Effects of intravenous infusion of glycerol on blood parameters and urinary glycerol concentrations.

In sports, the oral intake and intravenous administration of glycerol as a potential masking agent have been prohibited. The effect of glycerol on blood parameters was investigated by comparing the intravenous administration of glycerol (20g/200mL) with that of an electrolyte (8g glucose/200mL) as a comparator (n=7, fixed-dose-rate i.v. infusion, 200mL in 1h). This study was also designed to evaluate whether the urinary concentrations reached the positivity threshold after the intravenous infusion of glycerol. Significant decreases of the haemoglobin (HGB, g/dL), haematocrit (HCT, %) and OFF-h Score (OFF-score) values were observed after the infusion of glycerol (P<0.05 at 1-6h). The differences in the HGB, HCT and OFF-score between pre- and post-administration were -0.49±0.23g/dL (2h), -1.54±0.73% (2h) and -3.89±3.66 (2h), respectively. Glycerol infusion significantly increased the plasma volume by 12.1% (1h), 6.3% (2h) and 5.7% (3h) compared with the initial values. The infusion of the comparator also increased the plasma volume by 9.6% (1h), 5.8% (2h) and 4.9% (3h) compared with the values before infusion. There were no significant differences in the change of the plasma volume between the intravenous infusions of glycerol and the glucose-based electrolyte (as the comparator) (P≥0.05). This finding might indicate that glycerol itself only exhibited limited effects on the expansion of plasma. After administration of glycerol, the urinary glycerol concentrations increased from 0.0013±0.0004mg/mL to 6.86±2.86mg/mL at 1h and 6.45±3.08mg/mL at 2h. The intravenous infusion of glycerol can most likely be detected using the current urine analysis; however, the dependence of the concentration of urinary glycerol on the urine volume should be considered.

Comparison of urine analysis and dried blood spot analysis for the detection of ephedrine and methylephedrine in doping control.

When the misuse of stimulants is determined in doping control tests conducted during the in-competition period, athletes are asked to account for the violation of the rules. This study was designed to evaluate whether the urinary threshold values (10 µg/mL) for ephedrine and methylephedrine set by the World Anti-Doping Agency (WADA) can be exceeded after the oral administration of each substance (25 mg). In addition, the study describes the validity of a liquid chromatography-tandem mass spectrometric method using dried blood spot testing to detect ephedrine and methylephedrine by comparing it to a quantitative laboratory urine assay. After administration of ephedrine, the urinary concentration of ephedrine did not exceed the threshold at 4-10 h in two subjects, whereas the threshold was exceeded in both the subjects at 12 h after administration. For methylephedrine, the urinary concentrations of all the subjects failed to reach the threshold for up to 10 h after administration. The concentrations reached the threshold at 12-24 h after administration in some volunteers. In contrast, the blood concentrations of ephedrine and methylephedrine reached their maximum levels at 2-8 h after administration. The blood concentrations showed a low inter-individual variability, and the results suggested that the urinary excretion of ephedrine and methylephedrine can be strongly affected by urine pH and/or urine volume. These facts suggest that urinary concentrations cannot reflect the psychoactive level of ephedrines in circulation. Thus, dried blood analysis might be suitable for the adequate detection of stimulants during in-competition testing.

Possibility of analytical finding of glycerol caused by self-catheterization in doping control.

Glycerol is listed on the World Anti-Doping Agency (WADA) prohibited list as a masking agent principally because the administration of glycerol increases plasma volume and decreases the concentration of haemoglobin and the value of haematocrit in blood. Glycerol is a naturally occurring substance; therefore, the threshold is set as 1.0 mg/mL in the WADA technical document (WADA TD2013DL). In a WADA-accredited doping control laboratory, three doping control urine specimens collected from impaired athletes were determined to contain a high concentration of glycerol (>1.0 mg/mL); two of these specimens were considered adverse analytical findings (AAFs). Self-catheterization is necessary for athletes with neurological disorders such as neurogenic bladder dysfunction. We conducted a simple simulation of self-catheterization as an experimental test using urethral catheters with an antiseptic agent containing glycerol to confirm the influence of this antiseptic agent on the quantitative value of glycerol in doping control analysis. Some users employ a catheter with glycerol solution (ca. 1 mL) to avoid pain during use. The urine sample passed through such a catheter exhibited a glycerol concentration (4.94 mg/mL) greater than the threshold level. In September 2014, the threshold for glycerol will change from 1.0 to 4.3 mg/mL (WADA TD2014DL); however, a possibility exists for the quantitative value of glycerol in doping control analysis to exceed the threshold because of the use of an antiseptic agent containing glycerol for self-catheterization. The AAF for glycerol for impaired athletes, particularly those who participate in Paralympic sports, should account for the use of a catheter with glycerol.

