NO170392B - MATERIALS FOR PROTECTION OF DENTAL ELEMENTS AGAINST ACID CAUSAL DESCALING - Google Patents
MATERIALS FOR PROTECTION OF DENTAL ELEMENTS AGAINST ACID CAUSAL DESCALING Download PDFInfo
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- NO170392B NO170392B NO860853A NO860853A NO170392B NO 170392 B NO170392 B NO 170392B NO 860853 A NO860853 A NO 860853A NO 860853 A NO860853 A NO 860853A NO 170392 B NO170392 B NO 170392B
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- Prior art keywords
- mucins
- accordance
- synthetic analogues
- human
- sugar
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- 239000002253 acid Substances 0.000 title description 13
- 230000001364 causal effect Effects 0.000 title 1
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Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Dental Preparations (AREA)
Description
Den foreliggende oppfinnelse vedrører et materiale til beskyttelse av tenner mot avkalking forårsaket av virkningen av syre. The present invention relates to a material for protecting teeth against decalcification caused by the action of acid.
Et av problemene som regelmessig påtreffes innen tann-legevitenskapen er den syreforårsakede avkalking av tenner. Formålet med den foreliggende oppfinnelse er å komme med et bidrag til løsning av dette problem. One of the problems regularly encountered in dentistry is the acid-induced decalcification of teeth. The purpose of the present invention is to make a contribution to solving this problem.
Dette formål oppnås ved et materiale som er kjennetegnet ved at det inneholder muciner i form av glukoproteiner med molvekt 0,1-2 megadalton, isolert fra spytt avsondret fra humane slimhinnespyttkjertler, eller syntetiske analoger av humane muciner i form av 1) polypeptidkjeder bestående av minst 500 serin- og/eller treoninaminosyrer, og som er glukosylert ved hjelp av N-acetylgalaltosamin, eller 2) polyfosfazenbaserte syntetiske polymerer. This purpose is achieved by a material which is characterized by containing mucins in the form of glucoproteins with a molecular weight of 0.1-2 megadaltons, isolated from saliva secreted from human mucosal salivary glands, or synthetic analogues of human mucins in the form of 1) polypeptide chains consisting of at least 500 serine and/or threonine amino acids, and which are glucosylated by means of N-acetyl galaltosamine, or 2) polyphosphazene-based synthetic polymers.
Fortrinnsvis er materialet ifølge oppfinnelsen i form av en tannpasta. Preferably, the material according to the invention is in the form of a toothpaste.
Andre foretrukne utførelser av materialet ifølge oppfinnelsen fremgår av krav 3-10. Other preferred embodiments of the material according to the invention appear from claims 3-10.
Det har overraskende vist seg at muciner fra humant spytt, sammenlignet med dyremuciner, ikke bare adsorberes meget sterkt til hydroksyapatitt (HAP) og i denne forbindelse neppe, om i det hele tatt, hindres av nærværet av fosfatholdige proteiner, men også gir meget god beskyttelse av tannemalje mot syrevirkning på grunn av at de til forskjell fra proteiner i spytt i slimhinnen med en forholdsvis sterk adsorpsjon til HAP, såsom staterin og prolinrike proteiner, hindrer syreforårsaket avkalking av tenner. It has surprisingly been shown that mucins from human saliva, compared to animal mucins, not only adsorb very strongly to hydroxyapatite (HAP) and in this respect are hardly, if at all, hindered by the presence of phosphate-containing proteins, but also provide very good protection of tooth enamel against acid action due to the fact that, unlike proteins in saliva in the mucosa with a relatively strong adsorption to HAP, such as statherin and proline-rich proteins, they prevent acid-induced decalcification of teeth.
Fra hollandsk patentskrift 69.163 er det kjent tann-midler, såsom tannpasta og munnvann, som inneholder (ekstrakter av) spyttkjertler og deres gunstige virkning på tenner, men røper ikke innsikten at muciner separert fra humant spytt har særlige egenskaper, nemlig en mye sterkere adsorpsjon til hydroksyapatitt enn dyremuciner, en adsorpsjon som i tillegg er mye mindre ømfintlig for konkurranse med fosfatholdige proteiner, og en utmerket beskyttelse av tannemalje mot syrepåvirkning. I det hollandske patentskrift beskrives det bare anvendelse av spyttkjertler (eller ekstrakter derav) fra dyr, f.eks. storfe. From Dutch patent document 69,163 there are known dental agents, such as toothpaste and mouthwash, which contain (extracts of) salivary glands and their beneficial effect on teeth, but does not reveal the insight that mucins separated from human saliva have special properties, namely a much stronger adsorption to hydroxyapatite than animal mucins, an adsorption which is also much less sensitive to competition with phosphate-containing proteins, and an excellent protection of tooth enamel against acid attack. The Dutch patent only describes the use of salivary glands (or extracts thereof) from animals, e.g. cattle.
PCT-patentsøknad WO81/02977 er noe mer relevant ved at ordet muciner benyttes, men også denne er begrenset til ikke-humant materiale ("ikke-human mucin fra pattedyr"), såsom mucin fra grisemage (PGM) og mucin fra storfespytt (BSM). PCT patent application WO81/02977 is somewhat more relevant in that the word mucins is used, but this too is limited to non-human material ("non-human mucin from mammals"), such as mucin from pig stomach (PGM) and mucin from bovine saliva (BSM ).
