Clinica Chimica Acta 444 (2015) 154–155
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Letter to the Editor Determination of cortisol and cortisone in human mother's milk Keywords: Mother's milk Cortisol Cortisone LC–MS/MS
Dear Editor,
Human mother's milk is recommended as the standard nutrition for neonates, due to its widely acknowledged benefits. Besides being a source of nutrients, milk contains a variety of non-nutritive bioactive and immunomodulatory components [1]. Glucocorticoids are found in mother's milk, bound mainly to corticosteroid binding globulin and albumin [2]. Glucocorticoids are suggested to induce proliferation and differentiation of glandular cells in the mammary gland, and also to influence the neonate via the mother's milk [2,3]. Despite efforts to optimize the contents of formula feeding, human milk is still recommended for its widely acknowledged benefits. Maternal glucocorticoids might be one of the factors contributing to the advantages of breast milk over formula feeding and are therefore worthwhile to investigate. In order to be able to measure glucocorticoids in human milk we developed a reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine cortisol and cortisone in mother's milk. In this letter, we describe this method and the results of the validation of our assay, including experiments that investigated the stability of cortisol and cortisone in human milk. Donor mother's milk from 13 healthy mothers who donated between 8 to 28 weeks postpartum to the Dutch Human Milk Bank of the VU University Medical Center, Amsterdam was used. All samples were stored in polypropylene vials at −20 °C. Preparation of the samples was initiated by the addition of 2H4 labeled cortisol (Cambridge Isotope Laboratories) and 2H8 labeled cortisone (CDN Isotopes Inc.), both serving as internal standards, to 200 μL thawed milk and thorough mixing. To remove undesired lipids, the milk was washed by the addition of 2 mL hexane [2]. Capped tubes were mixed for 2 min in a multivortexer and centrifuged for 2 min, at 19 °C, 1900 g, resulting in the separation of the lipid layer from the aqueous layer. Thereafter the sample was frozen in a −60 °C CO2 ice bath, which enabled the liquid hexane to be decanted from the frozen milk. This washing procedure was completed three times, in total. Forty μL of the washed milk was injected onto a Symbiosis online solid phase extraction (SPE) system (Spark Holland, Emmen, The Netherlands). Online SPE with C8 cartridges (Spark Holland) was performed for further purification of the samples. The analyte was eluted from the cartridge with methanol–water and focused on the Synergi Hydro RP column (Phenomenex, Utrecht, The Netherlands), which was equipped with a C18 guard column (Phenomenex). A linear binary
http://dx.doi.org/10.1016/j.cca.2015.02.015 0009-8981/© 2015 Elsevier B.V. All rights reserved.
gradient from 55 to 61% methanol containing 0.1% formic acid and 2 mmol/L ammonium acetate was applied, after which the methanol content was increased to 100%. Cortisol, cortisone and their internal standards eluted at retention times of 5.2 and 4.8 min, respectively. The total run time was 7.5 min. A Quattro Premier XE tandem mass spectrometer (Waters Corp., Milford, MA), which operated in the electrospray positive ionization mode, served as detection instrument. Capillary voltage was 0.5 kV and the source temperature was 120 °C. Argon was used as collision gas. Cortisol and [2H4]cortisol were measured using the transitions (Q1 N Q3)m/z 363.2 → 121.1 and m/z 367.2 → 121.1, respectively. Cortisone and [2H8]cortisone were measured using the transitions (Q1 N Q3)m/z 361.2 → 163.1 and m/z 369.2 → 124.1, respectively. Table 1 in the supplement shows that the intra-coefficients of variation (CV%) were 4% and 5% at a level of 7 and 23 nmol/L, respectively, for cortisol and 5% at a level of 8 and 33 nmol/L for cortisone and that the inter-CV% was b9% for both cortisol and cortisone. Recoveries, as judged by the recovery of spiked analytes, were 97–102% for cortisol and 98– 106% for cortisone. Linearity was shown by 2-, 4- and 8-fold dilutions. The difference between expected and observed values varied between 93 and 106%. The Lower Limit of Quantitation (LLOQ) with a total allowable intra-assay CV of 15% was 0.5 nmol/L for cortisol and 0.25 nmol/L for cortison, determined using diluted milk samples. In addition, the stability of cortisol and cortisone in human milk under various conditions was evaluated. We determined the stability of cortisol and cortisone in milk stored at room temperature by aliquoting one sample of fresh human milk and storing these for 0, 2, 4, 6, 8, 10, 12, 24 and 36 h at 4 °C and 20 °C. After these time periods all aliquots were directly stored at - 20 °C. Subsequently, all samples were measured in one run. The analysis showed that both cortisol and cortisone were stable (b10% decrease) during 36 h storage at 4 °C and 20 °C in comparison to direct storage at -20 °C. Finally, we evaluated the influence of