In the Laboratory
W
Infrared Spectroscopy in the Study of Renal Lithiasis Jesús Fernández-Almeida Hospital Ramón y Cajal. Carretera de Colmenar Viejo, km. 9,100, E-28034 Madrid, Spain
Ana Fernández-Gacio and Carlos F. Marcos* Departamento de Química Orgánica, Facultad de Veterinaria, Universidad de Extremadura, Avenida de la Universidad s/n, E-10071 Cáceres, Spain; *
[email protected] Maira Fernández-Gacio Instituto Português Oncológico “Francisco Gentil”, Centro Regional do Porto, Rua Dr. António Bernardino de Almeida, 4200 Porto, Portugal
Chemistry often becomes an arduous subject for nonchemistry-major college students to study. The numerous implications of chemistry in life sciences do not always appear obvious for medical and veterinary students, and it is the chemistry teacher’s responsibility to illustrate these implications with proper and useful applications that can stimulate students’ interest. Chemistry not only forms the basis of the processes that occur in biological systems, but also constitutes a useful tool for the study and diagnosis of abnormal or pathological functions. Unfortunately it is difficult to find practical applications of current interest in human or veterinary medicine that are also easily performed in the undergraduate laboratory. Renal lithiasis (1) constitutes a remarkable example of the connection between chemistry and medicine. Its appearance is the consequence of urine supersaturation, which leads to the production of a precipitate in the urinary tract. This precipitate usually forms crystalline or amorphous conglomerates of variable sizes, frequently constituted essentially of a single organic or inorganic compound, or a mixture of two or more substances. The specific composition is influenced by genetic factors of the individual, and also, in different degrees, by age, sex, diet, geographic area, and social habits. Renal lithiasis is an important cause of morbidity, probably older than humankind, which equally affects humans and other animals with kidneys. Urinary stones were found in Egyptian (2) and pre-Columbian (3) mummies dated more than 4000 years old. Nowadays, prevalence in humans is estimated to be 2–3%, and the relapse incidence is very high, reaching about 85%. Determination of the chemical composition of urinary calculi is of vital importance in order to decide the proper dietary or therapeutic measures that must be taken to reduce the possibility of a relapse. Furthermore, urinary lithiasis is often a symptom of a hidden disease, and chemical analysis of calculi may be a powerful tool in diagnosis. Infrared spectroscopy, commonly used in the characterization of organic compounds (4), has also proved to be useful for the qualitative and semi-quantitative analysis of inorganic salts possessing polyatomic anions (5), and is therefore a valuable tool in renal stone analysis. Actually, FTIR spectroscopy in KBr pellets, complemented with binocular lens observation of macroscopic and microscopic features, is currently used as a routine method for the determination of calculi composition (6). IR is preferred to qualitative traditional wet chemical techniques, as these are not always unequivocal, and often lead to wrong conclusions (7). We have successfully used
both FTIR spectrometers and old continuous wave spectrometers in the analysis of renal stones from humans, dogs, and other domestic animals. IR analysis is rapid, precise, and requires 2 mg of sample or less. In mixed composition calculi, the different components can also be identified (8). Veterinary (or medical) undergraduates attending a general chemistry course can easily determine the chemical composition of renal stones by comparison of their IR spectra with the spectra taken from pure samples. The experiment has been designed to analyze actual calculi of human or animal origin, although the authors are aware that these may only be easily available in medical or veterinary schools associated with clinical hospitals. In other cases, the laboratory procedure can be easily modified to record the spectra of pure commercial compounds commonly present in urinary calculi, which can be used as standards to compare with the spectra of calculi handed out by the instructor. On the other hand, pure compounds can be mixed to emulate mixed composition calculus, or to compare with real samples of doubtful composition. Procedure Infrared spectra are performed using the KBr pellet technique (9). We use a standard hydraulic press, but any other simple methods previously described in this Journal (10) may also be suitable. In our case, spectra are recorded in a Bruker Vector 22 FTIR spectrometer and subsequently transformed to JCAMP-DX format (11), which makes it easier to manipulate and compare the different spectra with the standards on a PC. Otherwise, the spectra can be printed out and compared with printed copies of the IRs of pure samples handed out by the demonstrator. The complete process of preparing the KBr pellet, measuring the spectrum, transforming it to JCAMP-DX format, and copying it to a floppy disk takes no more than 15 minutes. In a two-hour session each group of students can then analyze several calculi of different composition. Analysis on old continuous wave spectrometers is also possible, though it needs larger amounts of sample, and the process requires longer times. Moreover, resolution is lower, and very weak or out of scale spectra are obtained if the precise amount of sample is not used. Students are also asked to analyze and write down the main external features (12) of the calculi, such as color, size, shape, hardness, crystallographic characteristics, and structural organization (concentric or not, homogeneous or heterogeneous). If the stone presents a heterogeneous structure
