We report the results from blood sampling taken for the first time during doping control in athletics. The study includes samples from 99 athletes tested during IAAF-meetings in 1993-94. Blood doping with allogenic blood was not detected. The distribution of haemoglobin levels in athletes did not differ markedly from that found in controls. Erythropoietin (EPO) values were markedly lower in athletes than in controls, and 58% had EPO lower than the detection limit for the assay. This may be due to high-altitude residence prior to testing. Measurements of growth hormone (GH) and insulin-like growth factor 1 did not suggest GH-misuse in any athlete tested. One third of the male athletes had testosterone levels that were lower than the normal reference interval. This may at least partly be due to the combination of sampling at night and after strenuous exercise. One female athlete was found to have a grossly elevated testosterone level. In conclusion, the present results show the importance of taking into account the special circumstances during sampling when interpreting results from blood testing in athletes. Future research should focus on developing more sensitive and specific tests to detect doping with endogenous substances such as GH and EPO.

Six laboratories in six countries collaborated to investigate the analytical method for estimating the testosterone to epitestosterone ratio (T/E) in urine by gas chromatography/mass spectrometry in the context of detecting the application of T as a doping agent in sport. The protocol specified many but not all details of reagents and instrument conditions. The design included the distribution and analysis of four urines with different T/E values, three replicates per value, and one standard. The ranges of mean T/E values for the four urines estimated by peak area (PA) were 0.32-0.42, 0.72-0.94, 0.91-1.14 and 3.19-5.48. The analyses of variance for these data and for the peak height (PH) data were significant for the laboratory factor (p < 0.0001). In addition there was a significant interaction between the urine factor and the laboratory factor which indicates the complexity of the analysis. T/E calculated using PA was not significantly different from that using PH. For within-laboratory precision all values for PH and PA were < 8.3%, and for between-laboratory precision all values were < 11.7% except for one (20.1%). The data represent a baseline for future experiments designed to elucidate the sources of within-and between-laboratory variance, and to harmonize estimates of T/E.

The ratio of urinary testosterone (u-T) to epitestosterone (T/EpiT) is used to disclose testosterone (T) administration in the doping control of sports, and a ratio greater than 6 constitutes an offence. Nevertheless, the possibility of biological outliers must not be discounted, and the use of ketoconazole has been suggested for a dynamic test to distinguish between such athletes and those using T. In this investigation, ketoconazole was administrated to three groups of T-pretreated and two groups of untreated healthy male subjects. The subjects in one of the pretreated groups were patients with mild hypogonadism. One untreated group consisted of athletes that had been tested three times with high urinary T/EpiT levels. The effects of ketoconazole administration on serum T (s-T) level and urinary T/EpiT ratio were monitored every 2 h for an 8-h period and clearly separated T-pretreated and untreated subjects into two clusters (P < 0.0001). The T/EpiT ratio increased and the s-T level remained unchanged in pretreated individuals during the ketoconazole test, whereas T/EpiT decreased by 60% and s-T by almost 90% in untreated subjects. The statistical power of the test increased by using several time points and combining the urinary T/EpiT with the s-T data. In conclusion, the ketoconazole test is suitable as a supportive dynamic test for the urinary T/EpiT ratio measurements in the doping control of athletes.

Anabolic-androgenic steroids are widely misused in human sports and are also used as growth promoters in livestock. Athletes who consume meat containing such hormone residues may risk failing a sports drug test. Prompted by an athlete's defense case, we questioned whether the consumption of small livestock given doses of anabolic steroid, orally or intramuscularly, could generate positive results in samples tested by our analytical procedures. We analyzed urine from eight men who consumed chickens that had been either fed with methenolone acetate (1 mg/day) from day 0 to 21 or injected with methenolone heptanoate depot (1 mg/intramuscular injection) on days 0, 7, and 14 and slaughtered on day 22. No methenolone or characteristic major metabolite was detected in samples from subjects who ate meat from the orally dosed chickens. However, 50% of the samples collected 24 h after consumption of the intramuscularly dosed chickens were confirmed positive. Hence, eating meat containing small amounts of injected hormone may constitute a serious liability to the athlete.

Determination of the ratio of testosterone to epitestosterone (T/E) in urine is used to detect testosterone administration in athletes, with a ratio > 6 considered as evidence of an offense. We show that administration of ketoconazole, which inhibits testosterone biosynthesis, may be useful for differentiating between an athlete who is using testosterone and one who naturally gives a ratio > 6. In a control subject pretreated with testosterone, ketoconazole caused the ratio to increase; conversely, it caused a decrease in the ratio in an athlete under investigation. Repeated administration of ketoconazole to two normal men caused a decrease in the ratio due to a large decrease in the urinary excretion rate of testosterone relative to epitestosterone. Stimulation with human chorionic gonadotropin exacerbated the differences in excretion rates. A single administration of ketoconazole to six normal men caused the T/E ratios to decrease significantly within 8 h, a suitable time scale for use in a dynamic test.

