Isopycnic Ultracentrifugation Research Articles

Characterization and Immunohistochemical Localization of the 15 kD Protein Isolated from Rat Lung Lamellar Bodies

We have characterized a protein of approximately 15 kD (lb15) derived from rat lung lamellar bodies, and then sequenced the first 42 residues. Following the normal isopycnic sucrose gradient ultracentrifugation, we diluted the band containing the crude lamellar body fraction with an equal volume of cold distilled water and further centrifuged it at 2,000 x g for 30 min to pellet a fraction of lamellar bodies. Under the electron microscope, this fraction appeared intact and highly purified. When this fraction was subjected to polyacrylamide gel electrophoresis, the major protein was one of 15 kD, regardless of whether the fraction was extracted or unextracted, reduced or unreduced; only a small amount of 35 kD protein was detected with Coomassie Blue staining. Disruption of lamellar bodies revealed that the limiting membrane was particularly enriched with lb15. Immunohistochemistry indicated that lb15 was present in lamellar bodies and tubular myelin, suggesting it was secreted along with the lipid. Amino acid analysis revealed a protein with 13.5% basic and 10.6% acidic residues. The N-terminal appeared particularly highly charged, with 32% of the charged residues in the first 14 amino acids. The lb15 protein is identical to rat lysozyme for the first 23 residues, with the important exception of residue 6, which is histidine in lb15 and cysteine in lysozyme. Residue 24 was not identified. Lb15 was also present in lavage material. We conclude that lb15 is the major protein in rat lung lamellar bodies, has a highly charged N-terminal, and shares some sequence homology with rat lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence and subcellular localization of tyrosine aminotransferase and p-hydroxyphenyllactate dehydrogenase in epimastigotes of Trypanosoma cruzi.

Cell-free extracts of epimastigotes of Trypanosoma cruzi contain tyrosine aminotransferase (TAT) and p-hydroxyphenyllactate dehydrogenase (pHPLDH). The TAT activity could be separated from aspartate aminotransferase (ASAT) by polyacrylamide gel electrophoresis or DEAE-cellulose chromatography; the latter procedure also allowed complete separation of pHPLDH. The subcellular localization of both T. cruzi enzymes, as determined by digitonin extraction, subcellular fractionation by differential centrifugation, and isopycnic ultracentrifugation in sucrose gradients, was mainly cytosolic, with low mitochondrial activities.

Comparison of the cytolytic effects in vitro on Trypanosoma brucei brucei of plasma, high density lipoproteins, and apolipoprotein A-I from hosts both susceptible (cattle and sheep) and resistant (human and baboon) to infection.

The African trypanosome, Trypanosoma brucei brucei causes a fatal wasting disease in livestock but does not ordinarily infect humans, apparently because this unicellular parasite is lysed by high density lipoproteins (HDL) in human serum. To assess whether there is a specific active constituent in trypanolytic HDL, we have systematically compared the cytotoxic action on T.b.brucei in vitro of native and delipidated HDL, and of individual apolipoproteins, from nonpermissive hosts (human and baboon) with their counterparts from susceptible hosts (cattle and sheep). When suspensions of trypanosomes were incubated for 2 h at 37 degrees C with human or baboon plasma most cells were lysed, but not with bovine or sheep plasma. Similarly, HDL isolated from human and baboon plasma were trypanolytic (typically about 95% and 60% lysis, respectively, at 1 mg protein/ml), whereas bovine and sheep HDL were benign (less than 8% lysis). Subfractionation of human HDL by serial isopycnic ultracentrifugation and by heparin-Sepharose affinity chromatography established that the denser and smaller particles had greater trypanolytic activity both in vitro and in vivo. When human HDL was delipidated, the trypanocidal activity was associated with the water-soluble protein (apolipoprotein) fraction and not with the lipid constituents. Bovine apolipoproteins were also weakly trypanolytic in free solution (20-40% lysis), but not when complexed with cholesterol-phospholipid liposomes (less than 10% lysis). The major apolipoprotein of human HDL, apolipoprotein (apo) A-I had full trypanolytic activity (89-95% lysis at 1 mg protein/ml) when purified, whether in solution or incorporated into liposomes, but other apolipoproteins isolated from human HDL, including apoA-II, apoC, and apoE, were nontrypanolytic. Purified baboon apoA-I was also trypanolytic, though less potent than human apoA-I, but apoA-I from permissive hosts (cattle and sheep) was inactive when presented in liposomes. Incubation of bovine or sheep HDL with purified human apoA-I, and subsequent separation of the HDL by ultracentrifugation, produced chimeric HDL containing significant amounts of the human apolipoprotein; these particles showed appreciable trypanolytic activity. By contrast, human HDL particles in which about 70% of the apoA-I had been displaced with apoA-II had markedly reduced lytic properties compared to the native HDL (30% versus 80% lysis at 0.6 mg total protein/ml). We tentatively conclude that the trypanolytic activity of native human or baboon plasma resides in the apoA-I content of the HDL particles and that, conversely, bovine and sheep plasma are inactive because the apoA-I polypeptide present in their HDL lacks trypanocidal activity.

