eA ENTS oy para i - | ' ; ’ en 4 , + THE EFFECT OF SURFACE-ACTIVE SUBSTANCES ON THE FUCHSIN REACTION OF HIGHER FATTY ALDEHYDES* Br GABRIELE EHRLICH, HARRIETT E. TAYLOR, anp HEINRICH WAELSCH From the Departments of Biochemistry, New York State Psychiatric Institute and the College of Physicians and Surgeons, Columbia University, New York) (Received for publication, September 26, 1947) Higher fatty aldehydes are present in considerable quantity in the lipide ‘action of muscle and brain and may be intermediates in lipide metabolism 1,2). The meager information concerning the quantitative distribution {the higher fatty aldehydes in tissues has been acquired exclusively by quantitative evaluation of the Schiff reaction, as in the procedure of Feul- --n and Griinberg (3) or modifications of it (4). Serious doubt as to the seliability of the method arose when it was found that added palmitalde- ayde or stearaldehyde or their acetals could not be estimated quantitatively n tissue extracts (4). The experiments reported in this paper show that the result of the quan- -tative fuschsin reaction for the determination of higher fatty aldehydes pends to a large degree on the presence of surface-active lipides in the cue extract. Naturally occurring lipides or synthetic surface-active -zents inhibit the color development if added at the beginning of the reac- son, and destroy the color already formed if added later. This effect of tface-active agents can be suppressed to a lurge degree by reducing the xater content of the medium through the use of a high concentration of setic acid. EXPERIMENTAL The Schiff reaction was employed in three forms, two at low and one at ‘sh concentrations of acetic acid. In all expriments palmitaldehyde gly- «vl acetal was used as reference substance (4). Reactions at Low Acetic Acid Concentrations (Reactions S. F. and S. F. -')}—The Schiff reaction for the determination of higher fatty aldehydes » carried out previously in our laboratory (4) differed in three respects frorn procedure used by Feulgen and Griinberg (3): (a) 1 ml. of 1 w HCl was -led to the reaction mixture consisting of 10 ml. of fuchsin reagent, mer- ie chloride solution, and 1 ml. of glacial acetic acid containing the com- “ands or tissue components to be tested. It was found that increased “The higher fatty aldehydes, IV. This work was supported by grants from the -vah Macy, Jr., Foundation and the United States Public Health Service. 547 J Beek, CHEN. ITS. S47-SS1, IS 548 FUCHSLN REACTION OF PATTY ALDEHYDES acidity resulted in a greater precision. (b) The color was developed at 37° for 18 to 24 hours. (c) The color complex was extracted with capryl (4, 5) instead of amyl alcohol (3). In the present experiments this procedure (Reaction S. F. HCl) was compared with a procedure (Reaction S. F.) in which no HCl was added. The reaction mixtures contained 8.9 percent (Re- action S. F.) and 8.1 per cent (Reaction S. F. HCl) acetic acid respectively. Reaction at High Acetic Acid Concentration (Reaction S. A. A.)—~2 gm. of basic fuchsin (National Aniline Division) were dissolved in 50 ml. of gla- cial acetic acid. 10 gm. of sodium bisulfite, 100 ml. of 0.1 x HCl, and 50 ml. of water were added in succession. The reagent was used after it had stood for several hours. The bisulfite did not decolorize the solution ap- preciably; the final reagent retained a reddish brown color. To 1 ml. of gla- cial acetic acid containing the compounds or tissue components to be tested 2 ml. of glacial acetic acid and 1 ml. of fuchsin reagent were added. The color was developed in sealed glass tubes (9 mm. inside diameter, 10 ml. capacity) at 50° for 18 to20 hours. When cool, the sealed tubes were opened, 2 ml. were transferred to a 25 ml. graduated cylinder (glass-stoppered), and 10 ml. of an aqueous solution were added, containing 5 gm. of sodium bi- sulfite and 5 ml. of concentrated HC] in 100 ml. In the blank samples the color faded within 10 minutes to a light yellow. The sohitions were extracted with 10 ml. of capryl alcohol exactly 10 minutes after the ad- dition of the sulfite solution, and the alcoholic solution was cleared by centrifuging as described previously (4). Substrates—The preparation of palm“‘aldehyde and stearaldehyde and their acetals was described previously (4). As synthetic surface-active substances the non-ionic detergents, Tweens and Spans, of the Atlas Pow- der Company (mono- and polyesters of sorbitan with long chain fatty acids and their polyalkylene derivatives) were used. These substances pro- duced small and consistent color values. The crude egg yolk phospha- tides were prepared according to the method of Feulgen and Grinberg (3) and dried to constant weight. The samples gave fuchsin color values (Re- action 8. F. HCl) corresponding to as much as 720 mg. of palmitaldehyde per 100 gm. of lipide. In the experiments with brain extract the residue of an alcohol-ether extract of finely minced brain was dissolved in the ap- propriate amount of glacial acctic acid. In all experiments reported in this paper the color density was deter- mined with a Coleman junior spectrophotometer, model 6, in cuvettes No. 6-302 at 545 my. The values obtained in Reaction 8. A. A. were doubled, since only half of the reaction mixture was extracted with capryl alcohol. RESULTS AND DISCUSSION The addition of Span 20, egg yolk phosphatides, or brain lipides to pal- mitaldehyde or its glyceryl acetal resulted in an inhibition of the color de- G. EHRLICH, H. FE. TAYLOR, AND H. WAELSCH 549 _elopment if the Schiff reaction was carried out in a medium of approxi- nately 90 per cent water (Reactions S. F. and 8. F. HCl) (Table I). By arrying out the Schiff reaction in a medium containing 80 per cent acetic Tass I schiff Reaction of Palmitaldehyde and Its Glyceryl Acetal in Presence of Lipides and Synthetie Surface-Active Agents Schiff reaction” optical Analytical recovery Source of Amount Addition Amount Reac- | Rea- | Reac- | Reac | Re | Rene tion ee tion | tion ev tion S.F. HCI S.A.A.t] S.F. HCI S. A.A. ¥ meg. per cent | per cent} per cent valmitalde- | 30 0.11 | 0.24 hyde 30 | Span 20 4 0.02 | 0.23 18 96 | 30 “20 8 0 | 0.22 0 | 93 ; 40 0.16 | 0.16 | 0.24 40 Span 20f 20 | 0.09 | 0.03 | 0.25 | 56 20 105 35 0.26 35 Phosphatides | 16 0.20 77 20 0.12 | 0.09 20 Phosphatides | 20 | 0.05 | 0.02 40 22 20 0.07 | 0.12 Brain lipides 14§ 0.07 | 0.24 20 “ “ 14 0.10 | 0.32 44 66 Palmitalde- | 82 0.31 | 0.28 | 0.68 hyde gly- | 82 Span 20 20 | 0.02 | 0.05 | 0.64 6 18 94 ceryl 30 0.26 acetal 30 Phosphatides ' 15 | 0.26 100 | 40 | 0.16 _ 40 | Phosphatides | 1 0 0 ' 61 0.54 : Brain lipides | 11 0.32 61 “ “e | 11 | 0.78 85 * Tn all experiments in which Span 20 or egg phosphatides were added the values * .¢ correeted for the densities given by these substances alone. ; t Optical density for palmitaldehyde glyceryl acetal: 20.4 y, 0.17; 40.8 7, 0.35; 1 12-7, 0.52; 81.6 +, 0.68; 102 y, 0.82; for palmitaldehyde 20.2 y, 0.15; 40.5 y, 0.30; \" 7, 0.54; 101 y, 0.66. - ; t Span added 5 hours after the start of the reaction. ° | § Weight of wet brain. ; . “id (Reaction S. A. A.) the effect of the surface-active agents was mini- : ; zed and the recoveries of added aldehyde or acetal amounted to 66 to ‘U0 per cent. The recovery of total color resulting from the aldehyde or -cetal plus that from various compounds which were added amounted in i. neactions S. F. and S. F. HCI to 0 to 74 per cent and in Reaction 8. A. A. : ;° 88 to 100 per cent. 550 FUCHSIN REACTION OF FATTY ALDEHYDES The color reaction at high acetic acid concentration is approximate!: twice as sensitive as that carried out at low acetic acid concentrations. (- the latter reactions the one with the higher acidity (Reaction S. F. HC; is slightly less sensitive and more susceptible to the action of surface-actiy: agents than the one with lower acidity (Reaction S. F.). In agreement with previous observations, equivalent quantities of differ. ent aldehydes or of the same aldehyde on different days did not yield tt. same color density. The same aldehyde sample tested in different con. centrations does not follow Beer’s law (cf. foot-note, Table I) probably t« cause of the difficulty in obtaining monomeric aldehydes, and therefor: the results of experiments carried out on these substrates are variable With acetal a better linear relation is obtained (ef. foot-note, Table I). In an