High-precision iron isotope analysis of whole blood,erythrocytes, and serum in adults

BackgroundIron isotopic composition serves as a biological indicator of Fe metabolism in humans. In the process of Fe metabolism, essential carriers of Fe circulate in the blood and pass through storage organs and intestinal absorptive tissues. This study aimed to establish an analytical method for high-precision Fe isotopic measurement, investigate Fe concentration and isotopic composition in different parts of whole blood, and explore the potential of Fe isotopic composition as an indicator for Fe status within individuals.Analytical methodsA total of 23 clinically healthy Taiwanese adults of Han descent were enrolled randomly and Fe isotopic compositions of their whole blood, erythrocytes, and serum were measured. The Fe isotopic analysis was performed by Neptune Plus multiple-collector inductively coupled plasma mass spectrometry with double-spike technique. The precision and reproducibility of the Fe isotopic analysis were monitored by international biological and geological reference materials.Main findingsHigh-precision Fe isotopic measurements were achieved alongside with high consistency in the isotopic data for well-characterized reference materials. The Fe isotopic signatures of whole blood and erythrocytes were resolvable from that of serum, where both whole blood and erythrocytes contained significantly lighter Fe isotopic compositions compared to the case of serum (P = 0.0296 and P = 0.0004, respectively). The δ^56/54Fe value of the serum sample was 0.2‰ heavier on an average than those of whole blood or erythrocytes. This isotopic fractionation observed in different parts of whole blood may indicate redox processes involved in Fe cycling, e.g. erythrocyte production and Fe transportation. Moreover, the δ^56/54Fe values of whole blood and serum significantly correlated with the hemoglobin level (P = 0.0126 and P = 0.0020, respectively), erythrocyte count (P = 0.0014 and P = 0.0005, respectively), and Mentzer index (P = 0.0055 and P = 0.0011, respectively), suggesting the Fe isotopic composition as an indicator of functional Fe status in healthy adults. The relationships between blood Fe isotopic compositions and relevant biodemographic variables were also examined. While the average Fe concentration of whole blood was significantly higher in males than in females (P = 0.0028), females exhibited a heavier Fe isotopic composition compared to that of males in whole blood (P = 0.0010) and serum (P < 0.0001). A significantly inverse correlation of the whole blood δ^56/54Fe value with body mass index of individuals (P = 0.0095) was also observed.ConclusionThe results presented herein reveal that blood Fe isotopic signature is consequentially linked to baseline erythrocyte parameters in individuals and is significantly affected by the gender and body mass index in the adult population. These findings support the role of Fe isotopic composition as an indicator for the variance of Fe metabolism among adult individuals and populations and warrant further study to elucidate the underlying mechanisms.     

Pyruvate shuttling during rest and exercise before and after endurance training in men.

We describe the isotopic exchange of lactate and pyruvate after arm vein infusion of [3-(13)C]lactate in men during rest and exercise. We tested the hypothesis that working muscle (limb net lactate and pyruvate exchange) is the source of the elevated systemic lactate-to-pyruvate concentration ratio (L/P) during exercise. We also hypothesized that the isotopic equilibration between lactate and pyruvate would decrease in arterial blood as glycolytic flux, as determined by relative exercise intensity, increased. Nine men were studied at rest and during exercise before and after 9 wk of endurance training. Although during exercise arterial pyruvate concentration decreased to below rest values (P < 0.05), pyruvate net release from working muscle was as large as lactate net release under all exercise conditions. Exogenous (arterial) lactate was the predominant origin of pyruvate released from working muscle. With no significant effect of exercise intensity or training, arterial isotopic equilibration [(IE(pyruvate)/IE(lactate)).100%, where IE is isotopic enrichment] decreased significantly (P < 0.05) from 60 +/- 3.1% at rest to an average value of 12 +/- 2.7% during exercise, and there were no changes in femoral venous isotopic equilibration. These data show that 1). the isotopic equilibration between lactate and pyruvate in arterial blood decreases significantly during exercise; 2). working muscle is not solely responsible for the decreased arterial isotopic equilibration or elevated arterial L/P occurring during exercise; 3). working muscle releases similar amounts of lactate and pyruvate, the predominant source of the latter being arterial lactate; 4). pyruvate clearance from blood occurs extensively outside of working muscle; and 5). working muscle also releases alanine, but alanine release is an order of magnitude smaller than lactate or pyruvate release. These results portray the complexity of metabolic integration among diverse tissue beds in vivo.     

