BONE AND BONE HEALTH
BENEFICAL ROLE OF CITRUS
EXECUTIVE SUMMARY
P.F. CANCALON
I- Bone physiology
Bone functions bio-mechanically to protect vital structures, anchor muscles, and absorb the impacts
arising from activity. It serves also as the body reservoir of calcium and phosphate, and bone
remodeling allows serum concentrations of these ions to be tightly controlled.
Bone is a living, growing tissue controlled by two types of cells with opposite functions. Osteoblasts are
bone-forming cells and osteoclasts are bone-resorbing cells. The processes of bone formation by
osteoblasts and bone resorption by osteoclasts produce opposite results B formation produces bone
mass and resorption reduces bone mass. Peak bone mass is the maximum mass of bone achieved by an
individual at skeletal maturity, typically between ages 25 and 35. After peak bone mass is attained, both
men and women lose bone mass over the remainder of their lifetimes. Because of the subsequent bone
loss, peak bone mass is an important factor in the development of osteoporosis. Osteoporosis is one of
the major problems affecting bone health
Type I-Postmenopausal Osteoporosis or high turnover osteoporosis occurs in some women aged 50-75
due to the sudden decrease in estrogen as a result of menopause. This causes rapid calcium loss from
the bones, making the women susceptible to hip, wrist, forearm, and spinal compression fractures. Type
II Senile Osteoporosis or low turnover osteoporosis occurs when bone loss and formation are not equal
and more bone is broken down than replaced. Senile osteoporosis is associated with normal processes
of aging, and a gradual decline in the number and activity of osteoblasts, and not primarily with an
increase in osteoclast activity.
1
II- Beneficial effects of citrus on bone health
Several citrus compounds including vitamin C, folic acid, flavonoids such as hesperidin and naringin,
carotenoids such as beta-cryptoxanthin act on bones to increase the anabolic process and increase bone
production. They act during the early part of life when maximal bone mass is reached and later in life
during senescent and post-menopausal osteoporosis. Citrus may also contribute to bone health by
limiting inflammation during the entire life and may have a beneficial effect on the various forms of
arthritis. Furthermore, potassium organic acids in citrus may limit blood acidity and slow calcium
leakage. Studies published in the last few years have shown that several compounds in citrus may act
synergistically to promote bone health
III- Conclusion of the effect of citrus on bone
Citrus appear to provide significant bone health benefits. They contain a wide array of compounds
acting on several targets that promote bone growth and maintenance and at the same time limit the
destruction part of the bone life cycle.
For a more detailed and referenced review of this topic, please continued reading
below
2
BONES AND BONE HEALTH
BENEFICIAL ROLE OF CITRUS
Literature Review
P.F. Cancalon
I- Bone anatomy and physiology
I-1 Bone structure
I-1-1 Cortical Bone
I-1-2 Trabecular bone
I-1-3 Bone Marrow
I-1-4 Bone cells
I-1-5 Cartilage
I-1-5-a Hyaline cartilage.
I-1-5-b White fibrocartilage.
I-1-5-c Elastic cartilage.
I-2 Bone homeostasis
I-2-1 Cellular and intracellular calcium and phosphorous metabolism
I-2-1-a Calcium metabolism
I-2-1-b Phosphorus metabolism
I-2-2 Skeletal metabolism
3
I-3 Bone formation, development, growth and remodeling
I-3-1 Bone Formation and growth
I-3-1-a Flat bones
I-3-1-b Long bones
I-3-2 Skeletal repair.
I-4 Bone Modeling and Remodeling
I-4-1 Bone modeling
I-4-2 Bone remodeling
I-5 Mineral homeostasis
I-5-1 Bone-blood transfer
I-5-2 Hormonal control of bone homeostasis
I-5-2-a Effect of parathyroid hormone on bone remodeling
I-5-2-b Effect of vitamin D on bone remodeling
I-5-2-c Effect of calcitonin on bone remodeling
I-5-2-d Effect of sex hormones and other hormones on bone remodeling
II- Bone disorders
II-1 Bones and mineral homeostasis impairment
II-1-1 Mineral absorption
II-1-1-a Calcium
II-1-1-b Phosphorus
II-1-1-c Minerals and nutrition
II-1-2 Mineral reabsorption
II-1-3 Chronic acidemia
4
II-1- 4 Renal osteodystrophy
II-2 Osteoporosis
II-2-1 Types of Osteoporosis
II-2-1-a Type I.
II-2-1-b Secondary Osteoporosis
II-2-1-c Idiopathic Juvenile Osteoporosis
II-2-2 Causes of osteoporosis
II-2-2-a Peak Bone Mass
II-2-2-b Bone losses
II-2-3 Post menopausal osteoporosis
II-2-3-a Menopause
II-2-3-b Women specific osteoporosis
II-2-3-c Senile osteoporosis
II-3 Osteoporosis and the immunity processes
II-3-1 Inflammation and immunity
II-3-1-a Innate immunity (Inflammation)
II-3-1-b Inflammation and pain
II-3-1-c Acquired or Adaptive Immunity
-Specific immunity
-Specific immune response
-Humoral (antibody-mediated) Response -B-cells
-Cell Mediated Response - T Cells
-Summary
5
II-3-2 Osteoporosis, arthritis and inflammation markers
II-3-2-a Bones and Inflammation markers
II-3-2-b Bones and homocysteine
6
II-3-3 Osteoporosis and immunity
II-3-4 Aging, osteoporosis and immunity
II-4 Arthritis
II-4-1 Osteoarthritis
II-4-2 Rheumatoid arthritis
III- Beneficial effects of citrus on bone health
III-1 Relation between the skeleton and other organs
III-1-1 Relation between the skeleton and other organs
III-1-2 Inflammation and the skeleton
III-2 Vitamins and the skeleton.
III-2-1 Beneficial effects of ascorbic acid on the skeleton
III-2-1-a Role of ascorbic acid as vitamin C
III-2-1-B Other roles of ascorbic acid
III-2-2 Beneficial effects of folic acid on the skeleton
III-3 Flavonoids and the skeleton.
II-3-1 Flavonoids and inflammation
II-3-2 Flavonoids and bone loss
II-3-2-a Flavonoids
II-3-2-b Citrus Flavonoids
III-4 Carotenoids and the skeleton.
III-4-1 Effect of beta cryptoxanthin and other carotenoids on osteoporosis and bone
formation
III-4-2 Effect of beta-cyptoxanthin and anti-oxidants on rheumatoid arthritis
III-5 Limonoids and the skeleton.
III-6 Organic acids and the skeleton.
III-5 -1 Calcium citrate and malate and bones
7
III-5 -2 Potassium citrate and malate and bones
III-7 Oligosaccharides and bones
IV- Conclusion
IV-1 Bone physiology
IV-1-1 Mineral homeostasis
IV-1-1 Bone maintenance
IV-2 Bone losses
IV-2-1 Bone losses and immunity processes.
IV-2-2 Bone degradation diseases
IV-2-2-a Osteoporosis
IV-2-2-b Bones diseases associated to the immune processes
IV-2-2-c Bones diseases associated with kidney activity
IV-3 Beneficial effects of citrus.
IV-3-1 Ascorbic acid, vitamin C
IV-3-2 Folic acid
IV-3-3 Flavonoids and polymethoxylated flavones
IV-4-4 Carotenoids
IV-4-5 Organic acids
IV-4-6 Conclusions
V- Considerations
V-1 Mineral homeostasis
V-1-1 Mineral bioavailibility
V-1-2 Mineral loss by the bones
V-2 Age related changes in bone physiology
V-3 Bone diseases
8
V-3 -1 Bone promoting properties of citrus compounds.
V-3-2 Bone conserving properties of citrus compounds.
V-4 Bone and inflammation
VI- Summary: Bone and citrus
VII- General consideration on health and citrus
VIII-References
9
I-1 Bone structure
The skeleton is a complex tissue whose properties are influenced by a broad array of factors. The skeleton
interacts with many other organs of the body and particularly, the gastrointestinal tract, the blood system and
the kidneys. It serves as the body reservoir of c alcium and phosphate, and bone remodeling allows serum
concentrations of these ions to be tightly controlled. Superimposed on its role in mineral homeostasis, bone
also functions bio-mechanically to protect vital structures, anchor muscles, and absorb the impacts arising
from activity. Bone is a living, growing tissue. It is a porous mineralized structure, made up of cells, vessels,
crystals of calcium compounds (hydroxyapatite). The human skeleton is actually made up of two types of
bones: the cortical bone and the trabecular bone (Baron,1999)(Fig.1).
Fig.1
I-1-1 Cortical Bone
Cortical bone represents nearly 80% of the skeletal mass. It is also called compact bone, because it forms a
protective outer shell around every bone in the body. Cortical bone has a slow turnover rate and a high
resistance to bending and torsion. The cortical bone (Fig.1) is made of a series of adjacent and overlapping
formations called osteons or Harvesian systems. Each osteon is composed of a central vascular channel
surrounded by a kind of tunnel, called the Harvesian canal. The canals contain capillaries, aterioles, venules,
nerves. Between each osteon are interstitial lamellae (concentric layers of mineralized bone). Lamellar bone
gets it strength from its plywood-like construction: parallel layers of bone alternate in orientation by 90
degrees. Mature compact bone is by weight 70% inorganic salts (mainly calcium and phosphate in the form
of hydroxyapatite crystals) and 30% organic components (of which 90% is collagen and 10% proteoglycans
and glycoproteins important for calcification.) Osteoblasts initially secrete a product called osteoid,
containing type I collagen, proteoglycans, and various glycoproteins including one called osteocalcin, which
is involved in binding calcium ions (Einhorn, 1996)
10
I-1-2 Trabecular bone
This kind of bone only represents 20% of the skeletal mass, but 80% of the bone surface. Trabecular bone is
less dense, more elastic and has a higher turnover rate than cortical bone. It is found in the epipheseal and
metaphysal regions of long bones and throughout the interior of short bones. Trabelcular bone constitutes
most of the bone tissue of the axial skeleton: bones of the skull, ribs and spine. It is formed in an intricate
and structural mesh. Trabecular bone forms the interior scaffolding (Fig.1), which helps bone to maintain
their shape despite compressive forces. Trabecular bone is rigid but appears spongy , it is composed of
bundles of short and parallel strands of bone fused together. The center of the bone contains red, yellow
marrow, bone cells and other tissues (Shier et al, 1996)
I-1-3 Bone Marrow
Fig.2a Fig2b
Bone marrow (Baron, 1999, Sakuma et al, 2006) is the tissue comprising the center of large bones (Fig.2a).
It is the place where new blood cells are produced. Bone marrow contains two types of stem cells:
hemopoietic (which can produce blood cells) and stromal (which can produce fat, cartilage and bone).
Stromal stem cells have the capability to differentiate into many kinds of tissues. Hematopoietic stem cells
give rise to the three classes of bl ood cells: leukocytes, red blood cells (erythrocytes), and platelets
(thrombocytes) (Fig.2b).
11
I-1-4 Bone cells (Fig.3)
Fig.3 -Schematic Representation of Osteoclast and Osteoblast Lineages Schematic representation of the osteoclast (top)
and osteoblast (bottom) lineages. The two lineages are distinct, but there is regulatory interaction among the cells (vertical
arrows). Osteoclastsoriginate from a hematopoietic stem cell that can also differentiate into a macrophage, granulocyte,
erythrocyte, megakaryocyte, mast cell, B-cell, or T-cell. Osteoblasts originate from a mesenchymal stem cell that can also
differentiate into a chondrocyte, myocyte, fibroblast,or adipocyte.Skeletal metabolism is regulated by bone cells and their
progenitors. Among the population of bone cells are osteoblasts, osteocytes, osteoclasts, and lining cells
(Marcus,1994;Liam,1996,1999).
Fig.4
12
Osteoblasts (bone-forming cells) work at bone surfaces where they secrete osteoid. The major product of
osteoblasts is type 1 c ollagen, which along with other proteins, forms the organic osteoid matrix that is
mineralized to hydroxyapatite and influences the activity of osteoclasts. Osteoblasts express receptors to
many bone active agents such as Parathyroid hormone ( P TH). P arathyroid hormone-related protein
(PTHrP), vitamin D metabolites, gonadal and adrenal steroids, and certain cytokines and growth factors
(Liam, 1996,1999).
Osteoclasts (bone-resorbing cells) are responsible for the resorption (destruction) of old, worn out bone,
which is necessary for the repair of bone surfaces and the remodeling of bone. They are terminally
differentiated, large, multinucleated giant cells that arise from hematopoietic marrow precursors under the
influences of hormones, growth factors, and cytokines (Bruder, 2001). The osteoclast resorbs bone by
attachment with a ruffled border through adhesion molecules and by secretion of hydrogen and chloride ions
that dissolve mineral, they also promote lytic proteases, notably lysosomal proteases active at low pH,
metalloproteinases and cysteine proteinases that dissolve the bone protein matrix. In contrast to the
osteoblasts, mature osteoclasts have few receptors, except for calcitonin ( CT). After completing their
function, the terminally differentiated osteoclast undergoes apoptosis (cell death). (Fig.4)
Osteocytes are osteoblasts which have become embedded within the mineralized regions of bone. They are
involved in the sensing and translation of information about the internal bone environment.
Bone lining cells are flat, elongated cells that cover inactive bone surfaces. Their function is unknown, but
they may be osteoblast precursors or function to clean up resorption and formation debris.
Monocytes and macrophages mediate certain aspects of skeletal metabolism.
Marrow cells contribute to the population of bone cells.
Mast cells can be seen at sites of bone resorption and may also participate in this process. These immune
system cells play a key role in bone metabolism, especially resorption, by their interactions with bone cells.
13
14
Fig.5
I-1-5 Cartilage
Cartilage (Fig.5) is not part of the bone per se, but is usually found in close association with bones in the
body. It is a type of connective tissue which is tough, semi-transparent, elastic and flexible. The matrix or
ground substance of cartilage consists mainly of glycoprotein material (association of sugar and protein).
The cartilage cells (chondrocytes) are scattered in the matrix. Cartilage is covered by a dense fibrous
membrane, the perichondrium. No nerves or blood vessels are present in the cartilage (Baron, 1999).
I-1-5-a Hyaline cartilage.
Hyaline cartilage is semi-transparent and appears bluish-white in color. It is extremely strong, and very
flexible and elastic. Hyaline cartilage also contains chondrocytes, which are situated far apart in fluid-filled
spaces, the lacunae. There is an extensive amount of rubbery matrix between the cells and the matrix
contains a number of collagenous fibers. Hyaline cartilage occurs in trachea, the larynx, the tip of the nose,
in the connection between the ribs and the breastbone and also the ends of bone where they form joints.
Temporary cartilage in mammalian embryos also consists of hyaline cartilage. Hyaline cartilage is
responsible for the longitudinal growth of bone in the neck regions of the long bones.
I-1-5-b White fibrocartilage.
White fibrocartilage is an extremely tough tissue. The orientation of the bundles depends upon the stresses
acting on the cartilage. The collagenous bundles take up a direction parallel to the cartilage. Fibrocartilage is
found as discs between the vertebrae between the pubic bones in front of the pelvic girdle and around the
edges of t he articular cavities such as the glenoid cavity in the shoulder joint. The cartilage between the
adjacent vertebrae absorbs the shocks that will otherwise damage and jar the bones while we run or walk.
15
I-1-5-c Elastic cartilage.
Basically elastic cartilage is similar to hyaline cartilage, but in addition to the collagenous fibers, the matrix
of the elastic also contains an abundant network of branched yellow elastic fibers. They run t hrough the
matrix in all directions. This type of cartilage is found in the lobe of the ear, the epiglottis and in parts of the
larynx.
I-2 Bone homeostasis
As seen in Figure 6, the skeleton homeostasis is closely associated with that of several other organs. It is the
reservoir of calcium for many physiological functions, and it serves a similar but not so unique role for
phosphorus and magnesium ( Bruder et al, 2001; Brown, 2001). Skeletal calcium is controlled through the
regulatory pathways of the gastrointestinal (GI) tract and the kidney, and this regulation is mediated in bone
by the bone forming cells ( os teoblasts) and the bone resorbing cells (osteoclasts). Calcium reaches the
skeleton by being absorbed from the diet in the GI tract. Absorbed dietary calcium then enters the
extracellular fluid (ECF) space and becomes incorporated into the skeleton through the process of
mineralization of the organic matrix of bone. Calcium is filtered by the kidney at a rate of about six grams
16
per day, where up to 98 percent of it is reabsorbed (Fig.7).
17
Fig.7 - Schematic Representation of Calcium and Skeletal Metabolism Abbreviations: A, absorption; S,
secretion; ECF, extracellular fluid; GF, glomerular filtration; TR, tubular reabsorption. The dark vertical
line between bone and ECF represents bone surface and bone-lining cells. Shaded area represents labile
skeletal calcium.
The regulation of bone and bone mineral metabolism results from the interactions among three hormones parathyroid hormone (PTH), calcitonin (CT), and vitamin D (VD) - at these three target organs - bone,
kidney, and GI tract - to regulate bone minerals - calcium and phosphorus. Other hormones and minerals
described in Figs. 6,7 and Table I are also involved in maintaining bone homeostasis (Deftos, 1998; Brown
2001; Bruder et al, 2001)
18
Table I
I-2-1 Cellular and intracellular calcium and phosphorous metabolism
Calcium, phosphorous, and magnesium, are transported to blood from bone, renal, and GI cells, and vice
versa ( Drezner,2002; Yanagawa and Lee,1992; Favus 1992). These transport mechanisms can be through
cells (transcellular) and around cells (pericellular). The cellular transport is mediated by the membrane
structures illustrated in Figure 8 a nd by binding transport proteins (Reuss 2000, Be rger 2000). T he
pericellular transport is generally passive and mediated by mineral gradients. These mechanisms also involve
corresponding co-transportation and exchange-transportation with other ions, notably sodium, potassium,
chloride, hydrogen, and bicarbonate, some of which are powered by ATP hydrolysis. Similar mechanisms
allow for the intracellular distribution of calcium, where it partitions primarily between the mitochondria and
cytosol (Fig.8).
