SUMMARY OF THE INVENTION

Triglyceride substitutes (pseudotriglycerides or PSTG) have been identified for use in clinical chemistry controls, calibrators, standards and related preparations. The substitute materials are mixtures of medium carbon length ( C ₃ - C ₁₈ ) fatty acids esterified to glycerol that are relatively inexpensive, making them useful for the control materials. In addition, single components used in different chemistry areas, were found to be usable in the instant application without the need for emulsifiers. This invention also relates to the process of using the substitute materials.

BACKGROUND OF THE INVENTION

The measurement of both lipase and triglycerides in clinical chemistry is important as an indicator of the presence of pancreatic disease, hyperlipidemia, coronary artery occlusive disease, etc. Since lipase is the enzyme which hydrolyzes

triglycerides, one assay can be used for determining both of these factors. If the lipase component is kept constant, the test can be used to determine triglyceride in the sample. On the other hand, if triglyceride is kept constant, lipase level can be determined.

Since glycerides are specific substrates for the enzyme lipase (pancreatic lipase, EC 3.1.1.3, triacylglycerol acylhydrolase), procedures for measurement of lipase require the use of triglyceride, e.g., purified olive oil, a naturally occurring glycerol ester of oleic acid (triolein), for measurement of enzyme activity. Although triglyceride oils like olive oil are not expensive, they are not waier-soluble and will separate from a protein matrix on standing, yielding grossly unstable readings when used in controls or calibrators.

There is considerable literature on the use of triglycerides for clinical chemistry applications. These triglycerides are extracted from egg yolk, or isolated from animal or human blood. However, there are inherent difficulties in using these materials such as:

1. They are unstable to freeze-thaw processing.

2. They can precipitate, and this precipitation concomitantly affects other analytes such as calcium and phosphate.

3. They tend to become easily and quickly contaminated by microbes, such contamination adversely affecting the products in which they are used.

4. They are usually poorly characterized mixtures causing reproducibility problems.

5. Their assayed values are not stable and continue to rise over time.

Some clinical chemistry control manufacturers attempt to circumvent these problems by substituting glycerol for the triglyceride to mimic the chemistry of the true triglyceride. This approach has met with limited success because the hydrolysis step which is essential in certain assays is eliminated, and, for some assays requiring measurement of hydrolyzed fatty acids, glycerol is totally unsuitable. Other control materials utilize various surfactants to keep the normally insoluble triglycerides in solution or emulsified.

Other materials which work well as substrates for lipase have been identified in the past by others, and so would possibly be useful as triglyceride replacements in controls, calibrators, standards and similar materials. Some of these synthetic triglyceride analogs are 2,3-dimercaptopropan-l -ol tributyrate, beta-naphthyl laurate, beta-naphthyl myristate, phenyl laurate and sorbitan esters, methylumbelliferone- and N-methylindoxyl myristate. While each of these compounds has been used as a substrate of lipase and so would serve theoretically as a substitute for natural triglycerides in controls, each is vastly too expensive and/or insoluble in serum to be practically useful.

This invention relates to the new and innovative use of substitute materials for human, animal or egg yolk triglycerides in clinical chemistry assay controls, calibrators, standards and related preparations. Unexpectedly these materials, which had previously found application in unrelated areas of chemistry, were found to be useful substrates for lipase. The substitute materials include medium carbon length ( C ₃ - C _{l g} ) fatty acids esterified to glycerol to form mono- and di- and tri-glyceride mixtures, which are currently commercially used as vehicles for water-insoluble, oil- soluble pharmaceuticals and as emollient oils for facial creams and cosmetics. They are sparingly soluble in water and water-based protein solutions and yield stable triglyceride measurements on standing, without extraneous additional stabilizers required in other preparations. Similar materials which can 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 tributrate. The use of these materials has avoided the problems of freeze-thaw instability, precipitation, microbial contamination, and poor characterization (and hence reproducibility) which are encountered with the previous materials. These new materials also are much less expensive to use than the previous materials and methods, thus making them practical for use in the manufacture of the 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 PSTG ^* s 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 the substitute materials. The preferred PSTG's were found to be those that were inexpensive, making them practical for usage in manufacturing the commercial control materials. These PSTG's are also safe to handle. These inexpensive materials were generally mixtures, rather than pure, single component items.

