Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 1392
Vitamin A and Osteoporosis
Experimental and Clinical Studies
BY
SARA JOHANSSON
ACTA UNIVERSITATIS UPSALIENSIS
UPPSALA 2004
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LIST OF PAPERS
This thesis is based on the following papers, which will be referred to in the
text by their Roman numerals.
I
Johansson S, Melhus H. (2001) Vitamin A Antagonizes Calcium
Response to Vitamin D in Man. J Bone Miner Res 16(10):18991905.
II
Johansson S, Lind PM, Hakansson H, Oxlund H, Orberg J, Melhus H. (2002) Subclinical Hypervitaminosis A Causes Fragile
Bones in Rats. Bone 31(6):685-689.
III
Lind PM, Johansson S, Melhus H. (2004) Subclinical Hypervitaminosis A: Effects on Fat-Soluble Vitamins and Bone Mineral
Density. Submitted.
IV
Jacobson A, Johansson S, Branting M, Melhus H. (2004) Vitamin A Differentially Regulates RANKL and OPG Expression in
Human Osteoblasts. Biochem Biophys Res Commun
322(1):162-167.
Reprints were made with the publishers’ permission.
CONTENTS
INTRODUCTION ..........................................................................................9
BONE .........................................................................................................9
OSTEOPOROSIS.....................................................................................14
CALCIUM, VITAMIN D AND BONE...................................................15
VITAMIN A – A GENERAL BACKGROUND .....................................17
VITAMIN A TOXICITY AND BONE ...................................................23
CONCLUDING REMARKS ...................................................................28
AIMS OF THE PROJECT............................................................................29
MATERIALS AND METHODS..................................................................30
RESULTS AND DISCUSSION ...................................................................35
PAPER I ...................................................................................................35
PAPER II and III ......................................................................................36
PAPER IV ................................................................................................38
GENERAL DISCUSSION AND FUTURE PERSPECTIVES ....................39
CONCLUSIONS ..........................................................................................42
ACKNOWLEDGEMENTS..........................................................................43
SUMMARY IN SWEDISH..........................................................................45
REFERENCES .............................................................................................50
ABBREVIATIONS
Į-MEM
ANOVA
BMC
BMD
cDNA
CRABP
CRBP
CrossLaps
CYP
DNA
DMSO
ELISA
FAO
FBS
FGF
GAPDH
HPLC
IGF
IL
LRAT
M-CSF
mRNA
NF-țǺ
NHANES
OPG
PBS
PDGF
PTH
PCR
pQCT
RA
RAE
RANK
RANKL
RAR
RARE
alpha modification of minimum essential medium Eagle
analysis of variance
bone mineral content
bone mineral density
complementary deoxyribonucleic acid
cellular retinoic acid-binding protein
cellular retinol-binding protein
C-telopeptide of type I collagen
cytochrome P450 enzyme
deoxyribonucleic acid
dimethyl sulfoxide
enzyme-linked immunosorbant assay
Food and Agriculture Organization of the United Nations
fetal bovine serum
fibroblast growth factor
glyceraldehyde 3-phosphate dehydrogenase
high-performance liquid chromatography
insulin-like growth factor
interleukin
lecithin-retinol acyltransferase
macrophage-colony stimulating factor
messenger ribonucleic acid
nuclear factor kappa-beta
National Health and Nutrition Examination Survey
Osteoprotegerin
phosphate buffered saline
platelet-derived growth factor
parathyroid hormone
polymerase chain reaction
peripheral quantitative computed tomography
retinoic acid
retinol activity equivalents
receptor activator of NF-țǺ
receptor activator of NF-țǺ ligand
retinoic acid receptor
retinoic acid responsive element
RBP
RDA
RDI
RE
REs
RIA
RNA
RXR
RXRE
S
TGF-ȕ
TNF-Į
TRAP
VDR
WHO
10 × C, 50 × C
1,25-(OH)2vitamin D3
retinol-binding protein
recommended dietary allowance
recommended dietary intake
retinol equivalent
retinyl esters
radio-immuno assay
ribonucleic acid
retinoid X receptor
retinoid X responsive element
serum
transforming growth factor-beta
tumor necrosis factor-alpha
tartrate-resistant acid phosphatase
vitamin D receptor
World Health Organization
10 times control, 50 times control
1,25-dihydroxy-vitamin D3
INTRODUCTION
BONE
The skeleton gives stability to the soft parts of the body. It protects the brain
and the internal organs of the thorax, and it provides muscular attachments
and levers, thus enabling movement. The cavities of the long bones and vertebrae contain the bone marrow, where blood cells are formed. Bone tissue is
also an important reservoir of calcium and other minerals and is therefore
central in mineral homeostasis.
There are two types of mature bone, cortical and cancellous bone, which
differ morphologically and functionally. Cortical bone is dominating in the
diaphyses of the long bones. It is compact, and its most important task is to
give mechanical strength to the skeleton. Cancellous bone is found in the
trabecular bone network in the vertebrae, pelvis and in the epiphyses of the
long bones. Despite its smaller volume, the surface of the cancellous bone is
larger than the cortical surface. It is in close contact with the bone marrow,
and is considered to be metabolically more active than cortical bone (1).
Osteoblasts, osteoclasts and osteocytes are the bone cells of major importance for production and maintenance of bone tissue, and for regulation of its
metabolic function. The precursors of the bone cells originate from the bone
marrow cavity and, when needed, move to skeletal sites for differentiation
and proliferation.
Osteoblasts and bone formation
Osteoblasts, the cells responsible for bone formation, are of mesenchymal
origin. Bone formation starts with the secretion of an extracellular matrix,
consisting mainly of type I collagen (90-95%), but also of several proteoglycans and non-collagenous proteins such as osteocalcin and bone sialoprotein
(2). The deposition of hydroxyapatite crystals of calcium and phosphate salts
converts the matrix into hard bone tissue. This combination of collagen and
mineral salts makes bone tissue both elastic and strong.
The differentiated, mature, active osteoblasts lie together at the bone surface, and are characterized by a cuboidal shape, high alkaline phosphatase
activity and expression of bone matrix proteins (2).
When the osteoblast has enclosed itself in mineralized matrix, it flattens
and remains in its lacunae as an osteocyte, connected to other osteocytes and
to blood vessels via cytoplasmic processes through bone canaliculi (1). The
9
osteocyte is active in the maintenance of the bone tissue, and is proposed to
be a sensor and mediator of the beneficial effect of mechanical stress on
bone mass (3). The lining cells are inactive, flat cells resting on the bone
surface. An ability of lining cells to differentiate into active osteoblastic cells
upon mechanical stimulation has been reported (1,4).
Osteoclasts and bone resorption
Bone resorption is initiated by the activation of osteoclasts. They are derived
from progenitor cells from the monocyte/macrophage lineage, which fuse
and differentiate into multinucleated giant cells. Upon activation, the osteoclast cytoskeleton is rearranged and forms a tightly sealed compartment between the ruffled border, which is a highly folded part of the plasma membrane, and the bone surface. By the transport of H+ ions and the secretion of
proteolytic enzymes, e.g. tartrate-resistant acid phosphatase (TRAP) and
cathepsin K, into this resorption pit, the underlying bone is eroded (2). Degradation products are endocytosed, and transported via the osteoclast cytoplasm to the circulation (2).
The differentiated osteoclast is large and extensively branched. It lies on
the bone surface and is characterized by its multiple nuclei and by the ruffled
border. Measurement of TRAP activity is used as an osteoclast marker (5).
Bone growth and remodeling
In the beginning of life, bone formation occurs via two different mechanisms. Intramembranous ossification is a direct transformation of mesenchymal cells into osteoblasts, which synthesize bone tissue. The flat bones of
the skull and a few other bones are formed this way. In endochondral ossification, the mesenchymal cells first develop into chondrocytes. They produce
a cartilage matrix which is mineralized and used as a template for the new
bone. After apoptosis of the chondrocytes, the cartilage is invaded by osteoblasts and transformed into bone tissue by the deposition of bone matrix.
The long bones, spine and ribs are formed this way. Cartilage remains in two
places: the articular cartilage and the epiphyseal plate, which connects the
bony epiphyses with the diaphysis. By growth of the epiphyseal plate, the
long bones grow longitudinally. The width of the long bones increases when
formation of new bone on the periosteal, external surface of the diaphysis
exceeds resorption on the endosteal side, a process called apposition. After
closure of the epiphyseal plates at the end of puberty, longitudinal growth is
not possible but apposition can still occur.
In the growing skeleton of children and adolescents, bone formation
dominates over resorption, the metabolic activity is high and bone mass is
increased. Peak bone mass is acquired around the age of 25-30 years. Later
in life, bone mass is continuously removed and rebuilt in a process called
bone remodeling (see Figure 1). Resorption always precedes formation, but
the phases are tightly linked. Remodeling, which is going on simultaneously
10
at numerous sites throughout the skeleton, permits maintenance of bone
shape during growth and repair of injuries, and is the prerequisite of the tight
regulation of serum (S) calcium levels. The net effect of bone remodeling is
determined by the balance between the activity of osteoclasts and osteoblasts, which is physiologically controlled by a complex network of
A
B
C
D
E
Figure 1. The remodeling sequence. Resting bone with the bone surface covered
with lining cells (A). When bone resorption is initiated, the lining cells retract. Osteoclast precursor cells fuse and become activated osteoclasts, which move in and
start to resorb bone (B). Meanwhile, osteoblastic precursors differentiate into osteoblasts. The osteoclasts are removed from the resorption pit, and are replaced by
mature osteoblasts (C). The osteoblasts synthesize new bone matrix, which is subsequently mineralized (D). Some osteoblasts are captured in the bone tissue (osteocytes) and some remain at the bone surface (lining cells) (E).
11
endocrine hormones as well as paracrine and autocrine signaling (6,7). Bone
mass is preserved in healthy young adults, but in the middle-aged and elderly, resorption dominates over formation, leading to a loss of bone mass.
