In spite of its strong familiality, gene identification for coronary artery disease (CAD) has not yielded a consistent picture. One major reason for this is that families or cases and controls were not recruited from a homogeneous population. We, therefore, attempted to map genes underlying 10 quantitative traits (QTs) that are known precursors of CAD in a homogeneous population (Marwari) of India. The QTs are apolipoprotein B (ApoB), C-reactive protein (CRP), fibrinogen (FBG), homocysteine (HCY), lipoprotein (a) (LPA), cholesterol - total (CHOL-T), cholesterol - HDL (CHOL-H), cholesterol - LDL (CHOL-L), cholesterol - VLDL (CHOL-V) and triglyceride (TG). We assayed 209 SNPs in 31 genes among members of Marwari families. After log-transformation and covariate-adjustment of the QTs, a two-step analysis was performed. In Step-1, data on unrelated individuals were analysed for association with the SNPs. In Step-2, for validation of Step-1 results, a quantitative transmission-disequilibrium test on parent- offspring data was performed for each SNP found to be significantly associated with a QT in Step-1 on an independent sample set drawn from the same population. Statistically significant results found for the various QTs and SNPs were: rs3774933, rs230528, rs230521, rs1005819 and rs1609798 (intronic, NFKB1) with APOB; rs5361 (Missense, R greatr than S, SELE) and rs4648004 (Intronic, NFKB1) with FBG; rs4220 (Missense, K greater than R, FGB) with HCY; and rs3025035 (Intronic, VEGFA) with CHOL-H. SNPs in SELE, VEGFA, FGB and NFKB1 genes impact significantly on levels of quantitative precursors of CAD in Marwaris.

Apolipoprotein R (apoR) is a 23-kDa protein found on very low-density lipoprotein (VLDL), on chylomicrons, and in the d>> 1.21 g/mL fraction of pig plasma. The plasma concentration of apoR is 5.1 micrograms/mL, with 11.5% of apoR found on VLDL. In vitro, apoR can transfer from the d>> 1.21 g/mL infranatant onto artificial lipid emulsions or human chylomicrons but not onto human VLDL. An apoR cDNA was isolated from a pig liver lambda gt11 expression library. DNA sequence analysis of the apoR cDNA revealed 67% identity with the 3'-terminal region of human C4b-binding protein alpha-chain cDNA (C4BP alpha). C4BP alpha is a 70-kDa glycoprotein that regulates both the coagulation and the complement cascades. In plasma, C4BP alpha exists as disulfide-linked multimers consisting of seven C4BP alpha chains and a single C4BP beta chain. Like C4BP, apoR forms high molecular weight disulfide-linked complexes in plasma. However, unlike C4BP alpha, apoR complexes do not appear to contain C4BP beta. ApoR mRNA was detected in pig liver, spleen, lung, bone marrow, and lymph node, but was absent in intestine and white blood cells. This distribution is consistent with the production of apoR in terminally differentiated macrophages but not in blood monocytes. ApoR mRNA was not detected in RNA isolated from human liver or lung. ApoR may be a lipoprotein-borne regulator of either the coagulation or the complement cascades.

A laser immunonephelometric procedure for the determination of human apolipoproteins A-I, B, and E in serum and lipoproteins was developed. Coefficients of variation were 2.3%-3.4% within-run, 4.7%-4.9% between-run. Serum apolipoprotein A-I values were similar in normal and hyperlipoproteinaemic subjects with a correlation coefficient of r = 0.585 to HDL-cholesterol levels. LDL-apolipoprotein B values were highly (r = 0.935), VLDL-apolipoprotein B values less strongly (r = 0.472) correlated to the corresponding cholesterol levels. Serum apolipoprotein E concentrations were distinctly elevated in Type III and not correlated to cholesterol values.

We evaluated a dual-precipitation method for determining cholesterol in high-density lipoprotein (HDL) and its subfractions HDL2 and HDL3. After total HDL was isolated by precipitation of very-low-density (VLDL) and low-density (LDL) lipoproteins with polyethylene glycol (Mr 8000), HDL2 was isolated from total HDL by precipitation with dextran sulfate (Mr 15,000), leaving HDL3 in the supernate. Concentration of total HDL cholesterol after precipitation of VLDL and LDL with PEG showed significant proportional and constant biases of -3.8% and 0.04 mmol/L, respectively, when compared with a phosphotungstic acid-based comparison method, although results by the two methods were correlated highly (r = 0.99, P less than 0.001). HDL2 and HDL3 cholesterol concentrations measured with the present technique were not different from those obtained by density-gradient ultracentrifugation or by combined precipitation-ultracentrifugation.

