Understanding mineralization process: the role of TNSALP and Matrix Vescicles.

  

Alkaline phosphatases

Alkaline phosphatase (ALP) was discovered in 1923 by Robert Robinson in young rats and rabbits within ossifying bone and cartilage. However , never Robinson referred to this enzyme as “alkaline” phosphatase, term introduced only later. For most of the past eight decades physicians have recognized the important clinical insight that can come from measurement of ALP activity in serum. Detection and monitoring of hepatobiliary and skeletal disease are generally possible. In fact, since 1930 ALP detection and quantification in serum has been routine in hospital laboratories.

Nevertheless, the physiologiocal function of ALP, is largely unknown.

At the end of 1960s, electron microscopy helped to rejuvenate Robinson’s hypothesis, when the earliest site of hydroxyapatite crystal deposition in the developing skeleton was noted by E. Bonucci and HC Anderson to be within novel extracellular structures called matrix vescicles (1969). These vescicles were found to be rich in ALP activity and later they have been demonstrated to be replenished by many enzymes and constituents such as:

1. Inorganic pyrophosphatase (PPi-ase)
2. ATPase
3. phospholipids
4. polysaccarides
5. glycolipids

During early phase (primary) of mineralization , hydroxyapatite crystals appear and grow within these structres. Soon after , the vescicles rupture and extravescicular (secondary) mineralization occurs as crystal propagation continues.

Actually the proposed biological roles of ALP in mammals are numerous including:

- hydrolysis of phosphate esters to supply the nonphosphate moiety

- transferase action for the synthesis of phosphate ester

- regulation of Pi metabolism

- maintenance of steady-state levels of phosphoryl metabolites

- action on phoshoprotein pshosphatases

-

At plasmamembrane level it has been proposed that ALP can function not only such as an active P transporter but also for:

- calcium muvement regulator

- Na+/K+ exchange regulator

- Fat exchange

- Protein exchange

- Carbohydrate exchange

Interestingly sequence analysis of ALP demonstrated that this enzyme can be coupled with other proteins, for example adhering to collagen, and it has been suggested that this physical property of ALP should be considered when we examined the action for example of ALP on skeletal matrix such as phosphoprotein phosphatase.

It has to be outlined that ALP function mainly such as a cell surface enzyme, but at some stages during embryo formation ALP may acts also intracellularly.

The role of ALP in skeletal mineralization should be resumed considering ALP as “inhibitor” of pyrophosphate deposition (a potent inhibitor of hydroxyapatite formation):

1. Locally increase P concentration
2. Destruction of inhibitors of hydroxyapatite crystal growth
3. Transport of P
4. Calcium binding protein ( used by cells such as uptake enzyme for calcium)
5. Ca++/Mg++ ATPase
6. Tyrosine specific phosphoprotein phosphatase

Recently these different activities previously attribute only to Alkaline Phosphatase activity, have been studies in more details.

It is interesting to note that in normal adolescents 13 to 14 years old pyridoxal-5-phosphate concentrations have been reported to be approximately 40 nmol/L, also if this developmental stage is associated with high alkaline phosphatase activity. Low levels of Pyridoxal-5-phosphate are also observed in patients with hypophosphatemic rickets and the researchers attributed this data to increased activity of alkaline phosphatase enzymes. However most of alkaline phosphatase values are within the normal range for children. It has also been suggested the presence of so called “functional hypophosphatasia” in patients affected by renal osteodystrophy were normal alkaline phosphatase levels are coupled with high serum inorganic phosphate levels. In other words in these pathophysiological conditions no correlation exists between alkaline phosphatase activity and pyridoxal-5-phosphate concentrations. However in these conditions plasma levels of inorganic phosphate are also higher than normal suggesting that the main factor in decreased pyridoxal 5 phosphate concentration would be low phosphate concentration rather than high levels of alkaline phosphatase.

An emerging role in Pyrophosphate production has been recently attributed to Ectonucleotide Pyrophosphatase/phosphodiesterase 1 (NPP1), previously referred such as plasma cell membrane glycoprotein 1. This enzyme has been found in mineralizing tissues such as bones and teeth. Mutations in NPP1 cause the generalized arterial calcification of infancy due to inability of vascular cells to form pyrophosphate. Moreover, mutations in NPP1 have also been reported as a second cause of autosomal recessive Hypophosphatemic Rickets, the first being attributed to mutations in Dentin Matrix Protein 1 (DMP1). The role of NPP1 would be the hydrolyis from Adenosine Triphosphate (ATP) of Pyrosphosphate. NPP1 clearly has a role in PPi generation at the level of chondrocyte and osteoblast membranes, whereas at level of Matrix Vecicles NPP1 does not use ATP efficiently.

Another pathway for generation of pyrophosphate production is the secretion from cells by the transmembrane spanning cell surface protein Ankylosis Human homologue of the mouse progressive ankylosis protein (ANKH). Two autosomal dominant human diseases have to date been reported:

- Craniometaphyseal Dysplasia

- Chondrocalcinosis-2

Data conerning the possible presence of an autosomal recessive form linked to a mutation on exon 6 of the 12 exons constituting ANKH gene has to be clarified by further studies. Anyway ANKH protein seems to be important for mediating intracellular to extracellular channeling of pyrophosphate.

Interestingly another protein called Phosphatase PHOSPHO-1, first identified in chick as a member of the haloacid dehalogenase (HAD) superfamily of Magnesium dependent hydrolases, is expresed at levels 100-fold higher in mineralizing tissues compared to nonmineralizing ones. PHOSPHO-1 shows high phosphohydrolase activity toward Phosphoetanolamine (PEA) and Phosphocholine (PCho); it is active inside chondrocytes and osteoblast derived Matrix Vescicles. The role of PHOSPHO-1 is to maintain the concentration of inorganic Pyrophosphate (PP i) so that the ratio of inorganic phosphate to inorganic pyrophosphate would be permissive of a normal mineralization process. Inside Matrix Vescicles (MV) soluble phosphatase PHOSPHO-1 , with specificty for phosphoethanolamine and phosphocholine, increases the local intravescicular concentration of inorganic phosphate (P i) to change the Pi/PPi ratio in favor of precipitation of hydroxyapatite seed crystals.

In summary given the role of FGF23/Klotho pathway in inorganic phosphorus metabolism, as well’s of Vitamin D3 metabolites, more important role should be attributed to inorganic phosphate concentration that to enzymatic phosphatase activity for study derangements in bone mineralization.

ALP is found in nearly all plants and animals. In humans, four ALP isoenzymes are encoded by four separate genes. Three of these are expressed in a tissue-specific manner are they are called:

- placental

- intestinal

- germ-cell (placental-like)

- Tissue Non Specific

The fourth ALP isoenzyme is ubiquitous, but expecially abundant in hepatic, skeletal and renal tissues (liver/Bone/kiney ALP) and it is called tissue non specific ALP (TNSALP). Interestingly TNSALP is a family of “secondary” isoenzymes (isoforms), with the same polypeptide sequence, encoded by one gene (TNSALP) but different each other only by posttranslational modification involving a different glycosylation pattern (carbohydrate). TNSALP is located on chromosome 1p36.1-34 near the end of shot arm; the genes coding for placental, intestinal and germ-cells ALP are found near the tip of the long arm of chromosome 2q34-37. The TNSALP chromosome structure is represented by 12 exons, 11 of which are translated into a 507 aminoacid nascent enzyme. The promoter region of TNSALP is located within 610 nucleotides 5’ to the transcription start site and it contains TATA box and an Sp1 binding site acting as regulatory elements. It is believed that basal levels of TNSALP expression reflect inherent “housekeeping” promoter effects, whereas differential expression in various tissues should be mediated by a postranslational mechanism. Interestingly 5’ untranslated region differ between the bone and liver TSNALP isoforms. From phylogenetic point of view, the TNSALP should represent an ancestral gene, whereas the tissue-specific ALPs is likely originated from a series of gene duplications. Human ALP isoenzymes gene sequence indicates that the nascent polypeptide has a short signal sequence of 17 or 21 aminoacids residues and a hydrophobic domain at its c terminal site. The active site is coded by six exons and it is composed by 15 aminoacid residues with a nucleotidic sequence well conserved throught nature. ALPs is a metalloenzyme linking Zinc atom, the link of Zn++ atom stabilizes the tertiary structure. In summary the structure of these enzymes is formed to link a dinuclear metal cofactor structure so that a common cathalytic mechanism for enzymes involved in phosphotransfer reactions has been identified involving spin-coupled metal binding site formed by a scaffold structure at active metal linking site constituted by the same repeated tertiary spatial construct.

Β sheet – α helix - Β sheet - α helix - Β sheet

The 3 β strands of this structure form a parallel sheet that is capped by intervening α helices. Two metal ions are positioned at the apex of this fold forming a dinuclear metal center with 3.0 - 4.0 Ǻ between metal ions, with 4 of the metal ligands provided by residues in the loops between β sheets and α helices. ALP in E. Coli has been extensively studied and a Mg++ with a Zn-Zn dinuclear center reminiscent of the dinuclera metal site of seine/threonine phosphatase has been identified. In E.coli His 372 forms an hydrogen bond with Asp 327 , an aminoacid involved into didentate Zn stabilization) and it is thought to lower the pKa of the Zn atom involved to binding a water molecule. Cathalytic activity require multimeric configuration of identical subunits, each monmer having an active site and two Zn atoms. The role of Zn atoms is probably those of allowing the formation of a nucleophil reactant by hydroxyl group of serine residue located on cathalitic site, that attract the phosphoric group disrupting the esteric link. The mechanism of enzymatic reaction in ALP present in E. Coli has been elucidated for phosphate ester hydrolysis forming first an intermediate phosphoenzyme. In particular ALP of E. Coli cathalizes the transfert of phosphoryl group throught the formation of a transient link with a Serine residue located on active catalitic site. Later this phosphate group is released and the cathalitic site left free to react with anoter phosphoester group. If ALP in serum is present as a dimer with α/β topology with a 10 –stranded beta sheets in its center, ALP at membrane level is linked as a homotetramer. ALP is linked to plasmamembrane surface, through a polar head group of a phosphatidylinositol glycan and it can be released by a specific phospholipase. Intracellualr degradation of ALPs can involve proteasomal structures. Release from plasma membrane could involve phosphatidase C or D.

Clearance of circulating ALP, as for many plasma proteins is assumed to occur via uptake by the liver.

