J Bone Miner Metab (2004) 22:561–568 DOI 10.1007/s00774-004-0524-0
© Springer-Verlag 2004
Effect of dietary-induced metabolic acidosis and ovariectomy on bone mineral density and markers of bone turnover Jennifer M. MacLeay1, Jerry D. Olson2, and A. Simon Turner1 1 2
Department of Clinical Sciences Colorado State University, 300 West Drake Road, Fort Collins, CO 80523, USA Independent Consultant, Fort Collins, CO, USA
Abstract Dietary-induced metabolic acidosis (DIMA) has been implicated as a significant confounder in the development of osteoporosis. Twenty-four mature ewes were randomly assigned to four groups of six sheep. Group 1 consumed a control diet (ND); group 2 consumed a normal diet (ND) and had ovariectomy (OVX), group 3 consumed a diet that induced metabolic acidosis (MA), without OVX, and group 4 consumed a diet that induced MA, with OVX. The study was conducted over 180 days and the sheep were maintained on the assigned diet throughout. Sheep were weighed and bone mineral density (BMD) was measured, using dualenergy X-ray absorptiometry (DEXA), on days 0 and 180. Serum bone alkaline phosphatase (BAP), urine deoxypyridinoline (DPD), and fractional excretions (FE) of Ca and P were determined on days 0, 90, and 180. Arterial blood pH was determined on day 180. Analysis consisted of a two-way analysis of variance for repeated measures with significance set at P 0.05. Body weights, serum BAP, and urine DPD were not influenced by either diet or OVX status. DIMA did significantly increase urinary FE of Ca and P and significantly decreased lumbar BMD and arterial pH. Arterial pH remained within physiologic normal limits. DIMA was a more potent cause of calcium wasting than OVX over the time frame of this study. Sheep appear to be sensitive to DIMA and will therefore be a useful animal model to study the influence of diet on the development of osteoporosis. The specific mechanisms through which DIMA exerts its influence are still unknown and are the subject of ongoing studies. Key words animal models · nutrition · osteoporosis · bone turnover markers · acid base · metabolic acidosis
Introduction Osteoporosis is a common and severe metabolic disease, such that the lifetime risk of having an Offprint requests to: J.M. MacLeay (e-mail:
[email protected]) Received: January 13, 2004 / Accepted: April 19, 2004
osteoporotic-related fracture is close to 40% in postmenopausal women residing within the United States [1]. Osteoporosis is a multifactorial disorder, being influenced by lifestyle, life-stage, genetic, and dietary factors. While all factors influencing the development of osteoporosis are important, dietary acid has gained attention recently. Several authors have implicated a dietary-induced metabolic acidosis as a contributing factor in the development of osteoporosis [2–5]. Metabolic acidosis induces increased calcium excretion without an adequate compensatory increase in calcium absorption from the gut, and therefore an overall decrease in total body calcium ensues. This is different from the rat model, where there appears to be a concomitant decrease in intestinal calcium excretion to compensate for increased urinary calcium loss in response to metabolic acidosis [6]. Therefore, dietaryinduced metabolic acidosis, in combination with other factors, appears to have a serious impact on the development of osteoporosis for susceptible individuals. Osteopenia induced by metabolic acidosis is typically thought of in the context of pathologic conditions such as chronic renal insufficiency; however, a relative excess of strong anions compared to strong cations in the diet may also induce metabolic acidosis in the face of normal renal function. This is because strong ions are absorbed directly from the intestine and therefore immediately impact acid-base balance. Depending on the composition of the diet, the imbalance may be slight or substantial, leading to a corresponding degree of metabolic acidosis [7]. Overall blood pH is maintained within the normal physiologic range due to the combined effect of renal excretion of excess ions and mobilization of calcium from bone to act as a buffer. The influence of diet on acid-base balance is accentuated in older human patients, where decreased or impaired renal function is common. However, the effect of diet on inducing metabolic acidosis and calciuria in individuals with normal renal function is well documented [4].