En artikkel av Prakobphol et al. i Carbohydrate Research, Vol. 108, 1982, p. 111-122 omtaler en mucin fra spytt fra den sublinguale kjevespyttkjertel, men det gis ingen indikasjoner på at dette humane materiale ville kunne være en egnet bestanddel i materialer til beskyttelse av tenner mot syreforårsaket avkalking og at det er mye bedret egnet for dette formål enn dyremuciner. An article by Prakobphol et al. in Carbohydrate Research, Vol. 108, 1982, p. 111-122 mentions a mucin from saliva from the sublingual mandibular salivary gland, but no indication is given that this human material could be a suitable component in materials for protecting teeth against acid-induced decalcification and that it is much better suited for this purpose than animal mucins.
Chemical Abstracts Vol. 99, 1983, abstract No. 187.710r inneholder ingen data om opprinnelsen fra mucinene som anvendes i forsøkene. I tillegg synes resultatene å antyde en preferanse for karboksymetylcellulose overfor mucin som en bestanddel i kunstig spytt. Chemical Abstracts Vol. 99, 1983, abstract No. 187.710r contains no data on the origin of the mucins used in the experiments. In addition, the results seem to suggest a preference for carboxymethylcellulose over mucin as a constituent of artificial saliva.
Chemical Abstracts Vol. 95, 1981, abstract No. 36.228f inneholder overhodet ingen antydning om anvendelse av særlig human mucin som en bestanddel i beskyttende materialer. Chemical Abstracts Vol. 95, 1981, abstract No. 36.228f contains absolutely no suggestion of the use of human mucin in particular as an ingredient in protective materials.
I en artikkel i Adv. Exp. Med. Biol., Vol. 144, 1982, p. 179-181 (Chemical Abstracts Vol. 96, abstract No. 213.674s), gis en sammenligning mellom noen fysisk-kjemiske egenskaper for atskillige muciner (HSL, MSL, HSM, MSM), men beskriver ikke essensen av den foreliggende oppfinnelse, nemlig de overraskende fordeler med humane muciner i forhold til dyremuciner til beskyttelse av tenner mot syreforårsaket avkalking. In an article in Adv. Exp. Med. Biol., Vol. 144, 1982, p. 179-181 (Chemical Abstracts Vol. 96, abstract No. 213,674s), a comparison is given between some physico-chemical properties of several mucins (HSL, MSL, HSM, MSM), but does not describe the essence of the present invention, namely the surprising advantages of human mucins over animal mucins in protecting teeth from acid-induced decalcification.
Oppfinnelsen vil bli nærmere forklart i det etter-følgende under henvisning til de medfølgende tegninger, hvor: The invention will be explained in more detail below with reference to the accompanying drawings, where:
Fig. 1 viser et eksempel på formelen for mucin fra kjevespyttkjertelen hos sau (OSM). Fig. 2 viser de mest viktige typer av oligosakkaridkjeder hos forskjellige typer muciner, nemlig mucin fra kjevespyttkjertelen hos sau (OSM), mucin fra kjevespyttkjertelen hos gris (PSM), mucin fra grisemage (PGM) samt mucin fra humant spytt (HWSM). Fig. 3 er et diagram som viser forholdet mellom mengden mucin som adsorberes til hydroksyapatitt (HAP) og begynnelses-mucinkonsentrasjonen av atskillige typer muciner. Fig. 4 er et diagram som viser forholdet mellom mengden mucin adsorbert til HAP og begynnelsesfytatkonsentrasjonen for atskillige typer muciner. Fig. 5 viser et eksempel på et syntetisk, mucinaktig produkt. Fig. 1 shows an example of the formula for sheep salivary gland mucin (OSM). Fig. 2 shows the most important types of oligosaccharide chains in different types of mucins, namely sheep salivary gland mucin (OSM), pig salivary gland mucin (PSM), pig stomach mucin (PGM) and human salivary mucin (HWSM). Fig. 3 is a graph showing the relationship between the amount of mucin adsorbed to hydroxyapatite (HAP) and the initial mucin concentration of various types of mucins. Fig. 4 is a graph showing the relationship between the amount of mucin adsorbed to HAP and the initial phytate concentration for several types of mucins. Fig. 5 shows an example of a synthetic, mucin-like product.
Humant spytt inneholder forskjellige typer polypeptider, omfattende enzymer som amylase som er involvert i fordøyelsen, prolinrike proteiner som er involvert i adsorpsjonen til tannemalje og som opprettholder overmetningen av spytt med Ca 2+, Human saliva contains different types of polypeptides, including enzymes such as amylase involved in digestion, proline-rich proteins involved in the adsorption to tooth enamel and maintaining the supersaturation of saliva with Ca 2+ ,
samt muciner som bevirker viskositeten i spytt. as well as mucins that affect the viscosity of saliva.
Mucinene utgjør en gruppe biopolymerer som er av stor viktighet for å beholde helsen i munnhulen, både tennene og epitelvevet. Muciner i spytt produseres og avsondres av atskillige typer slimhinnespyttkjertler, hvorav de største er: glandula Submandiburalis (SM) og glandula Sublingualis (SL). I tillegg er der utallige mindre slimhinnespyttkjertler i epitelvevet i tungen, ganen, kinnet og leppene, som også av-sondrer muciner. Imidlertid produseres og avsondres ingen muciner av serøse spyttkjertler, såsom glandula parotis (Par). Denne sistnevnte spyttkjertel produserer hovedsakelig amylase og prolinrike proteiner. Faktisk er rent spyttkjertel spytt ikke viskøst. The mucins form a group of biopolymers that are of great importance for maintaining the health of the oral cavity, both the teeth and the epithelial tissue. Mucins in saliva are produced and secreted by several types of mucosal salivary glands, the largest of which are: glandula Submandiburalis (SM) and glandula Sublingualis (SL). In addition, there are countless smaller mucosal salivary glands in the epithelial tissue of the tongue, palate, cheek and lips, which also secrete mucins. However, no mucins are produced and secreted by serous salivary glands, such as the parotid gland (Par). This latter salivary gland mainly produces amylase and proline-rich proteins. In fact, pure salivary gland saliva is not viscous.
