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Journal of the American College of Nutrition, Vol. 26, No. 6, 645-649 (2007)
Published by the American College of Nutrition

Acute Effect of Poly-{gamma}-Glutamic Acid on Calcium Absorption in Post-Menopausal Women

Hiroyuki Tanimoto, MSc, Tom Fox, PhD, John Eagles, LRSC, Hitoshi Satoh, MSc, Hiroko Nozawa, BSc, Atsushi Okiyama, PhD, Yasushi Morinaga, PhD and Susan J. Fairweather-Tait, DSc

Institute of Food Research, Norwich Research Park, Colney (H.T., T.F., J.E.)
Diet and Health Group, School of Medicine, Health Policy and Practice, University of East Anglia (S.J.F.-T.)
Norwich Norfolk, UNITED KINGDOM, Food Research & Development Laboratories, Ajinomoto Co., Inc., Kawasaki-ku, Kawasaki, JAPAN (H.T., H.S., H.N., A.O., Y.M.)

Address reprint requests to: Hiroyuki Tanimoto, Health Foods Business Group, Seasonings Dept., Ajinomoto Co., Inc., 15-1, Kyobashi 1-Chome, Chuo-ku, TOKYO 104-8315, JAPAN. E-mail: hiroyuki_tanimoto{at}ajinomoto.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: Poly-{gamma}-glutamic acid (PGA) increases calcium (Ca) solubility in vitro and in vivo, and is associated with reduced bone loss in post-menopausal Japanese women. This study is the first to examine the effect of PGA on Ca absorption in humans.

Methods: A single-blind, randomized, crossover study with a 3–4 week wash-out was performed to determine the effect of PGA (80.6% glutamic acids) on Ca absorption measured by the double stable isotope method. Twenty-four healthy, non-smoking, postmenopausal women (mean age: 56.4 ± SE 0.9) were given 200 g of orange juice containing 200 mg Ca as Ca-44 enriched CaCO3, with or without 60 mg of PGA, after an overnight fast. The two tests were separated by 3–4 weeks. An intravenous injection of Ca-42 (CaCl2 solution) was given 30 min after consuming the drink and a complete urine collection carried out from 24–48 h post-dosing. Ca absorption was calculated from the Ca isotope ratios measured by thermal ionization quadrupole mass spectrometry (TIQMS).

Results: Mean Ca absorption with PGA was significantly higher (P < 0.01) than without PGA, 39.1 (SE 1.6) % and 34.6 (SE 1.9) %, respectively. The effect of PGA on increasing Ca absorption was more marked in a sub-group of subjects whose baseline Ca absorption (without PGA) was lower than the population mean value.

Conclusion: Postmenopausal women who received a single dose of PGA increased their intestinal Ca absorption particularly those individuals with lower basal absorptive capacity.

Key words: poly-{gamma}-glutamic acid, natto, fermented soybeans, stable isotopes, calcium absorption, post-menopausal women


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
A number of food constituents have a positive [1–3] or negative [4–6] effect on intestinal Ca absorption in humans. Ca solubility is increased in the small intestine of rats given poly-{gamma}-glutamic acid (PGA), a polymer in which a large number of glutamic acid molecules are combined by {gamma}-linkages [7]. PGA is a component of natto mucilage obtained from fermented soybeans, a traditional Japanese food; estimates of natto consumption in Japan suggest that the daily intake of PGA is approximately 16 mg/day. PGA can also be produced by fermentation of Bacillus natto in a liquid medium [8] and it has been considered as a candidate compound for functional foods aimed at promoting bone health. A recent report suggests that the consumption of natto is associated with reduced bone loss in postmenopausal Japanese women [9]. We hypothesise that the mode of action of PGA is increased Ca solubility in the gut lumen [7] thereby increasing paracellular Ca absorption in the lower intestine.

The effect of PGA on human intestinal Ca absorption is unknown. We therefore undertook a double stable isotope study in which we measured Ca absorption from 200mg Ca, given with or without 60 mg PGA, in a group of healthy, non-smoking postmenopausal European Caucasian women. Orange juice was selected as the food vehicle because it does not contain substances that significantly affect Ca absorption.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Test Material
PGA was produced by fermentation of Bacillus natto in a liquid medium at a pilot scale plant (Kawasaki Factory, Ajinomoto Co., Inc., Kawasaki, Japan). The solution was purified by heating under acidic condition (sterilization and reduction of viscosity) and a dried product was prepared by freeze-drying (average molecular weight of PGA was 11,000). The PGA was 80.6% glutamic acids (w/w) and the remainder was moisture and ash.

Subjects
Healthy, non-smoking postmenopausal women (n = 24) were recruited from staff working at institutions on the Norwich Research Park and from local residents. All women had completed their menopause, had not taken hormone replacement therapy for at least 1 year, and were not taking any medication that could affect intestinal Ca absorption at entry to or during the study. The onset of their last menses ranged from 1.5 to 25 (mean 6.8) years.

