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Spectroscopic Characterization of Bendazac and Benzydamine: Possible Photochemical Modes of Action
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
221, 266–270 (1996)
0584
Spectroscopic Characterization of Bendazac and Benzydamine: Possible Photochemical Modes of Action Joseph M. Jez,* Jane M. Vanderkooi,*,1 and Alan M. Laties† *Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and †Department of Opthalmology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received February 26, 1996 The involvement of near-UV light in cataract development suggests that potential anti-cataract drugs may display unusual spectroscopic properties. As bendazac impedes certain effects associated with lens opacification, we have characterized the singlet and triplet states of bendazac and its analog, benzydamine, by fluorescence and phosphorescence methods. These compounds have much shorter triplet state lifetimes compared to the triplet state lifetimes observed in proteins. Our results raise the possibility that the photoprotective action of these compounds may result from their ability to dissipate energy through the triplet state. We propose alternative modes for the photoprotective actions of these compounds. © 1996 Academic Press, Inc.
Cataract has a number of causes, all ultimately leading to production of aggregates of lens proteins that scatter light and dim sight (1). Cataract development correlates with exposure to near-UV radiation and generation of triplet state tryptophan and reactive oxygen species which mediate damaging photochemical reactions (2). Although the detailed mechanism of how these reactive species damage lens proteins is not known, they represent points of attack in developing anti-cataract drugs. Bendazac (Fig. 1) has been shown as potentially useful in slowing the opacification process. Studies of bendazac describe the prevention of UV-induced denaturation of crystallins (3); reduction of X-ray induced damage to rabbit eye lens (4); prevention of cyanate-induced phase separation opacities (5); and inhibition of glycosylation of soluble lens proteins (6). Pre-treatment of rats with bendazac reduces the level of retinal damage following exposure to intense UV light (7). Also, bendazac inhibits denaturation of bovine serum albumin (BSA) in vitro, but does not inhibit reduction of cytochrome c in a xanthine oxidase system (7–9). These observations indicate that direct interaction between bendazac and protein can occur. The involvement of near-UV light in cataract development suggests that the action of bendazac could result from its spectroscopic properties, which have not been fully studied. Direct interaction between bendazac and excited state tryptophans of lens proteins could provide an additional mechanism to explain its photoprotective action. This work investigates the singlet and triplet state characteristics of bendazac by fluorescence and phosphorescence methods. For comparative purposes, benzydamine, a bendazac analog (Fig. 1), was included in these experiments. Our results suggest two new mechanisms to explain how bendazac protects against photochemical damage. MATERIALS AND METHODS Samples of bendazac and benzydamine were acquired from Angelini Pharmaceuticals. Trizma buffer and sodium chloride were obtained from Sigma. All experiments were conducted in the same buffer solution [1 mM Tris, pH 7.0; 10 mM NaCl]. Fluorescence excitation and emission spectra of bendazac and benzydamine were obtained with a Perkin-Elmer LS5 spectrophotometer equipped with an ATT6300 personal computer. The program FLURO (Softways) digitized the spectral data. The room-temperature singlet state lifetimes in the nanosecond time scale were measured with a time-correlated single
1
To whom correspondence should be addressed. Fax: (215) 573-2042. 266
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Chemical formula of bendazac and benzydamine. Bendazac R 4 CH2COOH; and Benzydamine R 4 (CH2CH2CH2)N(CH3)2. photon counting instrument, previously described (10). The Global Unlimited program provided data analysis of the decay curves (11). Phosphorescence spectra of bendazac and benzydamine were determined on the LS5 spectrophotometer with temperature regulation provided by a liquid nitrogen cold-finger Dewar. Triplet state lifetime determination used a previously described in-house phosphorimeter design with temperature regulation, as above (12).
