Determination of serum lipoprotein lipase using a latex particle-enhanced turbidimetric immunoassay with an automated analyzer
Introduction
Lipoprotein lipase (LPL) plays a major role in the metabolism and transport of lipids and lipoproteins [1], [2]. It is the enzyme responsible for the hydrolysis of core triglycerides (TG) in chylomicrons (CM) and very low density lipoproteins (VLDL), producing CM remnants and VLDL remnants, respectively. Determination of LPL in plasma has typically been routinely carried out by ELISA after the intravenous injection of heparin (with its activity and concentration). However, it is also known that a comparatively high LPL concentration (ranging approximately 30–100 ng/ml in normal controls) is found in the pre-heparin serum, with an undetectable level of LPL activity, indicating that the majority of circulating LPL is catalytically inactive, but still a ligand for the receptors [3], [4], [5], [6].
The LPL concentration and activity in the post-heparin plasma have been clinically used for the detection of LPL deficiency [2], but in general not for the diagnosis of lipid disorders or the risk of cardiovascular disease. This is because heparin injection dissociates LPL from the blood vessel endothelium, so the result does not necessarily reflect the physiological or pathophysiological concentration of circulating LPL.
An LPL-ELISA assay using specific monoclonal antibodies was reportedly developed previously by Brunzell et al. [7] and Ikeda et al. [8] for the detection of LPL in human plasma, which involved the administration of a heparin injection to the patients before the measurement of the plasma LPL concentration. Considering the assay time and the technical steps required for the quantitative measurement by ELISA, this method is not suitable for large-scale epidemiological studies or routine clinical laboratory assay.
Therefore, there remains a need for a reliable, rapid and automated assay for the LPL concentration that has both good sensitivity and good calibrator stability, in particular if the measurement of the pre-heparin serum LPL concentration is going to be clinically meaningful and useful. LPL concentrations in the pre-heparin serum have been intensively investigated by Shirai and his colleagues the last decades using LPL-ELISA, revealing the clinical significance of the pre-heparin LPL concentration in cardiovascular and diabetic diseases [9], [10], [11], [12], [13], [14], [15], [16].
We recently showed the possibility that the LPL concentration in the pre-heparin serum is replaceable with the LPL activity in the post-heparin plasma based on a comparison between them [17]. Therefore, the measurement of the LPL concentration in the pre-heparin serum will be able to provide more practical clinical applications in TG-rich patients without the need of a heparin injection using an automated LPL assay. As the serum pre-heparin LPL concentration is sufficiently high so as to measure it with a latex assay system, we developed a rapid and sensitive latex particle-enhanced turbidimetric immunoassay (LTIA) using latex bead-immobilized LPL-specific antibodies. The performance of the LTIA was evaluated on a Hitachi H7700 P automated analyzer. We compared its analytical properties with a commercially available ELISA assay [18] in normal volunteers, with and without heparin injection.
Section snippets
Reagents
Polystyrene latex particles were obtained from Fujikura Corporation and bovine serum albumin (BSA) from Sigma, respectively. Interfering reagents, containing bilirubin F and C, hemoglobin, triglycerides and rheumatoid factor, were from Sysmex. All of the chemicals and reagents were of the highest available grade.
Preparation of blood samples
The study was conducted in relatively healthy young volunteers (some were overweight or obese) in a male (n = 19) and female (n = 21) population (Caucasian 25, Asian 5, Hispanic 4, African
Linearity
Six concentrations of recombinant LPL (0, 50, 100, 200, 400 and 800 ng/ml) were used to assess the calibration curve. After dilution (up to 10 times) with saline, serial dilutions were measured with three replicates per specimen and the linearity was evaluated. Within the measuring range of 0–200 ng/ml and 0–800 ng/ml, the deviations from theoretical values did not exceed 5%, indicating no lack of parallelism and showing good linearity (Fig. 1A and B). These results suggest a good linearity within
Discussion
The main purpose for the development of the LPL latex assay system was to determine the pre-heparin serum LPL concentration for routine clinical laboratory use in a manner that does not require heparin injection before blood withdrawal. Heparin injection has been a great barrier for the determination of LPL in clinical practice. Although LPL in the pre-heparin serum is known to not have any activity, its concentration is high enough to be detected by a latex assay system as well as by ELISA. To
References (23)
- et al.
The low density lipoprotein receptor-related protein/a2-macroglobulin receptor binds and mediates catabolism of bovine milk lipoprotein lipase
J Biol Chem
(1992) - et al.
Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies
J Lipid Res
(1992) - et al.
A sandwich-enzyme immunoassay for the quantification of lipoprotein lipase and hepatic triglyceride lipase in human postheparin plasma using monoclonal antibodies to the corresponding enzymes
J Lipid Res
(1990) - et al.
The level of pre-heparin serum lipoprotein lipase mass at different stages of pregnancy
Clin Chim Acta
(2003) - et al.
Low lipoprotein lipase mass in preheparin serum of type 2 diabetes mellitus patients and its recovery with insulin therapy
Diabetes Res Clin Pract
(2002) - et al.
Enhancement of preheparin serum lipoprotein lipase mass by bezafibrate administration
Atherosclerosis
(2000) - et al.
Preheparin serum lipoprotein lipase mass is negatively related to coronary atherosclerosis
Atherosclerosis
(2000) - et al.
Preheparin serum lipoprotein lipase mass might be a biomarker of metabolic syndrome
Diabetes Res Clin Pract
(2007) - et al.
Lipoprotein lipase mass and activity in severe hypertriglyceridemia
Clin Chim Acta
(1993) - et al.
A novel method for measuring human lipoprotein lipase and hepatic lipase activities in postheparin plasma
J Lipid Res
(2008)
Cited by (23)
Quantification of soluble very low-density lipoprotein receptor in human serum using a sandwich enzyme-linked immunosorbent assay
2023, Practical Laboratory MedicineDevelopment and characterization of a latex turbidimetric immunoassay using rabbit anti-CRP single-chain Fv antibodies
2023, Journal of Immunological MethodsChylomicronemia from GPIHBP1 autoantibodies
2020, Journal of Lipid ResearchThe antagonic behavior of GPIHBP1 between EAT and circulation does not reflect lipolytic enzymes levels in the tissue and serum from coronary patients
2020, Clinica Chimica ActaCitation Excerpt :Remnant lipoproteins cholesterol (RLP-C) was calculated as Total cholesterol – LDL-C – HDL- C. LPL mass was determined by a rapid and sensitive latex particle-enhanced turbidimetric immunoassay (LTIA) using latex bead-immobilized anti-LPL monoclonal antibodies, the assay was performed on a Hitachi 7700P [25]. EL mass was measured using an ELISA with monoclonal antibodies specific to human plasma EL [26].
Increased plasma lipoprotein lipase activity in males with autism spectrum disorder
2020, Research in Autism Spectrum Disorders