Direct pH Measurements Research Articles

Basolateral Na+-H+ exchanger-1 in rat taste receptor cells is involved in neural adaptation to acidic stimuli.

The role of basolateral Na(+)-H(+) exchanger isoform-1 (NHE-1) was investigated in neural adaptation of rat taste responses to acidic stimuli, by direct measurement of intracellular pH (pH(i)) in polarized taste receptor cells (TRCs) and by chorda tympani (CT) taste nerve recordings. In TRCs perfused with CO(2)/HCO(3)(-)-free solution (pH 7.4), removal of basolateral Na(+) decreased pH(i) reversibly and zoniporide, a specific NHE-1 blocker, inhibited the Na(+)-induced changes in pH(i). The spontaneous rate of TRC pH(i) recovery from NH(4)Cl pulses was inhibited by basolateral zoniporide with a K(i) of 0.33microm. Exposure to basolateral ionomycin, reversibly increased TRC Ca(2+), resting pH(i), and the spontaneous rate of pH(i) recovery from an NH(4)Cl pulse. These effects of Ca(2+) on pH(i) were blocked by zoniporide. In in vivo experiments, topical lingual application of zoniporide increased the magnitude of the CT responses to acetic acid and CO(2), but not to HCl. Topical lingual application of ionomycin did not affect the phasic part of the CT responses to acidic stimuli, but decreased the tonic part by 50% of control over a period of about 1 min. This increased adaptation in the CT response was inhibited by zoniporide. Topical lingual application of 8-CPT-cAMP increased the CT responses to HCl, but not to CO(2), and acetic acid. In the presence of cAMP, ionomycin increased sensory adaptation to HCl, CO(2), and acetic acid. Thus, cAMP and Ca(2+) independently modulate CT responses to acidic stimuli. While cAMP enhances TRC apical H(+) entry and CT responses to strong acid, an increase in Ca(2+) activates NHE-1, and increases neural adaptation to all acidic stimuli.

Impaired gastric secretion and lack of trophic responses to hypergastrinemia in M 3 muscarinic receptor knockout mice

Background & Aims : The physiologic significance of the M 3 muscarinic receptor is unclear due to an absence of specific ligand. In the present study, M 3 receptor knockout (KO) mice were used to elucidate the role of M 3 receptors in gastric acid secretion and gastric mucosal integrity. Methods : M 3 KO versus wild-type mice aged 1 month to 2 years were included. Gastric acid secretion was assessed by both direct intragastric pH measurement and pylorus ligation. Serum gastrin and gastric mucosal histamine levels were determined by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. Morphologic analysis was performed by both immunohistochemistry and transmission electron microscopy. Results : Fasted M 3 KO mice exhibited higher intragastric pH, lower acid output after pylorus ligation, a lower proportion of active parietal cells, and higher serum gastrin levels than fasted wild-type mice. Acid secretion in response to carbachol, histamine, gastrin 17, and 2-deoxy- d-glucose was impaired in the mutant mice. Although carbachol was still able to cause ∼30% acid output in M 3 KO mice, the acid secretion was inhibited by pirenzepine or famotidine. Despite remarkable hypergastrinemia in M 3 KO mice, there were no trophic responses in the oxyntic mucosa with respect to the mucosal thickness, proliferation rate, and numbers of parietal and enterochromaffin-like cells. Cholecystokinin type 2 receptor antagonist YM022 was without the effect in M 3 KO mice. Conclusions : The present study shows that M 3 receptors are essential for basal acid secretion, a fully acid secretory response to histamine and gastrin, and the trophic responses of oxyntic mucosa to gastrin.

