The Effect of Brucellosis on Lipid Profile and Oxidant-Antioxidants Status

Iraqi J Pharm Sci , Vol.18 (Suppl.), 2009 Brucellosis on lipid and oxidant-antioxidants

The Effect of Brucellosis on Lipid Profile and Oxidant-Antioxidants

Status

Amal H. Ali
*,1

Department of Clinical Laboratory Science , College of Pharmacy, University of Baghdad, Baghdad, Iraq.

Abstract
The activation of inflammatory cells, the release of their mediators, and the excessive production

of free radicals may affect circulating lipids, but no evidence supports a role for peroxidation in the

pathogenesis of Brucellosis disease. The aim of this work is to study the effect of Brucellosis on lipid

profile concentration and oxidant-antioxidant status. We studied 20 Brucellosis patients (18 Females

and 2 males) and 15 healthy controls (age average from 16 to 60 years old). Significant differences

were noted between the serum lipids of Brucellosis patients and control group. Mean total cholesterol

and low density lipoprotein cholesterol (LDL-cholesterol) concentrations were higher in patients than

in control group (mean  SE 197.05  44.7, 165  37.6). (P≤0.004, P≤1.59×l0
-11

) respectively.

Whereas, high density lipoprotein cholesterol (HDL-cholesterol) and triglyceride show significant

lower concentration in patients than control groups (mean  SE 15.12  3.4 , 81.22  18.45) .

(P≤ 9×10
-6

, P≤ 9.3×10
-7

) respectively. Table l , Figure 1.While circulating concentration of glutathione

(GSH), soluble antioxidants were higher in Brucellosis patients than in control groups (mean  SE 2.49

 0.4). Additionally, increased oxidative stress was observed in the serum of patients with Brucellosis

as evidenced by higher malondialdehyde (MDA) concentrations than in control groups (mean  SE

270  9) (Figure 2, Table 1). In conclusion. Disturbances in the lipid profile, and oxidant-antioxidant

status occur in Brucellosis patients which may increase incidence of metabolic and cardiovascular

events.

Key words: Brucellosis disease, Lipid profile, oxidant-antioxidant status.

الخالصة
الؤ التؤاريز ى ؤ ي ؤىن الؤد لكؤه ال يى ؤد يؤدي ان تىشيط الخاليا االلتهابيت وافزاس مسبباتها وتكىيه مشيد مه الجذور الحزة

ىىد مزظ الحمؤ الزا ةؤت او ىمؤ مال ؤار لدرااؤت تؤاريز الحمؤ (Peroxidation)يليل في ى م االمزاض يدىم ىم يت فىق االكسدة

مؤه 81مؤه مزظؤ الحمؤ الزا ةؤت مؤزيط 20تؤم يرااؤت ى تزكيش مكىواث ي ىن وىىامل االكسدة ومعاياث االكسدةر الزا ةت

ىؤؤدة لؤؤىى ر ء كمز ؤؤ االصؤؤحا االشؤؤخا مؤؤه 81 ( و اؤؤىت 16 الؤؤ 81 بؤؤيه اىمؤؤار م تتؤؤزاو الؤؤذكىر مؤؤه 2 االوؤؤاو و

ىيؤؤو و ؤؤد ان االصؤؤحاء االشؤخا مؤؤ بالمقؤؤراروت الزا ةؤت الحمؤؤ مزظؤؤ ىىؤد الؤؤد ىن مصؤؤل اختالفؤاث اا زيؤؤت فؤؤي تزكيؤش

مؤؤ بالمقاروؤؤت الزا ةؤؤت الحمؤؤ مزظؤؤ ىىؤؤد وسؤؤبيا ىؤؤالي كىليسؤؤتيزوا -LDLالكىليسؤؤتيزوا الك ؤؤي والجؤؤرش ي تزكيؤؤش مصؤؤل

10 ( بالتةاقؤ mean  SE 197.05  44.7, 165  37.6 االصؤحاء االشؤخا
-11

) ×1.59 P≤ , P≤ 0.004بالتةاقؤ ر )

