Apolipoprotein B and A-I values in 147 576 Swedish males and females, standardized according to the World Health Organization–International Federation of Clinical Chemistry First International Reference Materials

¹ CALAB Research and CALAB Medical Laboratories, S:t Göran Hospital, S-112 81 Stockholm, Sweden.

² Department of Medicine, Northwest Lipid Research Laboratories, University of Washington, 2121 N 35th St., Seattle, WA 98103.

³ King Gustaf V Research Institute, Karolinska Institute, S-10401 Stockholm, Sweden, and ASTRA HÄSSLE AB, S-43183 Mölndal, Sweden.

⁴ Institute for Medical Statistics, Ullevål Sykehus, P.O. Box 6, 0407 Oslo 4, Norway.
^a Author for correspondence. Fax 46-86673418;

	Abstract

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Serum concentrations of apolipoprotein (apo) B and apo A-I were measured from 1985–1996 in a Swedish population sample of 83 112 males and 64 464 females, ages <20 to >80 years, using an automated immunoturbidimetric method calibrated against fresh pools of human serum and commercial calibrators. All values were recalculated in 1997 after calibration against the WHO-IFCC First International Reference Materials. The recalculation factor was 1.059 for apo B, whereas for apo A-I, the correction was: y = 0.989x + 0.101. The total CVs for both apo B and A-I were generally <7%. The mean value (± SD) for apo B was 1.31 ± 0.35 g/L in all males and 1.22 ± 0.36 g/L in all females. The mean apo A-I concentration was 1.36 ± 0.22 g/L in males–10% lower than in females (1.51 ± 0.24 g/L). The mean value of apo B increased up to 60 years of age in males and up to 70 years of age in females. apo A-I concentrations changed only slightly with age in both males and females. apo A-I concentrations among Swedes are nearly identical to those reported recently by two American studies and those obtained in a Finnish population sample. Mean apo B concentrations differ somewhat between the populations but mirror–as expected–differences in total cholesterol concentrations. The highest values were noted in Swedish subjects. The Swedish sample population is, to our knowledge, the largest describing the distribution of apo B and A-I in a general population of adult males and females of all ages determined with procedures standardized and traceable to the WHO-IFCC First International Reference Materials.

	Introduction

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In clinical practice, lipid, lipoprotein, or apolipoprotein (apo)¹ concentrations are measured to detect and diagnose lipid disorders (dyslipidemias), to assess the associated cardiovascular risk, and to decide subsequent treatments (1)(2). Sniderman and co-workers (3)(4) recently proposed that measurements of apo B should be used in routine clinical practice and should replace calculation of LDL-cholesterol by the Friedewald formula (5). These authors (3)(4), as well as ourselves and others (6)(7)(8), have argued that now that the measurements of apo B and apo A-I are standardized (9)(10)(11)(12), they should be more widely used in clinical settings. Furthermore, apo B appears to give a more accurate predictive estimate of atherogenic risk than total cholesterol (TC) or LDL-cholesterol (13)(14)(15). apo B and apo A-I can also be determined with high precision and accuracy, and the measurements are easily automated (16)(17), reducing labor costs. In the past, a wider clinical use of apolipoprotein assays was limited by lack of standardization and, consequently, by lack of common population-based reference values. However, recently two American studies have been published. Contois and co-workers (18)(19) presented reference intervals for apo B and apo A-I, primarily in adults, derived from the population-based Framingham Offspring Study. Bachorik et al. (20) measured the concentration of the two apos in Phase 1 (1988–1991) of the Third National Health and Nutrition Examination Survey (NHANES III). The latter presented the distributions of apo A-I and apo B in persons 4 years of age or older, which were the first measured in a probability sample of the noninstitutionalized US civilian population. Both these American studies, as well as a Finnish study in 1995 by Leino et al. (21), used the new WHO-IFCC International Reference Materials as the basis for accuracy. All three investigations were, however, performed on a smaller number of subjects: 3824, ~11 400, and 575, respectively.

Since 1985, one of our group (I.J.) implemented automated methods for the determination of apo B and apo A-I, applying them in screening programs of large populations to improve information about the composition and degree of dyslipoproteinemias. Measurements of apo B and A-I in 43 000 Swedes (6) and the basic data of the prospective study AMORIS (Apolipoprotein-related Mortality Risk) on 300 000 Swedes were reported in 1992 (7). The aim of the present study–after increasing more than threefold the investigated population and after recalculating earlier apolipoprotein measurements to the new WHO-IFCC standard–was to provide a large basis for apo B and apo A-I reference intervals.

