Yizhou Wang

Michael R Blatt
email: michael.blatt@glasgow.ac.uk

ANION CHANNEL SENSITIVITY TO CYTOSOLIC ORGANIC ACIDS IMPLICATES A CENTRAL ROLE FOR OXALOACETATE IN INTEGRATING ION FLUX WITH METABOLISM IN STOMATAL GUARD CELLS

Keywords: Stomatal guard cell / stomatal movement, diurnal / organic acid metabolism / anion channel, voltage-gated / voltage clamp / Ca 2+ concentration, cytosolic-free Biochemical

Stomatal guard cells play a key role in gas exchange for photosynthesis and in minimizing transpirational water loss from plants by opening and closing the stomatal pore. The bulk of the osmotic content driving stomatal movements depends on ionic fluxes across both the plasma membrane and tonoplast, the metabolism of organic acids, primarily malate (Mal), its accumulation and loss. Anion channels at the plasma membrane are thought to comprise a major pathway for Mal efflux during stomatal closure, implicating their key role in linking solute flux with metabolism. Nonetheless, little is known of the regulation of anion channel current (I Cl ) by cytosolic malate or its immediate metabolite oxaloacetate (OAA). We have examined the impact of Mal, OAA, and of the monocarboxylic acid anion acetate in guard cells of Vicia faba L. and report that all three organic acids affect I Cl , but with markedly different characteristics and sidedness to their activities. Most prominent was a suppression of I Cl by OAA within the physiological range of concentrations found in vivo. These findings indicate a capacity for OAA to coordinate organic acid metabolism with I Cl through the direct effect of organic acid pool size. Our findings also add perspective to in vivo recordings using acetate-based electrolytes.

INTRODUCTION

Stomata are pores that provide the major route for gas exchange across the impermeable cuticle of leaves and stems [START_REF] Hetherington | The role of stomata in sensing and driving environmental change[END_REF]. They open and close in response to exogenous and endogenous signals and thereby control the exchange of gases, most importantly water vapour and CO 2 , between the interior of the leaf and the atmosphere. The guard cells surrounding the stomatal also find that Mal as well as its precursor oxaloacetate (OAA) suppress I Cl in a voltageindependent manner when present on the cytosolic side of the membrane, the effect of OAA evident well-within its physiological concentration range. These findings point to control of the anion channels by OAA, indicating the capacity for feedback control from the cellular metabolic activity and a much more subtle 'fine tuning' of the anion channels by metabolism than previously anticipated. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

MATERIAL AND METHODS

Plant growth and preparation

Vicia faba L. cv. Bunyard Exhibition was grown on potting mix and perlite (70:30) at 22 o C and 60% relative humidity with 200 μmol m -2 sec -1 photosynthetic photon flux density under a 16/8 h day/night cycle. Epidermal strips were prepared from newly expanded leaves of 4-to 6-weekold plants as described previously [START_REF] Blatt | K + channels of stomatal guard cells: characteristics of the inward rectifier and its control by pH[END_REF]. Epidermal peels were affixed to the glass bottom of the experimental chamber after coating the chamber surface with an optically clear and pressuresensitive silicone adhesive and all operations were carried out on an Axiovert S100TV microscope (Zeiss, Oberkochen, Germany) fitted with Nomarski differential interference contrast optics. Measurements were conducted in continuous flowing solutions controlled by a gravityfed system at a rate of 20 chamber volumes min -1 . The standard perfusion medium contained 15 mM TEA-Cl, 15 mM CsCl and 5 mM 2-(N-morpholino)propanesulphonic acid (MES), titrated with Ca(OH) 2 to pH 6.1 ([Ca 2+ ] = 1 mM). The Na + salts of organic acids and other chemicals were from Sigma (Poole, UK) unless otherwise specified.

Electrophysiology

Surface area and volume of impaled epidermal cells were calculated assuming a cylindrical geometry using Henry IV EP software (www.psrg.org.uk/Software/Henry.htm). Single and double--barrelled microelectrodes were prepared and coated with paraffin to reduce capacitance as described previously [START_REF] Blatt | K + channels of stomatal guard cells: characteristics of the inward rectifier and its control by pH[END_REF][START_REF] Blatt | Electrical characteristics of stomatal guard cells: the contribution of ATP-dependent, "electrogenic" transport revealed by current-voltage and difference-currentvoltage analysis[END_REF][START_REF] Blatt | Concepts and techniques in plant membrane physiology[END_REF]. Current-and voltage-recording barrels were filled with 100 mM CsCl with additions as indicated. For measurements including organic acids in the microelectrode, K + salts of the acids were used and the electrolyte solution was adjusted to pH 7.5 with CsOH and HCl as necessary to avoid cytosolic acid or alkaline loading. Microelectrodes were connected to amplfier headstages via 1 M KCl | Ag-AgCl halfcells, and a 1 M KCl agar bridge served as the reference electrode. Voltage clamp recordings were carried out using Henry IV EP software using a two-electrode clamp circuit [START_REF] Blatt | Electrical characteristics of stomatal guard cells: the contribution of ATP-dependent, "electrogenic" transport revealed by current-voltage and difference-currentvoltage analysis[END_REF][START_REF] Blatt | Concepts and techniques in plant membrane physiology[END_REF] with an additional μP amplifier (Y-Science). Current records were filtered using an eight-pole active Bessel filter (f c , 0.3 kHz) and clamp current and voltage data were digitized at 1 kHz. Free-running voltages between voltage clamp recordings were digitized at 10 Hz.

Numerical analyses

All recordings were analyzed and leak currents subtracted using standard methods [START_REF] Blatt | Concepts and techniques in plant membrane physiology[END_REF] with Henry IV software. Where appropriate ata are presented as means ±SE of n observations, and differences validated by Student's T-test or ANOVA. Curve fittings were by nonlinear leastsquares using a Marquardt-Levenberg algorithm [START_REF] Marquardt | An algorithm for fleast-squaires estimation of nonlinear parameters[END_REF]. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

RESULTS

Previous studies indicated an activation of anion currents by extracellular Mal in isolated protoplasts [START_REF] Hedrich | Malate-induced feedback regulation of plasma membrane anion channels could provide a CO 2 sensor to guard cells[END_REF], but supporting evidence from intact guard cells has been equivocal [START_REF] Raschke | The slow and the quick anion conductance in whole guard cells: their voltage-dependent alternation, and the modulation of their activities by abscisic acid and CO 2[END_REF]. We recorded I Cl with a two-electrode voltage clamp in intact guard cells, bathing the cells in 15 mM CsCl and 15 mM tetraethylammonium chloride (TEA-Cl), and using microelectrodes filled with 100 mM CsCl. Diffusion of Cs + from the microelectrode and its presence with TEA + outside blocks all K + currents and Cl -diffusion from the microelectrode provides an effective 'substrate clamp' loading the cytosol within the first 5-10 min following impalements to enhance current carried by the anion [START_REF] Blatt | KCl leakage from microelectrodes and its impact on the membrane parameters of a nonexcitable cell[END_REF][START_REF] Blatt | Electrical characteristics of stomatal guard cells: the ionic basis of the membrane potential and the consequence of potassium chloride leakage from microelectrodes[END_REF][START_REF] Grabov | Parallel control of the inward-rectifier K + channel by cytosolic-free Ca 2+ and pH in Vicia guard cells[END_REF]. Under these conditions, the membrane was dominated by I Cl similar to that reported previously with instantaneous current comprising both anion channels and characteristics in the steady-state predominantly of the S-type anion current [START_REF] Schroeder | Two types of anion channel currents in guard cells with distinct voltage regulation[END_REF][START_REF] Grabov | Parallel control of the inward-rectifier K + channel by cytosolic-free Ca 2+ and pH in Vicia guard cells[END_REF][START_REF] Chen | Dynamic regulation of guard cell anion channels by cytosolic free Ca 2+ concentration and protein phosphorylation[END_REF].

Biphasic activation of I Cl by external Mal

To test the effects of Mal outside, after impalements we superfused guard cells with malate concentrations up to 20 mM. Figure 1A summarises results from 10 independent experiments, for clarity showing data only for the 0, 1 and 10 mM Mal, including recordings from one guard cell at the three Mal concentrations (insets below). Clamp voltage steps to +40 mV led to a slow rise in outward current over 10 s (not shown), and subsequent clamp steps to voltages negative of approximately -40 mV yielded substantial inward-directed and instantaneous I Cl that relaxed to steady-state values near -5 to -10 μA cm -2 within 3-5 s. Both mean instantaneous and, to a lesser extent, steady-state (inset above) I Cl were enhanced by 1 mM Mal. Amplitudes of the mean instantaneous current increased by 70-75% at voltages negative from -100 mV (from -52±7 to -91±9 μA cm -2 at -220 mV) and near the negative maximum at -75 mV the mean steady-state current increased by approximately 60% from -13±1 to -22±3 μA cm -2 . Analogous to previous results [START_REF] Hedrich | Malate-induced feedback regulation of plasma membrane anion channels could provide a CO 2 sensor to guard cells[END_REF], we observed roughly a -30 mV displacement in the voltage giving maximum steady-state I Cl in 1 mM Mal and a marginally greater displacement in 10 mM Mal (inset above). However, in 10 mM Mal both instantaneous and steady-state I Cl were reduced close to control values in the absence of Mal, and similar results were obtained with 20 mM Mal (not shown). To assess further Mal action on I Cl gating, we fitted I Cl relaxations to derive the halftime, t 1/2 , for current deactivation at voltages negative of -40 mV for all three sets of data. Figure 1B shows the voltage-dependence of t 1/2 for I Cl [START_REF] Grabov | Parallel control of the inward-rectifier K + channel by cytosolic-free Ca 2+ and pH in Vicia guard cells[END_REF] and underscores the biphasic action of Mal on the current: whereas 1 mM Mal resulted in an increase in t 1/2 consistent with a slowing of its relaxation, especially at move negative voltages (inset), additions of 10 mM Mal yielded t 1/2 values statistically indistinguishable from the control at all voltages.

