J. Phys. Chem. 1996, 100, 6845-6848
6845
Carbon Dioxide in Tissues, Cells, and Biological Fluids Detected by FTIR Spectroscopy† Christian P. Schultz, Hans H. Eysel, Henry H. Mantsch, and Michael Jackson* Institute for Biodiagnostics, National Research Council Canada, 435 Ellice AVenue, Winnipeg, Manitoba, Canada R3B 1Y6 ReceiVed: NoVember 3, 1995X
Previous studies from this lab have demonstrated the presence of a novel absorption in infrared spectra of synovial fluid films (2337-2343 cm-1) which was suggested to arise from CO2 trapped within the organic matrix left after drying. In the present paper, we establish the presence of this absorption in a wide range of biological fluids, tissues, and cell suspensions. Results of studies with a range of common biological materials suggest that the CO2 interacts nonspecifically with the protein components of cells and fluids. Experiments with 13C-labeled glucose clearly demonstrate that this ubiquitous absorption is attributable to CO2 produced by glucose breakdown within cells, presenting the interesting possibility of monitoring cellular metabolism by infrared spectroscopy.
Introduction Fourier transform infrared spectroscopy is an established tool for the structural characterization of biological molecules, particularly lipids,1 proteins,2 and nucleic acids.3 Using this powerful technique, it is possible to obtain information relating to membrane architecture and packing, protein structure, lipidprotein interactions, and nucleic acid conformation. We have recently applied the knowledge gained from such studies to the IR spectroscopic investigation of human tissue and fluids4 and the abnormal biochemistry of tissue and fluids in disease states. To date, we have identified spectroscopic features which may be used, together with pattern recognition techniques, to characterize neurological disorders such as Alzheimer’s disease5 and multiple sclerosis6 and arthritic disorders, including osteoarthritis and rheumatoid arthritis.7,8 During the course of our investigations, it has become apparent that the spectral region between 2100 and 2600 cm-1, generally believed to be devoid of detectable absorptions in most biological systems, does in fact contain a number of interesting, if weak, spectroscopic signatures arising from SH and CO2 vibrations.9,10 Of particular interest are absorptions at around 2340 cm-1, which we have observed in spectra of tissues, fluids, and dry films formed from fluids. Preliminary studies on synovial fluid films10 led us to assign absorptions in this region to CO2 enclathrated in the organic matrix left upon drying of the film, based upon comparisons with CO2 clathrates formed within ice. In this paper, we will demonstrate the existence of such CO2 clathrates in films of a number of biological fluids and the presence of spectroscopically detectable solution-phase CO2 in a variety of human tissues and cells. In addition, we will provide evidence which suggests that proteins are responsible for enclathration of CO2 in films of biological fluids. Finally we will speculate on the source of the CO2 present within human tissues and fluids and comment on possible applications of these findings. Experimental Section All human fluids and tissues were obtained from local hospitals. No fluids or tissues were obtained specifically for this study. Broncho-alveolar lavage and synovial fluid samples † X
Issued as NRCC No. 34763. Abstract published in AdVance ACS Abstracts, April 1, 1996.
0022-3654/96/20100-6845$12.00/0
were used as soon as possible after they were collected. All other fluids were stored at 4 °C prior to use. The viscous broncho-alveolar lavage samples were homogenized prior to use to ensure representative sampling. Dry films were prepared by placing 20 µL of the relevant fluid on a CaF2 window and drying under moderate vacuum (25 Torr) for 5 min. For studies on yeast, dried yeast cells were suspended in noncarbonated mineral water (2.4 mg/mL) and incubated for 10 min at room temperature before adding glucose (1.7 mg of glucose/mg of yeast). The yeast suspension was then injected into a flow-through cell comprised of a pair of CaF2 windows separated by a path length of 50 µm. Infrared spectra were measured on a range of Biorad Fourier transform infrared spectrometers (FTS-7, FTS-40, FTS-40A, and FTS-60) continuously purged with dry air and equipped with DTGS or MCT detectors. For each spectrum, 256 interferograms were collected and Fourier-transformed to generate spectra with 2-cm-1 nominal resolution. Results and Discussion The infrared spectra of a sample of native human synovial fluid and a film formed by drying of the synovial fluid onto a CaF2 window are shown in Figure 1A and 1B, respectively. As expected, the major absorptions in the fluid spectrum arise from water and are assigned to the O-H stretching (broad absorption at 2800-3700 cm-1) and O-H bending (1644 cm-1) vibrations of water. A series of weak absorptions between 1000 and 1700 cm-1 arise from the complex biological materials present in synovial fluid (proteins, proteoglycans, polysaccharides, etc.). In biological systems, the region between 1800 and 2800 cm-1 is generally devoid of absorptions, with the exception of a broad feature at around 2100 cm-1, generally referred to as the water combination band (attributed to the combination of the hindered rotational absorption and the O-H bending vibration of water) and a broad absorption arising from poorly compensated atmospheric CO2 at 2350 cm-1. Spectra of films formed by drying synovial fluid are dominated by absorptions which arise from proteins (32003600, 1655, and 1550 cm-1) but also contain significant absorptions from the CH stretching and bending vibrations of lipids, proteins, and carbohydrates (absorptions in the ranges 2800-3000 and 1450-1500 cm-1, respectively) and C-C as well as C-O stretching vibrations of carbohydrates (1000© 1996 American Chemical Society
6846 J. Phys. Chem., Vol. 100, No. 16, 1996
Figure 1. Infrared spectra of native human synovial fluid (A) and films formed by drying human synovial fluid (B), human plasma (C), and human cerebrospinal fluid (D). Insets show the region 2300-2400 cm-1 expanded by the factor noted.
