Literntiire Cited
F a w , L. Arch. Met. Ge0ph.w. Biokhn. 8, 229 (1955); Conzpte Rend. Acad. Sci. Paris 246, (102), 3161 (1958). Hatch, T. F., Gross, P., “Pulmonary Deposition and Retention of Inhaled Aerosols,” Academic Press, New York, N.Y.. 1964. Levich, V. G., “Physicochemical Hydrodynamics,” p. 115, Prentice-Hall, Englewood Cliffs, N.J., 1962. Seeley, L. E.3 469 377 Waldmann, L., Schmitt, K., in “Aerosol Science,” C. N. Davies, Ed., Academic Press, New York, N.Y., 1966. Weibel. E.. “Morohometrv of the Human Lung.” D. 68. Springer-Verlag, ‘Berlin, i963.
Best, C., Taylor, N., “The Physiological Basis of Medical Practice,” 7th ed., Sect. 111, Williams & Wilkins, Baltimore, Md., 1961. Brock, J. R., J . Colloid Interface Sci. 27, 95 (1968). Davies, C. N., in Inhaled Particles and Vapours, Vol. 11, p. xii, Pergamon Press, London, 1967. Derjaguin, B. V., Dukhin, S. S., Dokladj, Akad Nauk S S S R 111, 613 (1956).
Received NoGember 20, 1968. Accepted April 24, 1969. Presented at the Symposium on Surface Chemistry o f Air and Water Pollution, Dicision of Colloid and Surface Chemistry, 156th Meeting, ACS, Atlantic City, N.J., September 1968. The work was supported by the National Science Foundation through its contract with the National Center for Atmospheric Research, and by the US.Public Health Service, Grant No. AP 00479-03.
appreciable compared with Brownian diffusion only for 0.1micron or larger particles inhaled during periods of heavy exercise. Here again, however, deposition of particles exceeding about 1 micron in radius present in the alveoli will be controlled by gravitational settling, which is much stronger than diffusiophoresis for such particles. It is likely that diffusiophoresis proceeds towards the alveolar walls because of the stronger influence of oxygen diffusion on the particles.
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I
END OF SYMPOSIUM
Blue Tetrazolium Reduction by Whole Tobacco Smoke and Gas Phase Components Miasnig Hagopian Mason Research Institute. 21 Harvard Street, Worcester, Mass. 01608
rn A simple colorimetric method has been developed and applied to monitor tobacco smoke effluents. This sensitive method is based on the reduction of blue tetrazolium by constituents distributed throughout the gas phase and particulate phase of smoke. Data are presented to show the yield of blue tetrazolium reducing substances trapped from mainstream smoke of various commercially available cigarettes smoked by machine under predetermined, reproducible conditions. On an equivolume basis, the reducing power in the particulate phase was approximately ten times that of the gas phase for most of the brands tested. For those brands with filter tips, the total level and the proportion of blue tetrazolium-reducing substances in both phases was a very useful measure of filtration efficiency and selectivity. The applicability of this method in related areas is discussed.
