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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1568-1572.)
© 1996 American Heart Association, Inc.

Articles

Lipoprotein(a) in Stored Plasma Samples and the Ravages of Time

Why Epidemiological Studies Might Fail

Florian Kronenberg; Evi Trenkwalder; Hans Dieplinger; Gerd Utermann

the Institute of Medical Biology and Human Genetics, University of Innsbruck (Austria).

Correspondence to Dr Florian Kronenberg, Institute of Medical Biology and Human Genetics, University of Innsbruck, Schopfstr 41, A-6020 Innsbruck, Austria. E-mail Florian.Kronenberg@uibk.ac.at.

	Abstract

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Prospective case-control studies investigating lipoprotein(a) [Lp(a)] as a risk factor for atherosclerosis have measured Lp(a) in samples stored frozen up to nearly 20 years. We therefore prospectively examined the influence of long-term plasma sample storage on measured values, depending on the molecular weight of apolipoprotein(a) [apo(a)] isoforms. Apo(a) phenotyping was performed in 310 plasma samples, and Lp(a) was measured after 3 and 28 months of storage at -80°C. The values of both measurements correlated significantly for both low- and high-molecular-weight apo(a) phenotypes (r=.97 and r=.98, respectively, P<.001). Nevertheless, we detected on average a small decrease of 4.83% from mean±SD (median) 21.24±23.54 (11.10) mg/dL to 20.02±21.72 (10.55) mg/dL, which was statistically significant (P<.001). The absolute and relative Lp(a) decrease over time became larger with a decreasing number of kringle IV repeats of apo(a) (P<.05), and Lp(a) decreased markedly more in subjects with low-molecular-weight compared with those with high-molecular-weight apo(a) isoforms (-3.26 versus -0.46 mg/dL, P<.05). More than 70% of the absolute Lp(a) decrease in the total sample was caused by samples with low-molecular-weight apo(a) isoforms, which represented only 27% of the sample. Low-molecular-weight apo(a) isoforms are reportedly more frequent in patients with atherothrombotic disease compared with control subjects. Measurement of Lp(a) in several-year-old frozen samples is therefore likely to result in a preferential decrease and false lower Lp(a) concentrations in patient groups compared with control groups. The negative results of some prospective studies with retrospective measurement of Lp(a) may be caused by such an artifact.

Key Words: lipoproteins • apolipoproteins • sample storage • epidemiological studies • glycoproteins

	Introduction

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Lipoprotein(a) differs in structure from LDL by the highly polymorphic glycoprotein apo(a), which is covalently linked to the apolipoprotein B of LDL.¹ The wide range of Lp(a) plasma concentrations from <0.1 to >300 mg/dL within a population is controlled mainly by variation at the apo(a) gene locus, which is polymorphic in size due to a variable number of plasminogen-like kringle IV repeats.^{2 3} There exists an inverse correlation between the number of kringle IV repeats of apo(a) and the Lp(a) plasma concentrations.^{2 3 4}

During recent years, Lp(a) appreciably became a focus of interest because of its relationship to atherosclerosis. Several but not all retrospective and prospective case-control studies described an association between high Lp(a) plasma concentrations and CHD (for reviews see References 5 and 6). In one of these studies⁷ apo(a) phenotypes and Lp(a) plasma concentrations were determined in six ethnically different populations. The LMW apo(a) isoforms F, B, S1, and S2 (11 to 22 kringle IV repeats), with their associated high Lp(a) plasma concentrations occurred significantly more frequently in patients with CHD in all six populations.

Most prospective case-control studies measured Lp(a) in plasma or serum samples stored at -20°C or -80°C for up to nearly 20 years. Only few and conflicting data are available on the influence of long-term storage on the plasma concentration of Lp(a).^{8 9 10 11 12 13 14 15} Nothing is known about possible apo(a) phenotype–specific changes in Lp(a) concentrations caused by long-term storage of plasma. Such effects, if any, could be extremely important for the interpretation of prospective studies performed on frozen specimens.

The aim of this study was to investigate the stability and reproducibility of Lp(a) measurements in relation to apo(a) isoform size. We analyzed a large number of frozen plasma samples during a mean observation period of 25 months and observed an isoform-dependent change in measured Lp(a) over time.

	Methods

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Ethylenediamine tetraacetic acid blood was collected from 310 subjects. After low-speed centrifugation for 15 minutes at 1500g and 4°C, plasma was separated and stored in five aliquots at -80°C until analysis. Three months after blood withdrawal, Lp(a) was measured from thawed aliquots. Seventeen to 36 months (on average, 25±6 months) later, Lp(a) was measured again from an aliquot never thawed before. Time difference between the first and the second measurements for plasma samples with LMW and HMW apo(a) isoforms was similar (24.5 versus 24.9 months, NS). The person who performed the Lp(a) measurement was blinded to apo(a) phenotype grouping.

