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.

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.

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.

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.

Carbohydrate oxidation: do we eat ourselves or our food?

In post-absorptive man, energy is derived solely from oxidation of body stores, mainly by oxidation of fat rather than glycogen. Eating changes this pattern so that carbohydrate (CHO) oxidation predominates. If during feeding energy intake exceeds energy expenditure, the energy needs of the whole body can in theory be met entirely from the diet. However, it is not clear whether the CHO utilised in the fed state does come directly from the absorbed diet, or whether some continues to be removed from body stores.

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.

Cytokinin oxidase fromZea mays kernels andVinca rosea crown-gall tissue.

Cytokinin oxidase has been partially purified from matureZea mays kernels and fromVinca rosea corwn-gall tissue. The molecular weights of the two enzymes, determined by gel filtration, are very different: 94,400 (±10%) forZ. mays and 25,100 (±10%) forV. rosea. Specificity studies have been performed using a large number of synthetic and naturally occurring cytokinins. Only a small number of these compounds serve as substrates and both enzymes exhibit similar substrate specificity. In agreement with other workers, a Δ(2) double bond in the N(6) side chain is essential for activity. The presence of glucosyl or ribosyl groups in the 7-or 9-position or an alanyl group in the 9-position of the purine moiety have little effect on their susceptibility to cytokinin oxidase, but O-glucosyl derivatives are resistant to oxidation. The relevance of these enzyme systems to studies on cytokinin metabolism and to the endogenous cytokinins is discussed.

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.

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.

Zeatin-9-glucoside, a major endogenous cytokinin of Vinca rosea L. crown gall tissue.

Cultured crown gall tissue of Vinca rosea L. was found to contain, in addition to the previously reported cytokinins zeatin, zeatin riboside, and the 0-glucosides of these two compounds, relatively high levels of zeatin-9-D-glucopyranoside. This is the first conclusive identification of an endogenous cytokinin 9-glucoside.

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.

Selected ion monitoring/isotope dilution mass spectrometric determination of 1-aminocyclopropane-1-carboxylic acid levels in ripening tomato fruit.

A new method is described for the quantitation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene in plants. [2,2,3,3,-2H4]ACC has been synthesized and used as an internal standard for selected ion monitoring/isotope dilution quantitation of this compound in ripening tomato fruit. These data are compared with those derived from the widely used indirect oxidative ACC assay (which underestimated the ACC levels by between two- and fourfold). The greater accuracy, sensitivity (100X), and specificity of the mass spectrometric method will be of considerable benefit to those interested in factors which control ACC and ultimately ethylene levels since it is believed that ACC synthesis and its oxidative metabolism to ethylene are the key points at which ethylene biosynthesis is regulated.

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)

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)

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.

Mass-spectrometric quantitation of cytokinins in tobacco crown-gall tumours induced by mutated octopine Ti plasmids of Agrobacterium tumefaciens.

The levels of the major cytokinins, zeatin, zeatin riboside, zeatin riboside-5'-monophosphate and zeatin-7-glucoside were measured in tobacco (Nicotiana tabacum L.) crown-gall tissues carrying insertion and deletion mutations in the T-DNA. Measurements were made by combined gas chromatography-mass spectrometry using selected ion monitoring with (15)N- and (2)H-labelled internal standards. The results demonstrate that, relative to wild-type tumour tissue, cytokinin levels are considerably elevated in tissues lacking functional T-DNA auxin-biosynthetic genes. From a detailed analysis of the major cytokinin metabolites it is concluded that a reduction in the extent of cytokinin degradation via N(6)-side-chain cleavage is an important factor leading to increased cytokinin levels in these tissues.

Apical localization of 1-aminocyclopropane-1-carboxylic acid and its conversion to ethylene in etiolated pea seedlings.

The biosynthetic basis for the high rates of ethylene production by the apical region of etiolated pea (Pisum sativum L.) seedlings was investigated. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was quantified in extracts of various regions of seedlings by measuring isotopic dilution of a (2)H-labelled internal standard using selected-ion-monitoring gas chromatography/mass spectrometry. The ACC levels in the apical hook and leaves were much higher than in the expanded internodes of the epicotyl. The capacity of excised tissue sections to convert exogenous ACC to ethylene was also much greater in the apical region, reflecting the distribution of soluble protein in the epicotyl.