Presence and metabolism of natural steroids in meat-producing animals: current knowledge and potential strategies for detecting their abuse

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1 Presence and metabolism of natural steroids in meat-producing animals: current knowledge and potential strategies for detecting their abuse James Scarth, HFL, UK October 2007

2 Overview of presentation Context of the current project Background to the literature survey General trends in the data Summary of occurrence in each species Recommendations of the review Future work Questions

3 Context of the current project: A study on the natural steroid hormone levels in meat producing animals Sponsor: DEFRA (Dept of Environment, Food and Rural affairs) Project milestones over the next three years: 1) A review of the existing literature, culminating in a peer reviewed report 2) Agree details of the experimental phase based on the issues raised in milestone 1 3) Develop appropriate method/s 4) Validate method/s in GLP fashion 5) Obtain appropriate samples and analysed using validated method/s 6) Apply appropriate statistical approaches to data generate in milestone 5 7) Produce final project report

4 Milestone 1: a review of the existing literature Much research in the area of natural hormone residue analysis has been carried out, although no general consensus document on the topic exists The aim was therefore to review the literature and identify A) areas where sufficient data exists to make firm recommendations and B) areas where more research is needed This involved gathering as much published and unpublished data on individual animal results and population summary statistics as possible. In all, around 1000 published papers in the area were collected Laboratories that supplied extensive data were: LGC and HFL UK LABERCA France Uni of Ghent Belgium The Community Reference Laboratory The Netherlands The Canadian Food Inspection Agency The Australian Dept of Agriculture, Fisheries and Forestry

5 Which steroids were considered? Both natural and synthetic steroids may be used. The detection of synthetic steroids is relatively easy to achieve, but the grey area is the detection of natural steroids (the focus of the current project) Classes of natural steroids involved and their major catabolic transformations: Precursors of some of other natural steroids that may also be considered when designing methods to control steroid abuse:

6 Which species were considered? Mammalian (no poultry): Bovine Ovine Porcine Equine Caprine Cervine

7 Species differences in circulating estradiol and progesterone levels

8 Species differences in the route of excretion of different steroids Excretion of radioactivity in different waste products following intravenous injection Adapted from Martin 1966, Calvert et al. 1975, Velle 1976 and Palme et al. 1996

9 Methods used for analysing natural hormones in the published bovine literature

10 Matrices used for analysing natural hormones in the published bovine literature

11 Publication dates for natural hormones in the published bovine literature reviewed

12 Natural hormone population data A significant challenge in natural hormone analysis is the number of resulting values that are below the limit of detection (LOD). A normal distribution of the population data is rarely possible, so non-parametric statistical approaches are usually required A typical natural hormone population histogram; levels of epinandrolone (ngml -1 ) in sheep urine (from Casson et al. 2006)

13 Natural steroids in the bovine Testosterone - ubiquitous in males and females. One report of naturally high levels during the normal estrous cycle (immunoassay - unconfirmed). Artificial selection has been shown to affect testosterone levels in some breeds. Lack of data for pregnant animals. Major urinary metabolites include epitestosterone, epietiocholanolone, 5β-androstane-3α,17β-diol and 5β-androstane-3β,17α-diol Nandrolone epinandrolone endogenous during pregnancy in females. Some reports of natural levels in males subsequent to casualty (more work required), but not in normal males or females. Major urinary metabolites include epinandrolone, 5β-estrane-3α,17β-diol, 5β-estrane-3α,17α-diol and 5α-estrane-3β,17α-diol Boldenone not detected in plasma/serum, but urine and faeces have been shown to contain boldenone related compounds, possible secondary to their formation in the gut by bacteria. Major urinary metabolites include epiboldenone, 17α-hydroxy-5β-androst-1-en-3-one, 6β-hydroxyepiboldenone, 17β-hydroxy-5β-androst-1-en-3-one and 3α-hydroxy-5β-androst-1-en-17-one.

14 Natural steroids in the bovine Estradiol - ubiquitous in males and females. One report of naturally high estradiol levels following liver infection (immunoassay unconfirmed). One report stating that peak estradiol levels were higher in heifers than lactating cows during the summer, but not during the winter. One report showing that artificial selection can affect estrogen levels. Levels increase during pregnancy. Major urinary metabolite is epiestradiol Progesterone ubiquitous in males and females. Artificial selection has been shown to affect progesterone levels in some breeds. One report stating that peak progesterone levels were higher in heifers than lactating cows during the summer, but not during the winter. Levels in crease during pregnancy. Lack of metabolism studies mean that major urinary metabolites cannot be listed Cortisol ubiquitous in males and females. Stress is a major determinant on cortisol levels, although diet, medication and disease may also play a role. Lack of metabolism studies mean that major urinary metabolites cannot be listed

15 Natural steroids in the ovine Testosterone - ubiquitous in males and females. Testosterone levels vary with the season in male animals. Levels increased in freemartins and after naloxone treatment in male animals. One metabolism study found epitestosterone as a major urinary metabolite, although more work needed Nandrolone several reports of epinandrolone in pregnant females. One report of nandrolone and epinandrolone at a high frequency in male and female urine, although the population was not controlled. Lack of metabolism studies mean that major urinary metabolites cannot be listed Boldenone general lack of data. Two of 961 urines from Australian national monitoring programme contain low levels of epiboldenone Estradiol data collected, but not yet analysed in detail Progesterone data collected, but not yet analysed in detail Cortisol data collected, but not yet analysed in detail

