18078
2006, 110, 18078-18080 Published on Web 08/29/2006
trans-Sodium Crocetinate and Diffusion Enhancement Amanda K. Stennett,† Gail L. Dempsey,‡ and John L. Gainer* Department of Chemical Engineering, UniVersity of Virginia, 102 Engineers Way, CharlottesVille, Virginia 22904-4741 ReceiVed: July 9, 2006; In Final Form: August 16, 2006
trans-Sodium crocetinate (TSC) increases the diffusion coefficient of glucose through water by about 25-30%. This is the same percentage increase that TSC causes in the diffusivity of oxygen through water. TSC is also found to induce order in the surrounding water structure through increased hydrogen bonding of the water molecules, and molecular simulations suggest that the increase in diffusivity occurs only in these ordered regions.
Introduction Diffusion Enhancement. It has been suggested that transsodium crocetinate (TSC) can be used to increase the amount of oxygen getting to hypoxic cells and tissues.1,2 The chemical structure of TSC is similar to that of crocetin, a naturally occurring carotenoid. Both crocetin and TSC are thought to increase tissue oxygenation by increasing the diffusion coefficient (also called the diffusivity) of oxygen through blood plasma1. The mechanism by which TSC might exert such an effect has been investigated previously using molecular dynamics simulations3, and it was concluded that, in a water-TSCoxygen mixture, oxygen diffuses more slowly when next to a TSC molecule but, at a distance of 4-5 Å away from the TSC molecule, the oxygen diffuses considerably faster. The authors suggest that a rearrangement of water molecules within a solvation shell at that distance from the TSC molecule is responsible for the increased diffusivity.3 This simulation only considered oxygen diffusing in TSCwater mixtures, suggesting that a change in water structure is responsible for the increased diffusivity of oxygen, with no direct interactions of TSC and oxygen. This strongly suggests that the diffusivities of other solutes in water should also increase with TSC. This has been investigated using glucose as the diffusing solute. Changes in water structure caused by TSC have also been studied. Water was chosen for this study rather than plasma since the computer simulations were based on it, and they suggested that it is the hydrophobic interactions of TSC and water that are important. Experimental Methods Diffusion. To determine how TSC affects the diffusion of a small nongaseous solute, the diffusivity of glucose through water was measured with and without TSC. This was done using a microinterferometric method,4 in which a wedge is formed by * Corresponding author. Phone: 434-220-0718. Fax: 434-220-0722. E-mail:
[email protected]. † Current address: Fresenius Medical Care, Lexington, MA. ‡ Current address: Idexx Laboratories Inc., Greensboro, NC.
10.1021/jp064308+ CCC: $33.50
two microscope slides that have a partial coating of aluminum on the inner slides. The wedge is placed on a microscope stage and illuminated from below using a helium-neon laser. As the light passes through the wedge, the partial coating of aluminum allows some light to be transmitted, while some is reflected. This creates constructive and destructive lines known as fringes. The interference fringes formed are magnified by a microscope and recorded with a camera. As molecules diffuse along a concentration gradient created within the wedge, the refractive index of the solution at each point changes with time, as determined from the time behavior of the fringe pattern. Since the refractive index is proportional to the concentration of glucose, one can then calculate the diffusion coefficient, or diffusivity, from the fringe profile at each given time. The concentration of TSC in water ranged from 0.5 to 350 µM in these experiments. The measurements were conducted at a temperature of 25 °C, and the accuracy of the apparatus was determined by measuring the diffusivity of glucose in pure water. The value obtained was (6.9 ( 0.3) × 10-6 cm2/s, which closely corresponds to the CRC Handbook5 value of 6.7 × 10-6 cm2/s. In addition, the diffusivity of oxygen through water was also measured over the same TSC concentration range as that used for the glucose studies. These experiments were conducted in an apparatus commonly used for oxygen diffusing in liquids.6 In this experiment, the rate of movement of oxygen across a liquid layer was determined by measuring the concentration change over time, using an oxygen electrode, at the opposite boundary. All of these measurements were performed at 25 °C. Viscosity. The viscosities of water and solutions containing various concentrations of TSC were determined at 10, 20, 30, and 40 °C. The measurements were made using an Advanced Rheometer 2000 (TA Instruments, New Castle, DE). A double concentric cylinder geometry was used in the rheometer to increase the surface contact between the fluid and the instrument since the solutions had low viscosities. The shear stress was kept constant in each run, at 0.5 Pa for measurements made at 10 and 20 °C, 0.3 Pa for measurements at 30 °C, and 0.2 Pa for measurements at 40 °C. The viscosity of each solution at © 2006 American Chemical Society
Letters
Figure 1. Diffusion of glucose and oxygen in TSC-water mixtures.
