Flow-mediated dilation, carotid wall thickness and HDL function in subjects with hyperalphalipoproteinemia.

BACKGROUND AND AIMS: The relationships between very high plasma HDLc and subclinical atherosclerosis are still a matter of debate. METHODS AND RESULTS: Twenty subjects with primary hyperalphalipoproteinemia (HAL, with HDLc in the highest 10th percentile and absence of overt secondary causes of this condition), aged 30-65 years, were compared with 20 age and sex-matched controls. Lipid determination, lipoprotein particle distribution (Lipoprint(®)), Cholesterol Efflux Capacity (CEC), plasma adhesion molecule, analyses of CETP, SRB1 and LIPG genes and of different markers of subclinical vascular disease (ankle-brachial index, ABI; carotid intima-media thickness, cIMT; brachial-artery flow mediated dilation, FMD) were performed. Fasting HDLc levels were 40 mg/dl higher in HAL subjects while LDLc concentration was comparable to control group. CETP gene analysis in HAL subjects identified one novel rare Single Nucleotide Polymorphism (SNP, Asp131Asn), possibly damaging, while the common SNP p.Val422Ile was highly prevalent (50% vs. 27.4% in a control population). No rare mutations associated with HAL were found in SR-B1 and LIPG genes. Polyacrylamide gel electrophoresis in HAL subjects disclosed larger and more buoyant HDL particles than in controls, while LDL profile was much more similar. ABI, cIMT and arterial plaques did not differ in cases and controls and the two groups showed comparable FMD at brachial artery examination. Similarly, ABCA1 and ABCG1 HDL-mediated CEC, the most relevant for atheroprotection, did not discriminate between the groups and only ABCG1 pathway seemed somewhat related to arterial reactivity. CONCLUSIONS: HDL dimension, function and genetics seem scarcely related to subclinical atherosclerosis and vascular reactivity in middle-aged HAL subjects.

Variable phenotypic expression of homozygous familial hypobetalipoproteinaemia due to novel APOB gene mutations.

Homozygous familial hypobetalipoproteinaemia (Ho-FHBL) is a rare co-dominant disorder characterized by extremely low levels of low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (apoB). Most patients with Ho-FHBL have mutations in APOB gene resulting in truncated apoBs. Some patients are asymptomatic, while others have fatty liver, intestinal fat malabsorption and neurological dysfunctions. We investigated three adult subjects with severe hypobetalipoproteinaemia and a family history of FHBL. Proband FHBL-47 had liver cirrhosis with hepatocarcinoma and a renal carcinoma but no clinical manifestations related to FHBL. He was a compound heterozygote for a 7-bp deletion in exon 21 and a base insertion in exon 26 resulting in truncated apoBs (apoB-22.46/apoB-66.51). Proband FHBL-53, with severe hepatic steatosis and fibrosis, had a nonsense mutation in exon 19 resulting in a truncated apoB (apoB-20.61) and a rare nucleotide substitution in intron 14 (c.2068-4T>A). The latter was also present in her daughter, found to have low plasma LDL-C and apoB. Proband FHBL-82 had chronic diarrhoea and steatorrhoea. She was found to be homozygous for a nonsense mutation in exon 24 resulting in a truncated apoB (apoB-26.65). In adult subjects, the presence of chronic liver disease and chronic diarrhoea, when associated with severe hypobetalipoproteinaemia, may lead to the diagnosis of Ho-FHBL.

Changes in apolipoprotein A-I mRNA level in the liver of rats with experimental nephrotic syndrome.

