Effects of corn syrup solids containing maltobionic acid (maltobionic acid calcium salt) on bone resorption in healthy Japanese adult women: A randomized double‐blind placebo‐controlled crossover study

Abstract Maltobionic acid is known to have an inhibitory effect on the differentiation of osteoclasts, and it has also been reported in an intervention trial that ingestion of corn syrup solids containing maltobionic acid maintained and increased the bone density of postmenopausal women. However, there is no information on whether maltobionic acid improves bone metabolism in humans. Therefore, we evaluated the influence of corn syrup solids containing maltobionic acid (maltobionic acid calcium salt) on bone resorption markers in healthy Japanese women. Forty‐one individuals were selected from 68 participants and assigned to two groups: 21 individuals in the test food antecedent group and 20 individuals in the placebo food antecedent group; individuals in the first group ingested 4 g of corn syrup solids containing maltobionic acid, and subjects in the second group ingested 4 g of placebo (hydrous crystalline maltose and calcium carbonate) for 4 weeks. Bone resorption marker levels (DPD and u‐NTx) were evaluated by urinalysis. Forty subjects completed the study, and no adverse events related to the test food were observed. Fourteen subjects were excluded prior to the efficacy analysis because of conflict with the control criteria; the remaining 33 subjects were analyzed. Consumption of corn syrup solids containing maltobionic acid was maintained; DPD and u‐NTx values were improved (p < .05). These results indicate that corn syrup solids containing maltobionic acid might contribute to suppress bone resorption and improve bone metabolism in postmenstrual women. (UMIN‐CTR ID: UMIN000034257; Foundation: San‐ei Sucrochemical Co., Ltd.).


| INTRODUC TI ON
According to the NIH, osteoporosis is "A disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk" (NIH Consensus Development Panel, 2001). In the ultra-aging society of Japan, the number of patients with osteoporosis has been increasing year by year with current estimates suggesting that 12.8 million Japanese citizens over 40 years old are affected, 70% of whom are women (Yoshimura et al., 2010(Yoshimura et al., , 2009. Bone density peaks at puberty in both sexes, and decreases with age thereafter (Carrié Fässler & Bonjour, 1995;Ilich, Badenhop, & Matkovic, 1996). Women, in particular, are known to experience a sharp decrease in bone mass after menopause (Orito, Kuroda, Onoe, Sato, & Ohta, 2009).
Bone is a tissue composed mainly of hydroxyapatite, a type of calcium phosphate, and type I collagen. Within the bone, old bone is lysed and resorbed by osteoclasts (bone resorption) and new bone is formed by osteoblasts (osteogenesis) in a continuous cycle (Frost, 1964;Parfitt, 1994). It has been reported that an imbalance in bone metabolism, where an increase in bone resorption by osteoclasts overtakes bone formation, leads not only to a decrease in bone density and an increased risk of osteoporosis, but also drives the risk of additional diseases, such as rheumatoid arthritis (Haugeberg, Uhlig, Falch, Halse, & Kvien, 2000) and periodontitis (Mohammad, Hooper, Vermilyea, Mariotti, & Preshaw, 2003;Penoni et al., 2016). Estrogen deficiency, which also occurs frequently in postmenopausal women, induces osteoclast activation (Zebaze et al., 2010). Therefore, in addition to keeping the bone healthy, suppression of excessive bone resorption is important for maintaining quality of life (Q OL) (Silverman, Minshall, Shen, Harper, & Xie, 2001).
Maltobionic acid, a disaccharide in which glucose is α-1,4-bonded to gluconic acid, is a component of honey that has been a part of the human diet since ancient times ( Figure 1). Although it is a saccharide, it has a mildly sour taste and high water solubility despite ion binding with calcium, a feature that leads to a stable salt formation with inorganic cations (Suehiro, Okada, Fukami, Otuka, Nakagawa, & Hayakawa, 2017). Previously, we have reported using rat models that maltobionic acid enhances calcium and magnesium absorption by maintaining the solubilized state of minerals throughout the intestinal tract and increasing the amount of calcium in the femur (Suehiro et al., 2017;Suehiro, Okada, Fukami, Nakagawa, & Hayakawa, 2019). In vitro studies with cultured cells confirmed that maltobionic acid has an inhibitory effect on the differentiation of osteoclasts (manuscript in preparation). In addition, a 24-week intervention trial revealed that the ingestion of corn syrup solids containing maltobionic acid (maltobionic acid calcium salt) maintained and increased the bone density in postmenopausal women (Fukami, Suehiro, & Takara, 2019). However, there is no information on whether maltobionic acid improves bone metabolism in human clinical trials. Therefore, this study has investigated the effect on bone resorption markers in Japanese adult women up on ingesting maltobionic acid calcium salt.