RESULTS AND DISCUSSION Fluorescence spectroscopy was used to characterize the singlet excited state of bendazac and benzydamine. The fluorescence spectra of bendazac and benzydamine are identical with excitation lmax 4 306 nm and emission lmax 4 362 nm (Fig. 2). The emission spectra of bendazac and
FIG. 2. Fluorescence excitation and emission spectra of bendazac and BSA. The emission spectrum of bendazac (−) was obtained with excitation at 306 nm; the excitation spectrum of bendazac used a detection wavelength of 360 nm. The emission spectrum of BSA (− −) was obtained with excitation at 280 nm; the emission spectrum used a detection wavelength of 350. All spectra were determined with the excitation and emission bandpass set at 15 nm and 3 nm, respectively. Luminescence intensities were scaled for comparison. 267
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1 Summary of 293°K Fluorescence Data Compound
Excitation lmax
Emission lmax
Lifetime
Bendazac Benzydamine BSA (13) HEWL (13)
306 nm 306 nm 278 nm 284 nm
362 nm 362 nm 350 nm 345 nm
16.9 ns 15.5 ns 3.3–4.6 ns 2.6 ns
benzydamine are broad and devoid of vibrational resolution. The excitation and emission spectra of BSA are shown for comparison and demonstrates the overlap between the absorption spectrum of bendazac and the fluorescence emission spectrum of tryptophan in BSA. Bendazac and benzydamine differ from tryptophan in the duration of their room temperature fluorescence lifetimes. The lifetimes of both compounds are four to five times longer than that of tryptophan in BSA or hen egg-white lysozyme (HEWL), as noted in Table 1. The x2 values of the fits were 1.15 for bendazac and 1.05 for benzydamine indicating a rigorous single exponential decay for each compound. The long lifetime and single exponential decay suggest these drugs have
FIG. 3. 77°K Phosphorescence excitation and emission spectra of bendazac. The excitation spectrum used 460 nm as the observed wavelength and the emission spectrum 310 nm as the excitation wavelength. For each spectra, the first 0.1 ms of emission was gated out to limit the contribution of prompt fluorescence; also, excitation and emission bandpass were set at 15 nm each. Luminescence intensities were scaled for comparison. Inset: Phosphorescence lifetime decay of bendazac at 77°K: Determination of the lifetime decay utilized an in-house phosphorimeter exciting at 317 nm and observing emission at 369 nm with gate and bandpass as above. 268
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2 Summary of 77°K Phosphorescence Data Compound
Emission lmax, nm
Lifetime
Bendazac Benzydamine Free tryptophan (13) BSA (13) g crystallin (14)
467 nm 468 nm 440 nm 437 nm 442 nm
2.9 sec 3.1 sec 6.8 sec 6.0–6.4 sec 5.1 sec
a higher quantum yield than tryptophan and lack excited state photoreactions within the conditions used. The triplet state characteristics of bendazac and benzydamine were investigated with phosphorescence methods. Bendazac’s excitation lmax 4 317 nm with phosphorescence emission resolved into three distinct components—a major peak at 467 nm and two secondary peaks at 439 nm and 502 nm (Fig. 3). Benzydamine produces similar spectra (not shown). The two compounds also have similar triplet state lifetimes at 77°K—2.8 seconds for bendazac and 3.1 seconds for benzydamine (Fig. 3 inset). Both fit to a single exponential decay with R2 4 0.999. As summarized in Table 2, the triplet state lifetimes of bendazac and benzydamine are somewhat shorter than those of free tryptophan or tryptophan found in either BSA, HEWL, or lens g-crystallin. Previous studies demonstrate that protein-drug interactions play a role in bendazac’s photoprotective effect (7–9), and that triplet state tryptophan mediates photodamage (1, 14). Based on our spectroscopic characterization of bendazac and benzydamine, new mechanisms explaining bendazac’s protective action against UV-induced photodamage can be postulated. These