A set of measuring devices for transcutaneous CO₂ absorption, a plastic CO₂ adaptor and transcutaneous CO₂ absorption-accelerating hydrogel, (Formulation: carbomer (0.65%), glycerin (5.00%), sodium hydroxide (0.18%), sodium alginate (0.15%), sodium dihydrogen phosphate (0.15%), methylparaben (0.10%), and deionized water (balance)) were obtained from NeoChemir Inc., Kobe, Japan (International patent publication number: WO2004/002393). Pure CO₂ gas was purchased from Kobe Sanso Inc., Kobe, Japan. The actual application to humans is shown in Figures 1A and 1B. When we applied CO₂ transcutaneously using this system, sweating and redness of the skin were noted. In addition, the blood flow to the fingers increased, as shown by a Laser Doppler study (Data not shown).

Subjects: Six Sprague–Dawley rats were purchased (CLEA Japan, Tokyo, Japan). The animal experiment plan was reviewed and approved by the Animal Research Committee of Kobe University Graduate School of Medicine. The approval ID is P00220.

A set of measuring devices for transcutaneous CO₂ absorption consisting of (1) a container filled with CO₂ absorbing solution (600 mL of pure water) with a pH meter (D52: HORIBA, Kyoto, Japan) and a magnetic stirrer and (2) an airtight CO₂ gas chamber, with a 5-cm diameter hole covered by a skin specimen positioned at the bottom of the chamber, was used to observe the permeability of CO₂ gas thorough the rat skin specimen covered with, or without, the CO₂ hydrogel. (Figure 2) The CO₂-absorbing solution absorbs CO₂ gas through the skin specimen, and the pH of the solution decreases depending on the volume of the absorbed CO₂ (H₂O + CO₂ → H⁺ + HCO₃⁻). The skin from depilated Sprague–Dawley rats was harvested in 10×10-cm sections. Immediately after harvesting, a skin specimen was positioned over the hole of the chamber, in contact with the CO₂-absorbing solution, the depilated surface facing upwards. CO₂ hydrogel (0.5 g) was applied to the skin specimen in the Gel (+) groups but not in the Gel (−) groups, and the chamber was filled with pure CO₂ gas. After filling, the pH of the solution was measured in 30-s intervals for 15 min. by a pH meter.

Subject: Five healthy male volunteers with no history of respiratory or vascular disease participated in this study. Subjects were 23–38 years old (average: 33.0±6.6). This study was approved and permitted by the Ethical Committee of Kobe University Graduate School of Medicine and informed consent was obtained by written from all subjects before the start of the study. The approval ID is 997.

³¹P-MRS: The intramuscular pH was measured in the triceps surae muscle. All MR studies were performed with a 1.5-T superconducting imaging system (Gyroscan NT-Intera; Philips Medical Systems, Best, The Netherlands) and a surface coil. CO₂ hydrogel was applied to the subject's lower leg. A plastic CO₂ adaptor was then attached to the subject's lower leg, and the surface coil was positioned over the adaptor. After measurement preparations, pure CO₂ gas was flowed into the adaptor. Data were collected before infusion of CO₂ and every 5 min. after infusion. Quantification of the ³¹P-MRS metabolite data was reported before [20], and pHi was determined from the chemical shift of Pi with respect to PCr.

Subjects: Seven healthy male volunteers with no history of respiratory or vascular disease participated in this study. Subjects were 27–40 years of age (average: 32.0±4.6). This study was approved and permitted by the Ethical Committee of Kobe University Graduate School of Medicine and informed consent was obtained by written from all subjects before the start of the study. The approval ID is 619.

A near-infrared spectroscopy (NIRS), (NIRO-200 with multi-fiber adaptor: Hamamatsu Photonics. K. K. Hamamatsu, Japan), was used for Hb concentration measurement. Changes in oxygenated and deoxygenated Hb concentrations were measured using 3 channels by focusing on the differences in absorption of light at 775, 810, and 850 nm [21]–[23]. The recording probe was attached to the inner side of the subject's forearm. A pneumatic tourniquet system (ATS2000, Zimmer patient care division, Dover, OH) commonly used in orthopaedic surgery was used for avascularization of the arm.