Cytosolic Mal suppresses I Cl

Although implicit in the connection to cellular metabolism, to date almost nothing is known about the influence of Mal on I Cl from the cytosolic side of the membrane. Guard cells of many species, including Vicia, accumulate high concentrations of Mal to balance K + uptake during stomatal opening [START_REF] Willmer | [END_REF][START_REF] Raschke | Availability of chloride affects balance between potassium chloride and potassium malate in guard cells of Vicia faba L[END_REF][START_REF] Vankirk | Presence of chloride reduces malate production in epidermis during stomatal opening[END_REF]. Much of this Mal is distributed to the vacuole, but concentrations of 1-8 mM have been estimated in the cytosol [START_REF] Willmer | [END_REF][START_REF] Martinoia | Malate compartmentation -responses to a complex metabolism[END_REF]. To examine the effect of Mal on I Cl from inside, we impaled guard cells with double-barrelled microelectrodes as before, but with concentrations between 1 mM and 20 mM Mal in addition to 100 mM CsCl filling the microelectrode barrels to load the cytosol by diffusion from the microelectrode. Figure 2 summarises data from 10 or more independent experiments with each loading solution, in each case allowing at least 10 min following impalements for loading from the microelectrode [START_REF] Blatt | KCl leakage from microelectrodes and its impact on the membrane parameters of a nonexcitable cell[END_REF][START_REF] Blatt | Electrical characteristics of stomatal guard cells: the ionic basis of the membrane potential and the consequence of potassium chloride leakage from microelectrodes[END_REF][START_REF] Grabov | Parallel control of the inward-rectifier K + channel by cytosolic-free Ca 2+ and pH in Vicia guard cells[END_REF]. Recordings from guard cells impaled with 1 mM Mal in the microelectrodes showed a statistically significant increase in I Cl between -40 mV and -100 mV in the steady-state (Fig. 2A), A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

but the effect on the instantaneous current amplitude was marginal (inset) and no effect was evident in mean t 1/2 values for I Cl deactivation at any voltage (Fig. 2B). By contrast, recordings from guard cells impaled with 10 mM Mal in the microelectrodes yielded a reduction in instantaneous I Cl compared to the control, albeit with a smaller effect on the steady-state current (Fig. 2A) and greatly reduced t 1/2 (Fig. 2B) independent of membrane voltage. Similar results were obtained with 20 mM Mal (not shown), and fittings to a hyperbolic function for the t 1/2 and amplitude of I Cl (see Fig. 8) yielded apparent K i s for Mal of 3±1 mM and 10±3 mM, respectively. Thus, Mal suppressed I Cl on transition to negative voltages, accelerating deactivation and reducing the magnitude of the current, but the effect on current amplitude was evident primarily at the upper end of the phyisological range of Mal concentrations.

Cytosolic oxaloacetate blocks I Cl

Oxaloacetate (OAA) is the four-carbon precursor in the formation of cytosolic malate from phosphoenolpyruvate and immediate product of malate oxidation; it is an intermediate in several metabolic pathways -including glycolysis, amino acid synthesis and in the mitochondrial tricarboxylic acid cycle -and it exchanges with malate across both chloroplast and mitochondrial membranes [START_REF] Willmer | [END_REF][START_REF] Martinoia | Malate compartmentation -responses to a complex metabolism[END_REF]. Direct measurements of steady-state OAA concentration in the cytosol are not available, but estimates based on energy charge and Mal/OAA exchange transport equilibria suggest values of 0.1-0.5 mM and possibly as high as 3 mM in the light [START_REF] Heineke | Redox transfer across the inner chloroplast envelope membrane[END_REF]. Given its central importance to Mal balance, we examined the effects on I Cl of OAA additions both outside and inside the guard cells. We found that OAA concentrations as high as 10 mM added outside had no appreciable effect on instantaneous or steady-state I Cl (Fig. 3A), nor did it alter channel gating when quantified using current relaxations on deactivation at negative voltages (Fig. 3B). By contrast, cytosolic OAA loads introduced from the microelectrodes had a substantial effect on all three parameters. Adding 1 and 10 mM OAA inside suppressed the instantaneous I Cl approximately 58% and 70%, respectively, compared to the control and independent of membrane voltage (Fig. 4A). A similar block of steady-state I Cl was observed in each case (Fig. 4A, inset) and I Cl kinetics showed a substantial acceleration in current deactivation, notable even with 1 mM OAA and again largely independent of membrane voltage (Fig. 4B). Fitting to a hyperbolic function (see Fig. 8) yielded apparent K i s for OAA in accelerating I Cl deactivation and reducing the current amplitude of 0.17±0.08 mM and 0.21±0.05 mM, respectively, that is well within the physiological range of OAA concentrations. Additions of OAA, both inside and outside the guard cells had no significant effect on the reversal voltage for I Cl -as evidenced by a lack of significant difference in currents recorded either side of the apparent reversal voltage (see Fig. 4 legend) -indicating that the relative permeability of the channels for OAA is very low compared to that for Cl -and Mal.

Given the potentially counteractive effects of the organic acids in the cytosol we also examined whether OAA suppression of I Cl was rescued in the presence of Mal. In this case, guard cells were impaled with microelectrodes containing 1 mM OAA and 1 mM Mal and I Cl recorded as before. Figure 5 summarises results from 11 independent experiments with both organic acids. We found that OAA suppressed both instantaneous and steady-state I Cl (Fig. 5A) and accelerated current deactivation (Fig. 5B). This effect was only partially ameliorated by the presence of low Mal concentrations that enhanced I Cl (see Fig. 2) and, again, the influence of both organic acids was voltage independent (Fig. 5B, inset). These results suggest that the immediate metabolic partner of Mal may have an important role in control of I Cl under normal physiological conditions. A c c e p t e d M a n u s c r i p t Finally, we explored the impact of acetate (Ac) on I Cl . The two-carbon acid anion is a substrate for the tricarboxylic acid cycle and fatty acid synthesis, but in a thioester complex with Coenzyme A and normally occurs as the free acid at concentrations of 0.5-1.5 mM [START_REF] Roughan | Acetate concentrations in leaves are sufficient to drive in vivo fattyacid synthesis at maximum rates[END_REF]. Nonetheless, previous electrophysiological studies have used the free acid anion in microelectrode filling solutions to avoid cytosolic Cl -loading and its consequences for solute balance and background Cl -leakage across the membrane [START_REF] Blatt | KCl leakage from microelectrodes and its impact on the membrane parameters of a nonexcitable cell[END_REF][START_REF] Blatt | Electrical characteristics of stomatal guard cells: the ionic basis of the membrane potential and the consequence of potassium chloride leakage from microelectrodes[END_REF][START_REF] Blatt | Reversible inactivation of K + channels of Vicia stomatal guard cells following the photolysis of caged inositol 1,4,5-trisphosphate[END_REF][START_REF] Garcia-Mata | Nitric oxide regulates K + and Clchannels in guard cells through a subset of abscisic acidevoked signaling pathways[END_REF][START_REF] Roelfsema | Ion channels in guard cells of Arabidopsis thaliana (L) Heynh[END_REF]. In the absence of information on the Ac-sensitivity of I Cl , we challenged guard cells with 1 and 10 mM Ac by additions outside and to the microelectrode filling solution. Figures 6 and7 summarise results of 13 or more independent experiments for each treatment. We found in every case that increasing Ac concentrations suppressed the instantaneous and steady-state I Cl , in the latter case most noticeably at voltages between -40 and -100 mV, and it accelerated current deactivation. Unlike the effects of Mal and OAA, however, Ac showed a pronounced voltage dependence in its effect on I Cl kinetics, the reduction in t 1/2 being most apparent at voltages near and positive of -100 mV and when Ac was added to the cytosolic side of the membrane (Figs. 6B and7B), and it led to a shift in the apparent reversal potential for I Cl (Fig. 6A and 7A, insets). These results indicate a more complex interaction of Ac with the anion channels and we return to this point below.

Acetate electrolytes impact on I Cl

DISCUSSION

Both inorganic ion transport and organic acid metabolism make major contributions to the changes in osmotic content of the guard cells that drives stomatal movements [START_REF] Schroeder | Guard cell signal transduction[END_REF][START_REF] Blatt | Cellular signaling and volume control in stomatal movements in plants[END_REF][START_REF] Roelfsema | In the light of stomatal opening: new insights into 'the Watergate[END_REF][START_REF] Shimazaki | Light regulation of stomatal movement[END_REF]. During the diurnal cycle of stomatal opening and closing, the guard cells of most plant species accumulate and subsequently lose significant quantities of K + , Cl -and Mal, the latter offsetting Cl -especially later in the daylight period [START_REF] Raschke | Availability of chloride affects balance between potassium chloride and potassium malate in guard cells of Vicia faba L[END_REF][START_REF] Talbott | Sugar and organic acid accumulation in guard cells of Vicia faba in response to red and blue light[END_REF][START_REF] Talbott | Central roles for potassium and sucrose in guard cell osmoregulation[END_REF][START_REF] Asai | The effect of osmotic stress on the solutes in guard cells of Vicia faba L[END_REF]. Much of the Mal synthesized in the light is transported and accumulates in the vacuole [START_REF] Martinoia | Malate compartmentation -responses to a complex metabolism[END_REF], but is later transported out of the vacuole to be metabolised or released across the the plasma membrane when the stomata close [START_REF] Vankirk | Presence of chloride reduces malate production in epidermis during stomatal opening[END_REF][START_REF] Dittrich | Malate metabolism in isolated epidermis of Commelina communis L. in relation to stomatal functioning[END_REF]. Thus transport, notably of Cl -and Mal, must be regulated in concert with metabolism, both as Mal is synthesized for transport to the vacuole and again as it re-enters the cytosol for export and to feed into the metabolic pools of other organic acids. Very little is known of the mechanisms behind this regulation, although it is clearly central to the osmotic homeostasis of guard cells. We have examined the impact of cytosolic Mal, its immediate metabolite OAA, and of Ac on I Cl , the current carried by anion channels at the plasma membrane of Vicia guard cells. Although we cannot discount some degree of metabolic conversion of the acid anions during these experiments, both the extracellular medium and the microelectrode present virtually infinite volumes, the latter exchanging with the cytosol directly. Thus, at steady-state the membrane surface will have been exposed to organic acid concentrations effectively 'clamped' close to that of the corresponding solutions [START_REF] Blatt | KCl leakage from microelectrodes and its impact on the membrane parameters of a nonexcitable cell[END_REF][START_REF] Blatt | Electrical characteristics of stomatal guard cells: the ionic basis of the membrane potential and the consequence of potassium chloride leakage from microelectrodes[END_REF]. We found that all three organic acids affected I Cl , but with markedly different characteristics and sidedness to their activities. Most prominent was the suppression of I Cl with OAA evident even at low millimolar concentrations. These findings indicate a capacity for coordinating organic acid metabolism with I Cl through the direct effect of organic acid pool size and they suggest that the effects may be exerted as much by Mal metabolites as by Mal itself. They also add perspective to in vivo recordings using Ac-based electrolytes.

