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J Bone Miner Metab (2004) 22:561568

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: jmacleay@colostate.edu)
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 [25].
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 [3,5,810]. 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 Stewarts 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
150175 mEq
9001100 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 0C until analyzed. Serum samples were analyzed
for BAP, creatinine, calcium, and phosphorus. Urine
samples were collected as freely voided samples and
then stored at 0C 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

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

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

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 [2227]. 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 Universitys
College Research Council and Stryker Biotech, Inc. for their financial
support.

567

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