Immunochemical assay of rat postheparin plasma triacylglycerol lipases

363

Atherosclerosis, 35 (1980) 363-374 @ Elsevier/North-Holland Scientific Publishers, Ltd.

IMMUNOCHEMICAL TRIACYLGLYCEROL

ASSAY OF RAT POSTHEPARIN LIPASES

PLASMA

T. KUUSI, C. EHNHOLM and E.A. NIKKILA Third Department of Medicine, Helsinki (Finland)

University of Helsinki and Central Public Health Laboratory,

(Received 10 April, 1979) (Revised received 26 November, 1979) (Accepted 26 November, 1979)

Summary Immunochemical methods for selective measurement of lipoprotein lipase and hepatic lipase activities in rat postheparin plasma are described and validated. Lipoprotein lipase was measured using a substrate containing 10% serum and 0.1 M NaCl after inactivation of hepatic lipase with a specific antiserum. Hepatic lipase was measured at 1.0 M NaCl with a serum-free substrate. The heparin dose-response curve indicated maximum release of both activities at a heparin dose of 500 IU/kg. The lipase activities in rat postheparin plasma were 3 to 4-fold higher than those in human postheparin plasma. The LPL activity in female rats was significantly higher than in males whereas there was no sex difference for hepatic lipase. Key words:

Hepatic lipase -Immunoassay

-Lipoprotein

lipase - Postheparin plasma

Introduction Lipoprotein lipase and hepatic lipase (E .C. 3.1.1.3.) are the main triacylglycerol lipases in postheparin plasma [l]. LPL has a well-defined role in the metabolism of triglyceride-rich lipoproteins [ 2,3], but the physiological function of HL has not yet been established. Address correspondence to: Timo Kuusi. M.D., Third Department of Medicine, L2-20, Haartmaninkatu 4,00290 Helsinki 29. Finland. Abbreviations: LPL = lipoprotein lipase,HL = hepatic lipase, VLDL = very low density lipoprotein (d < 1.006 g/ml), LDL = low density lipoprotein (d = 1.006-1.063 g/ml). HDL = high density lipoprotein (d = 1.063-1.21 g/ml), NRS = normal rabbit serum.

364

Several methods have been developed for the selective measurement of the two lipases in postheparin plasma. These are based on the different properties of LPL and HL with respect to inhibitors [ 451 and substrates [6--81, on their different affinities for heparin-Sepharose [9], or on the use of specific antisera [lO,ll]. The immunological methods are superior to the others in specificity and reliability. We report here a simple, accurate and highly specific assay method for the selective measurement of the two lipases in rat postheparin plasma. Optimal assay conditions were investigated using specific antisera for both lipases. Materials and methods Materials

All the reagents were of analytical grade. Tri[l-14C]oleylglycerol with a specific activity of 57 /..&i/~mol was purchased from the Radiochemical Centre, Amersham, Bucks., Great Britain. The corresponding nonradioactive lipid was from Sigma Chemical Co., St. Louis, MO, U.S.A. Bovine serum albumin was obtained from Armour Pharmaceuticals, Eastbourne, Sussex, Great Britain, and heparin from Medica Ltd., Helsinki, Finland. Enzyme

sources

Postheparin plasma was prepared from nonfasted male and female SpragueDawley rats weighing 350-400 g and 250-300 g, respectively. The animals were anesthetized with urethane (1.2 g/kg body weight), the jugular veins were exposed and 500 IU heparin/kg body weight was injected. Two min later blood was taken from the opposite jugular vein, the plasma was separated by centrifugation at +4”C and stored at -20°C. Crude lipoprotein lipase was obtained as a heparin eluate of adipose tissue, heart and skeletal muscle. Each of the tissues was incubated for 15 min in a medium containing 4 g bovine serum albumin, 10 ml glycerol and 500 IU heparin in 100 ml of Krebs-Ringer phosphate buffer, pH 7.4. Two ml of this medium was used for 300 mg of fresh tissue. Supradiaphragmatic rats were prepared according to the technique described by Bezman-Tarcher and Robinson [ 121. Postheparin plasma was obtained from these rats by the method described above for the intact rats. Antisera

against lipoprotein

lipase and hepatic

lipase

Anti-LPL serum was prepared in white New Zealand rabbits by immunizing them with purified LPL of bovine milk [ 131. Five to 10 pg of LPL was mixed with Freund’s complete adjuvant and injected subcutaneously at 4-week intervals until the antiserum had a high titer for inhibition of LPL. Anti-HL serum was prepared in a similar manner using hepatic lipase, purified from heparin containing perfusates of rat liver [ 141, as antigen. For the immunization, 5-10 I.cgof HL was used with complete Freund’s adjuvant. Assay of lipase activity

