14C Tracer Technique Research Articles

Patterns of gas exchange, photosynthate allocation, and root growth during a root growth capacity test

Shoot and root growth, net assimilation, leaf conductance and transpiration rates, and carbon allocation were measured on 1.5-year-old Scots pine (Pinussylvestris L.) and Norway spruce (Piceaabies (L.) Karst.) seedlings during a 28-day root growth capacity test. The aim was to determine whether current rates of assimilation, carbon allocation, or shoot growth were related to root growth during the test. Carbon allocation was determined using 14C tracer techniques by exposing the seedlings to, 14CO2 for 24 h, while gas exchange was measured with a LI-COR 6200 meter seven times during the 28-day test. Both species broke bud and displayed substantial root growth. However, the species displayed very different gas exchange and allocation patterns during the test period. In Scots pine, assimilation and leaf conductance increased to a peak at 14–21 days and then began to decrease. Norway spruce showed almost constant and low net assimilation rates and leaf conductance. In both species, the 14C fraction transported to the root increased throughout the 28-day test. Correlations between gas exchange and 14C allocation were insignificant. The correlation between 14C accumulation in roots and increase in number of new roots was significant starting on day 14 in pine and day 23 in spruce.

The role of inhibition of pyruvate kinase in the stimulation of gluconeogenesis by glucagon: A reevaluation

We have reexamined the concept that glucagon controls gluconeogenesis from lactate-pyruvate in isolated rat hepatocytes almost entirely by inhibition of flux through pyruvate kinase, thereby making gluconeogenesis more efficient. 1. 1. We tested and refined the 14C-tracer technique that has previously yielded the opposite conclusion, that is, that inhibition of pyruvate kinase is a relatively unimportant mechanism. The tracer procedure, as used by us, was found to be insensitive to the size of the pyruvate pool, and experiments using modifications of the technique to obviate a number of other potential errors support the earlier conclusion that control of pyruvate kinase is not the predominant mechanism. 2. 2. Any stimulation of formation of glucose that results from inhibition of pyruvate kinase is the consequence of elevation of the steady-state concentrations of phosphoenolpyruvate and all subsequent intermediates in the gluconeogenic pathway. During ongoing stimulation of glucose synthesis by glucagon in isolated hepatocytes, the concentrations of all measured intermediate compounds between phosphoenolpyruvate and glucose were elevated except triose phosphates and fructose 1,6-bisphosphate. The failure of these compounds to rise above control levels indicates that not all gluconeogenic reactions beyond pyruvate kinase were accelerated thermodynamically as would occur with predominant control at pyruvate kinase. We conclude, therefore, that although glucagon inhibits flux through the pyruvate kinase reaction, this does not account for most of the stimulation of gluconeogenesis. Major control sites are also within the pyruvate-phosphoenolpyruvate segment and the fructose 1,6-bisphosphate Cycle.

Lactate transport in isolated mouse muscles studied with a tracer technique—kinetics, stereospecificity, pH dependency and maximal capacity

Lactate transport across the sarcolemma of isolated mouse muscles was studied with a 14C tracer technique. The cellular tracer uptake could be inhibited by unlabelled L-lactate (and pyruvate) and to a lesser extent by D-lactase. The stereospecific fraction had a Km of 3.5 mM, and made up 50% of the total transport. The tracer uptake was unaffected by 0.05 mM DIDS and 0.2 mM amiloride, but was inhibited by cinnamate (Ki = 8 mM) and PCMBS (Ki = 0.8 mM). With high concentrations of the latter inhibitor compounds or with high concentrations of unlabelled L-lactate, the tracer uptake was inhibited 80%, which indicates that the main part of the transport involves facilitated diffusion. The remaining fraction (20%) was non-saturable, reduced at high pH, and could not be inhibited; it is probably mediated by diffusion of undissociated lactic acid. Lactate transport was pH-dependent, which is consistent with a lactate-H+ symport. The maximal transport capacity, as calculated from the pH changes measured with pH-sensitive micro-electrodes while the lactate gradient was 30 mM, was 11.8 mmol kg-1 min-1 (pH 6.2).

