Isotope Dilution Studies Research Articles

A nitrogen‐15 isotope dilution study of ammonium production and consumption in a marsh sediment1

Gross rates of ammonium production and consumption by microbes were estimated from isotope dilution of extractable 15NH4+ in surface sediment samples from a tidal freshwater marsh in Massachusetts. At 13°C gross ammonium production was 11.6 ± 2.9 nmol N · (cm3 fresh sediment)−1 · h−1. This rate equaled or exceeded gross ammonium consumption in the absence of plant root uptake [6.4 ± 4.0 nmol N ·( cm3 fresh sediment)−1 · h−1] so that ammonium accumulated linearly for 50–70 h in the sediment samples [5.2 ± 1.6 nmol N · (cm3 fresh sediment)−1 ·h−1]. In the field, this excess ammonium is presumably available for plant uptake. Ammonium production and consumption rates were highest in surface sediments and fell rapidly with depth. Temperature has a strong influence on both rates (Q10 = 2.6). Substrate quality and availability, both of which generally decrease with sediment age or depth, are probably more important than end‐product inhibition as rate‐controlling factors in these sediments. In particular, the relationship between ammonium production and consumption rates at different depths and sediment ligin content is clearer than between these rates and dry weight or ash‐free dry weight sediment.The annual, net rate of ammonium production in the top 10 cm of unmixed, surface sediments was estimated from short term ammonium accumulation rates to be about 1.7 mol N · m−2. However, net ammonium production severely overestimated the true turnover time of ammonium as estimated by isotope dilution (5 days). Most important, while isotope dilution of ammonium is a good indicator of ammonium turnover rates, it may be a poor indicator of total nitrogen turnover in sediments (and so of total microbial activity) because it is insensitive to the nitrogen that microbes assimilate directly from the solid substrates to which they adhere.

Possible evidence for novel metabolism of nitrogen gas by a green alga

We report the isolation of a cukaryotic green alga (Chlorella, strain WPI‐2) which accumulates large stores of nitrogen (N) during growth in N‐free medium and seems to incorporate14N2, yet does not reduce acetylene to ethylene. Total N accumulation during growth on N‐free medium and in gases free of combined N was measured by three methods: Kjeldahl, oxidative pyrolysis via chemiluminescence (Antek N analyzer), and Dumas (Coleman N analyzer). Increases in N ranging from 22–64%± 1% were observed. Isotope dilution studies using cells labelled with 15NO 3‐ and then shifted to 14N2 in N‐free medium showed dilution of the 15N isotope by 14N from 5.67 to 5.32%± 0.05%. Using a variety of conditions, we were unable to demonstrate the reduction of acctylene to ethylene by WPI‐2, although diazotrophic cyanobacteria gave positive results. Although the data on WPI‐2 are not conclusive in establishing this alga as a diazotroph, the data do suggest that within the Chlorophyceae there may exist a novel form of nitrogen gas metabolism.

Growth and gibberellin-A1 metabolism in normal and gibberellin-insensitive (Rht3) wheat (Triticum aestivum L.) seedlings

Growth parameters were determined for tall (rht3) and dwarf (Rht3) seedlings of wheat (Triticum aestivum L.). Plant statures and leaf length were reduced by 50% in dwarfs but root and shoot dry weights were less affected. Leaves of dwarf seedlings had shorter epidermal cells and the numbers of cells per rank in talls and dwarfs matched the observed relationships in overall length. Talls grew at twice the rate of dwarfs (2.3 compared with 1.2 mm h(-1)). [(3)H]Gibberellin A1 ([(3)H]GA1) was fed to seedlings via the third leaf and metabolism was followed over 12 h. Immature leaves of tall seedlings transferred radioactivity rapidly to compounds co-chromatographing with [(3)H]gibberellin A8 ([(3)H]GA8) and a conjugate of [(3)H]GA8, whereas leaves of dwarf seedlings metabolised [(3)H]GA1 more slowly. Roots of both genotypes produced [(3)H]GA8-like material at similar rates. Isotopic dilution studies indicated a reduced 2β-hydroxylation capacity in dwarfs, but parallel estimates of the endogenous GA pool size, obtained by radioimmunoassay, indicated a 12-15 times higher level of GA in the dwarf immature leaves. Dwarfing by the Rht3 gene does not appear to operate through enhanced, or abnormal metabolism of active gibberellins and the act of GA metabolism does not bear an obligate relationship to the growth response.

