Impact of Adrenal Hormone Supplementation on Bone Geometry in Growing Teens with Anorexia Nervosa

Abstract

Purpose:

Adolescents with anorexia nervosa (AN) have decreased dehydroepiandrosterone (DHEA) and estrogen concentrations that may contribute to skeletal deficits. We sought to determine whether DHEA + estrogen replacement (ERT) prevented bone loss in young adolescents with AN.

Methods:

We recruited females with AN (n=70, ages 11–18 y) into a 12-month, randomized, double-blind placebo-controlled trial. Participants were randomized to oral micronized DHEA 50 mg + 20 mcg ethinyl estradiol/0.1 mg levonorgestrel daily (n=35) or placebo (n=35). Outcomes included serial measures of bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA; total body, hip, spine) and peripheral quantitative computed tomography (pQCT; tibia). Magnetic resonance imaging of T1-weighted images of the left knee determined physeal status (open/closed).

Results:

Sixty-two subjects completed the trial. Physeal closure status was the strongest predictor of aBMD changes. Among girls with open physes, those who received DHEA+ERT showed a decline in BMD-Z-scores compared to those receiving placebo, while there was no effect in those with at least one closed physis. Treatment did not affect any pQCT measures, regardless of physeal closure status.

Conclusions:

Combined DHEA + ERT did not significantly improve DXA or pQCT BMD measurements in young adolescent girls with AN, in contrast to an earlier trial showing benefit in older adolescents and young women. In girls with open physes, the mean change in the placebo arm was greater than that of the DHEA+ERT group. We conclude that DHEA+ERT is ineffective for preserving bone health in growing young adolescents with AN at the dose and route of administration described in this report.

Introduction

Adolescents with anorexia nervosa (AN) are known to have a striking suppression of bone formation, loss of bone mineral density (BMD) and skeletal strength, and are at increased risk for fracture [1–5]. The consequences of these threats to bone health are especially devastating for this young population. Adolescents are still attaining their peak bone mass [6,7], and the loss of BMD and skeletal strength in these patients is thought to be irreversible. The mechanisms of bone loss in AN include impaired bone formation and accelerated bone resorption [8–10] resulting from a combination of hypogonadism, hypercortisolism, low body mass, and poor nutrition, and are possibly related to alterations in bone marrow composition [11].

The implications for lifelong skeletal health are substantial; patients with AN have a seven-fold increased incidence of fractures, which may occur at multiple sites [3]. Effective therapies for preventing bone loss in patients with AN have remained elusive [4,12–14]. We previously conducted a randomized trial to investigate the effects of combination dehydroepiandrosterone (DHEA) + estrogen replacement therapy (ERT) in older adolescents and young women with AN (mean age 18 years) [15]. Whole body and lumbar spine BMD Z-scores were preserved in the DHEA+ERT group even after controlling for weight gain, but decreased over 18 months in the placebo group. Bone resorption markers (N-telopeptides) were suppressed in the treatment group alone. However, the impact of DHEA + ERT on bone density, geometry and strength in growing adolescents is not yet known. The underlying mechanism of action of this therapy remains unclear, as does the effect on the appendicular skeleton and potential impact of physeal closure.

While dual-energy X-ray absorptiometry (DXA) has been the most widely used clinical tool to assess bone mass, bone strength depends on skeletal geometry, bone material properties, and internal architecture which are not reflected directly in DXA measurements. Additionally, cortical and trabecular bone mineral content (BMC) are superimposed within a DXA measure, which may conceal distinct disease and treatment effects on the separate components of BMC. Peripheral QCT (pQCT) allows for direct selective measurement of trabecular and cortical volumetric BMD (vBMD), and additional cross-sectional geometry measures that are highly correlated with fracture load [16].

Building upon our previous work we sought to determine the impact of 12 months of DHEA + ERT on both the axial and appendicular skeleton, as reflected by measures of bone density, skeletal strength, and geometry in a cohort of young adolescent girls with AN. We hypothesized that aBMD of the total body and lumbar spine and vBMD of the radius and tibia would be maintained in adolescent girls with AN receiving DHEA+ERT, while losses would be observed in participants randomized to placebo.

