Effects of blocking plasma lipid transfer protein activity in the rabbit.

Plasma lipid transfer protein activity was completely blocked in rabbits for up to 48 h by infusion with goat antibody to rabbit lipid transfer protein. Lipid transfer protein activity in plasma of control animals, infused with antibody from a non-immune goat, decreased during the experiment but was never less than 50% of pre-infusion levels. During the period that lipid transfer protein activity was completely blocked, there were changes in high-density lipoprotein composition (expressed as % by weight) with a reduction in triacylglycerol from 8.4 +/- 2.4% to 1.0 +/- 0.2% (P less than 0.05) and an increase in esterified cholesterol from 10.7 +/- 1.7% to 14.5 +/- 0.3% (P less than 0.1). In conjunction with the observed changes in high-density lipoprotein composition, there was an increase in high-density lipoprotein particle size from a mean radius of 4.7 to 5.4 nm. The change in composition and particle size was not observed in high-density lipoproteins from control animals. There was a change in the distribution of plasma cholesterol in control animals, with a fall in the proportion of cholesterol in high-density lipoproteins (P less than 0.02) and consequently an increase in the proportion of cholesterol in low-density lipoproteins (P less than 0.02). However, the distribution of plasma cholesterol in animals in which lipid transfer protein activity was inhibited was maintained at original levels during the period of inhibition. Consequently, in these animals, there was a less atherogenic distribution of cholesterol during the period of lipid transfer protein inhibition when compared with control animals. The changes observed in lipoproteins, in the absence of lipid transfer protein activity, demonstrate that lipid transfer protein modifies lipoproteins in vivo and appears to contribute to a more atherogenic lipid profile.

Net mass transfer of esterified cholesterol from human low density lipoproteins to very low density lipoproteins incubated in vitro.

In 37 degrees C incubations of either human or rabbit very low density lipoproteins (VLDL), low density lipoproteins (LDL) and lipoprotein-free plasma thee was a net mass transfer of esterified cholesterol from LDL to VLDL which in the human studies resulted in an approximate doubling of the VLDL esterified cholesterol concentration after 24 h. There was also a net mass transfer of triacylglycerol in the reverse direction, from VLDL to LDL.

Immunoprecipitation of lipid transfer protein activity by an antibody against human plasma lipid transfer protein-I.

Two lipid transfer proteins, designated lipid transfer protein-I (Mr 69 000) and lipid transfer protein-II (Mr 55 000), each of which facilitates the transfer of radiolabelled cholesteryl ester, triacylglycerol and phosphatidylcholine between plasma lipoproteins, were purified from human plasma. Immunoglobulin G was prepared from goat antiserum to human lipid transfer protein-I (i.e., anti-human LTP-I IgG). The progressive addition of anti-human LTP-I IgG to buffered solutions containing either a highly purified mixture of human lipid transfer protein-I and lipid transfer protein-II, or highly purified rabbit lipid transfer protein (Abbey, M., Calvert, G.D. and Barter, P.J. (1984) Biochim. Biophys. Acta 793, 471-480) resulted in specific immunoprecipitation and the removal of increasing amounts, up to 100%, of cholesteryl ester, triacylglycerol and phosphatidylcholine transfer activities. However, similar precipitation studies on human and rabbit lipoprotein-free plasma resulted in the progressive removal of all cholesteryl ester and triacylglycerol transfer activities but only 30% (human) or 20% (rabbit) of phosphatidylcholine transfer activity. In all cases more anti-human LTP-I IgG was required to precipitate rabbit lipid transfer activity than human lipid transfer activity. These results suggest that lipid transfer protein-I and lipid transfer protein-II have antigenic sites in common, allowing precipitation of both proteins by specific antibody to lipid transfer protein-I. Most plasma phosphatidylcholine transfer activity is mediated by a protein (or proteins) other than lipid transfer protein-I and lipid transfer protein-II. In lipoprotein-free plasma all cholesteryl ester and triacylglycerol transfer activity, and some phosphatidylcholine transfer activity, is mediated by lipid transfer protein-I (or lipid transfer protein-I and an antigenically similar protein, lipid transfer protein-II.