Formation of ursodeoxycholic acid from chenodeoxycholic acid in the human colon: studies of the role of 7-ketolithocholic acid as an intermediate.

The formation of ursodeoxycholic acid from chenodeoxycholic acid and the role of 7-ketolithocholic acid as an intermediate in this biotransformation were studied in vitro in fecal incubations as well as in vivo in the human colon. [24-14C]-Labeled 7-ketolithocholic and chenodeoxycholic acids were studied at various concentrations, and the biotransformation products were analyzed by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry. There was rapid colonic conversion of 7-ketolithocholic acid to ursodeoxycholic acid and, to a lesser extent, to chenodeoxycholic acid. The reduction of 7-ketolithocholic to ursodeoxycholic acid proceeded significantly faster anaerobically and at acid pH than under aerobic and alkaline conditions. When chenodeoxycholic acid was incubated in vitro or instilled into the colon, various amounts of 7-ketolithocholic and ursodeoxycholic acids were formed. The formation of 7-ketolithocholic acid was favored by alkaline conditions. Isotope dilution studies, in which trace amounts of labeled 7-ketolithocholic acid were incubated with unlabeled chenodeoxycholic acid, indicate 7-ketolithocholic acid to be the major intermediate in the intestinal bacterial conversion of chenodeoxycholic to ursodeoxycholic acid.

Spectroscopic studies of the ν 2 mode of methyl iodide by Raman spectroscopy

Polarized Raman studies of the ν 2 mode in methyl iodide at 1 and 1600 bar have confirmed, through an independent route, the results previously found by isotopic-dilution studies. By using a cubic-close-packed approximation of the liquid state and a 1/ r 6 interaction potential for dipolar dephasing between molecules, the data may be reconciled. This result is interpreted to be strong evidence for the dominance of the intermolecular transition-dipole dephasing mechanism.

ChemInform Abstract: AN INFRARED STUDY OF THE DYNAMICS OF WEAKLY BOUND WATER IN CRYSTALLINE HYDRATES

Isotopic dilution studies of water molecules in weakly bound crystalline hydrates reveal a strong dependence of the infrared spectra on temperature. The infrared stretching and bending bands of dilute HDO in NaClO4⋅H2O and LiI⋅3H2O show multiple bands at low temperature which broaden and move together as the temperature is raised. In contrast, only single narrow bands are seen in the very similar LiClO4⋅3H2O. The potential in which the waters move is calculated from a simple electrostatic model. The stretching frequencies are taken to be a function of the librational coordinate of the water and the observed frequencies are then averages over the libration. This simple prescription for calculating the observed bands is that given by Redfield theory. We conclude that the observed band collapse in NaClO4⋅H2O is due primarily to the increase with temperature of the in‐plane water libration. At higher temperatures, the 180° flipping motion also contributes to the collapse. For LiI⋅3H2O, the motion is a complex one involving locally ordered domains of the water molecules in the crystal.

