Salivary cortisol and administration of concentrated oral glucose in

Scand J Clin Lab Invest 2004; 64: 113 – 118

Salivary cortisol and administration of concentrated oral glucose in newborn infants: improved detection limit and smaller sample volumes without glucose interference E. MORE LIUS*, N. NEL SON* & E. THEODORSSON { Departments of *Molecular and Clinical Medicine/Division of Pediatrics and {Biomedicine and Surgery/Division of Clinical Chemistry, University Hospital, Linkoping, Sweden

Morelius E, Nelson N, Theodorsson E. Salivary cortisol and administration of concentrated oral glucose in newborn infants: improved detection limit and smaller sample volumes without glucose interference. Scand J Clin Lab Invest 2004; 64: 113–118. Newborn infants are subject to repetitive painful and stressful events during neonatal intensive care. When the baby attempts to cope with a stressful situation the hypothalamus-pituitary-adrenal axis is activated, releasing cortisol. The free cortisol response is optimally measured in saliva and saliva samples can be taken easily and without pain. However, saliva is very scarce in infants and saliva stimulants can interfere with analytical methods. Nowadays, sweet solutions are frequently administered to neonates prior to a disturbing procedure in order to reduce pain. The possible interference of sweet solutions with the measurement of salivary cortisol has not yet been documented. The aims of the present study were to further improve the detection limit of the radioimmunoassay used for cortisol analysis and to determine the degree of interference of high concentrations of glucose with the analytical method. By decreasing incubation temperature and prolonging the incubation time it was possible to improve the detection limit of the radio immunoassay (RIA) to 0.5 nmol/L at the same time as the sample volume was decreased to 10 mL saliva. Saliva was collected from full-term and preterm babies and was sufficient for analysis in 113 out of 116 (97%) samples. Glucose in the concentrations and amounts commonly used for pain relief did not interfere with the RIA method. In conclusion, it is feasible to collect microlitre volumes of saliva and analyse even very low concentrations of cortisol in newborns. It is also possible to offer the baby oral glucose prior to a painful procedure and still reliably measure salivary cortisol. Key words: Glucocorticoid; neonate; pain; radioimmunoassay; saliva; stress; stress hormone; sweet solution Evalotte Morelius, Department of Molecular and Clinical Medicine/Division of Pediatrics, University Hospital, SE-581 85 Linkoping, Sweden. Tel. z46 13 221 378, fax. z46 13 221 377, e-mail. [email protected] DOI 10.1080/00365510410004452

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I N T R O D U C T I ON Infants in neonatal intensive care units (NICUs) are commonly exposed to painful and stressful situations as a part of the hospitalization and essential medical procedures [1]. Pain experienced in the newborn period can cause short- or long-term complications [2, 3]. Acute (patho) physiologic responses caused by painful or stressful stimuli may be important in the causation or subsequent extension of early intraventricular haemorrhage (IVH) or the ischaemic changes leading to periventricular leucomalacia (PVL) [4]. When the baby attempts to cope with a stressful situation the hypothalamus-pituitary-adrenal axis is activated in which cortisol is the most important hormone. The ability to respond with cortisol secretion is necessary for survival. For the preterm baby, cortisol promotes stabilization of blood pressure, maturation of the lungs and suppresses inflammation [5 – 8]. On the other hand, long-term side effects from high levels of cortisol can cause insulin resistance, hyperlipidaemia and destructive changes in the hippocampus area [9 – 11]. It is therefore likely to be beneficial to identify preterms that are less capable of coping with stress at an early stage and thus give them optimal and individualized care. Reliable methods are therefore needed to evaluate stress, in relation to the different procedures that infants are exposed to in the NICU. Many investigators have previously pointed out the advantages of using salivary cortisol as a marker for stress in adults [12], children [13], infants [14, 15] and preterm babies [16]. Measuring cortisol in saliva rather than in plasma or urine carries several advantages besides measuring the free cortisol concentrations; the sampling procedure is painless, noninvasive and is easily performed [15]. Measuring the concentrations of cortisol in saliva using radioimmunoassay (RIA) has proved to be a promising method for monitoring different aspects of stress in newborns admitted to NICUs [17]. Since there is an increased need for monitoring stress in preterm infants admitted to the NICU, improved methods for obtaining samples of saliva and analysing low concentrations of cortisol in small volumes are needed. Administration of oral glucose prior to a

painful procedure has been proven to minimize the stressful condition [18, 19]. Since the glucose solution has an analgesic effect before it even reaches the stomach, its effects are presumably mediated by neurohumoral mechanisms involving endogenous opioids. The full effect of orally administered glucose is not obtained when administered through a naso-gastric tube [20]. Previously, it has been reported that certain substances in formula and breast milk could interfere with the measurement of salivary cortisol in infants [21]. Furthermore, oral stimulants used to increase saliva excretion have the potential to lower sample pH, increase sample osmolality and affect the cortisol results by interfering with the antibody binding [22]. Today, glucose is frequently distributed to infants but the possible analytical interference of glucose with the RIA method has so far not been elucidated. The aims of the present study were, first, to lower the detection limit of salivary cortisol RIA even more in order to reduce the volume of saliva needed for the assay and, secondly, to investigate whether glucose in amounts used to cope with pain interferes with the analytical method.

