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OPEN

The effects of bathing in neutral
bicarbonate ion water
Tomoe Yamazaki1,6, Ryoko Ushikoshi‑Nakayama1,6, Supriya Shakya1, Daisuke Omagari1,
Naoyuki Matsumoto1, Chiyoko Nukuzuma4, Tomoko Komatsu2,3, Masaichi Chang‑il Lee2,
Hiroko Inoue5 & Ichiro Saito1*
Percutaneously absorbed carbon dioxide enhances blood flow. The mechanism by which it does so is
unclear, but we hypothesized that it involves bicarbonate ions. BALB/c mice were bathed in neutral
bicarbonate ionized water (NBIW) and showed increased blood bicarbonate levels and blood flow
via phosphorylation of peripheral vascular endothelial nitric oxide synthase (eNOS) and productionof nitric oxide (NO). Phosphorylation of eNOS and NO production were also increased in humanumbilical vein endothelial cells cultured in medium containing NBIW, and NBIW showed reactive
oxygen species scavenging activity. In a double-blind, randomized study in men and women aged
30 to 59 years with subjective cold intolerance, bathing in NBIW elevated body temperature fasterthan bathing in a control solution and improved chills and sleep quality. Taken together, our results
show that percutaneously absorbed carbon dioxide changes to bicarbonate ions, which act directly on
endothelial cells to increase NO production by phosphorylation of eNOS and thus improve blood flow.
Hot-spa therapy, i.e., bathing in mineral-rich hot water, is popular in many regions of the world and is believed to
improve mental and physical discomfort and help maintain good health. Improving blood circulation and elevating body temperature are known not only to prevent circulatory d­ isorders1 but also to cure t­ rauma2. Recent studies showed that hydrogen sulfide in sulfurous mineral water is diffused into the body via the skin and ­mucosa3.
In the body, hydrogen sulfide has antioxidant effects and enhances vascular circulation by vasodilation that isdependent on adenosine triphosphate-sensitive potassium (KATP) ­channels4,5. The potential of hydrogen sulfide
to prevent age-related diseases and support treatment of cardiovascular diseases has been attracting a­ ttention6,7.
Carbon dioxide (­ CO2)-enriched spas, which have been used since the Roman era for recuperation and treatment, are found abundantly all over the world. Many s­ tudies8–12 found that percutaneous absorption of carbon
dioxide has therapeutic effects by promoting blood flow and vascular regeneration in vascular disorders, such as
peripheral capillary regression in patients with d­ iabetes13 and severe limb ischemia in patients with peripheralarterial disease, and in skin d­ iseases14. ­CO2 bathing induces vascular endothelial growth factor (VEGF) synthesis
and nitric oxide (NO)-dependent capillary angiogenesis at the site of i­schemia15. Furthermore, percutaneousabsorption of C
­ O2 increases mitochondria in skeletal muscle and induces switching of muscular fibers; therefore, percutaneously absorbed C­ O2 was assumed to enhance muscle s­ trength16 and was expected to be useful forrehabilitation in older people, after operations, and in sports medicine.
The mechanism by which percutaneously absorbed ­CO2 enhances blood flow was previously thought to be
attributable to the Bohr e­ ffect17, in which the absorbed C­ O2 increases C­ O2 pressure at subcutaneous capillaries, causing ­O2 to be released from hemoglobin. However, many aspects of the actual mechanism remain to beelucidated.
When dissolved in water, C­ O2 usually exists as dissolved inorganic carbon (DIC), which is transformed
into 3 types—carbonic acid, bicarbonate ions, and carbonate ions—depending on the pH of a s­ olution18,19.
Carbonic acid is the most prevalent DIC type in weakly acidic solutions, bicarbonate ions in neutral to weakly
alkaline solutions, and carbonate ions in alkaline s­ olutions18. ­CO2 is percutaneously transferred from the skinsurface to the capillary vessels via the transappendageal route, i.e., through appendages such as hair follicles and1

