Method of measuring cholesterol, diagnosing vascular disease, and raising HDL cholesterol levels

Abstract: A method is provided for measuring the net HDL cholesterol in the blood. The LDL and VLDL moieties are precipitated out and the remaining cholesterol content of the supernatant is measured. The free cholesterol content of the supernatant is separately measured and substracted from the total to obtain net HDL cholesterol. This value serves as a useful indicator for diagnosing vascular disease. A diet supplement and method for raising serum HDL is provided.

Description: This invention relates to the measurement of various forms of cholesterol in the bloodstream of mammals, enabling the diagnosis of vascular disease or atherosclerosis from such measurements. Serum total cholesterol consists of both esterified and unesterified cholesterol fractions bound in varying amounts to several lipoprotein carriers. Approximately 71-75% of the total cholesterol is esterified, i.e. bonded to fatty esters. Unesterified (free) cholesterol, an insoluble, abrasive waxy alcohol, makes up about 25-29% of the total. Chylomicrons, very large particles containing essential lubricating lipids, are produced in the duodenum and jejunum and play a role in the absorption of the essential fatty acids and phospholipids needed to form lipoprotein particles. Chylomicrons transport triglycerides (TG) from the intestines to the liver. Low-density lipoprotein (LDL), the major cholesterol carrier, is a spherical particle consisting of about 50% cholesterol, 20% protein and 25% phospholipids. Very low-density lipoprotein (VLDL), a liver-generated precursor of LDL, is a large particle composed of 15-25% cholesterol and, mainly, triglycerides. LDL and VLDL have been implicated in atherosclerosis. VLDL particles typically have a life span of 4-5 hours, while LDL particles last 2-3 days. High-density lipoprotein (HDL), a small particle containing about 20% cholesterol, 50% protein, and 25% phospholipids, appears to play a major role in the exchange of free cholesterol between cells, the liver, and other lipoprotein moieties. Free cholesterol is needed in red blood cells, lipoprotein moieties, and most cell structures. High levels of HDL are believed to lower the incidence of cardiovascular disease. Blood tests to determine total serum cholesterol are commonly used to assess the risk of heart disease. However, since not all types of cholesterol indicate high risk, total serum cholesterol is not a very reliable indicator below 300 mg/dl. Consequently, attempts have been made to separately measure the HDL fraction. Typically, the LDL and VLDL fractions are precipitated out of solution and cell the cholesterol entities remaining in the supernatant are measured, as described in Moshides, J.S., (1987) Arteriosclerosis 7 :262-266. These entities are assumed to be HDL associated cholesterol. However, due to the presence of free cholesterol, the supernatant liquid contains a mixture of HDL associated and free cholesterol particles. Since the cholesterol in the supernatant is not all HDL, the measured HDL value is erroneous. The amount of HDL reported may be from 15 to 30% higher than the true value. Because this error is not constant, conventional HDL measurement techniques do not provide an accurate guide for comparing the percentage of HDL in the total serum cholesterol of various subjects. Those who are perceived to have a high risk of heart disease due to high cholesterol levels are often treated with a drug, e.g. lovastatin, to reduce their cholesterol levels. In addition, a low-cholesterol diet is generally suggested. The drug treatment is believed to work by suppressing cholesterol production in the liver. This treatment has several drawbacks: high cost; possible liver damage; and, other side effects. Because of the drawbacks, drug treatment is not recommended for most people in the moderate to high risk categories. Treatment is limited, in many cases, to recommending a low-cholesterol diet. While such a diet may be helpful, it is often not enough, particularly where a patient has difficulty staying on a diet. In accordance with a first aspect of the invention, there is provided a method of determining the net concentration of cholesterol associated with HDL particles in blood serum, comprising the steps of: (a) treating a sample of the serum with a precipitating agent which will combine with the LDL and VLDL particles in the serum; (b) centrifuging the treated serum sample until the LDL- and VLDL-containing precipitate is spun down, leaving a