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Clinical Science and Molecular Medicine (1978), 54, 201-203

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Lipolytic activities in post-heparin plasma in man measured with different substrate emulsions B. VESSBY, J. BOBERG AND H. LITHELL Department of Geriatrics, University of Uppsala, Uppsala, Sweden

(Received 24 June 1977; accepted 12 September 1977)

Summary 1. Post-heparin lipolytic activity in man has been studied by using a triglyceride substrate emulsion containing different emulsifiers. 2. The lipolytic activity measured was pro­ foundly influenced by the type of emulsifier used in the substrate. Substrates stabilized by synthetic emulsifiers give higher lipolytic activity than Intralipid, which contains egg phospholipids as emul­ sifiers. This difference was solely explained by higher salt-resistant lipase activities found with emulsions containing synthetic emulsifiers. The salt-inhibited lipase activity, which has properties as a lipoprotein lipase, was not influenced by the type of emulsifier. 3. When used under specified conditions Intralipid seems to be virtually specific for extrahepatic post-heparin lipolytic activity. Key words: emulsifier, lipoprotein lipase, postheparin lipolytic activity, triglyceride substrate. Introduction Post-heparin plasma contains at least two different triglyceride lipase activities. By using affinity chromatography of post-heparin plasma on heparin-Sepharose eluted with a linear salt gradient, Ehnholm, Shaw, Greten, Langfelder & Brown (1974) were able to demonstrate one saltresistant activity which did not need a serum cofactor for optimum activity and one serumCorrespondence: Dr Bengt Vessby, Department of Geriatrics, University of Uppsala, Box 641, S-751 27 Uppsala, Sweden.

activated triglyceride lipase activity (EC 3.1.1.3, triacylglycerol lipase, glycerol ester hydrolase) (Ehnholm et ah, 1974b). Although the latter enzyme activity showed inhibition characteristics similar to those of lipoprotein lipase (EC 3.1.1.34), e.g. from adipose or muscular tissue, the salt-resis­ tant lipase activity has been shown to originate in the liver (La Rosa, Levy, Windmueller & Fredrickson, 1972; Assman, Krauss, Fredrickson & Levy, 1973; Ehnholm, Bensadoun & Brown, 1973). The conflict between the results of earlier assays of post-heparin lipolytic activity may have been due to variation in amounts of more than one triglyceride lipase in plasma. Different assay systems may vary in specificity for the separate triglyceride lipase activities. The results are influen­ ced by the composition and pH of the assay mix­ ture (Ehnholm, Greten & Brown, 1974a), the dose of heparin injected (Boberg, 1972; Krauss, Levy & Fredrickson, 1974), the time between heparin in­ jection and blood sampling (Boberg, 1972; Krauss et al, 1974) and the properties of the substrate (Datta & Wiggins, 1964; Boberg & Carlson, 1964; Kraussei al., 1974). This study was undertaken to characterize how the type of emulsifier used in the substrate may influence the measurements of triglyceride lipase activities. Materials and methods Triglyceride substrates Soya-bean-oil emulsions containing 10% triglycerides and different emulsifiers were generously supplied by AB Vitrum, Stockholm, Sweden 201

202

B. Vessby, J. Boberg andH. Lithell

TABLE 1. Assay of post-heparin plasma lipolytic activity using triglyceride substrate emulsions containing 10% triglycerldes and different emulsifiers The concentration of each emulsifier is given in parentheses. Salt-resistant lipase activity is triglyceride lipase activity not inhibited by NaCl (1 mol/1) and salt-inhibited lipase activity is triglyceride lipase activity inhibited by NaCl (1 mol/1). Triglyceride lipase activity Qanol/min)

Type of emulsifier

Assay at 37°C

Assay at 27°C

Phospholipids (1-2%) (Intralipid) Myrj 52 (1%) Tween 20 (0· 195%)/Span 80 (0-455%) Triton X-100 (1%) Brij 35 (1%)