In previous studies we had shown that: one of the most specific feature of hyperlipoproteinemia found in rats with experimental nephrotic syndrome is the accumulation of apolipoprotein A-I-rich HDL in plasma and this disorder is associated with an overproduction of apolipoprotein A-I by the liver. The present study was designed to investigate whether the increased hepatic synthesis of apolipoprotein A-I was due to an accumulation of functionally active apolipoprotein A-I mRNA in liver of nephrotic rats. Hepatic mRNA was translated in vitro by rabbit reticulocyte lysate in the presence of [35S]methionine and in vitro synthesized apolipoprotein A-I, albumin and apolipoprotein E were immunoprecipitated by specific rabbit IgG. In nephrotic rats the amount of in vitro synthesized apolipoprotein A-I was almost twice that found in the controls, suggesting that functionally active apolipoprotein A-I mRNA was increased in liver of nephrotic rats. To confirm that this difference in apolipoprotein A-I mRNA activity was due to an actual increase of hepatic apolipoprotein A-I mRNA sequences, we performed nucleic acid hybridization experiments (northern blot) using several cloned cDNA probes (rat and human apolipoprotein A-I, rat apolipoprotein E and apolipoprotein A-II). The results indicate that in nephrotic rats the amount of hybridizable apolipoprotein A-I mRNA sequences was about 3-fold higher than that in controls. In contrast, there was no difference in the amount of hybridizable apolipoprotein A-II and apolipoprotein E mRNA sequences, indicating that the change in apolipoprotein A-I mRNA induced by the nephrotic state was specific for this mRNA.

Isolation of a cDNA clone for chick intestinal apolipoprotein AI (Apo-AI) and its use for detecting apo-AI mRNA expression in several chick tissues.

Three cDNA clones for chick apolipoprotein AI (Apo-AI), the major protein component of plasma high-density lipoproteins, have been isolated. The identity of the clones has been established first by screening a cDNA library in the pEX1 expression vector with anti-Apo-AI antibodies, second by Western blot analysis of the proteins expressed by positive clones. The use of the clone containing the largest, presumably full-size, cDNA insert (apo5C12) in molecular hybridization experiments confirms that apo-AI mRNA is expressed mainly in chick small intestine and liver. Furthermore, we provide evidence that brain, heart and skeletal muscle also synthesize significant amounts of apo-AI mRNA. The Southern-blot hybridization pattern of the restriction-enzyme-digested chick DNA with the apo5C12 DNA is consistent with there being a single copy of the apo-AI gene.

The complete sequence of chick apolipoprotein AI mRNA and its expression in the developing chick.

The nucleotide (nt) sequence analysis of a full-length cDNA for chick apolipoprotein AI (Apo-AI) shows an open reading frame (ORF) of 792 nt, coding for a 264-aa protein. RNase mapping and sequence analysis of the 3' end show that apo-AI mRNA consists of at least two different species of 985 and 996 nt, respectively. During the embryonic life of the chick apo-AI mRNA is found in high concentration only in the liver, while its level in the intestine, the major Apo-AI producing organ in the adult, becomes significant only after hatching. This switch from liver to intestine, as primary site of apo-AI mRNA synthesis, takes place about ten days after hatching. The developmental control of the tissue levels of apo-AI mRNA is particularly evident in the skeletal muscle, where this mRNA species is present at high level only immediately after hatching. Preliminary evidence suggests that the time-limited rise in muscle apo-AI mRNA might be due to an increased rate of transcription.

The protective effect on Cu2+- and AAPH-mediated oxidation of human low-density lipoproteins depends on glycosaminoglycan structure.

