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Technical Briefs |
1
University Hospital of Freiburg, Department of Clinical Chemistry, Hugstetter Strasse 55, 79106 Freiburg i. Br., Germany, and
2
University Hospital of Heidelberg, Department of Endocrinology, D-69115 Heidelberg, Germany;
a author for
correspondence: fax 49-761-270 3444, e-mail manauck{at}mzl200.ukl.uni-freiburg.de
Familial type III hyperlipoproteinemia (HLP) is characterized by
the accumulation of cholesterol-rich remnants (ß-VLDL). The
differential diagnosis of type III HLP is clinically important because
patients with type III HLP develop premature coronary artery disease
(CAD) and peripheral atherosclerosis and because type III HLP responds
well to dietary treatment and fibric acid derivatives(1)(2). Pathogenetically, type III HLP is
related to dysfunctional isoforms of apolipoprotein (apo) E. At the
APOE gene locus, three common alleles exist,
designated 
2, 3, and 4 (3)(4)(5). apoE2 is
defective in its binding to lipoprotein receptors(6)(7). Because of the impaired catabolism of
chylomicron and VLDL remnants, individuals homozygous for apoE2 reveal
detectable amounts of ß-VLDL in their plasma. ß-VLDLs are
atypical lipoproteins with a density <1.006 kg/L and ß-mobility on
agarose gel electrophoresis. Compared with normal VLDL, ß-VLDLs are
cholesterol-enriched; compared with normal LDL, they are relatively
enriched in triglycerides. More than 90% of patients with type III HLP
are homozygous for apoE2, but only ~1 in 20 individuals carrying the
E2/2 phenotype finally develops type III HLP (8). Those
homozygous carriers of apoE2 having small amounts of ß-VLDL in their
plasma not sufficient to produce overt hyperlipidemia have been
classified as suffering from normolipidemic
dysbetalipoproteinemia. The term type III HLP, in contrast, is
applied to hyperlipemic individuals only.
Clinical characteristics such as palmar, tendon, and/or tubero-eruptive xanthomas do not occur in all individuals with type III HLP. To establish biochemically the diagnosis of type III HLP, the following criteria have been applied in this study: (a) presence of increased cholesterol and triglycerides at 2500 mg/L or more, (b) an increased ratio of VLDL-cholesterol (VLDL-C) to VLDL-triglycerides (VLDL-TG; >0.4), and (c) an increased ratio of VLDL-C to total triglycerides (>0.3) (9)(10)(11).
We studied in total 1317 sera from women and men, ages 20–65 years. Among the participants with apoE phenotypes other than E2/2 (n = 1288) were 468 CAD patients recruited at the University Hospital, Freiburg or at the Benedikt-Kreutz-Klinik, Bad Krozingen, and 820 clinically healthy individuals, recruited at the Rheintalklinik, Bad Krozingen or employees of the BASF, Ludwigshafen. Twenty-nine apoE2 homozygotes were studied, including 21 samples obtained at the University Hospital of Heidelberg. In 8 of the 12 individuals with type III HLP, signs of atherosclerosis were present (66%). Two of the 17 apoE2 homozygotes without manifest type III HLP showed CAD (12%). Informed consent was obtained from each individual participating in this study; all procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 1983.
Blood was drawn after an overnight fast and allowed to clot at room temperature; serum was obtained by centrifugation at 1500g for 15 min. All analyses were performed within 2 days after blood collection at the University Hospital of Freiburg. Cholesterol and triglycerides, without blanking for glycerol, were determined enzymatically (Boehringer Mannheim) using a Hitachi type 747 (total cholesterol and triglycerides) or a Wako 30R analyzer (supernates of precipitation reactions), respectively. The total CVs for the methods were <2%.
LDL-like lipoproteins were isolated using 100 µL of serum and 1000 µL of dextran sulfate (DS)/MgCl2 precipitation reagent (Quantolip®; Immuno GmbH) and incubated for at least 10 min. The resulting precipitate was separated by a 5-min centrifugation, and the cholesterol and triglyceride content of the soluble lipoproteins (mainly VLDL and HDL) was measured in the supernate. Cholesterol and triglycerides associated with the precipitated lipoproteins (LDL-CDS and LDL-TGDS, respectively) were calculated as total cholesterol or triglycerides minus supernate cholesterol or triglycerides, respectively.
A combined ultracentrifugation (UC) and precipitation assay was used as the comparison method (12)(13). In this method, the recoveries of the bottom fraction (LDL plus HDL) was between 97% and 102%, whereas the recoveries of the top fraction (VLDL) was between 75% and 103%. All lipid measurements of one sample—lipids and lipoprotein fractions—were performed in the same analytical runs. The between-day CVs for LDL-CDS, LDL-TGDS, LDL-CUC, and HDL-CUC were below 3% and 4%, respectively.
apo E phenotyping was performed by isoelectric focusing on agarose and immunofixation (14)(15). Regression analyses were performed using the method of Passing and Bablok (16).