Department of Pathology, Tsurumi University School of Dental Medicine, 2‑1‑3 Tsurumi, Tsurumi‑ku,
Yokohama, Kanagawa 230‑8501, Japan. 2Kanagawa Dental University Graduate School of Dental Medicine,
Yokosuka-Shonan Disaster Oral Health Research Center & Oxidative Stress/ESR Laboratories, 82 Inaoka‑cho,
Yokosuka, Kanagawa 238‑8580, Japan. 3Division of Dentistry for the Special Patient, Department of Critical
Care Medicine and Dentistry, Kanagawa Dental University Graduate School of Dental Medicine, 82 Inaoka‑cho,
Yokosuka, Kanagawa 238‑8580, Japan. 4THERMOCELL Clinic, Tokyo Design Center 2F, 5‑25‑19 Higashi‑Gotanda,
Shinagawa‑ku, Tokyo 141‑0022, Japan. 5Department of Pharmaceutical Sciences, Nihon Pharmaceutical
University, 10281 Komuro, Ina‑machi, Kitaadachi‑gun, Saitama 362‑0806, Japan. 6These authors contributed
equally: Tomoe Yamazaki and Ryoko Ushikoshi-Nakayama. *email: saito-i@tsurumi-u.ac.jp
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sweat glands. Blood has a neutral pH range, so bicarbonate ions are the most prevalent DIC type in ­blood20.
Therefore, we evaluated the possibility that much of the C­ O2 transferred from the skin to the blood would havea biologically beneficial, physiological effect by forming bicarbonate ions. In fact, one example of direct actionof bicarbonate ions on cells is increased antibacterial activity because, when cultured in a medium containing
more sodium bicarbonate than usual (which ionizes into bicarbonate ions and sodium ions in an aqueous solution), macrophage cell lines stimulated by lipopolysaccharide and interferon gamma showed enhanced NO­production21,22. In addition, Buckley et al. demonstrated in children with single-ventricle physiology that bolus
injection of N
­ aHCO3 enhanced cerebral blood flow in a dose-dependent m­ anner23.
On the basis of the above findings, we hypothesized that bicarbonate ions act directly on endothelial cells toenhance NO production in the blood, which improves vascular flow via vasodilation. Therefore, we investigatedthe effect of bathing in neutral bicarbonate ion water (NBIW) in mice, human umbilical vein endothelial cells(HUVEC), and humans.

Results

Effect of NBIW bathing in mice. All animals were included in the analysis. NBIW was prepared by adjusting sodium bicarbonate and citric acid to obtain a solution containing bicarbonate ions at 2500 mg/L under
pH 7.4. Mice under anesthesia that were bathed in NBIW at 37 °C for 20 min (n = 6) showed significant foldchanges in blood flow from before to after bathing compared with control mice (n = 6) bathed in ultrapure water(p = 0.042*). On the other hand, mice bathed in sodium bicarbonate water (SBW; n = 5) showed nonsignificant
changes compared with control mice (p = 0.244) (Fig. 1a). The bicarbonate ion concentration in the blood after
bathing tended to be higher in the NBIW group than in the control group but tended to be lower in the SBWgroup than in the controls (NBIW: p = 0.113, Hedge’s d = 0.83 [95% CI − 0.46 to 2.124]; SBW: p = 0.300, Hedge’s
d = -0.44 [95% CI: − 1.892 to 1.003]) (Fig. 1b). The amount of NO in the homogenized femoral vessel of the mice
was significantly higher in the NBIW group than in the control group (p = 0.027*) and numerically higher in the
SBW group than in the control group (p = 0.244). To clarify the involvement of NO, we administered an intraperitoneal injection of L-NAME, an inhibitor of nitric oxide synthetase (NOS), to a group of mice (n = 4) before
NBIW bathing and found that the amount of NO in the vascular homogenate showed a significantly greater
decrease than in the NBIW group (p = 0.01*) (Fig. 1c). Western blotting analysis showed a significant increase in
the amount of eNOS in the vascular tissue in both the NBIW and SBW groups compared with the control group(NBIW: n = 5, p = 0.039*, SBW: n = 5, p = 0.041*; control: n = 6) (Fig. 1d,e). The phospho-eNOS level tended to behigher in both the NBIW and SBW groups than in the control group, whereby the effect size was greater in the
NBIW group (NBIW: p = 0.075, Hedge’s g = 0.95 [95% CI: − 0.299 to 2.205]; SBW: p = 0.281, Hedge’s g = 0.36, 95%
CI [− 0.833 to 1.56]) (Fig. 1f).
The measurements of the pH, ­PCO2, and bicarbonate volume of the NBIW used in the study showed nosignificant changes from before to during and after the study (see Supplementary Information).
In vitro experiment in HUVEC.

To clarify the effect of bicarbonate ions on vascular endothelial cells, wecultured HUVEC in a medium with NBIW that contained bicarbonate ions at 2.5 ppb. After 5 min of culture,
the eNOS phosphorylation level was increased (Figs. 2a,b).

Reactive oxygen species scavenging activity of NBIW.

Figures 2c,d show the analysis results ofelectron spin resonance (ESR) performed to assess the anti-oxidation activity of NBIW. The mean (SD) ROS
scavenging rate for hydroxy radicals generated by ultraviolet irradiation and aqueous hydrogen peroxide solution was 27.9 (0.21) in NBIW and 18.9 (2.81) in SBW, both of which were clearly higher than the 0.0 (4.85) inultrapure water as a control. NBIW showed significantly higher hydroxy radical scavenging activity than SBW(p = 0.02*). The mean (SD) scavenging rate for superoxides was 20.0 (0.66) in NBIW and 19.7 (1.74) in SBW, bothof which were also higher than in the control 0.0 (2.61).

Clinical trial.