Mechanisms for the uptake of inorganic carbon by two species of symbiont-bearing foraminifera

The mechanisms for uptake of inorganic carbon (Ci) for photosynthesis and calcification of a perforate foraminifer, Amphistegina lobifera Larsen, and an imperforate species, Amphisorus hemprichii Ehrenberg, from the Gulf of Eilat, Red Sea were studied in 1986–1987 using 14C tracer techniques. Total Ci uptake of A. lobifera and photosynthetic carbon uptake of A. hemprichii fit the Hill-Whittingham equation that describes the overall rate of enzymatic reactions that are provided with their substrate through a diffusion barrier. This suggests that diffusion is the rate limiting step for total Ci uptake in A. lobifera. Photosynthesis by the isolated symbionts and uptake of CO32- for calcification obey Michaelis-Menten kinetics indicating that enzymatic reactions determine the rate of the separate processes. Both photosynthesis and calcification can be inhibited without affecting each other. Calcification rates in A. lobifera were optimal at Ca levels around normal seawater concentration and were sensitive to inhibitors of respiratory adenosine triphosphate (ATP) generation and Ca-ATP-ase. This indicates that Ca uptake is also active. Calcification rates of A. hemprichii increased linearly as a function of external Ci concentration over the entire experimental range (0 to 4 mM Ci). In contrast, photosynthetic rates showed Hill-Whittingham type kinetics. The dependence of calcification on the CO32- concentration was also linear, suggesting that its diffusion is the rate limiting step for calcification in A. hemprichii. Increasing Ca concentrations yielded higher calcification rates over the entire range measured (0 to 40 mM Ca). Calcification in A. hemprichii was less sensitive to inhibitors of ATP generation than in A. lobifera, suggesting that in A. hemprichii energy supply is less important for this process.

Extent and nature of extracellular organic production by the marine coccolithophorid Hymenomonas carterae

Production of extracellular dissolved organic substances by cultures of the marine coccolithophorid Hymenomonas carterae SMBA 254 was investigated by the 14C-tracer technique. In 4 h incubations, extracellular 14C production represented 20 to 64% of 14C incorporation into cell material for cultures nearing, or during stationary growth; for rapidly growing cultures with relatively low cell densities the 14C-production was only 4 to 10% of incorporation. Intra-and extra-cellular material was fractionated by ion-exchange membrane electrodialysis. Ionic compounds accounted for 40 to 60% of extracellular material; in contrast only 16 to 27% of intracelular metabolites were in this category. Of a range of methods which were investigated, separation of compounds by gel-filtration in conjunction with two-dimensional thin-layer chromatography proved most effective. Up to 80% of extracellular material was of less than 1 800 mol. wt and included carbohydrates and amino acids, with glutamic acid predominating. The intracellular material was more heterogeneous, with a substantially greater proportion of high molecular weight material. The quite distinct compositions show clearly that extracellular production observed in these experiments arose through excretion and was not attributable to cell lysis.

Extracellular organic production by a picoplankton, Stichococcus bacillaris

A marine isolate of the picoplankton Stichococcus bacillaris was cultured in an artificial seawater medium, and production of extracellular dissolved organic substances investigated by the 14C-tracer technique. Extracellular 14C production represented up to 10% of 14C incorporation into cell material and fractionation by ion-exchange membrane electrodialysis showed that up to 98% of this material was acidic. In contrast, intracellular material was dominated by neutral components, ionic species representing no more than 34%. The predominant extracellular product was glycollic acid, with negligible amounts of other compounds.

Measurements of the diurnal OH cycle by a 14C-tracer method

The hydroxyl free radical, OH, is generally recognized as the primary oxidant for the removal of pollutants from the Earth's atmosphere. Measurements of OH concentrations are needed to test models of atmospheric photochemistry. We have used the 14C-tracer technique to measure five ambient diurnal OH concentration cycles in relatively pure and in polluted air. The early-to mid-October maximum midday OH concentrations for pure and polluted air were found to be, respectively, 2.4 × 106 and 9.5 × 106 radicals cm–3 (10–13–4 × 10–13 relative to air). Night-time OH concentrations of less than 2 × 105 radicals cm–3 were measured. Estimates of OH concentrations from photochemical models and trace gas lifetimes are consistent with our observations.