Subcellular localization of glutamate dehydrogenases and alanine aminotransferase in epimastigotes ofTrypanosoma cruzi

The subcellular localization of NAD- and NADP-linked glutamate dehydrogenases (GDH-NAD and GDH-NADP), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) in epimastigotes of Trypanosoma cruzi was studied by digitonin extraction from whole cells, subcellular fractionation by differential centrifugation. A and isopycnic ultracentrifugation. All enzymes presented both a cytosolic and a mitochondrial form; in addition, GDH-NADP seems to have a third, still undefined, localization. The results are compatible with the existence of two pathways for the production of l-alanine linked to the reoxidation of glycolytic NADH, one operative in the mitochondrion and the other in the cytosol, and perhaps responsible for the existence of the two alanine pools detected by 13C-nuclear magnetic resonance (B. Frydman et al., Eur. J. Biocbem. 192 (1990) 363–368).

Isopycnic ultracentrifugation: A simple and inexpensive demonstration of its princples

This demonstration allows one to visualize density gradient separations and to understand the differences between the principles of equilibrium and sedimentation velocity techniques.

Rapid isolation of vesicular and micellar carriers of biliary lipids by ultracentrifugation.

A simple, rapid, and new method has been developed to isolate and to quantitate the vesicular carrier of biliary lipids by isopycnic ultracentrifugation. The method combines the use of Metrizamide, as an inert centrifugation media to change the density of bile for isopycnic separation of vesicles, and a vertical rotor, to decrease both the time of centrifugation and the pressure of the hydrostatic column in the ultracentrifuge tube. Vesicles harvested from bile-Metrizamide density gradients were identified by negative staining electron microscopy. The buoyant densitites of biliary vesicles varied between 1.010 and 1.030 g/ml. The diameter of vesicles in fractions with d less than 1.020 g/ml was 82 +/- 10 nm and in fraction with d approximately 1.030 g/ml was 57 +/- 8 nm. Gel filtration chromatography with Ultrogel AcA 34 was used to validate the quantitive isolation of vesicles by the ultracentrifugal method. In experiments with bile-Metrizamide continuous preformed density gradients, greater than 93% of vesicular cholesterol was found in fractions with d less than 1.030 g/ml after 285 min of centrifugation at 50,000 rpm in a VTi vertical rotor (Beckman Instruments, Inc.). When 16% Metrizamide was dissolved in bile and centrifuged for 120 min, greater than 96% of total vesicular cholesterol was found in the top 0.4 ml of the 5-ml centrifuge tube, as assessed by gel filtration chromatography. This fraction contained less than 8% of cholesterol carried in micelles, as assessed by gel filtration chromatography. The variation coefficient of this short ultracentrifugal method to isolate biliary vesicles was 4.6%.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of lipid-binding regions in human apolipoprotein B-100.

