determinations. Thus, contamination in the ordinary urine sample encountered was considered unimportant. Typical values for urinary aldosterone in patients on a normal sodium intake of 100 meq. per day averaged 5.33 y per 24 hours with a range of 1.5 to 11.7. Individual values are s h o m in Table 111. Reproducibility. Six aliquots of a pooled urine extract (from patients on a low salt diet) were determined by the salicyloyl hydrazide procedure. The average aldosterone found was 17.9 y =t standard deviation 1.37 y for the 6-hour aliquots. The individual figures are shown in Table 11. DISCUSSION
Relatively pure steroid is required, necessitating chromatographic or other separation techniques for biological samples or crude mixtures. The present application to urinary aldosterone determination resembles several previous chemical methods in utilizing paper chromatographic purification, but differs in the final method of quantitation. Thus, others have employed sodium hydroxide fluorescence on paper (I,??, I S ) , reduction of alkaline blue tetrazolium on paper (12, IS) and in the test tube (IO, 14), ultraviolet absorption
(9, I d ) , and reaction with 2,4-dinitrophenylhydrazine (9). Inherent in any paper chromatographic separation are losses n-hich prevent quantitative recovery. Autoradiographic and Geiger-counting techniques revealed that chromatographically homogeneous radioactive cortisol was partially left at the origin and streaked to a lesser extent between the origin and the main bulk of the steroid. These losses are primarily due to adsorption and become serious with extremely small quantities of steroid. Thus, in any method based on paper chromatography, constant losses must be assumed as is done here, or correction made for losses by isotopic methods (2, 16). For precise results with quantities of steroid less than a few micrograms, the latter is mandatory. The distinctive shape and pH shift of the ultraviolet absorption spectra of salicyloyl hydrazones render salicyloyl hydrazide a useful reagent for identification purposes. This reagent offers greater specificity than many available steroid micromethods and can be readily used with ordinary analytical equipment. ACKNOWLEDGMENT
The technical assistance of Moses B. Middleton is acknowledged.
LITERATURE CITED
(1) Allen, W.M., J . Clin. Endocrinol. 10,
71 (1950). Agers, P. J., Garrod, O., Simpson, S. A., Tait, J. F., Biochem. J . 65, 639 (1957). (3) Bondi, S., 2. physiol Chem. 52, 170 (1907). (41 Bush, I. E., Biochem. J . 50, 370 (1952). (5) Camber, B., Xature 174, 1107 (1954). (6) Chen, P. S.,ASAL. CHEW 31, 296 (1959). (7) Eberlein, W. R., Bongiovanni, A. RI., Arch. Biochem. Biophys. 59, 90
(2)
(19.5,5\.
(8j-@&nzen, Hartwig, Eichler, T., J . prakt. Chem. 78, 157 (1908). (9) Gornall, A. G., Gwilliam, Cynthia, Can. J . Biochem. Phusiol. 35. 71 (1957). (10) Hernando-Avendano. L.’. crabbe. ’ J., ROSS, E. J., Reddy,‘ W. J., Renoldj A. E., Nelson, D. H., Thorn, G. W., Metabolism, Clin. and Exptl. 6 , 518 /,nc-t \ l t r c J l ,I.
(11) Liddle, G. W., Cornfield, J., Clasper, .4.G. T., Bartter, F. C., J . Clin. Invest. 34, 1410 (1955). (12) Seher, R., Wettstein, A., Acta Endocrinol. 18, 386 (1955). (13) Yeher, R., Wettstein, A., J . Clin. Incest. 35, 800 (1956). (14) Kowaczynski, W. J., Koiw, Erich, Genest, J., Can. J . Biochem. Physiol. 35, 425 (1957).
(15) Peterson, R. E., personal communication. (16) Peterson, R. E., Karrer, Aurora, Guerra, S. L., AXAL. CHEM.2 9 , 144 (1957). RECEIVEDfor review March 15, 1 15’. Accepted October 14, 1958.
Fluorescence of Some Salicyloyl Hydrazones PHILIP S. CHEN, Jr. Section o f Clinical Endocrinology, National Heart Institute, National Institutes of Health, Bethesda, Md.
