Prebeta-1 HDL in plasma of normolipidemic individuals: influences of plasma lipoproteins, age, and gender.

Prebeta-1 HDL is a molecular species of plasma HDL of approximately 67 kDa mass that contains apolipoprotein A-I, phospholipids, and unesterified cholesterol. It participates in a cyclic process involved in the retrieval of cholesterol from peripheral tissues. In this cycle, unesterified cholesterol from cells is incorporated into prebeta-1 HDL, providing a substrate for esterification of cholesterol by lecithin:cholesterol acyltransferase. Prebeta-1 HDL then becomes incorporated into larger HDL species of alpha mobility as esterification proceeds and is regenerated during the transfer of cholesteryl esters from alpha HDL particles to acceptor lipoproteins. Thus the steady state level of prebeta-1 HDL in plasma reflects the relative efficiencies of the major metabolic processes involved in its generation and removal. We have used an isotope dilution technique to measure prebeta-1 HDL levels in the plasmas of 136 normolipidemic individuals (46 M, 90 F). The mean absolute concentration of prebeta-1 HDL as apolipoprotein A-I was 68 +/- 40 microg/ml for women, and 84 +/- 49 m/ml for men. Prebeta-1 HDL represented 5.5 +/- 3.3% of total apolipoprotein A-I in women, and 7.2 +/- 4.0% in men. The distributions of both absolute and percent prebeta-1 HDL are highly asymmetric, with skew toward higher values. However, the skew appears not to be attributable to either plasma cholesterol or triglyceride levels which are also skewed in population samples. The percent prebeta-1 HDL was negatively correlated with HDL cholesterol levels (P < 0.0001), whereas absolute levels of prebeta-1 HDL were positively correlated with apolipoprotein A-I and negatively correlated with HDL cholesterol (P, for both, < 0.0001). Multiple linear regression analysis revealed effects of age and gender, but no association with lipoprotein fractions other than HDL. Lower levels of prebeta-1 HDL were associated with female gender in all models.

Effects of estrus cycle, ovariectomy, and treatment with estrogen, tamoxifen, and progesterone on apolipoprotein(a) gene expression in transgenic mice.

Plasma levels of lipoprotein(a) (Lp(a)), are regulated by the synthetic rate of apolipoprotein(a) (apo(a)), a major protein component of this atherogenic lipoprotein. Exogenously administered sex steroid hormones are potent regulators of plasma Lp(a) concentrations. We utilized a recently developed apo(a) yeast artificial chromosome (YAC) transgenic mouse model to study the effects of ovariectomy, estrus cycle, and exogenous administration of ethinyl-estradiol, the partial estrogen receptor agonist, tamoxifen, and progesterone on circulating apo(a) plasma levels. Analysis of liver RNA revealed that estrogen and tamoxifen exerts their plasma apo(a) lowering effect at the level of apo(a) mRNA. This action of estrogen and tamoxifen may contribute to their antiatherosclerotic and cardiovascular protective effect.

Measurement of prebeta-1 HDL in human plasma by an ultrafiltration-isotope dilution technique.

Prebeta-1 HDL is a 67-kDa species of plasma high-density lipoproteins (HDL) that contains two copies of apolipoprotein A-I. It functions in a metabolic cycle of cholesterol retrieval and may be formed during lipolysis in plasma. We have found that centrifugal ultrafiltration using a membrane with a permeability limit of 100 kDa discriminates categorically between the 67-kDa species and larger HDL particle species. Thus, the ultrafiltrate samples the pool of prebeta-1 HDL in plasma. We have developed a technique using the dispersal of purified prebeta-1 HDL, labeled covalently with tritium, in plasma samples, to label the prebeta-1 HDL pool. Subsequent determination of the specific activity of prebeta-1 HDL in the ultrafiltrate provides a means of calculating the content of prebeta-1 HDL in plasma by the isotope dilution principle. We employ a modification of an enzyme-linked immunosorbent assay technique for apolipoprotein A-I that allows the equal detection of that protein in prebeta-1 HDL and in other HDL particle species for determination of the fraction of total apolipoprotein A-I that is present in the prebeta-1 HDL particle species. The mean level of prebeta-1 HDL-associated apolipoprotein A-I in plasma samples from 86 normolipidemic adults was 74 +/- 43 microg/ml (+/-SD), representing an average of 6.6% of the total apolipoprotein A-I in plasma.

C/T polymorphism in the 5' untranslated region of the apolipoprotein(a) gene introduces an upstream ATG and reduces in vitro translation.

Elevated plasma levels of lipoprotein(a) [Lp(a)] are a significant-independent risk factor for arteriosclerosis. Interindividual levels of Lp(a) vary nearly 1000-fold and are mainly due to inheritance that is linked to the locus of the apolipoprotein(a) [apo(a)] gene. A search was made for sequence variants in the 5' flanking region of the apo(a) gene that affect its expression. A C to T transition at position +93 from the transcription start site was found with a frequency of 14% in the study population. In transient transfection assays in HepG2 cells, luciferase reporter gene constructs with a T at this position were associated with a 58% reduction in luciferase activity compared with the more common allele. This single base variant had no significant effect on the binding of nuclear regulatory proteins; however, it introduced an additional upstream ATG initiation codon with its own in-frame stop codon. Furthermore, equivalent levels of mRNA were produced in HepG2 cells transfected with reporter gene constructs containing either a T or a C at position +93. In vitro translation experiments using transcripts derived from either variant apo(a) promoter revealed a 60% reduction in translation associated with the T allele. Hence, the additional ATG created by the T at position +93 in the 5' flanking region of the apo(a) gene impairs the efficiency of translation from the bona fide ATG initiation codon.

