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Chemical and photolytical transformation of biomedically significant compounds in the presence of deuterated solvents
Journal of Photochemistry and Photobiology B: Biology 70 (2003) 91–97 www.elsevier.com / locate / jphotobiol
Chemical and photolytical transformation of biomedically significant compounds in the presence of deuterated solvents a, b b F.M. Salih *, A.E. Pillay , A. Al-Hamdi a
Department of Clinical and Biomedical Physics, College of Medicine, Sultan Qaboos University, P.O. Box 35, Al Khoud 123, Oman b Department of Chemistry, College of Science, Sultan Qaboos University, P.O. Box 36, Al Khoud 123, Oman Received 5 March 2002; received in revised form 3 December 2002; accepted 31 March 2003
Abstract The effect of the nature of solvent on the properties of biomedically important compounds is of particular importance. The conversion of certain biomedical compounds with deuterated solvents is an area of research that has not been accorded adequate recognition in the literature. We explored this area in the interest of shedding some light on the possible effects of solvent on the nature of the solute. The transformation of specific medically important compounds such as bilirubin, thymine, uracil, dehydrocholesterol (7-DHC) and vitamin D 3 was observed in the presence of deuterated solvents such as heavy water and deuterated chloroform. The products of the relevant reactions were confirmed spectrophotometrically. An additional feature to our investigation involved the photolysis of the aforementioned compounds by solar irradiation. The pure samples were dissolved in solutions of the deuterated solvents, corresponding to concentrations of typically 10 22 mM, and exposed to sunlight for about 15–30 min. The deuterated solvents caused chemical transformation in all chemical compounds tested, and produced intense characteristic absorbance maxima between 200 and 700 nm. Sunlight exposure was also effective in either augmenting the effects of deuterated solvent as in bilirubin and 7-DHC or reducing it as with thymine or having no effect as with uracil or completely changing it as in vitamin D 3 . It has been shown that the use of deuterated solvents produces unique chemical and photochemical conversions of bilirubin, 7-DHC, thymine, uracil and vitamin D 3 . This was attributed to the fact that deuterated compounds display a somewhat different chemistry to their ordinary counterparts and that possibly thermodynamic considerations could be responsible for the novel transformations. 2003 Elsevier B.V. All rights reserved. Keywords: Spectrophotometry; Deuterated solvents; Biomedical compounds; Sunlight; Transformation
1. Introduction Our investigation essentially focused on the influence of the nature of the solvent on the properties of certain biomedically important compounds. The conversion of these compounds with deuterated solvents is a relatively unexplored area of research. Our work with deuterated solvents started by initially observing marked transformations of these compounds when they were prepared in such media for specific instrumental studies. Such observations subsequently led to a wider exploration where different solvents were applied to a range of biomedical compounds. Certain compounds, such as thymine and uracil, are of immense medical significance because they play vital roles in research associated with DNA, RNA [1,2]. Other compounds, such as bilirubin, stimulate considerable clini*Corresponding author. Tel.: 1968-515-110. E-mail address: [email protected] (F.M. Salih). 1011-1344 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S1011-1344(03)00059-9
cal interest because of the association with neonatal jaundice in newborn babies [3–7], while dehydrocholesterol (7-DHC) and vitamin D 3 are essential in the metabolism of minerals, particularly calcium [7–9]. The chemical and photolytical conversion of these compounds continues to attract attention because such studies could lead to links associated with the aetiology of disease and disorders. The biological and clinical significance of tested chemical compounds lies in their vital role in maintaining growth and metabolism and ultimately in supporting a healthy status of the body. Thymine and uracil are the building blocks of DNA and RNA, respectively. They are vital for their replication [2]. They also play a major role in the process of protein synthesis. Any damage or change to their integrity will impair the process of DNA and RNA replication and hence this would affect the formation of particular proteins that are coded by these compounds. Many factors can create different types of damage to these chemicals, namely, radiation, UV and chemicals which
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would alter the genetic make up and lead to genetic mutations [10–12]. Vitamin D 3 is an indispensable compound. It is required to assist in calcium absorption from the intestine. Calcium is deposited in bones with the aid of vitamin D 3 . Such deposition is important to maintain the rigidity and strength of the bone. Vitamin D 3 is usually available in many nutritional components and our body can also photosynthesize it by solar conversion of 7-DHC. Deficiency of this vitamin originates mainly from either lower uptake or less exposure to sunlight [8,9]. However, it is documented that excessive exposure to sunlight may damage vitamin D 3 through reversible transformation to its photoisomers [13]. Bilirubin, on the other hand, is a by-product produced during the process of red blood cell degradation. It is normally converted by the liver to a water-soluble product and excreted by the kidney. When the rate of bilirubin formation increases either by excessive haemolysis or due to liver malfunctioning, as in many hepatic diseases or where the liver is not yet mature enough to carry out this process efficiently, as in neonates, bilirubin concentration in the blood rises. A clinical condition called hyperbilirubinemia results. This leads to the deposition of bilirubin in brain cells. Such a deposition makes a hyperbilirubinic person more susceptible to central nervous system damage [4–7]. Hence, bilirubin must be kept down to levels below 10–15% [7]. A preliminary study involving the conversions of these compounds in the presence of deuterated solvents produced some interesting observations. Further studies, mainly with heavy water and deuterated chloroform, revealed that the chemical and photolytical transformations that ensued were not previously observed, and no adequate recognition in the literature has been conferred on work of this nature. Our investigation could contribute to synthetic aspects of specific in vitro clinical and biochemical research, and may play a role in characterization studies involving these compounds and their precursors [14–16]. The essence of our research, therefore, examined the products and magnitude of in vitro transformation associated with the chemical and photochemical reactions of the aforementioned compounds in the presence of CDCl 3 and D 2 O. The relevant absorption spectra revealed prominent peaks between 200 and 700 nm indicating that significant yields were produced. These yields were compared with those originating from the reaction and photolysis of the solutes in ordinary water and regular chloroform. Thus, the work was semi-quantitative in nature, and involved a differential study of the relative chemical and photochemical yields. The recorded results form an interesting and unique addendum to biological and clinical science.