To investigate the effect of NBIW in humans, we conducted a placebo-controlled, doubleblind, randomized, parallel-group comparative trial with NBIW and control bath tablets. The ingredients of the
NBIW and control bath tablets are shown in Table 1.
Fifty-four participants (n = 27, 2 groups) were recruited; however, one person did not meet the selection
criteria and was excluded, so the trial was performed in 53 participants. None of the participants discontinued
or dropped out during the study, but one participant’s usage of the bath tablets met the exclusion criteria, so they
were excluded from the analysis. Data from 25 participants in the NBIW group (mean [SD] age: 47.3 [8.4] years,
12 men and 13 women) and 27 participants in the control group (mean [SD] age: 46.8 [6.5] years, 14 men and 13
women) were available for analysis (Fig. 3). No intergroup differences were found in any demographic factors,
including age, sex, alcohol intake, smoking, and exercise, or in the use rate of the study product or bathing time
during the study period (Table 2).
After 2 weeks, only the NBIW group showed a significant increase in body temperature on waking (p = 0.030*)
and one hour after bathing (p = 0.002**) compared with before use of the test sample, but body temperature
before bedtime was significantly increased in both groups compared with before use (NBIW: p = 0.005*, Control:
p = 0.016*). After 4 weeks, body temperature was significantly higher than before the trial in both groups at alltime points (Table 3).
A stratified analysis of the participants whose temperature on waking was below 36 °C showed a significant
increase in temperature before going to bed in the NBIW group (n = 8) compared with the control group (n = 6)
in both week 1 (p = 0.039*, Hedge’s g = 1.32 [95% CI: 0.19 to 2.59]) and week 2 (p = 0.002**, Hedge’s g = 1.55 [95%
CI: 0.38 to 2.88]), but no intergroup differences were observed from week 3 onward (Table 4).

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Figure 1.  Effects of neutral bicarbonate ion water in vivo. (a) Fold change in blood flow in BALB/c mice after
bathing. Flow was higher in the neutral bicarbonate ionized water (NBIW) group than in the control group(p = 0.042*). (Control: ultrapure water bathing, n = 6; NBIW, n = 6; SBW: sodium bicarbonate water bathing,n = 6). (b) Blood bicarbonate ion level after bathing. NBIW: p = 0.113 vs control, Hedge’s g = 0.83 (95% CI, − 0.46
to 2.124); SBW: p = 0.300 vs control, Hedge’s g = − 0.44 (95% CI, − 1.892 to 1.003). (Control: n = 5, NBIW: n = 5,
SBW: n = 3). (c) Level of nitric oxide (NO) in femoral vessel tissue after bathing. The increase was significantly
higher in the NBIW than in the control ­group† (p = 0.027*). L-NAME (100 mg/kg) significantly suppressed the
NBIW-associated NO increase (p = 0.01*). (d) Representative Western blot analysis of endothelial nitric oxide
synthase (eNOS) and phospho-eNOS (p-eNOS) in a blood vessel in each group (protein carried per lane: 20 μg)
in samples from all animals. The β-actin blot is from a different part of the gel blotted with eNOS. Blot imagesof all ­animals† are shown in the Supplementary Information. (e) The eNOS level in the femoral vessel tissue
was significantly higher in the NBIW and SBW groups than in the control ­group† (NBIW: p = 0.039*, SBW:
p = 0.041*). (f) The p-eNOS level tended to be higher in the NBIW and SBW groups than in the control group(NBIW, p = 0.075; d = 0.95; 95% CI, − 0.299 to 2.205; SBW, p = 0.281; d = 0.36; 95% CI, − 0.833 to 1.56) and wasnumerically higher in the NBIW group than in the SBW group. (g) Western blot images of all tested animals.
†(c–g) Control: n = 6, NBIW: n = 5, SBW: n = 5, NBIW + L-NAME: n = 4. For (a,b,e,f) Student’s t test was used,
and for c, Mann–Whitney U Test (* p < 0.05).
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Figure 2.  Effect of neutral bicarbonate ion water in human umbilical vein endothelial cells. (a,b) Effect ofneutral bicarbonate ion water (NBIW) in human umbilical vein endothelial cells (HUVEC). HUVEC wereserum starved for 6 h, NBIW equivalent to a bicarbonate ion level of 2.5 ppb was added in culture medium,and the cell lysate was analyzed 5 min later. (a) Typical example of Western blotting showing endothelial nitricoxide synthase (eNOS) and phospho-eNOS (p-eNOS) levels. The protein carried per lane was 20 μg. The β-actin
blot is from a different part of the gel blotted with p-eNOS. eNOS was reblotted onto the membrane blottedwith p-eNOS. The full blot is provided in the Supplementary Information. (b) eNOS phosphorylation level incells was higher in cells exposed to NBIW than in those exposed to the control solution. (c,d) Reactive oxygen
species (ROS) scavenging activity of NBIW. Hydroxy radical scavenging ability of NBIW was determined byelectron spin resonance (ESR). The mean (SD) scavenging rate for a hydroxy radical generated by ultraviolet
irradiation/hydroxy peroxide solution was 0.0 (4.85) for ultrapure water (control), 27.9 (0.21) for NBIW, and18.9 (2.81) for sodium bicarbonate water without citric acid (SBW). The mean (SD) superoxide scavenging rate
was 0.0 (2.61) for control, 20.0 (0.66) for NBIW, and 19.7 (1.74) for SBW. Each condition was repeated 3 times
and analyzed by Student’s or Welch’s t-test. *p < 0.05, †p < 0.01, ‡p < 0.005.