Materials and Methods:

From 2012–2015, we screened 191 adolescents (Supplemental Figure) for participation in a multiple-site, randomized, double blind, placebo-controlled trial (Clinicaltrials.gov, ). Effects on bone marrow composition in the appendicular skeleton by magnetic resonance imaging (MRI) have previously been published [17]. Eligible patients were age 11–17 years; were post-menarchal or had a bone age ≥13 years; and met DSM-5 criteria for AN or atypical AN. Subjects were excluded if they had other medical conditions known to affect bone health or if they were receiving medications known to affect the skeleton. The study was approved by the Boston Children’s Hospital Institutional Review Board; written informed consent was obtained from the parent/guardian, and assent from the adolescent.

Of those screened, 155 met eligibility criteria and 76 female adolescents were enrolled and assigned sequential randomization codes, interpretable as drug or placebo only by the pharmacy staff according to a key prepared by the statistician. The sequence of assignments was randomly permuted in blocks of 2, 4, and 6 to ensure unpredictability as well as temporal balance. The pharmacy dispensed drug or placebo in identical gelatin capsules indistinguishable by patients or staff. Assignments were not revealed until the entire trial was complete [17].

The treatment arm (n=35) received 12 months of oral micronized DHEA (50 mg daily; Belmar Pharmacy; IND 52192) [4,15,18]. DHEA was given with conjugated equine estrogens (0.3mg daily; Premarin®, Wyeth) for the first 3 months to minimize estrogen-related side effects, a regimen that has been well tolerated in our clinical practice and previous trials [15]. Subsequently, subjects in the treatment arm received 9 months of DHEA+ERT (20µg ethinyl estradiol/ 0.1mg levonorgestrel; Alesse®, Wyeth). The other group (n=35) received placebo for the entire 12 months. All participants received routine care including medical, nutritional, and psychological monitoring. Participants returned for assessments at 3, 6, 9, and 12 months. Medication compliance was measured at each visit with pill counts. All subjects were advised to consume the recommended daily intake of calcium (1300 mg) and vitamin D (400 IU). If participants were found to be vitamin D deficient at baseline (25OHD <20 ng/mL), they received a treatment dose of vitamin D3 2000 IU daily for 3 months. After follow up 25OHD measurements at the 3 month study visit, participants resumed a supplementation dose of vitamin D daily.

Skeletal Assessments

The primary outcomes were measurements through serial cross-sections of the left tibia obtained at baseline, 6 months, and 12 months by pQCT using a Stratec XCT 3000 pQCT device (Orthometrix, White Plains, NY) with a 12-detector unit, voxel size of 0.4mm, slice thickness of 2.3mm, and scan speed of 20 mm/s. Scans were obtained using established measures [19]. A scout view was first obtained to place a reference line at the proximal border of the fused distal tibia growth plate. The location of measurements was determined using percentages of the tibia length proximal to this reference line to avoid issues related to height. At the 3% metaphyseal site, the site of maximal cortical thickness, scans were analyzed for trabecular vBMD (mg/cm³). At the 38% meta-diaphyseal site, scans were analyzed for cortical vBMD, cortical BMC (mg), cortical cross-sectional area (CSA, mm²), periosteal circumference (mm), endosteal circumference (mm), and polar section modulus (Zp, mm³). Calf muscle and fat CSA (mm²) were assessed at the 66% diaphyseal site. As previously described [20,21], pQCT measure of muscle density (mg/cm³) was also obtained. Scans were analyzed using Stratec software, Version 6.20. The manufacturer’s phantom was scanned daily for quality assurance. The average coefficient of variation (CV) ranged from 0.2 to 0.6%.

DXA (Discovery A, Hologic, Inc., Bedford, MA) measurements were obtained for the total body less head (TBLH; i.e., with skull excluded), posteroanterior lumbar spine (L1-L4), and total hip on the same model scanner in Boston and Providence. Skeletal outcomes included BMC (g) and areal BMD (g/cm²). Measurements were compared with age- and sex-matched controls and aBMD Z-scores (number of standard deviations away from the average value of the reference group) were calculated using pediatric software [22]. Scans of the phantom were performed daily. The CV was <1.0%. Image analysis was performed using QDR for Windows XP 12.4 software (Hologic, Inc).

