Effects of Different Concentrations of Melatonin on the Time-course of Nitrite–induced Oxidation of Hemoglobin: In vitro Study

Iraqi J Pharm Sci , Vol.18 (1) , 2009 Melatonin inhibits erythrocyte oxidation

Effects of Different Concentrations of Melatonin on the Time-course

of Nitrite–induced Oxidation of Hemoglobin: In vitro Study
#

Saad A.Hussain
*, 1

, Shaima M. Mohammed
**

, Intesar T. Numan
*

and Ihab I. Abdulwahhab
*

*
Department of Pharmacology and Toxicology,College of Pharmacy,University of Baghdad,Baghdad, Iraq.

**
Department of Clinical Laboratory Sciences, College of Pharmacy,University of Baghdad, Baghdad, Iraq.

Abstract
Melatonin is a potent scavenger of reactive oxygen species or free radicals like superoxide and

hydroxyl radicals. The oxidation of hemoglobin to methemoglobin (meth-Hb) by oxidizing compounds

has been widely studied. The present work was designed to evaluate the ability of different

concentrations of melatonin to inhibit nitrite–induced oxidation of hemoglobin. Blood samples were

obtained from apparently healthy individuals from which erythrocyte hemolysate was prepared.

Different concentrations of melatonin (10
-9

-1.0 mg/ml) were incubated for 10 min with the hemolysate,

then to the resultant mixture 1 ml of sodium nitrite (final concentration 0.6 mM) was added, and the

formation of meth-Hb was measured by monitoring absorbance of light at 631 nm each min for 30

min. Control samples without melatonin were utilized for comparison. Nitrite caused rapid oxidation

of hemoglobin to meth-Hb in control samples; in the presence of melatonin, the oxidation process was

delayed in a dose–dependent manner. The effect of melatonin on the time course of nitrite-induced

oxidation of Hb showed that melatonin has a protective effect initiated early after addition along with

nitrite. Melatonin also affect the time required for the formation of meth-Hb, the time required to

convert 50% of the available Hb to meth-Hb was 4 min in the absence of melatonin, and became 17,

22, 26, 30, 114 and 383 min with increasing melatonin concentrations (10
-9

, 10
-6

, 0.001, 0.01, 0.1, and

1.0 mg/ml respectively). In conclusion, melatonin in a concentration and time dependent manner can

protect Hb from oxidation by nitrite; melatonin delays the onset of autocatalytic stage and the

protective effect extended over long period of time.

Key words: melatonin, erythrocytes oxidation

الخالصة
توو رااتوتهب بلووا واتوته وتهودر الدااتوة الحبليوة الوً تريوين هروداة وغلوىبيي دوديوغلىبيي وتحىله الً هيتهياى عولية اكسدة اله

لً هٌت أو تأخير حدوث عولية األكسدة بوبرة ًبيتراي الصىريىم. تن الحصوى علوً عيٌوبم رم هوي عتراكيز هختلفة هي هبرة الويالتىًيي

وعتوودة هوي د وا ايخوريي. تون هوزل هحلوى وغلوىبيي هوي الوريوبم الوتحللوة وحسور اللور اليأشخبص أصحبء وتحضير هحلوى هوي اله

11-1الهيوغلىبيي هت تراكيز هختلفة هي هبرة الويالتىًيي )
- 9

ردوبق تون بعوداب اةوبلة هللتور واحود هوي هوبرة ًوبيترام 11دة ووهلغن/هوا ل

ببتوتخدام هليوبر األشوعة كا رديرةوغلىبيي الوتوىى يالصىريىم كعبها هؤكسد. تو هتببعة عولية التأكسد هي خال ديبس هستىي الويته

لى ال ٌفسجية. أظهرم الٌتبقج اى للويالتىًيي الرداة علً توأخير تأكسود الهيوغلوىبيي بصوىاة تعتوود علوً التركيوز ولتورة الخلو . ويوووي

ولترة الوزل. األتتٌتبل ببى الويالتىًيي بأهوبًه حوبية الهيوغلىبيي هي التأكسد بىاتلة ًبيترام الصىريىم وبصىاة تعتود علً التركيز

Introduction

Recently, many experimental data

provided unequivocal evidence about the

formation and role of free radicals in

biological systems.
(1)