UDP-glucuronosyltransferase 2B17 genotyping in Japanese athletes and evaluation of the current sports drug testing for detecting testosterone misuse.

Ethnicity has been found to influence urinary testosterone glucuronide to epitestosterone glucuronide (T/E) ratios among athletes. Uridine diphospho-glucuronosyltransferase 2B17 (UGT2B17) is the most active enzyme in testosterone glucuronidation. UGT2B17 polymorphism analysis is rarely performed in Japanese athletes, and the influence of testosterone administration on steroid profiles and carbon isotope ratios, according to gene polymorphisms, in Asians remains unknown. The prevalence of UGT2B17 genotypes and urinary androgenic steroid profiles, classified according to UGT2B17 genotypes, was investigated in Japanese athletes (255 male and 256 female). Testosterone enanthate (100 mg) was administered intramuscularly to Japanese female volunteers (del/del: n = 6, del/ins: n = 3, ins/ins: n = 1). The distribution rates of the UGT2B17 del/del genotype in Japanese male and female athletes were 74.5% and 60.2%, respectively. The ins/ins genotype was detected in only three male (1.2%) and seven female (2.7%) athletes. The prevalence of the UGT2B17 deletion genotype was extremely high in Japanese athletes. The T/E ratio in the del/del group was significantly lower than that in the other groups. After testosterone was administered to female volunteers, the T/E ratios for the del/del individuals failed to reach the positivity criterion of 4. By contrast, in all of the del/del subjects, the gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS) analysis successfully fulfilled the positivity criterion. The overall result has demonstrated the limited effectiveness of population-based T/E ratios in screening tests for testosterone use. Subject-based steroid profiling with UGT2B17 genotyping will be an effective strategy for detecting testosterone misuse.

Effectiveness of GH isoform differential immunoassay for detecting rhGH doping on application of various growth factors.

The analytical method for detecting growth hormone (GH) doping, the so-called GH isoform differential immunoassay, is currently approved by the World Anti-Doping Agency (WADA). Anti-doping laboratories often face challenges by athletes' lawyers and need to have various types of scientific evidence against the claim that the adverse analytical finding (AAF) result was caused by excess ectopic or abnormal excretion. In this work, a population study of Japanese athletes (255 male and 256 female) and administration studies of recombinant human GH (rhGH) in Japanese females were conducted to confirm the applicability of GH isoform differential immunoassay. The present paper describes the effectiveness of the GH isoform differential immunoassay under abnormal excretion of endogenous GH as determined by administration studies of GH releasing hormone (GHRH(1-44)) and insulin-like growth factor-1 (IGF-1). No false positive findings were found in Japanese athletes. The GH isoform differential immunoassays could detect application of rhGH for approximately 12-24 h. The administration of GHRH(1-44) and IGF-1 as well as ghrelin receptor agonists did not affect the isoform ratio (no false positives). We conclude that the GH isoform differential immunoassay is a highly specific method for detecting rhGH doping. Subject-based profiling (i.e. athlete biological passport) very likely will represent a highly sensitive approach for detecting rhGH doping.

Influence of intravenous administration of growth hormone releasing peptide-2 (GHRP-2) on detection of growth hormone doping: growth hormone isoform profiles in Japanese male subjects.

Administration of exogenous 22 kDa recombinant human growth hormone (rhGH) suppresses the non-22 kDa pituitary growth hormone (GH) secretion by negative feedback; then, the elevated 22 kDa GH to non-22 kDa GH ratio (Rec/Pit ratio) can be utilized to detect doping with rhGH (isoform differential immunoassay). The influence of intravenous administration of growth hormone releasing peptide GHRP-2 on the isoform differential immunoassay for detecting rhGH doping has been investigated.In this study, a reference population (n=100) was used, with 0.04 mg/kg rhGH subcutaneous administration (n=5), 100 µg of GHRP-2 intravenous administration (n=10) and 0.04 mg/kg rhGH combined with 100 µg of GHRP-2 (n=10) in Japanese male subjects. The results indicated that the low dose (0.04 mg/kg) of rhGH led to significantly increased Rec/Pit ratio compared with the Japanese reference limit (P < 0.001). Because GHRP-2 dose led to increases in concentrations of both recombinant GH (recGH) and pituitary GH (pit GH), no significant change in the Rec/Pit ratio was observed (P > 0.05). In a combined administration study, after GHRP-2 dose the Rec/Pit ratios decreased to 39.9-43.9% compared with the elevated ratio caused by the rhGH dose.The results indicated that GHRP-2 administration cannot only be detected by the isoform differential immunoassay but also masks rhGH doping. The analysis of GHRP-2 was found to be suitable for compensating for the disadvantages of the isoform differential immunoassay because GHRP-2 and its metabolite (AA-3) in urine could be detected during the periods of masking of the Rec/Pit ratio by means of liquid chromatography/tandem mass spectrometry.