A non-radioactive method for measuring Cu uptake in HepG2 cells.

At present, all data on Cu uptake and metabolism have been derived from radioactive uptake experiments. These experiments are limited by the availability of the radioactive isotopes 64Cu or 67Cu, and their short half-life (12.5 and 62 h, respectively). In this paper, we investigate an alternative method to study the uptake of Cu with natural isotopes in HepG2 cells, a liver cell line used extensively to study Cu metabolism. In nature, Cu occurs as two stable isotopes, 63Cu and 65Cu (63Cu/65Cu = 2.23). This ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP-MS). In initial experiments, we attempted to measure the time course of Cu uptake using 65Cu. The change in the 63Cu/65Cu ratio, however, was too small to allow measurement of Cu uptake by the cells. To overcome this difficulty, the natural 63Cu/65Cu ratio in HepG2 cells was altered using long-term incubation with 63Cu. This had a significant effect on Cu concentration in HepG2 cells, changing it from 81.9 +/- 9.46 pmol microg DNA(-1) (week 1) to 155 +/- 8.63 pmol microg DNA(-1) (week 2) and stabilising at 171 +/- 4.82 pmol microg DNA(-1) (week 3). After three weeks of culture with 2 microM 63Cu the 63Cu/65Cu changed from 2.18 +/- 0.05 to 15.3 +/- 1.01. Cu uptake was then investigated as before using 65Cu. Uptake was linear over 60 min, temperature dependent and consistent with previous kinetics data. These observations suggest that stable isotope ICP-MS provides an alternative technique for the study of Cu uptake by HepG2 cells.

A high oat-bran intake does not impair zinc absorption in humans when added to a low-fiber animal protein-based diet.

Oat bran has a high phytate content and a low or inactivated phytase activity. A high intake of oat bran could therefore result in an impaired absorption of trace elements. The effect of a mean daily intake of 142 g of oat bran (102 g/10 MJ) on absorption of zinc was evaluated by the use of stable isotopes and fecal monitoring in 12 healthy subjects (6 males and 6 females). Each subject participated in two separate diet periods each of 21 d with identical low-fiber diets and with oat bran added in one of the periods. The oat bran was incorporated into bread and served at three daily main meals. The intake of zinc and phytate per 10 MJ was 138 micromol (9.0 mg) and 0.5 mmol, respectively, in the low-fiber period and 225 micromol (14.7 mg) and 4.0 mmol, respectively, in the oat bran period. Stable isotopes of zinc ((70)Zn) were added to the diets at d 7 of each period. The fractional absorptions (means +/- SD) of zinc from the low-fiber and oat bran diets were 0.48 +/- 0.11 and 0.40 +/- 0.15 (P = 0.07), respectively. The higher zinc content in the oat bran period resulted in a greater amount of zinc absorbed (64 +/- 19 micromol and 99 +/- 51 micromol, respectively, P = 0.009). Balance data suggest that the higher absorbed amount of zinc resulted in correspondingly higher intestinal endogenous excretion of zinc. In conclusion, the absorption of zinc was high and not affected by addition of oat bran.

Stable isotope dilution-mass spectrometry for determining total selenium levels in plants, soils and sewage sludges.

Quantitation of selenium in plants, soils and sludges was achieved by isotope dilution-mass spectrometry using a benchtop instrument. Samples for analysis were spiked with (76)Se isotope solution. Plant material was digested on a heating block at 150 degrees C using a mixture of nitric acid and hydrogen peroxide. Selenium in soils and sludges was released by treatment with nitric acid followed by digestion with nitric and hydrofluoric acids. Selenium in the digests was reduced to Se(IV) with hydrochloric acid and derivatised with nitro-1, 2-phenylenediamine to 5'-nitropiazselenol. Analysis by gas chromatography-mass spectrometry using selected ion monitoring was validated using certified reference materials (CRMs) and gave results within the certified range with a low standard deviation. The CRMs plant (Chinese cabbage leaves) and soil (Chinese soil) were found to contain (+/-95% confidence limits) 0.091(+/-0.007) mug g(-1) and 1.67(+/- 0.04) mug g(-1)Se respectively. The certified values were 0.083(+/-0.008) mug g(-1) and 1.56(+/-0.12) mug g(-1) respectively. The selenium content of four different freely drained acid Scottish soils under grasslands was in the range 0.5-0.8 mug g(-1) air-dried soil. Sewage sludges were found to contain measurably more selenium than the soils, and samples of three sludges taken from sites in the UK contained between 1.1 and 3.5 mug g(-1) dry matter.

