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Urinary constituents of infants of diabetic and nondiabetic mothers
652 The
]ournal of P E D I A T R I C S
Urinary constituents of infants of diabetic and nondiabetic mothers I. I 7 - H y d r o x y c o r t i c o s t e r o i d
excretion in premature
infants
Urinary total r 17-hydroxycorticosteroids were measured serially during the first week o[ Ii[e in 41 premature in[ants. Well premature in[ants o[ nondiabetlc and diabetic mothers had lower excretion values o[ urinary 17-hydroxycorticoids than normal [uU-term infants. These two groups o[ premature in[ants responded differently to the respiratory distress syndrome; in[ants o[ diabetic mothers had a significant increase in urinary 17-OHCS, whereas in[ants o[ nondiabetic mothers did not. Several possible explanations [or these observations are discussed.
Elizabeth K. Smith, Ph.D.,* Helen S. Reardon, M.D., and Sacha H. Field, M.D. SEATTLE~ WASH.
P R E s ~ N T knowledge of the functional capacity of the adrenal cortex in the neonate remains incomplete. Several investigators ~-8 have reported differences in urinary corticosteroid excretion between various groups of newborn infants. Investigation in this area has been limited by nonspecificity
From the Departments o[ Pediatrics, The Children's Orthopedic Hospital and Medical Center and Temple University School o[ Medicine. Investigation supported in part by Grants AM-05903 and GM-07327, National Institutes o[ Health. Material presented in part at Western Society [or Pediatric Research, Seattle, November 6, 1962. *Address, The Children's Orthopedic Hosl~tal and Medical Center, 4800 Sand Point Way; N.E., Seattle 5, Wash.
of chemical methods available for measurement of adrenocorticosteroids, and by the difficulties of obtaining accurate urine collections on adequate numbers of control groups of infants. Venning, Randall, and Gy6rgyfl using a bioassay procedure, reported no difference between full-term and premature infants during the first 5 days of life, and a gradual increase in adrenocorticosteroids after the tenth day of life. BjSrklund 1 and Farquhar s raised the question of the influence of hyperadrenocorticism on the development of the fetus of the diabetic mother and reported an elevation in urinary formaldehydrogenic corticoids of infants of diabetic mothers during the first week of life. Yenning 6 reported that 2 infants of diabetic mothers with atelectasis had slightly
Volume 64 Number 5
17-Hydroxycorticosteroid excretion in the premature
increased urinary corticosteroids which decreased with improvement in the clinical condition. Hillman 8 recently stated that newborn infants with the respiratory distress syndrome excreted approximately twice as much total 17-hydroxycorticosteroids (Porter-Silber chromogens) on the first day of life as did healthy infants, and that excretion values for both groups doubled on the second and third days of life. Cranny and Crannyg, 10 measured total Porter-Silber chromogens 11 in newborn premature infants with varying degrees of neonatal stress and suggested that relative adrenal insufficiency might contribute to the clinical findings in premature infants subjected to stress. Ulstrom and colleagues 12 recorded comprehensive serial studies of urinary 17hydroxycorticosteroids (17-OHCS) in normal full-term infants. These authors noted that the values for total steroids were generally lower t h a n w o u l d be predicted on the basis of small body size alone, and that the free fraction accounted for a higher percentage of the total steroids than is commonly found in adult man. Since infants of diabetic mothers and premature infants of nondiabetic mothers have an increased incidence of the respiratory distress syndrome, further investigation of the relationship of adrenocortical function to the clinical findings in these two groups of infants seemed warranted. Therefore, the purposes of this paper are to present preliminary observations of serial measurement of urinary 17-OHCS in premature infants during the first week of life and to evaluate the influence of illness, especially idiopathic respiratory distress, on the output of urinary 17-OHCS in premature infants of diabetic and nondiabetic mothers. SUBJECTS
AND PROCEDURES
Forty-one newborn premature infants were studied. Eighty urine specimens were obtained from 18 premature infants of nondiabetic mothers and 71 specimens were obtained from 23 infants of diabetic mothers. The 18 infants of nondiabetic mothers were classified i n t o three groups: (1) 6 healthy
65 3
premature infants, (2) 9 infants who had respiratory distress, and (3) 3 infants with various types of illness classified as "miscellaneous." The 23 infants of diabetic mothers were divided into two groups: (1) 10 healthy infants and (2) 13 sick infants with respiratory distress. Infants were considered to be well if they had an uneventful neonatal course or if only one abnormal finding was present for no longer than 2 hours. Infants considered to be sick were of two categories: (1) infants with miscellaneous causes of neonatal illness unrelated to respiratory distress, and (2) infants with idiopathic respiratory distress. Criteria for idiopathic distress were the presence of the following signs: (1) costal retractions, (2) expiratory grunt, (3) flare of alae nasi, (4) tachypnea, and (5) cyanosis. Infants with 2 or more of these signs for 2 consecutive hourly periods were classified as being critically ill with the respiratory distress syndrome. Infants who had only 1 sign of respiratory difficulty, usually tachypnea, for 2 hours or more were designated as having mild respiratory distress. The majority of the critically ill infants had significant clinical abnormalities within 30 minutes a f t e r delivery. Table I summarizes some o f the pertinent clinical findings. Eighty per cent of the critically ill infants and 50 per cent of the mildly ill infants of diabetic mothers were delivered by cesarean section, whereas all infants of nondiabetic mothers were delivered by the vaginal route. All infants of diabetic mothers were considered premature according to gestational a g e (less than 38 weeks) but full term according to weight (greater than 2,500 grams). The infants of nondiabetic mothers were premature both by gestational age and birth weight. Urine was collected for periods of 24 hours by placing the infants on the special collecting bed of Hepner and Lubchenco 18 which could be used e i t h e r inside Isolette or Armstrong incubators or in a crib. Infants were placed on the collecting bed as soon after birth as possible, usually within 4 hours. Timing of the 24 hour collection
654
Smith, Reardon, and Field
May 1964
T a b l e I. C l i n i c a l r e c o r d of p r e m a t u r e i n f a n t s
Well
Misc. I D M*
No. Gestation period (weeks, average) Birth weight (grams, average) Delivery by cesarean section (%). Clinical findings within 3~0 minutes of delivery (%)
illness Misc.
I nD M t
Sick Idiopathic respiratory distress Critically ill (survived) Mild illness RDS RD IDM I InDM IDM I InDM
10,
6
3
5
6
8
3
38
36
34
36
32
37
34
3,377
1,926
1,941
3,304
1,496
3,285
1,653
40
0
0
80
0
50
0
0
0
0
80
83
25
0
*I D M--Infants of diabetic mothers. t I nD M--Infants of nondiabetic mothers.
Table II. Urinary 17-OHCS nondiabetic mothers
e x c r e t i o n i n well p r e m a t u r e
In[ant
Age (days)
Weight (grams)
(M.e)
Wl-3 WI-6 WI-2 Wl-1 W1-3 W1-5 W1-6 Wl-2 W1-4 WI-1 W1-5 W1-6 W1-2 W1-4 WI-1 Wl-3 W1-6 W1-2 WI-4 WI-1 W1-3 WI-5 W1-4 WI-1 WI-3 W1-2 W1-4 WI-1 W1-3 W1-5 W1-2
1 1 1 2 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 5 5 5 5 6 6 6 6 7 7 7 8
1,751 1,439 2,155 1,983
0.14 0.12 0.15 0.15
2,133
0.16
2,105
0.15
S,A.
i n f a n t s of
Volume (mi./24 hr.)
Creatinine (mg./24 hr.)
17-Hydroxycorticosteroids total (mcg./24 hr.)
50 30 114 80 120 52 72 157 40 70 128 40 265 195 72 240 62 280 23O 106 26O 133 170 178 240 245 237 150 26O 172 173
11 5 21 19 2O 34 10 27 26 21 34 12 37 32 17 37 12 42 37 17 27 37 27 23 30 42 31 18 32 38 4O
12 22 39 54 47 89 29 39 38 54 I00 46 81 104 48 74 37 57 83 34 73 54 75 89 69 66 91 6O 8O 116 5O
Volume 64 Number 5
17-Hydroxycorticosteroid excretion in the premature
began with placement on the bed. If the infants were removed from the bed for feeding or technical purposes, they were partially enveloped in a plastic bag to insure continued collection of urine. All urine which was contaminated with fecal material was discarded. Urine was collected under oil in an iced container and upon completion of each 24 hour collection, a large aliquot was frozen immediately and preserved at -15 ~ C. These aliquots were packed in dry ice and shipped from Philadelphia to Seattle, where they were stored at -15 ~ C. until assayed. Data for blood and urine electrolytes obtained from these infants are being reported separately? * CHEMICAL
METHODS
Aliquots of the 24 hour collections were thawed and analyzed for total 17-OHCS according to the method of Glenn and Nelson. 15 Aliquots of urine were adjusted to p H 5 with acetic acid; acetate buffer p H 5, beta-glucuronidase, ~' 500 units per milliliter, and penicillin were added, and the mixture incubated at 37 ~ C. O u r earliest studies on some of the infants of diabetic mothers were carried out with 20 ml. aliquots of urine and a 28 hour incubation period. Subsequent assays were performed with aliquots of 100 ml. or a 12 hour equivalent. For the larger volumes, an incubation period of 48 hours was found necessary to obtain complete hydrolysis. Comparison of duplicate assays by both methods showed good agreement when the absolute concentration was high enough to be measured accurately with the color reaction. Aliquots were extracted 4 times for 15 minutes with one-half volume freshly distilled chloroform. The chloroform extracts were washed twice with one-sixth volume 0.1 N N a O H and once with one-sixth volume 0.1 N HC1, filtered through a minim u m quantity of anhydrous sodium sulfate, and evaporated to dryness with dry filtered air at 45 ~ C. Purification was performed with a Florisil column, and Porter-Silber ~Ketodase, furnished in part by Warner-Ghilcott Laboratories, Morris Plains, N. Y.