The significance of isotopic and paleontologic results on Quaternary calcareous nannofossil assemblages from Caribbean core P6304-4

The calcareous nannofossils in Late Quaternary Caribbean core P6304-4 are examined with respect to their stable isotope composition, biostratigraphy, and taxonomy. Using relations between the various nannofossil taxa and oxygen isotopic stages, a number of correlations are identified that indicate the value of nannofossils in Quaternary isotope stratigraphy. Three globally-synchronous datums previously identified are substantiated for the Caribbean. Variations in abundances of the most common species of nannofossils are not affected by glacial-interglacial changes and thus do not contribute to fluctuations seen in the oxygen isotopic record. However, nannofossil assemblages were more diverse during glacial stages. The carbon isotopic record of nannofossils remains enigmatic. Eventually it may be possible to recognize the various isotopic stages of the Quaternary based solely on the characteristics of the nannofossil assemblage.     

Temporal variation in trophic relationships among three congeneric penguin species breeding in sympatry

Penguins are a monophyletic group in which many species are found breeding sympatrically, raising questions regarding how these species coexist successfully. Here, the isotopic niche of three sympatric pygoscelid penguin species was investigated at Powell Island, South Orkney Islands, during two breeding seasons (austral summers 2013–2014 and 2015–2016). Measurements of carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope ratios were obtained from blood (adults) or feather (chicks) samples collected from Adélie Pygoscelis adeliae, chinstrap P. antarctica, and gentoo P. papua penguins. Isotopic niche regions (a proxy for the realized trophic niches) were computed to provide estimates of the trophic niche width of the studied species during the breeding season. The isotopic niche regions of adults of all three species were similar, but gentoo chicks had noticeably wider isotopic niches than the chicks of the other two species. Moderate to strong overlap in isotopic niche among species was found during each breeding season and for both age groups, suggesting that the potential for competition for shared food sources was similar during the two study years, although the actual level of competition could not be determined owing to the lack of data on resource abundance. Clear interannual shifts in isotopic niche were seen in all three species, though of lower amplitude for adult chinstrap penguins. These shifts were due to variation in carbon, but not nitrogen, isotopic ratios, which could indicate either a change in isotopic signature of their prey or a switch to an alternative food web. The main conclusions of this study are that (1) there is a partial overlap in the isotopic niches of these three congeneric species and that (2) they responded similarly to changes that likely occurred at the base of their food chain between the 2 years of the study.     

Compartmentation of carbohydrate metabolism in vascular smooth muscle: evidence for at least two functionally independent pools of glucose 6-phosphate

Previous work has indicated that there are at least two functionally independent Embden-Meyerhof pathways within the vascular smooth muscle of porcine carotid artery. We tested this hypothesis by analyzing the isotopic equilibrium between medium glucose and intracellular glucose 6-phosphate under basal conditions and after 30 min of mechanical activation, during which time the rate of glycogenolysis has been found to be substantial. Under basal conditions, the specific activity of glucose 6-phosphate equilibrated to a level which was not in isotopic equilibrium with medium glucose suggesting that there is a significant pool of glucose 6-phosphate which is not readily accessible to medium glucose. After 15 min of mechanical activation, the specific activity of intracellular glucose 6-phosphate was found to decrease significantly from its apparent steady-state distribution, indicating that glycogen was likely to be a significant source for glucose 6-phosphate. Since the specific activity of lactate was unaltered from its equilibrium distribution under similar stimulus conditions, these findings substantiate the existence of at least two independent pools of glucose 6-phosphate.     

