Quantitative Determination of Serum Triglycerides by the Use of Enzymes

Transcription

1 CLIN. CHEM. 19/5, (1973) Quantitative Determination of Serum Triglycerides by the Use of Enzymes Giovanni Bucolo and Harold David We describe a novel method for determining serum triglycerides, in which an enzymatic hydrolysis replaces the more commonly used saponification procedure. Under the conditions of the assay, the enzymatic hydrolysis can be completed in less than 10 mm by the combined action of a microbial lipase and a protease. We have been able to demonstrate complete hydrolysis of triglycerides by thin-layer chromatography of the reaction products, by recovery of glycerol from sera of known triglycerides content, and by comparison of triglyceride assays on a number of sera assayed by our method vs. the AutoAnalyzer procedure. The hydrolysis is directly coupled to the enzymatic determination of glycerol, and is followed through absorbance changes at 340 nm. The assay is simple, rapid, and requires only 50 tl or less of sample. Because the enzymes used do not release glycerol from other compounds in serum, the hydrolysis can be considered specific for triglycerides. Additional Keyphrases: R. delemar Iipase #{149}a-chymotrysin #{149}determination of glycerol by coupled enzymatic reaction #{149}thin-layer chromatography #{149}AutoAnalyzer The most widely used methods for determining serum triglycerides require at least a chemical hydrolysis (1-7), usually carried out on a solvent extract of the serum lipids. Compounds that may interfere with the final determination of the hydrolysis products must, in most procedures, be removed by appropriate purification procedures. All these methods yield precise and accurate results, but they are cumbersome, time consuming, and require considerable skill. On the other hand, hydrolysis of triglycerides by one or more enzymes under mild conditions would eliminate many of the deficiencies and complexities From the Biochemical Research Laboratories, Calbiochem, North Torrey Pines Road, Calif Received May 23, 1972; accepted Feb. 13, of the chemical procedures. It would be simple and specific. Errors or losses possible in more complex techniques would be minimized if prior treatment of the sample was not required and if the complete determination was performed in a single reaction vessel. We have developed a simple, precise, and reproducible procedure that links the enzymatic hydrolysis of serum triglycerides to the simultaneous enzymatic determination of glycerol. In our opinion, the self-evident advantages of the new technique render it the method of choice for the assay of serum triglycerides. Materials Apparatus A Model DBG Spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif ), a Model 2000 Spectrophotometer (Gilford Instruments, Inc., Oberlin, Ohio 44704) and a Model 330 Spectrophotometer (G. K. Turner Assoc., Palo Alto, Calif ) were used for the spectrophotometric assays. A single-channel AutoAnalyzer Sampler II System (Technicon Instrument Corp., Tarrytown, N. Y ) with a Model 111 Fluorometer (G. K. Turner Assoc.) was used for the automated determination of serum triglycerides. Matched Pyrex cuvets (Calbiochem, La Jolla, Calif ) were used for all spectrophotometric readings at 340 nm. Oxford Samplers (Calbiochem) were used in all assays. Chemicals Calbiochem products used were: a-chymotrypsin (peptidylpeptide hydrolase; EC ), elastase (pancreato-peptidase E; EC ), glycerol kinase (ATP: glycerol phosphotransferase; EC ), lactate dehydrogenase (L-lactate : NAD oxidoreductase; EC ), lipase (glycerol ester hydrolase; EC ) from hog pancreas ( Pronase ), pyruvate 476 CLINICAL CHEMISTRY, Vol. 19, No.5, 1973

