MXPA97001485A - Triglycerides substituted stable to be used in controls or calibrators of clinical chemistry analysis and process for preparation - Google Patents

Abstract

The present invention relates to materials and compositions for producing triglyceride controls, calibrators and standards, the materials being mono-, di- and triglycerides of medium length fatty acids mixed in human serum compositions or other protein solutions to form stable solutions, miscibles, suitable for use as controls, calibrators and standards in clinical chemistry for measurement and quality control in analysis for triglycerides. The materials described have been used as vehicles for oil-soluble drugs, insoluble in water, and as facial emollient oils for cosmetics, as such, they are safe, functional, stable, resistant to becoming rancid and very cheap compared to pure synthesized materials or described previously

Description

TRIGLYCERIDES SUBSTITUTES STABLE TO BE USED IN CONTROLS OR CALIBRATORS OF CLINICAL CHEMISTRY ANALYSIS AND PROCESS FOR YOUR PREPARATION COMPENDIUM OF THE INVENTION Triglyceride substitutes (pseudotriglycerides or PSTG) have been identified for use in controls, calibrators, standards, and related clinical chemistry preparations. The substitute materials are mixtures of medium length fatty acids (C3.C.sup.-8) esterified for giiceroi which are relatively inexpensive, making them useful for control materials. In addition, it was found that single components, used in different areas of chemistry, can be useful in the present application without needing emulsifiers. This invention also relates to the process for using the substitute materials.

BACKGROUND OF THE INVENTION The measurement of both lipase and proteins in clinical chemistry is important as an indicator of the presence of pancreatic disease, hyperlipidemia, occlusive disease of coronary arteries, etc. Since the raisin is the enzyme that hydrolyzes tpglicépdos, an analysis can be used to determine both of these factors. If the lipase component is kept constant, the test can be used to determine triglycerides in the sample. On the other hand, if the triglyceride is kept constant, the level of lipases can be determined. Since glycerides are substrates specific for the enzyme lipase (pancreatic lipase, EC 3.1.1.3, triacylglycerol acylhydroxysa), procedures for measuring lipase require the use of triglycerides, eg, purified olive oil, a glycerol ester which is present in the nature of oleic acid (triolein), to measure the enzymatic activity. Although triglyceride oils such as olive oil are not expensive, they are not soluble in water and will separate from a protein matrix when resting, producing excessively unstable readings when used in controls or calibrators. There is considerable literature on the use of triglycerides for clinical chemistry applications. These trigiicépdos are extracted from yolk of egg, or isolated of animal or human blood. However, there are inherent difficulties in using these materials such as: 1. They are unstable for freeze-thaw processing 2. They can be precipitated, and this precipitation concomitantly affects other anaerobes such as calcium and phosphate 3. They tend to be easily contaminated and rapidly by microbes, said contamination adversely affecting the products in which they are used. 4. They are usually poorly characterized mixtures, causing reproductive capacity problems. 5. Their analyzed values are not stable and continue to rise over time. Some manufacturers of clinical chemistry controls, try to avoid these problems by replacing glycerol with triglyceride to mimic the chemistry of the real triglyceride. This approach has met with limited success since the step of hydrolysis that is essential in certain analyzes is eliminated, and, for some analyzes that require measurement of hydrolyzed fatty acids, glycerol is totally inadequate. Other control materials use various surfactants to keep the triglycerides normally insoluble in solution or emulsified. In the past, others have identified other materials that work well as substrates for lipase, and therefore, would possibly be useful as triglyceride replacements in controls, calibrators, standards and similar materials. Some of these triguceride analogues are 2,3-dimercaptopropan-1-ol tributyrate, beta-naphthyl laurate, beta-naphthyl myristate, phenyl laurate and sorbitan esters, methylumbelliferone miptate and N-methylindoxyl. While each of these compounds has been used as a substrate of ¡pasa and therefore could theoretically serve as a substitute for natural triglycerides in controls, each one is too expensive and / or insoluble in serum to be practically useful. This invention relates to the novel and innovative use of substitute materials for human, animal or egg white triglycerides in controls, calibrators, standards and related preparations of clinical chemistry analysis. Substitute materials include fatty acids of average carbon length (C3-C? ß) esterified for glycerol in order to form mixtures of mono-, di- and triglycerides, which are currently used commercially as vehicles for water insoluble, soluble drugs. in oil and as emollient oils for facial creams and cosmetics. They are sparingly soluble in water and water-based protein solutions and produce stable triglyceride measurements when at rest, without additional extra stabilizers being required in other preparations. Similar materials that may also be used include, but are not limited to, glycerol monolinoleate, glycerol tripropionate, mono-, di- or tri-glycerides of caprylate and caprate, monocapryloyl glycerol, and glyceryl tributyrate. The use of these materials has avoided the problems of freezing-thawing instability, precipitation, microbial contamination, and poor characterization (and therefore, reproducibility) encountered with the previous materials. These new materials are also much less expensive to use than previous materials and methods, making them practical for use in the manufacture of clinical control materials. The invention covers not only the identification of the material, but also techniques for its use.