Metabolic flux analysis in Escherichia coli by integrating isotopic dynamic and isotopic stationary 13C labeling data

The novel concept of isotopic dynamic 13C metabolic flux analysis (ID-13C MFA) enables integrated analysis of isotopomer data from isotopic transient and/or isotopic stationary phase of a 13C labeling experiment, short-time experiments, and an extended range of applications of 13C MFA. In the presented work, an experimental and computational framework consisting of short-time 13C labeling, an integrated rapid sampling procedure, a LC-MS analytical method, numerical integration of the system of isotopomer differential equations, and estimation of metabolic fluxes was developed and applied to determine intracellular fluxes in glycolysis, pentose phosphate pathway (PPP), and citric acid cycle (TCA) in Escherichia coli grown in aerobic, glucose-limited chemostat culture at a dilution rate of D = 0.10 h(-1). Intracellular steady state concentrations were quantified for 12 metabolic intermediates. A total of 90 LC-MS mass isotopomers were quantified at sampling times t = 0, 91, 226, 346, 589 s and at isotopic stationary conditions. Isotopic stationarity was reached within 10 min in glycolytic and PPP metabolites. Consistent flux solutions were obtained by ID-13C MFA using isotopic dynamic and isotopic stationary 13C labeling data and by isotopic stationary 13C MFA (IS-13C MFA) using solely isotopic stationary data. It is demonstrated that integration of dynamic 13C labeling data increases the sensitivity of flux estimation, particularly at the glucose-6-phosphate branch point. The identified split ratio between glycolysis and PPP was 55%:44%. These results were confirmed by IS-13C MFA additionally using labeling data in proteinogenic amino acids (GC-MS) obtained after 5 h from sampled biomass.     

The effect of soil water status on fertiliser, topsoil and subsoil phosphorus utilisation by wheat

Background and aims

Crop phosphorus (P) content is controlled by P uptake from both banded P fertiliser and from P throughout the soil profile. These P supply factors are in turn controlled by soil, climatic and plant factors. The aim of this experiment was to measure the contribution of fertiliser, topsoil and subsoil P to wheat plants under wet and dry growing season conditions.

Methods

An isotopic tracer technique was used to measure P uptake from fertiliser at seven agricultural field sites under wet and dry growing season conditions. At three of these sites a dual isotopic technique was used to distinguish between wheat uptake of P from fertiliser, topsoil (0–15 cm) and subsoil (below 15 cm).

Results

The amount of P fertiliser used by wheat was in the order of 3–30% of the P applied and increased with increasing rainfall. Topsoil P was the most important P source, but when sufficient P was present in the subsoil, P fertiliser addition stimulated the use of subsoil P.

Conclusions

Most crop P uptake was from the topsoil, however P fertiliser banded below the seed increased plant P uptake and stimulated the use of subsoil P in one soil type in a decile 7 (above average rainfall) growing season.     

Differential δC and δN signatures among scallop tissues: implications for ecology and physiology

There have been several studies where the isotopic composition of organisms has been determined seasonally, but fewer have examined separate organs. In this context, separate organs (e.g. gonad, digestive gland and muscle) of a suspension-feeder, the scallop Pecten maximus, were used to assess seasonal changes of both stable isotopes and biochemical components. Our study used multiple indicators [stable carbon and nitrogen isotope ratios, biochemical components and seston chlorophyll-a (chl a)] to track nutritive activity and energy allocation in P. maximus from the Bay of Brest (France). In addition to seasonal variation in the isotopic composition of P. maximus tissues, we found strong differences in the mean isotopic signatures of different organs. This has serious implications for interpretation of animal diets and potential use in animal physiology. Furthermore, we present evidence that seasonal variations of metabolism will cause changes in the isotopic composition not related to changes in the diet. Interpretation of isotopic data may require consideration of values from several separate organs. Finally, δ¹⁵N appears powerful to track metabolite fates in the scallop P. maximus.     

A simple procedure for the synthesis of [32P]phosphoenolpyruvate via the pyruvate kinase exchange reaction at equilibrium

A one step procedure is presented for the preparation of [³²P]phosphoenolpyruvate from [γ-³²P]ATP using pyruvate kinase. The reaction is carried out at chemical equilibrium and involves only an exchange of isotope between ATP and phosphoenolpyruvate. The initial phosphoenolpyruvate/ATP ratio in the reaction mixture determines the degree of ³²P incorporation into phosphoenolpyruvate when isotopic equilibrium is achieved.     

