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cUN. CiEM. 21/6.

703-707(1975)

Direct Determination of Total Serum Cholesterol

by Use of Double-Wavelength Spectrophotometry

Philip B. Sommers,’ Peter I. Jatlow, and David Seilgson

We describe a simple, accurate method for direct deter- In accord with previously published observations
mination of total cholesterol in serum. Systematic inves- (derived from use of Liebermann-Burchard re-
tigation of a previously described modified Liebermann- agents), we have observed substantial interference
Burchard reagent has Indicated the necessity of ac- from bilirubin, amounting to an apparent increase of
counting for both bilirubin Interference and decreased 4 to 5 mg of cholesterol per deciliter for each mg of
specificity owing to exothermia. Double-wavelength
bilirubin per deciliter. The observed increase in ab-
spectrophotometry was used to optically null out biliru-
sorbance attributable to bilirubin clearly arises from
bin as an interfering factor, whereas adding serum to
the cold reagent increases ‘its specificity for the choles- the reaction of bilirubin with Liebermann-Burchard
terol color reaction. Comparison of 106 cholesterol reagent. We have studied the spectral characteristics
values with those obtained by the procedure of Abell et of this interference and have been able to utilize dou-
al. [J. Bk’!. Chem. 195, 357 (1952)] yIelded a correla- ble-wavelength spectrophotometry to greatly mini-
tion coefficient greater than 0.99; our inter-run coeff I- mize it.
dent of variation for pooled laboratory serum was Double-wavelength spectrophotometry-not to be
1.7%. confused with double-beam spectrophotometry-was
first described by Chance (8). A double-wavelength
Additional Keyphrases: comparison with Abel! method spectrophotometer passes light at two different
billirubin Interference #{149}reagent specificity
wavelengths through a single cuvette and automati-
It is generally accepted that “direct” 2 cholesterol cally measures the difference in absorbance between
methods, while highly desirable in terms of expe- the two preset wavelengths. With this technique it is
dience, tend to lack specificity when compared with possible to null out absorbance caused by an interfer-
procedures involving extraction or purification of ing chromophore if one can determine a pair of wave-
serum cholesterol. Consequently, critical reviews of lengths at which the interfering chromophore has an
cholesterol methodology for accuracy (1-4) recom- equal absorbance. Thus, by measuring the difference
mend use of procedures such as that of Abell et al. (5, in absorbances (absorbance X2 - absorbance X1) it is
6), which involve extraction of cholesterol. We have possible to resolve a two-component system and ob-
investigated the modified Liebermann-Burchard re- tain a measurement that is proportional to the con-
agent described by Huang et al. (7), to determine centration of a single component.
which factors alter the specificity or usefulness of Cholesterol reacts with the Huang et al. (7) reagent
this reagent. to yield an absorbance maximum at 618 nm. We in-
vestigated bilirubin as an interfering component in
Department of Laboratory Medicine, Yale University School of the Liebermann-Burchard reaction and observed
Medicine, New Haven, Conn. 06510. that the absorbance attributable to bilirubin has a
1 National Institutes of Health Postdoctoral Fellow, nearly identical value at 618 and 730 nm. Therefore,
2 Direct addition of serum to reagent without extraction of cho-
lesterol. by using double-wavelength spectrophotometry to
Received Jan. 17, 1975; accepted Jan. 30, 1975. automatically determine delta absorbance (absorb-

