Mineral bioavailability and bone mineral contents in pigs given calcium carbonate postprandially

Bone Vol. 17, No. 4 October 1995:357-362 ELSEVIER

Mineral Bioavailability and Bone Mineral Contents in Pigs Given Calcium Carbonate Postprandially A. P O I N T I L L A R T , C. C O L I N , H. C. L A C R O I X , and L. G U I ~ G U E N Laboratoire de Nutrition et S~curit~ Alimentaire, INRA, Jouy-en-Josas, France

bones react rapidly to nutritional changes. 19,2o Bones were therefore collected at slaughter to measure their mineral contents and breaking load and to correlate these parameters with the balance data. We also studied various biochemical parameters related to bone metabolism, especially the plasma osteocalcin concentration.

We have further investigated the "meal effect" on mineral bioavailability in pigs by mineral balance studies and measurements of bone ash contents and bending moment. A group of seven pigs (CAA) was given all its dietary Ca as CaCO a 5 h after the first daily meal for 8 weeks. The control group of seven pigs received CaCO a in the meal. Both groups were given normal P within the meals. Ca and P absorption and retention were evaluated by a 10-day balance trial. Several bones were collected at slaughter to determine bone ash, Ca, and P contents and bending moment (three-point bending test). Ingesting Ca after the meal did not affect Ca bioavailability or phosphorus absorption, but did reduce P retention, which in turn decreased the bone scores. Osteopenia, indicated by decreased total mineral contents of bones (and decreased ash:bone volume ratio), was associated with elevated plasma osteocalcin in the CAA group. Thus, CaCO a need not be incorporated into a meal for high Ca absorption, provided that Ca is given after a meal, but simultaneous intakes of Ca and P are required for the best mineral retention. (Bone 17:357-362; 1995)

Materials and Methods Animals and Feeding

Fourteen 53-day-old male crossbred pigs (Cepra, Vermenton, France), weighing 15 --- 0.3 kg, were randomly assigned to two groups: control (C) and separate Ca (CAA group). They were fed the appropriate diet for 8 weeks, stunned by electronarcosis, and killed by exsanguination. All the animals were given similar basal diets which consisted mainly of cereals and soyameal formulated to contain 19% protein (1.1% lysine), 3400 kcal digestible energy, 0.5% phosphorus, and 1000 IU of Vitamin D3/kg. These diets (Table 1) met the requirements for growing pigs. Feed intake was adjusted every 3-4 days and the growth rate was evaluated every week. Pigs were kept in individual pens and pair-fed. The main source of Ca was CaCO 3 (limestone, 38.1% Ca) (Vitacarb 28, Soci6t6 MEAC, Chartres, France) in the two diets; it was incorporated (2.2% of feed) into the control diet, and given separately to the CAA pigs. Because the plant ingredients contain very little Ca (0.11%), most of the Ca ingested was provided by the limestone. The adjusted Ca intakes of the CAA pigs were checked daily by measuring the amounts of food actually ingested by the control group, so that the two groups ingested similar daily amounts of Ca (overall mean 13.8 --- 0.00 g/day). Limestone was given every day at 1:30 P.M., 5 h after the beginning of the first meal (8:30 A.M.). This meal was eaten rapidly (less than 1 h). A second meal was given in the afternoon (at 5:30 P.M.). The limestone was mixed with a small quantity (25-50 g) of wheat meal (0.06% Ca, 0.34% P) and readily (15-20 min) eaten by the pigs. A "meal effect" was avoided by preconditioning the pigs to this mode of feed distribution before the experiment started. The control group was also given a small quantity of wheat meal, but without the limestone.

Key Words: Ca bioavailability; Bone mineral content; Meal effect; Osteocalcin; Phosphorus.