Uptake of inorganic carbon and internal carbon cycling in symbiont-bearing benthonic foraminifera

Incorporation rates of inorganic carbon and its distribution between the organic matter and the skeleton have been measured using 14C tracer techniques on two species of symbiont-bearing benthonic foraminifera in the Gulf of Elat: Amphistegina lobifera (a perforate species) and Amphisorus hemprichii (an imperforate species). Under constant experimental conditions, incorporation rates of the radiotracer become linear with time after several hours in A. hemprichii and after one day in A. lobifera. A. lobifera showed a lag time of 24 h for skeletal incorporation, whereas in A. hemprichii uptake into the skeleton started within 2 h. Pulse-chase incubations in radioactive seawater, followed by unlabelled incubations, demonstrate transfer of photosynthetically acquired 14C into the skeleton of A. lobifera. No such transfer was found in A. hemprichii. The total 14C uptake by A. lobifera increased during the first 24 h of cold chase incubation. This increase suggests the existence of an internal inorganic carbon pool that was lost (probably evaporated) during the analysis of pulse incubations. However, during the following chase incubations, the 14C in this pool was incorporated mainly into the skeleton and retained during analysis, causing the increase in the total uptake. No such increase was found in A. hemprichii. Additional 14C uptake experiments on other species of the genera Operculina, Heterostegina and Borelis suggest that the differences in pathways for incorporation of carbon between A. lobifera and A. hemprichii can be generalized to the perforate and imperforate foraminiferal groups. In perforate species, respired carbon originally taken up through photosynthesis is partly recycled into the skeleton. In imperforate species such a transfer has not been demonstrated. Perforate species seem to have a large internal inorganic carbon pool which serves mainly for calcification and possibly also for photosynthesis, while imperforate species may take up carbon for calcification directly from seawater or have a very small inorganic carbon pool.

Effect of growth conditions on enzyme activities, intracellular kinetics and biomass yields of a new RuMP-type methylotroph (T15)

A new methylotrophic strain (T15), which employs the ribulose monophosphate (RuMP) cycle of formaldehyde assimilation, was isolated on the basis of high in vitro activities of formaldehyde and formate dehydrogenases (19 and 678 mU per mg protein, respectively). Serial subculturing of the strain in batch cultures, on 4 g/l CH3OH for 6 months, led to loss of substantial percentages of the NAD-linked formaldehyde (25%) and formate (98%) dehydrogenases. The activities of these two enzymes were partially recovered when cells were grown continuously at very low dilution rate (0.03 h−1). We found large variations (40 to 1000%) in the activities of other key enzymes of carbon-substrate oxidation (both linear and cyclic) and assimilation, in batch cultures with pure and mixed substrates, and in continuous cultures of different dilution rates. Key intracellular reaction rates, including those of the cyclic and linear substrate oxidation, were measured in vivo using a 14C-tracer technique in both continuous and batch cultures. The results indicate significant variations in these reaction rates, particularly those of linear and cyclic carbon oxidation. Overall, the cyclic oxidation appears to be employed to a larger (although not predominant) extent in strain T15 compared with another RuMP strain (L3) we have previously examined. T15 exhibits high biomass yields (up to 0.63 g cells per g CH3OH) and growth rates (up to 0.46 h−1) on CH3OH in batch cultures. CH3NH2 can also be utilized as a substrate. In continuous culture, T15 could be grown at dilution rates up to 0.36 h−1 with a corresponding biomass yield of 0.4. Examination of a large number of data on the biomass yields of strains T15 and L3 reveals that the large variations in yields derive from the variable branching of carbon flow between linear and cyclic oxidation and assimilation, rather than changes in the biosynthetic efficiency of carbon incorporation into biomass.

Studies on hydrogen transfer mechanisms in coal liquefaction by means of 3H and 14C tracer techniques

The liquefaction of Taiheiyo coal using 3H and 14C labeled solvent or 3H labeled gaseous hydrogen has been conducted. The effects of NiMoAl 2O 3 catalyst, reaction times (0–300 min) and solvents were examined. The 3H and 14C concentrations in the reaction products were determined by a liquid scintillation counter to study the coal liquefaction mechanism. The hydrogen and carbon material balances were calculated and the amounts of the hydrogen addition and exchange among gas phase, coal products and solvent were estimated. In the process of the primary liquefaction, the coal was thermally decomposed and hydrogenated by hydrogen atoms mainly released from the solvent, tetralin. In the process of the hydrocracking of the primary coal liquid, hydrocracking took place by gas phase hydrogen on the catalyst. When naphthalene was used as the liquefaction solvent in the presence of the catalyst, the hydrogen transfer process through hydrogenation—dehydrogenation of the solvent, naphthalene → tetralin → naphthalene, was very important in the hydrogenation of coal by gaseous hydrogen.

Transformation and decomposition of photosynthetic products of lake phytoplankton.