Mucinene er oppbygget av en lang polypeptidkjede (500-4000 aminosyrer) hvortil en kort eller noe lenger karbohydratkjede er bundet kovalent til aminosyrene serin (Ser) og treonin (Thr) via N-acetylgalaktosamin (GalNAc). Mucinene er kjennetegnet ved sine endesukrer: a. sialinsyre (=neuraminsyre) inneholder en karboksyl-gruppe og gir som følge av dette en negativ ladning til mucinene: sure muciner. The mucins are made up of a long polypeptide chain (500-4000 amino acids) to which a short or somewhat longer carbohydrate chain is covalently bound to the amino acids serine (Ser) and threonine (Thr) via N-acetylgalactosamine (GalNAc). The mucins are characterized by their terminal sugars: a. sialic acid (=neuraminic acid) contains a carboxyl group and, as a result, gives the mucins a negative charge: acidic mucins.
b. fukose inneholder ingen ladet gruppe: nøytrale muciner. b. fucose contains no charged group: neutral mucins.
Både sialo- og fukomuciner kan inneholde sulfatgrupper. Both sialo- and fucomucins can contain sulfate groups.
En mucins fysikalske og biologiske egenskaper bestemmes i høy grad av polypeptidkjedens lengde og sammensetning. Disse bestemmer antallet mulige karbohydratkjeder. på grunn av at karbohydratkjedene er meget hydrofile har mucinene et stort vannhylster rundt seg, hvorved de opptar et stort volum i vandig løsning. Vannhylsteret bevirker slimegenskapene. A mucin's physical and biological properties are largely determined by the length and composition of the polypeptide chain. These determine the number of possible carbohydrate chains. due to the fact that the carbohydrate chains are very hydrophilic, the mucins have a large water envelope around them, whereby they occupy a large volume in aqueous solution. The water envelope causes the slime properties.
Mucinene har forskjellige funksjoner i munnhulen: The mucins have different functions in the oral cavity:
De gjør spyttet viskøst slik at det ikke flyter lett, They make the saliva viscous so that it does not flow easily,
de fukter tannoverflaten og hinnen og beskytter derved munnen fra å tørke opp, they moisten the tooth surface and membrane and thereby protect the mouth from drying up,
de beskytter slimhinnen mot bakterieinfeksjon ved at de danner et slimlag som er vanskelig å penetrere for bakterier, they protect the mucous membrane against bacterial infection by forming a mucus layer that is difficult for bacteria to penetrate,
de beskytter tennene mot avkalking og syreangrep, they protect the teeth against decalcification and acid attack,
de danner en barriere mot H+<->ionene, og they form a barrier against the H+<->ions, and
de aggregerer visse orale bakterier og gjør derved disse uskadelige. they aggregate certain oral bacteria and thereby render them harmless.
Humant slimhinnespytt oppnås ved å stimulere avsondring ved hjelp av noen få sitronsyrepellets i tungen mens kanal-åpningene for glandola parotis dekkes med en Lashley-kopp som er forbundet med utløpsrør. Dette resulterer i meget viskøst spytt som særlig kommer fra de store spyttkjertler glandula sublingualis og glandula submandibularis, slik at det betegnes SM-SL-spytt. Dette slimhinnespytt oppsamles i et iskjølt glassbeger som inneholder 3 0 Human mucosal saliva is obtained by stimulating secretion using a few citric acid pellets on the tongue while covering the parotid gland canal openings with a Lashley cup connected to an outlet tube. This results in very viscous saliva that particularly comes from the large salivary glands glandula sublingualis and glandula submandibularis, so that it is termed SM-SL saliva. This mucosal saliva is collected in an ice-cooled glass beaker containing 3 0
0,1% fenylmetylsulfonsyrefluorid (PMSF) per 3 0 ml anvendes som en inhibitor av proteolytisk dekomponering. Spyttet holdes ved -20°C inntil tilstrekkelig utgangsmateriale er blitt oppsamlet. 0.1% phenylmethylsulfonic acid fluoride (PMSF) per 30 ml is used as an inhibitor of proteolytic decomposition. The saliva is kept at -20°C until sufficient starting material has been collected.
På grunn av mucinenes høye molekylvekt Because of the mucins' high molecular weight
(500.000-2.000.000 dalton) kan de separeres fra de kuleformede spyttproteiner med hjelp av ultrasentrifugering, se Roukema et (500,000-2,000,000 daltons) they can be separated from the globular salivary proteins with the help of ultracentrifugation, see Roukema et
al., Biochim. Biophys.Acta, Vol. 428, 1976, p. 432-440 og Roukema and Nieuw Amerongen i Saliva and Dental Caries, 1979, p. 68-80. al., Biochim. Biophys.Acta, Vol. 428, 1976, pp. 432-440 and Roukema and Nieuw Amerongen in Saliva and Dental Caries, 1979, pp. 68-80.
