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Chronic metabolic acid load induced by changes in dietary electrolyte balance increased chloride retention but did not compromise bone in growing swine

Department of Animal Sciences, University of Wisconsin, Madison 53706

¹ Correspondence:
1675 Observatory Dr. (phone: 608-263-4423; fax: 608-262-5157; E-mail:
crenshaw{at}calshp.cals.wisc.edu).

	Abstract

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The effects of chronic dietary acid loads on shifts in bone mineral reserves and physiological concentrations of cations and anions in extracellular fluids were assessed in growing swine. Four trials were conducted with a total of 38 (8.16 ± 0.30 kg, mean ± SEM) Large White x Landrace x Duroc pigs randomly assigned to one of three dietary treatments. Semipurified diets, fed for 13 to 17 d, provided an analyzed dietary electrolyte balance (dEB, meq/kg diet = Na⁺ + K⁺ - Cl^-) of -35, 112, and 212 for the acidogenic, control, and alkalinogenic diets, respectively. Growth performance, arterial blood gas, serum chemistry, urine pH, mineral balance, bone mineral content gain, bone-breaking strength, bone ash, and percentage of bone ash were determined. Dietary treatments created a range of metabolic acid loads without affecting (P > 0.10) growth or feed intake. Urine pH was 5.71, 6.02, and 7.65 ± 0.48 (mean ± SEM) and arterial blood pH was 7.478, 7.485, and 7.526 ± 0.006 for pigs fed acidogenic, control, and alkalinogenic treatments, respectively. A lower dEB resulted in an increased (P < 0.001) apparent Cl^- retention (106.6, 55.4, and 41.2 ± 6.3 meq/d), of which only 1.6% was accounted for by expansion of the extracellular fluid Cl^- pool as calculated from serum Cl^- (105.5, 103.4, 101.6 ± 0.94 meq/L (mean ± SEM) for pigs fed acidogenic, control, and alkalinogenic treatments, respectively. A lower dEB did not decrease (P > 0.10) bone mineral content gain, bone-breaking strength, bone ash, percentage of bone ash, or calcium and phosphate balance. In conclusion, bone mineral (phosphate) was not depleted to buffer the dietary acid load in growing pigs over a 3-wk period.

Key Words: Bones • Calcium • Chlorides • Electrolytes • Phosphates • Pigs

	Introduction

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Physiological mechanisms invoked to compensate dietary acid loads are not well established in growing swine. Typical corn-soybean meal diets fed to swine in the United States induce a metabolic acid load, exemplified by an acidic urine (Crenshaw, 1992). In humans, approximately 60% of dietary acid load is thought to be compensated by renal ammonium ion excretion and the remainder by renal phosphate excretion (Pitts, 1964). Chronic dietary acid loads in lambs and rats decreased bone formation and increased bone resorption (Kraut et al., 1986; Damir et al., 1991). Upon resorption, bone mineral can buffer metabolic acid loads. Resorption of bone hydroxyapatite consumes 9.2 mol of acid for every 10 mol of Ca⁺⁺ released (Brosnan and Brosnan, 1982). The resorption of HPO₄⁼ from hydroxyapatite buffers acid by consumption of a proton (H⁺) under physiological conditions and excretion ofH₂PO₄^-. Resorption of bone also releases stored carbonate as an additional buffer. If the skeleton contributes to buffering of a dietary acid load by increased resorption of bone, feeding a more alkalinogenic diet may conserve mineral reserves and help reduce lameness in swine.

Van der Wal et al. (1986) evaluated the effect of dietary electrolyte balance (dEB) on clinical lameness and radiological osteochondrosis lesions in swine. Clinical symptoms of lameness increased, but the severity of radiological osteochondrosis lesions was reduced in pigs fed diets with dEB of 231 and 261 meq/kg of diet for grower and finisher pigs, respectively, compared with pigs fed diets with alkaline loads of 296 and 300 meq/kg diet. Patience and Chaplin (1997) found Ca⁺⁺ balance decreased in swine fed barley-soybean meal diets with a dEB of -20 meq/kg of diet compared with animals fed diets with 104 and 163 meq/kg. The purpose of this experiment was to assess shifts in bone mineral reserves and concentrations of extracellular cations and anions in response to a chronic dietary acid load in growing swine.

