Study of the adrenocortical reserve function in paediatric cholera patients

Bull. Org. mond. Sante 1972, 46, 27-32Bull. Wld Hlth Org. Study of the adrenocortical reserve function in paediatric cholera patients I. TAKAKURA,1 K. KATO,2 T. KOSHIMIZU,2 R. CAMACHO 3 & C. UYLANGCO 3 The adrenocortical reserve function of 20 cases of paediatric cholera was studied by intravenous ACTH tests. The findings indicate the presence of adrenocortical hyper- activity, and the poor response to ACTH stimulation shows that the adrenocortical reserve in these cases had been already stimulated to the maximum extent by salt depletion at the time of admission. These findings do not support the value of adrenocortical hormone treatment in cholera. Cholera is an acute infectious disease characterized by severe vomiting and diarrhoea. Because of severe dehydration consequent on these clinical symptoms the patient falls into a so-called " shock stage ". It is therefore interesting and important to understand adrenocortical function in the acute stage of the disease and to study the clinical effects of adrenocorticosteroid therapy. Such studies have been made for several years but it appears that no definite findings have been published. Only the occurrence of hyperactivity of the hypophyseo- adrenocortical system has been reported on the basis of pathological and histochemical findings (Choud- hury, 1969), but no systematic study has yet been made. The purpose of this study was to determine the adrenocortical reserve function of paediatric cholera patients by the intravenous ACTH injection method (Moncloa et al., 1966). MATERIALS AND METHOD Altogether, 20 paediatric cholera patients (under 10 years of age) who were admitted to the San Lazaro Hospital, Manila, Philippines, were studied. Immediately after their admission, the first blood samples were taken from the femoral vein of the patients before they received intravenous fluid therapy. Then, 0.25 mg of synthetic ACTH (1-24 1 WHO Inter-Regional Cholera Team. Present address: Department of Paediatrics, School of Medicine, Keio Uni- versity, Tokyo, Japan. ' Department of Paediatrics, School of Medicine, Keio University. 3San Lazaro Hospital, Manila, Philippines. peptide, plain) diluted with 5 ml of normal saline was given intravenously. This injection was followed by intravenous fluid therapy with lactated Ringer's solution. After 1 hour, the second blood samples were taken and the plasma-free 1 I-hydroxycortico- steroid (11-OHCS) levels of these sample pairs were determined. The method for determining plasma- free 11-OHCS is shown in Table 1. A group of 9 paediatric cholera patients who were not given ACTH intravenously but were given the same intravenous fluid therapy as the study group were studied as controls. Blood samples from these patients were taken on admission and 1 hour after the start of intravenous fluid therapy. Both groups were given the same antibiotic (tetracycline) treat- ment and the same intravenous fluid therapy (lactated Ringer's solution). RESULTS The results for the ACTH group are shown in Table 2 and Fig. 1, and those for the control group in Table 3 and Fig. 2. The plasma-free 1 -OHCS levels of these groups were higher than those of normal healthy children (Table 4 and Fig. 3) (Taka- kura, 1968a). The levels on admission ranged from 5.8 ,tg/1O0 ml to 88.2 ,ug/100 ml in the two groups, and the mean for the two groups was 36.0 ,tg/100 ml, which is much higher than the normal value (mean +2 standard deviations= 13.76+2.83 x 2=19.42 ,ug/ 100 ml). Of 29 cases, only 2 showed normal values. This result might be due to haemoconcentration, since almost all patients had severe dehydration on admission. The plasma specific gravity on admission ranged from 1.025 to 1.039 and the mean was 1.034 2778 -27- I. TAKAKURA AND OTHERS Table 1. Determination