Introduction The most widely used form of calcium supplement is alkalineinsoluble calcium carbonate. Its bioavailability in humans has often been correlated with the meal-stimulated secretion of gastric H C I . 1"1°'17'2°'21'27 Thus, we and others have observed a beneficial "meal effect" on the absorption of CaCO 3 in animals and humans, lO,2O.21 In a previous study, 2° we showed that giving calcium carbonate to fasting pigs 3 h before feeding resulted in a considerably lower (60-90%) absorption of calcium than when the calcium was given with a meal. This beneficial "meal effect" was attributed to the secretion of gastric HC1.17'27 But other authors have used different methods (for review see Wood and Serfaty-Lacrosniere 27) to show that insoluble Ca sources are normally absorbed in achlorhydric subjects when given with a meal. As part of our program to determine when calcium is best absorbed using our pig model, we have therefore studied Ca use (absorption and retention) from C a C O 3 (limestone) in growing pigs given this Ca source a few h (5 h) after a meal to better understand the "meal effect" on mineral bioavailability. Pig

Balance Study and Bone Measurements

The apparent Ca and P absorption and retention were evaluated in a 10-day balance trial, on the last 10 days, immediately before slaughter. The 14 animals were kept in individual cages and fed the same amounts of feed (1.9 +-- 0.00 kg/day). A 5% sample was taken of each 24-h urine collection and these samples pooled for the 10 days. Similarly, all the 24-h feces samples for each

Address for correspondence and reprints: Dr. A. Pointillart, LNSAINRA, 78352 Jouy-en-Josas C6dex, France.

© 1995 by ElsevierScience Inc.

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8756-3282/95/$9.50 SSDI 8756-3282(95)00242-6

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A. Pointillart et al. Postprandial calcium carbonate in pigs

Bone Vol. 17, No. 4 October 1995:357-362

HLPIGPLASMA.-e~--

Table 1. Diet composition (%)

Wheat Soya-meal-50 Corn oil Mineral-vitamin mixa NaC1 NaH2PO 4 • 2H20 Limestone Dietary Ca and P contents (analyzed) In-meal Ca Nonmealb Ca Phosphorus

Control

Ca apart (CAA)

72.1 23 1.5 0.35 0.17 0.75 2.2

74.7 23 1 0.35 0.17 0.75 --

0.97 -0.49

0.11 0.88 0.48

aSame as used previously (Ref. 20). bLimestone 38.1% Ca. animal were pooled and mixed. The Ca and P in these pooled samples was assayed. At slaughter, the tibia and main metatarsals (fingers III and IV) were excised from the right hind leg and dissected free of soft tissues. The whole, fresh tibia and metatarsal bones were then used to measure bending moment (threepoint bending test at the fracture point) and the apparent density (weight/volume of the whole bones). Bone bending moments were determined with a universal testing machine (AP 4000 Erichsen, Villetaneuse, France) according to the formula: bending moment = F x L/4, where F (failure load) is the force (newtons [N]) applied to the midpoint of the shaft until breaking and L the length (meters [m]) between the two fulcrum points that the bone rested on, and were expressed as N × m (SI units). Stiffness (N/mm) was determined by the slope of the forcedeformation curve (elastic region) obtained during the bending test. Volume was determined according to the Archimedes principle by weighing whole bones in air and in water, and then apparent bone density (weight-to-volume ratio) was calculated. Ash, Ca, and P contents were measured on the external (IV) and internal (III) metatarsals and ash-to-bone volume was calculated. Bones were dried in a 105°C oven (Prolabo, Paris) for 2 days to determine bone dry matter. Ash percentage was determined by placing the dried bones in a 550°C muffle furnace (Heraeus, 91 les Ulis, France) for 12 h. Ashes were then treated with 0. IN nitric acid followed by reagent grade 12N hydrochloric acid over heat until dissolved and then quantitatively transferred into volumetric flasks for mineral analysis. Ca and P measurements (balance parameters and bones) have been described in detail. ~9 Bone resorption was estimated by determining hydroxyproline excretion in urine samples collected during the balance period. 26