-
-
b o Hydroxybenzaldehyde, p hydroxybenzaldehyde, and p-dimethylaminobenzaldehyde form salicylol hydrazones with relatively specific fluorescence characteristics as compared with those of other salicyloyl hydrazones. The other, often closely related, compounds tested either did not fluoresce or fluoresced with excitation and fluorescence maxima similar to those of salicyloyl hydrazide. Fluorescence spectra of the three compounds listed could b e obtained in the presence of excess salicyloyl hydrazide, utilizing the greatly increased specificity afforded b y the spectrophotofluorometer. p-Aminoacetophenone could b e similarly determined, if a suitable excitation wave length were chosen.
T
development of a spectrophotofluorometer (1) featuring continuously variable excitation and fluoresHE
296
ANALYTICAL CHEMISTRY
cence wave lengths has prompted an evaluation of fluorescent derivatives of salicyloyl hydrazide. This compound has recently been proposed (9) as a useful fluorometric reagent for carbonylcontaining compounds. I n studying the uses of salicyloyl hydrazide, o-hydroxybenzaldehyde, phydroxybenzaldehyde, p-dimethylaniinobenzaldehyde, and p-aminoacetophenone were observed to form salicyloyl hydrazones with excitation-fluorescence spectra different from that of salicyloyl hydrazide. This permits the determination of their spectra even in the presence of a large excess of salicyloyl hydrazide. This paper surveys the fluorescent properties of a number of salicyloyl hydrazones. EXPERIMENTAL
Preparation of Derivatives. From 1 t o 10 mg. of the compounds were
dissolved in approximately 3 mi. of ethyl alcohol and placed in 15 X 150 mm. borosilicate glass test tubes. T o each tube, 10 mg. of salicyloyl hydrazide (Versatile Chemicals Corp., Los h g e l e s , Calif., melting point 147.5’C.) in 2.5 ml. of ethyl alcohol was added, followed by 0.1 ml. of glacial acetic aEid. The mixtures were heated to boiling in a water bath for 10 to 60 minutes, the time depending upon the reactivity of the compound. Reaction products were separated by crystallization in the cold, and further purified by recrystallization, generally from aqueous ethyl alcohol, but not exhaustively characterized further. The hydrazones mere dissolved in ethyl alcohol, except those of p-acetamidobenzaldehyde, p-isopropylbenzaldehyde, and m-nitrobenzaldehyde, which were dissolved in 20% dichloromethane in ethyl alcohol. Instrumentation. The AmincoBowman spectrophotofluorometer with a Hanovia 150-W xenon arc source and 1P28 multiplier photo-
tube n-as used. The excitation and fluorescence gratings (Bausch & Lomb Optical Co. reflectance gratings, 600 lines per mm.) were blazed to give maximum response a t 300 and 500 mp? respectively. Values reported are un-
50
A
'I
B
RESULTS
v)
E
corrected readings as obtained by the apparatus. Essential instrumental features (1) and the method of determining fluorescence characteristics (3, 4) have been adequately described. Fluorescence measurements of the compounds studied were conducted in 0.l.M carbonate-bicarbonate buffer a t pH 10.
40
' I ,
> 30 CL a a
em 2 0 a
a io 600
400
200
mP Figure 1. drazide
Spectra of salicyloyl hy-
A. Excitation spectrum 8. Fluorescence spectrum 10 y per mi. a t p H 10
Salicyloyl hydrazide fluoresces mahimall>- a t 425 mp and is excited masimally a t 350 mp. Typical excitation and fluorescence spectra for salicyloyl hydrazide are shown in Figure 1. The salicyloyl hydrazones (of oliydroxybenzaldehyde, p-hydroxybenzaldehyde, and p-dimethylaminobenzaldehyde) were excited niaxinially at 390 mp and fluoresced maximally a t 470 nip, Fluorescence features of these derivatives were sufficiently different from those of salicyloyl hydrazide that their fluorescence spectra could be obtained directly on a reaction mixture after adjustment of pH, n-ithout interference from excess salicyloyl hydrazide reagent. Figure 2 shows the fluorescence characteristics of the p-dimethylamino-
C A'O
400
600-
400
600
mP Figure 2. A. 8.
