benzene shows that the double bond in the styrene produces 0.68 division per micron increase in sensitivity. T h e addition of 0.68 to the molar sensitivity observed for Tetralin gives a n estimated sensitivity of 2.63 for indene, in good agreement with the observed value of 2.76. A further prediction can be made concerning the effects of substitution on the saturate ring compared with a substitution on the aromatic ring of a compound having condensed saturate and aromatic rings. T h e substitution on the saturate ring should have a negligible effect on the molar sensitivity, while a substitution on the aromatic ring should have a n effect similar to the effect of substitution of the group on a benzene ring. Unfortunately, no experimental data are available here to test this prediction. Although the discussions above have been concerned with the prediction of sensitivities from information on electron-accepting and -donating properties of substituent groups, one can now use the established generalizations to obtain useful structural information about relatively pure compounds. T h e type and positions of many substituents can be estimated from observed sensitivities. Because the correlations observed apply only to sensitivities on a per molecule basis, all data have been discussed in terms of molar sensitivities. The data are easily converted to sensitivities per unit liquid volume. as used in a practical analytical scheme, by multiplying by a factor containing liquid density/ molecular weight. Table I1 contains sensitivities converted to liquid volume units for all compounds studied.
CONCLUSIONS
The following set of structura1 correlations for predicting low voltage molar sensitivities has resulted from this study: T h e effect of a single substitution can be predicted from a tabulation, such as Table I, of relative electron-accepting and -donating properties. Increasing the length of a n alkyl substitution beyond two or three carbons produces no change in sensitivity. Molar sensitivities increase with increasing number of donor substituents, T h e position of donor substituents on disubstituted benzenes produces a sensitivity change increasing in the order ortho, meta, and para. Condensed saturate rings on an aromatic have the same effect on sensitivity as two alkyl substitutions. I n a compound having condensed saturate and aromatic rings, substitution on the saturate ring has little effect on the sensitivity. Sensitivities increase with an increase in the number of condensed aromatic rings per molecule. Donor substitution on the position of p-naphthalene produces a larger increase in sensitivity than a similar substitution in the alpha position. Higher condensed aromatics probably shox similar changes with position of substitution,
for obtaining the mass spectrometric experimental data. LITERATURE CITED
(1) Brewster, R. Q., “Organic Chemistry,” 2nd ed., p. 497, Prentice-Hall,
New York, 1954. ( 2 ) Chamberlain, K.F., ANAL.CHEM.31, 56 i1959). ( 3 ) Clerc, k. J., Hood, A,, O’Neal, 31. J., Ibid., 27, 868 (1955). (4) Coggeshall, N. D., J . Chem. Phys., in press. (5) Corio, P. L., Dailey, D. P., J . Am. Chem. Soc. 78, 3043 (1956). (6) Field, F. H., Franklin, J. F., “Electron Impact Phenomena,” p. 119, Academic Press, Kew York, 1957. ( 7 ) Ibid., p. 270. (8) Field, F. H., Hastings, S. H., AZIAL. CHEX.28, 1248 (1956). (9) Glasstone, S., “Textbook of Physical Chemistry,” 2nd ed., p. 591, Van Nostrsnd, Princeton, N. J., 1916. (10) Hastings, S. H., Johnson, B. H., Lumpkin, H. E., AXAL.CHEXI. 28, 1243 (1956). (11) Honig, R. E., J . Chem. Phys. 16, 105 ( I 948). ( 1 2 ) Ingold, C. K., “Structure and hlechanism in Organic Chemistry,” Chap. 6, Cornell University Press, Ithaca, S . Y., 1053. ( 1 3 ) Kearns, G. L., Ilaranowski, K. C., Crable, G. F., ANAL.CHEX. 31, 1646 (1959). (14) Lossing, F. P., Tickner, A. W., Bryce, K. A , , J . Chem. Phys. 19, 1254 (1951). 115) Lumokin. H. E., ASAL. CHEW 30. 321 (1968). ’ (16) Sicholson, A. J. C., J . Chern. Phys. 20. 1x12 - _ - - (1Q.m). (lfj Sharkey, A. G., Jr., Robinson, C. F., Friedel, R. A., 4STM Committee E-14, Conference on Mass Spectrometry, New Orleans, La., May 1958. RECEIVED for review June 29, 1959. Accepted October 1, 1959. Seventh Meeting of ASTRI Committee E-14 on Mass Spectrometry, Los Sngeles, Calif., May 1959. I
ACKNOWLEDGMENT
The authors thank K. D. Coggeshall and D. E. O’Reilly for a number of interesting discussions of this work and P. TT. hfazak, J. P. Klems, and D . J. Clancy
\ - - - - ,
Modifkation of S pectrofIuorometric Determinuti on of Aminochromes in Human Plasma A. N. PAYZA and MARGARET MAHON Psychiafric Research Unit, Deparfmenf o f Public Health, University Hospital, Saskatoon, Saskatchewan, Canada
b In connection with the assay method for adrenochrome (2,3-dihydro-3hydroxy-N-methylindole 5,6 quinone), originated in this laboratory, a modification has been developed which allows the method to b e used for whole tissues-Le., red blood cells, etc. A theoretical objection to the earlier method has been met in that the present procedure has a stable blank. A large series of indoles was also tested and found not to interfere with the basic method.
