Journal of the American College of Nutrition
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The effect of carbohydrates in milk on the absorption of calcium by postmenopausal women. S A Schuette, N J Yasillo & C M Thompson To cite this article: S A Schuette, N J Yasillo & C M Thompson (1991) The effect of carbohydrates in milk on the absorption of calcium by postmenopausal women., Journal of the American College of Nutrition, 10:2, 132-139, DOI: 10.1080/07315724.1991.10718137 To link to this article: http://dx.doi.org/10.1080/07315724.1991.10718137
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The Effect of Carbohydrates in Milk on the Absorption of Calcium by Postmenopausal Women Sally A. Schuette, PhD, Nicholas J. Yasillo, and Charlotte M. Thompson, MS
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Clinical Nutrition Research Unit and Department of Radiology, University of Chicago, Chicago The purpose of this investigation was to determine if the presence of carbohydrate in milk, either lactose or its hydrolysis products, enhance the bioavailability of calcium (Ca) in milk. Two studies were performed. In study A, fractional Ca absorption was measured in 11 lactose-tolerant postmenopausal women after an oral dose of 47Ca-equilibrated milk formula containing no carbohydrate (NOCHO), lactose (LACTOSE), or an equivalent amount of glucose plus galactose (SUGAR); all participated in three absorption studies in random order. The NOCHO formula contained 10.0 g protein and 217 mg Ca from a combination of milk mineral and protein isolates; the LACTOSE and SUGAR formulae contained in addition 12 g lactose or 6 g glucose plus 6 g galactose, respectively. In study B, fractional Ca absorption was measured in five postmenopausal women after an oral dose of 47Ca-equilibrated skim milk (217 mg Ca) and lactase-treated milk, each with sufficient carbohydrate added to equal 12 g. For both studies, the increase in forearm radioactivity 4 and 8 hours after oral 47Ca administration relative to the increase observed after IV administra tion was used to estimate fractional Ca absorption. The addition of lactose but not glucose plus galactose to the NOCHO formula enhanced Ca absorption (p < 0.05). Fractional absorption at 4 hours was 0.386 from the LACTOSE formula compared with 0.310 for both the NOCHO and SUGAR formulae. Those individuals with the lowest absorp tion in the absence of carbohydrate had the greatest increase with lactose. In contrast, Ca absorption was the same from skim milk as from lactase-treated skim milk (study B). The results of these studies suggest that lactose may enhance Ca absorption in situations where Ca solubility or active Ca absorption is limiting. Abbreviations: Ca = calcium, NOCHO = Ca-equilibrated milk fornitila contain ing no carbohydrate, 250HD = 25-hydroxyvitamin D.
INTRODUCTION Teleologically speaking, the possibility that lactose or "milk sugar" could specifically enhance calcium (Ca) absorption is appealing. The ß-l,4-glycosidic linkage found in lactose is unique among carbohydrates that can be digested by mammalian enzymes. The unique struc ture of lactose and its presence only in milk has long suggested a positive role for lactose in calcium absorp tion. A number of investigators have shown that the sub stitution of lactose for other carbohydrates in the diets of laboratory animals can improve Ca absorption and in crease the retention of Ca in bone [1-4]. In some instan ces the presence of lactose improved Ca absorption and retention despite the fact that the large amounts of lac
tose fed produced diarrhea and increased fecal loss of other nutrients such as protein and fat [5]. Lactose and Ca must be present in the intestine simultaneously in order for the positive effects of lactose on Ca absorption or bone mineralization to be manifest [6]. Based in large part on the results of animal studies, it has generally been accepted that lactose enhances Ca absorption in man unless the individual is lactase-deficient. However, a critical review of this literature reveals that the data are less than convincing. Debongnie et al [7] found significantly more Ca in ileal aspirates (less Ca absorbed) from four lactose-tolerant subjects given milk vs lactase-treated milk. Lactase-deficient subjects ab sorbed less Ca than normals from both milks but also showed the negative effect of lactose on Ca absorption. Greenwald et al [8] found no positive effect on Ca ab-
The work was presented, in part, at the 28th Annual Meeting of the American Society for Clinical Nutrition, Washington, D.C. April 28-30,1988. Address reprint requests to Dr. S. Schuette, University of Chicago, Clinical Nutrition Research Unit, S841 South Maryland, Box 223, Chicago, Illinois 60637.
Journal of the American College of Nutrition, Vol. 10, No. 2, 132-139 (1991) © 1991 John Wiley & Sons, Inc.
CCC 0731-5724/91/020132-08$04.00
Carbohydrates and Ca Absorption Table 1. Composition of Basic Milk Formula (Study A) Compared to Skim Milk Normalized to the Same Calcium Content1 CHO(g)
Ca(mg)
Mg (mg)
P(mg)
9.9 0.1 10.0
0.002 0.004 0.006
95 122 217
3 3 6
50 108 158
7.1
8.31
217
21
167
Protein (g) Basic milk formula TMP1200.il g Alamin 2000, 0.4 g Total Skim milk, 170 g 2
'Protein values based on manufacturer's information, others by analysis. This is the composition of the skim milk used in study B before the addition of extra carbohydrate (CHO).
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2
sorption or balance of adding lactose to an otherwise constant diet of older adults. However, only three patients with various complaints were studied in this in vestigation, and total Ca intake was high (1500 mg). Recently, Tremaine et al [9] reported mat fractional Ca absorption, measured by a dual-isotope technique, was the same from normal milk and lactase-treated milk. Similarly, Griesson et al reported that Ca absorption was the same from milk containing lactose or glucose as the carbohydrate source in lactose-tolerant adults [10]. On the positive side, Mills et al [11] found that six of seven young boys (4—7 years old) retained more Ca when 36 g lactose was added to their basal diet Also in children, Ziegler and Fomon [12] found better Ca ab sorption from formula containing lactose compared to an identical formula containing sucrose and com starch hydrolysate. Two investigations have demonstrated a positive effect of lactose on Ca absorption in adult man [13,14]. Kocian et al [13] reported more rapid and slight ly greater total values for 47Ca absorption from "milk" than from lactose-free "milk." However, the "milks" compared were really milk-based formulas which dif fered somewhat in their composition, including fortifica tion with different Ca salts. The most convincing study to show a positive effect of lactose on Ca absorption in adults was done by Cochet et al [14]. These investigators used a dual-isotope technique to determine fractional Ca absorption in the presence or absence of 50 g lactose. Total fractional Ca absorption was enhanced by 60% in the presence of lactose compared to water alone; in this investigation lactose was added to a solution of CaCl2 in water. Thus, although some evidence is available to suggest lactose may have a positive effect on Ca absorption in man, the data can hardly be considered definitive. Addi tionally, the results of several recent studies showing a positive effect of other readily available carbohydrates
on Ca absorption in man [15-18] suggest that "lactose enhancement" of Ca absorption may not be specific to lactose but may be a general effect of simple car bohydrates. The purpose of mis investigation was to determine if the presence of carbohydrate in milk, either lactose or its hydrolysis products glucose and galactose, significantly enhance the bioavailability of Ca in milk.