19
Fig. 8. Schematic representation of cellular transport of bone minerals. The model can be applied to transport of calcium,
magnesium, and phosphorus for cells of the renal tubules, gastrointestinal tract enterocytes, and bone cells. The mineral
transport can be with or against a gradient. Lumen refers to GI and renal tracts; for bone, it can refer to bone marrow,
blood, and/or matrix space. The site of the indicated membrane transport structures is schematic. Microsomes designate
other intracellular organelles such as secretory vesicles and endoplasmic reticulum.
Mineral homeostasis requires the transport of calcium, magnesium, and phosphate to their target cells in
bone, intestine, and kidney. The pericellular transport is usually diffusional, down a gradient, and is not
20
hormonally regulated. Transport across cells is more complex and usually against a gradient. This is an
active transport that requires energy. Once through the lumenal cell membrane, the minerals can cross the
cell into the extracellular fluid compartment. For bone cells, the corresponding compartments are marrow
and blood (Daftest, 1998; Ruder et al, 2001). Ca lcium is transported by the interaction among a family of
proteins that include calmodulin, calbindin, integral membrane protein, and alkaline phosphatase; the latter
three are vitamin D dependent (Favus 1992). Exit from the cell is regulated by membrane structures similar
to those that mediate entry.
I-2-1-a Calcium metabolism
Serum and extracellular calcium concentrations in mammals are closely regulated within a narrow
physiological range that is optimal for the many normal cellular functions affected by calcium in many
tissues ((Deftos, 1998; Bruder et al, 2001). Intracellular calcium, which serves as second messenger in many
signal transduction pathways, is also tightly controlled, but at concentrations several orders of magnitude
lower than extracellular calcium. Extraskeletal calcium accounts for only 1% of the total body calcium and is
primarily sequestered in bone. Approximately 10 g/day are filtered at the glomerulus (kidney) and most is
reabsorbed by the renal tubules, with only a few hundred milligrams appearing in the urine each day. The
skeleton turns over about 250 mg/day of calcium. The turnover is mediated by bone-forming osteoblasts and
bone resorbing osteoclasts.
21
I-2-1-b Phosphorus metabolism
Phosphorus is widely distributed and serves a variety of biological functions. Eighty five % of the
phosphorus is present in the skeleton as hydroxyapatite, the remaining 15 % is distributed among
extraskeletal sites like phosphoproteins, phospholipids, and nucleic acids. The regulation is not as tight as it
is for calcium. Phosphorus comes from foods. Absorption takes place at a site distal to the duodenum and
utilizes both calcium dependent and calcium independent mechanisms that can be active or passive. The
most significant quantitatively is postprandial passive absorption. Approximately 60-80% is absorbed
primarily by a diffusional process without a significant saturable component; however, there is regulation by
the calcitropic hormones, especially Vitamin D, whose active metabolites increase absorption. Renal
phosphate reabsorption controls the concentration of phosphate in the serum. Urinary phosphate is the major
route of homeostatic regulation (Yanagawa and Lee,1992). About 90% is filtered, with reabsorption being
the major regulatory step, primarily at the proximal tubule, where 80% is reabsorbed, mostly in the
convoluted segment. Parathyroid hormone (PTH) and calcitonin (CT), increase phosphorus excretion, and
Vitamin D decreases its excretion. The most important factors regulating proximal phosphate reabsorption
are PTH and the dietary phosphate intake. By resetting the renal phosphate threshold, PTH allows the kidney
to prevent increases in serum phosphate with calcium. Other factors that regulate phosphate reabsorption
include growth hormone and insulin which increase proximal phosphate reabsorption.
I-2-2 Skeletal metabolism
The metabolic function of bone is to provide a homeostasis mineral reservoir for calcium, magnesium and
phosphorus ( Daftest, 1998; Brown, 2001; Ruder et al, 2001). These bone minerals can be mobilized to
maintain systemic mineral homeostasis. This metabolic function of bone prevails over its structural function
in that calcium and other minerals are removed from and replaced in bone to serve systemic homeostatic
needs irrespective of loss of skeletal structural integrity. As seen previously bone consists of a mineral phase
and an organic phase (Brown , 2001). The major component of the mineral phase is hydroxyapatite crystal
and the major component of the organic phase is type 1 collagen which, with other bone proteins, comprises
the osteoid matrix of bone. The organic components of bone are products of the osteoblast. Bone minerals
are present in two forms in the skeleton. Hydroxy apatite crystals, represented by the formula
Ca10(PO4)6(OH)2, are the major forms and occur in mature bone. Amorphous calcium phosphate comprises
the remainder; it occurs in areas of active bone formation and matures through several intermediate stages to
hydroxyapatite. The end result is a highly organized amalgam of protein, primarily collagen, and mineral,
primarily hydroxyapatite, that has sufficient structural integrity to serve the mechanical functions of the
skeleton. Upon completion of this process, the osteoblast becomes encased in bone and becomes an
osteocyte. Cortical bone comprises approximately 80% of the skeleton. However, its surface area is only one
fifth that of trabecular bone, so trabecular bone is metabolically more active than cortical bone, with an
annual turnover (remodeling) of approximately 20% to 30% for the former and 3% to 10% for the latter. A
given skeletal site in the adult is remodeled approximately every three years. Bone mass is acquired up to the
fourth to fifth decade, with a rapid phase during adolescent growth. Most of peak bone mass is genetically
determined. Women have approximately 30% less peak bone mass than men and experience an accelerated
loss after the menopause. Both genders experience age-related loss of bone mass.
22
I-3 Bone formation, development, growth and remodeling
I-3-1 Bone Formation and growth
There are two major mechanisms of bone formation, involving either flat or long bones (Lian et al 1999).
I-3-1-a Flat bones
Intramembranous ossification makes "flat bones" (parietal bones of the skull and bones of the jaws). The
following steps can be summarized. a) Condensation of mesenchyme cells, which then differentiate directly
into osteoblasts. b) Bone matrix (osteoid) is produced by osteoblasts from many points in the condensed
mesenchyme and these ultimately fuse into a bone. c) Blood vessels ultimately invade this bone, bringing in
osteoclasts for remodeling and a marrow cavity is produced
I-3-1-b Long bones
Endochondral ossification forms the long bones and all other bones using hyaline cartilage models (Fig.9).
Fig.9
23
Growth in length of long bones (Fig.10). a) Within the growth plate, the cell proliferation rate
roughly equals the rate of chondrocyte death, so that the growth plate cartilage stays the same size. b) New
bone growth occurs in the hypertrophic cartilage. This is where length is added to the growing bone. c)
Growth stops when the growth plate cartilage disappears. Adults no longer have growth plates in their long
bones. Increased estrogen level is believed to play a role in the closing of the growth plate
Growth in bone diameter occurs at the same time as length growth by an "appositional growth
mechanism in which osteoprogenitor cells and osteoblasts continue to form in the periosteum and produce
lamellae of new compact bone beneath the periosteum which is soon remodeled.
Fig. 10
24
I-3-2 Skeletal repair.
Besides growth that stops with adulthood, bones are able to repair most damages including fractures. A
series of mechanisms is initiated to allow skeletal repair to occur ( Marcus 1994, Pagani et al, 2005 ). 1-Bone
repair
- Breakage of the calcified matrix in bone results in the breakage of vasculature. This causes release
of platelets and formation of a blood clot around the broken area. -The blood clot is invaded by
fibroblasts/mesenchymal precursor cells from periosteum and endosteum that produce connective
tissue or cartilage to rejoin the broken bone.
-Within the connective tissue, endochondral and intermembranous bone formation occurs and a
bone callus forms (primary or woven bone),
-This primary bone is then remodeled to secondary bone. 2- Repair of cartilage is much more
difficult
-Cartilage is avascular except for the perichondrium and so repair is very slow due to
lack of nutrients and oxygen. Nutrition and O2 supply comes from perichondrium if
present, or from exchange of synovial fluid in cartilage without a perichondrium.
-Damaged cartilage is slowly invaded by perichondrial cells that differentiate into chondroblasts and
synthesize matrix and gradually repair the cartilage. However, cartilage matrix is not reformed or
repaired efficiently and repaired cartilage that is produced in the area is often malformed.
-Repair of cartilage that does not have a perichondrium (such as articular cartilage) is
very
poor due to lack of a source for new chondroblasts
I-4 Bone Modeling and Remodeling
The processes of bone formation by osteoblasts and bone resorption by osteoclasts produce opposite results –
formation produces bone mass and resorption reduces bone mass (Aguila and , Rowe, 2005). These
processes can work in two ways. 1- They can work at the same time on different surfaces; the net effect is an
increase in bone. This process is called modeling and is responsible for shaping or sculpting the skeleton
during growth. 2- They can work together on the same area but at different times to renew bone; the net
effect is no change or a net loss. This process is called remodeling and is responsible for removing old bone
and forming new bone (Einhorn, 1996).
I-4-1 Bone modeling
Modeling begins during infancy and childhood, whereas remodeling continues during adulthood. During
infancy and childhood, bones grow and attain mature size and shapes. The osteoblast usually does more
work than the osteoclast because the net effect is an increase in bone mass. Modeling is age dependent. It
slows during adolescence and is nonexistent by the mid-twenties. At this point, remodeling activity
increases. Old bone is continually resorbed and new bone tissue is formed in its place (Einhorn, 1996). Like
the skin, bone replaces itself throughout life. As a person ages, repeated strain or stress on bone from
ordinary mechanical use, such as physical activity, results in the development of microdamage.
Accumulation of microdamage can reduce the strength of bone, and therefore, remodeling is necessary to
repair the damage. Remodeling allows the microdamage of worn or injured bone to be replaced and helps to
maintain skeletal strength. Any substantial decrease in the rate of remodeling may increase the risk of
spontaneous fractures because the skeleton's ability to repair itself is decreased (Pagani et al, 2005 ).
25
I-4-2 Bone remodeling
Bone density and structure is maintained through a balance of bone resorption by osteoclasts and bone
deposition by osteoblasts (Fig. 11). T he combination of simultaneous resorption and deposition creates
continual remodeling of bone while excess osteoclast activity leads to an imbalance and a loss of bone
density, causing osteoporosis. RANK (receptor activator of NF-kB ligand) is a receptor in the TNF receptor
gene family that is involved in osteoclast differentiation. RANK-ligand, also called osteoprotegerin-ligand,
binds to RANK, induces receptor dimerization, and activates downstream signaling. Osteoprotegerin is a
decoy receptor for RANK-ligand that suppresses osteoclast activity and bone remodeling, helping to
maintain balanced bone remodeling. RANK-ligand and osteoprotegerin are produced by osteoblasts and
some factors regulate osteoclast activity indirectly through their action on the expression of these factors by
osteoblasts. Binding of RANK-ligand to RANK activates signaling through TNF receptor-associated factor 6
(TRAF-6). TRAF-6 induces several downstream signaling events, including activation of NF-kB, c-Fos (one
of a family of intermediate early genes) and the kinase JNK1. Remodeling involves both the osteoclasts and
osteoblasts. Osteoclasts are stimulated to resorb bone at specific sites, and osteoblasts are activated to replace
the bone. The two processes are "coupled" (bone resorption is followed by bone formation). Postmenopausal
women not taking estrogen have net bone loss because resorption is greater than formation. In cortical bone,
osteoclasts create resorption tunnels parallel to the long axis of the bone and the osteoblasts fill in the tunnel
with new bone. In trabecular bone, osteoclasts create shallow excavations toward the center, which the
osteoblasts then fill in with new bone. In remodeling, bone formation by the osteoblast always follows
osteoclastic resorption at the same resorption site (Einhorn, 1996).
In trabecular bone it takes 3-6 months to complete a remodeling cycle, with about one month for resorption
followed by five months for formation (Baron, 1999). It is estimated that the remodeling cycle takes longer
in cortical bone (Einhorn, 1996). The number of remodeling units activated within a given space over a
given period of time determines the rate of bone turnover. Although trabecular bone represents only 20% of
skeletal mass, it represents 80% of bone turnover because of its large surface area-to-mass ratio. This is a far
greater percentage of bone turnover compared with cortical bone, which makes up 80% of skeletal mass and
accounts for only 20% of total bone turnover in a given period. Remodeling is estimated to account for
replacement of 20% of the adult skeleton each year and can be divided into five distinct phases: activation,
resorption, reversal, formation and resting.
Fig. 11. Normal bone remodeling cycle. The schematic diagram shows (1) the creation by osteoclasts of a
resorption cavity; (2) smoothing of erosion cavities by mononuclear cells (reversal); (3) differentiation of
osteoblasts within erosion cavities followed by the onset of matrix synthesis and mineralization; and (4) a
prolonged resting phase.
26
I-5 Mineral homeostasis
A steady supply of calcium and phosphorus are needed to perform the daily functions of the body. A
sufficient supply of calcium must be maintained in the blood at all times for normal muscle, bone, and nerve
function, and sufficient levels of calcium must be maintained in the bone for growth and repair (Gatton and
Hall, 2000). This process of maintaining adequate supplies of calcium and other minerals is called mineral
homeostasis.
I-5-1 Bone-blood transfer
The skeletal and circulatory systems maintain an intimate association. Bone tissue requires a steady supply
of nutrients which are transported by the blood vessels that penetrate the bone and branch into capillaries.
Besides supplying nutrients to the osteocytes, the capillaries carry away waste products and minerals. The
osteocytes may release calcium and phosphorus into the extracellular fluid space between themselves and the
capillaries. Calcium is an essential substance in body metabolism as well as an important part of bone
structure. It is necessary for proper muscle contraction and nerve impulse conduction. Even a slight decline
in the circulating level of calcium can cause the nerves and muscles to spasm. Thus, sufficient calcium must
be available for normal body functions (Guyton and Hall, 2000). C alcium absorption increases during
27
growth periods and decreases with advancing age. Also, not all forms of calcium are equally absorbed. The
gastrointestinal (GI) system ingests and processes food containing calcium and other nutrients. The calcium
that is absorbed into the bloodstream is then circulated through the kidneys, whereas the remainder is
excreted with waste products of the intestinal tract. The primary purpose of t he kidneys is to filter waste
substances from the blood and excrete them in the urine. The bulk of the calcium that normally enters the
kidneys is reabsorbed back into the bloodstream with small amounts of calcium being excreted in the urine.
However, the kidneys can exert significant control over calcium levels in the blood by reabsorbing more or
less calcium. T he primary sites of calcium metabolism and homeostasis are bone, intestine, and kidney
(Fig.12).
28
I-5-2 Hormonal control of bone homeostasis
I-5-2-a Effect of parathyroid hormone on bone remodeling
The parathyroid glands secrete an hormone called parathormone (PTH), it causes an increase in blood
calcium levels and a decrease in blood phosphate levels. There are four parathyroid glands located on the
posterior surface of the thyroid gland. Each parathyroid gland consists of tightly packed secretory cells that
are associated with capillaries. PTH is secreted into the bloodstream and transported to the target tissues in
response to a decrease in blood calcium levels. It causes a subsequent increase in blood calcium levels.
Figure 13 summarizes the effects of PTH. The secretion of PTH by the parathyroid glands is regulated by a
negative feedback
mechanism based
on the blood
calcium level. As
the calcium level
increases, less
PTH is secreted;
as the calcium
level decreases,
more PTH is
released (Shier et
al., 1996]). PTH
causes increased
osteoclastmediated bone
resorption and
movement of
calcium into the
extracellular fluid.
Paradoxically, the receptors for PTH are on osteoblasts rather than osteoclasts. It is believed that PTH
activation of osteoblasts results in their release of factor(s) that activate osteoclasts.
29
Fig. 13.
PTH causes the kidneys to conserve calcium and raise the blood calcium level by excreting less calcium in
the urine. At the same
time, PTH
diminishes phosphate
reabsorption and
promotes excretion of
phosphate in the
urine (Guyton and
Hall, 2000). PTH
secretion results in the
release of calcium
from bone and an
increase in blood
calcium levels. PTH
also enhances
intestinal absorption
of both calcium
and phosphorus by
influencing the
metabolism of
vitamin D. PTH
stimulates the kidneys
to produce the
active form of vitamin
D – 1,25-
dihydroxycholecalciferol. Vitamin D controls the mechanism by which calcium is absorbed from the
intestine. Therefore, PTH increases vitamin D, which increases intestinal calcium absorption.
I-5-2-b Effect of vitamin D on bone remodeling
Vitamin D, or cholecalciferol, plays an important role in the intestinal absorption of calcium, thus enhancing
bone formation. Vitamin D can be obtained directly from the diet or synthesized in the body. It is considered
to be a hormone and a vitamin. The synthesis of vitamin D begins with cholesterol, which is obtained in the
diet or synthesized endogenously. Cholesterol is converted into provitamin D (dehydrocholesterol) and
stored in the skin. When the skin is exposed to sunlight, the provitamin is converted to vitamin D. Most
people who live in temperate climates have adequate exposure to sunlight and fulfill their daily requirement
for vitamin D in this manner. Elderly people who become housebound are at risk for hip fractures in part
because of low vitamin D levels (decreased sunlight or intake). Vitamin D is metabolized in the liver and
kidney. In the liver, vitamin D is changed to 25hydroxycholecalciferol. The kidney converts it to the active
form of vitamin D, 1,25dihydroxycholecalciferol. The active form can only be made in the presence of PTH.
The active form of vitamin D controls the mechanism by which calcium is absorbed from the intestine. Thus,
PTH indirectly regulates intestinal calcium absorption by causing the kidneys to produce the active form of
vitamin D (Guyton and Hall, 2000). The role of vitamin D is summarized in Figure 14
30
Fig.14.
