- Email: [email protected]
Estradiol treatment of acromegaly Reduction of immunoreactive somatomedin-C and improvement in metabolic status
Estradiol
Treatment
of Acromegaly
Reduction of ,Immunoreactive Somatomedin-C Improvement in Metabolic Status
DAVID R. CLEMMONS, M.D.* LOUIS E. UNDERWOOD, M.D. Chapel Hill, North Carolina ELI C. RIDGWAY, M.D. BERNARD KLIMAN, M.D. RAYMOND N. KJELLBERG, Boston, Massachusetts
M.D.
JUDSON J. VAN WYK. M.D.+ Chapel Hill, North Carolina
From the Department of Pediatrics and Medicine, University of North Carolina, Chapel Hill, North Carolina and the Departments of Medicine and Neurosurgery, Massachusetts General Hospital, Boston, Massachusetts. This work was supported by the U.S. Public Health Service Research Grant AM0122. The Massachusetts General Hospital Clinical Research Center is supported by Grant RR-01066 from the General Clinical Research Center’s Program of the Division of Research Resources. Requests for reprints should be addressed to Dr. David R. Clemmons, Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514. Manuscript accepted May 2.1960. * Recipient of a National Research Service Award AM05991. + Recipient of a USPHL Research Center Award 4K06A AM14115.
and
Administration of estrogens to acromegalic patients has been shown to reduce the serum concentrations of bioassayable somatomedin and to cause improvement in clinical status. These effects appear not to result from an effect on the secretion of growth hormone since growth hormone concentrations are not consistently reduced. Using a sensitive radioimmunoassay for somatomedin-C, we have assessed the relationship between the estrogen-induced reduction of somatomedin and changes in several indices of disease activity in five acromegalic patients. Statistically significant reductions in serum somatomedin-C (p
October
1980 The hmerican Journal of Medicine
Volume
69
571
ESTROGEN
IN
ACROMEGALY-CLEMMONS
ET AL.
Laboratory Methods. Serum phosphorus, blood glucose and urinary total hydroxyproline, calcium, phosphate and creatinine were quantitated by previously published methods [12].
it possible for us to examine the relationship between the estrogen-induced reduction in somatomedin-C, change in serum growth hormone concentrations and the effects of estrogen on several indices of disease activity in acromegalic patients.
Serum human growth hormone was measured by radioimmunoassay using HS 840A as a standard [13]. Somatomedin-C was measured by radioimmunoassay using modifications reported previously [14]. The somatomedin-C standard is a commercially derived pool of serum from a large number of normal adults (Ortho 1778-5, Ortho Diagnostics, Raritan, NJ). This standard was assigned a potency of 1.66 U/ml [15]. Probably because of the presence in serum of binding proteins (161, which diminish the availability of somatomedin-C, the radioimmunoassay measures only 30 to 40 percent of the immunoreactive somatomedin-C present in unextracted serum [17]. This immunoreactivity referred to as the “available” somatomedin-C appears to reflect accurately the growth hormone secretory status and, unless otherwise indicated, is the fraction of somatomedin-C quantitated in this study. The “total” somatomedin content of serum is the e&i&ate made on samples which have been exposed to acid, a manipulation which alters the relationship between somatomedin-C and its
METHODS Patients and Study Methods. The participants in the study were five acromegalic adults, two male and three female between 20 and 68 years of age, who gave their informed written consent (Table I). Despite previous therapy, all exhibited signs of mild to moderate disease activity as evidenced by elevated basal growth hormone levels (four patients), elevated growth hormone levels 1 hour after the oral administration of 100 g of glucose (five patients), hypertension (two patients], elevated fasting blood sugar levels (two patients) and elevated serum phosphorus levels (two patients). Following admission to the clinical research unit of the Massachusetts General Hospital, each patient was given a diet containing 400 mg of elemental calcium, 1,000 mg of phosphate and, depending on home dietary intake, 2,200 to 3,000 calories/day. Each patient’s intake of hydroxyproline was held constant throughout the study. After a 48 hour adjustment period, daily 24 hour urine collections were begun for the determination of phosphorus, .calcium, hydroxyproline and creatinine. In addition, fractional 3 hour urine collections for phosphate and creatinine determinations were collected each day between 0800 hours and 1100 hours. Phosphorus and creatinine values determined from these collections were used to calculate the phosphate clearance ratio* and were added to the result obtained from urines collected during the remaining 21 hours of the day. Daily fasting blood samples were obtained at 0800 hours for the measurement of serum phosphorus, creatinine, growth hormone and somatomedin-C. Following the 48 hour adjustment period and a two day period of basal collections, 1 mg ethinyl estradiol was administered orally each day for five days. Because of nausea, one patient was withdrawn from the study after four days of estradiol therapy. No other patients experienced complications of treatment.