1200 cm-1), particularly hyaluronic acid. Spectra of films formed by drying of plasma (Figure 1C) share many of the features of spectra of synovial fluid films, the major difference being a reduction in the intensity of the C-O stretching absorptions due to the absence of hyaluronic acid in plasma. Spectra of cerebrospinal fluid, on the other hand, are markedly different from spectra of plasma and synovial fluid films, reflecting the unusual composition of this fluid (Figure 1D). The major absorptions in the spectra of cerebrospinal fluids arise from proteins, urea, glucose, lactate, and bicarbonate ions. Despite the varied composition of the synovial fluid, plasma, and cerebrospinal fluid, films of each fluid exhibit an unusual absorption in a region of the spectrum which is not normally populated in biological systems, around 2337 cm-1. We have also observed such an absorption in films of whole blood, amniotic fluid, and broncho-alveolar lavage fluid (not shown). Interestingly, upon reexamination of the spectra of the fluids from which the films discussed above were prepared, it is apparent that a weak absorption is present in the original fluids, although at the higher frequency of 2343 cm-1. In addition, an absorption at 2343 cm-1 is also observed in the IR spectra of almost all tissues (tissue must be sampled as soon as possible after it is obtained to detect this absorption). Spectra of tissue obtained from the left ventricle of a rat heart illustrating this absorption are presented in Figure 2. An additional weaker absorption is present at 2550-2560 cm-1 in heart tissue, which is assigned, by comparison with the spectrum of haemoglobin, to the S-H stretching vibration of cysteine residues of haemoglobin.9 The assignment of the absorptions at 2337 and 2343 cm-1 is less intuitive. The possibility that they arise from inadequate compensation of atmospheric CO2 may be ruled out based upon the shape of the absorption and the fact that the intensity of the absorption remains stable upon prolonged purging of the spectrometer with CO2-free air. A number of observations strongly suggest that these absorptions do, however, arise from condensed phase CO2: (i) Comparison of synovial fluid spectra with the spectrum of solutions of CO2 in water11 suggests that the absorption at 2343 cm-1 arises from CO2 in aqueous solution (Figure 3). (ii) In addition to the major absorption at 2343 cm-1, a second, much weaker absorption is seen 64 cm-1 lower in
Schultz et al.
Figure 2. Infrared spectra of rat ventricular tissue (A) and haemoglobin (B). Insets show second derivative spectra between 2200 and 2700 cm-1.
Figure 3. Infrared spectra of carbonated water (top) and a film formed by drying human synovial fluid (bottom).