	Materials and Methods

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The experiment included four trials with a total of 38 approximately 4-wk-old (Large White x Landrace x Duroc) pigs (Trial 1, n = six gilts; Trial 2, n = 12 barrows; Trials 3 and 4, n = 10 barrows each). Pigs were housed at the University of Wisconsin-Madison Livestock Laboratory and the experimental protocol was approved by the University of Wisconsin-Madison Research Animal Resource Committee. Pigs were weaned at either 2 (Trial 1 and 2) or 3 (Trials 3 and 4) wk of age into pens with four to five pigs per pen for a 1- to 2-wk pretrial adaptation period. The adaptation period was used to allow pigs to recover from the postweaning lag period. During the last 2 to 3 d of the adaptation period, pigs were placed in individual (1.2 x 1.2 m) pens. During the adaptation period, the pigs were allowed ad libitum access to a corn-soybean-whey based diet with 20% protein, 0.8% Ca⁺⁺, and 0.7% total phosphorus.

The pigs were randomly allotted within trial to one of three treatment groups. Treatments involved diets that were either acidogenic, control, or alkalinogenic based on the cation-anion balance. A semipurified diet (Table 1) was formulated to control quantity and availability of minerals. Treatment diets were formulated to meet or exceed requirements for 10-kg swine (NRC, 1998). The nutrient concentration was adjusted to account for the higher energy density of the diet. Casein was added at a concentration to meet the first-limiting amino acid (methionine + cysteine) requirement. In earlier work with similar diets (Golz and Crenshaw, 1990), pig growth responses to dietary cation-anion balance were attributed to K⁺ and Cl^-, but not Na⁺ concentrations. Thus, in the current experiment, changes in cation-anion balance were achieved by adjusting K⁺ and Cl^- levels in the diet using calcium chloride, calcium carbonate, potassium chloride, potassium citrate, sodium chloride, and sodium citrate while Na⁺ concentrations were held constant. Pigs were allowed ad libitum access to the treatment diet and water. For Trials 2 to 4, pigs remained in their (1.2 m²) pens until the final 4 d of the treatment period (collection period), and then they were placed in individual (21.5 x 97 cm) crates for collection of urine and fecal excreta. Excreta were not collected from pigs in Trial 1. Duration of trials was 13, 15, 17, and 15 d, respectively for Trials 1 to 4. Pigs were weighed at the beginning and end of the trial, and for Trials 2 to 4 at the beginning of the collection period. Feed intake was recorded for the entire treatment and collection period. Feed was subsampled each time feed was added during the collection period.

Surgical Procedure

Arterial blood gas concentrations were determined on pigs in Trials 2 to 4 during the collection period. One to five days before pigs were placed in metabolism crates, a catheter was placed in the femoral artery using a procedure similar to that described by Jackson et al. (1972). Pigs were anesthetized as described above, the right lumbar and inguinal areas clipped, scrubbed, and draped. Pigs were positioned in dorsal recumbency with hind limbs abducted. An approximately 5-cm incision was made in the right inguinal area. The femoral artery was exposed by blunt dissection of overlying fascia. The artery was ligated with 2-0 chromic gut approximately 1 cm distal to the anterior femoral artery, an adjustable ligature was placed just distal to the anterior femoral artery, and an incision was made between the ligatures with an 18-gauge needle. A sterile catheter approximately 75 cm in length was made from Tygon microbore tubing, 1.02 mm i.d., 1.78 mm o.d. (Norton Performance Plastics, Akron, OH). The catheter was beveled on one end to aid in insertion into the vessel, filled with 100 IU heparin/mL of normal saline, and sealed on the other end to prevent blood loss during insertion. The catheter was advanced cranially 10 cm into the femoral artery, the adjustable suture was tied, and another suture was placed around the femoral artery and catheter to secure the catheter in place. The catheter was sutured to the aponeurosis of the external abdominal oblique muscle for additional security. The pig was then rolled into left lateral recumbency, a small incision was made in the skin of the aseptically prepared right lumbar area, and a bitch urethral catheter was passed subcutaneously into the inguinal incision. The femoral artery catheter was inserted into the urethral catheter and pulled subcutaneously through the lumbar incision. Both incisions were then closed. The seal was cut from the femoral artery catheter, a blunted 18-gauge needle attached to a three-way stopcock was inserted into the catheter tubing and secured with wire. The portals of the stopcock were sealed with intermittent infusion plugs, and the stopcock assembly and external tubing of the catheter were placed into a canvas pouch loosely sutured and glued adjacent to the lumbar incision. Catheter placement in pigs used in Trial 2 was problematic (see footnote "a" of Table 2), partially due to pig (artery) size. Fewer problems were experienced with the larger pigs used in Trial 3 and 4. Pigs were given 250 mg of ampicillin i.m. in Trial 2 and 500 mg of ampicillin i.m. in Trials 3 and 4 to prevent infection. Catheters were maintained by daily flushing with 1 mL of 100 IU heparin/mL of normal saline.

