TracerKineticsof15-(Ortho-'2311311-Phenyl ...jnm.snmjournals.org/content/31/10/1608.full.pdf · over(7)acorrectionforthewater-soluble123!inblood andtissue(4,8)isneeded....

over ( 7) a correction for the water-soluble 123!in bloodand tissue (4,8) is needed.

To avoid a correction for labeled catabolites, 15-(para-'231-phenyl)-pentadecanoic acid (pPPA) was developed (9,10). pPPA behaves metabolically quite similarly to PA (11,12) although at a slower speed. pPPAis mainly catabolized to ‘23I-benzoicacid (13), which israpidly removed from the blood as iodo-hippuric acidafter intrahepatic conjugation with glycine (12).

When PPA is radioiodinated by electrophilic substitution, two-thirds of the product is in the form of thepara-isomer and one-third in the form of the orthoisomer. Initial animal experiments with the ortho-isomer 15-(ortho-I-phenyl)-pentadecanoicacid (oPPA) revealed a different metabolic behavior compared topPPA in rat myocardium: oPPA cardiac uptake wasslightly lower and its elimination rapid (11).

In humans, the kinetics of oPPA was surprisinglydifferent: oPPA is readily taken up and retained withstrikingly prolonged half-times (t½= 200 mm) (14).Serum samples taken from examined patients showeda significantly reduced level of iodinated water-solublecatabolites (15) in comparison to pPPA (12) during thefirst20 mm after injection.

The long elimination half-time of oPPA suggested amyocardial trapping of the label. The low level ofiodinated water-soluble catabolites, compared withpPPA, indicated an impairment in the fl-oxidation ofoPPA. To find the trappingsite ofoPPA, the metabolicpathway of physiologic fatty acids had to be considered(16—18).We investigated the metabolic pathway ofoPPA by two animal studies, one in vitro enzyme test,and two human studies.

MATERIALSAND METHODSRadiopharmaceuticals

Phenyl-(2-'4C)-pentadecanoic acid (407 MBq) with a specific activity of 2 GBq/mmol was provided by Amersham

The human myocardiumretains oPPA as opposed topPPA. Therefore turnoverof oPPA was compared withthat of pPPAin rat heartsand in man,the latter by usingsubstratesdouble-labeledwith 123/1311and 14C.Moreover,substratebindingto coenzyme-Awas tested in vitro. Inrats,oPPA remainedmainlyinthe poolof free fatty acids,as opposedto pPPA,which was metabolizedby mitochondnal @9-oxidation.Binding to coenzyme-A at maximumwas 62% for oPPA,81% for pPPAand 90% for palmiticacid.

In man, after i.v. and intracoronaryinjectionof doublelabeledoPPA,the two radionuclidesreappearedtogetherin venousbloodand in coronarysinusrespectively,in anunchangedratiobut at a significantlylowerrate than withpPPA. It can be concludedthat oPPA is boundto coenzyme-Aandis retainedin the cytosoliclipidpool,whilepPPAis metabolizedby [email protected] dualtracerapplicationof oPPA and pPPA has the potentialofbeing a specific probe for the function of the carnitineshuttle.

J NucIMed 1990;31:1608—1616

he first scintigraphic imaging with a radioiodinatedfatty acid, iodine- 131(1311)oleate, was described in 1965(1). In 1973, labeling ofheptadecanoic acid with 1231inthe omega position was initiated in our laboratory.Iodine-123-heptadecanoic acid (‘231-IHA)showed themost physiologic behavior(2,3) compared to other fattyacid analogs and has now been used in the diagnosis ofcoronary artery disease (4) and in alcoholic and dilatative cardiomyopathies (5,6). Still, for obtaining uptake and elimination curves reflecting fatty acid turn

ReceivedMay1, 1989; revisionaccepted Mar.27, 1990.For reprints contact: Prof. Dr. Ludwig E. Feinendegen, Institute of

Medicine,NuclearResearch Center, P.O. Box 1913, D-5170JUIich,WestGermany.

1608 TheJournalof NuclearMedicine•Vol.31 •No. 10 •October1990

Tracer Kinetics of 15-(Ortho-'2311311-Phenyl)-Pentadecanoic Acid (oPPA) and 15-

123/ 131I-Phenyl)-Pentadecanoic

Acid (pPPA) in Animals and ManKlaus P. Kaiser, Bernhard Geuting, Kurt Grol3mann, Ernst Vester, Benno Lösse, Mohamed A. Antar,Hans-J. Machulla, and Ludwig E. Feinendegen

Institute ofMedicine, Nuclear Research Center, Jülich,FRG; Department ofCardiology, University of DüsseldorfDüsseldorfFRG; Univeristy of@onnecticut and VA Medical Center, Newington, Connecticut; and Institute of MedicalRadiation Physics, University ofEssen, FRG

Buchler, Buckinghamshire, England. Our laboratory performed the iodination of the phenyl ring with stable iodineand the separation of para- and ortho-isomers by means ofHPLC.

A second paired aliquot of oPPA and pPPA was labeledwith 123!for i.v. injection in humans and 131!for the otherstudies.Thelabelingwasperformedby electrophilicsubstitution as described in detail elsewhere (9). The specific activitywas‘@830GBq/mmol.

Forthe dual-tracerstudies,the above-mentioned l5-(orthoor para-'27I-phenyl@2-'4C)-pentadecanoic acid was mixedwith the corresponding ‘23―311-labeledisomer resulting in aspecific activity of2.0 GBciJmmol for both ‘4Cand 123/1311.

