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The role of dietary protein in the pathogenesis of osteoporosis

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Peer-reviewed scientific article on s nt

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The role of dietary protein
in the pathogenesis of osteoporosis
SA’EED HALILU BAWA
Sa’eed Halilu Warsaw University of Life Sciences – SGGW, Department of Dietetics
Bawa Faculty of Human Nutrition and Consumer Sciences
Nowoursynowska Street 159C, Warsaw, 02776, Poland

ABSTRACT: It has long been known that systemic acidosis brings about depletion of the skeleton via negative calcium balance.
In vivo, acidosis can occur systemically due to renal, bronchial or gastrointestinal disease, diabetes, severe (anaerobic) exercise,
excessive protein intake, ageing, or menopause. The oxidation of proteins containing sulphur and phosphorus ultimately yields
H+ residues corresponding to sulphuric and phosphoric acids, which must be excreted via the kidneys. The average Western
diet has been estimated to generate an inorganic H+ residue of about 0.1 mol/day, which is equivalent to about 8 ml of
concentrated hydrochloric acid. Recent data indicate that excess acid generated from high protein intakes increases calcium
excretion and bone resorption, assessed as urinary pyridinoline and deoxypyridinoline. However, epidemiological studies have
also found a significant positive relationship between protein intake and bone mass or density. Similarly, isotopic studies in
humans have also demonstrated greater calcium retention and absorption by individuals consuming high-protein diets,
particularly when the calcium content of the diet was limiting. High-protein intake may positively impact bone health by several
mechanisms, including calcium absorption, stimulation of the secretion of insulin-like growth factor-1, and enhancement of
lean body mass. The perception that high intake of dietary protein brings about a large enough shift in systemic pH to increase
osteoclastic bone resorption seems weak..

INTRODUCTION mechanism proposed by this hypothesis is that increased

Diet and nutrition play an important role in the development
and maintenance of bone structures resistant to usual
mechanical loadings. Besides sufficient supply of dietary
calcium, and an adequate vitamin D intake, dietary
protein intake, especially of animal origin, because of the
sulfur amino acid content and its acid-ash nature, leads
to an increased glomerular filtration rate, reduced renal
reabsorption of calcium, hypercalciuria and thus leaching
of calcium out of the bone (5-9). This gradual dissolution of

protein and sodium exert impact on bone health. The
results of many investigations have shown that increasing
dietary protein increases urine calcium excretion such that
for each 50 g increment of protein consumed and extra 60
mg of urinary calcium is excreted (1-3). It follows that the
higher the protein intake, the more urine calcium is lost
and the more negative calcium balance becomes. It is
believed that the consumption of animal proteins would
result in a substantial metabolic acid load which in turn
would cause the dissolution of bone mineral as manifested
by increased calciuria.
On the other hand, well controlled experiments demonstrate
that a selective deficiency in dietary proteins, that is, without
any associated insufficiency in other macronutrients, total
energy, calcium and vitamin D, causes a rapid and marked
alteration in bone mass, microarchitecture and strength,
which can increase the risk of osteoporosis. Moreover, High-
protein intake may positively impact bone health by several
mechanisms, including calcium absorption, stimulation of the
secretion of insulin-like growth factor-1, and enhancement
of lean body mass (3). The purpose of this review is to analyse
the evidence that dietary protein can positively as well as
negatively affect bone health by increasing or decreasing
the risk for the development of osteoporosis.
NEGATIVE EFFECTS OF EXCESS PROTEIN ON BONE HEALTH
Increasing dietary protein increases urine calcium excretion
such that for each 50 g increment of protein consumed and
extra 60 mg of urinary calcium is excreted. It follows that the
higher the protein intake, the more urine calcium is lost and
the more negative calcium balance becomes (1-3).
Wachman and Bernstein (4) first hypothesized the alleged
negative effect of dietary protein on bone calcium. The
Focus on Proteins, Peptides, Aminoacids - Supplement to AgroFOOD industry hi-tech - Nov/Dec 2011 - vol 22 n 6
bone mineral and its loss through the kidneys over time is
often implicated in the etiology of osteoporosis (10-16).
In most individuals, the source of net acid load is from the
metabolism of protein (when consumed in large amounts)
and long-chain fatty acids (when they comprise more
than 20 percent of calories in the diet). A marker of net
acid production is the extent of degradation of sulfur-
containing amino aids: cysteine, cystine, and methionine.
Moreover, the breakdown of any of the seven acidic amino
acids (aspartate, glutamate, cysteine, cystine, proline/
hydroxyproline, serine, and threonine), plus the keto-acids
produced from amino acid metabolism, contribute to the
body’s fixed, organic acid load (17). The metabolism of
these amino acids produces H+ without buffering partners.
These H+ accumulate and must be neutralized by matching
buffering elements from the body. The buffering elements
include the organize anions (usually as K+ or other mineral
salts) in fruits, vegetables, lentils/pulses, herbs, and spices.
These also include metabolically alkaline forming citrate,
malate, succinate, and fumarate. In addition, short- and
medium-chain fatty acids reduce net acid burden by
“soaking up” acetate and 2-carbon acidic units in the cells.
The major recognized sources of net acid load in the body
are: a) large intakes of protein, especially of animal origin,
b) dietary phosphate/phosphoric acid, c) dietary sulphate,
d) excess consumption of long-chain fatty acids, e) distress
(excess cortisol and adrenaline), f) delayed immune system
reactions. Many studies have shown that bone responds to
an acid load by dissolving its basic buffering mineral salts. The
average adult skeleton contains a large but finite amount of
Ca2+ (50-65,000 mEq, 99 percent of total body stores) and
Mg2+ (1,060-1,600 mEq, 50 to 80 percent of body stores).
Bone minerals serve as a sizeable reservoir of buffer, usable
in the control of plasma pH (18, 19). Diets of the citizens of
industrialized nations commonly produce an excess load