Measurement of lipase activity was based on hydrolysis of a sonicated emulsion of [ l-‘4C]acyl-labelled triolein and the assay of free oleic acid radio-

365

activity. The sonication of triolein was carried out under standardized conditions. One hundred mg of tri[l-‘4C]oleylglycerol with a specific activity of 42 nCi/pmol, stored in benzene, was dried under nitrogen. After adding 7.5 ml of 5% (w/v) gum arabic the solution was sonicated with a Branson SonifierCell disuruptor type B15 (Branson Instruments, Co., Danbury, CT, U.S.A.). The microtip was placed 0.5 cm below the surface of the solution held in an ice bath and sonication was continued for 5 min at setting 4. A fresh batch was prepared each day and used within 2 h of the sonication. The substrate mixture contained 3.2 mmol [l-14C]triolein, 50 g of bovine serum albumin and 50 g gum arabic in one liter of 0.1 M Tris-HCI buffer, pH 8.6. Sodium chloride was added to a final concentration of either 0.1 M (low salt) or 1.0 M (high salt). For the assay of LPL activity normal human serum was added to the substrate in a ratio 1 : 10. In the assay system, 2.5 1.11of the sample was incubated with the sonicated emulsion for 90 min at +28”C. The reaction was terminated by the addition of 3.25 ml chloroform-methanolheptane (1.41 : 1.25 : 1). This was followed by 0.75 ml 0.14 M borate buffer, pH 10.5, after which the tubes were mixed on a Vortex mixer at maximum speed for 10 set and centrifuged for 15 min at 2800 X g,, [15]. One ml of the upper phase containing the free fatty acids was counted in a Packard Tricarb 3003 liquid scintillation counter using 10 ml of 0.4% (w/v) diphenyloxazone (PPO), 0.02% (w/v) p-bis-(2-(5-phenyloxazoyl)) benzene (POPOP), 6% naphthalene, 10% (v/v) methanol and 2% (v/v) ethyleneglycol in dioxane. Each assay was carried out in duplicate and each series contained two blanks and two reference standards of human or rat postheparin plasma stored at -20” C. The radioactivity detected in the free fatty acid fraction was used to calculate the amount of FFA released during the incubation. Lipase activity was expressed as pmoles FFA released per hour by 1 ml of the sample @moles FFA * h-’ - ml-‘). 0 ther methods Rat VLDL, LDL and HDL were isolated by ultracentrifugation according to Have1 et al. [ 161. Triglyceride concentrations were determined in a Technicon Auto-Analyzer II (Technicon Instruments Corp., Tarrytown, NY). [17]. Cholesterol was measured by an enzymatic method (Boehringer No. 187313). Results Specificity of the hepatic lipase (HL) antiserum To test the specificity of the anti-HL serum, 100 ~1 aliquots of heparin eluates of rat adipose tissue, heart and skeletal muscle, or 50 1.11of heparin perfusate of rat liver were incubated with 10 ~1 of anti-HL serum or with 10 /..d of normal rabbit serum (NRS) for 2 h at +4”C. Thereafter the lipase activity was assayed under conditions optimal for LPL (low salt substrate with added serum). The results are shown in Table 1. LPL activity in the heparin eluates of the extrahepatic tissues was similar both in the presence of anti-HL serum and with normal rabbit serum, indicating that the antiserum did not react with the LPL of the three tissues tested. On the other hand, of the lipase activity present in liver perfusate, only 1.2% was left after incubation with HL antiserum. This

366 TABLE

1

SPECIFICITY Enzyme

OF RAT HEPATIC

LIPASE

ANTISERUM

Lipolytic activity (~moles FFA . h-l

source

. g-l

after preincubation

with

% inhibition by anti-HL serum

NRS

Anti-HL

1 2

16.62 5.35

17.59 5.65

0

Heart muscle

1 2

11.46 5.04

11.12 4.73

3.0 6.2

Skeletal muscle

1 2

0.76 1.54

0.91 1.53

0 0

Liver perfusate

1 2

11.79 9.36

0.13 0.12

98.9 98.7

Adipose

tissue

serum 0

experiment confirmed the specificity of the antiserum virtually no LPL activity was present in heparin perfusates Inhibition

of postheparin

plasma lipolytic

and it showed of rat liver.

