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Storage of human plasma samples leads to alterations in the lipoprotein distribution of apoC-III and apoE

2004; Elsevier BV; Volume: 45; Issue: 8 Linguagem: Inglês

10.1194/jlr.d300041-jlr200

ISSN

1539-7262

Autores

Jeffrey S. Cohn, Claudia Rodriguez, Hélène Jacques, Michel J. Tremblay, Jean Davignon,

Tópico(s)

Diabetes, Cardiovascular Risks, and Lipoproteins

Resumo

The effect of frozen storage on lipoprotein distribution of apolipoprotein C-III (apoC-III) and apoE was investigated by measuring apoC-III and apoE by ELISA in HDL and apoB-containing lipoproteins of human plasma samples (n = 16) before and after 2 weeks of frozen storage (−20°C). HDLs were separated by heparin-manganese precipitation (HMP) or by fast-protein liquid chromatography (FPLC). Total plasma apoC-III and apoE levels were not affected by frozen storage. HDL-HMP apoC-III and apoE levels were significantly higher in frozen versus fresh samples: 7.7 ± 0.7 versus 6.7 ± 0.7 mg/dl (P < 0.05) and 2.0 ± 0.1 versus 1.2 ± 0.1 mg/dl (P < 0.001), respectively. HDL-FPLC apoC-III and apoE, but not triglyceride (TG) or cholesterol, levels were also higher in frozen samples: 12.0 ± 1.2 versus 7.5 ± 0.6 mg/dl (P < 0.001) and 2.7 ± 0.2 versus 1.6 ± 0.2 mg/dl (P < 0.001), respectively. Frozen storage led to a decrease in apoC-III (−17 ± 9%) and apoE (−19 ± 9%) in triglyceride-rich lipoprotein. Redistribution of apoC-III and apoE was most evident in samples with high TG levels. HDL apoC-III and apoE levels were also significantly higher when measured in plasma stored at −80°C.Our results demonstrate that lipoprotein distribution of apoC-III and apoE is affected by storage of human plasma, suggesting that analysis of frozen plasma should be avoided in studies relating lipoprotein levels of apoC-III and/or apoE to the incidence of coronary artery disease. The effect of frozen storage on lipoprotein distribution of apolipoprotein C-III (apoC-III) and apoE was investigated by measuring apoC-III and apoE by ELISA in HDL and apoB-containing lipoproteins of human plasma samples (n = 16) before and after 2 weeks of frozen storage (−20°C). HDLs were separated by heparin-manganese precipitation (HMP) or by fast-protein liquid chromatography (FPLC). Total plasma apoC-III and apoE levels were not affected by frozen storage. HDL-HMP apoC-III and apoE levels were significantly higher in frozen versus fresh samples: 7.7 ± 0.7 versus 6.7 ± 0.7 mg/dl (P < 0.05) and 2.0 ± 0.1 versus 1.2 ± 0.1 mg/dl (P < 0.001), respectively. HDL-FPLC apoC-III and apoE, but not triglyceride (TG) or cholesterol, levels were also higher in frozen samples: 12.0 ± 1.2 versus 7.5 ± 0.6 mg/dl (P < 0.001) and 2.7 ± 0.2 versus 1.6 ± 0.2 mg/dl (P < 0.001), respectively. Frozen storage led to a decrease in apoC-III (−17 ± 9%) and apoE (−19 ± 9%) in triglyceride-rich lipoprotein. Redistribution of apoC-III and apoE was most evident in samples with high TG levels. HDL apoC-III and apoE levels were also significantly higher when measured in plasma stored at −80°C. Our results demonstrate that lipoprotein distribution of apoC-III and apoE is affected by storage of human plasma, suggesting that analysis of frozen plasma should be avoided in studies relating lipoprotein levels of apoC-III and/or apoE to the incidence of coronary artery disease. Patients with coronary artery disease (CAD) tend to have increased levels of plasma triglyceride (TG) (1Goldstein J.L. Hazzard W.R. Schrott H.G. Bierman E.L. Motulsky A.G. Hyperlipidemia in coronary heart disease, I: lipid levels in 500 survivors of myocardial infarction.J. Clin. Invest. 