Metabolism of calcium and phosphates
Laboratory diagnostics (Ca,
phosphates, PTH, PTHrP, vitamin D,
paraproteins)
Calcium metabolism
• Body of adult human (young) contains 1000 – 1100 g of calcium
• skeleton (98 – 99 %)
• 1 – 2 % extraosseous, particularly extracellularly
• Very small amounts of calcium intracellularly
• 55 % ER, the rest namely in mitochondria
• The cytoplasmic concentration level 10-7 mol.L-1 versus blood plasma level 10-3 mol.L-1
(normal concentration around 2.5 mmol.L-1)
• The necessity of strict regulation - signaling role of calcium ions
• Muscle contraction, neurotransmission, secretion mechanisms, cell cycle and proliferation, cell
death, coagulation, etc.
• Ligand-gated or voltage-gated channels (type T – transient/type L – long lasting), eventually
channels activated mechanically
• Ca2+/H+ ATPase
• Antiport driven by Na+ gradient
Calcium intake
• Daily intake is about 1.0 g per day and increases during pregnancy,
lactation, growth, etc. (up to 1.5 g)
• Under physiological conditions it absorbed about 25 to 40% of
received calcium (duodenum - 15%, jejunum - 20%, ileum - 65%)
• Paracelullular/transcelular transport
• Paracelullar transport – claudin 2 and claudin 12
• Role of 1,25-dihydroxycholecalciferol!
• Decreased levels of plasma Ca2+ increases the synthesis of 1,25dihydroxycholecalciferol
and vice versa
2.5–7.5 mmol/day
Duodenum + jejunum – 90 %
! Compensatory mechanism (diet poor in calcium) –– ileum a jejunum
Kopic, S., and J.P. Geibel. 2013. GASTRIC
ACID, CALCIUM ABSORPTION, AND THEIR
IMPACT ON BONE HEALTH. Physiological
Reviews 93:189-268.
NCX - sodium-calcium
exchanger
PMCA - plasma membrane
calcium ATPase
TRPV6 - transient receptor
potential cation channel
subfamily V member 6
Kopic, S., and J.P. Geibel. 2013. GASTRIC
ACID, CALCIUM ABSORPTION, AND THEIR
IMPACT ON BONE HEALTH. Physiological
Reviews 93:189-268.
Doherty, A.H., C.K. Ghalambor, and
S.W. Donahue. 2015. Evolutionary
Physiology of Bone: Bone Metabolism
in Changing Environments. Physiology
30:17-29.
An interplay between mitochondria, endoplasmic reticulum and cytoplasm
in handling ROS and calcium ions. Briefly, endoplasmic reticulum is the
crucial and major site for calcium storage in cell. Sarco-/endoplasmic
reticulum Ca2+-ATPase represents the most important transport
mechanism for influx of calcium ions. On the other hand, mitochondria
represent the second most important calcium store in the cell. However,
these two organelles are closely connected in calcium handling, mainly in
response to ROS. Increase in ROS levels in the mitochondria, where the
respiratory chain (RCH) represents the major site for creation of ROS,
triggers the ER to release calcium and sensitizes a calcium-releasing
channel in the ER membrane, sending a feedback signal. On the other
hand, process of folding proteins contributes significantly to creation ROS
directly in ER. When incorrect disulfide bonds form, they need to be
reduced by GSH, resulting in a further decrease of GSH/GSSG ratio, altering
the redox state within the ER. Alternatively, misfolded proteins can be
directed to degradation through ER-associated degradation machinery.
Accumulation of misfolded proteins in the ER initiates the unfolded protein
response, which includes involvement of protein kinase RNA-like
endoplasmic reticulum kinase (PERK), inositol-requiring enzyme (IRE), and
activating transcription factor 6 (ATF6). All these proteins influence cellular
responses at different levels (transcription, translation, antioxidant
defence). Calcium ions released from ER during these processes (inositol
1,4,5-trisphosphate receptors (IP3R) and ryanodine receptors (RyR) –
accumulated in membranes with close connection with mitochondria - in
mitochondrial associated membranes (MAMs) trigger mitochondrial ROS
stimulation via stimulation of the tricarboxylic acid cycle. Mitochondria
release calcium ions via mitochondrial sodium/calcium exchanger (mNCX),
influx of calcium ions from cytoplasm in provided by voltage-dependent
anion channel (VDAC) and calcium uniporter. Increased load of
mitochondria with calcium ions stimulate release of cytochrome c and proapoptic
factors via mitochondrial permeability transition pore (mPTP).