Evidence that sabinene is an essential precursor of C(3)-oxygenated thujane monoterpenes

The volatile oil of immature Artemisia absinthium L. leaves contains sabinyl acetate (42%), 3-thujone (32%), sabinene (12%), and α-thujene (3%) as major constitutents, and label from the acyclic precursor [1- 3H]geraniol was incorporated, under aerobic conditions, into these thujane-type monoterpenes in proportion to their natural abundance in this tissue. Light had little effect on the synthesis of these monoterpenes from exogenous geraniol; however, at reduced oxygen levels, label from geraniol accumulated in the olefin sabinene while much less sabinyl acetate and 3-thujone were formed, suggesting a route to the ester and ketone by the allylic, nonphotochemical, oxygenation of sabinene. Supporting evidence for the intermediary role of the olefin was provided by isotopic dilution studies in which sabinene, but not α-thujene, blocked formation of the oxygenated derivatives from the labeled precursor. [10- 3H]Sabinene was incorporated directly as a substrate in A. absinthium leaves into both [10- 3H]sabinyl acetate and 3-[10- 3H]thujone. Furthermore, [ 3H]sabinene was specifically incorporated into 3-thujone in Tanacetum vulgare and into the diastereomeric ketone 3-isothujone in Salvia officinalis, confirming the role of this bicyclic olefin as the essential precursor of C(3)-oxygenated thujane monoterpenes.

An infrared study of the dynamics of weakly bound water in crystalline hydrates

Isotopic dilution studies of water molecules in weakly bound crystalline hydrates reveal a strong dependence of the infrared spectra on temperature. The infrared stretching and bending bands of dilute HDO in NaClO4⋅H2O and LiI⋅3H2O show multiple bands at low temperature which broaden and move together as the temperature is raised. In contrast, only single narrow bands are seen in the very similar LiClO4⋅3H2O. The potential in which the waters move is calculated from a simple electrostatic model. The stretching frequencies are taken to be a function of the librational coordinate of the water and the observed frequencies are then averages over the libration. This simple prescription for calculating the observed bands is that given by Redfield theory. We conclude that the observed band collapse in NaClO4⋅H2O is due primarily to the increase with temperature of the in-plane water libration. At higher temperatures, the 180° flipping motion also contributes to the collapse. For LiI⋅3H2O, the motion is a complex one involving locally ordered domains of the water molecules in the crystal.

Human testis does not secrete oestrone sulphate.

The concentrations of five steroids in samples of spermatic venous blood collected from 17 men undergoing ligation of varicocoeles were compared with those in samples from the antecubital vein. There was evidence of testicular secretion of testosterone, androstenedione, oestradiol-17 beta and oestrone, since the ratios of the mean concentrations in spermatic venous plasma to those in peripheral venous plasma were 77.2, 9.1, 28.7 and 1.6 respectively. The testicular secretion of oestrone sulphate was minimal; the ratio of the mean concentrations in spermatic and peripheral plasma was 1.07. These results support the view derived from isotope dilution studies that almost all oestrone and oestrone sulphate in the circulation is derived from peripheral conversion of other precursor steroids.

Isotope-dilution analysis of the effects of deoxyguanosine and deoxyadenosine on the incorporation of thymidine and deoxycytidine by hydroxyurea-treated thymus cells.

It is presumed that the dGTP and dATP needed for replicative DNA synthesis can be formed by way of either ;salvage' pathways or biosynthesis de novo. This was examined by adding hydroxyurea to cultures of rat thymus cells to inhibit ribonucleoside diphosphate reductase, a key enzyme of the ;de novo' pathway. Most of the inhibition of the incorporation of [Me-(3)H]thymidine and deoxy[5-(3)H]cytidine by low concentrations of hydroxyurea (100-500mum) was prevented by substrates of the salvage pathway (400mum-deoxyguanosine and, to a lesser extent, 200mum-deoxyadenosine). However, isotope-dilution studies indicated that the purine deoxyribonucleosides prevented inhibition by decreasing pyrimidine deoxyribonucleotide competitor pools. Evidence was obtained that a hydroxyurea-induced increase in the thymidine-competitor pool (probably dTTP) was prevented to an equal extent by deoxyguanosine and by the inhibitor of thymidylate synthase, deoxy-5-fluorouridine. These compounds had almost identical effects on hydroxyurea dose-response curves and on thymidine isotope-dilution plots. The evidence suggests that exogenous purine deoxyribonucleosides cannot prevent the inhibition by hydroxyurea of thymus-cell DNA synthesis. This could mean that, with respect to the metabolism of purine deoxyribonucleotides, ribonucleoside diphosphate reductase is tightly coupled to DNA polymerase in a multienzyme complex. The complex would not permit entry of exogenous metabolic intermediates into the ;de novo' pathway, but would still be subject to the regulatory effects of these intermediates. Thus dGTP and dATP formed from exogenous purine deoxyribonucleosides by salvage pathways might deplete pyrimidine deoxyribonucleotide competitor pools by inhibiting relatively hydroxyurea-insensitive activities of ribonucleoside diphosphate reductase.

