Potential of the Diffuse Reflectance Infrared Fourier Transform (DRIFT) Method in Paleontological Studies of Bones Y. D A U P H I N Laboratoire de Pdtrologie Sddimentaire et PalSontologie, URA 723 du CNRS, Bdt. 504, Universitd Paris Xl-Orsay, F 91405 Orsay Cedex, France

The possible range of variability in fossil bones is not well understood. The purpose of the present study is to demonstrate the usefulness of infrared spectrometry for quantitative and qualitative measurements of the relative abundance of preserved proteins. These criteria, correlated with the crystallinity of bones, can enable one to understand certain diagenetic processes. Index Headings: FT-IR; DRIFT; Fossil bone; Organic matrix; Preservation.

INTRODUCTION It is well known that bones are a complex mixture of a phosphatic mineral phase and an organic matrix. Fossil bones are abundant in sediments, but preservation and diagenetic processes are still subject to question. Previous electronprobe microanalyses (EDS) of bones and teeth have shown alterations in fossil microvertebrates. T M The problem of the relative abundance of organic matrix within fossil bones is one of the most commonly encountered problems. Such information is usually obtained via gas chromatography or pyrolysis, but these approaches are time-consuming and destructive. Infrared spectroscopy has proven to be a useful tool in investigations of synthetic or natural apatites, with special emphasis on the biological apatites: bone, enamel, and dentine2 -~° Proteins, including collagen, have also been studied. 1~-13 In spite of the availability of these methods, relatively few attempts have been made to apply them to paleontological materials. The purpose of this report is to suggest the usefulness of FT-IR techniques, especially the DRIFT method, in the study of bones. EXPERIMENTAL All spectra were recorded at 4 cm -1 resolution with 64 scans (measurement time > 4 min) with a strong NortonBeer apodization on a Perkin-Elmer Model 1600 Fourier transform infrared spectrometer (FT-IR), from 4000 to 450 cm -1. The spectrometer was equipped with a diffuse reflectance accessory which permits DRIFT measurements with high sensitivity on powders. All spectra were corrected by the Kubelka-Munk function. The system was purged and permanently maintained under nitrogen to reduce the atmospheric C02 and H20 absorptions. All samples and KBr were ground with an electric mortar for 10 min to obtain homogeneous granulometry. They were dried in an oven at 38°C for one night. Pow-

Received 8 J u n e 1992; revision received 20 August 1992.

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Volume 47, Number 1, 1993

dered bones and KBr were mixed (about 5 % powdered bone in KBr) and loaded into the sample cup (3 mm depth). A thin glass strip was pulled across the top to produce a flat surface. Increasing density by applying pressure to the sample surface increases the response of the spectra, 14 and thus no supplementary handling was made. According to Okada e t al., 15 proteins are denatured when included in KBr pellets for transmission studies of FT-IR spectra. According to these authors, it seems that the DRIFT method is not adequate for the IR measurements of solid type I collagen. However, the purpose of the present paper is to study not the structure of the collagen, but the composition of fossil bone (preservation of the organic matrix) with a nondestructive method. DRIFT spectra of type I collagen have been obtained with the Perkin-Elmer system; they demonstrate the main bands usually mentioned in the literature. Thus, the DRIFT method has been chosen for this study, certain practical advantages being considered later. Before a spectrum was run, the height of the sample cup was adjusted by using the alignment routine provided by Perkin-Elmer (energy equal or higher than 5 % ), so that a maximal signal throughput was obtained. A background spectrum was measured for pure KBr. Sample spectra were automatically ratioed against background to minimize C02 and H20 bands. Correlation coefficients between two spectra of the same samples are about 99 %. Recent bones were from rodents of the Paris Basin (France). They were cleaned only with water. Pellet bones were collected at Tighenif (Algeria). They were cleaned with water--with hairs, feathers, etc., being removed by ultrasonication. According to morphoscopic studies, the bird was a hawk. 3 Scanning electron probe x-ray emission microanalyses (EDS) have shown selective alteration of bones and teeth, is Fossil bones were collected at Tighenif (Pleistocene, Algeria). This site, previously known as Palikao or Ternifine (700,000 B.P.), has yielded the earliest hominid remains in North Africa. 17,1s Several sedimentary levels contain fossil rodents. EDS analyses performed on fossil bones have shown that bones and teeth are variously altered. For fresh, pellet, or fossil samples, several bones were mixed before grinding. RESULTS AND DISCUSSION DRIFT Spectra of Bones. Good spectra were obtained which allowed organic and mineral bands to be identified in fresh bones, the main bands coinciding fairly well with those observed in KBr pellets. The wavenumbers of the bands are listed in Table I and are comparable to those

