Anal. Chem. 1997, 69, 3674-3678
Glutamine Biosensors for Biotechnology Applications, with Suppression of the Endogenous Glutamate Signal Marcel B. Maˇdaˇras¸ ,*,† Robert B. Spokane,‡ Jay M. Johnson,‡ and John R. Woodward‡,§
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, and YSI Inc., 1725 Brannum Lane, Yellow Springs, Ohio 45387
Glutamine membranes for amperometric measurements are described. The interference of the endogenous glutamate is greatly diminished by using a supplementary “anti-glutamate” layer consisting of immobilized glutamate oxidase and catalase on top of the glutamine-sensitive layer having co-immobilized glutaminase and glutamate oxidase. The use of polycarbonate membranes with different permeability characteristics for the control of the substrate’s access to the enzyme layers is presented, as well as the effect of the density of the enzyme layer on the sensitivity of these membranes. The fabricated membranes have good operational stability (at least 5 days) and very good linearity (up to 10 mM glutamine). Using an appropriate choice of membranes and cross-linking conditions, membranes with good rejection of glutamate have been fabricated (less than 6% RSD for a 5 mM glutamine sample containing 5 mM glutamate as interferent). These membranes are suitable for monitoring of glutamine levels in mammalian cell cultures without the need of a separate measurement for glutamate. In the bioprocess industry and in many research laboratories, mammalian cells are cultivated in vitro for the production of different biomolecules such as glycosylated proteins having therapeutic value.1 The cell culture generally includes serum, vitamins, growth factors, minerals, and a mixture of amino acids. Glutamine is an essential amino acid for the cell growth because it represents the major energy source (30-65% of the requirements) and is also an important nitrogen source for the synthesis of proteins, nucleotides, and amino sugars.2 Glutamine is added in excess to the starting cell culture because it is metabolized by the cells and it is also chemically decomposed to form pyrrolidonecarboxylic acid and ammonia. When the amount of glutamine is too high, the levels of ammonia become toxic for the cells, inhibiting their growth.3 There is, therefore, an increasing demand for analytical methods for the monitoring and control of mammalian cell cultures. On-line control is preferred, since the
rapid determination of the glutamine level in the culture can be used as a feed-back loop for control of the cell’s productivity. Determination of glutamine in mammalian cell cultures is usually done by HPLC,4 but the technique is time consuming and expensive and requires specialized operators; thus, it is not optimally suited for on-line monitoring. Optical methods such as near-infrared spectroscopy (near-IR)5 and chemiluminescence6 have also been recently described. These methods have high sensitivity, but matrix interferences in the case of near-IR and background fluorescence in the case of chemiluminescence can degrade the selectivity of the measurements. Several methods involving biosensors have been proposed for glutamine measurement. Potentiometric sensors based on NH3 detection using immobilized glutaminase from E. coli,7 mitochondria,8 and pig kidney tissue9 have been described. Glutamine is hydrolyzed to glutamic acid by glutaminase, with the formation of NH3, which is then detected at an NH4+ ion-selective membrane. The interferent, endogenous NH4+ in the sample, was removed using an anion exchange membrane10 or by operating the sensor in the sub-Nernstian, linear response range.11 The amperometric approach is to use a bienzyme membrane, containing glutaminase and glutamate oxidase, attached to a suitable transducer: L-glutamine L-glutamate
glutaminase
+ H2O 98 L-glutamate + NH3 (1) glutamate oxidase
+ O2 98 R-ketoglutarate + NH3 + H2O2 (2)
The amperometric sensor can monitor either the O2 consumption with a Clark-type oxygen electrode12 or the enzymatically generated H2O2. The latter option was usually preferred by many investigators,2,13-20,22 because the first approach implies control
* To whom correspondence should be addressed. Present address: YSI Inc., 1725 Brannum Ln., Yellow Springs, OH 45387. E-mail:
[email protected]. † UNC-Chapel Hill. ‡ YSI Inc. § Present address: GLI International, Inc., 9020 West Dean Rd., Milwaukee, WI 53224. (1) Romette, J. L.; Cooney, C. L. U.S. Patent 4,780,191, 1988. (2) Cattaneo, M. V.; Luong, J. H. T.; Mercille, S. Biosens. Bioelectron. 1992, 7, 329-34. (3) Oh, G. S.; Izuishi, T.; Inoue, T.; Hu, W. S.; Yoshida, T. J. Ferment. Bioeng. 1996, 81, 329-36.
