(4)R. G. Bates, "Determination of pH". 26 ed., John Wlley and Sons, New York, 1973,Chap. 4. (5)H. S.Harned and R. W. Ehlers, J. Am. Chem. Soc.. 54, 1350 (1932);55, 2179 (1933). (6)R. G.Bates and V. E. Bower, J. Res. Natl. Bur. Stand., 53, 283 (1954).
(17)Reference 4,Chap. IO. (18)R. G.Bates, E. A. Guggenheim, H. S. Harned, D. J. G. Ives, G. J. Janz, C. B. Monk, J. E. Prue, R. A. Robinson, R. H. Stokes, and W. F. K. WynneJones, J. Chem. Phys., 25,361 (1956);26,222 (1957). (19) R. Gary, In "Electrochemical Analysis: Studies of Acids, Bases, and Salts
(7) L. G. Sillen, in "Equilibrium Concepts in Natural Water Systems", W.
by Emf, Conductance, Optical, and Kinetic Methods", R. G. Bates, Ed., NaN.
Stumm, Ed., Adv. Chem. Ser., 67,45-56 (1967). (8)D. Dryssen and L. G. SillBn, Tellus, 19, 113 (1967). (9) P. J. Reilly, R. H. Wood, and R. A. Robinson, J. Phys. Chem., 75, 1305 (1971). (IO) R. A. Robinson and R. H. Wood, J. Solution Chem., 1, 481 (1972). (11)K. S.Pltzer, J. Phys. Chem., 77,268(1973). (12)M. Whitfield, In "Chemical Oceanography", J. P. Riley and G. Skirrow, Ed., 2d ed., Vol. 1, Academic Press, New York, 1975,pp 43-171. (13) I. Hansson, S.Ahrland, R. G. Bates, G. Biedermann, D. Dyrssen, E. Hogfeldt,
Bur. Stand. (US.) Tech. Note, 271, 28 (1965). (20)R. A. Robinson, J. Mar. Biol. Assoc. U.K., 33, 449 (1954). (21)J. B. Macaskill, unpublished measurements, University of Florida, 1976. (22)C. Culberson, R. M. Pytkowlcz, and J. E. Hawley, J. Mar. Res., 28, 15 (1970). (23)D. Dyrssen and I. Hansson, Mar. Chem., 1, 137 (1973). (24) I. Hansson, Deep-sea Res., 20, 479 (1973). (25) G. Bledermann, in "The Nature of Seawater", E. D. Goldberg. Ed., "Physical and Chemical Research Rpt. l", Dahlem Konferenzen, Berlin, 1975,pp 339-362.
A. E. Martell, J. J. Morgan, P. W. Schlndler, T. B. Warner, and M. Whitfield, in "The Nature of Seawater", E. D. Goldberg, Ed., "Physical and Chemical Research Rpt. l", Dahlem Konferenzen, Berlin, 1975,pp 263-280. (14)D. R. Kester, Thesis, Oregon State University, 1970. (15)D. R. Kester and R. M. Pytkowlcz, Geochim. Cosmochim.Acta, 34, 1039
(1970). (16)W. C. Somerville and A. D. Campbell, Mikrochim. Acta, 991 (1963).
RECEIVEDfor review August 18,1976. Accepted September 20, 1976. This work was supported in part by the National Science Foundation under Grant DES75 03635.
Bioassay of Nystatin Bulk Material by Flow Microcalorimetry Anthony E. Beezer," Roger D. Newell,' and H. J. Valentine Tyrrell Chemistry Department, Chelsea College, Manresa Road, London S W3 6LX, England
A flow mlcrocalorimetrlc procedure has baen developed for the bloassay of nystatin. The antlblotic is assayed by monitoring thb effect of the antibiotic on a respiring culture of Saccharomyces cerevisiae. The yeast inocula are recovered for use from storage in liquld nitrogen. When compared wlth the classlcal agar plate dlffuslon method, the microcalorimetrlc procedure is more reproducible (f3.5% compared with f5-10%), more sensitive (0.5 unit mi-' compared with 20 units ml-'), more rapld ( 1 h compared wlth 16 h per sample) and, In addition, is simple and capable of automation.
