nitrogen content of dental pulp

7/29/2019 Nitrogen Content of Dental Pulp

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lNITROGEN CONTENT OF DENTAIIPULP

IIARVTN L. RTEDESEL, B.A., M.S.andALTON K. FISHER, 8.S., D.D.S.l)epartment of Oral Pathology, College ofDentistry, State Lrniversity of Ion.a.Iorva City, Iol.a

Reprintecl fromJOURNAL OF DENTAI, RESEARCH

St. LouisVol.33, No.5, Pages 632-636, October,1954

(Printed in the U. S. A.)


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NITROGDN CONTENT OF DENTAI, PUI,PMARVTN L. RTEDESEL, B.A., M.S., AND ALTON r{. rrsHER, 8.S., D.D.S.

Degtartment of OraI PathologE, Cotlege of Dentistry, Stat? aniaersity of loua'Iowa Ci'19, IowaINTRODUCTION

f)OSSIBITY beeause of its small size, the indiyidual dental pulp has notI been the subject of extensive quantitative physiologic study. Pincuslreported his observations relating to tissue respiration anal certain aspects ofchemical composition as cletermined from pooled pulps of freshly extracteclhuman teeth. However, the use of poolecl pulps obliterates individuaipeculiarities that might exist ancl that might be of significance from a normalphysiologic or pathologic standpoint. Furtherrnore, gross inethods necessitat-ing pooled samples impose a sometimes clifficult requirement of sufficientindividual specimens to permit even a minimum of su.ch studies. It is highlydesirable, therefore, to clevelop or adapt micro and ultramicro methods to therestrictions inherent in the pulp itself.If respiratory actir.ity of the individual dental pulp is to be stuclied,,micromanometric procedures are necessary. Ilorver.er, the total volume oftissue requirecl in these procedures' is sr-rfflcientiy small that clifflculty isencountered in routine gravimetric detelminations of the mass. Nevclthe-Iess, an accurate measrlre of mass, or its equivalent, is impcrative as a referenceunit to rvhich respiratory phenomcna crn be relatecl. Total nitrogen has beenused as such a measure ancl also has been recommentlecl as a stanilarcl ofreference to which oxygen consurnption can be relatetl.3' aPincusl repolted the nitrogen content of human tooth pulp as 10.1 percent of the dry weight. This value presuirrably rvas cletet:mined by the macro-Kjelclahl method. It was clecidecl, therefore, to inyestigate this pro.[lemfurther by the same method ancl then to attempt sirnilar cletermina.tions bythe micro methocl with the hope that ultrainicro methocls would becomefeasible eventually. Because variotts types of hnm:rrr clental pulp are notalways available for analytic pulposes, it was consiclered aclr.isable toinvestigate also the possible effects on nitrogen cletermination that mightresult from presen ation of this tissue in 10 per cellt aqueous formalin solution.

I{ATER,IAI,S AND,I{ETHODSThe tissues investigatecl were the pulps of bovine and human teeth. Thebovine material came flom animals between 2 ancl 3 years of age. The molarsBased on a thesis submitted by Marvin L. Riedesel in partial fulflllment of the require-ments for the degree of X{aster of Science in the Departrnent of Physiology in the GraduateCollege of the State Lhiversity of Ios'a.This investigation rvas supported in part by resealch grant D-126 from the United StatesPublic Health Service.Received for publication, Auei. 3, 1953.

632


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Volume 3 3Number 5 NITR,OGEN CONTENT OT' DENTAL PULPwere extracted and placed in cold Ringer's solution as soon as killing ancldecapitation had been accomplished. The teeth were then taken to thelaboratory, where they were cracked open and the pulps removed. The pulpswere carefully examined uncler a stereoscollic microscope in order to removeall fragments of enamel and dentin. Some of the pulps were pleserved ina 10 per cent aqueous formalin solution, and others were dried immediately,as indicated in Tables I and II. The pulps were cut into small pieces ofapproximately equal size to facilitate even dehydration and rvere placed in aconstant-temperature drying oven. The samples of Groups A, B, C, and Dof Table I and Group A of Tabie II were dried to a constant weight attemperatures between 95o C. and 100o C. The samples in the remaininggroups were dried. to a constant weight at 560 C. The longer drying at thelower temperature is recommendecl by the Association of Official AgriculturalChemists.'