655
chromogens were estimated in the 25 per cent methanol in chloroform eluate from the column. Color was developed with acid phenylhydrazine for one hour at 60 ~ C. A "sample blank" was measured with each sample. Readings were made in the Beckman D U or DB Spectrophotometer at 370, 410, and 450 m/x. A cortisoI standard was chromatographed and measured with each group of samples. Calculations were made by subtracting the O.D. readings for the sample blank and by utilizing the Allen correction equation 16 to express the results in micrograms of cortisol equivalent. The lower limits of the standard curve recommended by Glenn and Nelson were extended and concentrations as low as 5 mcg. were found to obey Beer's Law; however, a concentration of 10 meg. cortisol was found to be the lower limlt for obtaining a peak absorption at 410 m/x. All the assays for total 17-OHCS satisfied these criteria. The fraction referred to as "total" includes only the glucuronide-conjugated and unconjugated corticosteroids which are extractable with chloroform and does not include all the more polar corticosteroids such as 6-flhydroxycortisol. Creatinine was measured on all specimens for validity of 24 hour collection by the use of a modification 17 of the method of Jaffe. All urines were tested for glucose with Testape and found to be negative. RESULTS
Tables II, I I I , I V and V list the individual daily values during the first 8 days of life, for urine volume, creatinine, and total 17-OHCS. Table V I presents mean values of total 17-OHCS from various groups of premature infants of diabetic and nondiabetic mothers for a 1 to 8 day time period. These data are compared with values obtained by Ulstrom and co-authors lz from a group of normal full-term infants. T h e method of assay used by Ulstrom is similar to our method, hence direct comparison is considered justifiabIe. It is apparent that the mean 17-OHCS excretion for the well premature infants, namely 31.7 mcg. per
65 6
Smith, Reardon, and Field
Table III. Urinary diabetic m o t h e r s
May 1964
1 7 - O H C S excretion in well p r e m a t u r e infants of
In[ant
Age (days)
Weight (grams)
S.A. (M. e)
W51-1 W51-2 W51-3 W51-4 W51-6 W52-3 W51-5 W51-7 W51-4 W52-3 W51-7 W51-1 W52-1 W51-3 W51-4 W52-2 W51-6 W52-3 W51-7 W51-I W52-1 W52-2 W52-3 W52-3 W51-2 W51-7 W51-6 W52-3
1 1 1 1 1 1 1 1 2 2 2 3 3 3 3 3 3 3 3 4 4 4 4 5 6 6 7 7
2,736 2,020 4,125 3,452 4,040 3,133 3,473 3,313
0.18 0.15 0.23 0.21 0.23 0.20 0.21 0.20
3,005 3,133
0.20 0.20
3,756 3,912
0.22 0.23
3,700
0.21
3,019 3,133
0.20 0.20
3,586
0.22
3,175
0.20
k i l o g r a m p e r 24 hours, is m u c h lower t h a n the m e a n v a l u e of 59.9 mcg. p e r k i l o g r a m p e r 24 hours observed in well f u l l - t e r m infants. 12 T h i s difference also is a p p a r e n t when values a r e expressed in relation to surface a r e a instead of weight. T h e r e l a t i o n of illness to changes in outp u t of total 1 7 - O H C S in p r e m a t u r e infants of n o n d i a b e t i c a n d d i a b e t i c m o t h e r s was analyzed. Excretion values for infants of n o n d i a b e t i c mothers w i t h r e s p i r a t o r y distress who were critically ill d i d n o t differ significantly from those who were m i l d l y ill, hence, the d a t a for the two groups are combined. Fig. 1 is a s c a t t e r g r a m of the d a t a o b t a i n e d d a i l y for the first week of life from 6 well infants, 9 r e s p i r a t o r y distressed infants, a n d 3 infants w i t h miscellaneous illness delivered of n o n d i a b e t i c mothers. M e a n values for the total 1 to 8 d a y p e r i o d are shown in T a b l e V I a n d i n d i c a t e t h a t the
Urine volume (mi./24 hr.) 197 140 155 111 108 73 180 173 29O 233 159 325 236 120 290 223 200 195 196 345 260 408 244 324 235 264 480 332
Creatinine (mg./24 hr.) 25 38 45 24 46 36 55 35 51
17-Hydroxycorticosteroids (total meg.~24 hr.) 64 519 115 156 65 102 208 169 185 192 16,0,
39 58 47 29 39 45 42 46 48 57 53 68 34 46 65
90 1~00 84 182 84 1,34, 182 104, 62 110 108 2t}7 3116
109, 12P3 187 3165
1 7 - O H C S excretion for infants with respiratory distress a p p a r e n t l y was somewhat lower (29.0 mcg. p e r k i l o g r a m p e r 24 hours) t h a n the m e a n value of well p r e m a t u r e infants (31.7 mcg. p e r k i l o g r a m p e r 24 hours), b u t the differences Were not significant. Statistical analysis for significance of differences between groups of infants was performed, using student's " t " test. is I n f a n t s with o t h e r "miscellaneous" cases of illness h a d an average excretion of 41.9 meg. p e r kilogram per 24 hours w h i c h was significantly higher than the m e a n value observed for well p r e m a t u r e infants (P = < 0.001). I n contrast, prem a t u r e infants with r e s p i r a t o r y distress who were b o r n to diabetic m o t h e r s h a d a larger daily excretion of t o t a l 1 7 - O H C S t h a n did well infants of diabetic mothers, as shown in Fig. 2 a n d T a b l e V I . T h e m e a n value for distressed infants of diabetic m o t h e r s (59.7 mcg. p e r k i l o g r a m p e r 24 hours) was
Volume 64
Number 5
17-Hydroxyeortieosteroid excretion in the premature
s i g n i f i c a n t l y g r e a t e r t h a n f o r well i n f a n t s of d i a b e t i c m o t h e r s (45.7 m c g . p e r k i l o g r a m p e r 24 h o u r s ) Comparison Table
IV.
(P = < 0.001). of t h e e x c r e t i o n
Urinary
values
for
17-hydroxycorticosteroids
65 7
1 7 - O H C S i n r e s p i r a t o r y d i s t r e s s e d i n f a n t s of d i a b e t i c a n d n o n d i a b e t i c m o t h e r s , Fig. 3 a n d T a b l e V I , s h o w s t h a t t h e i n f a n t s of d i a b e t i c m o t h e r s e x c r e t e d 59.7 m c g . p e r k i l o g r a m p e r in
sick
infants
of
nondiabetic
mothers Urin~
Age Weight In[ant (days) (grams) A. In[ants with respiratory distress Sxl-3 Sxl-5 Sxl-4 DI-2 DI-1 SI-1 Sxl-3 Sxl-5 Sxl-6 Sxl-2 Sxl-4 DI-1 D 1-3 $I-I Sxl-3 Sxl-5 Sxl-6 Sxl-2 Sxl-4 D1-2 D1-3 Sl-1 Sxl-3 Sxl-5 Sx 1-6 Sxl-2 Sxl-4 D1-3 Sxl-3 Sxl-4 D1-2 Sxl-3 D 1-2 DI-1 SI-1
1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 4 4 4 4 4 4 5 5 5 6 6 6 6
S~
(M. e)
1,091 1,573 1,694" 1,970 1,488 1,892
0.11 0.13 0.13 0.15 0.12 0.14
992 1,935
0.11 0.14
1,603
0.13
17-Hydroxycortieosteroids (total mcg./24 hr.)
volume (ml./24 hr.)
Creatinine (mg./24 hr.)
32 2,8 17 2,7 18 23 43 60 46 55 110 116 163 128 45 86 50 172 50 97 183 110 43 96 5,3 126 45 178 70 100 173 77 2130 9.0 187
5 13 7 5 7 5 9 9 24 20 14 16 19 24 10 14 22 21 14 24 15 18 5 29 9 25 14 12 14 15 26 12 28 15 22
28 21 32 11 36 16 20 43 34 78 63 47 52 74 28 71 29 98 45 52 33 40 14 93 19 . 87 53 37 33 101 51 28 55 24 68
83 154 90 ~ 2~34 120 200 1,7:3 810 174 165 204 1713 213:0 176
15 34 39 41 23 34 33 22 31 30 45 23 41 21
52 59 80 65 103 59 154 40 54 111 70 108 70 101
B. In[ants with miscellaneous illness M1-3 M1-2 MI-1 M 1-2 M1-3 M1-2 M1-3 MI-1 M1-2 MI-3 M1-2 M1-3 M 1-2 M1-3
1 2 3 3 3 4 4 4 5 5 6 6 7 7
1,850 2,041 1,936
0.15 0.15 0.16
6 5 8 Smith, Reardon, and Field
May 1964
O
WELL
0
SICK,
RD+RDS
A
SICK,
MISC.
20C
ISC
~t oJ o E
A
,0c
00
0 8
9 00~
00 9 0 @ 0
i% 0 9
i
9 0~,
.... 99
o 0
I
A
8
9 Or, G
SC
A
0
~o
2
3
9149 9 O~
9149
A 9
0 ~
9
0 ~
9
9
0 ~
0
o
Oo
00
4
5
/ ~ DAYS
6
OF
"r
8
LIFE
Fig. l. Total urinary 17-OHCS in premature infants of nondiabetic mothers 9 RD refers to infants with respiratory distress, and RDS, to infants with the respiratory distress syndrome 9 Misc. refers to infants with miscellaneous, nonrespiratory illness.
24 hours and infants of nondiabetic mothers excreted 29.0 mcg. per kilogram per 24 hours. These differences are highly significant (P = < 0.001) and similar relationships exist when values are corrected for surface area as in Fig. 4. Therefore, increase in body size alone does not account for the higher excretion of 17-OHCS observed in
stressed infants of diabetic mothers as compared with stressed infants of nondiabetic mothers. Change in excretion of 17-OHCS with increase in age was evaluated 9 Mean values for the first 3 days and for the next 4 to 5 days, presented in Fig. 5, indicate that the output of total 17-OHCS increased very 9
WELL
O SICK, R D * R D S
0 400
0 0
O
m
0
?
300
9
0
s
8 200
9.
O9
o~
O
O0
09
~o~
09
o9 I
9
o
o.~
0
oO
2
0
O0
9 0
t I O0
O O
3
o o'
|
"o 4
o O0
5
6
7
8
DAYS OF L I F E
Fig. 2. Total urinary 17-OHCS in premature infants of diabetic mothers. Symbols same as in Fig. 1.