Plasma Parameters Ca, inorganic P, total and bone alkaline phosphatase isoenzyme activity, and osteocalcin were determined on venous blood samples collected at slaughter during exsanguination. Plasma total alkaline phosphatase activity was analyzed as previously. 7 Plasma bone isoenzyme alkaline phosphatase was determined using a kit (Tandem-R Ostase, Hybritech, Trappes, France) based on a radioimmunometric assay. ~ This has recently been shown to be a valuable indicator in human bone disease. 9 In the pigs, total plasma alkaline phosphatase was closely correlated (r = 0.87, p < 0.001) with the plasma bone isoenzyme concentration. Plasma osteocalcin (BGP) was measured using a modified

B/B~ 1

0.156 0.625 2.5 5 lo 0.0~,8 '0.312' 1.'25' " '

0.75

r

~

0.500.25"

00~.01 ........0.1 . ........ ,1 ........10 . S T A N D A R D BOVINE O S T E O C A L C l N ng/ml -c~

Figure 1. Radioimmunoassay of osteocalcin (BOP).

radioimmunoassay kit (OSTK-PR, Oris, Gif sur Yvette, France) with purified bovine BGP as standard and tracer, and rabbit antiserum to bovine BGP. Porcine and bovine BGP differ by only four amino acids. 12 This minor difference did not prevent the antiserum raised to bovine BGP recognizing porcine osteocalcin. Thus, the bovine BGP standard inhibition curve and the curve of serial dilutions of a porcine plasma were parallel (Figu r e 1) between 0.156 and 2.5 ILL porcine plasma (0.37~5.12 ng/mL equivalent bovine BGP). The high binding capacities of the antiserum (62-70%) allowed greater antiserum dilution than that proposed by the manufacturer. The antiserum was diluted 1.5-fold (2 volumes antiserum + 1 volume buffer). At this dilution, the sensitivity was maximal and the antiserum bound 50% of 15,000 cpm 125I-bovine standard BGP with nonspecific binding (NSB) near 5%. Sensitivity, defined as the smallest amount of standard BGP distinguishable from control buffer, was 0.15 ng/mL. Standard porcine plasma samples were diluted 1:5 to 1:640. The internal porcine standard consisted of three pooled plasma samples from growing pigs of similar age to those used in the experiment. Unknown porcine plasma samples were diluted 1:50 in 0.05 mol/L borate buffer (which contained 1% Table 2. Ca and P balances Control Calcium Intake (g/day) In-meal Nonmeal Refusals Total Ca Absorbed (g/day) Absorption (% intake) Urinary (g/day) Retained (g/day) Retention (% intake) Phosphorus Intake (g/day) Absorbed (g/day) Absorption (% intake) Urinary (g/day) Retained (g/day) Retention (% intake)

19 -+ 0.0 --19 ± 0.0 9.0 -+ 0.3 47 -+ 2 0.2 ± 0.04 8.8 ± 0.3 46 ± 2 9.4 5.3 56 0.4 4.9 52

-+ 0.0 ± 0.1 ± 2 ± 0.1 ± 0.1 -+ 1

CAA

2.1 16.9 1.1 17.9 8.7 49 0.2 8.5 48

+ 0.0 ± 0.0 -+ 0.4 -+ 0.4 a ~ 0.5 ± 4 ~ 0.03 ± 0.5 ± 3

9.1 5.2 57 1.3 3.9

± 0.0 -+ 0.2 -+ 2 ± 0.2 ~ ± 0.1 a

42 ±

Means - SEM (n = 7). Rounded values given for percentages. ap < 0.01 (Student's t-test).

1~

Bone Vol. 17, No. 4 October 1995:357-362

A. Pointillart et al. Postprandial calcium carbonate in pigs

359

Table 3a. Metatarsal data--ash contents

Fresh weight (g) Control CAA Volume (cm3) Control CAA Ash weight (g) Control CAA Ash % dry matter Control CAA Ash:bone volume (g/100 cm3) Control CAA

Overall effects a Treatment Bone

MTE

MTI

18.9 (0.5) 17.7 (0.9)

17.7 (0.6) 16.8 (0.8)

n.s.

n.s.

