Evaluation of Oxidative-Antioxidative Balance in Serum of Patients With Non Acute Hepatitis Virus Type B

Rasha Hasan Jasim

Chemistry Department, College of Education for Girls, University of Kufa

Abstract. Oxygen free radicals play an important role in the pathogenesis of tissue damage in many pathological conditions, including liver diseases. Aim of the present study focused on the investigation the possible relationship between serum malondialdehyde level, an index of lipid peroxidation, and ceruloplasmin levels, as protective agents against lipid peroxidation, in hepatitis B virus. A group of 26 hepatitis virus type B patients enrolled in the study, while; control group consisted of 20 healthy subjects. In the present study, total proteins, malondialdehyde (µmol/L), ceruloplasmin oxidase activity (U/L) and ceruloplasmin concentration (g/L) were measured in sera samples of patients with non acute hepatitis B virus as well as in the healthy controls. Non significant variation (p>0.05) of total serum protein and malondialdehyde levels, while, highly significant variations were found for ceruloplasmin oxidase activity (p<0.000) and ceruloplasmin concentration (p<0.001) in patients with hepatitis B virus when compared with those of healthy individuals. The results revealed a significant elevation (p<0.000) of copper level in patients with hepatitis B virus when they were compared with healthy controls group, on the other hand, non significant variations (0.784) were observed when iron levels in patients group were compared with healthy individuals group. The levels of malodialehyde and celoplasmin oxidase activity in sera of pateints as well as in healthy controls group failed to illustrate a significant statistically correlation. We can conclude that a raise in the ceruloplasmin oxide activity is not reflex to the ambulance in the oxidation – antioxidation status, but it is produced as one of the defense system’s proteins against the initial viral infection.

Key words: lipid peroxidation, malondialdehyde, ceruloplasmin

1. Introduction Hepatitis B is a viral illness causing inflammation of the liver, resulting from infection with a DNA-type

virus (Hepadnaviridae). This virus consists of an inner core surrounded by an outer capsule. The inner core contains the core antigen (HBcAg) and the antigen (HBeAg) also known as the "e" antigen. The outer capsule contains the hepatitis B surface antigen (HBsAg). Hepatitis B virus can be acute or chronic. It is a major public health concern worldwide which can lead to acute and chronic liver diseases including cirrhosis and hepatocellular carcinoma [1]. However, worldwide, about 400 million people have the virus, with most of these people living in Asia. Clearly, this is a significant public health and medical problem, about 5-10% of adult patients and 80-90% of children carriers became chronic carriers of the virus (basing to the World Health Organization at 2000) [2]. Moreover; 1.2 million people die from hepatitis virus type B and its related diseases every year [3].

Oxidative stress is a term denoting an imbalance between the production of oxidants and the respective defense systems of an organism [4, 5]. Lipid peroxidation occurs at low levels in all cells and tissues. In health, oxidation by free radicals and neutralization by antioxidants remain in balance, when the reactive oxygen species (ROS) are in abundance, oxidative stress occurs [6].Oxygen free radicals play an important role in the pathogenesis of tissue damage in many pathological conditions, including liver diseases [7].

Ceruloplasmin is a ferroxidase enzyme that in humans is encoded by the CP gene [8-10].Ceruloplasmin binds copper; appears to be more important as a copper storage pool than as a transport protein; integrates iron and copper homeostasis [11]. In addition to that, it have a protective effect as an antioxidant agent through its ability to prevent oxidative damage, using copper (II) centers[7, 12].

237

2011 International Conference on Chemistry and Chemical Process IPCBEE vol.10 (2011) © (2011) IACSIT Press, Singapore

Variousactivities. Efunctions, pinterferon ththe most tra

Copper including cyof 75% the study focusof lipid perovirus.