Animal StudiesIn VitroRat Heart Perfusion.In total, eight heartsfrom

male fasted Wistar rats were studied. Thirty minutes afterinduction of pentobarbital anesthesia (120 mg/kg, i.p.) andanticoagulation pretreatment with heparin (250 U, i.v.), theheartswereprepare4fora retrogradeperfusion,circumventinglonger periods of ischemia by an operative procedure describedin detail elsewhere(19).

The heartswerestimulatedat a constant rate of280 beats!mm and perfused according to the standard Langendoriftechniquewithout recirculationofthe perfusionmedium (20).Aftera 20-mmequilibrationperiod, tracer infusionwasinitiated. The tracer was complexed to albumin in solution andpalmiticacid added in a physiologicconcentration.About 37kBciJmin of oPPA or pPPA was infused for 30 mm. Arterialblood pressurewas registeredand indicated uncompromisedmyocardial performance in the experiments. Influx samplesand effluates were collected at 1-mm time intervals. Finally,the hearts were frozen between precooled aluminum blocks.

Aliquots of the influx and of the collected effluate werecounted and total infused and effluate activities were calculated.

The hearts wereextractedwith chloroform-methanol(2:1)(21) and the homogenates were filtered into pointed glasstubes. The filters were washed with 5 ml of the same solventand an equal volume ofdouble-distilled waterwas added. Theextractswereagitatedfor 1.5mm and centrifugedanother 20mm at 3000 rpm, providing an upper aqueous and a lowerorganic phase. The filters containing the solid residue of thehearts and aliquots ofthe obtained extracts ofthe hearts werecounted and total heartactivity was calculated.

The extractsof the hearts wereevaporatedto drynessandredissolved in a small volume of solvent. Samples of theeffluate of the different time intervals were extracted in thesameway.

Samplesofthese concentratesofthe effluatesand the heartextracts and unlabeled reference substances (iodobenzoic acid,iodophenylpropionic acid, dipalmitin, tripalmitin, and cholesteryl oleate) were treated by thin-layer chromatography (TLC)using the following solvent systems: 1)ethyl acetate; 2) diethylether/benzene/ethanol/acetic acid (40:50:2:0,2); and 3) diethyl ether/hexane (6:94). The second and third solvent systems were used for the separation of different lipid classes todemonstrate a possible incorporation of the label into theselipids (22). The bands of the reference substances were detected and marked under UV light.

The activity on the plates was counted with a BertholdMulti-Tracemastersystem, activity peaks were marked, and

the relative distribution of activity on the plates between thedifferent lipid fractions and the short-chain metabolites werecalculated.

In Vivo Rat Hearts. 2-[5-(4-chlorophenyl)pentyl]-oxirane2-carboxylate(POCA,obtained from Byk-OuldenGermany,Konstanz,FRG) inhibitscarnitine-acyltransferase(23,24).

Male Wistar rats weighing 200-300 g were studied with thiscompound after 24 hr of fasting. Twenty minutes after induction of pentobarbital anesthesia, POCA (30 mg/kg bodyweight) was injected intraperitoneally. Controls received saline. Two hours later, 2.6—2.9MBq of oPPA or pPPA wereinjected into the jugular vein. For each time interval (1, 2, 3,4, 5, 7, 10, and 20 mm) five animals were studied; their heartswereremovedand immediatelyfrozen in liquid nitrogen. Atthe same time, blood samples were taken from the inferiorvena cava. The frozen hearts were weighed, homogenized, andextractedwithchloroform-methanol(21),as describedabove.This provided fractions of total lipids, aqueous phase, andsolid residue. Radioactivity in each fraction was counted andcalculated as relative units or as a percentage of administereddose. The percent myocardial uptake was related to a standardized heart weight of 1 g.

Total lipidswerefurther separatedby TLC in parallelwithstandard lipid substances,such as phospholipids,cholesterol,cholesterol esters, tripalmitate, PA, oPPA, and pPPA (13).

In VitroTest for Bindingof FattyAcidsto CoAoPPA,pPPA,PA,andIHAweredissolvedin triton-X-l00

(300mg/i) in triplicates.The extentofactivation ofthese fattyacids by acyl-CoA synthetase was analysed by a colorimetncenzymatic assay (Boehringer Mannheim, GmbH, Mannheim,FRG) for determinationoffree fattyacids(25). At 30, 60, 90,and 120 mm of incubation of each solution, the generation ofenoyl-CoA from acyl-CoA in the presence ofperoxidase, withconcomitant appearance of a red dye, was measured in thevisible range at 546 nm.

HumanStudieswith Double-LabeledoPPAandpPPA

Intravenous Injection (Experimental Protocol). Eight patients (4 oPPA, 4 pPPA) with low probability of coronaryheart disease were examined with informed consent. Aftersimultaneous i.v. administration of 185 kBq pPPA or oPPAlabeled with carbon-14 (‘4C)and 100 MBq ofpPPA or oPPAlabeled with 123!,respectively, a dynamic planar study of 60mm was acquired with the gamma camera in the anteriorposition. At 0, 3, 5, 10, 20, 30, 40, 50, 60, and 70 mm, venousblood samples were taken.The blood samples were analysedfor ‘231-labeledwater-soluble compounds and 1231-labeledcompounds soluble in organic solvents. In addition, the cxhaledair wasmeasured,and both respiratoryvolume/minuteand specific activity ofexhaled ‘4C02were determined.