562
J.M. MacLeay et al.: Dietary metabolic acidosis in an ovine model
Total dietary cation-anion difference in the diet may be estimated by knowing the strong ion content of the diet as a whole or by calculating it from individual multiple feedstuffs. Multiple articles have examined and formulated methods for determining cation-anion balance in the diet [3,5,8–10]. Nevertheless, the most important strong ions in the diet are sodium, potassium, chloride, and sulfur, with magnesium and calcium playing a lesser role. Therefore, from dietary analysis, the overall dietary cation-anion difference (DCAD) may accurately, albeit roughly, be estimated by the equation: DCAD mEq/kg dry matter [Na K (0.15)Ca (0.15)Mg] [Cl (0.2)S (0.3)P]
The ions are measured in mEq/ kg dry matter (DM) through analysis of the diet. The aforementioned equation measures the acidifying capacity of a particular diet per kilogram of those feedstuffs. The influence of a particular diet on acid-base status depends on the quantity of a particular diet eaten. Therefore, when total daily intakes are known, the daily cation-anion difference consumed per day can also be calculated [9]. Metabolic acidosis may arise from endogenous production of pathologic acids (lactic acidosis), decreased ability to excrete acid, and/or absorption of acids from the intestine. Those molecules that act as acids and bases and therefore have the greatest impact on acidbase status, according to Stewart’s theories, include three independent variables; the strong ions (or strong ion difference), total protein, and PCO2, the partial pressure of carbon dioxide [11,12]. Decreased ability to excrete acid occurs in patients with respiratory disease (decreased ability to expire CO2), chronic renal disease, or renal tubular acidosis and in elderly patients with an age-related decline in renal capacity to excrete acid (decreased ability to excrete acids and strong ions in the urine) [4,13]. In the absence of pathology, a relative excess of strong anions compared to strong cations in the diet may also induce metabolic acidosis in the face of normal renal function [4,14]. Strong ions are those that are completely dissociated at physiologic pH and include Na, K, and Cl. This is because strong ions are absorbed directly from the intestine into the blood and therefore immediately impact acid-base balance, whereas weaker ions are absorbed at lesser efficiencies. Depending on the composition of the diet, the imbalance may be slight or substantial, leading to a corresponding degree of metabolic acidosis [7]. In human beings, dietary acid imbalance arises through the consumption of a diet that is relatively low in potassium and high in sulfur containing amino acids that comprise animal proteins [15]. In response to chronic dietary acid loads, the body seeks to maintain neutrality by mobilizing calcium, phosphate, and car-
bonate from bone as buffers. The mechanism by which bone is resorbed is both by physicochemical dissolution (in the acidic environment) and cell-mediated resorption, as osteoclasts are stimulated in an acidic microenvironment. [13,16,17]. Heritable differences in titratable acid excretion by the kidney may also play a role [18]. However, the exact mechanisms through which dietary metabolic acidosis leads to decreased bone mineral density (BMD) are unknown. In addition, in human medicine, because of the number of confounding factors, dietary acidosis alone as a cause of decreased BMD has not been shown. Unfortunately, the potential acid load of a particular diet has been described differently in the veterinary and human literature. The veterinary literature has focused on analysis of the diet to determine strong ion content of the diet per kilogram feedstuff, whereas the human literature has focused on urinary acid excretion (net endogenous acid production) or on calculating the potential renal acid load of foods. This discrepancy in scientific methods has made direct comparison between species difficult. However, in one human study, the daily DCAD could be calculated from the data provided by the authors. In this study a typical omnivore diet of 2700 (16) kcal per day had a mean DCAD of 27 mEq (range, 74 to 128 mEq) per day, using a simplified equation of (Na ) (Cl S) dietary cation-anion difference [19]. The purpose of this study was to examine the effects of metabolic acidosis induced by diet and determine if an interaction occurs between diet and estrogen depletion via ovariectomy. Bone mineral density of the lumbar spine, serum bone alkaline phosphatase (BAP), urine deoxypyridinoline (DPD), arterial pH, and urinary fractional excretions of calcium and phosphorus were used as markers of bone turnover.