The effect of various glycosaminoglycans on Cu(2+)- and AAPH-induced oxidation of human low-density lipoprotein (LDL) was studied by monitoring conjugated diene formation. Heparin (Hep) increased the lag phase (t(lag)) of LDL oxidation, and fast moving and slow moving Hep species modified the kinetics of LDL oxidation to the same extent. Beef spleen heparan sulfate (HS) sample produced a significant increase of the t(lag) and a decrease of the conjugated diene formation of LDL whilst beef kidney HS species modified LDL oxidation kinetics to a lower extent. Dermatan sulfate (DS) from different sources caused a significant increase of the t(lag) and a decrease of the conjugated diene formation of LDL. Hyaluronic acid had no effect. Chondroitin sulfate (CS) from beef trachea produced a very strong protective antioxidant effect evaluated by increasing of the t(lag) and decreasing of the conjugated diene formation. Hep was completely ineffective in protecting LDL from 2, 2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation, whilst DS was moderately effective. Beef trachea CS showed a very strong ability to protect LDL oxidation induced by 1 mM AAPH. The different protective effect on Cu(2+)- and AAPH-induced LDL oxidation by glycosaminoglycans is discussed considering their various structures and properties, and their capacity to interact to different extents with hydrophobic regions of LDL protein is confirmed by measuring the LDL-tryptophan fluorescence kinetics.

Separation of the isoprotein forms of apoprotein A-I of rat, rabbit and human HDL by combined isoelectrofocusing and SDS-polyacrylamide gel electrophoresis.

The distribution and the relative content of the isoprotein forms (isoforms) of apoprotein A-I (apo A-I) of HDL isolated from rat, rabbit and human plasma were studied by combined isoelectrofocusing and SDS-polyacrylamide gel electrophoresis. Rat apo A-I consists of seven isoforms having the same molecular weight (27,000) and moving in the 6.44-5.58 pH range. Isoforms 4, 5 and 6 are the major ones. Both rat HDL2 (1.090-1.210 g/ml) and purified rat apo A-I contain additional minor bands (designated 4a, 5a and 6a) which have the same isoelectric point as isoforms 4-6 but higher molecular weight (27,900). It is suggested that they might represent precursors of the main apo A-I isoforms. Rabbit apo A-I contains five isoforms focusing in the 5.69-5.34 pH range. Isoform 4 accounts for about 90% of apo A-I mass. Human apo A-I consists of five isoforms focusing in the pH range 5.91-5.0. Isoforms 3 and 4 are the main ones: their respective contents show high degrees of individual variation.

Isoforms of rat apolipoprotein A-I isolated from the lipoproteins of hepatic Golgi apparatus and plasma.

We compared apo A-I isolated from the lipoproteins of the Golgi apparatus of rat liver with apo A-I found in plasma lipoproteins. Golgi apo A-I consists of 3 main isoforms with a molecular weight of approximately 28000 and isoelectric points (pI) of 5.97, 5.88 and 5.76, respectively. Plasma apo A-I consists of 4 major and 3 minor isoforms with a molecular weight of 27000. The pI of the major isoforms (numbered 4-7) is 5.88, 5.80, 5.70 and 5.60, respectively. In order to investigate which of the plasma isoforms derived directly from Golgi apo A-I, [35S]methionine was injected into the portal vein and Golgi and plasma apo A-I were isolated shortly thereafter. While all Golgi isoforms were labelled only 3 isoforms of plasma apo A-I (namely isoforms 5, 6 and 7) were found to be labelled. The major plasma isoform (isoform 4 which accounts for more than 60% of apo A-I mass of plasma HDL) was found to be unlabelled. However, when 35S plasma lipoproteins newly secreted by the liver were incubated in vitro in the presence of heparinized plasma, labelled isoform 4 appeared suggesting that heparinized plasma contained some factor capable of converting isoforms 5-7 into isoform 4. This plasma factor appears to be a protease as the in vitro formation of isoform 4 is prevented by protease inhibitors.

Heavy metals and experimental atherosclerosis. Effect of lead intoxication on rabbit plasma lipoproteins.