Microcalorimetric methods of analysis have been extensively reviewed recently (1--4), particularly with respect to biochemical systems. However, the potential of analytical applications of microcalorimetry in the microbiological area has yet to be fully realized. Reports exist of several successful investigations into the enumeration of viable bacteria by microcalorimetry including diaghosis of bacteriuria (5); investigation of bacterial contamination of stored milk (6); identification of bacterial contamination of vacuum packed food (7);preparation, storage, and assay of frozen inocula (8). In addition, evidence has been produced to demonstrate that microcalorimetric methods can be used in the identification of bacteria and yeasts by virtue of the complex but reproducible thermograms obtained from growth in complex media as foreshadowed by Rubner in 1906 (9) and later by Prat (10). The modern microcalorimetric technique (for a review see (11)) has been used to study the family Enterobacteriacea (12, 13), and the thermograms of 200 clinically significant microorganisms have been reviewed (14). However, no reports exist on the analytical use of the microcalorimetric response of a microorganism to some applied interacting agent, e.g., antibiotic, detergent, etc. It has been shown (10) that addition of streptomycin to growing cultures of Escherichia coli reduced the heat output by about a half within a period of 2 h. Further qualitative results on this system have been summarized by Present address, Department of Oral Medicine and Pathology, Guy's Hospital Medical School, London SE19RT,England. 34
ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
Calvet and Prat (15).More recently, it has been shown that the addition of ampicillin to a growing culture of Escherichia coli in a complex medium significantly affects the thermogram derived from a flow microcalorimetric experiment within 1 h (16).These reports suggest the possibility of an analytical bioassay procedure, based upon microcalorimetry, for antibiotics. However, any technique based upon flow microcalorimetry would have to demonstrate significant advantages over the current well-established procedures. Factors of importance are reproducibility, precision, simplicity, and the speed of assay. A further possibility is that of automation, inherent in any flow procedure. The heterogeneous group of antibiotics known as the polyenes is characterized by a macrolide ring containing a number of conjugated double bonds (from 3-7) and a varying number of hydroxyl groups (17). Both these features are essential for biological activity. These antibiotics are active against fungi, particularly yeast forms and eukaryotic organisms, but are not generally active against prokaryotic cells (this distinguishes the polyene macrolides from other macrolide antibiotics). Many polyenes have been isolated, but the majority are too toxic for clinical use and only nystatin, amphotericin B, candidin, and hamycin have found useful clinical application. The polyenes are believed to bind with the sterol present in the membranes of sensitive cells (18) leading to the formation of pores (19, 20). The subsequent death of the microorganism is preceded by the leakage of cellular material. The extent of membrane disruption is dependent on the specific polyene antibiotic, its concentration, the preparation conditions of the inoculum and the experimental conditions used. The material leaked from the cell ranges from simple constituents of small size, such as K+ and NH4+ (21)to larger, more complex molecules such as nucleotides and proteins (22). The overall effect of the addition of antibiotic to a yeast inoculum was felt likely to be more rapid than that which would be observed with other groups of antibiotics such as those which, for example, interfere with protein synthesis (23).The polyenes were, therefore, regarded as particularly suitable for a detailed investigation of antibiotic assay by flow microcalorimetry. The degenerate heptaene nystatin (a tetraene
Oxygenated im-
Heot wtplt
rate
l v I
OF
1
0
10
20
30
40 50 Time (minutes)
r eimnutes1 Figure 2. Typical thermograms obtained under assay conditions
Figure 1. Thermograms of respiring yeast cells in glucose buffer
linked by a methylene bridge to a diene) was used in this study. The existence of standard liquid nitrogen frozen inocula of Saccharomyces cerevisiae (8) guaranteed the reproducibility of the responsive organism in the flow microcalorimetric experiment.
