Langmuir 1998, 14, 4359-4363
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Aggregation Kinetics of Sodium Deoxycholate in Aqueous Solution A. Jover, F. Meijide, E. Rodrı´guez Nu´n˜ez, and J. Va´zquez Tato* Universidad de Santiago, Campus de Lugo, Facultad de Ciencias, Departamentos de Quı´mica Fı´sica y Fı´sica Aplicada, 27002 Lugo, Spain Received November 20, 1997. In Final Form: April 27, 1998 The aggregation kinetics of sodium deoxycholate in aqueous solution has been studied at pH values close to neutrality. An induction time is observed and taken as inversely proportional to the rate constant for the aggregation of two small aggregates to form a larger one but one that is smaller than a critical size. When the critical size is reached, the length of the polymer-like structure allows the entanglement of the aggregates, accounting for the gel behavior of the solution. After the induction time, it is observed that the weight average aggregation number increases linearly with time for more than 50% of the aggregation. This is analyzed through the Smoluchowski equation in its simplest form. The influence of the temperature on the kinetics suggests the existence of a kinetic-thermodynamic relationship. It is deduced that the aggregation rate is limited by physical interactions, and the role of hydrogen bond in the aggregation rate is discussed. The influences of pH and phosphate concentration are also studied and discussed.
Bile salts are involved in different metabolic processes:1 they are the main products of cholesterol metabolism, and they have secretory and regulatory properties, complex cations, etc.2 Therefore, it is not surprising that bile salts have been extensively studied by using different experimental techniques to determine their physicochemical properties.3-5 The common bile acids-salts have a characteristic steroid structure (see Figure 1 for sodium deoxycholate, NaDC) with a mobile side chain at C-17, methyl groups at C-10 and C-13, and a different number of hydroxyl groups at C-3, C-7 (absent in NaDC), and C-12. At C-23, they have a carboxylic acid which may be conjugated with amino acids such as taurine and glycine. Therefore, they have hydrophobic and hydrophilic regions, a fact which explains their detergent-like properties since they self-associate to form aggregates (usually denoted micelles) and are surface active.6 In the absence of added electrolytes such as NaCl and at high pH values, the aggregation number of bile salts is rather low, ranging from 3 for trihydroxy derivatives to 6 for deoxy ones.4 For NaDC the aggregation number increases dramatically when the pH value is acidic and close to neutrality.7,8 In fact, the solution acquires a gel aspect which was first noticed by Sobotka and Czeczowizcza.9 At pH ) 7.3, Small7 deduced a value of 552 from ultracentrifugation measurements. Sugihara et al.10 studied the sol-gel transition at high pressures and (1) Hofmann, A. F. Hepatology 1984, 4, 4S. (2) (a) Hofmann, A.; Mysels, K. J. Colloids Surf. 1988, 30, 145. (b) Hofmann, A. F.; Small, D. M. Annu. Rev. Med. 1967, 18, 333. (c) Hofmann, A. F. In Bile Salt Metabolism; Schiff, L., Carey, J. B., Jr., Dietschy, J., Eds.; Thomas: Springfield, IL, 1969. (3) Kratohvil, J. P. Hepatology 1984, 4, 85S. (4) Coello, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. J. Pharm. Sci. 1996, 85, 9, and references therein. (5) Carey, M. C. In Sterols and Bile Acids; Danielsson, H., Sjo¨vall, J., Eds.; Elsevier: Amsterdam, 1985. (6) Small, D. M. In The Bile Acids, Chemistry, Physiology, and Metabolism; Nair, P. P., Kritchevski, D., Eds.; Plenum Press: New York, 1971. (7) Small, D. Adv. Chem. Ser. 1968, 84, 31. (8) Sugihara, G.; Ueda, T.; Kaneshina, S.; Tanaka, M. Bull. Chem. Soc. Jpn. 1977, 50, 2542. (9) Sobotka, H.; Czeczowizcza, N. J. Colloid Sci. 1958, 13, 188. (10) Sugihara, G.; Ueda, T.; Kaneshina, S.; Tanaka, M. Bull. Chem. Soc. Jpn. 1977, 50, 604.
