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1.P.396 A new method of assaying the high density lipoprotein-cholesterol
Monday
6 October 1997: Posters Methods
spectrometer. Thereafter, the leucine content in the pellet is determined by GC-MS using an internal standard. Knowing the number of leucine molecules in one molecule of apo B, the plasma concentration of apo B in the different lipoproteins can be calculated. The variation coefficients of apo B-100 measurements of VLDL (d < 1.006), VLDL + IDL (d < 1.019) and VLDL + IDL + LDL (d < 1.063) for normolipemic, hypertriglyceridemic and hypercholestemlemic plasma were less than 9%. The variation coefficients of other methods such as ELISA, electroimmunoassay and isopropanol precipitation combined with protein measurement according to the Lowry procedure were higher than those using GC-MS. Compared to the GC-MS procedure, ELISA and electroimmunoassay methods underestimate lipoprotein apo B concentrations. The combination of isopropanol precipitation and GC-MS method appears to be a precise and reliable method.
A new method of assaying the high density lipoprotein-cholesterol
I 1 P.396
D. Erlich, B. Charm, Y. Goussault. Laboratoire de Biochimie, Hopital Saint-Louis, 1 avenue Claude Vellefaux, 75475 Paris Cedex 10, France A new method of assaying the cholesterol of high-density lipoproteins is described. It consists in the use of magnetic particles coated with dextran-sulfate + magnesium chloride. They pull away precipitated lipoproteins (very low density and low density lipoproteins) by the use of a magnet located at the bottom
I 1 P.397
Identification of a potential celhdar HDL assembly mechanism dependent on a liver mitochondrial protein
Y. Fuiiwara, Australia
N. Fidge. Baker
Medical
Research
Institute,
Prahran,
Mctoria,
Whereas the biosynthesis of VLDL is well documented, particularly aspects of intracellular assembly and the involvement of a microsomal triglyceride transfer protein in the process, formation of nascent HDL is controversial and not well understood. Having found evidence that rat liver cells may assemble HDL intracellularly, we sought further details using cell free preparations of liver. The system comprised an immobilized lipid source (14C-phophatidylcholine (PC) bound to celite), liver fractions and the presence or absence of apoprotein AI (h apoA1). The addition of subcellular fractions of rat or pig liver homogenates stimulated phospholipid (PL) transfer in the presence of apoA1 compared to controls and most transfer activity was associated with mitochondria of both pig and rat liver. We partially purified a mitochondrial protein using alkaline extraction, ammonium sulfate precipitation and S-300 sephacryl chromatography which increased specific activity 3 times over crude mitochondria. Analysis of the partially purified pig mitochondrial protein by SDS-PAGE showed many minor but two major bands of MW < 14,000. The product formed by incubating the pig mitochondial partially purified protein, PL and apoAI was investigated by gel filtration and density gradient ultracentrifugation. The complex containing apoA1 and PL was present in the density 1.18-1.21 g/ml fraction. The apoAI:PL molar ratio of this complex was 4: 1. This data shows that a liver factor, concentrated in the mitochondrial fraction promotes “HDL’ formation in vitro and consequently may play an important regulatory role in the biosynthesis of HDL.
CLI1 P 398
The aim of this study is to compare this two analytical methods, one based on selective precipitation of some lipoproteins and the other using modified enzymes with poliethilenglicol, allowing to measure HDL-Cholesterol directly, without precipitation. Materials and Methods: 200 serum samples of patients of our Hospital have been processed, determining HDL-Cholesterol levels by two different methods: (1) HDL-Cholesterol determination by precipitation with phosphotungstic acid-Manganese Clorur and later total Cholesterol determination by an enzymatic method (CHOD-PAP) in a HITACHI 717 Autoanalyzer. (2) Direct HDL-Cholesterol determination in semm using cholesterolesterase and cholesterol-oxidase modified by P.E.G. and 4-aminofenazone. The determination is adapted to an HITACHI 717 Autoanalyzer. Results: The least-squares regression analysis for HDL-Cholesterol serum concentratiom showed a proportional systematic error: y = 0.4693
t 0.9068x,
Confidence
Intervals
Comparative study of two methods to measure HDL-cholesterol levels
Measurement of blood HDL-Cholesterol levels has a great clinical importance due to the inverse correlation between its concentration and development of atherosclerosis. Many different methods have been developed nowadays to determine HDL-Cholesterol. Among these methods, the most common in laboratory routine is that of precipitation. Lately a direct method has been developed, quicker and safter, with no need to precipitate lipoprotein fractions, and that improves laboratory organization and efficiency. I Ith International
Symposium
n = 200.
a (-0.5301,
1.4688)
b (0.8894.0.9242).