Microanalysis of C and O isotopes of azooxanthellate and zooxanthellate corals by ion microprobe

We have determined the δ18O and δ13C values of azooxanthellate (Lophelia pertusa) and zooxanthellate (Porites lutea) corals at a micrometer scale using an ion microprobe (SIMS—secondary ion mass spectrometry). In P. lutea, centers of calcification are small (10 to 15 μm) and difficult to locate during measurements. In L. pertusa, they are large (50 μm) and arranged in lines of centers of calcification. Our results show that centers of calcification in L. pertusa have a restricted range of variation in δ18O [-2.8±0.3‰ (PDB)], and a larger range in δ13C [14.3 to 10.9‰ (PDB)]. Surrounding skeletal fibers exhibit large isotopic variation both for C and O (up to 12‰), and δ13C and δ18O are positively correlated. The C and O isotopic compositions of the center of calcification deviate from this linear trend at the lightest δ18O values of the surrounding fibers. Ion microprobe results on P. lutea demonstrate also a large range of variation for the δ18O values (up to 10‰). No correlation is found with C isotopes that exhibit, in comparison with L. pertusa, a small range of variation (2‰). This variation of δ18O at a micrometer scale is probably the result of two processes: (1) an isotopic equilibrium calcification with 1 pH unit variation in the calcification fluid as indicated by direct measurements of coelenteron pH in the coral Galaxea fascicularis (Al-Horani et al. 2003) and (2) a kinetic fractionation. The δ13C apparent disequilibrium in P. lutea may be the result of mixing between metabolic CO2 (respiration) and dissolved inorganic carbon (DIC) coming directly from seawater.

Apparatus for direct pH measurement of supercritical aqueous solutions

A flow-through electrochemical cell was developed for direct pH measurement of supercritical aqueous solutions, based on our previously reported cell [Sue et al., Rev. Sci. Instrum. 72 (12) (2001) 4442]. The cell consisted of two high-temperature and high-pressure flow-through platinum hydrogen electrodes. The distance between tips of the two electrodes was minimized to reduce the streaming potential and to simplify the experimental procedure. Flow characteristics were considered in the design by placing orifices near the center of the cell to reduce concentration fluctuations around the electrodes. With the newly-designed cell, pH measurements were made for HCl+NaCl aqueous solutions at flow rates from 0.5 to 6.0 g/min, temperatures from 19.6 to 392.9 °C and pressures from 0.1 to 29.8 MPa. Comparison of the experimentally determined and theoretically calculated pH was better than ±0.02 logarithmic units over the range of conditions studied.

Modulation of rat chorda tympani NaCl responses and intracellular Na+ activity in polarized taste receptor cells by pH.

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH (pHi) and Na+ activity ([Na+]i) in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the lingual surface with NaCl solutions adjusted to pHs ranging between 2.0 and 10.3 increased the magnitude of NaCl CT responses linearly with increasing external pH (pHo). At pH 7.0, the epithelial sodium channel (ENaC) blocker, benzamil, decreased NaCl CT responses and inhibited further changes in CT responses induced by varying pHo to 2.0 or 10.3. At constant pHo, buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HCO3 −/CO2 inhibited NaCl CT responses relative to CT responses obtained with NaCl solutions buffered with HEPES. The carbonic anhydrase blockers, MK-507 and MK-417, attenuated the inhibition of NaCl CT responses in HCO3 −/CO2 buffer, suggesting a regulatory role for pHi. In polarized TRCs step changes in apical pHo from 10.3 to 2.0 induced a linear decrease in pHi that remained within the physiological range (slope = 0.035; r2 = 0.98). At constant pHo, perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO3 −/CO2 decreased resting TRC pHi, and MK-507 or MK-417 attenuated the decrease in pHi in TRCs perfused with HCO3 −/CO2 buffer. In parallel experiments, TRC [Na+]i decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical Na+, and (d) acid loading the cells with NH4Cl or sodium acetate at constant pHo. Diethylpyrocarbonate and Zn2+, modification reagents for histidine residues in proteins, attenuated the CO2-induced inhibition of NaCl CT responses and the pHi-induced inhibition of apical Na+ influx in TRCs. We conclude that TRC pHi regulates Na+-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures.

Sensors and measurements in solid state fermentation: a review

Solid state fermentation (SSF), biofiltration and soil bioremediation are processes involving microorganisms grown on solid or semi-solid substrates or supports. Monitoring these processes requires the measurement of environmental parameters (temperature, pH, water content and activity) and the carbon cycle (biomass, substrate concentration, CO 2). However, given the complexity and heterogeneity of the solid medium, these variables are not easily accessible and measurable. This paper reviews current on-line methods and innovative applications of methods with a potential to measure parameters in SSF. Direct measurements of temperature, pH, and water content are considered employing classical sensors, and indirect measurements of the biomass by respirometry or pressure drop (PD). More recent methods such as: aroma sensing, infrared spectrometry, artificial vision, and tomographic techniques (X-rays, Magnetic Resonance Imaging or MRI) are then explored.