EXPERIMENTAL Inocula. Frozen inocula of Saccharomyces cerevisiae (NCYC 239) were prepared, stored, and assayed as previously described (8). Nystatin Solutions. Nystatin bulk material (E. R. Squibb and Sons Ltd.) was stored in a desiccator at 260 K until required. Weighed portions of nystatin were dissolved in DMF and made up to the required concentration to be applied by addition of buffer solution. The final concentration of DMF in all solutions was 0.3%. Buffer Solution. Nystatin has been reported (24) to have pK,'s of -2.6 and -9.7. Preliminary experiments showed that the buffer system used in the official procedure (potassium phosphate buffer system pH 6) was unsatisfactory in the calorimetric procedure. Indeed, accounts of respiration of yeast cells in buffered glucose (21) after treatment with polyene antibiotics indicate that addition of "I+, K+, or Rb+ at pH's above 5.8 may reduce the effects of the antibiotic. At pH's 55.5, glycolysis and respiration cannot be reestablished by addition of these monovalent cations since there is an internal acidification of the cell (25). Experimentally a buffer pH of 4.5 yielded good assay results. The buffer finally chosen was therefore potassium hydrogen phthalate (0.05 M)/sodium hydroxide (0.01 MI. Calorimeter. The calorimeter used in this work was the LKB Flow Microcalorimeter (LKB type 10700-1,LKB Produkter AB, S-16125 Bromma 1, Sweden) designed by Monk and Wadso (26). This heat leak instrument employs two calorimetric cells operated within a constant temperature air bath. The calorimeter was operated in the flow through mode (26,27)at 298 K in a room maintained at 298 f 0.5 K. Procedure. Yeast Incubation i n Calorimeter. For a typical calorimetric incubation glucose buffer (10 mM glucose) is passed through the calorimeter cell (52 ml h-1) to establish a steady baseline deflection using an amplifier sensitivity equivalent to 10 p V for full scale deflection on a recorder (Phillips PM 8000). This baseline is recorded on the chart recorder and for 500 s on a numerical data recording system (printer driver unit, LKB 10758; digital voltmeter, Systemteknik,DPM-S-1016and printer, Addo-X, Model 13-0353-00). From the initiation of the printer driver recording, the following operations took place to a strict timetable. The liquid nitrogen stored ampoule was thawed at 313 K for 3 min. The yeast suspension (1ml) was inoculated into glucose buffer (50 ml) 2 rnin after the completion of thawing; 2 min later the nystatin solution (or buffer-DMF control, 1 ml) was added to the reaction medium; and 30 s after this addition, the inlet tube to the calorimeter was inserted into the incubation. Anaerobic incubation conditions were achieved by flushing the incubation vessel with nitrogen prior to inoculation and during the incubation. The flowing incubation medium reached the calorimeter chamber 2 min, 55 s after the inlet tube insertion. At this instant the thermogram was commenced; the recording of the baseline having been terminated at some convenient point after the elapse of 500 s from the initiation of thawing. The effluent from the calorimeter was recycled into the incubation vessel 8 min 45 s after the inoculation. The results of control incubations are as shown in Figure 1. The thermograms resulting from nystatin assay experiments are as described by Figure 2.
RESULTS AND DISCUSSION Thermograms obtained from yeast inocula respiring in buffered glucose varied with the oxygen tension within the calorimetric cell. The thermograms derived from nitrogen gas saturated incubations correspond to simple zero-order kinetic processes. The departure from zero-order type thermograms is small if the yeast suspension is simply stirred with its surface in contact with air in the incubation vessel. In the latter case, any air present is effectively removed by the yeast before the flowing suspension reaches the calorimeter cell itself. Under these conditions, the heat output rate becomes independent of time within 10 rnin of the observation of the first thermal response. Addition of small quantities of nystatin normally gave a curve of type b, Figure 2 , where the heat output rate initially exceeded that of the control but eventually fell below it. This initial excess heat output rate is probably due to the easier access of glucose substrate through pores (19, 20), formed by the interaction of the membrane with nystatin, leading to a small, temporary increase in the rate of metabolism. At higher concentrations of nystatin this phenomenon is not seen, probably because the system has passed through this phase before the flowing suspension has reached the calorimetric cell. In all subsequent work, anaerobic conditions were maintained by saturation of medium and fermentor with nitrogen gas. There is clearly a relationship between drug dose and the rate of decay of the metabolic