the use of stable isotopes for diagnosis and clinical research · methods for the direct analysis...

Clinical Science (1982) 63,485-496 485

EDITORIAL REVIEW

The use of stable isotopes for diagnosis and clinical research

D. H A L L I D A Y * AND M . J . R E N N I E T

University College Hospital Medical School, London *Clinical Research Centre, Harrow, Middlesex, UX., and Rayne Institute,

Introduction

Stable, non-radioactive isotopes, so long over- shadowed by radionuclides, have recently become much more widely used in clinical science. This renewed interest has been stimulated by improvements both in the increased availability and diversity of stable-isotope- labelled compounds and in mass-spectrometric methods for their quantitative analysis. In addition, there is an increasing awareness of the advantages of stable-isotope techniques for patient safety and applicability to clinical problems. The object of this review is to outline methods of stable-isotopic analysis and to survey the results of the application of 2H, 13C and 15N-labelled compounds in clinical research. Previous reviews on this topic have considered the use of 2H for the quantification of total body water and intercompartmental water movements, 13C for absorption and incorporation studies using fatty acids and triacylglycerols and 15N for metabolic studies with ammonia, urea, creatinine, uric acid and for quantitative haematological studies 11, 21. It is hoped that the present review will stimulate clinical interest in the potential use and scientific advantages of stable isotopes. In addition, we wish to emphasize the possible benefits of increased patient accept- ability and applicability in cases where radio- isotope measurements are impossible.

Radionuclides versus stable isotopes The increasing awareness of the possibility of

biological damage at dose levels of radioactivity previously considered acceptable, has resulted in a gradual restriction in the use of long-lived radionuclides (e.g. I4C, 3H, 32P), except in studies involving adults older than 55 years. This rules out scientifically and clinically valuable studies in

Correspondence: Dr D. Halliday, Clinical Research Centre, Watford Road, Harrow HA1 3UJ, Middx., U.K.

neonates, children, young adults and during pregnancy.

Scintillation counting, especially when used to distinguish multi-labelling within a single molecule, falls short of the precision and practical ease with which stable-isotope quantification can be achieved using selected ion monitoring/mass spectrometry (see below). In addition, nitrogen ( 15N) and oxygen ( 1 7 0 , lag) have no radioactive equivalents of sufficiently long half-life to under- take human metabolic studies of several hours duration.

Even when the use of radioisotopes is both practical and ethically acceptable, the permitted doses exclude the possibility of monitoring tissue protein incorporation of amino acids, except when large tissue samples are obtainable. For example, in studying the incorporation of [ 14C]- leucine into human muscle protein, an infusion of 100 pCi lasting 10 h would produce a plasma free-[ 14C]leucine value of only 180 c.p.m. ml-' of blood. This value reflects the relatively high whole-body leucine turnover rate of some 200 pmol h-' kg-'. As body proteins in general contain approx. 8% leucine and human mixed muscle protein synthesis is of the order of 2% per day, then 1 g of tissue would only provide 18 c.p.m. For good precision and sensitivity most workers would not be happy with less than five times this level of counts, which would require 5 g of tissue. Clearly, only surgical biopsies could satisfy this requirement. By contrast, by using stable isotopes, muscle protein synthesis can be estimated using needle-biopsy tissue samples [31 of only 60-100 mg.

There are some major disadvantages to the use of stable isotopes as biological tracers. These are (a) the natural background against which estimation of label must be made (1.1% in the case of 13C), (b) the cost of the isotope-labelled substances (e.g. [l-13C]leucine is about E250 g-') and (c) the necessity for high-technology measuring equipment, which is itself very expensive.

0143-5221/82/120485-12$01.50 0 1982 The Biochemical Society and the Medical Research Society

486 D. Halliday and M . J. Rennie

However, set against advantages of patient applicability and of increased scope of investigation the cost is not prohibitive. A complete stable-isotope measuring facility consisting of a dual-inlet/double-collector isotope-ratio instrument and a suitable gas chromatograph/mass spectrometer would cost about &200 000 (U.S. $360000) at present, i.e. about the cost of a topical nuclear-magnetic-resonance (n.m.r.) machine, two electron microscopes or half the cost of a computerized X-ray tomograph.

Isotope analysis

Methods for the direct analysis of the deuterium content of aqueous fluids or of organic molecules (via oxidative combustion) include the falling- drop method [4], freezing-point elevation 151, infrared spectroscopy [61, gas chromatography [7] or mass spectrometry [81. Quantification of 15N by emission spectrometry relies on the property of nitrogen gas at low pressures to emit light of characteristic wavelengths when activated by radio or micro waves. The presence of a 15N nucleus (possessing an extra neutron) causes shifts in the energy transitions of a 15N2 molecule which result in changes in the band wavelengths. These band intensities are directly proportional to their nitrogen isotope content. Though the precision of this mode of analysis is some two orders of magnitude less than that provided by mass spectrometry, the ability to estimate the 15N content of 2-10 pg of N has obvious advantages. The technique has found application in the study of amino acid absorption and flux rates and also in monitoring the distribution of 15N-labelled glycine, ammonium chloride or L-aspartate in fasted subjects [9, 101.

Clear-cut absorption frequency differences exhibited by 12C02 and 13C0, permit measurement of the heavier isotope by infrared spectroscopy [ l l l . The recent incorporation of a tuneable diode laser source into this analytical system should provide the sensitivity and precision required to estimate either the oxidation rate of an intravenously infused 13C-labelled substrate, or the ”CO, ensuing from an oral 13C breath test. This analysis requires no preliminary purification of expired air. N.m.r. techniques have assumed importance in biosynthetic studies owing to the relative ease with which the precise position of heavy atoms can be detected in the intact molecule [12, 131. The possibility exists with the modern topical n.m.r. machine of monitoring the metabolism of 13C compounds in uiuo [141.

Nevertheless, the major quantitative analvtical advances have been realized through mass

spectrometry either in the form of conventional isotope-ratio instruments (isotope-ratio mass spectrometry, i.r.m.s.) or, more recently, quadrupole tuneable field instruments linked to a gas chromatograph (g.c.) (which constitutes the sample-inlet system) and a computer-controlled data system to handle the mass of information acquired [the g.c./m.s./d.s. (data-system) facility]. Descriptions of both types of instrument, relevant sample preparative techniques and application to clinical investigations have been recently reviewed [15-171.

Isotope-ratio mass spectrometry (i.r.m.s.)

Briefly, the isotope-ratio mass spectrometer is a dual-inlet double-collector fixed-magnetic-field instrument designed for precise heavy-isotope measurement of low-molecular-weight permanent gases which retain their molecular identity under the conditions of analysis. The compound containing the labelled atom of interest thus requires initial ‘clean-up’, isolation, purification and thence wet chemistry, combustion or enzymatic degradation to provide a gaseous product suitable for isotope analysis. This type of instrument, capable of detecting 1 molecule of ZH2, 13C0, or 15N, in lo5 unlabelled molecules, requires 0.1-0.5 mg of gas for accurate isotopic assay, and is thus ideal where sample size is not limiting but high- precision isotopic measurements are essential. For example, measurements of 13C in expired CO, or of amino acid incorporation into tissue protein, with sample sizes in excess of 20 pg of carbon can easily be achieved by this method. However, relatively large volumes of plasma would be needed to measure isotope enrichments (13C or 15N) of individual plasma amino acids and corresponding intracellular measurements would be impossible.

Selected - ion - monitoring gas chromatography1 mass spectrometry (s.i.m.-g.c.1m.s.)

Many of the analytical problems which limit the application of i.r.m.s. can be solved by the use of s.i.m.-g.c.m.s. The g.c./m.s./d.s. can examine the isotopic enrichment of a particular atom within the molecular or fragment ion, the ionization of which is accomplished in the mass- spectrometer ‘source’. After the initial ‘clean-up’ of a sample mixture and its treatment to form volatile derivatives, individual compounds are separated on a g.c. and sequentially transferred into the mass spectrometer. Rapid switching of the electric field in a quadrupole mass spectrometer permits measurement of the ion intensities

Use of stable isotopes 487

of labelled and unlabelled fragments (s.i.m.). Isotopic enrichment is calculated from the relative ion current intensities. Whilst lacking the sensitivity and precision of i.r.m.s., the major advantage of g.c./m.s. operated in the s.i.m. mode is the requirement of only picogram or nanogram quantities of sample for isotope analysis. Thus kinetic studies using stable- isotope-labelled metabolites in plasma or intracellular water can be contemplated. In terms of sample size and ultimate precision of isotopic enrichment these two analytical procedures are complementary rather than mutually exclusive.

