1. 31 P-MRS muscolo scheletrico Glicogenolisi e glicolisi Metabolismo ionico Trasporto del Pi nel mitocondrio Efflusso di H + dal citosol Respirazione mitocondriale pH intracellulare (citosolico) [Mg 2+ ] libero del citosol G ATP idrolisi

2. ATP + H 2 OADP + Pi functional ATPases PiPCr [ADP] PCr + ADP + H + Cr + ATP Creatine kinase PCr + H 2 OCr + Pi sum

3. Stress metabolico

7. Esercizio muscolare Respirazione mitocondriale Glicogenolisi Trasporto del Pi nel mitocondrio Glicolisi

8. PCr + ADP + H + Cr + ATP

9. 6.70 6.80 6.90 7.00 7.10 cytosolic pH 60120 0 recovery time (s) rest workrecovery

10. Recupero dallesercizio Respirazione mitocondriale Glicogenolisi Trasporto del Pi nel mitocondrio Glicolisi

12. 60120180 240 0 0 40 20 10 30 Tempo di recupero (s) (uniotà arbitrarie MRS) PCr Livello a riposo a b ultimo livello di lavoro Y = a (1 – e ) - TC t TC = 26 s [PCr]

13. PCr (mM) Glycolytic test 0 10 CUFFON OFF REST RECOVERY 30 20 WORK 96 3 015 time (min)

14. 60120180 240 0 0 40 20 10 30 Recovery time (s) MRS arbitrary units Pi resting level b last level of work Y = b e - TC t TC = 28 s [Pi]

15. 6.70 6.80 6.90 7.00 7.10 cytosolic pH 60120 0 recovery time (s) rest workrecovery minimum pH

16. PCr + ADP + H + ATP + Cr

17. PCr + ADP + H +

18. 6.70 6.80 6.90 7.00 7.10 cytosolic pH 60120 0 recovery time (s) rest workrecovery 418 J/min 270 J/min minimum pH

20. La velocità di recupero di PCr e di Pi sono inversamente proporzionali al valore di pH carrier mitocondriale di Pi (rate limiting) Driving force: gradiente di pH (pH 8.0 matrice; pH 6.0 citosol) bassa K M for HPO 4 2- Affinità del Carrier: (pK2 = 6.68)

21. 6.26.46.66.87.0 Minimum pH 0 20 60 40 80 TC Pi (s) ( rate of PCr recovery ) Reference range (95% confidence interval)

22. calf muscle 31 P MRS in DMD/BMD carriers

23. Condizioni per la valutazione della velocità di recupero di PCr dallesercizio pH minimo minore di 6,90 Eccesso di substrati: Alto ADP (> 60 M) Alto Pi (> 25 mM) Alto O 2 (iperemia) __________

24. 6.26.46.66.87.0 Minimum pH 0 20 60 40 80 TC PCr (s) ( rate of PCr recovery ) Reference range (95% confidence interval)

25. 6.26.46.66.87.0 Minimum pH 0 20 60 40 80 TC PCr (s) ( rate of PCr recovery ) Reference range (95% confidence interval) mtDNA mutation at bp 117788

26. 0 6.26.46.66.87.0 Minimum pH 20 60 40 80 TC PCr (s) ( rate of PCr recovery ) Reference range (95% confidence interval) case 1 case 2 case 3 before training after training

27. Chronic Progressive External Ophtalmoplegia (CPEO) mtDNA deletion/s (Cox deficit), RRF Lebers Hereditary Optic Neuropathy (LHON) 11778 bp mtDNA mutation (Complex I) Mitochondrial myopathy (MM) mtDNA deletion/s (Cox / SDH deficit), RRF Mitochondrial encephalomyopathies MEM MELAS (3243 mtDNA mutation (tRNA-leu), RRF, SCR deficit,) MERRF (Myoclonal epilespsy w. RRF) - 8993 mtDNA mutation (tRNA-lys), RRF mitochondrial cytopathies

