Validation and application of a yeast bioassay for screening androgenic activity in calf urine and feed
Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening. Previously we described the construction and properties of a rapid and robust yeast androgen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP),...
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Published in | Analytica chimica acta Vol. 637; no. 1; pp. 225 - 234 |
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Main Authors | , , , , , |
Format | Journal Article Conference Proceeding |
Language | English |
Published |
Amsterdam
Elsevier B.V
01.04.2009
Elsevier |
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Abstract | Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening. Previously we described the construction and properties of a rapid and robust yeast androgen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. In the present study this yeast androgen bioassay was validated as a qualitative screening method for the determination of androgenic activity in calf urine and animal feed. This validation was performed according to EC Decision 2002/657. 20 blank samples were spiked with testosterone, 17α-methyltestosterone, 19-nortestosterone, 17β-trenbolone, 17β-boldenone or 17α-methylboldenone at 2 or 15
ng
mL
−1 in urine and 50 or 100
ng
g
−1 in feed. All blank and spiked samples fulfilled the CCα and CCβ criterions, meaning that all 20 blank samples gave signals below the determined decision limits CCα and were thus classified as compliant (
α
=
1%). For each component, at least 19 out of the 20 spiked samples gave a signal above the CCα and were thus classified as suspect (
β
=
5%). The method was specific, and high amounts of dexamethasone did not interfere with the outcome of the test. Although high levels of 17α-ethynylestradiol can significantly inhibit the response obtained with low amounts of androgens, that situation is not relevant in veterinary practice. When stored at their specific conditions, the androgens in feed were stable for at least 91 days. Real urine samples from a national control program were screened and a representative part of the compliant and suspect samples were confirmed by gas chromatography–tandem mass spectrometry. |
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AbstractList | Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening. Previously we described the construction and properties of a rapid and robust yeast androgen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. In the present study this yeast androgen bioassay was validated as a qualitative screening method for the determination of androgenic activity in calf urine and animal feed. This validation was performed according to EC Decision 2002/657. 20 blank samples were spiked with testosterone, 17alpha-methyltestosterone, 19-nortestosterone, 17beta-trenbolone, 17beta-boldenone or 17alpha-methylboldenone at 2 or 15 ng mL-1 in urine and 50 or 100 ng g-1 in feed. All blank and spiked samples fulfilled the CCalpha and CCbeta criterions, meaning that all 20 blank samples gave signals below the determined decision limits CCalpha and were thus classified as compliant (alpha = 1%). For each component, at least 19 out of the 20 spiked samples gave a signal above the CCalpha and were thus classified as suspect (beta = 5%). The method was specific, and high amounts of dexamethasone did not interfere with the outcome of the test. Although high levels of 17alpha-ethynylestradiol can significantly inhibit the response obtained with low amounts of androgens, that situation is not relevant in veterinary practice. When stored at their specific conditions, the androgens in feed were stable for at least 91 days. Real urine samples from a national control program were screened and a representative part of the compliant and suspect samples were confirmed by gas chromatography-tandem mass spectrometry. Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening. Previously we described the construction and properties of a rapid and robust yeast androgen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. In the present study this yeast androgen bioassay was validated as a qualitative screening method for the determination of androgenic activity in calf urine and animal feed. This validation was performed according to EC Decision 2002/657. 20 blank samples were spiked with testosterone, 17 alpha -methyltestosterone, 19-nortestosterone, 17 beta -trenbolone, 17 beta -boldenone or 17 alpha -methylboldenone at 2 or 15 ng mL[super]-1 in urine and 50 or 100 ng g[super]-1 in feed. All blank and spiked samples fulfilled the CC alpha and CC beta criterions, meaning that all 20 blank samples gave signals below the determined decision limits CC alpha and were thus classified as compliant ( alpha = 1%). For each component, at least 19 out of the 20 spiked samples gave a signal above the CC alpha and were thus classified as suspect ( beta = 5%). The method was specific, and high amounts of dexamethasone did not interfere with the outcome of the test. Although high levels of 17 alpha -ethynylestradiol can significantly inhibit the response obtained with low amounts of androgens, that situation is not relevant in veterinary practice. When stored at their specific conditions, the androgens in feed were stable for at least 91 days. Real urine samples from a national control program were screened and a representative part of the compliant and suspect samples were confirmed by gas chromatography-tandem mass spectrometry. Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening. Previously we described the construction and properties of a rapid and robust yeast androgen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. In the present study this yeast androgen bioassay was validated as a qualitative screening method for the determination of androgenic activity in calf urine and animal feed. This validation was performed according to EC Decision 2002/657. 20 blank samples were spiked with testosterone, 17α-methyltestosterone, 19-nortestosterone, 17β-trenbolone, 17β-boldenone or 17α-methylboldenone at 2 or 15 ng mL −1 in urine and 50 or 100 ng g −1 in feed. All blank and spiked samples fulfilled the CCα and CCβ criterions, meaning that all 20 blank samples gave signals below the determined decision limits CCα and were thus classified as compliant ( α = 1%). For each component, at least 19 out of the 20 spiked samples gave a signal above the CCα and were thus classified as suspect ( β = 5%). The method was specific, and high amounts of dexamethasone did not interfere with the outcome of the test. Although high levels of 17α-ethynylestradiol can significantly inhibit the response obtained with low amounts of androgens, that situation is not relevant in veterinary practice. When stored at their specific conditions, the androgens in feed were stable for at least 91 days. Real urine samples from a national control program were screened and a representative part of the compliant and suspect samples were confirmed by gas chromatography–tandem mass spectrometry. Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening. Previously we described the construction and properties of a rapid and robust yeast androgen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. In the present study this yeast androgen bioassay was validated as a qualitative screening method for the determination of androgenic activity in calf urine and animal feed. This validation was performed according to EC Decision 2002/657. 20 blank samples were spiked with testosterone, 17alpha-methyltestosterone, 19-nortestosterone, 17beta-trenbolone, 17beta-boldenone or 17alpha-methylboldenone at 2 or 15 ngmL(-1) in urine and 50 or 100 ngg(-1) in feed. All blank and spiked samples fulfilled the CCalpha and CCbeta criterions, meaning that all 20 blank samples gave signals below the determined decision limits CCalpha and were thus classified as compliant (alpha=1%). For each component, at least 19 out of the 20 spiked samples gave a signal above the CCalpha and were thus classified as suspect (beta=5%). The method was specific, and high amounts of dexamethasone did not interfere with the outcome of the test. Although high levels of 17alpha-ethynylestradiol can significantly inhibit the response obtained with low amounts of androgens, that situation is not relevant in veterinary practice. When stored at their specific conditions, the androgens in feed were stable for at least 91 days. Real urine samples from a national control program were screened and a representative part of the compliant and suspect samples were confirmed by gas chromatography-tandem mass spectrometry. |
Author | Heskamp, Henri H. Nielen, Michel W.F. Lasaroms, Johan J.P. Bor, Gerrit Sanders, Marieke B. Bovee, Toine F.H. |
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CitedBy_id | crossref_primary_10_1002_dta_3024 crossref_primary_10_1016_j_foodcont_2011_03_027 crossref_primary_10_1002_dta_3049 crossref_primary_10_1080_19440049_2012_745098 crossref_primary_10_3390_s21030893 crossref_primary_10_1080_19440049_2011_609492 crossref_primary_10_1021_ac900874m crossref_primary_10_1016_j_foodchem_2022_133610 crossref_primary_10_4155_bio_09_136 crossref_primary_10_1016_j_scitotenv_2014_05_100 crossref_primary_10_1016_j_tiv_2012_06_003 crossref_primary_10_3390_s130202148 crossref_primary_10_1016_j_aca_2010_11_016 crossref_primary_10_3920_WMJ2014_1845 crossref_primary_10_1016_j_chroma_2009_03_045 crossref_primary_10_1002_etc_618 crossref_primary_10_1080_09593330_2016_1144797 crossref_primary_10_1016_j_foodchem_2016_11_116 crossref_primary_10_1016_j_tiv_2012_04_012 crossref_primary_10_3390_biom11020167 crossref_primary_10_1039_c0an00515k crossref_primary_10_1016_j_jsbmb_2009_10_007 crossref_primary_10_1007_s00216_010_4401_5 crossref_primary_10_1080_10408444_2019_1576588 crossref_primary_10_1007_s12161_018_1164_7 crossref_primary_10_1016_j_trac_2010_06_010 crossref_primary_10_1007_s00216_010_3605_z crossref_primary_10_1007_s00216_009_3265_z |
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Keywords | Validation Androgens Hormone abuse Bioassay Steroids Human Hormone Steroid Urine Biological indicator Protein Gas chromatography Mass spectrometry MS/MS |
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Snippet | Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening. Previously we described the construction and properties of a rapid... |
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SubjectTerms | Analytical chemistry Androgens Androgens - analysis Androgens - metabolism Androgens - urine Animal Feed - analysis Animals Applied sciences Beta Bioassay Biological Assay - methods Blanks Cattle - urine Chemistry Chromatographic methods and physical methods associated with chromatography Dexamethasone Exact sciences and technology Gas chromatographic methods Global environmental pollution Green Fluorescent Proteins - metabolism Hormone abuse Humans Luminescent Agents - metabolism Pollution Receptors, Androgen - metabolism Reproducibility of Results Screening Spectrometric and optical methods Steroids Substance Abuse Detection - methods Time Factors Urine Validation Veterinary Yeast Yeasts - metabolism |
Title | Validation and application of a yeast bioassay for screening androgenic activity in calf urine and feed |
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