Implications of the variation in biological 18 O natural abundance in body water to inform use of Bayesian methods for modelling total energy expenditure when using doubly labelled water.

RATIONALE: Variation in 18 O natural abundance can lead to errors in the calculation of total energy expenditure (TEE) when using the doubly labelled water (DLW) method. The use of Bayesian statistics allows a distribution to be assigned to 18 O natural abundance, thus allowing a best-fit value to be used in the calculation. The aim of this study was to calculate within-subject variation in 18 O natural abundance and apply this to our original working model for TEE calculation. METHODS: Urine samples from a cohort of 99 women, dosed with 50 g of 20% 2 H2 O, undertaking a 14-day breast milk intake protocol, were analysed for 18 O. The within-subject variance was calculated and applied to a Bayesian model for the calculation of TEE in a separate cohort of 36 women. This cohort of 36 women had taken part in a DLW study and had been dosed with 80 mg/kg body weight 2 H2 O and 150 mg/kg body weight H2 18 O. RESULTS: The average change in the δ18 O value from the 99 women was 1.14‰ (0.77) [0.99, 1.29], with the average within-subject 18 O natural abundance variance being 0.13‰2 (0.25) [0.08, 0.18]. There were no significant differences in TEE (9745 (1414), 9804 (1460) and 9789 (1455) kJ/day, non-Bayesian, Bluck Bayesian and modified Bayesian models, respectively) between methods. CONCLUSIONS: Our findings demonstrate that using a reduced natural variation in 18 O as calculated from a population does not impact significantly on the calculation of TEE in our model. It may therefore be more conservative to allow a larger variance to account for individual extremes.

Decaffeinated coffee improves insulin sensitivity in healthy men.

Epidemiological studies have found coffee consumption is associated with a lower risk for type 2 diabetes mellitus, but the underlying mechanisms remain unclear. Thus, the aim of this randomised, cross-over single-blind study was to investigate the effects of regular coffee, regular coffee with sugar and decaffeinated coffee consumption on glucose metabolism and incretin hormones. Seventeen healthy men participated in five trials each, during which they consumed coffee (decaffeinated, regular (containing caffeine) or regular with sugar) or water (with or without sugar). After 1 h of each intervention, they received an oral glucose tolerance test with one intravenous dose of [1-13C]glucose. The Oral Dose Intravenous Label Experiment was applied and glucose and insulin levels were interpreted using a stable isotope two-compartment minimal model. A mixed-model procedure (PROC MIXED), with subject as random effect and time as repeated measure, was used to compare the effects of the beverages on glucose metabolism and incretin parameters (glucose-dependent insulinotropic peptide (GIP)) and glucagon-like peptide-1 (GLP-1)). Insulin sensitivity was higher with decaffeinated coffee than with water (P<0·05). Regular coffee with sugar did not significantly affect glucose, insulin, C-peptide and incretin hormones, compared with water with sugar. Glucose, insulin, C-peptide, GLP-1 and GIP levels were not statistically different after regular and decaffeinated coffee compared with water. Our findings demonstrated that the consumption of decaffeinated coffee improves insulin sensitivity without changing incretin hormones levels. There was no short-term adverse effect on glucose homoeostasis, after an oral glucose challenge, attributable to the consumption of regular coffee with sugar.

Bayesian hierarchical methods to interpret the (13)C-octanoic acid breath test for gastric emptying.

BACKGROUND: The (13)C-octanoic acid breath test is a convenient method for assessing gastric emptying (GE). Success depends on obtaining a well-characterized time profile of the excretion of label in breath, which may not be the case if GE is delayed. AIMS: To use Bayesian techniques in conjunction with hierarchical modelling as a method to increase the success of the modelling process. METHODS: Retrospective analysis of 164 individual breath tests using the WinBUGS program. The approach was tested by analysing the complete dataset simultaneously, and also as individual studies. RESULTS: The time required for Bayesian modelling was comparable with that needed for the usual methods. The results obtained were almost identical to those obtained from conventional modelling for well-behaved breath tests, but much more realistic in cases where the experimental data was poor, or when GE was delayed. CONCLUSIONS: The use of Bayesian estimation of the parameters of the (13)C-octanoic acid breath test is demonstrated. By adopting a hierarchical model, realistic values for the lag phase and half-emptying time were obtained in situations when conventional parameter estimation failed. This is particularly relevant when GE is unexpectedly delayed. We recommend that WinBUGS become the method of choice for analysing breath test data.

