Abstract
A simplified radioimmunoassay for measurement of plasma testosterone was established. Antiserum for testosterone was prepared in rabbit by immunization with testosterone-3-oxime linked to bovine serum albumin. The antiserum was diluted to 1: 10,000 with a borate buffer containing 0.1 % BSA. Plasma samples (0.1-0.5ml) were washed with 5 volumes of n-hexane and then extracted with 10 volumes of ether-hexane (2: 8) solvent. Extraction residue was dried directly in an assay tube when a higher value of plasma testosterone was expected. Further purification procedure with alumina column chromatography was desirable in samles with lower levels of plasma testosterone. Testosterone containing fractions were dried and incubated for 12 hours at 4° with 0.5ml 1/10,000 antitestosterone antiserum and testosterone-3H (20,000 dpm) in 0.5ml buffered saline. To each tube, 0.5 ml of dextran coated charcoal solution (250mg of Norit A and 250mg of Dextran T-80/100 ml buffer) were added ; mixture was shaken, incubated for 10 minutes at 4° and centrifuged for 10 minutes at 2,500 rpm, and the supernatant was taken for liquid scintillation counting. Duplicate testosterone standards (0, 50, 100, 200, 500, 1,000pg) were obtained in a similar fashion through the entire procedure and a standard curve was traced. Sensitivity of this method was 10ng/100ml when 0.5ml of plasma sample was assayed. Coefficient of variation was about 10 percent.
Plasma testosterone levels were measured in normal men and patients with pituitary Leydig cell dysfunction by using this assay procedure. Plasma LH levels were also measured simultanously by radioimmunoassay. The normal range of plasma testosterone in adult men were 450 to 1,150ng/100ml, which showed little diurnal variation, reduced to the half values upon 2 day oral administration of 15mg of fluoxymesterone and returned to the normal level upon an additional injection of 1,000 IU of human chorionic gonadotropin (HCG). The plasma testosterone levels in aged men (60-80 years old) did not show any significant difference from those in younger men (20-40 years old).
Responsiveness of Leydig cells to HCG was estimated by measuring the plasma testosterone levels after injection of HCG. Three thousands IU of HCG were injected intramusculary for 4 days to 4 young men, 3 aged men and 6 patients with hypogonadism. An average of the plasma testosterone values before HCG injection in young men, aged men and patients with hypogonadism were 680±120, 580±80 and 170±80ng/100ml (Mean±SD), respectively. The plasma testosterone response to HCG showed a 100 (1) increase after 4 day successive injection in young men, a 50 96 increase in aged men and no increase in hypogonadism.
One hundred microgram of LH-releasing hormone (LH-RH) was injected intravenously to 12 young men, 11 aged men and 8 patients with hypogonadism. In young men, the plasma testosterone levels reached to the peak of 50% increment in 120 minutes after injection of LH-RH, while the plasma LH levels were increased by more than 5 times values in 30 minutes after injection. In aged men, no increase in the plasma testosterone levels by administration of LH-RH were shown despite plasma LH were increased to the same levels as those in young men by LH-RH injection. In hypogonadic patients, neither plasma testosterone nor plasma LH showed any response to LH-RH.
The plasma testosterone and LH levels showed no significant difference between young men and aged men. However, responsiveness to HCG and to endogenous LH were cleary decreased. These findings suggest that the control mechanism of hormonal secretion in hypothalamo-pituitary-gonadal axis may be changed by aging.
Assay of plasma testosterone using a simplified radioimmunoassay may prove to be valuable as an indicator of Leydig cell function, especially upon stimulation with HCG and LH-RH.
