Anal. Chem. 1982, 5 4 , 2247-2250
tention indexes in GLC and of several kinds of partition coefficients on the bask of these factors is rather good and could perhaps be used a5 a tool for several purposes. Theoretical approaches of the intermolecular energies involved in solutions could perhaps be improved on the basis of this empirical approach. ACKNOWLEDGMENT The authors acknowledge Jacques Adda (41),Kjell Doving (42),Ernest Polak (43),and Erik von Sydow (44)for providing some unusual pure subskmces used in this experimental study.
(25) (26) (27) (28)
LITERATURE CITED (1) Laffort, P.; Pane, F. J . Chromatogr. 1976, 726, 625-639. (2) Karger, B. L.; Snyder, I_.R.; Eon, C. J . Chromatogr. 1976, 125, 71-88 (3) Karger, B. L.; Snyder, L. R.; Eon, C. Anal. Chem. 1978, 5 0 , 2126-2136. (4) Dravnieks, A., Ilaffort, P. "Olfaction and Taste IV"; Schneider, D., Ed.; Wissens-Verlag-MBH: Istuttgart, Germany, 1972; pp 142-148. (5) Laffort, P.; Patte, F.; Elcheto M. Ann. N . Y . Acad. Scl. 1974, 237, 193-208. (6) McReynoids, W.O.,Ceianese Chem. Co., Bishop, TX, personal communication, 1970. (7) Rohrschneider, L. J . Chromatogr. 1986, 22, 6-22. (8) Claverie, P. Institut de Bioiogie Physico-chiniique, 13, rue Pierre et Marie Curie, 75005 Paris, France, personal communication, 1978. (9) Etcheto, M. dipi6me de i'Ecole Pratique des Hautes Etudes, Paris, France, 1975, 77 p. (10) Laffort, P. "Structure-Activity Relationships in Chemoreception"; Benz, G., et ai. Eds.; Information Retrieval Limited: London, 1976; pp 185-195. (11) McReynoids, W. 0. J . Chromatogr. Scl. 1970, 8 , 685-691. (12) Van Deemter, J. J.; Zwyderweg, F. J.; Klinkenberg, A. Chem. Eng. Scl. 1956, 5 , 271-289. (13) Purneii, J. H. J . Chem. Soc. 1960, 1268-1274. (14) Peterson, M. L.; Hirsch, J. J . Lipid. Res. 1959, 7 , 132-134. (15) Kovats, E. Chkn. Acta 1958, 41, 1915-1935!. (16) Weast, R. C., Ed. "Handbook of Chemistry and Physics", 50th ed.;The Chemical Rubber Co.: Cleveland, OH, 1969. (17) Hansch, C.; Giave, W. R. Mol. fharmacol. 1871, 7 , 337-354. (18) Hansch, C.; Lion, E. J.; Helmer, F. Arch. Biochem. Biophys. 1960, 128, 319-330
(29) (30) (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44)
2247
Leo, A.; Hansch, C.; Eikins, D. Chem. Rev. 1971, 71, 525-616. Leo, A.; Hansch, C.; Church, C. J . Med. Chem. 1969, 12, 766-771. Butler, J. A. V. Trans. Faraday Soc. 1937, 33, 229-238. McAullffe, C. Nature (London) 1963, 200, 1092-1093. Pascal, P.; Dupont, G. "Constantes physico-chlmiques. Techniques de I'Inggnieur"; Monteil, C., Ed.; Paris, 1955. Pierotti, G. J.; Deai, C. H.; Derr, E. L. Ind. Eng. Chem. 1959, 51, 95-102. Pierotti, G. J.; Deai, C. M.; Derr, E. L. 1958, N o . 5782, 159 p (addendum to ref 24). Seidell, A. "Solubilities of Organic ComDounds"; Van Nostrand, New York 1941; 926 p. Stephen, H.; Stephen, T. "Solubilities of Inorganic Compounds"; Pergamon Press: Oxford, 1963, Voi. I. "A.P.I., Selected Values of Properties of Hydrocarbons, No. 2: Vapor Pressure Data for Hydrocarbons"; American Petroleum Institute Division of Refinina Research. 1964. Droiect 44. Appeil, L. Am .-ferfum. Cosmet'.'1964, 79 (5), 29-41. Jordan, T. E. "Vapor Pressure of Organic Compounds"; Interscience: New York, 1954. Timmermans, J. "Physico-chemical constants of Pure Organic Compounds"; Eisevier: New York, 1950. Patte, F. Thesis, UniversitB Claude Bernard-Lyon I,69621 Villeurbanne, France, 1978; 171 p. Exner, 0. Collect. Czech. Chem. Commun. 1966, 31. 3222-3251. McReynoids, W. 0. "Gas chromatographic retention data"; Preston Technical Abstracts Co.: Evanston, IL, 1966; 335 p. Lenfant, G.; Chastrette, M.; Dubois, J. E. J . Chromatogr. Sd. 1971, 9 , 220-226. ChrBtien, J. R.; Dubois, J. E. J . Chromatogr. 