Alternative analytical instruments

Isotope-ratio-monitoring gas chromatography/ mass spectrometry (i.r.m.-g.c./m.s.) combines many of the individual advantages of the two previously described analytical systems [ 18, 191. This analytical package relies on the gas- chromatographic separation of volatile derivatives of the compounds under investigation before their combustion to carbon dioxide, water, and oxides of nitrogen and sulphur. After selective trapping of unwanted gases and reduction of nitrogen oxides to nitrogen, carbon or nitrogen isotope ratios are measured using a modified conventional isotope-ratio mass spectrometer. It is likely that this approach will be developed considerably in the next few years, since potentially it provides a large range of sensitivity combined with a relatively low sample size and is thus ideally suited to biological and clinical work.

Quantitative analysis by isotope-dilution and related methods

The use of conventional isotope-ratio mass spectrometry for precise determination of deuterium isotopic content has been over- shadowed to a large extent recently by s.i.m.- g.c./m.s. analytical techniques. However, major improvements have been made in total-body- water (TBW) estimation by deuterium isotope dilution, requiring only 2-4 g of 2H,0 in adults [20]. Applications of the method have included serial estimates of TBW during pregnancy and post partum [211 and the assessment of milk intake in breast-fed babies over a 10-12-day period [221. It is surprising that current interest in energy balance and obesity [231 has as yet failed to stimulate the application of a method using 2H,180 for determining total CO, production (and hence energy expenditure) over prolonged periods in free-living human subjects. Its theoretical basis

was originally propounded some 25 years ago [241; the economic feasibility of its application to man and potential errors have been discussed in detail [25, 261.

Growing interest in quantitative analytical methods using g.c.1m.s. has resulted in a spate of investigations utilizing deuterium-labelled compounds in toxicology, pharmacology and clinical chemistry; several excellent reviews on these topics have appeared [27-291. Definitive methodology for a specific assay is offered by the use of s.i.m.-isotope-dilution mass spectrometry and the application of this technique to clinically important compounds has been reviewed [301. The technique involves the addition of a precise amount of isotopically labelled form of the analyte as an internal standard to an accurately measured aliquot of the sample. Recently, more precise methods for determining ion intensity ratios have been introduced [311. Similar g.c./ m.s. techniques have been employed to validate the radioimmunoassay of progesterone [321 and melatonin 1331 and to differentiate between endogenous and exogenous testosterone in plasma and urine 1341.

Kinetic analysis of material transfer within the body

End-product analysis

Many attempts to investigate metabolic problems are hampered not by lack of techniques capable of providing the answers, but by the suitability of the techniques in the clinical situation. This is particularly true of the study of children and of studies carried out in the field. It is not surprising, therefore, that much effort has been invested in making measurements on end products of metabolism excreted from the body, which are available without the need for invasive methods of collection. Such end products are CO, (and potentially water and NH,) in breath, and urinary NH,, urea, creatinine and uric acid.

Urinary lsN end-product analysis and protein metabolism. The theoretical basis of end-product methods applied to estimates of whole-body protein turnover is that the proportion of lSN- labelled tracer amino acid excreted in urine or incorporated into protein should reflect equivalent movements of the total nitrogen flux. Biased involvement in end-product formation or in protein synthesis would produce a weighted estimate. In practice, ammonia and urea have both been employed as end-product indicators of protein metabolism, though the choice of urea presents practical and theoretical problems that

488 D. Halliday and M. J. Rennie

relate to pool size, turnover rate and the enterohepatic recycling of 15N label.

Two methods for calculating whole-body protein turnover from end-product 15N data have been developed. Early workers based their cal- culations on a compartmental analysis of the time course of 15N-labelling in an end product following a single dose of 15N-labelled amino acid [351. Metabolic nitrogen pool size, turnover rate and the rate of incorporation of label into body protein could be derived. Examination of urea and ammonia 15N isotope decay curves, following a pulse dose of [l5N1alanine, has permitted the calculation of protein flux using a computer- generated four-compartmental model [361. As- sumptions involved in this study have been to some extent validated by comparison of the results with those obtained by the simultaneous application of a method using [ l-14C]leucine, given by constant infusion [371.

Following the lead of J. C. Waterlow and his colleagues [381, many workers have realized the advantages of a stochastic approach to treatment of end-product data. In this method, rates of protein turnover are calculated from the plateau enrichment in the end product, achieved during a constant infusion of tracer, or alternatively, from the cumulative excretion of isotope subsequent to a prime dose of label. One disadvantage is that the method does not permit estimation of the metabolic nitrogen pool. A comprehensive and critical analysis of these two approaches has been undertaken [391.

Compartmental analysis has provided values for the effect on protein turnover of variations in protein and energy (calorie) intake [40, 411, of the administration of tri-iodothyronine in myxoedema [421 and of the effects of immobilization [431 and sepsis 1361.

Calculation of protein turnover by stochastic analysis of measurements of 15N urinary urea enrichment at plateau during constant intravenous or nasogastric infusion of [15N1- glycine has found wide application. The technique, modified to repetitive oral feeding of [ l5N1- glycine, has been used productively in measuring protein flux in neonates [441 and infants [451, in young adults at two levels of protein intake [461 and in geriatric subjects [47, 481. These studies indicate that whole-body protein synthesis and breakdown is highest in the newborn, declines rapidly during normal growth to adulthood, and slowly declines as adults age. The greatest discrepancies between different studies in ab- solute rates of protein turnover were found in preterm low-birthweight infants [45, 49-5 11. Whilst analytical differences may partially ac-

count for the observed variation, the failure of [ 15N]glycine to label urinary urea represent- atively in all infants is important and requires further investigation [5 11. Close agreement in estimates of protein flux in adults derived from [ 15N]glycine or [ l4Clleucine infused simultaneously has bolstered the theoretical basis of both methodologies, as they are based on different, independent assumptions [481.

Stochastic 15N end-product methods have also been employed to study alterations in protein flux components resulting from stress and trauma, including bum injury [521, malignancy [531 and the effects of minor [541 and major [551 surgery. An important use of the method was in the demonstration of an increase in protein synthesis with no change in breakdown during amino acid infusions in overnight-fasted healthy subjects supplemented with glucose [561. The method has been applied to the study of strenuous exercise to show a fall in whole-body protein synthesis and a marked increase in protein breakdown [571. Accumulation of relatively large amounts of data, stemming from the use of the stochastic method, has permitted general relationships between protein flux and basal energy expenditure, dietary requirements, nitrogen balance and body weight to be defined 158,591.

A major limitation of this methodology, is the 48-60 h required to attain isotopic equilibrium in adults. Urinary urea I5N ‘plateau’ can however be achieved in approx. 12- 18 h if a priming dose of glycine is administered before the continuous infusion [601. Even so, acute or short-term repetitive measurements are precluded. In an attempt to provide this facility, Waterlow and his colleagues [6 1, 621 have systematically investigated the potential use of I5N urinary ammonia andlor urea as end-product indicator of protein flux following a single dose of [l5N1- glycine in normal, malnourished and obese subjects. The extent of the changes in protein turnover invoked by dietary manipulations was similar, irrespective of the mode of tracer administration (intravenous infusion of [ 1-l4C1- leucine, repeated oral dose or single oral dose of [ l5N1glycine). In another study, using the stochastic, end-product methodology it was found that obese women subjected to energy restriction but with adequate protein (3.3 MJ, 95 g of protein) exhibited a reduction in metabolic rate and protein turnover (synthesis and degradation). Daily administration of triiodothyronine (120 pg) reversed both these dietary responses [631. Vaccination of normal subjects against typhoid/cholera to cause short- term pyrexia produced an increase in both


protein synthesis (37%) and degradation (55%) as measured by the [ 15Nlglycine ammonia end-product method [641.

