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Clinical Intensive Care 1995; 6: 283-285Review paper

Use of tracer techniques in intensivecare research. Part I

H ENSINGER, J VOGT, K TRÄGER, M GEORGIEFF, P RADERMACHER

University Clinic of Anaesthesiology, University Hospital, Ulm, GermanyH Ensinger MD, J Vogt PhD, K Träger MD, M Georgieff MD, P Radermacher MD

Correspondence to: Dr H Ensinger, Universitätsklinik für Anästhesiologie,Steinhövelstraße 9, D-89075 Ulm (Donau), Germany.

In the first part of this review we will describe the methodsand principles of tracer technology in research in humans.In the second part of the review we will give some exampleson the use of stable isotope tracer technology in the field ofintensive care research.

The use of stable isotopes as metabolic tracers dates back60 years.1-5 With the advent of an expanded knowledge intoresearch and production of non-stable, radioactive isotopesand the ease of measuring tracer amounts of radio-labelledcompounds, stable isotope tracers were outdated in the 1950sand 1960s. In the 1970s the resurgence of the use of stable,non-radioactive labelled tracers began as the technology formeasuring stable isotopes had improved and awareness of thehealth hazards in the use of non-stable radioactive tracers hadincreased.

Tracers are used in research in medicine and biology mainlyfor elucidation of the kinetic of metabolites, hormones, neuro-transmitters and even atoms in vivo. An ideal tracer shouldhave no different properties compared to the tracee, with theexception that the tracer can be discriminated from the traceein the analytical process. During the production of a tracer,one or more atoms of a molecule are substituted by an isotope.The abundance of this isotope in the manufactured tracer ismany times higher than the natural abundance. With non-stable isotope tracers the specific radioactivity of the compoundis detected. With stable isotope tracers the enrichment ismeasured. A prerequisite for tracer studies is that the labelledcompound is handled in the same way as the naturally occurringcompound by the living organism or the biological system, ieno difference should be caused by introducing the stableisotope in the compound under investigation. However, theremay be a detectable isotope effect. The isotope effect is relatedto the difference in mass of the isotopes, which is inverselyrelated to the mass of the unlabelled atom. Thus, a deuteriumor tritium label, which doubles or triples the mass of hydrogen,may have a larger isotope effect than carbon or nitrogenwhere the mass increase brought about by the stable orradioactive isotope is smaller. However, even with the use oftritium the isotope effect is usually rather small. Trendelenburget al6 showed that for 3H-(-)-7, 3H-(-)-7,8 and 3H-(-)-2,5,6,-noradrenaline, neuronal uptake and metabolism are impaired

Figure 1 The single-compartment model for steady-stateconditions.

with an increase in the number of tritium labels introduced inthe molecule, with the exception that the tritium label inposition 8 of noradrenaline grossly inhibits deamination bymonoamine oxidase. For most of the other pathways (theneuronal uptake and enzymatic degradation process), the rateswere depressed by less than 50%. For stable tracers there isalso an isotope effect. The deuterium carbon-bond has differentproperties compared to a proton carbon-bond. Thus, if thereaction which leads to the tracer clearance involves a cleavageof this hydrogen-carbon bond, an isotope effect can beexpected. This has been shown with the deuterium-ring labelledphenylalanine. Its hydroxylation is about half that of theunlabelled compound.7 The isotope effect may be ofimportance when the absolute metabolic rates are measured.However, they are of minor importance when changesinduced by physiological, biochemical or pharmacologicalmanipulation are investigated.

Another prerequisite is that the amount of tracer infusedshould not contribute to the total pool concentration and doesnot influence the endogenous release of the tracee. Radioactivetracers are usually considered as massless. However, infusionof stable isotope tracers usually result in an enrichment of0.5-2 % and may thus contribute to the total pool concentration.This fact must be considered in both calculation andinterpretation.

The basic assumption in the use of a labelled metaboliteunder steady-state condition (Figure 1) is:

F =

[Tracer] (1)Ra [Tracee]

POOL

F [Tracer]

Ra [Tracee] Rd

F =

[Tracer]Ra [Tracee]

→

→

→

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CLINICAL INTENSIVE CARE, Vol. 6 No. 6, 1995284

where F is the infusion rate of the labelled metabolite (tracer)and Ra is the rate of appearance of the endogenous metabolite(tracee). The concentrations of both the tracer and the traceeare determined in the compartment of interest into which thetracer is infused and from which aliquots are sampled. Steady-state conditions mean that the concentration of both tracer andtracee are constant during the interval of sampling and thepool size does not change. The fraction of tracer/tracee is theenrichment:

E = [Tracer] (2)

[Tracee]

When using radioactive tracers the specific activity is theanalog for the enrichment. Rearranging and substituting gives:

Ra = F (3)

E

The rate of appearance is the fraction of the tracer infusion/enrichment. In steady state the rate conditions of appearanceequals the rate of disappearance, ie the rate at which the traceeleaves the pool. The clearance of the tracee under steady-stateconditions is calculated as:

Cl = Rd (4)

[M]

where Cl is clearance, Rd is rate of disappearance and M thetotal concentration of the metabolite.