JChemEd.chem.wisc.edu • Vol. 80 No. 8 August 2003 • Journal of Chemical Education
909
In the Laboratory
(e.g., a light-colored nucleus and a dark external layer, or a region with a different color or structure), it is necessary to sample and record IR spectra of the different regions. We must emphasize not only the analytical value of the technique, but also use this context to discuss the basic concepts of IR spectroscopy, and molecular vibrations as the origin of IR bands. Finally, students are required to write a short report including a discussion of results, with mention of the main characteristic bands shown on the recorded IR spectra. In summary, this experiment not only allows undergraduates to learn a spectrometric technique with current use in life sciences, but also prompts the discussion of interesting questions regarding organic and inorganic constituents of living beings, molecular absorption spectra, pH, solubility product, crystallization processes, and allomorphic forms. Hazards Biological samples should be handled with proper precautions; gloves and safety glasses must be worn when handling calculi. The pellet press (for the KBr) works under high pressures; the safety shield must be down when operating the press. W
Supplemental Material
Representative spectra of the most commonly occurring calculi in humans and domestic animals and their interpretations are provided in this issue of JCE Online. Acknowledgment We gratefully acknowledge financial support from the Consejería de Educación de la Junta de Extremadura y Fondo Social Europeo (ref. IPR00C043). Literature Cited 1. General articles on lithiasis, in humans and domestic animals, respectively, include: (a) Williams, H. E. N. Engl. J. Med. 1974, 290, 33; and (b) Franti, C. E.; Ling, G. V.; Ruby, A. L.; Johnson, D. L. Am. J. Vet. Res. 1999, 60, 29. 2. Modlin, M. S. Afr. Med. J. 1980, 58, 652. 3. See for example: Streitz, J. M.; Aufderheide, A. C.; El Najjar, M.; Ortner, D. J. Urol. 1981, 126, 452. 4. Reeder, D. M.; Sridharan, S. J. Chem. Educ. 1982, 59, 503.
910
5. (a) Ackermann, M. N. J. Chem. Educ. 1970, 47, 69; (b) Kalbus, G. E.; Kalbus, L. H. J. Chem. Educ. 1966, 43, 314; (c) Miller, F. A.; Wilkins, C. H. Analyt. Chem. 1952, 24, 1253. 6. (a) Oliver, L. K.; Sweet, R. V. Clin. Chim. Acta 1976, 72, 17; (b) Lafaut, J. P.; Daudon, M.; Hardeman, P.; Werbrouck, P. J. Urologie 1992, 98, 152. 7. Gault, M. H.; Ahmed, M.; Karla, J.; Senciall, I.; Cohen, W.; Churchill, D. Clin. Chim. Acta 1980, 104, 349. 8. (a) Berthelot, M.; Cornu, G.; Daudon, M.; Helbert, M.; Laurence, C. Clin. Chem. 1987, 33, 2070; (b) Volmer, M.; Wolthers, B. G.; Metting, H. J.; de Haan, T. H. Y.; Coenegracht, P. M. J.; van der Silk, W. Clin. Chem. 1994, 40, 1692. 9. Many experiments involving IR spectroscopy have been published in this Journal: cf refs 4 and 5a–5b. Recent publications include: (a) Hill, M. A. J. Chem. Educ. 2001, 78, 26; (b) Houghton, T. P.; Kalivas, J. H. J. Chem. Educ. 2000, 77, 1314; (c) Barker, B.; Owen, N. L. J. Chem. Educ. 1999, 76, 1706; (d) Dragan, S.; Fitch, A. J. Chem. Educ. 1998, 75, 1018; (e) Heuer, W. B.; Koubek, E. J. Chem. Educ. 1997, 74, 313. 10. Kalberg, C.; Ogren, P. J. J. Chem. Educ. 2000, 77, 391; and references therein. 11. JCAMP-DX is a standard format for exchange of spectra in computer readable form, which is currently the responsibility of the IUPAC Committee on Printed and Electronic Publications (CPEP). For a description of JCAMP-DX for IR see: (a) Grasselli, J. G. Pure & Appl. Chem. 1991, 63, 1781; (b) JCAMP-DX files can be viewed using a MDL CHIME plugin, freely available from the MDL Web site http://www.mdli.com/ chemscape/chime/chime.html (accessed May 2003); (c) For an application of MDL CHIME and JCAM-DX files for the interactive visualization infrared spectra and molecular models, see: Lahti, P. M.; Motyka, E. J.; Lancashire, R. J. J. Chem. Educ. 2000, 77, 649. 12. Impressive photographs of urinary calculi from the collection kept in Hospital Ramón y Cajal in Madrid, as well as a complete discussion on calculi analysis by IR spectroscopy can be found in: (a) Ávila Padilla, S.; Cuervo Herrero, C.; Ripoll Sevillano, E.; Villar Palasí, J. “Guía Práctica para el Reconocimiento Químico-Clínico de Cálculos de las Vías Urinarias”. Edited by Hospital Ramón y Cajal. Madrid, 1998. (b) Ávila Padilla, Sergio. “Aproximación al estudio bioquímico y epidemiológico de la litiasis urinaria a través de un nuevo sistema de clasificación de los cálculos urinarios”. PhD thesis. Universidad Complutense de Madrid. Facultad de Farmacia. Madrid, 1998. (c) Reproductions of photographs published in refs 12a and 12b can be found at this Internet site: http:// www.unex.es/qoceres/lithiasis.htm (accessed May 2003).
Journal of Chemical Education • Vol. 80 No. 8 August 2003 • JChemEd.chem.wisc.edu