of plasma-free 11 -OHCS by a modification of De Moor's (1960) method Sample plasma, 2 ml; distilled water, 2 ml Blank distilled water, 4 ml Standard cortisol (2 ,.g/ml), 1 ml; distilled water, 3 ml (100 Mg/100 ml concentration) add 12 ml of petroleum ether and shake for 30 seconds remove the petroleum ether and recover 3 ml add 15 ml of dichloromethane and mix without shaking wash the dichloromethane layer with 1 ml of N/10 sodium hydroxide dehydrate with anhydrous sodium sulfate and recover 8 ml add 8 ml of a 3: 1 concentrated sulfuric acid/ethanol mixture, shake for 15 seconds, and remove the dichloromethane examine by fluorometry a a The instrument used was a spectrophotometer (series 186) manufactured by the Farrand Optical Co., Inc. (filter wave lengths: 1 st, 470 nm; 2nd, 520 nm). bu 50 E = 40 2 30 - ;. E 20 10 Admission After 1 hour *o 10783 Fig. 1. Results of intravenous ACTH tests in paediatric cholera cases. cnc . 50 E (= 40 C-, o 30 0 20 10_ Admission After 1 hour WHO 10784 Fig. 2. Results of plasma-free 11 -OHCS determinations in paediatric cholera cases. Intravenous fluid therapy - ACTH 0.25 mg n en I1 28 uu ADRENOCORTICAL RESERVE FUNCTION IN PAEDIATRIC CHOLERA PATIENTS Table 2. Results of intravenous ACTH tests in paediatric cholera cases using 0.25-mg doses of synthetic ACTH 1 Specific Plasma 1 1 -OHCS (ug/100 ml)Patient | (years) Sex of plasty Before Adjusted After _______ _______ _of plasma Before_ value ihour T. P. 4 F 1.036 37.8 26.2 55.8 B.C. 5 M 1.029 36.1 31.1 40.0 A.V. 6 F 1.030 34.4 28.6 19.6 G.A. 4 M 1.038 31.8 21.6 23.8 T.Z. 4 F 1.033 34.4 26.1 34.4 L.C. 4 F 1.031 47.6 38.4 21.3 I.D. 6 M 1.030 44.2 36.8 32.8 R. M. 3 M 1.035 37.8 27.0 23.8 M. B. 3 M 1.033 25.4 19.3 19.6 J.D. 1 F 1.025 14.5 14.5 15.1 F.P. 3 M 1.036 40.8 28.3 22.6 N.S. 5 F 1.035 44.1 31.5 36.3 B.J. 2 F 1.032 31.1 24.3 28.2 L. D. 2 F 1.034 29.8 21.9 31.4 D.F. 5 M 1.039 30.7 19.7 23.4 B.C. 2 M 1.032 29.0 22.6 24.2 S.S. 3 F 1.030 25.8 21.5 17.8 R.G. 2 F 1.033 21.8 16.5 20.2 M.L. 1 F 1.036 35.5 24.6 36.3 J.S. 6 M 1.034 26.9 19.7 33.1 average j 1.033 J 33.0 25.0 28.0 in thetwo groups. Therefore, to discover the exact state of the adrenal function, it is necessary to adjust these values. The adjustment was calculated by consider- ing that the elevation of the plasma specific gravity from the normal value of 1.025 was due to dehydra- tion. After the adjustment, even though the mean for 29 cases fell to 26.7 ,tg/100 ml, only 4 of 29 first samples were within normal limits (mean ±2 stan- dard deviations; 13.76- 5.66) and 24 of them (83 %) were higher than the normal upper limit. Some samples showed levels as high as 56.5 and 42.1 ,ug/ 100 ml. Only one case had a level lower than the normal lower limit. Of 20 cases in the ACTH group, 14 had elevations of plasma 1 1-OHCS in the second samples but 7 of them did not reach the normal lower limit (mean-2 standard deviations; 31.01 ,ug/100 ml); another 7 cases did exceed this level but their first samples all gave higher readings than the normal upper limit, and the elevation in each case was not large. Therefore, it is not correct to say that the adrenal cortex of these patients could respond to ACTH stimulation. RESULTS The high plasma-free 11-OHCS levels of the first (admission) samples indicate hyperactivity of the adrenal cortex. This shows that in paediatric cholera patients, the severe dehydration due to vomiting and diarrhoea causes undue stress and stimulates the adrenal cortex to hyperactivity. 