freeze–thaw cycles and saw that at least six cycles did not influence (b5% decrease) cortisol and cortisone concentrations in human milk (N = 4). In order to determine reliable reference values a larger sample size should be used for measurements. The number of days postpartum and time of the day might influence milk cortisol concentration and should be taken into account [2]. In our study, based on 13 samples of healthy mothers collected at different moments during the day and on varying days up till 28 weeks postpartum, cortisol concentrations ranged from 4 to 23 nmol/L, cortisone concentrations ranged from 11 to 33 nmol/L and the cortisol:cortisone ratio ranged from 0.2 to 0.6. In the literature, a wide range of human milk cortisol concentrations is reported, from 0 to 1700 nmol/L (Table 1). This might have been caused by the variety of methods, including the potential cross reactivity with other steroids in the immunoassays, used in 4 out of 7 reported studies [2,4,6,8]. Nowadays, for reliable steroid hormone analysis LC– MS/MS is preferred, due to its superiority in specificity compared to immunoassays. Moreover the variety in reported concentrations might have been caused by the use of enzymatic conjugates, in 3 out of 7 reported studies [4,5,7]. Conjugated steroids are biologically inactive and
Letter to the Editor
155
Table 1 Literature overview of used methods and reported concentrations of human milk cortisol and cortisone. Reference
Subjects
Method
Concentration cortisol (nmol/L)
Concentration cortisone (nmol/L)
Grey et al. [4] (2013) Xu et al. [5] (2011) Kulski and Hartmann [2] (1981) Hart et al. [6] (2004) Sahlberg and Axelson [7] (1986) Groer, Humenick and Hill [8] (1994) Öst et al. [9] (1985) Van der Voorn et al. (Current study)
52 full term mothers; 3 months postpartum 8 full term mothers; 0–3 days postpartum 11 full term mothers; 1 month before birth–13 months postpartum 40 full term mothers; 1 week postpartum 6 full term mothers; b1 month postpartum 34 preterm and 29 full term mothers; 5 days postpartum 6 healthy women; 4–6 days postpartum 13 full term mothers; 8–28 weeks postpartum
Chemiluminescent immunoassay after enzymatic deconjugation LC–MS/MS after enzymatic deconjugation Radioimmunoassay
Mean 6.1
–
Mean ± SD 3.4 ± 1.7
Mean ± SD 28 ± 9.4
Postpartum: Range: 0.6–88
–
Mean ± SD 13.2 ± 13.0 Range: 0–38.6 Range: 0–0.4
–
–
HPLC
Preterm milk Mean 1700 Full term milk Mean 1600 Range ≤ 41
LC–MS/MS
Range: 4–23
Range: 11–33
Fluorometric immunoassay GC–MS after enzymatic deconjugation Radioimmunoassay
need to be hydrolyzed by sulfatases and glucuronidases to become reactivated and absorbed by the intestinal mucosal cells [10]. These enzymes are absent at birth in the human gut and are increasingly present with age due to bacterial colonization [10]. Therefore incubating the milk with conjugate enzymes could give falsely high levels, not reflecting the clinically relevant, biologically available glucocorticoid compound to the neonate. Furthermore, maternal, perinatal stress experienced in the first days postpartum could have caused higher milk cortisol concentrations. However, in relation to reported cortisol concentrations in other studies who used immunoassays with [2] or without [4,6] enzymatic deconjugation and studies who used samples collected during the first days postpartum [5,6] the extremely high concentrations (~1700 nmol/L) reported by Groer, Humenick and Hill [8] cannot be explained. In conclusion, in this study we present a reliable LC–MS/MS method to measure cortisol and cortisone in human milk, using a relatively easy sample work-up which requires only a small amount of milk. Moreover, we showed that cortisol and cortisone in human milk are stable during 36 h of storage at room temperature and during at least six freeze–thaw cycles. In addition, an overview of human milk cortisol and cortisone concentrations reported in literature is presented. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2015.02.015. References [1] Bernt KM, Walker WA. Human milk as a carrier of biochemical messages. Acta Paediatr Suppl 1999;88:27–41. [2] Kulski JK, Hartmann PE. Changes in the concentration of cortisol in milk during different stages of human lactation. Aust J Exp Biol Med Sci 1981;59:769–78. [3] Sullivan EC, Hinde K, Mendoza SP, Capitanio JP. Cortisol concentrations in the milk of rhesus monkey mothers are associated with confident temperament in sons, but not daughters. Dev Psychobiol 2011;53:96–104. [4] Grey KR, Davis EP, Sandman CA, Glynn LM. Human milk cortisol is associated with infant temperament. Psychoneuroendocrinology 2013;38:1178–85. [5] Xu L, Zhang L, Zhang Y, Sheng Q, Zhao A. Qualitative and quantitative comparison of hormone contents between bovine and human colostrums. Int Dairy J 2011;21: 54–7. [6] Hart S, Boylan LM, Border B, Carroll SR, Mcgunegle D, Lampe RM. Breast milk levels of cortisol and secretory immunoglobulin A (SIgA) differ with maternal mood and infant neuro-behavioral functioning. Infant Behav Dev 2004;27:101–6.