TABLE INrrnocnx CorvrrNr oI FnEsnty#J"rit+ltilii?3"."t*r

PULP TrssuE DrtnnnrNnnrrEAN I neNcc I(MG. N PER | 1mc. w "nn Il\rc- Dnv I uc. nnv I st-tNr',rnu\vErG.r.rr) | wnrcnr; Iouur,r.,osISSIIEA !'resh Macro-KjeltlahlB !'orrne.lin Macro-KjeklahlC Fresh Nessleri-zationD }'ormalin Nessieri-zationE Fresh X{icro-KjeldahlE Tornalin X{iolo-Kieldahl

METIIODot'IN.\T,\SISNO. OFDETERI{I-NATIONS

1411

7227525

R.\NGE OI'SAMPLE(MG. DriYwnrGlr!')150-4071 1 1-4071.3-2.67.3-2.(i20-2920-29

STANDAR,DDEVIATIONOF ME-I\N0.1257 0.7225- 0.0014 0.00040.128.10.1231-0.12860.08-0.150.08-0.150.1198-0.1336().1l77 -0.14;J7

0.12560.1200.1170.1273rl 1tlo

0.001_6 0.00050.013 0.0010.015 0.0020.0054 0.00110.0072 0.0014

TABLE IINrrnocrN Cowrrnr oF BovrNE AND HUMAN DDNTAT, PULP AS DETEB,MTNED BYI


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634 RIEDESEL AND FISHFJR J. D. Res.Ocrober.195,1rn accordance with the varying lirnitations of each analytic method, thesample sizes were of different magnitudes. rn some cases, as in Groups A andB of rable r, 1 to 3 bovine molar pulps were required to provide a singlesample. In other instances a single pulp 'lvoulcl furnish enough tissue for 1to 3 samples. fn Grou.ps O, D, E, and F of Table I and Groups B and C of

Table II a singie pulp was the source of from 1 to 10 samples.The human material r,vas of two types. The flrst type was from 6 infantheads obtained at autopsy (3 infants were born at full term and died shortiyafter birth, 3 were stillborn after eight months of gestation) and preservecl in10 per cent formalin solution from two months to four years. The toothgerms were clissected from the jaws, and the pulps were removed with theaid of a stereoscopic microscope. Each sample in Group D, Table rr, requiredthe pooling of from 4 to 9 pulps.The second type consistecl of the pulps of freshly extracted, noncariousteeth which were placerl in 10 per cent formalin solution immecliately afterextraction. Twerrt).tu,'o patients whose ages rangeil from 14 to 48 yearscontributed 35 teeth. The majority of the teeth were removed because of thenecessities of orthodoirtic or periodontic treatment. \Vithin several hours ofextraction, the teeth were cracked open and the pulps removed and dricd aspreviously described. The numbel of pulps requirecl to make up a siriglesample varied from 1 to 17.The dry weight of most of the samples was determined on a ChristianBecker chainoinatic analytical balance with a sensitivity of 0.1 mg. The dryweight measurements of human and bovirie samples smaller than 15 mg.,which were analyzecl by the micro-Kjeldahl method, were determined on anAinsworth keyboarcl-operated weight-carrier microbalance with a sensitivityof 2.5 pg.Freshly prepared conductivity water as clescribed by Fales and Kenny6was employed in the preparation of all solutions used.IIacro-KjeIdahl determinations were made according to the methoddescribed by Campbell,' with the exceptions that boric acid was used tocollect the distilled ammonias and bromcresol green-methyl red indicator wasused.e By employing the boric acid solution in place of a standard acid thereis one less solution to standardize and thus one less chance for error. Thebromcresol green-methyl red indicator gir.es a \rery distinct eolor changefrom blue to colorless.The nesslerization method described by Hawk and Bergeimlo was em-ployed in the analyses of Groups C and D of Table I. The colorimetriccleterminations were ma,de on a Klett-Summerson photoelectric colorimeter.The micro-Kjeldahl methotl used was that described by Beattyll with theaddition of 30 per cent hydrogen peroxide to the digestion mixture as sug-gesteil by Hawk and Bergeim.lo lMhen analyzing samples of 2.2 Lo 3.5 mg. ofdry weight, proportionately smaller quantities of reagents were employed.The apparatus employed was essentialiy that described by Keys1, except thata vacuum was not employed.


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-V,OIUMC 3.3 NITROGEN CONTENT OF DENTAL PULPNumber 5Before any method of analysis lvas employecl in the coliection of datain this series, it was tried experimentally until confidence in its use andproficiency in its technics were attainecl. The accuracy of each methocl wascheckecl by determining the nitrogen content of measured quantities of knownsolutions of reagent grade ammonium sulfate.

ITES U LTSThe observations reported were derived from a total of 304 cleterminationsof total nitrogen by the several methods referred to previously and asindicated in Tables I and II. \Vhen fresh bovine dental pulp and that pre-served in 10 per cent formalin solution were analyzed by the macro-Kjeldahl,micro-Kjeldahl, and nesslerization methods, there was substantial a,greementin the nitrogen yields of each type of tissue (Table I). While the meannitrogen values for formalin-preservecl tissue appear slightly lower than forthose of fresh tissue, this difference is not statistically significant.\\rhen comparing mean nitrogen cotrtent as determined by the variousanalytic methods, it may be observed from Table I that the lowest mean yield

is obtained in using the nesslerization technic. Since the macro-Kjeldahlmethod is presumed to be productive of the most accrlrate results, it servetlas a guide in evaluation of the other methocls. I,Iany laboratory workers areof the opinion that the lower values obtained by nesslerization are charac-teristic of the method. l\rhen this quality became clearly evident, the methodwas abandoned.Inspection of Tables I ancl II shows close correspondence between themean nitrogen values obtained by the macro- and micro-Kjeldahl methods.As might be expected, the range of individual values is greater when workingon the micro level. The mean total nitrogen of bovine dental pulp, computedfrom all data derivecl from both macro- and micro-Kjeldahl analyses, wasobserved to be 0.1258 mg. per milligram of dried tissue (Table III).