1973; 52: 1533-1543Crossref PubMed Scopus (456) Google Scholar, 2Lewis B. Chait A. Oakley C.M.O. Wootton I.D.P. Krikler D.M. Onitiri A. Sigurdsson G. February A. Serum lipoprotein abnormalities in patients with ischaemic heart disease: comparisons with control population.BMJ. 1974; 3: 489-493Crossref PubMed Scopus (90) Google Scholar, 3Castelli W.P. Doyle J.T. Gordon T. HDL cholesterol and other lipids in coronary heart disease. The cooperative lipoprotein phenotyping study.Circulation. 1977; 55: 767-772Crossref PubMed Scopus (1172) Google Scholar). However, multivariate analyses of prospective data have often failed to identify plasma TG as an independent risk factor for CAD (4Castelli W.P. The triglyceride issue: a view from Framingham.Am. Heart J. 1986; 112: 432-437Crossref PubMed Scopus (574) Google Scholar, 5Austin M.A. Plasma triglyceride and coronary artery disease.Arterioscler. Thromb. 1991; 11: 2-14Crossref PubMed Google Scholar). This is because plasma TG levels are closely related to other lipid (e.g., HDL cholesterol) as well as nonlipid risk factors (e.g., obesity, diabetes, and cigarette smoking) (6Davignon J. Cohn J.S. Triglycerides: a risk factor for coronary heart disease.Atherosclerosis. 1996; 124: 57-64Abstract Full Text PDF PubMed Scopus (118) Google Scholar). In addition, not all triglyceride-rich lipoproteins (TRLs) are equally atherogenic, and partially catabolized TRLs (i.e., remnant lipoproteins) are believed to promote the onset and development of atherosclerosis to a greater extent than do large, lipid-rich TRLs (7Cohn J.S. Marcoux C. Davignon J. Detection, quantification, and characterization of potentially atherogenic triglyceride-rich remnant lipoproteins.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2474-2486Crossref PubMed Scopus (140) Google Scholar). Studies have therefore been conducted to identify components of large and small TRLs that might be better able to predict risk of CAD. Apolipoprotein C-III (apoC-III) and apoE are two candidate CAD risk factors that are known to play a key role in regulating plasma TRL metabolism. ApoC-III inhibits TRL catabolism by reducing the lipolysis and uptake of TRL (8Jong M.C. Hofker M.H. Havekes L.M. Role of apoCs in lipoprotein metabolism: functional differences between apoC1, apoC2, and apoC3.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 472-484Crossref PubMed Scopus (437) Google Scholar, 9Shachter N.S. Apolipoproteins C-I and C-III as important modulators of lipoprotein metabolism.Curr. Opin. Lipidol. 2001; 12: 297-304Crossref PubMed Scopus (249) Google Scholar). ApoE, on the other hand, enhances TRL catabolism by: a) promoting conversion of VLDL to IDL and LDL, and b) acting as a ligand for receptor-mediated uptake of TRL remnants (10Weisgraber K.H. Apolipoprotein E: structure-function relationships.Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). Overexpression of human apoC-III in mice thus leads to severe hypertriglyceridemia (11Ito Y. Azrolan N. O'Connell A. Walsh A. Breslow J.L. Hypertriglyceridemia as a result of human apo CIII gene expression in transgenic mice.Science. 1990; 249: 790-793Crossref PubMed Scopus (453) Google Scholar) and increased development of atherosclerosis (12Masucci-Magoulas L. Goldberg I.J. Bisgaier C.L. Serajuddin H. Francone O.L. Breslow J.L. Tall A.R. A mouse model with features of familial combined hyperlipidemia.Science. 1997; 275: 391-394Crossref PubMed Scopus (122) Google Scholar), whereas apoE protects mice from hyperlipidemia and atherosclerotic lesion formation (13Kashyap V.S. Santamarina-Fojo S. Brown D.R. Parrott C.L. Applebaum-Bowden D. Meyn S. Talley G. Paigen B. Maeda N. Brewer Jr., H.B. Apolipoprotein E deficiency in mice: gene replacement and prevention of atherosclerosis using adenovirus vectors.J. Clin. Invest. 