An improved ultrafiltration method for determining free testosterone in serum.

Abstract

In this method, we use the Amicon MPS-1 centrifugal ultrafiltration device and the YMB membrane in measuring free testosterone in serum. Two independent assays are combined: total testosterone and the ultrafiltrable fraction of added [3H]testosterone. The unbound fraction is determined in 0.15-0.5 mL ultrafiltrates of 0.6 to 1 mL of variably diluted serum that has been equilibrated with [3H]testosterone at 37 degrees C. The assay is rapid (less than 1 h), practicable (requires 0.6 mL of serum), and reproducible (CV 3.2% within assay, 3.9% between assays). Accuracy was evaluated as the fraction of free testosterone in the ultrafiltrate of dialyzed serum vs that in a prior dialysate; they were the same confirming the validity of the free testosterone measurement. Samples from ostensibly healthy men and women and from hirsute and pregnant women gave results that agreed with those obtained by equilibrium dialysis. Total testosterone concentrations for normal and hirsute women showed considerable overlap, but data on free testosterone concentrations in these populations were better resolved.

Testosterone release rate and duration of action of testosterone pellet implants

“…Plasma LH (Axsym, Abbott Laboratories, IL, USA; CV 5·0–7·4%), FSH (Axsym, Abbott Laboratories; CV 3·5–7·4%), SHBG (Immulite, DPC, Los Angeles, CA, USA; 6·1–7·9%) and total testosterone (Immulite, DPC; 7·8–12·7%) were measured by commercial immunoassays as described previously (Ly et al ., 2001; Liu et al ., 2002). Free testosterone was measured by an in‐house centrifugal ultrafiltration assay (Vlahos et al ., 1982) using Centrifree columns (Millipore, Bedford, MA, USA) and tritiated testosterone to estimate percentage unbound testosterone from which actual free testosterone (CV 9·6–11·7%) is calculated using total testosterone.…”

Marked Decline in Trabecular Bone Mineral Content in Healthy Men with Age: Lack of Association with Sex Steroid Levels

“…Free testosterone levels were measured by the ultrafiltration method of Vlahos et al, 21 utilizing (1,2,6,7,‐ 3 H) testosterone (specific activity 102 Ci/mmol, purified on celite columns using iso‐octane: ethylacetate to 99% purity). A single YMT membrane and Amicon MPS‐1 ultrafiltration device (Amicon Corp., Danvers, Massachusetts) were used for each observation.…”

Testosterone Replacement Therapy for Treatment Refractory Cluster Headache

“…Unless reported otherwise, testosterone blood levels were measured utilizing ultrafiltration technique to measure percentage free, and the free fraction was calculated from the total testosterone result. 7 This technique was validated in house and found to correlate well with equilibrium dialysis. LH and FSH were measured by two-site immunochemiluminometric assay (ICMU) using reagents and automated platform (E170) from Roche Diagnostics Corporation (Indianapolis, IN).…”

“…The only reports, in fact, commented on the inadvisability of this approach due to behavioral side effects. [7][8][9] Functional neuroimaging studies, including PET and BOLD fMRI, have recently confirmed the suprachiasmatic nucleus (SCN) as the site of increased metabolic activity in both induced and spontaneous cluster headaches. Newer data now also support anterior hypothalamic loci as areas responsible for related trigeminal autonomic cephalalgias.…”