Use of a stable copper isotope (65Cu) in the differential diagnosis of Wilson's disease.

1. 65Cu/63Cu stable-isotope ratios have been measured in blood serum after oral administration of the stable isotope 65Cu. The incorporation of the isotope into the plasma protein pool was followed at various times for up to 3 days. The resulting patterns of enrichment in healthy control subjects, in Wilson's disease patients and in heterozygotes for the Wilson's disease gene, were similar in appearance to those found by others using copper radioactive isotopes. After an initially high enrichment at 2 h after dosage, the Wilson's disease cases, in contrast to the control subjects, did not show a secondary rise in isotope enrichment of the plasma pool after 72 h, demonstrating a failure to incorporate copper into caeruloplasmin. The Wilson's disease heterozygotes had variable degrees of impairment of isotope incorporation, not always distinguished from those of control subjects. 2. The stability of the isotope also permits the copper tracer to be followed for a longer period. Ten healthy subjects were studied for over 40 days, allowing the biological half-time of an oral dose of copper to be determined (median 18.5 days, 95% confidence interval 14-26 days). Known heterozygotes for the Wilson's disease gene were found to have a significantly increased biological half-time for removal of copper from the plasma pool (median 43 days, 95% confidence interval 32-77 days). 3. The incorporation of 65 Cu in patients with diseases of the liver (other than Wilson's disease) was found to be similar to that in control subjects, aiding differential diagnosis.

The comparison of capillary zone electrophoresis and atomic spectroscopy for the determination of the cation content of a standard reference material IAEA-A-11 milk powder.

Alkali and alkaline earth metals were separated and quantified by Capillary Zone Electrophoresis using a previously developed method. CZE combined with indirect detection has received considerable attention over the last few years. Methods for the determination of inorganic and organic cations and anions have been reported although few of these include quantitative application to real samples. All of these methods are characterized by high efficiencies and high capacity. Application of the method to the determination of the major cation content of an International Atomic Energy Agency (IAEA) standard reference material is described. Complete resolution and quantitation of the ions (Na(+), K(+), Ca(2+), Mg(2+)) was achieved with calibration curves, for the individual components, having correlation coefficients (r(2)) ranging from 0.996 to 0.999 and detection limits (two times the baseline noise) of 10 ppb for potassium and magnesium and of 2.5 ppb for sodium and 2 ppb for calcium were achieved. Comparable results were achieved when employing analysis by Atomic Spectroscopy. The accuracy of this method was tested by comparison with standard flame Atomic Absorption cation analysis. Statistical analysis of the instrumental results indicate that there is no evidence suggesting systematic differences between the methods. In addition, a number of potential advantages of CZE for cation analysis are discussed.

Validation of the doubly labeled water method in growing pigs.

The CO2 production (rCO2) of eight growing pigs was determined by continuous collection of CO2 over 21 days and simultaneously estimated using the doubly labeled water (DLW) method. The aim was to assess the accuracy of the method before and after correction for known sources of error and to test for any residual discrepancy arising from as yet unidentified sources of error. Mass spectrometer accuracy was verified by analyzing serial dilutions of the dose material in the form of an artificial decay curve; no significant bias was detected. The physiological errors were linearly dependent on weight gain. DLW-derived rCO2 (corrected only for fractionated water loss) underestimated the true value by 0.270 l CO2/g wt gain or -8% in the restricted (group R) and -16% in the ad libitum-fed (group AL) groups. Known sources of error accounted for -0.006 (methane), -0.032 (fecal 2H losses), -0.108 (fat synthesis), and -0.146 (changing pool size) l CO2/g wt gain. After correction for these sources of error the DLW-derived rCO2 differed from the true value by -2 +/- 3% in group R and 0 +/- 3% in group AL. Thus there was no significant bias in the DLW method after correction for known sources of error, even during rapid weight gain or at weight stability with or without correction. The precision estimates include both dose and background errors and uncertainty in the correction factors used. Strategies for optimizing precision are presented.