mechanisms center on the shorter triplet state lifetime of the drug versus the triplet state of tryptophan observed in protein (Table 2). The first mechanism involves bendazac acting as an excited state quencher. On interaction between bendazac and protein, energy transfer from the singlet state protein to the compound could occur, based on the overlap of the fluorescence spectra of BSA and bendazac (Fig. 2) (15). Since the energy required for a protein to enter the triplet state comes from the singlet state, binding and subsequent energy transfer may bleed energy away from the singlet state resulting in a decrease in the protein triplet state. Bendazac can then dissipate the accepted energy more rapidly than the protein. The phenyl group of bendazac provides a greater amount of vibrational freedom than the constrained tryptophan structure (16). This motion translates spectroscopically into the release of energy through vibrations rather than phosphorescence, thereby shortening the excited state lifetime and possibly limiting the time in which damaging reactions can occur (Table 2). Alternatively, bendazac may act as “chemical sunglasses” similar to p-aminobenzoic acid and cinnaminic acid in sunscreens (17). Bendazac spectroscopically resembles tryptophan and absorbs in the same region. This additional absorption may act like a “sunblock” for the protein, reducing the amount of UV energy available to cause photodamage without the requiring any protein-drug interaction. These additional ideas on the photoprotective action of bendazac do not preclude the already reported actions of this compound (3–9). It is possible that a combination of actions allows bendazac to reduce protein photochemical damage. Since unquenched triplet state tryptophan mediates opacification, excited state species may provide additional targets for potential anticataract drugs. The development of compounds with unique spectral properties to act as anticataract compounds has not been pursued, but could provide new avenues in treating the most common eye lens disease. ACKNOWLEDGMENTS This work was supported by NIH Grant GM34448 awarded to J.M.V. and by Research to Prevent Blindness, New York, NY and The National Retinitis Pigmentosa Foundation, Inc. to A.M.L. 269
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. Lerman, S. (1983) in Radiant Energy and the Eye, pp. 131–162, MacMillian, NY. 2. Bochow, T. W., West, S. K., Azar, A., Munoz, B., Somme, Munoz, A., Sommer, B., and Taylor, H. (1989) Arch. Opthal. 107, 369–372. 3. Silvestrini, B., Catanese, B., Barillari, G., Iorio, E., and Valeri, P. (1983) Int. J. Tissue React. 5, 217–255. 4. Pandolfo, L., Livrea, M. A., and Bono, A. (1986) Exp. Eye Res. 42, 167–175. 5. Lewis, B. S., Rixon, K. C., and Harding, J. J. (1986) Exp. Eye Res. 43, 973–979. 6. Lewis, B. S., and Harding, J. J. (1988) Exp. Eye Res. 47, 217–225. 7. Barcellone, P. S., Campana, A., Caranti, S., D’Onafrio, E., and Silvestrini, B. (1991) Pharm. Res. 24, 105–111. 8. Musci, G., and Silvestrini, B. (1987) Drugs Exp. & Clin. Res. 13, 289–291. 9. Balfour, J. A., and Clissold, S. P. (1990) Drugs 39, 575–596. 10. Holton, G. R., Trommsdorff, H. P., and Hochstrasser, R. M. (1986) Chem. Phys. Letters 131, 44–50. 11. Knutson, J. R., Beechem, J. M., and Brand, L. (1983) Chem. Phys. Letters 102, 501–507. 12. Green, T. P., Wilson, D. F., Vanderkooi, J. M., and DeFeo, J. D. (1988) Anal. Biochem. 174, 73–79. 13. Longworth, J. W. (1971) in Excited States of Proteins and Nucleic Acids (Steiner, R. F., and Wernaugh, J., Eds.), pp. 319–484, Plenum Press, NY. 14. Berger, J. W., and Vanderkooi, J. M. (1988) Biochemistry 28, 5501–5508. 15. Forster, T. (1951) in Fluoreszenz Organischer Verbindungen, Vandenhoeck and Rupt, Gottingen, FRG. 16. Robinson, G. W. (1974) in Excited States (Linn, E. C., Ed.) Vol. 1 pp. 1–34, Academic Press, NY. 17. Guercio-Hauer, C., MacFarlane, D. F., and Deleo, V. A. (1994) Amer. Fam. Phys. 50, 327–332.