1995; 96: 1612-1620Crossref PubMed Scopus (123) Google Scholar). Interestingly, patients with CAD have increased levels of both apoC-III and apoE in their TRL (14Luc G. Fievet C. Arveiler D. Evans A.E. Bard J.M. Cambien F. Fruchart J.C. Ducimetiere P. Apolipoproteins C-III and E in apoB- and non-apoB-containing lipoproteins in two populations at contrasting risk for myocardial infarction: the ECTIM study. Etude Cas Temoins sur 'Infarctus du Myocarde.J. Lipid Res. 1996; 37: 508-517Abstract Full Text PDF PubMed Google Scholar), consistent with a direct inhibitory effect of apoC-III on TRL catabolism and an intolerance or insensitivity to the lipid-clearing effects of apoE. Both angiographic and prospective coronary event studies have demonstrated that increased levels of apoC-III or apoE in TRL (or reduced levels of apoC-III in HDL) are independently associated with the progression or severity of CAD (15Blankenhorn D.H. Alaupovic P. Wickham E. Chin H.P. Azen S.P. Prediction of angiographic change in native human coronary arteries and aortocoronary bypass grafts. Lipid and nonlipid factors.Circulation. 1990; 81: 470-476Crossref PubMed Scopus (258) Google Scholar, 16Hodis H.N. Mack W.J. Azen S.P. Alaupovic P. Pogoda J.M. LaBree L. Hemphill L.C. Kramsch D.M. Blankenhorn D.H. Triglyceride- and cholesterol-rich lipoproteins have a differential effect on mild/moderate and severe lesion progression as assessed by quantitative coronary angiography in a controlled trial of lovastatin.Circulation. 1994; 90: 42-49Crossref PubMed Scopus (310) Google Scholar, 17Koren E. Corder C. Mueller G. Centurion H. Hallum G. Fesmire J. McConathy W.D. Alaupovic P. Triglyceride enriched lipoprotein particles correlate with the severity of coronary artery disease.Atherosclerosis. 1996; 122: 105-115Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 18Sacks F.M. Alaupovic P. Moye L.A. Cole T.G. Sussex B. Stampfer M.J. Pfeffer M.A. Braunwald E. VLDL, apolipoproteins B, CIII, and E, and risk of recurrent coronary events in the Cholesterol and Recurrent Events (CARE) trial.Circulation. 2000; 102: 1886-1892Crossref PubMed Scopus (415) Google Scholar, 19Lee S.J. Campos H. Moye L.A. Sacks F.M. LDL containing apolipoprotein CIII is an independent risk factor for coronary events in diabetic patients.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 853-858Crossref PubMed Scopus (149) Google Scholar). In the Cholesterol-Lowering Atherosclerosis Study, the predominant factor predicting the probability of global coronary progression in subjects treated with colestipol plus niacin was a decrease in the concentration of HDL apoC-III (15Blankenhorn D.H. Alaupovic P. Wickham E. Chin H.P. Azen S.P. Prediction of angiographic change in native human coronary arteries and aortocoronary bypass grafts. Lipid and nonlipid factors.Circulation. 1990; 81: 470-476Crossref PubMed Scopus (258) Google Scholar). In the Monitored Atherosclerosis Regression Study, predominant risk factors for severe lesions in lovastatin-treated patients were LDL cholesterol, the LDL-C to HDL-C ratio, and apoB, while predominant risk factors for mild/moderate lesions were TG levels and the concentration of apoC-III in apoB-containing lipoproteins (16Hodis H.N. Mack W.J. Azen S.P. Alaupovic P. Pogoda J.M. LaBree L. Hemphill L.C. Kramsch D.M. Blankenhorn D.H. Triglyceride- and cholesterol-rich lipoproteins have a differential effect on mild/moderate and severe lesion progression as assessed by quantitative coronary angiography in a controlled trial of lovastatin.Circulation. 1994; 90: 42-49Crossref PubMed Scopus (310) Google Scholar). A nested case control analysis of samples from the Cholesterol and Recurrent Events trial found that VLDL-apoB, apoC-III in VLDL + LDL, and apoE in HDL were significant independent predictors of coronary events (18Sacks F.M. Alaupovic P. Moye L.A. Cole T.G. Sussex B. Stampfer M.J. Pfeffer M.A. Braunwald E. VLDL, apolipoproteins B, CIII, and E, and risk of recurrent coronary events in the Cholesterol and Recurrent Events (CARE) trial.Circulation. 