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concentrations of triglyceride-rich lipoproteins, the insoluble Note: Precision and accuracy in the precipitation procedure can
complexes may either remain suspended in the solution or be monitored by analyzing control materials that are similar to
float to the surface. On the other hand, other plasma proteins patients specimens in analyte and matrix properties. Many of the
materials available commercially do not meet these specifica-
may coprecipitate with the lipoproteins, thereby increasing tions.
the density of the insoluble complex and facilitating sedi-
mentation even in specimens with high triglyceride concen- Prepare control materials by pooling fresh specimens
trations. The extent of protein coprecipitation is a function (plasma with EDTA as anticoagulant or serum) from donors
of the method and conditions of precipitation. Cholesterol or that remaining after laboratory analysis (22). Exclude
remaining in the supernatant solution can be considered to turbid or hypertriglyceridemic specimens so that the total
represent HDL, if sedimentation of LDL/VLDL is complete triglyceride value is less than 1 g/L. The use of two pools, one
and no HDL has precipitated. withHDL cholesterol in the low normal (200-400 mg/L) range
Colorimetric methods involving either strong acid or en- and one in the mid (500 mg/L) range, is recommended. Plasma
zymic reagents have generally been used to quantitate cho- may be treated with thrombin to minimize subsequent fibrin
lesterol (reviewed in 21). Methods may be direct, i.e., applied formation. Transfer aliquots to vials and seal. Quick-freeze
directly to specimens without pretreatment, or may include the contents and store in a freezer with a stable temperature,
a preliminary solvent extraction of the lipids from other in- preferably at or below -60 #{176}C. To allow verification of method
terfering constituents. Because approximately 80% of cho- accuracy, obtain a target value by requesting analysis of ali-
lesterol in HDL from humans is in form of the esters, a pre- quota from an experienced laboratory such as those of the
liminary hydrolysis step may be used to cleave the ester Lipid Research Clinics Program. Alternatively, a commercial
linkage. With enzymic assays hydrolysis is necessary, and may material (Lipid Fraction Control Serum, Hyland Diagnostics,
be accomplished by adding a cholesterol ester hydrolase to the Division of Travenol Laboratories, Inc., Deerfield, IL 60015)
reaction mixture. The 3/3-hydroxy group of free cholesterol is currently available that has an accurate reference value for
is then oxidized by cholesterol oxidase with liberation of hy- HDL cholesterol.
drogen peroxide, which can be coupled to a colorimetric re-
action.
Reagents for Enzymic Cholesterol Analysis
Note: The precipitation procedure described here appears to be
Materials and Methods compatible with any of the available cholesterol assays, provided
their accuracy and reproducibility are adequate. We have analyzed
Precipitation Reagents cholesterol in supernates by using Liebermann-Burchard reagent
according to the Lipid Research Clinics method (9,23); this is both
1. Dextran sulfate. Material (Mr 50 000 5000) with ac- accurate and precise but may not be practical for many clinical
ceptable quality can be obtained from SOCHIBO, SA, Bou- laboratories. We have also used with good results a manual enzymic
logne, France, 92100. Store preferably in a refrigerated des- assay as reported by Cooper et al. (24), which we describe below in
iccator. Prepare a stock solution as follows: Add 2.0 g of dex- detail, because the published procedure may not be available to
tran sulfate to 80 mL of de-ionized water in a beaker, and some readers.
adjust the pH if necessary to 7.0 with dilute HC1. Transfer
quantitatively to a volumetric flask and adjust the final vol- Microbial cholesterol ester hydrolase (EC 3.1.1.13) was
timeto 100 mL to givea concentration of 20 g/L. Store at 4 #{176}C. obtained from Boehringer-Mannheim Biochemicals, India-
A preservative solution containing, per liter, 50 g of NaN3 (J.T. napolis,IN 46250, and microbial cholesterol oxidase (EC
Baker Chemical Co., Phillipsburg, NJ 08865), 1.0 g of chlor- 1.1.3.6) was obtained from Beckman Microbics,Carlsbad,CA
amphenicol, and 0.5 g of gentamicin sulfate (both from Sigma 92005. PIPES buffer (1,4-piperazinediethanesulfonate) was
Chemical Co., St. Louis, MO 63178) can be added to this so- obtained from Research Organics, Cleveland, OH 44125.
