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Technical Briefs |
a address correspondence to this author at: Institut für Klinische Chemie und Laboratoriumsmedizin, Klinikum der Universität Regensburg, D-93042 Regensburg, Germany
The determination of HDL- and LDL-cholesterol has become very important because most therapeutic recommendations are based on the LDL-cholesterol concentration and consider HDL-cholesterol as a risk factor (1)(2). Furthermore, in fasting patients with normal or only moderately increased triglycerides (<4000 mg/L) and presumably normal VLDL-composition, LDL-cholesterol can be calculated with reasonable accuracy by the Friedewald formula if the concentrations of the total cholesterol, HDL-cholesterol, and triglycerides are known (3)(4). In fact, because there are few acceptable alternatives, this is probably the most common method for determining LDL-cholesterol. Thus, accurate determination of HDL-cholesterol is of critical importance in the diagnosis of disturbances of lipid metabolism.
The availability of homogeneous methods for determining HDL-cholesterol
without any additional sample pretreatment dramatically simplifies
automation of the assay. These methods have been evaluated widely
(5)(6)(7), and the protocol that uses polyethylene glycol
(PEG)-modified enzymes and 
-cyclodextrin, in particular, has been
shown to meet the NCEP requirements (7)(8).
Triglyceride interference was thoroughly analyzed and found to be
negligible and nonsystematic as long as triglycerides were <10 000
mg/L (9). We provide evidence that this is not the case for
sera from patients with type III hyperlipoproteinemia.
Cholesterol and triglycerides were measured by enzymatic methods
(CHOD-PAP and GPO-PAP; Boehringer Mannheim) on a Hitachi 717 analyzer.
HDL-cholesterol was determined either by a homogeneous method that uses
PEG-modified enzymes and -cyclodextrin (lyophilized reagent;
Boehringer Mannheim) on a Hitachi 717 analyzer or after precipitation
of apolipoprotein B-containing lipoproteins with phosphotungstic
acid/MgCl2 (PTA/Mg) with a commercial reagent
(Boehringer Mannheim). The day-to-day imprecision (CV) for the
homogeneous HDL-cholesterol and PTA/Mg precipitation methods in the
laboratory was <3% and <5%, respectively. In addition,
HDL-cholesterol was measured with both methods after removal of VLDL by
ultracentrifugation at a density of 1.006 kg/L. This approach was also
used to determine VLDL-cholesterol and LDL-cholesterol in the type III
patients (10). ß-VLDL was isolated from one type III
patient by preparative ultracentrifugation of serum at a density of
1.006 kg/L at 10 °C and 145 000g/min for 18 h in
a Beckman L70 ultracentrifuge using a Beckman 50.4 rotor.
Four patients with newly diagnosed type III hyperlipoproteinemia were
analyzed. Criteria for the diagnosis of type III hyperlipoproteinemia
were fasting cholesterol and triglycerides >2000 mg/L, apoE2
homozygosity, and a VLDL-cholesterol:total triglyceride ratio >0.3.
The patients were not on lipid-lowering medications at the time of
lipid analysis. Their lipid and lipoprotein values are shown in Table 1
. In three patients, the HDL-cholesterol in whole serum was
determined to be higher by the homogeneous method than by PTA/Mg
precipitation. After the VLDL was removed by ultracentrifugation,
HDL-cholesterol determined either by the homogeneous method or by
PTA/Mg precipitation was markedly lower than that determined by the
homogeneous method in whole serum. Thus, HDL-cholesterol was
overestimated by the homogeneous method in whole serum. Depending on
the HDL concentration, the bias ranged between 23% and 115% compared
with HDL determination after ultracentrifugation, even though
triglycerides were far below 10 000 mg/L and therefore well within the
reportedly acceptable range for the homogeneous assay. There was no
clearcut correlation between the relative or absolute bias and the
triglyceride or VLDL-cholesterol concentration.