Decomposition and transformation processes of cellular and extracellular products of lake phytoplankton photosynthesis were determined using a 14C tracer technique under laboratory conditions. The cellular organic matter was consumed by phytoplankton respiration in the early stage of incubation and decreased to 19% of the initial amount for 30 days at 25°C. A shift occurred in the chemical constitution of the predominant cellular organic matter : hot ethanol soluble and hot water soluble components changed into hat water insoluble ones. After decomposition for 85 days, 14.7% and 1.5% of the cellular products were transformed to particulate and dissolved organic matter, respectively. The extracellular products, which were 9.3% of the total photosynthetic products, were easily metabolized and decomposed by lake bacteria. After 133 days, 19.4% and 7.5% of the initial extracellular products changed to particulate and dissolved organic matter, respectively. During the course of the decomposition, a marked difference was observed in gel filtration profile of the dissolved organic matter derived from the extracellular products.

Reproducibility aspects in specific migration measurements: A collaborative study

Considerable variations in specific migration measurements are often observed when comparing results from different laboratories on apparently identical plastic/additive/liquid systems. In order to determine the influence of the various factors on reproducibility and repeatability in specific migration tests, and to develop an appropriate methodology, a collaborative study was undertaken with the participation of nine laboratories from the member countries of the EEC. Penta‐erythrityl tetrakis‐(3‐(3,5‐di‐tert.butyl‐4‐hydroxyphenyl) propionate)—further referred to as Irganox 1010—and dihydroxybiphenyl (DHBP), were incorporated in high density polyethylene (HDPE). A water‐ethanol mixture, olive oil and a synthetic fat were selected as test liquids for contact with the plastics under well‐defined storage conditions. The study was carried out in four phases: testing of a conventional h.p.l.c. method;testing and verification of a 14C tracer technique;study of repeatability and reproducibility values in one‐sid...

Methanogenesis from Methanol and Methylamines and Acetogenesis from Hydrogen and Carbon Dioxide in the Sediments of a Eutrophic Lake

C-tracer techniques were used to examine the metabolism of methanol and methylamines and acetogenesis from hydrogen and carbon dioxide in sediments from the profundal and littoral zones of eutrophic Wintergreen Lake, Michigan. Methanogens were primarily responsible for the metabolism of methanol, monomethylamine, and trimethylamine and maintained the pool size of these substrates below 10 muM in both sediment types. Methanol and methylamines were the precursors for less than 5 and 1%, respectively, of the total methane produced. Methanol and methylamines continued to be metabolized to methane when the sulfate concentration in the sediment was increased to 20 mM. Less than 2% of the total acetate production was derived from carbon dioxide reduction. Hydrogen consumption by hydrogen-oxidizing acetogens was 5% or less of the total hydrogen uptake by acetogens and methanogens. These results, in conjunction with previous studies, emphasize that acetate and hydrogen are the major methane precursors and that methanogens are the predominant hydrogen consumers in the sediments of this eutrophic lake.

ChemInform Abstract: STERICALLY HINDERED ALKANES. II. 1,1,1,3,3,3‐HEXAPHENYLPROPANE

AbstractDie aus dem Radikal (I) und Diazomethan unter N2‐Entwicklung entstehende Titelverbindung (II) bildet monokline Kristalle der Struktur C2/c (Z=4).

Sterically Hindered Alkanes, II 1,1,1,3,3,3-Hexaphenylpropane

The structure of the title compound 3, C39H32, obtained for the first time by Schlenk and Bornhardt (1912) from triphenylmethyl (1) and diazomethane (2) has been determined by 14C-tracer technique, NMR spectroscopy and X-ray analysis. It crystallizes in the monoclinic space group C2/c with Z = 4, a = 9.943(6), b= 19.054(3), c = 14.708(2) Å and β = 101.80(2)°. The structure was solved by direct methods and refined by full-matrix least squares to R = 0.046 for all 2625 reflexions (|F0| >0). On account of steric repulsions between the phenyl rings at both termini the central propane bond angle φ is stretched to 127.2°, whereas the bond lengths are not much affected. The X-ray results are compared with those of Blount et al. (1982), obtained on a triclinic crystal modification (P1) of 3. A fair agreement of bond lengths and angles is observed, the slopes of the homochiral propellers, however, aro different in both modifications. The measurement of the N2- evolution shows that the mechanism of formation of 3 from 1 and 2 is different in the photochemical and dark reaction. A carbene respectively a one-electron transfer mechanism is suggested. In spite of the remarkably normal CC-propane bond lengths homolytic cleavage of these bonds to give 1 is observed at 200-220 °C using the ESR technique

Studies on drug metabolism by use of isotopes. XVII. Mass spectrometric quantification of urinary metabolites of deuterated l-ephedrine in man.

