Lena Ekström

Doping Test Results Dependent on Genotype of Uridine Diphospho-Glucuronosyl Transferase 2B17, the Major Enzyme for Testosterone Glucuronidation

CONTEXT Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone (T/E) ratio, levels above 4.0 being considered suspicious. The large variation in testosterone glucuronide (TG) excretion and its strong association with a deletion polymorphism in the uridine diphospho-glucuronosyl transferase (UGT) 2B17 gene challenge the accuracy of the T/E ratio test. OBJECTIVE Our objective was to investigate whether genotype-based cutoff values will improve the sensitivity and specificity of the test. DESIGN This was an open three-armed comparative study. PARTICIPANTS A total of 55 healthy male volunteers with either two, one, or no allele [insertion/insertion, insertion/deletion, or deletion/deletion (del/del)] of the UGT2B17 gene was included in the study. INTERVENTION A single im dose of 500 mg testosterone enanthate was administered. MAIN OUTCOME MEASURES Urinary excretion of TG after dose and the T/E ratio during 15 d were calculated. RESULTS The degree and rate of increase in the TG excretion rate were highly dependent on the UGT2B17 genotype with a 20-fold higher average maximum increase in the insertion/insertion group compared with the del/del group. Of the del/del subjects, 40% never reached the T/E ratio of 4.0 on any of the 15 d after the dose. When differentiated cutoff levels for the del/del (1.0) and the other genotypes (6.0) were applied, the sensitivity increased substantially for the del/del group, and false positives in the other genotypes were eliminated. CONCLUSIONS Consideration of the genetic variation in disposition of androgens will improve the sensitivity and specificity of the testosterone doping test. This is of interest not only for combating androgen doping in sports, but also for detecting and preventing androgen abuse in society.

Studies on the Transcriptional Regulation of Cholesterol 24-Hydroxylase (CYP46A1) MARKED INSENSITIVITY TOWARD DIFFERENT REGULATORY AXES

Mammalian CNS contains a disproportionally large and remarkably stable pool of cholesterol. Despite an efficient recycling there is some requirement for elimination of brain cholesterol. Conversion of cholesterol into 24S-hydroxycholesterol by the cholesterol 24-hydroxylase (CYP46A1) is the quantitatively most important mechanism. Based on the protein expression and plasma levels of 24S-hydroxycholesterol, CYP46A1 activity appears to be highly stable in adults. Here we have made a structural and functional characterization of the promoter of the human CYP46A1 gene. No canonical TATA or CAAT boxes were found in the promoter region. Moreover this region had a high GC content, a feature often found in genes considered to have a largely housekeeping function. A broad spectrum of regulatory axes using a variety of promoter constructs did not result in a significant transcriptional regulation. Oxidative stress caused a significant increase in transcriptional activity. The possibility of a substrate-dependent transcriptional regulation was explored in vivo in a sterol-deficient mouse model (Dhcr24 null) in which almost all cholesterol had been replaced with desmosterol, which is not a substrate for CYP46A1. Compared with heterozygous littermates there was no statistically significant difference in the mRNA levels of Cyp46a1. During the first 2 weeks of life in the wild-type mouse, however, a significant increase of Cyp46a1 mRNA levels was found, in parallel with an increase in 24S-hydroxycholesterol level and a reduction of cholesterol synthesis. The failure to demonstrate a significant transcriptional regulation under most conditions is discussed in relation to the turnover of brain and neuronal cholesterol.

