1122
Specialia
c e n t r a t i o n s u p to 0.5 m M , giving n e a r l y 100% r e a c t i v a tion, b u t h i g h e r Zn 2+ c o n c e n t r a t i o n s c a u s e d a c o n s i d e r able inhibition. Ca s+ was c o n s i d e r a b l y less effective, a n d m u c h h i g h e r c o n c e n t r a t i o n s were required. The small a m o u n t of purified e n z y m e o b t a i n e d did n o t allow t h e d e t e r m i n a t i o n of t h e m e t a l c o n t e n t s of t h e m e t a l l o p r o t e i n . Since CaC1 s was r e q u i r e d for t h e p r o d u c t i o n of t h e protease, it m i g h t be s u p p o s e d t h a t t h e d i v a l e n t c a t i o n p r e s e n t in t h e m e t a l l o e n z y m e was Ca s+. H o w e v e r , as s h o w n in F i g u r e 2, o - p h e n a n t r o l i n e a t c o n c e n t r a t i o n s u p t o 1 m M i n h i b i t e d t h e e n z y m e e v e n in t h e p r e s e n c e of 10 m M CaCls, t h u s i n d i c a t i n g t h a t t h e d i v a l e n t c a t i o n
100~ %
o
o
50 ,,=,
' 0'.5 110 m/"/ Concentration of o-phenantro[ine Fig. 2. Inhibition of the extracellular protease from Ps. ]luorescens by o-phenantroline. 0.1 mI of purified enzyme preparation, previously exhaustively dialyzed against 20 mM Tris-tlC1 buffer (pH 7.6), was assayed as described under Methods except for the buffer, which was 30 mM Tris-acetate (pH 6.6), and o-phenantroline, which was directly added to the assay mixtures at the concentrations stated on the abcissa, in the absence (O) or in the presence (9 of 10 mM CaCI~. h~ order to avoid interference by o-phenaatroIine, after centrifugation a 1 ml-aliquot of the supernatant of the assay mixture was added 2 ml of 1 M NaOH and 0.5 ml of Folin-Ciocalteau's phenol reagent, diluted 1/a15. After 30 min at room temperature, the absorbance of the solutio~ at 750 am was read and taken as an expression of the enzyme activity. Blanks, to which trichloroacetie acid was added before the enzyme, were substracted. The enzyme activity is expressed as percent of the enzyme activity in the absence of the inhibitor (A A75o~,Imit~ = 0.11).
Occurrence of Trichochromes G. PROTA,
H. RORSMAN
a c t i n g as p r o s t h e t i c g r o u p is n o t Ca 2+, being p r o b a b l y one of t h e c a t i o n s able to f o r m s t r o n g c o m p l e x e s w i t h o - p h e n a n t r o l i n e , such as Co s+ or Zn s+. A similar f i n d i n g h a s b e e n r e p o r t e d for t h e alkaline p r o t e a s e of Ps. aeru-
ginosa t3. CaCI2, w h i c h is k n o w n to stabilize s o m e e x t r a c e l l u l a r protease*, did n o t p r o t e c t t h e Ps./luorescens e n z y m e , w h i c h was a l m o s t c o m p l e t e l y i n a c t i v a t e d a f t e r i n c u b a t i o n a t 50 ~ for 10 rain, e i t h e r in t h e a b s e n c e or in t h e p r e s e n c e of 5 m M CaC1 s. The results p r e s e n t e d in t h i s c o m m u n i c a t i o n s h o w t h a t Ps. fluorescens p r o d u c e s a n e x t r a c e l l u l a r p r o t e a s e able to digest casein w i t h i n a p H r a n g e w i d e r t h a n t h a t r e p o r t e d for t h e e n z y m e s purified f r o m o t h e r Pseudomonas 3,5. T h e molecular w e i g h t of t h e e n z y m e is similar to t h a t rep o r t e d for t h e p r o t e a s e s f r o m Ps.maltophilia 4 and Ps. ]ragi ~, b u t s i g n i f i c a n t l y l o w e r t h a n t h a t of t h e alkaline p r o t e a s e of Ps. aeruginosa 1~. T h e Ps./luorescens p r o t e a s e is a m e t a l l o e n z y m e , as t h e e n z y m e s p r o d u c e d b y o t h e r Pseudomonas 3, 4, 5, b u t in our case a stable a p o e n z y m e was o b t a i n e d b y dialysis a g a i n s t E D T A for r a t h e r long p e r i o d s of time. The lack of e n z y m e r e a c t i v a t i o n b y dialysis a g a i n s t distilled w a t e r suggests t h a t in t h e case of t h e Ps.fluorescens p r o t e a s e t h e m e t a l p r o s t h e t i c g r o u p was a c t u a l l y r e m o v e d , a n d n o t s i m p l y m a s k e d , b y t h e c h e l a t i n g agent. The second p o s s i b i l i t y is t h o u g h t t o be t r u e for t h e Ps. aeruginosa 14 e n z y m e . The d i v a l e n t c a t i o n b o u n d to t h e a p o e n z y m e m i g h t be Co s+ or Zn s+, as sugg e s t e d b y t h e g r e a t e r effectiveness of t h e s e cations for t h e r e c o n s t i t u t i o n of t h e h o l o e n z y m e (Figure 1) a n d b y t h e i n h i b i t i o n b y o - p h e n a n t r o l i n e in t h e p r e s e n c e of CaC1 s (Figure 2). Since o n l y one p r o t e o l y t i c e n z y m e w a s d e t e c t e d in t h e c u l t u r e s u p e r n a t a n t s of Ps. [luorescens, a n d it was able to digest casein a t n e u t r a l p H , t h e p r o t e a s e described here can be c o n s i d e r e d r e s p o n s i b l e for t h e gelatine h y d r o l y s i s w h i c h is one of t h e p h e n o t y p i c c h a r a c t e r s of Ps.fluo~,.escens
g .
13 t{. MORIHARAand H. TSUZUKI, Agrie. biol. Chem. 38, 621 (1974). 14 I(. MORIHARAand H. TsUzuKI, Biochim. biophys. Acta 92, 351 (1964). t5 J. C. P~eKUPand D. B. HoPI~, Biochem..7- t23, 153 (~971).
in the Urine of a Melanoma
2, A.-M. ROSENGREN
EXPERIENTIA 32/9
Patient 1
and E. I~OSENGREN
Department o/ Organic Chemistry, University o/ Naples, Napoli (Italy), and Department o/ Dermatology, Biochemistry and Pharmacology, University o/Lund, S-227 85 Lund (Sweden), 29 March 1976. Summary. The p r e s e n c e of t w o p h a e o m e l a n i c p i g m e n t s , t r i c h o c h r o m e B a n d C, was d e m o n s t r a t e d in t h e urine of a patient with malignant melanoma metastases. T r i c h o c h r o m e s 3-5, f o r m e r l y n a m e d trichosiderins, are a u n i q u e g r o u p of a m i n o acidic p i g m e n t s possessing a A 2 , 2 ' - b i - ( 2 H - 1 , 4 - b e n z o t h i a z i n e ) c h r o m o p h o re (Figure), w h i c h a c c o u n t s for t h e i r c h a r a c t e r i s t i c p H - d e p e n d e n t visible s p e c t r a . T h e y are t h e s i m p l e s t group of p h a e o m e l a n i c p i g m e n t s , a n d are f o r m e d in m e l a n o c y t e s b y a d e v i a t i o n of t h e e u m e l a n i n p a t h w a y i n v o l v i n g as k e y s t e p t h e 1,6a d d i t i o n of c y s t e i n e to d o p a q u i n o n e to give 5-S- a n d 2S-cysteinyldopa.
