Printed in Great Britain
`
`Biochem J 1990 266 863868
`The effects of serum and human albumin on calcium
`hydroxyapatite crystal growth
`
`863
`
`Jane GARNETT and Paul DIEPPE
`Rheumatology Unit University of Bristol Department of Medicine Bristol Royal Infirmary Marlborough Street
`Bristol BS2 8HW UK
`
`ultrafiltration
`
`The effects of potential serum inhibitors upon the growth of calcium hydroxyapatite HAP crystals were
`studied in vivo using a pHstat system Whole serum caused a marked decrease in crystal growth in a dose
`dependent manner At a protein concentration of 13 itgml whole serum reduced the initial
`rate of crystal
`growth from 84 innol of KOHh to 48 umol of KOHh Serum components were
`by
`separated
`10000 Da cutoff The high molecular mass fraction containing serum proteins gave an
`rate of crystal growth of 48 tanol of KOHh compared with 64 ttmol of KOHh given by the low
`initial
`molecular mass components Thus twothirds of the inhibitory activity was associated with proteins and
`the remainder of the activity was associated with the low molecular
`other serum macromolecules whilst
`rate of crystal growth of 59 pmol of KOHh
`mass components Albumin depleted serum showed an initial
`whilst albumin purified by affinity chromatography
`rate of crystal growth of 56 itmol of
`gave an initial
`KOHh at the same protein concentration Albumin therefore not only accounts
`for half of the protein
`the high molecular mass
`concentration in serum but also contributes half of
`the inhibitory activity of
`fraction Heat denaturation of albumin dramatically enhanced the inhibition of HAP seeded growth with
`rate of crystal growth falling to 27 umol of KOHh after treatment compared with 62 umol of
`the initial
`KOHh before denaturation Isoelectric focusing indicated that
`the tertiary and secondary structure and
`hence the distribution of surface charge of albumin are altered by heat denaturation Gels showed a mixture
`of species with isoelectric points ranging from 60 to 50 compared with the native protein value of 47 These
`that adsorption of serum proteins to the growing HAP crystals is one mechanism of growth
`data suggest
`is also clear that the most abundant serum protein albumin is an important mediator of this
`inhibition It
`
`process
`
`INTRODUCTION
`
`process of biological mineral de
`The fundamental
`position is under chemical control with nucleation and
`growth of crystals energetically regulated by levels of
`saturation and local concentrations of co precipitating
`ions Mann 1988 Nucleation
`and growth are also
`interactions of the precipitating
`controlled by specific
`mineral phases with organic andor inorganic molecules
`which function
`as promoters or inhibitors of crystal
`growth This leads to the selective deposition of specific
`and morphological mineral phases Weiner
`structural
`1986 Addadi
`Weiner 1985 The specificity of the
`type of
`thus determined by the
`mineral
`phase
`of promoter
`the concentration
`promoterinhibitor
`inhibitor and temporal control of the transport of all of
`these molecules to the site of mineralization
`A number of macromolecules which inhibit crystal
`growth have been identified and characterized in human
`body fluids For example salivary statherin Schlesinger
`Hay 1977 Aoba etal 1984 and prolinerich peptides
`secreted by the parotid gland Oppenheim et al 1971
`function as inhibitors of calcium phosphate formation in
`saliva In urine calcium oxalate monohydrate precipi
`tation is controlled by a glycoproteincrystallization
`inhibitor Nakagawa etal 1983 Worcester etal 1987
`
`is
`
`Calcium carbonate formation in pancreatic secretions is
`regulated by an acidic phosphoprotein de Caro et al
`1984 A number of macromolecular
`complexes which
`inhibitory activity in vitro have also been isolated
`exhibit
`et al 1985 Fisher
`tissues Hunter
`from connective
`Termine 1985 Chen
`Boskey 1986 Boskey 1989 In