Figure 1. Molecular structure of NaDC.
concluded that the formation of the gel implies the adsorption of a proton with the interchange of a counterion Na+. More recently, Jover et al.11 have carried out steadystate and time-resolved fluorescence studies on the NaDC gelation. During the gelation process, the formation of a pyrene excimer as a result of the aggregation of two smaller clusters carrying probes was observed. It was also concluded that the aggregates in the gel have a structure the same as that of the small aggregates of NaDC at high pH values.4 Blow and Rich12 and Sugihara et al.8 have studied the change of the solution viscosity with time, and from a polymer-like formation-degradation model, they deduced the aggregation number. However, since the gel is thixotropic,12 the stress applied for the measurement of viscosity can modify the gel structure, and the results are dubious. Therefore, the present study was undertaken. Here we present the experimental results for the aggregation kinetics obtained from static light scattering measurements. Although static light scattering has been extensively used to measure physicochemical properties of bile salt-aqueous solutions systems,7,13 it has not been applied to study the NaDC gelation process. It is also important that it is not an invasive technique and that the system remains unperturbed. Experimental Section NaDC was purchased from Sigma and was purified following the method published for NaC.14 Recrystallization was from methanol/water. NaOH was purchased from Merck, NaH2PO4 (11) Jover, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J.; Mosquera, M.; Rodrı´guez Prieto, F. Langmuir 1996, 12, 1789. (12) Blow, D. M.; Rich, A. J. Am. Chem. Soc. 1960, 82, 3566.
S0743-7463(97)01275-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/15/1998
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weight average molecular weight, A2 is the second virial coefficient, and P(q) is the particle-scattering factor. In the limit of small qRg where q is the magnitude of the scattering-wave vector, Rg is the radius of gyration of the scattering particle, and independently of its shape, P(q) is approximated by
P(q) ) 1 -
Figure 2. Critical micelle concentration determination for NaDC at 20 °C by fluorescence of 2-(4′-piridil)bencimidazol at 380 nm. λexc ) 333 nm. was purchased from Fluka, and the water was Milli-Q grade. 2-(4′-Piridil)bencimidazol was synthesized, following literature methods.15 The pH was measured on a radiometer pHM-82 with a Radiometer combined electrode GK4201C. Temperature was kept constant with a Haake thermostat. Steady-state fluorescence measurements were recorded on a Perkin-Elmer model 650-40. A Hitachi (model F-3010) was used to measure the static light scattering; the technique has been described elsewhere.16 The wavelength used was 436 nm, and the slit was 1.5 nm. Experiments were carried out in square quartz cells from Hellma. Cells were cleaned for 24 h with concentrated nitric acid to prevent catalysis caused by “seeding” of gel formed in a previous experiment.12 Solutions were prepared directly in the measuring cell by mixing the filtered solutions. A value of 2.89 mM was obtained for the cmc from fluorescence intensities of 2-(4′-piridil)bencimidazol (see Figure 2). The value is in the range of most published values at 0.1-0.2 M Na+ (217 to 3 mM13f,17,18).