Whereas in children ALP plasma activity is mainly of bone origin and the remaining is of intestinal isotype; interestingly an old data report that blood type (0 and B are secretors) influences the level of placental isoenzyme of ALP in the blood after an ingestion of a fatty meal. In adult blood, ALP activity reflects equal amounts of hepatic and bone isotypes. Interestingly only recently on 2000 the crystal structure of placental isoform of ALP was isolated and studied on X ray crystallography.

TNSALP has a major role in two kind of reactions involved into mineralization process:

- Pyrophosphatase : hydrolizing pyrophosphate into two inorganic phosphate ions

- ATPase/ADPase: hydrolizing Adenosin triphosphate into Adenosin bisphosphate and one molecule of inorganic phosphate.

Accordingly TNSALP partecipates in the calcification process both by restricting the concentration of extracellular inorganic pyrophosphate PPi and by contributing to the inorganic phosphate (Pi) pool available for calcification.

ATP > ADP > AMP + 2 Pi

PPi > Pi

The working model in bone and cartilage supposed that bone mineralization is first initiated within the lumen of Matrix Vescicles (MVs). In a second time, hydroxyapatite crystals grow beyond the confines of the MVs and become exposed to the extracellular milieu, where they continue to propagate along collagen fibrils. Hydroxyapatite seed crystals are formed in the sheltered interior of MVs favored by the Pi-generating activity of PHOSPHO-1 fosfatase enzyme,as well’s by the transport function of Pyrophosphate (PPi) transporters, such as ANKH. The keys rate limiting step seems to be the ratio between Pi/PPi concentrations:

Pi/PPi > Mineralization

In other words an increased concentration of inorganic pyrophosphate (PPi) inhibits the crystalization process of hydroxyapatite, whereas increase increased concentration of inorganic phosphate ions (Pi) promote both crystalization and nucleation processes.

 

Human diseases characterized by an abnormal decrease or increase in ALP blood levels are called respectively:

- Hypophosphatasias

- Aphosphatasia

- Hyperphosphatasias or Paget disease of bone

- Familial Expansile Osteolysis

- Expansile skeletal Hyperphosphatasias

- Early onset Paget’s disease of bone in Japan

- Hereditary Hyperphosphatasia (Juvenile Paget’s disease)

Hypophosphatasia

In 1948 a Canadian pediatrician John Campbell Rathbun coined the term hypophosphatasia reporting a boy who developed and died from severe rickets with epilepsy, whose ALP activity in serum, bone and other tissues was paradoxically subnormal.

Present in all races, however this condition is expecially frequent in inbred Mennonite families from Mannitoba, Canada, where about 1 every 25 individuals is a carrier and 1:2500 newborns manifests severe disease.

Six forms of hypophosphatasias have been individuated, the earlier is the presentation of symptoms and more severe is the skeletal disease and the biochemical manifestations:

1. Perinatal : autosomal recessive
2. Infantile : autosomal recessive
3. Childhood : autosomal dominant or recessive
4. adult: autosomal dominant or recessive
5. odontohypophosphatasia: autosomal dominat or recessive
6. pseudohypophosphatasia

Laboratory findings include elevated values of phosphoethanolamine, pyridoxalphosphate, inorganic pyrophosphate.

Hypophosphatasia is a rare heritable disordercaused by a loss-of-function mutation in the ALP gene encoding for the tissue non specific alkaline phosphatase (TNSALP). It is characterized by deficiency in serum and bone alkaline phosphatse and defective bone and tooth mineralization.

Nearly all babies with perinatal hypophosphatsia die in utero or shortly after birth.

Those with infantile form present before 6 months of age with rickets, failure to thrive, or vitamin B6-dependent seizures, and approximately 50% die for respiratory failure because of poor lung development or progressive hypomineralization of the rib cage.

Adult hypophosphtasia typicaly manifests during middle age as recurrent, slowly healing metatarsal fractures, followed by painful nonhealing proximal femur fractures or pseudofractures.

The bone symptoms are highly variable in their clinical expression, which ranges from stillbirth without mineralized bone to pathological fractures developing only late in adulthood.

Odontohypophosphatasia is characterized by premature exfoliation of primary teeth with roots intact and/or several dental caries, not associated with abnormalities of the skeletal system.

Severe forms of the disease such as perinatal and infantile forms are transmitted as an autosomal recessive trait, whereas both autosomal recessive and autosomal dominant transmission may be found in milder forms, especially odontohypophosphatasia.

The tissue nonspecific ALP (TNSALP) gene is localized on chromosome 1p36.1 and it consits of 12 exons distributed ober 50 Kbases. More than 160 mutations have been described to date in the TNSALP gene. In North American, Japanese, and European patients, indicating a very strong allelic heterogeneity in the disease. This variety of mutations results in highly variable clinical expression and a great number of compound heterozygous genotypes with missense mutations that account for 82% of mutations. The remaining mutations are:

- missense mutations (82%)

- microlesions (11%)

- splicing mutations (4%)

- nonsense mutations (3%)

- a nucleotide substitution on major transcription initiation site

- a denovo mutation on heterozygous carrier of a missense mutation

The affected individuals carry one or two loss-of-function mutations within the TNSALP gene alleles. This experiment of the nature, inherited as either an autosomal dominant or autosomal recessive trait, reveals a crucial role for TNSALP in skeletal mineralization. There is no established medical treatment for hypophosphatasia. Augmenting circulating alkaline phosphatase activity into or even above the normal range for several months using intravenously administered ALP from various tissues sources has had no convincing beneficial effects. Also transplantation therapy with cultured osteoblasts and bone fragments was quite unsuccessful and experiments suggested that we must lower PPi at mineralization sites. Accordingly TNSALP activity must be increased at mineralization sites more than at plasma level.

Recently a recombinant fusion protein including TNSALP ectodomain, the constant region of IgG1 Fc domain, and the terminal deca-aspartate motif has been admnistered in 11 patients with perinatal or infantile forms of hypophosphatasia. Treatment was associated with healing of skeletal manifestations of hypophosphatasia as well’s with improvement in respiratory and motor functions. Improvement is still being observed in patients receiving treatment for more than 3 years.

References

Robinson R. The possible significance of hexosephosphoric esters in ossification. Biochem 1923;17:286-293.

Anderson HC. Vescicles associated with calcification in the matrix of epiphyseal cartilage. J Cell Biol 1969;41:59-72.

deBernard B, Bianco P, Bonucci E et al. Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca++ -binding glycoprotein. J Cell Biol 1986;103:1615-23.

Coleman JE. Structure and mechanism of alkaline phosphatase. Ann Rev Biophys Biomol Struct 1992;21:441-83.

Coleman JE, Gettins P. Alkaline phosphatase p, solution structure, and mechanism . Adv Enzymol 1983;55:381-452.

Kim EE, Wyckoff HW. Reaction mechanism of alkaline phosphatase based on crystal structures: two metal ions catalysis. J Mol Biol 1991;218:449-64.

Xu X, Qin XQ, Kantrowitz ER. Probing the role of histidine-372 in zinc binding and the cathalitic mechanism of escherichia coli alkaline phsosphatase by site-specific mutagenesis. Biochemistry 1994;33:2279-84.

Whyte MP. Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization. Endocr Rev 1994;15:439-61.

Le Due HM, Stigbrand T, Taussig MJ et al. Crystal structure of alkaline phosphatase from human placenta at 1.8 Ǻ resolution. J Biological Chem 2000;275:9158-65.

Levy-Litan V, Hershkovitz E, Avizov L et al. Autosomal recessive hypophosphatemic rickets is associated with an inactivating mutation in the ENPP1 gene. Am J Hum Genet 2010;86:273-8.

Hessle L, Johnson KA, Anderson HC et al. Tissue non specific alkaline phosphatase and plasma cell membrane glycoprotein1 are central antagonistic regulators of bone mineralization. Proc Nat Acad Sci USA 2002;99:9445-9.

Lorenz-Depiereux B, Schnabel D, Tiosano D et al. Loss-of-function ENPP1 mutations cause both generalized arterial calcification of infancy and autosomal recessive hypophosphatemic rickets. Am J Hum Genet 2010;86:267-72.

Collins MT, Boehm M. It ANKH necessarily so. J Clin Endocrinol Metab 2011;96:72-4.

Ciancaglini P, Yadav MC, Simao AMS et al. Kinetic analysis of substrate utilization by native and TNAP, NPP or PHOSPHO1-Deficient matrix vescicles. J Bone Miner Res 2010;25:716-23.

Yadav MC, Simao AMS, Narisawa S et al. Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and Alkaline Phosphatase function: a unified model of the mechanisms of initiation of skeletal calcification. J Bone Miner Res 2011;26:286-97.

Whyte MP, Obrecht SE, Finnegan PM et al. Osteoprotegerin deficiency and Juvenile Paget’s disease. N Engl J Med 2002;347:175-84.

Whyte MP, Hughes AE. Expansile skeletal hyperphosphatasia is caused by a 15 base pair tandem duplication in TNFRSFIIA encoding RANK and is allelic to Familial Expansile Osteolysis. J Bone Min Res 2002;17:26-9.

Taillandier A, Sallinen SL, Brun-Heath P et al. Childhood hypophosphatasia due to a de novo missense mutation in the tissue nonspecific alkaline phosphatase gene. J Clin Endocrinol Metab 2005;90:2436-9.

Cahil RA, Wenkert D, Perlman SA et al. Infantile hypophosphatasia: transplantation therapy trial using bone fragments and cultured osteoblasts. J Clin Endocrinol Metab 2007;92:2923-30.

Schalin-Jantti C, Mornet E, Lamminen A et al. Parathyroid hormone treatment improves pain and fracture healing in adult hypophosphatasia. J Clin Endocrinol Metab 2010;95:5174-9.

Whyte MP, Greenberg CR, Salman NJ et al. Enzyme replacement therapy in life threatening hypophosphatasia. N Engl J Med 2012;366:904-13.

Thanks to Ivy for her support.

 

VITAMIN D: how a vitamin becomes a true hormone.

    

 

Calcium intake is well know to represent a very important factor affecting bone mass, bone biomechanical alteration and consequntly bone fracture incidence. Low calcium intake in particular with consequent decrease in intestinal calcium absorption is the gatekeeper in bone disease induction. It has been demonstrated from basic studies on osteoporotic patients that low intestinal calcium absorption is striclty related to loss or decrease activity of steroid hormonal compounds such as estrogens and activated form of Vitamin D3 (1,25 dihydrixyVitamin D3) at epithelial intestinal level. With aging in particular there is a decline in calcium absorption efficiency, related to intestinal loss of Vitamin D3 receptors or resistance/lower actictivity of these receptors to the action of 1,25 dihydroxyVitamin D3. Also seasonal variations in sunlight exposure profundly affect the level of Vitamin D3 synthetized in our skin by irradiation with ultraviolet wave length of our skin tissue. Pre Vitamin D2 and Pre Vitamin D3 better called ergocalciferol and colecalciferol, introduced with the diet respectively from vegetable and from animals foods, are efectively transformed on skin tissue in true Vitamin D2 and Vitamin D3 by automaintained non enzymatic reaction due only to exposure of our skin tissue to sunlight ultraviolet irradiation.