7
 of fixed acids of 100 to 200 mEq per day (20, 21). Remer
and Manz (21) found that a diet containing 120 grams of
protein yielded a net acid excretion of 135.5 mEq/day. Two
“moderate” protein diets (95g/day protein) yielded net acid
excess (NAE) of 69 to 112 mEq/day. A lactovegetarian “low”
protein diet (49g/day) gave an NAE of 24 mEq/day. Therefore,
the type of diet consumed influences net acid production.
High-protein diets produce a six-fold (600 percent) increase
in NAE. This results in low first-morning urine pH, indicating
that buffering functional reserve is deficient, and that the risk
of metabolic acidosis is correspondingly increased (21). In
a recent study, Jajoo et al. (22) demonstrated that a diet-
induced increase in NAE is associated with an increase in
bone resorption and urinary calcium excretion over a 60-
day period in healthy elderly men and women with relatively
high protein intakes. Their study suggests that changes
observed in 7 to 18 day metabolic studies may persist for
up to 60 days and the study also raises the possibility that
the diet-induced increase in bone resorption may involve
PTH-dependent and independent mechanisms. It should
be underlined that besides high-protein diets, acidosis may
also be the result of excessive intakes of beverages, such as
colas. Barzel and Massey (18) demonstrated that the pH of
colas with phosphoric acid is 2.8 to 3.2. However, the kidney
cannot excrete urine with a pH much lower than 5, without
significantly damaging the gentourinary tract. To achieve a
urinary pH of 5, a 12 oz. (330mL) can of cola would have
to be diluted 100-fold, requiring an additional 33 litres of
urine. Otherwise, a corresponding amount of buffer must
be drawn from the body to neutralize the excess acid. The
body routinely buffers the acidic beverage with sodium and
potassium if reserves permit, then with a corresponding loss
of calcium, magnesium, and other minerals, as available.
Although several clinical trials (5, 7, 8, 23-29) have attempted
Focus on Proteins, Peptides, Aminoacids - Supplement to AgroFOOD industry hi-tech - Nov/Dec 2011 - vol 22 n 6
to test the hypothesis proposed by Wachman and Bernstein
(4), the effects of dietary protein on calcium retention and
bone health remain unclear. The earliest studies testing
the hypothesis that dietary protein leads to calciuria and
decrease BMD used purified proteins (for example, casein
or lactalbumin) rather than common sources of protein
(e.g., meat or milk) and found that purified proteins indeed
do induce hypercalciuria (30-31) and this effect does not
adapt over time (32). This distinction between purified and
common dietary protein sources is important because the
latter contain a substantial amount of phosphorus, which
blunts the calciuric effect observed with purified proteins
(28). In fact, when common sources of protein were tested,
hypercalciuria and a negative calcium balance were
observed only when the phosphorus contents of the diets
were equalized (33) but not when phosphorus was allowed
to vary with the dietary protein content (33, 34).
Many nutritionists argue that both dietary protein and sodium
increase significantly increase calcium requirements. Table
1 reveal the extent of which protein, particularly of animal
origin as well as sodium increase the needs for calcium.
Equilibrium value
Group
mmol
Young adults
Young adults + skin
Menopause + skin
+ skin + 50 mmol Na
+ skin + 40 g protein
Young adults + skin less 40 g protein
+ skin less 40 g protein less 50 mmol Na
Table 1. Estimated calcium intakes at which absorbed calcium comes into equilibrium
with calcium losses in different groups on different diets (34).
8
ACIDOSIS AND OSTEOCLAST FUNCTION
Many studies have shown that osteoclasts (OC) can
be stimulated easily by acidosis. The sensitivity of OC
to extracellular H+ is such that pH reductions of only a
few hundredths of a unit cause a doubling of resorptive
activity (35, 36). This effect is not subject to tachyphylaxis
(or “escape”) in longer-term cultures: acid-activated
osteoclasts continue to form resorption pits over periods