How much human milk do infants consume? Data from 12 countries using a standardized stable isotope methodology.

The WHO has developed new growth curves based on breast-fed infants. Recommendations for energy intake have been adopted based on measurements of total energy expenditure. Data on human milk (HM) intake are needed to estimate the energy intake from this food source. However, objective HM data from around the world have not been available, because these measurements are difficult to obtain. Stable isotope methods have been developed to provide objective measurements over a 14-d period. A pooled analysis of 1115 data points of HM intake, obtained using the dose to the mother deuterium oxide turnover method, was undertaken in infants aged 0-24 mo from 12 countries across 5 continents. A hierarchical model was needed to estimate mean HM intake and its variance within and between countries given the complexity of the data. The overall mean HM intake was 0.78 (95% CI = 0.72, 0.84) kg/d, and the age-specific estimates indicated that intake increased over the first 3-4 mo and remained above 0.80 kg/d until 6-7 mo. The variability of intake increased in late infancy. Boys consumed 0.05 kg/d more than girls (P < 0.01). HM intake was strongly, inversely associated with non-HM water intake [r = -0.448 (95% CI -0.511 to -0.385); P < 0.0001]. These objective isotope values of HM intake improve our understanding of the magnitude and variability of HM intake within and across populations and help to estimate nutrient intakes in breast-fed infants.

A novel stable isotope approach for determining the impact of thickening agents on water absorption.

Research on the bioavailability of water from thickened fluids has recently been published and it concluded that the addition of certain thickening agents (namely, modified maize starch, guar gum, and xanthan gum) does not significantly alter the absorption of water from the healthy, mature human gut. Using xanthan gum as an example, our "proof of concept" study describes a simple, accurate, and noninvasive alternative to the methodology used in that first study, and involves the measurement and comparison of the dilution space ratios of the isotopes (2)H and (18)O and subsequent calculation of total body water. Our method involves the ingestion of a thickening agent labeled with (2)H 1 day after ingestion of (18)O. Analyses are based on the isotopic enrichment of urine samples collected prior to the administration of each isotope, and daily urine samples collected for 15 days postdosing. We urge that further research is needed to evaluate the impact of various thickening agents on the bioavailability of water from the developing gut and in cases of gut pathology and recommend our methodology.

Recent progress in stable isotope methods for assessing vitamin metabolism.

PURPOSE OF REVIEW: Recent developments in mass spectrometric methodology, in particular the widespread adoption of liquid chromatography/mass spectrometry, have presented investigators with new opportunities to investigate micronutrient absorption and metabolism. This review focuses on recent reports of the use of stable isotope techniques to facilitate research into vitamin uptake and utilization in humans. RECENT FINDINGS: Stable isotopes are used principally in two ways, as analytical standards in isotope dilution assays and as tracers in studies of physiology. There have been a number of advances in both fields recently for almost all of the vitamins. In particular, the effects of food preparation and meal composition on vitamin bioavailability are being probed more widely than before. SUMMARY: A considerable amount of method development has been reported. We now have the opportunity to consolidate our understanding of vitamin metabolism to better inform the dietary recommendations for optimal health.

A novel derivative for the assessment of urinary and salivary nitrate using gas chromatography/mass spectrometry.

