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The evaluation of a new rapid plasma lipase assay system
BIOCHEMICAL
MEDICINE
The
2,
413-426
Evaluation
( 1969)
of a New
Lipase ROBERT Department
Assay
D. MACKENZIE
Plasma
System
AND
EDWARD
M. AUXIER
The Wm.
of Biochemistry,
Division of Richardson-Merrell Received
Rapid
Inc.,
November
S. Merrell Company, Cincinnati, Ohio 45215
13, 1968
One of the conditions thought to be required for the development of atherosclerosis is hyperlipemia. There have been many reports (1, 2) that circulating lipoproteins perfuse the arterial wall and cause the deposition of lipid. On heparin administration Hahn in 1943 (3) first observed a clearing of lipemic plasma. It was later shown that post-heparin plasma contains a lipase (4, 5). Kom (4) reported on the properties and activities of the tissue lipase released by heparin, while Robinson and French (5) reviewed the properties of the lipase ( s) found in plasma after heparin administration. Heparin has been used to control lipemia in man. The assay of lipase activity should play an important role. in therapy. Fredrickson et al. (6) concluded that such an assay properly standardized. would be useful for wide application in the study of hyperglyceridemic subjects and their relatives. There is an abundance of literature on assay systems for plasma hpase. A large variety of lipid substrates, such as Ediol (3, 6, 7), emulsified olive oil (8)) lipoprotein fraction of plasma, or lipemic plasma ‘( 9), .have been used. Kom (4), Doizaki and Zieve (lo), Eiber et al. (II ), Baskys et al. ( 12, 13), and Tietz et al. (8) h ave reported on different substrates and other conditions for improvement of lipase assay. Many systems, especially those that use purified lipase, require an add,ed fatty acid acceptor such as albumin or calcium ion. Albumin, the best acceptor, must be reasonably pure and should have most of the free fatty acids removed by methods as described by Goodman (14)) or Chen ( 15). Change in turbidity, glycerol release, or free fatty acid liberation have been used in assay methods. The determination of the released free fatty acids is the most preferred assay. Many systems have been devised for such an assay. Dole and Meinertz (16) developed an extraction and two phase titration system, 413 @ 1969 by
Academic
Press,
Inc.
414
MACKIWZIE
AND
AUXJER
while Trout et al. (17) modified this system to improve its specificity. Calorimetric procedures have also been devised, such as those reported by Duncombe ( 18)) Mendelsohn ( 19)) and Novak (20). We have developed a calorimetric assay system for free fatty acids (21) that can be used for determining lipase activity. This system is simple, and a large number of samples can be assayed in a short time. The purpose of this report is to give the details of this new assay system and cite data obtained from the investigation of several properties of postheparin plasma lipase. MATERIALS
AND
METHODS
Reagents Edid. A fat emulsion made of 50% coconut oil. This material was made by Schenlabs, and obtained from Riker Laboratories, Northridge, California, in 1961. Lipostrute-CB. A fat emulsion similar to Ediol made by Calbiochem, Los Angeles, California. 2% B@er. Buffer solutions made up of Tris hydroxymethyl amino methane, Fisher Primary Standard, and various amounts of hydrochloric acid for various pH systems. The ionic strength was 0.15. Heparin. Heparin, sodium, U. S. P., 150 and 162 units/ mg batches from Wilson Laboratories, Chicago, Illinois were used in this study. Sodium Citrate. Fisher Certified Reagent, made up as a 3.8% solution. Methylal. Matheson, Coleman, and Bell, Cincinnati, Ohio. Petrobum Ether. Redistilled, 40-60”. Isopropyl Alcohol. Reagent grade. Neptune. Redistilled, 98-99”. Sulfuric Acid. A 0.0% solution. Rhodumine B Hydrochkvide. British Drug Houses*, Poole, Dorset, England, distributed in the United States by Gallard-Schlesinger Chemical Mfg. Corp., Carle Place, L. I., N. Y., an aqueous solution, 1 mg/ml. Uranyl Acetate. Fisher Certified Reagent, a 1% aqueous solution. Sodium Hydroxi&. A 0.02 N solution, carbonate free. Zeolite. W. A. Taylor & Co., Baltimore, Maryland, ground to 100 mesh and dried in an oven at 110” overnight. Nile B&e A. Matheson, Coleman, and Bell, OX in distilled water, washed with heptane until free of extractable colored impurities. Phosphoric Acid. Fisher Certified Reagent, 85%. Instrumentation Spectrophotometer. A Bausch and Lomb Spectronic 20. All absorption measurements were made at 545 mp. Mechanical Shaker. An E&&a& reciprocating type. Centrifuge. International, Size I Model SBR. Water Bath. A Blue M Magna Whirl. 1 Rhodamine
B from
one other
supplier
was not satisfactory
for
this
use,
A RAPID
PLASMA
LIPASE
ASSAY
SYSTEM
415
General Methods Plasma Isolation. Blood was taken by heart puncture from anesthetized rats and from the cephalic vein in dogs. The blood was collected with a 5-ml syringe that had been rinsed with a heparin-isotonic saline solution (0.5 mg/ml). In several experiments sodium citrate was used as the anticoagulant by adding 1 part citrate solution for every 9 parts of blood. The blood was centrifuged at 9OOg for 10 minutes and plasma was isolated. Zncufxztion System for Lipase Assay. The plasma was removed from the centrifuge tube by pipette and 0.5 ml placed in two separate tubes ( 16 x 125mm screw cap culture tubes, with a Teflon-lined cap). One of these plasma samples (blank) was placed in the refrigerator. The other sample (test) had the following materials added: 0.1 ml of a 5% Ediol solution and 0.5 ml of Tris buffer pH 8.1. Usually these two solutions were combined 1 part 5% Ediol solution to 5 parts Tris buffer and 0.6 ml of the mixture was added to the plasma. This test mixture was incubated in a water bath at 37”. At the sametime, a mixture of Ediol solution and Tris buffer ( 1:5) was also incubated. At the end of the incubation 0.6 ml of the Ediol-Tris mixture was added to the blank plasma sample, from the refrigerator, and both plasma-EdioI-Tris sampIes were extracted. Extraction and Assay of Free Fatty Acids. The extraction of fatty acids from plasma and their assay are described by MacKenzie et al. (21). The assay is based on a colored complex soluble in toluene between Bhodamine B, uranyl ion, and the fatty acid anion. The amount of free fatty acid found in the blank sample was subtracted from the amount found in the incubated (test) sample. The difference is the amount of fatty acid liberated due to lipase activity. RESULTS
A. Effect of Heparin
on Lipase Actiuity-Fatty
Acid Release
(1) Comparison of a Cokwimetric Fatty A.& Assay with Titration Albino rats were given 1 or 10 mg/kg (162 or 1620 units/kg) of heparin by subcutaneous injection. The rats were anesthetized with ether and blood was removed by heart puncture 30 minutes after heparin administration. Post-heparin plasma was pooled from a group of six animals and incubated with Ediol-Tris buffer mixture, pH 8.1. As soon as these ingredients were mixed, an aliquot was removed and used as the blank sample. The rest of the plasma-fat emulsion system was incubated at 37” and aliquots were removed at 10, 30, 60, and 126 minutes. All aliquots were extracted by the methylal-methanol-petroleum ether system (21),
Method.