Volume 64 Number 5
17-Hydroxycorticosteroid excretion in the premature
]
659
WELL INFANTS NON-DIABETIC MOTHERS SICK INFANTS, ROB NON-DIABETIC MOTHERS
] ]
SICK INFANTS, M i n c a l l o n e o u l NON-DIABETIC MOTHERS
]
SICK INFANTS, ROB DIABETIC MOTHERS
WELL INFANTS DIABETIC MOTHERS
&
I0O
t
Meen
4- SEM
.r .,r r
~.
75
MEAN VALUES
] ~ 8 DAYS
u
E
50
Number
31
35
28
14
43
Fig. 3. Total urinary 17-OHCS in premature infants of nondiabetic and diabetic mothers expressed as microgram per kilogram body weight per 24 hours. M e a n values for first 8 days of life. Lines at the top of each bar represent standard error of the mean. Figures at base of each bar represent numbers of observations in each category. RDS includes infants with respiratory distress and the respiratory distress syndrome.
] ] 1200
[] B
IOOO
"r q. ea
...
~
.800
5/L :/%
600
]
WELL IN FANTB NON-DIABETIC MOTHERS SICK INFANTS, ROB NON-DIABETIC MOTHERS SICK INFANTS, MI,cellone(~um NON--DIABETIC MOTHERS WELL INFANTS DIABETIC MOTHERS SICK INFANTS, ROB Of ABETIC MOTHERS
M,=a '=' BI~U
MEAN VALUES
L-- 8 DAYS
~4
400
t
200'
Number
~/~
31
35
14
28
43
Fig. 4. Total urinary 17-OHCS in premature infants of nondiabetic a n d diabetic mothers, expressed as micrograms per square meter surface area per 94 hours. Refer to Fig. 3 for explanation.
660
Smith, Reardon, and Field
May 1964
slightly with age in three of the groups of infants in w h o m sufficient data were available for comparison. Average daily variations are shown in Fig. 6. O u r results agree with those of U l s t r o m 12 who reported a questionably significant increase in corticosteroid excretion d u r i n g the first week of life in n o r m a l full-term infants a n d with those of C r a n n y a n d C r a n n y 9' 10 who reported a highly significant gradual increase d u r i n g the first m o n t h of life in p r e m a t u r e
infants; they differ from the results of Salmi, Pekkarinen, a n d Heikkila 4 who, using a different assay procedure, f o u n d high values on the first a n d second days of life in prem a t u r e a n d full-term infants, b u t a decrease d u r i n g the whole first month. T h e increase in excretion values in all groups of infants, sick a n d well, on the second day compared to the first day of life corroborates the findings of HillmanS; b u t contrary to the findings of this author, the increase was not m a i n -
T a b l e V. U r i n a r y 17-hydroxycorticosteroids in sick infants of diabetic mothers
Age Weight In[ant (days) (grams) In[ants with respiratory distress $51-2 Sx51-4 Sx52-6 Sx52-8 Sx51-6 Sx52-9 Sx52-7 $51-2 $52-1 $51-1 Sx51-3 Sx51-4 Sx52-6 Sx52-8 Sx51-6 Sx52-9 Sx52-7 $51-2 $52-1 $51-1 Sx52-5 Sx52-6 Sx52-8 Sx51-6 Sx52-9 Sx52-7 Sx52-1 $51-2 $51-1 Sx52-2 Sx52-8 $51-1 Sx51-4 Sx52-8 Sx51-6 $51-1 Sx52-8 Sx51-2 Sx51-6 Sx52-1 Sx52-2 Sx51-6
1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 5 5 5 5 6 6 6 6 8 8 8
S.A.
(m.~)
4,763 3,544 2,608 2,658 3,090 3,310 4~068
0.25 0.22 0.17 0.18 0.18 0.16 0.23
3,664 2,523 3,628
0.22 0.18 0.22
3,430
0.21
3,423
0.21
2,551
0.17
Urine Uoiume (mi./24 hr.)
Creatinine (rag.~24 hr.)
17-Hydroxycorticosteroids (total mcg./24 hr.)
99 106 110 45 128 98 150 186 176 133 232 280 118 109 193 66 275 335 84 176 132 113 219 143 100 270 145 320 313 176 220 233 272 255 255 243 225 280 267 220 240 355
57 36 21 12 22 26 46 65 80 18 50 66 18 40 30 28 47 58 33 44 31 16 30 39 28 44 19 59 42 25 29 42 24 41 40 37 40 40 37 33 36 40
320 126 63 55 205 112 383 432 227 154 140 120 56 238 232 97 239 437 205 285 81 48 204 250 107 211 50 386 279 90 179 283 144 167 277 263 146 140 260 120 170 313
Volume 64 Number 5
17-Hydroxycorticosteroid excretion in the premature
66 1
Table VI. Urinary 17-OHCS in newborn infants (mean values, first 8 days of life)
No. of infants Infants of diabetic mothers Premature, well Premature, sick RDS Infants of nondiabetic mothers Premature, well Premature, sick RDS Premature, sick misc. Full-Term, Normal (Ulstrom1~)
No. of specimens
Total mcg./24 hr.
Total mcg./Kg./24 hr.
Mean I S.D,
Mean t S.D.
10 13
2~8 43
146.9 2105.4
73.0 102.9
45.7 59.7
23.5 24.4
6 9 3
31 35 14
6,1.8 416.4 80..3
24.9 24.2 31.1
31.7 20'.0 41.9
11.5 12.2 19.4
51
219.5~
217
~Mean values calculated from " u n c o r r e c t e d "
Total mcg./M.e/24 hr. Mean ] S.D. 794.9 298.8 1,00121.4 427.8 42,51.6 353.5 532.6
167.8 1712.8 209.0'
9,35.2~
59.9*
data in original article.
Table VII. Relationship between urine volume and 17-OHCS concentration (mean values, 1 to 8 days) Infants of nondiabetic mothers
We. Urine volume ml./2r hr. ml./Kg. Total 17-OHCS mcg./24 hr. mcg,/Kg. meg,/ml.
143 69.4 6.1.8 3.1.7 0.52
I
ROS 82 57.0 46.4 29.0 0.67
tained to the third day of life in all groups, nor were the values in respiratory distressed premature infants different from those for well premature infants. Also, we are in essential agreement with the observations of Farquhar 3 who obtained higher values on the first day of life in infants of diabetic mothers than in healthy newborn infants of normal mothers. The possible relationship of urine volume to the output of 17-OHCS was evaluated. The data presented in Table V I I demonstrate that, although the absolute values for urine volume were much greater for the sick infants of diabetic mothers, values expressed in terms of body weight (milliliters per kilogram) were not significantly different than for the sick infants of nondiabetic mothers. On the other hand, values for total 17-OHCS expressed as micrograms per milliliter were approximately twice as high in sick infants of diabetic mothers as in well
I
Misc
I Infants of diabetic mothers
I
151 77.6 80.3 41.9 0.54
Well
t
ROS
230 73.0
201 61.3
146.9 45.7 0.70
205.4 59.9 1.11
or respiratory distressed infants of nondiabetic mothers, a relationship similar to that found for total 17-OHCS expressed as micrograms per kilogram. Although measurements of creatinine excretion were made primarily as a guide to completeness of collection, the data obtained were used as an additional parameter for evaluation of changes in the 17-OHCS output. Fig. 7 shows that the mean output of 17-OHCS expressed as micrograms per milligram creatinine for 1 to 8 days also is approximately twice as high in respiratory distressed infants of diabetic mothers (5.33) as in welt infants of diabetic mothers (3.36), well infants of nondiabetic mothers (2.44), or respiratory distressed infants of nondiabetic mothers (3.14). DISCUSSION The data indicate a significantly higher corticosteroid output in distressed premature
6 6 2 Smith, Reardon, and Field
May 1964
]
]
9
m J=
ioo
"-
75
E
50
Number
WELL INFANTS NON-DIABETIC MOTHERS SICK INFANTS, ROS NON- DIABETIC MOTHERS WELL INFANTS DIABETIC MOTHERS SICK tNFANT$, RDS DIABETIC MOTHERE
M.d.
14
2 :> I--3
19 Days
27
17
13 4-8
9
+_ S E .
16
Days
Fig. 5. Total urinary 17-OHCS in premature infants of nondiabetic and diabetic mothers, expressed as micrograms per kilogram body weight per 24 hours. Mean values for 1 to 3 days compared to 4 to 8 days of life9 Explanation same as for Fig. 3. infants of diabetic mothers as compared with distressed infants of nondiabetic mothers. An explanation for the differences in steroid excretion between the two groups of infants is not readily apparent. Possibilities include the influence of the diabetic state of one group of mothers on cortisol production and metabolism in the newborn in addition to differences in degree of prematurity of infants, mode of delivery, renal function, and the degree of steroid binding of plasma cortisol, which would alter the quantity of cortisol metabolites presented f o r excretion by the kidney. The question of hyperadrenocorticism in infants of diabetic mothers has been raised previously?, a, 6 Farquhar ~ particularly emphasized the possibility of hyperadrenocorticism in the fetus as a cause of certain morphologic features observed in the newborn infant of a diabetic mother. By using the urinary excretion of formaldehydrogenic steroids as an index of adrenocortical activity, Farquhar recorded elevated values in infants of diabetic mothers during the first 3
days of life. Of interest in this connection is the report by Baird and Bush 19 of no significant difference in the cortisol and cortisone content of amniotic fluid obtained from diabetic women as compared to that of normal women. 'In this preliminary study, groups of infants were not closely matched as to degree of immaturity. Infants of diabetic mothers generally are not delivered at very early gestational ages, as in the case of m a n y premature infants of nondiabetic mothers. Also, the former are notoriously large for their degree of immaturity. Expression of results in terms of body weight and surface area was considered the best approach to the problem until additional data became available. Differences in 17-OHCS content of cord blood between infants delivered by the vaginal route and by cesarean section have been documented. 2~ In this study, we have not attempted to correlate the infant's mode of delivery with the urinary 17-OHCS excretion rates. No data concerning renal function
Volume 64 Number 5
17-Hydroxycorticosteroid excretion in the premature
9
WELL INFANTS NON-O)AeETIC
t-~
SICK INFANTS, ROB NON-DIABETIC MOTHERS
MOTHERS
250
9
WELL INFANTS DIABETIC MOTHERS
[]
SICK INFANTS, RDS DIABETIC MOTHERS
66 3
4
/ /
-%
3 ----[]
200 .1:
150 E
I00
50
I
2
:5
4
5
6
7
8
Fig. 6. Total urinary 17-OHCS in premature infants of nondiabetic and diabetic mothers, expressed as micrograms per 24 hours. Mean daily changes during first 8 days of life. Figures above each point represent numbers of infants studied on each day.
t
.'A.
