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Technical Briefs |
1
Servei de Bioquímica and
2
Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau, C/Antoni M. Claret 167, 08025 Barcelona, Spain;
3
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 0825 Barcelona, Spain;
a author for correspondence: fax 34-93-2919196,
Mice have become important models in lipoprotein and atherosclerosis research (1)(2). Certain strains of mice, such as C57BL/6, develop hyperlipidemia—as a result of the accumulation in plasma of cholesterol-rich remnant particles—and aortic atherosclerosis when fed a high fat, high cholesterol diet (atherogenic diet) (1)(2). Apolipoprotein E-deficient [apoE(-)] mice also develop hyperlipidemia—as a result of the accumulation of remnant lipoproteins that float mainly as VLDL and intermediate-density lipoprotein—and massive atherosclerosis even when they are fed a diet of regular chow, which is only 4% fat (1). These two mouse models currently are being used to study the effect of gene expression in atherosclerosis with at least two main goals: to understand the mechanisms underlying the genesis and progression of atherosclerosis, and to investigate the feasibility of different genetic and pharmacological interventions to stop or regress atherosclerosis and prevent its complications.
Previous studies have already been instrumental in defining the role of the different HDL particles with respect to atherosclerosis (3)(4). Indeed, any study of this kind requires accurate and reproducible measurement of the cholesterol concentrations associated with the different lipoprotein fractions. For these purposes, fast protein liquid chromatography (FPLC) has been used to isolate HDL and to measure its cholesterol content (HDL-C) (5). This procedure can be performed using smaller volumes of plasma than with ultracentrifugation, which usually requires the use of plasma pooled from different mice. However, both preparative methods are labor-intensive. Because current experiments in mice require an increasingly larger number of animals, HDL has been also isolated by precipitation of apolipoprotein B (apoB)-containing lipoproteins using a variety of reagents (3). Recently, several new direct methods for testing HDL-C have been developed and adapted to many clinical chemistry laboratories (6). These methods eliminate the need for precipitation and separation steps and use smaller volumes of plasma than the precipitation-based methods (6). Therefore, they could be more suitable and practical for measurement of plasma HDL-C in mouse models of hyperlipidemia and atherosclerosis than those using precipitating reagents. The goal of this study was to compare the results of HDL-C measured after precipitation of apoB-containing lipoproteins with those obtained with direct HDL-C measurements.
We previously reported the procedures used in our laboratory to isolate
HDL by FPLC (5). Precipitation of apoB-containing
apolipoproteins was accomplished using a commercial
phosphotungstic-MgCl2 (PT) method (Boehringer
Mannheim). Total cholesterol was measured by the CHOD-PAP method
(Boehringer Mannheim)adapted to a 911 automated analyzer (Boehringer
Mannheim). Triglyceride determinations were performed using the GPO-PAP
method (Boehringer Mannheim) and corrected for the glycerol content
present in plasma (Sigma Diagnostics). The PEGME method (Boehringer
Mannheim), a direct HDL-C assay in which the combination of
polyethylene glycol-modified enzymes, 
-cyclodextrin sulfate, and
magnesium chloride provides selectivity for the determination of HDL-C
(7), and an additional direct HDL-C method (PPD method;
Daiichi; supplied to us as a gift from Izasa, Barcelona, Spain)
(8) that uses a mixture of polymers and polyanions to
complex and block apoB-containing lipoproteins were also adapted to the
911 analyzer (Boehringer Mannheim). In both cases, noncomplexed HDL was
measured using the CHOD-PAP method. All animal procedures were in
accordance with published recommendations for the use of laboratory
animals (9). All investigations in human patients were in
accordance with the Helsinki Declaration of 1975 (as revised in 1983).