1 A new homogeneous method for determination of LDL cholesterol
Mitsuhiro Nakamura’ Koichi Hino’, Mitsuaki Yamanoto’ , Mitsuhisa MrxKiichiro kobor?, Takashi Kanno*. ‘Daiichi Pure Chemicals Co., Ltd., Tokyo; 2Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan We have developed a new homogeneous method based on a technology which uses detergents to control the enzyme reactions with respective lipoprotein classes. First. lipoproteins other than LDL are reacted with cholesterol oxidase and cholesterol esterase in the presence of a detergent which specifically solubilizes these lipoproteins. The generated hydropemxide is consumed by a peroxidase reaction with 4-aminoantipyrine, yielding a colorless product. Subsequently, a specific reaction between LDL-C and the cholesterol enzymes is initiated and detected by the addition of a coupler and another detergent capable of solubilizing LDL. Assays were performed on a Hitachi 717 automated analyzer. Value assignment to the calibrator used for the assays was performed by the beta-quantification method (n = 3). Two hundred twenty patients’ sera were randomly selected and analyzed by the homogeneous method and beta-quantification method with a slight modification. The highest cholesterol level was 566 mg/dl, and the triglyceride level ranged from 14 to 1816 mg/dl. Linear regression analysis gave a correlation of 0.988 and an equation of y (homogeneous method) = 1.032 x (beta-quantification method) -0.584. Also, a mean absolute bias of 2.42 mg/dl was obtained. In addition, an analysis of forty eight sera obtained from patients diagnosed as hyperlipidemias (types I, II a, II b, III and IV) gave a correlation of 0.982 and an equation of y (homogeneous method) = 0.835 x (beta-quantification method) + 17.4. No significant discrepancies were observed between the homogeneous and beta-quantification methods, except one patient serum with type1 hyperlipidemia. On the other hand, comparison between Friedewald equation and the beta-quantification method showed significant discrepancies for type I and IV hyperlipidemia, providing negative values (below 0) for seven patients’ sera with triglyceride values greater than 800 mg/dl. We conclude that our method meets the requirements for routine analysis of LDL-C in clinical laboratories in terms of its accuracy and laboratory efficiency.
1 1 .P.400 1 Comparison of new methods for HDL-cholesterol determination with the phosphotungstic acidA3gCl2 precipitation procedure
A. Gavarr6, E. Berlanga, F. Campos, A. Veraguas, C. Llimiiiana, M. Torra. Hospital de Sabadell, Laboratory Service, Part Tat&s/n 08208Sabade II, Barcelona, Spain
Introduction:
r = 0.9837, of 95%:
Discussion: When we have compared both methods, no statistical differences have been obtained, with a very acceptable correlation index. These allows to conclude that both methods present similar results, and therefore it is possible to determine HDL-Cholesterol directly, with no need to precipitate lipoprotein fractions and improving routine laboratory organization, [ 1 .P.399
of the test-tubes.
Cholesterol is assayed in the supematant by conventional methods. The main advantage of this procedure is the suppression of the centrifugation step, making it fast and time-saving. This method is accurate and well-correlated with the sodium phosphotungstate-magnesium chloride method (r = 0.97).
101
M. Nauck, W. M&z, Hospital of Freiburg,
H. Wieland. Department FRG, Germany
ofclinical
Chemistry,
Univ.
