Clinica Chimica Acta, 203 (1991) 177-182 8 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8Y81/91/SO3.50

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Determination of apolipoprotein E phenotypes from stored or postmortem serum samples Terho Lehtimiki Department of Biomedical Sciences, Unic~ersityof Tampere, Tampere (Finland)

(Received 1 February 1991; revision received 19 August 1991; accepted 21 August 1991) Kf? words: Apolipoprotein

E; Postmortem change; Storage; Isoelectric focusing; lmmunoblotting

Summary In order to assess the validity of Apo k phenotyping from stored specimens, phenotypes determined from fresh serum samples were compared with those from stored (8 years at - 20°C) samples from the same individuals (n = 42). The effect of early postmortem period on Apo E phenotype determinability was studied by taking four duplicate blood samples from eight cadavers 2, 6, 12, and 24 h after death. Apo E phenotyping was performed directly from serum by isoelectric focusing and immunoblotting. From the cadavers, the same Apo E phenotypes were obtained 2, 6, 12, and 24 h after death. After eight years’ storage five out of ten Apo E4/4 phenotypes were falsely recorded as Apo E4/3 and one out of siu Apo M/3, one out of 12 Apo E4/2 were falsely interpreted as Apo E3/3. Phenotypes Apo E2/2 (n = 21, Apo E3/2 (n = 101, and Apo E3/3 (n = 2) were correctly assessed after 8 years of storage. In the total material, 17% (7/42) of Apo E phenotypes were incorrectly assessed after the storage.

Introduction Apolipoprotein E is a structural and functional constituent of plasma chylomicrons and very low density lipoproteins (VLDL) and their lipolytic degradation products, i.e., chylomicron remnants and intermediate density lipoproteins (IDL) [ 1,2]. Apo E serves as ligand for the specific uptake of lipoprotein particles by the liver [2-41.

Correspondence and requests for reprints to: Terho LehtimHki, B.M., Department Sciences, University of Tampere, P.O. Box 607. SF-33101 Tampere 10, Finland.

of Biomedical

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The genetic polymorphism of Apo E is caused by three alleles, ~2, ~3, and ~4, at a single genetic locus, coding for respective Apo E isoforms, E2, E3, and E4 [5,6]. Consequently, six different Apo E phenotypes can be detected in serum: E2/2, E3/2, E3/3, E4/2, E4/3, and E4/4. In several populations plasma total and LDL cholesterol concentrations increase according to Apo E phenotype in the order: E2/2 < E3/2 < E3/3 < E4/3 < E4/4 [7-121. There is also growing evidence that the Apo E4 isoform is associated with increased risk of coronary heart disease (CHD) [13-181. Epidemiological interest in Apo E phenotype raises the question whether the phenotyping could be carried out from stored or postmortem specimens [19]. There are no previous reports on the determinability of Apo E phenotype after death. The present study was carried out to examine the determinability of Apo E phenotype from serum samples kept frozen for 8 yr or taken after different time intervals postmortem. Subjects and methods Fasting venous blood samples were obtained from 42 individuals (age 3-18 years) in 1980 in connection with the Study of Cardiovascular Risk in Young Finns [20,21]. Apo E phenotypes were determined in 1988 after eight years’ storage at -20°C. The samples had been thawed and refrozen 2 to 7 times during the storage. New blood samples were taken from the same persons in 1986 and were phenotyped within 20 months of storage at -20°C. Havekes et al. have reported [19] that Apo E phenotypes can be reliably determined from four-year-old serum samples stored at -20°C. The Apo E phenotypes determined from the ‘fresh’ samples and the numbers of individuals were: E2/2 (n = 21, E3/2 (n = lo), E4/2 (n = 12), E3/3 (n = 2), E4/3 (n = 6) and E4/4 (n = 10). Blood samples were drawn after an overnight fast and were allowed to clot at room temperature. The serum was separated by centrifugation (2500 X g 15 min, 4°C) and stored at -20°C both in 1980 and 1986. All Apo E phenotypes were determined from delipidated serum with cysteamine-treated duplicates by isoelectric focusing (IEF) and immunoblotting, as described in detail by Lehtimaki et al. [12]. The recognition of Apo E phenotypes in the blots was based on comparison with IEF patterns from known Apo E phenotypes and with earlier published IEF patterns. The results were interpreted by two people independently, with 100% concordance. Fresh blood specimens were taken after an overnight fast from 15 healthy, normolipidemic medical students who volunteered for the study. Their Apo E phenotypes were: E3/2 (n = 41, E3/3 (n = 41, E4/3 (n = 4) and E4/4 (n = 3). The effect of repeated freezing-thawing cycles was studied using these samples (Table I). Venous blood was taken from corpses of eight men (six Apo E3/3 and two Apo E4/3), aged 31 to 74 years, who had died suddenly of myocardial infarction (n = 6), shotgun trauma (n = l), and choking (n = 1). The bodies were transferred to 4°C within 0.5 to 1 h after death. From the femoral or brachial veins four

