Exchangeable Sodium and Aldosterone Secretion in Children with Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency

Home , Pregnanetriolone

Pediat. Res. 4: 145-156 (1970) Aldosterone 21-hydroxylase congenital 11-ketopregnanetriol adrenal pregnanetriol hyperplasia sodium metabolism Exchangeable Sodium and Aldosterone Secretion in Children with Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency

B. LORAS, F. HAOUR and J. BERTRAND[34]

I.N.S.E.R.M., Unitt de Recherches Endocriniemes et Mttaboliques chez l'Enfant, HGpital Debrousse, Lyon, France

Extract Aldosterone secretion rates (ASR) and exchangeable body sodium (Na,) have been measured simultaneously in 25 cases of congenital adrenal hyperplasia (CAH) with and without salt loss, both during normal and during low salt intake. The degree of 21 -hydroxylase deficiency was assessed from the cortisol secretion rate (CSR) and urinary excretion of pregnanetriol and 11-ketopregnanetriol. In cases with salt loss, the sodium equilibrium was unstable, even with optimum treatment and increased dietary salt intake. Clinical evidence of salt-losing crisis appeared when 15-25% of Na, was lost. In these subjects, a parallel decrease in ASR and CSR usually occurred. The ASR were somewhat elevated in the nonsalt-losing form.

Speculation A salt-excreting factor is probably present in CAH, even in the nonsalt-losing form, since the ASR is raised, while the Na, remains normal. Salt loss due to 21-hydroxylase deficiency decreases with age. This appears to be due to modifications in the distribution of body sodium and to an absolute increase in the dietary salt intake rather than to modifications in adrenal secretion.

Introduction between aldosterone production and disordered sodium balance. Sodium levels in plasma and sodium About one-third of all cases of congenital adrenal excretion in urine, as well as the values for exchange- hyperplasia (CAH) due to 21-hydroxylase deficiency able sodium, have been measured in different situa- present with a salt-losing syndrome 1321. This may tions, namely, during normal dietary sodium intake, become evident by day 7-day 12 of life in the most and during the administration of a low salt regime, severe form of the disorder, may occur at a later age, The exchangeable sodium (Na,) represents the stock or, in some cases, may only arise during a situation of of sodium within the organism that can readily be stress. mobilized to maintain electrolytic homeostasis. In the Although it has been shown in the salt-losing form adult, about 30% of the total body sodium is fixed of CAH that the secretion of aldosterone is deficient, within the integral structure of bone and is not avail- there has been less investigation of concomitant chan- able for this purpose. In the child, however, almost all ges in body sodium content. The present study was the body sodium is exchangeable [17]. The Na, value undertaken in the hope of delineating the interrelation in the adult remains fairly constant at about 146 LORAS, HAOUR and BERTRAND

40 mEqjkg body weight [2]. The value is higher in steroid therapy was stopped for from 3 days to 1 month the child, particularly in the young child. Values prior to the investigations. The salt-losing cases were obtained for Na, in healthy newborn infants are of maintained in good general health during this period the order of 80-90 mEq/kg body weight, but by 6 by appropriate supplementation of their diet with salt. months of age a rapid fall has occurred and the values range from 55 to 65 mEq/kg. Thereafter, the Sodium Na, values in healthy children show a much more The sodium content of the diet was calculated from gradual fall until they reach adult levels, at about age standard tables [27] for the older children and, in the 12 or 13 [ll, 12, 18,261 (table I). case of the infants, the sodium content of the milk was calculated from the values given by the manufacturer. Table I. Values for exchangeable sodium in normal In a number of cases, these values were verified by subjects related to age1 direct analysis of the dietary intake. Sodium levels in plasma and urine were measured

Age Nae, mEq/kg by flame photometry. Tritiated aldosterone (2-3 pCi) and 1 pCi/kg of 24 Newborns 80-90 Na were injected simultaneously. Following 24 h of 6 months 55-65 equilibration, during which time the urine was collect- 2-5 yr 50-60 ed for measurement of the secretion rate of aldosterone 5-10 yr 45-55 and the excretion of labeled sodium, the specific activ- 12 yr and more = 40 ity of the plasma sodium and the value for the exchangeable sodium were measured by the isotope dilu- 1 From references 11, 12, 18, 26. tion method [18]. The exchangeable sodium was calculated by the Ideally, one would wish to measure the Na, in milli- formula: equivalents per kilogram of lean body mass since adi- 24Na injected — 24Na excreted Na, = pose tissue contains very little sodium. This circum- specific activity of plasma Na stance sometimes makes it difficult to define the normal limits with precision and it is certainly normal for an where: specific activity = obese subject to have an Na, value per kilogram total 24Na (in pCi) per 100 ml plasma body weight that is less than that of lean subjects. 23Na (in mEq) per 100 ml plasma In most of our cases, in addition to measuring aldo- 23Na being naturally occurring sodium. sterone secretion, we have also measured cortisol secretion, and the excretion of pregnanetriol in the urine. Aldosterone In this way, it has been possible to assess the degree of The secretion rate of aldosterone was measured by enzymatic block and to study the correlation between the double isotope derivative assay [24] modified as a deficit in cortisol and aldosterone production and a follows: deficit in body sodium. a) Aldosterone, liberated by hydrolysis at pH 1, was chromatographed in the E2B system (isooctane-terti- ary butanol-water, 100:50:90) for 20-22 h. If the Clinical Data and Methodology extract remained pigmented, it was rechromato- graphed in the Bush 5 system (benzene-methanol- Twenty-five children with congenital adrenal hyper- water, 100:50:50) for 7 h. plasia (CAH) due to deficiency of 2I-hydroxylase were b) Following acetylation, the diacetate was chroma- studied. The ages of the subjects varied from 6 days tographed twice in the system cyclohexane—benzene— to 14 years, and the clinical features showed all degrees methanol-water, 4:4:4:1, and once in the system of severity of the defect. In some cases, the salt loss cyclohexane-dioxan-methanol-water, 4:4:2:1. The presented as a crisis; in others, the clinical features did initial chromatography was repeated to secure the not arouse suspicion in the minds of the parents until removal of excess 14C. the children had reached 3-5 years of age. c) The oxidation product was chromatographed The clinical data are summarized in table 11. In twice, once in each of the two systems mentioned above. this table, the degree of salt loss has been estimated on The purity of the labeled aldosterone that was in- clinical criteria only. jected was checked by means of the E2 B system. Eight children showing salt loss and two children WELDON et al. [31] found the ASR to be 72f29.5 with the benign form of CAH had been studied prior ,ug/24 h for 22 normal subjects whose ages ranged from to the institution of any therapy. In the other children, 8 days to 1 year, the salt intake being normal. In 17 Exchangeable sodium and aldosterone secretion in children 147