We first wished to compare the results for LDL-C obtained with the DS precipitation method with those obtained with UC. In this comparison, we excluded 45 samples in which a recently developed algorithm indicated that the DS precipitation was incomplete because of fatty acid concentrations exceeding 2 mmol/L (17)(18)(19). Alternatively, samples can be diluted with bovine serum albumin before precipitation, which will overcome the effect of free fatty acids, as described for lipoprotein electrophoresis [Nauck et al., unpublished results, and Ref. (20)].
In the remaining 1243 samples with apoE phenotypes other than apoE2/2, LDL-CDS showed a good agreement to LDL-CUC (y = 1.07x - 154 mg/L; r = 0.969)(19). Recent work demonstrated that DS/MgCl2 precipitation co-precipitates VLDL(19).
In apoE2 homozygotes with normolipidemic dysbetalipoproteinemia, the two methods corresponded with each other (r = 0.953; y = 0.79x + 304 mg/L), indicating that co-precipitation of ß-VLDL does not markedly affect apparent LDL-CDS values. In individuals with type III HLP, however, virtually no correlation between LDL-CDS and LDL-CUC was observed (r = -0.126). The LDL-CDS was on average twice as high as LDL-CUC, suggesting the amounts of ß-VLDL.
The accumulation of triglyceride-enriched ß-VLDL can be detected by
analyzing the triglyceride content of the DS precipitate. We compared
the ratio of LDL-TGDS to
LDL-CDS between individuals with type III HLP and
other types of HLP. Eight samples with type III HLP were matched with
16 samples with other types of HLP according to total cholesterol
and total triglycerides. The ratio of LDL-TGDS to
LDL-CDS was significantly higher in type III HLP,
but this did not allow unequivocal diagnosis of type III HLP because
this ratio may be increased in other types of HLP as well (Table 1
).
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In Fig. 1
A, absolute values of LDL-TGDS are plotted
vs LDL-CDS, including values for 18 samples from
apoE2 homozygotes. All samples from individuals with type III HLP
(n = 8) had increased LDL-TGDS >950 mg/L
and LDL-CDS >1750 mg/L, whereas all samples from
subjects with normolipidemic dysbetalipoproteinemia did not meet these
criteria. Twenty-one samples of 1243 samples with phenotypes other than
apoE2/2 (1.69%) exceeded the two threshold concentrations. The
proportion of actual type III HLP among the suspicious samples thus was
28% (8 of 29). When calculated on the basis of all samples, the
sensitivity to detect type III HLP by applying the combined thresholds
of LDL-TGDS >950 and
LDL-CDS >1750 mg/L was thus 100%, and the
specificity was 98.3%. The specificity dropped to 92% if only samples
with LDL-CDS >1750 mg/L were considered. To
verify these results, we analyzed another 11 completely independent
serum samples from other individuals homozygous for apoE2 with and
without type III HLP (Fig. 1B
). All individuals were classified
correctly when we applied the above threshold values to these samples.
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According to these results, a sample should be considered suspicious of type III HLP if (a) both total cholesterol and triglycerides exceed 2500 mg/L, (b) the LDL-CDS concentration is > 1750, and (c) the LDL-TGDS concentration is >950 mg/L.
However, only ~30% of the suspicious cases really had type III HLP, whereas in the remaining cases other types of HLP existed. Because our diagnostic criteria have been chosen in favor of sensitivity rather than specificity, we recommend confirming the diagnosis of type III HLP in suspicious samples using apoE phenotyping and/or apoE genotyping along with preparative UC and/or lipoprotein electrophoresis.
In conclusion, the advantage of the proposed procedure is that the screening for increased LDL-C concentrations includes the option to detect high concentrations of atherogenic LDL-like particles with an abnormal composition by simultaneous determination of triglycerides and cholesterol after precipitation with DS and Mg2+ ions. Without this information, type III HLP will frequently be overlooked. Our procedure is inexpensive and can be performed in every laboratory because it needs no specialized equipment.
Acknowledgments
We thank Brigitte Haas, Gabi Herr, Isolde Friedrich, Sabine von Karger, Brigitte Kreisel, Sibylle Rall, and Susanne Schuler for excellent technical assistance. We thank Prof. Dr. H. Just, Prof. Dr. H. Roskam, Dr. Frölich, and Prof. Dr. Dr. A. Zober for their cooperation in the recruitment of patients and healthy individuals.
References
The following articles in journals at HighWire Press have cited this article:
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  D. J. Blom, F. H. O'Neill, and A. D. Marais Screening for Dysbetalipoproteinemia by Plasma Cholesterol and Apolipoprotein B Concentrations Clin. Chem., May 1, 2005; 51(5): 904 - 907. [Full Text] [PDF]
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M. Nauck, G. R. Warnick, and N. Rifai Methods for Measurement of LDL-Cholesterol: A Critical Assessment of Direct Measurement by Homogeneous Assays versus Calculation Clin. Chem., February 1, 2002; 48(2): 236 - 254. [Abstract] [Full Text] [PDF]
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