Previous gas chromatography/mass spectrometry (GC/MS) methods for determining nitrate in biological samples involve either hazardous chemicals or produce multiple isomers that can be difficult to quantitate. Modification of these methods, by the nitration of mesitylene instead of benzene and in the presence of trifluoroacetic anhydride rather than sulphuric acid, should enable simple isotopic quantitation for use in tracer studies, for example, in the measurement of nitric oxide production. Desiccated urine and saliva samples, in addition to aqueous labelled and unlabelled nitrate standards, were treated with trifluoroacetic anhydride and mesitylene at 70 degrees C for 1 h, cooled, then sequentially washed with deionised water and aqueous sodium bicarbonate. The solution of nitromesitylene in mesitylene was separated, dried and analysed by GC/MS. The full mass spectra exhibited abundant ions at m/z 165 and 166 corresponding to the unlabelled and labelled molecular species of nitromesitylene, respectively. Selected ion monitoring of these masses for a series of gravimetrically prepared standards indicated good agreement with isotopic enrichments in the range 0.0625-5 mole % excess, and at nitrate concentrations within the physiological range of 0.078-2 mmol/L. Derivatised samples were stable with respect to isotopic enrichments and nitrate concentrations at -20 degrees C for up to 21 days and exhibited excellent repeatability. Nitration of mesitylene proved to be a simple and rapid method for the measurement of isotope ratios in aqueous nitrates by GC/MS, which has applications in tracer studies and in concentration determinations by isotope dilution techniques for nitric oxide production.

Insulin administration and rate of glucose appearance in people with type 1 diabetes.

OBJECTIVE: To assess whether prandial insulin, in addition to basal insulin, has an effect on the rate of glucose appearance from a meal in people with type 1 diabetes. RESEARCH DESIGN AND METHODS: The rate of glucose appearance from a mixed meal (Ra(meal)) was investigated in six adult (aged 24 +/- 2 years), lean (BMI 23.6 +/- 1.5 kg/m(2)) subjects with well-controlled type 1 diabetes (duration 7.9 +/- 6.9 years, A1C 7.6 +/- 0.9%) with/without prandial insulin. Actrapid was infused to maintain euglycemia before meals were consumed. Subjects consumed two identical meals on separate occasions, and Ra(meal) was measured using a dual isotope method. [6,6-(2)H(2)]glucose was incorporated into the meal (0.081 g/kg body wt), and a primed constant/variable rate infusion of [1,2,3,4,5,6,6-(2)H(2)]glucose was administered. In the tests with prandial insulin, an additional bolus dose of Actrapid was given 20 min before the meal at 0.1 units/kg body wt. RESULTS: Insulin concentration with prandial insulin was significantly higher than during basal insulin studies (119 +/- 16 vs. 66 +/- 15 pmol/l, P = 0.03 by paired t test). Despite differences in insulin concentration, there were no differences in total glucose appearance (3,398 +/- 197 vs. 3,307 +/- 343 micromol/kg) or time taken for 25% (33.1 +/- 3.3 vs. 31.7 +/- 3.5 min), 50% (54.6 +/- 3.5 vs. 54.1 +/- 4.7 min), and 75% (82.9 +/- 7.1 vs. 82.8 +/- 5.8 min) of total glucose appearance. The fraction of the glucose dose appearing in the circulation was the same for basal (73 +/- 8%) and prandial (75 +/- 4%) study days. CONCLUSIONS: These results suggest that meal glucose appearance is independent of prandial insulin concentration in people with type 1 diabetes.

The hydration ability of three commercially available sports drinks and water.

This paper compares the hydration ability of three commercially-available sports drinks with water under conditions of rest and exercise, using a deuterium dilution technique. For the rest group, 0.05g/kg of body weight of deuterium, contained in gelatine capsules, was ingested with one of the test solutions and saliva samples were collected every five minutes for an hour while the subject remained seated. The deuterium was administered as above for the exercise group but sample collection was during one hour of exercise on a treadmill at 55% of the subject's maximum heart rate. The enrichment data for each subject were mathematically modelled to describe the kinetics of hydration and the parameters obtained were compared across drinks using a basic Anova. At rest, significant differences were found for t(1), t(1/2), and the percent of drink absorbed at t(1). The differences between drinks were not significant for t(2) or the maximum absorption rate. For the exercise group, the only significant difference was found between water and the sports drinks at t(1). Therefore, we conclude that labelling with a deuterium tracer is a good measure of the relative rate ingested fluids are absorbed by the body. Because of the lack of differences found at t(2), which is indicative of the 100% absorption time, both at rest and during exercise, it may be speculated that, compared to water, the sports drinks studied in this paper did not hydrate the body at a faster rate.