`comparison to these body fluids human serum has not
`been characterized to the same extent Plasma ultrafiltrate
`containing low molecular mass inorganic ions has been
`studied in detail and citrate magnesium and pyro
`phosphate have been shown to inhibit HAP precipitation
`reviewed in Fleisch 1981 Rufenacht
`Fleisch 1984
`Fleisch 1984ab Eidelman et al 1988 How
`Meyer
`ever despite the identification of a range of biological
`inhibitors there is limited knowledge of
`the precise
`mechanisms by which the process of crystal growth is
`inhibited by these macromolecules Williams
`Sallis
`1979 1982 The present study therefore aimed to identify
`the macromolecules of serum responsible for inhibition
`promotion of HAP crystal growth using a pHstat assay
`of seeded crystal growth
`Preliminary work from this unit has suggested
`the
`existence of inhibitors of HAP crystallization
`in serum
`and synovial fluid Sera from patients with osteoarthritis
`commonly associated with crystals were compared with
`sera from patients with rheumatoid arthritis in the pH
`
`used HAP hydroxyapatite PBS phosphate buffered saline Ro initial
`rate of crystal growth
`Abbreviations
`To whom all correspondence should be sent at present address Oxford GlycoSystems Ltd Unit 4 Hitching Court Blacklands Way Abingdon
`Oxon OX I 4 IRG UK
`
`Vol 266
`
`Abraxis EX2052
`Actavis LLC v Abraxis Bioscience LLC
`1PR201701101 1PR201701103 1PR201701104
`
`

`

`864
`
`J Garnett and P Dieppe
`
`stat crystal growth assay and both groups showed similar
`The
`fluid from these
`inhibitory potential
`synovial
`arthritic patients also caused inhibition of crystal growth
`but
`to serum showed
`a wide range of
`in contrast
`inhibitory potential Campion et al 1988
`Numerous rheumatic diseases involve the deposition
`of inorganic crystalline phases including hydroxyapatite
`in articular tissues which may lead to chronic joint
`damage and disability or acute inflammatory responses
`Dieppe et al 1988 Schumacher 1988 The mechanisms
`for the formation of these pathological mineral deposits
`are poorly understood Smith 1982 In the long term an
`the molecular mechanisms is vitally
`understanding of
`important for the development of rational approaches
`to
`prevention and treatment of pathological mineral de
`Calvert 1983
`position Dieppe
`Our hypothesis is that normal body fluids contain
`inhibitors of crystal growth which are
`macromolecular
`in regions of ectopic
`depleted
`or absent
`ineffective
`mineral deposition This work was undertaken to give
`the growth of HAP in the
`detailed information about
`presence of normal human serum and to identify the
`macromolecules
`influence crystal growth These
`that
`data from normal human sera are an essential prerequisite
`testing our hypothesis during comparisons with
`for
`fluids Campion et al
`sera and synovial
`pathological
`1988
`
`MATERIALS AND METHODS
`
`titrating
`
`Preparation of seed crystal
`Synthetic HAP used to seed the growth of crystals in
`was
`ortho
`prepared
`by
`solution
`slowly
`phosphoric acid into a suspension of calcium oxide 20 g
`prepared by heating calcium carbonate at 1000 °C for
`24 h to a CaP final molar ratio of 167 McDowell et al
`1977 The 1
`litre suspension was attached
`to a reflux
`condenser kept under a nitrogen atmosphere stirred
`continuously and maintained at 90 + 1 °C for 48 h The
`precipitate was allowed to settle the liquid phase was
`and
`the precipitate was
`removed by centrifugation
`three times by stirring in 2 litres of distilled
`washed
`water settling and centrifuging then similarly washed in
`1 mmorthophosphoric acid and dried at 110 °C for 24 h
`under nitrogen This HAP showed sharp ir and Xray
`diffraction
`characteristic of well crystallized
`patterns
`HAP without carbonate The crystal mass was crushed
`sieved and those particles between 250 um and 750 ium
`collected The specific surface area of these crystals was