30. CPEO (Cox deficit)control

31. normal control MELAS patient PCr Pi

32. 2 34576 resting Pi (mM) patients controls reference values (95% confidence interval) 4.822.43

33. 60120180 240 0 0 40 20 10 30 Recovery time (s) PCr recovery (MRS arbitrary units) Control TC = 38s; pH = 6.58 TC = 58s; pH = 6.62 CPEO (Cox deficit)

34. 6.26.46.66.87.0 Minimum pH Reference range (95% confidence interval) 0 20 60 40 80 TC PCr (s) ( rate of PCr recovery ) 100

35. Multiple logistic regression applied to two independent indicators: 1. [Pi] at rest, 2. rate of PCr recovery PrD ( probability of disease ) = 1 + e -(0.538 * Ds + 4.312 P – 23.729) 1 Ds = rate of PCr recovery (sec)P = [Pi] at rest

36. Rest only 15/31 48.4 % Recovery only 26/31 83.9 % Assessment condition Muscle abnormality revealed by 31P-MRS Analysis by multiple logistic regression 31/31 100.0 % SENSITIVITY 100%; SPECIFICITY 100%

37. HEADACHE (over 120 patients studied) (brain – occipital lobes) Complicated migraine 2.91 + 0.25 39.5 Migraine with aura 3.73 + 0.30 55.3 Migraine withot aura 3.39 + 0.30 50.3 Cluster headache 3.54 + 0.36 52.1 Healty volunteers 4.45 + 0.27 83.7 [Phosphocreatine] (mM) Phosphorylation potential (mM -1 ) Failure of mitochondrial energy transductions in the brain of all patiens with headache is a favouring background for the triggering of headache

38. HEADACHE (over 120 patients studied) skeletal muscle (gastrocnemius) The rate of PCr recovery after exercise is a measure of mitochondrial funtionality dashed area defines the reference interval Failure of skeletal muscle energy tranductions lead us to put forward the hypothesis that headache is a systemic disease primarely involving energy metabolism A = MS; B = MwA; C = MwoA; D = CH

39. Glicogenosi muscolari

40. McArdle UDPGPLD glycogen glucose-1-P glucose -6-P fructose -6-P fructose -1,6-P glycerldehyde-3-P (2) 3-P-glyceroyl phosphate (2) 3-P-glycerate (2) 2-P-glycerate (2) Phosphoenolpyruvate (2) Pyruvate (2) Lactate (2)

41. Deficit di PFK UDPGPLD glycogen glucose-1-P glucose -6-P fructose -6-P fructose -1,6-P glycerldehyde-3-P (2) 3-P-glyceroyl phosphate (2) 3-P-glycerate (2) 2-P-glycerate (2) Phosphoenolpyruvate (2) Pyruvate (2) Lactate (2) patientcontrol No pH variation during work and recovery

42. Deficit di PGAM UDPGPLD glycogen glucose-1-P glucose -6-P fructose -6-P fructose -1,6-P glycerldehyde-3-P (2) 3-P-glyceroyl phosphate (2) 3-P-glycerate (2) 2-P-glycerate (2) Phosphoenolpyruvate (2) Pyruvate (2) Lactate (2) controllo paziente No pH variation during work and recovery

43. Valutazione delleffetto della terapia trattamento con CoQ pazienti con citopatie mitocondriali

45. Reference range 6.56.77.1 minimum pH 6.9 TC PCr (s) 20 40 80 60 100 0 6 7 9 5 1 2 3 4 10 8 oral CoQ (150 mg/day) for 6 months Mitochondrial myopathies

46. 31 P MRS Abnormal BLOOD ANALYSIS Biochemistry, molecular genetic MUSCULAR BIOPSY Histochemistry, biochemistry, molecular genetic Normal Symptoms and clinical examination suggesting metabolic myopathy 31 P MRS Diagnosis confirmation Therapy follow-up Assessment of evolution Screening of families Bicycle exercise test Ischaemic test EMG