Plasma lipolytic activity after subcutaneous administration of heparin and a low molecular weight heparin fragment

THROMBOSIS RESEARCH 46; 697-704, 1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.

PLASMA LIPOLYTICACTIVITYAFTER SUBCUTANEOUSADMINISTRATION OF HEPARINAND A Low MOLECULARWEIGHT HEPARIN FRAGMENT

E. Persson,J. Nordenstrijm*, L. Hagenfeldt+& P. Nilsson-Ehle' * Departmentof Anaesthesia,Karol&nska Hospital,Stockholm, Surgery,Huddinge UniversityHosy$ital, Huddinge, ClinicalChemistry,KarolinskaHospital, Stockholmand ClinicalChemistry,UniversityHospital,Lund, Sweden. (Received 14.11.1986; Accepted in revised form 5.2.1987 by Editor B. Wiman) (Received in final form by Executive Editorial Office 18.3.1987) ABSTRACT

The effect of heparin and a low molecularweight heparin fragment (I&WI,mean molecularweight 4000-6000) on plasma anticoagulation and lipolysiswas studied in eight healthymen. The activitiesof antifactorXa (antiFXa),lipoproteinlipase (LPL),hepatic lipase (HL) and plasma levelsof free fatty acids (FFA)were analysedafter the injectionof 5000 antiFXaunits of heparinor IMWH subcutaneously.In comparisonwith heparin,the administrationof I&lWHresultedin a significantlyhigher antiFXa activity (p < O.OOl)buta lower release of LPL and I-IL (p < O.OOl), which did not increaseplasma FFA. It is concludedthat subcutaneousinjectionof IMWH in men elicitsan adequate anticoagulanteffect measuredas antiFXa activitybut has a negligibleeffect on plasma lipolyticactivity.

INTRODUCTION The anticoagulanteffect of heparin involvesinhibitionof severalactivated coagulationfactors,the result of which is a prolongationof the clotting time. This action has been shown to depend on the molecularweight (MW) of the heparin preparation.In contrastto standardheparin (MW 12,000 low molecularweight heparin fragments (IMWH)with an MW of about - lS,OOO),

Key words: Heparin,hepatic lipase, lipoproteinlipase, low molecularweight heparin.

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4,000 have a littleeffect on the activatedpartial thromboplastintime (APTI') (1,2).The inhibitionof activatedfactor X is independentof the MW of the heparin,however.Thus, LMWH exerts its anticoagulanteffect mainly by increasingantifactorXa (antiFXa)(3).Recent studieson patientswith deep vein thrombosis (4) and on postoperativethrombo-embolism(5) have shown an antithromboticeffect of I&lWHtreatmentby the subcutaneousroute which is comparableto conventionalheparin.I&lWHhas also been shown to appear safe as an anticoagulantagent to patientsundergoinghaemodialysisand haemofiltration (6). The bioavailabilityof heparin after subcutaneousadministrationis also dependenton its MW. Comparedto standardheparin,UIWH has been shown to have a higher and more durable antiFXa activity (7,8). Apart from its anticoagulanteffect,heparin also exerts a lipolyticeffect on circulatingtriglyceride-rich particlesin plasma by releasinglipoprotein lipase (LPL)and hepatic lipase (HL) from the vascularendothelium(9).We have shown in a previousstudy that the plasma activitiesof LPL and HL are lower after intravenousinjectionof ti than after standardheparin (10). The aim of this study was to determinewhether I&lWH,with its weaker lipolytic potential,still elicits a strongerlipolyticactivitythan heparin after subcutaneousinjectionowing to the better bioavailabilityof UlWH by this route.