lution at the rate of 10 mL/L before final volume adjust- Horseradish peroxidase, Type VI (EC 1.11.1.7), 4-aminoan-
ment. tipyrine, and sodium cholate were purchased from Sigma
2. Stock Mg2 solution. Reagent-grade MgCl2 . 6H20, Chemical Co.; phenol was obtained from J.T.Baker Chemical
which is hydroscopic, should be stored tightly closed or in a Co., and Triton X-100 from Rohm and Haas, Philadelphia,
desiccator to minimize water uptake. Prepare a stock solution PA 1105. The enzyme solutions were stored at 4 #{176}C.
of 1.0 mol/L by dissolving 20.3 g of MgC12 6H2O in 80 mL of
. Prepare the following reagents:
de-ionized water in a beaker; adjust the pH to 7.0 with a dilute 1. PIPES buffer, 50 mmol/L, pH 6.9. Into a 1000-mL glass
NaOH solution. Transfer quantitatively to a 100-mL volu- beaker containing approximately 800 mL of distilled water,
metric flask and adjust the final volume to io#{246} mL. Mg2 add 17.7 g of PIPES buffer and stir for approximately 10 mm
We prepared a suitable secondary standard as follows: Note:Alternatively, 50 L each of the separate dextran sulfate and
MgC12 stock solutions can be added sequentially to the 1.0-mL
Reconstitute Lipid Fraction Control Serum (lot no. 4610 specimens, with thorough mixing after each addition.
YOO2A; Hyland Diagnostics), according to the manufacturers
instructions. Dilute with an equal volume of 60 g/L bovine 4. Allow the tubes to stand at room temperature for 10 mm
serum albumin, Cohn Fraction V (Sigma Chemical Co.), in before sedimenting the insoluble lipoproteins by centrifuga-
0.15 mol/L NaCl solution. Freeze aliquots in sealed vials for tion.
subsequent use as a secondary calibrator for assay of choles- Note: Centrifugation for 30 mm with a refrigerated centrifuge (4
terol in supernates. The reported target value for this pool is #{176}C)
attaining 1500 X g is preferred. However, results by this method
1640 mg/L as determined by the modified Abell-Kendall are nearly the same as those obtained with centrifugation in an
method (29) at the Clinical Chemistry Standardization Sec- unrefrigerated benchtop centrifuge at 1000 X g for 15 mm.
tion of CDC. The dilution yields a value of 820 mg/L, which
5. Remove tubes from the centrifuge and inspect super-
is appropriate for assaying HDL specimens. Another suitable
nates for turbidity. Obtain an aliquot of the clear supernates
secondary calibrator is Standard Reference Material 909, a for cholesterol analysis, or transfer the supernatant solution
human serum pool available from the National Bureau of with a Pasteur pipette to a second labeled vial for later anal-
Standards, Washington, DC 20234. This material has a target ysis.
value for cholesterol assigned by an assay involving isotope
dilution-mass spectrometry, which is a candidate Definitive Note: Any turbidity or cloudiness in the supernates indicates in-
Method for cholesterol (30). Alternatively, an in-house pool complete sedimentation of LDL/VLDL and consequent contami-
nation and overestimation of HDL. This is usually observed in
of human donor or pooled serum or plasma can be prepared
specimens with high triglyceride values.
as described previously (22); dilute with albumin solution (60
g/L) to obtain a total cholesterol value in the 800-1000 mg/L 6. Turbid supernates can be conveniently cleared by one
range. A target value can be established by a suitable reference of the following methods:
method. (a) Without separating the turbid supernate from the
precipitate, add to the separation tube 1.0 mL of 0.15 mol/L
Collection of Specimens NaCl solution and another 100 zL of combined precipitant
Collect blood from the antecubital vein of seated subjects reagent. Mix thoroughly with a vortex-type mixer, then cen-
Table 1. Lipoprotein Precipitation by Dextran Sulfate and Mg2 (DSM) Compared with Heparin and
Mn2 (HM)
Concn, mg/I, in supernate C
ApoB-assoclated
Concn, mg/L, Cholesterol (LRC) cholesterol Precipitate
total plasma, mean SD DIfference ApoA-l, mg/L
Specimens n Cholesterola Triglycerldeb HM DSM (HM - DSM) HM DSM HM DSM
Overall 48 2497 564 1797 1768 493.7 475.8 179d 10.3 0.75 31.2 47.4
Normolipidemic
Males 20 2184387 1132 482 488.8 468.6 20.2 6.6 0.7 21.6 33.0
Females 9 2266 284 800 212 671.7 655.7 16.0 10.5 2.1 25.0 50.2
Hypercholesterolemic 9 3098 268 1472 518 460.7 447.3 13.4 13.0 0.8 38.2 48.2
Hypertriglyceridemic 9 2315 289 3400 1110 324.1 322.7 1.4 2.9 0 3599 458g
Hypercholesterolemic and 4 3510396 56923598 446.2 400.1 46.1 32.1 0 43.5 88.8
hypertriglyceridemic
a Plasma cholesterol range, 1340 to 3820 mg/L. Plasma triglyceride range, 45010 10 750 mg/L. Separations were performed in duplicate. Values are the
means for duplicate determinations, Paired SD 21.3. Students paired f-test 5.09, significant at p < 0.00 1. a Quantitated by radial immunodiffusion assay (11,
12, 36). Quantitated by radial immunodiffusion assay (11, 12, 37).9 ApoA-l not measured in some lipemic specimens because of losses associated with filtration
of turbid supernates.