DETAILED DESCRIPTION OF THE INVENTION This invention deals with the identification of novel sources of PSTGs for use in clinical chemistry controls, calibrators, standards and related preparations (together referred to as control materials). This invention also relates to the use of substitute materials. It was found that the preferred PSTGs are those that were not expensive, making them practical for use in the manufacture of commercial control materials. These PSTGs are also safe to handle. These inexpensive materials were generally mixtures, rather than articles of single, pure components. PSTGs were analyzed in protein-based matrices to determine performance characteristics, as shown in the following examples. In order to analyze the solutions, commercially available clinical analyzes, v. Gr., Ektachem (manufactured by Kodak), D-Dimension, were used. 380 (from DuPont), Express (from Ciba Corning), and ACÁ (from DuPont) to read serum typing levels. The procedures involve the specific measurement of glyceroi after the fatty acid nidróhsis containing portions. Substitutes included mono-, di- or triglycerides of medium carbon chain length (C3-Cie) fatty acids: Capmul MCM and Capmul MCM-90 (brands of Karlshamns Inc.), 1 -modecanoyl-rac-glycerol, tributyrate of glycerol (tributyryl), glycerol tripropionate (tripropionate), monocapryloyl glycerol (Sigma Chemical Co.) and linoleic acid monoglyceride (eg, Myverol from Eastman Chemical Co.). It was found that mixtures of C 3 -C 8 mono-, di- or triglycerides, not isolated from animal, human or other natural sources, are particularly useful in the present. The above is a representative but not exhaustive list of possible substitutes. It has been found that the solubility of these compounds is affected not only by the number of glycerol substituents, but also by the chain length of each substituent. For example, if the three hydroxyl groups in the glycerol are esterified, the solubility of glycerides of substituents exceeding 6 carbons (Ce) in length, becomes sufficiently insoluble so that they become ineffective without the addition of emulsifying agents (surfactants), whereas if only one hydroxyl group is esterified, leaving two hydroxyl groups to aid solubility, glycerides with fatty acid substituent chains can be used as well as C? 8 As indicated above, the materials used herein, They are often mixtures. For example, Capmul MCM is a mixture of > 80% monoglicepdo and < _20% glyceride. The fatty acid composition thereof is a non-specific additional mixture of caprylic and capric acids esterified for glycerol, in order to form the above glyceride composition. It contains < 1% free fatty acid and < 1% free glycerol. Capmul MCM-90 is a mixture of the following composition: > . 90% monoglyceride and < 10% diglyceride. The fatty acid composition thereof is a non-specific additional mixture of caprylic and capric acids esterified for glycerol in order to form the above glyceride composition. It contains < 1% free fatty acid and < 1% free glycerol. Triglyceride levels in serum scale of human fasting pattern of 44-210 mg / dL. To determine whether the standards of triglyceride standards and pattrons can be simulated by the use of substitute materials, serial dilutions of a concentrated solution of Capmul MCM were prepared in human serum (See Example 3). The results of these tests showed linearity and quantitative recovery of PSTG in a human serum base. (See Example 3). This quantitative recovery does not seem to be time dependent (See Example 1). In addition, additional work was done to determine that quantitative PSTG recoveries are consistent over most clinical analyzes and on a scale of PSTG materials (See Examples 4-7). To be useful in clinical materials, it must comply with minimum stability criteria, eg, after 10 days of storage in refrigeration (2-8 ° C), a recovery of plus or minus 10% must be obtained when the PSTG is introduced in a base of proteins at normal or abnormal levels. The results of stability studies indicated that these PSTGs could be commercially useful as controls, calibrators or standards (See Example 2). Also, to be useful, the samples must be linear when diluted, since the clinical samples of humans are diluted at high concentrations, to obtain the concentration scale analyzed for the test. (Example 3). Once adequate quantitative recovery and stability for the PSTG was shown when it was introduced into a protein base, the material was used in a number of applications where triglycerides isolated from egg yolk or human or animal blood have been used by other laboratories, namely, to develop materials that could be used as controls for clinical analyzes for the quantitative and qualitative measurement of triglycerides in human serum (See Examples 4 and 8). These materials are usable not only in human serum, but also in matrices composed of serum from other animals, human or animal albumin or mixtures thereof, urine, spinal fluid, saliva, or other fluids containing protein or mixtures of any of the fluids mentioned above. The foregoing describes the best mode contemplated by the inventors for the use of PSTG