PSTG's were analyzed in protein based matrices to determine performance characteristics, as shown in examples hereafter. To analyze the solutions, commercially available clinical analyzers were utilized e.g., Ektachem (manufactured by Kodak), Dimension D-380 (from DuPont), Express (from Ciba Corning), and ACA (from DuPont) to read serum triglyceride levels. Procedures involve the specific measurement of glycerol after the hydrolysis of the fatty acid - containing moieties. The substitutes included mono- di- or tri- glycerides of medium carbon chain length fatty acids (C ₃ - C, ₈ ): Capmul MCM and Capmul MCM-90 (trademarks of Karlshamns Inc.), 1-mondecanoyl-rac -glycerol, glycerol tributyrate (tributyrin), glycerol tripropionate (tripropionate), monocaproyloyl glycerol (Sigma Chemical

Co.) and monoglyceride of linoleic acid (e.g., Myverol from Eastman Chemical Co.). Mixtures of C ₃ - C| _g mono-, di-, or tri-glycerides, not isolated from animal, human or other natural sources, were found to be particularly useful herein. The above is a representative but not exhaustive list of the 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 all three hydroxyl groups on glycerol are esterified, the solubility of glycerides of substituents exceeding 6 carbons (C ₆ ) in length become sufficiently insoluble as to be rendered useless without addition of emulsifying agents (surfactants), while if only one hydroxyl group is esterified, leaving two hydroxyl groups to help solubility, glycerides with substituent fatty acid chains as long as C _{1 g} can be used.

As indicated above, the materials used herein are frequently mixtures. For example, Capmul MCM is a mixture of >80% monoglyceride and < 20% diglyceride. The fatty acid composition thereof is a further unspecified mixture of caprylic and capric acids esterified to glycerol 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 further unspecified mixture of caprylic and capric acids esterified to glycerol to form the above glyceride composition. It contains < 1% free fatty acid and < 1% free glycerol.

Triglyceride levels in normal fasting human serum range from 44-210 mg/dL. To determine if normal and abnormal triglyceride levels could be simulated by use of the substitute materials , serial dilutions were prepared from a concentrated solution of Capmul MCM in human serum (See Example 3). The results of these tests showed the linearity and quantitative recovery of PSTG in a human serum base. (See Example 3.) This quantitative recovery does not appear to be time dependent (See Example 1).

In addition, further work was done to determine that quantitative recoveries of PSTG are consistent over the majority of clinical analyzers and over a range of PSTG materials (See Examples 4 - 7).

To be useful in clinical materials, minimal stability criteria must be met, e.g. after 10 days refrigerated storage (2-8° C), a recovery of plus or minus 10% should be obtained when the PSTG is spiked into a protein base at normal or abnormal levels. The results of stability studies indicated these PSTG's would be commercially useful as controls, calibrators or standards (See Example 2). Also, to be useful, samples must be linear upon dilution, since high concentration human clinical samples are diluted to get within the assayed concentration range for the test. (Example 3.)

Once quantitative recovery and adequate stability was shown for the PSTG when spiked into a protein base, the material was utilized in a number of applications where triglycerides isolated from egg yolk or human or animal blood had been used by other laboratories, namely, to develop materials which could be used as controls for clinical assays for the quantitative and qualitative measurement of triglyceride 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 aforementioned fluids.

The above describes the best mode contemplated by the inventors for the use of the PSTG materials. However, it is contemplated that the PSTG's could be used in place of triglyceride 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, utilize either a labelled antigen or a labelled antibody. PSTG antigen or antibody could be labelled using various established techniques, for example, the addition of a radioactive label, an enzymatic label, a fluorescent label, a

chemiluminescent label or other labels which would make the material useful in an immunochemical analytical technique. The label would serve as the reporting groups in the immunoassays. It is also contemplated that PSTG ^' s might be purified and utilized, or perhaps even utilized without purification, in other analytical techniques where triglycerides isolated from egg yolk, or isolated from animal or human blood, might currently be used.

It is further contemplated that the PSTG's could be used as immunogens to develop an antibody. Polyclonal, monoclonal or other antibodies could be raised against the 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.)

Either the PSTG's or antibody produced therefrom could be immobilized on a solid support. Numerous supports could be used, for example agarose resins (Sepharose etc.), glass beads, etc. An immobilized antibody to triglyceride could act as a rapid and efficient purification tool to obtain pure triglyceride from crude sources. Likewise, pure antibody could be obtained utilizing immobilized PSTG material. These immunoaffinity chromatographic methods are well established in the literature.

