By isoelectric focusing of delipidated sera followed by immunoblotting we studied the apolipoprotein (apo) E polymorphism in 2018 randomly selected 35-years-old males from three different areas in the Netherlands. Comparison of the APOE allele (E*2, E*3, and E*4) frequencies estimated in this study with those reported for several other population samples showed that there are marked differences between the Dutch population and the populations of Japan, New Zealand, Finland, and the United States. These differences in APOE allele frequencies appeared to be mainly due to differences in frequencies of the E*2 allele (decreased in Japan and Finland; increased in New Zealand) and the E*4 allele (increased in Finland; decreased in Japan and the United States). No difference in APOE allele frequencies was found between the Dutch population and the populations of West Germany and Scotland. Measurements of plasma cholesterol and apo B and E concentrations showed that the E*4 allele is associated with elevated plasma cholesterol and apo B levels and with decreased apo E concentrations, whereas the opposite is true for the E*2 allele. In the Dutch population, the sum of average allelic effects of the common APOE alleles on plasma cholesterol and apo B levels is 6.8% and 14.2%, respectively, of the total population mean. The total average allelic effect on plasma apo E concentrations was more pronounced (50.1%), suggesting that the APOE alleles primarily affect apo E concentrations rather than plasma cholesterol and apo B levels. This hypothesis is sustained by the observation that for plasma apo E levels the genetic variance associated with the APOE gene locus contributed about 18% to the total phenotypic variance. For plasma cholesterol and apo B this contribution was only 1.4% and 2.3% and is relatively low as compared with that reported for other population samples.

ABSTRACT. In this study the lipid and apoprotein profiles were investigated in newborns at 0, 7, and 30 days of life. The plasma lipoproteins were separated both by ultracentrifugation and gel filtration in order to compare the patterns obtained by the two techniques. At birth, the apo E concentration is comparable to that measured in adults, but its distribution among lipoproteins is significantly different as more than 80% of the plasma apo E belongs to high-density lipoproteins (HDL). At 7 and 30 days the plasma apo E concentrations are close to the values at birth, but a significant redistribution occurs from HDL to very low-density lipoproteins. By analogy with apo B, the plasma apo CIII concentration is low at birth and increases between 0 and 7 days by a factor of about two. Plasma triglycerides increase significantly during the first week of life so that the apo CIII increase is most pronounced in very low-density lipoproteins. These lipoproteins therefore become enriched in apo E, apo CIII and triglycerides between 0 and 7 days. At birth, a distinct HDL fraction, enriched in apo E, apo A11 and cholesterol (HDLE), could be detected. To compensate for the low LDL levels, this HDLE fraction might function as an additional source for cholesterol delivery to peripheral tissues via the apo (B, E) receptor. At later age, low-density lipoprotein synthesis is enhanced, apo E is transferred to very low-density lipoproteins, and cholesterol delivery via the HDL, becomes less important. These data demonstrate that significant differences occur in the plasma concentration and distribution of the apo CIII and E proteins during the initial period of life, and that these apoproteins fulfill an important metabolic role. (Pediatr Res 20: 324-328, 1986) Abbreviations HDL, high-density lipoproteins VLDL, very low-density lipoproteins LDL, low-density lipoproteins During the perinatal period, major shifts in nutritional supply cause changes in substrate utilization by the fetus and newborn. Unimpeded substrate flow from placenta to fetus is required to meet the energy requirements for growth and for fuel storage, primarily of glycogen and fat. During the intra-and postpartum periods, this constant flow is interrupted so that glycogen is required to maintain serum glucose levels. Glycogen stores are then depleted and active glycogenesis starts. In the perinatal period, fatty acid and ketone body oxidation become important energy sources, and during this period an adaptation to a high fat milk diet becomes therefore necessary.These adaptations are controlled by changes in substrate flow and hormonal milieu of the fetus and newborn. Such changes n substrate result in rapid shifts in the synthesis and metabolism of lipid transporting particles, whose composition then changes from that before birth.In newborns, plasma lipid and lipoprotein patterns also are significantly different from those observed in adults, both in their concentration and distribution. Newborn plasma lipoproteins consist mostly of HDL, while VLDL and LDL are...

