Schematic of the fluorescence-coupled GABA assay.

<p>The GABase preparation, a combination of ABAT and ALDH5A1 enzymes from <i>P. fluorescens</i> converts GABA to succinate through the conversion of alpha-ketoglutarate to glutamate and NADP to NADPH. Although NAD or NADP can potentially be used as substrates by ALDH5A1, NADP is conventionally used as a cofactor for this preparation. Succinic semialdehyde (SSAL), an intermediate of GABA metabolism is also metabolized by the GABase preparation. Diaphorase oxidizes the NADPH and reduces resazurin to fluorescent resorufin. Application of 2-aminoethyl hydrogen sulfate (2-AEHS), an inhibitor of ABAT can be used to determine the contribution of SSAL to the total signal generated from GABA and SSAL in cell lysates.</p

Quantitation of GABA, SSAL, and glutamate in PNEC cells following 24 hours of pH stress and nutrient deprivation.

<p>The effects of 24-hour glucose and glutamine deprivation on <b>A.</b> GABA, <b>B.</b> SSAL, and <b>C.</b> glutamate levels in PNEC cells were measured. Nutrient deprivation experiments were performed in substrate-deficient DMEM/F12 media containing dialyzed fetal calf serum supplemented with 6 mM glutamine and/or 17.5 mM glucose for 24 hours. The effects of 24-hour pH stress at pH 6.5 and 8.5 relative to physiologic pH 7.4 on <b>D.</b> GABA, <b>E.</b> SSAL, and <b>F.</b> glutamate levels in PNEC cells were measured. Metabolite levels in the cell lysates was assayed and normalized to total cellular protein. * Significance relative to control (p<0.05). Data represents quadruplicate measurements.</p

Limitations of the GABase assay.

<p><b>A.</b> Inhibitors of the GABase assay. Titrations of potential inhibitors of the GABase assay were carried out and signal was measured after 30 minutes. The assay demonstrated inhibition by the heavy metals cobalt (IC₅₀ = 1 mM) and zinc (IC₅₀ = 400 µm) at high concentrations. There is weak inhibition by hydrogen peroxide at high sample concentrations (1 mM). Data were obtained in triplicate. <b>B.</b> Reaction mix decay kinetics. Following preparation of complete reaction mix, the mix was stored in the dark at either 4°C or 25°C. Following the times listed, the reaction mix was added to a fixed concentration of GABA standard. Signal obtained at different time points was normalized to the signal obtained at time zero. Data were obtained in triplicate.</p

Enzyme activity measurements of GAD1, GLUL, and ALDH5A1 in PNEC cells following pH stress.

<p>The effects of 24-hour acid and alkaline stress on <b>A.</b> GAD1, <b>B.</b> GLUL, and <b>C.</b> ALDH5A1 levels in PNEC cells were measured. D. Protein expression of enzymes correlates with enzyme activities. Beta actin (ACTB) is provided as a loading control. Enzyme activity in the cell lysates was assayed and normalized to reaction time and total cellular protein. * Significance relative to control (p<0.05). Data represents quadruplicate measurements.</p

Mechanics of the GABase assay.

<p><b>A.</b> Representative kinetics for the coupled GABase assay using 25 µM GABA. Plateau kinetics are seen after 30 minutes. The kinetic curve represents an average of triplicate measurements. <b>B.</b> Linearity of the assay. GABA and SSAL standards (10 µL) at the concentrations indicated were added to reaction mix (90 µL) and measured after 30 minutes. The limit of detection for the assay was 0.78 µM GABA and 0.41 µM SSAL. The limit of quantitation was 2.6 µM GABA and 1.4 µM SSAL. The Z’ factor for the assay was 0.8 for GABA and 0.9 for SSAL. <b>C.</b> Standard curve of [GABA] using a conventional NADPH fluorescence assay. This assay demonstrated lower sensitivity than the coupled resazurin assay when carried out under optimum conditions in an optical plate. The limit of detection for the assay was 67 µM. The limit of quantitation for the assay was 223 µM. The Z’ factor for the assay was 0.9. Fluorescence signal is measured in arbitrary units (A.U.) and is corrected against the sample blank. Data were obtained in triplicate.</p

Specificity of the GABase assay.