As part of problems on the applicability of a drug labeled with deuterium to metabolic studies in man, an analytical method for urinary metabolites by deuterium-tracer technique was investigated and its reliability was compared with that of 14C-tracer technique. l-Ephedrine [arom.-2H5] or l-ephedrine [α-14C] was subcutaneously injected to rabbits and the main urinary metabolites (1-phenyl-1, 2-propanediol and hippuric acid) excreted for 24 hr after dosing were analyzed. A deuterated metabolite such as the diol absent in normal urine was submitted to the inverse isotope dilution analysis using the unlabeled compound both as an internal standard and as a carrier. The molar ratio of the unlabeled to the deuterated compound was determined by mass spectrometry. The amount of deuterated hippuric acid diluted with the endogenous material present in urine was determined by using both the direct isotope dilution analysis and the inverse isotope dilution analysis. It was found that reliability of the deuterium-tracer technique was comparable with that of the 14C-tracer technique on the basis of data obtained from both techniques.

Diurnal Variation of Dissolved Inorganic Carbon and its Use in Estimating Primary Production and CO2 Invasion in Lake 227

Standard in situ measurements of phytoplankton production and 14C bottle bioassays gave erroneous results when applied to lake 227, a eutrophic softwater lake in the Canadian Shield. Errors were found to be due to diurnal variations in the degree of carbon limitation of phytoplankton, and to invasion of CO2 from the atmosphere and hypolimnion.A method based on diurnal measurements of dissolved inorganic carbon, community respiration, and invasion of CO2, using gas chromatography, is described. Production by phytoplankton in lakes fertilized with nitrogen and phosphorus was found to be several times higher than in natural lakes of the area. Net production during summer stratification was found to equal invasion of CO2 from the atmosphere.The new technique should have application in other eutrophic low carbon lakes, where 14C tracer techniques are encumbered by serious technical complications.

The validity of measurement of mean whole body intracellular hydrogen ion activity using 5,5-dimethyl-2,4-oxazolidinedione.

1. To study the validity of mean whole-body intracellular hydrogen ion activity measurements ([H+]i) by the distribution of 14C-labelled 5,5-dimethyl-2,4-oxazo-lidinedione ([14C]DMO), eight anaesthetized dogs received three separate injections of iso-osmotic buffered chemical DMO, in cumulative doses of 50–2000 mg/kg body weight, following a control [H+]i determination by the 14C-tracer technique. 2. A correlation between dose of chemical DMO and measured [H+]i was found with a regression line of −Δ[H+]i = 23.27 + 16.45 [log DMO dose −2.41]. 3. Although the results are consistent with active transport of DMO across cell membranes, other possible explanations such as altered cell buffering capacity by DMO loading and variable sequestration of DMO in the gastrointestinal tract must be considered. 4. There is no evidence for cellular binding of DMO.

Removal of alpha-methyldopa (aldomet) in man by dialysis.

SummaryDialytic removal of alpha-methyldopa (Aldomet) has been investigated in chronic uremic patients undergoing either hemodialysis or peritoneal dialysis, using 14C tracer techniques. A mean of 60% of the estimated absorbed radioactivity was recovered in the dialysate of 3 patients undergoing 4 hemodialyses; the mean relative dialysance of radioactivity from alpha-methyldopa and its products was 0.34. A similar amount of radioactivity was recovered in the dialysate of 2 patients undergoing peritoneal dialysis.The antihypertensive action of administered alpha-methyldopa and its major metabolite (a mono-O-sulfate conjugate) may be diminished by hemodialysis. This effect could contribute to the reemergence of hypertension occasionally observed in the postdialysis period in patients receiving alpha-methyldopa therapy.

Distribution of radio activity in trismethylene‐trinitrobenzene obtained from 14C‐labelled 1,3,5‐trinitrobenzene: Nucleophilic aromatic addition V

AbstractThe previously suggested mechanism for the formation and the stepwise degradation of “trismethylene‐trinitrobenzene” (I) is confirmed by application of a 14C tracer technique. It is shown that all transformations occur without hydrogen shift.In this paper besides compound II a stereoisomer was considered, with a different position of the two cyclopropyl‐groups towards one another. Further investigation in collaboration with Mr. H. P. Venker has shown that formula II is most likely. However this may be, it is of no consequence for the conclusions reached in the present paper.