Unlike eumelanin, w h i c h occurs in several t y p e s of pigm e n t e d tissues, t r i c h o c h r o m e s h a v e so far b e e n f o u n d o n l y in c e r t a i n red hair a n d feathers, a n d it has b e e n s u g g e s t e d t h a t t h e i r f o r m a t i o n is s o m e h o w r e s t r i c t e d t o t h e s e k e r a t i u i z e d s t r u c t u r e s . T h e r e c e n t finding of large a m o u n t s of 5 - S - c y s t e i n y l d o p a in t h e urine of m e l a n o m a patients~, ~ led us to look for t r i c h o c h r o m e s in t h e urine of a p a t i e n t w i t h m e l a n o m a m e t a s t a s e s a n d m a r k e d l y
15.9. 1976
OH
HO2C
1123
Specialia
H
OH
NH2
OH
HO2C
NH2
1 R = CO2H 3 : R =H
OH
NH2
CO2H
H2N
CQ2H
OH
HO2C
5
H2N
4:R=H
:
HO2C
OH
2 - R-- CO2H
H2N
OH
H
H2N
NH2 6
1. Trichochrome B. 2. Trichochrome C. 3. Decarboxytrichochrome B. 4. Deearboxytrichochrome C. 5. Triehochrome E. 6. Trichochrome F.
e l e v a t e d u r i n a r y e x c r e t i o n of 5 - S - c y s t e i n y l d o p a a n d related metabolites. M a t e r i a l a n d methods. U r i n e was o b t a i n e d f r o m a male p a t i e n t aged 46 y e a r s w i t h r e d - b l o n d hair. H e h a d b e e n o p e r a t e d on for a c u t a n e o u s m e l a n o m a , a n d h a d wides p r e a d m e t a s t a s e s . 24-hour s p e c i m e n s were collected in plastic b o t t l e s c o n t a i n i n g 50 m l of acetic acid a n d 1 g of s o d i u m m e t a b i s u l p h i t e . D e t e r m i n a t i o n of 5-S-cysteinyld o p a a n d d o p a + d o p a m i n e was carried o u t b y m e t h o d s p r e v i o u s l y d e s c r i b e d 8, ~. A u t h e n t i c s a m p l e s of d e c a r b o x y t r i c h o c h r o m e B a n d C were o b t a i n e d as follows, 22 m g of a m i x t u r e of 2-S- a n d 5 - S - c y s t e i n y l d o p a (molar r a t i o c. 1:9) was dissolved in a small v o l u m e of 0.1 N HC1. The solution was p a s s e d on a c o l u m n (2 • 10 cm) of D o w e x 50W'-X4, 200-400 mesh, H+ form. A f t e r w a s h i n g w i t h w a t e r (200 ml), t h e c o l u m n was t r e a t e d w i t h 0.5 N N a O H , a n d yielded a b r o w n i s h yellow eluate w h i c h was acidified t o p H 1 w i t h 6 N HC1 a n d boiled for 20 min. T h e r e d d i s h - b r o w n s o l u t i o n so o b t a i n e d was r e - c h r o m a t o g r a p h e d on a D o w e x 50W c o l u m n e q u i l i b r a t e d w i t h 0.1 N HCI. A f t e r w a s h i n g as before, elution was first p e r f o r m e d w i t h 0.2 M s o d i u m a c e t a t e , w h i c h r e m o v e d m o s t of t h e coloured material, a n d finally w i t h 0.1 N N a O H . The alkaline eluate was a d j u s t e d to p H 3 a n d t h e p r e c i p i t a t e d p i g m e n t s were collected b y c e n t r i f u g a t i o n , dissolved in 0.1 N HC1, a n d I r a c t i o n a t e d b y p a p e r c h r o m a t o g r a p h y on W h a t m a n 3MM w i t h i s o p r o p a n o l - f o r m i c acid - c o n c e n t r a t e d HC1 (30:70:1, v/v). The red b a n d s c o r r e s p o n d i n g to decarb o x y t r i c h o c h r o m e B (lower R f value) a n d d e c a r b o x y t r i c h o c h r o m e C were c u t o u t a n d e l u t e d w i t h 0.1 N N a O H . Q u a n t i t a t i o n of t h e p i g m e n t s so o b t a i n e d was d o n e b y spectrophotometry. I s o l a t i o n of t r i c h o c h r o m e s f r o m t h e m e l a n o m a urine was carried o u t b y t h e following procedures. 1. A urine s a m p l e (10 ml) was acidified to p H 1 w i t h 6 N HC1 a n d boiled for 10 m i n t o c o n v e r t t r i c h o c h r o m e s B a n d C into
the corresponding decarboxy derivatives. Isopropanol (10 ml) was t h e n a d d e d a n d t h e m i x t u r e was s a t u r a t e d w i t h NaC1. A f t e r s h a k i n g for 5 min, t h e i s o p r o p a n o l l a y e r was w i t h d r a w n a n d e v a p o r a t e d to dryness. T h e residue was dissolved in a small v o l u m e of 0.1 N HC1 a n d a n a l y z e d d i r e c t l y b y TLC on cellulose using i s o p r o p a n o l - f o r m i c acidc o n c e n t r a t e d HCI (30: 70: 1) as eluent. 2. A 24-hour urine s a m p l e was acidified to p H 1 w i t h 6 N HC1 a n d p a s s e d on a c o l u m n (2 • 15 cm) of D o w e x 50W-X4, H + form. A f t e r w a s h i n g w i t h N HC1 (450 ml), 2 N HC1 (1200 ml), a n d w a t e r (200 ml) to r e m o v e cysteinyld o p a s a n d r e l a t e d m e t a b o l i t e s , t h e c o l u m n w a s eluted w i t h 0.5 N N a O H , a n d a b r o w n i s h - y e l l o w f r a c t i o n cont a i n i n g t r i c h o c h r o m e s was collected, acidified to p H 1, a n d boiled for 15 min. The r e s u l t i n g r e d d i s h - b r o w n so-
i Supported by grants from the Swedish Cancer Society (No. 626B75 04XA), 869-B75-01P, the Swedish Medical Research Council (No. B76-04X-00036-12), and the Walter, Ellen and Lennart Hesselman Foundation for Scientific Research. Professor PROTA was a visiting scientist of the Swedish Cancer Society (No. 626B75 04U). Reprint requests to: Professor HaNS RORSMAN, Department of Dermatology, Lasarettet, S 221 85 LUND, Sweden. a G. PROTA, in Pigmentation: Its Genesis and Biological Control (Ed. V. RILEY: Appleton-Century-Crofts, New York 1972), p. 615. a R. H. THOMSON,Angew. Chem. Int. Edit. 13, 305 (1974). s G. PROTAand R. H. THOMSON, Endeavour 35, 32 (1976). 6 H. RORSIVIAN, A.-M. ROSENGREN and E. ROS~NGREN, Yale J. Biol. Med. 46, 516 (1973). G. AGRUP, P. AGRUP, T. ANDERSSON, B. FALCK, J.-A. HANSSON, S. JACOBSSON, H. RORSMAN, A.-]3/[. ROSENGREN and E. ROSEN-
GREN, Acta dermatovener, Stoekhohn 55, 337 (1975). 8 H. RORSMAN, A.-M. ROSENGREN and E. ROSENGREN, Acts derma-
tovener, Stockholm 53, 248 (1973). 9 A. H. ANTON and D. F. SAYRE, J. Pharmac. exp. Ther. 145, 326 (1964).
