Reaction of Dihydroxyacetone (Dha) with Human Skin Callus and Amino Compounds* Eva Wittgenstein and Helen K

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REACTION OF DIHYDROXYACETONE (DHA) WITH HUMAN SKIN CALLUS AND AMINO COMPOUNDS* EVA WITTGENSTEIN AND HELEN K. BERRY, M.A.

Dihydroxyacetone (DHA), a keto-sugar, Solutions of DHA and fructose (10%) were CH2OHCOCH2OH, has been known for atreated with ammonia, amino acids, or related number of years as an intermediate product incompounds. These results are shown in Table 1. normal carbohydrate metabolism in humans andArginine and DHA reacted almost immediately to produce a yellow color. A stable brown color animals (1, 2, 3, 4). Recently it was shown thatdeveloped within 30 minutes. Other basic amino solutions of DHA are able to stain human skinacids and glycine reacted more slowly with a (5). The present studies were undertaken toyellow or brown color after 6 to 12 hours. Further investigate the chemical mechanism responsibletests were performed on arginine and some sub- for the "tanning" phenomenon. stituted guanidine compounds to test whether the reactivity of the guanido group in arginine might METHODS AND RESULTS be modified by substituents. Aminoguanidine Callus from human skin was treated with solu-bicarbonate when combined with DIIA solution tions of DHA of varying concentration. By usingturned dark brown within 3 hours. 1 ,3-diphenyl- ground callus the reactive surface area was en-guanidine showed a faint color reaction with DHA larged. Control samples of callus were treated withwhen the mixture was allowed to stand 4 days normal saline. Specimens were also treated withat 3° C. 1,2,3-triphenylguanidine and DHA de- fructose solutions for comparison. Fructose isveloped a slight yellow color only after three also a keto-sugar which, like DHA, possessesweeks at 30° C. Guanidine nitrate and DHA re- hydroxyl groups on carbons adjacent to the car-acted to produce a faint yellow color after 3 weeks. bonyl carbon. The callus developed a dark brownNo color formation occurred when DHA was com- color at room temperature a few hours after beingbined with urea. The reactivity of the DHA- painted with 10% solution of DnA. A brown colorguanidine mixture appears to be directly related developed when unpainted callus was ground intoto the availability of the guanidine hydrogens a powder and mixed with solutions of DHA. Nofor reaction. immediate reaction occurred when callus was The reaction mixtures of DHA with ammonia treated with 10% fructose solution. Ground callusand amino acids were subjected to paper chroma- treated with fructose solution developed a deeptography and qualitative chemical tests for func- brown color when heated on a steam bath. Piecestional groups. In Figure 1 are shown the positions of callus immersed in concentrated fructose solu-on chromatograms of DHA and intermediate tion turned brown when heated in a boiling waterproducts in the reaction between DHA and am- bath for 10 to 15 minutes. monia. Untreated DHA (300 jig) was added to the Solutions of DHA dried on filter paper at roomorigin. The chromatogram was resolved in an temperature turned brown when exposed to theammoniacal atmosphere during which reaction laboratory atmosphere for several days. Theof DHA and ammonia occurred. After removal brown substance was eluted from the filter paper.of the first solvent, the reaction products were Spot tests using an equilibrium solution of zincseparated in butanol-acetic acid-water solvent. oxinate on the eluant showed the presence ofThree stable intermediate substances were pro- ammonia (6). Control papers kept in an ammoniaduced. Two of these no longer gave reactions free atmosphere in tightly closed wrappings or incharacteristic of sugars (see Table 2), but reacted a desiccator over sulfuric acid did not developwith Pauly reagent (diazotized sulfanilic acid- color after several weeks. Eluant from thesesodium carbonate) to produce red color. If solu- papers gave negative tests for ammonia. tions containing the isolated intermediate compounds were heated, a brown color developed. *FromThe Children's Hospital Research Foundation and the Department of Pediatrics,When mixtures of DHA and amino acids were University of Cincinnati, College of Medicine,chromatographed, in addition to the unchanged Cincinnati, Ohio. amino acid, substances were detected which were With assistance of a grant-in-aid, No. MA-1175, from the National Institutes of Health, U. S.positive both with ninhydrin (amino acid) and Public Health Service. aniline-phthalate (sugar) reagents (7). The nature Received for publication June 29, 1960. of these compounds produced by interaction of 283 284 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

TABLE 1 Reactions of DHA and fructose with amino compounds

DHA Fructose

Ammonia vapor immediate formation of a dark brownbrown colored syrup after approx. 1 hr. syrupy compound Arginine immediate yellow; dark brown afterbrown color after 12 hr. at 30° or sev- 30 minutes eral minutes at 100° Glycine yellow after 60 mm; dark brown afterbrown color after 72 hr. at 30° 12 hr. Histidine Same as glycine Lysine Same as glycine Tryptophane yellow after 12 hr. no color reaction after 12 hr. Alanine faint yellow after 12 hr. Valine, Leucine, no color change after 12 hr. no color reaction Phenylalanine Amino guanidine- dark brown within 3 hr. no color reaction bicarbonate 1 ,3-diphenyl- faint yellow-gray after 96 hr. at 30° guanidine 1,2,3-triphenyl- faint yellow after 3 weeks at 30° guanidine Guanidine nitrate faint yellow after 3 weeks at 30° Urea no color formation at 300 or 1000 no color formation

0 which never became as dark as under the influence of DHA alone. Callus painted with formaldehyde or ground callus mixed with formaldehyde gave 1! no color reaction even upon heating. Addition of formaldehyde to the reaction mixture of DHA and arginine inhibited the color formation.