Changes in lipid and apolipoprotein composition of pig lipoproteins facilitated by rabbit lipid transfer protein.

A lipid transfer protein has been purified from the lipoprotein-free fraction of rabbit plasma. Rabbit lipid transfer protein, which was purified 600-700-fold with a 4% recovery, has an apparent Mr of 68 000 and facilitates the transfer of isotopically labelled cholesteryl ester, triacylglycerol and phosphatidylcholine between low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Rabbit lipid transfer protein, which appears to be very similar to the cholesteryl ester exchange protein previously purified from human plasma, was incubated with pig plasma at 37 degrees C for up to 6 h. Analysis of very-low-density lipoprotein (VLDL, d less than 1.006 g/ml), LDL (d 1.019-1.063 g/ml) and HDL (d 1.090-1.21 g/ml) after incubation showed that lipid transfer protein had a marked effect on the composition of the lipoprotein classes. The VLDL became enriched with cholesteryl ester and depleted of triacylglycerol. The LDL and HDL became enriched with triacylglycerol. In addition to these changes in lipid composition there were also changes in apolipoprotein composition. The most prominent change in apolipoprotein distribution was a marked increase in the apolipoprotein E content of LDL which was observed only after incubation in the presence of lipid transfer protein.

Pathways for the incorporation of esterified cholesterol into very low density and low density lipoproteins in plasma incubated in vitro.

In vitro incubations of human or pig plasma containing a tracer amount of [3H]cholesterol have been performed to determine which lipoprotein fractions are the immediate recipients of the esterified cholesterol formed in the reaction catalysed by lecithin: cholesterol acyltransferase. In pig plasma, which is deficient in activity of the protein which promotes transfer of esterified cholesterol between different lipoprotein fractions, 87-90% of the lecithin: cholesterol acyltransferase-derived esterified cholesterol was incorporated into the high density lipoprotein (HDL) fraction. In human plasma there was an initial recovery of more than 80% in HDL, although the proportion recovered in very low density lipoproteins (VLDL) and low density lipoproteins (LDL) became progressively greater with increasing duration of incubation, consistent with a transfer from an HDL -esterified cholesterol pool of increasing specific activity. Nevertheless, as in the pig plasma incubations, there was evidence that some 10-15% of the esterified cholesterol formed in the lecithin: cholesterol acyltransferase reaction was incorporated directly into human VLDL and LDL. In quantitative terms, however, it was found that most of the esterified cholesterol delivered to human VLDL and LDL was the result of transfers from HDL rather than as a direct incorporation from its site of synthesis.

Detection of lipid transfer protein activity in rabbit liver perfusate.

Cholesteryl ester, triacylglycerol and phospholipid transfer activity was detected in rabbit liver perfusate after 2 h perfusions in situ. Lipoproteins were removed from the perfusate plasma by ultracentrifugation prior to hydrophobic interaction chromatography of the lipoprotein-free perfusate. The hydrophobic protein, eluted with water from a Phenyl-Sepharose column, facilitated the transfer of radiolabelled cholesteryl ester, triacylglycerol and phosphatidylcholine from low-density lipoprotein to high-density lipoprotein during 3 h incubations at 37 degrees C. These results suggest that rabbit plasma lipid transfer protein is produced by the liver.

Dietary variables and glucose tolerance in pregnancy.

