CORTISOL PRODUCTION RATE

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CORTISOL

PRODUCTION

RATE

Iv.

Infants

Born of Steroid-Treated

Mothers

and of Diabetic

Mothers.

Infants

with

Trisomy

Syndrome

and with

Anencephaly

Frederic M. Kenny, M.D., Chawalit Preeyasombat, M.D., John S. Spaulding, M.D., and

Claude J. Migeon, M.D., with the technical assistance of Betty Lawrence and

Catherine Richards, B.S.

University of Pittsburgh School of Medicine and the Children’s Hospital of Pittsburgh; and the Harriet Lane Service of the Children’s Medical and Surgical Center,

Johns Hopkins Hospital and University, Baltimore

(Submitted November 12, 1965; accepted for publication January 4, 1966.)

This work was supported by U.S. Public Health Service Research Grants NB 04963-02, AM 00180,

and the Renziehausen Fund. Dr. Preeyasombat was a Renziehausen Fellow. Dr. Spaulding was a fellow under traineeship grant Ti-AM 5219 of USPHS.

ADDRESS: (C.P.) Sirfraj University School of Medicine, Bangkok, Thailand; (J.S.S.) Department of

Pediatrics, University of Kansas; (F.M.K.) Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania 152i3; (C.J.M.) Johns Hopkins Hospital, Baltimore 5, Maryland.

I

N AN earlier paper we have defined the

normal range of cortisol production rate (CPR) during the neonatal period and during childhood.1 In the present paper

at-tention is directed to some conditions in

which abnormal adrenocortical function has been suspected. These include infants born of steroid-treated mothers, infants born of

diabetic mothers, and anencephalic babies. Two infants with the 13-15 and 17-18

tn-somy syndrome were also studied.

METHODS

Experimental Subjects

INFANTS Bonrc OF

Momiis

(

CASES 1-5) : The maternal

dis-orders which accounted for the

glucocorti-coid treatment of the mothers, the type of steroid used, and its dosage prior to and during pregnancy are shown in Table I. In Case 2 the dose of prednisone administered

0 The following trivial names and abbreviations

have been used-Cortisol (F): 11,17a,21-trihy-droxy-4-pregnene-3,20-dione; 63-hydroxycortisol (6f1-F):

6f,1117a,21-tetra-hydroxy-4-pregnene-3,20-dione; Tetrahydrocortisol (THF): 3a,11,17a, 21-tetra-hydroxy-pregnane-20-one;

Tetrahydrocor-tisone (THE): 3a,17a,21-trihydroxy-pregnane-11,

20-dione; Allotetrahydrocortisol (Allo-THF): 3a,

1 113,17a,21-tetra-hydroxy-allo-pregnane-20-one;

17-hydroxycorticosteroids (17-OHCS):

17,21-dihy-droxy-20-ketosteroids.

during pregnancy was equal to or slightly higher than that used for replacement therapy. In the other four cases, the dosage was equal to two to four times replacement

levels.

In all cases treatment was administered until the time of delivery with no increased

dosage during labor and delivery. The five infants were delivered by the vaginal route without complications.

The age of the infants at the time of the

test and the dose of radio-labeled cortisol injected are shown in Table II. Two of the infants (Cases 3 and 5) were premature

by age of gestation and by weight. Serum

electrolytes were normal in all five babies, and the blood glucose never fell below 30 mg/100 ml in the first four cases. A blood

sugar of 20 mg/100 ml was obtained on

Case 5 during our test, and was accom-panied by tremors of the extremities. This

infant took oral feedings well, and a repeat blood sugar was 76 mg/100 ml 1 week later.

INFANTS BORN OF DIABETIC Momiiis

(

CASES 6, 7, 8 AND 9

)

AND A PROBABLE

PEE-DIABETIC

(

CASE 10

)

Moi’imii: The mothers

of Cases 8 and 9 had the onset of diabetes

prior to puberty and at the time of delivery were well controlled on 85 and 70 units I

day of insulin, respectively. The mothers of Cases 6 and 7 had adult onset diabetes; they

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TABLE I

STEROID TREATED MOTHERS

Case

No.