each temperature was measured 25 times, and the data was averaged. Near-Infrared Spectroscopy. The near-infrared (NIR) spectra of water and aqueous solutions of various concentrations of TSC were measured at room temperature. The spectra were obtained using an FTS-60A Fourier transform infrared spectrometer (Bio-Rad, Hercules, CA). A demountable liquid transmission cell (Harrick, Ossining, NY) was used with the spectrometer. It contained ZeSe windows and had a transmission path length of 200 µm. The source was a halogen bulb, and the detector was PbSe. A 12% neutral density filter was also used and was placed in the optical path just before the sample cell. A spectrum was obtained over the wavenumber range of 40008000 cm-1, and 500 spectra were recorded and averaged for one experimental run. An average spectrum was recorded for each solution six times. The peak positions in each spectrum were calculated by nonlinear least-squares regression. Results and Discussion The Effect of TSC on Diffusivity. The diffusivities of glucose and oxygen were measured in TSC-water solutions, with the results shown in Figure 1. The data are presented as the percentage change from the value obtained when there was no TSC present. Error bars have been omitted in this figure to
J. Phys. Chem. B, Vol. 110, No. 37, 2006 18079 simplify the graph; however, the increases seen are statistically significant (Student t-test, p < 0.05).7 Although the diffusivity values for oxygen through water and for glucose through water are different, above a TSC concentration of 1.3 µM, the diffusion coefficients for both solutes increased by the same percentage (approximately 30%) in the presence of TSC, until reaching a concentration of 150 µM. At that point, the diffusivities of both solutes began to decrease to their values in pure water. Computer Simulations. The molecular dynamics simulation done previously utilized almost 15 000 water molecules3. To obtain results faster, a smaller simulation was performed containing approximately 1400 water molecules, using Tinker for the molecular dynamics program.8 The larger simulation showed that TSC caused a 30% increase in the oxygen diffusivity through water, the same as seen experimentally (Figure 1). Although the computed value for the diffusivity in the smaller simulation did not exactly match the experimental results, it still showed that the addition of TSC caused the diffusivity of oxygen through water to increase. Both simulations indicated that TSC results in an increase in the diffusivity of oxygen because TSC causes a change in the water structure and that TSC exerts no direct effect on the oxygen molecules themselves. In fact, the TSC appears to slow the diffusion when oxygen is nearby by blocking its pathway. Experimental Studies If TSC is a “structure maker”, it should result in a decrease in the entropy of an aqueous system when added to that system. The decrease in entropy would result in a heat flow from the system, decreasing the temperature of the system. Using basic calorimetry methods, it was estimated that 250 kcal is lost from the solution per mole of TSC added to water.8 This indicates that TSC is a strong structure maker. Another study performed to determine the effect of TSC on water structure measured the viscosities of aqueous solutions containing various concentrations of TSC at different temperatures. The presence of TSC in water results in a slightly increased viscosity (Figure 2), and how that varies with temperature can offer some insight. At 10 °C, water is close to the freezing point and is closer to being solid ice and more ordered. At that temperature, the presence of TSC has little
Figure 2. The effect of TSC on the viscosity of water at various temperatures.