2. Mater

2.1 IndivThe stu

years who wNajaf, Iraq, course of dThe control70 (range of

All seraare shown ihabits, withPatients ansocioeconombiochemicasubjects in evacuated tu

2.2 DetermA total

Manufactursodium-potaserum albumwith 2.5 mabsorbance

2.3 MeasMalond

Abridgment1ml of thiobheated at 1

s trace elemEssential micprotein syntherapy respoace elements

is essential ytochrome ocopper is ased on the invoxidation, an

rials and

viduals of thdy group comwere admitte between Jun

drugs. The pl group compf 51), the rat

Fig. 1: Ge

a were collecin the table hout history nd control smic status anl, histologicaaccordance wubes without

mination ol serum proring Companassium tartramin was usel Biuret reag was measur

urement ofdialdehyde let, 150 µl of tbarbituric ac00°C for 15

P

ments are recronutrients athesis, oxid

onse regulatio importancetrace metal

oxide, superosociated withvestigation tnd ceruloplas

Methods

he Study mprised 26 ped consecutivne and Noveatients groupprised 20 hetio of male to

ender Distribu

cted in the m1. The healtof viral hep

subjects resnd similar dial and virolowith standart anticoagula

f Total Serotein was eny in a conate (16 mMed as a standgent, then thred at 540 nm

f Serum Mevel is measuthe serum said (0.6 %). U

5 minutes in

11Male

Patients42%

esponsible fare involved ative damagons and alter [13]. which is a c

oxide dismutah specific cothe possible rsmin levels,

patients withvely to the Lember 2010.p consisted oalthy volunte

o female was

ution of Patien

morning afterthy volunteepatitis, routiiding in thiet habits. Pa

ogical evidenrd procedureation agents.

rum Proteinstimated us

ntainer contaM), potassiumdard protein. he mixture wm.

Malondialdeured by the ample was mUsing the vorn the water

15 Female Patients

58%

for many bin many me

ge and antirations of the

component oase, tyrosine

opper-bindingrelationship as protective

h newly diagnLiver and Dig

All patients of 15 femaleseers that inc

s shown in fig

nts with Hepat

r fasting 10 ers were seleing clinical

he same geoatients with

nce in the sam, 6-10 ml of

ns Levels ing Biuret

ains 100 ml,m iodide (15

The procedwas incubate

hyde Levelthiobarbituri

mixed with 1rtex, the finabath. After

biochemical, etabolic pathi-oxidant dee virus genom

of a wide rane, dopamine hg protein, cerbetween ser

e agents agai

nosed hepatitgestive Tractwere enrolle

s and 11 malluded 11 femgure 1.

titis B Virus, a

hour featureected on the check up duographical achronic hepa

me medical cf blood was c

method [15, that consis mM), and

dure includeded at room t

l ic acid-react ml of trichl

al mixture wathe mixture

9HealthMale45%

immunologhways in the efense, immumes, copper

nge of intrachydroxylase ruloplasmin

rum malondiinst lipid per

tis B virus, bt Center of Aed in the stules and their males and 9

and Healthy In

e of the subjebasis of no

uring the enarea, and thatitis B diagcity. Blood scollected fro

5]. Biuret rest of sodiumcupric sulfa

d mixing of temperature

ting substancloro acetic aas mix, the re

was cooled

11 HealtFema

55%

hy es

%

gical, and pliver, such aunological cand iron are

cellular metaand lysyl ox[14]. Aim oaldehyde levroxidation, in

between the aAl-Sader Meddy before rerange age wmales aged

ndividuals.

ects in the palcoholic an

ntire period hey were in

gnosed basedsamples wereom vein and

eagent suppm hydroxide ate (6 mM). 50 µl serumfor 30 minu

ces (TBARS)cid (TCA) (eaction mixtud with tap w

thyales%

physiologicalas enzymaticcompetence,example for

alloenzymes,xidase, moref the present

vel, an indexn hepatitis B

age of 21-75dical City ineceiving the

was 54 years.between 19-

present studynd smokingof research.n the same

d on clinical,e taken fromprotected in

plied by the(100 mM),The bovine

m or standardutes, and the

) assay [16].17.5 %) andure was then

water, it was

l c , r

, e t x B

5 n e . -

y g . e ,

m n

e , e d e

. d n s

238

extracted with 1 ml TCA (70 %), the mixture was stand for 20 minutes at 25°C, and centrifuged at 3000 xg for 15 minutes. The organic phase was measured by use of a spectrophotometer with a wavelength of 534 nm.

2.4 Determination of Ceruloplasmin Oxidase activity The activity of ceruloplasmin oxidase was determined in serum using the modified Rice method [17].