The preceding illustrates how immobilized ligands can be utilized but should not be construed to limit their usefulness. For example, immobilized triglyceride substitute antibody could be used as a stripping agent to obtain triglyceride free serum.

The materials and processes described herein can also be used to determine lipase level. In this case, the triglyceride would be kept constant, and the measurement would include:

1. treating serum containing lipase to be measured by adding porcine colipase (a lipase cofactor which speeds up the hydrolysis reaction of lipase) and phenylmethylsulfonylfluoride (which inhibits nonlipase esterase which would

otherwise also react with substrate -see, for example, Teitz, N.W.. "Textbook of Clinical Chemistry", W.B. Saunders Company, Philadelphia (1986) p. 739)

2. adding reagent comprising a triglyceride or triglyceride substitute comprised of C ₃ - C ₁₈ fatty acids esterified to glycerol to form mono-, di-, or tri¬ glycerides not isolated from animal, human or other natural sources or mixtures thereof , in a protein solution, along with one or more indicator reagents, and

3. measuring one of the hydrolysis reaction products (either glycerol or fatty acids) by accepted methods (see, for example, Peace, A.J. and Kaplan, L.A.,

"Methods in Clinical Chemistry", C.V. Mosby Company, St. Louis (1987) p. 849) after a fixed period of reaction time and comparing the amount so measured to the amount produced by a standardized amount of lipase in the same time. Lipase activity in the sample will be proportionate to the amounts of these reaction products. Variations in this lipase assay procedure may be anticipated by those with skill in the art.

The following examples describe aspects of the stability and usefulness in various instruments of the PSTG. These materials and the products produced therefrom are also useful in manual techniques. (For a general discussion of procedures for triglyceride, see, for example, "Methods in Clinical Chemistry" , A. J.

Pesce et al, C. V. Mosby Co., St. Louis, 1987, Chap. 18, pp. 1215-1227.) However, these examples are not intended to limit the usefulness of the PSTG's or techniques for utilization thereof.

EXAMPLE 1

Effect of Time Upon Dissolution :

Using human serum as the matrix of choice, 7.5 mg of PSTG from Karlshamn

(Capmul® MCM, lot # 30418-6 ) was added to 30 mL of serum in a glass bottle, and mixed by tumbling at room temperature. The concentration of Capmul added was 25

mg/dL. No additional solubilizing agents were used. Samples of the solution were periodically taken and assayed for triglyceride concentration using a lipase-containing assay specific for triglyceride on a Ciba Corning Express 550 Clinical Chemistry Analyzer. The following table shows that the material yields a triglyceride concentration with the Express analyzer, that dissolution is complete by 94 minutes and that the concentrations measured remain stable with time. Also, the measured concentration on the Express 550 was 212% of the amount added, demonstrating that PSTG reacts as if it were more potent than endogenous triglyceride. (The variation in the data for concentrations is caused by imprecision of the method of measurement and is well within the precision expected from the Express 550.)

Table

Triglyceride Net PSTG

Time concentration Added (minutes) ⁽ mg/dL) (mg/d )

Endogenous

Triglycerides

(baseline) 138

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 assayed for triglyceride concentration on the Express 550, and separate samples were placed at 5° C, 23° C and 30° C. The samples were assayed for triglyceride concentration at intervals for up to 12 days. The following table shows the results. All of the concentrations are in mg/dL. The data show that the control materials stored at 5° C are stable for approximately 3 years, based on extrapolation of the accelerated 30° storage stability studies shown below.

Table 2.

Time Storage (a) 5° C Storage (a).23° 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

* 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 Effect of Concentration on PSTG Activity:

To 30 mL of human serum was added 32 mg of PSTG. The mixture was rotated 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 ACA III Clinical Analyzer. The following table presents the data obtained. Units of concentration are mg/dL.

Table 3

Total PSTG PSTG

Serum Cone. Baselin. : Net Calc.

Dilution ACA Cone. Cone. Cone. R,

undiluted 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 21 1 130 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 baseline concentration is the triglyceride activity due to endogenous material in the human serum, and the PSTG calculated concentration is the actual concentration based on the amount of PSTG that was weighed out. It is clear from this table that throughout the concentrations evaluated , the ratio, which is the total concentration minus the baseline concentration divided by the calculated concentration, remains fairly constant at about 2.9. This ratio may be different depending on the analyzer doing the

measurement, or the specific PSTG used, but it still remains constant.