Range: 0–17
[7] Sahlberg BL, Axelson M. Identification and quantitation of free and conjugated steroids in milk from lactating women. J Steroid Biochem 1986;25:379–91. [8] Groer M, Humenick S, Hill P. Characterizations and psychoneuroimmunologic implications of secretory immunoglobulin A and cortisol in preterm and term breast milk. J Perinat Neonatal Nurs 1994;7:42–51. [9] Öst L, Wettrell G, Björkhem I, Rane A. Prednisolone excretion in human milk. J Pediatr 1985;106:1008–11. [10] Kent T, Fischer L, Marr R. Glucuronidase activity in intestinal contents of rat and man and relationship to bacterial flora. Proc Soc Exp Biol Med 1972;140:590–4.
Bibian van der Voorn Department of Pediatric Endocrinology, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands Frans Martens Department of Clinical Chemistry, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands Nathasja S. Peppelman Joost Rotteveel Department of Pediatric Endocrinology, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands Marinus A. Blankenstein Department of Clinical Chemistry, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands Martijn J.J. Finken Department of Pediatric Endocrinology, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands Annemieke C. Heijboer Department of Clinical Chemistry, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands Corresponding author at: Department of Clinical Chemistry, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands. Tel.: +31 20 4443872. E-mail address: [email protected]. 22 January 2015 Available online 14 February 2015
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Letter to the Editor Determination of cortisol and cortisone in human mother's milk Keywords: Mother's milk Cortisol Cortisone LC–MS/MS
Dear Editor,
Human mother's milk is recommended as the standard nutrition for neonates, due to its widely acknowledged benefits. Besides being a source of nutrients, milk contains a variety of non-nutritive bioactive and immunomodulatory components [1]. Glucocorticoids are found in mother's milk, bound mainly to corticosteroid binding globulin and albumin [2]. Glucocorticoids are suggested to induce proliferation and differentiation of glandular cells in the mammary gland, and also to influence the neonate via the mother's milk [2,3]. Despite efforts to optimize the contents of formula feeding, human milk is still recommended for its widely acknowledged benefits. Maternal glucocorticoids might be one of the factors contributing to the advantages of breast milk over formula feeding and are therefore worthwhile to investigate. In order to be able to measure glucocorticoids in human milk we developed a reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine cortisol and cortisone in mother's milk. In this letter, we describe this method and the results of the validation of our assay, including experiments that investigated the stability of cortisol and cortisone in human milk. Donor mother's milk from 13 healthy mothers who donated between 8 to 28 weeks postpartum to the Dutch Human Milk Bank of the VU University Medical Center, Amsterdam was used. All samples were stored in polypropylene vials at −20 °C. Preparation of the samples was initiated by the addition of 2H4 labeled cortisol (Cambridge Isotope Laboratories) and 2H8 labeled cortisone (CDN Isotopes Inc.), both serving as internal standards, to 200 μL thawed milk and thorough mixing. To remove undesired lipids, the milk was washed by the addition of 2 mL hexane [2]. Capped tubes were mixed for 2 min in a multivortexer and centrifuged for 2 min, at 19 °C, 1900 g, resulting in the separation of the lipid layer from the aqueous layer. Thereafter the sample was frozen in a −60 °C CO2 ice bath, which enabled the liquid hexane to be decanted from the frozen milk. This washing procedure was completed three times, in total. Forty μL of the washed milk was injected onto a Symbiosis online solid phase extraction (SPE) system (Spark Holland, Emmen, The Netherlands). Online SPE with C8 cartridges (Spark Holland) was performed for further purification of the samples. The analyte was eluted from the cartridge with methanol–water and focused on the Synergi Hydro RP column (Phenomenex, Utrecht, The Netherlands), which was equipped with a C18 guard column (Phenomenex). A linear binary
http://dx.doi.org/10.1016/j.cca.2015.02.015 0009-8981/© 2015 Elsevier B.V. All rights reserved.
