A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: Application of the CD3/CD28 assay in lymphocytes

Abstract

Abbreviated title: An improved assay for LDL receptor activity using anti-CD3/CD28 beads Abbreviations: DiI, 3,3"--dioctadecylindocarbocyanin; FH, familial hypercholesterolemia; NARC-1, neural apoptosis-regulated convertase 1; FITC, fluorescein isothiocyanate; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; LDLR, low-density lipoprotein receptor; LPDS, lipoprotein deficient serum; rIL-2, recombinant interleukin-2; MF, mean fluorescence; TC, total cholesterol; CV, coefficient of variation. 3 AbstractBackground: The objective of this study was to develop a new and simple method for measuring low-density lipoprotein receptor (LDLR) activity using peripheral lymphocytes enabling us to clinically diagnose familial hypercholesterolemia (FH) and ascertain the involved mutations (such as K790X mutation), that might not be clearly detected in the conventional method.Methods: Our method comprised the following 2 features: First, we used anti-CD3/CD28 beads to stimulate T-lymphocytes to obtain a uniform fraction of lymphocytes and maximum up-regulation of LDLR. Second, we excluded the possibility of overestimation of lymphocyte signals bound only to its surface, by adding heparin to the cultured lymphocytes used for the LDLR assay.Results: Based on the genetic mutation, the FH subjects were divided into two groups, K790X, (n = 20) and P664L, (n = 5), and their LDLR activities was measured by this method, which was found to be 55.3 ± 8.9% and 63.9 ± 13.8%, respectively, of that of the control group (n = 15). In comparison, the LDLR activity was 86.1 ± 11.6% (K790X) and 73.3 ± 6.3% (P664L) of that of the control group when measured by the conventional method, indicating that impairment of LDLR function in FH K790X subjects was much more clearly differenciated with our method than with the conventional method (paired t-test, p < 0.0001). The levels of LDLR expression also showed similar tendencies, that is, 89.4 ± 13.2% (K790X) and 76.9 ± 17.4% (P664L) of that of the control group when measured by the conventional method, and 78.1 ± 9.7% (K790X) and 70.3 ± 26.5% (P664L) when measured by our new method. In addition, we confirmed that there was little influence of statin treatment on LDLR activity among the study subjects when our method was used.Conclusion: These results demonstrate that our new method is applicable for measuring LDLR 4 activity, even in subjects with an internally defective allele, and that T-lymphocytes of the FH K790X mutation possess characteristics of that allele.

Altered Metabolism of Low-Density Lipoprotein and Very-Low-Density Lipoprotein Remnant in Autosomal Recessive Hypercholesterolemia

“…ApoB-100 direct removal occurs from VLDL2 (k(0,13)), VLDL remnant (k(0,12)), IDL (k(0,21)), IDL remnant (k(0,22)), and LDL (k(0,31)). To make the model identifiable, the rate constant from VLDL1 to VLDL2 (k (13,11)), representing delipidation, was constrained to be equal to that from VLDL2 to IDL (k (21,13)). For comparison between 2 groups (ARH patient and controls) the VLDL1, VLDL2, and VLDL remnant data were presented as VLDL delipidation rate and VLDL FCR, which represents the sum of delipidation and direct removal rate.…”

“…12 LDLR activity was measured by 2 methods, both of which used peripheral lymphocytes; The first was commercially available binding assay and the second was our original assay, which was described in detail elsewhere. 13 Briefly, we could measure accurate LDLR activity by using heparin to exclude the overestimation signals only bound at the surfaces of lymphocytes, even in the case with internalization defective type of disease. …”

Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment

“…Current methodologies, which determine LDLR functionality ex vivo , are based on the use of radioactivity or flow cytometry. LDL uptake and degradation of 125 I-labeled LDL is commonly determined in radioactivity-based methods, and very similarly, binding and uptake of LDL are determined by flow cytometry when using fluorescent-labelled LDL [6], [12]–[17]. Both methods are used indistinctly, probably depending on the research laboratory facilities or because there is a traditional used methodology in the laboratory.…”

An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations

“…The main problem of these functional assays was the bad separation between residual LDLR activity from FH patients and controls. The separation of different leukocyte populations obtains more accurate results ( 8 ); the selection of viable lymphocytes by stimulation with a mitogen or by fl ow cytometry gating improves the discrimination between patients and controls although it does not allow complete discrimination of FH heterozygote patients from healthy controls ( 9,10 ).…”