CYP27A1 hydroxylase

After sunlight activation Vitamin D3 is transported into our body linked to protein carriers on the first time to the liver, where it is hydroxylated by a cytocrome P450 dependent enzyme called 25 hydroxylase CYP27A1. At this level a first selection is performed by hydroxylating electively the choleclaciferol derivatives (i.e. Vitamin D3) more than ergocalciferol derivatives (i.e. Vitamin D2). From this first evidence is not effective the therapeutical administration of vegetable form of vitamin D, leading on the contrary to adecrease in levels of active hydroxylated forms of Vitamin D3. Measurement of plasma levels of 25-hydroxy Vitamin D3 is universally considered the main index of individual body reserve of Vitamin D3. However, many controversies exist, and recently published Guidelines outlined this topic, about optimal plasmatic levels achievable in human in order to prevent Vitamin D3 deficiency. Accordingly, a seasonal fluctuation in hormonal vitamin D3 are present in humans related to sunlight exposure with greater levels reached during spring and summer seasons, and lower levels during fall and winter seasons. Again, interindividual variations between individuals are present according to proportion of time spent in indoor and outdoor activities. Moreover, also variations according to latitute degree of countries where people live are present if we consider that sunlight irradiation is most importantat near tropical area of the world compared to northern countries.

CYP27B1 hydroxylase

After liver activation 25 hydroxyVitamin D3 is transported to the kidney proxymal convoluted tubules in order to be fully activated by 1-alfa hydroxylase enzyme cytocrome P450 CYP27B1. This hydroxylative enzyme has been profoundly studied thanks to the more evident regulation by hormonal axis devoted to calcium-phosphate homeostasis. However, as we can see later, 1 alfa hydroxylase activity is not so relevant to know the individual needs of Vitamin D3 supplementation with fortified foods. On the contrary, it is important for the synthesis of active hormonal Vitamin D3 hydroxylated in 25 and 1 alfa carbons allowing a global interaction with zinc fingher sites of intranuclear Vitamin D receptors present in target tissues. In particular , it has been largely demonstrated both in vitro and in vivo that 1 alfa hydroxylase is sensitive to stimulating action of parathyroid hormone, estrogens and probably androgenic steroids hormones, Growth Hormone/Insulin growth factor 1.

CYP24A1 hydroxylase

Responsible for catabolism of Vitamin D3 into liver secreted “ calcitroic acid “ 24 hydroxylase enzyme CYP24A1 has been only recently subjected to an accurated studies. In particular the genetic demonstrations of genetic polymorphisms leading to inactive mutated forms of CYP24A1 hydroxylase has been recently confirmed also in clinical background as responsible of Vitamin D3 toxicity. In particular the Idiopathic Infantile Hypercalcemia ( milder form ) also called Lightwood type (OMIM 143880) has been demonstrated in 3 affected patients linked to mutations in CYP24A1 gene expression. It is possible that clinically noted interindividual variations in Vitamin D3 need by fortified foods in humans could be due to inactivating mutations in CYP24A1 gene. Interestingly a new hormonal axis has been invoved into regulation of CYP24A1 expression: called FGF23/KLOTHO axis. Klotho is a protein with glycosidase activity able to modify the FGF Receptor present at kidney level for FGF23. FGF23 as bee demonstrated to be a potent stimulator of CYP24A1 and a potent inhibitor of CYP27B1 suggesting in this way a more complex picture compared to the past, involving two homeostatic system for phospho-calcium metabolism:

      PTH/ Vitamin D3 axis: CYP27B1

      FGF23/ KLOTHO axis: CYP27B1 and CYP24A1

References

Koro-o M, Matsumura Y, Aizawa H et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 1997;390:45-51.

Urakawa I, Yamazaki Y, Shimada T et al. Klotho converts canonial FGF receptor into a specific receptor for FGF23. Nature 2006;444:770-4.

Makishima M, Lu TT, Xie W et al. Vitamin D erceptor as an intestinal bile acid sensor. Science 2002;296:1313-6.

Kurosu H, Yamamoto M, Clark JD et al. Suppression of aging in mice by the hormone Klotho. Science 2005;309:1829-33.

Chang Q, Hoefs S, van der Kemp AW et al. The beta glucosidase Klotho hydrolyzes and activates the TRPV5 Channel. Science 2005;310:490-3.

Imura A, Tsuji Y, Murata M et al. Alfa Klotho as a regulator of calcium homeostasis. Science 2007;316:1615-6.

Cai Q, Hodgsan SF, Kao PC et al. Inhibition of renal phosphate transport by a tumor product in a patients with a oncogenic osteomalacia. N Engl J Med 1994;330:1645-9.

Econs MJ, Drezner MK. Tumor induced osteomalacia – unveiling a new homone. N Engl J Med 1994;330:1679-81.

Kronenberg HM. NPT2a – The key to phosphate homeostasis. N Engl J Med 2002;347:1022-4.

Priè D, Huart V, Bakouh N et al. Nephrolithiasis and osteoporosis associated with hypophosohatemia caused by mutations in the type 2a sodium-phosphate cotransporter. N Engl J Med 2002;347:983-91.

Johnsson KB, Zahradnik R, Larsson T et al. Fibroblast growth factor 23 in oncogenic osteomalacia and X linked hypophosphatemia. N Engl J Med 2003;348:1656-63.

Karim Z, Gérard B, Bakouh N et al. NHERF1 mutations and responsiveness of renal parathyroid hormone. N Engl J Med 2008;359:584-92.

Priè D, Friedlander G. Genetic disorders of renal phosphate transport. N Engl J Med 2010;362:362:2399-2409.

Schlingmann KP, Kaufmann M, Weber S et al. Mutations in CYP24A1 and Idiopathic Infantile Hypercalcemia. N Engl J Med 2011;365:410-21.

FRAX Index and osteoporosis prevention: clinical application of risks stratification studies.

        

Vertebral fractures

 

Accurate detection of vertebral fracture is essential for risk assessment of individual patients in clinical practice, for determining the drug efficacy in clinical trials and for evaluating the prevalence and incidence of osteoporotic disease in a given population.

A prevalent vertebral fracture is the strongest predictor of subsequent vertebral fracture as well’s of any subsequent osteoporotic fracture.

A vertebral fracture results in a 4.4 fold increase risk of future vertebral fracture in people with a prevalent fracture.

Although bone mass is an important component of the risk of fracture, other abnormalities occur in the skeleton that contribute to fragility fractures.

In addition, a variety of nonskeletal factors, such as the liability to fall and force of impact, contribute to fracture risk. In this view an accurate assessment of fracture risk should ideally take into account other readly measured indices of fracture risk, including in particular those adding more informations to that provided by Bone Mineral Density measurement.

It has been suggested that the ability of Bone Mineral Density to predict a fracture is comparable to the use of blood pressure measurements to predict stroke and better than serum cholesterol levels to predict myocardial infarction.

At the age of 50 years, the proportion of women with osteoporosis who will fracture their hip, spine, forearm or proximal humerus in the next 10 years is about 45%. However the detection rate of these fractures (i.e. sensitivity) is low, and 90% of such fractures would occur in women without osteoporosis.

Low sensitivity is one of the reasons why widespread population-based screening is not widely recommended in women at the time of menopause.

Vertebral fractures assessment

The problem is besides all the definition of vertebral fracture, at present there’s no Consensus Giudelines on osteoporotic vertebral fracture definition.

Identification of vertebral fracture can be very difficult, because the shape of normal vertebral bodies varies widely between individuals. Vertebral bodies can be present abnormal features because non-osteoporotic deformities and errors in radiological projection can induce a misdiagnosis of fractured body.

We have to remember that about 50% of vertebral fractures are asymptomatic and therefore are only casually identified. They are not the source of pain !Even when chest radiographs or vertebral images are correctly obtained only 35 to 50% of all radiographic vertebral fractures are correctly reported. It has been estimated that only 19% of these fractures reach clinical attention and can be correctly treated with a antiosteoporotic treatment. In view of high radiation exposure routine chest and lumbar radiographs are not recommended, but the availability of vertebral imaging using DEXA take the advantage of utilize an image of near radiographic quality available with a low fraction of the radiation dose.

Imaging vertebral fractures using DXA is called Vertebral Fracture Assessment (VFA).

The disadvantage of VFA is poor image resolution compared to conventional radiography, CT or MRI and the increased difficulties in imaging the thoracic spine, expecially above T7. Between 5 to 15% of thoracic vertebrae can be visualized only by conventioonal radiography.

The sensitivity and specicificty of this approach compared with conventional radiography varies with the kind of approach used to definy a vertebral fracture:

- Morphometric

- Semiquantitative (SQ)

- Visual identification

Irrespective of the approach, sensitivity is only moderated for for mild fractures corresponding to Genant grade 1 vertebral fracture showing a sensitivity of 54%, due to low imge resolution of this technique.

The sensitivity for identifying moderate to severe vertebral fractures (corresponding to Genant grade 2 and 3 fractures ) is substantial higher ranging from 90 to 94%.

Specificity is high, with a result between 94 to 99%, compared to conventional radiography.

Morphometric and visual identification using DXA images in children can be especially problematic because currently available software cannot detect the vertebrae in most children. That’s why we use total body DXA evaluation in children.

One advantage of DXA imaging is that the scan are not subjected to the same degree of projection distortion as conventional radiography because the X-ray beam is always orthogonal to the spine. Reducing the X ray diffraction effect.

Moreover DXA reduces the frequency that soft tissue obscure the endplates compared to single energy mode.

Side-by-side viewing facilitates the identification of incidental vertebral fractures.

Morphometrical analysis

It uses the measurement of vertebral height to define vertebral fractures. A normative daatabase is established against which the vertebrae are compared, There are a number of different morphometric approaches that vary with the criteria by which they define a vertebral fracture and in the reference data used, The most widely used approaches to identify prevalent and incidental vertebral fractures are the two different algorithms proposed by McCloskey et al (3) and Eastell R et al (4). Morphometric analysis has a high sensitivity and moderate high specificity in discriminating between normal vertebrae and fractured vertebrae. Moreover, all the morphometric approaches for defining prevalent and incident vertebral fractures are correlated with clinical risk factors for vertebral fractures.