~. S : "
D
WELL
INFANT8 NON-DIABETIC
MOTHERB
]
DICK
INFANTE NON-DIABETIC
MOTNBNB
WELL
INFANTE DIABETIC MOTH[RE
lICK
INFANTE DIABETIC
MOTHERB
C
(.3 7
E ...
6
:
5
E 4
Number,
14 27 19 27 I-3 Days
17
22 .4-8
9 16 Days
Fig. 7. Total urinary 17-OHCS in premature infants of nondiabetic and diabetic mothers, expressed as micrograms per milligram daily creatinine excretion. Explanation same as for Fig. 3.
664
Smith, Reardon, and Field
or steroid binding of plasma cortisol are available for our groups of infants. Our findings concerning the influence of stress from the respiratory distress syndrome on the urinary excretion of 17-hydroxycorticosteroids in premature infants are contrary to those outlined by Hillman. s He found significantly higher excretion values in premature infants with respiratory distress of nondiabetic mothers than in healthy infants. We have no reasonable explanation for this descrepancy. Differences in clinical classification a n d / o r treatment may be related; the method for assay of urinary 17OHCS was similar, but not identical, in the two studies. Several studies in which highly specific methods for assessing adrenocortical function in infants were used have been reported in recent years? 1-2~ Although differences have been noted between newborn infants and adults, TM 23 limited studies have shown no significant differences between full-term and premature infants 24 or between normal full-term infants and infants of diabetic mothersl2~ Since the level of urinary excretion of 17-OHCS is not necessarily correlated with adrenal function, interpretation of our results for urinary 17-OHCS in various groups of infants in terms of differences in adrenal function is not warranted. It is hoped that the data on urinary 17-OHCS will provide valuable comparative information and suggest avenues of further investigation. Since our study was initiated, several reports have appeared describing significant alterations in the patterns of steroid excretion in the neonate. One prominent finding is the reduction in glucuronide conjugation12, 23.24, 27 indicated by a high percentage of free 17-OHCS and consistent with the recognized immaturity of the enzyme systems involved in glucuronide conjugation in the newborn infant. Another alteration in the pattern of excretion is the presence of highly polar corticosteroids such as 6-/3hydroxycortisol, which accounts for a large proportion o f the unconjugated corticosteroidsY3, 26 Extraction of these polar cor-
May 1964
tisol metabolites from urine is incomplete with chloroform; a more polar solvent such as ethyl acetate i s required. 26' 28 Experiments involving ethyl acetate extraction of urine from premature infants are in progress. 'The possibility that the relative proportion of individual urinary metabolites comprising the total may change significantly during the first few days of life has been suggested by Ulstrom. 12 Likewise, the possibility exists that the relative proportion may change during the last few weeks of fetal life, and, therefore, the excretory pattern of corticosteroids may be different in premature infants from that in full-term infants and may be altered by diabetes in the mother or by unknown etiologic factors, such as those associated with the respiratory distress syndrome. Further studies of changes in individual cortisol metabolites comprising the steroid excretion pattern in newborn infants are planned. SUMMARY
Urinary total chloroform-extractable 17OHCS (Porter-Silber chromogens) have been measured serially during the first week of life in 41 premature infants born to nondiabetic and diabetic mothers. Well premature infants of nondiabetic mothers excreted significantly lower quantities of total 17-OHCS than did the normal full-term infants studied by Ulstrom. 12 With essentially similar clinical findings and management of the respiratory distress syndrome, infants of nondiabetic mothers showed a slight decrease, and infants of diabetic mothers showed a twofold increase, in total 17OHCS excretion expressed as micrograms per kilogram per 24 hours, compared to well infants of nondiabetic mothers. A similar twofold increase obtained when the data were expressed as micrograms per square meter surface area, micrograms per milliliter urine volume, or micrograms per milligrams creatinine excreted. We express our gratitude for valuable assistance provided by Dr. Norman Kendall and Dr. Herta Schrom of the attending staff, and Miss
Volume 64 Number 5
17-Hydroxycorticosteroid excretion in the premature
Lois Berglund and Miss Pauline King of the Nursing Staff of Temple University Hospital. Technical help in performing the chemical analyses, provided by Miss Carolyn Klabunde and Mrs. Felicidad Arguelles of The Children's Orthopedic Hospital and Medical Center, is greatly appreciated.
REFERENCES
1. BjSrklund, S. I.: Infants of diabetic mothers with special reference to neonatal adrenocorticoids and tests using adrenocorticotrophic hormones, Acta Endocrinol. 15: 25, 1954. 2. Hoet, J. P.: Adrenocortical function in infants of diabetic mothers, Cold Spring Harbor Symposium on Quantitative Biology 19: 182, 1954. 3. Farquhar, J. W.: The possible influence of hyperadrenocorticism on the fetus of the diabetic woman, Arch. Dis. Childhood 31: 483, I956. 4. Salmi, T., Pekkarinen, A., and Heikkila, S.: The excretion of total 17-hydroxycorticostefolds into the urine of newborn prematures and infant boys during the first two days and the first month, Ann. Pediat. Fenniae 3: 70, 1957. 5. Matson, C. F., and Longwell, B. B.: The excretion of neutral lipid-soluble reducing substances by infants, J. Clin. Endocrinol. 9: 646, 1949. 6. Venning, E. I'i., Randall, J. P., and GySrgy, P.: Excretion of glucocorticoids in the newborn, Endocrinology 45: 430, 1949. 7. Read, C. H., Vennlng, E. H., and Ripstein, M. P.: Adrenocortical function in newly born infants, J. Clin. Endocrinol. 10: 845, 1950. 8. Hillman, D. A.: Urinary 17-hydroxycorticosteroid excretion in newborn infants with respiratory distress syndrome, A. M. A. J. Dis. Child. 102: 569, 1961. 9. Cranny, R. L., and Cranny, C. L.: The urinary excretion of Porter-Silber chromogens by premature infants, A. M. A. J. Dis. Child. 95: 401, 1958. 10. Cranny, R. L., and Cranny, C. L.: The urinary excretion of 17-hydroxycorticosteroids by premature infants, A. M. A. J. Dis. Child. 99: 344, 1960. 11. Reddy, W. J.: Modification of the ReddyJenkins-Thorn method for the estimation of 17-hydroxycorticoids in urine, Metabolism 3: 489, 1954. 12. Ulstrom, R. A., Colle, E., Burley, J., Gunville, R.: Adrenoeortical steroid metabolism in newborn infants. I. Urinary excretion of free and conjugated 17-hydroxycorticosteroids in normal full-term infants, J. Clin. Endocrinol. 20: 1066, 1960. 13. Hepner, R., and Lubchenco, L.: A method for continuous urine and stool collection in young infants, Pediatrics 26: 828, 1960.
6 65
14. Field, S. H., Reardon, H. S., and Smith, E. K.: Urinary constituents of infants of diabetic and non-diabetic mothers. II. Electrolyte and water excretion in newborn infants (in preparation). 15. Glenn, E. M., and Nelson, D. H.: Chemical method for the determination of 17-hydroxycorticosteroids and 17-ketosteroids in urine following hydrolysis with fl-glucuronidase, J. Clin. Endocrinol. 13: 91I, 1953. 16. Allen, W. M.: A simple method for analyzing complicated absorption curves, of use in the colorimetric determination of urinary steroids, J. Clin. Endocrinol. i0: 71, 1950. 17. Clark, L. C., Jr., and Thompson, H. L.: Determination of creatine and creatinine in urine, Anal. Chem. 21: 1218, 1949. 18. Dixon, W. J., and Massey, F. J., Jr.: Introduction to statistical analysis, ed. 2, New York, 1957, McGraw-Hill Book Company, Inc., p. 119. 19. Baird, C. W., and Bush, I. E.: Cortisol and cortisone content of amniotic fluid from diabetic and non-diabetic women, Acta Endocrinot. 34: 97, i960. 20. Migeon, C. J., Nicolopoulos, D., and Cornblath, M.: Concentrations of 17-hydroxycorticosteroids in the blood of diabetic mothers and in blood from the umbilical cords of their offspring at the time of delivery, Pediatrics 25: 605, 1960. 21. Bongiovanni, A. M., Eberlein, W. R., Westphal, M., and Boggs, T.: Prolonged turnover rate of hydrocortisone in the newborn infant, J. Clin. Endocrinol. 18: 1127, 1958. 22. Cranny, R. L., Kirschvink, J. F., and Kelley, V. C.: The half-life of hydrocortisone in normal newborn infants, A. M. A. J. Dis. Child. 99: 437, 1960. 23. ReynoIds, J. W., Colle, E., and Ulstrom, R. A.: Adrenocortical steroid metabolism in newborn infants. V. Physiologic disposition of exogenous cortisol loads in the early neonatal period, J. Clin. Endocrinol. 22: 245, 1962. 24. Kenny, F. M., Malvaux, P., and Migeon, C. J.: Cortisol production rate in newborn babies, older infants and children, Pediatrics 31: 360, 1963. 25. Aarskog, D.: Cortisol production rate in newborn infants of diabetic mothers, J. PEOIAT. 62: 807, 1963. 26. Ulstrom, R. A., Colle, E., Burley, J., and Gunville, R.: Adrenocortical steroid metabolism in newborn infants. II. Urinary excretion of 6/3-hydroxycortisol and other polar metabolites, J. Clin. Endocrinol. 20: 1080, 1960. 27. Migeon, C. J.: Cortisol production and metabolism in the neonate, J. PEDIAT. 55: 280, 1959. 28. Frantz, A. G., Katz, F. N., and Jailer, J. W.: 6~-hydroxycortisol and other polar corticosteroids, measurement and significance in human urine, J. Clin. Endocrinol. 21: 1290, 1961.