Misuse of drugs and methods of doping in connection with various physical activities have become serious problems for sports organizations and may seriously impair the health of the misusers. The Norwegian Confederation of Sports has banned doping, and carries out doping controls at competitions as well as out-of-competition tests. Doping controls and laboratory analyses are performed according to approved procedures. An athlete who is found guilty of doping will be excluded from organized sport for a specified period of time. Doctors have a special duty to keep themselves updated on the doping problem, and support the anti-doping work by their own practices.

A 37-year-old woman presented with a history of secondary amenorrhoea and hirsutism for 4 years. She had elevated serum levels of testosterone and dihydrotestosterone, and decreased serum levels of sex hormone binding globulin and oestradiol. Almost daily use of a testosterone-containing ointment in the vulvar region for 6 years was.disclosed as the cause of the hyperandrogenism. Serum testosterone, testosterone excretion rate in urine and testosterone/epitestosterone ratio in urine were determined at fixed intervals 24 h before and 48 h after application of the testosterone-containing ointment. There was a rapid increase in serum testosterone, with a peak level after 4-6 h. The testosterone excretion rate and the testosterone/epitestosterone ratio in urine peaked after 2-4 h. After 48 h the serum testosterone level was still about twice the basal value. The testosterone/epitestosterone level was over the 'doping limit' of 6 for 28 h. We conclude that determination of the testosterone/epitestosterone ratio in urine would have disclosed exogenous testosterone administration in this patient. We recommend this test for patients in whom exogenous testosterone administration is suspected.

The analysis of body fluids for drugs takes place in two distinctly different environments: the medical model and the penalty model. In the former, and in the context of patient care, the physician requests the test and the patient is both fully co-operative with the sample collectors and highly motivated to provide the ideal sample. In the latter case, the request usually comes from a non-medical authority and it may not be in the best interest ofthe individual to submit a valid sample. In the medical model the information obtained from the test is used to assist the physician in the care and management of the patient. In contrast, in the penalty model the results are used to impose some penalty to the individual. This fundamental difference, together with legal imperatives, has broad implications for the overall design and implementation of a drug testing programme.

An increased ratio between urinary testosterone (T) and epitestosterone (epiT) has been accepted by the International Olympic Committee as a marker for T doping. However, in a few subjects, we and others have observed constantly above-normal urinary T/epiT ratios that are unlikely to be related to exogenous T administration. To find a better test for T doping, we studied several serum and urinary androgens and androgen precursors, estrogens, and luteinizing hormone (LH) in seven healthy volunteers for 35 days after an intramuscular injection of 250 mg of testosterone enanthate. Among urinary analyses, only the T/epiT ratio was a suitable marker of T doping; of the serum assays, 17-alpha-hydroxyprogesterone (17OHP), T/17OHP ratio, LH, and T/LH ratio were fair to good markers of T doping. The serum T/17OHP ratio was the best marker of those tested, with all seven subjects having above-normal values for this in the first 3 days of the observation period. No other marker showed abnormal values in all subjects at any time. Moreover, the T/17OHP ratio was affected by neither diurnal variation nor physical stress. The value of this marker for T doping was further supported by the finding of normal T/17OHP ratios in a subject with increased urinary T/epiT ratios caused by an abnormally low testicular epiT production, probably related to genetic factors.

Changes in the testosterone concentrations after single sessions of endurance and strength training were measured in seven well trained men, experienced in both forms of training. Both training sessions were rated as hard to very hard on the Borg scale. Blood samples for testosterone measurements were taken before, immediately after, and 2, 4 and 6 h after the training sessions as well as the next morning. The mean tes tosterone concentration increased 27% (P<0.02) and 37% (P< 0.02) during the strength and endurance training session, respectively. Two hours after the training sessions the mean testosterone concentration had re turned to the pre-training level and remained at that level for the length of the observation period. There were no significant differences in the changes in testosterone concentration after strength and endurance training but there were large differences in the testosterone response at the level of the individual. A high correlation (r=0.98;P<0.001) for individuals was found between increases in testosterone concentration after strength and after endurance training. It was concluded that the changes in mean testosterone values followed the same timecourse after single sessions of strength and endurance training of the same duration and perceived exertion. The interindividual differences in tes tosterone response may be of importance for individual adaptation to training.

We have previously demonstrated the presence of cytochrome P-450 in a soluble preparation of bovine brain mitochondria (Oftebro, H., Størmer, F.C., and Pedersen, J.I. (1979) J. Biol. Chem. 254, 4331). In the present work we show that this preparation, in the presence of NADPH, adrenodoxin and adrenodoxin reductase catalyzes omega-hydroxylation of a number of C27-steroids that are intermediates in bile acid biosynthesis. The rates of hydroxylation were 1-2 order of magnitudes lower than reported for similar preparations from rat and human liver. No significant activity was detected with cholesterol as substrate. The physiological significance of brain mitochondrial cytochrome P-450 is discussed.