416
MACKENZIE
AND
AUXIEH
and six aliquots of each extract assayed, three by titration (IS), and three by the calorimetric method (21). Figure 1 shows the average values for fatty acids liberated by both methods plotted versus incubation time. There was excellent agreement between methods. 12.0
1
6.0.
: 5 E IO
I
30 60 INCUBATION TIME,
120 MINUTES
FIG. 1. Comparison of titration method with calorimetric method for post-heparin plasma lipase activity. ( 0 ) Calorimetric method, 16.2 units/kg; ( 0 ) titration method, 162 u&s/kg; (0) calorimetric method, 1620 units/kg; ( 0 ) titration method, 1620 units&. ‘Ediol was used as substrate.
The calorimetric method was used in all further work. (2) Effect ofI ncu bat ion Time on Fatty Acid Liberation. Post-heparin rat plasma was isolated after the injection of either 1 or 10 mg/ kg ( 162 or 1626 units/kg) of heparin to rats as described above. When Ediol was used as a substrate for the assay of the post-heparin plasma lipase, the liberation of fatty acids was found to be linear with time on a semilog plot between 5 minutes to at least 3 hours (Fig. 2). (3) Enzyme Concentration Versus Fatty Acid Liberation. Post-heparin plasma was obtained from a blood sample taken 20 minutes after a beagle dog was given 10 mg/kg (1500 units/kg) of heparin intravenously. Prior to heparin administration, a control plasma sample was obtained from the same dog. The post-heparin plasma was diluted to concentrations of 75, 50, and 25%with the control plasma. Aliquots from all five samples were incubated with Ediol-Tris at pH 8.1 for 3 hours. Aliquots were removed
A RAPID
PLASMA
LIPASE
ASSAY
417
SYSTEM
ii 8.0-
0
I 2 ,,, 6.0+ z
o;
5
IO INCUBATION TIME.
30 MINUTES
100
180
FIG. 2. Relationship of FFA release to time iu Ediol-post hepariu plasma-T& buffer system. Calorimetric FFA assay. Upper curve, 1620 heparin units/kg rat; lower curve, 162 units/kg.
at different intervals and free fatty acid was determined by calorimetric method. The results are shown in Table 1. Figure 3 shows the values obtained at 30 minutes and 3 hours incubation when plotted as PM fatty acid liberated per ml plasma versus log concentration of lipase, in terms of percentage maximum concentration of the post-heparin plasma. A linear TABLE 1 EFFECT OF LIPASE CONCENTRATION ON FREE
FATTY
ACID
PM Free fatty acid liberated/ml Incubation time (minutes) -0 5 10 15 20 30 60 120 180
Percentage concentration
LIBERATION
plasma
post-heparin
plasma
25
50
75
100
9 1.72” 2.15 2.00 2.22 2.50 3.94 5.37 5.65
0 1.93 2.57 2.72 2.93 3.87 5.01 6.87 7.58
0 1.93 2.85 3.00 3.43 3.93 5.50 6.93 8.13
0 1.86 2.43 2.72 3.43 4.01 6.51 7.50 8.49
(1Average of three determinat,ions;
Ediol used as substrate.
418
MACKENZIE
0
AND
AUXIER
A_1
25 PERCENT
POST
SO HEPARIN
7’5
lb0
PLASMA
FIG. 3. Effect of lipase concentration on fatty acid liberation. PM Fatty acid liberated from Ediol after: ( 0 ) 30 minutes incubation; (0) 3 hours incubation, the equation for the line is y = 4.94x + 1.7.
response was obtained at 3 hours. For incubations of 30 minutes or less, linear responseswere not obtained. (4) Effect of Heparin on Fatty Acid Liberation. Three dogs were given heparin at 0.1, 0.2, 0.3, 1.0, 3.0, and 10 mg/kg (15-1500 units/kg) by intravenous injection. The different doseswere given to the same three dogs about 4 days apart. Blood was taken 15-20 minutes after heparin administration and plasma was isolated and pooled. The plasma was assayed for lipase activity as described. Lipostrate-CB was used as the source of triglyceride. Assays were made from duplicate aliquots of the assay mixture starting at 5 minutes of incubation and at various intervals up to 3 hours. A single aliquot was taken at 0, 1, 2, 3, and 4 minutes. The results are tabulated in Table 2. Figure 4 shows the dose response plotted as PM fatty acid liberated per ml plasma versus log dose for the 30-minute and 3-hour values. The data obtained from the 3-hour values gave a very good linear dose response from 0.1 through 3 mg/kg. Ten mg/kg of heparin gave a lower activity. The best dose response was obtamed with the 3-hour values; at the lower incubation times linearity and sensitivity were not so good. At the beginning of incubation there is a lag period in which no or little fatty acid is liberated. This lag period varies from 5-15 minutes in differ-
A RAPID
PLASMA
LIPASE
TABLE EFFECT
OF HEPARIN
DOSE
ASSAY
SYSTEM
FATTY
ARID
2
ON FREE
PM Free fatty acid liberate& Incubation time (minutes) 0 1” 2 3 4 5"
10 15 30 60 90 120
180
419
LIBERATION
ml plasma
Dose of heparin (mg/kg) 0.1
0.2
0 .:3
0 0 0 0 0.11 0.03 0.05 0.09 0.17 0.24 0.44 0.81 1.15
0 0 0 0.14 0.17 0.24 0.30 0.47 0.54 1.24 2.06 2.48 3.45
0 0.01 0.03 0 14 0.30 0.38 0.42 0.64 0.95 1.63 2.44 3.96 4.48
1.0 0 0.12 0.23 0.37 0 3.5 0.71 1.76 2.36 3.34 4.78 5.76 6.63 7.26
3.0
10.0
0 0.06 0.10 0.14 0.51 0.68 1.48 2.56 3.80 5.84 7.26 8.65 10.02
0.42 0.45 0.58 0.69 0.78 0.88 1.10 2.62 4.38 5.23 6.35 7.33
0
a One determination only for l-5 minutes of incubation time. b Average of duplicate assays for S-180 minutes of incubation time. c Lipostrate-CB used as substrate.
0
DOSE
OF HEPARIN
(mO/kOi
FIG. 4. Effect of heparin dose on fatty acid liberation. PM Fatty acid liberated from Lipostrate-CB after ( 0) 30 minutes incubation; ( 0) 3 hours incubation.
420
MACKENZIE
AND
AUXIER
ent experiments. This is the main reason for the best dose response occurring at 3 hours and not at a shorter interval. This lag period is probably due to the need for activation of the substrate with the lipoproteins present in the plasma sample. B. Effect
of Various
Conditions
on Lipolytic
Activity
(1) Effect of pH. Post-heparin plasma was pooled from three dogs given 1 mg/kg (150 units/kg) of heparin by subcutaneous injection. Post-heparin plasma was also pooled from 12 rats given 2 mg/kg of heparin by subcutaneous injection, Lipolytic activity was determined by using T&-buffered Ediol emulsion in the pH range of 7.0-9.0. The 3-hour incubation mixtures were assayed for fatty acid liberation. Determinations were made in quadruplicate. Figure 5 illustrates that the optimum pH for post-heparin plasma lipase activity is 8.1 in both species. 6.0
1
INCUBATION FIG. liberated
5. Effect of buffer from Ediol after
pH on post-heparin 3 hours incubation,
pH plasma lipase (0) Dog, (n)
activity. Rat.