2 kinase (ATP: pyruvate phosphotransferase; EC ), trypsin ( peptidylpeptide hydrolase; EC ), adenosine-5 -diphosphate (ADP), adenosine-5 -triphosphate (ATP), bovine serum albumin, L-a-glycerophOSphate, lecithin, linolenic acid, lysolecithin, methyl linoleate, methyl linolenate, methyl oleate, reduced nicotinamide adenine dinucleotide (NADH), oleic acid, P-enolpyruvate (PEP), and tnolein. 1,3-Diolein was purchased from Applied Sciences Laboratory, Inc., State College, Pa Lipases (glycerol ester hydrolase; EC ) from R. delemar, A. niger and G. candidum were products of Miles Laboratories, Inc., Kankakee, Ill All other chemicals were either Analytical Reagent or the otherwise best available grade. Samples of serum were obtained from hospital and commercial laboratories and from donors. All samples and pooled sera were assayed, subdivided, and maintained frozen or lyophilized until needed. In our experience (to be reported in a future communication) triglycerides in serum are stable under these conditions. Reagents and Procedures Triglycerides were assayed with the AutoAnalyzer according to the procedure of Kessler and Lederer (4). Triglycerides were hydrolyzed to glycerol and fatty acid either enzymatically with a mixture of R. delemar lipase and a-chymotrypsin or by saponification with potassium hydroxide in ethanol. The resulting free glycerol was then determined enzymatically by the following series of reactions: Pyruvate + NADH + H glycerol lactate kinase Glycerol + ATP glycerol-i-p + ADP pyruvate kinaoe ADP #{247} PEP -. ATP #{247} pyruvate dehydrogenaae - Lactate + NAD The decrease in absorbance at 340 nm is proportional to the concentration of glycerol. The glycerol assay reagent contains, in a final volume of 3 ml: 5 mol of magnesium, 0.9 mol of ATP, 0.9 zmol of PEP, 6 U of pyruvate kinase, 2 U of lactate dehydrogenase, NADH to a final absorbance at 340 nm of (approx. 0.4 mg) in potassium phosphate buffer (ph 7, 0.1 mol/liter). The triglyceride reagent has the same composition as the glycerol reagent; per assay, it contains additionally 400 U of R. delemar lipase, 30 U of a-chymotrypsin, and 5 mg of bovine serum albumin. A glycerol kinase solution containing 2 U in 50 zl is prepared by diluting the enzyme in potassium phosphate buffer (ph 7, 0.1 mol/liter) containing 0.1 g of bovine serum albumin per ml. The glycerol and the triglycerides reagents have proven to be stable for at least one year in dry form in the refrigerator ( Triglycerides and Glycerol Stat- Pack, Calbiochem, La Jolla, Calif ). After reconstitution with water and storage at 4#{176}C, these reagents can be used for at least 24 h. The same glycerol reagent is used for the enzymatic determination of glycerol after alkaline hydrolysis, but the final volume of the reagent is 2.5 ml instead of 3 ml. The following additional reagents are needed for the alkaline hydrolysis: Potassium hydroxide, approx. 0.7 mol/liter. Dissolve 4 g of potassium hydroxide in 10 ml distilled water. When cool, dilute to ml with ethanol. Magnesium sulfate, approx mol/liter. Dissolve 2.17 g of anhydrous magnesium sulfate in water and dilute to ml. Free GyceroI in Serum 1. To 3 ml of glycerol reagent in a cuvet with a 10-mm light path, add 0.2 ml of serum. Mix. Incubate for about 5 mm at 30#{176}C, to consume endogenous substrates. 2. Read the absorbance (A0) at 340 nm, with distilled water as a blank. Add 50 l of the glycerol kinase solution, mix, and incubate at 30#{176}C. 3. Read the absorbance of the sample 10 mm (A10) after the addition of glycerol kinase. Obtain ixa by subtracting A10 from Ao. Calculations: M x 23.7 = mg glycerol in ml of serum; LA x 228 = mg glycerol in ml of serum, expressed as triolein. Triglycerides in Serum A. Enzymatic hydrolysis. 1. To 3 ml of triglycerides reagent in a cuvet with a 10-mm light path, add 50 cl of serum, mix, and incubate at 30#{176}C for about 10mm. 2. Read the absorbance (A0) of the sample at 340 nm, with distilled water as a blank. Add 50 l of glycerol kinase, mix, and incubate at 30#{176}C. 3. Read the absorbance at 340 nm 10 mm (A10) after the addition of glycerol kinase. 4. Obtain &4 by subtracting A10 from A0. 5. Calculations:.A X 883 = mg triglycerides in ml of serum, expressed as triolein. B. Alkaline hydrolysis. 