2000; 102: 1886-1892Crossref PubMed Scopus (415) Google Scholar). The long time course and large number of patients in multicentre studies often require that plasma samples be stored at −20°C or −80°C for later analysis. Therefore, in most of the aforementioned studies (15Blankenhorn D.H. Alaupovic P. Wickham E. Chin H.P. Azen S.P. Prediction of angiographic change in native human coronary arteries and aortocoronary bypass grafts. Lipid and nonlipid factors.Circulation. 1990; 81: 470-476Crossref PubMed Scopus (258) Google Scholar, 16Hodis H.N. Mack W.J. Azen S.P. Alaupovic P. Pogoda J.M. LaBree L. Hemphill L.C. Kramsch D.M. Blankenhorn D.H. Triglyceride- and cholesterol-rich lipoproteins have a differential effect on mild/moderate and severe lesion progression as assessed by quantitative coronary angiography in a controlled trial of lovastatin.Circulation. 1994; 90: 42-49Crossref PubMed Scopus (310) Google Scholar, 18Sacks F.M. Alaupovic P. Moye L.A. Cole T.G. Sussex B. Stampfer M.J. Pfeffer M.A. Braunwald E. VLDL, apolipoproteins B, CIII, and E, and risk of recurrent coronary events in the Cholesterol and Recurrent Events (CARE) trial.Circulation. 2000; 102: 1886-1892Crossref PubMed Scopus (415) Google Scholar, 19Lee S.J. Campos H. Moye L.A. Sacks F.M. LDL containing apolipoprotein CIII is an independent risk factor for coronary events in diabetic patients.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 853-858Crossref PubMed Scopus (149) Google Scholar), lipoprotein distribution of apoC-III and/or apoE was determined in plasma that had been previously frozen and thawed. In contrast, metabolic studies in our laboratory have always involved the measurement of apoC-III and/or apoE in lipoproteins from freshly isolated plasma (20Cohn J.S. Tremblay M. Amiot M. Bouthillier D. Roy M. Genest Jr., J. Davignon J. Plasma concentration of apolipoprotein E in intermediate-sized remnant-like lipoproteins in normolipidemic and hyperlipidemic subjects.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 149-159Crossref PubMed Scopus (89) Google Scholar, 21Fredenrich A. Giroux L-M. Tremblay M. Krimbou L. Davignon J. Cohn J.S. Plasma lipoprotein distribution of apoC-III in normolipidemic and hypertriglyceridemic subjects: comparison of the apoC-III to apoE ratio in different lipoprotein fractions.J. Lipid Res. 1997; 38: 1421-1432Abstract Full Text PDF PubMed Google Scholar, 22Batal R. Tremblay M. Barrett P.H.R. Jacques H. Fredenrich A. Mamer O. Davignon J. Cohn J.S. Plasma kinetics of apoC-III and apoE in normolipidemic and hyperlipidemic subjects.J. Lipid Res. 2000; 41: 706-718Abstract Full Text Full Text PDF PubMed Google Scholar, 23Marcoux C. Tremblay M. Fredenrich A. Davignon J. Cohn J.S. Lipoprotein distribution of apolipoprotein C-III and its relationship to the presence in plasma of triglyceride-rich remnant lipoproteins.Metabolism. 2001; 50: 112-119Abstract Full Text PDF PubMed Scopus (36) Google Scholar). Our experience is that frozen storage does not affect the total plasma concentration of apoC-III and/or apoE, but that it can affect the relative amount of these apolipoproteins in different lipoprotein fractions. To document this effect, we carried out the present study, in which human plasma samples were stored under various conditions and lipoprotein distribution of apoC-III and apoE was determined before and after storage. Blood samples were obtained after an overnight fast from patients being treated at the lipid clinic of the Clinical Research Institute of Montreal. Patients were selected on