Lp(a) quantification was performed as described in detail¹⁰ with a double-antibody enzyme-linked immunosorbent assay with an affinity-purified polyclonal apo(a) antibody for coating and the horseradish peroxidase–conjugated monoclonal 1A2 for detection. This anti-apo(a) antibody recognizes the repeat epitope motif YYPN in kringle IV type 2.¹⁶ An Lp(a)-positive serum from Immuno (Vienna, Austria) with the same apo(a) isoforms served as standard throughout the whole study. Each sample was analyzed in duplicate, and intra-assay and interassay coefficients of variation were 2.7% and 6%, respectively. The stability of our assay was secured by four control plasma samples used in each run. Apo(a) phenotyping was performed by sodium dodecyl sulfate–agarose gel electrophoresis under reducing conditions, as outlined,¹⁷ with slight modifications. Eighty-three subjects (26.8%) had an LMW apo(a) phenotype and 227 (73.2%) showed only HMW apo(a) isoforms.

Statistical Analysis
The paired Wilcoxon test was used to investigate the changes of Lp(a) values over time in samples stored 3 and 28 months, on average. The Spearman rank correlation coefficient was calculated between the Lp(a) values measured in samples stored for 3 and 28 months as well as between the number of kringle IV repeats and the absolute Lp(a) changes over time.

Subjects were divided into five groups according to the number of kringle IV repeats of the smaller apo(a) isoform.^{18 19} Eleven to 19 kringle IV repeats correspond to the originally described isoforms⁴ F, B, and S1; 20 to 22 repeats to S2; 23 to 25 repeats to S3; and >25 repeats to S4. The Kruskal-Wallis ANOVA by ranks was used to test the hypothesis that mean changes in Lp(a) over time differed significantly among individuals from various apo(a) isoform groups.

We excluded the phenomenon "regression toward the mean"²⁰ by the following procedure: after logarithmic transformation of Lp(a) values, plasma samples were divided into quartiles according to log-transformed Lp(a) values. Pearson's ² test was used to compare the frequencies of plasma samples showing a decrease of Lp(a) in the four groups of samples.

Statistical analysis was performed with SPSS for Windows, Release 6.0.1. (Chicago, Ill). A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Lp(a) was measured in 310 plasma samples after 3 and 28 months of storage at -80°C. On average, we detected a decrease of 4.83% from 21.24±23.54 mg/dL (median, 11.10) to 20.02±21.72 mg/dL (median, 10.55), which, though small, was statistically significant (P<.001). Two thirds of the samples showed a decrease and the remaining samples no change or a slight increase of Lp(a) over time. Lp(a) measured in samples stored for 28 months correlated significantly with values measured after 3 months of storage for both LMW and HMW apo(a) phenotypes (r=.97 and r=.98, respectively, P<.001) (Fig 1). Despite these high correlations, the decrease in Lp(a) was markedly greater in the 83 subjects with LMW apo(a) isoforms (11 to 22 kringle IV repeats) compared with the 227 individuals with HMW phenotypes (>22 kringle IV repeats). This is supported by the different slope of the regression lines for LMW and HMW apo(a) phenotypes in Fig 1 and by the different mean absolute and relative changes of Lp(a) for LMW and HMW apo(a) phenotypes of -3.26 mg/dL versus -0.46 mg/dL and -8.1% versus -3.3%, respectively (P<.05). More than 70% of the absolute Lp(a) decrease in the total sample was caused by samples with LMW apo(a) isoforms, which represented only 27% of the sample. When we considered only samples with Lp(a) plasma concentrations 30 mg/dL, 37 of 54 samples (74%) with LMW apo(a) isoforms and 17 of 34 (50%) with HMW apo(a) phenotypes showed a decrease of Lp(a) over time, which is statistically significant (P<.05).

View larger version (18K): [in this window] [in a new window]   Figure 1. Comparison between Lp(a) concentrations measured in plasma samples stored at -80°C for 3 or 28 months. Data are given separately for subjects with HMW and LMW apo(a) phenotypes.

We calculated the frequencies of plasma samples showing a decrease of Lp(a) in each quartile of logarithmic-transformed Lp(a) values to investigate whether the observed changes are an effect of regression toward the mean. A similar frequency of samples with decreasing Lp(a) values was observed from the lowest to the highest quartile: 67%, 69%, 55%, and 66% (P>.2).