1976, 126, 171-189. ChrBtien, J. R.; Dubois, J. E. Anal. Chem. 1977, 49, 747-756. ChrBtien, J. R.; Dubois, J. E. J . Chromatogr. 1976, 158, 43-56. Barton, A. F. M. Chem. Rev. 1975, 75, 731-753. TiJssen, R.; Biiiiet, H. A. H.; Schoenmakers, P. J. J . Chromatogr. 1976, 122, 185-203. Adda, J., INRA, Dijon, France, personal communication, 1976. Doving, K. B., Universitet IOslo, Blindern, Oslo 3, Norway, personal communication, 1973. Polak, E., EPHE-CENFAR, 92260 Fontenay-aux-Roses, France, personal communication, 1977. Von Sydow, E., Pharmacia AB, 75104, Uppsaia Sweden, personal communication, 1977.
RECEIVED for review December 7 , 1981. Resubmitted and Accepted June 14, 1982.
Measurement of Urinary Estriol by Immobilized Enzymes and Boxcar Chromatography Peter R. Johnson and Larry D. Bowers* Department of Laboratory ,Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455
A system Is described In which urlnary estrlol conjugates are hydrolyzed on-line with @-glucuronidaseimmobillzed on controlled pore glass. The chromatographlc analysis is accompllshed by using column switching to slmuitaneously separate the analyte of Interest frlom several samples. Urine samples were Injected dlrectly a1 a rate of 9 samples/h. The Immobilized enzyme reactor is the llmiting component with respect to the sample throughput rate. The parameters which llmlt thls rate are discussed, as are the potenlial llmltlng characteristics of the other system components;.
In many biological and pharmacological separation problems, sample preparation has become a major contributor to the time of analysis. Sample extraction, modification, andlor concentration are required not only to eliminate interferences and potentially disasterous degeneration of column performance by matrix components but also to preconcentrate trace level constituents for accurate quantification. In some cases,
the native material is either undetectable or difficult to chromatograph in the presence of the matrix, in which case chemical modification is mandated. The desire for rapid analysis of one or two compounds in a system which is inherently a multicomponent technique exacerbates these problems, since chromatographic speed occurs a t the cost of resolution. This in turn requires faster and better sample preparation. These problems have been the subject of a great deal of research. A number of techniques have recently addressed the problem of extraction and preconcentration in both "off-line" (1, 2) and "on-line" ( 3 , 4 ) modes. The "on-line" precolumn extraction approach has been reviewed by Frei (5). Preliminary sample preparation can therefore be simplified and the use of large volumes of solvents minimized. The more efficient use of HPLC columns in the analysis of a few components of a mixture has been addressed by Snyder and his colleagues with the development of "boxcar chromatography" (6, 7). We (8), and others (9-11), have recently begun to explore the usefulness of immobilized enzymes as an addition
0003-2700/82/0354-2247$01.25/00 1982 American Chemical Society
2248
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982 /
G
7I
waste
Schematic diagram of the coupled HPLC-immobilized enzyme system. Pump A1 supplies aqueous buffer required for enzyme hydrolysis while pump A2 supplles chromatographic eluent to the analytical column. The upper figure shows the system for a urine injection where the estriol produced by the IMER (C) is trapped on the precolumn (D) and eluent is directed to waste. When all estriol is trapped, valve E Is switched and the estriol is eluted onto the analytical column by the chromatographic eluent. When all estriol is transferred, valve E is returned to the initial position. Flgure 1.