All subjects underwent magnetic resonance imaging (MRI; Trio systems, Siemens Medical Inc., Erlangen, Germany) of the right knee at baseline, using previously published methods.[17] Coronal T1 weighted images (TR 600 msec, TE 9.7 msec, FOV 140mm, 3mm slice thickness, 0.3mm gap) were obtained through the knee. Distal femoral and proximal tibial physes were assessed as open or closed as a marker of skeletal maturation. Closed physes by MR were defined as the absence of cartilage signal intensity between the epiphysis and metaphysis with marrow signal intensity extending between those two structures. We defined “open physes” as the presence of open physes at both the tibia and the femur.

Biochemical Assessments

Biomarkers of bone formation [serum levels of osteocalcin (OC) and bone-specific alkaline phosphatase (BSAP)] and bone resorption [serum C-telopeptides (CTx)] were measured every 3 months. OC was measured by ELISA (ALPCO Diagnostics, Salem, NH; %CV 5.0–6.5%); CTx was measured by immunoradiometric assay (Immunodiagnostic Systems, Tyne and Wear, United Kingdom; %CV 5.2–6.8%); and BSAP was measured by electrochemiluminescence immunoassay (ECLIA; Beckman Coulter, Brea, CA; %CV 1.5–2.6%). Serum 25-hydroxyvitamin D [25OHD; RIA (Diasorin, Inc., Stillwater, MN); %CV ≦8.3%] was measured at baseline.

Hormonal measures included serum estradiol and testosterone concentrations [CIA (Beckman Coulter, Fullerton, CA); %CV ≤ 8.0% for estradiol and ≤ 7.0% for testosterone], leptin [RIA (Millipore, Billerica, MA); human (total) ghrelin [RIA (Millipore), %CV 5.2–7.2%]; adiponectin [RIA (Millipore); %CV 6.9–9.3%]; cortisol [CT RIA [Diagnostics Systems Laboratories (DSL), Webster, TX]; %CV 6.1–12.0%], and DHEAS [double-antibody ELISA (DSL), %CV 4.8–5.3%]. Insulin-like growth factor I (IGF-I) concentrations were measured in the Maine Medical Center Laboratory in Scarborough, ME using ELISA (Immunodiagnostic Systems, Inc, Fountain Hills, AZ; %CV 4.6–7.2%). Safety studies included liver function tests (AST, ALT) every six months and a lipid panel at the beginning and end of the study.

Additional Study Measurements

Height (cm) was measured using standardized procedure with a wall-mounted stadiometer. Weight (kg) was measured in the morning, post-voiding, with subjects wearing a hospital gown. BMI was calculated as Weight (kg) / [Height (m)]². CDC growth data were used to calculated percentage median body mass index and BMI Z-score for each participant.[23]. Body composition measures [total body lean mass (kg), total body fat mass (kg), percentage body fat] were obtained by DXA. All participants responded to a structured interview to obtain demographic information and health history, which included information about menstrual history, smoking status, personal history of fracture, and family history of osteoporosis.

Statistical Analysis

To estimate power, we used data from our prior trial on the variability and pre-post correlation of aBMD Z-score (standard deviation0.89, Pearson r=0.93). A target of 60 completers provided 80% power to detect a difference between treatment arms of 0.25 for change in total body aBMD Z-score over 12 months, an effect smaller by 23% than that observed in the prior trial. All analyses followed the intention-to-treat principle, ascribing the randomly assigned treatment to each participant regardless of compliance. We analyzed each outcome using repeated-measures analysis of variance, with an unstructured covariance matrix and with interative reweighting to minimize the influence of outliers. Outcomes with severely skewed distribution were log-transformed for analysis. Models were adjusted for concurrent weight (time-varying) and baseline anthropometrics (age, height), menarchal status, duration of amenorrhea, and physeal status (open at both tibia and femur or closed at one site or both) by adding appropriate covariate terms to the repeated-measures model. Arm × visit interaction was tested to address the primary hypothesis of treatment effect. Arm × visit × physeal status interaction was additionally tested to determine whether physeal closure modified the treatment effect. Adjusted closure-specific means and differences were constructed from parameters of the fitted model. For each stratum of treatment and physeal status we constructed a contrast testing H₀: Baseline = 6 mo = 12mo, yielding an F-statistic with 2,61 df and a level of statistical significance that we denote P_change. For each stratum of physeal status we constructed a contrast testing H₀: parallel time course in Active and Placebo, yielding an F-statistic with 2,61 df and a level of statistical significance that we denote P_diff. We took P<0.05 to indicate a statistically significant effect. We used SAS software (Cary, NC, version 9.4) for all computations.