PM
Fatty
acid
(2) Substrate Effects. The Ediol obtained from Schenlabs (Riker) was compared with the new emulsion Lipostrate-CB. The thin-layer chromatographic separation of the lipids according to Freeman and West (21) showed the same lipid pattern except for free fatty acid levels, Our Ediol, which had been stored for 7 years, contained much larger amounts of free fatty acid than Lipostrate, 64 PM/ml versus 7.2 PM/ml for Lipostrate. Post-heparin plasma was pooled from two dogs given 1 mg/kg (150
A RAPID
PLASMA
LIPASE
ASSAY
421
SYSTEM
unit/kg) of heparin intravenously. Each triglyceride emulsion was diluted to 5% (v/v) with distilled water and mixed 1:5 with Tris buffer pH 8.1. The mixtures were incubated and duplicate aliquots were removed for assay at various intervals up to 3 hours. The average values are plotted in Fig. 6. The data indicate equivalent fatty acid liberation up to 2 hours. The 3-hour values are significantly different. Lipostrate was the better substrate. Its superiority is possibly due to: (I) Superior emulsification lO.O-
a 9.0. I cn a 2 8.0. ,E g
7.0.
2 4
6.0.
3 2 6.0. : G
E 4.0. a 3.01 15
FIG. 6. Comparison plasma lipase activity.
30 INCUBATlON
of Ediol ( 0 ) Ediol,
60 90 120 TIME (minutes)
and Lipostrate-CB ( 0 ) Lipostrate-CB.
as substrates
180 for
post-heparin
because it is a fresher preparation, and (2) lower product concentration initially (fatty acids). The higher fatty acid level would inhibit the enzymatic reaction (23, 24), especiahy since there is a limited amount of carrier for the product present in the incubation system. In other experiments such as under A 2, Ediol gave linear response at lower total free fatty acid liberation. (3) Enzyme Stability. Post-heparin plasma was collected and pooled from three dogs given 1 mgf kg ( 150 units/ kg) of heparin subcutaneously. It was also isolated from 12 rats given 2 mg/kg of heparin subcutaneously. Blood was taken from the rats 45-66 minutes after heparin administration and 30-45 minutes after administration to dogs. Both
422
MACKENZIE
AND
AUXIIZR
pooled samples were separated into five groups of four equal aliquots (0.5 ml). These five groups consisted of samples to be assayed fresh, after freezing for 24 and 48 hours and refrigerated for 24 and 48 hours. The lipolytic assay was made with Ediol as source of triglyceride. The data are reported in Table 3. No great loss in activity occurred in 2 days ( Q 22%). If frozen for 24 hours no significant loss occurred. (4) Inhibition of Lipase Activity by NaCE. Post-heparin plasma was obtained by giving 3 mg/kg (450 units/kg) of heparin intravenously to a dog. Blood was collected 15 minutes after heparin administration. The plasma was incubated with a Tris buffer-Lipostrate mixture. One incubation tube contained buffer at pH 8.6 and another tube at pH 8.1. One tube at each pH had NaCl added to a concentration of 1 molar. The fifth TABLE THE
3
DETERMIRTAT~OIS OF POST-HEPARIN RAT AND DOG PLASMA SJYABILITY ON REFRIGERATIOX ASD FREEZING, USING EDIOL AS SUBSTRATE
Rat
Dog Pooled samples Fresh Frozen-24 hours Refrigerated-24 hours Frozen48 hours Refrigerated48 hours
Percentage of fresh samples
/.a FFA liberated 3.90” 3.96 3.70 3.56 3.44
+ 0.15 31 0.05 * 0.07 f 0.06 zk 0.06
0 Average of four determinations b Average of four determinations
LIPASE
100
102 95 91 88
pi FFA liberated 4.93b 4.53 4.60 3.97 3.83
Percentage of fresh samples
f 0.09 *
0.11
31 0.08 i 0.04 + 0.16
100 93 93 81 78
+ SEM from pooled sample (3 dogs). + SEM from pooled sample (12 rats).
incubation system had the plasma preincubated for 15 minutes with the 1 M NaCl before the Tris buffer-Lipostrate pH 8.1 was added. Aliquots were removed at various intervals up to 3 hours and the quantity of free fatty acid liberated determined. The data are shown in Table 4. There was a slightly greater percentage inhibition at pH 8.1 than at 8.6, though the total amount of fatty acid liberated in 3 hours in the NaCl containing systems was the same at both pH values. There was slightly more inhibition in the tube preincubated for 15 minutes, but this was not significant. Since Kom (4) has shown that 1 M NaCl inhibits lipoprotein lipase lo@ the data indicate that about 15%of the lipase activity is due to some other enzyme. (5) Eflect of Anticoagulunt Type on Lipase Activity. When postheparin plasma is isolated from blood, an anticoagulant must be used. Usually we have used small quantities of heparin, but citrate as an exam-
A RAPID PLASMA
INHIBITORY
EFFECT
LJPASE
acid
Condition
pH (1)
0 15 30 60 120 180 Percentage o NaCl
8.6
0 1.14 2.36 3.58 5.58 6.36 inhibition incubated
pH 8.6 +lM KaCI (11) 0 1.04 1.21 1.04 1.04 1 57
the plasma
mixture
+l
0 1.92’ 2.79 4.71 7.81 9 33
LIPASE
plasma
of incubation
75 with
PLASMA
liberated/ml
pH x.1 (III)
423
SYSTEM
TABLE 4 OF 1 M NaCl ON POST-HEPARIN 1~ Fatty
Incubation time (min)
ASSAY
pH X.1 M NaCl (IV,
pH 8.1 + 1 M NaCla 09
0 0.69 0.87 0.96 1.57 1.57
0 0.43 0.70 0.87 1.31 1.39
83 for
15 minutes
before
Ediol-Tris
85 buffer
added.