1124
Specialia
l u t i o n was p a s s e d o n a smaller c o l u m n (2 • cm) of D o w e x 50W w h i c h was t h e n w a s h e d w i t h N HC1 (100 ml), w a t e r a n d 0.2 M s o d i u m a c e t a t e u n t i l t h e p H of t h e eff l u e n t rose to 5. S u b s e q u e n t e l u t i o n w i t h 0.1 N N a O H g a v e a b r o w n i s h - y e l l o w f r a c t i o n which, ~after acidification a n d e v a p o r a t i o n , was f u r t h e r p u r i f i e d b y c h r o m a t o g r a p h y on a c o l u m n (1.5 • 20 cm) of S e p h a d e x L H - 2 0 u s i n g m e t h anol-2 N HC1 (95:5) as eluent. U n d e r t h e s e c o n d i t i o n s , a well-defined red b a n d a p p e a r e d w h i c h was collected a n d e v a p o r a t e d to dryness. T h e residue was dissolved in a m i n i m u m v o l u m e of 0.1 N HC1. T h e r e s u l t i n g s o l u t i o n was first a n a l y z e d s p e c t r o p h o t o m e t r i c a l l y to d e t e r m i n e t h e t o t a l yield of t h e p i g m e n t s , a n d was s u b s e q u e n t l y fractionated by paper chromatography on Whatman 3MM w i t h i s o p r o p a n o l - f o r m i c a c i d - c o n c e n t r a t e d HC1 (30:70:1). 3. A s a m p l e of u r i n e (100 ml) was a d j u s t e d t o p H 13 w i t h 5 N N a O H a n d left a t r o o m t e m p e r a t u r e for 72 h under an oxygen current to convert cysteinyldopas into stable, colourless b e n z o t h i a z i n e d e r i v a t i v e s 10. T h e oxidized u r i n e was t h e n acidified t o p H 1 w i t h 6 N HC1 a n d passed o n a c o l u m n (2 • 10 cm) of D o w e x 50 W, H + form. A f t e r w a s h i n g w i t h N HC1 (150 ml) a n d water, t h e c o l u m n was e l u t e d w i t h 0.5 N N a O H to give a t r i c h o c h r o m e - c o n r a i n i n g f r a c t i o n w h i c h was w o r k e d u p as described in m e t h o d 2. As a c o n t r o l t o m e t h o d 3, a s o l u t i o n of 5 - S - c y s t e i n y l d o p a in 0.1 N N a O H was t r e a t e d e x a c t l y as t h e m e l a n o m a urine. Results. B e c a u s e of t h e p r e s e n c e of large a m o u n t s of 5-S-cysteinyldopa and related metabolites, examination of t h e m e l a n o m a u r i n e was carried o u t b y t h e 3 proced u r e s d e s c r i b e d a b o v e , all of w h i c h g a v e similar results. 1. Isopropanol extraction o/ trichochromes. C h r o m a t o g r a p h i c a n a l y s i s of t h e i s o p r o p a n o l e x t r a c t o b t a i n e d f r o m m e l a n o m a u r i n e r e v e a l e d t h e p r e s e n c e of 3 red p i g m e n t s , 2 of w h i c h s h o w e d p H - d e p e n d e n t colours a n d R f v a l u e s i d e n t i c a l to t h o s e of d e c a r b o x y t r i c h o c h r o m e B a n d C. T h e 3rd s p o t s h o w e d n o colour c h a n g e w h e n exposed to a m m o n i a v a p o u r , a n d s h o w e d a n 1Rf v a l u e w h i c h did n o t c o r r e s p o n d to a n y h i t h e r t o k n o w n t r i c h o c h r o m e .
2. Isolation of trichochromes a/ter removal o/ pigment precursors by ionic exchange resin. F r a c t i o n a t i o n of t h e m e l a n o m a u r i n e on D o w e x 50"vV c o l u m n s followed b y c h r o m a t o g r a p h y Of t h e c r u d e p i g m e n t m i x t u r e on a S e p h a d e x L H - 2 0 c o l u m n w i t h m e t h a n o l - 2 N HC1 (95:5) g a v e a single red b a n d w i t h a b s o r p t i o n m a x i m a a t 533 n m in 2 N HC1 a n d 460 n m in 0.1 N N a O H , c o r r e s p o n d i n g to d e c a r b o x y t r i c h o c h r o m e C + B . T h e t o t a l a m o u n t of t h e s e p i g m e n t s , d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y , was f o u n d t o be 5 m g / 2 4 h. S u b s e q u e n t c h r o m a t o g r a p h y of t h e m i x t u r e o n W h a t m a n 3 MM p a p e r led t o t h e isolation of p u r e d e c a r b o x y t r i c h o c h r o m e C a n d B, a l o n g w i t h t r a c e a m o u n t s of t r i c h o c h r o m e E a n d F identified b y t h e i r chromatographic and spectral properties.