D
uring studies involving the evaluation of cigarette smoking machines and animal exposure chambers, a reliable analytical method was required to monitor fresh tobacco smoke effluents. Many techniques are available for measuring individual components or groups of components of the complex tobacco smoke mixture (Wynder and Hoffmann, 1967). However, no reasonably simple, rapid, and reproducible method has previously been described which could be applied to detect and measure a family of constituents representative of a cross section of the total gaseous and particulate material in mainstream tobscco smoke. Many of the organic constituents of cigarette smoke are products of pyrolysis, pyrosynthesis, and incomplete oxidation of tobacco plus various additives. Among these are phenols, ketones, aldehydes, and polyhydroxylic compounds, a number of which are reducing substances. The simultaneous determination of such smoke components would, in effect, permit
relative estimates of smoke yields from various tobacco products smoked under various conditions. After considering commonly used reagents for the detection of reducing substances, blue tetrazolium (BT) was chosen because of its simplicity, sensitivity, and general applicability to this problem. The application of tetrazolium reagents to quantitative measurements have been limited largely to the analyses of alpha-ketols. That tetrazolium salts are also reduced by a variety of nonketolic substances have been shown by Weiner (1948), Rosenkrantz (1959), and Salim, Manni et al. (1964). This report presents the results of a typical application of this method to evaluate differences in the content of BT-reducing substances in the smoke effluent of commercially available cigarettes. Experimental Apparatus. A filamatic automatic filling unit (Model AB) fitted with a 50-ml. syringe and a variable interval timer (National Instrument Co., Inc.) was used as the source of suction for smoking the cigarettes (O’Keeffe and Lieser, 1958). The smoke particulates were trapped on a Cambridge CM-113 filter pad in a lucite holder (Phipps and Bird, Inc.), and the gas phase was in a Midget Impinger bubbler trap (Gelman Instrument Co.). These units were connected in series, and the entire cigarette smoking assembly was essentially the same as that described by Wartman, Cogbill, et al. (1959). A Coleman Jr. spectrophotometer was used for absorbance measurements in 10 X 75 mm. borosilicate glass cuvettes. Reagents. Blue tetrazolium reagent solution-the (3,3’-dimethoxy- 4,4‘ - biphenylene)bis[2,5- diphenyl - 2H - tetrazolium chloride] (Dajac Laboratories)-was dissolved in U.S.P. absolute ethanol (150 mg. per 100 ml.). Tetramethylammonium hydroxide reagent solution--3 ml. of 10% aqueous solution (Eastman)-was diluted to 100 ml. with absolute ethanol. Volume 3, Number 6, June 1969 567
Procedure. Commercially available cigarettes, selected at random from freshly opened packages, were smoked on the automatic smoking machine adjusted for a 35-ml. puff volume of 2 seconds duration with a 58-second interval between puffs. The particulate matter was collected on a Cambridge filter pad, and the gas phase material not retained by the filter was passed through 15 ml. of absolute ethanol in the bubbler trap. The Cambridge filter is designed to remove essentially all particles with greater than a 0.3-micron diameter and has become accepted as providing an empirically defined division between the gaseous and particulate phases of smoke. Some components, however, will be distributed between the two phases because the gaseous phase of each puff of smoke passes through the previously collected sample. The trapped material on the filter pads was extracted four times with approximately 7-ml. portions of absolute ethanol each time, and the final volume was adjusted to 30 ml. In some experiments, the Cambridge filter and its holder were weighed before and after smoke sample collection to obtain the weight of the total particulate matter. As little time as possible was allowed to elapse between collection and extraction of the particulate matter on the Cambridge filters. Samples of the extracts were analyzed on the day of collection although it was observed that the reducing power of the extracts in ethanol remained essentially unchanged for five days after collection. To appropriate aliquots (usually 0.1 to 0.5 ml.) of the ethanolic “smoke” solutions were added 0.25 ml. of blue tetrazolium solution followed by 0.25 ml. of tetramethylammonium hydroxide solution. This was incubated at 45 O C. for 15minutes in a constant temperature water bath. Optical densities were read at 530 mp. against a reagent blank. Results and Discussion The data listed in Table I are a direct measure of blue tetrazolium reduction in terms of absorbance units, A , per
Table I. Reactivity of Blue Tetrazolium with Reducing Constituents in Cigarette Smoke in Relationship to the Number of Puffs Absorbance, A , of 1/30 aliquot No. of Ratio Cigarette PMb GPc brands0 puffs PM/GP 12 2.1 0.18 A 2 3.8 0.41 9 A 4 7.3 0.68 11 A 6 10.2 0.98 8 10 A 12 14.5 1.20 A 10 3.5 0.37 9 4 B 11 3.4 0.30 4 C 10 4.4 0.45 C 6 3.2 0.30 11 D 4 9 4.4 0.47 D 6 9 8.1 0.88 D 10 11 2.7 0.26 4 E 10 2.5 0.26 F 4 4 1.2 0.34 G 4 4 3.5 1 .oo 8 G 26 2.3 0.09 4 H 30 8.5 0.28 8 H a Brand A is 70 mm. long, without filter tip; all other brands are 85 mrn. with filters. b PM. Particulate matter reducing components trapped on a Cambridge filter. c GP. Gas phase reducing components passing a Cambridge filter and recovered in an ethanol bubbler trap.