The distribution of lipid-binding regions of human apolipoprotein B-100 has been investigated by recombining proteolytic fragments of B-100 with lipids and characterizing the lipid-bound fragments by peptide mapping, amino acid sequencing, and immunoblotting. Fragments of B-100 were generated by digestion of low-density lipoproteins (LDL) in the presence of sodium decyl sulfate with either Staphylococcus aureus V8 protease, pancreatic elastase, or chymotrypsin. Particles with electron microscopic appearance of native lipoproteins formed spontaneously when detergent was removed by dialysis from enzyme digests containing fragments of B-100 and endogenous lipids, or from incubation mixtures of delipidated B-100 fragments mixed with microemulsions of exogenous lipids (cholesteryl oleate and egg phosphatidylcholine). Fractionation of the recombinant particles by isopycnic or density gradient ultracentrifugation yielded complexes similar to native LDL with respect to shape, diameter, electrophoretic mobility, and surface and core compositions. Circular dichroic spectra of these particles showed helicity similar to LDL but a somewhat decreased content of beta-structure. Most of the fragments of B-100 were capable of binding to lipids; 12 were identified by direct sequence analysis and 14 by reaction with antisera against specific sequences within B-100. Our results indicate that lipid-binding regions of B-100 are widely distributed within the protein molecule and that proteolytic fragments derived from B-100 can reassociate in vitro with lipids to form LDL-like particles.

Purification and characterization of pre-S-containing hepatitis B surface antigens produced in recombinant mammalian cell culture.

Heterogeneous, pre-S-rich HBsAg particles were expressed in recombinant mammalian cell culture and purified to near homogeneity. The purification process comprises: concentration of cell culture medium, protein precipitation by poly(ethylene glycol), gel filtration column chromatography, isopycnic ultracentrifugation by KBr and sucrose density gradient ultracentrifugation. The resulting HBsAg product was greater than 98% pure, and contained much of pre-S1 and pre-S2 components. Scanning densitometry analysis of the silver-stained HBsAg product showed approximately 70-80% S protein, approximately 10-20% pre-S2 protein, and approximately 5-15% pre-S1 protein. It was estimated that the amount of HBV-specific DNA present the final product was less than 7 pg mg-1 HBsAg. Further biochemical analysis has demonstrated that the HBsAg particles are very heterogeneous in charge and density. Charge heterogeneity was quite random among the particles, but density heterogeneity could be related to the different amounts of pre-S2 component in the particles.

Purity, antigenicity and immunogenicity of the hepatitis B surface antigen purified by five different methods

Five published methods for the purification of HBsAg from plasma were compared for specific activity (SA), degree of purification, and yield. The SA value was determined by dividing the reciprocal of the end point dilution per milliliter as determined using a commercial radioimmunoassay (AUSRIA II; Abbott Laboratories, North Chicago, IL) by the protein concentration quantitated by the Lowry method. HBsAg purified by two consecutive isopycnic ultracentrifugation separations in KBr and one rate-zonal separation in sucrose using a zonal rotor (Ti-14, Beckman, Palo Alto, CA) yielded a preparation which gave the highest SA value, degree of purification and yield as compared to four other methods. Each purified preparation was adsorbed to alum adjuvant and injected into mice to determine the immunogenic dose at which 50% of the animals elicited an anti-HBs response (ID 50). The zonal rotor method resulted in the lowest ID 50 value (365 ng/ml) supporting the highest SA value. Furthermore, SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis revealed that this preparation had the greatest number of HBsAg-specific polypeptides ( N = 7) and the fewest contaminating polypeptides ( N = 5). The contaminating proteins were identified as alpha-2-macroglobulin, heavy chains of IgG and IgM, immunoglobulin kappa chain, and albumin.