CLINICAL CHEMISTRY, Vol. 21, No. 6, 1975 703


ance at 618 - absorbance at 730 nm) a measurement AAx lO

can be obtained that is proportional to cholesterol 401

concentration and is relatively independent of biliru-


bin.
301
Addition of serum (containing water) to the Lie-
bermann-Burchard reagent results in a rapid tem-
perature rise, which does not occur when cholesterol 201

standards (in nonaqueous solvents) are added to the


same reagent. This temperature rise distorts absorp- lOl
tion spectra for serum samples and consequently ele-
vates the observed absorbance at 618 nm. Addition of
sera to reagent at -15 #{176}C prevents this temperature 0 tOO 200 300 400
rise and yields absorption spectra that are identical CHOLESTEROL,mq/dI

in shape to the spectrum for pure cholesterol. Fig. 1. Absorbance dIfference (A618 - A730) as a function of
cholesterol concentration
Materials and Methods Delta absorbance values Obtained15 mmafter addItion of standards to cold
reagent
Reagents
Primary cholesterol standards (J. T. Baker “Ul-
trex” cholesterol) were prepared in absolute ethanol.
All other chemicals were reagent grade and used
0.420
without further purification.
The modified Liebermann-Burchard reagent (7) 0.410

consists of acetic anhydride/glacial acetic acid/con- 0.400 ‘.., A


centrated sulfuric acid (60:30:10 by vol) and 2 g of an-
0.390
hydrous sodium sulfate per deciliter. To prepare 1
liter of reagent, add 300 ml of glacial acetic acid to 0.380

600 ml of acetic anhydride. Gently swirl this solution 0.370


and carefully add 100 ml of concentrated sulfuric 5
0 0.360
acid followed by 20 g of anhydrous sodium sulfate.
Vigorous mixing is required to dissolve the salt. 0.210
B
When stored at 4 #{176}C this reagent is stable for at least
0.200
a month. Storage at room temperature for longer
than one day is not recommended. 0,190

0.110
Equipment
0.100
Spectrophotometric measurements were made C
with a double-wavelength spectrophotometer (Model 0.0 90

156, Perkin-Elmer Corp., Norwalk, Conn. 06856). Al-


ternatively, a conventional spectrophotometer, using 0 tO 20 30 40
MINUTES
two readings at separate wavelengths, can be used
FIg. 2. Absorbance difference (A618 A730) as a function of -
(although less conveniently). time for various concentrations of cholesterol
A. 400 mg/dI cholesterol standard; B, pooled laboratory serum wIth a choles-
Procedure terol concentration of 196 mg/dI; C, 100 mg/dI cholesterol standard
Serum or standard, 100 l, is added directly to a
screw-capped vial containing 3.6 ml of cold (-15 #{176}C)
reagent. Storage of reagent in a conventional freezer
will accomplish this. This solution is vortex-mixed color stability. When solutions are incubated at 30

and then placed in a 30 #{176}C bath for 15 mm. Immedi- #{176}C, the value for delta absorbance becomes constant

ately after incubation, delta absorbance (A618 nm - 15 mm after serum is added and is stable (not greater

A730 nm) is recorded. Standard curves are constructed than 1% decrease) for 5 mm thereafter (Figure 2). It

by plotting delta absorbance vs. cholesterol concen- is also evident that in terms of color stability, those

tration (400, 300, 250, 200, and 100 mg/dl cholesterol using a single-wavelength (narrow band-pass) spec-

standards). trophotometer could reasonably tolerate a 10 mm

delay between the two (618 and 730 nm) readings if

Results they wish to read a batch of specimens at one wave-

Figure 1 shows a typical standard curve, The rela- length (618 nm) and then, after resetting the blank,

tionship of delta A to cholesterol is linear to a con- re-read them at the second wavelength (730 nm).

centration of 400 mg/dl. Color stability is a function Bilirubin. A spectrum obtained after bilirubin was

of cholesterol concentration. Figure 2 shows that added to Liebermann-Burchard reagent is compared

higher cholesterol values result in slightly decreased with that of pure cholesterol standard in Figure 3. Al-

