Comparison of behaviour of very low density lipoproteins of type III hyperlipoproteinaemia on electrophoresis on paper and on agarose gel with a note on a late (slow) pre-beta VLDL liproprotein.

The diagnosis of type III hyperlipoproteinaemia is based on the presence of a very low density lipoprotein (VLDL) that on electrophoresis has beta instead of the usual pre-beta mobility. This definition is based on paper electrophoresis. Lipoproteins of type III sera were studied by simultaneous electrophoresis on paper and on agarose gel. On paper the ultracentrifugally isolated VLDL had beta mobility, but on agarose gel their mobility was slightly more rapid than beta. It is thus important to consider the electrophoretic conditions in the diagnosis of type III. The diagnosis of type III is further complicated by the presence of agarose gel electrophoresis - contrary to paper - of a second, a 'late pre-beta VLDL' lipoprotein (LPbeta) in 20-30 per cent of both normal and hyperlipoproteinaemic sera. Quantitative lipoprotein analysis of 609 consecutive sera showed that when LPbeta was present, both in normo- and hyperlipoproteinaemia, certain significant lipoprotein changes occurred. Thus VLDL had a high cholesterol content and a raised cholesterol to triglyceride ratio. Furthermore, the triglyceride content of low density lipoproteins (LDL) was increased. These lipoprotein abnormalities are also seen in type III hyperlipoproteinaemia. With regard both to chemical composition of VLDL and LDL and to electrophoretic mobility on agarose gel of VLDL, type III hyperlipoproteinaemia and LPbeta are similar. The possibility of a metabolic relation is discussed.     

Exclusion of pre-beta lipoproteins from agarose gel electrophoresis in the presence of chylomicrons.

Chylomicrons present in the fasting serum of patients with type V hyperlipoproteinaemia have been found to exclude pre-beta lipoproteins from entering into agarose gel during lipoprotein electrophoresis. It is recommended that when chylomicrons are present they be removed prior to diagnostic and quantitative lipoprotein electrophoresis in agarose gel.     

Comparison of the Behaviour of Very Low Density Lipoproteins of Type III Hyperlipoproteinaemia on Electrophoresis on Paper and on Agarose Gel with a Note on a Late (Slow) Pre-β VLDL Lipoprotein

The diagnosis of type III hyperlipoproteinaemia is based on the presence of a very low density lipoprotein (VLDL) that on electrophoresis has /J instead of the usual pre-β mobility. This definition is based on paper electrophoresis. Lipoproteins of type III sera were studied by simultaneous electrophoresis on paper and on agarose gel. On paper the ultracentrifugally isolated VLDL had β mobility, but on agarose gel their mobility was slightly more rapid than β. It is thus important to consider the electrophoretic conditions in the diagnosis of type III. The diagnosis of type III is further complicated by the presence in agarose gel electrophoresis—contrary to paper—of a second, a ‘late pre-β VLDL’ lipoprotein (LPβ) in 20–30 per cent of both normal and hyperlipoproteinaemic sera. Quantitative lipoprotein analysis of 609 consecutive sera showed that when LPyβ was present, both in normo- and hyperlipoproteinaemia, certain significant lipoprotein changes occurred. Thus VLDL had a high cholesterol content and a raised cholesterol to triglyceride ratio. Furthermore, the triglyceride content of low density lipoproteins (LDL) was increased. These lipoprotein abnormalities are also seen in type III hyperlipoproteinaemia. With regard both to chemical composition of VLDL and LDL and to electrophoretic mobility on agarose gel of VLDL, type III hyperlipoproteinaemia and LPβ are similar. The possibility of a metabolic relation is discussed.     

Agarose gel electrophoresis of plasma lipoproteins using the Durrum cell.