The procedure included two glass tubes, test (A) and blank (B), 1ml of substrate buffer was added to each tube, then incubated at 37ºC for 5 min. A 100μl of serum sample was added to tube A then incubated at 37ºC for 15 min. A volume of 3 ml of cold working inhibition solution was added to all of A and B tubes; at last 100μl of deionized water was added to tube B. The absorbance was measured at λ=540 nm. Reagents

Preparation of Substrate Buffer: Two gram of p-phenylenediamine was dissolved in the smallest volume of absolute ethanol, then filtered through double filter paper (Whitman number 1). Gently and gradually, concentrated hydrochloric acid was added. The pink precipitate was filtered and washed with methanol, then the product salt (p-phenylenediamine-2HCl) was dried at 70ºC. To purification of p-phenylenediamine-2HCl, the salt was dissolved in a minimum volume of hot water (60ºC), charcoal was added and left for 5 min, and then the mixture was filtered while hot. The purified salt was cold and precipitated from the filtrate by the addition of cold acetone until the turbidity was appeared (for the perfect results, all these step must be done in the ice bath). The mixture was refrigerated for several hours, filtered off the crystals, then it was dried in the dark in a vacuum desiccators over anhydrous calcium. To prepare substrate buffer, 0.1g of crystal p-phenylenediamine-2HCl was dissolved in 100ml of acetate buffer (0.4M, pH 5.2, containing 0.4 μM EDTA).

Working inhibition solution: This solution was prepared by diluting 3 ml of stock inhibition solution (0.1 M of sodium azide and 0.5 M of sodium chloride) to 100 ml with deionized water, stored at 4ºC, and used cold [18].

Ceruloplasmin Oxidase Activity = The Absorbance of A–B tubes×349.04

Ceruloplasmin oxidase concentration was determined by measuring the absorbance of A and B tubes at wavelength = 605 nm.

Ceruloplasmin Oxidase Concentration = The Absorbance of A–B tubes×87.5.

2.5 Determination of Serum Copper and Iron Levels The levels of serum copper and iron were determined by flame atomic absorption spectrophotometry

(GBC-933plus).

2.6 Statistical Analysis The findings were expressed as the mean ± standard deviation (S.D.). The data were analyzed with

Student’s independent t test. All statistical analyses were performed with the program Statistical Package for the Social Science (SPSS for Windows, Version 14.0). Pearson’s correlation was applied to determined the relations among the laboratory parameters of the present study, significance was determined regression. A p-value of <0.05 was accepted as statistically significant.

3. Results In the present study, total proteins, malondialdehyde (µmol/L), ceruloplasmin oxidase activity (U/L) and

ceruloplasmin concentration (g/L) were measured in sera samples of patients with non acute hepatitis B virus as well as in the healthy controls. Table 1, shows that no significant variation (p > 0.05) of total serum protein levels in patients with hepatitis B virus when compared with those of healthy individuals. With same manner, the statistical evaluation failed to exhibit significant variation (p=0.180) for serum malondialdehyde when patients of hepatitis B virus were compared with those of healthy controls. On the other hand, when the comparison was carried out for hepatitis B virus patients and control group, highly significant variations were found for ceruloplasmin oxidase activity and ceruloplasmin concentration (p<0.000 and p<0.001 for ceruloplasmin oxidase activity and ceruloplasmin concentration; respectively).

239

Table 1: Lev

In

P

C

p

The prethe serum twere studieand total se0.05) when (A and B).

Fig. 2: Corr

Figure 3to the serucorrelation

Fig. 3:

Hepatiticeruloplasm(figure 4 A

vels (glL) of TConcen

dividuals

M

Patients

(n=26)

4

Controls

(n=20)

4

p-value

esent study intotal proteinsd. Significan

erum proteinsthe relation

relation of Ag

3 A, illustratum malondia(r = 336 at p

Correlation o

is B virus min oxidase a

and B).

y = -0R

0

15

30

45

60

75

0

Age

(Yea

rs)

y =

0

15

30

45

60

75

0

Age

(Yea

rs)

TSP, (µmol/L)ntration in pa

Age (Years)

Mean ± S.D. Min-Max

Range 49.000±16.122

21-75 54

41.850±15.24619-70

51 0.715

nvolved a ws, malondialdnt negative cs, while lesswas between

ge to the Total

tes a significaldehyde levp < 0.05) betw

f Age to the S

patients demactivity (r =

0.992x + 114.6R² = 0.417

20 40Total Serum P

= 7.338x + 6.783R² = 0.400

2 4Serum Malonad

) of MAD, (U/atient of Hepat

TSP Lev(g/L)

Mean ± SMin-Ma

Range2 66.120±10

42.000-8240.000

6 74.050±659.000-83

24.0000.064

wide age patiedehyde, cerucorrelation (r of this resun healthy ind

l Serum Protei

cant positivevel, in contrween control

Serum Malond

monstrated s0.716 at p <

60 80Protiens (g/L)

6 8dildehyde (µmol/

/L) of Ceruloptitis B Virus a

vel ) S.D.

