1. GARLIC AND HEALTH in vivo Metabolism of DADS 4 th October 2001 Study of in vivo Diallyl disulfide Metabolism Aims of the study : –To determine the pharmacokinetic parameters of DADS –To determine the metabolites of DADS : bio-availability of DADS and of the metabolites Experimental conditions –in rat –DADS administrated by force feeding –Analysis instruments :  DADS, AM, AMS, AMSO, AMSO 2 : volatile compounds studied by GC-MS (gas chromatography mass spectrometry) and quantified by using the technique of Selected Ion Monitoring (SIM)  AGS: non-volatile compounds studied by HPLC-UV (254nm)

2. GARLIC AND HEALTH in vivo Metabolism of DADS 4 th October 2001 Metabolism in vitro of DADS S S DADS,diallyldisulfide S S O DADSO,allicin SH AM, Allyl mercaptan S S-glutathion AGS,Allylglutathionylsulfure AMSO 2,Allylmethylsulfone S O O Allyl methyl sulfur AMSO, allyl methyl sulfoxide O S S

3. GARLIC AND HEALTH in vivo Metabolism of DADS 4 th October 2001 DADS i.g. 200 mg/kg KidneyPlasma Liver Stomach Intestine Urine Method and experimental protocol Sacrifice â 0.15, 0.3, 0.6, 1, 2, 4, 6, 24 hours âevery single days during 2 weeks. Sample preparation 4homogenization, p cymene (internal standard) 4protein precipitation (TCA, 30%) 4Extraction with CH 2 Cl 2 (3 times) 4organic phase : nonane (standardization for concentration) GC-MS analysis

4. GARLIC AND HEALTH in vivo Metabolism of DADS 4 th October 2001 General Observations Toxicity: –8% toxicity (4/52 of treated rats dead during the first 24 hours) Evaluation of GC-MS analytical procedures –Minimum detection : AM=1 E-4µg/ml AMS=8E- 4µg/ml DADS=1E-3µg/ml –Linearity : AM =7.E-3µg/ml -100µg/ml, AMS=3 E-3µg/ml - 87µg/ml DADS= 3 E-3µg/ml - 110µg/ml Quantitative analysis of sulfur compounds levels performed by integrating standards levels (GC-MS) sulfur compounds â u.a g-1 = p cymene X nonane X organ weight â No DADSO detected in liver and plasma extracts No AGS in liver extracts HPLC-UV

5. GARLIC AND HEALTH in vivo Metabolism of DADS 4 th October 2001 Early (<24 hours) 1- Detected in each organ 2- Maximum in liver Long-time (<15 days) 1- Quantifiable in stomach and in intestine until the 3rd day 2- Very low levels in other organs Repartition of DADS stomach = 20 000ua.g -1

6. GARLIC AND HEALTH in vivo Metabolism of DADS 4 th October 2001 Metabolites in plasma 1- Early 2- long time - Very low levels of AM and AMS - Rapid appearance of AMSO and AMSO 2 (10 min.) - Peaks at the second day

7. GARLIC AND HEALTH in vivo Metabolism of DADS 4 th October 2001 Metabolites in liver - The highest level of AMS, AMSO and AMSO 2 - Levels of AM, AMS, AMSO and AMSO 2 associated - Ratio AMSO 2 /AMSO : liver > plasma 1- Early 2- long time

8. GARLIC AND HEALTH in vivo Metabolism of DADS 4 th October 2001 Metabolites in stomach - Highest level of AM (vs other organs) - No association between all sulfur compounds levels - Essentially similar pattern in intestine 1- Early 2- long time

9. GARLIC AND HEALTH in vivo Metabolism of DADS 4 th October 2001 Metabolites in kidney - Similar to plasma (distribution or storage ?) 1- Early 2- long time

10. GARLIC AND HEALTH in vivo Metabolism of DADS 4 th October 2001 Metabolites in urine - Moderate levels of compounds (predominant way of elimination ?) - Complete elimination by the 11 th day 1- Early 2- long time

11. GARLIC AND HEALTH in vivo Metabolism of DADS 4 th October 2001 Conclusions Absolute quantification is required (purified oxidized compounds are now available) Oral administration of DADS induces AM, AMS, AMSO, AMSO 2 formation âConfirmation of the in vitro hepatic metabolism. âBut no DADSO and AGS are detectable Two steps : – a rapid biotransformation and disappearance of DADS < 6 hours – a long period of elimination for oxidized metabolites <13 days Perspectives – study of in vivo DADSO metabolism â implication of AGS