In cases where the experimental protocol has included an estimate of both urinary [l5N1- ammonia and [l5N1urea labelling, it has been repeatedly demonstrated that the concept of a single homogeneous metabolic nitrogen pool, a necessary assumption of the method, is invalid. Indeed, a range of experimental conditions have indicated gross compartmentation in the isotopic enrichment of the two end products. Averaging the measured I5N enrichment of both end products (ammonia and urea) and accounting for any change in the urea pool size during the period of the estimate may fortuitously negate some of these problems in practical terms, but the approach is purely empirical. Nevertheless, it seems that it may provide a more accurate estimate of whole-body protein turnover, un- weighted in terms of a specific organ or tissue, and also reduce the effects of compartmentation [651. WO, breath tests. The theoretical basis of

diagnostic breath tests is the use of a specifically labelled substrate containing a 'target bond', which on enzymatic cleavage results in the release of a functional group destined to produce labelled CO, as a metabolic end product. Oral administration of substrates may be used to test for abnormal absorption, hepatic transformation and distribution, before metabolism by other tissues. Total specificity and unequivocal diagnostic interpretation is possible only if one of the metabolic steps is known to be rate-limiting. Practical details and relevant theoretical considerations relating to expired 13C0, collection, sample handling, storage and automatic measurement of isotopic enrichment have been documen- ted [66-701. Attention has also been drawn to the need to correct rates of "CO, production and substrate oxidation when unlabelled substrate or food is given during the study [71].

Clinical applications of 13C breath tests have to date been largely restricted to defining certain aspects of gastrointestinal malfunction. Thus [ 13Cltrioctanoin (trioctanoylglycerol) has been used to provide positive discrimination of fat malabsorption from other possible causes of steatorrhea in patients in whom more than 25% of dietary fat is excreted; positive discrimination was also possible in a majority of patients in whom fat excretion was 10-20% day-' [721. Of the patients studied, those with cystic fibrosis showed an excellent correlation between the cumulative 3 h excretion of I3CO, and faecal fat excretion as estimated by the classical 72 h

balance method. Clearly the 13C breath-test approach is considerably easier, faster and more acceptable. There is evidence that [ "CI- tripalmitin (tripalmitoylglycerol) or triolein (trioleoylglycerol) may provide greater diagnostic discrimination in general fat malabsorption studies [731 than trioctanoin.

The use of [13Clglycocholate has been validated against its radioactive equivalent as an aid in the diagnosis of upper-intestinal bacterial overgrowth or ileal dysfunction [741. Though breath T O , analysis alone offers no chance to differentiate between bile acid deconjugation and malabsorption, this distinction may be achieved from I3C faecal analysis [751. 13C breath tests have also been used in the assessment of hepatic microsomal drug metabolism in normal subjects and patients with both mild and severe liver disease 761. Induction of hepatic microsomal demethylation has been monitored in patients with primary biliary cirrhosis before and during phenobarbital administration [77]. The toxicity of aminopyrine in terms of possible agranulocytosis has prompted the development of an analogous breath test using P4Clphenacetin [781, which could equally be performed with the 13C-labelled compound. The 14C test demonstrated signifi- cantly different levels of excretion of 14C0, between normal and cirrhotic subjects. Good discrimination between degrees of severity of alcoholic cirrhosis can be achieved within If h of [ 13Clgalactose administration by analysis of 13C0, production in breath [791, which reflects the intracellular oxidation of the sugar rather than simply disappearance from the plasma.

Techniques developed for assessing the isotopic enrichment of expired I3CO, are applicable irrespective of the substrate oxidized. Glucose, amino acid and keto acid oxidation rates have been studied in this manner using artificially labelled substrate. Naturally "C-labelled maize glucose has been extensively employed to appraise oxidation of an oral glucose load.

The variable concentration of 13C in plant sugars results from a selective isotope fraction- ation occurring during reactions of photo- synthesis, particularly primary carboxylation, i.e. the initial fixation of atmospheric CO, [801. This fixation involves either ribulosebisphosphate car- boxylase (EC 4.1.1.39), where the initial stable product is a 3-carbon compound (Calvin-Benson or C, pathway), or phosphoenolpyruvate carboxy- lase (EC 4.1.1.31), in which instance a 4-carbon acid is initially formed (Hatch-Slack or C, pathway). In practical terms so-called 'C4 plants' (e.g. maize, sugar cane) produce carbohydrates that contain some 15 p.p.t. (parts per thousand)


more 13C than in C, plants (beet, potatoes). Oral administration of 100 g of maize glucose to an adult will increase the isotopic content of breath I3CO, by some 3-8 p.p.t. which can be easily detected. Unfortunately plasma glucose kinetics cannot be monitored using naturally labelled glucose, owing to insufficient enrichment achieved by the plasma pool. It is worth considering that less than 25 mg of 90% [U-13C]glucose would elicit a similar 13C0, signal and, whilst the cost of enriched compounds 1s not inconsiderable, used at this level it represents a negligible percentage of the overall cost of mass-spectrometric isotopic analysis. In addition there are major advantages accruing from the use of highly enriched glucose in that it provides a means of estimating glucose turnover and endogenous glucose oxidation simultaneously. Of course the same applies to any other substrate present in the blood which can be artificially labelled with 1°C.

Advantage has been taken of 13C-labelled maize glucose to expand the normal oral glucose tolerance test to include estimation of the substrate oxidation in normal, obese and diabetic obese subjects [8l , 821. Within the latter patient group, a slight but significant increase in the oxidation of an oral glucose load was demonstrated following 2 weeks treatment with butyl- biguanide. Studies .of normal subjects during exercise (40-50% VoZmax., where Vo, is oxygen uptake per minute) indicated the almost complete oxidation of 100 g of exogenous glucose load within 4 h [831. Less strenuous exercise (20% VoZmax.) has highlighted Merences in substrate utilizacon between obese and normal individuals [841. Results from this study support the concept that, in obese subjects, exercise stimulates carbo- hydrate utilization, producing an acute improve- ment in glucose tolerance.

Kinetic methods based upon measurements of tracer enrichments of plasma constituents

The theoretical basis for the stochastic analysis of the turnover of substances found in the blood by labelling them with intravenous infusion of tracer is well-founded and has been extensively described [39, 851. The methods all aim to achieve a plateau of labelling of the substance of interest in the blood, a circumstance in which the net entry and exit of the tracee are equal to each other. Knowledge of the plateau labelling and the dosage rate of the tracer enables the flux (i.e. net entry or exit rates) to be calculated by dividing the second by the first. The methods are inevitably invasive and at their simplest require the placing of intravenous cannulae in at least

two veins. If the measurements are combined with the analysis of an end product (e.g. CO,, NH, or urea) the extra information brings a large bonus in the increased extent to which data can be used to calculate the rates of metabolic processes. For example, measurement of 13C0, output during the infusion of [ l-13C]leucine not only glows the measurement of the oxidation rate of leucine but also, if the net rate of exit (i.e. the flux) of leucine from the plasma is known (from the plasma leucine labelling), the other compo- nent of the flux besides oxidation, i.e. the synthesis of body protein, can be calculated.

Glucose turnover. Using [U-13Clglucose, glucose turnover has been estimated in malnourished and hypoglycaemic children [86], whilst the use of 1 l-13Clglucose has provided data concerning endogenous glucose production rate in pregnant women at term 1871 and in newborn infants [88]. The latter study was extended to investigate the magnitude of 13C recycling using l-13C-labelled glucose when compared with similar estimates of glucose production obtained in newborn subjects with non-recycling [6,6- ZHlglucose 1891. The direct comparison suggested that approx. 10% of the 13C of the l-position is re-utilized via the Cori cycle. In circumstances where both oxidation (from expired 13C0,) and glucose production rate are of interest, the latter can be obtained using the U-”C-labelled compound and monitoring the [MI and [ M + 61 molecular ion in the g.c.1m.s. to eliminate the problem of recycling. Optimal glucose requirements following burn injury have been defined using [U-13Clglucose in terms of the upper limit at which no increase in the rate of glucose oxidation or protein synthesis could be effected 1901. Using deuterated glucose, a linear relationship between glucose production rate and brain weight throughout life has been demonstrated [911.

In the course of these and related studies, analytical techniques have been developed for isotopic-enrichment determinations of both ,H- and 13C-labelled glucose. Sequentially linked enzyme decarboxylation [921, glucose oxidase treatment followed by wet combustion of the formed gluconic acid 1861 and classical oxidative combustion of isolated glucose [931 all provide gaseous 13C0, for i.r.m.s. analysis. [ ZH]glucose measured in the g.c.1m.s. requires initial anionic/ cationic resin treatment to exclude polar molecules and subsequent derivatization of the glucose. The derivative of choice will be dictated by production in the mass spectrometer of the particular fragment ion that includes the deuterium atom of interest [94,951.

