or genetic factors might be involved. There were no interracial differences in the composition of plasma cholesterol ester fatty acids of the tested women and neonates. This finding suggests that dietary fat does not account for the interracial difference in CHD, nor does the cause appear to be due to genetic differences in lipid profiles, as there was no significant difference between values for plasma triglycerides, total cholesterol, high density lipoprotein (HDL) cholesterol, apo-I, apo-II, apo B or cholesterol ester fatty acids in the cord blood of each racial group. Blood samples were collected from 69 nonpregnant and 71 postpartum, fasted Negro and Indian women. Also taken were 71 umbilical cord blood samples. The mean triglyceride level was significantly lower in the Negro nonpregnant and postpartum women than in the Indians. HDL cholesterol and apo-I values were lower in the Indian women. There were no significant differences in the total cholesterol and apo B measurements. The triglyceride values for postpartum women were higher than those of the nonpregnant Negroes and Indians (75% and 47%, respectively), whereas the total cholesterol and HDL cholesterol, apo A-I and apo A-II ranged from 9% to 29% higher in the postpartum women. Apo B was about 40% higher postpartum in both ethnic groups. The high CHD rate of Indians in Trinidad cannot be explained by dietary factors, plasma total cholesterol or fatty acid composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of commercial kits for apoprotein A-I and apoprotein B with standardized apoprotein A-I and B radioimmunoassays performed at the Northwest Lipid Research Center.

There has recently been a proliferation of commercial kits available for apoproteins A-I and B. Since reference procedures for apoproteins have not yet been established we have elected to compare apoprotein kit methods with highly standardized apoprotein B and A-I radioimmunoassays developed at the Northwest Lipid Research Center. Commercial radial immunodiffusion kits for apoproteins A-I and B were obtained from three separate companies, Calbiochem, Daiichi Pure Chemicals, and Tago, and a commercial radioimmunoassay kit for apoprotein A-I was obtained from Ventrex Laboratories. Considerable differences were observed between the commercial kit methods and the Northwest Lipid Research radioimmunoassay methods. Some of the differences between methods were related to the assigned value of the reference materials. Other differences between methods were clearly method-dependent.

Comparison of two commercial nephelometric methods for apoprotein A-I and apoprotein B with standardized apoprotein A-I and B radioimmunoassays.

Normotriglyceridemic and hypertriglyceridemic samples were analyzed for apoproteins A-I and B using the Beckman Array System and the Behring Nephelometer, and the nephelometric values were compared to values obtained by highly standardized radioimmunoassays developed at the Northwest Lipid Research Center. Although the means of the apoA-I values obtained by each method were similar, comparison of sample values by least-squares regression analysis revealed large differences (Sy = 20 mg/dl for Beckman, Sy = 18 mg/dl for Behring) (Sy = standard error of the estimate) regardless of whether the comparison included hypertriglyceridemic samples. For normotriglyceridemic samples, there was good agreement between apoB values obtained by the Behring Nephelometer and those obtained by RIA (r = 0.91, m = 1.03, Sy = 12 mg/dl). However, significantly higher apoB values were obtained on hypertriglyceridemic samples by the Behring Nephelometer. ApoB values for normotriglyceridemic samples obtained by the Beckman System and RIA showed fairly good correlation (r = 0.86, m = 0.71, Sy = 14 mg/dl). However, the nephelometric values for normotriglyceridemic samples averaged 29% lower than those obtained by RIA. This difference could largely be accounted for by the low apoprotein B value assigned to the Beckman calibrator. Significantly lower apoprotein B values were obtained on hypertriglyceridemic samples by the Beckman Nephelometer even after correction for calibration differences. Apoprotein values obtained by nephelometric methods may be inaccurate, particularly if the samples are hypertriglyceridemic.

Injuries in adolescent female players in European football: a prospective study over one outdoor soccer season.

In this prospective study, injuries in 153 adolescent female soccer players were recorded during one outdoor season (April-October). The overall injury incidence rate was 6.8 per 1000 h soccer (games and practice) and the incidence rate of traumatic injury 9.1 and 1.5 per 1000 player-hours in games and practice, respectively. Sixty-three players (41%) sustained 79 injuries. Sixty-six percent of the injuries were traumatic and 34% were overuse injuries. Most of the traumatic injuries occurred during games. Eighty-nine percent of the injuries were located in the lower extremities and 42% occurred in the knee or ankle. The most frequent type of injury was ankle sprain (22.8%). Forty-one percent of the traumatic injuries and 56% of the ankle sprains were re-injuries. Most of the injuries were of moderate severity (52%), while 34% were minor and 14% were major. Most of the major injuries were traumatic such as knee ligament injuries and ankle sprains.