704 CLINICAL CHEMISTRY, Vol. 21, No. 6, 1975


A
0.4
,
0.6

400
I
450 500
I
550
I
600 600
I
700
I
750 600 #{176}
‘ .e
I I I I
nm
350 600 650 TOO 150 800
nm
Fig. 3. Absorption spectra resultIng from the reaction of biliru-
bIn(10 mg/dI) and cholesterol (300 mg/dl) with the Huang et Fig. 5. Effect of reagent temperature upon spectral shape
al.reagent Spectrum A. serum with a cholesterol concentration of 310 mg/dI added to
Upper spectrum Is that of bIlfrubln. Spectra were Obtained 15 mm after addi- room temperature reagent; spectrum B. addition of same serum to -15 #{176}C
tion of blllrubln or cholesterol to cold reagent. Wavelengths noted are 618 reagent; spectrum C, 300 mg/dl cholesterol standard added to -15 #{176}C
re-
(X2)and 730 nm (X1) agent. All spectra recorded at 30 #{176}C,
15 mm after serum or standard was
added

70
bin per deciliter. It should also be noted that in terms
of magnitude, the same cholesterol error is observed
for solutions of bilirubin in chloroform as is seen for
icteric sera.
Temperature effects. Addition of sera to the modi-
fied Liebermann-Burchard reagent at room tempera-
ture results in a rapid temperature rise to about 42
#{176}C.
Figure 5 indicates that this exothermic process
tends to decrease specificity for the cholesterol color
reaction by artifactually elevating the observed ab-
sorbance at 618 nm (spectrum A, Figure 5). However,
if serum is added directly to reagent that has been
0 O IS 20 25 30 35 stored at -15 #{176}C, one will observe an absorption
TOTh1. 8IURUBIN, mq/dI spectrum (spectrum B, Figure 5) that is not distorted
-25
when compared with that of pure cholesterol (spec-
trum C, Figure 5). Our standards (cholesterol in ab-
Fig. 4. Effect of bilirubin on cholesterol values determined by
both sIngle- (upper line) and double- (lower line) wavelength solute ethanol) are anhydrous and thus addition to
spectrophotometry the Liebermann-Burchard reagent does not result in
Delta cholesterol refers to the dIfference between a measured cholesterol a temperature rise, or distortion of spectral shape.
value and that value determined by the reference method (6). All values ob-
tained with lcterlc sera, except those noted as 0 or 0, whIch were mea- Hemoglobin. Although hemoglobin has been con-
sured by using solutions of bllirubinIn chloroform sidered as a possible source of interference in the di-
rect determination of cholesterol (9, 13), we have ob-
served that a hemoglobin concentration as high as
200 mg/dl, which would represent unusually severe
though the bilirubin substantially elevates absorb- hemolysis, results in a cholesterol error of less than
ance values recorded at the cholesterol maximum of 10 mg/dl. The effect of gamma globulin is also negli-
618 nm (X2) it has an almost identical absorbance gible. A concentration of 8 gm/dl (rarely seen) would
value at 730 nm (X1). Consequently, delta A is pro- elevate calculated cholesterol values by less than 5
portional to cholesterol concentration and indepen- mg/dl.
dent of bilirubin. Lipemia. Addition of lipemic sera to the modified
Figure 4 shows a comparison between direct cho- Liebermann-Burchard reagent produces mild tur-
lesterol values as determined on icteric sera by both bidity that, in our experience, always clears upon
double-wavelength and conventional single-wave- standing. However, even if turbidity occurred, dou-
length (618 nm) measurements and values obtained ble-wavelength spectrophotometry would correct for
by the reference method of Abell et al. (6). For a bili- it.
rubin concentration of 20 mg/dl, the conventional Inter-method comparison. Figure 6 is a compari-
single wavelength determination (upper line, Figure son of 106 cholesterol values calculated by use of
4) results in a positive error in cholesterol of about double-wavelength spectrophotometry with those
100 mg/dl, which agrees with previous reports (9-12), values obtained by using the Abell-Kendall method
indicating an error of 4 to 6 mg of cholesterol per de- (5, 6). The calculated correlation coefficient is great-
ciliter for each milligram of bilirubin per deciliter. er than 0.99.
Use of double-wavelength spectrophotometry (lower We are routinely using the proposed method in our
line, Figure 4) reduced the error to about 1 mg of laboratory and have observed the following day-to-
cholesterol per deciliter for each milligram of biliru- day precision for 40 determinations run on pooled