Maternal Disease Steroid Used

Dose Estimated.

tortzsone Equivalent per

day During

Pregnancy

Pre-pregnancy Dunng. Pregnancy

1

2

3

4

5

Asthma

Disseminated lupus erythematosus

Sarcoidosis; uveitis and

lymphadenopathy

Allergic dermatitis

Prednisonet

Prednisone

Prednisone

Triamcino1one

30 mg/day for several years

10 to 30 mg/day for 1.5 years

10 to 40 mg/day for 2

years

10 to 20 mg/day

in-termittently for S years

None

40 mg/day throughout 10 mg/day

throughout

30 mg/day

throughout

20 mg/day

throughout

15 mg/day for last

6months

200mg

50mg

150mg

100mg

t

V-cortisone.

16a-hydroxy-9a-fluoro-&-cortisone.

were controlled on 15 units/day and by diet

alone, respectively.

Infant

7

was the smaller of twins. A single blood sugar determination during

the CPR test was 60 mg/100 ml, and there were never symptoms of hypoglycemia. The

blood sugar was 46 mg/100 ml in infant

8

just prior to the simultaneous start of our test and the initiation of the intravenous

Usher regimen2 for treatment of respira-tory distress. No symptoms of hypoglycemia were noted and the baby survived.

Tremors were noted on one or two oc-casions in both infants 6 and 9. Blood sugars

of 18 and 62 mg/100 ml were found in in-fant 9 on the day before the CPR test, but no blood sugars were obtained on either infant during the CPR tests as they took

feedings well and were otherwise asympto-matic.

We considered the mother of infant 10 to be probably prediabetic because of a posi-tive family history (her sister has diabetes) and because she delivered this 5,050 gm

baby whose appearance was typical of babies of diabetic mothers. Infant 10 was very large with a chubby face; she was

plethoric (hematocnit 81% on the day of

birth) and became cyanotic with crying.

She was moderately jaundiced on the third

day of life, but did not require exchange transfusion. Hypoglycemic symptomatology was absent and no blood sugar

determina-tions were made.

Tiis 13-15, AND 17-18 TEISOMY SYNDROMES

(CASES No. 11 AND 12): Patient 11 was

found to have a 13-15 trisomy by

karyotyp-ing. He presented various abnormalities,

including abnormally shaped skull, micro-phthalmia, beakilke nose, polydactyly, and probable ventricular septal defect.

Patient 12 had 47 chromosomes with a

17-18 trisomy and several congenital

ab-normalities: low set ears, receding mandi-ble, tightly flexed fingers with the second

and fifth digits overlapping the third and

fourth, and cardiac anomaly.

At the time of the determination of the

CPR the two patients were in fairly good condition, but, later on, failure to thrive resulted in death at 3 and 7 weeks, respec-lively. A third tnisomic patient, reported

previously,3 is included in Figure 1.

ANENCEPHALIC INFANTS (CAsES

No.

13

AND 14) : Patient 13 was in fair to poor

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0 STEROID MOTHER

CPR #{149}DIABETIC MOTHER

ABNORMAL CONDITIONS g PREDIABETIC

IN THE NEWBORN PERIOD TRISOMY

A ANENCEPHALY

- 0

:

‘

I 25-I

:

____

I ;

AGE IN DAYS ments of the extremities and had two

seizures, as well as episodes of cyanosis. A

single blood sugar of 22 mg/100 ml was ob-tamed on the second day of life. Electrolyte determinations were not made. At autopsy this patient was found to have typically

small adrenal glands, being approximately 1/10 of the normal size, and the adrenal cortex was narrow with absence of the fetal zone. Ten grams of brain and a small pituitary were present.

Patient 14 was in fair condition during

the period of the test, the serum electrolytes being normal and blood sugar being 35 mg/100 ml. He died during a generalized

convulsion at 98 hours of age. At autopsy

the adrenal glands weighed 620 mg and

a small amount of pituitary tissue was found

in the sella turcica.

Cortisol Production Rate; Urinary

17-Hydroxycorticosteroids; Blood Glucose

The in vivo isotope dilution technique for estimation of the cortisol production rate

was described in previous publications.1 In the studies performed in Pittsburgh,

4-C’-cortisol (specific activity 25 jtc/milli-mole) was obtained from the New England

Nuclear Company. In the studies per-formed in Baltimore, 1, 2-H3-cortisol (specific activity 50 zc per 1 jtg) was

gra-ciously provided by the Endocrinology Study Section, Division of Research Grants,

National Institutes of Health, Bethesda,

Maryland.