16 Natural steroids in the porcine Testosterone - ubiquitous in males and females. Testosterone levels vary with the season and time of day in male animals. Lack of metabolism studies mean that major urinary metabolites cannot be listed 16-androstenes these compounds are produced in several species, but at particularly high levels in boars and contribute to boar taint. They are not liable to abuse as anabolic agents, but are down-regulated after administration of exogenous steroids due to negative feedback loops Nandrolone - nandrolone and 19-norandrostenedione detected in animals of all sexes. 19-nor steroids detected in urine may be sample preparation degradation artefacts of 19-carboxylic acids. A-ring reduced metabolites not detected endogenously, but after administration major metabolites are 19- norepiandrosterone, 19-noretiocholanolone and 5β-estrane-3α,17β-diol Boldenone detected in urine and some other matrices of boars, barrows, gilts and cryptorchids, but not sows or intersex animals. Lack of metabolism studies mean that major urinary metabolites cannot be listed Estradiol, progesterone + cortisol data collected, but not yet analysed in detail

17 Natural steroids in the equine Testosterone ubiquitous in males and females. Major urinary metabolites are 5α-androstane-3β,17β-diol, 5α-androstane-3β,17α-diol + 3β-hydroxy-5α-androstan-17-one Nandrolone detected in stallions and pregnant mares, but not geldings or fillies. The majority of 19-nor steroids detected in urine are sample preparation artefacts of 19-carboxylic acids. Major urinary metabolite is 5α-estrane-3β,17α-diol Boldenone - detected at low levels in the urine of stallions, but not geldings or fillies. Major urinary metabolite is epiboldenone. Estradiol data collected, but not yet analysed in detail Progesterone data collected, but not yet analysed in detail Cortisol data collected, but not yet analysed in detail

18 Natural steroids in the caprine Testosterone - ubiquitous in males and females. Data collected, but not yet analysed in detail Nandrolone epinandrolone, but not nandrolone, detected in urine during pregnancy, while neither analyte detected in non-pregnant females. Studies on endogenous levels in males is lacking. Lack of metabolism studies mean that major urinary metabolites cannot be listed Boldenone data on endogenous levels or metabolism following exogenous administration in this species is lacking Estradiol data collected, but not yet analysed in detail Progesterone data collected, but not yet analysed in detail Cortisol data collected, but not yet analysed in detail

19 Natural steroids in the cervine Testosterone - ubiquitous in males and females. Data collected, but not yet analysed in detail Nandrolone One study reported detecting epinandrolone in the urine of a pregnant red deer, but no published studies have looked at non-pregnant females or males or the metabolism following exogenous administration. However, 1 of 35 urines (sex of individual urine unknown) from an Australian national monitoring programme contained epinandrolone at 4.5 ng/ml, but no nandrolone was detected in this sample above the LOD of 1 ng/ml Boldenone no published studies regarding the endogenous presence of boldenone or its metabolism following exogenous administration. However, all 35 urines from an Australian national monitoring programme were negative for boldenone and epiboldenone at an LOD of 1 ng/ml Estradiol data collected, but not yet analysed in detail Progesterone data collected, but not yet analysed in detail Cortisol data collected, but not yet analysed in detail

20 Existing control measures in the bovine Age/sex of animal Female (nonpregnant) Male (< 6-months) Male (> 6-months) EEC decision limit in plasma (ngml -1 ) Estradiol Testosterone Sex of animal Male Female (nonpregnant) UK nandrolone threshold limit in urine (sum of nandrolone + epinandrolone in ngml -1 ) Any level of β-boldenone conjugates in urine is evidence of abuse in the EEC if faecal contamination of urine is avoided during sampling GC-C-IRMS and the detection of intact steroid esters in hair is used in some countries for some endogenous steroids

21 Recommendations of the literature survey report Univariate thresholds for some analyte/matrix combinations in the bovine and ovine were proposed Several physiological situations were found that could lead to false positive results Areas where more significantly more research is needed were highlighted Constantly lower LODs for analytes inevitably means that steroids will be found as endogenous in more species and matrices as methods become more sensitive

22 Future work in the UK Development of validated high sensitivity quantitative multiresidue methods in urine using GC-MS/MS Collection and analysis of large populations of bovine, ovine and porcine urine from animals with known medication history Statistical analysis of generated data with a view to setting uni/multi-variate thresholds for controlling the abuse of testosterone, nandrolone, boldenone, estradiol and progesterone Publication of a final report on the DEFRA website

23 Acknowledgements: VMD, UK Jack Kay RIVM, NL Leen Van Ginkel LABERCA, FR Bruno Le Bizec Ghent Uni, BL Hubert DeBrabander, Johan Van Hende National Residues Survey AU Wolfgang Korth Canadian Food Inspection Agency Jim McNeill LGC, UK John Points AFBINI, UK Glenn Kennedy CSL, UK Alistair Murray, Roy Macarthur, Sara Stead HFL, UK Phil Teale, Clive Pearce, Simon Biddle

National Institute for Public Health and the Environment Annual CRL workshop 22 October Update on natural Hormone studies

National Institute for Public Health and the Environment Annual CRL workshop 22 October Update on natural Hormone studies 2008 Annual CRL workshop 22 October 2008 Update on natural Hormone studies Natural hormone studies: update Possible approaches - C12/C13 ratio: can result in proof of abuse - Determination of intact esters