2. Materials and methods
2.1. Sample preparation and irradiation The chemical and photochemical reactions were ex-
amined by preparing 5.30310 25 M bilirubin, 6.50310 24 M 7-DHC, 2.86310 25 M vitamin D 3 , 3.97310 24 M thymine and 2.85310 24 M uracil (Fluka, Buchs, Switzerland) in the relevant solvent and homogenizing. These concentrations are generally implemented to prevent any unwanted chemical effects such as precipitation [16]. For purposes of comparison, regular solvents (H 2 O and CHCl 3 ) and their deuterated counterparts (D 2 O and CDCl 3 ) were used. The use of different (deuterated) solvents depended on the solubility of the relevant compound in the selected medium. After the chemical reaction was monitored, the solutions were exposed to sunlight for about 15–30 min, which was adequate for completion of the relevant photochemical reactions [15–17]. The sunlight intensity (5.8310 2 W m 22 ) was monitored regularly using a Research Radiometer IL 1700 (International Light, Newburyport, MA, USA). Irradiating the sample at noon in a thin-walled quartz cuvette attained optimum intensity. The irradiated specimen was immediately subjected to spectrophotometric spectral analysis. The unirradiated sample represented the control.
2.2. Absorption spectra A UVPC Personal Spectroscopy spectrophotometer (Shimadzo, Tokyo, Japan) was employed for observing the absorption spectra. Blank measurements were made in the absence of the sample of interest. In the deuterated solvents only the products are displayed in absorption spectra. Spectra were recorded on disc for measurements off-line.
3. Results and discussion
3.1. Studies with CDCl3 3.1.1. Bilirubin Bilirubin is a reduced derivative of biliverdin. In blood circulation bilirubin is normally unconjugated, lipid-soluble compound. When its concentration in blood of neonates exceeds the capacity of the liver to convert it into conjugated, water-soluble bilirubin (i.e., to be excreted in the urine), the permeability of the blood–brain barrier will be increased and bilirubin will be deposited in the brain cells [4–6]. Such a deposition makes the infants with hyperbilirubinemia (neonatal jaundice) more susceptible to central nervous system damage [7]. Reducing blood bilirubin concentration is needed to avoid any possible complications. Light (450–500 nm) was successfully used in hyperbilirubinic infants to reduce the plasma concentration of bilirubin to safe levels by enhancing the photooxidation of bilirubin. A highly efficient process is required to reduce bilirubin concentration. Therefore, the effect of using deuterated solvents as light mimicking agents and sunlight on transformation of bilirubin was investigated. The chemical basis for this has been given elsewhere [14,15],
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but it is worthwhile mentioning some features of this phenomenon. It was found that there was a striking resemblance between the chemical reaction of bilirubin in CDCl 3 and the photochemical reaction associated with bilirubin in CHCl 3 [15]. An appropriate comparison of the rate constants of the reaction in the deuterated solvent and the photochemical reaction in CHCl 3 was investigated, and found to be 0.17 and 0.15 s 21 , respectively [15]. These data supported the proposal [14] that the chemical reaction could be used as a suitable substitute for the photochemical one to generate appreciable yields of biliverdin. In regular chloroform, bilirubin has a typical absorption maximum at 453 nm. The addition of the unconjugated specimen of bililrubin to CDCl 3 produced a greenish– brown solution. The reaction itself was instantaneous and a colour change was observed within minutes of adding the sample to the reagent [14]. The absorption spectrum produced two major peaks in the region of 660 nm and at 365 nm—characteristic of biliverdin (Fig. 1). The photochemical reaction of bilirubin was studied in regular chloroform and CDCl 3 (Fig. 1). In the case of regular chloroform, weak absorption bands were seen at 365 and 660 nm. These lines were much more intense in the presence of CDCl 3 , demonstrating that the deuterated solvent could be usefully applied to the generation of appreciable quantities of biliverdin. Alternatively, from the perspective of bilirubin degradation, the photochemical results for CHCl 3 were more promising. The recorded observations suggested that CDCl 3 reacts with bilirubin to produce biliverdin as a by-product. No
Fig. 1. Absorption spectra of bilirubin dissolved in (1) CHCl 3 , (2) CDCl 3 , (3) CHCl 3 plus solar irradiation, (4) CDCl 3 plus solar irradiation.
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equivalent reaction was perceived with CHCl 3 . The effect of the deuterated solvent at a molecular level has been discussed elsewhere [14]. A straightforward comparison of the biliverdin yields obtained with sunlight photolysis and the CDCl 3 reaction revealed that the yield from the reaction with CDCl 3 is an appreciable fraction of that of the photochemical reaction [14], and demonstrated that it is possible to use the chemical reaction for the simple preparation of micro-quantities of biliverdin, if a suitable separation process is implemented. The implication for chemistry is clear: both components at 365 and 660 nm could be appropriately separated for purposes of employment as precursors in other studies [16]. The clinical implications are more significant and indicate that we have developed a rapid test for in vitro identification of bilirubin in the presence of CDCl 3 , especially where bilirubin cannot be directly identified by spectrophotometry because of the presence of spectral interferences [14,15].