1. Into a screw-capped culture tube, pipet 0.2 ml of serum and 0.5 ml of potassium hydroxide solution, mix, cap the tube and place it in a water bath at 60#{176}-70#{176}C for 30 mm. Cool to room temperature. 2. Add 1 ml of magnesium sulfate solution, mix, and centrifuge. Use the clear supernatant fluid in the following assay. 3. To 2.5 ml of the glycerol reagent in a cuvet with a 10-mm light path, add 0.5 ml of supernatant fluid (from step 2), incubate for 5 mm at 30#{176}C, and read the absorbance (A0) of the sample at 340 nm, with distilled water as a blank. 4. Add 50 tl of glycerol kinase solution. Mix and incubate at 30#{176}C. Read the absorbance 10 mm (A10) after the addition of glycerol kinase. 5. Obtain A by subtractinga1o from Ao. 6. Calculations: Corrected M x 738 = mg triglycerides in ml of serum, as triolein. CLINICAL CHEMISTRY, Vol. 19. No. 5,

3 For the methods described, the following considerations should be noted: 1. A blank determination, in which a volume of water is used that is equivalent to the volume of sample, must be performed at least once for every batch of reagent to compensate for contaminants (substrates or enzymes) in the components of the assay. 2. No correction has been made in the calculations to account for the dilution made when 50 l of glycerol kinase is added to the reaction mixture. The decrease in initial absorbance (Ao) resulting from this dilution can be obtained by multiplying A0 by the factors for glycerol or for triglycerides, after enzymatic or alkaline hydrolysis. 3. Preformed free glycerol in the sample must be determined and the results (expressed as tniolein) subtracted from the total triglycerides value. 4. The factors used in the calculation are based on a molar absorptivity for NADH of 6.22 X 10. Thin-Layer Chromatography Chloroform:methanol (2:1 by vol) was used to extract the lipids from incubation mixtures (8). The solvent was evaporated at low temperature (less than 40#{176}C) under a stream of nitrogen. The residue was dissolved in a volume of chloroform:methanol (2:1) equivalent to one-tenth the original volume of the serum. Standard solutions were prepared to contain 10 g of triglycerides (or equivalent weight of other compounds) per liter. A 5-l sample of each solution was applied to an Eastman Kodak Silica Gel No Chromagram, 1 cm above the bottom edge. The ascending chromatogram was developed (9, 10) in diethyl ether: benzene: ethanol: acetic acid (40:50: - 2:0.2) until the solvent front had reached a height of 4 cm above the point of sample application. The chromatogram was then dried and developed in the same direction in hexane:diethyl ether:acetic acid (180:30:2). The spots were made visible by exposure to iodine vapors in a closed tank. Evaluation of Lipases Lipases were evaluated by one or more of the following procedures: 1. Titration of fatty acids liberated from an emulsion of olive oil (11). Units of lipase are expressed as microequivalents of fatty acid liberated per minute by this procedure at 30#{176}C. 2. Determination of the glycerol liberated by various amounts of lipase from the same olive oil emulsion (11). The reaction was stopped at selected times by adding an equal volume of trichloroacetic acid, 5 g in ml of water. The acid and the unreacted olive oil were removed by extracting twice with four volumes of chloroform and once with four volumes of diethyl ether. The last traces of ether were removed by bubbling nitrogen through the solution. This treatment left a clear aqueous solution containing glycerol in amounts proportional to the extent of hydrolysis. Glycerol was determined in a suitable fraction of this solution by the procedure described. 