270
221, 266–270 (1996)
0584
Spectroscopic Characterization of Bendazac and Benzydamine: Possible Photochemical Modes of Action Joseph M. Jez,* Jane M. Vanderkooi,*,1 and Alan M. Laties† *Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and †Department of Opthalmology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received February 26, 1996 The involvement of near-UV light in cataract development suggests that potential anti-cataract drugs may display unusual spectroscopic properties. As bendazac impedes certain effects associated with lens opacification, we have characterized the singlet and triplet states of bendazac and its analog, benzydamine, by fluorescence and phosphorescence methods. These compounds have much shorter triplet state lifetimes compared to the triplet state lifetimes observed in proteins. Our results raise the possibility that the photoprotective action of these compounds may result from their ability to dissipate energy through the triplet state. We propose alternative modes for the photoprotective actions of these compounds. © 1996 Academic Press, Inc.
Cataract has a number of causes, all ultimately leading to production of aggregates of lens proteins that scatter light and dim sight (1). Cataract development correlates with exposure to near-UV radiation and generation of triplet state tryptophan and reactive oxygen species which mediate damaging photochemical reactions (2). Although the detailed mechanism of how these reactive species damage lens proteins is not known, they represent points of attack in developing anti-cataract drugs. Bendazac (Fig. 1) has been shown as potentially useful in slowing the opacification process. Studies of bendazac describe the prevention of UV-induced denaturation of crystallins (3); reduction of X-ray induced damage to rabbit eye lens (4); prevention of cyanate-induced phase separation opacities (5); and inhibition of glycosylation of soluble lens proteins (6). Pre-treatment of rats with bendazac reduces the level of retinal damage following exposure to intense UV light (7). Also, bendazac inhibits denaturation of bovine serum albumin (BSA) in vitro, but does not inhibit reduction of cytochrome c in a xanthine oxidase system (7–9). These observations indicate that direct interaction between bendazac and protein can occur. The involvement of near-UV light in cataract development suggests that the action of bendazac could result from its spectroscopic properties, which have not been fully studied. Direct interaction between bendazac and excited state tryptophans of lens proteins could provide an additional mechanism to explain its photoprotective action. This work investigates the singlet and triplet state characteristics of bendazac by fluorescence and phosphorescence methods. For comparative purposes, benzydamine, a bendazac analog (Fig. 1), was included in these experiments. Our results suggest two new mechanisms to explain how bendazac protects against photochemical damage. MATERIALS AND METHODS Samples of bendazac and benzydamine were acquired from Angelini Pharmaceuticals. Trizma buffer and sodium chloride were obtained from Sigma. All experiments were conducted in the same buffer solution [1 mM Tris, pH 7.0; 10 mM NaCl]. Fluorescence excitation and emission spectra of bendazac and benzydamine were obtained with a Perkin-Elmer LS5 spectrophotometer equipped with an ATT6300 personal computer. The program FLURO (Softways) digitized the spectral data. The room-temperature singlet state lifetimes in the nanosecond time scale were measured with a time-correlated single
1
To whom correspondence should be addressed. Fax: (215) 573-2042. 266
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Chemical formula of bendazac and benzydamine. Bendazac R 4 CH2COOH; and Benzydamine R 4 (CH2CH2CH2)N(CH3)2. photon counting instrument, previously described (10). The Global Unlimited program provided data analysis of the decay curves (11). Phosphorescence spectra of bendazac and benzydamine were determined on the LS5 spectrophotometer with temperature regulation provided by a liquid nitrogen cold-finger Dewar. Triplet state lifetime determination used a previously described in-house phosphorimeter design with temperature regulation, as above (12).