EXPERIENTIA 32/9
T h e m e t h o d i n v o l v e s p r e l i m i n a r y p u r i f i c a t i o n of t h e u r i n e on s t r o n g l y c a t i o n i c e x c h a n g e resin, a n d s u b s e q u e n t h e a t ing of t h e p i g m e n t m i x t u r e in acid m e d i u m t o c o n v e r t t h e yellow u n s t a b l e t r i c h o c h r o m e B a n d C i n t o t h e corres p o n d i n g d e c a r b o x y d e r i v a t i v e s , w h i c h possess more favourable analytical properties. U s i n g t h i s p r o c e d u r e large a m o u n t s (up to 30 m g / 2 4 h) of d e c a r b o x y t r i c h o c h r o m e C a n d B along w i t h smaller q u a n t i t i e s of t r i c h o c h r o m e F a n d E were o b t a i n e d from t h e u r i n e of s e v e r a l p a t i e n t s w i t h m e l a n o m a m e t a s t a s e s a n d h i g h u r i n a r y e x c r e t i o n of 5 - S - c y s t e i n y l d o p a . S u b s e q u e n t e x p e r i m e n t s , h o w e v e r , r e v e a l e d t h a t p a r t of t h e t r i c h o c h r o m e s i s o l a t e d f r o m u r i n e could be a r t i f a c t s arizing f r o m aerial o x i d a t i o n of 5 - S - c y s t e i n y l d o p a a n d r e l a t e d m e t a b olites d u r i n g t h e w o r k - u p p r o c e d u r e . I n d e e d , w h e n a s o l u t i o n of p u r e 5 - S - c y s t e i n y l d o p a was used in t h e a b o v e m e n t i o n e d p r o c e d u r e i n s t e a d of m e l a n o m a u r i n e a good yield of d e c a r b o x y t r i c h o c h r o m e C was o b t a i n e d a~. T h e s a m e e x p e r i m e n t u s i n g a m i x t u r e of 5-S- a n d 2-S-cysteiny l d o p a also led to t h e f o r m a t i o n of d e c a r b o x y t r i c h o c h r o m e B. I n fact, t h e p r o c e d u r e p r o v e d to be ideal as a b i o g e n e t i c - t y p e s y n t h e s i s for p r e p a r i n g d e c a r b o x y t r i c h o chrome B and C from cysteinyldopas. I n t h e l i g h t o f t h e a b o v e findings, we t h e n w e n t on to develop t h e 3 a n a l y t i c a l p r o c e d u r e s r e p o r t e d , w h i c h p r o v e d to b e of v a l u e for a s c e r t a i n i n g t h e a c t u a l exc r e t i o n of t r i c h o c h r o m e s in m e l a l l o m a u r i n e w i t h o u t a r t i f a c t f o r m a t i o n d u e to t h e co-presence of p i g m e n t precursors. A n a l y s i s of t h e u r i n e w i t h m e t h o d s 1 or 3 s h o w e d o n l y t h e p r e s e n c e of t r i c h o c h r o m e B a n d C, w h e r e a s u s i n g m e t h o d 2 s m a l l a m o u n t s of t r i c h o c h r o m e E a n d F were also o b t a i n e d . I t s h o u l d be n o t e d , h o w e v e r , t h a t large a m o u n t s of t r i c h o c h r o m e E a n d F are p r o d u c e d b y aerial o x i d a t i o n of c y s t e i n y l d o p a s p r e s e n t in t h e u r i n e d u r i n g acid e l u t i o n of t h e D o w e x c o l u m n l a . A l t h o u g h t h e b u l k of t h e s e p i g m e n t s is Muted b y p r o l o n g e d w a s h i n g w i t h 2 N HC1, some m a t e r i a l e v i d e n t l y r e m a i n s o n t h e resin a n d is t h e n f o u n d in t h e alkaline eluate. T h e s m a l l a m o u n t s of t r i c h o c h r o m e E a n d F d e t e c t e d in t h e u r i n e w i t h m e t h o d 2 m u s t t h e r e f o r e b e r e g a r d e d as artifacts. T h e f i n d i n g of t r i c h o c h r o m e B a n d C in t h e u r i n e of a p a t i e n t w i t h m e l a n o n l a h a s i m p o r t a n t i m p l i c a t i o n s for t h e b i o c h e m i s t r y of m a m m a l i a n p i g m e n t a t i o n , a n d h a s also p o t e n t i a l clinical significance. I n d e e d , u n t i l n o w in m a m m a l s t r i c h o c h r o m e s h a d b e e n f o u n d o n l y in red hair, a n d i t s e e m e d t h a t t h e i r f o r m a t i o n was in some w a y d e p e n d e n t on t h e p a r t i c u l a r b i o c h e m i c a l e n v i r o n m e n t of t h e h a i r follicle m e l a n o c y t e . T h e p r e s e n t f i n d i n g s s t r o n g l y s u g g e s t t h a t t r i c h o c h r o m e s are f o r m e d also in o t h e r t y p e s of m e l a n o c y t e s , a n d t h a t t h e y are e x c r e t e d as a result of t h e p a t h o l o g i c a l a c t i v i t y of m a l i g n a n t m e l a n o c y t e s .
3. Isolation o/ trichocheomes after removal o/ pigment precumors by oxidation in alkaline medium. W o r k - u p of the melanoma urine by this procedure, involving initial o x i d a t i o n of t h e p i g m e n t p r e c u r s o r s p r e s e n t w i t h o x y g e n a t p H 13, led a g a i n to t h e isolation of d e c a r b o x y t r i c h o c h r o m e C a n d B, w h e r e a s n o t r i c h o c h r o m e E or F was o b t a i n e d . T h e t o t a l a m o u n t of d e c a r b o x y t r i c h o c h r o m e C+B e x c r e t e d was e s t i m a t e d s p e c t r o p h o t o m e t r i c a l l y a f t e r p u r i f i c a t i o n o n S e p h a d e x LH-20, a n d f o u n d to be 9 rag/24 h . Discussion. D u r i n g t h e i n i t i a l p h a s e of o u r work, s e a r c h for t r i c h o c h r o m e s in t h e m e l a n o m a u r i n e was carried o u t b y a m o d i f i c a t i o n of t h e p r o c e d u r e p r e v i o u s l y d e s c r i b e d for t h e isolation of p i g m e n t s f r o m r e d h a i r a n d f e a t h e r s ~.
10 G. PROTA, G. SCHERILLO a n d R. A. NICOLAUS, Gazz. Chim. ital.
98,495 (1968). 11 G. PROTA and R. A. NICOLAUS,in Advances in Biology o/ Skin (Eds. W. MONTAGNAand F. Hu; Pergamon Press, Oxford 1967), vol. 8, p. 323. 13 Decarboxytrichochrome C was also obtained simply by oxidation of 5-S eysteinyldopa in 0.1 N NaOH with atmospheric oxygen for a few minutes, followed by acidification and boiling of the reaction mixture. 13 Work at our laboratory has demonstrated the presence of large amounts of 2-S-cysteinyldopa in the urines of melanoma patients.