METHODS Experimental Design Study A Fractional Ca absorption was measured in 11 postmenopausal women after an oral dose of "Ca-equilibrated milk formula containing little carbohydrate (NOCHO), lactose (LACTOSE), or an equivalent amount of glucose plus galactose (SUGAR); all 11 women participated in three separate absorption studies; one with each of the milk formulae. The milk formulae were made from milk protein isolate, containing both casein and whey proteins, plus a milk mineral con centrate (TMP 1220 and Alamin 2000, respectively, both from New Zealand Milk Products, Ine, Petaluma, CA). The protein, carbohydrate, Ca, phosphorus, and mag nesium contents of the basic formula are given in Table 1 and compared to skim milk. The milk formulae used for the absorption tests were as follows: NOCHO: LACTOSE: SUGAR:
basic formula (10.0 g protein and 217 mg Ca in 200 ml H20) + 74 kBq 47Ca basic formula + 12 g lactose + 74 kBq 47Ca basic formula + 6 g glucose + 6 g galactose + 74 kBq 47Ca
The formulae were flavored with 0.8 ml chocolate extract and a few drops of artificial sweetener and were
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Carbohydrates and Ca Absorption Table 2. The Effect of Lactase Treatment and the Addition of Sugars on the Carbohydrate Content of Skim Milks Used in Study B per 170 g Carbohydrate Milk Untreated skim milk MILK+LAC1
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Lactase-treated skim milk MILK+SUGAR 2
Lactose 8.21 12.00 0.05 0.05
Galactose (or glucose) 0 0 4.10 6.00
'Lactose was added as monohydrate; as such, 3.88 g lactose monohydrate was added to yield 3.69 g of lactose. 2 Galactose content was determined en2ymatically. Since the hydrolysis of lactose yields equimolar amounts of glucose and galactose, glucose content was assumed and not determined directly. Galactose and glucose, anhydrous, were added at the level of 1.90 g each.
indistinguishable by the subjects. The formulae were ad ministered in a random manner; however, since 11 sub jects participated, one of six possible orders of ad ministration was used once instead of twice. Study B Fractional Ca absorption was measured twice in each of five postmenopausal women after an oral dose of 47 Ca-equilibrated skim milk or lactase-treated skim milk, each with sufficient carbohydrate added to be equal in sugar content to the milk formula used in study A; lac tose was added to skim milk (MILK+LAC) and glucose plus galactose to the lactase-treated milk (MILK+ SUGAR). Lactose treatment was performed using ßgalactosidase enzyme (LactAid, Pleasantville, NJ) at double the manufacturer's recommended dose for 48 hours at refrigerated temperature. The protein, car bohydrate, Ca, phosphorus, and magnesium contents of the milk used were as shown in Table 1. The effect of lactase treatment and the final carbohydrate contents are shown in Table 2. The milks were administered in a random manner. Again the milks were flavored with chocolate extract and artificial sweetener and were indis tinguishable by the subjects. Both studies had been approved by the Human Use Radioisotope and Clinical Investigation Committees of the University of Chicago. Informed written consent was obtained from each participant. A single blood sample was collected from each subject at the beginning of the study for measurement of serum 25-hydroxyvitamin D (250HD).
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Subjects A total of 16 postmenopausal women were subjects in these investigations. Menopause was defined as com plete cessation of menses for at least 2 years. Women fitting this criterion underwent a standard lactose breath H2 test using 25 g lactose to determine lactose tolerance. The H2 content of expired air was determined at 0, 1, 2, and 3 hours after dosing. Lactose intolerance was defined as a breath H2 level > 20 ppm above baseline at one or more timepoints. Only lactose-tolerant women participated as subjects in these investigations. In study A, the 11 postmenopausal women who par ticipated ranged in age from 38 to 66 years and were 3-17 years postmenopause. In study B, these same char acteristics for the postmenopausal subjects were 39-61 and 3-30 years, respectively. Those subjects taking medications prescribed by their personal physicians at the beginning of the study continued these medications throughout. Subject characteristics are listed individually in Table 3.
Analytical Methods Radioabsorption Test Fractional Ca absorption was determined using a modification of the forearm counting method described by Wills et al [19]. Details of the radiocalcium absorp tion test are as follows: on the first morning and after an overnight fast, the subjects received 37 kBq (1 μ ο ) of
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Carbohydrates and Ca Absorption Table 3. Subject Characteristics1
Subject
Age
Years postmenopause
1A
51
3a
2A 3A 4A 5A 6A 7A 8A 9A 10A 11A
58 59 57 58 66 49 62 55 42 38
10 17 3 12 17 9s 9 5 3a 3a
IB
61
30*
2B 3B 4B 5B
40 55 39 42
9" 3 9a
Serum 250HD (ng/ml)
Medications
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Study A Indomethacin, 75 mg bid Medroxyprogesterone, 10 mg qd Conjugated estrogens, 1.25 mg qd None None None None None None Propranolol, 20 mg qd Chlorpropamide, 250 mg qd None None
33.5
42.6 25.2 27.2 30.4 26.8 27.3 21.6 13.0 34.7 22.0
Study B
15
Conjugated estrogens, 0.3 mg qd Theophylline, 200 mg qd Enalaprii, 5 mg qd None None Conjugated estrogens, 0.625 mg qd None
2
— — — —
Normal range for serum 250HD level is 10-60 ng/ml. Superscripted a denotes surgical menopause. Subjects 10A and 2B had only a partial hysterectomy and had ovaries intact. Serum samples from these subjects were accidentally discarded.
Table 4. Fractional Calcium Absorption from the Various Milk Formulae, Study A1 NOCHO
LACTOSE
SUGAR
0.310 ±0.037 a
0.386 ±0.030 b
0.310 ±0.025 a
Values are given as the mean ± SEM for all subjects at 4 hours. Values with different superscripts are significantly different, p < 0.05.
Table 5. Fractional Calcium Absorption from Milk or Lactase-Treated Milk, Study B 1 MILK+LAC
MILK+SUGAR
At 4 hours
0.209 ±0.029
0.194 ±0.037
At 8 hours
0.239 ± 0.032
0.222 ± 0.037
Values are given as the mean ± SEM for all subjects.