Regulation of bone remodeling –the RANK/RANKL/OPG system
Bone remodeling begins with the activation of osteoclast progenitor cells to
form osteoclasts. Fusion of the precursor cells and subsequent differentiation
requires stimulation by M-CSF (8), but it has long been known that cell-cellinteraction with cells of the osteoblastic lineage is also needed. In 1997-98
the molecular constituents of this interaction were identified. Receptor activator of NF-țǺ ligand (RANKL) is expressed by osteoblastic cells, and
binds to its receptor RANK present on the surface of the osteoclastic progenitors (9-12), thereby stimulating the proliferation and differentiation of
the progenitors and the activation of the mature osteoclasts. Osteoprotegerin
(OPG), belonging to the same TNF receptor superfamily as RANK, is a
soluble protein also produced by osteoblastic cells. It is secreted, and acts via
binding to RANKL, thereby blocking its interaction with RANK and neutralizing its osteoclast activating effects (figure 2) (13-15). The importance of
these factors as regulators of osteoclastogenesis is apparent. Over-expression
of OPG in the liver of transgenic mice, causing high circulating levels of
OPG generates an osteopetrotic phenotype (13) while the OPG knock-out
mice have severe, early onset osteoporosis (16,17). Administration of
RANKL increases bone resorption in mice, and is associated with systemic
hypercalcemia (10), and both the RANKL and RANK knock-out mice have
profound osteopetrosis and lack osteoclasts (18,19). After RANKL activation, RANK interferes with TNF receptor-associated family members
(TRAFs), thereby stimulating intracellular signaling pathways leading to
activation of transcription factors and transcription of osteoclast specific
genes (11,12). The ability of pre-osteoblastic/osteoblastic cells to produce
RANKL and activate osteoclastogenesis is greatest in the less differentiated
cells. The more differentiated osteoblasts instead increase the production of
OPG, thereby avoiding the stimulation of resorption at the moment when
formation is about to begin (20).
Apart from the RANK/RANKL/OPG system, there are other molecules
that act locally in bone. For example, the inflammatory cytokines IL-1 and
TNF-Į are potent inducers of bone resorption and they can also inhibit bone
formation (21). On the endocrine level, several factors such as PTH, vitamin
D, sex steroids, cortisol and thyroid hormone can affect the remodeling balance, with apparent effects on bone mass (22-28). However, the
RANK/RANKL/OPG system constitutes the essential regulatory components in the paracrine signaling between bone cells. According to current
knowledge, these molecules are the final regulators of bone remodeling and
most of the other cytokines and hormones are considered to be modulators of
bone remodeling rather than necessary factors.
12
Most of the modulators, though signaling via osteoblasts, primarily affect
osteoclast differentiation and resorptive activity. Less is known about the
regulation of bone formation. Direct stimulation of the proliferation and
differentiation of osteoblastic progenitors is exerted by growth factors such
as FGFs, IGF-1 and TGF-ȕ released from the eroded bone or produced by
the bone cells (29,30), and a regulatory effect of bone matrix proteins is also
likely (31). Leptin was first described as neuro-endocrine inhibitor of bone
formation, but may also act as an autocrine factor in bone tissue in a way
that promotes bone formation (32). Mechanical stress has a positive effect
on bone mass via mechanisms only partly known (33,34).
Mineralization, which completes the remodeling cycle, is not well characterized with regard to its regulation. A calcium-binding role for the bone
matrix proteins, e.g. osteocalcin has been established (35).
Fusion
Differentiation
Activation
Osteoclast
precursor
RANK
RANKL
Mature
osteoclast
OPG
Osteoblastic cell
Figure 2. Cells of the osteoblastic lineage stimulate osteoclast precursor cells to
fuse and differentiate via production and cell surface exposure of receptor activator
for NF-țǺ ligand (RANKL), which binds to its receptor RANK on the osteoclast
precursor cells. Osteoprotegerin (OPG) is a secreted protein produced by osteoblastic cells. OPG acts via binding to RANKL, thereby blocking its interaction
with RANK and neutralizing its osteoclast activating effects.
13
OSTEOPOROSIS
The bone remodeling process is essential for maintenance of the skeletal
functions, but also makes the bone sensitive to disturbances leading to disease. Osteoporosis reflects an imbalance in the remodeling process in favor
of bone resorption, leading to “low bone mass, microarchitectural deterioration of bone tissue and a consequent increase in fracture risk” (36). The increased risk of fracture after low-energy trauma is the major complication of
the disease.
The hip, spine and distal radius are typical sites for osteoporotic fractures
with hip fractures being particularly associated with functional impairment
and increased mortality for the individual, and high costs for society (37,38).
The absolute number of hip fractures in Sweden is around 18,000 per year,
and the calculated cost is around 1.1% of the total direct medical care costs
or 2-3 billion kronor per year (39). The incidence of hip fracture is remarkably high for both men and women in the Scandinavian countries (40-42). In
Sweden, the life-time risk for a 50 year old person of suffering an osteoporotic fracture is approximately 50% for women, and 25% for men (39).
In 1994, WHO defined female osteoporosis as a bone mineral density
(BMD) of 2.5 standard deviations or more below the mean value for healthy,
young adults (43). However, low BMD alone can not explain all osteoporotic fractures (44). According to the Study of Osteoporotic Fractures,
total hip BMD can predict only 28% of hip fractures (45), and elderly individuals have a several-fold increased hip fracture risk compared to young
individuals with the same BMD (46). Thus, there are other factors of interest
in the search for variables that explain and predict osteoporotic fractures.
Among these are the intrinsic mechanical quality (material properties) of the
bone tissue and its spatial distribution (geometry) (44). An increased rate of
bone remodeling, apart from bringing about a loss of bone mass, is itself a
risk factor for fracture, and has been proposed as a major cause of bone fragility (47). Also the quality of bone matrix and its degree of mineralization
can be of importance (48,49).
Postmenopausal osteoporosis is caused by multiple factors, both genetic
and environmental. It is a multigenetic disease, i.e. several genes with modest effects on bone mass are involved. Around 50–90% of the interindividual variance in BMD is considered to be genetically determined, but
the genetic influence is less pronounced with increasing age (50-52). Advanced age and female sex are risk factors for osteoporosis which can, at
least in part, be attributed to postmenopausal estrogen deficiency. However,
men are also at risk for osteoporosis (53), and both men and women are dependent on estrogen for maintenance of bone mass (54). Other risk factors
are height (39), low body weight (55), smoking (56), low physical activity or
immobilization (34,57), low exposure to sunlight (58) and dietary factors
such as calcium and vitamin intake (see below). Secondary osteoporosis can
14
be induced by corticosteroid treatment, hyperthyroidism, alcohol abuse and
skeletal malignancies, among other factors (53). The fracture risk is also
dependent on the tendency to fall, which is influenced by loss of balance,
muscle weakness, visual impairment, and the environment (59,60).
CALCIUM, VITAMIN D AND BONE
Calcium
Calcium is important for cells throughout the body, as a co-factor for proteins, for control of neuronal excitability and as an intracellular 2nd messenger. Therefore the calcium level of the extracellular compartment is kept
constant through delicate homeostasis. In the skeleton, deposition of calcium
salts simultaneously increases bone strength and provides a store of calcium,
and about 99% of the body’s calcium content is found in the tissues of bone
and teeth. Calcium absorption and preservation in the body is not very efficient. Intestinal absorption can be as low as 15% (61), and the ability to
adapt to a low calcium intake is reduced after menopause and in elderly people (62). Renal conservation of calcium also deteriorates after menopause
(63).
The skeleton, intestine and kidney are central in the regulation of calcium
homeostasis. Calcium absorption in the intestine is facilitated by vitamin D,
especially under conditions of low calcium intake (64). Via a brush border
calcium channel and the intracellular calcium-binding protein calbindinD9k,
both largely vitamin D-dependent, calcium is actively absorbed and transported to the circulation (65). If abundant, calcium is deposited in bone tissue via mineralization. On the contrary, a low calcium level leads to an instantaneous release of PTH from the parathyroid glands. PTH increases activation of vitamin D in the kidney, with secondary positive effects on intestinal calcium absorption. PTH also increases reabsorption of calcium from the
urine and mobilizes calcium via direct effects on bone (66). Via these
mechanisms, the overall goal to increase serum calcium to normal is
achieved. Since the need for calcium mobilization overrides other functions
of the skeleton, calcium homeostasis is of major importance for the balance
of bone remodeling (30).
Vitamin D
Vitamin D is produced endogenously in the skin where 7-dehydrocholesterol
is converted to cholecalciferol (vitamin D3) in response to exposure to ultraviolet light (67). Vitamin D3 and the less common ergocalciferol (vitamin
D2) can also be obtained from the diet, via absorption in the small intestine
and transport in chylomicrons to the circulation. During the darker period of
the year, people in Sweden and other countries at high latitude have a low
15
endogenous production of vitamin D, and hence become more dependent on
dietary intake. Young people can use stores of vitamin D produced during
the summer, but elderly people have a reduced capacity for endogenous production and therefore reduced storage capacity, and are at risk of vitamin D
deficiency (64).
The active compound 1,25-(OH)2-vitamin D3 (calcitriol) is produced via
successive 25- and 1Į-hydroxylation in the liver and kidney, respectively
(67). Since 25-hydroxylation in the liver is poorly regulated, 25-OH-vitamin
D is commonly used as an indicator of vitamin D status. A 24-hydroxylase,
present in the kidney, liver and target tissues, can also convert 1,25-(OH)2vitamin D3 into an inactive metabolite, thus initiating the major catabolic
pathway (68). 1,25-(OH)2-vitamin D3 exerts vitamin D activity via binding
to the genome in complex with the vitamin D receptor (VDR), thereby regulating the expression of certain genes. For some very rapid responses to vitamin D treatment, among them the rapid stimulation of intestinal calcium
absorption, an alternative signaling pathway including cell surface receptors
has been proposed (69). The classic vitamin D target tissues include the intestine, the kidney, the parathyroids and bone. Vitamin D also has nonclassic
actions in many other organs where it regulates cell differentiation and proliferation (64).
Despite the usage of vitamin D for the treatment of osteoporosis and other
metabolic bone diseases, the mechanism by which it is beneficial for bone is
not exactly known. 1,25-(OH)2-vitamin D3 can induce differentiation of osteoclastic cells, but indirectly via osteoblastic cells (64). Interestingly, a
physiological vitamin D dose inhibits bone resorption while a pharmacological dose stimulates resorption (70-72). The characteristic disease of vitamin
D deficiency is rickets or, in the adult, osteomalacia, i.e. incomplete mineralization. However, the skeletal symptoms of the VDR knock-out mice –
hypocalcemia and rickets – are rescued by a high calcium diet (73,74).
Therefore it has been assumed that the function of vitamin D is to provide
calcium levels sufficient for mineralization rather than acting directly in the
mineralization process.
Intake levels in relation to bone health
Several randomized trials have shown a positive effect of calcium supplementation on BMD ((75,76) and references therein) and animals fed a calcium-deprived diet suffer from a PTH-dependent loss of bone mass (61). It is
suggested that an insufficient calcium intake leads to decreased bone gain
during growth and bone loss in the adult (75). Despite this, a large number of
observational studies have come to different conclusions about the effect of
dietary calcium intake on bone health (77), and there is no consensus about
the optimal level of calcium intake. Though an increased dietary intake of
vitamin D has been associated with decreased risk of hip fracture, the few
clinical trials about vitamin D supplementation and fracture risk are conflict16
ing ((78) and references therein). Since no study has proven an effect of calcium supplementation independent of vitamin D on osteoporotic fractures, a
combination of calcium and vitamin D supplement is recommended as osteoporosis treatment in many countries, including Sweden (39).
