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 patients 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

SERUM TOTAL LIPIDS

103

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 lipid
=

0.73(l.69)

+ 0.27 c+

P + T=

1.5037 C

P+

l)

The

formula

assumes

the total
cholesterol,

cholesterol
phospholipids,

content
and

to represent
will be

73%
expressed

1n all equations, total lipid, in milligrams per 100 mL

triglycerides

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. the the

8 I
Ui

PHENOL

METHOD

2 S.D.
IUi

20
CD

Fig.
PIICCS

1. Mean of phenol

percent method

differfrom

0
I(I)

gravitnetric method and the limits imposed by 2 S.D. of the mean ((op curves) is compared to the mean of the differences of calculated method from gravinietric nietliod 2 S.D.
(beer durres).

10

ii
C

CALCJ.A.ATED METHOD

2 S.D.

J
501-600 601-700
C

701-800 801-900 m9/100 ml Total Lipid

I I

901-1000

30

60 NUMBER

47 OF SUBJECTS

25

25

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 gravimetrie totallipid (2)

was 97.3% and ranged from The mean percent recovery

92 to 102%. of the cholesterol

FC + EC fractionation)

fractions
(3)

(hefore

was 96% with

a range

of 93 to 100%.

Vol. 15, No. 2

1969

SERUM TOTAL

LIPIDS

105
D.&TA Mean (mg./100 ml.) S.D. (mg/ZOO

Table
Sample

1.

SYNOPSIS

OF LIPID

No.

Range (mg./lOO ml.)

ml.)

Total lipid < 700 Gravimetric Calculated Total lipid > 700 Gravimetric Calculated All total lipids Gravinietric Calculated Lipid fractions Cholesterol Phospholipid Triglyceride

174 174 154 154 328 328 328 328 328

--

599.7
603. 1 833.4 834.3 709.4 711.7 225.6 238.8 131.3

70.32 75.29 136.28 133.03 106.3 106.2 45.03 43.5 79.7

371-1750 372-1750 112-369 130-382 16-846

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
=

and calculated total ml. was found to be of total lipid values calculated total lipid the larger total lipid

1.5037

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 C

A least-squares the cholesterol

fit of the chemically content was

P
=

determined

phospholipid

level

to

77.5 + 0.715

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
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). 1.