Malate, oxaloacetate and I Cl regulation

One of the most surprising of our findings was that I Cl is substantially more sensitive to OAA in the cytosol than it is to Mal (Fig. 8). Much as previously reported [START_REF] Hedrich | Malate-induced feedback regulation of plasma membrane anion channels could provide a CO 2 sensor to guard cells[END_REF][START_REF] Schmidt | Anion selectivity of slow anion channels in the plasma membrane of guard cells -large nitrate permeability[END_REF], we observed that low concentrations of Mal outside affected the voltage dependence of the current. We also observed an enhancement of I Cl with 1 mM Mal in the cytosol, although elevation of cytosolic Mal to 10-20 mM led to measurable declines in I Cl relative to the control (Fig. 2). Additions even of 1 mM OAA, however, resulted in a significant block of I Cl consistent with an apparent K i near 0.1 mM and well within the dynamic concentration range for OAA in vivo [START_REF] Heineke | Redox transfer across the inner chloroplast envelope membrane[END_REF]. Mal synthesis in the cytosol of guard cells is fed largely from the pools of phosphoenolpyruvate and OAA [START_REF] Willmer | [END_REF]. The flux through these metabolites will be balanced in part with Mal transport across the tonoplast and can lead to large accumulations of Mal in the vacuole especially in the second half of the diurnal cycle [START_REF] Willmer | [END_REF][START_REF] Talbott | Sugar and organic acid accumulation in guard cells of Vicia faba in response to red and blue light[END_REF]. By contrast, stomatal closure at the end of the day or following stimulation with abscisic acid must be accompanied by export of much of the Mal in the vacuole and its elimination through metabolism and loss across the plasma membrane [START_REF] Willmer | [END_REF][START_REF] Roelfsema | In the light of stomatal opening: new insights into 'the Watergate[END_REF]. These observations are generally consistent with variations in the low millimolar range previously reported for the pool of cytosolic Mal in leaves [START_REF] Martinoia | Malate compartmentation -responses to a complex metabolism[END_REF]. However, such 'channelling' of Mal, in the first instance into the vacuole and in the second to the apoplast, implies a substantial hysteresis in Mal flux control overall and a fine control over Mal export both at the tonoplast and at the plasma membrane during the course of the day.

If the cytosolic Mal pool remains within the bounds of 0.5-10 mM, how might I Cl be regulated to prevent undue Mal efflux during stomatal opening yet provide a major pathway for its loss during stomatal closure? One obvious mechanism rests with increasing [Ca 2+ ] i that enhance the activity of I Cl and both R-and S-type anion channels [START_REF] Schroeder | Two types of anion channel currents in guard cells with distinct voltage regulation[END_REF][START_REF] Hedrich | Malate-induced feedback regulation of plasma membrane anion channels could provide a CO 2 sensor to guard cells[END_REF][START_REF] Chen | Dynamic regulation of guard cell anion channels by cytosolic free Ca 2+ concentration and protein phosphorylation[END_REF][START_REF] Grabov | Alteration of anion channel kinetics in wild-type and abi1-1 transgenic Nicotiana benthamiana guard cells by abscisic acid[END_REF][START_REF] Geiger | Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca 2+ affinities[END_REF], in effect promoting Mal (and Cl -) efflux across the plasma membrane. This mechanism is certainly important in the response of guard cells to ABA and drought stress [START_REF] Schroeder | Guard cell signal transduction[END_REF][START_REF] Blatt | Cellular signaling and volume control in stomatal movements in plants[END_REF], but it is more difficult to reconcile the diurnal cycle of [Ca 2+ ] i with that of Mal synthesis and accumulation in the day and release at night. Guard cells undergo diurnal changes in [Ca 2+ ] i , but the range of free concentrations in the steady-state [START_REF] Dodd | The Arabidopsis circadian clock incorporates a cADPR-based feedback loop[END_REF][START_REF] Dodd | The plant clock shows its metal: circadian regulation of cytosolic free Ca 2+[END_REF] is substantially below the K 1/2 of 600-700 nM for I Cl activation by [Ca 2+ ] i [START_REF] Chen | Dynamic regulation of guard cell anion channels by cytosolic free Ca 2+ concentration and protein phosphorylation[END_REF]. Furthermore, the diurnal oscillation in [Ca 2+ ] i is 180 o out of phase with the changes that might be expected to modulate Mal in guard cells [START_REF] Dodd | The plant clock shows its metal: circadian regulation of cytosolic free Ca 2+[END_REF]: [Ca 2+ ] i increases in the day and declines at night, whereas the opposite would be required to facilitate Mal accumulation in the day and its efflux through the anion channels at night. In short, the diurnal changes in Mal do not sit well with the characteristics of I Cl modulation by [Ca 2+ ] i in guard cells.

The sensitivity of I Cl to OAA does lend itself to another mechanism however. It is generally thought that much of the fixed carbon in guard cells is imported and passes through glycolysis before condensation with bicarbonate to form OAA and Mal [START_REF] Willmer | [END_REF][START_REF] Asai | Role of malate synthesis mediated by phosphoenolpyruvate carboxylase in guard cells in the regulation of stomatal movement[END_REF][START_REF] Outlaw | Integration of cellular and physiological functions of guard cells[END_REF]. OAA is formed through the action of phosphoenol pyruvate carboxylase, which activated by its phosphorylation in the light thus suppressing its sensitivity to inhibition by Mal [START_REF] Outlaw | Requirements for activation of the signal-transduction network that leads to regulatory phosphorylation of leaf guard-cell phosphoenolpyruvate carboxylase during fusicoccinstimulated stomatal opening[END_REF][START_REF] Cotelle | Potential strong regulation of guard cell phosphoenolpyruvate carboxylase through phosphorylation[END_REF]. Thus, we might anticipate that sucrose uptake and metabolism to form Mal in the light will be accompanied by an elevation in the cytosolic OAA pool, even if NAD-malate dehydrogenase is activated by light [START_REF] Gotow | Light activation of NADP-malate dehydrogenase in guard cell protoplasts from Vicia faba L[END_REF]; conversely, carbon passage through Mal and OAA to PEP and pyruvate and transfer of both Mal and pyruvate to the mitochondria at the end of the day [START_REF] Willmer | [END_REF] is likely to be accompanied by a relative depletion of this same OAA pool. In other words, diurnal variations in cytosolic OAA are potentially in phase with the expected oscillation between Mal retention and stomatal opening during the day and I Cl -mediated Mal release and stomatal closure at night. The K m for OAA of cytosolic NAD-malate dehydrogenase [START_REF] Gotow | Light activation of NADP-malate dehydrogenase in guard cell protoplasts from Vicia faba L[END_REF] is very close to the apparent K i for OAA of I Cl in Vicia guard cells (Figs. 4 and8), thereby ensuring a close coordination between the two kinetic processes. We note, too, that low millimolar OAA inside suppresses I Cl even in the presence of Mal (Figs. 4 and5). So it is conceivable that diurnal variations in the cytosolic OAA could play an important role in regulating I Cl and Mal efflux across the plasma membrane. Indeed, in this context it is of interest to note that the diurnal oscillation of the OAA pool in various CAM species is also in phase with stomatal movements [START_REF] Chen | A comparative study on diurnal changes in metabolite levels in the leaves of three crassulacean acid metabolism (CAM) species, Ananas comosus, Kalanchoe daigremontiana and K. pinnata[END_REF], albeit with the higher OAA concentration and stomatal opening at night.

Electrophysiological studies with acetate electrolytes

Finally it is of interest that I Cl showed a sensitivity to Ac. The monocarboxylic acid is normally present at low concentrations [START_REF] Roughan | Acetate concentrations in leaves are sufficient to drive in vivo fattyacid synthesis at maximum rates[END_REF] relative to its action on the current (Figs. 6 and7), and is therefore unlikely to have an impact on I Cl activity in vivo. Effects of weak-acid loading with Ac can also be ruled out because, counter to the the evidence of Figs. 6 and7, cytosolic acidification enhances anion channel activity [START_REF] Schulzlessdorf | GCAC1 recognizes the pH gradient across the plasma membrane: a pH-sensitive and ATP-dependent anion channel links guard cell membrane potential to acid and energy metabolism[END_REF]. The action of higher Ac concentrations is relevant, however, because the caboxylic acid anion is commonly used as an electrolyte substitute to avoid the consequences of Cl -loading via the microelectrode [START_REF] Blatt | KCl leakage from microelectrodes and its impact on the membrane parameters of a nonexcitable cell[END_REF][START_REF] Blatt | Electrical characteristics of stomatal guard cells: the ionic basis of the membrane potential and the consequence of potassium chloride leakage from microelectrodes[END_REF][START_REF] Blatt | Reversible inactivation of K + channels of Vicia stomatal guard cells following the photolysis of caged inositol 1,4,5-trisphosphate[END_REF][START_REF] Garcia-Mata | Nitric oxide regulates K + and Clchannels in guard cells through a subset of abscisic acidevoked signaling pathways[END_REF][START_REF] Roelfsema | Ion channels in guard cells of Arabidopsis thaliana (L) Heynh[END_REF]. It is clear that this practice does more than simply avoiding the effects of elevating Cl -in the cytosol: Ac loading leads to a block of I Cl . Our results -including parallel effects for Ac added outside and inside, the apparent shift in instantaneous current and current reversal voltage, and the strong voltage dependence to its action on t 1/2 (Figs. 6 and7) -implies a complex dependence on Ac residence within the channel pore itself; furthermore, the voltage-dependence to Ac action (Figs. 6B and7B) and the more pronounced effect on instantaneous I Cl when added outside (Fig. 6A) may indicate a greater sensitivity of the R-type anion channels to Ac. Regardless, however, the block of I Cl has had the serendipitous advantage of simplifying the kinetic analysis of the guard cell K + channels and other currents through their isolation from anion flux through I Cl .

From a physiological standpoint, the block of I Cl by Ac will not influence other membrane currents under voltage clamp. However, it may well affect the balance of ionic currents under free-running (non-clamped) conditions. For example, suppressing I Cl might be expected to promote Ca 2+ entry and [Ca 2+ ] i elevation initially by favouring membrane hyperpolarisation, even with low millimolar concentrations of K + outside [START_REF] Blatt | Cellular signaling and volume control in stomatal movements in plants[END_REF][START_REF] Pandey | Roles of ion channels and transporters in guard cell signal transduction[END_REF][START_REF] Blatt | Hormonal control of ion channel gating[END_REF]. Raising [Ca 2+ ] i above 500-600 nM normally promotes a burst in I Cl activity [START_REF] Chen | Dynamic regulation of guard cell anion channels by cytosolic free Ca 2+ concentration and protein phosphorylation[END_REF], but this activity is likely to be much suppressed by an Ac load with a consequent loss of the 'damping' effect of I Cl in promoting membrane depolarisation. In short, Ac loading may have led to an overestimate of the normal dynamic range for [Ca 2+ ] i in its coupling with oscillations with membrane voltage [START_REF] Grabov | Membrane voltage initiates Ca 2+ waves and potentiates Ca 2+ increases with abscisic acid in stomatal guard cells[END_REF][START_REF] Sokolovski | Protein phosphorylation is a prerequisite for intracellular Ca 2+ release and ion channel control by nitric oxide and abscisic acid in guard cells[END_REF]. Indeed, preliminary analysis using quantitative kinetic modelling of guard cell ion transport and homeostasis has suggested that [Ca 2+ ] i oscillations are likely to be constrained with an upper limit near 600 nM largely as a result of I Cl activation [START_REF] Hills | Quantitative Dynamic Systems Modelling of Guard Cell Membrane Transport[END_REF].

In conclusion, we find that the anion channel current of Vicia guard cells shows a compound dependence, from the cytosolic side of the membrane, on several organic acids. Most notable, suppression of I Cl by OAA is consistent with a role for this dicarboxylic acid in modulating channel activity for a diurnal cycle of Mal accumulation and release that parallels the cycle of stomatal opening and closing. Additionally, block of I Cl by the monocarboxylic acid Ac demonstrates that its use in electrophysiological studies has serendipitous consequences for the study of other ion channels at the guard cell plasma membrane.