3.1.2. 7 -DHC The significance of vitamin D 3 for the body is no doubt very high due to its contribution in many vital activities. Vitamin D 3 is available in many natural sources in addition to the ability of our body to photosynthesize it. However, the readily available quantity varies and sometimes it does not meet the daily requirement. Additional sources are usually explored for possible enrichment of vitamin D 3 . However, there is still a need for a handy source to keep up the supply. Natural sources are under high demand but need to be supported by additional sources. In the present investigation deuterated solvent and sunlight were implemented for possible beneficial transformation of 7-DHC to vitamin D 3 . The chemical reactions of 7-DHC in the presence of CHCl 3 and CDCl 3 appear in Fig. 2. A peak of high intensity was observed at about 285 nm, which represents the absorption of the compound in regular chloroform. A considerable difference in spectral features was recorded when the solvent was changed to CDCl 3 . In the latter case a broad ‘‘hump’’ with a maximum at about 360 nm was observed, demonstrating that a novel complex is formed in the deuterated solvent. The absorption spectrum recorded after solar irradiation also appears in Fig. 2. Common absorption maxima occurred at the same wavelength for both solutions, about 311 nm. This was supported in an article by McLaughlin et al. [18], which describes the association of 7-DHC and previtamin D 3 (plus side products) whose spectral character reveals an absorption maximum at about 300 nm, subsequent to UV irradiation. From previous works [19,20] we confirmed that previtamin D 3 corresponds to the assignment around 285 nm, and the maxima around 310 nm refer to lumisterol [19,20]. The reactive wavelengths fall between 248 and 300 nm, and it is difficult to pinpoint the exact position of each compound because of the complex nature of the mixture. The production of lumisterol as the foremost component is interesting and the
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of possible hydrogen-bond like ‘‘conjugation’’ [20] of the complexes in equilibrium with each other, which leads to interaction of their p electron systems, producing hypochromicity characterized by the broad bands shown in the spectrum [20].
Fig. 2. Absorption spectra of 7-DHC dissolved in (1) CHCl 3 , (2) CDCl 3 , (3) CHCl 3 plus solar irradiation, (4) CDCl 3 plus solar irradiation.
3.1.3. Vitamin D3 Vitamin D 3 is a photosensitive compound. It reverts to its photoisomers when exposed to light and loses its biological activity [13]. A study of its photolysis in the presence of deuterated solvent may reveal a possible application that may contribute to minimizing this deteriorating photoeffect. The spectrum of vitamin D 3 is presented in Fig. 3. Two monumental peaks at about 307 and 325 nm were assigned to the dissolved solute in CHCl 3 and the corresponding study in sunlight, respectively. The assignment at 325 nm exhibits satellite peaks at 310 and 320 nm. The breadth of the 325 nm peak indicates that it covers a multiple of complexes which can be attributed to photoisomers of vitamin D 3 [18]. The implementation of the deuterated solvent, CDCl 3 , produced an assignment at about 285 nm, which is highly suggestive of previtamin D 3 . However, solar irradiation of the deuterated solvent generated a broad hump ranging from about 325–400 nm. This is perhaps the most striking feature of the spectrum because it represents the disappearance or ‘‘destruction’’ of the original vitamin D 3 , and this
photolysis encountered with the deuterated solvent generated a peak of about thrice the intensity as that observed with CHCl 3 . This demonstrates that the improved production of lumisterol, originating from solar irradiation of 7-DHC in CDCl 3 , is useful for preparing appreciable levels of the compound for use on a laboratory and commercial scale. The photolytic transformation of 7-DHC generally leads to vitamin D 3 via the following process: 7-HYDROCHOLESTEROL jhn LUMISTEROL ↔ PREVITAMIN D 3 ↔ TACHYSTEROL j VITAMIN D 3
The formation of vitamin D 3 involves three light dependent steps [19,20] of which previtamin D 3 occupies the central position. The final product is reached via a comparatively slow reaction which is strongly temperature dependent. Lumisterol and tachysterol are sideproducts that are in photochemical equilibrium with previtamin D 3 . There are few examples in the literature showing absorption spectra of all four compounds in equilibrium. Terenetskaya et al. [19] displayed the spectrum of previtamin D 3 only after separation, and their spectrum shows a minor resemblance (Fig. 2), although the structural features in Fig. 2 are much more pronounced. This is probably a result
Fig. 3. Absorption spectra of vitamin D 3 dissolved in (1) CHCl 3 , (2) CDCl 3 , (3) CHCl 3 plus solar irradiation, (4) CDCl 3 plus solar irradiation.
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particular phenomenon could have strong medical implications. As aforementioned, light exposure of vitamin D 3 could lead to side products, which are in equilibrium with each other. The conversion of vitamin D 3 is also temperaturedependent [20]. Here again there is the possibility of ‘‘conjugation’’ of these complexes by ‘‘mixing’’ of their p systems, which could lead to some form of hypochromicity thus producing the broad band visible in the spectrum [20]. It should be mentioned that our work focused on the equilibrium mixture, which could be used in characterization studies.
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3.2.1. Thymine Transformation of thymine alters its nature and it would no longer be functional. This creates a faulty base in the DNA that will perturb the DNA double helix and interfere with accurate DNA replication. In this case a wrong message will be coded and a different protein may be produced. Changes like this, if not repaired, will produce a mutant cell or the cell may not be able to survive. Moreover, intact DNA is needed to maintain the genetic characteristics of the living organism. The spectrum of thymine in the presence of H 2 O and D 2 O is presented in Fig. 4. In water, there are strong absorption bands at about 209 and 264 nm. The intense
line at 264 nm is a commonly observed absorption of thymine [20,21],and the band at 209 nm is characteristic of the hydrated form [22]. The spectrum recorded in the deuterated solvent was quite interesting, producing a ‘‘shift’’ of about 25 nm. This results in the appearance of the relevant absorption bands at 236 and 292 nm, which reflected the unusual nature of heavy water [14], and could possibly explain why the intensity of the former band is almost as intense as the one at 264 nm. The absorption band around 264 nm is due to the 5–6-double bond of thymine [20]. Similar features are seen in D 2 O, at 292 nm, which takes into account the wavelength shift [23]. The irradiated spectrum shows no sign of the dimer, but only produces a strong band at 299 nm. Nothing of consequence was observed with irradiation using normal water. Several ‘‘blips’’ above the noise level were recorded between 200 and 295 nm, but these were comparatively too low to be of any significance. The spectral features of thymine deserve further comment. According to Keifer [20], the band at 209 nm represents an overlap of thymine and its dimer, which creates difficulty in distinguishing the two in H 2 O. Due to difficulties created by this overlap we did not irradiate the specimen in water. We wish to emphasize that the dimer was not observed with solar irradiation of the sample in D 2 O, and only the band at 299 nm was recorded. We can use this information for purposes of characterizing thymine.