Analysis ofBlood Samples, ‘231-LabeledCompounds (AnalyticalProcedure).Blood samples(5 ml each) were takenfrom a peripheralvein with hepannized syringes. Two milliliters of serum were taken and extracted with chloroformmethanol, as describedabove (21).

The organicand water-solublephaseswere separated andthe paper filters, containing the solid residue of the serum,were counted for 123!with a gamma scintillation counter. Theactivityof the phaseswasexpressedin percent ofthe injecteddose (%ID).

1609Myocardial Fatty Acid Metabolism •Kaiser et al

TABLEITotalActivity Distribution in the Effiuates and TotalHeartin IsolatedPerfusedRatHeartStudypPPA0PPASign.

test(ps)Benzoic

acidin effluates5.7 ±1.30.2 ±0.00.01Phenyl-Propionicacidin effluates1 .5 ±0.30.3 ±0.10.01Free

fatty acid in effluates82.2 ±1.697.7 ±0.30.001Heartactivity9.4 ±0.61 .3 ±0.20.0001

Analysis of Exhaled Air: ‘4C02.The exhaled air was ledthrough a two-way-breathing mask and a tube to a gas meterfor determining the respiratory volume/minute. At 0, 2, 5,10, 15, 20, 30, 40, 45, and 50 mm postinjection, a definedpart of the exhaled air was led through a vial filled with adefined amount of methylbenzethonium hydroxide (Hyamine) (26); an indicator changed color when C02-saturation(1 mmol) was reached. After adding the scintillator solution,Insta-Gel (Canberra-Packard, Frankfurt/Main, FRG), the‘4C02activitywas counted.The specific‘4Cactivityof theexhaled air was calculated as Bq/mmol CO2.

Intracoronary Injection (Experimental Protocol). For investigating the human myocardium exclusive of other organs,another study was designed. Seven patients were examined (3with oPPA and 4 with pPPA) after obtaining informed consentconcerning the procedure. All patients underwent coronaryangiography because of valvular heart disease. They had noapparent coronary heart disease. In addition to the left heartcatheterization, another catheter was positioned in the venoussinus from the right atrium. oPPA or pPPA labeled with ‘4Cand ‘@‘I(37 kBq for both) as described above were injecteddirectly into the left coronary artery. Blood samples, of 5 mleach, were taken simultaneously at 0, 1, 2, 3.5, 5, 7, 10, and15 mm postinjection with heparinized syringes from the coronary sinus and the aorta. The latter samples were obtainedfor the correction for recirculating labeled catabolites.

The blood samples were analyzed for uncatabolized ‘4C-labeled fatty acids, ‘4C02,uncatabolized ‘31I-labeledfattyacids, and ‘@ ‘I-labeledcatabolites, i.e., ‘@ ‘I-benzoicacid and‘311-phenylpropionicacid. In the case of one patient, whoreceived pPPA, only the ‘4C-datawere available.

Analytical Procedure: ‘4C02.The ‘4C02was collected directly by a diffusion method (27). Two milliliters of eachblood sample were placed in the outer well of a doublechambered glass-flask, especially designed for that purpose.The samples were slightly acidified with lactic acid and thetightly closed flasks were agitated on a shaker table for 24 hr.The center well of the flask was filled with 3 ml of 1 MHyamine that trapped the released ‘4CO2.In our laboratory,the recovery of 4C02 from NaH'4C03 added to whole bloodin vitro by this method is more than 98%. The Hyaminesolution with the trapped “CO2was mixed with the scintillatorInsta-Gel and counted.

Carbon-14-Labeled Fatty Acid. The ‘4C-labeledfatty acidwas determined by counting the ‘4C-activityofserum samples,which had been freed from ‘4C02by addition of lactic acidand agitation on a shaker table for 24 hr. The count rates ofthe serum were corrected for the@@ ‘I-activityof these samples.

Jodine-131-Labeled Compounds. Two aliquots of 200 @ilserum of each sample were counted in a gamma-scintillationcounter for determination of total ‘@‘Iactivity. The distribution ofthe label between catabolites and non-catabolised fatty

acids was determined by TLC. For this purpose, the remainingserum of each samplewas extractedwith chloroform-methanol (21). The extracts were filtered, evaporated to dryness,and redissolved with a small volume of solvent, as describedabove.

Probes of each sample were separated by TLC, includingchromatography of unlabeled reference substances, as describedabove.

The activity on the plates was counted with a BertholdMulti-Tracemaster system; the regions of interest were determined by the corresponding reference substances, and therelative distribution ofactivity on the plates between differentlipid fractions and ‘31I-labeledcatabolites was calculated.

Activity for ‘4C02,‘4C-fattyacid, ‘31I-catabolite,and 131J@fatty acid was expressed in cpm/ml whole blood. When serumwas analyzed, the results were corrected with the hematocrit.

Welch's t-test was applied throughout for determination ofstatistical significance of difference of means.

RESULTS

AnimalStudiesIn Vitro Rat Heart Perfusion. The total amount of

‘31I-benzoicacid, ‘311-phenylpropionicacid, and ‘@‘Iphenylated fatty acid as found in the effluates during a30-mm continuous infusion of pPPA and oPPA, expressed as percent of the infused activity, and theamount of the total activity retained in the hearts afterthe tracer infusion was analyzed (see Table 1).