the basis of their fasting TG concentration at a previous visit—from low to moderately elevated (<8 mmol/l). They agreed to have their blood used for scientific purposes through signed consent. Blood was drawn from an arm vein into Vacutainer tubes containing EDTA (final concentration, 1.5 mg/ml). Samples were centrifuged immediately (15 min, 3,000 rpm, 4°C). Plasma was collected by aspiration, and samples were immediately immersed in crushed ice. Plasma samples (1.5 ml aliquots) were then stored at 4°C in a Uni-Therm refrigerator (Grand Haven, MI) until analysis (within 4 h) or were frozen by being placed in a Revco −20°C (Asheville, NC) or Baxter −80°C (Asheville, NC) laboratory freezer. In some experiments, plasma samples were rapidly frozen by immersion in liquid nitrogen. After 2 weeks, frozen samples were thawed for analysis by being left at room temperature (1 h, 24°C). In some experiments, samples were thawed by being incubated for 30 min at 37°C. HDLs were separated from apoB-containing lipoproteins by heparin-manganese precipitation (HMP) according to the Lipid Research Clinics procedure (24Lipid Research Clinics Program Manual of Laboratory Operations. 1974. DHEW (NIH) Publication No. 75-628.Google Scholar), as modified by Warnick and Albers (25Warnick G.R. Albers J.J. A comprehensive evaluation of the heparin-manganese precipitation procedure for estimating high density lipoprotein cholesterol.J. Lipid Res. 1978; 19: 65-76Abstract Full Text PDF PubMed Google Scholar). Heparin-MnCl2 solution was added to plasma at a ratio of 0.1:1.0 (v/v). After 30 min at room temperature, samples were centrifuged (60 min, 3,000 rpm, 4°C). Supernates were aspirated and stored at 4°C until analysis (<24 h). Lipoproteins were also separated from plasma samples by automated gel filtration chromatography on a Pharmacia (Pharmacia LKB Biotechnology, Uppsala, Sweden) fast-protein liquid chromatography (FPLC) system, as described previously (20Cohn J.S. Tremblay M. Amiot M. Bouthillier D. Roy M. Genest Jr., J. Davignon J. Plasma concentration of apolipoprotein E in intermediate-sized remnant-like lipoproteins in normolipidemic and hyperlipidemic subjects.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 149-159Crossref PubMed Scopus (89) Google Scholar, 21Fredenrich A. Giroux L-M. Tremblay M. Krimbou L. Davignon J. Cohn J.S. Plasma lipoprotein distribution of apoC-III in normolipidemic and hypertriglyceridemic subjects: comparison of the apoC-III to apoE ratio in different lipoprotein fractions.J. Lipid Res. 1997; 38: 1421-1432Abstract Full Text PDF PubMed Google Scholar). Plasma samples (1 ml) were manually transferred to a 2 ml sample loop with two washes of 0.5 ml saline solution. They were programmed (Liquid Chromatography Controller LCC-500 Plus) to be loaded and separated on a 50 cm column (16 mm internal diameter) packed with cross-linked agarose gel (Superose 6 prep grade, Pharmacia). The column was eluted with 0.15 mol/l NaCl (0.01% EDTA, 0.02% sodium azide, pH 7.2) at a rate of 1.0 ml/min, and 25 min after addition of sample, 80 × 1 ml fractions were collected sequentially. Total run time for each sample, including pre- and postwashes was 150 min. Sample elution was monitored spectrophotometrically at an optical density of 280 nm. Three peaks of TG or cholesterol were identifiable for each plasma sample, corresponding to TRL, IDL + LDL, and HDL lipoprotein fractions. FPLC profiles were analyzed by measuring analytes in every second tube (from fractions 4–56), or pooled fractions were assayed: TRL (5Austin M.A. Plasma triglyceride and coronary artery disease.Arterioscler. Thromb. 1991; 11: 2-14Crossref PubMed Google Scholar, 6Davignon J. Cohn J.S. Triglycerides: a risk factor for coronary heart disease.Atherosclerosis. 