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A Summation
Technic
for Serum Total Lipids
Comparison of Methods
Chandler
S. Cheek and Dorothy F. Wease
An efficient summation technic for the quantitation of total lipid from the results
of the cholesterol, phospholipid,and triglycerides determinations is compared to the
phenol turbidity and gravimetric method. Also considered is the total lipid value
calculated from the cholesteroland triglyceride concentrations.
THE
AVAILABLE
SEMIAUTOMATED
METHODS
for the precise
and
accurate
quantitation
of serum cholesterol,
phospholipids,
and glycerides
have
vastly
reduced
the labor,
laboratory
equipment,
space,
and typical
errors attendant
to performing
these studies. Still needed, however,
is a
procedure
for total lipid assay which retains
the analytic
integrity
of
the gravimetric
method
and, at the same time, can be conveniently
applied
to quantitating
a large number
of serum samples.
One solution
to this problem
is to perform
the cholesterol,
phospholipid, and triglyceride
assays,
and then calculate
the total lipid concentration,
from the values of the lipid fractions.
The purpose
of this paper, then, is to evaluate
this summation
method as a useful tool in providing
accurate
and precise
total lipid values
for clinical use.
Methods
Subject Preparation
Total cholesterol
(C), free cholesterol
(FC),
esterified
cholesterol
(EC), phospholipid
(P), triglyceride
(T), and total lipid determinations
were performed
on serum samples
from 328 adult males. The subjects
were outpatients
who arrived
1 or 2 days before
the samples
were
collected.
Prior to the patient’s
visit, he received
a letter
of instructions which directed
him to eat food ad libitum during the 3 days prior
to his appointment
at the laboratory.
Specifically,
he was directed
to
include a minimum
of 2 oz. of meat, 3 oz. of potatoes,
1-2 cups of coffee
From the Clinical Pathology
Branch,
U. S. Air Force School of Aerospace
Medicine
Brooks Air Force Base, Tex. 78235.
Received
for publication
Jan. 25, 1968; accepted
for publication
May 20, 1968.
102
(APSe),
Vol. 15, No. 2, 1969
103
SERUM TOTAL LIPIDS
with sugar,
3-5 candy
bars, and (i slices of bread
daily.
Alcoholic
beverages
were prohibited
during
this period.
Furthermore,
the subject was directed
to take nothing but water by mouth after the midnight
prior to reporting
to the laboratory.
Laboratory Analyses
Fasting
blood samples
were obtained
by venipuncture
between
0730
and 0800 hours (7:30 and 8:00 A.M.) on the day the subjects
reported
to the laboratory.
Blood samples
were permitted
to clot, and the serum
was promptly
separated.
The extraction
method
is essentially
that of Folch as modified
by
Sperry
(1). Lipids were extracted
from 1 ml. of serum with a chloroform-methanol
mixture,
purified
of nonlipid
material
by washing
with
distilled
water, and dried. The dried extract
was redissolved
in 3 ml.
of petroleum
ether and quantitatively
transferred
through
a Buchner
funnel
into a tared beaker.
The petroleum
ether was evaporated
at
room temperature
by passing
air over the top of the beaker. The beaker
containing
the dried extract
was placed in a desiccator
over indicating
silica gel, and the air in the decissator
was replaced
with nitrogen
to
preclude
possible
oxidation
of the lipids which would effect errors
in
the cholesterol
and phospholipid
determinations
performed
on the
extract.
The desiccator
was covered
to protect
the contents
from light and
was allowed to remain
undisturbed
overnight.
The beakers
were carefully removed
and weighed
to obtain the total lipid value. After weighing, the extract
was redissolved
in petroleum
ether. A 5-mi. aliquot
of
the petroleum
ether was removed
for the total cholesterol
determination,
and 20 ml. was pipetted
onto a prepared
column of silicic acid from
which cholesterol
esters, nonesterified
cholesterol,
glycerides,
and phospholipids
were separately
eluted
and quantitated
spectrophotometrically. The analytic
methods
employed
previously
have been described
by Wease
(2).
In addition,
207 of the 328 samples
were analyzed
for
total lipid content by the phenol turbidity
method
of Kunkel
et ci (3).