Table II. Congenital adrenal hyperplasia: clinical data Case nos. Age Sex Pseudoherma- Clinical Observations uhrodism salt loss1 Prader 1,CS F Type I11 0 Two affected siblings 2,JC 57i2 yr M 0 3,SM 1O3/1zYr F Type IV 0 'Salt-craving' quoted by family 4,JMC 102/1, Yr M 0 Brother of MC, case no. 6 5,PD F Type I1 0 One affected sibling 6,MC F Type I1 0 Sister ofJMC, case no. 4 7, DC 6 days F Type I1 First signs of salt loss when 10 days old; two affected siblings 8,NL 8 days Type I11 First signs of salt loss when 15 days old; Sister of PL, case no. 12 9,HC 10 days F Type IV First signs of salt loss when 8 days old F Type I11 First signs when 10 days old; 10, OM 11 days M crisis at 1 month Breast fed for 8 days; first signs when 11, SD 18 days 8 days old; crisis at 15 days M Breast fed for 15 days; first signs when 12, PL 6 wk 15 days old; crisis at 1 month and 3'3/12 during episode of otitis media; Doca stopped at 2j/,, 13, MD 9 wk F Type I11 Doca stopped at 7 months 14, SF 4 months M First signs when 10 days old; crisis at 3 months during an infectious episode 15, PC 4 months F Type I11 Breast fed for 2 months; first signs of 3 salt loss very early; Doca stopped at 2 /1, 16, RB 11 months M First signs when 12 days old; crisis at 20 days during episode of otitis media 17, DJ SV.yr F Type I11 Crisis at 1 and 2 months; crisis at 36/1, during a low salt diet trial; Doca stopped at 42/1, 18, CF 47i2 yr F Type IV First signs when 8 days old; crisis at 2 months, 3 months, 26/,, during 6 infectious episodes; Doca stopped at 4 /1, 4 19, PP 5V12yr M Crisis at 2 /12 during infectious episode; Doca stopped at 4 20, JI 5'/,« vr M Crisis at 1 month, 9 months, 210/1,, and y 3 1, without known precipitating factor; one affected sibling 9 10 21, AA TypeV Doca stopped at 3 /12; crisis at 4 /1, without known precipitating factor 1° 22, MC F Type IV Doca stopped at 6 /,, 23, AG S^yr F Type IV One affected sibling; first signs of salt loss when 1 month old; Doca and salt stopped at 4 24, BB 9yr M First signs at 1 month; crisis at 3 and 6 months; Doca stopped at 7; crisis during a low salt diet trial 25, GR 10 yr M First signs when 15 days old; crisis durinc low salt diet 1 0: no clinical evidence of salt loss; + : slight salt loss as witnessed by relatively late presentation of the salt loss, absence of recurrence of salt loss during treatment on the occasion of illness or other stress, a low require- ment of deoxycorticosterone acetate (Doca) and of salt supplementation, and tolerance of a low salt regime, either for a few days, or for longer periods with advancing age; + + : tendency to severe salt loss. 148 LORAS, HAOUR and BERTRAND

normal subjects, in the age range of 1-15 years, the value was 91130.4 ,ug/24 h. On a low sodium diet, the mean percentage increase of ASR was 319% in infants and children. Some of the results contributing to these values were obtained in this laboratory. 20

Cortisol Cortisol secretion rates were determined as follows: 3 d

Two-to-four microcuries of 1,2 H cortisol were in- te jected and estimates were made on a one-fifth portion e

of the 24-h urine output from which free steroids had excr a been removed by a chloroform extraction. Glucosid- M uronate conjugates were hydrolyzed by means of p- -0 glucuronidase (Ketodase), and the resulting free steroids extracted with chloroform. After washing with Normal children CAH with CAH without 0.1 N NaOH, the extracts were purified by Florisil (25) salt loss salt loss column chromatography with collection of the 25 % methanol fraction in chloroform. The fraction was Fig. 1. Percentage of 2Wa excreted in the 24-h urine chromatographed on paper in the Bush system (ben- during normal • and low salt 0 regimes. zene-methanol-water, 100:55:45) for 20 h and in the E2B system for 8 h. The radioactivity was measured in the three major metabolites THF, a110 THE, and 400- ASR |jg/24h THE [33] and quantitative estimation of these compounds was achieved by the PORTER-SILBER [19] tech- 300- nique. In some cases of CAH, THE remained contaminated 200- even at this stage and gave a specific activity which was less than that found with the other two metabolites. 100 - In such cases, the secretion rate was calculated from ^^ fill the specific activities of THF and a110 THF only. J II i...iiin l The mean normal value obtained in this laboratory NormaEll l + + + salt loss is 11 1*3 mg/m2/24 h for all ages, save the neonatal children CAH period [5]. Under ACTH stimulation (25 mg per- 2 fused Over 8 h, the values rise t' 3040 mg/m /24 h. Fig. 2. Aldosterone secretion rates (pg/24 h) in normal In all cases, when secretion rates were being measur- children and in cases of CAH less than 1 year old, on ed, the total radioactivity in the urine was estimated to normal . and low salt D regimes. shaded zone, ASR verify that there had not been an appreciable loss of ofsD) for normal infants aged 8 days-l year on urine. a normal salt intake (30).