Fremgangsmåten ved isolering av muciner fra humant spytt er følgende: is av spyttet og tilsett N-acetylcystein til slutt-konsentrasjonen på 0,02 M, The procedure for isolating mucins from human saliva is as follows: freeze the saliva and add N-acetylcysteine to the final concentration of 0.02 M,
omrør med magnetrører i 3 minutter, stir with a magnetic stirrer for 3 minutes,
sentrifuger ved 10.000 x g i 10 minutter, centrifuge at 10,000 x g for 10 minutes,
dialyser den oppå liggende væske natten over, dialyze the supernatant overnight,
sentrifuger bunnfallet ved 100.000 x g i 2 timer. Som følge av lav pH (ca. 3) er mucinene høyviskøse og sedimenteres . centrifuge the pellet at 100,000 x g for 2 hours. As a result of the low pH (approx. 3), the mucins are highly viscous and sediment.
Suspender sedimentet i en buffer med pH på 7,15, bestående av 0,11 M NaCl + 0,014 M Na2HP04 + 0,056 M NaH2P04, etterfulgt av sentrifugering ved 100.000 x g i 1 time, Suspend the sediment in a buffer of pH 7.15, consisting of 0.11 M NaCl + 0.014 M Na2HP04 + 0.056 M NaH2PO4, followed by centrifugation at 100,000 x g for 1 hour,
sentrifuger deretter den klare oppå liggende væske ved 100.000 x g i 24 timer for å sedimentere mucinene, then centrifuge the clear supernatant at 100,000 x g for 24 hours to sediment the mucins,
suspender bunnfraksjonen i en Na-acetatbuffer med pH 4,5, og varm den ved 100°C i 10 minutter og sentrifuger den i en bordsentrifuge, suspend the bottom fraction in a Na-acetate buffer of pH 4.5 and heat it at 100°C for 10 minutes and centrifuge it in a benchtop centrifuge,
overfør den oppå liggende væske til sentrifugerings-bufferen med pH 7,15 og sentrifuger ved 100.000 x g i 24 timer, samt transfer the supernatant to the centrifugation buffer with pH 7.15 and centrifuge at 100,000 x g for 24 hours, and
suspender sedimentet i destillert vann og dialyser grundig. suspend the sediment in distilled water and dialyze thoroughly.
Bunnfallet tørrfryses og inneholder nå den totale mucin-fraksjon i humant spytt, benevnt HSWM: totale humane spyttmuciner. Når denne fraksjon ble anvendt til eksperimenter ble det først testet på fravær av forurensende proteiner ved hjelp av polyakrylamid-gelelektroforese. The precipitate is freeze-dried and now contains the total mucin fraction in human saliva, called HSWM: total human salivary mucins. When this fraction was used for experiments, it was first tested for the absence of contaminating proteins by means of polyacrylamide gel electrophoresis.
Som anført ovenfor består muciner av en lang polypeptidkjede hvorom det befinner seg en tett pakking av kortere eller lengre oligosakkarider som beskytter polypeptidkjeden fra proteolytisk dekomponering. Grovt regnet består mucinene av 10-30% protein og 70-90% sukker. HWSM består av ca. 18% protein ifølge aminosyreanalyse. De viktigste aminosyrer i HSWM er treonin (14,7%), valin (12,6%), serin (10,8%) og pro-lin (8,4%), se tabell A. En karbohydratkjede kan være kovalent bundet til hydroksylgruppen i treonin- og serinaminosyrene via bindingssukkeret N-acetylgalaktosamin (GalNac). Den kjemiske sammensetning hos humane spyttmuciner er angitt i tabell B. As stated above, mucins consist of a long polypeptide chain around which there is a dense packing of shorter or longer oligosaccharides that protect the polypeptide chain from proteolytic decomposition. Roughly speaking, the mucins consist of 10-30% protein and 70-90% sugar. HWSM consists of approx. 18% protein according to amino acid analysis. The most important amino acids in HSWM are threonine (14.7%), valine (12.6%), serine (10.8%) and proline (8.4%), see table A. A carbohydrate chain can be covalently linked to the hydroxyl group in the threonine and serine amino acids via the binding sugar N-acetylgalactosamine (GalNac). The chemical composition of human salivary mucins is given in Table B.
Karbohydratkonsentrasjonen i HWSM er 74%, omfattende 5,1% sialinsyre og 17,4% fukose som endegruppesukker. Sialinsyre er av viktighet for den negative ladning, som følge av at dette sukker inneholder en ionisert COO~-gruppe, til forskjell fra fukose. I tillegg inneholder HSWM som negativ ladete funksjonelle grupper sulfat (1,4%) og fosfat (0,14%) samt også karboksylgrupper av aminosyrene asparaginsyre (6,1%) og glut-aminsyre (7,3%) . The carbohydrate concentration in HWSM is 74%, comprising 5.1% sialic acid and 17.4% fucose as end group sugar. Sialic acid is important for the negative charge, as a result of this sugar containing an ionized COO~ group, unlike fucose. In addition, HSWM contains negatively charged functional groups sulfate (1.4%) and phosphate (0.14%) as well as carboxyl groups of the amino acids aspartic acid (6.1%) and glutamic acid (7.3%).
Når det gjelder mucinenes struktur består en mucin av en lang, strukket polypeptidkjede hvori 1 i 3-4 aminosyrer er treonin eller serin hvortil det kan være bundet en kort karbohydratkjede. Bindingssukkeret til disse to hydroksyaminosyrer er alltid N-acetylgalaktosamin (GalNac). Karbohydratkjedene er bestemt av typen mucin. Ytterligere et (ende) sukker kan være bundet til N-acetylgalaktosaminet, f.eks. aminet sialinsyre (NANA) i mucin fra underkjeven hos sau (OSM), men der kan foreligge flere sukker, såsom i HWSM. Regarding the structure of mucins, a mucin consists of a long, stretched polypeptide chain in which 1 in 3-4 amino acids is threonine or serine to which a short carbohydrate chain may be attached. The binding sugar of these two hydroxyamino acids is always N-acetylgalactosamine (GalNac). The carbohydrate chains are determined by the type of mucin. A further (terminal) sugar can be attached to the N-acetylgalactosamine, e.g. amino sialic acid (NANA) in mucin from the lower jaw of sheep (OSM), but there may be more sugars, such as in HWSM.
