Neonatal salivary cortisol in response to heelstick

Scand J Clin Lab Invest 2001; 61: 287–292

Neonatal salivary cortisol in response to heelstick: method modi cations enable analysis of low concentrations and small sample volumes N . NE LS ON , K. AR BR I NG & E. TH EO DO RS SON Scand J Clin Lab Invest Downloaded from informahealthcare.com by Linkoping University on 01/02/11 For personal use only.

Departments of Paediatrics and Clinical Chemistry, University Hospital, Linko¨ping, Sweden Nelson N, Arbring K, Theodorsson E. Neonatal salivary cortisol in response to heelstick: method modi cations enable analysis of low concentrations and small sample volumes. Scand J Clin Lab Invest 2001; 61: 287–292. Measuring cortisol in saliva oŒers important advantages compared to measurement in plasma or serum. However, the sampling procedure and also the detection limit cause problems, especially in paediatric and neonatal care. We describe a simple and e cient sampling procedure, together with a modi cation of a radioimmunoassay, which enables analysis of low (down to 1 nmol/ L) concentrations of salivary cortisol (10 times lower detection limit than in the original procedure). This setting was used in studying salivary cortisol concentrations before and after heelstick on healthy newborn infants. A signi cant rise (median 81%; p < 0.01) in salivary cortisol as response to this invasive stressor was noted. Key words: Adrenal hormone; newborn infant; radioimmunoassay; saliva; stressors; glucocorticoids Nina Nelson, Department of Paediatrics, University Hospital, SE-581 85 Linko¨ping, Sweden. Tel. + 46 13 221 328, fax. + 46 13 148 265, e-mail. [email protected]

I N TR OD UC TI O N Measuring concentrations of the ‘‘stress hormone’’ cortisol has recently become a promising alternative when monitoring diŒerent aspects of stress in neonatal care [1–4]. Most studies of cortisol concentrations in response to stress rely on measuring the concentrations in serum or plasma [1]. Blood sampling, however, has the disadvantages of being: (a) painful and stressprovoking on its own, (b) less representative of the physiologically active (free) cortisol, and (c) complicated by repetitive sampling due to the risk of confounding the study result and adding to blood loss, which can be of importance in the case of the very premature baby. Measuring the concentration of cortisol in saliva is not connected with the same problems, and has been found to correlate well with concentrations in serum [3, 5–7]. Of the several alternative methods

published on how to collect saliva from newborns we have not been able to make any of them work smoothly in practice in the common setting of neonatal wards and in the routine laboratory. Various stimulants, mainly citric acid, have been used to obtain enough saliva required for the analysis of cortisol [6–11]. The amount of residual stimulants in the samples has not been elucidated and the in uence on the pH and ionic strength, etc., of the nal incubation mixtures in the radioimmunoassay has not been thoroughly studied. It is well known that changes in pH, ionic strength and concentrations of macromolecules may seriously in uence the concentrations measured in all immunochemical methods, particularly in the competitive radioimmunoassays (RIA) most commonly used to measure concentrations of cortisol. Our own studies show that the amount of citric acid required (and commonly used) to 287

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stimulate salivary ow seriously inhibits the binding of radioligand to antibodies in the RIA and results in false high concentrations of cortisol. This eŒect can be seen already at 60 mg/ ml citric acid. Therefore a collection technique not requiring citric acid stimulation was chosen in the present study. A second problem is that very low concentrations of cortisol are common in neonatal intensive care settings, and so an analytical method capable of detecting very low concentrations is needed. The purpose of the present study is to describe a sampling procedure and highly ‘‘sensitive’’ method by which to solve these problems. The model for testing our methods in practice was one examining the cortisol response to heelstick in 11 healthy newborn infants. Serum cortisol response to heelstick has earlier been investigated for diŒerent age categories of full-term healthy infants [12].

safely oŒthe bottom of the tube. In order to extract the absorbed saliva from the cotton tip the tube was centrifuged (5700 rpm) for 5 min. The sample was immediately frozen and stored in the refrigerator (–20°C ) for less than 3 months until analysis.

Determination of the detection limit The de nition of the lowest detectable concentration (statistical detection limit) used in the present paper is the concentration in serially diluted spiked saliva samples which, using Student’s t-test, was statistically diŒerent (p < 0.01) from the RIA buŒer or saline blank (n = 10). The practical limit of detection is taken to be the lowest concentration determined diŒerent from buŒer or saline blank in duplicate samples.