1. A low cost method for lipoprotein electrophoresis in agarose gel is described. The basic procedure, described by Noble, has been modified for use with a Durrum cell which is commonly used for paper electrophoreis. 2. Up to 28 samples can be analyzed per Durrum cell in 4 hours. 3. A staining technique, which involves filtration of a suspension of Sudan Black B, allows for rapid staining and provides satisfactory reproducibility from run to run. 4. In a small number of patients a double pre-beta band is seen, and is different from sinking pre-beta and floating beta lipoproteins.     

Increased frequency of late pre-β lipoproteins (LP β) in isolated serum very low density lipoproteins in uraemia

Abstract. Hypertriglyceridaemia, which is caused by raised levels of the triglyceride rich serum very low density lipoproteins (VLDL), is common in uraemia. Here we report qualitative studies on VLDL in this condition. VLDL were isolated from serum of 137 uraemic patients by ultracentrifugation, subjected to agarose electrophoresis and stained for lipids. An increased frequency of presence of electrophoretically slow migrating, discrete VLDL band, the so-called late pre-β lipoproteins, LPβ, was found, 76% in uraemia versus 30% in controls. Patients on haemodialysis had a higher frequency of LPβ than those not on dialysis, 94% versus 69%. Furthermore the cholesterol/trigly-ceride ratio of VLDL was raised by about 30% in uraemia. Since the cholesterol enriched LPβ is believed to represent an end stage of the catabolism of VLDL these data suggest that there is a delayed clearance of VLDL remnants in uraemia. In haemodialysis patients heparin might have contributed to the higher frequency of LPβ.     

Secretion of Cholesterol-Rich Lipoproteins by Perfused Livers of Hypercholesterolemic Rats

Rats maintained on a high-fat diet supplemented with propylthiouracil develop a hypercholesterolemia, an increased serum level of apolipoprotein (apo) E, abnormal very low density lipoproteins (VLDL) and low density lipoproteins (LDL), and a fatty liver which contains cholesterol ester as its major lipid. The fatty liver secretes apoE into a recirculating perfusate at a significantly higher rate and produces cholesterol ester-rich, apoC-deficient VLDL with slower electrophoretic mobility than the triacylglycerol-rich VLDL produced by perfused normal livers. LDL, secreted in significant quantities by the perfused fatty liver, but not by the normal liver, is also cholesterol rich and contains apoE as well as apoB. The incorporation of [(3)H]leucine into apoVLDL and apoLDL secreted by the livers of the hypercholesterolemic animals and the apoVLDL secreted by the normal liver corresponds to the pattern visualized when the apoproteins are separated by polyacrylamide gel electrophoresis. Similar patterns are noted when non-recirculating perfusates are studied. These results indicate that the cholesterol ester-rich, apoC-deficient VLDL and the apoE-containing LDL found in the serum of hypercholesterolemic rats are not solely catabolic remnants of VLDL and chylomicrons but are secreted by the liver. Separation of the perfusate lipoproteins by agarose gel filtration revealed that most of the apoE secreted by the livers of hypercholesterolemic rats is found in the VLDL and LDL, whereas apoE secreted by the normal livers is distributed equally between VLDL, high density lipoproteins, and a low molecular weight fraction which corresponds to the virtually delipidated apoprotein. Thus the distribution of apoE among the lipoprotein fractions may be related to the total amount of cholesterol being transported in the circulation.     

Cord blood cholesterol,triglycerides and lipoprotein electrophoresis in 124 Italian infants

Summary Total cholesterol (TC), triglycerides (TG), and lipoproteins (by electrophoresis on agarose gel) were determined in the cord blood of 124 Italian infants (Verona area). The mean TC and TG values, when compared with the values reported in other studies, turned out to be remarkably uniform, suggesting common genetic determinants in the modulation of blood lipids; no sex differences were observed. The TG distribution curve was skewed, overlapping the adult pattern. Cord blood TC and TG were not modified by the presence of perinatal factors. Both β and α bands (on agarose gel electrophoresis) were present in all the cases. The pre-β band was clearly detectable in 90 % of the cases; it was barely visible for TG values below 20 mg/100 ml; occasionally a discrepancy between the intensity of the pre-β band and the cord blood TG was observed, indicating a variable lipid composition of very low density lipoproteins (VLDL). In 4 % of the cases a small band at the origin of the electrophoretic run was observed, consistent with the presence of chylomicrons. In the serum of both a newborn infant and its mother we detected a double band migrating in the pre-β region. This finding confirms the hypothesis of a familial transmission of this abnormality.     