568 Environmental Science & Technology
indicated fractional portion of the smoke from one or more puffs per cigarette. The relative standard deviation of the method for an individual determination repeated 10 times was + 13% for the particulate phase and =t11 % for the gas phase. This includes sampling and smoking variations in addition to analytical error, No attempt was made to relate or normalize these values to any “typical” standard reducing component in smoke because of the wide range of reducing potentials among the compounds contributing to the total reaction. Tests of some of the hundreds of compounds that have been identified in smoke (Stedman, 1968) revealed a reactivity with blue tetrazolium ranging from negative for acetone, acetonitrile, acrylonitrile, acetaldehyde, propionaldehyde, methyl ethyl ketone, and hydrogen cyanide; and moderate to extreme reactivity for acrolein, furfural, 2,3-butanedione (0.3 A per pg.) and hydroquinone (0.5 A per pg.). The relationship of the number of puffs of smoke from various cigarettes to the concentration of blue tetrazolium reducing constituents is shown in Table I. A plot of the absorbance values for brand A shows a linear response with increasing numbers of puffs above four for both particulate and gas phases. These data closely parallel the relationship between the number of puffs and the yield of particulate matter determined gravimetrically by Keith and Tesh (1965). The sensitivity of the blue tetrazolium test, estimated from the data in Table I, in terms of puff volume, is such that the reducing capacity present in 1 ml. of mainstream smoke (brand A) gives an absorbance of approximately 1.0 for the particulate phase and 0.1 for the gas phase. On an equivolume basis, therefore, the reducing power of the partidate matter retained by the Cambridge filter is about 10 times greater than that of the gas phase passing through the filter. This absorbance ratio of PM/GP remains fairly constant, at approximately 10, for most of the filtered brands (Table I) evaluated in this study. Very significant deviations from this ratio, however, were observed for cigarette brands G and H. Cigarette brand G is equipped with a very efficient filter for “tars” and nicotine [among the lowest in the Federal Trade Commission (FTC) “tar” determinations] and brand H contains activated charcoal in its filter. The relatively low PMiGP ratio of 4 for brand G reflects its high efficie-q for filtration of aerosols from the smoke but without a proportional reduction of the gaseous phase. The selective removal of a larger proportion of gas phase reducing substances, arld presumably other organic vapors, from the charcoal filtered smoke (brand H) gives rise to a 3 times higher PMIGP ratio. Apparently, the application of this same, simple test to both phases of fresh mainstream smoke provides data for more complete evaluation of the smoke effluent yield from various tobacco products. The simultaneous determination of the reducing constituents in both phases also facilitates comparison of differential efficiency of various filter types. The mounting concern about the presence of components in the gaseous phase of tobacco smoke with potential biological activity (Green and Carolin, 1967; Kensler and Battista, 1963; Wakter and Kiefer, 1966) lends further support to the value of a readily applicable method to detect and measure gas phase constituents. In those instances where the weight of the total particulate matter was determined, the results indicate that for a given brand the absorbance of the particulate phase of the smoke effluent varies directly with the mass yield. For brand A in Table I, the TPM weights were 4.3, 7.4, 13.3, 19.4, and 26.6 mg. for 2, 4, 6, 8, and 10 puffs respectively. Accordingly, the particulate phase yields as determined from the level of blue tetrazolium reduction by most of the brands tested were in es-
sentially the same relative sequence as that determined for “tar” levels for the same brands by the FTC. The application of this analytical technique to biological studies will be the subject of a further communication. The purpose of one such study is to determine the retention of reducing substances in the respiratory system during inhalation of fresh smoke. Finally, it is suggested that the blue tetrazolium method might be applied to monitor atmospheric samples in air pollution studies where smoke from the incomplete combustion of natural products is a major source of pollutants.