The total lipid concentration
was also determined
by calculation
for
each of the 328 serum samples
from the chemically
determined
concentrations
of the cholesterol,
phospholipid,
and triglyceride
concentrations.
The formula
used was :
Total
The
formula
lipid
=
0.73’(l.69)
assumes
1n all equations,
total lipid,
in milligrams
per 100 mL
C
+ 0.27 c+
the total
cholesterol,
P + T=
cholesterol
phospholipids,
+
1.5037 C
content
and
P+
T
l)
to represent
triglycerides
will
be
73%
expressed
104
CHEEK
& WEASE
Clinical
Chemiafry
esterified
cholesterol,
27% free cholesterol,
and tile molecular
weight
of the esterified
cholesterol
to be 1.69 times the molecular
weight
of
the free form (4). The concentrations
of phospholipid
and triglyceride
were added to account for the remaining
major lipid fractions.
Results
The mean percent
differences
of the phenol method
data from
gravimetric
method data and of the calculated
method results froni
gravimetric
method values are graphically
illustrated
in Fig. 1.
8I
PHENOL
the
the
METHOD
Ui
±2 S.D.
I-
Fig.
Ui
20
PIICCS
CD
1. Mean
of phenol
percent
method
differfrom
gravitnetric
method
and
the
limits imposed
by 2 S.D. of the
mean ((op curves)
is compared
0
‘I(I)
to the mean of the differences
of
calculated
method
from
gravinietric
nietliod
± 2 S.D.
10
(beer
ii
CALCJ.A.ATED METHOD
durres).
±2 S.D.
J
C
501-600
601-700
701-800
801-900
m9/100
ml Total Lipid
C
30
60
NUMBER
I
I
47
25
901-1000
25
OF SUBJECTS
The range,
mean,
and standard
deviation
of tile gravimetric
total
lipid, calculated
total lipid, cholesterol,
phospholipid,
and triglyceride
values are listed in Table 1.
The mean percent
recovery
of the sum of the four fractions
from the
gravimetric
total lipid value, calculated
by:
FC+(l.69XEC)+T+P
(2)
gravimetrie totallipid
was 97.3% and ranged
from
The mean percent
recovery
92 to 102%.
of the cholesterol
FC + EC
(hefore
was 96% with
a range
fractionation)
of 93 to 100%.
fractions
(3)
Vol. 15, No. 2
1969
SERUM TOTAL
Table
1.
Sample
SYNOPSIS
OF LIPID
D.&TA
Range
(mg./lOO
ml.)
No.
Total lipid < 700
Gravimetric
Calculated
Total lipid > 700
Gravimetric
Calculated
All total lipids
Gravinietric
Calculated
Lipid fractions
Cholesterol
Phospholipid
Triglyceride
105
LIPIDS
Mean
(mg./100
ml.)
S.D.
(mg/ZOO
-
599.7
--
603. 1
70.32
75.29
-
833.4
834.3
136.28
133.03
328
328
371-1750
372-1750
709.4
711.7
106.3
106.2
328
328
328
112-369
130-382
16-846
225.6
238.8
131.3
174
174
154
154
-
ml.)
45.03
43.5
79.7
Discussion
The wide disagreement
of the phenol
turbidity
method
and the
gravimetric
method
for total lipid (Fig. 1) was sufficient
justification
for the authors
to discard
the idea of using this easily performed
total
lipid test as a substitute
for the gravimetric
method.
While the mean
percent
difference
of the values
of tile calculated
method
from the
gravimetric
method
was always
less than 5.0%, the differences
of the
phenol method
values from the gravimetric
was always
greater
than
10.0%.
Calculated Total Lipid
The mean differences
of the calculated
total lipid value (Equation
2)
from the gravimetric
total lipids for each set (gravimetric
total lipid
<700 and
700) were not significantly
different
from zero (p <0.05).
The variance
of tile differences
of the gravimetric
lipid values for the set greater
than 700 mg./100
significantly
greater
than the variance
for the set
less than 700 mg./100 ml. Thus, it appears
that the
varies more from tile gravimetric
total lipid for
values.