Fig. 4. Absorption spectra of thymine dissolved in (1) water, (2) D 2 O, (3) D 2 O plus solar irradiation.
3.2.2. Uracil Maintaining the integrity of nucleic acid components is important to assure healthy cells. Uracil is one of the four bases of RNA. Any damage or change in its nature may lead to faulty coding and wrong translation of genetic information for protein synthesis. This may produce unwanted protein that can lead to biochemical and physiological changes. Uracil in H 2 O produced strong absorption bands at about 202 and 258 nm (Fig. 5). The corresponding study in D 2 O showed that a novel complex was formed at about 300 nm. This represented a complete transformation of the uracil in the latter solvent. The metamorphosis of uracil in heavy water could be used to characterise the compound. Solar irradiation in H 2 O produced a minor peak at about 286 nm. This was attributed to the hydrate formation [20,21]. In D 2 O a similar weak line at 286 nm was seen, superimposed on the peak at about 300 nm. The exact identification of the 300 nm line is as yet not determined and is the subject of an extended investigation. The spectral features of uracil are noteworthy because, as mentioned above, a completely different assignment is observed around 300 nm under conditions of deuterated solvation. The compound is therefore, transformed with the use of this heavy solvent, and this transformation represents a procedure for degrading the compound. This
3.2. Studies with D2 O
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and the presence of D 2 O abolishes it (Fig. 4), this may earn a practical application such as getting rid of the thymine dimers in a cell either by reverting them to normal status (monomerization) or by removing them from the chain of DNA, which the excision repair mechanism does. Accordingly, D 2 O and sunlight could then be of possible biomedical application like preventing thymine dimer formation. If this is the case, then D 2 O may be useful for the treatment of individuals with Xeroderma pigmentosum.
3.3. Solvent isotope effects
Fig. 5. Absorption spectra of uracil dissolved in (1) water, (2) D 2 O, (3) water plus solar irradiation. When uracil in D 2 O was exposed to sunlight the spectrum exactly superimposes spectrum 2.
The recorded observations suggest that CDCl 3 and D 2 O generate products that are more thermodynamically stable [24,25]. The significantly different features in the spectra of the deuterated solvents compared with their counterparts demonstrate conclusively that the behaviour of the deuterated compounds produce transformations that are unique and unprecedented, particularly the use of D 2 O in biomedical studies. Other authors have suggested that the isotope-effect, especially with light elements, tends to completely transform the chemistry of the isotopically heavier compounds [26,27]. Itzhaki and Evans [26] suggested that there was a dependence of hydrophobic interaction on solvent isotopic composition. This phenomenon is validated by several other studies in the literature [28,29].
particular technique can be usefully employed in certain synthetic studies for purposes of removing uracil. 4. Conclusion
3.2.3. Biomedical significance of thymine and uracil Generally, if we consider that the chemical transformation of thymine and uracil produced in vitro is expected to be produced in living cells, then possible DNA and RNA damage may result. Accordingly, surviving cells may be genetically altered (mutated) [2]. When multiple mutations (genetic events) accumulate within the DNA of a single somatic cell, the cell loses growth control [2]. Therefore, DNA base damage by deuterated solvents has biological and medical consequences which are needed to be highly appreciated, taking into account the present findings that D 2 O and CDCl 3 tremendously changed the nature of the treated compound particularly thymine and uracil. It should always be remembered that when mutations are induced in germ cells, changes in the off-springs’ characteristics are very much expected. Thymine dimers are produced when the cell is exposed to UV light. Normally, the cell gets rid of this dimer by the excision repair mechanism [1,2]. If a cell is deficient in the repair of this UV-induced DNA damage a possible defect may result. It seems very likely, therefore, that the high incidence of individuals with Xeroderma pigmentosum (extensive skin tumours that develop after exposure to sunlight) is due to a deficiency in DNA damage repair. Since sunlight produces thymine dimers in H 2 O [20,21],
It has been shown that the use of deuterated solvents produce unique chemical and photolytical conversions of bilirubin, vitamin D 3 , 7-DHC, thymine and uracil which can have far-reaching medical applications. This was attributed to the fact that deuterated compounds display somewhat of a different chemistry to their ordinary counterparts and that possibly thermodynamic considerations could be responsible for the novel transformations.
