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The Journal of Nutrition Vol. 129 No. 1 January 1999,
pp. 170-173
Purdue University, West Lafayette, IN 47907-1264, * Creighton University, Omaha, NE 68178 and Georgetown University Medical Center, Washington, DC 20007
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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Calcium absorption is thought to occur only if calcium is in a soluble or dissociated form, although experimental evidence is lacking. The intestinal absorption of calcium oxalate, a small, neutral and virtually insoluble calcium salt, was elucidated in the whole body of awake rats. Suspensions of 45Ca ascorbate, 14C-oxalic acid and doubly labeled 45Ca-[14C]-oxalate were given by gavage to separate groups of rats. Following dosing, blood samples were drawn for up to 240 min through a previously inserted intravenous catheter. Serum was assayed for radioactive tracers, and data were then plotted as fraction of dose over time. Calcium absorption was 15% [with a loading of 0.3 mmol (15 mg) calcium], oxalic acid absorption was 22% and Ca-oxalate absorption was <2%. Appearance of 45Ca from calcium ascorbate and 14C from oxalic acid differed, whereas 45Ca and 14C from doubly labeled Ca-oxalate had identical serum appearance profiles. Therefore, we conclude that calcium oxalate was absorbed intact. Addition of excess, unlabeled calcium to the doubly-labeled calcium oxalate did not alter the relationship of the serum level of the two tracers, confirming absorption of calcium oxalate as the intact salt. Thus, calcium bound as a small, neutral, calcium salt such as calcium oxalate does not have to be dissociated prior to absorption. Possibly other small compounds would be similarly absorbed. These results alter our current understanding of calcium bioavailability from foods and therapeutic agents.
KEY WORDS: calcium absorption · calcium bioavailability · calcium oxalate · rats
Previously we proposed that calcium oxalate is absorbed as an intact complex, probably by a paracellular route (Weaver and Heaney 1991 Recent experiments on rats provided additional evidence for the hypothesis that small, neutral calcium complexes can be absorbed intact. Andon et al. (1993) Although these experiments show evidence that strongly supports intact absorption of small neutral salts, direct experimental proof is needed to further test the hypothesis that calcium absorption does not always require prior dissociation. This manuscript discusses the evidence of absorption of doubly labeled 45calcium- [14C]-oxalate salt as an intact complex in rats, by comparing calcium-oxalate kinetics with models developed for calcium and oxalic acid in the companion paper Hanes et al. (1999) Animals.
Young adult male Sprague-Dawley rats, age 56-68 d and weighing 175-200 g, were purchased from Harlan Industries (Indianapolis, IN). Animal care and use procedures during all phases of the study were approved by the Purdue University Animal Care and Use Committee. Upon receipt, rats were individually housed in suspended, stainless steel cages in an environmentally controlled room (19-26°C; relative humidity 40-70%; 12 h light-dark cycle). The rats were given free access to nonpurified diet (Purina Rat Chow #5001, Purina Company, St. Louis, MO), containing 10 g calcium/kg, and distilled water.
Surgery.
Prior to all experimentation, rats underwent surgery to catheterize the jugular vein. This surgery technique allowed direct measurement of tracer absorption in the blood over time for the intact awake rat. Rats weighing approximately 230 g were anesthetized with a mixture of 90 mg ketamine/kg and 10 mg xylazine/kg given intraperitoneally. Silastic catheters were then inserted into the left jugular vein through a midline neck incision (ID = 0.05 cm), exteriorized out the back of the head and plugged. Lines were flushed daily with isotonic saline containing 0.1 U heparin /L to prevent clotting. The 0.09 mg of the analgesic butorphanol tartrate/kg was given intramuscularly immediately after surgery and at the beginning of the dark cycle on that day. Rats were given free access to nonpurified diet before and after surgery until no less than 6 h prior to experimentation. Rats were allowed to recover to at least their presurgery body weight before an experiment was performed.
Absorption of calcium oxalate.
Doubly-labeled calcium oxalate was precipitated from 45CaCl2 and [14C]-oxalic acid. The insoluble salt containing 0.38 mmol Ca and 0.56 mBq 45Ca and 0.56 mBq 14C (Amersham, Arlington Heights, IL) was given by gavage to rats (n = 9) in a deionized water suspension after Kinetic modeling.