Leucocyte Electrolytes in the Syndrome of Inappropriate Antidiuretic Hormone Secretion: A Comparison with Values Derived from Isotope Dilution Studies

Leucocyte Electrolytes in the Syndrome of Inappropriate Antidiuretic Hormone Secretion: A Comparison with Values Derived from Isotope Dilution Studies

Determination of the pool size and synthesis rate of bile acids by measurements in blood of patients with liver disease.

1. A simplified technique for the measurement of bile-acid pool size and synthesis rate has been developed in patients with liver disease. Isotope dilution studies in blood and bile were performed after intravenous injection of [24-14C]cholic acid with radioimmunoassay for the measurement of the bile-acid concentration. The interpolated pool sizes and synthesis rates, determined from results from both blood and bile, were not significantly different. The concentration of bile acids in the blood of healthy controls was not sufficiently elevated to permit application of this technique. 2. Three out of six patients with cirrhosis had a markedly reduced pool size compared with that of controls, whereas those with cholestasis had an unchanged pool size. The daily synthesis rate was reduced in both groups. Liver disease caused a redistribution (0.5-16%) of the bile-acid pool to the blood, which was associated with enhanced urinary excretion of cholic acid and its metabolites.

Indices of stress in housed experimental sheep

Six trained sheep were used in isotope dilution studies to determine the relations between glucose and lactate metabolism. The animals were judged by their behaviour and by their arterialplasma glucose and cortisol concentrations to be unstressed before and during each experiment. In each animal, however, the concentration of lactate in an arterial plasma sample taken immediately before an experiment was greater than the mean arterial plasma lactate concentration found during the course of the experiment. This increase in the concentration of lactate may be a sensitive index of apprehension in sheep which are well accustomed to being handled and which by other criteria appear to be unstressed.

Physics in nuclear medicine

Following an introduction to the physical processes of radioactivity and radiation detection, the subject of diagnostic nuclear medicine is discussed under the headings nontomographic imaging, emission computerised tomography, metabolic and isotope dilution studies, radioimmunoassay and activation analysis.

The synthesis of oxalate from hydroxypyruvate by isolated perfused rat liver the mechanism of hyperoxaluria in L-lyceric aciduria

Hydroxypyruvate and glycolate inhibited the oxidation of [U- 14C]glyoxylate to [ 14C]oxalate in isolated perfused rat liver, but stimulated total oxalate and glycolate synthesis. [ 14C]Oxalate synthesis from [ 14C]glycine similarly inhibited by hydroxypyruvate, but conversion of [ 14C 1]glycolate to [ 4C]oxalate was increased three-fold. Pyruvate had no effect on the synthesis of [ 14C]oxalate or total oxalate. The inhibition studies suggest that hydroxypyruvate is a precursor of glycolate and oxalate and that the conversion of glycolate to oxalate does not involve free glyoxylate as an intermediate. [ 14C 3]Hydroxypyruvate, but not [ 14C 1]hydroxypyruvate, was oxidized to [ 14C]oxalate in isolated perfused rat liver. Isotope dilution studies indicate the major pathway involves the decarboxylation of hydroxypyruvate forming glycolaldehyde which is subsequently oxidized to oxalate via glycolate. The oxidation of serine to oxalate appears to proceed predominantly via hydroxypyruvate rather than glycine or ethanolamine. The hyperoxaluria of L-glyceric aciduria, primary hyperoxaluria type II, is induced by the oxidation of the hydroxypyruvate, which accumulates because of the deficiency of D-glyceric dehydrogenase, to oxalate.