Ingredients

NBIW

Control

Sodium bicarbonate

75%

0%

Sodium carbonate

5%0%Citric acid

15%

0%

Magnesium sulphate

0%

48%

Sodium sulphate

0%

48%

Other

5%

4%

Table 1.  Ingredients of the bath tablets. NBIW neutral bicarbonate ionized water.

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Figure 3.  Flow diagram of randomized, double-blind, parallel-group study. NBIW neutral bicarbonate ionwater.

Participants
p value

Items

NBIW

Control

Number of participants

26

27

–

Male/female

13/13

14/13

0.893

Age, mean (SD), y

46.8 (8.5)

46.8 (6.5)

0.676

Drinks alcohol, yes/no

12/15

16/10

0.213

Current smoker, smoker/nonsmoker (of which smoked in the past)

1/24 (3)0/27 (5)0.304

Regular exercise, yes/no

9/18

13/13

0.218

Percentage use of bath tablets, mean (SD)98.2 (4.2)

99.6 (1.5)

0.240Bathing time, mean (SD), min

22.8 (7.4)

24.4 (6.6)

0.254

Table 2.  Background of the participants and use of bath tablets. NBIW neutral bicarbonate ionized water.
No significant difference was found between control and NBIW groups in any item. Age, use of test sample,
bathing time: Wilcoxon rank sum test. Other items: Chi-square test.

Intervention (week)
Items
On waking1 h after bathing
Before going to bed

SampleBefore intervention

1

2

3

4

NBIW

36.12 (0.35)

36.22 (0.32)

36.23 (0.34)*

36.24 (0.34)*

36.29 (0.31)**

Control

36.12 (0.3)

36.13 (0.28)

36.18 (0.29)

36.24 (0.29)**36.22 (0.31)*

NBIW

36.42 (0.33)

36.47 (0.26)

36.55 (0.23)**36.55 (0.23)*

36.57 (0.25)*

Control

36.45 (0.3)

36.45 (0.44)

36.51 (0.25)

36.57 (0.28)*

36.57 (0.25)*

NBIW

36.29 (0.25)

36.35 (0.28)

36.42 (0.21)*

36.39 (0.27)

36.44 (0.27)*

Control

36.23 (0.25)

36.26 (0.29)

36.32 (0.24)*

36.38 (0.29)**36.37 (0.29)**

Table 3.  Body temperature. NBIW neutral bicarbonate ionized water. NBIW: n = 25, Control: n = 27. Values
are shown as mean (SD). Within-group comparison: Significant differences were found compared with before
the intervention (* p < 0.05, ** p < 0.01: Wilcoxon signed rank test). Between-group comparison: No significant
differences were found compared with the control group (Wilcoxon rank sum test).

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Intervention (week)
Items
On waking1 h after bathing
Before going to bed

SampleBefore intervention

1

2

3

4

NBIW

35.71 (0.26)

35.96 (0.41)*

35.95 (0.45)

35.97 (0.42)

36.08 (0.37)*

Control

35.67 (0.14)

35.74 (0.09)

35.87 (0.27)

35.91 (0.26)*

35.81 (0.22)

NBIW

36.33 (0.42)

36.47 (0.23)

36.5 (0.24)

36.54 (0.21)

36.48 (0.18)
36.52 (0.26)

Control

36.42 (0.30)

36.59 (0.31)

36.48 (0.27)

36.51 (0.29)

NBIW

36.31 (0.31)

36.38 (0.25)#

36.43 (0.20)##36.37 (0.28)

36.41 (0.23)

Control

36.09 (0.22)

36.08 (0.14)

36.13 (0.15)

36.15 (0.26)

36.2 (0.17)*

Table 4.  Body temperature (stratified analysis by participants with body temperature below 36 °C on waking).
NBIW neutral bicarbonate ionized water. NBIW: n = 8, Control: n = 6. Values are shown as mean (SD). Withingroup comparison: Significant differences were found compared with before the intervention (* p < 0.05:
Wilcoxon signed rank test). Between-group comparison: Significant differences between group (# p < 0.05, ##p < 0.01: Wilcoxon rank sum test).

p value
Item
PSQI-J scores

SampleBefore intervention

End point of intervention

Within-group

NBIW

4.6 (2.0)

3.4 (1.2)**

0.003

Control

3.9 (1.9)

3.0 (1.5)**

0.002

Between-group
0.249

Table 5.  Japanese version of Pittsburgh Sleep Quality Index (PSQI-J) scores. NBIW neutral bicarbonate
ionized water. NBIW: n = 25, Control: n = 27. Values are shown as mean (SD). Within-group comparison:
Significant difference compared with before intervention (**p < 0.01: Wilcoxon signed rank test). Betweengroup comparison: No significant difference between-group (Wilcoxon rank sum test).