MATERIALSAND METHODS Eight healthy men were investigated.Their mean (range)age, weight and height were 28 (22-44)years, 71 (61-79)kg and 181 (178-186)cm. Each subject was investigatedtwice with one week's interval.Serum triglyceride levelswere within normal ranges in all subjectsat both occasions (1.1+ 0.1 ml/l and 1.3 + 0.1 nnnol/lrespectively).The investigations were perfo%ed in the morning after an overnightfast. No oral intake was permittedduring the experiment. An indwellingintravenouscatheterwas insertedin the antecubitalvein for blood samplingusing the vacutainersystem.After initialblood sampling, an injectionof 5,000 antiFXa units (U) of heparin or LMWH was given subcutaneouslyin the thigh in a randomizedsequence. The IMWH preparation which was providedfrom KabiVitrumAR, Sweden (Kabi 2165) had an MW of 4,000-6,000and a specificactivityof 160 antiFXaunits/mg or 40 APTT units/mg.The standardheparinpreparation(KabiVitrumAD) had a mean MW or 12,000-15,000.Its specificactivitywas about 160 antiFXa units/n-g or about 160 APTT units/q. After the injections,blood sampleswere taken at regular intervalsaccording to the experimentalprotocol.The sampleswere immediatelyplaced on ice; plasma was separatedby centrifugationat 3,000 g during 10 min. in the cold state and extractedfor analysisof free fatty acids (FFA)in triplicateby the method of Hagenfeldt (11).APTT was checked imnediatelyusing a standard method (Cephotest,Nyegaard & Co, Norway).A portion of the plasma was frozen to -2OOC until analysedfor LPL and HL activitiesas describedby NilssonEhle and Ekman (12).AntiFXa activitywas analysedin titratedplasma using the chromogenicsubstrateS-2222 (Coatest,KabiVitrumAD). The statisticalcalculationscomprisedanalysisof variancewith a randomized three block design for comparisonsof the time course of the curves. Data concerningAPTT were analysedby Student'st-test.All values are given as the mean + SEM.

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RESULTS

Four hours after subcutaneousinjection,APIT was prolongedonly in the LMWH group (TableI). The inhibitionof factor Xa was five times stronger after injectionof IMWH than after heparin (p < 0.001, Fig. 1). The maximal release of LPL activitywas seen one hour after I&lWHinjection and two hours after heparin (Fig.2). The LPL activitymeasuredin plasma over the study period was greater after heparin administration(p < 0.001). The peak of HL activitywas reached two hours after administrationof I&lWH and four hours after heparin (Fig.3). The releaseof HL activitywas considerablygreater after the heparin injection (p < 0.001). An increasein plasma levelsof FFA was seen at the end of the study period after heparin administration(p < 0.01). After IMWH a small but not significant increasein FFA was noted (Fig.4).

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Figure1 The effectofsubcutaneousadministrationof 5,000 U of heparin (o-o) and I&lWH(o--o) on antifactorXa activity (U/ml).Mean values + SEM, p < 0.001 heparin vs. IMWH. TABLE I APTI'(seconds)before and four hours after subcutaneous administrationof 5,000 U of heparin and IJIWH. Mean values -+ SEM. HEPARIN Before After

33 -+ 1

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* 36 -+ 1 * p < 0.05 before vs. after 33 -+ 1

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Figure2 Plasma lipoproteinlipase activity (mu/ml)after subcutaneousadministration of 5,000 U of heparin (o-o) and LNWH (o--o). Mean values + SEM, p < 0.001 heparin vs. L&WI.

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Figure3 Plasma hepatic lipase activity (mu/ml)after subcutaneousadministrationof 5,000 U of heparin (o-o) and LMWH (o--o). Mean values + SEN, p < 0.001 heparin vs. LMWH.

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Figure4 Plasma levelsof FFA (ml/l) after SubcutaneOUSinjectionof 5,000 U of heparin (o-) (p < 0.01 comparedto pre-heparinvalue) and IJQIH(o- -01 (not significant).Mean values -+ SEM.