Of the infused activity, 9.4% was taken up by theheart during pPPA infusion, 1.3% in the case of oPPA.In the effluate, 5.7% of infused pPPA and 0.2% ofoPPA were found as iodo-benzoic acid, and 1.5% ofpPPA and 0.3% of oPPA activity as iodo-phenylpropionic acid. The free fatty acids in the effluate amountedto 82.9% of infused pPPA and 97.7% of oPPA (Table1).

Table 2 summarizes the activity distribution withinthe myocardium; the major difference between pPPAand oPPA is in the fraction of the free fatty-acids (only1.0% for pPPA but 4 1.5% for oPPA). pPPA was mainlyfound esterified to phospholipids (34.3%) and triglycerides (49.5%). oPPA was esterified to a lower degree:24.7% asphospholipidsand 24.6% astriglycerides.Theactivity of short-chain catabolites in the myocardiumwas relatively low for both pPPA and oPPA, whichindicates a rapid elimination of these catabolites intothe effluates.

Figure 1 shows iodo-benzoic acid in the effluates in

1610 The Journal of Nuclear Medicine •Vol. 31 •No. 10 •October1990

TABLE2Activityin Different Lipid Fractions and Benzoic AcidinIsolated

PerfusedRat HeartStudy(%Heart Activity, mean ±s.d., n =4)pPPA

0PPA Sign.test(ps)Phospholipids

34.3±3.5 24.7±1.00.01Benzoicacid 3.5 ±2.2 2.3 ±0.2n.s.Phenyl-Propionic

acid 0.1 ±0.1 0.5 ±0.6n.s.Freefattyacid 1.0±0.2 41.5±1.00.0001Diglycerides

1.1 ±0.4 0.0±0.00.02Triglycerides49.5±2.5 24.6±0.70.0001Solid

residue 9.8 ±1.1 6.4 ±0.8 0.01

tracer was found mainly in the total lipids while activityin the water-soluble phase did not exceed a level of0. 16% during the time of observation. After POCApretreatment, the activity of the water-soluble phasewas lower than that in control hearts for pPPA through

out the study, and to a lesser degree for oPPA at timeslater than 4 mm.

Table 4A gives the data of the chromatographicanalysis of the total lipids; in the control group afterpPPA, there was a rapid activity decrease of the freefatty acid fraction to undetectable amounts after 1 mm.The main part of tracer activity was initially found inthe triglycerides and phospholipids. In the case of oPPA,the activity was mainly in the unesterified free fattyacids during the time of observation.

In animals pretreated with POCA (see Table 4B),pPPA was mainly esterified to triglycerides, and freefatty acids could now be detected up to 10 mm insignificantly larger amounts compared to controls. Inthe case of oPPA, POCA slightly diminished the lowtracer incorporation into complex lipids and increasedthe tracer in the free fatty acid pool.

In Vitro Test for Binding of Fatty Acids to CoAThe results of this enzyme test are listed in Table 5.

PA and IHA showed rather constant high levels ofactivation (‘@-90%for both) during the two hours ofobservation. The rates of activation of the phenyl fattyacids were much lower at the beginning of incubationand increased with time for pPPA from 48% to 81%and for oPPA from 32% to 62%.

HumanStudieswithDouble-LabeledoPPAandpPPAIntravenous Injection. The total radioiodine activity

and the distribution between lipids and water-solublefractions of blood serum after i.v. injection of double

percent of the total effluate activity during the time ofinfusion. In the case ofpPPA, up to 8.4% was eventuallyfound as iodo-benzoic acid. In contrast, during oPPAinfusion only little, if any, iodo-benzoic acid appeared.Similar results were obtained for iodo-phenylpropionicacid: a maximum activity of 2% and 0.44% of theeffluate activity for pPPA and oPPA respectively.Therefore, there was little catabolism of oPPA.

In Vivo Rat Hearts. The myocardial uptake of pPPA(Fig. 2) showed a peak of 3.6% of injected dose pergram of tissue at 3 mm with activity decreasing rapidlyto 0.9% per gram oftissue at 20 mm. oPPA had a lowerpeak of 2.8% per gram of tissue after 1 mm and alsorapidly decreased to 0.5% per gram oftissue at 20 mm.After POCA treatment, pPPA showed progressivelyincreasing uptake and retention within the myocardiumand reached a plateau (3.5%) at 10 to 20 mm. oPPAkinetics after POCA treatment, however, was onlyslightly different compared to the control group.

As shown in Table 3, after pPPA, normal rat heartsshowed a maximum of ‘@‘iactivity ofO.93% of injecteddose in the total lipid fraction and 0.66% in the watersoluble catabolites at 3 mm. In the case of oPPA, the

wC)

WI

4W

0<

@.LL

FIGURE 1Iodine-i31-benzoic acid in percent ofeffluate 1311activityafter continuousinfusion of pPPA or oPPA in isolatedperfusedrathearts(n= 4).TIME (mm)

1611Myocardial Fatty Acid Metabolism •Kaiseret aI

Time(mm)ControlpPPAAnimals oPPAPOCA pPPAAnimals oPPAContr.

vpPPAs.