A single glucose derivative suitable for gas chromatography/mass spectrometry and gas chromatography/combustion/isotope ratio mass spectrometry.

The incorporation of stable isotopes improves the assessment of glucose metabolism and, with some researchers using two tracers, (2)H-glucose assessed by gas chromatography/mass spectrometry (GC/MS) and (13)C-glucose by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), a common derivative for both is advantageous. The most commonly used derivatives for GC/MS are inappropriate for GC/C/IRMS as additional functional groups dilute the label. We therefore considered the suitability of six derivatives for both GC/MS and GC/C/IRMS. Glucose alkylboronates were prepared by adding the appropriate alkylboronic acid (butyl- or methylboronic acid) in pyridine to desiccated glucose. The derivatisation was completed by reacting this with either (a) acetic anhydride or trifluoroacetic anhydride (acetate derivatives) or (b) bis(trimethylsilyl)trifluoroacetamide BSTFA (TMS derivatives). All six derivatives were assessed using GC/MS and (13)C GC/C/IRMS. Neither TMS derivative exhibited any signal intensity in the molecular ion, although a M-15 ion showed good agreement between experimental and theoretical data and, whilst still low in intensity, could be suitable for isotope work. Similarly, none of the acetate derivatives showed any intensity at the molecular ion although three key fragmentation series were identified. The most attractive sequence, initiated by the loss of 1,2 cyclic boronate, resulted in the main fragment ion of interest, m/z 240, corresponding to the fluorinated methylboronate derivate. Minimal carbon and hydrogen atoms are added to this derivative making it an excellent choice for stable isotope work, while proving suitable for analysis by both GC/MS and GC/C/IRMS.

Analysis of isotope ratios in vitamin K1 (phylloquinone) from human plasma by gas chromatography/mass spectrometry.

Vitamin K(1) is a fat-soluble vitamin required for the gamma-carboxylation of vitamin K-dependent proteins. Recent work has suggested an important role for vitamin K(1) in bone health beyond its more established function in the control and regulation of blood coagulation. However, current UK recommended intakes do not reflect this recent evidence. The use of stable isotopes provides a powerful tool to investigate vitamin K kinetics, turnover and absorption in man, although published methods have reported difficulties in the extraction and analysis of isotope ratios of vitamin K in human plasma. In this paper, we report a new methodology for the extraction and measurement of isotope ratios in vitamin K(1). Sample clean-up is achieved with liquid-liquid extraction, enzyme hydrolysis with lipase and cholesterol esterase, and solid-phase extraction. Isotopic analysis of the pentafluoropropionyl derivative of vitamin K(1) is performed by gas chromatography/mass spectrometry (GC/MS). The limit of quantitation is equivalent to at least 0.3 nmol/L and the method is demonstrated to be linear over a range of enrichments. This method provides a robust alternative to previous work requiring the use of semi-preparative high-performance liquid chromatography (HPLC).

Measurement of gastric emptying by the 13C-octanoate breath test--rationalization with scintigraphy.