Excitation and adaptation in the detection of hydrogen ions by taste receptor cells: a role for cAMP and Ca(2+).

The role of intracellular cAMP and Ca(2+) in the excitation and adaptation of taste responses by HCl was investigated by direct measurement of intracellular pH (pH(i)) in polarized taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the tongue with HCl concentrations between 1 and 30 mM caused a dose-dependent increase in CT responses that were insensitive to voltage clamp of the lingual receptive field and to amiloride. At a fixed HCl concentration (20 mM) topical lingual application of 8-chlorophenylthio(CPT)-cAMP increased the magnitude of HCl-induced CT response by twofold under zero current clamp. The magnitude of the CT response increased further at -60 mV and decreased at +60 mV lingual voltage clamp but remained amiloride insensitive. In untreated polarized TRCs, apical stimulation with HCl concentrations between 1 and 30 mM HCl induced sustained decreases in TRC pH(i). The magnitude of pH(i) decrease increased with increasing HCl concentration. Following treatment of the basolateral membrane with 8-CPT-cAMP the decrease in pH(i) due to apical 1 mM HCl application was significantly increased. Treatment with cAMP alone decreased resting TRC pH(i) and inhibited the recovery of pH(i) from a basolateral NH4Cl pulse by 46%. Topical lingual application of ionomycin, a Ca(2+) ionophore, did not affect the initial CT response to 20 mM HCl +10 mM CaCl2, but the response declined rapidly to 50% of its initial level within 2 min. In polarized TRCs, basolateral exposure to ionomycin increased TRC pH(i) and activated pH(i) recovery from NH4Cl pulse by 388%. Apical HCl stimulation induced a transient decrease in resting TRC pH(i) followed by spontaneous recovery. The data suggest that cAMP enhances the sour taste of strong acids by activating a Ca(2+)- and amiloride-insensitive H(+) conductance and inhibiting pH(i) recovery in TRCs. However, an increase in [Ca(2+)]i stimulates pH(i) recovery, which, in turn, increases sensory adaptation to acids.

Iron deficiency-associated changes in the composition of the leaf apoplastic fluid from field-grown pear (Pyrus communis L.) trees.

Experiments have been carried out with field-grown pear trees to investigate the effect of iron chlorosis on the composition of the leaf apoplast. Iron deficiency was associated with an increase in the leaf apoplastic pH from the control values of 5.5-5.9 to 6.5-6.6, as judged from direct pH measurements in apoplastic fluid obtained by centrifugation and fluorescence of leaves incubated with 5-CF. The major organic acids found in leaf apoplastic fluid of iron-deficient and iron-sufficient pear leaves were malate, citrate and ascorbate. The total concentration of organic acids was 2.9 mM in the controls and increased to 5.5 mM in Fe-deficient leaves. The total apoplastic concentration of inorganic cations (Ca, K and Mg) increased with Fe deficiency from 15 to 20 mM. The total apoplastic concentration of inorganic anions (Cl-, NO3-, SO4(2-) and HPO4(2-)) did not change with Fe deficiency. Iron concentrations decreased from 4 to 1.6 microM with Fe deficiency. The major Fe species predicted to exist in the apoplast was [FeCitOH](-1) in both Fe-sufficient and deficient leaves. Organic acids in whole leaf homogenates increased from 20 to 40 nmol x m(-2) with Fe deficiency. The accumulation of organic anions in the Fe-deficient leaves does not appear to be associated to an increased C fixation in leaves, but rather it seems to be a consequence of C transport via xylem.

Differences in pH between interstitial fluid and arterial blood in water-breathing and air-breathing vertebrates.