Materials and methods Animals and diet Twenty-four skeletally mature (4- to 7-year-old) Rambouillet-Columbia cross ewes were used and all procedures were approved by the Colorado State University institutional animal care and use committee. Twelve sheep underwent ovariectomy (OVX) and 12 sheep did not. Following surgery, sheep were assigned to consume one of two diets (Table 1). One diet was designed to induce metabolic acidosis, whereas the other was not. Therefore, the investigative groups were composed of 6 animals each; the groups were: metabolic acidosis (MA) induction diet with and without OVX (OVX MA and noOVX MA) and control diet (ND) with and without OVX (OVX ND and noOVX ND).
J.M. MacLeay et al.: Dietary metabolic acidosis in an ovine model
563
Table 1. Dietary composition
Diet
Compositions
Estimated amount consumed/ day per sheep at 3.3% of body weight/day
MA
Grain mix Grass hay Grass hay
1.26 kg 1.0 kg 2.7 kg
ND
No. of kcal offered/ day per sheep (total)
Dietary cationanion difference mEq/kg dry matter
1900 1100 (3000) 2970
765 308 308
Dietary cation-anion difference of total diet consumed/ day per sheep if consumed between 2600 and 3300 kcal/day 150–175 mEq 900–1100 mEq
MA, low dietary cation-anion difference; ND, normal dietary cation-anion difference; DM, dry matter
The control diet consisted of free-choice grass hay, and the MA diet consisted of a specially formulated pellet that provided adequate amounts of all nutrients, including calcium, in addition to a limited amount of grass hay (Table 2). Metabolic acidosis was induced primarily by limiting the amount of potassium and adding magnesium, sodium, sulfur, and chloride to the diet. The diets were analyzed to determine their cationto-anion difference by quantifying their content of strong ions; sodium, potassium, chloride, and sulfur in addition to magnesium, sulfur and phosphorus. From this data the strong ion difference or DCAD was determined. DCAD mEq/kg dry matter [Na K (0.15)Ca (0.15)Mg] [Cl (0.2)S (0.3)P]
The control diet had a DCAD of approximately 308 mEq/kg DM, which was normal for this species and was designated ND. If it was assumed that the sheep consumed approximately 3000 kcal/day, then their daily DCAD was approximately 1000 mEq/day. The MA diet had a DCAD of 465 mEq/kg DM, or for an intake of approximately 3000 kcal/day, they consumed 160 mEq/day (Table 1) All sheep were weighed on day 0 and on day 180. All sheep underwent dual-energy X-ray absorptiometry (DEXA) of the lumbar vertebrae on days 0 and 180. All sheep were humanely euthanized, using an intravenous overdose of 8 g of pentobarbital, on day 180 [20]. Ovariectomy General anesthesia was induced using 7.5 mg of valium and 250 mg of ketamine intravenously and maintained using isoflurane at 1%–3% and oxygen at 2 l/min. Ovariectomy was performed via midline laparotomy. Ovaries were exteriorized, the pedicles were ligated, and the ovaries were removed. After standard midline threelayer closure the sheep were moved to the DEXA machine for BMD and bone mineral content (BMC) measurements. Sheep that were not ovariectomized were not subjected to sham surgery, but were anesthetized similarly to the OVX sheep and received DEXA