OBJECTIVE: To investigate relationships between dietary macronutrient intakes and glucose tolerance in pregnancy RESEARCH DESIGN AND METHODS: Nulliparous pregnant Chinese women diagnosed with gestational diabetes mellitus (GDM) (n = 56) were compared to age-, gestational age-, height-, and parity-matched groups with normal glucose tolerance (n = 77) and glucose intolerance (IGT) (n = 38) based on the results of an oral glucose tolerance test (National Diabetes Data Group criteria), performed between 24 and 28 weeks of pregnancy. A 24-h recall dietary assessment was also obtained at the time of screening. RESULTS: Subjects with IGT and GDM were significantly heavier (66.1 +/- 1.4 and 68.6 +/- 1.2 kg, respectively, mean +/- SEM) (P < 0.0001) than the normal group (61.2 +/- 1.8 kg) and had a higher BMI. Overall energy intake was similar between groups, as were the intakes of each macronutrient (%kcal). However, there was a highly significant reduction in polyunsaturated fat intake in the IGT and GDM groups whether expressed as %kcal, % of total fat, or fat kcal. This effect was independent of body weight or BMI whether assessed by ordinal logistic regression or by analysis of a weight- and BMI-matched subgroup of the subjects (P = 0.002 for %kcal; n = 47 normal, 26 IGT, and 43 GDM subjects). In logistic regression analysis of the complete data set, increased body weight (P < 0.0001) and decreased polyunsaturated fat intake (P = 0.0014) were both independent predictors of glucose intolerance (IGT and GDM), as were increased body weight and a low dietary polyunsaturated to saturated fat ratio. CONCLUSIONS: Increased polyunsaturated fat intake is associated with a reduced incidence of glucose intolerance during pregnancy. This finding may have major implications for dietary management of women with or at risk of developing GDM.

Adaptive effects of dietary ethanol in the pig: changes in plasma high-density lipoproteins and fecal steroid excretion and mutagenicity.

Six young mature male pigs were maintained on a high fat, low fiber "Western" type diet. Substitution of ethanol for sucrose raised plasma total cholesterol, an increase that was solely due to a rise in high-density lipoproteins. Plasma triacylglycerols and apo-B concentrations were unchanged and although apo-A1 rose with ethanol, this was not statistically significant. Ethanol did not alter total fecal steroids but both bile acids and the ratio of bile acids/neutral sterols were increased. In fecal extracts from these animals, mutagenic activity in the Ames bacterial test was also raised. The data are discussed in relation to the relationships between dietary ethanol and coronary heart disease and colorectal cancer.

Effects of dietary saponins on fecal bile acids and neutral sterols, plasma lipids and lipoprotein turnover in the pig.

Four young mature male pigs, 110 to 120 kg of body weight, were maintained on a low (0.01%) cholesterol diet. A double changeover design was used so that at any time two pigs received additionally 20 g/day of saponins as a 0.33% solution in drinking water. Saponins raised concentrations of fecal bile acids and neutral sterols and increased the contribution of primary acids to excretion. Neither the concentration of total plasma cholesterol nor low-density and high-density lipoprotein cholesterol were affected by saponins. There was also no change in either absolute or fractional catabolic rate of low-density or high-density lipiproteins. The data are discussed in relation to the effects of cholestyramine on plasma cholesterol and bile acid excretion in the pig and to the possible role of saponin-containing foods in the control of plasma cholesterol in man.

Properties of two pig low density lipoproteins prepared by zonal ultracentrifugation.

