Neurochemical measurement of adenosine in discrete brain regions of five strains of inbred mice

Abstract

Adenosine (ADO), a non-classical neurotransmitter and neuromodulator, and its metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP), have been shown to play an important role in a number of biochemical processes. Although their signaling is well described, it has been difficult to directly, accurately and simultaneously quantitate these purines in tissue or fluids. Here, we describe a novel method for measuring adenosine (ADO) and its metabolites using high performance liquid chromatography with electrochemical detection (HPLC-ECD). Using this chromatographic technique, we examined baseline levels of ADO and ATP, ADP and AMP in 6 different brain regions of the C57BL/6J mouse: stratum, cortex, hippocampus, olfactory bulb, substantia nigra and cerebellum and compared ADO levels in 5 different strains of mice (C57BL/6J, Swiss-Webster, FVB/NJ, 129P/J, and BALB/c). These studies demonstrate that baseline levels of purines vary significantly among the brain regions as well as between different mouse strains. These dissimilarities in purine concentrations may explain the variable phenotypes among background strains described in neurological disease models.

Figure 1. Identification of optimal parameters for simultaneous detection of ADO, ATP, ADP and AMP.

(A) To determine the optimum voltage necessary to fully oxidize ADO, ATP, ADP and AMP, we measured the number of oxidized molecules at voltages ranging from +700–+1700 mV. Construction of a hydrodynamic voltamogram (HDV), necessary to determine the optimal signal/noise (s/n) ratio, showed that +1300 mV was optimum for the simultaneous detection of the compounds. (B) To determine the accurate range and linearity of detection of ADO, ATP, ADP, and AMP, 0–100 ng of each standard was injected into the HPLC. Electrochemical detection of the oxidized molecules shows a linear slope for each of the compounds. (C) Representative chromatograms of simultaneous separation of ADO, ATP, ADP and AMP demonstrating linearity of the injected analytes.

Figure 2. Determination of elution time for Purines.

Sample chromatograms showing elution time for (A) ADO, (B) ATP, (C) ADP, (D) AMP, (E) a combined solution containing all four compounds and (F) Olfactory bulb from a 24 month C57BL/6J mouse. The elution time for each of the compounds is distinct and allows for the simultaneous detection of ADO, ATP, ADP and AMP.

Figure 3. Quantitation of ADO in 7 regions of the brain in 5 strains of mice.

ANOVA demonstrated significant differences between mouse strains in each region examined. Post-hoc Bonferroni comparisons are shown for each brain region. N = 10 for each measurement.