Quantitative Analysis of Eumelanin and Pheomelanin in Humans, Mice,

and Other Animals: a Comparative Review

SHOSUKE ITO and KAZUMASA WAKAMATSU
Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan
*Address reprint requests to Prof. Shosuke Ito, Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi 470-
1192, Japan. E-mail: sito@fujita-hu.ac.jp
Received 21 May 2003; in nal form 2 June 2003
The color of hair, skin, and eyes in animals mainly depends on
the quantity, quality, and distribution of the pigment melanin,
which occurs in two types: black to brown eumelanin and
yellow to reddish pheomelanin. Microanalytical methods to
quantify the amounts of eumelanin and pheomelanin in
biological materials were developed in 1985. The methods are
based on the chemical degradation of eumelanin to pyrrole-
2,3,5-tricarboxylic acid and of pheomelanin to amino-
hydroxyphenylalanine isomers, which can be analyzed and
quantitated by high performance liquid chromatography.
This review summarizes and compares eumelanin and pheo-
melanin contents in various pigmented tissues obtained from
humans, mice, and other animals. These methods have become
valuable tools to study the functions of melanin, the con-
trol of melanogenesis, and the actions and interactions of
pigmentation genes. The methods have also found applications
in many clinical studies. High levels of pheomelanin are found
only in yellow to red hairs of mammals and in red feathers of
birds. It remains an intriguing question why lower vertebrates
such as shes do not synthesize pheomelanin. Detectable levels
of pheomelanin are detected in human skin regardless of race,
color, and skin type. However, eumelanin is always the major
constituent of epidermal melanin, and the skin color appears to
be determined by the quantity of melanin produced but not by
the quality.
Key words: Eumelanin, Pheomelanin, Aminohydroxyphenyl-
alanine, Pyrrole-2,3,5-tricarboxylic acid, High performance
liquid chromatography
INTRODUCTION
The color of hair, skin, and eyes in animals mainly depends
on the quantity, quality, and distribution of the pigment
melanin. Melanocytes are responsible for the synthesis of
melanin within membrane-bound organelles (melanosomes)
and the transport of melanosomes to surrounding epidermal
cells (keratinocytes). Melanocytes in mammals and birds
produce two chemically distinct types of melanin pigments,
the black to brown eumelanin and the yellow to reddish
pheomelanin (14). Eumelanin is a highly heterogeneous
polymer consisting of 5,6-dihydroxyindole (DHI) and 5,6-
dihydroxyindole-2-carboxylic acid (DHICA)-derived units,
while pheomelanin consists mainly of sulfur containing
benzothiazine derivatives.
Eumelanin and pheomelanin both derive from the com-
mon precursor dopaquinone, which is formed following the
oxidation of tyrosine by tyrosinase. Dopaquinone is a highly
reactive intermediate, and in the absence of thiol compounds,
it undergoes intramolecular cyclization, leading eventually to
the production of eumelanin. However, the intervention of
thiols, such as cysteine, with this process gives rise to the thiol
adducts 5-S-cysteinyldopa (5-S-CD) and 2-S-cysteinyldopa
(2-S-CD). Further oxidation of these CD isomers leads to the
production of pheomelanin. In reality, most of the melanin
pigments present in the hair and skin, and in melanomas are
not homopolymers of a single monomer unit, but rather they
are complex heteropolymers made up of both eumelanin and
pheomelanin building blocks (1).
Melanin pigments play several diverse and important roles,
including thermoregulation in lower vertebrates, camouage,
and sexual attraction in virtually all species, as well as
photoprotection of the skin in higher mammals. Eumelanin
and pheomelanin appear to play rather dierent roles in
Abbreviations AHP, aminohydroxyphenylalanine; 3-AHP, 3-amino-4-hydroxyphenylalanine; 4-AHP, 4-amino-3-hydroxyphenylalanine;
DHI, 5,6-dihydroxyindole; DHICA, 5,6-dihydroxyindole-2-carboxylic acid; MC1R, melanocortin 1 receptor; MSH, melanocyte stimulating hormone;
PDCA, pyrrole-2,3-dicarboxylic acid; PTCA, pyrrole-2,3,5-tricarboxylic acid; PUVA, psoralen plus ultraviolet A; 2-S-CD, 2-S-cysteinyldopa;
5-S-CD, 5-S-cysteinyldopa
PIGMENT CELL RES 16: 523531. 2003 Copyright Blackwell Munksgaard 2003
Printed in UKall rights reserved ISSN 0893-5785
Pigment Cell Res. 16, 2003 523
those functions (e.g., eumelanin is photoprotective while
pheomelanin is believed to be carcinogenic after UV
radiation) (5). In order to study the regulation of melano-
genesis as well as the biological roles of melanin pigment, it is
thus essential to analyze the contents of eumelanin and
pheomelanin in biological samples. This review summarizes
and compares eumelanin and pheomelanin contents in
various pigmented tissues obtained from humans, mice, and
other animals. These data have accumulated over the last
20 yr, since the quantitative analysis of eumelanin and
pheomelanin in biological samples was rst reported in
1983 (6).
DEVELOPMENT OF ANALYTICAL ASSAYS
A group in Italy led by Nicolaus, Prota, and Fattorusso
carried out extensive degradative studies on eumelanin
isolated from Sepia and pheomelanin isolated from chicken
feathers (1). Those methods were adopted and modied when
the microanalytical methods to quantify the amounts of
eumelanin and pheomelanin in biological materials were rst
developed in 1985 (7). They are based on the chemical
degradation of the melanin polymer and high-performance
liquid chromatography (HPLC) analysis of their specic
degradation products. The eumelanin polymer is degraded
into pyrrole-2,3,5-tricarboxylic acid (PTCA) by KMnO
4
oxidation (Fig. 1). PTCA is formed from DHICA-melanin
in ca. 2.8% yield, while it is produced only in 0.03% yield
from DHI-melanin (2, 3). Thus, PTCA serves as a specic
marker of DHICA-derived eumelanin. In contrast, the
reductive hydrolysis of pheomelanin with hydriodic acid
(HI) splits the sulfurcarbon bonds in benzothiazine units of
pheomelanin to give rise to aminohydroxyphenylalanine
(AHP) isomers. Pheomelanin monomer units derived from
5-S-CD or from 2-S-CD produce 4-amino-3-hydroxypheny-
lalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine
(3-AHP), respectively (Fig. 1). The combined yield of AHP
isomers (total AHP) from various pheomelanin preparations
is approximately 20% (7).
The HPLC conditions were such that 4- and 3-AHP eluted
in a single peak (total AHP). However, small amounts of
background values of total AHP were found in hairs from
tyrosinase-negative albino mice (8, 9). These situations
prompted us to investigate improving the HPLC conditions
and the usefulness of 4-AHP as a marker of pheomelanin.
HPLC conditions, which enabled the direct injection of the
HI reduction products into the HPLC, were developed in
2002 (10) and allowed complete separation of 4- and 3-AHP
(10, 11). Considerable and variable amounts of 3-AHP are
produced from precursors other than pheomelanin, most
likely 3-nitrotyrosine-containing proteins (10; Fig. 1).
The contents of PTCA and total AHP (in some cases,
4- and 3-AHP separately), instead of those of eumelanin and
pheomelanin to make direct comparison of the contents in
diverse samples easier, are reported in the present paper.
However, when one wishes to convert those PTCA and AHP
contents to eumelanin and pheomelanin, the following
conversion factors may be used (3). For rough estimation
