31
Clink-x Chimica Acta, 88 (1978) 31-36 @ Elsevier/North-Holland Biomedical Press
CCA 9450
EVALUATION FOR APO-LDL D.J. NAZIR
OF AN AUTOMATED FOR PHENOTYPING
*, PHYLLIS
IMMUNOASSAY PROCEDURE TYPE II HYPERLIPOPROTEINEMIA
L. BROWN and M.J. McQUEEN
Clinical Chemistry, Department of Laboratory Medicine, Hamilton General Hospital, Hamilton (Canada) and Department of Pathology, McMaster University, Hamilton (Canada) (Received
January
20th, 1978)
Summary
Apo-low density lipoproteins were determined by an automated immunoassay procedure on serum samples from 88 normolipidemic individuals and 84 hyperlipoproteinemic subjects, to establish whether this method was useful in the routine detection of type II hyperlipoproteinemia. The results obtained were compared with the cholesterol levels of the same specimens. In subjects with type II hyperlipoproteinemia, the apo low density lipoprotein levels, as well as the ratio of low density lipoprotein cholesterol/ape-low density lipoprotein were higher, as expected, than in normals or in subjects with other types of hyperlipoproteinemia. However, there was considerable overlap in individual values of both these parameters, between patients with type II hyperlipoproteinemia and normals or subjects with other types of hyperlipoproteinemia, suggesting that apo low density lipoprotein levels alone were not sufficiently discriminatory for the laboratory determination of type II hyperlipoproteinemia.
Introduction
Several recent reports have suggested that a strong correlation exists between raised low density lipoprotein (LDL) levels in plasma and coronary artery disease [ 1,2] and ischemic heart disease [ 3,4]. Low density lipoproteins have usually been quantitated by measuring the cholesterol content of LDL. It has also been suggested that LDL may be responsible for the regulation of cholesterol synthesis via interaction with specific membrane receptors and concomitant inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, which is the rate controlling enzyme in cholesterol biosynthesis [5]. In view of this suggestion, we investigated the possibility that the measurement of apo-LDL could * Please address all correspondence to: Dr. D.J. Nazir. Clinical Chemistry, Medicine, Hamilton General Hospital. Hamilton. Ontario, LSL 2X2. Canada.
Department
of Laboratory
32
perhaps be a more reliable indicator cholesterol.
of type II hyperlipoproteinemia
than LDL
Material and methods A total of 172 subjects were examined for this study. They were phenotyped according to the classification proposed by Fredrickson et al. [6,7]. Further information on the subjects selected for this study is provided in Table I. Cholesterol [8] and triglycerides [9] were determined on an Abbott ABA100 by enzymatic methods. Low density lipoprotein cholesterol was determined by the method suggested by Wilson and Spiger [lo] using a dual precipitation technique. High density lipoprotein (YDL) cholesterol was determined after precipitating the very low density (VLDL) and LDL lipoproteins with heparin and MnCl, [ll] while VLDL alone was precipitated with sodium dodecylsulfate. Apo-LDL was determined on an Autoanalyzer II system by an automated immunoprecipitin method described in detail in a Technicon publication [12]. The antiserum used in these experiments was specific for the protein portion of the /3-lipoprotein molecule (apo-LDL) and not for the entire LDL (basic antigenic protein + associated lipid fractions). Results and discussion The accuracy and precision of the automated immunoassay procedure for the determination of apo-LDL was established by using a control apo-LDL antigen supplied by Atlantic Antibodies. By appropriate dilutions, solutions theoretically containing 38.0, 76.0, 114.0, 152.0 and 190.0 mg/lOO ml of apoLDL were obtained. Results obtained on 34-41 separate runs indicate that the mean values obtained were within about 1 mg/lOO ml of the theoretical value and the standard deviations varied between 4.0 and 4.7 mg/lOO ml throughout the concentration range studied. The coefficients of variation were 10.3, 6.2, 3.7, 3.1 and 2.1% respectively, showing the expected improvement of precision of the method at higher concentrations. One hundred and twenty specimens could be analyzed per hour; a rate satisfactory for routine use. However, it was necessary to run a blank for every sample, due to differences in the inherent light scattering properties of the samples undergoing analysis. Richie and Clark [13] stated that some samples exhibit.ed considerable light scattering ability, which was not related to turbidity assessed visually. The effect of storage on the estimation of apo-LDL was studied by storing aliquots of serum at room temperature (24°C for 2 h), in the refrigerator (4°C) for 1, 3 and 7 days, and in the freezer (-20°C) for 1 and 3 days (n = 32). An excellent correlation was obtained under all conditions of storage studied in these experiments (r = 0.94-0.99). The slope of the regression line ranged from 0.96 to 1.29 for the different storage conditions. However, on storage for 3 days at both 4°C and -2O”C, the intercepts on the Y-axis were -42.62 and -35.24 respectively, suggesting that storage under these conditions resulted in apo-LDL values somewhat lower than those obtained with fresh specimens (intercept = 6.34). Somewhat unexpectedly, the intercept was only -0.76 on the 7th day of storage at 4°C. A possible explanation for these results could be