Bers, D.M. 2008. Calcium cycling and signaling in
cardiac myocytes. In Annual Review of Physiology.
Annual Reviews, Palo Alto. 23-49.
Bers, D.M. 2008. Calcium cycling and
signaling in cardiac myocytes. In Annual
Review of Physiology. Annual Reviews,
Palo Alto. 23-49.
Serum calcium
• Concentration 2.5 mmol.L-1 , resp. 2.2 – 2.6 mmol.L-1 (100 mg.L-1)
• The upper limit compatible with life 4 – 5 mmol.L-1
• The lower limit 1 mmol.L-1
• About 60 % in diffusible form:
• filtered by the kidneys
• 50 % ionized – free (Ca2+) = biologically active form (1.1 – 1.3 mmol.L-1)
• 10 % in low-molecular complexes (citrates, phosphates, hydrogen carbonates)
• About 40 % in nondiffusible form:
• Calcium ions bound to proteins
• Albumin 90 %, globulin 10 %
• Cannot be filtered
• Biologically inactive form, BUT may be released at hypocalcemia
• hypoalbuminemia – fraction bound to albumin decreases (decrease for 10 g.L-1 causes
no changes in the concentration of ionized calcium)
• hyperproteinemia (multiple myeloma) – increase in total calcemia, again without
changing in the concentration of free calcium
• pH:
CASR
Kopic, S., and J.P. Geibel. 2013. GASTRIC ACID, CALCIUM
ABSORPTION, AND THEIR IMPACT ON BONE HEALTH.
Physiological Reviews 93:189-268.
DiGirolamo, D.J., T.L. Clemens, and S. Kousteni. 2012.
The skeleton as an endocrine organ. Nature Reviews
Rheumatology 8:674-683.
Bone tissue and
three types of cells
BMU - basic multicellular unit
90 – 130 days
Doherty, A.H., C.K. Ghalambor, and
S.W. Donahue. 2015. Evolutionary
Physiology of Bone: Bone Metabolism
in Changing Environments. Physiology
30:17-29.
Factors affecting bone remodeling
• Genetic factors
• 60-80% of the amount of bone tissue is genetically determined
• The differences between the races (most Negroes, Asians least)
• Mechanical factors
• Remodeling of bone structure according to the mechanical requirements
• Physical activity is essential for proper bone development
• Vascular / neural factors
• Vascularization necessary for proper bone development, especially for ossification
• Innervation - neuropeptides and their receptors
• Nutritional factors
• calcium intake
• Addictions - coffee, smoking, alcohol, excess salt - risk factors for osteopenia
• The function of the endocrine system
• Besides the above mentioned also:
• Androgens - anabolic effect, stimulation of osteoblasts, bone density modification
• Estrogens - estrogen receptors found in osteoblasts, osteocytes, and osteoclasts, dual effect - stimulation of of osteoblasts,
increased levels of osteoprotegerin, reduction of bone resorption
• Progesterone - anabolic effect on bone, osteoblasts - receptors - direct / indirect effect (competes with glucocorticoids)
• Insulin - stimulating the creation of matrix
• Glucocorticoids - differentiation during development, but postnatally inhibit bone formation, inhibition of of IGF-1 / BMP-2,
which are important for osteoblastogenesis
• Growth hormone - direct effect (stimulation of osteoblasts), the indirect effect via increasing IGF-1/2 = stimulation of
osteoblast proliferation and differentiation
- Diet poor in calcium
- Diet poor in vitamin D
- Exposition to sun!
Local remodeling of bone tissue
• Especially growth factors and cytokines
• Growth factors:
• Polypeptides produced by bone tissue or extraosseously
• Modulation of growth, proliferation, differentiation
• IGF-1/2
• Liver / osteoblasts
• In high concentration in the bone matrix
• Stimulation of collagen synthesis
• Regulation of interactions between osteoblasts and osteoclasts
• IGF-2 – embryogenesis
• TGF-b (Transforming growth factor - b)
• Inhibition of bone resorption by inhibiting osteoclast differentiation and apoptosisStimulace tvorby kostní tkáně, indukce diferenciace a proliferace
osteoblastů
• Inhibition of the synthesis of matrix protease (MMP)
• BMP
• PDGF (Platelet - derived growth factor)
• stimulation of protein synthesis
• Fibroblast proliferation, neovascularization, collagen synthesis
• FGF (Fibroblastic growth factor) – mitogenic effect on osteoblasts, mutations in receptors - e.g. Apert syndrome (premature closure of
sutures, syndactyly, extension of cranium - turricephaly)
• EGF (Epidermal growth factor)
• VEGF (Vascular endothelial growth factor) – stimulation of angiogenesis and proliferation of endothelium, important especially in the
early stages of regeneration (fracture)
• GM-CSF (Granulocyte / macrophage - colony stimulating factor) – osteoclastogenesis, osteopetrosis
• M-CSF (Macrophage - colony stimulating factor) – osteoclastogenesis first phase, without any direct effect on osteoclasts
• TNF (Tumor necrosis factor) – stimulation of bone resorption
Local remodeling of bone tissue
• Cytokines – immune cells, a number of functions (immune response, inflammation, hematopoiesis,
autocrine / paracrine effect, pleiotropic effect)
• Osteoprotegerin!
