A new method for measuring pancreatic phospholipase A2

Clinica Chimica Acta, 125 Elsevier Biomedical Press

(I 982) 359-365

359

CCA 2308

Brief technical

note

A new method for measuring pancreatic phospholipase A 2 Nigel

C. Bird

*

University Surgrcal Unit, Royal Hallamshire (Received

March

Hosprtal, Sheffield (UK)

18th; revision July 19th, 1982)

Introduction Phospholipase A, (EC 3.1.1.4) is an enzyme, produced by the pancreas, which selectively catalyses the hydrolysis of the 2-acyl group from sn-3-phosphoglycerides causing the release of a fatty acid with the formation of a lysophosphoglyceride. This hydrolysis occurs when the enzyme molecule and the substrate micelles form a stable complex. A considerable amount of work has been done on the nature of lipid-protein interaction in general and the mode of action of phospholipase in particular [l-3]. Studies by Verger et al [4] have shown that phospholipase A, contains an interface recognition site, distinct from its enzymatically active site, which is responsible for the interaction of the lipid/water interface. The presence of calcium ions is essential for activity; they are believed to be responsible for stronger binding to the micelles and therefore increase the rate of breakdown of substrate [3]. Pancreatic phospholipase is known to be secreted as a zymogen [5] consisting of a single polypeptide chain 130 amino acids long and containing 6 disulphide bridges (to which it owes its remarkable physical stability). Limited proteolysis by trypsin at Arg-7-Ala-8 produces the active enzyme, which only then is able to carry out hydrolysis at the lipid/water interface [6]. The problems in attempting to measure phospholipase A, activity lie partly in the difficulties of achieving satisfactory suspension of lipid substrate in aqueous media, and partly in those of measuring the products of reaction. Most commonly the assay has been done by titrating the free fatty acids released from an egg lecithin substrate. Modifications from the original method of Vogel and Zieve [7] have reduced the assay time considerably, but still utilise the titration of released fatty acids as an indication of enzyme activity. Presented here is a novel and rapid method for measuring pancreatic phospholipase A, activity which is sensitive, specific and suitable for the assay of large batches of samples. The substrate used is L-u-dipalmitoyl phosphatidylcholine and the palmitic acid released is measured by gasAddress Hospital,

for correspondence: Mr. N.C. Sheffield SlO 2JF, UK.

0009-8981/82/0000-0000/$02.75

Bird,

University

Surgical

0 1982 Elsevier Biomedical

Press

Unit,

Floor

K, Royal

Hallamshire

360

liquid chromatography of the methyl ester. Flash methylation in the injector body with (m-trifluoromethylphenyl) trimethyl ammonium hydroxide [8.9] (TTAH) dispenses with the need for multiple extraction and derivatisation steps. Materials and methods Chemicals and reagents All chemicals and reagents were analytical grade. Tris (hydroxymethyl) methylamine, n-heptane and ethylene diamine tetracetic acid (sodium salt) were obtained from BDH Chemicals, Poole, UK. Deoxycholic acid (sodium salt), L-cY-dipalmitoyl phosphatidylcholine and heptadecanoic acid were obtained from Sigma (London) Chemical Co. Ltd., Poole, UK. The (m-trifluoromethylphenyl) trimethyl ammonium hydroxide was prepared as described by MacGee and Allen [8]. Phospholipase A, preparation was obtained from BCL, Lewes, UK. Preparation of samples Duodenal juices were obtained by aspiration following duodenal intubation, the tip of the tube being screened by fluoroscopy until it was positioned at the ligament of Treitz. Samples were collected in crushed ice and frozen at - 20°C until assayed. Preparation of substrate A 9.0 mmol/l solution of L-a-dipalmitoyl phosphatidyl choline was prepared in 100 mmol/l Tris/HCl buffer pH 8.2, containing 4.8 mmol/l sodium deoxycholate. In order to achieve complete dispersion of the substrate it was necessary to vortex for 30 s and then heat the mixture to 60°C well above the transition temperature [lO,ll] for 10 min and then allow it to cool to 37°C before use. Method Samples were diluted 1 in 5 in working buffer and heated at 60°C for 15 min. 1 ml of substrate solution and 0.05 ml of diluted sample was incubated at 37°C for 15 min in stoppered glass tubes. The reaction was stopped by the addition of 0.1 ml EDTA (10 mmol/l). 0.1 ml of 1 mmol/l heptadecanoic acid added as the internal standard, followed by 5 ml of n-heptane and then the tubes shaken by hand for 30 s. (Vortex mixing produces a gel which is difficult to separate.) After centrifuging at 1500 X g for 1 min, 4 ml of supernatant were aliquoted into a stoppered conical centrifuge tube. 25 ~1 of UTAH was added and the tubes vortexed for 30 s. After allowing the layers to separate, 2 ~1 of the lower aqueous TTAH phase were injected into the gas chromatograph. Gas-liquid chromatography The gas chromatograph was a Varian Model 3700 equipped with a flame ionisation detector and glass column, 2 m X 4 mm I.D., packed with 12% Stabilised Diethylene Glycol Succinate (DEGS) on Anakrom AS 80- 100 mesh (Field Instru-

361

ments Ltd., Surbiton, detector 24O’C and the 30 ml/mm, the flame Recording, integration reporting integrator.

