Plasma apolipoproteins A-I and B in survivors of myocardial infarction and in a control group

¹ Laboratorio di Chimica Clinica e di Ematologia, Ospedale Civile Maggiore, piazza Stefani, 1 37126 Verona, Italy.

² Divisione Clinicizzata di Cardiologia, Università di Verona, Verona, Italy.

³ Servizio di Patologia Clinica, Ospedale di Desio, Milano, Italy.

⁴ University of Washington, Northwest Lipid Research Laboratories, Seattle, WA.
^a Author for correspondence. Fax 39-45-8072026; e-mail maristella.graziani{at}iol.it.

	Abstract

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The values of apolipoproteins (apo) A-I and B were determined in a population sample of hospital outpatients with a standardized method to verify if the cutpoints calculated in a cross-sectional study in the US are usable with other populations. We also tested the apolipoproteins' ability to discriminate between healthy people and survivors of myocardial infarction. In the studied population the apo A-I value corresponding to the HDL-cholesterol decisional centile is 1.12 g/L for males and 1.17 g/L for females; the apo B value corresponding to the LDL-cholesterol decisional centile is 1.23 g/L for males and 1.14 g/L for females. These values are quite close to the cutpoints proposed for the American population (1.20 g/L for both apolipoproteins). In comparison with the LDL- and HDL-cholesterol decisional concentrations, the cutpoints for apolipoproteins allow a correct classification of a greater percentage of postmyocardial infarction patients (16% higher for apo B and 5% for apo A-I). Standardized assays coupled with a reference database allow a better clinical use of apolipoprotein measurements.

	Introduction

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Several epidemiological studies have confirmed that coronary heart disease (CHD) is associated with lipid abnormalities (1)(2), and intervention studies have demonstrated that a reduction of total and LDL-cholesterol (LDL-C) and an increase of HDL-cholesterol (HDL-C) decrease the risk of CHD (3)(4).¹ As a result of these studies, the National Cholesterol Education Program (NCEP) published guidelines and defined decisional concentrations for total cholesterol, LDL-C, and HDL-C (5).

It has been postulated for many years that apolipoproteins could be better predictors than lipids for CHD, and many case-control studies have shown that apolipoproteins A-I and B (apo A-I and B) are good markers for CHD (6); however, prospective data confirming these findings are still lacking.

There are substantial reasons to suppose that apolipoproteins could be of help in improving our capability of classifying subjects regarding their risk, because the variety of roles played by apolipoproteins in lipid metabolism suggests that the metabolic fate of a lipoprotein is regulated by its protein rather than its lipid content (7)(8). In spite of the potential utility of apolipoproteins, the transfer of these measurements from research to clinical medicine has been hampered by the lack of standardization and suitable reference data.

To improve the standardization of apolipoprotein assays, the IFCC Committee on Apolipoproteins has successfully produced suitable reference materials for apo A-I and apo B aimed at assuring comparability between different methods (9)(10). These reference materials were endorsed by the WHO as the WHO-IFCC First International Reference Material for apo A-I and apo B (11)(12). Therefore, clinical chemistry laboratories can now use commercial calibrators traceable to these International Reference Materials to produce comparable apo A-I and apo B data. Even though other potential sources of variability among methods are still present, the IFCC study has demonstrated that with the use of calibrators having values traceable to the reference materials, comparable apo A-I and apo B results can be obtained by different methods in different laboratories (11)(12).

Recently, the distribution of apo A-I and apo B values obtained with a standardized method in a well-defined population (the Framingham Offspring) was published (13)(14). This was the first study designed to establish reference ranges for apolipoproteins and determine the apolipoprotein concentrations associated with CHD. Because the obtained values are traceable to the WHO-IFCC International Reference Material, the suggested decisional values could be widely used to classify subjects according to their cardiovascular risk, if apolipoprotein values are likewise obtained with a standardized method.

These studies represent a fundamental step for the clinical use of apolipoprotein measurements because from now onwards, standardized methods, population studies, and defined cutoff concentrations will be available for these indicators.

The present study describes the value distribution of apo A-I and apo B obtained in two populations: subjects living in a medium-sized town in Northern Italy who were hospital outpatients, and a group of patients with myocardial infarction (MI) from an epidemiological study called GISSI-Prevention study. The aim of this study is twofold: first to determine the distribution of apo A-I and apo B in an Italian population sample of hospital outpatients, comparing it with that obtained in an American population, and second to compare apolipolipoprotein values in this control population with those found in post-MI patients.