Most cells are bathed by interstitial fluid, but extracellular pH measurements are mostly for arterial plasma. Whole-body mean pH differences between the two fluids have been estimated in terms of a simple model. This relates to the diffusive exchange of carbon dioxide and oxygen and utilizes literature data, for 22 vertebrate species, on arterial and mixed-venous tensions of both gases. Uncertainties arise because the carbon dioxide reaction in blood may sometimes be in disequilibrium and because carbon dioxide diffusion is facilitated to unknown degrees in the presence of buffers. Nevertheless, the model suggests that the pH difference should tend to vary inversely with arterial carbon dioxide tension. In some species, this may aid interstitial pH homeostasis, but a clearer implication is that the difference should be generally greater in water breathers than in air breathers. It has previously been found that arterial pH in water-breathing teleosts also tends to be higher than in air-breathing tetrapods (when allowance is made for temperature and plasma sodium concentration) and to a comparable extent. Thus, mean interstitial pH may be more nearly similar in the two groups than is arterial pH. Direct measurements of interstitial pH do not yet suffice to test the model.

Pharyngeal pH measurements in patients with respiratory symptoms before and during proton pump inhibitor therapy

Background: Pharyngeal pH monitoring is a diagnostic tool used to identify Gastroesophageal reflux disease (GERD) as an etiology of respiratory symptoms. We performed pharyngeal pH monitoring on 14 patients with respiratory symptoms thought to be induced by GERD. Methods: Symptoms and pH monitoring (esophageal and pharyngeal) were assessed prior to and 3 months after the initiation of double-dose proton pump inhibitor therapy. Results: Symptoms included cough, hoarseness, and throat clearing. Ten patients had at least one episode of pharyngeal reflux (PR+) and 4 patients had no pharyngeal reflux (PR−). Pharyngeal reflux episodes in PR+ patients decreased from 3.5 to 0.9 ( P <0.05) per day with 8 of 10 (80%) patients having elimination or reduction of such episodes. Eight of 9 PR+ patients (89%) with suppressed pharyngeal reflux on medical therapy had resolution of respiratory symptoms. Three of 4 PR− patients (75%) had persistent symptoms on medical therapy. Conclusions: Proton pump inhibitor therapy improves clinical symptoms and decreases pharyngeal reflux episodes in patients with respiratory symptoms related to GERD. Direct measurement of pharyngeal pH is helpful in the identification of patients likely to respond to antireflux therapy.

A novel low-noise measurement principle for LAPS and its application to faster measurement of pH

A novel measurement principle for the light-addressable potentiometric sensor (LAPS) system is proposed in this paper. With several features like low-noise and low-background, this measurement scheme extends the application of LAPS in faster high-sensitive direct monitoring of ionic concentration and small change in the surface potential without the need of costly instruments like potentiostat, lock-in amplifier. Using two out-of-phase modulated light sources applied at two different points of the Si surface of the heterostructure, a kind of differential LAPS response is obtained. Due to the addition of the two out-of-phase photocurrents, low-frequency components associated with the photocurrents are canceled out and a low-noise and hence highly sensitive response is expected. As an application of the new principle, the pH measurement of an electrolyte solution is investigated and a new instrumentation for the direct, high-sensitive, and faster measurement of pH is proposed.

Proton involvement in the transition from the "peroxy" to the ferryl intermediate of cytochrome c oxidase.

In the absence of any external electron donor, the "peroxy" intermediate of cytochrome c oxidase (CcO-607) is converted to the ferryl form (CcO-580) and subsequently to oxidized enzyme. The rate of conversion of CcO-607 to the CcO-580 form is pH dependent between pH 3.0 and pH 7.6. A plot of the logarithm of the rate constant for this conversion is a linear function of pH with a slope of -0.92, implying the involvement of a single proton in the transition. Upon rapidly lowering the pH from 8.1 to 5.8, the uptake of one proton was observed by direct pH measurement, and the kinetics of proton uptake coincide with the spectral conversion of CcO-607 to CcO-580. We interpret the slow endogenous decay of CcO-607 to CcO-580 to be the result of proton transfer to a deprotonated group generated in the binuclear cavity during CcO-607 formation. This group is not freely accessible to protons from the medium, and its pK(a) is probably higher than 9.0.