Max e

M(µm

MeanMin

Ra0.5002.0000

5.7533.440

5.2.492

3.0000

5.3053.440

3.4 0.

ents range, auloplasmin or = - 0.646 alt was noteddividual�s a

ins in A: Patie

e correlationrast to this l’s age and se

dialdehyde in Individuals

significant p< 0.005), how

100

A0

15

30

45

60

75

Age

(Yea

rs)

10/L)

A0

15

30

45

60

75

Age

(Yea

rs)

plasmin Oxidaand control sub

MAD mol/L) n ± S.D.n-Max ange

C

MM

±1.3900-8.650210

10955.

±0.9870-7.240130

43.15

180

according to oxidase activt p < 0.005)

d (moderate nage and total

ents of Hepati

(r = 0.632 atresult, figur

erum malond

A: Patients of

positive corrwever those o

y = -1.204x +R² = 0.2

0

5

0

5

0

5

0 20Tota

y = 5.203x + R² = 0.11

0 2Serum M

ase Activity, abjects (mean ±

p. Activity (U/L)

Mean ± S.D. Min-Max

Range 9.130±37.544 491-199.279 143.788

.573±11.822 .705-63.169

47.464 0.000

that the corvity and ceru

was observnegative corrserum prote

tis B Virus, an

t p < 0.005) re 3 B, shodialdehyde le

f Hepatitis B V

relation of tof healthy in

+ 131.0263

40 60al Serum Protien

14.2413

4Malonadildehyde

and (glL) of C± S.D.)

Cp. Leve(g/L)

Mean ± S.Min-Max

Range 19.981±6.610.063-33.5

23.450 9. 492±3.22.363-14.0

11.637 0.001

rrelation of culoplasmin cved between relation, r = ins, as show

nd B: Healthy

of hepatitis pows that no evel.

Virus, and B:

their age tondividuals fai

80 100ns (g/L)

B

6 8e (µmol/L)

B

Ceruloplasmin

el

D.x

612513

285000

case’s age tooncentrationpateints age- 513 at p <n in figure 2

y Individuals

patient’s agestatistically

Healthy

o the serumiled to do so

o n e < 2

e y

m o

240

Fig. 4: Cor

Highly concentratiocorrelation t

Fig. 5: C

Accordi[19], in the the levels osignificant ewith healthywhen iron le

Table 2: Se

In ordeinfection wi

rrelation of A

significant on (r = 0.76to the serum

Correlation of A

ing to the facsame time t

of these metaelevation (p y controls gevels in patieerum Copper

er to find thith B virus o

y =

0

15

30

45

60

75

0

Age

(Yea

rs)

Ser

y =

0

15

30

45

60

75

0

Age

(Yea

rs)

Seru

Age to the Seru

positive cor64 at p < 0.

m ceruloplasm

Age to the Ser

ct that coppethe level of cals were me< 0.000) of roup (table 2ents group wand Iron Leve

Indivi

Pati

(n=

Cont

(n=p-va

e possible rof hepatitis, t

0.240x + 22.77R² = 0.513

40 80 12rum Ceruloplasm

1.342x + 22.17R² = 0.583

8 16

um Ceruplasmin

um CeruloplasHe

rrelation wa.0005) as sh

min concentra

rum CeruloplaHe

er level basincopper metaasured usingcopper level2). On the o

were compareels (µg/ml) in

iduals

Cu LM

M

ients

=26)

1.0.

trols

=20)

0.0.

alue

relation betwthe correlatio

20 160 200min Activity (U/

24 32

n Concentration

smin Oxidase ealthy Individu

as also obsehown in figuation of heal

asmin Concenealthy Individu

ng on the actl control andg flame atoml in patients other hand, ned with healtPatient of He

Level (µg/ml)Mean ± S.D.

