To investigate the role of 2,3-butanediol in myocardial ischaemia we analysed this compound in pig's myocardium and blood. Ischaemia was induced by ligation of a coronary artery. In the first study we found significantly higher levels of 2,3-butanediol in the homogenate of ischaemic myocardium than in non-ischaemic myocardium. The lactate concentration was also significantly elevated. In the second study, where ischaemia was similarly induced, and where reperfusion was achieved by re-opening the ligated coronary artery after 20 min, 2,3-butanediol in peripheral blood was found to increase significantly. In the pigs in which the coronary artery was not re-opened, the 2,3-butanediol level in peripheral blood was unchanged. We conclude that in pigs' anaerobic myocardia accumulation of 2,3-butanediol occurs; if the myocardium is reperfused this metabolite also appears in the bloo

ALTHAUS, U.

HEER, K. R.

METTLER, D.

SCHILT, W.

THOELEN, H.

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European Heart Journal (1991) 12, 654-6562,3-butanediol in experimental myocardial ischaemia in pigsK. R. HEER, U. ALTHAUS, D. METTLER, W. SCHILT AND H. THOELENDepartement Forschung Kantonsspital Basel, Klinikfur Thorax-, Herz- und Gefasschirurgie Inselspital Bern,SwitzerlandKEY WORDS: Myocardial ischaemia, intermediary metabolism, 2,3-butanediol, pigs.To investigate the role of 2,3-butanediol in myocardial ischaemia we analysed this compound in pig's myocardium andblood. Ischaemia was induced by ligation of a coronary artery. In the first study we found significantly higher levels of 2,3-butanediol in the homogenate ofischaemic myocardium than in non-ischaemicmyocardium. The lactate concentration wasalso significantly elevated. In the second study, where ischaemia was similarly induced, and where reperfusion was achievedby re-opening the ligated coronary artery after 20 min, 2,3-butanediol in peripheral blood was found to increase signifi-cantly. In the pigs in which the coronary artery was not re-opened, the 2,3-butanediol level in peripheral blood wasunchanged. We conclude that in pigs' anaerobic myocardia accumulation of 2,3-butanediol occurs; if the myocardium isreperfused this metabolite also appears in the blood.Introduction2,3-butanediol is a product from the intermediary metab-olism; its precursors are pyruvate, acetaldehyde andacetoin'1'2'. Similar to lactate, the formation of this productis accelerated under anaerobic conditions'31. Abnormallactate extraction does occur in ischaemic myocardiumand abnormal levels of lactate are found in coronary sinusbut not in peripheral blood'41. To investigate the possiblerole of 2,3-butanediol as a peripheral biochemical markerfor anaerobic metabolism, we analysed it in ischaemic andnon-ischaemic myocardium of five pigs (tissue study). In asecond study (plasma study) we compared plasma levels intwo groups of pigs: in the first five pigs the occluded coron-ary artery was reopened after 20 min, whereas in the secondfive the occlusion was maintained during the study.MethodsANIMAL PREPARATIONSPigs were chosen because their coronary distributions,unlike other species, are not subjected to wide variations.The animals, weighing from 20 to 25 kg, were premedi-cated intramuscularly with ketamine hydrochloride(10 mg kg"') and intravenously with atropine sulfate(1 mg kg"1). Anaesthesia was induced with intravenousazaperone (2mg kg"1) and metomidate (5mg kg"1).Following tracheal intubation, respiration was controlledusing a volume ventilator supplying oxygen-enrichedroom air; anaesthesia was maintained by adding fluo-thane to the gas mixture (0-5—1 -5%). Venous bloodsampling was performed from a superior caval catheterand arterial blood pressure was measured by a Stathamtransducer by cannulating the carotid artery.Through a left thoracotomy the left anterior descend-ing coronary artery (LAD) was dissected distally to theSubmitted for publication on 1 November 1988. and in revised form 24November 1989.Correspondence: Dr Kaspar Heer, Spezialarzt fur Innere Medizin. Spez.Herzkrankheiten. Kasernenstr. 