UK). The temperatures were, for injector 24O’C. The carrier gas was gases were hydrogen at 30 ml/min and calculations were made on a

the column 175”C, the nitrogen at a flow rate of and air at 300 ml/min. Hewlett-Packard 3390A

Results The substrate was initially prepared by the method of Menashe et al [12]. However, the technique described was simpler and no difference in enzyme activities could be detected. Fig. 1 demonstrated the linear relationship between activity and time under the conditions of the method. The rather broad optimum pH band observed by de Haas et al [5] was not so apparent in this assay system, in which it was at pH 8.1-8.2 (Fig. 2). Similarly the optimum temperature was a sharply defined 37°C (Fig. 3). The effect of increasing the concentration of Ca2+ from l- 10 mmol/l was in agreement with the findings of Figarella and Ribeiro [16] in that whilst Ca2+ were necessary in the reaction mixture, at concentrations above 2 mmol/l inhibition of the enzyme occurred (Fig. 4). Initial standardisation was achieved by preparing an aqueous solution of palmitic

I

I

8.0 Time

(min)

1

I

1

I

1

8.1

8.2

8.3

8.4

8.5

PH

Fig. 1. Plot of activity against incubation time. 0.8 tag phosphohpase A, preparation was incubated with 25 mg substrate in 5 ml buffer. At 10 min intervals, I ml was removed and aliquoted to a test tube containing 0.1 mg 10 mmol/l EDTA. Fig. 2. The effect of increasing pH on the enzyme activity. 0.16 pg phospholipase A, preparation was incubated with 1 ml substrate for 15 min at 37°C. The reactions were stopped with 0.1 mg 10 mmol/l EDTA.

362

60

1

10 -

I

,

,

,

,

,

,

, I

20 25 30 35 40 45 50 =C

I

I

5 mmol/l

10

Ca”

Fig. 3. Effect of increasing temperature on enzyme activity. 0.16 ug phospholipase A, preparation was incubated with 1 ml of substrate at 25. 30, 35. 37, 40 and 45°C. the reactions being terminated by the addition of 0.1 ml 10 mmol/l EDTA. in the reaction mixture. 0.16 pg phospholipase A, Fig. 4. Effect of increasing Ca2+ concentrations preparation was incubated with 1 ml of substrate mixtures containing 0, I, 2. 3. 5. 7 and 10 mmol/l Ca2+.

acid at a concentration of 100 l.~.mol/l in the assay buffer, and by using this solution in the substrate mixture determining response factors and recovery of the palmitic acid in the test system (Table I). The heptadecanoic acid as an internal standard was able to correct for variable extraction and methylation by the TTAH, since the reproducibility of injection and methylation was good (Table II). The specificity and reproducibility of the assay are shown in Tables III and IV.

TABLE

I

RECOVERY OF PALMITIC ACID STANDARD IN AQUEOUS SYSTEM IN 6 SAMPLES OF PANCREATIC PHOSPHOLIPASE Substance

Palmitic acid standard Test samples Test sample plus palmitic acid standard

Result (mean)

Recovery (X)

100 216

_ _

318

102

SOLUTION

FROM

THE

TEST

363

TABLE

II

REPRODUCIBILITY FLASH

Fatty

METHYLATED

acid

Heptadecanoic Methyl ester Palmitic acid Heptadecanoic

TABLE

OF 20 INJECTIONS PALMIC

ACID

OF HEPTADECANOIC

AND

HEPTADECANOIC

ESTER

AND

OF TWO PHOSPHOLIPASE

A,

Mea,n area

SD

cv

METHYL

4611860 3 365 750 21395 400

238 502 84919 623 302

5.2 2.52 2.27

acid,

acid

III

REPRODUCIBILITY OF TEN (BETWEEN BATCH) CONTROLS AND A PALMITIC ACID STANDARD Standard

ANALYSES

Mean

Palmitic acid (100 U/l Phospholipase A2 Phospholipase A2

TABLE

ACID

ACID

equivalent)

101.5 99.4 194.4

SD

cv

(U/l)

(%)

1.76 6.6 9.0

1.74 6.7 4.6

IV

SPECIFICITY

OF PHOSPHOLIPASE

A, IN THE REACTION

Sample activity

Phospholipase activity

(U/I)

(U/l)

SYSTEM Az

Phospholipase Az activity after incubation at 60°C (U/l)

Phospholipase Az Phospholipase C Phospholipase D Amylase Lipase Cholesterol esterase

loo 100 100

100 16 2

98 5 2

1000

0

0

1000 1000

8 5

2 5

From this it was possible to express the activity of phospholipase A, in units, one unit being defined as that amount of phospholipase A, causing the release of 1 umol of palmitic acid from L-cy-dipalmitoyl phosphatidylcholine per min at 37°C and pH 8.2. Discussion

