CO2 Hydroxylation Research Articles

Mineralogical and environmental effects on the δ13C, δ18O, and clumped isotope composition of modern bryozoans

Extensive bryozoan fossil records date back to the early Ordovician, forming an important part of sedimentary archives, yet the applicability of classical 18‑oxygen thermometry to bryozoan carbonate is still a matter of debate, with mineralogical and biological issues still hindering paleoclimate reconstructions. Complementing other methods (i.e., δ18O, Mg/Ca), clumped-isotope thermometry (Δ47) could provide more accurate paleotemperature estimates and help identify potential biotic factors influencing the isotopic composition of bryozoans. Here we report on the first investigation of clumped-isotope thermometry applied to bryozoan carbonate, spanning a broad range of modern species living in different environments from two localities (Atlantic Ocean and Mediterranean Sea). We confirm that bryozoan δ13C and δ18O records are affected by biotic and abiotic factors susceptible to bias in growth condition estimates. Our Atlantic bryozoans yield Δ47 derived temperatures (T47) consistent with spring/fall seawater temperature (but only after correcting for minor mineralogical effects), reflecting seasonal growth bias or, more likely, moderate isotopic disequilibrium. By contrast, Mediterranean samples display large positive offsets from Δ47 equilibrium values. We propose that this stronger disequilibrium is related to the higher salinity at this site, decreasing Carbonic Anhydrase activity, favoring CO2 hydroxylation over hydration, and slowing down DIC (dissolved inorganic carbon) equilibration reactions at the precipitation site. Our findings highlight how “vital effects” in bryozoans is not only species-specific as often assumed for other biocalcifiers, but could also depend on mineralogy and environmental factors.

Triple oxygen isotope systematics of CO2 hydroxylation

Kinetic isotope effects occurring during carbonate precipitation impact the accuracy of paleoclimate records. One rate-limiting process is CO2 absorption, which, at high pH, primarily proceeds through hydroxylation, where aqueous CO2 and OH− react to form HCO3−. In this study, we investigated the triple oxygen isotope fractionation (18O/16O and 17O/16O) associated with the hydroxylation reaction. We experimentally determined the extent of the kinetic isotope effects superimposed on the reacting CO2 and OH− during hydroxylation, using high-pH precipitation reactions. These results were compared to the equilibrium triple oxygen isotope fractionation between H2O and OH− modeled using quantum chemical computations.Our quantum chemical modeling confirms the previously suggested equilibrium θeq.H2O/OH- of 0.5296 at 25 °C. Our empirical data show a kinetic isotope effect superimposed on the OH− involved in the hydroxylation reaction of approximately −16.4‰ for the fractionation of 18O/16O and a corresponding slope of 0.514 in triple oxygen isotope space. The θeffectiveH2O/OH- for the fractionation between water and the reacting OH− is 0.523.In order to identify and potentially correct kinetic isotope effects in carbonates, it is important to understand the fractionations occurring during precipitation. Our results enable a more accurate modeling of kinetic isotope effects in triple oxygen isotope space, particularly the slope of hydroxylation. The θhydroxylation is dependent on the isotope composition of the ambient water, CO2, and the temperature. We estimate that θhydroxylation is approximately 0.532 for carbonates growing in 25 °C seawater.

A robust methodology for triple (∆47, ∆48, ∆49) clumped isotope analysis of carbonates