Direct measurement of ph within marine biofilms on passive metals

Direct measurements of the pH within natural marine biofilms grown on platinum have been made with iridium/iridium oxide solid‐state microelectrodes. The data show that the pH is highly variable, with steep gradients both perpendicular to and along the metal surface. Measured values ranged from 8 in the outer portions of the biofilm to below 2 in localized areas near the metal surface. It was verified that bulk water flowing along the hydrophilic outer sheath of the micro‐electrode could effect the measured pH values. However, meaningful data could still be taken because the response time of the electrode was sufficiently less than the time for water ingression. The data presented are consistent with recent heterogeneous biofilm models and with the idea that localized areas of low pH within biofilms can be a contributing factor in biologically influenced corrosion processes.

Experimental Indications of Effects of Surface Deprotonation on Na-Bentonite Porewater Chemistry in a Geological Repository

ABSTRACTBentonite-water interaction was studied using a simple equilibrium model based on experimental measurements in order to describe bentonite porewater chemistry. Direct pH measurements for highly compacted bentonite and batch-type bentonite-water interaction experiments were performed under anaerobic conditions. In the direct pH measurements, resin particles doped with a pH indicator were sandwiched between a pair of bentonite columns immersed in a test solution. The experimental results showed that the solution compositions in equilibrium with bentonite depended on the bentonite to liquid ratio (B/L) and the initial solution composition. An equilibrium model assuming only fast equilibration processes between the bentonite minerals and the solution could be used to calculate the trends of pH and other ion concentrations with B/L. This study indicates that the surface deprotonation of smectite is a very important factor influencing the porewater chemistry in highly compacted bentonite.

Direct measurement of cytosolic calcium and pH in living Chlamydomonas reinhardtii cells

Intracellular free Ca2+ and H+ were quantified in Chlamydomonas reinhardtii, using the fluorescent ion indicators Fura-2 and BCECF. We demonstrate that both indicators can be loaded into living cells as acetoxymethylesters. The esters were hydrolyzed intracellularly to genuine Fura-2 and BCECF capable of indicating changes in Ca2+i and H+i. Fura-2 accumulated in the cytoplasm to a concentration of 50 microM, whereas BCECF reached a concentration of 200 microM. The average Ca2+i was estimated to be 180 +/- 40 nM and the average pHi was 7.4 +/- 0.1. To document the applicability of the ion indicators in Chlamydomonas, we tested their responses to several stimuli. We observed increases in cytoplasmic Ca2+ in response to elevated external Ca2+ on membrane-permeable acids, which are known to induce flagellar excision in Chlamydomonas. The membrane-permeable acids caused a decrease in cytoplasmic pH. Pulses of photosynthetically active light lead to transient pHi changes. Finally, concomitant measurements of rhodopsin-triggered and voltage-sensitive photocurrents indicated that Ca2+ influx is accompanied by a transient depolarisation of the plasmalemma. These experiments document that Fura-2 and BCECF are versatile dyes for studying various ionic processes in Chlamydomonas.

Investigation of the Electrospray Plume by Laser-Induced Fluorescence Spectroscopy

Laser-induced fluorescence spectroscopy has been employed to probe the electrospray plume through measurement of the emission spectra of fluorescent dyes in the spray stream. The system is evaluated with respect to sensitivity and reproducibility using solutions of Eosin Y under typical electrospray conditions. Application of the technique for monitoring spray-induced pH changes is demonstrated using fluorescence signals from a pH-sensitive fluorophore, carboxyseminaphthorhodafluor-1 (C.SNARF-1). To our knowledge, this is the first direct measurement of pH within the electrospray plume.

Determination of the pH gradient across the stratum corneum.

The objective of this work was to determine the pH gradient profile across hairless mouse stratum corneum (SC) using ratiometric laser scanning confocal microscopy imaging and direct pH measurement with a flat surface electrode. Dual-emission ratiometric imaging used the fluorophore, carboxy seminaphthorhodafluor-1, which displays a pH-dependent shift in its emission spectrum. The assay developed was unsuccessful, however, because (i) the pKa of seminaphthorhodafluor (approximately 7.5) makes it insufficiently sensitive to the normal pH gradient (4-7.4) across mammalian SC, and (ii) the unexpectedly high buffering capacity of the skin precluded a meaningful calibration of the system. In the second method, pH measurements with a flat surface electrode were recorded as the hairless mouse SC was progressively tape stripped. In the superficial SC, the pH remained relatively constant (approximately 5.9); further removal of the barrier resulted in a steady increase in pH to approximately 7. In conclusion, improved methodology is clearly required to characterize precisely the pH profile across the SC.