Mass spectrometric methods for studying nutrient mineral and trace element absorption and metabolism in humans using stable isotopes. A review.

Mass spectrometric methods for determining stable isotopes of nutrient minerals and trace elements in human metabolic studies are described and discussed. The advantages and disadvantages of the techniques of electron ionization, fast atom bombardment, thermal ionization, and inductively coupled plasma and gas chromatography mass spectrometry are evaluated with reference to their accuracy, precision, sensitivity, and convenience, and the demands of human nutrition research. Examples of specific applications are described and the significance of current developments in mass spectrometry are discussed with reference to present and probable future research needs.

Validation in sheep of the doubly labeled water method for estimating CO2 production.

Carbon dioxide production (rCO2) was estimated in four sheep over a period of 10 days using doubly labeled water (2H and 18O) and was compared with simultaneous respiration chamber measurements of CO2. The excess 2H and 18O measurements were corrected for the empirically determined effects of isotope rebreathing within the confines of the chambers. A weighted monoexponential curve was then fitted to the data from which isotope flux rates and ultimately rCO2 and water turnover (rH2O) estimates were made. The curve fits were weighted assuming a Poisson model. Selection of this weighting policy did not bias the results, and curvature in the data also appeared to have little effect on the rCO2 estimates. Fractionated evaporative water loss expressed as a fraction of rH2O (X) was estimated from water balance and breath water production estimates; the mean X was 0.145 and ranged from 0.108 to 0.183. Corrections for 2H loss in fecal solids reduced the mean rH2O (4,746 g/day) by 35.5 g/day and increased the mean rCO2 (332.3 l/day) by 21.2 l/day. Further corrections to account for 2H loss in methane (mean production rate 27.2 l/day) reduced rH2O by 33.8 g/day and increased rCO2 by 20.3 l/day. The final isotopic estimates of rH2O were 14.6 +/- 6.59% (n = 4) lower than direct measurements and the mean rCO2 was 3.5 +/- 14.48% (n = 4) lower than the chamber measured rCO2. However, in one of the animals studied the rCO2 deviated markedly from the chamber-derived value, and this discrepancy has yet to be explained. When this animal was excluded from the comparisons, the standard deviation was greatly reduced (+/- 3.6, n = 3) and the mean overall error on rCO2 was +3.6%.

Molybdenum-induced changes in the epiphyseal growth plate.

Molybdenum (Mo), at high concentrations, induces changes in the epiphyseal growth plate through its effects on copper (Cu) metabolism but it is unclear whether or not Mo can induce changes independent of its effects on copper status. To this end, the effect of Mo on longitudinal bone growth was examined in rats. Dietary Mo was given either as ammonium heptamolybdate or as ammonium tetrathiomolybdate, the latter producing a marked Cu deficiency. There was a significant reduction in longitudinal bone growth in both groups; however, growth plate width was increased only in the Cu-deficient animals due to an increase in the width of the zone of transitional/hypertrophic chondrocytes. Both glucose 6-phosphate dehydrogenase activity and cell proliferation (assessed by bromodeoxyuridine incorporation) were markedly decreased in the proliferating zone of the growth plate in both Mo-treated groups. These changes were not apparently related to changes in circulating vitamin D metabolites or insulin-like growth factor-1. The results indicate that excess Mo impairs cell proliferation within the growth plate, whereas the effects of copper deficiency are more related to chondrocyte differentiation. Thus, Mo can induce changes in longitudinal bone growth which are distinct from those resulting from Cu deficiency.