Electrophoretic and immunoblot analysis of Cryptosporidium oocysts.

Cryptosporidium oocysts were recovered by density gradient centrifugation from diarrhoeal faeces of four human patients and one goat kid. Goat-derived oocysts were further treated with excystation medium and the excysted oocyst walls purified by isopycnic ultracentrifugation. Soluble extracts from intact oocysts and the oocyst wall preparation were analysed by SDS-PAGE. Fifty-one polypeptide bands were detected in intact oocyst preparations: 48 were in the range 14,000-200,000 molecular weight (MW), two bands were less than 14,000 MW and one band was above 200,000 MW. Twenty-one bands were detected in the oocyst wall preparation, all within the range 14,000-200,000 MW. Immunoblot analysis of Cryptosporidium polypeptides using acute or convalescent human and goat sera revealed a large number of reactive bands. Varying degrees of heterogeneity were observed within and between the two serum groups. Nine of the 10 human sera and all of the goat kid sera reacted with a 23,000 MW and 32,000 MW antigen. A 15,500 MW antigen was also detected by all the goat and four of the 10 human sera. Both serum groups reacted with various antigens above 40,000 MW. Surface labelling of three human isolates of Cryptosporidium oocysts with 125I was performed using the Bolton and Hunter reagent. The solubilized preparations were separated by SDS-PAGE on 12% and 18% slab gels and autoradiographed. Common bands were seen at 15,500, 32,000, 47,500, 79,000 and 96,000 MW. Some variation in the molecular weight of polypeptides labelled with 125I was observed among the three isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and separation of cysts and epimastigotes of Blastocrithidia triatomae Cerisola et al., 1971 (Trypanosomatidae)

Developmental stages of the flagellate Blastocrithidia triatomae were isolated according to differences in density or in surface charge from dry feces or the intestinal content of reduviid hosts or from in vitro cultures which contained host cells. Isopycnic ultracentrifugation on isoosmotic Percoll gradients separated cysts ( ϱ = 1.145 g/cm 3) from epimastigotes ( ϱ = 1.075 g/cm 3), representing the two major morphological stages of B. triatomae. Moreover, a continuous shift in buoyant density of encystating and/or excystating stages from the level of mature cysts to lower densities displayed by epimastigotes was observed. This characteristic excluded the isolation of pure epimastigotes by density gradients. Nevertheless, approximately 1 × 10 8 mature cysts per gradient were obtained by this procedure. Employing DEAE-ion exchange chromatography for cyst isolation, approximately 2 × 10 10 cysts could be purified per column. In addition, DEAE-chromatography was used for the isolation of pure epimastigote fractions. The succesful isolation of B. triatomae-stages from three completely different sources suggests that the methods derived in our study may be easily adapted for the isolation of other insect trypanosomatids.

Surfactant-associated 15- and 35-kDa proteins are concentrated in different organelles in rat lung tissue.

We have used isopycnic gradient ultracentrifugation to isolate a total lamellar body fraction (total-lb) from rat lung and then further subfractionated this using differential centrifugation to obtain two distinct subpopulations of organelles. When the total-lb was diluted to 0.25 M with sucrose and centrifuged at 8000 X for 30 min we obtained a fraction (lbA) that contained primarily intact classic-appearing lb. When the supernatant was then centrifuged at 80,000 X g for 60 min we obtained a vesicular fraction (lbB). Whereas both fractions had an identical phospholipid composition, their enzyme profiles differed markedly. The lbA had a higher level of beta-glycerophosphatase, while lbB had more 5'-nucleotidase. Moreover, lbB had a phospholipid:protein ratio of 9.2 while lbA had one of 6.3. An examination of the specific activity-time curves revealed that lbA had a curve that was broader and reached a peak earlier than lbB, but the downslopes of both curves were identical; they did not bear a classic precursor-product relationship to one another. The two fractions differed very significantly in their protein profiles. Whereas lbA contained a large amount of a 15-kDa protein with very small amounts of 35-, 37-, 38-, 45-, and 60-kDa proteins, lbB contained predominantly a 35-kDa protein with smaller amounts of 15-, 23-, 26-, 37-, 38-, 45-, and 60-kDa proteins. We suggest that lbB is surfactant taken back up into the alveolar type II cell, or a second release form of tissue surfactant, or a mixture of the two.