Use of stable isotopes 49 1

Amino acid turnover and metabolism. One of the greatest benefits in the use of recently developed g.c.1m.s. techniques to study turnover of body constituents has come in the area of amino acid kinetics. Theoretically and practically it is possible to measure the 13C or 15N content of individual plasma amino acids by i.r.m.s. [961, though the method is cumbersome and requires 30 ml of blood for each sample. Clearly this would be unacceptable in most studies on ill patients. This method of isotope analysis has been superseded by the development of sensitive g.c./m.s. techniques capable of estimating 13C or 15N enrichment in individual plasma amino acids from less than 0.5 ml of blood [19,97,981.

Amino acid pool size and turnover rate for glycine and alanine have been reported in man and rabbits [99, 1001. The direct conversion of [ "Nlglycine into serine and subsequent entry into and distribution from the amino acid transamination pool has been measured [ 10 1 ]. Similar g.c.1m.s. techniques involved in these studies (selected ion monitoring of specific molecular fragments) have permitted the assessment of urea pool size and flux in human subjects 1021.

13C-labelled amino acids, only recently available, have already been profitably exploited. Both the estimation of gluconeogenesis in normal adults, and the demonstration of the capability of infants to produce glucose within 6 h of birth, have been achieved using [2,3-l3C1alanine 195, 1031. Amino acid flux studies in patients present- ing with burn injuries of differing severity 1901 were performed with constant infusions of [ 1- 13Clleucine to plasma plateau using a recently described g.c.1m.s. method [1041. If doubly labelled [l-13C,a-1SN]leucine is infused it is possible to measure both its transamination and subsequent decarboxylation of the keto acid [ 1051. This information could not be obtained in any other way. In addition to flux measurements, 13C-labelled amino acids have been used as internal standards to estimate both plasma and urinary amino acids [106, 1071.

It has been suggested that keto acid analogues of essential amino acids may have a therapeutic use in limiting nitrogen intake, maintaining protein synthesis and thus improving nitrogen balance both in patients suffering from chronic renal failure [ 1081 and in starving obese patients [log]. Experimental support for these ideas has come from stable-isotope studies to strengthen the circumstantial evidence of the in vivo transamination of keto acids [110]. The efficiency of this conversion has indicated the therapeutic potential for at least two of these keto acids [ 1 1 1, 1121.

Protein synthesis estimated by incorporation of labelled amino acids. Only when the precursor pool of protein synthesis has achieved isotopic equilibrium with respect to the infused amino acid can incorporation into specific proteins per unit time, and hence fractional synthesis rates, be calculated. Albumin synthesis in patients of various ages has been reported, using urinary urea to measure the enrichment of the precursor for albumin synthesis [113]. The required assumption in these studies is that since the liver produces both urea and albumin, presumably from closely related amino acid pools or even the same pool, the urea 15N enrichment should reflect the precursor pool labelling. Incorporation of label into plasma albumin was monitored via the guanidine lSN of arginine in albumin.

The rate of incorporation of [15Nllysine into muscle protein during a prolonged constant infusion of labelled lysine has been reported [961. Measurements of protein bound 15Nllysine were performed by i.r.m.s. following muscle protein hydrolysis and subsequent lysine separation by ion-exchange chromatography. By using a more sophisticated and rapid approach, muscle protein synthesis has been measured by means of the enrichment of muscle protein with [ l-13Clleucine using both isotope-ratio and g.c.1m.s. techniques to measure the enrichment of amino acids and CO,. In this latter investigation a primed constant infusion of [13Clleucine was used and the time period of the investigation was much shorter than in the lysine study. Protein synthesis in muscle could be measured in a total investigation time of 7 h, allowing this procedure to be applied in the investigation of patients with Duchenne dystrophy and other muscle diseases 11 141. A similar protocol can be easily adapted to the study of protein synthesis in a variety of tissues obtained at surgery so long as the infusion of the tracer is carried out for some time before the patient is brought to the operating theatre. The method has been validated in terms of the rise to plateau of the plasma leucine labelling and also of the labelling of a-ketoisocaproate (oxoisohexanoate; (rKIC), leucine's transamination product. It appears from studies of the intracellular labelling of leucine that under most circumstances this is very close to the labelling of plasma e K I C and therefore the necessity for making repeated biopsies in order to follow the time course of the intracellular precursor pool is obviated by the measurement of the enrichment of mKIC. Protein synthesis appears to be linear over time in human skeletal muscle, and it is likely that the same situation occurs in other tissues. The results show that human muscle tissue in the fed state


accounts for 53% of the whole-body protein synthetic rate and that it is more sensitive than the other tissues to feeding or fasting (M. J. Rennie, R. H. T. Edwards, D. Halliday, D. E. Matthews, S. L. Wolman & D. J. Millward, unpublished work). The synthetic ' rate of the other tissues can be obtained in toto by subtrac- tion of the muscle synthetic rate from the whole-body synthetic rate. Rather surprisingly, by contrast with the synthetic rate of non-skeletal muscle tissues in animals, in man these tissues only appear to turn over at rates of up to four times the muscle rate. This much smaller differen- tial rate of non-skeletal muscle protein synthesis above skeletal muscle protein synthesis in man has been confirmed by direct measurements of gut and skin (and some other tissues) in surgical patients [ 1151.

Metabolic-pathway elucidation

Intestinal tyrosine metabolism [ 1161 and phenylalanine-tyrosine interactions [ 1 171 have been followed using deuterium tracers, as have the pathways resulting in the incorporation of deuterium into urinary organic acids [ 1181. Deuterated substrates have similarly been used as tracers in the study of inherited metabolic diseases to demonstrate decreased alanine flux in children with maple-syrup disease [ 1191, to investigate glucose homoeostasis in severe cyanotic congenital heart disease [1201, and to accomplish the prenatal diagnosis of proprionic and methylmalonic acidaemia [ 1211. Ring- deuterated-phenylalanine has allowed the in vivo estimation of phenylalanine 4-hydroxylase activity, thus obviating the need for the assay of liver biopsy material or the insensitive, indis- criminatory, phenylalanine loading test. Thus measurement by s.i.m.-g.c./m.s. incorporation of deuterium into tyrosine, after a loading dose of ring-deuterated phenylalanine provides an index of phenylalanine 4-hydroxylase activity [ 1221. An alternative approach has relied on measurement of the liberation of deuterium from the deuterated phenylalanine and its trapping into body water [ 1231. Isolated reports have described the use of deuterium labels to study bile-salt synthesis in the neonate 11241 and of leucine turnover 11251.

The use of multi-labelled substrates has already provided substantial information about interconversion rates of closely related substances such as leucine and cc-ketoisocaproate [lo51 and glucose and alanine [95, 1031. This type of investigation will become increasingly attractive as equipment for measurement

becomes more widely available and should provide us with insights which hitherto have been unobtainable with radioisotopes.

Metal isotopes

Trace metals are currently receiving considerable attention as a result of their nutritional signifi- cance [1261. The kinetics of lead metabolism in five adult subjects have been comprehensively investigated using *04Pb [1271. Using a three- compartmental model derived from isotopic analysis of blood, urine, sweat, faeces, hair, nails and bone, and the diet, the rate of movement of lead between the defined pools was calculated. Methods have been developed using neutron- activation analysis to study the absorption of 58Fe, W u and 70Zn during oral contraceptive administration [ 1281 and to investigate calcium metabolism using 46Ca and 48Ca [1291. Mass- spectrometric techniques have been reported for the estimation of a range of alkaline earth metals and transition metals including 54Fe, 57Fe and 58Fe [130] as volatile chelates [131]. All groups of investigators have realistically considered the potential application of these isotopes to human studies and the analytical problems involved 11321.

Summary

The use of stable-isotope-labelled substances for metabolic investigation in man is only now, owing to great technological improvements, beginning to fulfil the promise envisaged by the pioneers in the field some 40 years ago. The next such period ought to see the use of stable-isotope technology become indispensable to the study of a range of metabolic problems, including those which would be insoluble by traditional techniques. It is hoped that the present review will stimulate clinical scientists to ask how their fields of study would benefit from the application of these powerful techniques, and to help make the case for the provision of suitable analytical facilities.