only. Thereafter all sheep recovered and were moved to similar but separate outdoor group pens for housing. No complications with ovariectomy or general anesthesia were noted during the course of the study. Bone densitometry Bone densitometry (DEXA) measurement of the lumbar vertebrae was performed on days 0 and 180, using a Hologic Delphi QDR dual-energy X-ray absorptiometer and software version 11.1 (Hologic, Bedford, MA, USA). All measurements were performed by the same operator (A.S.T.). The BMD of the last four lumbar vertebrae was measured in a standard dorsoventral view twice and the total BMD was then averaged between the two measurements. Serum and urine samples Jugular venous blood and urine samples were collected at 0, 90, and 180 days. Serum was drawn off and held at 0°C until analyzed. Serum samples were analyzed for BAP, creatinine, calcium, and phosphorus. Urine samples were collected as freely voided samples and then stored at 0°C until analyzed for deoxypyridinoline, creatinine, calcium, and phosphorus. Urine and serum samples were both collected in the morning at approximately 9 a.m. Arterial blood samples were obtained on day 180 into heparinized syringes and immediately analyzed, using a standard blood gas analyzer (Radiometer ABL 505; SenDx Medial, Carlsbad, CA, USA). Urine DPD was determined using a commercially available competitive enzyme immunoassay (Metra DPD EIA kit; Quidel, Santa Clara, CA, USA), which normalized DPD values to urine creatinine; and serum BAP concentrations were determined using a commercially available immunoassay (Metra BPA EIA kit; Quidel). Serum and urine calcium, phosphorus, and creatinine concentrations were determined on a standard chemistry analyzer (Hitachi, Tokyo, Japan). Urine was acidified to dissolve any calcium crystals that may have been present prior to analysis. Urinary fractional excretions, as a percentage, were calculated by normal-
Unknown
izing urine and serum values for calcium and phosphorus to urine and serum values for creatinine. Statistical analysis
0.26%
Statistical analysis consisted of a two-way analysis of variance for repeated measures, and was conducted for the total BMD of the last four lumbar vertebrae, BAP, urine DPD, urinary fractional excretion of calcium and phosphorus, and animal weights. Arterial pH was compared between groups, using a two-way analysis of variance for a single measurement. Analysis was conducted using commercially available software (SPSS, Chicago, IL, USA).
0.18%
27 6 Unknown 14.5 3.5 5.24 6 0.5a 3.6
Sodium (g)
Sulfur (g)
Chloride (g)
J.M. MacLeay et al.: Dietary metabolic acidosis in an ovine model
0.18% 0.38%
0.8%
8 4 3.6 5.5 4 2.8
31 45 16
There was no significant effect of diet (P 0.07) or ovariectomy status (P 0.872) on animal weights throughout the study. There was a significant effect of diet (P 0.05), but not of ovariectomy status (P 0.15) on BMD of the lumbar spine (Fig. 1). There was neither a significant effect of diet (P 0.21) nor of ovariectomy status (P 0.19) on serum BAP concentration over time (Fig. 2). Similarly, there was neither a significant effect of diet (P 0.13) nor of ovariectomy status (P 0.9) on urine DPD (Fig. 3). There was a significant affect of diet on increased urinary fractional excretion of phosphorus (P 0.05), but there was not a significant affect induced by ovariectomy status (P 0.9; Fig. 4). Urinary fractional excretion of calcium was increased by both diet (P 0.05) and ovariectomy status (P 0.05; Fig. 5). In addition,
0 Days 180 Days
1.200
1.150
1.100
BMD g/cm2
These sheep did have access to a NaCl salt-lick source a
0.82%
304 208 122
8 11 2.7
Mean Bone Mineral Density (g/cm2)
MA ND Sheep nutrient requirements for 80 kg BW Sheep nutrient requirements % of diet dry matter
Calcium (g)
Phosphorus (g)
Magnesium (g)
Potassium (g)
Results
Adjusted crude Protein (g)
Table 2. Major mineral content for each diet, assuming sheep on the MA and ND diets consumed the offered amounts listed in Table 1. Sheep nutrient requirements are offered for comparison. Sheep nutrient requirements listed are those established by the Subcommittee on Sheep Nutrition, Committee on Animal Nutrition, Board of Agriculture, National Research Council, and published as Nutrient requirements of sheep. 6th Edn; 1985, by National Academy Press