Homozygous familial hypercholesterolemia (HoFH) represents the most severe lipoprotein disorder, generally attributable to mutation(s) of the low-density lipoprotein receptor (LDL-R), i.e. autosomal dominant hypercholesterolemia type 1 (ADH1). Much lower percentages are due to alterations of apolipoprotein B (ADH2), or gain-of-function mutations of proprotein convertase subtilisin/kexin type 9 (PCSK9) (ADH3). In certain geographical areas a significant number of patients may be affected by an autosomal recessive hypercholesterolemia (ARH). Mutations may be also combined (two mutations of the same gene, compound heterozygosity), or two in different genes (double heterozygosity). Among the most innovative therapeutic approaches made available recently, inhibitors of the microsomal transfer protein (MTP) system have shown a high clinical potential. MTP plays a critical role in the assembly/secretion of very-low-density lipoproteins (VLDL), and its absence leads to apo B deficiency. MTP antagonists dramatically lower LDL-cholesterol (LDL-C) in animals, although a reported increase of liver fat delayed their clinical development. Lomitapide, the best-studied MTP inhibitor, reduces LDL-C by 50% or more in HoFH patients, with modest, reversible, liver steatosis. Recent US approval has confirmed an acceptable tolerability, provided patients adhere to a strictly low-fat regimen. There are no clinical data on atherosclerosis reduction/regression, but animal models provide encouraging results.

We compared the serum lipoprotein cholesterol concentrations in subgroups of children (n = 360), ages 5-17 years, as measured by the heparin-Ca2+ and preparative ultracentrifugation methods. Children were grouped from the total population on the basis of their previous results for serum beta- and pre-beta-lipoprotein cholesterol (Group I: low beta- and low pre-beta-; Group II: high beta- and low pre-beta; Group III: high beta- and high pre-beta-; Group IV: low beta- and high pre-beta-). The values for very-low-density (VLDL) cholesterol by ultracentrifugation method were 44, 53, 15, and 10 mg/L greater than the values for pre-beta-lipoprotein cholesterol by the heparin-Ca2+ method in Groups I, II, III, and IV, respectively; the differences were not significant in Group IV. The values of low-density (LDL) cholesterol were 64, 137, 144, and 73 mg/L less than the values for beta-lipoprotein cholesterol in Groups I, II, III, and IV, respectively (p less than 0.005). On the other hand, high-density (HDL) cholesterol concentrations in the respective four groups were 10, 37, 93, and 52 mg/L greater than alpha-lipoprotein cholesterol concentrations; the differences were significant for Groups II, III, and IV (p less than 0.005). Overall, the values for LDL-cholesterol correlated highly with beta-lipoprotein cholesterol (r = 0.94), whereas correlations for VLDL- and HDL-cholesterol values with pre-beta-lipoprotein cholesterol (r = 0.76) and alpha-lipoprotein cholesterol (r = 0.77) were somewhat lower. The differences between these two methods may result from their different operational definitions for measuring serum lipoproteins and the possibility that without appropriate corrections the values obtained by preparative ultracentrifugation do not serve as reference values.

The acute effects of intravenous heparin administration (50 U/kg body weight) on apolipoprotein (apo)B-48 and apoB-100-containing lipoproteins in relation to postheparin lipase activities were studied in ten healthy normolipidemic volunteers. Five subjects returned to receive sham injections with saline. Lipoproteins were separated from plasma by density gradient ultracentrifugation at baseline, 3, and 20 min postheparin. ApoB-48 and apoB-100 in d < 1.006 g/mL and 1.006 < d < 1.019 g/mL fractions were quantitatively measured after electrophoresis on 5% SDS polyacrylamide gels and Coomassie-blue staining. No significant changes were observed after saline injections. Heparin administration released lipoprotein lipase (LPL) and hepatic lipase (HL) activities after 20 min, and significantly reduced apoB-48 concentrations in d < 1.006 g/mL fractions only. ApoB-100 concentrations showed a trend to decrease in d < 1.006 g/mL fractions and to increase in 1.006 < d < 1.019 g/mL fractions. LPL activity was related to the percentual disappearance of apoB-48 (r = 0.81, P = 0.004) and apoB-100 (r = 0.91, P < 0.001) in d < 1.006 g/mL fractions. When little LPL was released (LPL activity < 120 mU/mL) by heparin, apoB-48 was preferentially eliminated over apoB-100. However, when abundant LPL was released (LPL activity>> 140 mU/mL), comparable percentual reductions for apoB-48 and apoB-100 were seen. Pharmacokinetic analysis revealed first-order kinetics for the clearance of apoB-48 in d < 1.006 g/mL fractions, but zero-order kinetics for apoB-100 clearance. Under conditions of artificially enhanced lipolysis, the first catabolic step of apoB-48-containing lipoproteins and hepatic VLDL showed different pharmacokinetics. ApoB-48-containing lipoproteins were the preferred substrate for LPL, and only when abundant LPL was present, clearance of hepatic VLDL occurred.