Biochemical

  Journal Immediate Publication. Published on 11 Jul 2011 as manuscript BJ20110845 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110845

Biochemical

  Journal Immediate Publication. Published on 11 Jul 2011 as manuscript BJ20110845 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110845

Biochemical

  Journal Immediate Publication. Published on 11 Jul 2011 as manuscript BJ20110845 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110845

Biochemical

  Journal Immediate Publication. Published on 11 Jul 2011 as manuscript BJ20110845 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110845

  Biochemical Journal Immediate Publication. Published on 11 Jul 2011 as manuscript BJ20110845 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110845A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure legends: Fig. 1 .

 1 Figure legends: Fig. 1. External Mal shows a biphasic modulation of I Cl in Vicia guard cells. (A) Instantaneous anion current as a function of voltage from 10 independent experiments as means ±SE. Measurements were carried out in 5 mM Ca 2+ -MES buffer, pH 6.1, with 15 mM TEA-Cl and 15 mM CsCl alone and with additions of 1-20 mM Mal. Data for 0 (), 1 () and 10 mM (-) Mal only shown for clarity. Voltages clamped from a conditioning voltage of +40 mV to voltages between +40 and -220 mV in 10-s steps. Inset (below): representative current traces with 0 indicated on left. Scale: 50 μA cm -2 (vertical), 5 s (horizontal). Inset (above): Steady-state current-voltage curves derived from the same measurements (crossreferenced by symbol). (B) Current relaxation halftimes (t 1/2 ) as a function of voltage. Bars are means ±SE from the same 10 independent experiments (above) for 0 mM (solid), 1 mM (open) and 10 mM Mal (diagonal-hashed). Inset: Relaxation halftimes plotted relative to the control without Mal for 1 mM () and 10 mM () Mal.

Fig. 2 .

 2 Fig. 2. Internal Mal shows a marginal stimulation of I Cl at low millimolar concentrations in Vicia guard cells. (A) Steady-state anion current as a function of voltage as means ±SE from 10 or more independent experiments at each Mal concentration. Measurements were carried out in 5 mM Ca 2+ -MES buffer, pH 6.1, with 15 mM TEA-Cl and 15 mM CsCl. Guard cells were impaled with double-barrelled microelectrodes filled with 100 mM CsCl alone () and with additions 1-20 mM Mal; data for 1 mM () and 10 mM (-) Mal only shown for clarity (see also Fig. 8). Voltages clamped from a conditioning voltage of +40 mV to voltages between +40 and -220 mV in 10-s steps as in Fig. 1. Inset (above): Instantaneous current-voltage curves derived from the same measurements (cross-referenced by symbol). (B) Current relaxation halftimes (t 1/2 ) as a function of voltage. Bars are means ±SE from the same experiments (above) for 0 mM (solid), 1 mM (open) and 10 mM Mal (diagonalhashed). Inset: Relaxation halftimes plotted relative to the control without Mal for 1 mM () and 10 mM () Mal.

Fig. 3 .

 3 Fig. 3. I Cl in Vicia guard cells shows no appreciable sensitivity to external OAA. (A) Instantaneous anion current as a function of voltage from 15 independent experiments as means ±SE. Measurements were carried out in 5 mM Ca 2+ -MES buffer, pH 6.1, with 15 mM TEA-Cl and 15 mM CsCl alone and with additions of 1-10 mM OAA. Data for 0 (), 1 () and 10 mM (-) Mal only shown for clarity. Voltages clamped from a conditioning voltage of +40 mV to voltages between +40 and -220 mV in 10-s steps as in Fig. 1. Inset (above): Steady-state current-voltage curves derived from the same measurements (cross-referenced by symbol). (B) Current relaxation halftimes (t 1/2 ) as a function of voltage. Bars are means ±SE from the same experiments (above) for 0 mM (solid), 1 mM (open) and 10 mM Mal (diagonalhashed). Inset: Relaxation halftimes plotted relative to the control without OAA for 1 mM () and 10 mM () OAA.

Fig. 4 .

 4 Fig. 4. Internal OAA suppresses I Cl at low millimolar concentrations in Vicia guard cells. (A) Instantaneous anion current as a function of voltage as means ±SE from 10 or more independent experiments at each OAA concentration. Measurements were carried out in 5 mM Ca 2+ -MES buffer, pH 6.1, with 15 mM TEA-Cl and 15 mM CsCl. Guard cells were impaled with double-barrelled microelectrodes filled with 100 mM CsCl alone () and with

additions 1 -

 1 10 mM OAA; data for 1 mM () and 10 mM (-) Mal only shown for clarity (see also Fig.8). Voltages clamped from a conditioning voltage of +40 mV to voltages between +40 and -220 mV in 10-s steps as in Fig.1. Statistical analysis by ANOVA indicated that currents recorded at -15 and +12 mV were not significantly different at P<0.2, indicating a lack of discernable difference in the reversal voltages for the current under the three experimental conditions. Inset (above): Steady-state current-voltage curves derived from the same measurements (cross-referenced by symbol). (B) Current relaxation halftimes (t 1/2 ) as a function of voltage. Bars are means ±SE from the same experiments (above) for 0 mM (solid), 1 mM (open) and 10 mM Mal (diagonalhashed). Inset: Relaxation halftimes plotted relative to the control without Mal for 1 mM () and 10 mM () Mal.

Fig. 5 .

 5 Fig. 5.Internal OAA suppresses I Cl in the presence of Mal in Vicia guard cells. (A) Instantaneous anion current as a function of voltage as means ±SE from 11 or more independent experiments for each combination of treatments. Measurements were carried out in 5 mM Ca 2+ -MES buffer, pH 6.1, with 15 mM TEA-Cl and 15 mM CsCl. Guard cells were impaled with double-barrelled microelectrodes filled with 100 mM CsCl alone () and with additions 1 mM Mal without () and with1 mM OAA (-). Voltages clamped from a conditioning voltage of +40 mV to voltages between +40 and -220 mV in 10-s steps as in Fig. 1. Inset (above): Steady-state current-voltage curves derived from the same measurements (cross-referenced by symbol). (B) Current relaxation halftimes (t 1/2 ) as a function of voltage. Bars are means ±SE from the same experiments (above) for 0 mM Mal and OAA (solid), 1 mM Mal without (open) and with 1 mM OAA (diagonal-hashed). Inset: Relaxation halftimes plotted relative to the control without additions for 1 mM Mal alone () and with 1 mM OAA ().

Fig. 6 .

 6 Fig. 6. I Cl in Vicia guard cells show a sensitivity to external Ac. (A) Steady-state anion current as a function of voltage from 13 independent experiments as means ±SE. Measurements were carried out in 5 mM Ca 2+ -MES buffer, pH 6.1, with 15 mM TEA-Cl and 15 mM CsCl alone () and with additions of 1 mM () and 10 mM (-) Ac. Voltages clamped from a conditioning voltage of +40 mV to voltages between +40 and -220 mV in 10-s steps as in Fig. 1. Inset (above): Instantaneous current-voltage curves derived from the same measurements (cross-referenced by symbol). (B) Current relaxation halftimes (t 1/2 ) as a function of voltage. Bars are means ±SE from the same experiments (above) for 0 mM (solid), 1 mM (open) and 10 mM Ac (diagonal-hashed). Inset: Relaxation halftimes plotted relative to the control without Ac for 1 mM () and 10 mM () Ac.

Fig. 7 .V 5 BVBiochemicalB

 75 Fig. 7. I Cl in Vicia guard cells is suppressed by internal Ac. (A) Steady-state anion current as a function of voltage from 15 or more independent experiments as means ±SE. Measurements were carried out in 5 mM Ca 2+ -MES buffer, pH 6.1, with 15 mM TEA-Cl and 15 mM CsCl alone () and with additions of 1 mM () and 10 mM (-) Ac. Voltages clamped from a conditioning voltage of +40 mV to voltages between +40 and -220 mV in 10-s steps as in Fig. 1. Inset (above): Instantaneous current-voltage curves derived from the same measurements (cross-referenced by symbol). (B) Current relaxation halftimes (t 1/2 ) as a function of voltage. Bars are means ±SE from the

5 B 5 ABiochemical 8 Biochemical

 558 Fig 5

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Acknowledgements

We thank Amparo Ruiz-Prado for preparing plant materials and Dr. Zhonghua Chen for helpful discussion during the preparation of the manuscript. This work was supported by BBSRC grants BB/F001673/1 and BB/F001630/1 to MRB. YW is holds China Scholarship Council Studentship with the University of Glasgow. Biochemical Journal Immediate Publication. Published on 11 Jul 2011 as manuscript BJ20110845 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110845

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.


Iwan V Kityk

Mikhail G Brik

Leszek ; Jaroszewicz

Artur ; Wojciechowski

Andrzej Majchrowski

Spectroscopy of YAl3(BO3)4:Cr3+ crystals following first

Keywords:

come

Introduction

The rare earth doped YAl 3 (BO 3 ) 4 (YAB)single crystals are of particular interest due to existence of an interesting physical properties like incommensurate phase [1]; possibility to obtain nanopowder phosphors [2], defect -induced phase transitions [3] existence of up-and down-conversions [4].

Particular interest present the Cr doped YAB crystals. In particularly in the Ref. 5 it was found the piezo-induced second-order nonlinear optical effect in polyvinyl alcohol/YAB:Cr nanocomposites and influence of YAB:Cr 3+ nanocrystallite sizes [6]. These materials are of special interest for the light emitting diodes [7]. These crystals have a broad fluorescence ranging from the red to near-IR spectral region, and their lifetimes were measured as 170 µs and 87 µs, respectively. The emission cross sections are estimated to be 0.8 ×10 -20 and 1.6 ×10 -20 cm 2 , respectively. The vibronic levels of their ground state were estimated to be ≈1400 cm -1 , so they can provide a four-level system [8]. The optical gain of Cr 3+ :YAB crystal was measured in the range from 744 nm to 852.5 nm. The maximum single-pass gain was 1.9 at 830 nm. Second harmonic generation in the UV region (375 nm) was obtained by a Cr 3+ :YAB Cr 3+ :YAB crystal and Cr 3+ :YGAB crystal have potential as new optically operated materials [9].

To vary the properties of the crystals in the desired direction it is necessary to have the parameters of the band energy structure for the crystals doped by the dopants. This can help to clarify the transfer of the carrier during the interaction with laser beam. In the Ref. 10. it was performed their band structure calculations. However, it would be of great interest to perform the calculation with taking into account of the doping by Cr ions.

In the present work we will do such kind of calculations taking into account of the influence of Chromium ions on the band structure. In the second part are presented the methods of crystal growth and principal features of the crystals. Section 3 describes first methods of principle calculations of the band energy structure. The principal results of the band energy structure calculations and corresponding photoinduced experimental data are given in the Section 4.

Crystal growth and crystal features.