Acknowledgments

M.J.R. is supported as a Wellcome Senior Lecturer at University College Hospital Medical School, London. We should like to thank the Medical Research Council, the Muscular Dys- trophy Group of Great Britain, the Muscular Dystrophy Association of America, and Action Research for the Crippled Child for grant support for certain aspects of the work described in this review.


References 111 CRANE, C.W. (1978) In: Recent Advances in Clinical

Biochemistry, vol. 1, pp. 175-200. Ed. Alberti, K.G.M.M. Churchill Livingstone, Edinburgh.

121 HAUIDAY, D. & LOCKHART, I.M. (1978) In: Progress in Medicinal Chemistry, vol. 15, pp. 1-86. Ed. Ellis, G.P. & West, G.B. North-Holland Publishing Company, Amsterdam.

131 EDWARDS, R.H.T., YOUNG, A. & WILES, M. (1980) Needle biopsy of skeletal muscle in the diagnosis of myopathy and the clinical study of muscle function and repair. New England Journal ofMedicine, 302,261-271.

[41 SCHLOERB, P.R., FRIIS-HANSEN, B.J., EDELMAN, I.S., SOLOMON, A.K. & MOORE, F.D. (195 1) The measurement of total body water in the human subject by deuterium oxide dilution. Journal of Clinical Investigation, 29,1296-13 10.

I51 GARRY, P.J., NELSON, C.E. & OWEN, G.M. (1968) Measure- ment of deuterium oxide in plasma water by determining freezing point elevation. Journal of Laboratory and Clinical Medicine, 71,709-714.

I61 STANSELL, M.J. & MOJICA, L. (1968) Determination of body water content using trace levels of deuterium oxide and infrared spectrophotometry. Clinical Chemistry, 14, 11 12- 1124.

[71 MENDEZ, J., PROKOP, E., PICOU-REATEGUI, E., AKERS, R. & BUSKIRK, E.R. (1970) Total body water by D,O dilution using saliva samples and gas chromatography. Journal of AppliedPhysiology, 28,354-357.

[8l SOLOMON, A.K., EDELMAN, I.S. & SOLWAY, S. (1950) The use of the mass spectrometer to measure deuterium in body fluids. Journal of Clinical Investigation, 29.13 11-13 19.

191 SALTER, D.N. (1981) Emission spectrometric analysis of "N. Proceedings of the Nutrition Sociely, 40,335-342.

1101 STEIN, T.P., LESKIW, M.J. & WALLACE, H.W. (1976) Equilibration of L5N-labeled amino compounds in man. American Journal of Physiology, 230,13261330.

I1 11 HIRANO, S., KANAMATSU, T., TAKAQI, Y. & ABEI, T. (1979) A simple infrared spectroscopic method for the measurement of expired "COP Analytical Biochemistry, %,64-69.

1 121 OLDFIELD, E. & ALLERHAND, A. (1975) Studies of individual carbon sites of hemoglobins in solution by natural abundance carbon- 13 nuclear magnetic resonance spectroscopy. Journal of Biological Chemistry, 250,6403-6407.

I131 TANAKA, K., ARMITAGE, I.M., RAMSDELL, H.S., HSIA, Y.E., LIPSKY, S.R. & ROSENBERG, L.E. (1975) lL3ClValine metabolism in methylmalonicacidemia using nuclear magnetic resonance. Proceedings of the National Academy of Sciences of the United States ofAmerica, 12,3692-3696.

1141 ALOER, J.R., SILLERUD, L.O., BEHAR, K.L., GILLIES, R.J., SHULMAN, R.G., GORDON, R.E., SHAW, D. & HANLEY, P.E. (1981) In vivo carbon-13 nuclear magnetic studies in mammals. Science, 214,660-662.

1151 BIER, D.M., MA-ITHEWS, D.E. & YOUNG, V.R. (1981) In: Nitrogen Metabolism in Man, p. 289. Ed. Waterlow, J.C. & Stephen, J.M.L. Applied Science, London.

1161 HALLIDAY, D. & READ, W.C.C. (1981) Mass spectrometric assay of stable isotopic enrichment for the estimation of protein turnover in man. Proceedings of the Nutrition Society,

I171 HALLIDAY, D. (1981) In: Nitrogen Metabolism in Man, p. 295. Ed. Waterlow, J.C. & Stephen, J.M.L. Applied Science, London.

(181 SANO, M., YOTSUI, Y., ABE, H. & SASAKI, S. (1976) A new technique for the detection of metabolites labelled by the isotope "C using mass fragmentography. Biomedical Mass Spectrometry, 3, 1-3.

1191 M A ~ W S , D.E. & HAYES, J.M. (1978) Isotope-ratio- monitoring gas chromatography-mass spectrometry. Analytical Chemistry, 50,1465-1473.

1201 HALLIDAY, D. & MILLER, A.G. (1977) Precise measurement of total body water using trace quantities of deuterium oxide. Biomedical Mass Spectrometry, 4,8247.

I211 PIPE, N.J.G., SMITH, T., HALLIDAY, D., EDMONDS, L.J., WILLIAMS, C. & COLTART, T.M. (1979) Changes in fat, fat free mass and body water in normal human pregnancy. British Journal of Obstetrics and Gynaecology, 86,929-940.

l22l COWARD, W.A., WHITEHEAD, R.G., SAWYER, M.B., PRBNTICE, A.M. & EVANS, J. (1979) New method for measuring milk intakes in breast-fed babies. Lancet, U, 13-14.

40,321-334.

1231 GARROW, J.S. (1978) Energy Balance and Obesity in Man, p. 113. Elsevier/North-Holland, Amsterdam.

1241 LIFSON, N., GORDON, G.B. & MCCLINTOCK, R. (1955) Measurement of total carbon dioxide production by means of D,018. Journal ofAppliedPhysiology, I , 704-710.

1251 LIPSON, N., LIITLE, W.S., LEVIIT, D.G. & HENDERSON, R.M. (1975) Oxygen-18 deuterium oxide method for carbon dioxide output in small mammals and economic feasibility in man. Journal of Applied Physiology, 39,657-664.

1261 NAGY, K.A. (1980) CO, production in animals: analysis of potential errors in the doubly labeled water method. American Journal of Physiology, 238,1R466-R473.

1271 LAWSON, A.M. & DRAFFAN, G.H. (1975) In: Progress in Medicinal Chemistry, vol. 12, p. 1. Ed. Ellis, G.P. & West, G.B. North-Holland Publishing Company, Amsterdam.

1281 BLAKE, M., CRESPI, H.L. & KATZ, J.J. (1975) Studies with deuterated drugs. Journal of Pharmaceutical Science, 64, 307-39 I.

I291 LAWSON, A.M. (1975) The scope of mass spectrometry in clinical chemistry. Clinical Chemistry, 21,803-824.

I301 TIETZ, N.W. (1979) A model for a comprehensive measurement system in clinical chemistry. Clinical Chemistry, 25, 833-839.

1311 LAWSON, A.M., LIM, C.K., RICHMOND, W., SAMSON, D.N., SETCHELL, K.D.R. & THOMAS, A.C.S. (1980) In: Current Developments in the Clinical Applications of HPLC GC and MS. Clinical Research Centre Symposium No. I , pp. 135-153. Ed. Lawson, A.M., Lim, C.K. & Richmond, W. Academic Press, London.

1321 DEHENNIN, L., REIFSTECK, A. & SCHOLLER, R.A. (1974) A quantitative method for the estimation of testosterone and progesterone in human plasma, using gas chromatography- mass spectrometer combination with single ion monitoring. Journal of Steroid Biochemistry, 5.81-86

I331 KENNAWAY, D.J., FRITH, R.G., PHILLIPOU, G., M A ~ E W S , C.D. & SEAMARK, R.F. (1977) A specific radioimmunoassay for melatonin in biological tissue and fluids and its validation by gas chromatography-mass spectrometry. Endocrinology, 101,119-127.

1341 BABA, S., SHINOHARA, Y. & KASUYA, Y. (1980) Dif- ferentiation between endogenous and exogenous testosterone in human plasma and urine after oral administration of deuterium-labelled testosterone by mass fragmentography. Journal of Clinical Endocrinology and Metabolism, SO, 889-894.

I351 SAN PIETRO, D.P. & RI'ITENBERG, D. (1953) A study of the rate of protein synthesis in humans. 11. Measurement of the metabolic pool and the rate of protein synthesis. Journal of Biological Chemistry, 201,457473.