While the formula:
Total
lipid
=
1.5037
and calculated
total
ml. was found to be
of total lipid values
calculated
total lipid
the larger
total lipid
C + P + 1’
(4)
is the reasonable
calculation
of the total lipid value from the three
known lipid fractions,
a least-square
prediction
equation
fitted to the
gravimetrically
determined
total lipids was found to be:
Total
lipid
=
74.5
+ 1.53 C + 0.607
P + 1.13 T
(5)
106
CHEEK & WEASE
Clinical
Chemistry
The relationship
in Equation
4 was considered
as a standard,
and the
relationship
in Equation
5 was tested
to determine
if the constant
(74.5) was different
from zero, if the coefficient
of cholesterol
was
different
from 1.5037, and if the coefficients
of phospholipid
and triglyceride
were different
from one. The coefficient
of cholesterol
was
found not to be significantly
different
from 1.5037 at the 0.05 significance level, but the other three were significantly
different
from their
hypothetical
values
(p < 0.01). The coefficients
of cholesterol,
phospholipid,
and triglyceride
levels were also found
to be significantly
different
from zero (p < 0.01). This implies
that using all three variables to predict
the total lipid value does significantly
better
than
using any two of the variables
and omitting
one of the three fractions.
The linear correlation
coefficient
between
the gravimetric
total lipid
values and the data obtained
through
the use of Equation
2 was 0.971.
The correlation
between
the gravimetric
values
and results
from
Equation
3 was 0.975. The best estimation
for these gravimetric
total
lipid data by Equation
3 is not an appreciably
better estimation
than
Equation
2; there is insufficient
justification
to use Equation
3 rather
than the theoretically
preferred
Equation
2.
The relatively
difficult total phosphorus
determination
prompted
the
authors
to investigate
an alternative
approach,
which would be to
perform
only the cholesterol
and triglyceride
determinations
and
calculate
the phospholipid
concentration
from the cholesterol
value.
The validity
of this alternate
approach
was evaluated
by testing
the
formula
for phospholipid
as described
by Man et al. (5):
P =90.5+0.735
A least-squares
the cholesterol
fit of the chemically
content was
P
=
C
determined
77.5 + 0.715
phospholipid
level
to
C
Assuming
Equation
6 to be a standard,
the constant
and coefficient
of
the cholesterol
level in Equation
7 are not significantly
different
from
the standard
values.
Since no significant
differences
were detected
in
the tests assuming
Equation
6 to be a standard,
no differences
would be
detected
in comparing
Equations
6 and 7 if an estimate
of error were
available
for the coefficients
in Equation
6 and if Equation
6 were not
considered
as a standard.
However,
the use of either
one of these
substitutions
for the phospholipid
value in Equations
2 or 3 for total
lipid would result in a significant
decrease
in the predictability
of the
total lipid value. This is borne out in the significance
of the difference
from zero of the coefficient of phospholipid
concentration.
Vol. IS, No. 2, 1969
SERUM TOTAL
LIPIDS
107
Conclusion
The calculation
of total lipid from the analytic
results
of the cholesterol, phospholipid,
and triglyceride
values is an efficient and acceptable
technic for clinical use. The calculation
of total lipid from any two of
the three
lipid fractions
results
in a decrease
in predictability
of
total lipid.
References
1.
Sperry, W. M., Lipid
Analysis:
Methods
of Biochemical
Analysis
(Vol. 2), Click, D. Ed.
Interscience,New York, 1955, p. 83.
2. Wease, D. F., A unified approach to the analysis of human serum lipids for clinicalinvesti.
gation. U. S. Air Force School of Aerospace Medicine Publication No. SAM-TR65-45,
1965.
3. Kunkel,
H. C., Ahrens, E. H., and Eisenmenger, W. J., Application of turbidimetric methods for the estimation
of gamma
globulin
and total lipid to the study of patients
with
liverdisease. Gastroenterology
11, 499 (1948).
4. Sperry, W. M., The relationship between total and free cholesterol in human blood serum.
J. Biol. Chem. 114, 125 (1936).
#{182}an,
E. B., Karten, B. C., Durlacher, S. H., and Peters, J. P., The lipids of serum and
liver in patients with hepatic diseases.J. Gun. Invest.
24, 623 (1945).