Anna van der Veen (Presenter)
University Medical Center Groningen
Bio: My name is Anna van der Veen and I’m in the second year of my PhD. I’m working in the laboratory of professor Ido Kema (University Medical Center Groningen, The Netherlands, Department of Special Chemistry). This laboratory is specialized in the analysis of low molecular weight biomarkers, with special focus on neuroendocrine biomarkers, such as biogenic amines for routine patient care and research projects. My focus is on optimization and implementation of mass spectrometry assays for routine diagnostics and projects with special interest in androgens and the tryptophan pathway.
Authorship: A. VAN DER VEEN (1) , M. VAN FAASSEN (1), W.H.A. DE JONG (1), M.N. KERSTENS (2), I.P. KEMA (1)
(1) Department of Laboratory Medicine and (2) Department of Endocrinology, University Medical Center Groningen, University of Groningen, The Netherlands.
Recently, in our laboratory an isotope dilution mass spectrometric method was developed and extensively validated for the simultaneous analysis of six steroids including testosterone (T) and dihydrotestosterone (DHT). Plasma samples were analyzed by online SPE coupled to LC-MS/MS in 240 healthy individuals. Reference intervals were defined as the central 95% of the population. Differences in T, DHT and DHT/T ratio levels between age decades were compared using Kruskal-Wallis analysis. Reference intervals for T were 11-35nmol/L in men and 0.34-2.0nmol/L in women. For DHT, 0.9-3.2nmol/L in men and 0.1-0.8nmol/L in women and for DHT/T ratio 0.06-0.14 in men and 0.03-0.79 in women. Only in women significant differences between age decades were found for T, DHT and DHT/T ratio, not in men. This method illustrates the significant added value of mass spectrometry in the field of endocrinology.
Introduction: Isotope dilution mass spectrometry has established itself as the technique of choice for steroid hormone measurement. We recently developed and validated an isotope dilution mass spectrometric method for the simultaneous quantification of the following androgens in plasma: progesterone, 17-OH-progesterone, dehydroepiandrosterone, androstenedione, testosterone (T) and dihydrotestosterone (DHT). Measuring several steroids in combination with determining their ratios, as prediction for enzyme activity, can and will improve diagnostic procedures and outcomes. Here we present the results from an age- and gender-specific reference interval study for plasma total T, DHT and the DHT/T ratio.
Methods: Apparently healthy individuals (n=240; male-to-female ratio 1:1) from the Lifelines cohort aged 20-80 years were included, stratified with 20 males and 20 females per age decade group. Females using oral contraceptives were excluded in this study.
Plasma samples were analyzed by online SPE coupled to an isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method. In short, 200µL plasma was mixed with 25µL of internal standard solution (13C3-labeled T and DHT) and 25µL of protein disrupting buffer. After 30 minutes incubation, the samples were filled up to 1mL with water. After centrifugation, 40µL was injected onto an automated solid-phase extraction system (Waters). XBridge C8 OSM cartridges (Waters) were used in combination with a Kinetex C18 column (Phenomenex) and a Waters Xevo-TQS mass spectrometer. The run-time from injection to injection was 6 minutes. For this method, the interassay CVs for T were 3.1% at 0.37nmol/L, 2.6% at 3.8nmol/L and 3.2% at 49.7nmol/L. For DHT the interassay CVs were 7.8% at 0.12nmol/l, 4.5% at 1.6nmol/L and 3.5% at 5.3nmol/L. The limit of quantification (LOQ) for total T and DHT was 0.04 and 0.1 nmol/L, respectively. There was an excellent linearity for all analytes over a broad range (r2 = 0.99).
Reference intervals were defined as the central 95% of the population. Differences in T, DHT and DHT/T ratio levels between the age decades were compared with Kruskal-Wallis analysis and pairwise comparison using the adjusted p-values (Dunn-Bonferroni corrected).
Results: The reference intervals, represented as the 2.5-97.5th percentiles, for T were 11-35nmol/L in men and 0.34-2.0nmol/L in women. For DHT in men, the 2.5-97.5th percentiles were 0.91-3.2nmol/L. For women aged 20-49 years the DHT reference intervals were 0.10-0.86nmol/L and for women >50 years <0.39nmol/L. The 2.5-97.5th percentiles for the DHT/T ratio in men were 0.06-0.14 and in women aged 20-49 they were 0.11-0.86. For women over 50 years the reference interval for DHT/T ratio was <0.69.
Kruskal-Wallis analysis showed significant differences between age decades for T, DHT and DHT/T ratio in women, H(5) = 12.5, p = 0.03, H(5) = 55.1, p = <0.001 and H(5) = 56.3, p = <0.001, respectively. For men, no differences were found for T, DHT and DHT/T ratio between the age decades, H(5) = 5.9, p = 0.31, H(5) = 1.7, p = 0.89 and H(5) = 5.1, p = 0.41, respectively.
In women pairwise comparisons, with adjusted p-values, between the different decades was performed (post-hoc analysis). For DHT and DHT/T ratio, clear significant differences were found when the age decades 20-29, 30-39 and 40-49 were compared with the age decades 60-69 and 70-79. This is in line with the observed decline in DHT and DHT/T ratio values with increasing age. For T in women, the pairwise comparison with adjusted p-values showed that only a borderline significant difference could be found between the age decades 20-29 and 60-69, despite the significant overall effect of T after Kruskal-Wallis analysis.
Conclusion: Online-SPE coupled to LC-MS/MS enables accurate simultaneous quantification of six plasma androgens including DHT. Here we report reference intervals for plasma total T, DHT and the DHT/T ratio in healthy adults. Using Kruskal-Wallis analysis, we found a clear significant difference between age decades for DHT and DHT/T ratio in women, but not in men. The decline of DHT and therefore also the decline of the DHT/T ratio was apparent in women aged 50-79 years. The explanation and clinical relevance of this finding is yet to be unraveled.
The described method illustrates the significant added value of mass spectrometry in the field of endocrinology, as related steroids can be analyzed in one single run.
References & Acknowledgements:
IP Royalty: no
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