As a first step to achieve the goal of this study, we compared the
cholesterol concentrations measured by the conventional CHOD-PAP method
in HDL isolated by PT precipitation of apoB-containing lipoproteins or
after FPLC. Table 1
shows these results obtained from plasma of C57BL/6 control
mice and apoE(-) mice fed either regular chow diet or an atherogenic
diet. HDL-C obtained after PT precipitation was -5.5% to +11.0% with
respect to the results obtained with FPLC; these values were not
statistically different when analyzed with the Student
t-test. Similar results have also been reported by others
(10), who compared mouse plasma HDL-C concentrations
obtained using PT precipitation and ultracentrifugation. PT
precipitation is, therefore, useful for measuring HDL-C in these mouse
models of hyperlipidemia and atherosclerosis. The results for HDL-C
obtained after PT precipitation and by the PEGME method were then
compared. The HDL-C concentrations were very similar when plasmas from
chow-fed control C57BL/6 mice were measured. The regression equation
was: y = 0.93x + 0.14, r =0.94,
P <0.001. However, the similarity in HDL-C results
disappeared when plasmas from control C57BL/6 mice fed an atherogenic
diet and from chow-fed apoE(-) mice were compared (Fig. 1
, A and B). The differences between the mean HDL-C plasma
concentrations obtained with PEGME and those obtained with the PT
precipitation-based method were very important. The PEGME method gave
results in control C57BL/6 mice fed an atherogenic diet that were 72%
higher than those obtained after PT precipitation; this difference
increased to 228% in chow-fed apoE(-) mice (in both cases,
P <0.001, Student paired t-test). Interestingly,
the discordance between the two methods seemed to be associated with
the degree of hyperlipidemia because plasma VLDL-cholesterol (VLDL-C)
+ LDL-cholesterol was higher in chow-fed apoE(-) mice than in
C57BL/6 mice fed an atherogenic diet. Because these two mouse models
share the plasmatic accumulation of remnant lipoproteins, interference
was investigated by adding increasing concentrations of VLDL isolated
by ultracentrifugation from the plasma of C57BL/6 control mice fed an
atherogenic diet or from chow-fed apoE(-) mice. VLDL did not
substantially interfere with the precipitation-based method, but the
PEGME HDL-C value increased as more VLDL was added, especially when it
originated from apoE(-) mice (Fig. 1C
). Because the mean VLDL-C
concentration in the plasma of control C57BL/6 and apoE(-) mice was
3.03 and 4.52 mmol/L, respectively, it is very likely that
intermediate-density lipoprotein and LDL, which in mice are also
largely remnant-like particles containing apoB-48, also cause
interference with the PEGME measurement of HDL-C.
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HDL-C results obtained with the PPD method and the PT precipitation-based method were highly correlated when plasmas of chow-fed control C57BL/6 were measured (r = 0.94; P <0.001). Similar to the results obtained with the PEGME, the HDL-C of chow-fed apoE(-) mice was 100% (P <0.001) higher when measured with the PPD method than after PT precipitation; in this case, VLDL also interfered when added to the plasma (data not shown). Therefore, the VLDL present in these mouse models of hyperlipidemia interfere with at least two different direct HDL-C methods. In addition, the plasma of other mouse models of hyperlipidemia and atherosclerosis, such as human apoA-II transgenic mice or mice deficient in LDL receptor, presented similar interferences (data not shown). Other methods of direct HDL-C measurements were not studied. The reasons included similarity with the ones tested previously (11) and the use of antibodies to human apolipoproteins that would not react with their mouse counterparts (1)(2)(3)(4)(5). In our opinion, therefore, it is very likely that these methods would not be useful for HDL-C measurement in most mouse models of hyperlipidemia and atherosclerosis.