كىليستيزوا والتزايك يسيزايد ق ت واظحت ومهمت ىىؤد مزظؤ الحمؤ الزا ةؤت بالمقاروؤت مؤ االشؤخا – HDLبيىما ااهز تزكيش الر

10 (mean  SE 15.12  3.4 , 81.22  18.45)االصؤحاء
-7

) ×10
-6

, P≤ 9.3 ×(P≤ 9 ر8والزاؤم 8 بالتةاقؤ ر الجؤدوا

( (mean  SE 2.49  0.4الي ىىؤد مزظؤ الحمؤ الزا ةؤترىؤ (Glutathione)اوؤ خخؤرز و ؤد ان تزكيؤش معؤاي االكسؤدة مؤه

اءرررررررؤراكثز مما فؤي االشؤخا االصح (Malondialdehyde)ت في تزكيش المدكسد ررررررررررررراية م حىاررررررررررررررافت ال سيررررباالظ

(mean  SE 270  9) رلمىاقشؤت ؤذا المىظؤىظ ااهؤزث الدرااؤت اخؤتال وىؤد اوتةؤا وسؤبت الؤد ىن 8والجؤدوا 2 الزاؤم)

والمدكسداث ومعاياث االكسدة ىىد مزظ الحم الزا ةت مما يدي ال سياية مدرزة في التأيط وتص الشزاييهر

Introduction
Brucellosis was firstly described by

Bruce in 1887 with the discovery of the

bacterial species subsequently named Brucella

melitensis. It is a type of febrile illness

characterized by regular remissions or

intermissions has been recognized in the

Mediterranean region for centuries. Many

names have been applied to it, often relating to

the locations in which it was particularly

prevalent. (Malta fever, Mediterranean fever,

Gibraltar fever or Rock fever and Undulant

fever) are probably the best known
(1,2)

Brucellosis is the major bacterial zoonosis and

an important cause of a serious debilitating

disease in human, and abortion and sterility in

domestic animals. It is classified into two

subdivision of proteobacteria, (small gram-

negative and facultative intracellular

pathogens) that can multiply within

professional and non professional phagocytes
(3,4)

. The genus brucella consists of seven

species according to antigenic variation and

primary host, B.melitensis (sheep and goats),

B. suis (hogs), B. abortus (cattle), B. ovis

(sheep), B. canis (dogs), B. neotanac (wood

rats), and B. maris (marine mammals)
(5)

1
Corresponding author E-mail : Amal Hamadah AL Hadithy 49 @ yahoo.com .

Received : 17/2/2009

Accepted : 24/5/2009

Iraqi J Pharm Sci , Vol.18 (Suppl.), 2009 Brucellosis on lipid and oxidant-antioxidants

In contrast to other intracellular pathogens,

brucella species do not produce exotoxins,

antiphagocytic capsules, thick cell wall,

resistance forms or fimbriae and do not show

antigenic variation
(6)

. A key aspect of the

virulence of brucella species is their ability to

proliferate within professional and non

professional phagocytic host cells, therefore

successfully by passing the bactericidal effects

of phagocytes. Their virulence and chronic

infections are thought to be due to their ability

to avoid the killing mechanisms within host

cells
(7,8)

. Lipid profiles include serum

cholesterol, serum triglycerides, the fractions

of break up of various cholesterol like low

density lipoprotein, cholesterol (LDL-

cholesterol), high density lipoprotein

cholesterol (HDL-cholesterol), and the ratio of

LDL/HDL
(10)

. Total cholesterol has been found

to correlate with cardiovascular mortality in

30-50 years age group. Cardiovascular

mortality increases 9% for each 10mg/dl

increase in total cholesterol over the base line

value of l80mg/dl.HDL-cholesterol is a good

cholesterol in that of cardiovascular disease

decrease with increase of HDL
(11)

. Triglyceride

level is a risk factor independent of the

cholesterol level, triglycerides are important as

risk factors only if they are not part of the

chylomicron fraction
(12)

. Lipid profile is

Known to alter in patients with sever sepsis,

but few studies regarding the status of lipid

levels in brucellosis are available
(13)