Analysis of ∆48 and ∆47 values of CO2 released from phosphoric acid digestion of carbonates enables the rate-limiting kinetic processes involved in carbonate mineralization to be identified and formation temperatures to be determined by accounting for these kinetic biases. This method has the benefit of only requiring analysis of a single carbonate phase. To resolve temperature from the kinetic information, mass spectrometric measurements of highest accuracy and precision are required.Here, we outline a robust methodology for high-precision triple (∆47, ∆48, ∆49) clumped isotope analysis using turbo-pumped gas and carbonate preparation lines, a common acid bath and the dual inlet of a Thermo Scientific 253plus gas source mass spectrometer, equipped with additional m/z47.5 half mass cup. By analysis of >400 replicates each, we report long-term (50 months) ∆47, CDES 90 and ∆48, CDES 90 values for three internationally accepted (ETH-1, -2 and -3) and two internal carbonate standards (GU1, Carrara) unbiased by scale compression. In order to obtain highest accuracy and precisions closest to shot noise limit it is essential to account for a sample size effect, correct raw intensities for a negative background utilizing the half mass cup, avoid pressure imbalances between sample and reference gas and distribute samples evenly across a given analytical session.We confirm ∆47, CDES 90 values that were recently assigned to ETH-1 and ETH-2. Our long-term average ∆47, CDES 90 value of 0.6032 ‰ for ETH-3, on the contrary, occurs 10 ppm lower than its recently assigned value. As a consequence, ∆47, CDES 90 values of samples having relatively low temperatures of formation are not consistent with ∆47, I-CDES values. We show that GU1, a synthetic carbonate deriving from the hydroxylation of CO2 and exhibiting a strong anti-clumped ∆48, CDES 90 value, appears to be homogeneous in its isotopic composition. If used in combination with ETH-1, −2 and − 3, it may allow projection of background corrected raw data without any bias into ∆47-∆48 CDES 90 space, provided our long-term dual clumped isotope values for these carbonate anchors are considered.Stability of mass spectrometric measurements is related to filament age and/or room temperature fluctuations and can be monitored using reference gas m/z49 intensity. Under the most stable analytical conditions, the ∆49, CDES 90 values of equilibrated gases and carbonates reveal a trend with formation temperature. Using data from the most stable sessions, we present a temperature calibration for ∆49, CDES 90 in CO2 evolved from phosphoric acid digestion of carbonates.

The mechanisms and drivers of lithification in slag‐dominated artificial ground

AbstractUnconsolidated artificial ground is an ever‐increasing feature on the Earth's surface but it poses various challenges such as pollutant release and ground instability. The process of lithification could be an important factor in changing the properties of artificial ground and ameliorating these challenges. In this study, a lithified deposit of a furnace slag associated with a former iron and steel works in Scotland was analysed to determine the mechanisms and drivers of lithification. Scanning electron microscope analysis showed that Ca leached from around the edges of clasts of slag through reaction of the chemically unstable slag with water from an adjacent water body. Dissolution of Ca (and OH−) from the slag caused the water in contact with the slag to become hyperalkaline, facilitating ingassing and hydroxylation of CO2 from the atmosphere (fingerprinted through carbon isotope analysis). Reaction of the dissolved Ca and CO2 led to precipitation of calcite. Scanning electron microscope analysis showed the calcite is distributed between slag clasts, forming rims around clasts and cementing clasts together into a solid rock‐like mass. Understanding the mechanisms and drivers of lithification in artificial ground will be important, given its widespread nature particularly in urban areas where artificial ground is the substrate of most development.

Dual clumped isotope thermometry of coral carbonate

The stable (δ18O and δ13C) and clumped (Δ47) isotope compositions of coral carbonate are valuable archives for paleoclimate reconstructions. However, the Δ47-temperature relationships of warm and cold-water corals deviate from that of inorganic carbonate precipitated at equilibrium. Dual clumped isotope thermometry of carbonates (i.e., simultaneous Δ47 and Δ48 measurements on a single carbonate) has the potential to achieve more accurate paleotemperature reconstruction, identifying and correcting for kinetically driven isotopic disequilibrium. Here we present the first extensive dual clumped isotope dataset of coral carbonate, spanning a broad range of cold and warm-water coral species. We confirm that corals are enriched in Δ47 and depleted in Δ48 relative to equilibrium, a pattern corresponding to the mixing of an equilibrium DIC pool with kinetically derived HCO3– produced by hydration and hydroxylation of CO2. Dual clumped isotope measurements of cold-water corals fall on the initial linear portion of model (IsoDIC) predicted departure from equilibrium. The dual clumped isotope composition of cold-water corals, corrected by the model-predicted Δ47/Δ48 offset slope (−0.78), yield accurate reconstruction of coral growth temperature with a precision of <3 °C at the 68% confidence level. In contrast, disequilibrium offsets in the Δ47 and Δ48 of warm-water corals correspond to precipitation from a more equilibrated DIC pool, which we attribute to the action of carbonic anhydrase in the calcifying fluid. It may be possible to correct warm-water coral growth temperatures, using an empirically derived correction (Δ47/ Δ48 offset slope of −0.4). Dual clumped isotope thermometry of coral carbonate opens new possibilities to reconstruct both sea surface temperatures and ocean dynamics of intermediate to deep water masses.