that

activities by the lipase antisera

Figures 1A and B demonstrate that the lipolytic activity of postheparin plasma was inhibited to the same extent by HL and LPL antiserum irrespective of the order of addition (79.4 and 77.9 I.tmol * h-’ * ml-’ by anti-LPL serum, and 66.8 and 68.3 pmol - h-’ - ml-’ b y anti-HL serum in Figs. 1A and B, respectively). The lipolytic activity remaining after incubation with both antisera was less than 5% of that initially present, indicating that almost all triacylglycerol lipase activity in rat postheparin plasma was either LPL or HL. Since the anti-HL serum did not cross-react with LPL of peripheral tissues, it seemed appropriate to use it for selective assay of both lipases in rat postheparin plasma. Optimal conditions for the assay system were therefore worked out. 200 -

@

0

100

":-; ANTI-LPLO 5 10 15 20

50 50 50

~----------3

II 5 10 10 10

10

10

0 0

20

50

0

5 10

Fig. 1. Inhibition of rat post-heparin plasma tdacylglycerol Iipase activities by anti-LPL (A) and anti-HL (B) sera. 10 ~1 aliquots of post-heparin plasma from a normal rat were preincubated for 2 h at +9’C with the indicated amounts of hepatic Iipase antiserum (anti-HL) and lipoprotein Iipase antiserum (antiLPL) made up to a volume of 70 ~1 with normal rabbit serum. Thereafter the Iipase activity was determined using the low salt substrate containing 10% (v/v) of human serum. It should be noted that the order of addition of antisera is different in the two experiments.

367

Effect of sodium chloride on postheparin plasma lipase activities Increasing concentrations of sodium chloride were added to the substrate mixture and the inhibition of postheparin plasma lipase activity was tested in the presence of either anti-HL or anti-LPL serum or normal rabbit serum. As illustrated in Fig. 2, the lipase activity remaining after treatment with anti-LPL serum was completely resistant to 1.0 M NaCl but was slightly inhibited by 2.0 M NaCl. The lipase activity of anti-HL serum treated plasma, on the other hand, was inhibited by as much as 82% by 1.0 M NaCl. Thus it was evident that HL could be measured at 1.0 M NaCl without significant interference by LPL. Serum activator required for full activity of LPL was present in all substrates used in the experiments shown in Fig. 2. At all salt concentrations the sum of the two lipase activities (activity remaining after preincubation with anti-HL and anti-LPL sera) was equal to the measured total lipolytic activity of rat postheparin plasma (preincubated with NRS) (Fig. 2). Effect of serum concentration Since LPL is activated by serum apolipoproteins we tested the effect of increasing concentrations of serum on the postheparin plasma lipase activities. Addition of either human or rat serum increased the LPL activity, the optimal amount being 10% of the volume of the final substrate mixture (Fig. 3). The HL activity, on the other hand, was not influenced by the addition of human serum but was inhibited by higher concentrations of rat serum.

200

NaCI,M Fig. 2. The effect of ionic strength on the determination of hepatic lipase and lipoprotein lipase in rat postheparin Plasma. Triacylglycetol lipase activity of rat postheparin plasma was measured with a substrate containing 10% (v/v) human serum at the NaCl concentration indicated, after preincubation with NRS (a-). anti-HL serum (O------O) or anti-LPL serum (*B ). The sum of activities after pretreatment with anti-HL and anti-LPL sera is also given (X -X).

368

VOLUME PER CENT SERUM IN ASSAY MIXTURE Fig. 3. Effect of serum on rat postheparin plasma triacylglycerol lipases. Postheparin plasma hepatic lipase was measured at a concentration of 1.0 M NaCl without preincubation with antiserum. Lipoprotein lipase activity was measured at 0.1 M NaCl after preincubation with antiserum against rat hepatic lipase. The effect of the different amounts of rat serum (o-----c ) or human serum (@A 1 added to the substrate mixture on the activities of lipoprotein lipase (A) and hepatic lipase (B) is shown.