`measured by the modified BET method Brunauer
`et al 1938 using nitrogen adsorption after degassing at
`120°C and was 308 + 06 m2g n = 3 Area meter
`Strahlein Gmbh Co
`Preparation of solutions
`All solutions were made with reagent grade chemicals
`BDH Ltd using distilled water Solutions super
`to HAP were prepared from stock
`saturated with respect
`solutions and mixed to final concentrations of 197 mm
`CaC12 096 mmK1121304 and 150 mmKC1 to mimic the
`Tomazic
`concentrations found in serum Nancollas
`1974 Eidelman et al 1988 KC1 an inert electrolyte
`was included to maintain ionic strength and was es
`sentially constant during the experiment The saturation
`ratio which is related to the thermodynamic
`driving
`force for crystal growth Neilsen 1984 was calculated
`
`to
`
`as 1952 with respect of HAP 252 with respect
`octacalcium phosphate and 077 with respect
`to dicalcium
`phosphate dihydrate by using the computer program
`of Shellis 1988 For serum the ratios are 140150 for
`HAP and 1828 for octacalcium phosphate Eidelman
`et al 1988
`A water jacketed
`glass vessel was used for the pre
`cipitation reactions The metastable calcium phosphate
`solutions 120 ml were maintained at 37 °C continually
`stirred using a magnetic stirrer and were purged with
`carbon dioxide The pH of
`the
`nitrogen to exclude
`reaction solution was measured in situ with a combination
`glass electrode Metrohm AG and adjusted to pH 740
`by addition of 25 mmKOH using a pHstat assembly
`Metrohm Combititrator Metrohm AG Herisau
`Switzerland The solution was maintained at pH 740
`during crystal growth and the volume of alkali added
`Mohan
`1970
`recorded continuously Nancollas
`Varughese 1981 The control experiments
`Moreno
`began with the addition of 15 mg of HAP seed crystal
`suspended in 05 ml of HAP saturated distilled water
`had nothing else added and ran for 60 min The final
`precipitate was collected by filtration onto 022 Am filters
`Millipore Ltd and dried at 37 °C for 60 min Samples
`under investigation were added to the reaction solution
`in 02 ml of phosphate buffered saline PBS Oxoid Ltd
`during the initial pH adjustment after the solution had
`been adjusted to greater than pH 700 The addition of
`02 ml of PBS alone to the reaction solution showed
`reaction
`growth
`identical
`kinetics
`to
`the
`control
`Measurements were repeated a minimum of three times
`and a maximum of seven times The SD of the mean and
`The
`inhibition of crystal growth were calculated
`latter values varied over the range 154
`
`BlueSepharose chromatography
`Serum from a healthy individual 2 ml was applied in
`PBS to a column of BlueSepharose Pharmacia Ltd
`30 ml 15 mm diameter flow rate 30 mlh equilibrated
`in PBS The absorbance was monitored at 280 nm and
`The
`single peak of absorbance
`collected
`fractions
`representing the unbound proteins was collected Proteins
`adsorbed to the column were eluted with PBS contain
`15 mKC1 Affinity Chromatography
`booklet
`ing
`Pharmacia Ltd and dialysed against PBS for a total of
`16 h at 4°C to remove KC1 Both fractions were
`by vacuum dialysis collodion bags Sar
`concentrated
`torius GmbH to a protein concentration of 80 mgml
`as measured by a dye binding assay standardized against
`human serum albumin BioRad Ltd The
`protein
`profiles of the two fractions were examined by Coomassie
`Blue stain of a 12 SDSpolyacrylamide gel Laemmli
`1970
`
`Heat denaturation of albumin
`
`fraction V Sigma 80 mg was
`Purified albumin
`suspended in distilled water 100 ml and heated at 90 °C
`Addadi 1988
`in a closed vial for 16 h PerleTreves
`removed by centrifugation
`Aggregated protein was
`10000 g for 15 min the supernatant collected
`and the
`at 280 nm
`protein content measured by absorbance
`
`RESULTS
`
`Crystal growth
`Crystal growth was induced in a metastable calcium
`phosphate solution by inoculation with seed HAP crystals
`
`1990
`
`

`

`Effects of serum and human albumin on hydroxyapatite