	Subjects and Methods

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Subjects
Swedish males and females (147 576) ranging in age from <20 years to >80 years were studied. The Swedish healthcare system has focused on general health screening performed during the last 20 years as a service to almost all employees of most companies, as well as to adult males and females in the general community (virtually all native Swedes). Calab has served as a major laboratory for the >3000 physicians involved in general healthcare. The present clinical material comprises all blood samples sequentially sent to Calab without any limitations because of age, possible diagnosis, or ongoing treatment with diet and/or drugs. Thus, no sample/subjects were excluded from this population study. Because our database does not contain any information about the use of drugs such as beta blockers and estrogens, the possible effects of such drugs on lipids and apo composition could not be analyzed in this study. The large majority of the subjects were subjectively healthy. The major part of the studied population comprised males and females 35–65 years of age, as presented in our previous study (7). No samples were obtained from hospitalized patients or from subjects participating in clinical trials. Most subjects lived in the greater Stockholm area (~20% were from other parts of Sweden). The original AMORIS population investigated during 1985–1989 comprised 300 000 subjects (7). The study has recently been extended to include data collected in 1990–1996. The data base now comprises 581 839 subjects (309 251 males, mean ± SD age, 43.6 ± 13.8 years; and 272 588 females, mean age, 44.7 ± 15.4 years).

Here we describe findings from a subsample of the whole study population. These subjects had their first complete profile of apo B apo A-I TC triglycerides (TGs) taken simultaneously (n = 147 576; mean age, 46.8 ± 13.2 years for males and 49.5 ± 15.2 years for females). The subjects 20–79 years of age are classified in decades. The classes <20 or 80 years of age have mean ages of 17 ± 2.4 and 83 ± 4.5 years, respectively; the number of males equaled the number of females. About two-thirds of the subjects (n = 90 537) were fasted overnight (n = 52 433 for males and n = 38 104 for females). The others had a light morning meal (n = 40 690), or the nutrition status was unknown (n = 19 349).

methods
The methods are the same as described earlier (6). apo B and A-I were determined by an immunoturbidimetric method according to Riepponen et al. (22), using polyclonal antisera from Orion Diagnostica (commercial assay). The company is one of the manufacturers that participated in the WHO-IFCC Standardization Program (9)(10)(11)(12). TC was determined with the cholesterol oxidase/peroxidase (CHOD-PAP) assay and TGs with the glycerol phosphate oxidase/peroxidase (GPO-PAP) assay, using enzymatic methods (reagents from Boehringer Mannheim) for the PRISMA® instrument and from Bayer Diagnostics GmbH for the DAX(TM) instrument. All four methods were from the outset highly automated; from 1985 to 1992, the Multichannel AutoChemist®-PRISMA (New Clinicon) was used and since 1993, the Multichannel DAX-96 (Technicon/Bayer Corp.) has been used. All analyzers were computerized with systems for automatic calibration.

An extensive quality-control scheme was used throughout the study in determining serum apos. Table 1 shows the long-term precision data from materials used as controls. The total CV (CV total) was the same as reported earlier (6) for the initial (1985–1989) part of the study: generally <7% for apo B, apo A-I, and TGs, and <3% for TC. For apo B and A-I, "zero point" and one-level calibration were done using fresh pools of human serum because of the lack of suitable calibrators for daily use. A pool of human serum was prepared each day from ~300 clear samples from healthy controls. By using this large number of sera, the same mean values were expected (23). The technique was implemented from the start of the measurements in 1985. The pool material was replaced as calibrator as of 1993 by the commercially available Seronorm Lipid (Nycomed Pharma) supplemented by the Apolipoprotein Calibration Set SPQ(TM) (Incstar Corp.) to maintain stable quality performance.

Daily samples from fresh or frozen pools of human sera were used as controls; since 1993 commercially available materials like Seronorm Lipid and Beckman control sera were utilized (Triad® LINK level 1–3). The inaccuracy for TC and TGs was checked against material from the National Institute of Standards and Technology, Gaithersburg, MD, or by analyses performed by lipid reference laboratories certified by the CDC.