Results and Discussion For micellar systems, in which the micelles exist only above the cmc, the Debye equation for light scattering experiments may be written as
1 1 ∝ + 2A2(c - cmc) I Mw P(q)
(1)
where I is the intensity of the light scattered, Mw is the (13) (a) De Moerlose, P.; Ruysse, R. J. Pharm. Belg. 1958, 95. (b) Furusawa, T. Fukuoka Igaku Zasshi, 1962, 53, 124. (c) Olson, J. A.; Herron, J. S. Proc. Int. Congr. Biochem. 6th 1964, 7, 588. (d) Kratohvil, J. P.; Dellicolli, H. T. Can. J. Biochem. 1968, 46, 945. (e) Juna, K.; Sugano, T. Nippon Kagaku Zasshi 1969, 90, 463. (f) Benzonana, G. Biochim. Biophys. Acta 1969, 176, 836. (g) Fontell, K. Kolloid Z. Z. Polym. 1971, 244, 253. (h) Chang, Y.; Cardinal, J. R. J. Pharm. Sci. 1978, 67, 174 and 994. (i) Oakenfull, D. G.; Fisher, L. R. J. Phys. Chem. 1978, 82, 1443. (j) Mazer, N. A.; Carey, M. C.; Kwasnick, R. F.; Benedek, G. B. Biochemistry 1979, 18, 3064. (k) Schurtenberger, P.; Mazer, N.; Ka¨nzig, W. J. Phys. Chem. 1983, 87, 308. (l) Kratohvil, J. P.; Hsu, W. P.; Jacobs, M. A.; Aminabhavi, T. M.; Mukunoki, Y. Colloid Polym. Sci. 1983, 261, 781. (m) Murata, Y.; Sugihara, G.; Nishikido, N.; Tanaka, M. Solution Behavior of Surfactants; (Mittal, K. L., Bothorel, P., Eds.; Plenum Press: New York, 1984. (n) Kratohvil, J. P.; Hsu, W. P.; Kwok, D. I. Langmuir 1986, 2, 256. (14) Coello, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. J. Phys. Chem. 1993, 97, 10186. (15) Rios Rodrı´guez, M. C. Doctoral Thesis, Universidad de Santiago de Compostela, 1994. (16) (a) Mouga´n, M. A.; Coello, A.; Jover, A.; Meijide, F.; Va´zquez Tato, J. J. Chem. Educ. 1995, 72, 284. (b) Coello, A.; Meijide, F.; Mouga´n, M. A.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. J. Chem. Educ. 1995, 72, 73. (17) Kawamura, H.; Murata, Y.; Yamaguchi, T.; Igimi, H.; Tanaka, M.; Sugihara, G.; Kratohvil, J. P. J. Phys. Chem. 1989, 93, 3321. (18) Roda, A.; Hofmann, A. F.; Mysels, K. J. J. Biol. Chem. 1983, 258, 6362.
q2 Rg2 + ... 3
(2)
The intensity-time experimental results were transformed in aggregation number vs time plots as follows. Esposito et al.19 have proposed that NaDC aggregates have a helical structure; for an aggregation number of 60, the helix radius is 1.05 nm, and the height 11.6 nm. It is expected that the maximum radius of gyration corresponds to the maximum aggregation number; accepting the value of 552 given by Small7 and that the helix is equivalent to a rigid rod, it can be deduced that the maximum radius of gyration is 31 nm. This implies that the term q2Rg2/3 in eq 2 is 0.23. However, if the maximum value obtained from some fluorescence measurements1 is accepted (lower than 200), then the radius of gyration term is only 0.03. The aggregation number from static fluorescence was determined according Turro and Yekta,20 accepting that the quencher, Q, and the fluorescent probe are entirely in the micellar phase. The gelation process was followed by measuring the change in the fluorescence intensity of pyrene with time at different quencher (cetylpyridinium chloride) concentrations. An intensity decrease was observed with time at wavelengths lower than an isosemission wavelength at 450 nm, while increases were observed at higher wavelengths due to the formation of an excimer.11 At high time values, the intensity remained constant which means that the gel was completely formed. The obtained results are in semiquantitative agreement with those obtained from light scattering measurements. However, since the validity of the Turro and Yekta equation has been questioned,21 they are not presented here. Furthermore, compared to fluorescence, light scattering experiments require much less experimental effort, and the influence of different experimental variables (temperature, concentration, pH, ...) on the gelation kinetics are much easier studied by this technique. Therefore, we can estimate an error lower than 10% if we accept that in eq 2, P(q) (at a scattering angle of 90°) is equal to 1. On the other hand, Chang and Cardinal13h have determined a value of 7.8 × 10-4 mL mol/g2 for the second virial coefficient of NaDC micelles. Finally, the proportionality constant in eq 1 can be deduced, accepting the value of 8 obtained by Kratohvil et al.13n for the aggregation number of NaDC (in experimental conditionss temperature and ionic strengthsvery similar to those used in this work) as the aggregation number at zero time. As it might be expected, the gel formation process implies the increase of the average aggregation number of aggregates. A typical experiment is plotted in Figure 3 where a sigmoidal curve of the average aggregation number vs time can be observed. It shows the existence of an induction time which is related to the seeding effect12 since, when a small amount of a previously formed gel was added at zero time, no induction time was observed. (19) Esposito, G.; Giglio, E.; Pavel, N. V.; Zanobi, A. J. Phys. Chem. 1987, 91, 356. (20) (a) Turro, N. J.; Yekta, A. J. Am. Chem. Soc. 1978, 100, 5951. (b) Rodrı´guez Prieto, M. F.; Rios, M. C.; Mosquera, M.; Rios, A. M.; Mejuto, J. C. J. Chem. Educ. 1995, 72, 662. (21) Jover, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J.; Mosquera, M. Langmuir 1997, 13, 161.