29 1. TAKAKURA AND OTHERS Table 3. Plasma-free 1 1 -OCHS of paediatric cholera cases i A e l Sex lSpecific Plasma 11-OHCS (gg/100 ml)Ptet Age Sex graityfic____ ______ |(years) | of plasma Before Adjusted 1 hour A.P. 5 F 1.034 27.4 20.1 23.4 R.G. 8 M 1.038 5.8 3.3 20.6 F.D. 7 M 1.036 60.8 42.1 15.7 J.V. 3 M 1.037 41.2 27.9 28.4 R.M. 4 M 1.039 88.2 56.5 47.0 T.C. 3 F 1.034 53.0 39.0 31.4 J.C. 4 F 1.030 21.6 18.0 23.4 J.B. 6 F 1.026 33.4 32.1 35.2 R. R. 8 M 1.037 53.8 36.1 41.2 average [ 1.035 42.8 30.6 29.6 fn 50- healthy children. ~ ~ ~ ~ ~ ~ Mea witou appACTH0.25 mgl: 0=30 C? '45 20- ~10 C Admission After 1 hour w)10 10785 Fig. 3. Results of intravenous ACTH tests in normal healthy children. Choudhury (1965) showed similar results in 20 cho- lera cases using plasma 17-OHCS as an indicator without applying ACTH stimulation. An absence of increase in the 11-OHCS levels of the second samples shows that the adrenocortica reserve function of the paediatric cholera patients had already been stimulated to the maximum extent by water and salt depletion at the time of admission, and it was impossible to demonstrate an additional response to ACTH. Another explanation for this finding is that if the hyperactive stage of the adrenal cortex had lasted for a certain period before admis- Table 4. Results of intravenous ACTH tests in normal healthy children using 0.25-mg doses of synthetic ACTH Age Plasma-free 1 1 -OHCS (14g/100 ml) _ujc (years) [Be foex_Subec exBefore |After 1 hour T.H. 14 F 12.2 38.4 M.K. 13 F 14.0 51.8 M.O. 12 M 10.5 38.4 T.T. 11 F 11.2 41.8 T.S. 11 M 14.6 48.8 T.T. 9 M 15.8 45.1 R.S. 7 F 11.5 32.6 E.T. 7 M 15.5 49.5 T.Y. 5 F 11.9 37.2 H.M. 4 M 20.4 48.3 mean i standard deviation 13.76 ± 2.83 43.19 ± 6.09 T + 2 S.D. 30 ADRENOCORTICAL RESERVE FUNCTION IN PAEDIATRIC CHOLERA PATIENTS sion, the adrenal cortex could already have been exhausted at the time of admission, even though the plasma 1l-OHCS levels were still high. The dis- appearance of free 11-OHCS from the plasma depends on liver function, i.e., glucuronidation. Therefore, in cases of circulatory insufficiency, such as occurs in the acute stage of cholera, glucuronida- tion would be inhibited and removal of l1-OHCS would take longer. However, no actual evidence for this hypothesis has been demonstrated and therefore in this study adrenocorticosteroid therapy in the treatment of cholera cannot be recommended. DISCUSSION Adrenocorticalfunction tests and ACTH Various methods have been tried as tests of adrenocortical function. The adrenocortical reserve function tests, using exogenous ACTH as the stimul- ating factor, are one of the most important and widely applied methods. Natural ACTH is composed of a mixture of peptides, which have 39 amino acids as their main components. Amino acids 1-19 of the peptides have the original corticotropic activity and amino acids 25-32 are the site of species differences (Overbeek, 1967). The disadvantage of using natural ACTH for adrenocortical function tests lies in the risk of ACTH allergy. It is said that in children the fre- quency of ACTH allergy is lower than in adults (Rosenblum & Rosenblum, 1964). However, some cases of this allergy have been reported, and we have also experienced three ACTH anaphylactic reactions in approximately 250 patients who were treated with ACTH (Takakura et al., 1968b). Therefore, synthetic ACTI4 is more suitable for adrenocortical function tests. When we gave synthetic ACTH intravenously to patients who had anaphylactic reactions against natural ACTH, there were no side-effects. Plasma-free JJ-OHCS as an ind;cator Total urinary 17-OHCS and plasma-free 17-OHCS have been widely used as indicators in adreno- cortical function tests. However, they are not ideal for the purpose of rapid testing; plasma-free 1 -OHCS is ideal for this purpose because collection of the specimens takes only 1 hour and determination of the samples takes only about 3 hours. The results obtained by this method are comparable with those from other methods that use plasma-free 17-OHCS as an indicator (Tsuchiya, 1963; Nozaki, 1967). Adjustment of the reading The results were adjusted, taking the effect of haemoconcentration on