George W. Moll

Preserving adult height potential in girls with idiopathic true precocious puberty

We designed a prospective study of height potential in girls with idiopathic precocious puberty, comparing the presenting features of girls with and without evidence of reduced adult height potential. The 14 girls with impaired adult height prognoses (group 1) were reexamined after treatment with a gonadotropin releasing hormone agonist, nafarelin. The seven girls with the prognosis of unimpaired height (group 2) were followed without therapy. We found that the group could be distinguished at initial examination by the greater bone age/height age ratio of group 1 (mean +/- SEM: 1.4 +/- 0.06 vs 1.0 +/- 0.05; p less than 0.005) and by the greater difference between predicted height and target height in group 1. The mean predicted height in group 1 was significantly less than the mean target height (150.7 +/- 2.1 vs 165.4 +/- 3.0 cm; p less than 0.005), whereas the mean predicted and target heights in group 2 were similar (165.4 +/- 3.0 vs 164.3 +/- 2.1 cm). Initial estradiol levels were also greater in group 1 than in group 2 (21.6 vs 10.6 pg/ml; p less than 0.05), although this difference was not sustained during follow-up. In group 1, nafarelin therapy suppressed the pituitary-gonadal axis, and although there was a transient reduction in height potential in girls with the youngest bone ages during the first 6 months of therapy, 2 years of treatment slightly improved predicted heights from 150.7 +/- 2.1 to 152.7 +/- 2.0 cm (p less than 0.05). Height predictions also increased without therapy during the 2-year observation period in group 2, from 165.4 +/- 3.0 to 168.7 +/- 4.1 cm (p less than 0.05). Our data indicate that gonadotropin releasing hormone agonist therapy preserves height potential in girls with an initially impaired height prognosis, and that height potential is preserved without therapy in those with a good initial height prognosis.

Plasma free testosterone in the diagnosis of adolescent polycystic ovary syndrome.

We have employed plasma free testosterone concentrations to diagnose polycystic ovary syndrome in adolescence after demonstrating that mature plasma free androgen levels are achieved by midpuberty. Among 43 female volunteers, 10 to 16 years of age, followed up in a mixed longitudinal-cross-sectional study, we found that three (7%) had elevated plasma free testosterone levels in the absence of symptoms or signs of hyperandrogenemia. Of two who were followed up, the abnormality resolved on completion of sexual maturation in one, and in the other polycystic ovary syndrome was subsequently diagnosed. We diagnosed this syndrome in 14 adolescents as the basis of menstrual disorders associated with hirsutism, acne, or obesity. The diagnosis was based on finding elevated free testosterone values, which did not decrease after adrenal suppression by dexamethasone. Serum concentrations of luteinizing hormone were elevated in most but not all. In some of these patients further confirmation of polycystic ovary syndrome was obtained by laparoscopy or ultrasound examination. These studies demonstrate that measurement of plasma free testosterone before and after administration of dexamethasone appears to be the most sensitive single method for detecting polycystic ovary syndrome.

A flow dialysis technique for the measurement of free testosterone in human plasma under physiologic conditions.

Abstract A technique is described for measuring the percentage of free (unbound) testosterone under physiologic conditions (37°C in whole plasma) without disturbance of the equilibrium state. The technique employs 10 ml of whole plasma with a tracer amount of tritiated testosterone in a flow dialysis cell. The precision of the technique is 5–10%. The percentage of free testosterone values so obtained for several samples of human plasma are in general agreement with previous estimates. The technique permits re-evaluation of individual plasma samples several times, and it can be easily generalized to the study of other steroids in whole blood or plasma. The free testosterone is generally believed to be the biologically active portion of the plasma testosterone (1, 2). Free testosterone is that portion of the total which is not bound to plasma binding species. Practically speaking, the free testosterone fraction is that portion of testosterone in plasma which is capable of diffusing through a dialysis membrane. A technique has been described by Colowick and Womack (3) for the measurement of the percentage of diffusible molecules at equilibrium without significantly disturbing the equilibrium state. An apparatus is described here which is a modification of that used by Colowick and Womack and which permits measurement of the percentage of free steroid in plasma under physiologic conditions.