heat output rate, but it is not obvious how the form of such a relationship could be established. The decay curves shown in Figure 2 do not fit t o a simple first- or second-order kinetics; consequently it is not practicable to extract from the curves a single rate parameter which could be related t o drug dose. Several other possible parameters were tested including the total area beneath the decay curve. A suitable parameter should show good discrimination between doses of close but not identical concentration and depend in a simple manner, e.g., linearly, on some simple function of the dose. After some exploration of possible measures the parameter finally selected was the time required from the moment (To)a t which the first calorimetric response is detected for the signal to rise and fall to some arbitrary level ( x % ) over the baseline, i.e., to B,. The response parameter can also be related to a % reduction in heat output rate from the level of the control respiring cells although this is essentially the same as the baseline dependent parameter. The baseline dependent parameter in addition t o proving analytically satisfactory, has the advantage of being recorded automatically in a simple fashion €allowing any automation of the described procedure. The measurement of time from To to some point B, permits many measurements t o be recorded for each sample, whereas in the classical agar plate diffusion technique only one measurement is made-namely that of the diameter of the final ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
35
‘‘*I
Table I. Sensitivity Range toward Nystatin of Yeast Cells of Varying Age of Preparation Age of cells, h
Sensitive range, units ml-l
4 6 8 12
0.5- 5 1.5-10 3 -15 10 -75
OB-
Table 11. Assay Results for Nystatin Bulk Material and Heat Treated Nystatin Samples
06-
Potency, units ml-1 R w c mI d
Flgure 3. Plot of a representativeset of data for the microcalorimetric bioassay of nystatin (8-h cell preparation: cell density 8.4 X lo6 cells
rn1-l)
inhibition zone. This is the pattern expected when comparing a physical chemical means of end-point detection with a purely chemical (e.g., indicator) method. There are many possible measurements of “time to B,” parameters for each assay experiment. The standard deviations of such parameters are f2.7% for the control respiration and, at worst, f 3 . 5 %for a nystatin assay experiment. Figure 3 shows a representative set of data based on this parameter. Figure 3 shows that the linear relationship between log dose and response usually found in diffusion assays (29) is followed here. The concentrations of nystatin described by the figure are based upon the potency of the bulk material as measured in the classical agar plate diffusion procedure. It is apparent from Figure 3 that the Bzo parameter deviates from linearity at the low concentration end of the range and is thus not satisfactory. The parameters involving useful antibiotic concentration ranges and convenient, relatively rapid response times are the B30 and B40 parameters. The method was initially tested by determination of different nystatin concentrations using two different procedures. (i) High-Low extrapolation. The relationship between log dose and response is linear over a reasonable range of nystatin concentrations. Therefore “high” and “low” standard solutions were prepared and tested. Intermediate “unknowns” were assayed by interpolation from the assumed linear relationship between response and log dose for the “high” and “low” controls. (ii) These same intermediate concentrations were also determined on the basis of the standard logarithmic calibration curve referred to earlier. Both procedures yielded accurate and precise results. However, the “high-low” extrapolation procedure is more flexible, being applicable throughout a wide range of nystatin concentrations. Similar analytical results were obtained with cell preparations of differing incubation age prior to freezing. The range of useful sensitivity toward nystatin is shown in Table I. As can be seen from Table I, yeast cells are more susceptible to nystatin, as the preparation age decreases. Yeast cells of low preparation age are, however, less able to survive the freezing process (recovered viabilities; 12-h cells, 94%; 6-h cells, 63%). A change in the cell wall with age, namely a more tightly cross-linked wall structure has been suggested (28)to explain the decreased sensitivity of Candida a1bicans toward the modified polyene antibiotic amphotericin B methyl ester. Parallel assays were performed by both the calorimetric procedure and by the classical agar plate diffusion procedure tests (8 X 8 latin square, 2 2 cavity type (29));performed by E. R. Squibb & Sons Ltd. on the same bulk nystatin ma-
+
36
ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
Microcal.
Material
assay
Microcal.