22a. 4410 Liestal. Switzerland.origin of the first diagonal branch and a suture passedaround the dissected blood vessel, in order to obtain apermanent or temporary occlusion. Ventricular arrhyth-mias were treated with intravenous lignocaine (20 mg)and electrical defibrillation was carried out if ventricularfibrillation occurred.Experimental protocolSTUDY A (TISSUE STUDY)Coronary occlusion was maintained for 10 min in fivepigs. The animals were then sacrificed and the heartimmediately removed. Two specimens of 1 g were excised,both from ischaemic and non-ischaemic areas of myo-cardium and prepared for analysis of 2,3-butanediol andlactate.STUDY B (PLASMA STUDY)Two pigs from the same litter were used for each exper-iment. The animals were randomized and divided into twogroups. In five pigs the dissected coronary arteryremained ligated permanently (group A) and in anotherfive coronary perfusion was restored following a 20 minocclusion (group B). In the latter group the end of thethread passed around the LAD was drawn through asmall and rigid rubber tube to cause temporary occlusionwithout kinking'5'. In both groups the chest was closed inlayers and the animals were allowed to recover.For serial determinations of 2,3-butanediol, samples ofvenous blood were periodically drawn from the cavalcatheter prior to, and during, the 2 h following permanentor temporary occlusion. Twenty-four h after the surgicalprocedure the animals were anaesthetized with intra-venous sodium pentobarbital (30mg kg"') and put todeath. The heart was rapidly excised and deep frozen tofacilitate slicing.ANALYSIS OF 2,3-BUTANEDIOL AND LACTATE2,3-butanediolStudy A. One g of excised muscle was treated with 4 mlof phosphate buffer and homogenized. 10 ml of 10%0195-668X91 -050654 + 03 S03.00,0 © 1991 The European Society of Cardiology2,3-butanediol in experimental myocardial ischaemia 655trichloroacetic acid and 20 ml of water were added. Themixture was shaken and centrifuged. The supernatantsolution was decanted and deep frozen.Study B. Samples of 10 ml venous blood were treatedwith 10 ml of 10% sodium tungstate and 10 ml of sul-phuric acid. 20 ml of water was added: the mixture wasthen centrifuged and the supernatant solution decantedand deep frozen.2,3-butanediol was determined using the methodsdescribed by Westerfeld'6' and Happold and Spencer'71.Values are given in umolg"1 (study A) and umoll"'(study B). In our laboratory the relative coefficient of vari-ation is less than 5%. Lactate was analysed using themethod described by Passonneau'8'.ASSESSMENT OF INFARCT SIZEThe frozen ventricles were cut into parallel slices andincubated in a solution of nitro-benztoluene as described inour previous report'5'. Tetrazolium in nitro-benztoluene isreduced by the dehydrogenase systems of the intact mito-chondria to insoluble diformazane, staining normal myo-cardium dark-blue and leaving infarcted areas almostwhite. This technique permitted a clear identification ofnecrotic myocardial tissue and allowed quantitativeassessment of the infarcted area using a morphometricpoint counting method.STATISTICSPaired and unpaired t-tests were used for statisticalanalysis.ResultsSTUDY A (TISSUE STUDY)Distributions of 2,3-butanediol were: 18-74 +1-75 umolg"1 in ischaemic and 14-29 + 0-75 umolg"1in non-ischaemic myocardium (/>< 0-001), and lactate:27-93 + 2-31 mmol g"1 in ischaemic and 16-67 +4-62mmolg~' in non-ischaemic myocardium (P <001)(Fig. 1).STUDY B (PLASMA STUDY)In those pigs in which the occlusion was maintained foronly 20 min (group B), 2,3-butanediol increased graduallyand significantly from 7-83+ 1-87 umol I"1 to11-44 + 2-31 umol I"1 in samples taken 40 min after re-opening (P<002). This elevated level was also signifi-cantly higher than the 60 min level in pigs where the arteryremained occluded (group A, P<002). The decline afterthe peak level was gradual; 100 min after re-opening, 2,3-butanediol was in the range of the initial values. In pigs inwhich the occlusion was maintained during the entirestudy (group A), 2,3-butanediol remained unchanged:803+1-68 umol I"1 and 7-85 ± 1-45 umol I"1 before and60 min after occlusion respectively (Fig. 2).INFARCT SIZEIn pigs in which the occlusion was re-opened after20 min, no infarct could be documented histochemicallyand morphometrically; in pig hearts with