(4) Kurokawa, H.; Park, Y. S.; Iijima, S.; Kobayashi, T. Biotechnol. Bioeng. 1994, 44, 95-103. (5) Chung, H.; Arnold, M. A.; Rhiel, M.; Murhammer, D. W. Appl. Spectrosc. 1996, 50, 270-6. (6) (a) Blankenstein, G.; Preuschoff, F.; Spohn, U.; Mohr, K. H.; Kula, M. R. Anal. Chim. Acta 1993, 271, 231-7. (b) Cattaneo, M. V.; Male, K. B.; Luong, J. H. T. Biosens. Bioelectron. 1992, 7, 569-74. (7) Guilbault, G. G.; Shu, F. R. Anal. Chim. Acta 1971, 56, 333-8. (8) Arnold, M. A.; Rechnitz, G. A. Anal. Chem. 1980, 52, 1170-4. (9) Rechnitz, G. A.; Arnold, M. A.; Meyerhoff, M. E. Nature 1979, 28, 466-7. (10) Rosario, S. A.; Cha, G. S.; Meyerhoff, M. E.; Trojanowicz, M. Anal. Chem. 1990, 62, 2418-24. (11) Matuszewski, W.; Rosario, S. A.; Meyerhoff, M. E. Anal. Chem. 1991, 63, 1906-9. (12) Romette, J. L.; Cooney, C. L. Anal. Lett. 1987, 20, 1069-81.
3674 Analytical Chemistry, Vol. 69, No. 18, September 15, 1997
S0003-2700(97)00173-X CCC: $14.00
© 1997 American Chemical Society
of the O2 level in the reactor, whereas the H2O2 approach only requires the O2 to be above a specific threshold value. The main problem with the amperometric method involves the bias in the sensor signal due to the endogenous glutamate in the cell culture. To overcome this problem, the glutamate response at a second (glutamate) sensor is subtracted from the total current due to glutamate and glutamine at the glutamine sensor.2,16,18,21 Other approaches for the glutamate signal removal have used an external acetate anion exchange resin column,14 an immobilized glutamate dehydrogenase (GDH) reactor,17 or co-immobilization of glutamate oxidase and catalase on aminopropyl glass beads column.6b Villarta and co-workers13 have used a membrane containing glutamate oxidase and catalase to eliminate the signal caused by free glutamate in serum samples. A similar approach was selected for our work. It enables the measurement of glutamine in the presence of glutamate without the need for a second sensor or external removal of glutamate from the sample. The real advantage of this approach is that it allows the use of a second probe on the YSI 2700 biochemistry analyzer for the simultaneous measurement of glucose. Glucose is the major carbohydrate source for animal cell cultures; therefore, its monitoring and control is of highest interest in the biotechnology industry. EXPERIMENTAL SECTION Reagents. L-Glutamate oxidase (EC 1.4.3.11, from Streptomyces sp. X119-6, 6.8 units/mg) was from Yamasa Shoyu Co. (Chiba, Japan), glutaminase (EC 3.5.1.2, from Bacillus sp., 0.27 unit/mg) was from Applied Enzyme Technologies Ltd. (Leeds, UK), and catalase (EC 1.11.1.6, from Aspergillus niger, 1420 units/mg) was from Genzyme Diagnostics (Cambridge, MA). Glutaraldehyde (25% solution) was purchased from Polysciences Inc.(Warrington, PA). L-Glutamine (GLN, >99%), L-glutamic acid (GLU, min 99%) and D-(+)-cellobiose were purchased from Sigma Chemical (St. Louis, MO). Polycarbonate membranes were from Poretics Products (Livermore, CA). Phosphate buffer (PB) solution (100 mM, pH 7.2) was used for all the enzyme mixture preparations. Cellulose acetate (CA) sheets were prepared using a proprietary procedure. Caution: Handling of the 25% glutaraldehyde solution should be performed with necessary eye and hand protection equipment, since it is a potential hazard. Preparation of Enzyme Membrane Biosensors. The membrane “sandwich” for the glutamine detection consists of two enzyme layer films physically separated by a polycarbonate membrane (separator). The structure of this laminate is presented (13) Villarta, R. L.; Palleschi, G.; Suleiman, A.; Guilbault, G. G. Electroanalysis 1992, 4, 27-31. (14) Male, K. B.; Luong, J. H. T.; Tom, R.; Mercille, S. Enzyme Microb. Technol. 