CLINICAL CHEMISTRY, Vol. 21, No. 6, 1975 705


*00 made at 540 nm where, despite greater color stability,
0. 1.009)X) 0.894
interference from bilirubin (Figure 3) and hemoglo-
550
r #{149}
0 996 bin are considerably greater than are seen at 618 nm.
#{163}
300 N 06
Although the original Huang et al. reagent was
250 used at room temperature, Huang et al. (15) later
000 stated that the reagent “is stored in the refrigerator
a
SO
and used without warming.” Our preliminary experi-
ments indicate that the exothermia resulting from di-
ISO
rect addition of serum to room temperature reagent
50
tends to increase measured cholesterol values by
0 50 00 00 200 200 300 350 400
about 4%.
ABELL- KENDALL, g/dI
Use of any Liebermann-Burchard reagent natural-
FIg. 6.Comparison of measured cholesterol values ly raises a question concerning the equivalence of
The proposed method ( vs. that of Abell et at. (6) (X) color development by cholesterol esters, relative to
that for nonesterified cholesterol (16-18). Carr and
Drekter (19), however, clearly showed that esters
laboratory serum: mean = 183 mg/dl, SD = 3.15 mg/ yield more color than nonesterified cholesterol only
dl, CV = 1.7%. when the Liebermann-Burchard reaction is carried
out in chloroform. We have determined the choles-
Discussion terol content of solutions of cholesterol acetate and
In theory, double-wavelength spectrophotometry found that measured values do not differ significant-
should null out one component of a two-component ly from the known concentration of cholesterol. Fur-
system, assuming that one can determine two wave- thermore, the close correspondence between values
lengths at which the interfering component has an by our procedure and by that of Abell et al. (which
equivalent absorbance. Figure 4 indicates that al- measures cholesterol after hydrolysis) indicates that
though we have substantially decreased bilirubin in- measured cholesterol values are not influenced by the
terference, it has not been possible to eliminate it degree of esterification.
completely. This is explained by our observation that In summary, elevated serum bilirubin is the most
the correct wavelength (730 nm) needed to null out serious obstacle to overcome when using Lieber-
the bilirubin chromophore(s) at 618 nm is, to a small mann-Burchard reagent to determine ‘cholesterol
degree, a function of bilirubin concentration. Choice values directly on serum. By use of both double-
of 730 nm as a second wavelength has decreased the wavelength spectrophotometry. to null out bilirubin
cholesterol error caused by bilirubin four- to six-fold. and cold reagent to minimize artifacts caused by
It should be noted that several points drawn about exothermia, it is possible to obtain accurate choles-
the lower line in Figure 4 are actually a reflection of terol values on unextracted serum. Furthermore, it is
precision for the two methods rather than a true bili- possible to use pure cholesterol solutions as primary
rubin effect. The Abell-Kendall procedure, with standards for the proposed method. A secondary
more analytical steps, has a poorer precision than the serum standard is not necessary.
proposed method.
The rapid temperature rise resulting from addition
of serum to room temperature Liebermann-Bur- This work was supported in part by Training Grant 2-TOl-
GM00696-13 from the U.S. Public Health Service.
chard reagent appears to promote color development
by formation of a secondary cholesterol chromophore
that has an absorbance maximum near 670 nm and References
thus artifactually enhances the observed absorbance
1. Tonks, D. B., The estimation of cholesterol in serum: A classifi-
at 618 nm. Addition of serum to cold reagent renders cation and critical review of methods. Clin. Biochem. 1, 12 (1967).
the color reaction more specific, apparently by sup- 2. Morris, T. G., A comparison of methods for the estimation of
pressing color development by this secondary choles- serum cholesterol and values in random samples of populations in
terol chromophore. Addition of cholesterol standards the 55-64 age group. J. Clin. Pathol. 12,518 (1959).
3. Hollinger, N. F., Austin, E., Chandler, D., and Lansing, R. F.,
(in alcohol) to reagent that has been heated (42 #{176}C)
Studies of cholesterol methodology and application to population
so as to mimic the exothermia caused by addition of surveys. Clin. Chem. 5, 458 (1959).
serum, results in the same characteristic distortion of 4. Vanzetti, G., Methods photom#{233}triquesde dosage du cholesterol
the spectrum. This suggests that color development dans le serum. Clin. Chim. Acta 10,389 (1964).
at 670 nm results primarily from the altered reactivi- 5. Abell, L. L., Levy, B. B., Brodie, B. B., and Kendall, F. E., Cho-
lesterol in serum. Stand. Methods Clin. Chem. 2, 26 (1958).
ty of cholesterol with Liebermann-Burchard reagent 6. Abell, L. L., Levy, B. B., Brodie, B. B., and Kendall, F. E., A
at higher temperature, rather than from reaction of a simplified method for the estimation of total cholesterol in serum
noncholesterol component present in serum. and demonstration of its specificity. J. Biol. Chem. 195, 357
Ness et al. (14) also noted improved accuracy with (1952).
7. Huang, T. C., Chen, C. P., Wefler, V., and Raftery, A., A stable
a cold reagent, but were not able to overcome inter- reagent for the Liebermann-Burchard reaction. Anal. Chem. 33,
ference from bilirubin. Their measurements were 1405 (1961).