Urine collections were made for 48

hours, and analyses were carried out on to 3 of the total 48-hour collection. The aliquots were extracted with ethyl acetate to recover relatively “polar” unconjugated steroids. The residue was incubated at pH 4.6 with f3-glucuronidase for 15 hours

at 47#{176}C.Extraction with chloroform was

performed to recover “non-polar” con-jugated metabolites.

The unconjugated and glucuronide frac-tions were washed with 0.1 N NaOH and 0.1 N HC1, chromatographed on flonisil columns and eluted with 50% ethanol in

Fic. 1. Cortisol production rate in abnormal con-ditions of the newborn period. The shaded areas

show the value of mean ± 2 S.D. in normal in-fants. The values of the 30-day-old trisomic patient

have been reported previously.’

chloroform for the unconjugated fraction

and 25% ethanol in chloroform for the glucuronide fraction. The dried residues were taken up with a few drops of chloro-form and ethanol and chromatographed in

a Bush B5 system for 19 hours. Approproate

standards of 6f3-hydroxycortisol, THF and

THE were chromatographed on each side

of the sample. The strips corresponding to the reference steroids were sprayed with an equal mixture of 0.2% Blue Tetrazolium in ethanol and 10% NaOH solution. Appro-priate areas of the chromatogram were eluted with ethanol and the radioactivity measured and Porter-Silber chromogens

determined. In Pittsburgh, a Nuclear Chi-cago C 115 low background gas flow counter was used to measure radioactivity.

Background was about 1.6 counts per minute, and counting efficiency about 30%. In Baltimore, a Packard Tri-Carb Scintilla-tion counter was used.

Urinary 17-hydroxycorticosteroids were

measured as previously ri by a

modification of the method of Glenn and Nelson.4

Glucose concentration in capillary blood

samples was determined by the method of

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964

Interpretation of the Results of

Our Studies in Newborns

Less than 48 Hours of Age

At present, there is no method by which

one can measure separately cortisol pro-duced by the newborn ithant and that produced by the mother. Therefore, when

our studies were initiated under 48 hours

of age, it is likely that maternal

contnibu-lion of steroids played a role in the findings presented. After 48 hours of age, it is

un-likely that maternally derived steroid af-fected our studies, since it is well docu-mented that even very early in life only negligable amounts of isotope are excreted later than 48 hours following an

RESULTS AND COMMENTS

Fourteen patients were studied, and the results are shown in Table II and Figure 1. In the figure, the mean and range of normal cortisol production rates at various ages are taken from our previous publication.1

For vaginally delivered infants under 5 days of age the values were 3.7 ± 0.8

mg/24 hours (18.7 ± 3.7 mg/M2/24 hours).

For infants from 5-20 days of age the values were 2.8 ± 0.7 mg/24 hours (13.9 ±

2.9 mg/M2/24 hours). The values for infants delivered by elective cesarean section did

not differ significantly from those of the vaginally delivered babies. For subjects in the age range 4 months to 20 years the

CPR was 12.1 ± 2.9 mg/M/24 hours.

Infants Born of Steroid-Treated Mothers

None of the babies had symptoms sug-gestive of adrenal insufficiency, and the

CPR in every baby was within the normal

range. Nevertheless, pediatricians should continue to be concerned about the optimal management of infants whose mothers

re-ceive steroid therapy with various gluco-corticoid preparations during pregnancy, and assessment of adrenal function should be made in more of these babies.

Oppen-heimer has reported a neonatal death which may have been attributable to steroid therapy and secondary fetal adrenal

atro-phy.8 On the other hand, Bongiovanni and McPadden reviewed 260 pregnancies

dur-ing which pharmacologic doses of cortisone

or its analogs had been used for variable

periods of time.9 Only one infant was thought to have adrenocortical insufficiency, but this was not documented adequately.

Radioactive cortisol administered to pregnant women crosses the placenta but

the ratio maternal : fetal concentrations is

approximately 3: 1 and this could partially

protect the fetus against high maternal

con-centrations. On the other hand, transcortin

levels are low during the fetal and neonatal

periods.10 A small increase in total plasma cortisol could result in a proportionally large increase in unbound cortisol. Since

only the unbound steroid is biologically active,1’ such an increase could suppress endogenous cortisol secretion of the fetus resulting in adrenocortical insufficiency

dur-ing neonatal life. Finally, a large fraction of

total plasma 17-OHCS of the neonate is cortisone rather than cortisoP2 and this

could be another factor contributing to steroid homeostasis in infancy.