VITAMIN A – A GENERAL BACKGROUND
Vitamin A is a group of fat-soluble nutrients which are essential for cell
proliferation and differentiation in many different tissues. They are involved
in important physiological processes during embryogenesis, in the immune
system, in reproduction, and for maintenance of the skin and mucous membranes. In the eye, the retinol metabolites have a unique role in the visual
cycle (79).
In the diet, two main groups of vitamin A can be distinguished: retinoids
or preformed vitamin A, including retinol and its metabolites, and carotenoids or provitamin A (see figure 3). Retinoids are found in animal products
such as liver and dairy products, and vitamin supplements are also an important source (80-82). Carotenoids, for example ȕ-carotene, come from vegetable sources (83).
Nomenclature
Biochemically, the term retinoid is defined as a substance that can exert biologic vitamin A activity, via binding to and activation of a specific receptor,
“with the program for the biologic response of the target cell residing in the
retinoid receptor rather than in the ligand itself” (84). This definition includes the dietary retinoids but also synthetic retinoids, substances that are
often more active than the naturally occurring compounds. Concerning the
carotenoids, only about 10% of them can exert vitamin A activity (85), and
only after conversion in the body to retinol. They are therefore not included
in the term retinoids.
In this thesis, both “vitamin A” and “retinoids” will be used as alternate,
general terms for the group of naturally occurring substances that can readily
exert retinoid activity, i.e. not including the synthetic retinoids or the carotenoids. Specific metabolites will be denoted by their chemical names.
Definition of vitamin A activity
FAO/WHO defined the golden standard for calculation of vitamin A activity
in the diet. The unit 1 µg retinol equivalent (RE) was defined as equal to the
activity of 1 µg retinol or 3.33 international units (IU) of vitamin A activity
from retinol (86). Moreover 1 µg RE was defined as equal to 6 µg of betacarotene or 12 µg of other carotenoids. Because of increasing knowledge
about the poor bioconversion of provitamin A compounds, a new definition
termed retinol activity equivalents (RAEs) has been proposed. One micro17
gram RAE equals 1 µg retinol = 6 µg supplemental beta-carotene = 12 µg
dietary beta-carotene. Still RE is most commonly used for dietary recommendations.
Beta - carotene
CH2OH
Retinyl
esters
All - trans - retinol
CHO
All - trans - retinal
COOH
All - trans - retinoic acid
Figure 3. Vitamin A metabolites
Recommended Dietary Intake
The recommended dietary intake (RDI) of vitamin A stated by FAO/WHO is
600 µg RE/d for men and 500 µg RE/d for women (87). However, there is an
uncertainty about the adequate intake, and therefore quite large differences
between countries. The Nordic recommendations are 900 µg RE/d for men
and 800 µg RE/d for women (88). The actual intake in the population also
varies considerably. In the Nurses’ Health Study, 14% of the women had an
intake below the recommended dietary allowance (RDA, used in the United
States) of 700 µg RAE/d, while 21% had an intake exceeding the tolerable
18
upper limit which is set at 3,000 µg RAE/d (82,89). The average vitamin A
intake in Sweden is 1,300 µg RE/d for men and 1,100 µg RE/d for women
(90), which is high compared to countries in southern Europe (91). In Norway, the intake is even higher (1,900 µg/d for men and 1,600 µg/d for
women (92)).
Vitamin A metabolism
The uptake, transport and storage of vitamin A are well-regulated, with the
goal of providing the body with correct amounts of retiniods even when dietary intake fluctuates. Retinoids occur in the diet mainly as retinyl esters
(REs). They are hydrolyzed to retinol and free fatty acids in the intestine and
absorbed from the lumen. Carotenoids, mainly ȕ-carotene, diffuse into the
epithelial cells where they are cleaved by a dioxygenase (93). The resulting
two retinal molecules are reduced to retinol by a retinal reductase (94). In the
luminal epithelial cell, retinol is bound to the cellular retinol-binding proteinII (CRBP-II) (95). Retinol is re-esterified, mainly with palmitate, by lecithinretinol acyltransferase (LRAT) (96), incorporated into chylomicrons and
transported via the lymph to the blood circulation (97). The chylomicron
remnants are cleared by the liver where the REs are hydrolyzed and reesterified by hepatic hydrolases and LRAT, respectively. During these processes, the free retinol is bound to CRBP-I, and transported to stellate cells
where re-esterification and storage of the resulting REs occur. Some other
organs, such as the kidneys, also have storage capacity (98). CRBP-I shares
structural, genetic and biochemical properties with CRBP-II but is expressed
in various tissues (99). CRBP-II is almost exclusively expressed in the cells
of the small intestine where it is one of the most abundant proteins. Both
proteins have a function in the enzymatic conversions of vitamin A metabolism, and CRBP-II possibly also in the absorption of retinol from the intestinal lumen (100). Apart from this generally accepted description of retinoid
uptake and storage, there are also reports that small amounts of retinoic acid
can be transported from the intestine via the portal vein (98,100).
When there is a need for retinoids in the body, the stored REs are hydrolyzed and released to the circulation as retinol bound to retinol-binding protein (RBP) (101). RBP has been considered to protect the body from toxic
effects of free retinol. However, RBP-knockout mice were essentially unaffected (102). In the target tissues, retinol is transported across the plasma
membrane through a mechanism that is still unclear. In the eye, retinol is
transformed stepwise to 11-cis-retinal, which together with opsin forms
rhodopsin, the visual cycle chromofore in photoreceptor cells (103). In most
other tissues, including bone, the active metabolite is retinoic acid (RA).
19
Intestinal lumen and epithelial cell
Retinyl
esters
Retinol
Circulation
Liver
Chylomicronsretinyl esters
Retinyl
esters
Retinyl
esters
Retinol
Retinol
Retinal
Betacarotene
Betacarotene
RBPretinol
Storage of
retinyl esters
Target tissue
RARRXR
Retinoic
acid
Retinal
Retinol
Figure 4. Vitamin A metabolism. Retinoids (retinyl esters) from the diet are hydrolyzed to retinol and free fatty acids in the intestinal lumen before absorption. Carotenoids (beta-carotene) are cleaved to retinal after absorption, and further reduced
to retinol. Retinol from both sources is esterified and incorporated into chylomicrons, which are transported via the lymph to the circulation. In the liver, retinyl
esters are hydrolyzed to retinol, which is either released to the circulation bound to
retinol-binding protein (RBP) or re-esterified and stored in the liver as retinyl esters. In the target tissues, retinol is converted to the active metabolite retinoic acid,
which binds the retinoic acid receptor-retinoid X receptor (RAR-RXR) dimer. The
receptor-ligand complex acts as a transcription factor in the nucleus.
In RA synthesis, retinoids bound to CRBP-I are believed to be the dominating substrates. A retinol dehydrogenase oxidizes retinol to retinal, which
is further oxidized by retinal dehydrogenase to RA (104). The last step is
irreversible, and ends with the transferal of RA from CRBP-I to cellular
retinoic acid-binding protein (CRABP) (105). Tissue RA is inactivated by
hydroxylation and oxidation to more polar and less active substances. The
20
conversion is catalyzed by cytochrome P450 enzymes (98), of which the
most important is CYP26 whose expression is also induced by RA
(106,107). RA and retinol can also be converted to retinoyl ȕ-glucoronide
and retinyl ȕ-glucoronide, respectively, mainly in the liver and intestine
(105). These compounds are less toxic and are secreted in the bile, but the
physiological importance of this route of inactivation is uncertain.
Serum levels of vitamin A metabolites
Retinol, bound to RBP, is the most abundant of the retinoid metabolites in
serum. The normal value is poorly defined, but is usually considered to be
approximately 1-2 µM (108). S-retinol (< 0.7 µM) is used as a marker of
retinoid deficiency, though it is not very sensitive. Not until liver levels of
vitamin A are very low does S-retinol decrease (109). S-RA levels are low,
around 4-14 nM (98), but may contribute to the delivery of retinoids to target
tissues (110). S-REs normally increase postprandially, and there is a fivefold interindividual difference in clearance from serum (half-life 1.54-9.90 h
after a single intake of 50,000 IU) (111). In cases of vitamin A intoxication,
S-REs are elevated for extended periods. It is generally accepted that the
normal S-RE level is <5-10% of circulating vitamin A and that S-RE >10%
is an indication of chronic vitamin A toxicity (83) In absolute numbers, this
corresponds to a normal value <244 nM (112).
Molecular action of vitamin A – regulation of gene transcription
The identification of the first retinoid receptor in 1987 was a breakthrough in
the vitamin A field (113,114). Since then, five more receptors have been
cloned and the retinoid receptors known today include retinoic acid receptor
(RAR) Į, ȕ and Ȗ, and the retinoid X receptor (RXR) Į, ȕ and Ȗ (115). However, many more isoforms exist, due to different splicing and promoter usage. Both the all-trans-RA and 9-cis-RA metabolites can bind to the RARs
while the RXRs bind only 9-cis-RA (116).
RARs and RXRs are members of the steroid-thyroid hormone receptor
family, which also includes the vitamin D receptor (VDR) and the thyroid
hormone receptor. These receptors act as heterodimers, and RXR is the
common dimerization partner, acting together with RAR, VDR or any other
receptor in the family (117). Binding of ligand, for example RA binding to
RAR, activates the dimer complex. The RXR ligand 9-cis-RA has been
shown to be a modulator of heterodimer action in vitro, and has been proposed as a possible modulator of vitamin D, thyroid and steroid hormone
signaling (118). However, the physiological relevance of these findings is
controversial. A recent in vivo study on mouse embryos indicates that transcriptional regulation by 9-cis-RA is a pharmacological phenomenon, but
that 9-cis-RA is not necessary under physiological conditions (119). Even
the existence of 9-cis-RA in vivo is questioned, since it has been undetect21
able after its initial discovery. RA will hereon be used to refer to all-transRA, unless otherwise is stated.
In the classical signaling pathway, ligand-binding activates the RARRXR complex, leading to conformational changes of the heterodimer (115).
The activated complex binds to specific DNA sequences called retinoid response elements (RAREs or RXREs), which are transcription enhancers
upstream or downstream the transcription start site. Ligand-activated RARRXR functions as a transcription factor with transactivating or, less commonly, transrepressive effects (115). In the absence of ligand, the heterodimer can repress transcription of target genes (120). Transcriptional
control involves interaction with co-activator and co-repressor proteins
(121).
The genes of many proteins of importance for retinoid metabolism, such
as RARs, CRBPs and CRABPs, contain RAREs (115,122). However, some
genes also lack RAREs, even though their expression is affected by retinoids. This regulation is called indirect, as opposed to the direct regulation
via the classical RA pathway. Indirect regulation can entail activation of
receptors other than RAR-RXR, influence on mRNA stability, interference
with other transcription factors or regulation of an intermediary factor (122).