PH Is Regulated Differently by Glucose in Skeletal Muscle from Fed and Starved Rats: A Study Using 31P-NMR Spectroscopy ,

The aim of this study was to determine whether exogenous glucose metabolism influences the pH in superfused EDL muscle from growing rats fed or starved for 48 h (body weight 55 and 45 g, respectively). Energy state and intracellular pH of muscle were repeatedly monitored by 31P-nuclear magnetic resonance spectroscopy (31P-NMRS); glycogen and other energy metabolites were assayed enzymatically in muscle extracts at the end of the experiment. In EDL muscles from starved rats superfused with glucose for 4 h, intracellular pH was elevated (7–7.3), lactate concentration low, glycogen repletion very intense and citrate synthase activity high. We conclude that glucose was routed mainly toward both oxidative phosphorylation and glycogen synthesis in EDL muscles after food deprivation of rats. In contrast, the major pathway in muscles from fed rats may be glycolysis because the glycogen pool remained constant throughout the experiment. The additional and minor pH component (in the range of 6.5 to 6.8) seen in muscles from fed rats, even in the presence of exogenous glucose, might be due to impaired glucose utilization because this component appears also in muscles from starved rats superfused without glucose or with a nonmetabolizable analog of glucose. Consequently, direct pH measurement by 31P-NMR may be considered to be a precise criterion for evaluation of differences in metabolic potentialities of muscle studied ex vivo in relation to the nutritional state of rats.

Hydrolysis of Zinc Ion and Solubility of Zinc Oxide in High-Temperature Aqueous Systems

Hydrolysis constants of the zinc ion were measured at 25, 50, 75, 185, 200, and 225°C through the direct measurement of pH using pH sensors, especially of the yttria-stabilized zirconia membrane-type in the case of high temperatures over 185°C, and evaluation was done on the temperature dependence of the hydrolysis constants of the zinc ion. Solubilities of zinc oxide in pure oxygenated water were measured at 150, 200, and 250°C. Equilibrium constants of zinc oxide dissolution and the values of ΔGf0(Zn2+) at each temperature were estimated by thermodynamic analysis applying the estimated hydrolysis constants to the solubility data of ZnO.

The regulation of intracellular pH in the diabetic myocardium.

Time for primary review 25 days. . In myocardial cells, as in any given cell, steady-state intracellular pH (pHi) is strictly maintained within a narrow range at relatively alkaline values. Resting pHi (7.1–7.2) is determined by the algebraic sum of acid-loading and acid-extruding processes. Whenever acid-loading exceeds acid extrusion, pHi falls. The degree to which pHi changes is inversely related to the intracellular buffering power (βi). The role of intracellular buffering power, which is the first line of defence of a cardiac cell against an intracellular acid–base disturbance, is to moderate pHi changes produced by acute acid (or alkali) load [1]. However, buffering mechanisms cannot prevent a change in pHi, but only reduce its amplitude. The regulation of pHi then largely depends upon the activity of plasma membrane carrier-mediated transport of acid/base equivalents [2, 3]. Intracellular pH is important for the activity of a number of enzymes with optimal pH within the physiological pH range, as well as for the conductivity of ion channels [4], calcium homeostasis, and the efficiency of contractile elements [5, 6]. Despite a vast literature demonstrating myocardial metabolic changes associated with diabetes (for reviews, see Refs. [7, 8]), until recent years relatively few studies have addressed the effects of diabetes on intracellular pH. Yet, disturbances in intracellular pH or in the processes regulating intracellular pH may be expected to occur in diabetic hearts as a result of either altered cellular metabolism and/or cellular and subcellular membrane changes. This review provides a summary of the data obtained with both multicellular and single cell preparations, as well as with isolated hearts from chemically-induced diabetic rats. The first study that allowed direct measurement of intracellular pH in cardiac cells of streptozotocin (STZ)-induced diabetic rat hearts was … * Tel.: +33 (1) 69 15 78 98; fax: +33 (1) 69 15 68 41.