Application of accelerator mass spectrometry (AMS) for high-sensitivity measurements of 14CO2 in long-term studies of fat metabolism

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Appl. Radiat. lsot. Vol. 47, No. 4, pp. 417-422, 1996

Pergamon

Copyright 'g) 1996 ElsevierScience Ltd Printed in Great Britain. All rights reserved 0969-8043/96 $15.00 + 0.00

Application of Accelerator Mass Spectrometry (AMS) for High-sensitivity Measurements of 14CO2 in Long-term Studies of Fat Metabolism K R I S T I N A S T E N S T R O M I*, S I G R I D L E I D E - S V E G B O R N 2, B E N G T E R L A N D S S O N I, R A G N A R H E L L B O R G 1, S O R E N M A T T S S O N 2, L A R S - E R I K N I L S S O N 3, B E R T I L N O S S L I N 2, G O R A N S K O G 4 a n d ANDERS WIEBERT l ~Department of Nuclear Physics, University of Lund, S61vegatan 14, S-223 62, Lund, Sweden 2Department of Radiation Physics and 3Department of Clinical Physiology, University of Lund, Malta6 University Hospital, S-205 02, Malm6, Sweden 4Radiocarbon Dating Laboratory, Department of Quaternary Geology, University of Lund, Tornavfigen 13, S-223 63, Lund, Sweden (Received 18 June 1995)

Long-term measurements of ~4C in CO,_ expired after ingestion of ~4C-labelled triolein were performed using accelerator mass spectrometry (AMS). About 30% of a given amount of ~4C-labelled triolein was catabolized rapidly, while the remaining 70% had a very slow turnover. The study shows the potential of the AMS technique for the study of the long-term biokinetics of ~4C-labelled pharmaceuticals. The AMS technique allows the administered activity to be reduced by several orders of magnitude without compromising the study. It may also allow studies of rare drug metabolites.

Introduction There are considerable uncertainties in the current estimates of absorbed doses to man from 14C-labelled radiopharmaceuticals mainly due to its long physical half-life (5730 yr) and the difficulties of making long-term, high-sensitivity measurements of the retention of 14C in the body. Some studies are considered to give comparatively high absorbed doses. Organic compounds labelled with 14C are used in clinical medicine to demonstrate abnormalities in metabolism, such as malabsorption, increased turnover or disturbed excretion. ~4C-labelled compounds are also used to study liver function and to demonstrate abnormal activity of gastrointestinal bacteria. The catabolic end-product carbon dioxide is expired and can easily be collected for measurement. This is the basis for various "breath tests" in clinical use (Hepner, 1974). The radioactive decay of the radionuclide is usually measured by liquid scintillation counting (LSC) or gas flow counting. Clinically useful information is obtained from samples taken a *To whom all correspondence should be addressed. ARI47/~-C

few hours after the administration of the test compound, even if the total turnover time is much longer. In contrast, a complete biokinetic study, needed for such purposes as the calculation of the radiation dose, requires sampling over a much longer t i m e - - u p to several months or even longer. Standard methods of measurement used in medical applications, such as LSC, are capable of detecting increased levels of ~4C in expired air only for a few days after ingestion. Even using high activity of ~*C, LSC is unable to detect activity in some metabolites. There is thus a need for a much more sensitive technique for a complete study. Therefore we have chosen to test accelerator mass spectrometry (AMS) (Kutschera, 1993; Felton et al., 1990; Vogel et al., 1990) for the analysis of samples taken up to several months after the ingestion of a compound labelled with 14C. In this study we report long-term measurements of ~4C in humans after ingestion of 14C-labelled triolein, which is used to demonstrate and quantify the degree of fat malabsorption (Newcomer et al., 1979). Two earlier studies (Malmendier et al., 1974; Pedersen and Marqversen, 1981), using standard measuring techniques, have indicated the existence of a significant,

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slowly metabolized fraction with a biological half-life of the order of several hundred days. This fraction, if it exists, will account for the main part of the absorbed dose (ICRP, 1991). The aim of this work was to use AMS to study the magnitude and elimination of this remaining fraction and to explore the possibilities of using extremely small amounts of activity and, thus, reduce the radiation dose for the test by employing the AMS technique.

a liquid scintillation counter (1217 Rackbeta, Wallac Sweden AB). The data were presented as expiratory ~4CO2 (percentage of the administered activity per hour). A constant CO2 expiration of 9 mmol/kg body weight/h was assumed (Winchell et al., 1970). Seven measurements were carried out for each volunteer (viz. of the detector background, expired CO, before administration, after 2, 4, 5, 6 h and a standard). The counting time was 1 h/sample. Sampling for A M S