Min-Max Range

788±0.668 .887-3.232

2.345

678±0.291 047 -1.022

0.975 0.000

ween the oxion between m

0 240/L)

A0

15

30

45

60

75

Age

(Yea

rs)

40

(g/L)

A0

15

30

45

60

75A

ge (Y

ears

)

Activity in Auals

erved for pare 5 A, no lthy individu

ntration in A: uals

ivity and cond affect the lmic absorptiowith hepatiti

non significathy individuapatitis B Viru

Fe Level (Mean ±

Min-MRang

1.476±00.900-2.

1.198

1.472±00.808-1.

1.1420.784

idation and malondialdeh

y = 0.097x +R² = 0.0

0 15Serum Cerulo

y = 1.912x + R² = 0.16

0 2 4Serum Ceru

A: Patients of H

atients age tsuch findin

uals was stud

Patients of He

ncentration olevel of iron on techniqueis B virus whant variationsals group, as us and Healthy

µg/ml)S.D.

Max ge .391 .098 8

.413 .950 2 4

antioxidationhyde as a pr

+ 37.62005

30 45oplasmin Activit

23.6969

6 8 10plasmin Concen

Hepatitis B Vi

to serum ceg was notice

died (figure 5

epatitis B Viru

of ceruloplasmetal [20, 2

e. The resulthen they wers (0.784) weshown in tab

y Controls (me

n processes rofile to the p

60 75ty (U/L)

B

12 14 16ntration (g/L)

B

irus, and B:

eruloplasmined when the5 B).

us, and B:

smin enzyme21], for that,ts revealed are comparedere observedble 2. ean ± S.D.)

through theperoxidation

n e

e , a d d

e n

241

process, anstudied. As of pateints a

Fig. 6: Co

4. DiscuThe ou

depends upoAccording tas well as current stud

Lipid pextracted frdamage to progressive This relatiorelation betwfinding. Onhealthy indistage of infmalonaldehother studie

Accordiof acute anceruloplasmthe cerulopassociated wplasma in asResults of tB comparisphase of undefense frugby the liverthe disease serum, It ma

Highligconcentratioceruloplasmliteratures r

nd ceruloplasshown in fig

as well as in

rrelation of S

ussion utcome of heon the patiento the previothis alteratio

dy agreed witperoxidation rom the methcell structurrise of malo

onship was hween serum

n the other hividuals; accfection with

hyde level as es [30], and eing to the facnd chronic i

min levels in plasmin levewith possiblessociation wthe current ston to healthy

ncomplicatedgalities of acr). This respo

progresses ay be explain

ght on the reson can be

min concentrarecorded tha

y = 0R

0

2

4

6

8

10

0

Seru

m M

alon

adild

ehyd

e (µ

mol

/L)

Se

smin enzymgure 6 A andhealthy cont

Serum Malond

epatitis B innt’s age at inous studies, non in the prth these findioccurs whe

hylene carbores [26]. In ondialdehydehighly signif

malondialdehand; maloncording to thi

hepatitis va reflex for

especially thoct that ceruloinflammationpatients’ serls [33-35]. e oxidation

with transferritudy illustraty controls. T

d hepatitis (hcute phase ponse is stimufrom chronined by the results of coppexplained aation as defe

at raises of th

.008x + 4.813R² = 0.054

50 100rum Ceruloplas

me (cerulopld B , the levetrols group fa

dialdehyde to tVirus, and

nfection parnfection and normally; tootein levels ings.

en lipids areon side chainprevious stu

e level in serficant (r=0.6ehyde level aaldehyde levis observatiovirus type Blipid peroxid

ose with liveoplasmin is on, basing tora. This resuCeruloplasmof ferrous irin, which canted that rise This raise refhepatitis viruroteins respo

ulated by cytc hepatitis telease of copper and iron ls a reflex tense protein he iron level

150 200min Activity (U/

lasmin oxidels of malodifailed to illust

the Ceruloplad B: Healthy I

rameters (whimmune stat

otal serum prwas recorde

e attacked byn, initiating udy (personarum samples8 at p<0.005and the agevel in sera oon the lipid pB. This resul

dation in numer diseases [3one of the acuo that, hepatult agreed wimin exhibits ron into ferrin only carry of copper lefers to the alus type B), tonse ; whichtokines and ro liver cirrh

pper from damlevels in the to the increagainst chrols can be refe

250/L)