33
that, for some unexplained reason, the results obtained on day 3 were uniformly lower than those obtained on all other days. Richie and Clark [13] reported an excellent correlation (r= 0.97) for LDL protein before and after freezing, although they did not provide any information regarding the temperature and duration of freezing. The LDL cholesterol ranged from 190 to 241 mg/lOO ml for type IIA and from 191 to 205 mg/lOO ml for type IIb on the subjects studied in these experiments (Table II). The mean f 1 S.D. for types IIA and IIB HLP ranged from 192 to 224 and from 195 to 205 mg/lOO ml respectively. An analysis for the LDL cholesterol values in normals and in subjects having other types of hyperlipoproteinemias revealed that only one out of two type II HLP’s and three out of fifty-eight subjects with type IV HLP had LDL cholesterol values a192 mg/ 100 ml. The values obtained for apo-LDL in type IIA and IIB HLP ranged from 98 to 149 and from 131 to 150 mg/lOO ml respectively (Table II). Calculations for the mean + 1 S.D. gave values ranging from 112 to 148 mg/lOO ml for type IIA HLP and from 135 to 147 mg/lOO ml for type IIB HLP. Out of the 88 normal subjects examined in this study, 27 had an apo-LDLvalue >112mg/lOO ml. Both type III subjects, 30 out of the 58 type IV HLP’s and 1 out of 4 type V HLP subjects also showed apo-LDL concentrations 2112 mg/lOO ml. Thus in our experience, apo-LDL concentrations were not very useful in phenotyping hyperlipoproteinemias. The ratio of LDL cholesterol/ape-LDL ranged from 1.36 to 1.94 for type IIA and from 1.33 to 1.55 for type IIB, with mean ? lS.D. ranges from 1.42 to 1.82 and from 1.35 to 1.49 respectively (Table III). Of the 88 normal subjects studied, 25 had a ratio a1.35. One out of two type III HLP, 21 out of 58 type IV HLP, and 2 out of 4 type V HLP also had ratios a1.35, indicating that like apo-LDL, this ratio was not very useful in differentiating type II HLP from normals, and subjects having other types of HLP. The correlation coefficient between LDL cholesterol and apo-LDL was 0.84 in normals, and dropped to 0.58, 0.15 and 0.78 for types IIA, IIB and IV respectively (Table III). The correlation coefficient between total cholesterol and apo-LDL was not any better; the value for normals was 0.82, and 0.25, 0.17 and 0.77 for types IIA, IIB and IV respectively. In the 88 normal subjects examined in this study, a mean value of 1.24 with a SD. of kO.24 was obtained for the LDL-cholesterol/LDL protein ratio, similar to the value reported by Thompson et al. [14]. Lees [15] and Albers et al. [16] reported a ratio of 1.7 in normal subjects. In type II (A and B) hyperlipoproteinemic subjects, Lees [ 151 and Albers et al. [16] reported a ratio of 1.7-1.8. In the present study the mean ratios were 1.62 for type IIA HLP and 1.42 for type IIB HLP, values which are somewhat lower than those reported in the literature. The LDL molecule is stated to contain about 45% cholesterol and 20% protein [ 17,181. The most widely used method for phenotyping type II HLP is to obtain a value >190 mg/lOO ml for LDL cholesterol [6]. Assuming that the ratio of LDL-cholesterol/ape-LDL is reasonably constant in normal and hyperlipidemic subjects [16], and increase in LDL-cholesterol in type II HLP should be accompanied by an increased concentration of apo LDL, then measurement
No. of subjects
88 9 11 2 58 4
Type of HLP
Normal IIA IIB III IV V
LDL CHOLESTEROL
TABLE If
88 9 11 2 58 4
172
Total
group Normal IIA IIB HI XV V
N
38 5 8 1 15 -
16-79 50-68 44-77 68 24-69 -
16-79
35-190 190-241 191-205 112-252 56-206 83-137
37 16 5 34
117 208 200
139 118
105-173
80-l 54 192-224 195-205
38-137 98-149 131-150 119-167 39-l 50 51-l 14
Range
Range
Mean t 1 S.D.
APO-LDL (m&l00
Mean
110 87
95 130 141
Meen
ml)
24
24 18 6
S.D.
72-258 273-293 270-291 312 151-274
86-134
71-119 112-148 135-147
Mean + 1 S.D.
34-171 80-l 56 188-339 322 158-396 -
34-396
(range)
(range) 72-312
Triglycerides (mg/lOO ml)
Cholesterol (mg/lOO ml)
SUBJECTS
Age (range)
AND HYPERLIPOPROTEINEMIC
44-181 loo-146 183-223 177 154-591 411-674
67
N
Females
LDL cholesterol (mg/lOO ml) S.D.