Results:

Among the randomized subjects, 6 became ineligible or withdrew before completion of the baseline visit, resulting in a final sample of 70 (35 DHEA+ERT, 35 placebo), aged 15.5 ± 1.9 yr (mean ± SD) [17]. The treatment groups did not differ with respect to baseline characteristics (Table 1). Subjects had a low BMI (18.7 ± 1.7 kg/m²; range 12.8–22.4) and percentage median body weight (%MBW), consistent with their AN. Most were Caucasian and non-Hispanic. The median duration of amenorrhea was 4 months (range 1–18 months). Girls who had not yet had their first menses (n=11) were not included in the calculation of duration of amenorrhea. No subjects were spontaneously menstruating; those designated as amenorrheic for 1 month were 13 participants receiving oral contraceptive pills at recruitment who discontinued them ≥1 month before study participation. Changes in bone mineral content Z-score over 12 months were different in subjects who had used oral contraceptives compared to subjects who did not; we controlled for the duration of amenorrhea in our analyses to account for these differences. Three subjects receiving DHEA+ERT and 5 receiving placebo dropped out for reasons unrelated to the study; 62 adolescents completed the trial.

Table 1:

Baseline characteristics of a sample of 70 adolescents with AN receiving 12 months of either DHEA+ERT or placebo

	DHEA + ERT (n=35)	Placebo (n=35)	P*
	Mean ± SD
Age, y	15.5 ± 1.9	15.5 ± 2.0	0.97
Height, cm	159.5 ± 7.4	161.5 ± 8.5	0.30
Weight, kg	48.0 ± 6.2	48.8 ± 6.9	0.60
BMI, kg/m2	18.8 ± 1.6	18.7 ± 1.8	0.69
BMI Z-score	−0.6 ± 0.7	−0.7 ± 0.9	0.58
Percentage of mBBMI, %	94 ± 8	93±10	0.76
	Median (Min-Max)
Time since AN diagnosis, mo	4 (1–36)	6 (1–60)	0.17
Duration of amenorrhea, moǂ	4 (1–17)	4 (1–18)	0.67
DHEAS, mcg/dL	185 (46–445)	125 (39–431)	0.01
Estradiol, pg/mL	29 (1–208)	26 (2–274)	0.52
	N (%)
Race:   Caucasian       Other	30 (86) 5(14)	31 (89) 4(11)	1
Ethnicity: Hispanic       Non-Hispanic	3 (9) 32 (91)	1 (3) 34 (97)	0.61
Previous fracture: Yes       No	10 (29) 25 (71)	12 (34) 23 (66)	0.80
Family history of osteoporosis       Yes       No/Unknown	9 (26) 26 (74)	8 (23) 27 (77)	1
Vitamin D status (25(OH)D, ng/mL)       Deficient (<20)       Insufficient (20–30)       Sufficient (>30)	4 (11) 12 (34) 19 (54)	3 (9) 16 (46) 16 (46)	1
Physeal closure status:       Both open       Femur open, tibia closed       Both closed	5 (14) 9 (26) 21 (60)	12 (34) 6 (17) 17 (49)	0.17

AN, anorexia nervosa; DHEAS, dehydroepiandrosterone sulfate; ERT, estrogen replacement therapy; 25(OH)D, 25-hydroxy-vitamin D; BMI, body mass index; mBMI, median body mass index;

^*From Student t, Wilcoxon rank-sum, or Fisher Exact test, testing for equal distribution in DHEA+ERT and placebo arms.