Pig plasma lipoproteins were separted into four density classes (very low density, two low density and high density lipoproteins, VLDL, LDL1, LDL2 and HDL respectively) from 670 ml plasma by ultracentrifugation in a continuous density gradient using the Spinco Ti15 zonal rotor. LDL1 and LDL2 were partly characterised. LDL1 and LDL2 are beta-migrating lipoproteins of different size and hydrated density; they are similar to human LDL2 and LDL3 respectively. Pig plasma contains about twice as much LDL1 as LDL2. LDL1 migrates at Sf 4.9 (modal value), and has a mean diameter of 217 A and a modal density of 1.035 g/ml (range 1.03-1.04 g/ml). LDL2 migrates at Sf 1.8 and has a mean diameter of 195 A and a density of 1.050 g/ml. Both lipoproteins are precipitated by heparin and Mn++ or by dextran sulphate and Ca++. The apoproteins of LDL1 and LDL2 are both largely insoluble in 8 M urea solution. When dissolved in 1% sodium dodecyl sulphate solution and electrophoresed on polyacrylamide gel at pH 7.0, the apoproteins of LDL1 and LDL2 formed a pattern of multiple bands of high molecular weight similar to that obtained from the apoprotein of human LDL. Both LDL1 and LDL2 share a major antigen with each other and with VLDL; in this respect again they resemble human LDL. The amino acid compositions of LDL1 and LDL2 are very similar. We concluded that the apoprotein moieties of pig plasma LDL1 and LDL2 are probably identical, and similar to apoprotein B in human serum. Zonal ultracentifugation has proved to be a rapid and effective method for isolating large quantities of these two lipoprotein classes for further metabolic studies. This method allows rapid bulk preparation of lipoproteins, and provides a record of their distribution and quantity in a continuous density gradient.

In vivo metabolism of esterified cholesterol and apoproteins in rabbit plasma low density lipoproteins.

Preparations of rabbit low density lipoproteins (LDL) labelled with 3H in the esterified and free cholesterol moieties and with 125I in the apoprotein moiety were injected intravenously into other rabbits. A substantial proportion of the esterified [3H]cholesterol removed from LDL during the first 30 min was recovered in the high density lipoprotein (HDL) fraction, while no such transfer of labelled apoprotein was observed. There was a clear cut differential in the decay of the different LDL components, slowest for the apoprotein, intermediate for esterified cholesterol and fastest for free cholesterol.

The plasma and tissue turnover and distribution of two radio-iodine-labelled pig plasma low density lipoproteins.

Two classes of pig plasma low density lipoprotein (LDL1 and LDL2) with different densities and molecular sizes were isolated by zonal ultracentrifugation and were further purified by flotation. The peptide component was iodinated with 125I, and the labelled lipoprotein was injected intravenously. 125I-LDL1 turnover studies were performed on 22 3-4 month old female Large White pigs, and 125I-LDL2 turnover studies on 4 similar pigs. A biological screening experiment confirmed that the shape of the plasma activity curve was not a function of protein denaturation. The pattern of radioactivity decline in plasma was not affected by the degree of LDL iodination. 125I-LDL1 turnover: The curve of plasma radioactivity plotted against time over the first 5 days after injection could be resolved into two exponentials. The plasma biological half-life (T 1/2) was calculated from the slower exponential predominant from the second day. The mean T 1/2 over 2-5 days was 22.9 hr (range 17.2-28.5 hr). Multicompartmental analysis of the plasma decay curve using an open mammillary model gave a mean fractional catabolic rate per day for LDL1 of 1.4 (range 0.9-1.9). The mean T 1/2 was 0.26-0.31 times and the fractional catabolic rate 3.0-3.9 times those values found in two studies on adult humans. The tissue distribution of 125I was analysed in a series of 20 animals killed from 1.0 to 33.8 days after 125I-LDL1 injection. Most tissue 125I (86-89%) was protein bound. An appropriate correction was made to the 125I counts for retained plasma in liver and spleen (using 131I-albumin); retained plasma in other tissues was negligible. Highest 125I tissue levels were found in the liver, supporting other evidence that the liver may be the major site of LDL1 catabolism. After 2.06 and 4.06 days the livers in two animals contained 1.6% and 0.7% respectively of the total injected 125I, equal to 33% and 54% of the total plasma 125I at those times. The skin contained about one-third to one-ninth the 125I in the liver at various times. Distribution in other organs was quantitatively minimal. Higher levels of radioactivity were found in the intima and inner media of the aorta than in the outer media. These results suggest that plasma LDL in the pig diffuses through the endothelial surface into the arterial wall. These findings are confirmed by autoradiography. 125I-LDL2 turnover: Parallel studies of plasma 125I-LDL2 turnover and tissue distribution were performed. The plasma biological decay curve was multi-exponential, suggesting that LDL2 metabolism is complex, and possibly more rapid than that of LDL1 (LDL2 is smaller and denser than LDL1). The tissue distribution of 125I-LDL2 in these pigs was very similar to that of 125I-LDL1. As LDL1 and LDL2 differ in the amount of lipid they contain, they may have different roles to play in lipid transport, and there may be interconversion of one into the other at different sites. This hypothesis remains conjectural.