Of course this distinction is not easily made for many of the known RAregulated genes.
It is largely unknown how retinoid signaling is regulated. The uptake of
retinol in the target cell, the rate of retinol esterification to REs or oxidation
to RA, the expression of receptors and retinoid binding proteins are all possible regulative stages. In this perspective, Raldh2 and other retinal dehydrogenases have been pointed out as key factors during development (123).
Regulation of of LRAT and CYP26 expression can serve to maintain retinoid homeostasis (124). Cell-specific action can be achieved by variation of
receptor expression, isoforms and co-factors.
The physiological role of vitamin A in bone
In general, a major role of vitamin A is to regulate proliferation and differentiation of cells, and this is a likely role of vitamin A in bone tissue as well.
Both osteoblastic and osteoclastic cells express retinoid receptors. RARĮ,
RARȕ, RARȖ and RXRĮ have been found in osteoblasts (125), which also
express CRBP-I, CRABP-I and low levels of CRABP-II. In osteoclasts, the
expression of RARĮ and RXRȕ has been demonstrated (126). A direct, upregulatory effect of RA is reported for at least the RARĮ, ȕ and Ȗ, CRABP-II
and CRBP-I genes (122,127,128). The generation of mice with combined
knock-outs of the retinoid receptors has emphasized the importance of vitamin A during development, but since the mutants died in utero or shortly
after birth, our knowledge about how they function in the adult skeleton was
hardly increased (reviewed in (129)).
22
In bone cells, known targets for RA gene activation in vitro are the genes
for alkaline phosphatase (130,131), osteocalcin (132), osteopontin (133) and
osteonectin (134) which are induced in osteoblasts, and the cathepsin K/OC2 (126) and osteopontin (135) genes, which are induced in osteoclasts. RA
treatment also influences the expression of collagen and collagenase
(136,137), and vitamin A stimulates bone resorption and osteoclast number
and activity both in vitro and in vivo.
The expression and regulation of genes involved in retinoid signaling and
metabolism, and the induction of bone-specific genes by RA indicate a
physiological role for vitamin A in bone tissue.
VITAMIN A TOXICITY AND BONE
Both deficiency and overdose of vitamin A are hazardous for the body. Vitamin A deficiency usually does not occur in well-nourished populations, but
in the rest of the world it is one of the major nutritional problems. The opposite situation, vitamin A intoxication or hypervitaminosis A, with special
regard to skeletal toxicity, is the subject of the rest of this thesis. Since carotenoids do not cause hypervitaminosis A in animal experiments, and there
are no reports about vitamin A toxicity even after massive overdose of carotenoids (138,139), the following work will focus on the toxicity of retinoids.
General vitamin A toxicity
The first description of vitamin A intoxication, written in 1596, tells about a
group of Dutch polar explorers complaining of headache, dizziness, and
vomiting after eating polar bear liver (81). There are several more reports
about acute toxic reactions, and even one death, after ingestion of bear and
seal liver. Today the common cause is overdose of vitamin A supplements.
However, acute toxicity is relatively rare. It occurs within a short time period
(hours up to a few days) after ingestion of one single high dose, in adults
usually of 500,000 IU or more (over 150 times the RDI) (140). Children are
more sensitive. The symptoms include headache, nausea, mental confusion,
fatigue, dry skin, bulging of the fontanel, hydrocephalus and toxic hepatitis
(140). Chronic toxicity presents more insidiously, but with similar symptoms: dryness and desquamation of the skin and mucous membranes, alopecia, nausea and vomiting, loss of appetite and weight, fatigue, headache and
bone and joint pain (81).
Because of the accumulation of retinoids in the liver, chronic toxicity can
arise at intake levels much lower than those causing acute toxicity. The duration of intake is crucial, and chronic toxicity occurs after months or years of
increased consumption (139). The lowest dose causing chronic hypervitaminosis A in humans is not known. It usually appears at doses of 100,000 IU
(>30 times RDI) per day and above (81), but has been reported at intake
23
levels as low as 25,000-50,000 IU daily (141-143). The suggested threshold
dose for teratogenicity is 3 mg or 10,000 IU per day (144). Vitamin A in
water-miscible, emulsified and solid forms are more toxic than oil-based
preparations or retinol in liver, and result in higher peak plasma values,
higher liver concentrations and lower fecal losses (145).
The evaluation of reported human data is complicated. Intake of other nutrients or toxins (e.g. high alcohol intake), and the storage capacity of the
liver, which is affected by liver disease and prior vitamin A intake, influence
the dose and time needed for development of toxicity symptoms (139,145).
Moreover, it is difficult to estimate the dietary intake of vitamin A, and in
many studies the results have been blurred because the researchers have not
differentiated between retinoid and carotenoid intake. Subclinical adverse
effects are probably evident at intake levels considerably lower than those
reported for general, clinical toxicity.
Bone toxicity of vitamin A in rats
In 1925, the adverse effects of hypervitaminosis A in rat were described for
the first time by Takahashi et al who treated rats with fish-oil-concentrates in
daily doses around 10,000 times the levels required (146). Takahashi et al
described the general vitamin A toxicity syndrome, including paralysis of the
legs, hemorrhages, weight loss and alopecia of the head. The true skeletal
harmfulness of vitamin A was evident in 1933 when spontaneous fractures
were confirmed by X-ray examination in young rats receiving very high
doses of fish-oil concentrates (147,148). Moore and Wang carried out the
first study on intoxication with pure vitamin A (purified retinyl acetate) in
1945, thereby confirming that the fish-oil toxicity was caused by retinoids
(149). They also reported spontaneous fractures as the most prominent
symptom. Skeletal lesions have also been reported in dogs, pigs, rabbits and
chickens (150).
Fractures, which are found mainly at the diaphysis of the long bones
(147), seem to develop earlier in young rats than in adults, and the lowest
daily dose known to lead to fractures is around 3 mg (10,000 IU) in young
rats and 7.5 mg (25,000 IU) in adult rats (150). Other bone lesions include
thinning of the long bones (151), thinning of the cortex, premature epiphyseal closure and accelerated bone remodeling (reviewed in (150) and (143)).
More recent studies, using histomorphometry, have shown that the number of osteoclasts is increased and osteoid surface decreased in rats treated
with 50,000 IU per day for 6 weeks (152) and in rats treated with 10,00025,000 IU per day for 3 weeks (153). Increased S-alkaline phosphatase and
urinary hydroxyproline excretion, indicating increased bone turnover, were
also seen (153).
The effect of moderately elevated doses of vitamin A has rarely been
evaluated. Li et al saw no significant changes in bone ash content or bone
areas in adult rats fed 300 IU retinyl acetate per day for 14 months, although
24
histomorphometry showed changes in both fractional resorption and formation surfaces (154). This dose is around 5 times the calculated daily amount
needed for normal growth of the rat (155). Decreased whole body BMD, as
measured by DPX-Į bone scanning and ash weight of femur, has been reported after administration of RA at doses ranging from 10 to 100 µg/day for
4 months in young rats (156). Since it is not known how IUs correspond to a
certain amount of RA, it is difficult to compare the doses in this study with
previous experiments.
Bone toxicity of vitamin A in vitro
A bone resorptive effect of vitamin A has been demonstrated both as shrinking of long bones of mouse and chick embryos cultivated in vitamin A-rich
medium (157) and as increased 45Ca release from cultured fetal rat and
mouse calvaria (153,158). Both stimulation of osteoclast numbers and activity (159,160) and decreased growth of osteoblasts (161) have been reported.
RA can regulate the expression of the genes for alkaline phosphatase, osteocalcin, and osteonectin in osteoblastic cells (130-132,134), and cathepsin K
and osteopontin in osteoclasts (126,135). In embryonic chick calvaria, collagen expression is decreased (136) and collagenase synthesis and collagen
degradation increased (137) by retinol.
It is difficult to compare the in vitro effects of vitamin A because of the
variety of experimental systems (e.g. fetal calvarial cultures, resorption pit
assays), cell types (e.g. fetal or adult, different species) and methods for isolation of osteoclasts (pieces of whole bone, bone cell suspensions including
both osteoblastic and osteoclastic progenitors, isolated osteoclasts). One
study even shows that RA inhibits bone resorption (162). However, altogether the in vitro data strongly indicate that vitamin A possesses bone tissue
activity, and RA is generally considered to be a bone resorbing agent. The
expression of RARs, RXRs, and retinoid binding proteins in bone cells, and
their regulation by RA, support this statement (127,128).
Bone toxicity of vitamin A in humans – case reports
Several reports have described skeletal pain and abnormalities seen in accidental cases of chronic vitamin A intoxication (163-166). Severe bone pain,
hypercalcemia, periosteal calcifications, exostoses and increased bone resorption are among the reported symptoms, and radiographic osteopenia has
been seen. Patients with hypercalcemia have normal or decreased PTH levels, indicating that the high calcium level has arisen through a direct effect of
vitamin A on bone rather than via PTH (166,167). Hypercalcemia does not
always develop, but the reason for this is unclear.
The amount of ingested vitamin A differs between the cases, but is usually about 85,000-125,000 IU per day or above. For water-miscible, emulsified and solid forms of vitamin A, the lowest dose known to induce chronic
toxicity is as low as around 43,000 IU per day (0.2 mg/kg per day) (145).
25
The duration of intake has usually been one or more years, but much shorter
periods are reported for water-miscible, emulsified and solid preparations
(143,145). One case of fracture in a 1 year old girl has been reported after
intake of 50,000 IU daily for 7 months (168).
Bone toxicity of vitamin A in humans – epidemiological studies
A number of epidemiological studies have addressed the question of vitamin
A and bone health. The information from some of these studies is limited,
since they are too small or have not distinguished between retinoid and carotenoid intake.
The first study in which a separate analysis of retinoid and carotenoid intake was performed, came from Melhus et al in 1998 (169). They reported an
association between a high intake of dietary vitamin A and reduced BMD
and increased risk for hip fracture. BMD was reduced by 10% at the femoral
neck and the risk for hip fracture was doubled when the group with an intake
exceeding 1.5 mg retinol per day was compared with the group with an intake of 0.5 mg per day or less. This finding has been confirmed by others,
using either dietary retinoid intake or S-retinol as a measure of vitamin A
exposure (77,82,170,171). In one study, it was shown that men with a Sretinol level exceeding 3.6 µM had a relative risk of 7.14 for any fracture
(77).
There are also studies which do not find an association (112,172,173).
One explanation to this failure may be the difficulty to correctly estimate
vitamin A intake, leading to miscalculations (174). The serum metabolites
are not very sensitive markers of high vitamin A intake either. Another explanation for the inconsistency is the fact that vitamin A intake actually varies between populations. Accordingly, mean intake in the different studies
varies considerably, and this is likely to affect the outcome. In two studies
the association is U-shaped (170,171), and this dual relationship may also
explain the failure to demonstrate a linear association in some studies.