materials. However, it is contemplated that PSTGs could be used in place of triglycerides isolated from egg yolk, or isolated from animal or human blood, in all analytical procedures, including, without limitation, radioimmunoassay, ELISA, and other analytical techniques. For example, most immunoassays, for the identification of an antigen, use either a labeled antigen, or a labeled antibody. The PSTG antigen or antibody could be labeled using several established techniques, for example, the addition of a radioactive label, an enzymatic label, a fluorescent label, a chemiluminescent label or other labels that could form the material useful in an immunochemical analytical technique. The label could serve as reporting groups in the immunoassay. It is also contemplated that PSTGs can be purified and used, or perhaps still used without purification, in other analytical techniques where triglycerides isolated from egg yolk or isolated from animal or human blood, can currently be used. It is further contemplated that PSTGs can be used as immunogens to develop an antibody. Polyionates, mono-or other antibodies, could arise against triglyceride substitutes. The technology for production of antibodies (polyclonal or monoclonal) has been well established. (See, for example, Immunology, Second Edition, I. Roitt et al., Gower Medical Publishing, London, 1989, page 8.2). Both the PSTGs and the antibody produced therefrom could be immobilized on a solid support. Numerous supports can be used, for example, agarose resins (Sepharose etc.), glass beads, etc. An immobilized antibody for triglyceride could act as a fast and efficient purification tool to obtain pure trigiichéride from crude sources. Likewise, the pure antibody could be obtained using immobilized PSTG material. These chromatographic immunoaffinity methods are well established in the literature. The above illustrates the way in which immobilized ligands can be used, but should not be considered as limiting their utility. For example, the immobilized triglyceride substitute antibody can be used as a separating agent to obtain triglyceride-free serum. The materials and processes described herein may also be used to determine the level of lipase. In this case, the triglyceride could remain constant and the measurement could include: 1. treat serum containing iipase to be measured by adding porcine colipase (a cofactor of lipases that accelerates the hydrolysis reaction of lipase) and phenylmethylsulfonyl fluoride (which inhibits esterase without lipase that could also react in some way with the substrate, see for example, Teitz, NW, "Textbook of Clinical Chemistry," WB Saunders Company, Philadelphia (1986) p.739). 2. adding reagent comprising a triglyceride or triglyceride substitute comprised of C3C? 8 fatty acids esterified for glycerol in order to form mono-, di- or t-glycerides or isolates from animal, human or other natural sources, or mixtures of the same, in a protein solution, together with one or more indicator reagents, and 3. measure one of the hydrolysis reaction products (either glycerol or fatty acids) by accepted methods (see, for example, Peace, AJ and Kaplan, LA, "Methods in Clinical Chemistry", CV Mosby Company, St. Louis (1987) page 849) after a fixed period of reaction time and comparing the amount so measured with the amount produced by a standardized amount of lipase in the same time. The lipase activity in the sample will be provided to the amounts of these reaction products. Variations in this lipase analysis procedure can be anticipated by those with experience in the field. The following examples describe aspects of stability and utility in various PSTG instruments. These materials and the products produced from them are also useful in manual techniques. (For a general discussion of triglyceride procedures, see, for example, "Methods in Clinical Chemistry," A.J. Pesce et al., C.V. Mosby Co., St. Louis, 1987, Chapter 18, pp. 1215-1227). However, these examples are not intended to mimic the utility of PSTGs or techniques for using them. EXAMPLE 1 Effect of Time on Dissolution. Using human serum as the matrix of choice, 7.5 mg of PSTG from Karlshamn (Capmul® MCM, lot # 30418-6) were added to 30 ml of serum in a glass bottle, and mixed by stirring at room temperature. The concentration of added Capmul was 25 mg / dL. No additional solubilization agents were used. Samples of the solution were taken and analyzed periodically for triglyceride concentration using an analysis containing triglyceride-specific lipase in a Clinical Chemistry Analyzer Express 550 from Ciba Corning. The following table shows that the material produces a concentration of triglycerides with the Express analyzer, that the solution is completed for 94 minutes and that the concentrations of measurements remain stable over time. Also, the concentration measured on the Express 550 was 212% of the amount added, demonstrating that PSTG reacts as if it were more potent than the endogenous triglyceride. (The variation in the data for concentrations is caused by the inaccuracy of the measurement method and also within the expected accuracy of the Express 550).

