Five enzymes are involved in the conversion of cholesterol to testosterone (1). The initial reaction in this process involves side chain cleavage of cholesterol to form pregnenolone. The subsequent conversion of pregnenolone to testosterone involves an ordered series of enzymatic reactions, including side chain cleavage, reduction of 17-keto groups and A-ring oxidation. 17-OH-Progesterone is one of the steroids involved in the course of that process, and also a major step in the biosynthetic pathway leading to cortisol, by hydroxylation at both C₁₁ and C₂₁.

C₂₁-hydroxylase deficiency is the most common cause of ambiguous genitalia in infants as well as the most common form of congenital adrenal hyperplasia (CAH). Most CAH (2,3,4) result from an enzyme block producing very high plasma concentrations of 17-OH-Progesterone and androgen precursors, including androstenedione. Urinary excretion of pregnantriol, the metabolite of 17-OH-Progesterone, will also be increased. The diagnosis of C₂₁-hydroxylase deficiency should always be considered in :

- patients with ambiguous genitalia.

- infants presenting severe dehydrated conditions.

- males and females with signs of virilization before puberty.

17-OH-Progesterone circulates bound to both transcortin and albumin. The elevation in concentration of plasma 17-hydroxyprogesterone is such a distinctive marker of 21- hydroxylase deficiency that prenatal diagnosis has been attempted by measuring its concentration in amniotic fluid (5).

Testes, ovaries, adrenals and placenta can produce 17-OH-Progesterone. Direct assays were described for the determination of 17-OH-Progesterone in small volumes of serum, saliva or blood spots (6-9). It should be kept in mind that some interference of steroids (17-OH-Pregnenolone,...) in some newborn sera might occur with radioimmunological determinations of 17-OH-Progesterone (10-11).

Radioimmunoassay is based on the ability of a limited quantity of antibody to bind a fixed amount of radiolabelled antigen(¹²⁵I-Ag). The percentage of bound radiolabelled antigen is inversely related to the increasing concentration of unlabelled analyte in the sample. Separation of the bound and free radiolabelled antigen is necessary in order to determine the quantity of unlabelled antigen. The Bio-Line 17-OH-Progesterone kit utilizes the coated tubes methodology. The quantity of unlabelled antigen in an unknown sample is then determined by comparing the remaining radioactivity in the coated tubes with data established using known standards in the same assay system.

Kit contains sufficient reagents for 100 determinations.

Sera should be separated from blood cells immediately after collection. Sera are stable for at least 7 days at 4^o C and for longer periods of time when stored frozen.

Bring reagents to room temperature and mix before use. Label tubes for total counts (Tc), standards, control sera and unknowns.

Determine the average counts for each set of duplicate tubes. Divide this value by the average net counts of the Bo, and multiply by 100 to yield the % B/Bo

Plot % B/Bo for each standard vs its concentration in ng/ml on semi-log graph paper. The concentration of 17-OH-Progesterone in the unknown samples may be read directly from the standard curve.

Range (ng/ml)

cord blood 7 - 20

newborn 1 - 7

adult male 0.3 - 2

adult female Foll. phase 0.1 - 1.2

adult female LH peak > 1

luteal phase 0.3 - 3

postmenopausal 0.1 - 0.8

children with CAH > 50

Each laboratory should analyze normal samples to establish its own normal ranges.

Conversion factor: 1 ng/ml = 3 nmol/l

10. Specific performance characteristics:

1. Specificity:

The relative percent of cross-reactivity by weight of 17-OH-Progesterone and various related compounds was evaluated for the antibody used in this assay. Cross-reactivities are expressed as the amount of 17-OH-Progesterone required to reduce the binding of ¹²⁵I-17-OH-Progesterone by 50%, relative to the amount of a related compound required to do the same.