The doubly labeled water method: errors due to deuterium exchange and sequestration in ruminants.

The doubly labeled water (DLW) technique allows the CO2 production (rCO2) of free living animals to be estimated from the difference between the turnover of 2H2O and H218O in the body water. A fundamental assumption of this technique is that neither of the isotopes used are lost in products other than CO2 and H2O. We found, however, that 2H was lost in both exchangeable and nonexchangeable positions in the feces of sheep. Negligible amounts of 18O were lost in exchangeable positions. 2H losses led to a 0.75% (SE 0.06, n = 4) overestimation of the measured 2H2O flux, leading to an average error in rCO2 estimates of 20.3 l/day. For a typical rCO2 rate of 370 l/day, this would amount to an error of approximately 5% (range -7.0 to -4.3%, n = 4). Correction factors to account for this loss were presented. The error in rCO2 due to 2H sequestration into fat was calculated to be at most 2.1 l/day or about -0.66% in lambs with a rCO2 of 320 l/day. In a triply labeled water (TLW) study the maximum error in the estimation of fractionated evaporative water loss (X) would lead to a 0.81% underestimation of rCO2. We recommend that during a DLW study involving ruminant animals the correction factors presented here be used to compensate for 2H loss in feces. This may be particularly important where the diet has a high roughage content leading to a significant fecal dry matter production.

Turnover rates of different collagen types measured by isotope ratio mass spectrometry.

The rates of collagen turnover in different tissues have been estimated in growing rats previously exposed to gaseous 18O2. The abundance of the stable isotope was measured using isotope ratio mass spectrometry following combustion of isolated collagen-derived hydroxyproline. Using this method, problems of label reutilization associated with radiolabelling methods are avoided. In general the results confirm the slow turnover rates with half-lives of total collagen in skin, muscle and gut of 74, 45 and 244 d, respectively. The use of cyanogen bromide digests of whole tissues followed by isolation of collagen type-specific peptides has allowed the comparison of turnover rates of collagen types I and III, indicating that collagen type III is turned over more rapidly than type I.

Water hydrogen incorporation into body fat in pigs: effect on double/triple-labeled water method.

A basic assumption of the doubly labeled water (DLW) and triply labeled water (TLW) methods for measuring water flux (rH2O), CO2 production (rCO2), and fractionated water loss (X) is that the H of body water only leaves the body as water. Any loss of isotopes in other products will introduce an error into these techniques. The body fat represents the largest potential sink for water H. 2H sequestration into the carcass fatty acids was investigated in eight pigs labeled with 2H2O for 21 days. rCO2 was measured simultaneously in respiration chambers to allow an accurate assessment of the effect of 2H sequestration on the estimated rCO2. The fat content of the diet (1.63%), level of intake, and stage of maturity were all designed to give the widest possible range of sequestration effects. Four animals were restricted to their estimated maintenance requirement and four were allowed to feed ad libitum giving a range of weight gain from 100 to 650 g/day. This was reflected in the estimated error on rH2O (+0.42% in the restricted group and +2.52% in the fast-growing animals) and on rCO2 (-1.30 and -7.59%, respectively). The error on the calculation of X using TLW was +0.03 units in the restricted group and +0.20 units in the fast-growing animals. The error of +0.2 on X propagates through to an underestimate of rCO2 of approximately 4%, and since this is additive with the error on DLW the ultimate error on rCO2 using TLW would be approximately -12%.(ABSTRACT TRUNCATED AT 250 WORDS)

Body fat in lean and overweight women estimated by six methods.