`
`crystal growth
`
`865
`
`40
`
`30
`
`Li
`
`20
`
`10
`
`o c
`
`0
`
`5
`
`10
`Serum mg of protein
`
`15
`
`Fig 3 Doseresponses of serum 0 and dialysed serumA on
`
`calcium phosphate precipitation
`
`Serial dilutions were made in PBS prior to the addition to
`the reaction solution and are expressed as the total amount
`of protein added to the 120 ml assay
`
`reaction solution Nancollas Mohan 1970 Nancollas
`Tomazic 1974 Initially the rate of base addition was
`linear with time and to calculate the initial
`rate of crystal
`growth data points were measured over the first 14 min
`Due to the depletion of calcium and phosphate with time
`the reactions were routinely limited to 60 min Examin
`ation of seed crystals after 60 min gave characteristic
`patterns for crystalline HAP by ir and Xray diffraction
`the addition of HAP
`analyses data not shown Without
`seed crystals no uptake of hydroxyl
`ions was recorded
`indicating that serum alone did not
`induce nucleation
`and growth
`
`Inhibitory effects of serum
`
`The addition of normal serum has an inhibitory effect
`on HAP crystal growth as shown in Fig 2 In the control
`experiment only HAP seed crystal was added and showed
`growth rate of 84 mot of KOHh but after
`an initial
`the addition of serum at a protein concentration
`at
`13 1ugm1 the initial growth rate fell
`to 48 Amol of
`KOHh a 43
`decrease Serum was dialysed to remove
`the low molecular mass components and
`caused
`the
`rate of crystal growth to fall
`to 57 umol of
`initial
`KOHh a 32 decrease from control values
`The inhibition of crystal growth by serum was dose
`dependent as serum was diluted the inhibition of crystal
`growth decreased Fig 3 However
`very dilute serum
`inhibition during which further
`reached
`a plateau of
`dilution does not decrease the inhibition 040013 mg
`of protein120 m1 This effect
`could be due to two
`processes one operating in a protein dependent manner
`until a concentration of 04 mg total protein120 ml was
`reached and below this value another effect
`that
`is not
`protein concentration dependent To test this hypothesis
`the doseresponse of dialysed serum was measured and
`showed inhibition decreasing with decreasing amounts of
`rate Fig 3
`reached the control
`dialysed serum until
`the low and high
`to compare directly
`In order
`of serum these were
`molecular mass components
`separated by ultrafiltration Both fractions were able to
`decrease the rate of HAP crystal growth Table 1 The
`serum macromolecules retained by the filter were twice as
`
`it
`
`80
`
`60
`
`40
`
`0
`
`10
`
`20
`15
`Seed HAP mg120 ml
`
`25
`
`fKOHh
`
`rateµmolo
`
`precipitation
`
`Initial
`
`Fig 1 Initial
`rates of crystal growth are proportional
`amount of seed HAP used to initiate the precipitation
`
`to the
`
`60
`
`50
`
`z 40
`
`ow
`
`77
`
`co
`
`30
`
`° 20
`
`10
`
`0
`
`10
`
`20
`
`40
`
`50
`
`60
`
`30
`Time min
`Fig 2 Comparison of the inhibitory effect of serum and dialysed
`serum
`The effects are shown by the amount of KOH required to
`maintain a constant pH 740 during the precipitation of
`calcium phosphate at 37 °C in a 120 ml assay At time zero
`15 mg of HAP seed crystals were added to initiate crystal
`growth The control
`did not contain
`experiment
`any
`The protein content of each serum was
`macromolecules
`02 ml were added to
`measured and 16 mg of proteins
`the assay The values are the means of seven normal sera
`three dialysed sera and four controls The error bars
`represent SD of the mean Inhibition
`
`0 serum 0 43 dialysed serum A 32
`
`control 0
`
`and the rate of crystal growth was followed by hydroxyl
`The quantity of seed HAP crystals
`ion consumption
`rate of crystal growth R
`added was characterized for each batch of HAP made
`Fig 1 shows that the initial
`area of seed HAP
`was linearly related to the surface
`available Since the seed HAP used is constant within
`any one batch the weight of seed HAP is proportional