DISCUSSION The relationshipbetween the MW of heparin and its anticoagulanteffect is well documented (1,2).The influenceon AE?l?T decreaseswith decreasingMW, while antiFXa activityremains unchangedor even increases (3). During the last few years it has been found that heparinsof high and low MW interacton the vascularendotheliumdifferently.O'Brien et al have reported that the potency of IMWH to mobilizeplateletfactor 4 from the endothelium is weaker than that of unfractionatedheparin (13).The abilityof heparin to releaseLPL and HL from the vascularendotheliumis also dependent on MW. In a previous study we have shown that the intravascularlipolytic effect after intravenousinjectionof IklWHis less pronouncedthan after injectionof heparin (10). In the present study antiFXa activitywas about five times greaterwith I&lWHthan with heparin after subcutaneousadministration.Similar results have been obtainedby Berggvistet al, who found that the heparin fragment has a more inmediateeffect, a higher peak activityand a longerdurationof

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antiFXa activitythan native heparinwhen given subcutaneously(8).The antiFXa levelsobtainedafter I.MWHin this study were well within the therapeutic range for the treatmentof deep vein thrombosis (4) and preventionof postoperativethrombosis (5).A slight prolongationof APTT was also seen with IMWH. These observationsare probablydue to faster absorptionof the smaller moleculesfrom the subcutaneousdepot into the blood stream.Thus, even though heparin pa ne has a strongerlipolyticpotentialthan LMWH, this effect may be outweighedby the better bioavailabilityof IMWH when given subcutaneously. LPL is the key enzyme in the plasma delipidationprocess of chylomicrons and very low density lipoproteinsto FFA and glycerol (9).When injectedintravenously,heparin releasesLPL from the capillaryendotheliumwithin minutes (10).In this study, with subcutaneousadministration,the peak of LPL activity was reached one to two hours after IMWH and heparin.The lipaseactivity over the study period was more prolonged,however,and of greater magnitude after heparin as comparedto I&lWH.This differencewas marked only during the second half of the study period during which time a significantincreasein plasma levelsof FFA was seen in the heparin group. It is thereforelikelyto assume that this increasein FFA reflectsthe lipolyticeffect of heparin. In the clinicalsituation,an increasein plasma FFA levelsafter injection of heparin is considereddisadvantageousor even harqul. Cardiac arrhythmias during myocardialischaemiahave been reportedto be caused by heparin-induced FFA elevationin plasma (14,15).There have also been reports on displacement of drugs from albuminbinding sites by high concentrationsof FFA (16,17). Althoughthe risk of such potentialadverse effects is greatestafter intravenous injectionof heparin,the risk of side effects after subcutaneousheparin administrationshould be consideredsince this also causes an increasein plasma FFA. The risk of an accumulativeeffect after repeatedheparin administrationshould also be kept in mind. Despite an increasein plasma lipase activityafter I.MWHinjection,this activitywas not high enough to result in a significantelevationof plasma FFA. From this study it can thereforebe concludedthat comparedto standard heparin,subcutaneousadministrationof LMWH results in a much higher antiFXa activitybut a considerablyweaker lipolyticeffect with unchangedplasma levels of FFA. From the obtaineddata it can be calculatedthat the ratio of LPL activityand antiFXa activitybetween heparin and IMWH is about 7.5. Thereforesubcutaneousadministrationof LMWH offers a clinicaladvantageover standardheparinwhen an anticoagulantand antithromboticeffect is desired and an elevationof plasma FFA is consideredundesirable.

ACKNCWLEi3GEMENTS We are gratefulto MS Karin S&derberg,KabiVitrumAB, for performingthe antiFXa assays.This work was supportedby grants from the SwedishMedical Research Council (projectsno. 722, 4201 and 4966).

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15. TANSEY M.J.B., OPIE L.H. Relationbetween plasma free fatty acids and arrhythmiaswithin the first twelve hours of acute myocardialinfarction. Lancet,ii, 419-422,1983. 16. CRAIG W.A., SUH B. Changes in protein binding during disease.Scand.J. Infect.Dis.,14, 239-244,1978. 17. RUDMAN D., BIXLER T.J., DEL RIO A.E. Effect of free fatty acids on binding of drugs to bovine serum albuminby human serum albumin and by rabbit serum. J.Pharmacol.Exp.Therap., 176, 261-272,1971.