supernatant liquid having esterified HDL cholesterol, free cholesterol associated with the surface of said HDL particles, and free supernatant cholesterol; (c) separating the supernatant liquid from the precipitate; (d) treating a first sample of the supernatant liquid with cholesterol esterase or lipase in sufficient quantity to break down all the HDL particles into free cholesterol and fatty acids, resulting in a cholesterol-containing fluid having no esterified cholesterol; (e) treating the cholesterol-containing fluid with cholesterol oxidase in sufficient quantity to oxidise all the cholesterol present, forming hydrogen peroxide and cholest-4-en-3-one; (f) further treating the fluid with peroxidase, 4-aminoantipyrine and a chromogen in sufficient amounts to completely react all the hydrogen peroxide formed in step (e) to produce a quinoneimine; (g) measuring the electromagnetic radiation absorbance of the quinoneimine-containing fluid produced in step (f) at a wavelength at which the quinoneimine exhibits significant absorbance; (h) performing steps (d) through (g) on each of one or more standard cholesterol-containing fluids; (i) calculating the combined concentration of de-esterified cholesterol from HDL particles, free cholesterol associated with the surface of the HDL particles, and free supernatant cholesterol according to the formula (de-esterified cholesterol from HDL + free cholesterol associated with surface of HDL + free supernatant cholesterol concentration) = standard concentration x 2 (A supernatant ) A standard where A supernatant represents the absorbance measured for the supernatant and A standard represents the absorbance measured for the standard cholesterol-containing fluid; (j) performing steps (e) through (g) on a second sample of the supernatant liquid, and on one or more standard cholesterol-containing fluids, (k) calculating the concentration of free supernatant cholesterol from the absorbance values obtained in step (j) according to the formula free supernatant cholesterol concentration = 2 (A supernatant ) A standard x standard concentration; and (l) calculating the net concentration of cholesterol associated with HDL particles from the results of steps (i) and (k) according to the formula net concentration of cholesterol associated with HDL = (de-esterified cholesterol from HDL + free cholesterol associated with surface of HDL + free supernatant cholesterol concentration) - free supernatant cholesterol concentration. In accordance with a further aspect of the invention, there is provided a method for determining the percentage of total serum cholesterol constituting cholesterol associated with HDL particles, comprising: determining the net concentration of cholesterol associated with the HDL particles according to the method described above in a first portion of a serum sample; determining total serum cholesterol concentration by treating a second portion of the serum sample according to steps (d) through (h) above, and calculating the total serum cholesterol concentration according to the formula total serum cholesterol concentration = standard concentration X A serum A standard where A serum represents the absorbance measured for the serum, and A standard is defined above; and calculating the percentage of cholesterol associated with HDL particles according to the formula % HDL cholesterol = 100 X (net HDL cholesterol) (total serum cholesterol) . Net HDL cholesterol is measured by separately measuring: the free supernatant cholesterol; and, the combined HDL and free cholesterol in the supernatant. The net HDL value is obtained by subtraction. The measurement of the combined HDL and free cholesterol fractions is performed in two main steps. First, the LDL and VLDL components are precipitated out of the serum by using an appropriate reagent, e.g. polyethylene glycol (PEG), and centrifuging until the LDL and VLDL are pulled down, leaving a supernatant liquid containing both HDL and free cholesterol. Second, the total cholesterol content of the supernatant is measured by adding a reagent comprising POD, CO, an appropriate enzyme such as a cholesterol esterase or lipase, 4-aminoantipyrine, and a chromogen to a sample of the supernatant liquid; a quinoneimine is produced and its concentration is measured spectroscopically. To measure the free cholesterol, a sample of the supernatant is treated with a reagent containing peroxidase (POD), cholesterol oxidase (CO), 4-aminoantipyrine, and a chromogen. A quinoneimine is produced chemically in proportion to the amount of free cholesterol