]ournal of P E D I A T R I C S
Urinary constituents of infants of diabetic and nondiabetic mothers I. I 7 - H y d r o x y c o r t i c o s t e r o i d
excretion in premature
infants
Urinary total r 17-hydroxycorticosteroids were measured serially during the first week o[ Ii[e in 41 premature in[ants. Well premature in[ants o[ nondiabetlc and diabetic mothers had lower excretion values o[ urinary 17-hydroxycorticoids than normal [uU-term infants. These two groups o[ premature in[ants responded differently to the respiratory distress syndrome; in[ants o[ diabetic mothers had a significant increase in urinary 17-OHCS, whereas in[ants o[ nondiabetic mothers did not. Several possible explanations [or these observations are discussed.
Elizabeth K. Smith, Ph.D.,* Helen S. Reardon, M.D., and Sacha H. Field, M.D. SEATTLE~ WASH.
P R E s ~ N T knowledge of the functional capacity of the adrenal cortex in the neonate remains incomplete. Several investigators ~-8 have reported differences in urinary corticosteroid excretion between various groups of newborn infants. Investigation in this area has been limited by nonspecificity
From the Departments o[ Pediatrics, The Children's Orthopedic Hospital and Medical Center and Temple University School o[ Medicine. Investigation supported in part by Grants AM-05903 and GM-07327, National Institutes o[ Health. Material presented in part at Western Society [or Pediatric Research, Seattle, November 6, 1962. *Address, The Children's Orthopedic Hosl~tal and Medical Center, 4800 Sand Point Way; N.E., Seattle 5, Wash.
of chemical methods available for measurement of adrenocorticosteroids, and by the difficulties of obtaining accurate urine collections on adequate numbers of control groups of infants. Venning, Randall, and Gy6rgyfl using a bioassay procedure, reported no difference between full-term and premature infants during the first 5 days of life, and a gradual increase in adrenocorticosteroids after the tenth day of life. BjSrklund 1 and Farquhar s raised the question of the influence of hyperadrenocorticism on the development of the fetus of the diabetic mother and reported an elevation in urinary formaldehydrogenic corticoids of infants of diabetic mothers during the first week of life. Yenning 6 reported that 2 infants of diabetic mothers with atelectasis had slightly
Volume 64 Number 5
17-Hydroxycorticosteroid excretion in the premature
increased urinary corticosteroids which decreased with improvement in the clinical condition. Hillman 8 recently stated that newborn infants with the respiratory distress syndrome excreted approximately twice as much total 17-hydroxycorticosteroids (Porter-Silber chromogens) on the first day of life as did healthy infants, and that excretion values for both groups doubled on the second and third days of life. Cranny and Crannyg, 10 measured total Porter-Silber chromogens 11 in newborn premature infants with varying degrees of neonatal stress and suggested that relative adrenal insufficiency might contribute to the clinical findings in premature infants subjected to stress. Ulstrom and colleagues 12 recorded comprehensive serial studies of urinary 17hydroxycorticosteroids (17-OHCS) in normal full-term infants. These authors noted that the values for total steroids were generally lower t h a n w o u l d be predicted on the basis of small body size alone, and that the free fraction accounted for a higher percentage of the total steroids than is commonly found in adult man. Since infants of diabetic mothers and premature infants of nondiabetic mothers have an increased incidence of the respiratory distress syndrome, further investigation of the relationship of adrenocortical function to the clinical findings in these two groups of infants seemed warranted. Therefore, the purposes of this paper are to present preliminary observations of serial measurement of urinary 17-OHCS in premature infants during the first week of life and to evaluate the influence of illness, especially idiopathic respiratory distress, on the output of urinary 17-OHCS in premature infants of diabetic and nondiabetic mothers. SUBJECTS
AND PROCEDURES
Forty-one newborn premature infants were studied. Eighty urine specimens were obtained from 18 premature infants of nondiabetic mothers and 71 specimens were obtained from 23 infants of diabetic mothers. The 18 infants of nondiabetic mothers were classified i n t o three groups: (1) 6 healthy
65 3
premature infants, (2) 9 infants who had respiratory distress, and (3) 3 infants with various types of illness classified as "miscellaneous." The 23 infants of diabetic mothers were divided into two groups: (1) 10 healthy infants and (2) 13 sick infants with respiratory distress. Infants were considered to be well if they had an uneventful neonatal course or if only one abnormal finding was present for no longer than 2 hours. Infants considered to be sick were of two categories: (1) infants with miscellaneous causes of neonatal illness unrelated to respiratory distress, and (2) infants with idiopathic respiratory distress. Criteria for idiopathic distress were the presence of the following signs: (1) costal retractions, (2) expiratory grunt, (3) flare of alae nasi, (4) tachypnea, and (5) cyanosis. Infants with 2 or more of these signs for 2 consecutive hourly periods were classified as being critically ill with the respiratory distress syndrome. Infants who had only 1 sign of respiratory difficulty, usually tachypnea, for 2 hours or more were designated as having mild respiratory distress. The majority of the critically ill infants had significant clinical abnormalities within 30 minutes a f t e r delivery. Table I summarizes some o f the pertinent clinical findings. Eighty per cent of the critically ill infants and 50 per cent of the mildly ill infants of diabetic mothers were delivered by cesarean section, whereas all infants of nondiabetic mothers were delivered by the vaginal route. All infants of diabetic mothers were considered premature according to gestational a g e (less than 38 weeks) but full term according to weight (greater than 2,500 grams). The infants of nondiabetic mothers were premature both by gestational age and birth weight. Urine was collected for periods of 24 hours by placing the infants on the special collecting bed of Hepner and Lubchenco 18 which could be used e i t h e r inside Isolette or Armstrong incubators or in a crib. Infants were placed on the collecting bed as soon after birth as possible, usually within 4 hours. Timing of the 24 hour collection
654
Smith, Reardon, and Field
May 1964
T a b l e I. C l i n i c a l r e c o r d of p r e m a t u r e i n f a n t s
Well
Misc. I D M*
No. Gestation period (weeks, average) Birth weight (grams, average) Delivery by cesarean section (%). Clinical findings within 3~0 minutes of delivery (%)
illness Misc.
I nD M t
Sick Idiopathic respiratory distress Critically ill (survived) Mild illness RDS RD IDM I InDM IDM I InDM
10,
6
3
5
6
8
3
38
36
34
36
32
37
34
3,377
1,926
1,941
3,304
1,496
3,285
1,653
40
0
0
80
0
50
0
0
0
0
80
83
25
0
*I D M--Infants of diabetic mothers. t I nD M--Infants of nondiabetic mothers.
Table II. Urinary 17-OHCS nondiabetic mothers
e x c r e t i o n i n well p r e m a t u r e
In[ant
Age (days)
Weight (grams)
(M.e)
Wl-3 WI-6 WI-2 Wl-1 W1-3 W1-5 W1-6 Wl-2 W1-4 WI-1 W1-5 W1-6 W1-2 W1-4 WI-1 Wl-3 W1-6 W1-2 WI-4 WI-1 W1-3 WI-5 W1-4 WI-1 WI-3 W1-2 W1-4 WI-1 W1-3 W1-5 W1-2
1 1 1 2 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 5 5 5 5 6 6 6 6 7 7 7 8
1,751 1,439 2,155 1,983
0.14 0.12 0.15 0.15
2,133
0.16
2,105
0.15
S,A.
i n f a n t s of
Volume (mi./24 hr.)
Creatinine (mg./24 hr.)
17-Hydroxycorticosteroids total (mcg./24 hr.)
50 30 114 80 120 52 72 157 40 70 128 40 265 195 72 240 62 280 23O 106 26O 133 170 178 240 245 237 150 26O 172 173
11 5 21 19 2O 34 10 27 26 21 34 12 37 32 17 37 12 42 37 17 27 37 27 23 30 42 31 18 32 38 4O
12 22 39 54 47 89 29 39 38 54 I00 46 81 104 48 74 37 57 83 34 73 54 75 89 69 66 91 6O 8O 116 5O
Volume 64 Number 5
17-Hydroxycorticosteroid excretion in the premature
began with placement on the bed. If the infants were removed from the bed for feeding or technical purposes, they were partially enveloped in a plastic bag to insure continued collection of urine. All urine which was contaminated with fecal material was discarded. Urine was collected under oil in an iced container and upon completion of each 24 hour collection, a large aliquot was frozen immediately and preserved at -15 ~ C. These aliquots were packed in dry ice and shipped from Philadelphia to Seattle, where they were stored at -15 ~ C. until assayed. Data for blood and urine electrolytes obtained from these infants are being reported separately? * CHEMICAL
METHODS
Aliquots of the 24 hour collections were thawed and analyzed for total 17-OHCS according to the method of Glenn and Nelson. 15 Aliquots of urine were adjusted to p H 5 with acetic acid; acetate buffer p H 5, beta-glucuronidase, ~' 500 units per milliliter, and penicillin were added, and the mixture incubated at 37 ~ C. O u r earliest studies on some of the infants of diabetic mothers were carried out with 20 ml. aliquots of urine and a 28 hour incubation period. Subsequent assays were performed with aliquots of 100 ml. or a 12 hour equivalent. For the larger volumes, an incubation period of 48 hours was found necessary to obtain complete hydrolysis. Comparison of duplicate assays by both methods showed good agreement when the absolute concentration was high enough to be measured accurately with the color reaction. Aliquots were extracted 4 times for 15 minutes with one-half volume freshly distilled chloroform. The chloroform extracts were washed twice with one-sixth volume 0.1 N N a O H and once with one-sixth volume 0.1 N HC1, filtered through a minim u m quantity of anhydrous sodium sulfate, and evaporated to dryness with dry filtered air at 45 ~ C. Purification was performed with a Florisil column, and Porter-Silber ~Ketodase, furnished in part by Warner-Ghilcott Laboratories, Morris Plains, N. Y.
