The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 12 6087-6091
Copyright © 2004 by The Endocrine Society
Elevated Urine Pregnanetriolone Definitively Establishes the Diagnosis of Classical 21-Hydroxylase Deficiency in Term and Preterm Neonates
Keiko Homma,
Tomonobu Hasegawa,
Eiko Takeshita,
Kiyoaki Watanabe,
Makoto Anzo,
Takio Toyoura,
Kazuhiko Jinno,
Toya Ohashi,
Takashi Hamajima,
Yukihiro Takahashi,
Takao Takahashi and
Nobutake Matsuo
Department of Laboratory Medicine (K.H., E.T., K.W.), and Department of Pediatrics (T.Has., M.A., T.Ta., N.M.), Keio University School of Medicine, Tokyo 160-8582, Japan; Tokyo Metropolitan Kiyose Children’s Hospital (M.A.), Tokyo 204-0022, Japan; Department of Pediatrics (T.To.), Tokyo Medical and Dental University, Tokyo 113-8519, Japan; Department of Pediatrics (K.J.), Division of Medical Intelligence and Informatics, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan; Department of Pediatrics (T.O.), The Jikei University School of Medicine, Tokyo 105-8471, Japan; Department of Endocrinology (T.Ham.), Aichi Children’s Health and Medical Center, Aichi 474-0031, Japan; Division of Neonatal Intensive Care (Y.T.), Center of Perinatal Medicine, Nara Medical University Hospital, Nara 634-8522, Japan; and National Center for Child Health and Development (N.M.), Tokyo 157-8535, Japan
Address all correspondence and requests for reprints to: Keiko Homma, Department of Laboratory Medicine, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: keikoh{at}happy.email.ne.jp.
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Abstract
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Elevated blood 17
-hydroxyprogesterone (17OHP) level, although widely used for the screening of classical 21-hydroxylase deficiency (21OHD) in neonates, has frequently been found in some neonates without classical 21OHD, particularly preterm neonates. We studied the diagnostic value of the metabolite of 21-deoxycortisol (pregnanetriolone, Ptl) and the metabolite of 17OHP (pregnanetriol, PT) in identifying 21OHD in term and preterm neonates with elevated blood 17OHP on the newborn screening.
Spot urine samples from 59 classical 21OHD neonates (50 term, 9 preterm), 83 neonates without 21OHD having transiently elevated blood 17OHP (non-21OHD) (49 term, 34 preterm), and 62 control term neonates were studied using gas chromatography/mass spectrometry in selected ion monitoring analysis for Ptl, PT, 5ß-tetrahydrocortisone (ßTHE), and 5-tetrahydrocortisone (THE).
Ptl and Ptl/(ßTHE+THE) showed no overlap between 21OHD and non-21OHD, and 21OHD and controls, respectively (Ptl was 0.46–124 mg/g creatinine in 21OHD term, 0.80–26.9 mg/g creatinine in 21OHD preterm, 
0.08 mg/g creatinine in non-21OHD term, 0.06 mg/g creatinine in non-21OHD preterm, and 0.07 mg/g creatinine in controls). PT and PT/(ßTHE+THE) showed significant overlap between 21OHD and non-21OHD.
The above data indicate that spot urine Ptl is a highly specific marker of 21OHD with a cutoff value of 0.1 mg/g creatinine, yielding an unambiguous separation between 21OHD and non-21OHD in term and preterm neonates.
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Introduction
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NEWBORN SCREENING FOR 21-hydroxylase deficiency (21OHD) was implemented in 1977 by Pang et al. (1) using a microfilter paper technique. This method uses blood 17-hydroxyprogesterone (17OHP), as measured by the RIA, as the marker for 21OHD and is currently widely used throughout the world because the reliability and feasibility of the method have been confirmed in various populations (2, 3, 4). However, the method lacks sufficiently high specificity, although it retains high sensitivity in the diagnosis of 21OHD in term and preterm neonates (5, 6, 7).
Thus, alternative steroid markers for 21OHD have been studied in both term and preterm neonates by various investigators (8, 9, 10, 11, 12). These include urine 17OHP metabolites such as pregnanetriol (PT), 17-hydroxypregnanolone, and 15ß,17-dihydroxypregnanolone, and urine 21-deoxycortisol (21DOF) metabolites such as pregnanetriolone (Ptl), each measured by gas chromatography/mass spectrometry in selected ion monitoring (GC/MS-SIM) on a spot or 24-h urine sample (Fig. 1
). The results of such studies indicate that each metabolite is a more accurate marker for 21OHD when compared with blood 17OHP and clearly differentiates neonates with classical 21OHD from control neonates. However, no previous studies have addressed whether such 17OHP and 21DOF metabolites may differentiate neonates with classical 21OHD from neonates without classical 21OHD having transiently elevated blood 17OHP in term and preterm neonates.
The major focus of the present paper is to determine the sensitivity and specificity of urine PT and Ptl measurements in the differential diagnosis of term and preterm neonates with elevated 17OHP as detected by the newborn screening.