Glutathione peroxidase are major enzymes that

remove hydrogen peroxide generated by SOD

in cytosol and mitochondria by oxidizing the

tripeptide glutathione (GSH) in to its oxidized

form (GSSG). The glutathione peroxidase that

removes hydrogen peroxide contains selenium

(essential for catalytic function) at its active

site
(14)

Subjects and Methods
Twenty patients (18 Females and 2

males, age average from 16 to 60 years old)

with brucellosis were recruited from the clinic

of Al-Khadmia Teaching Hospital. The

diagnosis of the disease was based on standard

clinical, histological features, and serological

test which is established by positive rose

Bengal plate agglutination test with antibody

titer more than 1/320
(15)

. The severity of the

disease was evaluated by the severity of

abdominal pain accompanied by nausea and

vomiting
(l6)

. Gastrointestinal symptoms are

noted in 40% of patients with brucellosis with

anorexia, weight loss and hepatosplenomegaly

are the most common. History of fever

generalized myalgia and arthralgia, low back

pain and sweating
(17)

. Study was also

conducted on fifteen apparently healthy

individual as a control groups, blood samples

were collected after subjects had fasted for 12

hours overnight, serum was separated

immediately by low speed centrifugation and

stored pending analysis.

Lipid profile analysis
Serum concentration of total cholesterol

and triglycerides were measured enzymatically

with a commercial kit (boehringer, Mannheim

montreal). According to the following

principle
(18)

The cholesterol present in the sample originates a coloured complex.

CHE

Cholesterol esters + H2O cholesterol + fatty acids

CHOD

Cholesterol + O2 4-cholestadien + H2O2

POD

2 H2O2 + phenol + 4-Aminophenazone Quinonimine + 4 H2O

Triglyceride was measured by GPO-POD enzymatic colorimetric method as the following principle
(19)

LPL

Triglyceride + H2O Glycerol + free fatty acids

Glycero kinase

Glycerol + ATP G3P + ADP

GPO

G3P + O2 DAP + H2O2

POD

H2O2 + 4-AP + p-Chlorophenol Quinone + H2O

Iraqi J Pharm Sci , Vol.18 (Suppl.), 2009 Brucellosis on lipid and oxidant-antioxidants

HDL-cholesterol was measured after

precipitation of very low density lipoprotein

(VLDL) and LDL-cholesterol with

phosphotungstic acid, LDL-cholesterol

measured by Friede Wald equation as the

following:

LDL-cholesterol = total cholesterol − (HDL + Trigly/5)

When triglyceride levels are less than 400

mg/dl
(20)

Oxidant and antioxidant analysis
Serum free malondialdehyde concentrations

were measured according to a modified

method of Chirico (1994)
(2I)

. Proteins were

first precipitated with a 10% NaSO4 solution.

The protein free supernatant was then reacted

with an isovolume of a 5% thiobarbiuric acid

solution at 95°C for 30 min. after the reaction

component were cooled to room temperature,

pink chromagene was extracted with n-butanol

then dried over a stream of nitrogen at 37°C.

The dry extract was then resuspended in

a mobile phase of KH2PO4 – methanol

(70:30) before malondialdehyde detection by

HPLC
(21)

Glutathione was measured by Titze's

technique
(14)

. According to this equation:

2GSH + H2O2 GSSG + 2H2O

Statistical analysis
All values were expressed as mean ± SE.

statistical differences were assessed by

student's two tailed t-test. P. values ≤0.05 were

considered significant.

Results

Lipid profile

Significant differences were noted between the

serum lipids of patients with brucellosis and

control group. Where mean total serum

cholesterol concentration was higher in

patients than in control group. Total

cholesterol was characterized by higher LDL-

cholesterol in brucellosis patients (mean  SE

197.05  44.7, 165  37.6) (P≤0.004,

P≤1.59×10
-11

respectively) with a significant

lower HDL-cholesterol and triglyceride

concentration (mean  SE 15.12  3.4, 81.22

18.45) (P≤ 9×10
-6

, P≤ 9.3×10
-7

respectively).

(Table 1 , Figure 1).

Table 1 : Mean±SE (mg/dl) of serum lipid profile (total cholesterol, LDL-cholesterol,

Triglyceride, HDL-cholesterol, Malondialdehyde and Glutathione) levels (nmol/l) and

LDL/HDL ratio in patients with brucellosis and control groups.