The approach used can ahve a significant impact on the prevalence of vertebral fractures identified, varying from 3% to 90%.

A loss of vertebral height of 20 to 25 % is usually used to define an incident vertebral fracture; using this definition comparable ability to identify any vertebral frcture is present irrespective of aproach used to define a baseline fracture.

As mentioned above VFA is more effective in identifying moderate to severe deformities with a sensitivity of 81.6% for grade 2 deformities, whereas mild grade 1 deformities identifiation has a sensitivity as low as 22%.

Finally , the precision error is small if compared with the reduction in vertebral height of 20 to 25% threshold used to define vertebral fractures and it is less using conventional radiology than using VFA.

Semiquantitative analysis

SQ analysis combines measurements of vertebral height with subsequent evaluation of all vertebrae with a short vertebral height by an expert reader. This combined approach enables the identification of non-osteoporotic fracture vertebral deformities, which are not identified using morphometric analysis alone. As a consequence, SQ analysis is able to reduces false positive results.

The most widely used SQ analysis is that of Genant HK (6). Baseline or prevalent vertebral fractures are graded from “0” equal to normal to “3” equal to severe fracture, and incident fractures are defined as an increase of more than or equal than 1 grade on follow-up radiographs.

Genant grade 1 corresponds to an 20 to 25% reduction in anterior, middle or posterior height

Genant grade 2 corresponds to a 25 to 40% reduction in any height

Genant grade 3 corresponds to more than 40% reduction in any vertebral height

Mild grade 1 SQ vertebral deformities are frequently not associated with low BMD values.

The interobserver agreement for conventional radiographs or DXA images is similar with a K score of 0.53 and 0.51 respectively.

This approach is currently those recommended by International Society of Clinical Densitometry for diagnosing vertebral fractures with VFA.

Visual Identification by an expert reader (Algorithm Based Qualitative Approach)

ABQ approach differs from SQ analysis because the last one is based only on variations of vertebral height; not considering variations on endplates cracks or breaks as the primary event with a subsequent evaluation of vertebral height. ABQ focus more attention on the vertebral endplate alterations rather than on short vertebral height. Using ABQ we have a greater association with low BMD and interobserver agreement for radiography and DXA images of 0.74 and 0.65 respectively. So that mild vertebral fractures identified with ABQ are more strngly associated with osteoporosis than when this mild fractures are identified with SQ method.

The definition of vertebral fractures includes the presence of breaks in the cortex of vertebral body; these breaks always occurs in the center or either the superior or inferior endplates that are the weakest area of endplate because it is more distant from the strong outer vertebral ring. As a consequence, the endplate buckles or collapses under pressure because of interventebral disc and it results in a concave appearance to the superior and/or inferior endplate. If the concavity extend beyond the inner border of the vertebral ring , it is unlikely to represent an osteoporotic fracture. A vertebral fracture initially involves a crack of the superior or inferior endplate with or without the simultaneous loss of vertebral height. As severity of the fracture progresses, the vertebral ring fractures resulting in loss of height and buckling of the anterior, lateral and occasionally posterior cortex. It is important to outlined these aspects because there is considerable variation in vertebral shape resulting in osteoporotic and non osteoporotic deformities that can result in considerable intraobserver error even among expert readers.

Commonly we can see wedge deformity fracture associated with endplate fracture where is present a fracture of the anterior cortex of vertebral body.

A true compression fracture associated with endplate fracture is an osteoporotic compression fracture of superior endplate associated with fracture of anterior and posterior cortex of vertebral body.

It is also important to identify the different characteristics of high trauma burst fractures. There is usually an history of high trauma injury (such as a car accident, fall from a significant height) immediately resulting in acute, severe, localized back pain with localized tenderness.

Also the presence of intravertebral edema during MRI, used in the studies under discussion is not universally accepted target of vertebral osteoporotic fracture. Such as CT scan, also MRI, are usually only required in the presence of localized pain, focal neurological signs, or symptoms suggesting cord compression or a radiculopathy, or the clinical suspicion of primary or metastatic lesions, but not in osteoporotic patients.

Osteoporotic fractures are rare above T4 and in this settings, it is very important to consider metastatic lesions and , if appropriate to investigate for a primary neoplastic lesion.

Variations in shape or size of the vertebrae or of the vertebral endplate can be caused by degenerative diseases (osteoarthrosis), congenital deformities and metastatic lesions.

In addition, the aging skeleton, particularly in women, may develop slight wedging because of remodelling without depression or break in the endplate or cortex.

Scheuermann’s disease is frequently associated with a short anterior vertebral height in combination with irregularity of the whole superioe and/or inferior endplates. It appears to be isolated in a single vertebral body or involving adjacent vertebrae.

Schmorl’s nodes consist of a rounded flash-like break in the superior or inferior endplatein either the anteroposterior or lateral view, which rarely affects more than 25% of the endplate. They are found in about 35-75% of population and are formed by extrusion or erniation of the nuclear material from the interventebral disk into the vertebral body.

Degenerative osteomalacic changes are present in a vertebral body involved by an uniform or symmetrical concavity of the superior and inferior endplates. It is associated with a generalized thinning and reduced density of all vertebral bodies.

Clinical recommendations for screening for vertebral fractures

The current recommendations for using fracture assessment through DXA imaging (VFA) by the International Society of Clinical Densitometry are:

1. When the results may influence clinical management
2. If BMD is indicated then consider performing VTA if clinically indicated in:

- Documented height loss greater than 2 cm

- Historical height loss greater than 4 cm since young adult

- History of fracture after 50 years old

- Commitment to long term oral or parental glucocorticoid therapy

- History or findings suggestive of vertebral fracture not documented by previous radiographic imaging

Althoough risk factors can provide guidance to identify which patients require screening for osteoporosis, very few subjects would be prepared to initiate long-term therapy to prevent a fracture without confirmation of a diagnosis of osteoporosis using DXA scan.

If a patient has osteopenia and a fragility fracture at any site, the majority of physicians would intervene with therapy.

Therefore, it is reasonable to screen all patients with osteopenia using VFA, if it will alter the management of the patient. In a study at Mayo Clinic 16% of patients 60 to 69 years old and 45% of those older than 70 years had a previously undiagnosed vertebral fracture on VFA.

Fracture Risk Quantification

Concurrent considerations of risk factors that operate independently of BMD improuve evaluation of fracture risk.

The best example is age. The same T-score (i.e. the number of Standard Deviations from BMD found on people 35 years old) has different significance at different ages. For any BMD value, fracture risk is much higher in the elderly than in the young people. This is because age contributes to fracture risk indepently of BMD.

In general, risk factor scores show relative poor specificity and sensitivity in predicting either BMD or fracture risk. However, some risk factors vary in importance according to age. For example risk factors for falling, such as reduced mobility, sedatives use, visual impairment are more strongly predictive of fracture in the elderly than in younger individuals.

A series of meta-analyses has been undertaken to identify clinical risk factors that could be used in case finding strategies with or without the use of BMD measurement:

1. Low body mass index (BMI): A low BMI is a significant risk factors for hip fracture. 20 kg/m2 vs 25 kg/m2 shows a RR of 1.27, whereas 30 Kg/m2 vs 25 Kg/m2 shows a RR of 0.89.
2. Fragility fracture after 50 years of age : RR 1.86. In other words the presence of prior vertebral fracture approximately doubles the risk of having another fracture.
3. Parental history of hip fracture : RR 1.54 an increase in risk independent from BMD value.
4. Smoking is a RR 1.29
5. Ever use of corticosteroids: RR 1.65
6. Alcohol intake shows an increase in fracture risk that is dose dependent. Where the alcohol intake is on average 2 Units or less daily,there is no increase in risk. Intakes of 3 or more Units daily are associated with a dose-dependent increase in risk. RR 1.38
7. Rheumatoid Arthritis RR 1.56. In contrast to many causes of secodary forms of osteoporosis rheumatoid arthritis causes an increase in fracture risk indenpendently of BMD and the use of glucocorticoids.

The multiplicity of these risk factors poses problems in the units of risk to be used. Also if the Relative Risk can be used, the best suited for clinician is the absolute risk ( or probability ) of fracture. The absolute risk dependes on age and life expenctancy, as well’s from the current relative risk. In general lifetime risk of fracture decreases with age, in particular after 70 years of age, because the risk of death with age outweight the increasing incidence of fracture. Estimates of lifetime risk are less relevant in assessing individual clinical risk of fracture in order to choose a therapeutic intervention. So that it has been recommended the use of a short term absolute risk ( i.e. a probability over 10-year interval), 10 years interval covers the likely duration of treatment and average life expectancy in elderly 60 year or older.

What about heart failure ?

Recently Heart failure has been identified such a risk factor for fragility fractures in two large studies by Ezekowitz JA on 2008 and by Carbone L on 2010.

Heart Failure is a leading cause of hospitalization and mortality in Europe and North America. Successfully enhanced treatment rates of hypertension and survival after myocardial infarction have produced a delay in the incidence in heart failure. So that the median age of heart failure patients in clinical trials and large epidemiological studies ranges between 65 and 75 years of age. Such patients are notably at risk for other co-morbid conditions causally related or not such as bone fracture.

Osteoporosis is one of such co-morbidity affecting 1 in 4 women and 1 in 8 men over 50 years old and it is known to be clinically evident with fragility fractures.

A central unanswered question is now evident: does heart failure lead to osteoporosis and fragility fractures or is it a passive participant in a population at risk of both diseases ?

A first answer comes on 1997 in a description of 101 patients with endstage heart failure awaiting cardiac transplantation, low bone mass was common, as vitamin D deficiency and Hyperparathyroidism.

What are the possible links between osteoporosis, fragility fractures, and heart failure ? Many scientists point to shared risk factors for both diseases such as older age, smoking, diabetes, renal dysfunction, inactivity, and poor nutrition.

An interesting role in linking bone loss to heart failure should be hyperaldosteronism always present in heart failure.

Elevated aldosterone levels have been associated with urinary magnesium and calcium wasting, causing secondary hyperparathyroidism.

Heart failure patients treated with spironolactone, a known aldosterone antagonist, showed few fractures compared to matching heart failure affected patients. It is not established if the consequent reduction in hyperparathyroidism is the cause of increased bone repair, increased mineralization, or mineral retentions in particular calcium and magnesium.

Future researches are needed in order to examine biomarkers, imaging, and clinical outcomes related to bone health after carefully clinically phenotyping patients with heart failure.