Table 1 Time Concentration of PSTG Net triglyceride Added (minutes) (ma / dl) (mq / dl) Triglycerides 138 0 endogenous (base line) 0 191 53 94 194 56 331 187 49 1424 189 51 EXAMPLE 2 Effect of Time and Temperature on PSTG Activity: Similar to Example 1 above, 15 mg of PSTG was added to 30 ml of human serum in amber bottles. The solutions were analyzed for triglyceride concentration on the Express 550, and separated samples were placed at 5 ° C, 23 ° C and 30 ° C. Samples were analyzed for triglyceride concentration at intervals of up to 12 days. The following table shows the results. All concentrations are in mg / dl. The data show that the control materials stored at 5 ° C are stable for approximately 3 years, based on the extrapolation of the 30 ° accelerated storage stability studies, shown below.

Table 2. Storage Time Storage Storage < a > 5th C < 3 > 23 ° C (S) 30 ° C (days) Control PSTG Control PSTG Control PSTG 0 140 245 135 243 131 248 0. 25 128 241 128 243 131 251 0. 50 128 241 128 242 131 243 2 128 243 132 241 135 248 4 135 245 137 253 143 267 6 136 250 143 262 152 272 11 145 256 159 282 176 301 12 172 282 191 300 206 322 EXAMPLE 3 The Concentration Effect on ASTG Activity: To 30 ml of human serum, 32 mg of PSTG was added. The mixture was stirred for 60 hours at 5 ° C, after which aliquots were taken and further diluted with human serum. The triglyceride activity of the final solutions was determined on a DuPont AC III Clinical Analyzer. The following table presents the obtained data. The concentration units are mg / dl.