Materials and Methods Volunteers Three healthy, male volunteers (A, B and C), aged 73, 67 and 50 yr, were studied after approval from the ethical committee of Lund University and after informed consent had been obtained. They followed the same routines as patients studied for malabsorption of fat at the Malm6 University Hospital. An additional series of measurements was also made. After an overnight fast, the volunteers were given 74 kBq ~4C-triolein (CFA 258, Amersham Sweden AB, Solna) evaporated on a piece of sugar, followed by 100 mL of a fat-containing test meal, Intralipid ~ (Pharmacia AB, Stockholm, Sweden). The fat content was 20 g (corresponding to 840 k J). The volunteers then had to drink a glass of water to remove residual activity from their mouths. The volunteers were at rest, and neither smoking nor eating was allowed during the first 4 h of the experiment, but small amounts of water were permitted. After 4 h, a light lunch accompanied by tea or coffee was offered. Using current estimates (ICRP, 1991) the study will result in an effective dose of 0.15 mSv. A fourth male volunteer (D), age 62, was given 50 times less ~4C-triolein, 1.48 kBq, in order to test the AMS method with lower amounts of ~4C than those administered in the ordinary malabsorption test. The test on volunteer D was repeated 8 months later. Sampling and measurements for L S C In order to absorb the expired carbon dioxide, the volunteers were required to breathe through a plastic tube and a chamber of a drying agent, into a glass vial containing 4.0 mL Hyamin 10-X (Packard Instruments B.V., The Netherlands) and one drop of phenolphthalein solution (0.4% in ethanol). The volunteers breathed into this until saturation of the solution was indicated by a colour change. 2-4 min o f normal breathing was required. The binding capacity of the Hyamin solution was about 2 mmol CO_,. The exact capacity was determined by titration against 0.1 N HC1. A standard containing 1% of the administered activity was prepared from the stock solution. 15 mL of scintillation liquid (Hionic Fluor, Packard Instruments B.V., The Netherlands) were added to each vial and the samples were measured in

The volunteers breathed through the same type of equipment as described above, but the liquid scintillation vial was replaced by a glass vial containing NaOH on a solid support, Ascarite" (Thomas Scientific, Swedesboro, N J). This was used to trap the CO2. The volunteers were required to make five maximal expirations through the system for each sample. To establish the normal ~4C content in the expired air, samples were also taken before the intake of the ~4C-labelled fat. For the first 6 h after ingestion, AMS samples were taken at the same time (within 5 min) as some of the LSC samples, in order to compare the two methods. Thereafter, samples were taken for AMS at longer intervals, up to 363 days for volunteers A and B, and 265 days for volunteer C. The activity expired by volunteer D was followed for the first 6 h after ingestion in the first test and for 28.5 h after the second test. As part of the study, complementary measurements were performed to find out whether variations in food intake influenced the excretion of ~4C. One of the volunteers, C, fasted for 32 h (only drinking water), and then had a proper meal. Breath samples were taken before, during and after the fasting period. A M S measurements The AMS measurements were performed at the Pelletron tandem accelerator at the Department of Physics, University of Lund (Skog et al., 1992). The accelerator is equipped with an ion source which requires samples in a solid, compact form in order to produce a negative and intense ion beam. Consequently, the carbon compounds in the expired air samples have to be extracted and converted into graphite. The sample preparation system. The preparation of ion source graphite samples is a two-stage process undertaken in vacuum (Stenstr6m et al., 1994). In the first step, the Ascarite sample, containing the carbon compounds from the expired air as carbonate, is mixed with about 3 mL of phosphoric acid (H3PO4), to re-constitute carbon dioxide. In the second step this carbon dioxide is catalytically reduced to graphite using about 5 mg iron powder as catalyst (Vogel et al., 1984). The iron powder is placed in a horizontal glass tube and heated to about 650°C. When the carbon dioxide and hydrogen gas are introduced into this tube, solid carbon is produced on

