**3. Discussion**

The present study revealed an association between *H. pylori* infection and the absorption of fucoidan. Specifically, fucoidan absorption was significantly diminished among *H. pylori*-positive subjects aged ≥40 years who ate mozuku at least once monthly, whereas no association was found among *H. pylori*-negative subjects irrespective of the frequency of mozuku consumption and age. In addition, fucoidan absorption was not diminished among *H. pylori*-positive subjects who ate mozuku once every 2–3 months or less; therefore, mozuku consumption affects the absorption of fucoidan. Although the precise mechanisms by which *H. pylori* infection and mozuku consumption reduce fucoidan absorption have not been determined, a few possibilities have been postulated.

As ΔMax fucoidan values were similar between *H. pylori*-negative and *H. pylori*-positive subjects among participants aged ≥40 years, *H. pylori* was less likely to directly diminish the absorption of fucoidan in this age group. Excluding *H. pylori*-positive participants aged ≥40 years who ate mozuku regularly, ΔMax fucoidan values were similar between *H. pylori*-positive and *H. pylori*-negative subjects. In addition, the frequency of mozuku consumption among *H. pylori*-positive participants was similar between subjects aged <40 years and those aged ≥40 years; therefore, the frequency of mozuku consumption is not directly associated with the absorption of fucoidan. Given that *H. pylori* positivity and regular mozuku consumption were associated with diminished fucoidan absorption among subjects aged ≥40 years but not among younger subjects, the duration of *H. pylori* infection and the frequency of mozuku appear important for fucoidan absorption.

How do frequent mozuku consumption and *H. pylori* infection disturb fucoidan absorption in subjects aged ≥40 years? *H. pylori* can change the secretion and acidification functions of the stomach because it penetrates into this organ. Although nutrient absorption does not occur in the stomach, *H. pylori* infection can affect the digestion and absorption of nutrients such as vitamin B12, vitamin C, vitamin A, vitamin E, and folate [19–21]. Shibata et al. [7] reported that mozuku fucoidan can bind to *H. pylori* and inhibit its attachment to the gastric mucosa at pH 2.0 and 4.0, but not at pH 7.4. It is well known that *H. pylori* rarely causes atrophic gastritis in young people (<40 years old), whereas *H. pylori*-induced atrophic gastritis tends to be rather common in the elderly [15–18]. When hypochlorhydria occurs after *H. pylori*-induced atrophic gastritis, intragastric pH increases, consequently inhibiting the ability of *H. pylori* to bind to fucoidan. However, fucoidan absorption was not diminished in *H. pylori*-positive subjects aged ≥40 years who rarely ate mozuku, suggesting the influence of a long duration of mozuku ingestion on fucoidan absorption. Amornlerdpison et al. reported that fucoidan present in mozuku acts as an antagonist of the H2 receptor (similar to cimetidine), decreasing the acidity of gastric acid and raising the pH in the stomach [23]. Taken together, *H. pylori*-induced atrophic gastritis and a long duration of mozuku ingestion may markedly decrease acid secretion, consequently leading to the failure of *H. pylori* to bind fucoidan and reductions of its absorption in the small intestine in people aged ≥40 years. Thus, the possible mechanism by which *H. pylori* infection leads to reduced gastric acid secretion and fucoidan absorption has not been sufficiently investigated to draw definite conclusions, and other mechanisms other than hypochlorhydria following *H. pylori* infection are possible.

Of note, significant positive correlations of the basal fucoidan levels with both *H. pylori* titers and ΔMax fucoidan values were revealed in *H. pylori*-positive subjects aged <40 years who frequently consumed fucoidan. Such correlations were not found in the corresponding group of participants aged ≥40 years, nor were they observed in *H. pylori*-positive subjects who rarely ate mozuku or in *H. pylori*-negative subjects. Because the significance of the positive correlation observed in *H. pylori*-positive subjects aged <40 years who frequently consumed fucoidan is unclear, further research is necessary to clarify the relevance of *H. pylori* infection and mozuku intake to fucoidan absorption using a large number of subjects.

Interestingly, basal fucoidan levels were significantly increased by *H. pylori* infection and mozuku consumption. As *H. pylori* is known to affect the absorption of various nutrients, this stomach bacterium may participate in basal fucoidan absorption. In recent years, "the nutrition-gut microbiome-physiology axis" has attracted substantial attention [24–28]. Because *H. pylori* can induce drastic alterations in the variety of the gastrointestinal microbiota [29–31], the microbe is speculated to increase basal fucoidan levels by modifying the gastrointestinal microbiota. Basal fucoidan levels were also significantly higher in *H. pylori*-negative subjects who regularly consumed mozuku than in their counterparts who rarely ate mozuku, which confirmed our previous findings [13]. We speculated that Japanese people may have acquired digestive enzymes from mozuku because the seaweed is extensively consumed within this area. Because of the limited evidence, the overall significance of *H. pylori* infection and mozuku consumption to basal fucoidan levels is unclear.

This study had several limitations. First, subjects who received eradication therapy for *H. pylori* and underwent gastrectomy prior to the study were not excluded. The study also did not exclude subjects who used complementary and alternative medicines, which can affect the absorption of fucoidan.

Second, a urine-based ELISA kit (URINELISA) was used to assay *H. pylori* infection, and the high accuracy of this test was certified by several investigators [32,33]. A disadvantage of this test is that proteinuria can cause false-positive results; therefore, urine protein levels should be measured in future research. We plan to study the relationship of *H. pylori* infection with fucoidan absorption using serum-based ELISA kits or the 13C urea breath test (13C-UBT) in future research.

In addition, the specificity of our fucoidan ELISA was limited. We assayed urinary fucoidan levels using a polyclonal antibody for Okinawa mozuku fucoidan, which weakly cross-reacted with *Fucus vesiculosus* fucoidan [22]. Because the brown seaweeds of kombu (*Laminaria japonica*) and wakame (*Undaria pinnatifida*) are traditional foodstuffs in Japan, fucoidan contained in these seaweeds may cross-react with our antibody. Further studies are necessary to elucidate the effects of mozuku consumption on the intestinal absorption of fucoidan using ELISA with a monoclonal antibody.