Table 3 Dilution Conc. Conc. Conc. Conc. Ratio of total line serum from Cale Net, ACA base PSTG PSTG no difference 436 130 306 106.7 2.86 1: 2 284 130 154 53.3 2.89 1: 3 234 130 104 35.6 2.92 1: 4 211 130 81 26.7 3.04 1: 5 198 130 68 21.3 3.18 1: 6 181 130 51 17.8 2.87 In this table, the concentration of the base line is the triglyceride activity due to the endogenous material in the Human serum, and the calculated PSTG concentration, is the actual concentration based on the amount of PSTG that was weighed. From this it is clear that through the concentrations evaluated, the ratio, which is the total concentration minus the concentration of the base line divided by the calculated concentration, remains totally constant at approximately 2.9. This relationship may be different depending on the analyzer that performs the measurement, or the specific PSTG used, but still remains constant. In Table 3 of Example 3, it is shown that the recovery of the triglyceride measured using PSTG is quantitative and reproducible, by calculating a ratio that remains constant at about 2.9. This relationship will probably be different depending on which PSTG is selected to be used, but, for each PSTG, some fixed relationship will be obtained. This then allows the PSTG to be used as a standard or calibration material as well as a control. A calibrator or standard could be prepared as follows: to achieve a calibration value of 200 mg / dl, add to a liter of divided serum of endogenous triglycerides, 689.7 mg of PSTG (specifically in this case Capmul MCM). The calculation is as follows: 689.7 mg / l x 2.9 x 0.1 L / dl = 200 mg / dl To achieve other values of the calibrator, use proportionally more or less PSTG per liter of separated serum. For other PSTGs in addition to Capmul MCM, or other analytical procedures, the appropriate factor could be used to achieve the desired concentration of "triglyceride". EXAMPLE 4 The PSTG Activity as Measured on the Dupont D-380 Analyzer: When several types of PSTG are added to a final concentration of 210 mg / dl in human serum, which has an endogenous triglyceride value of approximately 90 mg / dl, and is mixed for 6 hours at 5 ° C, they produce stable PSTG values. . A sample of these solutions is then stored at 5 ° C, and analyzed for triglyceride activity over time. The following table shows the data of these PSTGs when analyzed in a Dupont D-380 Analyzer.

Table 4 PSTG Markings Time Control Myverol Tripro Capmul Mono C-8 Tribut (days) 0 86 237 248 228 256 234 2 95 230 265 216 245 222 95 234 289 221 242 225 9 94 234 293 225 248 222 27 87 239 306 233 256 234 Myverol is a distilled glyceryl monolinoleate, Tppro is glyceryl tripropionate, Capmul is a mixture of mono and diglycerides of caprylate and caprate, Mono C-8 is monocapryloyl glyceroi, and Tpbut is glyceryl tributyrate. EXAMPLE 5 The PSTG Activity was measured on the DuPont ACL III: The samples were mixed as described above in Example 4. The following table shows the data for these PSTGs when analyzed in a DuPont ACAll Analyzer.

Table 5 PSTG Markings Time Control Myverol Tripro Capmul Mono C-8 Tribut (days) 0 100 252 269 250 274 251 2 97 257 297 250 275 253 102 258 321 257 275 253 9 104 257 326 257 281 257 EJ EM PLO 16 The PSTG Activity was measured in Express 550 from Ciba Corning: The samples were mixed as described in Example 4 above. The following table shows the data for these PSTGs when they were analyzed in the Ciba Corning Express 550. Table 6 PSTG Marks Time Control Myverol Tripro Capmul Mono C-8 Tribut (days) 0 86 247 263 244 271 249 2 83 246 300 240 272 240 90 261 321 239 272 250 9 90 246 323 240 267 248 EXAMPLE 7 The PSTG Activity was Measured on a Kodak Ekctachem 700XRC Analyzer: The samples were mixed as described above in Example 4. The following table shows the data for these PSTGs when analyzed in the Kodak Ektachem 700XRC Analyzer. Here only one point is shown, but it serves very well to show that these PSTGs also work very well in this analyzer. Table 7 PSTG marks Time Control Myverol Tripro Capmul Mono C-8 Tribut (days) 23 91 268 335 258 287 260 EXAMPLE 8 The use of a PSTG (Capmul) in a Complete Chemical Control: sufficient Capmul MCM (a PSTG) was added to a human serum based on complete chemical control to give a final triglyceride value of approximately 200 mg / dl when the control was analyzed in a Du Pont D-380. The chemical control contains approximately 50 separate analytes and the following table shows few representative analytes together with the PSTG acting as the majority of the triglyceride component. The control material was maintained at 5 ° C, and the activity of several analytes was determined at the times shown.