the first samples and haemo- dilution on the second samples into account simply by supposing that the elevation of the plasma specific gravity was due only to water loss. The values for the first samples were adjusted by estimat- ing how their plasma 1 I-OHCS levels would fall when diluted with water to a specific gravity of 1.025. However, in dehydrated cases with marked haemo- concentration, such as occurs in cholera, the percen- tage of non-liquid substances in plasma becomes larger. Therefore, if the results are represented as the value per litre of plasma, they will be lower than the exact value per litre of plasma plus water. Thus in case of electrolyte concentration, it is recom- mended (Watten et al., 1959) that the value per litre of plasma plus water should be used because it represents the exact concentration of electrolytes without being affected by haemoconcentration. The results in this study before the adjustment might show the exact state of adrenocortical activity of the patients or might be even lower than the exact value. However, when we compare the levels before and after ACTH treatment, the effect of haemodilution due to intravenous fluid therapy is much more significant than the effect of dehydration because large amounts of fluid are given intra- venously to patients in the first hour of the treatment. It is therefore better to show the adjusted values, taking the effect of haemodilution into consideration even though a control group with the same intrave- nous fluid therapy but without ACTH stimulation was available. ACKNOWLEDGEMENTS This study was carried out under the Joint Philippines-Japan-WHO Cholera Research Project. The authors are grateful to Dr J. C. Azurin, overall co-ordinator of the project, and to Dr Y. Ichihashi, Professor of Paediatrics at Keio University, Japan, who made this study possible. 31 32 I. TAKAKURA AND OTHERS RtSUMI ETUDE DE LA FONCTION CORTICO-SURRENALE DE RESERVE CHEZ DE JEUNES ENFANTS ATTEINTS DE CHOLERA Dans la phase aigue du cholera, les malades presentent un etat de choc consecutif a la deshydratation grave due a la diarrh&e et aux vomissements. II etait interessant d'explorer la fonction cortico-surrfnale chez ces patients et de voir si I'administration d'hormone adrenocortico- trope (ACTH) ou de steroldes cortico-surrenaux a des effets cliniques sur 1'evolution de la maladie. Vingt jeunes choleriques ont subi, des leur admission a 1'h8pital, un prelevement de sang. On leur a ensuite injecte par voie intraveineuse 0,25 mg d'ACTH, puis on a recueilli, apres une heure, un deuxieme echantillon de sang. La reponse hormonale du cortex surrenal a e appreciee par la mesure, dans chaque dchantillon, du taux plasmatique du 11-hydroxycorticost6rolde (11- OHCS). Neuf autres malades (groupe temoin) n'ont pas recu d'ACIH. Les enfants des deux groupes ont par ailleurs et6 traites de fa$on identique par administration de liquide de remplacement et par antibiotiques. La quasi-totalit6 des enfants presentaient, lors de I'admission, des taux 6leves de 1 -OHCS. Apres injection d'ACIH, on n'a constate chez la majorite d'entre eux aucune hausse des taux. On peut en conclure que chez les jeunes chol6riques la fonction cortico-surrenale est d6ja stimulee au maximum en raison de la d6pletion en eau et en electrolytes de sorte que l'injection d'ACTH ne suscite aucune reponse suppl6mentaire. Les r6sultats de la pre- sente etude ne plaident nullement en faveur de I'efficacite d'un traitement du cholera par les hormones cortico- surrenales. REFERENCES Choudhury, R. P. (1965) Calcutta med. J., 62, 397-411 Choudhury, R. P. (1969) J. trop. Med. Hyg., 72, 179-184 De Moor, G. J. et al. (1960) Acta Endocr., 33, 297-307 Moncloa, F. et al. (1966) J. clin. Endocr., 26, 482-483 Nozaki, M. (1967) J. Jap. ped. 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