16.6 (0.4) 15.9 (0.8)

15.5 (0.6) 14.9 (0.7)

n.s.

n.s.

4.5 (0.1) 3.9 (0.2)

4.4 (0.1) 4.0 (0.2)

P < 0.004

n.s.

40.7 (0.2) 39.9 (0.5)

42.8 (0.4) 42.9 (0.3)

n.s.

p < 0.005

27.1 (0.6) 24.5 (0.4)

28.3 (0.4) 27.1 (0.6)

P < 0.003

p < 0.004

There was no significant treatment × bone interactions for any parameters. MTE = external metatarsal; MTI = internal metatarsal bone, right hind leg; n.s. = not significant. aTwo-way (treatment × bone) ANOVA, means and SEM, n = 7. human serum albumin), pH 7.5, and assayed in triplicate. Diluted unknown plasma (50 txL) was mixed with 100 txL of diluted antiserum (1.5 in borate buffer) and preincubated for 6 h at 4°C. Tracer (200 IxL, 15,000 cpm) was then added and incubation continued for 12 h at 4°C. The final assay volume was 0.35 mL. The assay was terminated by precipitating the rabbit antibody with 1 mL of diluted sheep antirabbit ",/-globulin antiserum (from the kit) to separate free and antibody-bound fractions. The 125I-labeled BGP bound to rabbit antibody was collected by centrifugation at 2000g for 15 min, and the supernatant was discarded. NSB was measured by incubating labeled BGP and normal rabbit serum (NSB for standard curve) or pig plasma (NSB for unknown samples) instead of specific antiserum followed by the usual second antibody precipitation. Total and antibodybound 125I-BGP were determined by counting in a LKB gammacounter (LKB-Pharmacia, St. Quentin en Yvelines, France). The BGP intra-assay variation evaluated, by measuring three

different plasma samples seven times each, was < 5 % ; interassay variation from repeated measurements of porcine samples was < 13% for eight assays. Several recovery tests were performed at porcine plasma sample final dilutions of 1:40 and 1:640. The average recovery was 106 - 8% (n = 5). The detection limit was 0.15 ng/mL equivalent bovine BGP at a dilution of 1:50 for 1 txL porcine plasma.

Statistical Methods Ca and P balance data and plasma parameters were compared by Student's t-test. A two-way (treatment and bone kind) variance analysis (ANOVA) was performed using commercial software 1 to determine any significant differences in bone parameters, either between the two groups (overall treatment effect on a given bone criteria) or between the two (or three, bone density only)

Table 3b. Metatarsal d a t a ~ a and P contents a

Total Ca (g) Control CAA Ca % dry matter Control CAA Ca:bone volume (g/100 cm3) Control CAA Total P (g) Control CAA P % dry matter Control CAA P:bone volume (g/100 cm3) Control CAA

MTE

MTI

1.5 (0.04) 1.3 (0.06)

1.5 (0.04) 1.3 (0.07)

Overall effects Treatment Bone

P < 0.01

n.s.

13.8 (0.2) 13.5 (0.2)

14.2 (0.2) 14.4 (0.2)

n.s.

9.4 (0.3) 8.4 (0.2)

9.4 (0.1) 9.1 (0.2)

P < 0.006

n.s.

0.8 (0.02) 0.7 (0.03)

0.8 (0.03) 0.7 (0.03)

P < 0.02

n.s.

7.1 (0.1) 7.0 (0.1)

7.3 (0.1) 7.4 (0.1)

4.7 (0.1) 4.4 (0.1)

4.9 (O.1) 4.7 (0.1)

aSee footnote to Table 3a for explanations.

n.s. P < 0.05

p < 0.001

p < 0.01 n.s.