Such reactive species

may bring about oxidative damage to virtually

all cell compartments, eventually leading to

various pathologies and aging.
(2)

These

studies prompted research on physiological

antioxidant systems and molecules, and

stimulated the development of natural or

synthetic compounds that prevent oxidative

stress and damage mediated by an enhanced

formation of free radicals.
(3)

After the

discovery of its radical-scavenging properties,

melatonin (N-cetyl-5-methoxytryptamine) has

been considered as a putative biological

antioxidant but it has been questioned to

whether it may have a real antioxidant

function under physiological conditions;
(4)

its

molecular mechanisms of action remain to be

clarified. Interactions of melatonin

contributing to its antioxidant effects in vivo

may be lost during in vitro experiments; when

it behaves in vitro as an electron donor, many

electrophilic compounds, such as the hydroxyl

radical, Fe
+3

, or carbon centered radicals may

act as acceptors in one-electron transfer

reactions, which convert the indolamine to the

indolyl cation radical.
(5)

Reactivity of

melatonin with oxygen centered radicals, such

as peroxyl or alkoxyl radicals, as well as a

moderate activity towards lipoperoxyl

radicals, has also been demonstrated.
#
Based on oral presentation in the seventh scientific conference of the College of pharmacy /University

of Baghdad held in 26-27 November 2008.

1 Corresponding author E-mail : saad_alzaidi@yahoo.com

Received : 3/1/2009

Accepted : 8/4/2009

mailto:saad_alzaidi@yahoo.com

Iraqi J Pharm Sci , Vol.18 (1) , 2009 Melatonin inhibits erythrocyte oxidation

Although the exact relationship between such

activity and the concentrations required to

perform it is not clarified, its ability to

scavenge a broad spectrum of radicals could

allow melatonin to behave as an antioxidant in

various and possibly complex ways.
(6)

This

study was designed to investigate the

antioxidant activity of melatonin in different

concentrations using an in vitro model of

nitrite-induced hemoglobin oxidation.

Material and method
Blood samples were obtained from

apparently healthy individuals, and were

centrifuged at 2500 rpm and 4°C for 10 min to

remove plasma and the buffy coat of white

cells. The erythrocytes obtained were washed

thrice with phosphate-buffered saline and

lased by suspending in 20 volumes of 20mM

phosphate buffer pH 7.4 to yield the required

hemolysate concentration of 1:20. Different

concentrations of melatonin were incubated

for 10 min with the hemolysate starting with

stock solution (melatonin 1mg/ml) from which

serial dilutions were made to give

concentrations of 0.1, 0.01, 0.001, 10
-6

and 10
-

9
mg/ml melatonin solution. Then to these

incubated mixtures 1ml of sodium nitrite (final

concentration 0.6 mM) were added and the

formation of methemoglobin was measured by

monitoring absorbance at 631 nm each min for

30 min using a spectrophotometer.
(7)

In the

second part of the study, melatonin was added

either before or at various time intervals (5

min and 10 min) after the addition of sodium

nitrite to the hemolysate solution, and the

formation of methhemoglobin was measured

by monitoring the absorbance of light at 631

nm, and the results were compared with

control samples without melatonin; all

experiments were performed in triplicate and

repeated many times.

Results
Nitrite causes a rapid oxidation of

hemoglobin to methemoglobin, as shown in

control curve (figure 1). In the presence of

melatonin, the oxidation process was delayed

in a dose-dependent manner. Figure1 describes

the effect of different melatonin concentrations

on the time- course of nitrite oxidation of

hemoglobin; without melatonin, the time-

course of oxidation shows a characteristic

pattern of slow initial transformation followed

by a rapid autocatalytic process; in presence of

melatonin there is slow increase in absorbance

related to reduced levels of methemoglobin

formation in all test samples. Figure 2 showed

that addition of melatonin to the incubation

mixture, at different time intervals (after 5 and

10 min) during the autocatalytic phase, did not

affect its ability to decrease meth-Hb

formation. The time required to convert 50%

of the available hemoglobin to met

hemoglobin was (4 min) in the absence of

melatonin, whereas with 1 mg/ml melatonin

solution the time was increased to 383 min

(6.4 hr) (table 1).

Figure 1. Effect of different melatonin concentrations on the time-course of nitrite–induced

oxidation of hemoglobin.