Pregnanetriol and ll-Ketopregnanetriol Pregnanetriol and Il-ketopregnanetriol were meas- 500" ASR ug'24h ured by gas-liquid chromatography (GLC). Following hydrolysis with B-glucuronidase, steroids 400- were extracted with toluene and separated by paper chromatography with the system hexane-benzene- methanol-water, 70:30:80:20 for 12 h. Pregnanetriol 300 - and 11-ketopregnanetriol were located from reference standards run in parallel and were eluted. The results 200- obtained following GLC were not corrected for losses. n 100 - Fig. 3. Aldosterone secretion rates (pg/24 h) in normal if 11l 1 children and in cases of CAH aged 2-14 years, on I •Illillh 1 normal m and low salt • regimes. Shaded zone, ASR Normal + + + 0 salt loss (meanks~) for normal subjects aged 1-15 years on a children CAH normal salt intake (31). Exchangeable sodium and aldosterone secretion in children 149

The 17-ketosteroids and the total ketogenic corti- reduction in sodium intake; clinically she had a mild coids were measured by the technique of APPLEBY et form, and was able to double her ASR under a low al. [l] and FEW 1151. Na diet. In two infants, reduction of the salt supplements did not give rise to any significant increase in the ASR and this regimen was poorly tolerated (case Results nos. 15 and 16). The ASR values before and after the Results are given in table 111. Abnormalities of sodium salt reduction were 38 and 36 ,ug/24 h in one case and balance in CAH were demonstrated by measurement 44 and 56 pg/24 h in the other. of the percentage of labeled sodium excreted in the In some of the older salt losers (case nos. 18 and 21), urine of patients (24-h period) (fig. 1). Normal sub- the base-line ASR values (44 and 106 ,ug/24 h) were jects excreted 3-7 % sodium on normal salt intake and normal; however, during salt restriction, their ASR less than 1 % when on a low salt intake [26]. Patients values were frankly subnormal. In other children, the with CAH who had no salt loss responded to a low salt base-line ASR values were low and there was little or diet by a reduction in their sodium excretion and by a no response to the low salt diet (case nos. 17, 19, 20, reduction in the percentage of 24Na excreted in the 24, and 25). 24-h urines. Salt losers showed a variable excretion of Children with CAH who were not salt losers showed 24Na per 24 h, from 1.8 to 25%. In the majority of ASR values that were normal or above normal (124— these subjects, this value does not decrease during ad- 258 ,ug/24 h) and invariably responded to a low salt ministration of a low sodium diet. regime (188-455 ,ug/24 h) (figs. 2 and 3). In the salt losers, the sodium levels in plasma were frequently low, even when salt supplements were given Pregnanetriol and 11-Ketofiregnanetriol (case nos.10, 11, 15, and 21). Low plasma sodium The level of pregnanetriol excreted in the urine was levels also may be found in the simple form of the raised in all cases and was further increased following disorder, on a low salt regime or on a normal salt ACTH administration. In the youngest subjects, 11- intake (case nos. 13, 21, and 22), and lowered plasma ketopregnanetriol was excreted in greater quantity sodium may even be found in cases who do not show than pregnanetriol itself. This was noted in only one clinical evidence of salt loss (case nos. 3 and 5). of the older subjects (case no.25) who remained a severe salt loser. Exchangeable Sodium 17-Ketosteroids and total ketogenic corticoids were Patients without salt loss had a Na, value that was excreted in considerable amounts by all subjects. In normal under conditions of normal sodium intake, two salt losers in whom treatment had been stopped with little change during administration of a low salt for only 3 and 5 days, respectively (case nos. 23 and 24) diet. In normal subjects, a reduction of 6-8% -is ob- at the time of the first estimations, the values were al- served on a low salt regime [26]. These children with ready extremely high and near their maximum. On CAH on a low salt regime, however, although they do clinical grounds, however, case no. 23 did not have a not show salt loss frequently show a loss of weight severe defect. Almost all the investigations were carried (case nos. 3, 4, and 6) and a reduction in their plasma out when the adrenal glands of the patients had return- sodium levels (case nos. 3, 5, and 6). ed to what we assumed was their full degree of activity, In all salt losers under 1 year of age, the Na, value although, due to the passage of time, this might not was below normal whether on normal sodium intake necessarily be the degree of activity prior to treatment. or receiving sodium supplements. A low Na, was also found in two older salt losers (case nos. 20 and 24). In Cortisol Secretion Rates (CSR) the other salt losers, the Na, values were normal or Children with the salt-losing form of CAH showed low normal, but these values were likely to have been cortisol secretion rates that were widely scattered, influenced because they received salt supplements. with values ranging from immeasurably low to sub- Typical salt losers cannot conserve sodium during salt normal. The response to ACTH was equally variable, restriction; the exceptions were patients with a mild being none (case nos. 12, 16, and 20), inadequate (case form of the disorder (case nos. 13 and 22). A clinically no. 23), or even paradoxical with a lowered CSR follow- evident crisis state of salt losing corresponds to a reduc- ing stimulation (case nos. 24 and 25). The correlation tion in the pool of exchangeable sodium of 15-20%. between a low cortisol secretion rate and a high degree of salt loss is fairly good. Aldosterone Secretion Rates (ASR) Cases of CAH without salt loss showed CSR values The ASR in salt-losing infants (under 1 year of age) that were low or normal. They showed a response to were all low, ranging from 9 to 44 pg/24 h. Only one ACTH with an increase in cortisol secretion, but this infant (case no. 13) was able to support a significant response was invariably less than normal. Table III. Congenital adrenal hyperplasia: biological data en Cortisol A 0 secretion a rate Urinary an Exchange- V M X &x X 0 "So