Each subject entered the environmental chamber, which was maintained at an ambient temperature of 26°C and relative humidity of 45%. NIRS probes were attached to the subjects' forearms. A tourniquet was wound around the upper arms, and then Hb concentration was measured. After confirming that the oxy-/deoxy-Hb ratio had stabilized, the tourniquet was inflated to a pressure of 250 mmHg, a commonly used pressure level in surgeries to avoid bleeding from the forearm (Figure 3). Eight minutes after the inflation, CO₂ hydrogel was applied to the subject's forearm, and the entire arm was enclosed by a CO₂ adaptor. Ten minutes after the inflation, pure CO₂ gas or air (control) was allowed to flow into the adaptor. The relative concentrations of the oxy- and deoxy-Hb were measured at 2-s intervals using NIRS. The duration of tourniquet inflation was limited to a maximum of 20 min to avoid ischemic damage to subjects' tissues.

Paired t-tests were used to compare all the variables in the control and CO₂ groups. All values were analysed with measurement analysis of variance (ANOVA), followed by analysis of simple main effects. All data are presented as mean ± S.E.M. P<0.05 was considered statistically significant.

Before we used this system in humans, the validity of the CO₂ hydrogel was confirmed in vitro using a measuring device for transcutaneous CO₂ absorption (Figure 2). Four groups of rat skin specimens with or without CO₂ hydrogel that had been filled with CO₂ gas or air were used in the experiment. The CO₂-absorbing solution receives CO₂ gas through the skin specimen, and the pH of the solution decreases, depending on the volume of the absorbed CO₂. The pH of the solution decreased time-dependently in the CO₂ (+) groups, and the pH values were significantly lower in the CO₂ (+) Gel (+) group compared to the CO₂ (+) Gel (−) group after 3.5 min (Figure 4). These results showed that CO₂ hydrogel actually enhanced CO₂ gas permeation through the rat skin.

Next, to test this system in the human body, we measured the pH change in the muscle during transcutaneous application of CO₂. We used phosphorus-31 magnetic resonance spectroscopy (³¹P-MRS) to measure the intracellular acid–base status in the subject's triceps surae muscle [20]. The room temperature was 25°C. CO₂ hydrogel was first applied to the subject's lower leg, and the CO₂ adaptor was attached to seal the lower leg; then the surface coil was applied over the adaptor adhering it to the calf. After preparing the subject for measurement, pure CO₂ was infused into the adaptor. The measurements were performed before CO₂ infusion and every 5 min. after infusion. The intracellular pH of the triceps surae muscle decreased significantly 10 min. after transcutaneous application of CO₂ (Figure 5). The results showed that the intramuscular pH decreased by transcutaneous application of CO₂ using this system in vivo. Although the pH change was expected to be buffered by body fluid and active blood flow, buffering was not enough to prevent the pH change. From these results, we confirmed that our novel system allowed transcutaneous penetration of CO₂ in vivo.

The oxy- and deoxy-Hb concentrations at all time points during the experiment are shown in Figure 6A. The oxy-Hb concentration decreased and the deoxy-Hb concentration increased after halting the blood flow (resting O₂ consumption). The relative concentrations of oxy- and deoxy-Hb changed gradually, almost reaching a maximum after 8 min. Both the decrease in oxy-Hb and the increase in deoxy-Hb were greater in the CO₂-applied arms than in the control arms.

Oxy- and deoxy-Hb concentrations at 8 min. after inflation were assigned to be the control values. The mean values of 5 points around every second minute of measurement are shown in Figure 6B and Table. 1. The data show that the oxy-Hb concentration decreased significantly 4 min. after CO₂ application (The relative changes of the oxy-Hb concentrations were −33.4±23.9% in the CO₂ applied group and −11.8±8.0% in the air applied (control) group), and that deoxy-Hb concentration increased significantly 2 min. after CO₂ application in the CO₂-applied group compared to the control group (The relative changes of the deoxy-Hb concentrations were 21.9±3.6% in the CO2 applied group and 9.1±1.6% in the control group). Thus, it was confirmed that transcutaneous CO₂ application facilitates a decrease in oxy-Hb and an increase in deoxy-Hb in the human body, providing evidence of O₂ dissociation of Hb resulting from transcutaneous application of CO₂ in vivo.