POCAoPPApPPA

vControlss.

oPPAPOCA10.32

±0.10.03 ±0.010.12 ±0.040.05 ±0.020.01n.s.0.010.0220.47±0.20.05 ±0.020.10 ±0.020.09 ±0.030.020.050.01n.s.30.66±0.20.08 ±0.030.12 ±0.040.05 ±0.020.01n.s.0.010.0240.28±0.20.04 ±0.020.09 ±0.020.04 ±0.01n.s.n.s.n.s.0.0150.33±0.10.05 ±0.030.10 ±0.010.02 ±0.010.01n.s.0.0020.000170.32±0.10.16 ±0.10.06 ±0.020.02 ±0.000.010.050.050.02100.21±0.10.05 ±0.010.05 ±0.020.01 ±0.000.050.0010.050.02200.1

1 ±0.030.03 ±0.010.06 ±0.010.01 ±0.000.050.010.010.001

4

FIGURE2Totalheartactivityafteri.v.injectionof131Il@@@ pPPA or oPPA in rats withandwithoutpretreatmentwith POCA(phenylalkyl-oxyrane-carboxylicacid),which inhibits the carnitine fatty acidshuttle from the cytosol into mitochondna. TIME

injection, while for oPPA a maximum of46.5% for ‘@‘Icatabolites was only reached after 15 mm.

After pPPA injection, ‘4CO2reached 68.3% after 7mm and 84% after 15 mm, while only 22.3% of the‘4C-activitywere found as ‘4CO2in the case of oPPAafter 15 mm. For both pPPA and oPPA, “CO2appearedlater than the ‘31I-catabolites.

In all, significantlyless ‘31I-catabolitesand ‘4CO2wereproduced in parallel, after oPPA injection, comparedto pPPA.

DISCUSSION

oPPA is readily taken up and retained in the humanmyocardium, as seen in dynamic gamma-camera studies with ‘231-labeledoPPA (Fig. 7) (14,15).

oPPA- and pPPA-catabolites that were measured inthe peripheral blood after i.v. injection could be produced by the liver or other organs with a high lipidturnover, e.g., skeletal muscle. Still, measurement ofserum activity distribution following i.v. injection ofboth tracers revealed a significantly higher concentraton of pPPA-catabolites compared to oPPA.

labeled pPPA and oPPA, expressed as %ID are shownin Figure 3.

In the case of pPPA, an early rise of water-solubleiodine catabolites could be observed. With oPPA, asignificantly lower amount of iodine catabolites appeared.

Figure 4 (upper panel) shows the specific activity of14CO2exhaled in air after intravenous injection. Therate ofappearance of “CO2after oPPA was significantlyless than after pPPA from the tenth minute after injection. The fraction of water-soluble ‘231-catabolitesinserum after oPPA was significantly smaller than in thecase of pPPA, as shown in Figure 4 (lower panel).

Intracoronary Injection. Figure 5 shows the appearance oftotal ‘@‘I-and ‘4C-activityin the coronary sinus,expressed as %ID/ml. There is a significant differencebetween the curves for pPPA and oPPA indicative of alower initial extraction of oPPA by the myocardium.

The distribution of the @@‘I-and the ‘4C-labeledcatabolites in the coronary sinus blood is shown in Figure6. In the case of pPPA, the ‘31I-catabolitesreached,..-90% of the total activity of the samples 5 mm after

TABLE 3Water-solubleActivityafter pPPAand oPPA in POCA Pretreatedand ControlRats: In Vivo Heart Study

[% Dig tissue, mean ±s.d., (n = 5)] Significancetest (ps)

1612 TheJournalof NuclearMedicine•Vol.31 •No. 10 •October1990

Time(mm)Free

FattyAcidsTriglyceridesPhospholipidspPPAoPPApPPAoPPApPPAoPPA10.18

±0.0011 .57 ±0.120.35 ±0.020.0 ±0.00.30 ±0.010.0 ±0.020.0±0.01 .11 ±0.090.55 ±0.030.0 ±0.00.21 ±0.010.0 ±0.030.0±0.00.89 ±0.060.23 ±0.010.02 ±0.0050.30 ±0.010.07 ±0.00540.0±0.00.77 ±0.050.41 ±0.020.05 ±0.010.08 ±0.0030.03 ±0.00250.0±0.00.63 ±0.030.40 ±0.020.14 ±0.010.07 ±0.0030.03 ±0.00370.0±0.00.49 ±0.020.39 ±0.020.13 ±0.020.08 ±0.0040.17 ±0.01100.0±0.00.23 ±0.010.46 ±0.030.05 ±0.010.08 ±0.0030.02 ±0.002200.0±0.00.10 ±0.0010.38 ±0.010.13 ±0.010.07 ±0.0040.03 ±0.002

Time(mm)Free

FattyAcidsTriglyceridesPhospholipidspPPAoPPApPPAoPPApPPAoPPA10.72

±0.051 .44 ±0.010.29 ±0.010.0 ±0.00.24 ±0.010.0 ±0.020.36±0.021 .41 ±0.010.64 ±0.040.0 ±0.00.1 5 ±0.010.0 ±0.030.22±0.011 .25 ±0.010.87 ±0.060.0 ±0.00.1 1 ±0.010.0 ±0.040.16±0.011.17 ±0.010.89 ±0.060.0 ±0.00.10 ±0.010.0 ±0.050.14±0.010.91 ±0.0081.08 ±0.090.02 ±0.0010.11 ±0.010.0 ±0.070.1

1 ±0.010.76 ±0.0071 .25 ±0.110.02 ±0.0010.15 ±0.010.0 ±0.0100.06±0.0010.68 ±0.0051.14 ±0.090.07 ±0.0020.07 ±0.0030.0 ±0.0200.0±0.00.33 ±0.0021.62 ±0.120.10 ±0.010.0 ±0.00.0 ±0.0

TABLE 4AActivity in Different Lipid Fractions after pPPA and oPPA in Control Rats: In Vivo Heart Study

(% ID/g Tissue, mean ±s.d., n = 5)

The special experimental design of intracoronarytracer application in humans allowed a correction forrecirculating labeled catabolites.

