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Technical Briefs |
1
Biochemistry Department, Tenon Hospital, 75020 Paris, France;
2
Biochemistry Department, CHU, BP 217, 38043 Grenoble cedex 09, France;
3
Biochemistry Department, Avicenne Hosp, 93009 Bobigny, France;
Lipoprotein(a) [Lp(a)] is an LDL-like particle whose apo B100
is disulfide-linked to an apolipoprotein A [apo(a)]. Apo(a) is a
glycoprotein containing 23% carbohydrate by weight (1).
Apo(a) possesses a high degree of polymorphism because of a variable
number (11–52) of the repetitive structural unit called "kringle"
IV (K IV; Mr 
17 kDa), which leads to a wide
range of molecular weights, from almost 200 kDa to 800 kDa
(2). The apo(a) K IV shares a strong sequence homology with
plasminogen K IV, and apo(a) has been shown to inhibit fibrinolysis in
vitro. (3)
A high Lp(a) plasma concentration is an independent risk factor for cerebro- and cardiovascular atherosclerosis; furthermore, low molecular mass isoforms are more frequent in patients with high Lp(a) concentrations (4). In addition, the antifibrinolytic effect of Lp(a) is inversely correlated with the size of these isoforms (5). Accurately determining the number of apo(a) Ks is, therefore, important.
The molecular weight determination of apo(a) is complicated by its anomalous mobility in SDS gels, which differs from the mobility of most commercially available molecular weight markers (1). In addition, because of the relatively small size of a K, electrophoretic techniques for apo(a) phenotyping must provide high resolution.
The aims of our study were to develop an accurate and simple method for apo(a) phenotyping by measuring the number of Ks, to validate this method by comparison with a genotyping method, and to determine, through this technique, the frequency of occurrence of the common apo(a) isoforms in healthy subjects.
We mixed EDTA-treated plasma samples from 238 healthy Caucasian subjects with antiproteases (1 µmol/L D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, Calbiochem 520 222; 0.5 mmol/L aminoethyl-benzenesulfonyl fluoride from Sigma diluted in isopropanol; 100 000 U/L aprotinin, Bayer Pharma 92807) and stored them frozen at -80 °C until they were phenotyped.
Plasma Lp(a) was measured by an immunonephelometric assay (Beckman).
Apo(a) genotyping was performed on 15 samples by pulsed-field gel electrophoresis of KpnI-digested genomic DNA as described by Lackner et al. (6).
Electrophoresis was carried out according to a method modified from Kamboh et al. (7) and using 1.5% agarose gels containing sodium dodecyl sulfate (SDS). A 1.5-mm thick gel slab containing ultra pure Seakem LE® dissolved in 90 mmol/L Tris, 90 mmol/L boric acid, 2 mmol/L Na2EDTA, and 1 g/L SDS, pH 8.5, was cast in a 14 x 16- cm vertical cell (LKB). A commercially available calibrator (Immuno®) was prepared by mixing different plasmas; the calibrator contained five apo(a) isoforms (35, 27, 23, 19, and 14 Ks). For the first sample run, the sample was applied undiluted so that faint isoforms in heterozygous subjects could be detected. When necessary, the sample was reanalyzed after the apo(a) mass load onto the gel was adjusted to ~25 ng. Before electrophoresis, 20 µL of plasma was mixed with 60 µL of reducing buffer (1:2:10, by volume, of ß-mercaptoethanol, 5 g/L bromphenol blue in 50 mL/L glycerol, and 50 g/L SDS); the mixture was then heated for 1 min at 100 °C. The mixture (3 µL) was loaded into 6 mm-wide wells cast into the gel. Gels were run at 12 °C for 2 h at a constant power of 10 W and an initial voltage of 250 V. Proteins were then pressure-transferred for 2 h to a 0.2-µm nitrocellulose (NC) membrane. The NC membrane was incubated for 15 min in a 10 g/L bovine serum albumin solution to block any remaining protein binding sites and then immersed overnight in monospecific polyclonal rabbit anti-human apo(a) antiserum from Beckman (1 mL antiserum/L buffered milk). This step can be shortened to 2 h by incubating the NC membrane in the anti-apo(a) antiserum diluted to 5 mL/L buffered milk. After the filter was washed, it was incubated with a goat anti-rabbit IgG conjugated with peroxidase (Sigma A4914; 0.5 mL/L buffered nonfat milk). After the filter was washed extensively, the apo(a) banding pattern was visualized through chemical staining for peroxidase with diaminobenzidine.
In our method, the dye in the samples runs off the gel during electrophoresis; therefore, we measure the absolute distance of migration. A standard curve was thus generated by plotting the distance of migration of the standard bands against the log of the K number, using least-square regression analysis.
The detection limit of the method was determined by analyzing a sample with a known Lp(a) concentration at different dilutions [100, 10, and 1 ng apo(a)].