18080 J. Phys. Chem. B, Vol. 110, No. 37, 2006 effect. As the temperature of the system increases, however, the addition of TSC causes more of an increase in viscosity. This suggests that TSC causes a more ordered structure and that this effect is larger the less ordered the water is without the TSC (i.e., at higher temperatures). TSC and Hydrogen Bonding. Structural changes in water are frequently related to the formation/breaking of hydrogen bonds. NIR spectroscopy can be used to investigate the changes in hydrogen bonding in aqueous solutions. The NIR spectrum of water has two absorption peaks that represent overtones of the O-H vibrations, which are located near the wavelengths of 1450 and 1900 nm (corresponding to wavenumbers of 6900 and 5200 cm-1). These peaks display a characteristic shift toward higher wavelengths (or lower wavenumbers) when hydrogen bonds are strong. (This is especially pronounced in the case of ice, where hydrogen bonds are at maximal strength.9) This phenomenon occurs because the hydrogen bond is weakened with increasing temperature, which, in turn, strengthens the covalent O-H bond and consequently causes the water to vibrate at higher frequencies,10 which can be detected using NIR spectroscopy. TSC caused the O-H overtone peak center to shift to a lower wavenumber. The shift implies stronger hydrogen bonding, and is proportional to the TSC concentration. The slope of the concentration dependence of the peak center was -277 cm-1/ M, which is 33 times larger than that found previously for strong structure makers.11 Conclusions TSC has been suggested to increase the diffusivity of oxygen through blood plasma. Experiments show that this same effect occurs in pure water and, further, that the diffusivity of glucose is also increased by the same percentage as oxygen. Molecular dynamics simulations suggest that the increased diffusivity of oxygen does not occur adjacent to the TSC molecule but, instead, takes place in a “water solvation shell”, which lies several angstroms from it. Calorimetry and viscosity data also suggest that TSC causes a more ordered water structure to form, and NIR studies indicate that TSC promotes increased hydrogen bonding among the water molecules. Apparently, this increased structure corresponds to a decrease in density, which allows for an increase in the diffusion of solutes through the medium.12 However, greater increases in hydrogen bonding from
Letters further TSC additions may instead eventually result in a disordering, which causes the diffusivity to decrease. TSC has been shown to cause physiological effects in hypoxic situations such as hemorrhagic shock.1,2 These effects correlate with increased tissue oxygen consumption, which is thought to result from an increase in the diffusion of oxygen through plasma. The current results indicate that TSC exerts a diffusionenhancing effect by causing a change in water structure. This appears to be a totally novel mechanism of action for a pharmacological agent. Since it apparently works through a physicochemical change rather than a biological one, it may provide new insight into basic physiological processes. Acknowledgment. Financial aid for this study was provided by the Office of Naval Research, Arlington, VA, through Grant N00014-95-1-0213 References and Notes (1) Roy, J. W.; Graham, M. C.; Griffin, A. M.; Gainer, J. L. A novel fluid resuscitation therapy for hemorrhagic shock. Shock 1998, 10, 213217. (2) Giassi, L. J.; Gilchrist, J.; Graham, M. C.; Gainer, J. L. Trans sodium crocetinate restores blood pressure, heart rate, and plasma lactate after hemorrhagic shock. J. Trauma 2001, 51, 932-938. (3) Laidig, K. E.; Gainer, J. L.; Daggett, V. Altering diffusivity in biological solutions through modification of solution structure and dynamics. J. Am. Chem. Soc. 1998, 120, 9394-9395. (4) Secor, R. M. The effect of concentration on diffusion coefficient in polymer solutions. AIChE J. 1965, 11, 452-456. (5) CRC Handbook of Chemistry and Physics; Lide, D. R., Ed.; CRC Press: Boca Raton, FL, 1998; p 6-181. (6) Golkdstick, T. K.; Ciuryla, V. T.; Zuckerman, L. Diffusion of oxygen in plasma and blood. AdV. Exp. Med. Biol. 1976, 75, 183-190. (7) Stennett, A. K. Mechanism of action of TSC. Ph.D. Dissertation, University of Virginia, Charlottesville, VA, 2005. (8) Dempsey, G. Molecular dynamics of oxygen in water with TSC or CSC. Ph.D. Dissertation, University of Virginia, Charlottesville, VA, 2003. (9) Galinski, E. A.; Stein, M.; Amendt, B.; Kinder, M. The kosmotropic (structure-forming) effect of compensatory solutes. Comp. Biochem. Physiol. 1997, 117A, 357-365. (10) Segtnan, V.; Sasic, S.; Isaksson, T.; Ozaki, Y. Studies on the structure of water using two-dimensional near-infrared correlation spectroscopy and principle component analysis. Anal. Chem. 2001, 73, 31533161. (11) Lever, M.; Randall, K.; Galinski, E. A. Near infrared spectra of urea with glycine betaine or trimethylamine N-oxide are additive. Biochim. Biophys. Acta 2001, 1528, 135-140. (12) Gainer, J. L. Altering diffusivities in dilute polymeric and biological solutions. I&EC Res. 1994, 33, 2341-2344.