On d 0 of the collection period, a male external catheter (Cida, Topeka, KS) was secured over the prepuce of the pig and attached to Tygon tubing extending into a 4-L container for urine collection. Urine was collected every 24 h. Throughout the collection period, urine collection containers were surrounded with ice to prevent loss of bicarbonate, ammonia, and bacterial release of urea nitrogen. In preliminary tests, no changes were found in urine pH or total N between a fresh urine sample and the same sample after 24 h of storage in a vessel surrounded by ice.

For fecal collection a 16.5 x 25.4-cm colostomy bag (Bard Patient Care Division, Murray Hill, NJ) was glued to a 10 x 10 cm skin barrier (Pfizer Hospital Products Group, Inc., Guelph, Ontario, Canada) cut with a center hole to fit around the tail and anus of the pig. Bags were changed as needed during the 4-d collection period.

In Trials 3 and 4, pigs were electrically stunned and exsanguinated prior to the final BMC scan. After the scan was complete, the right and left fifth ribs and humeri were dissected free of soft tissue, sealed in plastic bags to maintain moisture, and stored at -10°C until used for mechanical tests. Crenshaw et al. (1981) found mechanical properties of the femur, humerus, and rib were more sensitive to alterations in dietary Ca⁺⁺ and phosphate concentrations in pigs approximately 2 mo of age than metatarsal, metacarpal, or vertebrae. The femur was not used since there was uncertainty as to whether all pigs continued to bear weight normally on their hindlimbs once the femoral artery catheter was placed.

Blood gas samples were taken during 3 d of the collection period in Trial 2, and on 2 d of the collection period in Trials 3 and 4. The variation among pigs within treatment was greater than individual pig variation from day to day; therefore, little additional information was gained by sampling the third day. Blood samples for serum chemistry analysis were taken during 2 d of the collection period when blood was sampled for blood gas determinations.

Sample Analysis

Arterial blood was analyzed for pH, pCO₂, pO₂, bicarbonate (HCO₃^-), and base excess (BE) using a blood gas analyzer (248 pH/Blood Gas Analyzer, Ciba Corning Diagnostics Ltd., Halstead, Essex, U.K.). For blood gas determination, 0.5 to 1.0 mL of blood was drawn into a 1-mL, heparin-coated syringe. Any air bubbles were immediately tapped out, and the syringe was sealed and placed into an ice bath until analysis. All blood gas samples were analyzed within 30 min of collection.

Serum Na⁺, K⁺, Cl^-, total calcium, phosphate, and Mg⁺⁺ were analyzed with a blood chemistry analyzer using dry-slide technology (Vitros 250, Ortho-Clinical Diagnostics, Inc., Rochester, NY). Analysis of Na⁺, K⁺, and Cl^- were done by potentiometry, and total calcium, phosphate, and Mg⁺⁺ by colorimetric analysis. For blood chemistry analysis, 3 mL of blood was collected into vacutainer tubes without anticoagulant; samples were allowed to clot for approximately 1 h, centrifuged for 10 min at 1,000 x g, the serum separated, and refrigerated until analysis.

Daily urine collections were analyzed immediately for pH and HCO₃^-. A composite based on a weighted percentage of daily urine samples was made for analysis of ammonia N, total N, Na⁺, K⁺, Cl^-, Ca⁺⁺, phosphate, and SO₄⁼. Daily urine collections were weighed, mixed, and pH determined using a pH meter with temperature correction (ORION model 811, Cambridge, MA). Urine was sampled for HCO₃^- analysis, and aliquots (based on a weighted percentage of daily excreta) of fresh and acidified urine saved for analysis. Urine was acidified with 0.2 mL of concentrated H₂SO₄/30 mL of urine, which decreased sample pH to <2 to prevent bacterial release of urea N and loss of ammonia. A collection period composite was made of the daily fresh and acidified urine aliquots based a weighted percentage of urine collected each day during the collection period. Average urine pH over the 4-d collection period was calculated from the total hydrogen ion excretion in each of the daily collections. Urine was stored refrigerated until analysis.

