Commonly used method for chylomicron isolation catches less than 0.1% of chylomicrons from whole plasma

Health effects of orally ingested bioactive compounds can only occur if bioactive molecules are absorbed and transported to target tissues. Paradoxically, many foods are rich in micronutrients but intestinal absorption is often limited. Immediately after absorption, lipid soluble nutrients are packaged into chylomicrons (CM), therefore quantification of the micronutrient content in CM has been used as tool to evaluate bioavailability(1). For bioavailability studies the isolated CM fraction must not contain liver derived lipoproteins (VLDL, LDL) since only intestine derived CM carry recently absorbed lipids and lipophilic micronutrients. As isolated CM can be contaminated with liver derived lipoproteins, this study evaluated the purity of collected CM and VLDL fractions. Each CM contains one apolipoprotein B-48 (apoB-48) and each VLDL contains one apolipoprotein B-100 (apoB-100) on its surface(2) therefore purity of CM and VLDL fractions was evaluated via the presence/absence of apoB-100 and apoB-48.CM and VLDL fractions were isolated as previously described(1,3)from whole plasma collected at 0, 2, 4 or 6 h after participants consumed a lipid (15.4g) and carotenoid (36mg) rich smoothie (480mL). Plasma density was adjusted to 1.1g/mL with KBr (0.14g/mL), placed in ultracentrifugation tubes (38 mL, thickwall) and overlaid with 3 solutions containing NaCl, KBr and Na-EDTA with densities of 1.020, 1.065 and 1.006g/mL.CM fraction was collected after ultracentrifugation at 25,000 rpm for 34 min in a Beckman Coulter Optima XE-90 with SW32Ti swinging bucket rotor. VLDL fraction was collected after additional 102 min at 25,000 rpm.ELISA analysis revealed that apoB-48 and apoB-100 were present in both CM and VLDL fractions. Less than 0.1% of plasma apoB-48 (< 98pmol/L) was present in CM or VLDL fraction. Less than 0.3% (< 2,425pmol/L) and 4% (< 54,124pmol/L) of plasma apoB-100 was present in CM and VLDL fractions, respectively. Low recoveries of apoB-48 and apoB-100 in CM and VLDL fraction suggests that ultracentrifugation neither concentrated nor isolated CM or VLDL. While ourapoB-48 and apoB-100 results agree with previously reported values in plasma, CM and VLDL(3,4), no previous study evaluated apoB-48 and apoB-100 recovery from plasma.

Comparison of Ultracentrifugation and Nuclear Magnetic Resonance Spectroscopy in the Quantification of Triglyceride-Rich Lipoproteins after an Oral Fat Load

Background: The measurement of triglyceride (TG)-rich particles after an oral fat challenge has been used to provide a measure of risk for coronary artery disease independent of the fasting plasma triglyceride concentration. The analytical “gold standard” for measuring TG-rich lipoproteins uses density gradient ultracentrifugation; however, this technique is labor-intensive. Because of our need to perform numerous postprandial analyses of TG-rich lipoproteins for a large interventional study (Genetics of Lipid Lowering Drugs and Diet Network), we evaluated the use of nuclear magnetic resonance (NMR) spectroscopy for measuring TG-rich particles. Methods: EDTA-blood samples were obtained 0, 3.5, 6, and 8 h after ingestion of an oral fat meal (89% of calories from fat) in 20 apparently healthy individuals. The plasma TG concentrations of chylomicron and chylomicron remnant/VLDL fractions were analyzed by ultracentrifugation and NMR spectroscopy. Results: Comparison of all values (n = 78) by ultracentrifugation (x) and NMR (y) produced a linear regression equation of y = 0.979x − 0.035 mmol/L (R2 = 0.90) for chylomicrons and y = 1.398x + 0.067 mmol/L (R2 = 0.96) for the fraction containing chylomicron remnants and VLDL. Postprandial response of chylomicrons and chylomicron remnant/VLDL was similar, with maximum response occurring between 3.5 to 6 h regardless of method of measurement. Conclusion: Chylomicron and chylomicron remnant/VLDL fraction measurements obtained by NMR have a high degree of correlation with results produced by ultracentrifugation. NMR may therefore be suitable as an alternative method for the measurement of postprandial TG-rich lipoproteins in individuals consuming a high-fat meal.