Trajectory and timescale of oxygen and clumped isotope equilibration in the dissolved carbonate system under normal and enzymatically-catalyzed conditions at 25 °C

The abundance of 18O isotopes and 13C-18O isotopic “clumps” (measured as δ18O and Δ47, respectively) in carbonate minerals have been used to infer mineral formation temperatures. An inherent requirement or assumption for these paleothermometers is mineral formation in isotopic equilibrium. Yet, apparent disequilibrium is not uncommon in biogenic and abiogenic carbonates formed in nature and in synthetic carbonates prepared under laboratory settings, as the dissolved carbonate pool (DCP) from which minerals precipitate is often out of δ18O and Δ47 equilibrium. For this, a complete understanding of both equilibrium and kinetics of isotopic partitioning and 13C-18O clumping in DCP is crucial. To this end, we analyzed Δ47 of inorganic BaCO3 samples from Uchikawa and Zeebe (2012) (denoted as UZ12), which were quantitatively precipitated from NaHCO3 solutions at various times over the course of isotopic equilibration at 25 °C and pHNBS of 8.9. Our data show that, although the timescales for δ18O and Δ47 equilibrium in DCP are relatively similar, their equilibration trajectories are markedly different. As opposed to a simple unidirectional and asymptotic approach toward δ18O equilibrium (first-order kinetics), Δ47 equilibration initially moves away from equilibrium and then changes its course towards equilibrium. This excess Δ47 disequilibrium is manifested as a characteristic “dip” in the Δ47 equilibration trajectory, a feature consistent with an earlier study by Staudigel and Swart (2018) (denoted as SS18). From the numerical model of SS18, the non-first-order kinetics for Δ47 equilibration can be understood as a result of the difference in the exchange rate for oxygen isotopes bound to 12C versus 13C, or an isotope effect of ~25‰. We also developed an independent model for the Exchange and Clumping of 13C and 18O in DCP (ExClump38 model) to trace the evolution of singly- and doubly-substituted isotopic species (i.e., δ13C, δ18O and Δ47). The model suggests that the dip in the Δ47 equilibration trajectory is due largely to kinetic carbon isotope fractionation for hydration and hydroxylation of CO2. We additionally examined the BaCO3 samples prepared from NaHCO3 solutions supplemented with carbonic anhydrase (CA), an enzyme known to facilitate δ18O equilibration in DCP by catalyzing CO2 hydration (UZ12). These samples revealed that, while CA effectively shortens the time required for Δ47 equilibrium in DCP, the overall pattern and magnitude of the dip in the Δ47 equilibration trajectory remain unchanged. This suggests no additional isotope effects due to the CA enzyme within the tested CA concentrations. With the ExClump38 model, we test various physicochemical scenarios for the timescales and trajectories of isotopic equilibration in DCP and discuss their implications for the Δ47 paleothermometry.

Isotopic fractionation accompanying CO2 hydroxylation and carbonate precipitation from high pH waters at The Cedars, California, USA