The apolipoprotein C-II variant apoC-IILys19-->Thr is not associated with dyslipidemia in an affected kindred.

The rare apolipoprotein C-II (apoC-II) mutation, apoC-IILys19-->Thr, also known as apoC-II-v, has been found previously in association with hyperlipoproteinemia. From a lipid clinic screening we identified three unrelated individuals who had the apoC-IILys19-->Thr mutation. Among eight family members of one proband, we have found another four who were affected. None of the individuals in this kindred is dyslipidemic and there is no difference in lipid levels between affected and unaffected family members. Therefore, we conclude that the presence of this apolipoprotein variant by itself has no effect on lipoprotein levels. In addition, the apolipoprotein E (apoE) isoform, apoE4 does not have a synergistic effect on lipoprotein levels in this kindred, in contrast to observations on the interaction of apoE4 with another apoC-II mutant (apoC-IIToronto). The single nucleotide substitution-that causes the apoC-IILys19-->Thr variant introduces a previously unrecognized restriction site (for Mae III), that provides for easy screening.

Four new mutations in the apolipoprotein B gene causing hypobetalipoproteinemia, including two different frameshift mutations that yield truncated apolipoprotein B proteins of identical length.

Familial hypobetalipoproteinemia can be caused by mutations in the apolipoprotein (apo)B gene that interfere with the translation of a full-length apoB molecule. Frequently, a truncated apoB molecule can be detected in the plasma lipoproteins of affected subjects. In this report, we characterize four different apoB gene mutations causing hypobetalipoproteinemia that are associated with the synthesis of truncated apoB proteins. Two of the mutations are nonsense mutations caused by single nucleotide substitutions; these mutations are associated with the production of apoB-32.5 (1473 amino acids) and apoB-82 (3733 amino acids). The other two mutations are single nucleotide deletions (of apoB cDNA nucleotides 7295 and 7359, respectively). The altered reading frames created by these different frameshift mutations terminated with the same stop codon, and both therefore yielded a truncated protein of identical size: apoB-52.8 (2395 amino acids). The two apoB-52.8 proteins differ, however, in the number of novel carboxyl-terminal amino acids introduced by the frameshift. The buoyant density of lipoproteins containing the truncated apoBs was inversely related to the length of the truncated apoB. ApoB-32.5 was present only in high density lipoproteins (HDL) and the d > 1.21 g/ml fraction, whereas apoB-82 was present almost exclusively in very low density lipoproteins (VLDL). ApoB-52.8 was present primarily in VLDL, intermediate density lipoproteins (IDL), and low density lipoproteins (LDL); trace amounts were observed in the HDL.

5' control regions of the apolipoprotein(a) gene and members of the related plasminogen gene family.

Elevated blood levels of apolipoprotein(a), the component of lipoprotein(a) that distinguishes it from low density lipoprotein, are a major risk factor for atherosclerosis. The apolipoprotein(a) gene is highly similar to the plasminogen gene and to at least four other genes or pseudogenes. The 5' untranslated and flanking sequences of these six genes contain extensive regions of near identity and share sequence elements involved in the initiation of transcription and translation. About 1000 base pairs of flanking DNA of each gene are sufficient to promote transcription in cultured hepatocytes. The apolipoprotein(a) gene promoter contains functional interleukin 6-responsive elements, consistent with the reported acute-phase response of apolipoprotein(a). Flanking genomic fragments of the apoliprotein(a) gene from two individuals with vastly different plasma apolipoprotein(a) concentrations have sequence differences that are reflected in differences in the rate of in vitro transcription.

Molecular cloning and characteristics of a new apolipoprotein C-II mutant identified in three unrelated individuals with hypercholesterolemia and hypertriglyceridemia.

A new rare mutant form of apolipoprotein C-II (apoC-II), designated apoC-IISF, was identified in three unrelated hyperlipidemic patients. The first was a Caucasian male with a total cholesterol (TC) of 313 mg/dl and total triglyceride (TG) of 282 mg/dl, the second an African-American female (TC 345 mg/dl, TG 203 mg/dl) and the third, an African-American male (TC 345 mg/dl, TG 1000 mg/dl). Each subject was found to be heterozygous for a G to A substitution in the codon for residue 38, resulting in a Lys for Glu exchange. This accounts for the increased pl value of 5.3. The third patient, in addition to apoC-IISF, had apoC-II2, another charge variant. This was determined by DNA sequencing, confirming the Gln for Lys change at residue 55 previously predicted by analysis of peptide fragments in this laboratory. Similar Michaelis constants of activation and activation energies were observed when the ability of apoC-IISF to activate lipoprotein lipase was compared to normal apoC-II. This indicates that major changes in charge around residue 38 lack effect on the activation properties. The variant may be altered in some other property, such as lipid binding, but since the distribution of apoC-IISF revealed no simple co-inheritance with lipid levels, it is unclear to what extent it plays a role in the observed hyperlipidemia. The presence of other factors acting together with the variant may predispose to elevated lipid levels.