Previous studies have suggested that exposure to heavy metals may be a risk factor in coronary atherosclerotic heart disease in humans as well as in experimental animals. Little is known however on the mechanism underlying the effect of heavy metals on the development of atherosclerosis. In this study we tried to ascertain whether exposure to lead might: (a) alter plasma lipoprotein in normally fed rabbits; and (b) aggravate the hyperlipidemia usually found in cholesterol-fed animals. Rabbits were fed a normal diet or a diet containing 1% cholesterol in the presence or in the absence of 0.5% of lead subacetate for 45 days. This produced an accumulation of lead in plasma and bone. While in cholesterol-fed rabbits, lead exposure did not modify the plasma lipoprotein pattern, in normally fed animals it induced a striking elevation of cholesterol esters. This was associated with an increased concentration of VLDL (1.006 g/ml), LDL1 (1.006-1.020 g/ml), LDL2 (1.020-1.050 g/ml) and HDL1 (1.050-1.210 g/ml). These lipoproteins had an elevated content of cholesterol esters and apolipoprotein B. It is suggested that some of these lipoproteins may be important in the development of atherosclerosis in subjects chronically exposed to lead.

Influence of chondroitin sulfate charge density, sulfate group position, and molecular mass on Cu2+-mediated oxidation of human low-density lipoproteins: effect of normal human plasma-derived chondroitin sulfate.

The effects of chondroitin sulfate samples with decreasing charge densities, different 4-sulfate/6-sulfate ratios, and various molecular masses on Cu2+-induced oxidation of human low-density lipoprotein (LDL) were evaluated by monitoring conjugated diene formation and the tryptophan fluorescence kinetics. Low-sulfated chondroitin sulfate (CS) from beef trachea had a very strong protective antioxidant effect. Quite similar behavior was observed for CS from pig trachea, and a fructose-containing polysaccharide with a chondroitin backbone from Escherichia coli was also strongly protective as to LDL oxidation. CS samples with decreasing charge densities proved effective in inhibiting LDL oxidation. A totally desulfated sample still exhibited a great capacity to protect LDL against oxidation. CS-4-sulfate samples (sulfate to carboxyl ratio of 0.62, about 65% 4-sulfate groups and 5% 6-sulfate groups) retained great ability to inhibit the Cu2+-mediated human LDL oxidation. CS fractions with different molecular masses were examined as possible inhibitors of LDL oxidation. Samples with molecular masses lower than about 8,570 (13-15 disaccharide units) were unable to protect human LDL from Cu2+-induced oxidation. Similar results were obtained on studying the degradation of tryptophan residues of the LDL protein moiety resulting from Cu2+ complexation through amino acid residues. A low-sulfated CS (sulfate to carboxyl ratio of 0.41, a molecular mass of about 15,600) having effective anti-oxidant properties as to metal-induced LDL oxidation was isolated from normal human plasma. The protective capacity as to Cu2+-mediated LDL oxidation of CS is discussed in relation to its structure, also considering the physiological role of plasma CS in relation to factors that can alter its properties.

Cholesterol synthesis in isolated rat hepatocytes: effect of homologous and heterologous serum lipoproteins.

The present study investigated the effect of serum lipoproteins on sterol synthesis by isolated rat hepatocytes. These cells were maintained in culture medium for 24 hr and incubated for the same period of time with increasing concentrations of serum lipoproteins (5-150 microgram of lipoprotein-protein per ml) isolated from different animal species. The viability of the cells was ascertained by their ability to synthesize cholesterol and protein and to secrete serum proteins into the medium. Rat VLDL and LDL did not alter sterol synthesis, which was stimulated instead by HDL. Rat serum chylomicrons were also ineffective. Human LDL significantly reduced the synthesis of sterols from both acetate and tritiated water; this effect was also induced by human VLDL to a reduced extent. VLDL isolated from hypercholesterolemic rabbit (VLDLC) strongly inhibited sterol synthesis from acetate but not from mevalonate. Cholesteryl-ester-rich VLDL isolated from a patient with type III hyperlipidemia (type III VLDL) were more effective than normal VLDL in suppressing sterol synthesis from acetate. The implications of these findings are discussed with regard to the possible role of cholesteryl-ester-rich lipoproteins on the in vivo regulation of sterol synthesis in the liver.