of 7 days or more, amplifying the effects of modest pH
differences. Acidosis is required for the initiation of resorption;
once activated, OC can be further stimulated by factors
such as RANKL, 1,25(OH)2 vitamin D, PTH and ATP (37, 38);
note that pro-resorptive agents such as RANKL and PTH are
inactive on osteoclasts at pH 7.4 or above). Thus, osteoclast
stimulation is a 2-step process, with acid-activation as
the key initial requirement – and extracellular H+ may be
regarded as the long-sought “OC activation factor” (OAF).
As demonstrated by many investigations, acidosis
stimulates resorption in calvarial bone organ cultures
similarly. In addition, H+-stimulated Ca2+ release from
calvaria is almost entirely osteoclast-mediated, with a
minimal physicochemical component (39, 40). This finding is
consistent with the fact that mineralized bone surfaces are
normally covered by living cells, and are thus not directly
exposed to ion-exchange phenomena. These observations
suggest that the effects of acidosis on bone loss in vivo are
likely to be mostly cell-mediated.
ACIDOSIS AND OSTEOBLAST FUNCTION
Bushinsky et al. (40, 41) showed that acidosis inhibited
osteoblast function by decreasing expression of
extracellular matrix genes, including collagen. Brandao-
Burch et al. (42) investigated the effects of pH on osteoblast
function using bone nodule-forming primary rat osteoblast
cultures. They found that abundant, matrix-containing
mineralized nodules formed at pH 7.4, but acidification
progressively reduced mineralisation of bone nodules, with
complete abolition at pH 6.9. We also found that osteoblast
proliferation and collagen synthesis were unaffected by pH
in the range 7.4 to 6.9; moreover, no effect of acidification
on collagen ultrastructure and organisation was evident.
However, osteoblast alkaline phosphatase activity, which
peaked strongly near pH 7.4, was reduced 8-fold at pH 6.9.
Reducing pH to 6.9 also down regulated mRNA for alkaline
phosphatase, but up regulated mRNA for matrix Gla protein,
an inhibitor of mineralisation. The same pH reduction is
associated with 2- and 4-fold increases in Ca2+ and PO43−
solubility for hydroxyapatite, respectively (42).Results of
studies performed by Wrong et al. (43) show that acidosis
exerts a selective, inhibitory action on matrix mineralization
that is reciprocal with the OC activation response. Thus, in
uncorrected acidosis, the deposition of alkaline mineral
in bone by OB is reduced, and OC resorptive activity
is increased in order to maximize the
availability of hydroxyl ions in solution
to buffer protons (44). It is possible that
mg
14.5 580 these results could help to account
21.0 840 for the osteomalacia that sometimes
accompanies acidosis in renal disease.
26.0 1040
35.0 1400
44.0 1760 Positive effects of protein on bone health
14.5 580 Both under-consumption and excess
12.0 480 intakes of protein can increase the risk
of osteoporosis. The mechanism whereby
a low protein intake has adverse effects
on bone may be due to inadequate
production of IGF-1, which exerts anabolic effects on bone
mass, not only during growth, but also during adulthood (3, 45).
Dietary protein supplies the necessary substrates for the
formation and remodelling of the highly proteinaceous
organic matrix of bone. Dietary protein may modulate a
favourable systemic hormonal milieu for bone formation by
increasing the circulating levels of insulin-like growth factor-1
(IGF-1), an osteotrophic hormone (3, 46, 47). This enhancing
effect of dietary protein on serum IGF-1 was previously
demonstrated in elderly subjects supplemented with milk
(48) or protein supplements (49). This peptide hormone
functions both at the level of the kidneys by stimulating
renal transport of inorganic phosphate and production of
1,25 dihydroxyvitamin D, and at the level of the osteoblast
by stimulating proliferation, differentiation and phosphate
transport of these cells (28). IGF-1 may also modulate some
of the anabolic effects of parathyroid hormone (PTH) on
bone and might be a coupling factor for PTH-mediated