-
T
-
HE procedure for determining the reagent blank in a previous method
(4)was criticized by Feldstein ( 1 ) on the basis of interference of zinc acetateascorbic acid fluorescence, Kith the fluorescence arising from the aminochrome-zinc acetate complex. The present reaction of aminochrome with zinc acetate does not use ascorbic acid, the source of the unstable reagent blank. MATERIALS AND METHOD
T h e chemicals described in the previous method were used (4). Yew compounds tested were: 5-hydroxyindole, 5-hydroxyindole-3-acetic acid (Regis Chemical Co.), 3,4-dihydroxyphenyl-
serine (Bios Laboratories), 3-(2-aminoethyl)-5-indolol (serotonin) (Nutritional Biochemicals), and N-isopropyladrenalone sulfate, ephedrine of unknown origin. The following additional compounds were studied: N-ethylnoradrenochrome, AT-isopropylnoradrenochrome, 3-iOdOadrenochrome, 2-iodo-2-methylnoradrenochrome, 2-iodo-N-ethylnoradrenochrome, 2-iodo-N-isopropylnoradrenochrome, adrenochrome methyl ether, adrenochrome ethyl ether (3-ethylepinchrome) , leucoadrenochrorne (5>6dihydroxy-hr-methylindole), 2-iodoleucoadrenochrome, and adrenolutin (l-methyl-3,5,6-indoletriol), Reagents. 1. Trichloroacetic acid VOL. 32,
NO. 1, JANUARY 1960
17
(TCA), 10% in distilled water. 2. Trichloroacetic acid, 10% in acetone. 3. 1M zinc acetate in trichloroacetic acid, 10% in distilled water. Procedure. Heparinized blood (5 ml.) was mixed with 2 parts of reagent 1 and filtered. In four test tubes (10ml. capacity), marked AI, BI, SI, and Sz, 2 ml. of clear filtrate were placed. Distilled water (5 ml.) was used (as above) instead of blood for the reagent blank in test tubes marked As and Bz. To the SI and Sz tubes 0.1 ml. of adrenochrome (2/3 y) solution in distilled water was added as the internal standard. T o the other tubes 0.1 ml. of distilled water was added. T o all tubes, 2 ml. of reagent 2 Table I.
were added, follo\T ed by 1 ml. of reagent 3 to tubes AI, Az, and SI. One milliliter of reagent 1 was added by pipet to tubes B1, Bz, and Sz and then the tubes were mixed. Ten minutes after the addition of reagent 3 to the tubes, the fluorescence was read in the Farrand spectrofluorometer (Model 104293 using a 5-1iil. quartz cuvette) a t 4 1 0 - m ~ excitation and 500-mfi emission at the 0.1 range of the microammeter. The difference in the fluorescence readings between the BI and Bz tubes was not more than two units in any deterniination.
CALCULATION.
