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D. Qujeq / Steroids 67 (2002) 1071–1077
dehydrogenase activity has been available. Because of the
low activities of 3 beta-hydroxy-delta 5-steroid dehydro-
genase, the spectrophotometric method suffers from poor
sensitivity. The radiochemical assay, although sensitive, is
cumbersome in addition to being expensive, requiring ra-
dioactive chemicals and sophisticated instrumentation. The
purpose of this investigation was to develop a new colori-
metric, rapid, and sensitive method for the determination
of 3 beta-hydroxy-delta 5-steroid dehydrogenase activity in
rat testis by use of nitro blue tetrazolium (NBT).
was stable for 30 min. A standard curve was prepared by
plotting NADH concentration versus absorbance.
2.5. Animals
The animals used in these studies were adult male rats,
which were fed an estrogen free diet ad libitum. The rats
weighed between 250 and 300 g. All animals survived the
study without signs of illness. All experimental manipula-
tion was carried out with the animals under ether inhalation
anesthesia.
2. Materials and methods
2.6. Preparation of homogenates
Adult male rats were killed by ether anesthesia, and their
testes were excised. Excess fat was removed from the testes.
They were then chilled on ice, blotted, and weighed. At least
10–12 testes from five to six rats were pooled as the gland
size was small (70 mg), homogenized using ground glass
homogenizers with 10.0 ml of 0.15 M Tris–HCl buffer (pH
7.25), and centrifuged at 10,000 × g at 5 ◦C. The lipid layer
was removed by aspiration, and the supernatant was used as
the enzyme extract.
2.1. Chemicals
Pregnenolone (5-pregne-3-ol-2-one) and testosterone
(17-hydroxyandrostan-4-en-3-one) were purchased from
Sigma Chemical Co. (St. Louis, MO). Nicotinamide ade-
nine dinucleotide (NAD) and the reduced form of nicoti-
namide adenine dinucleotide (NADH) were obtained from
Boehringer Mannheim (Germany). NBT, nicotinamide
adenine dinucleotide phosphate (NADP), and phenazine
methosulfate (PMS) were purchased from Merck (Darm-
stadt, Germany). All other reagents were of highest purity,
and available solvents were freshly redistilled prior to use.
2.7. Assay procedure
3-HSDH activity was determined by measuring the rate
of conversion of pregnenolone into progesterone. The iso-
merase reaction is rapid, so it is customary to refer to the
assay in terms of the rate-limiting dehydrogenation step.
The enzyme was assayed in 1.5 ml of 0.15 M Tris–HCl
buffer (pH 7.25) containing 1.5 ml of 400 M NAD and
0.2 ml of 200 M pregnenolone in a total volume of 3.20 ml.
The reaction was started by adding the enzyme (100 l) and
incubated at 37 ◦C for 60 min. A control incubation of the
rat testis homogenate and NAD, without addition of preg-
nenolone was carried out. At the end of the incubation pe-
riod, the reaction was stopped by the addition of 2.0 ml of
phthalate buffer (pH 4.25). The turbidity was removed by
centrifugation at 5000 × g for 30 min, and the supernatant
was read at 570 nm in a spectrophotometer. Enzyme activity
was calculated from the standard curve of NADH and ex-
pressed as nmol NADH formed/h mg protein. An enzyme
unit is defined as the amount of enzyme that catalyzes the
release of 1 nmol NADH/min at 37 ◦C. Specific activity is
expressed in terms of units per mg of protein. The pro-
tein contents of various enzyme extracts relative to standard
solution of bovine serum albumin were determined by the
Lowry et al. method [12].
2.2. Reagents
Phthalate buffer (70 mmol, pH 4.25): 3.57 g of potassium
hydrogen phthalate was dissolved in a mixture of 50 ml of
0.15 M HCl and 3.0 ml of Tween 20, and the pH was adjusted
to 4.25. The volume was made up to 300 ml with distilled,
deionized water. Then, Tris–HCl buffers (0.15 M, pH 7.25)
and NAD (10 mm) were added, and the contents were mixed.
2.3. Color reagent
Fifty milligram NBT, 15 mg PMS, and 1.0 ml Tween 20
were dissolved in 50 ml distilled water for the standard curve.
For the enzyme assay, PMS was omitted from the reagent.
The reagent containing PMS was stored in a dark bottle.
The substrate (Pregnenolone, 50 mM) was first dissolved in
0.5 ml of dimethyl formamide, and the stock solution (1 mm)
was prepared in 50 or 100 ml Tris–HCl buffer (0.15 M, pH
7.25).
2.4. Standard curve
A 1 mM solution of NADH was freshly prepared in dis-
tilled water, and the contents were mixed. Aliquots of graded
concentrations of NADH (0–2 nm) were reacted with the
color reagent (0.5 ml), and after the color formed, 2.0 ml of
phthalate buffer was added to each tube, and the absorbance
of standard against blank was read at 570 nm. The coloration
2.8. Measurement of the conversion of radioactive
pregnenolone to progesterone
3-HSDH activity was measured by a modification of
the procedure described by Armstrong and Wells [13]. A
solution of the substrate, [4-14C] pregnenolone (200 M;