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APPLIED SPECTROSCOPY

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FIG. 1. (A) Calculation of the amount of organic matrix from the band intensities in DRIFT spectra of recent and fossil bones. (B) Calculation of the composition of the organic matrix from the band intensities in DRIFT spectra of recent and fossil bones. (C) Calculation of the percentage of crystallinity of the same bones (SF). FR: fresh recent bones; PEL: pellets; 2, 3, etc.: successive levels in the Tighenif site.

already mentioned in the literature, s,~9 Changes in the bone absorption bands for fresh and various fossil levels are also illustrated in Table I and Fig. 1. However, a table cannot give a complete idea of the complexity of bone spectra (see Fig. 2). Pellet bone spectra also show the main bands correlated with the two components (Fig. 2). The correlation coefficient between the recent and pellet samples is 0.90. Since both samples contain mainly collagen, the spectra show the characteristics of this protein. All fossil bone spectra are different from the recent bones (Table I). Amide B and amide III bands are absent, as are other protein bands. Intensities of bands also differ in recent and fossil bones (Fig. 2). Content of the Organic Matrix. In recent bone, the mineral phase is about 60 %, the organic matrix is 30 %, and cells + H20 are 10 %. The organic matrix is mainly collagen (about 20 % ). Thus, the organic matrix/mineral phase ratio is equal to 0.5. The intensity ratio of two bands due to the amide A and PO4 (I~mideA//_PO4)calculated from DRIFT spectra is about 0.50 in rodent fresh bone. In pellet bones, the I~m~dei/Ip04 ratio is higher than that

of the fresh bones. In all fossil bones, this ratio is lower than in fresh and pellet bones (Fig. 1A). Alteration of the Organic Matrix. In addition to the determination of the organic/mineral ratio, the DRIFT technique enables one to detect the alteration of the organic matter in pellet and fossil bones. The use of Iamide I/ Iamide A and Iamide I(1654)/IamideII(1550)ratios shows the diagenetic changes of the organic matrix composition (Fig. 1B). The two diagrams are parallel, except for level 2 in the Tighenif sample, where the amide II band is absent (Table I). Crystallinity Ratio. It is possible to estimate the percentage of crystallinity of bone from IR spectra, without previous extraction of the organic component) ,s Splitting fraction (SF) measurement of the crystallinity in bioapatites, based on area ratios, gives the SF index. A welldefined doublet between 500 and 600 cm -1 is indicative of high crystallinity, whereas a poorly defined doublet indicates amorphous calcium phosphate (SF = 0). 5 According to this method, crystallinity has been calculated for each fossil level and recent bones (Fig. 1C). The lowest crystallinity ratio is that of fossil level 3 of the Tighenif sample, whereas the highest ratio is that of the pellet bones. DISCUSSION The organic matrix and mineral phase have several IR bands. Among the possible combinations, several have been tested in order to achieve the best organic/mineral ratio. The result of the I~mideA(3290)/Ipo4(1030)ratio seems fairly similar to the usual data. This ratio has been calAPPLIED SPECTROSCOPY

53

TABLE I. Synthesis of the main infrared bands in fresh bone according to previous literature 8 (left columns) and DRIFT bands observed in the recent fresh bones (FR), pellet bones (PEL), and fossil bones from Tighenif (right columns). Lev. 2, Lev. 3, etc.: successive levels in the Tighenif site. a

Literature

1743 1659 s 1649 s 1559 1547 s 1515 1480 1470 sh 1450 s 1410 s 1342-4 sh 1238 w 1233 w 1250 910 960w 878 871 598 s 557 s