Chronic treatment with bark infusion fromCroton cajucaralowers plasma triglyceride levels in genetic hyperlipidemic mice

Aqueous infusion and preparations containing dehydrocrotonin (DHC) and essential oil from Croton cajucara bark were tested for plasma lipid-lowering effects in genetically modified hyperlipidemic mice. Two mouse models were tested: 1) primary hypercholesterolemia resulting from the LDL-receptor gene knockout, and 2) combined hyperlipidemia resulting from crosses of LDL-receptor knockout mice with transgenic mice overexpressing apolipo protein (apo) CIII and cholesteryl ester-transfer protein. Mice treated with bark infusion, DHC, essential oil, or placebos for 25 days showed no signals of toxicity as judged by biochemical tests for liver and kidney functions. The bark infusion reduced triglyceride plasma levels by 40%, while essential oil and DHC had no significant effects on plasma lipid levels. The bark infusion treatment promoted a redistribution of cholesterol among the lipoprotein fractions in combined hyperlipidemic mice. There was a marked reduction in the VLDL fraction and an increase in the HDL fraction, in such a way that the (VLDL + LDL)/HDL ratio was reduced by half. The bark infusion treatment did not modify cholesterol distribution in hypercholesterolemic mice. In conclusion, C. cajucara bark infusion reduced plasma triglycerides levels and promoted a redistribution of cholesterol among lipoproteins in genetically combined hyperlipidemic mice. These changes modify risk factors for the development of atherosclerotic diseases.Key words: hyperlipidemia, transgenic mice, Croton cajucara, dehydrocrotonin, cholesterol.

Biphasic transmittance waveform in the APTT coagulation assay is due to the formation of a Ca++-dependent complex of C-reactive protein with very-low–density lipoprotein and is a novel marker of impending disseminated intravascular coagulation

A decrease in light transmittance before clot formation, manifesting as a biphasic waveform (BPW) pattern in coagulation assays, was previously correlated with the onset of disseminated intravascular coagulation (DIC). In this study of 1187 consecutive admissions to the intensive care unit, the degree of this change on admission predicts DIC better than D-dimer measurements. Additionally, the BPW preceded the time of DIC diagnosis by 18 hours, on average, in 56% (203 of 362) of DIC patients. The BPW is due to the rapid formation of a precipitate and coincident turbidity change on recalcification of plasma. The isolated precipitate contains very-low–density lipoprotein (VLDL) and C-reactive protein (CRP). The addition of CRP and Ca++ to normal plasma also causes the precipitation of VLDL and IDL, but not LDL or HDL. The Kd of the CRP/VLDL interaction is 340 nM, and the IC50 for Ca++ is 5.0 mM. In 15 plasmas with the BPW, CRP was highly elevated (77-398 μg/mL), and the concentration of isolated VLDL ranged from 0.082 to 1.32 mM (cholesterol). The turbidity change on recalcification correlates well with the calculated level of the CRP–VLDL complex. Clinically, the BPW better predicts for DIC than either CRP or triglyceride alone. The complex may have pathophysiological implications because CRP can be detected in the VLDL fraction from sera of patients with the BPW, and the VLDL fraction has enhanced prothrombinase surface activity. The complex has been designated lipoprotein complexed C-reactive protein.

Lipid traffic between high density lipoproteins and Plasmodium falciparum-infected red blood cells.