to the chemical modification armamentarium (12) available for reversed-phase chromatography. Coupling of high-performance liquid chromatography and immobilized enzymes should be synergistic for a number of reasons. The group specificity of enzyme reactions provides additional selectivity for the modification and detection of a desired group of analytes. Some catalyzed reactions require a single reactant, the analyte, and thus immobilized enzymes could maintain the high system efficiencies associated with HPLC. In addition, there is no consumption of catalyst. The use of column purification before the immobilized enzyme reactor (IMER) could separate the analyte and any inhibitors present to allow more rapid and complete conversion of the analyte to product. There are, of course, limitations. For precolumn modification, the reaction must be stoichiometric since fractional conversion will result in multiple peaks for each component in the sample. The separation conditions must also be compatible with the enzyme characteristics such as p H optima, pressure sensitivity, and stability in organic solvents. The sample preparation of the metabolites of many drugs, hormones, and environmental toxins present in urine requires acid- or enzyme-catalyzed hydrolysis of glucuronide and sulfate conjugates prior to their measurement. We have reported earlier on a coupled (3-glucuronidaseIMER-HPLC system (8). In this study, the principles of column switching and “boxcar chromatography” (6)have been used to develop a rapid, direct analysis of glucuronide conjugates of estriol from urine. In the case of urinary estriol, a sample throughput rate of 9 samples/h was achieved.
EXPERIMENTAL SECTION Apparatus. The separations were carried out on the system illustrated in Figure 1. The solvent delivery system was a Perkin-Elmer Series 3 which supplied a 0.1 M phosphate buffer, pH 6.8, to the catalytic system components such as the IMER from pump 1 and the isocratic chromatographic eluent of methanol/water (57/43, v/v) from pump 2. Valves B and E were both standard six-port valves, Rheodyne 7125 and 7000, respectively. The precolumn (D) was a 50 x 4.6 mm column packed with Whatman CoPell C-18 using a modified bump and tap technique. The immobilized enzyme reactor (IMER, C) was a 50 X 2.1 mm column which was wet-slurry packed with p-glucuronidase immobilized on controlled pore glass (37-74 pm) (13). A 150 X 4.6 mm column packed in our laboratory with Nucleosil C-18 (7.5 pm) bonded phase packing was used as the analytical column (F). The fluorescence detector was a Kratos Model 970 (G). With the system in the configuration shown in the upper drawing, buffer passes through the IMER and precolumn and to waste. Under these conditions, pump 2 supplies mobile phase
directly to the analytical column. After a sample is injected, sufficient buffer is allowed to flow to assure that the analyte is hydrolyzed, eluted, and trapped on the precolumn. Valve E is then switched (lower drawing) and chromatographic mobile phase elutes the analyte from the precolumn onto the analytical column. Following a time interval sufficient to assure elution from the precolumn, valve E is repositioned to allow the precolumn to equilibratewith the buffer prior to introduction of the next sample. It should be noted that the next sample is injected before the analyte from the first sample is eluted from the analytical column. Thus, several sample aliquots occupy different regions of the analytical column simultaneously (6). System Characterization. The relationship between conversion of estriol conjugate and flow rate was studied by increasing the flow rate through the reactor system and trapping 10 mL of effluent on the precolumn. The estriol and estriol-16a-glucuronide were then chromatographed and the peak heights used to quantitate the fraction of estriol conjugate converted. Total recovery from the IMER was monitored by trapping the estriol from increasing volumes of effluent on the precolumn. Recovery was considered complete when the peak area was constant and equal to the area of an equimolar solution of estriol. Breakthrough volumes for the precolumn were measured by connecting it directly to the detector and injecting estriol into it. The breakthrough volume was defined as the volume of buffer where estriol was first observed in the effluent. Reagents and Standards. Water was deionized with a Milli Q system. Methanol was HPLC grade solvent. The K2HP04and KH2P04used for the buffer solution were HPLC grade. Estriol and estriol-16a-glucuronidewere obtained from Sigma. All other chemicals were reagent grade. Urine samples were obtained from the University of Minnesota Hospitals.
