Lactulose for osteoporosis

Animal studies have shown that calcium absorption is increased by lactulose, a synthetic disaccharide. Therefore, the effect of lactulose on calcium absorption has been measured in postmenopausal women, who may benefit from the enhancing effect of lactulose on calcium absorption. Twelve postmenopausal women took 5 or 10 g of lactulose or reference substance per 100 ml. water at breakfast for 9 days. Three treatments were completed on a randomized, double-blind, cross-over schedule separated by a 19-day washout period. On day 8 of each treatment period ⁴⁴Ca dissolved in orange juice was drunk immediately after the solution with the test substance before the usual breakfast with 162 mg. calcium capsule. After half an hour, ⁴⁸Sa was administered intravenously. Based on the isotope ratio measured in urine taken before and after 36 hours of isotope administration, the true fractional calcium absorption was calculated. Calcium absorption during treatment with the reference substance, 5 g and 10 g lactulose was (mean SD (mean ± deviation) 27.7 ± 7.7, 30.0 ± 7.6 and 32.2 ± 7.0, respectively. A significant difference in calcium absorption was seen between the highest dose of lactulose and reference treatment (p<0.01) An important linear trend was seen between the dose of lactulose and its positive effect on calcium absorption In conclusion, in postmenopausal women, 9 days of lactulose supplementation increases calcium absorption in a dose dependent manner. study of lactulose's stimulation of calcium absorption and whether it can improve calcium balance and/or attenuate bone loss with age (J Bone Miner Res 1999;14;1211-1216).

Lactulose is a synthetic disaccharide (4-0-β-D-galactopyranosyl-D-fructose, molecular weight 342.3) that does not exist in nature. It is obtained in small quantities during the heat treatment of milk. In large volumes, lactulose can be produced from lactose by alkaline isomerization, in which galactose binds to fructose as a 1→4 glycoside(1,2). 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 decreases. (3)

Animal studies have shown that calcium (Ca) absorption is increased by lactulose, which appears to be more effective than lactose (4) and pectin (5). In addition to the possible effect of lactulose on the absorption of minerals in the small intestine due to the improved solubility of Ca (6), the absorption pathway in the large intestine is also of interest. Fermentation of undigested food components and hence lower PH levels (through the formation of short-chain fatty acids by the microflora) may have a positive effect on Ca absorption from the distal intestine (7).

Increasing Ca absorption may be of particular interest to postmenopausal women. The reciprocal effect of age on Ca absorption has been correlated with a decrease in serum levels of 1.25-dihydroxyvitamin D(8), and thus the active component of absorption is reduced. Through the ingestion of non-digestible ingredients such as lactulose, the level of passive absorption of Ca in the small or large intestine can be increased and the efficiency of Ca absorption is restored in later life.

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

	placebo	5 g lactulose	10 g lactulose
Lactulose (g.) Aspartame (mg.) Quinoline yellow (mg.) Benzoic acid (mg) Water (ml)	— 12.5 0.04 100 100	5 6,25 0,02 100 100	10 — — 100 100

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

The test subjects were recruited from the Food and Nutrition Research Institute's TNO volunteer team and through an ad in a local newspaper.

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

At baseline, their age ranged between 56 and 64 years (mean age 60.5 years) and their body mass ranged between 20.7 and 27.8 (mean 25.0). Eleven test subjects have E values₂ were <20 pg/ml. E levels₂  and FSH in 7 subjects were 60 pg/mL and 51 IU/L, respectively. FSH values of all subjects ranged between 51 and 121 IU/L (mean 89.6 IU/L). All subjects received a medical certificate of good general health from a staff physician and gave informed consent to participate in the study after the entire procedure of the study was explained to them. The study protocol was approved by the TNO's external medical ethics committee.

The study was conducted in accordance with the amended Declaration of Helsinki (Somerset West, South Africa, 1996) and the ICH (International Conference on Harmonization) guidelines for good clinical practice (ICH subject matter E6, adopted 01-05-1996 and enforced 17-01- 1997). Subjects were instructed to take the test substance at breakfast for 9 days in addition to their usual meal.

The test substances consisted of 5 g 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 shown in table 1.

Aspartame was chosen as placebo; it was not intended to interfere with Ca absorption due to the small amount relative to lactulose and Ca and because aspartame is completely digested in the stomach/small intestine, so it is completely absent from the colon to avoid interaction with Ca. Quinoline yellow was added to give the three solutions the same color. Benzoic acid has been added as a preservative. The touch panel did not show any difference in bitterness or sourness between the different test substances. The 5 g lactulose solution was considered the sweetest solution, followed by the placebo and the 10 g lactulose solution.

Three treatments of 9 days each were completed on a double-blind, randomized cross-over schedule, separated by a 19-day drop-out period. The timeline is shown in Figure 1.

During the first 7 days of each treatment period, subjects received home-delivered test substances. On the last 2 days, the subjects were placed in the metabolic department of the institute and analyzed for Ca absorption.

