**3. Results**

120 Basic and Clinical Endocrinology Up-to-Date

OAT activity was measured in whole kidneys. Samples were mixed at 4°C with a Turrax homogenizer in a buffer (0.33 M sucrose, 5 mM HEPES, 1 mM EGTA, 1 mM DTT, and 0.5% Triton X 100). Homogenates were centrifuged 21,000 x *g* for 10 min at 4°C. The protein concentration was determined on the supernatant using the Bradford protein assay. OAT activity was determined using the enzyme assay previously described (Herzfeld & Knox, 1968; Peraino & Pitot, 1963). Briefly, the supernatant was incubated with a buffer composed of 75 mM potassium phosphate pH 8.0, 20 mM L-ornithine, 0.45 mM pyridoxal phosphate, 5 mM α-aminobenzaldehyde, and 3.75 mM α-keto-glutarate for 15 or 30 min at 37°C. Blanks did not contain α-keto-glutarate. The reaction was stopped by adding trichloroacetic acid 40%. Samples were centrifuged 21,000 x *g* for 3 min at 4°C and absorbance was measured on the clear supernatant at 440 nm on a Hitachi U-1100 spectophotometer (Meylan, France).

Testosterone was measured by radioimmunoassay after extraction by organic solvant and partition chromatography of the plasma samples as previously described (Dechaud et al, 1989). Corticosterone concentration was measured, after diethylether extraction, by radioimmunoassay using 1,2,6,7 [3H]-corticosterone and rabbit anti-corticosterone

The calculations were as follows: for each group of mice and for each protein, the mean intensity optical densitometry (IOD) of the bands was calculated. The mean IOD of the untreated (group I) and sham-operated (group V) mice were used as a reference (control). For each mouse, the IOD value of a given protein was divided by the mean IOD value of the control group. Consequently, this ratio value is 1 in each control group. Then, these ratios

OAT activity was expressed in absorbance per 15 min per mg soluble protein because we were unable to find a supplier to buy P5C as standart to convert the absorbance of P5C into molar unit. Depending on the material used or the physiological interest of expressing the data in other units, OAT activity is expressed in absorbance per 15 min per mg soluble

Results are expressed as means ± SE. Non-parametric statistical tests were used to analyze the data. Where appropriate, statistical differences were assessed using the Kruskal-Wallis test at level significance of 95% and this test was followed by the U-Mann-Whitney test at level significance of 95% (StatView SE+Gr). For correlation analyses, the correlation coefficient *r2* was calculated with Microsoft Excel, and *P* was determined from tables at the

Salts and most chemicals, Ponceau S solution, secondary anti-IgG antibodies, Kodak X-MAT

were purchased from Boehringer Mannhein (Strasbourg, France). Agarose Seakem GTG was purchased from TEBU (Le Perray-en-Yvelines, France). ECL Western Blotting Kits, and ImagerMaster Total Lab v2.01 program were purchased from Amersham

Quentin Fallavier, France). Protease inhibitor cocktail

Duplicate or triplicate samples and blanks were performed for all experiments.

**2.6 Determination of testosterone and corticosterone levels** 

**2.5 Measurement of OAT activity** 

polyclonal antibody (Filipski et al, 2002).

**2.7 Calculations and statistical analyses** 

were related to those of β-actin and/or G3PDH.

proteins.

95% level of significance.

(Buckinghamshire, UK).

film were purchased from Sigma (St

**2.8 Chemicals** 