maintained2,3-butanediolfj.mo\ g~lLactatemmol g~'Figure 1 2,3-butanediol and lactate in homogenate of pig hearts'ischaemic O and non-ischaemic ( • ) myocardium.7 10-Q .oJ*CD O§• Io _S> .2HrO20 40 60(min)80 120Figure 2 Time-related pattern of 2,3-butanediol in pigs' blood withand without permanent occlusion of the coronary artery (group Aversus group B).656 K. R. Heer et al.Table 1 Infarct size (%) in group A and group BGroup A, Occlusion ofleftcoronary arteryGroup B, Occlusion ofleftcoronary artery; reopeningafter 20 minPiglPig 2Pig 3Pig 4Pig 514-4%16-7%18-9%18-6%—PiglPig 2Pig 3Pig 4Pig 50%0%0%0%0%occlusion, the infarct size varied between 14-4 and 18-9%.Figures are given for four pigs only; the fifth died in theexperiment (Table 1).col, however, did not allow us to measure the ischaemicarea during occlusion; from the infarct study we assumethat this area was nearly the same in all five pigs (between14-4 and 18-9%). The decline after the peak value was alsogradual and 100 min later initial values were achieved. Thisfinding is in accordance with Soling et al. who concludedfrom their in vivo infusion study that 2,3-butanedioldisappears from the blood within hours'9'.We conclude that in pigs, anaerobic conditions inthe myocardium can lead to the accumulation of 2,3-butanediol in myocardial tissue; if the myocardium isreperfused 2,3-butanediol also appears in the blood.The results suggest that 2,3-butanediol is a marker forischaemic metabolism in heart muscle.We gratefully acknowledge the technical assistance of Mr FerencToth and the staffof the experimental unit of the Inselspital Bern.DiscussionLittle is known about the metabolic pathway of 2,3-butanediol. From in vitro studies it has been proposedthat its precursor is mainly a condensation product fromtwo molecules of pyruvate; it is then reduced by a butane-dioldehydrogenase to form 2,3-butanediol[u21. Underanaerobic conditions it can be expected that this diol-formation is increased similar to the production of lactate.In our homogenate study (study A) we were able to con-firm this hypothesis: in ischaemic myocardium we foundsignificantly more 2,3-butanediol and lactate than in non-ischaemic myocardium in the same pig heart (Fig. 1).Although we could not undertake studies with a shorterischaemic time, we assume that this diol formation takesplace very rapidly. The products are then accumulated inthe ischaemic cell; the further metabolism within the cell isunknown. In the second study (study B) we demonstratedthat by re-opening the occluded vessel, these productscould be washed out into the blood. Forty min after re-opening a coronary artery which had been occluded for20 min we found a gradual and significant increase of 2,3-butanediol (/><0-02, Fig. 2). The concentration maydepend on the extension of the ischaemic area; the proto-References[1] SchreiberG, KohlhawG, GoedeHW, HolzerH. Die Biosynthesevon Acetoin in Schweineherzmuskel. Biochem Zeitschrift 1963;339: 83-93.[2] Veech RL, Gitomer WL, Casazza JP. Metabolic pathways leadingto diol formation. Genet Alcohol 1987; 185-99.[3] Jarnefelt J. Studies on the enzymatic synthesis and breakdown ofacetoin in the animal organism. Ann Acad Sci Fenn 1955; V:1-78.[4] Opie LH, Owen P, Thomas M, Samson R. Coronary sinus lactatemeasurements in assessment of myocardial ischaemia. Am JCardiol 1973; 32: 295-305.[5] Althaus U, Gurtner HP, Baur H, Hamburger S, Roos B. Conse-quences of myocardial reperfusion following temporary coron-ary occlusion in pigs; effects on morphologic, biochemical andhemodynamic findings. Eur JClin Invest 1977; 7:437.[6] Westerfeld WW. Colorimetric determination of blood acetoin. JBiolChem 1945; 161:495-502.[7] Happold FC, Spencer CP. The bacterial formation of acethyl-methylcarbinol and 2,3-butylene glycol. Biochim Biophys Acta(AMST)1952;8: 18-29.[8] Passonneau JV. Bestimmung von Lactat. In: Bergmeyer HU, ed.Methoden der enzymatischen Analyse. Chemie. Weinheim,1974;1515-7.[9] Soling HD, Kohlhaw G, Schnermann J, Holzer H, CreutzfeldtW. Zur Bedeutung des Acetoins fur die Pathogenese des Comahepaticum 1964; 89:457-63.

2,3-butanediol in experimental myocardial ischaemia in pigs

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