M A T E R I A L S A N D ME T H O D S Radioimmunoassay of cortisol The method gently to collect saliva samples from newborns and analyse them with a modified commercially available kit has previously been described by Nelson et al. [15]. By diluting the ingredients with 0.1 mol/L phosphate buffer, pH 7.4 containing 0.02% bovine serum albumin (BSA) and 0.01% triton X-100, they were able to modify the commercially available kit and reach a detection limit of as low as 1.0 nmol/L using 25 mL saliva [15]. In the present study, we diluted the ingredients even further with the same phosphate buffer as that described above. Antiserum was diluted in the ratio 1:40 (instead of 1:20), the radioligand was diluted until 100 mL representing 3000 counts per minute (CPM). The antiserum volume was 100 mL, radioligand volume was 100 mL (~3000 CPM), calibrator and sample volumes were each 10 mL. In order to lower the limit of detection further, the addition of

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radioligand was delayed and the incubation time prolonged. The samples were initially incubated with the antiserum at z4‡C for 48 h before the radioligand was added and then additionally 24 h before the bound and free fractions were separated using solid-phase, bound anti-rabbit antibodies. The assays were analysed in a gamma counter 1277 from Wallac (Turku, Finland). Salivary cortisol Saliva was collected on 116 different occasions from 24 full-term and 5 preterm babies at 2 – 18 days of age (mean age 7.7 days). The fullterm babies were all healthy with birthweights appropriate for gestational age (mean weight 3665 g, range 2560 – 4660 g). The preterm babies were born at a mean gestational age of 28 weeks (range 26 – 34 weeks) with a mean birthweight of 1050 g (range 560 – 2085 g). The preterm babies were all cared for in the NICU. The local ethics committee approved the study and informed consent was obtained from all the parents. Glucose concentrations in saliva Ten healthy full-term newborn babies with birthweights appropriate for gestational age were included. The local ethics committee approved the study and informed consent was obtained from all parents. The babies were each given 1 mL oral glucose 300 mg/mL (Fresenius Kabi) administered with a syringe. Saliva was collected just before the glucose was given (baseline) and after 30 min. The reason for testing glucose concentration in saliva after 30 min was because that is the time when cortisol in saliva usually peaks after an induced stressor. Saliva was collected using cottontipped pins (Fig. 1) [15]. The cotton pins were centrifuged at 1500 g for 5 min and the saliva was immediately frozen and stored at 270‡C until analysed. All samples were analysed for glucose concentration within one month. Before analysis the saliva was thawed and diluted in appropriate solution (sodium chloride 154 mmol/L, sodium fluoride 48 mmol/L and water). Analysis of glucose in saliva was done using an enzymatic assay on a Hitachi 917 (Roche Diagnostics Scandinavia AB) using reagents from Roche.

FIG. 1. Saliva collecting technique. Two cottontipped pins are kept together with a sewing thread and a piece of surgical tape. Saliva is gently collected when holding the pins in the baby’s mouth for a couple of minutes. The two cotton-tipped pins are placed in a tube with the sewing thread on the outside of the tube and centrifuged for 5 min at 1500 g. After centrifugation, the pins are removed from the tube using the sewing thread and the saliva is immediately frozen and stored at 270‡C until analysis.

Test of in vitro interference of high concentrations of glucose in the cortisol radioimmunoassay To analyse possible interference, glucose was added to the RIA. Glucose at 1660 mmol/L was serially diluted at 1:2 to 1:512. Ten replicates of each glucose concentration were added to the assay and analysed. The experiment was repeated on four different occasions in different immunoassays. Statistics Statistical analysis was performed using nonparametric tests as appropriate (Wilcoxon, MannWhitney and Friedman tests). A p-value v0.05 was considered significant.

RESULTS Radioimmunoassay of cortisol By prolonging the incubation time and decreasing incubation temperature, it was possible to decrease the amount of saliva needed for the analysis by more than 50% and to lower the practical limit of detection by 50%. Only a 10-mL sample volume was

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FIG. 2. Calibration curves for the radioimmunoassay for cortisol. Open circles represent the original procedure, closed circles the modified procedure reported earlier, and the triangles represent the new procedure reported in the present paper.

required. The limit of detection was as low as 0.5 nmol/L (Fig. 2). The 50% inhibitory concentration (IC50) for the present assay was 1.7 nmol/L compared with 10 nmol/L using the non-modified procedure. The intra-assay coefficient of variation was 12% at 2.0 nmol/L and 6% at 10 nmol/L.

