9 1987 by The Humana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/87/120(G0375502.00

Distribution of Trace Elements Within Bones in Nasal Cavity and Labyrinth in Humans V. VALKOVI(~* AND M. JAK~14: Ruder Bogkovid Institute, POB 1O16, Zagreb, Yugoslavia and JELENA KRMPOTI(~-NEMANI(~ Faculty of Medicine, University of Zagreb, Zagreb, Yugoslavia ABSTRACT Measurements of trace element concentrations within bones in nasal cavity and labyrinth have shown large variations, both with a single bone and between different bones of a same individual. Factors that influence trace element levels include: metabolic activity, environmental effects, sex, and age. Detection of characteristic X-rays has been shown to be a convenient method for the measurement of concentration profiles, micropixe for micrometer variations, and X-ray fluorescence for millimeter variations. Index Entries: Trace elements; bone, trace elements in; nasal cav-

ity; labyrinth.

INTRODUCTION Studies of trace e l e m e n t s in biological materials have resulted in significant i m p r o v e m e n t s of our u n d e r s t a n d i n g of the functioning of biological systems. M u c h information has been a c c u m u l a t e d about essential trace e l e m e n t s a n d the r e s p o n s e of living systems to their concentration levels. What is usually m e a s u r e d are whole body, organ, or blood trace elem e n t levels. H o w e v e r , m a n y tissues have distributions of concentration patterns reflecting function or metabolic activity as well as b o d y stores of s o m e elements. In the case in which tissue has an i n h e r e n t variation in *Author to whom all correspondence and reprint requests should be addressed. Biological Trace ElementResearch

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Valkovi4, Jak~i~, and KrmpotiG Nemani~

element concentration, the whole-tissue concentration value will not have much meaning. It is expected that the knowledge of the distribution of chemical elements on a microscopic scale in biological systems may be of help in understanding basic phenomena in these systems. This information can be obtained using the detection of characteristic X-rays excited by focussed beams of charged particles and photons. Trace element distributions can be measured with a beam spot size of 1-10 ~,m on the scale of 1-2 mm with a proton micropixe. Recently, the first analytical microprobe using synchrotron radiation excitation was established with a pinhole size of 2 ~m (1). Beam spot sizes of 1-2 mm for the measurements of concentration profiles of trace elements on the scale of several centimeters are available in proton-induced X-ray emission spectroscopy (PIXE) and tube or radioactive source fluorescence (XRF). In this presentation we shall describe two examples of trace element concentration profiles in bones; one on the scale of micrometers and the other on the scale of millimeters.

METHODS The experimental results described in this paper were obtained with an X-ray Mo-tube excited system at the Ruder Bo~kovi4 Institute, Zagreb, and proton microbeam setup at the Free University, Amsterdam. The details of the Free University proton microbeam setup are described in several papers [see, for example, ref. (2)]. All measurements described here were done with 3 MeV protons, current 10-15 pA. For the scanning, the target was moved by a set of piezo-electric crystals controlled by a microprocessor; the average speed of target was usually around 80 p~m/s. The sample preparation consisted of embedding the bone material into araldite (AY 103 with hardener HY 956). Targets were prepared in the form of 30-~m thick coupes, using a microtome. An aluminum layer of approximate thickness of 5 nm was evaporated on each target to prevent charging of the target and to reduce heating of the samples. The element concentrations were deduced from the measured ratio of detected characteristic X-rays and the number of backscattered protons (3,4). Prepared samples were inspected by microscope before and after irradiation. X-ray spectra were measured at the 32 positions along the track passing across the separating wall between the fundus and the basal coil of the cochlea. Trace element levels in the bones from nasal cavity were determined using an XRF setup at the Ruder Bo~kovi4 Institute. Secondary target excitation using Mo and Zr radiators on Mo-tube (V = 34 keV and I = 26 mA) was used. No sample preparation was done, all bones were analyzed as received. Biological Trace Element Research