(a1 - B,) -
(A2
- B2) = R
Comparison of Fluorescence Formation by Adrenochrome-Zinc Acetate Complex and Stability of Adrenolutin Fluorescence
Time, hlin. 3 5 8
10 12 16
A1
Bi
52 64 72 78
10 10 10 10 10 10
80
77
3 5
140 94
Fluorescence, Arbitrary Units" a1 bi A? Bz a? b? Adrenochrome 12 10 12 10 24 10 12 10 27 10 12 10 12 10 27 10 12 10 10 12 10 27 10 12 12 10 27 10 12 10 12 10 27 10 12 10 Adrenolutin 19 17 25 21 19 17 23 21 .. 16 15 .. 18 16 19 17 8 6 .. 410-mu excitation maximum, 500-mp emission maximum.
140 94 70 8 io 10 25 26 100 X 0.1 range readings a t Flasks contain: AI, B1: 0.005 pmole adrenochrome in sample. Az, Bz:0.001 &moleadrenochrome in sample. A,, al, A*, az: 1 ml. TCA lo%, 1 ml. in distilled xater, 2 ml. TCA 10% in acetone, 1 ml. 134 zinc acetate in 10% TCA in distilled water. B,, bl, BB,b,: 1 ml. TCA 1 0 ~ o1, ml. in distilled water, 2 ml. TCA 10% in acetone, 1 ml. lOYc T C 9 in distilled water. Table II.
Production of Fluorescence by Different Aminochromes with Zinc Acetate
FluoConcentrarescence, Maxima, h'lp tion in Sample, Arbitrary Reading ExcitaUnitso at Max. tion .Emission Compounds umole 78 105 390 480 Noradrenochrome (2,3-dihydro-3- 0.005 16 20 hydroxyindole-5,6-quinone) 0.001 70 ... 410 500 Adrenochrome (2,3-dihydro-30.005 hydroxy-iV-methylindole-5,6-quin- 0.001 15 ... one) 127 ... 410 500 A'-Ethylnoradrenochrome 0.005 25 ... 0.001 410 500 N-Isopropylnoradrenochrome 0.005 115 0.001 25 15 ... 410 0.005 500 2-Iodoadrenochrome 3 ... 0.001 19 ... 410 500 2-Iodo-oxoadrenoc hrome 0.005 . . , 4 0,001 43 ... 410 500 2-Iodo-h'-ethylnoradrenochrome 0.005 10 ... 0.001 91 ... 410 500 2-Iodo-N-isopropylnoradrenochrome 0,005 20 ... 0,001 10 ... 410 500 2- hlethylnoradrenochrome 0.005 2 0.001 ... 440 2-Carboxynoradrenochrome (Dopa- 0.005 1 27 325 0 5 chrome) 0.001 10 ... 410 Adrenochrome bisulfite complex 0.005 500 0.001 2 ... a. Reagent blank readings subtracted; 100 x 0.1 range, 410-m~excitation, 500-m~ emission. 18
ANALYTICAL CHEMISTRY
micrograms of aminochrome (expressed as adrenochrome) per liter
STABILITY
OF
FLCORESCENCE.
Adrenochrome reacts with zinc acetate in the presence of acetone, trichloroacetic acid, and distilled water and produces a stable fluorescence within 3 to 5 niinutes a t 410-mu excitation and 500-mp emission maxima as reported ( 4 ) . The fluorescence produced by zinc acetate with blood coincides with the same maxima. Blood samples and trichloroacetic acid extracts of blood nere stable without loss of aminochromes for 90 minutes' storage a t 4' C before determination. After reagent 3 ~1as added to the blood extracts the fluorescence was stable for 60 minutes a t low concentrations and 15 minutes a t high concentrations of adrenochrome (Table I). EXPERIMENTAL COMPOrrh-DS T H A T PRODUCE FLUORESCENCE WITH ZINC ACETATE. The
compounds listed in Table I1 produce fluorescence T\ ith zinc acetate n ith different intensity and excitation and emission maxima in the presence of trichloroacetic acid, acetone, and distilled Rater. The effect of other zinc compounds TI as reported (4). Other acetate salts have no effect on fluoreecence formation by aminochromes.