of eumelanin and pheomelanin, the PTCA and AHP (total
AHP) contents can be multiplied by factors of 50 and
5, respectively (7). For more accurate estimation, factors of
45 and 160 for PTCA in mice and humans, respectively, and
N
S
(HOOC)
N
S
(HOOC)
HOOC
HOOC
HI
+
4-AHP
COOH
COOH
COOH
COOH
COOH COOH
NH
2
NH
2
NH
2
HO
HO
HO
HO
HO
HO
HO
HO
HO H
2
N
NH
2
NH
2
O
2
N
H
2
N
3-AHP
Benzothiazine intermediates
3-Nitrotyrosine
HI HI 11% yield
DHI-derived eumelanin
DHICA-derived eumelanin
KMnO
4
/H
+
KMnO
4
/H
+
2.8% yield
0.03% yield
PTCA
COOH
N
H
N
H
N
H
Fig. 1. Chemical degradation of eumelanin and pheomelanin. Note that the yield of PTCA from DHI-derived eumelanin is extremely low,
compared with that from DHICA-derived eumelanin. PTCA is thus a specic degradation product of DHICA-derived eumelanin (24). Reductive
hydrolysis of pheomelanin with hydriodic acid gives two isomeric 4-AHP and 3-AHP derived from the benzothiazine units. 3-AHP also arises from
3-nitrotyrosine-containing proteins (3, 10).
524 Pigment Cell Res. 16, 2003
a factor of 9 for 4-AHP (3) were used. The rather large
difference in the conversion factors for PTCA reects a
difference in the relative proportion of DHICA-derived
units, which is much higher in mice than in humans (12).
QUANTITATIVE ANALYSIS OF EUMELANIN
AND PHEOMELANIN IN HUMANS
Hair
Hair color is the most obvious and diverse characteristic
among pigmentation phenotypes. The rst application of the
HPLC method appeared in 1983 when Jimbow et al. (13)
reported high levels of AHP in three of ve red-haired
subjects (Table 1). Their study stressed that the phenotype of
hair color does not always reect the type of melanogenesis
in human red hair. More recent studies using chemical
analysis of human hair of various colors showed that PTCA
and AHP values correlated well with the phenotype (4, 14).
Black, brown, light brown, and blond hairs differed only in
their eumelanin content, while red hair was exceptional in
that it contained fourfold greater contents of pheomelanin
compared with light brown and blond hairs.
A very recent study using 4-AHP as a more specic
pheomelanin marker showed that median 4-AHP to PTCA
ratios were 2.7, 0.27, and 0.042 in red, fair, and dark hairs,
respectively (10). It should be noted that in fair and dark-
colored hairs, the 4-AHP levels are much lower than the
3-AHP levels. Thus, most of the 3-AHP in those hairs appears
to derive from nitrotyrosine-containing proteins (10).
The method described in the study has been applied to
clinical studies by analyzing yellow hairs from tyrosinase-
positive albinism patients (15) and light brown hairs from
patients with Hermansky-Pudlak syndrome (16). Other
applications include the analysis of segmented heterochromic
hair (17) and yellow hair from a congenital hypomelanosis
patient (18; data not shown).
Skin
Thody et al. (19) reported the rst application of this method
to study the human skin pigmentation. Eumelanin and
pheomelanin were found in all epidermal samples and their
relative proportions correlated well with those found in
samples of hair taken from the same subjects. Following
psoralen and ultraviolet A (PUVA) therapy (for treatment of
psoriasis or mycosis fungoides) increases in the amounts of
both pigments were observed, but eumelanin levels showed a
better correlation to the PUVA-induced tanning. Salopek
et al. (20) examined the type and amount of melanin
synthesized in 31 melanocytic nevi excised from 27 patients
and compared that with amounts in the surrounding normal
skin. They found that dysplastic melanocytic nevi lesions
contain signicantly higher amounts of pheomelanin than do
either common melanocytic nevi or normal skin. This nding
suggested that epidermal melanocytes in dysplastic melano-
cytic nevi undergo deranged melanogenesis.
Tobin et al. (21) compared changes in PTCA/AHP ratios
following ve daily suberythemal doses of UVB. Skin types
I/II showed a decrease in that ratio from 0.20 to 0.11, while
skin types III/IV showed an increase from 0.33 to 0.40.
Hunt et al. (22) compared eumelanin and pheomelanin
levels in epidermis from 11 Europeans (lightly pigmented)
and from eight non-Europeans (deeply pigmented). Eumel-
anin and pheomelanin contents were more than 10-fold
higher in non-Europeans, but there was an excellent
correlation between the levels of PTCA and AHP for all
19 subjects (r 0.97). Those results indicate that variations
in skin pigmentation derive mostly from dierences in the
levels of melanin pigment but not from the relative ratios of
eumelanin and pheomelanin. Nakagawa and Imokawa (23)
examined melanin contents in epidermis from 19 Japanese
(light colored to tanned skin). They found that the
eumelanin to pheomelanin ratio varied individually, but
showed a good correlation with the ratio of ellipsoidal to
spheroid melanosomes (r 0.749).
Recently, using the more specic pheomelanin assay,
Tadokoro et al. (24) examined the relationships between
melanin content and DNA damage induced by UV expo-
sure in normal skin of different racial/ethnic groups. The
results showed that after exposure to one minimal erythema
dose of UV, the skin of subjects from all groups suffered
signicant DNA damage, and that increasing contents of
constitutive melanin correlated inversely with the amount of
DNA damage. Moreover, the average contents of eumela-
nin and pheomelanin did not change appreciably in human
skin over a 7-day observation period following UV expo-
sure.
Another clinical application of these melanin assays in the
skin was reported by Parsad et al. (25). Levels of eumelanin
and pheomelanin were measured in depigmented as well as
repigmented patches of vitiligo during PUVA therapy.
Depigmented lesions had low levels of both types of
melanin with relatively high proportions of pheomelanin,
whereas repigmented lesions after PUVA had predominantly
eumelanin.
Melanoma, Melanoma Cells, and Melanocytes
Few systematic studies of eumelanin and pheomelanin
contents in human melanoma tissues can be found except
for the study by Morishima and Fukada (26), which showed
that eumelanin to pheomelanin ratios were highly variable
among melanoma tissues. del Marmol et al. (27) were the
rst to examine eumelanin and pheomelanin contents in
cultured human melanoma cells and human melanocytes.
They found that visible pigmentation of those cells is
observed only when eumelanin is detectable, and even when
substantial amounts of pheomelanin is present there can be
no visible pigmentation. Hunt et al. (28) examined the effects
of a synthetic homologue of melanocyte stimulating hormone
(a-MSH) on levels of eumelanin and pheomelanin in cultured
human melanocytes. Synthetic a-MSH induced signicant
increases in the level of eumelanin but had lesser and varied
eects on the level of pheomelanin. Hunt et al. (22) also
showed that cultured melanocytes derived from non-Euro-
peans had more than 10-fold higher levels of eumelanin and
pheomelanin than did those derived from Europeans, but
there was no difference in the pheomelanin to eumelanin
ratios in the two groups. It should be noted that the
Pigment Cell Res. 16, 2003 525
T
a
b
l
e
1
.
C
o
n
t
e
n
t
s
o
f
e
u
m
e
l
a
n
i
n
(
P
T
C
A
)
a
n
d
p
h
e
o
m
e
l
a
n
i
n
(
A
H
P
)
i
n
v
a
r
i
o
u
s
t
i
s
s
u
e
s
o
f
h
u
m
a
n
s
a
R
e
f
e
r
e
n
c
e
S
a
m
p
l
e
S
o
u
r
c
e
n
P
T
C
A
T
o
t
a
l
A
H
P
(
o
r
4
-
a
n
d
3
-
A
H
P
)
U
n
i
t
J
i
m
b
o
w
e
t
a
l
.
(
1
3
)
H
a
i
r
R
e
d
c
o
l
o
r
5
7
.
0