gradient from 55 to 61% methanol containing 0.1% formic acid and 2 mmol/L ammonium acetate was applied, after which the methanol content was increased to 100%. Cortisol, cortisone and their internal standards eluted at retention times of 5.2 and 4.8 min, respectively. The total run time was 7.5 min. A Quattro Premier XE tandem mass spectrometer (Waters Corp., Milford, MA), which operated in the electrospray positive ionization mode, served as detection instrument. Capillary voltage was 0.5 kV and the source temperature was 120 °C. Argon was used as collision gas. Cortisol and [2H4]cortisol were measured using the transitions (Q1 N Q3)m/z 363.2 → 121.1 and m/z 367.2 → 121.1, respectively. Cortisone and [2H8]cortisone were measured using the transitions (Q1 N Q3)m/z 361.2 → 163.1 and m/z 369.2 → 124.1, respectively. Table 1 in the supplement shows that the intra-coefficients of variation (CV%) were 4% and 5% at a level of 7 and 23 nmol/L, respectively, for cortisol and 5% at a level of 8 and 33 nmol/L for cortisone and that the inter-CV% was b9% for both cortisol and cortisone. Recoveries, as judged by the recovery of spiked analytes, were 97–102% for cortisol and 98– 106% for cortisone. Linearity was shown by 2-, 4- and 8-fold dilutions. The difference between expected and observed values varied between 93 and 106%. The Lower Limit of Quantitation (LLOQ) with a total allowable intra-assay CV of 15% was 0.5 nmol/L for cortisol and 0.25 nmol/L for cortison, determined using diluted milk samples. In addition, the stability of cortisol and cortisone in human milk under various conditions was evaluated. We determined the stability of cortisol and cortisone in milk stored at room temperature by aliquoting one sample of fresh human milk and storing these for 0, 2, 4, 6, 8, 10, 12, 24 and 36 h at 4 °C and 20 °C. After these time periods all aliquots were directly stored at - 20 °C. Subsequently, all samples were measured in one run. The analysis showed that both cortisol and cortisone were stable (b10% decrease) during 36 h storage at 4 °C and 20 °C in comparison to direct storage at -20 °C. Finally, we evaluated the influence of freeze–thaw cycles and saw that at least six cycles did not influence (b5% decrease) cortisol and cortisone concentrations in human milk (N = 4). In order to determine reliable reference values a larger sample size should be used for measurements. The number of days postpartum and time of the day might influence milk cortisol concentration and should be taken into account [2]. In our study, based on 13 samples of healthy mothers collected at different moments during the day and on varying days up till 28 weeks postpartum, cortisol concentrations ranged from 4 to 23 nmol/L, cortisone concentrations ranged from 11 to 33 nmol/L and the cortisol:cortisone ratio ranged from 0.2 to 0.6. In the literature, a wide range of human milk cortisol concentrations is reported, from 0 to 1700 nmol/L (Table 1). This might have been caused by the variety of methods, including the potential cross reactivity with other steroids in the immunoassays, used in 4 out of 7 reported studies [2,4,6,8]. Nowadays, for reliable steroid hormone analysis LC– MS/MS is preferred, due to its superiority in specificity compared to immunoassays. Moreover the variety in reported concentrations might have been caused by the use of enzymatic conjugates, in 3 out of 7 reported studies [4,5,7]. Conjugated steroids are biologically inactive and
Letter to the Editor
155
Table 1 Literature overview of used methods and reported concentrations of human milk cortisol and cortisone. Reference
Subjects
Method
Concentration cortisol (nmol/L)
Concentration cortisone (nmol/L)
Grey et al. [4] (2013) Xu et al. [5] (2011) Kulski and Hartmann [2] (1981) Hart et al. [6] (2004) Sahlberg and Axelson [7] (1986) Groer, Humenick and Hill [8] (1994) Öst et al. [9] (1985) Van der Voorn et al. (Current study)
52 full term mothers; 3 months postpartum 8 full term mothers; 0–3 days postpartum 11 full term mothers; 1 month before birth–13 months postpartum 40 full term mothers; 1 week postpartum 6 full term mothers; b1 month postpartum 34 preterm and 29 full term mothers; 5 days postpartum 6 healthy women; 4–6 days postpartum 13 full term mothers; 8–28 weeks postpartum
Chemiluminescent immunoassay after enzymatic deconjugation LC–MS/MS after enzymatic deconjugation Radioimmunoassay