Cross-reactivity of x = 100 x conc. 17-OH-P /conc. compound  at 50% B/B_o

                          Compound x Cross-reactivity (%)

17-OH-Progesterone 100 %

17-OH-Pregnenolone < 2 %

Progesterone < 1 %

11-Deoxycortisol < 1 %

Pregnenolone < 0.50 %

Cortisone < 0.05 %

Corticosterone < 0.05 %

Deoxycorticosterone < 0.05 %

DHEA < 0.05 %

DHEA-S < 0.05 %

Estriol < 0.05 %

Estrone < 0.05 %

Hydroxycortisone < 0.05 %

Testosterone < 0.05 %

2. Sensitivity:

The lowest detectable concentration of 17-OH-Progesterone that can be reliably distinguished from zero with this kit has been evaluated to be £ 0.09 ng/ml.

3. Precision and reproducibility:

Assays variations of two serum samples are mentioned in the following table.

                                            Sample 1                                 Sample 2

                                            Mean 2.9 ng/m                         10.2 ng/ml

Within assay variation         3.8 %                                         4.3 %

Between assay variation    4.2 %                                         7.9 %

4. Linearity:

The results obtained when diluting a serum with elevated 17-OH-Progesterone concentration with a 17-OH-Progesterone- free serum are summarized in the following table

Dilution factor         Expected values             Experimental values

1:1                                 19.92 ng/ml                             -

1:2                                 9.96 ng/ml                         9.82 ng/ml

1:4                                 4.98 ng/ml                         5.07 ng/ml

1:8                                 2.49 ng/ml                         2.84 ng/ml

11. Bibliography:

1. Yanaihara T.,Troen P. Studies of the human testis. I. Biosynthetic pathways for androgen formation in human testicular tissue in vitro. J Clin Endocrinol Metab 1972; 34: 783-792.

2. New MI, Levine LS, Pang S, et al. Adrenal components in abnormal sexual differentiation. In: Serio M, Zanisi M, eds. Sexual Differentiation: Basic and Clinical Aspects. New York: Raven Press, 1984: 321-349.

3. Godo B, Visser HKA, Degenhart JH. Plasma 17-OH-Progesterone in fullterm and preterm infants at birth and during the early neonatal period. Horm Res 1981; 15:65-71.

4. de Piretti E, Forest M. Pitfalls in the etiologic diagnosis of congenital adrenal hyperplasia in the early neonatal period. Horm Res 1982; 16: 10-20

5. Frasier SD, Thorneycroft IH, Weiss BA, et al. Elevated amniotic fluid concentration of 17 hydroxyprogesterone in congenital adrenal hyperplasia. (Letters to the editor.) J Pediatr 1975; 86: 310-311.

6. Tsuji A, Maeda M, Arakawa, et al Fluorescence and chemiluminescence enzyme immunoassays of 17 hydroxyprogesterone in dried blood spotted on filter paper. J Steroid Biochem 1987; 27: 33-40.

7. El-Gamal BA, Eremin SA, Smith DS, Landon J. Development of a direct fluoroimmuno-assay for serum levels of 17-OH-Progest. Ann Clin Biochem 1988; 25:35-41

8. Dyas J, Read GF, Guha-Maulik T, Hughes IA, Riad-Fahmy D. A rapid assay for 17 OH-progesterone in plasma, saliva and amniotic fluid using a magnetisable solid-phase antiserum. Ann Clin Biochem 1984; 21: 417-24.

9. Wallace AM, Beastall GH, Cook B, et al. Neonatal sreening for congenital adrenal hyperplasia: a programme based on a novel direct immunoassay for 17 hydroxyprogesterone in blood spots. J Endocrinol 1986; 108: 299-308.

10.Makela S.K., Ellis G. False positive elevetionof serum 17-OH-Progesterone in neonates, as measured by a direct radioimmunoassay. Clin. Chem; 1987,33: 886.

11.Masako Maeda et al. Enzyme-linked immunosorbent assay for 17-OH-Progesterone in dried blood spotted on filter paper. Clin. Chem.1987,33: 761.