Studies of esterified cholesterol in sub-fractions of plasma high density lipoproteins.

With human lipoproteins and lipoprotein-free plasma incubated at 37 degrees C as a source of esterified cholesterol transfer activity, there was a molecular exchange of esterified cholesterol between the high density lipoprotein (HDL) subfractions, HDL2 (density 1.063-1.125 g/ml) and HDL3 (density 1.125-1.21 g/ml). A transfer of esterified cholesterol from both HDL2 and HDL3 to very low density lipoproteins (VLDL) was also observed. When human plasma was incubated with [3H]cholesterol at 37 degrees C, the newly formed esterified [3H]cholesterol became distributed among all plasma lipoprotein fractions. After 24 h the relative specific activities of esterified cholesterol in HDL3, HDL2, low density lipoproteins (LDL) and VLDL were 1.0, 0.81, 0.77 and 0.79, respectively. In comparable 24-h incubations of the plasma of pigs, a species found to have a very low level of plasma esterified cholesterol transfer activity, the relative esterified cholesterol specific activities in HDL3, HDL2, LDL and VLDL were 1.0, 0.31, 0.12 and 0.01, respectively. However, the addition of rabbit lipoprotein-free plasma as a source of exogenous esterified cholesterol transfer activity to incubations of pig lipoproteins resulted in a distribution of the newly formed esterified [3H]cholesterol which was very similar to that in incubated human plasma. It has been concluded that the formation of plasma esterified cholesterol occurs in the HDL3 subfraction. In man, who possesses adequate levels of esterified cholesterol transfer activity, the esterified cholesterol so formed becomes distributed among all plasma lipoprotein fractions. In pigs, however, which lack transfer activity, the esterified cholesterol formed in HDL3 is only minimally transferred to other fractions and remains predominantly in HDL, resulting in an increase in the HDL particle size with a consequent conversion of HDL3 to the larger HDL2.

Role of dietary factors: macronutrients.

Insulin resistance is an important early marker of the metabolic syndrome disease cluster. Our understanding of the role of dietary macronutrients in the etiology of insulin resistance is currently limited by a paucity of credible intervention studies in humans. In contemplating such studies there are many issues that need consideration from actual study design (e.g., duration of intervention, study population, cross-over or not, nutrient formulation) to practical issues such as palatability and compliance (i.e., that terribly important issue of achievability because realistically individuals must be "free range" in order to complete studies of sufficient duration). Initiatives to support well-designed multicenter studies on diet and insulin resistance would have a major impact on our ability to treat, but more importantly to prevent, the metabolic syndrome diseases.

Attracting and training more chemical pathologists in the United Kingdom.

I have attempted to define the function of the medical graduate in the clinical biochemistry laboratory and have examined data on recrutiment in the United Kingdom into clinical biochemistry. If trainee pathologists were encouraged to become proficient in both a branch of clinical medicine and in research techniques, the resulting chemical pathologists should be able to improve the consultative and investigative functions of the laboratory. To this end I have suggested some changes in the training regulations and in the role of the chemical pathologists.

Does dietary fat influence insulin action?