The serum data obtained from the experiments following oral or intraveneous administration of calcium oxalate were analyzed by kinetic modeling using the SAAM (Simulation Analysis and Modeling) program as described in the companion paper Hanes et al. (1999) Statistics.
Group means for absorption were compared by Student's t test with a P value of 0.05 to determine significant differences (Mendenhall 1983 To contrast calcium oxalate absorption profiles with the respective ions, the serum profiles from 45Ca and [14C]-oxalic acid are compared in the same figure (Fig. 1). The appearance in serum of administered equimolar amounts of [14C]-oxalate and 45Ca ascorbate following gavage are clearly different. Calcium was absorbed to a similar extent as oxalic acid but remained in the blood throughout the study period, whereas the fraction of oxalate that was absorbed was quickly cleared from the serum (Table 1).
The pattern of serum appearance of 45Ca-[14C]-oxalate in the serum of all rats was most consistent with that of soluble 45Ca at the same molar load and the passive mode of transport via the paracellular route. The serum profiles of both soluble 45Ca and insoluble 45Ca-[14C]-oxalate were markedly different from that of soluble [14C]-oxalic acid, which was absorbed and cleared more rapidly.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). In women, calcium absorption from intrinsically labeled calcium oxalate averaged 10 ± 4.3%, a third of that from milk on the same load (Heaney and Weaver 1989). Subsequently, the exchangeability of 45Ca from calcium oxalate when coingested with intrinsically labeled 47Ca milk was measured (Weaver and Heaney 1991). The complete absence of tracer exchange in the case of 45Ca oxalate suggested that no ionic dissociation occurred prior to absorption.
determined the bioavailability of calcium carbonate, a highly insoluble calcium salt (solubility constant, Ksp = 0.87 × 10
8 at 25°C), in adult male Sprague-Dawley rats by measuring whole body retention of 47Ca following oral administration of 6 mg calcium as intrinsically labeled 47CaCO3. Whole body retention of 47Ca at 72 h postdose showed that 32% of the calcium from calcium carbonate was bioavailable. Kanerva et al. (1993) repeated this experiment with a comparable calcium load but administered the dose intraduodenally, thus bypassing the stomach and possible gastric dissolution of the salt. Whole body retention of 47Ca from 47CaCO3 was 19.2%, or 60% of that which traveled through the entire gastrointestinal tract. Due to the neutral conditions of the intestine, 47CaCO3 would not have dissociated. Thus, a significant amount of the calcium from calcium carbonate administered orally was likely absorbed in the intestine as the intact salt. Furthermore, calcium absorption is normal in achlorhydric patients and not affected by the absence of acid secretion (Bo-Linn et al. 1984, Knox et al. 1991, Recker 1985). Calcium oxalate is even less soluble (approximately 0.04 mmol/L) than CaCO3, reducing further the opportunity for stomach dissolution.
. Calcium absorption kinetics from calcium oxalate have not been previously studied, likely because it has been presumed that all calcium salts required dissociation prior to absorption (Allen 1982).
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
6 h of food deprivation. This load of calcium is ~1/5 of the daily intake for a rat. To determine distribution of calcium oxalate, 0.025 mmol of 45Ca-[14C]-oxalate containing 0.56 mBq 45Ca were given intravenously (IV) through the catheter (n = 5). Following administration of tracer, 600 µL blood samples were drawn through the previously inserted catheter at each of six time points over a 4 h period. From each 600 µL blood aliquot, two 100 µL serum aliquots were obtained. The first aliquot was bleached and counted for total 14C and 45Ca radioactivity. The second aliquot was ashed at 600°C for 16 h, then assayed for 45Ca radioactivity. 14C radioactivity was determined by difference. Plots were made for each rat of fractional dose of both radiolabels per L serum vs. time in minutes.
. Absorption was calculated by the ratio of tracer that moves from the gastrointestinal tract into the plasma divided by the sum of calcium moving out of the gut compartment. To confirm absorption of the intact salt, in two rats the experiment was repeated with the addition of an equimolar amount of nonlabeled calcium ascorbate to the gavage dose. Ten-fold higher levels of radioactivity were used. Excess calcium would depress fractional absorption of a dissociated calcium moiety, but not of the nondissociated calcium bound to oxalate.
