BACKGROUND OF THE INVENTION
The field of the present invention is an analytical procedure for quantitatively determining the amount of cholesterol in a sample to be analyzed.
Cholesterol, either in the free form or as an ester, is an essential constituent of all animal and plant cells, and is often accompanied by its derivatives, dihydrocholesterol and 7-dihydrocholesterol. Blood cholesterol, both esterified and free, is one of the major blood lipids and is present together with total fatty acids, triglycerides, and phosphatides. Cholesterol in the blood is distributed almost equally between the plasma and corpuscles, with higher concentrations in the leukocytes. Human blood plasma normally contains 100 to 320 mg. of total cholesterol, with a mean of 160 mg. per 100 cc. of plasma which is usually so distributed that about one-fourth is free cholesterol and the remaining three-fourths is the cholesterol ester of fatty acids. Since the sum of the free cholesterol and the cholesterol esters in the serum is usually referred to as total cholesterol, for convenience, this term will be so applied hereinafter.
Every human being maintains a constant total serum cholesterol level and large deviations from this level do not ordinarily occur. However, research directed to the effects of abnormally high total cholesterol blood levels has made it increasingly apparent that the total cholesterol blood level should be controlled and should be maintained within the limits considered normal for the particular individual. The total cholesterol level in the blood plasma tends to be high, for example, in hyperglycemia and in uncontrolled diabetes. Cholesterol has also been found to be the principal lipid in the intima of the arterial wall in atherosclerosis. Plasma cholesterol levels are also high in icterus, naphritis, alcoholism, anesthesia, pregnancy, syphilis, and following excision of the adrenals. Total serum cholesterol is also elevated in diseases of the liver associated with obstruction of bile flow, in familial hypercholesterolemia, in hypothyroidism and in the nephrotic syndrome. Low blood cholesterol is found in severe liver disease, wasting illness, malnutrition, and hyperthyroidism. Accordingly, the accurate determination of serum cholesterol levels is important for modern diagnostic procedures.
Numerous reagents have been employed in methods for the determination of total cholesterol in biological fluids such as serum or plasma or the like. In all of these methods, the amount of total cholesterol in a sample of a biological fluid is normally determined by contacting the biological fluid with a reagent which combines with cholesterol in the fluid to form a colored reaction product. The reagent is mixed with the fluid in such a way that the depth or intensity of color produced is proportional to the amount of cholesterol present in the sample of biological fluid. The concentration of cholesterol in the sample is then determined by measuring the depth or intensity of color, usually with a colorimeter or a spectrophotometer. By use of conversion charts or comparisons to standard solutions, the measurement of the color produced by the use of the reagent can be converted to give the concentration of total cholesterol in the sample.
Among the most widely accepted methods typical of the prior art determinations is the method of Abell et al., J. Biol. Chem., 195, 357 (1952). In the method of Abell et al., the serum sample is first treated with alcoholic potassium hydroxide to liberate cholesterol from lipoprotein complexes and saponify cholesterol esters. The sample is then extracted with a predetermined volume of petroleum ether. The ether extract is evaporated and the residue is then treated with a reagent comprising sulfuric acid, glacial acetic acid and acetic anhydride to form a colored product. The intensity of color produced is then measured in a colorimeter or spectrophotometer to give a value which is proportional to the concentration of cholesterol in the sample.
The saponification and extraction steps make this procedure lengthy and time-consuming. Direct methods employing the same reagent have reduced the time required for analysis, but have necessitated a correction of the results obtained to account for interference caused by the presence of bilirubin in the sample. The correction depends on the amount of bilirubin present in the sample. Also, the reagent employed is not stable in storage; thus, frequent preparation of fresh reagent has been necessary.
Various direct methods have employed a reagent comprising p-toluenesulfonic acid, acetic anhydride, glacial acetic acid and sulfuric acid. See, for example, Van Boetzelaar and Zondag, Clin. Chim. Acta, 5,943 (1960). The methods employing p-toluenesulfonic acid have required corrections for bilirubin. Other reagents have been employed, including a mixture of ferric chloride, sulfuric acid and glacial acetic acid as described by Zlatkis et al., J. Lab. Clin. Med., 41, 486 (1953) and Furst et al., Scand J. Clin. Lab. Invest., 6, 60 (1954).
A significant drawback associated with prior art direct measurements utilizing glacial acetic acid, acetic anhydride and sulfuric acid is that the water used to dilute a serum sample interferes with the analysis. In connection with the foregoing, it should be noted that automated blood analyzers normally analyze blood serum that has been diluted with water before the analysis.