Spirometric, biochemical, and endocrine responses during a maximal ergometer cycle test and during three runs (10 km, 21.1 km, and 42.2 km) were investigated in one female with cystic fibrosis (CF) 27 years of age and in two healthy control females 26 and 29 years of age. One of the controls ran as a companion to the CF woman, while the other ran at her own speed. The CF woman has a chronic respiratory Pseudomonas aeruginosa infection, and her spirometric values were 50%-70% of predicted values at the time of the study. For the last years she has been training almost daily with aerobics, running, cycling, or skiing. She completed the four types of exercise without major problems. Her spirometric values increased transiently following the cycle test and for several hours following the three races (maximal 20%-30% increase of spirometric values), while the controls had transient decreases of the same variables in response to the runs. The biochemical and the hormonal changes were similar in the CF woman and the control who ran at her own speed, while the control who was a less stressed companion showed smaller changes. This study demonstrates that well-trained females with CF may participate in strenuous prolonged exercise without untoward effects.

This report describes two methods for the measurement of 1,25-dihydroxyvitamin D [1,25(OH)2D] in serum: A modified radio receptor assay (RRA), employing a 1,25(OH)2D receptor from calf thymus, and selected ion monitoring (SIM) with combined capillary gas chromatography (GC)-mass spectrometry (MS). The intra-assay coefficient of variation was close to 13% for both methods, and the inter-assay coefficients of variation were 14.0 and 6.5% for RRA and SIM (GC-MS), respectively. Aliquots of 2 ml (RRA) and 20 ml (SIM) serum were used, and the limits of detection were 10 and 6 pmol/l, respectively. The analytical recovery of each method was assessed, and a maximum deviation from the expected value of 10 and 2% was found for RRA and SIM, respectively. A correlation coefficient of 0.93 (slope 0.97) was obtained when 27 different serum samples were analyzed by both methods. Included in this study were serum samples from healthy subjects and patients with subnormal as well as supranormal 1,25(OH)2D levels. This result showed that the RRA accurately measured the serum levels of 1,25(OH)2D and therefore should be useful in the diagnosis and control of vitamin D dependent diseases.

The purpose of this study was to compare biochemical changes and endocrine responses during an incremental maximal bicycle test in three well-trained 18-year-old patients with cystic fibrosis (CF) and in three healthy controls. Although the blood concentration at rest of the white cell count, haptoglobin, phosphorus, urea, creatinine, and uric acid were somewhat different in the two groups, the CF patients had similar biochemical changes in response to the exercise compared with the healthy men. The endocrine responses to exercise seemed to be different between the two groups with regard to changes in Cortisol, growth hormone, and testosterone concentrations. The differences, however, were probably caused by differences in age, training situation, and psychological stress reaction rather than by pathological mechanisms.

Biochemical changes and endocrine responses during the New York Marathon (42195 m) were investigated in three 18-year-old male adolescents with cystic fibrosis (CF) and three healthy men who accompanied the CFs during the race. The ambient temperature was 20 degrees-28 degrees C and the relative humidity 98%-75% during the run. The CF patients, who had Shwachman scores of 60, 85 and 95 completed the run without major problems in 6.10, 4.42, and 4.32 h, respectively. Serum concentrations of Na and Cl decreased slightly, but the values were still within normal range. Na and Cl excretions in the urine decreased to very low levels in the CF adolescents during the run. All the other biochemical changes were similar to the changes in the controls. Aldosterone concentration increased to a higher level and maintained this increase for a longer time after the race in the CFs. Testosterone concentration decreased more in the CFs during the race compared with the controls. Growth hormone concentration showed individually varying changes in response to the exercise. This study demonstrates that patients with CF may participate in strenuous prolonged exercise even in humid and hot conditions, without untoward effects. The observed differences in hormonal responses to exercise might be explained by differences in age, training status, and relative exercise intensity rather than by hormonal or other disturbances in CF.

To study changes in the rate of bone loss before and after the menopause, 19 normal premenopausal women of the same age were followed with annual measurements of appendicular bone mass for 8 yr. Their serum estrone, estradiol, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and vitamin D-binding protein levels also were measured annually. The serum estrogen levels declined before the menopause, and the rate of bone loss more than doubled at the time of the menopause. There were no significant changes in the serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin LD or vitamin D-binding protein levels from the pre- to the postmenopausal period. We conclude that changes in vitamin D metabolite levels are not associated with early postmenopausal bone loss.

Vitamin D3 25-hydroxylase activity was measured in subcellular and submitochondrial fractions of human liver. Quantitation of 25-hydroxyvitamin D3 was based on high performance liquid chromatography. Vitamin D3 25-hydroxylase activity was detected in the mitochondrial fraction only. The mitochondrial 25-hydroxylase activity was linear with time up to 60 min and with mitochondrial protein up to 1 mg/ml. An apparent Km value of about 10(-5) M was found. Substrate satuation level was not reached. In the presence of 2.4 X 10(-4) M vitamin D3, the rate of 25-hydroxyvitamin D3 formation was 0.19 nmol X mg of protein-1 X h-1 After fractionation of the mitochondria, 86% of the 25-hydroxylase activity was recovered in the mitoplast fraction. The outer membrane fraction was devoid of activity. It is concluded that human liver contains only one detectable vitamin D3 25-hydroxylase enzyme localized to the mitochondrial inner membrane.