A simple CO2 equilibration method for measuring blood oxygen isotope compositions.

RATIONALE: Blood water oxygen isotope compositions can provide valuable insights into physiological processes and ecological patterns. While blood samples are commonly drawn for medical or scientific purposes, blood fractions are infrequently measured for oxygen isotopic compositions (δ18 O) because such measurements are time consuming and expensive. METHODS: We sampled blood from sheep, goats, and iguanas raised in field and animal laboratories into serum, EDTA, heparin, and uncoated plastic vials commonly used in medical and scientific research, then separated red blood cell (RBC) and plasma or serum blood fractions. These were injected into helium-flushed Exetainer tubes where they naturally outgassed endogenous CO2 (goat blood), or into He- and CO2 -flushed tubes (iguana blood). The CO2 gas was sampled on a GasBench II system, and δ18 O was measured by an isotope ratio mass spectrometer (IRMS). RESULTS: Repeated δ18 O measurements were stable over multiple days. The addition of desiccated blood solids to water standards had little impact on their δ18 O measurements, suggesting that organic molecular constituents within blood serum and plasma do not interfere with blood water δ18 O values. We observed slight but statistically significant δ18 O offsets between plasma, serum and RBC fractions. Mass-dependent body water turnover times for iguanas were derived from the data. CONCLUSIONS: We demonstrate that a simple blood-CO2 equilibration method using the GasBench can quickly, reliably and accurately characterize water δ18 O in the plasma, RBC, and whole blood fractions of mammalian and reptilian blood samples (precision ≤ 0.1‰). This method will expand the application of blood stable isotope analysis in physiological and medical research.

Determinants of blood water δ 18O variation in a population of experimental sheep: implications for paleoclimate reconstruction.

Mammalian body, blood and hard tissue oxygen isotope compositions (δ 18O values) reflect environmental water and food sources, climate, and physiological processes. For this reason, fossil and archaeological hard tissues, which originally formed in equilibrium with body chemistry, are a valuable record of past climate, landscape paleoecology, and animal physiology and behavior. However, the environmental and physiological determinants of blood oxygen isotope composition have not been determined experimentally from large herbivores. This class of fauna is abundant in Cenozoic terrestrial fossil assemblages, and the isotopic composition of large herbivore teeth has been central to a number of climate and ecological reconstructions. Furthermore, existing models predict blood water, or nearly equivalently body water, δ 18O values based on environmental water sources. These have been evaluated on gross timescales, but have not been employed to track seasonal variation. Here we report how water, food, and physiology determine blood water δ 18O values in experimental sheep (Ovis aries) subjected to controlled water switches. We find that blood water δ 18O values rapidly reach steady state with environmental drinking water and reflect transient events including weaning, seasons, and snowstorms. Behavioral and physiological variation within a single genetically homogenous population of herbivores results in significant inter-animal variation in blood water δ 18O values at single collection times (1 s.d. = 0.1-1.4 ‰, range = 3.5 ‰) and reveals a range of water flux rates (t1/2 = 2.2-2.9 days) within the population. We find that extant models can predict average observed sheep blood δ 18O values with striking fidelity, but predict a pattern of seasonal variation exactly opposite of that observed in our population for which water input variation was controlled and the effect of physiology was more directly observed. We introduce to these models an evaporative loss term that is a function of environmental temperatures. The inclusion of this function produces model predictions that mimic the observed seasonal fluctuations and match observations to within 1.0 ‰. These results increase the applicability of available physiological models for paleoseasonality reconstructions from stable isotope measurements in fossil or archaeological enamel, the composition of which is determined in equilibrium with blood values. However, significant blood δ 18O variation in this experimentally controlled population should promote caution when interpreting isotopic variation in the archaeological and paleontological record.

Caution on the use of NBS 30 biotite for hydrogen-isotope measurements with on-line high-temperature conversion systems.

RATIONALE: The supply of NBS 30 biotite is nearly exhausted. During measurements of NBS 30 and potential replacements, reproducible δ(2)HVSMOW-SLAP values could not be obtained by three laboratories using high-temperature conversion (HTC) systems. The cause of this issue has been investigated using the silver-tube technique for hydrogen-isotope measurements of water. METHODS: The δ(2)HVSMOW-SLAP values of NBS 30 biotite, other biotites, muscovites, and kaolinite with different particle sizes, along with IAEA-CH-7 polyethylene, and reference waters and NBS 22 oil that were sealed in silver-tube segments, were measured. The effect of absorbed water on mineral surfaces was investigated with waters both enriched and depleted in (2)H. The quantitative conversion of hydrogen from biotite into gaseous hydrogen as a function of mass and particle size was also investigated. RESULTS: The δ(2)HVSMOW-SLAP values of NBS 30 obtained by three laboratories were as much as 21 ‰ too high compared with the accepted value of -65.7 ‰, determined by conventional off-line measurements. The experiments showed a strong correlation between grain size and the δ(2)HVSMOW-SLAP value of NBS 30 biotite, but not of biotites with lower iron content. The δ(2)HVSMOW-SLAP values of NBS 30 as a function of particle size show a clear trend toward -65.7 ‰ with finer grain size. CONCLUSIONS: Determination of the δ(2)HVSMOW-SLAP values of hydrous minerals and of NBS 30 biotite by on-line HTC systems coupled to isotope-ratio mass spectrometers may be unreliable because hydrogen in this biotite may not be converted quantitatively into molecular hydrogen. Extreme caution in the use and interpretation of δ(2)HVSMOW-SLAP on-line measurements of hydrous minerals is recommended.