I-5-2-c Effect of calcitonin on bone remodeling
The hormone calcitonin, which the thyroid gland produces, acts as the physiologic antagonist to PTH. The
major site of calcitonin action is the bone where it decreases osteoclast activity through calcitonin receptors
on the osteoclast.These effects of calcitonin on blood calcium levels are mainly temporary, lasting for a few
hours to a few days. Calcitonin also has minor effects on the kidney and intestinal tract. Again, these effects
are opposite to those of PTH. Calcitonin, like PTH operates on a negative feedback system. When the blood
calcium level rises, more calcitonin is secreted. However, calcitonin disappears rapidly from circulation, and
may function primarily in
short-term regulation.
In the long term, the more
powerful
PTH
mechanism overrides the
action of calcitonin.
The exact role of calcitonin
in
adults
remains
unknown (Kanis, 1994).
31
I-5-2-d Effect of sex hormones and other hormones on bone remodeling
As mentioned previously, the skeleton is comprised of living tissue that continually renews itself throughout
life. The osteoclasts function to dissolve older bone and the osteoblasts produce new bone. Osteoblasts and
osteoclasts require hormonal guidance to function properly. T he female hormone (estrogen) and the male
hormone (testosterone) are involved in maintaining bone homeostasis in both men and women. Osteoblasts
depend primarily on progesterone and testosterone, while osteoclasts need estrogen. Estrogen, the primary
female sex hormone, regulates the activity of osteoclasts, which results in the slowing of the process of older
bone dissolution. Estrogens affect bone at several levels (Zarcone et al. 1997; Castelo-Branco 1998): They
stimulate bone formation by direct action on os teoblasts (bone-producing cells), increase the level of
transforming growth factor ( TGF-b) a regulator of growth and proliferation of cells which seems to inhibit
osteoclasts (bone-resorbing cells). They also act on os teoclasts by inhibiting their function, promoting
apoptosis and acting on bone angiogenesis (The process of developing new blood vessels).
Progesterone promotes the production of osteoblasts (Prior 1990; Prior et al. 1994) w hich are required to
affect new bone formation. Natural progesterone has been shown to stimulate osteoblastmediated new bone
formation which is required to prevent and reverse osteoporosis. Testosterone and dihydrotestosterone help
maintain adequate bone mineral density. Several other hormones also act on bone, although their roles are
less clearly understood. Thyroid hormone is necessary for normal bone growth and remodeling (Venken et
al, 2006; Nair and Saag, 2006).
32
II- Bone disorders
II-1 Bones and mineral homeostasis impairment
The permanent process of remodeling mediated by osteoblasts and osteoclasts is one of the main properties
of the skeleton. The activity of these cells is regulated by many factors. One well-studied regulatory system
involves the maintenance of calcium homeostasis through a network whose main regulatory components
include ionized calcium, phosphate, parathyroid hormone and active vitamin
D. Bone health to a large extent depends on mineral homeostasis. Minerals are absorbed through the
digestive tract, then eventually are eliminated or reabsorbed through the kidneys.
II-1-1 Mineral absorption
Diseases of calcium, phosphate, and skeletal metabolism are most common (Bronner, 1998). T hey can
involve abnormalities in the serum concentrations of the two minerals, especially calcium; abnormalities of
bone; and abnormalities of the major regulating organ systems, especially the parathyroid gland, kidney and
gastrointestinal (GI) tract. The serum calcium concentration can be abnormally high, as in malignancy and
primary hyperparathyroidism, or abnormally low as it is in renal failure and hypoparathyroidism. The
skeleton can have low bone density, as occurs in osteoporosis and osteomalacia, or high bone density as
Paget's disease and osteopetrosis. The GI tract can exhibit low calcium absorption, as in mal absorptive
states, or high calcium absorption, as in vitamin D intoxication and the milk-alkali syndrome. The kidneys
can fail to excrete calcium, as occurs in some hypercalcemic disorders; over excrete calcium, as in some
cases of nephrolithiasis; under excrete phosphorus, as in renal failure; and over excrete phosphorus, as in
some renal tubular disorders. Corresponding events occur for magnesium.
The vast bulk of mineral absorption occurs in the small intestine. The mechanisms of absorption of calcium
and iron have been extensively studied. Minerals are re quired for health, but most may be toxic when
present at higher than normal concentrations. In many cases intestinal absorption is a key regulatory step in
mineral homeostasis (Bronner, 1998; Yanagawa and Lee,1992; Favus,1002; Reuss, 2000).
II-1-1-a Calcium
The quantity of calcium absorbed in the intestine is controlled by how much calcium has been in the diet
during recent periods of time. Calcium is absorbed by two distinct mechanisms, and their relative magnitude
of importance is set by how much of the mineral has been provided by the diet. Active, trans-cellular
absorption (Fig.15) occurs only in the duodenum when calcium intake has been low. This process involves
import of calcium into the enterocyte, transport across the cell, and export into extracellular fluid and blood.
Calcium enters the intestinal epithelial cells through voltage-insensitive channels and is pumped out of the
cell via a calcium-ATPase.
33
Fig. 15.
The rate limiting step in
absorption is transport across
greatly enhanced by the carrier
synthesis of which is totally
Passive pericellular absorption
ileum, and, to a much lesser
dietary calcium levels have
this case, ionized calcium
junctions into the basolateral
and hence into blood. Such
having higher concentrations of
intestinal lumen than in blood.
transcellular
calcium
the epithelial cell, which is
protein
calbindin,
the
dependent on vitamin D.
occurs in the jejunum and
extent, in the colon when
been moderate or high. In
diffuses through tight
spaces around enterocytes,
transport
depends
on
free calcium in the
(Bronner, 1998).
II-1-1-b Phosphorus
Phosphorus is predominantly absorbed as inorganic phosphate in the upper small intestine. Phosphate is
transported into the epithelial cells by cotransport with sodium, and expression of t hese transporters is
enhanced by vitamin D (Bronner, 1998).
II-1-1-c Minerals and nutrition
Nutrition is an important "modifiable" factor in the development and maintenance of bone mass and in the
prevention of osteoporosis. The improvement of calcium intake in prepuberal age translates into gain in
bone mass and into higher Peak Bone Mass (PBM) value at completion of physiological growth.
Individuals with higher PBM achieved in early adulthood will be at lower risk for developing
osteoporosis later in life. Once the PBM is reached, it is important to maintain the bone mass gained and
reduce the loss. The diet must be nutritionally balanced with a caloric intake adequate for each individual.
Bacciottini and Brandi (2004) showed that adequate nutrition may represent a way to prevent bone diseases.
An adequate intake of alkali-rich foods may help promote a favorable effect of dietary protein on the
skeleton. The diet should contain food with high amounts of calcium, potassium, magnesium and low
amounts of sodium (2g/day or l ess). Other vitamins (Vit. A, C, E , K) and minerals (phosphorus, fluoride,
iron, zinc, copper and boron) are required for normal bone metabolism (Miggiano and Gagliardi,2005).
Tucker (2004) reported the effect of a variety of nutrients on bone status. They include minerals, magnesium,
potassium, copper, zinc, silicon and sodium; and vitamins, C, K ,B12 and A; and macronutrients: protein,
fatty acids and sugars. In addition, foods and food components, including milk, fruits and vegetables have
been examined. The evidence suggests that prevention of bone loss through diet is complex and involves
many nutrients.
34
Calcium is absorbed in the intestine by both active and passive mechanisms. T he active component
dependents on vitamin D. and is mainly located in the upper intestine. P assive absorption is more
specifically located in the lower intestine. However, the gut reduces active calcium absorption in response to
a high-calcium delivery, but maintains active transport i n response to a low-calcium diet. Therefore, the
efficiency of calcium absorption is inversely related to intake. Dietary data and indirect measures of bone
health indicate that bioavailability is important when habitual intakes are low, especially during periods of
bone growth or loss. Yamada and Inaba (2004) pointed out that the intake of magnesium and trace elements,
such as zinc and copper is important for the regulation of bone metabolism.
Poor bone health has also been associated with impaired GI mineral absorption. Osteoporosis is a frequent
finding in patients with Crohn's disease and ulcerative colitis. The prevalence of vertebral fractures in those
patients with significantly reduced bone mineral density is up to 22% (Reinshagen and Von Tirpitz, 2004).
II-1-2 Mineral reabsorption
The maintenance of calcium homeostasis is established through a link between bone and kidney. One of the
kidney's endocrine functions is the activation of vitamin D, while electrolyte homeostasis is one of its
excretory functions. (Mondry et al, 2005).
Fig. 16.
35
-28-
36
++
++
Many physiological functions rely on the precise maintenance of body calcium (Ca ) and magnesium (Mg )
balance, which is tightly regulated by the concerted actions of intestinal absorption, renal reabsorption, and
exchange with bone. The kidney plays an important role in the homeostasis of divalent ions. Most Ca and
Mg reabsorption occurs in the proximal tubules and the thick ascending limb of Henle's loop via a passive
paracellular pathway (Fig.16). At the level of the distal convoluted tubule (DCT) and the connecting tubule
(CNT), Ca and Mg are reabsorbed via an active transcellular route. Reabsorption of divalent cations in
these latter segments is regulated in a Ca , Mg -specific manner and determines the final excretion in the
urine. Dysregulation or malfunction of these influx pathways has been associated with renal Ca and Mg
losses ( Thebault, 2006)
++
++
++
++
++
++
++
++
II-1-3 Chronic acidemia
Chronic acidemia or high blood acidity is generated by the oxidation of excess sulfur containing amino acids
to sulfate ions, and is related to the dietary protein level. To neutralize the acid the body may mobilize its
calcium reserve (mainly from bone) and the kidneys neutralize the fixed acidity by generating calcium
bicarbonate. Low-grade metabolic acidosis can have a deleterious effects on bone calcium status ( Demigne
et al, 2004 a,b, 2006; Sabboh et al 2005,2006).
Bone matrix is deposited and mineralized by osteoblasts and it is resorbed by osteoclasts, multinucleate cells
that excavate pits on bone surfaces. It has been known since the early 20th century that systemic acidosis
causes depletion of the skeleton, an effect assumed to result from physicochemical dissolution of bone
minerals. A rnett (2003) showed that resorption pit formation by cultured osteoclasts was absolutely
dependent on extracellular acidification; these cells are inactive at pH levels above 7.3 and show maximum
stimulation at a pH of about 6.9. Bone resorption is most sensitive to changes in H+ concentration at a pH of
about 7.1 (w hich may be close to the interstitial pH in bone). In this region pH shifts of < 0.05 uni ts can
cause a doubling or ha lving of pit formation. In whole-bone cultures, chronic HCO3- acidosis results in
similar stimulations of osteoclast-mediated Ca release, with a negligible physico-chemical component. In
vivo, severe systemic acidosis (pH change of about -0.05 to -0.20) often results from renal disease; milder
chronic acidosis (pH change of about -0.02 to -0.05) can be caused by excessive protein intake, acid feeding,
prolonged exercise, ageing, airway diseases or m enopause. Acidosis can also occur locally as a result of
inflammation, infection, wounds, tumors or diabetic ischaemia. Cell function, including that of osteoblasts, is
normally impaired by acid; the unusual stimulatory effect of acid on osteoclasts may represent a primitive
'fail-safe' that evolved with terrestrial vertebrates to correct systemic acidosis by ensuring release of alkaline
bone minerals when the lungs and kidneys are unable to remove sufficient H+ equivalent. The present
results suggest that even subtle chronic acidosis could be sufficient to cause appreciable bone loss over
time.
++
Similarly, Krieger et al. ( 2004) showed that the effect of pH on acid-induced changes in bone minerals is
consistent with a role for bone as a proton buffer. In response to metabolic acidosis in an in-vitro bone organ
culture system, we observed a fall in mineral sodium, potassium, carbonate and phosphate, which each
buffer protons and in vivo should increase systemic pH toward the physiologic normal. Initially, metabolic
acidosis stimulates physicochemical mineral dissolution and subsequently cell-mediated bone resorption.
Acidosis suppresses the activity of bone-resorbing cells, osteoblasts, decreasing gene expression of
specific matrix proteins and alkaline phosphatase activity. There is concomitant acid stimulation of
prostaglandin production by osteoblasts, which acting in a paracrine manner increases synthesis of the
37
osteoblastic receptor activator of nuclear factor kappa B ligand (RANKL). The acid induction of
RANKL then stimulates osteoclastic activity and recruitment of new osteoclasts to promote bone resorption
and buffering of the proton load. Both the regulation of RANKL and acid-induced calcium efflux from bone
are mediated by prostaglandins. Metabolic acidosis, which occurs during renal failure, renal insufficiency or
renal tubular acidosis, results in decreased systemic pH and is associated with an increase in urine calcium
excretion. The apparent protective function of bone to help maintain systemic pH, which has a clear survival
advantage for mammals, will come partly at the expense of its mineral stores.
38
II-1-4 Renal osteodystrophy
Impaired renal function leads to disturbances in this regulatory system, resulting in the complex syndrome of
renal osteodystrophy that affects the majority of patients with chronic renal failure. (Mondry et al, 2005)
Renal osteodystrophy is a bone disease that occurs when your kidneys fail to maintain the proper levels of
calcium and phosphorus in your blood. It's a common problem in people with kidney disease and affects 90
percent of dialysis patients. Renal osteodystrophy is most serious in children because their bones are still
growing. The condition slows bone growth and causes deformities. One such deformity occurs when the legs
bend inward toward each other or outward away from each other; this deformity is referred to as "renal
rickets." Another important consequence is short stature. Symptoms can be seen in growing children with
renal disease even before they start dialysis. The bone changes from renal osteodystrophy can begin many
years before symptoms appear in adults with kidney disease. For this reason, it's called the "silent crippler."
If left untreated, the bones gradually become thin and weak, and a person with renal osteodystrophy may
begin to feel bone and joint pain. There's also an increased risk of bone fractures (Malluche and MonierFaugere, 2006).
II-2 Osteoporosis
Fig.17.
39
To function properly the skeleton requires a delicate equilibrium between anabolic and catabolic processes.
Many bone diseases occur when the homoeostatic equilibrium is broken. The major bone impairment is a
loss of strength leading to micro and macro fractures (Phelps, 2000).
Osteoporosis is a thinning and weakening of the bones, usually associated with the aging process. With
osteoporosis, the amount of calcium present in the bone slowly decreases. The structural integrity of
trabecular bone is impaired. Cortical bone becomes more porous and thinner and more likely to fracture. A
committee of the World Health Organization (WHO) has defined osteoporosis based on bone density. Using
standardized bone density measurements of the total hip, "normal" bone density is greater than 833 mg/cm .
In "Osteopenia" it is between 833 and 648mg/cm . Osteoporosis is characterized by a density lower than
648mg/cm , and "severe (established) osteoporosis" is when there has been a fragility fracture.(NIH Consens
Statement 2000 , Looker 1995,1997). Although these criteria are widely used, they were devised in
Caucasian females so there will be some differences when these levels are applied to non Caucasian females
or to males in general. Despite this flaw, measurement of BMD is used daily and has proven to be very
helpful in all groups. Some men will be subject to increased fracture rates when they have significantly less
BMD than the predicted fracture level for women. In other words, some men will be at increased risk for
fracture even when they have osteopenia. Osteoporosis is different from most other diseases or common
illnesses in that there is no one single cause. The overall health of a person's bones is a function of many
things ranging from how well the bones were formed as a youth, to the level of exercise the bones have seen
over the years. During the first 20 years of life, the formation of bone is the most important factor, but after
that point it is the prevention of bone loss which becomes most important. Anything which leads to
decreased formation of bone early in life, or loss of bone structure later in life will lead to osteoporosis and
fragile bones which are subject to fracture (Reed, 2000).
2
2
2
Pasco et al (2006) followed 616 postmenopausal women aged 60-94 years for about six years to determine
the incidence of fractures according to age, BMD and the presence of a prior fracture. Based on WHO
criteria, 37.6% of the women had normal total hip BMD, 48.0% had osteopenia and 14.5% had osteoporosis.
The incidence of fracture during follow-up was highest in women with osteoporosis, but only 26.9% of all
fractures arose from this group; 73.1% occurred in women without osteoporosis (56.5% in women with
osteopenia, 16.6% in women with normal BMD). Decreasing BMD, increasing age and prior fracture
contributed independently to increased fracture risk.
II-2-1 Types of Osteoporosis
There are different types of osteoporosis
II-2-1-a Type I Osteoporosis
Type I - postmenopausal osteoporosis or high turnover osteoporosis occurs in some women aged 50-75
due to the sudden decrease in estrogen as a result of menopause (Fig18). This causes rapid calcium loss from
the bones, making the women susceptible to hip, wrist, forearm, and spinal compression fractures.
40
Fig. 18
Postmenopausal osteoporosis affects the trabecular, rather than cortical bone, and is hormonal in origin.
After
menopause,
women's
estrogen
levels fall and as
estrogen
is
responsible
for
regulating
bone
growth and absorption
this has an immediate
effect upon bone
density.
Post
Menopausal women
can lose as much as 2-3% of their overall bone mass each year, making women six times more likely to
develop osteoporosis than men. Often after the first ten years of accelerated bone depletion the rate begins to
slow down until it is on a par with that of men, however often if intervention does not occur to prevent the
bone loss, by the age of 70 or 80, i n some women, bone loss of up to 50% can have occurred. Because type
one osteoporosis affects trabecular bone, it is associated with the collapse of the spine (a dowagers hump),
fractures of the hip, wrist and forearms where there is larger concentrations of trabecular bone than cortical
bone (Eastell,1999).