1993, 15, 26-32. (15) Schalkhammer, T.; Lobmaier, C.; Ecker, B.; Wakolbinger, W.; Kynclova, E.; Hawa, G.; Pittner, F. Sens. Actuators B 1994, 18-19, 587-91. (16) White, S. F.; Turner, A. P. F.; Bilitewski, U.; Bradley, J.; Schmid, R. D. Biosens. Bioelectron. 1995, 10, 543-51. (17) Huang, Y. L.; Khoo, S. B.; Yap, M. G. S. Anal. Lett. 1995, 28, 593-603. (18) Moser, I.; Jobst, G.; Aschauer, E.; Svasek, P.; Varahram, M.; Urban, G.; Zanin, V. A.; Tjoutrina, G. Y.; Zharikova, A. V.; Berezov, T. T. Biosens. Bioelectron. 1995, 10, 527-32. (19) White, S. F.; Turner, A. P. F.; Bilitewski, U.; Schmid, R. D.; Bradley, J. Anal. Chim. Acta 1994, 295, 243-51. (20) Renneberg, R.; Trott-Kriegeskorte, G.; Lietz, M.; Ja¨ger, V.; Pawlowa, M.; Kaiser, G.; Wollenberger, U.; Schubert, F.; Wagner, R.; Schmid, R. D.; Scheller, F. W. J. Biotechnol. 1991, 21, 173-86. (21) YSI Inc., Application Note 325, 1995. (22) Mulchandani, A.; Bassi, A. S. Biosens. Bioelectron. 1996, 11, 271-80.
Figure 1. Structure of the membrane “sandwich” (laminate). Table 1. Characteristics of Different Polycarbonate Membranes Used for the Laminate Structure Fabrication membrane code
pore diameter (Å)
pore density (cm-2)
thickness (µm)
nominal air flow (mL min-1 cm-2)
A B C D E
160-215 200-400 100-300 100-150 50-100
3 × 109 6 × 108 3 × 109 6 × 108 3 × 109
2 6 6 6 6
80-170 30-60 20-30 6-15 15-25
in Figure 1. The glutamine-sensitive layer (EL-2) was produced by a proprietary immobilization system with glutamate oxidase (GLO) and glutaminase (GMN). The final mixture was spread onto the surface of the cellulose acetate membrane. A piece of polycarbonate membrane (PC) was laid on the top of the enzyme mixture, which was then spread out using light pressure from the edge of a piece of stiff wax paper. The laminate was covered with absorbent tissue (Kimwipes) and rolled firmly using an ink roller. The laminate was then air-dried overnight. The “antiglutamate” layer (EL-1) was formed on top of the laminate by mixing the appropriate amounts of GLO and catalase (CAT) with a dilute solution of cellobiose (0.42% in PB). After complete homogenization, the necessary amount of a 1% glutaraldehyde solution was added to the mixture, the mixture was stirred for 1 min, and the final mixture was spread on top of the separator. The procedure was then completed as described above for EL-2. After the complete “sandwich” was dried for about 2 h in air, O-rings were glued onto the outer PC side using a cyanoacrylatebased glue (Permabond 101), and the membranes were punchedout using a sharpened metal tube cutter. The membranes were stored desiccated at 4 °C until used. Several PC membranes (AE) were tested as separators and outer membranes, and their characteristics are presented in Table 1. Amperometric Measurements. The enzyme membranes were mounted on the surface of a YSI hydrogen peroxide sensor, and the working Pt electrode was polarized at +0.7 V vs Ag/AgCl incorporated in the probe. Calibration of the sensors was performed with either 5 mM GLU (aqueous solution preserved with sodium benzoate and potassium EDTA) or 5 mM GLN (in BES buffer solution preserved with sodium benzoate and potassium EDTA) depending on the case. For linearity studies, 8 and 10 mM GLN and 10 mM GLU solutions have been used. All the solutions were kept at 4 °C when not in use. RESULTS AND DISCUSSION Optimization of the “Anti-Glutamate” Layer (EL-1). The first step in the design of the membrane “sandwich” consisted of Analytical Chemistry, Vol. 69, No. 18, September 15, 1997
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Figure 2. Effect of catalase (CAT) on the response of the glutamate membranes; 5 mM GLU was used as analyte, with a 40 µL sample size.