RESULTS AND DISCUSSION The use of selectivity of chromatography combined with the speed of analysis associated with automated analysis systems has long been a goal in bioanalytical chemistry. We have reported an on-line precolumn modification for reversed-phase chromatographic analysis of estrogens using immobilized (3-glucuronidase (8). Because of the poor organic solvent stability of the enzyme, a variety of manipulations were required which limited the speed of analysis. I t became obvious that increased sample throughput could be achieved if the enzyme hydrolysis and separation processes could occur in parallel rather than in series. Snyder, Dolan, and van der Wal (6) have introduced the technique of “boxcar chromatography” in which a small separating column is used to achieve a crude separation of the components of a mixture. All of the effluent is directed to waste, except for that portion which contains the compounds of interest. This is directed to the analytical column via stream switching. Since the sample now occupies only a small fraction of the analytical column, aliquots from subsequent samples can be coupled to the first, thus the separation of several samples is occurring simultaneously throughout the column. This results in substantial savings in analysis time since the entire column is utilized. We have extended this concept to a system using on-line sample modification and the use of a two-solvent system. Optimization of the IMER. One of the goals of our present studies was to achieve complete hydrolysis of urinary glucuronide conjugates in a single pass through a small column of immobilized enzyme. For a fixed bed IMER, the amount of product can be simplistically derived from the substrate rate-limiting form of the integrated Michaelis-Menten equation
Where [PI and [SI, are the product and initial substrate concentrations, respectively, [E] is the apparent enzyme
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982
L1
0
-2
2249
1 4 -5
3
Flowrate (mltmin)
Flgure 2. Conversion of estriol-l6a-glucuronicle to estriol by immobilized @-glucuronidaseas a function of flow rate. The percentage hydrolysis was calculated from both the decrease of the E16G peak
(0)and the increase of the estriol peak (0).
concentration, K , is the Michaelis constant, lzz is the rate constant of enzyme-substrate complex dissociation, F is the volumetric flow rate, and V , is the volume of the reactor. Adachi and his co-workers (14) have provided a more accurate assessment of the product concentration profiles with pulse sample introduction into an IMER. Nevertheless, in either case it can be shown that fractional hydrolysis of the conjugates can be adjusted for analytical purposes by the enzyme characteristics (Kmand K 2 ) ,the amount of enzyme ([E] and VJ, and the flow rate. The selection of the enzyme is important. In the case of p-glucuronidase, the enzyme derived from E. coli has its greatest reaction rate with estrogen glucuronides (15)while that, from P. vulgata is most catalytic for morphine conjugates (16). In the present study we have used only the E. coli enzyme, although those derived from other organisms can be readily immobilized. The K , of the enzyme is a function of the particular enzyme-substrate system involved and, in imlmobilized enzyme technology, of diffusion and the flow rate as well (17). For the enzyme used in these studies, the K , wider enzyme rate limitations was 40% of that measured under diffusion limiting conditions. Thus, under the conditions used for analysis, the rate of the enzyme reaction is significantly lower than that expected from solution phase experiments. In the present applic,ation,this decrease in rate is a liability. The amount of enzyme coupled to a solid support is a function of surface ama, immobilization site density, and immobilization chemistry (18). We have described the characteristics of @-glucuronidasebound to controlled pore glass (CPG) (13). It is of interest that we can achieve up to 10-fold greater loadings of enzyme on aminopropyl-CPG prepared in our llaboratory than on comimercially available derivatized CPG. Enzyme loadings of approximately 300 000 IU/g of support have been reproducibly achieved. Although the amount of enzyme bound is important, the observed reaction rate is a convolution of the catalytic rate and the rate of mass transfer i(17). For the @-glucuronidaseimmobilized on 37-74 pm particles used here, the enzyme-limiting rate is 2.5-fold greater than the diffusion-limited rate observed a t flow rates of 1 mL/min. Diffusion-limited reaction rates do have analytical advantages (17). For precolumn modification purposes, the reaction must be complete. Figure 2 illustrates the effect of flow rate on the conversion of estriol-16a-glucuronide to estriol. For the IMER tested, flow rates of greater than 1 mL/min resulted in incomplete hydrolysis. It should be noted that the hydrolysis must occur in about 6 s at the flow rate of 1 mL/min. In addition to its role in catalysis, @-glucuronidaseacts as a biospecific adsorbent for its substrates and/or products (19). The elution profile is extremely asymmetric ( a / b > 10). Preliminary studies into the analytical value of this biospecific adsorption indicated that neither inhibitors such as saccha-
Flgure 3. IMER-boxcar chromatography based analysis of urinary estriol. See text for discussion.
