Glucosinolate Analysis. The procedure followed was that of Minchinton (1). Dried broccoli sprouts (80 mg) were mixed with 8 ml of 80% boiling ethanol and boiled for a further 5 min in a water bath. The mixtures were subsequently homogenized by using a Polytron tissue homogenizer (Brinkmann) and centrifuged for 3 min at 3,000 × g. The supernatants were pipetted into microcentrifuge tubes, and the leftover plant material received an additional 1 ml of boiling 80% ethanol and were placed in the boiling water bath for 5 min more and then centrifuged. The supernatants from each sample were combined, and ethanol was evaporated by placing tubes into the carriage of a Reacti-Therm Heating module that was set at a temperature of 40°C and allowing nitrogen gas to flow over the tubes until samples were dry. The concentrated extracts were redissolved in 2 ml of distilled and deionized water, and 0.5 ml of 0.5 M lead–barium acetate was added to precipitate proteins. The samples were vortexed and centrifuged, and supernatants were placed on a DEAE-Sephadex A-25 (pyridine acetate form; Sigma–Aldrich) column. A 3 mg/ml aryl sulfatase H-1 (Sigma–Aldrich) solution was prepared in water. To the DEAE-Sephadex A-25 columns, 75 m l of the 3 mg/ml aryl sulfatase solution was applied and left for 24 h at room temperature. After the incubation period, 2 ml of distilled-deionized water was placed onto each of the columns, eluting the desulpho analogues. The obtained eluates were boiled in a water bath for 3 min to inactivate any residual galactosidase activity. The boiled eluates were subsequently filtered through 0.2-m nylon Acrodisc filters (Gelman). A 1 m g/m l (3.3 m M) internal standard solution, o-nitrophenyl-D-galactopyranoside (Sigma–Aldrich) was prepared in water. To 180 m l of each of the filtered desulphoglucosinolate samples, 20 m l of the internal standard solution was added.

For desulfoglucosinolate separation and identification, a 1100 series liquid chromatograph (Hewlett–Packard), controlled by a HP CHEMSTATION software (version 4.01) was used. The UV detector was set at 226 nm. A Hypersil ODS column (250 × 4.6 mm, 5 M particle size) with a 4.0-mm i.d. ODS-Hypersil 5 m M guard column was used to separate the desulphoglucosinolates. Aliquots of 100 m l were injected onto the column set at a temperature of 25°C. The flow rate of the eluent was 1.0 ml/min. The solvent program consisted of 2% o-phosphoric acid (analytical grade; BDH) flowing isocratically for 10 min, followed by a 0–12% linear gradient of acetonitrile (HPLC grade; EM Science) over 55 min, finishing with 12% acetonitrile flowing in an isocratic mode for 25 min.

We also collected the 19.2 min peak from a number of HPLC runs, evaporated the fractions collected and determined that the collected fractions contained only desulfoglucoraphanin. By applying known amounts to the HPLC column, we determined the relationship of the area under the desulfoglucoraphanin curve to the area under the curve of the internal standard. This information was used to quantify the glucoraphanin in our broccoli sprout samples.

Animal Tissue Preparation. At the end of study, the animals, while under halothane anesthesia, were perfused through the ascending aorta with 0.05 M heparinized phosphate buffer saline and tissues, including aorta, carotid artery, heart, and kidney, were removed. Some aorta tissues were used immediately after isolation for isometric tension studies, and the remaining tissues were frozen immediately in liquid nitrogen. The frozen tissues were directly transferred into a special metal container and ground frozen with a homogenizer (Mickro-Dismembrator; Braun, Melsungen, Germany) for 4 × 1 min at the frequency of 3,000 rpm. The homogenized tissue powders were stored at –80°C until used for glutathione analysis, enzyme assays, and Western blotting.

The rats used for immunohistochemical study were perfused with heparinized PBS and then continued with the same solution containing 4% paraformaldehyde. The tissues including aorta, carotid artery, heart, and kidney were dissected out immediately, kept in same fixative overnight, incubated in a 30% sucrose solution for 3 days at 4°C for cryoprotection, and embedded in Optimal Cutting Temperature (OCT) compound (Somagen Diagnostics, Inc). Frozen sections (10–12 m m) were cut, collected on gelatin-chrome alum-coated slides and stored at –20°C until further processing.

Isometric Tension Studies. The thoracic aortae collected following buffered saline perfusion were cleaned of adipose tissue and cut into 2- to 3-mm rings. The rings were suspended under 1.0 g of tension in a 10-ml organ bath chamber filled with Krebs’ saline solution aerated with 95%O₂/5%CO₂, as described (2). We added 1 m mol/l indomethacin to Krebs’ saline that was comprised of 115 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO₄, 1.2 mM NaH₂PO₄, 25 mM NaHCO₃, 11 mM D-glucose and 1.8 mM CaCl₂. Phenylephrine (3 × 10^–7 M) was used to induce contraction and in the stable tonic contraction, acetylcholine (ACh) or sodium nitroprusside was introduced into the bath solution. The isometric tension was measured at 37°C with FT 03 force-displacement transducers (Grass Instruments, Quincy, MA). Data acquisition and analysis were accomplished with a Biopac system (Biopac Systems, Goleta, CA).

1. Minchinton, I., Sang, J., Burke, D. & Truscott, R. J. (1982) J. Chromatogr. 247, 141–148.

2. Wu, L., Wang, Z. & Wang, R. (2000) Can. J. Physiol. Pharmacol. 78, 696–707.