The (13)C-octanoate breath test has not achieved universal acceptance for the measurement of solid phase gastric emptying, largely because the results are not comparable with those from direct methods such as scintigraphy. To convert breath-test data to their scintigraphic equivalent scale corrections are applied which have been obtained from population studies, but there is no guarantee that these are applicable in all cases. We propose an alternative method applicable on an individual basis based upon a simple physiological model which does not require any change to the breath-test protocol. It is demonstrated by comparison with scintigraphy and the octanoate saliva test. Results from an existing dataset of simultaneous breath test, saliva test and scintigraphic determinations of solid phase gastric emptying from nine healthy subjects were re-analysed. The corrected breath tests gave gastric emptying curves which were shown to be not significantly different to those obtained from scintigraphy. The method provides a simple but effective way of generating gastric emptying curves from breath-test data that are directly comparable with direct measurement methods, which is advantageous since it allows the whole of the gastric emptying profile to be generated, not just values for the lag phase and half-emptying times.

A stable isotope minimal model protocol with oral glucose administration.

A protocol for investigating glucose metabolism whereby stable isotope tracer is given intravenously after an oral glucose challenge is described. Frequent sampling of plasma glucose and insulin allows the tracer disappearance to be interpreted on the basis of established minimal models. We have investigated the glucose effectiveness and insulin sensitivity parameters and their reproducibility in a group of six healthy adults, each studied twice. A mono-compartmental description of glucose distribution did not provide a physiological description of glucose kinetics, whereas a two-compartment model gave adequate results in every case. The estimates of glucose effectiveness and insulin sensitivity were 2-3 times higher than those obtained in similar populations using the conventional protocol of the frequently sampled intravenous glucose tolerance test, and this appeared to be related to the kinetics of transport of glucose from accessible to remote pools. The indices of insulin sensitivity obtained in this way were highly reproducible, with a between-test correlation of 93%.

13C- and 2H-labelled glucose compared for minimal model estimates of glucose metabolism in man.

In the present study, we have investigated the use of 1-[(13)C]glucose and GC/combustion/isotope-ratio MS as an alternative to 6,6-[(2)H(2)]glucose and GC/MS in the determination of parameters of glucose metabolism using the IVGTT (intravenous glucose tolerance test) interpreted by labelled (hot) minimal models. The study has been done in four populations, normoglycaemics (subdivided into lean and obese individuals), subjects with impaired glucose tolerance and those with diabetes mellitus. Although the use of carbon label may in some circumstances be compromised by substrate recycling, our hypothesis was that this would not be an issue under the condition of suppression of hepatic glucose production during the short timescale of an IVGTT. In all four groups, we found that the methodology employing the carbon label gave equivalent results to those obtained using the conventional deuterated material, but the sensitivity of the measurement technique in the new approach was sufficient to allow an approx. 15-fold reduction in the quantity of isotope administered. In addition to the clear cost advantages, this represents a significant scientific advance in that true tracer status is more nearly attained in these measurements with near-physiological tracee loads.

Measurement of gastric emptying by octanoate metabolism.

PURPOSE OF REVIEW: Since its introduction just over 10 years ago, there have been a number of studies that have used the octanoate breath test to assess gastric emptying. Although use of the method is on the increase (the number of gastric emptying studies published on PubMed using the octanoate breath test has doubled between the periods 1997-2000 and 2001-2004 compared with a drop of approximately 20% in the use of scintigraphy over the same periods), the methodology has not achieved universal acceptance, primarily because it can provide results comparable to established techniques only indirectly. RECENT FINDINGS: Recent methods for overcoming this difficulty are reviewed, including modified methods for breath test interpretation and the application of the related saliva test. The latter promises to be useful as a non-invasive proxy for established techniques, such as scintigraphy, for further validation of the breath test. Recent applications of octanoate-based methods are briefly considered. SUMMARY: A novel approach detailed in this review for breath test interpretation, where the bicarbonate pool is modelled as a single compartment, could prove useful for obtaining breath test gastric emptying parameters that are directly comparable with those obtained from the gold standard, gamma scintigraphy. In combination with the saliva test, this could add credence to use of the octanoate breath test as a clinically accepted diagnostic tool, in addition to its potential in research.

The 'anomalous' absorption of labelled and unlabelled vitamin C in man.

