Growth Hormone – Possible to Detect?

GH

• ”The most expensive, most fashionable and least understood of the new athletic drugs.”

(Underground Steroid Handbook 1983)

Could be – but does it work?

GH increases muscle mass in animals…

… 4 months GH replacement in an adult GH-deficient patient

GH substitution reduces Abdominal fat

Clin Endocrinol1996;45:681-8

Clinical Endocrinology (2009) 71, 860–866

Isometric muscle strength

Mata Analysis of Placebo-Controlled Trials

Clinical Endocrinology (2009) 71, 860–866

Impact of GH substitution on exercise capacity and muscle strength in GH-deficient adults:

a meta-analysis of blinded, placebo controlled trials

Aerobic exercise capacity

Ann Intern Med. 2010;152(9):568-577 • Placebo-controlled,

double blind GH trial (2 mg/d) in 96 recreationally trained athletes (63 men and 33 women) for 8 weeks + 6 weeks washout

• Measurements included body composition, muscle strength, VO2-max, sprint capacity (wingate value)

• Supported by WADA

3.9% (P = 0.05)

8.3% (P < 0.005)

Sprint Capacity (Wingate Value)

17

Challenges in GH abuse detection

• rhGH is almost identical to pituitary GH

• GH is secreted in a pulsatile manner

– Exercise & stress increase GH secretion

• Urine testing is not feasible • The presence of a prohibited substance or its

metabolites or markers in an athlete’s bodily specimen’’…constitutes an offence (WADA).

Hartman et al, J Clin Endocrinol Metab 1992; 74: 757

GH secretion is pulsatile and episodic!

24 h profile of serum GH concentration and deconvolution-resolved GH secretory rate

GH secretion is stimulated by exercise In an intensity-based manner

Endocrine Review 1991;12:424-449

Endogenous GH comes in many forms!

• Narrow window of opportunity (< 24 h) • Detects only biosynthetic 22k doping • Does not detect GH secretagogs

”Isoform method”

GH 2000 • UK, Sweden, Italy, Denmark Joint Study

supported by EU-IOC (1997-2000) • Protocols:

– High dose GH in healthy subjects – GH and related markers in athletes

• Cross-sectional • Prospective • Before, during and after competition

• Results: – 20 peer-review publications – A method…

Males

Females

Discriminant analysis: The combination of IGF-I and P-III-P Provided the best model Corrections for age and gender A pre-defined specificity of 1:10000 Sensitivity: Males: 86% Females: 60%

GH & IGF Res 2007;17:220-226

25

Treatment 0.06 IU/kg/day GH Placebo

Detection of dopers with GH-2000 formula

GH

tre

atm

ent

6 . 0 4 . 8 3 . 7 2 . 4 1 . 2 0 . 0 - 1 . 2

0 3

5 7 9

11 13 16

19

Visit day of study

GH-2000 formula score

“Non-doped” “Doped”

Overall sensitivity

90%

Erotokritou-Mulligan GH& IGF Research 2007 17(5):416-23. Epub 2007 Jun 20

IOC Rome Workshop 1999

• It is possible to detect exogenous GH administration for up to 2 weeks after the last dose

– Principle was scientifically sound

– Sensitivity and specificity reasonable

• Blood testing and immunoassays will be necessary

• Results obtained were in a predominantly white European population

• More data on the effect of injury needed

…and so the GH-2004 project was established

Ethnicity 1200

1000

800

600

400

200

0

10 20 30 40 50 60 70

Age (years)

IGF-I (ng/ml)

Caucasian (n=493) Oriental (n=25) Afro-Caribbean (n=191) Indo-Asian (n=19)

* Other (n=29)

Injury

Number of Days since Injury

2

0

-2

-4

7±3

14±3

21±3

84±7 28±3 0-3

42±7

GH-2000 score

Men (n=127)

Further work: Adolescents

• GH-2000 score increases during puberty

• No false positives

Current state according to the inventor • The Biomarker is formally approved by WADA/IOC at

the London Olympic Games and caught two para-olympians who were missed by the iso-form test. Both admitted doping and went home.

• The test is currently in limbo as there are commercial assay problems…

• Maybe measurement of IGF-I and PIIINP with MS may solve the problem?