Sample size was tested by noncentral t distribution before stratified analysis by using the week 2 temperatures
before going to bed, and the power was found to be 0.739.
The score on the Japanese version of the Pittsburgh Sleep Quality Index (PSQI-J) was significantly lower inboth the NBIW and control groups in Week 4 compared with baseline (NBIW: p = 0.003**, control: p = 0.002**),
but no significant intergroup difference was observed (Table 5).
A psychological test with the simplified Profile of Mood States Second Edition-Adults Short (POMS2-A Short)showed a significant increase in vigor–activity (VA) in the NBIW group from before to after the intervention(Table 6), and the scores of all items related to the subjective symptom of cold intolerance were significantly
lower in both groups after the intervention (Table 7). No serious adverse events or adverse reactions attributable
to the use of the study product were observed.

Discussion

In this study, we hypothesized that the mechanism of the blood flow enhancement effect of bathing in mineralrich water is related to percutaneous absorption of ­CO2 and the subsequent formation of bicarbonate ions, i.e.,
we assumed that C­ O2 migrates to capillaries via the transappendageal route of the skin and exerts its blood
flow-enhancing effect by directly acting on vascular endothelial cells as bicarbonate ions in the neutral pH
environment in blood.
We reproduced the components of natural carbonated spring water rich in bicarbonate ions and referred to it
as NBIW. We set the composition ratio of bicarbonate and citric acid in NBIW so that a solution with 2500 ppm
of bicarbonate ions was obtained under a neutral pH environment when ­CO2 gas was generated by the neutralization reaction of sodium bicarbonate and citric acid. The results of our in vivo experiment in which mice
were bathed in NBIW showed a significant increase in blood flow proportional to the increased blood level ofbicarbonate ions resulting from NBIW bathing. The finding that the pH, P­ CO2, and bicarbonate volume of the
NBIW used in the study showed no significant changes from before to during and after the study indicated thatthe concentration of bicarbonate ions in the solution remained stable.
This study adjusted the ratio of sodium bicarbonate to citric acid so that the pH of NBIW was 7.4, i.e.,equivalent to that of blood. At this pH level, dissolved inorganic carbonic acid is mostly present as bicarbonate
ions (Fig. 4a). Moreover, when carbonic acid is percutaneously absorbed into blood, approximately 90% of the
carbonic acid is known to be present as ­HCO3–20. Therefore, in our mice study we assume that bicarbonate ionswere percutaneously absorbed during bathing with NBIW and that carbonic acid had less influence.
NO, a free radical with a short half-life that is synthesized in the human body, was discovered as a vascularendothelium-derived ­vasodilator24 that has a vasoprotective effect by causing vasodilation, retention of blood
flow, suppression of neutrophil adhesion, suppression of platelet aggregation, active oxygen scavenging, andsuppression of cell g­ rowth25. In vascula r endothelial cells, NO is synthesized by eNOS after it is activated via­phosphorylation26. Given that the amount of eNOS, its phosphorylation level, and the amount of NO wereincreased in the mouse femoral vessels after NBIW bathing, our results show that NBIW bathing activates eNOS
in vascular endothelial cells, increasing NO production and inducing vasodilation.
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p value
Items
AH (Anger-Hostility)
CB (Confusion-Bewilderment)
DD (Depression-Dejection)
FI (Fatigue-Inertia)
TA (Tension-Anxiety)
VA(Vigor-Activity)
F (Friendliness)
TMD (Total Mood Disturbance)

SampleBefore intervention

End point of intervention

Within-group

NBIW

41.4 (4.6)

42.8 (7.4)

0.336

Control

42.7 (5.2)

43.0 (3.8)

0.624

NBIW

42.4 (3.9)

43.2 (5.5)

0.292

Control

44.6 (6.0)

43.3 (4.8)

0.189

NBIW

43.6 (3.3)

43.9 (3.4)

0.550

Control

44.6 (4.3)

44.0 (3.8)

0.350

NBIW

41.1 (5.2)

41.7 (6.9)

0.886

Control

42.4 (5.7)

40.3 (3.9)

0.057

NBIW

42.2 (6.2)

42.3 (6.7)

0.983

Control

44.9 (8.1)

42.9 (7.0)

0.054

NBIW

51.8 (8.7)

56.6 (10.0)

0.008**

Control

55.9 (8.6)

56.3 (6.9)

0.667

NBIW

50.8 (11.5)#

52.1 (11.4)

0.296

Control

56.0 (9.5)

53.2 (9.8)

0.185

NBIW

41.2 (4.3)

40.9 (6.2)

0.732

Control

42.1 (6.3)

40.7 (5.0)

0.095

Between-group
0.246
0.659
0.897
0.692
0.883
0.706
0.790
0.854

Table 6.  Profile of mood states 2nd edition-adult short (POMS2-A short) scores. NBIW neutral bicarbonate
ionized water. Control: n = 27, NBIW: n = 25. Values are shown as mean (SD). Within-group comparison: A
significant difference was found compared to before intervention (** p < 0.01: Wilcoxon signed rank test).
Between-group comparison: Before intervention, in “Friendliness” a significant difference was found comparedwith the control group (# p < 0.05); At the end point of intervention, no significant between group (Wilcoxon
rank sum test).

p value
Location of cold intolerance
Fingertips
Back of the hand
Whole hand
AbdomenLower backLegs to toes
Whole body

SampleBefore intervention

End point of intervention

Within-group

NBIW

2.2 (1.1)