1996; 124: 57-64Abstract Full Text PDF PubMed Scopus (118) Google Scholar, 7Cohn J.S. Marcoux C. Davignon J. Detection, quantification, and characterization of potentially atherogenic triglyceride-rich remnant lipoproteins.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2474-2486Crossref PubMed Scopus (140) Google Scholar, 8Jong M.C. Hofker M.H. Havekes L.M. Role of apoCs in lipoprotein metabolism: functional differences between apoC1, apoC2, and apoC3.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 472-484Crossref PubMed Scopus (437) Google Scholar, 9Shachter N.S. Apolipoproteins C-I and C-III as important modulators of lipoprotein metabolism.Curr. Opin. Lipidol. 2001; 12: 297-304Crossref PubMed Scopus (249) Google Scholar, 10Weisgraber K.H. Apolipoprotein E: structure-function relationships.Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 11Ito Y. Azrolan N. O'Connell A. Walsh A. Breslow J.L. Hypertriglyceridemia as a result of human apo CIII gene expression in transgenic mice.Science. 1990; 249: 790-793Crossref PubMed Scopus (453) Google Scholar, 12Masucci-Magoulas L. Goldberg I.J. Bisgaier C.L. Serajuddin H. Francone O.L. Breslow J.L. Tall A.R. A mouse model with features of familial combined hyperlipidemia.Science. 1997; 275: 391-394Crossref PubMed Scopus (122) Google Scholar, 13Kashyap V.S. Santamarina-Fojo S. Brown D.R. Parrott C.L. Applebaum-Bowden D. Meyn S. Talley G. Paigen B. Maeda N. Brewer Jr., H.B. Apolipoprotein E deficiency in mice: gene replacement and prevention of atherosclerosis using adenovirus vectors.J. Clin. Invest. 1995; 96: 1612-1620Crossref PubMed Scopus (123) Google Scholar, 14Luc G. Fievet C. Arveiler D. Evans A.E. Bard J.M. Cambien F. Fruchart J.C. Ducimetiere P. Apolipoproteins C-III and E in apoB- and non-apoB-containing lipoproteins in two populations at contrasting risk for myocardial infarction: the ECTIM study. Etude Cas Temoins sur 'Infarctus du Myocarde.J. Lipid Res. 1996; 37: 508-517Abstract Full Text PDF PubMed Google Scholar), IDL + LDL (15–35), and HDL (36–56). Recovery of lipoproteins was 80–90%, and all concentrations were corrected to give 100% recovery. Plasma and lipoprotein TG and cholesterol concentrations were determined enzymatically on an autoanalyzer (Cobas Mira, Roche). ApoC-III and apoE were measured by ELISA using immunopurified polyclonal antibodies (20Cohn J.S. Tremblay M. Amiot M. Bouthillier D. Roy M. Genest Jr., J. Davignon J. Plasma concentration of apolipoprotein E in intermediate-sized remnant-like lipoproteins in normolipidemic and hyperlipidemic subjects.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 149-159Crossref PubMed Scopus (89) Google Scholar, 21Fredenrich A. Giroux L-M. Tremblay M. Krimbou L. Davignon J. Cohn J.S. Plasma lipoprotein distribution of apoC-III in normolipidemic and hypertriglyceridemic subjects: comparison of the apoC-III to apoE ratio in different lipoprotein fractions.J. Lipid Res. 1997; 38: 1421-1432Abstract Full Text PDF PubMed Google Scholar). Mean data were compared before and after storage by Student's paired t-test. Means that were different with a probability of less than 5% that this was by chance (P < 0.05) were taken to be significantly different. Plasma samples varied in TG concentration from 0.68 to 7.63 mmol/l and in total cholesterol concentration from 3.82 mmol/l to 8.14 mmol/l. Mean plasma TG concentration of samples (n = 16) before storage was 2.29 ± 0.45 mmol/l, and mean plasma cholesterol concentration was 5.59 ± 0.31 mmol/l. As shown in Table 1, frozen storage led to a small (3.9%) but consistent (P < 0.01) increase in the measured concentration of plasma TG, as described previously (26Kronenberg F. Lobentanz E-M. König P. Utermann G. Dieplinger H. Effect of sample storage on