a LRc. Lipid Research Clinics assay with Liebermann-Burchard reagent (9, 23);enzymic, the enzymic assay of Cooper et al. (24)as described in Procedure:
15 determInations each in separate analytical runs performed over a three-month period. b Determined in low total-cholesterol pooled serum (MQ3).
I
600
-j
I
I
550
500
Co
450
I
0 200 400 600 800 1000
1384 CLINICALCHEMISTRY,Vol.28,No.6,1982
protein precipitation was constant, with mean cholesterol
values between 414 and 416 mg/L. At higher or lower pH, Ii-
Table3.Accuracy ofthe Enzymic Cholesterol
poprotein precipitation was slightly less: mean cholesterol by Primary or Secondary
Assay withCalibration
values were 427 mg/L at pH 6.0,425 mg/L at pH 7.5, and 432 Standards
mg/L at pH 8.0. Virtually all of this pH dependence was ob- Enzymlc cholesterol, a mg/L
served in the three hypertriglyceridemic specimens, however. LRC b Secondary standard
In the normolipidemic and hypercholesterolemic specimens, cholesterol, Primary C LIpid fractiond Frozen
supernatant cholesterol values did not vary significantly over mg/L standard control serum plasma
pH 6.0-8.0. On the basis of these results we recommend ad- Assay materials (each n = 12)
justing the precipitant reagent to neutral pH. AQ6 437 378 414 407
The ionic strength of the precipitant reagent solution in-
MQ3 523 490 538 526
fluenced lipoprotein precipitation. The higher the ionic
strength, the less the tendency for lipoprotein precipitation.
Plasma pool 740 647 708 687
When the dextran sulfate solution was prepared in 0.15 mol/L Mean 567 505 553 543
NaC1 solution rather than water, supernatant cholesterol Dextran sulfate_Mg2+ supernates (n = 26)
values averaged approximately 15 mg/L (3%) higher. There- 488 431 479 468
fore, the solutions should be prepared in high-quality water
to obtain the indicated level of accuracy under these condi- a Enzymic assay as described in FcerA,e. 5Assay by Lipid Research Clinics
tions. Also, reagent-grade MgC12 .6 H20 and dextran sulfate
Method with Liebermann-Burchard reagent (9, 10). Accuracy comparablewith
of high quality should be used. reference method (29). Preciset 500 mg/L; Biodynamics/bmc, Indianapolis,
Lipoprotein separation by some precipitation methods is IN 46250. d HylandDiagnostics,Deerfield,IL 60015,dilutedtwofold as described
reportedly a function of the temperature before and during in Ptvcedure. In-house material of donor plasma dilutedfivefold as described
centrifugation. This does not appear to be the case with the inProcedure.
dextran sulfate-Mg24 method. Values for supernatant cho-
lesterol were not significantly different (6 mg/L) when the Accuracy in cholesterol quantitation is in large measure a
incubation and centrifugation steps were done at either 4 or function of the calibration technique. Table 3 illustrates ac-
23 #{176}C.