M AT ER I AL A ND M ET HO DS Eleven babies at 3–4 days of age (7 girls and 4 boys) were included. All the mothers were healthy and with normal pregnancies and vaginal deliveries. All babies were full term and with birth weight appropriate for gestational age (mean weight 3450 g, range 2630–4050 g). Apgar scores 5 min postnatal were > 7 for all babies. The local ethics committee approved the study and informed consent was obtained from all parents. The heelstick for blood sampling was performed for a metabolic screening, which all newborns in Sweden are submitted to. The babies were not fed 1 h preceding saliva sampling, since it is known that high concentrations of immunoreactive cortisol may be present in formula and breast milk. The baseline sample was taken just before heelstick and the stimulated sample was taken 30 min after the heelstick. A visual inspection of the baby’s mouth was made to minimize the risk of milk contamination, after which two sterile cotton tipped applicators (‘‘earpins’’) with cotton tips were introduced gently into the mouth and placed under the tongue. The pins were held in place for 3–4 min. The mouth and insides of the cheeks were gently wiped with the pins before being removed. The pins were thereafter taped together with a sewing thread and placed in a plastic tube. By holding the thread tightly while the cork was screwed on, the pins were kept

Radioimmunoassay of cortisol Cortisol was analysed using a modi ed commercially available competitive radioimmunoassay from Orion Diagnostica, Espoo, Finland. Taking the binding of cortisol as 100%, the antiserum cross-reacts 137% with 5a -dihydroxycortisol, 35.9% with 21-deoxycortisol, 35.9% with prednisolone but less than 1% with endogenous steroids. Diluting the ingredients with 0.1 mol/ L phosphate buŒer, pH 7.4 containing 0.02% BSA and 0.01% Triton X-100 modi ed the commercially available kit. The radioligand was diluted 1/ 4 and the antiserum was diluted 1/ 8. Calibrator and sample volume was 25 l L, radioligand volume was 100 l L and antiserum volume was 100 l l. Incubation was at 37°C for 60 min and the bound and free fractions were separated using solid-phase bound anti-rabbit antibodies. The 50% inhibitory concentration for the modi ed assay was 10 nmol/ L, at least an order of magnitude lower than the non-modi ed procedure. The detection limit was 0.31nmol/ L. The intra- and inter-assay coe cients of variation of the original assay were 2.6% (261 nmol/ L) and 6.5% (813 nmol/ L), respectively. The intra-assay coe cient of variation of the modi ed method was 7.2% at 10 nmol/ L.

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R ES ULT S The sampling procedure was uncomplicated, and it was easy to collect su cient sample material for analysis by centrifuging the cotton pins. None of the babies cried or showed signs of discomfort during the collection of saliva. Reducing the amount of antiserum and radioligand in the reaction mixture lowered the limit of detection of the measurement procedure by more than an order of magnitude. This enabled the detection of cortisol concentration in all samples where the sample volume was su cient (statistical limit of detection 0.31 nmol/ L and practical limit of detection 1.0 nmol/ L) (Figs 1 and 2). Furthermore, a minute sample volume of only 25 l L was all that was required. Delayed addition of radioligand or incubation at + 4°C, which lower the limit of detection still further, were not necessary for the present purpose.

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The volume of collected saliva was su cient for analysis in 21 out of 22 samples (total sample volume > 25 l L). The measured concentrations of cortisol are shown in Fig. 3. The median concentration increased from 3.7 to 14.9 nmol/ L in response to the pain of the heelstick, a median percent increase of 81% (p < 0.01, Wilcoxon signed rank test). The concentration increased in 9 of 10 patients where complete measurement series were obtained.

DI S CU SSI O N The present results show that non-stimulated salivary samples can be collected from neonates using widely available inexpensive sample collection material (simple cotton ear swab pins). Furthermore, the commercially available competitive RIA-kit for measuring cortisol in serum and urine was readily modi ed to measure the low concentrations and small sample volumes common in salivary samples from neonates. Many attempts have been made to nd suitable methods for collecting saliva and for

Fig. 1. Calibration curves for the RIA, open circles denote the original procedure and closed circles the modi ed procedure.

Fig. 2. Determination of the detection limit of the RIA for cortisol. The mean and SD of 10 replicates of each dilution are shown.