RESULTS AND DISCUSSION Fluorescence spectroscopy was used to characterize the singlet excited state of bendazac and benzydamine. The fluorescence spectra of bendazac and benzydamine are identical with excitation lmax 4 306 nm and emission lmax 4 362 nm (Fig. 2). The emission spectra of bendazac and
FIG. 2. Fluorescence excitation and emission spectra of bendazac and BSA. The emission spectrum of bendazac (−) was obtained with excitation at 306 nm; the excitation spectrum of bendazac used a detection wavelength of 360 nm. The emission spectrum of BSA (− −) was obtained with excitation at 280 nm; the emission spectrum used a detection wavelength of 350. All spectra were determined with the excitation and emission bandpass set at 15 nm and 3 nm, respectively. Luminescence intensities were scaled for comparison. 267
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1 Summary of 293°K Fluorescence Data Compound
Excitation lmax
Emission lmax
Lifetime
Bendazac Benzydamine BSA (13) HEWL (13)
306 nm 306 nm 278 nm 284 nm
362 nm 362 nm 350 nm 345 nm
16.9 ns 15.5 ns 3.3–4.6 ns 2.6 ns
benzydamine are broad and devoid of vibrational resolution. The excitation and emission spectra of BSA are shown for comparison and demonstrates the overlap between the absorption spectrum of bendazac and the fluorescence emission spectrum of tryptophan in BSA. Bendazac and benzydamine differ from tryptophan in the duration of their room temperature fluorescence lifetimes. The lifetimes of both compounds are four to five times longer than that of tryptophan in BSA or hen egg-white lysozyme (HEWL), as noted in Table 1. The x2 values of the fits were 1.15 for bendazac and 1.05 for benzydamine indicating a rigorous single exponential decay for each compound. The long lifetime and single exponential decay suggest these drugs have
FIG. 3. 77°K Phosphorescence excitation and emission spectra of bendazac. The excitation spectrum used 460 nm as the observed wavelength and the emission spectrum 310 nm as the excitation wavelength. For each spectra, the first 0.1 ms of emission was gated out to limit the contribution of prompt fluorescence; also, excitation and emission bandpass were set at 15 nm each. Luminescence intensities were scaled for comparison. Inset: Phosphorescence lifetime decay of bendazac at 77°K: Determination of the lifetime decay utilized an in-house phosphorimeter exciting at 317 nm and observing emission at 369 nm with gate and bandpass as above. 268
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2 Summary of 77°K Phosphorescence Data Compound
Emission lmax, nm
Lifetime
Bendazac Benzydamine Free tryptophan (13) BSA (13) g crystallin (14)
467 nm 468 nm 440 nm 437 nm 442 nm
2.9 sec 3.1 sec 6.8 sec 6.0–6.4 sec 5.1 sec
a higher quantum yield than tryptophan and lack excited state photoreactions within the conditions used. The triplet state characteristics of bendazac and benzydamine were investigated with phosphorescence methods. Bendazac’s excitation lmax 4 317 nm with phosphorescence emission resolved into three distinct components—a major peak at 467 nm and two secondary peaks at 439 nm and 502 nm (Fig. 3). Benzydamine produces similar spectra (not shown). The two compounds also have similar triplet state lifetimes at 77°K—2.8 seconds for bendazac and 3.1 seconds for benzydamine (Fig. 3 inset). Both fit to a single exponential decay with R2 4 0.999. As summarized in Table 2, the triplet state lifetimes of bendazac and benzydamine are somewhat shorter than those of free tryptophan or tryptophan found in either BSA, HEWL, or lens g-crystallin. Previous studies demonstrate that protein-drug interactions play a role in bendazac’s photoprotective effect (7–9), and that triplet state tryptophan mediates photodamage (1, 14). Based on our spectroscopic characterization of bendazac and benzydamine, new mechanisms explaining bendazac’s protective action against UV-induced photodamage can be postulated. These