II-2-1-b Senile Osteoporosis
Type II-senile osteoporosis or low turnover osteoporosis . It occurs when bone loss and formation are not
equal and more bone is broken down than replaced. S enile osteoporosis is associated with normal
processes of aging, a gradual decline in the number and activity of osteoblasts, and not primarily with an
increase in osteoclast activity. It typically occurs in patients > 60 yr and is twice as common in women as in
men. Type II osteoporosis affects trabecular and cortical bone, often resulting in fractures of the femoral
neck, vertebrae, proximal humerus, proximal tibia, and pelvis. (Marcus, 1996)
II-2-1-c Secondary Osteoporosis
Secondary Osteoporosis is a term applied only when osteoporosis is caused by the use of a drug or as a
complication of another condition. These secondary conditions may include (Kanis, 1994):
Medical conditions Serious kidney failure
41
Bone marrow tumors
Scurvy
Cushing's disease (a tumor of the pituitary gland, responsible for secreting some of the body's
hormones
Hyperthyroidism (a condition that results in rapid heart beat and increased rate of metabolism)
Liver impairment
Anorexia
Secondary cancer in a site away from the site of the original cancer (metastatic cancer)
Leukemia
Diabetes
Rheumatoid arthritis
Nutritional disorders
Multiple Sclerosis
Chronic Obstructive Pulmonary Disease (COPD)
Medications or chemicals
Cigarette smoking
Steroid therapy
Alcohol abuse
Lithium (used to treat many psychiatric disorders)
Aluminum
Barbiturates
Antacids containing aluminum
II-2-1-d Idiopathic Juvenile Osteoporosis
Although osteoporosis affects mainly the older generation, it can sometimes affect children or
42
adolescents for no known reason, starting between the ages of 8 to14. In children where there is no
identifiable cause of the condition, it is called idiopathic juvenile osteoporosis. Idiopathic juvenile
osteoporosis is extremely rare (Kanis, 1994).
II-2-2 Causes of Osteoporosis
II-2-2-a Peak Bone Mass
Peak bone mass is the maximum mass of bone achieved by an individual at skeletal maturity, typically
between ages 25 and 35. After peak bone mass is attained, both men and women lose bone mass over the
remainder of their lifetimes. Because of the subsequent bone loss, peak bone mass is an important factor in
the development of osteoporosis (Kanis, 1994).
Several factors determine an individual's peak bone mass (Kanis, 1994)
genetics – demonstrated in studies of twins and studies establishing a strong relationship between the bone
mass of mothers and daughters
gender – men have greater peak bone mass than women
ethnic origin (e.g., people of African origin generally have higher bone mass than those of northern
European origin)
nutritional factors (e.g., calcium, vitamin D, protein intake)
hormonal factors
weight-bearing exercise
Other environmental factors (e.g., tobacco and alcohol consumption).
The stages of bone mass for women are shown below. Figure 19 depicts the time during which active bone
growth occurs and peak bone mass is attained. This is followed by a period of very slow and gradual
decline in bone mass until the time of menopause when the rate of bone loss accelerates for several years.
After about age 60, the rate of bone loss decreases (Wasnich et al., 1989). The stages of bone loss for men
and women follow the same progression. However, unlike men, women attain a lower peak bone mass and
experience a s harp decline following menopause. The difference between men and women who have
osteoporotic fractures and those who do not is related to the amount of bone they have between ages 25 and
35 (i.e., peak bone mass) and the rate of bone loss thereafter, plus other risk factors.
43
Fig. 19.
II-2-2-b Bone losses
The underlying problem in osteoporosis is an imbalance between bone resorption and bone formation. In
osteoporosis, bone resorption takes place to a greater extent than bone formation, so a negative balance
occurs and results in a net loss of bone. This imbalance might occur as a result of one or both of the
following factors: increased bone resorption and/or decreased bone formation (incomplete coupling)
(Eastell, 1999).
Trabecular bone is metabolically more active than cortical bone. It also has a larger surface area because of
the trabecular network of bony plates and spaces. Because bone remodeling depends on surface area and
trabecular bone has an extensive surface, trabecular bone has a more rapid turnover than cortical bone
(Einhorn, 1996). Vertebrae, which have a large proportion of trabecular bone, are commonly the first sites to
show bone loss in osteoporosis. Normal vertebrae contain a dense honeycomb structure with thick
trabeculae, whereas osteoporotic bone has fewer and thinner trabeculae with loss of intertrabecular
connections. The vertebrae, proximal femur, and distal radius are particularly prone to osteoporotic fracture
because these parts of the skeleton contain a large proportion of trabecular bone.
II-2-3 Post menopausal osteoporosis
II-2-3-a Menopause
The female reproductive system matures in a continuous process from menarche to menopause, as the finite
numbers of oocytes produced during fetal development are gradually lost to ovulation and senescence.
Menopause is defined as the permanent cessation of menses (Delmas, 2002) There is evidence that the
timing of natural menopause is genetically programmed. Genetic variability adds to the complexity of the
actions and interactions of the reproductive steroids and to the timing and extent of menopausal symptoms.
44
Menopause is characterized by significant hormonal changes. Subtle rise in the concentration of folliclestimulating hormone (FSH) is the earliest hormonal change noted in studies of reproductive aging.
Luteinizing hormone (LH) levels remain normal initially, but they eventually become elevated as ovarian
steroid secretion falls and gonadotropin-releasing hormone (GnRH) increases. As the absence of ovulation
persists, FSH and LH remain chronically elevated, there is a 10- to 20-fold increase in the FSH level and a 3
to 5 fold increase in the LH level, and estradiol levels fall below 50 pg/ml (Figs. 20,21).
Fig. 20 Approximate average serum concentrations of estradiol, estrone, FSH, LH, and total testosterone
during the menopausal transition and postmenopause. A subtle rise in FSH occurs first, followed by a rise in
LH and a decline in estradiol and estrone. There are no abrupt changes in testosterone, but a gradual
continuous decline occurs that begins before the menopausal transition (Lobo et al,2000).
45
Fig. 21 Multiple hormonal changes associated with reproductive aging.
46
II-2-3-b Women specific osteoporosis
Postmenopausal women are most vulnerable, and about four times more likely to develop the disease as
men. Women who have recently experienced menopause have greater osteoclast activity than premenopausal women. Menopause results in a decreased production of estrogen which is also vital for the
regulation of the female bone remodeling cycle. Hence postmenopausal women can experience a sudden and
rapid loss of bone, placing them in a position that could stimulate the development of osteoporosis. Peak
bone mass is achieved by both men and women in their 20s and 30s. After that time, there begins a period of
net bone loss. With the onset of menopause, women begin an accelerated period of bone loss that may
increase by tenfold so that they may lose bone at the rate of 3-6% per year (Christiansen 1994). Osteoclasts,
the bone cells that break down bone, are activated by a loss of estrogen and a subsequent elevation of
inflammatory factors that stimulate bone resorption (removal of bone from the body with transport to the
blood).
Known risk factors for osteoporosis are:
Being female Thin or small frame Family history of osteoporosis Postmenopausal, including surgical
menopause (i.e., hysterectomy including ovariectomy) History of anorexia or bulimia Prolonged amenorrhea
(absence of menstrual periods) Low calcium diet Lack of exercise Cigarette smoking Excessive alcohol use
Excessive caffeine use
47
II-3 Osteoporosis and the immunity processes II-3-1 Inflammation and immunity
Inflammation and immunity are very closely related processes. Their main goal is to protect the body from
foreign invasion.. The two mechanisms are intertwined and generate many of the same markers and
mediators (Yun, and Lee, 2004).
II-3-1-a Innate immunity (Inflammation)
Fig. 22.
Inflammation can be divided into acute and chronic forms. The acute phase is associated with wounds or
infection and lasts only a short time. It is, in many cases, beneficial as it is part of the healing process. Acute
inflammation lasts from a few minutes to a few days. It is characterized by redness, swelling, heat production
and pain (Fig. 22).
If the acute phase cannot be resolved and recovery does not occur the inflammation process evolves into a
chronic phase. Chronic inflammation, tends to occur over a longer time. It can be the continuation of acute
inflammation if healing does not occur, but autoimmune diseases, such as rheumatoid arthritis, are
characterized by chronic inflammation without without a preceding acute phase.
The general features of chronic inflammation, have been recently described (Cancalon 2005) a nd are
characterized by infiltration of mixed inflammatory cell types containing predominantly macrophages,
lymphocytes and plasma cells (compare with the predominance of neutrophils in acute inflammation).
48
Lymphoid cells can proliferate at the site of inflammation as well as in local lymph nodes. In many cases the
inflammation persists for years. Constant inflammation eventually results in damage to the affected organs.
49
II-3-1-b Inflammation and pain
Inflammation, such as the one resulting from RA, may be accompanied by extensive and prolongated pain.
After tissue injury, there is an upregulation of cyclooxygenase-2(COX-2). Cyclooxygenase (COX) is an
enzyme naturally present in our bod y. There were two forms of t his CO X enzyme: COX-1 and COX-2
enzymes. The COX-2 isoenzyme leads to prostaglandin production. Prostaglandins bind to specific receptors
and lead to production of intracellular protein kinases, which, in turn, phosphorylate the sodium channels
that are responsible for impulse propagation down the neuron (Fig.23). The neuronal impulse, generated by
COX2, eventually reaches the brain and produces pain (Vane et al.1998; Carey et al. 2003).Therefore,
inhibiting COX-2 is an option for muscle pain management.
Fig. 23.
COX-1 enzymes are
produced
widely
throughout the body and are involved in the regulation of many cellular and metabolic activities such as
maintaining stomach lining integrity, regulating blood flow within the kidneys and balancing platelet
function. COX-1 enzymes are always present in the body and should not be inhibited.
50
In rheumatology patients the us e of non s elective, nonsteroidal, anti-inflammatory drugs (NSAIDs), for
example Aspirin, effectively reduces pain and inflammation by inhibiting COX-2, but also inhibit COX-1.
They inhibit homeostatic enzymes responsible for gastric cytoprotection. The use of non selective NSAIDs
is, therefore, limited by poor tolerability, particularly ulceration in the upper GI tract. Cyclooxygenase-2
(COX-2) specific inhibitors preserve COX-1 activity, and have demonstrated both efficacy and GI
tolerability. The use of COX-2 specific inhibitors re duce central COX-2 activity, and block the rise in
prostaglandins, however because of side effects several have been withdrawn from the market. Cox2
inhibitors have been linked with increase risk of heart attack or stroke. It has been suggested that patients
taking these drugs chronically double the risk of a heart attack (Carey et al.2003).
II-3-1-c Acquired or Adaptive Immunity
-Specific immunity
The central feature of the specific immune system is the ability to distinguish between self and nonself. Every cell has complex molecules (proteins and glycoproteins) on its surface membrane which act
as recognition devices. The immune system usually does not attack them.
However, breakdown of the recognition system can lead to autoimmune disease such as AIDS and
rheumatoid arthritis, which result in self-destruction of body parts. Foreign molecules are called non-self
antigens. An antigen induces the s ynthesis of specific proteins called immunoglobins (antibodies). An
antibody has a specific molecular structure capable of recognizing a complementary molecular structure on
the antigen.
The most common human immunoglobin, immunoglobin G (IgG), consists of two long "heavy" chains (A
and B) and two short "light" chains (C and D). These are in the shape of a Y bonded together with disulfide
bonds (Fig. 24). There are several major areas of globular tertiary structure on the chains. The globular
structure on the ends of the chains may be variable and accounts for some of the differences in specificity for
different antigens. There are also two carbohydrate chains in between the A and B chains.
Fig.24.
51
-Specific immune response
When a foreign organism enters the body, the foreign antigens on the invading cells activate an immune
response. The immune system produces antibodies and specialized cells that attempt to destroy foreign
elements. There are two types of responses: Humoral(antibody) response (involving B cells) and cell
mediated immunity (involving T cells). The two responses are summarized in Figs.25 and 26).
-Humoral (antibody-mediated) Response -B-cells
The humoral immune response is initiated by an activation phase. When a macrophage engulfs a foreign
substance, some of the digested antigens are displayed on the surfaces of the macrophages (called epitopes).
This display provides other cells of the immune system with an opportunity to recognize the invader and
Fig.25.
become activated: this is called antigen presentation. T he antigen-presenting cells (APCs) are called
dendritic cells (DCs). There are several subsets of them: some produced by monocytes and others derived
from a progenitor cell that gives rise to both them and monocytes. Dendritic cells can also present intact
antigen directly to B cells. In this case, the engulfed antigen is not degraded in lysosomes but is returned to
the cell surface for presentation to B c ells bearing BCRs of the appropriate specificity. D uring antigen
presentation, the macrophage selects T-helper cells and B-cells that have membrane receptors that are
complementary in shape to the antigens exposed. This is known as clonal selection. T-helper cells recognize
and bind to the displayed antigens. In the next phase, called the effector phase activated T cells trigger
specific B-cells to proliferate and release antibodies. These antibodies bind to the invader and fight infection.
When confronted with an antigen for the first time, B cells produce memory cells as well as plasma cells;
this is called the primary response. The primary response is usually slow, taking days or e ven weeks to
recruit enough plasma cells to bring an infection under control. However, when a second invasion occurs, the
response is quicker. Memory cells are involved in the secondary response and stick to and destroy antigens.
h
52
-Cell Mediated Response - T Cells
The cell-mediated response involves cells that are specific to the antigens on t he invading pathogens. The
cells involved are lymphocytes, called T cells, which mature in the thymus. In the thymus the T cells
develop surface receptors called T-cell receptors where they become programmed for the antigen of their
specific enemy. After encountering a specific foreign antigen, T cells reproduce rapidly, however they do
not produce antibodies like B cells. Cytotoxic (killer) T cells attack body cells that have been infected by
virus, bacteria or fungus. T cell identify an antigen, such as a viral protein coat left outside the infected cell,
and kill the infected cell before the virus has time to replicate
c
In conclusion, the innate and the adaptive immune systems do not act in an independent way:
antibodies make infectious agents more susceptible so that phagocytes recognize and engulf their
targets more effectively; activated T lymphocytes produce cytokines and some of these cytokines
stimulate phagocytes to destroy infectious agents in a more efficient way; T lymphocytes help the socalled B lymphocytes to produce antibodies.
53
-44
54
II-3-2 Osteoporosis, arthritis and inflammation markers
II-3-2-a Bones and Inflammation markers
Inflammation and immunological mechanisms play an important role in most bone ailments. Although
osteoporosis is not typically considered an immunological disorder, recent data have indicated overlapping
pathways between bone biology and biology of inflammation (Kong et al, 2000; Goldring, 2003; Siggelkow
et al, 2003; Pfeilschifter 2003). Cl inical observations reveal coincidence of osteoporosis with period of
systemic inflammation as well as co-localization of regional osteoporosis with areas of regional
inflammation (Yun and Lee, 2004). D ifferent epidemiologic studies report an increase in the risk of
developing osteoporosis in various inflammatory conditions (Mitra,2000; Haugeberg, et al 2004; Bultinket al
2005; Mikuls et al 2005). Immunological dysfunctions, a utoimmune and chronic inflammatory diseases
(Walsh and Gravallese, 2004), rheumatoid arthritis (Jensen, 2004) and inflammatory bowel diseases are
associated with osteoporosis. Erosions seen in conditions such as gout, osteomyelitis, rheumatoid arthritis,
ankylosing spondylitis, and psoriatic arthritis, are typically associated with inflammation in the joints. Proosteoclastic cytokines, such as tumor necrosis factor (TNF)-and interleukin (IL)-6, are elevated in these
conditions and local cytokine profile is consistent with the cytokines that modulate bone resorption (Ishihara,
2002; Moschen, 2005). C-reactive protein (CRP) production in the liver is upregulated by IL-1, IL-6 and
TNF-, and is regarded as a sensitive marker of systemic inflammation (Koh , 2005; Muller 2002). An
association between circulating high sensitive (hs)CRP level and bone mineral density has been
observed in several immune and inflammatory diseases, as well as in healthy individuals, suggesting a
relationship between subclinical systemic inflammation and osteoporosis (Ganesan, 2005). Certain proinflammatory cytokines play potential critical roles both in the normal bone remodeling process and in the
pathogenesis of perimenopausal and late-life osteoporosis (Manolagas and Jilka, 1995). For example,
interleukin (IL)-6 promotes osteoclast differentiation and activation (Manolagas, 2000). IL-1 is another
potent stimulator of bone resorption (Wei et al 2005) that has been linked to the accelerated bone loss seen in
idiopathic and postmenopausal osteoporosis (Pacifici et al, 1989). T NF- is implicated in tumor-induced
bone resorption and non-tumor-induced osteopenia (Moffett, 2005). Anti-TNF drugs, currently used in the
therapy of several immunological disorders, are also useful in preventing and/or reversing systemic bone loss
associated to the disease, as they target both the bone and the inflammatory process (Saidenberg-Kermanach,
2004). The production of IL-1, IL-6, and/or TNF- by peripheral blood monocytes has been positively
correlated with bone resorption or s pinal bone loss in healthy pre- and postmenopausal women (ScheidtNave, et al. 2004). T he inflammatory mediator nitric oxide (NO) is also involved in the pathogenesis of
osteoporosis. The activation of the inducible NO synthesis (iNOS) pathway by cytokines, such as IL-1 and
TNF-, inhibits osteoblast function in vitro and stimulates osteoblast apoptosis (Armour, et al. 2001).