X ; sodium able sodium V -*i 0 •* > •* : i l e 1 - 2 8 2 P. x •a , . f h h m n y sa d , 4 4 g o l ,

0^ a % -s m g e mg/ mg/ 1 nos 3. •* mg/2 , , , an e k

_ 9 10 days 3.37 Never treated 15-20 15 -20 15-20 -20 15-20 15 1.4 13.1 18.7 6 months 5.82 Stopped for 15 days Norma1+ 25 a1 0.2 4.7 2.9 1+25

10 11 days 3.56 Never treated 17 1 4.0 134 210 60.0 9 4.0 14.4 ACTH 68 1.0 4.8 7.8 29.7 W |xj 3 months 4.80 Treated Normal+34 95 24.5 153 381 78.6 0.1 2.9 o 18 months 10.9 Treated Normal+34 25 3.0 121 503 49.0 1.4 5.6 &T>r + + 3 aq 10.2 Stopped for 15 days Normal+34 16 16.6 4.9 5.2 43.1 a 11 18 days 3.3 Never treated 65-70 130 19 2.3 4.9 8.0 19.6 cr + + 3.3 65-70 2.3 2.3 8.9 2.3 rT 3.3 ACTH 65-70 126 3.7 4.1 8.2 27.9 o 8£"' 12 6 weeks 3.55 Never treated 85 143 26 0.6 2.4 2.4 17.0 3 ++ ACTH 85 134 not measurable 5.2 26.0 13 9 weeks 5.3 Never treated 13 134 4.8 17.2 1.4 1.4 2.3 7.5 3 + 13 9 3.6 132 290 54.7 39 2.1 8.6 aSL- 5.4 4-5 0.7 294 5.4 96 5.4 3.7 ACTH 13 2.2 3.8 3.7 13.6 1* 3 14 4 months 3.74 Stopped for 10 days 85 68 23.5 144 244 62.5 3 1.5 6.3 3.7 15.3 3 3.40 ACTH 85 1.6 8.1 5.9 42.2 a n 6 months 5.25 Treated 85 55 19.0 140 325 63.9 1.3 2.1 o

15 4 months 4.9 Never treated 85 60 22.0 136 286 58.4 85 8.2 2.2 7.6 29.7 5' + + 4.7 50 50 20.0 120 228 46.6 36 4.5 17.0 B" 4.2 70 2.0 7.3 4.0 16.0 3 16 11 months 6.4 Stopped for 25 days Normal+72 1.2 3.5 2.9 12.1 g- ++ 6.4 Doca, ACTH Normal+72 2.0 6.1 5.1 26.6 n> 6.3 Doca 75 68 9.4 407 64.6 6.3 Doca stopped for 7 days 75 61 16.0 139 384 61.0 44 4.2 22.9 6.3 40 39 10.0 120 314 49.8 56 6.3 13.4 5.0 34.3 17 37l2 10.9 Stopped for 15 days3 Norma1+54 80 14.0 133 601 56.0 1+ 1.7 18.2 ++ 10.8 Normal+ 54 10.1 4.0 3.1 18.5 3 18 4%, 13.35 Stopped for 8 days Normalf85 149 19.0 146 694 52.0 60 4.1 18.7 ++ 12.35 15-25 15 2.9 143 637 51.6 15-25 15-25 8.4 20.2 174.5 , , 19 57u 13.6 Stopped for 15 days3 Normal 94 131 712 51.0 44 61.1 6.4 15.3 87.0 i—• ++ 13.4 6-10 119 33 40.7 17.6 13.9 80.9 Table III. (Continued)

13 Cortisol j . a Ol u o secretion •a 43 NO ( Urinary rate •* /lit Exchange- §> xT CM cr si u bo sodium W able sodium di i : l

