The Ratio of Free to Esterified Cholesterol in Serum
A New Discriminant in Correlating Lipid Metabolism with Disease State
ELEANOR L SAIER, M.S., EGON NORDSTRAND, MARIAM W. JUVES, M.S., AND ROBERT J. HARTSOCK, M.D.
Saier, Eleanor L., Nordstrand, Egon, Juves, Mariam W., and
Hartsock, Robert J.: The ratio of free to esterified cholesterol
in serum. A new discriminant in correlating lipid metabolism
with disease state. Am J Clin Pathol 71: 83-87. A normal
range has been established for the ratio of free to esterified
cholesterol in serum. It has been observed that whereas this
ratio remains constant in clinically healthy subjects and is not
influenced by sex or age, it is markedly altered in certain
disease states. The present study is concerned with the correlation of the normal free:esterified cholesterol ratio with
ratios determined for 148 patients hospitalized for various
reasons. This study has allowed correlations to be made with
respect to various groups of diseases, such as cardiac
disease and cancer. Changes in the free:esterified cholesterol
ratios offer a new and incisive discriminant in correlating
the relationships between cholesterol and various diseases. The
methods used were gas-liquid chromatography for determination of "free" and "total" cholesterol and the new enzymatic
methods for "free" and "total" cholesterol. (Key words:
Cholesterol; Lipid metabolism.)
Department of Laboratory Medicine, William H. Singer
Laboratory of the Allegheny General Hospital, Pittsburgh,
Pennsylvania
Clinical investigation of the causes of these discrepancies led to studies of a possible correlation of the
abnormal free:esterified cholesterol values with cardiac
disease, cancer, and other clinical diseases and conditions. One hundred forty-eight hospitalized patients
who were tested routinely for serum cholesterol
levels were included in this survey.
The use of the ratio between free cholesterol and
esterified cholesterol offers a new and incisive discriminant in correlating the relationships between
cholesterol metabolism and various diseases.
Methods and Materials
STUDIES IN THE FIELD of lipid metabolism have
been concerned with the relationship between either
total cholesterol and free cholesterol or total cholesterol and esterified cholesterol. 5,6
In plotting the normal values of the free cholesterol
versus the total cholesterol, it was found that the intercept was far from zero. However, in plotting the
values of fre$ cholesterol versus esterified cholesterol
the intercept was found to be near zero, thus better
reflecting the physiologic state. It was, therefore,
decided to calculate the ratio of free cholesterol to
esterified cholesterol and to establish a normal range
for this value. This ratio was observed to be very constant in healthy ("normal") individuals regardless of
age or sex. An additional advantage of using the free:
esterified cholesterol ratio is that it remains relatively
constant through the years, even though the absolute
values for free cholesterol and esterified cholesterol
may change.
In working with patient samples, we found some
ratios that were outside our established normal range.
Fasting blood samples were obtained for all determinations. Assays for both free and total cholesterol
were performed by gas-liquid chromatography.
Chemical methodologies, which changed several times
during the period of the study, were also used for the
assay of total cholesterol. They were (1) the manual
method 9 on serum, using the Liebermann-Burchard
reaction; (2) a semiautomated Technicon method 10 on
extracted serum with the Liebermann-Burchard
assay; (3) the completely automated enzymatic
method, 1 using the Abbott ABA-100.*
Each time the chemical methodology for the total
cholesterol determinations was changed, we ran correlation studies with our gas-liquid chromatographic
method (Table 1), which was not altered during the
period of the study.
The gas-liquid chromatographic method used in this
laboratory was developed by one of us (E.N.) and
consists essentially of a methylene chloride-methanol
extraction of serum to which has been added a known
amount of epicoprostanol as the internal standard. 4,7
This extract is assayed directly by gas-liquid chromatography for free cholesterol using a 3% OV 225
Received August 18, 1977; received revised manuscript and
accepted for publication November 30, 1977.
Address reprint requests to Dr. Hartsock: Department of Laboratory Medicine, William H. Singer Laboratory of the Allegheny
General Hospital, Pittsburgh, Pennsylvania 15212.
* Abbott Laboratories, 820 Mission Street, South Pasadena,
Cal. 91030.
0002-9173/79/0100/0083 $00.75 © American Society of Clinical Pathologists
83
84
100-1
SAIER ETAL.