References

Cameron JR, Sorenson J. Measurement of bone mineral in vivo: an improved method. Science 1963;142:230-2.

Smith R, Walker R. Femoral expansion in aging women: implications for osteoporosis and fractures. Science 1964;217:945-8.

Tinetti ME. Clinical Practice. Preventing falls in elderly persons. N Engl J Med 2003;348:42-9.

Ahlborg HG, Johnell O, Turner CH et al. Bone loss and bone size after menopause. N Engl J Med 2003;349:327-334.

Khosla S, Melton LJ III. Osteopenia. N Engl J Med 2007;356:2293-300.

Abendschein W, Hyatt GW. Ultrasonic and selected physical properties of bone. Clin Orthop 1970;69:294-301.

Ahlborg HG, Nguyen ND, Nguyen TV et al. Contribution of hip strength indeces to hip fracture risk in elderly men and women. J Bone Min Res 2005;20:1820-27.

Bolotin HH, Sievanen H. Inaccuracies inherent in dual-energy X-ray absorptiometry in vivo bone mineral density can seriously mislead diagnostic/prognostic interpretations of patients-specific bone fragility. J Bone Miner Res 2001;16:799-805.

Looker AC, Beck TJ, Orwoll ES. Does body size account for gender differences in femur bone density and geometry? J Bone Miner Res 2001;16:1291-9.

Richards JB, Leslie WD, Joseph L et al. Changes to osteoporosis prevalence according to method of risk assessment. J Bone Miner Res 2007;22:228-34.

Lewis CE, Ewing SK, Taylor BC et al. Predictors of non-spine fracture in elderly men: the MrOS study. J Bone Miner Res 2007;22:211-9.

Mussolino ME, Looker AC, Madans JH et al. Risk fators for hip fracture in white men: the NHANES I epidemiologic follow up study. J Bone Miner Res 1998;13:918-24.

Melton LJ III, Atkinson EJ, O’Connor MK et al. Bone density and fracture risk in men. J Bone Miner Res 1998;13:1915-23.

Amin S, Zhang Y, Felson DT et al. Estradiol, testosterone, and the risk for hip fractures in elderly men from the Framingham study. Am J Med 2006;119:426-33.

McCloskey EV, Spector TD, Eyres KS et al. The assessment of vertebral deformity: A method for use in population studies and clinical trials. Osteoporosis Int 1993;3:138-47.

Eastell R, Cedel SL, Wahner HW et al. Classification of vertebral fractures. J Bone Miner Res 1991;6:207-15.

Genant HK, Jergas M, Palermo L et al. Comparison of semiquantitative visual and quantitative morphometric assessment of prevalent and incident vertebral fractures in osteopororsis. The Study of Osteoporotic Fractures Research Group. J Bone Miner Res 1996;11:984-96.

Rea JA, Li J, Blake GM et al. Visual assessment of vertebral deformity by X-ray absorptiometry: A highly predictive method to exclude vertebral deformity. Osteoporosis Int 2000;11:660-8.

Siminoski K, Jiang G, Adachi JD et al. Accuracy of height loss during prospective monitoring for detection of incident vertebral fractures. Osteoporosis Int 2005;16:403-10.

Siminoski K, Warshawski RS, Jen H et al. The acuracy of historical height loss for the detection of vertebral fractures in postmenopausal women. Osteoporosis Int 2006;17:290-6.

Schousboe JT, Ensrud KE, Nyman JA et al. Cost-effectiveness of vertebral fracture assessment to detect prevalent vertebral deformity and select postmenopausal women with a femoral neck T-score lower than 2.5 SD for alendronate therapy: A modeling Study. J Clin Densitom 2006;9:133-43.

Hiu SL, Slemenda CW, Johnston CC. Age and bone mass sd predictor of fracture in a prospective study. J Clin Invest 1988;81:1804-9.

Kanis JA, McCloskey EV. Evaluation of the risk of hip fracture. Bone 1996;18:127-32.

Kanis JA, Johnell O, Oden A et al. Ten-year risk of osteoporotic fracture and the effect of risk factors on screening strategies. Bone 2001;30:251-8.

Kanis JA. Diagnosis of osteoporosis and assessment of fracture risk. Lancet 2002;359:1929-36.

Shane E, Mancini D, Aaronson K et al. Bone mass, vitamin D deficiency, and hyperparathyroidism in congestive heart failure. Am J Med 1997;103:197-207.

Jankowska EA, Jakubaszko J, Cwynar A et al. Bone mineral status and bone loss over time in men with chronic systolic heart failure and their clinical and hormonal determinats. Eur J Heart Fail 2009;11:28-38.

Van Diepen S, Majumdar SR, Bakal JA et al. Heart failure is a risk factor for orthopedic fracture: a population-based analysis of 16.294 patients. Circulation 2008;118:1946-52.

Carbone L, Buzkova P, Fink HE et al. Hip fractures and heart failure: findings from the Cardiovascular Health Study. Europ Heart J 2010;31:77-84.

Carbone LD, Cross JD, Raza SH et al. Fracture risk in men with congestive heart failure risk reduction with spironolactone. J Am Coll Cardiol 2008;52:135-8.

Klotho more than “ninphae”

        

    

 

From Greek mythology “Klotho” was one of three Moirae. She is responsible for spinning the thread of human life, so that she mades major decisons when a person is born. She controls who born through his life she also decide who has to be saved or put to death.

Two other sisters , Lachesis and Atropos, are responsible of human destiny and influence their misery and suffering.

Clotho assisted Hermes to create the alphabet, and forced the goddess Afrodite into making love with other gods, killed the Titan Typhon with poison fruits and persuaded Zeus to kill Asclepius with a bolt of lightning.

As you know Asclepius, the Roman Esculapius, is the god of Medicine and Healing. Asclepius has a daughter Epione ( Goddess of soothin of pain) and he his the father of : Hygieia (Hygiene), Panacea (Universal Remedy), Aceso (Goddess of Healing process), Leso or Laso (Goddess of recuperation from illness), Aglaea or Aglaia (Shining one, splendor, brillant, Healthy Glow) wife of Hephaesto and mother of Eucleia (good repute), Eupheme (Acclaim), Euthenia (Prosperity), Philophrosyne (Welcome).

The rod of Asclepius, a snake-entwined staff, remains a symbol of medicine today, also if sometimes a staff with two snakes (the caduceus) is mistakenly used instead.

He was one of the Apollo’s sons.

Asclepius

God of Medicine

Is KLOTHO, an anti-ageing hormone ?

The KLOTHO gene was identified serendipitously through a hypomorphic allele that results in severe early degenerative changes and short lifespan ( Nature 1997). The homozygous mutant animals develop normally until 3 weeks of age, then exhibit severe growth retardation, osteoporosis, ectopic calcification, aterosclerosis, emphysema and atrophy of the skin, thymus, testes and ovaries, and die at an average age of 61 days.

KLOTHO prolong lifespan at least in part by inhibiting insulin-IGF-1 signalling. KLOTHO may be secreted by KIDNEY cells blocking both IGF-1 and Insulin receptor action at adipocytes and target tissue levels.

Levels of calcium and phosphate are elevated in KLOTHO deficient mice, demonstrating that this protein has a role in calcium and phoshate homeostasis. The altered calcium and phoshate levels are due to elevated 1,25 OH Vitamin D3 levels, which result from increased expression of 1alfa hydroxylase gene activity at kidney level.

Normalization of 1,25 OH Vitamin D3 with a Vitamin D deficient diet partially rescued some of the KLOTHO deficient phenotypes, including slow growth, ectopic calcification and early death. This suggests that the putative pro-ageing effect of KLTHO deficiency is not associated with ageing itself, but rather that the pathology is related to altered Vitamin D metabolism.

FGF23 KO mice share many features with the KLOTHO deficient mouse, including hypercalcemia, hyperphosphatemia, ectopic calcification, hypoglycaemia, infertility, and very short lifespan.

FGF23 is a circulating factor that is produced in the bone and inhibits phosphate transport in renal proximal tubular cells. FGF23 deficiency results in phosphate retention and hyperphosphatemia, constitutively elevated expression of 1 alfa hydroxylase, elevated levels of 1,25 OH Vitamin D3 and hypercalcaemia.

Interestingly without KLOTHO the functions of FGF23 is literally abolished.

Many tissues express FGF Receptors subtypes that interact with the KLOTHO-FGF23 complex, therefore it is possible that KLOTHO exerts his anti-ageing action through the activation of his enzymatic activity i.e. beta glucoronidation.

We know that all steroids enzymes including all lipophilic vitamins such as Vitamin D, with a steroid like structure, can be glucuronized in order to achieve a better hydrophility and flow into the blood vessels. The pool of glucuronide-linked steroids hormones is an inactive quote of hormones, those destiny is in normal condition to be metabolized further into hepatic cells or recycled by endocrine organs.

KLOTHO can hydrolyse STEROID GLUCURONIDES, including estradiol, estrone, estriol and vitamin D so that some effects of KLOTHO can occur through processing of inactive streroid glucuronides to active steroids hormones. Steroid hormones could have a role in the regulation of ageing in mammals.

 

 

References

Arking DE, Krebsova A, Macek M Sr et al. Association of human aging with a functional variant of klotho. Proc Natl Acad Sci USA 2002;99:856-61.

Duce JA, Podvin S, Hollander W et al. Gene profiling analysis implicates klotho as an important contributor to aging changes in brain white matter of rhesus monkey. Glia 2008;56:106-117.

Arking DE, Becker DM, Yanek LR et al. KLOTHO allele status and the risk of early-onset occult coronary artery disease. Am J Hum Genet 2003;72:1154-61.

Mitani H, Ishizaka T, Aizawa T et al. In vivo klotho gene transfer ameliorates angiotensin II-induced renal damage. Hypertension 2002;39:838-43.

Ogata N, Matsumura Y, Shiraki M et al. Association of klotho gene polymorphism with bone density and spondylosis of the lumbar spine in postmenopausal women. Bone 2002;31:37-42.

Yamada Y, Ando F, Niino N et al. Association of polymorphisms of the androgen receptor and klotho genes with bone mineral density in Japanese women. J Mol Med 2005;83:50-7.

Mullin BH, Wilson SG, Islam FM etr al. Klotho gene polymorphisms are associated with osteocalcin levels but not bone density of aged postmenopausal women. Calcif Tissue Int 2005;77:145-51.

Kawano K, Ogata N, Chiano M et al. Klotho gene polymorphisms associated with bone density of aged postmenopausal women. J Bone Miner Res 2002;17:1744-51.