14C]oxalate synthesis from [ U- 14C]xylitol: In vivo and in vitro studies

[ 14C]Oxalate was produced in vivo from [ U- 14C]xylitol, [ U- 14C]sorbitol, [1- 14C]glyoxylate, and [1- 14C]glycolate and also in isolated rat hepatocytes. Pyridoxine deficiency increased oxalate synthesis from all compounds, both in vivo and in vitro. Control animals excreted 12-fold more oxalate after xylitol injections, compared with sorbitol treatment. In hepatocytes, maximal conversion of xylitol to oxalate occurred at a xylitol concentration of 1 m m. A comparison between fructose, sorbitol, glucose, glycerol, and xylitol at this concentration demonstrated that xylitol was the most significant oxalate precursor, producing threefold more oxalate than fructose, the next most effective compound. Metabolic studies, including the use of an artificial electron acceptor, revealed that an oxidized cellular redox state was essential for maximal conversion of xylitol to oxalate. Furthermore, the detection of [ 14C]glycolate in the urine of xylitol-treated rats, together with isotope dilution studies in hepatocytes, indicate that this compound is the key intermediate in the pathway from xylitol to oxalate.

Suitability of [11,12-3H2]chenodeoxycholic acid and [11,12-3H2]lithocholic acid for isotope dilution studies of bile acid metabolism in man

Studies were carried out to assess the stability of the label in [11,12-(3)H(2)]chenodeoxycholic acid and [11,12-(3)H(2)]lithocholic acid during enterohepatic cycling and thus define the suitability of these tracers for isotope dilution studies of bile acids in man. To do this, [11,12-(3)H(2)]-chenodeoxycholic acid and [24-(14)C]chenodeoxycholic acid were administered simultaneously to six healthy adults. The (3)H/(14)C ratio of biliary chenodeoxycholic acid and the specific activity of biliary chenodeoxycholic acid were determined to permit calculation of pool size and turnover rate by the conventional isotope dilution procedure of Lindstedt (1957. Acta Physiol. Scand. 40: 1). Excretion of label in urine was measured as well as the (3)H/(14)C ratio of fecal bile acids. Similar studies were carried out with [11,12-(3)H(2)]lithocholic acid and [24-(14)C]lithocholic acid. With [11,12-(3)H(2)]chenodeoxycholic acid, 10-15% of the label was lost during the first 24 hours, after which the (3)H/(14)C ratio of biliary chenodeoxycholic acid remained constant. The loss of (3)H caused a consistent overestimate of pool size by 10-15%, but since the fractional turnover rate of the two isotopes was identical, the calculated rate of synthesis was 10-15% greater when determined using (3)H. The loss of (3)H was attributed to the presence of (3)H in positions other than 11 and 12 occurring during the labeling procedure when the unsaturated precursor was exposed to carrier-free tritium gas. The (3)H/(14)C ratio of feces was consistently below that of bile, suggesting bacterial removal of additional (3)H during distal intestinal passage. In contrast, [11,12-(3)H(2)]lithocholic acid lost (3)H continuously, so that by 2 days after administration, the (3)H/(14)C ratio had fallen to nearly half of that of the administered mixture. It is concluded that isotope dilution studies may be carried out with one preparation of [11,12-(3)H(2)]chenodeoxycholic acid with an error of 10-15%; possibly a more stable preparation can be prepared with a gentler tritiation procedure. One preparation of [11,12-(3)H(2)]lithocholic acid was unsatisfactory for isotope dilution studies in man.