frequency in the spectra of soda water. The ratio of the integrated intensities of these two bands is in accordance with the relative abundance of 13CO2 and 12CO2, (i.e., 1% and 99% of naturally occurring carbon, respectively), suggesting that this second absorption arises from 13CO2. In tissues, biological fluids and films of biological fluids, giving rise to a prominent absorption at 2337-2343 cm-1, a second weaker absorption is also seen at a frequency 64 cm-1 lower than the main band. Again the ratio of the integrated intensities of these two bands is in accordance with the relative abundance of 13CO2 and 12CO2. The similarities between the absorptions arising from the two isotopic forms of CO2 in soda water and the tissues, fluids, and films discussed here strongly support our assignment of these absorptions in tissues and biological fluids to CO2. (iii) Absorptions at frequencies similar to those reported here for biological fluid films have been reported in the spectroscopic studies of CO2 enclathrated (trapped) within ice.12 (iV) Of more physiological relevance, similar absorptions attributed to CO2 within germinating seeds have been reported.13 Thus, we confidently assign the absorptions seen at 2343 cm-1 in the IR spectra of biological fluids (including synovial fluid, broncho-alveolar lavage, cerebrospinal fluid, amniotic fluid, whole blood, and plasma) and tissues to dissolved CO2,
CO2 in Tissues, Cells, and Biological Fluids
J. Phys. Chem., Vol. 100, No. 16, 1996 6847
Figure 4. Infrared spectra of films formed by drying carbonated solutions of albumin (A), haemoglobin (B), γ-globulin (C), and concanavalin A (D).
Figure 5. Time dependence of the infrared spectra of yeast incubated with glucose and of the integrated intensity of the absorption band at 2343 cm-1 (inset).
while the absorption seen at 2337 cm-1 in dry films prepared from fluids is attributed to CO2 trapped within the matrix formed upon drying. The fact that the CO2 remains trapped within dry films is somewhat surprising. Particularly surprising is the observation that CO2 is retained in films formed under reduced pressure (25 Torr). It may be expected that CO2 would be lost under such conditions. This is evidently not the case. How, then, does the CO2 remain within the film? Obviously, as the film dries, some type of interaction must take place between CO2 and one or more of the nonaqueous molecular species present in the fluid. During the course of our initial studies on dry films of synovial fluid, we suggested that the unusual composition of synovial fluid was responsible for the presence of CO2 in films and speculated that the enclathration site may be the specialized proteoglycan and hyaluronic acid components of synovial fluid.10 However, the presence of CO2 in dry films of fluids as diverse as cerebrospinal fluid, whole blood, plasma, amniotic fluid, and synovial fluid suggests that CO2 either interacts with a wide variety of biological materials to form stable complexes or interacts with a small number of materials common to all fluids. We have investigated potential sites for interaction of CO2 within the films by drying aqueous solutions/suspensions of lipids, proteins, and carbohydrate saturated with CO2. These materials were chosen as they represent the most widespread classes of biological molecules and would be expected to be found in all of the fluids we have studied. Films prepared from CO2-containing solutions of simple carbohydrates or suspensions of phospholipids showed no absorptions between 2330 and 2350 cm-1, indicating that phospholipids and simple carbohydrates are unable to trap CO2 (not shown). On the other hand, films formed by drying carbonated solutions of a range of proteins with varying secondary structural characteristics all exhibited a weak absorption at 2337 cm-1 (Figure 4), suggesting that proteins present within biological fluids represent the main site of interaction with CO2. The fact that secondary structural characteristics do not appear to significantly affect the interaction with CO2 suggests that this interaction is highly nonspecific. Furthermore, this interaction appears to take place in solution as well as in dry films, as CO2 is not lost from biological fluids and carbonated protein solutions with time, whereas CO2 is lost from carbonated water with time.