ple of the decalcifying agents could also be used. The effect of both types and their combination was determined. No significant difference was observed in lipase activity, when heparin at 20 units/ml, sodium citrate at 3.3% ( 1 part to 9 parts of blood), or the combination of both anticoagulants was used to prevent coagulation. (6) General 0bse~oation.s. When heparin activates lipase in different species, it does not have the same potency. In our studies the rat and dog were used. The dog has by far the more responsive system to heparin administration. Dog plasma also has only one fifth of the endogenous lipase activity that rat plasma contains. These observations agree with the report of Monkhouse and MacKneson (25). DISCUSSION
The method described for the assay of lipase in plasma is a simple and rapid procedure. The plasma is used not only for a source of lipase but also for the accepters of the free fatty acids, albumin, and calcium ion. The incubation mixture, therefore, contains the minimum ingredients required for such a test; plasma, the source of lipase, lipoproteins, albumin, and calcium ion; fat emulsion, source of substrate for enzyme action; buffer, to maintain optimum pH of system. The amount of anticoagulant (heparin) used in the isolation of plasma should be limited to only that required to prevent clotting. Large quantities can inhibit the system (26). The incubation time for a good dose response relationship is 3 hours, though any time after 1 hour may be used with reasonable results. It was
424
MACKENZIE
AND
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found that the earlier times did not give a good dose response even though zero order kinetics should occur initially. In our experiments we found a lag time, which is interpreted as the time required for the lipoproteins present in the plasma to activate the fat emulsion. The experiments on the relationships of lipase concentration and heparin dose to rate of fatty acid liberation agree with those reported by Wenke et al. (27). Since these produced linear relationships, the method can be used for quantitative estimations. By comparing the effect of various inhibitors, Hollett ( 28 ) reported that tissue lipase and plasma lipase released by heparin are not the same enzyme. Baskys et al. (12) did report on the optimum pH of post-heparin plasma lipase showing it to be 8.1. We have confirmed this. According to Korn (4) and others tissue Iipase activity is optimum at pH 8.5. This also indicates that the lipases are different. Ediol is a substrate used by many investigators (7, 11, 23, 29), but other substrates have also been reported: Intralipid (30)) olive oil emulsion ( 8)) and lipemic plasma (9). A new form of Ediol called LipostrateCB, available from Calbiochem, Inc., was compared with the Ediol made by Schenlabs, Lipostrate has the same formula as Ediol, but our old Ediol contained 9 times the concentration of free fatty acid as did Lipostrate. The use of Lipostrate showed similar fatty acid liberation up to 2 hours, after which there was greater liberation when Lipostrate was used. Van Den Bosch et al. (23) explained this as an effect on surface tension by the liberated free fatty acids. The lowered surface tension inhibits the lipase activity. Fielding (24) suggests that a complex is formed between free fatty acid and lipase. He suggests that the complex stabilizes the lipase so that it will not “inactivate.” His method, however, was not designed to indicate inactivity due to this stabilizing effect, What appears quite clear in this area of research is that there needs to be an international method standardization for the assay of post heparin plasma lipase. At this time no two laboratories can compare their data because different methods are used. Many investigators at present are using suboptimum, nonphysiological pH values from 8.5 to as high as 8.8, though optimum is 8.1. Not only does pH or buffer vary but also substrate. There is a great need for a standard substrate that all investigators can use. Ideally, lipoprotein should be the substrate. Since such a standard would be difficult to mass produce and would not be stable, a standard fat emulsion must be suggested. SUMMARY
A rapid, simple, and sensitive method is described for the assay of post-heparin plasma lipase. The incubation system contains only the in-
A RAPID
PLASMA
LIPASE
ASSAY
SYSTEM
425
gredients required for the assay: plasma, as the source of lipase, lipoprotein, and albumin; fat emulsion as substrate, and Tris buffer, pH 8.1. With a S-hour incubation period, liberation of fatty acid is proportional to log of either heparin dose or lipase concentration. The lipase assay utilizes a new free fatty acid assay procedure (21) developed in this laboratory. This procedure depends on the formation of a complex soluble in toluene between Rhodamine B, uranyl ion, and the fatty acid anion. The method was used to investigate the effects of pH, 1 M NaCI, and substrate on the assay system. The stability of the enzyme on refrigeration and freezing was also investigated. A need for an international standardized method is mentioned. ACKNOWLEDGMENTS We acknowledge the encouragement, guidance, and patience given us by Dr. T. R. Blohm. We thank Drs. N. L. Wiech and J. W. Newbeme (Pathology-Toxicology Department), and Ft. Hannah (Medical Research) for their helpful suggestions in the preparation of this manuscript. REFERENCES 1. WATTS, H. F., in “Role of Lipoproteins in the Formation of Atherosclerosis; Evolution of the Atherosclerosis Plaque” (R. J. Jones, ea.), pp. 117-132. University of Chicago Press, Chicago, Illinois, 1963. 2. CONSTANTIIWDES, P., “Experimental Atherosclerosis,” Elsevier, Amsterdam, 1965. 3. HAHN, P. F., Science 98,19 (1943). 4. KORN, E. D., in “Methods of Biochemical Analysis,” Vol. 7, (D. Click, ea.), pp. 145-192. Wiley (Interscience) New York, 1959. 5. ROBINSON, D. S., AND FRENCH, J. E., Pharmuool. Reu. 12, 241 (1966). 6. FREXXWXSON, D. S., ONO, K., AND DAVIS, L. L., J. Lipid Res. 4, 24 (1963). 7. HOLLETT, C. R., Biochim. Biophys. Acta 98, 53 (1965). 8. TIETZ, N. W., BORDEN, T., AND STEPLETON, J. D., Am. J. Clin. Pathol. 31, 148 (1959). 9. SALAMAN, M. R., AND ROBINSON, D. S., Biochem. J. 99,640 ( 1966). 10. DOIZAKI, W. M., AND ZIEVE, L., Proc. Sot. ErptZ. Biol. Med. 122, 606 (1966). 11. EIBER, H. B., PAYZA, A. N., AND GOLDBERG, B., Biochim. Biophys. Acta 116, 256 (1966). 12. BASKYS, B., KLEIN, E., AND LEVER, W. F., Arch. Biochem. Biophys. 99, 25 (1962). 13. BASKYS, B., KLEIN, E., AND LEVER, W. F., Arch. Biochem. Biophys. 99, 31 (1962). 14. GOODMAN, D. S., Science 125,1296 (1957). 15. CHEN, R. F., J. BioZ. Chem. 242,173 (1967). 16. DOLE, V. P., AND MEINERTZ, H., J. BioZ. Chem. 235,2595 (1960). 17. TROUT, D. L., ESTES, E. M., AND F-ERG, S. J., J. Lipid Res. 1, 199 (1960). 18. DUNCOMB, W. G., Biochem. 1. 88,7 (1963). 19. MENDELSOHN, D. S., S. Ajricun J. Med. Sci. 23,75 (1958). 20. NOVAK, M., J. Lipid Res. 6, 431 ( 1965).
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21. MACKENZIE, R. D., BLOHM, T. R., AUXIER, E. A., AND LUTHER, A. C., J. Lipid Res. 8, 589 ( 1967). 22. FREEMAN, C. P., AND WEST, D., J. Lipid Res. 7,324 ( 1966). 23. VAN DEN BOSCH, J., EVRARD, E., DESOMER, P., AND JOOSENS, J. V., Arch. Intern. Phurmucodyn. 130,64 ( 1961). 24. FIELDING, C. J., Biochim. Biophys. Actu 159, 94 (1963). 25. MONKHOUSE, F. C., AND MACKNESON, R. G., Can. J. Biochem. Physid. 36, 1065 (1958). 26. PAYZA, A. N., EIBF.R, H. B., AND DANISHEFSK~, I., Biochim. Biophys. Actu 111, 159 ( 1965). 27. WENKE, M., WENKEOVA, J., MUHLBACHOVA, E., AND HYNJE, S., Arch. Intern. Pharmucodyn. 134,417 (1961). 28. HOLLETT, C. R., Arch. Biochem. Biophys. 108,244 ( 1964). !?I% PASTA, A. N., EIEIER, H. B., AND WALTERS, S., Proc. Sot. Erptl. Biol. Med. 125, 188 (1966). 30. MUIR, J. R., Clin. Chim. Acfu 17,312 (1967).