e a fi 2 i - x 2 , . f h h m

y CM , sa

d 2 £i 4 4 g o l h h , a m

1 ^ 17-Ketost 17-Ketoge 11-Ketoo triol clinica loss Therap Sodiu mEq/2 mEq/k mg/2 Pregnane triol mg/2 corticoid Cas degre Wt,k intake mEq/2 Plasm sodium qE Ag < s I 20 5'/l! 16.9 Treated 140 39 5.0 140 758 44.6 7.6 25.6 + + 16.9 Treated 140 187 140 140 721 43.0 4.5 13.7 16.3 Doca stopped for 3 days 140 216 1 5.3 140 7.9 28.8 16.3 Doca stopped for 3 days 210 182 26.0 138 683 43.0 3.8 15.6 16.2 Stopped for 6 days 210 200 125 2.5 3.5 88.7 39.2 10 174.5 16.0 Stopped for 6 days 90 125 74 20.5 8.9 7.5 40.7 15.9 Doca, ACTH 210 210 2.6 3.6 43.6 37.3 210 128.0 o 3 21 6u/i2 21.3 Stopped for 11 days Normal+50 4.8 5.7 36.9 17.5 29.0 100.8 V + + 21.3 Normal+50 206 126 100 27.3 15.5 25.6 73.9 21.7 Normal 167 14.0 133 1,050 48.4 106 36.3 22.0 25.5 — o 21.0 6-10 29 2.3 124 887 41.8 136 29.1 19.6 29.2 110.0 § 21.0 Treated for 4 days Normal 0.9 — 5.0 6.7 3 22 79/i. 25.9 Stopped for 9 days Normal+35 118 1.8 138 1,100 42.5 100 27.3 5.5 9.3 67.4 fa- + 25.5 ACTH Normal+35 48.7 5.0 14.0 127.0 w 25.7 Normal 167 14.0 137 1,083 42.1 112 28.1 — 10.7 66.5 H 24.8 6-10 10 0.7 128 1,083 43.7 234 35.6 5.3 9.7 77.8 Treated for 5 days Normal 1.2 0.3 — — D 2 3 23 8 /12 29.7 Stopped for 5 days Normal. 107 7.3 145 1,412 48.0 99 13.4 79.1 + 6-10 6.6 6.6 30.2 24.1 10 78.0 6-10 10 2.9 137 1,217 40.7 188 10 170.0 ACTH Normal 26.4 26.4 78.3 29.6 20.6 127.0 24 9 26.6 Stopped for 3 days3 Normal 109 12.0 131 923 34.7 54 67.1 27.3 19.0 195.0 + + Normal 19.3 20.9 31.0 241.0 ACTH Normal 11.7 13.7 76.8 32.2 44.5 400.0 Treated for 15 days Normal 0.2 — 0.7 3.3 25 10 40.4 Stopped for 12 days3 Normal 209 2.3 140 1,611 39.9 35 50.7 73.3 25.4 169.1 ++ 40.5 Normal 14.8 12.6 60.4 83.4 26.0 171.0 39.6 40 6 0.1 134 1,359 34.0 40 8.9 31.4 39.9 ACTH Normal+68 4.1 3.6 23.7 142.0 1 See table 11, footnote 1 for description of clinical salt loss. 2 Percentage of 24Na excreted in the urine in 24 h. 3 Interval between cessation of treatment and the start of the investigation. Exchangeable sodium and aldosterone secretion in children 153

Discussion duction, demonstrable on restricting salt. Although the Na, was below normal and the plasma sodium was Pregnanetriol and 11-Ketofiregnanetriol on the low side, the clinical condition at the time was The predominance of II-ketopregnanetriol over satisfactory and the excretion of sodium in the urine pregnanetriol in infants under 1 year of age [14, 151 was less than the dietary intake. is probably a manifestation of a more general phe- In the newborns (case nos. 8, 9, and lo), and in the nomenon that is also found in the 17-ketosteroids of other subjects during salt restriction (case nos. 15, 16, children with CAH [16, 171. 21, and 25), clinical signs of salt loss appeared. In all It was of interest to note that this infantile pattern of these cases, the loss of sodium reached or surpassed of adrenal secretion persisted in case no. 25 and, to a 15-20% of the Na, value of the child in optimum con- limited extent. in case no. 20 who showed an increased dition. Salt loss in the urine is indicated by the insuffi- proportion of 11-ketopregnanetriol during stimulation cient reduction in urinary 24Na. In many cases, the with ACTH and during salt restriction. It may be institution of a low salt diet led to increased excretion significant that these subjects were two of the three of total ketogenic corticoid (case nos. 16, 18, 21, 22, and children in the series who remained severe salt losers 23), and the increased excretion of pregnanetriol itself despite the passage of time. was indicated in case no. 22. Thus, a low salt intake, In general, the older patients continued to excrete even though it does not lead to a clinically evident salt- 11 -ketopregnanetriol, but in proportionately smaller losing crisis, may still create a situation of stress for amounts. FINKELSTEIN [16] found measurable excre- these subjects and provoke an increased secretion of tion of 11-ketopregnanetriol in 44 of 50 cases of CAH, endogenous ACTH. In contrast, in case nos. 20 and 25 and the 6 subjects not excreting this compound pro- in whom the introduction of a low salt regime had led bably sutiered from the form of CAH due to deficiency to a frank salt-losing crisis, the steroid excretion was of 11 p-hydroxylase. 11-Ketopregnanetriol is not ex- diminished. A probable interpretation of this result is creted by normal subjects in measurable quantities that the adrenals had become exhausted. and would appear to arise only during intense stimula- In children with the nonsalt-losing form of CAH, tion of the adrenals with ACTH. FINKELSTEIN found in the absence of treatment the Na, is seen to be normal this compound in the urine of patients suffering from for the age and body build, although in case no. 1 the Cushing's syndrome associated with adrenal hyper- value was rather low. Likewise, the percentage excre- plasia but not in cases of adrenal tumor. tion of 24Na was normal for dietary conditions, but In CAH, it is possible that 11 p-hydroxylase deprived these normal values were associated with aldosterone of its normal 21-hydroxylated substrates, acts upon secretion rates that tended to be higher than those of those steroids formed in great quantity, particularly normal children. 17-hydroxyprogesterone. During treatment capable of maintaining the salt The abnormal excretion of pregnanetriol is reduced losers in good general health the Na, values were found rapidly by therapy. Case nos.4, 21, and 22 show that to be normal (case nos. 8, 10, and 14) or still low (case after only 46 days of treatment the urinary levels are no. 10). These findings and the great variation in so- low. It is emphasized, however, that in subjects with dium excretion suggest that sodium equilibrium re- CAH under 1 year of age, assay of urinary pregnane- mains rather unstable even during optimum thera- trio1 alone is inadequate because of the importance of peutic conditions. the 11-keto form, and could lead to serious underesti- mation of the quantity of adrenal precursor compounds Factors Involved in Salt Loss secreted. Three hypotheses have been put forward to explain the presence or absence of salt loss in CAH due to 21- Sodium hydroxylase deficiency [8]. Firstly, it has been sug- The four newborns that were studied before clinical gested that there may be two adrenal 21-hydroxylases evidence indicated salt losing (case nos. 7, 8, 9, and 10) acting on two types of substrates, steroids with an showed low sodium levels in plasma, and in the three hydroxyl group at C-17, and precursors of aldosterone. cases in which it was studied, the Na, showed a severe In the simple form of the disorder only the first enzyme deficit. The negative sodium balance in these infants would be involved, and in the salt-losing form both would, therefore, appear to arise very early and, pro- would be deficient. bably, as suggested by BONGIOVANNI et al. [8] dates Secondly, some authors hold that the limited ca- from the time of birth. pacity for aldosterone secretion is in itself sufficient to Case no. 13 is of interest in that she showed a value explain the salt loss and that the degree of deficiency for Na, that was less than normal during normal salt of a single enzyme is responsible for the varying degrees intake despite a capacity for further aldosterone pro- of severity of the syndrome. 154 LORAS, HAOUR and BERTRAND