1361 LONG, C.L., JEEVANANDAM, M., KIM, B.M. & KINNEY, J.M. (1977) Whole body protein synthesis and catabolism in septic man. American Journal of Clinical Nutrition, 30,1340-1344.

1371 BIRKHAHN, R.H., LONG, C.L., FITKIN, D., JEEVANDAM, M. & BLAKEMORE, W.S. (198 1) Whole-body protein metabolism due to trauma in man as estimated by L-115Nlalanine. American Journal of Physiology, 241, E64-E7 1.

1381 PICOU, D. & TAYLOR-ROBERTS, T. (1969) The measurement of total protein synthesis and catabolism and nitrogen turnover in infants in different nutritional states and receiving different amounts of dietary protein. Clinical Science, 36, 283-296.

I391 WATERLOW, J.C., GARLICK, P.J. & MILLWARD, D.J. (1978) Protein Turnover in Mammalian Tissues and in the Whole Body. chapter 7. North-Holland Publishing Company, Amsterdam.

1401 TSCHUDY, D.P., BACCHUS, H., WEISSMAN, S., WATKIN, D.M., EUBANKS, M. & WHITE., J. (1959) Studies of the effect of dietary protein and caloric levels on the kinetics of nitrogen metabolism using 15N baspartic acid. Journal of Clinical Investigation, 38,892-901.

141) KASSENMR, A., DE GRAEFF, 1. & KAWENHOVEN, A.T. (1960) "N-glycine studies of protein synthesis during refeeding in anorexia nervosa. Metabolism, Clinical and Experimental, 9,83 1-837.

1421 CRISPELL, K.R., PARSON, W. & HOLLIFIELD, G. (1956) A study of the rate of protein synthesis before and during the administration of L-triiodothyronine to patients with myxe- dema and healthy volunteers using N-15 glycine. Journal of Clinical Investigation, 35, 164-169.


1431 SCHBNHYDER, F., HEILSKOV, N.C.S. & OLESEN, K. (1954) Isotope studies on the mechanism of negative nitrogen balance produced by immobilization. Scandinavian Journal of Laboratory Inuestigafion, 6,178-188.

I441 PENCHARZ, P.B., STEPFEE, W.P., COCHRAN, W., SCRIMSHAW, N.S., RAND, W.M. & YOUNG, V.R. (1977) Protein metabolism in human neonates: nitrogen-balance studies, estimated obligatory losses of nitrogen and whole- body turnover of nitrogen. Clinical Science and Molecular Medicine, 52,485-498.

1451 GOLDEN, M.H.N., WATERLOW, J.C. & PICOU, D. (1977) Protein turnover, synthesis and breakdown before and after recovery from protein-energy malnutrition. Clinical Science and Molecular Medicine, 53,473-477.

I461 STEFFEE, W.P., GOLDSMITH, R.S., PENCHARZ, P.B., SCRIMSHAW. N.S. & YOUNG. V.R. (19761 Dietarv Drotein

1471

1481

I491

1501

I511

I521

I531

1541

. , I . .~

intake and dynamic aspects of whole bcdy nitrogen metabolism in adult humans. Metabolism. Clinical and Experi- mental, 25,28 1-297. WINTERER, J.C., STEFFEE, W.P., DAW, W., PERERA, A., UAUY, R., SCRIMSHAW, N.S. & YOUNG, V.R. (1976) Whole body protein turnover in aging man. Experimental Gerontology, I I, 79-87. GOLDEN, M.H.N. & WATERLOW, J.C. (1977) Total protein synthesis in elderly people: a comparison of results with lrJN1glycine and [14Clleucine. Clinical Science and Molecular Medicine, 53,277-288. NICHOLSON, J.F. (1970) Rate of protein synthesis in premature infants. Pediatric Research, 4,389-397. DUFFY, B., GUNN, T., COLLMGE, J. & PENCHARZ, P. (1981) The effect of varying protein quality and energy intake on the nitrogen metabolism of parenterally fed very low birthweight (i 160 g) infants. Pediatric Research, 15, 104Cb1044. JACKSON, A.A., SHAW, J.C.L., BARBER, A. & GOLDEN, M.H.N. (1981) Nitrogen metabolism in preterm infants fed human donor breast milk: the possible essentiality of glycine. PediatricResearch, IS, 1454-1461. KIEN, C.L., YOUNG, V.R., ROHRBAUGH, A.M. & BURKE, J.F. (1978) Increased rates of whole body protein synthesis and breakdown in children recovering from bums. Annals of Surgery, 187,383-391. NORTON, J.A., STEIN, T.P. & BRENNAN, M.F. (1981) Whole body protein synthesis and turnover in normal man and malnourished patients with and without known cancer. Annals of Surgery, 194,123-128. KIEN, C.L., YOUNG, V.R., ROHRBAUGH, D.K. &BURKE, J.F. (1978) Whole body protein synthesis and breakdown rates in children before and after reconstructive surgery of the skin. Metabolism, Clinical and Experimental, 27,27-34.

1551 CRANE, C.W., PICOU, D., SMITH, R. & WATERLOW, J.C. (1977) Protein turnover in patients before and after elective orthopaedic surgery. Brifish Journalof Surgery, 64,129-133.

I561 SIM, A.J.W., YOUNG, V.R., WOLF, B.M., CLARKE, D. & MOORE, F.D. (1979) Glucose promotes whole-body protein synthesis from infused amino acids in fasting man. Lancet, i, 68-72.

1571 RENNIE, M.J., EDWARDS, R.H.T., KRYWAWYCH, S., DAVIES, C.T.M., HALLIDAY, D., WATERLOW, J.C. & MILLWARD, D.J. (1981) Effect of exercise on protein turnover in man. Clinical Science, 61,627-639.

1581 YOUNG, V.R., STEFFEE, W.P., PENCHARZ, P.B., WINTERER, J.C. & SCRIMSHAW, N.S. (1975) Total human body protein synthesis in relation to protein requirements at various ages. Nature (London), 253,192-193.

1591 WATERLOW, J.C. & JACKSON. A.A. (1981) Nutrition and protein turnover in man. British Medical Bulletin, 37,5-10.

I601 SIM, A.J.W., SUGDEN, B., YOUNG, V.R. & MOORE, F.D. (1981) Nitrogen turnover measurement by IJN glycine priming prior to continuous infusion in healthy man. Proceedings of the Nutrition Society, 40,90A.

1611 WATERLOW, J.C., GOLDEN, M.H.N. & GARLICK, P.J. (1978) Protein turnover in man measured with IJN: comparison of end products and dose regimes. American Journal of Physiology, 235, E165-EI74.

I621 GARLICK, P.J., CLUGSTON, G.A. & WATERLOW, J.C. (1980) Influence of low-energy diets on whole-body protein turnover in obese subjects. American Journal of Physiology, 238, E2354244.

1631 NAIR, K.S., HALLIDAY, D. & GARROW, J.S. (1980) Protein metabolism in obese patients on caloric restriction and on T, therapy. Alimenfazione Nutrizione Metabolismo, I, 326.

I641 GARLICK, P.J., MCNURLAN, M.A., FERN, E.B., TOMKINS, A.M. & WATERLOW, J.C. (1980) Stimulation of protein synthesis and breakdown by vaccination. British Medical Journal, 281,263-264.

l65l FERN, E.B., GARLICK, P.J., MCNURLAN, M.A. & WATERLOW, J.C. (1981) The excretion of isotope in urea and ammonia for estimating protein turnover in man with ["Nlglycine. Clinical Science, 61,217-228.

I661 CASPARY, W.F. (1978) Breath tests. Clinics in Gastro- enterology, 7,35 1-374.

1671 BARR, R.G., PERMAN, J.A., SCHOELLER, D.A. & WATKINS, J.B. (1978) Breath tests in pediatric gastrointestinal disorders: new diaenostic oooortunities. Pediatrics. 62.393401.

I . . -. 1681 SCHOELLER, D.A., SCHNEIDER, J.F., SOLOMONS, N.W.,

WATKINS, J.B. & KLEIN, P.D. (1977) Clinical diagnosis with the stable isotope in CO, breath tests: methodology and fundamental considerations. Journal of Laboratory and Clinical Medicine, 90,4 1 2-42 1.