Urine HCO₃^- was determined by barium carbonate precipitation (Leng and Leonard, 1965). Ten milliliters of fresh urine was injected into 1.5 mL of 30% tricarboxylic acid in the bottom of a McCartney bottle. The CO₂ released from the urine was trapped in a test tube containing 0.5 mL of 2 N NaOH within the McCartney bottle. The BaCO₃ was precipitated with the addition of 0.5 mL of 2 M NH₄Cl and 1.5 mL of 0.4 M BaCl₂ to the test tube after the bottles had been shaken for at least 48 h. The precipitate was filtered, dried, and weighed. Recovery for KHCO₃-spiked samples averaged 100.6%.

A composite was made of the fecal samples collected during the 4-d collection period. Fecal and diet composites were analyzed for total N, Na⁺, K⁺, Cl^-, Ca⁺⁺, phosphate, Mg⁺⁺, and DM. Feces and diet samples were stored frozen at -10° C.

Calcium, Mg⁺⁺, Na⁺, and K⁺ were determined using atomic absorption spectrophotometry (Perkin-Elmer 2280, Perkin-Elmer Corp., Norwalk, CT). For Ca⁺⁺ and Mg⁺⁺ analysis, all samples were digested using a 1:3 ratio of 60% perchloric acid:70% nitric acid in Teflon tubes at 90°C for 90 min then 190°C for 210 min. Digests were assayed in a 1% lanthanum and 5% HCl solution. Sodium and K⁺ were analyzed in a 1,500 ppm of cesium chloride solution using the perchloric-nitric acid digested samples for feces and diet, and the fresh composite for urine analysis. Recoveries for the perchloric-nitric acid digest and Ca⁺⁺, Mg⁺⁺, Na⁺, and K⁺ assays were 101.3, 97.3, 98.5, 101.4, and 99.6%, respectively, for samples spiked with atomic absorption reference solutions.

Total N, ammonia N, phosphate, and Cl^- were analyzed by spectrophotometry (Gilford Spectrophotometer 260, Gilford Instrument Laboratories, Inc., Oberlin, OH). A phenol-hypochlorite assay (Broderick and Kang, 1980) was used to determine total and ammonia N. Total urine, fecal, and diet N were assayed using samples digested in a sulfuric acid-hydrogen peroxide mixture (Brotz and Schaefer, 1984). Urine ammonia N was assayed using the acidified urine composite. Average assay recovery was 101.0% for (NH₄)₂SO₄-spiked samples, and average digest recovery was 101.6% for L-tyrosine spiked samples. A molybdovanadate assay (AOAC, 1980) was used to determine phosphate. Sulfuric acid-hydrogen peroxide digests of samples were used for fecal phosphate analysis. Perchloric-nitric acid digested diet samples and acidified urine composites were used for diet and urine total phosphate determinations. The ratio of monobasic (H₂PO₄^-) and dibasic(HPO₄⁼) phosphate concentration in each urine sample was calculated using the Henderson-Hasselbalch equation: pH = pK_a + log [(HPO₄⁼)/(H₂PO₄^-)], with an assumed pKa of 6.8 and the measured urine pH. Absolute amounts of H₂PO₄^- and HPO₄⁼ excreted in urine were determined using the calculated ratio and the analyzed total phosphate. Recoveries for the phosphate assay averaged 102.1% for samples spiked with KH₂PO₄. A colorimetric ferric ammonium sulfate-alcoholic and mercuric thiocyanate assay (Jeffrey and Hutchinson, 1981) was used to determine Cl^-. Diet and feces were first fused with alcoholic potassium hydroxide at 550°C for 6 h then extraction of Cl^- using hot deionized water. A fresh urine composite was used for the urine Cl^- analysis. Recoveries for the fusion and assay were 102.2 and 106.1%, respectively, in samples spiked with NaCl.

Urine SO₄⁼ was measured by a barium sulfate precipitation. Five milliliters of a fresh urine composite was acidified with 0.5 mL of 12 N HCl and heated to 80°C for at least 30 min to remove HCO₃^-, and then BaSO₄ was precipitated with the addition of 1.5 mL of 0.4 M BaCl₂. The precipitate was filtered, ashed at 525°C for 2 h, and weighed. Average recovery was 101.8% for a Na₂SO₄ standard solution.