Some inflammation markers are specific to bone diseases. T he TNF-family molecule RANKL
(Osteoprotegerin ligand [OPGL]), a secreted glycoprotein under the TNF ligand super family, mediates its
signals through its association to its receptors which are members of TNFR super family
- RANK (receptor activator of NF-kappa-B), type I transmembrane protein. RANKL and its receptor RANK
(receptor activator of NFkB ligand) have been specifically implicated in the bone loss in rheumatoid arthritis
(Romas et al 2002). They are key regulators of bone remodeling and are essential for the development and
activation of osteoclasts. Intriguingly, RANK/RANK interactions also regulate T cell/dendritic cell
communication, dendritic cell survival and lymph node formation (Theill et al 2002). Ca lciotropic factors
such as vitamin D3, PGE2, IL-1, IL-11, TNF-a and glucocorticoid induce RANKL expression on
55
osteoblasts (Eghbali-Fatourechi et al 2003). RA NKL binding to the RANK expressed on ha ematopoietic
progenitors activates a signal transduction cascade that leads to osteoclast differentiation. Moreover,
RANKL stimulates bone resorbing activity in mature osteoclasts via RANK. When RANK is activated, it
sends signals into the cells through tumor necrosis factor receptor-associated factors (TRAFs), mainly
TRAF6. These RANK associated molecules, through downstream pathways such as NF-kB, JNK/SAPK
and Akt/PKB, regulate bone resorption, activation, survival, and differentiation of osteoclasts and dendritic
cells known as osteoclastogenesis inhibitory factors, function as a soluble decoy receptor to RANKL and
compete with RANK for RANKL binding. TGF released from bone during active bone resorption has been
suggested as a feedback mechanism by upregulating OPG level. Estrogen can enhance OPG production on
osteoblasts which is a possible explanation of postmenopausal osteoporosis following estrogen withdrawal
(Theill et al 2002). Since the expression of RANKL/RANK can be controlled by sex hormones, it is possible
to speculate that this system may control gender specific differences in immunity and could be involved in
the higher incidence of autoimmune diseases and osteoporosis in women. RANKL, RANK and OPG
therefore provide a molecular link between bone remodeling, immunity and inflammation.
56
II-3-2-b Bones and homocysteine
As seen previously, (Cancalon, 2005), all ailments linked with inflammation are associated with elevated
homocysteine levels. S imilarly, there is evidence that a high level of homocysteine is associated with
increased risk of osteoporotic (fragile bone) fractures (van Meurs, et al.,2004; McLean et al, 2004). The risk
of fracture was analyzed based on data from over 2000 patients over the age of 55. Researchers found that
those in the quarter with the highest levels of plasma homocysteine had almost two times the risk of fracture
than those in the other quarters. The risk seemed to be independent of other factors, such as bone mineral
density. In the other study, data from nearly 2000 patients ages 59 to 91 were examined. These researchers
found that men and women in the highest quarter for plasma homocysteine had almost four and two times,
respectively, the risk of hip fracture than those in the lowest quarter. It was concluded t hat elevated
homocysteine is an indicator of high risk for fractures among older people (van Meurs, et al., 2004; van
Meurs and Uitterlinden 2005). However, as in any case of hyperhomocysteinemia, the question remains
whether the high levels of homocysteine are causative – affecting bone fragility – or merely associated
somehow with bone fractures (Herrmann et al, 2005 a,b,c). For example, homocysteine is elevated in
individuals who suffer from poor nutrition, such as those who are folate-deficient, and there have been some
studies linking folate deficiency with low bone mineral density. It, therefore, could be the nutritional deficit
causing the fractures, not the elevated homocysteine (Raisz, 2004).
57
II-3-3 Osteoporosis and immunity
The interaction between the bone and the immune system has been termed osteoimmunology (Yun
and Lee 2004). The activity of immune cells affects the balance of bone mineralization and resorption
carried out by the opposing actions of osteoblasts and osteoclasts (Teitelbaum, 2000). Dendritic cells,
specialized in presenting antigens, and osteoclasts, specialized in resorbing bone, share the same bone
marrow precursors of the monocyte lineage and exhibit parallel lifecycles, regulated by a variety of
cytokines, transcription factors and inflammatory mediators. Molecules that regulate osteoclastogenesis are
key factors in many immunological functions. For example, TRAF6 functions as a molecular bridge
spanning adaptive immunity, innate immunity and osteoimmunology (Wu and Arron (2003). TRAF6deficient mice lack osteoclasts and concomitantly have defects in cytokine production and T cell stimulation
(Lomaga et al, 1999). Data have shown that immune cells interact with bones through the expression of
inflammation markers (Cancalon, 2005). Activated T cells affect bone physiology by producing cytokines
that lead to RANKL expression on osteoblasts. Moreover, activated T cells directly express and produce
RANKL that induces osteoclast formation and activation through its specific receptor ( Theill et al, 2002).
There are multiple mechanisms and interactions by which cytokines regulate bone resorption. IL-6
contributes to RANKL upregulation in osteoblastic cells. IL-1 and TNF- may not only promote osteoclast
generation, but they also appear to stimulate mature osteoclasts to perform more resorption cycles through
modulation of RANKL activity. IL-1 is further involved in bone metabolism as an osteoblast activator.
Osteoblasts secrete RANKL which promotes survival and differentiation of the osteoclast precursors to
mature osteoclasts through RANK. IL-1 and IL-6 also directly enhance osteoclast activity by RANKLindependent mechanisms. They may directly extend the lifespan of the osteoclasts by inhibiting osteoclast
apoptosis. Both TNF- and IL-1 inhibit collagen synthesis in osteoblasts and enhance degradation of the
extracellular matrix (McLatchie et al 1998). In inflammatory or autoimmune disease states, activated T cells
produce RANKL and pro-inflammatory cytokines, all of which can induce RANKL expression in
osteoblasts (Hofbauer et al, 1998)]. However, the constant activity of T cells does not usually cause
extensive bone loss. Multiple T cell-derived cytokines, such as IL-12 and IL-18, might be able to interfere
with RANK signaling and therefore with osteoclastogenesis and osteoclast functions. A crucial counterregulatory mechanism whereby activated T cells can inhibit osteoclast development and activation is through
the action of the antiviral cytokine interferon (IFN)-.. IFN- activates the ubiquitin-proteasome pathway
within the osteoclasts, resulting in the degradation of TRAF6 (Wu and Arron (2003).
58
Fig. 27 Control of bone remodeling by RANKL and OPG. RANKL is a potent inducer of osteoclast
formation. Osteotropic factors—1,25-dihydroxyvitamin D , parathyroid hormone, prostaglandin E and
interleukin 11—induce the formation of osteoclasts by up-regulating RANKL expression on the surface of
marrow stromal cells and immature osteoblasts. RANKL binds to its receptor, RANK, on the surface of
osteoclast precursors and signals to induce osteoclast formation and promote osteoclast survival. A decoy
receptor, osteoprotegerin, inhibits RANKL binding to RANK. (Roodman. 2004).
3
2,
II-3-4 Aging, osteoporosis and immunity
Inflammation-aging may, at least partially, be a common mechanism for the development of lower bone
mass and other age-related disorders (Wallin et al, 2001; Hirose et al, 2003). Senility is in fact notable for
acceleration of diseases that are increasingly attributed to inflammation, such as atherosclerosis, Alzheimer's
disease and asthma (Bruunsgaard and Pedersen, 2003; Ginaldi et al, 2005). Many cytosines, including IL-6,
TNF- IL-1, are elevated during senescence and play direct roles in the pathogenesis of these diseases
(Bruunsgaard, 2002). All these cytokines are stimulators of osteoclast activity as well. Associations between
atherosclerosis and osteopenia have been well documented ( Hak et al, 2000; Jorgensen et al 2001). These
findings suggest a potential causal relationship between systemic inflammation that is observed in the
elderly and the prevalence of generalized age-related osteoporosis (Wu and Arron (2003). Moreover, the
increased catabolic signal, driven by inflammation also in the absence of clinically diagnosable inflammatory
diseases, could be able to induce osteoblast apotosis (Roubenoff, 2003), as well as apoptosis of muscle cells,
leading to age-related osteoporosis. An interesting aspect of immunosenescence is the increased production
of pro-inflammatory cytokines with age and a close link between age-related systemic inflammatory process
(inflammation-aging) and osteoporosis. During aging, under the influence of the lifelong exposure to
chronic antigenic load and oxidative stress, the physiological counter-regulatory process which inhibits bone
resorption following T cell activation is likely impaired. This would contribute, together with the age-related
systemic low-grade inflammation, to the increasing incidence of osteoporosis during senescence.
59
The rate extent of bone loss during senescence varies widely between individuals. It could be
postulated that this may be in part due to individual differences in cytokine activity. In support of this
hypothesis it has been demonstrated that certain IL-6 polymorphisms are able to influence the risk of
osteoporosis in postmenopausal women (Ferrari et al 2003). Similarly, IL-1 and IL-1 receptor antagonist
(IL-1Ra) gene polymorphisms are associated with reduced bone mineral density and predispose women to
osteoporosis at the lumbar spine (Chenet al, 2002). Moreover, the sex hormonal decline which accompanies
aging contributes to the pathogenesis of senile osteoporosis through immunologically mediated mechanisms.
It has been postulated that estrogens exert their effect on bone not only by direct action per se, but
also by inhibiting IL-6 gene expression. A similar relationship between androgen and IL-6 gene
expression also exists
(Liu et al, 2005). The decline in ovarian function is associated with decreased OPG production and
spontaneous increases in proinflammatory and pro-osteoclastic cytokines such as IL-6, TNF-a, and IL-1
(Pfeilschifter et al, 2002; Ershler et al 1997).
II-4 Arthritis
The various forms of arthritis have been reviewed by Valim and Barros, (2006). The two major forms are
explained in Fig.28.
60
II-4-1 Osteoarthritis
Osteoarthritis (OA) also known as degenerative arthritis or degenerative joint disease is a condition in
which low-grade inflammation results in pain in the joints, caused by wearing of the cartilage that covers the
inside of the joints and acts as a cushion. Osteoarthritis is characterized by the breakdown of joint cartilage
and may affect any joint in your body, including those in your fingers, hips, knees, lower back and feet.
Initially osteoarthritis may strike only one joint. But if your fingers are affected, multiple hand joints may
become arthritic. As the bone surfaces become less well protected by cartilage, the patient experiences pain
upon weight bearing, including walking and standing. Due to decreased movement because of the pain,
regional muscles may atrophy, and ligaments may become more lax. OA is the most common form of
arthritis.
I
I
4
2
R
h
e
u
m
a
t
o
i
d
a
r
thritis
Fig.29.
Rheumatoid arthritis (RA) is a typical example of the link between inflammation and osteoporosis. (Yun and
Lee 2004). RA pathogenic includes significant cellular elements of innate immunity (fibroblastic
synoviocytes, macrophages, mastocytes . . . ) and adaptive immunity (T and B lymphocytes) (Loeser,
2006) (Fig.29). These molecules interact through the production of specific (cytokines, chemokines and
auto-antibodies) and non-specific (prostaglandins, nitrous oxide [NO] complement, proteases) mediators.
RA may be genetically inherited, however, some infectious agents such as viruses, bacteria, and fungi or
factors in the environment may also trigger the immune reaction. RA initiation could be the consequence of a
precocious activation of the innate immunity, induced by bacterial agents or debris. The activation of the
61
synoviocytes and the macrophages via specific receptors unleashes an intense inflammatory reaction that
triggers a cascade of events. The ongoing nature of this synovitis leads to the intraarticular recruitment of
different immunity cells. This cellular afflux amplifies the macrophagic and synoviocytic activation and
proliferation. All of these interactive phenomena end in the production of large quantities of proinflammatory cytokines (TNFa, IL1, IL6, IL15, IL17, IL18) but also other pathogenic mediators (autoantibodies, complement, prostaglandins, nitrous oxide). This synovitis persists, as it is no longer regulated by
a sufficient production of physiological regulators (soluble receptors and inhibitors of cytokines). The
consequence of this intense inflammation and synovial proliferation leads to osteo-articular destruction by
the production of proteases and the activation of osteoclasts by the RANK/RANK-ligand pathway under the
effect of cytokines (TNFa, IL5, IL1, IL6, IL17) and other mediators (prostaglandins) liberated by
synoviocytes, macrophages and lymphocytes. (Sibilia, 2005; Walsh et al, 2005 )(Fig.30). One cytokine
gaining recognition for its importance in RA inflammation is macrophage migration inhibitory factor (MIF).
MIF has the ability to induce inflammation and plays a role in both innate and adaptive immunity. In RA,
increased MIF levels have been demonstrated in serum, synovial fluid and tissue. In vitro, MIF induces
production of key proinflammatory genes operative in arthritis, including IL-1, TNF, IL-6, IL-8, COX-2,
phospholipase A2 and matrix metalloproteinases. In vivo, MIF antagonism or MIF deficiency result in
decreased disease severity in animal models of RA further confirming a role for MIF in joint inflammation
(Santos,2006).
62
RA is a chronic, inflammatory autoimmune disease that primarily targets the synovial tissues, but
bone loss occurs both in the joints and throughout the skeleton as a result of the release of proteinase
(metalloproteinases) and proinflammatory cytokines (IL-1, TNF-), which are responsible for
cartilage and bone destruction( Saidenberg-Kermanach et al, 2004).
63
Fig.30 Mechanism of arthritic bone destruction: the critical role of RANKL on synovial fibroblasts and T
cells. Activated T cells stimulate the macrophages to secrete proinflammatory cytokines such as TNF-α and
IL-1, which strongly induce RANKL on synovial fibroblasts. In addition, T cells themselves also express
RANKL. In constrast, there is a very low level of IFN-γ, a potent RANKL inhibitor produced by T cells. This
imbalance may be responsible for the aberrant activation of osteoclast formation in arthritis.
64
III- Beneficial effects of citrus on bone health
III-1 Relation between the skeleton and other organs
III-1-1 Relation between the skeleton and other organs
The properties of the skeleton reviewed previously indicate that bones are not physiologically isolated and
that their health depends greatly on the proper interaction with other organs particularly the gastrointestinal
tract and the kidneys. These interactions are under a complex hormonal control. Bones are the body’s
reservoir of calcium, phosphorus and magnesium used for many physiological functions (Brown, 2001;
Bruder et al, 2001). Calcium is absorbed from the diet in the GI tract. It is filtered and reabsorbed by the
kidneys. Within the bones, the anabolic and catabolic actions of the osteoblasts and osteoclasts assure the
maintenance of the bone mass. Early in life, up to about thirty, the bone steady state favors active bone
growth and peak bone mass is attained. This is followed, after age fifty, by a period of gradual decline in
bone mass (Wasnich et al., 1989). The stages of bone loss for men and women follow the same progression.
However, unlike men, women attain a lower peak bone mass and experience a sharp decline following
menopause. Therefore, adult bone health is determined by two factors: the level of peak bone mass
achieved and the rate of bone loss occurring with aging (Kanis, 1994). Any food that has a beneficial
contribution to bone health will be much more effective if it can act on both anabolic and catabolic
processes.
III-1-2 Inflammation and the skeleton
Many skeletal impairments have been shown to be, at least in part, related to inflammation processes.
Osteoporosis is currently attributed to various endocrine, metabolic and mechanical factors. Clinical and
molecular evidence suggests that inflammation also exerts significant influence on bone turnover, inducing
osteoporosis. Numerous proinflammatory cytokines have been implicated in the regulation of osteoblasts and
osteoclasts. Chronic inflammation and the immune system changes which are characteristic of ageing, as
well as of other pathological conditions commonly associated with osteoporosis, may be determinant
pathogenetic factors. Osteoarthritis (OA), or degenerative arthritis caused by wearing of the cartilage is a
condition in which low-grade inflammation results in pain in the joints (Loeser, 2006). Rheumatoid arthritis
(RA) is a chronic, inflammatory autoimmune disease that primarily targets synovial tissues (Santos and
Morand, 2006). Coxam and Davicco (2005) and Coxam (2006) extensively reviewed the effects of diet on
bone metabolism and osteoporosis. Citrus contain many compounds ( ascorbic acid, folic acid,
flavonoids, carotenoids, limonoids) that may play a role in limiting or even reversing inflammation
(Cancalon, 2005). Such compounds may have beneficial effects on bone diseases by affecting the
extent of the inflammation processes.
III-2 Vitamins and the skeleton.
Vitamins are essential to maintain normal metabolic processes and homeostasis within the body. The amount
of a specific vitamin required by an individual varies considerably with body size, growth rate, and physical
activity. Vitamins A and D may be stored in sufficient amounts to maintain an individual without any intake
for 5 to 10 months and 2 to 4 months, respectively. However, a deficiency of vitamin B compounds (except
65
vitamin B12) may be noted within days, and the lack of vitamin C will manifest within weeks and may result
in death in 5 to 6 months. The current recommended dietary allowance (RDA) of vitamin C is 75 mg for
woman and 90 mg for men, based on the need for protection from deficiency. High intakes of the vitamin C
may be needed to insure a proper antioxidant activity, however, a Tolerable Upper Level (TUL) was recently
set at 2 g based on gastrointestinal upset that sometimes accompanies excessive dosages. Several populations
warrant special attention with respect to vitamin C requirements. These include patients with periodontal
disease, smokers, pregnant and lactating women, and the elderly (Bsoul and Terezhalmy, 2004). Hirota and
Hirota (2004) reviewed problems associated with vitamins deficiency in elderly patients with osteoporosis.
Improving these vitamins status may help to treat and prevent osteoporosis in elderly people. Deficiency of
vitamin K, C, or B (Carinci et al, 2005) are modifiable risk factors for osteoporosis and bone fracture. The
authors concluded that a diet rich in fruit and vegetables together with fish and meat could fulfill a
balance among these vitamins and should be recommended for prevention or treatment of
osteoporosis.