Eksempel på strukturen til OSM er vist i fig. 1. An example of the structure of OSM is shown in fig. 1.
Karbohydratkjedene er for det meste mer komplekse i sammensetning. Fig. 2 viser skjematisk et antall karakteri-stiske oligosakkaridkjeder i en rekke muciner. De anvendte forkortelser har følgende betydninger: The carbohydrate chains are mostly more complex in composition. Fig. 2 schematically shows a number of characteristic oligosaccharide chains in a number of mucins. The abbreviations used have the following meanings:
OSM, mucindisakkarid fra underkjeven hos sau, OSM, mucin disaccharide from the lower jaw of sheep,
PSM, mueinpentasakkarid fra underkjeven hos gris, PSM, mueinpentasaccharide from pig mandible,
PGM, mucinpentasakkarid med sulfat fra grisemage, PGM, mucin pentasaccharide with sulfate from pig stomach,
HWSM, muciner fra humant spytt. Fig. 2 viser et eksempel på en kompleks kjede. Det finnes andre komplekse oligosakkaridkjeder med sialinsyreendegrupper og også med- sulfat-bundne sukre. HWSM, mucins from human saliva. Fig. 2 shows an example of a complex chain. There are other complex oligosaccharide chains with sialic acid end groups and also co-sulfate-linked sugars.
I betydelig grad bestemmer karbohydratkjedene de spesi-fikke fysisk-kjemiske og biokjemiske egenskaper til en mucin, såsom dens bidrag til viskositet, vedheft til tannoverflaten, aggrering av bakterier etc. Tabell C angir en oversikt over To a significant extent, the carbohydrate chains determine the specific physicochemical and biochemical properties of a mucin, such as its contribution to viscosity, adhesion to the tooth surface, aggres- sion of bacteria, etc. Table C provides an overview of
den kjemiske sammensetningen til noen muciner. the chemical composition of some mucins.
I tillegg til at der er en forskjell i den kjemiske sammensetning hos muciner gjelder dette også deres stereostruktur. Der er muciner oppbygget av en polypeptidkjede (f.eks. OSM), men der er også muciner som via S-S-bindinger mellom cysteinkjeden i polypeptidkjeden er bundet sammen til dannelse av større kompleks, f.eks. PGM. Denne mucin er oppbygget av fire underenheter på hver 500.000 dalton, med en total molekylvekt på 2x10 dalton. Denne stereostruktur hos et mucin har også en virkning på de fysisk-kjemiske egenskaper, særlig viskositeten. In addition to the fact that there is a difference in the chemical composition of mucins, this also applies to their stereostructure. There are mucins made up of a polypeptide chain (e.g. OSM), but there are also mucins which via S-S bonds between the cysteine chain in the polypeptide chain are bound together to form a larger complex, e.g. PGM. This mucin is made up of four subunits of 500,000 daltons each, with a total molecular weight of 2x10 daltons. This stereostructure of a mucin also has an effect on the physico-chemical properties, especially the viscosity.
For bestemmelse av kvantitative data vedrørende adsorpsjon av HWSM til hydroksyapatitt, som en modell for tannemalje, ble utført som følger: 10 mg hydroksyapatitt (HAP) av Biorad (spesifikt areal 72,5 m 2 per g) ble vasket tre ganger med destillert vann, For the determination of quantitative data regarding the adsorption of HWSM to hydroxyapatite, as a model for tooth enamel, was carried out as follows: 10 mg of hydroxyapatite (HAP) of Biorad (specific area 72.5 m 2 per g) was washed three times with distilled water,
adsorbat (HWSM) løses i 2 mM K-fosfatbuffer (pH 6,3 eller 7,0) og tilsettes til HAP, adsorbate (HWSM) is dissolved in 2 mM K-phosphate buffer (pH 6.3 or 7.0) and added to HAP,
inkubering ved romtemperatur i 4 timer i lukkede rør ved kontinuerlig rotasjon av disse ved seks omløp per minutt, incubation at room temperature for 4 hours in closed tubes by continuous rotation of these at six revolutions per minute,
etter avslutning av inkuberingen sedimenteres HAP umiddelbart ved sentrifugering, after the end of the incubation, the HAP is sedimented immediately by centrifugation,
mengden adsorbert HWSM bestemmes ved hjelp av protein-og sialinsyrebestemmelse i den oppå liggende væske. the amount of adsorbed HWSM is determined by protein and sialic acid determination in the overlying liquid.
Ut fra disse data kan den maksimale bindingskapasitet for HWSM (N-verdi i ug adsorbater/m 2HAP) beregnes og bestemmes og bestemmes grafisk ved hjelp av Langmuir's adsorp-sjonsligning: From this data, the maximum binding capacity for HWSM (N-value in ug adsorbates/m 2HAP) can be calculated and determined and determined graphically using Langmuir's adsorption equation:
hvor where
np = likevektskonsentrasjon av adsorbat (mg/ml) A = adsorbert mengde (mg/m <2>) np = equilibrium concentration of adsorbate (mg/ml) A = adsorbed amount (mg/m <2>)
N = maksimal bindingskapasitet (mg/m 2) N = maximum binding capacity (mg/m 2 )
K = affinitetskonstant (ml/mg) K = affinity constant (ml/mg)
I tabell D er N-verdiene for atskillige muciner angitt. In Table D, the N values for several mucins are given.