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[18] J.A. MacLaughlin, R.R. Anderson, M.F. Holick, Spectral character of sunlight modulates photosynthesis of previtamin D 3 and its photoisomers in human skin, Science 216 (1982) 1001–1003. [19] I.P. Terenetskaya, O.G. Dmitrenko, A.M. Eremenko, Conformational control in previtamin D photochemistry by heterogeneous reaction media, Res. Chem. Intermed. 21 (1995) 653–664. [20] J. Kiefer, Biological Radiation Effects, Springer-Verlag, Berlin, 1990. [21] L.I. Grossweiner, Photochemistry, in: K.C. Smith (Ed.), The Science of Photobiology, 2nd Edition, Plenum Press, New York, 1989, pp. 47–76. [22] B. Schneider, D.M. Cohen, L. Schleifer, A.R. Srinivasan, W.K. Olson, H.M. Berman, A systematic method for studying the spatial distribution of water molecules around nucleic acid bases, Biophys. J. 65 (1993) 2291–2303. [23] D.A. Skoog, F.J. Holler, T.A. Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, PA, 1998. [24] G.A. Ropp, Effect of isotope substitution on organic reaction rates, Nucleonics 10 (1952) 22–27. [25] G.D. Chase, J.L. Rabinowitz, Principles of Radioisotope Methodology, Burgess Publishers, Philadelphia, PA, 1962. [26] L.S. Itzhaki, P.A. Evans, Solvent isotope effects on the refolding kinetics of hen egg-white lysozyme, Protein Sci. 5 (1996) 140–146. [27] M. Some, S. Svensson, J.O. Hoog, A. Helander, Studies on the interaction between ethanol and serotonin metabolism in rat, using deuterated ethanol and 4-methylpyrazole, Biochem. Pharmacol. 15 (2000) 385–391. [28] V.L. Hsu, I.M. Armitage, Solution structure of cyclosporin A and a nonimmunosupressive analogue bound to fully deuterated cyclophilin, Biochem. J. 31 (1992) 12778–12784. [29] L.D. Williams, B.R. Shaw, Protonated base pairs explain the ambiguous pairing properties of O6-methylguanine, Proc. Natl. Acad. Sci. USA 84 (1987) 1779–1783.
Chemical and photolytical transformation of biomedically significant compounds in the presence of deuterated solvents a, b b F.M. Salih *, A.E. Pillay , A. Al-Hamdi a
Department of Clinical and Biomedical Physics, College of Medicine, Sultan Qaboos University, P.O. Box 35, Al Khoud 123, Oman b Department of Chemistry, College of Science, Sultan Qaboos University, P.O. Box 36, Al Khoud 123, Oman Received 5 March 2002; received in revised form 3 December 2002; accepted 31 March 2003
Abstract The effect of the nature of solvent on the properties of biomedically important compounds is of particular importance. The conversion of certain biomedical compounds with deuterated solvents is an area of research that has not been accorded adequate recognition in the literature. We explored this area in the interest of shedding some light on the possible effects of solvent on the nature of the solute. The transformation of specific medically important compounds such as bilirubin, thymine, uracil, dehydrocholesterol (7-DHC) and vitamin D 3 was observed in the presence of deuterated solvents such as heavy water and deuterated chloroform. The products of the relevant reactions were confirmed spectrophotometrically. An additional feature to our investigation involved the photolysis of the aforementioned compounds by solar irradiation. The pure samples were dissolved in solutions of the deuterated solvents, corresponding to concentrations of typically 10 22 mM, and exposed to sunlight for about 15–30 min. The deuterated solvents caused chemical transformation in all chemical compounds tested, and produced intense characteristic absorbance maxima between 200 and 700 nm. Sunlight exposure was also effective in either augmenting the effects of deuterated solvent as in bilirubin and 7-DHC or reducing it as with thymine or having no effect as with uracil or completely changing it as in vitamin D 3 . It has been shown that the use of deuterated solvents produces unique chemical and photochemical conversions of bilirubin, 7-DHC, thymine, uracil and vitamin D 3 . This was attributed to the fact that deuterated compounds display a somewhat different chemistry to their ordinary counterparts and that possibly thermodynamic considerations could be responsible for the novel transformations. 2003 Elsevier B.V. All rights reserved. Keywords: Spectrophotometry; Deuterated solvents; Biomedical compounds; Sunlight; Transformation
1. Introduction Our investigation essentially focused on the influence of the nature of the solvent on the properties of certain biomedically important compounds. The conversion of these compounds with deuterated solvents is a relatively unexplored area of research. Our work with deuterated solvents started by initially observing marked transformations of these compounds when they were prepared in such media for specific instrumental studies. Such observations subsequently led to a wider exploration where different solvents were applied to a range of biomedical compounds. Certain compounds, such as thymine and uracil, are of immense medical significance because they play vital roles in research associated with DNA, RNA [1,2]. Other compounds, such as bilirubin, stimulate considerable clini*Corresponding author. Tel.: 1968-515-110. E-mail address: [email protected] (F.M. Salih). 1011-1344 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S1011-1344(03)00059-9
cal interest because of the association with neonatal jaundice in newborn babies [3–7], while dehydrocholesterol (7-DHC) and vitamin D 3 are essential in the metabolism of minerals, particularly calcium [7–9]. The chemical and photolytical conversion of these compounds continues to attract attention because such studies could lead to links associated with the aetiology of disease and disorders. The biological and clinical significance of tested chemical compounds lies in their vital role in maintaining growth and metabolism and ultimately in supporting a healthy status of the body. Thymine and uracil are the building blocks of DNA and RNA, respectively. They are vital for their replication [2]. They also play a major role in the process of protein synthesis. Any damage or change to their integrity will impair the process of DNA and RNA replication and hence this would affect the formation of particular proteins that are coded by these compounds. Many factors can create different types of damage to these chemicals, namely, radiation, UV and chemicals which
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would alter the genetic make up and lead to genetic mutations [10–12]. Vitamin D 3 is an indispensable compound. It is required to assist in calcium absorption from the intestine. Calcium is deposited in bones with the aid of vitamin D 3 . Such deposition is important to maintain the rigidity and strength of the bone. Vitamin D 3 is usually available in many nutritional components and our body can also photosynthesize it by solar conversion of 7-DHC. Deficiency of this vitamin originates mainly from either lower uptake or less exposure to sunlight [8,9]. However, it is documented that excessive exposure to sunlight may damage vitamin D 3 through reversible transformation to its photoisomers [13]. Bilirubin, on the other hand, is a by-product produced during the process of red blood cell degradation. It is normally converted by the liver to a water-soluble product and excreted by the kidney. When the rate of bilirubin formation increases either by excessive haemolysis or due to liver malfunctioning, as in many hepatic diseases or where the liver is not yet mature enough to carry out this process efficiently, as in neonates, bilirubin concentration in the blood rises. A clinical condition called hyperbilirubinemia results. This leads to the deposition of bilirubin in brain cells. Such a deposition makes a hyperbilirubinic person more susceptible to central nervous system damage [4–7]. Hence, bilirubin must be kept down to levels below 10–15% [7]. A preliminary study involving the conversions of these compounds in the presence of deuterated solvents produced some interesting observations. Further studies, mainly with heavy water and deuterated chloroform, revealed that the chemical and photolytical transformations that ensued were not previously observed, and no adequate recognition in the literature has been conferred on work of this nature. Our investigation could contribute to synthetic aspects of specific in vitro clinical and biochemical research, and may play a role in characterization studies involving these compounds and their precursors [14–16]. The essence of our research, therefore, examined the products and magnitude of in vitro transformation associated with the chemical and photochemical reactions of the aforementioned compounds in the presence of CDCl 3 and D 2 O. The relevant absorption spectra revealed prominent peaks between 200 and 700 nm indicating that significant yields were produced. These yields were compared with those originating from the reaction and photolysis of the solutes in ordinary water and regular chloroform. Thus, the work was semi-quantitative in nature, and involved a differential study of the relative chemical and photochemical yields. The recorded results form an interesting and unique addendum to biological and clinical science.