360

A. Pointillart et al. Postprandial calcium carbonate in pigs

Bone Vol. 17, No. 4 October 1995:357-362

Table 3c. Metatarsal data--mechanical parametersa

MTE Failure load (N) Control CAA Stiffness (N/mm) Control CAA Bending moment (N x m) Control CAA

MTI

Overall effects Treatment Bone

620 508

(28) (14)

697 658

(24) (32)

P < 0.007

p < 0.001

51 45

(3) (2)

79 63

(4) (2)

P < 0.002

p < 0.001

p < 0.007

p < 0.001

5.4 (0.2) 4.4 (0.1)

6.1 (0.2) 5.8 (0.3)

aSee footnote to Table 3a for explanations. bones (whatever treatment). This could be done because there was no significant treatment x bone interaction. A correlation matrix was also determined. Results

The nutritional treatment had no effect on general performance since slaughter body weight (49.2 -+ 0.6 vs. 49.1 ± 0.8 kg) average daily weight gain (0.71 ± 0.01 vs. 0.70 - 0.01 kg), and feed-to-gain ratio (1.9 ± 0.03 vs. 2 ± 0.03) for the two group~ (control vs. CAA) were almost identical. The Ca and P balance data are shown in T a b l e 2. Nonmeal Ca (Ca given 5 h after the meal) was as well absorbed and retained as calcium incorporated into the meal. The urinary Ca for the two groups of pigs was identical. The manner in which the calcium was given had no effect on phosphorus absorption, but phosphorus retention (in terms of grams per day or retention:intake ratio) was lower (p < 0.01) in the nonmeal Ca group. The urinary P excretion of the nonmeal Ca pigs was nearly threefold greater (p < 0.01) than that of the controls. The bone measurements data are shown in Tables 3 (parts a-c) and 4. Dietary treatment did not change bone fresh weights (or bone volumes), but ash bone weights were lower (t9 < 0.004) in the nonmeal Ca pigs than in the controls (as were total Ca and P per bone, 0.02 > p < 0.01). Ash, Ca, and P to bone dry matter were unchanged, whereas ash, Ca, and P to bone volume (0.05 > p < 0.003) of the metatarsal bones were decreased in the pigs on nonmeal Ca. Failure load (p < 0.007), stiffness (p < 0.002), and bending moments (p < 0.007) of the metatarsal bones were decreased in the pigs on nonmeal Ca. Bone apparent density (metatarsals + tibia, overall mean: 115 +-- 1 controls vs. 113 ± 1 g/100 cm 3 CAA; p < 0.02) was decreased in the nonmeal Ca pigs. There was no effect of dietary treatment on the bending moment of the tibia (control 28.6 ± 1.5 N × m vs. CAA 28.1 ± 1.4 N × m). There were some differences between the external and internal metatarsal parameters: ash-, Ca-, and P-tobone dry matter, ash-to-bone volume, and bending moment (and the other mechanical parameters) were generally higher (0.01 > p < 0.001) in the internal than in the external metatarsals, whatever the treatment. The density of the tibia was greater (p < 0.05) than that of the two other bones. The retained P (grams per day) measured during the balance period was highly and significantly (p < 0.05) correlated with the total ash contents (r = 0.65) (Figure 2), with the total Ca (r = 0.58) and P (r - 0.56) contents (as well as with the sum of

Ca + P of both bones, r = 0.58, p < 0 . 0 5 ) o f metatarsal bones, and with the average bone density (three bones) (r = 0.56). The total ash content (r = 0.76) and ash-to-bone volume (r = 0.63) of the metatarsals were correlated (p < 0.01) with their bending moment. Urinary hydroxyproline excretion was similar for both groups (control, 200 + 33; CAA, 221 + 44 mg/day; NS). Plasma osteocalcin concentration was elevated (p < 0.01) in the nonmeal Ca group, whereas the other plasma parameters (Ca, P, and alkaline phosphatase) were unchanged (Table 5). Discussion