• Interleukin 1
• Direct stimulation of osteoclastic bone resorption
• Stimulation of proliferation and differentiation of preosteoblasts
• Inhibition of apoptosis of osteoclast
• Interleukin 6
• Stimulation of bone resorption
• Role in the Paget's disease
• The initial phase of osteoclastogenesis
• Interleukin 11
• Bone marrow, stimulation of osteoclastogenesis
• Prostaglandins
• Especially PGE2
• Stimulation of bone resorption
• Experimentally – inhibition of COX2 = inhibition of bone formation in the dependence on the mechanical stress
• Leukotrienes
• Role in the bone remodeling
Development schema of haematopoietic precursor cell differentiation into mature osteoclasts, which are fused polykaryons arising from multiple (10–20) individual
cells. Maturation occurs on bone from peripheral blood-borne mononuclear cells with traits of the macrophage lineage shown below. M-CSF (CSF-1) and RANKL are
essential for osteoclastogenesis, and their action during lineage allocation and maturation is shown. OPG can bind and neutralize RANKL, and can negatively regulate
both osteoclastogenesis and activation of mature osteoclasts. Shown below are the single-gene mutations that block osteoclastogenesis and activation. Those
indicated in italic font are naturally occurring mutations in rodents and humans, whereas the others are the result of targeted mutagenesis to generate null alleles.
Shown above are the single-gene mutant alleles that increase osteoclastogenesis and/or activation and survival and result in osteoporosis. Note that all of these
mutants represent null mutations with the exception of the OPG and sRANKL transgenic mouse overexpression models (in blue-outlined boxes).
Schematic representation of the mechanism of action of a, pro-resorptive and calcitropic factors; and b, anabolic and anti-osteoclastic factors. RANKL expression is
induced in osteoblasts, activated T cells, synovial fibroblasts and bone marrow stromal cells, and subsequently binds to its specific membrane-bound receptor RANK,
thereby triggering a network of TRAF-mediated kinase cascades that promote osteoclast differentiation, activation and survival. Conversely, OPG expression is induced
by factors that block bone catabolism and promote anabolic effects. OPG binds and neutralizes RANKL, leading to a block in osteoclastogenesis and decreased survival
of pre-existing osteoclasts.
Endocrine
regulation of
bone
metabolism
Kopic, S., and J.P. Geibel. 2013. GASTRIC
ACID, CALCIUM ABSORPTION, AND THEIR
IMPACT ON BONE HEALTH. Physiological
Reviews 93:189-268.
Přímý efekt na kostní tkáň? (osteoblasty)
Kopic, S., and J.P. Geibel. 2013. GASTRIC
ACID, CALCIUM ABSORPTION, AND THEIR
IMPACT ON BONE HEALTH. Physiological
Reviews 93:189-268.
Vitamin D
Parathormon
Barret, K.E., Boitano, S., Barman, S.M., Brooks, H.L.
Ganong´s Review of Medical Physiology. 23rd Ed.
McGraw-Hill Companies 2010
PTH1R versus PTH2R
A, PTH secretion by dispersed
normal human parathyroid
cells in culture in response to
varying concentrations of
extracellular calcium. B, The
four-parameter model
describing the inverse
sigmoidal relationship
between extracellular
calcium and PTH secretion.
Parameter 1 is the maximal
secretory rate, parameter 2 is
the slope of the curve at the
midpoint, parameter 3 is the
set point, and parameter 4 is
the minimum secretory rate.
Kopic, S., and J.P. Geibel. 2013. GASTRIC
ACID, CALCIUM ABSORPTION, AND THEIR
IMPACT ON BONE HEALTH. Physiological
Reviews 93:189-268.