On the 8th day after a 12-hour overnight fast, they drank orange juice with ⁴⁴Ca immediately after the test substance just before the start of a regular breakfast with 162 mg. Ca in capsule as measured by atomic absorption spectrometry. After half an hour of oral administration ⁴⁴Sa, ⁴⁸Ca was administered intravenously. Before and after the bolus infusion, blood pressure and pulse rate were measured for safety. The average number of isotopes obtained by each method, which was calculated by weighing the ampoules or syringes before and after ingestion, was 13.9 mg. (in the range of 13.2 -15.1 mg.) ⁴⁴Ca and 1.15 mg. (in the range of 1.10 -1.17 mg.) ⁴⁸Sa. From the ratio of measurement readings ⁴⁴Ca/⁴³Sa and ⁴⁸Ca/⁴³Ca in the urine taken before the dose was calculated fractional absorption of Ca in accordance with the formula indicated by Van Dokkum.

Persistent isotopes were obtained from NEDRAY (Bunschoten, The Netherlands) in the form of Ca carbonate. The content of various Ca isotopes according to analysis by inductively coupled plasma mass spectrometry (ICP-MS) was: 3.39% ⁴⁰Ca, 0.06% ⁴² Ca, 0.03 % ⁴³Sa, 95% ⁴⁴Ca, <0.01 % ⁴⁶Ca, 0.02% ⁴⁸Ca for Ca enriched in ⁴⁴Sa, and 8,96% ⁴⁰Ca, 0.09% ⁴²Ca, 0.02% ⁴³Ca, 0.24% ⁴⁴Ca, <0.01% ⁴⁶Ca, 90.69% ⁴⁸Ca for Ca enriched in ⁴⁸Sa. Carbonate ⁴⁴Ca was converted to the chloride salt diluted with deionized water adjusted to pH 5. The same procedure was carried out for the carbonate ⁴⁸Ca, only saline solution was used instead of deionized water. After filtration, the solution was distributed in 10 ml. ampoules for injection and sterilized for 25 minutes.

Although ICMP-MS analysis has several advantages over other methods, it sometimes suffers from molecular interference from different sources. The most obvious is isobaric interference at low masses, especially ⁴⁰ Ar from plasma with ⁴⁰Sa. Therefore, the ratios of Ca isotopes  ⁴⁴Ca/⁴³Sa and ⁴⁸Ca/⁴³Urinary Ca was measured by ICMP-MS after protein precipitation in 3,5% trichloroacetic acid, Ca precipitation in saturated ammonium oxalate, and dissolution of Ca oxalate formed in 1.2 M HCL. (10)

The Ca concentration in the HCL solution was measured by atomic absorption spectrometry and, if necessary, was diluted to the norm of 10 μg/ml Ca. Local urine samples and 36 hours of urine samples taken before and after the isotope administration of the same subject were analyzed in 1 day along with empty and standard 10 μg/ml Ca. All values were consistent with minor deviations from standard Ca solutions with accepted natural ratios. All samples were measured twice.

Differences in Ca absorption among treatments were tested by latin square fluctuation analysis. The presence of linear and square tendencies was also checked. (eleven)

If the variation analysis indicated a treatment outcome (p<0.05), control treatment and lactulose treatment were compared by Student's t-test. Regression analyzes were used to assess closeness among variables.

All subjects were tested. Based on the number of ampoules returned without test substance and the compliance check through a questionnaire, the compliance appeared to be very good. None of the test subjects ever forgot to drink orange juice with the test substance.

There were no significant complaints about the functioning of the gastrointestinal tract. During treatment with 10 g of lactulose, two cases of increased flatulence and one case of constipation were noted. During treatment with 5 g of lactulose, one case of flatulence or diarrhea was noted. Such complaints did not occur during the control treatment.
Because Subject 6's primary urine sample taken on Day 8 was too small, ICP-MS analysis of this urine was not performed twice. Analysis of all other urine samples was duplicated. Coefficient of variation (CV) ratio between duplicate samples for  ⁴⁴Ca/⁴³Ca was 0.26%, and the KV ratio for   ⁴⁸Ca/⁴³Ca was 0.33%.

Average Base Ratio ⁴⁴Ca/⁴³Ca (n = 36) was 15,⁴⁴9 (CV 0.37%) and the ratio ⁴⁸Ca/⁴³Ca was 1.393 (CV 0.74%). Table 2 shows the average percentage enrichment of the ratios ⁴⁴Ca/⁴³Sa and ⁴⁸Ca/⁴³Sa for one course of treatment.

Calcium absorption during treatment with the reference substance, 5g. and 10 g lactulose (mean ± CO) 27.7 ±7.7, 30.0 ±7.6 and 32.2. ± 7.0 respectively. Figure 2 Individual changes in Ca uptake (standard error of 1.3). Ca absorption was significantly higher during 10 g lactulose per day than during placebo treatment (p < 0.01). A significant linear trend was found between lactulose dose and its positive effect on Ca absorption (p < 0.01).

A significant relationship was found between Ca absorption and total Ca excreted in 36 h urine (y = 5.8x + 51.6; r = 0.51 p < 0.01). Total Ca excretion from 36 hours urine did not differ significantly between treatments (See Table 2; p=0.69).