Radioimmunoassay of human plasma Lp(a) lipoprotein.

A quantitative immunodiffusion assay demonstrated Lp(a) lipoprotein in 91% (911 of 1000) of subjects. In order to quantitate Lp(a) in all plasma, a sensitive and specific double antibody radioimmunoassay was developed. The between-assay coefficient of variation was 8%. Lp(a) levels by radioimmunoassay were highly correlated with those obtained by the less sensitive radial immunodiffusion method (r = 0.98, n = 51). All but one of the 89 Lp(a) "negative" subjects by immunodiffusion had detectable levels of Lp(a) by radioimmunoassay. The one subject without detectable Lp(a) had abetalipoproteinemia (without detectable apolipoprotein B by radioimmunoassay). Furthermore, Lp(a) was detected in all three non-human primates examined: patas monkey, baboon, and pig-tail monkey. Quantitation of Lp(a) levels in 90 male myocardial infarction (MI) survivors and their spouses showed that the distribution of Lp(a) levels of MI survivors was significantly higher above the 50th percentile cut-point (P < 0.02) and exceeded that of the spouses. Furthermore, the Lp(a) distribution at and above the 50th percentile for the MI survivors who had an MI at age <50 (n = 36) was shifted to values higher than those having an MI at age >50. Thus, high levels of Lp(a) may be associated with premature coronary disease. We conclude that Lp(a) is present in all individuals with apolipoprotein B and that apolipoprotein B appears necessary for the plasma transport of the Lp(a) lipoprotein. Consistent with this hypothesis, quantitative immunochemical precipitation of (125)I-Lp(a) indicated that essentially all individual molecules of six purified Lp(a) preparations contain both the Lp(a) antigen and apolipoprotein B.

Familial lecithin-cholesterol acyltransferase: identification of heterozygotes with half-normal enzyme activity and mass.

Lecithin-cholesterol acyltransferase (LCAT) mass and activity was measured in Canadian kindred of Italian and Swedish descent with familial LCAT deficiency. Four subjects had LCAT mass of 5.21 +/- 0.87 micrograms/ml (mean +/- SD) and LCAT activity of 98.8 +/- 12.0 nmol/h/ml, well within their respective normal ranges. Five family members, including the parents, the maternal grandmother, and two of four siblings of the LCAT deficient subjects, had enzyme mass (2.85 +/- 0.32 micrograms/ml) and activity (50.8 +/- 6.3 nmol/h/ml) approximately one-half that of normal levels. These presumed heterozygotes had normal levels of apolipoproteins A-I, A-II, B and D. The two subjects with LCAT deficiency had no detectable LCAT mass (below 0.1 microgram/ml) or LCAT activity (below 0.76 nmol/h/ml), apolipoprotein A-I and D levels approximately 50% of normal, and apolipoproteins B and A-II levels only 30-35% of normal. LCAT deficiency in this family is determined by an autosomal recessive mode. Furthermore, LCAT levels and activity are determined by two autosomal codominant alleles, LCATn, the normal LCAT gene, and LCATd, the LCAT deficiency gene.

Lecithin:cholesterol acyltransferase (LCAT) mass; its relationship to LCAT activity and cholesterol esterification rate.