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Trace Elements in Bones

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RESULTS In the study of trace elements in the human labyrinth, the investigated material included 11 macerated temporal bones from individuals, ranging from 2 to 75 yr of age, of both sexes. In all measured spectra, peaks associated with P, Ca. Fe, Zn, and Sr could be easily identified. As an example, Fig. 1 shows concentration profiles for Ca, Fe, and Zn. Although Ca shows uniform distribution across the bone material, Zn and Fe are preferentially concentrated on the surfaces. Zinc and calcium distribution appear age dependent. In very young (2 yr) and young (third decade) individuals calcium is found in the whole thickness of the wall in high concentrations. Zinc makes an appearance on the wall surface toward the fundus. With advancing age (fourth to sixth decade), the concentration of zinc becomes higher on the bone surface, but it appears also within the wall, i.e., in the region of the nerve channels. In the seventh decade, zinc piles up also on the opposite bone surface, i.e., toward the basal coil of the cochlea. Simultaneously, the concentration of calcium is reduced, although it is present in the whole thickness of the wall. Figure 2 shows the schematic of air flow through the nasal cavity and positions of septum, concha media, and concha inferior. In addition, trace element levels were measured in maxilla, lamina, and a few other bones. Experimental results for different subjects for elements Fe and Pb are shown in Fig. 3, and for elements Zn and Sr in Fig. 4. From the presented data it can be concluded that iron and lead concentrations in the nasal cavity depend on the direction of the air flow. Under normal condi-

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Schematic presentation of air flow through the nasal cavity.

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T r a c e E l e m e n t s in B o n e s 300-

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T r a c e E l e m e n t s in B o n e s

TABLE 1 Zinc (ppm) in Some Bones from Nasal Cavity Subject Sex--Age, yr M--28 M--32 M--33 M~36 M--53 F--53 M---54 F--57 M--60 F--62

Septum

Concha media

Concha inferior

360 232 53 522 202 263 281 251 855 265

1010 652 195 964 260 305 636 543 635 440

1060 344 100 1113 270 535 398 350 969 349

tions the highest concentration of Fe a n d Pb is in the m i d d l e concha. If the variations of the nasal cavity deflect the air flow, the concentrations of e l e m e n t s also c h a n g e their localization in the three s t u d i e d structures: s e p t u m a n d both conchae. Numerical values for zinc a n d lead for s e p t u m , concha media, a n d concha inferior for different subjects are s h o w n in Tables 1 a n d 2. In s o m e subjects very high Pb levels are found.

DISCUSSION Results p r e s e n t e d in this p a p e r s h o w that there are great differences in trace e l e m e n t concentration levels in different bones of the same b o d y TABLE 2 Lead (ppm) in Some Bones from Nasal Cavity Subject Sex--Age, yr M--28 M--32 M--33 F--53 M--53 M--54 F--57 M--60 F--62

Biological Trace Element Research

Septum

Concha media

Concha inferior

11 64 128 74 570 21 97 28 202

13 93 211 52 370 90 281 64 104

11 42 200 122 840 39 184 90 101

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Valkovi4, dak~i4, and

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CONCENTRATION/pprn Fig. 5. Distribution of measured values of Pb, Sr, Fe, and Zn in all bones from nasal cavities for all the individuals studied. region (nasal cavity). In addition, studies of the labyrinth indicate that some elements are concentrated only in specific locations in the bone, indicating their role in metabolic processes. Some authors (5) have suggested that Zn is associated with calcifying margin in bone and that zinc is detected in the layer of preosseous tissue whose calcification is imminent (6). Distributions of measured values of Pb, Sr, Fe, and Zn concentrations in all bones from nasal cavity for all the individuals studied are s h o w n in Fig. 5. The spread in values is at least order of magnitude, whereas for lead this is more than two orders of magnitude. This indicates the importance of environmental effects in lead incorporation into bone material.

REFERENCES 1. P. Horowitz and J. Howell Science 178, 608 (1972). 2. J. C. den Ouden, A. J. J. Bos, R. D. Vis, and H. Verheul, Nucl. Instr. Meth. 181, 131 (1981). 3. A. J. J. Bos, The Amsterdam Proton Microbeam, Ph. D. thesis, Free University, Amsterdam 1984. 4. A. J. J. Bos, C. C. A. H. v.d. Stap, V. ValkoviG R. D. Vis, and H. Verheul, Nucl. Instr. Meth. in Phys. Res. B3, 654 (1984). 5. C. W. Asling and L. S. Hurley, Clin. Orthop. 27, 213 (1963). 6. S. Haumont, J. Histochem. Cytochem. 9, 141 (1961).

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