Adsorpsjonen av OSM, PGM og HWSM er vist i fig. 3. The adsorption of OSM, PGM and HWSM is shown in fig. 3.
Mucinene fra humant spytt adsorberes ekstremt sterkt til hydroksyapatitt sammenlignet med dyremuciner. Den maksimale bindingskapasitet (N) for HWSM er 2640 ug/m 2HAP. De testede spyttmuciner av dyreopprinneIse (gris, PSM, sau OSM, storfe BSM) har imidlertid 10-20 ganger lavere maksimal bindingskapasitet. På den annen side inneholder spytt fra spyttkjertlene også proteiner som har en sammenlignbar maksimal bindingskapasitet som HWSMs kapasitet, f.eks. staterin- og prolinrike proteiner. Proteinene i spyttkjertelspytt. beskytter imidlertid ikke tannemaljen mot innvirkning av syre, noe HWSM gjør. The mucins from human saliva adsorb extremely strongly to hydroxyapatite compared to animal mucins. The maximum binding capacity (N) for HWSM is 2640 ug/m 2HAP. However, the tested salivary mucins of animal origin (pig, PSM, sheep OSM, bovine BSM) have a 10-20 times lower maximum binding capacity. On the other hand, saliva from the salivary glands also contains proteins that have a comparable maximum binding capacity to that of HWSM, e.g. staterin- and proline-rich proteins. The proteins in salivary gland saliva. however, it does not protect tooth enamel from the effects of acid, which HWSM does.
Som følge av at spytt inneholder mange typer proteiner, som hvert kan separeres separat til HAP, særlig fosfatholdige proteiner, er det blitt undersøkt om der er noen konkurranse når to komponenter samtidig inkuberes med HAP. Som en modell for en fosfatkomponent ble det valgt fytat, som utøver en sterk binding HAP (N = 700 ug/m 2 HAP). Fytat er heksakisfosfat av myoinositol, som kan isoleres fra korn. Det har en molekylvekt på 924. As a result of the fact that saliva contains many types of proteins, each of which can be separated separately into HAP, especially phosphate-containing proteins, it has been investigated whether there is any competition when two components are simultaneously incubated with HAP. As a model for a phosphate component, phytate was chosen, which exerts a strong binding HAP (N = 700 ug/m 2 HAP). Phytate is the hexakisphosphate of myoinositol, which can be isolated from grains. It has a molecular weight of 924.
Fytat er i stand til å hindre adsorpsjonen av OSM, selv ved meget lave konsentrasjoner. Når 3 00 ug OSM inkuberes med HAP vil 3 0 ug fytat nedsette adsorpsjonen av OSM til en verdi på helt ned til 50%, mens 300 ug fytat vil hindre adsorpsjonen av OSM fullstendig (fig. 4). Phytate is able to prevent the adsorption of OSM, even at very low concentrations. When 300 ug OSM is incubated with HAP, 30 ug phytate will reduce the adsorption of OSM to a value as low as 50%, while 300 ug phytate will prevent the adsorption of OSM completely (fig. 4).
Adsorpsjonen av PGM kan senkes til 3 0% fytat, men inhi-beringen av adsorpsjonen er mye langsommere enn i tilfellet med OSM. På den annen side har fytat bare en meget liten virkning eller ingen i det hele tatt på adsorpsjonen av HWSM. Selv ved høy fytatkonsentrasjon (600 ug) er minskningen i adsorpsjon av HWSM bare meget liten. Tilsynelatende fester HWSM seg mye sterkere til HAP enn det OSM og PGM gjør, noe som er i overenstemmelse med de beregnede N-verdier. The adsorption of PGM can be lowered to 30% phytate, but the inhibition of adsorption is much slower than in the case of OSM. On the other hand, phytate has very little or no effect on the adsorption of HWSM. Even at high phytate concentration (600 µg) the reduction in adsorption of HWSM is only very small. Apparently, HWSM adheres much more strongly to HAP than OSM and PGM do, which is consistent with the calculated N values.
Årsaken til forskjellen i adsorpsjonsbeteende kan skyldes forskjellen i kjemisk sammensetning og i funksjonelle grupper og i molekylenes konformasjon. The reason for the difference in adsorption behavior may be due to the difference in chemical composition and in functional groups and in the conformation of the molecules.
Tabell E gir en oversikt over antallet fosfat-, sulfat-og sialinsyregrupper per molekyl i de testede muciner med molekylvekt på 500.000 dalton. Table E gives an overview of the number of phosphate, sulfate and sialic acid groups per molecule in the tested mucins with a molecular weight of 500,000 daltons.
Som vist har PSM og OSM et meget stort antall sialinsyregrupper, men intet sulfat ojj fosfat. PGM har et lite antall sialinsyregrupper og inneholder 62 sulfatgrupper. På den annen side har HWSM et beskjedent antall sialinsyregrupper, men dessuten det høyeste antall sulfatgrupper og i tillegg 7 fosfatgrupper. Muligens resulterer kombinasjonen av sialinsyre - sulfat - fosfat i den ekstremt sterke adsorpsjon av HWSM til HAP. As shown, PSM and OSM have a very large number of sialic acid groups, but no sulfate or phosphate. PGM has a small number of sialic acid groups and contains 62 sulfate groups. On the other hand, HWSM has a modest number of sialic acid groups, but also the highest number of sulfate groups and additionally 7 phosphate groups. Possibly the combination of sialic acid - sulfate - phosphate results in the extremely strong adsorption of HWSM to HAP.