2
.
2
2
1
8

6
9
n
g
/
m
g
S
a
i
t
o
e
t
a
l
.
(
1
5
)
H
a
i
r
T
y
r
o
s
i
n
a
s
e
-
p
o
s
i
t
i
v
e
a
l
b
i
n
i
s
m
3
3
2

9
.
3
5
.
2

1
.
1
n
g
/
m
g
I
t
o
e
t
a
l
.
(
4
)
H
a
i
r
B
l
a
c
k
c
o
l
o
r
4
1
0
3

2
2
4
4

3
3
n
g
/
m
g
D
a
r
k
b
r
o
w
n
t
o
b
r
o
w
n
c
o
l
o
r
5
5
9

1
6
5
0

2
8
L
i
g
h
t
b
r
o
w
n
4
2
1

9
.
7
1
1
1

3
7
B
l
o
n
d
5
1
4

4
.
9
9
3

2
0
R
e
d
5
1
3

4
.
9
3
4
8

6
8
H
o
r
i
k
a
w
a
e
t
a
l
.
(
1
6
)
H
a
i
r
H
e
r
m
a
n
s
k
y
-
P
u
d
l
a
k
s
y
n
d
r
o
m
e
1
8
.
7
3
6
n
g
/
m
g
W
a
k
a
m
a
t
s
u
e
t
a
l
.
(
1
0
)
H
a
i
r
D
a
r
k
c
o
l
o
r
2
8
6
0

2
9
4
-
A
H
P
,
2
.
2

1
.
0
;
3
-
A
H
P
,
1
5

1
2
n
g
/
m
g
F
a
i
r
c
o
l
o
r
1
3
2
3

1
4
4
-
A
H
P
,
8
.
1

9
.
0
;
3
-
A
H
P
,
1
8

8
R
e
d
c
o
l
o
r
3
6
1
3

7
4
-
A
H
P
,
6
3

6
0
;
3
-
A
H
P
,
6
4

3
4
T
h
o
d
y
e
t
a
l
.
(
1
9
)
S
k
i
n
S
k
i
n
t
y
p
e
I

I
I
I
,
u
p
p
e
r
a
r
m
1
3
0
.
8

0
.
6
4
.
9

2
.
6
n
g
/
m
g
w
e
t
w
t
S
a
m
e
s
u
b
j
e
c
t
,
a
f
t
e
r
P
U
V
A
1
3
2
.
2

1
.
2
7
.
6

3
.
6
S
a
l
o
p
e
k
e
t
a
l
.
(
2
0
)
S
k
i
n
C
a
u
c
a
s
i
a
n
,
n
o
r
m
a
l
s
k
i
n
2
5
0
.
7

0
.
5
6
.
8

8
.
6
n
g
/
m
g
w
e
t
w
t
S
a
m
e
s
u
b
j
e
c
t
,
c
o
m
m
o
n
n
e
v
u
s
1
3
2
.
3

2
.
4
3
4
.
5

3
3
.
3
S
a
m
e
s
u
b
j
e
c
t
,
d
y
s
p
l
a
s
t
i
c
n
e
v
u
s
1
8
1
.
3

1
.
4
4
3
.
4

4
5
.
1
T
o
b
i
n
e
t
a
l
.
(
2
1
)
S
k
i
n
S
k
i
n
t
y
p
e
I

I
V
6
4
.
5

3
.
1
1
5
.
3

7
.
0
n
g
/
m
g
d
r
y
w
t
S
a
m
e
s
u
b
j
e
c
t
,
a
f
t
e
r
U
V
R
6
6
.
4

4
.
7
2
1
.
4

2
.
6
H
u
n
t
e
t
a
l
.
(
2
2
)
S
k
i
n
N
o
n
-
E
u
r
o
p
e
a
n
,
v
a
r
i
o
u
s
p
a
r
t
s
8
5
6

3
9
1
2
3

1
1
0
n
g
/
m
g
d
r
y
w
t
E
u
r
o
p
e
a
n
,
v
a
r
i
o
u
s
p
a
r
t
s
1
1
3
.
9

4
.
4
1
1
.
7

7
.
9
N
a
k
a
g
a
w
a
a
n
d
I
m
o
k
a
w
a
(
2
3
)
S
k
i
n
J
a
p
a
n
e
s
e
,
v
a
r
i
o
u
s
p
a
r
t
s
1
9
1
3
.
2

6
.
2
1
8
.
0

1
9
.
1
n
g
/
m
g
d
r
y
w
t
T
a
d
o
k
o
r
o
e
t
a
l
.
(
2
4
)
S
k
i
n
B
l
a
c
k
,
b
a
c
k
5
3
0

9
4
-
A
H
P
,
1
5

6
;
3
-
A
H
P
,
1
1

7
n
g
/
m
g
d
r
y
w
t
S
a
m
e
s
u
b
j
e
c
t
,
7
d
a
y
s
a
f
t
e
r
U
V
R
5
3
2

1
6
4
-
A
H
P
,
1
3

1
1
;
3
-
A
H
P
,
6
.
3

2
.
7
A
s
i
a
n
,
b
a
c
k
3
1
1
.
6

2
.
5
4
-
A
H
P
,
9
.
3

4
.
1
;
3
-
A
H
P
,
5
.
0

2
.
6
S
a
m
e
s
u
b
j
e
c
t
,
7
d
a
y
s
a
f
t
e
r
U
V
R
3
9
.
2

2
.
1
4
-
A
H
P
,
8
.
4

4
.
0
;
3
-
A
H
P
,
5
.
0

1
.
8
W
h
i
t
e
,
b
a
c
k
7
4
.
5

1
.
5
4
-
A
H
P
,
6
.
3

4
.
0
;
3
-
A
H
P
,
3
.
9

2
.
0
S
a
m
e
s
u
b
j
e
c
t
,
7
d
a
y
s
a
f
t
e
r
U
V
R
7
4
.
9

1
.
6
4
-
A
H
P
,
4
.
7

4
.
2
;
3
-
A
H
P
,
5
.
6

5
.
2
P
a
r
s
a
d
e
t
a
l
.
(
2
5
)
S
k
i
n
V
i
t
i
l
i
g
o
p
a
t
i
e
n
t
,
d
e
p
i
g
m
e
n
t
e
d
s
k
i
n
5
4
.
1

3
.
3
4
-
A
H
P
,
2
5

3
7
;
3
-
A
H
P
,
1
8

1
9
n
g
/
4
m
m
p
i
e
c
e
S
a
m
e
s
u
b
j
e
c
t
,
r
e
p
i
g
m
e
n
t
e
d
s
k
i
n
5
1
0
.
4

4
.
1
4
-
A
H
P
,
7
.
5

4
.
1
;
3
-
A
H
P
,
1
1

2
.
1
M
o
r
i
s
h
i
m
a
a
n
d
F
u
k
a
d
a
(
2
6
)
M
e
l
a
n
o
m
a
P
r
i
m
a
r
y
a
n
d
m
e
t
a
s
t
a
t
i
c
1
0
6
3

5
8
7
1
9

4
6
4
n
g
/
m
g
w
e
t
w
t
d
e
l
M
a
r
m
o
l
e
t
a
l
.
(
2
7
)
C
e
l
l
s
N
o
n
-
p
i
g
m
e
n
t
e
d
m
e
l
a
n
o
m
a
c
e
l
l
s
4
<
1
2
1
3