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Figure 3. Weight aggregation number vs time for NaDC at 20 °C and pH ) 6.79. [NaDC] ) 0.0217 M, [NaH2PO4] ) 0.02 M.
Similar induction times have been observed for other aggregation processes such as in silicalite nucleation22 and in the formation of gold colloids in the presence of intermediate concentrations of pyridine.23 After the observed induction period, it can be considered that the weight aggregation number of aggregates grows almost linearly with time for more than 50% of the final aggregation number value. Twomey et al.22 have studied the silicalite nucleation and growth by light scattering techniques and analyzed the results in a similar way to the one presented here. Figures 4-6 show the dependence of the ordinate (obtained for the minimum value of the aggregation number which here is considered to be 8) and the slope of the dependence of Nw with time, at different experimental conditions. For instance, Figure 4a,b show that both the slope and intercept can be considered practically constant with phosphate buffer concentration. The linear dependence after the induction period is analyzed through Smoluchowski’s equation.24 By accepting that aggregation is an irreversible process, this kinetic equation is given by eq 3.
dck dt
) 1/2
∑
k(i,j)cicj -
i+j)k
k(j,k)cjck ∑ j)1
(3)
The first summation represents the formation of aggregates of size k from smaller aggregates, and the second one represents the reaction of clusters of size k to form larger aggregates. k(i,j) is an element of the reaction kernel that describes the aggregation process. Unfortunately, Smoluchowski’s equation has exact solutions for very simple situations, and only approximate solutions are available25 which do not cover all the concentration or time ranges. However, a quite simple result is obtained when all the kinetic constants are identical to a common value k. In this case, the Smoluchowski set of differential equations predicts a linear dependence of the weight average aggregation number with time (eq 4)
Nw ) 1 + 2T
(4)
(22) Twomey, T. A. M.; Mackay, M.; Kuipers, H. P. C. E.; Thompson, R. W. Zeolites 1994, 14, 162. (23) Weitz, D. A.; Huang, J. S. In Kinetics of aggregation and gelation; Family, F., Landau, D. P., Eds.; Elsevier: Amsterdam, 1984. (24) (a) von Smoluchowski, M. Z. Phys. 1916, 17, 585. (b) von Smoluchowski, M. Z. Phys. Chem. 1917, 92, 129. (c) Ziff, R. M. In Kinetics of aggregation and gelation; Family, F., Landau, D. P., Eds.; Elsevier: Amsterdam, 1984. (d) Leyvraz, F. In Kinetics of aggregation and gelation, Family, F., Landau, D. P., Eds.; Elsevier: Amsterdam, 1984.
Figure 4. Dependence of the induction time (a) and the slope (b) of the aggregation kinetics of NaDC with phosphate buffer concentration at 20 °C and different pH values. [NaDC] ) 0.0217 M.
