An Increase in Diffusion or Migration Current. In all investigations described above, the magnetic field effect on polarography has been observed as a decrease in the maximum wave current, and has been explained using the theory of magnetohydrodynamics. However, it has recently been found that the limiting current of migration or diffusion is also affected by the magnetic field. Contrary to the previous case, the present effect is observed as an increase in the migration or diffusion current as shown in Figure 8, and is small in magnitude compared with the previous one. Favorable conditions for observing this effect are a higher mercury reservoir height, the absence of surface active substances, and a lower concentration of supporting electrolyte. A possible mechanism can be obtained through the analogy of a magnetic mercury cathode (16)-namely, the magnetic field causes the electrolyte or the mercury drop to rotate or agitate like a rotor in a motor when the polarographic current flows. The third condition mentioned above may concern the removal of the tangential motion. However, explanations for the other two conditions remain to be worked out. (16) E. J. Center, R. C. Overbeck, and D. L.Chase, ANAL.CHEM., 23, 1134 (1951).
CONCLUSION By the use of a theory of magnetohydrodynamics, the mechanism of the effect of magnetic field on the second kind of maxima is explained in terms of the retardation of tangential motion of the mercury surface. Using the magnetic field effect as a criterion, it is concluded that motion of electrolytic solutions accompanying maxima of the first kind is classified into two types of different origin. The first type of motion, originating at the surface of a mercury drop, is affected appreciably by a magnetic field. On the contrary, the second type of motion which arises in electrolytic solutions near the mercury drop, is little influenced by a magnetic field. ACKNOWLEDGMENT The authors thank Hiroki Haraguchi for his valuable discussion and help in the experimental work and Yoji Arata for his helpful comments. RECEIVED for review April 2, 1968. Accepted August 12, 1968.
Fluorescence of Prostaglandin C. L. Gantt, L. R. Kizlaitis, D. R. Thomas, and J. G. Greslin Clinical Research Center, Department of Medicine, Unieersity of Illinois College o j Medicine, Chicago, 111. 60612
PROSTAGLANDINS, originally isolated and identified from sheep seminal fluid, comprise a large group of naturallyoccurring compounds found in most body organs and fluids. These are of great potential biologic interest because most have profound effects at very small dosages. Several recent reviews on the nomenclature, chemistry, physiology, and pharmacology of prostaglandins have appeared (1-5). The structure of prostaglandin El (9-Keto-1 la,l5a-dihydroxyprost-13-enoic acid) is shown in Figure 1. Because the structure of the prostaglandins suggested the possibility for several resonance forms of the molecule, this study was undertaken to determine their possible fluorescence. EXPERIMENTAL Prostaglandin El (PGEJ was weighed and dissolved in methanol at a concentration of 1 mg/ml and stored at 4 "C. Freshly-prepared dilutions of the stock solution were made in methanol at concentrations of 5 pg/ml and less. Aliquots of these were evaporated to dryness in 2-rnl test tubes, under nitrogen, with a sand bath at 40 "C. One-half milliliter of -
(1) E. W. Horton and C. J. Thompson, Brit. J . Pharmacol Chemofher., 22, 183 (1964). (2) S. Bergstrom and B. Samuelsson, J. Bid. Chem., 237, PC3005
(1962). ( 3 ) M. Hamberg and B. Samuelsson, ibid., 241, 257 (1966). (4) K. Green and B. Samuelsson, J. Lipid Res., 5, 117 (1964). (5) S. Bergstrom, Science, 157, 382 (1967).
2 198
e
ANALYTICAL CHEMISTRY
20
OH
Figure 1. Structure of PGEl sulfuric acid-water reagent (70%, 30% v/v) was pipetted into these tubes which were then mechanically swirled. The samples and appropriate blanks of sulfuric acid were closed with ground glass stoppers and incubated for one-half hour at 65 "C. They were immediately immersed in 15 "C water for one-half hour. Fluorescence was determined on an Aminco-Bowman spectrophotofluorometer with quartz micro-cuvettes, previously acid cleaned and stored in concentrated nitric acid. The samples were activated at 366 mp with a Xenon lamp source and read at 420 mp. The stability of the instrument is checked with a standard solution of hydrocortisone. All glassware was cleaned in concentrated sulfuric aciddichromate solution and rinsed thoroughly with distilled water. Meticulous care and cleanliness were necessary to obtain reproducible and accurate results when measuring small quantities of the prostaglandins. In these studies, the turret of the spectrophotofluorometer was set at 3 and the sensitivity at 30.