The relationship between plasma lecithin:cholesterol acyltransferase mass and enzyme activity and between mass and plasma cholesterol esterification rate was determined in 25 adult volunteers without overt disease (14 normolipidemic and 11 hyperlipidemic). Furthermore, the relationship of lecithin:cholesterol acyltransferase mass and cholesterol esterification rate to lipids, apoproteins, age, and ideal body weight was assessed. Lecithin:cholesterol acyltransferase mass determined by radioimmunoassay was highly correlated with enzyme activity assayed using a heated plasma substrate (r = 0.636) and with the molar cholesterol esterification rate determined either by radioassay (r = 0.809) or by measurement of the decrease of unesterified cholesterol (r = 0.621). Lecithin:cholesterol acyltransferase mass was also positively correlated with total cholesterol (r = 0.608), unesterified cholesterol (r = 0.562), age (r = 0.544), and percent ideal body weight (r = 0.619), but was not significantly correlated with log triglyceride, high density lipoprotein cholesterol, or apolipoproteins A-I, A-II, or D. Plasma cholesterol esterification rate by both methods was highly positively correlated with total cholesterol, unesterified cholesterol, log triglyceride, and age, but was inversely correlated with high density lipoprotein cholesterol. Upon partial correlation analysis with lecithin:cholesterol acyltransferase mass kept constant the cholesterol esterification rate remained significantly positively related to total cholesterol, unesterified cholesterol, and log triglyceride and inversely related to high density lipoprotein cholesterol. Two subjects had normal lecithin:cholesterol acyltransferase but approximately half normal molar cholesterol esterification rate. Measurement of lecithin:cholesterol acyltransferase mass and activity along with plasma cholesterol esterification rate will permit differentiation of abnormalities of enzyme from qualitative or quantitative substrate or cofactor abnormalities. Also, the finding that the regression line between LCAT mass and the plasma esterification rate by direct determination of unesterified cholesterol passes through the origin suggests that all immunodetectable LCAT in plasma is active in normal subjects.-Albers, J. J., C-H. Chen, and J. L. Adolphson. Lecithin:cholesterol acyltransferase (LCAT) mass; its relationship to LCAT activity and cholesterol esterification rate.

Familial lecithin-cholesterol acyltransferase deficiency in a Japanese family: evidence for functionally defective enzyme in homozygotes and obligate heterozygotes.

Lecithin-cholesterol acyltransferase (LCAT) mass and activity were measured in a Japanese family with familial LCAT deficiency. The two LCAT-deficient subjects had LCAT mass approximately 40-46% of normal (2.65 and 2.31 micrograms/ml respectively, as compared with normal levels of 5.76 +/- 0.95 microgram/ml in 19 Japanese subjects) and enzyme activity less than 10% of normal (9.1 and 8.3 nmol/h/ml respectively, as compared with normal levels of 100 nmol/h/ml). All obligate heterozygotes examined, including the father of the two LCAT-deficient subjects, and all five children of the deficient subjects had LCAT mass approximately 72-80% of the normal LCAT mass (4.12, 4.38, 4.45, 4.48, 4.49, 4.61 micrograms/ml, respectively) and LCAT activity approximately half normal (51.9, 52.4, 54.2, 56.6, and 57.2 nmol/h/ml). We conclude that the two LCAT-deficient subjects of this family have functionally defective enzyme. Furthermore, the data suggest that the plasma of the obligate heterozygotes contain both normal and functionally defective enzymes.

Evaluation of the measurement of B protein of plasma low density lipoprotein by radial immunodiffusion.

Radial immunodiffusion (RID) has been used for determination of low density lipoprotein (LDL) B protein in plasma. During measurement of B protein in plasma and the d less than and d greater than 1.019 g/ml plasma fractions by RID in 1.0%, 1.5%, 2.0%, and 2.5% agarose, the d less than 1.019 g/ml lipoproteins diffuse in the agarose and produce precipitin rings. Among normotriglyceridemic subjects, the B protein values in whole plasma obtained by RID using 1.5 to 2.5% agarose were only slightly higher than the values in the d greater than 1.019 g/ml fraction obtained by RID and closely approximated the values obtained in the d greater than 1.019 g/ml fraction by radioimmunoassay. However, among the hypertriglyceridemic subjects, the RID measurement of B protein in plasma using 1.0 to 2.5% agarose overestimated the LDL B protein levels in plasma. The RID procedure at agarose concentrations of 1.5% to 2.5% can be used to estimate plasma LDL B protein levels in normotriglyceridemic subjects. However, measurement of LDL B protein by RID in plasma of hypertriglyceridemic subjects must be interpreted with caution; the LDL B protein is overestimated by this procedure because of the contribution by the d less than 1.019 g/ml lipoproteins to the B protein value.

Effects of lyophilization of serum on the measurement of apolipoproteins A-I and B.