Synthetic retinoids in clinical use
Pharmacological use of synthetic retinoids has been of great benefit for the
treatment of dermatological and malignant diseases. However, this use also
has an established skeletal toxicity, as evident from several reports of bone
pain and artralgia, calcifications of ligaments and tendons, osteophytes, premature epiphyseal closure and radiographic changes in patients on etretinate
or isotretinoin treatment (175-180). Hypercalcemia is reported after longterm treatment (181), but Kindmark et al found a transient decrease of Scalcium as well as of several markers of bone turnover during the first 14
days of isotretinoin treatment (182). Regarding effects on bone mass, this
and some other studies found no harmful effects of isotretinoin (182-184),
etretinate (185) or acitretin (186) while some studies show decreased BMD
after treatment with etretinate (184,187) or isotretinoin (188). Large, pro26
spective studies, which are needed to thoroughly investigate the effects of
synthetic retinoids on bone mass, have not been published yet.
In the rat, administration of 5 mg/kg (about 0.8 mg) etretinate daily for 15
days led to decreased tibial breaking strain (-11%) and spontaneous fractures
in 1 of 5 rats, without affecting body weight gain (189,190). After administration of 50 mg/kg (about 8 mg) isotretinoin for 15 days, tibial breaking
strain was reduced (-15%) but no spontaneous fractures were seen.
Interaction between vitamin A and vitamin D
An interaction between vitamin A and D has long been suggested, in part
based on clinical observations and in part on the basis of the VDR-RXR
heterodimer mediating the molecular action of vitamin D. Accordingly, allosteric receptor ligand interactions have been demonstrated in vitro (118),
and in vitro studies have variously indicated antagonistic, additive or synergistic interaction between the vitamins (191). The recent finding that 9-cisRA exhibits receptor-binding activity in vivo only at pharmacological doses,
and the failure to isolate 9-cis-RA in vivo (119) makes a direct ligandmediated interaction with the VDR-RXR complex less likely, but an antagonism can arise via competition between RAR and VDR for formation of
RXR heterodimers. Many other mechanisms for interaction, during intestinal
absorption, transport, metabolism or storage of the vitamins, are of course
possible. For example, RA can induce expression of a 24-OH-hydroxylase
which is a key catabolic enzyme for 1,25-(OH)2-vitamin D3 (192).
In vivo, a high level of vitamin A intake has been shown to reduce the
toxicity associated with hypervitaminosis D in the rat (193) and the turkey
poult (194) and to increase the need for dietary vitamin D in the chicken
(195) and the poult (194). Hypervitaminosis D similarly diminishes vitamin
A toxicity symptoms, such as reduced bone ash, in the chicken (196). In rats,
an antagonistic relationship has been demonstrated in bone and intestine by
Rohde et al 1999 (197). The possible interaction between vitamin A and D in
man had not been investigated when this project was initiated. In 2003, a
review of all hitherto published case reports of vitamin A intoxication found
that the mean dose causing chronic toxicity was lower for patients taking
vitamin A only, compared to patients taking supplements containing both
vitamin A and vitamin D (145). This finding is in agreement with competitive antagonism between the vitamins.
27
CONCLUDING REMARKS
In summary, the skeletal toxicity of vitamin A is well documented in animal
studies. The potential of vitamin A to affect the human skeleton is evident
from case reports of human vitamin A intoxications, and several epidemiological studies have shown an association between an excessive dietary intake of vitamin A and increased fracture risk. However, our knowledge
about how vitamin A functions in bone tissue is still limited. Also, the lowest
level for vitamin A toxicity is not known.
The possible mechanisms for vitamin A action on bone tissue can be
roughly categorized as direct or indirect. Direct effects on the skeleton are
likely, since both osteoblastic and osteoclastic cells express retinoid receptors and other proteins involved in vitamin A metabolism. RA can regulate
several bone specific proteins and stimulate bone resorption in vitro, but the
molecular mechanisms of RA action in bone tissue are still largely unknown.
Among indirect mechanisms, the interaction between vitamin A and D
has been in focus. I animals, an antagonistic relationship has been established. The corresponding relationship in man has not been investigated previously.
28
AIMS OF THE PROJECT
The starting point for this thesis was the publication in 1998 by my tutor
Håkan Melhus, who had found that an excessive intake of dietary vitamin A
was associated with increased risk of hip fracture. This finding naturally
raised the question of a causal relationship, i.e. can vitamin A be involved in
the pathogenesis of osteoporosis?
In this project, the effect of vitamin A on bone metabolism and bone quality in vitro and in vivo has been studied. The general aim was to identify
possible mechanisms of vitamin A action and to investigate the lowest level
for vitamin A toxicity. In specific, the aims were:
x to examine whether vitamin A can antagonize the effect of vitamin
D in humans by studying the acute effects of vitamin A and D on
calcium homeostasis.
x to study if a long-term excessive vitamin A intake at a level that
does not cause general toxicity can adversely affect the rat skeleton
and, if so, begin to investigate at what level of intake skeletal lesions manifest.
x to examine how BMD and bone geometry were affected in rats
with subclinical hypervitaminosis A.
x to study in vitro the effect of RA on the expression of OPG and
RANKL - central molecules in the regulation of osteoclastogenesis.
29
MATERIALS AND METHODS
The following is a brief summary of materials and methods used in this thesis. A detailed description can be found in the individual papers.
Subjects (Paper I)
Four men and five women (mean age 28 years) were recruited. Their healthy
status was assessed by an interview and the presence of any undiagnosed
parathyroid disease was ruled out by the biochemical analyses of serum performed on the first reference blood sample (see Design). Informed consent
was obtained from all subjects volunteering for the study.
Animals (Paper II and III)
Forty-five female Sprague-Dawley rats, 3 months of age, were obtained
from Möllegaards breeding centre Ltd. (Skensved, Denmark). They were
kept in polycarbonate cages with hardwood chips as bedding, using a 12
hour light:dark cycle, and they had free access to water and commercial pellet diet (Lactamin R36, Stockholm, Sweden). Prior to use they were allowed
to acclimatize for a minimum of 23 days.
Cell Cultures (Paper IV)
Cells from the human osteosarcoma cell line MG-63 and primary human
osteoblast-like cultures were used. Primary cultures were isolated from bone
fragments taken from patients undergoing surgery. Trabecular bone was cut
into pieces, thoroughly rinsed and vortexed in PBS five times. Both cell
types were cultured in Į-MEM supplemented with 10% FBS, 2 mM Lglutamine and antibiotics. The osteoblastic phenotype of cells in the primary
culture was verified by use of biochemical markers as previously described
(198). RA was dissolved in 95% ethanol in a dark room under flow of nitrogen. The stock solution was stored at –70 °C and shielded from light until
use. The high affinity pan-RAR antagonist (AGN 194310) and the RAR-ȕ,Ȗagonist (AGN 190299) were dissolved in dimethyl sulfoxide (DMSO).
Study Design (Paper I-IV)
All studies were approved by the Uppsala University ethics committee.
30
Paper I
Nine healthy subjects were included in a double-blind, randomized, crossover study. On four different study occasions, every subject took one of four
different oral vitamin preparations (Table 1).
Table 1. Vitamin preparations
Occasion Vitamin intake
1.
2.
3.
4.
15 mg retinyl palmitate
2 µg 1,25-(OH)2-vitamin D3
15 mg retinyl palmitate plus 2 µg 1,25-(OH)2-vitamin D3 (combined intake)
Placebo
The vitamin intake was always at 22.00, and the effect was monitored by
urine and blood samples the following day (Table 2). All samples were analyzed for S-calcium, S-albumin, S-parathyroid hormone and the degradation
product of C-telopeptide of type I collagen (S-CrossLaps), and urine calcium
and creatinine. Analyses of S-1,25-(OH)2-vitamin D3 and S-REs were performed for the eight o’clock fasting samples only.
Table 2. Vitamin intake schedule
Time
Day 1
Day 2
08.00
10.00
12.00
14.00
16.00
Reference blood sample (fasting)
Blood and urine samples (fasting)
Blood sample
Blood sample
Blood sample
Blood sample
22.00
Vitamin intake
Paper II and III
Forty-five mature, female Sprague-Dawley rats were divided into three
groups and fed diets with increasing amounts of vitamin A, in the form of
retinyl palmitate and retinyl acetate. The diet was either a standard diet containing 12 IU vitamin A/g pellet (control, C) or standard diet supplemented
with 120 IU (“10 × C”) or 600 IU (“50 × C”) vitamin A/g pellet. Clinical
examination was performed weekly. Body weight was measured at the beginning and at the end of the study. After three months the rats were killed.
Internal organs and serum samples were analyzed for retinoid content. The
bone quality and strength was tested with three-point bending analysis of the
femur and with peripheral quantitative computed tomography (pQCT) of the
humerus, and humeral dimensions and composition were determined.
31
Paper IV
The human osteosarcoma cell line MG-63 and primary cultures were cultured in cell medium which was changed two times a week until subconfluence was achieved. Prior to stimulation with RA, cells were incubated in
serum-free medium for 24 hours. RA, at final concentrations ranging from
10-6 to 10-10 M, or vehicle (ethanol, not exceeding 0.1%), RAR antagonist or
RAR-ȕ,Ȗ-agonist was added in fresh serum-free medium. For measurement
of OPG secretion, medium aliquots were collected after 24 hours and the
number of cells in each well was counted manually. For RNA isolation, cultures were harvested 2, 4, 8 and 24 hours after stimulation. All sets of experiments were repeated twice.
Biochemical analysis (Paper I-III)
The biochemical analyses listed in table 3 are described in paper I, II and III,
respectively.
Table 3. Biochemical analyses
Analysis
Method
S-1,25-(OH)2-vitamin D3
S-calcium, albumin
S-parathyroid hormone
S-CrossLaps
U-calcium
U-creatinine
S- RE
Retinoids in liver and kidney
25-OH-vit D3, Į-tocopherol,
phylloquinone
immunoextraction and RIA
colorimetric spectrophotometry
RIA
ELISA
atomic absorption spectrophotometry
colorimetric spectrophotometry
HPLC
diisopropyl ether extraction, HPLC
Paper I
Paper I
Paper I
Paper I
Paper I
Paper I
Paper I and II
Paper II
HPLC
Paper III
Peripheral quantitative computed tomography (Paper II and III)
The peripheral quantitative computed tomography (pQCT) system Stratec
XCT 960A with version 5.20 software (Norland Stratec Medizintechnik,
Pforzheim, Germany) was used. For cortical measurement, the humeral
diaphysis was scanned at midshaft just distal to the deltoid tuberosity. For
trabecular measurement, the humeral epiphysis was scanned 5 mm from the
proximal end of the bone, an area rich in trabecular bone (figure 4). The left
humerus was placed horizontally inside a glass tube and scanned using a
voxel size of 0.148 × 0.148 × 1.25 mm and an attenuation threshold coefficient of 0.930 cm-1 for definition of cortical bone.