| Study design and participants
This was a randomized, double-blind, placebo-controlled, crossover trial. The study participants were publicly recruited and included Japanese adult women who worked at Chubu University (Aichi, Japan). A preliminary questionnaire was administered to those who gave written informed consent confirming their wish to participate in the study. Sixty-eight women aged 40-69 years were enrolled in the study. The following exclusion criteria were used to select the final study participants by excluding those with: (a) a medical history of osteoporosis, malignant tumor, heart failure, or myocardial infarction; (b) the presence of other diseases (osteoporosis, arrhythmia, liver dysfunction, kidney dysfunction, cerebrovascular disease, rheumatism, diabetes, dyslipidemia, hypertension, or other chronic disease); (c) the regular use of pharmaceuticals (including Kampo) or supplements; (d) the regular ingestion of foods for specialized health use or with functional claims; (e) weekly ingestion of calcium, vitamin D, vitamin K, magnesium, isoflavones (including daidzein, genistein, equal) and all other supplements, foods for specialized health use, foods with functional claims, and foods with nutritional function claims that could affect bone metabolism; and (f) allergies (pharmaceuticals and foods related to the test foods in this study). The subjects in their premenopausal period or those who had undergone premature menopause due to genetics, illness, or medical procedures were also excluded.  Mori, Sagara, Ikeda, Miki, and Yamori (2004) and studies by Uesugi, Fukui, and Yamori (2002).

| Selection, randomization, and blinding
Forty-one postmenopausal women who had undergone natural menopause were selected from 68 participants and assigned to two groups: 21 individuals in the test food antecedent group and 20 individuals in the placebo food antecedent group, where those in each group did not differ greatly in age or BMI. Group allotments were conducted by an intermediary study controller using StatLight #11 (Yukms Co., Ltd.).
The group allotments were unknown to study participants, principal investigator, outcome assessors, and all other staff involved in this study; none of these individuals were involved in the allotment process.

| Test food
The test food used in the intervention was corn syrup solids containing maltobionic acid (SourOligo C, San-Ei Sucrochemical Co., Ltd.) packaged in stick-shaped packet form (4 g/packet

| Outcome measures
Examinations were performed a total of four times before and after the intervention.

Primary outcome: Urinary bone resorption marker
Approximately 10 ml of first morning urine was collected from test participants. Two bone turnover markers, urinary deoxypyridinoline (DPD) and urinary type I collagen-cross-linked-N-telopeptide (u-NTx), were measured and corrected by urinary creatinine content.

Diet survey
Calcium (Xu, McElduff, D'Este, & Attia, 2004), potassium (Macdonald et al., 2008), vitamin D (Matsumoto et al., 2005), and vitamin K (Feskanich et al., 1999) are known to maintain bone density and bone metabolism; the test foods in this study are expected to have similar effects. To correctly assess the effects of the test foods, a diet survey was performed using a brief-type self-administered diet history questionnaire (BDHQ) before and after the interventions began. The BDHQ questionnaire-based on a computer algorithm that takes into account the energetics and selected nutrients consumed to estimate the intake-was used to investigate the ingestion of 56 foods and beverages over the last month (Kobayashi et al., 2012(Kobayashi et al., , 2011.

| Statistical analysis
All outcomes were presented as mean ± SD. The primary outcome, DPD, and u-NTX, were tested for carryover and aging effects, thereby confirming that the crossover design was adequate.
Results were examined in intragroup and intergroup comparisons.  Figure 2 shows a follow-up flowchart for the study participants.

| Analysis of subjects
Of the 68 individuals who consented to participate in this study, 27 were excluded during interviews with the principal investigator or because of the inclusion/exclusion criteria. Finally, 41 individuals were enrolled in this study and assigned to the test food antecedent group (21 individuals) and the placebo food antecedent group (20 individuals). We excluded one dropout from the study and seven violators of compliance (unsubmitted diaries, ingested food consumption rates of 80% or less, and inadequacies at the time of urine sample submission). Though the study participants whose urinary bone resorption markers (DPD and u-NTx) in the urinalysis before and after the intervention deviated from the standard values specified in the guideline (Nishizawa et al., 2019) were to be excluded, none of the participants were eligible.
Therefore, the object of the analysis was to carry out the analysis in the "per protocol set," which amounts to 33 subjects (mean age, 52.3 ± 5.3 years).