Pre-beta high-density lipoprotein determined by immunoblotting with chemiluminescent detection.

We describe a novel assay of pre-beta high-density lipoprotein (HDL), a unique apolipoprotein A-I (apo A-I)-containing lipoprotein particle. The pre-beta and alpha lipoproteins are separated by electrophoresis in agarose and transferred onto a membrane by capillary blotting. The membrane blot is sequentially incubated with sheep anti-human apo A-I antiserum and then with a conjugate of rabbit anti-sheep immunoglobulin and horseradish peroxidase. Chemiluminescence formed by the peroxidase-catalyzed oxidation of luminol in the presence of an enhancer is captured on photographic film, and the pre-beta HDL band is quantified by transmission densitometry. The assay is calibrated with standards prepared from a reference serum diluted in 9 mol/L urea. Within-batch precision (CV) at pre-beta HDL concentrations of 22.1 and 44.3 mg/L was 7% and 4.9% respectively. Pre-beta HDL contained 1.6% (0.65-2.6%, mean and range) of total serum apo A-I in 30 normolipidemic subjects.     

Lipoprotein electrophoresis on acrylamide-agarose plates, with discontinuous acrylamide gradient]

We describe a modified lipoprotein electrophoresis on acrylamide gel. The lipoproteins, prestained with Sudan Black, are separated by running on acrylamide-agarose gels, with two different concentrations of acrylamide and a constant concentration of agarose. The sera are deposited in the first gel (2% acrylamide). Chylomicrons remain at origin and other lipoproteins run through the first gel to the second one (3% acrylamide gel). VLDL stays at the junction of both gels, LDL and HDL are separated in the 3% acrylamide gel. By this technique, we were able to detect an increase of Lp(a) and to identify the different types of hyperlipoproteinemia. After running, the plates can be treated for storage.     

Isolation and characterization of hepatic Golgi lipoproteins from hypercholesterolemic rats.

The feeding of cholesterol-rich diets alters the serum lipoproteins of a number of mammalian species. These lipoproteins are characterized by the presence of several classes of particles enriched in cholesteryl esters and apolipoprotein E (apo E). It was the aim of this study to determine whether one or more of these particles arises by de novo hepatic synthesis by characterizing nascent lipoproteins isolated from the hepatic Golgi apparatus of hypercholesterolemic rats. Characterization of these lipoproteins afforded the opportunity to assess morphologic, biochemical, and biophysical properties of newly synthesized lipoproteins before enzymatic alterations and apoprotein transfer known to occur after secretion into the plasma compartment. Golgi very low density lipoproteins (VLDL, d < 1.006 g/ml) from hypercholesterolemic rats contained nearly four times the total cholesterol mass found in control Golgi VLDL. They exhibited electrophoretic mobility intermediate between beta and pre-beta and were devoid of apo C. A second population of hepatic Golgi lipoproteins was isolated from hypercholesterolemic rats at 1.006--1.040 g/ml d. These low density lipoproteins were smaller than VLDL, displayed beta electrophoretic mobility, were enriched in cholesteryl esters, and contained apo E as well as apo B. The fatty acid composition of the core lipids of the nascent lipoproteins was found to reflect that of dietary triglyceride. The liver of the hypercholesterolemic rat thus plays an active role in dietary-induced hypercholesterolemia by synthesizing a modified VLDL and a low density lipoprotein resembling serum low density lipoprotein.     

Automated microdensitometry and quantification of lipoproteins by agarose gel electrophoresis.