564
a
a
a
b
a
b
a
b
1.050
1.000
0.950
0.900
0.850
0.800
NoOVX ND
OVX ND
No OVX MA
OVX MA
Fig. 1. Mean bone mineral density (BMD [g/cm2]) for the last four lumbar vertebrae, as measured by dual-energy X-ray absorptiometry (DEXA) for each group of sheep on day 0 and day 180 (July to January). Standard deviation bars are shown. OVX, ovariectomy; ND, control diet; MA, metabolic acidosis induction diet. Unlike letters are significantly (P 0.05) different from each other
J.M. MacLeay et al.: Dietary metabolic acidosis in an ovine model
565 Mean Fractional Excretion of Phosphorus (%)
Mean Serum Bone Alkaline Phosphase (U/L) Day 0 BAP
18.000
Day 90 BAP
16.000
16.000
Day 180 BAP
14.000
FE P Day 90
14.000
12.000
12.000
10.000
10.000
8.000
8.000
6.000
6.000
FE P Day 0
18.000
20.000
FE P Day 180
4.000
4.000
2.000
2.000
0.000
0.000
No OVX ND
No OVX ND
OVX ND
No OVX MA
OVX ND
No OVX MA
OVX MA
OVX MA
Fig. 2. Serum bone alkaline phosphatase (BAP [U/l]), as measured on days 0, 90, and 180 for each group of sheep. Standard deviation bars are shown. OVX, ovariectomy; ND, control diet; MA, metabolic acidosis induction diet. There was no significant influence of time, ovariectomy status, or dietary treatment on BAP
Fig. 4. Urinary fractional excretion of phosphorus (FE P [%]) as measured on days 0, 90, and 180 for each group of sheep. Standard deviation bars are shown. OVX, ovariectomy; ND, control diet; MA, metabolic acidosis induction diet. There was a significant (P 0.05) effect of diet on urinary fractional excretion of phosphorus, but not of ovariectomy status
Mean Fractional Excretion of Calcium (%)
Mean Urine Deoxypyridinoline (nmol/L) Day 0 16
12.000
FE Ca Day 0
Day 90
14
Day 180
12
FE Ca Day 90 10.000 FE Ca Day 180 8.000
10
6.000 8
4.000
6 4
2.000
2
0.000 0
No OVX ND NoOVX ND
OVX ND
noOVX MA
OVX ND
No OVX MA
OVX MA
OVX MA
Fig. 3. Urine deoxypyridinoline (DPD [nmol/l]) as measured on days 0, 90, and 180 for each group of sheep. Standard deviation bars are shown. OVX, ovariectomy; ND, control diet; MA, metabolic acidosis induction diet. There was no significant influence of time, ovariectomy status, or dietary treatment on DPD
there was a trend towards an interaction between time, ovariectomy status, and diet (P 0.055). For fractional excretion of calcium, all groups except ND OVX had low excretion rates at time 0. The elevated excretion at time 0 was due to three sheep in this group with significantly elevated fractional excretions of calcium that was not repeated in the following months. Therefore, it is likely that these findings were due to a spurious error. Due to limited quantities of the samples, the tests could not be rerun to verify the results. Arterial pH values were significantly lower due to diet (P 0.05), but not due to ovariectomy status (P
Fig. 5. Urinary fractional excretion of calcium (FE Ca [%]), as measured on days 0, 90, and 180 for each group of sheep. Standard deviation bars are shown. OVX, ovariectomy; ND, control diet; MA, metabolic acidosis induction diet. Fractional excretion of calcium was significantly (P 0.05) influenced by both diet and ovariectomy status, with a trend towards an interaction between time, ovariectomy status, and diet (P 0.05)
0.91). However, there was a tendency for an interaction between diet and ovariectomy status (P 0.051; Fig. 6). No group had a mean arterial pH that was outside what would be considered the normal physiologic range (pH 7.4 0.2) [21].