Specialia
c e n t r a t i o n s u p to 0.5 m M , giving n e a r l y 100% r e a c t i v a tion, b u t h i g h e r Zn 2+ c o n c e n t r a t i o n s c a u s e d a c o n s i d e r able inhibition. Ca s+ was c o n s i d e r a b l y less effective, a n d m u c h h i g h e r c o n c e n t r a t i o n s were required. The small a m o u n t of purified e n z y m e o b t a i n e d did n o t allow t h e d e t e r m i n a t i o n of t h e m e t a l c o n t e n t s of t h e m e t a l l o p r o t e i n . Since CaC1 s was r e q u i r e d for t h e p r o d u c t i o n of t h e protease, it m i g h t be s u p p o s e d t h a t t h e d i v a l e n t c a t i o n p r e s e n t in t h e m e t a l l o e n z y m e was Ca s+. H o w e v e r , as s h o w n in F i g u r e 2, o - p h e n a n t r o l i n e a t c o n c e n t r a t i o n s u p t o 1 m M i n h i b i t e d t h e e n z y m e e v e n in t h e p r e s e n c e of 10 m M CaCls, t h u s i n d i c a t i n g t h a t t h e d i v a l e n t c a t i o n
100~ %
o
o
50 ,,=,
' 0'.5 110 m/"/ Concentration of o-phenantro[ine Fig. 2. Inhibition of the extracellular protease from Ps. ]luorescens by o-phenantroline. 0.1 mI of purified enzyme preparation, previously exhaustively dialyzed against 20 mM Tris-tlC1 buffer (pH 7.6), was assayed as described under Methods except for the buffer, which was 30 mM Tris-acetate (pH 6.6), and o-phenantroline, which was directly added to the assay mixtures at the concentrations stated on the abcissa, in the absence (O) or in the presence (9 of 10 mM CaCI~. h~ order to avoid interference by o-phenaatroIine, after centrifugation a 1 ml-aliquot of the supernatant of the assay mixture was added 2 ml of 1 M NaOH and 0.5 ml of Folin-Ciocalteau's phenol reagent, diluted 1/a15. After 30 min at room temperature, the absorbance of the solutio~ at 750 am was read and taken as an expression of the enzyme activity. Blanks, to which trichloroacetie acid was added before the enzyme, were substracted. The enzyme activity is expressed as percent of the enzyme activity in the absence of the inhibitor (A A75o~,Imit~ = 0.11).
Occurrence of Trichochromes G. PROTA,
H. RORSMAN
a c t i n g as p r o s t h e t i c g r o u p is n o t Ca 2+, being p r o b a b l y one of t h e c a t i o n s able to f o r m s t r o n g c o m p l e x e s w i t h o - p h e n a n t r o l i n e , such as Co s+ or Zn s+. A similar f i n d i n g h a s b e e n r e p o r t e d for t h e alkaline p r o t e a s e of Ps. aeru-
ginosa t3. CaCI2, w h i c h is k n o w n to stabilize s o m e e x t r a c e l l u l a r protease*, did n o t p r o t e c t t h e Ps./luorescens e n z y m e , w h i c h was a l m o s t c o m p l e t e l y i n a c t i v a t e d a f t e r i n c u b a t i o n a t 50 ~ for 10 rain, e i t h e r in t h e a b s e n c e or in t h e p r e s e n c e of 5 m M CaC1 s. The results p r e s e n t e d in t h i s c o m m u n i c a t i o n s h o w t h a t Ps. fluorescens p r o d u c e s a n e x t r a c e l l u l a r p r o t e a s e able to digest casein w i t h i n a p H r a n g e w i d e r t h a n t h a t r e p o r t e d for t h e e n z y m e s purified f r o m o t h e r Pseudomonas 3,5. T h e molecular w e i g h t of t h e e n z y m e is similar to t h a t rep o r t e d for t h e p r o t e a s e s f r o m Ps.maltophilia 4 and Ps. ]ragi ~, b u t s i g n i f i c a n t l y l o w e r t h a n t h a t of t h e alkaline p r o t e a s e of Ps. aeruginosa 1~. T h e Ps./luorescens p r o t e a s e is a m e t a l l o e n z y m e , as t h e e n z y m e s p r o d u c e d b y o t h e r Pseudomonas 3, 4, 5, b u t in our case a stable a p o e n z y m e was o b t a i n e d b y dialysis a g a i n s t E D T A for r a t h e r long p e r i o d s of time. The lack of e n z y m e r e a c t i v a t i o n b y dialysis a g a i n s t distilled w a t e r suggests t h a t in t h e case of t h e Ps.fluorescens p r o t e a s e t h e m e t a l p r o s t h e t i c g r o u p was a c t u a l l y r e m o v e d , a n d n o t s i m p l y m a s k e d , b y t h e c h e l a t i n g agent. The second p o s s i b i l i t y is t h o u g h t t o be t r u e for t h e Ps. aeruginosa 14 e n z y m e . The d i v a l e n t c a t i o n b o u n d to t h e a p o e n z y m e m i g h t be Co s+ or Zn s+, as sugg e s t e d b y t h e g r e a t e r effectiveness of t h e s e cations for t h e r e c o n s t i t u t i o n of t h e h o l o e n z y m e (Figure 1) a n d b y t h e i n h i b i t i o n b y o - p h e n a n t r o l i n e in t h e p r e s e n c e of CaC1 s (Figure 2). Since o n l y one p r o t e o l y t i c e n z y m e w a s d e t e c t e d in t h e c u l t u r e s u p e r n a t a n t s of Ps. [luorescens, a n d it was able to digest casein a t n e u t r a l p H , t h e p r o t e a s e described here can be c o n s i d e r e d r e s p o n s i b l e for t h e gelatine h y d r o l y s i s w h i c h is one of t h e p h e n o t y p i c c h a r a c t e r s of Ps.fluo~,.escens
g .
13 t{. MORIHARAand H. TSUZUKI, Agrie. biol. Chem. 38, 621 (1974). 14 I(. MORIHARAand H. TsUzuKI, Biochim. biophys. Acta 92, 351 (1964). t5 J. C. P~eKUPand D. B. HoPI~, Biochem..7- t23, 153 (~971).
in the Urine of a Melanoma
2, A.-M. ROSENGREN
EXPERIENTIA 32/9
Patient 1
and E. I~OSENGREN
Department o/ Organic Chemistry, University o/ Naples, Napoli (Italy), and Department o/ Dermatology, Biochemistry and Pharmacology, University o/Lund, S-227 85 Lund (Sweden), 29 March 1976. Summary. The p r e s e n c e of t w o p h a e o m e l a n i c p i g m e n t s , t r i c h o c h r o m e B a n d C, was d e m o n s t r a t e d in t h e urine of a patient with malignant melanoma metastases. T r i c h o c h r o m e s 3-5, f o r m e r l y n a m e d trichosiderins, are a u n i q u e g r o u p of a m i n o acidic p i g m e n t s possessing a A 2 , 2 ' - b i - ( 2 H - 1 , 4 - b e n z o t h i a z i n e ) c h r o m o p h o re (Figure), w h i c h a c c o u n t s for t h e i r c h a r a c t e r i s t i c p H - d e p e n d e n t visible s p e c t r a . T h e y are t h e s i m p l e s t group of p h a e o m e l a n i c p i g m e n t s , a n d are f o r m e d in m e l a n o c y t e s b y a d e v i a t i o n of t h e e u m e l a n i n p a t h w a y i n v o l v i n g as k e y s t e p t h e 1,6a d d i t i o n of c y s t e i n e to d o p a q u i n o n e to give 5-S- a n d 2S-cysteinyldopa.