A.J.C.P. • January 1979
NORMAL F/E « 0.371.04
y» 0.35 x* 3.16
TJ
9 80
E
O
(E 6 0
UJ
I(0
UJ
O 40
X
FIG. 1. Relationship of free cholesterol to
esterified cholesterol in the sera of clinically healthy individuals.
o
Ul
g 20
u.
—i—
— I —
20
40
—I—
— I —
—i
120
60
80
100
140
ESTERIFIED CHOLESTEROL mg/dl
—I—
r
column. The coefficient of variation (CV) of this
method for free cholesterol is 0.5%.
Total cholesterol is determined by hydrolyzing with
alkaline ethanol another portion of the same serum
sample, containing the internal standard. This is followed by extraction and assay for total cholesterol on
the same 3% OV 225 column. The CV of this method
for total cholesterol is 1.4%.
Esterified cholesterol is calculated as the difference
between total and free cholesterol.
From a practical standpoint, it was not always possible to do the gas-liquid chromatographic analysis
for cholesterol at the same time the chemical determinations were performed. It was decided, therefore,
to split some of these samples and freeze one portion.
They were stored at 20 C for periods no longer than
six weeks. We found that when the gas-liquid
chromatographic analyses were done immediately after
thawing, the results correlated with the chemical determinations within the experimental error of the determinations shown in Table 1.
- 1 —
160
180
200
Cholesterol determinations by classic chemical
methods have been both tedious and subject to nonspecificity. In the late fall of 1976, an enzymatic
method using the A-GENT cholesterol on the Abbot
ABA-100 for determination of total cholesterol was
introduced in this laboratory.
At the same time, Abbott Diagnostics had available
a small amount of the reagent made without cholesterol
ester hydrolase for the determination of free cholesterol, which they supplied to us.
Correlation studies were performed for free and total
cholesterol by both gas-liquid chromatography and
the enzymatic method. These results are also summarized in Table 1.
Results
Normal Values
A normal range was established for the free:esterified
cholesterol ratio by examining specimens from 38 clini-
Table 1. Correlation of Methodologies
Year
Cholesterol
Number of
Samples
Method I
1970
Total
47
Gas-liquid chromatography
Manual
Liebermann-Burchard assay
0.865
1972-73
Total
49
Gas-liquid chromatography
Technicon
Liebermann-Burchard assay on extracted serum
0.991
1972-73
Total
46
Gas-liquid chromatography
(frozen sera)
Technicon
Liebermann-Burchard assay on extracted serum
0.977
1975
Total
39
Gas-liquid chromatography
(frozen sera)
Technicon
Liebermann-Burchard assay on extracted serum
0.988
1976
Total
23
Gas-liquid chromatography
(frozen asera)
Enzymatic
ABA-100
0.995
1976
Free
24
Gas-liquid chromatography
(frozen sera)
Enzymatic
ABA-100 using A-GENT cholesterol without cholesterol ester hydrolase
0.993
Method II
Correlation
Coefficient8
85
FREE/ESTERIFIED CHOLESTEROL RATIO
Vol. 71 • No. 1
NORMAL MEAN F/E RATIO « 0.37 ± 0 . 0 4
0.90
0.800.70
0.60
FIG. 2. Free:esterified cholesterol ratios for patients with diagnosed atherosclerosis plotted in relation to the normal range.
2 0.50 H
cc
w 0.40
• • •
h-*5
fc
• •
a
s
r-*.
••
•
•
•—•—
0.30-
a
„aa
"
•
s
ft
•"
2S.D.
MEAN
MEM
•
2S.D.
0.200.10I I I I | I I I I 1 I I I I | I I I I | I II I | II I I | I I II | I II I 1 I II
5
10
15
20
25
30
35
40
PATIENT NUMBER
cally healthy individuals. Their ages ranged from 20 to
60 years. Twenty-five were female and 13 male. The values are based on the results of gas-liquid chromatographic
determinations of free and total cholesterol in serum.