YAl 3 (BO 3 ) 4 single crystals belong to the R32H space group with lattice constants (in Å): a = 9.293, c = 7.236, γ = 120° [11]. The YAB:Cr single crystals were synthesized by means of spontaneous crystallization. YAB decomposes at 1280°C into YBO 3 and AlBO 3 due to incongruent melting [12], so crystallization had to be carried out from high-temperature solution. Potassium trimolybdate K 2 Mo 3 O 10 with addition of 3wt% of B 2 O 3 was used as a solvent [13]. The starting composition of the melt was 20wt% of YAB doped with 1at% of Cr 3+ ions and 80wt% of the molybdate solvent. The growth was carried out from 200 g of the melt placed in a 100 ml Pt crucible covered with a Pt lid under conditions of low-temperature gradients. The crucible was placed in a resistance furnace isolated thermally to ensure proper temperature gradients. The heating zone of the furnace was controlled with use of Eurotherm 916S programmer. The soaking temperature of the melt was 1100°C, after 24 hours the temperature was lowered at a rate of 10 K/h to 1050°C that is the saturation temperature of the melt.

Next the temperature was lowered at a rate of 0.1K/h for 58 days and then the furnace was cooled at a rate of 10 K/h to room temperature. The as-grown YAB:Cr 3+ single crystals (up to 10x10x10 mm) were extracted from the solidified melt by dissolution of the solvent in hot water. According to Ref.

[14], the distribution coefficient of Cr 3+ ions between YAB crystal and the melt is very high and The effect shows its non-sensitivity to the photoinducing beam. The light scattering was controlled by the additional sphere spectrophotometer and it was less than 3%.

The absorption measurements were done by spectrophotometer Ocean Optics with spectral resolution 0.2 nm.

3. First-principles calculations for pure and Cr 3+ -doped YAl 3 (BO 3 ) 4

The CASTEP module [15] of Materials Studio package was used for calculations of the structural, electronic and optical properties of pure and Cr 3+ -doped YAB. The exchange-correlation effects were treated within the generalized gradient approximation (GGA) with the Perdew-Burke-Ernzerhof functional [16]. The Monkhorst-Pack scheme k-points grid sampling was set at 3×3×4 for the Brillouin zone (BZ). The plane-wave basis set energy cutoff was set at 340 eV; ultrasoft pseudopotentials were used for all chemical elements. The convergence parameters were as follows:

total energy tolerance -1×10 -5 eV/atom, maximum force tolerance 0.03 eV/nm, maximal stress component 0.05 GPa and maximal displacement 0.001 Å.

Results of calculations.

The calculated lattice constants after optimization of the crystal lattice structure were as follows states appear within the forbidden energy band gap. They possess some dispersion which is maximal Moreover, it is possible to distinguish between the t 2g and e g states, which arise from the crystal field splitting of the Cr 3+ 3d states and which are separated by about 2 eV (Fig. 3). The maximal dispersion of these levels is observed for F -Q -Z direction. The localized Cr impurities are sensitive to the crystalline anisotropy. Such a separation is denoted by 10Dq and its value can be determined from the absorption spectra by the position of the first strong spin-allowed transition, which corresponds to the 4 T 2 state. Fig. 4 shows the PDOS diagrams. The composition of the calculated electronic bands is the same as for pure YAB, but two very narrow deep bands appear at about -44 eV and -72 eV; they are due to the chromium 3p and 3s states, respectively. For an analysis of the absorption spectra of the Cr 3+ -doped YAB, crystal field theory (namely, exchange charge model, ECM [START_REF] Malkin | Spectroscopy of solids containing rare-earth ions[END_REF]) has been used.

Since detailed description of this model can be found in the literature [START_REF] Malkin | Spectroscopy of solids containing rare-earth ions[END_REF], we shall not give here description of the model and its main equations, but restrict ourselves to mentioning the details of calculations and obtained results. To calculate the crystal field parameters, a large cluster of 31793 ions located at the distances up to 73 Å from the central ion was considered. The overlap integrals between the Cr 3+ and O 2-ions were calculated previously [27]. The calculated values of the crystal field parameters (in Stevens normalization, all in cm -1 ) are as follows:

Values of the crystal field and 4 spanned by all LS wave functions of the LS terms of the 3d 3 electron configuration of Cr 3+ ion. The Racah parameters B and C were chosen to be 700 and 3163 cm -1 , respectively. The calculated energy levels are given below (all in cm -1 ). The spin-quartet energy levels: 4 The character of splitting of the orbital triplets and doublets suggests a low symmetry of the Cr 3+ position (C 1 ). However, splitting of the orbital doublets is very small, and in all triplet states it is possible to find a group of two very closely located energy levels and a single level, lying far apart from this group. Such a splitting pattern suggests that the local symmetry of the Cr 3+ position can be approximately described as a trigonal, when a triplet state is split into the doublet and singlet. Such a conclusion can be also verified by the values of the crystal field parameters: the main contribution comes from the Fig. 5 below shows the experimental absorption spectrum of YAB:Cr 3+ , in comparison with the above-given calculated energy levels. Following the performed X-ray micoranalysis these spectra correspond to the 0.85 % of Cr 3+ ions. However , there exists some non-uniformity through the sample's surface (from 0.65 % up to 1.05 %). So we present the averaged spectrum of the absorption.

Following this figure, agreement between the positions of the calculated energy levels and main features of the absorption spectrum is good. In particular, narrow peaks at about 14800 -15100 cm -1 nicely agree with the calculated positions of the spin doublets 2 E and 2 T 1 . Calculated splitting of the 4 T 2 state (between 16400 and 17250 cm -1 ) agrees with the width of the first spin-allowed absorption band. Moreover, the ECM calculated position of the 4 T 2 state is in a very good agreement with the estimation of the 10Dq parameter obtained from the band structure calculations for YAB:Cr 3+ (Fig. 3).

Two wide intensive peaks at about 33000 and 37000 cm -1 are due to the superposition of transitions from the 4 A 2 ground state to the 4 T 1 (

  higher conversion efficiency compared with LBO crystal. Consequently

reaches 9 . 7 ,

 97 what caused non-uniform distribution of the dopant in as-grown crystals. The Cr 3+ concentration in the central parts of YAB:Cr 3+ crystals was much higher than in the starting composition, and then it decreased towards the faces of the crystals. The photoinduced 532 nm cw laser at wavelength possesses Gaussian-like sequence beam with diameter about 1.5 mm. Its intensity

( 4 Fig. 1 .Fig. 2 .

 412 Fig. 1. Calculated band structure of pure YAB. The coordinates of the special points of the Brillouin zone are (in terms of the unit vectors of reciprocal lattice: Γ(0, 0, 0); A (0, 0, 1/2), H(-1/3, 2/3, 1/2); K(-1/3, 2/3, 0); M(0, 1/2, 0); L(0, 1/2, 1/2).

6 Fig. 3 .

 63 Fig. 3. Calculated band structure of the YAB:Cr 3+ crystal. The special points of the Brillouin zone are:Γ(0, 0, 0); A (0, 0, 1/2), H(-1/3, 2/3, 1/2); K(-1/3, 2/3, 0); M(0, 1/2, 0); L(0, 1/2, 1/2). The highest occupied state is taken as zero of energy.

Fig. 4 .

 4 Fig. 4. Calculated partial and total density of states for YAB:Cr 3+ .

4 -B 4 -

 44 =1422.0;3

  are characteristic for a trigonal crystal field. Moreover several contribution here should gie the broadening caused by electron-phononinteractions and occurrence of non-homogenuities.

Fig. 5 .Fig. 6 .

 56 Fig.5. Experimental absorption spectrum of YAB:Cr 3+ (solid line) and calculated energy levels (vertical lines). Assignment of the dense group of the spin doublet states in the region above 30000 cm - 1 is ambiguous.Following the absorption spectra we have verified their sensitivity to the illumination by 300 mW SHG of the Nd:YAG laser with wavelength 532 nm. Following the Fig.6one can say that the changes are very small. So these crystal may be promising for the high intensive nonlinear optical interactions. The obtained parameters show that the Cr ions change not only the energy positions of the bands, however cause an influence on the band dispersion. Additional monitoring of the photocrystallization does not show any signs of crystallization.

  A 2 -0; 4 T 2 -16439, 16451, 17254; 4 T 1 ( 4 F) -22672, 24155, 24171; 4 T 1 ( 4 P) -35855, 35947, 39538. The spin-doublet energy levels: 2 E -14471, 14489; 2 T 1 -15112, 15129, 15677; 2 T 2 -20994, 22383, 22498; 2 A 1 -28958; then very dense

	group of energy levels arising from the orbital triplets and doublets: 30884, 30927, 31052, 31464,
	31673, 31818, 33612, 33724 cm -1 .

  4 F) state and a very dense group of the spin doublet states, which are mixed up with the 4 T 1 ( 4 F) state by spin-orbit interaction.

					Wavelength, nm
		1000	900	800	700	600	500	400	300
				4 T 2	4 T 1 (	4 F)		4 T 1 ( 4 P)
	Absorption, arb. units	F o r P 2 E 2 T 1	2 T 2	2 A 1
			e
				e r
		10000	15000	R 20000 e 25000 e i v Energy, cm -1	30000	35000	40000
						w
							O n l
								y
									9

http://mc.manuscriptcentral.com/pm-pml Philosophical Magazine & Philosophical Magazine Letters

CONCLUSIONS

Following the experimental measurements and theoretical simulations for the YAB:Cr crystals we avhe established a good agreement between the positions of the calculated energy levels and main features of the absorption spectrum is good. In particular, narrow peaks at about 14800 -15100 cm -1 nicely agree with the calculated positions of the spin doublets 2 E and 2 T 1 . Calculated splitting of the 4 T 2 state (between 16400 and 17250 cm -1 ) agrees with the width of the first spin-allowed absorption band. Moreover, the ECM calculated position of the 4 T 2 state is in a very good agreement with the estimation of the 10Dq parameter obtained from the band structure calculations for YAB:Cr 3+ (Fig. 3).

The localized Cr 3+ impurities are sensitive to the crystalline anisotropy. Two wide intensive peaks at about 33000 and 37000 cm -1 are due to the superposition of transitions from the 4 A 2 ground state to the 4 T 1 ( 4 F) state and a very dense group of the spin doublet states, which are mixed up with the 4 T 1 ( 4 F) state by spin-orbit interaction. Additional treatment by the 300 mW cw 532 nm wavelengths does not caused any significant changes of the absorption.

Aknowledgements

The authors appreciate the financial support of the Polish Ministry of Sciences and Higher Education, Key Project POIG.01.03.01-14-016/08 "New Photonic Materials and their Advanced Applications".


MD Thierry V Scohy
email: t.scohy@erasmusmc.nl

Hanna D Golab

Mohamud Egal

MD Johanna J M Takkenberg

MD, PhD b . Ad J J C Bogers

insulin infusion during pediatric cardiac surgery for congenital heart disease

Keywords: congenital heart disease < Cardiac, glycemic control, intraoperative intraoperative glucose control, pediatric cardiac surgery

Intraoperative glycemic control without insulin infusion

during pediatric cardiac surgery for congenital heart disease.