MEDICINE
The
2,
413-426
Evaluation
( 1969)
of a New
Lipase ROBERT Department
Assay
D. MACKENZIE
Plasma
System
AND
EDWARD
M. AUXIER
The Wm.
of Biochemistry,
Division of Richardson-Merrell Received
Rapid
Inc.,
November
S. Merrell Company, Cincinnati, Ohio 45215
13, 1968
One of the conditions thought to be required for the development of atherosclerosis is hyperlipemia. There have been many reports (1, 2) that circulating lipoproteins perfuse the arterial wall and cause the deposition of lipid. On heparin administration Hahn in 1943 (3) first observed a clearing of lipemic plasma. It was later shown that post-heparin plasma contains a lipase (4, 5). Kom (4) reported on the properties and activities of the tissue lipase released by heparin, while Robinson and French (5) reviewed the properties of the lipase ( s) found in plasma after heparin administration. Heparin has been used to control lipemia in man. The assay of lipase activity should play an important role. in therapy. Fredrickson et al. (6) concluded that such an assay properly standardized. would be useful for wide application in the study of hyperglyceridemic subjects and their relatives. There is an abundance of literature on assay systems for plasma hpase. A large variety of lipid substrates, such as Ediol (3, 6, 7), emulsified olive oil (8)) lipoprotein fraction of plasma, or lipemic plasma ‘( 9), .have been used. Kom (4), Doizaki and Zieve (lo), Eiber et al. (II ), Baskys et al. ( 12, 13), and Tietz et al. (8) h ave reported on different substrates and other conditions for improvement of lipase assay. Many systems, especially those that use purified lipase, require an add,ed fatty acid acceptor such as albumin or calcium ion. Albumin, the best acceptor, must be reasonably pure and should have most of the free fatty acids removed by methods as described by Goodman (14)) or Chen ( 15). Change in turbidity, glycerol release, or free fatty acid liberation have been used in assay methods. The determination of the released free fatty acids is the most preferred assay. Many systems have been devised for such an assay. Dole and Meinertz (16) developed an extraction and two phase titration system, 413 @ 1969 by
Academic
Press,
Inc.
414
MACKIWZIE
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while Trout et al. (17) modified this system to improve its specificity. Calorimetric procedures have also been devised, such as those reported by Duncombe ( 18)) Mendelsohn ( 19)) and Novak (20). We have developed a calorimetric assay system for free fatty acids (21) that can be used for determining lipase activity. This system is simple, and a large number of samples can be assayed in a short time. The purpose of this report is to give the details of this new assay system and cite data obtained from the investigation of several properties of postheparin plasma lipase. MATERIALS
AND
METHODS
Reagents Edid. A fat emulsion made of 50% coconut oil. This material was made by Schenlabs, and obtained from Riker Laboratories, Northridge, California, in 1961. Lipostrute-CB. A fat emulsion similar to Ediol made by Calbiochem, Los Angeles, California. 2% B@er. Buffer solutions made up of Tris hydroxymethyl amino methane, Fisher Primary Standard, and various amounts of hydrochloric acid for various pH systems. The ionic strength was 0.15. Heparin. Heparin, sodium, U. S. P., 150 and 162 units/ mg batches from Wilson Laboratories, Chicago, Illinois were used in this study. Sodium Citrate. Fisher Certified Reagent, made up as a 3.8% solution. Methylal. Matheson, Coleman, and Bell, Cincinnati, Ohio. Petrobum Ether. Redistilled, 40-60”. Isopropyl Alcohol. Reagent grade. Neptune. Redistilled, 98-99”. Sulfuric Acid. A 0.0% solution. Rhodumine B Hydrochkvide. British Drug Houses*, Poole, Dorset, England, distributed in the United States by Gallard-Schlesinger Chemical Mfg. Corp., Carle Place, L. I., N. Y., an aqueous solution, 1 mg/ml. Uranyl Acetate. Fisher Certified Reagent, a 1% aqueous solution. Sodium Hydroxi&. A 0.02 N solution, carbonate free. Zeolite. W. A. Taylor & Co., Baltimore, Maryland, ground to 100 mesh and dried in an oven at 110” overnight. Nile B&e A. Matheson, Coleman, and Bell, OX in distilled water, washed with heptane until free of extractable colored impurities. Phosphoric Acid. Fisher Certified Reagent, 85%. Instrumentation Spectrophotometer. A Bausch and Lomb Spectronic 20. All absorption measurements were made at 545 mp. Mechanical Shaker. An E&&a& reciprocating type. Centrifuge. International, Size I Model SBR. Water Bath. A Blue M Magna Whirl. 1 Rhodamine
B from
one other
supplier
was not satisfactory
for
this
use,
A RAPID
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415
General Methods Plasma Isolation. Blood was taken by heart puncture from anesthetized rats and from the cephalic vein in dogs. The blood was collected with a 5-ml syringe that had been rinsed with a heparin-isotonic saline solution (0.5 mg/ml). In several experiments sodium citrate was used as the anticoagulant by adding 1 part citrate solution for every 9 parts of blood. The blood was centrifuged at 9OOg for 10 minutes and plasma was isolated. Zncufxztion System for Lipase Assay. The plasma was removed from the centrifuge tube by pipette and 0.5 ml placed in two separate tubes ( 16 x 125mm screw cap culture tubes, with a Teflon-lined cap). One of these plasma samples (blank) was placed in the refrigerator. The other sample (test) had the following materials added: 0.1 ml of a 5% Ediol solution and 0.5 ml of Tris buffer pH 8.1. Usually these two solutions were combined 1 part 5% Ediol solution to 5 parts Tris buffer and 0.6 ml of the mixture was added to the plasma. This test mixture was incubated in a water bath at 37”. At the sametime, a mixture of Ediol solution and Tris buffer ( 1:5) was also incubated. At the end of the incubation 0.6 ml of the Ediol-Tris mixture was added to the blank plasma sample, from the refrigerator, and both plasma-EdioI-Tris sampIes were extracted. Extraction and Assay of Free Fatty Acids. The extraction of fatty acids from plasma and their assay are described by MacKenzie et al. (21). The assay is based on a colored complex soluble in toluene between Bhodamine B, uranyl ion, and the fatty acid anion. The amount of free fatty acid found in the blank sample was subtracted from the amount found in the incubated (test) sample. The difference is the amount of fatty acid liberated due to lipase activity. RESULTS
A. Effect of Heparin
on Lipase Actiuity-Fatty
Acid Release
(1) Comparison of a Cokwimetric Fatty A.& Assay with Titration Albino rats were given 1 or 10 mg/kg (162 or 1620 units/kg) of heparin by subcutaneous injection. The rats were anesthetized with ether and blood was removed by heart puncture 30 minutes after heparin administration. Post-heparin plasma was pooled from a group of six animals and incubated with Ediol-Tris buffer mixture, pH 8.1. As soon as these ingredients were mixed, an aliquot was removed and used as the blank sample. The rest of the plasma-fat emulsion system was incubated at 37” and aliquots were removed at 10, 30, 60, and 126 minutes. All aliquots were extracted by the methylal-methanol-petroleum ether system (21),
Method.
