A high-throughput screening assay of ascorbate in brain samples

Abstract

Ascorbate is a vital reductant/free radical scavenger in the CNS, whose content defines - to a large extent - the redox status and the antioxidant reserves. Quick, reliable and specific methods for its measurement in brain samples are highly desirable. We have developed a new high-throughput screening assay for measurements of ascorbate using a fluorescence plate-reader. This assay is based on a direct reaction of ascorbate with a nitroxide radical conjugated with a fluorogenic acridine moiety, 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl radical (AC-TEMPO), yielding fluorescent hydroxylamine product (AC-TEMPO-H). The reaction was monitored over time using fluorescence and electron spin resonance techniques. The appearance of fluorescent AC-TEMPO-H was linear within the range of 3.75-75μM AscH(-) in the sample (0.5-10μM AscH(-) in the well). Assay was validated with high performance liquid chromatography method. The concentration of ascorbate in murine tissue samples, including brain samples after traumatic brain injury and hemorrhagic shock, was measured.

Fig. 1

Measurement of ascorbate by AC-TEMPO. A – Time-course of AC-TEMPO ESR signal decay produced by ascorbate (0–46.7 μM). Insert: initial ESR signal. N=3. B – Dose-dependence of AC-TEMPO ESR signal at 25 min after addition of ascorbate. Insert – Blue: superposition of Asc• and TEMPO•. Red – simulated spectra. The reaction mixture contained 100 μM AC-TEMPO. N=3. C – Time-course of accumulation of fluorogenic product (AC-TEMPO-H) after reaction of AC-TEMPO with ascorbate. Conditions: AC-TEMPO – 50 μM, ascorbate concentration (in μM) indicated. Note, that after 40 min the reaction reaches saturation. N=3. D – Typical calibration curve utilized for ascorbate measurement in biological samples. AC-TEMPO – 20 μM, AscH⁻ 3.75–75 μM (0.5–10 μM in the well). N=18. E – Calibration curves of freshly prepared ascorbate standards (−MPA) and standards treated the same way as the tissue samples (+MPA). N=6. F Fluorescence spectra of AC-TEMPO-H at different pH measured at 30 min. Conditions: AC-TEMPO – 50 μM, ascorbate 10 μM. G –. Specificity of AC-TEMPO assay. Time course of accumulation of fluorogenic product (AC-TEMPO-H) after reaction of AC-TEMPO with ascorbate in the presence of dopamine (DA). Conditions: AC-TEMPO – 50 μM, ascorbate – 10 μM, DA (in μM) as indicated. Note that in the absence of ascorbate, DA did not significantly affect AC-TEMPO reduction by ascorbate. I –Typical HPLC profile of ascorbate measurement in rat right cortex (RC). Arrow indicates ascorbate peak in standard and RC sample. Note that ascorbate peak completely disappears in AO-treated sample.

Fig. 2

A, B – Assessments of ascorbate (A) and GSH (B) loss in murine brain samples after controlled cortical impact (CCI) and CCI + HS followed by fluid resuscitation with Hextend® or polynitroxylated albumin (PNA). Significant difference between naïve and other groups indicated by * (p value < 0.05, N=4–5).