Finally, the presence of a factor promoting salt ex- also been found in two of three cases by JAILER et al. cretion has been suggested, a factor which is probably [23], and high values have also been reported by Ko- found among the precursors of cortisol and which is WARSKI et al. [25] and by BARTTER et al. [3]. secreted in considerable quantity as a result of the The evidence of an elevated ASR in the absence of enzymatic block. This factor is believed to act by com- steroid therapy suggests that a salt-losing factor is petitive inhibition with aldosterone at the level of the present but is compensated. An explanation is requir- renal tubule and not as a primary agent prompting ed, however, for the inconstant nature of this phe- sodium excretion. This theory suggests that some nomenon. In previous studies [3, 211 a few ASR values affected children compensate for the tendency to salt were within normal limits or only slightly elevated. loss by increasing their secretion of aldosterone and The five cases of DEGENHART et al. [14] and the four these are the nonsalt losers. The others, in whom the cases of NEW et al. [29] have ASR values considered capacity for aldosterone secretion is limited, cannot normal and one can find neither correlation between overcome the effects of the factor and these cases are, these varying values for aldosterone secretion and the clinically, the salt losers. 17-ketosteroid values nor correlation between ASR and The investigations described here do not permit us the other variables as they are presented, namely pres- to differentiate between a one- or a two-enzyme defect. ence or absence of a treatment period before investiga- The most that can be said is that, in general, the tion, sodium intake, chronological age, sex, and age of severity of the defect in cortisol secretion corresponds the child when treatment was first begun. A possible with that in aldosterone secretion. This does not sup- exception would be urinary pregnanetriol values. port the idea of two enzymes or of two separate genes GODARD et al. [21] suggested that the capacity to being involved. secrete aldosterone is limited, even in the nonsalt- Among the factors concerned in salt loss there is no losing subjects. ULICK [30] was of the same opinion in doubt that the limited capacity to secrete aldosterone view of his finding of an abnormally high excretion of is important. Our observations support those already 11, 18-dihydroxypregnanetriol, and 18 OH-THA [33], reported [lo, 14,21,25,291. suggesting 'that aldosterone deficiency in the salt- The most severely affected salt losers have the lowest losers was a more severe expression of a biosynthetic CSR. This relation is already known [7] but it does defect which could occur, in compensated form, in non- not explain the interrelation between sodium and salt-losers as well'. In our cases of nonsalt losers (case cortisol. These children also have a very low ASR; nos. 1-6) the ASR response to a low salt regime was thus, although the cortisol insufficiency associated with certainly not greater than normal (yet the pool of Na, the salt loss could be a causative factor due to lack of was reasonably well maintained). It is, therefore, diffi- the 'permissive' effect of cortisol [8], and although the cult to understand why some cases show only a normal presence of a salt-excreting factor in the precursors or subnormal rise in ASR with a low salt intake while could play a role, the major factor may well be, quite others [3] show a very striking rise (to 3,244 pg/24 h). simply, the inadequate ASR which is a constant asso- If patients without salt loss can exhibit reasonable ciation. satisfactory aldosterone production and Na, conser- There are several arguments in support of the idea vation, however, they can also show evidence of some of a salt-losing factor. I) Steroid therapy brings a fall minor abnormality such as loss of weight during a low in the aldosterone secretion rate (3, 21, 25, and caw salt regime or even a lowering of the sodium level in nos.3 and 6). 2) A positive relation between the excre- plasma. The institution of a low salt regime can there- tion of pregnanetriol in urine and that of aldosterone fore be regarded as a stress situation in the nonsalt has been described by HALL and HOKFELT [22] and losers, as in the salt losers, but of much less severity. by MATTOX et al. 1281 in both types of CAH. We have This may be explained by a limitation in the capacity found the same relation between ASR and either to secrete aldosterone as well as by the concept of a urinary pregnanetriol or urinary total 17-ketogenic salt-losing factor. corticoids in nonsalt-losing patients, although the re- These two concepts are in no way in opposition and lation has not been defined on a mathematical basis. it seems reasonable to presume the coexistence of two The existence of this positive relation is strongly in factors—the salt-excreting factor and the insufficiency favor of the presence of a salt-losing factor since even of both cortisol and aldosterone—their respective im- if inexact, urinary pregnanetriol is a good index of the portance being inversely proportional and varying quantity of adrenal precursor steroids that have been according to the gravity of the deficit. An increasing formed. 