Statistical analyses were performed with Statview IV Software (Alsyd 38240). The relationship between the number of Ks and the Lp(a) concentrations was estimated using polynomial regression. Lp(a) concentrations in men and women were compared using an ANOVA in which log Lp(a) concentrations were entered because of the skewness of the Lp(a) concentration distribution. Lp(a) concentration frequency distributions in men and women were compared by plotting Lp(a) concentration percentiles in men and in women. The number of subjects in each class of apo(a) isoforms defined by the K number was very low. Therefore, to compare the data with those previously described, apo(a) isoforms were categorized into six groups corresponding to those originally reported (8). Groups B, S1, S2, S3, S4, and >S4 include apo(a) isoforms <17, 17–19, 20–22, 23–25, 26–30, and >30 Ks, respectively.
A typical blot is shown in Fig. 1
A. The resolution accuracy of adjacent apo(a) isoforms was
estimated to be within 2 Ks, and the lower limit of detection was
almost 1 ng apo(a)/band. The CV of the calibration slopes was ~5%.
We compared our phenotyping method with genotyping (Fig. 1B
). Despite
differences between the number of Ks determined by the two methods,
these differences averaged 1.3 Ks and did not exceed 2 Ks in 90% of
the samples or 1 K in 68% of the samples.
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Thirty apo(a) isoforms from 12 to 41 Ks were identified in 238 healthy
subjects. The relative frequency of the apo(a) phenotypes in this
healthy French population is given in Table 1
. The distributions of apo(a) phenotypes in men and women were
not markedly different. The number of phenotypes observed was 102,
which is less than the 465 calculated from the number of alleles
identified. The frequency of single-band phenotypes was 48%. The
numbers of apo(a) Ks in one-banded subjects were inversely related to
Lp(a) concentrations (Fig. 1C
). In heterozygous subjects, plasma Lp(a)
concentrations (y) correlated negatively with the number of
Ks in the smallest isoform (x) [y (mg/L) =
627.59 - 32.14x 0.47 x;
r = 0.284; P = 0.0065], but there was
no substantial correlation between Lp(a) concentrations and the number
of Ks in the largest isoform.
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These data indicate that our SDS-agarose method allows a simpler and more rapid determination of the apo(a) number of Ks than in previously described methods (7)(9)(10)(11)(12)(13).
The comparison between DNA genotyping and protein phenotyping confirmed that apo(a) calibrators and plasma apo(a) have the same electrophoretic behavior (14). Moreover, the use of molecular weight markers other than the apo(a) molecules themselves led to a high systematic error (1)(12).
The resolution of our technique is the same as that reported by Csako et al. (10). The number of isoforms we detected is comparable with that obtained by Marcovina et al. (13). The percentage of unbanded subjects, which depends on the detection limit, is in the range of the more recently described methods, and 52% of double-banded subjects were identified, the same percentage as in other studies using phenotyping methods (11)(12)(15). However, when only one isoform is observed, one can reanalyze the sample at a lower apo(a) concentration because an isoform at a high concentration could mask another isoform of similar size that is present in a very low concentration. On the other hand, the sample can also be reanalyzed at a higher concentration to increase the apo(a) mass of the weaker isoform loaded onto the gel to above the detection limit (16).
The main advantage of this method is its simplicity in terms of its praticability and its rapidness. The migration time compared with the Kamboh et al. method (7) was shortened from almost 20 h to 2 h; the shortened time avoids excessive diffusion of apo(a) molecules during the electrophoresis step and improves the resolution. The capillary blotting has been reduced to 2 h as well. Therefore, this method may be carried out in approximately one day, making this method faster than all others previously cited.
As described in other Caucasian populations, high molecular weight isoforms, i.e., with >23 Ks IV, were the most frequent in our French population. However, a high percentage of unbanded subjects in some studies makes the comparison uncertain (4)(9)(17)(18)(19)(20). Marcovina et al. (21) used SDS-polyacrylamide gel electrophoresis to show that 76.8% of Caucasians were carrying almost one allele with >22 Ks. Kraft et al. (4) found a similar percentage (82.7%) in Tyroleans, and we found that 80.0% of our subjects carried one or two alleles with at least 23 Ks.
In conclusion, this apo(a) phenotyping method is rapid, simple, accurate, and sensitive, which makes it suitable for large-scale epidemiologic and clinical studies.
Footnotes
and * address for correspondence: Biochemistry Department, Tenon Hosp, 4 rue de la Chine, 75020 Paris, France
fax 33 1 40 30 78 40, e-mail remy.couderc{at}tnn.ap-hop-paris.fr
References
The following articles in journals at HighWire Press have cited this article:
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  E. Anglés-Cano, S. Loyau, G. Cardoso-Saldaña, R. Couderc, and P. Gillery A novel kringle-4 number-based recombinant apo[a] standard for human apo[a] phenotyping J. Lipid Res., February 1, 1999; 40(2): 354 - 359. [Abstract] [Full Text]
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