This invention relates to a method for determining the concentration of total lipids in animal fluids by a colorimetric method based upon the use of a phospho-vanillin reagent.
Presently, it is believed that abnormal serum lipid concentrations are associated with diseases of the heart such as atherosclerosis. The serum lipids such as cholesterol, phospho-lipids and triglycerides are present in the blood stream both alone and in combination with proteins in the form of lipoproteins. In current analytical practice, the test for total lipid concentration functions as a screening test in that when the concentration is found to be abnormally high, subsequent tests for specific lipids are performed to determine their concentrations in the serum. A common method for determining total lipid concentration involves reacting a serum sample first with sulfuric acid and then with a phospho-vanillin reagent to form a chromogen, the light absorbance of which is measured. The concentration then is determined in accordance with Beer's law from a standard curve relating lipid concentration as a function of light absorbance of the chromogen.
One procedure for carrying out a test for total lipids based upon the phospho-vanillin reagent is described by one of us in the American Journal of Clinical Pathology, Vol. 53, Pages 89-91 (1970). In this procedure, 2 ml. of concentrated sulfuric acid is mixed with a 0.10 ml. of blood serum and heated for about 10 minutes. A 0.10 ml. aliquot of the mixture then is transferred to a second container and 5.0 ml. of a phospho-vanillin reagent is added thereto, mixed and heated for about 15 minutes at 37.degree. C. The mole ratio of vanillin to phosphoric acid in the phospho-vanillin reagent is less than 1.12 .times. 10.sup.-.sup.3. The light absorbance of the test sample is measured at 540 mu. and the concentration determined from a standard curve. The phospho-vanillin reagent employed is prepared by admixing 800 ml. concentrated phosphoric acid and 200 ml. of a 0.6% (W/V) aqueous vanillin solution.
This procedure as well as other present procedures employing a phospho-vanillin reagent have a number of disadvantages which have limited their use. The prime disadvantage is that these tests require the transfer of a specific volume of the sulfuric acid-serum mixture to a second container prior to adding the phospho-vanillin reagent. This transfer introduces inaccuracies in the test since there will be inaccuracies in the liquid volume measured. The required transfer step also renders their test procedures undesirable for use in automatic testing apparatus wherein serum samples are mixed with reagents and treated on a continuous basis. Furthermore the above-described procedure follows Beer's law only up to a lipid concentration of about 1000 mg/dl and requires a relatively large volume of serum sample of at least 0.10 ml.
It would be highly desirable to provide a test procedure for measuring total lipid concentration in micro quantities of serum which eliminates the use of the present sample transfer procedure since such a procedure would be linear to higher lipid concentrations than present procedures and could be adapted easily for use in automated analysis processes.