After oPPA intracoronary injection, a small production rate of ‘311-labeledcatabolites, i.e., iodo-benzoicacid and iodo-phenylpropionic acid, and of ‘4CO2wasfound. In contrast, a significantly higher productionrate ofcatabolites was seen after pPPA injection. If ‘@‘ilabeled fragments of oPPA had been retained in mitochondria, a higher rate of ‘4CO2-productioncomparedwith that of ‘31I-cataboliteswould have been expected.However, with both fatty acids a similar ratio of ‘@‘Icatabolites and ‘4C02was found. Therefore, the slowelimination of oPPA from the human myocardial cellis caused by a retention of the entire fatty acid in thecytosolic lipid pool. Moreover, the delayed appearanceof ‘4CO2in the coronary sinus following intracoronaryarteryinjection of pPPA and oPPA in comparison with‘311-catabolitespoints to some intracellular reutilizationof ‘4CO2or rapid transport of ‘311-labeledcatabolitesfrom the mitochondria into the circulatingblood.

There is a species-related difference in the myocardialkinetics of oPPA; rat heart does not show a prolongedretention, in contrast to the human heart. This appearsto be due to the inhibition of esterification to complexlipids and a high rate of backdiffusion of unchangedoPPA in rats. The animal studies using POCA help to

better understand the phenomena. Inhibition of a partof the enzyme system carrying fatty acids into themitochondria, i.e., of the carnitine-acyl-transferase I(CPT I), with POCA, a specific CPT I-inhibitor, alteredthe metabolic behavior of oPPA only slightly as cornpared to pPPA. In fact, there was a further slight diminution of oPPA esterification into complex lipids. Onthe other hand, in the case of pPPA, CPT I-inhibitionresulted in a significant change ofkinetics with a dirninished fl-oxidation and an augmented storage in thecytosolic lipid pool, mainly in triglycerides. The relatively rapid turnover of oPPA in the rat heart afterPOCA injection is likely due to backdiffusion from themyocardiurn into the vascular system.

In the continuously perfused rat heart, pPPA wasrapidly catabolized, whereas oPPA was less well retamed, again probably because of enhanced backdiffusion and lower rate of esterification to phospholipidsand triglycerides.

The kinetic difference between pPPA and oPPAmight be due to a change ofthe substrate conformationby the ortho iodine substitution on the phenyl ring (28).This has in principle also been observed for othersubstituted benzoic acid derivatives (29).

In accordancewith these observations,the catabolismof oPPA by 13-oxidation was shown to be highly reduced, probably because oPPA might be blocked from

TABLE 4BActivity in Different Upid Fractions after pPPA and 0PPA in POCA Pretreated Rats: In Vivo Heart Study

(% ID/g Tissue, mean ±s.d., n = 5)

1613Myocardial Fatty Acid Metabolism •Kaiser et al

TABLE5InVitroTest for Bindingof DifferentFattyAcidstoCoA(Percent

of Binding to CoA, mean ±s.d.)Time

ofincubation(mm)

PA IHAt pPPA*oPPA@30

90.5 ±3.5 83.2 ±2.2 47.5 ±2.2 31 .7 ±2.56089.2±2.5 87.0±2.0 58.4±2.4 43.2±3.19089.2±2.4 87.2±2.5 75.3±2.3 57.2±2.812090.5 ±3.6 90.8 ±2.7 80.8 ±2.5 61.7 ±2.8.

PA = palmiticacid.t

IHA = 17-1-Heptadecanoic acid.* pPPA = 1 5-(p&a-l-Phenyl)-pentadecanolc acid.

. oPPA = 15-(ortho-l-Phenyl)-pentadecanoic acid.

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binding with the carnitine-acyl-transferase, the carrierenzyme of fatty acids into the mitochondria.

A second important finding of our in vitro rat heartperfusion study is that only small amounts of the totalinfused oPPA and pPPA activity were found in theheart as short chain catabolites, indicating that in the

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FiGURE 4Specificactivityof 14CO2in exhaledair (upperpanel)andwater-soluble123I-catabolitesinvenousbloodsamples(serum)(lower panel) after i.v. injection of dOUbe-IabaledPPPA oroPPAinman(n= 4).

rat the catabolites of these phenylated fatty acids arenot retained in the myocardium. As this is probablyalso true in humans, the externally detectable activityafter i.v. administration of oPPA and pPPA should notbe influenced by retained labeled catabolites.

Another interesting detail of oPPA metabolism isgiven by the in vitro comparison of the activation ofdifferent fatty acids by acyl-CoA synthetase. pPPA andoPPA were not as quickly activated as IHA and PA.This data indicates that the phenylated fatty acids areto beusedin man with caution iftheir metabolicuptakeinto the myocardium is to be studied.

30 40

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FIGURE3Iodine123activity distributionin venousbloodsamples(serum)after i.v. injection of dOuble-labeledPPPA (upper panel) oroPPA(lowerpanel)in man(n= 4).Thesampleswereseparated into organic(not catabolizedpPPAor oPPA)and watersolublephase(fattyacidcatabolites).