Kuro-o M, Matsumura Y, Aizawa H et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 1997;390:45-51.

Roth GS, Lane MA, Ingram DK et al. Biomarkers of caloric restriction may predict longevity in humans. Science 2002;297:811.

Kurosu H, Yamamoto M, Clark JD et al. Suppression of aging in mice by the hormone Klotho. Science 2005;309:1829-33.

Chang Q, Hoefs S, van der Kemp AW et al. The beta glucuronidase Klotho hydrolyzes and activates the TRPV5 channel. Science 2005;310:490-3.

Russell SJ, Kahn CR. Endocrine regulation of aging. Nat Rev Mol Cell Biol 2007;8:681-91.

Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science 1996;273:59-63.

Ingram DK, Cutler RG, Weindruch R et al. Dietary restriction and aging: the initiation of a primate study. J Gerontol 1990;45:B148-B163.

Sohal RS, Argarwal S, Candas M et al. Effect of age and caloric restriction on DNA oxidative damage in different tissues of C57BL/6 mice. Mech Ageing Dev 1994;76:215-24.

Lee C-K, Klopp RG, Weindruch R et al. Gene expression profile of aging and its retardation by caloric restriction. Science 1999;285:1390-3.

Kujoth GC, Hiona A, Pugh TD et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science 2003;309:481-4.

Weindruch R, Sohal RS. Caloric intake and aging. N Engl J Med 1997;337:986-94.

Priè D, Friedlander G. Genetic disorders of renal phosphate transport. N Engl J Med 2010;362:2399-2409.

High Resolution peripheral Quantitative Tomography and bone quality.

 

  

 

 

Bisphosphonates

Bisphosphonates (BP) are synthetic analogues of inorganic pyrphosphate with a central carbon instead of an oxigen element that protect BPs from biological degradation.

P – O – P inorganic pyrophosphate

P – C – P bisphosphonates

Thos P-C-P backbone is identical across all types of BPs, and two sidechains account for their biological diversity: a hydroxyl residue at the R1 side chain enhances the affinity to bone, whereas nitrogen residues at the R2 side chain account for their potency, mechanism of action, and side effects. Developed and traditionally used to soften water in irrigation systems in the 19^th century, BPs were introduced into clinical medicine in the 1970s and 1980s in the treatment of Paget’s disease of bone and hypercalcemia of malignancy. Bisphosphonates (BP) may act via many signalling pathways, some of which are specific for a given BP.

First of all two groups of BPs have been identified to date acting in a different ways:

1. Non-amino bisphosphonates act through ATP block producing toxic analogs of ATP and causing cells death.
2. Amino bisphosphonates act through inhibition of an enzyme called farnesyl pyrophosphate synthase, an enzyme present in in the 3 hydroxymethyl glutaryl Co A reductase pathway.

Other oossible intracellular pathways have been proposed some of wich are specific for some bisphosphonates. These includes:

- Altering key apoptotic proteins, specifically increasing BAX and decreasing Bcl-2.

- Activating mitochondrial pathway via translocation of apoptosis iducing factor.

- Inhibiting mitochondrial adenine nucleatide translocase (ANT), known to be involved in causing apoptosis

- Inducing ApppI ( an densoine triphosphate analog), which triggers direct apoptosis through blockade of mitochondrial ANT.

- Inhibiting metalloproteinases necessary for proteolytic degradation of the extracellular matrix (ECM).

- Inhibiting cancer cell adhesion ( ICMA-1, VCAM-1) and prevents cancer cells spreading at lower concentration than those required to cause apoptosis.

Main characteristics of BPs are:

- Poor oral bioavailability

- High affinity for, and accumulation in bone

- Target FPP synthase in osteoclasts

- Efficiency across a broad spectrum of osteoclast mediated diseases

Stimulated by the launch of alendronate, the first potent oral aminobisphosphonate, the mechanisms of this drug class were elucidated in the ’90. After parenteral and oral administration in which less than 1% is absorbed, BPs bind to hydroxyapatite crystals and concentrate at skeletal sites where active remodeling takes place. Following embedding into the skeleton, BPs inhibit osteoclasts activity and, under acidic conditions in resorption lacunae, are incorporated into osteoclasts.Nitrogen-containing BPs, the most widely used class of antiosteoporosis drugs, which includes alendronate, risendronate, ibandronate, and zolendronic acid interfere with the mevalonate pathway and inhibit Farnesyl Pyrophosphate synthase (FPPS). FPPS is the enzyme that generates Farnesyl Pyrophosphate and Geranyl-geranyl Pyrophosphate (GGPP), essential for post-translational isoprenylation reaction of small GTPases. These enzymes are able to modulate and coordinate subcellular protein trafficking, cell survival, and cytoskeletal integrity (called Ras, Rho, Rac, Rap).The potency of BPs depends upon the inhibitory effect on FPP synthase activity and the affinity for mineral bone.

In theory, the enzyme hydroxymethy glutaryl (HMG) Coenzyme A reductase inhibitors ( also called “statins”), used usually in therapy for reducing plasma cholesterol, which inhibit the production of mevalonate, also have an osteotropic effect. Due to their lipophilic properties, they preferentially target the liver, but not the skeletal tissue. Inhibition of FPPS by BPs results in decreased osteoclast activity, enhanced osteoclast apoptosis and a profound antiresorptive affect. A relevant finding obtained from bone biopsies of patients treated for long term with bisphosphonates is the increased number of giant, hypernucleated osteoclasts that are detached from bone lacunae and undergone slowly a protracted apoptotic process. (see Manolagas NEJM ). Apart from their specific antiosteoclastic activity, BPs protect osteoblasts and osteocytes against apoptosis, enhancing osteoblastic differentiation, and increasing osteoblastic production of Osteoprotegerin.Since the first BP, alendronate, was approved in 1995 these agents have been the first therapy for treating postmenopausal osteoporosis, male osteoporosis, glucocorticoid-induced osteoporosis, Paget’s disease of bone, Hypercalcemia of malignancy, multiple myeloma of bone, and skeletal metastases.

Bone loss associated with aromatase inhibitor therapy in women with breast cancer, which is associated with very low estrogen levels, has been treated with zolendronic acid administered twice per year.In malignant skeletal diseases, intravenous BPs ( such as zolendronic acid 4 mg, or pamidronate 90 mg) are administered every 4 weeks or also more frequently. The shorter therapy interval is required in order to control excessively enhanced bone resorption in malignant conditions. Under this regimen, the rate of side effects is considerably higher, including renal toxicity and the development of osteonecrosis of the jaw (ONJ), particularly in patients with myeloma or breast cancer following dental procedures. The reported decrease in hip fracture rates in long term clinical studies reported for osteoporosis treatment results from multiple factors. BP effectively reduce fracture risk in postmenopausal women over a period of at least 10 year, but preclinical studies demonstrated that they also negatively affect bone quality.

Bone quality

Not all fractures have the same pathogenesis or structural abnormalities that cause bone fragility. Some fractures are associated with reduced tissue mineral density; in others, there is a reduced density of osteocytes.Women with fractures may have high, normal, or low rates of remodeling. Some women with fractures have a negative balance in the bone multicellular units owing to reduced bone formation, increased bone resorption, or both; other women with fractures have no negative balance in the bone multicellular unit balance.The heterogeneity of mechanisms suggests that all patients with fragility fractures should not be treated in the same way.In most postmenopausal women, the remodeling rate is high; in other words a large number of bone multicellular units excavate cavities while other units are at various stages involved in the completion of remodeling. When an antiresorptive agents is given, this steady state is perturbed. The birth rate of new bone multicellular units decreases quickly when treatment is started, whereas the many bone multicellular units at various stages in the remodeling cycle complete the remodeling process by depositing a volume of new bone that reduces the depth of the escavated site.The newly deposite bone undergoes primary mineralization during the deposition of osteoid (normally a rapid process) and then slower secondary mineralization with enlargement of newly yet formed crystal occurred thereafter.The increased tissue mineral density and reduced porosity slightly improve bone strength. During treatment with antiresorptive agents, the slow remodeling rate and the reduced depth of a decreased number of excavated sites produces bone loss and structural decay also if more slowly than before, and bone fragility reemerges.Fractures continue but are less frequent than in untreated controls with a rapid remodeling and a negative balance in bone multicellular units exponentially increase bone fragility.

Antiresorptive agents finally slow the progression of fragility by suppressing the rate of remodeling and reducing the depth of resorption in each of the reduced number of bone multicellular units engaged into remodeling bone.Since remodeling is slow during treatment with antiresorptive agents, more time is available for secondary mineralization of new mineral bone both in sites actively resorbing before drug exposure and in sites distant from endosteal surface. So that slower remodeling allows increased bone mineral density with more homogenous distribution of mineral between adjacent regions.

However greater secondary mineralization rate increases tissue stiffness, thereby predisposing to bone microdamage.

Whereas the greater homogeneity in tissue density offers less resistance to the propagation of cracking.

Reducing remodeling may also reduce removal of microdamage in bone.

Studies in dogs shows a nearly 30% decline in material toughness ( the normalized energy to fracture ) over 3 years of reatment at doses that stimulate those used in treating osteoporosis in postmenopausal women. This creates a material more brittle that untreated bone, facilitating microdamage, which, combined with the natural suppression of remodeling to repair it, significantly increases its burden in bone.Micro-damage accumulation is likely a consequence of the increased brittleness and reduced toughness, not its cause.

HR-pQCT

Advances in non invasive techniques are likely to provide insights into the effects of these therapeutic agents on bone structure and increasingly accurate information concerning the structural heterogeneity of bone fragility from patient to patient and so may improve the sensitivity of the prediction of fracture risk.Whereas DEXA has become the most commonly used technique worldwide to predict fracture risk and assess response to therapy, based on a two dimensional interpretation of skeletal tissue; it’s increasingly evident that it provides limited interpretation of three dimensional skeletal properties and so on its structural charateristics.Other imaging modalities such as CT and MRI offer considerable greater charaterization of bone architecture, but their software and technical evolution has not been validated until recently.

High Resolution three dimensional peripheral QuantitativeTomography (HR-pQCT) device has been developed by Xtreme CT, Scanco Medical AG, Bassersdorf, Switzerland in order to provides measures of bone microachitecture and micro Finite Element analysis software for numerical quantification of mechanical properties of bone in vivo.In the following diagram we can see the difference between osteopenic postmenopausal women and osteporotic women with fracture using DXA (BMD) and HR-pQCT parameters. Clearly the differences between two groups is more evident with the later technique.