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Carbohydrates and Ca Absorption
.60
i0.
cr O
g.40. < a o _i < z o
j= .20 ü
7.4 MBq/mg or 200 μθϊ/ιτ^; Amersham, Chicago); the IV dose approximates 100% absorption and is necessary for calculation of fractional Ca absorption. Right forearm radioactivity was determined 4 and 8 hours later in a large volume gamma counter built specifically for this purpose (consists of four 5" diameter by 5" Nal crystals with associated counting electronics and is completely shielded). The subjects remained fasted until after the 4 hours forearm count. After this they were allowed to eat lunch but had been instructed on how to choose a lowCa lunch. On subsequent days, at least 48 hours apart, a similar procedure was followed except that the subjects received 74 kBq (2 μθί) of 47Ca orally, in milk or milk formula containing 217 mg Ca. For all oral doses, the 47CaCl2 was added to the milk or milk formula the afternoon before. After the first isotope administration, a baseline forearm count was performed just prior to administration of the next oral dose of 47Ca to correct for residual radioactivity. Fractional Ca absorption was calculated as: forearm radioactivity after oral 47Ca dose xlOO forearm radioactivity after IV Ca dose
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after correction for dose administered, background radioactivity, and decay. Laboratory Analyses Ca and magnesium contents of samples of the milk protein isolate, milk mineral concentrate, and skim milk were determined by atomic absorption spectrophotometry (Model 5000, Perkin-Elmer, Norwalk, CT). For Ca and magnesium determinations, samples were first dried in a muffle furnace for 3 hours at 150°C and then ashed for 12-15 hours at 450"C. Following this, 1 ml of nitric acid was added to the ash and the samples were returned to the muffle furnace for 1-3 hours at 150'C. The final ash was dissolved in nitric acid and deionized water. The phosphorus content of milk protein and mineral plus skim milk were determined colorimetrically [20] after wet ashing with nitric and perchloric acid [21]. Skim milk standard (National Bureau of Standards refer ence material #1549) analyzed in parallel with our samples was found to contain 97-106% of the expected values for magnesium, Ca, and phosphorus. Lactose and galactose content of the milk formulae and skim milk were determined enzymatically using a
VOL. 10, NO. 2
Carbohydrates and Ca Absorption commercially available kit (Cat #176303, Boehringer Mannheim GmbH, Mannheim, Germany). Serum 250HD levels were determined by a modification of the competitive binding assay of Belsay et al [22] using nor mal human serum as the standard. Statistical Analysis For study A, fractional absorption of Ca from the various milk formulae was compared by two-way ANOVA (subject and milk formula). If a significant milk formula effect was observed, mean absorption values were compared using Tukey's multiple comparison pro cedure (p < 0.05) [23]. For study B, Ca absorption of the subjects from MILK+LAC vs MELK+SUGAR was com pared using a t-test for paired observations.
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RESULTS Study A As shown in Table 4, the addition of lactose but not glucose plus galactose to a milk formula containing neg ligible amounts of carbohydrate significantly enhanced fractional Ca absorption. At 4 hours, fractional Ca ab sorption was 0.386 from the LACTOSE formula com pared with 0.310 for both the NOCHO and SUGAR for mulae; this represents a 25% increase in Ca absorption with lactose. Fractional Ca absorption calculated from the 8- vs 4-hr data was slightly but not statistically greater for all of the milk formulae used, and the dif ferences between formulae were also significant at 8 hours. The 4-hr data are presented in Table 3 and Figure 1 because only the 4-hr data is available for all subjects on all three treatments; subject 1A forgot to return for her 8-hr forearm count during the LACTOSE absorption test. The mean 4- and 8-hr fractional absorption values, exclusive of data from subject 1A, were 0.393 vs 0.400, 0.324 vs 0.338, and 0.314 vs 0.326 for the LACTOSE, NOCHO, and SUGAR formulae, respectively. The individual fractional absorption values for all subjects from the LACTOSE and NOCHO formulae at 4 hours are depicted in Figure 1. There are several interest ing observations to be made from looking at the in dividual responses. Generally, those individuals with the lowest absorption values in the absence of carbohydrate had the greatest increase in the presence of lactose; the converse was also true. In fact, there was a significant negative correlation between an individual's fractional absorption of Ca from the NOCHO formula and the in crease in absorption seen with the addition of lactose (r = 0.609, p < 0.05). For example, subject 1A had a fractional absorption value of only 0.166 in the absence of carbohydrate and the value increased to 0.320 with
lactose, whereas subject 10A had an absorption value of 0.585 without lactose and the value decreased slightly to 0.552 with lactose. For the small number of subjects studied in this inves tigation, no significant correlation was found between fractional Ca absorption from the NOCHO formula and years postmenopause. In addition, no significant correla tion was found between age of subject or years postmenopause and the increase in Ca absorption ob served with lactose. All subjects had serum 250HD levels within the normal range (Table 1).
Study B As shown in Table 5, lactose equivalent to the amount present in the LACTOSE formula of study A did not enhance Ca absorption from skim milk compared to an equimolar amount of glucose plus galactose. In addition, fractional Ca absorption appeared to be lower from the skim milk with added carbohydrate (study B) than from the comparable milk formulae (study B); fractional Ca absorption at 4 hours from the LACTOSE formula vs MILK+LAC and the SUGAR formula vs MILK+SUGAR was 0.386 vs 0.209 and 0.310 vs 0.194, respectively (p < 0.02). Although these differences were statistically significant, the small num ber of subjects studied in study B makes the biological significance of this difference questionable. No dif ference in the age of the subjects, years postmenopause, or proportion of subjects taking medications is apparent between the two groups. Serum 250HD levels are not available for the subjects of study B.
DISCUSSION The results of this investigation suggest that lactose can enhance the absorption of Ca by postmenopausal women under some, but not all, conditions. Lactose caused a 25% increase in Ca absorption when present in an artificial milk formula compared to an equimolar amount of glucose plus galactose or the absence of car bohydrate. However, the same amount of lactose in fresh skim milk did not enhance Ca absorption compared with its constituent sugars — glucose plus galactose. The milk formula used was similar to skim milk in total mineral composition and was made from protein and mineral fractions isolated from milk but was dif ferent from milk in some potentially important charac teristics. The proportions of mineral and protein isolate used were chosen to yield a Ca:P ratio similar to skim milk, but neither isolate component was completely soluble in water. As a result, our milk formula formed a suspension from which some portion of the mineral and
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Carbohydrates and Ca Absorption protein fractions would slowly settle out.* This is clearly different from skim milk. In addition, in our milk for mula a smaller portion (55%) of the Ca came from the protein isolate than is normally associated with the col loid protein fraction of skim milk (70%) [24]. It is pos sible that lactose enhanced the solubility of Ca in the milk formula or during the digestion process and thus resulted in greater Ca absorption. Lactose has been shown to form a more stable chelate with Ca than glucose or galactose [25]. The same situation did not exist for skim milk, where all the Ca was either soluble or associated with colloidal protein. The fact that lactose enhancement of Ca absorption from the milk formula was more pronounced in women with low fractional absorption in the absence of car bohydrate is in keeping with several recent reports, which all suggest that some individuals with low absorp tion respond positively to the presence of various dietary factors by mechanisms which are not clearly understood. This was first described by Kelly et al [26] and led to a series of investigations into the effect of glucose and glucose polymers on Ca absorption [15,16]. More recently, Heaney and co-workers have observed sig nificantly meal effect on Ca absorption which is remarkably more pronounced in some individuals than others [27,28]. In addition, Knowles et al [29] recently demonstrated a clear dose response for the effect of glucose on Ca in seven of 10 subjects, whereas three showed no effect. Each of these studies may represent a specific nutrient/meal effect on Ca absorption (i.e., in the present investigation lactose, which is absorbed more distally than glucose and galactose, may effectively en hance Ca absorption only in those individuals with poor active absorption in the more proximal gut) or all may be related via a general mediator(s) of gastrointestinal func tion (i.e., via CCK, secretion, vasoactive intestinal peptide, etc.) and lead to observable enhancement of Ca absorption only when absorption is low in the absence of enhancer.