To see whether direct HDL-C methods could be subjected to interferences
in patients with type III hyperlipidemia, we retrospectively selected
20 patients from our clinical lipid laboratory whose analyses at least
once presented a ratio of VLDL-C/triglycerides >0.50 and nonsecondary
hyperlipidemia. These patients were subjected to clinical
re-examination and to new venipuncture. We demonstrated a genetic
defect underlying the described biochemical alteration in only in four
of these patients. Two of these cases presented the
E2/E2 genotype, and another two presented an
Arg136
Ser mutation (12). Of these four individuals, only
one E2/E2 patient remained hyperlipidemic
(cholesterol, 7.12 mmol/L; triglycerides, 4.93 mmol/L) and maintained a
ratio of VLDL-C/triglyceride >0.65 upon re-analysis although he was
receiving specific drug treatment. In this patient, the HDL-C values
obtained with PT precipitation, PEGME, and PPD were 0.98, 0.99, and
1.05 mmol/L, respectively. There also were no differences in the
results for HDL-C when these three different methods were used to
analyze blood from the remaining three individuals with apparent
genetic susceptibility to type III hyperlipidemia but who were
normolipidemic at the time of the re-analysis. Thus, the sample size
was too low to draw a valid conclusion and our results in humans cannot
be compared with the recent findings of Lackner and Schmitz
(13) who, using the PEGME direct HDL-C method in four
E2/E2 nontreated patients severely affected by
type III hyperlipidemia, reported similar data to ours in
hyperlipidemic mice. However, our results in mice and theirs in
patients with type III hyperlipidemia (13) show that the
remnant lipoprotein particles of two different animal species are
partially recognized by reagents that should recognize only HDL. This
produces an important interference in at least some of the direct HDL-C
methods used at present.
In summary, at least two direct methods of HDL-C measurement are not suitable for studies in at least several mouse models of hyperlipidemia and atherosclerosis. If not otherwise demonstrated, great caution should be exercised before applying direct HDL-C measurements to the plasma of other animal models of hyperlipidemia and atherosclerosis in which remnant particles are generated, such as the diet-induced hypercholesterolemic rabbit.
Acknowledgments
This study was supported in part by grants from the Fundació d'Investigació Cardiovascular-Marató de TV3 1995 (F.B-V.) and CICYT SAF 98-0097 (F.G-S.). During this study, J.C.E-G. was a predoctoral fellow of the Ministerio de Educación y Cultura.
References
The following articles in journals at HighWire Press have cited this article:
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  S. Acin, M. A. Navarro, J. M. Arbones-Mainar, N. Guillen, A. J. Sarria, R. Carnicer, J. C. Surra, I. Orman, J. C. Segovia, R. d. l. Torre, et al. Hydroxytyrosol Administration Enhances Atherosclerotic Lesion Development in Apo E Deficient Mice J. Biochem., September 1, 2006; 140(3): 383 - 391. [Abstract] [Full Text] [PDF]
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 G. R. Warnick, M. Nauck, and N. Rifai Evolution of Methods for Measurement of HDL-Cholesterol: From Ultracentrifugation to Homogeneous Assays Clin. Chem., September 1, 2001; 47(9): 1579 - 1596. [Abstract] [Full Text] [PDF]
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 J. C. Escolà-Gil, J. Julve, A. Marzal-Casacuberta, J. Ordóñez-Llanos, F. González-Sastre, and F. Blanco-Vaca ApoA-II expression in CETP transgenic mice increases VLDL production and impairs VLDL clearance J. Lipid Res., February 1, 2001; 42(2): 241 - 248. [Abstract] [Full Text]
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J. Ordonez-Llanos, A. M. Wagner, R. Bonet-Marques, J. L. Sanchez-Quesada, F. Blanco-Vaca, and F. Gonzalez-Sastre Which Cholesterol Are We Measuring with the Roche Direct, Homogeneous LDL-C Plus Assay? Clin. Chem., January 1, 2001; 47(1): 124 - 126. [Full Text] [PDF]
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) and PT-based (
) methods
and effects of VLDL isolated by sequential ultracentrifugation from
plasma of chow-fed apoE(-) mice on the direct HDL-C (•) and PT-based
(
) methods.