Jonas Ceponis (Presenter)
Los Angeles Biomedical Institute at Harbor-UCLA
Bio: I received my M.D. in Kaunas University of Medicine (Kaunas, Lithuania) in 2004. I worked as an intern in Kaunas District Hospital in 2004-2005 and a resident in Endocrinology at Kaunas University of Medicine (Kaunas, Lithuania) 2005-2009. Upon completion of residency I worked as a consultant endocrinologist. As a PhD student at Lithuanian University of Health Sciences in Kaunas, Lithuania (2009-2014) I studied metabolic and psychological associations with andrological status in healthy and hypogonadal men. I obtained my PhD in Medicine in 2014. Since 2010 I have worked in Lithuanian University of Health Sciences as a faculty member in the Department of Endocrinology; since 2011 I worked as a junior researcher in the Laboratory of General Endocrinology in the Institute of Endocrinology. Since July, 2015 I am a visiting research scholar at Los Angeles Biomedical Institute
Authorship: Jonas Ceponis*, Andrew Leung*, Feng Bai, Atherine Dolom, Laura Hull, Ronald Swerdloff, Christina Wang. (* These authors contribute equally to the abstract)
Division of Endocrinology, Department of Medicine, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute, 1000 Carson Street, Torrance, CA 90509
| Short Abstract Our aim was to determine the most appropriate blood collection tube for measurement of DMAU and DMA in blood after oral administration of Dimethandrolone Undecanoate (DMAU), a new androgen being developed as a potential male hormonal contraceptive. In vitro experiment showed that when venous blood was transferred into six types of blood collection tubes and known amounts of DMAU were added to each tube, NaF+Oxalate, NaF+EDTA and P800 tubes showed the least interference in measuring DMA when kept for 30 min at 4C. In vivo experiment in 6 subjects showed that when serial blood samples were collected over 24h after oral administration of DMAU and kept for 30 min at 4C, DMA level differences between the different blood collection tubes were within the imprecision of the assays. The pronounced interference seen in vitro was not reflected in samples collected after oral administration of DMAU. |
Long Abstract
Introduction: Dimethandrolone undecanoate (DMAU) is being developed as a potential male contraceptive [1]. It is hydrolyzed to the active compound dimethandrolone (DMA) in the liver. Our laboratory has developed methods to measure DMAU and DMA in serum. Plain tubes with no preservatives or anticoagulants have been used for blood collection to determine serum steroid hormone levels using LC-MS/MS. New challenges were discovered with introduction of novel synthetic hormone esters (DMAU) because of the presence of non-specific esterases in red cell. These esterases may hydrolyze steroid esters (DMAU) into the steroid (DMA) itself in the blood collection tube before centrifugation to collect serum or plasma leading to inaccurate DMA results.
Study aim: Our aim was to determine the most appropriate blood collection tube for measurement of DMAU and DMA in blood after oral administration of DMAU.
Methods: Our study consisted of two experiments. In vitro experiment: 50 ml of venous blood were collected from a healthy volunteer and 2 ml were transferred immediately into duplicate tubes of six different types of blood collection tubes (BD Diagnostics, Franklin Lakes, NJ): plain tube, P-800 tube (with esterase inhibitor), EDTA, NaF (10mg) +EDTA, NaF (10mg) +Oxalate, and NaF (30 mg). Known amounts of DMAU (0 ng/ml, 300 ng/ml, and 600 ng/ml, respectively) were added to each. Prior to centrifugation the tubes were stored at 4°C for 30, 60 minutes or 60 minutes at room temperature. In vivo experiment: 10 serial blood samples were collected over 24 h after oral administration of DMAU 200 mg in 6 subjects. Blood was collected into the different tubes.
Levels of DMA and DMAU were measured by LC-MS/MS using the Shimadzu high-performance LC 20 series system (Columbia, MD) with an Applied Biosystems API 5500 with ESI source (Foster City, CA). The within run and between run imprecision was 9.7% and 9.5% for DMA and 5.6% and 10.6% for DMAU, respectively, while accuracy ranged from 93.4% to 118.4% for DMA and 91.9% to 102.2% for DMAU [2].
Results: When DMAU was added to the different tubes, keeping the tubes at 4°C for 30 minutes resulted in the least hydrolysis of DMAU into DMA. When DMAU was added to a plain tube, this led to the largest overestimation of DMA compared to all other tubes in the experiment. EDTA tubes did not prevent the overestimation of DMA after addition of DMAU. Both tubes with NaF and P800 resulted in the least conversion of DMAU to DMA in vitro while the tubes were kept at 4°C for 30 to 60 minutes but did not prevent DMAU hydrolysis to DMA at room temperature. There appeared to be no significant difference between these three collection tubes in preventing non-specific esterase hydrolyzing DMAU to DMA in vitro.
However, after oral administration of DMAU, serum DMAU and DMA levels rose. Both DMAU and DMA levels were consistently higher in serum collected in the plain tubes for all subjects, while NaF (30 mg) tube resulted in the lowest DMA levels. The results of DMA in all the other tubes were similar to the plain tube suggesting the interference of blood collection tubes in the measurement of DMA and DMAU was minimal.
Conclusions: In vitro experiments showed that NaF (10mg) +Oxalate, NaF (10mg) +EDTA and P800 tubes showed the least interference in the measurement of DMA after addition of DMAU if the tubes are kept for 30 min. at 4°C. After oral administration of oral DMAU in vivo, the differences in DMA levels between the collection tubes were minimal and within the imprecision of the assays. Thus the pronounced interference by adding DMAU to blood collection tubes is not reflected in the samples collected after oral administration of DMAU.
References & Acknowledgements:
References
1. Attardi B J, Hild S A & Reel J R. (2006) Dimethandrolone undecanoate: a new potent orally active androgen with progestational activity. Endocrinology 147, 3016-3026.
2. Surampudi P, Page S T, Swerdloff R S, Nya-Ngatchou J J, Liu P Y, Amory J K, Leung A, Hull L, Blithe D L, Woo J, Bremner W J & Wang C. (2014) Single, escalating dose pharmacokinetics, safety and food effects of a new oral androgen dimethandrolone undecanoate in man: a prototype oral male hormonal contraceptive. Andrology 2, 579-587.
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