originally present in the supernatant; the quantity of quinoneimine produced is measured by uv-visible absorbance spectroscopy. The net HDL in the specimen may be calculated by subtracting the measured value for free cholesterol from the measured value of the combined HDL and free cholesterol. This net HDL value is a useful indicator for assessing a patient's risk of heart disease. The level of HDL in the bloodstream may be increased by intake of a diet supplement, mixed to form a non-separating suspension, comprising: polyunsaturated lipids, e.g. safflower oil; phospholipids containing essential fatty acids; anti-oxidants, e.g. Vitamins C or E; and polysaccharides. The supplement is generally given once each morning. The optimum amount of the diet supplement will vary depending upon individual need. Serum cholesterol comprises several components, as shown by the following expression: Total cholesterol = HDL + LDL + VLDL + free cholesterol. Unesterified (free) serum cholesterol may be measured by taking advantage of the following chemical reactions: The free cholesterol is oxidized to cholest-4-en-3-one and hydrogen peroxide in a reaction catalyzed by cholesterol oxidase (CO). The peroxide then reacts with 4-aminoantipyrine and the chromogen, in a reaction catalyzed by peroxidase (POD), to form a quinoneimine and water. The amount of quinomeimine produced is directly proportional to the amount of free cholesterol oxidized in the first reaction, and may be measured spectroscopically. Typically, low-sensitivity chromogens such as phenol or p-hydroxybenzoate are used. However, any suitable chromogen may be employed. Preferably, the absorbance at 510 nm is measured and compared to the absorbance of reference standards at that wavelength to determine the quinoneimine concentration. Total serum cholesterol may be measured in a similar fashion by converting all the esterified cholesterol, found in the HDL, LDL and VLDL particles, to free cholesterol by using the catalyst lipase to break down the cholesterol esters into free cholesterol and fatty acids. Once these esters are broken down, all the serum cholesterol is in the free form, and thus the total serum cholesterol may be measured by the method described above for measuring free cholesterol. The HDL cholesterol component may be measured by first separating it from the LDL and VLDL fractions. The serum is treated with Polyethylene glycol (PEG) and then centrifuged, pulling down a precipitate containing the LDL and VLDL. Much of the free serum cholesterol is also pulled down. The supernatant liquid contains the HDL and some free cholesterol. This liquid is then treated with an enzyme such as lipase to break down the HDL and the cholesterol content is measured as described above. This measurement yields the total cholesterol in the supernatant, i.e. HDL and free supernatant cholesterol. The amount of free cholesterol in the supernatant is measured by treating a sample of supernatant according to the method described above for measuring free serum cholesterol. However, since the amount of free cholesterol in the supernatant is much less than the amount in a serum sample, it has been found that the use of a high-sensitivity chromogen, e.g. 3-hydroxy-2,4,6-triiodobenzoic acid and the like, is preferred for the free supernatant cholesterol measurement. The net HDL cholesterol in the serum specimen may be calculated by subtracting the measured value of free cholesterol in the supernatant from the measured value of the total cholesterol in the supernatant. The fraction of cholesterol bound in HDL particles may be calculated by dividing the net HDL value by the measured value for the serum total cholesterol. The risk of vascular disease is inversely proportional to the percentage of net HDL. A net HDL level of less than 15% of the total serum cholesterol is associated with high risk whereas levels above 15% are associated with a decreased risk of vascular disease. The reagents used in the cholesterol measurements described above are preferably buffered. The PEG precipitating reagent is buffered to a pH of approximately 10.0. A free cholesterol reagent containing CO, POD, chromogen and 4-aminoantipyrine is preferably buffered to a pH of about 7.5. Any suitable buffers may be used; standard phosphate buffers and Good buffers have been found to work well. The HDL particle is composed of approximately 20% cholesterol, 50% protein, and 25% phospholipids. Absorption of sufficient quantities of essential fatty acids (EFA) and long-chain phospholipids is vital to the production