Bone mechanical properties of the fifth ribs and humeri were determined from a three-point bending test and the area moment of inertia (MI) measurements (Crenshaw et al., 1981). The three-point bending tests to determine the force and deformation at the yield and maximal points on a load deformation curve were conducted with a materials testing machine (MTS Bionics 858). Load was applied at 5 mm/min. Bones were thawed to room temperature and removed from plastic bags before testing. Ribs were loaded from the medial side, whereas humeri were loaded from the lateral side. After mechanical testing, a transverse section, approximately 2-mm thick, was cut at the midpoint of load application using a diamond band saw (model C, Gryphon Corp., Burbank, CA). Broken bone cross-sections were glued together before image collection. Bone cross-sections were positioned on a lighted table, the image viewed through a camera attached to a monitor, and traced three times each with Optimus 3.1 software. The area MI was calculated from the image using the SLICE program (Nagurka and Hayes, 1980). Average coefficient of variation for repeated MI measurements on the same section was 0.65%. After cutting, the remaining bone sections were extracted with ether for 2 wk and then dried for 16 h at 110°C to determine the fat-free dry bone weight. Bones were ashed at 750°C for at least 8 h to determine ash weight. An ashing temperature of 750°C for at least 8 h was required in our laboratory for complete removal of carbon while allowing 99 to 101% recovery of Ca⁺⁺ from hydroxyapatite (Schneider et al., 1997). The percentage of ash was calculated as ash weight per unit of dry fat-free weight. The average of right and left bones from individual animals was used in final statistical analysis.

Statistical Analysis

Data were analyzed for main effects of treatment, trial, and treatment x trial interactions using the GLM procedure of SAS (SAS Inst., Inc, Cary, NC). Type III sums of squares were used to appropriately handle unequal subclass numbers. The sum of squares for treatment x trial was pooled into the error since no significant treatment x trial interactions were detected. Data were assessed for normality using the UNIVARIATE procedure of SAS and for equal variance using the HOVTEST=BF procedure of SAS. Differences were considered significant if P < 0.05. Contrasts for linear and quadratic effects were used to detect differences among dietary treatments.

	Results

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Changing dEB from -35 to 212 meq/kg diet did not alter pig growth performance (Table 2). Over the entire trial period, ADG was not affected by dietary treatment; but as expected, ADG of pigs during the 4-d collection period was higher than ADG over the entire trial. Average daily feed intake and feed efficiency likewise were not different among dietary treatment groups. Differences in growth and feed intake were detected among trials (Table 3), but no trial x treatment interactions were detected. The lack of differences in growth and feed consumption among pigs confirms the ability of pigs to compensate for dietary loads of acids or bases and simplifies interpretation of retention data. Analyzed mineral composition of diets was consistent with calculated composition (Table 1) except for minor differences in Cl^- and Ca⁺⁺ contents. The analyzed Cl^- content of the acidogenic diet was slightly higher than the formulated value, resulting in a wider range in dEB than calculated. The analyzed Ca⁺⁺ content of all diets was less than formulated values, but tended to increase as dEB decreased. This disparity is consistent with an instability of CaCl₂ and lower Ca⁺⁺ content in the CaCO₃ source than assumed. Ingredients were not analyzed. The magnitude of differences in dietary Cl^- and Ca⁺⁺ are not sufficient to compromise the experiment objective.

View larger version (39K): [in this window] [in a new window]   Figure 1. Summation of urinary cations (+) and anions (-) excreted by pigs fed either acidogenic, control, or alkalinogenic diets. Values are based on averages of nine pigs per dietary treatment group. Urine excretion values are shown in Table 5 except for ammonium (NH4+), undetermined anion (UDA-), bicarbonate (HCO3-), and sulfate (SO4=). The values for UDA- were calculated as the difference between cations and anions. The amounts of monobasic (H2PO4-) and dibasic phosphate (HPO4=) were calculated as described in the methods section.

View larger version (40K): [in this window] [in a new window]   Figure 2. Apparent retention and urinary and fecal excretion of minerals and nitrogen are expressed as a percentage of responses by pigs fed the control diet. Each bar within the mineral groups represents values from pigs fed acidogenic (Ac), control (C), or alkalinogenic (Al) diets, respectively. Urinary nitrogen excretion is shown as urea and ammonium.

Serum Cl^- reflected the change in Cl^- retention and increased as dietary levels and retained Cl^- increased. Changes in serum levels would be expected with a change in Cl^- retention since Cl^- is primarily an extracellular anion. However, based on calculations of the total extracellular pool, the change in serum Cl^- could only account for 1.5% of the difference in the quantity of Cl^- retained over the treatment period between acidogenic and alkalinogenic treatment groups. Serum Cl^- would need to change by approximately 250 meq/L between acidogenic and alkalinogenic treatment groups over the entire treatment period (compared to the actual change of 4 meq/L) to account for the change in Cl^- retention. Calculations were based on assumptions that 60% of BW is water and 40% of body water is in interstitial fluid and serum. Chloride concentration in serum was assumed to be in equilibrium with interstitial fluid, and no volume changes were assumed in body fluid compartments due to treatment. Patience and Chaplin (1997) concluded that increased serum Cl^- was associated with increased metabolic acid load, but was not affected by dietary Cl^- level.