The value for esterified cholesterol is calculated as
the difference between total and free cholesterol. The
results obtained for these determinations are in agreement with those reported by others using similar
methodologies.213
The mean and standard deviation8 for total
cholesterol were calculated for this group of healthy
subjects and found to be 195 ± 36 mg/dl. Free cholesterol was found to be 53 ± 10 mg/dl, and calculated
esterified cholesterol, 142 ± 26 mg/dl. Free:esterified
cholesterol ratios were determined for each individual,
Table 2. Values Obtained at Irregular Intervals for
Five Clinically Healthy Subjects
Year
Total
Free
Esterified
Free:
Esterified
Cholesterol
Ratio
S.L.
1970
1973
213
226
57
61
156
165
0.37
0.37
E.S.
1970
1972
1975
203
219
182
82
62
50
151
157
132
0.34
0.39
0.38
E.N.
1970
1972
1976
210
245
214
54
58
62
153
187
152
0.37
0.31
0.41
G.S.
1972
1975
1976
254
226
241
81
62
69
173
164
172
0.46
0.38
0.40
E.C.A.
1970
1972
141
146
40
46
101
100
0.40
0.46
Cholesterol, mg/dl*
Subject
1
Determined by gas-liquid chromatography.
Table 3. Values Obtained During Pregnancy
Patient
Total
Free
Esterified
Free:
Esterified
Cholesterol
Ratio
R.R. (pre-pregnancy)
R.R. (1 month
pregnant)
E.C. (2 months
pregnant)
E.B. (3 months
pregnant)
S.S. (7 months
pregnant)
T. W. (8 months
pregnant)
R.S. (1 day postpartum)
169
43
126
0.26
197
49
148
0.33
163
121
42
0.34
130
96
34
0.35
225
157
68
0.43
204
144
60
0.42
192
142
50
0.35
Cholesterol, mg/dl
and the mean and standard deviation were calculated
for the entire group. The ratio for our healthy subjects was 0.37 ± .04. The range for this ratio at two
standard deviations was 0.29-0.45.
The relationship of free cholesterol to esterified
cholesterol is shown graphically in Fig. 1. The regression equation8 for this line, derived from the normal
data, is y = 0.35x + 3.16.
It was observed that the free:esterified cholesterol
ratio was not appreciably influenced by either sex or
age within our normal population. The possibility of
variation in the ratio over a prolonged period was also
evaluated. Table 2 summarizes the results obtained for
five individuals at different intervals during this
study. While slight changes occurred for individual
cholesterol values over a period of years, it is pertinent
that the free:esterified cholesterol ratios remained
within the normal range.
86
A.J.C.P. • January 1979
SAIER ETAL.
NORMAL MEAN F/E RATIO = 0.37 ± 0.04
1.00
0.900.80
0.70-
0.60
O
|*0.50H
- _D
ii 0.40
MEAN
0.30-
FIG. 3. Free:esterified cholesterol ratios
for patients with diagnosed malignant diseases plotted in relation to the normal
range.
2S.D.
0.200.101 1 1 1 ~rt
1 1 j-rr F T | I 1 1 1 [ 1 1 1 1 1 I 1 1 1 1 |" 111 "
IO
20 •
25
30
35
15
5«
PATIENT NUMBER
Cardiac Disease
The free:esterified cholesterol ratios were determined for 43 patients who had coronary atherosclerosis,
Table 4. Types of Malignant Disease
Represented in Figure 3
Diagnosis
Carcinoma of the lung
Number
of Cases
21
Patient Number
1 , 3 , 5 , 6 , 8 , 9 , 10, 15,20,
24,28,29,31,32,33,34,
39, 42, 44, 47, 48
Glioblastoma
2
2,30
Carcinoma of the breast
3
4, 18, 35
Carcinoma of the kidney
1
7
Carcinoma of the
esophagus
2
11,27
Carcinoma of the cervix
2
12, 19
Basal-cell carcinoma of
the skin
1
36
Carcinoma of the rectum
4
14, 25, 37, 49
Carcinoma of the urinary
bladder with metastases
2
16, 17
Acute myeloblastic
(granulocytic) leukemia
1
21
Ovarian carcinoma
2
22,23
Malignant lymphoma
1
41
Squamous-cell carcinoma
of the larynx
2
38,43
Metastatic carcinoma,
unknown origin
3
26, 40, 46
Carcinoma of the penis
1
45
Adenocystic carcinoma of
the salivary gland
1
13
1 1 | 1 11 1 | 1
45
40
established by clinical studies and coronary angiography. These values are plotted in Figure 2 in relation
to our established normal range for free:esterified
cholesterol ratios. It is apparent that the ratios were
not significantly altered by their disease, and they fall,
for the most part, into the normal pattern. It is also
interesting that of these 43 patients, seven (16%) had
high total cholesterol values, i.e., above 260 mg/dl.