708 CLINICAL CHEMISTRY, Vol. 21, No. 6, 1975


8. Chance, B., Rapid and sensitive spectrophotometry. III. A dou- a recently reported stable Liebermann-Burchard reagent and its
ble beam apparatus. Rev. Sci. Instrum. 22,634 (1951). use for the direct determination of serum total cholesterol. Clin.
Chim. Acta 10, 229 (1964).
9. Kim, E., and Morris, G., Serum cholesterol assay using a stable
Liebermann-Burchard reagent. Clin. Chem. 15, 1171 (1969). 15. Huang, T. C., Wefler, V., and Raftery, A., A simplified spectro-
photometric method for determination of total and esterified cho-
10. Moline, C., and Barron, E. J., Effect of bilirubin and lipemia lesterol with tomatine. Anal. Chem. 35, 1757 (1963).
on an automated method for serum cholesterol. Clin. Chem. 16,
16. Ireland, J. T., The colorimetric estimation of total cholesterol
521 (1969).
in whole blood, serum, plasma, and other biological material. Bio-
11. VanBoetzelaer, G.L., and Zondag, H. A., A rapid modification chem. J. 35,283 (1941).
of the Pearson reaction for t.otal serum cholesterol. Clin. Chim. 17. Kenny, A. P., The determination of cholesterol by the Lieber-
Acta 5,945 (1960). mann-Burchard reaction. Biochem. J. 52,611(1952).
12. Zurkowski, P., A rapid method for cholesterol determination 18. Kerr, L., and Bauld, W. S., The chromatographic separation of
with a single reagent. Clin. Chem. 10,451 (1964). free and combined plasma cholesterol. Biochem. J. 56,872 (1953).
13. Watson, D., A simple method for the determination of serum 19. Carr, J. J., and Drekter, I. J., A simplified rapid technic for the
cholesterol. Clin. Chim. Acta 5,637 (1960). extraction and determination of serum cholesterol without saponi-
14. Ness, A. T., Pastewka, J. A.,and Peacock, A. C., Evaluation of fication. Clin. Chem. 2,353 (1956).

CLINICAL CHEMISTRY, Vol. 21, No. 6, 1975 707

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