Ingesting calcium from CaCO 3 (limestone) 5 h after the meal does not appear to affect Ca bioavailability, or modify phosphorus absorption, but it does markedly reduce phosphorus retention and hence decreases bone mineralization. The finding that calcium absorption is not affected when calcium carbonate is given 5 h after the meal, but that absorption is altered when the calcium carbonate is given to fasting pigs 3 h before the meal 2° may indicate that gastric acidity is not the only factor involved in the absorption of insoluble calcium, and/or that the meal effect continues for at least several hours. Some authors have reported that hypochlorhydric subjects normally absorb Ca from insoluble sources given in a meal 14 and that partial inhibition of gastric HC1 secretion does not reduce the Ca absorbed from CaCO 3 also given with a meal. 4 CaCO3 is also well absorbed by atrophic gastritis patients, if it is given with a meal. 27 The critical points seem to be the gastroduodenal (and jejunal) pH and the " m e a l effect." CaCO 3 is readily soluble at pH 6 or below. ~7 This pH seems to be reached in achlorhydric and hypochlorhydric subjects (for review see Ref. 27) who are able to absorb insoluble Ca, provided it is given with (or within) a meal. Thus, according to Bo-Linn et al. 4 the bulk luminal postprandial pH of the jejunum is usually around 6, which they showed in vitro would release 37% of the calcium from an oyster shell (CaCO3) supplement within 2 h. Nevertheless, the " m e a l effect" is clearly important for the absorption of insoluble Ca by humans, rats, and pigs. m'2°'2~ In pigs, absorption efficiency dropped by at least 60% when CaCO 3 was given 3 h prior to the meal, 2° in contrast to the efficient Ca absorption by the pigs given CaCO 3 5 h postprandially. As in humans, digestion continues several hours after ingestion in pigs. ~6 The antral pH is quickly buffered at pH 5 by the meal, and then takes several hours to return to its initial value (pH 1), whereas duodenal (postpylorus) pH oscillates between 7 (prior to a meal) and 5; the average pH is around 6 during the hours following a meal (as is the jejunal pH). 22 This pH is very similar to that of the proximal small intestine in humans, where substantial amounts of soluble calcium are detected after a meal. 24 Although we did not meaTable 4. Apparent density (grams per 100 cubic centimeters) of the three bones

Control CAA

MTE

MTI

T

113 (1) 111 (1)

114 (1) 113 (1)

117 (1) 115 (1)

Overall effectsa Treatment Bone b P < 0.02

p < 0.001

MTE - external metatarsal; MTI = internal metatarsal, right hind leg; T = tibia, right hind leg. ~Two-way (treatment × bone) ANOVA, means and SEM, n = 7. There was no significant treatment x bone interactions. ~Yibia greater than both metatarsals, Newman-Keuls test: p < 0.05.

Bone Vol. 17, No. 4 October 1995:357-362

A. Pointillart et al. Postprandial calcium carbonate in pigs

5 0

.C

0

0

[] 0

0

C3

0

o4 C o

,o

0 []

0

3

h

3

g

~,

retained phosphorus, g/day Figure 2. Relationship between daily retained phosphorus measured during the balance study (x-axis: grams per day) and the total ash contents of metatarsal bones (average of external and intemal bones; y-axis: grams) y = 0.421x + 2.371 (r = 0.648, p < 0.05).