655
chromogens were estimated in the 25 per cent methanol in chloroform eluate from the column. Color was developed with acid phenylhydrazine for one hour at 60 ~ C. A "sample blank" was measured with each sample. Readings were made in the Beckman D U or DB Spectrophotometer at 370, 410, and 450 m/x. A cortisoI standard was chromatographed and measured with each group of samples. Calculations were made by subtracting the O.D. readings for the sample blank and by utilizing the Allen correction equation 16 to express the results in micrograms of cortisol equivalent. The lower limits of the standard curve recommended by Glenn and Nelson were extended and concentrations as low as 5 mcg. were found to obey Beer's Law; however, a concentration of 10 meg. cortisol was found to be the lower limlt for obtaining a peak absorption at 410 m/x. All the assays for total 17-OHCS satisfied these criteria. The fraction referred to as "total" includes only the glucuronide-conjugated and unconjugated corticosteroids which are extractable with chloroform and does not include all the more polar corticosteroids such as 6-flhydroxycortisol. Creatinine was measured on all specimens for validity of 24 hour collection by the use of a modification 17 of the method of Jaffe. All urines were tested for glucose with Testape and found to be negative. RESULTS
Tables II, I I I , I V and V list the individual daily values during the first 8 days of life, for urine volume, creatinine, and total 17-OHCS. Table V I presents mean values of total 17-OHCS from various groups of premature infants of diabetic and nondiabetic mothers for a 1 to 8 day time period. These data are compared with values obtained by Ulstrom and co-authors lz from a group of normal full-term infants. T h e method of assay used by Ulstrom is similar to our method, hence direct comparison is considered justifiabIe. It is apparent that the mean 17-OHCS excretion for the well premature infants, namely 31.7 mcg. per
65 6
Smith, Reardon, and Field
Table III. Urinary diabetic m o t h e r s
May 1964
1 7 - O H C S excretion in well p r e m a t u r e infants of
In[ant
Age (days)
Weight (grams)
S.A. (M. e)
W51-1 W51-2 W51-3 W51-4 W51-6 W52-3 W51-5 W51-7 W51-4 W52-3 W51-7 W51-1 W52-1 W51-3 W51-4 W52-2 W51-6 W52-3 W51-7 W51-I W52-1 W52-2 W52-3 W52-3 W51-2 W51-7 W51-6 W52-3
1 1 1 1 1 1 1 1 2 2 2 3 3 3 3 3 3 3 3 4 4 4 4 5 6 6 7 7
2,736 2,020 4,125 3,452 4,040 3,133 3,473 3,313
0.18 0.15 0.23 0.21 0.23 0.20 0.21 0.20
3,005 3,133
0.20 0.20
3,756 3,912
0.22 0.23
3,700
0.21
3,019 3,133
0.20 0.20
3,586
0.22
3,175
0.20
k i l o g r a m p e r 24 hours, is m u c h lower t h a n the m e a n v a l u e of 59.9 mcg. p e r k i l o g r a m p e r 24 hours observed in well f u l l - t e r m infants. 12 T h i s difference also is a p p a r e n t when values a r e expressed in relation to surface a r e a instead of weight. T h e r e l a t i o n of illness to changes in outp u t of total 1 7 - O H C S in p r e m a t u r e infants of n o n d i a b e t i c a n d d i a b e t i c m o t h e r s was analyzed. Excretion values for infants of n o n d i a b e t i c mothers w i t h r e s p i r a t o r y distress who were critically ill d i d n o t differ significantly from those who were m i l d l y ill, hence, the d a t a for the two groups are combined. Fig. 1 is a s c a t t e r g r a m of the d a t a o b t a i n e d d a i l y for the first week of life from 6 well infants, 9 r e s p i r a t o r y distressed infants, a n d 3 infants w i t h miscellaneous illness delivered of n o n d i a b e t i c mothers. M e a n values for the total 1 to 8 d a y p e r i o d are shown in T a b l e V I a n d i n d i c a t e t h a t the
Urine volume (mi./24 hr.) 197 140 155 111 108 73 180 173 29O 233 159 325 236 120 290 223 200 195 196 345 260 408 244 324 235 264 480 332
Creatinine (mg./24 hr.) 25 38 45 24 46 36 55 35 51
17-Hydroxycorticosteroids (total meg.~24 hr.) 64 519 115 156 65 102 208 169 185 192 16,0,
39 58 47 29 39 45 42 46 48 57 53 68 34 46 65
90 1~00 84 182 84 1,34, 182 104, 62 110 108 2t}7 3116
109, 12P3 187 3165
1 7 - O H C S excretion for infants with respiratory distress a p p a r e n t l y was somewhat lower (29.0 mcg. p e r k i l o g r a m p e r 24 hours) t h a n the m e a n value of well p r e m a t u r e infants (31.7 mcg. p e r k i l o g r a m p e r 24 hours), b u t the differences Were not significant. Statistical analysis for significance of differences between groups of infants was performed, using student's " t " test. is I n f a n t s with o t h e r "miscellaneous" cases of illness h a d an average excretion of 41.9 meg. p e r kilogram per 24 hours w h i c h was significantly higher than the m e a n value observed for well p r e m a t u r e infants (P = < 0.001). I n contrast, prem a t u r e infants with r e s p i r a t o r y distress who were b o r n to diabetic m o t h e r s h a d a larger daily excretion of t o t a l 1 7 - O H C S t h a n did well infants of diabetic mothers, as shown in Fig. 2 a n d T a b l e V I . T h e m e a n value for distressed infants of diabetic m o t h e r s (59.7 mcg. p e r k i l o g r a m p e r 24 hours) was
Volume 64
Number 5
17-Hydroxyeortieosteroid excretion in the premature
s i g n i f i c a n t l y g r e a t e r t h a n f o r well i n f a n t s of d i a b e t i c m o t h e r s (45.7 m c g . p e r k i l o g r a m p e r 24 h o u r s ) Comparison Table
IV.
(P = < 0.001). of t h e e x c r e t i o n
Urinary
values
for
17-hydroxycorticosteroids
65 7
1 7 - O H C S i n r e s p i r a t o r y d i s t r e s s e d i n f a n t s of d i a b e t i c a n d n o n d i a b e t i c m o t h e r s , Fig. 3 a n d T a b l e V I , s h o w s t h a t t h e i n f a n t s of d i a b e t i c m o t h e r s e x c r e t e d 59.7 m c g . p e r k i l o g r a m p e r in
sick
infants
of
nondiabetic
mothers Urin~
Age Weight In[ant (days) (grams) A. In[ants with respiratory distress Sxl-3 Sxl-5 Sxl-4 DI-2 DI-1 SI-1 Sxl-3 Sxl-5 Sxl-6 Sxl-2 Sxl-4 DI-1 D 1-3 $I-I Sxl-3 Sxl-5 Sxl-6 Sxl-2 Sxl-4 D1-2 D1-3 Sl-1 Sxl-3 Sxl-5 Sx 1-6 Sxl-2 Sxl-4 D1-3 Sxl-3 Sxl-4 D1-2 Sxl-3 D 1-2 DI-1 SI-1
1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 4 4 4 4 4 4 5 5 5 6 6 6 6
S~
(M. e)
1,091 1,573 1,694" 1,970 1,488 1,892
0.11 0.13 0.13 0.15 0.12 0.14
992 1,935
0.11 0.14
1,603
0.13
17-Hydroxycortieosteroids (total mcg./24 hr.)
volume (ml./24 hr.)
Creatinine (mg./24 hr.)
32 2,8 17 2,7 18 23 43 60 46 55 110 116 163 128 45 86 50 172 50 97 183 110 43 96 5,3 126 45 178 70 100 173 77 2130 9.0 187
5 13 7 5 7 5 9 9 24 20 14 16 19 24 10 14 22 21 14 24 15 18 5 29 9 25 14 12 14 15 26 12 28 15 22
28 21 32 11 36 16 20 43 34 78 63 47 52 74 28 71 29 98 45 52 33 40 14 93 19 . 87 53 37 33 101 51 28 55 24 68
83 154 90 ~ 2~34 120 200 1,7:3 810 174 165 204 1713 213:0 176
15 34 39 41 23 34 33 22 31 30 45 23 41 21
52 59 80 65 103 59 154 40 54 111 70 108 70 101
B. In[ants with miscellaneous illness M1-3 M1-2 MI-1 M 1-2 M1-3 M1-2 M1-3 MI-1 M1-2 MI-3 M1-2 M1-3 M 1-2 M1-3
1 2 3 3 3 4 4 4 5 5 6 6 7 7
1,850 2,041 1,936
0.15 0.15 0.16
6 5 8 Smith, Reardon, and Field
May 1964
O
WELL
0
SICK,
RD+RDS
A
SICK,
MISC.
20C
ISC
~t oJ o E
A
,0c
00
0 8
9 00~
00 9 0 @ 0
i% 0 9
i
9 0~,
.... 99
o 0
I
A
8
9 Or, G
SC
A
0
~o
2
3
9149 9 O~
9149
A 9
0 ~
9
0 ~
9
9
0 ~
0
o
Oo
00
4
5
/ ~ DAYS
6
OF
"r
8
LIFE
Fig. l. Total urinary 17-OHCS in premature infants of nondiabetic mothers 9 RD refers to infants with respiratory distress, and RDS, to infants with the respiratory distress syndrome 9 Misc. refers to infants with miscellaneous, nonrespiratory illness.
24 hours and infants of nondiabetic mothers excreted 29.0 mcg. per kilogram per 24 hours. These differences are highly significant (P = < 0.001) and similar relationships exist when values are corrected for surface area as in Fig. 4. Therefore, increase in body size alone does not account for the higher excretion of 17-OHCS observed in
stressed infants of diabetic mothers as compared with stressed infants of nondiabetic mothers. Change in excretion of 17-OHCS with increase in age was evaluated 9 Mean values for the first 3 days and for the next 4 to 5 days, presented in Fig. 5, indicate that the output of total 17-OHCS increased very 9
WELL
O SICK, R D * R D S
0 400
0 0
O
m
0
?
300
9
0
s
8 200
9.
O9
o~
O
O0
09
~o~
09
o9 I
9
o
o.~
0
oO
2
0
O0
9 0
t I O0
O O
3
o o'
|
"o 4
o O0
5
6
7
8
DAYS OF L I F E
Fig. 2. Total urinary 17-OHCS in premature infants of diabetic mothers. Symbols same as in Fig. 1.