Summarvborn infants were selected and neonates with history of chronic Lipoproteins in newborn plasma were isolated from a minimal sample amount (0.3 ml) by a single-step ultracentrifugation in a density gradient, spanning the density range 1.02-1.20 g/ml. After 66 h ultracentrifugation in a swinging-bucket rotor, the content of the tube was eluted and collected in 0.4 ml fractions. Cholesterol and apoproteins AI, AII, and B were assayed in each fraction yielding both the distribution and composition of the very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL2, and HDL3). Newborn plasma was characterized by a low amount of triglyceride-poor and cholesterol ester-rich VLDL and high content of HDLz and HDL3. The VLDL and LDL concentrations increased drastically between 0 and 7 days together with the triglyceride content of the VLDL.At 30 days the lipid composition of VLDL was similar to that of adults, whereas the lipid/protein content remained low both in VLDL and LDL. The composition of HDL2 and HDL3 remained constant during this period, the percentage of HDL2 being higher in newborns than in adults. These compositional changes were reflected in the microviscosity of the lipoproteins, specially in the VLDL fraction. The investigation of plasma lipoprotein profiles in newborns is mostly limited by the available volume of the samples. Conventional isolation techniques, by sequential ultracent;ifugation, for VLDL, LDL, HDL and their subfractions require 5 ml plasma whereas only 1-2 ml blood can easily be drawn by venapuncture in newborns. Recently, Nillson et al. (10) have adapted the singlespin ultracentrifugation procedure, previously described by Foreman et al. (5), to the analysis of 200-1000 p1 plasma. We have applied this procedure to the quantitation of the lipoprotein profiles in newborns at 0, 7 and 30 days in order to obtain the compositional analysis of the various lipoprotein fractions. As previously shown (17, 18) the lipoprotein patterns in newborns are profoundly modified during the first month of life when several synthetic and catabolic pathways are developing.The single-spin ultracentrifugation procedure enables further investigation of these metabolic events and of their impact on the plasma lipoprotein distribution and composition.maternal-foetal problems such as diabetes or hypeiension were not included in the study. All infants were born after uneventful delivery. All subjects were investigated after informed consent had been obtained from their parents. A blood sample was obtained, immediately after delivery and clamping of the umbilical cord, from the umbilical vein. Blood samples at 7 and 30 days were drawn by venapuncture after 7 h fasting. Blood was allowed to clot at room temperature for about 30 min and the serum separated at 4°C at 10,000 rpm. Serum samples were stored at 4OC in the presence of 0.05% EDTA pH 7 for maximum 5 days.Density gradient ultracentrifugation. The single-spin density ultracentrifugation was based on the method proposed...

Siunmary:A sandwich enzyme linked immunosorbent assay (ELISA) for apolipoprotein C-III was developed. Anti apolipoprotein C-III immunoglobulins were used both for coating of microtitre plates and for the preparation of an anti apolipoprotein C-III horse-radish peroxidase conjugate.Under optimized assay conditions, the sensitivity lies around 0.3 ng apolipoprotein C-III with a working ränge of l to 6 ng. Standard curves are parallel for purified apolipoprotein C-III, for untreated plasma and for lipoprotein fractions. Delipidation did not affect the content of apolipoprotein C-III in plasma. The assay was evaluated by comparison with an immunonephelometric assay for apolipoprotein C-III, yielding a correlation coefficient of 0.982 (n = 79). The mean intra-and interassay CV for the whole working ränge of the assay were 3.6% and 4.2% respectively. Enzyme linked immunosorbent assay (ELISA) für menschliches Apolipoprotein C-III