CONCLUSIONS In agreement with other recent reports [9,10], we ob served that the lactose content of milk has little effect on
*This characteristic of the milk formula did not result in poor equilibra tion of the radiolabel. Determination of the specific activity of both the soluble and suspension fractions of a milk formula identical to that used in the absorption study but equilibrated with 45Ca showed that the radiolabel was even distributed. The specific activity of the two fractions were: soluble—631 ± 5 cpm/mmol; suspension—637 ± 52 cpm/mmol.
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Ca absorption from milk in lactose-tolerant adult humans. However, we also observed that lactose, like glucose, glucose polymer, and other nonspecific meal effects, may significantly enhance Ca absorption under some conditions. A better understanding of the possible relationship between these observed effects may lead to more specific recommendations for the most effective way of providing highly bioavailable Ca for individuals with increased needs.
ACKNOWLEDGMENTS The authors acknowledge the support of this work by WMMB grant #IV-39 and Clinical Nutrition Research Center grant AM 26678. In addition, the authors would like to thank Dr. Malcom Cooper and Ms. Erma Peterson for their helpful assistance in the preparation and ad ministration of the 47Ca doses, as well as Ms. Lynette McCall for her assistance in preparation of this manuscript
REFERENCES 1. Bergeim O: Intestinal chemistry. V. Carbohydrates and calcium and phosphorus absorption. J Biol Chem 70:3545, 1926. 2. Evans JL, Ali R: Calcium utilization and feed efficiency in the growing rat as affected by dietary calcium, buffering capacity, lactose and EDTA. J Nutr 92:417-424, 1967. 3. Schaafsma G, Visser R: Nutritional interrelationships be tween calcium, phosphorus and lactose in rats. J Nutr 110:1101-1111, 1980. 4. Miller SC, Miller MA, Omura T: Dietary lactose improves endochondrial growth and bone development and mineralization in rats fed a vitamin D-deficient diet. J Nutr 118:72-77,1988. 5. Leichter J, Tolensky AF: Effect of dietary lactose on the absorption of protein, fat and calcium in the postweaning rat. Am J Clin Nutr 28:238-241,1975. 6. Lengemann FW: The site of action of lactose in the enhan cement of calcium utilization. J Nutr 69:23-27, 1959. 7. Debongnie JC, Newcomer AD, McGill DB, Phillips SF: Absorption of nutrients in lactase deficiency. Dig Dis Sci 24:225-231, 1979. 8. Greenwald E, Samachson J, Spencer H: Effect of lactose on calcium metabolism in man. J Nutr 79:531-538, 1963. 9. Tremaine WJ, Newcomer AD, Riggs BL, McGill DB: Cal cium absorption from milk in lactase-deficient and lactasesufficient adults. Dig Dis Sci 31:376-378, 1986. 10. Griessen M, Cochet B, Infante F, Jung A, Bartholdi P, Donath A, Loizeau E, Courvoisier B: Calcium absorption from milk in lactase-deficient subjects. Am J Clin Nutr 49:377-384,1989.
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Carbohydrates and Ca Absorption 11. Mills R, Breiter H, Kempster E, McKey B, Pickens M, Outhouse J: The influence of lactose on calcium retention in children. J Nutr 20:467-476,1940. 12. Zeigler E, Fomon S: Lactose enhances mineral absorption in infancy. 3 Pediatr Gastroenterol Nutr 2:288-294, 1983. 13. Kocian J, Skala I, Bakos K: Calcium absorption from milk and lactose-free milk in healthy subjects and patients with lactose intolerance. Digestion 9:317-324, 1973. 14. Cochet B, Jung A, Griessen M, Bartholdi P, Schaller P, Donath A: Effects of lactose on intestinal calcium absorp tion in normal and lactase-deficient subjects. Gastroenterology 84:935-940, 1983. 15. Bei L, Wood RJ, Rosenberg IH: Glucose polymer in creases jejunal calcium, magnesium and zinc absorption in humans. Am J Clin Nutr 44:244-247, 1986. 16. Wood RJ, Gerhardt A, Rosenberg IH: Effects of glucose and glucose polymers on calcium absorption in healthy subjects. Am J Clin Nutr 46:699-701, 1987. 17. Norman DA, Morawski SG, Fordtran JS: Influence of glucose, fructose, and water movement on calcium absorp tion in the jejunum. Gastroenterology 78:22-25, 1980. 18. Schuette SA, Knowles JB, Ford HE: The effect of lactose or its component sugars on jejunal calcium absorption in adult man. Am J Clin Nutr 50:1084-1087, 1989. 19. Wills M, Zisman E, Wortsman J, Evens RG, Pak CYC, Banter FC: The measurement of intestinal calcium absorp tion by external radioisotope counting: application to study of neophrolithiasis. Clin Sci 39:95-106, 1970. 20. Fiske C, Subbarow Y: The colorimetrie determination of phosphorus. J Biol Chem 66:375-400,1925.
21. Destruction with nitric and perchloric acids. In Jolly S (ed): "Official Standard and Recommended Methods of Analysis." Cambridge, MA: W Haffer and Sons, pp 1011, 1963. 22. Belsey R, DeLuca HF, Potts JT: Competitive binding assay for vitamin D and 25-hydroxyvitamin D. J Clin Endocrinol Metab 33:554-557,1971. 23. Winer BJ: "Statistical Principles in Experimental Design." New York: McGraw-Hill, pp 196-201, 1962. 24. Bloomfield VA, Mead RJ: Structure and stability of casein micelles. J Dairy Sci 58:592-601, 1975. 25. Charley P, Saltman P: Chelation of calcium by lactose: its role in transport mechanisms. Science 139:1205-1206, 1963. 26. Kelly SE, Chawla-Singh K, Sellin JH, Yasillo NJ, Rosen berg IH: Effect of meal composition on calcium absorp tion: enhancing effect of carbohydrate polymers. Gastroenterology 87:596-600, 1984. 27. Recker RR: Calcium absorption and achlorhydria. New Engl J Med 313:70-73, 1965. 28. Heaney RP, Smith KT, Recker PP, Hinders SM: Meal ef fects on calcium absorption. Am J Clin Nutr 49:372-376, 1989. 29. Knowles JB, Wood RJ, Rosenberg Hi: Response of frac tional calcium absorption in women to various coadministered oral glucose doses. Am J Clin Nutr 48:14711474, 1988.
Received June 1989; revision accepted September 1990.