0.2

0.4

0.6

0.8

1.2

1.4

1.6

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

Control

1mg/ml

l
0.1mg/ml

l
0.01mg/ml

l
10

-3
mg/ml

mg/ml
10

-6
mg/ml

mg/ml
10

-9
mg/ml

mg/ml

b
s
o
rb

a
n

c
e

Time (min)

Iraqi J Pharm Sci , Vol.18 (1) , 2009 Melatonin inhibits erythrocyte oxidation

Figure 2. Effect of melatonin on the time course of methhemoglobin formation at various time

intervals from nitrite addition.

Table( 1) : Time to form 50% Meth-Hb in

presence of different concentrations of

melatonin.

Melatonin

concentration

mg/ml

formation

of Meth-

Time to form

50% Meth-

Hb (min)

Control 100 4.0

10
-9

mg/ml 96.1 17.0

10-6 mg/ml 86.9 22.0

0.001 mg/ml 50.5 26.0

0.01 mg/ml 37.9 30.0

0.1 mg/ml 29.9 114.3

1.0 mg/ml 16.6 383.0

Discussion
The oxidation of Hb to Meth-Hb by

nitrite has been widely studied,
(7-9)

formation

of Meth-Hb occurs in two stages; there is a

slow initial stage followed by a rapid

autocatalytic stage, which carries the reaction

to completion.
(10)

The present study has

shown that melatonin can protect hemoglobin

from oxidation by sodium nitrite in

hemolysate, and there are two suggested

theories for the mechanism through which

melatonin produces this protective role;

erythrocytes are utilized as a traditional target

for studying oxidative damage, when exposed

to high oxygen tensions and in presence of

high iron contents (transition metal promoting

the formation of oxygen free radicals)

oxidative damage occur due to both

endogenous and exogenous insults. Sodium

nitrite as a prooxidant induces a primary

extensive methemoglobin formation as a result

of generation of several free radical species

like super oxide anion, peroxynitrite, and nitric

dioxide, which are generated during the course

of nitrite–induced oxidation of hemoglobin.
(11)

After the discovery of radical-scavenging

properties of melatonin, it has been considered

a putative biological antioxidant, but it has

been questioned whether it may have a real

antioxidant function under physiological in

vivo conditions.
(6)

The molecular mechanisms

of actions of melatonin remain to be better

clarified; it is capable to prevent the onset of

the autocatalytic stage since superoxide is

implicated in the autocatalytic stage , and the

fact that melatonin is a potent scavenger of

superoxide anion,
(5)

the results of the present

study suggests that the protective action of

melatonin might be due to its scavenger effect

and not due to reduction of methemoglobin to

hemoglobin, since it fails to reverse the

oxidation of hemoglobin; additionally, direct

interaction between nitrite and melatonin as a

reason for protection can be ruled out because

the concentrations of melatonin which protect

erythrocytes is very low.
(11)

Kinetic evidence

indicates that melatonin delays oxidative

denaturation of Hb through it's reaction with

Hb-derived oxoferryl radicals, and this may

0.2

0.4

0.6

0.8

1.2

1.4

1 3 5 7 9 11 13 15 17 19 20 22 24 26 28 30

Time (min)

A
b

s
o

rb
a

n
c
e

CONTROL

After 5 min

After 10 min

ZERO

Iraqi J Pharm Sci , Vol.18 (1) , 2009 Melatonin inhibits erythrocyte oxidation

explain the reported antioxidant effects;

Tesoriere et al (2001) studied the reaction of

melatonin with hemoglobin-derived oxoferryl

radicals and the inhibition the oxidant effects

of hydroxyl peroxide-induced hemoglobin

denaturation in red blood cells, they found that

the basic requirement for oxidative

denaturation of Hb by hydroperoxides is the

transient formation of the perferryl-Hb;
(12)

perferryl-Hb, which includes a hypervalent-

iron oxoferryl heme group and a radical

species, localized in the globin is a strong

oxidant towards the globin moiety, which

leads to Hb denaturation with the formation of

hemichrome and heme release.
(13)

The

perferryl species, generated from met-Hb and

H2O2,
(14)

comprises a radical localized on the

globin, possibly an aromatic amino acid

radical , and an oxoferryl heme group.
(12)