Finally, the absolute Lp(a) changes over time correlated significantly with the number of kringle IV repeats (r=.24, P<.001): with increasing number of repeats the absolute and relative Lp(a) decrease over time became smaller (P<.05, Kruskal-Wallis ANOVA) (Fig 2).

View larger version (42K): [in this window] [in a new window]   Figure 2. Mean absolute and relative changes in Lp(a) concentrations in relation to the molecular weight (number of kringle IV repeats) of the smaller apo(a) isoform in the samples during a storage period of 25 months (P<.05 by Kruskal-Wallis ANOVA). n is the number of samples investigated.

	Discussion

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The first prospective study to investigate Lp(a) as a risk factor for CHD was published a few years ago.²¹ In the meantime, data on nearly a dozen prospective studies have been made available.^{9 21 22 23 24 25 26 27 28 29 30} Some of these were negative and have caused considerable confusion.^{9 28 30} Most of the studies measured Lp(a) in long-term frozen samples without thorough evaluation of the influence of long-term sample storage on the Lp(a) assay used.

Studies of time-dependent changes of Lp(a) in long-term frozen plasma or serum samples are conflicting.^{8 9 10 11 12 13 14} None of these studies investigated whether apo(a) isoform–dependent changes of Lp(a) occur over time. This issue is of potential importance since it has been shown that LMW apo(a) isoforms are overrepresented in patients with CHD and other atherosclerotic disorders.^{7 31 32 33 34 35} If Lp(a) of various apo(a) isoforms decreases differently over time, this might severely bias the results of studies using frozen specimens.

We therefore measured Lp(a) in frozen samples of 310 subjects 3 and 28 months after blood withdrawal. A markedly higher absolute and relative decrease of Lp(a) over time was observed in plasma samples with LMW compared with HMW apo(a) isoforms (Figs 1 and 2). If such a disproportionately greater decrease of Lp(a) in plasma samples with LMW apo(a) isoforms continues over the next few years, the measured Lp(a) values of samples containing LMW apo(a) isoforms will come closer to those containing HMW isoforms. Accordingly, differences in Lp(a) between groups caused by different isoform frequencies will vanish. LMW apo(a) isoforms are more frequent in CHD patients.^{7 31 32} A measurement of Lp(a) in several-year-old samples will result in disproportionately lower false Lp(a) values in the CHD group compared with the control group and therefore blur a possible association of high Lp(a) values with CHD. We simulated such a scenario using Lp(a) values and apo(a) phenotype frequencies from a study that had demonstrated a significant association of Lp(a) concentration and apo(a) isoform size with carotid plaques.³⁴ In this study, patients with plaques showed significantly higher Lp(a) levels compared with patients without plaques (29.3 versus 19.7 mg/dL, P<.05), and the plaque-affected group included three times as many patients with LMW apo(a) isoforms than did the plaque-free group (26.9% versus 8.5%, P<.05). Extrapolating from the data in Fig 2 and assuming a linear decrease of Lp(a) over time, we calculated that the significant difference in Lp(a) levels of 9.6 mg/dL [which reflects a higher frequency of LMW apo(a) isoforms] would virtually disappear after 8 years of storage (Fig 3). Some of the prospective studies used samples stored for up to 20 years.²⁵ We are aware that the assumption of a linear decrease of Lp(a) over time, which underlies our simulation, cannot be proven directly in our study because this would call for each sample to be measured several times during the longitudinal observation period. Our assumption, however, is supported by a study by Craig et al,⁸ who observed a nearly linear decrease in Lp(a) immunoreactivity of 46% during 6 months of storage. The data from another study showed an even higher decrease after a longer storage period of about 600 days, and Lp(a) values in stored samples were 75% lower than in fresh samples (6.8±6.8 mg/dL versus 29.5±24.5 mg/dL).¹⁴ Although our assay was less sensitive to sample storage conditions than were other reported assays,^{8 14} a significant difference in Lp(a) plasma concentrations caused by different apo(a) phenotype frequencies between two groups would disappear nevertheless after some years of sample storage (see Fig 3). If these changes are also phenotype-associated in other assays, which show substantially higher changes of Lp(a) immunoreactivity with storage over time,^{8 14} or if these phenotype-specific changes are more pronounced, significant differences between two groups would disappear even faster.

View larger version (19K): [in this window] [in a new window]   Figure 3. Simulation of the apo(a) phenotype–associated decrease of Lp(a) values caused by long-term sample storage. Lp(a) concentrations and apo(a) isoform frequency distribution in a group of patients with and a group without carotid plaques from Reference 34 was used for the calculations. The plaque-affected group included three times as many patients with LMW apo(a) isoforms than did the plaque-free group.34 Extrapolating from the data in Fig 2 and assuming a linear decrease of Lp(a) over time, we calculated that the significant difference in Lp(a) levels between the two groups would virtually disappear after 8 years of sample storage.

