A rapid modification of the Pearson reaction for total serum cholesterol

SHORT COMMUNICATIONS

943

A rapid modification of the Pearson reaction for total serum cholesterol Numerous

methods

for the determination

of serum cholesterol

have been pub-

lished, but most are either time consuming or inaccurate. However, reliable results can be obtained within 5 min by the method described by ABELL’ with ECHOL’S cholesterol reagent*. The composition of this reagent has not been published and it is too expensive for routine use in Europe. This communication describes a method, using a similar reagent which enables a precise and simple determination of total serum cholesterol within IO min or less. Extraction and deproteinisation are omitted, pipetting is limited to one step and the color development period is reduced to a minimum. Thus this seems to us a real improvement for clinical routine. The green Liebermann-Burchard color is produced rapidly after addition

of serum to the reagent ; it remains

constant

during the

following 4 or 5 min (about 5-10 min after addition of serum) and then decreases slowly. The extinction produced (measured in a Beckman DU spectrophotometer at 615 rnp, slit width 0.02 mm, in r-cm cuvettes) using 0.1 ml of serum, follows Beer’s Law strictly up to IOOOmg of cholesterol per IOO ml of serum (optical density D = 0.107 per IOO mg o/0cholesterol). The blank, with water instead of serum added to the reagent, has the same extinction as water. Calibration can be done in various ways, provided that addition of standard solution to the reagent is followed by addition of 0.1 ml of serum in the way described for the test sample. Standard solutions of cholesterol or cholesterylesters in various nonaqueous solvents react only very slowly with the reagent (exept cholesteryl oleate, which reacts promptly), but upon addition of 0.1 ml of serum the temperature rises to about 35” and the reaction starts in the normal way. The optical density of serum, subtracted from the optical density of serum + standard, gives the optical density of the standard. With 0.1 ml of water instead of serum the results obtained are too low. Contamination of the reagent with water is critical in respect to the measured extinction. Serum can be replaced by Dade’s Lab-Trol**, a synthetic serum containing no cholesterol or bilirubin and giving no extinction at 615 m,u with the reagent. As bilirubin

produces a green chromogen

(with absorption

in the 6I5-m,u region)

with the reagent, a correction is necessary. This correction can be established easily for the instrument in use by measuring the extinction of serum before and after addition of a known quantity of pure bilirubin. It was found that with the Beckman DU spectrophotometer I mg bilirubin/Ioo ml gives the same optical density as 5 mg cholesterol/Ioo ml. Thus it is possible to make a correction for icteric sera. For a serum with normal bilirubin content this correction can be neglected. Good agreement was found with other methods 2y 3. Thirty consecutive determinations on Hyland’s Clinical Chemistry Control Serum with various lots of reagent and under normal experimental conditions showed a standard deviation of 2.7%. Normal values were estimated by the described method in 120 donors (60 men and 60 women) of the Red Cross Blood Transfusion Service: male, average 215 mg% * The Standex Standard Laboratories, 305 X. l’romenade, ** Dade Reagents, Inc., Miami, Florida (U.S.A.).

Halletsville, Clin. Chim.

Texas (U.S.i\.).

Acta,

5 (1960)

943-944

SHORT COMMUNICATIONS

944 (150-306 46 mg%.

mg%), Further

S.D. 36 mg%; female, average 203 mg”/o (117-325 details of the method will be published elsewhere4.

mg%),

S.D.

MATERIALS AND METHOD Cholesterol reagent A Dissolve 45 g of $-toluene sulfonic acid (I aq., Merck, for chromatography) in 600 ml of acetic anhydride (reagent grade) and add 400 ml of glacial acetic acid (gg-roo”/o, reagent grade). This mixture must be practically clear and colorless. A new batch of reagent must be checked before use with a serum of known cholesterol content.

The solution

Cholesterol

is stable at room temperature.

reagent B

Mix reagent

A with concentrated

sulfuric

acid (g5-g7%,

reagent

grade) in the

ratio 5 : I. For safety reasons, do not mix more than 35 ml of reagent A and 7 ml of sulfuric acid at a time. Cool the mixture to room temperature. This mixture must be clear and colorless. It is stable for several days or longer when stored in a flasks or in rubber stoppered tubes. Careful handling is recommended when pouring this mixture away after use. Procedure Place 5 ml of reagent B in a test-tube. Blow out strongly 0.1 ml of serum with a MacLean pipette and mix immediately by bubbling air through the mixture with the pipette optical

for at least 5 sec. Rinse the pipette twice with the mixture. Measure the density at 615 rnp (or with a convenient filter). Within 5-10 min maximum

extinction is reached. Read the cholesterol concentration on a graph. A correction is made with icteric samples. A control serum of known cholesterol content (Hyland Clinical Chemistry Control Serum* or any other frozen serum) is analysed every time to check the method. Measurements can be made against water at 100% transmission; the optical density of the reagent blank (5 ml reagent B + 0.1 ml H,O) is zero against water. Clinical Chemical Laboratory, Haarlem (The Netherlands)

St. Elisabeth’s

Hospital,

G. L.

VAN

1 E. ABELL, Am. J. Med. Technol., 25 (1959) 65. 2 T. J. TURNER AND L. EALES, Stand. J. Clin. & Lab. Invest., 9 (1957) 210. s C. H. DICKHOUT AND E. G. M. TH. ASBERG, Ned. Tijdschr. Geneesk., ‘04 (1960) 4 G. L. VAN BOETZELAER AND H. A. ZONDAG, Tijdschr. Med. 4nalysten, 15 (1960)

Received

July

rgth,

BOETZELAER

H. A.

ZONDAG

390. 119 (in English).

rg6o

* Hylancl Laboratories,

Los Angeles, California

(U.S.A.). Clin. Chim. Acta, 5 (x960) 943-914