Equations 1-4 hold for a single pool model during steadystate and the continuous tracer infusion technique. Whensteady state is not likely, Ra can be calculated for a single poolmodel:

Ra = F–pV[(C

2+C

1)/2][(E

2–E

1)/(t

2–t

1)] (5)

(E2+E

1)/2

where C is the concentration of the tracee, t is time and theindices correspond to the two samples. V is the volume ofdistribution and p is a correction factor. Under non-steadystate conditions Rd is calculated as:

Rd = Ra – pV(C

2–C

1) (6)

t2–t

1

However, a living organism consists of various compartmentsand time constants. Thus, a single volume of distributiondetermined during steady state will not be correct during non-steady state conditions. For this reason the correction factor pis introduced. The value of p is an estimate based onexperimental experience. However, the product pV not onlyhas the drawback of being an estimate, but it may also be achanging variable under some circumstances. For glucose,the term pV is estimated at about 40-200 ml/kg bodyweight.However, Allsop et al found that the product pV changedwhen Ra was changed rapidly.8

In analogy to radioactive tracers, the rate of appearance canbe estimated by giving the tracer as a bolus and analysing the

data using non-compartmental models.9 These models use the‘area under the tracer concentration-time curve’ and thusrequire measurements of the entire response of the tracerplasma enrichment. The response can last several hours inorder to obtain the decay of the slow compartments. This maybe impossible in rapidly evolving diseases such as sepsis.These models are based on the assumption that the endogenousmetabolite production yields into the blood into which thetracer is infused. This latter assumption holds only for a fewmetabolites since many metabolites appear in the cytosol andonly part of the metabolite which appears in the cytosoldiffuses into the plasma. To accommodate for endogenousproduction sites that are different from the blood samplingsite, multi-compartmental models have been used to determineendogenous production of ketone bodies10 or the release ofleucine from protein breakdown, the conversion toketoisocaproic acid and the subsequent oxidation.11 Thesemodels are based on the assumption that the plasmaconcentration of the endogenously derived tracee does notchange during the experiment. This restricts the tracer bolusto amounts which allow the interaction of the tracer boluswith the metabolic system to be ignored. Otherwise the needwould arise to define how a change in a metabolic reaction islinked to changes in the plasma concentration of the metaboliteof interest. This is difficult for reactions in compartmentswhich are not identical with the sampling site, which is mostcommonly blood.

As can be seen in Equation 3, it is not necessary to measurethe absolute concentration of tracer and tracee to derive Ra. Itis sufficient to measure the fraction tracer/tracee over theenrichment. Gas chromatography/mass spectrometry (GCMS)in the selective ion monitoring mode allows measurement ofthis ratio without the need to determine the absoluteconcentration of tracer and tracee. The mass spectrometer ismore expensive and difficult to handle compared to thedetection of a radioactive compound. However, GCMS permitsmeasurement of the enrichment in the same probe without theneed for correction, for example recovery. The absoluteconcentration of tracer and tracee can be measured by GCMSusing internal standards. By contrast, using radioactive tracersthe absolute concentration of tracer and tracee have to bemeasured using, at least in part, different methods which haveto be corrected for recovery, quench, counter efficiency, etc.Thus the analytical procedure with radioisotopes introducessome variance in the measurement of the specific activity ofwhich stable isotope technology is devoid. In this regard, theuse of stable isotopes is preferable to the use of radioactivetracers.

Another advantage in the use of stable isotopes is the easewith which the enrichments of labels on different positions onthe molecule of interest can be separated. This allows thedetermination of the enrichments of different tracers in thesame compound and provides the tool for more complexmetabolic studies. For example, the conversion of lactate toglucose can be determined when tracers for both metabolitesare administered and the enrichment of the infused glucosetracer and the enrichment of the lactate-derived glucose tracerare measured. These approaches allow estimation of the rateof biosynthesis from a specific precursor. To date these have

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USE OF TRACER TECHNIQUES IN INTENSIVE CARE RESEARCH. PART I 285

135L: 789-90.5. Wood HG, Werkman CH, Hemingway A, Nier AO.

Degradation of propionic acid synthesized by propionicbacterium. Iowa State Coll J Sci 1941; 15: 213-14.

6. Trendelenburg U, Stefano FJE, Grohmann M. The isotopeeffect of tritium in 3H-noradrenaline. Naunyn SchmiedebergsArch Pharmacol 1983; 323: 128-40.

7. Marchini JS, Castillo L, Chapman TE, Vogt JA, Ajami A,Young VR. Phenylalanine conversion to tyrosine: comparativedetermination with L-[ring-2H5]phenylalanine andL-[1-13C] phenylalanine as tracers in man. Metabolism 1993;42: 1316-22.

8. Allsop JR, Wolfe RR, Burke JF. The reliability of rates ofglucose appearance in vivo calculated from constant tracerinfusions. Biochem J 1978; 172: 407-16.

9. Cobelli C, Toffolo G. Compartmental versus noncompartmentalmodeling for two accessible pools. Am J Physiol 1984; 247:R488-96.

10. Cobelli C, Toffolo G, Bier DM, Nosadini R. Models to interpretkinetic data in stable isotope studies. Am J Physiol 1984; 247:E551-61.