The study protocol was approved by Norwich District Ethics Committee. Written informed consent was obtained from all volunteers.

Blood Sampling
Before acceptance onto the study, screening blood samples were taken from each volunteer for general health evaluation, and to measure plasma 25-hydroxyvitamin D (25-OHD) concentration. Samples were stored at –25°C and plasma concentration of 25-OHD was measured by competitive radioimmunoassay (RIA) using a 25-OHD-125I RIA Kit (INCSTAR Corporation, Stillwater, Minnesota, USA).

Study Protocol
Estimation of Daily Ca Intake.
Each subject who had passed the simple screening test completed a food frequency questionnaire designed to estimate habitual daily intakes of Ca [10].

Measurement of Ca Absorption.
Ca absorption was determined using the double stable isotope method [11]. The study was a single-blind, randomized, crossover design. After an overnight fast (approximately 12 h) each volunteer attended the Human Nutrition Unit at the Institute of Food Research and a cannula was inserted into the ante-cubital vein of the forearm.

A test drink was prepared from commercially available orange juice (Pure Orange Juice, Sainsbury's, UK). Calcium carbonate was labelled with Ca-44 (200 mg of Ca containing 28–42 mg of Ca-44; the isotope dose was calculated in relation to body weight to ensure sufficient isotope enrichment in the urine) and added to 200 g of orange juice. To this mixture was added 60 mg of PGA, or not, depending on the randomization schedule for each volunteer. The test drink was consumed and exactly 30 min later 1.5–2.0 mL of calcium chloride solution (containing 5–8 mg of Ca-42, depending on body weight, see below) mixed with 20 mL of 0.9 % saline solution was infused slowly via the cannula. The isotopes Ca-44 (96.5 % enrichment) and Ca-42 (80.7 % enrichment) were purchased from Europe Scientific Ltd. (Crewe, UK).

The intravenous (i.v.) dose did not at any time exceed 5% of the estimated size of the rapidly miscible pool of Ca for adults [12]. The time for infusion was approximately 20 min and afterwards the cannula was withdrawn. The quantity of both stable isotopes administered was adjusted for individual body weight; the doses of Ca-44 and Ca-42 were calculated as 0.5 and 0.1 mg per kg body weight of the subjects, respectively. Volunteers were not allowed any food or drink for 3 h after the i.v. infusion and then they returned to their usual diet.

A complete 24-hour urine collection was made during the period 24-48h after the test meal (from the second void on the day after the test meal and including the first urine on the following day) in acid-washed bottles. The urine sample was mixed well, and 150 mL was transferred into an acid-washed bottle and stored at –25 °C until analysis.

After an interval of approximately 3–4 weeks subjects consumed the second test drink and the procedure to measure Ca absorption was repeated.

Sample Preparation and Analysis.
Sub-samples of urine (50–100 mL) were digested using a microwave oven (CEM Model MD5-2000 Microwave Sample Preparation System; CEM Corporation, Matthews, NC, USA), the digestate was purified by ion exchange chromatography using AG 50W-X8 resin (200–400 mesh, Hydrogen form, Biotechnology Grade, Bio-Rad Laboratories, Hercules, CA, USA) and the Ca-enriched fractions were collected and dried under a 1 KW lamp [13].

The dried samples were dissolved in 0.2 M HNO3 (to give approx. 1 mg/mL) and Ca isotope ratios determined by TIQMS (THQ, Finnegan-Mat GmbH, Bremen, Germany) using the double filament technique [11]. The calculation of Ca absorption was based on the assumption that both intravenously and orally administered Ca isotopes were metabolized at the same rate once a state of equilibrium was reached. The percentage absorption from the oral dose was determined according to the following equation [13]:

Formula 1(1)
where na is the natural abundance of the two isotopes (atom %), i.v. and oral are the exact dose administered (mM), and {Delta}%XS is the degree to which a particular ratio differs from the natural ratio.

Statistics
A paired t-test was used to assess the difference in Ca absorption with and without PGA. Differences in mean values between the two groups were considered significant at p < 0.05. The paired t-test was calculated using Arcus Quikstat (Longman Software Publishing, Cambridge, UK).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
A summary of the characteristics of the volunteers is given in Table 1. Mean (± SE) plasma 25-OHD was 28.5 ± 1.3 ng/mL. There was no significant difference in plasma 25-OHD between the subjects taking vitamin D tablets and those not taking them (30.8 ± 2.2 ng/mL and 27.5 ± 1.6 ng/mL, respectively). However, the seven subjects taking vitamin D supplements were asked to refrain from taking them for the last week before the Ca absorption tests to avoid any possible effects of oral vitamin D tablets on Ca absorption. The plasma levels of 25-OHD in the study subjects were within the range reported by Hine and Roberts [14].