Several studies report that the occurrence of hyperglycemia in the postoperative period is associated with increased morbidity and mortality rates in children after cardiac surgery for congenital heart disease [START_REF] Wintergerst | Association of hypoglycemia, hyperglycemia, and glucose variability with morbidity and death in the pediatric intensive care unit[END_REF][START_REF] Polito | Association between intraoperative and early postoperative glucose levels and adverse outcomes after complex congenital heart surgery[END_REF][START_REF] Vlasselaers | Intensive insulin therapy for patients in paediatric intensive care: a prospective, randomised controlled study[END_REF][START_REF] Yates | Hyperglycemia is a marker for poor outcome in the postoperative pediatric cardiac patient[END_REF]. However, no association with intraoperative management or complexity of congenital heart disease has yet been assessed.

Lately there is concern that glycemic control in the peri-operative period, aiming at avoiding hyperglycemia while maintaining a strict euglycemic target, could place patients at risk for hypoglycemia and hereby enhance the risk for adverse outcome [START_REF] Wintergerst | Association of hypoglycemia, hyperglycemia, and glucose variability with morbidity and death in the pediatric intensive care unit[END_REF][START_REF] Polito | Association between intraoperative and early postoperative glucose levels and adverse outcomes after complex congenital heart surgery[END_REF][START_REF] Ulate | Strict glycemic targets need not be so strict: a more permissive glycemic range for critically ill children[END_REF][START_REF] Finfer | Intensive versus conventional glucose control in critically ill patients[END_REF][START_REF] Floyd | Con: Tight perioperative glycemic control[END_REF]. In addition, the NICE-sugar Study [START_REF] Finfer | Intensive versus conventional glucose control in critically ill patients[END_REF] found that postoperative intensive glucose control (81 to 108 mg/dl; 4.5 to 6.0 mmol/l) increased mortality and a moderate blood glucose target <180mg/dl (<10mmol/l) resulted in lower mortality in adults.

Infants often have reduced glycogen reserve, especially in the setting of physiological stress, and insulin therapy in this setting may place them at risk for developing hypoglycemia [START_REF] Piper | Real-time continuous glucose monitoring in pediatric patients during and after cardiac surgery[END_REF]. To our knowledge there is only one published report on adverse outcome and intraoperative glucose levels during complex congenital heart surgery in children [START_REF] Polito | Association between intraoperative and early postoperative glucose levels and adverse outcomes after complex congenital heart surgery[END_REF].

In this light we report on our pediatric cardiac anesthesiological management and the blood glucose levels in 204 consecutive children during open cardiac surgery for congenital heart disease, exploring the association between blood glucose and complexity of congenital heart disease patient and preoperative characteristics.

Methods

The study population consisted of 204 consecutive pediatric patients aged from 3 days to 15.4 years undergoing open cardiac surgery for congenital heart disease between June 2007 and January 2009. The retrospective study was approved by the Erasmus MC internal review board and the need for informed consent was waived. Pre-operatively children were not allowed any food at least four hours before induction of anesthesia, but were allowed glucose containing clear liquids until two hours before anesthesia. None of the patients received premedication. Before induction of anesthesia, all patients were monitored with a five-lead, two-channel electrocardiogram, non-invasive blood pressure measurement, and pulse oximetry. All patients without a peripheral intravenous (iv) access had an inhalation induction with sevoflurane 8%. After insertion of iv catheter, midazolam 0.3 mg/kg, sufentanil 2 mcg/kg, pancuronium 0.15 mg/kg and 40mg/kg cefazoline were administrated and sevoflurane was stopped. Patients were nasotracheally intubated and pressure controlled ventilated after an alveolar recruitment strategy [START_REF] Scohy | Alveolar recruitment strategy and PEEP improve oxygenation, dynamic compliance of respiratory system and end-expiratory lung volume in pediatric patients undergoing cardiac surgery for congenital heart disease[END_REF]. Anesthesia was maintained with midazolam 0.1 mg/kg/h. Invasive monitoring via 20G arterial line in the femoral artery, an internal jugular central venous catheter, a Foley bladder catheter, a rectal temperature probe and the Oldelft MicroMultiplane TEE probe (Oldelft, Delft, The Netherlands) were routinely inserted [START_REF] Scohy | Intraoperative evaluation of micromultiplane transesophageal echocardiographic probe in surgery for congenital heart disease[END_REF].

Before incision we administered another 0.2mg/kg midazolam, 2mcg/kg sufentanil together with 30mg/kg magnesium sulfate, 30mg/kg methylprednisolone, 1 mg/kg ranitidine and 1mg/kg furosemide. All patients received a continuous infusion of NaCl 0.9% during the preincision period. Glucose containing fluids were not administrated intraoperatively. In case of normovolemic hypotension we administered 1 mcg/kg phenylephrine, in case of hypertension sevoflurane was used. Before initiation of CPB we administered 2mcg/kg sufentanil, 0.1 Pediatric CPB circuits consisted always of hollow-fiber oxygenator with hard-shell reservoir, a roller pump and arterial filter. The circuits were phosphorylcholine coated. Prime volumes varied depending on patient's body weight; for neonates and infants up to 10 kg -300 ml, for patients up to 25 kg -450 ml and for patients up to 45 kg -850 ml. Prime was composed of homologous red blood cells (RBCs), fresh-frozen plasma (FFP) and Gelofusine (B.Braun, Melsungen, Germany) and was always completed with 0.5 g/kg BW mannitol, 0.5 g/kg BW human albumin 20% solution (Sanquin, Amsterdam, The Netherlands), 4.2 IU heparine / mL priming volume and 2-5 ml NaHCO 3 8.4%.

Administration of RBCs, FFP, crystalloids or colloids during CPB was based upon the system working volumes and target values for hematocrit during the CPB (not lower than 0.28 L/L for acyanotic as well as cyanotic patients). Human albumin was added to maintain the colloid oncotic pressure ≥15 mmHg. In accordance with protocol no modified ultrafiltration and no antifibrinolitic medication was used. Activated clotting time was monitored during the bypass and maintained above 480 sec.

Nonpulsatile CPB, with mild hypothermia of 28 °C to 32° C, was performed with blood flow rates between 1.8 L/min/m 2 to 3.2 L/min/m 2 to maintain venous oxygen saturation above 70% and mean arterial pressure between 40 to 60 mmHg. In case of a deep hypothermic circulatory arrest (DHCA) patients were cooled till 18 ° C nasopharyngeal and 21 ° C rectal temperature.

Anterograde cerebral perfusion was utilized when appropriate. During CPB α -stat regulation was employed together with target value for arterial oxygen tension of 20 kPa. Myocardial protection was achieved with non-glucose containing crystalloid cardioplegia. Cardioplegic After weaning from CPB another 2 mcg/kg sufentanil and 0.1 mg/kg midazolam was administered. All patients received a total of 10 mcg/kg of sufentanil throughout the whole surgical procedure.

Glucose levels were measured before CPB, 10 minutes after initiation of CPB, every hour on CPB, 10 minutes after administrating 4 mg/kg Protamine and on arrival at Intensive Care Unit (ICU).

In line with previous publications, we defined the following blood glucose categories: severe hypoglycemia (< 30mg/dl; 1.7 mmol/l), moderate hypoglycemia (30-60mg/dl; 1.7-3.3 mmol/l)), euglycemia (60-125 mg/dl; 3.3-6.9 mmol/l), mild hyperglycemia (126-139 mg/dl; 6.9-7.7 mmol/l), moderate hyperglycemia (140-179 mg/dl; 7.7-9.9 mmol/L), or severe hyperglycemia (>180 mg/dl, >9.9 mmol/l) [START_REF] Polito | Association between intraoperative and early postoperative glucose levels and adverse outcomes after complex congenital heart surgery[END_REF][START_REF] Vlasselaers | Intensive insulin therapy for patients in paediatric intensive care: a prospective, randomised controlled study[END_REF][START_REF] Yates | Hyperglycemia is a marker for poor outcome in the postoperative pediatric cardiac patient[END_REF][START_REF] Ulate | Strict glycemic targets need not be so strict: a more permissive glycemic range for critically ill children[END_REF][START_REF] Preissig | Glycemic control for postoperative pediatric cardiac patients[END_REF].

For reasons of safety a 2 ml/kg glucose 10% solution was given when blood glucose level dropped under 50 mg/dl (2.8 mmol/l).

Statistical analyses

All statistical analyses were performed with SPSS 15.0 (SPSS Inc, Chicago, IL, USA).

Continuous variables are displayed as mean (SD), or median and interquartile range, categorical variables are displayed as proportions. The One-Sample Kolmorogov-Smirnov Test was used to analyse distribution of continuous variables. In case of a normal distribution comparison of continuous variables was done using the independent samples T-test, otherwise the Mann-Whitney U-test was used. Univariable linear regression was applied to assess influence of cyanotic/acyanotic CHD, age, weight, Aristotle complex score, RACHS score, CPB time, aortic cross clamp time (AoX), lowest intraoperative body temperature, intraoperative blood glucose levels (<60 mg/dl), intraoperative blood glucose levels (<75 mg/dl), moderate intraoperative blood glucose levels (>140 mg/dl) and severe intraoperative blood glucose levels (>180 mg/dl) as independent variables on LOS, ICU days, extubation time and mortality as dependent variables.

Univariable binary logistic regression analysis was used to study potential determinants of blood glucose level > 180 mg/dl on arrival at ICU. The following factors were considered as potential determinants: patient age, weight, Aristotle score, RACHS score, CPB time, AoX time, lowest intraoperative body temperature, blood glucose level before CPB as covariates was used.

Graphs were constructed with Graphpad software package (version 4.0, Graphpad Software Inc.,San Diego, USA).

Results

204 consecutive pediatric patients from 2.4 kg to 45 kg (130 male/ 74 female) (cyanotic versus acyanotic lesions 95/109) operated for congenital heart disease on CPB at the Thoraxcenter Rotterdam The Netherlands were included. Patients demographics, surgical procedure code by Aristotle Complexity Score [START_REF] Lacour-Gayet | The Aristotle score: a complexity-adjusted method to evaluate surgical results[END_REF] Box plots of per-operative and on arrival at ICU blood glucose levels are displayed in figure 1.

No patients received insulin during surgery.

Fifty-seven of the 204 patients (27.9%) had at least one intraoperative glucose level > 180 mg/dl (10 mmol/l), 132/204 patients (64%) had at least one intraoperative glucose level > 140 mg/dl (7.7 mmol/l), but only 12 patients (5.8%) had a glucose level > 180 mg/dl (10 mmol/l) on arrival at ICU (table 3), 59 patients (28.9%) had a glucose level > 140 mg/dl (7.7 mmol/l) on arrival at ICU.