^ǂExcluding 11 subjects with primary amenorrhea (4 DHEA+ERT, 7 placebo)

Physeal closure status was the strongest predictor of changes in aBMD (Figure 1). In participants with open physes at baseline, changes in total body BMD-Z score differed significantly between participants receiving DHEA+ERT vs. placebo subjects (Table 2; P_diff =0.02). Similar results were seen at the lumbar spine and total hip, where subjects with baseline open physes receiving active drug showed a decline in BMD-Z score compared with those receiving placebo. In contrast, in participants with closed physes, no treatment differences in BMD Z-scores were detected at any measured site (Figure 1; P_diff >0.05 for all). The same pattern was demonstrated in measures of BMC Z-scores at the total body, total hip, and lumbar spine (Table 2).

Figure 1:

Changes in measurements of areal bone mineral density Z-score (aBMD-Z) in adolescents randomized to receive either DHEA+ERT (n=35; represented by •) or placebo (n=35; represented by ×) over 12 months, by baseline physeal closure status. Figure 1A shows values obtained for the total body, while Figure 1B demonstrates those at the lumbar spine.

Table 2.

Changes in bone measures over 12 months in 70 girls with AN in response to treatment with active drug (DHEA+ERT) or placebo.

Measure	Treatment	Change from Baseline*
		Open physes (both sites)			Closed physes (1 site or both)
		12-mo change ± SE	Pchange	Pdiff	12-mo change ± SE	Pchange	Pdiff
DXA Measurements
aBMD-Z, total body	Active Placebo	−0.30 ± 0.15 0.14 ± 0.10	0.06 0.35	0.02	−0.13 ± 0.07 −0.22 ± 0.08	0.10 0.006	0.43
aBMD-Z, total body less head	Active Placebo	0.01 ± 0.15 0.14 ± 0.10	>0.99 0.30	0.73	−0.02 ± 0.07 −0.13 ± 0.08	0.79 0.019	0.04
aBMD-Z, spine	Active Placebo	−0.35 ± 0.17 0.14 ± 0.11	0.020 0.40	0.009	−0.11 ± 0.07 −0.20 ± 0.09	0.10 0.08	0.62
aBMD-Z, hip	Active Placebo	−0.22 ± 0.16 0.13 ± 0.10	0.005 0.42	0.004	−0.15 ± 0.07 −0.25 ± 0.08	0.12 0.013	0.45
BMC-Z, total body less head	Active Placebo	−0.21 ± 0.17 0.14 ± 0.10	0.26 0.16	0.06	−0.08 ± 0.07 −0.16 ± 0.09	0.17 0.14	0.18
BMC-Z, spine	Active Placebo	−0.75 ± 0.30 −0.20 ± 0.19	0.008 0.56	0.05	−0.21 ± 0.13 −0.35 ± 0.16	0.17 0.04	0.76
BMC-Z, hip	Active Placebo	−0.43 ± 0.20 −0.09 ± 0.13	0.002 0.40	0.003	−0.12 ± 0.09 −0.15 ± 0.11	0.16 0.32	0.41
pQCT Measurements
Trabecular vBMD-Z, 3%	Active Placebo	0.32 ± 0.19 0.13 ± 0.12	0.23 0.04	0.47	−0.11 ± 0.08 −0.03 ± 0.11	0.35 0.85	0.67
Cortical vBMD-Z, 38%	Active Placebo	0.05 ± 0.22 0.25 ± 0.14	0.24 0.10	0.60	−0.17 ± 0.09 0.12 ± 0.12	0.21 0.53	0.16
Section modulus, 38%	Active Placebo	−0.27 ± 0.13 −0.06 ± 0.09	0.13 0.47	0.32	−0.07 ± 0.06 −0.13 ± 0.07	0.24 0.22	0.62

AN, anorexia nervosa; DHEA, dehydroepiandrosterone; ERT, estrogen replacement therapy; DXA, dual energy X-ray absorptiometry; aBMD-Z, areal bone mineral density Z-score; BMC-Z, bone mineral content Z-score; pQCT, peripheral quantitative computed tomography; vBMD-Z, volumetric bone mineral density Z-score

^*From repeated-measures analysis over the time course of the study (baseline, 6 mo, 12 mo), adjusted for concurrent weight and baseline anthropometrics (age, height), menarchal status, and duration of AN. Estimate ± standard error is shown for 12-mo. change from baseline. P_change tests for change within treatment group over the three measurement times. P_diff tests for treatment effect, i.e., difference in time course between Active and Placebo.