2
3
6
n
g
/
1
0
6
c
e
l
l
s
P
i
g
m
e
n
t
e
d
m
e
l
a
n
o
m
a
c
e
l
l
s
2
2
9

2
1
1
4
2

5
5
N
o
r
m
a
l
m
e
l
a
n
o
c
y
t
e
s
3
1
1
9

7
1
1
3
8
0

2
7
1
H
u
n
t
e
t
a
l
.
(
2
8
)
C
e
l
l
s
F
r
o
m
f
o
r
e
s
k
i
n
o
f
n
o
n
-
C
a
u
c
a
s
i
a
n
3
2
1

1
7
1
4
5
0

1
0
4
0
n
g
/
1
0
6
c
e
l
l
s
S
a
m
e
s
u
b
j
e
c
t
,
a
f
t
e
r
s
y
n
t
h
e
s
i
s
a
-
M
S
H
3
6
3

6
9
1
9
6
0

2
0
0
0
F
r
o
m
f
o
r
e
s
k
i
n
o
f
C
a
u
c
a
s
i
a
n
3
3
.
8

5
.
0
1
8
3

1
8
8
S
a
m
e
s
u
b
j
e
c
t
,
a
f
t
e
r
s
y
n
t
h
e
s
i
s
M
S
H
3
6
.
9

4
.
8
1
8
8

1
0
2
H
u
n
t
e
t
a
l
.
(
2
2
)
C
e
l
l
s
F
r
o
m
s
k
i
n
o
f
n
o
n
-
E
u
r
o
p
e
a
n
7
5
8

4
7
2
1
0
0

1
8
3
0
n
g
/
1
0
6
c
e
l
l
s
F
r
o
m
s
k
i
n
o
f
E
u
r
o
p
e
a
n
9
3
.
9

3
.
4
1
2
6

1
4
4
S
c
o
t
t
e
t
a
l
.
(
2
9
)
C
e
l
l
s
F
r
o
m
f
o
r
e
s
k
i
n
o
f
d
a
r
k
c
o
l
o
r
4
1
5
9

5
0
3
3
7

7
7
n
g
/
1
0
6
c
e
l
l
s
F
r
o
m
f
o
r
e
s
k
i
n
o
f
l
i
g
h
t
c
o
l
o
r
7
9
.
2

4
.
5
1
4
8

8
6
W
a
k
a
m
a
t
s
u
e
t
a
l
.
(
3
0
)
S
e
r
u
m
C
o
n
t
r
o
l
s
u
b
j
e
c
t
3
6
N
D
4
-
A
H
P
,
4
5

2
1
;
3
-
A
H
P
,
3
2
9

2
9
6
n
m
o
l
/
l
M
e
l
a
n
o
m
a
p
a
t
i
e
n
t
w
i
t
h
o
u
t
m
e
t
a
s
t
a
s
i
s
9
2
N
D
4
-
A
H
P
,
8
0

7
5
;
3
-
A
H
P
,
3
3
9

3
8
9
M
e
l
a
n
o
m
a
p
a
t
i
e
n
t
w
i
t
h
m
e
t
a
s
t
a
s
i
s
2
4
N
D
4
-
A
H
P
;
3
0
6

6
2
7
;
3
-
A
H
P
,
1
2
0
0

2
1
1
0
T
a
k
a
s
a
k
i
e
t
a
l
.
(
3
1
)
U
r
i
n
e
C
o
n
t
r
o
l
s
u
b
j
e
c
t
2
N
D
4
-
A
H
P
,
0
.
2
1