Effect of substrate concentration The effect of substrate concentration on the activity of LPL and HL of postheparin plasma is shown in Fig. 4. The apparent KM values for both LPL and HL were 0.3. Maximal reaction rates were attained at about 1.5 mM triolein.

0

0.5

1.0

1.75

TRIGLYCERIDE

30

,mM

Fig. 4. Effect of substrate concentration on lipoprotein lipase and hepatic lipase activities in rat postheparfn plasma. The triglyceride concentration of the substrate was varied as indicated on the abscissa. The hepatic lipase activity (o---+ ) was determined at 1.0 M NaCl and without addition of serum cofactor. The assay for lipoprotein lipase (o----Q) was performed with preincubation with anti-HL serum and subsequent incubation with a substrate containing 0.1 M NaCl and human serum added as co-factor.

369

Effect of incubation time, pH, serum lipoproteins and sample volume The hydrolysis of triolein by both LPL and HL continued at a linear rate for 120 min at +28”C. The optimal pH was 8.6. Addition of purified rat serum lipoproteins to the sample did not significantly influence the rate of either lipase reaction (Table 2). The measurement of LPL present in increasing amounts of postheparin plasma was linear up to 50 ~1. The measurement of HL (1 .O M NaCl and no serum added) was linear only up to 10 ,ul of postheparin plasma as shown in Fig. 5. Therefore a sample volume of 2.5 or 5 1.c1was adopted for the HL assay. Figure 5 further illustrates the identity of the salt resistant lipase with hepatic lipase, since anti-HL serum inhibits all the triacylglycerol lipase activity in postheparin plasma which is measured with 1.0 M NaCl in the absence of serum. Selective assay of LPL and HL in rat postheparin plasma On the basis of the above experiments the following method was selected. It is based on the inhibition of HL by anti-HL serum and of LPL by 1.0 M NaCl and by the absence of serum as activator. For the assay of LPL, postheparin plasma is incubated with an appropriate amount (2-10 ~1) of anti-HL serum for 2 h at +4”C. Thereafter 500 ~1 of substrate mixture containing 0.1 M NaCl and 10% (v/v) human serum is added and the incubation is continued for 90 min at +28”C. In the assay of HL, 2.5 ~1 of postheparin plasma is incubated with 500 ~1 of substrate containing 1.0 M NaCl, for 90 min at +28”C. After the incubation the mixture is extracted and the radioactivity of FFA is determined as described under Materials and Methods. To assess the specificity of the assay method described above, postheparin plasma from supradiaphragmatic rats and heparin-containing perfusates of rat livers were used (Table 3). These enzyme

TABLE 2 EFFECT LIPASE

OF VLDL.

LDL AND HDL ON THE ACTIVITY

OF LIPOPROTEIN

LIPASE AND HEPATIC

Aliquots of postheparin plasma, 2.5 ~1, were mixed with purified preparations of VLDL. LDL and HDL. and the Iipase activity was determined with the standard procedure. Addition

Lipase activity (pmoles FFA

. h-l . ml-* )

Lipoprotein Iipase

Hepatic Iipase

VLDL a

0.00 1.39 2.76 5.52

84.73 78.78 85.20 83.93

85.76 81.88 82.09 82.57

LDL b

0.00 0.96 1.92 3.84

87.28 79.23 80.30 84.73

85.35 84.73 83.35 80.62

HDL b

0.00 0.71 1.54 3.08

73.09 68.27 71.90 70.97

66.42 61.30 57.77 57.95

a Final concentration of VLDL triglyceride in the sample (mM). b Final concentration of LDL or HDL cholesterol in the sample (mM).

5

10

20 PI POST-HEPARIN

30

40

50

PLASMA

Fig. 5. Immunotitration of rat postheparin plasma salt resistant lipase with anti-HI, serum. Various amounts of rat postheparin plasma were preincubated with 2 ~1 of anti-HL serum (e----O) or with normal rabbit serum (O------C ) for 2 h at +4’C!. Thereafter. lipase activity was determined with a substrate containing 1.0 M NaCl without addition of serum.