32
Dimensions and composition of humerus (Paper III)
Total length (defined as the distance from the upper edge of the humeral
head to the medial epicondyle) and waist (defined as the diameter of the
narrowest part of the humerus) were measured using an electronic sliding
caliper. Bone volume of the right humerus was measured using Archimedes
principles of displacement. Determination of bone composition was made
with the bone ash method. The bones were dried at 100°C for 24 hours and
finally burned to ashes at 800°C for 48 hours. The weight was determined at
each step.
Measurement of OPG secretion (Paper IV)
The levels of OPG were analyzed using ELISA. A MaxiSorb micro titer
plate was coated with anti-human OPG mouse capture antibody. After incubation for 2 hours with sample or standard recombinant human OPG protein,
detection was performed with biotinylated anti-human OPG goat detecting
antibody. After developing, the plate was read at 450 nm in a microplate
reader. Protein concentrations were normalized to the number of cells, and
expressed as pg/ml per 106 cells.
RNA isolation and quantitative real time PCR analysis (Paper IV)
Total ribonucleic acid was isolated using RNeasy Midi kit. RNA (1
µg/sample) was reverse transcribed to cDNA with Superscript II. All RNA
samples were transcribed at the same time and each cDNA was analyzed in
duplicate. Real-time PCR analysis was performed using TaqMan 7700 with
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as endogenous control. Gene-specific primers for RANKL and OPG were purchased as Assayon-Demand from Applied Biosystems.
Statistical analysis (Paper I-III)
The StatView 4.5 or 5.0 software (Abacus Concepts, Berkeley, USA) was
used for all statistical analyses. In every case, a P value less than 0.05 was
considered significant.
Paper I
To analyze the effect of vitamin intake on S-calcium, the five serum samples
from day two (8.00 to 16.00) were used. As a substitute for measurement of
the area under curve, the mean level of S-calcium was calculated. This was
done separately for each vitamin intake and for placebo. The effect of vitamin intake was defined as the mean S-calcium level after vitamin intake
minus the mean level after placebo. Thus, for every individual three values,
corresponding to the effect of retinyl palmitate intake, 1,25-(OH)2-vitamin
D3 intake and combined intake, respectively, were calculated. These effect
values were statistically analyzed using the Wilcoxon signed rank test. The
33
effect of vitamin intake on S-PTH and S-CrossLaps was analyzed in exactly
the same way. The effect of vitamin intake on S-1,25-(OH)2-vitamin D3 and
S-RE, and on urine calcium excretion was evaluated by the Wilcoxon signed
rank test.
Paper II and III
The data were evaluated by one-way ANOVA followed by post-hoc Fisher´s
PLSD.
34
RESULTS AND DISCUSSION
PAPER I
Vitamin A antagonizes calcium response to vitamin D in man.
The effects of single doses of 1,25-(OH)2-vitamin D3 and retinyl palmitate
on the S-calcium level were investigated. As expected, intake of 2 µg 1,25(OH)2-vitamin D3 resulted in increased S-calcium and concomitantly decreased S-PTH. After the combined intake of 1,25-(OH)2-vitamin D3 and
retinyl palmitate, the rise in S-calcium was significantly lower compared to
the rise after intake of 1,25-(OH)2-vitamin D3 alone. Thus, the intestinal calcium response to vitamin D was antagonized by retinyl palmitate.
After intake of retinyl palmitate only, S-calcium but not S-PTH, was significantly decreased compared to placebo. Since the study subjects were not
vitamin D depleted before the experiment, the decrease in S-calcium after
intake of retinyl palmitate only may be due to an antagonistic effect on
physiological vitamin D levels, and does not necessarily prove a direct effect
of vitamin A on calcium homeostasis.
Possible mechanisms for the interaction are at the level of intestinal calcium absorption, vitamin absorption, degradation or transport, bone resorption or renal calcium reabsorption. In this study, the interaction is not obviously exerted in bone or kidney, or at the level of vitamin absorption since
neither the bone resorption marker Cross-Laps nor urine calcium was significantly affected, and serum levels of retinyl palmitate and 1,25-(OH)2vitamin D3 were not significantly affected after combined intake. In a study
on rats, a similar antagonistic effect of retinyl acetate on the S-calcium response to a low dose of vitamin D2 was eliminated in rats fed a rachitogenic
diet (197), suggesting that the antagonism was exerted at the level of intestinal calcium absorption. Our data, although not conclusive, are in agreement
with this study.
35
PAPER II and III
Subclinical hypervitaminosis A causes fragile bones in rats
Subclinical hypervitaminosis A: effects on fat-soluble vitamins and BMD
In this study on rats, the excessive vitamin A intake in the 10 × C group (diet
supplemented with 120 IU retinyl palmitate/g pellet) and the 50 × C group
(diet supplemented with 600 IU/g pellet) led to dose-dependently increased
levels of serum and liver retinoids, indicating that intestinal uptake was adequate. In the 50 × C group kidney retinoids were also increased. No typical
signs of general toxicity were evident, i.e. the intoxication was subclinical.
No effect on the bone was seen in the 10 × C group but in the 50 × C group,
bone strength was reduced as measured by three-point bending breaking
force (-10.3%, p < 0.01). In this group, pQCT showed decreased cortical
area and reduced diameter of the diaphysis of the long bones, i.e. bone
changes typical for hypervitaminosis A (199). Thus, bone toxicity is evident
at this level of subclinical hypervitaminosis A.
This is the first demonstration of increased bone fragility in rats with subclinical hypervitaminosis A. According to the literature, the lowest dose
known to induce chronic toxicity and fractures in adult rats is around 25,000
IU/day, but the doses used are often even higher (150). Our data indicate that
the negative effect on bone strength appears at a vitamin A intake of somewhere between 1,800 IU/day and 9,000 IU/day, a level considerably lower
than previously reported. Similarly, a decreased serum vitamin D at this
level of vitamin A intake has not been reported before. Previously published
data show that administration of 50,000 IU vitamin A three times per week
to rats decreased the level of 25-OH-vitamin D3 (152) while no effect on S1,25-(OH)2-vitamin D3 was seen in rats fed 10,000 IU vitamin A per day for
3 weeks (153) or 300 IU per day for 14 months (154). In our study administration of approximately 9,000 IU per day (50 × C group) for 12 weeks
resulted in a reduction of both S-25-OH-vitamin D2 and S-25-OH-vitamin
D3.
It is important to note that vitamin A is rapidly absorbed but slowly
cleared from the circulation. It is accumulated in the liver until the storage
capacity of the liver is exceeded. Therefore, toxicity is dependent on both
dose and duration of vitamin A administration. Interestingly, in the 10 × C
group, S-25-OH-vitamin D2 and also S-Į-tocopherol (vitamin E) were decreased, suggesting that subclinical toxicity is developing already at this
level of vitamin A intake. Prolonged exposure would most likely result in
adverse skeletal effects in this group as well, although this was not found in
our study.
Like vitamin D2 and vitamin D3, S-vitamin E was decreased in the 50 × C
group. A general antagonistic interaction among the fat-soluble vitamins has
36
previously been demonstrated in the chicken (200,201). Our data suggest
that a similar general interaction among fat-soluble vitamins is also present
in the rat. Although the decrease in S-phylloquinone (vitamin K) only
reached marginal statistical significance, an interaction between vitamin K
and vitamin A seems likely. Clinical reports of bleedings and hypothrombinemia after intoxication with vitamin A and vitamin E, respectively, support this hypothesis (202,203). Vitamin D has well-known beneficial effects
on the skeleton, and similar effects are proposed for vitamins K and E
(204,205). Therefore, our findings suggest possible indirect mechanisms of
vitamin A toxicity.
The antagonism causing reduced serum levels of other vitamins can occur
during vitamin absorption in the intestine, or during transport, storage or
degradation of the vitamins. Previous studies have suggested a competitive
interaction at the level of intestinal absorption (195,206), and our data are in
agreement with this hypothesis (see also discussion in paper III). However,
other levels of interaction must also be considered. For example the 24-OHhydroxylase, a key catabolic enzyme for 1,25-(OH)2-vitamin D3, is upregulated by RA (192).
The effect of vitamin A on bone mass was evaluated by the bone ash
method and pQCT. No detectable changes in BMD could explain the increased bone fragility. However, the external diameter and cortical thickness
were reduced. These variables are of major importance for determination of
bone strength, since the bending strength is proportional to the fourth power
of the radius (207).
In the 50 × C group, bone ash measurement even showed a slightly increased BMD (ash weight divided by bone volume). This increase is due to
the fact that the volume decrease is larger than the ash weight decrease and
does not reflect a true increase in bone mass. PQCT also showed decreased
total bone mineral content at the thinned diaphysis but the corresponding
BMD was not decreased. Thus, early vitamin A toxicity in the rat seems to
affect bone geometry and bone strength before any negative effect is detectable by density measurements. For evaluation of early hypervitaminosis A in
the rat, measurements other than BMD must be recommended. Since the
diagnosis osteoporosis is based on BMD, further studies are warranted to
examine whether this is also true in humans.
37
PAPER IV
Vitamin A Differentially Regulates RANKL and OPG Expression in
Osteoblasts
In the human osteosarcoma cell line MG-63, OPG protein expression, measured by ELISA, was down-regulated by RA in a dose-dependent way. With
quantitative real-time PCR, a similar down-regulation of OPG mRNA levels
was detected. In both cases, the maximum effect was seen at a dose of 10-6
M. The down-regulation was evident in primary cultures of human osteoblast-like cells as well. Time-course experiments with 10-6 M RA showed
a maximum effect at 4 hours. RANKL mRNA levels were up-regulated by
RA stimulation in primary cultures. The maximum effect was at 10-6 M RA,
after 8 hours. In MG-63 no RANKL expression was detected. This is consistent with previous studies (208). In summary, it was demonstrated that OPG
is down-regulated and RANKL is up-regulated in osteoblastic cells by RA.
OPG and RANKL are essential regulators of osteoclastogenesis. Their effect on osteoclast formation and activation is determined principally by the
relative ratio of RANKL/OPG in the bone marrow microenvironment
(9,209). In our in vitro system, the RANKL/OPG ratio was increased by RA
stimulation. This differential regulation may be an important mechanism by
which vitamin A induces bone resorption in vivo.