Body fat content of seven lean women (body mass index (BMI) 20.6 (SD 1.8) kg/m2) and seven overweight women (BMI 31.1 (SD 3.3) kg/m2) was estimated by six different methods: underwater weighing (UWW), body-water dilution (BWD), whole-body counting (40K), skinfold thickness (SFT), bioelectrical impedance (BEI) and magnetic resonance imaging (MRI). Using UWW as the reference method, the differences between percentage fat by each other method and the percentage fat by UWW were calculated for each subject. The mean difference was lowest for SFT and highest for BWD. MRI showed the lowest variability in individual results, and 40K the highest. 40K and BWD methods used in combination gave better agreement with UWW results than either 40K or BWD methods alone. There was a weak negative correlation between the difference from the UWW results and percentage fat in the SFT measurements, but not in the BWD, 40K, BEI or MRI measurements, suggesting that for these methods the assumptions involved produced no greater inaccuracy in the overweight women than in the lean women. In all subjects the BEI offered little improvement over the traditional SFT measurements. The agreement between MRI and UWW estimates in both lean and overweight women suggests that MRI may be a satisfactory substitute for the more established methods of body fat estimation in adult women.

Nutrient oxidation patterns and protein metabolism in lean and obese subjects.

The immediate metabolic response to eating has been compared in a group of grossly obese subjects (W/H2 = 45) with that in lean controls (W/H2 = 22). Dietary intake of energy for obese subjects was based on their estimated basal energy expenditure for ideal body weight (given at an hourly rate of 3 X BMR over a 4-h period). Lean subjects were measured twice: control 1 with the same intake of energy as the obese in terms of ideal body weight and control 2 with the same energy intake in relation to each subject's measured resting energy expenditure (2.2 X REE). The changes in energy expenditure and nutrient disposal with the onset of eating have been assessed by a method of combined respiratory gas analysis and intravenous infusion of 13C-labelled leucine. Leucine kinetics were used to quantitate rapid changes in protein oxidation and to assess protein synthesis and degradation. 1) Total energy expenditure was 20-30 per cent greater in obese subjects than lean subjects in fasting and feeding. Energy expenditure expressed per kg fat-free mass, from D2O dilution, was similar in obese and lean subjects in both fasting (5.8 v. 5.5 kJ/kg FFM/h) and feeding [6.7 v. 6.3 (Control 2) kJ/kg FFM/h]. 2) The onset of eating was associated with increased carbohydrate and protein oxidation with decreased fat oxidation in both lean and obese individuals. In obese subjects, however, both the decrease in fat oxidation and the increase in protein oxidation were significantly smaller (P less than 0.05) than the corresponding increments in lean subjects (Control 2). 3) The rate of protein synthesis was significantly (P less than 0.05) higher in obese subjects both in the fasting state (99 v. 84 mumols leucine/kg FFM/h) and in the fed state [94 v. 67 (Control 2) mumols leucine/kg FFM/h]. The rate of protein degradation was also higher in obese individuals in fasting (117 +/- 6 v. 106 +/- 4 mumol leucine/kg FFM/h) and feeding [65 +/- 4 v. 54 +/- 6 (Control 2) mumol leucine/kg FFM/h] though these differences are not statistically significant (P greater than 0.05). 4) The observed differences between obese and lean individuals in protein and energy metabolism in the fasted state and in the immediate response to eating do not support a hypothesis of greater metabolic efficiency in obesity.

Effects of exercise intensity on absorption of ingested fluids in man.

Plasma 2H accumulation was measured in six male volunteers after ingestion of drinks containing trace amounts of 2H2O. Subjects fasted overnight and remained seated at rest or exercised on a cycle ergometer at 42, 61 or 80% of their maximum oxygen uptake (VO2, max). The rate of plasma 2H accumulation was faster at rest than during exercise at 61 or 80% of VO2, max (P less than 0.05), and was faster at 42 and 61% than at 80% of VO2, max (P less than 0.05). The time to peak plasma 2H concentration was longer during exercise than at rest. This suggests that strenuous exercise may reduce the availability of fluid ingested during exercise.

Methane production in ruminants: its effect on the doubly labeled water method.