66
III-2-1 Beneficial effects of ascorbic acid on the skeleton
III-2-1-a Role of ascorbic acid as vitamin C
The importance of vitamin C on bone health is clearly revealed in patients suffering from scurvy (Fain,
2005). Scurvy occurs in individuals who eat inadequate amounts of fresh fruit or v egetables. Asthenia,
vascular purpura, bleeding, and gum abnormalities are the main symptoms. In 80% o f cases, the
manifestations of scurvy include musculoskeletal symptoms consisting of arthralgia, myalgia, hemarthrosis,
and muscular hematomas. Vitamin C depletion is responsible for structural collagen alterations,
defective osteoid matrix formation, and increased bone resorption (Fain, 2005). Imaging studies may
show osteolysis, joint space loss, osteonecrosis, osteopenia, and/or periosteal (bone membrane) proliferation.
Trabecular and cortical osteoporosis becomes common. Vitamin C s upplementation ensures prompt
resolution of the symptoms.
Vitamin C, acts as a cofactor required for the function of several enzymes particularly hydroxylases.. It is
essential for hydroxylation of major collagen amino acids (prolyl and lysyl residues) needed for bone
collagen matrix formation. Without the proper matrix, the subsequent steps in bone formation cannot occur.
Furhtermore, Carinci et al (2005) showed that vitamin C induces embryonic stem cells to differentiate into
osteoblasts. The mechanism by which vitamin C s ustains pre-osteoblast proliferation and commitment is
mediated through the synthesis of collagen type I, interaction with alpha2- and beta1-integrin, activation of
the mitogen activated protein kinase pathway, and phosphorylation of osteoblast-specific transcription
factors. MC3T3-E1 mouse cell line is a w ell-defined in vitro model of pre-osteoblast differentiation, and
vitamin C i s essential for the proliferation and differentiation of MC3T3-E1. By using DNA micro-arrays
containing 15,000 genes, the authors identified several genes in MC3T3-E1 cultured with vitamin C for 24h
whose expression was significantly up or downregulated. The differentially expressed genes covered a broad
range of functional activities: (1) cell growth; (2) metabolism; (3) morphogenesis;(4) cell death; (5) cell
communication. These data show that vitamin C plays a role in the early stage stimulation of preosteoblasts,
and reveals the molecular mechanism of pre-osteoblast proliferation and commitment.
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Carinci et al, (2005) concluded that study of the molecular mechanism of osteoblast differentiation has
revealed that vitamin C accelerates bone growth and regeneration.
Munday et al, (2005) tested the hypothesis that collagen cross linkage, essential for bone formation, varies
with ascorbate status in man. The collagen cross linkage ratios were compared between British and Gambian
prepubertal boys, mean age 8.3 years, and in Gambian boys between two seasons with contrasting ascorbate
availability. The mean cross-links ratio in 216 British boys was 4.36 , significantly greater than in sixty-two
Gambian boys: 3.83. In the Gambians the cross-links ratio was significantly higher in the dry season (with
high ascorbate intake) than in the rains (with low intake).
III-2-1-b Other roles of ascorbic acid
Ascorbic acid (AA) is also a major physiological antioxidant, repairing oxidatively damaged biomolecules,
preventing the formation of excessive reactive oxygen species or scavenging these species (Coxam, 2005).
Wang et al, (2006) showed that ascorbic acid can modulate the proliferation and osteogenic differentiation of
bone marrow-derived human mesenchymal stem cells (MSCs). Ascorbic acid synergistically promoted and
retained the ability of MSCs to respond to osteogenic stimulation over extensive cell expansions in vitro. AA
plays a key role in the down regulation of the differentiation and activation of osteoclasts (OCL). Xiao et al
(2005) reported that AA might induce the formation of OCL in cocultures of mouse bone marrow cells and
ST2 cells (osteoblast precursors). The percentage area of resorption lacunae induced by RANKL was
decreased when AA was added to the cultures. The results demonstrate that AA inhibits RANKL-induced
differentiation of OCL precursor cells into mature OCL and reduces the formation of bone resorption pits in
vitro.
The stromal cell line ST2, derived from mouse bone marrow was examined by Otsuka et al, (1999). The
cells differentiated into osteoblast-like cells in response to ascorbicacid. Ascorbic acid induced alkaline
phosphatase (ALPase) activity and the expression of mRNAs for proteins that are markers of osteoblastic
differentiation. These results suggest that ascorbic acid might promote the differentiation of ST2 cells into
osteoblast-like cells by inducing the formation of a matrix of type I collagen,with subsequent activation of
the signaling pathways that involveBMPs. However, AA may also have some negative effects. AA has been
shown to activate latent transforming growth factor (TGF) beta ( Kraus et al,2004). Prolonged intraarticular
exposure to TGF beta has been shown to cause spontaneous osteoarthritis -like changes. The authors
reported the expression of active TGF beta in osteophytes in association with ascorbic acid treatment. Thus,
the deleterious effects of prolonged ascorbic acid exposure may be mediated in part by TGF beta.
Although dehydroascorbate (DHA) has been shown to have vitamin C a ctivity (Bsoul and Terezhalmy
,2004), its physiological value has been questioned since it could act as a prooxidant. McNulty et al (2005a)
provided evidence that chondrocytes transport DHA via the glucose transporters and that this transport
mechanism is modestly selective for L-DHA. In the setting of upregulated DHA transport at low oxygen
tensions, DHA would contribute 26% of the total intracellular Asc in OA chondrocytes and 94% of that in
RA chondrocytes. These results demonstrate that DHA is a physiologically relevant source of ascorbate for
chondrocytes, particularly in the setting of an inflammatory arthritis, such as RA. Furthermore, the authors
(McNulty et al 2005b) provided evidence that a functional isoform of the sodium-dependent vitamin C
transporter 2 mediates an active and concentrative transport of ascorbic acid in human chondrocytes
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One of the main role of vitamin C is to promote collagen formation. Since the formation of a collagen
scaffold is the first step in bone formation, vitamin C play a major role in bone development. Ascorbic
acid, mainly through its antioxidant activity may play a role in limiting osteoclast activity and
promoting osteoblast formation.
III-2-2 Beneficial effects of folic acid on the skeleton
Elevated homocysteine is known to be a risk factor for heart attack and stroke and homocysteine testing is
often included in cardiac risk assessment. It also is used to determine if a patient is folateor B12-deficient
since homocysteine has been found to be increased in both of these conditions.
Several studies have revealed a correlation between elevated homocysteine and the risk of fractures. A
high level of homocysteine appears to be associated with osteoporosis. Raisz (2004) analyzed the risk of
fracture based on data from more than 2000 patients over the age of 55. The author found that those in the
quartile with the highest levels of plasma homocysteine had almost 2 times the risk of fracture than those in
the other quartiles. The risk seemed to be independent of other factors, such as bone mineral density. Data
from nearly 2000 patients ages 59 to 91 were examined by Van Meurs et al (2004). These researchers found
that men and women in the highest quarter for plasma homocysteine had almost 4 and 2 times, respectively,
the risk of hip fracture than those in the lowest quarter. It was concluded that elevated homocysteine is an
important indicator of high risk for fractures among older people. The authors raised the question whether
the high levels of homocysteine are causative – affecting bone fragility – or merely associated somehow
with bone fractures. For example, homocysteine is elevated in individuals who suffer from poor nutrition,
such as those who are folate-deficient, and there have been some studies linking folate deficiency with low
bone mineral density. It, therefore, could be the nutritional deficit causing the fractures, not the elevated
homocysteine. Ravaglia et al ( 2005 ), in an epidemiological study, concluded that low serum folate is
responsible for the association between homocysteine and risk of osteoporotic fracture in elderly persons and
that folate, but not homocysteine, predicts the risk of fracture. Whether a reduction in homocysteine levels,
such as through changes in diet, can reduce the incidence of fractures has yet to be proven (McLean,2004).
Gjesdal et al (2006) reported that plasma ho mocysteine (Hcy) was associated with hip fracture but not
directly with bone mineral density (BMD). Elevated Hcy and low folate levels were associated with reduced
BMD in women but not in men. These findings suggest that Hcy may be a potential modifiable risk factor
for osteoporosis in women. In order to determine if Hcy adversely affects bone metabolism Herrmann et al
(2005 a) analyzed the relation between Hcy and biochemical markers of bone metabolism and bone mineral
density (BMD). T he authors investigated 143 pe riand post-menopausal women. A ll subjects underwent
medical examination, measurement of bone mineral density at lumbar spine (BMD-LS) and total hip (BMDHIP), fasting venous blood and urine sampling. Osteocalcin (OC), serum calcium (Ca), urinary
desoxypyridinoline cross-links (DPD), osteoprotegerin (OPG) and soluble receptor activator of NF-kappaB
ligand (sRANKL) were studied. No significant relations could be observed between Hcy levels and the
various markers of bone resorption. The results demonstrate that homocysteine levels appear to be
poorly associated with bone resorption. The same authors (Herrmann et al , 20 05b) suggested that
homocysteine (Hcy), folate, vitamin B6 and vitamin B12 affect bone metabolism, bone quality and fracture
risk in humans. They added that since circulating Hcy depends on folate, vitamin B6 and vitamin B12, Hcy
could be suitable as a risk indicator for micronutrient-deficiency-related osteoporosis and that there are
indications that Hcy is not only a risk indicator, but also a player in bone metabolism. However, existing data
open speculation that folate, vitamin B6 and vitamin B12 act not only via Hcydependent pathways, but also
69
via Hcy-independent pathways. It was concluded that more studies are needed to clarify the mechanistic role
of Hcy, folate, vitamin B6 and vitamin B12 i n bone metabolism. The same group (Herrmann et al, 2006)
showed that short-term folic acid supplementation does not affect biochemical bone markers in nonosteoporotic subjects with a low folate status.
70
As seen previously with inflammation and brain diseases (Cancalon 2005,2006) the role of folate and
homocysteine in human diseases is still unclear. High homocysteine could be the result of various
metabolic problems such as B12 and B6 impairment.
III-3 Flavonoids and the skeleton.
II-3-1 Flavonoids and inflammation
Inflammation is one important component of the majority of bone impairment diseases including
osteoporosis, osteoarthritis and rheumatoid arthritis. Aging and sex hormones related changes lead to
inflammatory and oxidative conditions, which are involved in the pathogenesis of osteoporosis. Aspirin and
other non-steroidal anti-inflammatory drugs (NSAIDs) have long been used to combat inflammation.
Recently, cyclooxygenase (COX) inhibitors have been developed and recommended for treatment of
rheumatoid arthritis (RA) and osteoarthritis (OA). However, two COX-2 i nhibitors have been withdrawn
from the market due to side effects. Polyphenols, including flavonoids, a re found in many dietary plant
products, including fruits and vegetables. These compounds have been shown to be able to positively affect
inflammation (Cancalon,2005). E pidemiological and experimental evidence of dietary polyphenols antiinflammatory properties were reviewed by Yoon and Baek,.(2005). The anti-inflammatory properties of
flavonols (quercetin, rutin and morin) and flavanones (hesperetin and hesperidin) were examined by Rotelli
et al, (2003) in animal models of acute and chronic inflammation.
II-3-2 Flavonoids and bone loss
II-3-2-a Flavonoids
Some flavonoids not present in citrus will be reviewed, since their chemical structures are very similar to
some of the major citrus compounds. Q ian er al, (2006)showed that the flavonoids of Herba epimedii
(HEF) are bone anabolic agents. Z hang et al, (2006) showed that flavonoids inhibited bone resorption,
stimulated bone formation, and accordingly prevented osteoporosis and enhanced intestinal calcium
absorption. Puel et al (2005) assessed the effect of phloridzin (phloretin 2'-O-glucose) a flavonoid found in
apple, on bone metabolism in ovariectomized (OVX) rats with and without inflammation. Daily phloridzin
intake prevented OVX-induced bone loss. The authors concluded that phloridzin consumption may provide
protection against OVX -induced osteopenia under inflammation conditions by improving inflammation
markers and bone resorption. A related compound phlorin (3,5-dihydroxyphenyl beta-D-glucopyranoside) is
found in significant amounts in citrus and could have a similar activity. Epigallocatechin-3-gallate (EGCG)
mostly present in tea was found by Ahmed et al (2004) to act on the expression and activity of matrix
metalloproteinases (MMPs) , a family of zinc metalloendopeptidases secreted by cells, responsible for much
of the turnover of matrix components and on t he activities of transcription factors NF-kappaB and AP-1.
These results also suggest that EGCG or c ompounds derived from it may be therapeutically effective
inhibitors of IL-1beta-induced production of matrix-degrading enzymes in arthritis. Catechins, were shown
by Adcocks et al (2002) to inhibit proteoglycan and type II collagen breakdown and concluded that catechins
are chondroprotective.
71
Humpel et al, (2005) studied 8-prenylnaringenin (8-PN). In adult OVX rats it can protect from ovariectomy
induced bone-loss while exhibiting minimal, trophic effects on uterus and endometrium. Studies (Kim et al,
2005) indicate that the flavonoid luteolin blocks LPS-induced NF-kappaB signalin G and pro-inflammatory
gene expression in intestinal epithelial cells and dendritic cells. Modulation of innate immunity by natural
plant products may represent an attractive strategy to prevent intestinal inflammation associated with
dysregulated innate immune responses. Horcajada-Molteni et al (2000) examined the effects of rut in
(quercetin-3-O-glucose rhamnose) on bone metabolism in OVX rats. These results indicate that rutin (and/or
its metabolites), which appeared devoid of any uterotrophic activity, inhibits OVX-induced trabecular bone
loss in rats, both by slowing down resorption and increasing osteoblastic activity.
II-3-2-b Citrus Flavonoids
Citrus are rich in phenolic compounds. They were re viewed by Augustin et al (2005) and Horowitz and
Gentilli (1977). Citrus flavonoids include flavonoid glycosides such as neohesperidin, naringin, hesperidin,
rutoside, sinensetin and polymethoxylated flavones such as nobiletin, tangeretin. A t least 44 different
flavone glycosides were inventoried in citrus by Berhow et al,1998.
Hesperidin
Hesperedin , t he main orange flavonoid, has been implicated in the inhibition of the various enzymes
involved in several diseases: phospholipase A2, lipoxygenase, HMG-CoA reductase and cyclooxygenase
(Bok et al,1999, Harita et al, 2005). As a result, hesperidin may have antioxidant, anti-inflammatory,
hypolipidemic, vasoprotective, anticarcinogenic and cholesterol lowering properties (Horcajada and Coxam ,
2004).
Furthermore, studies have shown that hesperidin may play an important role in maintaining bone health,
particularly in the area of postmenopausal osteoporosis. A study in ovariectomized (OVX) mice, an animal
model of postmenopausal osteoporosis, by Chiba et al (2003), showed that hesperidin added to the diet not
only lowered serum and hepatic cholesterol, but also inhibited bone loss by decreasing osteoclast number. In
OVX mice, the bone mineral density of the femur was lower than in the sham group and this bone loss was
significantly prevented by dietary hesperidin or alphaglucosylhesperidin. The Ca, P and Zn concentrations in
the femur were significantly higher in the hesperidin-fed group. Furthermore, hesperidin decreased the
osteoclast number of the femoral metaphysis in OVX mice.
72
Horcajada and Coxam, (2004). investigated the effect of hesperidin on bone metabolism in intact and OVX
rats. Twenty rats were sham operated (SH), while the others were ovariectomized. Among each group, 10
sham-operated (TSH) and 10 o variectomized (TOVX) rats were fed a standard diet for 90 da ys following
surgery, and the 20 re maining animals received the same regimen but with 0.5% he speridin added (10
HpSH, 10 H pOVX). Hesperidin consumption totally prevented the ovariectomy induced demineralization
and significantly improved femoral diaphyseal density in both SH and OVX rats, while an improvement of
bone strength was only observed in ovariectomized animals. Plasma osteocalcin concentrations were higher
in HpOVX than HpSH. Urinary deoxypyridinolin excretion was reduced in both HpSH and HpOVX rats
compared to their controls. In a somewhat different area, Hosseinimehr and Nernati (2006) showed that.
hesperidin has powerful protective effects on the radiation-induced DNA damage and on the decline in cell
proliferation in mouse bone marrow.
These results indicate that, hesperidin consumption can improve bone mass acquisition in normal,
unoperated rats and exhibited a significant protection against ovariectomy-induced bone impairment.
The preventive and therapeutic effects of hesperidin on the development of collagen-induced arthritis (CIA),
were reported by Kawaguchi et al (2006) on a mouse model of rheumatoid arthritis (RA). Mice were
administered hesperidin orally three times a week starting from either the onset (day 21) of secondary
immunization or on day 31, when the CIA development had reached a plateau. In both cases, treatment with
hesperidin resulted in a significant suppression of clinical scores and improvement of histological features.
These results suggest that oral administration of hesperidin could be effective for treating human RA
patients.
The pain associated with RA inflammation is mainly induced through COX-2 enzymes and prostaglandin
production which affect nerve activity. In the concentration range 250-500 microM, hesperetin and
hesperidin showed potent inhibition of lipopolysaccharides (LPS)-induced expression of the COX-2 gene in
RAW 264.7 cells, a macrophage precursor of osteoclasts, suggesting the anti-inflammatory activity of these
compounds. The ability of hesperetin and hesperidin to suppress COX2 gene expression may be a
consequence of their antioxidant activity (Hirita et al, 2005). S akata et al (2003) also investigated the
inhibitory effect of hesperidin on LPS-induced over-expression of various inflammatory factors: COX-2
inducible nitric oxide synthase (iNOS) proteins, overproduction of prostaglandin E2 (PGE2) and nitric oxide
(NO) using mouse macrophage cells. Treatment with hesperidin suppressed production of PGE2, nitrogen
dioxide (NO2), and expression of iNOS protein. The data indicated that hesperidin may act as a COX-2
and iNOS inhibitor, which might explain the anti-inflammatory efficacy of this flavonoid. However,
further studies are needed to see if the COX-2 suppression could be associated with pain decrease.