AMS for long-term studies of fat metabolism the iron catalyst. The water vapour also produced is removed by an ethanol-dry ice cold trap, in order to obtain a complete reduction. When, after 3-4 h, the process is complete, the remaining hydrogen gas is evacuated from the system. The Fe-C mixture is pressed into the well of a copper probe. The total amount of solid carbon required for the AMS measurements is a few milligrammes. The accelerator system. The copper probe containing the carbon from the sample of expired air is placed in the ion source of the accelerator system, where it is converted into a negative ion beam at an initial energy of a few tens of keV. The negative carbon current produced (mainly consisting of ~2C-) was about 5 #A. A first dipole magnet selects the ion mass to be injected into the accelerator. Together with the ~4C--ions, other mass-14 ions, such as t2CHf and ~3CH-, will also be injected into the accelerator. However, by using a tandem accelerator and thus a negative ion source, the isobar 14N is suppressed (because of the negative value of the electron affinity, it is not possible to form 14N- ions). When the negative ion beam is accelerated towards the high-voltage terminal of the accelerator and passes through a thin carbon foil (2/,tg/cm 2) at the terminal, electrons are stripped off the ions, resulting in a positive ion beam with ions of different charges. The positive ions thus produced will be further accelerated as they are repelled from the high-voltage terminal, gaining energy proportional to their charge state. The distribution of the different charge states depends on the velocity o f the ions and hence the terminal voltage. The stripping process is of great importance since it breaks up molecular ions by Coulomb explosion, in the process removing three or more electrons from each carbon ion. If a high charge state is selected by means of a Second dipole magnet, the molecular isobars can be eliminated. For the Lund Pelletron accelerator, carbon ions with charge state 3 + are suitable for AMS experiments. The terminal voltage employed was 2.4 MV, since for carbon ions the 3 + state dominates at this voltage (Wiebert et al., 1994). When the 14C3+ions have passed the second dipole magnet (which is tuned to accept only ions with the correct ratio between momentum and charge) a velocity analyzer acts as a last filter to exclude ions that have changed charge state through collisions with rest molecules along the accelerator tube and thereby managed to slip through the second dipole magnet. In the velocity analyzer ions with incorrect velocities are deflected out of the beam. A rectangular

419

aperture in front of the detector accepts only ions with the correct velocity. To establish the activity of the carbon sample, the number of ~4C atoms is measured relative to the known amount of stable ~3Catoms in the sample. The number of incoming ~4C3+ ions is measured in a 10 × 20 mm windowless photodiode detector (Hamamatsu $2744-04). By setting the accelerator system to accept only mass 13 and charge state 3 + , the current of ~3C3÷ ions can be measured using a Faraday cup, situated just in front of the photodiode detector. To minimize the error introduced in the measured ~4C/~3C-ratio, the system (computer-controlled to a large extent) alternates between mass 13 and mass 14 several times during each measurement cycle (Stenstr6m et al., 1993): Calculations Carbon samples made from the NBS oxalic acid standard (Stuiver and Polach, 1977) were used as reference for the AMS measurements. The absolute activity in 1950 of this standard has been determined by gas proportional counting to be 14.27 ___0.07 disintegrations/min/g carbon (the uncertainty denotes estimated statistical error) (Karl6n et al., 1964). In 1995 this corresponds to an activity of 0.2365 Bq/ gcarbon.Samples of !4C-free anthracite, processed in the sample preparation system, were also measured to provide the background of the sample preparation and accelerator systems. T h e activity of the samples is given by: N s - Nb As = A o x ' - Nox-- Nb where As and Aox are the sample activity and the oxalic acid standard activity, respectively. Ns, Nox and Nb are the ~4C/13C count rates of the sample, oxalic acid and anthracite, respectively. The anthracite background Nb was always less than 5% of the oxalic acid count rate Nox.

Results The activity concentration of 14C in samples taken within 6 h after ingestion, measured by the two methods, is shown in Table 1. In order to achieve a suitable !4C count rate in the AMS particle detector, the samples measured were diluted with ~4C-free carbon dioxide before conversion to solid carbon. The dilution was performed in a manner that introduced an uncertainty of about _ 1 0 % . The low-level samples taken later and the samples from

Table I. Comparisonbetweenthe results obtainedwith the AMS and LSC methods for three volunteerseach given 74 kBq R4C-triolein Volunteer (wt) Time after ingestion(h) AMS: ~4C-activity(Bq/g¢,rbon) LSC: ~4C-activity(Bq/g~,r~o,) A (76 kg) 5 252 333 B (71 kg/ 6 508 428 C (94 kg) 4 196 228 6 185 224