What is clear from the research thus far is that dietary fat intake does influence insulin action. However, whether the effect is good, bad, or indifferent is strongly related to the fatty acid profile of that dietary fat. The evidence has taken many forms, including in vitro evidence of differences in insulin binding and glucose transport in cells grown with different types of fat in the incubation medium, in vivo results in animals fed different fats, relationships demonstrated between the membrane structural lipid fatty acid profile and insulin resistance in humans, and finally epidemiological evidence linking particularly high saturated fat intake with hyperinsulinemia and increased risk of diabetes. This contrasts with the lack of relationship, or even possible protective effect, of polyunsaturated fats. In particular, habitual increased n-3 polyunsaturated dietary fat intake (as fish fats) would appear to be protective against the development of glucose intolerance. It is reassuring that the patterns of dietary fatty acids that appear beneficial for insulin action and energy balance are also the patterns that would seem appropriate in the fight against thrombosis and cardiovascular disease. Mechanisms, though, still need to be defined. However, there are strong indicators that defining the ways in which changes in the fatty acid profile of membrane structural lipids are achieved, and in turn influence relevant transport events, plus understanding the processes that control accumulation and availability of storage lipid in muscle may be fruitful avenues for future research. One of the problems of moving the knowledge gained from research at the cellular level through to the individual and on to populations is the need for more accommodating research designs. In vitro studies may provide in-depth insights into intricate mechanisms, but they do not give the "big picture" for practical recommendations. On the other hand, correlational studies tend to be fairly blunt instruments, requiring large numbers that are very often not feasible if a greater depth of understanding of the biological processes is to be incorporated. There may be benefit in turning to the clinical case study as a framework for a more comprehensive analysis of the links between dietary fats and insulin action. The real challenge is to keep the depth of analysis rigorous enough to be able to explain and accommodate individual variation (i.e., the diversity of both environmental and genetic backgrounds) while at the same time satisfying the cultural need to provide appropriate overall dietary guidelines. Finally, David Kritchevsky brought to our attention a delightful quote from Mark Twain: "There is something fascinating about science. One gets such a wholesale return of conjecture for such a trifling investment of fact." In the field of dietary fats and the Metabolic Syndrome, this quotation is, unfortunately, apt. Much more research is necessary to define how dietary fats really work to affect insulin action. Well designed, long-term studies in "free range" humans must be undertaken if dietary guidelines for the Metabolic Syndrome are to be based on anything more than a "trifling" amount of "fact."

A physiologic role for the esterified cholesterol transfer protein: in vivo studies in rabbits and pigs.

The observation that pig plasma is deficient in esterified cholesterol transfer activity has been exploited in an attempt to establish an in vivo role for the esterified cholesterol transfer protein. The plasma high density lipoproteins (HDL) of pigs and also of rabbits (a species known to possess an active esterified cholesterol transfer protein) were labeled with 3H in the esterified cholesterol moiety and with 125I in the apoprotein moieties and reinjected into the respective species. In both rabbits and pigs, the removal of 125I from the recipient HDL fraction was parallel to that from the whole plasma, with negligible 125I appearing in other plasma lipoprotein fractions. In the pig, the removal of esterified 3H-cholesterol from the recipient HLD was very similar to that of 125I; there was only minimal appearance in other lipoproteins. In the rabbit, however, there was a major in vivo transfer of esterified 3H-cholesterol from HDL to other fractions. It has been concluded that an active esterified cholesterol transfer protein is probably necessary to achieve the in vivo transfer of esterified cholesterol from HDL to other plasma lipoproteins.

Fatty acids, triglycerides and syndromes of insulin resistance.