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To further assess this issue, VLDL isolated from patient III was added
to serum from a fasting, healthy donor. The VLDL fraction contained
14 680 mg/L triglycerides and 10 200 mg/L cholesterol. This
composition is typical for ß-VLDL. The serum from the healthy donor
contained 1850 mg/L cholesterol, 1130 mg/L triglycerides, and 550 mg/L
HDL-cholesterol. Fifty microliters of undiluted ß-VLDL, ß-VLDL
diluted with 9 g/L NaCl, or 9 g/L NaCl were added to 100 µL of serum
from a healthy donor. The addition of 50 µL of undiluted ß-VLDL led
to approximately the same ß-VLDL concentration in the sample as in
the serum of patient III, whereas addition of 9 g/L NaCl gave final
concentrations of 375 mg/L HDL-cholesterol and 690 mg/L triglycerides
in the sample. HDL-cholesterol in the supplemented samples was
determined by the homogeneous method. The results of this experiment
are shown in Fig. 1
, which also shows the final triglyceride concentrations of
those samples. All concentrations represent duplicate determinations,
with the difference between the single results being 
5% in all
cases. At triglyceride concentrations of ~3000 mg/L, the bias for
HDL-cholesterol was close to 10%. At the highest concentration of
ß-VLDL, the triglycerides were 5710 mg/L and HDL-cholesterol was
overestimated by >20%. The same experiment was performed with VLDL
isolated from a patient with hypertriglyceridemia. This preparation
contained 11 350 mg/L triglycerides and 2650 mg/L cholesterol. The
maximal triglyceride concentration after the addition of ß-VLDL to
serum from a healthy donor as described above was 4600 mg/L. The
HDL-cholesterol was determined to be 15 mg/L higher than in the sample
to which only NaCl had been added, which is within the imprecision of
the method. Overall, no systematic effect on the homogeneous HDL
determination was observed in the range analyzed. These experiments
confirm that VLDL from type III sera causes a positive bias when
HDL-cholesterol is measured by the homogeneous method.
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In summary, our results show that the presence of ß-VLDL, as is typically seen in patients with type III hyperlipoproteinemia, interferes with the newly developed homogeneous HDL-cholesterol determination based on PEG-modified enzymes. In these patients, HDL-cholesterol will be overestimated at triglyceride concentrations far below 10 000 mg/L, which is within the range at which no triglyceride interference would be expected according to published data (7)(9). The simplest explanation would be that the cholesterol associated with ß-VLDL might be a substrate for these enzymes, although it reacts much less than HDL-cholesterol. However, because the concentration of ß-VLDL, and thus ß-VLDL-cholesterol, in type III patients is high, this leads to a positive bias when HDL-cholesterol is determined from whole serum. This finding could have further implications if one assumes that whole serum samples from other patients with abnormal VLDL or remnant particles, for example, diabetic patients, may cause similar problems in the homogeneous assay. To date, this has not been specifically investigated.
Acknowledgments
We gratefully acknowledge the expert technical assistance of Dieter Nutz, who brought this phenomenon to our attention.
Footnotes
Institute for Clinical Chemistry and Laboratory Medicine, University Hospital of Regensburg, D-93042 Regensburg, Germany
fax 49 941 944 6202, e-mail karl.lackner{at}klinik.uni-regensburg.de
References
The following articles in journals at HighWire Press have cited this article:
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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. 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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M. Esteban-Salan, A. Guimon-Bardesi, J. M. de la Viuda-Unzueta, M. N. Azcarate-Ania, P. Pascual-Usandizaga, and E. Amoroto-Del-Rio Analytical and Clinical Evaluation of Two Homogeneous Assays for LDL-Cholesterol in Hyperlipidemic Patients Clin. Chem., August 1, 2000; 46(8): 1121 - 1131. [Abstract] [Full Text] [PDF]
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J. C. Escola-Gil, O. Jorba, J. Julve-Gil, F. Gonzalez-Sastre, J. Ordonez-Llanos, and F. Blanco-Vaca Pitfalls of Direct HDL-Cholesterol Measurements in Mouse Models of Hyperlipidemia and Atherosclerosis Clin. Chem., September 1, 1999; 45(9): 1567 - 1569. [Full Text] [PDF]
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HDL-Cholesterol, the difference between serum
to which ß-VLDL was added and serum to which 50 µL of 9 g/L NaCl
solution was added; % ß-VLDL, the final concentration of
ß-VLDL in the serum to which ß-VLDL was added. The triglyceride
(Trig) concentrations of the serum samples to which ß-VLDL
was added are also indicated.