Analysis of circulating immune complexes from patients with ankylosing spondylitis by gel electrophoresis and immunoblotting using antiserum against a psoriasis associated retrovirus-like particle.

Circulating immune complexes (CIC) were isolated from patients with ankylosing spondylitis (AS) and healthy blood donors by isopycnic ultracentrifugation in sucrose gradients. The CIC were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The major components of the CIC were identified as albumin, immunoglobulins, and complement factors. A 70 kD component and several low molecular weight components (Mr 19 kD and 14 kD (doublet] were detectable only in CIC from patients with AS. An antiserum raised against the envelope glycoprotein, gp70, of a psoriasis associated retrovirus-like particle was applied to check for cross reacting activity. This antiserum reacted with both a 70 kD and a 40-45 kD component in CIC from three out of six patients but not with CIC from any of the blood donors.

Evidence for heterogeneity of low-density lipoprotein metabolism in the cynomolgus monkey

Preliminary studies were performed to establish whether there was kinetic heterogeneity in the metabolism of subclasses of low-density lipoproteins (LDL) in the cynomolgus monkey. Previous studies of the effects of inhibition of hepatic triglyceride lipase in this species had shown an increase in the mass of lighter LDL ( S f ⪢ 9) and a decrease in the mass of denser LDL. LDL (1.019 < d < 1.063) were subdivided into two subfractions LDL, (1.019 < d < 1.035) and LDL 2 (1.035 < d < 1.063) by ultracentrifugation. The lipoproteins in these these two fractions could be shown to have different floatation by analytic and isopycnic ultracentrifugation. When tracer amounts of homologous 125I-labeled very-low-density lipoproteins (VLDL) were injected into chow-fed cynomolgus monkeys, apoB radioactivity appeared in LDL 1 prior to its appearance in LDL 2. [ 125I)LDL 1 injected into the monkey was removed from the LDL 1 density subclass with a half-life of 5.5–10.3 h. Much of the radioactivity injected as LDL 1 was converted to denser LDL (LDL 2). Labeled LDL 2 injected into the monkey was not converted to LDL 1. Thus, at least two kinetically distinct subpopulations of LDL circulate in the plasma of this species. The lighter LDL is to a large extent a metabolic precursor of the more dense LDL (LDL 2).

Salmonella cytotoxin: a component of the bacterial outer membrane

Salmonella cytotoxin present in cell-free sonic lysates causes rounding and detachment of Chinese hamster ovary (CHO) cells. Although the precise role of this toxin in the pathogenesis of salmonellosis is unclear, cytotoxin production by Salmonella could account for tissue damage or possibly, facilitate invasion. A variety of other bacteria (e.g. Shigella, Escherichia, Legionella) have been shown to form soluble cytotoxins, many of which may be involved in pathogenesis. The data in this report indicate that the Salmonella cytotoxin in cell-free sonic lysates is firmly associated with cell membrane fragments that can be pelleted by ultracentrifugation (270000 g for 2.4 h). Furthermore, lysozyme treatment of filter-sterlized sonic extracts of Salmonella species followed by isopycnic sucrose gradient ultracentrifugation allowed separation of the outer and inner membrane components. The outer membrane (OM) peak contained the cytotoxic activity when assayed for detachment of CHO cells. The importance of these data resides in the observation that the Salmonella cytotoxin is an outer membrane component. Its mere location places it in a position of direct contact with host cells and suggests a possible role in cell damage and/or invasion. Furthermore, ultracentrifugation provides a method by which much of the Salmonella cytotoxin in sonic extracts can be removed allowing expression of the Salmonella enterotoxin, whose CHO cell elongation effect is usually obscured by the presence of the cytotoxin causing cell rounding and detachment.