1614 The Journal of Nudear Medicine •Vol. 31 •No. 10 •October1990

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FIGURE5Total 1311and 14Cactivity in blood samples taken from thecoronarysinus after intracoronaryinjectionof double-labeledpPPA (upper panel)and oPPA (lower panel) in man (n = 3,14C-valuesof pPPAn = 4).

The present data show that once oPPA and pPPAare incorporated into the metabolic chain, their retentions depend on specific subsequent metabolic reactions. Since the kinetics of oPPA deviates from that ofpPPA, PA or IHA, clearly at the carnithine shuttle,resulting in trapping in the lipid pool in man, theexternallymeasureddifferencebetween respectiveturnover curves may be exploited for probing the carnitineshuttle.

In conclusion, oPPA uptake in human myocardiumseems to depend on local perfusion, regional myocardialfree fatty acid extraction, and on a retention in thecytosol after coenzyme-A-activation. Irrespective of theuncertainty ofthe mode ofthis retention in the cytosol,oPPA deserves special interest for in vivo myocardialstudies, because oPPA uptake not only depends onmyocardial perfusion, but also serves as an indicator ofthe early steps of fatty acid metabolism in the myocardium.

The long retention of oPPA in the human myocardium is advantageous for time-consuming SPECT studies, where the image quality is aided by the idealgamma-energy of 1231(159 keV).

Most important, the use of oPPA in conjunction

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FIGURE6Iodine-i31-labaled catabolites and 14C02in blood sampiestaken from the coronarysinus after intracoronaryinjectionofdouble-labeledpPPA(upperpanel)and oPPA(lower panel)inman(n= 3, 14C-valuesof pPPAn = 4).

with a suitably labeled fatty acid analog that is nottrapped in the cytosol but undergoes fl-oxidation, likepPPA or IHA, i.e., an in vivo dual-tracer analysis, hasthe potential of evaluating fatty acid transport into

FIGURE7Time-activitycurves from humanmyocardiumafter i.v. injection of 140 MBq 123ll@(@JpPPA or oPPA from dynamicstudiesover60mm,2 frames/mm,inanteriorpositionwithoutzoom. (Y-axis:counts/30 see).

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1615Myocardial Fatty Acid Metabolism •Kaiser et al

10. Daus Hi. Reske K. Vyska K, Feinendegen LE. Pharmakoki

netics of 5-(p-'23I-phenyl)-pentadecanoic acid in heart. In:Schmidt HAE, Wolf F, Mahlstedt J, eds. Nuk/earmedizinNziklcarmedi:in im interdis:iplinãrenBe:ug. Stuttgart-NewYork: F.K. Schattauer-Verlag:1981:108—il1.

I 1. Beckurts TE, Shreeve WW, Schieren R, Feinendegen LE.

Kinetics ofdifferent 231and ‘4C-labeledfatty acids in normaland diabetic rat myocardium in vivo.NuclMed Comm 1985;6:415—424.

12. Dudczak R. Myokardszintigraphie mit jod-l23-markiertenfeusauren. WienKIm Wschr 1983;65(suppl 143):1—35.

13. Schmitz B, Reske SN, Machulla Hi, Egge H, Winkler C.Cardiac metabolism of omega-(p-iodo-phenyl)-petadecanoicacid: a gas-liquid chromatographic-mass spectrometric analysis.J LipidRes 1984;25:1102—1108.

14. Feinendegen LE. Nuclear medicine, science and the future.In: Schmidt HAE, Eli Pi, Brinon KE, eds. Nuk/earmedizin—.Nuklearmedizin inforschung undpraxis Stuttgart-New York:Schattauer; 1986:XLI-IL.

I 5. Antar MA, SpohrG, Herzog HH, et al. 15-(ortho-'23I-phenyl)-

pentadecanoic acid, a new myocardial imaging agent forclinical use. NucI Med Comm 1986; 7:683—696.

16. Stremmel W, Strohmeyer G, Borchard F, Kochwa 5, BerkPD. Isolation and partial characterisation of a fatty acidbinding protein in rat liver plasma membranes. Proc NatIAcadSci USA 1985;82:4—8.

17. Gloster J, Harris P. Fatty acid binding to cytoplasmic proteins

of myocardium and red and white sceletal muscle in the rat.A possible new role for myoglobin. Biochem Biophys ResCommun 1977; 74:506—513.

18. Taegtmeyer H. Myocardial metabolism. In: Phelps M, Mazziotta i, Schelbert H, eds. Positron emission tomography andautoradiography:principles andapplicationsfor the brain andheart. New York: Raven Press; 1986:149—195.

19. Doring Hi, Dehnert H. Das isolierte perfundierte Hera nachLangendorif. BVM-BiomeBtechnik Heft V. BiomeBtechnikVerlagMarch GmbH, 1985.

20. LangendorifO. Untersuchungen am BberlebendenSaugetierherzen.PflUgersArchivGes Physiol1895;61:291—332.

21. Foich J, Lees M, Sloane-Stanley H. A simple method for the

isolation and purification of total lipids from animal tissues.J Biol Chem 1957; 226:497—509.

22. Freeman CP, West D. Complete separation oflipid classes on

a singlethin-layer plate. J Lipid Res 1966;7:324—327.23. Wolf HPO, Eistetter K, LudwigG. Phenylalkyloxyranecar

boxylic acids, a new class of hypoglycemic substances: hypoglycemic and hypoketonaemic effects of sodium 2-5-(4-chlorophenyl)pentyl-oxyrane-2-carboxylate (B 807-27) in fastedanimals. Diabetologia 1982;22:456—463.