Palaeogene isotopic temperatures of western India

Oxygen isotopic measurements of larger benthic foraminifers from western India suggest that the Palaeogene temperature varied between 22deg;C and 32deg;C in this region. The warm climate of the Palaeocene and the Early Eocene, hovering around 32°C, deteriorated in late Middle Eocene (corresponding to planktic zones P13-P14) when the temperature dropped by 6deg;C. With progressive cooling through the Late Eocene, the temperature reached 22deg;C during Early Oligocene times. The cooling trend set in the Middle Eocene seems to have been terminated towards the end of the Palaeogene when the temperature rose to 25°C. □ Palaeotemperature, Palaeogene, oxygen isotopes, larger foraminifiers, India.     

Photoproducts from DNA pyrimidine bases and polycyclic aromatic hydrocarbons

The major photoproduct formed between benzo[a]pyrene and thymine is identified as 1-(benzo[a]pyren-6-yl)-thymine by means of spectroscopic analysis and isotopic syntheses. Irradiation of 1-methylcytosine hydrochloride and anthracene gives two isolable photoproducts of which one is assigned the structure 5-(anthracen-9-yl)-1-methylcytosine.     

Technical behaviours and subsistence patterns of lower middle palaeolithic men the case of the site of Payre in the Rhône valley (South-East of France)

Excavations at Payre, between 1990 and 1998, yielded lithic artefacts and bones in four levels. Men came during the isotopic stage 5 interglacial (Eemian) and isotopic stages 6 and 7. Sites of this age are not many in this part of the Rhône valley and this cave is an important element for the oldest Middle Palaeolithic and its diversity. The men lived in the cave before/after departure of bears and hyenas or wolves, probably for short occupations. Sedimentological, palynological, palaeontological and geochronological studies carry much informations about human life in this cave. The artefacts belong to the Lower Middle Palaeolithic culture, which shows originalities as compared to the South-West of France and also sites of the South-East for example Orgnac 3 dating to isotopic stage 9. The South-East of France shows more and more its important place in the knowledge of the Mousterians and could become in future a rich country for informations.     

周口店北京猿人洞骨化石铀系年龄数据——混合模式

铀系混合模式得出北京猿人在第一地点生活的年代是距今50万年到23万年。通过周口店猿人洞堆积物骨化石中 Th~(230)/U~(234)和 U~(234)/U~(232)的测定,建立铀系混合模式,给出下列年龄值:第1—3层距今23万年,6—7层距今35万年,8—9层大于40万年,12层距今50万年前或更早。一些中间层位的Th~(230)/U~(234)比值测定年龄偏低,可能是近20万年以来铀发生迁移造成的。     

The kinetics of rabbit muscle pyruvate kinase. Initial-velocity, substrate- and product-inhibition and isotopic-exchange studies of the reverse reaction.

1. An assay, based on the transfer of label from [gamma-32P]ATP to [32P]phosphoenolpyruvate, suitable for a steady-state kinetic analysis of pyruvate kinase in the reverse direction (i.e. phosphoenolpyruvate synthesis), is described. 2. This assay was used in a kinetic investigation of the rabbit muscle enzyme including initial-rate and product-inhibition experiments, at a pH of 7.4 and constant concentrations of total K+ and free Mg2+. 3. These studies indicate that there is a random release of ADP and phosphoenolpyruvate from the enzyme and that there is a competitive substrate inhibition by ATP. Some of the results were suggestive that the rapid-equilibrium assumption, generally used for this enzyme was not valid. 4. Techniques were developed to measure the rate of isotopic exchange between all the substrate-product pairs. 5. By using these techniques the rates of isotopic exchange at chemical equilibrium were measured. The results indicate that this enzyme does not catalyse a truly rapid-equilibrium random mechanism, although in the forward reaction all initial-rate data obtained to date are consistent with this assumption.     

Evaluation of radioactive and nonradioactive gene probes and cell culture for detection of poliovirus in water samples.