ro-1,4-lactone (20) nor substrates were selective eluents or effective peak shape modifiers. Since the catalytic buffer constitutes an extremely weak chromatographic eluent, the analyte band shape will be improved during adsorption onto the head of the precolumn. Nevertheless, it was necessary to flush sufficient buffer through the IMER to elute all of the product onto the precolumn. Studies with the reactor indicated that 6 mL (58 column volumes) were required to completely remove the estriol from the IMER. This fact, combined with the flow rate limitation noted above, determines the minimum time between injections. The adsorption also prevents the use of larger reactors (increased V,) since it would offset any advantage of decreased hydrolysis time. We have been unable to ascertain the effect of increased enzyme loading on the adsorption, but since both active and denatured enzyme bind the substrate and product, we infer that increased loading also increases biospecific adsorption. At present, the optimization process is empirical. Optimization of Precolumn Performance. The purpose of the precolumn in the present system is to trap the estriol in a narrow band on the head of the column while allowing the hydrophilic components of urine to elute to waste. The size of the precolumn is limited by two factors. I t must be large enough to provide resolution of the analyte from the matrix components. Greater enhancement of analysis rate, however, is obtained with smaller precolumns (7). In the present application, the precolumn must be only large enough to retain the estriol during the IMER wash step while allowing the hydrophilic components to elute. With a 4.6 X 50 mm precolumn, breakthrough of estriol required more than 12 mL (24 column volumes) of catalytic buffer. Since elution of the IMER required 6 mL, breakthrough was not a significant problem. The ability of the precolumn to direct a majority of the urinary interferences to waste is demonstrated in Figure 3. The precolumn also acts to negate any band broadening due to the IMER. In order to assure this narrowing, the analyte must be rapidly eluted from the precolumn after the eluent is switched from the catalytic buffer system to the chromatographic mobile phase. In the case of estriol, the analyte is eluted with less than 0.6 mL (about 1 column volume) of mobile phase. Measurement of the chromatographic plate count with and without the IMER in the system indicated no significant differences in system efficiency. The
2250
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982
precolumn therefore effectively removes any deleterious effects of the IMER. One final consideration is reequilibration of the precolumn since it is exposed to both buffer and the chromatographic mobile phase. Passage of one column volume of aqueous buffer was sufficient to obtain reproducible estriol retention. Optimization of System Performance. The data output of the system is shown in Figure 3. For reference purposes, a chromatogram from our previous work is shown in the inset. The total analysis time was 50 min, and the estriol peak is marked with an arrow. The lower traces show an equivalent amount of time using the switching system to emphasize the enhancement in throughput rate. The traces are from urine samples injected directly into the chromatographic system. Figure 3A shows the waste effluent from the precolumn, monitored a t 280 nm. Most of the water-soluble matrix components are directed from the precolumn to waste and thus never enter the analytical column. After 6 min, the precolumn is switched into the chromatographic eluent stream and the estriol is separated from interferences on the analytic column. Figure 3B shows the analytical column effluent using fluorescencedetection. The beginning of the trace corresponds to the time in trace A at which the switching took place. The precolumn is in the chromatographic eluent stream for 1min, after which it is returned to the catalytic eluent stream. While the separation of the first sample occurs in the analytical column, a second sample is injected and processed through the IMER and precolumn. Due to the 6 mL wash required by the IMER, only two samples occupy the analytical column at the same time (e.g., the "boxcar" concept). We are actively working to reduce this limitation and thus further increase sample throughput. With aqueous estriol standards, a throughput rate of 20 samples/h can be achieved, in which four sample fractions occupy the 15-cm analytical column simultaneously. Quantitation of Urinary Estriol. The use of on-line cleavage of the estriol glucuronidase should improve both the speed and precision of analysis. Since each specimen is handled identically, there was no need for addition of an internal standard. The coefficient of variation for repeated injections (n = 10) of aqueous standards was 0.9% at 25 mg/L. The linear dynamic range is 0.1-200 mg/L. Comparison of the concentration of estriol in 15 samples determined with the present method vs. those obtained with a manual hydrolysis-HPLC technique yielded a linear regression slope of 0.86 (=k0.09),an intercept of 3.75 (f1.70), a Syxof 3.2, and a correlation coefficient of 0.986. Another advantage of this system is that a high-pressure pump is not required to perfuse the reactor-precolumn system since the pressure drop is less than 10 bar. We have used a peristaltic pump for this purpose. No interferences were observed, since the "boxcar" approach maintains resolution while increasing throughput.