RARĮ, RARȕ and RARȖ are all expressed in osteoblasts (125,210,211)
and signaling via these receptors is the most likely signaling pathway for
vitamin A activity in bone. In this study, the addition of a RAR antagonist,
which inhibits the action of RARĮ,-ȕ and -Ȗ, could inhibit the effect of 10-6
M RA, while an agonist for RARȕ and -Ȗ could mimic the RA effect. This
was evident on both OPG and RANKL expression in both cell types, indicating that the RA effect is mediated by these receptors. To confirm that these
are indeed transcriptional effects and do not represent a change in message
stability, further experiments such as a reporter assay using RANKL and
OPG upstream regulatory sequences will be needed.
38
GENERAL DISCUSSION AND FUTURE
PERSPECTIVES
Since the identification of vitamin A in 1913, its structure and function have
been thoroughly investigated. In some fields, such as the phototransduction
in the eye, this has led to detailed knowledge. In contrast, our knowledge
about how vitamin A functions in bone tissue is still limited, even though it
has been known for almost a century that high doses of vitamin A are severely toxic to the rat skeleton. The potential of vitamin A to affect the human skeleton is evident from case reports of human vitamin A intoxications.
Since the publication by Melhus et al in 1998 (169), the possible relationship
between high vitamin A intake and osteoporosis has gained increasing interest.
One question, maybe the most important in view of the pathogenesis of
osteoporosis, is whether toxic effects of hypervitaminosis A occur at levels
close enough to physiologically high intakes, i.e. of relevance for human
nutrition. This is suggested by the epidemiological studies, showing that the
level of vitamin A intake associated to increased fracture risk in humans is
below the tolerable upper limit of 3,000 µg/d or only about two times the
Swedish RDI. The average vitamin A intake in Sweden is between one and
two times the RDI for both women and men (88,90). I have shown that adverse skeletal effects of vitamin A are demonstrable in rats where no clinical
signs of toxicity are evident. Although the doses are many times the “RDI”
for rats, it is likely that subclinical skeletal effects may arise at much lower
doses. This field needs to be further investigated. As discussed previously,
the duration of intake is crucial for vitamin A toxicity. In the pathogenesis of
human osteoporosis, both peak bone mass and later bone losses are of importance, and exposure time can potentially be life-long. Therefore, when studying the lowest safe level of vitamin A intake, longer exposure times are recommended.
The relevance of animal models for human disease pathogenesis can naturally be questioned. In general, the rat is considered to be an appropriate
model for human postmenopausal osteoporosis (212). In the experiments
demonstrating spontaneous fractures in rats, the doses were extremely high.
In the severe cases of human vitamin A intoxication, the dose per kg bodyweight was lower, but instead continued for a longer time. In one patient,
who ingested 500,000 IU per day for several years, x-rays showed os39
teopenia (163). In another patient, with lower doses and shorter exposure
time, x-rays were normal but bone biopsy showed increased resorptive surfaces (164). When the lower dose is taken into account, these symptoms are
consistent with the fractures seen in rats.
I report that RA can increase the RANKL/OPG ratio in osteoblastic cells
in vitro. Considering the importance of the RANK/RANKL/OPG system for
osteoclastogensis and bone resorption, the regulation of these molecules is a
likely signaling pathway for the direct effects of vitamin A in bone. It would
be of interest to elucidate the effects on osteoclasts in a co-culture in vitro
system, but in vivo studies are needed for certain confirmation.
An interaction between vitamin A and D in humans was also demonstrated, where the calcium response elicited by vitamin D was antagonized
by simultaneous intake of vitamin A. In the rats with subclinical hypervitaminosis A, the serum levels of other fat-soluble vitamins were reduced, suggesting a general antagonistic effect on fat-soluble vitamins. These findings
are consistent with previous literature and argue in favor of indirect effects
in development of vitamin A toxicity.
It has previously been assumed from animal studies that the antagonistic
action between vitamin A and D in bone is weak, since it is evident at high
levels of vitamin A intake (193-195). However, at marginal dietary intake of
vitamin D, the antagonistic effects occur at lower vitamin A doses (197). In
vitamin D-depleted rats with a marginal dietary intake of phosphorous or
calcium, the negative effect of vitamin A on bone ash was aggravated when
vitamin D was added to the diet, compared to controls with a vitamin Ddeficient diet (156). These data suggest that at least in certain conditions of
limited intake of vitamin D and calcium and/or phosphorous, the antagonistic effect on vitamin D and other fat-soluble vitamins may also be of importance.
In humans, these indirect effects can possibly be more pronounced in the
elderly, and in other individuals with poor intake of calcium and/or vitamin
D. In Scandinavia, the average daily intake of vitamin D is below recommended levels in many groups (213-216) and the limited exposure to
sunlight further increases the risk for hypovitaminosis D. I hypothesize that
the high intake of vitamin A in Scandinavia may further aggravate the effect
of hypovitaminosis D on calcium absorption and, possibly, contribute to the
high incidence of osteoporosis.
Several new questions are raised: For which doses of vitamin A and D is
the antagonistic effect evident in humans? How long after intake does vitamin A exert an antagonistic effect? What are the effects of long term high
vitamin A intake on vitamin K and vitamin E in humans? It would also be
interesting to see if the outcome of epidemiological studies would be different if, in addition to the level of vitamin A intake, consideration was taken to
the vitamin D status of the individuals. Answers to these questions would
40
help in the effort to more precisely pinpoint the physiological relevance of
the antagonistic effects in humans.
Although further studies are needed to clarify whether the past and current daily intake of vitamin A in Sweden and other countries has contributed
to the high incidence of osteoporosis, the question is not if vitamin A is toxic
for the skeleton, but rather at what level of intake this toxicity begins (143).
It has become clear that vitamins can not be considered magic pills that bring
only benefit to health, and that the widespread misconception – “the more
vitamins, the better” - is a fallacy.
41
CONCLUSIONS
In this project, it was found that:
x the serum calcium-increasing effect of vitamin D is antagonized by
simultaneous intake of vitamin A in humans.
x in rats with increased intake levels of vitamin A but no clinical
signs of vitamin A toxicity, bone fragility is increased. This bone
toxicity appears at somewhere between 10 and 50 times the intake
of vitamin A in the control rats.
x in rats with increased bone fragility, the bones have reduced diameter and cortical area, but BMD is not decreased. Moreover, levels
of other fat-soluble vitamins are reduced.
x in osteoblastic primary cultures and in the osteosarcoma cell line
MG-63, stimulation with RA decreases OPG levels while RANKL
is increased.
In summary, these findings indicate that vitamin A can increase bone fragility in the rat at doses considerably lower than previously shown. Since geometrical variables, but not BMD, were negatively affected, measurements
other than BMD are suggested for evaluation of early hypervitaminosis A.
The regulation of RANKL/OPG in vitro provides a mechanistic explanation
for the bone resorbing activity of vitamin A, and is a likely pathway for direct effects of vitamin A in bone in vivo. An antagonistic effect of vitamin A
on vitamin D action has for the first time been demonstrated in humans, suggesting indirect mechanisms of vitamin A toxicity.
42
ACKNOWLEDGEMENTS
I wish to thank all the people who, in different ways, have contributed to this
thesis, and among them especially:
my tutor Håkan Melhus, who introduced me (most of us) to the exciting
world of vitamin A and bone, for your support and inspiring enthusiasm, for
your persistent belief in this project, and for lifting coffee break discussions
with your creative analyses of everything from Nobel prizes to Robinson.
my co-tutor and co-author Monica Lind, for your go-ahead spirit, for your
generous encouragement and for good collaboration in the rat project.
Andreas Kindmark, my co-tutor, for valuable input on my manuscript and
for creating a cheerful atmosphere when you are around.
Annica Jacobson, my co-author and co-worker, for stimulating collaboration,
for discussions of science and everything else, for trying to keep coffee breaks regular, and for being an extremely considerate person.
my co-worker Maria Branting, for help and co-operation in numerous ways,
for your loyalty and for significantly increasing the comfort and well-being
at lab 22.
the lab’s philosopher Fredrik Stiger, for sharing your thoughts of life, the
universe and everything, and for trying to teach me how to play billiards.
the previous hypertensive part of our research group, Julia Karlsson and Lisa
Kurland, for cheering up our lab 22 with inspiring hair colors, delicate muffins and nice company.
Birgitta Wilén for introducing me into the laboratory and the world of pipetting.
Anna-Lena Johansson and Thomas Björkman for your friendly technical
guidance and help.
43
the “lab 21” group; Filip Sköldberg, Gennet Gebre-Medhin, Håkan Hedstrand, Olov Ekwall and Olle Kämpe (and Gunnel Nordmark joining) for
coffee discussions in the crowded red-sofa-corner, and for inviting me to
your gourmet lunch seminars.
fellow PhD students at the “Forskningsavdelningen”, all of you through the
years from Jonas Andersson and Juán Ramón López to Gunilla Englund and
Elin Grundstrand, for laughter, small talk and helping hands.
Åsa Hallgren, Margareta Ericson, Reza Dadnehal and everybody else at
“Forskninsavdelningen” who has brightened up my time here.
the secretaries and “computer persons” of the institution, for your help, and
especially Birgitta Sembrant for always giving me the feeling that nothing is
troublesome.
and in the world outside…
my friends – you know who you are – for good fun and for sharing life and
reminding me of what is really important.
my family, for always supporting me and always being there, and especially
Olof for providing me with a place to live, lunch boxes and considerate friendship this last year.
and Peter for your patience, proof-reading and much more.
Uppsala, October 28th 2004
This thesis has been conducted at the Department of Medical Sciences, Uppsala University. Financial support was generously provided from Uppsala
University and from the Foundation for Geriatric Research, the Swedish
Medical Research Council, the Göran Gustafsson Foundation, the Astrid
Karlsson Foundation, and the Torsten and Ragnar Söderberg Foundation.
44
SUMMARY IN SWEDISH
Vitamin A och osteoporos
Kliniska och experimentella studier
Utgångspunkten för den här avhandlingen var en artikel som min handledare
Håkan Melhus publicerade 1998. Det var en epidemiologisk studie som visade på att ett högt intag av vitamin A i kosten var kopplat till en minskad
bentäthet och en ökad risk för höftfraktur. Dessa resultat kan inte säkert kan
bevisa ett orsakssamband, men har ändå väckt frågan: kan utvecklingen av
sjukdomen osteoporos, som kännetecknas av minskad bentäthet och stor
frakturrisk, påverkas av vitamin A?
Kort om ben och osteoporos
Skelettet är ett organ med många olika uppgifter. Det ger stadga och skydd
åt kroppen, innehåller benmärgen och, inte minst, är en reservoar för kalcium och fosfat som är viktiga ämnen i kroppen. Ben produceras av benbildande celler, osteoblaster, som producerar ett nätverk av proteiner där kalcium och fosfat kan inlagras i en process som kallas mineralisering. Därigenom blir benet hårt och hållfast. I benet finns också osteoklaster som bryter
ned (resorberar) ben. Efter att skelettet har vuxit klart fortsätter osteoblasterna och osteoklasterna att arbeta, och kan genom ett finjusterat samarbete
som kallas ”remodellering” bryta ned och bygga upp ben (se figur 1, sid 11).