Five nonradioactive probe assays were evaluated by using chemiluminescent and colormetric signals, along with two isotopic assays and cell culture, for the detection of poliovirus in concentrated water samples. In environmental samples, a 100% correlation existed between digoxigenin and single-stranded [32P]RNA probes. All probe assays detected more positive samples than the cell culture did.     

Penicillin acyltransferase in Penicillium chrysogenum

Isotopic exchange of (35)S between penicillins and 6-amino-penicillanic acid (6-APA) was observed in cell-free extracts of Penicillium chrysogenum. Sulfhydryl-containing compounds were required for activity. Dithiothreitol, dithioerythritol, mercaptoethanol, and glutathione served as activators. The acyltransferase was purified threefold by adsorption on calcium phosphate gel at pH 6 and elution at pH 8. The partially purified enzyme showed maximal activity at pH 8. The enzyme was stable at 25 C for at least 30 min at pH 8. Dissociable inhibitors or activators, other than the sulfhydryl-containing compounds, could not be demonstrated in the enzyme preparation. An apparent Michaelis constant of 1.5 +/- 0.5 mm was determined for penicillin G at a 6-APA concentration of 5 mm. The enzyme did not appear to possess penicillin amidase activity. The exchange mechanism probably involves an acyl-enzyme intermediate. Penicillins V, G, K, X, and dihydro F showed isotopic exchange with (35)S-6-APA. Penicillin N, methylpenicillin, and phenyl-penicillin did not show exchange. The level of acyltransferase in P. chrysogenum 51-20F3 was measured at times during the fermentation. The level of activity increased threefold between 40 and 55 hr, remaining high until about 90 hr.     

Temperature effect on leaf water deuterium enrichment and isotopic fractionation during leaf lipid biosynthesis: results from controlled growth of C3 and C4 land plants

The hydrogen isotopic ratios ((2)H/(1)H) of land plant leaf water and the carbon-bound hydrogen of leaf wax lipids are valuable indicators for climatic, physiological, metabolic and geochemical studies. Temperature will exert a profound effect on the stable isotopic composition of leaf water and leaf lipids as it directly influences the isotopic equilibrium (IE) during leaf water evaporation and cellular water dissociation. It is also expected to affect the kinetics of enzymes involved in lipid biosynthesis, and therefore the balance of hydrogen inputs along different biochemical routes. We conducted a controlled growth experiment to examine the effect of temperature on the stable hydrogen isotopic composition of leaf water and the biological and biochemical isotopic fractionations during lipid biosynthesis. We find that leaf water (2)H enrichment at 20°C is lower than that at 30°C. This is contrary to the expectation that at lower temperatures leaf water should be more enriched in (2)H due to a larger equilibrium isotope effect associated with evapotranspiration from the leaf if all other variables are held constant. A hypothesis is presented to explain the apparent discrepancy whereby lower temperature-induced down-regulation of available aquaporin water channels and/or partial closure of transmembrane water channel forces water flow to "detour" to a more convoluted apoplastic pathway, effectively increasing the length over which diffusion acts against advection as described by the Péclet effect (Farquhar and Lloyd, 1993) and decreasing the average leaf water enrichment. The impact of temperature on leaf water enrichment is not reflected in the biological isotopic fractionation or the biochemical isotopic fractionation during lipid biosynthesis. Neither the biological nor biochemical fractionations at 20°C are significantly different from that at 30°C, implying that temperature has a negligible effect on the isotopic fractionation during lipid biosynthesis.     

Phosphorus and nitrogen co-limitation of forest ground vegetation under elevated anthropogenic nitrogen deposition