0
.
1
5
;
3
-
A
H
P
,
0
.
3
3

0
.
4
0
l
m
o
l
/
l
M
e
l
a
n
o
m
a
p
a
t
i
e
n
t
4
9
N
D
4
-
A
H
P
,
2
7
.
1

6
4
.
2
;
3
-
A
H
P
,
1
2
.
6

3
1
.
4
N
D
,
n
o
t
d
e
t
e
r
m
i
n
e
d
.
a
V
a
l
u
e
s
a
r
e
m
e
a
n

S
D
.
526 Pigment Cell Res. 16, 2003
pheomelanin to eumelanin ratios were much higher in
cultured melanocytes than in the corresponding epidermis
(see Comments below). Scott et al. (29) compared the ability
of cultured human melanocytes with known MC1R gene
mutations to respond to a-MSH and/or to UV radiation. The
mean eumelanin to pheomelanin ratio of melanocytes from
dark-colored foreskins was sevenfold higher than that from
light-colored foreskins.
Serum and Urine
For clinical studies, it is often preferable to use serum or
urine specimens to monitor the degree of pigmentation.
Recently, the specic pheomelanin assay method was applied
to analyze pheomelanin in the serum and urine from
melanoma patients. Wakamatsu et al. (30) reported that
serum levels of 4-AHP in metastatic melanoma patients were
sevenfold higher than in control subjects and they correlated
well (r 0.887) with serum levels of 5-S-CD. Similarly,
Takasaki et al. (31) showed good correlations of 4-AHP
(r 0.862) and 3-AHP (r 0.907) in melanoma urine with
the urinary levels of 5-S-CD. These results suggest that levels
of 4-AHP in serum and urine could be used to monitor the
production of pheomelanin in human skin. It will be
interesting to follow changes in the 4-AHP levels in serum
and urine of normal subjects in response to UV exposure.
QUANTITATIVE ANALYSIS OF EUMELANIN
AND PHEOMELANIN IN MICE
Hair
Pigmentation has been most extensively studied among
animals in mice because nearly 100 pigmentation genes are
known to directly or indirectly aect coat color in mice (32).
More than 30 genes have now been cloned and their
functions elucidated.
Burchill et al. (33) rst reported the application of analyt-
ical methods to study the regulation of melanogenesis in mice
(Table 2). Viable yellow mice at the pubertal stage produce
dark hair. Administration of a-MSH increased tyrosinase
activity in the skin which resulted in the production of dark
hair with a twofold increase in eumelanin levels. In contrast,
bromocriptine (a dopamine agonist) decreased tyrosinase
levels and eumelanin content (to 10% of the original levels)
and increased the pheomelanin content (twofold). This study
indicated an active role of tyrosinase in the switch between
production of eumelanin and pheomelanin. A similar study
was conducted on newborn viable yellow mice (34; data not
shown).
Ozeki et al. (8) examined the effects of various coat-color
genes on the chemical properties of melanins synthesized in
congenic mice. They demonstrated that (i) hairs of lethal
yellow and recessive yellow mice contain almost pure pheo-
melanin and (ii) melanins from hairs of brown, light, silver,
and pink-eyed dilution mice share chemical properties in
common that are characterized by partial solubility in strong
alkali. Extending the study by Ozeki et al. (8), Lamoreux
et al. (9) analyzed the eumelanin and pheomelanin contents
of hairs from mice mutant at one or more of the major
pigment loci. Chinchilla mice produce a mutant tyrosinase
whose activity is one-third of the wild type. The results
indicate that such a reduction in tyrosinase activity aects
pheomelanogenesis more profoundly compared with eumel-
anogenesis.
Lehman et al. (35) examined mice with mutations at the
underwhite (uw) locus for eects on pigmentation. All three
uw mutants, uw/uw, uw
d
/uw
d
, and Uw
dbr
/Uw
dbr
showed
greatly reduced levels of eumelanin, much less than one-
tenth of that found in the C57BL/6 wild-type black mice.
Mutations of the murine Attractin (formerly mahogany)
gene are recognized because they suppress agouti pigment
type switching. Gunn et al. (36) examined the effects of
three Atrn alleles, mg-3J, mg, and mg-L, on pigmentation.
All homozygous and compound heterozygous Atrn mutants
revealed an AHP content that was nearly 10-fold lower than
wild-type C3H/HeJ agouti mice. Other examples of melanin
assays in murine hair include bcl-2-decient mice (37) and a
series of agouti mutant mice that resulted from germline
damage caused by chemicals and radiation (38; data not
shown).
Wakamatsu et al. (10) examined the contents of 4- and
3-AHP separately in hairs of various coat color mice (Table 2)
and found that in black hair and in albino hair, most of the
3-AHP was derived from sources other than pheomelanin,
most likely from 3-nitrotyrosine-containing proteins.
Skin and Other Samples
Tamate et al. (39) analyzed eumelanin and pheomelanin
contents in skins from ve congenic strains of mice with
different coat colors (Table 2). The eumelanin contents in
agouti and brown mice were 3040% of that in B10 black
mice, while those in albino and pink-eyed dilution mice were
reduced to <5%. The pheomelanin content was increased
remarkably in agouti mice, but most of that pheomelanin was
assumed to come from contaminating follicular melanocytes
(T. Hirobe, personal communication).
Melanin contents in melanoma tissues and melanosomes
isolated from B16 and from Harding-Passey melanomas
were analyzed in a previous study (7). B16 melanosomes
produce almost pure eumelanic pigment while Harding-
Passey melanosomes produce pigment containing some
pheomelanin.
Hirobe et al. (40) derived primary cultures of melanocytes
from neonatal skins of C57BL/10JHir (black) mice and
congenic mice carrying agouti, brown, pink-eyed dilution,
and/or albino genes. The eumelanin contents in agouti and
brown melanocytes were about two-thirds and one-third of
that in black, respectively, while those in pink-eyed and albino
melanocytes were reduced to <0.5% of black. The pheome-
lanin contents did not dier signicantly among those
melanocytes. Hirobe et al. (41) then examined the effects of
l-tyrosine on the melanin contents in pink-eyed dilution
epidermal melanocytes (data not shown). Graham et al. (42)
examined the effects of agouti signal protein on B16F1
melanoma cells. They showed that agouti signal protein
inhibited melanogenesis in the presence or absence of
a-MSHand also caused dose-related decreases in the synthesis
of both eumelanin and pheomelanin (data not shown).
Pigment Cell Res. 16, 2003 527
Bartels et al. (43) analyzed eumelanin and pheomelanin
contents in the stria vascularis of C57BL/6J black, lethal
yellow, and albino mice. No elevated level of pheomelanin
was found in the yellow mice, indicating the absence of
pheomelanogenesis in strial melanocytes.
QUANTITATIVE ANALYSIS OF EUMELANIN
AND PHEOMELANIN IN OTHER ANIMALS
Ink sacs of cuttlesh (Sepia ofcinalis) contain large amounts
of pigment granules. Analysis of that pigment showed that it
consists of pure eumelanin (7; Table 3). Melanin pigments in
shes have not been yet thoroughly analyzed, barring the
skin and eyes of medaka sh (44). The results show that
melanin pigments in shes are essentially eumelanic. It is not
clear at present whether shes actually produce pheomelanic
pigments.
Haase et al. (45) analyzed melanin contents in feathers
from wild and from domestic pigeons. Uniformly red
feathers from e/e birds contain pheomelanic pigment, while
uniformly black feathers from wild birds contain eumelanic
pigment. The same group (46) also examined the effects of
sex, castration, and androgens on the eumelanin and