Estradiol inhibition of pituitary luteinizing hormone release is antagonized by serum proteins

We tested the influence of protein binding upon the rapid 17 beta-estradiol (E2) inhibitory effect on luteinizing hormone (LH) responsiveness to LH releasing hormone (LHRH) in perifused rat anterior pituitaries. LHRH pulses were given before 1 h after changing the perifusion medium to contain various combinations of protein and E2. In the absence of albumin an E2 concentration of about 27 pg/ml inhibited LH secretion in response to LHRH by 50% (judged from the change in secretion ratio, i.e LH response to LHRH pulse no. 2 divided by that to pulse no. 1). Albumin (1 g/dl) and human plasma enhanced the self-priming effect of LHRH (P less than 0.1) and blunted the slope of the dose-response relationship between E2 and LH secretion. These effects suggest a stimulatory effect of proteins on LH secretion which is independent of E2. However, proteins appear to antagonize the E2 effect in part by binding E2. A total E2 dose of 270 pg/ml or more was required to achieve about 50% inhibition of the LH secretion ratio in the presence of albumin. Since 20% of E2 was unbound in the presence of albumin, the 50% effective dose of free E2 was about 54 pg/ml. This closely approximates the comparably effective dose of E2 in the absence of protein. Although a total E2 concentration of 216 pg/ml was required for a 50%-inhibitory E2 effect in the presence of heat-inactivated plasma, this was equivalent to between 28 and 37 pg/ml of free E2 in these experiments. The effect of E2 in a total dose of 216 pg/ml was attenuated more by the use of TEBG-positive plasma than of TEBG-negative (heat-inactivated) plasma (P less than 0.01). Our data indicate that the E2 inhibitory effect upon LH responsiveness to LHRH is attenuated by albumin and TEBG to approximately the extent expected by binding of E2 to these proteins. Our data are certainly contrary to those expected if either albumin or TEBG augmented this E2 action. Our data support the concept that the bioavailable fraction of plasma sex steroids is that which is free from binding to plasma proteins.

24 ADOLESCENT POLYCYSTIC OVARY SYNDROME-OUT-PATIENT DIAGNOSIS

We have expanded the endocrine diagnostic criteria for PCOS and have identified it in 8 adolescents. PCOS consists of a spectrum of functional ovarian hyperandrogenic states ranging from cases without histologic ovarian abnormalities through classic Stein-Leventhal ovaries. Diagnostic criteria developed for adults are: elevated plasma free (unbound) testosterone (T) (≥12 pg/ml) which does not fall to ≤ 8 upon adequate adrenal suppression by dexamethasone (dex). In addition, we found bioactive LH (B-LH) to be more often significantly elevated in PCOS than radioimmunoassayable LH (I-LH).We first established that adult female levels of total and free T and LH are normally achieved prior to the time of menarche.The 8 PCOS patients were 12.5-17 years old. 5 had menstrual disturbances (4 oligo- or amenorrhea × 1.5-2.5 yrs and 1 menometrorrhagia × 1.5 yrs), 6 had hirsutism, and 5 were obese. I with subclinical problems was detected in a school screening program. A high free T level was found in each (15-25 pg/ml), though total T (51-133 ng/dl) and free T (10.1-25) were intermittently normal in some. Free T fell only to 9-28 pg/ml on dex. B-LH was elevated in 4/6 (>620 ng/ml), while I-LH was only elevated in 2/6 (>91 ng/ml). 1 patient with persistently high I-LH (260-410 ng/ml) underwent laparoscopy which revealed sclerocystic ovaries. Estrogen-progestin (BCP) (n=3) normalized free T (≤8 pg/ml).PCOS, as the basis of teenage menstrual difficulties or hirsutism--sometimes minor, can be diagnosed from elevated free T levels pre and post dex and elevated B-LH without invasive procedures.

LUPRON TREATMENT OF PRECOCIOUS PUBERTY (CPP) HAS NOT PRODUCED LOSS OF BONE MINERAL

We tested the hypothesis that GnRH agonist treatment of CPP is associated with loss of bone mineral density (BMD) and content (BMC) after young women treated with GnRH agonists were reported to lose up to 6% BMD in the first treatment year similar to that seen during menopause. We determined lumbar spine (L1-L4) BMD (g/cm2) and BMC (g) by dual photon absorptiometry and measured growth parameters and blood Ca (mg/dl), alk. phos. (AP, mU/ml) and integrated growth hormone (GH, ng/ml) on CPP patients before and after (3 of our 6 patients to date) six months treatment with the GnRH agonist LUPRON (TAP Pharm., 4-8 ug/kg SC qD). Our patients (3 girls 5-8 yr, 9-10 yr bone age [BA]; 3 boys 3-8 yr, 7-10 yr BA) were Stage 3-4 upon entering the study with informed consent.All 6 had similar basal BMD (.724±.05, SEM). Despite reduction of V or T toward prepubertal levels, growth rates and BMC did not decline and GH and BMD increased while Ca and AP decreased.We conclude that early, partial suppression of CPP with LUPRON is not associated with loss of BMD or BMC. Followup with further suppression of CPP should help to distinguish between variable time courses of end organ responses or therapeutic selectivity.