binding proteins, thus making additional endogenous serum somatomedin available to react with antibody [17,18]. This represents the best method currently available to measure “total” somatomedin-C in serum. Samples are prepared for “total” somatomedin-C determination by mixing serum with an equal volume of 0.1 M glycine hydrochloride buffer, pH 3.3. This mixture, which has a pH of 3.6, is allowed to stand at 24’C for 48 hours, lyophilized to dryness and reconstituted in a 0.03 M phosphate buffer, pH 7.4, prior to assay. In addition to acidification, the total somatomedin content of acid-exposed serums taken before and during estrogen treatment was assessed by gel chromatography. Following exposure to acid and neutralization, samples were chromatographed on a 78 by 1.6 cm Sephadex G 200 (Pharmacia) column. The column was run at pH 7.4 in 0.05 M phosphate buffer, and the sum of the immunoreactivity of all’fractions was determined. RESULTS
In contrast to normal adults in whom somatomedin-C concentrations are between 0.4 and 2.0 U, the somatomedin-C values in the acromegalic patients ranged between 3.6 and 10.2 U/ml (Table I). The administration of estrogens caused prompt, significant (p <0.02 by
* The phosphate clearance ratio was obtained by the following formula: (U PO4 X S creat)/(U Creat X S PO4); where U is the urine concentration in mg/liter and S is the serum concentration in mg/dl. TABLE
I
Clinical
Features
of Patients Growth
Sex
Patient
and b (yr)
Heel Pad Thickness (mm)
Fasllng Glucose WdU
Serum Phosphate OWW
Growth Hormone Wml)
HOnlIOM alter GIllCOW Wml)
Bed
Previous maw
ouretion ol Qmpt= (Yd
Blood Pressure (mm Hg)
Serum somatomadiM &J/ml)
1 2
F,48 M.20
Proton beam Proton beam, hywphy-
9 6
140190 122176
27 33
104 98
4.5 5.0
32 88
23 84
4.9 10.2
3 4 5
M.50 F,58 F,88
Proton beam Proton beam Medroxyprogesterone
8 8 11
1621102 138186 114J70
30 33 22
92 108 87
4.2 3.7 3.5
12 18 7
20 20 30
3.6 10.1 3.6
sectomy
572
October
1980
The American
Journal of Medicine
Volume
69
ESTROGEN
two-tailed Student’s t test] declines in somatomedin-C concentrations to approximately 45 percent of the values observed in the control days (Figure 1). Concurrently, there were significant reductions in urinary hydroxyproline (p <0.05) and phosphorus clearance ratio (p
II
Metabolic
Effects of Estradlol
Serum phosphorus (mg/dl) Urine calcium (mg/g creatinine) Urine hydroxyproline (mg/g creatinine) Phosphorus creatinine clearance ratio Serum growth hormone (rig/ml) Serum somat0medin-C (U/ml) Mean
of five patients
f
standard
4.2 226.6 48.2 0.122 31 6.4
ACROMEGALY-CLEMMONS
ET AL.
100 .
40
A------A M A---A O---O
_
1b
Calcium pLy-Aj Hydraxypraline Phosphate Clearance Ratio Samatamedin-C
I 3
-
I 4
I 5
Treatment Day Control Day -_ -. Figure 1. Changes in serum somatomedinC concentrations and metabolic indices after the administration of estradiol. Calcium, phosphorus, hydroxyproline and creatinine levels were determined on 24 hour urine samples and the somatomedinG level was determined on fasting serum samples from five acromegalic subjects during two control days. Ethinyl estradiol, 1 mg daily, then was administered orally and measurements were repeated on days 3, 4 and 5 of treatment. Urinary calcium, phosphorus and hydroxyproline values were standardized by the grams of creatinine excreted and are expressed as the percentage of control values obtained on the initial study day. Each point is the mean of values for five acromegalic patients f 1 standard deviation.
fleeted a true reduction in somatomedin content or an increase in binding protein concentration. In unextracted serum, estradiol treatment caused a 51 percent reduction in the mean somatomedin-C values. In the same serum samples, which had been acid-treated, estradiol caused a 47 percent decline in the total somatomedin-C content [Table III]. In addition, when the percent decline in nonacid-treated serum from each patient is compared to the decline in acid-treated serum, the individual differences are minimal. Acidification of both pre- and postestradiol samples caused an approximate twofold increase in immunoreactive somatomedin-C. When serum samples taken before and during estrogen therapy were treated with acid, then chromatographed on Sephadex G-200 under neutral conditions, there was no significant discrepancy bePatients
Before Estrogen
Variable
l
In Acromegalic
IN
After Estrogen
f 0.55’ f 114.6 f 20.96 f 0.024 f 31.3 f 3.2
3.8 171.5 35.66 0.071 22.9 3.2
f f f f f f
0.58’ 69.3 15.46 0.013 19.1 2.0
F Value
P Value
3.21 4.98 14.60 37.60 2.47 16.30
NS NS -Co.05
NS
deviation.