Discussion The sheep has been shown to be an adequate animal model to study estrogen depletion and the onset of
566
J.M. MacLeay et al.: Dietary metabolic acidosis in an ovine model Mean Arterial pH
7.56
a
a
b
b
7.54 7.52 7.50 7.48 7.46 7.44 7.42 7.40 7.38 noOVX ND
OVX ND
noOVX MA
OVX MA
Fig. 6. Arterial pH, as measured on day 180 for each group of sheep. Standard deviation bars are shown. OVX, ovariectomy; ND, control diet; MA, metabolic acidosis induction diet. Arterial pH was significantly lower (P 0.05) due to dietary treatment but not due to ovariectomy status. There was a tendency for an interaction between diet and ovariectomy status (P 0.051). Unlike letters are significantly different from one another. Normal physiologic range (7.4 0.2) [21]
osteopenia [22–27]. The purpose of this study was to examine the potential role of dietary-induced metabolic acidosis in the face of estrogen depletion on BMD, as measured by DEXA. Markers of bone turnover were also examined, and included serum BAP, urine DPD, and urinary fractional excretion of calcium and phosphorus. Arterial blood pH was measured on day 180 and confirmed that a state of compensated metabolic acidosis existed in the MA diet-treated groups. Bone alkaline phosphatase (BAP) is generally increased in osteoporosis and is a sign of increased bone turnover as it is released from osteoblasts. Osteoclast activity can be approximated using urinary DPD. Approximately 90% of the organic matrix of bone is type 1 collagen. Crosslinks of mature type 1 collagen in bone are the pryidinium crosslinks, pyridinoline and DPD. Urine DPD is released into the circulation during the bone resorption process and is excreted unmetabolized in urine. In this study, despite the decrease in BMD observed in the MA diet groups, we did not document a significant increase in serum BAP or urine DPD. Serum BAP showed no real trends over the course of this study within each group, but treated groups tended to have higher values than the control group. Urine DPD appeared to generally decrease. The changes over time for BAP and DPD may reflect a seasonal component, as the study was conducted from summer to winter and therefore days were shorter at the end of the study. This may be supported by the decrease in fractional excretion of calcium in the ND groups at day 180. However, this is in contrast to human studies that found an increase in
BAP during winter months [28]. As significant differences were not found, it is important not to overinterpret apparent trends in the BAP and DPD results. The variance may also reflect the relatively small group size of this study. Neither serum BAP nor urine DPD appeared to adequately reflect the changes in BMD that were observed by DEXA, and as such may not be ideal markers for use in the ovine model. Urinary fractional excretions of calcium and phosphorus did increase and were found to be significantly associated with the MA diet and, therefore, may be better indicators of calcium and phosphorus balance within the body. Whether the mechanism by which calcium and phosphorus were liberated from bone was primarily physicochemical or cell-mediated has yet to be determined and is an area of future research. Other markers of bone turnover, such as osteocalcin, parathyroid hormone (PTH), and vitamin D were not examined in this study and may be determined to be useful markers in future studies. One other drawback of this study was the relatively larger proportion of sodium in the MA diet compared to the control diet. The role of sodium in calcium wasting is controversial [29,30]. Some studies have found that increased salt intake is associated with calciuria. However, in these studies it is unclear whether the overall diet induced metabolic acidosis or not. Other researchers argue that, as sodium intake is generally consumed as sodium chloride, that it is the other cations and anions in the diet that are more important influencers of acid-base balance and, therefore, calciuria is more important than actual NaCl intake [31]. Further studies are necessary to settle this controversy. It is difficult in human studies to examine the effect of diet on bone metabolism without considering other confounders. Using this ovine animal model, we were able to examine the influence of diet and ovariectomy status in a similarly treated, housed, and aged population. We found that diet was a potent influence on BMD. We did not see strong evidence for a synergistic effect between diet and ovariectomy status, but one limit of this study was its short duration of 180 days. Previous studies in the sheep typically required at least 12 months to witness a significant decline in BMD of the lumbar spine in the face of estrogen depletion alone [27]. Nevertheless, the potent influence of diet on BMD in a large animal model has heretofore not been described in the literature. It is interesting to note that the calcium content of both diets was in excess of minimum requirements for this species and considerably higher than that recommended for humans, and yet significant calcium wasting still occurred. Therefore the mechanism by which dietary metabolic acidosis causes calcium wasting appears to be at least partially independent of calcium intake.