A major obstacle in the application of quantitative microelectrophoresis has been tedious manipulations and calculations. To overcome these difficulties, we have developed an automatic system for the microdensitometry and calculations as part of a quantitative agarose gel electrophoresis facility. Results are internally standardized by serum cholesterol and/or triglyceride measurements. The hardware consists of a densitometer, an analog to digital converter, a cathode ray tube terminal, a teleprinter, and a small computer. A program in 4K words allows sample coding, electrophoretic scan display, indexing, and systematic identification of each peak. Data are acquired from scans of electrophoretic patterns of serum alone or in combination with the 1.006 gm/ml VLDL top and/or bottom preparative lipoprotein fractions. As many as 30 scans can be stored in 4K words of memory and then sent via high-speed telephone line to a larger computer for remote processing. The analysis corrects for baseline drifts and pre-beta asymmetry and will properly identify and quantify the amount of VLDL, LDL, and HDL with corrections for "sinking pre-beta" and "floating beta" in LDL and VLDL, respectively. Results are given in milligrams per 100 milliliter as well as percentile rank and standard deviation score ranking of each lipoprotein class as compared to an appropriate normal reference population. The latter data are in a form more meaningful to the physician and patient and provide a quantitative dimension to lipoprotein phenotyping.     

Characterization of polypeptides serologically and structurally related to hexosaminidase in cultured fibroblasts

Human fibroblasts synthesize several polypeptides that assort into the various forms of hexosaminidase (hex). We report here the occurrence of three newly identified, hexosaminidase-related polypeptides resolved by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of immunoprecipitates from [35S]methionine-labeled cell extracts. These polypeptides, called band 2 (75,000), band 3 (70,000), and band 4 (63,000), were immunoprecipitated by an antiserum specific to placental hex I2. They are distinct from pre-alpha- (60,000) and pre-beta- (58,000) precursor polypeptides and the alpha- (56,000), beta a- (27,000), and beta b- (27,000) polypeptides of the mature hex A (alpha beta a beta b) and hex B (2[beta a beta b]). When fibroblast extracts were chromatographed on DEAE-Sepharose, bands 2, 3, and 4 were eluted together in fractions before hex A, in a position characteristic of serum and placental hex I2 and serum hex P. This suggests that bands 2, 3, and 4 might represent the polypeptides of a fibroblast hex I. The analysis of partial proteolytic digests of the radioactively labeled polypeptides revealed that bands 2 and 3, pre-beta, and beta a had several peptides in common, suggesting that they are structurally related to each other. However, bands 2, 3, and 4 were present in extracts of Tay-Sachs (pre-alpha and alpha deficiency) and Sandhoff cells (pre-beta, beta a, and beta b deficiency) and appeared later than pre-beta in pulse-chase experiments. These results suggest that bands 2 and 3 occur independently of pre-beta and beta a and are probably specified by different mRNA, whether from the same gene or distinct but homologous genes.     

Very low density lipoproteins in intestinal lymph: origin, composition, and role in lipid transport in the fasting state

The transport of endogenous lipids in the lipoproteins of mesenteric lymph was studied in fasting rats with mesenteric lymph fistulas. The lymph was found to contain, in addition to chylomicrons (S(f) >400), a significant amount of another, more dense, triglyceride-rich fraction, the very low density lipoproteins (VLDL), which showed a peak S(f) of 102. The VLDL differed from chylomicrons not only in flotation, but also in per cent lipid composition and electrophoretic mobility in agarose gel. The VLDL fraction was found to contain 47% of the triglyceride and 54% of the cholesterol of fasting lymph and, in the fasting state, was the major lipoprotein species present.When cholestyramine resin was administered intraduodenally, or bile flow was acutely diverted from the intestine, it was demonstrated that the lipids in lymph VLDL, like those in chylomicrons, were derived from the intestine and bile. These data indicate that the VLDL in intestinal lymph are not derived from the plasma but are of intestinal origin.Because certain properties of lymph VLDL were similar to those reported for plasma VLDL (per cent lipid composition, flotation coefficient, and continuing entry into plasma in the fasting state), additional comparisons between these fractions were made. Although lymph VLDL moved to the alpha(2) region in agarose gel, when they were mixed with VLDL-free serum immediately before electrophoresis they showed the alpha(2) mobility of rat serum VLDL. Furthermore, immunoelectrophoretic comparison of partially delipidated lymph and serum VLDL revealed that these fractions shared in common their major apoprotein, and possibly others as well. The fatty acid composition of lymph and serum triglycerides, as determined by gas-liquid chromatography, revealed that although they were generally similar, differences existed which most likely reflected the presence in serum of triglycerides of hepatic origin.These experiments demonstrate the importance of intestinal VLDL in the transport of endogenous lipids in mesenteric lymph in the fasting state. The similarities between intestinal lymph VLDL and plasma VLDL suggest that the latter may be derived in part from the former.     