The Cedars ultramafic block hosts alkaline springs (pH > 11) in which calcium carbonate forms upon uptake of atmospheric CO2 and at times via mixing with surface water. These processes lead to distinct carbonate morphologies with “floes” forming at the atmosphere-water interface, “snow” of fine particles accumulating at the bottom of pools and terraced constructions of travertine. Floe material is mainly composed of aragonite needles despite CaCO3 precipitation occurring in waters with low Mg/Ca (<0.01). Precipitation of aragonite is likely promoted by the high pH (11.5–12.0) of pool waters, in agreement with published experiments illustrating the effect of pH on calcium carbonate polymorph selection.The calcium carbonates exhibit an extreme range and approximately 1:1 covariation in δ13C (−9 to −28‰ VPDB) and δ18O (0 to −20‰ VPDB) that is characteristic of travertine formed in high pH waters. The large isotopic fractionations have previously been attributed to kinetic isotope effects accompanying CO2 hydroxylation but the controls on the δ13C-δ18O endmembers and slope have not been fully resolved, limiting the use of travertine as a paleoenvironmental archive. The limited areal extent of the springs (∼0.5 km2) and the limited range of water sources and temperatures, combined with our sampling strategy, allow us to place tight constraints on the processes involved in generating the systematic C and O isotope variations.We develop an isotopic reaction–diffusion model and an isotopic box model for a CO2-fed solution that tracks the isotopic composition of each dissolved inorganic carbon (DIC) species and CaCO3. The box model includes four sources or sinks of DIC (atmospheric CO2, high pH spring water, fresh creek water, and CaCO3 precipitation). Model parameters are informed by new floe Δ44Ca data (−0.75 ± 0.07‰), direct mineral growth rate measurements (4.8 to 8 × 10−7 mol/m2/s) and by previously published elemental and isotopic data of local water and DIC sources. Model results suggest two processes control the extremes of the array: (1) the isotopically light end member is controlled by the isotopic composition of atmospheric CO2 and the kinetic isotope fractionation factor (KFF (‰) = (α − 1) × 1000) accompanying CO2 hydroxylation, estimated here to be −17.1 ± 0.8‰ (vs. CO2(aq)) for carbon and −7.1 ± 1.1‰ (vs. ‘CO2(aq) + H2O’) for oxygen at 17.4 ± 1.0 °C. Combining our results with revised CO2 hydroxylation KFF values based on previous work suggests consistent KFF values of −17.0 ± 0.3‰ (vs. CO2(aq)) for carbon and −6.8 ± 0.8‰ for oxygen (vs. ‘CO2(aq) + H2O’) over the 17–28 °C temperature range. (2) The isotopically heavy endmember of calcium carbonates at The Cedars reflects the composition of isotopically equilibrated DIC from creek or surface water (mostly HCO3-, pH = 7.8–8.7) that occasionally mixes with the high-pH spring water. The bulk carbonate δ13C and δ18O values of modern and ancient travertines therefore reflect the proportion of calcium carbonate formed by processes (1) and (2), with process (2) dominating the carbonate precipitation budget at The Cedars. These results show that recent advances in understanding kinetic isotope effects allow us to model complicated but common natural processes, and suggest ancient travertine may be used to retrieve past meteoric water δ18O and atmospheric δ13C values. There is evidence that older travertine at The Cedars recorded atmospheric δ13C that predates large-scale combustion of fossil fuels.

Equilibrium and kinetic isotopic fractionation in the CO2 hydration and hydroxylation reactions: Analysis of the role of hydrogen-bonding via quantum mechanical calculations

The formation of H2CO3 and HCO3− from CO2 is common in aqueous environmental systems and occurs via the CO2 hydration and hydroxylation reactions. The reactions are especially important in the context of CaCO3 mineral precipitation. The reactions carry with them equilibrium and kinetic isotope fractionations that impact paleoclimate proxy records. Herein, quantum mechanical calculations of dissolved inorganic carbon species embedded in H2O clusters are analyzed under a transition state theory framework to predict kinetic isotope fractionations associated with the reactions. Experimental measurements of reaction energetics and equilibrium isotope fractionations are well-reproduced by the models which suggests the computational methods are justified.The CO2 hydration reaction discriminates against 13C by 19–23‰ and against 18O by 14–15‰ at 25 °C. The CO2 hydroxylation reaction discriminates against 13C by 26–31‰ and against 18O by 27–30‰ at 25 °C. Our analyses of the model isotopic fractionations lead to the conclusion that hydrogen-bonds and C(CO2)-O(H2O or OH−) distances influence the calculated values. We infer that when the model H-bond patterns more realistically represent the aqueous species, the accuracy and precision of equilibrium fractionation factor prediction is improved.Implications for coral carbonate paleothermometry are discussed. Results indicate that hydration and hydroxylation δ13C-δ18O slopes bound the values of slopes form corals and abiotic precipitation experiments, albeit with key uncertainties related to precipitation fractionations. The process of regressing molecular model fractionation results against H-bond patterns as presented here is a promising way to improve isotope fractionation calculations in aqueous systems.