The question remains as to the source of this CO2. An exogenous source is ruled out, as the CO2 level in films prepared from the same fluid at different time points is stable. Furthermore, spectra of suspensions of human cells (cultured breast tumor cells or isolated lymphocytes) exhibit a significant CO2 absorption. However, following gentle centrifugation the CO2, absorption is not observed in the spectra of the supernatant but only appears in the spectra of the pelleted cells. This suggests an intercellular location for the CO2, while CO2 from an exogenous source would be expected to be dissolved in the supernatant. The most likely source of CO2 in tissues, biological fluids, and cell suspensions is metabolic activity. Synovial fluid CO2 absorptions would therefore arise as a result of metabolism of the cellular elements of the fluid, the synovial membrane, and cartilage. The CO2 absorption seen in spectra of plasma would represent the end point of glucose metabolism in all of the cells with which the blood has been in contact. Cerebrospinal fluid would be expected to contain CO2 from at least three sources: (i) the metabolic activity of the cellular elements of the cerebrospinal fluid, (ii) metabolic activity of the central nervous system tissue which the fluid bathes, and (iii) CO2 which has diffused across the blood-brain barrier from the blood into the cerebrospinal fluid. To confirm our hypothesis that metabolic activity is indeed the source of the observed CO2, we obtained spectra of yeast suspended in mineralized, non-carbonated water containing sufficient glucose to allow cell metabolism and monitored the intensity of the absorption at 2343 cm-1 as a function of time (Figure 5). Time t ) 0 min represents the stage at which glucose was added to the yeast suspension. Measurements conducted prior to this stage show no apparent absorption at 2343 cm-1 (not shown). After incubation with glucose at 20 °C, there is a progressive increase in the intensity of the absorption at 2343 cm-1, which plateaus after about 15 h. As expected, the spectra also show a small increase in the intensity of the absorption from 13CO2. To confirm that the observed increase in CO2 arises directly from glucose metabolism, yeast cells were incubated with 13Clabeled glucose. If the CO2 is indeed produced by breakdown of glucose, then under these conditions, a large increase in the intensity of the 13CO2 absorption (2279 cm-1) should be
6848 J. Phys. Chem., Vol. 100, No. 16, 1996
Schultz et al. attributed to CO2, most likely present as the end point of glucose metabolism. While the presence of this absorption is interesting, the question remains as to the practical advantages of being able to detect CO2 in biological fluids and tissues. If detection could be achieved in vivo, then the advantages are obvious: noninvasive monitoring of metabolism. While this is currently not feasible, there are a number of potential in vitro applications of CO2 monitoring. For example, monitoring of viability of seeds based upon CO2 production has already been postulated.13 A logical extension of this application would be monitoring of fermentation reactions and viability of cells in cultures. Acknowledgment. We thank Lin-P’ing Choo and Jing Wang for the spectra of cerebrospinal fluid and broncho-alveolar lavage fluid, respectively. References and Notes
Figure 6. Time dependence of the infrared spectra of yeast incubated with 13C-labeled glucose and of the integrated intensity of the absorptions band at 2279 and 2343 cm-1 (inset).
observed. As can be seen from Figure 6, incubation with 13Clabeled glucose results in a marked increase in the intensity of the 13CO2 absorption, again reaching a maximum after about 15 h. As the only possible source of this 13CO2 is oxidative phosphorylation of the 13C-labeled glucose, this result suggests that the source of the endogenous CO2 in spectra of human fluids, tissues, and cells is metabolic activity. In addition to an increase in the intensity of the 13CO2 absorption, the 12CO2 absorption also increases in intensity during incubation with labeled glucose, presumably as a result of the incorporation of nonlabeled substrates present in the cells into the metabolic cycle. Conclusions It is now clear that the absorption we have observed in tissues, cell suspensions, and biological fluids and their films is
(1) Jackson, M.; Mantsch, H. H. Spectrochim. Acta ReV. 1993, 15, 53. (2) Jackson, M.; Mantsch, H. H. CRC Crit. ReV. Biochem. Mol. Biol. 1995, 30, 95. (3) Taillandier, E.; Liquier, F. Meth. Enzymol. 1992, 211, 306. (4) Fabian, H.; Jackson, M.; Murphy, L. C.; Watson, P. H.; Fichtner, I.; Mantsch, H. H. Biospectroscopy 1995, 1, 37. (5) Choo, L.-P.; Mansfield, J. R.; Pizzi, N.; Somorjai, R. L.; Jackson, M.; Halliday, W. C.; Mantsch, H. H. Biospectroscopy 1995, 1, 141. (6) Choo, L.-P.; Jackson, M.; Halliday, W. C.; Mantsch, H. H. Biochim. Biophys. Acta 1993, 1182, 333. (7) Eysel, H. H.; Jackson, M.; Mantsch, H. H. U.S. Patent 5,473,160, 1995. (8) Shaw, R. A.; Kotowich, S.; Eysel, H. H.; Jackson, M.; Thomson, G. T. D.; Mantsch, H. H. Rheumatol. Int. 1995, 15, 159. (9) Alben, J. O.; Bare, G. H. Appl. Opt. 1978, 17, 2985. (10) Eysel, H. H.; Jackson, M.; Mantsch, H. H.; Thomson, G. T. D. Appl. Spectrosc. 1993, 47, 1519. (11) Falk, M.; Miller, A. G. Vibr. Spectrosc. 1992, 4, 105. (12) Fleyfel, F. ; Devlin, J. P. J. Phys. Chem. 1991, 95, 3811. (13) Sowa, S; Towill, L. E. Plant Physiol. 1991, 95, 610.
JP953254T