	Subjects and Methods

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subjects
The procedures used in this study are in accordance with the Helsinki Declaration of 1975, as revised in 1983.

Control population
consisted of people from the outpatient section of the Clinical Chemistry Laboratory of a large hospital in a town situated in Northern Italy (Ospedale Civile Maggiore, Verona, Italy). Subjects were selected according to sex and age on the basis of the data obtained from the 1992 Italian census. The dwellers of the geographical area served by the hospital at the time of this census approximated 600 000; to have an estimate of the distribution of apolipoprotein values representative of the entire population, we planned to examine 1:1000 people. The number of the subjects recruited (n = 616) was proportional to the distribution of the census for sex and age classes; pregnant women and subjects in hypolipidemic treatment were excluded. Blood was collected in the fasting state, allowed to clot at room temperature, and the serum was stored at -80 °C until the examination. The laboratory tests included total cholesterol, LDL-C and HDL-C, triglycerides, apo A-I and apo B, and the erythrocyte sedimentation rate (as a marker of inflammation).

Patients with MI
were those enrolled in the GISSI-Prevention study (15). The GISSI study is a preventive intervention study on the atherosclerotic and thrombotic component of post-MI risk and is being carried out by the Mario Negri Institute of Pharmacological Sciences and ANMCO (an Italian Society of Cardiologists). Its aim is to verify whether pharmacological treatments in post-MI patients are able to achieve both a decrease in total cholesterol concentration and a reduction in cardiovascular mortality. The study involved about 12 000 patients from all over the country; for the present study, a small subset of patients (n = 553) was used. We selected patients living in the north of Italy whose samples were stored at -80 °C until the examination. The blood was collected 6 months after MI before any pharmacological treatment was started.

methods
Total cholesterol
and triglycerides were determined with enzymatic methods; the analytical system for cholesterol was traceable to the National Reference System (16).

HDL-C
was determined by measuring cholesterol after precipitation of apo-B-containing lipoproteins (17); the accuracy of the method was assessed with frozen serum pools with values assigned by the designed Reference Method (18).

LDL-C
was determined with the Friedewald formula (19).

Apo A-I and apo B
were measured with a rate nephelometric method (20) on the Array 360 (Beckman Instruments); the assays were performed according to the manufacturer's instructions with manufacturer-provided calibrators and antisera. The manufacturer claimed that the values of the calibrator were assigned on the basis of the WHO-IFCC International Reference Materials for apo A-I and apo B.

All the samples were kept frozen at -80 °C until they were analyzed; the maximum time of storage was 15 months.

Quality control (QC) (accuracy and precision) for total cholesterol and HDL-C and triglycerides was performed with two frozen serum pools (low and high), at different concentrations of the analytes, assayed in duplicate, before and after each analytical run. The values of the pools were assigned by one of the laboratories of the Cholesterol Reference Method Laboratory Network (Ospedale S. Raffaele-Milano).

QC for apo A-I and apo B was performed with fresh frozen pools at three different concentrations of the analytes with values assigned against the WHO-IFCC materials; the pools were kindly supplied by the Northwest Lipid Research Laboratory.

The QC scheme based on frozen serum pools allows the direct transfer of the results obtained on control samples to those obtained on patient's specimens.

statistical analysis
The distribution of the continuous variables was described by mean, SD, median value, and 5th, 10th, 25th, 75th, 90th, and 95th percentiles. The comparison between groups was performed by analysis of variance, including gender and age class in the model. The comparison between the mean values of the groups was done by Student's unpaired t-test.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Table 1 reports the QC for lipids and apolipoproteins. The total errors (TE) (CV x 1.96 bias) for total cholesterol, HDL-C, LDL-C (high concentration), and triglycerides are within the limits established by NCEP (21), and so the patients' values obtained in this study can be regarded as adequately precise and accurate. For apolipoproteins, definite criteria to verify the precision and the accuracy of the methods used in clinical laboratories have not yet been established, so the analytical performances can only be described. Apo B measurements show lower CVs than apo A-I measurements; regarding accuracy, apo B presents negative bias, whereas apo A-I shows positive bias. Apo A-I TE are much higher than apo B at any concentration of the analyte.

The distributions of apo A-I, HDL-C, apo B, and LDL-C in the control population are given in Tables 2–5.

Subjects with erythrocyte sedimentation rate >30 mm/h (n = 62) have been included in the evaluation because their apolipoprotein concentrations were not significantly different from the remaining population (apo A-I 1.50 ± 0.21 and 1.49 ± 0.23 g/L; apo B 1.25 ± 0.35 and 1.11 ± 0.32 g/L).