Improved electrophoretic resolution of human serum alkaline phsophatase isoenzymes in agarose gel by Triton X-100.

Triton X-100 caused the liver isoenzymes of alkaline phosphatase in some serum samples to separate into two fractions on agarose gel electrophoresis. One of these fractions migrated at the rate of the original one, and one migrated more slowly. The latter fraction corresponded to a fast-moving component on electrophoresis in Cellogel and seems to be identical with a slowly moving isoenzyme in starch and polyacrylamide gel electrophoresis. The migration rates of the bone, placental, and intestinal isoenzymes were unaltered, but the fractions appeared sharper.     

Uranyl acetate as an excellent fixative for lipoproteins after electrophoresis on agarose gel

A study was made of the effectiveness of the fixatives most commonly used for lipoproteins after electrophoresis on agarose gel. The results prove that acetic acid in water or in alcohol does not fix, but allows the removal of the albumin together with the middle part of the alpha lipoprotein band during drying. A double alpha band, therefore, arises artificially. After fixation with methanol or ethanol, not only the alpha, but also the greater part of the beta lipoprotein group remains soluble.Both trichloroacetic acid + formaldehyde, and picric acid, proposed earlier in the literature for the fixation of serum proteins, proved to be effective for lipoproteins, but uranyl acetate was found to be by far the best of all the fixatives yet investigated.     

High density lipoprotein infusion and partial plasma exchange in Tangier disease

High density lipoprotein (HDL) infusion and partial plasma exchange were undertaken in two patients homozygous for Tangier disease. Serum samples and ultracentrifugally isolated serum fractions were analysed over a period of 7 days post infusion by agarose electrophoresis, two-dimensional immunoelectrophoresis (employing antibodies to HDL, HDL3, Apoprotein A-I, and Apoprotein A-II), Apoprotein A radioimmunoassay, and analytical polyacrylamide electrophoresis. The following observations were made: (a) immediately after HDL substitution the broad-beta band, normally visible upon agarose electrophoresis of Tangier plasma, resolved into a distinct beta and pre-beta band; (b)as HDL was catabolized, an abnormal alpha-migrating lipoprotein was generated which contained Apoprotein A-II as protein constituent; and (c) there was a proferential loss of Apoprotein A-I from HDL and the plasma compartment in the course of HDL catabolism. The results suggest that the defect in Tangier disease resides with enhanced catabolism or defective synthesis of Apoprotein A-I.     

Serum lipoproteins and apolipoproteins in rats with streptozotocin-induced diabetes.