Because of their reduced effect on Na retention, prednisone, triamcinolone, and

other synthetic products are often used for therapy, but no data are available on their

transplacental transfer. An absence of transfer or a significant transfer but with immediate recovery of adrenal function

after birth could explain our results.

Symptomatic adrenal insufficiency

occur-ring after cessation of prolonged courses

of glucocorticoids is not observed in all

adult patients13 and our series could be too small to have included such occurrence.

At the present time, one must therefore

recommend that blood concentrations of

true glucose be followed serially in infants

of steroid-treated mothers. If the values were to fall below 30 mg/100 mi,14 glucose infusion in amounts sufficient to maintain

normal concentrations should be the first

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Infants Born of Diabetic Mothers and a

Probable Prediabetic Mother

When corrected for surface area, all of these babies had normal CPR’s, although

the largest baby, infant 8, had a high ab-solute value: 8.3 mg/24 hours.

The babies of diabetic mothers have been described as “Cushingoid”15 and there has been considerable interest in attempts to assess their adrenal function. Klein and

Taylonil found that babies born to “poorly controlled” diabetic mothers were exposed to higher levels of placentally-transferred

corticoids at the time of birth than were comparable controls, but that these exces-sive levels rapidly disappeared in most

infants. However, Migeon, et al.17 found

levels of 17-OHCS in the plasma of diabetic mothers at term and of their offspring which

were not significantly different from those of corresponding controls.

Serum 17-OHCS are altered by

imma-turity and dysfunction of the liver and

kidneys and may not be an accurate index of adrenal function in this group of babies. Since the CPR technique depends on the

ratio of labeled to unlabeled steroids rather than on the serum or urinary level, it is not subject to variations due to hepatic or renal aberrations. Aarskog1 estimated the mean CPR in five normal babies to be

22 mg/M2/24 hours, and the mean for eight babies of diabetic mothers to be 27 mg/M2/24 hours. There was no statistically

significant difference between these two means.

The CPR estimations were begun after 60 hours of age in Aarskog’s material. Four of our five subjects were injected before 36 hours of age. The mean CPR in our group of patients was 15.3 mg/M2/24

hours; this is not significantly different from the mean of 18.3 mg/M/24 hours for the normal babies whom we studied in this age group.’

Autosomal Trisomy

The mesonephros contributes to the de-velopment of the kidneys, gonads, and

adrenal. Renal and gonadal anomalies have

been described in autosomal tnisomy, but no adrenal malformation has been

docu-mented. Normal function of the adrenal cortex with regard to CPR was found in the three patients whom we have studied

(two

patients in the present paper and one from previous works).

Anencephaly

In patient 13, the CPR of 1.9 mg/24 hours was below the normal range;

how-ever, when the surface area correction was applied, the value of 12.4 mg/M2/24 hours was within the normal range. The values for patient 14 were also in the normal range, although at its lower limit.

That the CPR were not lower is sur-prising in view of the typically small adrenals at autopsy. In experimental

ani-mals, decapitation or hypophysectomy dur-ing fetal life have also produced hypo-adrenocorticism.1#{176}

The concentration of plasma 17-OHCS in cord plasma of anencephalic infants has

been shown to be similar to that of normal infants.#{176} Since cortisol readily crosses the

placenta, the cortisol found in the cord blood of anencephalic babies could be of maternal origin. It is possible that some of the cortisol “produced” by patients 13 and

1 4 came from their mother, since the studies were begun in early life (at 12 and 24 hours of age). However, maternally derived

corti-sol could account for no more than about

0.5 mg. of the total CPR of the babies, as-suming a cord plasma cortisol plus cortisone concentration of 20 g/100 ml of plasma,12 and a maximum possible distribution

vol-ume of cortisol of 2.5 liters in these two infants. A possible explanation is that the

small amounts of pituitary tissue which are always found in anencephalic infants are

sufficient for the maintenance of a

sub-normal cortisol secretion but not of the “fetal zone” of the adrenal cortex.

SUMMARY

Treatment of pregnant mothers with

pharmacologic doses of prednisone and

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966 CORTISOL PRODUCTION OF INFANTS

of their offspring. Babies of diabetic

mothers and infants with autosomal trisomy had normal CPR’s. Two infants with

anen-cephaly had a CPR in the lower range of normal.