Under anvendelse av to forskjellige fremgangsmåter er det blitt testet hvorvidt HWSM kan hindre avkalking (de-mineralisering) av tenner: Using two different methods, it has been tested whether HWSM can prevent decalcification (de-mineralization) of teeth:
når HWSM adsorberes til HAP løses bare lite Ca av mineralsyren. Med fosfatholdige forbindelser, såsom fytat, løses imidlertid mye mer Ca , noe som vil fremgå av tabell F nedenfor. when HWSM is adsorbed to HAP, only a little Ca is dissolved by the mineral acid. However, with phosphate-containing compounds, such as phytate, much more Ca is dissolved, which will appear from table F below.
tannemalje fra menneske og storfe ble etter innleiring og polering inkubert med: After embedding and polishing, tooth enamel from humans and cattle was incubated with:
a. SM-SL-spytt, dialysert, a. SM-SL saliva, dialyzed,
2+ b. SM-SL-spytt, dialysert, deretter tilsetning av Ca til 1 mM, 2+ b. SM-SL saliva, dialyzed, then addition of Ca to 1 mM,
c. spyttkjertelspytt, uten dialyse, med og uten til- c. salivary gland saliva, without dialysis, with and without
2+ 2+
setning av Ca statement of Ca
Inkubering foregikk i 7 dager med 20 ml spytt/tann. Spyttet ble oppfrisket hver dag. Deretter ble tennene inkubert i 1 prosentig sitronsyreløsning i 1 minutt for å bevirke avkalking. Denne kan bestemmes ved overflatemåling. Det er bare SM-SL-spyttet hvortil det etter dialyse er blitt tilsatt ImM Incubation took place for 7 days with 20 ml saliva/tooth. The spit was refreshed every day. The teeth were then incubated in 1 percent citric acid solution for 1 minute to effect decalcification. This can be determined by surface measurement. It is only the SM-SL saliva to which ImM has been added after dialysis
2+ 2+
Ca som gir en beskyttelse mot avkalking opp til 100%. Tilsynelatende er Ca 2+ vesentlig for å hindre avkalking. Ca which provides protection against descaling up to 100%. Apparently, Ca 2+ is essential to prevent decalcification.
I tillegg er det blitt gjort sammenligningsforsøk med 0,1 prosentig HWSM-løsning i ImM Ca 2 +, pH 7,0. Fra så kort-varig inkubering som 3 0 minutter kan det påvises tydelig in-hibering av avkalkingen, noe som øker til 100% med tiden. In addition, comparison tests have been carried out with a 0.1 percent HWSM solution in ImM Ca 2 +, pH 7.0. From as short-term incubation as 30 minutes, a clear inhibition of the descaling can be demonstrated, which increases to 100% with time.
Disse forsøk viser at adsorpsjonen av proteiner til tannemalje ikke nødvendigvis betyr at tennene også beskyttes mot avkalking. Proteiner fra spyttkjertler adsorberes, men hindrer ikke avkalking, til forskjell fra HWSM. These experiments show that the adsorption of proteins to tooth enamel does not necessarily mean that the teeth are also protected against decalcification. Proteins from salivary glands are adsorbed, but do not prevent decalcification, unlike HWSM.
Spytt inneholder forskjellige høymolekylære proteiner, som er i stand til å aggregere forskjellige typer mikroorganismer i munnhulen, slik at disse ikke lenger er i stand til å danne kolonier. Etter aggregering føres disse mikroorganismer videre til fordøyelseskanalen. Saliva contains various high-molecular proteins, which are able to aggregate different types of microorganisms in the oral cavity, so that these are no longer able to form colonies. After aggregation, these microorganisms are carried on to the digestive tract.
En av disse høymolekulære spyttbestanddeler har vist seg å være HWSM. Muciner fra humant spytt er allerede i lav kon-sentrasjon i stand til å forårsake aggregering av forskjellige typer mikroorganismer i munnhulen. One of these high molecular weight salivary constituents has been shown to be HWSM. Mucins from human saliva are already in low concentration able to cause aggregation of different types of microorganisms in the oral cavity.
For å fremstille tilstrekkelige kvanta kommersielt til-gjengelige muciner for anvendelse i tannpasta er det ønskelig å kunne fremstille dem syntetisk. Siden strukturen til en mucin er forholdsvis enkel er dette en realistisk mulighet. For dette formål kan polyserin og polytreonin, som består av kjeder på 500-4000 aminosyrer, en molekylvekter på fra 50.000 til 400.000, anvendes som utgangsmaterialer for polypeptidkjeden. Deretter koples bindingssukkeret N-acetylgalaktosamin til serin- og treonin. Dette kan utføres enten ved hjelp av en organisk kjemisk reaksjon eller ved hjelp av en enzymatisk reaksjon under anvendelse av N-acetylgalaktosamintransferase. In order to produce sufficient quantities of commercially available mucins for use in toothpaste, it is desirable to be able to produce them synthetically. Since the structure of a mucin is relatively simple, this is a realistic possibility. For this purpose, polyserine and polythreonine, which consist of chains of 500-4000 amino acids, a molecular weight of from 50,000 to 400,000, can be used as starting materials for the polypeptide chain. The binding sugar N-acetylgalactosamine is then linked to serine and threonine. This can be carried out either by means of an organic chemical reaction or by means of an enzymatic reaction using N-acetylgalactosamine transferase.