TAB],8 IIIC O I{ PAR' SO N O F MTAN NITRO GH,S"#iT:f,,XT,:J' il.'.iil3"5u MAN D ENTAL PUIP S

RANGE OFSAT[PLE(r[c. DRY$'ErcHT)ISSI]ENO. OFDETERMI.N,\TIONS

MEAN(Mc. N PERMG. DRYWEIGHT)R,ANGE(Mc. N PERMG. DR,YWEIGHT) STANDARDDEVIATION

STANDAIiDDEVIATIONOF MEANBovinefresh andf ormalinHumanf ormalin

2.2-407

2.5-20.80.1258

0. 1 050

0.1.177-0.7137

0.1000-0.11400.0037 0.000420.0031 0.0006

Ifuman dentai pulp preserved in 10 per cent formalin solutioh wasanalyzed by the micro-Kjeldahl method only (Table II). The mean totalnitrogen values of infant and more mature tissue were observed to be almostidentical. The mean nitrogen content of human dental pulp was found to be0.1050 mg. per milligram of dried tissue (Table III).


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RII]DXS]IL AND FISHER J. D. Res.October,1954DISCUSSIONA comparison of mcan total nitrogen of bovine and human pulp tissue(Table III) rel'eals a significant differcnce. Sincc the series of human materialshows no age differcnces, and the macro- and micro-Kjeldahl methods comparevery favorably as to reliability, it is suggested that this difference is an ex-pression of species peculiarity.A critical comparison of mean nitrogen content of human dental pulpobserved in this investigation with that reported by Pincus' is difflcr:Llt bccausehe does not elaborate on his rncthods in this regard. But since he does 1'cportthat "nitrogen (Kjeldahl) rvas 10.1 per cent on a moisture and. ash frecbasis. .," the results of this study suggest that the nitrogen value he ob-tained is too low.SUM}[ARY

1. The mean total nitrogen (Kjeldahl) of hnman clental pulp was obselvedto be 0.1050 mg. per milligram of clried tissue and that of bovine pulp 0.1258mg. It is suggested that this difference is a species peculiarity'2. Age of the indiviclual does not appear to influence the nitrogen contentof human dental pulp.3. Preservation of clental pulp in 10 per cent aqueous formalin solutiondoes not significantly alter its suitability for nitrogen analysis by the Kjelcltrhlmethod.

REX'ERENCES1. P.incus, P.: Some Pirysiologir Dare on Tluman Deillal Pulp, Br.it. D. J.89:_143, 1950.l. Lirrtlerslrom-Lang, K.: Oi the 'I heory of the Cartesian Dir er Xlicro Xespilometer'Compt. rendl'd. tral). d,w tab. Carlsberg, stri,e chemi'r1we 24: 333' 1943-3. Burris, R. H., and wilson, P. w.: Measures of Respiratory Activity with Resting cells,Proa. Sob. Euper. B'ioL & Med. +5i 721' 1939.4. Moulton, C. R.: -Units of Reference foi'Basal l{etabolism and Their Interrelation-ships, ./. Bi,ol. Chem.2+i 299,1916.5. Lepper, -I{. A.: Official Nlethods of the Association of Ofrcial Agricultural Chemists,''ed. 7. \\'ashirston. 1950, Tlre Assor'jation of Official Agricullulal Chemists, p. .159.6. Fales, i{.'A., and Keniy, F.: Inorganic Quanlitaiive -A.nalysis, nerv eclition, Nerv York,1939, D. Appleton-Century Company, Inc., p. 56'7. Campbell,' W. R.; ai-d Htmna;M. f;r 'The Delermination of Nitrogen by Modifieci Kjel--dahl Metlrods, J. Biol. Cliem. lI9: 1, 1C37.8. nleeker, E., and Wagner, E. C.: Tili'rtjon of -Ammonia jn Presence of Boric Aci,1,Indust. S Engin. Chen. (AttalAt. Ed.) 5:396, 1933.9. Sobel, A. E.,'Me;,er, A. 1\[., and Gottfried, s. P.: A convenient Titrometric LTltlamicro-riethocl' for- the Estimation of Urea and Kjeldahi Nitrogen, J. BioI. Chem- 756:355, 791+.10. Hawk, P. B., and Bergeim, o.: Practical Physiological chemistry, eci. 11, Philadelphia,.1937, P. Blakiston's Son & Co.' p. 70_7-.11. Beatty, C., III: Anhydrous Coppei Sulfate in the Kjelt1ahl Nitrogen Determinations"Indust. $ Engin. Chem. (Analyt. Ed'.) L5: 476, 1943.12. Keys, A.: A Rapitl Xficro-Kjeldahl Method, J. BioI. Chem.732:181, 1940.

nitrogen content of dental pulp

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