Treatment did not affect any pQCT measures over the 12-month trial, regardless of physeal closure status (Table 2). No significant changes in trabecular vBMD, cortical vBMD, section modulus, or muscle area occurred between girls receiving DHEA + ERT and those receiving placebo (P_diff >0.05 for all).

Bone turnover markers again responded differently based upon baseline physeal closure. In girls with open physes, BSAP increased transiently in girls receiving DHEA + ERT, while subjects taking placebo showed no change (Figure 2A; P_diff =0.02). No treatment differences were seen in girls with closed physes (P_diff =0.72). Girls with open physes receiving DHEA + ERT showed small, non-significant increases in osteocalcin over 12 months; however, the trajectory of change was the same in girls receiving placebo (Figure 2B; P_diff =0.40). Again, no differences occurred in girls with closed physes. Changes in CTx were also not different between the two treatment groups, regardless of physeal closure status (Figure 2C).

Figure 2:

Changes in serum measures of bone turnover over 12 months in adolescents randomized to receive either DHEA+ERT (n=35; represented by •) or placebo (n=35; represented by ×). (A) bone specific alkaline phosphatase (BSAP), (B) osteocalcin, and (C) C-telopeptides (CTx)

Safety data were also reviewed (Supplemental Table). Both treatment groups gained weight over the course of the study (median change in BMI Z-score 0.20 SD over 12 mos, p=0.03). Change in BMI did not differ on the basis of physeal closure status, nor on the basis of treatment assignment. The observed changes in BMI Z-score over the course of the study were not correlated with any bone measurement change over time. Liver function tests remained within the normal range for all participants in the trial over the 12 months. Both self-reported compliance and compliance measured by pill count were excellent. Median self-reported compliance was 97.8% at 3 months (range 67–100%) and 99.4% at 12 months (range 50–100%); by pill counts, compliance ranged from 98.3% at 3 months (range 96.7–100%) to 100% at 12 months (range 50–100%). Despite these measures, serum measurements of DHEAS concentration suggest that subjects were compliant for the first 6 months of participation, but may have waned in their medication adherence over the last 6 months (Figure 3; P_diff <0.001 for both open and closed physes). No treatment group differences in serum measurements of estradiol, total testosterone, leptin, or ghrelin were observed (Supplemental Table; P_diff >0.05 for all).

Figure 3:

Changes in serum measures of dehydroepiandrosterone sulfate (DHEAS) over 12 months in adolescents randomized to receive either DHEA+ERT (represented by •) or placebo (represented by ×). The left side of Figure 3 represents girls with both physes open, while the right side represents girls with one or both physes closed.

Discussion

In young adolescents with AN and open physes, replacement of adrenal and gonadal hormones led to significant decreases in markers of bone health over 12 months of treatment. Physeal closure status was an important effect modifier of the impact of hormonal therapy on these bone outcomes. Subjects with open physes at baseline showed declines in areal BMD and BMD Z-scores in response to this therapy. In subjects with closed physes, no change, either positive or negative, occurred over 12 months of treatment. Skeletal outcomes measured by pQCT did not change over time in response to DHEA+ERT in either group. The data suggest that the potential skeletal benefits derived from this therapy apply only to older adolescents and young women who have completed their growth and have closed epiphyses.