73
Naringin
Naringin is the main grapefruit flavonoid. Wong and R abie (2006) compared the amount of new bone
produced by bone grafts with naringin in collagen matrix to that produced by bone grafts and collagen matrix
alone. Quantitative analysis of new bone formation revealed that 284% and 490% more new bone was
present in defects grafted with naringin in collagen matrix than those grafted with bone and collagen.
Naringin may improve collagen matrix formation and consequently increase new bone formation.
Nobelitin
Ito et al. (1999) and Ishiwa et al. (2000) investigated the chondroprotective effect of citrus flavonoids,
especially nobiletin, using cultured rabbit synovial fibroblasts and articular chondrocytes. The authors
examined the effects of citrus flavonoids on the production and gene expression of matrix metalloproteinases
(MMP) and prostaglandin E2 (PGE2) in rabbit synovial fibroblasts. Six flavonoids including tangeretin, 6demethoxytangeretin, nobiletin, 5-demethylnobiletin, 6demethoxynobiletin, and sinensetin suppressed the
interleukin 1 (IL -1) induced production of proMMP-9/progelatinase B i n rabbit synovial cells in a dose
dependent manner. Nobiletin most effectively suppressed proMMP-9 production along with the decrease in
its mRNA. Nobiletin also reduced IL-1 induced production of PGE2 in the synovial cells, but did not modify
the synthesis of total protein. These suppressive effects of nobiletin were also observed in rabbit articular
chondrocytes. Nobiletin inhibited proliferation of rabbit synovial fibroblasts in the growth phase.
These results suggest nobiletin is an antiinflammatory compound that has the potential to inhibit
PGE2 production, and matrix degradation of the articular cartilage in osteoarthritis and rheumatoid
arthritis.
Quercitin and Kaempferol
Pang et al, (2006) examined the flavonols, quercetin and kaempferol, as protective agents against
postmenopausal bone loss. T hey reported that kaempferol exerts profound antiosteoclastogenic effects by
acting on both osteoblasts and osteoclasts. Kaempferol, but not quercetin dose-dependently inhibited tumor
necrosis factor alpha (TNFalpha)-induced production of the osteoclastogenic cytokines interleukin-6 (IL-6)
and monocyte chemoattractant protein-1 (MCP-1/CCL2) in osteoblasts. The effect on IL -6 was
posttranscriptional, whereas kaempferol reduced MCP-1 mRNA levels. In addition, in mouse primary
calvarial osteoblasts, kaempferol but not quercetin blocked TNFalpha-induced translocation of the nuclear
factor kappaB (NF-kappaB) subunit p65 from the cytoplasm to the nucleus. However, TNF-alpha-stimulated
intracellular reactive oxidative species production was unaltered by kaempferol. In RAW264.7 cells, a
monocyte/macrophage precursor for osteoclasts, both kaempferol and quercetin dose-dependently inhibited
the receptor activator of NFkappaB ligand (RANKL)-induced immediate-early oncogene c-fos expression at
6 h. A fter 3-5 days, both flavonols robustly inhibited RANKL-induced expression of the osteoclastic
differentiation markers, RANK and calcitonin receptor. Consistent with down regulation of these
osteoclastic differentiation markers, both flavonols strongly attenuated the RANKL-induced formation of
multinucleated osteoclasts. However, kaempferol was more potent than quercetin in inhibiting RANKLstimulated effects on RAW264.7 cells. Thus, the data indicate that kaempferol exerts profound
antiosteoclastogenic effects by specifically antagonizing TNF receptor family action on bone cells at two
distinct levels, by disrupting production of osteoclastogenic cytokines from osteoblasts and attenuating
osteoclast precursor cell differentiation.
74
The role of quercitin in preventing bone loss by affecting osteoclastogenesis and regulating many systemic
and local factors including hormones and cytokines was studied by Son et al, (2006). They examined how
quercetin acts on t umor necrosis factor-alpha (TNFalpha)-mediated growth inhibition and apoptosis in
MC3T3-E1 osteoblastic cells. Apoptosis assays revealed an accelerating effect of quercetin on TNF-alphainduced apoptosis in MC3T3-E1 cells. In addition, Fas activation and poly (ADP ribose) polymerase
cleavage are thought to be closely associated with the TNF-alpha-induced apoptosis and its acceleration by
the quercetin treatment in the cells. This study showed that quercetin accelerates the TNF-alpha-induced
growth inhibition and apoptosis in MC3T3-E1 osteoblastic cells.
Quercetin and kaempferol may have beneficial effects on bone health, although they are present in
relatively small amounts in citrus.
III-4 Carotenoids and the skeleton.
III-4-1 Effect of beta cyptoxanthin and other carotenoids on
osteporosis and bone formation
The vast majority of the work on the effects of citrus carotenoids on bones was performed by a single group,
Uchiyama and Yamaguchi, who studied extensively the activity of beta-cryptoxanthin (BC). Yamaguchi and
Uchiyama ,(2003) compared the e ffects of various carotenoids and rutin on calcium content and alkaline
phosphatase activity in the femoral-diaphyseal and femoral-metaphyseal tissues of young rats in vitro. The
presence of BC c aused a significant increase in calcium content and alkaline phosphatase activity in the
-8
femoral tissues. A t the lowest concentration (10 M) beta cryptoxanthin caused a significant increase in
diaphyseal and metaphyseal calcium content. Lutein, lycopene or rutin did not have a significant effect on
bone calcium content and alkaline phosphatase activity. The study suggested that beta-cryptoxanthin has
a unique anabolic effect on bone calcification in vitro.
Recently, Uchiyama and Yamaguchi (2006a), ex amined the effects of BC on osteoclastic cells in mouse
marrow culture system in vitro. O steoclastic cells were cultured in medium containing either vehicle or
beta-cryptoxanthin in the presence of macrophage colony-stimulating factor M-CSF and receptor activator
of NF-kappaB ligand (RANKL) for 4 days with or without M-CSF and RANKL for 24-72 h. Osteoclastic
cells were significantly decreased with culture of beta-cryptoxanthin indicating that the carotenoid induces
apoptotic cell death. Apoptosis related gene expression was determined using reverse transcriptionpolymerase chain reaction (RT-PCR). Cathepsin K mRNA expressions were significantly decreased with
culture of beta-cryptoxanthin in the presence or absence of M-CSF and RANKL. This study demonstrates
that beta cryptoxanthin has stimulatory effects on apoptotic cell death and suppressive effects on
osteoclastic cell function
and therefore should limit bone destruction. In a further study, Uchiyama and Yamaguchi (2006 b)
administered BC (5 or 1 0 microg/100 g body weight) orally once daily for 3 m onths to ovariectomized
(OVX) rats. OVX induced a significant increase in body weight and a significant decrease in serum calcium
and inorganic phosphorus concentrations as compared with those of sham-operated (control) rats. These
alterations induced by OVX were minimized by the administration of beta-cryptoxanthin (5 or 10
microg/100 g). Moreover, OVX induced a significant decrease in calcium content and alkaline phosphatase
activity in the femoral-diaphyseal and -metaphyseal tissues and deoxyribonucleic acid (DNA) content in the
metaphyseal tissues. These decreases were significantly prevented by the administration of beta75
cryptoxanthin (5 or 10 microg/100 g). This study demonstrates that betacryptoxanthin has a preventive effect
on OVX-induced bone loss in vivo. Yamaguchi et al (2006), further examined the effects of combined betacryptoxanthin and zinc on bone components in the femoraldiaphyseal and showed that the oral
administration of the combination of zinc at lower doses synergistically enhances beta-cryptoxanthininduced anabolic effects on bone components in the femoral tissues of rats in vivo. Similar effects were
demonstrated in vitro by Uchiyama et al, (2005).
76
Diabetes has detrimental effects on m any organs and particularly bones. It decreases calcium and DNA
content as well as alkaline phosphatase activity. Uchiyama and Yamaguchi (2005c) examined the femoral
tissue of streptozotocin (STZ)-diabetic rats. The decreases were significantly prevented by the
administration of beta-cryptoxanthin (5 or 10 microg/100 g) for 14 d. And even in normal rats the
administration of beta-cryptoxanthin f or 14 d caused a significant increase in calcium content, alkaline
phosphatase activity, and DNA content in the femoral-diaphyseal and -metaphyseal tissues.
This study demonstrated that the intake of beta-cryptoxanthin has a preventive effect on bone loss in
diabetic rats and has also a positive activity in normal animals.
Yamaguchi and Uchiyama (2003, 2004) demonstrated that beta-cryptoxanthin has a direct stimulatory effect
on bone formation and an inhibitory effect on bone resorption in tissue culture in vitro. The authors assumed
that bone resorption inhibitory action of beta-cryptoxanthin may partly involve in a newly synthesized
protein component which is related to RANKL stimulation in osteoclastogenesis. The in vitro studies were
confirmed in rats in vivo by Uchiyama et al (2004). Gene expression in osteoblastic cells was examined in
vitro by Uchiyama and Yamaguchi (2005a, b) using reverse transcription polymerase chain reaction (RTPCR). Results indicated that the mode of action of beta-cryptoxanthin differs from that of vitamin A. BC
stimulates runt -related transcription factor 2 (Runx2) type 1 a nd alpha1 collagen mRNA levels, protein
content, and alkaline phosphatase activity. Prolonged culture with beta-cryptoxanthin for 3 to 21 days caused
a significant increase in cell number, deoxyribonucleic acid (DNA) content, protein content, and alkaline
phosphatase activity in osteoblastic cells, suggesting that beta-cryptoxanthin stimulates cell proliferation and
differentiation. Moreover, culture with beta-cryptoxanthin caused an increase in mineralization.
These studies demonstrated that beta cryptoxanthin has an important anabolic effect on bone
development which may be important early in life when the body approches its peak bone density.
Other carotenoids may also have a beneficial effect on bone physiology. Plasma levels of beta-carotene and
other carotenoids were measured in free-living, non-supplemented, elderly women with or without severe
osteoporosis. Plasma levels of all carotenoids tested, with the exception of lutein, were consistently lower in
osteoporotic than in control women. The study suggests a bone sparing effect of some carotenoids (Maggio
et al, 2006). However, vitamin A may have adverse effects on bone health at a level of intake about twice
the current recommendation for adult females (Crandall, 2004).
77
III-4-2 Effect of beta-cyptoxanthin and anti-oxidants on rheumatoid arthritis
Epidemiologic studies suggest that the antioxidant potential of dietary carotenoids may protect against the
oxidative damage that can result in inflammation. Pattison et al, (2005) through epidemiological studies
showed that a modest increase in beta-cryptoxanthin intake, equivalent to one glass of freshly squeezed
orange juice per day, is associated with a reduced risk of developing inflammatory disorders such as
rheumatoid arthritis (RA). The same authors (Pattison et al 2004 a,b, c) reported that there was evidence of a
protective effect of higher consumption of olive oil, oil-rich fish, fruit, vegetables and beta-cryptoxanthin.
The beneficial effects of certain food and particularly citrus compounds are linked to a decrease in
inflammation since lower serum concentrations of antioxidants were associated with an increased risk
of RA
III-5 Limonoids and the skeleton.
There is very little information on the influence of limonoids on the skeleton. Li et al (1998) examined water
extracts of Japanese herbal supplement Kampo containing various compounds including limonin and noted
an inhibitory effect on bone resorption.
III-6 Organic acids and the skeleton.
As seen previously, despite its apparent static condition, the skeleton undergoes a permanent process of
remodeling. This activity is regulated by many factors, one of the most important being the maintenance of
calcium homeostasis. This calcium equilibrium is largely controlled by food intake and excretion. Demigne
et al, (2006) showed that mice chronically fed a westernized diet (W), characterized by high intakes of
processed and red meats, refined grains, sweets, and desserts, as a model of obesity develop the metabolic
syndrome and osteoporosis. The authors studied wild type outbred mice to evaluate the validity of a model
of (W) diet reproducing the eating habits of Western countries, In contrast to the standard (S) diet, the W
diet triggered a marked increase in adiposity with some characteristics of metabolic syndrome
(hypercholesterolemia, hyperinsulinemia.). There was an heterogeneity in the propensity to become obese
upon exposure to the W diet in female mice. Overweight mice also presented some disturbances of renal
function, such as hyperalbuminuria and hypocitraturia. Mice adapted to the W diet showed a reduction of
bone mineral density, especially the non-obese ones. The link between bone and kidney was recently
reviewed by Mondry et al,( 2005). The authors pointed out that one of the kidney's endocrine functions is the
activation of vitamin D, while electrolyte homeostasis is one of its excretory functions. Impaired renal
function leads to disturbances in this regulatory system, resulting in the complex syndrome of renal
osteodystrophy that affects the majority of patients with chronic renal failure.
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III-6 -1 Calcium citrate and malate and bones
Proper calcium intake from the diet is very important for bone health. Many reports have shown the superior
calcium bioavailability from calcium citrate as compared with calcium carbonate (Harvey et al, 1988).
Kochanowski (1990), demonstrated that calcium citrate-malate is better-absorbed than calcium from
calcium carbonate (oyster shell) in humans and in rats. Calcium citrate-malate is a more bioavailable
calcium source than calcium carbonate. Furthermore, there is concern that ingestion of oyster shell calcium
supplements and antacids might contribute to an increased risk of lead poisoning (Zerwekh and Pak , 1998).
Citrus juices are rich in citric and malic acid, when taken with the proper source of calcium they should
increase biovailibility. Furthermore, orange juice is often supplemented with calcium. Heaney et al.(2005)
compared the bioavailability of calcium from two fortification systems used in orange juice and reported that
calcium citrate malate was 48% better absorbed than tricalcium phosphate/calcium lactate. The citrus acids,
citric and malic, may help the absorption of calcium in the intestinal tract.
III-6 -2 Potassium citrate and malate and bones
Although citrus are not rich in calcium, they contain significant amounts of potassium (K). Many reports
have shown that potassium citrate may improve calcium balance by conferring an alkali load. If there is not
enough bicarbonate available, the body takes calcium carbonate from the bones to balance the acid excess
and that can lead to bone loss. Calcium supplementation slows postmenopausal bone loss by inhibiting
parathyroid hormone PTH secretion. In postmenopausal women, combined treatment with potassium citrate
and calcium citrate, inhibits bone resorption by providing alkali load and increasing absorbed calcium. The
importance of dietary organic anions in preventive nutrition were reviewed by Demigne et al, (2004a).
Organic anions are supplied by plant foods, as partially neutralized K salts mainly pot assium citrate and
potassium malate. Citrate and malate anions are absorbed in the upper digestive tract, while a substantial
proportion is metabolized in the splanchnic area. Potassium citrate is metabolized into potassium bicarbonate
which is used by the kidneys to neutralize fixed acidity. This acidity is essentially generated by the oxidation
of excess sulfur containing amino acids to sulfate ions, and is related to the dietary protein level. The
authors concluded that, the failure to neutralize acidity leads to low-grade metabolic acidosis, with
possible long-term deleterious effects on bone Ca status and on protein status. Furthermore, low-grade
acidosis is liable to affect other metabolic processes, such as peroxidation of biological structures.
These metabolic disturbances could be connected with the relatively high incidence of osteoporosis and
muscle-protein wasting problems observed in aging individuals in Europe and North America.
Providing a sufficient supply of K organic anions through fruit and vegetable intake should be
recommended to limit calcium release from bones.
Sakhaee et al, (2005) s howed that the administration of calcium citrate and potassium citrate produces a
more pronounced inhibition of bone loss, due to the effect of calcium citrate (parathyroid suppression) and of
potassium citrate (alkali load) in directly reducing osteoclastic bone resorption. The authors reported that
women who were getting 1,560 mg a day of potassium cut their daily loss of calcium in the urine by 50 mg
in just two weeks, compared to when they received a placebo. Marangella et al, (2004) studied whether
potassium citrate favorably affects bone turnover markers in postmenopausal females with low bone density.
79
Women, aged 50 to 65 years old received potassium citrate supplementation (0.08-0.1 g/kg daily). All were
evaluated for electrolyte and acid-base balance-related parameters, bone turnover markers and renal function.
A significant decrease in net acid excretion was observed upon c itrate supplementation, and this was
paralleled by a significant decrease of urinary deoxypyridinolines and hydroxyproline-to-creatinine ratios.
Percent variations of urine citrate were inversely related to those of deoxypyridinolines and hydroxyproline.
No change occurred in the control group. The results suggest that treatment with an alkaline salt, such as
potassium citrate, can reduce bone resorption and may reverse the potential adverse effects caused by
chronic acidemia of protein-rich diets. Treatment with potassium citrate was shown by Pak et al, (2002)
to stabilize spinal bone density among patients with recurrent calcium oxalate nephrolithiasis and may avert
age dependent bone loss. Spinal bone density increased in most patients when it normally decreases. High
dietary sodium chloride increases urine calcium excretion and bone turnover markers in postmenopausal
women. Sellmeyer et al,(2002) determined that the addition of oral potassium citrate to a high-salt diet
prevented the increased excretion of urine calcium and the bone resorption marker caused by a high salt
intake. The authors concluded that increased intake of dietary sources of potassium alkaline salts from fruits
and vegetables, may be beneficial for postmenopausal women at risk for osteoporosis, particularly those
consuming a diet generous in sodium chloride
80
These conclusions were supported by several animal studies. Sabboh et al, (2005) reported that, in rats, a
diet with a slight protein excess (20 % casein) and non-alkalinizing salts ( high Na:K ratio) induced an acidic
urine (pH 5.5) and a high rate of divalent cation excretion, especially Mg. Potassium bicarbonate or malate
reduced Mg and Ca excretion and alkalinised urine pH (up to pH 8). This animal model of low-grade
metabolic acidosis indicates that potassium malate may be as effective as potassium bicarbonate to
counteract urine acidification and to limit divalent cation excretion in urine. The same group (Demigne et al
2004b) concluded that fruits and vegetables contain K/organic anion salts (malate, citrate), which exert
alkalinizing effects, through KHCO(3)(-) generation, which serves to neutralize fixed acidity in urine.