OPG – osteoprotegrin; RANK - receptor activator of nuclear factor kB
Kopic S, Geibel JP: GASTRIC ACID,
CALCIUM ABSORPTION, AND THEIR
IMPACT ON BONE HEALTH. Physiol Rev
2013, 93(1):189-268.
DiGirolamo, D.J., T.L. Clemens, and S.
Kousteni. 2012. The skeleton as an
endocrine organ. Nature Reviews
Rheumatology 8:674-683.
DiGirolamo, D.J., T.L. Clemens, and S. Kousteni. 2012. The
skeleton as an endocrine organ. Nature Reviews
Rheumatology 8:674-683.
Parathyroid hormone-related peptide (PTHrP)
and hypercalcemia
• “ectopic” production by cancers of peptide hormones (ACTH, PTH?)
• PTH? – hypercalcemia, hypophosphataemia (bone metastases, renal
cancer, lung cancer, some neuroendocrine tumors)
• Radioimmunoassays – PTHrP (↓ PTH versus ↑ PTHrP)
• Physiological functions of PTHrP?
• auto-/para-/endocrine
• affecting endochondral bone formation - blocks maturation of chondrocytes
• growth and differentiation of mammary gland, skin and pancreatic islets
• smooth muscle relaxation
• transepithelial transport of calcium (placenta)
Martin, T.J. 2016.
PARATHYROID HORMONERELATED
PROTEIN, ITS
REGULATION OF
CARTILAGE AND BONE
DEVELOPMENT, AND ROLE
IN TREATING BONE
DISEASES. Physiological
Reviews 96:831-871.
Martin, T.J. 2016. PARATHYROID HORMONE-RELATED PROTEIN, ITS REGULATION OF CARTILAGE AND BONE DEVELOPMENT, AND ROLE
IN TREATING BONE DISEASES. Physiological Reviews 96:831-871.
Martin, T.J. 2016.
PARATHYROID HORMONERELATED
PROTEIN, ITS
REGULATION OF
CARTILAGE AND BONE
DEVELOPMENT, AND ROLE
IN TREATING BONE
DISEASES. Physiological
Reviews 96:831-871.
Intrakrinní funkce – regulace
buněčné proliferace a apoptózy?
Martin, T.J. 2016. PARATHYROID HORMONERELATED
PROTEIN, ITS REGULATION OF
CARTILAGE AND BONE DEVELOPMENT, AND ROLE
IN TREATING BONE DISEASES. Physiological Reviews
96:831-871.
Martin, T.J. 2016.
PARATHYROID
HORMONE-RELATED
PROTEIN, ITS
REGULATION OF
CARTILAGE AND
BONE
DEVELOPMENT, AND
ROLE IN TREATING
BONE DISEASES.
Physiological Reviews
96:831-871.
Martin, T.J. 2016.
PARATHYROID
HORMONE-
RELATED
PROTEIN, ITS
REGULATION
OF CARTILAGE
AND BONE
DEVELOPMENT,
AND ROLE IN
TREATING
BONE
DISEASES.
Physiological
Reviews 96:831-
871.
Martin, T.J. 2016.
PARATHYROID
HORMONE-
RELATED
PROTEIN, ITS
REGULATION OF
CARTILAGE AND
BONE
DEVELOPMENT,
AND ROLE IN
TREATING BONE
DISEASES.
Physiological
Reviews 96:831-
871.
Martin, T.J. 2016. PARATHYROID HORMONERELATED
PROTEIN, ITS REGULATION OF
CARTILAGE AND BONE DEVELOPMENT, AND
ROLE IN TREATING BONE DISEASES.
Physiological Reviews 96:831-871.
cAMP response element
binding (CREB) protein
Calcitonin
Russell, F.A., R. King, S.J. Smillie,
X. Kodji, and S.D. Brain. 2014.
CALCITONIN GENE-RELATED
PEPTIDE: PHYSIOLOGY AND
PATHOPHYSIOLOGY.
Physiological Reviews 94:1099-
1142.
Deduced model for the roles of CT and α-CGRP in bone formation.
Based on the work of several investigators it is likely that CT inhibits
bone resorption through a direct effect on osteoclasts, and that α-CGRP
activates bone formation through a direct effect on osteoblasts (solid
lines). The negative influence of CT on bone formation however, may
be indirectly mediated by the hypothalamus or by osteoclasts (dashed
lines).
Huebner, A.K., J. Keller, P. Catala-Lehnen, S.
Perkovic, T. Streichert, R.B. Emeson, M. Amling,
and T. Schinke. 2008. The role of calcitonin and
alpha-calcitonin gene-related peptide in bone
formation. Archives of Biochemistry and
Biophysics 473:210-217.