Decreased dEB resulted in an increase (P < 0.05) in serum total calcium (Table 4). Although the analyzed Ca⁺⁺ content of diets (Table 1) differed from formulated values, such small differences in dietary concentrations are unlikely to induce differences in total serum calcium independent of the dEB differences in the diet. Lemann et al. (1967) and Newell and Beauchene (1975) reported total serum calcium decreased with metabolic acid loads. However, in other experiments, no changes were detected in serum calcium (Lemann et al., 1966; Barzel and Jowsey, 1969; Damir et al., 1991). Urine Ca⁺⁺ excretion increased (P < 0.0001) with decreased dEB (Table 5) an observation consistent with increased acid loads (Lemann et al., 1967; Newell and Beauchene, 1975; Patience and Chaplin, 1997). Analyzed Ca⁺⁺ intake tended (P < 0.10) to increase with decreased dEB. Neither fecal Ca⁺⁺, retained Ca⁺⁺, nor apparent absorption of Ca⁺⁺ amounts differed among dietary treatment groups.

Skeletal mineral reserves offer ample resources of counter-ions as a site for retention of the excess Cl^-. Whether these mineral reserves are compromised by storage of excess Cl^- is more difficult to access. Bone mineral content measurements determined by repeated scans of pigs were used to measure bone mineral content gain (Table 6). Bone mineral content gain doubled over the treatment period (15 d); however, BMC gain was not affected by dietary treatment. Assuming 99% of the body Ca⁺⁺ is located in skeleton tissue and that hydroxyapatite is composed of 39% Ca⁺⁺, the daily gain in body Ca⁺⁺ over 15 d [(11.2 g of BMC/0.99) x 0.39 = 4.4 g of Ca⁺⁺/d] is less than the gain in Ca⁺⁺ predicted from apparent Ca⁺⁺ retention data based on urine and fecal collections (174 mmol/d x 40.08 = 6.97 g of Ca⁺⁺ retained/d). These differences are not surprising since retention estimates were made over the last 4 d of the 15-d trial, but BMC gain was based over the entire 15-d period. Over the 15-d trial, pigs consumed an average of 6.82 g of Ca⁺⁺ each day, resulting in a 66% efficiency of Ca⁺⁺ use based on BMC scans. During the collection period, efficiency of Ca⁺⁺ use was 94%, an unreasonably high efficiency. The contrast between efficiency calculations based on excreta collections and BMC scans illustrate the inherent errors associated with apparent retention values based on short-term collection periods.

	Discussion

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In the current experiment, diet-induced acid loads were related the Cl^- content of the diets. The most apparent impact of feeding the acidogenic diets was the excess retention of Cl^- (Figure 2). Very little research has focused on responses to dietary Cl^- content. Golz and Crenshaw (1991) found Cl^- retention increased with increased Cl^- intake in young swine, but the increased Cl^- retention was not related to dEB. However, Patience and Chaplin (1997), found Cl^- retention decreased in swine with increased Cl^- intake. The loss of Cl^- was accounted for in urinary excretion. Adams et al. (1979) found no change in Cl^- balance when Cl^- or acid intake was altered in humans. Understanding how the pig responds to excess Cl^- will offer insights to adaptive responses to diet-induced acid loads. Because of physiological regulation of osmotic balance, excess dietary Cl^- must either be excreted with a counterbalance of cations or stored in a body compartment with a replacement of another anion. A logical compartment for storage of excess Cl^- is bone mineral.

If an increase in metabolic acid load was buffered by increased bone resorption or decreased bone formation, the following might be expected to decrease as the dEB decreased: retained Ca⁺⁺ and phosphate, BMC gain, bone ash, and bone-breaking strength. The current data do not support this type of bone involvement. Dietary treatments were imposed for only approximately 2 wk, but in pigs at this age, the treatment period was sufficient to allow BW and BMC to nearly double. Dietary differences, if present, should be detectable. The mineral balance data were collected only over the last 4 d of the treatment period. Balance data are subject to inherent errors involving variation in voluntary passage of excreta during a short-term collection period. These errors were apparently equally distributed across treatment groups, but may provide part of the explanation for the high percentage of Ca⁺⁺ apparently retained. Measurements of BMC by DEXA and calculation of Ca⁺⁺ retention over the entire trial period, as presented in the results, offer a more accurate estimate of Ca⁺⁺ retention than the balance data. However, both DEXA and balance estimates lead to the same conclusion. Gross decrements in bone mineral reserves were not induced by a dietary acid load as imposed in this experiment.

