Lactulose stimulates Ca absorption

Lactulose stimulates calcium absorption in postmenopausal women.

Olen J.H.M., Van Den Guevel, Theo Muijs, Wim Van Dokkum, Gertchen Schaafsma 1 TNO

(Netherlands Organization for Applied Scientific Research)

Food and Nutrition Research Institute, Department of Physiology, Zeist, The Netherlands. DMV International (International Dairy Company) Wegel, The Netherlands.

Animal studies have shown that calcium absorption increases with the use of lactulose, a synthetic disaccharide.

Therefore, the effect of lactulose on calcium absorption was measured in postmenopausal women who may benefit from the increased effect of lactulose on calcium absorption. Twelve postmenopausal women took 5 or 10 g of lactulose or a reference substance in 100 ml. of water with breakfast for 9 days. Three courses of treatment were completed in a randomized, double-blind, crossover schedule separated by a 19-day washout period. On the 8th day of each treatment period, 44 Ca dissolved in orange juice was drunk immediately after the solution with the test substance before a normal breakfast of 162 mg. of calcium in a capsule. Half an hour later, 48 Ca was administered intravenously. Based on the isotope ratio measured in urine collected before and after 36 hours after isotope administration, the true fractional calcium absorption was calculated. Calcium absorption during treatment with the reference substance, 5 g and 10 g of lactulose was (mean SD (standard deviation) 27.77.7, 30.07.6 and 32.27.0 respectively). A significant difference in calcium absorption was observed between the highest dose of lactulose and the reference treatment (p < 0.01). An important linear trend was observed between the dose of lactulose and its positive effect on calcium absorption. Thus, in postmenopausal women, 9 days of lactulose administration increased calcium absorption in a dose-dependent manner. Further study of the stimulation of calcium absorption by lactulose and whether it can improve calcium balance and/or attenuate bone loss with age would be warranted. (J Bone Miner Res 1999;14;1211-1216)

Introduction.

Lactulose is a synthetic disaccharide (4-0- D-galactopyranosyl - D-fructose, molecular weight 342.3), which does not occur in nature. It is obtained in small quantities by heat treatment of milk. In large quantities, lactulose can be produced from lactose by alkaline isomerization, in which galactose is linked to fructose as a 14-glycoside. It is not digested in the stomach/small intestine, but is fermented in the large intestine by native microflora. Due to fermentation, as one of the factors, short-chain fatty acids are formed and the pH level is reduced.

Animal studies have shown that calcium ( Ca ) absorption is increased by lactulose, which was found to be more effective than lactose and pectin. In addition to the possible effect of lactulose on mineral absorption in the small intestine due to improved Ca solubility, the absorption pathway in the large intestine is also interesting. Fermentation of undigested food components and therefore a lower pH (due to the formation of short-chain fatty acids by the microflora) may have a positive effect on Ca absorption from the distal intestine.

Increased Ca absorption may be of particular interest to postmenopausal women.

The inverse effect of age on Ca absorption has been associated with a decrease in serum 1,25-dihydroxyvitamin D (8), thus reducing the active component of absorption. By taking indigestible ingredients such as lactulose, the level of passive Ca absorption in the small or large intestine can be increased and the efficiency of Ca absorption in later life is restored.

Due to the lack of human studies on the effect of lactulose on Ca absorption, a study was conducted in a group of people (postmenopausal women) who may benefit from a possible enhancement of Ca absorption by lactulose. The primary aim of this study was to investigate the possible positive effect of lactulose consumption on true fractional Ca absorption in healthy postmenopausal women.

Table 1. Composition of the studied substances.

A secondary objective was to compare the effect of taking 5 g versus 10 g of lactulose on actual Ca absorption.

Materials and methods

Subjects were recruited from the TNO volunteer team at the Food and Nutrition Research Institute and through advertisements in a local newspaper.

Twelve women who had been postmenopausal for at least 5 years were selected based on high follicle-stimulating hormone (FSH) and low estradiol (E2) levels.