Salivary cortisol

Test of in vitro interference of high concentrations of glucose in the cortisol radioimmunoassay The possible in vitro interference of glucose on the radioimmunoassay was tested in the whole range of concentrations observed in vivo and up to an order of magnitude higher than the highest observed in vivo concentration (1.8 – 1660 mmol/L). In four separate experiments, we

The volume of collected saliva was sufficient for analysis in 90 out of 92 (98%) samples taken from full-term babies and in 23 out of 24 (96%) samples taken from preterm babies. The measured concentrations of cortisol ranged from 0.9 to 223.6 nmol/L for the full-term babies (mean: 12.2 nmol/L, median: 4.5 nmol/L, lower quartile (Q1) to upper quartile (Q3): 2.5 – 8.6 nmol/L). For the preterm babies, the concentrations ranged from 4.9 to 118.7 nmol/L (mean: 40.6 nmol/L, median: 43 nmol/L, Q1 – Q3: 15 – 54.6 nmol/L).

Glucose concentrations in saliva The study included seven boys and three girls, aged 3 – 4 days at the time of the study. Median (Q1 – Q3) of baseline glucose concentrations was 2.8 mmol/L (1.6 – 4.7). After 30 min there was a significant increase in salivary glucose. The median (Q1 – Q3) concentrations of glucose was 51.1 mmol/L (29.7 – 76.1) (Fig. 3).

FIG. 3. Glucose concentrations in saliva. Glucose concentrations in the saliva of newborn infants (n~10) before and 30 min after administration of 1 mL oral glucose (1660 mmol/L).

Salivary cortisol in newborn infants did not find any significant interference in concentrations v830 mmol/L. However, two of the experiments indicated marginally affected binding (possible false high concentrations of cortisol) at 1660 mmol/L (Fig. 4).

DISCUSSION There is no doubt about the practical and ethical advantages of measuring cortisol in saliva in favour of serum or urine in the newborn period. Still, to collect saliva from preterm infants can be difficult. In several studies citric acid was used to stimulate saliva excretion in young infants [14, 16]. Our method enables measurement of cortisol in saliva in newborn infants exposed to stress without the need to use confounding citric acid to stimulate the production of saliva. Fortunately, the concentration of salivary cortisol is not dependent on salivary flow rate [23, 24]. Larger volumes needed for analysis lead to a longer time period being required for collecting saliva, which in turn increases the risk of disturbing the baby and thereby confounding the results. Furthermore, not only the preterm infant may

FIG. 4. Test of in vitro interference of high concentrations of glucose in the cortisol radioimmunoassay. Increasing concentrations of glucose were added to samples containing buffer only (0-reference) and the percentage decrease in binding of the radioligand to the antibody was measured.

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have low excretion of saliva; even healthy, fullterm infants often show a low excretion during there first days of life before they are fed sufficient breast milk and whereby they change from a catabolic to an anabolic state. Furthermore, it is known from studies of adults that stressful events may decrease the saliva excretion [25]. Our success rate of 97% in both preterm and full-term babies confirms the usefulness of a low-volume technique. Our method does not require aspiration of saliva with a catheter, as described elsewhere [13, 14, 26]. A possible confounder using the aspiration technique is that mucus is readily aspirated as well, which is known to contaminate the salivary cortisol analyses. Instead, we used sterile cotton-tipped pins which gently collect saliva with smooth strokes inside the babies mouth, as previously described [15]. It has been reported that cotton-based sample collection methods can interfere with the salivary immunoassay results for different biomarkers, but cotton proved not to interfere with the cortisol analysis [27]. The results of the present study showed that the glucose concentration in saliva was still higher than baseline 30 min after oral glucose was given but considerably lower than the concentrations for interference with the RIA. This contrasts with the results of Smith et al., who could not detect any sugar 3 min after sucrose administration when they used sugar reagent strips to determine the presence of sugar in saliva in young children and adults [28]. This contrast is probably due to the use of different analytical methods. Children and adults also have the ability to swallow larger amounts than infants do. Since glucose nowadays is frequently given to infants before painful procedures have to be carried out [18, 19] and many parents as well as medical stuff are well informed and familiar with the concept of sugar as a pain reliever, they usually expect its use. The results of this study exclude the risk of interference with oral glucose ordinarily given to the infant and the saliva sample collected to reflect the cortisol response.

C O N CL U S I O N S We present a reliable method to analyse salivary cortisol using very small volumes of saliva and

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still with an extremely low limit of detection. This enables us to collect saliva from preterm as well as full-term infants and other individuals with low saliva excretion. We also show that it is possible to offer the baby oral glucose prior to a painful procedure and still measure salivary cortisol as a stress response. This opens up new possibilities for research in the field of pain and stress.

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Received: 27 October 2003 Accepted: 22 December 2003