Familial combined hyperlipidemia (FCH) is a heritable lipid disorder that is associated with an increased risk of premature cardiovascular disease. An elevated plasma apolipoprotein (apo) B concentration is reported to be a diagnostic feature of the disorder. Recently we demonstrated a strong relation between plasma apoB concentrations and the cholesterol concentration in VLDL plus LDL, both elevated in FCH families. Therefore, examination of the inheritance of elevated plasma apoB levels in FCH families may reveal important information about the mechanism responsible for the aggregation of elevated plasma lipids in FCH. This study included 663 Dutch family members in 40 families ascertained through FCH probands. Plasma apoB concentration correlated significantly with apoB-related cholesterol both in the probands and the relatives (r=.83 and r=.90, respectively). Adjustment for age, sex, body mass index, and smoking habits accounted for 35.7% of the variation in apoB levels, and there was strong familial aggregation in adjusted apoB levels in these families. Complex segregation analysis was performed to determine the mechanism of inheritance behind this familial aggregation. The aggregation of elevated apoB levels was best explained by a major gene effect inherited by a codominant mechanism. Estimated mean apoB levels for the three supposed genotypes AA, AB, and BB were 111.5, 126.7, and 165.7 mg/dL, respectively, with relative frequencies of 43.5%, 44.9%, and 11.6%, respectively. In conclusion, despite assumed metabolic and genetic heterogeneity of FCH, there is clear evidence for a single gene effect on apoB concentrations in families ascertained through FCH. Linkage studies based on this analysis may further clarify the molecular basis of the apoB regulation in these families.

Hypopituitarism is associated with hyperlipidemia, the mechanisms of which are not fully known. One possible mechanism is an increased hepatic secretion of very-low-density lipoprotein (VLDL) apolipoprotein B100 (apo B100). To investigate this, 13 hypopituitary patients (seven women and six men; age, 46 +/- 3 years [mean +/- SEM]; body mass index [BMI], 29 +/- 2 kg/m2) and 13 matched controls (seven women and six men; age, 43 +/- 3 years; BMI, 28 +/- 2 kg/m2) were investigated in a stable-isotope study. [1-(13)C]leucine (1 mg/kg body weight) was administered, followed by a continuous 6-hour infusion of [1-(13)C]leucine (at a rate of 1 mg/kg/h). Patients had a similar fractional secretion rate (FSR) of VLDL apo B100 versus controls (0.37 +/- 0.05 v 0.38 +/- 0.06 pools/h, respectively), but they had a significantly larger pool size (3.4 +/- 0.3 v 1.9 +/- 0.3 mg/kg) and higher absolute secretion rate ([ASR] 27.8 +/- 2.9 v 16.0 +/- 2.5 mg/kg/d). The increase in hepatic VLDL production may explain the lipid abnormalities found in hypopituitarism. Fasting circulating nonesterified fatty acids (NEFAs) were decreased in the patients (284 +/- 26 v 664 +/- 92 micromol/L, P < .001) despite the increase in VLDL secretion. An inverse relationship was observed between the NEFA level and VLDL apo B100 FSR in the patients (r(s) = -.85, P < .005).

We investigated the reactivities of cholesteryl ester transfer protein (CETP) in Japanese white rabbits fed either a low-cholesterol diet containing 0.1% cholesterol (Control group) or a diet containing 0.1% cholesterol plus 17.5% omega-3 eicosapentaenoic acid (omega-3 20:5, EPA) of 4.5% (w/w) total lipid (EPA group) for 6 weeks. The plasma total and LDL cholesterol levels and aortic cholesterol content were all significantly higher in the EPA group than in the control group. The aortic cholesterol content significantly correlated with LDL cholesterol (r = 0.81). HDL cholesterol levels tended to be lower in the EPA group compared with control group, which was not statistically significant. The plasma VLDL cholesterol levels did not differ significantly between the groups. In addition, no significant differences were observed in the plasma CETP activity or lecithin:cholesterol acyltransferase (LCAT) activity between the groups. However, the cholesteryl ester (CE) mass transfer from fractionated HDL in the EPA group to excess VLDL and/or LDL as acceptors by purified CETP increased significantly compared with the control group, even if the acceptors were fractionated from either the EPA or the control group. Fatty acid analyses of CE showed that the omega-3 18:3, 20:4 or omega-3 20:5 fatty acid acyl groups in CE of HDL were significantly more transferred to apo B-containing lipoproteins compared with the 14:0,16:0, 18:0, 18:1 or 18:2 fatty acid acyl groups in CE of HDL during the incubation period. The amount of CE in HDL containing omega-3 18:3 and omega-3 20:5 fatty acid acyl groups was greater, while the amount of CE containing 18:2 fatty acid acyl groups was smaller in the EPA group than in the control group. These results show that although CETP itself did not change, the transfer of CE in HDL to apo B-containing lipoproteins by CETP increased in the rabbits fed a diet containing EPA as the HDL is modified by the diet, which may partly explain why atherogenicity was thus found to progress in the rabbits fed a cholesterol plus EPA diet.

