# Pigment Melanin Scavenges Nitric Oxide In Vitro: Possible Relevance to Keloid Formation

Authors: Julian M. Menter; Danita Eatman; Mohamed Bayorh; Ahmad M. Dawaghreh; Isaac Willis
Journal: Research Letters in Physical Chemistry
 (2008)

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## Abstract

Recently, nitric oxide (NO) has been implicated in the formation of keloids, preferentially formed in dark-skinned persons, and we suspected that pigment melanin itself may play a direct role by adsorbing NO. We tested the ability of cuttlefish sepia melanin to scavenge (adsorb) NO, generated in situ by 2-(N.N Diethylamino) diazeneolate-2-oxide (DEA/NO), through a dialysis membrane. NO was measured asNO2_ and NO3_ by the Griess method and as N2O3 by trapping experiments with the fluorogenic substrate 4,5-diaminofluorescein (DAF-2). Initial NO2_ and NO3_ concentrations were significantly lower in the test dialyzates than in controls. Scavenging of NO was rapid enough to compete with DAF adduct formation. Both analytical methods gave comparable results. Adsorbed NO and/or its oxidized products may undergo interactions with melanin, adsorbed O2, and/or dermal material that may lead to keloid formation.


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## Body

## 1. Introduction

Melanin pigments are responsible for
epidermal skin coloring in humans. Melanin’s broad optical absorption and
stable free radical properties and binding capability make it an effective in vivo antioxidant [1, 2] photoprotective agent [1, 3], electron transfer agent  [4–6],  with semiconductor properties [6].  People
of color are particularly susceptible to keloids, a recalcitrant consequence of
aberrant wound healing, characterized by excessive collagen deposition that
extends beyond the original wound [7–9].
Recently, nitric oxide (NO) has been implicated as a key player in the
formation of keloids and hypertrophic scars [10]
by stimulating over-expression of inducible nitric oxide synthase (iNOS)
[11] which is in proximity to melanocytes
and dermal collagen [11]. That NO can
diffuse through biological membranes [12] suggests that these reactive species
can reach the melanosomes within nearby melanocytes, so that NO adsorption
to melanin and its sequelae may play a role in keloid formation, particularly
in dark-skinned individuals.


## 2. Materials and Methods

Sepia melanin, [13], (MelanInk@) was predialyzed through a
Spectropore membrane (MW cutoff 6–8 kD) into 100 mL 0.1 M phosphate buffer, pH 7.4/0.1 M EDTA, followed by two changes of
0.1 M buffer alone. As a source of exogenous NO, we used DEA/NO (see
Figure 1) (Sigma Chemical Co., Mo, USA). 200 mL of freshly  made 0.9 mM DEA/NO stock solution (0.18 μmole) was placed into each of two stirred 25 mL graduated
cylinders containing dialysis bags (Spectrum Laboratories, Inc., Calif, USA, MW
cutoff 6–8 Kd) filled with
(a) 3 mL of a 90 mg melanin buffer suspension (“melanin bag”) or (b) 3 mL buffer
alone “control bag.”  We measured DEA/NO
generated—NO in 200 μL aliquots (1.44 nmole) of dialyzate
test and control samples t=0–90 minutes as
nitrite and nitrate by the Griess method with a SOFT maxPRO NO—measuring kit
(Molecular Devices). Control experiments confirmed that no melanin escaped into
the dialyzate, and there was no significant contamination by nitrite or nitrate
prior to addition of DEA/NO.Figure 1
Structure of DEA/NO. The sodium salt (R=1) was used in the Griess analysis experiments. The diethylammonium salt (R=2) was used for the trapping of NO with the fluorogenic DAF-2 to form the highly fluorescent DAF-2T.NO was also detected by its ability
of its oxidation product,N2O3, to form highly fluorescent
triazoles (DAF-2T) from 4, 5-diaminofluorescein (DAF-2) in the presence
of molecular O2 [14] (see Figure 2).
Initially, 10.0 nmole of DAF-2 in buffer solution was mixed in disposable
fluorescence cuvettes with the appropriate amount of added buffer to make a
total volume of 2.0 mL. After further addition of 1.0 mL of DEA/NO (diethylammonium
salt), the fluorescence intensity of melanin and control dialyzates was monitored as functions of time on a Perkin-Elmer 650–40 fluorescence
spectrophotometer (λex=495nm,λem=515nm). The fluorescence
scavenging ratio was
the ratio of fluorescence intensities of melanin and control bag dialyzates
under steady-state conditions.  In some
experiments, a 20 mM melanin suspension in the absence of a dialysis membrane
was analyzed as before. These latter results were corrected for the absorption
and emission of melanin [15].Figure 2
Reaction of DAF with NO to form
the highly fluorescent DAF-2T.T-tests were conducted to determine
the statistical significance of the results. The determined P-values
assume normal distributions for each group.