sources should contain either LPL or HL, respectively. The results show that about 2% of the triacylglycerol lipase activity measurable with the LPL assay can be detected with the HL assay. Further evidence for the specificity of the immunochemical method is the fact that less than 1% of the hepatic lipase is measured with the assay for LPL. The release of lipolytic activities in vivo In order to determine the dose of heparin that gives the maximal release of both triacylglycerol lipases, rats were injected with different doses of heparin. Plasma was collected 2 min later. Thereafter the rats were given a massive dose of heparin (2000 III/kg body weight), and 2 min later a new plasma sample was taken to assess the maximal lipolytic activity which could be released with heparin. The percentage of the maximal lipase activity released with each dose of heparin is shown in Fig. 6. Similar dose-response curves were obtained for LPL and HL activities. A heparin dose of 500 III/kg body weight seems to give maximal response. The time course of the lipase activities in rat postheparin TABLE

3

SELECTIVE MEASUREMENT OF HL AND LPL IN POSTHEPARIN MATIC RATS AND HEPARIN-CONTAINING LIVER PERFUSATES

PLASMA

Enzyme

* h-l * ml-‘)

source

Lipase activity Lipoprotein

Supradiaphragmatic rats (n = 3) Perfused rat livers (n = 3)

37.33 f 2.63 0.04 * 0.01

@moles

assay

FFA

OF SUPRADIAPHRAG-

Hepatic lipase 0.87 f 0.07 4.49 f 0.36

with assay for

371

HEPARIN, I.U./KG Fig. 6. Release of postheparin plasma lipoprotein lipase and hepatic llpaae as a function of the heparin dose injected. Rats were injected with the dose to be tested followed by a massive dose (2000 NJ/kg body weight). The activity released with the fist dose is given as percentage of the activity released with the lipoprotein lipase. Each point is a mean f SEM of 3 hepatic lipase; 04 second dose. ?? A experiments.

plasma after the injection of different amounts of heparin is illustrated in Figs. 7A and B. Maximal activities for both lipases were measured 2 min after the injection of heparin. This time interval was chosen for routine assays. Figure 7 also demonstrates that the amount of lipase activity remaining in the circulation after different time intervals is a function of the heparin dose injected.

100

IO0

75

75 -

50

26

25 -

2

15

30

60

120

2

180

15

30

60

120

MIN Fig. 7. Disappearance of postheparin plasma llpoproteln llpase and hepatic llpase after injections of various doses of heparin. Rats were injected with heparin under anesthesia and the percentage disappear100 IU. .250 IU. ance of postheparin plasma LPL (A) and HL (B) was monitored. o-----O 2000 IU heparin/kg body weight. a500 IU and A-

372

LPL and HL activities in rat postheparin plasma In order to study the relative contribution of HL and LPL activities to the total postheparin plasma lipase activity, LPL and HL were measured in postheparin plasma taken from 8 male and 8 female nonfasted rats 2 min after the injection of 500 IU of heparin per kg body weight. The mean LPL activity was 52.2 f 4.1 E.tmol - h-l - ml- ’ for male rats and 75.5 + 3.6 pmol - h-l - ml-’ for female rats (mean ? SEM). The corresponding HL activities were 74.8 f 4.2 and 65.8 f 5.0 E.cmol - h-l - ml -l. The difference between the LPL values was statistically significant (P < O.OOl), whereas the values for HL were not significantly different. Discussion The rat has been widely used to study lipid metabolism. We have therefore optimized an immunochemical assay for the selective measurement of the triacylglycerol lipases in rat postheparin plasma. The availability of specific antisera against both HL and LPL allowed us to assay one of the lipase activities after selective immunoprecipitation of the other. The specificity of the anti-HL serum was studied using enzymes from different sources. The antiserum did not cause inhibition of lipase activity eluted from adipose tissue, heart or skeletal muscle. Neither did it inhibit the lipase activity in the postheparin plasma of supradiaphragmatic rats, but it caused inhibition of lipase activity in heparin-containing perfusates of rat livers and of purified rat heparin lipase (not shown). Thus, the antiserum is specific and well suited for the assay of postheparin plasma LPL activity. This was further validated with the aid of anti-LPL serum. The triacylglycerol lipase activity remaining in postheparin plasma after preincubation with anti-HL serum, was dependent on the serum co-factor for activity and could be inhibited by high ionic strength, characteristics typical of LPL. This activity was abolished by anti-LPL serum, obtained by immunizing rabbits with purified LPL of bovine milk. There are conflicting reports about the presence of LPL activity in liver perfusates [18-211. In our studies we found no evidence for the presence of LPL activity in rat liver perfusates. The triacylglycerol lipase activity found in the latter was salt resistant, did not require co-factor for full activity and was not affected by anti-LPL serum. The apparent discrepancy between these results and those reported by others may reflect differences in assay conditions or even species variation. In the determination of HL from rat postheparin plasma, the LPL component adds less than 3% to the activity measured, as judged from the experiments with postheparin plasma from supradiaphragmatic rats (Table 3). Also the absence of any effect of anti-LPL serum added to the HL assay of postheparin plasma, indicates that no LPLactivity is measured with the high salt substrate in the absence of serum co-factor. The assay for hepatic lipase was linear only to about 10 ~1 of postheparin plasma: this should be kept in mind when assaying serum samples with high HL activity. A similar apparent saturation was not observed in the LPL assay. The reason for this is not known at present. The amount of human serum required as co-factor in the LPL assay

