Beruflich Dokumente
Kultur Dokumente
703-707(1975)
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-
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
0.110
Equipment
0.100
Spectrophotometric measurements were made C
with a double-wavelength spectrophotometer (Model 0.0 90
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-
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
higher cholesterol values result in slightly decreased with that of pure cholesterol standard in Figure 3. Al-
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