Attribution Amide A Amide B

Amide I

FR. 3294 3066 2930 2874 1734 1653

Amide II

1558 1542

1733 1653 1636 1559 1542

1473 1457 1419 1340 1242

1474 1458 1419 1339 1237

1038 962 873 604 568

Protein CO3 Protein Amide III PO4 PO4 CO3 P04

PEL. 3300 3068 2970

Lev. 2 3230

Lev. 3 3270

Lev. 4 3260

Lev. 5 3242

Lev. 6 3282

Lev. 7 3400

Lev. 8 3215

1734 1653 1636 1559 1540

1734 1654 1636 1558 1541

1734 1654 1637 1558 1540 1472 1458 1419

1734 1654 1636 1558 1540

1558 1540

1734 1654 1636 1558 1540

1418

1458 1419

1458 1419

1458 1419

1458 1420

1458 1420

1037 963 872

1057

1059

1052

1060

1055

1053

866

867

867

867

1047 963 868

603 570

603 569

604 576

602 569

605 569

604 568

605 577

1652

1654

604 570

871

Note: s = strong; sh = shoulder; w = weak. culated f r o m a b s o r b a n c e intensities. T h e 3290-cm-' a m ide b a n d has b e e n chosen because it is p r e s e n t in b o t h pellet a n d fossil bones. O r g a n i c / m i n e r a l ratios calculated f r o m o t h e r protein b a n d s are very different f r o m the value of 0.5 in fresh bones. T h e a m i d e I I I b a n d is p r e s e n t only in recent bones. Consideration of this b a n d increases the difference b e t w e e n fossil a n d recent bones. Because the a m i d e I I I b a n d is a b s e n t in all fossil bones examined, slight differences b e t w e e n samples are not highlighted. T h e r e f o r e , the a m i d e I I I b a n d was not used. T h e PO4 1030-cm -1 b a n d has b e e n chosen as r e p r e s e n t a t i v e of the m i n e r a l phase. T o c o m p a r e the o r g a n i c / m i n e r a l ratio a n d t h e change in the organic m a t t e r , the a m i d e I b a n d at 1654 cm -1 has b e e n chosen for the second ratio. Comparison of the o r g a n i c / m i n e r a l ratio (Fig. 1A) a n d the c o m p o s i t i o n of the organic m a t t e r (Fig. 1B) d e m o n s t r a t e s t h a t the decrease of the organic c o n t e n t is not directly correlative to the alteration of the organic m a t t e r composition. T h e s e results s u p p o r t the view t h a t fossilization is a complex process. CONCLUSIONS Results can be divided in two m a i n categories: (1) knowledge of the processes of fossilization a n d (2) the m e t h o d . W i t h respect to category 1, the c u r r e n t viewp o i n t is t h a t the fossil r o d e n t a c c u m u l a t i o n s are due to p r i m a r y a c c u m u l a t i o n of r a p t o r regurgitation pellets. Alt h o u g h it is clear t h a t this stage modifies the recent bones '-3 (Table I a n d Fig. 1), the m e c h a n i s m s involved in these changes are poorly understood. Moreover, the identification of this digestive stage on fossil bones is uncertain. E v e n if the IamideA/Ipo4 ratio is not an accurate m e a s u r e m e n t of the organic c o n t e n t in bone, it gives a suitable calibration for the state of p r e s e r v a t i o n of fossil

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Volume 47, Number 1, 1993

bones. T h e c o m p a r i s o n b e t w e e n the crystallinity ratio a n d IamideA/Ip04 ratio p e r m i t s b e t t e r precision of certain alteration processes. I t m a y be s u p p o s e d t h a t the organic m a t r i x of bone is m o r e easily digested b y the r a p t o r t h a n is the m i n e r a l phase. However, the effects of digestion on bones d e p e n d on the bird, t h e age of the bird, the f r e q u e n c y of meals, etc. 2° T h e crystallinity ratio (SF) is highest a n d the organic m a t r i x m o s t a b u n d a n t in pellet bones. I t a p p e a r s t h a t the digestion b y hawks p r e f e r a b l y r e m o v e s a m o r p h o u s bone. Organic m a t t e r r e m a i n s a b u n d a n t in fossil levels 6, 4, a n d 7 a t Tighenif. T h e organic c o m p o s i t i o n of bones in level 4 is similar to t h a t of recent bones. Crystallinity is high in level 7. I t should be noticed t h a t these t h r e e levels are especially rich in fossil rodents. T h e r e a p p e a r s to be a relationship b e t w e e n the a b u n dance of bones a n d their composition. Consideration of category 2 d e m o n s t r a t e s t h a t the D R I F T t e c h n i q u e is a very effective m e a n s of obtaining i n f o r m a t i o n concerning the c o m p o s i t i o n a n d relative c o n c e n t r a t i o n of organic m a t e r i a l a n d the crystallinity of bones. Moreover, this m e t h o d is n o n d e s t r u c t i v e , a n d the s a m p l e s can be recovered a f t e r the e x p e r i m e n t . For example, it is possible to r e m o v e soluble K B r with ultrafiltration. T h e bone powder t h e n can be decalcified for later analysis, such as gel filtration, a m i n o - a c i d analysis, etc. T h e variability of diagenesis in fossil bones is very high, even within the s a m e bone. Hence, we m u s t use the s a m e s a m p l e for various analyses. T h i s is n o t possible with K B r pressed pellets. D a t a on crystallinity are also available. Moreover, a fossil is the only one of its kind a n d is n o t o b t a i n a b l e in vitro. T h u s its " d e s t r u c t i o n " b y grinding or o t h e r m e t h o d s m u s t provide m a x i m u m data. T h e s t u d y of the purified soluble a n d insoluble m a t r i x of bones b y infrared s p e c t r o s c o p y a n d o t h e r t e c h n i q u e s should provide m o r e detail concerning protein alteration.