the evaluation of the optimum ratio of CAT to GLO in the outermost enzyme layer. A series of glutamate membranes (batch 1) with different ratios of CAT to GLO were fabricated, using CA as an inner membrane and C as an outer membrane. CAT catalyzes the decomposition of H2O2 formed in reaction 2, according to the following reaction: CAT
2H2O2 98 2H2O + O2
(3)
The effect of CAT on the response of the glutamate membranes is presented in Figure 2. The glutamate response (current) of different membranes containing CAT was normalized to the response of a glutamate membrane that does not contain any CAT. A ratio of activities CAT/GLO (units/units) of around 200 was found to be the best for the glutamate signal suppression. The activity numbers were those of the lyophilized preparations according to the manufacturers, and no attempt at estimating the remaining activities of the enzymes in the immobilized state was done. The response of the membranes was always recorded on day 1 (about 24 h) after the installation on the probes, unless otherwise mentioned. This way, the membranes have time to “break-in”, and the reproducibility of the measurements is improved. The membrane with the best GLU signal suppression was then studied for 4 days. The average plateau current (PL) for a 5 mM GLU sample (using a 40 µL sample) was always less than 0.1 nA, an encouraging sign that the GLU rejection could be maintained for a longer period of time. Selection of the Appropriate Separator Membrane(s). The purpose of the next step was to evaluate the efficiency of the GLU rejection in a complete two-layer structure (“two-layer” refers here to the enzyme layers), as depicted in Figure 1. A series of membranes (batch 2) were fabricated using the same EL-1 composition and the same outer membrane (E), and only the separator was different from one membrane to another. A ratio CAT/GLO ) 226 was used based on the above-described optimization study (batch 1) and other preliminary studies (not presented) that showed no improvement in the GLU rejection by increasing this ratio above approximately 225. The results in Figure 3 show that, by decreasing the permeability of the separator (represented by the nominal air flow values), the GLU rejection is improved. The GLU rejection is expressed as the percentage 3676 Analytical Chemistry, Vol. 69, No. 18, September 15, 1997
Figure 3. Influence of the separator on the GLU rejection (n ) 3 membranes of each type). The air flow values are the average values of the data from Table 1.
ratio of the plateau currents for 5 mM GLU and 5 mM GLN (GLU/ GLN, %). Each point in this figure is the average value for three membranes of each type. No clear trend could be observed for the response to GLN of various membranes from this batch, a fact which made us conclude that, probably, the overall diffusion rate for GLN is controlled by the external layers (EL-1 + outer membrane). In this particular experiment, the outer membrane was one with low flow (E), so it is reasonable to believe that the response to GLN is controlled to a higher degree by this outer membrane and not by the separator. However, the fact that the ratio GLU/GLN is influenced by the separator is probably caused by the diffusion of the unreacted GLU through the separator into the inner enzyme layer. If the separator is not restrictive enough, some GLU can pass through the EL-1 without reacting with GLO and give a signal due to its reaction in EL-2. From this experiment, we concluded that E or D membranes will be best suited as separator. Because the membrane with the highest flow (A) had clearly the worst behavior in rejecting GLU, no further studies using this particular PC membrane were performed. Effect of the Outer Membrane. A new set of membranes (batch 3) was fabricated, using the same EL compositions as for those described in the preceding section (batch 2), this time keeping the separator (D) identical for all membranes in the batch and changing the outer membrane. Again, by decreasing the permeability of the outer membrane, the GLU rejection is improved, as shown in Figure 4. Unfortunately, the fabrication of D/E (separator/outer membrane) was not successful for this batch. To confirm the trend observed, another series of membranes was fabricated, using similar enzyme layer composition and membranes (batch 4). In this batch, the fabrication of D/C was not successful. The GLU rejection dependence on the air flow of the outer membrane is also illustrated in Figure 4. Surprisingly, the improved rejection with decreased flow is not observed for D/D, the membrane with the lowest flow. However, replotting these data as a function of the pore size of the membranes (rather than the air flow), the trend is restored (Figure 5). This fact indicates that the pore size of the membrane is a major controlling factor of diffusion of the substrates into the layer. The diffusion of GLU and GLN takes place in the liquid-filled pores of the PC membranes. The nominal air flow values presented in Table 1 are measured using an idealized approach (flow of air through the pores) which only approximates the real behavior of these membranes once in contact with the sample.