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The effect of carbohydrates in milk on the absorption of calcium by postmenopausal women. S A Schuette, N J Yasillo & C M Thompson To cite this article: S A Schuette, N J Yasillo & C M Thompson (1991) The effect of carbohydrates in milk on the absorption of calcium by postmenopausal women., Journal of the American College of Nutrition, 10:2, 132-139, DOI: 10.1080/07315724.1991.10718137 To link to this article: http://dx.doi.org/10.1080/07315724.1991.10718137
Published online: 02 Sep 2013.
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Date: 21 June 2016, At: 04:20
The Effect of Carbohydrates in Milk on the Absorption of Calcium by Postmenopausal Women Sally A. Schuette, PhD, Nicholas J. Yasillo, and Charlotte M. Thompson, MS
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Clinical Nutrition Research Unit and Department of Radiology, University of Chicago, Chicago The purpose of this investigation was to determine if the presence of carbohydrate in milk, either lactose or its hydrolysis products, enhance the bioavailability of calcium (Ca) in milk. Two studies were performed. In study A, fractional Ca absorption was measured in 11 lactose-tolerant postmenopausal women after an oral dose of 47Ca-equilibrated milk formula containing no carbohydrate (NOCHO), lactose (LACTOSE), or an equivalent amount of glucose plus galactose (SUGAR); all participated in three absorption studies in random order. The NOCHO formula contained 10.0 g protein and 217 mg Ca from a combination of milk mineral and protein isolates; the LACTOSE and SUGAR formulae contained in addition 12 g lactose or 6 g glucose plus 6 g galactose, respectively. In study B, fractional Ca absorption was measured in five postmenopausal women after an oral dose of 47Ca-equilibrated skim milk (217 mg Ca) and lactase-treated milk, each with sufficient carbohydrate added to equal 12 g. For both studies, the increase in forearm radioactivity 4 and 8 hours after oral 47Ca administration relative to the increase observed after IV administra tion was used to estimate fractional Ca absorption. The addition of lactose but not glucose plus galactose to the NOCHO formula enhanced Ca absorption (p < 0.05). Fractional absorption at 4 hours was 0.386 from the LACTOSE formula compared with 0.310 for both the NOCHO and SUGAR formulae. Those individuals with the lowest absorp tion in the absence of carbohydrate had the greatest increase with lactose. In contrast, Ca absorption was the same from skim milk as from lactase-treated skim milk (study B). The results of these studies suggest that lactose may enhance Ca absorption in situations where Ca solubility or active Ca absorption is limiting. Abbreviations: Ca = calcium, NOCHO = Ca-equilibrated milk fornitila contain ing no carbohydrate, 250HD = 25-hydroxyvitamin D.
INTRODUCTION Teleologically speaking, the possibility that lactose or "milk sugar" could specifically enhance calcium (Ca) absorption is appealing. The ß-l,4-glycosidic linkage found in lactose is unique among carbohydrates that can be digested by mammalian enzymes. The unique struc ture of lactose and its presence only in milk has long suggested a positive role for lactose in calcium absorp tion. A number of investigators have shown that the sub stitution of lactose for other carbohydrates in the diets of laboratory animals can improve Ca absorption and in crease the retention of Ca in bone [1-4]. In some instan ces the presence of lactose improved Ca absorption and retention despite the fact that the large amounts of lac
tose fed produced diarrhea and increased fecal loss of other nutrients such as protein and fat [5]. Lactose and Ca must be present in the intestine simultaneously in order for the positive effects of lactose on Ca absorption or bone mineralization to be manifest [6]. Based in large part on the results of animal studies, it has generally been accepted that lactose enhances Ca absorption in man unless the individual is lactase-deficient. However, a critical review of this literature reveals that the data are less than convincing. Debongnie et al [7] found significantly more Ca in ileal aspirates (less Ca absorbed) from four lactose-tolerant subjects given milk vs lactase-treated milk. Lactase-deficient subjects ab sorbed less Ca than normals from both milks but also showed the negative effect of lactose on Ca absorption. Greenwald et al [8] found no positive effect on Ca ab-
The work was presented, in part, at the 28th Annual Meeting of the American Society for Clinical Nutrition, Washington, D.C. April 28-30,1988. Address reprint requests to Dr. S. Schuette, University of Chicago, Clinical Nutrition Research Unit, S841 South Maryland, Box 223, Chicago, Illinois 60637.
Journal of the American College of Nutrition, Vol. 10, No. 2, 132-139 (1991) © 1991 John Wiley & Sons, Inc.
CCC 0731-5724/91/020132-08$04.00
Carbohydrates and Ca Absorption Table 1. Composition of Basic Milk Formula (Study A) Compared to Skim Milk Normalized to the Same Calcium Content1 CHO(g)
Ca(mg)
Mg (mg)
P(mg)
9.9 0.1 10.0
0.002 0.004 0.006
95 122 217
3 3 6
50 108 158
7.1
8.31
217
21
167
Protein (g) Basic milk formula TMP1200.il g Alamin 2000, 0.4 g Total Skim milk, 170 g 2
'Protein values based on manufacturer's information, others by analysis. This is the composition of the skim milk used in study B before the addition of extra carbohydrate (CHO).
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2
sorption or balance of adding lactose to an otherwise constant diet of older adults. However, only three patients with various complaints were studied in this in vestigation, and total Ca intake was high (1500 mg). Recently, Tremaine et al [9] reported mat fractional Ca absorption, measured by a dual-isotope technique, was the same from normal milk and lactase-treated milk. Similarly, Griesson et al reported that Ca absorption was the same from milk containing lactose or glucose as the carbohydrate source in lactose-tolerant adults [10]. On the positive side, Mills et al [11] found that six of seven young boys (4—7 years old) retained more Ca when 36 g lactose was added to their basal diet Also in children, Ziegler and Fomon [12] found better Ca ab sorption from formula containing lactose compared to an identical formula containing sucrose and com starch hydrolysate. Two investigations have demonstrated a positive effect of lactose on Ca absorption in adult man [13,14]. Kocian et al [13] reported more rapid and slight ly greater total values for 47Ca absorption from "milk" than from lactose-free "milk." However, the "milks" compared were really milk-based formulas which dif fered somewhat in their composition, including fortifica tion with different Ca salts. The most convincing study to show a positive effect of lactose on Ca absorption in adults was done by Cochet et al [14]. These investigators used a dual-isotope technique to determine fractional Ca absorption in the presence or absence of 50 g lactose. Total fractional Ca absorption was enhanced by 60% in the presence of lactose compared to water alone; in this investigation lactose was added to a solution of CaCl2 in water. Thus, although some evidence is available to suggest lactose may have a positive effect on Ca absorption in man, the data can hardly be considered definitive. Addi tionally, the results of several recent studies showing a positive effect of other readily available carbohydrates
on Ca absorption in man [15-18] suggest that "lactose enhancement" of Ca absorption may not be specific to lactose but may be a general effect of simple car bohydrates. The purpose of mis investigation was to determine if the presence of carbohydrate in milk, either lactose or its hydrolysis products glucose and galactose, significantly enhance the bioavailability of Ca in milk.