At the start of the study, their ages ranged from 56 to 64 years (mean age 60.5 years) and their body mass index ranged from 20.7 to 27.8 (mean 25.0). Eleven subjects had E2 levels <20 pg/mL. Seven subjects had E2 and FSH levels of 60 pg/mL and 51 IU/L, respectively. All subjects had FSH levels between 51 and 121 IU/L (mean 89.6 IU/L). All subjects received a medical report on their general health from a staff physician and gave informed consent to participate in the study after the entire study procedure was explained to them. The study protocol was approved by the external medical ethics committee at TNO.

Research plan and research implementation

The study was conducted in accordance with the revised Declaration of Helsinki (Somerset West, South Africa, 1996) and the ICH (International Conference on Harmonization) guidelines for good clinical practice (ICH discussion paper E6, adopted 01-05-1996 and implemented 17). The subjects were informed that the test substance would be taken with breakfast for 9 days in addition to their usual meals.

The test substances consisted of 5 or 10 g of lactulose powder (Solvay Pharmaceuticals GmbH, Hannover, Germany) or placebo dissolved in 100 ml of water with benzoic acid. The exact composition is given in Table 1.

Aspartame was chosen as placebo; It was not expected to interfere with Ca absorption due to its small amount relative to lactulose and Ca and because aspartame is completely digested in the stomach/small intestine, thus completely absent in the colon to avoid interaction with Ca. Quinoline yellow was added to give the three solutions the same color. Benzoic acid was added as a precaution. The sensory panel did not show any difference in bitterness or acidity of the different test substances. The 5 g lactulose solution was considered the sweetest solution, followed by placebo and the 10 g lactulose solution.

During the first 7 days of each treatment period, the subjects received the test substances delivered to their homes. On the last 2 days, the subjects were admitted to the metabolic department of the institute and Ca absorption tests were performed.

On day 8 after a 12-hour overnight fast, orange juice with 44 Ca was drunk immediately after the test substance before starting a regular breakfast with 162 mg. Ca in a capsule as measured by atomic absorption spectrometry. After half an hour of oral intake of 44 Ca, 48 Ca was administered intravenously. Blood pressure and pulse rate were measured before and after the bolus infusion for safety. The average amount of isotopes obtained by each method, which was calculated by weighing the ampoules or syringes before and after intake, was 13.9 mg. (In the range of 13.2 -15.1 mg.) 44 Ca and 1.15 mg. (In the range of 1.10 -1.17 mg.) 48 Ca. From the ratio of the 44Ca/43Ca and 48Ca/43Ca measurements of urine taken before the dose, the fractional absorption of Ca was calculated according to the formula specified by Van Dokkum.

Preparation of stable isotope solutions

Stable isotopes were obtained from NEDRAY (Bunschoten, The Netherlands) in the form of Ca carbonate. The content of the different Ca isotopes according to inductively coupled plasma mass spectrometry (ICP-MS) analysis was: 3.39% 40 Ca, 0.03% 43 Ca, 95% 44 Ca, 4.8 40 Ca, 0.09% 42 Ca, 0.02% 43 Ca, 0.24% 44 Ca, <0.01% 46 Ca, 90.69% 48 Ca for Ca enriched in 48 Ca. The 44 Ca carbonate was converted to the chloride salt, diluted with deionized water adjusted to a pH of 5. The same procedure was performed for the 48 Ca carbonate, only saline solution was used instead of deionized water. After filtration, the solution was distributed into 10-ml injection ampoules and sterilized for 25 minutes.

Stable isotope analysis

Although ICMP-MS analysis has several advantages over other methods, it sometimes suffers from molecular interference from various sources. The most obvious is isobaric interference at low masses, especially 40 Ar from plasma with 40 Ca. Therefore, the Ca isotope ratios 44 Ca/ 43 Ca and 48 Ca/ 43 Ca in urine were measured by ICMP-MS after protein precipitation in 3.5% trichloroacetic acid, Ca precipitation in saturated ammonium oxalate, and dissolution of the Ca2 M2 oxalate. The Ca concentration in the HCl solution was measured by atomic absorption spectrometry and, if necessary, diluted to a standard of 10 g/mL Ca. Spot urine samples and 36 h urine samples taken before and after the administration of the isotopes from the same subject were analyzed on the same day together with blanks and 10 g/mL Ca standards. All values ​​were consistent with minor deviations from standard Ca solutions with accepted natural relationships. All samples were measured twice.