DISCUSSION I Ammonia and its derivatives such as amines have long been known to react readily with 2 carbonyl compounds (10). Ketones generally are 3OO' less reactive than aldehydes. Reducing sugars Butonol-GlaciatAcetic Acid —water (80—20-20) readily form hydrazones, oximes, ureides, and Fia.1. Two dimensional chromatogram ofsemicarbazones, for example. The interaction of DHA and unknown substances formed in reactionamino acids and proteins with carbohydrates has between DHA and aminonium hydroxide. been shown to be the cause of much of the browning that occurs during manufacture and DHA and amino acids or ammonia is also understoring of foods (8). Richards (9) investigating investigation. the interaction between D-glucose and glycine, Fructose and ammonia reacted to give a brownsucceeded in isolating an intermediate in the color after an hour. Fructose and arginine gave abrowning reaction, the enolic form of N-(carboxy- brown color after 12 hours at 30° C, or after several minutes at 100° C. methyl)-amino-1-deoxyfructose, which gave re- When callus was treated with formaldehydeactions of both a sugar and an amino acid. several minutes prior to the application of DHA,Carbonyl compounds with strongly electron- no color was observed. When equimolar amountsattracting groups are highly reactive. In the of DHA and formaldehyde were mixed in aqueouscase of DHA, the normal electrophilic reactivity solution and the callus was painted with thisof the carbonyl group is enhanced by the hydroxyl mixture, a yellow or light brown color developedgroups on adjacent carbon atoms. Arginine with DHA AND HUMAN SKIN CALLUS 285

TABLE 2

UV Light Ammontum Aniline molybdate Phospho molybdate plitilalate Pauly reagent

DHA (spot 2) yellow-white + + + — Unknownl — — — — + Unknown3 — — — + Unknown 4 faintyellow ft+ ft+ ft+ yellow Fructose yellow-white + + (slow) + — Reactions of unknown intermediate compounds produced by combination of DHA and ammonium hydroxide. Compared with DHA and fructose. its nucleophilic guanido group reacts readily SUMJL&RY with DIIA, to form a stable dark brown color The reaction of dihydroxyacetone (DHA) with after 30 minutes. amino acids and with callus from human skin The formation of stable intermediates in thehas been studied in relation to the "tanning" reaction of DHA and ammonia suggests aeffect of DHA on human skin. Ground human similarity in the mechanism to that describedskin was stained brown by solutions of DHA at by Richards in interaction between glucose androom temperature; callus immersed in fructose glycine. The reaction of fructose with arginineturned brown when heated at boiling for 15 and skin to produce a brown color indicates thatminutes. Arginine and DHA react rapidly to the browning of skin may be a general reactionform a brown color. Fructose and arginine react characteristic of amino acids and sugars. Thesimilarly but at a much slower rate at room rapid development of color which occurs whentemperature. Chromatographic studies revealed skin is treated with DHA results from the highthe presence of several intermediate products reactivity both of DHA and the basic groupswhich may be responsible for the browning of arginine. The decreased color noted whenphenomenon. From the point of view of these formaldehyde was added as a third reactant isstudies, argiine is one of the most important consistent with the observation that formalde-amino acid constituents of human skin. Com- hyde is an unusually active carbonyl compoundbination of DIIA and the basic groups of and reacts readily with amino acids by substitu- arginine is probably responsible for the browning tion of the amino group. It also combines withwhich is observed when human skin is treated ammonium hydroxide to produce a brownwith DHA. syrupy substance. In the reaction of DHA with skin or arginine, formaldehyde probably com- ACKNOWLEDGMENT petitively inhibits color formation. Arginine is present in proteins of the skin in We are grateful to Dr. R. M. Delcamp, high concentration. It seems likely that thisProfessor of Chemistry, University of Cincinnati, amino acid is responsible for most of the "tan-for samples of substituted guanidine compounds ning" effect of DHA. Variations in color develop-used in this study and for helpful discussion. ment and the relation to individual variations in REFERENCES amino acid composition of skin should be in- 1. RABIN0wITCH, I. M. (with the assistance of vestigated. The relatively slow rate of reaction Althea B. Frith and Eleanor V. Baxin): Blood sugar time curves following the of DHA on the skin (6 to 8 hours when applied ingestion of dihydroxyacetone. J. Biol. as a cosmetic) as compared to the rapid rate of Chem., 65: 5—58, 1955. reaction of DHA and arginine in vitro (less than 2. LINNEWEH, F.: Zur Differentialdiagnose kindlicher Lebertumoren. Mschr. Kinder- 30 minutes) might be due to any one of several heilk., 67: 422—428, 1936. factors, such as availability of free amino groups 3. MASON, E. H. (with the technical assistance of Miss Eleanor Hill): Dihydroxyacetone in skin protein, pH of skin, or presence of inhibi- studies. I. Its respiratory and carbohy- tors. Further investigation will be required to drate metabolism in normal men. J. Clin. clarify details of the staining mechanism. Invest., 2: 521—544, 1925. 286 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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