We evaluated four new commercial methods for HDL-C determination. Three completely homogeneous assays were from Boehringer Mannheim (BM), Genzyme (GZ) and WAKO (WA). In a further method non-HDL lipoproteins are removed using dextran sulfate-coated magnetic beads and Mgc+ (provided by Reference Diagnostics, RD). We compared these methods with the conventional phosphotungstic acid/MgClz precipitation procedure (PTA).
on Atherosclerosis,
Paris,
October
1997
6 October 1997: Posters Methods
spectrometer. Thereafter, the leucine content in the pellet is determined by GC-MS using an internal standard. Knowing the number of leucine molecules in one molecule of apo B, the plasma concentration of apo B in the different lipoproteins can be calculated. The variation coefficients of apo B-100 measurements of VLDL (d < 1.006), VLDL + IDL (d < 1.019) and VLDL + IDL + LDL (d < 1.063) for normolipemic, hypertriglyceridemic and hypercholestemlemic plasma were less than 9%. The variation coefficients of other methods such as ELISA, electroimmunoassay and isopropanol precipitation combined with protein measurement according to the Lowry procedure were higher than those using GC-MS. Compared to the GC-MS procedure, ELISA and electroimmunoassay methods underestimate lipoprotein apo B concentrations. The combination of isopropanol precipitation and GC-MS method appears to be a precise and reliable method.
A new method of assaying the high density lipoprotein-cholesterol
I 1 P.396
D. Erlich, B. Charm, Y. Goussault. Laboratoire de Biochimie, Hopital Saint-Louis, 1 avenue Claude Vellefaux, 75475 Paris Cedex 10, France A new method of assaying the cholesterol of high-density lipoproteins is described. It consists in the use of magnetic particles coated with dextran-sulfate + magnesium chloride. They pull away precipitated lipoproteins (very low density and low density lipoproteins) by the use of a magnet located at the bottom
I 1 P.397
Identification of a potential celhdar HDL assembly mechanism dependent on a liver mitochondrial protein
Y. Fuiiwara, Australia
N. Fidge. Baker
Medical
Research
Institute,
Prahran,
Mctoria,
Whereas the biosynthesis of VLDL is well documented, particularly aspects of intracellular assembly and the involvement of a microsomal triglyceride transfer protein in the process, formation of nascent HDL is controversial and not well understood. Having found evidence that rat liver cells may assemble HDL intracellularly, we sought further details using cell free preparations of liver. The system comprised an immobilized lipid source (14C-phophatidylcholine (PC) bound to celite), liver fractions and the presence or absence of apoprotein AI (h apoA1). The addition of subcellular fractions of rat or pig liver homogenates stimulated phospholipid (PL) transfer in the presence of apoA1 compared to controls and most transfer activity was associated with mitochondria of both pig and rat liver. We partially purified a mitochondrial protein using alkaline extraction, ammonium sulfate precipitation and S-300 sephacryl chromatography which increased specific activity 3 times over crude mitochondria. Analysis of the partially purified pig mitochondrial protein by SDS-PAGE showed many minor but two major bands of MW < 14,000. The product formed by incubating the pig mitochondial partially purified protein, PL and apoAI was investigated by gel filtration and density gradient ultracentrifugation. The complex containing apoA1 and PL was present in the density 1.18-1.21 g/ml fraction. The apoAI:PL molar ratio of this complex was 4: 1. This data shows that a liver factor, concentrated in the mitochondrial fraction promotes “HDL’ formation in vitro and consequently may play an important regulatory role in the biosynthesis of HDL.
CLI1 P 398
The aim of this study is to compare this two analytical methods, one based on selective precipitation of some lipoproteins and the other using modified enzymes with poliethilenglicol, allowing to measure HDL-Cholesterol directly, without precipitation. Materials and Methods: 200 serum samples of patients of our Hospital have been processed, determining HDL-Cholesterol levels by two different methods: (1) HDL-Cholesterol determination by precipitation with phosphotungstic acid-Manganese Clorur and later total Cholesterol determination by an enzymatic method (CHOD-PAP) in a HITACHI 717 Autoanalyzer. (2) Direct HDL-Cholesterol determination in semm using cholesterolesterase and cholesterol-oxidase modified by P.E.G. and 4-aminofenazone. The determination is adapted to an HITACHI 717 Autoanalyzer. Results: The least-squares regression analysis for HDL-Cholesterol serum concentratiom showed a proportional systematic error: y = 0.4693
t 0.9068x,
Confidence
Intervals
Comparative study of two methods to measure HDL-cholesterol levels
Measurement of blood HDL-Cholesterol levels has a great clinical importance due to the inverse correlation between its concentration and development of atherosclerosis. Many different methods have been developed nowadays to determine HDL-Cholesterol. Among these methods, the most common in laboratory routine is that of precipitation. Lately a direct method has been developed, quicker and safter, with no need to precipitate lipoprotein fractions, and that improves laboratory organization and efficiency. I Ith International
Symposium
n = 200.
a (-0.5301,
1.4688)
b (0.8894.0.9242).