Volume 64 Number 5
17-Hydroxycorticosteroid excretion in the premature
]
659
WELL INFANTS NON-DIABETIC MOTHERS SICK INFANTS, ROB NON-DIABETIC MOTHERS
] ]
SICK INFANTS, M i n c a l l o n e o u l NON-DIABETIC MOTHERS
]
SICK INFANTS, ROB DIABETIC MOTHERS
WELL INFANTS DIABETIC MOTHERS
&
I0O
t
Meen
4- SEM
.r .,r r
~.
75
MEAN VALUES
] ~ 8 DAYS
u
E
50
Number
31
35
28
14
43
Fig. 3. Total urinary 17-OHCS in premature infants of nondiabetic and diabetic mothers expressed as microgram per kilogram body weight per 24 hours. M e a n values for first 8 days of life. Lines at the top of each bar represent standard error of the mean. Figures at base of each bar represent numbers of observations in each category. RDS includes infants with respiratory distress and the respiratory distress syndrome.
] ] 1200
[] B
IOOO
"r q. ea
...
~
.800
5/L :/%
600
]
WELL IN FANTB NON-DIABETIC MOTHERS SICK INFANTS, ROB NON-DIABETIC MOTHERS SICK INFANTS, MI,cellone(~um NON--DIABETIC MOTHERS WELL INFANTS DIABETIC MOTHERS SICK INFANTS, ROB Of ABETIC MOTHERS
M,=a '=' BI~U
MEAN VALUES
L-- 8 DAYS
~4
400
t
200'
Number
~/~
31
35
14
28
43
Fig. 4. Total urinary 17-OHCS in premature infants of nondiabetic a n d diabetic mothers, expressed as micrograms per square meter surface area per 94 hours. Refer to Fig. 3 for explanation.
660
Smith, Reardon, and Field
May 1964
slightly with age in three of the groups of infants in w h o m sufficient data were available for comparison. Average daily variations are shown in Fig. 6. O u r results agree with those of U l s t r o m 12 who reported a questionably significant increase in corticosteroid excretion d u r i n g the first week of life in n o r m a l full-term infants a n d with those of C r a n n y a n d C r a n n y 9' 10 who reported a highly significant gradual increase d u r i n g the first m o n t h of life in p r e m a t u r e
infants; they differ from the results of Salmi, Pekkarinen, a n d Heikkila 4 who, using a different assay procedure, f o u n d high values on the first a n d second days of life in prem a t u r e a n d full-term infants, b u t a decrease d u r i n g the whole first month. T h e increase in excretion values in all groups of infants, sick a n d well, on the second day compared to the first day of life corroborates the findings of HillmanS; b u t contrary to the findings of this author, the increase was not m a i n -
T a b l e V. U r i n a r y 17-hydroxycorticosteroids in sick infants of diabetic mothers
Age Weight In[ant (days) (grams) In[ants with respiratory distress $51-2 Sx51-4 Sx52-6 Sx52-8 Sx51-6 Sx52-9 Sx52-7 $51-2 $52-1 $51-1 Sx51-3 Sx51-4 Sx52-6 Sx52-8 Sx51-6 Sx52-9 Sx52-7 $51-2 $52-1 $51-1 Sx52-5 Sx52-6 Sx52-8 Sx51-6 Sx52-9 Sx52-7 Sx52-1 $51-2 $51-1 Sx52-2 Sx52-8 $51-1 Sx51-4 Sx52-8 Sx51-6 $51-1 Sx52-8 Sx51-2 Sx51-6 Sx52-1 Sx52-2 Sx51-6
1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 5 5 5 5 6 6 6 6 8 8 8
S.A.
(m.~)
4,763 3,544 2,608 2,658 3,090 3,310 4~068
0.25 0.22 0.17 0.18 0.18 0.16 0.23
3,664 2,523 3,628
0.22 0.18 0.22
3,430
0.21
3,423
0.21
2,551
0.17
Urine Uoiume (mi./24 hr.)
Creatinine (rag.~24 hr.)
17-Hydroxycorticosteroids (total mcg./24 hr.)
99 106 110 45 128 98 150 186 176 133 232 280 118 109 193 66 275 335 84 176 132 113 219 143 100 270 145 320 313 176 220 233 272 255 255 243 225 280 267 220 240 355
57 36 21 12 22 26 46 65 80 18 50 66 18 40 30 28 47 58 33 44 31 16 30 39 28 44 19 59 42 25 29 42 24 41 40 37 40 40 37 33 36 40
320 126 63 55 205 112 383 432 227 154 140 120 56 238 232 97 239 437 205 285 81 48 204 250 107 211 50 386 279 90 179 283 144 167 277 263 146 140 260 120 170 313
Volume 64 Number 5
17-Hydroxycorticosteroid excretion in the premature
66 1
Table VI. Urinary 17-OHCS in newborn infants (mean values, first 8 days of life)
No. of infants Infants of diabetic mothers Premature, well Premature, sick RDS Infants of nondiabetic mothers Premature, well Premature, sick RDS Premature, sick misc. Full-Term, Normal (Ulstrom1~)
No. of specimens
Total mcg./24 hr.
Total mcg./Kg./24 hr.
Mean I S.D,
Mean t S.D.
10 13
2~8 43
146.9 2105.4
73.0 102.9
45.7 59.7
23.5 24.4
6 9 3
31 35 14
6,1.8 416.4 80..3
24.9 24.2 31.1
31.7 20'.0 41.9
11.5 12.2 19.4
51
219.5~
217
~Mean values calculated from " u n c o r r e c t e d "
Total mcg./M.e/24 hr. Mean ] S.D. 794.9 298.8 1,00121.4 427.8 42,51.6 353.5 532.6
167.8 1712.8 209.0'
9,35.2~
59.9*
data in original article.
Table VII. Relationship between urine volume and 17-OHCS concentration (mean values, 1 to 8 days) Infants of nondiabetic mothers
We. Urine volume ml./2r hr. ml./Kg. Total 17-OHCS mcg./24 hr. mcg,/Kg. meg,/ml.
143 69.4 6.1.8 3.1.7 0.52
I
ROS 82 57.0 46.4 29.0 0.67
tained to the third day of life in all groups, nor were the values in respiratory distressed premature infants different from those for well premature infants. Also, we are in essential agreement with the observations of Farquhar 3 who obtained higher values on the first day of life in infants of diabetic mothers than in healthy newborn infants of normal mothers. The possible relationship of urine volume to the output of 17-OHCS was evaluated. The data presented in Table V I I demonstrate that, although the absolute values for urine volume were much greater for the sick infants of diabetic mothers, values expressed in terms of body weight (milliliters per kilogram) were not significantly different than for the sick infants of nondiabetic mothers. On the other hand, values for total 17-OHCS expressed as micrograms per milliliter were approximately twice as high in sick infants of diabetic mothers as in well
I
Misc
I Infants of diabetic mothers
I
151 77.6 80.3 41.9 0.54
Well
t
ROS
230 73.0
201 61.3
146.9 45.7 0.70
205.4 59.9 1.11
or respiratory distressed infants of nondiabetic mothers, a relationship similar to that found for total 17-OHCS expressed as micrograms per kilogram. Although measurements of creatinine excretion were made primarily as a guide to completeness of collection, the data obtained were used as an additional parameter for evaluation of changes in the 17-OHCS output. Fig. 7 shows that the mean output of 17-OHCS expressed as micrograms per milligram creatinine for 1 to 8 days also is approximately twice as high in respiratory distressed infants of diabetic mothers (5.33) as in welt infants of diabetic mothers (3.36), well infants of nondiabetic mothers (2.44), or respiratory distressed infants of nondiabetic mothers (3.14). DISCUSSION The data indicate a significantly higher corticosteroid output in distressed premature
6 6 2 Smith, Reardon, and Field
May 1964
]
]
9
m J=
ioo
"-
75
E
50
Number
WELL INFANTS NON-DIABETIC MOTHERS SICK INFANTS, ROS NON- DIABETIC MOTHERS WELL INFANTS DIABETIC MOTHERS SICK tNFANT$, RDS DIABETIC MOTHERE
M.d.
14
2 :> I--3
19 Days
27
17
13 4-8
9
+_ S E .
16
Days
Fig. 5. Total urinary 17-OHCS in premature infants of nondiabetic and diabetic mothers, expressed as micrograms per kilogram body weight per 24 hours. Mean values for 1 to 3 days compared to 4 to 8 days of life9 Explanation same as for Fig. 3. infants of diabetic mothers as compared with distressed infants of nondiabetic mothers. An explanation for the differences in steroid excretion between the two groups of infants is not readily apparent. Possibilities include the influence of the diabetic state of one group of mothers on cortisol production and metabolism in the newborn in addition to differences in degree of prematurity of infants, mode of delivery, renal function, and the degree of steroid binding of plasma cortisol, which would alter the quantity of cortisol metabolites presented f o r excretion by the kidney. The question of hyperadrenocorticism in infants of diabetic mothers has been raised previously?, a, 6 Farquhar ~ particularly emphasized the possibility of hyperadrenocorticism in the fetus as a cause of certain morphologic features observed in the newborn infant of a diabetic mother. By using the urinary excretion of formaldehydrogenic steroids as an index of adrenocortical activity, Farquhar recorded elevated values in infants of diabetic mothers during the first 3
days of life. Of interest in this connection is the report by Baird and Bush 19 of no significant difference in the cortisol and cortisone content of amniotic fluid obtained from diabetic women as compared to that of normal women. 'In this preliminary study, groups of infants were not closely matched as to degree of immaturity. Infants of diabetic mothers generally are not delivered at very early gestational ages, as in the case of m a n y premature infants of nondiabetic mothers. Also, the former are notoriously large for their degree of immaturity. Expression of results in terms of body weight and surface area was considered the best approach to the problem until additional data became available. Differences in 17-OHCS content of cord blood between infants delivered by the vaginal route and by cesarean section have been documented. 2~ In this study, we have not attempted to correlate the infant's mode of delivery with the urinary 17-OHCS excretion rates. No data concerning renal function
Volume 64 Number 5
17-Hydroxycorticosteroid excretion in the premature
9
WELL INFANTS NON-O)AeETIC
t-~
SICK INFANTS, ROB NON-DIABETIC MOTHERS
MOTHERS
250
9
WELL INFANTS DIABETIC MOTHERS
[]
SICK INFANTS, RDS DIABETIC MOTHERS
66 3
4
/ /
-%
3 ----[]
200 .1:
150 E
I00
50
I
2
:5
4
5
6
7
8
Fig. 6. Total urinary 17-OHCS in premature infants of nondiabetic and diabetic mothers, expressed as micrograms per 24 hours. Mean daily changes during first 8 days of life. Figures above each point represent numbers of infants studied on each day.
t
.'A.