ACKNOWLEDGMENTS I wish to thank Dr. C. Denys (U.R.A. 327 C.N.R.S., Universit6 de Montpellier 2) for the loan of the samples ground for this study. I also thank Pr. Dr. B. H. Purser (U.R.A. 723 C.N.R.S., Universit6 Paris X I Orsay) for reviewing the English manuscript. 1. C. Denys, Bull. Mus. natn. Hist. nat. Paris 4~ s6r., 7, Sect. A, 4, 879 (1985). 2. P. Andrews, Owls, Caves and Fossils (Natural History Museum Publishers, London, 1990). 3. C. Denys and M. Mahboubi, Bull. Mus. natn. Hist. nat. Paris, Sect. A, 1 (in press). 4. Y. Dauphin and C. Denys, M6m. Soc. g6ol. Fr. 160 (in press). 5. A. S. Termine and A. S. Posner, Nature 211, 268 (1966). 6. A. Shemesh, Geochim. Cosmochim. Acta 54, 2433 (1990). 7. R. Z. LeGeros, W. P. Shirra, M. A. Miravite, and J. P. Legeros, "Amorphous Calcium Phosphates: Synthetic and Biological," in Physicochimie et Cristallographie des Apatites d'Intdrdt biologique (Colloques Intern. C.N.R.S., France, 1975), No. 230, p. 105. 8. H. Furedi and A. G. Walton, Appl. Spectrosc. 22, 23 (1968). 9. J. C. Elliot, "Infrared and Raman Spectroscopy of Calcified Tissues," in Methods of Calcified Tissue Preparation, G. R. Dickson, Ed. (Elsevier, Amsterdam, 1984), p.413. 10. T. Sakae, H. Mishima, and Y. Kozawa, "Proboscidea Fossil Teeth Suggest the Evolution of Enamel Crystals," in Mechanism and Phylogeny of Mineralization in Biological Systems, S. Suga and H. Nakahara, Eds. (Springer-Verlag, New York, 1991), p. 477.

11. H. Susi, J. S. Ard, and R. J. Carroll, Biopolymers 10, 1597 (1971). 12. T. Miyazawa, "Infra-red Studies of the Conformations of Polypeptides and Proteins," in Aspects of Proteins Structure, G. N. Ramachandran, Ed. (Academic Press, New York, 1963), p. 257. 13. S. Krimm and J. Bandekar, "Vibrational Spectroscopy and Conformation of Peptides, Polypeptides, and Proteins," in Advances in Protein Chemistry, C. B. Anfinsen, J. T. Edsall, and F. M. Richards, Eds. (Academic Press, New York, 1986), Vol. 38, p. 181. 14. D. M. Hembree, Jr., and N. R. Smyrl, Appl. Spectrosc. 43, 267 (1989). 15. K. Okada, Y. Ozaki, K. Kawauchi, and S. Muraishi, Appl. Spectrosc. 44, 1412 (1990). 16. Y. Dauphin, C. Denys, and A. Denis, Bull. Mus. natn. Hist. nat. Paris 4~ s6r., 11, Sect. A, l, 253 (1989). 17. C. Arambourg and R. Hoffstetter, Arch. Inst. Pal. Hum. 32, 9 (1963). 18. D. Geraads, J. J. Hublin, J. J. Jaeger, H. Tong, S. Sen, and P. Toubeau, Quatern. Res. 25, 380 (1986). 19. M. Suzuki, "Studies on the Physicochemical Nature of Hard Tissue. Infrared, N.M.R., X-ray Diffraction Investigation of Hydroxylradical, Crystalline Water and Carbonate Substitution in Biological Apatites," in Physicochimie et CristaUographie des Apatites d'Intdrdt Biologique (Colloques Intern. C.N.R.S., France, 1975), No. 230, p. 77. 20. P. Leprince, G. Dandrifosse, and E. Schoffeniels, Chem. Syst. Ecol. 7, 223 (1979).

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