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  		Table 1. Subject Characteristics (mean ± SEM)

 
Daily Ca intake, as calculated from the questionnaire, ranged from 694 to 1968 mg (mean = 1141 mg). These results are relatively high compared to the Reference Nutrient Intake for Ca in UK adults of 700 mg/day, but are similar to the results previously reported for people living in or near Norwich, namely 1233 mg/day [10]. One of the reasons for the high daily Ca intake is the significant contribution made by tap water in Norwich (11.3 mg/100 mL used for the calculation). The mean Ca intake from drinks including coffee and tea was calculated to be 181 mg/day. The main Ca source was from dairy products (48 % of daily Ca intake).

Ca Absorption
Fractional Ca absorption (%) with and without PGA measured from the 24–48h urine sample is shown in Fig. 1. Mean absorption with PGA was significantly greater (39.1 ± 1.6%) than when the subjects were not receiving PGA (34.6 ± 1.9; mean ± SE) (mean of differences = 4.5, 95% confidence interval = 1.2 to 7.8, P < 0.01).


Figure 1
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  		Fig. 1. Calcium absorption in post-menopausal women measured from a complete 24–48h urine collection after consuming 200mg Ca in orange juice with and without poly-{gamma}-glutamic acid (PGA). The significance of the difference between Ca absorption with and without PGA was evaluated by paired t-test (mean of differences = 4.5, 95% confidence intervals = 1.2 to 7.8, P = 0.0094). Each value is expressed as the Mean ± SEM.

 

   DISCUSSION
 
This is the first report of a human study investigating the effect of PGA on Ca absorption. We do not know the site of effect of PGA and although the first 24h urine is used to measure Ca absorption [15,17], this time period may not include all of the oral isotope associated with absorption from the large intestine. Furthermore, earlier studies indicate that it may take up to 24 h for the retained isotopes to equilibrate within the body [18], therefore we collected the 24–48 h urine samples because this combines upper and lower intestinal absorption. The results clearly demonstrated that a single dose of PGA significantly increased Ca absorption in post-menopausal women, which supports the earlier observations in rats [7] and the recent epidemiological data on natto consumption and risk of osteoporosis in Japanese women [9].

The ability of PGA to stimulate Ca absorption was assessed when volunteers were stratified by the ratio of their Ca absorption with and without PGA, as shown in Fig. 2. Points above 1.0 on the Y-axis mean that Ca absorption was greater with PGA. In the sub-group of volunteers (n = 13) whose Ca absorption without PGA was less than the mean value of 34.6 % (the left side of the x-axis), the effect of PGA was marked. In the other sub-group of volunteers (n = 11) whose Ca absorption without PGA was more than 34.6 % (the right side of the x-axis), there was little effect of PGA. This indicates that PGA had a greater enhancing effect on Ca absorption in volunteers with a lower efficiency of Ca absorption.


Figure 2
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  		Fig. 2. The ratio of Ca absorption with PGA and without PGA was plotted against Ca absorption without PGA. The dotted line on the X-axis shows the mean value (34.6%) for Ca absorption without PGA. The solid line on the Y-axis shows the ratio of with to without PGA as 1.0.

 
Serum 25-OHD, intestinal transit, and urinary Ca to creatinine ratio are reported to be significantly and positively correlated with efficiency of Ca absorption [19]. However, we measured plasma 25-OHD from the screening blood, and found no correlation between plasma 25-OHD and Ca absorption without PGA (P = 0.945).

It is generally accepted that intestinal calcium absorption involves two processes [20], a saturable (active and transcellular) transport that is regulated by activated vitamin D and takes place in the upper small intestine, and a non-saturable (passive and paracellular) transport that is dependent on soluble Ca concentration and takes place throughout the entire length of the intestine [21] but predominates in the lower small intestine [22]. Although it is not yet clear by which mechanism PGA enhances Ca absorption, preliminary studies suggest that PGA may enhance paracellular transport, since it increases Ca solubility in the lower small intestine in rats [7].


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
ACKNOWLEDGMENTS
 REFERENCES
 
Postmenopausal women given a single dose of PGA demonstrated a significant increase in intestinal Ca absorption, particularly in individuals with a baseline efficiency of Ca absorption below the mean.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
REFERENCES
 
This study was funded by the Ajinomoto Company, Japan. The authors thank Rob Foxall, Institute of Food Research, Norwich, UK, for statistical support. The authors thank Dr. David P. Katz, Consultant to Ajinomoto USA, for his review and suggestions for the paper.

Received April 7, 2006. Accepted December 21, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 

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This Article
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Right arrow Articles by Tanimoto, H.
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