~. S : "
D
WELL
INFANT8 NON-DIABETIC
MOTHERB
]
DICK
INFANTE NON-DIABETIC
MOTNBNB
WELL
INFANTE DIABETIC MOTH[RE
lICK
INFANTE DIABETIC
MOTHERB
C
(.3 7
E ...
6
:
5
E 4
Number,
14 27 19 27 I-3 Days
17
22 .4-8
9 16 Days
Fig. 7. Total urinary 17-OHCS in premature infants of nondiabetic and diabetic mothers, expressed as micrograms per milligram daily creatinine excretion. Explanation same as for Fig. 3.
664
Smith, Reardon, and Field
or steroid binding of plasma cortisol are available for our groups of infants. Our findings concerning the influence of stress from the respiratory distress syndrome on the urinary excretion of 17-hydroxycorticosteroids in premature infants are contrary to those outlined by Hillman. s He found significantly higher excretion values in premature infants with respiratory distress of nondiabetic mothers than in healthy infants. We have no reasonable explanation for this descrepancy. Differences in clinical classification a n d / o r treatment may be related; the method for assay of urinary 17OHCS was similar, but not identical, in the two studies. Several studies in which highly specific methods for assessing adrenocortical function in infants were used have been reported in recent years? 1-2~ Although differences have been noted between newborn infants and adults, TM 23 limited studies have shown no significant differences between full-term and premature infants 24 or between normal full-term infants and infants of diabetic mothersl2~ Since the level of urinary excretion of 17-OHCS is not necessarily correlated with adrenal function, interpretation of our results for urinary 17-OHCS in various groups of infants in terms of differences in adrenal function is not warranted. It is hoped that the data on urinary 17-OHCS will provide valuable comparative information and suggest avenues of further investigation. Since our study was initiated, several reports have appeared describing significant alterations in the patterns of steroid excretion in the neonate. One prominent finding is the reduction in glucuronide conjugation12, 23.24, 27 indicated by a high percentage of free 17-OHCS and consistent with the recognized immaturity of the enzyme systems involved in glucuronide conjugation in the newborn infant. Another alteration in the pattern of excretion is the presence of highly polar corticosteroids such as 6-/3hydroxycortisol, which accounts for a large proportion o f the unconjugated corticosteroidsY3, 26 Extraction of these polar cor-
May 1964
tisol metabolites from urine is incomplete with chloroform; a more polar solvent such as ethyl acetate i s required. 26' 28 Experiments involving ethyl acetate extraction of urine from premature infants are in progress. 'The possibility that the relative proportion of individual urinary metabolites comprising the total may change significantly during the first few days of life has been suggested by Ulstrom. 12 Likewise, the possibility exists that the relative proportion may change during the last few weeks of fetal life, and, therefore, the excretory pattern of corticosteroids may be different in premature infants from that in full-term infants and may be altered by diabetes in the mother or by unknown etiologic factors, such as those associated with the respiratory distress syndrome. Further studies of changes in individual cortisol metabolites comprising the steroid excretion pattern in newborn infants are planned. SUMMARY
Urinary total chloroform-extractable 17OHCS (Porter-Silber chromogens) have been measured serially during the first week of life in 41 premature infants born to nondiabetic and diabetic mothers. Well premature infants of nondiabetic mothers excreted significantly lower quantities of total 17-OHCS than did the normal full-term infants studied by Ulstrom. 12 With essentially similar clinical findings and management of the respiratory distress syndrome, infants of nondiabetic mothers showed a slight decrease, and infants of diabetic mothers showed a twofold increase, in total 17OHCS excretion expressed as micrograms per kilogram per 24 hours, compared to well infants of nondiabetic mothers. A similar twofold increase obtained when the data were expressed as micrograms per square meter surface area, micrograms per milliliter urine volume, or micrograms per milligrams creatinine excreted. We express our gratitude for valuable assistance provided by Dr. Norman Kendall and Dr. Herta Schrom of the attending staff, and Miss
Volume 64 Number 5
17-Hydroxycorticosteroid excretion in the premature
Lois Berglund and Miss Pauline King of the Nursing Staff of Temple University Hospital. Technical help in performing the chemical analyses, provided by Miss Carolyn Klabunde and Mrs. Felicidad Arguelles of The Children's Orthopedic Hospital and Medical Center, is greatly appreciated.
REFERENCES
1. BjSrklund, S. I.: Infants of diabetic mothers with special reference to neonatal adrenocorticoids and tests using adrenocorticotrophic hormones, Acta Endocrinol. 15: 25, 1954. 2. Hoet, J. P.: Adrenocortical function in infants of diabetic mothers, Cold Spring Harbor Symposium on Quantitative Biology 19: 182, 1954. 3. Farquhar, J. W.: The possible influence of hyperadrenocorticism on the fetus of the diabetic woman, Arch. Dis. Childhood 31: 483, I956. 4. Salmi, T., Pekkarinen, A., and Heikkila, S.: The excretion of total 17-hydroxycorticostefolds into the urine of newborn prematures and infant boys during the first two days and the first month, Ann. Pediat. Fenniae 3: 70, 1957. 5. Matson, C. F., and Longwell, B. B.: The excretion of neutral lipid-soluble reducing substances by infants, J. Clin. Endocrinol. 9: 646, 1949. 6. Venning, E. I'i., Randall, J. P., and GySrgy, P.: Excretion of glucocorticoids in the newborn, Endocrinology 45: 430, 1949. 7. Read, C. H., Vennlng, E. H., and Ripstein, M. P.: Adrenocortical function in newly born infants, J. Clin. Endocrinol. 10: 845, 1950. 8. Hillman, D. A.: Urinary 17-hydroxycorticosteroid excretion in newborn infants with respiratory distress syndrome, A. M. A. J. Dis. Child. 102: 569, 1961. 9. Cranny, R. L., and Cranny, C. L.: The urinary excretion of Porter-Silber chromogens by premature infants, A. M. A. J. Dis. Child. 95: 401, 1958. 10. Cranny, R. L., and Cranny, C. L.: The urinary excretion of 17-hydroxycorticosteroids by premature infants, A. M. A. J. Dis. Child. 99: 344, 1960. 11. Reddy, W. J.: Modification of the ReddyJenkins-Thorn method for the estimation of 17-hydroxycorticoids in urine, Metabolism 3: 489, 1954. 12. Ulstrom, R. A., Colle, E., Burley, J., Gunville, R.: Adrenoeortical steroid metabolism in newborn infants. I. Urinary excretion of free and conjugated 17-hydroxycorticosteroids in normal full-term infants, J. Clin. Endocrinol. 20: 1066, 1960. 13. Hepner, R., and Lubchenco, L.: A method for continuous urine and stool collection in young infants, Pediatrics 26: 828, 1960.
6 65
14. Field, S. H., Reardon, H. S., and Smith, E. K.: Urinary constituents of infants of diabetic and non-diabetic mothers. II. Electrolyte and water excretion in newborn infants (in preparation). 15. Glenn, E. M., and Nelson, D. H.: Chemical method for the determination of 17-hydroxycorticosteroids and 17-ketosteroids in urine following hydrolysis with fl-glucuronidase, J. Clin. Endocrinol. 13: 91I, 1953. 16. Allen, W. M.: A simple method for analyzing complicated absorption curves, of use in the colorimetric determination of urinary steroids, J. Clin. Endocrinol. i0: 71, 1950. 17. Clark, L. C., Jr., and Thompson, H. L.: Determination of creatine and creatinine in urine, Anal. Chem. 21: 1218, 1949. 18. Dixon, W. J., and Massey, F. J., Jr.: Introduction to statistical analysis, ed. 2, New York, 1957, McGraw-Hill Book Company, Inc., p. 119. 19. Baird, C. W., and Bush, I. E.: Cortisol and cortisone content of amniotic fluid from diabetic and non-diabetic women, Acta Endocrinot. 34: 97, i960. 20. Migeon, C. J., Nicolopoulos, D., and Cornblath, M.: Concentrations of 17-hydroxycorticosteroids in the blood of diabetic mothers and in blood from the umbilical cords of their offspring at the time of delivery, Pediatrics 25: 605, 1960. 21. Bongiovanni, A. M., Eberlein, W. R., Westphal, M., and Boggs, T.: Prolonged turnover rate of hydrocortisone in the newborn infant, J. Clin. Endocrinol. 18: 1127, 1958. 22. Cranny, R. L., Kirschvink, J. F., and Kelley, V. C.: The half-life of hydrocortisone in normal newborn infants, A. M. A. J. Dis. Child. 99: 437, 1960. 23. ReynoIds, J. W., Colle, E., and Ulstrom, R. A.: Adrenocortical steroid metabolism in newborn infants. V. Physiologic disposition of exogenous cortisol loads in the early neonatal period, J. Clin. Endocrinol. 22: 245, 1962. 24. Kenny, F. M., Malvaux, P., and Migeon, C. J.: Cortisol production rate in newborn babies, older infants and children, Pediatrics 31: 360, 1963. 25. Aarskog, D.: Cortisol production rate in newborn infants of diabetic mothers, J. PEOIAT. 62: 807, 1963. 26. Ulstrom, R. A., Colle, E., Burley, J., and Gunville, R.: Adrenocortical steroid metabolism in newborn infants. II. Urinary excretion of 6/3-hydroxycortisol and other polar metabolites, J. Clin. Endocrinol. 20: 1080, 1960. 27. Migeon, C. J.: Cortisol production and metabolism in the neonate, J. PEDIAT. 55: 280, 1959. 28. Frantz, A. G., Katz, F. N., and Jailer, J. W.: 6~-hydroxycortisol and other polar corticosteroids, measurement and significance in human urine, J. Clin. Endocrinol. 21: 1290, 1961.