En egnet reaksjonsblanding for N-acetylgalaktosamintransferase er: A suitable reaction mixture for N-acetylgalactosamine transferase is:
1,3 mg serumalbumin 1.3 mg of serum albumin
0,29 nmol UDP-N-acetylgalaktosamin 0.29 nmol UDP-N-acetylgalactosamine
0,375 u mol ditiotreitol 0.375 u mol dithiothreitol
1,2 5 mol MnCl21.2 5 mol MnCl2
0,5 mg polyserin og polytreonin 0.5 mg polyserine and polythreonine
7,5 u mol HEPES-buffer, pH 6,8 7.5 µmol HEPES buffer, pH 6.8
Det totale volum av reaksjonsblandingen er 125 ul. Inkuberingen utføres ved 37°C i 60 minutter. (KO og Raghupathy, Biochem. Biophys. Res. Comm., Vol. 46, 1972, p. 1704). The total volume of the reaction mixture is 125 µl. The incubation is carried out at 37°C for 60 minutes. (KO and Raghupathy, Biochem. Biophys. Res. Comm., Vol. 46, 1972, p. 1704).
Deretter koples en sianinsyrerest til N-acetylgalaktosaminet ved hjelp av et spesifikt enzymsialyltransferase, som finnes i spyttkjertlene. A cyanic acid residue is then linked to the N-acetylgalactosamine by means of a specific enzyme sialyltransferase, which is found in the salivary glands.
En egnet sialyltransferasereaksjonsblanding er: A suitable sialyltransferase reaction mixture is:
1 mg polyserin-N-acetylgalatosamin eller polytreonin-N-acetylgalaktosamin 1 mg polyserine-N-acetylgalactosamine or polythreonine-N-acetylgalactosamine
1 mg sialyltransferase fra spyttkjertler 1 mg sialyltransferase from salivary glands
800 |i g serumalbumin fra storfe 800 µg bovine serum albumin
80 u mol Trismaleat-buffer pH 6,8 80 u mol Trismaleate buffer pH 6.8
2,9 u mol CMP-NANA 2.9 µmol CMP-NANA
Inkubering finner sted ved 37°C i 3-6 timer. (Van den Eijnden et al., Biochem., Biophys. Res. Comm., Vol. 92, 1980, p. 839-845. Incubation takes place at 37°C for 3-6 hours. (Van den Eijnden et al., Biochem., Biophys. Res. Comm., Vol. 92, 1980, p. 839-845.
Ved hjelp av denne reaksjon kan sialinsyre bindes til N-acetylgalaktosamin via en cK-(2-6)-binding. Et eksempel på strukturen til det derved oppnådde produkt er vist i fig. 5. By means of this reaction, sialic acid can be bound to N-acetylgalactosamine via a cK-(2-6) bond. An example of the structure of the thus obtained product is shown in fig. 5.
Om ønskelig kan det utføres variasjoner i sukkerkjeden. Fordelen med en slik kunstig mucin er at den vil være bio-logisk nedbrytbar som følge av at den inneholder naturlige peptid- og glykosidbindinger. If desired, variations can be made in the sugar chain. The advantage of such an artificial mucin is that it will be biologically degradable as a result of the fact that it contains natural peptide and glycosidic bonds.
Anvendelse Application
Tannpasta Toothpaste
De foreslåtte muciner, som bidrar til beskyttelse av tannemaljen mot avkalking kan tilsettes til en tannpasta. Av vesentlig viktighet for aktiviteten til muciner er at de ikke bindes til poleringsmidlet (ofte CaCO^) i tannpastaen. Det har overraskende vist seg at muciner tilfredsstiller denne betingelse. The proposed mucins, which contribute to the protection of tooth enamel against decalcification, can be added to a toothpaste. Of essential importance for the activity of mucins is that they do not bind to the polishing agent (often CaCO^) in the toothpaste. It has surprisingly been shown that mucins satisfy this condition.
En tannpasta som har vist seg å være egnet har følgende sammensetning (i vektdeler): A toothpaste that has proven to be suitable has the following composition (in parts by weight):
En slik tannpasta som inneholder HWSM eller syntetiske analoger på basis av polyserin eller polytreonin i lav kon-sentrasjon, har vist seg effektivt å hindre avkalkingen av tennene som følge av syreløsning. I tillegg er HWSM i stand til å aggregere forskjellige typer mikroorganismer i munnhulen og derved hindre kolonidannelse av disse. Dette resulterer i at mindre syre dannes i munnhulen. Such a toothpaste containing HWSM or synthetic analogues based on polyserine or polythreonine in low concentration has been shown to effectively prevent the decalcification of the teeth as a result of acid solution. In addition, HWSM is able to aggregate different types of microorganisms in the oral cavity and thereby prevent their colonization. This results in less acid forming in the oral cavity.
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NO860853A NO170392C (en) | 1986-03-07 | 1986-03-07 | MATERIALS FOR PROTECTION OF DENTAL ELEMENTS AGAINST ACID CAUSAL DESCALING |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NO860853A NO170392C (en) | 1986-03-07 | 1986-03-07 | MATERIALS FOR PROTECTION OF DENTAL ELEMENTS AGAINST ACID CAUSAL DESCALING |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| NO860853L NO860853L (en) | 1987-09-08 |
| NO170392B true NO170392B (en) | 1992-07-06 |
| NO170392C NO170392C (en) | 1992-10-14 |
Family
ID=19888790
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO860853A NO170392C (en) | 1986-03-07 | 1986-03-07 | MATERIALS FOR PROTECTION OF DENTAL ELEMENTS AGAINST ACID CAUSAL DESCALING |
Country Status (1)
| Country | Link |
|---|---|
| NO (1) | NO170392C (en) |
-
1986
- 1986-03-07 NO NO860853A patent/NO170392C/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| NO170392C (en) | 1992-10-14 |
| NO860853L (en) | 1987-09-08 |
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