pheomelanin contents of feathers from wild mallards (data
not shown).
Several species of mammals in addition to humans and
mice have been analyzed for melanin contents. Black, yellow,
and white areas of tortoise-shell guinea-pig hair were
analyzed (7), and the eumelanin and pheomelanin contents
were found to correspond to visual expectations. Aliev et al.
(47) analyzed melanin contents in various colored wools
obtained from several strains of sheep. The black phenotype
results from eumelanin content, while tan and brown to
red phenotypes result from mixtures of eumelanin and
Table 2. Contents of eumelanin (PTCA) and pheomelanin (AHP) in various tissues of mice
a
Reference Sample Source n PTCA
Total AHP
(or 4- and 3-AHP) Unit
Burchill et al. (33) Hair Viable yellow (A
vy
/A
vy
), Pubertal 3 272 49 832 220 ng/mg
Same animal, after a-MSH 3 403 26 534 68
Same animal, after bromocriptine 3 29 14 1740 420
Ozeki et al. (8) Hair B10-a/a, black 2 1560 43 ng/mg
B10-A/A, agouti 2 1270 837
B10-b/b, brown 2 459 89
B10-A
y
/a, lethal yellow 2 22 3900
B10-e/e, recessive yellow 2 13 4000
B10-p/p, pink-eyed 2 70 91
B10-c/c, albino 2 2 18
Lehman et al. (35) Hair C57BL-uw/uw, underwhite 2 29 80 ng/mg
C57BL-uw
d
/uw
d
, underwhite 2 89 108
C57BL-Uw
dbr
/Uw
dbr
, underwhite 2 85 114
Lamoreux et al. (9) Hair C57BL-a/a, black 2 1590 49 ng/mg
C57BL-slt/slt, slaty 2 247 54
C57BL-si/si, silver 2 1270 41
C57BL-c-ch/c-ch, chinchilla 2 804 53
C57BL-e/e, recessive yellow 2 37 2500
C57BL-e/e c-ch/c-ch,
ressive yellow chinchilla
2 20 407
Gunn et al. (36) Hair C3H/HeJ-+/+, agouti 2 1640 49 471 51 ng/mg
C3H/HeJ-mg-3J/mg-3J, mahogany 2 980 325 36 1
C3H/HeJ-mg/mg, mahogany 2 1280 269 75 19
C3H/HeJ-mg-L/mg-L, mahogany 2 799 69 69 2
Wakamatsu et al. (10) Hair Lethal yellow (A
y
/a) 1 ND 4-AHP, 1600; 3-AHP, 450 ng/mg
Recessive yellow (e/e) 1 ND 4-AHP, 1200; 3-AHP, 450
Black (a/a) 1 ND 4-AHP, 10; 3-AHP, 28
Albino (c/c) 1 ND 4-AHP, 5; 3-AHP, 13
Tamate et al. (39) Skin B10-a/a, black 3 33.0 4.6 2.3 0.4 ng/mg wet wt
B10-A/A, agouti 3 13.0 0.6 53.0 7.5
B10-b/b, brown 3 11.0 0.6 2.3 1.2
B10-p/p, pink-eyed dilution 3 1.2 0.3 1.3 0.4
B10-c/c, albino 3 0.2 0.1 0.4 0.1
Ito and Fujita (7) Melanoma B16 3 106 59 8.9 1.3 ng/mg wet wt
Harding-Passey 3 16 4.7 51 5.9
Ito and Fujita (7) Melanosomes B16 1 1530 68 ng/mg
Harding-Passey 1 800 260
Hirobe et al. (40) Melanocytes From B10-a/a, black 3 1660 715 7.1 3.5 ng/10
6
cells
From B10-A/A, agouti 3 1190 314 5.5 0.8
From B10-b/b, brown 2 654 95 31 11
From B10-p/p, pink-eyed dilution 3 6.3 5.7 12.9 4.0
From B10-c/c, albino 2 6.2 6.6 7.4 5.6
Bartels et al. (43) Inner ear C57BL-a/a, black, stria vascularis 4 0.5 0.1 1.9 0.7 ng/stria
C57BL-A
y
/a, yellow, stria vascularis 4 0.5 0.1 2.2 0.9
C57BL-c/c, albino, stria vascularis 2 <0.1 1.4 0.4
ND, not determined.
a
Values are mean SD.
528 Pigment Cell Res. 16, 2003
pheomelanin in varying ratios and levels. Similar results have
been reported for goats and llamas (48; data not shown).
Sponenberg et al. (49) analyzed hair samples of various
colors of horses for eumelanin and pheomelanin contents.
The results indicate that hairs in phenotypically black areas
of horses are eumelanic while red to yellow hairs are because
of pheomelanic pigments of mixed type.
The wild-type agouti-banding pattern of hair is well
characterized in lower mammals such as mice. The switch
between eumelanin and pheomelanin in those bands in hair
results from the interaction of a-MSH and agouti signal
protein on the melanocortin 1 receptor (MC1R) of melano-
cytes. Ito et al. (50) analyzed the melanin contents in agouti
hairs of baboons where those bands are quite large. The hairs
were excised with scissors into the black or the yellow bands.
In fact, hairs also oscillate between a eumelanic band and a
pheomelanic band.
COMMENTS
In this review, the contents of eumelanin and pheomelanin
are summarized. Several laboratories have adopted our
methods for eumelanin and pheomelanin assays (15, 23,
26, 5157). Others use one of the melanin assay methods
(5862) combined with spectrophotometric methods (12, 63)
or an alkaline hydrogen peroxide oxidation method which
degrades eumelanin into PTCA (58, 62).
Prota et al. analyze the contents of eumelanin and pheo-
melanin in various tissues including hairs of mice (58) and
eyes of human beings (64). Although these data are valuable
from a comparative standpoint, direct comparison with our
data seems to be inappropriate, as the alkaline hydrogen
peroxide method they used articially produces PTCA (3, 58,
65). Nevertheless, the alkaline hydrogen peroxide method
does have a certain advantage over the acidic permanganate
method (66, 67). The latter method does not produce
appreciable amounts of pyrrole-2,3-dicarboxylic acid
(PDCA) from DHI-derived units in eumelanin, while the
former method gives practically good yields of PDCA from
DHI-rich melanin such as dopamine-melanin (62, 65, 66).
Thus, the alkaline hydrogen peroxide oxidation method
appears to be suitable for the characterization of neuromel-
anin (68). Furthermore, Napolitano et al. (67) suggested that
a new benzothiazole amino acid produced by alkaline
hydrogen peroxide oxidation of human hair represents a
new biogenetic marker for predicting individuals at high risk
for skin cancer and melanoma.
The interpretation of melanin content in cultured melan-
oma cells and melanocytes requires some caution. del
Marmol et al. (69) reported that the deprivation of l-cysteine
from melanoma cells promotes eumelanogenesis, and they
suggested that the eumelanin to pheomelanin ratio is
governed by the concentration of l-cysteine (supplied in the
form of l-cystine) in the culture medium. The concentration
of l-tyrosine in the medium also inuences the type and
content of melanin synthesized in cultured cells (41, 59, 70).
In this regard, it should be repeated that the pheomelanin to
eumelanin ratios were much higher in cultured melanocytes
than in the corresponding epidermis (22). A similar phenom-
enon was reported by Prota et al. (64) in irides of human
eyes: analysis of cultured iridial melanocytes in the growing
stage showed a signicant shift to pheomelanic pigmentation
when compared with those in iris pigment epithelium.
Another caution is necessary when levels of PTCA and
AHP (total AHP or 4-AHP) are compared between dierent
species. This is because the relative proportion of
DHICA-derived units in eumelanin varies from one species
to another (12, 14), and the contribution of AHP levels to the
color intensity also differs among species (12, 14).
Finally, it should be pointed out that melanin contents in
humans are highly variable, especially in skin and cellular
samples. The dierences in constitutive pigmentation in the
skin might be caused by dierences in preparation of skin
samples and also those resulting from dierences in ethnic
origin. The dierences in pigmentation in cultured melano-
cytes might arise from dierences in culture conditions.
CONCLUSIONS
1 Our methods for the assay of eumelanin and pheomelanin
have become valuable tools to study the functions of
melanin, the control of melanogenesis, and the actions
Table 3. Contents of eumelanin (PTCA) and pheomelanin (AHP) in various tissue of other animals
a
Reference Animal Sample Source n PTCA Total AHP Unit
Ito and Fujita (7) Cuttlesh Pigment granules Inc sacs of Sepia ofnalis 1 12 800 <10 ng/mg