I691 SCHOELLER, D.A. & KLEIN, P.D. (1978) A simplified technique for collecting breath CO, for isotope ratio mass spectrometry. Biomedical Mass Spectromefry, 5,29-3 I.

1701 SCHOELLER, D.A. & KWIN, P.D. (1978) A microprocessor controlled mass spectrometer for the fully automated analysis of breath carbon dioxide. Biomedical Mass Spectrometry, 6, 35Cb355.

[711 SCHOELLER, D.A., KLEIN, P.D., WATKINS, J.B., HEIM, T. & MACLEAN, W.C. (1980) 'IC abundances of nutrients and the effect of variations in isotopic abundances of test meals formulated for "CO, breath tests. American Journal of Clinical Nutrition, 33,2375-2385.

1721 WATKINS, J.B., SCHOELLER, D.A., KLEIN, P.D., On, D.G., NeWcoMER, A.D. & HOFMANN, A.F. (1977) "C-trioctanoin: a non-radioactive breath test to detect fat malabsorption. Journal of Laborafory and Clinical Medicine, 90,422-430.

1731 NEWCOMER, A.D., THOMAS, P.H. & HOFMANN, A.F. (1976) Breath tests for detecting steatorrhea: comparison of labeled trioctanoin, triolein and tripalmitin. Gastroenferology. 70, 923-929.

1741 SOLOMONS, N.W., SCHOELLER, D.A., WAGONFELD, J.B., Om, D., ROSENBERG, I.H. & KLEIN, P.D. (1977) Ap- plication of a stable isotope ("Cjlabelled glycocholate breath test to diagnosis of bacterial overgrowth and ileal dysfunction. Journal of Laboratory and Clinical Medicine, 30,43 1-439.

I751 FROMM, H. & HOFMA", A.F. (1971) Breath test for altered bile-acid metabolism. Lancet, ll,62 1-625.

1761 SCHNEIDER, J.F., BAKER, A.L., HAINES, N.W., HATFIELD, G. & BAYER, J.L. (1978) Aminopyrine N-demethylation: A prognostic test of liver function in patients with alcoholic liver disease. Gastroenterology, 79, 1145-1 150.

I771 LAUTERBURG, B.H. & BIRCHER, J. (1976) Expiratory measurement of maximal aminopyrine demethylation in vivo: effect of phenobarbital, partial hepatectomy, portacaval shunt and bile duct ligation in the rat. Journal oJPharmacology and Experimental Therapeutics, 196,50 1-509.

1781 BPEEN, K.J., DESMOND, P.V., BURY, R., CALDER, I. & MASHFORD, M.L. (1977) A I4C-phenacetin breath test in the assessment of hepatic function. Gastroenterology, 72,

1791 SHREEVE, W.W., SHOOP, J.D., On, D.G. & MCINTEER, B.B. (1976) Test for alcoholic cirrhosis by conversion of Il'CCl or [llCl galactose to expired CO,. Gastroenterology, 71, 98- 101.

(801 BLACK, C.C. (1973) Photosynthetic carbon fixation in relation to net CO, uvtake. Annual Review of Plant Phvsiolorv. 24.

A- 10/ 1033.

I811

I821

1831

- . , I, I

253-286. LACROIX, M., MOSORA, F., PONTUS, M., LEFEBVRE, P., LUYCKX, A. & LOPEZ-HABIB, G. (1973) Glucose naturally labeled with carbon-13: use for metabolic studies in man. Science, 181,445-446. LEFEBVRE, P., MOSORA, F., LACROIX, M., LUYCKX, A., LOPEZ-HABIB, G. & DUCHESNE, J. (1975) Naturally labeled llC-glucose: Metabolic studies in human diabetes and obesity. Diabefes, 24,185-189. PIRNAY, F., LACROIX, M., MOSORA, F., LUYCKX, A. &


LEPEBVRE, P. (1977) Glucose oxidation during prolonged exercise evaluated with naturally labeled glucose. Journal ofAppliedPhysiology, 43,258-26 1.

[841 RAWSSIN, E., PAKUD, P., THELIN-DOERNER, A., ARNAUD, M.J. & JEQUIER, E. (1980) Substrate utilization during prolonged exercise after ingestion of llC-glucose in obese and control subjects. International Journal of Obesity, 4, 235- 242.

1851 Riffis, D.S. (1963) The Mathematical Approach to Physio- lo~ical Problems: a Critical Primer. Williams and Wilkins.

[861

[871

1881

[a91

Batimore. KERR, D.S., STEVENS, M.C.G. & Prcou, D.I.M. (1978) Fasting metabolism in infants: 11. The effect of severe undernutrition and infusion of alanine on glucose production estimated with U-llC-glucose. Metabolism. Clinical and Experimental, 27,83 1-848.

P.A.J. (1979) Glucose production in pregnant women at term gestation. Journal of Clinical Investigation, 63,388-394. KALHAN, S.C., Sam, S.M. & ADAM, P.A.J. (1976) Measurement of glucose turnover in the human newborn with glucose- l-I3C. Journal of Clinical Endocrinology and Metabolism, 43,704-707. KALHAN, S.C., BIER, D.M., SAWN, S.M. & ADAM, P.A.J. (1980) Estimation of glucose turnover and "C recycling in the human newborn by simultaneous Il-'3Clglucose and [6,6-1H,]glucose tracers. Journal of Clinical Endocrinology

KALHAN, S.C., D'ANOELO, L.J., SAVIN, S.M. & ADAM,

and Mehbolism, 50,456-460. 1901 BURKE, J.F., WOLFE, R.R., MULLANY, C.J., MAITHEWS,

D.E. & BIER, D.M. (1979) Glucose requirements following burn injury. Annals of Surgery, 190,274-283.

I911 BIER, D.M., LEAKE, R.D., HAYMOND, M.W., ARNOLD, K.J., GRUENKE, L.D., SPERLINO, M.A. & KIPNIS, D.M. (1977) Measurement of "true" glucose production rates in infancy and childhood with 6,6-dideuteroglucose. Diabetes, 26, 1016-1023.

1921 KALHAN, S.C., SAVIN, S.M. & ADAM, P.A.J. (1977) Estimation of glucose turnover with stable tracer glucose- l-']C. Journal of Laboratory and Clinical Medicine, 89, 285-294.

[931 WOLPE, R.R., ALLSOP, J.R. & BURKE, J.F. (1979) Glucose metabolism in man: Responses to intravenous glucose infusion. Metabolism, Clinical and Experimental, 2% 2 10- 220.

[941 WIECKO, J. & SHERMAN, W.R. (1976) Boroacetylation of carbohydrates. Correlations between structure and mass spectral behaviour in monoacetylocose cyclic boronic esters. Journal of the American Chemical Society, 98,763 1-7637.

1951 BIER, D.M., ARNOLD, K.J., SHERMAN, W.R., HOLLAND, W.H., HOLMES, W.F. & KIPNIS, D.M. (1977) In vivo measurement of glucose and alanine metabolism with stable isotopic tracers. Diabetes, 26,1005-1015.

I961 HALLIDAY, D. & MCKERAN, R.O. (1975) Measurement of muscle protein synthetic rate from serial muscle biopsies and total body protein turnover in man by continuous intravenous infusion of ~-1h~~NIlysine. Clinical Science and Molecular Medicine, 49,581-590.

[971 M A ~ E W S , D.E., BEN-GALIM, E. & BIER, D.M. (1979) Determination of stable isotope enrichment in individual plasma amino acids by chemical ionization mass spectrometry. Analytical Chemistry, 51,8&84.

1981 BIER, D.M. & CHRISTOPHERSON, H.L. (1979) Rapid micro- method for determination of "N enrichment in plasma lysine: application to measurement of whole body protein turnover. Analytical Biochemistry, 94,242-248.

[991 LAPIDOT, A. & NISSIM, I. (1980) Regulation of pool sizes and turnover ratio of amino acids in humans: lsN-glycine and IJN-alanine single dose experiments using gas chromatography-mass spectrometry analysis. Metabolism, Clinical and Experimental, 29,230-239.

11001 IRVINO, C.S., NISSIM, 1. & LAPIDOT, A. (1978) The determination of amino acid pool sizes and turnover rates by gas chromatographic-mass spectrometric analysis of stable isotope enrichment. Biomedical Mass Spectrometry, 5 , 117-122.