(here T is the time in k-1 units). This means that the aggregation rate between two clusters or aggregates is independent of the aggregation number (i.e., size) of the involved aggregates. It is known that deoxycholic acid can form helical structures in the crystalline state26 in which hydrogen bonding plays a leading role. X-ray diffraction studies confirm the existence of helices in the crystalline state and in the solid fibers formed in the gel phase.26a In aqueous solution, polymer-like aggregates have also been proposed.27 Therefore, it is reasonable to accept that the thickening and gelling phenomenon is due to the entanglement of long (helical or otherwise) polymer-like aggregates of NaDC. Gel networks formed from surfac(25) (a) Wright, H.; Muralidhar, R.; Ramkrishna, D. Phys. Rev. A 1992, 46, 5072. (b) Broide, M. L.; Cohen, R. J. J. Colloid Interface Sci. 1992, 153, 493. (c) Meakin, P. Croat. Chem. Acta 1992, 65, 237. (d) Meakin, P. Phys. Scr. 1992, 46, 295. (e) Ball, R. C.; Weitz, D. A.; Witten, T. A.; Leyvraz, F. Phys. Rev. Lett. 1987, 58, 274. (f) van Dongen, P. G. J. J. Phys. A: Math. Gen. 1987, 20, 1889. (g) van Dongen, P. G. J.; Ernst, M. H. J. Phys. A: Math. Gen. 1985, 18, 2779. (h) Ziff, R. M.; McGrady, E. D.; Meakin, J. Chem. Phys. 1985, 82, 5269. (i) Villarica, M.; Casey, M. J.; Goodisman, J.; Chaiken, J. J. Chem. Phys. 1993, 98, 4610. (j) Leyvraz, F. In On Growth and Form; Stanley, H. E., Ostrowsky, N. Eds.; NATO ASI Series E: Applied Sciences, No. 100; Martinus Nijhoff: Dordrecht, The Netherlands, 1986. (26) (a) Conte, G.; Di Biasi, R.; Giglio, E.; Paretta, A.; Pavel, N. V. J. Phys. Chem. 1984, 88, 5720. (b) Campanelli, A. R.; De Sanctis, S. C.; Chiessi, E.; D’Alagni, M.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1989, 93, 1536. (27) Murata, Y.; Sugihara, G.; Fukushima, K.; Tanaka, M.; Matsushita, K. J. Phys. Chem. 1982, 86, 4690.
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Figure 5. Dependence of the induction time (a) and the slope (b) of the aggregation kinetics of NaDC with temperature. [NaH2PO4] ) 0.02 M, [NaDC] ) 0.0217 M, pH ) 6.72.
tants are normally heterogeneous materials formed by interconnected organized microdomains.28 Our kinetic results clearly show that, during the gel formation, there is an increase in the average size of aggregates. Therefore, the induction time observed can be understood as the time necessary for the formation of a polymer-like aggregate with a minimum or critical length under which the entanglement is not possible. The aggregation is seen as a two-step process: the first one corresponding to the induction time during which the growth rate of aggregates is rather slow. The length of this induction period may be taken as inversely proportional to the rate constant for the aggregation of two small aggregates to form a larger one but one that is smaller than a critical size. This interpretation is supported by the fluorescence studies carried out previously. From steady-state and timeresolved fluorescence studies, Jover et al.11 have concluded that the formation of a pyrene excimer is the result of the formation of a larger aggregate from two smaller ones carrying probes. Furthermore, the constancy of the ratio for first and third pyrene vibronic peaks, I1/I3, during the gelation process suggests that the aggregates in the gel have the same structure as that of the small aggregates of NaDC at high pH values. After the induction period, two processes are occurring simultaneously, a faster growth of aggregates and the entanglement of the aggregates which accounts for the gel behavior of the solution. This interpretation is similar to the one given by Twomey et al.22 to explain the silicalite nucleation and growth. However, the dependence upon the temperature of the kinetic constant is contrary to that of the one observed for the zeolite system. In the later (28) Terech, P.; Smith, W. G.; Weiss, R. G. J. Chem. Soc., Faraday Trans. 1996, 92, 3157.
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Figure 6. Dependence of the induction time (a) and the slope (b) of the aggregation kinetics of NaDC with pH at 20 °C. [NaH2PO4] ) 0.02 M, [NaDC] ) 0.0217 M.