mechanisms center on the shorter triplet state lifetime of the drug versus the triplet state of tryptophan observed in protein (Table 2). The first mechanism involves bendazac acting as an excited state quencher. On interaction between bendazac and protein, energy transfer from the singlet state protein to the compound could occur, based on the overlap of the fluorescence spectra of BSA and bendazac (Fig. 2) (15). Since the energy required for a protein to enter the triplet state comes from the singlet state, binding and subsequent energy transfer may bleed energy away from the singlet state resulting in a decrease in the protein triplet state. Bendazac can then dissipate the accepted energy more rapidly than the protein. The phenyl group of bendazac provides a greater amount of vibrational freedom than the constrained tryptophan structure (16). This motion translates spectroscopically into the release of energy through vibrations rather than phosphorescence, thereby shortening the excited state lifetime and possibly limiting the time in which damaging reactions can occur (Table 2). Alternatively, bendazac may act as “chemical sunglasses” similar to p-aminobenzoic acid and cinnaminic acid in sunscreens (17). Bendazac spectroscopically resembles tryptophan and absorbs in the same region. This additional absorption may act like a “sunblock” for the protein, reducing the amount of UV energy available to cause photodamage without the requiring any protein-drug interaction. These additional ideas on the photoprotective action of bendazac do not preclude the already reported actions of this compound (3–9). It is possible that a combination of actions allows bendazac to reduce protein photochemical damage. Since unquenched triplet state tryptophan mediates opacification, excited state species may provide additional targets for potential anticataract drugs. The development of compounds with unique spectral properties to act as anticataract compounds has not been pursued, but could provide new avenues in treating the most common eye lens disease. ACKNOWLEDGMENTS This work was supported by NIH Grant GM34448 awarded to J.M.V. and by Research to Prevent Blindness, New York, NY and The National Retinitis Pigmentosa Foundation, Inc. to A.M.L. 269
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. Lerman, S. (1983) in Radiant Energy and the Eye, pp. 131–162, MacMillian, NY. 2. Bochow, T. W., West, S. K., Azar, A., Munoz, B., Somme, Munoz, A., Sommer, B., and Taylor, H. (1989) Arch. Opthal. 107, 369–372. 3. Silvestrini, B., Catanese, B., Barillari, G., Iorio, E., and Valeri, P. (1983) Int. J. Tissue React. 5, 217–255. 4. Pandolfo, L., Livrea, M. A., and Bono, A. (1986) Exp. Eye Res. 42, 167–175. 5. Lewis, B. S., Rixon, K. C., and Harding, J. J. (1986) Exp. Eye Res. 43, 973–979. 6. Lewis, B. S., and Harding, J. J. (1988) Exp. Eye Res. 47, 217–225. 7. Barcellone, P. S., Campana, A., Caranti, S., D’Onafrio, E., and Silvestrini, B. (1991) Pharm. Res. 24, 105–111. 8. Musci, G., and Silvestrini, B. (1987) Drugs Exp. & Clin. Res. 13, 289–291. 9. Balfour, J. A., and Clissold, S. P. (1990) Drugs 39, 575–596. 10. Holton, G. R., Trommsdorff, H. P., and Hochstrasser, R. M. (1986) Chem. Phys. Letters 131, 44–50. 11. Knutson, J. R., Beechem, J. M., and Brand, L. (1983) Chem. Phys. Letters 102, 501–507. 12. Green, T. P., Wilson, D. F., Vanderkooi, J. M., and DeFeo, J. D. (1988) Anal. Biochem. 174, 73–79. 13. Longworth, J. W. (1971) in Excited States of Proteins and Nucleic Acids (Steiner, R. F., and Wernaugh, J., Eds.), pp. 319–484, Plenum Press, NY. 14. Berger, J. W., and Vanderkooi, J. M. (1988) Biochemistry 28, 5501–5508. 15. Forster, T. (1951) in Fluoreszenz Organischer Verbindungen, Vandenhoeck and Rupt, Gottingen, FRG. 16. Robinson, G. W. (1974) in Excited States (Linn, E. C., Ed.) Vol. 1 pp. 1–34, Academic Press, NY. 17. Guercio-Hauer, C., MacFarlane, D. F., and Deleo, V. A. (1994) Amer. Fam. Phys. 50, 327–332.
270