Previous studies of vitamin C absorption in man using stable isotope probes have given results which cannot easily be reconciled with those obtained using non-isotope measurement. In order to investigate some of the apparent paradoxes we have conducted a study using two consecutive doses of vitamin C, one labelled and one unlabelled, given 90 min apart. Compatibility of the experimental results with two feasible models was investigated. In Model 1, ingested vitamin C enters a pre-existing pool before absorption, which occurs only when a threshold is exceeded; in Model 2, ingested vitamin C is exchanged with a pre-existing flux before absorption. The key difference between these two models lies in the predicted profile of labelled material in plasma. Model 1 predicts that the second unlabelled dose will produce a secondary release of labelled vitamin C which will not be observed on the basis of Model 2. In all subjects Model 1 failed to predict the observed plasma concentration profiles for labelled and unlabelled vitamin C, but Model 2 fitted the experimental observations. We speculate on possible physiological explanations for this behaviour, but from the limited information available cannot unequivocally confirm the model structure by identifying the source of the supposed flux.

Stable isotope-labelled vitamin C as a probe for vitamin C absorption by human subjects.

Factors affecting absorption of physiological doses of vitamin C in man have not been widely studied, partly because few suitable tools exist to distinguish recently absorbed vitamin C from endogenous vitamin. Stable isotope-labelled vitamin C provides such a tool. Fifteen healthy non-smoking subjects aged 26-59 years were studied. Each received 30 mg l-[1-(13)C]ascorbic acid orally on two occasions, 3-4 weeks apart. The ascorbate was given alone or with Fe (100 mg as ferrous fumarate) or with red grape juice, which is rich in polyphenols. Blood was collected at frequent intervals for 1 h, and then each hour for a further 3 h. Total concentration of vitamin C was measured fluorometrically and its (13)C-isotope enrichment was measured by GC-MS after conversion to volatile trimethylsilyl esters. Peak plasma enrichment occurred within 25-50 min. No kinetic variables were significantly altered by the iron fumarate supplement. Grape juice attenuated vitamin C absorption, reaching significance at the 20 min time point. There were weak correlations between isotope enrichment and body weight or endogenous ascorbate concentration. The increment in total plasma ascorbate was smaller if calculated from isotope enrichment than from vitamin C concentration increase. The dilution pool was much larger than the plasma ascorbate pool. Further studies are needed to resolve these paradoxes. Stable isotope-labelled ascorbate is potentially useful for measurement of vitamin C absorption by human subjects.

Use of isotopically labelled octanoic acid to assess the effect of meal size on gastric emptying.

It has been proposed that the (13)C-octanoic acid breath test (OBT) provides a safe, non-radioactive means of measuring gastric emptying. However, deuterated octanoic acid provides a better marker when compared with scintigraphy, as the kinetics are less complex than those of the (13)C label. The appearance of (2)H in saliva is modelled as a two-compartment body water system, using an asymmetric triangular gastric emptying function. This study compared the (2)H-octanoic acid saliva test (OST) with the OBT in measuring altered states of gastric emptying in the nutritional context of diet manipulation. Gastric emptying was measured using the OST and OBT in a three-way crossover study involving 12 healthy male and female subjects (mean BMI = 23.4 kg/m(2), aged 24-57 years). Following an overnight fast, subjects were given an egg meal, labelled with 10 microL/kg body weight (2)H-octanoic acid and 100 microL (13)C-octanoic acid. The meal was nutritionally manipulated to provide a 1 MJ, 2 MJ or 3 MJ meal. Breath and saliva samples were collected at regular intervals for 6 h, with further saliva samples being collected over four subsequent days. (2)H isotopic enrichment in saliva and (13)C isotopic enrichment in breath were analysed using isotope ratio mass spectrometry and the data fitted to the respective gastric emptying models. The half excretion time (T(1/2) (D)), time to maximum emptying rate (T(1) (D)) and time when emptying is complete (T(2) (D)) were calculated from the (2)H saliva test data, and the lag time (T(lag) (C)), half excretion time (T(1/2) (C)), latency phase (T(lat) (C)) and ascension time (T(asc) (C)) were calculated from the (13)C breath test data. Overall, the OBT correlated well with the OST, with a significant relationship between T(1/2) (C) and T(1/2) (D), a significant relationship between T(lat) (C) and T(1) (D) and finally a significant relationship between T(asc) (C) and T(2) (D). Gastric emptying measured using the OST was significantly faster with the 1 MJ meal (DeltaT(1/2) (D) = -0.77 h vs. 2 MJ, p = 0.004). Increases were also seen when the meal size was increased from 2 MJ to 3 MJ (DeltaT(1/2) (D) = +0.44 h vs. 2 MJ), but these were not significant. These trends were mirrored in the OBT data, with significant differences between 1 MJ and 2 MJ (DeltaT(1/2) (C) = -0.63 h vs. 2 MJ, p = 0.013) and non-significant increases with the larger 3 MJ meal (DeltaT(1/2) (C) = +0.10 h vs. 2 MJ). Total meal calorie content was shown to have an effect on gastric emptying using both the OBT and the OST. The deuterium method allows the direct calculation of the gastric emptying function and could be used as an alternative to gamma scintigraphy, allowing further validation of the (13)C-octanoic acid breath test.