Hirose et al. (44) Medaka Skin Wild type 1 19 2.0 ng/mg wet wt
Eye Wild type 1 238 4.5
Hasse et al. (45) Pigeon Feather Wild type, uniformly black 1 507 78 ng/mg
e/e, Uniformely red 1 55 4360
Ito and Fujita (7) Guinea pig Hair Black area 3 102 18 320 270 ng/mg
Yellow area 3 4.5 1.4 1420 210
White area 3 2.4 0.8 4.5 1.7
Aliev et al. (47) Sheep Wool Tajik, black 3 1000 106 20 10 ng/mg
Tajik, dark red 6 66 46 1040 460
Tajik, light tan 4 6 4 160 80
Sponenberg et al. (49) Horse Hair a/a, Grulla, body 1 408 18 ng/mg
e/e, Red chestnut, body 1 13 725
e/e, Chestnut, body 1 41 876
e/e, Blond sorrel, body 1 13 358
Ito et al. (50) Baboon Hair Black bands of agouti pattern 3 145 25 157 28 ng/mg
Yellow bands of agouti pattern 3 8 2 523 170
a
Values are mean SD.
Pigment Cell Res. 16, 2003 529
and interactions of pigmentation genes. These methods
have also found applications in many clinical studies.
2 High levels of pheomelanin are found only in yellow to
red hairs of mammals and in red feathers of birds. It
remains an intriguing question why lower vertebrates like
shes do not synthesize pheomelanin. The usefulness of
serum or urinary pheomelanin levels for assessing normal
pigmentation remains to be established.
3 Detectable levels of pheomelanin are found in human
skin regardless of race, color, and skin type. However,
eumelanin is always the major constituent of epidermal
melanin, and the skin color appears to be determined by
the quantity of melanin produced but not by the quality.
This is particularly important when possible etiological
roles of pheomelanin in inducing skin cancer are dis-
cussed.
Acknowledgements This review is dedicated to the memory of the late
Prof. Giuseppe Prota. S. Ito wishes to deeply acknowledge that his career
as a pigment chemist was very much developed when he worked with
Prof. Prota in 1976 at the Stazione Zoologica di Napoli, Italy.
REFERENCES
1. Prota G. Melanins and Melanogenesis. New York: Academic Press;
1992. pp. 1290
2. Ito S. Advances in chemical analysis of melanins. In: Nordlund JJ,
Boissy RE, Hearing VJ, King RA, Ortonne JP, eds. The Pigmentary
System: Physiology and Pathophysiology. New York: Oxford Uni-
versity Press; 1998. pp. 439450
3. Wakamatsu K, Ito S. Review: Innovative technology. Advanced
chemical methods in melanin determination. Pigment Cell Res
2002;15:174183
4. Ito S, Wakamatsu K, Ozeki H. Chemical analysis of melanins and its
application to the study of the regulation of melanogenesis. Pigment
Cell Res 2000;13 (Suppl. 8):103109
5. Harsanyi ZP, Post PW, Jeannie P, Brinkmann P, Chedekel MR,
Deibel RM. Mutagenicity of melanin from human red hair.
Experientia 1980;36:291292
6. Ito S, Jimbow K. Quantitative analysis of eumelanin and pheomel-
anin in hair and melanomas. J Invest Dermatol 1983;80:268272
7. Ito S, Fujita K. Microanalysis of eumelanin and pheomelanin in hair
and melanomas by chemical degradation and liquid chromatogra-
phy. Anal Biochem 1985;144:527536
8. Ozeki H, Ito S, Wakamatsu K, Hirobe T. Chemical characterization
of hair melanins in various coat-color mutants of mice. J Invest
Dermatol 1995;105:361366
9. Lamoreux ML, Wakamatsu K, Ito S. Interaction of major coat color
gene functions in mice as studied by chemical analysis of eumelanin
and pheomelanin. Pigment Cell Res 2001;14:2331
10. Wakamatsu K, Ito S, Rees JL. Usefulness of 4-amino-3-hydroxyph-
enylalanine as a specic marker of pheomelanin. Pigment Cell Res
2002;15:225232
11. Kolb AM, Lentjes EGWM, Smit NPM, Schothorst A, Vermeer BJ,
Pavel S. Determination of pheomelanin by measurement of amino-
hydroxyphenylalanine isomers by high-performance liquid chroma-
tography. Anal Biochem 1997;252:293298
12. Ozeki H, Ito S, Wakamatsu K, Thody AJ. Spectrophotometric
characterization of eumelanin and pheomelanin in hair. Pigment Cell
Res 1996;9:265270
13. Jimbow K, Ishida O, Ito S, Hori Y, Witkop CJ Jr, King RA.
Combined chemical and electron microscopic studies of pheomelan-
osomes in human red hair. J Invest Dermatol 1983;81:506511
14. Ozeki H, Ito S, Wakamatsu K. Chemical characterization of
melanins in sheep wool and human hair. Pigment Cell Res
1996;9:5157
15. Saito N, Morishima T. Eumelanin and pheomelanin contents in hair
and 5-S-cysteinyldopa and 5-hydroxy-6-methoxyindole-2-carboxylic
acid levels in urine in Japanese oculocutaneous albinism. Arch
Dermatol Res 1991;283:79
16. Horikawa T, Araki K, Fukai K, Ueda M, Ueda T, Ito S, Ichihashi M.
Heterozygous HPS1 mutations in a case of Hermansky-Pudlak
syndrome with giant melanosomes. Brit J Dermatol 2000;143:635640
17. Sato S, Jitsukawa K, Sato H, Yoshino M, Seta S, Ito S, Hayashi Y,
Anzai T. Segmented heterochromia in black scalp hair associated
with iron-deciency anemia. Canities Segmentata Sideropaenica.
Arch Dermatol 1989;125:531535
18. Fukai K, Ishii M, Kadoya A, Hamada T, Wakamatsu K, Ito S.
Nevus depigmentosus systematicus with partial yellow scalp hair due
to selective suppression of eumelanogenesis. Pediatric Dermatol
1993;10:205208
19. Thody AJ, Higgins EM, Wakamatsu K, Ito S, Burchill SA, Marks
JM. Phaeomelanin as well as eumelanin is present in human
epidermis. J Invest Dermatol 1991;97:340344
20. Salopek TG, Yamada K, Ito S, Jimbow K. Dysplastic melanocytic
nevi contain high levels of pheomelanin: quantitative comparison of
pheomelanin/eumelanin levels between normal skin, common nevi,
and dysplastic nevi. Pigment Cell Res 1991;4:172179
21. Tobin D, Quinn AG, Ito S, Thody AJ. The presence of tyrosinase
and related proteins in human epidermis and their relationship to
melanin type. Pigment Cell Res 1994;7:204209
22. Hunt G, Kyne S, Ito S, Wakamatsu K, Todd C, Thody AJ.
Eumelanin and phaeomelanin contents of human epidermis and
cultured melanocytes. Pigment Cell Res 1995;8:202208
23. Nakagawa H, Imokawa G. Characterization of melanogenesis in
normal human epidermal melanocytes by chemical and ultrastrac-
tural analysis. Pigment Cell Res 1996;9:175178
24. Tadokoro T, Kobayashi N, Zmudzka BZ, Ito S, Wakamatsu K,
Yamaguchi Y, Korossy KS, Miller SA, Beer JZ, Hearing VJ. UV-
induced DNA damage and melanin content in human skin diering
in racial/ethnic origin. FASEB J 2003;17:11771179
25. Parsad D, Wakamatsu K, Kanwar AJ, Kumar B, Ito S. Eumelanin
and phaeomelanin contents of depigmented and repigmented skin of
vitiligo patients. Br J Dermatol 2003 (in press)
26. Morishima T, Fukuda E. Quantitative analysis of eumelanin and
pheomelanin in human malignant-melanoma tissues. Arch Dermatol
Res 1985;277:248250
27. del Marmol V, Ito S, Jackson I, Vachtenheim J, Berr P, Ghanem G,
Morandini R, Wakamatsu K, Huez G. TRP-1 expression correlates
with eumelanogenesis in human pigment cells in culture. FEBS Lett
1993;327:307310
28. Hunt G, Kyne S, Wakamatsu K, Ito S, Thody AJ. Nle
4
DPhe
7
a-
melanocyte-stimulating hormone increases the eumelanin: phaeomel-
anin ratio in cultured human melanocytes. J Invest Dermatol
1995;104:8385
29. Scott MC, Wakamatsu K, Ito S, Kadekaro AL, Kobayashi N,
Groden J, Kavanagh R, Takakuwa T, Virador V, Hearing VJ,
Abdel-Malek ZA. Human melanocortin 1 receptor variants, receptor
function and melanocyte response to UV radiation. J Cell Sci
2002;115:23492355
30. Wakamatsu K, Yokochi M, Naito A, Kageshita T, Ito S. Compar-
ison of phaeomelanin and its precursor 5-S-cysteinyldopa in the
serum of melanoma patients. Melanoma Res 2003 (in press)
31. Takasaki A, Nezirevic D, A