Intraoperatively only 1 patient (a 2 year old boy with ASD) had a blood glucose level < 50 mg/dl (2.8 mmol/l). We measured 34.2 mg/dl and after administration of 2ml/kg glucose 10% blood glucose level rose to 48.6 mg/dl where after another glucose bolus was administrated and blood glucose level rose to 120 mg/dl. Nine out of 204 patients had Deep Hypothermic Cardiac Arrest (DHCA), of which 5/9 (56%) had blood glucose levels > 180 mg/dl (10 mmol/l) on arrival at ICU, while only 7 of 195 (3.5%) of the patients who did not undergo DHCA had blood glucose levels > 180 mg/dl on arrival at ICU.

Of the 204 patients 142 had representative extubation times (table 1), the other 62 patients received supplemental sedation for early transportation to the children ICU (logistic problem), or needed supplemental sedation because of their actual clinical condition or both. 30 days mortality was 1.5% (3/204); one sudden death 23 days postoperative after good recovery from TOF repair, one death a week after Norwood procedure due to respiratory infections complicated with necrotic enterocolitis and 1 death due to a mediastinale hemorrhage after Norwood procedure which needed postoperative ECMO support.

Factors potentially associated with hyperglycemia (blood glucose level > 180 mg/dl) on arrival at ICU are shown in table 4. Patients age < 30 days had higher glucose levels on ICU arrival compared to patients age > 30 days (156.1 + 54.1 versus 126.2 + 30.5; p=0.001).

Patients weighing 5 kilos or less had higher glucose levels on ICU arrival compared to patients weighing more than 5 kilos (139.0 + 44.1 versus 125.5 + 29.8; p=0.008).

Table 5 displays the correlation results between intraoperative blood glucose levels and LOS, ICU days, extubation time and mortality. All correlations were either nonsignificant or at the most very weak (Spearman´s Rho ‹ 0.30).

Linear regression suggested that neither lower intraoperative blood glucose levels (<60 or <75 mg/dl) nor higher intraoperative blood glucose levels (>140 or >180mg/dl) had influence on 6). Blood glucose levels at ICU arrival were associated with longer ICU stay (p=0.04) (Figure 2). The study shows that with a conventional (no insulin, no glucose) pediatric cardiac anesthetic approach there is little risk for intraoperative hypoglycaemia during pediatric cardiac surgery for congenital heart disease. However, frequent blood glucose monitoring is appropriate. Since all patients received 30mg/kg methylprednisolone iv before going on CPB and since steroids use in pediatric cardiac surgery is still controversial [20,21], additional benefit in preventing hyperglycemia might be achieved by withholding steroids in pediatric cardiac surgery. In addition, severe hyperglycemia on ICU arrival was uncommon, but comparable with the paper of DeCampli et al. [START_REF] Decampli | Perioperative hyperglycemia: effect on outcome after infant congenital heart surgery[END_REF], younger patients (< 30 days) and lower weights (< 5 kilos) were associated with a higher risk for hyperglycemia on ICU arrival.

High doses of opioids (sufentanil 10mcg/kg) attenuate the physiologic response to stress in infants undergoing cardiac surgery [START_REF] Duncan | Reducing stress responses in the pre-bypass phase of open heart surgery in infants and young children: a comparison of different fentanyl doses[END_REF][START_REF] Anand | Halothane-morphine compared with high-dose sufentanil for anesthesia and postoperative analgesia in neonatal cardiac surgery[END_REF][START_REF] Ellis | Fentanyl dosage is associated with reduced blood glucose in pediatric patients after hypothermic cardiopulmonary bypass[END_REF]. On the other hand there is concern that high dose opioids may influence extubation time and ICU stay. But, even with high dose opioids, all our 142 pediatric patients, that did not received additional sedations, were extubated within 21.1 hours after arrival at ICU. High doses of sufentanil together with nonglucose iv fluids contributed to the high percentage (95%) of our patients that had glucose level < 180 mg/dl (10 mmol/l) on arrival at ICU. These findings together with the NICE-SUGAR study [START_REF] Finfer | Intensive versus conventional glucose control in critically ill patients[END_REF] results, which showed that a blood glucose target of < 180 mg/dl (<10 mmol/l) resulted in lower mortality in adult patients than did a more strict blood glucose target. Endorse the belief that a conventional (no insulin, no glucose) anesthetic management seems sufficient in the vast majority of patients [START_REF] Finfer | Intensive versus conventional glucose control in critically ill patients[END_REF]. Special attention should be paid to neonates who need DHCA for surgical repair and are at an increased risk of postoperative hyperglycemia (5 out of 9 DHCA patients had blood glucose levels > 180 mg/dl at ICU arrival). In these patients a more strict intraoperative glycemic target ((90-140 mg/dl; 5-7.7 mmol/l) [START_REF] Ulate | Strict glycemic targets need not be so strict: a more permissive glycemic range for critically ill children[END_REF], (110-126 mg/dl; 6.1-7 mmol/l) [START_REF] Polito | Association between intraoperative and early postoperative glucose levels and adverse outcomes after complex congenital heart surgery[END_REF]) might be useful, requiring insulin infusions and Real-Time continuous glucose monitoring [START_REF] Piper | Real-time continuous glucose monitoring in pediatric patients during and after cardiac surgery[END_REF]. Balancing between perioperative hypoglycaemia and hyperglycemia Our study did not support the hypothesis that lower-or higher intraoperative blood glucose levels would be associated with a longer duration of postoperative hospitalisation.

According to the results of Polito et al. [START_REF] Polito | Association between intraoperative and early postoperative glucose levels and adverse outcomes after complex congenital heart surgery[END_REF] 23% of our patients are at greater risk for adverse outcome (mortality and composite morbidity variable) because of an intraoperative glucose level < 75 mg/dl (4.1 mmol/l). Our results do not support this, patients whose minimum intraoperative glucose level was < 75 mg/dl or even < 60 mg/dl did not show any association with LOS, ICU stay, extubation time or mortality. Avoiding lower glucose levels (< 75 mg/dl) can only be achieved by administrating glucose containing fluids in the pre CPB period, which will increase the occurrence of hyperglycemic periods [START_REF] Bell | The effect of intravenous dextrose infusion on postbypass hyperglycemia in pediatric patients undergoing cardiac operations[END_REF].

Twenty-eight percent of our patients had at least one intraoperative glucose level > 180 mg/dl (10 mmol/l) and 64% had at least one episode of blood glucose levels > 140 mg/dl which, in accordance to the results of Polito et al. [START_REF] Polito | Association between intraoperative and early postoperative glucose levels and adverse outcomes after complex congenital heart surgery[END_REF], is not associated with adverse events.

We only found a weak association between blood glucose levels at ICU arrival and ICU days.

On the other hand various other reports have shown an association between postoperative hyperglycemia and enhanced extubation times, LOS and mortality [START_REF] Wintergerst | Association of hypoglycemia, hyperglycemia, and glucose variability with morbidity and death in the pediatric intensive care unit[END_REF][START_REF] Vlasselaers | Intensive insulin therapy for patients in paediatric intensive care: a prospective, randomised controlled study[END_REF][START_REF] Yates | Hyperglycemia is a marker for poor outcome in the postoperative pediatric cardiac patient[END_REF].

Finally in our study we observed an overall 30 days postoperative mortality of 1.5% (3/204), if we only take into account the patients < 10 kg then mortality would be 3/131 (2.3%), while the now reported mortality in the European Association for Cardio-thoracic Surgery and the Society of Thoracic Surgeons Congenital Heart Surgery Database is 4% [START_REF] Jacobs | Cyanotic lesions/ acyanotic lesions 95/109 CPB (min): n=204 Median (IQR) 82 (52.5-107) AOX (min): n=204 Median (IQR) 47 (26-67.5) DHCA (min): n=9 Median (IQR) 53 (21.5-70.5) CPB temp (Celsius): n=204 Median (IQR) 30.3 (28.7-33.5) Extubation (hours): n=142 Median (IQR) 7.1 (4[END_REF] .

Limitations

This is a non-blinded retrospective study. Because of logistic problems at our ICU F o r P e e r R e v i e w 13 30% of our patients had to be transported to another department for which they received additional sedation, the reason for additional sedation whether it was for transportation or for medical reasons was not traceable.

Conclusions and recommendations

Since the optimal intraoperative blood glucose target range in pediatric patients during cardiac surgery for congenital heart disease remains unclear: "Conventional intraoperative high dose opioids and withholding glucose in intravenous fluids permits for a safe and moderate glucose control during pediatric cardiac surgery for congenital heart disease".

F

  midazolam, 300 IU heparine and 20mg/kg cefazoline,. Another bolus of 2mcg/kg sufentanil and 0.1 mg/kg midazolam was administrated during rewarming phase of CPB and dobutamine 4 mcg/kg/min was started. After declamping of the aorta enoximone 0.2mg/kg was administrated.

  sucked into the cell-saving device (CS). Perioperative blood loss was collected and processed by CS device, together with residual volume of the CPB circuit. The CS product was always considered first line blood replacement therapy.

7 The

 7 Spearman Correlation test was applied to assess correlation between blood glucose level on arrival at ICU, Length of stay in the hospital (LOS), ICU days, highest intraoperative blood glucose against age, weight, Aristotle complex score, RACHS score, CPB time, aortic cross clamp time (AoX), lowest intraoperative body temperature, and cyanotic/acyanotic CHD.

Figure 1 :

 1 Figure 1: Intraoperative and ICU arrival blood glucose levels.

  stay, mortality or extubation time (Table

Figure 2 :

 2 Figure 2: Blood glucose levels at ICU arrival versus ICU days.

Table 1 :

 1 , RACHS (risk adjustment for surgery for )[START_REF] Jenkins | Consensusbased method for risk adjustment for surgery for congenital heart disease[END_REF], CPB time, AoX time and lowest body temperature, blood glucose levels, LOS, IC stay are listed in Table 1. According to the Kolmogorov-Smirnov Test all groups of Table 1 are not normally distributed except for blood glucose level on arrival at ICU. Operations by RACHS-1 category are shown in table 2. Patients demographics and operative details SD= standard deviation; IQR= Interquartile range; RACHS= risk adjustment for surgery for congenital heart disease; CPB= cardiopulmonary bypass; AoX= aortic cross clamp time; DHCA= deep hypothermic cardiac arrest; ICU= intensive care unit, LOS= length of stay in hospital , IC= intensive care.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 :

 2 Individual procedures by RACHS-1 categoryASD=atrial septal defct, CAVSD=complete atrioventricular septal defect, TAPVR=Total anomalous pulmonary venous return, TGA=transposition of great arteries, TOF=tetralogy of Fallot, VSD=ventricular septal defect.

Table 3 :

 3 Data of patients with hyperglycemia > 180mg/dl at ICU arrival. septal defect; Switch= arterial switch operation; ALCAPA= abnormal left coronary artery from the pulmonary artery; VSD= ventricular septal defect; CoA=aortic coarctation; PDA= patent ductus arteriosus; AP-window=aortic pulmonary window; TOF= tetralogy of Fallot; corr. Ao Arch= correction aortic arch, RACHS= risk adjustment for surgery for congenital heart disease, CPB= cardiopulmonary bypass, AoX= Aortic crossclamping, DHCA= deep hypothermic cardiac arrest, ACP= antegrade cerebral perfusion, glc IC= blood glucose on Intensive Care arrival.

