l

NITROGEN CONTENT OF DENTAII PULP

IIARVTN L. RTEDESEL, B.A.,
and M.S.

ALTON K. FISHER, 8.S., D.D.S. l)epartment of Oral Pathology, College of Dentistry, State Lrniversity of Ion.a. Iorva City, Iol.a
Reprintecl from

JOURNAL OF DENTAI, RESEARCH St. Louis

Vol.33, No.5, Pages

632-636, October,1954

(Printed in the U. S. A.)

NITROGDN CONTENT OF DENTAI, PUI,P MARVTN L. RTEDESEL, B.A., M.S., AND ALTON r{. rrsHER, 8.S.,
Iowa
Ci'19,

D.D.S.

Degtartment of OraI PathologE, Cotlege of Dentistry, Stat? aniaersity of loua'

Iowa

INTRODUCTION

f)OSSIBITY beeause of its small size, the indiyidual dental pulp has not been the subject of extensive quantitative physiologic study. Pincusl reported his observations relating to tissue respiration anal certain aspects of chemical composition as cletermined from pooled pulps of freshly extractecl human teeth. However, the use of poolecl pulps obliterates individuai peculiarities that might exist ancl that might be of significance from a normal physiologic or pathologic standpoint. Furtherrnore, gross inethods necessitating pooled samples impose a sometimes clifficult requirement of sufficient individual specimens to permit even a minimum of su.ch studies. It is highly desirable, therefore, to clevelop or adapt micro and ultramicro methods to the

restrictions 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 of tissue requirecl in these procedures' is sr-rfflcientiy small that clifflculty is encountered in routine gravimetric detelminations of the mass. NevcltheIess, an accurate measrlre of mass, or its equivalent, is impcrative as a reference unit to rvhich respiratory phenomcna crn be relatecl. Total nitrogen has been used as such a measure ancl also has been recommentlecl as a stanilarcl of reference to which oxygen consurnption can be relatetl.3' a Pincusl repolted the nitrogen content of human tooth pulp as 10.1 per cent of the dry weight. This value presuirrably rvas cletet:mined by the macroKjelclahl method. It was clecidecl, therefore, to inyestigate this pro.[lem further by the same method ancl then to attempt sirnilar cletermina.tions by the micro methocl with the hope that ultrainicro methocls would become feasible eventually. Because variotts types of hnm:rrr clental pulp are not always available for analytic pulposes, it was consiclered aclr.isable to investigate also the possible effects on nitrogen cletermination that might result from presen ation of this tissue in 10 per cellt aqueous formalin solution.
I{ATER,IAI,S AND,I{ETHODS

The tissues investigatecl were the pulps of bovine and human teeth. The bovine material came flom animals between 2 ancl 3 years of age. The molars
Based on a thesis submitted by Marvin L. Riedesel in partial fulflllment of the requirements for the degree of X{aster of Science in the Departrnent of Physiology in the Graduate College of the State Lhiversity of Ios'a. This investigation rvas supported in part by resealch grant D-126 from the United States

Public Health Service.

Received

for publication, Auei. 3, 1953.

632

Number

Volume

33 5

NITR,OGEN CONTENT OT' DENTAL PULP

were extracted and placed in cold Ringer's solution as soon as killing ancl decapitation had been accomplished. The teeth were then taken to the laboratory, where they were cracked open and the pulps removed. The pulps were carefully examined uncler a stereoscollic microscope in order to remove all fragments of enamel and dentin. Some of the pulps were pleserved in a 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 of approximately equal size to facilitate even dehydration and rvere placed in a constant-temperature drying oven. The samples of Groups A, B, C, and D of Table I and Group A of Tabie II were dried to a constant weight at temperatures between 95o C. and 100o C. The samples in the remaining groups were dried. to a constant weight at 560 C. The longer drying at the lower temperature is recommendecl by the Association of Official Agricultural
Chemists.'
TABLE

I
PULP TrssuE DrtnnnrNnn

Nrrnocnx CorvrrNr oI FnEsn

ty#J"rit+ltilii?3"."t*r
R.\NGE OI'
SAMPLE

rrEAN I neNcc
(MG. N PER | 1mc. w

METIIOD

TISSIIE

ot' IN.\T,\SIS

NO. OF DETERI{INATIONS

(MG. DriY

wnrGlr!')
150-407
1 1

"nn I uc. nnv I st-tNr',rnu \vErG.r.rr) | wnrcnr; Iouur,r.,os

l\rc- Dnv

STANDAR,D

DEVIATION

OF

ME-I\N

A !'resh B !'orrne.lin
C Fresh

MacroMacro-

14
11

Kjeltlahl
1-407

0.1257 0.7225- 0.0014

0.128.1

0.0004 0.0005 0.001 0.002 0.0011 0.0014

0.1256 0.120 0.117

Kjeklahl
722
75 25

D }'ormalin

E Fresh
E Tornalin

Nesslerization Nessierization
X{icro-

1.3-2.6 7.3-2.(i 20-29 20-29

0.1273 rl 1tlo

Kjeldahl Kieldahl
TABLE

X{iolo-

0.12310.1286 0.080.15 0.080.15 0.11980.1336 ().1l77 0.14;J7

0.001_6

0.013 0.015 0.0054 0.0072

II

NrrnocrN Cowrrnr

oF BovrNE AND HUMAN DDNTAT, PULP AS DETEB,MTNED BY I<JET,DAHL Muruols

F,ANGE OI'

MTTIIOD
OF

NO.

