Reference Intervals for Steroid Hormones in Healthy 6- to 15-Year-Old Girls Based on Liquid Chromatography-Tandem Mass Spectrometry in China
The evaluation of adrenal and gonadal function in adolescent girls is crucial for understanding their development and diagnosing potential disorders. Steroid hormone levels serve as key biomarkers in this assessment. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a powerful analytical tool for measuring steroid hormones due to its high sensitivity, specificity, and ability to separate complex biological mixtures. This study aimed to establish reference intervals for six steroid hormones in healthy Chinese girls aged 6 to 15 years using LC-MS/MS, filling a significant gap in the existing literature.
The study enrolled 981 girls from middle and elementary schools in Shunyi, Beijing, using a cluster sampling method. After excluding 265 participants due to severe diseases, medication use, or elevated body mass index, 716 healthy girls were included in the analysis. The participants were divided into four age groups: 6 to <9 years, 9 to <11 years, 11 to <13 years, and 13 to 15 years. Pubertal development was assessed using Tanner staging for breast (B) and pubic hair (PH) development by trained pediatricians through on-the-spot interviews.
Fasting serum samples were collected between 7:30 and 8:30 AM, processed by centrifugation, and stored at -80°C until analysis. Free testosterone was measured using chemiluminescence immunoassay (CLIA) with a Maglumi 2000 automatic immunoassay analyzer. The remaining five hormones – pregnenolone, 17α-hydroxyprogesterone, corticosterone, dehydroepiandrosterone, and androstenedione – were quantified using LC-MS/MS with an Agilent 1200 Series HPLC system coupled to an AB Sciex API5000 tandem mass spectrometer. The method demonstrated excellent precision, with intra- and inter-assay coefficients of variation below 10% for all analytes.
Statistical analysis was performed using SPSS version 23.0. Due to the non-normal distribution of hormone levels, the 2.5th, 25th, 50th, 75th, and 97.5th percentiles were calculated for each age group and Tanner stage. Interclass variance was analyzed using the Kruskal-Wallis test, with P values <0.05 considered statistically significant.
The study revealed significant age-related changes in five of the six steroid hormones measured. Pregnenolone levels remained stable until age 9, then increased significantly, with median values rising from 0.20 mg/L in the 6 to <9 years group to 0.46 mg/L in the 13-15 years group. Similarly, 17α-hydroxyprogesterone showed a gradual increase from 0.42 ng/mL to 0.79 ng/mL across the same age range. Dehydroepiandrosterone demonstrated a continuous increase from 0.61 mg/L to 3.80 mg/L, while androstenedione levels rose from 0.14 mg/L to 0.97 mg/L. Free testosterone exhibited a progressive increase from 3.00 pg/mL to 3.80 pg/mL across the age groups. In contrast, corticosterone levels remained relatively stable, with median values ranging from 1.27 mg/L to 1.58 mg/L across all age groups.
The study also examined hormone levels in relation to pubertal development, as assessed by Tanner stages of breast development. Significant increases were observed for all hormones except corticosterone as puberty progressed. For instance, pregnenolone levels increased from 0.22 mg/L at Tanner stage I to 0.48 mg/L at stages IV-V, while dehydroepiandrosterone levels rose from 0.72 mg/L to 4.54 mg/L across the same Tanner stages. Free testosterone showed a similar pattern, increasing from 2.90 pg/mL at Tanner stage I to 3.90 pg/mL at stages IV-V.
The observed hormonal changes reflect the physiological development of the adrenal glands and gonads during childhood and adolescence. The increase in pregnenolone and 17α-hydroxyprogesterone after age 9 corresponds to the adrenal gland’s response to adrenocorticotropic hormone (ACTH), with the D5 pathway predominating during early adrenarche. Dehydroepiandrosterone, a key marker of adrenarche, showed a continuous increase from age 6, reflecting the development and differentiation of the adrenal gland and changes in steroid hormone synthesis pathways. The progressive rise in androstenedione and free testosterone levels can be attributed to the increasing number of 3β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome b5 (Cyt-b5) double-positive hybrid cells in the adrenal cortex, along with increased ovarian production during puberty.
The stability of corticosterone levels across age groups is consistent with its role as a precursor to aldosterone, which is primarily regulated by the renin-angiotensin system rather than developmental changes. This finding underscores the specificity of the observed hormonal changes to the adrenal and gonadal axes.
The establishment of these reference intervals using LC-MS/MS provides a valuable tool for clinicians and researchers. The high sensitivity and specificity of the method ensure accurate measurement of steroid hormone levels, which is particularly important in pediatric populations where hormone concentrations are often lower than in adults. These reference values will aid in the diagnosis and management of various pediatric endocrine disorders, including precocious puberty, congenital adrenal hyperplasia, and adrenal insufficiency.
Furthermore, the study highlights the importance of using age- and puberty-specific reference intervals when interpreting steroid hormone levels in children and adolescents. The significant variations observed across different age groups and Tanner stages emphasize the need to consider developmental stage when assessing adrenal and gonadal function.
The data also provide insights into the normal progression of adrenarche and gonadarche in Chinese girls. The observed patterns of hormonal changes can serve as a baseline for future studies investigating the impact of environmental factors, nutrition, and lifestyle on pubertal development in this population.
In conclusion, this study provides comprehensive reference intervals for six steroid hormones in healthy Chinese girls aged 6 to 15 years, measured using the gold-standard LC-MS/MS method. The data demonstrate significant age- and puberty-related changes in most hormones, reflecting normal adrenal and gonadal development. These reference values will enhance the accuracy of clinical assessments and contribute to a better understanding of normal endocrine development in Chinese children and adolescents.
doi.org/10.1097/CM9.0000000000000771
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