Familial hypobetalipoproteinemia: analysis of three Spanish cases with two new mutations in the APOB gene.

Extremely low LDL-cholesterol concentrations are very unusual and generally related with comorbidities accompanying malnutrition. Less frequently low LDL-cholesterol levels result from mutations in the APOB, PCSK9, ANGPTL3, SAR1B and MTTP genes (primary hypobetalipoproteinemia). We investigated three patients with plasma LDL-cholesterol levels below the fifth percentile of the Spanish population. We recorded data on demographic and anthropometric characteristics, life style habits, physical examination, liver ultrasound and lipid and lipoprotein levels, in the probands and their first-degree relatives. Secondary causes of hypocholesterolemia were ruled out by clinical study, complementary tests and follow-up. The APOB, MTTP and SAR1B genes were sequenced. Patients were found to be heterozygotes for point mutations located in the exon 26 of the APOB gene. One patient, with fatty liver, carried a previously described mutation (c.7600C>T) (Arg2507X), causing the formation of truncated Apo B-55.25. The other two mutations producing truncations are new. One asymptomatic patient carried the Arg3672X (Apo B-80.93) and the other with fatty liver and steatorrhea carried the Ser2184fsVal2193X (Apo B-48.32). Our study reinforces the concept that in the heterozygous carriers of truncated Apo Bs, the clinical manifestations of FHBL are dependent on the size of the truncations.

Lung involvement in Niemann-Pick disease type C1: improvement with bronchoalveolar lavage.

Progressive lung infiltration is a major cause of death in Niemann-Pick disease type A and B (NPA, NPB) and in the recently defined type C2. In type C1 (NPC1), the main manifestations are neurological. We report a patient with a classic, neurological, late infantile form of NPC1 disease, carrying the mutation P474L and the variant I642M in the NPC1 gene, who suffered recurrent respiratory manifestations. Bronchoalveolar lavage of a lung segment due to deteriorating respiratory condition revealed many foamy macrophages and was followed by an improvement in symptoms. Pneumopathy may therefore be considered a feature of NPC1 disease for which a partial bronchoalveolar lavage could be a useful treatment.

Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma.

We report a very rapid and simple isocratic reversed-phase HPLC separation of malondialdehyde (MDA) in normal human plasma without previous purification of the MDA-2-thiobarbituric acid (TBA) complex. The separation of MDA-TBA complex was performed using a 250x4.6 mm Nucleosil-5C18 column with a mobile phase composed of 35% methanol and 65% 50 mM sodium phosphate buffer, pH 7.0. Samples of 50 microl (composed of 100 microl plasma mixed with 1.0 ml of 0.2% 2-thiobarbituric acid in 2 M sodium acetate buffer containing 1 mM diethylenetriaminepentaacetic acid, pH 3.5, and 10 microl of 5% 2,6-di-tert.-butyl-4-methylphenol in 96% ethanol, incubated at 95 degrees C for 45 min [K. Fukunaga, K. Takama and T. Suzuki, Anal. Biochem., 230 (1995) 20] were injected into the column. The MDA-TBA complex was eluted at a flow-rate of 1 ml/min and monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm. Analysis of groups of normal male and female volunteers gave plasma levels of MDA of 1.076 nmol/ml with a coefficient of variation of about 58%. No significant statistical differences were found between male and female groups, and no correlation was discovered on the age.

Sequential expression during postnatal development of specific markers of junctional and free sarcoplasmic reticulum in chicken pectoralis muscle.