[loll MAITHEWS, D.E., CONWAY, J.M., YOUNO, V.R. & BIER, D.M. (1981) Glycine nitrogen metabolism in man. Metabolism, Clinical and Experimenlal, 30, 886-893.

11021 NISSIM, I., YUDKOPP, M., YANO, W., TERWILLIGER, T. & SEGAL, S. (1981) Rapid gas chromatographic-mass spectrometric analysis of ["NI urea: application to-human metabolic studies. Clinica Chimica Acta, 109,295-304.

11031 F-R, T.E., KARL, I.E., HILLMAN, L.S. & Bmn, D.M. (1981) Direct measurement of gluconeogenesis from 12,3- llCllalanine in the human neonate. American Journal of Physiology, 240, E615-E621.

[lo41 MATIMEWS, D.E., MOTIL, K.J., ROHRSAUGH, D.K., BURKE, J.F., YOUNO, V.R. & BIER, D.M. (1980) Measurement of leucine metabolism in man from a primed continuous infusion of L-[ l-13Clleucine. American Journal of Physiology, 238, E473-E479.

[lo51 M A ~ E W S , D.E., Bmn, D.M., RENNIE, MJ., EDWARDS,

(1981) Regulation of leucine metabolism in man: a stable isotopestudy. Science, 214, 1129-1131.

11061 KINOSTON, E.E. & DUPPIELD, A.M. (1978) Plasma amino acid quantitation using gas chromatography chemical ionisation mass spectrometry and amino acids as internal standards. Biomedical Mass Spectrometry, 5,621-626.

11071 FINLAYSON, P.J., CHRISTOPHER, R.K. & DUPPIELD, A.M. (1980) Quantitation of fourteen urinary hamino acids using isohutane gas chromatography chemical ionisation mass spectrometry with 13C amino acids as internal standards. Biomedical Mass Spectrometry, 7,450-453.

[lOSl WALSER, M., COULTE.R, A,, DIQHE, S. & CRANTZ F.R. (1973) The effect of keto-analogues of essential amino acids in severe chronic uremia. Journal of Clinical Investigation, 52,678-690,

11091 SAPIR, D.G., OWEN, O.E., POZEPSKY, T. & Wasen , M. (1974) Nitrogen sparing induced by a mixture of essential amino acids given chiefly as their keto-analogues during prolonged starvation in obese subjects. Journal of Clinical Investigation, 54,974-980.

Ill01 RICHARDS, P., ELL, S. & HALLIDAY, D. (1977) Direct evidence for synthesis of valiie in man. Lancet, i, 112-1 14.

R.H.T., HALLIDAY, D., MUWARD, D.J. & CLUGSTON, G.A.

11111

I1121

[1131

11141

11151

HALLIDAY, D., MADIOAN, M., CHALMERS, R.A., PURKISS, P., ELLS, S., BEROSTROM, J., F~~RsT, P., NEUHAUSER, M. & RICHARDS, P. (1981) The degree of conversion of hketo acids to valiie and phenylalanine in health and uraemia. Quarterly Journal of Medicine, 50,5342. MITCH, W.E., WALSER, M. & SAPIR, D.G. (1981) In: Metabolism and Clinical Implications of Branched Chain Amino and Ketoacids, p. 349. Ed. Walser, M. & Williamson, J.R. Elsevier/North-Holland, Amsterdam. GERSOWZ, M., MUNRO, H.N., UDALL, J. & YOUNG, V.R. (1980) Albumin synthesis in young and elderly subjects using a new stable isotope methodology: Response to level of protein intake. Metabolism, Clinical and Experimental, 29, 1075-1086. RENNIE, M.J., EDWARDS, R.H.T., MILLWARD, D.J., WOLMAN, S.L., HALLIDAY, D. & M A ~ E W S , D.E. (1982) Effects of Duchenne muscular dystrophy on muscle protein synthesis. Nature (London), 2%. 165-167. READ, M.A., READ, W.W.C., HALLIDAY, D., BATES, P.C., MILLWARD, D.J., WARD, M.W.N., PETERS, J. & RENNIE, M.J. (1982) Incorporation of l-L3C-leucine into protein in human skeletal muscle, skin and gut. 3rd European Congress on Parenteral and Enteral Nutrition, Maastricht. (In press.)

11161 CURTIUS, H.-CH. & Memen. M. (1976) Study of the intestinal tyrosine metabolism using stable isotopes and gas chromatography-mass spectrometry. Journal of Chromatography, 126,569-580.

11171 CURTIUS, H.€H., V~LLMIN, J.A. & BAE~UOCKER, K. (1972) The use of deuterated phenylalanine for the elucidation of the phenylalanine-tyrosine metabolism. Clinica Chimica Acta, 37,277-285.

[ 1181 FELL, V., HOSKINS, J.A. & POLLITT, R.J. (1978) The labelling of urinary acids after oral doses of deuterated L-phenylalanine and L-tyrosine in normal subjects. Clinica Chimica Acta, 83,259-269.

[1191 HAYMOND, M.W.. BeN-GALIM, E,& Sntoseh K.E. (1978) GTucose and alan-ine metabolism in children with maple syrup urine disease. Journal of Clinical Investigation, 62, 398-405.

11201 HAYMOND, M.W., STRAUSS, A.W., ARNOLD, K.J. & BIER, D.M. (1979) Glucose homeostasis in children with severe


cyanotic congenital heart disease. Journal of Pediatrics, 95, 220-227.

11211 NAYLOR, G., SWEETMAN, L., NYHAN, W.L., HORNBECK, C., GRIPPITHS, J., M ~ R C H , L. & BRANDANOE, S. (1980) Isotope dilution analysis of methylcitric acid in amniotic fluid for the prenatal diagnosis of proprionic and methylmalonic acidemia. Clinica Chimica Acta, 107,175-183.

11221 CURTIUS, H.-CH., ZAQALEK, M.J., BAERLOCHER, K., SCHAUB, J., LEIMBACHER, R.W. & REDWEIK, V. (1977) In vivo studies of the phenylalanine-4-hydroxylase system in hyperphenylalaninemics and phenylketonurics. Helvetia Pediafrica Acta, 3 8 4 6 1 4 6 9 .

11231 MILSTEM, S. & KAUPMAN, S. (1975) Studies on the phenylalanine hydroxylase system in vivo. Journal of Biological Chemistry, 250,47824705.

[I241 WATKINS, J.B., INGALL, D., SZCZEPANIK, P., KLEIN, P.D. & LESTER, R. (1973) Bile salt metabolism in the newborn. New England Journal of Medicine, 288,43 1 4 3 4 .

11251 HAYMOND, M.W., HOWARD, C.P., MILES, J.M. & GEIUCH, J.E. (1980) Determination of leucine flux in vivo by gas chromatography-mass spectrometry utilising stable isotopes for trace and internal standard. Journal of Chromatography, 183,403409.

[I261 GOLDEN, M.H.N. & GOLDEN, B.E. (1981) Trace elements,

potential importance in human nutrition with particular reference to zinc and vanadium. British Medical Bulletin, 37,

11271 R A B I N O W ~ M.B., WETHERILL, G.W. & KOPPLE, J.D. (1976) Kinetic analysis of lead metabolism in healthy humans. Journal of Clinical Investigation, 56,26&270.

I1281 KING, J.C., RAYNOLDS, W.L. & MARORN, S. (1978) Absorption of stable isotopes of iron, copper and zinc during oral contraceptive use. American Journal of Clinical Nutrition, 31,l198-1203.

[I291 JANGHORBANI, M., SUNDARESAN, A. & YOUNG, V.R. (1981) Accurate measurement of stable isotopes “Ca and ‘“a in human feces, plasma, and urine in relation to human nutrition of calcium. Clinica Chimica Acta, 113,267-280.

I1301 MILLER, D.D. & VAN CAMPEN, D. (1979) A method for the detection and assay of iron stable isotope tracers in blood serum. American Journal of Clinical Nutrition, 32, 2354- 2361.

11311 HACKEY, D.L., BLAIS, J.-C. & KLEIN, P.D. (1980) High precision isotopic ratio analysis of volatile metal chelates. Analytical Chemistry, 52,1131-1135.

[ 1321 RISBY, T.H. (1980) Use of negative chemical ionization mass spectrometry for the trace analysis of metals. Environmental Health Perspectives, 3 6 , 3 9 4 6 .

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the use of stable isotopes for diagnosis and clinical research · methods for the direct analysis...

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