REFERENCES

1. Kenny, F. M., Preeyasombat, C., and Migeon,

C. J.: Cortisol production rate. II. Normal infants, children, and adults. PEDIATRICS,

37:34, 1966.

2. Usher, R.: Reduction of mortality from respira-tory distress syndrome of prematurity with early administration of intravenous glucose and sodium bicarbonate. PEDIATRICS, 32:966,

1963.

3. Kenny, F. M., Malvaux, P., and Migeon, C. J.:

Cortisol production rate in newborn babies, infants and children. PEDIATRICS, 31:360,

1963.

4. Glenn, E. M., and Nelson, D. H.: Chemical method for the determination of

17-hydroxy-corticosteroids and i7-ketosteroids in urine following hydrolysis with 13-glucuronidase.

J. Clin. Endocr., 13:911, 1953.

5. Somogyi, M. : A new reagent for the

determi-nation of sugars. J. Biol. Chem., 160:61,

1945.

6. Nelson, N.: Photometric adaptation of Somogyi

method for determination of glucose. J. Biol.

Chem., 153:375, 1944.

7. Migeon, C. J., Bertrand, J., and Wall, P. E.:

Physiological disposition of 4-C”-cortisol

during late pregnancy. J. Chn. Invest.,

36:1350, 1957.

8. Oppenheimer, E. : Lesions in the adrenals of an infant following maternal corticosteroid therapy. Bull. Hopkins Hosp., 114:146, 1964.

9. Bongiovanni, A. M., and McPadden, A. J.:

Steroids during pregnancy and possible fetal consequences. Fertil. Steril., 11:181, 1960.

10. Sandberg, A. A., and Slaunwhite, W. R., Jr.:

Transcortin: a cortico-steroid-binding

pro-tein of plasma. II. Levels in various condi-tions and the effects of estrogens. J. Clin.

Invest., 38:1290, 1959.

11. Slaunwhite, W. R., Jr., Lockie, C. N., Black, N., and Sandberg, A. A.: Inactivity in vivo

of transcortin-bound cortisol. Science,

135:3508, 1962.

12. Hillman, D. A., and Giroud, C. J. P.: Plasma cortisone and cortisol levels at birth and during the neonatal period. J. Clin. Endocr., 25:243, 1965.

13. Robinson, B. H. B., Mattingly, D., and Cope,

C. L. : Adrenal function after prolonged corticosteroid therapy. Brit. Med. J., 1:1579,

1962.

14. Cornblath, M., and Reisner, S. H.: Blood glu-cose in the neonate and its clinical signifi-cance. New Eng. J. Med., 273:378, 1965. 15. Nelson, W. E. : Textbook of Pediatrics, ed. 8.

Philadelphia: W. A. Saunders, p. 392, 1964. 16. Klein, R., and Taylor, P.:

17-hydroxycorticos-teroids in blood of diabetic mothers and their offspring. PEDIATRICS, 26:333, 1960. 17. Migeon, C. J., Nicolopoulus, D., and

Corn-blath, M. : Concentrations of 17-hydroxycor-ticosteroids in the blood of diabetic mothers and in blood from the umbilical cords of their offspring at the time of delivery.

PEDI-ATRICS, 25:605, 1960.

18. Aarskog, D. : Cortisol production rate in new-born infants of diabetic mothers. J. Pediat.,

62:807, 1963.

19. Jost, A.: Problems of fetal endocrinology: the adrenal gland. Recent Progr. Hormone Res., in press.

20. Nichols, J., Lescure, 0. L., and Migeon, C. J.:

Levels of 17-hydroxycorticosteroids and 17-ketosteroids in maternal and cord plasma

in term anencephaly. J. Clin. Endocr., 18:444, 1958.

Acknowledgment

The authors wish to express their gratitude to Dr. Richard L. Day for his interest in this work,

and to Dr. Paul Taylor for his cooperation.

CORRECTION

In Passarge, E., and Lenz, W. : Caudal Regression in Infants of Diabetic Mothers

(Pimirmcs, 37:672, 1966) Case 43 of Table

I, “hypoglycemia” should read

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1966;37;960

Pediatrics

Frederic M. Kenny, Chawalit Preeyasombat, John S. Spaulding and Claude J. Migeon

of Diabetic Mothers. Infants with Trisomy Syndrome and with Anencephaly