Figure 4. Influence of the air flow of the outer membrane on the GLU rejection. Each point is the average of two tested membranes. Legend: [, batch 3; 9, batch 4.
Figure 5. Dependence of the GLU rejection on the pore size of the outer membrane. Each point is the average of two tested membranes. The pore diameter numbers are the average values of the data from Table 1. Same legend as in Figure 4.
Figure 6. Time dependence of the response of the glutamine membranes with GLU rejection layer; response to 5 mM GLN (40 µL sample size). Legend: [, D/B; 9, D/C; ×, D/D.
The membranes had initially lower sensitivity for GLN, and as the membranes are used in time, the sensitivity increases, as shown in Figure 6 for some typical membranes from batch 3. It was determined that a sample size of 40 µL will give minimum acceptable plateau currents (5 nA) for the YSI analyzer from day 1 for all the membranes, and this value was used for all subsequent tests, unless otherwise specified. GLU rejection of the membranes from batch 3 is presented in Figure 7. It is clear that the laminate having the higher flow (D/ B) becomes increasingly more responsive in time to GLU. The
Figure 7. Time dependence of the GLU rejection of the glutamine membranes. The GLU rejection is expressed as the percentage ratio of the plateau currents for 5 mM GLU and 5 mM GLN. Same legend as in Figure 6.
best GLU rejection was achieved in this batch by the combination D/D, which showed very good rejection of GLU (GLU/GLN < 5%) for up to 4 days. Very good rejection was also observed for D/C; however, this was rather an exception for this combination: efforts to reproduce this result in further batches were not successful. Linearity of D/D and D/C was very good. The membranes were linear up to at least 10 mM GLN, even after 5 days of testing. This extended linearity is caused by the improved diffusion control of glutamine in the thicker “two-layer” laminated structure. Comparing again the results from batches 3 and 4 presented in Figure 5, it is evident that, although the trend is the same for both batches, all the rejection values are worse in batch 4 in comparison with batch 3. This variability is probably caused by EL-1 preparation and made us suspect that this layer permeability contributes significantly to the overall transport resistance. If the layer is more “open”, a larger proportion of GLU can reach the EL-2 through the separator without reacting with GLO in EL-1. This behavior is enhanced by a more open outer membrane, as seen for D/B in Figures 4 and 7. Two more batches (batches 5 and 6) were fabricated to further evaluate the effect of the separator and the outer membrane. Although the reproducibility of the same type of laminate from batch to batch was only moderate, E/E- and D/D-type membranes were more consistent and provided acceptable GLU rejection. This fact helps to confirm that, by using more diffusion-restrictive membranes, the variability in the sensor response due to the preparation of the enzyme layers could be reduced. Effect of the Cross-Linking of the “Anti-Glutamate” Layer. Another experiment (batch 7) was designed to check the hypothesis that the glutamate rejection layer (EL-1) density is an important factor in the overall diffusion control behavior of the membrane. Two different configurations were used, D/D and D/B, and two different enzyme mixtures, one with the previously used amount of glutaraldehyde (normal, N) and one with an increased amount of cross-linking agent (high, H). The composition of EL-2 was identical for all the membranes in this batch. The extent of cross-linking of EL-1 was characterized by the crosslinking factor, f, which is the ratio of the amounts of glutaraldehyde and total protein (CAT + GLO) in the enzyme preparation, expressed in % weight. The normal values for f used in all the previous preparations were around 0.013-0.014. For this experiAnalytical Chemistry, Vol. 69, No. 18, September 15, 1997
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Table 2. Effect of Cross-Linking of the “Anti-Glutamate” Layer on the Membrane’s Sensitivity to Glutamine laminate structurea D/D-N
(nA)b
for 5 mM GLU PL PL (nA)b for 5 mM GLN GLU/GLN (%) a
D/D-H
D/B-N
D/B-H
first
second
first
second
first
second
first
second
0.20 5.00 4.0
0.17 4.83 3.5
0.14 3.35 4.2
0.11 1.53 7.2
0.43 6.80 6.3
0.75 9.28 8.1
0.41 5.36 7.6
0.37 4.63 8.0
Two membranes were tested from each combination (first and second). b Average plateau currents on day 1 (n ) 5 repetitive measurements).