The same authors (Sabboh et al, 2006) further examined the casein dietary model of protein excess and K
anion salt deficit on the occurrence of metabolic acidosis. Rats were provided with diets containing either 13
or 26% casein, together with mineral imbalance, through lowering K/increasing sodium/omitting alkalinizing
anions. For each protein level, a group of rats was supplemented with K citrate. Dietary K citrate resulted in
neutral urinary pH, whatever the protein level. Urea excretion was higher in rats adapted to 26% casein than
13% casein diets, but K citrate enhanced this excretion and suppressed ammonium elimination. No citraturia
could be observed in acidotic rats, whereas K citrate greatly stimulated citraturia and 2-ketoglutarate
excretion. In conclusion, low-grade metabolic acidosis can occur with a moderate protein level in the diet. K
citrate was apparently less effective in rats adapted to the 26% casein level than in those adapted to the 13%.
A slightly negative point was raised by Rafferty et al (2005) who reported that a high K diet (i.e., one rich in
fruits, vegetables, and dairy products) has multiple health benefits and clearly lowers urine Ca, but it does
not seem to exert any appreciable net influence on the Ca economy, largely because the reduced calciuria is
offset by reduction of intestinal absorption. This may suggest that calcium and potassium rich food should be
ingested at different times
81
Demigne et al, (2004b) pointed out that fruits and vegetables should receive great attention in a
strategy to increase the nutritional value of meals especially reducing calcium losses from bones. The
need to ensure a 2.5- to 3.5-g daily K+ supply from fruits and vegetables represents a strong rationale
for the "5-10 servings per day" recommendations.
III-7 Oligosaccharides and bones
The addition of soluble fibers to diets (specifically pectins, gums, resistant starches, lactulose, oligofructose
and inulin) has been found in various studies to add viscosity to the gut contents, promote fermentation and
the production of volatile fatty acids in the cecum, have a trophic effect on the ceca of animals and increase
serum enteroglucagon. As a consequence, the addition of soluble forms of fiber to diets has been found to
improve absorption of minerals. This may reflect absorption of electrolytes from the large intestine (Greger,
1999). Short chain fructooligosaccharides (sc-FOS) cannot be digested in the small intestine but can be
fermented in the colon. Sc-FOS are converted into short chain fatty acids (SCFAs) by intestinal bacteria in
the colon and improves gastrointestinal (GI) condition. Besides improvement of GI health, dietary sc-FOS
may improve calcium and magnesium absorption in the colon( Tokunaga, 2004, Zafar et al, 2004). Raschka
and Daniel (2005) showed that inulin-type fructans at dietary levels of 10 % (w /w) i ncrease mineral
absorption, retention and accumulation in bone in the case of Ca, Mg and Zn, but only when the basic diet
for the control group contains no intrinsic fructans and when the mineral demand is particularly high as
during growth. In a similar study Hirama et al (2003) showed that gastrectomy-evoked osteopenia in the
femoral metaphysis of rats can be prevented by the consumption of fructooligosaccharides (FOS).
These studies have been conducted mainly with fructooligosaccharides. However Fukunaga et al, ( 2 003)
showed that pectin supplementation resulted in significant increase in short chain fatty acid level . It remains
to be seen if citrus pectins could have a similar effect and increase mineral absorption. Recently Zdunczyk et
al, (2006) suggested that grapefruit flavonoids, such as naringin promote short chain fatty acids production
and could possibly promote mineral uptake. It could be asked if citrus may play a part in the development of
probiotic foods.
82
IV- Conclusions
IV-1 Bone physiology
IV-1-1 Mineral homeostasis
Bones are a living, growing tissue. The skeleton is not physiologically isolated and its health depends
greatly on t he proper interaction with other organs particularly the gastrointestinal tract and the kidneys.
Bones are a reservoir of calcium, phosphorus and magnesium for many physiological functions. The main
bone mineral, calcium, is absorbed from the diet in the GI tract and is filtered back by the kidneys. This
process of maintaining adequate supplies of calcium and other minerals is called mineral homeostasis and is
under a complex hormonal control. In many cases intestinal absorption is a key regulatory step in mineral
homeostasis. Impaired renal function leads to disturbances in this regulatory system, resulting in the complex
syndrome of renal osteodystrophy that affects the majority of patients with chronic renal failure.
A steady supply of calcium and phosphorus is needed to perform the daily functions of the body.
IV-1-1 Bone maintenance
Within the bones, the anabolic and catabolic actions that assure the maintenance of the bone mass are
mediated by the bone-forming cells (osteoblasts) and the bone resorbing cells (osteoclasts). The processes of
bone formation produce opposite results – formation produces bone mass and resorption reduces bone mass.
The two processes are "coupled" (bone resorption is followed by bone formation) and are called bone
remodeling.
Early in life, up to about thirty, the bone steady state favors active bone growth and peak bone mass is
attained. Peak bone mass is the maximum mass of bone achieved by an individual at skeletal maturity,
typically between ages 25 and 35. After peak bone mass is attained, both men and women lose bone mass
over the remainder of their lifetimes.
Since, it is not possible to prevent bone losses later in life, it is important to reach a peak bone mass as
high as possible during the early part of life. The difference between men and women who have
osteoporotic fractures and those who do not is related to the amount of bone they have between ages
25 and 35 (i.e., peak bone mass) and the rate of bone loss thereafter. Studies have shown that citrus
compounds may act on various targets of the body and affect both bone formation by stimulating
osteoblast and bone losses by limiting osteoclasts activity and slowing kidney mineral release.
83
IV-2 Bone losses
IV-2-1 Bone losses and immunity processes.
Both innate and acquired immunities are closely associated with bone degradation diseases from
osteoporosis to rheumatoid arthritis (RA). Furthermore, inflammation and immune systems do not act in an
independent way. Most of the cells involved in the immune response release inflammation markers such as
interleukin and cytokines which are actively involved in bone degradation. There is an intimate interplay
between the bone and the immune system which has been termed osteoimmunology.
Inflammation (innate immunity) can be divided into acute and chronic forms. The acute phase is
associated with wounds or infection and lasts only a short time. It is in many cases beneficial as it is part of
the healing process. If the acute phase cannot be resolved and recovery does not occur, the inflammation
process evolves into a chronic phase which tends to occur over a longer time.
The acquired immune system produces antibodies and specialized cells that attempt to destroy foreign
elements that have entered the body. There are two types of responses: Humoral (antibody) response
(involving B cells) and cell mediated immunity (involving T cells). When a foreign organism enters the
body, the foreign antigens on the invading cells activate an immune response that leads to the destruction of
the intruder. Under certain conditions, the immune system, mistakenly, assumes that a part of the body is
foreign and attempts to destroy it. These errors lead to serious autoimmune diseases such as RA.
IV-2-2 Bone degradation diseases
IV-2-2-a Osteoporosis
The stages of bone loss for m en and women follow the same progression. However, unlike men, women
attain a lower peak bone mass and experience a sharp decline following menopause. T he underlying
problem in osteoporosis is an imbalance between bone resorption and bone formation. In osteoporosis, bone
resorption takes place to a greater extent than bone formation, so a negative balance occurs and results in a
net loss of bone. This imbalance might occur as a result of one or a combination of the following factors:
increased bone resorption within a remodeling unit and/or decreased bone formation within a remodeling
unit (incomplete coupling).
Type I - Postmenopausal osteoporosis occurs in some women aged 50-75 as a result of menopause.
Women who have recently experienced menopause have greater osteoclast activity than premenopausal
women. Menopause results in a decreased production of estrogen hormones which is also essential to
the regulation of the female bone remodeling cycle. Osteoclasts, the bone cells that break down bone,
are activated by a loss of estrogen and a subsequent elevation of inflammatory factors that
stimulate bone resorption (removal of bone from the body with transport to the blood).
84
Type II osteoporosis or senile osteoporosis. It occurs when bone loss and formation are not equal and more
bone is broken down than replaced. It affects both men and women. Type II osteoporosis is associated with
normal processes of aging, with a gradual decline in the number and activity of osteoblasts and not
primarily with an increase in osteoclast activity.
IV-2-2-b Bone diseases associated with the immune processes
Osteoarthritis (OA), also known as degenerative arthritis, is a condition in which low-grade
inflammation results in pain in the joints, caused by wearing of the cartilage that covers and acts as a
cushion inside joints.
Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that primarily targets the
synovial tissues. RA may be genetically inherited, but some infectious agents such as viruses, bacteria, fungi
and certain environmental factors may also trigger the immune system to attack the body's own tissues. It
should be pointed out that with RA, the damage is not limited to the synovial tissue, but also bone loss
occurs both in the joints and throughout the skeleton as a result of the release of proteinase
(metalloproteinases) and proinflammatory cytokines (IL-1, TNF-), which are responsible for
cartilage and bone destruction.
IV-2-2-c Bone diseases associated with kidney activity
Renal osteodystrophy. Impaired renal function occurs when kidneys fail to maintain the proper levels of
calcium and phosphorus in the blood. It affects the majority of patients with chronic renal failure.
Chronic acidemia or high blood acidity is produced by the oxidation of excess sulfur containing amino acids
to sulfate ions, and is related to excessive protein ingestion. To neutralize the acid the body may mobilize its
calcium reserve (mainly from bone) and the kidneys neutralize the fixed acidity by generating calcium
bicarbonate. Low-grade metabolic acidosis can have a deleterious effects on bone calcium status and
lead to bone dissolution.
IV-3 Beneficial effects of citrus.
In recent years many studies have shown that a wide array of citrus compounds have positive activities in
promoting bone growth and preventing bone degradation
85
IV-3-1 Ascorbic acid (vitamin C)
The importance of vitamin C on bone health has been known for a long time from patients suffering from
scurvy. Vitamin C de pletion is responsible for structural collagen alterations, defective osteoid matrix
formation, and increased bone resorption.
Ascorbic acid (AA) is also a major physiological antioxidant, repairing oxidatively damaged
biomolecules, and preventing the formation of excessive reactive oxygen species or scavenging these
species. AA therefore can have a beneficial effect in moderating inflammation processes associated
with bone ailments.
Dehydroascorbic acid (the oxidized form of AA) is a physiologically relevant source of AA for
chondrocytes, particularly in the setting of inflammatory arthritis, such as RA.
Ascorbic acid can modulate the proliferation and osteogenic differentiation of bone cells. Furthermore, AA
plays a key role in the down regulation of the differentiation and activation of osteoclasts. AA plays a role
in the early stage of pre-osteoblasts stimulation, and promotes the molecular mechanism of preosteoblast proliferation.
AA has been reported to play a role in activating bone cell differentiation and limiting the activity of
bone destroying cells. As vitamin C it promotes the formation of bone collagen matrix, necessary for
calcium deposition.
IV-3-2 Folic acid
Elevated homocysteine ( Hcy) and low folate levels are associated with reduced bone mineral density in
women but not in men. This suggests that Hcy may be a potential modifiable risk factor for osteoporosis in
women. However, as seen previously more studies are needed to clarify the role of Hcy, folate, vitamin B6
and vitamin B12 in bone metabolism. It can be concluded that folate contributes to lowering Hcy and
that this amino acid is associated with low bone density. However, as seen in all cases involving Hcy.
However, the cause-effect relationship remains to be determined.
IV-3-3 Flavonoids and polymethoxylated flavones
86
There is now a wide array of evidences showing that flavonoids: polymethoxylated flavones and
polyphenols in general may have a beneficial role on bone health. These compounds may act on t wo
different targets.
1- In a broad manner, flavonoids decrease inflammation. Inflammation is an important component in the
majority of bone impairment diseases including osteoporosis, osteoarthritis and rheumatoid arthritis.
2- Polyphenols appear also to act more specifically on both anabolic and catabolic bone processes.
Hesperidin may prevent bone demineralization (Ca, P and Zn) in both normal and postmenopausal
patients.
Hesperidin may promote the development of bone-forming osteoblasts.
Hesperidin increases the concentration of osteocalcin a protein found in bone that plays a role in
mineralization and calcium ion homeostasis.
Hesperidin may limit the development of collagen-induced arthritis (CIA) and could be effective for
treating human RA patients.
Naringin may promote collagen matrix formation and as a result increase new bone formation.
Kaempferol and hesperidin may prevent the formation of osteoclasts (bone destroying cells)
.
Quercetin may prevent the death of osteoblastic cells.
Flavonoids, and especially hesperidin consumption can improve bone mass acquisition. This is
important during the entire life but particularly during the early years when peak bone mass is being
reached.
Hesperidin and other polyphenols may also play an important role in limiting bone losses in postmenopausal and rheumatoid arthritic patients. This is important later in life when bone loss becomes
a major health problem.
87
Nobiletin is a polymethoxylated flavone with anti-inflammatory properties that may have the potential to
inhibit matrix degradation of the articular cartilage in osteoarthritis and rheumatoid arthritis.
IV-4-4 Carotenoids
Beta-cryptoxanthin has an important anabolic effect on bone development which may be important early in
life when the body tries to reach its peak bone density and increase calcium content.
Beta-cryptoxanthin may stimulate osteoblast proliferation and differentiation and improve bone development
and density.
Beta-cryptoxanthin may also induce osteoclastic cell death and may limit the catabolic effects of osteoclasts.
Studies have demonstrated that beta-cryptoxanthin has an important anabolic effect on bone
development which may be important early in life when the body tries to reach its peak bone density.
The carotenoid may also limit bone losses associated with menopause.
IV-4-5 Organic acids
Proteins generate blood acidity, as a result, the body dissolves some bone calcium to produce the
neutralizing agent, calcium carbonate and eliminate the excess acid. Citrus contain potassium organic salts
(malate, citrate),which exert an a lkalinizing effects through the formation of potassium carbonate. This
potassium salt neutralizes fixed acidity in urine, resulting in neutral urinary pH.
Alkaline salt, such as potassium citrate and malate, can prevent the loss of bone calcium, reducing
bone resorption and may reverse the potential adverse effects caused by chronic acidemia of proteinrich diets.
IV-4-6 Conclusions
Several citrus compounds including vitamin C, folic acid, flavonoids such as hesperidin and naringin,
carotenoids such as beta-cryptoxanthin act on bones to increase the anabolic process and increase
bone production. They act during the early part of life when maximal bone mass is reached and later
in life during senescent and post-menopausal osteoporosis as well as with the various forms of arthritis
to limit bone losses. They also contribute to bone health by limiting inflammation during the entire
life. Furthermore potassium organic acids in citrus limit blood acidity and slow calcium leakage.
88
V- Considerations
V-1 Mineral homeostasis
V-1-1 Mineral bioavailibility
The bioavailabilty of minerals and particularly calcium is very important. M any forms of calcium
particularly calcium carbonate (oyster shell) are very poorly absorbed. Organic calcium salts found in citrus
(citrate and malate) are well absorbed. Citrus juice fortified with calcium provides a very good source of
bioavailable calcium.
V-1-2 Mineral loss by the bones
Citrus juices are rich in potassium citrate and malate. When the blood becomes too acid, calcium is
released from the bone and used to neutralize the acidity. Potassium citrate and malate can neutralize
blood acidity and prevent the release of bone calcium. Minimizing mineral losses is important in both
post-menopausal and senescent osteoporosis.
V-2 Age related changes in bone physiology
In both sexes the equilibrium leans toward bone formation until age thirty. At this time, the bone mass peak
density is reached. During the rest of the life, the equilibrium leans toward destruction(senescent
osteoporosis). In women hormonal changes associated with the menopause accelerate bone loss (postmenopausal osteoporosis).
To achieve the best bone health, it is necessary to maximize bone density early in life and limit bone
losses later. Citrus provide compounds that may be beneficial in these two areas.
V-3 Bone diseases
Most degenerative bone diseases are the result of an impairment between bone formation and destruction
equilibrium. Citrus compounds may be able to help restore bone homeostasis.
V-3 -1 Bone promoting properties of citrus compounds.
89
The main areas directly involved in preserving bone density are:
Collagen matrix formation.
Mineral deposition in the collagen matrix.
Cartilage generation and maintenance.
Osteoblast differentiation and activation.
Ascorbic acid as vitamin C and as ascorbate, flavonoids, particularly hesperidin and nobelitin, and the
carotenoid beta -cryptoxanthin appear to have positive effects in these various areas. They improve
collagen formation, mineral deposition and osteoblast differentiation. Anabolic processes are
important during the entire life, but particularly during the early years of bone development.
V-3-2 Bone conserving properties of citrus compounds.
Osteoclasts are important in eliminating old bone cells. However, later in life and with several diseases like
osteoporosis and the various forms of arthritis, their influence becomes major and lead to bone loss.
Ascorbic acid as vitamin C and as ascorbate, flavonoids, particularly hesperidin and nobelitin and the
carotenoid beta-cryptoxanthin appear to have positive effects in preventing excessive catabolic activity
of osteoblasts.
V-4 Bone and inflammation
Most degenerative bone diseases including osteoporosis, have an important inflammation component.
Inflammation is a major consequence of arthritis and particularly rheumatoid arthritis (RA). RA is also an
auto-immune disease. The immune reaction generates many inflammation markers. All citrus compounds
moderating inflammation ( ascorbic acid, folic acid, flavonoids, carotenoids, limonoids) may have a
positive effects on bone ailments.
VI-Summary: Bone and citrus
Citrus appear to provide significant bone health benefits. They contain a wide array of compounds
acting on several targets that promote bone growth and maintenance and at the same time limit the
destruction part of the bone life cycle. The bone health values of citrus may be the best health
properties of citrus reported so far.
VII- General consideration on health and citrus
90
It should also be emphasized that all the reviews which have been completed so far, indicate very
strongly that the beneficial effects of citrus are maximized when citrus are ingested during the entire
life.
91
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