A0

2

4

6

8

Seru

m M

alon

adild

ehyd

e (µ

mol

/L)

dase activity)ialehyde andtrate a signif

asmin OxidaseIndividuals

hich evaluattus as well aroteins leveled in numer

y free radica cascade ofality work ins for individu5). In the prprogression;

of patients dperoxidationt is agreed wmerous disea

31, 32]. ute-phase retitis viral inith several sta copper-de

ic iron, thereiron in the fevel in serumlterations of that meaningh leads to incraised of the hosis, coppermaged necropatients’ ser

eases in theonic hepatitisfer to the dam

y = 0.029x + 4R² = 0.126

0

2

4

6

8

0 20Serum

) as an agend celoplasminficant statisti

e Activity in A

ted in the pas the level ol decrease wrous diseases

al species af free ROS tn our laborauals (patientresent work,; this result a

didn’t elevatestatus didn’twith severalases [28, 29]

actant [12], infection maytudies whichependent oxefore assistinerric state [36

m of patients copper meta

g an elevatiocrease de novhormones c

r will exhibiotic hepatocyra illustrates e ceruloplasms B infectionmage of liver

4.0106

40m Ceruloplasmin

nt of antioxn oxidase actcally correla

A: Patients of H

present reseaof hepatitis Bwith the age s [22-25]. T

and hydrogenthat can cauatory) [27], ts and health, there was aagreed with ed than thost occurred inl studies th], while it dis

it is elevatedy cause an h recorded anxidase activitng in its tran6, 37]. with hepatit

abolism durion may be reva synthesis ortisol and git high conceytes [13, 14, 3

that the elevmin oxide

n. On the othr cells [20, 3

60 80n Activity (U/L)

B

xidation wastivity in sera

ation.

Hepatitis B

arch) largelyB replication.

progression;The result of

n atoms areuse oxidativeit showed a

hy) with age.a significantthe previous

se in sera ofn the primaryhat measuredsagreed with

d in the caseselevation ofn increase inty, which isnsport in the

is virus typeng the acuteesulted from(principally

glucagon. Asentrations in38]. vated copperactivity and

her hand, the9], while; in

s a

y . ; f

e e a . t s f y d h

s f n s e

e e

m y s n

r d e n

242

the present study the levels of iron in patients’ sera were within normal value, the matter can support the hypothesis that in the early stage of infection with hepatitis virus type B the liver stay hale.

In order to prove the hypothesis of liver cells safety during the prim infection period with hepatitis virus type B, the relationship of ceruloplasmin oxidase activity to the malondialdehyde levels. The study outcome showed there is no significant relation between these parameters; for that, we can conclude that a raise in the ceruloplasmin oxide activity is not reflex to the ambulance in the oxidation – antioxidation status, but it is produced as one of the defense system’s proteins against the initial viral infection.

5. References [1] E. Passarge. Color Atlas of Genetics. 3rd Eddition. Thieme, Stuttgart. New York, 2007.

[2] G. A. Ghorbani, S. M. Alavian, and H. R. Ghadimi. Long Term Effect of One or Two Doses of Hepatitis B Vaccine in Adult after Five Years. Pakistan Journal of Biological Sciences. 2008, 11(4): 660-663.

[3] G. I. Saravanamuttu, M. J. Samreen, M. Ramsay, G. Jonathan. International Public Health Repository for Hepatitis B. Nucleic Acid Research. 2007, 35(2): 367-370.

[4] S. Guimarães, A. Aragão, J. Santos, O. Kimura, P. Barbosa, and P. de Vasconcelos. Oxidative Stress Induced by Torsion of the Spermatic Cord in Young Rats. Acta Cirúrgica Brasileira. 2007, 22(1): 30-33.

[5] S. Nikam, P. Nikam, and S. K. Ahaley. Role of Free Radical and Antioxidant Imbalance in Pathogenesis of Parkinson's Disease. Biomedical Research. 2009, 20 (1): 55-58.

[6] S. Sahu, R. Abraham, R. Vedavallir, and M. Daniel. Study of Lipid Profile, Lipid Peroxidation, and Vitamin E in Pregnancy Induced Hypertension. Indian Journal of Physiology Pharmacology. 2009, 53 (4): 365–369.