Detta sker hela tiden i liten skala men på en stor mängd ställen i benet, som
ett slags underhållsarbete. Man räknar med att cirka 10% av benmassan byts
ut varje år på det här sättet.
Lite förenklat kan man säga att ju äldre man blir, desto mer dominerande blir
bennedbrytningen, och alla människor tenderar att förlora benmassa när de
blir äldre. Detta blir särskilt tydligt hos kvinnor efter klimakteriet, eftersom
östrogen är en viktig faktor för att hålla remodelleringen i god balans. Sjukdomen osteoporos innebär en minskad benmassa, men också förändrad
struktur i benet, så att hållfastheten minskar och risken för fraktur ökar kraftigt. De typiska osteoporos-frakturerna är fraktur i kotkropparna, handleden
och höften. Särskilt höftfrakturen innebär mycket lidande och besvär för
patienten, en minskad överlevnad och stora kostnader för samhället.
45
Vitamin A
Vitamin A är en grupp fettlösliga ämnen som är nödvändiga för många fysiologiska processer i kroppen, bland annat under fosterutvecklingen, i hunden
och för immunförsvaret. De verkar genom att binda till speciella receptorer,
som i sin tur påverkar vilka gener som uttrycks (används) i en cell. På så sätt
är vitamin A ett – av många – ämnen som reglerar hur celler delar sig och
mognar.
Vitamin A kan inte tillverkas i kroppen, utan måste tillföras med kosten. Den
mest aktiva formen av vitamin A, retinoiderna, finns i ett fåtal livsmedel som
lever, mejeriprodukter och fet fisk. Modersubstansen retinol förekommer i
kosten framför allt som ester, bunden till en fettsyra (retinyl palmitat). Karotenoider, däribland beta-karoten, är en mindre aktiv form av vitamin A som
finns i röda och gröna grönsaker, och som måste brytas ned till retinol för att
kunna tas upp i kroppen (se figur 4, sid 20). Vårt exakta behov av vitamin A
är inte säkerställt, och kostrekommendationerna varierar följaktligen mellan
olika länder. I Sverige är RDI (rekommenderat dagligt intag) 900 µg/d för
män och 800 µg/d för kvinnor, vilket är högre än vad t ex WHO rekommenderar. Medelintaget av vitamin A ligger ännu högre, ca 1300 µg/d för män
och 1100 µg/d för kvinnor. Tillsammans med Norge och Island ligger vi
högst i Europa vad gäller A-vitaminintag, delvis på grund av den utbredda
användningen av vitamintillskott med ett högt innehåll av vitamin A och,
framför allt i Norge, intag av fiskleverolja. Sverige berikar också bland annat
lättmejeriprodukter med ett överskott av vitamin A, vilket kan bidra till vår
höga konsumtion.
Vitamin A och ben
Vad finns då känt om hur vitamin A påverkar skelettet? Redan i början av
1900-talet, när vitamin A nyligen hade identifierats och man hade börjat
undersöka effekten av över- och underdosering av olika vitaminer, visade det
sig att råttor som fick mycket höga doser av vitamin A fick spontana frakturer. Hittills är vitamin A det enda ämne som har visat sig ge spontana frakturer hos råtta, och tillsammans med blödningar var detta det mest framträdande symtomet. Senare studier har visat att benen hos vitamin A-intoxikerade
råttor är förtunnade, och har ett ökat antal osteoklaster. Man har också visat
att vitamin A kan påverka olika benceller, och styra regleringen av flera benspecifika proteiner, och att bennedbrytningen ökar i benbitar som odlats i en
vitamin A-rik miljö.
Hos människor som av misstag ätit för mycket av vitamin A (detta gäller
dock inte överintag av karotenoider) under en längre tid, oftast i form av
46
kosttillskott, utvecklas ett förgiftningssyndrom med torr hud, huvudvärk,
illamående, trötthet och aptitlöshet. Värk i ben och leder är också ett vanligt
symtom, och vid röntgenundersökning ses ofta olika abnormaliteter som
bennabbar och förkalkningar, och – hos de med allra högst intag – tecken på
ökad benresorption. Många patienter har också förhöjda calciumnivåer i
blodet. Någon form av skelettpåverkan verkar alltså finnas även hos människor med vitamin A-intoxikation.
På senare år har betydelsen av vitamin A för skelettet fått ökad uppmärksamhet, efter att flera (dock inte alla) epidemiologiska studier har påvisat ett
samband mellan högt A-vitaminintag och risken att få minskad bentäthet och
osteoporos-frakturer. Med tanke på det höga intaget av vitamin A i Skandinavien har frågan om vitamin A kan bidra till utvecklingen av osteoporos
fått ytterligare tyngd. Vår kunskap om hur vitamin A påverkar skelettet är
dock begränsad, och det är inte känt – varken för råtta eller människa – vilken som är den lägsta toxiska dosen.
Mål
Målet med avhandlingsarbetet har varit dels att studera olika mekanismer för
hur vitamin A kan påverka benet, dels att undersöka gränsen för hur låga
doser som är skadliga för skelettet.
Projekt I
En mekanism för vitamin A toxicitet skulle kunna vara att motverka effekten
av vitamin D, som är av känd positiv betydelse för skelettet. Detta har påvisats hos djur, t ex råtta och kyckling, men har aldrig tidigare undersökts hos
människa. I det här projektet har jag undersökt hur vitamin A och vitamin D
påverkade nivån av kalcium i blodserum (S-kalcium).
Nio friska försökspersoner fick äta olika kombinationer av engångsdoser av
vitaminerna vid fyra olika tillfällen. Följande dag fick försökspersonerna
lämna blodprov varannan timme, samt ett urinprov.
Efter intag av endast vitamin D steg nivån av S-kalcium under dagen, en
väntad effekt eftersom den viktigaste funktionen för vitamin D är att öka
upptaget av kalcium i tarmen. Kombinationen av vitamin A och D gjorde att
ökningen av S-kalcium blev signifikant lägre än efter enbart vitamin D-intag,
och efter intag av enbart vitamin A sjönk kalciumnivån jämfört med placebo.
Slutsatsen blir att vitamin A kan motverka den kalciumhöjande effekten av
vitamin D, och att en tänkbar indirekt mekanism för hur vitamin A-effekter
kan medieras har påvisats hos människa.
47
Projekt II och III
I de här projekten studerade jag råttor som under 3 månader fått extra tillskott av vitamin A i kosten. Tre grupper med 15 råttor/grupp fick antingen
standardfoder (kontroll, C), standardfoder med ett ca 10 gånger ökat vitamin
A-innehåll (10 × C) eller standardfoder med ett ca 50 gånger ökat vitamin Ainnehåll (50 × C).
I tidigare studier där man påvisat spontana frakturer hos råttor har man använt mycket höga doser, som även medfört allmän avtackling och död. I
projekt II visade jag att råttornas skelett kunde påverkas negativt av vitamin
A, trots att råttorna inte uppvisade några allmänna tecken på vitamin Aintoxikation (subklinisk toxicitet). Nedsatt hållfasthet i skelettet (-10,3%)
påvisades med 3-punkts-böjnings-test.
I vårt försök verkade den lägsta skadliga nivån ligga någonstans mellan
intaget i 10 × C- och 50 × C-gruppen. I det här sammanhanget är det av betydelse att levern har en stor förmåga att ta upp och lagra vitamin A från
blodet. Detta innebär ett skydd både mot enstaka för höga intag av vitamin A
och mot vitamin A-brist i tider av ett ojämnt intag. Det gör dock också att
det kan ta lång tid innan toxiska symtom utvecklas vid medelhöga doser,
eftersom det sker först när leverns förmåga till lagring har överskridits. Man
kan anta att även råttorna i 10 × C-gruppen skulle få toxiska symtom om
exponeringen fortsatte tillräckligt länge. Fler och längre studier krävs därför
för att kunna utvärdera den nedre toxiska gränsen bättre.
I projekt III undersöktes råttornas ben vad gäller form och bentäthet. Det
visade sig att benen i 50 × C gruppen var förtunnade, vilket är typiskt för
vitamin A-intoxikation. Bentätheten däremot var inte minskad, trots att benets hållfasthet var nedsatt. Detta resultat antyder att andra faktorer än bentäthet, som t ex benets geometri, kan vara av betydelse för hållfastheten.
Detta är i samstämighet med tidigare studier om osteoporos, som visat att
endast en del av alla osteoporosfrakturer kan förklaras av låg bentäthet.
I projekt III fann jag också att nivåerna av andra fettlösliga vitaminer i blodet, var nedsatta hos råttor med förhöjt vitamin A-intag. Detta skulle kunna
bero på att höga doser vitamin A motverkar upptaget i tarmen av övriga vitaminer, eller t ex på att deras nedbrytning ökats. Liksom i projekt I antyder
de här resultaten indirekta mekanismer för vitamin A-toxicitet.
48
Projekt IV
I djurförsök och i experiment med benbitar har man sett att vitamin A kan
öka bennedbrytningen, men den exakta mekanismen för hur detta går till är
inte känd. Eftersom osteoklastens aktivitet är avgörande för graden av bennedbrytning undersökte jag i det här projektet hur vitamin A påverkar två
signalsubstanser, RANKL och OPG, av stor betydelse för regleringen av
osteoklasterna. Både RANKL och OPG produceras av osteoblaster. RANKL
stimulerar osteoklasternas tillväxt och aktivitet medan OPG motverkar denna
stimulering, och därför hämmar osteoklastaktivitet och bennedbrytning (se
figur 2, sid 13).
I försöket odlade jag osteoblaster, som stimulerades med vitamin A. Stimulering med vitamin A medförde att osteoblasternas produktion av RANKL
ökade medan OPG-produktionen minskade. Detta är en förändring som skulle innebära en ökad stimulering av osteoklasterna i benvävnaden. Slutsatsen
blir att reglering av nivåerna av RANKL och OPG är en trolig mekanism för
hur vitamin A kan inducera benresorption.
Sammanfatting
Jag har visat att vitamin A-intoxikation ger minskad hållfasthet i skelettet på
råttor vid doser betydligt lägre än vad som tidigare varit känt. Jag har också
undersökt olika mekanismer för hur vitamin A kan påverka skelettet: dels
genom att påverka hur de bennedbrytande osteoklasterna fungerar, dels genom att motverka effekten av vitamin D. Mina resultat tyder på att vitamin A
är skadligt för skelettet, men för att säkert kunna besvara frågan om vitamin
A har bidragit till den höga förekomsten av osteoporos i Sverige krävs fler
studier.
49
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