The effects of triglycerides (TG) from tallow (1.21 and 2.13 g TG/kg of body weight (BW) per meal, diets R and B respectively) and from tallow plus cream (2.50 g TG/kg of BW per meal, diet L) with or without L-methionine (2.6 g/kg dry matter) on hepatic apparent secretion of very low density lipoproteins (VLDL) were investigated in 3 groups of 4 preruminant calves fitted with chronic catheters and with electromagnetic blood-flow probes implanted in their hepatic vessels. Increasing TG concentrations stimulated the apparent VLDL secretion by the liver (1.02, -0.36 and -1.51 mg VLDL mass/min per kg of BW in diets L, B and R, respectively). L-Methionine increased this secretion when associated with the lipid-restricted (diet R; 0.25 and -1.51 mg VLDL/min per kg of BW) and basal (diet B; 0.35 and -0.36 mg VLDL/min per kg of BW) diets (non-significant). However, the VLDL apparent secretion decreased with the lipid-enriched diet (diet L), which suggests an insufficient dose of L-methionine compared with the level of TG intake, and a possible competition between liver and intestine for utilization of L-methionine for the synthesis of TG-rich lipoproteins.

In order to determine the contribution of the low density lipoprotein receptor (LDL-R) to the removal of apoB-containing native lipoproteins by macrophages, we compared the uptake of beta-VLDL in peritoneal macrophages (MPM) from wild type mice and mice lacking the LDL-R. The d<1.006 g/ml lipoproteins obtained from apoE deficient mice fed a high fat diet were poorly degraded by macrophages and caused only a slight formation of CE in macrophages from both types of mice. On the other hand, d<1.006 g/ml lipoproteins obtained from LDL-R deficient mice fed a high fat diet, beta-VLDL with apoE, were avidly taken up by and markedly stimulated CE formation in wild type macrophages, but not in macrophages lacking the LDL-R. The degradation of 125I-labeled-apoE-containing beta-VLDL by wild type MPM was poorly inhibited by unlabeled human LDL, and beta-VLDL without apoE had no effects. In conclusion, we propose that the in vitro uptake of native apoE-enriched lipoproteins by murine macrophages is primarily mediated by the LDL receptor and not by other apoE-recognizing receptor systems such as: the LDL receptor related protein, the VLDL receptor or the triglyceride-rich lipoprotein receptor.

To investigate the role of various lipoproteins in plasma to promote cholesterol efflux from cell membranes, potencies of lipoproteins in normolipidemic fasting and postprandial (PP) plasmas to accept additional cholesterol molecules from cell membranes were determined. We used red blood cells (RBCs) and lipoproteins in fresh blood as donors and acceptors of cell membrane cholesterol, respectively. When fresh fasting plasma (n=24) containing active lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer proteins (CETP) was incubated with a 3-fold excess of autologous RBCs at 37 degrees C for 18 hours, plasma cholesterol levels increased by 19.6% (38.5+/-14.2 mg/dL) owing to an exclusive increase in the CE level. Very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) fractions retained 48.1%, 26.3%, and 25.6% of the net cholesterol mass increase in fasting plasma, resulting in 91%, 8%, and 21% increases in their cholesterol contents, respectively. The PP plasma was 1.3-fold more potent than fasting plasma in promoting cholesterol efflux from RBCs by associating excess cholesterol with chylomicrons, resulting in a 356% increase in the cholesterol content of chylomicrons. These increases in lipoprotein cholesterol content indicate that chylomicrons were about 3.9x, 44x, and 17x more potent than fasting VLDL, LDL, and HDL, respectively, in accepting additional cholesterol molecules released from RBCs. The capacity of PP plasma to promote cholesterol efflux from RBCs was significantly correlated with plasma cholesterol levels (r=0.60, P<0.005), triglycerides (r=0.68, P<0.001), chylomicrons (r=0.90, P<0.001), VLDL (r=0.65, P<0.001), and LDL (r=0.47, P<0.025) but not with the levels of HDL (r= -0.34, P<0.20). In fasting plasma containing a low level of VLDL and HDL, isolated chylomicrons supplemented to the plasma were approximately 9x more potent than HDL in boosting the capacity of plasma to promote cholesterol efflux from RBCs. This study indicates that chylomicrons in PP plasma are the most potent ultimate acceptors of cholesterol released from cell membranes and that a low HDL level is not a factor that limits the ability of PP plasma to promote cholesterol efflux from cell membranes. Our data obtained from an in-vitro system suggest that PP chylomicrons may play a major role in promoting reverse cholesterol transport in vivo, since the transfer of cholesterol from cell membranes to chylomicrons will lead to the rapid removal of this cholesterol by the liver. HDL in vivo may promote reverse cholesterol transport by enhancing the rapid removal of chylomicrons from the circulation, since the rate of clearance of chylomicrons is positively correlated with the HDL level in plasma.

