An immunochemical method for the selective measurement of two triglyceride lipases in human postheparin plasma

335

Clinica Chimica Acta, 63 (1975) 335-347 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

CGA 7242

AN ~MMUNOCH~MICAL METHOD FOR THE SELECTIVE MEASUREMENT OF TWO TRIGLYCERIDE LIPASES IN HUMAN POSTHEPARIN PLASMA

JUSSI K. HUTTUNEN”, and ESKO A. NIKKILX

CHRISTIAN EHNHOLM, PAAVO K.J. KINNUNEN

Third Department of Medicine and Department of Helsinki, Helsinki (Finland)

of Serology and Bacteriology, University

(Received March 18,1975)

Summary A new method for the selective measurement of postheparin plasma lipoprotein lipase and hepatic lipase is described and validated. The activity of lipoprotein lipase is determined at 0.1 M NaCl after removal of hepatic lipase by specific ~tise~m, and the hepatic lipase is assayed in a medium containing 1.0 M NaCl but no additional serum. The optimal conditions for the determination of the two postheparin plasma triglyceride hydrolases were shown to be similar to those described for the purified enzymes. The new assay methods are simple, accurate and highly specific for the two lipase activities. VLDL and LDL do not interfere with the measurement, making the methods suitable for studies of patients with various hyperlipidemias. More than 90% of the total triglyceride hydrolase activity in postheparin plasma is precipitated with antisera raised against purified human postheparin plasma hepatic lipase and bovine milk lipoprotein lipase . The time and dose dependence of the two postheparin plasma lipase responses differ. For optimal activity of both enzymes, plasma taken 15 minutes after intravenous administration of 100 I.U./kg of heparin, should be used. The activity of postheparin plasma lipoprotein lipase and hepatic lipase in 12 young, healthy males is reported.

Removal of circuIating triglycerides occurs in man in the capillary bed of peripheral tissues by a triglyceride hydrolase, lipoprotein lipase [l] . This en* Address corres&xmdenoe to: Dr Jussi K. Huttunen, Third Department of Medicine, LMversity of Helsinki, Ha~tm~~~tu 4.00290 Helsinki 29, Finland.

336

zyme requires a specific serum factor, apolipoprotein C II (apo-Lp Glu) for full activity [2,3] . Lipoprotein lipase is released into plasma after intravenous administration of heparin and it was believed earlier that all triglyceride lipase activity in postheparin plasma is due to lipoprotein lipase [4]. However, a second triglyceride lipase activity has been recently described in plasma after heparin injection in man [ 5-71, and in normal but not in hepatectomized experimental animals [ 8-101. In contrast to lipoprotein lipase, this activity is stimulated by high NaCl concentrations and does not require the presence of serum factors [6,11]. A similar lipase activity is present in liver plasma membranes [12] and in heparin perfusates of rat liver [13] suggesting that this activity originates from the liver. Selective measurement of lipoprotein lipase* and hepatic lipase* in postheparin plasma has been reported by methods employing various inhibitors [ 141 and by affinity chromatography on heparin-Sepharose [ 15 ] . In this paper we describe a new highly specific assay method for the two enzymes which is based on the use of antiserum prepared towards purified hepatic lipase. Serum lipoproteins do not interfere with the measurement of the lipase activities making this new method useful in clinical studies of patients with various types of hyperlipoproteinemia. It has been so far applied by our group to studies of postheparin plasma lipases in normal subjects [16] , in obese patients [17] and in hyperlipidemic patients treated with oxandrolone [ 181. Material and methods Subjects Healthy male and female laboratory workers, all with normal plasma cholesterol and triglyceride concentrations (<260 and 170 mg%, respectively) served as donors of postheparin plasma used in the studies of the assay conditions and in the isolation of postheparin lipases. All subjects fasted 12-14 hours before blood samples were withdrawn. Postheparin plasma All blood samples were collected in tubes containing 5 I.U. heparin/ml of blood. The tubes were immediately placed in ice. Plasma was separated by centrifugation at +4”C and stored frozen at -20°C. In the standard heparin test venous samples were withdrawn before and 5 and 15 min after injection of 100 I.U. of heparin per kg of body weight. In special experiments the effect of heparin doses from 10 to 200 I.U./kg was studied and the time course of lipase activity was measured at intervals between 1 and 120 min after heparin injection. Preparation of antisera against postheparin hepatic lipase Postheparin plasma obtained by plasmapheresis from healthy volunteers after injection of 100 I.U. of heparin/kg of the body weight was used as the

* In this paper lipoprotein lipase represents a triglyceride lipase which is stimulated by serum cofactors and is inhibited by 1 M NaCI. Hepatic lipase represents a triglyceride lipase which is stimulated by 1 M NaCl. does not require the presence of serum cofactors, and is precipitated by antibodies against purified salt-resistant triglyceride lipase from human postheparin plasma t7.191.

