in the general circulation. Conversely, values obtained by measuring oxidation of protein thiol groups showed an opposite trend, i.e. reached a minimum after 90 min. Both parameters returned to baseline 24 hours thereafter. Therefore, it appears that RI can exert a local and a rapid systemic effect due to absorption of ROS and LOPs generated by the interaction of ozone with biomolecules present in the luminal content. The quantity of absorbed ROS and LOPs are however unpredictable due to the variable content of fecal material. Figure 6 attempts to suggest that ozone dissolves rapidly in the luminal water, but, in comparison to oxygen, it is not absorbed because it partly reacts with mucoproteins lining the mucosa, partly may react with fecal material and partly can be reduced by antioxidants. LOPs, like oxygen, pass through the muscularis mucosa (MM) and enter the circulation via lymphatic and venous capillaries. This conclusion is relevant and would support the contention that the beneficial effect of RI in chronic limb ischaemia may Chapter 6 50 The Actual Six Therapeutic Modalities be similar or equivalent to major AHT. If this result can be confirmed in a controlled, randomised clinical trial, it will be helpful for patients because they will be able to do automedication and avoid repeated venous punctures. Moreover it does explain why prolonged (up to 13 weeks) RI in aged subjects cause an increase of both ATP and 2,3-DPG in erythrocytes (Viebahn, 1999a,b). These results are the more surprising because, in comparison to the precise volumes and ozone concentrations in major AHT, we know very well how imprecise the application of ozone can be and particularly the volume of gas retained and effectively acting in the gut lumen. Figure 6. A schematic view of the transfer of the O2-O3 gas mixture from the colonic lumen into the submucosa. Both gases dissolve in the luminal mucous layer, but ozone reacts immediately and decomposes into a number of ROS and LOPs. These are absorbed with water via venous and lymphatic capillaries in the submucosa below the muscularis mucosae (MM). 51 This leads to the discussion of some technical details in terms of gas volume, ozone concentration and schedule of administration. RI should be done after defecation or after an enema, when the rectal ampulla is empty. The patient must lie on one side and try to relax; often he/she prefers to personally insert the disposable, oil-lubricated polyethylene (rubber must never be used) catheter (30-40 cm long). The insertion is easy and it should not stimulate peristalsis. To this end, the gas has to be introduced slowly and in steps of 50-100 ml every 1-2 min. If it is done quickly, the gas will be expelled at once. The gas can be introduced via: a) a manual two-way silicone pump connected to the gas just collected in a polyethylene bag, or with b) a 50 ml silicone-coated syringe, clamping the catheter each time after insufflation. We can obtain good compliance if we start with 150 ml and slowly scale up to about 450-600 ml depending on the patient’s tolerance. This volume can easily be retained for at least 20-30 min. Knock et al. (1987) insufflated up to 800 ml in 1 min, but I cannot confirm this and it is likely that the patient would rapidly expel most of the gas. Carpendale et al. insufflated from 700 to 1300 ml of gas (up to 30 mg ozone daily) in AIDS patients, hoping the gas would diffuse into the whole colon. This was a desperate, almost useless enterprise because Cryptosporidium contaminates the whole gastro-intestinal and bile ducts. The patient should be left to rest for at least 15 min after RI to avoid rapid gas expulsion and to allow the reaction of ozone with the luminal contents.