Table 8 Time ACP ALP Bilirubin CK AST ALT TRIG (days) 0 3.76 165 7.55 251 222 147 212 1 3.86 178 7.46 233 214 142 21 8 2 3.83 1 81 7.28 227 214 143 227 4 3.77 1 79 7.01 231 21 8 147 233 6 3.74 171 6.79 232 221 144 21 7 9 3.71 1 71 6.31 21 8 21 5 1 37 238 The data in this table clearly show the stability of this PSTG in the presence of numerous other components. For those skilled in the relevant art, additional variations in the development of control materials comprising triglyceride substitutes will be apparent.

Claims (17)

1. CLAIMS 1. A process for producing a clinical chemistry control product used in an assay for detecting the presence or amount of a triglyceride wherein said control product comprises C3-C? 8 fatty acids esterified for glycerol, in order to forming mono-, di- or triglycerides, not isolated from animal, human and other sources, or mixtures thereof which are substrates of iipase and soluble in a protein solution.
2. 2. A process of claim 1, wherein the control material is a control, calibrator or clinical standard.
3. 3. A process of claim 1, wherein said triglyceride substitute is selected from the group consisting of glyceryl monolinoleate, glycerol tripropionate, monocapryloyl glycerol, glyceryl tributyrate, and mixtures of mono- and diglycerides of caprylate and caprate. .
4. 4. A process of claim 3, wherein the control material is a mixture of caprylic and capric acids esterified for glycerol in order to form an additional mixture of mono- and diglycerides.
5. The process of claim 1, wherein the protein solution comprises human or other animal serum.
6. The process of claim 1, wherein the protein solution comprises albumin of human or other animal.
7. 7. The process of claim 1, wherein the protein solution comprises fluids containing proteins from human or other animal.
8. 8. The process of claim 7, wherein said fluid is urine, spinal fluid or saliva.
9. 9. A process for producing a chemical control comprising a triglyceride component, said component containing one or more fatty acids of medium chain length, said fatty acids varying from C3-C? 8, esterified for glycerol in order to form mixtures of mono-, di- and triglycerides, said mixtures being lipase substrates soluble in solutions of aqueous proteins, wherein said process comprises. a) the selection of said glycerides, b) the addition of said glycerides to an aqueous environment of proteins, and c) the addition of any remaining constituents.
10. 10. A process for producing a multi-constituent clinical control, which comprises a triglyceride component, wherein said component is selected from the group consisting of glyceryl monolinoleate, glycerol tripropionate, glyceryl tributyrate and mixtures of caprylate mono- and diglycerides and caprate, wherein said process comprises: a) the selection of said mono-, di- and triglycerides, which are soluble in an aqueous environment of proteins and a substrate for the enzyme lipase, b) the addition of said mono-, di- and triglycerides; and triglycerides to an aqueous protein environment, and c) the addition of any remaining constituents to form the multi-constituent clinical control.
11. 11. A control, calibrator or clinical chemistry pattern comprising a substitute of triglycerides containing C3-C18 fatty acids esterified for glycerol in order to form mono-, di- or non-isolated triglycerides from animal, human or other natural sources , or mixtures thereof which are substrates of iipase and soluble in a protein solution.
12. 12. A control, calibrator or standard of claim 11, wherein the protein solution comprises human or other animal serum.
13. 13. A control, calibrator or standard of claim 11, wherein the protein solution comprises human or other animal albumin.
14. 14. A control, calibrator or standard of claim 11, wherein the protein solution comprises fluids containing human or other animal protein.
15. 15. A control, calibrator or pattern of claim 14, wherein said fluid is urine, spinal fluid or saliva.
16. 16. A control, calibrator or standard of claim 11, wherein said triglyceride substitute is selected from the group consisting of glyceryl monolinoleate, glycerol tripropionate, glycerol monocapryloyl, glyceryl tributyrate, and mixtures of mono and diglycerides of caprylate and caprate.
17. 17. A control, calibrator or standard of claim 1, wherein said triglyceride substitute is a mixture of caprylic and capric acids esterified for glycerol in order to form an additional mixture of mono and diglycerides.