Summary:A specific, sensitive and accurate, non competitive enzyme-linked immuriosorbent assay was developed for the quantitätion of human apolipoprotein C-II. Using apolipoprotein C-II and apolipoprotein C-III immunosorbent columns, monospecific anti-apolipoprotein C-IFantibodies were prepared for coating and for the preparation of a peroxidase-antibody conjugate. The assay is sensitive down to 0.25 ng apolipoprotein C-II per assay and precise, with mean intra-and inter-assay coefficients of Variation of 3.1% and 7.9% respectively. The apolipoprotein C-II concentrations in normolipaemic and hyperlipaemic plasma were not affected by delipidation, and increased only slightly after treatment with detergents or urea.The mean plasma apolipoprotein C-II concentration in a group of 30 normolipaemic subjects, was 33.1 ± 7.5 mg/1. All hypertriglyceridaemic subjects had significantly elevated apolipoprotein C-II plasma concentrations, which were most pronounced in Fredrickson type III and type V patients.The apolipoprotein C-II profiles, obtained by column fractionation of 6 normolipaemic and 11 hypertriglyceridaemic plasmas, demonstrated a shift of apolipoprotein C-II towards the triglyceride-rich lipoproteins in hypertriglyceridaemic subjects. Bestimmung von Apolipoprotein C-II vom Menschen mit einem Sandwich-Immunosorbent-AssayZusammenfassung: Ein spezifischer, empfindlicher und genauer nicht-kompetitiver Enzyme-Linked-Immunosorbent-Assay wurde für die Quantifizierung von menschlichem Apolipoprotein C-II entwickelt. Mit Apolipoprotein C-II und Apolipoprotein GJII Immunosorbent-Säulen wurden monospezifische anti-Apolipoprotein C-II-Antikörper für die Beschichtung sowie ein Peroxidase-Antikörper-Konjugat hergestellt. Die Empfindlichkeit des Bestimmungsverfahrens beträgt bis zu 0,25 ng Apolipoprotein C-II. Der Assay ist präzis mit mittleren Variationskoeffizienten von 3,1% innerhalb der Serie und 7,9% von Tag zu Tag.Der Apolipoprotein C-II-Gehalt in normolipämischen und hyperlipämischen Plasmen wurde von der Lipidabtrennung nicht beeinflußt und nahm nach Behandlung mit Detergentien oder Harnstoff nur ein wenig zu. Die mittlere Apolipoprotein C-II-Konzentration im Plasma einer Gruppe von 30 normolipämischen Probanden war 33,1 ± 7,5 mg/1. Alle hypertriglyceridämischen Probanden hatten signifikant erhöhte Apolipoprotein C-II Konzentrationen im Plasma, besonders bei Fredrickson Type III und Type V. Die Apolipoprotein C-IIProfile, die durch Saulenfraktionierung von 6 normolipämischen und 11 hypertriglyceridämischen Plasmen erhalten wurden, zeigten eine Verschiebung von Apolipoprotein C-II zu den triglyceridämischen Lipoproteinen in hypertriglyceridämischen Plasmen.

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This new, sensitive, specific "sandwich"-type enzyme-linked immunosorbent assay (ELISA) for quantifying lipoprotein(a) [Lp(a)] in human serum and in ultracentrifugal lipoprotein fractions is based on use of a monoclonal antibody raised against apolipoprotein(a) as coating protein and a polyclonal antibody, raised against either apo B or against Lp(a) and conjugated with peroxidase, for detection of bound Lp(a). Mean intra- and interassay CVs for assay of 16 samples were 3.0% and 5.6%, respectively. Sample pretreatment with urea did not enhance Lp(a) immunoreactivity, and treatment with nonionic detergents decreased binding to the monoclonal antibody. Results correlated well (r = 0.99, n = 38) with those by radial immunodiffusion (RID). The ELISA assay, however, detects amounts corresponding to Lp(a) contents of 10 to 1000 mg/L in plasma samples diluted 1000-fold, compared with 100-500 mg/L for RID. For 92 normolipidemic subjects, the mean Lp(a) concentration was 120 (SD 130) mg/L. In patients undergoing coronary angiography, Lp(a) concentrations increased with the severity of the disease but were not correlated with either HDL cholesterol, triglycerides, apo A-I, or apo B, and only weakly with plasma cholesterol and apo A-II. These two correlations were even weaker in normal subjects, and only the correlation with total cholesterol was valid. Lp(a), measured at birth and at seven days and six months, steadily increased with age. This assay is well suited for measuring Lp(a) in plasma and in lipoprotein fractions and also for screening programs evaluating this significant genetic risk factor for the development of atherosclerosis.

A specific and sensitive "sandwich"-type enzyme-linked immunosorbent assay (ELISA) has been developed for quantifying human apo A-IV. Using apo A-IV immunosorbent columns, we isolated monospecific anti-apo A-IV antibodies for coating the ELISA plates and for preparing peroxidase-antibody conjugate. The assay can detect as little as 0.20 ng of apo A-IV, with mean intra- and interassay CVs of 3.6% and 8.2%, respectively. The apoA-IV concentrations in normolipemic and hyperlipemic plasma were unaffected by either delipidation or treatment with detergents or urea. To validate the ELISA assay we compared it with an immunoelectrophoretic technique. ApoA-IV concentrations in plasma from normo- and dyslipemic subjects compared well by the two assays (r = 0.89). The mean apo A-IV concentration, measured by ELISA in plasma from 50 normolipemic subjects, was 143 (SD 52) mg/L; values for dyslipemic subjects were not significantly different. We also used this new assay to monitor apo A-IV profiles of normolipemic and hypertriglyceridemic plasma after chromatographic fractionation.