Unlike eumelanin, w h i c h occurs in several t y p e s of pigm e n t e d tissues, t r i c h o c h r o m e s h a v e so far b e e n f o u n d o n l y in c e r t a i n red hair a n d feathers, a n d it has b e e n s u g g e s t e d t h a t t h e i r f o r m a t i o n is s o m e h o w r e s t r i c t e d t o t h e s e k e r a t i u i z e d s t r u c t u r e s . T h e r e c e n t finding of large a m o u n t s of 5 - S - c y s t e i n y l d o p a in t h e urine of m e l a n o m a patients~, ~ led us to look for t r i c h o c h r o m e s in t h e urine of a p a t i e n t w i t h m e l a n o m a m e t a s t a s e s a n d m a r k e d l y
15.9. 1976
OH
HO2C
1123
Specialia
H
OH
NH2
OH
HO2C
NH2
1 R = CO2H 3 : R =H
OH
NH2
CO2H
H2N
CQ2H
OH
HO2C
5
H2N
4:R=H
:
HO2C
OH
2 - R-- CO2H
H2N
OH
H
H2N
NH2 6
1. Trichochrome B. 2. Trichochrome C. 3. Decarboxytrichochrome B. 4. Deearboxytrichochrome C. 5. Triehochrome E. 6. Trichochrome F.
e l e v a t e d u r i n a r y e x c r e t i o n of 5 - S - c y s t e i n y l d o p a a n d related metabolites. M a t e r i a l a n d methods. U r i n e was o b t a i n e d f r o m a male p a t i e n t aged 46 y e a r s w i t h r e d - b l o n d hair. H e h a d b e e n o p e r a t e d on for a c u t a n e o u s m e l a n o m a , a n d h a d wides p r e a d m e t a s t a s e s . 24-hour s p e c i m e n s were collected in plastic b o t t l e s c o n t a i n i n g 50 m l of acetic acid a n d 1 g of s o d i u m m e t a b i s u l p h i t e . D e t e r m i n a t i o n of 5-S-cysteinyld o p a a n d d o p a + d o p a m i n e was carried o u t b y m e t h o d s p r e v i o u s l y d e s c r i b e d 8, ~. A u t h e n t i c s a m p l e s of d e c a r b o x y t r i c h o c h r o m e B a n d C were o b t a i n e d as follows, 22 m g of a m i x t u r e of 2-S- a n d 5 - S - c y s t e i n y l d o p a (molar r a t i o c. 1:9) was dissolved in a small v o l u m e of 0.1 N HC1. The solution was p a s s e d on a c o l u m n (2 • 10 cm) of D o w e x 50W'-X4, 200-400 mesh, H+ form. A f t e r w a s h i n g w i t h w a t e r (200 ml), t h e c o l u m n was t r e a t e d w i t h 0.5 N N a O H , a n d yielded a b r o w n i s h yellow eluate w h i c h was acidified t o p H 1 w i t h 6 N HC1 a n d boiled for 20 min. T h e r e d d i s h - b r o w n s o l u t i o n so o b t a i n e d was r e - c h r o m a t o g r a p h e d on a D o w e x 50W c o l u m n e q u i l i b r a t e d w i t h 0.1 N HCI. A f t e r w a s h i n g as before, elution was first p e r f o r m e d w i t h 0.2 M s o d i u m a c e t a t e , w h i c h r e m o v e d m o s t of t h e coloured material, a n d finally w i t h 0.1 N N a O H . The alkaline eluate was a d j u s t e d to p H 3 a n d t h e p r e c i p i t a t e d p i g m e n t s were collected b y c e n t r i f u g a t i o n , dissolved in 0.1 N HC1, a n d I r a c t i o n a t e d b y p a p e r c h r o m a t o g r a p h y on W h a t m a n 3MM w i t h i s o p r o p a n o l - f o r m i c acid - c o n c e n t r a t e d HC1 (30:70:1, v/v). The red b a n d s c o r r e s p o n d i n g to decarb o x y t r i c h o c h r o m e B (lower R f value) a n d d e c a r b o x y t r i c h o c h r o m e C were c u t o u t a n d e l u t e d w i t h 0.1 N N a O H . Q u a n t i t a t i o n of t h e p i g m e n t s so o b t a i n e d was d o n e b y spectrophotometry. I s o l a t i o n of t r i c h o c h r o m e s f r o m t h e m e l a n o m a urine was carried o u t b y t h e following procedures. 1. A urine s a m p l e (10 ml) was acidified to p H 1 w i t h 6 N HC1 a n d boiled for 10 m i n t o c o n v e r t t r i c h o c h r o m e s B a n d C into
the corresponding decarboxy derivatives. Isopropanol (10 ml) was t h e n a d d e d a n d t h e m i x t u r e was s a t u r a t e d w i t h NaC1. A f t e r s h a k i n g for 5 min, t h e i s o p r o p a n o l l a y e r was w i t h d r a w n a n d e v a p o r a t e d to dryness. T h e residue was dissolved in a small v o l u m e of 0.1 N HC1 a n d a n a l y z e d d i r e c t l y b y TLC on cellulose using i s o p r o p a n o l - f o r m i c acidc o n c e n t r a t e d HCI (30: 70: 1) as eluent. 2. A 24-hour urine s a m p l e was acidified to p H 1 w i t h 6 N HC1 a n d p a s s e d on a c o l u m n (2 • 15 cm) of D o w e x 50W-X4, H + form. A f t e r w a s h i n g w i t h N HC1 (450 ml), 2 N HC1 (1200 ml), a n d w a t e r (200 ml) to r e m o v e cysteinyld o p a s a n d r e l a t e d m e t a b o l i t e s , t h e c o l u m n w a s eluted w i t h 0.5 N N a O H , a n d a b r o w n i s h - y e l l o w f r a c t i o n cont a i n i n g t r i c h o c h r o m e s was collected, acidified to p H 1, a n d boiled for 15 min. The r e s u l t i n g r e d d i s h - b r o w n so-
i Supported by grants from the Swedish Cancer Society (No. 626B75 04XA), 869-B75-01P, the Swedish Medical Research Council (No. B76-04X-00036-12), and the Walter, Ellen and Lennart Hesselman Foundation for Scientific Research. Professor PROTA was a visiting scientist of the Swedish Cancer Society (No. 626B75 04U). Reprint requests to: Professor HaNS RORSMAN, Department of Dermatology, Lasarettet, S 221 85 LUND, Sweden. a G. PROTA, in Pigmentation: Its Genesis and Biological Control (Ed. V. RILEY: Appleton-Century-Crofts, New York 1972), p. 615. a R. H. THOMSON,Angew. Chem. Int. Edit. 13, 305 (1974). s G. PROTAand R. H. THOMSON, Endeavour 35, 32 (1976). 6 H. RORSIVIAN, A.-M. ROSENGREN and E. ROS~NGREN, Yale J. Biol. Med. 46, 516 (1973). G. AGRUP, P. AGRUP, T. ANDERSSON, B. FALCK, J.-A. HANSSON, S. JACOBSSON, H. RORSMAN, A.-]3/[. ROSENGREN and E. ROSEN-
GREN, Acta dermatovener, Stoekhohn 55, 337 (1975). 8 H. RORSMAN, A.-M. ROSENGREN and E. ROSENGREN, Acts derma-
tovener, Stockholm 53, 248 (1973). 9 A. H. ANTON and D. F. SAYRE, J. Pharmac. exp. Ther. 145, 326 (1964).