Pressure baseline correction and high-precision CO2 clumped-isotope (∆47) measurements in bellows and micro-volume modes.

RATIONALE: CO(2) 'clumped-isotope' measurements (tracking enrichment of (16)O(13)C(18)O, reported as ∆(47) values, on CO(2) derived from carbonate minerals or the atmosphere) are becoming central to a wide range of geochemical investigations. We present a novel approach to address problems with instrument stability, external precision, and the analysis of small samples that have hampered the advancement of Δ(47) measurements. METHODS: We measured Δ(47) values on CO(2) gases introduced via dual inlet to an isotope ratio mass spectrometer. We developed a method for determining the 'pressure baseline' and integrating a correction to ion beam intensity measurements during analysis. We then tested this approach for both bellows and micro-volume modes of sample introduction. Heated gas and equilibrated gas lines (Δ(47) vs. δ(47)) established the effectiveness of this correction. RESULTS: We have determined that drift in instrument calibration that compromises Δ(47) measurements results from a shift in the baseline signal on sensitive collectors (m/z 47, 48, and 49) that occurs when gas is admitted to the ion source. Applying a 'pressure baseline' (PBL) correction significantly stabilizes ∆(47) measurements and reduces the dependence of ∆(47) values on δ(47) values by up to an order of magnitude. CONCLUSIONS: PBL-corrected heated gas and equilibrated gas calibrations in bellows and micro-volume modes are nearly identical and stable through time. Introduction of the PBL correction, a revision to the absolute reference frame approach to determining Δ(47) values, dramatically improves the external precision of Δ(47) measurements to near instrumental analytical uncertainty (6-8 ppm (1σ) in bellows mode; 10-12 ppm in micro-volume mode).

Half-sandwich iridium complexes for homogeneous water-oxidation catalysis.

Iridium half-sandwich complexes of the types Cp*Ir(N-C)X, [Cp*Ir(N-N)X]X, and [CpIr(N-N)X]X are catalyst precursors for the homogeneous oxidation of water to dioxygen. Kinetic studies with cerium(IV) ammonium nitrate as primary oxidant show that oxygen evolution is rapid and continues over many hours. In addition, [Cp*Ir(H(2)O)(3)]SO(4) and [(Cp*Ir)(2)(µ-OH)(3)]OH can show even higher turnover frequencies (up to 20 min(-1) at pH 0.89). Aqueous electrochemical studies on the cationic complexes having chelate ligands show catalytic oxidation at pH > 7; conversely, at low pH, there are no oxidation waves up to 1.5 V vs NHE for the complexes. H(2)(18)O isotope incorporation studies demonstrate that water is the source of oxygen atoms during cerium(IV)-driven catalysis. DFT calculations and kinetic experiments, including kinetic-isotope-effect studies, suggest a mechanism for homogeneous iridium-catalyzed water oxidation and contribute to the determination of the rate-determining step. The kinetic experiments also help distinguish the active homogeneous catalyst from heterogeneous nanoparticulate iridium dioxide.

Evidence against bicarbonate bound in the O2-evolving complex of photosystem II.

The oxidation of water to molecular oxygen by photosystem II (PSII) is inhibited in bicarbonate-depleted media. One contribution to the inhibition is the binding of bicarbonate to the non-heme iron, which is required for efficient electron transfer on the electron-acceptor side of PSII. There are also proposals that bicarbonate is required for formation of O 2 by the manganese-containing O 2-evolving complex (OEC). Previous work indicates that a bicarbonate ion does not bind reversibly close to the OEC, but it remains possible that bicarbonate is bound sufficiently tightly to the OEC that it cannot readily exchange with bicarbonate in solution. In this study, we have used NH 2OH to destroy the OEC, which would release any tightly bound bicarbonate ions from the active site, and mass spectrometry to detect any released bicarbonate as CO 2. The amount of CO 2 per PSII released by the NH 2OH treatment is observed to be comparable to the background level, although N 2O, a product of the reaction of NH 2OH with the OEC, is detected in good yield. These results strongly argue against tightly bound bicarbonate ions in the OEC.

Speciation of the catalytic oxygen evolution system: [MnIII/IV2(mu-O)2(terpy)2(H2O)2](NO3)3 + HSO5-.

[MnIII/IV2(-O)2(terpy)2(OH2)2](NO3)3 (1, where terpy = 2,2':6'2' '-terpyridine) + oxone (2KHSO5 x KHSO4 x K2SO4) provides a functional model system for the oxygen-evolving complex of photosystem II that is based on a structurally relevant Mn-(-O)2-Mn moiety (Limburg, J.; et al. J. Am. Chem. Soc. 2001, 123, 423-430). In this study, electron paramagnetic resonance, ultraviolet-visible spectroscopy, electrospray ionization mass spectrometry, X-ray absorption spectroscopy, and gas-phase stable isotope ratio mass spectrometry were utilized to identify the title compounds in the catalytic solution. We find that (a) O2 evolution does not proceed through heterogeneous catalysis by MnO2 or other decomposition products, that (b) O atoms from solvent water are incorporated into the evolved O2 to a significant extent but not into oxone, that (c) the MnIII/IV2 title compound 1 is an active precatalyst in the catalytic cycle of O2 evolution with oxone, while the MnIV/IV2 oxidation state is not, and that (d) the isotope label incorporation in the evolved O2, together with points a-c above, is consistent with a mechanism involving competing reactions of oxone and water with a "MnV=O" intermediate in the O-O bond-forming step.