Traceability to the WHO-IFCC International Reference Materials.
Recalculation of collected apolipoprotein data was done in 1997 in collaboration with the Northwest Lipid Research Laboratories, University of Washington, Seattle, WA. This laboratory was the one that organized and coordinated the IFCC standardization program (9)(10)(11)(12). They provided Calab with three concentrations of their apo B and apo A-I fresh-frozen quality-control samples prepared in-house, with values assigned against the WHO-IFCC Reference Materials. During 5 days, 36 determinations of apo A-I and apo B were performed on each quality-control sample, and data were sent to Northwest Lipid Research Laboratories for statistical evaluation. On the basis of the results of this analysis, a correction factor of 1.059 for apo B and the correction y = 0.989x 0.101 for apo A-I was used to ensure that values were traceable to the WHO-IFCC International Reference Materials.

Accredited laboratory facilities.
All analyses were done at Calab Medical Laboratories, Stockholm, Sweden. For almost every determination of apos and lipids, 20 other laboratory analyses were performed as a basis for clinical evaluation. In 1994, the laboratory received an international accreditation according to European Norm 45001 by the Swedish Board for the Technical Accreditation (Borås, Sweden). Calab also received a Statement of Good Laboratory Practice compliance (Läkemedelsverket, Uppsala, Sweden) in 1994. The laboratory performed >4 000 000 analyses in 1996 and is accredited in clinical chemistry, hematology, immunology, and microbiology.

statistical analysis
Data are presented as means (and SDs), 95% ranges, medians, or selected, theoretically calculated log-normal percentile limits. For between-group mean differences, 95% confidence limits (95% CL) are used.

	Results

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mean values and distributions of apolipoproteins
The results of the first complete profile data (apo B, apo A-I, apo B/A-I ratio, TC, and TGs) from 147 576 subjects are shown in Table 2 Selected percentile values of apo B and apo A-I as well as means and SDs of the other variables for males and females in each decade and in the total population are presented. These values were obtained by pooling results from fasting and nonfasting subjects, as in NHANES (20). The mean apo B concentration (Table 2A ) was 1.31 ± 0.35 g/L in males (n = 83 112)–significantly higher than the mean for the 64 464 females [1.22 ± 0.36 g/L; 95% CL for difference, 0.090 (0.086, 0.094)]. The mean apo A-I concentration (Table 2B ) in males was 1.36 ± 0.22 g/L–10% lower than in females [1.51 ± 0.24 g/L; 95% CL for difference, 0.15 (0.148, 0.152)]. apo B and A-I concentrations showed an almost gaussian distribution.

For the various age and sex strata, the differences between the mean and the median values ranged from 0.03 to 0.05 g/L (apo B) and from 0.01–0.04 g/L (apo A-I), suggesting that the distributions of apo B and A-I were virtually gaussian. The mean apo B/A-I ratio (Table 2C ) was 0.99 ± 0.32 in males and 0.83 ± 0.29 in females [95% CL for difference, 0.16 (0.157, 0.163)], whereas the 95% interval was 0.50–1.75 in males and 0.40–1.51 in females, including all ages. The distributions of the apo B/A-I ratio were also virtually gaussian, the differences between the mean and the median values ranging from 0.02 to 0.05 (data not shown).

apo b and apo a-i in fasting and nonfasting subjects
A comparison between samples collected from subjects reported to be in a fasting state or in nonfasting state showed for apo A-I no difference, whereas the values for apo B were slightly lower. The maximal deviation was 3.3% for apo A-I and 7.7% for apo B (data not shown).

apo b and apo a-i by age
Median apo B concentrations in adult males were 0.99–1.36 g/L, and the increase continued from those 20–59 years of age and tended to decrease after age 60 (Fig. 1 A). The median apo B concentrations in adult females were 0.92–1.38 g/L, and the increase continued in those in the age group 20–69 years, with a more pronounced increase in the age group 40–59 years, and then decreased after age 70 (Fig. 1A ). In both males and females, apo B ultimately reached about the same concentration (1.36 g/L for males and 1.38 g/L for females); however, in males this occurred 10 years earlier than in females. apo B values in younger age cohorts were higher among males than among females, whereas the cohorts at 60 years of age showed a reverse relation. The 95% interval for apo B was 0.73–2.18 g/L for males and 0.65–2.08 g/L for females, including all ages.

