Zero electrophoretic mobility of very low density lipoproteins in type III hyperlipidemic patients during treatment.

In some cases of type III hyperlipoproteinemia reduction of lipid levels by diet and/or drugs results in a complete loss of mobility of the VLDL fraction on paper electrophoresis.     

Separation and partial characterization of new apoproteins from human plasma high density lipoproteins.

In four patients receiving parenteral fluids, human plasma high density lipoproteins were obtained by sequential ultracentrifugation and delipidated. Apoproteins were resolved by gel filtration on Sephadex G-200, DEAE-cellulose chromatography, and polyacrylamide gel electrophoresis. Sephadex-Fraction V was unusually large (11--33% compared to 3--7% in normal subjects) and was found to contain seven new apoprotein components of an apparent molecular weight (by SDS gel electrophoresis) between 8000 and 11 000. Amino acid analysis showed that all these peptides had a high glycine and arginine content and a very low content of threonine and valine. Isoelectric focusing gave isoelectric points ranging from 5.00 to 8.00. In two patients injected with L-[1-17C]-leucine, incorporation of radioactivity into these peptides gave specific activities of the same order of magnitude as apo C-II and apo C-III. IT may be postulated that these peptides increased significantly when proteins are deficient in the diet or have low levels in plasma. The structural and functional significance of these peptides remains to be determined.     

Cholinesterase in serum and low density lipoprotein of hyperlipidemic patients.

Cholinesterase activity in the low density lipoprotein fraction of serum is increased in types IIa, IIb and IV hyperlipoproteinemic patients, whereas only types IIb and IV show increases in serum cholinesterase activity. In obese patients, cholinesterase activity is increased both in the serum and low density lipoprotein fraction only when hyperlipidemia is present. Cholinesterase activity is also found to increase in proportion with increases in low density lipoprotein, cholesterol, and triglycerides both in the serum and low density lipoprotein fraction. We suggest on the basis of these findings that cholinesterase has a function in lipid and lipoprotein metabolism.     

Electrophoretic mobility in agarose of very low density lipoprotein subfractions in type III hyperlipoproteinaemia.

The electrophoretic mobilities in agarose gel of the very low density lipoprotein (VLDL) subfractions of Sf greater than 100, 60-100 and 20-60 from six subjects with type III hyperlipoproteinaemia (HLP) have been compared with those from eight normal volunteers. In type III HLP beta or near-beta (slower VLDL) electrophoretic mobility was not necessarily confined to the VLDL fraction of Sf 20-60 in which it may normally be detected.     

The composition of low (LDL) and very low (VLDL) density lipoprotein subfractions in type III hyperlipoproteinaemia: comparison with normal subjects.

The lipid and protein composition of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) subfractions (Sf greater than 100, 60--100 and 20--60 VLDL and Sf 10.4--20, 5.7--12 and 3.5--6.5 LDL) in six subjects with type III hyperlipoproteinaemia (HLP) was compared to that of 12 normal subjects. In type III HLP all VLDL subfractions contained increased concentrations of cholesterol and triglycerides and were relatively enriched in cholesterol. VLDL of Sf 20--60 also contained and increased concentration of B-protein. The tetramethylurea (TMU) soluble apolipoproteins of the VLDL subfractions were separated by polyacrylamide disc gel electrophoresis. In the subjects with type III HLP the proportion of arginine rich protein (ARP) was increased in all subfractions. The concentrations of cholesterol and triglycerides were increased in the LDL subfraction of Sf 10.4--20 and cholesterol was decreased in LDL of Sf 5.7--12, but the ratios of cholesterol to triglycerides were not significantly different from those in the LDL subfractions of the normal subjects and the protein composition was also similar. These results provide further evidence that in type III HLP abnormalities are not confined to the stage of conversion of VLDL to LDL, but occur throughout the VLDL spectrum.     

A method for measurement of low levels of guanosine 3′,5′-cylic monophosphate in blood plasma

A very sensitive method is described for the assay of guanosine 3',5'-cyclic monophosphate (cGMP) based on a competitive protein-binding technique. The procedure is simple to carry out, and does not require the purchase of expensive reagents. The binding-protein used is extracted from rat lungs and the bound and unbound cGMP are separated by ammonium sulphate precipitation. Although prior extraction of the cGMP is required, the time for this extra step is easily compensated for by the shortness of the assay time. The mean cGMP found in the blood plasma of normal young males and females is 9.9 ± 2.4 (S.D.) nmol/l (range 5.9–14.3 nmol/l) and 9.5 ± 2.1 (S.D.) nmol/l (range 6.9–12.7 nmol/l), respectively.     

Triglyceride lipase activity in postheparin plasma and plasma lipoproteins in liver disease.