Literature Cited
Acknowledgment
Green, G. M., Carolin, D., New Eng. J. Med. 276, 422-7 (1967). Keith, C. H., Tesh, P. G., Tobacco Sci. 9, 61-4 (1965). Kensler, C. J., Battista, S . P., New Eng. J . Med. 269, 1161-6 (1963). O’Keeffe, A. E., Lieser, R. C., Tobacco Sci. 2, 73-6 (1958). Rosenkrantz, H., Arch. Biochem. Biophys. 81, 194-203 (1959). Salim. E. F.. Manni. P. E.. Sinsheimer. J. E.. J . Pharnz. Sci. 53, ’391-4 (1964). ’ Stedman, R. L., Chem. Rem. 68, 153-207 (1968). Walker. T. R.. Kiefer. J. E.. Science 153. 1248 (1966). Wartman, W.’ B., Jr.; Cogbill, E. C., Harlow,’ E. S., Anal. Chenz. 31, 1705-9 (1959). Weiner, S., Chemist Analyst 37, 56-9 (1948). Wynder, E. L., Hoffmann, D., “Tobacco and Tobacco Smoke,” pp. 317-416, Academic Press, New York, 1967.
The helpful suggestions by Harris Rosenkrantz and the technical assistance of Joan Graham are acknowledged.
Receiced for review October I O , 1968. Accepted March 13, 1969.
Kinetics of Substrate Uptake in Pure and Mixed Culture R. I. Matelesl and S. K. Chian2 Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass. 021 39
Recent research dealing with kinetics of substrate uptake with mixed substrate is reviewed with particular reference to natural populations. Even with carbon source limitation, excretion of acetate during continuous culture was observed. This phenomenon appears to be of some generality and can also be readily observed in batch cultures.
M
onod (1942, 1950) provided evidence that the growth rate of a microorganism in pure culture, on a single carbon source, can be related to the concentration of that source by Equation 1, in which p is the specific
growth rate (hr. -I); pLmis the maximum specific growth rate for the organism and medium, s is the concentration of limiting nutrient; and K, is a constant numerically equal to the substrate concentration at which p = % p m . This equation is formally identical to the Michaelis-Menten equation, but should not be considered as implying anything concerning the mechanisms limiting growth rate. It is merely a convenient, and not particularly exact, correlation of the data. These early investigations of Monod revealed why previous workers using batch cultures were unable to observe any influence of substrate concentration on growth rate: the constant K , is of the Present address, Institute of Microbiology, Hebrew University-Hadassah Medical School, P.O. Box 1172, Jerusalem, Israel Present address, Abcor, Inc., 341 Vassar St., Cambridge, Mass. 02139
order of 10 mg. per liter for many carbon compounds, and when the substrate concentration decreases to that value at the end of a batch culture, its concentration is changing so rapidly that it is not possible to measure it accurately. Using continuous culture, however, accurate measurements are possible, although difficult. Equation 1 predicts that growth rate should be more or less independent of substrate concentration when the substrate concentration is several times greater than K,. This, in fact, is regularly seen when the phenomenon of log phase growth is observed. As shown in Equation 2,
during log phase growth the rate of substrate consumption per unit mass of cells is also independent of the substrate concentration (with Y being the yield constant) when the substrate concentration is much greater than K , (StunimZollinger, 1966). Thus, one would expect to observe zero-order kinetics of substrate uptake at substrate concentrations much higher than the value of K,, while at concentrations much lower than K , the rate of uptake should be of the first order. At intermediate concentrations, Equation 1 would apply. These equations have been reasonably well confirmed for pure cultures on single substrates (Powell, 1967). When nongrowing activated sludge systems are examined, confirmation of zero-order kinetics can be obtained. Figures 1 and 2 show zero-order kinetics for sludge metabolizing various compounds, alone or together. Figure 3, the most interesting, shows zero-order kinetics for the disappearance of the substrate, but first-order kinetics for the disappearance of BOD, thus indicating that there are metabolites other than the known substrates contributing to the BOD. Volume 3, Number 6, June 1969 569