October
1980
The American
Journal
of Medicine
Volume
69
573
ESTROGEN
IN
I Control
ACROMEGALY-CLEMMONS
2”3 Day
4 Treatment
ET
5 Day
AL.
Control
Day
Treotment
tween the summed immunoreactivity of the chromatography fractions and the activity of the original, acid-treated samples. These results suggest that a hidden pool of somatomedin is not unmasked by chromatography and that exposure to acid alone is sufficient to provide an estimate of “total” somatomedin. The decrease in immunoreactive somatomedin-C observed following the administration of estradiol to acromegalic patients confirms previous findings by bioassay [5,6,10] and strengthens the conclusion that estrogens act to reduce circulating somatomedin-C levels directly rather than to induce an inhibitory substance which interferes with the bioassay. In all patients, the decrease in somatomedin-C values occurred almost entirely within 48 hours of the beginning of estradiol administration. This suggests that estrogens cause either rapid cessation of somatomedin-C synthesis or an increase in its metabolic clearance rate. An estrogeninduced reduction in somatomedin-C synthesis would be in keeping with its inhibitory effect on synthesis of certain other proteins [19,20]. Pharmacologic levels of estrogen might inhibit production of somatomedin-C directly or interfere with the action of stimulatory factors, such as growth hormone or amino acid substrate availability. III
Effect of Exposure
Patient
Before Estrogen
1 2 3 4 5
4.9 10.2 3.6 10.1 3.6
Mean NOTE:
574
to Acid on Serum Somatomedin-C No Acid Rx After Estrogen
6.5
Minus
sign
October
3.2
in parentheses
1980
indicates
The American
that there
(U/ml)
(-) (-) (-) (-) (-)
46.5 34.0 77.0 62.6 49.9
(-)
51.3
was a reduction
Journal of Medicine
Acid Rx After Estrogen
Before Estrogen
% Difference
2.6 6.8 0.8 3.8 1.8
Doy
The declines in urinary hydroxyproline, cqlcium and phosphorus excretion with estradiol administration are in keeping with previously published results [4-61. Although this study provides no proof that the estrogeninduced decline in immunoreactive somatomedin-C is the cause of the changes in urinary hydroxyproline and phosphate clearance, the close temporal association of these changes and the lack of consistent changes in growth hormone levels suggest that somatomedin-C is more closely linked to these responses than is growth hormone. The question of somatomedin-C’s causal role can be answered only by direct in vivo administration of somatomedin-C, experiments which will require larger quantities of somatomedin-C than are presently available. The decline in “available” somatomedin-C, which results from estrogen treatment, correlates well with clinical responses to estrogen, diminished bioactive somatomedin [5] and reduced immunoreactive “total” somatomedin-C. The decline observed in “total” somatomedin-C supports the hypothesis that estrogen therapy actually reduces circulating concentrations of somatomedin-C rather than increasing the binding capacity of serum. The change in “available” somatomedin-C, therefore, reliably reflects the change in “total” somatomedin-C content which occurs as a result of estrogen therapy.
COMMENTS
TABLE
Figure 2. Changes in serum somatomedin-C and growth hormone levels in individual acromegalic patients following the administration of estradiol. Serum samples were drawn at 0800 while patients were fasting. O-O, patient 1; A-A, patient 2; O-O, patient 3; A-A, patient 4; D-0, patient 5.
6.9 15.5 a.2
16.1 8.5 11.0 in serum
Volume
89
somatomedin-C
content,.
% Diierence
4.6 11.8 3.0 7.4 3.6
(-) (-) (-) (-) (-)
34.0 24.4 63.0 55.0 58.0
6.4
(-)
47.0
ESTROGEN
The lack of correlation between growth hormone and somatomedin-C concentrations in acromegaly has been noted previously [14], and somatomedin-C has been shown to correlate better than growth hormone with indices of acromegalic activity, such as heel pad thickness, fasting blood sugar and 1 hour postprandial glucose. It is not surprising, therefore, that, somatomedin-C correlated better than growth hormone with metabolic effects of estrogen. These findings further support the contention that somatomedin-C levels more reliably reflect the growth hormone secretory status of patients with acromegaly than do periodic measurements of growth hormone itself. In a previous report, preliminary comparisons between changes in immunoreactive somatomedin-C and .results of surgical or proton beam treatment of acromegaly suggested that immunoreactive somatomedin-C
IN ACROMEGALY-CLEMMONS
ET AL.