337 enzyme source. Purification of hepatic lipase and lipoprotein lipase was carried out as described by Ehnholm et al. El9 ] , Briefly, postheparin plasma was incubated for 15 min at 37°C with l/20 vol. of 20% Int~ipidR (Vit~m, Sweden). After centrifugation at 60 000 X g for 60 min, the fat cake containing more than 50% of the triglyceride lipase activity was collected and the preparation was delipidated with acetone and diethylether. The residue was dissolved in 5 mM sodium-barbital buffer, pH 7.4, containing 20% glycerol, and subjected to chromato~aphy on Sepharose containing covalently bound heparin [20] . Elution of the enzymes was carried out with a linear NaCl gradient from 0.4 M to 1.5 M NaCl in 5 mM sodium-barbital buffer, pH 7.4, containing 20% glycerol. Three major peaks eluting at 0.7, 0.85 and 1.15 M NaCl were obtained. The activity in peaks emerging at 0.7 (I) and 0.85 (II) M NaCl was resistant to 1.0 M NaCl and did not require serum for full activity. The activity eluting at 1.15 M NaCl (III} was activated by serum and apolipopro~in CII, and was inhibited by high salt concentration. For immunization the proteins in peak I (50-100 ,ug) were mixed with complete Freund’s adjuvant and injected into New Zealand white rabbits. The immunization was continued at intervals of 2 weeks ‘until a good inhibitory titer against peak I lipase was obtained. In separate experiments it was shown that antiserum towards peak I lipase precipitated the activity present in peak II. All antisera were tested for inhibitor activity against peak III lipase activity (lipoprotein lipase) but no evidence for crossreaction was found. Preparation of triolein emulsions The triolein emulsion used as substrate in lipase assays was prepared under strictly standardized conditions. 100 mg of [a&-1-l 4 C J triolein (specific activity 0.070 &i/pmol), stored in benzene, was transferred into a conical 20 ml test-tube. The solvent was evaporated under nitrogen and 7.5 ml of 5% gum arabic was added. The microtip of a Branson Sonifer-Cell Disruptor (Branson Instruments Col., Danbury, Conn.) was centered 0.5 cm below the surface of the solution and the mixture was sonicated in an ice-bath at setting 4 for 4 min. A fresh batch was usually prepared for each day, although it was shown that the emulsion retained its properties at 4°C at least for 48 hours when briefly resonicated before use. standard assay of postheparin total lipolytic activity, hepatic lipase and lipoprotein tipase The selective measurement of lipoprotein lipase activity was based on inactivation of hepatic lipase with specific antiserum and subsequent assay of the remaining activity in the presence of serum activator. In the standard assay 10 1.11of postheparin plasma was incubated for 2 hours at 4°C with 10 ~1 of hepatic lipase antiserum (diluted if necessary with normal rabbit serum). Thereafter, 500 ~1 of the assay mixture containing 3.2 mM [acyl-l-‘4C] triolein emulsion, 40 mM Tris chloride buffer, pH 8.4,0.1 M NaCl, 2.5% bovine serum albumin and 50 ~1 of normal human serum was added, and the tubes were incubated at 28°C for 60 min. The released FFA radioactivity was determined by the liquid-liquid partitioning system described by Belfrage and Vaughan [21]. In the assay of hepatic lipase the activity of lipoprotein lipase was inhibited by

338

raising the NaCl concentration to 1.0 M and omitting the serum activator. The assay was carried out without preincubation in a reaction mixture of the same composition as that used for lipoprotein lipase, except that the NaCl concentration was 1.0 M and human serum was omitted. Total lipolytic activity towards triolein emulsion was determined under conditions used for lipoprotein lipase but without preincubation with antiserum.