 

This system, first described in detail by Andreas Laib on 1998, uses a two dimensional detector array in combination with a 0.08 mm point-focus X ray tube, enabling the simultaneous acquisition of a stack of 116 high-resolution parallel CT slices, using an effective energy of 40 keV, X-ray tube current of 95 mA, slice thickness of 89 μm, field of view of 90 mm, image matrix of 1536 x 1536 pixels, and pixel size of 82 μm (voxel size).Older 3-D pQCT devices used before have a voxel size of 165 μm, the need to have an higher resolution value is due to adequately solve the distance between the trabecular ridges ( about 300-500 μm) and not necessary to resolve individular trabeculae (100 μm or less ). At each site 110 CT slices were obtained, thus delivering a three dimensional representation of about 9 mm in the axial direction. The arm of leg of the patient was immobilized during the examination in an anatomically formed carbon fiber shell. An anteroposterior scout view was used to define the measurement region. Briefly, a reference line was manually placed at the endplates of the radius and the tibia. The first CT slice was 9.5 mm and 22.5 mm proximal to the reference line for the distal radius and tibia , respectively. The effective dose was less than 3 microSievert per measurement with a measurement time of 2.8 minutes.Quality control, based on Shewart rules, was monitored by daily scans of a phantom containing rods of HA ( densities of 0, 100, 200, 400, 800 mg HA/cm³ ) embedded in a soft tissue equivalent resin (QRM Moehrendorf, Germany).The entire volume of interest was automatically separated into a cortical and trabecular region using a threshold based algorithm. The threshold used to discrimite cortical from trabecular bone was set to one third of the apparent cortical bone density value (D cort). Mean cortical thicknes (CTh) was defined as the mean cortical volume diveded by the outer boe surface.Trabecular bone density (D trab) in gHA/cm³ was compouted as the average mineral density whitin the trabecular volume of interest.Trabecular bone volume (BV) fraction (BV/TV Trabecular volume %) was then expressed from trabecular density assuming fully mineralized bone to have a mineral density of 1.2 gHA/cm³

BV/TV %= 100 x 1200 mgHA/cm³

Because the thickness of every trabeculae cannot be measured accurately because of partial volume effects, a thickness independent algorithm was used to assess trabecular structure.First, a mid-axis transformation method was used to identify trabecular elements and the distance between them assessed threedimensionally using the distance transform method. Trabeculae cannot be resolved at their correct thickness because of partial volume effects, to avoid this problem the center point of every trabecula is detected in the gray-level image and called the 3-D ridges. Trabecular number is taken as the inverse of the mean spacing of the ridges.Trabecular number (TbN, mm^-1) was defined as the inverse of the mean spacing of the mid-axes and is thus truly three-dimensional and it does not depend on “a priori” assumptions regarding the plate or rod-like nature of the underlying nature.

TbN = 1/Mean Tb space = nTb/mm

Trabecular thickness (TbTh, μm) and separation (TbSp, μm) were derived from BV/TV and TbN using a standard methods from histomorphometry.

TbTh = (BV/TV)/TbN

TbSp = (1-BV/TV)/TbN

Distance transformation techniques also enable the calculation of intra-individual distribution of separation (TbSp SD, μm) quantified bt the umeber of Standard Deviation (SD) of the separation mean, a parameter reflecting the heterogeneity of trabecular network.For follow-up measurements, an algorithm automatically uses the cross-sectional area (CSA, mm² ) within the periosteal boundary of the radius and tibia to match the volumes of interest (VOI) on the baseline and fllow-up scans, and thus only the bone volume common to previous scans is used to assess density and microarchitectural measurements.Thus, of the initial 110 slices, on average 103 (range 93 to 108) were analyzed in the follow-up scans.

The outcome variables used in this analyses included volumetrical bone density (gHA/cm³) for entire (D_tot), trabecular (D_trab), and cortical (D_cort) regions; cortical thickness (CTh, μm), trabecular bone volume fraction (BV/TV, %), trabecular thickness (TbTh, μm), trabecular number (TbN*, mm^-1), trabecular separation (TbSp, μm), and intra-individual distribution of separation (TbSp SD, μm).

Interestingly trabecular and cortical densities obtained with HR-pQCT are only moderately related to each other and trabecular density is strongly correlated to trabecular achitectural measurements at both distal radius and tibia. On the contrary, cortical density is higly correlated with cortical thickness but weakly correlated with trabecular architecture in normal subject.Postmenopausal osteopenic and osteoporotic women show density and architectural parameters significantly different, with the exception of cortical density. Compared with those classified as osteopenic, osteoporotic women have lower bone density, decreased trabecular number and trabecular thickness, increased trabecular separartion and intra-individual distribution of separation, and decreased cortical thickness. Osteopenic women with and without an history of fracture did not differ with regard to BMD (measured with classic Hologic densitometr) at lumbar spine and femoral neck, nor in HR-pQCT measurements at the distal tibia. However, at the distal radius density and architectural parameters were significantly different in women with an history of fracture compared with those with no previous fractures.In men trabecular bone volume declines similarly as women over life, however the microstructural basis for the decrease in trabecular volume differ between sexes. In women there appear to be loss of trabeculae with decrease in trabecular number and incresed intertrabecular space, whereas in men the primary mechanism for the decrease in trabecular volume is trabecular thinning.

This mechanism in turn is likely to have a significant impact on age related changes in bone strength in women compared to men, because the reduction in trabecular number has a 2 or 5 times greater impact on bone strength compared with reduction in trabecular thickness that result in similar decreases in bone volume.

Micro-finite element analysis using HR-pQCT

Micro-finite element analysis (μFE) tecniques applied to HR-pQCT data sets provide an estimate of bone mechanical competence (stiffness) that distinguishes between groups of subjects with and without fractures.Each subvolume of HR-pQCT image oof the distal radius and distal tibia is converted to a micro-finite element (μFE) with an element size of 82 x 82 x 82 μm³.The HR-pQCT measurement, as writed above, include 116 slices, corresponding to a 9.02 mm sections along the axial skeleton, with a nominal voxel size of 82 μm.The mineralized phase was thresholded automatically, using Laplace-Hamming filter followed by a global threshold using a fixed value of 40% of the maximal grayscale value of the images.Using customized element-by-element pre-conditioned conjugate gradient solver, 6 μFEs were performed for each model, representing 3 uniaxial compression tests along 3 imaging axes and 3 uniaxial shear tests.The trabecular bone tissue is considered as an isotropic, linear elastic material with a Young’s modulus (E) of 15 Gpa and a Poisson’s ratio of 0.3 for all uniaxial model.The general anysotropic stifness of bone matrix is transformed into a new value through the calculation by means of appropriate algorithm, called Powell’s method, of full orthotopic stiffness tensor value by best orthotopic symmetry through the new chosed coordinate system formed by chosed 3 orthotopic axes ( X₁, X₂, X₃ ) representing the best orthotopic symmetry calculated using an optimization procedure.The elastic constants and stiffness matrix moduli were sorted so that E₁₁ was in the medial-to-lateral direction ( representing the lowest axial modulus), E₂₂ along the antero-posterior direction, whereas E₃₃ was in the direction of the highest axial direction.

Finally 6 elastic moduli were derived from the orthotopic system tensor value:

3 Young moduli: E₁₁ < E₂₂ < E₃₃ for unaxial compression tests

3 Shear moduli: G₂₃ < G₃₁ < G₁₂ for unaxial shear tests

Several studies have reported that HR-pQCT parameters discriminate between postmenopausal women with and without fractures, whereas BMD by DEXA did not.Melton LJ III and Delmas PJ groups reported that decreased vBMD, microstructure, and stiffness estimated by μFE of the radius are associted with forearm fracture in postmenopausal women.Patients studied with postmenopausal osteopenia, radius but not tibia HR-pQCT measurements discriminated between those with and without fractures. It is also important because tibia is a weight bearing bone and it would be predicted that mechanical loading would result in a relative sparing at this site. The study by Cohen A. confirms these data by providing the evidence of cortical and trabecular microarchitectural deterioration at both radius and tibia in premenopausal women with idiopatic osteoporosis, whether or not they have had fracture.Estimated stiffness was significantly lower in all directions at both radius and tibia. Noteworthy was the finding that trabecular bone microachitecture and stiffness were severely affected at radial site in women with low BMD (measured with standard Hologic densitometry) who had an adult low trauma fracture.

Bisphosphonates

The excessive suppression of bone remodeling by high doses of bisphosphonates is thought to compromise bone integrity by accumulation of microdamage (microcraks).However,the microdame ge accumulation has been demonstrated to peak during early period of high dose bisphosphonate treatment and the drugs does not continue to accumulate with longer treatment periods, Determinats of bone strength including ultimate load, stiffness, anergy to failure as well’s other material properties including bone maximum stress and modulus have been shown to be unaffected and preserved after three years of daily alendronate treatment also in preclinical animal models.The role of other material properties has been found to be altered by bisphosphonate treatment but they role in alteration of fracture stiffness is less evident. We talk mainly of alterations of bone mineralization quality, collagen ultrastructural quality, and mineral hydroxyapatite quality. Concerning the possible role of increased quantity of mineralized bone it is quite clear that higher bone mineralization is beneficial in increasing bone stiffness and reducing the incidence of new fractures at any site. The report of possible brittleness of new bone formed and the increasing report of new subtrochanteric and mid shaft femur fractures have not be considered osteoporotic fractures, so not related to disease treated by bisphosphonates, but related to intensity work load in a possible normal bone.

The change in bone tissue is more likely caused by larger accumulation of advanced glycation end-products, called AGEs and directly related to increased glucose levels.AGEs are the by-products of the formation of collagen cross-links by non-enzymatic processes, and naturally accumulates in bone as it ages. Undernormal bone turnover rates, AGEs are prevented from accumulating to high levels. When bone turnover is suppressed, however, they can accumulate, and laboratory studies show them to be associated with increased brittleness.The micro-damage accumulation, and possibly the build-up of AGEs in the bone extracellular matrix, can only be reversed by bone anabolic agents such as teriparatide.

Intravenous BPs and in particular zolendronic acid may be associated with hypocalcemia, renal toxicity, and an acute phase reaction with flulike symptoms during drugs infusion. The latter is thought to be due to extraskeletal effects of aminobisphosphonates, the release of cytokines from macrophages, and the activation of T lymphocytes linking to γδ T cell receptor (see NEJM letter).Accordingly, aminobisphosphonats, and in particular zolendronic acid, may also induce apoptosis in breast cancer cells, although the clinical relevance of this effect is not clear.We know that bone remodelling is a process involving T lymphocytes, bone marrow stromal cells, machrophages (antigen presenting cells) in a complex signalling pathway involving the activation of osteocytes, osteoblasts and finally osteoclasts through a signaling sequences very complexes and those more intensely studied require TNF alfa related factors and their receptors (RANK/RANKL/OPG). So that in any changes of bone turnover level a true inflammatory-like pathway is activated at bone marrow-trabecular interfaces.