Evidence for archaeal methanogenesis within veins at the onshore serpentinite-hosted Chimaera seeps, Turkey

Serpentinite-hosted ecosystems are potential sites where life may first have evolved on Earth. Serpentinization reactions produce strongly reducing and highly alkaline fluids that are typified by high concentrations of molecular hydrogen (H2) and methane (CH4), which can be used as an energy source by chemosynthetic life. Low-temperature serpentinization at slow-spreading mid-ocean ridges provides an ideal environment for rich microbial communities. Similar environments have also been discovered on land, where present-day low temperature serpentinization occurs during the circulation of groundwater through exposed ophiolites, triggering the production of CH4 and H2, as well as the precipitation of secondary carbonate minerals. The rock samples analyzed here are from the Chimaera seeps in Turkey, representing serpentinized peridotites that are cross-cut by veins composed of brucite and hydromagnesite. Hydromagnesite features a mean δ13C value of −19.8‰ caused by kinetic isotope fractionation during air-groundwater exchange of CO2, followed by CO2 hydroxylation to bicarbonate within the groundwater. Geochemical modeling revealed that mixing of Mg- and Ca-rich groundwaters is required for hydromagnesite formation at the expense of brucite. Within the carbonate-hydroxide veins the lipid biomarkers pentamethylicosane (PMI) and squalane with δ13C values of +10‰ and +14‰, respectively, and unsaturated derivatives thereof were identified. Archaeol, sn2-hydroxyarchaeol, and sn3-hydroxyarchaeol are other prominent archaeal biomarkers in the veins, also revealing high δ13C values from +6 to +13‰. These isotope patterns combined with the absence of crocetane – a biomarker for methanotrophic archaea – reveal that the microbial communities of the Chimaera seeps performed methanogenesis from a CO2-limited pool rather than methanotrophy. Moreover, bacterial dialkyl glycerol diethers (DAGEs) with unusually high δ13C values (−9 to −2‰) and minor monoalkyl glycerol monoethers (MAGEs) were identified, suggesting that bacterial sulfate reduction is also active at the Chimaera site. This study reveals that archaeal methanogenesis and bacterial sulfate reduction may be prominent at onshore peridotite-hosted sites, and that biogenic CH4 may contribute to abiotic CH4 emissions from terrestrial seeps.

Multi-isotope (Ba, C, O) partitioning during experimental carbonatization of a hyper-alkaline solution

Carbonates formed from hyperalkaline aqueous solutions at the Earth́s surface are known to bear the most extreme disequilibrium isotope signatures reported so far in nature. We present here the results for stable carbon (C), oxygen (O), and barium (Ba) isotope fractionation during the precipitation of witherite (BaCO3) induced by the chemical absorption of atmospheric carbon dioxide (CO2) into an aqueous hyper-alkaline solution (at 4° and 21 °C; 1 atm total pressure). Independent from temperature, the barium carbonate formation was associated with a substantial enrichment of the lighter C and O isotopes in the solid compared to the atmosphere (C, O), close to previous results found in experiments and nature. A new approach is introduced to explain oxygen isotope fractionation upon hydroxylation of CO2. With Ba isotope enrichment factors between −0.45 and −0.53‰ (138/134ε) or −0.34 and −0.40‰ (137/134ε), respectively, the synthesized BaCO3 displays the highest kinetic enrichment of the light Ba isotope in the carbonate solid reported so far.

Controls on the stable isotope compositions of travertine from hyperalkaline springs in Oman: Insights from clumped isotope measurements

Carbonate formation at hyperalkaline springs is typical of serpentinization in peridotite massifs worldwide. These travertines have long been known to exhibit large variations in their carbon and oxygen isotope compositions, extending from apparent equilibrium values to highly depleted values. However, the exact causes of these variations are not well constrained. We analyzed a suite of well-characterized fresh carbonate precipitates and travertines associated with hyperalkaline springs in the peridotite section of the Samail ophiolite, Sultanate of Oman, and found their clumped isotope compositions vary systematically with formation environments. Based on these findings, we identified four main processes controlling the stable isotope compositions of these carbonates. These include hydroxylation of CO2, partial isotope equilibration of dissolved inorganic carbon, mixing between isotopically distinct carbonate end-members, and post-depositional recrystallization. Most notably, in fresh crystalline films on the surface of hyperalkaline springs and in some fresh carbonate precipitates from the bottom of hyperalkaline pools, we observed large enrichments in Δ47 (up to ∼0.2‰ above expected equilibrium values) which accompany depletions in δ18O and δ13C, yielding about 0.01‰ increase in Δ47 and 1.1‰ decrease in δ13C for every 1‰ decrease in δ18O, relative to expected equilibrium values. This disequilibrium trend, also reflected in preserved travertines ranging in age from modern to ∼40,000years old, is interpreted to arise mainly from the isotope effects associated with the hydroxylation of CO2 in high-pH fluids and agrees with our first-order theoretical estimation. In addition, in some fresh carbonate precipitates from the bottom of hyperalkaline pools and in subsamples of one preserved travertine terrace, we observed additional enrichments in Δ47 at intermediate δ13C and δ18O, consistent with mixing between isotopically distinct carbonate end-members. Our results suggest that carbonate clumped isotope analysis can be a valuable tool for identifying and distinguishing processes not readily apparent from the carbonate bulk stable isotope compositions alone, e.g., kinetic effects or mixing of different carbonate end-members, which can significantly alter both the apparent formation temperatures and apparent radiocarbon ages. The isotope trends observed in these travertine samples could be applied more broadly to identify extinct hyperalkaline springs in terrestrial and extraterrestrial environments, to better constrain the formation conditions and post-depositional alteration of hyperalkaline spring carbonates, and to extract potential paleoclimate information.

