Comparison of a new method for the direct and simultaneous assessment of LDL- and HDL-cholesterol with ultracentrifugation and established methods.
BACKGROUND
Automated electrophoresis combined with enzymatic cholesterol staining might improve routine assessment of LDL- and HDL-cholesterol (LDLC and HDLC), as an alternative to the Friedewald equation and precipitation. A new method (Hydrasys; SEBIA) that adapts the cholesterol esterase/cholesterol oxidase reaction within urea-free gels was evaluated.


METHODS
Fresh sera from 725 subjects (512 dyslipidemics) were analyzed by electrophoresis, in parallel with sequential ultracentrifugation, beta-quantification, calculation, and precipitation.


RESULTS
Electrophoresis was linear up to 4 g/L cholesterol, with a detection limit of 0.042 g/L cholesterol/band. Within-run, between-run, between-batch, and between-operator imprecision (CVs) were 1.6%, 2.0%, 1.5%, and 2.7% for LDLC, and 3.9%, 4.3%, 5.5%, and 4.9% for HDLC, and remained unchanged up to 6.3 g/L plasma triglycerides (TGs). Precision decreased with very low HDLC (<0.25 g/L). Serum storage for 3-7 days at +4 or -80 degrees C did not interfere significantly with the assay. Agreement with beta-quantification was stable for LDLC up to 5.07 g/L (r = 0.94), even at TG concentrations >4 g/L (r = 0.91). Bias (2.88% +/- 12%) and total error (7.84%) were unchanged at TG concentrations up to 18.5 g/L. Electrophoresis predicted National Cholesterol Education Program cut-points with <0.04 g/L error, exactly and appropriately classified 79% and 96% of the subjects, and divided by 2.4 (all subjects) and 5.8 (TGs >1.5 g/L) the percentage of subjects underestimated by calculation. One-half of the patients with TGs >4 g/L had LDLC >1.30 g/L. For HDLC, correlation was better with precipitation (r = 0.87) than ultracentrifugation (r = 0.76). Error (-0.10% +/- 26%) increased when HDLC decreased (<0.35 g/L). Direct assessment of the LDLC/HDLC ratio detected 45% more high-risk subjects than the calculation/precipitation combination.


CONCLUSIONS
Electrophoresis provides reliable quantification of LDLC, improving precision, accuracy, and concordance over calculation, particularly with increasing plasma TGs. Implementation of methods to detect low cholesterol concentrations could extend the applications for HDLC assessment.
Benlian
P
P
Department of Biochemistry, Hôpital Saint Antoine, 75012 Paris, France. pascale.benlian@sat.ap-hop-paris.fr
Cansier
C
C
Hennache
G
G
Khallouf
O
O
Bayer
P
P
Duron
F
F
Carrat
F
F
Couderc
R
R
Chazouillères
O
O
Bardet
J
J
Bouchard
P
P
Poupon
R
R
Masliah
J
J
Béréziat
G
G
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001057:Apolipoproteins E / Q000097:blood; D001663:Bilirubin / Q000032:analysis; D011232:Chemical Precipitation; D002648:Child; D002789:Cholesterol Oxidase; D008076:Cholesterol, HDL / Q000097:blood*; D008078:Cholesterol, LDL / Q000097:blood*; D003124:Colorimetry; D004587:Electrophoresis, Agar Gel; D005260:Female; D006454:Hemoglobins / Q000032:analysis; D006495:Heparin, Low-Molecular-Weight / Q000097:blood; D006801:Humans; D017270:Lipoprotein(a) / Q000097:blood; D008297:Male; D008875:Middle Aged; D011446:Prospective Studies; D012680:Sensitivity and Specificity; D002787:Sterol Esterase; D014461:Ultracentrifugation
D001057:Apolipoproteins E; D008076:Cholesterol, HDL; D008078:Cholesterol, LDL; D006454:Hemoglobins; D006495:Heparin, Low-Molecular-Weight; D017270:Lipoprotein(a); D002789:Cholesterol Oxidase; D002787:Sterol Esterase; D001663:Bilirubin