Automatic determination of ligand purity and apparent dissociation constant in buffer solutions and the for anion binding in physiological solutions from -macroelectrode measurements

Kay, Jim; Steven, Rachel J.; McGuigan, John A. S.; Elder, H. Y.

doi:10.1016/j.compbiomed.2007.07.008

Cited by 12 publications

(7 citation statements)

References 13 publications

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“…The free Ca 2+ concentrations in the calibration solutions were determined using a Ca 2+ -selective electrode ( Kay et al, 2008 ; McGuigan et al, 1991 ). Calibration solutions contained (in mM): R max : 140 KCl, 2.5 KOH, 15 NaCl, 1 MgCl 2 , 5 HEPES, 10 CaCl 2 , and 0.05 fura-2; R min : 129.5 KCl, 13 KOH, 15 NaCl, 1 MgCl 2 , 5 HEPES, 4 EGTA, and 0.05 fura-2; R def : 129.5 KCl, 13 KOH, 10.3 NaCl, 4.7 NaOH, 1 MgCl 2 , 5 HEPES, 4 EGTA, 2.7 CaCl 2 , and 0.05 fura-2, yielding a free Ca 2+ concentration of 0.35 µM.…”

Section: Methodsmentioning

confidence: 99%

Energy imbalance alters Ca2+ handling and excitability of POMC neurons

Paeger

Pippow

Heß

et al. 2017

eLife

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Satiety-signaling, pro-opiomelanocortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus play a pivotal role in the regulation of energy homeostasis. Recent studies reported altered mitochondrial dynamics and decreased mitochondria- endoplasmic reticulum contacts in POMC neurons during diet-induced obesity. Since mitochondria play a crucial role in Ca2+ signaling, we investigated whether obesity alters Ca2+ handling of these neurons in mice. In diet-induced obesity, cellular Ca2+ handling properties including mitochondrial Ca2+ uptake capacity are impaired, and an increased resting level of free intracellular Ca2+ is accompanied by a marked decrease in neuronal excitability. Experimentally increasing or decreasing intracellular Ca2+ concentrations reproduced electrophysiological properties observed in diet-induced obesity. Taken together, we provide the first direct evidence for a diet-dependent deterioration of Ca2+ homeostasis in POMC neurons during obesity development resulting in impaired function of these critical energy homeostasis-regulating neurons.DOI: http://dx.doi.org/10.7554/eLife.25641.001

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Section: Methodsmentioning

confidence: 99%

Energy imbalance alters Ca2+ handling and excitability of POMC neurons

Paeger

Pippow

Heß

et al. 2017

eLife

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“…With the ISE data, computation in the original LOM was automated by defining an objective function and then minimizing it subject to constraints, as described by Kay, Stevens, McGuigan, and Elder (). The computer program described there is now obsolete.…”