## 3. Results

### 3.1. NO Measurement as Nitrite and Nitrate

Pigment melanin rapidly sequesters nitric
oxide (see Figures3(a) and 3(b)). At t=0, the mean±S.D. of 3
experiments afforded scavenging ratios of 0.503±0.232 (P<.03) for NO2− and
0.323±0.266 for NO3− (P<.02). Initial [NO3−]/ [NO2−]
ratios were ~1.5.(a) Determination
of NO asNO2− by Griess analysis in 0.1 M phosphate buffer, pH
7.4. Black circles: initial amount of NO=1.44 nmole in “melanin bag” (see
text). White circles: initial amount of 1.44 nmole in the absence of melanin
(“blank bag”; see text). Mean±S.D. of 3 determinations, P<.03.
(b) Determination of NO as NO3− by Griess analysis in 0.1 M phosphate
buffer, pH 7.4 under the same conditions as in Figure 3(a). Black circles: “melanin
bag.” White circles: “blank bag”; see text.  Mean±S.D. of 3 determinations, P<.02.
(a)(b)


### 3.2. Fluorescence Measurements

The fluorescence intensities (i.e., DAF-2T formation) of both control and test samples
increased to a steady-state value att∼20 minutes (see Figure 4). Melanin
competes successfully with DAF for NO as evidenced by a steady-state fluorescence
scavengingratio=0.706±0.103 (n=4; P<.0054).
These results were qualitatively the same whether dialysis systems or melanin
suspensions were used.Figure 4
Uptake of NO by
sepia melanin as determined by trapping with DAF in 0.1 M phosphate
buffer, pH 7.4 (see text). Black Circles: with added melanin (20μM in 3 mL). White circles: without added melanin Mean±S.D. of 4 determinations, P<.005.


## 3.1. NO Measurement as Nitrite and Nitrate

Pigment melanin rapidly sequesters nitric
oxide (see Figures3(a) and 3(b)). At t=0, the mean±S.D. of 3
experiments afforded scavenging ratios of 0.503±0.232 (P<.03) for NO2− and
0.323±0.266 for NO3− (P<.02). Initial [NO3−]/ [NO2−]
ratios were ~1.5.(a) Determination
of NO asNO2− by Griess analysis in 0.1 M phosphate buffer, pH
7.4. Black circles: initial amount of NO=1.44 nmole in “melanin bag” (see
text). White circles: initial amount of 1.44 nmole in the absence of melanin
(“blank bag”; see text). Mean±S.D. of 3 determinations, P<.03.
(b) Determination of NO as NO3− by Griess analysis in 0.1 M phosphate
buffer, pH 7.4 under the same conditions as in Figure 3(a). Black circles: “melanin
bag.” White circles: “blank bag”; see text.  Mean±S.D. of 3 determinations, P<.02.
(a)(b)


## 3.2. Fluorescence Measurements

The fluorescence intensities (i.e., DAF-2T formation) of both control and test samples
increased to a steady-state value att∼20 minutes (see Figure 4). Melanin
competes successfully with DAF for NO as evidenced by a steady-state fluorescence
scavengingratio=0.706±0.103 (n=4; P<.0054).
These results were qualitatively the same whether dialysis systems or melanin
suspensions were used.Figure 4
Uptake of NO by
sepia melanin as determined by trapping with DAF in 0.1 M phosphate
buffer, pH 7.4 (see text). Black Circles: with added melanin (20μM in 3 mL). White circles: without added melanin Mean±S.D. of 4 determinations, P<.005.


## 4. Discussion

Melanin
scavenging of NO takes place rapidly (see Figures3(a) and 3(b)). The lower steady-state
fluorescence intensity in the presence of melanin (see Figure 4) confirms that
scavenging competes successfully with the relatively slow [14] triazole
formation, and strongly suggests that NO itself is initially adsorbed to
melanin. Adsorbed NO can form a variety of active species in oxygen—containing
solution including NO2, N2O3, O2•−, and
ONOO−,
and could change melanin’s oxidation state [13, 16]. Any of these might
stimulate keloid formation and might offer a basis for the observation that
African-Americans, with higher and more robust
concentrations of melanosomes, are more susceptible to keloids than are
Caucasians.Reaction
of gaseous NO in solution withH2O2 and/or O2•− arising
from melanin autoxidation [17] is unlikely to be significant. Since melanin
acts as an efficient “pseudodismutase” [17], the steady-state [O2•−] is low
outside the melanin “cage.” Hydrogen peroxide does not react with NO [18].



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*Source: 210616-2008-09-23.xml*