The lipoproteins of rats fed a high sucrose diet and made diabetic by administration of 45 mg/kg of streptozotocin were studied. All lipoprotein classes were found to be present in increased concentrations. The apolipoprotein composition of the various lipoprotein fractions was studied by polyacrylamide-gel electrophoresis in the presence of 8 M urea, isoelectric focusing in the presence of 8 M urea, and sodium dodecyl sulfate gel electrophoresis in polyacrylamide gels. In the very low density lipoproteins (VLDL) of diabetic rats, there was a marked alteration in the relative amounts of C proteins by polyacrylamide-gel electrophoresis, and this was found by isoelectric focusing to be primarily a relative increase in C-III-3 apoprotein and a decrease in C-III-O. In addition, in the diabetic rats, the VLDL contained a protein of mol wt 46,000, the A-IV protein, which normally is only present in the high density lipoproteins. In the high density lipoproteins, (HDL) the same alterations in pattern of the C proteins seen in the VLDL were present. Furthermore, the arginine-rich and A-IV protein normally present in HDL could not be detected in the HDL, although the other apolipoproteins are present. Apolipoprotein concentrations were determined by quantitative immunoelectrophoresis. It was found that in the diabetic rats there was an increase in the total amount of apo-B in the plasma, with the increment divided proportionately between the VLDL and the low density lipoprotein (LDL). The total apo-C concentration of plasma increased minimally. The A-IV concentration of plasma increased by 27%; it decreased markedly in the HDL, but appeared in increased amounts in both VLDL and in the d greater than 1.21 fraction. The arginine-rich protein decreased by 63% in the plasma and decreased significantly in the HDL, but increased in VLDL, LDL, and in the d greater than 1.21 fraction. These alterations in apolipoprotein patterns in diabetic animals suggest that the apolipoproteins may play an important role in determining the concentration of various lipoprotein fractions, or may be the result of altered metabolism of the lipoproteins. These lipoproteins with altered apolipoprotein composition may have important biologic differences from normal lipoporteins. Nevertheless, the HDL, despite the fact that it is deficient in some of its major constituents, was unchanged in its cholesterol content.     

Apoprotein profile of plasma and chylous ascites lipoproteins.

Plasma and chylous ascites lipoproteins were compared in a rare case of exudative enteropathy associated with stenosis of the thoracic duct. All ascites lipoproteins separated by ultracentrifugation showed a much higher triglyceride/protein ratio and a lower cholesterol ester content than their plasma counterparts. Polyarcylamide gel electrophoresis of apoproteins before and after fractionation on Sephadex G-200 essentially showed, compared to normal plasma, a reduction of apoprotein C in VLDL and HDL particularly obvious for apoCIII1 and CIII2. Ascites lipoproteins were characterized by an increased apoA content in chylomicrons, VLDL and LDL and a reduced percentage of apo CII and CIII1, mostly in chylomicrons, VLDL and HDL. The differences in composition between plasma and ascites might be explained by different origins for the various apoprotein subfractions. The transsudation of chyle into the abdominal cavity might divert some peptides of intestinal origin from their normal entry into the circulation.     

Comparison of electrophoresis on agar gel and agarose gel in the evaluation of gamma-globulin abnormalities in cerebrospinal fluid and serum in multiple sclerosis

The band pattern found in the gamma region on investigation of concentrated CSF by agarose gel electrophoresis is denned. Extra gamma bands are found in multiple sclerosis about equally often when agar gel or agarose gel is used as a supporting medium for electrophoresis. Extra gamma bands can be demonstrated slightly more often when CSF from patients with other neurological disorders is investigated by agarose gel electrophoresis; in these instances the extra gamma bands may be recovered in the serum pattern. The simultaneous finding in CSF and serum of extra gamma bands must be judged with caution when a diagnosis of multiple sclerosis is suspected. A standardization of CSF electrophoresis with regard to the absolute amount of immunoglobulin G applied per mm of the application trough is recommended.     

Diagnosis of dyslipoprotein aemia by molecular sieve chromatography.

Agarose gel molecular sieve chromatography has been used as a tool in the diagnosis of dyslipoproteinaemias. The elution profile from control plasma contained three peaks, corresponding to very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). These accounted for 9.5%, 23.5% and 67% of the total lipoprotein absorption at 280 nm. Elution patterns from dyslipoproteinaemic serum showed alterations, not only in peak area, but also in peak migration rate through the agarose gel. These variations were reproducible and of such magnitude as to permit the use of molecular sieve chromatography as a tool in the diagnosis of dyslipoprotein-aemias of Type I, II (a + b), III, IV and V.