2. Materials and methods
2.1. Sample preparation and irradiation The chemical and photochemical reactions were ex-
amined by preparing 5.30310 25 M bilirubin, 6.50310 24 M 7-DHC, 2.86310 25 M vitamin D 3 , 3.97310 24 M thymine and 2.85310 24 M uracil (Fluka, Buchs, Switzerland) in the relevant solvent and homogenizing. These concentrations are generally implemented to prevent any unwanted chemical effects such as precipitation [16]. For purposes of comparison, regular solvents (H 2 O and CHCl 3 ) and their deuterated counterparts (D 2 O and CDCl 3 ) were used. The use of different (deuterated) solvents depended on the solubility of the relevant compound in the selected medium. After the chemical reaction was monitored, the solutions were exposed to sunlight for about 15–30 min, which was adequate for completion of the relevant photochemical reactions [15–17]. The sunlight intensity (5.8310 2 W m 22 ) was monitored regularly using a Research Radiometer IL 1700 (International Light, Newburyport, MA, USA). Irradiating the sample at noon in a thin-walled quartz cuvette attained optimum intensity. The irradiated specimen was immediately subjected to spectrophotometric spectral analysis. The unirradiated sample represented the control.
2.2. Absorption spectra A UVPC Personal Spectroscopy spectrophotometer (Shimadzo, Tokyo, Japan) was employed for observing the absorption spectra. Blank measurements were made in the absence of the sample of interest. In the deuterated solvents only the products are displayed in absorption spectra. Spectra were recorded on disc for measurements off-line.
3. Results and discussion
3.1. Studies with CDCl3 3.1.1. Bilirubin Bilirubin is a reduced derivative of biliverdin. In blood circulation bilirubin is normally unconjugated, lipid-soluble compound. When its concentration in blood of neonates exceeds the capacity of the liver to convert it into conjugated, water-soluble bilirubin (i.e., to be excreted in the urine), the permeability of the blood–brain barrier will be increased and bilirubin will be deposited in the brain cells [4–6]. Such a deposition makes the infants with hyperbilirubinemia (neonatal jaundice) more susceptible to central nervous system damage [7]. Reducing blood bilirubin concentration is needed to avoid any possible complications. Light (450–500 nm) was successfully used in hyperbilirubinic infants to reduce the plasma concentration of bilirubin to safe levels by enhancing the photooxidation of bilirubin. A highly efficient process is required to reduce bilirubin concentration. Therefore, the effect of using deuterated solvents as light mimicking agents and sunlight on transformation of bilirubin was investigated. The chemical basis for this has been given elsewhere [14,15],
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but it is worthwhile mentioning some features of this phenomenon. It was found that there was a striking resemblance between the chemical reaction of bilirubin in CDCl 3 and the photochemical reaction associated with bilirubin in CHCl 3 [15]. An appropriate comparison of the rate constants of the reaction in the deuterated solvent and the photochemical reaction in CHCl 3 was investigated, and found to be 0.17 and 0.15 s 21 , respectively [15]. These data supported the proposal [14] that the chemical reaction could be used as a suitable substitute for the photochemical one to generate appreciable yields of biliverdin. In regular chloroform, bilirubin has a typical absorption maximum at 453 nm. The addition of the unconjugated specimen of bililrubin to CDCl 3 produced a greenish– brown solution. The reaction itself was instantaneous and a colour change was observed within minutes of adding the sample to the reagent [14]. The absorption spectrum produced two major peaks in the region of 660 nm and at 365 nm—characteristic of biliverdin (Fig. 1). The photochemical reaction of bilirubin was studied in regular chloroform and CDCl 3 (Fig. 1). In the case of regular chloroform, weak absorption bands were seen at 365 and 660 nm. These lines were much more intense in the presence of CDCl 3 , demonstrating that the deuterated solvent could be usefully applied to the generation of appreciable quantities of biliverdin. Alternatively, from the perspective of bilirubin degradation, the photochemical results for CHCl 3 were more promising. The recorded observations suggested that CDCl 3 reacts with bilirubin to produce biliverdin as a by-product. No
Fig. 1. Absorption spectra of bilirubin dissolved in (1) CHCl 3 , (2) CDCl 3 , (3) CHCl 3 plus solar irradiation, (4) CDCl 3 plus solar irradiation.