A rapid, sensitive and specific method for the measurement of pancreatic phospholipase A, is presented. Precise control over the conditions of reaction are ensured

364

by the use of a pure homogenous substrate. Indeed, this is the most likely explanation for the sharply defined pH and temperature optima. The potentiometric titration methods [5,7,10,13,14] have long been established and developed to their maximum. They suffer from three main disadvantages. The first is that owing to the heterogenous nature of the substrate, optimum conditions have been difficult to establish [5]. Secondly, standardisation is difficult to achieve and activity is expressed as pmol of NaOH added per min. Thirdly, batch analysis is not possible whereas in the method presented batches may be analysed immediately or stored in the extracted n-heptane and measured at a later date. The use of organic quaternary ammonium salts for pyrolytic methylation in the injection port of the gas chromatograph was first described by Robb and Westbrook [ 151. Many modifications have been made to this original method in an attempt to reduce the severity of the conditions required for methylation. MacGee and Allen [8] described a method in which fatty acids extracted into n-hexane were then re-extracted into a small volume of (m-trifluoromethyl phenyl) trimethyl ammonium hydroxide. This is the basis of the method presented. Not only does it decrease the amount of time required to process samples but it also does not require the services of highly skilled and specialised personnel. The extraction into heptane and then the re-extraction into TTAH takes minutes and requires little skill. The method works well for the assay of phospholipase A, in samples of duodenal or pancreatic juice. Linearity is observed up to values of 1000 U/l and is suitable for batch analysis. Preliminary work suggests that the activity found in normal duodenal samples rises to approximately 2000 U/I after stimulation [ 161. References I Verger R. Interfacial enzyme kinetics of lipolysis. Ann Rev Biophys Bioeng 1976; 5: 77- 117. 2 Donne-Op der Kelder GM. Hille JDR. Dijkman R, de Haas GH, Egmond MR. Binding of porcine pancreatic phospholipase A, to various micellar substrate analogues. Involvement of histidine-48 and aspartic acid-49 in the binding process. Biochemistry 1981; 20: 4074-4078. studies 3 Hille JDR, Donne-Op der Kelder GM. Salve P. de Haas GH, Egmond MR. Physicochemical on the interaction of pancreatic phosphohpase A, with a micellar substrate analogue. Biochemistry 1981; 20: 406884073. 4 Verger R. Mieras MCE, de Haas GH. Action of phospholipase A at interfaces. J Biol Chem 1973; 248: 402334034. S De Haas GH, Postema NM, Nieuwenhuizen W. Van Deenen LLM. Purification and properties of phospholipase A from porcine pancreas. Biochim Biophys Acta 1968; 159: 103- 117. 6 De Haas GH. Slotboom AJ, Bonsen PPM. Studies on phospholipase A and its zymogen from porcine pancreas. I. The complete amino acid sequence. Biochim Biophys Acta 1970; 221: 31-53. Vogel WC, Zieve L. A lecithinase A in duodenal contents of man. J Clin Invest 1960; 39: 129S- 1301. MacGee J, Allen KG. Preparation of methyl esters from the saponifiable fatty acids in small biological specimens for gas-liquid chromatographic analysis. J Chromatogr 1974; 100: 35-42. Middleditch BS, Desiderio DM. Formation of fatty acid methyl esters during gas chromatography using trimethylanilinium hydroxide. Anal Lett 1972; 5: 605-609. Suurkuusk J. Lentz BR, Barenholz Y. Biltonen RL, Thompson TE. A calorimetric and fluorescent probe study of the gel-liquid crystalline phase transition m small, single-lamellar dipalmitoyl-phosphatidylcholine vesicles. Biochemistry 1976; 15: 1393- 1401. Op den Kamp JAF, De Gier J, Van Deenen LLM. Hydrolysis of phosphatidylcholine liposomes by

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12

13 14 15 16

pancreatic phospholipase A, at the transition temperature. Biochim Biophys Acta 1974; 345: 253-256. Menashe M, Lichterberg D, Gutierrez-Merino C, Biltonen RL. Relationship between the activity of pancreatic phospholipase A, and the physical state of the substrate. J Biol Chem 1981; 256(g): 4541-4543. Zieve L, Vogel WC. Measurement of lecithinase A in serum and other body fluids. J Clin Lab Invest 1961; 57: 586-599. Nieuwenhuizen W. Kunze H, de Haas GH. Phospholipase A, (phosphatide acylhydrolase EC 3.1.1.4) from porcine pancreas. Methods Enzymol 1974; 32(B): 147-154. Robb EW, Westbrook JJ. Preparation of methyl esters for gas liquid chromatography of acids by pyrolysis of tetramethylammonium salts. Anal Chem 1963; 35: 1644-1647. Figarella C. Ribeiro T. The assay of human pancreatic phospholipase A in pancreatic juice and duodenal contents. Stand J Gastroenterol 197 1; 6: 133- 137.