Several intraerythrocytic growth cycles of Plasmodium falciparum could be achieved in vitro using a serum free medium supplemented only with a human high density lipoprotein (HDL) fraction (d = 1.063-1.210). The parasitemia obtained was similar to that in standard culture medium containing human serum. The parasite development was incomplete with the low density lipoprotein (LDL) fraction and did not occur with the VLDL fraction. The lipid traffic from HDL to the infected erythrocytes was demonstrated by pulse labeling experiments using HDL loaded with either fluorescent NBD-phosphatidylcholine (NBD-PC) or radioactive [3H]palmitoyl-PC. At 37 degrees C, the lipid probes rapidly accumulated in the infected cells. After incubation in HDL medium containing labeled PC, a subsequent incubation in medium with either an excess of native HDL or 20% human serum induced the disappearance of the label from the erythrocyte plasma membrane but not from the intraerythrocytic parasite. Internalization of lipids did not occur at 4 degrees C. The mechanism involved a unidirectional flux of lipids but no endocytosis. The absence of labeling of P. falciparum, with HDL previously [125I]iodinated on their apolipoproteins or with antibodies against the apolipoproteins AI and AII by immunofluorescence and immunoblotting, confirmed that no endocytosis of the HDL was involved. A possible pathway of lipid transport could be a membrane flux since fluorescence videomicroscopy showed numerous organelles labeled with NBD-PC moving between the erythrocyte and the parasitophorous membranes. TLC analysis showed that a partial conversion of the PC to phosphatidylethanolamine was observed in P. falciparum-infected red cells after pulse with [3H]palmitoyl-PC-HDL. The intensity of the lipid traffic was stage dependent with a maximum at the trophozoite and young schizont stages (38th h of the erythrocyte life cycle). We conclude that the HDL fraction appears to be a major lipid source for Plasmodium growth.

Redistribution of cholesterol and β-sitosterol between Intralipid and rat plasma lipoproteins and red blood cells in vivo

Male Wistar rats were injected intravenously with 2 mL of Intralipid containing 7.5 × 105 counts per minute (cpm) [14C]cholesterol and 7.5 × 105 cpm β-[3H]sitosterol. Blood was withdrawn immediately and at 5, 10, 20, 60,120, and 1440 min after injection from different animals. Plasma and red cells were separated and washed by conventional centrifugation, while lipoprotein density classes corresponding to chylomicrons, very low (VLDL), low (LDL), and high density lipoproteins (HDL) were isolated by ultracentrifugation. Total lipid and sterol compositions were determined by thin-layer chromatography in combination with gas–liquid chromatography, whereas radioactivity was measured by scintillation counting. The ratio of [14C]cholesterol/β-[3H]sitosterol rose from 1 to 3.65 in the plasma VLDL fraction, whereas that in the LDL and HDL fractions were equilibrated at about 2, following an initial transient increase in favour of cholesterol. The appearance and disappearance of the radioactivity from LDL and HDL fractions exhibited precursor–product relationship owing probably to the conversion of the Intralipid into an intermediate lipoprotein-X-like particle, which possesses a density similar to that of LDL. The radioactive cholesterol and β-sitosterol were incorporated into the red blood cell membranes at nearly similar initial rates, while at later times the incorporation of cholesterol was much preferred.

Quinidine and propranolol binding to very low and low density lipoproteins of human plasma

The interaction of quinidine (Q) and propranolol (P) with human very low density lipoproteins (VLDL) and low density lipoproteins (LDL) was determined by ultrafiltration in phosphate buffer (pH 7.4) for Q and in phosphate and Tris buffers (pH 7.4–7.5) for P, respectively. The Scatchard plots of Q are curved and were best described by a model assuming two independent classes of binding sites on lipoproteins. When working with P, the Scatchard plots were also nonlinear, but positive cooperativity was observed for the VLDL fraction in phosphate buffer and apparently also for the LDL fraction in Tris buffer. These nonlinear curves were described by the model used for Q, excluding any data falling in the cooperating region. The binding parameters, primary (K1) and secondary (K2) affinity constants and the number of sites in each class of independent binding sites, were generated by a computer program. The results of this in vitro study suggest that drug binding (and possibly distribution) to lipoproteins may be affected by the nature and concentration of some ions present in serum.