bone remodelling (50). On the other hand, decreased
serum concentration of serum IGF-1 has been associated
with reduced bone breaking strength in rats and increased
fracture risk in humans (51). In the study by Dawson-Hughes
and Harris (52) no association between dietary protein
and serum IGF-1 was detected. However, caution must be
exercised when interpreting serum IGF-1 data, given that
the level of measured IGF-1 does not necessarily match
its bioactivity because some of its binding proteins (BP)
potentiate IGF-1 activity (for example, IGFBP-3 and IGFBP-5)
and some inhibit it (for example, IGFBP-1 and IGFBP-4). It has
been shown that in starvation, IGFBP-1 increases and binds
IGF-1 more avidly, thus inhibiting IGF-1 bioactivity (53).
Protein replenishment in patients with hip fracture can
improve not only BMD, but also muscle mass and strength.
These two variables are important determinants of the
likelihood and consequences of falling and thus incidence
of osteoporotic fractures. This observation underlines the
importance of weight-bearing in the maintenance of
bone mass (54). At the tissue level, immobilization results in
bone resorption being greater than bone formation. At the
cellular level, immobilization increases bone reabsorption
by osteoclasts associated with a decrease in osteoblastic
formation (55). The molecular signal(s) perceiving the
reduction in mechanical strain associated with
immobility has not been identified.
While excess protein intake is common for
the average American, consuming too little
protein, especially from animal sources, can
increase the risk for weakened bones and
osteoporosis (56). There is growing evidence
that a low protein diet has a detrimental
effect on bone. For example, Kerstetter
et al. (57) reported that in healthy young
women, acute intakes of a low-protein diet
(0.7 g protein/kg) decreased urinary calcium
excretion with accompanied secondary
hyperparathyroidism. The etiology of the
secondary hyperparathyroidism is due,
in part, to a significant reduction in
intestinal calcium absorption during a
low protein diet.
In a short-term intervention trial,
Kerstetter et al. (58) evaluated
the effects of graded
levels of dietary protein
(0.7, 0.8, 0.9, and 1.0 g
protein/kg) on calcium
homeostasis. Secondary
hyperparathyroidism
developed by day 4 of
the 0.7 and 0.8 g protein/kg diets (due to the decreased
intestinal calcium absorption), but not during the 0.9 or 1.0
g protein/kg diets in eight young women. There were no
significant differences in mean urinary calcium excretion
over the relatively narrow range of dietary protein intakes
studied, although the mean value with the 0.7-g/kg intake
was lower than that with the 1.0 g/kg intake by 0.25 mmol (10
mg). According to authors of this study, the lack of change
may be due to the small sample and the inherent variability
in urinary calcium excretion. Similarly, when Giannini et
al. (59) restricted dietary protein to 0.8 g protein/kg, they
observed an acute rise in serum parathyroid hormone (PTH)
in 18 middle-aged hypercalciuric adults. Taken together,
both of studies suggest, at least in the short term, that
the RDA for protein (0.8 g/kg) does not support normal
calcium homeostasis.
Many other studies have shown that although excess
protein intake is common for the average American and
citizens of the European Union, consuming too little protein,
especially from animal sources, can increase the risk for
weakened bones and osteoporosis (33, 37-40). In older
adults, protein intake was found to be positively correlated
with bone mineral density and inversely related to rates
of bone loss (56, 60-63). Bonjour et al. (45) carried out a
6-month, randomized, double-blind, placebo-controlled
trial in 82 older adults who had suffered a hip fracture
and found that increasing protein intake brought about
a decrease in loss of leg bone density by 50 percent and
an increase in blood levels of insulin-like growth factor,
which promotes bone gain, and reduced the stay in the
rehabilitation hospital by 10 days.