3. The final and most important consideration in the selection of a lipase was, of course, its ability to completely hydrolyze triglycerides within the parameters of our assay. Crude enzyme preparations, not suitable for direct spectrophotometnic assay, were evaluated before purification by a two-step procedure. In this case, serum was incubated with the iipase in the presence of a-chymotrypsin for 15 mm at 30#{176}C. Proteins were precipitated with perchloric acid and the assay mixture was centrifuged. The clear supernatant fluid was neutralized with solid potassium carbonate. The potassium perchlorate was removed by centrifugation and the glycerol in the supernatant fluid was assayed as described. Results and Discussion The most important considerations in the selection of a lipase for our spectrophotometnic assay were the extent of hydrolysis of triglycerides, the specificity for these compounds, and the period required for maximum activity. However, none of the lipases from different sources (e.g., from A. niger, G. candidum, hog pancreas, or R. delemar) catalyzed complete hydrolysis of serum triglycerides by itself. We assumed for these lipases a mode of action similar to that reported for pancreatic lipase. This enzyme has been shown to preferentially hydrolyze the esters at the primary hydroxyl groups of glycerol, while hydrolysis at the secondary hydroxyl group is very slow and limited (12-15). On the basis of this model, it was conceivable that a nonspecific esterase could complete the hydrolysis of the ester in the beta position. a-chymotrypsin was chosen as the auxiliary enzyme because of its well-known esterase activity (16); moreover, the enzyme is available in highly purified form, free of contaminants having an adverse effect on the other constituents of the glycerol reagent, and it is readily soluble. To test the validity of this assumption, various amounts of a-chymotrypsin were added to the glycerol reagent and the lipases were evaluated in the triglycerides assay. Among the various lipases, only the one from R. delemar (17) completely hydrolyzed triglycerides in the reagent supplemented with a- chymotrypsmn. The optimum concentration of the latter enzyme was found to be 30 U/3 ml of assay mixture. a-chymotrypsin exerts a similar action when added to lipases from other sources. For example, if 30 U of.chymotrypsin is added to 800 U of partially purified porcine pancreas lipase, recovery of free glycerol from serum triglycerides increases from 18% to 65%. Figure 1 demonstrates the increase in glycerol production by the R. delemar lipase in the presence of a-chymotrypsin from an olive oil emulsion as compared with glycerol liberated by lipase alone by the procedure described. An increase in glycerol liberated by R. delemar IIpase from serum triglycerides was also found when 478 CLINICAL CHEMISTRY, Vol. 19, No.5, 1973

4 mq GLYCEROL (AS IRIOLEIN) S IS IS Table 1. Effect of Proteases on the Hydrolysis of Serum Triglycerides by A. delemar Lipase (400 U) Protease added Amount Percent hydrolysis No addition 30-40% a-chymotrypsin 30 U % Elastase 15 U 90% Pronase 90 P.U.K. units 98% Trypsin 5000 N.F. units 73% UNITS OF LIPASEPERASSAY Fig. 1. Production of glycerol from an olive oil emulsion by R. delemar lipase, with and without s-chymotrypsin other proteases-such as elastase (15 U), trypsin (5000 N.F. units) (18), and Pronase (90 PUK units) (19)-were substituted for a-chymotrypsin (Table 1). Because of the common behavior of these enzymes, we believe that esterase activity is not their function. The mechanism of action of the proteases in our assay is currently under investigation. No lipase activity has been found in any of the proteolytic enzymes investigated, as glycerol is not released from triglycerides by these enzymes. Thinlayer chromatography (Figure 3) similarly shows the lack of activity toward triglycerides by a-chymotrypsin in the absence of lipase. One of our major concerns was the possibility that the lipase-protease combination could hydrolyze phospholipids. Under the conditions of our assay, no free glycerol was formed from lecithin, lysolecithin, or indeed from L-a-glycerol phosphate. We did not investigate whether fatty acids are liberated by our hydrolyzing enzymes from these compounds. This would be an important