1124
Specialia
l u t i o n was p a s s e d o n a smaller c o l u m n (2 • cm) of D o w e x 50W w h i c h was t h e n w a s h e d w i t h N HC1 (100 ml), w a t e r a n d 0.2 M s o d i u m a c e t a t e u n t i l t h e p H of t h e eff l u e n t rose to 5. S u b s e q u e n t e l u t i o n w i t h 0.1 N N a O H g a v e a b r o w n i s h - y e l l o w f r a c t i o n which, ~after acidification a n d e v a p o r a t i o n , was f u r t h e r p u r i f i e d b y c h r o m a t o g r a p h y on a c o l u m n (1.5 • 20 cm) of S e p h a d e x L H - 2 0 u s i n g m e t h anol-2 N HC1 (95:5) as eluent. U n d e r t h e s e c o n d i t i o n s , a well-defined red b a n d a p p e a r e d w h i c h was collected a n d e v a p o r a t e d to dryness. T h e residue was dissolved in a m i n i m u m v o l u m e of 0.1 N HC1. T h e r e s u l t i n g s o l u t i o n was first a n a l y z e d s p e c t r o p h o t o m e t r i c a l l y to d e t e r m i n e t h e t o t a l yield of t h e p i g m e n t s , a n d was s u b s e q u e n t l y fractionated by paper chromatography on Whatman 3MM w i t h i s o p r o p a n o l - f o r m i c a c i d - c o n c e n t r a t e d HC1 (30:70:1). 3. A s a m p l e of u r i n e (100 ml) was a d j u s t e d t o p H 13 w i t h 5 N N a O H a n d left a t r o o m t e m p e r a t u r e for 72 h under an oxygen current to convert cysteinyldopas into stable, colourless b e n z o t h i a z i n e d e r i v a t i v e s 10. T h e oxidized u r i n e was t h e n acidified t o p H 1 w i t h 6 N HC1 a n d passed o n a c o l u m n (2 • 10 cm) of D o w e x 50 W, H + form. A f t e r w a s h i n g w i t h N HC1 (150 ml) a n d water, t h e c o l u m n was e l u t e d w i t h 0.5 N N a O H to give a t r i c h o c h r o m e - c o n r a i n i n g f r a c t i o n w h i c h was w o r k e d u p as described in m e t h o d 2. As a c o n t r o l t o m e t h o d 3, a s o l u t i o n of 5 - S - c y s t e i n y l d o p a in 0.1 N N a O H was t r e a t e d e x a c t l y as t h e m e l a n o m a urine. Results. B e c a u s e of t h e p r e s e n c e of large a m o u n t s of 5-S-cysteinyldopa and related metabolites, examination of t h e m e l a n o m a u r i n e was carried o u t b y t h e 3 proced u r e s d e s c r i b e d a b o v e , all of w h i c h g a v e similar results. 1. Isopropanol extraction o/ trichochromes. C h r o m a t o g r a p h i c a n a l y s i s of t h e i s o p r o p a n o l e x t r a c t o b t a i n e d f r o m m e l a n o m a u r i n e r e v e a l e d t h e p r e s e n c e of 3 red p i g m e n t s , 2 of w h i c h s h o w e d p H - d e p e n d e n t colours a n d R f v a l u e s i d e n t i c a l to t h o s e of d e c a r b o x y t r i c h o c h r o m e B a n d C. T h e 3rd s p o t s h o w e d n o colour c h a n g e w h e n exposed to a m m o n i a v a p o u r , a n d s h o w e d a n 1Rf v a l u e w h i c h did n o t c o r r e s p o n d to a n y h i t h e r t o k n o w n t r i c h o c h r o m e .
2. Isolation of trichochromes a/ter removal o/ pigment precursors by ionic exchange resin. F r a c t i o n a t i o n of t h e m e l a n o m a u r i n e on D o w e x 50"vV c o l u m n s followed b y c h r o m a t o g r a p h y Of t h e c r u d e p i g m e n t m i x t u r e on a S e p h a d e x L H - 2 0 c o l u m n w i t h m e t h a n o l - 2 N HC1 (95:5) g a v e a single red b a n d w i t h a b s o r p t i o n m a x i m a a t 533 n m in 2 N HC1 a n d 460 n m in 0.1 N N a O H , c o r r e s p o n d i n g to d e c a r b o x y t r i c h o c h r o m e C + B . T h e t o t a l a m o u n t of t h e s e p i g m e n t s , d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y , was f o u n d t o be 5 m g / 2 4 h. S u b s e q u e n t c h r o m a t o g r a p h y of t h e m i x t u r e o n W h a t m a n 3 MM p a p e r led t o t h e isolation of p u r e d e c a r b o x y t r i c h o c h r o m e C a n d B, a l o n g w i t h t r a c e a m o u n t s of t r i c h o c h r o m e E a n d F identified b y t h e i r chromatographic and spectral properties.