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Subjects and Methods
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We recruited 59 neonates with classical 21OHD (48 salt wasting, 11 simple virilizing), 83 neonates without classical 21OHD having transiently elevated blood 17OHP (non-21OHD), and 62 control neonates from 1989 through 2002 at Keio University Hospital and 45 related hospitals throughout the country. The diagnosis of 21OHD was made in 22 neonates by CYP 21 gene analysis, and in 37 neonates by typical signs and symptoms coupled with persistently elevated blood 17OHP levels. The diagnosis of non-21OHD was made in all the 83 neonates in whom blood 17OHP level was found to be elevated by the ELISA direct method, and confirmed to be less than 5 (or 3) ng/ml by the repeated measurements by ELISA direct or extraction method. Of these 83 neonates, two showed a mild degree of female pseudohermaphroditism (one term and one preterm), 17 showed hyponatremia of various degrees (one term and 16 preterm), and the remaining 64 were asymptomatic. The 62 control neonates had normal blood 17OHP levels (<5 ng/ml) as screened by the ELISA direct method during the first week of age. All parents of the neonates gave informed consent.
Spot urine samples were collected for the study at age of 3–28 d and kept at –20 C until analyzed. There was no diurnal variation in urine steroid profile in neonates as reported by us (13). Table 1 summarizes baseline data of the study subjects. Each neonate was studied for urine Ptl, PT, and metabolites of cortisol [5ß-tetrahydrocortisone (ßTHE) and 5-tetrahydrocortisone (THE)], by using GC/MS-SIM analysis. The detailed method is available in our previous publication (13). In brief, 0.2–5 ml of urine sample was subjected to methyloxime-trimethylsilyl derivatization after enzymatic hydrolysis and organic solvent extraction, and the derivative was subjected to GC/MS-SIM analysis (an HP 5890II GC with an HP-ULTRA 1 fused silica column (25 m x 0.2 nm x 0.33 µm) coupled to an HP 5971 MS; Agilent Technologies, Palo Alto, CA). Each steroid was identified with retention time and ratio of its two characteristic mass ions, and quantified using stigmasterol as an internal standard (milligrams per gram creatinine). The interassay coefficient of variation was 4–8%, and the sensitivity was 5–20 pg/injection or 0.001 mg/g creatinine for Ptl, PT, and metabolites of cortisol. The turnaround time of the assay is 2 d, and the throughput for one GC/MS instrument is 75 samples/wk. The direct cost is estimated to be $250/sample.
Blood 17OHP was measured by ELISA (Bayer-Medical Ltd., Tokyo, Japan; or Eiken Chemical Co. Ltd., Tokyo, Japan) direct or extraction method on a filter paper blood spot sample. The turnaround time is 1–2 d, and the throughput is 300 samples/d. The direct cost is $4.50/sample.
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Results
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Urine Ptl in 21OHD, non-21OHD, and control neonates
Figure 2 and Table 2 show urine Ptl values (milligrams per gram creatinine) in 21OHD, non-21OHD, and control neonates. Urine Ptl values showed no overlap between 21OHD and non-21OHD, and 21OHD and controls, respectively. The sensitivity of urine Ptl for 21OHD and the specificity of urine Ptl for non-21OHD were both 100% in term and preterm neonates using a cutoff value of 0.1 mg/g creatinine. Ptl values relative to cortisol metabolites [Ptl/(ßTHE+THE)] showed no overlap between 21OHD and non-21OHD, and 21OHD and controls, respectively (Table 2).
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FIG. 2. Urine Ptl in neonates with 21OHD, non-21OHD, and control neonates. Urine Ptl values showed no overlap, with a cutoff value of 0.1 mg/g creatinine between 21OHD and non-21OHD, and 21OHD and controls, respectively. Triangle, Group median; 21OHD, classical 21OHD; non-21OHD, neonates without classical 21OHD having transiently elevated blood 17OHP; control, neonates without elevated blood 17OHP.
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Urine PT in 21OHD, non-21OHD, and control neonates
Figure 3 and Table 3 show urine PT values (mg/g creatinine) in 21OHD, non-21OHD, and control neonates. Urine PT values showed significant overlap between 21OHD and non-21OHD. The sensitivity of urine PT was 100% both in term and preterm neonates, and the specificity was 80% in term neonates and 35% in preterm neonates, respectively, using a cutoff value of 0.6 mg/g creatinine. Urine PT values relative to cortisol metabolites [PT/(ßTHE+THE)] showed significant overlap between 21OHD and non-21OHD (Table 3), although the degree of overlap in urine PT values relative to cortisol metabolites was smaller than that in urine PT values (milligrams per gram creatinine).
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FIG. 3. Urine PT in 21OHD, non-21OHD, and control neonates. Urine PT values showed significant overlap between 21OHD and non-21OHD. Triangle, Group median; 21OHD, classical 21OHD; non-21OHD, neonates without classical 21OHD having transiently elevated blood 17OHP; Control, neonates without elevated blood 17OHP.