Determination of protamine-sensitive

and protamine-resistant

triglyceride lipase

Protamine-sensitive and protamine-resistant triglyceride lipases of postheparin plasma were assayed as described by Krauss et al. [ 141 except that the FFA radioactivity was determined according to Belfrage and Vaughan [Zl] . 0

ther methods

Chylomicrons, VLDL and LDL were prepared with ultracentrifugation according to Hatch and Lees [22]. Triglyceride concentration was determined in a Technicon autoanalyzer (Technicon Instruments Corp., Tarrytown, N.Y.). Free fatty acids were assayed with a micromodification of the cobalt method described by Novak [23].

Statistical methods Standard deviation (S.D.), standard error of mean and linear regression were computed with an Olivetti desk computer. Precision was calculated from duplicates and is expressed as the relative S.D. (variation coefficient).

60 30 -

HLA: LPL AS

0 0

1 0

2 0

3 0

4 0

5 0

6 1

5 2

5 4 NaCl

ANTISERUM

(hl)

+‘I 1

Fig. 1. Effect of hepatic lipase antiserum and lipoprotein lipase antiserum on the activity of triglyceride lipase in postheparin plasma. Aliquots (10 ~1) of postheparin plasma were incubated with the indicated amounts of hepatic lipase antiserum (HL AS) and lipoprotein lipase antiserum (LPL AS) (antiserum against bovine milklipoprotein lipase was a generous gift from Drs Hernell, Egelrud and Olivecrona. Ume& Sweden [241) in a total volume of 20 ~1 (diluted if necessary with normal rabbit serum). The activity remaining in the solution was determined using the standard assay mixture at 0.1 M NaCl in the presence of serum or at 1 .O M NaCl in the absence of serum (0-j. (0 --O) Fig. 2. Effect of NaCl concentration on the total lipolytic activity and the activity of hepatic lipase and lipoprotein lipase in POSthepaIin plasma. Triglyceride lipase activity of postheparin plasma was measured 0) or lipoprotein lipase before (o-----C ) and after immunoprecipitation of hepatic lipase (o(e-----4) with specific antiserum. The NaCl concentration of the assay mixture was varied as indicated on the abscissa.

339

TIME

(mln)

Fig. 3. The relationship of postheparin lipoprotein lipase and hepatic lipase activities to incubation time. Aliquots (10 ~1) of postheparin plasma were assayed for the activity of lipoprotein lipase and hepatic lipase with the standard procedure, but varying the incubation time as indicated on the abscissa.

Results Effect of antisera against lipoprotein lipase and hepatic lipase on postheparin plasma triglyceride hydrolase activity Preincubation of postheparin plasma from normal subjects with antiserum against purified hepatic lipase removed 30-80% of triglyceride lipase activity assayed at 0.1 M NaCl (for a representative experiment, see Fig. 1). Incubation of postheparin plasma with antisera against both lipoprotein lipase and hepatic lipase lowered the activity to less than 5% of the original indicating that essentially all triglyceride lipase activity in postheparin plasma is due to lipoprotein lipase and hepatic lipase. The optimal NaCl concentration for the enzyme activity remaining in the solution after immunoprecipitation of the hepatic lipase was 0.1 M NaCl (Fig. 2). Raising the NaCl concentration to 1.0 M abolished more than 90% of the activity (Fig. 2). When the same experiments was carried out after removal of lipo-

Fig. 4. Effect of serum addition on the activity of postheparin lipoprotein lipase and hepatic hpaae. Aliquots of Postheparin plasma (10 ~1) were assayed for the activity of lipoprotein lipase (o----o) and hepatic lipase (*A ) with the standard assay system in the presence of various amounts of normal human serum.

340

protein lipase, an increase in salt concentration from 0.1 to 1.0 M enhanced the activity 1.5fold {Fig. 2). Similar enhancement has earlier been described for hepatic lipase purified from human and swine posthepa~n plasma [6,9]. Thus, in the absence of antisera both lipoprotein lipase and hepatic lipase contribute to the total lipolytic activity at 0.1 M NaCl. At 1.0 M NaCl most of the lipoprotein lipase is inhibited and the remaining activity is due to hepatic lipase.