According to my opinion all side-effects founded in long term studies using bisphosphonates and in particular after their parenteral administration one a months or yearly is due to an increase in local inflammatory tissutal answer, finally accounting for:

1. Osteonecrosis of the jaw (ONJ)
2. Atrial Fibrillation (AF)
3. Esophageal cancer
4. Musculoskeletal pain
5. Atypical fractures due to increased skeletal fragility at diaphyseal or subtrochanteric femur regions.

Since late 2003 there have been reports in the literature of a possible association between bisphosphonate use and the appearance of avascular necrosis of the jaw. Marx on 2003 described a group of 36 American patients who received either pamidronate or zoledronate iv for the management of bone disease associated with metastatic cancer, multiple myeloma and osteoporosis and who subsequently developed avascular necrosis of the jaws. In the majority of patients, the latter condition developed after dental extraction, but in about 30% of cases, it apparently occurred spontaneously.

Bisphosphonates related osteomyelitis (BON) and necrosis of the jaw possibly results from the inability of hypodynamic and hypovascular bone to meet an increased demand for repair and remodeling owing to physiological stress (mastication), iatrogenic trauma ( tooth extraction or denture induced local injury ), or tooth infection in a environment that is both trauma intense and plenty of bacteria.Cofactors may include the use of other medications with antiangiogenic properties such as glucocorticoids, diabetes mellitus, irradiation of jaw bone, peripheral vascular disease, hyperviscosity syndrome such as in multiple myeloma.Bisphosphonate related osteomyelitis (BON) is a true bone infection due to direct effect of bisphosphonates on bone turnover and subsequent physiolgical reaction even more increased if we look at region with increased work load and stress such as daily work activity we spend during mastication. A great work load per cm square is exerted on oral cavity bones so that these bone regions require a very intense answer by extracellular matrix structures.

Concerning the presence of Advanced glycation end products, we know that increasing the concentration of glucose, the link of glucose to all proteins present in our body increases from hemoglobin to proteins present in the ocular structures (both cornea and retinal epithelial cells). Glycation reactions changes the biochemical properties of enzymatic proteins, or receptor proteins, or structural proteins such as collagen fibers. We can postulate that also the presence of increased Atrial Fibrillation should be attributed to altered glycations and expression at myocardial level of proteins forming ion channels, and in the presence of altered calcium homeostasis ( as we have during osteoporotic bone resorption) we have an increased probability to develop myocardial depolarizarion leading finally to Atrial Fibrillation.Cellular electrophysiolgical studies have revealed a marked reduction in the densities of L-type volatage gated Calcium channels, transient outward Potassium currents, and ultrarapid delayed rectifier Potassium currents in atrial myocites of patients affected by Atrial Fibrillation.Interestingly similar ( but not identical ) changes are present in canine models of Atrial Fibrillation, where changes in ions currents are correlated with reduced expression of the underlying channels forming subunits.In both human and canine Atrial Fibrillation, reduced Calcium voltage currents seem to be enought to explaine the reduction action potentials in duration and effective refractory period characteristics of remodelling atria.In addition the sarcoplasmic expression of Calcium dependent ATPase is reduced in myocites, suggesting that calcium cycling is affected in atrial fibrillated myocites.

References

Seeman E, Delmas PD. Bone quality – The material and structural basis of bone strength and fragility. N Engl J Med 2006;354:2250-61.

Watts NB, Diab DL. Long term use of bisphosphonates in osteoporosis. J Clin Endocrinol Metab 2010;95:1555-65.

Liberman UA, Weiss SR, Bröll J et al. Effects of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. The Alendronate Phase III Osteoporosis Treatment Study Group. N Engl J Med 1995;333:1437-43.

Orwoll E, Ettinger M, Weiss S et al. Alendronate for the treatment of osteoporosis in men. N Engl J Med 2000;343:604-10.

Reid IR, Brown JP, Burkhardt P et al. Intravenous Zolendronic acid in postmenopausal women with low bone mineral density. N Engl J Med 2002;346:653-61.

Bone HG, Hosking D, Devogelaer JP et al. Ten years experience with alendronate for osteoporosis in postmenopausal women. N Engl J Med 2004;350:1189-99.

Black DM, Delmas PD, Eastell R et al. for the HORIZON Pivotal Fracture Trial. Once-yearly zolendronic acid for treatment of postmenopausal osteoporosis. N Engl J Med 2007;356:1809-22.

Cummings SR, Schwartz AV, Black DM. Alendronate and atrial fibrillation. N Engl J Med 2007;356:1895-6.

Lyles KW, Colòn-Emeric CS, Magaziner JS et al. HORIZON Recurrent fracture trial. Zolendronic acid and clinical fractures and mortality after hip fracture. N Engl J Med 2007;357:1799-809.

Lenart BA, Lorich DG, Lane JM. Atypical fractures of the femoral diaphysis in postmenopausal women taking alendronate. N Engl J Med 2008;358:1304-6.

Weinstein RS, Roberson PK, Manolagas SC. Giant osteoclast formation and long term oral bisphosphonate therapy. N Engl J Med 2009;360:53-62.

Black DM, Kelly MP, Genant HK et al. Bisphosphonates and fractures of the subtrochanteric or diaphyseal femur. N Engl J Med 2010;362:1761-71.

Shane E. Evolving data about subtrochanteric fractures and bisphosphonates. N Engl J Med 2010;362:1825-7.

Allen MR, Burr DB. Three years of alendronate treatment results in similar levels of vertebral microdamage as after one year of treatment. J Bone Miner Res 2007;22:1759-65.

Allen MR, Gineyts E, Leeming DJ et al. Bisphosphonates alter trabecular bone collagen cross-linking and isomerization in beagle dog vertebra. Osteoporosis Int 2008;19:329-337.

Saito M, Mori S, Mashiba T et al. Collagen maturity, glycation induced-pentosidine, and mineralization are increased following 3 yeras treatment with incadronate in dogs. Osteoporosis Int 2008;19:1343-54.

Wang X, Shen X, Li X et al. Age-related changes in the collagen network and toughness of bone. Bone 2002;31:1-7.

Khamaisi M, Yarom N, Avni B et al. Possible association between diabetes and bisphosphonate-related jaw osteonecrosis. J Clin Endocrinol Metab 2007;92:1172-5.

Aviles RJ, Martin DO, Apperson-Hansen C et al. Inflammation as a risk factor for atrial fibrillation. Circulation 2003;108:3006-10.

HR-pQCT

Muller R, Hildebrand T, Ruegsegger P. Non-invasive bone biopsy: a new method to analyse and display the three-dimensional structure of trabecular bone. Phys Med Biol 1994;39:145-64.

Van Rietbergen B, Weinans H, Huiskes R et al. A new method to determine trabecular elastic properties and loading using micromechanical finite-element model. J Biomech. 1995;28:69-81.

Laib A, Hildebrand T, Houselmann HJ et al. Ridge number density: a new parameter for in vivo bone structure analysis. Bone 1997;21:541-46.

Laib A, Hauselmann HJ, Ruegsegger P. In vivo high resolution 3D-QCT of the human forearm. Technol Health Care 1998;6:329-37.

Laib A, Ruegsegger P. Comparison of structure extraction methods for in vivo trabecular bone measurements. Comput Med Imaging Graph 1999;23:69-74.

Laib A, Ruegsegger P. Calibration of trabecular bone structure measurements of in vivo three-dimensional peripheral quantitative computed tomography with 28-micron-resolution microcomputed tomography. Bone 1999;24:35-39.

Ulrich D, van Rietbergen B, Laib A et al. The ability of three dimensional structural indices to reflect mechanical aspects of trabecular bone. Bone 1999;25:55-60.

Boutroy S, Bouxsein ML, Munoz F et al. In vivo assessment of trabecular bone microarchitecture by high resolution peripheral quantitative computed tomography. J Clin Endocrinol Metab 2005;90:6508-15.

Khosla S, Riggs BL, Robb RA et al. Relationship of volumetric bone density and structural parameters at different skeletal sites to sex steroid level in women. J Clin Endocrinol Metab 2005;90:5096-5103.

Khosla S, Melton LJ III, Robb RA et al. Relationship of volumetric BMD and structural parameters at different skeletal sites to sex steroid level in men. J Bone Miner Res 2005;20:730-40.

Khosla S, Riggs BL, Atkinson EJ et al. Effects of sex and age on bone microstructure at the ultradistal radius: a population based noninvasive in vivo assessment. J Bone Miner Res 2006;21:124-31.

Melton LJ 3^rd, Riggs BL, van Lenthe GH et al. Contribution of in vivo structural measurememnts and load/strength ratios to the determination of forearm fracture risk in postmenopausal women. J Bone Miner Res 2007;22:1442-8.

Boutroy S, Van Rietbergen B, Sornay-Rendu E et al. Finite element analysis on in vivo HR-pQCT images of the distal radius is associated with wrist fracture in postmenopausal women. J Bone Miner Res 2008;23:392-9.

Macneil JA, Boyd SK. Bone strength at the distal radius can be estimated from high-resolution peripheral quantitative tomography and the finite element method. Bone 2008;42:1203-13.

Cohen A, Liu S, Stein EM et al. Bone microarchitecture and stiffness in premenopausal women with idiopatic osteoporosis. J Clin Endocrinol Metab 2009;94:4351-60.

Wren TAL, Gilsanz V. Evolving role of imaging in the evaluation of bone structure. J Bone Miner Metab 2009;24:1943-45.

Burghardt AJ, Kazakia GJ, Ramachandran S et al. Age and gender related differences in the geometric properties and biomechanical significance of intracortical porosity in the distal radius and tibia. J Bone Miner Res 2010;25:983-93.

Smith RW, Walker RR. Femoral expansion in aging women: implications for osteoporosis and fractures. Science 1964;145:156-7.

Ruff CB, Hayes WC. Subperiosteal expansion and cortical remodeling of the human femur and tibia with aging. Science 1982;217:945-8.

Currey JD, Pitchford JW, Baxter PD. Variability of mechanical properties of bone, and its evolutionary consequences. J R Soc Interface 2007;4:127-35.