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Discussion
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Previous studies have shown that 24-h urine Ptl (micrograms per 24 h) (11), spot urine Ptl (milligrams per gram creatinine), and spot urine Ptl/cortisol metabolite ratio (12) as measured by GC/MS-SIM are useful diagnostic markers for classical 21OHD and are clearly separable between classical 21OHD and controls in neonates. However, these studies did not address whether or not their controls had elevated blood 17OHP.
In this study, we first showed that spot urine Ptl (milligrams per gram creatinine) and Ptl/cortisol metabolite ratio as measured by GC/MS-SIM are clearly separable between classical 21OHD and neonates without classical 21OHD having transiently elevated blood 17OHP (non-21OHD) in a large number of term and preterm neonates. These findings add further credence to the usefulness of urine Ptl measurement in the differential diagnosis of neonates with elevated blood 17OHP as detected by the newborn screening test. The measurement of Ptl may be particularly useful in a neonate with salt loss or ambiguous genitalia in whom elevated blood 17OHP is detected.
There are several limitations, however: 1) A relatively small number of neonates with 21OHD and non-21OHD were studied in the first week of age. 2) Only one very low birth weight neonate with 21OHD was studied. 3) Nonclassical 21OHD was not studied. The paucity of urine Ptl data in the first week of age and extreme prematurity may overestimate performance of the test (sensitivity and specificity of urine Ptl), because their hepatic 5ß-reductase, 3-hydroxysteroid dehydrogenase, and 20-hydroxysteroid dehydrogenase activities are likely to be depressed, and the low reductase activities will lead to compromised conversion of 21DOF to Ptl. Nonclassical 21OHD is known to have less elevated blood 21DOF levels. Thus, it is well conceivable that these subjects have lower urine Ptl levels as compared with those of classical 21OHD, though no one has studied such subjects for urine Ptl.
Before the use of urine Ptl as a diagnostic marker of 21OHD, urine PT had been used as the marker in clinical practice. However, sensitivity and specificity of urine PT as measured by GC/MS-SIM was not well defined in the differential diagnosis of 21OHD and controls. In 1997, Malunowicz (11) first reported that 24-h urine PT (micrograms per 24 h) and Ptl (micrograms per 24 h) as measured by GC/MS-SIM were useful markers for 21OHD in term and preterm neonates, although 24-h urine PT overlapped between 21OHD and controls. In 2002, Caulfield et al.(12) reported that spot urine PT (milligrams per gram creatinine), spot urine Ptl (milligrams per gram creatinine), spot urine PT/cortisol metabolite ratio, and Ptl/cortisol metabolite ratio as measured by GC/MS-SIM were equally useful markers for 21OHD in neonates, each being clearly separable between 21OHD and controls. Both studies did not refer to blood 17OHP levels of their control neonates, and the reason for apparently contradictory results between the two studies remains to be explained.
Our study shows that urine Ptl is a better marker of classical 21OHD as compared with urine PT, and that elevated urine Ptl unambiguously establishes diagnosis of classical 21OHD in term and preterm neonates. The reason why Ptl is a better marker is not entirely clear, but it is probably related to the fact that blood 21DOF increases only in 21OHD, whereas blood 17OHP increases in both 21OHD and non-21OHD. We suspect that blood 21DOF may increase in classical 21OHD through an activated 11ß-hydroxylation pathway of 17OHP by chronically elevated blood ACTH level, and that such an activation of 11ß-hydroxylation pathway of 17OHP does not occur in non-21OHD devoid of chronically elevated blood ACTH level.
Persistently elevated blood 17OHP occurs in 0.036% of neonates (approximately 400 neonates annually), and 21OHD affects 0.006% of neonates (66 neonates annually) in Japan (14). Thus, the added cost for implementing urine Ptl measurement as a second tier of our national newborn screening is estimated to be $100,000 ($250 x 400 neonates), or $1,515 to identify one patient with 21OHD. Given the relatively high incidence of 21OHD among neonates with persistently elevated blood 17OHP, such a modest cost for centralized testing would seem reasonable.
In summary, we suggest that spot urine Ptl measurement by GC/MS-SIM be routinely performed in each neonate with elevated blood 17OHP as detected by the newborn screening test, if the diagnosis of 21OHD remains uncertain.
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Acknowledgments
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We are indebted to Hisami Iri, M.D., and Ms. Teruko Uchida for their continued support; Dr. Makoto Ueki in Mitsubishi Kagaku Bio-Clinical Laboratories (Tokyo, Japan) for his technical advice; and Mr. Yoshikazu Hisano and Mr. Takuya Yanagawa for their collaboration. We also thank Dr. John I. Takayama in the National Center for Child Health and Development for his critical comments and language editing.
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Footnotes
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This work was supported by the Pfizer Fund for Growth and Development Research.
Abbreviations: 21DOF, 21-Deoxycortisol; GC/MS-SIM, gas chromatography/mass spectrometry in selected ion monitoring; 21OHD, 21-hydroxylase deficiency; 17OHP, 17-hydroxyprogesterone; PT, pregnanetriol; Ptl, pregnanetriolone; ßTHE, 5ß-tetrahydrocortisone; THE, 5-tetrahydrocortisone.
Received March 11, 2004.
Accepted September 9, 2004.
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References
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Abstract
Introduction