The aim of the present study was to delineate the mechanism(s) responsible for the increased secretion of VLDL (very-low-density lipoprotein) particles in patients with FCH (familial combined hyperlipidaemia). In 194 young adults (<25 years of age) recruited from families with FCH, we investigated how plasma lipids, (apo)lipoproteins and BMI (body mass index) varied with age. Furthermore, we performed a 5-year follow-up study of clinical and biochemical characteristics of a subset of this population (n=85) stratified by apoB (apolipoprotein B) levels (below or above the 75th percentile adjusted for age and gender). Plasma apoB concentration (r=0.45, P<0.0001), triacylglycerol (triglyceride) concentration (r=0.45, P<0.0001), LDL (low-density lipoprotein) subfraction profile (r=-0.46, P<0.0001) and BMI (r=0.51, P<0.0001) were significantly associated with age. Plasma apoB concentration in the hyperapoB group was already elevated at a young age, whereas other characteristics of FCH, as observed in adults, including triacylglycerol levels >1.5 mmol/l and/or small-dense LDL, were observed only sporadically. After the 5-year follow-up, BMI increased in both groups, and this increase was associated with changes in apoB (r=0.27, P<0.05), triacylglycerol (r=0.30, P<0.01), VLDL cholesterol (r=0.22, P<0.05), VLDL triacylglycerol (r=0.25, P<0.05) and high-density lipoprotein cholesterol (r=-0.27, P<0.05). In conclusion, we have found indirect evidence of a primary, presumably genetically determined, increase in plasma apoB concentration occurring early in life of offspring from families with FCH. However, aging-related post-maturation increases in adipose tissue mass also appear to contribute to an aggravation and/or modulation of this genetically determined apoB overproduction.

The aim of the present study was to investigate the relationship between circulating PCSK9 (proprotein convertase subtilisin kexin type 9) and FCHL (familial combined hyperlipidaemia) and, when positive, to determine the strength of its heritability. Plasma PCSK9 levels were measured in FCHL patients (n=45), NL (normolipidaemic) relatives (n=139) and their spouses (n=72). In addition, 11 FCHL patients were treated with atorvastatin to study the response in PCSK9 levels. PCSK9 levels were higher in FCHL patients compared with NL relatives and spouses: 96.1 compared with 78.7 and 82.0 ng/ml (P=0.004 and P=0.002 respectively). PCSK9 was significantly associated with both TAG (triacylglycerol) and apolipoprotein B levels (P<0.001). The latter relationship was accounted for by LDL (low-density lipoprotein)-apolipoprotein B (r=0.31, P=0.02), not by VLDL (very-low-density lipoprotein)-apolipoprotein B (r=0.09, P=0.49) in a subgroup of subjects (n=59). Heritability calculations for PCSK9 using SOLAR and FCOR software yielded estimates of 67-84% respectively (P<0.0001). PCSK9 increased from 122 to 150 ng/ml in 11 FCHL patients treated with atorvastatin (40 mg) once daily for 8 weeks (P=0.018). In conclusion, plasma PCSK9 is a heritable trait associated with both FCHL diagnostic hallmarks. These results, combined with the significant rise in PCSK9 levels after statin therapy, warrant further studies in order to unravel the exact role of PCSK9 in the pathogenesis and treatment of this highly prevalent genetic dyslipidaemia.