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MACKENZIE
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and six aliquots of each extract assayed, three by titration (IS), and three by the calorimetric method (21). Figure 1 shows the average values for fatty acids liberated by both methods plotted versus incubation time. There was excellent agreement between methods. 12.0
1
6.0.
: 5 E IO
I
30 60 INCUBATION TIME,
120 MINUTES
FIG. 1. Comparison of titration method with calorimetric method for post-heparin plasma lipase activity. ( 0 ) Calorimetric method, 16.2 units/kg; ( 0 ) titration method, 162 u&s/kg; (0) calorimetric method, 1620 units/kg; ( 0 ) titration method, 1620 units&. ‘Ediol was used as substrate.
The calorimetric method was used in all further work. (2) Effect ofI ncu bat ion Time on Fatty Acid Liberation. Post-heparin rat plasma was isolated after the injection of either 1 or 10 mg/ kg ( 162 or 1626 units/kg) of heparin to rats as described above. When Ediol was used as a substrate for the assay of the post-heparin plasma lipase, the liberation of fatty acids was found to be linear with time on a semilog plot between 5 minutes to at least 3 hours (Fig. 2). (3) Enzyme Concentration Versus Fatty Acid Liberation. Post-heparin plasma was obtained from a blood sample taken 20 minutes after a beagle dog was given 10 mg/kg (1500 units/kg) of heparin intravenously. Prior to heparin administration, a control plasma sample was obtained from the same dog. The post-heparin plasma was diluted to concentrations of 75, 50, and 25%with the control plasma. Aliquots from all five samples were incubated with Ediol-Tris at pH 8.1 for 3 hours. Aliquots were removed
A RAPID
PLASMA
LIPASE
ASSAY
417
SYSTEM
ii 8.0-
0
I 2 ,,, 6.0+ z
o;
5
IO INCUBATION TIME.
30 MINUTES
100
180
FIG. 2. Relationship of FFA release to time iu Ediol-post hepariu plasma-T& buffer system. Calorimetric FFA assay. Upper curve, 1620 heparin units/kg rat; lower curve, 162 units/kg.
at different intervals and free fatty acid was determined by calorimetric method. The results are shown in Table 1. Figure 3 shows the values obtained at 30 minutes and 3 hours incubation when plotted as PM fatty acid liberated per ml plasma versus log concentration of lipase, in terms of percentage maximum concentration of the post-heparin plasma. A linear TABLE 1 EFFECT OF LIPASE CONCENTRATION ON FREE
FATTY
ACID
PM Free fatty acid liberated/ml Incubation time (minutes) -0 5 10 15 20 30 60 120 180
Percentage concentration
LIBERATION
plasma
post-heparin
plasma
25
50
75
100
9 1.72” 2.15 2.00 2.22 2.50 3.94 5.37 5.65
0 1.93 2.57 2.72 2.93 3.87 5.01 6.87 7.58
0 1.93 2.85 3.00 3.43 3.93 5.50 6.93 8.13
0 1.86 2.43 2.72 3.43 4.01 6.51 7.50 8.49
(1Average of three determinat,ions;
Ediol used as substrate.
418
MACKENZIE
0
AND
AUXIER
A_1
25 PERCENT
POST
SO HEPARIN
7’5
lb0
PLASMA
FIG. 3. Effect of lipase concentration on fatty acid liberation. PM Fatty acid liberated from Ediol after: ( 0 ) 30 minutes incubation; (0) 3 hours incubation, the equation for the line is y = 4.94x + 1.7.
response was obtained at 3 hours. For incubations of 30 minutes or less, linear responseswere not obtained. (4) Effect of Heparin on Fatty Acid Liberation. Three dogs were given heparin at 0.1, 0.2, 0.3, 1.0, 3.0, and 10 mg/kg (15-1500 units/kg) by intravenous injection. The different doseswere given to the same three dogs about 4 days apart. Blood was taken 15-20 minutes after heparin administration and plasma was isolated and pooled. The plasma was assayed for lipase activity as described. Lipostrate-CB was used as the source of triglyceride. Assays were made from duplicate aliquots of the assay mixture starting at 5 minutes of incubation and at various intervals up to 3 hours. A single aliquot was taken at 0, 1, 2, 3, and 4 minutes. The results are tabulated in Table 2. Figure 4 shows the dose response plotted as PM fatty acid liberated per ml plasma versus log dose for the 30-minute and 3-hour values. The data obtained from the 3-hour values gave a very good linear dose response from 0.1 through 3 mg/kg. Ten mg/kg of heparin gave a lower activity. The best dose response was obtamed with the 3-hour values; at the lower incubation times linearity and sensitivity were not so good. At the beginning of incubation there is a lag period in which no or little fatty acid is liberated. This lag period varies from 5-15 minutes in differ-
A RAPID
PLASMA
LIPASE
TABLE EFFECT
OF HEPARIN
DOSE
ASSAY
SYSTEM
FATTY
ARID
2
ON FREE
PM Free fatty acid liberate& Incubation time (minutes) 0 1” 2 3 4 5"
10 15 30 60 90 120
180
419
LIBERATION
ml plasma
Dose of heparin (mg/kg) 0.1
0.2
0 .:3
0 0 0 0 0.11 0.03 0.05 0.09 0.17 0.24 0.44 0.81 1.15
0 0 0 0.14 0.17 0.24 0.30 0.47 0.54 1.24 2.06 2.48 3.45
0 0.01 0.03 0 14 0.30 0.38 0.42 0.64 0.95 1.63 2.44 3.96 4.48
1.0 0 0.12 0.23 0.37 0 3.5 0.71 1.76 2.36 3.34 4.78 5.76 6.63 7.26
3.0
10.0
0 0.06 0.10 0.14 0.51 0.68 1.48 2.56 3.80 5.84 7.26 8.65 10.02
0.42 0.45 0.58 0.69 0.78 0.88 1.10 2.62 4.38 5.23 6.35 7.33
0
a One determination only for l-5 minutes of incubation time. b Average of duplicate assays for S-180 minutes of incubation time. c Lipostrate-CB used as substrate.
0
DOSE
OF HEPARIN
(mO/kOi
FIG. 4. Effect of heparin dose on fatty acid liberation. PM Fatty acid liberated from Lipostrate-CB after ( 0) 30 minutes incubation; ( 0) 3 hours incubation.
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MACKENZIE
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ent experiments. This is the main reason for the best dose response occurring at 3 hours and not at a shorter interval. This lag period is probably due to the need for activation of the substrate with the lipoproteins present in the plasma sample. B. Effect
of Various
Conditions
on Lipolytic
Activity
(1) Effect of pH. Post-heparin plasma was pooled from three dogs given 1 mg/kg (150 units/kg) of heparin by subcutaneous injection. Post-heparin plasma was also pooled from 12 rats given 2 mg/kg of heparin by subcutaneous injection, Lipolytic activity was determined by using T&-buffered Ediol emulsion in the pH range of 7.0-9.0. The 3-hour incubation mixtures were assayed for fatty acid liberation. Determinations were made in quadruplicate. Figure 5 illustrates that the optimum pH for post-heparin plasma lipase activity is 8.1 in both species. 6.0
1
INCUBATION FIG. liberated
5. Effect of buffer from Ediol after
pH on post-heparin 3 hours incubation,
pH plasma lipase (0) Dog, (n)
activity. Rat.