METHODS Experimental Design Study A Fractional Ca absorption was measured in 11 postmenopausal women after an oral dose of "Ca-equilibrated milk formula containing little carbohydrate (NOCHO), lactose (LACTOSE), or an equivalent amount of glucose plus galactose (SUGAR); all 11 women participated in three separate absorption studies; one with each of the milk formulae. The milk formulae were made from milk protein isolate, containing both casein and whey proteins, plus a milk mineral con centrate (TMP 1220 and Alamin 2000, respectively, both from New Zealand Milk Products, Ine, Petaluma, CA). The protein, carbohydrate, Ca, phosphorus, and mag nesium contents of the basic formula are given in Table 1 and compared to skim milk. The milk formulae used for the absorption tests were as follows: NOCHO: LACTOSE: SUGAR:
basic formula (10.0 g protein and 217 mg Ca in 200 ml H20) + 74 kBq 47Ca basic formula + 12 g lactose + 74 kBq 47Ca basic formula + 6 g glucose + 6 g galactose + 74 kBq 47Ca
The formulae were flavored with 0.8 ml chocolate extract and a few drops of artificial sweetener and were
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Carbohydrates and Ca Absorption Table 2. The Effect of Lactase Treatment and the Addition of Sugars on the Carbohydrate Content of Skim Milks Used in Study B per 170 g Carbohydrate Milk Untreated skim milk MILK+LAC1
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Lactase-treated skim milk MILK+SUGAR 2
Lactose 8.21 12.00 0.05 0.05
Galactose (or glucose) 0 0 4.10 6.00
'Lactose was added as monohydrate; as such, 3.88 g lactose monohydrate was added to yield 3.69 g of lactose. 2 Galactose content was determined en2ymatically. Since the hydrolysis of lactose yields equimolar amounts of glucose and galactose, glucose content was assumed and not determined directly. Galactose and glucose, anhydrous, were added at the level of 1.90 g each.
indistinguishable by the subjects. The formulae were ad ministered in a random manner; however, since 11 sub jects participated, one of six possible orders of ad ministration was used once instead of twice. Study B Fractional Ca absorption was measured twice in each of five postmenopausal women after an oral dose of 47 Ca-equilibrated skim milk or lactase-treated skim milk, each with sufficient carbohydrate added to be equal in sugar content to the milk formula used in study A; lac tose was added to skim milk (MILK+LAC) and glucose plus galactose to the lactase-treated milk (MILK+ SUGAR). Lactose treatment was performed using ßgalactosidase enzyme (LactAid, Pleasantville, NJ) at double the manufacturer's recommended dose for 48 hours at refrigerated temperature. The protein, car bohydrate, Ca, phosphorus, and magnesium contents of the milk used were as shown in Table 1. The effect of lactase treatment and the final carbohydrate contents are shown in Table 2. The milks were administered in a random manner. Again the milks were flavored with chocolate extract and artificial sweetener and were indis tinguishable by the subjects. Both studies had been approved by the Human Use Radioisotope and Clinical Investigation Committees of the University of Chicago. Informed written consent was obtained from each participant. A single blood sample was collected from each subject at the beginning of the study for measurement of serum 25-hydroxyvitamin D (250HD).
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Subjects A total of 16 postmenopausal women were subjects in these investigations. Menopause was defined as com plete cessation of menses for at least 2 years. Women fitting this criterion underwent a standard lactose breath H2 test using 25 g lactose to determine lactose tolerance. The H2 content of expired air was determined at 0, 1, 2, and 3 hours after dosing. Lactose intolerance was defined as a breath H2 level > 20 ppm above baseline at one or more timepoints. Only lactose-tolerant women participated as subjects in these investigations. In study A, the 11 postmenopausal women who par ticipated ranged in age from 38 to 66 years and were 3-17 years postmenopause. In study B, these same char acteristics for the postmenopausal subjects were 39-61 and 3-30 years, respectively. Those subjects taking medications prescribed by their personal physicians at the beginning of the study continued these medications throughout. Subject characteristics are listed individually in Table 3.
Analytical Methods Radioabsorption Test Fractional Ca absorption was determined using a modification of the forearm counting method described by Wills et al [19]. Details of the radiocalcium absorp tion test are as follows: on the first morning and after an overnight fast, the subjects received 37 kBq (1 μ ο ) of
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Carbohydrates and Ca Absorption Table 3. Subject Characteristics1
Subject
Age
Years postmenopause
1A
51
3a
2A 3A 4A 5A 6A 7A 8A 9A 10A 11A
58 59 57 58 66 49 62 55 42 38
10 17 3 12 17 9s 9 5 3a 3a
IB
61
30*
2B 3B 4B 5B
40 55 39 42
9" 3 9a
Serum 250HD (ng/ml)
Medications
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Study A Indomethacin, 75 mg bid Medroxyprogesterone, 10 mg qd Conjugated estrogens, 1.25 mg qd None None None None None None Propranolol, 20 mg qd Chlorpropamide, 250 mg qd None None
33.5
42.6 25.2 27.2 30.4 26.8 27.3 21.6 13.0 34.7 22.0
Study B
15
Conjugated estrogens, 0.3 mg qd Theophylline, 200 mg qd Enalaprii, 5 mg qd None None Conjugated estrogens, 0.625 mg qd None
2
— — — —
Normal range for serum 250HD level is 10-60 ng/ml. Superscripted a denotes surgical menopause. Subjects 10A and 2B had only a partial hysterectomy and had ovaries intact. Serum samples from these subjects were accidentally discarded.
Table 4. Fractional Calcium Absorption from the Various Milk Formulae, Study A1 NOCHO
LACTOSE
SUGAR
0.310 ±0.037 a
0.386 ±0.030 b
0.310 ±0.025 a
Values are given as the mean ± SEM for all subjects at 4 hours. Values with different superscripts are significantly different, p < 0.05.
Table 5. Fractional Calcium Absorption from Milk or Lactase-Treated Milk, Study B 1 MILK+LAC
MILK+SUGAR
At 4 hours
0.209 ±0.029
0.194 ±0.037
At 8 hours
0.239 ± 0.032
0.222 ± 0.037
Values are given as the mean ± SEM for all subjects.