Apo A-I values tended to increase with age in both sexes; women in every age group showed higher apo A-I concentrations than men (Table 2 ). The analysis of variance showed that the effects of both age and gender on apo A-I concentrations were statistically highly significant (P <0.001). Apo B values tended to increase with age in both sexes; however, whereas men younger than 50 years presented apo B values higher than those of women, the values were reversed for gender in age groups 50–59, 60–69, and 70–79 years, with women showing higher values than those of men in the older age groups (Table 4 ). The analysis of variance demonstrated no gender effect on apo B concentrations, but the effect of age was highly significant (P <0.001).

In our control population, the NCEP decisional concentration for HDL-C (0.9 mmol/L) fell at the 7th percentile for males and at the 2nd for females; the apo A-I values corresponding to the same centiles were 1.12 g/L for males and 1.17 for females. Similarly, the NCEP decisional concentration for LDL-C (4.14 mmol/L) in our study fell at the 69th percentile for males and at the 61st for females; the apo B values corresponding to the same centiles were 1.23 g/L for males and 1.14 for females.

Table 6 presents the serum concentrations of lipids, apolipoproteins, and other characteristics for the control and the GISSI population.

The comparison between the control group and the post-MI patients revealed (Table 6 ) tat all indicators show significant differences between the two populations; however, after including age and gender in the model, the difference in total cholesterol was no longer significant.

The percentage of the post-MI patients classified at risk on the basis of the NCEP decisional concentration for LDL-C (4.14 mmol/L) is 48; if patients are classified at risk on the basis of the cutpoint suggested in the Framingham population for apo B (1.20 g/L), the percentage increases to 64. The protective indicators give the following figures: 28% if the NCEP decisional concentration for HDL-C (0.9 mmol/L) is adopted and 33% if the cutpoint suggested in the Framingham population for apo A-I (1.20 g/L) is considered.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Until recently, the clinical application of apo A-I and apo B measurement was seriously limited by the lack of comparability of data obtained by different methods and by the consequent lack of common reference ranges. To assure comparability between different methods, the IFCC Committee on Apolipoprotein Standardization has made available to manufacturers and reference laboratories the WHO-IFCC First International Reference Materials for apo A-I and apo B (9)(10). The results of studies performed with the reference materials (11)(12) demonstrated that apo A-I and apo B can be measured by a variety of immunochemical methods with the degree of accuracy and precision required for assessing the risk for coronary artery disease. Recently, population-based reference ranges for apo A-I and apo B obtained by a standardized method have been reported (13)(14). These studies provide critical cutpoints that can be used to assess the risk of CHD and may constitute the basis for widespread clinical use of apo A-I and apo B measurements.

This study describes the distribution of apo A-I and apo B values in an Italian free-living population of hospital outpatients and in post-MI patients. Despite the fact that the control subjects were stratified by age and sex as reflected in the Italian census, this cannot be considered a true population-based sample, since subjects were drawn from a particular subset of the population of Verona, i.e., hospital outpatients. These subjects, by virtue of their patient status, can differ in some ways from the parent population. Our recruitment strategy for the control group was therefore aimed at obtaining a group of subjects as representative as possible of the general population. Subjects in hypolipidemic treatment and pregnant women were not included because their percentage in a hospital outpatient group is very likely to be much higher than in the population at large. Some survivors of MI could have been included in our control population, but their number can be considered negligible because the subjects were randomly drawn from a large outpatient population (where it can be assumed that people with CHD are a small proportion). The post-MI patient samples, unlike the control group, were collected from different Centers; however, the geographical area (Northern Italy) where controls and patients live is of limited extension and the two groups share the same diet and life-style. The apo A-I and apo B assays that we used were standardized according to the WHO-IFCC materials; this allowed us to use the published cutpoints to classify patients and made comparisons between populations possible.

The analytical performances of the methods used for apolipoprotein measurement seem satisfactory both for precision and accuracy even if a certain amount of imprecision can be observed for apo A-I measurements.

In agreement with other reports (6)(22)(23)(24)(25), the differences between males and females and those related to age observed in the distribution of apo A-I and apo B in the control population indicate that apo A-I (and HDL-C) concentrations have a strong gender association and that apo B (and LDL-C) concentrations are associated with age.

The percentile distribution of lipids and apolipoproteins of the Italian control group we used showed lower values than those observed in the Framingham population (13)(14) for both proteins in both sexes; the Italian control group compared with the Framingham population presents a less favorable risk profile if we consider apo B and LDL-C and a more favorable one if we consider apo A-I and HDL-C.