That the recovery of measured triglyceride using PSTG is quantitative and reproduceable is shown in Table 3 of Example 3 by the calculation of a ratio which remains constant, at about 2.9. This ratio will likely be different depending upon which PSTG is selected for use, but. for each PSTG, some fixed ratio will be obtained.

This then permits the PSTG to be used as a standard or calibration material as well as a control. A calibrator or standard would be prepared as follows: to achieve a calibration value of 200 mg/dL, add to one liter of serum stripped of endogenous triglycerides 689.7 g 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 calibrator values, use proportionately more or less PSTG per liter of stripped serum. For other PSTG's aside from Capmul MCM, or other analytical procedures, the appropriate factor would be used to achieve the desired concentration of "triglyceride".

EXAMPLE 4

PSTG Activity as Measured on the Dupont D-380 Analyzer:

When various types of PSTG are added to a final concentration of 210 mg/dL in human serum, which has an endogenous triglyceride value of about 90 mg/dL, and is mixed for 6 hours at 5° C, they yield stable PSTG values. A sample of these solutions is then stored at 5° C, and assayed for triglyceride activity over time. The following table shows the data for these PSTG's when analyzed on a Dupont D-380 Analyzer.

Table 4

Brands of PSTG

Time (days) Control Myverol Tripro Capmul Mono C-8 Tribut

0 86 237 248 228 256 234 2 95 230 265 216 245 222

5 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, Tripro is glyceryl tripropionate, Capmul is a mixture of mono- and diglycerides of caprylate and caprate, Mono C-8 is monocapryloyl glycerol, and Tribut is glyceryl tributrate.

EXAMPLE 5

PSTG Activity as Measured on the DuPont ACA III:

The samples were mixed as described above in Example 4. The following table shows the data for these PSTG's when analyzed on a DuPont ACA III

Analyzer.

Table 5

Brands of PSTG Time (days) Control Myverol Tripro Capmul Mono C-8 Tribut

0 100 252 269 250 274 251

2 97 257 297 250 275 253

5 102 258 321 257 275 253

9 104 257 326 257 281 257

05

EXAMPLE 6

PSTG Activity as Measured on Ciba Coming's Express 550:

The samples were mixed as described in Example 4 above. The following table shows the data for these PSTG's when analyzed on the Ciba Coming

Express 550.

Table 6

Brands of PSTG Time (days) Control Myverol Tripro Capmul Mono C-8 Tribut

0 86 247 263 244 271 249

2 83 246 300 240 272 240

5 90 261 321 239 272 250

9 90 246 323 240 267 248

EXAMPLE7

PSTG Activity as Measured on Kodak's Ektachem 700XRC Analyzer:

The samples were mixed as described above in Example 4. The following table shows the data for these PSTG's when analyzed on Kodak's Ektachem 700XRC Analyzer. Only a single time point is shown here, but serves very well to show that these PSTG's work quite well on this analyzer also.

Table 7

Brands of PSTG Time (days) Control Myverol Tripro Capmul Mono C-8 Tribut

23 91 268 335 258 287 260

EXAMPLE 8

The Use of a PSTG(Capmul) in a Complete Chemistry Control:

Sufficient Capmul MCM (a PSTG) is added to a human serum based complete chemistry control to give a final triglyceride value of about 200 mg/dL when the control is assayed on a DuPont D-380. The chemistry control contains about 50 separate analytes, and shown in the following table are a few representative analytes along with the PSTG acting as most of the triglyceride component.

The control material was held at 5° C, and the activity of the various analytes was determined at the time intervals shown.

Table 8

Time (days) ACP ALP Bilirubin CK AST ALT TRIG

0 3.76 165 7.55 251 222 147 212

1 3.86 178 7.46 233 214 142 218

2 3.83 181 7.28 227 214 143 227

4 3.77 179 7.01 231 218 147 233

6 3.74 171 6.79 232 221 144 217

9 3.71 171 6.31 218 215 137 238

The data in this table clearly show the stability of this PSTG in the presence of numerous other components.

Further variations in the development of control materials comprising triglyceride substitutes will become apparent to those with expertise in the relevant art.