Zusammenfassung
Glucocorticoidhormone spielen eine wichtige Rolle im Energiehaushalt und in der Stressantwort des menschlichen Körpers und gehören zu den weltweit am meisten verschriebenen antiinflammatorisch und immunsuppressiv wirksamen Medikamenten. Trotz großem Erfolg in der Behandlung einer Vielzahl von Krankheiten entwickeln manche Patienten unter Glucocorticoidtherapie gravierende Nebenwirkungen, wohingegen andere auf die Therapie nicht ansprechen. Verschiedenartige pharmakodynamische und pharmakokinetische Vorgänge tragen möglicherweise zu diesen individuellen Unterschieden in der Glucocorticoidsensitivität bei. Routinemäßig werden antikörperbasierte Methoden wie RIA (Radioimmunoassay) und ELISA (Enzyme-linked immunosorbent assay) verwendet, um Serumkonzentrationen von Glucocorticoiden, meist Cortisol, zu bestimmen. Bei diesen Techniken wird allerdings zumeist die Gesamtmenge eines spezifischen Glucocorticoids gemessen und nicht zwischen proteingebundenen und frei verfügbaren (biologisch aktiven) Glucocorticoiden unterschieden. Zudem differenzieren diese Assays nicht zwischen endogenen und therapeutisch eingesetzten Glucocorticoiden, sodass nicht die Menge an aktiven Hormon bzw. das "Glucocorticoidmilieu" eines Patienten widergespiegelt wird. Die Möglichkeit, Glucocorticoidbioaktivität im Serum oder anderen Körperflüssigkeiten zu bestimmen, könnte bei der Identifizierung Glucocorticoid-sensitiver und -resistenter Patienten helfen und dazu beitragen, Erklärungen für das unterschiedliche Ansprechen einzelner Patienten zu finden. Aus diesem Grund entwickelten wir einen Glucocorticoidbioaktivitätsassay, der auf der Messung von Glucocorticoid-abhängiger Aktivität eines Reportergens beruht. Die Verwendung einer T-Zell-Leukämie-Linie, ausgestattet mit dem Glucocorticoidrezeptor und dem Fluoreszenzprotein Venus als Reporter (JurkatGR-MMTV-VNP), ermöglicht die Bestimmung von Glucocorticoidbioaktivität aus kleinen Mengen Serum oder anderen biologischen Flüssigkeiten. Der etablierte Assay ist sensitiv und reproduzierbar – ohne nennenswerte Kreuzreaktivität mit anderen Steroidhormonen – und kann ohne Aufwand im Routinelabor eingesetzt werden.
Summary
Glucocorticoids are hormones that play a major role in energy homeostasis and stress response of the body. As drugs they are most frequently used for immunosuppressive and anti-inflammatory purposes. Glucocorticoids are exploited successfully in the treatment of a wide variety of diseases; however, some patients develop side-effects, while others fail to respond to this form of therapy. Alterations in pharmacodynamic and pharmacokinetic actions might contribute to individual differences in glucocorticoid sensitivity. Antibody-based methods such as RIA (Radioimmunoassay) and ELISA (Enzyme-linked immunosorbent assay) are routinely used to determine glucocorticoid serum levels. However, as these techniques measure the total amount of a specific glucocorticoid and do not discriminate between protein-bound and freely available (i.e. biologically active) glucocorticoids, the results do not necessarily reflect the active levels of glucocorticoid, i.e. the "glucocorticoid milieu" in a patient. Being able to determine glucocorticoid bioactivity in serum or other body fluids could help identifying glucocorticoid-sensitive or -resistant patients and help finding explanations for different responses in individual patients. For this reason, we established a glucocorticoid bioactivity assay that is based on the measurement of glucocorticoid-dependent reporter gene activity. Making use of a human T-cell leukemia line, equipped with the glucocorticoid receptor and the fluorescence protein Venus as the assay's reporter (JurkatGR-MMTV-VNP), glucocorticoid bioactivity can be determined from small amounts of serum or other biologic fluids. The developed glucocorticoid bioassay is both sensitive and reproducible, without any relevant cross-reactivity with steroid hormones other than glucocorticoids and can be practically applied in daily laboratory routine.
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Oppl, B., Kofler, A., Schwarz, S. et al. Establishing a sensitive and specific assay for determination of glucocorticoid bioactivity. Wien Klin Wochenschr 123, 222–229 (2011). https://doi.org/10.1007/s00508-011-1562-y
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DOI: https://doi.org/10.1007/s00508-011-1562-y