View larger version (13K): [in this window] [in a new window]   Figure 1. Differences in apo B and apo A-I concentrations and ratios according to age group. Median concentrations (g/L) for apo B (A) and apo A-I (B) together with apo B/A-I ratio (C) at each 10-year interval of males and females. The classes <20 or 80 years have mean ages of 17 ± 2.4 and 83 ± 4.5 years, respectively.

Median apo A-I concentrations in adult males ranged from 1.30 to 1.36 g/L, with only a slight continuous increase from 20 to 59 years, followed by a plateau at 50–69 years, and then a decrease at >70 years (Fig. 1B ). Median apo A-I concentrations in adult females ranged from 1.45 to 1.52 g/L, and there was a first plateau in the age range 20–39 years, then an increase up to 59 years, followed by a second plateau in the age range 50–69 years, and a decrease at >70 years (Fig. 1B ). The 95% interval for apo A-I was 0.99–1.84 g/L for males and 1.09–2.04 g/L for females, including all ages. The highest apo B/A-I ratio for males was seen in males 50–59 years old, whereas the highest ratio for females was obtained in those 60–69 years old (Fig. 1C ).

tc and tg values
The mean TC (5.9 mmol/L) was the same for males and females (Table 2C ). There was no difference between fasting and nonfasting TC values (data not shown). The median TG concentration, including results from all individuals in fasting or nonfasting states, was 1.35 mmol/L for males (95th percentile, 3.64 mmol/L) and 1.04 mmol/L (95th percentile, 2.56 mmol/L) for females (data not shown); Table 2C shows mean values. The fasting median TG (data not shown) for males was 1.29 mmol/L (95th percentile, 3.44 mmol/L), and 1.00 mmol/L (95th percentile, 2.42 mmol/L) for females; fasting mean TG values are given for comparison (Table 3

comparison with other population studies traceable to the who-ifcc
This Swedish population sample was compared (Table 3 ) with characteristics for three other population samples traceable to the WHO-IFCC First International Reference Materials: two American (the Framingham Offspring Study (18)(19) and the NHANES III (20)) and one Finnish (21). These populations are from different ethnic origins and differ in sample size and TC concentrations. Their mean age was about the same. If differences in TC concentrations among the population samples are taken into account, the concentrations and percentile distributions of apo B are very similar and of apo A-I are almost identical.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The present study is the first large epidemiological study applying automated apo methods over several years to the health screening of males and females covering wide age ranges. However, compared with previously published population studies (18)(19)(20), our results have always been obtained on fresh samples, thus avoiding possible effects of long-term storage and of freezing and thawing of the samples. In 1992 (6), we presented the first large population report on apo determinations in 43 000 males and females. Now we have corrected our original results, so that they are traceable to the WHO-IFCC Reference Materials. Thus, our present results are directly comparable with those obtained in recently presented population studies. Determinations of apos may provide a more specific characterization of the type of lipoprotein abnormality, thus leading to a more accurate prediction of the risk of coronary artery disease than that made using the TC, TG, and HDL values alone, as discussed elsewhere (3)(4). To establish whether apo B and A-I, as well as the ratio apo B/A-I, are better predictors of atherogenic risk than TC and TGs, the AMORIS subjects (7) will be monitored over an extended period of time. Another major question that remains to be answered is how much apo B and apo A-I add to the determination of TC and TGs in prediction of total fatal risk. A second report of the AMORIS study, with extended mortality data in relation to initial TC and TG and especially to initial apo B and apo A-I concentrations, is under preparation.

Our present study shows that the measurements of apo B and apo A-I were performed with good precision and accuracy throughout the whole observation period. As controls, pools of both fresh and frozen samples of human serum (Table 1 ) were used. These gave results similar to those found by other authors (19)(21)(24). Of our originally presented data (6)(7), only minor corrections for both apo B and A-I were needed to harmonize the data to be traceable to the WHO-IFCC International Reference Materials. The errors of the methods are of the same order of magnitude or better than those suggested by Marcovina and Albers (25). They recommended that the between-run values should be <8%, and optimally <5%. Bachorik et al. (20) report that the CVs for apo A-I and B averaged <6% throughout the NHANES III study. Recent CV estimates of the overall biological variation of apo B and apo A-1 were 6–7% (24). In our study, there was virtually no difference (apo A-I) or only a slight (apo B) difference, when we compared values obtained in fasting state with values obtained in individuals having taken a light meal. Although the data were obtained from different individuals, the total number of subjects was large enough in each group of individuals to support the notion that a light meal does not change apo B or apo A-I concentrations (26)(27)(28). Bachorik et al. (20) also found in the NHANES study that there were no significant differences in apo B or apo A-I between fasting or nonfasting subjects; they therefore combined the data from both groups. Thus, apos may be regarded as "robust" measurements, at least regarding sampling conditions. This has considerable practical impact, both for the patient often traveling far to get his or her blood taken and for the physician in terms of the interpretation of the data.