Vitamin D3 supplementation in patients with frequent respiratory tract infections: a randomised and double-blind intervention study

Background Low serum levels of 25-hydroxyvitamin D3 are associated with an increased risk of respiratory tract infections (RTIs). Clinical trials with vitamin D3 against various infections have been carried out but data are so far not conclusive. Thus, there is a need for additional randomised controlled trials of effects of vitamin D3 on infections. Objective To investigate if supplementation with vitamin D3 could reduce infectious symptoms and antibiotic consumption among patients with antibody deficiency or frequent RTIs. Design A double-blind randomised controlled trial. Setting Karolinska University Hospital, Huddinge. Participants 140 patients with antibody deficiency (selective IgA subclass deficiency, IgG subclass deficiency, common variable immune disorder) and patients with increased susceptibility to RTIs (>4 bacterial RTIs/year) but without immunological diagnosis. Intervention Vitamin D3 (4000 IU) or placebo was given daily for 1 year. Primary and secondary outcome measures The primary endpoint was an infectious score based on five parameters: symptoms from respiratory tract, ears and sinuses, malaise and antibiotic consumption. Secondary endpoints were serum levels of 25-hydroxyvitamin D3, microbiological findings and levels of antimicrobial peptides (LL-37, HNP1–3) in nasal fluid. Results The overall infectious score was significantly reduced for patients allocated to the vitamin D group (202 points) compared with the placebo group (249 points; adjusted relative score 0.771, 95% CI 0.604 to 0.985, p=0.04). Limitations A single study centre, small sample size and a selected group of patients. The sample size calculation was performed using p=0.02 as the significance level whereas the primary and secondary endpoints were analysed using the conventional p=0.05 as the significance level. Conclusions Supplementation with vitamin D3 may reduce disease burden in patients with frequent RTIs.

Novel route for elimination of brain oxysterols across the blood-brain barrier: Conversion into 7α-hydroxy-3-oxo-4-cholestenoic acid

Recently, we demonstrated a net blood-to-brain passage of the oxysterol 27-hydroxycholesterol corresponding to 4–5 mg/day. As the steady-state levels of this sterol are only 1–2 μg/g brain tissue, we hypothesized that it is metabolized and subsequently eliminated from the brain. To explore this concept, we first measured the capacity of in vitro systems representing the major cell populations found in the brain to metabolize 27-hydroxycholesterol. We show here that 27-hydroxycholesterol is metabolized into the known C27 steroidal acid 7α-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7α-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7α-hydroxy-3-oxo-4-cholestenoic acid in plasma from the internal jugular vein and brachial artery of healthy volunteers. Calculation of the arteriovenous concentration difference revealed a significant in vivo flux of this steroid from the brain into the circulation in human. Together, these studies identify a novel metabolic route for the elimination of 27-hydroxylated sterols from the brain. Given the emerging connections between cholesterol and neurodegeneration, this pathway may be of importance for the development of these conditions.

Deletion polymorphism of the UGT2B17 gene is associated with increased risk for prostate cancer and correlated to gene expression in the prostate.

Metabolism of androgens includes glucuronidation, the major pathway of steroid elimination in several steroid target tissues. Glucuronidation is catalysed by UDP-glucuronosyltransferases (UGTs). UGT2B17 has been shown to be particularly active against androgens and is highly abundant in the prostate. Recently, we discovered that deletion of the UGT2B17 gene is associated with low or undetectable urinary testosterone levels. Here, we determined the phenotypic outcome of the deletion by quantifying the UGT2B17 mRNA expression in normal prostate tissues in individuals with different genotypes. Additionally, the frequency of UGT2B17 deletion polymorphism was studied in a Swedish population-based case–control study including 176 patients diagnosed with prostate cancer and 161 controls. We found that the individuals homozygous for the insertion allele expressed 30 times more UGT2B17 mRNA in prostate tissue than the heterozygotes. Carriers of the deletion allele had a significantly increased risk of prostate cancer (OR=2.07; 95% CI=1.32–3.25). In conclusion, these results show the UGT2B17 deletion polymorphism is associated with prostate cancer risk.

Rapid Single-Nucleotide Polymorphism Detection of Cytochrome P450 (CYP2C9) and Vitamin K Epoxide Reductase (VKORC1) Genes for the Warfarin Dose Adjustment by the SMart-Amplification Process Version 2

BACKGROUND Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (-1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required. METHODS We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 -1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h after blood collection. RESULTS We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance. CONCLUSIONS The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2-based diagnostics have key advantages.