Using a non-invasive stable isotope tracer to measure the absorption of water in humans.

The development of solutions that prevent dehydration or promote adequate re-hydration play a vital role in preventing fatigue during exercise, however, the methods commonly used to assess the hydration ability of such solutions are invasive and often assess the components of absorption separately. This paper describes using a non-invasive deuterium tracer technique that assesses gastric emptying and intestinal absorption simultaneously to evaluate the uptake of water during rest and exercise. The kinetics of absorption are further examined by mathematical modelling of the data generated. For the rest group, 0.05 g/kg of body weight of deuterium, contained in gelatine capsules, was ingested with ordinary tap water and saliva samples were collected every 5 min for one hour while the subject remained seated. The deuterium was administered as above for the exercise group but sample collection was during one hour of exercise on a treadmill at 55% of the subject's maximum heart rate. The enrichment data for each subject were mathematically modelled and the parameters obtained were compared across groups using an independent samples t-test. Compared with the rest condition, the exercise group showed delayed absorption of water as indicated by significant differences for the modelling parameters t2, t1/2, maximum absorption rate and solution absorption amount at t1. Labelling with a deuterium tracer is a good measure of the relative rate ingested fluids are absorbed by the body. Mathematical modelling of the data generates rates of maximum absorption and allows calculation of the percentage of the solution that is absorbed at any given time during the testing period.

Glycogenesis and glucose oxidation during an intravenous glucose tolerance test in man.

The quantity of deuterated glucose customarily given in labelled IVGTTs (intravenous glucose tolerance tests) changes the isotopic composition of the subject's body water enough to be detected by mass spectrometric techniques. Glucose undergoing direct glycogenesis does not contribute label to the body water pool, and isotope incorporated into it must have come from glucose that has either been oxidized or undergone indirect glycogenesis. By subtracting the amount of label found in body water from the total amount of glucose utilized, as calculated from the minimal model of glucose disappearance, it should be possible to study the partitioning of the dose given between direct glycogenesis in skeletal muscle and other metabolic pathways. To establish these principles, we used isotope ratio MS to determine body water composition in groups of healthy ( n =7; mean weight, 76 kg; fasting plasma glucose and insulin, 5.1 mmol and 40 pmol respectively) and Type II diabetic ( n =5; mean weight, 84 kg; fasting plasma glucose and insulin, 6.2 mmol and 75 pmol respectively) subjects undergoing IVGTTs. It was found that, for healthy subjects, 31% of the dose given was utilized in direct glycogenesis and this was decreased to 15% in diabetes. Defects in muscle glycogen synthesis in diabetes of the same order are well known from magnetic resonance studies. We conclude that measurement of label incorporation into body water is potentially useful for investigation of the metabolism of a glucose load in vivo during an IVGTT.