of HDL particles. Chylomicrons, very large particles containing essential fatty acids from the diet which are produced in the duodenum and jejunum and quickly cleared once they enter the bloodstream, are required to build lipoprotein moieties and cells. If chylomicrons are not produced in the intestinal mucosa then the system will not produce the types of cell membranes and lipoprotein moieties, e.g. HDL, that protect the organism from atherosclerotic disease. The diet appears to play a major role in determining chylomicron levels. For instance, excessive amounts of dietary saturated fats may play a role in EFA deficiencies. A pre-emulsified EFA food supplement can increase the quantity of EFA absorbed, and thus raise system-absorbed chylomicron levels. This in turn leads to the production of more HDL particles. Since HDL is believed to scavenge free cholesterol, a decrease in the overall level of cholesterol in the system may also result. The increased level of HDL, and the accompanying decrease in percent LDL and VLDL, may result in less risk of vascular disease. The dietary supplement referred to above may comprise: polyunsaturated lipids, preferredly lipids having 18 carbon atoms and two or three carbon-carbon double bonds; phospholipids containing essential fatty acids; polysaccharides; and antioxidants, e.g. Vitamin C and/or Vitamin E. This supplement is preferably administered once daily, more preferably in the morning without other foods. The amount of supplement administered will vary based upon individual requirements. One preferred supplement comprises the combination of apple pectin, safflower oil, liquid lecithin, ascorbic acid, and Vitamin E. The selection of the proper lipids is crucial to the effectiveness of the diet supplement in raising the percentage of HDL cholesterol. The polyunsaturated lipids selected are suitably oils containing predominantly one or more essential fatty acids having a carbon chain length of 18 and two carbon chain double bonds. These oils may optionally contain a significant amount of a fatty acid having an 18-carbon chain containing three double bonds. One preferred oil is safflower oil. Saturated lipids, monounsaturated lipids (i.e. oleic acid), and longer-chain lipids such as are found in marine animals are not believed to be effective for raising HDL levels. A preferred phospholipid is liquid lecithin. Similar unsaturated phospholipids may be substituted therefor. Saturated (solid) lecithins are not suitable. Apple pectin is a highly suitable polysaccharide for the practice of this invention. However, similar polysaccharides may be substituted therefor. Vitamins A and C are suitable antioxidants. However, those skilled in the art will appreciate that other antioxidants may be substituted. The inclusion of one or more vitamins is not essential to the effectiveness of the supplement; thus any compatible antioxidant(s) may be employed in the diet supplement. The diet supplement comprises an aqueous emulsion containing the ingredients described above. A typical dose of this supplement is about 85-140g (3-5 ounces) for an adult human. However, the amount will vary based upon individual need as determined by cholesterol test results. Dosing is typically done on a daily basis, with the number of days depending on individual need. Often a larger dose, e.g. 140g (5 ounces) is given for about 60 days and a smaller dose, e.g. 85g (3 ounces) is given thereafter. At 85g (three-ounce) portion of the diet supplement typically comprises: about from 0.07 to 0.12 g./kg. body weight of polyunsaturated lipids, preferably about 0.08-0.10 g./kg., and most preferably 0.09 g./kg; phospholipids in the amount of approximately 0.025-0.050 g./kg body weight, preferably 0.030-0.045 g./kg. and most preferably 0.039 g./kg.; polysaccharides in the amount of approximately 0.025-0.055 g./kg. body weight, preferably about 0.030-0.045 g./kg, and most preferably 0.039 g./kg.; about 400-800 mg. of antioxidants, preferably about 400-600 mg. Vitamin C and 25-50 IU vitamin E, most preferably about 500 mg. vitamin C and about 30-40 IU vitamin E; and water. The supplement may optionally be mixed with apple juice, or similar liquids, to make it more palatable. Emulsification of the supplement ingredients is crucial for proper absorption by the intestines. Therefore, the ingredients must be mixed well when making the diet supplement. The supplement should be frozen if it is to be stored for an extended period, to prevent undesirable oxidation or other chemical change. EXAMPLE I Free serum cholesterol was