experiment reported in Table I, 1 mg. of egg yolk phosphatide in. hibited completely the color developed by 40 + of acetal (Reaction 8. F. HCl). However, when 0.5 mg. of phosphatide was added, definite inhib:. tion of color development was found in some experiments, but in others thc color values coincided (within the error of the method) with the contri: value of acetal alone. This finding may explain the result reported pr- viously (4) that addition of 1 mg. of egg yolk phosphatides, prepared ac- cording to Feulgen and Griinberg, does not affect the 18 hour color valu: of acetal. In that experiment the phosphatide preparation used was nc: dried to constant weight and the actual amount may have been consider- ably less than 1 mg. The effect of surface-active agents can also be demonstrated on the alde- hydes present in tissue extracts. 20 mg. of Tween 85 were added to a se! ution of brain lipides in glacial acetic acid. Measurements at different time intervals from 30 minutes up to 18 hours after mixing the reagents gave color values corresponding to 10 to 16 per cent of the simultaneous! determined control values. Similar results were obtained in experiment: in which Span 20 was used as the surface-active agent. The addition of detergent after full color had developed with palmitalde hyde (Reaction S. F. HCI) caused fading. On the other hand if Reactiv: §. A. A. was used, full color was obtained when the detergent was adde. 5 hours after mixing of the reagents (Table I). The effect of surface-active agents on the Schiff reaction of the highe’ fatty aldehydes in aqueous medium supports the point of view express: previously (4), that analytical results obtained with the fuchsin meth probably do not represent the true concentrations of higher fatty aldehyde in tissue extracts. ‘The fact that consistent values have been obtained m>: be a reflection of relatively constant ratios between higher fatty aldehyde and surface-active lipides. An apparent variation, found by the fuchs" method in aqueous media, in the aldehyde concentrations in a tissue unde: G. EHRLICH, H. BE. TAYLOR, AND H. WAELSCH aol why -siological or pathological conditions may be the result of a change in -sncentration of aldehydes, of surface-active lipides, or of hoth (5, 6). rhe form in which the aldehy des are present in the extracts is not known vith certainty, and the lipide composition oceurring in an extract obtained .om tissues With an organic solvent cannot be duplicated experimentally. Iti is, therefore, difficult to decide how well model experiments with deter- nts, free aldehydes, and acetals approximate conditions in tissue ex- cracts. But it appears that the new procedure elaborated for the use of -he Schiff reaction as presented in this paper may climinate oneof the potential errors in the determination of the higher fatty aldehydes in tissue extracts SUMMARY Naturally occurring lipides and synthetic surface-active substances in- mbit the color development of the higher fatty aldehydes and their acetals n the fuchsin test as proposed by Feulgen and used in the original or modi- “ed form by others. If the surface-active substances are added after color ans developed, rapid fading occurs. Addition of synthetic detergents in the determination of aldehydes present in tissue lipides also suppresses the development of color to a marked degree. These findings cast serious doubt on the usefulness of the fuchsin method for the quentitative deter- mination of the higher fatty aldehydes as carried out with the Feulgen method or its modifications. The effect of surface-active, naturally occurring or synthetic agents is suppressed tu a large degree if the Schiff reaction is carried out in a medium rontaining a high concentration of acetic acid. BIBLIOGRAPHY . Anchel, M., and Waelsch, H., J. Biol. Chem., 145, 605 (1942). . Ehrlich, G., and Waelsch, H., J. Biol. Chem., 163, 195 (1946). . Feulgen, R., and Grinberg, H., Z. physiol. Chem., 257, 161 (1938). . Anchel, M., and Waelsch, H., J. Biol. Chem., 162, 501 (1944). Waelsch, H., Proc, Am. Soc. Biol. Chem., J. Biol. Chem., 140, p. exxxvi (1941). s. Anchel, M., and Waelsch, H., Federation Proc., 2, 57 (1943). —_ Wt ee ee ty