ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 14, No. 4 Copyright © 1984, Institute for Clinical Science, Inc. Analysis of Lead in Circumpulpal Dentin of Deciduous Teeth* PHILIPPE GRANDJEAN, M.D., PHD.,t OLE N0RBY HANSEN, M.P s y c h ., and KAREN LYNGBYE, D.D.M. Institute of Community Health, Odense University, Institute of Psychology, Aarhus University, and the Municipal School Dentistry Service, Randers, Denmark ABSTRACT Absorbed lead continuously adds to the lead deposited in the vascular­ ized circumpulpal dentin of the teeth. Thus, this dental tissue is expected to contain a lead concentration which reflects the integrated lead exposure during the time from completion of tooth formation to tooth extraction or shedding. A method has been developed to assess the lead level in the dentin surrounding the pulp chamber in deciduous teeth. Variation within the tooth is minimal, but upper medial incisors show a slightly lower lead level than do other incisors, as assessed in 714 teeth from first-grade Danish school children. This tendency was not confirmed, however, in a small number of paired teeth from the same children. The new method appears advantageous for epidemiological studies of lead neurotoxicity in children. Introduction used as an integrated measure of the total exposure during early life. Lead levels Determination of lead concentrations may vary, however, between different in shed deciduous teeth has become a tooth tissues and between different tooth useful measure of past lead exposures in types. Thus, the highest lead concentra­ children. This metal accumulates in cal­ tions are found in the inner and outer cified tissues owing to the formation of surfaces of the tooth, i.e., the outer layer sparingly soluble lead phosphate. The of the enamel and the circumpulpal development of deciduous teeth starts al­ dentin.1,6 The circumpulpal dentin, ready in prenatal life, and the average which is formed after the tooth formation lead level in the shed teeth has been has been completed, continues to accu­ mulate lead until the tooth is shed.6,11 * Presented in part at the 2nd International Con­ Primary dentin contains much lower lead ference on the Clinical Chemistry and Chemical Tox­ concentrations. In addition to variations icology of Metals, Montreal, Canada, July 19-22, within individual teeth, the lead concen­ 1983. tration in whole teeth (or crowns) tends t To whom reprint requests should be addressed at Institute of Community Health, J. B. Winsl0ws to decrease from the medial incisors to Vej 19, 5000 Odense, Denmark. the premolars.2,5 A more extensive report 270 0091-7370/84/0700-0270 $00.90 © Institute for Clinical Science, Inc. LEAD IN CIRCUMPULPAL DENTIN 271 recently concluded that the largest dif­ ference between incisor lead levels was found when comparing upper medial in­ cisors to lower lateral incisors.9 How­ ever, such variations have not been found in less extensive studies of circum- pulpal dentin levels.4 The possibility ex­ ists that the variation related to tooth type could be partly due to a differential proportion of dental tissues in the dif­ F i g u r e 1. Special vise designed to split deciduous ferent tooth types. teeth into two halves. Three different methods have been used previously: (1) whole tooth or groove is cut vertically in the midline of crown;2,3’5,911 (2) a 300 |xm fragment of the anterior and posterior surfaces by dentin from a 600 |xm thick section of the means of a thin diamond grinder. The tooth;7,8 and (3) circumpulpal dentin ob­ tooth is then placed in a special vise tained from the root canal with a which splits the tooth into two halves reamer.4’8 Each of these methods may (figure 1). Soft tissue residues are then suffer from drawbacks with regard to removed from the pulp chamber by speed of analysis, inter-tooth variability means of a dental explorer, followed by or biological interpretation. Circum­ a one-minute immersion in about one ml pulpal dentin attracts particular interest, of 10 percent hydrogen peroxide. Re­ because only this tissue has a blood maining surface contamination is re­ supply and, in principle, accumulates moved by subsequent washing of the lead shortly after the time of tooth erup­ tooth fragment for 20 seconds in an ul­ tion and until the tooth is shed (or ex­ trasonic bath with concentrated ethanol tracted). Owing to root resorption prior followed by drying. Clean laboratory to the shedding of deciduous teeth, little techniques are used throughout. The circumpulpal dentin remains apart from dentin bordering the pulp chamber is re­ the tissue lining the pulp chamber. A moved by a small wolfram carbide ro­ method has therefore been developed to sette burr.