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RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View larger version (12K):
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Fig 1.
Comparison of [14C]-oxalic acid and 45Ca serum profiles in representative rats following gavage of 0.375 mmol [14C]-oxalic acid or 45Ca ascorbate, respectively. Data taken from Hanes et al. (1998). The curve for oxalate has been multiplied by 10 to compare the two on the same figure.
View this table:
Table 1.
Calcium, oxalic acid and calcium oxalate absorption in rats gavaged with equimolar (0.38 mmol) radiolabeled
test solutions1
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Fig 2.
Serum 45Ca and [14C]-oxalate profiles in a representative rat following intravenous administration of 0.025 mmol 45Ca-14C-oxalate. Data points were connected to show that neither label changed over the time of study.
). Data were fitted by adding a delay (Compartment 29, Fig. 4) of about 40 min before the site of absorption (Compartment 24), likely to be the small intestine. Compartment 27 represented a delay of about 170 min before absorption of calcium from a second site (Compartment 26) in the large intestine. Absorption of calcium from the intact salt (1 ± 1% absorption) was significantly less than from calcium ascorbate. After the salt appeared to dissociate, an additional 7% calcium was absorbed, but no further absorption of oxalate occurred from this site. Although no loss of IV calcium oxalate was observed, following oral administration, oxalate was removed from serum during the study period in ~1/2 the rats.
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Fig 3.
Serum 45Ca and [14C]-oxalate profiles in representative rats following gavage of 0.375 mmol 45Ca-[14C]-oxalate in the absence (A) and presence (B) of the addition of 0.375 mmol cold calcium ascorbate.
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Fig 4.
Model for plasma appearance of 45Ca-[14C]-oxalate in rat serum following gavage of 0.375 mmol 45Ca-[14C]-oxalate, indicated by *(G). Compartment 21 represents plasma. Compartment 29 represents an initial delay prior to absorption from compartment 24. Compartment 24 likely represents the upper intestine and compartment 26 the lower intestine, with compartment 27 a delay compartment between the two absorption sites. Values next to the arrows are transfer coefficients (fraction/mL), mean ± SD. Volume of distribution was 50 mL.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
).
). Estimated absorption ranged between 1.7 and 4.0%. Oxalate excretion increased within 1-8 h after ingestion of the labeled meals. Urinary excretion patterns of 14C were similar regardless of form or presence of vegetable. However, meals were adjusted to 15 mmol calcium; thus, it is likely that calcium oxalate salt had formed in all meals prior to digestion. This would explain the apparent contrast to our rat data where oxalate absorption was much greater than calcium oxalate absorption. Although urinary excretion is an inexact approach for determining time of peak absorption, little 14C was found in the urine after 10 h, suggesting that absorption occurred prior to the colon. In patients with jejunoileal bypass surgery, [14C]-oxalate urinary excretion is elevated, presumably from colonic absorption (Hofmann et al. 1983). In the rat study, compartmental modeling showed Ca oxalate absorption from two sites in the intestine. In the colon, it appears that bacterial cleavage of the salt resulted in additional absorption of calcium, but not oxalate. Although calcium appearance in the serum diverged from oxalate after 170 min in all rats, future research at extended time points will be needed to affirm whether or not this is indeed physiological. Calcium absorption from the human colon was reported (Sandstrom et al. 1986). In contrast to observations in this study, oxalate was reported to be absorbed from human colon by passive diffusion (Binder 1974).
for calcium oxalate absorption and show that calcium does not have to be in soluble form to be absorbed in rats. Absorption of intact CaCO3 may explain the ability of subjects with achlorhydria to absorb calcium (Recker 1985). Moreover, absorption of intact complexes suggests possible therapeutic avenues using small calcium salts for individuals with impaired active calcium transport. More studies are required to examine candidate calcium salts in models with impaired active transport.
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FOOTNOTES |
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Manuscript received 13 January 1998. Initial reviews completed 3 April 1998. Revision accepted 9 September 1998.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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