C.
p-Dimethylaminobenzaldehyde salicyloyl hydrazone
Excitation spectrum, 1 2 y per ml. Fluorescence spectra, 1 2 y per ml. Excitation a t 400 mp (bottom) and 390 m p (top). Saiicyloyl hydrazide, 60 y per ml. does not alter curves Fluorescence spectra, 1, 2, 20, 5, 10 y per ml. Note self-depression a t 20 y per ml.
a 400
200
Figure 3. A. B.
C.
300
600
400
Table I. Salicyloyl Hydrazones Which Fluoresce
Excitation Fluorescence Maxima, Maxima, Salicyloyl hydrazide Butyraldehyde hcetophenone Strophanthidin Isoandrosterone Dehydroisoandrosterone Cortisone
Cortisol Corticosterone Aldosterone Progesterone Testosterone
Mfi
11p
350 350 330 340 340
425 430 440 430 440
340 350 330 330 330 330 330
440 425 430 430 430 430 430
benzaldehyde derivative. Fluorescence intensity a t 470 mp &-asproportional to concentration up to about 5 y per ml., above which self-depression occurred rrithout altering the shape of the curve. The presence of salicyloyl hydrazide in concentrations of 60 y per ml. did not interfere n ith fluorescence nieasurements when activated a t 390 or 400 mp. (In other experiments, salicyloyl hydrazide in eoncentrations of 200 y per nil. did not interfere.) Snother substance fluorescing at 470 nip was the derivative of p-aminoacetophenon?. This derivative, however, exhibited a loi~erexcitation maximum a t 360 m p , as shown in Figure 3. A t this excitation n-ave length, the presence of salicyloyl hydrazide will enhance the fluorescence spectra. HOKever, by shifting the excitation wave length t o a nonoptimum value of 370 m p and recording fluorescence a t 500 nip (also nonoptimum), salicyloyl hydrazide interference was eliminated. Several salicyloyl hydrazones fluoresced a t the same general wave length as salicyloyl hydrazide and were also excited very close to 350 mp. Thus, fluorescence spectra of these hydrazones could not be obtained in the presence of salicyloyl hydrazide because of fluorescence interference (Table I). The derivatives of other aldehydes tested which did not fluoresce a t all in concentrations of 10 t o 20 y per nil. a t pH 10 include acetaldehyde, benzaldehyde, o-nitrobenzaldehyde, m-niitrobenzaldehyde, m-hydroxybenzaldehyde, p-chlorohenzaldehyde, p-acetamidobenzaldehyde, p-isopropylbenzaldehyde, 2.3-diniethoxybenzaldehyde,and piperonal.
600
miJ p-Aminoacetophenone salicyloyl hydrazone
Excitation spectrum, 10 y per ml. Peak a t 360 m g Fluorescence spectra, 3.5 y per ml. Excitation a t 360 m p . Without (bottom) and with (top) 40 y per ml. salicyloyl hydrazide Fluorescence spectra, 3.5 y per ml. Excitation a t 370 m p . Without (bottom) and with (top) 100 y per ml. salicyloyl hydrazide
DISCUSSION
The shift in excitation and fluorescence spectra of the three benzaldehyde derivatives when condensed with salicyloyl hydrazide was remarkably specific. None of the other salicyloyl hyVOL. 31, NO. 2, FEBRUARY 1959
297
drazones tested exhibited a fluorescence peak a t 470 mp when excited at 390 to 400 n i ~ . p-Aminoacetophenone, while fluorescing a t this wave length, ivas excited maximally by a shorter excitation wive length. Fluorescence spectra of these four compounds were easily obtained even with an excess of salicyloyl hydrazide. A reaction mixture of one of the above-mentioned substances and salicyloyl hydrazide could be read directly in the spectrophotofluorometer, after condmsation in acetic acid-ethyl alco-
hol with heating. The mixture ncetltd only to be neutralized, brought to an alkaline pH with buffer, diluted properly, and read in the instrument. Fluorescent salicyloyl hydrazones which cannot be resolved by their fluorescence spectra could be assayed, if free salicyloyl hydrazide was removed after reaction. One simple method is to partition between purified dichloromethane (purified through silica gel) and 20% ethyl alcohol, 0.1M in hydrochloric acid. Salicyloyl hydrazide remains in the aqueous phase.