SUMMARY OF THE INVENTION
In accordance with the present invention, water and a reagent including glacial acetic acid, acetic anhydride and sulfuric acid is reacted with serum containing cholesterol to determine the amount of cholesterol present therein. In order to prevent interference and produce a complex which can be measured at 500 nm, the ratio of serum to water to reagent is maintained within the range of one unit volume of serum to 15-24 of water to 200 of reagent.
Accordingly, it is an object of the present invention to provide a new and improved method for direct determinations of the amount of cholesterol in blood serum.
A further object of the present invention is to provide a method for directly measuring the amount of cholesterol in blood serum utilizing a reagent which includes glacial acetic acid, acetic anhydride and sulfuric acid in which interfering reactions are eliminated.
A further object of the present invention is to provide a method for determining the amount of cholesterol present in blood serum which has been diluted with water in which the water is a reacting material which contributes to the development of a chromogen.
A further object of the present invention is to provide a new and improved method for the measurement of cholesterol which can be utilized in any automated system in which a dilution of a sample with water occurs prior to the analysis.
BRIEF DESCRIPTION OF THE DRAWING
The sole FIGURE of the drawing is a diagram illustrating the operating parameters of the process of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
At the outset, the process of the present invention is described in its broadest overall aspects with a more detailed description following. In the method of the present invention, cholesterol is treated with water and a reagent including glacial acetic acid, acetic anhydride and sulfuric acid to form colored substances. Most probably, the colored substances are composed of bis-cholestadiene disulfonic acids. The colored substances have a pink color, are proportional to cholesterol concentration and are measured at 500 nm.
One important factor that enables the formation of the pink colored substances is the amount of water that is present in the system. In accordance with the present invention, for each unit of volume of serum to by analyzed, 15-24 unit volumes of water to 200 unit volumes of reagent are utilized. The optimum volume ratio of serum to water to reagent is 1 to 19 to 200.
The method of the present invention can be performed manually. The determinations of appropriate parameters such as volume measurements, timing and temperature controls are well within the skill of those in this art. The preferred embodiment of the present invention, however, is performed in an automatic blood analyzer such as the type manufactured by Damon Corporation, Needham Heights, Massachusetts, under the name AcuChem Microanalyzer. Details of this type of automatic blood analyzer are set forth in U.S. application Ser. No. 191,884 entitled "Constituents-Measuring Chemical Analyzer having Sample-Processing Conduit Feeding Aliquot-Processing Conveyor System" by David I. Kosowsky, Andres Ferrari and Carl R. Hurtig, filed Oct. 22, 1971, the teachings of which are incorporated herein by reference.
The analytical process of this type of blood analyzer is illustrated by the operating parameter diagram set forth in the sole FIGURE of the drawing. The parameter diagram shows a circular layout of paired circles which forms a probe position plan for a given method of analysis. Each pair of circles represents the one or two positions in each segment of a probe support plate available for each method of analysis on the AcuChem Microanalyzer. On the plate itself, corresponding positions in each succeeding segment are reserved for that same method. The parameter diagram shows the function assigned for the methods as the test tubes pass under the probe support plate. In one important embodiment of the invention, prior to running an analysis with the AcuChem Microanalyzer, the instrument is calibrated with calibrating fluids. The calibrating fluids contain a known amount of the constituent of blood serum for which an analysis is sought. For example, when cholesterol is to be analyzed, the AcuChem instrument is calibrated by placing a known amount of cholesterol in the sample and analyzing that sample. The preferred method of calibrating the AcuChem Micro-analyzer is to utilize several samples of known constituents. Typically, the machine is calibrated with constituents of blood serum in three ranges. The ranges are selected on the basis of a low amount, a mid-range amount and a high amount of the particular constituent as compared to that amount which would be present in normal blood serum. At this point, it should be noted that the AcuChem instrument is capable of performing an analysis for more than one constituent of blood. Thus, the calibrating fluids contain more than one blood serum constituent. In effect, the calibrating fluids are prepared in the laboratory and contain all those constituents normally present in blood serum which are to be analyzed in the AcuChem Microanalyzer.