Statistics

Differences in Ca absorption among treatments were tested by Latin square analysis of variance. Linear and quadratic trends were also tested.

If variance analysis indicated a treatment effect ( p < 0.05), control and lactulose treatments were compared using Student's t-test. Regression analyses were used to assess the association between variables.

Results

All subjects completed the study. Based on the number of vials returned without the test substance and the compliance check by questionnaire, compliance was very good. None of the subjects ever forgot to drink the orange juice with the test substance.

No significant gastrointestinal complaints were noted. During the 10-year lactulose treatment, two cases of increased flatulence and one case of constipation were noted. During the 5-year lactulose treatment, one case of flatulence or diarrhea was noted. No such complaints occurred during the control treatment.

Because the main urine sample of subject 6, taken on day 8, was too small, ICP-MS analysis of this urine was not performed twice. The other urine samples were analyzed in duplicate. The coefficient of variation (CV) of the ratio between duplicate samples for 44 Ca/ 43 Ca was 0.26%, and the CV of the ratio for 48 Ca/ 43 Ca was 0.33%.

The mean baseline 44 Ca/ 43 Ca ratio (n = 36) was 15.449 (CV 0.37%) and the 48 Ca/ 43 Ca ratio was 1.393 (CV 0.74%). Table 2 shows the mean percent enrichment of the 44 Ca/ 43 Ca and 48 Ca/ 43 Ca ratios by one treatment course.

Calcium absorption during treatment with the reference substance, 5 g. and 10 g. lactulose (mean CO ) was 27.77.7, 30.0 7.6 and 32.2. 7.0, respectively. Figure 2 shows individual changes in Ca absorption (standard error of difference 1.3). Ca absorption was significantly higher with 10 g lactulose per day than with placebo treatment (p < 0.01). A significant linear trend was found between the dose of lactulose and its positive effect on Ca absorption (p < 0.01).

A significant relationship was found between Ca absorption and the total amount of Ca excreted in 36 h urine (y = 5.8 x + 51.6; r = 0.51 p < 0.01). Total Ca excretion in 36 h urine did not differ significantly between the treatment courses (see Table 2; p = 0.69).

Discussion

Measurement of fractional Ca absorption from 24-h urine collected after oral and intravenous administration of two different Ca isotopes has been shown to be an accurate and reliable method. In this study, this method was used to study the effect of lactulose on Ca absorption. However, since lactulose and other indigestible carbohydrates are unlikely to be absorbed in the small intestine but are fermented in the terminal ileum and colon, and since acid fermentation in the colon may enhance Ca absorption at this site, urine collection was prolonged.

To date, the effect of lactulose on Ca absorption has only been studied in rat studies, in which a positive effect of lactulose on Ca absorption was found. The positive effect of lactulose on Ca absorption is nonspecific and general for other indigestible carbohydrates, at least in rats.

In our study, the effect of 5g. or 10 g of lactulose on Ca absorption was compared with placebo treatment in 12 postmenopausal women who did not experience adverse side effects of lactulose. When the double stable isotope technique is used to measure Ca absorption, there is a 10% variation in any given absorption value in any individual subject, of which approximately two-thirds represents true biological variation in absorption. (14) Despite this biological variation, as shown in Fig. 2, the overall effect of lactulose was a significant increase in Ca absorption.

A significant linear increase in Ca absorption was found with the highest dose of lactulose. In rats, the effect of lactulose continued to increase with further increases in dietary concentration from 10% to 15%. A possible decrease in vitamin D-dependent active Ca transport may counterbalance the lactulose-induced increase in passive Ca absorption. In rats, 15% dietary lactulose could also adversely affect gastrointestinal function and cause diarrhea.