	ASD=atrial

Table 4 :

 4 Logistic regression analysis (blood glucose >180 mg/dl at ICU arrival as dependent variable) RACHS= risk adjustment for surgery for congenital heart disease, CPB= cardiopulmonary bypass, AoX= Aortic crossclamping, DHCA= deep hypothermic cardiac arrest, ACP= antegrade cerebral perfusion, glc IC= blood glucose on Intensive Care arrival. B=sample regression coefficient, S.E.= standard error, Sig.= two-sided P value or observed significance level, Exp(B)= exponentiation of the B coefficient, C.I.=confidence interval.

Table 5 :

 5 Correlation analysis results of intraoperative blood glucose versus LOS, ICU days, extubation time and mortality.

Table 6 :

 6 Linear regression analysis results: Lower (<60 and <75mg/dl) and higher intraoperative blood glucose levels (>140 and >180mg/dl) versus LOS, ICU stay, mortality or extubation time.

	B=sample regression coefficient, S.E.= standard error, Sig.= two-sided P value or observed significance level,
	Beta=standardized coefficient, C.I.=confidence interval.

Table 1 :

 1 Brunow dC, Fonseca MC: Steroids use in pediatric cardiac surgery: more questions. Pediatr Crit Care Med 2007, 8: 503-504. 21. Robertson-Malt S, Afrane B, El Barbary M: Prophylactic steroids for pediatric open heart surgery. Cochrane Database Syst Rev 2007, CD005550. Patients demographics and operative details. SD= standard deviation; IQR= Interquartile range; RACHS= risk adjustment for surgery for congenital heart disease; CPB= cardiopulmonary bypass; AoX= aortic cross clamp time; DHCA= deep hypothermic cardiac arrest; ICU= intensive care unit.

	o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 :

 2 individual procedures by RACHS-1 category. septal defct, CAVSD=complete atrioventricular septal defect, TAPVR=Total anomalous pulmonary venous return, TGA=transposition of great arteries, TOF=tetralogy of Fallot, VSD=ventricular septal defect.

	ASD=atrial

Table 3 :

 3 Data of patients with hyperglycemia > 180mg/dl at ICU arrival.

	ASD=atrial septal defect; Switch= arterial switch operation; ALCAPA= abnormal left coronary artery from the
	pulmonary artery; VSD= ventricular septal defect; CoA=aortic coarctation; PDA= patent ductus arteriosus; AP-
	window=aortic pulmonary window; TOF= tetralogy of Fallot; corr. Ao Arch= correction aortic arch, RACHS=
	risk adjustment for surgery for congenital heart disease, CPB= cardiopulmonary bypass, AoX= Aortic
	crossclamping, DHCA= deep hypothermic cardiac arrest, ACP= antegrade cerebral perfusion, glc IC= blood
	glucose on Intensive Care arrival.

Table 4 :

 4 Logistic regression analysisRACHS= risk adjustment for surgery for congenital heart disease, CPB= cardiopulmonary bypass, AoX= Aortic crossclamping, DHCA= deep hypothermic cardiac arrest, ACP= antegrade cerebral perfusion, glc IC= blood glucose on Intensive Care arrival. B=sample regression coefficient, S.E.= standard error, Sig.= two-sided P value or observed significance level, Exp(B)= exponentiation of the B coefficient, C.I.=confidence interval.

Table 5 :

 5 Correlation analysis results of intraoperative blood glucose versus LOS, ICU days, extubation time and mortality.B=sample regression coefficient, S.E.= standard error, Sig.= two-sided P value or observed significance level, Beta=standardized coefficient, C.I.=confidence interval.

	Table 6: Linear regression analysis results: Lower (<60 and <75mg/dl) and higher
	intraoperative blood glucose levels (>140 and >180mg/dl) versus LOS, ICU stay,
	mortality or extubation time.

Table : Individual procedures by RACHS-1 category

 :

	Risk Category 1 gender Age Weight (kg) Correlation Highest intraop Glc / ICU days sort B Linear Regression	S.E.	Aristotle Score Spearman's rho RACHS score 95.0% C.I.for Significance (2-tailed) 95% C.I. CPB time (min) AOX (min) CPB temp DHCA/ACP (min) Lower 0.199 0.005 Upper Sig. Exp(B) EXP(B) B Std Error Beta Sign. lower higher	(mg/dl) glc IC
	M F M M	ASD closure (29) Coarctation repair with left-left shunt at age > 30d (1) Partially anomalous pulmonary venous return repair (2) Sinus venosus atrial septal defect closue (2) Risk Category 2 1m3d 13.4 ASD 3 1 45 Weight -0.358 0.144 0.013 0.699 0.527 Highest intraop Glc / LOS 0.197 <60mg/dl intraop glc / LOS -1.949 4.285 -0.035 4d 2.5 Switch+ASD 13 4 148 Age_days -0.018 0.007 0.008 0.982 0.969 Highest intraop Glc / ICU glc 0.284 <60mg/dl intraop glc / ICU days 0.154 1.863 0.006 3m3d 5.9 ALCAPA 10 3 224 Aristotle_Score 0.317 0.101 0.002 1.373 1.126 Highest intraop Glc / Extubation time 0.259 <60mg/dl intraop glc / extubation 1.715 2.2 0.071 14d 3.8 VSD,ASD,CoA,PDA 15 3 104 CPB_time 0.006 0.003 0.07 1.006 1 RACHS_score 0.633 0.223 0.004 1.884 1.217 Highest intraop Glc / mortality 0.132 <60mg/dl intraop glc / mortality -0.004 0.038 -0.009	20 93 156 53	35.1 0.005 0.65 -10.399 0.927 26.2 0.995 0.0001 0.934 -3.519 25.8 1.675 0.0001 0.437 -2.635 26.1 1.012 2.916 NS 0.91 -0.08	6.501 4/0 3.828 6.066 21/0 0.071	181.8 196.2 185.4 199.8
	M M	Aortic-pulmonary window repair and ASD closure (1) Aortic valvuloplasty at age > 30d (1) 2.5 PDA, AP-window, AoXmin 2m 0.12 0.006 0.068 1.012 0.999 > 140 mg/dl intraop glc / LOS 0.14 <75mg/dl intraop glc / LOS 1.884 2.711 0.063 Atrial Septal Fenestartion. 9.8 2 65 gluc_pre 0.028 0.01 0.004 1.028 1.009 > 140 mg/dl intraop glc / ICU days 0.102 <75mg/dl intraop glc / ICU days 0.795 1.178 0.06 CPB temp -0.296 0.066 0 0.744 0.654 > 140 mg/dl intraop glc / extubation time 0.143 <75mg/dl intraop glc / extubation 0.741 1.169 0.667 Partial cavo-pulmonary connection (16) Pulmonary valvuloplasty (5) 1m16d 4.3 TOF 8 2 156 DHCA 0.4 0.014 0.004 0.5 1.012 > 140 mg/dl intraop glc / mortality 0.09 <75mg/dl intraop glc / mortality -0.025 0.024 -0.089	30 94	1.024 0.488 1.048 0.501 0.846 0.528 1.069 0.325	NS -3.461 28.4 NS -1.529 NS -1.572 28.6 NS -0.072	7.23 3.118 3.053 0.024	201.6 219.6
	M	5m2d	Pulmonary valve replacement (1) 5.2 TOF	8	2	85	45	30.7	190.8
	M M F M M	Total repair of TOF (21) VSD closure (29) VSD and ASD closure (9) VSD closure and pulmonary valvuloplasty (2) VSD closure and right vemtricle infundibulectomy (5) VSD closure and subaortic stenosis (3) Risk Category 3 Anomalous left coronary artery from pulmonary artery reimplantation (1) Aortic-and mitral valvuloplasty (1) Aortic-pulmonary window (2) Arterial switch operation (3) CAVSD repair (19) Coarctation repair and VSD closure (2) Heart transplantation (2) Intracardiac tumor excision (1) Left Ventricular Assist Device (1) Mitral valvuloplasty (1) Pulmonary artery banding and VSD closure (1) Pulmonary valve replacement and VSD closure (1) Ross procedure (1) Tricuspid valvuloplasty (1) Total cavo-pulmonary connection (16) Tricuspid valve repositioning for Ebstein anomaly at age > 30d (1) w w w w w TOF repair with pulmonary atresia (1) F o r P e e r R e v i e F o r P e e r R e v i e 6d 3.8 VSD+corr. Ao Arch 13 4 126 56 22.1 4d 3.4 Norwood 14.5 6 197 110 17.2 3m11d 5.4 TOF+corr. Ao Arch 14 5 111 66 21.5 7d 3 Norwood 14.5 6 162 100 25.5 13d 3.1 ASD+PDA 6 1 102 45 29.3 F o r P e e r R e v i e F o r P e e r R e v i e > 180 mg/dl intraop glc / LOS 0.137 NS > 180 mg/dl intraop glc / ICU days 0.147 0.038 > 180 mg/dl intraop glc / extubation time 0.203 0.015 > 180 mg/dl intraop glc / mortality 0.175 0.012 F >140mg/dl intraop glc / LOS 2.492 1.991 0.088 0.212 -1.435 >140mg/dl intraop glc / ICU days -0.943 1.214 -0.77 0.438 -3.337 >140mg/dl intraop glc / extubation 1.258 0.852 0.124 0.142 -0.426 >140mg/dl intraop glc / mortality 0.023 0.018 0.9 0.199 -0.12 F o r F o r o r P e >180mg/dl intraop glc / LOS 2.45 2.05 0.84 0.233 -1.593 >180mg/dl intraop glc / ICU days 0.015 0.01 0.118 0.126 -0.004 >180mg/dl intraop glc / extubation 1.319 0.877 0.126 0.135 -0.414 P P >180mg/dl intraop glc / mortality 0.027 0.17 0.106 0.11 -0.006 e e r e e r e r R e v i e R e v i e w 355x305mm (96 x 96 DPI) R e v i e w	82/59 24/0 54/34 6.418 1.45 2.942 0.58 6.493 0.062 3.053 0.06	187.2 275.4 181.8 286.2 196.2
		Risk Category 4
			Atrial septectomy (1)
			Arterial switch operation and VSD closure (6)
			Hypoplastic aortic arch repair and VSD closure (2)
			Interrupted aortic arch repair (1)
			TAPVR repair at age < 30d (5)
			TAPVR, CAVSD and TGA repair (1)
		Risk Category 5
			Damus-Kaye-Stansel procedure (1)
			Norwood operation (5)

ASD=atrial septal defct, CAVSD=complete atrioventricular septal defect, TAPVR=Total anomalous pulmonary venous return, TGA=transposition of great arteries, TOF=tetralogy of Fallot, VSD=ventricular septal defect. 318x201mm (96 x 96 DPI)

[START_REF] Polito | Association between intraoperative and early postoperative glucose levels and adverse outcomes after complex congenital heart surgery[END_REF][START_REF] Vlasselaers | Intensive insulin therapy for patients in paediatric intensive care: a prospective, randomised controlled study[END_REF]