A method for calculating the distribution of pH in tissues and a new source of pH error from the 31P-NMR spectrum

Abstract

The true distribution of the pH in tissues can be determined from the in vivo 31P-nuclear magnetic resonance (NMR) spectrum by converting the parts per million (PPM) axis of the pH responsive resonance to pH using the Henderson-Hasselbalch equation. In addition, the intensity axis of the resonance must be divided by the derivative of the Henderson-Hasselbalch equation to correct for the nonlinear relationship between pH and PPM. This nonlinear relationship causes the apparent center of the resonance in PPM to be dependent not only on the center of the pH distribution but also on its width and distance from the pKa, where Ka is the association constant. Therefore, the pH determined from uncorrected spectra may be in significant error, particularly if the pH distribution is distant from the pKa and is broad. The method was applied to the isolated perfused Morris hepatoma 5123C to determine the distribution of intracellular pH (pHi) using resonances from two intracellular compounds. The two resonances did not report the same pHi unless the spectral data were properly corrected. The method should be of interest to anyone interested in pHi.

A Study of Imidazole-Based Nuclear Magnetic Resonance Probes of Cellular pH

“…The harvested cells were embedded in agarose threads by the method of Foxall et al (13) and perfused at a rate of 2 ml/min in artificial cerebral spinal fluid (ACSF) containing 124 mM NaCl, 5 mM KCl, 1.2 mM MgSO 4 , 1.26 mM CaCl 2 , 10 mM glucose, 26 mM NaHCO 3 and bubbled with a 95%O 2 /5%CO 2 gas mixture. The perfusate used in low pH experiments on C6 cells contained 5 mM histidine instead of NaHCO 3 and was adjusted to pH 6.1 with NaOH. The perfusate for canine glioma samples was maintained at pH 7.4 Ϯ 0.1 by the bicarbonate buffering system.…”

“…However, a high signal-tonoise ratio for accurate P i measurement is not always achievable in a short period of time, particularly in normoxic tissue, and the interpretation of the P i lineshape is often complicated by heterogeneity in pH as well as in the production of P i in the sample. Though nonlinear curve fitting has been applied to the analysis of asymmetric or clearly multicomponent P i lineshapes, such analyses rely on several a priori assumptions about the number, fractional volumes, or continuous distributions of unique pH compartments present in the sample (1,(3)(4)(5). This would make P i data acquired during periods of continuous changes in pH and P i levels, such as during ischemia or hypoxia, particularly difficult to model accurately by lineshape analysis, even though fits to such data might appear quite good.…”

Absolute quantification of human liver metabolite concentrations by localized in vivo 31P NMR spectroscopy in diffuse liver disease

“…The cell membranes are composed of a bilayer of phospholipids. 31 P-MRS offers a non-invasive method to study the synthesis of these membranes and the energy state of the hepatocytes, and it also provides information about carbohydrate metabolism, intracellular pH [1] and free magnesium ion concentrations [2].…”

Simultaneous intracellular and extracelular pH measurement in the heart by 19F NMR of 6‐fluoropyridoxol

“…However, a subtle source of systematic bias may be introduced by the nonlinear sigmoidal titration curve, which produces greater uncertainty for values distant from the pKa. Indeed, the peak shape should be corrected to account for the nonlinearity of the titration curve (34). Applying the nonlinear correction, we have found errors as large as -0.13 pH units when pH is -7.1.…”