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equivalent reaction was perceived with CHCl 3 . The effect of the deuterated solvent at a molecular level has been discussed elsewhere [14]. A straightforward comparison of the biliverdin yields obtained with sunlight photolysis and the CDCl 3 reaction revealed that the yield from the reaction with CDCl 3 is an appreciable fraction of that of the photochemical reaction [14], and demonstrated that it is possible to use the chemical reaction for the simple preparation of micro-quantities of biliverdin, if a suitable separation process is implemented. The implication for chemistry is clear: both components at 365 and 660 nm could be appropriately separated for purposes of employment as precursors in other studies [16]. The clinical implications are more significant and indicate that we have developed a rapid test for in vitro identification of bilirubin in the presence of CDCl 3 , especially where bilirubin cannot be directly identified by spectrophotometry because of the presence of spectral interferences [14,15].
3.1.2. 7 -DHC The significance of vitamin D 3 for the body is no doubt very high due to its contribution in many vital activities. Vitamin D 3 is available in many natural sources in addition to the ability of our body to photosynthesize it. However, the readily available quantity varies and sometimes it does not meet the daily requirement. Additional sources are usually explored for possible enrichment of vitamin D 3 . However, there is still a need for a handy source to keep up the supply. Natural sources are under high demand but need to be supported by additional sources. In the present investigation deuterated solvent and sunlight were implemented for possible beneficial transformation of 7-DHC to vitamin D 3 . The chemical reactions of 7-DHC in the presence of CHCl 3 and CDCl 3 appear in Fig. 2. A peak of high intensity was observed at about 285 nm, which represents the absorption of the compound in regular chloroform. A considerable difference in spectral features was recorded when the solvent was changed to CDCl 3 . In the latter case a broad ‘‘hump’’ with a maximum at about 360 nm was observed, demonstrating that a novel complex is formed in the deuterated solvent. The absorption spectrum recorded after solar irradiation also appears in Fig. 2. Common absorption maxima occurred at the same wavelength for both solutions, about 311 nm. This was supported in an article by McLaughlin et al. [18], which describes the association of 7-DHC and previtamin D 3 (plus side products) whose spectral character reveals an absorption maximum at about 300 nm, subsequent to UV irradiation. From previous works [19,20] we confirmed that previtamin D 3 corresponds to the assignment around 285 nm, and the maxima around 310 nm refer to lumisterol [19,20]. The reactive wavelengths fall between 248 and 300 nm, and it is difficult to pinpoint the exact position of each compound because of the complex nature of the mixture. The production of lumisterol as the foremost component is interesting and the
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of possible hydrogen-bond like ‘‘conjugation’’ [20] of the complexes in equilibrium with each other, which leads to interaction of their p electron systems, producing hypochromicity characterized by the broad bands shown in the spectrum [20].
Fig. 2. Absorption spectra of 7-DHC dissolved in (1) CHCl 3 , (2) CDCl 3 , (3) CHCl 3 plus solar irradiation, (4) CDCl 3 plus solar irradiation.
3.1.3. Vitamin D3 Vitamin D 3 is a photosensitive compound. It reverts to its photoisomers when exposed to light and loses its biological activity [13]. A study of its photolysis in the presence of deuterated solvent may reveal a possible application that may contribute to minimizing this deteriorating photoeffect. The spectrum of vitamin D 3 is presented in Fig. 3. Two monumental peaks at about 307 and 325 nm were assigned to the dissolved solute in CHCl 3 and the corresponding study in sunlight, respectively. The assignment at 325 nm exhibits satellite peaks at 310 and 320 nm. The breadth of the 325 nm peak indicates that it covers a multiple of complexes which can be attributed to photoisomers of vitamin D 3 [18]. The implementation of the deuterated solvent, CDCl 3 , produced an assignment at about 285 nm, which is highly suggestive of previtamin D 3 . However, solar irradiation of the deuterated solvent generated a broad hump ranging from about 325–400 nm. This is perhaps the most striking feature of the spectrum because it represents the disappearance or ‘‘destruction’’ of the original vitamin D 3 , and this
photolysis encountered with the deuterated solvent generated a peak of about thrice the intensity as that observed with CHCl 3 . This demonstrates that the improved production of lumisterol, originating from solar irradiation of 7-DHC in CDCl 3 , is useful for preparing appreciable levels of the compound for use on a laboratory and commercial scale. The photolytic transformation of 7-DHC generally leads to vitamin D 3 via the following process: 7-HYDROCHOLESTEROL jhn LUMISTEROL ↔ PREVITAMIN D 3 ↔ TACHYSTEROL j VITAMIN D 3
The formation of vitamin D 3 involves three light dependent steps [19,20] of which previtamin D 3 occupies the central position. The final product is reached via a comparatively slow reaction which is strongly temperature dependent. Lumisterol and tachysterol are sideproducts that are in photochemical equilibrium with previtamin D 3 . There are few examples in the literature showing absorption spectra of all four compounds in equilibrium. Terenetskaya et al. [19] displayed the spectrum of previtamin D 3 only after separation, and their spectrum shows a minor resemblance (Fig. 2), although the structural features in Fig. 2 are much more pronounced. This is probably a result
Fig. 3. Absorption spectra of vitamin D 3 dissolved in (1) CHCl 3 , (2) CDCl 3 , (3) CHCl 3 plus solar irradiation, (4) CDCl 3 plus solar irradiation.
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particular phenomenon could have strong medical implications. As aforementioned, light exposure of vitamin D 3 could lead to side products, which are in equilibrium with each other. The conversion of vitamin D 3 is also temperaturedependent [20]. Here again there is the possibility of ‘‘conjugation’’ of these complexes by ‘‘mixing’’ of their p systems, which could lead to some form of hypochromicity thus producing the broad band visible in the spectrum [20]. It should be mentioned that our work focused on the equilibrium mixture, which could be used in characterization studies.