here that the cross-linking study (batches 7 and 8) was done considering the actual activity of GLO as 4.5 units/mg (assayed), and not 6.8 units/mg as specified by the manufacturer (Yamasa Co.), the value used in the calculations for batches 1-6. This way, the actual ratio of activities CAT/GLO for batches 7 and 8 was 185, lower than the 225 value previously used. This fact may have caused a slight degradation in the elimination of the GLU signal for the last two batches.
Figure 8. Glutamine membrane’s response to a mixture 5 mM GLN + 5 mM GLU (50 µL sample size). Legend: [, D/E-N; 9, D/D-N; 2, D/E-L; ×, D/D-L.
ment, normal f ) 0.013, and high f ) 0.030. The results in Table 2 illustrate that the membranes produced with a larger amount of glutaraldehyde (H) showed lower sensitivity for GLN in comparison with those produced using normal amount of crosslinking agent (N), a result expected due to a more dense enzyme layer. The best GLU rejection was achieved as before with D/D and normal f. In conclusion, increasing the glutaraldehyde amount in the enzyme mixture did not improve the membrane’s performance. The next step was to try to lower the glutaraldehyde content, to see if the GLN sensitivity can be improved without compromising the selectivity against GLU. This batch (batch 8) was fabricated in three configurations: D/E, D/D, and D/B, and with two enzyme mixtures, the normal f ) 0.0134 and a low (L) f ) 0.0068. Comparing similar pairs of membranes, the only clear difference could be observed for D/B (highest flow outer membrane), which showed higher sensitivity for both GLU and GLN for a lower f, which was expected for a more permeable layer. No relevant differences could be observed for the other two configurations by comparing normal and low f pairs, confirming that lower flow outer membranes will control the overall diffusion rate of the analyte, and the density of the enzyme layer will be less important in this case. The membranes were also tested with a mixture of GLU + GLN, to see the extent of GLU interference in the GLN signal in an experiment closer to the operating conditions to be encountered in real cell culture samples. Figure 8 shows that D/E-N gives the lowest GLU interference (less than 6% positive bias for a 5 mM GLN sample containing 5 mM GLU). D/D-L has a similar (even better at the beginning) behavior, but slowly becomes more sensitive to GLU. It is important to mention
3678 Analytical Chemistry, Vol. 69, No. 18, September 15, 1997
CONCLUSIONS The ultimate goal of this project is the ability to measure GLN in the presence of GLU in complex samples (cell cultures). Consequently, it is important to look at the results in Figure 8 from this perspective. Luong and co-workers2 have mentioned that, in batchwise cultivation, GLN is normally added at a concentration between 0.7 and 5 mM. In perfusion, fed-batch, or glutamine-pulsed cultures, the glutamine level is kept at or slightly below 2 mM, to minimize ammonia production. From the literature, the levels of GLU to be encountered in mammalian cell cultures range between 0.07 and 0.7 mM,16,17,22 with up to 1 mM values mentioned by Luong et al.2,14 This fact indicates that the results in rejecting GLU obtained by us with the mixture 5 mM GLU + 5 mM GLN are likely to be improved in real samples, where the concentration ratio GLU:GLN is almost always lower than unity and, in many cases, even lower than 0.5. From the results obtained to date, the D/D-type membranes are considered the best in terms of reproducibility, sensitivity, linearity (up to at least 10 mM GLN), and stability of GLU rejection in time. The present specifications for the commercial GLN membranes (YSI No. 2735) indicate a precision of (4% (for the simultaneous measurement of GLU and GLN with two probes) and a lifetime of 5 days. Some of the membranes fabricated with the GLU rejection layer approach these specifications. Further optimization of the fabrication method presented in this work might provide a commercially attractive alternative to the differential measurement avenue. ACKNOWLEDGMENT M.B.M. thanks DukesNorth Carolina NSFsEngineering Research Center for the Fellowship that made possible a part of his work at YSI, Inc. Received for review February 11, 1997. Accepted July 2, 1997.X AC970173F X
Abstract published in Advance ACS Abstracts, August 15, 1997.