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Carbohydrates and Ca Absorption
.60
i0.
cr O
g.40. < a o _i < z o
j= .20 ü
7.4 MBq/mg or 200 μθϊ/ιτ^; Amersham, Chicago); the IV dose approximates 100% absorption and is necessary for calculation of fractional Ca absorption. Right forearm radioactivity was determined 4 and 8 hours later in a large volume gamma counter built specifically for this purpose (consists of four 5" diameter by 5" Nal crystals with associated counting electronics and is completely shielded). The subjects remained fasted until after the 4 hours forearm count. After this they were allowed to eat lunch but had been instructed on how to choose a lowCa lunch. On subsequent days, at least 48 hours apart, a similar procedure was followed except that the subjects received 74 kBq (2 μθί) of 47Ca orally, in milk or milk formula containing 217 mg Ca. For all oral doses, the 47CaCl2 was added to the milk or milk formula the afternoon before. After the first isotope administration, a baseline forearm count was performed just prior to administration of the next oral dose of 47Ca to correct for residual radioactivity. Fractional Ca absorption was calculated as: forearm radioactivity after oral 47Ca dose xlOO forearm radioactivity after IV Ca dose
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after correction for dose administered, background radioactivity, and decay. Laboratory Analyses Ca and magnesium contents of samples of the milk protein isolate, milk mineral concentrate, and skim milk were determined by atomic absorption spectrophotometry (Model 5000, Perkin-Elmer, Norwalk, CT). For Ca and magnesium determinations, samples were first dried in a muffle furnace for 3 hours at 150°C and then ashed for 12-15 hours at 450"C. Following this, 1 ml of nitric acid was added to the ash and the samples were returned to the muffle furnace for 1-3 hours at 150'C. The final ash was dissolved in nitric acid and deionized water. The phosphorus content of milk protein and mineral plus skim milk were determined colorimetrically [20] after wet ashing with nitric and perchloric acid [21]. Skim milk standard (National Bureau of Standards refer ence material #1549) analyzed in parallel with our samples was found to contain 97-106% of the expected values for magnesium, Ca, and phosphorus. Lactose and galactose content of the milk formulae and skim milk were determined enzymatically using a
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Carbohydrates and Ca Absorption commercially available kit (Cat #176303, Boehringer Mannheim GmbH, Mannheim, Germany). Serum 250HD levels were determined by a modification of the competitive binding assay of Belsay et al [22] using nor mal human serum as the standard. Statistical Analysis For study A, fractional absorption of Ca from the various milk formulae was compared by two-way ANOVA (subject and milk formula). If a significant milk formula effect was observed, mean absorption values were compared using Tukey's multiple comparison pro cedure (p < 0.05) [23]. For study B, Ca absorption of the subjects from MILK+LAC vs MELK+SUGAR was com pared using a t-test for paired observations.
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RESULTS Study A As shown in Table 4, the addition of lactose but not glucose plus galactose to a milk formula containing neg ligible amounts of carbohydrate significantly enhanced fractional Ca absorption. At 4 hours, fractional Ca ab sorption was 0.386 from the LACTOSE formula com pared with 0.310 for both the NOCHO and SUGAR for mulae; this represents a 25% increase in Ca absorption with lactose. Fractional Ca absorption calculated from the 8- vs 4-hr data was slightly but not statistically greater for all of the milk formulae used, and the dif ferences between formulae were also significant at 8 hours. The 4-hr data are presented in Table 3 and Figure 1 because only the 4-hr data is available for all subjects on all three treatments; subject 1A forgot to return for her 8-hr forearm count during the LACTOSE absorption test. The mean 4- and 8-hr fractional absorption values, exclusive of data from subject 1A, were 0.393 vs 0.400, 0.324 vs 0.338, and 0.314 vs 0.326 for the LACTOSE, NOCHO, and SUGAR formulae, respectively. The individual fractional absorption values for all subjects from the LACTOSE and NOCHO formulae at 4 hours are depicted in Figure 1. There are several interest ing observations to be made from looking at the in dividual responses. Generally, those individuals with the lowest absorption values in the absence of carbohydrate had the greatest increase in the presence of lactose; the converse was also true. In fact, there was a significant negative correlation between an individual's fractional absorption of Ca from the NOCHO formula and the in crease in absorption seen with the addition of lactose (r = 0.609, p < 0.05). For example, subject 1A had a fractional absorption value of only 0.166 in the absence of carbohydrate and the value increased to 0.320 with
lactose, whereas subject 10A had an absorption value of 0.585 without lactose and the value decreased slightly to 0.552 with lactose. For the small number of subjects studied in this inves tigation, no significant correlation was found between fractional Ca absorption from the NOCHO formula and years postmenopause. In addition, no significant correla tion was found between age of subject or years postmenopause and the increase in Ca absorption ob served with lactose. All subjects had serum 250HD levels within the normal range (Table 1).
Study B As shown in Table 5, lactose equivalent to the amount present in the LACTOSE formula of study A did not enhance Ca absorption from skim milk compared to an equimolar amount of glucose plus galactose. In addition, fractional Ca absorption appeared to be lower from the skim milk with added carbohydrate (study B) than from the comparable milk formulae (study B); fractional Ca absorption at 4 hours from the LACTOSE formula vs MILK+LAC and the SUGAR formula vs MILK+SUGAR was 0.386 vs 0.209 and 0.310 vs 0.194, respectively (p < 0.02). Although these differences were statistically significant, the small num ber of subjects studied in study B makes the biological significance of this difference questionable. No dif ference in the age of the subjects, years postmenopause, or proportion of subjects taking medications is apparent between the two groups. Serum 250HD levels are not available for the subjects of study B.
DISCUSSION The results of this investigation suggest that lactose can enhance the absorption of Ca by postmenopausal women under some, but not all, conditions. Lactose caused a 25% increase in Ca absorption when present in an artificial milk formula compared to an equimolar amount of glucose plus galactose or the absence of car bohydrate. However, the same amount of lactose in fresh skim milk did not enhance Ca absorption compared with its constituent sugars — glucose plus galactose. The milk formula used was similar to skim milk in total mineral composition and was made from protein and mineral fractions isolated from milk but was dif ferent from milk in some potentially important charac teristics. The proportions of mineral and protein isolate used were chosen to yield a Ca:P ratio similar to skim milk, but neither isolate component was completely soluble in water. As a result, our milk formula formed a suspension from which some portion of the mineral and
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Carbohydrates and Ca Absorption protein fractions would slowly settle out.* This is clearly different from skim milk. In addition, in our milk for mula a smaller portion (55%) of the Ca came from the protein isolate than is normally associated with the col loid protein fraction of skim milk (70%) [24]. It is pos sible that lactose enhanced the solubility of Ca in the milk formula or during the digestion process and thus resulted in greater Ca absorption. Lactose has been shown to form a more stable chelate with Ca than glucose or galactose [25]. The same situation did not exist for skim milk, where all the Ca was either soluble or associated with colloidal protein. The fact that lactose enhancement of Ca absorption from the milk formula was more pronounced in women with low fractional absorption in the absence of car bohydrate is in keeping with several recent reports, which all suggest that some individuals with low absorp tion respond positively to the presence of various dietary factors by mechanisms which are not clearly understood. This was first described by Kelly et al [26] and led to a series of investigations into the effect of glucose and glucose polymers on Ca absorption [15,16]. More recently, Heaney and co-workers have observed sig nificantly meal effect on Ca absorption which is remarkably more pronounced in some individuals than others [27,28]. In addition, Knowles et al [29] recently demonstrated a clear dose response for the effect of glucose on Ca in seven of 10 subjects, whereas three showed no effect. Each of these studies may represent a specific nutrient/meal effect on Ca absorption (i.e., in the present investigation lactose, which is absorbed more distally than glucose and galactose, may effectively en hance Ca absorption only in those individuals with poor active absorption in the more proximal gut) or all may be related via a general mediator(s) of gastrointestinal func tion (i.e., via CCK, secretion, vasoactive intestinal peptide, etc.) and lead to observable enhancement of Ca absorption only when absorption is low in the absence of enhancer.