Amino acid nitrogen isotopic analysis is a relatively new method for estimating trophic position. It uses the isotopic difference between an individual’s ‘trophic’ and ‘source’ amino acids to determine its trophic position. So far, there is no accepted explanation for the mechanism by which the isotopic signals in ‘trophic’ and ‘source’ amino acids arise. Yet without a metabolic understanding, the utility of nitrogen isotopic analyses as a method for probing trophic relations, at either bulk tissue or amino acid level, is limited. I draw on isotopic tracer studies of protein metabolism, together with a consideration of amino acid metabolic pathways, to suggest that the ‘trophic’/‘source’ groupings have a fundamental metabolic origin, to do with the cycling of amino-nitrogen between amino acids. ‘Trophic’ amino acids are those whose amino-nitrogens are interchangeable, part of a metabolic amino-nitrogen pool, and ‘source’ amino acids are those whose amino-nitrogens are not interchangeable with the metabolic pool. Nitrogen isotopic values of ‘trophic’ amino acids will reflect an averaged isotopic signal of all such dietary amino acids, offset by the integrated effect of isotopic fractionation from nitrogen cycling, and modulated by metabolic and physiological effects. Isotopic values of ‘source’ amino acids will be more closely linked to those of equivalent dietary amino acids, but also modulated by metabolism and physiology. The complexity of nitrogen cycling suggests that a single identifiable value for ‘trophic discrimination factors’ is unlikely to exist. Greater consideration of physiology and metabolism should help in better understanding observed patterns in nitrogen isotopic values.     

Changes in the oceanic 13C/12C ratio during the last 140 000 years: High-latitude surface water records

Detailed records of the carbon and oxygen isotopic ratios of Neogloboquadrina pachyderma are compared between nine high-latitude sediment cores, from the Northern and Southern Hemispheres, covering the last 140 000 yrs. The strong analogies between the δ¹³C records permit to define a δ¹³C stratigraphic scale, with three clear cut transitions simultaneous with the oxygen isotopic transitions 6/5 (125 kyrs.), 5/4 (65 kyrs.), and 2/1 (13 kyrs.). The δ¹³C records of N. pachyderma in the high-latitude cores, which follow the changes in δ¹³C of the surface water TCO₂ near areas of deep water formation present trends similar to the benthic foraminifera δ¹³C records in cores V19–30 and M12-392, although amplitudes of the isotopic shifts are different. This implies that a large part of the observed variations represents global changes in the carbon distribution between biosphere and ocean.The ¹³C/¹²C ratios of N. pachyderma in the North Atlantic cores display larger regional variations at 18 kyrs. B.P. than at present. To explain these differences, we have plotted the 18 kyrs. B.P. δ¹³C values of N. pachyderma from 17 cores distributed N of 40°N. Comparison with published surface water temperature distribution at 18 kyrs. B.P. indicates that a strong divergent cyclonic cell, centered approximatively 55°N and 15°W, was active during most of the last ice-age maximum This hydrology, analogous to the present Weddell Sea, explains the published evidences of bottom water formation, if located on the northern flank of the gyre, and the strong polar front on the southern flank, probable location of intermediate water formation.     

Relevance of using whole-ring stable isotopes of black spruce trees in the perspective of climate reconstruction

Studies in dendroisotope chemistry suggested that latewood cellulose contains better climatic records than whole-ring cellulose. However, this approach has never been tested on northeastern Canadian spruce trees. This study compares dendroisotopic series of cellulose from late and whole ring, and analyses their statistical relationships with hydro-climatic variables with the aim of selecting the best suited protocol for future hydro-climatic reconstruction in the downstream sector of Churchill River basin of Labrador, Canada. To this end, δ¹³C and δ¹⁸O series from latewood (LW) and whole ring (WR) α-cellulose of black spruce trees (Picea mariana [Mill.] B.S.P.) were produced for the 1940–2010 period. The results show strong correlations between LW and WR isotopic series suggesting that there are no important variation in the isotopic ratios during the growing year and that black spruce trees use photosynthates of the current growing season to form their earlywood. Moreover, LW and WR δ¹³C and δ¹⁸O show similar relationships with both maximum temperature (T_max) and Churchill River discharge. Correlations are higher when combining δ¹³C and δ¹⁸O for LW and WR. Overall, those correlations support the indirect relationship between tree-ring isotopic series and river discharge, as they are integrators of several climatic variables and derived parameters (T_max, relative humidity, evapotranspiration, etc.). The LW and WR isotopic series give similar statistical relationships with hydro-climatic variables, and the WR treatment is faster (separation easier compared to LW). Thus, for black spruce the use of combined isotopic series in WR can be favored over LW for hydro-climatic reconstruction in the study region.