IDDM patients treated with conventional subcutaneous insulin have an abnormal increase in cholesteryl ester transfer (CET), the proatherogenic step in reverse-cholesterol transport that results in the enrichment of the apolipoprotein (apo) B-containing lipoproteins (VLDL, LDL) with cholesteryl ester (CE). This disturbance is closely linked to iatrogenic hyperinsulinemia and the nonphysiologic stimulation of lipoprotein lipase (LpL), a physiologic activator of CET, because lowering systemic insulin levels by administering insulin through the intraperitoneal insulin route normalizes LpL and CET. Hyperinsulinemia persists in IDDM patients who undergo successful pancreas-kidney transplantation (PKT) when their allografts are placed in the pelvis and drain into the iliac vein. Therefore, to determine whether hyperinsulinemia promotes CET in this setting, we studied CET, LpL, and insulin levels in 14 euglycemic normolipidemic IDDM PKT patients with near-normal kidney function (creatinine 1.5 +/- 0.4 mg/dl). Consistent with our prediction, the net mass of CE transferred from HDL to VLDL + LDL was significantly increased in the PKT group (P < 0.001) compared with nondiabetic renal transplant patients receiving the same immunosuppressive drugs and healthy control subjects. Both basal and arginine-stimulated insulin levels were increased above the kidney transplant group's levels and correlated with the mass of CE transferred at 2 h (r = 0.71, P < 0.05; r = 0.66, P < 0.05, respectively). Total basal LpL activities, LpL and hepatic triacylglycerol lipase activities, and LpL mass all tended to be higher than levels in healthy control subjects. Consistent with these changes in lipase activity, VLDL particle size was significantly reduced (P < 0.025) compared with that of control subjects. These findings indicate that PKT patients with systemically draining allografts have a persisting profile of potentially atherogenic disturbances in insulin levels, LpL, and CET that resemble IDDM patients treated with conventional subcutaneous insulin injections.

OBJECTIVE

Subcutaneous adipose tissue grows rapidly during the first months of life. Lipoprotein lipase (LPL) has a quantitatively important function in adipose tissue fat accumulation and insulin-like growth factor-I (IGF-I) is a determinant of neonatal growth. Recent studies showed that LPL mass in non-heparinized serum (LPLm) was an index of LPL-mediated lipolysis of plasma triacylglycerol (TG). The objective was to know the influence of serum LPL and IGF-I on neonatal subcutaneous fat growth, especially on catch-up growth in low birth weight infants.

METHODS

We included 47 healthy neonates (30 males, 17 females), including 7 small for gestational age. We measured serum LPLm and IGF-I concentrations at birth and 1 month, and analyzed those associations with subcutaneous fat accumulation.

RESULTS

Serum LPLm and IGF-I concentrations increased markedly during the first month, and positively correlated with the sum of skinfold thicknesses both at birth (r=0.573, P=0.0001; r=0.457, P=0.0035) and at 1 month (r=0.614, P<0.0001; r=0.787, P<0.0001, respectively). In addition, serum LPLm concentrations correlated inversely to very low-density lipoprotein (VLDL)-TG levels (r=-0.692, P<0.0001 at birth; r=-0.429, P=0.0052 at 1 month). Moreover, the birth weight Z-score had an inverse association with the postnatal changes in individual serum LPLm concentrations (r=-0.639, P<0.0001).

CONCLUSIONS

Both serum LPLm and IGF-I concentrations were the determinants of subcutaneous fat accumulation during the fetal and neonatal periods. During this time, LPL-mediated lipolysis of VLDL-TG may be one of the major mechanisms of rapid growth in subcutaneous fat tissue. Moreover, LPL, as well as IGF-I, may contribute to catch-up growth in smaller neonates.

The effects of freeze-dried soya milk (SM) and Bifidobacterium-fermented soya milk (FSM) on plasma and liver lipids, and faecal steroid excretion were estimated in hamsters fed on a cholesterol-free or cholesterol-enriched diet. Hamsters fed on the cholesterol-free diet containing 300 g FSM/kg had lower levels of plasma VLDL + LDL cholesterol than the animals fed on the control diet. SM in the diet produced a similar pattern without significant differences. In the cholesterol-enriched diet group, SM and FSM decreased the levels of plasma total cholesterol and VLDL + LDL-cholesterol. SM and FSM decreased the plasma triacylglycerol level in both the cholesterol-free and -enriched diet groups. The liver total cholesterol contents in the SM and FSM groups were lower than that in the control group, for hamsters fed on the cholesterol-free diet. The liver triacylglycerol content was not modified by SM or FSM in hamsters fed on either the cholesterol-free or -enriched diet. SM and FSM increased the total bile acid excretion and the proportion of cholesterol entering the cholic acid biosynthesis pathway in both the cholesterol-free and -enriched diet groups. SM and FSM did not affect neutral steroid excretion in the cholesterol-free or -enriched diet group. There was an inverse relationship between VLDL + LDL-cholesterol and faecal bile acid excretion in hamsters fed on the cholesterol-free (r -0.670, P < 0.01) and cholesterol-enriched (r -0.761, P < 0.001) diets respectively. These results indicated that SM had an anti-atherogenic effect, and that this effect was not diminished by prior fermentation.