~ha~~terization

of lipoprotein lipase and hepatic lipase of postheparin plasma

The hydrolysis of triglyceride by lipoprotein lipase and hepatic lipase was linear for at least 120 min under the conditions used (Fig. 3). Addition of 2050 1.11of preheparin plasma caused a significant rise in the activity of lipoprotein lipase but did not influence the activity of hepatic lipase (Fig. 4). Higher amounts of plasma caused an inhibition in the activity of both enzymes. The activity of both postheparin plasma lipases was proportional to the amount of added postheparin plasma up to 50 /..d/O.S ml of the assay mixture (Fig. 5). Above this concentration the activity of hepatic lipase did not increase and the activity of lipoprotein lipase was less than the expected value. The inhibition was not due to accumulation of reaction products, since a similar effect was obtained when increasing amounts of preheparin plasma were added to the assay mixture with a 10 ~1 sample of posthep~in plasma (Fig. 4). Furthermore, studies with purified enzymes indicated that the activity of both enzymes is directly proportional to the amount of enzyme in at least 2-fold excess to the amounts used in the experiment depicted in Fig. 5. The effect of substrate concentration on the activities of lipoprotein lipase and hepatic lipase is shown in Fig. 6. Maximal velocity was obtained at triolein concentrations exceeding 1.2 mM. With low substrate concentrations (less than 0.5 mM) the activity of lipoprotein lipase was relatively lower than that of

POSTHEPARlN

PLASMA

(#I)

TRIOLEIN

t mM)

Fig. 5. The relationship of the postheparin lipoprotein lipase and hepatic lipase activity to the amount of postheparin plasma. Various amounts of postheparin phsma were assayed for lipoprotein lipase (0 --0) and hepatic lipase (0 ---+) activity using the standard procedure. Fig. 6. Effect of substrate concentration on the activity of postheparin lipoprotein lipase and hepatic lipase. Aliquots of postheparin plasma (10 ~1) were assayed for the activity of lipoprotein lipase (O-----O) and hepatic lipase (*) with the standard system varying the trio&in concentration as indicated on the abscissa.

341

7 SONICATION

TIME

(min)

9

9

10

PH

Fig. ‘7. Effect of sonic&ion time on the properties of the lipase substrate. Aliquots of postheparin plasma (10 1.11) were assayed for lipoprotein lipase (O0) and hepatic lipase (*A ) activity with the standard assay system. The sonication time of the triolein emulsion was varied as indicated on the abscissa. Fig. 8. Effect of PH on the activity of postheparin lipoprotein lipase and hepatic lipase. Aliquots heparin plasma (10 ~1) were assayed for lipoprotein lipase (o-----c ) and hepatic lipase (0~) the standard procedure varying the pH of the assay mixture as indicated on the abscissa.

of postwith

hepatic lipase. A similar phenomenon was observed when triolein emulsions sonicated for different times were used as the substrate (Fig. 7). With substrate preparations sonicated less than 2 min the activity of lipoprotein lipase was low. Maximal activity of both enzymes was seen when triolein emulsions were sonicated for 3 minutes or longer. The pH dependence of the lipase activities is seen in Fig. 8. Under the conditions used the pH optimum of lipoprotein lipase was 8.4. The pH optimum of hepatic lipase was somewhat higher with a maximum at pH 9.2-9.6. The effect of human serum lipoproteins on postheparin plasma lipase activities was studied by mixing postheparin plasma with purified lipoprotein fractions and with plasma obtained from patients with Type II and Type IV hyperlipoproteinemia. Table I shows that concentrations of VLDL-triglyceride up to 10 mM and LDL-cholesterol up to 17 mM in the plasma sample did not interfere with the determination of the lipolytic activity. The stability of the enzyme activities during storage was studied by determining the lipase activities in 20 postheparin plasma samples before and after storage at -20°C for 6 months. No evidence for a decrease in the activity was observed. Precision and reproducibility of the lipase assays The precision of lipoprotein lipase and hepatic lipase determinations in 26 duplicates in three series of standard assays were 5.0 and 4.0%, respectively. The day to day variation was studied by including duplicate samples of standard postheparin plasma in 10 consecutive series of determinations. The interassay variations, expressed as the variation coefficient of the means in 10 consecutive series, were 14.4% and 14% for the two enzymes, respectively. The specificity of the assay of the two lipases was tested by adding purified preparations of both enzymes to postheparin plasma. The results in Table II demonstrate that plasma in the amounts used for the assay doe8 not interfere