[7] R. Moreno-Otero and M. Trapero-Marugán. Hepatoprotective Effects of Antioxidants in Chronic Hepatitis C. World Journal of Gastroenterol. 20101, 6(15): 1937-1938 .

[8] N.E. Hellman and J.D. Gitlin. Ceruloplasmin Metabolism and Function. Annu. Rev. Nutr. 2002, 22: 439–58.

[9] B. Mazumder, V. Seshadri, and P. L. Fox. Translational Control by the 3'-UTR: The Ends Specify the Means. Trends Biochemistry Science. 2003, 28 (2): 91–108.

[10] N. Giurgea, M.I. Constantinescu, and R. Stanciu. Ceruloplasmin - Acute-Phase Reactant or Endogenous Antioxidant? The Case of Cardiovascular Disease. Medical Science Monit. 2005, 11(2): 48–51.

[11] C. Smith, A. Marks, and M. Lieberman. Marks’ Basic Medical Biochemistry. A Clinical Approach: Oxygen Toxicity and Free Radical Injury. 2nd Edition. Lippincott Williama and Wilkins. 2009. p 829.

[12] H. Baghshani, S. Nazifi, M. Saeb, and S. Saeb. Influence of Road Transportation on Plasma Concentrations of Acute Phase Proteins, Including Fibrinogen, Haptoglobin, Serum Amyloid A, and Ceruloplasmin, in Dromedary Camels (Camelus Dromedarius). Comparative Clinical Pathology Journal. 2010, 19(2):193-198.

[13] R. Selvarjur, R. Ganapathirmanr, R. Naranaswamy, and R. Vallippan. Trace Element Analysis in Hepatitis in Hepatitis B Affected Human Blood Serum Inductively Coupled Plasmaatomic Emission Spectroscopy (ICP-AES). Romanian Journal of Biophysiology. 2009, 19(1): 35–42.

[14] A. Gaw, R. A. Cowan, D.J. O'Relly, M.J. Stewart, & J. Shepherd. Core Biochemistry. In: An Illustrated Colour Text Clinical Biochemistry. 2nd Edition. Published by Churchill Livingstone, 1999.

[15] A. Plummer. An Introduction to Practical Biochemistry. 1st Eddition. Mcgraw Hill. Book Company. London. U K. 1971.

[16] K. Yagi. Assay for Lipid Peroxide Level and Its Clinical Significance. In: Yagi K Editor Lipid Peroxides in Biology and Medicine, New York, 1982. p233–242.

[17] Rice E W. [1962]: Ceruloplasmin Assay in Serum: Standardization of Ceruloplasmin Activity in Terms of International Enzyme Unit "Standard Methods of Clinical Chemistry". 4th Eddition, Siligson D., New York, Academic press.

[18] R.M. Daoud. Study of Some Biochemical Changes in Serum & Saliva of Patients with Oral Epithelial Tumors. 2008. Baghdad University, College of Science, Chemistry Department. Treatise.

[19] J. Koolman and K. Roehm. Color Atlas of Biochemistry. 2nd Edition. Thieme. Stuttgart. New York, 2005. p277.

243

[20] S. Cherukuri, R. Potla, J. Sarkar, S. Nurko, Z.L. Harris, and P.L. Fox. Unexpected Role of Ceruloplasmin in Intestinal Iron Absorption. Cell Metabolism Journal. 2005, 2, 309-319.

[21] L. Jin, J. Wang, L. Zhao, H. Jin, G. Fei, Y. Zhang, M. Zeng, and C. Zhong. Decreased Serum Ceruloplasmin Levels Characteristically Aggravate Nigral Iron Deposition in Parkinson's Disease. European Parkinson's Disease Association. 2010

[22] R. Al-Faham. A Biochemical Study of Serum Sialic Acid and Lectins Activity as Tumor Markers for Gastrointestinal Tract Cancers. 2002. University of Kufa, College of Education, Chemistry Department. Thesis.

[23] G.T. Porter, A.M. Pou, F.B. Quinn, & M.W. Ryan. Nutritional Considerations for Head and Neck Cancer Patients. 2003. Grand Rounds Presentation, UTMB, Department of Otolaryngology.