Only one of these patients had a cholesterol value that
reached 508 mg/dl. However, in checking the triglycerides of these patients, 26 (60%) had triglyceride
levels above normal.
Pregnancy
Ratios determined in the serum of six patients in
various months of pregnancy were studied to determine whether these ratios were altered. Elevation of
free cholesterol in urine has been reported to occur
during pregnancy.11 Our results, shown in Table 3, indicate that in serum, free cholesterol values stay within
the normal limits, and that the free:esterified cholesterol ratios remain within the normal range. This observation is in agreement with results of a recent
study12 of lipids in pregnancy.
Malignant Disease
Free:esterified cholesterol ratios were determined
for 49 patients with various malignant neoplasms in
various stages of clinical evolution. These data are
presented graphically in relation to the normal range
in Figure 3. Table 4 lists the various types of malignant
neoplastic disease with which the patients were affected. Analysis of these cases indicates that patients
with elevated values tend to have metastatic disease,
whereas patients with normal values tend to have either
vol. 71 . No. l
FREE/ESTERIFIED CHOLESTEROL RATIO
87
Table 5. Miscellaneous Cases in Which Free:Esterified Cholesterol Ratios Were Abnormally High
Cholesterol, mg/dl
Patient
Total
Free
E.L.
C.W.
J.R.
M.L.
A.C.
G.W.
765
82
192
65
61
102
738
72
111
33
27.4
41
Esterified
Free:Esterified
Cholesterol
Ratio
Diagnosis
27
11
81
32
33
61
27.3
6.6
J.37
1.03
0.82
0.67
Jaundice, diabetes mellitus, cholecystectomy with choledocholithiasis
Viral hepatitis, type B
Alcoholic cirrhosis, jaundice, hepatitis, gastritis, duodenal ulcers
Alcoholic cirrhosis, pancreatic insufficiency, diabetes mellitus
Status post-jejunoileal bypass with electrolyte imbalance
Alcoholic cirrhosis and uremia
localized neoplasms or neoplasms that have been
altered by recent therapy. It should be noted that the
patients with malignant lymphoma, basal cell carcinoma of the skin, and carcinoma of the penis had
normal ratios.
Miscellaneous
During this study, free:esterified cholesterol ratios of
50 patients hospitalized for various reasons were calculated. All these patients had values in the normal
range except six, listed in Table 5. These unusually
high values seem to have been associated with lipid
imbalance, again illustrating the diagnostic importance
of the free:esterified cholesterol ratio.
Discussion
The present study is concerned with the correlation
of free:esterified cholesterol ratios in sera of clinically
healthy individuals with those found for patients hospitalized for various reasons. In this study, the normal
range for this ratio was established as 0.29-0.45. This
indicates that in the clinically healthy individual a
balance between the production of free and esterified
cholesterol is maintained.
Our findings so far indicate that, for the most part,
our patients who had cardiac disease did not have
high values for total cholesterol or free cholesterol,
which is in agreement with results of other studies3,5,6;
when free:esterified cholesterol ratios are calculated
from their data, the resulting values are in agreement
with ours.
The ratios for those patients in various stages of pregnancy were within the normal range. Since pregnancy
is a period of normal controlled growth of a fetus for a
nine-month term, it follows that a normal ratio should
be present, unless an abnormal condition upsets the
balance.
Patients with various neoplasms had free:esterified
cholesterol ratios above the normal range, except for
those patients who had recently had surgical treatment
or irradiation therapy.
We also found high ratios for patients with various
types of hepatic disease (Table 5). A high value was
also found for one patient who had an ileojejunal
bypass.
With the recent development of simple, rapid enzymatic methods for determination of free and esterified cholesterol, the determination of this ratio
becomes readily available to all who are interested.
Our studies indicate that the ratio of free to esterified
cholesterol may be an additional aid in the clinical
evaluation of patients who have various malignant
diseases and diseases of the liver.
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