EXPERIENTIA 32/9
T h e m e t h o d i n v o l v e s p r e l i m i n a r y p u r i f i c a t i o n of t h e u r i n e on s t r o n g l y c a t i o n i c e x c h a n g e resin, a n d s u b s e q u e n t h e a t ing of t h e p i g m e n t m i x t u r e in acid m e d i u m t o c o n v e r t t h e yellow u n s t a b l e t r i c h o c h r o m e B a n d C i n t o t h e corres p o n d i n g d e c a r b o x y d e r i v a t i v e s , w h i c h possess more favourable analytical properties. U s i n g t h i s p r o c e d u r e large a m o u n t s (up to 30 m g / 2 4 h) of d e c a r b o x y t r i c h o c h r o m e C a n d B along w i t h smaller q u a n t i t i e s of t r i c h o c h r o m e F a n d E were o b t a i n e d from t h e u r i n e of s e v e r a l p a t i e n t s w i t h m e l a n o m a m e t a s t a s e s a n d h i g h u r i n a r y e x c r e t i o n of 5 - S - c y s t e i n y l d o p a . S u b s e q u e n t e x p e r i m e n t s , h o w e v e r , r e v e a l e d t h a t p a r t of t h e t r i c h o c h r o m e s i s o l a t e d f r o m u r i n e could be a r t i f a c t s arizing f r o m aerial o x i d a t i o n of 5 - S - c y s t e i n y l d o p a a n d r e l a t e d m e t a b olites d u r i n g t h e w o r k - u p p r o c e d u r e . I n d e e d , w h e n a s o l u t i o n of p u r e 5 - S - c y s t e i n y l d o p a was used in t h e a b o v e m e n t i o n e d p r o c e d u r e i n s t e a d of m e l a n o m a u r i n e a good yield of d e c a r b o x y t r i c h o c h r o m e C was o b t a i n e d a~. T h e s a m e e x p e r i m e n t u s i n g a m i x t u r e of 5-S- a n d 2-S-cysteiny l d o p a also led to t h e f o r m a t i o n of d e c a r b o x y t r i c h o c h r o m e B. I n fact, t h e p r o c e d u r e p r o v e d to be ideal as a b i o g e n e t i c - t y p e s y n t h e s i s for p r e p a r i n g d e c a r b o x y t r i c h o chrome B and C from cysteinyldopas. I n t h e l i g h t o f t h e a b o v e findings, we t h e n w e n t on to develop t h e 3 a n a l y t i c a l p r o c e d u r e s r e p o r t e d , w h i c h p r o v e d to b e of v a l u e for a s c e r t a i n i n g t h e a c t u a l exc r e t i o n of t r i c h o c h r o m e s in m e l a l l o m a u r i n e w i t h o u t a r t i f a c t f o r m a t i o n d u e to t h e co-presence of p i g m e n t precursors. A n a l y s i s of t h e u r i n e w i t h m e t h o d s 1 or 3 s h o w e d o n l y t h e p r e s e n c e of t r i c h o c h r o m e B a n d C, w h e r e a s u s i n g m e t h o d 2 s m a l l a m o u n t s of t r i c h o c h r o m e E a n d F were also o b t a i n e d . I t s h o u l d be n o t e d , h o w e v e r , t h a t large a m o u n t s of t r i c h o c h r o m e E a n d F are p r o d u c e d b y aerial o x i d a t i o n of c y s t e i n y l d o p a s p r e s e n t in t h e u r i n e d u r i n g acid e l u t i o n of t h e D o w e x c o l u m n l a . A l t h o u g h t h e b u l k of t h e s e p i g m e n t s is Muted b y p r o l o n g e d w a s h i n g w i t h 2 N HC1, some m a t e r i a l e v i d e n t l y r e m a i n s o n t h e resin a n d is t h e n f o u n d in t h e alkaline eluate. T h e s m a l l a m o u n t s of t r i c h o c h r o m e E a n d F d e t e c t e d in t h e u r i n e w i t h m e t h o d 2 m u s t t h e r e f o r e b e r e g a r d e d as artifacts. T h e f i n d i n g of t r i c h o c h r o m e B a n d C in t h e u r i n e of a p a t i e n t w i t h m e l a n o n l a h a s i m p o r t a n t i m p l i c a t i o n s for t h e b i o c h e m i s t r y of m a m m a l i a n p i g m e n t a t i o n , a n d h a s also p o t e n t i a l clinical significance. I n d e e d , u n t i l n o w in m a m m a l s t r i c h o c h r o m e s h a d b e e n f o u n d o n l y in red hair, a n d i t s e e m e d t h a t t h e i r f o r m a t i o n was in some w a y d e p e n d e n t on t h e p a r t i c u l a r b i o c h e m i c a l e n v i r o n m e n t of t h e h a i r follicle m e l a n o c y t e . T h e p r e s e n t f i n d i n g s s t r o n g l y s u g g e s t t h a t t r i c h o c h r o m e s are f o r m e d also in o t h e r t y p e s of m e l a n o c y t e s , a n d t h a t t h e y are e x c r e t e d as a result of t h e p a t h o l o g i c a l a c t i v i t y of m a l i g n a n t m e l a n o c y t e s .
3. Isolation o/ trichocheomes after removal o/ pigment precumors by oxidation in alkaline medium. W o r k - u p of the melanoma urine by this procedure, involving initial o x i d a t i o n of t h e p i g m e n t p r e c u r s o r s p r e s e n t w i t h o x y g e n a t p H 13, led a g a i n to t h e isolation of d e c a r b o x y t r i c h o c h r o m e C a n d B, w h e r e a s n o t r i c h o c h r o m e E or F was o b t a i n e d . T h e t o t a l a m o u n t of d e c a r b o x y t r i c h o c h r o m e C+B e x c r e t e d was e s t i m a t e d s p e c t r o p h o t o m e t r i c a l l y a f t e r p u r i f i c a t i o n o n S e p h a d e x LH-20, a n d f o u n d to be 9 rag/24 h . Discussion. D u r i n g t h e i n i t i a l p h a s e of o u r work, s e a r c h for t r i c h o c h r o m e s in t h e m e l a n o m a u r i n e was carried o u t b y a m o d i f i c a t i o n of t h e p r o c e d u r e p r e v i o u s l y d e s c r i b e d for t h e isolation of p i g m e n t s f r o m r e d h a i r a n d f e a t h e r s ~.
10 G. PROTA, G. SCHERILLO a n d R. A. NICOLAUS, Gazz. Chim. ital.
98,495 (1968). 11 G. PROTA and R. A. NICOLAUS,in Advances in Biology o/ Skin (Eds. W. MONTAGNAand F. Hu; Pergamon Press, Oxford 1967), vol. 8, p. 323. 13 Decarboxytrichochrome C was also obtained simply by oxidation of 5-S eysteinyldopa in 0.1 N NaOH with atmospheric oxygen for a few minutes, followed by acidification and boiling of the reaction mixture. 13 Work at our laboratory has demonstrated the presence of large amounts of 2-S-cysteinyldopa in the urines of melanoma patients.