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volunteer D were not diluted and therefore the measured activity had an estimated uncertainty of __.3%. Considering the _+ 10% uncertainty, together with the other uncertainties in the AMS method and the LSC method, including differences in sampling conditions, the activity concentrations of ~4C measured by the two methods are considered to be acceptably similar. The precision and lowest detectable additional ~4C concentration of the two methods were determined by studying the results from a number of samples taken before ~4C-administration. The activity was measured by LSC in 10 samples of normal expiration from each of two normal persons. The results, corresponding to 44.2 and 44.3 "Bq/g~arbo~" (SD = 0.9 "Bq/g~,~bo~"), are totally dominated by the instrumental background since the natural ~4C-levelis only about 0.26 Bq/gc~rbo,. The lowest detectable concentration of additional ~4C (corresponding to 3 SD of the background signal) is 2.7 Bq/g¢,~o. for the LSC method. The lowest detectable concentration of additional ~4C for the AMS method was determined from the activity concentrations in eight samples taken from volunteer C prior to z4C-administration. The mean value of these activity concentrations was 0.258 Bq/gCarbon (SD=0.008Bq/g~a~bon) which is in good agreement with the present, natural 14C specific activity. In analogy with the estimation for the LSC method, the lowest detectable concentration of additional ~4C for the AMS method is thus 0.024 Bq/g~bo~, which is more than 100 times less than the LSC value. The contribution to the detection limit for a4C from the AMS system itself is lower. For the Lurid AMS system, it is about 0.005 Bq/g~,rbon. The 14C concentrations, measured by AMS, as a function of time after the intake of ~4C for the three volunteers given 74 kBq 14C (A, B and C) are shown in Fig. 1. The expired activity divided by the total administered activity was calculated, assuming a CO_, expiration of 9 mmol/kg body weight/h (Winchell e t a l . , 1970). For the first 24 h the expired fraction, calculated from the area under the curve, was 30% for A, 33% for B and 28% for C, corresponding to a biological half-life of about 2 days. For C, a further 7% was expired during the following 8 days, and for the next 254 days another 3% left the body. The activity concentration curve had its maximum at 4-6 h after ingestion and then rapidly fell to very low values, not measurable by the LSC technique (see Fig. 1) but definitely higher than the individual background value, measured by the AMS method. Even after several months, enhanced ~4C-levelscould be detected. On about day 72, volunteer C fasted for 32 h (only drinking water), which resulted in an increase in the expired 14COzconcentration, as seen in one of the enlarged parts of Fig. 1. The level of the normal activity concentration for volunteer C, 0.258 Bq/gc,~bo,, which was determined from the eight samples collected before intake of t4C, is indicated in the figure. This level cannot, however, be applied to

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Fig. 1. The activity concentration of ~4Cin expired CO_,as a function of time after the ingestion of 74 kBq ~4C-triolein for the three volunteers A, B and C. Concerning the enlarged parts of the curve, see the text. Note the change of timescale after 40 days.

the other volunteers, since the normal activity concentration may differ somewhat for each individual. For volunteer B only one sample was taken before 14C ingestion, showing 0.248 Bq/gc,ruo,. At 363 days after ingestion the value was 0.273 Bq/gcarbon. NO reliable samples of normal activity for volunteer A exist. Figure 2 shows the activity concentrations measured using AMS in the two test series on volunteer D, who was given 50 times less activity, namely, 1.48 kBq 14C-triolein. In the first test, samples were taken 2, 4, 5 and 6 h after

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AMS for long-term studies of fat metabolism