reflects the long-term clinical response to therapy [la]. The present study suggests that measurement of plasma somatomedin-C also accurately reflects drug-induced changes in acromegalic activity. The prompt reduction in somatomedin-C observed with estrogen treatment suggests that measurement of this hormone may be helpful in monitoring short-term changes induced by other drugs used in the treatment of acromegaly, such as bromocriptine [21]. ACKNOWLEDGMENT We wish to thank Ms. Diane Chapin, Eyvonne Bruton and Fran Foti for their technical assistance with this study. We also thank Ms. Cindy Sullivan for her invaluable assistance in the preparation of this manuscript.
REFERENCES Kirklin OL, Wilder RM: Follicular hormone administered in acromegaly. Mayo Clin Proc 1936; 11: 121. McCullagh EP,=BeckJC. Schaffenburg CA: Control of diabetes and other features of acromegaly following treatment with estrogens. Diabetes 1955; 4: 13. Hamwi GJ. Skillman TG, Tufts KC: Acromegaly. Am J Med 1960;29:690. Albright F. Reifenstein EC, Forbes AP, et al.: Effect of estrogen in acromegaly. In: Conference on metabolic aspects of convalescence. Transactions of the 40th meeting. New York: Josiah Macy Foundation. 1946: 102. 5. Wiedemann E, Schwartz E: Suppression of growth hormone-dependent human serum sulfation factor by estrogen. J Clin Endocrinol Metab 1972; 34: 51. 6. Schwartz E, Echemendia E, Schiffer M, et al.: Mechanisms of estrogenic action in acromegaly. J Clin Invest 1969; 48: 260. 7. Mintz DH, Finster JL, Josimovich JB: Effect of estrogen therapy on carbohydrate metabolism in acromegaly. J Clin Endocrinol Metab 1967: 27: 1321. 8. Deller JJ, Di Raimondo Vd, Grodsky GM, et al.: Acromegaly: the effects of various steroid hormones on insulin-induced growth hormone response. Calif Med 1966: 104: 1. 9. Wiedemann E, Schwartz E, Frantz AG: Acute and chronic estrogen effects upon serum somatomedin activity, growth hormone and prolactin in man. 1 Clin Endocrinol Metab 1976;42:942. 10. Almqvist S, Ikkos D, Luft R: Studies on sulfation factor (SF) activity of human serum: the effects of estrogen and x-ray therapy on serum SF activity in acromegaly. Acta Endocrinol (Copenh) 1961; 37: 138. 11. Bierich JR: Estrogen treatment of girls with constitutional tall stature. Pediatrics 1978; 62 (suppl): 1196. 12. Scully RE, Galdabini JJ, McNeely BV, eds.: Case records of
October
13.
14. 15.
16. 17.
18.
19.
20. 21.
1980
the Massachusetts General Hospital weekly CPC exercises. N Engl J Med 1980; 302: 37. Boden G, Soeldner JS: A sensitive double antibody radioimmunoassay for human growth hormone (hGH): levels of serum hGH following rauid tolbutamide infusion. Diabetologia 1967; 3: 413. - _ Clemmons DR, Van Wyk JJ, Ridgway EC, et al.: Evaluation of acromeealv bv measurement of somatomedin-C. N Ens1 J Med 19%: 30< 1138. Coneland KC. Underwood LE. Van Wvk 11: Induction of i*mmunoreactive somatomedin-C in “human serum by growth hormone: dose response relationships and effect on chromatographic profiles. J Clin Endocrinol Metab 1980: 50:690. Hintz RL, Liu F: Demonstration of specific plasma protein binding sites for somatomedin. J Clin Endocrinol Metab 1977;45:988. Blethen SL, Van Wyk JJ, Underwood LE, et al.: Somatomedin-C is generated in serum by a temperature, time, pH, and divalent cation deoendent nrocess. Presented at the 6lst meeting of the Endocrine Society, Anaheim, CA, June 13-15.1979. Chatelain PG. Blethen SL;Van Wyk JJ,et al.: Immunoreactive somatomedin-C in serum is increased by the in vitro action of serum proteases or exposure to acid. [Submitted for nublication.1 O’*Malley SW; Means AR: Female steroid hormones and targer cell nuclei, Science 1974; 183: 610. Doe RP, Mellinger GT, Swaim WT, et al.: Estrogen dosage effects on serum proteins: a longitudinal study. J Clin Endocrinol Metab 1967; 27: 1081. Wass JAH.,Thorner MO, Morris DV. et al.: Long-term treatment of acromegaly with bromocriptine. Br Med J 1977; 1: 875.
