Biomedical subjects

Frank Giton

Publications and source records attributed to Frank Giton.

5 recordsLinked to original sources

PubMed2005

Serum bioavailable testosterone: assayed or calculated?

BACKGROUND: Bioavailable testosterone (BT), circulating testosterone not bound to sex hormone-binding globulin (SHBG), is thought to easily penetrate cells. We compared BT measurements obtained by assays with those obtained by calculation with different testosterone association constants. METHODS: We obtained sera from 2 groups of hypogonadal men [group 1 (G1), 1421 samples; group 2 (G2), 170 samples] and a group of healthy men [group 3 (G3), 109 samples]. We added minute doses of [3H]testosterone to the sera, precipitated the SHBG-bound fraction of testosterone with ammonium sulfate (50% saturation), and then assayed serum BT (ABT) as %BT x total. Calculated BT (CBT) was determined with theoretical association constants of testosterone for SHBG (Ks = 1 x 10(9) L/mol) and albumin (Ka = 3.6 x 10(4) L/mol) and paired optimal Ks and Ka values obtained by use of Microsoft Excel software. RESULTS: CBT calculated with theoretical constants differed from ABT by >30% in 85.7% (G1), 84.1% (G2), and 77.9% (G3) of samples, and the mean CBT/ABT ratios were 1.57 (G1), 1.85 (G2), and 1.50 (G3) in spite of fairly good correlations. CBT calculated with paired optimal K(s) and K(a) differed from ABT by <30% in 87.4% (G1), 87.5% (G2), and 97.5% (G3) of samples, and mean CBT/ABT ratios were 0.95-1.04. CONCLUSIONS: To obtain CBT values as close as possible to ABT, optimal paired association constants determined for each studied population must be used instead of the theoretical association constants. Considering the uncertainty of calculating BT, however, use of the ammonium sulfate precipitation method for determining BT is advisable.

Androgens↗

PubMed2004

Development of a highly sensitive and specific new testosterone time-resolved fluoroimmunoassay in human serum.

A new time-resolved fluoroimmunoassay (TR-FIA) of testosterone in serum is described, using a biotinylated testosterone tracer, with a long spacer arm between biotin and testosterone, coupled to the C3 of the testosterone: a biotinylaminodecane carboxymethyloxime testosterone. This tracer affords a great sensitivity of the standard curve, because a amount of 0.3 pg of testosterone can be significantly measured on the testosterone standard curve. The "functional" sensitivity is at least equal to 21 pg/ml of serum. The specificity of the assay is insured by a celite chromatographic step on new minicolumns before immunoassay. The variation coefficient of inter-series reproducibility measured on low and normal testosterone levels in untreated and testosterone treated hypogonadal men were between 2.17 and 5.07%. The accuracy test, (overload and dilution tests) gave satisfying results. Moreover, in a comparison with GCMS, it appeared that the correlation coefficient was 0.992 and no significant difference could be exhibited between the two methods. Consequently, this specific, sensitive reproducible and easy to use method is well suited to the measurement of testosterone in clinical and pharmacological conditions.

Biotin↗

PubMed2003

Plasma 11beta-hydroxy-4-androstene-3,17-dione: comparison of a time-resolved fluoroimmunoassay using a biotinylated tracer with a radioimmunoassay using a tritiated tracer.

The plasma concentration of 11beta-hydroxy-4-androstene-3,17-dione (11beta) is very high in 21-hydroxylase deficiency, Cushing's syndrome, and hyperandrogenism of adrenal origin, and very low in congenital 11-hydroxylase deficiency and adrenal insufficiency. Thus, when plasma 4-androstenedione is elevated, it is useful to measure the plasma 11beta level in order to determine the adrenal or ovarian origin of the hyperandrogenism. To eliminate disadvantages related to the 11beta radioimmunoassay (RIA), which uses a tritiated tracer, as well as the high cost associated with scintillation proximity assay (SPA), we developed a non-isotopic 11beta assay that utilizes an 11beta-biotin conjugate synthesized in our laboratory to measure time-resolved fluorescence after addition of streptavidin-europium to microtitration wells. The analytical qualities of this assay are very similar to those of the radioimmunoassay using a tritiated tracer, and an extraction step followed by celite chromatography (which separates 11beta from interfering plasma steroids) prior to a final radioimmuno-competition step. The correlation coefficient between 11beta levels measured by time-resolved plasma 11beta fluoroimmunoassay (TR-FIA) and RIA was 0.965.Finally, the TR-FIA technique was more sensitive and of greater precision than the RIA method.

Androstenedione↗

PubMed2002

Development of a new sensitive and specific time-resolved fluoroimmunoassay (TR-FIA) of chlormadinone acetate in the serum of treated menopausal women.

We describe the development of a serum chlormadinone acetate (CMA) time-resolved fluoroimmunoassay (TR-FIA). We prepared haptens (3-CMO-chlormadinone acetate and 6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinate), biotinylated tracers (3(biotinylaminopropylamido) 3-CMO-chlormadinone acetate and 3-(6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinylamino)1-biotinylaminopropane), and immunogens necessary for eliciting two antibodies (anti-chlormadinone acetate 3-CMO/BSA and anti-chlormadinone 20-hemisuccinate/BSA). The specificity of the assay was rigorously studied to eliminate possible interference by polar metabolites of CMA, particularly 17 alpha-acetoxy-6-chloro-3beta-hydroxypregna-4,6-diene-20-one (3beta-hydroxy metabolite), employing an easy-to-use ethylene glycol chromatographic step prior to immunoassay, so as to separate the polar metabolites, in particular the 3beta-hydroxy-CMA metabolite, from the intact CMA. The choice of the anti-CMA antibody was guided by the high assay sensitivity obtained with the anti-CMA 3-CMO/BSA antibody. The detection limit was 51pg/ml. Interassay reproducibility CVs were between 2.6 and 4.5%. This TR-FIA thus appeared to be a sensitive, specific, precise, and consequently well-suited method for measurement of serum CMA during a pharmacokinetic study in women.

Chlormadinone Acetate↗

PubMed2002

Specific radioimmunoassay of estrone sulfate. Application to measurement in male plasma.

We described a new, specific and easy to use radioimmunoassay (RIA) of estrone sulfate (E1S) in males. After synthesis of an E1S-6-(O-carboxymethyl) oxime hapten then coupling to BSA, we obtained a specific anti-E1S antiserum. Although the cross-reactivity of DHEAS with our anti-E1S antiserum was low (CR=0.002%), we confirmed the absolute necessity of separating plasma DHEAS from plasma E1S, before E1S RIA, because in plasma, DHEAS is present at levels 3-6000-fold higher than E1S, which generally is ignored. Thus, we elicited an easy separation of DHEAS from E1S, by a fast chromatography on in-house minicolumns. This new RIA, was applied to the determination of E1S plasma normal values in males. In 27 young men (<35 years), mean+/-S.D. were 1.97nmol/l+/-1.07nmol/l and in 63 untreated healthy aged men (>55 years), 1.80nmol/l+/-1.21nmol/l. No significant difference was seen between young and older subjects. The ranges of E1S plasma levels in these subjects were rather large and the ratios between the highest and the lowest E1S plasma levels were seven in the young group and 23.4 in the older group. No decrease of E1S plasma levels was observed with ages. Contrary to large interindividual E1S plasma level variations, the intraindividual variations have been found to be no significant. Correlations between E1S and unconjugated estrogens, E2 and E1 were 0.22 (P=0.016) and 0.51 (0.001), respectively.

Adult↗