<p><b>A.</b> Metabolic standards (25 µM) were added to reaction mix and kinetically observed for 30 minutes. When compared to metabolites similar in chemical structure, GABA and SSAL are the only compounds that generate measurable signal in the coupled assay. Note break in scale to visualize residual activities. ND: No signal detected over background. Fluorescence signal is measured in arbitrary units (A.U.) and is corrected against the sample blank. <b>B.</b> Inhibition curve of 2-aminoethyl hydrogen sulfate, an inhibitor of the GABA transaminase (ABAT) component of the GABase preparation. At concentrations equal to or greater than 25 mM, the resorufin signal from GABA is completely abolished, allowing for the NADPH (hence resorufin signal) produced by succinic aldehyde dehydrogenase (ALDH5A1) component of the GABase preparation to be specific for succinic semialdehyde (SSAL). Data were obtained in triplicate and normalized to signal in the absence of inhibitor.</p

GABA synthetic and metabolic pathways in NE cells.

<p>GLUD: glutamate dehydrogenase; GLS: glutaminase; GLUL: glutamate-ammonia ligase; GAD1: glutamate decarboxylase; ODC1: ornithine decarboxylase; ABP1: diamine oxidase; ABAT: GABA transaminase; SSAL: succinic semialdehyde; ALDH5A1: succinic acid semialdehyde dehydrogenase; ALDH9A1: aldehyde dehydrogenase; SAT1: spermidine/spermine N-acetyltransferase.</p

GABA shunt gene expression in human cancers.

<p><b>A.... </b><i>GAD1</i> mRNA overexpression in prostate adenocarcinomas correlates with decreased disease-free survival. Tumors with a <i>GAD1</i> expression z-score greater than +2 display decreased disease-free survival (median 19.8 months) relative to tumors that do not have increased <i>GAD1</i> expression (median 110.3 months; p = 0.002). <b>B.... </b><i>GLUL</i> downregulation in prostate adenocarcinomas correlates with disease-free survival (median 27.9 months) relative to tumors without <i>GLUL</i> downregulation (median 110.3 months; p = 0.006). <b>C.... </b><i>GAD1</i> mRNA expression in prostate cancer cell lines measured with quantitative PCR. <i>GAD1</i> expression is detectable in castrate-resistant cell lines NCI-H660, PC3, and DU145, but not androgen-responsive cell lines LNCaP and LAPC4. 18S rRNA was used as an internal standard. ΔC_t is the difference of the threshold cycle of <i>GAD1</i> and the threshold cycle of 18S rRNA. ND: Not detected. <b>D.... </b><i>GAD1</i> mRNA overexpression in clear cell renal cell carcinomas correlates with reduced overall survival. Tumors with a <i>GAD1</i> z-score >+2 display decreased overall survival (median 52.5 months) relative to tumors that do not have increased <i>GAD1</i> expression (median 80.6 months; p = 0.01). Tumors with a <i>GAD1</i> z-score >+3 display a greater decreased median survival time (median 31.1 months) relative to the remainder of the tumors (median 78.4 months; p = 0.002).</p

Effects of sample preparation on GABA and SSAL stability and recovery.

<p>25 µM of GABA or SSAL standard was treated with the sample preparation protocol consisting of 50 mM sodium hydroxide followed by heating at 60°C for 40 minutes in excess 50 mM HCl and then neutralization with 400 mM Tris base. There was no significant difference in signal obtained from a 25 µM GABA or SSAL standard versus 25 µM GABA or SSAL treated with the sample preparation technique. To test recovery of GABA and SSAL from tissue, GABA or SSAL standard was added to the LNCaP prostate cancer cell sample at a final concentration of 25 µM and subjected to the sample preparation protocol. The signal recovered from the sample was subtracted from the signal obtained from an identical tissue sample without added standard, demonstrating quantitative recovery of GABA and SSAL from tissues using this technique. Fluorescence signal was measured and all data normalized to untreated standards. Data were obtained in triplicate.</p

Optimization curves for GABase-diaphorase-resazurin coupled assay.

<p>Each component of the reaction mix (<b>A–F</b>) was titrated to determine the optimal concentration to achieve the highest signal to background ratio. The final reaction mix based upon these curves was 6.25 µM resazurin, 0.063 U/mL GABase, 0.063 U/mL diaphorase, 100 µM NADP, 5 mM alpha-ketoglutarate, and 3.125 µM DTT. Data were obtained in triplicate.</p