Although the results do not support a change in bone resorption (or a decrease in bone accumulation) as a source of buffer for the metabolic acid load, bone may be involved in storage of retained Cl^-. An often overlooked compartment of bone, the hydration shell, may store or release ions that buffer metabolic acid loads (Green and Kleeman, 1991). The hydration shell of bone contains an electrostatic field of ions and water surrounding hydroxyapatite crystals, composed primarily of Ca⁺⁺ and HPO₄⁼, with traces of Na⁺, K⁺, Mg⁺⁺, Cl^-, and carbonate and citrate ions. Chloride could possibly be exchanged in the hydration shell for other anions or accumulated with a counter-cation, without altering the quantity and composition of the bone hydroxyapatite crystal structure. Bone crystals provide a large surface area per unit of mass, approximately 100 m²/g bone mineral (Nichols and Nichols, 1956) and bind a hydration shell 1.9 times its volume (Green and Kleeman, 1991).

Bone contains 99% of the total Ca⁺⁺ in the body, 80 to 85% of the phosphate, 50 to 67% of the Mg⁺⁺, 30 to 35% of the Na⁺, 15 % of the Cl^-, 2 % of the K⁺, 80% of the carbonate, and 80% of the citrate (Crenshaw, 1991; Green and Kleeman, 1991; Broadus, 1996). The K⁺ and Cl^- in bone are freely exchangeable with plasma (Triffitt et al., 1968) suggesting a location in the hydration shell only. However, K⁺ may have a small unexchangeable fraction (Norman, 1963a; Hartsuck et al., 1969). Sodium, Mg⁺⁺, carbonate are less exchangeable than K⁺ and Cl^- suggesting some incorporation into the crystal phase of bone (Edelman et al., 1954; Norman, 1963a; Triffitt et al., 1968). Calcium and phosphate are the least exchangeable minerals in bone suggesting they are mainly located within bone crystals (Triffitt et al., 1968).

A proposed (Neuman and Ramp, 1971; Bushinsky et al., 1989) "bone membrane" isolates the bone hydration shell from extracellular fluid (ECF) and could allow a concentration difference between plasma and the hydration shell. Levitt et al. (1956), Nichols and Nichols (1956), and Triffitt et al. (1968) found that the concentrations of K⁺ and Cl^- were in excess of what would be expected based on bone fluid volume. Potassium concentration in the hydration shell was as much as 23 times the concentration of ECF. The concentration of Ca⁺⁺ in plasma is supersaturated in respect to the solubility of Ca⁺⁺ from hydroxyapatite in hydration shell of bone crystals (i.e., if plasma ionized Ca⁺⁺ were in equilibrium with the hydration shell of bone crystals, the concentration in plasma would be around 10^-4 M instead of 10^-3 M) (Talmage, 1996). The composition of the "membrane" has not been determined; however, live bone cells are apparently required to maintain the compartment (Bushinsky et al., 1989).

Chloride could be exchanged for other stored anions in bone, such as carbonate, citrate, and even phosphate. Levitt et al. (1956) found an increase in bone Cl^- when HCl or NH₄Cl solutions were infused into rats. However, in other experiments, no changes were detected in bone Cl^- (Bergstrom and Wallace, 1954) or the additional Cl^- could be accounted for by changes in ECF Cl^- and changes in red blood cell intracellular Cl^- (Banus and Katz, 1927, Swan et al., 1955; Tobin, 1956). Despite evidence for excess Cl^- in the bone hydration shell, it is uncertain if the concentration would increase with increased retention of Cl^- or metabolic acidosis in the pig.