1 A new homogeneous method for determination of LDL cholesterol
Mitsuhiro Nakamura’ Koichi Hino’, Mitsuaki Yamanoto’ , Mitsuhisa MrxKiichiro kobor?, Takashi Kanno*. ‘Daiichi Pure Chemicals Co., Ltd., Tokyo; 2Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan We have developed a new homogeneous method based on a technology which uses detergents to control the enzyme reactions with respective lipoprotein classes. First. lipoproteins other than LDL are reacted with cholesterol oxidase and cholesterol esterase in the presence of a detergent which specifically solubilizes these lipoproteins. The generated hydropemxide is consumed by a peroxidase reaction with 4-aminoantipyrine, yielding a colorless product. Subsequently, a specific reaction between LDL-C and the cholesterol enzymes is initiated and detected by the addition of a coupler and another detergent capable of solubilizing LDL. Assays were performed on a Hitachi 717 automated analyzer. Value assignment to the calibrator used for the assays was performed by the beta-quantification method (n = 3). Two hundred twenty patients’ sera were randomly selected and analyzed by the homogeneous method and beta-quantification method with a slight modification. The highest cholesterol level was 566 mg/dl, and the triglyceride level ranged from 14 to 1816 mg/dl. Linear regression analysis gave a correlation of 0.988 and an equation of y (homogeneous method) = 1.032 x (beta-quantification method) -0.584. Also, a mean absolute bias of 2.42 mg/dl was obtained. In addition, an analysis of forty eight sera obtained from patients diagnosed as hyperlipidemias (types I, II a, II b, III and IV) gave a correlation of 0.982 and an equation of y (homogeneous method) = 0.835 x (beta-quantification method) + 17.4. No significant discrepancies were observed between the homogeneous and beta-quantification methods, except one patient serum with type1 hyperlipidemia. On the other hand, comparison between Friedewald equation and the beta-quantification method showed significant discrepancies for type I and IV hyperlipidemia, providing negative values (below 0) for seven patients’ sera with triglyceride values greater than 800 mg/dl. We conclude that our method meets the requirements for routine analysis of LDL-C in clinical laboratories in terms of its accuracy and laboratory efficiency.
1 1 .P.400 1 Comparison of new methods for HDL-cholesterol determination with the phosphotungstic acidA3gCl2 precipitation procedure
A. Gavarr6, E. Berlanga, F. Campos, A. Veraguas, C. Llimiiiana, M. Torra. Hospital de Sabadell, Laboratory Service, Part Tat&s/n 08208Sabade II, Barcelona, Spain
Introduction:
r = 0.9837, of 95%:
Discussion: When we have compared both methods, no statistical differences have been obtained, with a very acceptable correlation index. These allows to conclude that both methods present similar results, and therefore it is possible to determine HDL-Cholesterol directly, with no need to precipitate lipoprotein fractions and improving routine laboratory organization, [ 1 .P.399
of the test-tubes.
Cholesterol is assayed in the supematant by conventional methods. The main advantage of this procedure is the suppression of the centrifugation step, making it fast and time-saving. This method is accurate and well-correlated with the sodium phosphotungstate-magnesium chloride method (r = 0.97).
101
M. Nauck, W. M&z, Hospital of Freiburg,
H. Wieland. Department FRG, Germany
ofclinical
Chemistry,
Univ.
We evaluated four new commercial methods for HDL-C determination. Three completely homogeneous assays were from Boehringer Mannheim (BM), Genzyme (GZ) and WAKO (WA). In a further method non-HDL lipoproteins are removed using dextran sulfate-coated magnetic beads and Mgc+ (provided by Reference Diagnostics, RD). We compared these methods with the conventional phosphotungstic acid/MgClz precipitation procedure (PTA).
on Atherosclerosis,
Paris,
October
1997