Total activity

Salt-resistant activity

Salt-inhibited activity

Total activity

Salt-resistant activity

Salt-inhibited activity

60 100 92 112 92

16 68 60 64 52

44 32 32 48 40

84 136 156 148 120

0 60 64 48 48

84 76 92 100 72

Intralipid was completely inhibited by sodium chloride (1 mol/1) when the assay was performed at 37°C. The total triglyceride lipase activity measured with Intralipid at 27 °C was lower than at 37°C. However, a certain amount of the lipase activity was not inhibited by salt when measured at 27°C. The salt-inhibited lipase activity was approximately doubled when the incubation temperature was increased from 27°C to 37°C. When the plasma sample was preincubated with protamine sulphate (Krauss et al., 1974) for 10 min Assay of triglyceride lipase activity in post-heparin the lipase activity was inhibited to a similar extent plasma as with sodium chloride (1 mol/1). All emulsions stabilized by synthetic detergents For determination of post-heparin lipolytic ac­ showed similar properties both at 27°C and at tivity in plasma heparin (5000 i.u./ml; AB Vitrum) 37°C, which were clearly different from those of was given in a dose of 100 i.u./kg body weight the phospholipid-stabilized Intralipid. At 27°C intravenously to a healthy man who had fasted Intralipid showed an activity of salt-resistant tri­ overnight A venous blood sample was taken 40 glyceride lipase that was only 27% of the total min after heparin injection. Post-heparin lipolytic triglyceride lipase activity, and this ranged from activity was determined by the method of Boberg 57 to 68% with the other four emulsions. The total & Carlson (1964) and Boberg (1970). The assay salt-resistant activity measured with these emul­ was performed either at 27°C or 37°C in an sions at 27° C was approximately four times that ammonium buffer [31 vol. of aq. ammonia solution (0· 1 mol/1), 30 vol. of ammonium chloride solution measured with Intralipid as a substrate. However, (0· 1 mol/1)] containing 10% bovine albumin at pH Intralipid and the four emulsions stabilized with synthetic detergents showed a similar salt-inhibited 8-70. The concentration of the triglyceride sub­ triglyceride lipase activity. strate in the incubation mixture was 8 mmol/1. When the incubation temperature was 37°C the total triglyceride lipase activity was higher than at Results 27°C with all substrates. This was always due to Table 1 shows the results from one experiment an increase in salt-inhibited lipase activity, which performed at 27°C and 37°C with different sub­ approximately doubled when the incubation strate emulsions. Similar results were achieved in temperature was increased from 27°C to 37°C. several experiments. The assay was performed both With the emulsions stabilized by synthetic deter­ without extra salt and in the presence of sodium gents the amount of salt-resistant lipase activity chloride (1 mol/1) in the incubation mixture. measured at 37°C was virtually unchanged com­ pared with that at 27°C. The total lipolytic activity Post-heparin lipolytic activity measured with (Table 1). The detergents used were synthetic surfactants containing polyoxyethylene chains of diverging length in ether or ester bindings (Myrj 52, Tween 20/Span 80, Triton X-100, Brij 35). The emulsions were made in the same way as Intralipid (AB Vitrum) with the exception that no glycerol had been added. They were all made from the same batch of soya-bean oil and prepared at the same time during identical technical conditions.

Post-heparin lipolytic activity was 60-80% higher with emulsions with synthetic detergents than with Intralipid. This difference was solely explained by the higher salt-resistant activity that was present with the emulsions stabilized by synthetic emulsifiers. With Intralipid as a substrate virtually all lipolytic activity was inhibited by sodium chloride (1 mol/1) when measured at 37°C. With the other four emulsions the salt-resistant lipase activity amounted to 32-44% of the total activity at the same temperature.

203

also reported on a glycerol-based trioleylglycerol and phosphatidylcholine emulsion similar to Intra­ lipid, which seems to be specific for extrahepatic post-heparin lipolytic activity. Acknowledgments This work was supported by the Swedish Medical Research Council (grant no. B76-4679-02) and Vitrum AB, Sweden. References

Discussion In the past, different types of substrates have been used in assay systems for determination of lipoprotein lipase activity. The diverging results from studies where Triton X-100 (La Rosa et al., 1972; Krauss et al., 1974), Tween 60 (Datta & Wiggins, 1964), gum arabic (Ehnholm et al, 1974b) and phospholipids (Boberg & Carlson, 1964) have been used as emulsifiers, probably arise to some extent from the properties of the substrates. It is not clear why the type of emulsifier in the substrate so profoundly influences the determination of the enzyme activities. Electron-microscopy studies of the particle size of soya-bean-oil emulsions, of Intralipid type prepared with various emulsifying agents did not indicate that particle size varies with the emulsifying system used (Jeppson & Schoefl, 1974). We have not yet defined the physicochemical factors responsible for the differences in substrate properties between triglyceride emulsions containing different emulsifying agents. Intralipid stabilized by egg phospholipids thus seems to have unique properties as triglyceride lipase substrate. In an assay performed at 37°C, with a plasma sample drawn 40 min after heparin injection as enzyme source, the lipase activity was virtually completely inhibited by sodium chloride (1 mol/1) or by preincubation with protamine sulphate. This is in accordance with earlier reports by Boberg & Carlson (1964) and Boberg (1970). This assay thus seems to measure extrahepatic triglyceride activity very specifically with inhibition properties which are characteristic for lipoprotein lipase. Our results are supported by Rogers, Barnett & Robinson (1976), who used Intralipid as a substrate, finding that at most 10% of the total lipolytic activity could be attributed to the saltresistant lipase. Corey & Zilversmit (1977) have

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R.I. (1973) Characterization, subcellular localization and partial purification of a heparin-released triglyceride lipase from rat liver. Journal of Biological Chemistry, 248, 19921999. BOBERO, J. (1970) Quantitative determination of heparin released lipoprotein lipase activity in human plasma. Lipids, 5,452-456. BOBERG, J. (1972) Heparin-released blood plasma lipoprotein lipase activity in patients with hyperlipoproteinemia. Äcta Medtca Scandinavica, 191,97-102. BOBERG, J. & CARLSON, L.A. (1964) Determination of heparininduced lipoprotein lipase activity in human plasma. Clinlca ChlmicaActa, 10,420-427. COREY, J. & ZILVERSMIT (1977) Validation of a stable emulsion for the assay of lipoprotein lipase activity. Journal of Labora­ tory and Clinical Medicine, 89,666-674. DATTA, D.V. & WIGGINS, H.S. (1964) New effects of sodium chloride and protamine on human postheparin plasma 'lipo­ protein' lipase activity. Proceedings of the Society for Experimental Biology and Medicine, 115,788-792. EHNHOLM,

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BROWN, W.V. (1974b) Separation and charac­ terization of two triglyceride lipase activities from human post-heparin plasma. In: Atherosclerosis III, pp. 557-560. Ed. Schettler, G. & Weizel, A. Springer Verlag, Berlin. JEPPSON, R. & SCHOEFL, G.I. (1974) The ultrastructure of lipid particles in emulsions prepared with various emulsifiers. Australian Journal of Experimental Biology and Medical Science, 52, 697-702. KRAUSS, R.M., LEVY, R.I. & FREDRICKSON, D.S.

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