3.6 (1.1)**

0.00048

Control

2.0 (1.1)

3.6 (1.4)**0.00004

NBIW

3.0 (1.1)

4.5 (0.9)**0.00036

Control

2.7 (0.9)

4.4 (1.4)**0.00006

NBIW

2.5 (0.9)

4.1 (0.9)**0.00013

Control

2.4 (0.9)

3.9 (1.3)**

0.00009NBIW

3.0 (1.1)

4.6 (1.0)**

0.00009

Control

2.9 (1.1)

4.2 (1.2)**0.00025NBIW

2.9 (1.2)

4.6 (1.1)**

0.00003

Control

2.6 (1.0)

4.1 (1.2)**0.00020NBIW

1.4 (0.7)

3.3 (1.0)**

0.00003

Control

1.4 (0.6)

3.1 (1.2)**0.00004

NBIW

2.4 (0.9)

4.0 (0.9)**0.00009

Control

2.1 (0.9)

3.5 (1.2)**0.00043

Between-group
0.784
0.902
0.490
0.165
0.077
0.421
0.056

Table 7.  Subjective symptoms of cold intolerance. NBIW neutral bicarbonate ionized water. Control: n = 27,
NBIW: n = 25. Values are shown as mean (SD). Within-group comparison: Significant differences were foundcompared with before use (**p < 0.01: Wilcoxon signed rank test). Between-group comparison: No significant
differences between group (Wilcoxon rank sum test).

The results of our in vitro experiment in HUVEC also showed increased eNOS activity in the vascularendothelial cells cultured in a medium with NBIW. These results supported our hypothesis that bicarbonate ionspercutaneously transferred into the capillaries during bathing act directly on the endothelial cells and induce
NO production. The amount of bicarbonate ions in the NBIW used in this study was comparable to that in SBW.
Although SBW increased the amount of blood circulation and NO production more than ultrapure water (control) during in vivo experiments, the response to SBW was weaker than that to NBIW. The blood flow-enhancing
effect of SBW might have been weakened by oxidation of the produced NO. In the in vitro experiment, thehydroxy radical scavenging rate of NBIW determined by ESR was 1.5-fold higher than that of SBW. Therefore,
prevention of oxidation of the produced NO by hydroxy radicals in NBIW might have contributed to an efficient

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Figure 4.  Schematic diagram of the underlying mechanism by which bicarbonate ions enhance blood
flow. (a) Changes in the relative amounts of dissolved inorganic carbon (%) by pH. The pH of the neutral
bicarbonate ion water (NBIW) was equivalent to that of blood, i.e., 7.4 (vertical dotted line). Among thedissolved inorganic carbon ions, bicarbonate ions (­ HCO3−) were most abundant. Modified from References 18
and 19. (b) Bicarbonate ions ­(HCO3−) and carbonic acid ­(H2CO3) are absorbed percutaneously, and carbonicacid is changed into bicarbonate ions in vivo. Bicarbonate ions that enter endothelial cells phosphorylate
endothelial nitric oxide synthase (eNOS). Phosphorylated eNOS produces nitric oxide (NO), which relaxes thesmooth muscle in the outside of endothelial cells, resulting in dilation of the vessel. CO32− carbonic ion, eNOS
endothelial nitric oxide synthase, H+ hydrogen ion, H2CO3 carbonic acid, HCO3− bicarbonate ion, NO nitricoxide, p-eNOS phosphorylated eNOS.