373

10% (v/v) of the assay medium. In contrast to human serum, the activation seen with rat serum varied greatly between different sera and was optimal within a relatively narrow range. We therefore use human serum as activator. The appearance of both postheparin plasma lipases after the injection of heparin was fast, as previously observed by Jansen et al. [22]. Only 2 min after injection, maximal release of both activities was obtained irrespective of whether arterial or venous postheparin plasma was used [23]. With the heparin doses used there was no difference in the pattern of release of the two activities, a finding somewhat different from that previously reported [22]. This could be due to the fact that very low doses of heparin were not used in our study. The injection of different amounts of heparin also indicated that the higher the dose, the longer the apparent half-life of lipolytic activity in plasma (Fig. 7). Therefore, in order to get a reproducible assay, a standard dose of heparin (500 IU/kg body weight) should be injected and the postheparin plasma sample should be obtained 2 min thereafter. Compared to human postheparin plasma lipase values [2], the activities in the rat were much higher. The activity of the hepatic lipase was about 4-fold and that of LPL about 3.5-fold higher than the corresponding values in man. The physiological meaning of these high activities is at present unclear. It is however tempting to speculate that these high levels could be responsible for the efficient clearance of chylomicron and very low density lipoprotein triglycerides in the rat [ 2,241. The immunochemical method described for the selective measurement of both triacylglycerol lipase activities in rat postheparin plasma should be a powerful tool in clarifying the roles of HL and LPL in the lipoprotein metabolism. was

Acknowledgements The skilful technical assistance of Mrs. Hannele Linturi is gratefully acknowledged. This work was supported by the State Medical Research Council (Academy of Finland) and by the Finnish Foundation for Cardiovascular Research. References Ehnholm. C., Shaw. W., Greten, H.. Lengfelder, W. and Brown, W.V.. Separation and characterization of two triglyceride lipase activities from human postheparln plasma. In: G. Schettler and A. Weizel (Eds.), Atherosclerosis III (Proc. III Int. Symp. Atherosclerosis), Springer, Berlin, 1974, p. 557. Huttunen, J.K.. Ehnholm, C., Kekki, M. and Nikkilii. E.A., Post-heparin plasma lipoprotein Iipase and hepatic lipase in normal subjects and in patients with hypertriglyceridaemia - Correlations to sex, age and various parameters of triglyceride metabolism, Clln. Sci. Mol. Med., 50 (1976) 249. Kompiang, I.P.. Bensadoun, A. and Wang. M.W.W., Effect of an antilipoprotein lipase serum on plasma triglyceride removal, J. Lipid Res.. 17 (1976) 498. Krauss, R.M.. Windmueller. H.G., Levy, R.I. and Fredrickson, D.S.. Selective measurement of two different triglyceride lipase activities in rat postheparin plasma, J. Lipid Res., 14 (1973) 286. Baginsky, M.L., Wu, F. and Brown, W.V.. Assay of lipoprotein lipase in postheparin plasma after suppression of the hepatic triglyceride lipase with sodium dodecyl sulphate. In: H. Peeters (Ed.), The Lipoprotein Molecule, Plenum Press, New York, 1978, p. 169. Jansen, H. and Hillsmann, W.C.. On the hepatic and extrahepatic postheparln serum llpase activities and the influence of experimental hypercortisollsm and diabetes of these activities, Biochim. Biophys. Acta. 398 (1975) 337. Corey, J.E. and Zilversmit. D.B., Validation of a stable emulsion for the assay of lipoprotein lipase activity, J. Lab. Clln. Med., 89 (1977) 666.

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with

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