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3.2.1. Thymine Transformation of thymine alters its nature and it would no longer be functional. This creates a faulty base in the DNA that will perturb the DNA double helix and interfere with accurate DNA replication. In this case a wrong message will be coded and a different protein may be produced. Changes like this, if not repaired, will produce a mutant cell or the cell may not be able to survive. Moreover, intact DNA is needed to maintain the genetic characteristics of the living organism. The spectrum of thymine in the presence of H 2 O and D 2 O is presented in Fig. 4. In water, there are strong absorption bands at about 209 and 264 nm. The intense
line at 264 nm is a commonly observed absorption of thymine [20,21],and the band at 209 nm is characteristic of the hydrated form [22]. The spectrum recorded in the deuterated solvent was quite interesting, producing a ‘‘shift’’ of about 25 nm. This results in the appearance of the relevant absorption bands at 236 and 292 nm, which reflected the unusual nature of heavy water [14], and could possibly explain why the intensity of the former band is almost as intense as the one at 264 nm. The absorption band around 264 nm is due to the 5–6-double bond of thymine [20]. Similar features are seen in D 2 O, at 292 nm, which takes into account the wavelength shift [23]. The irradiated spectrum shows no sign of the dimer, but only produces a strong band at 299 nm. Nothing of consequence was observed with irradiation using normal water. Several ‘‘blips’’ above the noise level were recorded between 200 and 295 nm, but these were comparatively too low to be of any significance. The spectral features of thymine deserve further comment. According to Keifer [20], the band at 209 nm represents an overlap of thymine and its dimer, which creates difficulty in distinguishing the two in H 2 O. Due to difficulties created by this overlap we did not irradiate the specimen in water. We wish to emphasize that the dimer was not observed with solar irradiation of the sample in D 2 O, and only the band at 299 nm was recorded. We can use this information for purposes of characterizing thymine.
Fig. 4. Absorption spectra of thymine dissolved in (1) water, (2) D 2 O, (3) D 2 O plus solar irradiation.
3.2.2. Uracil Maintaining the integrity of nucleic acid components is important to assure healthy cells. Uracil is one of the four bases of RNA. Any damage or change in its nature may lead to faulty coding and wrong translation of genetic information for protein synthesis. This may produce unwanted protein that can lead to biochemical and physiological changes. Uracil in H 2 O produced strong absorption bands at about 202 and 258 nm (Fig. 5). The corresponding study in D 2 O showed that a novel complex was formed at about 300 nm. This represented a complete transformation of the uracil in the latter solvent. The metamorphosis of uracil in heavy water could be used to characterise the compound. Solar irradiation in H 2 O produced a minor peak at about 286 nm. This was attributed to the hydrate formation [20,21]. In D 2 O a similar weak line at 286 nm was seen, superimposed on the peak at about 300 nm. The exact identification of the 300 nm line is as yet not determined and is the subject of an extended investigation. The spectral features of uracil are noteworthy because, as mentioned above, a completely different assignment is observed around 300 nm under conditions of deuterated solvation. The compound is therefore, transformed with the use of this heavy solvent, and this transformation represents a procedure for degrading the compound. This
3.2. Studies with D2 O
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and the presence of D 2 O abolishes it (Fig. 4), this may earn a practical application such as getting rid of the thymine dimers in a cell either by reverting them to normal status (monomerization) or by removing them from the chain of DNA, which the excision repair mechanism does. Accordingly, D 2 O and sunlight could then be of possible biomedical application like preventing thymine dimer formation. If this is the case, then D 2 O may be useful for the treatment of individuals with Xeroderma pigmentosum.
3.3. Solvent isotope effects
Fig. 5. Absorption spectra of uracil dissolved in (1) water, (2) D 2 O, (3) water plus solar irradiation. When uracil in D 2 O was exposed to sunlight the spectrum exactly superimposes spectrum 2.
The recorded observations suggest that CDCl 3 and D 2 O generate products that are more thermodynamically stable [24,25]. The significantly different features in the spectra of the deuterated solvents compared with their counterparts demonstrate conclusively that the behaviour of the deuterated compounds produce transformations that are unique and unprecedented, particularly the use of D 2 O in biomedical studies. Other authors have suggested that the isotope-effect, especially with light elements, tends to completely transform the chemistry of the isotopically heavier compounds [26,27]. Itzhaki and Evans [26] suggested that there was a dependence of hydrophobic interaction on solvent isotopic composition. This phenomenon is validated by several other studies in the literature [28,29].
particular technique can be usefully employed in certain synthetic studies for purposes of removing uracil. 4. Conclusion
3.2.3. Biomedical significance of thymine and uracil Generally, if we consider that the chemical transformation of thymine and uracil produced in vitro is expected to be produced in living cells, then possible DNA and RNA damage may result. Accordingly, surviving cells may be genetically altered (mutated) [2]. When multiple mutations (genetic events) accumulate within the DNA of a single somatic cell, the cell loses growth control [2]. Therefore, DNA base damage by deuterated solvents has biological and medical consequences which are needed to be highly appreciated, taking into account the present findings that D 2 O and CDCl 3 tremendously changed the nature of the treated compound particularly thymine and uracil. It should always be remembered that when mutations are induced in germ cells, changes in the off-springs’ characteristics are very much expected. Thymine dimers are produced when the cell is exposed to UV light. Normally, the cell gets rid of this dimer by the excision repair mechanism [1,2]. If a cell is deficient in the repair of this UV-induced DNA damage a possible defect may result. It seems very likely, therefore, that the high incidence of individuals with Xeroderma pigmentosum (extensive skin tumours that develop after exposure to sunlight) is due to a deficiency in DNA damage repair. Since sunlight produces thymine dimers in H 2 O [20,21],
It has been shown that the use of deuterated solvents produce unique chemical and photolytical conversions of bilirubin, vitamin D 3 , 7-DHC, thymine and uracil which can have far-reaching medical applications. This was attributed to the fact that deuterated compounds display somewhat of a different chemistry to their ordinary counterparts and that possibly thermodynamic considerations could be responsible for the novel transformations.
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