COMPOUXDS THATDo KOTPRODLCE FLLORESCENCE WITH ZIKC ACETATE. Compounds that do not produce fluorescence n i t h zinc acetate a t 0.001- to 0.005-pmole quantities in the sample are: tryptophan, 5-hydroxytryptophan, tryptamine, 5-hydroxytryptamine, indole-3-acetic acid, 5-hydroxyindole-3acetic acid, 5-hydroxyindole, adrenolutin (1 - methyl - 3,5,6 - indoletriol). 2,3 - dihydro - 3 - methoxg - S - methylindole - 5,6 - quinone (adrenochrome methyl ether), 2,3-dihydro-X-methylindole-5,6-quinone (epinochrome), 3ethoxy - 2,3 - dihydroindole - 5,6quinone (3-ethoxyepinochronie), 5,Gdihydroxy-.V-methylindole (leucoadrenochrome), 2-iodo-5,B-dihydroxyK-niethylindole, 2-iodo-2-methylnoradrenochrome, X-isopropyloxonoradrenochrome, epinephrine [l-(3.4dihydroxphenyl) - 2 - niethylamii?t ethanol], norepinephrine [l-(3,4dihydroxyphenyl) - 2 - aminoethanol], adrenalone (3,4-dihydroxy-p-isopropj L amino-acetophenone), X-isopropylnoiadrenalone, epinine [A'-methyl-2-(3,4dihydroxyphen yl) -ethyl amine 1, DO PA [p - (3,4 - dihydroxyphenyl) - cy - alnnine]. dl-dopamine [4-(2-aminoeth>1)pyrocatechol], 3,4-dihydroxyphenylserine, ephedrine (c~-hydroxy-p-methylaminopropglbenzene), and CY - (3,4dihydroxypheng1)-a- hydroxy - p- aminopropape.
RECOVERY AND DETERMINATION O F -4DRENOCHROME I N AND PLASMA.
BLOOD
Intravenous injections of adrenochrome (10 mg.) in distilled water given to
Table 111.
Cases Nonschizophrenic mental patients
Aminochrome Determinations on Nonschizophrenic Patients
Fluorescence, Arbitrary Units" B a b Range, Range, Range, Mean i: iMean f Mean i 21.5 4.5 12.0 1 10.0 1 12.0 1 10.0 13.5 2.5 1 30.0 17.0 13.0 1 11.0 1 15.5 5.5 13.5 1 11.5 1 30.0 10.0 13.0 1 11.0 1 16.0 4.5 13.5 1 11.5 1
Yo. of A Individuals Range, Material Tested Mean f Blood 8 34.0 8 Plasma 3.5 3 18.0 Blood 3 40.0 17.0 Plasma 3 23.0 9.5 Blood 3 46.0 16.5 Plasma 3 29.5 11.5
iiminochrome, ?/Liter (Adrenochrome Equivalent) 375 120 290 196 500 376
Before adrenochrome injection in same group hfter intravenous injection of adrenochrome in distilled water a 0.1 range x 100. Flasks contain: -4, B: 2 ml. of 1Oyotrichloroacetic acid in distilled water, filtrate of blood ( 1 : 2). a, b: 2 ml. of distilled water, TCA, filtrate. A, B and a, b: 2 ml. of 10% TC.4 in acetone. A, a : 1 ml. of 1M zinc acetate in lOy0 TCA. B, b: 1 ml. of 10% TCA.
mental patients increased the fluorescence by zinc acetate (Table 111). Recovery of injected adrenochrome from total body fluids, 15 minutes after injection, was 63% for a subject who weighed approximately 65 kg. This may be due to absorption of adrenochrome by the tissues, decomposition ( d ) , or excretion. Recovery of added adrenochrome to the blood in vitro was also low (Table IV), whereas yields were high when adrenochrome was added to trichloroacetic acid extracts of blood.