The effect of carbonic anhydrase on the kinetics and equilibrium of the oxygen isotope exchange in the CO2–H2O system: Implications for δ18O vital effects in biogenic carbonates

Interpretations of the primary paleoceanographic information recorded in stable oxygen isotope values (δ18O) of biogenic CaCO3 can be obscured by disequilibrium effects. CaCO3 is often depleted in 18O relative to the δ18O values expected for precipitation in thermodynamic equilibrium with ambient seawater as a result of vital effects. Vital effects in δ18O have been explained in terms of the influence of fluid pH on the overall δ18O of the sum of dissolved inorganic carbon (DIC) species (often referred to as “pH model”) and in terms of 18O depletion as a result of the kinetic effects associated with CO2 hydration (CO2+H2O↔H2CO3↔HCO3−+H+) and CO2 hydroxylation (CO2+OH−↔HCO3−) in the calcification sites (so-called “kinetic model”). This study addresses the potential role of an enzyme, carbonic anhydrase (CA), that catalyzes inter-conversion of CO2 and HCO3− in relation to the underlying mechanism of vital effects. We performed quantitative inorganic carbonate precipitation experiments in order to examine the changes in 18O equilibration rate as a function of CA concentration. Experiments were performed at pH 8.3 and 8.9. These pH values are comparable to the average surface ocean pH and elevated pH levels observed in the calcification sites of some coral and foraminiferal species, respectively. The rate of uncatalyzed 18O exchange in the CO2–H2O system is governed by the pH-dependent DIC speciation and the kinetic rate constant for CO2 hydration and hydroxylation, which can be summarized by a simple mathematical expression. The results from control experiments (no CA addition) are in agreement with this expression. The results from control experiments also suggest that the most recently published kinetic rate constant for CO2 hydroxylation has been overestimated. When CA is present, the 18O equilibration process is greatly enhanced at both pH levels due to the catalysis of CO2 hydration by the enzyme. For example, the time required for 18O equilibrium is nearly halved by the presence of 3.7×10−9M of CA used for the experiments. Despite its significant influence on the oxygen isotope exchange kinetics, the equilibrium oxygen isotope fractionation between individual DIC species and H2O is unaffected by CA. Because many CaCO3-secreting organisms possess active CA, our findings imply that 18O equilibration of the CO2–H2O system is possible within realistic timescales of biogenic calcification.

Dual effect of carbonic anhydrase inhibitors on H+ transport by the turtle bladder.

Previous studies in isolated turtle bladder have demonstrated that high concentrations of carbonic anhydrase (CA) inhibitors limit H+ transport (JH) by reducing the catalyzed rate of CO2 hydroxylation and also by inhibiting some other step in the acidification process. One possibility is that these inhibitors alter the energy substrate requirement for JH. Recent work has demonstrated that JH may be dependent in part on glucose oxidation via the pentose shunt (PS). The present study was undertaken to determine whether CA inhibitors exert a direct effect on PS metabolism by turtle bladder. Acetazolamide and benzolamide at concentrations of 5 X 10(-4) M significantly reduced the rate of 14CO2 evolution from [1-14C]- but not [6-14C]glucose after JH was abolished by an adverse electrochemical gradient for JH+. These changes are consistent with a reduction in PS metabolism. These same sulfonamides also reduced glucose-6-phosphate dehydrogenase (G-6-PD) activity in mucosal cell homogenates. Acetazolamide decreased the Vmax of G-6-PD but not the Km and, therefore, appears to be a noncompetitive inhibitor of G-6-PD with an estimated Ki of 10(-4) M. The t-butyl analogue of acetazolamide, CL 13850, which is without CA inhibitory activity, had no measurable effect on G-6-PD activity. Accordingly, it is suggested the sulfonamide CA inhibitors may reduce JH by two modes of action, inhibition of CA and inhibition of G-6-PD.