The American
Journal of Medicine
Volume
89
575
Treatment
of Acromegaly
Reduction of ,Immunoreactive Somatomedin-C Improvement in Metabolic Status
DAVID R. CLEMMONS, M.D.* LOUIS E. UNDERWOOD, M.D. Chapel Hill, North Carolina ELI C. RIDGWAY, M.D. BERNARD KLIMAN, M.D. RAYMOND N. KJELLBERG, Boston, Massachusetts
M.D.
JUDSON J. VAN WYK. M.D.+ Chapel Hill, North Carolina
From the Department of Pediatrics and Medicine, University of North Carolina, Chapel Hill, North Carolina and the Departments of Medicine and Neurosurgery, Massachusetts General Hospital, Boston, Massachusetts. This work was supported by the U.S. Public Health Service Research Grant AM0122. The Massachusetts General Hospital Clinical Research Center is supported by Grant RR-01066 from the General Clinical Research Center’s Program of the Division of Research Resources. Requests for reprints should be addressed to Dr. David R. Clemmons, Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514. Manuscript accepted May 2.1960. * Recipient of a National Research Service Award AM05991. + Recipient of a USPHL Research Center Award 4K06A AM14115.
and
Administration of estrogens to acromegalic patients has been shown to reduce the serum concentrations of bioassayable somatomedin and to cause improvement in clinical status. These effects appear not to result from an effect on the secretion of growth hormone since growth hormone concentrations are not consistently reduced. Using a sensitive radioimmunoassay for somatomedin-C, we have assessed the relationship between the estrogen-induced reduction of somatomedin and changes in several indices of disease activity in five acromegalic patients. Statistically significant reductions in serum somatomedin-C (p
October
1980 The hmerican Journal of Medicine
Volume
69
571
ESTROGEN
IN
ACROMEGALY-CLEMMONS
ET AL.
Laboratory Methods. Serum phosphorus, blood glucose and urinary total hydroxyproline, calcium, phosphate and creatinine were quantitated by previously published methods [12].
it possible for us to examine the relationship between the estrogen-induced reduction in somatomedin-C, change in serum growth hormone concentrations and the effects of estrogen on several indices of disease activity in acromegalic patients.
Serum human growth hormone was measured by radioimmunoassay using HS 840A as a standard [13]. Somatomedin-C was measured by radioimmunoassay using modifications reported previously [14]. The somatomedin-C standard is a commercially derived pool of serum from a large number of normal adults (Ortho 1778-5, Ortho Diagnostics, Raritan, NJ). This standard was assigned a potency of 1.66 U/ml [15]. Probably because of the presence in serum of binding proteins (161, which diminish the availability of somatomedin-C, the radioimmunoassay measures only 30 to 40 percent of the immunoreactive somatomedin-C present in unextracted serum [17]. This immunoreactivity referred to as the “available” somatomedin-C appears to reflect accurately the growth hormone secretory status and, unless otherwise indicated, is the fraction of somatomedin-C quantitated in this study. The “total” somatomedin content of serum is the e&i&ate made on samples which have been exposed to acid, a manipulation which alters the relationship between somatomedin-C and its
METHODS Patients and Study Methods. The participants in the study were five acromegalic adults, two male and three female between 20 and 68 years of age, who gave their informed written consent (Table I). Despite previous therapy, all exhibited signs of mild to moderate disease activity as evidenced by elevated basal growth hormone levels (four patients), elevated growth hormone levels 1 hour after the oral administration of 100 g of glucose (five patients), hypertension (two patients], elevated fasting blood sugar levels (two patients) and elevated serum phosphorus levels (two patients). Following admission to the clinical research unit of the Massachusetts General Hospital, each patient was given a diet containing 400 mg of elemental calcium, 1,000 mg of phosphate and, depending on home dietary intake, 2,200 to 3,000 calories/day. Each patient’s intake of hydroxyproline was held constant throughout the study. After a 48 hour adjustment period, daily 24 hour urine collections were begun for the determination of phosphorus, .calcium, hydroxyproline and creatinine. In addition, fractional 3 hour urine collections for phosphate and creatinine determinations were collected each day between 0800 hours and 1100 hours. Phosphorus and creatinine values determined from these collections were used to calculate the phosphate clearance ratio* and were added to the result obtained from urines collected during the remaining 21 hours of the day. Daily fasting blood samples were obtained at 0800 hours for the measurement of serum phosphorus, creatinine, growth hormone and somatomedin-C. Following the 48 hour adjustment period and a two day period of basal collections, 1 mg ethinyl estradiol was administered orally each day for five days. Because of nausea, one patient was withdrawn from the study after four days of estradiol therapy. No other patients experienced complications of treatment.