Mean 6.1
–
Mean ± SD 3.4 ± 1.7
Mean ± SD 28 ± 9.4
Postpartum: Range: 0.6–88
–
Mean ± SD 13.2 ± 13.0 Range: 0–38.6 Range: 0–0.4
–
–
HPLC
Preterm milk Mean 1700 Full term milk Mean 1600 Range ≤ 41
LC–MS/MS
Range: 4–23
Range: 11–33
Fluorometric immunoassay GC–MS after enzymatic deconjugation Radioimmunoassay
need to be hydrolyzed by sulfatases and glucuronidases to become reactivated and absorbed by the intestinal mucosal cells [10]. These enzymes are absent at birth in the human gut and are increasingly present with age due to bacterial colonization [10]. Therefore incubating the milk with conjugate enzymes could give falsely high levels, not reflecting the clinically relevant, biologically available glucocorticoid compound to the neonate. Furthermore, maternal, perinatal stress experienced in the first days postpartum could have caused higher milk cortisol concentrations. However, in relation to reported cortisol concentrations in other studies who used immunoassays with [2] or without [4,6] enzymatic deconjugation and studies who used samples collected during the first days postpartum [5,6] the extremely high concentrations (~1700 nmol/L) reported by Groer, Humenick and Hill [8] cannot be explained. In conclusion, in this study we present a reliable LC–MS/MS method to measure cortisol and cortisone in human milk, using a relatively easy sample work-up which requires only a small amount of milk. Moreover, we showed that cortisol and cortisone in human milk are stable during 36 h of storage at room temperature and during at least six freeze–thaw cycles. In addition, an overview of human milk cortisol and cortisone concentrations reported in literature is presented. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2015.02.015. References [1] Bernt KM, Walker WA. Human milk as a carrier of biochemical messages. Acta Paediatr Suppl 1999;88:27–41. [2] Kulski JK, Hartmann PE. Changes in the concentration of cortisol in milk during different stages of human lactation. Aust J Exp Biol Med Sci 1981;59:769–78. [3] Sullivan EC, Hinde K, Mendoza SP, Capitanio JP. Cortisol concentrations in the milk of rhesus monkey mothers are associated with confident temperament in sons, but not daughters. Dev Psychobiol 2011;53:96–104. [4] Grey KR, Davis EP, Sandman CA, Glynn LM. Human milk cortisol is associated with infant temperament. Psychoneuroendocrinology 2013;38:1178–85. [5] Xu L, Zhang L, Zhang Y, Sheng Q, Zhao A. Qualitative and quantitative comparison of hormone contents between bovine and human colostrums. Int Dairy J 2011;21: 54–7. [6] Hart S, Boylan LM, Border B, Carroll SR, Mcgunegle D, Lampe RM. Breast milk levels of cortisol and secretory immunoglobulin A (SIgA) differ with maternal mood and infant neuro-behavioral functioning. Infant Behav Dev 2004;27:101–6.
Range: 0–17
[7] Sahlberg BL, Axelson M. Identification and quantitation of free and conjugated steroids in milk from lactating women. J Steroid Biochem 1986;25:379–91. [8] Groer M, Humenick S, Hill P. Characterizations and psychoneuroimmunologic implications of secretory immunoglobulin A and cortisol in preterm and term breast milk. J Perinat Neonatal Nurs 1994;7:42–51. [9] Öst L, Wettrell G, Björkhem I, Rane A. Prednisolone excretion in human milk. J Pediatr 1985;106:1008–11. [10] Kent T, Fischer L, Marr R. Glucuronidase activity in intestinal contents of rat and man and relationship to bacterial flora. Proc Soc Exp Biol Med 1972;140:590–4.
Bibian van der Voorn Department of Pediatric Endocrinology, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands Frans Martens Department of Clinical Chemistry, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands Nathasja S. Peppelman Joost Rotteveel Department of Pediatric Endocrinology, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands Marinus A. Blankenstein Department of Clinical Chemistry, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands Martijn J.J. Finken Department of Pediatric Endocrinology, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands Annemieke C. Heijboer Department of Clinical Chemistry, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands Corresponding author at: Department of Clinical Chemistry, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands. Tel.: +31 20 4443872. E-mail address: [email protected]. 22 January 2015 Available online 14 February 2015