process, both the nucleation process (first step) and the growth (second) step are considerably enhanced by an increase in the reaction temperature. This seemed to indicate that the nucleation and growth steps involve a chemical interaction rather than a physical one. The contrary temperature dependence is observed here. The induction time is reduced (the inverse kinetic constant increased) (Figure 5a), and the slope is enhanced by lowering the temperature (Figure 5b). This again suggests that physical processes limit the formation rate of the gel. This is in agreement with the type of superstructure commented on above in which the physical interactions are responsible for the formation and entanglement of aggregates. The temperature dependence also suggests a kineticthermodynamic relationship. Sugihara et al.10 have studied the sol-gel transition of NaDC at high pressures and different temperatures. At any pressure and temperature, negative values for the enthalpy, entropy, and volume changes of gel formation were obtained, all of them being more negative when temperature decreases. Therefore, the free energy can be deduced. The calculated values clearly show that the lower the temperature the more negative the value for the free energy. For instance, at a pressure of 500 atm, values of 0.24 (36 °C) and 0.59 (28 °C) are obtained for -∆G/kcal mol-1, while at 103 atm, the corresponding values are 0.52 and 1.2 kcal mol-1, respectively. Here, it has been observed that the reaction kinetics is faster (the induction time is smaller and the slope is higher) when the temperature decreases. Therefore, it can be concluded that the aggregation kinetics and the gel formation are faster when the sol-gel transition is more favorable from a thermodynamical point of view. It is important to remember here that Sugihara et al.11 have
Aggregation Kinetics of Sodium Deoxycholate
concluded that the gel formation is attributable to intermolecular hydrogen bonds and that their number tends to increase as the temperature is lowered, although partial hydrophobic interactions (back-to-back between steroid skeletons) also contribute to the formation of ordered polymer-like aggregates.27 All these facts reinforce the idea that physical interactions limit the aggregation rate. Other similar analogies can be found. From 11B NMR measurements, Murata et al.27 have concluded that the borate ion does not contribute to the formation of the polymer-like structures of NaDC at pH 7.8. Here, we have found that the phosphate ion does not modify the kinetics of aggregation since the induction time and the aggregation number-time slope are independent of its concentration (see Figure 4). Again, Murata et al. have concluded that the intermolecular hydrogen bonding and hydrophobic interactions are responsible for the formation of these polymer-like structures. Figure 5a,b clearly shows that the aggregation process is faster when the pH is reduced since the induction time decreases and the slope increases. It is necessary to take into account different facts to understand it. (i) Fontell29 has shown that bile salt aggregates are highly hydrated and the number of water molecules bonded per mole of bile salt are higher than 20 for NaDC and NaC aggregates. Similar results have been obtained by Lindman et al.30 (ii) Murata et al.27 have considered that the polymer-like aggregate is formed by the intermolecular hydrogen bonding between the hydroxyl group at the 3 position of the steroid group and at the carboxyl group of other molecule. In aqueous solution, this hydrogen bonding formation has to compete with the one corresponding to the interaction between the carboxylate group and water. (29) Fontell, K., Kolloid Z. Z. Polym. 1971, 246, 614. (30) (a) Lindman, B.; Kamenka, N.; Fabre, H.; Ulmius, J.; Wieloch, T. J. Colloid Interface Sci. 1980, 73, 556. (b) Lindman, B.; Kamenka, N.; Fabre, H.; Ulmius, J.; Wieloch, T. J. Phys. Chem. 1984, 88, 5048.
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(iii) Gustavsson and Lindman31 have proposed hydrogenbond interactions between the water of counterion hydration and carboxylate group to explain some differences between the counterion binding to octanoate and octyl sulfate micelles. (iv) Finally, observing that when gel formation takes place, the pH rises linearly with the logarithm of the reaction time, Sugihara et al.8 have concluded that the adsorption of proton on the micelle surface is necessary for the structure making of gel facilitating the formation of hydrogen bonds between deoxycholate molecules. All these facts and the experimental kinetic results presented here suggest that the reduction of pH facilitates the formation of hydrogen bonds between the aggregates, favoring the formation of polymerlike structures and their entanglement from both thermodynamical and kinetical points of view. It was also observed that the final aggregation number of the polymer-like structures is almost independent of pH and phosphate concentration, the average value being 250. Similar values have been deduced from fluorescence measurements. These values are lower than those published by Sugihara et al.8 who have estimated values in the range 625-1250 and Small7 who published a value of 552 at pH 7.3. The values published by Sugihara et al. depend on the aggregation number used for the primary or initial micelle which was 62, a number rather high if we take into account other published values.4 The difference can also arise from the fact that when the entanglement between the polymer structures is becoming important, as when the gel is fully developed, the hypotheses used in eqs 1 and 2 are no longer valid. Acknowledgment. We thank the DGICYT for financial support (project PB90-0758). A.J. thanks the Xunta de Galicia for a grant. LA9712754 (31) Gustavsson, H.; Lindman, B. J. Am. Chem. Soc. 1975, 97, 3923.