J.M. MacLeay et al.: Dietary metabolic acidosis in an ovine model
One theory for loss of bone mineral in humans is that dietary cation-anion difference or dietary-induced metabolic acidosis plays a significant role in bone turnover [3,4,32]. We felt that the combination of dietary manipulation and ovariectomy presented in this study may more realistically mimic the physiologic processes occurring in postmenopausal women than other experimental methods published in the literature. Based on data from Dwyer et al. [19], we could calculate the daily dietary cation-anion difference (DCAD (Na K) (Cl S)) of a typical human diet to be approximately 27 (74 to 128) mEq/day. Sheep typically consume a diet that is approximately 1000 mEq/day. In this study, we fed an experimental diet that had a DCAD of approximately 162 mEq/day and this produced a significant decline in BMD. Our experimental diet was relatively more alkalogenic than a typical human diet, yet we were able to induce rapid bone mineral loss at a rate not seen in humans consuming a similar diet. Therefore, we can conclude that sheep are well adapted to their typical diet and that when a more acidogenic diet than is typical is consumed, a negative calcium balance results. It has been suggested that the modern diet of humans is somewhat more acidic than that of Paleolithic man to which man would be most adapted, which is why the typical diet consumed by humans today contributes to the loss of bone mineral over time [15,31]. We cannot make assumptions as to the degree of pressure that any particular diet can induce within any particular species. Nor can we make any assumptions concerning the correlation between a particular degree of acidosis between species consuming diets of similar cation-anion differences. Because of their apparent sensitivity to dietary manipulation, we can conclude that the findings of this study support the use of sheep as an excellent model in which to study the mechanisms involved in bone loss related to acidifying diets. Areas of future study should include the study of the effects of less severe degrees of dietary metabolic acidosis on bone turnover and potentially the protective effect of alkalinizing diets on bone mineral loss in the face of ovariectomy over the long term. Elucidation of more ideal bone biomarkers in the sheep, such as vitamin D, PTH, and/or osteocalcin would also be useful. Determination of the primary cause for BMD loss, as arising from physicochemical or cell-mediated mechanisms must be made. Histomorphological and biomechanical studies of the bones from the sheep reported in this study are ongoing. Acknowledgments. The authors thank the Colorado State University’s College Research Council and Stryker Biotech, Inc. for their financial support.
567
References 1. Melton L, Chrischilles E, Cooper C, Lane A, Riggs B (1992) How many women have osteoporosis? J Bone Miner Res 7:1005– 1010 2. Lemann J, Litzow J, Lennon E (1966) The effects of chronic acid loads in normal man: further evidence for the participation of bone mineral in the defense against chronic metabolic acidosis. J Clin Invest 45:1608–1614 3. Frassetto L, Todd K, Morris RJ, Sebastian A (1998) Estimation of net endogenous noncarbonic acid production in humans from diet potassium and protein contents. Am J Clin Nutr 68:576–583 4. Sebastian A, Harris S, Ottaway J, Todd K, Morris RJ (1994) Improved mineral balance and skeletal metabolism in postmenopausal women treated with potassium bicarbonate. N Engl J Med 23:1776–1781 5. New S (2002) The role of the skeleton in acid-base homeostasis. Proc Nutr Soc 61:151–164 6. Whiting S, Draper H (1981) Effect of chronic high protein feeding on bone composition in the adult rat. J Nutr 111:178–183 7. Remer T, Manz F (1995) Potential renal acid load of