* About one percent of the prepare secondary dentin from the pulp tooth weight, i.e., an average of about chamber of shed deciduous teeth. 0.65 mg, is sampled in this way. The powdered dentin is weighed directly into Materials and Methods a sampling cup, and 100 |jl1 of concen­ Deciduous teeth are currently being trated nitric acid are added for overnight collected from children in first-grade dissolution. The following day, 400 jjlI of classes of schools in four municipalities redistilled water are added, and the so­ in Denmark. The 714 teeth used in the lution is analyzed in two independent present study all originated from six to runs by electrothermal atomic absorption seven-year-old children from the city of spectrometry, t Small amounts of dentin Aarhus. This epidemiological study is cause no discernible matrix effect; nitric coordinated by ONH; all teeth were acid is added to all standard solutions. examined and classified by KL, and anal­ The temperature program for a 20 jxl yses were performed at Odense Univer­ sample is: drying at 100°C for 25 seconds, sity, Denmark. Only teeth without fill­ ings were used. Some teeth could be * Borer rund 008, Hager & Meisinger GMBH, D- 4000 Düsseldorf 1, Federal Republic of Germany. split into two parts of similar size by use t Perkin-Elmer model 305A with an HGA-76 of a pointed or sharp instrument (pref­ graphite atomizer, autosampler AS-1, deuterium erably a mandrel). Otherwise, a small background corrector and recorder. 272 GRANDJEAN, HANSEN, AND LYNGBYE ashing at an increasing (rate 2) tempera­ TABLE I ture up to 700°C, atomization for three Day-to Day Variation of Micro seconds with gas stop at 2100°C followed Lead Analysis (yg/g) of "Reference Materials" by three seconds at maximum tempera­ Number of ture. By the technique described, an av­ Material Analyses Mean + S.D. erage of about 20 teeth can be analyzed per day. IAEA bone* 41 3.09 + 0.41 Tooth powderf 5 9.08 + 2.87 Three different solid “reference mate­ Hydroxyapatite 47 0.41 + 0.11 rials” were used. A sample of powdered tooth was kindly provided;^ this sample ♦Certified lead level, 3.1 ± 0.28 yg/g; variation of "accepted" levels in interlaboratory compar­ consisted mainly of particles of 0.1 to 0.5 ison, 3.0 ± 1.08 yg/g mm diameter.10The IAEA standard bone fMean of 13 laboratories, 8.1 yg/g; range, 4.3 - material, § which has a certified lead con­ 15.5 yg/g (reference 10) centration for aliquots of at least 100 mg, was also used. Inhomogeneity of these materials could cause some analytical in table II indicate that lead concentra­ variability when using aliquots of about tions decrease with subsequent sam­ 1 mg. Finally, commercial hydroxyapa- plings into the deeper dentin tissue. titell was used as a low-lead-level, dentin­ Thus, a homogeneous sample of circum­ like reference material. pulpal dentin must be as small as allowed by the sensitivity of the detection method. A sample of 0.5 mg seems suf­ Results ficient, because it will result in 0.02 ng Although day-to-day variations in lead of lead in the 20 |xl of solution injected levels of standard solutions were min­ into the graphite tube, if the dentin lead imal, larger variations occurred in the level is 1 |xg per g. solid materials (table I). For the bone and The total of 714 shed deciduous teeth tooth materials, some of the variation represents roughly one-half of the teeth may be due to inhomogeneity of the sam­ collected in Aarhus. A comparison of lead ples. levels in different incisors is shown in The reproducibility of the method was table III. Although the lead levels in assessed by determining the lead level of each of the tooth types varied widely (av­ circumpulpal dentin prepared indepen­ erage coefficient of variation, 56 per­ dently from both halves of each of twelve cent), each group of incisors showed deciduous teeth. The average lead con­ almost the same average lead level. How­ centrations varied between 0.54 and 16.6 ever, upper medial incisors showed lead |xg per g, and individual values for half­ levels which were lower than those of teeth differed by an average of 1.2 n-g per g from the tooth mean, i.e., an average of 10.7 percent, only one value being TABLE I I above 20 percent. Additional studies Lead Levels (yg/g) in Subsequent Layers of concerned five teeth where three sub­ Circumpulpal Dentin of Five Deciduous Teeth sequent samplings of about one mg from each tooth were made in order to pene­ First Second Third trate into deeper dentin layers. The data 3.3 1.6 1.6 4.3 2.2 3.1 5.2 3.1 2.9 $ Dr. M. Stack, MRC Dental Unit, Bristol, U.K. 6.3 1.8 2.1 § Reference Material H-5, IAEA, P.O.