IN NORMAL
86-242 262-344 274-292 196 113-270 151-199
44-614
(range)
(range) 86-344
Triglycerides (mg/lOO ml)
Cholesterol (IYwlOO ml)
AND APO-LDL CONCENTRATIONS
14-86 62-61 45-63 30 19-93 25-63
14-93
105
50 4 3 1 43 4
AiZe (range)
N
Males
DATA ON SUBJECTS
TYPO
BIOCHEMICAL
TABLE I
35 TABLE III RATIO
OF LDL
CHOLESTEROL
AND
APO-LDL
IN NORMAL
AND
HYPERLIPOPROTEINEMIC
s UBJECTS LDL cholesterollapo-LDL,
Normal Range Mean S.D. Mean f 1 S.D.
0.66-l .72 1.24 0.24 1.00-1.48
Type IIA 1.36-1.94 1.62 0.20 1.42-1.82
x = LDL cholesterol; Y = ape-LDL Slope of 0.54 0.66 regression line Intercept on 32.06 -6.64 y-axis Correlation 0.84 0.58 coefficient
ratio Type IIB 1.33-1.55 1.42 0.07 1.35-1.49
Type III
Type IV
0.94-1.52
0.76-1.90 1.29 0.22 1.07-1.51
0.17
0.56
105.99
31.94
0.15
0.78
Type V 0.97-2.41 1.48
x = total cholesterol; y = apo-LDL Slope of regression line Intercept on y-axis Correlation coefficient
-0.12
0.50
74.57
175.37
5.35
0.25
0.17
0.77
0.49
0.19
4.87 0.82
of apo LDL should be a useful method for the determination of type II HLP. However, the results presented in tables II and III indicate such an overlap between the apo-LDL of type II HLP and apo-LDL of either normal subjects or other types of hyperlipoproteinemias, as to suggest that apo-LDL is not a very useful indicator for the determination of type II HLP. Similarly, the ratio of LDL cholesterol/ape-LDL is not a useful index for type II HLP either. The data presented by Lees [15] and Albers et al. [16] support these conclusions. Richie and Clark [ 131 have suggested that the determination of LDL protein could be useful for the typing of hyperlipoproteinemias. However, the difficulty in separating type IIB, III, IV and V which all show high triglyceride and high protein values, would prove to be a serious drawback to the widespread use of LDL protein concentrations in the classification of hyperlipoproteinemias . Acknowledgement The authors gratefully acknowledge the support provided funds of the Hamilton Civic Hospitals (Grant No. 76-105). References 1 2 3 4 6
Levy. R.I. (1975) Postgrad. Med. J. 51 (Suppl. 8). 16-24 Jensen, J., Blankenhom, D.H. and Komemp. V. (1967) Circulation 36, 77 ‘-82 Slack. J. and Nevin, N.C. (1968) J. Med. Genet. 5.4-8 Slack, J. (1969) Lancet ii. 1380-1382 Brown, M.S. and Goldstein, J.L. (1974) Science 185.61-64
from the research
36 6 Fredrikcson, D.S., Levy, R.I. and Lees, R.S. (1967) New Eng. J. Med. 276,148-156 7 Fredrickson, D.S. and Levy, R.I. (1972) in Metabolic Basis of Inherited Disease (Stanbury. J.B., Wyngaarden. J.B. and Fredrickson, D.S.. eds.), PP. 545-614. McGraw Hill, New York 8 BMC Instrument Application Cat. No, 172626 9 Dow Reagent Systems for the ABA 100. Cat. No. 46676 10 Wilson. D.E. and Spiger. M.J. (1973) J. Lab. Clin. Med. 82, 473-482 11 Manual of Laboratory Operations (1974) in Lipid Research Clinics Program, Vol. 1, Lipid and Lipoprotein Analysis, pp. 56-59, National Heart and Lung Institute, N.I.H., Bethesda, Md. 12 Technicon Method No. SE4-0038 FE5 (1975) May 13 Richie, R.F. and Clark, C. (1972) in Automated Immuno Precipitin Reactions. Papers Presented at Colloquium on A.I.P., Dept. of Exptl. Med., Univ. of Louvain. Brussels, Belgium, May 2 and Technicon International Congress, New York, June 12. pp. 13-16 14 Thompson, G.R., Birnbaumer. M.E.. Levy. R.I. and Gotto, Jr., A.M. (1976) Atherosclerosis 24, 107118 15 Lees, R.S. (1970) Science 169.493-495 16 Albers, J.J.. Cabana, V.G. and Hazzard. W.R. (1975) Metabolism 24, 1339-1351 17 Blaton, V.H. and Peeters. H. (1972) in Blood Lipids and Lipoproteins (Nelson, G.J., ed.), PP. 281. Wiley Interscience, New York 18 Hatch, F.T. and Lees, R.S. (1968) in Advances in Lipid Research (Paoletti, R. and Kritchevsky. D., eds.), p. 4. Academic Press, New York