342 TABLE

I

EFFECT

OF VLDL

Aliquote

of postheparin

lipase activities

AND LDL ON THE ACTIVITY plasma (10 ~1) were mixed

in the mixtures

Addition

were determined

Lipase activity Hepatic

wmol

ml-l

lipase

with purified

LIPASE

preparations

with the standard

AND HEPATIC

of VLDL

and LDL,

LIPASE and the

procedure.

h-l) Lipoprotein

VLDL* 0.65 0.99 1.35 2.98 5.62 10.9

20.9 22.8 23.5 22.3 23.5 24.4

18.5 18.4 19.4 16.4 16.5 15.2

LDL** 6.0 10.0 14.6 25.3

19.6 16.9 21.1 18.4

15.4 15.7 17.5 17.0

* Final concentration ** Final concentration

OF LIPOPROTEIN

lipase

of VLDL triglyceride in the plasma sample (mmol/l). of LDL cholesterol in the plasma sample (mmol/l).

with the measurement. Only after addition of a several-fold excess of hepatic lipase a significant change was seen in the apparent activity of lipoprotein lipase. No evidence for interference with the hepatic lipase determination was observed when similar amounts of purified lipoprotein lipase were added to the samples prior to incubation. Time and dose dependence of postheparin plasma lipase responses The effect of different doses of heparin on the activities of lipoprotein lipases and hepatic lipase in postheparin plasma is shown in Fig. 9. Increasing the dose from 10 to 100 I.U./kg body weight induced a variable change in the lipoprotein lipase response, which was evident at all time points (only 15 minute TABLE

II

EFFECT PROTEIN

OF ADDITION OF PURIFIED LIPASE AND HEPATIC LIPASE

LIPASE PREPARATIONS ON THE IN POSTHEPARIN PLASMA

ACTIVITY

OF LIPO-

Purified hepatic lipase and lipoprotein lipase (see Material and Methods) were mixed with aliquots of postheparin plasma and the activity of lipoprotein lipase and hepatic lipase was determined with the standard procedure. Postheparin plasma @l)

10 10 10

Additions

Lipase activity

(~1)

(gmol

FFA ml-’

Lipoprotein lipase

Hepatic lipase

Lipoprotein lipase

Hepatic lipase

10 10 -

10

20.8 17.7 0.9 46.2 20.8

26.1 1.7 33.8 27.8 52.1

10

h-1)

343

E

2-i . .

HEPARIN

DOSE

0

50

100

(W/kg

body

weight)

150

20

) and hepatic lipase (O) at 16 minutes after Fig. 9. Postheptin plasma lipoprotein lipase (m administration of varying doses of heparin in four healthy young subjects. The heparin dose was sequentially increased at 34 day intervals.

values shown) after injection of heparin. On the other hand, only a small increase in the activity occurred, when heparin dose was raised from 100 to 200 I.U./kg, indicating that the amount of heparin used in the standard test (100 T.U./kg body weight) gives a near maximal response in the activity of lipoprotein lipase. A similar, although less pronounced increase was seen in the activity of hepatic lipase, when the dose of heparin was increased from 10 to 200 I.U./kg. Lipase activities in plasma of a healthy young male subject at various time intervals after injection of 100 I.U. heparin per kg body weight are shown in Fig. 10. The activity of hepatic lipase reached maximum at 2-5 minutes after heparin administration, whereas the peak activity of lipoprotein lipase was seen 15-20 minutes later. The activity of hepatic lipase declined slower than that of lipoprotein lipase and at 3 hours, when no lipoprotein lipase activity was any more detectable, significant amounts of hepatic lipase activity were still found in the circulation. Postheparin plasma lipase activities in normal human subjects The activity of hepatic lipase and lipoprotein lipase in a group of young

0

D

60

190

120 TIME

240

(min)

Fig. 10. Time course of the change in plasma triglyceride lipase activities in a healthy young male after lipoprotein lipase: l-, hepatic lipase. intravenous injection of 100 I.U./kg of heparin. o----O,

344 TABLE HI ACTIVITY YOUNG

OF

POSTHEPARIN

HEALTHY

PLASMA

LIPOPROTEIN

Lipase activity* Q.hnoi FFA nil-’

Subject

HM JV TH AN TL EH JK LL HH HI HH LI Mean ?: SE.