These results suggest the incubation step can be con- curacy results for a series of control materials analyzed by the
veniently performed at ambient temperatures, and centrifu- LRC Liebermann-Burchard method and by the enzymic
gation need not be performed in a refrigerated centrifuge. method, calibrated with one primary standard and two types
However, where possible, centrifugation at a consistent tem- of secondary standard. The primary standard contained
perature in a refrigerated centrifuge would be preferable. unesterified cholesterol in water solution with detergent. Use
The stability of the combined precipitant reagent was of the primary standard resulted in values for specimens that
evaluated over a period of four months. Combined solutions were low by approximately 10%, even though rate curves
prepared at weekly intervals and containing preservative as demonstrated the reactions had reached equilibrium during
described in the Materials and Methods section were accu- the 20-mm incubation. Calibration of the enzymic assay with
mulated over this period and stored in a refrigerator at 4 #{176}C. a commercial lyophilized human serum (Lipid Fraction
Each of the solutions was then used to precipitate normoli- Control Serum) or human plasma (in-house) gave results
pidemic, hypercholesterolemic, and hypertriglyceridemic similar to the LRC values. The Lipid Fraction Control Serum
specimens. Supernatant cholesterol results did not exhibit any is commercially available with a target value established by
trends that would indicate deterioration of the solutions over the modified Abell-Kendall reference procedure (29). The
the four-month period. The means of duplicate determina- Lipid Standardization Laboratory of the CDC kindly provided
tions on the three pools were all within 20 mg/L. a reference target value for the in-house pool. In other ex-
Mn2 produces a positive interference with many of the periments, not shown, the reaction rate in this enzymic assay
enzymic cholesterol assays (13) but does not interfere with was considerably slower for the primary standard of choles-
Liebermann-Burchard methods. We performed experiments terol with detergent in water solution than for the two sec-
to discover whether the dextran sulfate and Mg2 reagents ondary calibrators, which had reaction rates similar to those
described here produced interference with either the LRC of dextran sulfate-Mg2' supernates.
Liebermann-Burchard or the enzymic cholesterol assay. These results are consistent with reports of other investi-
Plasma, freed of LDL and VLDL by ultracentrifugation at d gators (25-27). The observed underestimation of specimen
= 1.063 g/L, was dialyzed against 0.15 molfL NaC1. The cholesterol has been attributed, at least in part, to incomplete
combined dextran sulfate-Mg2 precipitant solution was hydrolysis of esterified cholesterol in specimens by cholesterol
added in a volume of 0.1 mL/mL of specimen to give final ester hydrolase in the enzymic reagent. Approximately 70-80%
concentrations of 0.90 g of dextran sulfate and 45 mmol of of the cholesterol in specimens is present in the esterified
Mg24 per liter. A control specimen was prepared by diluting form, which, without hydrolysis, is not a suitable substrate for
the same LDL/VLDL-free plasma with water at 0.1 mL/mL. the cholesterol oxidase. Hence, cholesterol in specimens would
Cholesterol was analyzed in each solution 10 times by both the be underestimated to the extent of incomplete hydrolysis in
enzymic and the LRC assays as described. Cholesterol content relation to a primary standard of free cholesterol, which does
of the HDL solution containing dextran sulfate-Mg2- aver- not require hydrolysis. Also, the alcohol or detergent required
aged 325 and 322 mg/L by the LRC and enzymic assays, re- to solubilize unesterified cholesterol in a primary standard
spectively, while the control solution averaged 326 and 323 may interfere with the enzyme activity (28), producing reac-
mg/L by the two assays. Therefore, the dextran sulfate and tion rates for the primary standards that differ from those for
Mg2 reagents described here appear not to interfere with serum or plasma specimens.
either the LRC Liebermann-Burchard or this enzymic cho- Therefore, calibration with a secondary standard may be
lesterol assay. Dextran sulfate of M 500 000 has been re- more appropriate with the enzymic cholesterol assays. The
ported, however, to interfere with another enzymic cholesterol secondary standard should resemble specimens in the pro-
assay (38). The interference may be due to aggregation and portion of esterified and free cholesterol as well as matrix
partial inhibition of some pancreatic cholesterol ester hy- properties. In addition, a reliable target value is essential for
drolases by dextran sulfate (personal communication from obtaining accurate results with a secondary standard. In some
Garry Handelman). applications, the use of both a primary and a secondary cali-
This approach to calibration, for example, has resulted in Age, PercentIle Percentile
excellent accuracy and precision with the LRC cholesterol yr Mean 5th 95th Mean 5th 95th
a Heparln-Mn24precipitation and modified AbeIl-Kendall (29) cholesterol assay by Clinical Chemistry Standardization Section CDC. bCommercial enzymic
cholesterol assay andcalibratoron MultistatIll Micro Centrifugal Analyzer (Instrumentation Laboratory). C Enzymic cholesterol assay with secondary serum calibrator
(24).
1388 CLINICALCHEMISTRY,Vol.28,No.6.1982