the measurement of lipoprotein[a], apolipoproteins B and A-IV, total and high density lipoprotein cholesterol and triglycerides.J. Lipid Res. 1994; 35: 1318-1328Abstract Full Text PDF PubMed Google Scholar). Total plasma cholesterol, apoC-III, and apoE levels were not significantly affected by storage. HDLs were isolated by HMP before and after sample storage. HDL-HMP cholesterol levels were not affected by storage, but HDL-HMP TG levels were consistently lower (−9.9%) in plasma samples that had been frozen. As predicted, HDL-HMP apoC-III concentrations were significantly higher (25%, P < 0.05) in samples after storage. HDL apoE levels were also higher, and this increase (99%) was greater than that for apoC-III. To verify the aforementioned results, lipoproteins were separated by FPLC from the same plasma samples (n = 16) before and after storage. FPLC profiles for two samples are shown in Figs. 1, 2. The plasma in Fig. 1 had TG, cholesterol, apoC-III, and apoE concentrations of 2.30 mmol/l, 4.56 mmol/l, 18.5 mg/dl, and 5.1 mg/dl, respectively. The sample in Fig. 2 had TG, cholesterol, apoC-III, and apoE concentrations of 4.84 mmol/l, 5.56 mmol/l, 47.9 mg/dl, and 6.7 mg/dl, respectively. The sample shown in Fig. 2, therefore, had 2-fold higher TG and apoC-III levels than did that shown in Fig. 1. Frozen storage of these samples had qualitatively similar effects on the lipoprotein distribution of plasma lipids and apolipoproteins. In both cases, there were small increases in TRL TG and TRL cholesterol after storage at the expense of TG and cholesterol in large LDL or remnant particles. No change was observed in the size or position of HDL TG and HDL cholesterol peaks in samples after storage. In contrast, frozen storage had a marked effect on lipoprotein distribution of apoC-III and apoE, with considerably more apoC-III and apoE being detected in HDL and considerably less in TRL and/or IDL/LDL. The magnitude of these changes is depicted by mean data for all samples (n = 16) presented in Table 2. Total plasma apoC-III and apoE measurements were not significantly affected by storage. HDL-FPLC apoC-III levels were higher by 65% on average (P < 0.001), and HDL-FPLC apoE levels were higher by 104% (P < 0.001). At the same time, TRL apoC-III levels decreased by 17% on average (P < 0.001), and TRL apoE levels decreased by 19% (P < 0.05). IDL/LDL apoC-III and apoE levels also decreased, although only the change in IDL/LDL apoE was statistically significant (−25%, P < 0.001). Mean TRL TG concentrations before and after storage were 1.19 ± 0.36 and 1.32 ± 0.40 mmol/l (P < 0.01). Mean TRL cholesterol concentrations before and after storage were 0.37 ± 0.12 and 0.50 ± 0.16 mmol/l (P < 0.01). Frozen storage therefore caused significant increases in TRL lipids but significant decreases in TRL apoC-III and apoE. Mean HDL-FPLC cholesterol concentrations before and after storage were 1.13 ± 0.08 and 1.13 ± 0.16 mmol/l (not significant, P = 0.94). Mean HDL-FPLC TG concentrations before and after storage were 0.19 ± 0.02 and 0.19 ± 0.02 mmol/l (not significant, P = 0.67). HDL-FPLC lipid levels were therefore unaffected by storage despite marked increases in HDL-FPLC apoC-III and apoE.TABLE 1Total plasma and HDL lipid and apolipoprotein concentrations in samples before and after frozen storageBefore StorageAfter StoragePercentage DifferenceP aStatistical significance of difference due to storage.Total plasma TG2.29 ± 0.452.36 ± 0.46 3.9 ± 1.0<0.01 Cholesterol5.59 ± 0.315.72 ± 0.312.4 ± 1.5— apoC-III22.9 ± 2.724.8 ± 3.27.9 ± 3.3— apoE5.4 ± 0.45.5 ± 0.50.1 ± 3.2—HDL TG0.26 ± 0.030.22 ± 0.02−9.9 ± 2.8<0.05 Cholesterol1.19 ± 0.081.18 ± 0.08−0.5 ± 1.4— apoC-III6.7 ± 0.77.7 ± 0.725.4 ± 12.2<0.05 apoE1.2 ± 0.12.0 ± 0.199.0 ± 