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Allopregnanolone Assays

Montreal General Hospital Montreal, Canada H3G 1A4

To the editor:

In their recent article in JCEM, Luisi et al. (1) found a mean level of allopregnanolone to be about 50 ng/mL in late pregnancy. The method used by Luisi et al. (1), described in a previous publication (2) involves extraction into an organic solvent (diethyl ether) and passage through a C18 Sep-Pak cartridge, followed by RIA using an antibody made and characterized by Purdy et al. (3). No data were provided as to the specificity of this method.

The antibody used was made to an antigen conjugated at C11 and, although highly sensitive for allopregnanolone, cross-reacts 17% with progesterone, 50% with 5-tetrahydoprogesterone and 3-dihydroprogesterone, and to a minor extent with some other steroids (4). The original method of Purdy et al. (3) involved purification of an extract with high-performance liquid chromatography. However, Luisi et al. (1) used only C18 Sep-Pak cartridges for purification.

In our experience, passage through a Sep-Pak cartridge does not remove progesterone, 5-dihydroprogesterone, or many other steroids. Unless their Sep-Pak cartridges have properties differing from ours (also obtained from Waters Corp.), then progesterone, at a concentration of approximately 200 ng/mL in late pregnancy, would account for about 34 ng/mL of their 50 ng/mL value, while 5-dihydroprogesterone would account for an additional 3 ng/mL. Even in nonpregnant patients (2), competing steroids would be expected to give falsely high levels. Indeed, the values for nonpregnant women (3) and our values in nonpregnant women and men, as well as those in pregnancy (4), are lower than those found by Genazzani et al. (2).

As has been found previously for cortisol, it is essential to define the specificity of an assay for the particular milieu in which it is used. Thus, an assay validated for plasma cannot be assumed to be accurate for urine (5, 6); nor can an assay validated for plasma in nonpregnant adults be assumed to be satisfactory for that of newborns or pregnant women (7).

Received October 19, 2000.

References

1. Luisi S, Petraglia F, Benedetto C, et al. 2000 Serum allopreganolone levels in pregnant women; changes during pregnancy, at delivery, and in hypertensive patients. J Clin Endocrinol Metab. 85:2429–2433.[Abstract/Free Full Text]
2. Genazzani R, Petraglia F, Bernardi F, et al. 1998 Circulating levels of allopregnanolone in humans: gender, age, and endocrine influences. J Clin Endocrinol Metab. 83:2099–2103.[Abstract/Free Full Text]
3. Purdy RH, Moore Jr PH, Rao PN, et al. 1990 Radioimmunoassay of 3-hydroxy-5-pregnan-20-one in rat and human plasma. Steroids. 55:290–296.[CrossRef][Medline]
4. Murphy BEP, Allison CM. 2000 Determination of progesterone and some of its ring A-reduced metabolites in human serum. J Steroid Biochem Mol Biol. 74:137–142.[CrossRef][Medline]
5. Murphy BEP, Manor Okouneff L, Klein GP, Ngo S. 1981 Lack of specificity of cortisol determinations in human urine. J Clin Endocrinol Metab. 53:91–99.[Abstract/Free Full Text]
6. Murphy BEP. 1999 Lack of specificity of determinations of urinary free cortisol: why does it continue? (Letter). J Clin Endocrinol Metab. 84:2258–2259.[Free Full Text]
7. Murphy BEP. 1983 Human fetal serum cortisol levels: a review. Endocrine Rev. 4:150–154.[Abstract/Free Full Text]

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