PM
Fatty
acid
(2) Substrate Effects. The Ediol obtained from Schenlabs (Riker) was compared with the new emulsion Lipostrate-CB. The thin-layer chromatographic separation of the lipids according to Freeman and West (21) showed the same lipid pattern except for free fatty acid levels, Our Ediol, which had been stored for 7 years, contained much larger amounts of free fatty acid than Lipostrate, 64 PM/ml versus 7.2 PM/ml for Lipostrate. Post-heparin plasma was pooled from two dogs given 1 mg/kg (150
A RAPID
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ASSAY
421
SYSTEM
unit/kg) of heparin intravenously. Each triglyceride emulsion was diluted to 5% (v/v) with distilled water and mixed 1:5 with Tris buffer pH 8.1. The mixtures were incubated and duplicate aliquots were removed for assay at various intervals up to 3 hours. The average values are plotted in Fig. 6. The data indicate equivalent fatty acid liberation up to 2 hours. The 3-hour values are significantly different. Lipostrate was the better substrate. Its superiority is possibly due to: (I) Superior emulsification lO.O-
a 9.0. I cn a 2 8.0. ,E g
7.0.
2 4
6.0.
3 2 6.0. : G
E 4.0. a 3.01 15
FIG. 6. Comparison plasma lipase activity.
30 INCUBATlON
of Ediol ( 0 ) Ediol,
60 90 120 TIME (minutes)
and Lipostrate-CB ( 0 ) Lipostrate-CB.
as substrates
180 for
post-heparin
because it is a fresher preparation, and (2) lower product concentration initially (fatty acids). The higher fatty acid level would inhibit the enzymatic reaction (23, 24), especiahy since there is a limited amount of carrier for the product present in the incubation system. In other experiments such as under A 2, Ediol gave linear response at lower total free fatty acid liberation. (3) Enzyme Stability. Post-heparin plasma was collected and pooled from three dogs given 1 mgf kg ( 150 units/ kg) of heparin subcutaneously. It was also isolated from 12 rats given 2 mg/kg of heparin subcutaneously. Blood was taken from the rats 45-66 minutes after heparin administration and 30-45 minutes after administration to dogs. Both
422
MACKENZIE
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pooled samples were separated into five groups of four equal aliquots (0.5 ml). These five groups consisted of samples to be assayed fresh, after freezing for 24 and 48 hours and refrigerated for 24 and 48 hours. The lipolytic assay was made with Ediol as source of triglyceride. The data are reported in Table 3. No great loss in activity occurred in 2 days ( Q 22%). If frozen for 24 hours no significant loss occurred. (4) Inhibition of Lipase Activity by NaCE. Post-heparin plasma was obtained by giving 3 mg/kg (450 units/kg) of heparin intravenously to a dog. Blood was collected 15 minutes after heparin administration. The plasma was incubated with a Tris buffer-Lipostrate mixture. One incubation tube contained buffer at pH 8.6 and another tube at pH 8.1. One tube at each pH had NaCl added to a concentration of 1 molar. The fifth TABLE THE
3
DETERMIRTAT~OIS OF POST-HEPARIN RAT AND DOG PLASMA SJYABILITY ON REFRIGERATIOX ASD FREEZING, USING EDIOL AS SUBSTRATE
Rat
Dog Pooled samples Fresh Frozen-24 hours Refrigerated-24 hours Frozen48 hours Refrigerated48 hours
Percentage of fresh samples
/.a FFA liberated 3.90” 3.96 3.70 3.56 3.44
+ 0.15 31 0.05 * 0.07 f 0.06 zk 0.06
0 Average of four determinations b Average of four determinations
LIPASE
100
102 95 91 88
pi FFA liberated 4.93b 4.53 4.60 3.97 3.83
Percentage of fresh samples
f 0.09 *
0.11
31 0.08 i 0.04 + 0.16
100 93 93 81 78
+ SEM from pooled sample (3 dogs). + SEM from pooled sample (12 rats).
incubation system had the plasma preincubated for 15 minutes with the 1 M NaCl before the Tris buffer-Lipostrate pH 8.1 was added. Aliquots were removed at various intervals up to 3 hours and the quantity of free fatty acid liberated determined. The data are shown in Table 4. There was a slightly greater percentage inhibition at pH 8.1 than at 8.6, though the total amount of fatty acid liberated in 3 hours in the NaCl containing systems was the same at both pH values. There was slightly more inhibition in the tube preincubated for 15 minutes, but this was not significant. Since Kom (4) has shown that 1 M NaCl inhibits lipoprotein lipase lo@ the data indicate that about 15%of the lipase activity is due to some other enzyme. (5) Eflect of Anticoagulunt Type on Lipase Activity. When postheparin plasma is isolated from blood, an anticoagulant must be used. Usually we have used small quantities of heparin, but citrate as an exam-
A RAPID PLASMA
INHIBITORY
EFFECT
LJPASE
acid
Condition
pH (1)
0 15 30 60 120 180 Percentage o NaCl
8.6
0 1.14 2.36 3.58 5.58 6.36 inhibition incubated
pH 8.6 +lM KaCI (11) 0 1.04 1.21 1.04 1.04 1 57
the plasma
mixture
+l
0 1.92’ 2.79 4.71 7.81 9 33
LIPASE
plasma
of incubation
75 with
PLASMA
liberated/ml
pH x.1 (III)
423
SYSTEM
TABLE 4 OF 1 M NaCl ON POST-HEPARIN 1~ Fatty
Incubation time (min)
ASSAY
pH X.1 M NaCl (IV,
pH 8.1 + 1 M NaCla 09
0 0.69 0.87 0.96 1.57 1.57
0 0.43 0.70 0.87 1.31 1.39
83 for
15 minutes
before
Ediol-Tris
85 buffer
added.