Current status of membrane-bound carbonic anhydrase.

We have studied the kinetic properties, and susceptibility to inhibition, of cytoplasmic and membrane carbonic anhydrase from dog kidneys, and attempted to place the data in the context of earlier work on this subject. The cytoplasmic enzyme thus far seems the same as human red cell carbonic anhydrase C, on the basis of kinetics, inhibition, amino acid composition and immunochemistry. On the other hand, the membrane enzyme is quite a different protein from either the cytoplasmic, or human red cell B or C. This enzyme is found in both luminal (brush border) and antiluminal (basolateral) fractions, and there appear no differences between the two. The turnover number (kcat) lies between those of B and C, and susceptibility to sulfonamide inhibition is two to 135-fold less than for the cytoplasmic enzyme, depending on the drug used. The usual difference is about fivefold. The KI for acetazolamide against the membrane enzyme is 10(-7) M, so that at the renal concentrations achieved at the usual in vivo doses (approximately 20 mg/kg) or used in current in situ perfusion work (both 10(-4) M) the enzyme is 99.9% inhibited. A striking difference between the membrane carbonic anhydrase and cytoplasmic or red cell B or C is its resistance to inhibition by halions. At 0.5 M chloride, there is no effect, whereas for the other three types inhibition ranges from 70%-99%. The membrane renal enzyme is also immunologically distinct from the other three types. The membrane enzyme has activity in its native state, but can be solubilized without loss of activity by treatment with Triton or sodium dodecyl sulfate. The actions of the renal carbonic anhydrases are depicted in a scheme that takes into account the protolysis of water, the attraction of H+ and of OH (and HCO3-) to the luminal and antiluminal membranes respectively, and the catalytic hydration and hydroxylation of CO2.

Effect of acetazolamide on the otolith growth of goldfish.

In order to clarify the involvement of a functional carbonic anhydrase (CA) system in the otolith formation of the goldfish, Carassius auratus, acetazolamide, a specific CA inhibitor, was injected intraperitoneally every 3 or 4 days, and its inhibitory effect on the otolith growth was examined by means of a tetracycline labeling technique. Calcium-45 deposition on the otolith was also examind after a single injection of the drug. Given in multiple doses of 50mg per Kg of body weight, acetazolamide did not reduce the growth rate of the otolith on either dorsal or ventral side. With multiple doses of 100mg, however, the dorsal growth was significantly depressed by 17%.The ventral growth was not affected. Simialarly, calcium-45 deposition on the otolith was effectively reduced (39%) only when a dose of 100mg was given. These results suggest that, if involved, the enzyme-catalyzed hydration or hydroxylation of CO2 is not indispensable to the carbonate formation of the otolith.

Transport of H plus and other electrolytes across isolated gastric mucosa of the rabbit

The relationship of transmural and cellular phenomena to the rate of spontaneous luminal HH+ secretion by short-circuited gastric mucosa bathed in HCO3- and CO2-free Ringer solution was studied by the pH-stat technique. The bulk of luminal acidification can be accounted for by the appearance of H+ and not by organic acids. Acid secretion requires the presence of O2 and exogenous substrate but is not limited by endogenous CO2 production. The rate of H+ secretion is matched by the serosal appearance of alkali. The greater appearance of CO2 on the serosal side of the mucosa is consistent with hydroxylation of CO2 and greater permeability of the serosal border to HCO3-. Luminal changes in H+ concentrations affect H+ secretion but serosal changes do not, suggesting that the gradient produced by H+ secretion is at the luminal border. Steady-state isotopic Na+ and Cl- fluxes indicate net secretion of both ions, but net K+ transport is negligible.