3) Cases of CAH without salt loss tend to have maturity of renal function with age and the dietary elevated ASR values when untreated. The ASR values sodium intake must also be taken into consideration. of our six children can be regarded as on the high side. The syndrome of salt loss will appear when there is Above normal values for ASR in nonsalt losers have failure of equilibrium between the production of salt- Exchangeable sodium and aldosterone secretion in children 155 losing factor (illustrated by the ketogenic corticoid References and Notes levels) and the production of cortisol and aldosterone. The disequilibrium and the salt loss will be worse when 1. APPLEBY, J.E.; GIBSON, G.; NORYMBERSKI, J.K. the child is very young and the sodium intake is poor. and STUBBS, R. D.: Indirect analysis of corticosteroids. I. The determination of 17 hydroxycorti- The 'Recovery' costeroids. Biochem.J. 60: 453 (1955). The recovery from salt loss, which is known to occur 2. ARONS, W.A.; NUSIMOVICH, B.; VANDERLINDE, in the majority of cases, can be explained by allied R.J. and THORN, G.W.: Effect of exogenous adre- arguments. This 'recovery' usually occurs between 3 nocortical hormones and states of adrenal dysfunc- and 6 years of age, and in clinical practice means that tion on body sodium and potassium composition. one may reduce the frequency and later, stop com- J.clin.Endocrin. 18: 611 (1958). pletely, the administration of deoxycorticosterone ace- 3. BARTTER, I?. C.; HENKIN, R. I. and BRYAN, G.T.: tate (Doca) or other mineralocorticoids. Likewise, it Aldosterone hypersecretion in 'non salt-losing' is possible to gradually reduce the salt supplementation congenital adrenal hyperplasia. J. clin. Invest. 47: during the years after mineralocorticoids have been 1742 (1968). stopped. 4. BERGSTRAND, C. G.; BIRKE, G. and PLANTIN, L. 0. : The 'recovery' is not perfect. In situations of stress, Differences in corticosteroid excretion pattern be- the needs for Doca and for salt again arise, and, in the tween infants and children with the adrenogenital absence of stress, a strict low salt intake is poorly tolerat- syndrome. Acta endocrin., Kbh. 34: 508 (1960). ed. Our patients (case nos. 15,18, 21, and 22) went into 5. BERTRAND, J.; LORAS, B.; GILLY, R. et CAUTE- negative sodium balance on a low salt diet and could NET, B.: Contribution B l'ttude de la stcrttion et not conserve Na,. Nevertheless, the improvement on du mttabolisme du cortisol chez le nouveau-nt et the state occurring in the early months and years of le nourrisson de moins de trois mois. Path. Biol. 11: life was quite spectacular. 997 (1963). The mechanism of the recovery is not known, al- 6. BERTRAND, J.; ROUX, H. and COTTE, C.: Paper though newborns are known to have a most unstable chromatography of urinary 17 ketosteroids in chil- water and sodium control. dren. Acta endocrin., Kbh. 45: suppl. 89 (1963). An alteration in renal function with age, which per- 7. BONGIOVANNI, A. M. and EBERLEIN, W. R.: De- mits improved conservation of sodium under the in- fective steroidal biogenesis in congenital adrenal fluence of equivalent amounts of aldosterone, is very hyperplasia. Pediatrics 21: 661 (1958). unlikely. Cases of CAH respond well to aldosterone 8. BONGIOVANNI, A.M.; EBERLEIN, W.R.; GOLDMAN, from a very early age [20]. A. S. and NEW, M.: Disorders of adrenal steroids The possibility that the mechanism concerned with biogenesis. Recent Progr. Hormone Res. 23: 375 secretion of cortisol and aldosterone is improved with (1967). age has also been considered but this conjecture is 9. BONGIOVANN~, A.M.; EBERLEIN, W.R.; SMITH, highly unlikely. We have, in fact, repeated measure- J. D. and MCPADDEN, A.J.: The urinary excretion ments of secretion of aldosterone following a consider- of three C21 methyl corticosteroids in the adreno- able time interval to see if there were significant modi- genital syndrome. J.clin.Endocrin. 19: 1608 (1959). fications and the results in two cases show that there 10. BRYAN, G.T.; KLIMAN, B. and BARTTER, I?.C.: were not (case nos. 9 and 10). Impaired aldosterone production in 'salt-losing' The one remaining factor that alters with time is the congenital adrenal hyperplasia. J. clin. Invest. 44: sodium intake and this shows some relation with ASR 957 (1965). in normal subjects. Furthermore, the relative as well 11. CHRISTIAN, J. R.; TALSO, P.J.; KAREZERIS, M. S.; as the absolute sodium intake increases as the child SPAFFORD, N. R.; JACKSON, M. and LARSON, M.: grows. It would seem, therefore, that Doca can be Total body water and exchangeable sodium in stopped when the diet contains sufficient salt to ba- normal full term newborn infants. Amer.J. Dis. lance or to overcompensate for the limited degree of Child. 92: 325 (1956). aldosterone production of which the adrenals are 12. CORSA, L.; GRIBETZ, J. R.; COOK, C. and TALBOT, capable, and that this dietary sodium is a factor of N. B.: Total body exchangeable water, sodium and vital importance in producing the equilibrium. potassium in hospital normal infants and children. Pediatrics 17: 184(1956). 13. DAVID, R. R.; BERGADA, C. and MIGEON, C.J.: Effect of age on urinary steroid excretion in congenital adrenal hyperplasia. Bull. Johns Hopk. Hosp. 117: 16 (1965). 156 LORAS, HAOUR and BERTRAND