LIPASE

AND

HEPATIC

LIPASE

IN 12

MALES

Lipoprotein-- Iipase 5 min 16 min

Hepatic lipase 5 min 15min

10.8 18.0 23.0 19.3 16.2 15.0 19.8 20.7 20.1 7.4 14.7 9.9 16.2 + 1.4

23.1 40.0 28.9 31.3 19.9 27.6 53.9 11.5 23.7 10.0 19.9 22.3 26.5 i 3.4

13.6 22.5 21.3 26.1 24.7 19.4 27.7 26.1 28.5 8.3 18.8 24.1 21.7 f 1.7

Serum triglyceride concentration (mmol/l)

h-l)

25.1 41.1 30.2 30.4 22.2 27.0 56.2 15.0 25.1 14.0 22.1 25.5 27.8 f 3.3

1.19 0.95 0.26 0.20 0.47 0.99 1.26 0.69 0.90 1.19 0.72 1.01 0.82 * 0.10

Relative body weight (%I

102 111 81 108 112 101 110 92 106 120 102 113

* Lipase activity 5 and 10 minutes after injection of heparin (100 I.U.lkg body weight).

healthy males is given in Table III. The mean activity of lipoprotein lipase at 15 min afterheparin administration was 21.7 f. 1.7 pmol FFA ml-” h-’ and that of hepatic lipase 27.8 + 3.3 pmol FFA ml-’ h-’ , respectively. A highly significant correlation was observed between the 5 and 15 minute samples of both lipopro~in lipase (r = +0.75, p < 0.01) and hepatic Iipase (r = +0.97, p < 0.001) suggesting that the values obtained at these time intervals reflect the activity of a single pool of both enzymes.

Comparison of the present method with the protamine inactivation method of Krauss et al. [24] The results of lipase measurements

q p’ 001 r=+0.87

40. 30 20

10

P d

0

0

r, !z?

2% YE

0

0

procedure

s?$

0

0

with the present

2s iE

d 0

d

performed

L

10

20

PROTAMINE-

30

0

INACTIVATED

10

LIPASE

p’mole

FFA

ml-‘.

20

PROTAMINE

30

- RESISTANT

LIPASE hr.’

Fig. 11. Comparison of the present assay method with the protamine inactivation procedure described by Krauss et al. [141. Postheparin plasma samples from 10 subjects were assayed for the activity of hepatic Iipase and lipoprotein Iipase with the method described in this paper, and for the activity of protamineresistant and protamine-inactivated iipases by the method of Krause et aI. 1143.

345

were compared with those obtained with the protamine inactivation method described by Krauss et al. in plasma samples from 10 subjects with different levels of ~popro~~ lipase and hepatic hpase activity. As shown in Fig. 11, a highly significant correlation was present between the activities of protamineresistant lipase and hepatic lipase, and between the activities of protamine-inactivated lipase and lipoprotein lipase, respectively. With both activities, the absolute values obtained with the present method were slightly higher than those obtained with the procedure described by Krauss and his coworkers.

Discussion The recent demonstration that heparin releases at least two separate tiglyceride lipase activities into circulation [7,8,14] offers new possibilities of detecting specific enzyme deficiencies in patients with hypertriglyceridemia. The measurement of total postheparin plasma lipolytic activity (PHLA) must therefore be replaced by more specific assay methods. Selective measurement of postheparin lipases has been earlier reported using affinity chromatography on heparin-Sepharose [ 151 or differential inhibition of the two enzymes by protamine [14]. The new principle for the specific assay of posthep~n hepatic lipase and of lipoprotein lipase presented in this paper is based on immunoprecipitation of hepatic lipase and subsequent determination of lipoprotein lipase under specified optimal conditions. The hepatic lipase, on the other hand, is measured at 1.0 M NaCl, which completely inhibits the lipoprotein lipase. The method appears to be simple and highly reproducible under standardized conditions. It will permit a daily analysis of more than 20 duplicate plasma samples by one technician. The assay method produces accurate results for both enzymes even in plasma samples which contain several-fold excess of either of the two triglyceride lipases. The specificity of the new method was validated by several independent experiments. First, successive treatment of posthep~n plasma with specific antisera against lipoprotein lipase and hepatic lipase removed more than 95% of the triglyceride lipase activity indicating that the two enzyme activities are the major components of postheparin plasma triglyceride hydrolase activity. Second, addition of 1.0 M NaCl to the assay medium after immunoprecipitation of hepatic lipase always inhibited more than 90% of the residual lipase activity. The high degree of inhibition by salt is ch~cte~stic to the lipoprotein lipase isolated from milk, adipose tissue and myocardium [24-261. On the other hand, the activity remaining in solution after immunoprecipitation of lipoprotein lipase was 1.5fold higher at 1.0 than at 0.1 M NaCl, a behavior similar to that of hepatic lipase purified by affinity chromatography. The specificity and accuracy of the two lipase assays were further corroborated in experiments, where each of the purified enzyme fractions was added to plasma before measurement of activity. Recovery of both activities was complete and neither of the enzymes substantially interfered with the determination of the other. Experiments with VLDL and LDL added to plasma samples indicated that even high concentrations of these lipoproteins do not interfere with the assay of postheparin plasma lipases in the present method. This observation makes