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content accessible with conventional mass spectrometry is about a few ppm relative to the stable isotope ~2C, which is several orders of magnitude higher than required in this investigation. By extending conventional mass spectrometry to AMS, it is possible to measure down to a value of 10- ~4for the 14C concentration relative to ~2C. The AMS method fulfils the demands of this investigation. This method can measure a total activity concentration down to about 1% of the concentration of ~4C in CO,, in normal expired air (about 0.25 Bq/gcarbon) and there is in principle no upper detection limit. Each sample requires only a few mg of carbon, a quantity that can be collected from a few expirations, thus satisfying the demand of Discussion a fast and simple sample collection. AMS is The concentration of ~4C can be determined either furthermore very efficient since each sample takes by counting the number of decays within a certain only about 20 min to analyze. The sample preptime interval or by counting the number of atoms. aration, i.e. the production of solid carbon, is the Which of these two approaches is preferable depends most time- and labour-consuming step, taking about 4 h/sample. However, by using a suitable number of on a number of factors. For the method used in this study, it is required to preparation lines operating simultaneously, the input measure 14C activity concentrations spanning about of manpower can be minimized and the sample 0.25 Bq/gcarbo, tO over 500 Bq/goarbon. The sample preparation can be administered independently of the collection procedure should be simple and rapid, not AMS-analysis. The precision of the method is also taking more than a few minutes. This implies that the sufficient. The repeated measurements on volunteer C sample size is small even if an efficient carbon dioxide before ~4C ingestion, resulting in 0.258 _+ 0.008 Bq/ trap is used. Short measuring times are desirable, g,~bon, give a precision of about __.3%, This may be making it possible to measure a sufficient number of a combination of natural variations and variations in samples for a complete study within a feasible time. the AMS method. Other types of biological material, Another important matter is the precision in the such as fat and bone biopsies, can also be analyzed. determination of the ~4C concentration. After combustion, the CO2 produced is treated as To measure ~4C activities down to 0.25 Bq/gc~rbonby above. Figure 1 shows that the short- and long-term counting radioactive decays, low-level counting techniques have to be relied on. Two methods are elimination for the three volunteers A, B and C are commonly used, gas proportional counting (Ostlund very similar. The fraction of the administered activity and Engstrand, 1963) and LSC with extremely low expired during the first 24 h is for all volunteers about background (Polach, 1987). Although theoretically 30%. The initial elimination is very rapid, but leaves feasible, these methods imply at least three major about 70% in the body. Figure 1 also shows that disadvantages: (1) to measure low levels of ~4C AMS can be used to follow the t4CO2 concentration activities, a considerable amount of material is for months after an uptake test. The sampling was, needed. The sample has to contain c a 1 g of carbon, however, not standardized in relation to intake of which is about 1000 times more than required by the food. This is illustrated in the result for the sample AMS method; (2) the sample preparation procedure taken after 5.5 days (see one of the enlarged parts of is fairly complicated. Either a very clean gas of CO_, Fig. 1), when volunteer C had three proper meals or C_,H2 for gas proportional counting has to be within 4 h prior to sampling. This observation produced, or a complete benzene synthesis for LSC initiated the special study around day 72 when fasting has to be made; and (3) due to the low counting rates, gave a rapid increase in the amount of expired ~4CO,. the measuring time has to be relatively long, about During the fast, stored body fat is used to a higher degree and since most of the administered ~4C is still 24 h/sample. Because of the long half-life and low concen- stored in the body, the ~4C concentration in the trations of ~4C, it is often much more efficient to expired air will increase significantly. The total release actually count the number of ~4C atoms in the sample during the 32 h fasting period was, however, very relative to the stable carbon isotopes, than to measure small (about 0.1% of the administered ~4C). The the number of the B-decays. Especially when dealing results indicate the need to standardize the sampling. with small samples, atom counting is favourable, For future measurements we recommend that since for a reasonable measuring time many more samples are taken in the morning just before atoms survive than decay. Choosing the atom breakfast. counting approach, conventional mass spectrometry The ~4Cactivity expired from volunteer D, who was becomes feasible. However, the lowest detectable ~4C given 50 times less ~4C-triolein than the others, was

administration. The curve obtained did not show a maximum between 4 and 6 h, as would be expected. The test was therefore repeated 8 months later and samples were taken more frequently, and revealed a maximum activity concentration at 3 h after intake of the test compound. The fraction of the administered activity which was expired during the first day was approx. 30 and 43%, respectively, and is thus of the same order of magnitude as for the volunteers given 74 kBq. The activity concentration before the intake of the ~4C-labelled triolein was 0.244 and 0.247 Bq/gcarbon in the first and second tests, respectively.

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easily detectable by the A M S m e t h o d , a n d it is possible to use even 2-3 times lower a m o u n t s o f administered activity. As seen in Fig. 2, the two tests, performed 8 m o n t h s apart, gave rise to curves o f similar shape b u t different height. This p r o b a b l y reflects differences in the nutritional state, which was n o t standardized; it is k n o w n t h a t the supply of c a r b o h y d r a t e s clearly influences the rate of fat metabolism. However, the results show t h a t it is indeed possible to use A M S with extremely low a m o u n t s o f activity in f a t - m a l a b s o r p t i o n tests. In conclusion, o u r studies show t h a t a b o u t 30% of the administered triolein is catabolized rapidly with a biological half-life of a b o u t 2 days, while the r e m a i n i n g 7 0 % h a s a very slow t u r n o v e r with a half-life o f the order of several h u n d r e d days. T h e details o f the long-term retention p a t t e r n for the volunteers in this study will be the subject o f further investigations. The results a n d experience gained from this study will be used to investigate further the long-term retention o f ~4C after f a t - m a l a b s o r p t i o n tests. F o r instance, the u n c e r t a i n t y introduced by the dilution o f the samples of high activity will be reduced by more precise CO2 pressure measurements, a n d the samples will be t a k e n m o r e often for a better estimation o f the a m o u n t of expired 14C. The A M S technique is also suitable for detailed studies of the m e t a b o l i s m of o t h e r ~4C-labelled c o m p o u n d s a n d pharmaceuticals. Acknowledgements--This project was supported by the

Swedish Medical Research Council (B95-39X-11272-01A), Swedish Institute of Radiation Protection and Malm6 University Hospital.