Hepatic lipase activity and lipoprotein lipase activity were studied in postheparin plasma from 14 patients with various liver disorders. Plasma lecithin: cholesterol acyltransferase (LCAT) activity and lipoprotein composition and structure were also estimated. Five patients had lower hepatic lipase activity than the lowest control value, and in three of these no hepatic lipase activity was detected. Lipoprotein lipase was low in 5 patients, but in only one of them was hepatic lipase activity also low. Hepatic lipase was not significantly correlated to the concentration of plasma triglycerides, either in controls or in patients, whereas lipoprotein lipase was negatively correlated with plasma triglycerides both in controls and patients. Lipoprotein lipase and LCAT activity, but not hepatic lipase, was negatively correlated to the triglyceride content of the low density lipoproteins (density 1.019-1.063 g/ml) from the patients. No specific lipid or lipoprotein pattern was found in plasma from the patients with a low or without any hepatic lipase activity. The results suggest an important role of lipoprotein lipase and LCAT, for the increased content of triglycerides in the low density lipoproteins in patients with liver disease. The role of hepatic lipase remains unclear.     

Concentration and composition of human serum lipoproteins at birth

By ultracentrifugation of sera from 69 cord bloods very low (VLDL), low (LDL) and high density lipoproteins (HDL) were obtained and analysed for triglycerides (TG) and cholesterol (CHOL). Electrophoresis in agarose gel was performed on serum, VLDL and LDL + HDL. VLDL lipids were present in all newboms. VLDL contained 30 and 5% of serum TG and CHOL. The range of TG in VLDL was wide (0.02–0.62 mmol/l), but there was a constant ratio between TG and CHOL. The electrophoretic mobility of VLDL was slower than in adults. LDL contained 50% of both serum TG and CHOL, and HDL contained 15 and 50% of these serum lipids. A published formula for calculating CHOL in VLDL and LDL from serum CHOL and TG and CHOL in HDL gave values for CHOL in LDL which corresponded well with the determined values. For VLDL, however, the calculated values were too high.     

Stability of lipid and protein composition of very low-density lipoproteins during the storage after freezing of whole serum and isolated lipoproteins (author's transl)]

The lipid and protein composition of lipoprotein is now regarded with great interest for the identification of hyperlipoproteinemias. Because this relatively time-consuming study cannot be envisaged currently in clinical services, it seems useful to analyse a storage procedure which does not affect the composition of very-low density lipoproteins, whose proportion is very important among subjects with type IV hyperlipoproteinemia. Two studies were considered, one using whole sera stored at -20 degrees C and one using very low-density lipoproteins held in the same conditions. The lipid composition was determined for triglycerides and cholesterol concentrations of total proteins, proteins soluble in tetramethylurea (apoprotein C, "arginine-rich fraction", and apoprotein D), and peptides CII, CIII1 and CIII2 of apoprotein C. For both procedures considered, no significant changes were seen in lipid and protein composition for periods as long as 8 weeks.     

Determination of cholesterol and phospholipid concentrations in high-density lipoproteins by heparin-manganese chloride precipitation.

Cholesterol as well as phospholipids in high-density lipoproteins (HDL) have been measured in 60 serum samples after heparin-manganese chloride precipitation of the apolipoprotein B containing lipoproteins. The values obtained correlated well with values from ultracentrifugal separation (slope 1.11 for cholesterol, 0.92 for phospholipids). Furthermore, there was a significant correlation between the low-density lipoprotein (LDL) cholesterol estimated by the formula of Friedewald et al. and the ultracentrifugally separated LDL cholesterol (slope 1.02). By the additional determination of HDL phospholipids with a precipitation procedure, further information about the "antiatherogenic" is easily available.     

Cholesterol gas-liquid chromatographic microassay in serum lipoproteins separated by polyacrylamide gel electrophoresis

A new method of cholesterol assay in serum lipoprotein fractions separated by electrophoresis on polyacrylamide plates is described: each lipoprotein fraction was collected by cutting the gel and, after gel dissolution, cholesterol was extracted and assayed by gas-liquid chromatography.This method associates the specificity and sensitivity of gas-liquid chromatography with the resolution of polyacrylamide gel electrophoresis, a highly reliable technique for hyperlipoproteinemia phenotyping. It is precise (variation coefficient below 2%), fast and needs only 2.5 microlitres of serum. Direct assay of cholesterol is feasible not only on each of the three main fractions, very low density lipoproteins, low density lipoproteins and high density lipoproteins, but also on intermediary fractions such as Lp(a) lipoprotein. This method should allow a better evaluation of the relationship between the serum cholesterol fractions and development of atherosclerosis.     

An evaluation of quantitative agarose-gel electrophoresis of serum lipoproteins.

The method of Hatch et al. (Hatch, F.T., Lindgren, F.T., Adamson, G.L., Jensen, L.C. and Wong, A.W. (1973) J. Lab. Clin. Med. 81, 946-960) of quantitative determination of serum lipoproteins has been compared with preparative ultracentrifugation and chemical analysis of the lipoprotein fractions and a good concordance was demonstrated. The replacement of the coefficients proposed by Hatch et al. by the ones derived from the present study does not bring about relevant changes of the results when the harmonic mean of two calibration factors, derived from serum cholesterol and triglycerides, is used, thus indicating that this method of calculation minimizes the effect of variation of the chemical composition of lipoproteins. The method is sufficiently reliable and reproducible for practical pruposes and is recommended as the standard method for the clinical laboratory.     