LITERATURE CITED
( I ) Bowman, R. L., Caulfield, P. A , , Cdenfriend, Sidney, Science 122, 32-3 (1955). (2]-Camber, B., Nature 174, 1107 (1954). (3) Duggan, D. E., Bowman, R. L., Bradie, B. B., Udenfriend, Sidnev, Arch. ' Biochem. B i o p h y s . 68, 1-i4 (1957). (4) Udenfriend, Sidney, Duggan, D. E., Vasta, B. II.,Brodie, B. B., J . Pharmacol. Ezptl. Therap. 120, 26-32 (1957).
RECEIVED for revie\? January 18, 1958. Accepted October 14,1958.
Enzymatic Determination of Polyunsaturated Fatty Acids JOSEPH MacGEE Miami Valley laboratories, The Procter & Gamble Co., Cincinnati, Ohio
b A simple and rapid enzymatic method for the quantitative estimation of total cis-methylene-interrupted polyenoic acids has been devised. Linoleic, linolenic, and arachidonic acids, the more common acids of this group, were used to calibrate the method. The potassium salts of the fatty acids are oxidized b y atmospheric oxygen in the presence of the enzyme lipoxidase, and the absorption of the conjugated diene hydroperoxide is measured a t 234 mp. As little as 5 y of linoleic acid can b e quantitatively measured with good accuracy and precision. The total content of polyunsaturated fatty acids containing the cis-methylene-interrupted diene structure of fats, oils, hydrogenated oils, fatty acids, esters, blood plasma, microorganisms, and plant seeds has been measured directly by this method.
T
HL quantitative estimation of polyuiisaturated fatty acids in the fats and oils of commerce and in tissues has, a t best, been difficult. Before publication of the ultraviolets pectrophotometric method developed by NIitchell, Kraybill, and Zscheile (6), the principal method for the determination of these biologically important acids involved physical isolation of the individual fatty acids by means of their bromide derivatives ( 2 ) . As more information about their functions became available, the importance of polyunsaturated fatty acids in the fields of nutrition and medicine becanie more evident, and methods for their estimation were sought. The first big advance in this analytical problem was the development of the spectrophotometric method based on the absorption of ultraviolet light by tile conjugated unsaturation induced by
298
ANALYTICAL CHEMISTRY
alkaline isomerization of the polyunsaturated fatty acids (6). However, in addition to being strictly empirical, satisfactory application of the isomerization method has been limited to samples which are available in relatively large amounts (about 0.1 gram) as isolated lipides, and to materials with a limited trans-isomer content. The latter point is important, because knowledge of the cis-polyunsaturated fatty acid content is often desired. Although the alkali isomerization technique is quantitative for the cis-isomers, transisomers are partially measured. The need for a simple method which would be specific for cis-isomers and at the same time be applicable to smaller quantities of materials and to the analysis of body fluids and tissues for their polyunsaturated fatty acid content led to consideration of the specific reagent, lipoxidase. Lipoxidase is an oxidative enzyme found in a variety of plant tissues. 18
I7
I6
$5
13
14
I2
/I
10
9
8
Its only known substrates all contain single methylene-interrupted double bonds in which the double bonds are in the cis configuration. Three of the many natural substrates of lipoxidase are the polyunsaturated fatty acids, linoleic, linolenic, and arachidonic. The mode of action of lipoxidase on these acids involves a shift of one of the double bonds and addition of oxygen to form a conjugated diene hydroperoxide (5). Figure 1 illustrates the products from linoleic acid. The activity of the enzyme has been measured, by others, by means of the rates of oxygen uptake, hydroperoxide formation, and conjugated diene formation in the presence of excess substrate, but with limiting amounts of enzyme (4). A method based on ultraviolet absorption after complete conjugation of a limited amount of a substrate in the presence of an excess of enzyme is a logical extension. The alkali isomerization procedure
e
7
I
, ,
2
C H ~ C H ~ C H ~ C H ~ C H ~ CC H = ~CH H = CcHH ~ C H ~ C H ~ C H ~ C H ~ C H ~ C H ~ ~ O O H
Figure 1. Enzymatic oxidation and conjugation of linoleic acid
4 I.
I3
I*
,,
10
I
I
- CHzCH=CHCH=CHyHCH2 OOH
AIO' 12
,
9 - hydroperoxide
w
I,
E
+H10
0
I
.CH2$HCH = C H CH = C HCH2 ,I
OOH
A9,II , 13 -hydroperoxide