Biophysical properties and morphology of purified antigen associated with non-A, non-B hepatitis

The antigen from a non-A, non-B antigen-antibody system previously described was purified and, when tested by immunodiffusion, was shown to produce patterns of identity with all antigen-positive sera of the system. The sedimentation coefficient S20,w of the antigen was estimated by rate zonal ultracentrifugation to be 48. Isopycnic ultracentrifugation in CsCl gradient of non-A, non-B antigen generated a single sedimentation peak at the density of 1.28. SDS-polyacrylamide-agarose gel electrophoresis showed that I125-labelled antigen migrated as a single band (molecular weight 3.0 X 10(6)); in addition, labelled material also migrated to the albumin region. With SDS-PAGE under reducing conditions, the antigen was shown to consist of seven polypeptides. Spherical particles of 23 nm in diameter, demonstrated by electron microscopy, could be aggregated by purified non-A, non-B antibody. They probably represent the antigen and not a complete viral particle. The characteristics of this antigen associated with non-A, non-B hepatitis appear therefore to be close to those of hepatitis B surface antigen.

Hyperthyroidism Influences the Distribution and Apolipoprotein A Composition of the High Density Lipoproteins in Man

Hyperthyroidism has a different influence on the major high density lipoprotein (HDL) components cholesterol, apoprotein (apo) A-I, and apo A-II. To characterize in greater detail the alterations induced by hyperthyroidism within the HDL subclasses, we investigated HDL distribution and composition in 11 hyperthyroid women before and during treatment. The plasma concentrations of total cholesterol, HDL cholesterol, phospholipids, apo A-I, and apo B were decreased when the patients were hyperthyroid compared with the values during treatment. Apo A-II and apo C-III levels were only slightly lower in the hyperthyroid state. Triglyceride and apo E concentrations did not change significantly during therapy. Analysis of lipoprotein subclasses separated by isopycnic ultracentrifugation revealed 1) marked decreases in low density lipoprotein (LDL) cholesterol, phospholipids, and apo B; 2) less pronounced reductions in the very low density lipoprotein (VLDL) lipid and apo B concentrations; and 3) a consistent decrease in the HDL2b (density, 1.063-1.100 g/ml) fraction in the hyperthyroid patients. The reduction in HDL2b mass was associated with lower concentrations of HDL2b cholesterol, phospholipids, and apo A-I. The HDL2b apo A-II levels remained constant during treatment. Hyperthyroidism, therefore, modified the apo A composition of the HDL2b particles and resulted in a decreased molar apo A-I to apo A-II ratio within HDL2b. Further analysis of HDL particles differing in their apo A composition; i.e. HDL particles containing apo A-I only [(A-I)HDL] or containing both apo A-I and A-II [(A-I + A-II)HDL], by immunological procedures suggested that hyperthyroidism influenced the apo A content of HDL2b mainly by changing the proportions of (A-I)HDL and (A-I + A-II)HDL and the amount of apo A-I associated with (A-I)HDL. Treatment reversed the preferential decrease in (A-I)HDL within the HDL2b subclass. The particle sizes within HDL subfractions, measured by polyacrylamide gradient gel electrophoresis, were similar in the untreated and treated patients. Consequently, the decreased mass of apo A-I and lipids within HDL2b in the hyperthyroid patients could be attributed to a reduced number of identically sized particles within this fraction. These data demonstrate that the thyroid hormones are important regulators of HDL metabolism through their influence on the concentration and distribution of apo A-I.

Rapid purification of HBsAG from human plasma

A method is described for the rapid isolation of purified H BsAg from infected donor plasma. This method uses polyethylene glycol precipitation and a single isopycnic ultracentrifugation step in which the gradient is formed in situ. It works equally well using a small swingout rotor or a zonal rotor.