OF

DETER,l[1-

TISSUE

ANAT,YSIS

NAT]ONS

Si\MPI'E (MG. DRY wEIGHT)

R.{.NGD ME^N (MG. N PER (MG, N PER I{G. DRY MG. DR,Y STANDARD

STANDARD

wErcHT)
0.1256

wErclrT)
0.72250.1286

DEVIATION

DEVIATION OF MEAN

A B
C

Bovine Macrofresh and Kielilahl

f

177.107
50

0.0013 0.0065 0.0082 0.0025 0.0040

0.0002

ormalin ormalin
Micro5 18 o

1\{icroBovine fresh and Kieldahl

f

20-29
2.24-3.70 13.8-20.8

0.1261

0.rt7T0.1437 0.1200.127 0.1001-

0.0009

Bovine
f ormalin

0.7232
0.1051 0.1050

0.0041 0.0006 0.0014

l) I{umen E
infant
adult
Human

Kieldahl

MicroK.ieidahl Micro-

Kieldtrhl

2.5,$

0.1084
0.10000.1140

634

RIEDESEL AND FISHFJR

Ocrober.195,1

J. D.

Res.

rn accordance with the varying lirnitations of each analytic method, the sample sizes were of different magnitudes. rn some cases, as in Groups A and

B of rable r, 1 to 3 bovine molar pulps were required to provide a single sample. In other instances a single pulp 'lvoulcl furnish enough tissue for 1 to 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 infant at autopsy (3 infants were born at full term and died shortiy after birth, 3 were stillborn after eight months of gestation) and preservecl in 10 per cent formalin solution from two months to four years. The tooth germs were clissected from the jaws, and the pulps were removed with the aid of a stereoscopic microscope. Each sample in Group D, Table rr, required
heads obtained

of the pulps of freshly extracted, noncarious teeth which were placerl in 10 per cent formalin solution immecliately after extraction. Twerrt).tu,'o patients whose ages rangeil from 14 to 48 years contributed 35 teeth. The majority of the teeth were removed because of the necessities of orthodoirtic or periodontic treatment. \Vithin several hours of extraction, the teeth were cracked open and the pulps removed and dricd as previously described. The numbel of pulps requirecl to make up a sirigle sample varied from 1 to 17. The dry weight of most of the samples was determined on a Christian Becker chainoinatic analytical balance with a sensitivity of 0.1 mg. The dry weight measurements of human and bovirie samples smaller than 15 mg., which were analyzecl by the micro-Kjeldahl method, were determined on an Ainsworth keyboarcl-operated weight-carrier microbalance with a sensitivity of 2.5 pg. Freshly prepared conductivity water as clescribed by Fales and Kenny6 was employed in the preparation of all solutions used. IIacro-KjeIdahl determinations were made according to the method described by Campbell,' with the exceptions that boric acid was used to collect the distilled ammonias and bromcresol green-methyl red indicator was used.e By employing the boric acid solution in place of a standard acid there is one less solution to standardize and thus one less chance for error. The bromcresol green-methyl red indicator gir.es a \rery distinct eolor change from blue to colorless. The nesslerization method described by Hawk and Bergeimlo was employed in the analyses of Groups C and D of Table I. The colorimetric cleterminations were ma,de on a Klett-Summerson photoelectric colorimeter. The micro-Kjeldahl methotl used was that described by Beattyll with the addition of 30 per cent hydrogen peroxide to the digestion mixture as suggesteil by Hawk and Bergeim.lo lMhen analyzing samples of 2.2 Lo 3.5 mg. of dry weight, proportionately smaller quantities of reagents were employed. The apparatus employed was essentialiy that described by Keys1, except that a vacuum was not employed.

the pooling of from 4 to 9 pulps. The second type consistecl

-V,OIUMC Number

3.3
5

NITROGEN CONTENT OF DENTAL PULP

Before any method of analysis lvas employecl in the coliection of data in this series, it was tried experimentally until confidence in its use and
proficiency in its technics were attainecl. The accuracy of each methocl was checkecl by determining the nitrogen content of measured quantities of known solutions of reagent grade ammonium sulfate.
ITES U LTS

of total nitrogen by the several methods referred to previously and as indicated in Tables I and II. \Vhen fresh bovine dental pulp and that prein 10 per cent formalin solution were analyzed by the macro-Kjeldahl, micro-Kjeldahl, and nesslerization methods, there was substantial a,greement in the nitrogen yields of each type of tissue (Table I). While the mean nitrogen values for formalin-preservecl tissue appear slightly lower than for those of fresh tissue, this difference is not statistically significant. \\rhen comparing mean nitrogen cotrtent as determined by the various analytic methods, it may be observed from Table I that the lowest mean yield is obtained in using the nesslerization technic. Since the macro-Kjeldahl method is presumed to be productive of the most accrlrate results, it servetl as a guide in evaluation of the other methocls. I,Iany laboratory workers are of the opinion that the lower values obtained by nesslerization are characteristic of the method. l\rhen this quality became clearly evident, the method
served
was abandoned.

