Bibian van der Voorn

70 CHAPTER 5 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 wks 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 2 ). 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 . 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 4,7,8 . Conjugated steroids are biologically inactive and need to be hydrolyzed by sulfatases and glucuronidases to become reactivated and absorbed by the intestinal mucosal cells 9 . These enzymes are absent at birth in the human gut and are increasingly present with age due to bacterial colonization 9 . Therefore incubating the milk with conjugate enzymes could give falsely high levels, not reflecting the clinically relevant, biologically available glucocorticoid compound to the neonate. TABLE 2. Literature overview of used methods and reported concentrations of human milk cortisol and cortisone. Reference Subjects Method Concentration cortisol * Concentration cortisone * Grey et al. 4 52 full term mothers; 3 mo. pp Chemiluminescent immunoassay after enzymatic deconjugation Mean 6.1 - Xu et al. 7 8 full term mothers; 0-3 days pp LC-MS/MS after enzymatic deconjugation Mean ± SD 3.4 ± 1.7 Mean ± SD 28 ± 9.4 Kulski & Hartmann 2 11 full term mothers; 1 mo. before birth -13 mo. pp Radioimmunoassay Postpartum: Range: 0.6 – 88 - Hart et al. 5 40 full term mothers; 1 wk pp Fluorometric immunoassay Mean ± SD 13.2 ± 13.0 Range 0 – 38.6 - Sahlberg & Axelson 8 6 full term mothers; <1 mo. pp GC-MS after enzymatic deconjugation Range 0 - 0.4 Range 0 - 17 Groer et al. 6 34 preterm and 29 full term mothers; 5 days pp Radioimmunoassay Preterm milk Mean 1700 Full term milk Mean 1600 - Ost et al. 10 6 healthy women; 4-6 days pp HPLC Range ≤ 41 Van derVoorn et al. (Current study) 13 full term mothers; 8-28 wks pp LC-MS/MS Range 4 - 23 Range 11 - 33 *concentrations are reported in nmol/L. GC-MS = Gas chromatography–mass spectrometry; HPLC = High-performance liquid chromatography; LC-MS/MS = liquid chromatography - tandem mass spectrometry