For this study, we recruited only girls who were post-menarchal or who had attained an expected bone age of 13 years or more. We anticipated that these girls would have already gone through their growth spurt, and would be in the growth deceleration phase with 96% of final height attained [24]. The current findings are in contrast to our previous research which was conducted in older, more substantially malnourished adolescents and young women with closed physes [15]. Older adolescents and young women in our previous studies have shown an arrest of bone loss, as determined by DXA outcomes of BMD, after combined therapy with adrenal and gonadal hormone replacement. We also showed hip strength variables, which were derived from DXA measurements, to be improved after 18 months of the same ERT regimen [25]. Interestingly, in this same cohort, ERT led to arrest of bone marrow changes associated with mild to moderately severe AN [17]. Despite our recruitment strategy for the current study, almost half of participants had at least one open physis on MRI. In these girls, DHEA+ERT was not beneficial. The onset of AN prior to puberty leads to more profound skeletal deficits and in particular, compromised osteoblast activity, with lower BMD seen at both axial and appendicular sites[26]. In contrast, patients with onset of disease during adulthood are more likely exhibit defects solely in the axial skeleton.

Additionally, a young age of onset of the disease has the potential to more negatively impact peak bone mass, since the disease may persist for a greater duration of time. We previously studied a small cohort of adolescents admitted for hemodynamic stabilization and tracked bone biomarkers while on bed rest [27]. Similarly, we found the most compromised bone formation and suppressed bone formation markers in the youngest adolescents (i.e., age 11–14 vs. 15–17 yr), potentially due to the status of the physis [27].

Increases in bone specific-alkaline phosphatase (BSAP) were observed in those subjects who were growing (i.e., in those subjects who had open physes) and receiving DHEA+ERT. Markers in subjects receiving placebo and those with closed physes remained unchanged. The increases in BSAP suggest that the participants with open physes were still very receptive to bone growth. Osteocalcin, a measure of mature osteoblast function, also increased over time but was not different between girls receiving DHEA+ERT or placebo. Osteoclast activity (measured via serum C-telopeptides) did not change; this result is not surprising given that the primary bone insult in AN is a lack of osteoblastic activity. It is unclear why the increases in BSAP did not lead to other signs of bone formation, such as improvement in measures of bone density or bone geometry over time. Our findings are in contrast to our previous study evaluating marrow changes as a result of DHEA + ERT[17]. We hypothesize that the observed marrow changes may take longer to be reflected in clinical measures of skeletal health such as DXA. The differences between our DXA and pQCT outcomes in patients with open physes, with data diverging in treatment vs. placebo by DXA yet showing minimal change by pQCT, may highlight the limitations of DXA. In the pQCT measurements (which would not be confounded by bone size), no harmful changes were seen over time in the group with open physes receiving DHEA + ERT. However, we would not recommend further study or clinical use of DHEA + ERT in young adolescents, given the potential adverse effects in this young group. These questions will need to be addressed in future studies, as well as the effect of oral estrogen compared to transdermal regimens, examining both the effects on circulating bone biomarkers and bone density, geometry, and structure in the axial and peripheral skeleton.

Study limitations should be acknowledged. Our cohort includes subjects with AN diagnosed via DSM-5 criteria and patients with mild severity of malnutrition, in contrast to our earlier trials that met the more stringent weight criteria of the DSM-IV [4,15]. However, the goal of including these participants was to make our study findings more generalizable to the typical patients seen by clinicians in practice. It should also be acknowledged that the interpretation of bone biomarker data in growing adolescents is complex compared to adults or older adolescents with closed epiphyses.

Conclusion:

In conclusion, a combination regimen of oral DHEA + ERT for 12 months did not improve bone density, geometry, or strength compared to placebo in young adolescents with AN with open epiphyses. The benefits with respect to attenuation of bone loss and enhancements in bone strength appear to be limited to older adolescents and young women. Therefore, other therapeutic strategies such as transdermal estrogen [14] should be consideredin young patients affected with this disease, in addition to standard multidisciplinary care including medical, nutritional, and psychological support.

Supplementary Material

2

Supplemental Figure: CONSORT diagram of subject recruitment

Implications and Contribution:

In a 12-month randomized clinical trial, dehydroepiandrosterone and estrogen replacement therapy supplementation did not improve bone health in young adolescents with open physes who have anorexia nervosa.