measured using a working reagent having the following composition: Peroxidase more than 2000 IU/L Cholesterol Oxidase more than 100 IU/L 4-Aminoantipyrine 0.4 mM P-Hydroxybenzoate 4mM Sodium Cholate 3mM Buffer pH 7.5 A 2.0 ml. aliquot of working reagent was pipetted into each of the clean, dry test tubes to be used for reagent blank, standards, serum control or patients' serum. A 0.02 ml. aliquot of standards, serum control or patients' serum was pipetted into each of the tubes. The contents of each tube was mixed thoroughly and incubated at 37°C for five minutes. The absorbance at 510nm of the liquids in the serum tubes and in the standard tubes was measured against that of the reagent blank. EXAMPLE II The sum of the HDL cholesterol and the free supernatant cholesterol was measured using two working reagents. The first was a PEG reagent comprising 200 g./l. PEG buffered to pH 10.00. The second was a cholesterol reagent comprising: Peroxidase more than 2000 IU/L Lipase at least 150,000 IU/L Cholesterol Oxidase more than 150 IU/L 4-Aminoantipyrine 0.4mM Phenol 4mM Sodium Cholate 3mM Buffer pH 7.5 A 0.2 ml. aliquot of PEG reagent was pipetted into a small tapered centrifuge tube and 0.2 ml. serum was added. The contents were mixed thoroughly and incubated at 25°C for 10 minutes, then centrifuged at 2000g for ten minutes at 25°C. The LDL and VLDL was thus precipitated out by the PEG and spun down, leaving a supernatant liquid containing the HDL and free cholesterol, although at one-half the original serum concentration due to the dilution by the reagent. A 0.02 ml. portion of the supernatant liquid was pipetted into another test tube; 0.02 ml. of each of the standards, and of a serum control, were separately pipetted into other test tubes. Then 0.02 ml. of cholesterol reagent was added to each test tube. The fluids in the test tubes were thoroughly mixed and then incubated at 37°C for 10 minutes. Absorbance at 510 nm was measured against a reagent blank for each test solution. The absorbance value of the supernatant test solution was doubled to correct for the dilution effect of the PEG reagent. The concentration of the HDL plus free cholesterol in the supernatant could then be determined by comparing the corrected absorbance to the absorbance of the standard solutions. EXAMPLE III The free cholesterol content of the supernatant obtained in Example II was measured by using a cholesterol reagent containing: Peroxidase More than 2000 IU/L Cholesterol Oxidase More than 100 IU/L 4-Aminiantipyrine 0.4 mM 3-Hydroxy-2,4,6-triiodobenzoic acid 1.5 mM Sodium Cholate 3mM Buffer pH 7.5 A 2.0 ml aliquot of this reagent was pipetted into each test tube; 0.02 ml of supernatant, or of a standard solution, was then pipetted into each test tube. The contents of the tubes were mixed thoroughly and incubated at 25°C for 5 minutes. The absorbance of each tube was measured against a reagent blank, and the concentration of free cholesterol was calculated as in Example II. EXAMPLE IV The total cholesterol in the serum tested in Example II was measured using the same working cholesterol reagent as that used in Example II. A 2.0 ml. aliquot of reagent was pipetted into each test tube; 0.01 ml. of the serum tested in Example II was added to one test tube, and 0.01 ml. of standards and a serum control were individually added to the other tubes. All were mixed well and incubated at 37°C for 10 minutes. Absorbances were measured, and cholesterol concentration calculated, as in Example II, except that no correction for PEG dilution was needed. EXAMPLE V The net HDL concentration of a serum sample was calculated by subtracting the concentration of free supernatant cholesterol, as determined in Example III, from the concentration of HDL and free supernatant cholesterol, as determined in Example II. [HDL + free] - [free] = [net HDL] This value was then divided by the measured total serum cholesterol (see Example IV) to calculate the percentage of HDL in the total cholesterol: [net HDL] [Total Cholesterol] X 100% = % HDL EXAMPLE VI Ten human subjects were given a 5-ounce dose of an emulsified supplement, on a daily basis, comprising: Dietary supplement components Dosage apple pectin 5ml (one teaspoon) safflower oil 10ml (two teaspoons) lecithin 5ml (one teaspoon) ascorbic acid 500 mg. Vitamin E 30 IU After 60 days they were retested. The changes in serum cholesterol levels are shown in Table I, below. Subject # HDL/mg/dl) Free Cholesterol (mg/dl) Total Cholesterol (mg/dl) original final change original final change original final change 1 39 58 +19 290 278 -12 2 27 70 +43 92 89 -3 341 315 -26 3 30 45 +15 85 70 -15 257 250 -7 4 44 60 +16 78 62 -16 253 268 +15 5 40 50 +10 321 180 -141 6 40 56 +16 86 75 -11 299 280 -19 7 27 49 +32 41 46 +5 172 204 +32 8 36 52 +16 60 51 -19 207 180 -27 9 43 58 +15 60 55 -5 185 191 +6 10 47 75 +28 264 226 -38 Average +21 -9 -22