The doubly labeled water (DLW) technique for measuring CO2 production (rCO2) in free-living animals requires an assessment of the elimination of both 2H and 18O from the body over a long period of time. To calculate rCO2, it is necessary to calculate water turnover (rH2O) from the 2H flux rate. In ruminant animals, the accuracy of this calculation is affected by the loss of 2H in methane. We have quantified the effect of methane production (rCH4) on the 2H flux rate, determined in four sheep given 2H2O. The methane produced was depleted in 2H relative to the urine. A relationship between the enrichment of the methane and urine was established. The ratio of urine to methane enrichment was found on average to be 0.6536, and this value was unaffected by the level of rCH4 but showed some dependence on the absolute concentration of 2H in urine. For this reason, the ratio value obtained from four sheep not given 2H2O was different, a mean of 0.6886 was measured, this ratio was unaffected by changes in the diet supplied to the animals. Computer modeling was used to illustrate the dependence of the isotopically derived value for rCO2 on not only rCH4 but also the magnitude of rCO2 itself. The effect of rCH4 on the DLW method can be predicted from the observed ratio of rCO2 to rCH4 and the value of 0.6536 obtained for the ratio of methane to urine enrichment. With the use of data from several studies at this Institute, a limited range of 10 to 20 was found for rCO2/rCH4 in animals fed at or above maintenance.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of fractionated water loss and CO2 production using triply labelled water.

The doubly labelled water (DLW) method for measuring CO2 production has recently been the subject of much interest since no other technique gives integrated values for CO2 production over long periods by free-living subjects. The importance of evaporative water loss and fractionation factors to the calculation of CO2 production using this technique is described. Present methods of estimating evaporative water loss and the use of fractionation factors are summarized together with a discussion of their limitations. A novel technique is proposed whereby water labelled with three isotopes can be used to measure evaporative water loss and CO2 production in completely free-living subjects, and the feasibility of the method is tested in simulations using experimental data. This technique has three advantages over existing methods of estimating evaporative water loss: (1) it can be used in completely free-living subjects without any additional experimental procedures (e.g. water-balance studies or physical trapping of water vapour); (2) it gives a direct estimate of fractionated evaporative water loss in each subject, since non-fractionated water lost as vapour is automatically compensated for; and (3) the routes of water loss do not have to be known. The appropriate calculations are presented together with a discussion of the difficulties of measuring oxygen-17 by mass spectrometry. It is estimated that the maximum theoretical error on calculated CO2 production is +/- 0.3%. Practical ways of achieving this theoretical level of accuracy are suggested. We conclude that the proposed technique will allow correction for evaporative water loss to be made more exactly, thereby increasing the accuracy of the heavy water technique for measuring CO2 production in free-living subjects.

The applicability of evacuated serological tubes for the collection of breath for isotopic analysis of CO2 by isotope ratio mass spectrometry.

Stable isotope ratio mass spectrometry was used for the analysis of 13C and 18O in CO2 of human breath collected in evacuated serological tubes (Vacutainer and Venoject) and in gas sample bottles. There was a 1-2% difference between the delta 13 CPDB values obtained from breath CO2 collected in gas bottles and that collected in two batches of Vacutainer. delta 18OPDB differed between Vacutainers and gas bottles by as much as 14% and standard deviations within the two batches of Vacutainer were 0.7 and 1.8%. The poor reproducibility and accuracy for the delta 18OPDB values was caused by the presence of a contaminant which originated from the rubber septa of the Vacutainers. The results show that it is not possible to obtain delta 18OPDB values with a high degree of accuracy or precision for breath samples collected in Vacutainers. However, with selection of less contaminated or re-evacuated batches, delta 13CPDB analysis of breath CO2 in Vacutainers may provide acceptable accuracy and precision. delta 13CPDB and delta 18OPDB values measured in breath collected in non-sterile Venoject tubes were also significantly different from those obtained with gas bottles. However, the accuracy and precision of these determinations were considerably better than in sterilized Vacutainer tubes. As a result of these comparisons it is concluded that gas bottles are necessary for the full accuracy and precision of stable isotope ratio analysis mass spectrometers to be exploited in breath CO2 analysis. However, automated analysis of breath CO2 collected in non-sterile Venoject tubes will provide an accuracy and precision that is suitable for most biological studies.