increase in the amount of NO, which might explain the differences between NBIW and SBW in terms of NO
production and the blood flow-enhancing effects.
To elucidate whether enhancing blood flow by regular bathing in bicarbonate ions is effective in increasing
basal body temperature in humans and whether this elevated basal body temperature improves sleep and mental and physical conditions, the randomized, double-blind study in humans compared bath tablets containing
sodium bicarbonate with control bath tablets containing sulfate salts; these salts do not generate bicarbonate
ions. The results showed increased body temperature on waking, 1 h after bathing, and before going to bed in the
NBIW group from week 2 onwards; although this effect was seen also in the control group, it occurred earlier in
the NBIW group. Also, a stratified analysis of data from participants with a body temperature on waking below
36 °C showed a significant increase in body temperature in the NBIW group compared with the control group
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in weeks 1 and 2. In other words, NBIW bathing raised body temperature more quickly than bathing in a control
solution containing magnesium sulfate and sodium sulfate, which are known spa ingredients.
The blood plays an important role as a heat exchanger in thermoregulation in humans, and decreased bloodflow lowers body ­temperature27,28. The participants in our clinical trial were men and women aged 30 to 59 years
with cold intolerance, and some of them presented symptoms of mild hypothermia. Thermoregulatory function
declines with a­ ging29, and one of the causes is an aging-associated decrease in production of NO, which has avasodilator ­effect30. Some researchers reported that poorer thermoregulation with aging is related to suppressionof mitochondria metabolism, increased ROS production, and accumulated oxidative damage to c­ ells31. Thus,
because aging is related to cold intolerance, the results of this study can be generalized only to middle-aged andolder people. However, a limitation of this study is that it cannot clarify the effect of NBIW in people who do
not have cold intolerance or in young people.
In vivo experiments showed that bathing in NBIW increased NO production and improved blood flow byenhancing endothelial function. Also, NBIW activated eNOS and ROS scavenging activity in vitro. Therefore,
the finding in this study that bathing in NBIW improved hypothermia sooner than bathing in the control solution may be related to both direct endothelial activation and NO production, which protects cells from ROS.
Recent reports showed that infective respiratory diseases were more likely to occur in persons with a lower bodytemperature because of the negative effects of cold on the immune s­ ystem32,33. Additionally, 1 in 4 menopausal
women in Japan have cold intolerance, which makes them susceptible to many diseases and conditions and may
cause an imbalance of autonomic nervous system functions, such as sleep, and disturb their mental ­health34.
However, cold intolerance is a problem that is often overlooked because it is difficult to define and a medical
diagnosis and effective interventions are not available. Therefore, bathing in NBIW may have advantages forgeneral health and subsequently may even lengthen life. This issue requires further study.
In conclusion, the results of our in vivo and in vitro experiments indicate that bicarbonate ions in NBIW
are percutaneously absorbed during bathing and act directly on endothelial cells to increase NO production byphosphorylation of eNOS, leading to relaxation of vascular smooth muscle and enhanced blood flow. A schematic
diagram of the underlying mechanism is shown in Fig. 4b.
Consequently, we suggest that the known effects of C
­ O2-enriched spas on blood flow and vascular regeneration are also due to bicarbonate ions that form in the neutral pH environment of the blood after percutaneousabsorption of C
­ O2. In our clinical trial in middle-aged people, bathing in NBIW was confirmed to show a hyperthermal effect sooner than bathing in a control solution. The research presented here delivers new findings on
the mechanism of enhanced blood flow after bathing in NBIW and clarifies its usefulness.

Methods

Mice. The experiments in mice were approved by the Tsurumi University Animal Experiment Committee(approval number: 20A020) and conducted according to the related guidelines, laws, and regulations. The study
is reported in accordance with ARRIVE guidelines (https://​arriv​eguid​elines.​org).
Male BALB/c mice aged 11 to 14 weeks were reared in a husbandry facility with controlled temperature andhumidity under a 12-h light cycle. The mice had free access to feed and water. Randomization was not used toallocate animals to groups. No data were obtained from animals that died during the experiment. Confounders
were not controlled, and all study investigators were aware of group allocation during the experiments, assessment of outcome, and data analysis.
NBIW bathing . Sodium bicarbonate was diluted in ultrapure water at 3.8 mg/mL, and citric acid was added
to the solution at 0.5 mg/mL to obtain a solution that had a neutral pH and 2500 mg/L of bicarbonate ions.
Ultrapure water and SBW at 3.8 mg/mL were used as controls. The bicarbonate ion concentration was determined by titration with sulfuric acid. The BALB/c mice were anesthetized with 3 types of mixed anesthetic
agents (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol)35 and bathed in
NBIW (n = 6) or control solution at 37 °C for 20 min. For the NOS inhibitory experiment, L-NAME 100 mg/kg
was injected intraperitoneally into 4 mice 1 h before bathing. After bathing, blood flow in the mice was determined by the laser doppler blood flowmeter RBF-101 (Pioneer Corporation).
The other 2 mice were bathed under anesthesia and euthanized by cutting the carotid vein with a 5-mm
Glodenrod Animal Lancet (MEDIpoint, Inc.), and the released blood was collected in a 1.5-mL centrifuge tube.
The blood gas analyzer GASTAT-navi (Techno Medica Co., Ltd.) was used to measure the partial pressure ofoxygen, partial pressure of carbon dioxide, pH, and bicarbonate levels of the collected blood. The femoral arteries
and veins of the mice were also isolated and stored at − 80 °C. The isolated vessels were homogenized in purified
water, and the amount of NO was measured by the ­NO2/NO3 Assay kit-FX (Fluorometric) 2,3-Diaminonaphthalene Kit (Dojindo Laboratories).
The pH, ­PCO2, and bicarbonate in the NBIW used for the study were measured before, during, and after theexperiment.
Western blot analysis. The vessels isolated from the mice were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor and phosphatase inhibitor, and the tissue lysate was used
for Western blot analysis with anti-eNOS antibody (M221; ab76198, 1:1000, Abcam plc), anti-eNOS (phospho
S1177) antibody (EPR20991; ab230158, 1:1000, Abcam plc), and anti-β-actin antibody (HRP-60008, 1:5000,
Proteintech). eNOS, p-eNOS and β-actin in the samples were detected by C-DiGit® Blot Scanner (LI-COR Inc.)on the basis of the chemiluminescence intensity generated by the ECL Prime Western Blotting Detection Reagent (Cytiva) and were quantitatively assessed by Image Studio™ (LI-COR Inc). After SDS-PAGE, gels were separated at each target molecule range to normalize eNOS and p-eNOS expression level by β-actin, a housekeeping
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protein. Each gel was then transferred to a polyvinylidene difluoride membrane and hybridized with primary
antibody for eNOS and p-eNOS or β-actin.