Whereas apo B values for both sexes clearly increased with age, apo A-I values showed only minor age-related variation. Males have higher apo B values than females up to age 60. After menopause, apo B concentrations in females continued to rise. Females had ~10% higher apo A-I concentrations than males in all age groups. Similar age-related values for apo B and apo A-I were observed in 1992 (6). The present results for apo A-I are in close agreement with the 10% difference also found in the NHANES III Survey (20) and the 13% found in the Framingham Offspring Study (19) and the Finnish study (21) (Table 3 ). The results reported in these studies were all obtained by turbidimetric analysis with the exception of the NHANES III where rate immunonephelometry was used most of the time (they changed from a radial immunoassay when automated methods were becoming more widely used). The Framingham study used turbidimetric assay reagents and calibrators from Incstar Corp. The Swedish and Finnish studies both used the same turbidimetric method (22) and the same supplier of reagents (Orion Diagnostics).

The percentile distributions for the four studies were rather similar (apo B) and nearly identical (apo A-I). apo B is positively correlated with TC (6)(18)(21); therefore, in accordance with the cholesterol data, mean apo B was highest for the Swedes and lowest for the Americans. Mean apo A-I concentrations were virtually the same for all four population samples, with ranges of 1.34–1.38 g/L for males and 1.51–1.58 g/L for females (Table 3 ).

The often conflicting findings reported earlier for apo analysis have been mainly ascribed to differences in methodological approaches, sample handling and storage, and especially to the previous lack of common reference materials to which the calibration of the methods could be referred. In view of the results from the latest investigations, which all rely on the WHO-IFCC International Reference Materials, it can be concluded that the WHO-IFCC standardization has attained its purpose and that it is now possible to establish that there are only minor differences between different populations. Bachorik et al. (20) compared the results from their survey with the Framingham Offspring Study and reported only small differences between different ethnic groups.

Several authors, especially after the introduction of the WHO-IFCC Reference Materials, have discussed and suggested cutpoints for apo B (4)(8)(18)(20) and apo A-I (8)(19)(20) that may be used to assess coronary heart disease risk. Bachorik at al. (20) determined apo B concentrations in adults, ages 20 years, categorized by the National Cholesterol Education Program risk levels for LDL-cholesterol. They conclude that the question of whether apo B might eventually be used instead of LDL-cholesterol as the basis for assessing risk for coronary heart disease remains open. Because apo B varies with age and apo A-I varies with sex, age- and sex-standardized values should be used as markers for cardiovascular risk. However, the introduction of new markers for atherogenic risk that may be used instead of the established and accepted LDL and HDL determinations makes this new approach difficult from a practical standpoint. Too many cutoff values create practical problems related to lack of simplicity. Instead of using sex- and age-standardized values, the alternative approach would be to choose a few cutoff values and evaluate whether such values are clinically significant. We are in the process of analyzing AMORIS data relating baseline apo B, apo A-I, TC, and TG values to final risk/fatal myocardial infarction, using the approach of dichotomizing risk in relation to age and sex, as well as by age-and sex-standardized algorithms. The recommended cutoff value should be proven to discriminate future coronary heart disease risk in a stringent, predictive, and possibly simple way. The final decision of how to best define relevant risk and cutoff values must await outcome studies like AMORIS.

	Acknowledgments

 
This work was supported by grants from Gunnar and Ingmar Jungner Foundation for Laboratory Medicine and from Bure Hälsa & Sjukvård Ltd, Stockholm, to Calab Research.

	Footnotes

 
¹ Nonstandard abbreviations: apo, apolipoprotein; TC, total cholesterol; NHANES III, National Health and Nutrition Examination Survey III; AMORIS, Apolipoprotein-related Mortality Risk Study; TG, triglyceride; and CL, confidence limit.

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