consideration if triglycerides were determined by assaying free fatty acids produced, rather than glycerol. Thin-layer chromatography was used to separate and identify the components formed by the following incubation mixture: serum (0.5 ml) was incubated for 30 mm at 30#{176}C with 50 U of a-chymotrypsin or 1300 U of lipase, or both enzymes in 2 ml of potassium phosphate buffer (0.1 mol/liter, ph 7), or with the complete triglycerides reagent. The lipids were extracted from these mixtures and separated by thin-layer chromatography as described (Figures 2-4). Esters of cholesterol and of fatty acids are not hydrolyzed by the reagent (Figure 4). Free fatty acids were evident after a very short period of interaction of the reagent with serum (Figure 4, C). At this time the triglycerides spot was very faint. After 10 mm, this spot had completely disappeared. Glycerol monooleate and triglycerides are not hydrolyzed by chymotrypsin alone, but are rapidly attacked when lipase is added. Hydrolysis of triglycerides is complete when both enzymes are present; the other components of the glycerol reagent are not needed for the hydrolysis, as shown in Figure 3. A S S.: S. #{149} I C D E F G Fig. 2. Standards separated by thin-layer chromatography A, Cholesteryl oleate; B. Methyl linolenate; C, Triolein; D, Mixture of all standards; E, Linolenic acid; F, Diolein II. #{149} ,-& - S 4;#{149}r1 Fig. 3. Thin-layer chromatography of hydrolysis products from serum lipids A, Untreated serum; B, Standards without methyl linolenate; C, Serum treated with a-chymotrypsin; D, Serum treated with lipase; E. Standards without cholesteryl oleate; F, Serum treated with o-chymotrypsin and Iipase CLINICAL CHEMISTRY, Vol. 19. No.5,

5 A :t #{248}. I S. I 4 S I 4 * ii A #{149}0 I. J_ -.,.,_. - A -$t. #{149}-#{176}i f Fig. 4. Thin-layer chromatography of hydrolysis products from serum lipids after treatment with the total triglycerides reagent A, Untreated serum; B, Standards without cholesteryl oleate; C, Serum with reagent-30 seconds incubation; D. Standards without methyl 1mblenate; E, Serum with reagent after 10 minutes incubation; F. Reagent We found that there was generally a poor relationship between the specific activity of lipases as measured by titration of fatty acids, by the measurement of glycerol (Figure 1) produced from a olive oil emulsion, and by the concentration of enzyme required for complete hydrolysis of triglycerides. These parameters would be more directly related in a highly purified enzyme preparation; such a preparation is not required in our assay and would be very costly. For this reason, the amount of lipase needed per assay must be optimized in every new preparation; for R. delemar lipase, this amount varied between and 400 U. The glycerol reagent, even though based on a published procedure (3), was modified to perform in combination with the hydrolyzing enzymes. The course of events that guided us during this development is represented in Figure 5, which shows patterns of absorbance changes at 340 nm during the assay. When a-chymotrypsin is added to the assay with and without lipase (Figure 5, B, D, F, G) the serum clears, as indicated by a rapid initial fall in absorbance, which is barely visible at the beginning of the charts. This is a common phenomenon, observed to varying degrees with many samples, but more evident with lipemic samples. In one experiment, 200 l of lipemic serum was added to the glycerol reagent supplemented with 30 U of a-chymotrypsin..a, at 340 nm, before addition of glycerol kinase was 0.5; the change in the absence of a-chymotrypsin was negligible. The same clarification takes place with lipase alone (Figure 5, E), but after an initial lag period. In the absence of bovine serum albumin, turbidity develops in the system after the initial phase of clearing (Figure 5, F), as indicated by a slow increase in absorbance before the glycerol kinase is added. The absence of turbidity when albu S $6 1$ S $ 20 T. 1. MipuNs