The measurement of fractional Ca uptake from 24 h collected urine after oral and intravenous administration of two different Ca isotopes was found to be an accurate and reliable method. (12, 13) In this study, this method was used to study the effect of lactulose on Ca absorption. However, since lactulose and other non-digestible carbohydrates are hardly absorbed in the small intestine, but are fermented in the terminal ileum and large intestine, and since acidic fermentation in the large intestine can improve Ca absorption at this site (7), urine collection was extended to 36 hours

Up to this point, the effect of lactulose on Ca absorption has been studied only in experiments on rats, in which a positive effect of lactulose on Ca absorption was revealed. (4-6) The positive effect of lactulose on Ca absorption is non-specific and common to other non-digestible carbohydrates, at least in rats.(4)

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

As Bromage et al.,(4) we found a significant linear increase in Ca absorption with the highest dose of lactulose. In rats, the effect of lactulose further increased with further increases in dietary concentration from 10% to 15%. It is possible that the reduced transport of vitamin D-dependent active Ca may counterbalance the lactulose-induced increase in passive Ca absorption (4). In 15% rats, dietary lactulose could also adversely affect gastrointestinal function and cause diarrhea.

	placebo	5 g lactulose	10 g lactulose
Enrichment Ratio 44Ca/43Sa Ratio 48Ca/43Sa	3.70.8 13.0±1.9	4.2±1.1 13.4±1.7	4.5±0.8 13.7±2.2

 Bromage and Al. (4) found a positive acute effect of lactulose on Ca absorption in rats. 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. (5, 6) We found a significant positive effect of 10 g lactulose and a slight positive effect of 5 g lactulose on Ca absorption after 1 week of habituation. This 1 week may have slightly reduced the positive effect in Ca absorption at 5 g lactulose. There are two mechanisms of Ca absorption: “active” transcellular absorption (mainly in the duodenum and highly regulated by vitamin D) and “passive” paracellular transport through the small and large intestine. intestines. (15) Several theories have been put forward to explain the stimulatory effect on Ca absorption. These theories refer to trans- and/or paracellular Ca uptake in the small/large intestine. Observations that the effects of lactulose and calcitriol are additive indicate that lactulose stimulates the passive rather than the vitamin D-dependent, active component of Ca absorption. (4) Also, Demine and Remesy (16) attributed the increase in Ca absorption following a diet rich in fiber from indigestible carbohydrates to a passive process.

Transcellular transport of Ca can be stimulated by short chain fatty acids. In humans, increased serum short-chain fatty acid (SCFA) production (3) and increased serum acetate concentration have been found following lactulose supplementation. (17) The direct effect probably involves the diffusion of protonated SCFAs across the apical membrane. In the cell, the protonated SCFA molecule breaks down, leaving behind increased intracellular H+, which is released from the cell in exchange for Ca2+ from the distal colon. Outside the cell, H+ appears for SCFA protonation for diffusion into the cell. (18, 19) This stimulatory effect of SCFA seems to occur in the distal but not the proximal colon. (18) Whether this process involves passive or active transport is a matter for study.

Paracellular Ca uptake can be stimulated by a decrease in PH. A decrease in pH was found in …. Lactulose caused a decrease in ideal pH (from 7.5 to 7.0) which was inversely related to apparent mineral absorption. (6) In rats given indigestible oligosaccharides, this decrease was associated with an increased amount of soluble serum Ca. (20, 21) Increased solubility may cause 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 uptake is an increased amount of fluid in the lumen to maintain isotonicity. This accessory fluid can increase the swelling and permeability of intercellular junctions between enterocytes, thus increasing the passive paracellular uptake of Ca and other elements in the small intestine. (4) Increasing Ca absorption would be of great interest to postmenopausal women because absorption efficiency is dependent on both age and menopausal estrogen loss. The two influences of age and estrogen loss account for a 20-25 % deterioration in absorption rates for women aged 40 to 60 years. (22) This study indicates that this decrease may be entirely counterbalanced by 10 g/day of lactulose, which was found to increase Ca absorption by 16%. Since an increase in Ca absorption was not related to an increase in urinary Ca excretion, lactulose may also increase bone Ca absorption and/or delay bone resorption. A positive effect of indigestible carbohydrates on bone mineralization was also found in rats. (23–25) According to Nordin (26), one of the factors in osteoporosis is a negative Ca balance, caused by poor absorption of calcium and/or high indispensable excretion of calcium in the urine. In 41 studies of postmenopausal women who did not take estrogen, calcium balance averaged 43 mg/day. (27) The increase in urinary calcium at menopause is estimated at 20-40mg/day, which is consistent with bone loss after menopause (40mg/day is 1% of bone mineral per year).(26) Calcium balance can be increased by higher intake calcium or by increasing the bioavailability of calcium. With 1000mg/day of Ca, 10g of lactulose could increase the amount of calcium bioavailability by 50mg/day, an amount sufficient to substantially reduce negative Ca balance in postmenopausal women. Therefore, it seems that taking lactulose might help reduce the negative Ca balance and thus the development of osteoporosis.

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

July 1999, Volume 14, Number 7 Page 1211