AMINOCHROME LEVELSIN THE BLOOD. The mean aminochrome concentration (expressed as adrenochrome) in the blood of 11 nonschizophrenic mental patients was 332.5 f 149 and 158 f 71.5 y per liter for plasma (Table 111). Aminochromes already present in the blood or injected adrenochrome nere not equally distributed in the red cells and plasma and there were more in the red cells DISCUSSION
Fluorescence produced in blood by the zinc acetate reaction very likely originates from aniinochromes with the 2,3dihydro - 3 - hydroxyindole - 5,6quinone structure, and N-methyl, N-ethyl, and iV-isopropyl derivatives do not alter fluorescence. 2-Iodo, 2methyl. or 2-carboxy derivatives produce considerably less fluorescence. The 3-hydroxy group had to be present and free as 2,3-dihydro-iY-methylindole5)6-quinone (epinochrome) , 3-ethoxy-2, 3 - dihydro - N - methylindole - 5,6 quinone (3-ethoxyepinochrome), and 3 - methoxy - 2,3 - dihydro - N - methylindole - 5,6 - quinone (adrenochrome methyl ether) did not produce fluorescence. There was no interference b y other indoles and catecholamines with the fluorescence of aminochromes after reaction with zinc acetate. Harley-Mason had reported the conversion of 3hydroxy - 2,3, - dihydroindole - 5,6-
Table IV.
Recovery of Added Adrenochrome to Blood in Vitro
Arbitrary Fluorescence Units" Blank 3 Blood (stored) TCA 18 14 Blood (fresh) TCA 22 Blood (stored) adrenochrome TCA 14 (1 -/ per ml.) 28 Blood (stored) adrenochrome TCA (0.5 y per ml.) 23 14 TCA adrenochrome blood (stored) (1 y per ml.) 29 14 adrenochrome blood (stored) TC.4 23 (0.5 y per ml.) 14 Filtrate KO.2 and adrenochrome (1 y per ml.) 40 15 Filtrate No. 2 adrenochrome (0.5 y per ml.) 30 15 Distilled water adrenochrome + TCA (1 y per ml.) 57 10 Distilled viater adrenochrome TCA (0.5 y per ml.) 34 10 a 0.1 range x 100.
+ + + +
+
+ +
+ +
+
+ + +
+
quinone to 3,5,6 - trihydroxyindole by zinc and aluminum salts. The authors confirmed this finding that the reaction end product of 3-hydroxy-2,3-dihydroN-methylindole-5,6-quinone (adrenochrome) and zinc acetate had spectrofluorometric properties of pure l-methyl3,5,6-indoletriol (adrenolutin) (4). However, in the presence of trichloroacetic acid the rate of decomposition of the adrenolutin and adrenochrome reaction end product with zinc acetate was not identical (Table I), The zinc complex of l-methyl-3,5,6-indoletriol is more stable than l-methyl-3,5,6-indoletriol itself in trichloroacetic acid-acetonedistilled water mixture, which indicates they are not identical. Further evidence t h a t the fluorescence produced in blood b y zinc acetate is aminochrome will be reported in accordance with the findings of Osinskaya and Utevskil (3, 6).
Recovery, 5 8
..
70 ...
..
...
14
9
21.4
9
4
18.2
15
10
23.8
9
4
18.2
25
20
47.5
15
10
45.5
47
42
100
24
22
100
A
A - 1
ACKNOWLEDGMENT
The authors thank R. A. Heacock for the preparation of several of the standard compounds used in this investigation. LITERATURE CITED
(1) Feldstein, A,, Worcester Foundation for Experimental Biology, Shrewsbury, Mass., personal communication, June 19.58.
(2) Hoffer, A., Smith, C., Chwelos, N., Callbeck, M. J., Mahon, M., J . CEin. Ezptl. Psychopath~l.20 , 125-34 (1959). (3) Osinskaya, V. O., Biokhimiya 22, 537 (1957). (4) Payza, A. K.,Mahon, M. E., ANAL. CHEM.31,1170 (1959). (5) Utevskil, A. M., Osinskaya, V. O., Ukrain. Biokhim. Zhur. 27, 401 (1955).
RECEIVED for review June 29, 1959. Accepted September 28, 1959. Work supported by National Health Grants, Ottawa, and the Rockefeller Foundation, New York. VOL. 32, NO. 1, JANUARY 1960
19