rstrand K, Wakamatsu K, Ito S,

Ka gedal B. HPLC analysis of pheomelanin degradation products
in human urine. Pigment Cell Res 2003;16:480486
32. Silvers WK. The Coat Colors of Mice: A Model for Mammalian Gene
Action and Interaction. Basel: Springer-Verlag; 1979. pp. 1380
33. Burchill SA, Thody AJ, Ito S. Melanocyte-stimulating hormone,
tyrosinase activity and the regulation of eumelanogenesis and
pheomelanogenesis in the hair follicular melanocytes of the mouse.
J Endocrinol 1986;109:1521
34. Burchill SA, Ito S, Thody AJ. Eects of melanocyte-stimulating
homone on tyrosinase expression and melanin synthesis in hair
follicular melanocytes of the mouse. J Endocrinol 1993;137:189195
35. Lehman AL, Silvers WK, Puri N, Wakamatsu K, Ito S, Brilliant
MH. The underwhite (uw) locus acts autonomously and reduces the
production of melanin. J Invest Dermatol 2000;115:601606
36. Gunn TM, Inui T, Kitada K, Ito S, Wakamatsu K, He L, Bouley
DM, Serikawa T, Barsh GS. Molecular and phenotypic analysis of
Attractin mutant mice. Genetics 2001;158:16831695
37. Yamamura K, Kamada S, Ito S, Nakagawa K, Ichihashi M,
Tsujimoto Y. Accelerated disappearance of melanocytes in bcl-2-
decient mice. Cancer Res 1996;56:35463550
38. Miltenberger RJ, Wakamatsu K, Ito S, Woychik RP, Russell LB,
Michaud EJ. Molecular and phenotypic analysis of 25 recessive,
530 Pigment Cell Res. 16, 2003
homozygous-viable alleles at the mouse agouti locus. Genetics
2002;160:659674
39. Tamate HB, Hirobe T, Wakamatsu K, Ito S, Shibahara S, Ishikawa
K. Levels of tyrosinase and its mRNA in coat color mutants of
C57BL/10J congenic mice: eects of genic substitution at the agouti,
brown, albino, dilute, and pink-eyed dilution loci. J Exp Zool
1989;250:304311
40. Hirobe T, Wakamatsu K, Ito S. Eects of genic substitution at the
agouti, brown, albino, dilute, and pink-eyed dilution loci on the
proliferation and dierentiation of mouse epidermal melanocytes in
serum-free culture. Eur J Cell Biol 1998;75:184191
41. Hirobe T, Wakamatsu K, Ito S, Abe H, Kawa Y, Mizoguchi M.
Stimulation of the proliferation and dierentiation of mouse pink-
eyed dilution epidermal melanocytes by excess tyrosine in serum-free
primary culture. J Cell Physiol 2002;191:162172
42. Graham A, Wakamatsu K, Hunt G, Ito S, Thody AJ. Agouti protein
inhibits the production of eumelanin and phaeomelanin in the
presence and absence of a-melanocyte stimulating hormone. Pigment
Cell Res 1997;10:298303
43. Bartels S, Ito S, Trune DR, Nuttall AL. Noise-induced hearing loss:
the eect of melanin in the stria vascularis. Hearing Res
2001;154:116123
44. Hirose E, Ono H, Tsubokawa T, Yamamoto H, Ito S, Matsumoto J.
Melanogenesis in transgenic medaka sh bearing the gene for mouse
tyrosinase: a possible shift of their genesis to mouse type. Pigment
Cell Res 1999;12 (Suppl. 7):57 (abstract)
45. Haase E, Ito S, Sell A, Wakamatsu K. Melanin concentrations in
feathers from wild and domestic pigeons. J Hered 1992;83:6467
46. Haase E, Ito S, Wakamatsu K. Inuences of sex, castration, and
androgens on the eumelanin and pheomelanin contents of dierent
feathers in wild mallards. Pigment Cell Res 1995;8:164170
47. Aliev G, Rachkovsky M, Ito S, Wakamatsu K, Ivanov A. Pigment
types in selected color genotypes of Asiatic Sheep. Pigment Cell Res
1990;3:177180
48. Sponenberg DP, Ito S, Wakamatsu K, Eng LA. Pigment types in
sheep, goats, and llamas. Pigment Cell Res 1988;1:414418
49. Sponenberg DP, Ito S, Eng LA, Schwink K. Pigment types of various
color genotypes of horses. Pigment Cell Res 1988;1:410413
50. Ito S, Wakamatsu K, Matsunaga N, Hearing VJ, Carey KD,
Anderson S, Dooley TP. Cyclic oscillations in melanin composition
within hairs of baboons. Pigment Cell Res 2001;14:180184
51. Imokawa G. Analysis of initial melanogenesis including tyrosinase
transfer and melanosome dierentiation through interrupted mela-
nization by glutathione. J Invest Dermatol 1989;93:100107
52. Shimizu H, Shargill NS, Bray GA, Yen TT, Gesellchen PD. Eects
of MSH on food intake, body weight and coat color of the yellow
obese mouse. Life Sci 1989;45:543552
53. Odh G, Hindemith A, Carstam R, Paulson J, Rosengren E, Rorsman
H. Melanins in IGR1 melanoma cells. Pigment Cell Res 1994;7:419
427
54. Alena F, Dixon W, Thomas P, Jimbow K. Glutathione plays a key
role in the depigmenting and melanocytotoxic action of N-acetyl-4-S-
cysteaminylphenol in black and yellow hair follicles. J Invest
Dermatol 1995;104:792797
55. Kondoh H, Wilczek A, Narimizu S, Mishima Y. Mouse broblast
expressing human tyrosinase with DHICA-oxidase activity produces
predominantly pheomelanin deposit in lysosome. Zool Sci
1996;13:825831
56. Dorr RT, Dvorakova K, Brooks C, Lines R, Levine N, Schram K,
Miketova P, Hruby V, Alberts DS. Increased eumelanin expression
and tanning is induced by a superpotent melanotropin [Nle
4
-D-Phe
7
]-
a-MSH in humans. Photochem Photobiol 2000;72:526552
57. Alaluf S, Heath A, Carter N, Atkins D, Mahalingam H, Barrett K,
Kolb R, Smit N. Variation in melanin content and composition in
type V and VI photoexposed and photoprotected human skin: the
dominant role of DHI. Pigment Cell Res 2001;14:337347
58. Prota G, Lamoreux ML, Muller J, Kobayashi T, Napolitano A,
Vincensi MR, Sakai C, Hearing VJ. Comparative analysis of
melanins and melanosomes produced by various coat color mutants.
Pigment Cell Res 1995;8:153163
59. Smit NP, Van der Meulen H, Koerten HK, Kolb RM, Mommaas
AM, Lentjes EG, Pavel S. Melanogenesis in cultured melanocytes
can be substantially inuenced by L-tyrosine and L-cysteine. J Invest
Dermatol 1997;109:796800
60. Slawson MH, Wilkins DG, Rollins DE. The incorporation of drugs
into hair: relationship of hair color and melanin concentration to
phencyclidine incorporation. J Anal Toxicol 1998;22:406413
61. Kronstrand R, Fo rstberg-Peterson S, Ka gedal B, Ahlner J, Larson
G. Codeine concentration in hair after oral administration is
dependent on melanin content. Clin Chem 1999;45:14851494
62. Borges CR, Roberts JC, Wilkins DG, Rollins DE. Relationship of
melanin degradation products to actual melanin content: application
to human hair. Anal Biochem 2001;290:116125
63. Ito S, Wakamatsu K, Ozeki H. Spectrophotometric assay of
eumelanin in tissue samples. Anal Biochem 1993;215:273277
64. Prota G, Hu DN, Vincensi MR, McCormick SA, Napolitano A.
Characterization of melanins in human irides and cultured melano-
cytes from eyes of dierent colors. Exp Eye Res 1998;67:293299
65. Ito S, Wakamatsu K. Chemical degradation of melanins: application
to identication of dopamine-melanin. Pigment Cell Res
1998;11:120126
66. Napolitano A, Pezzella A, Vincensi MR, Prota G. Oxidative
degradation of melanins to pyrrole acids: a pilot study. Tetrahedron
1995;59:59135920
67. Napolitano A, Vincensi MR, Di Donato P, Monfrecola G, Prota G.
Microanalysis of melanins in mammalian hair by alkaline hydrogen
peroxide degradation: identication of a new structural marker of
pheomelanins. J Invest Dermatol 2000;114:11411147
68. Wakamatsu K, Fujikawa K, Zucca F, Zecca L, Ito S. The structure
of neuromelanin as studied by chemical degradative methods.
J Neurochem 2003 (in press)
69. del Marmol V, Ito S, Bouchard B, Libert A, Wakamatsu K, Ghanem
G, Solano F. Cysteine deprivation promotes eumelanogenesis in
human melanoma cells. J Invest Dermatol 1996;107:698702
70. Potterf SB, Virador V, Wakamatsu K, Furumura M, Santis C, Ito S,
Hearing VJ. Cysteine transport in melanosomes from murine
melanocytes. Pigment Cell Res 1999;12:412
Pigment Cell Res. 16, 2003 531