ple of the decalcifying agents could also be used. The effect of both types and their combination was determined. No significant difference was observed in lipase activity, when heparin at 20 units/ml, sodium citrate at 3.3% ( 1 part to 9 parts of blood), or the combination of both anticoagulants was used to prevent coagulation. (6) General 0bse~oation.s. When heparin activates lipase in different species, it does not have the same potency. In our studies the rat and dog were used. The dog has by far the more responsive system to heparin administration. Dog plasma also has only one fifth of the endogenous lipase activity that rat plasma contains. These observations agree with the report of Monkhouse and MacKneson (25). DISCUSSION
The method described for the assay of lipase in plasma is a simple and rapid procedure. The plasma is used not only for a source of lipase but also for the accepters of the free fatty acids, albumin, and calcium ion. The incubation mixture, therefore, contains the minimum ingredients required for such a test; plasma, the source of lipase, lipoproteins, albumin, and calcium ion; fat emulsion, source of substrate for enzyme action; buffer, to maintain optimum pH of system. The amount of anticoagulant (heparin) used in the isolation of plasma should be limited to only that required to prevent clotting. Large quantities can inhibit the system (26). The incubation time for a good dose response relationship is 3 hours, though any time after 1 hour may be used with reasonable results. It was
424
MACKENZIE
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found that the earlier times did not give a good dose response even though zero order kinetics should occur initially. In our experiments we found a lag time, which is interpreted as the time required for the lipoproteins present in the plasma to activate the fat emulsion. The experiments on the relationships of lipase concentration and heparin dose to rate of fatty acid liberation agree with those reported by Wenke et al. (27). Since these produced linear relationships, the method can be used for quantitative estimations. By comparing the effect of various inhibitors, Hollett ( 28 ) reported that tissue lipase and plasma lipase released by heparin are not the same enzyme. Baskys et al. (12) did report on the optimum pH of post-heparin plasma lipase showing it to be 8.1. We have confirmed this. According to Korn (4) and others tissue Iipase activity is optimum at pH 8.5. This also indicates that the lipases are different. Ediol is a substrate used by many investigators (7, 11, 23, 29), but other substrates have also been reported: Intralipid (30)) olive oil emulsion ( 8)) and lipemic plasma (9). A new form of Ediol called LipostrateCB, available from Calbiochem, Inc., was compared with the Ediol made by Schenlabs, Lipostrate has the same formula as Ediol, but our old Ediol contained 9 times the concentration of free fatty acid as did Lipostrate. The use of Lipostrate showed similar fatty acid liberation up to 2 hours, after which there was greater liberation when Lipostrate was used. Van Den Bosch et al. (23) explained this as an effect on surface tension by the liberated free fatty acids. The lowered surface tension inhibits the lipase activity. Fielding (24) suggests that a complex is formed between free fatty acid and lipase. He suggests that the complex stabilizes the lipase so that it will not “inactivate.” His method, however, was not designed to indicate inactivity due to this stabilizing effect, What appears quite clear in this area of research is that there needs to be an international method standardization for the assay of post heparin plasma lipase. At this time no two laboratories can compare their data because different methods are used. Many investigators at present are using suboptimum, nonphysiological pH values from 8.5 to as high as 8.8, though optimum is 8.1. Not only does pH or buffer vary but also substrate. There is a great need for a standard substrate that all investigators can use. Ideally, lipoprotein should be the substrate. Since such a standard would be difficult to mass produce and would not be stable, a standard fat emulsion must be suggested. SUMMARY
A rapid, simple, and sensitive method is described for the assay of post-heparin plasma lipase. The incubation system contains only the in-
A RAPID
PLASMA
LIPASE
ASSAY
SYSTEM
425
gredients required for the assay: plasma, as the source of lipase, lipoprotein, and albumin; fat emulsion as substrate, and Tris buffer, pH 8.1. With a S-hour incubation period, liberation of fatty acid is proportional to log of either heparin dose or lipase concentration. The lipase assay utilizes a new free fatty acid assay procedure (21) developed in this laboratory. This procedure depends on the formation of a complex soluble in toluene between Rhodamine B, uranyl ion, and the fatty acid anion. The method was used to investigate the effects of pH, 1 M NaCI, and substrate on the assay system. The stability of the enzyme on refrigeration and freezing was also investigated. A need for an international standardized method is mentioned. ACKNOWLEDGMENTS We acknowledge the encouragement, guidance, and patience given us by Dr. T. R. Blohm. We thank Drs. N. L. Wiech and J. W. Newbeme (Pathology-Toxicology Department), and Ft. Hannah (Medical Research) for their helpful suggestions in the preparation of this manuscript. REFERENCES 1. WATTS, H. F., in “Role of Lipoproteins in the Formation of Atherosclerosis; Evolution of the Atherosclerosis Plaque” (R. J. Jones, ea.), pp. 117-132. University of Chicago Press, Chicago, Illinois, 1963. 2. CONSTANTIIWDES, P., “Experimental Atherosclerosis,” Elsevier, Amsterdam, 1965. 3. HAHN, P. F., Science 98,19 (1943). 4. KORN, E. D., in “Methods of Biochemical Analysis,” Vol. 7, (D. Click, ea.), pp. 145-192. Wiley (Interscience) New York, 1959. 5. ROBINSON, D. S., AND FRENCH, J. E., Pharmuool. Reu. 12, 241 (1966). 6. FREXXWXSON, D. S., ONO, K., AND DAVIS, L. L., J. Lipid Res. 4, 24 (1963). 7. HOLLETT, C. R., Biochim. Biophys. Acta 98, 53 (1965). 8. TIETZ, N. W., BORDEN, T., AND STEPLETON, J. D., Am. J. Clin. Pathol. 31, 148 (1959). 9. SALAMAN, M. R., AND ROBINSON, D. S., Biochem. J. 99,640 ( 1966). 10. DOIZAKI, W. M., AND ZIEVE, L., Proc. Sot. ErptZ. Biol. Med. 122, 606 (1966). 11. EIBER, H. B., PAYZA, A. N., AND GOLDBERG, B., Biochim. Biophys. Acta 116, 256 (1966). 12. BASKYS, B., KLEIN, E., AND LEVER, W. F., Arch. Biochem. Biophys. 99, 25 (1962). 13. BASKYS, B., KLEIN, E., AND LEVER, W. F., Arch. Biochem. Biophys. 99, 31 (1962). 14. GOODMAN, D. S., Science 125,1296 (1957). 15. CHEN, R. F., J. BioZ. Chem. 242,173 (1967). 16. DOLE, V. P., AND MEINERTZ, H., J. BioZ. Chem. 235,2595 (1960). 17. TROUT, D. L., ESTES, E. M., AND F-ERG, S. J., J. Lipid Res. 1, 199 (1960). 18. DUNCOMB, W. G., Biochem. 1. 88,7 (1963). 19. MENDELSOHN, D. S., S. Ajricun J. Med. Sci. 23,75 (1958). 20. NOVAK, M., J. Lipid Res. 6, 431 ( 1965).
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21. MACKENZIE, R. D., BLOHM, T. R., AUXIER, E. A., AND LUTHER, A. C., J. Lipid Res. 8, 589 ( 1967). 22. FREEMAN, C. P., AND WEST, D., J. Lipid Res. 7,324 ( 1966). 23. VAN DEN BOSCH, J., EVRARD, E., DESOMER, P., AND JOOSENS, J. V., Arch. Intern. Phurmucodyn. 130,64 ( 1961). 24. FIELDING, C. J., Biochim. Biophys. Actu 159, 94 (1963). 25. MONKHOUSE, F. C., AND MACKNESON, R. G., Can. J. Biochem. Physid. 36, 1065 (1958). 26. PAYZA, A. N., EIBF.R, H. B., AND DANISHEFSK~, I., Biochim. Biophys. Actu 111, 159 ( 1965). 27. WENKE, M., WENKEOVA, J., MUHLBACHOVA, E., AND HYNJE, S., Arch. Intern. Pharmucodyn. 134,417 (1961). 28. HOLLETT, C. R., Arch. Biochem. Biophys. 108,244 ( 1964). !?I% PASTA, A. N., EIEIER, H. B., AND WALTERS, S., Proc. Sot. Erptl. Biol. Med. 125, 188 (1966). 30. MUIR, J. R., Clin. Chim. Acfu 17,312 (1967).