`to
`the surface area Amounts of seed HAP below 10 mg
`120 ml solution do not show the same linear relationships
`A seed concentration of 15 mg120 ml solution was
`chosen for the following experiments and the growth of
`HAP is shown in Fig 2 The rate of HAP crystal growth
`to the amount of KOH added to the
`is proportional
`
`Vol 266
`
`

`

`866
`
`J Garnett and P Dieppe
`
`z
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`addedpmol
`
`KOH
`
`0
`
`10
`
`20
`
`30
`Time min
`
`40
`
`50
`
`60
`
`Fig 4 Comparison of
`the inhibitory effects of human serum
`albumin and heat denatured albumin
`
`Each point
`represents the mean of triplicate samples and
`the SD values are shown by the vertical bars Each assay
`in 02 ml of PBS
`contained 16 mg of protein resuspended
`
`Inhibition c0 control 0 0 albumin P 24 heat
`denatured albumin 0 83
`
`structure to inhibition of HAP crystal growth by albumin
`was tested A solution of purified albumin was denatured
`by heating and its effect upon HAP crystal growth
`compared with native albumin in the pHstat assay Fig
`4 Denatured albumin reduced the initial
`rate of crystal
`growth to 27 umol of KOHh a much bigger decrease
`than native albumin at 62 gmol of KOHh When
`compared to the control 81 umol of KOHh these show
`and 24 inhibition of HAP crystal growth re
`83
`spectively The native albumin had an isoelectric point
`pI of 47 in contrast
`to the heat denatured
`albumin
`which showed a range of species with pI values ranging
`from 60 to 50 Phastgel 39 Phast System Pharmacia
`Ltd
`
`DISCUSSION
`
`These data demonstrate that human serum has
`a
`significant effect upon HAP crystal growth in the in vitro
`pHstat assay system The removal of the low molecular
`mass components mainly inorganic ions from serum
`reduces but does not abolish the inhibitory effect Fig 2
`and Table 1 The low molecular mass inorganic ions
`may be responsible for the inhibition when serum is very
`dilute <8 ug of proteinml Fig 3 The organic macro
`molecules of serum play a more important
`role in
`controlling the nucleation and growth of HAP than the
`inhibitory inorganic ions This was also the conclusion of
`Fleisch 1984b using an assay
`another study by Meyer
`In vivo organic and in
`at constant supersaturation
`organic molecules coexist both in serum and in bone
`mineral and so their effects also depend upon interactions
`interactions with HAP
`with one another
`as well as
`crystals During pathological mineralization specific inter
`actions between crystals and biological macromolecules
`in controlling the development of
`may be significant
`et al 1981 1983
`crystal deposition diseases Boskey
`Anderson 1983 PerleTreves
`Addadi 1988
`Previous studies using human and rat sera fractionated
`by gel permeation chromatography
`reported inhibitor
`150000 Da Meyer
`at 43000 and
`Fleisch
`
`activity
`
`1990
`
`low and high molecular mass
`Table I Comparison of
`components of serum on HAP precipitation
`
`the
`
`Serum 2 ml was fractionated by ultrafiltration
`through a
`membrane with a 10000 Da cutoff Amicon Microconcen
`trator10 The fractions were made up to equal volumes
`with PBS 2 ml and tested for their inhibitory activity by
`inclusion of 02 ml in the seeded crystal growth assay The
`rates of crystal growth Ro were calculated as
`initial
`described in the Results section
`
`Sample
`
`Control
`Serum
`
`Ultrafiltrate
`
`Retentate
`
`Ro
`
`ttmol of KOHh
`
`Inhibition
`
`795
`435
`640
`480
`
`0
`
`45
`
`19
`
`40
`
`Table 2 Inhibitory effect of purified human serum albumin and
`albumin depleted serum
`
`Serum was
`which
`fractionated
`BlueSepharose
`on
`effectively removes albumin and very few other proteins
`The
`two
`were
`assayed
`equal
`protein
`fractions
`at
`concentrations of 20 mg120 ml reaction volume
`
`Sample
`
`Control
`Flow through
`Eluate
`
`Ro
`
`amol of KOHh
`
`Inhibition
`
`810
`585
`555
`
`0
`28
`
`32
`