24. Seitelberger R, Kraupp 0, Beck A, Bacher 5, Raberger 0.Effects ofthe acylcarnitine-transferase blocking agent sodium2(5-(4-chlorophenyl)-pentyl)-oxirane-2-carboxylate(POCA)on cardiodynamics and myocardial metabolism in dogs. JCardiovasc Pharmacol 1984; 6:902—908.

25. Shimizu 5, Tani Y, Yamada H, Tabata M, Murachi T.Enzymatic determination of serum-free fatty acids: a colonmetric method. Anal Biochem 1980; 107:193—198.

26. Shreeve WW. Labeled carbon breath analysis. In: Harbert i,Roha AFO, eds. Textbook of nuclear medicine, volume I:basic science, Second edition. Philadelphia: Lippincott;1984:351—362.

27. HagenfeldtL. A simplifiedprocedure for the measurement of‘4C02in blood.ClinChemActa1967;18:320-321.

28. Loffler G, Petrides PE, Weiss L, Harper HA. Physiologische

chemie Berlin, HeidelbergNew York: Springer, 1979.29. Quick Al. The conjugation ofsubstituted benzoic acid. JBiol

Chem 1932;96:83—101.

mitochondria, i.e., the function ofthe carnitine shuttle,which may be of considerable clinical interest.

ACKNOWLEDGMENTSThe authors thank Prof. H. Reinauer (Department of Bio

chemistry, University of DUsseldorf@Düsseldorf,FRG) andDr. W.W. Shreeve(Nuclear Medicine Service,VA MedicalCenter, Northport, NY) for their valuable advice and discussion; Mrs. M. Adrian (Department of Biochemistry, University of DUsseldorf@Düsseldorf,FRG), Mr. W. Friedrich (Institute of Medicine, Nuclear Research Center, D-5 170, JUlich,FRG),W.E.Schultz(InstituteofMedicine, NuclearResearchCenter, D-5l70 Jülich,FRG), and D. Strung (Institute ofMedicine, Nuclear Research Center, D-5 170JUlich, FRG) fortheir dedicated technical assistance; and Mrs. D. Beaujean(Institute of Medicine, Nuclear Research Center, D-5 170,JUlich, FRG) for efficient secretarial help. They also thank theAmersham International Company, Amersham Place, LittleChalfront, Buckinghamshire HP7 9NA, England for theirgenerous supply of phenyl-(2-'4C)-pentadecanoic acid.

Data in this paper are obtained in part from the Thesisprepared by Mr. Geuting and Mr. Grossman for the degree ofmedical doctor.

REFERENCES1. Evans JR. Gunton RW, Baker RG, Beanlands DS, Spears JC.

Use of radioiodinated fatty acids for photoscans ofthe heart.CircRes 1965; 16:1—10.

2. Machulla HJ, StocklinG, KupfernagelC, et al. Comparativestudies ofdifferent labeled fatty acids for heart muscle metabolism. In: Schmidt HAE, Woldring M, eds. Nuklearmedizin.Stand und Zukunft. Stuttgart-New York: Schattauer;1978:212—215.

3. Freundlieb C, Hock A, Vyska K, Machulla HJ, Stocklin G,Feinendegen LE. Nuklearmedizinische Analyse des Fettsaureumsatzes in Myokard. In: OefT K, Schmidt HAE, eds.Nuk/earmedizin. Nuklearmedizin und Biokybernetik. Stuttgart-New York: Schattauer, Bd. I. 1978:415—419.

4. FreundliebC, Hock A, VyskaK, FeinendegenLE, MachullaHJ, StöcklinG. Myocardial imaging and metabolic studieswith l7@23I-heptadecanoic acid. J NucI Med 1980; 21:1043—1050.

5. Notohamiprodjo 0, Spohr G, Hock A, Freundlieb C,Schweitzer H, Feinendegen LE. Influence of acute ethanolingestion on myocaridal fatty acid metabolism. In: HöferR,Bergmann H, eds. Radioakiive isotope in klinik und forschung.Vienna: Egermann; 1984:755—764.

6. Hock A, Rafflenbeul D, Freundlieb C, et al. Myocardialuptake and elimination half-times of l7-'23I-heptadecanoicacid in patientswith earlystageofcongestivecardiomyopathy.In: Raynaud C, ed. Nuclear medicine and biology. Proceedings of the third world congress of nuclear medicine andbiology.Paris, New York: Pergamon Press; 1982:2526—2529.

7. Visser FC, van Eenige MJ, Duwel CMB, Roos JP. Radioio

dinated fatty acids; can we measure myocardial metabolism?EurfNuclMed 1986; 12:520—523.

8. Feinendegen LE, Vyska K, Freundlieb C, Machulla H-i,Kloster 0, Stöcklin0. Non-invasive analysis of metabolicreactions in body tissues, the case of myocardial fatty acids.EurfNuclMed 1981; 6:191—200.

9. Machulla H-i, Marsmann M, Dutschka K. Biochemicalconcept and synthesisofa radioiodinated phenylfattyacid for invivo metabolic studies of the myocardium. Eur J NucIMed1980;5:171—173.

1616 TheJournalof NuclearMedicine•Vol.31 •No. 10 •October1990