Ti.. I. MI..tss Fig. 5. Influence of the components of the hydrolysis mixture on the triglycerides reaction. Curve A, Albumin; Curve B, t-chymotrypsin; Curve C, Albumin + lipase; Curve D, Albumin + s-chymotrypsin; Curve E, Lipase; Curve F, Lipase + t-chymotrypsin; Curve G, Albumin + lipase + -chymotrypsin. Glycerol kinase added after 10 minute incubation period at 30#{176}C mm is present, as demonstrated by the constant rate of decrease in absorbance of the system before the addition of glycerol kinase (Figure 5, G), may be the result of the binding of fatty acids to this protein and thus to their solubilization and removal from the reactants. This was the principal reason for adding this protein to the reagent. The blank rate generated in the assay is due to phosphatases acting on PEP and ATP. The major sources of these contaminants are lipase, glycerol kinase, and possibly the sample itself. The rate owing to contaminating enzymes or loss in absorbance caused by contaminating substrates in the reagent can be determined for every batch by performing a blank assay with water in the place of the sample. Phosphate ions are known, to inhibit phosphatase activity (20), and their effect in reducing the activity of the contaminants in the reagent is shown in Table 2. The ph chosen was 7, even though the Table indicates that the phosphatase activity from the reagent is somewhat higher at this ph. On the other hand, it is important to inhibit alkaline phosphatases from serum, which may have higher activities than those contaminating the enzymes of the reagent. Under these conditions, and with this buffer, increase in blank rate caused by phosphatases in the sample has not been observed by us. Even when purified human intestinal or placental alkaline phosphatase was added to serum (2000 U per liter), the increase in blank rate of the system is negligible. The average increase noted at this concentration of enzyme was less than for 10 mm, equivalent to 13 mg of triglycerides per ml under the conditions of our assay. However, our experience in the application of this method has not yet been so extensive as to rule out increase in rate or inhibitors from sera in some pathological conditions or as a result of a particular medication. In these cases, individual 480 CLINICAL CHEMISTRY, Vol. 19, No. 5, 1973

6 Table 2. Effect of Various Buffers (0.1 mol/liter) and ph on the Phosphatase and ATPase Activity of Contaminants in the Triglycerides Reagent at 30#{176}C inhibition, Buffer ph Av A/mIn % Tris-maleate Tris-succinate Tris-succinate Potassium phosphate Potassium phosphate Potassium phosphate AUTO ANALYZER correction could be applied by taking a third absorbance reading 20 mm after addition of glycerol kinase, and subtracting the xa between 10 and 20 mm from that between 0 and 10 mm. Should the blank rate thus determined be lower than that found with the reagent alone, it would indicate a depletion in NADH or inhibition of some of the enzymes in the system. NADH may be depleted at high concentrations of triglycerides or as a result of high pyruvate content in the sample. This last compound may be present in high concentration in some control sera. The triglycerides reagent was modified according to these considerations and to the optimum concentration of the components as determined under the conditions of our assay. The reagent as finally developed contained: magnesium, 5 zmol; ATP, 0.9 zmol; PEP, 0.9 mol; bovine serum albumin, 5 mg; pyruvate kinase, 6 U; lactate dehydrogenase, 2 U; R. delemar lipase, 400 U; a-chymotrypsin, 30 U; NADH, 0.4 mg, all in a final volume of 3 ml in potassium phosphate buffer (ph 7, 0.1 mol/liter). Standard solutions of glycerol can be used to provide a control for the reagent. A solution containing 1 g of triolein in ml of 2-propanol can also be used. Aliquots of 5, 10, and 15 l of this solution will give a decrease in absorbance equivalent to that obtained by using 50 l of serum containing, respectively,, 200, and 