Changes in the distribution and composition of high-density lipoproteins in primary hypothyroidism

The distribution and composition of high-density lipoprotein (HDL) subclasses were investigated in 14 women with severe hypothyroidism who were studied before and during treatment. The plasma concentrations of triglycerides, total cholesterol, HDL cholesterol, and of the apoproteins (apo) A-I, B, and E were increased in the hypothyroid state, while the apo A-II levels did not change significantly. After normalization of the thyroid function tests, the lipid and apoprotein levels were similar to those of normal individuals. Isopycnic ultracentrifugation in the density range 1.020 to 1.210 g/mL showed increases of both cholesterol and apo B in very-low-density lipoprotein (VLDL) and in low-density lipoprotein (LDL). The distribution of the HDL subclasses was modified in the hypothyroid subjects; both the less dense HDL fraction (d 1.063 to 1.100 g/mL; HDL 2b), and the denser subclass (d 1.150 to 1.210 g/mL; HDL 3b+3c) were increased, while the intermediate density subfraction (d 1.100 to 1.150 g/mL; HDL 2a+3a) did not vary significantly. This redistribution of the HDL subfractions was associated with increased concentrations of cholesterol, phospholipid, and apo A-I in HDL 2b, and of phospholipid and apo A-I in DHL 3b+3c. Treatment of hypothyroidism decreased the concentrations of these fractions, and HDL 2a+3a became the major HDL subclass in the euthyroid state. The particle sizes within HDL subfractions, measured by polyacrylamide gradient gel electrophoresis, were identical in the untreated and treated patients. The increased mass of protein and lipid within HDL 2b and HDL 3b+3c could therefore be attributed to an accumulation of identical-sized particles. The overall lipid and protein composition of the HDL lipoproteins was similar before and during treatment. The apoprotein composition was however modified, due to the higher apo A-I to apo A-II ratio observed in the HDL subfractions. Analysis of the lipoprotein distribution of apo E by sepharose column chromatography revealed a marked increase in the apo E content of VLDL, while changes in HDL-associated apo E were less pronounced. The major effect of hypothyroidism on apo E metabolism therefore appears to be an enrichment of VLDL with apo E, resulting in a significant increase of the plasma apo E levels.

Lipoprotein distribution and composition in the human nephrotic syndrom

Plasma lipoprotein profiles were quantitated in 9 patients with the nephrotic syndrome. Six subjects were studied both during an active proteinuric phase and during a remission phase without proteinuria. During the proteinuric phase, the plasma triglyceride, cholesterol and apo B levels were markedly increased, whereas the HDL cholesterol, apo A-I, and apo A-II concentrations were normal. Analysis of the distribution and composition of the lipoprotein subclasses, separated by isopycnic ultracentrifugation, showed typical patterns characterized by: (1) elevated apo B-rich VLDL and LDL fractions, (2) the presence of a denser LDL subfraction, floating at d 1.053 g/ml, which contained about 35% of LDL cholesterol and apo B and (3) a redistribution among HDL subclasses. The HDL 2b (d 1.063–1.100 g/ml) fraction was markedly decreased, while the HDL2a+3a (d 1.100–1.150 g/ml) and HDL 3b + 3c ( d 1.150–1.210 g/ml) subclasses were moderately elevated. The decreased cholesterol and apo A-I contents of HDL 2b therefore counterbalanced their increase in HDL2a+3a and HDL 3b + 3c, resulting in normal plasma HDL cholesterol and apo A-I concentrations. When reinvestigated during a remission phase without proteinuria, the nephrotic patient's overall lipoprotein distribution and composition were similar to those in healthy controls. The combination of several factors such as the presence of elevated apo B-rich VLDL, IDL and LDL, together with decreased HDL 2 cholesterol and HDL 2 apo A-I suggests that nephrotic patients are at increased risk for atherosclerosis.