The AcuChem Microanalyzer will process samples at a rate of 60 per hour. All splits from a single sample, having been diluted 1:20, are simultaneously introduced through probes in segment No. 1 (see the sole FIGURE of the drawing) of the probe support plate into test tubes in the test tube plate. Each segment of the test tube plate, containing diluted serum portions from one patient, passes under the probe support plate at the rate of one segment per minute. Thus, the various functions of reagent dispensing, mixing, transfer to photometer, washing and drying are carried out in turn according to the probe position plan. The instrument design allows nine segments between sample addition and transfer to photometer; hence, a 10 minute dwell time. Thus, nine segments are available for carrying out the required steps in a particular chemistry. After the reacted sample has been transferred to the photometer and while the test tubes are still under segment No. 11, wash water is added. This is then aspirated and functions as a flow cell wash. Additional washing and drying of the test tubes is carried out as they pass under segments No. 12 - No. 24.
The process of the present invention is further illustrated by the following non-limiting examples.
Automatic Determination of Cholesterol
A sample of blood serum having been diluted with deionized water to yield a water-to-serum ratio of 20 to 1 is introduced into segment No. 1 of an AcuChem Microanalyzer. At segment No. 2, cholesterol color reagent is added to the sample. At segment No. 3, the reagent and sample are mixed. At segment No. 11, the absorbance of the sample at 500 nm is measured by a photometer. The cholesterol color reagent added at segment No. 2 is prepared as follows:
To make 1,000 ml. of cholesterol color reagent, 483 ml. of acetic anhydride (Baker No. 0018) is added to 276 ml. of glacial acetic acid (aldehyde-free, Baker No. 9511) with continuous mixing and cooling. To this mixture (while mixing and cooling continue), 241 ml. of concentrated sulfuric acid (H2 SO4, Fisher No. A-300C) is added slowly so as not to allow the temperature to rise above 30°C. The reagent is stored in amber glass or polyethylene bottle.
Various operating parameters for this analysis are given below.
______________________________________ Wavelength = 500 nm Rate = 60/hr Path length = 50 mm Incubation temp. = ambient Sample or Reagent Vol. (μl) Segment 1. Sample 100 Segment 2. Cholesterol 1000 color reagent ______________________________________
As is set forth above, the method of the present invention can be performed manually. Below is a non-limiting example illustrating a manual method for performing an analysis in accordance with the present invention.
Cholesterol--Nonautomated (Manual) Method
1. Using deionized water as the diluent, three 1:20 dilutions of calibration fluids containing known amounts of cholesterol in a low, mid and high range are prepared.
2. 1:20 dilutions of the unknown serums using deionized water as the diluent are prepared.
3. To 0.20 ml. of deionized water, 2.0 ml. of the cholesterol color reagent as prepared in Example 1 is added and mixed well. This is the reagent blank.
4. To 0.20 ml. of diluted unknowns or calibration fluids, 2.0 ml. of the cholesterol color reagent is added and mixed well.
5. 8 to 12 minutes after mixing the water and cholesterol reagent, the mixture is poured into a 10 mm light path cuvet and a photometer is adjusted (set at 500 nm) to zero.
6. Eight to twelve minutes after mixing diluted unknowns or calibration fluids and cholesterol reagent, the mixture is poured into a 10 mm light path cuvet and the absorbance is read.
7. With good quality graph paper, absorbances of the reacted calibration fluids is plotted on the ordinate versus their cholesterol concentrations on the abcissa.
8. Referring to the calibration curve, concentrations of unknowns is read off directly in mg/dl cholesterol.
Note: Unknowns and calibration fluids should be assayed in duplicate and the mean of the duplicates should be used for final preparation and use of the calibration curve.
Accordingly, by following the teachings of the present invention, a new and improved method for the quantitative analysis of cholesterol results. By controlling the serum to water to reagent ratio in accordance with the present invention, it is possible to measure cholesterol concentration directly without making any interference adjustments. In the prior art methods, when a cholesterol color reagent which included glacial acetic acid, acetic anhydride and sulfuric acid was utilized, interference adjustments were necessary. However, in accordance with the present invention, water in the proper proportion contributes toward the development of the chromogen and there is no significant interference. Furthermore, the process of the present invention produces a chromogen which is different from the prior art chromogen which occur with the same reagent, i.e., glacial acetic acid, acetic anhydride and sulfuric acid. In this regard, the process of the present invention is broadly directed to maintaining a proper ratio of serum to water to reagent to produce a pink substance which absorbs light in wavebands centered at 500 nm. Prior art processes utilizing glacial acetic acid, acetic anhydride and sulfuric acid as a color reagent produced a blue-green substance which absorbed light with wavebands centered between the range of 600 - 650 nm.
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.