31.1<0.001ApoC-III, apolipoprotein C-III; TG, triglyceride. Results represent means ± SE for 16 samples. Lipid levels are expressed in units of mmol/l and apolipoproteins in units of mg/dl. HDLs were separated by heparin-manganese precipitation (HMP).a Statistical significance of difference due to storage. Open table in a new tab Fig. 2Separation by FPLC of lipoproteins from fresh and frozen plasma of a male subject with a plasma TG concentration of 4.8 mmol/l and a plasma cholesterol concentration of 5.6 mmol/l. Results for fresh plasma are shown with filled circles and those for frozen plasma are shown with open circles. Triglyceride and cholesterol data are shown in the upper panels and apolipoprotein data are shown in the lower panels.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Total plasma and lipoprotein apoC-III and apoE concentrations in samples before and after frozen storageBefore StorageAfter StoragePercentage DifferenceP aStatistical significance of difference due to storage.apoC-III Total22.9 ± 2.724.8 ± 3.2 7.9 ± 3.3%— TRL7.1 ± 1.85.2 ± 1.3−17.1 ± 8.5%<0.001 IDL/LDL8.3 ± 1.17.6 ± 1.1−8.0 + 7.1%— HDL7.5 ± 0.612.0 ± 1.2 64.6 ± 14.9%<0.001apoE Total5.4 ± 0.45.5 ± 0.5 0.1 ± 3.2%— TRL1.3 ± 0.30.8 ± 0.2−19.0 ± 9.4%<0.05 IDL/LDL2.6 ± 0.31.9 ± 0.2−25.2 ± 4.8%<0.001 HDL1.6 ± 0.22.7 ± 0.2103.6 ± 30.4%<0.001IDL, intermediate density lipoprotein; TRL, triglyceride-rich lipoprotein. Results represent means ± SE for 16 samples. Apolipoprotein levels are expressed in units of mg/dl. Lipoprotein fractions were separated by fast-protein liquid chromatography (FPLC).a Statistical significance of difference due to storage. Open table in a new tab ApoC-III, apolipoprotein C-III; TG, triglyceride. Results represent means ± SE for 16 samples. Lipid levels are expressed in units of mmol/l and apolipoproteins in units of mg/dl. HDLs were separated by heparin-manganese precipitation (HMP). IDL, intermediate density lipoprotein; TRL, triglyceride-rich lipoprotein. Results represent means ± SE for 16 samples. Apolipoprotein levels are expressed in units of mg/dl. Lipoprotein fractions were separated by fast-protein liquid chromatography (FPLC). From the raw data, it was evident that samples with higher TG levels tended to have greater changes in lipoprotein apoC-III and apoE. To show this effect, samples were divided into quartiles on the basis of their TG concentration. Mean TG levels for the quartiles are shown in Table 3. Mean plasma apoC-III levels for the four quartiles were 13.9 ± 1.8, 16.3 ± 2.9, 24.2 ± 2.0, and 37.3 ± 4.6 mg/dl, respectively. Mean plasma apoE levels for the four quartiles were 4.4 ± 0.4, 4.2 ± 0.6, 5.4 ± 0.2, and 7.7 ± 0.4 mg/dl, respectively. Apolipoprotein levels determined in frozen samples were then expressed as a percentage of levels found in fresh samples, and mean data for the different quartiles were determined (Table 3). Independent of whether HDLs were separated by HMP or FPLC, samples with higher TG levels had greater increases in HDL apoC-III and apoE. These samples also had greater decreases in TRL apoC-III and apoE. Mean absolute increases in TRL TG for the four quartiles (Q1 to Q4) were 0.01 ± 0.01, 0.06 ± 0.01, 0.10 ± 0.01, and 0.35 ± 0.12 mmol/l, respectively, and mean absolute increases in TRL cholesterol for the four quartiles (Q1 to Q4) were 0.02 ± 0.01, 0.08 ± 0.01, 0.13 ± 0.01, and 0.32 ± 0.08 mmol/l, respectively. Thus, decreases in TRL apolipoproteins were reciprocally related to increases in TRL lipids.TABLE 3Effect of frozen storage on apoC-III and apoE levels in samples grouped according to their plasma TG concentrationQ1Q2Q3Q4PaStatistical significance of difference between groups (by ANOVA).Plasma TG (mmol/l)0.76 ± 0.031.

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