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Department of Clinical Biochemistry, Royal Prince Alfred Hospital, Missenden Rd., Camperdown, New South Wales, 2050, Australia.
1
Nonstandard abbreviations: LDL-C, LDL
cholesterol; CAD, coronary artery disease; apo, apolipoprotein; TG,
triglyceride; TC, total cholesterol; and AQAP, Australian Quality
Assurance Programme.
a Author for correspondence. Fax 61-2-95157931; e-mail davids{at}bioc.rpa.cs.nsw.gov.au
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: ultracentrifugation • triglycerides • precipitin tests • immunologic techniques
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In routine clinical practice, total apo B measurement and LDL-C estimated by the Friedewald equation (6) tend to be used, thereby avoiding ultracentrifugation. Total apo B may not distinguish between apo B48- and apo B100-containing particles. In addition, apo B methods that involve light-scattering techniques may be unreliable in hypertriglyceridemic samples. Estimation of LDL-apo B cannot be calculated by a process equivalent to the Friedewald formula, and the Friedewald equation is also inappropriate for samples from nonfasting individuals or those with plasma triglyceride (TG) concentrations >4.0 mmol/L (6).
We have assessed the use of a commercial immunoseparation kit as part of an alternative method for LDL-apo B quantification. The kit was developed for LDL-C measurement in samples from hypertriglyceridemic or nonfasting individuals that are unsuitable for LDL-C calculation by the Friedewald equation (7)(8).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ultracentrifugation
Lipoproteins of d <1.006 and d >1.006 kg/L
were fractionated from whole plasma of all participants by
ultracentrifugation. Plasma (3 or 4 mL) was dispensed into 6-mL
capacity Quick-Seal centrifuge tubes (Beckman Instruments, Palo Alto,
CA), tubes were filled with d = 1.006 kg/L solution
(0.15 mol/L NaCl, 0.1 g/L NaN3, and 0.1 g/L EDTA), and then
centrifuged at 199 800g for 16 h at 4 °C in a 50.4
Ti rotor and Optima L-80 ultracentrifuge (Beckman Instruments). The top
fraction (d <1.006 kg/L), containing VLDL, and the bottom
fraction (d >1.006 kg/L), containing IDL, LDL, and HDL,
were recovered after tube slicing. Both fractions were restored to the
original plasma volume with d = 1.006 kg/L solution.
The mean (SD) cholesterol recovery, based on the sum of cholesterol
concentrations in the two fractions relative to the total plasma
cholesterol, was 99 (3)%.
precipitation
HDL were isolated from plasma and the d >1.006 kg/L
plasma fraction after precipitation of apo B-containing lipoproteins
with dextran sulfate:magnesium chloride (9).
immunoseparation
LDL were isolated from plasma by using the Direct LDL-C
Immunoseparation reagent kit kindly provided by Genzyme Diagnostics
(Genzyme Corp., Cambridge, MA). This procedure involves a suspension of
polystyrene latex beads coated with goat polyclonal antibodies to human
apo AI and apo E. Plasma (30 µL) and reagent (200 µL) were mixed in
the inner tube of a dual-chamber centrifuge filter unit. After a 10-min
incubation at room temperature, the unit was centrifuged at
1500g for 5 min. Immunoprecipitated VLDL, IDL, and HDL
remained in the inner tube, being trapped by a filter at its base. LDL
passed freely through the filter, and an LDL-containing filtrate was
recovered from the outer tube. CVs were determined by measuring
cholesterol concentration in the filtrates of control samples provided
(1.8 mmol/L and 5.3 mmol/L). The intraassay CVs were generated by
repeated immunoseparation (n = 10) of both controls. These were
3.9% and 2.0%, respectively. The interassay CVs were generated by
immunoseparation of the same controls on 10 separate runs. These were
10.0% and 3.5%, respectively.
lipid, lipoprotein, and apolipoprotein quantification
TC and TG were measured by standard enzymatic techniques on an
automated analyzer (BM/Hitachi 747, Tokyo, Japan) by using Boehringer
Mannheim (Mannheim, Germany) reagents (CHOD-PAP for TC, GPO-PAP for
TG). The interassay CVs were 2.5% and 4.8%, respectively, whereas
external quality-assurance performance on Cycle 35 of the Australian
Quality Assurance Programme (AQAP) revealed CV = 1.9%, bias
= 0.13 mmol/L for TC and CV = 1.4%, bias = 0.23 mmol/L for
TG.