After exhaustion of H2O2, decay of the

perferryl to the oxoferryl form occurs, and

then the latter is slowly converted to met-Hb

by a so-called autoreduction reaction;
(14)

this

process involves intramolecular electron

transfer and modification of the globin moiety.
(13)

Such oxidative modifications of globin on

exposure to H2O2 may be avoided by the

presence of certain antioxidant compounds

such as melatonin, ascorbate or Trolox at the

time of reaction, suggesting that rapid

deactivation of the protein radical in the

perferryl species is crucial for protection;
(15)

the mentioned mechanisms may prove that

melatonin acts through its reducing activity

towards perferryl-Hb, and this may include

the reduction of the oxoferryl moiety or the

unpaired electron electrophile center at the

globin moiety by melatonin, or both.
(11)

Although in our study we did not investigate

the reactivity of melatonin as a reducing agent,

we can not exclude this effect and it needs

further investigations. In conclusion,

melatonin protects hemoglobin against nitrite-

induced oxidation and delay the formation of

meth-Hb in concentration dependent pattern.

Acknowledgment
The authors thank University of Baghdad for

supporting this project.

References
1- Tesoriere L, D'Arpa D, Conti S, et al.

Melatonin protects human red blood cells

from oxidative hemolysis: New insights into

the radical-scavenging activity. J Pineal Res

1999; 27: 195-205.

2- Harman D. Free radical involvement in
aging: Patho-physiology and therapeutic

implications. Drugs Aging 1993; 3: 60-80.

3- Rice-Evans CA, Diplock AT. Current status
of antioxidant therapy. Free Radic Biol Med

1993; 15: 77-96.

4- Loffler M. Melatonin as antioxidant. Use or
misuse? Exp Clin Endocrinol Diabetes

1996; 104: 308-310.

5- Poeggeler B, Saarela S, Reiter RJ, et al.
Melatonin. A highly potent endogenous

scavenger and electron donor: New aspects

of the oxidation chemistry of this indole

accessed in vitro. Ann N Y Acad Sci 1994;

738: 419-420.

6- Livrea MA, Tesoriere L, D’arpa D. et al.
Reaction of melatonin with lipoperoxyl

radicals in phospholipid bilayers. Free Radic

Biol Med 1997; 23: 706-711.

7- Doyle MP, Pickering RA, Dykatra RL, et al.
Nitrite-induced hemoglobin oxidation.

Biochem Biophys Res Commun 1982; 105:

1 -132.

8- Wallace W, Houtchens RA, Maxwell JC, et
al. Mechanisms of hemoglobin oxidation.

Biol Chem 1982; 257: 4966-4977.

9- Tomoda A, Tsuiji A, Yoneysnia Y.
Biochemical concequences of Hb oxidation.

Biochem 1981; 193: 169-179.

10- Venkatesan P, Unnikrishnan MK. Effect of
curcumin analogues on oxidation of

haemoglobin and lysis of erythrocytes. J

Current Sci 2003; 84: 74-78.

11- Tesoriere L, Allegra M, D’Arpa D, et al.
Reaction of melatonin with hemoglobin-

derived oxoferryl radicals and inhibition of

the hydroperoxide-induced hemoglobin

denaturation in red blood cells. J Pineal Res

2001; 31: 114-119.

12- Davies MJ. Detection of peroxyl and alkoxyl
radicals produced by reaction of

hydroperoxides with heme-proteins by

electron spin resonance spectroscopy.

Biochim Biophys Acta 1988; 964: 28-35.

13- Giulivi C, Cadenas E. Ferrylmyoglobin:
Formation and chemical reactivity toward

electron-donating compounds. In: Methods

in Enzymology, 1994; Packer L, (Ed), Vol.

233. Academic Press, San Diego, CA, 189-

202.

14- Patel RP, Svistunenko DA, Darley-Usmar
VM, et al. Redox cycling of human

methemoglobin by H2O2 yields persistent

ferryl iron and protein based radicals. Free

Radic Res Commun 1994; 25: 117-123.

15- Giulivi C, Romero FJ, Cadenas E. The
interactions of Trolox C, a water-soluble

vitamin E analog, with ferrylmyoglobin:

Reduction of the oxoferryl moiety. Arch

Biochem Biophys 1992; 299: 302-312.