346

the new method suitable for studies in patients with various types of hyperlipoproteinemia. On the other hand, it was shown that normal fasting plasma inhibited the activity of both lipases at concentrations exceeding 10% of the volume of the assay mixture. This finding is in agreement with early observations on the presence of inhibitors of clearing factor lipase in human postheparin plasma [27]. Although the possibility cannot be excluded that the inhibition is caused by apolipoproteins C-I and C-II [28], the lack of effect of VLDL added to plasma samples speaks for the presence of non-lipoprotein inhibitors. It remains to be determined whether abnormal plasma samples are inhibitory at smaller concentrations than the normal ones. Both lipases were maximally active at triolein concentration exceeding 1 mmol/l. The saturating concentration of triolein depends, however, on the properties of the triolein emulsion, since in reactions involving water-insoluble substrates the surface area, and not the absolute concentration, is the limiting factor [29]. The importance of the conditions used in the preparation of the substrate is demonstrated by the 3-fold increase in the enzyme activity when the sonication time was extended from 1 to 10 minutes at the same energy output. Furthermore, the length of the sonication had a different effect on the two enzymes: the relative activity of lipoprotein lipase was lower when short sonication times were used. The different sonication time probably also explains the somewhat lower absolute activities of both lipases in our earlier investigations [17]. The appearance of lipolytic activities into plasma after heparin injection was dependent on time and the dose of heparin. The release of the two enzymes occurred at different heparin dose levels. Thus, at 10 I.U./kg (0.1 mg/kg) the heparin released approximately 75% of the maximal activity of hepatic lipase but only about 30% of the corresponding activity of lipoprotein lipase. The dose of heparin needed for the release of maximal activity was 100 to 200 I.U./ kg for lipoprotein lipase but only about 50 I.U./kg for hepatic lipase. The dose employed in our standard test (100 I.U./kg) was selected to give a maximal response of both enzyme activities without causing undue risks to the subjects. The use of lower heparin doses [14] is not desirable, since the response of lipoprotein lipase is in a dose-sensitive range (see Fig. 9). This may increase the variation of the results and decrease the sensitivity of the assay in detecting subtle abnormalities in the activity of this enzyme. Furthermore, it is possible that the lipolytic activities released by small heparin doses are derived from different enzyme pools than those liberated by higher amounts of heparin. The activities found in postheparin plasma of healthy young male subjects varied from 15 to 25 pmol FFA ml’ h-l for lipoprotein lipase and from 15 to 45 E.cmoles FFA ml-’ h-l for hepatic lipase. These values are significantly higher than those reported by Krauss et al. for postheparin plasma protamine-sensitive and protamine-resistant lipase activities [14]. As we found a highly significant positive correlation between the activities of protamine-sensitive lipase and immunochemically determined lipoprotein lipase on the one hand, and between the protamine-resistant lipase and hepatic lipase on the other, it seems that the quantitative differences are accounted for by the smaller dose of heparin (10 I.U./kg) used by Krauss et al. [14] than by us (100 I.U./kg). The fact that the present method produced slightly higher activities also when applied to

347

same plasma samples (see Fig. 11) might be explained of the assay mixture used in the two methods.

by a different

composition

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