Claims: A method of determining the net concentration of chclesterol associated with HDL particles in blood serum, comprising the steps of: (a) treating a sample of the serum with a precipitating agent which will combine with the LDL and VLDL particles in the serum; (b) centrifuging the treated serum sample until the LDL- and VLDL-containing precipitate is spun down, leaving a supernatant liquid having HDL associated cholesterol , and free supernatant cholesterol; (c) separating the supernatant liquid from the precipitate; (d) treating a first sample of the supernatant liquid with cholesterol esterase or lipase in sufficient quantity to break down all the HDL particles into free cholesterol and fatty acids, resulting in a cholesterol-containing fluid having no esterified cholesterol; (e) treating the cholesterol-containing fluid with cholesterol oxidase in sufficient quantity to oxidise all the cholesterol present, forming hydrogen peroxide and cholest-4-en-3-one; (f) further treating the fluid with peroxidase, 4-aminoantipyrine and a chromogen in sufficient amounts to completely react all the hydrogen peroxide formed in step (e) to produce a quinoneimine; (g) measuring the electromagnetic radiation absorbance of the quinoneimine-containing fluid produced in step (f) at a wavelength at which the quinoneimine exhibits significant absorbance; (h) performing steps (d) through (g) on each of one or more standard cholesterol-containing fluids; (i) calculating the combined concentration of cholesterol associated with the HDL particles, and free supernatant cholesterol according to the formula ( cholesterol associated with HDL + free supernatant cholesterol concentration) = standard concentration x 2 (A supernatant ) A standard where A supernatant represents the absorbance measured for the supernatant and A standard represents the absorbance measured for the standard cholesterol-containing fluid; (j) performing steps (e) through (g) on a second sample of the supernatant liquid, and on one or more standard cholesterol-containing fluids, (k) calculating the concentration of free supernatant cholesterol from the absorbance values obtained in step (j) according to the formula free supernatant cholesterol concentration = 2 (A supernatant ) A standard x standard concentration; and (l) calculating the net concentration of cholesterol associated with HDL particles from the results of steps (i) and (k) according to the formula net concentration of cholesterol associated with HDL = (cholesterol associated with HDL + free supernatant cholesterol concentration) - free supernatant cholesterol concentration. A method according to claim 1, wherein the precipitating agent comprises polyethyleneglycol. A method according to claim 1 or claim 2, wherein steps (d), (e) and (f) are performed in essentially one step by treating the fluid with a quantity of a reagent comprising lipase, cholesterol oxidase, 4-aminoantipyrine, chromogen and peroxidase. A method according to any one of claims 1 to 3, wherein the chromogen is selected from phenol, p-hydroxy-benzoate and 3-hydroxy-2,4,6-triilodobenzoic acid. A method according to any one of claims 1 to 4, wherein absorbance is measured at a wavelength of 510 nm. A method for determining the percentage of total serum cholesterol constituting cholesterol associated with HDL particles, comprising: determining the net concentration of cholesterol associated with the HDL particles according to the method of claim 1 in a first portion of a serum sample; determining total serum cholesterol concentration by treating a second portion of the serum sample according to steps (d) through (h) of claim 1, and calculating the total serum cholesterol concentration according to the formula total serum cholesterol concentration = standard concentration X A serum A standard where A serum represents the absorbance measured for the serum, and A standard is in claim 1; and calculating the percentage of cholesterol associated with HDL particles according to the formula % HDL cholesterol = 100 X (net HDL cholesterol) (total serum cholesterol) .

Inventors: MAINES ROBERT Q, MAINES, ROBERT Q.
Applicants: MAINES ROBERT Q, MAINES, ROBERT Q.
Application year: 1990
Main classification: C12Q1
IPC codes: A23L1, C12Q1