Quantitative analysis of serum lipoproteins by micro-scale thin-layer chromatography.

A method has been devised for the complete chemical analysis of serum lipoproteins, in which the constituents are separated by thin-layer chromatography and then measured by means of a flame ionisation detector. Since the response of the detector differs for each constituent, it is necessary to use a previously prepared calibration curve for each one. A complete analysis can be obtained from a single run on about 20 microgram of lipoprotein. However, from 5--10 chromatograms are needed for an adequate degree of precision. The method, which could be adapted to the measurement of tissue lipids, takes less than 2 h to complete. This speed and simplicity seem to give the method considerable potential for the investigation of patients with disorders of lipid transport.     

Response of plasma lipoproteins and acute phase proteins to myocardial infarction.

Plasma concentrations of lipoprotein-lipids, apolipoprotein B (apoB) and of seven other proteins have been estimated serially in 27 patients up to three months following myocardial infarction. Results were compared with those from age- and sex-matched control subjects. At three months the mean total, low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol concentrations were higher than those of the control subjects, whereas very low density lipoprotein (VLDL) cholesterol, total and VLDL triglyceride, and total and LDL apolipoprotein B concentrations were not significantly different. Relative to concentrations at three months total and LDL cholesterol and apolipoprotein B concentrations fell markedly, and a slight fall occurred in HDL cholesterol following infarction. VLDL cholesterol and total and VLDL triglyceride were decreased only on day one. Albumin and transferrin concentrations were higher and alpha 1-acid glycoprotein was lower at three months than in the control subjects; alpha 2-macroglobulin, caeruloplasmin, haptoglobin and immunoglobulin IgM were not significantly different. Following infarction albumin and transferrin fell, alpha 2-macroglobulin did not change, and alpha 1-acid glycoprotein, caeruloplasmin, haptoglobin and IgM rose. The changes in both lipids and protein are probably part of the general metabolic response to trauma.     

Familial hyper-alpha-lipoproteinaemia. Further studies on serum lipoproteins and some serum enzymes.

Some new data are given concerning a family whose members are characterized by high levels of alpha-(HDL)-cholesterol. Data from the new members of the family, not previously studied, confirm that the defect is not associated with any cardiac or neurological defect nor with xanthomata. Studies performed to assess the polypeptide composition of HDL of the affected members resulted in completely normal results. The post-heparin lipolytic activity (PHLA), the liver lipase and the lecithin-cholesterol acyltransferase (LCAT) activities also proved to be within the normal limits.     

Quantification of plasma lipoprotein cholesterol: A simple procedure for enzymatic determination of cholesterol in electrophoretically separated lipoproteins

A method is described for the enzymatic determination of the cholesterol content of serum lipoproteins separated by electrophoresis on agarose. The method shows high precision and correlates well with quantitative lipoprotein electrophoresis based on polyanion precipitation.     

Immunological characteristics of a peroxidase protein in human neonatal cord serum.

Adult serum haptoglobin-hemoglobin complexes exhibit acid-stable peroxidase activity. The haptoglobin level in human neonatal cord sera, however, is reported to be very low. We have isolated a protein from the cord sera of neonates, which has acid-stable peroxidase activity analagous to adult serum haptoglobins-haemoglobin complexes, but does not show any immunological identity to adult serum haptoglobins. It differs from the latter in its molecular size and electrophoretic mobility in agarose and polyacrylamide gels. It is unique to cord sera and it bears immunological identity to human IgG.     

The role of lecithin : cholesterol acyltransferase in high density lipoprotein3/high density lipoprotein2 interconversion

Serum was incubated in vitro with and without inhibition of lecithin : cholesterol acyltransferase (LCAT, EC 2.3.1.43). High density lipoprotein₂ (HDL₂) and high density lipoprotein₃ (HDL₃) were separated by zonal ultracentrifugation and analysed for lipid and apoprotein contents. The incubation of fresh sera resulted in a time-dependent decrease in HDL₃ and an increase in HDL₂. At the end of 24 h incubation HDL₃ disappeared completely and the HDL₂ peak had reached its maximum. The newly formed HDL₂ was relatively enriched in total protein (apoprotein A-I, C-apoproteins) and cholesteryl esters, and depleted in phosphatidylcholine. Its migration in polyacrylamide gel electrophoresis was identical with HDL₂ contained in fresh serum or HDL₂ isolated from fresh serum by zonal ultracentrifugation. The generated HDL₂ particles exhibited the same electron microscopical characteristics as reference HDL₂ samples prior to incubation.Addition of Ellman's reagent to the incubation mixture or heat inactivation of the samples prior to incubation resulted in a complete inhibition of HDL₃/HDL₂ interconversion, whereas addition of 1 mol/l NaCl had no detectable influence. There was also a substantial increase in HDL₂ when VLDL-deficient serum was incubated at 37°C. Similarly, in fresh serum from a patient affected with familial lipoprotein lipase deficiency, HDL₃ was completely converted to HDL₂.Our experiments demonstrate that LCAT promotes HDL₃/HDL₂ interconversion in native serum irrespective of the presence or absence of triglyceride-rich lipoproteins and lipoprotein lipase.