The observations reported were derived from a total of 304 cleterminations

Inspection of Tables I ancl II shows close correspondence between the mean nitrogen values obtained by the macro- and micro-Kjeldahl methods. As might be expected, the range of individual values is greater when working on the micro level. The mean total nitrogen of bovine dental pulp, computed from all data derivecl from both macro- and micro-Kjeldahl analyses, was observed to be 0.1258 mg. per milligram of dried tissue (Table III).
TAB],8
C
O

III
il.'.iil3"5u
R,ANGE

I{ PAR'

SO

N O F MTAN NITRO GH,S"#iT:f,,XT,:J'

RANGE OF SAT[PLE MEAN

MAN D ENTAL PUIP

NO.
TISSI]E

OF

(Mc. N PER
MG.
DRY

DETERMI.

(r[c.

(Mc. N

PER STANDARD

DRY

N,\TIONS

$'ErcHT)
2.2-407

WEIGHT) 0.1258

MG. DR,Y WEIGHT) 0.1.177-0.7137

DEVIATION

STANDAIiD DEVIATION OF MEAN

Bovine fresh and

f

0.0037 0.0031

0.00042

ormalin
2.5-20.8
0. 1 050

Human f ormalin

0.1000-0.1140

0.0006

Ifuman dentai pulp preserved in 10 per cent formalin solutioh was analyzed by the micro-Kjeldahl method only (Table II). The mean total nitrogen values of infant and more mature tissue were observed to be almost identical. The mean nitrogen content of human dental pulp was found to be 0.1050 mg. per milligram of dried tissue (Table III).

RII]DXS]IL AND FISHER

DISCUSSION

J. D. Res. October,1954

comparison of mcan total nitrogen of bovine and human pulp tissue III) rel'eals a significant differcnce. Sincc the series of human material shows no age differcnces, and the macro- and micro-Kjeldahl methods compare very favorably as to reliability, it is suggested that this difference is an expression of species peculiarity. A critical comparison of mean nitrogen content of human dental pulp observed in this investigation with that reported by Pincus' is difflcr:Llt bccause he does not elaborate on his rncthods in this regard. But since he does 1'cport that "nitrogen (Kjeldahl) rvas 10.1 per cent on a moisture and. ash frec

(Table

basis. .," 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 obselved to be 0.1050 mg. per milligram of clried tissue and that of bovine pulp 0.1258 mg. It is suggested that this difference is a species peculiarity' 2. Age of the indiviclual does not appear to influence the nitrogen content
of human dental pulp.
does not significantly alter

3. Preservation of clental pulp in 10 per cent aqueous formalin solution its suitability for nitrogen analysis by the Kjelcltrhl
REX'ERENCES

method.
1. 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' 19433. Burris, R. H., and wilson, P. w.: Measures of Respiratory Activity with Resting cells, Proa. Sob. Euper. B'ioL & Med. +5i 721' 1939. -Units

4. Moulton, C. R.: 5. 6. 7. 8. 9.

of Reference foi'Basal l{etabolism and Their Interrelation-

10. Hawk, P. B., and Bergeim, o.: Practical Physiological chemistry, eci. 11, Philadelphia,. 1937, P. Blakiston's Son & Co.' p. 70_7-. Anhydrous Coppei Sulfate in the Kjelt1ahl Nitrogen Determinations" 11. Beatty, C., III:

ships, ./. Bi,ol. Chem.2+i 299,1916. -I{. A.: Official Nlethods of the Association of Ofrcial Agricultural Lepper, ''ed. 7. \\'ashirston. 1950, Tlre Assor'jation of Official Agricullulal Chemists, Chemists, p. .159. Fales, i{.'A., and Keniy, F.: Inorganic Quanlitaiive -A.nalysis, nerv eclition, Nerv York, 1939, D. Appleton-Century Company, Inc., p. 56' 'The Delermination of Nitrogen by Modifieci KjelCampbell,' W. R.; ai-d Htmna;M. f;r -dahl Metlrods, J. Biol. Cliem. lI9: 1, 1C37. 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. Sobel, A. E.,'Me;,er, A. 1\[., and Gottfried, s. P.: A convenient Titrometric LTltlamicroriethocl' for- the Estimation of Urea and Kjeldahi Nitrogen, J. BioI. Chem- 756: 355, 791+.

Indust. $ Engin. Chem. (Analyt. Ed'.) L5: 476, 1943. 12. Keys, A.: A Rapitl Xficro-Kjeldahl Method, J. BioI. Chem.732:181, 1940.