179 TABLE I The numbers of correct and incorrect interpretations serum Phenotype (n)

Number of correct interpretations

EZ/2 E3,‘2 E3,‘3 E4/2 E4/3 E4,‘4 All

2 10 2 11 5 5 35

(2) (IO) (2) (12) (61 (10) (42)

of Apo E phenotypes after eight years’ storage of

Number of incorrect interpretations

% of correct interpretation

1 1 5 7

100 100 100 92 83 50 83

duplicate samples were taken into tubes containing sodium citrate 2, 6, 12, and 24 h after death. After centrifugation (2500 X g, 15 min, 4”C), portions of plasma were stored at - 20°C until analyzed. The study protocol was approved by the ethical committees of the participating universities. Results A total of 42 serum samples were analyzed for Apo E phenotype both after a few months’ storage (‘fresh’ samples) and after 8 years’ storage at - 20°C. Figure 1 illustrates the effect of 8 years’ storage on representative isoelectric focusing patterns of different Apo E phenotypes. Additional minor bands (Fig. 1B) appeared in the isoelectric focusing patterns of Apo E isoforms, particularly between the Apo E4 and Apo E3 isoforms after eight years of storage of serum at -20°C. These additional minor bands were not seen when fresh serum samples were used (Fig. 1A). After storage, five out of ten Apo E4/4 phenotypes were erroneously interpreted as Apo E4/3 and one Apo E4/3 out of six and one Apo E4/2 out of 12 were interpreted as Apo E3/3. In phenotypes Apo E2/2, E3/2, or E3/3, storage appeared to cause no errors in the assessment of the phenotype (Table I). From serum of all the eight cadavers representing phenotypes E3/3 or E4/3 the same phenotypes were recorded 2, 6, 12, and 24 h after death (Table II). TABLE II The interpretation

of Apo E phenotype after different time intervals of death

Phenotype a

Hours after death

E3/3 E4/3 All

2

6

12

24

% of correct interpretations

W/3 (61 E4/3 (2) (8)

E3,‘3 (6) E4/3 (2) (8)

E3,‘3 (6) E4/3 (21 (8)

E3/3 (6) E4,‘3(2) (81

100 100 100

a The results were interpreted by two persons independently, with 100% concordance. The numbers in parentheses show the number of correct interpretations of Apo E phenotype.

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Fig. 1. Isoelectric focusing patterns of six representative serum samples after dehpidation. The bands were detected by immunoblotting. (A) ‘Fresh’ serum samples; (Bl samples from same persons, stored for 8 years at - 20°C. The untreated samples are to the left and the cysteamine treated (T) to the right. The locations of the main Apo E isoform bands are indicated. The cathode (-) is at the top and the anode ( +) at the bottom.