Plasma lecithin-cholesterol acyltransferase (LCAT) activity, lipoprotein composition, and lipoprotein concentrations were measured in 21 children with kwashiorkor before (day 1), during (day 10), and after treatment (day 30). Day 1 LCAT activity (78.2 mumol.L-1.h-1) was decreased with respect to day 10 (139.2 mumol.L-1.h-1, P less than 0.001) and day 30 (108.0 mumol.L-1.h-1, P = 0.08). Plasma total cholesterol (TC), cholesterol ester (CE), and lipoprotein CEs (VLDL, IDL, LDL, and HDL) were reduced relative to days 10 and 30 (P less than 0.001). Before treatment HDL composition was abnormal. On days 1, 10, and 30, the respective mean HDL-apolipoprotein A-I (apo A-I) concentrations were 23.33, 39.66, and 36.08 mumol/L. LDL-apo B concentrations were 0.40, 0.68, and 0.56 mumol/L (P less than 0.01, days 10 and 30 vs day 1). LDL particles on day 1 were decreased in number, depleted of CE, and laden with triacylglycerol and surface lipids. LCAT activity on day 1 correlated with LDL-CE linoleate (P less than 0.05, r = 0.48). Reduced plasma LCAT activity is an important factor related to abnormalities in lipoprotein composition and concentrations.

We evaluated a new homogeneous assay for quantifying high-density lipoprotein cholesterol (HDL-C). The assay included four reagents: polyethylene glycol for "wrapping" chylomicrons, very-low-density lipoproteins (VLDL), and low-density lipoproteins (LDL); antibodies specific for apolipoprotein (apo) B and apo C-III to produce aggregates of chylomicrons, VLDL, and LDL; enzymes for the enzymatic cholesterol determination of the noncomplexed lipoproteins with 4-aminoantipyrine as the color reagent; and guanidine salt to stop the enzymatic reaction and to solubilize the complexes of apo B-containing lipoproteins, which would otherwise interfere with the reading of absorbance. The total CVs of the new method ranged between 2.4% and 8.4%. The HDL-C values (y) were in good agreement with those by a comparison phosphotungstic acid/MgCl2 method (x): y= 0.987x + 17.2 mg/L (68th percentile of the residuals on the regression line= 21.49, r= 0.970). At triglyceride concentrations of 20 g/L (Intralipid) the homogeneous HDL-C concentrations increased by 2%. Hemoglobin markedly increased the results, whereas bilirubin reduced them. The homogeneous HDL-C assay was easy to handle and allows full automation. This test should considerably facilitate the screening of individuals at an increased risk of cardiovascular disease.

Hyperalphalipoproteinemia, characterized by increased plasma concentrations of apoA-I and of HDL lipid and protein, was observed in rats treated with triiodothyronine (T(3)) for 7 days. The increase in the plasma HDL apoproteins was general for apoC, apoE plus A-IV, and apoA-I, as determined by isoelectric focusing. Hypotriglyceridemia, characterized by decreased concentrations of VLDL and apoB, was also observed in the hyperthyroid state. Although in the mildly hypothyroid animals (propylthiouracil-treated), hepatic metabolism of free fatty acid is shifted toward esterification to triglyceride and VLDL formation, as we reported previously, plasma HDL and apoA-I concentrations were not different from control plasma values, while the d 1.006-1.063 g/ml (IDL + LDL) lipoprotein fraction tended to be increased. In general, the proportion of apoE in the (IDL + LDL) fraction of the hypothyroid rat was greater than in controls and hyperthyroid animals, while the proportion of apoE tended to be lower in VLDL from both hypo- and hyperthyroid rats than in VLDL from controls. An enhanced release of apoA-I by perfused livers isolated from rats treated with T(3) was also observed; this enhanced output of apoA-I may explain, in part, the hyperalphalipoproteinemia observed in these rats. The depressed net output of apoA-I in vitro by perfused livers from rats treated with propylthiouracil (PTU) was not expressed in a statistically significant diminished plasma concentration of HDL or apoA-I in the intact animals. Treatment with T(3) also resulted in modification of the content of essential fatty acids in various lipid classes. Linoleic acid residues were significantly reduced and arachidonic acid content was increased in plasma phospholipids and esterified cholesterol in T(3)-treated rats. However, the relative fatty acid composition of unesterified fatty acids and triglyceride fatty acids was not altered by T(3) treatment. PTU treatment had no effect on fatty acid distribution in any of the plasma lipids. Secretion of biliary lipids was increased in perfused livers from T(3)-treated rats, while treatment with PTU did not affect release of lipids in the bile. These observations suggest a regulatory role for thyroid hormones that determine concentration and composition of plasma HDL and other lipoproteins.-Wilcox, H. G., W. G. Keyes, T. A. Hale, R. Frank, D. W. Morgan, and M. Heimberg. Effects of triiodothyronine and propylthiouracil on plasma lipoproteins in male rats.