binding proteins, thus making additional endogenous serum somatomedin available to react with antibody [17,18]. This represents the best method currently available to measure “total” somatomedin-C in serum. Samples are prepared for “total” somatomedin-C determination by mixing serum with an equal volume of 0.1 M glycine hydrochloride buffer, pH 3.3. This mixture, which has a pH of 3.6, is allowed to stand at 24’C for 48 hours, lyophilized to dryness and reconstituted in a 0.03 M phosphate buffer, pH 7.4, prior to assay. In addition to acidification, the total somatomedin content of acid-exposed serums taken before and during estrogen treatment was assessed by gel chromatography. Following exposure to acid and neutralization, samples were chromatographed on a 78 by 1.6 cm Sephadex G 200 (Pharmacia) column. The column was run at pH 7.4 in 0.05 M phosphate buffer, and the sum of the immunoreactivity of all’fractions was determined. RESULTS
In contrast to normal adults in whom somatomedin-C concentrations are between 0.4 and 2.0 U, the somatomedin-C values in the acromegalic patients ranged between 3.6 and 10.2 U/ml (Table I). The administration of estrogens caused prompt, significant (p <0.02 by
* The phosphate clearance ratio was obtained by the following formula: (U PO4 X S creat)/(U Creat X S PO4); where U is the urine concentration in mg/liter and S is the serum concentration in mg/dl. TABLE
I
Clinical
Features
of Patients Growth
Sex
Patient
and b (yr)
Heel Pad Thickness (mm)
Fasllng Glucose WdU
Serum Phosphate OWW
Growth Hormone Wml)
HOnlIOM alter GIllCOW Wml)
Bed
Previous maw
ouretion ol Qmpt= (Yd
Blood Pressure (mm Hg)
Serum somatomadiM &J/ml)
1 2
F,48 M.20
Proton beam Proton beam, hywphy-
9 6
140190 122176
27 33
104 98
4.5 5.0
32 88
23 84
4.9 10.2
3 4 5
M.50 F,58 F,88
Proton beam Proton beam Medroxyprogesterone
8 8 11
1621102 138186 114J70
30 33 22
92 108 87
4.2 3.7 3.5
12 18 7
20 20 30
3.6 10.1 3.6
sectomy
572
October
1980
The American
Journal of Medicine
Volume
69
ESTROGEN
two-tailed Student’s t test] declines in somatomedin-C concentrations to approximately 45 percent of the values observed in the control days (Figure 1). Concurrently, there were significant reductions in urinary hydroxyproline (p <0.05) and phosphorus clearance ratio (p
II
Metabolic
Effects of Estradlol
Serum phosphorus (mg/dl) Urine calcium (mg/g creatinine) Urine hydroxyproline (mg/g creatinine) Phosphorus creatinine clearance ratio Serum growth hormone (rig/ml) Serum somat0medin-C (U/ml) Mean
of five patients
f
standard
4.2 226.6 48.2 0.122 31 6.4
ACROMEGALY-CLEMMONS
ET AL.
100 .
40
A------A M A---A O---O
_
1b
Calcium pLy-Aj Hydraxypraline Phosphate Clearance Ratio Samatamedin-C
I 3
-
I 4
I 5
Treatment Day Control Day -_ -. Figure 1. Changes in serum somatomedinC concentrations and metabolic indices after the administration of estradiol. Calcium, phosphorus, hydroxyproline and creatinine levels were determined on 24 hour urine samples and the somatomedinG level was determined on fasting serum samples from five acromegalic subjects during two control days. Ethinyl estradiol, 1 mg daily, then was administered orally and measurements were repeated on days 3, 4 and 5 of treatment. Urinary calcium, phosphorus and hydroxyproline values were standardized by the grams of creatinine excreted and are expressed as the percentage of control values obtained on the initial study day. Each point is the mean of values for five acromegalic patients f 1 standard deviation.
fleeted a true reduction in somatomedin content or an increase in binding protein concentration. In unextracted serum, estradiol treatment caused a 51 percent reduction in the mean somatomedin-C values. In the same serum samples, which had been acid-treated, estradiol caused a 47 percent decline in the total somatomedin-C content [Table III]. In addition, when the percent decline in nonacid-treated serum from each patient is compared to the decline in acid-treated serum, the individual differences are minimal. Acidification of both pre- and postestradiol samples caused an approximate twofold increase in immunoreactive somatomedin-C. When serum samples taken before and during estrogen therapy were treated with acid, then chromatographed on Sephadex G-200 under neutral conditions, there was no significant discrepancy bePatients
Before Estrogen
Variable
l
In Acromegalic
IN
After Estrogen
f 0.55’ f 114.6 f 20.96 f 0.024 f 31.3 f 3.2
3.8 171.5 35.66 0.071 22.9 3.2
f f f f f f
0.58’ 69.3 15.46 0.013 19.1 2.0
F Value
P Value
3.21 4.98 14.60 37.60 2.47 16.30
NS NS -Co.05
NS
deviation.