sure the gastrointestinal pH of the pigs given CaCO 3 5 h postprandially, their small intestine pH levels were probably sufficiently acidic 2"3 to dissolve CaCO3, thus allowing Ca absorption to be as high as in those given the limestone incorporated into the meal. In contrast, the low Ca absorption reported in our previous experiment2° for pigs given CaCO 3 3 h prior to the meal might be due to a reprecipitation of CaCO 3 dissolved in the stomach within the small intestine, where the pH was probably near 7,2'24 since these animals were fasting. Stimulation of active, Vitamin D-dependent, duodenal Ca absorption might be also involved in the pigs given the low Ca meal. We did not measure plasma calcitriol in these pigs, but in our previous experiment2° the levels in the control pigs (53 --- 7 pg/mL) and nonmeal Ca-fed pigs (68 --- 9 pg/mL, p > 0.20) were similar. Slaughter plasma Ca and inorganic P concentrations were also similar for both groups of pigs (Table 5). This, at least, does not suggest changes in Ca/P hormonal regulation. Although the dietary Ca levels did not influence P absorption, giving both minerals simultaneously did interfere with the retention of phosphorus. This was already observed in our previous experiment, 2° since pigs given Ca separate from meals retained P better when the phosphorus was given at the same time as the Ca. In fact, retained phosphorus was nearly 20% lower in the pigs given the Ca source after the meal which provided the dietary P. Ca and P are required simultaneously for optimal bone formation and mineralization. This is emphasized by the bone density data, the bending moment, ash-to-bone volume, and total ash contents of metatarsal bones. They reflect the lower mineralization of the bones from pigs given the nonmeal Ca. This is also supported by the good correlations between daily retained P and some of the bone mineral contents or breaking load parameters. Thus, P was better retained and bones were better mineralized when Ca and P were both given with a meal. This confirms our previous observations in pigs in which Ca and P were better retained and bones tended to be better mineralized when Table 5. Plasma parameters

Calcium (mg/100 mL) Phosphate (mg/100 mL) Alkaline phosphatase Total (IU/L) Bone (l~g/L) Osteocalcin (ng/mL)

Control

CAA

10.3 ± 0.2 8.1 ± 0.3

10.1 ± 0.2 7.9 ± 0.2

74 ± 5 46 ± 4 332 ± 20

71 _+ 6 39 + 5 417 ± 19a

ap < 0.01 (Student's t-test), means • SEM (n = 7).

361

Ca and P were given simultaneously, and preferably with a meal. 2° The urinary P excretion varied inversely with the efficiency of P retention, as previously described, z° The plasma osteocalcin concentration in pigs on nonmeal Ca was about 25% elevated. Increased osteocalcin has also been found in women given a low Ca diet. ts Moreover, we have also recorded increased plasma osteocalcin coexisting with decreased bone mineral contents in another experiment in pigs fed a Cadeficient regimen (unpublished data). Thus, the unavailability of minerals to bone led to osteopenia in pigs, a disorder often associated with elevated plasma osteocalcin in humans. 6 Both plasma BGP and the bone-isoenzyme alkaline phosphatase (BALP) are generally considered to be correlated with bone formation. 5'23 However, plasma B-ALP and total ALP were unchanged in the CAA group. Furthermore, we found no correlation between plasma B-ALP (or total ALP) and plasma BGP. This dissociation has also been reported in some clinical studies t3't8 and in one experimental study on rats, 8 although high correlations have been found in oophorectomized and in pregnant women, and in osteomalacic patients. 6"t8"25 We have no explanation for these discrepancies at the present time. The unchanged urinary hydroxyproline excretion does not suggest that bone resorption was stimulated in the pigs exhibiting lower bone scores. This already occurred in our previous study on the "meal effect. ''2° However, in contrast to this study, ash-to-dry matter was not altered in the pigs given Ca 5 h after the meal which provided the phosphorus. Thus, bone tissue was probably normally mineralized where the bone was formed, but less total bone was formed, as demonstrated by the lower bone density, lower ash-to-bone volume, and lower bone ash weights. In conclusion, calcium carbonate (as limestone) is well absorbed when given a few hours after a meal, but simultaneous distributions of Ca and P are required for the best mineral retention and the best bone scores.

Acknowledgments: This work was supported by the AlP "Nutriage" from INRA, managed by G. Pascal. We thank J. C. Bemardin and D. Besnard for animal care and H. Roy for help in making the diets. We also thank G. Charpigny for advice on adapting the osteocalcin RIA, and O. Parkes for editing the text.

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Date Received: December 9, 1994 Date Revised: April 18, 1995 Date Accepted: June 13, 1995