Russell, F.A., R. King, S.J. Smillie, X. Kodji, and S.D. Brain. 2014. CALCITONIN GENE-RELATED PEPTIDE: PHYSIOLOGY AND
PATHOPHYSIOLOGY. Physiological Reviews 94:1099-1142.
Estrogens/androgens
Manolagas, S.C., C.A. O'Brien, and M. Almeida. 2013. The role of estrogen and androgen receptors in bone health and disease. Nat. Rev.
Endocrinol. 9:699-712.
Glucocorticoids
Weinstein, R.S. 2011. Glucocorticoid-Induced Bone Disease. New England
Journal of Medicine 365:62-70.
- GnRH ! (androgens/estrogens)
- IGF-1
- Reduced resoprtion of calcium
Osteocalcin
Vitamin K is required for the formation of γ-carboxyglutamic acid
Booth, S. L. et al. (2012) The role of osteocalcin in human glucose metabolism: marker or mediator?
Nat. Rev. Endocrinol. doi:10.1038/nrendo.2012.201
Osteocalcin in bone
metabolism
Chapurlat, R.D., and C.B. Confavreux. 2016.
Novel biological markers of bone: from bone
metabolism to bone physiology. Rheumatology
55:1714-1725.
IGF-1 and
bone
metabolism
Leptin and bone
metabolism
Leptin and bone
metabolism
Karsenty, G., and F. Oury. 2012. Biology
Without Walls: The Novel Endocrinology of
Bone. In Annual Review of Physiology, Vol
74. D. Julius, and D.E. Clapham, editors.
87-105.
Karsenty, G., and F. Oury. 2012. Biology Without Walls: The Novel Endocrinology of Bone. In Annual Review of Physiology, Vol 74.
D. Julius, and D.E. Clapham, editors. 87-105.
Karsenty, G., and F.
Oury. 2012. Biology
Without Walls: The
Novel Endocrinology of
Bone. In Annual Review
of Physiology, Vol 74. D.
Julius, and D.E.
Clapham, editors. 87-
105.
Karsenty, G., and F.
Oury. 2012. Biology
Without Walls: The
Novel Endocrinology of
Bone. In Annual Review
of Physiology, Vol 74. D.
Julius, and D.E.
Clapham, editors. 87-
105.
Oxytocin and bone metabolism
Colaianni, G., L. Sun, M. Zaidi, and A. Zallone.
2014. Oxytocin and bone. American Journal of
Physiology-Regulatory Integrative and
Comparative Physiology 307:R970-R977.
Russell, J.T. 2011.
Imaging calcium signals
in vivo: a powerful tool in
physiology and
pharmacology. British
Journal of
Pharmacology
163:1605-1625.
In vivo
calcium
imaging
Russell, J.T. 2011.
Imaging calcium signals
in vivo: a powerful tool in
physiology and
pharmacology. British
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163:1605-1625.
Biochemical markers of bone turnover
Chapurlat, R.D., and C.B. Confavreux. 2016.
Novel biological markers of bone: from bone
metabolism to bone physiology. Rheumatology
55:1714-1725.
„New“ markers of bone metabolism
Chapurlat, R.D., and C.B. Confavreux. 2016. Novel biological markers of bone: from bone metabolism to bone physiology.
Rheumatology 55:1714-1725.
Huang, C.L., and O.W. Moe. 2011.
Klotho: a novel regulator of calcium
and phosphorus homeostasis.
Pflugers Archiv-European Journal
of Physiology 462:185-193.
Klotho:
 b-glukuronidáza
- Stárnutí
- Kostní metabolismus
- Abusus alkoholu
- Ateroskleróza
RIA
ELISA
HPLC
Paraproteins
- M proteins
- Immunoglobulin or its fragment
resulting produced by lymphoidclone
of plasma cells without a
distinct antibody function
- IgG, IgA, IgM, light / heavy chain(s)
- Hematologic malignancies, blood
diseases
A, Polyclonal pattern from a densitometer
tracing of agarose gel: broad-based peak of γ
mobility. B, Polyclonal pattern from
electrophoresis of agarose gel (anode on the
left). The band at the right is broad and extends
throughout the γ area.
A, Monoclonal pattern of serum protein as
traced by a densitometer after
electrophoresis on agarose gel: tall, narrowbased
peak of γ mobility. B, Monoclonal
pattern from electrophoresis of serum on
agarose gel (anode on the left): dense,
localized band representing monoclonal
protein of γ mobility.