October
1980
The American
Journal
of Medicine
Volume
69
573
ESTROGEN
IN
I Control
ACROMEGALY-CLEMMONS
2”3 Day
4 Treatment
ET
5 Day
AL.
Control
Day
Treotment
tween the summed immunoreactivity of the chromatography fractions and the activity of the original, acid-treated samples. These results suggest that a hidden pool of somatomedin is not unmasked by chromatography and that exposure to acid alone is sufficient to provide an estimate of “total” somatomedin. The decrease in immunoreactive somatomedin-C observed following the administration of estradiol to acromegalic patients confirms previous findings by bioassay [5,6,10] and strengthens the conclusion that estrogens act to reduce circulating somatomedin-C levels directly rather than to induce an inhibitory substance which interferes with the bioassay. In all patients, the decrease in somatomedin-C values occurred almost entirely within 48 hours of the beginning of estradiol administration. This suggests that estrogens cause either rapid cessation of somatomedin-C synthesis or an increase in its metabolic clearance rate. An estrogeninduced reduction in somatomedin-C synthesis would be in keeping with its inhibitory effect on synthesis of certain other proteins [19,20]. Pharmacologic levels of estrogen might inhibit production of somatomedin-C directly or interfere with the action of stimulatory factors, such as growth hormone or amino acid substrate availability. III
Effect of Exposure
Patient
Before Estrogen
1 2 3 4 5
4.9 10.2 3.6 10.1 3.6
Mean NOTE:
574
to Acid on Serum Somatomedin-C No Acid Rx After Estrogen
6.5
Minus
sign
October
3.2
in parentheses
1980
indicates
The American
that there
(U/ml)
(-) (-) (-) (-) (-)
46.5 34.0 77.0 62.6 49.9
(-)
51.3
was a reduction
Journal of Medicine
Acid Rx After Estrogen
Before Estrogen
% Difference
2.6 6.8 0.8 3.8 1.8
Doy
The declines in urinary hydroxyproline, cqlcium and phosphorus excretion with estradiol administration are in keeping with previously published results [4-61. Although this study provides no proof that the estrogeninduced decline in immunoreactive somatomedin-C is the cause of the changes in urinary hydroxyproline and phosphate clearance, the close temporal association of these changes and the lack of consistent changes in growth hormone levels suggest that somatomedin-C is more closely linked to these responses than is growth hormone. The question of somatomedin-C’s causal role can be answered only by direct in vivo administration of somatomedin-C, experiments which will require larger quantities of somatomedin-C than are presently available. The decline in “available” somatomedin-C, which results from estrogen treatment, correlates well with clinical responses to estrogen, diminished bioactive somatomedin [5] and reduced immunoreactive “total” somatomedin-C. The decline observed in “total” somatomedin-C supports the hypothesis that estrogen therapy actually reduces circulating concentrations of somatomedin-C rather than increasing the binding capacity of serum. The change in “available” somatomedin-C, therefore, reliably reflects the change in “total” somatomedin-C content which occurs as a result of estrogen therapy.
COMMENTS
TABLE
Figure 2. Changes in serum somatomedin-C and growth hormone levels in individual acromegalic patients following the administration of estradiol. Serum samples were drawn at 0800 while patients were fasting. O-O, patient 1; A-A, patient 2; O-O, patient 3; A-A, patient 4; D-0, patient 5.
6.9 15.5 a.2
16.1 8.5 11.0 in serum
Volume
89
somatomedin-C
content,.
% Diierence
4.6 11.8 3.0 7.4 3.6
(-) (-) (-) (-) (-)
34.0 24.4 63.0 55.0 58.0
6.4
(-)
47.0
ESTROGEN
The lack of correlation between growth hormone and somatomedin-C concentrations in acromegaly has been noted previously [14], and somatomedin-C has been shown to correlate better than growth hormone with indices of acromegalic activity, such as heel pad thickness, fasting blood sugar and 1 hour postprandial glucose. It is not surprising, therefore, that, somatomedin-C correlated better than growth hormone with metabolic effects of estrogen. These findings further support the contention that somatomedin-C levels more reliably reflect the growth hormone secretory status of patients with acromegaly than do periodic measurements of growth hormone itself. In a previous report, preliminary comparisons between changes in immunoreactive somatomedin-C and .results of surgical or proton beam treatment of acromegaly suggested that immunoreactive somatomedin-C
IN ACROMEGALY-CLEMMONS
ET AL.
reflects the long-term clinical response to therapy [la]. The present study suggests that measurement of plasma somatomedin-C also accurately reflects drug-induced changes in acromegalic activity. The prompt reduction in somatomedin-C observed with estrogen treatment suggests that measurement of this hormone may be helpful in monitoring short-term changes induced by other drugs used in the treatment of acromegaly, such as bromocriptine [21]. ACKNOWLEDGMENT We wish to thank Ms. Diane Chapin, Eyvonne Bruton and Fran Foti for their technical assistance with this study. We also thank Ms. Cindy Sullivan for her invaluable assistance in the preparation of this manuscript.