300 mg triglycerides in ml. This will serve as a control for the hydrolyzing enzymes as well as for the glycerol reagent. We prefer to use a serum control standardized by assay of glycerol after alkaline hydrolysis or by the AutoAnalyzer procedure. Results for 38 sera by the present method vs. the AutoAnalyzer procedure (4) produced a correlation coefficient (r) of and a linear regression of y = x - 4 (Figure 6). Three serum pools were assayed on 10 different days by the present method. The results appear in Table 3. Other methods for determining glycerol liberated in the hydrolysis of triglycerides are currently under investigation. In general, they are not as convenient as the one presented here because in most cases additional steps are required, and they do not lend themselves as readily to the use of a single reagent. PRESENT METHOD Fig. 6. Triglycerides assays of 38 serum samples by AutoAnalyzer and by present method Mean Figures are in mg/ ml Table 3. Results of 30 Assays on Each of Three Serum Pools Pool SD ± CV % We thank Drs. W. Drell and J. K. Pollard, Jr. for their suggestions, sound advice, and constant support. CLINICAL CHEMISTRY. Vol. 19. No.5,

7 References 1. Carlson, L. A., and Wadstrom, L. B., Determination of glycerides in blood serum. Clin. Chim. Acta 4, 197 (19). 2. Henry, R. J., Clinical Chemistry: Principles and Technics, Hoeber, New York, N. Y., 1964, p Eggstein, M., and Kreutz, F. H., Eine neue Bestimmung der Neutralfette in Blutserum and Gewebe. KIm. Wochenschr. 44, 262(1966). 4. Kessler, G., and Lederer, H., Fluorometric measurement of triglycerides. In Automation in Analytical Chemistry, Technicon Symposia. 1965; L. T. Skeggs, et al, Eds. Mediad, New York, N.Y., 1966,p Rice, E. W., Triglycerides ( neutral fat ) in serum. Stand. Methods Clin.Chem. 6,215(1970). 6. Soloni, F. G., Simplified manual micromethod for determination of serum triglycerides. Clin.Chem. 17,529(1971). 7. Chin, H. P., Abd El-Meguid, S. S., and Blankenhorn, D. H., An improved method for determination of serum and plasma triglycerides. Clin. Chim. Acta 31, 381 (1971). 8. Folch, J., Lees, M., and Sloane-Stanley, G. H., A simple method for the isolation and purification of total lipids from animal tissue. J. Biol.Chem. 226, 497 (17). 9. Freeman, C. P., and West, D., Complete separation of lipid classes on a single thin-layer plate. J. Lipid Res. 7,324(1966). 10. Louis-Ferdinand, R. T., Therriault, D. G., Blatt, W. F., and Mager, M., Application of thin-layer chromatography to the quantitation of plasma neutral lipids and free fatty acids. Clin. Chem. 13,773(1967). 11. Laboureur, P., and Labrousse, M., Lipase de Rhizopus arrhizus. Obtention, purification, et proprietes de Ia lipase de Rhizopus arrhizus var. delemar. Bull. Soc. Chim. Biol. 48, 747 (1966). 12. Willis, E. D., Lipases. Advan. Lipid Res. 3, 197 (1965). 13. Mattson, F. H., and Beck, L. W., The digestion in vitro of triglycerides by pancreatic lipase. J. Biol. Chem. 214, 115 (15). 14. Mattson, F. H., and Beck, L. W., The specificity of pancreatic lipase for the primary hydroxyl groups of glycerides. J. Biol. Chem. 219, 735(16). 15. Mattson, F. H., and Volpenheim, R. A., Hydrolysis of primary and secondary esters of glycerol by pancreatic juice. J. Lipid Res. 9,79(1968). 16. Desnuelle, P., Chymotrypsin. In The Enzymes, 2nd ed., 4, P. D. Boyer, H. Lardy, and K. Myrback, Eds. Academic Press, New York, N. Y., 1960, pp 111 and Fukumoto, J., Lwai, M., and Tsujisaka, Y., Studies on lipase. IV. Purification and properties of a lipase secreted by Rhizopus delemar. J. Gen. Appl. Microbiol. 10, 257 (1964). 18. National Formularv, 13th ed., American Pharmaceutical Association, Washington, D. C , p Brochure on Pronase by Kaken Chemical Co., Ltd., Tokyo, Japan. Distributed by Calbiochem, La Jolla, Calif tadtman, T. C., Alkaline phosphatases. In The Enzymes, 2nd ed., 5, P. D. Boyer, H. Lardy, and K. Myrback, Eds. Academic Press, New York, N. Y., 1960, p CLINICAL CHEMISTRY, Vol. 19, No.5, 1973