`the ultrafiltrate
`inhibitory as
`molecular mass components
`
`Effects of albumin
`
`containing
`
`the
`
`low
`
`Since half of the protein in serum is a single species
`upon HAP crystal growth was
`albumin its
`effect
`investigated Purified albumin 167 1ugm1 added to the
`rate of crystal growth of
`pHstat assay gave an initial
`62 umol of KOHh Compared to a control
`rate of
`inhibition of 24
`81 umol of KOHh this is an
`Inhibition of HAP crystal growth by purified human
`serum albumin also showed a doseresponse dependent
`upon protein concentration results not shown How
`ever purified albumin does not wholly represent albumin
`found in vivo which acts as a transport protein and is
`complexed to many other serum molecules Peters 1975
`KraghHansen 1981 Albumin was therefore removed
`from serum by dyeligand chromatography using Blue
`and
`Sepharose Polyacrylamide
`electrophoresis
`gel
`showed
`that albumin was not
`immunoelectrophoresis
`detectable in the fraction that did not bind to the matrix
`The serum fraction eluted from the matrix by high salt
`buffer was found to contain albumin and trace amounts
`of other proteins eg armacroglobulin The albumin
`depleted serum and the albumin enriched fraction were
`tested for their ability to inhibit HAP crystal growth and
`at equal protein concentrations showed similar inhibitory
`capacity Table 2
`The contribution
`
`of protein tertiary
`
`and secondary
`
`

`

`Effects of serum and human albumin on hydroxyapatite
`
`crystal growth
`
`867
`
`it
`
`that
`
`the
`
`1984a However
`no further analyses of these protein
`fractions were undertaken to identify the macromolecules
`involved The present study has clearly shown that whole
`the seeded growth of HAP Fig 2
`serum can inhibit
`Affinity purified human serum albumin has the capacity
`to inhibit HAP precipitation
`the re
`to that of
`equal
`maining proteins see Table 2 In view of these results
`the crystalprotein interactions which might mediate the
`inhibition of HAP growth by albumin were
`observed
`investigated
`Terkeltaub et al 1988 have shown that when HAP
`crystals are incubated in whole human serum a number
`are adsorbed and
`of proteins
`that albumin was
`a
`significant component of
`the adsorbed species
`The
`adsorption of proteins and polypeptides onto HAP has
`been correlated with the presence of specific functional
`groups in the organic molecule and specific adsorption
`sites on the HAP Williams Sallis 1979 1982 Fujisawa
`etal 1986 1987 From other studies of HAP inhibitors
`is known that besides functional groups the structural
`conformation of the inhibitor molecule is an important
`for potency Moreno et al 1984 Williams
`determinant
`Sallis 1982 Romberg etal 1985 1986 Myers 1987
`et al 1988 The changes
`in the surface
`Schlesinger
`charge of albumin demonstrated by changes in the pI
`the protein with HAP
`interactions of
`allow increased
`crystals and hence increased inhibition of crystal growth
`In a study of serum albumin binding to HAP Hlady
`FiirediMilhofer 1978 concluded
`that mutual
`inter
`action depended upon surface charge and was increased
`in pH temperature or ionic strength They also
`by a fall
`the conformation
`of albumin
`suggested
`at
`interface may not be the same as
`proteincrystal
`in
`solution with adsorbed albumin unfolding relative to its
`native state In the assay heat denatured albumin proved
`to be a threefold more potent inhibitor of HAP growth
`than native albumin Fig 4 which shows
`that
`the
`
`of
`conformation
`structural
`in
`this protein is critical
`mediating the interactions of the protein with the growing
`the threedimensional
`crystals Although
`structure of
`albumin is not well defined there is clear evidence that
`the single polypeptide is ordered into 18 a helices and that
`these are aligned into three parallel cylindrical domains
`Bloomfield 1966 Brown 1977b KraghHansen 1981
`Heat denaturation induces