Table I. Relationship of Fluorescence Intensity and PGEl Concentration in 70 Sulfuric Acid-Water (v/v) Incubated at 65 "Cfor 30 Min Fluorescence intensity, PGEi , No. of arbitrary units samples Mean Std. error
420 rnp
60
r
0.5 1.0 1.5 2.0 2.5
>-
[L
d
40
7
6 10 8 8 6
12.3 31.3 45.8 59.5
74.3
ztl.0 h1.2 f2.3 h2.1 3~2.6
t m a
a
20 10
c
200
300
400
500
600
700
-
WAVELENGTH m p
Figure 2. Uncorrected excitation ( A ) and emission spectra ( B ) of PGEl in 7 0 x sulfuric acid-water (VjV) heated at 65 "C for 30 min. Emission set at 420 mp to record excitation peak and excitation set at 365 mp to record emission spectrum. Reagent blank 70% aqueous sulfuric acid, lower curves (C) The sulfuric acid-water reagent deteriorated in a few days making it necessary to use a freshly-prepared, cooled reagent for very accurate work. The reason for the deterioration of the reagent was not studied but is very likely caused by hydration. Several batches and different commercial brands of reagent grade sulfuric acid were tested and blanks of these showed little variation in non-specific fluorescence. RESULTS iiND DISCUSSION
Fluorescence Spectrum. The uncorrected activation and emission spectra for prostaglandin El are shown in Figure 2. The activation scan was obtained with emission set at 420 mp. The emission scan was obtained with the activation at 365 mp. Increased concentrations of PGEl produced increased fluorescence as shown in Table I. With the equipment indicated, the limits of detection for accurate measurement are in the range of 0.2 to 0.5 pg of prostaglandin El. Factors Affecting Fluorescence Development and Decay. The effect of incubation temperature on maximal fluorescence development was studied at 45, 65, and 85 "C. 65 "C was definitely superior. The influence of various concentrations of sulfuric acid on fluorescence development, when the samples were incubated at 65 "C for one-half hour then read 30 minutes later, was investigated. There was little difference in fluorescence between 90 and 7 0 x with a sharp decline between 70 and 6 0 x sulfuric acid vjv in water. The lowest concentration of
sulfuric acid that could be used to reproduce the induced fluorescence was chosen because higher concentrations of sulfuric acid tend to increase non-specific fluorescence in other materials. Studies on the effect of time of determination of fluorescence after incubation at 65 "G in 70% sulfuric acid-water (viv) for one-half hour and cooled immediately in a 15 "C water bath indicated that any consistent arbitrary time could be used for reading all samples. For convenience, we chose the time of 30 minutes which was the earliest that we could get reproducible, concentration-related results. The fluorescence of PGE1, in 70% sulfuric acid-ethanol (v/v), was approximately one half of that observed with the corresponding aqueous sulfuric acid mixture. Fluorescence of Other Prostaglandins. Preliminary studies on PGE?, PGE3, PGA1, and PGBl indicate that these compounds fluoresce at absorption and emission spectra very similar to PGE1. The emission spectrum of PGFm appears about 30 mp longer than those of the other compounds. The relative fluorescence intensities (arbitrary units) of PGE1, PGE2, PGE3, PGA1, and PGB, appear similar with these same conditions. Techniques for the extraction of prostaglandins from organs and body fluids are available and chromatographic techniques (column, thin-layer, gas-liquid and mass spectrograph) for their separations have been elucidated (5, 6). Determination of the chromatographic thin-layer product by the relatively simple spectrophotofluorometric technique described herein could assist materially in these studies. Because of possible interfering fluorescent materials, suggestions are that the present technique be used only after isolation of relatively pure prostaglandins. For example, simple ether extracts of acidified (pH 3.0) human plasma contain several interfering compounds. ACKNOF7'LEDGMENT
The prostaglandins used in the study were kindly supplied by John E. Pike of the Upjohn Co., Kalamazoo, Mich. RECEIVED for review June 3, 1968. Accepted August 19, 1968. Work supported by Grant PHS M 0 1 FR 45-06 from the National Institutes of Health and by a grant-in-aid from G. D. Searle and Co., Chicago, Ill. (6) S. Bergstrom, F. Dressler, R. Ryhage, B. Samuelsson, and J. Sjovall, Ark. Kemi, 19, 563 (1962).
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