Skeletal muscle sarcoplasmic reticulum comprises two distinct membrane domains, i.e., the Ca(2+)-pump membrane, corresponding mainly to longitudinal tubules, and the junctional membrane of the terminal cisternae containing the ryanodine receptor/Ca(2+)-release channel. Additional minor proteins previously shown in rabbit fast-twitch skeletal muscle to fractionate selectively to each membrane domain comprise 160- and 53-kDa glycoproteins and 170-kDa low-density lipoprotein (LDL)-binding protein, respectively (Damiani and Margreth, 1991, Biochem. J. 277, 825-832). We report evidence in chicken pectoralis, a predominantly fast muscle, on two closely immunologically related glycoproteins, a minor component of 130-kDa and a major 53-kDa protein. In contrast to the seemingly highly conserved structure of this protein, our results show marked differences in mobilities for chicken 125I-LDL that were detected as a 130- to 116-kDa protein doublet after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, although being otherwise indistinguishable from rabbit 170-kDa protein in LDL-binding characteristics, as well as for preferential association to junctional terminal cisternae. Chicken Ca(2+)-ATPase, although being extensively homologous to rabbit Ca(2+)-ATPase, is shown to be less active and to differ slightly in electrophoretic properties. We have investigated the time course of expression of the specific protein components of longitudinal and of junctional sarcoplasmic reticulum in chick pectoralis muscle from late embryonic development up to 2 months after hatching. Coincident with the posthatching increase in membrane density of high-affinity [3H]ryanodine-binding sites in muscle, both calsequestrin and the species-specific LDL-binding protein(s) are detected in increasing amounts, using ligand blot techniques. In contrast, the appearance and steady accumulation in muscle of Ca(2+)-ATPase, like the time-correlated increase of sarcoplasmic reticulum glycoproteins, are relatively delayed, the most striking changes occurring from 1 week after hatching onward. The sequential expression in chick developing muscle of proteins selectively associated with the junctional terminal cisternae and with longitudinal sarcoplasmic reticulum, respectively, argues for a similar morphogenetic program in avian and mammalian species and, to account for that, for the existence of common epigenetic differentiating influences on the expression of sarcoplasmic reticulum protein genes.

Absence of apolipoprotein B-48 in the chick, Gallus domesticus.

This study was designed to investigate: a) whether multiple forms of apoB are present in chick plasma lipoproteins; and b) which forms of apoB are produced in vitro by liver and intestine at various stages of pre- and post-natal development. Plasma lipoproteins of d less than 1.019 g/ml, isolated from fasted and nonfasted chicks, contained exclusively the high molecular weight apoB form (apoB-100) that comigrated with human and rat apoB-100 on SDS-PAGE gel. No apoB-48 was detected either in overloaded Coomassie blue-stained gels or after immunoblotting. ApoB-100 but no apoB-48 was found in portomicrons, the triglyceride-rich lipoproteins equivalent to chylomicrons, that in the chick are transported via the porto-mesenteric venous system. To ascertain whether a minute amount of apoB-48 was present in chick plasma, [35S]methionine was injected intraduodenally and the 35S-labeled d less than 1.019 g/ml plasma lipoproteins were isolated 45 min later from the systemic and the porto-mesenteric circulation. Only apoB-100 was found to be labeled in these lipoproteins. Cholesterol feeding did not induce the appearance of apoB-48 in plasma despite a marked accumulation of cholesterol-rich d less than 1.040 g/ml lipoproteins in the plasma. In vitro synthesis of apoB forms was studied in liver and intestinal slices isolated from chick embryos (8 and 5 days before hatching), newly hatched chicks (2 and 7 days after hatching), and young chicks (21 days old) that were incubated in the presence of [35S]methionine. At each stage of development, liver slices secreted predominantly apoB-100. Intestinal slices of newly hatched and young chicks secreted two forms of apoB: apoB-100 and an additional form with an electrophoretic mobility similar to rat plasma apoB-95. No apoB-48 was synthesized or secreted by the intestine. Our results indicate that the absence of apoB-48 in chick plasma reflects the lack of synthesis of this peptide in the intestine. It is conceivable that in chick intestine the recently described molecular mechanism responsible for the co/posttranscriptional modification of apoB mRNA leading to the formation of apoB-48 is lacking or defective.

Cholesterol synthesis in freshly isolated human leukocytes.