[24] R.W. Raymond. Biochemistry of Cancer. In: Cancer Biology. Section 1. 2007. p 108-120.

[25] R.H. Jasim. Isolation, Purification, and Partial Characterization of Human Lectins from Patients of Kidney Tumors. 2009. Baghdad University, College of Science for Women, Chemistry Department. Treatise.

[26] C. Chen, M. Arjomandi, J. Balmes, I. Tager, and N. Holland. Effects of Chronic and Acute Ozone Exposure on Lipid Peroxidation and Antioxidant Capacity in Healthy Young Adults. Environmental Health Perspectives. 2007, 115(12): 1732–1737.

[27] R.H. Jasim. Serum Malondialdehyde and Lipid Profile Levels of Young Patients "Haven’t a Family History of Hypertension": A New Study for Cases in the Civic Society. Under publishing (Participated in the 1st International Conference of Chemistry and Its Role in Science). 2011.

[28] H. Aksoy, M. Koruk, and F. Akçay. The Relationship Between Serum Malondialdehyde and Ceruloplasmin in Chronic Liver Disease. Turkish Journal of Biochemistry. 2003, 28 (2): 32-34.

[29] L. McGrath, B. Mc Gleenon, S. Brennan, D. McColl, S. McIlroy, and A. Passmore. Increased Oxidative Stress in Alzheimer's Disease as Assessed with 4- Hydroxynonenal but not Malondialdehyde. Q J Med. 2001, 94: 485-490.

[30] U. Merle, K.H. Weiss, C. Eisenbach, S. Tuma, P. Ferenci, and W. Stremmel. Truncating Mutations in the Wilson Disease Gene ATP7B are Associated with Very Low Serum Ceruloplasmin Oxidase Activity and An Early Onset of Wilson Disease. BMC Gastroenterology. 2010, 10(8): 2-6.

[31] Ko W, Guo C, Yeh M, Lin L, Hsu G W, Chen P, Luo M, and Lin C. []: Blood Micronutrient, Oxidative Stress, and Viral Load in Patients with Chronic Hepatitis C. World Journal of Gastroenterology. 2005, 11(30):4697-4702.

[32] A.V. Janaia and K.M. Suprapaneni. Antioxidant Vitamins And Enzymes Status In Patients With Alcoholic Liver Disease. Journal of Clinical and Diagnostic Research. 2010, 4(4): 2742-2747.

[33] O. Virit, S. Selek, M. Bulut, H.A. Savas, H. Celik, O. Erel, and H. Herken. High Ceruloplasmin Levels are Associated with Obsessive Compulsive Disorder: A case Control Study. Behavior Brain Function. 2008, 4(52): 1-7.

[34] V. Lopez-Avila, W.H. Robinson, and K. Lokits. Ceruloplasmin Levels in Human Sera from Various Diseases and Their Correlation with Patient’s Age and Gender. Health Journal. 2009, 1(2): 104-110.

[35] G.J. Brewer, S.H. Kanzer, E.A. Zimmerman, D.F. Celmins, and S.M. Heckman. Copper and Ceruloplasmin Abnormalities in Alzheimer’s Disease. American Journal of Alzheimer’s Disease. 2010, 25 (6): 490-497.

[36] B.N. Patel, R.J. Dunn, S.Y. Jeong, Q. Zhu, J. Julien, and S. David. Ceruloplasmin Regulates Iron Levels in the CNS and Prevents Free Radical Injury. The Journal of Neuroscience. 2002, 22(15): 6578-6586.

[37] A.I. Bush. []: Iron-Export Ferroxidase Activity of β-Amyloid Precursor Protein Is Inhibited by Zinc in Alzheimer's Disease. Cell Journal. 2010,142(6): 857-867.

[38] B. Spee, P. Mandigers, B. Arends, P. Bode, G. Hoffmann, J. Rothuizen, and L. Penning. Differential Expression of Copper-Associated and Oxidative Stress Related Proteins in a New Variant of Copper Toxicosis in Doberman Pinschers. BMC Comparative Hepatology. 2005, 4(3):1-13.

[39] E. Aigner and C. Datez. Iron Perturbations in Human Non-Alcoholic Fatty Liver Disease (NAFLD): Clinical Relevance and Molecular Mechanisms. Hepatitis Monthly. 2008, 8(3): 213-220.

244