Jorgensen

Excuse me…I know the game is over, but just for the record, I don’t think my buzzer was

working properly

Validation in other populations/ labs

• Test worked in other studies

– Germany

– Australia

– Rome

34

Determining Decision limits to be used in anti-doping

• New limits with currently available assays that should allow introduction of test for anti-doping purposes

35

… as well as in man

39 High dose GH

Change in IGF-I and P-III-P with GH

0

0.5

1.0

1.5

2.0

2.5

3.0 P-III-P

Pre GH On GH 0

500

1000

1500

2000

2500

3000

Pre GH On GH

IGF-I

Low dose GH Placebo

P-III-P (U/ml)

IGF-I (ng/ml)

Eur J Endocrinol. 2010 Jul;163(1):45-54

40

Effect of Exercise on IGF-I

0

10

20

30

40

50

60

70

80

90

0 15 30 45 60 75 90 105 120 135 150

GH

Placebo

IGF-

I

Time (minutes)

Exercise

Wallace et al JCEM 1999. 84: 3591-601

41

Institut für Dopinganalytik und Sportbiochemie Kreischa GH Study

• 15 healthy male, non-competitive athletes • Age 21–33 yrs, mean 24 years • BMI 19.4–31.7 kg/m2

• Two treatment groups – placebo (n=5) – rhGH 0.06 IU/kg/day (n=10)

• 14 days therapy by injection • Blood samples taken prior to, during and until 4 weeks after

treatment.

Kniess et al Anal Bioanal Chem. 2003 Jul;376(5):696-700

42

Response of GH-dependent markers in an ethnically diverse

group

• 31 male & 13 female healthy volunteers recruited from gyms & University of Southampton sports clubs

• 19 Afro-Caribbean, 21 Indo-Asian, 4 Oriental

• Randomly assigned to three treatment groups: – Placebo

43

Effect of Injury

• No significant change in IGF-I

• 40-70% rise in P-III-P – Dependent on type and severity of injury

2

0

-2

-4

7±3

14±3

21±3

84±7 28±3 0-3

42±7

Number of Days since Injury

GH-2000 score

Men (n=127)

2

0

-2

-4

Women (n=40)

7±3

14±3

21±3

84±7 28±3 0-3

42±7

Erotokritou-Mulligan et al (2008) J Clin Endocrinol & Metab 93(7):2760-3

Fat

STAT5b is the major signaling protein

GH + − − + 60

4 + − 30

2 3 − +

30

1

+ − 60

5

+ − 30

6

60

Subject #

p

t

p

t

Muscle

Fat

AU

0,00

0,05

0,10

0,15

0,20

0,25

Muscle Saline GH Saline GH

P=0.001 P=0.07

Am J Physiol 2006;291:E899-E905

GHR-GH-GHR

JAK2

STAT5b

SOCS IGF-I

PI3-kinase PKB/Akt

MAPK ERK1/2

Src

JNK

GH signalling pathways documented in vivo in muscle and fat in human subjects

AJP 2006;291:E899-905 JCEM 2008;93:2842-50

Time (minutes)-50 0 50 100 150 200

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

GHSaline

0

20

40

60

80

100

120

(µg/l)

GH

FFA (mmol/l) P = 0.03

Am J Physiol 2006;291:E899-E905

Identified n = 137

Retrieved n = 49

Excluded n = 88

Excluded n = 23

Fulfilled Inclusion n = 26

New Articles n = 4

Non-Extractable

data n = 13

Meta-analysis n = 17

Effect of GH substitution on exercise capacity and muscle strength: a meta-analysis

Clinical Endocrinology (2009) 71, 860–866

V02-max (n=148)

Weighted mean±SE: 0,17 ± 0.02 l/min

(p < 0,0001)

Jorgensen 1996

Vahl

Wallymahmed

Whitehead

Jørgensen 1989

Total

Muscle Strength n = 113

weighted mean ± SE: 2,8 ± 2,2 % (p =0,20)

Juul et al. JCEM 1994, 78: 744-752

IGF-I is an important mediator of the anabolic GH effects (but serum IGF-I mainly reflects hepatic production)

Ethnicity

0 56 49 42 35 28 21 14 7 63 70 77 84

GH treatment High dose GH Low dose GH Placebo

Day

2500

2000

1500

1000

500

0

IGF-

I (μ

g/L

)