11. Matthews DE, Cobelli C. Leucine metabolism in man: lessonsfrom modelling. JPEN 1991; 15: 86-9S.

12. Jenssen T, Nurjhan N, Consoli A, Gerich JE. Dose-responseeffects of lactate infusions on gluconeogenesis from lactate innormal man. Eur J Clin Invest 1993; 23: 448-54.

13. Kelleher JK, Masterson TM. Model equations for condensationbiosynthesis using stable isotopes and radioisotopes. Am JPhysiol 1992; 262: E118-25.

14. Castillo L, Sanchez M, Vogt J et al. Plasma arginine, citrulline,and ornithine kinetics in adults, with observations on nitricoxide synthesis. Am J Physiol 1995; 268: E360-7.

15. Castillo L, Chapman TE, Sanchez M et al. Plasma arginine andcitrulline kinetics in adults given adequate and arginine-freediets. Proc Natl Acad Sci USA 1993; 90: 7749-53.

16. Esler M, Jennings G, Korner P, Blombery P, Sacharias N,Leonard P. Measurement of total and organ specificnorepinephrine kinetics in humans. Am J Physiol 1984; 247:E21-8.

17. Majewski H, Hedler L, Starke K. Evidence for a physiologicalrole of presynaptic alpha adrenoceptors: Modulation ofnoradrenaline release in the pithed rabbit. NaunynSchmiedebergs Arch Pharmacol 1983; 324: 256-63.

18. Majewski H, Hedler L, Starke K. The noradrenaline releaserate in the anaesthetized rabbit: Facilitation by adrenaline.Naunyn Schmiedebergs Arch Pharmacol 1982; 321: 20-7.

19. Ensinger H, Majewski H, Hedler L, Starke K. Neuronal andpostjunctional components in the blood pressure effects ofdopamine and bromocriptine in rabbits. J Pharmacol Exp Ther1985; 234: 681-90.

20. Wolfe RR. Radioactive and Stable Isotope Tracers inBiomedicine. Principles and Practice of Kinetic Aanalysis.New York, Wiley Liss Inc, 1992.

21. Wolfe RR, Jahoor F, Herndon DH, Miyoshi H. Isotopicevaluation of the metabolism of pyruvate and related substratesin normal adult volunteers and severly burned children: effectof dichloroacetate and glucose infusion. Surgery 1991; 110:54-67.

22. Wolfe RR, Herndon DN, Peters EJ, Jahoor F, Desai MH,Holland OB. Regulation of lipolysis in severely burned children.Ann Surg 1987; 206: 214-21.

been applied to estimation of gluconeogenesis12 andlipogenesis,13 and to probe the metabolism of amino acidssuch as production of plasma arginine from plasma ornithineand vice versa.14,15

Radioactive tracers have the advantage of the limit ofdetection usually being lower. Thus metabolites, hormones orneuro-transmitters can be traced with a radioactive-labelledcompound. An example is the rate of appearance (‘spill-overrate’) of noradrenaline from the peripheral sympathetic nervoussystem. This was achieved using tritium-labelled noradrenalinein humans16 and in animal experiments.17-19 Until now thekinetic of noradrenaline has not been investigated in vivo withthe use of a stable isotope tracer.

Due to the possible health hazards of non-stable radioactivetracers there are legal restrictions in their use in humans inmany countries. There are no health hazards known with theuse of stable isotope tracers at rates of application which arereasonable for research purposes. In Table 1 the daily intakeof stable isotopes is compared with the amount administeredduring a 4-8 hour study protocol. It can be seen that theamount of the less frequently occurring stable isotopes takenup with the daily food intake usually exceeds the amountadministered during an investigation. In animal experimentsharmful effects were found only when H

2O was replaced by

D2O to a degree greater than 20%.20 Stable isotope tracers are

regarded to be safe for use in pregnant women as well as inchildren.21,22

Table 1 Uptake of stable isotopes during daily foodintake as compared to stable isotopeadministration during an experiment.

Isotope Natural Body Body Normal Uptakeenrichmentcontent content uptake with

(%) (mg/kg) (mg/70 kg) with diet study(mg/kg) (mg/kg)

2H 0.015 15 1,050 6.9 0.04913C 1.107 1,980 138,600 99.90 0.10615N 0.366 111 7,700 0.15 0.12218O 0.204 130 9,100 133.40 0.147

References

1. Schoenheimer R, Rittenberger D. Deuterium as an indicator ina study of intermediary metabolism. Science 1935; 82: 156-7.

2. Schoenheimer R, Rittenberger D. Deuterium as an indicator instudy of intermediary metabolism: Role of fat tissue. J BiolChem 1935; 111: 175-81.

3. Schoenheimer R, Ruter S, Rittenberger D. Studies on proteinmetabolism: Metabolic activity of body proteins investigatedwith 1(-)-leucine containing 2 isotopes. J Biol Chem 1939;130: 730-2.

4. Wood HG, Werkman CH, Hemingway A, Nier AO. Heavy Cas a tracer in bacterial fixation of CO

2. J Biol Chem 1940;

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