Fig. 3. Cortisol concentration in the saliva of newborn infants before and after heelstick.


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measuring cortisol in saliva since Al-Ansari and co-workers presented their modi cations of a commercial kit for analysis of cortisol in serum and urine of children aged 4 years and above [13–15]. The advantages of measuring cortisol in saliva instead of serum or urine are obvious, especially when dealing with small children. Still, di culties are associated with collecting saliva from neonates, including the methods used for stimulating the secretion of saliva or the physical appliances used for collecting the secreted saliva [4, 8]. Collection methods, including chewing [16 ], may not be applicable for newborn infants. Magnano, Gardner and Karmel had to exclude 9 out of 47 infants due to inability to collect enough saliva [2]. With our modi ed method, sample volumes as reported earlier (50–1000 l L) were not needed [2, 3, 6, 8]. Furthermore, we did not have to use citric acid or other type of stimulant of salivary secretion, as have the overwhelming proportion of earlier reports [6–11]. Whether our method of collecting saliva will be su cient for pre-term infants remains to be studied. In agreement with Grauer, Francis and Ramsay, with their respective co-workers, we did not observe crying or any other sign of discomfort while the saliva was being collected [6, 10]. This enables us to use the method when studying diŒerent potential stressors for the neonate. As re ected in our results, as well as in those of others, individual base levels of cortisol vary a great deal in the neonatal period. Still, single basal levels are used when studying the ability of the neonatal HPA system to react on stress [1, 16 ]. Infants with signi cant diŒerences in response cortisol levels may still have similar basal levels [2]. To evaluate the stress-coping capacity of the HPA system a standard provocation test should be used. The heelstick is a known reliable stimulator of the adrenocortical system in newborn infants [3, 12, 17]. We were thus certain to use a type of stress that would test the baby’s capacity to further increase the cortisol secretion. Our material is almost as small as Francis’s when he argued for using salivary cortisol in assessing adrencortical activity in newborn infants [6 ]. However, our material is more homogenous than reported in other studies [8, 18] where, for example, circumcised infants were included. They also used a stress set-up including several potentially stressful manoeuvres, which can make it harder to

analyse what exactly elicits the documented responses. Cortisol concentrations in saliva closely re ect the concentration of free cortisol (not bound to corticosteroid-binding globulin or albumin) and are independent of the ow rate of saliva. Sampling saliva is both convenient and gives the concentration of the biologically active form of cortisol. CO NC LU SI O NS The present study shows that simple cotton ear pins can be used to obtain non-stimulated salivary samples from neonates. Commercial radioimmunoassay kits can readily be modi ed to cater for the minute sample amounts obtained and for the low concentrations of salivary cortisol encountered in neonates. The techniques described here are likely to be practical in most clinical environments in which the analysis of salivary cortisol is used as an indicator of stress in diŒerent clinical circumstances.

AC KN OW LE DG E ME NT S The expert technical assistance of Liselotte Jarl and Irja Va¨ina¨mo¨ is gratefully acknowledged. RE FE R EN CE S 1 Economou G, Andronikou S, Challa A, Cholevas V, Lapatsanis PD. Cortisol secretion in stressed babies during the neonatal period. Horm Res 1993; 40: 217–21. 2 Magnano C, Gardner JM, Karmel BZ. DiŒerences in salivary cortisol levels in cocaine—exposed and noncocaine—exposed NICU infants. Dev Psychobiol 1992; 25: 93–103. 3 Gunnar MR, Hertsgaard L, Larson M, Rigatuso J. Cortisol and behavioural responses to repeated stressors in the human newborn. Dev Psychobiol 1992; 24: 487–505. 4 Vaughn PR, Townsend SF, Thilo EH, McKenzie S, Moreland S, Kawato K. Comparison of continuous infusion of fentanyl to bolus dosing in neonates after surgery. J Pediatr Surg 1996; 31: 1616–23. 5 Burke PM, Reichler RJ, Smoth E, Dugaw K, McCauley E, Mitchell J. Correlation between serum and salivary cortisol levels in depressed and nondepressed children and adolescents. Am J Psychiatr 1985; 142: 1065–67. 6 Francis SJ, Walker RF, Riad-Fahmy D, Hughes D, Murphy JF, Gray OP. Assessment of adrenocortical activity in term newborn infants using

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Received: 14 July 2000 Accepted: 22 December 2000