We can rule out the possibility that the lower Lp(a) values at the time of the second measurement are only an effect of regression toward the mean.²⁰ In this case, we would expect a decrease of Lp(a) mainly in samples with concentrations above the mean and an increase in those below the mean. However, we observed a similar frequency of plasma samples with a decrease in each quartile of Lp(a). The absolute and relative decrease was highest in the upper quartile of Lp(a) because this group included a high frequency of patients with LMW apo(a) isoforms.

Some studies have tried to control the effect of storage by analyzing a fresh blood sample withdrawn several years after the first withdrawal that was stored frozen.^{28 36} They argued that a significant correlation of r=.94 between Lp(a) values measured in fresh and frozen samples proves that storage has no significant effect on the analysis and interpretation of data. As shown here, this method is inappropriate. Despite the higher correlation of r=.97 for LMW and r=.98 for HMW apo(a) phenotypes in our study, significant apo(a) isoform–specific changes of Lp(a) values can be observed over time.

Most antibodies used to measure apo(a) are likely to be directed against a repetitive epitope on kringle IV type 2, as recently shown to be the case for our monoclonal antibody 1A2.¹⁶ It is conceivable that the stability of Lp(a) measurement is related to a methodological problem because of the inverse relation between the number of kringle IV repeats and Lp(a) plasma concentration. On the one hand, epitopes could be lost for the antibody by degradation of apo(a), which would possibly result in a higher relative decrease in Lp(a) values over time for HMW apo(a) isoforms. On the other hand, degraded kringles are possibly more accessible for antibodies, which would cause increasing Lp(a) values over storage time. These speculative concepts must be evaluated by future studies with monoclonal antibodies directed against unique kringles of apo(a).

Aside from storage conditions, the validity of the methods and reagents used is of importance and explains the highly variable changes over time between various assays. Control plasma samples must be included in each assay run and the calibration standard used must have the same apo(a) isoforms during the entire study. This is especially important in assays with antibodies directed against repetitive epitopes of kringle IV, as shown by Marcovina et al.³⁷

Apo(a) isoform–specific changes of Lp(a) values caused by long-term sample storage should be considered in each study comparing Lp(a) values of groups with different frequency distributions of LMW and HMW apo(a) isoforms. Case-control studies relating Lp(a) to atherosclerosis risk are especially prone to misinterpretation as a consequence of storage. On the other hand, apo(a) isoform–specific changes of Lp(a) will have no effect on outcome in studies with a similar frequency distribution of apo(a) isoforms for compared groups.

We believe Lp(a) assays must be evaluated carefully before they can be applied to measure Lp(a) in frozen samples. In particular, it is important to know about apo(a) isoform–specific changes of Lp(a) over time for each particular assay. Such evaluations are time-consuming but necessary. Studies that do not include proper controls for storage effects should not be trusted. As in earlier epidemiological studies, the inclusion of apo(a) phenotyping^{7 31 32 33 34 35} or DNA phenotyping^{38 39} can add important information in the case that apo(a) isoform–specific changes in Lp(a) immunoreactivity cannot be excluded.

Our study provides a reasonable explanation for the failure of some prospective^{9 28 30 36} epidemiological studies to identify Lp(a) as a risk factor for atherothrombotic disease.

Note added in proof. In the meantime, the only prospective study that measured Lp(a) in freshly isolated sera in a subgroup of the PROCAM study has been published. This study found a significant relation between Lp(a) levels and the occurrence of major coronary events.⁴⁰

	Selected Abbreviations and Acronyms

 

apo(a) = apolipoprotein(a) CHD = coronary heart disease HMW = high-molecular-weight LMW = low-molecular-weight Lp(a) = lipoprotein(a)

	Acknowledgments

 
This study was supported by grants from the Austrian Nationalbank (Project 5553) to Florian Kronenberg and the Austrian "Fonds zur Forderung der wissenschaftlichen Forschung" to Hans Dieplinger (P-10090) and Gerd Utermann (S-7109). We thank Astrid Freudenstein for expert technical assistance.

Received February 7, 1996; revision received May 7, 1996;

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				T. Ohira, P. J. Schreiner, J. D. Morrisett, L. E. Chambless, W. D. Rosamond, and A. R. Folsom Lipoprotein(a) and Incident Ischemic Stroke: The Atherosclerosis Risk in Communities (ARIC) Study Stroke, June 1, 2006; 37(6): 1407 - 1412. [Abstract] [Full Text] [PDF]

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