14. DEGENHART, H. J.; VISSER, H. K. A.; WILMINKAND, plasia. A discussion of the theories on the patho- R. and CROUGHS, W.: Aldosterone and cortisol genesis of the salt-losing form of the syndrome. secretion rates in infants and children with con- J. clin. Invest. 44: 1505 (1965). genital adrenal hyperplasia suggesting different 21 26. LORAS, B.; HAOUR, F. et BERTRAND, J.: Mesure hydroxylation defects in 'salt-losers' and 'non salt- du sodium Cchangeable et du taux de sCcrCtion losers'. Acta endocrin., Kbh. 48: 587 (1965). d'aldosterone chez des enfants. C.R. Soc. Biol., 15. FEW, J.D.: A method for the analysis of urinary Paris 161: 2490 (1967). 17 hydroxycorticosteroids. J. Endocrin. 22: 31 27. MCCANCE, K.A. and WIDDOWSON, E.M: The (1961). chemical composition of food. Medical Research 16. FINKELSTEIN, M.: Estimation of pregnanetriolone, Council (Her Majesty's Stationary Office, London the estrogens and testosterone as an aid to clinical 1946). diagnostic; in: C.CASSANO: Research on steroids, 28. MATTOX, V.R.; HAYLES, A.B.; SALASSA, R.M. p. 67 (I1 pensiero scientifico, Rome 1964). and DION, F. R.: Urinary steroid patterns and loss 17. FORBES, G. B.: in: COMAR and BRONNER : Mineral of salt in congenital adrenal hyperplasia. J. clin. metabolism, vol.11, part. B, 2 (Academic Press, Endocrin. 24: 517 (1964). London/New York 1962). 29. NEW, M.I.; MILLER, B. and PETERSON, R.E.: 18. FORBES, G.B. and PERLEY, A.M.: Estimation of Aldosterone excretion in normal children and in total body sodium by isotope dilution. I. Studies children with adrenal hyperplasia. J. clin. Invest. in young adults. J.clin. Invest. 30: 566 (1951). 45: 412 (1966). 19. GLEEN, E. M. and NELSON, D. H.: Chemical meth- 30. ULICK, S.: Aldosterone biosynthesis in congenital od for the determination of 17 hydroxycorticoids and adrenal hyperplasia. Abstract no. 6. 49th Meeting 17 ketosteroids in urine following hydrolysis with of the Endocrin. Soc., June 1967. P glycuronidase. J. clin.Endocrin. 13: 911 (1953). 31. WELDON, V.V.; KOWARSKI, A. and MIGEON, C.J.: 20. GREENBERG, A. J.; MCNAMARA, H. and MCCRORY, Aldosterone secretion rates in normal subjects from W.W.: Renal tubular response to aldosterone in infancy to adulthood. Pediatrics 39: 884 (1966). normal infants and children with adrenal disorders. 32. WILKINS, L.: The diagnosis and treatment of endo- J.clin.Endocrin. 27: 1197 (1967). crine disorders in childhood and adolescence, p. 340 21. GODARD, C.; RIONDEL, A.M.; VEYRAT, R.; ME- (Thomas, Springfield, IL 1957). GEVAND, A. and MULLER, A. F.: Plasma renin 33. The following abbreviations have been used in activity and aldosterone secretion in congenital this paper: THF: 3 a, 11 j3, 17 a, 21-tetrahydroxy-5 adrenal hyperplasia. Pediatrics 41: 883 (1968). ,8-pregnane-20-one. a110 THF: 3 a, 11 ft 17 a, 22. HALL, K. and HOKFELT, B.: Clinical and steroid 21-tetrahydroxy-5 cc-pregnane-20-one. THE: 3 cc, metabolic studies in four siblings with congenital 17 cc, 21-trihydroxy-5 j3-pregnane-11, 20-dione. virilizing adrenal hyperplasia. Acta endocrin., 18 OH-THA: 3 a, 18,21-trihydroxy-5 j3-pregnane- Kbh. 52:535 (1966). 11, 20-dione. 23. JAILER, J.W.; ULICK, S. and LIEBERMAN, S.: Aldo- 34. We thank Professor M.JEUNE, Professor P. MON- sterone and hydrocortisone secretion rates in pa- NET, and Professor R. FRAN~OIS for allowing us tients with the salt-losing form of congenital adrenal access to their patients; Dr. CLAUDE J. MIGEON for hyperplasia. Trans. Ass. amer. Physicians 72: 149 his advice and criticism; and Dr. DAVID M. CA- (1959). THRO for translating this material. Informed con- 24. KLIMAN, B. and PETERSON, R.E.: Double isotope sent was obtained for all subjects in this study. derivative assay of aldosterone in biological ex- 35. Requests for reprints should be addressed to: tracts. J.biol. Chem. 235: 1639 (1960). J. BERTRAND, M.D., I.N.S.E.R.M., Unit6 de Re- 25. KOWARSKI, A.; FINKELSTEIN, J.W.; SPAUI.DING, cherche~ Endocrinitnnes et MCtaboliques chez e J. S.; HOLMAN, G.H. and MIGEON, C.J.: Aldoste- l'Enfant, HBpital Debrousse, Lyon 5 , France. rone secretion rate in congenital adrenal hyper- 36. Accepted for publication October 3, 1969.