Std dev, %
D1548 D 1770 HTS l b HTS 2 HTS 3 HTS 5
4160 4899 5700 5079 4594 4180
4217 5157 5765 5284 4609 4626
2.5 1.2 1.3 2.8 3.5 1.1
Plate
%
diff” +1.4 +5.3 +1.1 +4.0 0 f9.6
Represents the % difference between the plate assay result (taken as standard) and the microcalorimetric result. HTS = Heat Treated Samples.
terial. The results are as in Table 11. Both techniques used high-low extrapolation procedures; the calorimetric technique being useful in the range 3-40 units ml-l (although indications are that this range may be extended to 0.5-100 units ml-1 by suitable choice of cell preparation age), whereas the high and low standards in the diffusion procedure are 80 and 20 units ml-l, respectively. In addition to results on two different production batches of nystatin, Table I1 contains comparative assay data on four heat treated samples of nystatin. These heat treated samples are of considerable theoretical and practical importance. Nystatin is not a homogeneous material, being composed of at least two fractions designated AI and A2 plus a heptaene, nursemycin (30, 31). (There may be up to 6 components present in bulk nystatin). The heat treated samples may give information about stability and composition. Nystatin AI is the least stable component and the composition of nystatin will alter upon heating. The agar plate diffusion method has a reproducibility of a t best f 5 % , and f10% is frequently quoted (see for example Ref. 32). Thus compatible results were obtained if the values derived from each method are within the sum of the standard deviations of the individual measurements. The calorimetric and diffusion methods give such agreement in all instances, except for the heat treated sample No. 5 . The calorimetric potencies are, in the main, greater than those determined classically. The methodogical differences between the two methods are extreme and may be an explanation. The classical technique employs a solution of nystatin in 10%aqueous DMF added to the well in the agar plate. The inhibition zone measured in the growing yeast is produced by diffusion through the gel of nystatin from a micellar system. In contrast the microcalorimetric technique employs a homogeneous aqueous suspension containing only 0.3% DMF acting on glycolyzing cells. The relative concentrations of DMF required are a function of the sensitivities of the two techniques (minimum assayable concentrations of nystatin are 0.5 unit ml-l calorimetrically and 20 units ml-1 by agar plate diffusion). The increased sensitivity of the microcalorimetric bioassay,
although not important for the routine assay of bulk nystatin material, has obvious potential in the assay of nystatin (and other polyenes) in other situations, for example, body fluids, and in metabolic studies. The greatly improved reproducibility is clearly also an advantage in this context. The time of about 1h required for an individual assay may be compared with the 16 h period required for the agar plate diffusion assay. I t is evident that a large number of determinations can be carried out simultaneously using plate assays and its total throughput of determinations is greater than would be possible with present calorimeter designs. In principle a calorimeter having more than the current two flow chambers could be constructed, and a multichannel instrument of this kind coupled with a suitable data handling system would be able to process samples more rapidly. T h e advantage of this microcalorimetric technique in respect of reproducibility (f3.5% compared with f5-10%) and sensitivity (0.1 unit ml-1 compared with 20 units m1-I) could then be used more fully. The details of the method used could conceivably be improved since in the current investigation we have not been able to explore fully the affect of the many variables characteristic of such a complex biological system and thereby to optimize the operating conditions. It is, however, a viable assay technique with some distinct advantages and capable of further development. A subsequent paper will deal with the microcalorimetric bioassay of formulations of nystatin and the possible potentiation of nystatin potency by addition of another antibiotic.
ACKNOWLEDGMENT We thank E. R. Squibb and Sons Ltd. for the gifts of materials and Messrs. Ridgway, Fairbrother, Forster, and Cosgrove of E. R. Squibb for helpful discussions and for performing agar plate diffusion experiments referred to in the text. We are most grateful to R. P. Lipscombe for technical assistance. LITERATURE CITED (1)T. H. Benzinger, in "A Laboratory Manual of Analytical Methods of Protein Chemistry", P. Alexander and H. P. Lundgren, Ed., Pergarnon Press, Oxford,
1969.