Carbonate can be lost from bone during acid loads; however, in the experiments discussed below, bone Cl^- was not measured and no determination can be made relative to the potential exchange of Cl^- for carbonate. Metabolic acidosis resulted in efflux of carbonate from neonatal mouse calvariae cultures as soon as 3 h and continued for at least 48 h (Bushinsky et al., 1993). Bone carbonate decreased in dogs, rats, and guinea pigs given 15 to 20 meq of HCl or NH₄Cl/kg of BW each day for 3 to 48 d. Bone Ca⁺⁺ did not change, resulting in a decrease in the carbonate:calcium ratio (Irving and Chute, 1932; Burnell, 1971). Bone carbonate also decreased 1 d after acid treatment in rats. The decrease in carbonate could be attributed to the readily exchangeable pool (Bettice, 1984), but carbonate did not continue to decline with increased duration of acid treatment up to 8 d.

Urine citrate excretion decreased with increased dietary metabolic acid loads (Sakhee et al., 1983; Lemann et al., 1989). The exchange of citrate for Cl^- in bone is unlikely, unless citrate was metabolized to a much greater extent with metabolic acidosis.

Based on valence, phosphate could be depleted from bone and replaced by Cl^-. Phosphate loss as a replacement for Cl^- is not supported by the current data. Urine or fecal phosphate did not change in direct proportion to dEB. Retained phosphate actually tended (P = 0.12) to increase with decreased dEB.

Chloride could also accumulate in the hydration shell with a counter-cation, such as Ca⁺⁺, Na⁺, K⁺, or Mg⁺⁺. If Cl^- was stored as a counter-ion to Ca⁺⁺, the 65.5 mmol/d difference in Cl^- retention between pigs fed acidogenic and alkalinogenic treatments would require a 32.5 mmol/d of Ca⁺⁺ retention. Only 17.6-mmol/d of difference in retained Ca⁺⁺ was observed between acidogenic and alkalinogenic treatment groups, and these differences were not statistically significant. Bone Na⁺ decreased with metabolic acidosis (Levitt et al., 1956; Burnell, 1971; Bettice and Gambel, 1975) and bone K⁺ either decreased (Bergstrom and Wallace, 1954) or did not change (Norman, 1963b), making it unlikely that either Na⁺ or K⁺ would accumulate as a counter-cation to Cl^- in bone. Retention data in the current experiment do not support accumulation of Na⁺ or K⁺ as a counter-cation. The difference in retained Na⁺ was very small (4 mmol/d) compared with Cl^- retention, and K⁺ retention tended to decrease with increased acid load. Retained Mg⁺⁺ did not change with treatment. Thus, the counter-cation for approximately half of the retained Cl^- cannot be identified.

Other possible sites for Cl^- retention include soft tissues, but accumulation of Cl^- in these tissues is unlikely due to physiological regulation of osmotic equilibrium. Chloride concentrations in tendon, red blood cells (RBC), and muscle have also been studied in response to excess Cl^- loads. Tendon has a greater concentration of Cl^- than extracellular fluid and may be a potential site for accumulation of excess retained Cl^-. The Cl^- concentration in tendon did not change with injection of NH₄Cl or HCl (Levitt et al., 1956). The intracellular Cl^- concentration of RBC increased 11 (Levitt et al., 1956) and 22 mmol/L (Swan et al., 1955) with HCl or NH₄Cl injection, respectively; however, the fluid volume of RBC could only account for approximately 1.4 mmol/d of Cl^-, assuming a RBC volume of approximately 3% of body weight. Muscle Cl^- concentration did not change with NH₄Cl or HCl injection (Levitt et al., 1956). Thus, changes in soft tissue Cl^- concentrations are not expected, but were not assessed in the current experiment.

In conclusion, dietary changes in K⁺ and Cl^- concentrations resulted in a range of dietary acid loads in young, growing pigs. The results do not indicate that decreased bone formation or increased bone resorption are involved in buffering a chronic dietary acid load. Increased dietary Cl^- resulted in increased Cl^- retention, that could not be accounted for by changes in serum Cl^- concentrations. The potential for changes in ion concentrations in the bone hydration shell may accommodate the excess Cl^- retention. Ion concentrations in the bone hydration shell were not assessed in the current experiment, but this compartment offers the only major reserve available for storage of the excess Cl^- retained by pigs fed the acidogenic diet.

	Implications

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Typical corn-soybean meal diets fed to growing pigs impose an acid load that must be compensated by the animal. Concerns that these diets may deplete bone mineral reserves were not supported by the current study. Apparent mineral excretion and retention estimates based on fecal and urine collections did not reveal shifts in a counter-ion(s) to explain an apparent accumulation of Cl^- in pigs fed acidogenic diets.

Received for publication September 5, 2001. Accepted for publication September 17, 2002.

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Top Abstract Introduction Materials and Methods Results Discussion Implications Literature Cited

 


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