HDL-C was measured by standard enzymatic methods on a centrifugal analyzer (Multistat III Plus; Instrumentation Laboratory, Lexington, MA) with Australian Scientific Enterprise (Sydney, Australia) reagents (CHOD-PAP). The interassay CV was 2.0% and AQAP performance was CV = 9.0% and bias = 0.21 mmol/L.
LDL-C was determined by three methods: (a) In plasma with TG
4.0 mmol/L, LDL-C (range 1.6–8.2 mmol/L, n = 35) was estimated
by the Friedewald equation (6); (b) in the
d >1.006 kg/L fraction, LDL-C (range 0.9–8.5 mmol/L,
n = 46) was measured by subtracting HDL-C from d
>1.006 kg/L cholesterol; this measurement includes IDL-C (1.006<
d <1.019 kg/L); (c) in the immunoseparated
filtrate, LDL-C (range 1.5–8.2 mmol/L, n = 46) was measured
directly as the cholesterol concentration of the ultrafiltrate.
Apo B measurements were carried out on fractions from 40 subjects because sample volumes from 6 patients were insufficient. A Behring nephelometer and commercially available antibodies (Behringwerke, Marburg, Germany) were used. Apo B was determined in both the d >1.006 kg/L fraction, which includes IDL-apo B (range 0.52–2.24 g/L, n = 40), and the immunoseparated LDL filtrate (range 0.58–2.14 g/L, n = 40). The interassay CV was 4.0% and AQAP specimens based on an IFCC calibrator revealed CV = 2.8% and bias = 0.038 g/L.
statistical analyses
Pearson correlation coefficients were computed to assess the
association between parameters. All statistical tests were performed
with Fig.P software (Fig.P Software Corp., Durham, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In samples with plasma TG 4.0 mmol/L (n = 35), LDL-C estimated
by the Friedewald calculation (y) correlated well with
cholesterol measured directly in the d >1.006 kg/L fraction
after ultracentrifugation (x) (y =
0.99x + 0.07; r = 0.98; n = 34). The
95% confidence limits for the slope and intercept were 0.92–1.06 and
-0.29–0.43 respectively. Similar correlations were found between
LDL-C obtained by immunoseparation and ultracentrifugation
(y = 0.87x + 0.71; r = 0.98;
n = 46), and between immunoseparation and Friedewald calculation
(y = 0.88x + 0.61; r = 0.96;
n = 35). The 95% confidence limits for slope and intercept were
0.81–0.93 and 0.42–1.00 respectively for the correlation between
immunoseparation and ultracentrifugation, whereas those for the
correlation between immunoseparation and the Friedewald equation were
0.79–0.97 and 0.12–1.10 respectively.
To detect the between-method bias for the LDL-C methods under
comparison, the absolute difference was plotted against the mean for
each pair of measurements. The comparison between immunoseparation and
ultracentrifugation (Fig. 2
A) and the comparison between immunoseparation and the
Friedewald calculation (Fig. 2B
) both show a positive bias in favor of
immunoseparation at lower LDL-C concentrations, but a negative bias at
higher LDL-C concentrations.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2
. Although other centrifugation
techniques that isolate the 1.019< d <1.063 kg/L fraction
would avoid this problem, they are more cumbersome and, as a
consequence, have been used less frequently for the analysis of
clinical samples. Increased concentrations of LDL-apo B may identify patients with increased numbers of LDL in whom CAD risk is increased. LDL-C concentrations in these patients may not necessarily be increased. The original immunodiffusion technique for LDL-apo B measurement (5) has been difficult to standardize between laboratories and has not been widely adopted. Many clinical laboratories use total apo B concentrations as an indication of hyperapobetalipoproteinemia. The inclusion of VLDL and interference due to turbidity associated with d <1.006 lipoproteins is likely to confound this approach, whereas the presence of apo B48-containing lipoproteins is also a theoretical concern. Nevertheless, the direct measurement of LDL-apo B in immunoseparated LDL avoids all of these confounding factors. We believe that the measurement of apo B by immunoturbidimetry is reliable after the removal of d <1.006 kg/L lipoproteins because this removes apo B48-containing particles and minimizes problems with turbidity. However, we acknowledge that optimization of the direct measurement of LDL-apo B may involve alternative methods for apo B quantification.