In the Iowa Women’s Health Study of more than 32,000
women, higher intakes of protein, especially from animal
sources, were associated with reduced risk of hip fractures
Focus on Proteins, Peptides, Aminoacids - Supplement to AgroFOOD industry hi-tech - Nov/Dec 2011 - vol 22 n 6
in postmenopausal women (64). Women with hip fractures
consumed less red meat (beef, lamb, pork) than women
who did not suffer hip fractures.
Dietary calcium to protein ratio
The effect of protein intake on calcium balance is often
interpreted as negative, especially in the consumer
press. However, this is a classic case of a nutrient-nutrient
interaction and should be viewed in terms of the
calcium to protein intake ratio (65).
The calcium:protein ratio of the diet has been
found to be more closely related (positively)
to rate of bone gain than either calcium
intake (positive) or protein intake (negative)
alone (66).
The intakes of protein by Americans usually
exceed recommended levels, but the
consumption of calcium is well below
recommended dietary intake levels (67-69).
Based on current dietary recommendations
for calcium (68) and protein (69), a dietary
calcium to protein ratio of 16:1 (mg:g) or
higher likely provides adequate protection
for the skeleton and can be considered
optimal for adults (Table 2).
However, nutrient intake data from USDA’s
1994-96 Continuing Survey of Food
Intakes by Individuals (CSFII) (65,
66) reveal that calcium to protein
ratio of about half the optimal
ratio is achieved by adults
(Table 2). Similar findings
are observed from the Third
National Health and Nutrition
Examination Survey (66-67).
9
 Although protein intakes
are somewhat higher than Optimal Dietary
Sex/age Calcium Protein intake
recommended, low calcium calcium:protein calcium:protein
group intake (mg) (g)
intake is the real problem (19). ratio* (mg:g) ratio (mg:g)
Dietary protein may enhance Males
bone turnover by increasing 30-39 16 951 102.7 9.3
intestinal calcium absorption. It 40-49 16 876 95.3 9.2
is well known that dietary protein 50-59 19 791 90.3 8.8
stimulates gastric acid production. 60-69 19 796 83.5 9.5
This dietary protein-induced 70+ 19 746 72.9 10.2
increase in gastric acid production Females
may, in turn, improve calcium 30-39 20 661 65.3 10.1
bioavailability by increasing its
40-49 20 634 63.5 10.0
solubility. The mechanisms by which
50-59 24 630 64.1 9.8
dietary protein may affect skeletal
homeostasis are summarized in 60-69 24 604 60.4 10.0
Figure 1. These mechanisms are not 70+ 24 584 56.6 10.3
mutually exclusive (70). Table 2. Optimal versus actual dietary calcium:protein ratio (mg:g) for adults (Institute of
Medicine, 1997; National Research Council, 1989; USDA, 1997).
*Based on DRI for calcium (65) and RDA for protein (66).

CONCLUDING REMARKS

Both dietary calcium and vitamin D are

undoubtedly beneficial to skeletal health.
In contrast, despite intense investigation,
the impact of dietary protein on calcium
metabolism and bone balance remains
controversial. Dietary protein, both of plant
and animal origin appears to plays an
important role in the maintenance of bone
health. Besides their protein content, both
plant and animal foods provide other nutrients
that can exert positive influences on bone
health. Even in groups or among individuals
Focus on Proteins, Peptides, Aminoacids - Supplement to AgroFOOD industry hi-tech - Nov/Dec 2011 - vol 22 n 6

who are favourable to consuming foods

from animal sources, whether for economic
or palatability reasons, it is generally agreed
that a well-balanced diet includes the regular
consumption of fruits and vegetables. Besides
calcium and vitamin D, an adequate intake
of proteins should be recommended in the
prevention and treatment of postmenopausal
and age-dependent osteoporosis.

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Ph y s io lo g y : A Problem-Based Approac h, 3 r d Edi ti o n . People interested in the complete list of references and notes
Philadelphia: WB Saunders Company (1999). should contact the author at saeed_bawa@sggw.pl

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