Genetic aspects of epitestosterone formation and androgen disposition: influence of polymorphisms in CYP17 and UGT2B enzymes.

Objective Testosterone is a commonly abused androgen in sports and in the gym culture of the society. Its abuse is conventionally disclosed by urinary assay of the testosterone/epitestosterone (T/E) glucuronide ratio, which should not exceed 4. A noteworthy number of athletes, however, have higher natural ratios than 4, most likely because of decreased excretion of epitestosterone glucuronide. Falsely positive doping test results are of great concern for the legal rights of the sportsman. Our objective was to study the genetic aspects of epitestosterone formation, and to elucidate the impact of genetic variation in androgen-metabolizing enzymes. Methods Urine from different study populations was analysed for androgen glucuronides by gas chromatography-mass spectrometry. All men were genotyped for the uridine diphospho-glucuronosyltransferase (UGT) 2B17 deletion polymorphism and single nucleotide polymorphisms in the cytochrome P-450c17&agr; (CYP17), UGT2B15 and UGT2B7 genes. Expression of UGT2B15 mRNA in human liver samples was analysed using real-time PCR. Results A T>C (A1>A2) promoter polymorphism in the CYP17 gene was associated with the urinary glucuronide levels of epitestosterone and its putative precursor androstene-3&bgr;, 17&agr;-diol, resulting in 64% higher T/E ratios in A1/A1 homozygotes. Individuals devoid of UGT2B17 had significantly higher UGT2B15 mRNA levels in liver than individuals carrying two functional UGT2B17 alleles. Conclusion The CYP17 promoter polymorphism may partly explain high natural (>4) T/E ratios. Our data indicate that 5-androstene-3&bgr;, 17&agr;-diol is an important precursor of epitestosterone and that CYP17 is involved in its production. In addition, we found that lack of the UGT2B17 enzyme may be compensated for by increase in UGT2B15 transcription.

Radical prostatectomy: Influence on serum and urinary androgen levels

The role of the prostate as an active endocrine organ and the hormonal changes after radical prostatectomy (RP) has not been well studied. The objective of this study was to investigate the serum and urine hormonal changes after RP.

The UGT2B17 Gene Deletion is Not Associated With Prostate Cancer Risk

Deletion polymorphism of the UDP‐glucuronosyltransferase 2B17 (UGT2B17) gene has been associated with an increased prostate cancer risk in two previous independent studies. Here we determine the risk in a large‐scale population‐based case–control study.

Substantial advantage of a combined Bayesian and genotyping approach in testosterone doping tests

Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone (T/E) ratio, levels above 4.0 being considered suspicious. A deletion polymorphism in the gene coding for UGT2B17 is strongly associated with reduced testosterone glucuronide (TG) levels in urine. Many of the individuals devoid of the gene would not reach a T/E ratio of 4.0 after testosterone intake. Future test programs will most likely shift from population based- to individual-based T/E cut-off ratios using Bayesian inference. A longitudinal analysis is dependent on an individuals true negative baseline T/E ratio. The aim was to investigate whether it is possible to increase the sensitivity and specificity of the T/E test by addition of UGT2B17 genotype information in a Bayesian framework. A single intramuscular dose of 500mg testosterone enanthate was given to 55 healthy male volunteers with either two, one or no allele (ins/ins, ins/del or del/del) of the UGT2B17 gene. Urinary excretion of TG and the T/E ratio was measured during 15 days. The Bayesian analysis was conducted to calculate the individual T/E cut-off ratio. When adding the genotype information, the program returned lower individual cut-off ratios in all del/del subjects increasing the sensitivity of the test considerably. It will be difficult, if not impossible, to discriminate between a true negative baseline T/E value and a false negative one without knowledge of the UGT2B17 genotype. UGT2B17 genotype information is crucial, both to decide which initial cut-off ratio to use for an individual, and for increasing the sensitivity of the Bayesian analysis.