Table 2. Enrichment of the absorption ratios 44 Ca/ 43 Ca, 48 Ca/ 43 Ca and Ca treatment course (mean ± SD)

A positive acute effect of lactulose on Ca absorption in rats was found. This effect was lost after a few days of adaptation to 5% lactulose. Other groups found that 10% lactulose increased Ca absorption, even after 3 weeks of habituation. A significant positive effect of 10 g lactulose and a small positive effect of 5 g lactulose on Ca absorption were found after 1 week of habituation. This 1 week may have slightly reduced the positive effect on Ca absorption with 5 g lactulose.

There are two mechanisms of Ca absorption: “active” transcellular absorption (mainly in the duodenum and strongly regulated by vitamin D) and “passive” paracellular transport through the small and large intestines. Several theories have been proposed to explain the stimulating effect on Ca absorption. These theories relate to trans- and/or paracellular Ca absorption in the small/large intestine. The observation that the effects of lactulose and calcitriol are additive suggests that lactulose stimulates the passive rather than the vitamin D-dependent, active component of Ca absorption. Demine and Remezi also attributed the increase in Ca absorption after a diet high in fiber from indigestible carbohydrates to a passive process.

Transcellular Ca transport may be stimulated by short-chain fatty acids. In humans, increased serum short-chain fatty acid (SCFA) production and increased serum acetate concentrations have been found after ingestion of lactulose. The direct effect may involve diffusion of protonated SCFA across the apical membrane. In the cell, the protonated SCFA molecule dissociates, leaving behind increased intracellular H+, which is released from the cell in exchange for Ca2+ from the distal colon. Outside the cell, H+ is available to protonate the SCFAs for diffusion into the cell. This stimulatory effect of SCFAs appears to occur in the distal but not proximal colon. Whether this process involves passive or active transport is a question that needs to be studied.

Paracellular Ca absorption can be stimulated by a decrease in pH. Lactulose caused a decrease in ideal pH (from 7.5 to 7.0) that was inversely proportional to apparent mineral absorption. In rats given indigestible oligosaccharides, this decrease was associated with increased serum soluble Ca. The increased solubility may result in increased paracellular Ca transport in the distal small intestine and early colon.

Another hypothesis for the mechanism by which osmotically active sucrose in the small intestine stimulates paracellular Ca absorption is that it increases the amount of fluid in the lumen to maintain isotonicity. This auxiliary fluid may increase the distention and permeability of the intercellular junctions between enterocytes, thereby increasing passive paracellular absorption of Ca and other small intestinal elements. An increase in Ca absorption would be of great interest to postmenopausal women, because the efficiency of absorption depends on both age and estrogen loss at menopause. The two effects of age and estrogen loss cause a 20-25% decrease in absorption rates for women aged 40 to 60 years. This study indicates that this decrease can be completely counterbalanced by the intake of 10 g/day of lactulose, which was found to increase Ca absorption by 16%. Since the increase in Ca absorption was not related to an increase in urinary Ca excretion, lactulose may also increase Ca uptake in bone and/or delay bone resorption. The positive effect of indigestible carbohydrates on bone mineralization has also been shown in rats. According to Nordin, one of the factors in osteoporosis is a negative Ca balance, which is caused by poor calcium absorption and/or high urinary calcium excretion. In 41 studies of postmenopausal women taking estrogen, calcium balance averaged 43 mg/day.

The increase in urinary calcium in menopause is estimated to be 20-40 mg/day, which corresponds to bone loss after menopause (40 mg/day represents 1% bone mineral per year). Calcium balance can be increased by higher calcium intake or by increasing calcium bioavailability. With a calcium intake of 1000 mg/day, 10 g of lactulose could increase the amount of calcium bioavailability by 50 mg/day, an amount sufficient to significantly reduce the negative calcium balance in postmenopausal women. It therefore seems that lactulose could help reduce the negative calcium balance and thus the development of osteoporosis.

In conclusion, it should be noted that lactulose increases calcium absorption in postmenopausal women, without increasing urinary calcium excretion. The positive effect on calcium absorption is significantly related to the dose of lactulose. Further study is needed to investigate how lactulose stimulates Ca absorption and whether it may improve Ca balance in humans and/or reduce bone loss with age.

Animal studies have shown that calcium absorption is increased by the use of lactulose, a synthetic disaccharide.