In vitro experiments in HUVEC. HUVEC were starved for 6 h in a medium not containing serum or
VEGF supplement (KBM VEC-1; Kohjin Bio Co., Ltd). NBIW was diluted 1,000,000-fold in the culture medium
so that the bicarbonate ion content became 2.5 ppb. After 5 min, cell lysate was obtained in ultrapure water or
RIPA buffer, and the amount of NO and eNOS phosphorylation activity was evaluated as described above.
ESR analysis. The ROS scavenging activity of NBIW was determined by ESR. Superoxide generated byxanthine/xanthine oxidase, a major ROS, and a hydroxy radical generated by UV-irradiated hydrogen peroxide solution were trapped by 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide(CYPMPO) and determined as a CYPMPO ­adduct36. NBIW at a concentration 100-fold higher than that used
for the mouse bathing experiment was prepared, and its ROS scavenging activity was compared with that of
SBW at a concentration 100-fold higher than that used in the mouse bathing experiment and that of ultrapure
water. Each condition was repeated 3 times, and the data were analyzed.

Randomized, double‑blind, parallel‑group comparison study.

The clinical study was performed incompliance with the Declaration of Helsinki and CONSORT statement and was approved by the Chiyoda Paramedical Care Clinic Ethics Review Committee (IRB number: KRK171C1; approval number: HTT18C1) andregistered with the UMIN Clinical Trials Registry (UMIN000031026) on 29/06/2018. All participants provided
written informed consent to participate in the study. The study was conducted between December 2016 andApril 2017, and no major changes were made to the protocol after the start of the study. Data were collected at
the Chiyoda Oral Healthcare Clinic and Chiyoda Paramedic Care Clinic, and statistical analysis of the data was
performed by CPCC Company Limited, a contract research organization.
Participants consisted of male and female volunteers between 30 and 59 years of age with a subjective symptom of cold intolerance who fulfilled the inclusion criteria and did not meet the exclusion criteria. Inclusion criteria were subjective symptoms of cold intolerance or consistently low body temperature, menopause or a stable
menstrual cycle in women, and ability to bathe daily during the study period and to provide written informed
consent. Exclusion criteria included regular intake of medicine, regular consumption of foods that may improveblood flow and body temperature, history of serious illness or skin conditions, and pregnancy or breastfeeding.
The participants were divided into 2 groups by block randomization on the basis of sex and body temperature on
waking. The person responsible for allocating assigned participants to be given either NBIW tablets containing
bicarbonate ions as the main ingredient or control tablets containing magnesium sulphate and sodium sulphate.
Table 1 shows the composition of NBIW and control tablets. Both tablets were designed as white tablets of 15 geach that were indistinguishable. The randomization code was not disclosed to participants or the people responsible for collecting and analyzing trial data until the end of analysis. Participants were tested during a 1-week
pre-observation period and a 4-week intervention trial. They were instructed to dissolve 4 tablets in a bath with
hot water at a temperature below 41 °C once daily for 4 weeks and to soak in the water for at least for 15 min.
They were also told to not make significant changes to their lifestyle during the study period. Primary outcome
endpoints were body temperature on waking, sleep quality assessed by the Japanese version of Pittsburgh Sleep
Quality Index (PSQI-J)37,38, and score on the simplified POMS-2 for a­ dults39,40. Secondary outcomes were bodytemperature before lunch, before supper, before bathing, 1 h after bathing, and before going to bed and the presence or absence of a subjective symptom of cold intolerance. Participants were given a logbook and instructed
to use it daily to record the items specified in the protocol, such as use of bath tablets, time of bathing, and bodytemperature upon waking and at bedtime, from one week before to the end of the intervention.

Statistical analysis. Statistical analyses were performed with Mac statistical analysis (ESUMI Co., Ltd.)and StatPlus:mac (AnalystSoft Inc.). For the animal experiments, comparisons of 2 independent groups were
performed by Student’s t test for parametric data and the Mann–Whitney U test for non-parametric data. For
the clinical study, Wilcoxon rank sum test or Chi-square test was used for comparisons of 2 independent groups
and Wilcoxon signed rank test, for intragroup comparisons before and after the study. Hedge’s g with the 95% CI
was calculated as an index of impact of the ­effect41.

Data availability

All study data are available from the corresponding author.
Received: 23 April 2021; Accepted: 26 October 2021

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Author contributions

I.S., T.Y., and R.U.N. designed the study; T.Y., R.U.N., S.S., D.O., N.M., T.K., and C.N. contributed to the collection and analysis of study data; I.S., T.Y., R.U.N., M.C.L., and H.I. interpreted the data; and I.S. and R.U.N.
wrote the article. T.Y. and R.U.N. are co-first authors and contributed equally to this article. All authors approved
the final manuscript after critical revision of the manuscript and agree to accept responsibility for its scientificaccuracy and consistency.

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Funding

This research was supported by a research grant from the MEXT-Supported Program for the Strategic Research
Foundation at Private Universities (S1511018).

Competing interests

The authors declare no competing interests.

Additional information

Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​021-​01285-4.
Correspondence and requests for materials should be addressed to I.S.
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