| Efficacy assessment
The background characteristics of the 33 subjects in the subject population for the efficacy analysis are shown in Table 1. The efficacy assessment items (DPD and u-NTx) are shown in Table 2 and

Urinalysis
In the urinalysis, although items showing false positives (Pro, Bld) and slight positives (Ket) were occasionally observed, there was no significant difference between the groups, and the changes observed were not considered medically problematic.

Diet survey
In the dietary survey by BDHQ, there were no significant differences in the various nutrients ingested between the groups.

| D ISCUSS I ON
The purpose of this study was to evaluate the effect of maltobionic acid intake on bone metabolism in Japanese women who were 40-69 years old and who were more than 1 year past menopause. In order to evaluate bone metabolism, bone density and bone metabolism markers are usually utilized. Because bone metabolism changes from day to day, the metabolic state is different even at the same bone density value (Miller, Hochberg, Wehren, Ross, & Wasnich, 2005). Therefore, an observation period of 6 months to 1 year is required to make bone density a dynamic marker. Conversely, as the bone metabolism markers quantitatively reflect bone metabolism at the time of measurement, they are useful to predict future bone loss and fracture risks and are essential examination items in the diagnosis of osteoporosis (Soen et al., 2013). Elevated bone resorption markers have been identified as fracture predictors in prospective studies (Ivaska, Gerdhem, Väänänen, Åkesson, & Obrant, 2010) and have also been reported to be risk factors for fractures independent of bone mineral density (Gerdhem et al., 2004).
In this study, DPD and u-NTx were measured in urine specimens as bone resorption markers. As noninvasive markers, where blood collection is unnecessary, they are widely used in medical practice for the diagnosis of osteoporosis and confirmation of therapeutic effect (Eastell   Robins et al., 1991). In particular, DPD is mainly localized in bone and dental tissues, though dental tissues do not affect urinary excretion levels. Thus, bone can be regarded as the only tissue of origin for urinary DPD, which has been reported to be particularly useful as an indicator of bone resorption (Uebelhart et al., 1991). Note: The number of subjects with each result is shown.

TA B L E 4 Diet survey (BDHQ)
for the treatment of osteoporosis (Chesnut et al., 1995;Marttunen et al., 1999). Maltobionic acid has also been shown to maintain or increase bone mineral density in a 24-week intervention study in postmenopausal women , which may originate from the inhibition of bone resorption. Furthermore, in an animal study in rats, it was confirmed that maltobionic acid maintained the solubilization state of calcium throughout the intestine, thereby increasing the calcium retention rate . It has been reported that the bioavailability of calcium is closely related to bone density and bone metabolism (Cashman, 2002;Weaver, 1992). Maltobionic acid may have further contributed to the improvement of bone metabolism by enhancing the bioavailability of calcium.
Finally, the efficacy outcomes in this study were limited to the markers of bone resorption. For further assessment of the effects on bone turnover, markers of bone formation (e.g., OCs, BAPs) and bone matrix-related markers (e.g., ucOC, pentosidine) must also be included in the outcomes and analyzed comprehensively (Eastell et al., 2018;Eastell & Szulc, 2017). Additionally, quantification of serum parathyroid hormone and calcitonin, which are blood markers, is necessary to evaluate the increase in calcium bioavailability by maltobionic acid and their effects on markers of bone metabolism. Further research is necessary to clarify the mechanism underlying bone metabolism improvement effect by the intake of maltobionic acid.
In conclusion, continuous intake of maltobionic acid in healthy Japanese adult women was found to suppress bone resorption and improve bone metabolism, which may contribute to the prevention of osteoporosis.

ACK N OWLED G M ENTS
This research was partially supported by the Chubu University Grant (A). We would like to thank Editage (www.edita ge.com) for English language editing. At the same time, we would like to thank others who contributed to the experiments but have not been listed as authors.

E TH I C A L A PPROVA L
This study was approved by the Chubu University Certified Review Board on September 21, 2018 (no. 300019-2).

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.