CONCLUSIONS We have extended the concept of "boxcar chromatography" (6, 7) to a system involving on-line sample preparation. Several factors complicate the present system relative to that described earlier (6). We have used two separate mobile phases for the hydrolytic and chromatographic systems. Only the precolumn is exposed to both solvent systems. This changes the role of the precolumn. We have used a "step gradient" to elute the analyte from the precolumn which results in no change in the chromatographic efficiency. This is acceptable only if the components of interest are easily resolved on the precolumn. Finally, the sample throughput of the present system is limited by the sample preparation manipulations rather than chromatographic resolution. The combination of on-line sample preparation and boxcar chromatography is syngergistic in that time-consuming sample cleanup steps can be performed while achieving increased rates of analysis with HPLC. LITERATURE CITED Hukkinen, R.; Fotsis, T.; Aldercreutz, H. Clin. Chem. ( Winston-Salem, N . C . ) 1981, 27, 1186-1189. Williams, R. C.; Viola, J. L. J. Chromatogr. 1979, 185, 505-513. Bannister, S. J.; van der Wal, Si.;Dolan, J. W.; Snyder, L. R. Clin. Chem. (Winston-Salem, N.C.) 1981, 27,849-855. Hux, R. A.; Mohammed, H. Y.; Cantwell, F. F. Anal. Chem. 1982, 54, 113-117. Frei, R. W. Anal. Proc. 1980, 17,519-524. Snyder, L. R.; Dolan, J. W.; van der Wal, Si. J. Chromatogr. 1981, 203, 3-17. Snyder, L. R.; van der Wai, SI.,personal communication, June 1981. Bowers, L. D.; Johnson, P. R. Clin. Chem., (Winston-Salem, N.C.) 1981, 27, 1554-1557. Okuyama, S.;Kokubun, N.; Higashidate, S.;Uemura, D.; Hirata, Y. Chem. Lett. 1979, 72,1443-1446. Ogren, L.; Csiky, I.;Risinger, L.; Niisson, L. G.; Johansson. G. Anal. Chim. Acta 1980, 117, 71-79. Arisue, K.; Ogawa, 2.;Hosotsubo, H.; Furukawa, I.; Kohda, K.; Hayashi, C.; Mivai. K.; Ishida, Y. Proc. Svmr,. . . Chem. Physiol. Pathol. 1979, 19,.133-137. Knapp, D. R. "Handbook of Analytical Derivatization Reactions"; Wiley: New York, 1979. Bowers, L. D.; Johnson, P. R. Biochim. Biophys. Acta 1981, 661, 100-1 05. Adachi, S.;Hashimoto, K.; Matsumo, R.; Nakanishi, K.; Kamikubo, T. Biotechmol. Bloeng. 1980, 22, 779-797. Graef, V.; Furuya, E.; Nlshikage, 0. Clin, Chem. ( Winston-Salem, N . C . ) 1977, 23, 532-535. Combie, J.; Blake, J. W.; Nugent, T. E.; Tobin, T. Clin. Chem. (Winston-Salem, N.C.) 1982, 26,83-86. Carr, P. W.; Bowers, L. D. "Immobilized Enzymes in Analytical and Clinical Chemistry"; Wiley: New York, 1981; Chapter 7. Carr, P. W.; Bowers, L. D. "Immobilized Enzymes in Analytical and Clinical Chemistry"; Wiley: New York, 1981; Chapter 4. Bowers, L. D.; Johnson, P. R. Anal. Biochem. 1981, 776,111-115. Roy, A. B. I n "Chemical and Biological Aspects of Steroid Conjugation": Bernstein. S., Soloman, S.,Eds.; Springer-Verlag: New York, 1970; pp 74-130.
RECEIVED for review June 25,1982. Accepted August 2,1982. The authors gratefully acknowledge financial support from the NIH (Grant No. RO1-GMS 26214-02).