When fresh serum samples, representing phenotypes E3/2 (n = 4), E3/3 (n = 4), E4/3 (n = 4), and E4/4 (n = 3) were thawed and refrozen one to eight times, exactly same Apo E phenotyping results were achieved in all phenotype groups by both of the two persons that did the interpretation. Discussion During eight years of storage of serum at -20°C additional minor bands appeared in the isoelectric focusing patterns of Apo E isoforms, particularly between the Apo E4 and Apo E3 isoforms (Fig. 1). The extra bands were recognized by the polyclonal antibody to Apo E and are probably caused by proteolysis during storage, as also suggested by Havekes et al [19], or by repeated freezing-thawing cycles of the samples. According to Menzel and Utermann [22] one of the Apo E fragments produced by thrombin focuses close to the position of the Apo E3 isoform.

181

The proteolytic fragments of Apo E may confound Apo E pheno~ing, especially in phenotypes containing the Apo E4 isoprotein. If the intensity of Apo E4 isoform band on the blot is diminished after proteoIysis, homozygotes E4/4 may be wrongly taken as heterozygotes E4/3 (as was the case in five out of ten E4/4’s in our study) and E4/3 or E4/2 may be interpreted as E3/3 (as was the case in two out of 18 samples). Similar problems were not encountered in the assessment of Apo E3/3 or Apo E2/2 homo~gotes (Table I), which may be due to a different sensitivity of E2 and E3 isoproteins to proteoIysis as compared to E4. However, it seems more likely that proteolytic splitting of Apo E3/3 phenotype (n = 2) during the storage would have been observed if there had been more samples in this phenotype group. After eight freezing-thawing cycles of samples exactly same phenotype results were achieved. It seems likely that a substantial part of the errors in the interpretation of the Apo E phenotypes in the present study are due to the changes arising during storage. Hydrolytic splitting of proteins has also been observed during the early postmortem autolysis process [23,24]. These changes are of importance when concentrations of blood components after death are measured quantitatively [25-271. There are no previous reports on postmortem Apo E levels. Apolipoprotein A-I (APO A-I) concentrations have been reported to be measurabie 24 h after death [25]. Due to the similarities in biochemical structure of apoproteins E and A-I [2] it is likely that Apo E measured two hours of death is representative of the living person, In addition, knowledge of the absolute Apo E concentrations in serum is not required for Apo E phenotype analysis. In the present study exactly the same Apo E pheno~pes were obtained 2, 612, and 24 h after death in every case studied. Only a few additional minor bands, which did not impede Apo E phenotyping, were obtained in the blots from 6-, 12-, or 24-h-old postmortem serum samples.

The author thanks Professor Tapio Nikkari, Associate Professor Jorma Viikari, Dr. Teemu Moilanen and Dr. Christian Ehnholm for criticism of the manuscript, Dr. Seppo YlhHerttuala for the specimens of postmortem serum, and Miss Nina Peltonen for technical assistance. This study was supported by a grant from the Finnish Foundation for Cardiovascular Research and The Elli and Elvi Oksanen Fund of the Pirkanmaa Regionai Fund under the auspices of the Finnish Cultural Foundation. References Shore VG, Shore B. Heterogenei~ of human plasma very low density lipoproteins. Separation of species differing in protein components. Biochemistry 1973;12:502-507. Mahley RW, lnnerarity TL, Rail Jr SC, Weisgraber KH. Plasma lipoproteins: apolipoprotein structure and function. J Lipid Res 1984;25:1277-1294. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science 1986;232:34-47.