In the Framingham population, the cutpoints for apo A-I and apo B were chosen quite arbitrarily (13)(14), as the centiles of apo A-I and apo B distribution correspond to the centiles of NCEP decisional concentrations for HDL-C (0.9 mmol/L) and LDL-C (4.14 mmol/L) respectively. The apolipoprotein values thus obtained in the Framingham study are: for apo A-I 1.15 g/L (females) and 1.18 g/L (males), for apo B 1.11 g/L (females) and 1.18 g/L (males). These figures have been rounded off to 1.20 g/L for both apoproteins to generate a number easy to remember. It can be argued that, if differences between populations in the distribution of lipids do exist (as we observed in our population sample), then the cutpoints for apolipoproteins could change concurrently, making the use of the suggested cutpoints problematic. We verified that this is not the case because the cutpoints established on the basis of the lipid distribution in our control population (following the above-described procedure) are very close to those established in the Framingham population on the basis of their lipid distribution. The figures we obtained are: apo A-I 1.17 g/L for females and 1.12 g/L for males, apo B 1.14 g/L for females and 1.23 g/L for males, and can be similarly approximated to 1.20 g/L for both apoproteins. From these data, one can conclude that the proposed cutpoints can be used in other populations even in the presence of some differences in lipid distribution; however, decisional concentrations adopted on the basis of prospective studies could be of more general application.

Another study aimed at determining the apo A-I and apo B reference ranges in a population sample with a standardized assay was performed on a Finnish population (26). The distribution of values in that population showed a more risky lipoprotein profile compared with the control population used in this study. The cutpoints suggested in the Framingham study (1.20 g/L) correspond in the Finnish population to the 50th percentile for apo B and to the 25th for apo A-I (10th for females); this could be due to unfavorable dietary or genetic influences on that population (27).

Considering the mean values, lipids and apolipoproteins are equally able to discriminate between healthy subjects and post-MI patients; however, apolipoproteins are able to identify a greater proportion of patients correctly. It is possible to affirm that in the studied populations, apolipoproteins display a discriminatory power better than lipids. This has already been reported (6)(28). This is a cross-sectional study and it is impossible to affirm, on the basis of the data presented here, a superior clinical utility for the use of apolipoprotein measurements to assign risk to subjects; this will be more appropriately assessed by prospective studies.

Because LDL-C and HDL-C present some well-known analytical problems (21)(29), and apolipoproteins are easily determined on automated instruments with standardized reagents, it is possible to add to the considerations expressed above also some analytical advantages in favor of a widespread adoption of apolipoprotein determinations to assess a subject's risk (30).

On the basis of the data of the present study, we conclude that: (a) The distribution of apolipoprotein concentration in a Northern Italian population of hospital outpatients is comparable with that of the Framingham population, and the decisional concentrations established in American people will probably be suitable for use in our population; (b) apoprotein values are of help in discriminating post-MI patients from control subjects, and in so doing they seem superior to lipids; and (c) the standardization of apolipoprotein assays coupled with the definition of decisional concentrations will allow a better clinical use of apolipoprotein measurements.

	Acknowledgments

 
We acknowledge the cooperation of the following colleagues in the Clinical Chemistry Laboratories of the GISSI Centers, in collecting and sending the samples: F. Bordone, S. Ratibondi, H Regionale Aosta; V. Siclari, H Civile Belluno; R. Antinozzi, S. Pagani, P. Ostinelli, H S.Anna Como; P. Brambilla, D. Giannone, H Desio; P. Ferrante, M.G. Calvo, I. Don Gnocchi Milano; A. Berlusconi, C. Faravelli, Pio Albergo Trivulzio Milano; A. Marocchi, L. Prencipe, H Ca' Granda-Niguarda Milano; R. Ridolfi, L. DeAngelis, H Silvestrini Perugia; M. Colombo, Centro Riabilitazione Veruno; S. Petrella, M. Righini, H Civile Vizzolo-Predabissi; R. Pagni, A. Gariboldi, H Molinette Torino. We also thank Beckman Analytical SpA Milano (Italy) for the kind supply of the reagents used in measuring apo A-I and apo B.

	Footnotes

 
¹ Nonstandard abbreviations: CHD, coronary heart disease; LDL-C, LDL cholesterol; HDL-C, HDL cholesterol; NCEP, National Cholesterol Education Program; apo, apolipoprotein; MI myocardial infarction; QC, quality control; and TE, total error.

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