A common accuracy-based standardization program is indispensable for establishing reference intervals for the clinical use of apolipoproteins. The development and distribution of reference materials and quality-control materials that do not exhibit matrix effects between methods is essential to the standardization process. We examined the suitability of lyophilized material as a common reference material for the measurement of apolipoproteins A-I and B. We determined values for apolipoproteins A-I and B in frozen and lyophilized serum pools, using different immunochemical approaches. We found little or no differences in apolipoprotein A-I values between frozen and lyophilized pools as determined by the different methods. In contrast, values for apolipoprotein B in lyophilized samples were consistently lower than those obtained for frozen samples. After adjusting for the effect of dilution due to reconstitution, the difference in the apolipoprotein B values for lyophilized as compared with frozen samples ranged from -26% to 4%, depending upon the assay method. Evidently, serum pools in lyophilized from are not a suitable matrix for reference materials for apolipoprotein B measurements but can be used for apolipoprotein A-I measurements.

Screening for lipoprotein[a] elevations in plasma and assessment of size heterogeneity using gradient gel electrophoresis.

Plasma was screened for the presence of lipoprotein[a] using 2-16% nondenaturing, polyacrylamide gradient gel electrophoresis. Gels were scanned with a densitometer after staining with Sudan black B. Bands that migrated above low density lipoprotein bands were identified as lipoprotein[a] by immunoblotting with polyclonal and monoclonal antibodies to apolipoprotein[a]. Lipoprotein[a] was measured by gradient gel electrophoresis and by radioimmunoassay in 115 male patients with premature coronary artery disease and 132 control subjects. Lipoprotein[a] bands were detected in 96.7% of subjects with lipoprotein[a] values above 40 mg/dl; in 31.3% with values between 21 and 40 mg/dl, and in 6.5% with values below 20 mg/dl. This gel methodology is a simple and effective procedure for detecting elevated plasma lipoprotein[a] levels and for investigating size heterogeneity, but does not replace immunoassay for quantitation.

Effects of interleukin-2 (IL-2) on human plasma lipid, lipoprotein, and C-reactive protein.

Six patients with confirmed malignant disease received four consecutive weekly cycles of human recombinant interleukin-2 (IL-2) 4 days/week, continuous iv. infusion, 3 X 10(6) U/m2/day. Plasma cholesterol decreased a mean of 7% within 24 hours after IL-2 infusion and decreased by 33% within 4 days. Plasma cholesterol was significantly lower than baseline concentration by day 21 (-21%), and day 25 (-41%) was significantly lower than day 21. Decreased plasma cholesterol was the result of decreased HDL and LDL cholesterol concentrations. Plasma triglyceride demonstrated a mean increase of 46% after 4 days of therapy and remained greater than baseline concentrations at all time points analyzed. Apolipoprotein AI and AII decreased concomitantly with HDL-cholesterol concentrations, whereas apolipoprotein B after an initial mean decrease of 17% during the first cycle was not significantly different from baseline during the fourth cycle. Apolipoprotein E and Lp(a) were not significantly affected by IL-2 treatment. Plasma C-reactive protein (CRP) increased by 79% within 24 hours of therapy, increased by 254% on day 4, then decreased to baseline concentrations by day 21 after 3 days off of IL-2. Day 25 CRP was elevated compared to both baseline and day 21 concentrations. IL-2 induced plasma lipoprotein changes may be due in part to the induction of interferon gamma.

Complete cDNA encoding human phospholipid transfer protein from human endothelial cells.

Phospholipid transfer protein, with an apparent molecular mass of 81 kDa, was purified from human plasma. The NH2-terminal amino acid sequence of a 51-kDa proteolytic fragment obtained from phospholipid transfer protein allowed degenerate primers to be designed for polymerase chain reaction and the eventual isolation of a full-length cDNA from a human endothelial cDNA library. The cDNA is 1,750 base pairs in length and contains an open reading frame of 1,518 nucleotides encoding a leader of 17 amino acids and a mature protein of 476 residues. Northern blot analysis shows a single mRNA transcript of approximately 1.8 kilobases with a wide tissue distribution. The gene was mapped to chromosome 20 using a human/rodent somatic cell hybrid mapping panel. Phospholipid transfer protein was found to be homologous to human cholesteryl ester transfer protein, human lipopolysaccharide-binding protein, and human neutrophil bactericidal permeability increasing protein (20, 24, and 26% identity, respectively).