foods and its influence on urine pH. J Am Diet Assoc 95:791–797 8. Oetzel GR (1993) Use of anionic salts for prevention of milk fever in dairy cattle. Compendium 15:1138–1145 9. Riond J (2001) Animal nutrition and acid base balance. Eur J Nutr 40:245–254 10. Remer T, Manz F (1994) Estimation of the renal net acid excretion by adults consuming diets containing variable amounts of protein. Am J Clin Nutr 59:1356–1361 11. Stewart P (1981) How to understand acid base balance. In: Stewart P (ed) A Quantitative Acid-Base Primer for Biology and Medicine. Elsevier, New York 12. Stewart P (1983) Modern quantitative acid-base chemistry. Can J Physiol Pharmacol 61:1444–1461 13. Arnett T (2003) Regulation of bone cell function by acid-base balance. Proc Nutr Soc 62:511–520 14. Goff JP, Horst RL, Mueller FJ (1991) Addition of chloride to a prepartal diet high in cations increases 1,25-dihydroxyvitamin D response to hypocalcemia preventing milk fever. J Dairy Sci 74: 3863–3871 15. Boyd Eaton S, Cordain L (1997) Evolutionary aspects of diet: old genes, new fuels. World Rev Nutr Diet 81:26–37 16. Bushinsky D (1996) Metabolic alkalosis decreases bone calcium efflux by suppressing osteoclasts and stimulating osteoblasts. Am J Physiol 271:F216–F222 17. Bushinsky D (2001) Chronic acidosis: calcium release. Eur J Nutr 40:240–244 18. Vaitkevicius J, Witt R, Maasadam M, Walters K, Gould M, MacKenzie S, Farrow S, Lockette W (2002) Ethnic differences in titratable acid excretion and bone mineralization. Med Sci Sports Exerc 34:295–302 19. Dwyer J, Foulkes E, Evans M, Ausman L (1985) Acid/alkaline ash diets: time for assessment and change. J Am Diet Assoc 85: 841–845 20. AVMA Panel on Euthanasia (1993) J Am Vet Med Assoc 202: 229–249 21. Muir W, Hubbell J (1989) Handbook of Veterinary Anesthesia. CV Mosby, St. Louis, 340 22. Turner AS, Alvis M, Myers W, Stevens ML, Lundy MW (1995) Changes in bone mineral density and bone-specific alkaline phosphatase in ovariectomized ewes. Bone 17 (Suppl 4):395S– 402S 23. Turner AS, Alvis M, Mallinckrodt C, Bryant H (1995) Dualenergy X-ray absorptiometry in sheep: experiences with in vivo and in vitro studies. Bone 17 (Suppl):381S–387S 24. Turner AS, Alvis M, Mallinckrodt C, Bryant H (1995) Dose response effects of estradiol on bone mineral density in ovariectomized ewes. Bone 17 (Suppl):421S–427S 25. Turner AS (2001) Research in orthopedic surgery. J Surg Res
568
J.M. MacLeay et al.: Dietary metabolic acidosis in an ovine model
26. Turner AS (2001) Animal models of osteoporosis—necessity and limitations. Eur Cell Mater 1:66–81 27. Turner AS (2002) The sheep as a model for osteoporosis in humans. Vet J 163:1–8 28. Woitge H, Scheidt-Nave C, Kissling C, Leidig-Bruckner G, Meyer K, Grauer A, Scharla S, Ziegler R, Seibel M (1998) Seasonal variation of biochemical markers of bone turnover: results of a population-based study. J Clin Endocrinol Metab 83:68–75 29. Oh K, Oh E, Kim S, Lee D, Roh S, Song S, Kang M (2002) The Relationship Between Urinary Sodium Excretion and Bone Mineral Metabolism of Climacteric Women in Korea. American Society for Bone and Mineral Research, vol. 24. Society for Bone and Mineral Research, San Antonio, p S469
30. Harrington M, Cashman K (2003) High salt intake appears to increase bone resorption in postmenopausal women but high potassium intake ameliorates this effect. Nutr Rev 61 (5 pt 1): 179–183 31. Frassetto L, Morris RJ, Sellmeyer D, Todd K, Sebastian A (2001) Diet, evolution and aging: the pathophysiolgic effects of postagricultural inversion of the potassium-to-sodium and base-tochloride ratios in the human diet. Eur J Nutr 40:200–213 32. Barzel U (1995) The skeleton as an ion exchange system: implications for the role of acid-base imbalance in the genesis of osteoporosis. J Bone Miner Res 10:1431–1436