REFERENCES Kirklin OL, Wilder RM: Follicular hormone administered in acromegaly. Mayo Clin Proc 1936; 11: 121. McCullagh EP,=BeckJC. Schaffenburg CA: Control of diabetes and other features of acromegaly following treatment with estrogens. Diabetes 1955; 4: 13. Hamwi GJ. Skillman TG, Tufts KC: Acromegaly. Am J Med 1960;29:690. Albright F. Reifenstein EC, Forbes AP, et al.: Effect of estrogen in acromegaly. In: Conference on metabolic aspects of convalescence. Transactions of the 40th meeting. New York: Josiah Macy Foundation. 1946: 102. 5. Wiedemann E, Schwartz E: Suppression of growth hormone-dependent human serum sulfation factor by estrogen. J Clin Endocrinol Metab 1972; 34: 51. 6. Schwartz E, Echemendia E, Schiffer M, et al.: Mechanisms of estrogenic action in acromegaly. J Clin Invest 1969; 48: 260. 7. Mintz DH, Finster JL, Josimovich JB: Effect of estrogen therapy on carbohydrate metabolism in acromegaly. J Clin Endocrinol Metab 1967: 27: 1321. 8. Deller JJ, Di Raimondo Vd, Grodsky GM, et al.: Acromegaly: the effects of various steroid hormones on insulin-induced growth hormone response. Calif Med 1966: 104: 1. 9. Wiedemann E, Schwartz E, Frantz AG: Acute and chronic estrogen effects upon serum somatomedin activity, growth hormone and prolactin in man. 1 Clin Endocrinol Metab 1976;42:942. 10. Almqvist S, Ikkos D, Luft R: Studies on sulfation factor (SF) activity of human serum: the effects of estrogen and x-ray therapy on serum SF activity in acromegaly. Acta Endocrinol (Copenh) 1961; 37: 138. 11. Bierich JR: Estrogen treatment of girls with constitutional tall stature. Pediatrics 1978; 62 (suppl): 1196. 12. Scully RE, Galdabini JJ, McNeely BV, eds.: Case records of
October
13.
14. 15.
16. 17.
18.
19.
20. 21.
1980
the Massachusetts General Hospital weekly CPC exercises. N Engl J Med 1980; 302: 37. Boden G, Soeldner JS: A sensitive double antibody radioimmunoassay for human growth hormone (hGH): levels of serum hGH following rauid tolbutamide infusion. Diabetologia 1967; 3: 413. - _ Clemmons DR, Van Wyk JJ, Ridgway EC, et al.: Evaluation of acromeealv bv measurement of somatomedin-C. N Ens1 J Med 19%: 30< 1138. Coneland KC. Underwood LE. Van Wvk 11: Induction of i*mmunoreactive somatomedin-C in “human serum by growth hormone: dose response relationships and effect on chromatographic profiles. J Clin Endocrinol Metab 1980: 50:690. Hintz RL, Liu F: Demonstration of specific plasma protein binding sites for somatomedin. J Clin Endocrinol Metab 1977;45:988. Blethen SL, Van Wyk JJ, Underwood LE, et al.: Somatomedin-C is generated in serum by a temperature, time, pH, and divalent cation deoendent nrocess. Presented at the 6lst meeting of the Endocrine Society, Anaheim, CA, June 13-15.1979. Chatelain PG. Blethen SL;Van Wyk JJ,et al.: Immunoreactive somatomedin-C in serum is increased by the in vitro action of serum proteases or exposure to acid. [Submitted for nublication.1 O’*Malley SW; Means AR: Female steroid hormones and targer cell nuclei, Science 1974; 183: 610. Doe RP, Mellinger GT, Swaim WT, et al.: Estrogen dosage effects on serum proteins: a longitudinal study. J Clin Endocrinol Metab 1967; 27: 1081. Wass JAH.,Thorner MO, Morris DV. et al.: Long-term treatment of acromegaly with bromocriptine. Br Med J 1977; 1: 875.
The American
Journal of Medicine
Volume
89
575