182 4 Kowal RC, Herz J, Goldstein

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21 22 23 24 25

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JL, Esser V, Brown MS. Low density lipoprotein receptor-related protein mediates uptake of cholesteryl esters derived from apolipoprotein E-enriched lipoproteins. Proc Nat1 Acad Sci USA 1989; 865810-5814. Zannis VI, Just PW, Breslow JL. Human apolipoprotein E isoprotein subclasses are genetically determined. Am J Hum Genet 1981;33:11-24. Utermann G, Hees M, Steinmetz A. Polymorphism of apolipoprotein E and occurrence of dysbetalipoproteinaemia in man. Nature 1977;269:604-607. Ehnholm C, Lukka M, Kuusi T, Nikkilli E, Utermann G. Apolipoprotein E polymorphism in the Finnish population: gene frequencies and relation to lipoprotein concentrations. J Lipid Res 1986;27:227-235. Eto M, Watanabe K, Ishii K. Reciprocal effects of apolipoprotein E alleles (~2 and 64) on plasma lipid levels in normolipidemic subjects. Clin Genet 1986;29:477-484. Utermann G. Apolipoprotein E polymorphism in health and disease. Am Heart J 1987;113:433-440. Sing CF, Davignon J. Role of the apolipoprotein E polymorphism in determining normal plasma lipid and lipoprotein variation. Am J Hum Genet 1985;37:268-285. Ordovas JM, Litwack-Klein L, Wilson PWF, Schaefer MM, Schaefer EJ. Apolipoprotein E isoform phenotyping methodology and population frequency with identification of Apo El and Apo E5 isoforms. J Lipid Res 1987;28:371-380. Lehtimiiki T, Moilanen T, Viikari J, et al. Apolipoprotein E phenotypes in Finnish youths: a cross-sectional and six year follow-up study. J Lipid Res 1990;31:487-495. Menzel HJ, Kladetzky RG, Assmann G. Apolipoprotein E polymorphism and coronary artery disease. Arteriosclerosis 1983;3:310-315. Cumming AM, Robertson FW. Polymorphism at the apoprotein-E locus in relation to risk of coronary disease. Clin Genet 1984;25:310-313. Lenzen HJ, Assmann G, Buchwalsky R, Schulte H. Association of apolipoprotein E polymorphism, lowdensity lipoprotein cholesterol, and coronary artery disease. Clin Chem 1986;32:778-781. Utermann G, Hardewig A, Zimmer F. Apolipoprotein E phenotypes in patients with myocardial infarction. Hum Genet 1984;65:237-241. Davignon J, Gregg RE, Sing CF. Apolipoprotein E polymorphism and atherosclerosis. Arteriosclerosis 1988;8:1-21. Kuusi T, Nieminen MS, Ehnholm C, et al. Apoprotein-E polymorphism and coronary artery disease. Increased prevalence of apolipoprotein E-4 in angiographically verified coronary patients. Arteriosclerosis 1989;9:237-241. Havekes LM, de Knijff P, Beisiegel U, Havinga J, Smit M, Klasen E. A rapid micromethod for apolipoprotein E phenotyping directly in serum. J Lipid Res 1987;28:455-463. ;ikerblom HK, Viikari J, Uhari M, et al. Atherosclerosis precursors in Finnish children and adolescents. I. General description of the cross-sectional study of 1980 and an account of the children’s and families’ state of health. Acta Paediatr Stand 1985;Suppl. 318:49-63. ,&erblom HK, Viikari J, Riisiinen L, Kuusela V, Uhari M, Lautala P. Cardiovascular risk in young Finns, results from the second follow-up study. Ann Med 1989;21:223-225. Menzel H-J, Utermann G. Apolipoprotein E phenotyping from serum by Western blotting. Electrophoresis 1986;7:492-495. Enticknap JB. Lipids in cadaveric sera after fatal heart attacks. J Clin Path 1961;14:443-451. Enticknap JB. Fatty acid content of cadaveric sera in fatal ischaemic heart disease. Clin Sci 1962;23:425-431. SHrkioja T, YII-Herttuala S, Solakivi T, Nikkari T, Hirvonen J. Stability of plasma total cholesterol, triglycerides, and apolipoproteins B and A-I during the early postmortem period. J Foren Sci 1988;33:1432-1438. Marek Z, Jaegermann K, Ciba T. Atherosclerosis and levels of serum cholesterol in postmortem investigations. Am Heart J 1962;63:768-774. Albers JJ, Cheung MC, Wahl PW. Effects of storage on the measurement of apolipoproteins A-I and A-II by radial immunodiffusion. J Lipid Res 1980,21:874-878.

Determination of apolipoprotein E phenotypes from stored or postmortem serum samples.

In order to assess the validity of Apo E phenotyping from stored specimens, phenotypes determined from fresh serum samples were compared with those fr...
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