`a partial unfolding of
`the
`Addadi 1988 The
`helical conformation PerleTreves
`cylindrical domains have a hydrophobic
`interior and a
`polar exterior Brown 1977a and this alignment of the
`residues would favour electrostatic interactions
`polar
`domains on the protein and the
`between the charged
`exposed surfaces of HAP crystals By binding to the
`crystal surface where growth is taking place the adsorbed
`molecules would inhibit growth The question which
`remains to be answered
`is whether this inhibitory effect
`results from interactions
`between
`specific molecular
`faces and functional groups within
`motifs in the crystal
`the protein Moreno et al 1984 Williams Sallis 1982
`Addadi 1988 or whether the inhibition
`PerleTreves
`arises merely from less specific crystalprotein inter
`actions
`Transmission electron microscope analyses of HAP
`crystals grown in the presence of human serum albumin
`indicate that albumin mediated aggregation may be
`partly responsible for the observed growth inhibition
`Further studies using immunogold labelling techniques
`also suggest adsorption of the protein may be to specific
`
`Vol 266
`
`faces B Heywood
`J Garnett unpublished
`crystal
`work It
`the interaction of
`is interesting to note that
`im
`human serum albumin with another biologically
`portant mineral monosodium urate monohydrate is
`highly specific Using immunolocalization it has been
`to the 110 face of
`shown that albumin binds selectively
`Addadi 1988
`this monoclinic crystal PerleTreves
`In the present study we have investigated the possible
`role of serum components in the control of HAP crystal
`growth and demonstrated that a key serum component
`this process Abnormal
`inhibitor of
`acts as a potent
`tissue mineralization underlies a wide spectrum of patho
`logical disorders and the mineral phases associated with
`such conditions have been characterized Associated risk
`factors which influence their occurrence
`have
`been
`identified such as increasing age and tissue damage
`Dieppe
`Calvert 1983
`A range of normal and apatite containing tissues have
`and a
`unique set of proteolipids
`been
`analysed
`and
`proteolipidphospholipid
`calciumphospholipidphosphate
`complexes have been identified Boskey
`1982 Boyan 1986 These com
`al 1982 Wuthier
`plexes will nucleate HAP from metastable solutions
`role in biomineral
`important
`indicating a potentially
`ization Boskey
`Posner 1977 However
`it has also
`been
`demonstrated
`phospholipid
`that
`the
`acidic
`inhibit HAP growth when added
`solutions A L
`phosphatidylserine will
`to metastable calcium phosphate
`thus the role of
`Boskey
`personal communication
`phosphorylated membrane components associated with
`is unclear and there remains an
`mineralized tissues
`incomplete understanding of the processes controlling
`and
`nucleation
`growth
`in
`crystal
`pathological
`mineralization Knowledge of the molecular mechanisms
`and
`also
`growth
`directing
`crystal
`crystalprotein
`lead to a better understanding of the
`interactions will
`aberrations which induce
`pathological
`and provide a base for the design of
`crystallization
`therapeutic strategies in the treatment and management
`of crystal deposition diseases
`
`physiological
`
`et
`
`We are most grateful
`to Dr Peter Shellis for advice and
`the pHstat assay and synthesis of HAP
`encouragement about
`crystals Dr Brigid Heywood University of Bath Dr Chris
`and Dr Neil McGill
`discussions and
`Elson
`long
`for
`constructive criticism of the manuscript Our thanks go to Mrs
`Angela Swan for ir analyses Dr Anna Hayes University of
`analyses and Mr John Dimery
`Bath for Xray diffraction
`Department of Physical Chemistry for use of the Area meter
`This work was funded by the Nuffield Foundation Oliver Bird
`Fund for Rheumatology
`and a Young Investigators Award
`from the European Society for Calcified Tissue
`
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