472 ESTROGEN AT LOW DOSES RAPIDLY AUGMENTS GROWTH HORMONE (GH) RELEASE INDEPENDENTLY OF SOMATOMEDIN-C (SM-C)

We tested whether low doses of ethinyl estradiol (EE) would stimulate GH independently of SM-C inhibition, after we found EE 50 mcg/m2/d × 2d to augment peak GH responses to L-dopa testing (LDT) while lowering SM (Rich et al, Pediatr.Res. 13:329A, 1979).We first examined the effect of one bedtime dose EE(29±2(SEM) mcg/m2) upon GH reserve in 8 prepubertal short-normal children. L-dopa(0.3 gm/m2) stimulated GH to ≥7 ng/ml inconsistently (6/8) before EE. EE resulted in a significant increase in peak GH response (29±5 ng/ml)(p<.05) to LDT. EE (10-30mcg/m2) was then given for 2 days prior to repeat LDT in 15 prepubertal children with the following results.GH responses to LDT were <7 ng/ml in 3/15 prior to EE but were consistently ≥7 ng/ml after EE. Augmentation of the GH response by EE at 27±1 mcg/m2/d was of similar magnitude to that noted with 50 mcg/m2/d EE, but this low-dose EE did not act via lowering SM-C levels (.54±.09 vs .47±.07, n=8).We conclude that a single bedtime dose of EE as low as 29±2 mcg/m2 augments the peak GH response to LDT and enhances the discrimination of L-dopa testing for GH reserve. Our results suggest low-dose estrogen rapidly and directly augments GH release, consistent with its enhancing effect on growth.

CORTISOL BINDING GLOBULIN(CBG) IS DECREASED IN OBESE MALE ADOLESCENTS

We have measured the concentration of CBG in plasma from adolescents at various stages of pubertal development and assessed the influence of obesity upon plasma CBG levels.We found that plasma CBG concentrations did not vary significantly with pubertal stage in normal, nonobese male (CBG=250±50 nM [Mean±SD]) or female (CBG=240±70 nM) adolescents. In these normal adolescents CBG levels did not significantly correlate with an index of body mass (weight/[height]2.77).We compared the CBG plasma concentrations in obese adolescents with those of our nonobese normals. Among 9 obese males (11-17 y/o,145-246 lbs),4 had subnormal CBG levels (75-144 nM,p<0.05). Among 5 obese females (5-18 y/o, 75-338 lbs), none had abnormal CBG levels.We conclude that body mass, sex and pubertal stage do not normally influence CBG levels during adolescence. This contrasts with the recognized influence of these variables upon Testosterone-Estradiol Binding Globulin (TeBG) concentrations. In the presence of obesity, CBG levels are reduced in males. The normal interrelationships among body mass, CBG and TeBG levels appear to be altered in the presence of obesity in adolescence.

442 BIOACTIVE LH PITUITARY RESERVES DURING NORMAL/ABNORMAL MALE PUBERTY

We explored the possibility that the serum bioactive LH (B-LH) response to gonadotropin releasing hormone (GnRH) infusion might provide additional insight into normal and abnormal pubertal maturation. Controls consisted of 24 boys and 10 men; 1 true isosexual precocity (TIP) and 11 hypopituitary (11-53 yr) cases were studied. Maturationa. staging was by AM testosterone (T). B-LH was assayed by rat leydig cell T production, immunoreactive LH (I-LH) by RIA.Normally the GnRH-induced B-LH was related to maturational stage in a biphasic manner (cubic fit r=0.85): a) stimulated B-LH is minimal in the most immature children (rising only from 23 to 26 ng/ml in the youngest), b) undergoes an initial rise at T = 20-40 ng/dl (p < .05), c) peaks at a T level of 196 ng/dl, and d) then declines modestly (p< .05) as the T level rises further. B-LH was not so closely related to chronologic age, bone age, or Tanner stage. The pubertal peak in LH was not seen in I-LH data, but was in B-LH/I-LH (B/I), a measure of LH biopotency.The TIP case had elevated basal B-LH, stimulated-B-LH, and B/I, though I-LH was normal. The abnormalities were progestin-suppressible.Stimulated B-LH corresponded better than I-LH to the maturation achieved by hypogonadotropic men. B-LH responses to GnRH discriminated teenagers with delayed puberty from those with gonadotropin deficiency once T levels exceeded 30 ng/dl.In summary, the normal biphasic relationship of B-LH reserve to the baseline T level is an improved diagnostic criterion for distinguishing hypogonadotropinism from delayed puberty.