The exhaled breath condensate sampling device, developed at HYD LLC, is designed to collect condensate from exhaled air efficiently. The EBC sampling device consists of four parts, as shown in Figure 1(a) and Figure 1(b) .

The device, which weighs approximately 30 grams and measures 110 × 90 × 80 mm, was constructed from biocompatible PA12 polyamide powder using an SLS 3D printer.

The way of use and measurement of D concentration:

1) Water is filled into the inner part of the condensation unit ( Figure 1(a) ).

2) The cap is placed back on the top of the inner part of the condensation unit.

3) Part Yellow is placed into the outer part of the condenser unit.

4) The complete condensation unit (Parts Yellow, Green, and Blue) is placed into the stand and put in a freezer set at −18˚C.

5) When the water in Part Yellow freezes (within 4 - 5 hours), the sample vial is decapped and placed under the condensation unit. Now, the sampling device is ready to use. The subject can start to exhale through the stub of Part Blue.

6) Exhaled air flows between the outer and inner parts of the condensation unit, precipitating water on the cold surface of Part Yellow and draining into the vial. The test found that the cooling capacity (volume and temperature) of the ice in Part Yellow is far greater than what is required to condense 1 mL of exhaled vapor.

7) When EBC volume reaches 1 mL in the vial (usually in 6 - 8 minutes), it can be removed and closed. Before the next use, the parts of the condensation unit (Part Yellow) are wiped dry; a standard surface disinfectant disinfects the stub, and the device, after refrigeration, is ready for subsequent use. These steps show that the novel EBC sampler is easy to use and allows in-home sampling.

8) The EBC sample, now collected and prepared, is ready for measurement by a liquid-water isotope analyzer.

Given the ice volume and temperature, its cooling capacity is far greater than required to condense 1 mL of exhaled vapor. This is evidenced by the fact that most of the water remains frozen inside the device by the end of the sample collection, and the temperature of the surface of the inner part of the condensation unit does not exceed 0˚C.

The instrument (Liquid-Water Isotope Analyzer-24d, manufactured by Los Gatos Research Inc., San Jose, CA, USA) uses off-axis integrated cavity output spectroscopy to measure the absolute abundances of D-containing water molecules via laser absorption (IAEA, 2009). The deuterium concentration is given in ppm (with ±1 ppm accuracy as stated by the manufacturer).

Air humidity can potentially influence the D concentration of the collected samples if there is time for an exchange reaction. To exclude this impact, the sampling conditions were varied. The extensive exhalation in 4 minutes produced 0.6 mL of condensed water, and the slow exhalation in 16 minutes produced 1.5 mL. Measuring the D concentration of the samples obtained in the two ways showed no difference (131.2 ppm and 130.3 ppm, respectively). The device was also tested alone, allowing the air humidity to precipitate [ 16 ]. Still, less than 100 µL was collected during an hour, suggesting that air humidity does not impact the D concentration of EBC. This is in line with the data that at room temperature, the water content of one liter of air is less than 1 µL.

The device was used as described above by subjects of both sexes aged 8 to 56, with body weights ranging from 26 to 110 kg. Table 1 summarizes the data collected.

The samples were taken once a day (except for one subject; see below). Three persons provided new samples one year after giving the first ones. In two cases, there were no differences (144.82 - 144.45 ppm, 143.3 - 144.76 ppm); in one case, a difference of 5.2 ppm (138.3 - 143.5 ppm) was found. The difference between the minimal and maximal D levels in one volunteer’s samples was 1.07 to 5.81 ppm. Interestingly, the highest differences (3.48 ppm and 5.81 ppm) were detected in the two volunteers of age 8. Among the adults, the differences were between 1.07 and 5.2 ppm. Considering that the difference, and hence the SD value, for each volunteer was low, we can conclude that in persons consuming normal water, the D concentration is stable and independent of age, gender, body weight, and sampling time. The data also indicate that the device provided reliable, reproducible samples for measurement.

One person (female, 56 years old, weighing 78 kg) gave two samples daily, at 10 a.m. and 10 p.m., for a week. The EBC D concentration results (145.1 - 144.8 ppm ± 0.64) further confirmed the stability of body water D levels.

Based on all 59 samples, the average D concentration was 143.61 ppm ± 2.64. This aligns with our earlier findings, where 30 volunteers were enrolled in a clinical study (mentioned above), and their average serum D concentration before consuming DDW was 147.57 ppm. (146 - 150 ppm) [ 8 ].

Consuming exclusively DDW is the basic rule to maximize the anticancer effect of deuterium depletion. The subsequent study represents and follows the changes when the patient’s compliance is not adequate. The test person’s daily water intake of 1.5 L consisted of 0.5 L of deuterium-depleted (105 ppm D) and 1.0 L of regular water (148 ppm D), or oppositely, over 33 days, as shown in Table 2 . During this period, the overall decrease of D concentration in the EBC was 6.7 ppm. The changes of D concentration in the EBC corresponded to the varying proportions (2:1 to 1:2) of the two types of water, with a manifest decrease on the days of higher (cca. 2:1) DDW-to-normal water ratio but a temporary increase when the majority of the water consumed was normal ( Table 2 ).

The following data were obtained from a 64-year-old woman who consumed DDW with different D concentrations. The data show a correlation between the length of DDW consumption, the D concentration of the water consumed, and the measured D concentration in the exhaled air ( Figure 2 ).

Over 51 days of consuming exclusively DDW (except for two days), a 27.54 ppm decrease was detected (0.69 ppm per day). During these 51 days, the person consumed DDW of five different D concentrations. In period A, DDW with 105 ppm D concentration resulted in a 10.81 ppm decrease within 22 days. In periods B and D, the deuterium concentration of the DDW was 85 ppm, but in period C, she consumed regular water. There was a 5.71 ppm decrease in D concentration of EBC in 13 days (periods B and D) but no additional decrease during regular water consumption in period C (127.09 - 127.09 ppm). In periods E and F, the person consumed DDW with 65 ppm and 45 ppm D concentration, resulting in a 5.74 ppm decrease within ten days and 2.62 ppm within two days. The average decrease of D concentration was 0.47 ppm/day in period A, 0.92 ppm in period B, 0.57 in period E, and 1.31 in period F. The person stopped drinking DDW on the 51^st day. Starting to consume regular water (145 ppm) on the 52^nd day caused an increase in D concentration (period G). The increase was faster (18.29 ppm) in the first 12 days, while it was only 7.66 ppm in the subsequent 37 days when D concentration almost reached the original level (141.99 ppm).

Another subject, a 68-year-old male consumed DDW of 105 ppm. Figure 3 shows a rapid decrease in D concentration in the first 4 - 5 days, with an additional decrease in the following days. However, the D concentration reached equilibrium within three weeks, and no additional decrease was observed.

The next question addressed was whether significant deuterium (D) concentration changes could be observed within half a day after ingesting deuterium-depleted water (DDW) of 25 ppm D concentration. This would also help determine how quickly D concentration equilibrates in the body. Two individuals (one female and one male, with body weights of 92 kg and 77 kg, respectively) consumed DDW containing 25 ppm D between 7:30 a.m. and 8:30 p.m. Over the 13 hours, a decrease of 4.64 ppm and 4.03 ppm in D concentration was measured after consuming 1.2 liters and 1 liter of DDW, respectively.