Cholesterol synthesis has been studied in human leukocytes shortly after the isolation from healthy subjects. Not delipidated human serum reduced the cholesterol synthesis when added to the incubation medium. A similar effect was obtained when the leukocytes were incubated in the presence of physiological concentrations of low density lipoproteins.

Plasma and urine lipoproteins during the development of nephrotic syndrome induced in the rat by adriamycin.

The changes of plasma lipoproteins which occur during the development of nephrotic syndrome induced in the rat were investigated by the administration of the antineoplastic drug adriamycin. Rats received a single intravenous injection of the drug (7.5 mg/Kg) and were sacrificed 5, 10, 15, 20, 25, and 30 days after treatment. By monitoring plasma and urine albumin, four stages in the development of nephrosis were identified: (1) a prenephrotic stage, (2) a mild nephrosis with moderate albuminuria and hypoalbuminemia; (3) a severe nephrosis with massive albuminuria and severe hypoalbuminemia; and (4) a recovery stage in which plasma albumin showed the tendency to increase. Apart from a mild elevation of plasma triacylglycerols and VLDL observed as early as Day 5, no changes in plasma cholesterol and in the other lipoprotein classes were observed at the stage of mild nephrosis (Day 10). However, as the disease became more severe (Day 15-25) there was a striking increase of HDL1 (1.050-1.090 g/ml) and, above all, of HDL2 (1.090-1.210 g/ml). VLDL and LDL also increased but at a later stage. The elevation of HDL1 and HDL2 was associated with an increase of apolipoprotein A-I in plasma (fourfold increase). Moreover, the relative content of this apolipoprotein in HDL1 and HDL2 increased as the disease progressed from mild to severe, so that in severely nephrotic rats HDL1 and HDL2 contained almost exclusively A-I and C apolipoproteins. HDL enriched in apolipoprotein A-I were also found in urine of severely nephrotic animals. Since these findings are similar to those previously described in nephrotic syndrome induced by puromycin aminonucleoside (Gherardi, E., and Calandra, S. (1982). Biochim. Biophys. Acta 710, 188.) the following conclusions can be drawn: (1) the key signs of nephrotic syndrome (albuminuria and hypoalbuminemia) precede the elevation of plasma lipoproteins; (2) the pattern of nephrotic hyperlipoproteinemia evolves as a function of the severity of the disease; (3) the accumulation of HDL enriched in apolipoprotein A-I represents an early and specific feature of nephrotic hyperlipoproteinemia in the rat.

Synthesis and secretion of apolipoprotein A-I by chick skin.

Chick skin slices were incubated with [35S]methionine and labeled apoA-I was immunoprecipitated from incubation medium and tissue homogenate. ApoA-I accounted for approximately 13 and 2.5% of radioactive medium and cell proteins, respectively. After ultracentrifugation of the medium, 55% of labeled apoA-I was found as a constituent of lipoproteins (d less than 1.210 g/ml) and 45% in a lipid-poor form (1.210-1.260 g/ml). To ascertain whether this large proportion of lipid-poor apoA-I was due to a dissociation of this peptide from medium lipoproteins during ultracentrifugation, labeled incubation medium was applied to an anti-chick apoA-I immunoaffinity column. The material bound to the column was analyzed by nondenaturing polyacrylamide gradient gel electrophoresis and found to contain three subpopulations of lipoproteins with a particle size of 12, 11, and 9 nm, respectively. The radioactivity of these subpopulations accounted for 82% of total radioactive medium apoA-I. ApoA-I was localized by immunohistochemistry in the viable cells of the epidermis and in the stratum corneum. Rat skin slices were found to synthesize and secrete apoE but no apoA-I. ApoA-I and apoE secreted by chick and rat skin, respectively, may play a role in the secretion of lipids from the differentiating keratinocytes and thus contribute to the formation of the hydrophobic barrier of the skin.