Normolipidemic fasting VLDL stimulated the formation of cholesteryl ester in the human macrophage-like cell line, U937. VLDL from different persons exhibited heterogeneity over a 10-fold range in stimulating cholesterol esterification, and were 7% to 98% as active as human LDL. The effects of VLDL in U937 cells were highly correlated to those in normal human fibroblasts. VLDL did not induce cholesterol esterification in resident mouse peritoneal macrophages, nor in fibroblasts that lacked LDL receptors, suggesting that receptors for LDL, rather than for beta-VLDL, mediate the effects of VLDL. The active VLDL particles were found in the dense VLDL subfraction, Sf20 to 60, but not in Sf60 to 100 or Sf100 to 400. The activity of unfractionated VLDL was correlated directly with % cholesteryl ester in total or Sf20 to 60 VLDL, and inversely with VLDL triglycerides. Biologic variation of fasting VLDL activity in U937 cells was found in 13 normolipidemic ambulatory subjects. As compared to the correlation between the activity of two VLDL samples derived from a single aliquot of blood (r = 0.90), the correlation was lower (r = 0.60) between the activity of samples taken on successive days, and even less (r = 0.05) between VLDL samples obtained 2 weeks apart. Substantial biologic variation also occurred in VLDL composition except for VLDL unesterified cholesterol. These findings suggest an environmental influence on VLDL metabolism by macrophages.

Patients with type 1 diabetes (T1D) present increased risk of cardiovascular disease (CVD). The aim of this study is to improve the assessment of lipoprotein profile in patients with T1D by using a robust developed method 1H nuclear magnetic resonance spectroscopy (1H NMR), for further correlation with clinical factors associated to CVD. Thirty patients with T1D and 30 non-diabetes control (CT) subjects, matched for gender, age, body composition (DXA, BMI, waist/hip ratio), regular physical activity levels and cardiorespiratory capacity (VO2peak), were analyzed. Dietary records and routine lipids were assessed. Serum lipoprotein particle subfractions, particle sizes, and cholesterol and triglycerides subfractions were analyzed by 1H NMR. It was evidenced that subjects with T1D presented lower concentrations of small LDL cholesterol, medium VLDL particles, large VLDL triglycerides, and total triglycerides as compared to CT subjects. Women with T1D presented a positive association with HDL size (p<0.005; R = 0.601) and large HDL triglycerides (p<0.005; R = 0.534) and negative (p<0.005; R = -0.586) to small HDL triglycerides. Body fat composition represented an important factor independently of normal BMI, with large LDL particles presenting a positive correlation to total body fat (p<0.005; R = 0.505), and total LDL cholesterol and small LDL cholesterol a positive correlation (p<0.005; R = 0.502 and R = 0.552, respectively) to abdominal fat in T1D subjects; meanwhile, in CT subjects, body fat composition was mainly associated to HDL subclasses. VO2peak was negatively associated (p<0.005; R = -0.520) to large LDL-particles only in the group of patients with T1D. In conclusion, patients with T1D with adequate glycemic control and BMI and without chronic complications presented a more favourable lipoprotein profile as compared to control counterparts. In addition, slight alterations in BMI and/or body fat composition showed to be relevant to provoking alterations in lipoproteins profiles. Finally, body fat composition appears to be a determinant for cardioprotector lipoprotein profile.

BACKGROUND

The dyslipidemia of type 2 diabetic patients is characterized by high VLDL, abnormal LDL composition and low HDL cholesterol concentrations. The aim of this study was to establish whether the type of dietary fats affects LDL size and density and HDL cholesterol concentrations in these patients.

METHODS

Plasma phospholipid fatty acid composition, which reflects the type of dietary fatty acids, was quantified by gas chromatography. LDL relative flotation (LDL-Rf), a measure of LDL particle size and density, was determined by single vertical spin density gradient ultracentrifugation in 97 type 2 diabetic patients.

RESULTS

By linear regression analysis of the data, plasma fatty acids were associated neither with LDL-cholesterol levels nor with LDL-Rf. The HDL cholesterol concentrations were negatively related with saturated fatty acids (r = -0.23; p = 0.02) but positively related with monounsaturated fatty acids (r = +0.20; p = 0.00). Furthermore, higher HDL concentrations were associated with large and buoyant LDL particles (HDL cholesterol vs LDL-Rf: r = +0.47; p = 0.00). In the multiple regression analysis, the LDL-Rf was significantly related both to triglycerides (beta coefficient = -0.55, p = 0.000) and HDL cholesterol (beta coefficient = 0.19, p = 0.034) concentrations. In a stepwise regression analysis including both triglycerides and HDL cholesterol, triglycerides alone explained the 43.0% of the LDL-Rf variability.

CONCLUSIONS

A reduction of the dietary saturated fats and an increment of monounsaturated fats might increase HDL cholesterol concentrations in type 2 diabetic patients. Modifications of LDL composition might be expected from interventions aimed to reduce plasma triglycerides.