The role of LDL-C has become more important in the assessment of CAD
risk and is the basis of some public health guidelines for the
management of patients with dyslipidemia (National Cholesterol
Education Program II) (10). It could be argued that
quantification of atherogenic lipoproteins should also include IDL
because they are also extremely atherogenic. In cases of
dysbetalipoproteinemia and mixed hyperlipidemia, their contribution may
need to be assessed separately. The Genzyme LDL assay does not
distinguish between LDL and lipoprotein(a), but the Friedewald formula
(6) and ultracentrifugation techniques also fail to
separate these two lipoproteins. This is not of major practical
significance since both lipoproteins are likely to be highly
atherogenic. The only methods available to assess LDL-C directly
involve one of several ultracentrifugation procedures, all of which are
relatively cumbersome, labor intensive, time consuming, and expensive.
Friedewald et al. derived an equation to estimate the LDL-C so as to
avoid this problem (6). Our results on samples with plasma
TG 4.0 mmol/L confirm that the calculated Friedewald estimation
correlates very well with LDL-C measured after ultracentrifugation with
minimal inaccuracy. The disadvantages are that calculation of
Friedewald LDL-C in samples that have TG concentrations >4.0 mmol/L is
inappropriate. Furthermore, it is inconvenient to require patients to
fast for 14 to 16 h before blood collection, and samples from
nonfasting individuals will be inaccurate. The new technique was not
tested with postprandial samples, but it is unlikely that it would be
subject to interference from postprandial TG-rich lipoproteins.
Finally, the Friedewald equation does not provide information about
other aspects of LDL composition such as its apo B concentration. The
new method is simple and provides the extra benefit of allowing the
assessment of LDL composition (e.g., LDL cholesterol:apo B ratio),
which may reflect LDL size and density. LDL composition differs between
individuals with differing LDL subclass phenotype (11),
and increased concentrations of LDL-apo B may identify individuals with
increased CAD risk despite nonincreased LDL-C concentrations due to
increased numbers of small dense LDL (3). Other methods
for the assessment of LDL size and density include gradient gel
electrophoresis, which is qualitative rather than quantitative, and
sequential ultracentrifugation, which is extremely laborious.
In conclusion, immunoseparation offers a convenient technique for the direct estimation of not only LDL-C, but also LDL-apoB, even in hypertriglyceridemic samples. It requires only 30 µL of sample as compared with much larger volumes required for ultracentrifugation, and effectively eliminates cholesterol-containing lipoproteins other than LDL and lipoprotein(a). The removal of the TG-rich lipoproteins may reduce interference due to turbidity if light-scattering techniques are used. The measurement of both cholesterol and apo B by using the immunoseparation technique provides important additional clinical information because LDL composition reflects its size and density. Freezing and storage before immunoseparation causes a negative bias (7)(8), so this technique appears unsuitable for retrospective analysis of LDL-apo B in stored frozen samples. However, the convenience of this new technique may warrant the inclusion of this potentially useful measurement in clinical care and research.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  J. R. Burnett and G. F. Watts Estimating LDL ApoB: Infomania or Clinical Advance? Clin. Chem., May 1, 2008; 54(5): 782 - 784. [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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F. Gomez, J. Camps, J. M. Simo, N. Ferre, and J. Joven Agreement Study of Methods Based on the Elimination Principle for the Measurement of LDL- and HDL-Cholesterol Compared with Ultracentrifugation in Patients with Liver Cirrhosis Clin. Chem., August 1, 2000; 46(8): 1188 - 1191. [Full Text] [PDF]
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