Section: Calibration Of Ca2+/mg2+ Ises Fluorochromes and Aequorinmentioning

confidence: 99%

Ionized concentrations in Ca²⁺ and Mg²⁺ buffers must be measured, not calculated

McGuigan

Kay

Elder

2020

Experimental Physiology

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New Findings What is the topic of this review?The [Ca2+]/[Mg2+] in buffers are usually calculated using one of eight programs. These all give different values, thus [Ca2+]/[Mg2+] must be measured. What advances does it highlight?The ligand optimization method (LOM) using electrodes is an accurate method to do this. The limitations of the method are described. The LOM has been generalized to include calibration of fluorochromes and aequorin. It is the method of choice to measure intracellular equilibrium constants. Owing to the uncertainties for the values of resting [Ca2+], ∆[Ca2+] and the pK′ values for intracellular Ca2+/Mg2+ binding used in modelling, these values must now be re‐examined critically. Abstract Modelling intracellular regulation of Ca2+/Mg2+ is now an established part of physiology. However, the conclusions drawn from such studies depend on accurate knowledge of intracellular [Ca2+], ∆[Ca2+] and the pK′ values for the intracellular binding of Ca2+/Mg2+. Calculation of [Ca2+]/[Mg2+] in buffers is normal. The eight freely available programs all give different values for the [Ca2+]/[Mg2+] in the buffer solutions, varying by up to a factor of 4.3. As a result, concentrations must be measured. There are two methods to do this, both based on the ligand optimization method (LOM): (1) calibration solutions from 0.5 to 4 mmol l−1; and (2) calibration solutions from 0.1 µmol l−1 to 2 mmol l−1. Both methods can be used to calibrate Ca2+/Mg2+ electrodes. Only Method 2 can be used directly to calibrate fluorochromes and aequorin. Software in the statistical program R to calculate the [Ca2+]/[Mg2+] in buffers is provided for both methods. The LOM has now been generalized for use with electrodes, fluorochromes and aequorin, making it the ideal method to determine the pK′ values for intracellular binding of Ca2+/Mg2+. The [Ca2+]/[Mg2+] in buffers must be measured routinely, which is best done by calibrating electrodes with the LOM and software written in R. If [Ca2+]/[Mg2+] in buffers are calculated, the parameters used in modelling show the same degree of variability as the software programs. Uncritical acceptance of such parameters means that conclusions reached from such studies are relative, not absolute, and must now be re‐examined.

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“…[3]) was compared with the purity determined on the same batch of EGTA by the ligand optimization method using the Excel program ALE [6]. There was no significant difference between the value of 3.797 ± 0.099 mM (n = 6) obtained using the pH method and the value of 3.803 ± 0.057 mM (n = 7) measured using the ligand optimization method [6]. (Note that determination was carried out on a different batch of EGTA than in the experiments shown in Fig.…”

Section: Puritymentioning

confidence: 99%

“…There was no agreement between the values calculated using the constants of Refs. [8] and [9] or the program Chelator; these differences versus the measured Values for the pCa of these buffer solutions were then obtained using the ligand optimization method [3,6] to fit the data to the Nikolsky-Eisenman equation, yielding E = 57.190 + 28.533 Ã log (10 ÀpCa + 6.0554 -Ã 10 À8 ); r = 0.999743. values were on the order of 2. If purity was ignored, the values decreased by a maximum factor of one-third.…”

Section: Apparent Dissociation Constantmentioning

confidence: 99%

“…The total time for a complete calibration was approximately 70 min. Subsequently, the data were fitted with the Excel program ALE [6], which optimizes the values for [Ligand] T (total concentration of chelating ligand), the K app in the buffer solutions, and a parameter describing the nonlinearity of the electrode to obtain the best fit of the data to the Nikolsky-Eisenman equation (see Ref. [7], p. 68).…”

Section: Measurement Of [Ligand] T and K App Via Ligand Optimization mentioning

confidence: 99%

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Calculated and measured [Ca2+] in buffers used to calibrate Ca2+ macroelectrodes

McGuigan

Stumpff

2013

Analytical Biochemistry

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Automatic determination of ligand purity and apparent dissociation constant in buffer solutions and the for anion binding in physiological solutions from -macroelectrode measurements

Cited by 12 publications

References 13 publications

Energy imbalance alters Ca2+ handling and excitability of POMC neurons

Energy imbalance alters Ca2+ handling and excitability of POMC neurons

Ionized concentrations in Ca²⁺ and Mg²⁺ buffers must be measured, not calculated

Calculated and measured [Ca2+] in buffers used to calibrate Ca2+ macroelectrodes

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Automatic determination of ligand purity and apparent dissociation constant in buffer solutions and the for anion binding in physiological solutions from -macroelectrode measurements

Cited by 12 publications

References 13 publications

Energy imbalance alters Ca2+ handling and excitability of POMC neurons

Energy imbalance alters Ca2+ handling and excitability of POMC neurons

Ionized concentrations in Ca2+ and Mg2+ buffers must be measured, not calculated

Calculated and measured [Ca2+] in buffers used to calibrate Ca2+ macroelectrodes

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Ionized concentrations in Ca²⁺ and Mg²⁺ buffers must be measured, not calculated