Human skin biopsy collection
Punch skin biopsies (3 mm) were obtained from the distal thigh of healthy volunteers after anesthesia with 0.5 cc 2% lidocaine subcutaneous injection . The protocol was approved by the Johns Hopkins Institutional Review Board in compliance with the Helsinki declaration. Samples were placed in cold Tris buffer (pH 7.4) and GCP enzymatic activity was carried out within 1 h of collection.
Rodent drug dosing and paw and sciatic nerve sample collection
All experimental protocols were approved by the Institutional Animal Care and Use Committee of SoBran, Inc., Baltimore and adhered to all of the applicable institutional and governmental guidelines for the humane treatment of laboratory animals. Rats (male Wistar) were administered vehicle (HEPES saline, pH 7, 50 mM) or 2-PMPA (1, 10 and 100 mg/kg, i.p.) using a dosing volume of 2 mL/kg. There were 10 animals in each group. Animals were sacrificed 1 h after 2-PMPA or vehicle administration. 2-PMPA brain concentrations were previously shown to be highest 50 - 75 min after i.p. administration . Skin was collected from the planter hindpaw by 3 mm skin biopsy dissection and stored at -80°C until ready for analysis. In order to obtain sciatic nerve, 1-2 cm incisions were made on the skin on top of the mid thigh so that sciatic nerve, gluteus superficialis muscle and biceps femoris muscle became exposed. The three were then separated and 5 mm of sciatic nerve was dissected out.
Human skin biopsy and rodent paw and sciatic nerve sample preparation
Human skin biopsies were sonicated in Tris buffer (pH 7.4, 40 mM, 0.5 mL) for 1 min in ice. The mixture was centrifuged for 2 min at 16000 × g; the supernantant (containing cytosolic fraction) was removed and the resulting pellet (containing plasma membrane) was reconstituted in 70 μL assay buffer (Tris pH 7.4, 40 mM containing 1 mM CoCl2) and used as source of GCP in the activity assay. Rat paw pads and sciatic nerve isolated from vehicle and 2-PMPA treated animals were sonicated for 2 min in ice. The mixture was centrifuged for 2 min at 16000 × g and the resulting pellet was reconstituted similar to the pellets obtained from the human skin dissections.
Measurement of GCP activity in human skin biopsies and rodent paw pads
GCP activity measurements were carried out following published procedures [3, 25]. Briefly, the reaction mixture contained [3H]-NAAG (70 nM, 50 Ci/mmol) and reconstituted pellet (human skin, paw pad, or sciatic nerve) in Tris-HCl containing 1 mM CoCl2 in a total volume of 90 μL. The reaction was carried out at 37°C at different times as indicated, and stopped with ice-cold sodium phosphate buffer (pH 7.4, 0.1 M, 90 μL). When human skin was used as GCP source, the reaction was carried out in the presence and absence of the selective GCP inhibitor 2-PMPA (1 μM). When rat tissue was used from the ex vivo study, 2-PMPA was administered i.p. and the animals were sacrificed and their paw pads removed for GCP enzymatic determinations. In both cases, blanks were obtained by incubating the reaction mixture without pellet. Duplicate aliquots of 90 μL from each terminated reaction was transferred to a well in a 96-well spin column containing AG1X8 ion- exchange resin; the plate was centrifuged at 1000 rpm for 5 minutes using a Beckman GS-6R centrifuge equipped with a PTS-2000 rotor. [3H]-NAAG bound to the resin and [3H]-glutamate eluted in the flow through. Columns were then washed twice with formate (1 M, 90 μL) to ensure complete elution of [3H]-glutamate. The flow through and the washes were collected in a deep 96-well block; from each well with a total volume of 270 μL, a 200 μL aliquot was transferred to a glass scintillation vial, to which 10 ml of Ultima-Gold (Perkin Elmer) was added. The radioactivity in each vial corresponding to [3H]-glutamate was determined via a Beckman LS-6000IC scintillation counter. Radioactivity values in dpm were converted to fmoles of glutamate using the relation 1 pCi/2.2 dpm and the specific activity of [3H]-glutamate (same as that of [3H]-NAAG: 1 fmole/50 pCi). As a result, if 16711 dpm [3H]-glutamate were measured after incubating 10 mg tissue for 1 h, the normalized activity would be: 16711 dpm × (1 pCi/2.2 dpm) × (1 fmole/50 pCi)/10 mg tissue = 15 fmole/h/mg tissue.
Significant differences among measurements were determined using two-tailed distribution, equal variance student's t test.
Determination of 2-PMPA concentration in rodent paws by LC-MS/MS
Frozen samples were thawed in a water bath at ambient temperature and subjected to a liquid extraction using MeOH. Samples were placed in brown glass vials containing 500 μL of 100% MeOH. The vial was capped and mixed vigorously for 10 sec on a vortex-mixer followed by 30 min on an automated multitude shaker, followed by incubation for 24 h at 4°C. The top organic layer was transferred to a disposable borosilicate glass culture tube (13 × 100 mm) and evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was reconstituted in 100 μL acetonitrile-water (1:1, v/v) containing the internal standard, temazepam (50 μg/mL), by vortex mixing (30 sec) and immersion in an ultrasound bath (5 min). The sample was transferred to a 250 μL polypropylene auto sampler vial sealed with a Teflon crimp cap, and a volume of 50 μL was injected onto the HPLC instrument for quantitative analysis using a temperature-controlled auto sampling device operating at 10°C.
Chromatographic analysis was performed using a Waters ACQUITY UPLC (Milford, MA, USA). Separation of the analytes from potentially interfering material was achieved at ambient temperature using a Waters Altantis column (100 × 2.1 mm i.d.) packed with a 3 μm ODS stationary phase, protected by a guard column packed with 3.5 μm RP18 material (Milford, MA, USA). The mobile phase used for the chromatographic separation was composed of acetonitrile-water (60:40, v/v) containing 0.1% formic acid, and was delivered isocratically at a flow rate of 0.3 mL/min. The column effluent was monitored using an AB SCIEX TRIPLE QUAD 5500 triple-quadrupole mass-spectrometric detector (Applied Biosystems, Foster City, CA, USA). The instrument was equipped with an electrospray interface, operated in a positive mode and controlled by the Analyst version 1.5 software (Applied Biosystems). The spectrometer was programmed to allow the [MH+] ion of 2-PMPA at m/z 226.8 and that of the internal standard at m/z 301.1 pass through the first quadrupole (Q1) and into the collision cell (Q2). The daughter ions for 2-PMPA (m/z 191.1) and the internal standard (m/z 255.1) were monitored through the third quadrupole (Q3). Calibration curves were generated over the range of 200 to 10,000 ng/mL. Mouse paw pad samples were then quantitated in μg/g as: nominal concentration (ng/mL) × 0.0625 (standardized dilution) × sample weight (in mg).