CONCLUSIONS In agreement with other recent reports [9,10], we ob served that the lactose content of milk has little effect on
*This characteristic of the milk formula did not result in poor equilibra tion of the radiolabel. Determination of the specific activity of both the soluble and suspension fractions of a milk formula identical to that used in the absorption study but equilibrated with 45Ca showed that the radiolabel was even distributed. The specific activity of the two fractions were: soluble—631 ± 5 cpm/mmol; suspension—637 ± 52 cpm/mmol.
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Ca absorption from milk in lactose-tolerant adult humans. However, we also observed that lactose, like glucose, glucose polymer, and other nonspecific meal effects, may significantly enhance Ca absorption under some conditions. A better understanding of the possible relationship between these observed effects may lead to more specific recommendations for the most effective way of providing highly bioavailable Ca for individuals with increased needs.
ACKNOWLEDGMENTS The authors acknowledge the support of this work by WMMB grant #IV-39 and Clinical Nutrition Research Center grant AM 26678. In addition, the authors would like to thank Dr. Malcom Cooper and Ms. Erma Peterson for their helpful assistance in the preparation and ad ministration of the 47Ca doses, as well as Ms. Lynette McCall for her assistance in preparation of this manuscript
REFERENCES 1. Bergeim O: Intestinal chemistry. V. Carbohydrates and calcium and phosphorus absorption. J Biol Chem 70:3545, 1926. 2. Evans JL, Ali R: Calcium utilization and feed efficiency in the growing rat as affected by dietary calcium, buffering capacity, lactose and EDTA. J Nutr 92:417-424, 1967. 3. Schaafsma G, Visser R: Nutritional interrelationships be tween calcium, phosphorus and lactose in rats. J Nutr 110:1101-1111, 1980. 4. Miller SC, Miller MA, Omura T: Dietary lactose improves endochondrial growth and bone development and mineralization in rats fed a vitamin D-deficient diet. J Nutr 118:72-77,1988. 5. Leichter J, Tolensky AF: Effect of dietary lactose on the absorption of protein, fat and calcium in the postweaning rat. Am J Clin Nutr 28:238-241,1975. 6. Lengemann FW: The site of action of lactose in the enhan cement of calcium utilization. J Nutr 69:23-27, 1959. 7. Debongnie JC, Newcomer AD, McGill DB, Phillips SF: Absorption of nutrients in lactase deficiency. Dig Dis Sci 24:225-231, 1979. 8. Greenwald E, Samachson J, Spencer H: Effect of lactose on calcium metabolism in man. J Nutr 79:531-538, 1963. 9. Tremaine WJ, Newcomer AD, Riggs BL, McGill DB: Cal cium absorption from milk in lactase-deficient and lactasesufficient adults. Dig Dis Sci 31:376-378, 1986. 10. Griessen M, Cochet B, Infante F, Jung A, Bartholdi P, Donath A, Loizeau E, Courvoisier B: Calcium absorption from milk in lactase-deficient subjects. Am J Clin Nutr 49:377-384,1989.
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Carbohydrates and Ca Absorption 11. Mills R, Breiter H, Kempster E, McKey B, Pickens M, Outhouse J: The influence of lactose on calcium retention in children. J Nutr 20:467-476,1940. 12. Zeigler E, Fomon S: Lactose enhances mineral absorption in infancy. 3 Pediatr Gastroenterol Nutr 2:288-294, 1983. 13. Kocian J, Skala I, Bakos K: Calcium absorption from milk and lactose-free milk in healthy subjects and patients with lactose intolerance. Digestion 9:317-324, 1973. 14. Cochet B, Jung A, Griessen M, Bartholdi P, Schaller P, Donath A: Effects of lactose on intestinal calcium absorp tion in normal and lactase-deficient subjects. Gastroenterology 84:935-940, 1983. 15. Bei L, Wood RJ, Rosenberg IH: Glucose polymer in creases jejunal calcium, magnesium and zinc absorption in humans. Am J Clin Nutr 44:244-247, 1986. 16. Wood RJ, Gerhardt A, Rosenberg IH: Effects of glucose and glucose polymers on calcium absorption in healthy subjects. Am J Clin Nutr 46:699-701, 1987. 17. Norman DA, Morawski SG, Fordtran JS: Influence of glucose, fructose, and water movement on calcium absorp tion in the jejunum. Gastroenterology 78:22-25, 1980. 18. Schuette SA, Knowles JB, Ford HE: The effect of lactose or its component sugars on jejunal calcium absorption in adult man. Am J Clin Nutr 50:1084-1087, 1989. 19. Wills M, Zisman E, Wortsman J, Evens RG, Pak CYC, Banter FC: The measurement of intestinal calcium absorp tion by external radioisotope counting: application to study of neophrolithiasis. Clin Sci 39:95-106, 1970. 20. Fiske C, Subbarow Y: The colorimetrie determination of phosphorus. J Biol Chem 66:375-400,1925.
21. Destruction with nitric and perchloric acids. In Jolly S (ed): "Official Standard and Recommended Methods of Analysis." Cambridge, MA: W Haffer and Sons, pp 1011, 1963. 22. Belsey R, DeLuca HF, Potts JT: Competitive binding assay for vitamin D and 25-hydroxyvitamin D. J Clin Endocrinol Metab 33:554-557,1971. 23. Winer BJ: "Statistical Principles in Experimental Design." New York: McGraw-Hill, pp 196-201, 1962. 24. Bloomfield VA, Mead RJ: Structure and stability of casein micelles. J Dairy Sci 58:592-601, 1975. 25. Charley P, Saltman P: Chelation of calcium by lactose: its role in transport mechanisms. Science 139:1205-1206, 1963. 26. Kelly SE, Chawla-Singh K, Sellin JH, Yasillo NJ, Rosen berg IH: Effect of meal composition on calcium absorp tion: enhancing effect of carbohydrate polymers. Gastroenterology 87:596-600, 1984. 27. Recker RR: Calcium absorption and achlorhydria. New Engl J Med 313:70-73, 1965. 28. Heaney RP, Smith KT, Recker PP, Hinders SM: Meal ef fects on calcium absorption. Am J Clin Nutr 49:372-376, 1989. 29. Knowles JB, Wood RJ, Rosenberg Hi: Response of frac tional calcium absorption in women to various coadministered oral glucose doses. Am J Clin Nutr 48:14711474, 1988.
Received June 1989; revision accepted September 1990.
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