Muscle plays a major role in insulin-stimulated glucose disposal. There is now a range of evidence in humans and experimental animals demonstrating strong relationships between the fatty acid composition of structural membrane lipids and insulin action. The in vivo work is correlative but the in vitro studies suggest a causal relationship exists. Good insulin action is associated with an increased proportion of n-3 fatty acids, low saturates, a low n-6/n-3 ratio and possibly increased monounsaturates. What is reassuring is that there is a pleasing symmetry with the fatty acid pattern that might lead to decreased thrombosis. There is little argument about saturated fats with a reduction having a range of beneficial effects. However, the n-3 fatty acids might also be a key to amelioration of both insulin resistance and thrombosis. The sites of action of n-3s are multiple: decreased triglyceride and VLDL production; inhibition of thromboxane A2 production, increased thromboxane A3 and decreased platelet aggregation; reduction of triglyceride and VLDL concentration; improved blood rheology and membrane transport; action on the endothelium and proliferation of the intimal cells, and improvement of vascular tone. The data here are now strong and reasonably consistent. Similarly, after initial controversy, the evidence for n-3s playing a beneficial role in insulin action is now accumulating. The n-6 PUFAs are a bit of a worry: while arachidonic acid levels in muscle phospholipid has linked positively to insulin action in our studies, linoleic is negative. Linoleic acid, in high amounts, is known to inhibit the delta6 fatty acid desaturase enzyme and with the competition between n-6 and n-3 fatty acids for the enzymes of desaturation and elongation it does focus on a high n-6/n-3 ratio as a critical factor in both insulin resistance and atherosclerosis.

The effect of bile salts on the esterolytic assay of trypsin.

A bile salt mixture and pure sodium taurocholate were each shown to increase the esterolytic activity of trypsin in aqueous solution and in intestinal juice. rho-Toluene-sulphonyl-L-arginine methyl ester (TAME) was used as a substrate, and both a spectrophotometric and a potentiometric assay system were used. The maximal potentiation of the esterolytic activity of trypsin by bile salts was about 1.6 to 2.2 times the activity without bile salts (depending on the assay conditions and whether the trypsin was in aqueous solution or intestinal juice). The proteolytic activity of trypsin was decreased by the addition of bile salts. It seemed likely, therefore, that the potentiating effect of bile salts on trypsin esterolytic activity is primarily on the substrate (TAME) rather than trypsin itself. It was thought that TAME might be taken up into bile salt micelles and thus be more readily hydrolysed by trypsin, but we were unable to substantiate this hypothesis. The precision of the trypsin esterolytic assay was better when bile salts were not added. If however bile salts were to be used routinely in the trypsin assay, it would be useful to ensure that the concentration of calcium, included as activator, is sufficiently low to prevent the formation of a precipitate. This precipitate is probably a complex of calcium and bile salts.

Electroimmunoassay of a subunit protein in a macromolecular complex (apolipoprotein B in human plasma very low density lipoprotein); implications for other electroimmunoassay systems.

With an electroimmunoassay ("rocket") system for the apolipoprotein B component of the plasma very low density lipoprotein complex we obtained results which were similar to those obtained by a colorimetric tetramethylurea extraction method. Results were up to twice as high as those using radioimmunoassay. Low density lipoprotein containing apolipoprotein B as the only demonstrable protein component was used as the standard for these assays. This protein produced larger and higher rockets at pH 8.6 when the negative particle charge was increased by maleylation. The very low density lipoprotein complex has a higher negative charge at pH 8.6 than low density lipoprotein. These findings suggest that some apolipoprotein B in vary low density lipoprotein is not "recognised" by anti-apolipoprotein B antibodies, hence radioimmunoassay results are lower than those obtained with the tetramethylurea extraction method. The higher negative charge on very low density lipoprotein particles (compared with low density lipoprotein), as a factor tending to increase rocket area and height, is counterbalanced by reduced recognition by antiapolipoprotein B antibodies. The net result of these opposing tendencies is that the rocket electroimmunoassay of apolipoprotein B in very low density lipoprotein fortuitously gives valid results, under the specified assay conditions. We conclude that electroimmunoassays of complex proteins are not necessarily valid if protein subunits are used for standards. This has implications for the electroimmunoassay of other apolipoproteins.