Raman study of vibrational dephasing in liquid CH3CN and CD3CN

The Raman line shapes of the ν1(a1) C–H and C–D fundamentals in liquid acetonitrile and acetonitrile-d3 have been measured as a function of pressure up to 4 kbar within the temperature interval 30–120 °C. Densities have also been determined. From the isotropic component of the vibrational Raman band shape the vibrational relaxation times have been obtained as a function of temperature and pressure (density). The experimental results can be summarized as follows: (i) as T increases at constant density ρ, the vibrational relaxation rate (τvib)−1 increases; (ii) at constant T as density is raised τvib−1 increases; (iii) at constant pressure the T increase produces higher τvib−1, however, the change is more pronounced for the CD3CN liquid. Isotopic dilution studies of the CH3CN/CD3CN mixtures shows no significant effect on (τvib−1). The experimental data are interpreted in terms of the Kubo stochastic line shape theory and the dephasing model of Fischer and Laubereau. The results based on Kubo formalism indicate that dephasing is the dominant relaxation mechanism and that the modulation is fast. The isolated binary collision model proposed by Fischer and Laubereau for vibrational dephasing reproduces the essential features of the density and temperature dependence of the (τvib)−1 and suggests that pure dephasing is the dominant broadening mechanism for the isotropic line shapes studied. In the calculation the elastic collision times were approximated by the Enskog relaxation times.

Isotope-dilution studies of the effects of 5-fluorodeoxyuridine and hydroxyurea on the incorporation of deoxycytidine and thymidine by cultured thymus cells.

From experimentally induced changes in the slope (Vmax) and intercept (pool) of isotope-dilution plots inferences may be drawn on the position and regulation of rate-limiting steps affecting the incorporation of pyrimidine deoxynucleosides by intact cells. 5-Fluorodeoxyuridine (FdUrd; 1 μM) reduced the Vmax of radioactive labelling with deoxy[5-3H]cytidine; this was reversed by thymidine (19 μM) suggesting that FdUrd makes the concentration of deoxythymidine triphosphate (dTTP) rate-limiting for DNA synthesis. With deoxy[U-14C]cytidine the reversal of FdUrd inhibition by thymidine was only partial; this was in keeping with (i) deoxy[U-14C]cytidine labelling both cytosine and thymine in DNA, and (ii) a continuing inhibition of thymidylate synthetase by FdUrd in the presence of thymidine (19 μM).The deoxycytidine competitor pool was increased by cytidine (10–50 μM) and decreased by (i) thymidine (19 μM), (ii) hydroxyurea (50 μM) and (iii) deoxycytidine (12 μM, in the presence of FdUrd). It is suggested that these pool-decreasing agents, or their derivatives (e.g., dTTP), inhibit ribonucleotide reductase and hence prevent the entry of pyrimidine ribonucleotide derivatives into the deoxycytidine competitor pool; because of this pool decrease, radioactive labelling with deoxy[5-3H]cytidine was enhanced by thymidine (19 μM) and hydroxyurea (50 μM). However, at the latter hydroxyurea concentration, labelling with [Me-3H]thymidine was inhibited, due to a decrease in the Vmax of the rate-limiting step for thymidine incorporation (probably thymidine kinase). This sensitivity of labelling with [Me-3H]thymidine to inhibition by hydroxyurea (50 μM) was reduced by adding FdUrd to prevent the accumulation of dTTP. At high hydroxyurea concentrations (0.1–1.0 mM), labelling with deoxy[5-3H]cytidine was also inhibited, due to a decrease in Vmax of the rate-limiting step, which was probably at the level of DNA polymerase.The results suggest that hydroxyurea inhibits DNA synthesis by making the concentration of purine deoxynucleotides rate-limiting. Pyrimidine deoxynucleotides are formed in sufficient quantities from deoxycytidine by way of salvage pathways. Indeed, dTTP accumulates and inhibits thymidine kinase, thus amplifying the inhibitory effect of hydroxyurea on labelling with [Me-3 H]thymidine.

Determination of oxalic and glycolic acid with isotope dilution methods and studies on the determination of glyoxylic acid

Rapid and accurate isotope dilution methods for determination of oxalic and glycolic acids are described. Determination of glyoxylic acid by this approach leads to variable errors of unknown origin.