References Felton J. S., Turteltaub K. W., Vogel J. S., Balhorn R., Gledhill B. L., Southon J. R., Caffee M. W., Finkel D. E., Proctor I. D. and Davis J. C. (1990) Accelerator mass spectrometry in the biomedical sciences: applications in low-exposure biomedical and environmental dosimetry. Nucl. Instr. Meth. B52, 517. Hepner G. W. (1974) Breath analysis: gastroenterological applications. Gastroenterology 67, 1250. ICRP (1991) Radiation dose to patients from radiopharmaceuticals. Addendum 1 to Publication 53. Radiological Protection in Biomedical Research, 1CRP Publication 62. Annals o f the 1CRP 22(3). Pergamon Press, Oxford.

Karl6n I., Olsson I. U., Kfillberg P. and Kilicci S. (1964) Absolute determination of the activity of two t4C dating standards. Arkiv Geofvsik 4:22, 465. Kutschera W. (1993) Accelerator mass spectrometry: counting atoms rather than decays. Nucl. Phys. News 3, 15. Malmendier C. L., Delcroix C. and Berman M. (1974) Interrelations in oxidative metabolism of free fatty acids, glucose and glycerol in normal and hyperlipemic patients. A compartmental model. J. Clin. Invest. 54, 461. Newcomer A. D., Hofman A. F., Di Magno E. P., Thomas P. J. and Carlson G. L. (1979) Triolein breath test. A sensitive and specific test for fat malabsorption. Gastroenterology 76, 6. Pedersen N. T. and Marqversen J. (1981) Metabolism of ingested ~4C triolein. Estimation of radiation dose in tests of lipid assimilation using ~4C and 3H-labelled fatty acids. Eur. J. Nucl. Med. 7, 327. Polach H. A. (1987) Evaluation and status of liquid scintillation counting for radiocarbon dating. Radiocarbon 29, 1. Skog G., Hellborg R. and Erlandsson B. (1992) Accelerator mass spectrometry at the Lund Pelletron Accelerator. Radiocarbon 34, 468. Stenstr6m K., Erlandsson B., HeUborg R., H~.kansson K., Wiebert A. and Skog G. (1993) Development of a method to measure the concentration of ~4C in the stack air of nuclear power plants by accelerator mass spectrometry (AMS). LUNFD6/(NFFR-3061)/1-31/(1993) Laboratory report, Department of Nuclear Physics, University of Lund. Stenstr6m K., Erlandsson B., Hellborg R., Hfikansson K., Wiebert A. and Skog G. (1994) A sample preparation system for production of elemental carbon for AMS analyses. LUNFD6/(NFFR-3065)/1-33/(1994) Laboratory report, Department of Nuclear Physics, University of Lund. Stuiver M. and Polach H. A. (1977) Reporting of ~4C data. Radiocarbon 19, 355. Vogel J. S., Southon J. R., Nelson D. E. and Brown T. A. (1984) Performance of catalytically condensed carbon for use in accelerator mass spectrometry. Nucl. Instr. Meth. B5, 289. Vogel J. S., Turteltaub K. W., Felton J. S., Gledhill B. L., Nelson D. E., Southon J. R., Proctor I. D. and Davis J. C. (1990) Application of AMS to the biomedical sciences. Nucl. lnstr. Meth. B52, 524. Wiebert A., Erlandsson B., Hellborg R., Stenstr6m K. and Skog G. (1994) The charge state distributions of carbon beams measured at the Lund Pelletron Accelerator. Nucl. Instr. Meth. B89, 259. Winchell H. S., Stahelin H., Kusubov N. et al. (1970) Kinetics of COz-HCO3 in normal adult males. J. Nucl. Med. 11, 711. Ostlund H. G. and Engstrand L. G. (1963) Stockholm natural radiocarbon measurements V. Radiocarbon 5, 203.