(2)A. E. Beezer, in "MTP International Review of Science, Phys. Chem. Series l", Vol. 13, T. S. West, Ed., Butterworth and University Park Press, 1973. (3) R. N. Goldberg and G. T. Armstrong, Med. instrum., 8 , 30 (1974). (4)C. Spink and I. Wadso, in "Methods in Biochemical Analysis", Vol. 23,D. Glick, Ed., Wiley-lnterscience. New York, 1975. (5) A. E. Beezer, K. A. Bettelheim, R. D. Newell, and J. Stevens, Sci. Tools, 21, 13 (1974). ( 6 ) A. J. Cliffe, C. H. McKinnon, and N. J. Berridge, J. SOC.Dairy Techno/.,28,
209 (1973). (7)R. A. Larnpi, D. A. Mikelson, D. R. Rowley, J. J. Previte, and R. E. Wells, Food Technoi. Champaign, 28,52 (1974) (8) A. E. Beezer, R. D. Newell, and H. J. V. Tyrreii, J. Appl. Bacterioi., in press. (9)M. Rubner, Ark. Hygiene Bakteriol., 57, 193 (1906);57, 244 (1906). (IO) H. Prat, Rev. Can. Biol., 12, 19 (1953). (11) R. D. Newell, Ph.D. thesis, University of London, 1975. (12)B. R. Staples, E. J. Prosen and R. N. Goldberg. Nat. Bur. Stand. (US.)Rep.,
NBSlR 73-181 (1973). (13) E. A. Boling, G. C. Blanchard, and W. J. Russell, Nafure 241, 472
(1973). (14)W. J. Russell, S. R. Farling, G. C. Blanchard, and E. A. Boling, in "Mlcrobiology-l975", D. Schlessinger, Ed., American Society Microbiology, Washington, D.C., 1975. (15) E. Calvet and H. Prat. in "Recent Progress in Microcalorimetry", translated by H. A. Skinner, Ed., Pergamon Press, Oxford, 1963. (16) S. Delin, P. Monk, and I. Wadso, Sci. Tools, 16, 12 (1969). (17)J. M. T. Hamilton-Miller, BacferiolRev., 37, 166 (1973). (18)J. 0. Lampen, P. M. Arnow, 2. Borowski, and A. I.Laskin, J. Bacteriol.. 84,
1152 (1962). (19)B. de Kruyff and R . A. Demel, Biochim. Biophys. Acta, 339, 57 (1974). (20)A. Finkelstein and R. Hoiz, in "Membranes, Lipid Bilayers and Antibiotics", G. Eisenman, Ed., Vol. 2,Dekker, New York, 1973. (21)F. Marini, P. Arnow, and J. 0. Lampen, J. Gen. Microbioi., 24, 51 (1961). (22)0. D. Sutton, P. M. Arnow, and J. 0. Lampen, Proc. SOC.Exp. Biol. Med., 108, 170 (1961). (23)E. F. Gale, E. Cundliffe, P. E. Reynolds, M. H. Richmond and M. J. Waring, "The Molecular Basis of Antibiotic Action", J. Wiley and Sons, London,
1972. .~ (24)E. D. Etingov, E. L. Blashko, and N. I. Etingova, Anfibiotiki, 22, 678 (1974). (25)J. 0. Lampen, Am. J. Clin. Pafhol., 52, 138 (1969). (26) P. Monk and I. Wadso. Acta Chem. Scand., 23, 29 (1969). (27)A. E. Beezer and H. J. V. Tyrrell, Sci. Tools, 19, 13 (1972). (28)E. F. Gale, A. M. Johnson, D. Kerridge and T. Y. Koh, J. Gen. Microbiol., 87,20 (1975). (29)F. Kavanagh, Ed., "Analytical Microbiology", Academic Press, New York, 1963. (30) Y. Shenin, T. V. Kotenko, and 0. N. Exzernpiyarov. Anfibiotiki, 13, 387 (1968). (31)W. Mechiinski and C. P. Schaffner, J. Chromatogr., 99, 619 (1974). (32)J. W. Lightbrown, M. Kogut, and K. Vemura, Bull. W. H. 0. 29, 87 (1963).
RECEIVEDfor review July 9, 1976. Accepted September 21, 1976. One of us (R.D.N.) thanks E. R. Squibb and the Governors of Chelsea College for the award of a research studentship.
ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
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