Control n=15 Patients n=20

variable Mean ± SE Mean ± SE P

Total cholesterol 190.45 ± 4.3 197.05 ± 44.7 ≤ 0.004

Triglyceride 135.1 ± 30.7 81.22 ± 18.45 ≤ 9 ×10
-6

LDL-cholesterol 102.3 ± 23.25 165.45 ± 37.6 ≤ 1.59 × 10
-11

HDL-cholesterol 36.6 ± 8.3 15.12 ± 3.4 ≤ 9.3 ×10
-7

LDL/HDL ratio 2.79 10.9 ≤ 0.001

MDA 76 ± 17.5 270 ± 9 ≤ 0.001

GSH 1.9 ± 0.27 2.49 ± 0.4 ≤ 0.005

GSH – PX

Iraqi J Pharm Sci , Vol.18 (Suppl.), 2009 Brucellosis on lipid and oxidant-antioxidants

Figure1: Mean (±SE) serum lipid profile

(total cholesterol, LDL-cholesterol,

Triglyceride, and HDL-cholesterol) levels

and LDL/HDL ratio in fasting Brucellosis

patients (n= 20) and control groups (n= 15)

the deference is significant.

Oxidant- antioxidant status

Circulating concentrations of glutathione,

a key endogenous soluble antioxidant, were

higher in patients with brucellosis than in

control groups (mean  SE 2.49  0.4).

Moreover, increased oxidative stress was

observed in brucellosis patients as evidenced

by higher malondialdehyde concentrations

than in the control group (mean  SE 270  9).

(Table 1, Figure 2).

Figure 2: Mean (±SE) Malondialdehyde and

Glutathione concentration in serum of

patients with Brucellosis disease (n=20) and

control groups (n=15) the differences are

significant P< 0.005 for glutathione, and

p<0.001 for Malondialdehyde.

Discussion
The present study identified important

alterations in the lipid profile and oxidant-

antioxidants status in patients with brucellosis

compared with healthy control groups. Our

data documented abnormally high serum

concentrations of total cholesterol and LDL-

cholesterol in patients with brucellosis, with

concomitant decrease in triglyceride and HDL-

cholesterol The altered oxidant- antioxidant

status of brucellosis patients was shown by

higher glutathione concentration and higher

plasma malondialdehyde concentrations than

in control groups. This last finding is

consistent with abnormal lipid peroxidation in

the circulation of Crohn's disease patients
(13)

.Yet the little information is available on the

plasma lipid profile and lipoprotein

concentrations in patients with brucellosis.

Although our findings disclose various

changes in these biochemical indexes, no

obvious associations were noted between these

disturbances and selected clinical aspects of

the patients, such as disease activity, site of

disease, and current medications. Further

more, none of our patients smoked or

consumed alcohol, which are the two factors

that may influence antioxidant and lipoprotein

status. At this time, it's uncertain whether the

hypercholesterolemia noted in brucellosis

patients results from a consequence of high

carbohydrate consumption. Similarly, further

work is needed to explore whether the

hypotriglyceridemia in patients results from

intestinal malabsorption or malnutrition
(23,

24)
.The excessive local production of soluble

mediators from activated monocytes and

polymorphonuclear leukocytes has been

implicated in mediating the tissues injury
(25)

Important among these mediators are oxygen

free radicals. The chronic inflammatory cells

promote an imbalance between oxidant and

antioxidant mechanisms at the tissue level and

may even compromise circulating antioxidant

concentrations
(26,27)

.However, children with

CD (crohn's disease) were reported to have

higher plasma antioxidant concentrations than

healthy children
(28)

. In the present study,

glutathione is an important intracellular

antioxidant, show significant increase in

patients than in control groups, consistent with

other reports in adults with CD
(29,30)

.These in

consistencies in reports of circulating

antioxidants to date may be due to the patient's

degree of inflammation, the patient's

medication and supplement use, enteric losses,

altered mobilization as a result of inflamed

mucosa, malabsorption and decrease nutrient

100

150

200

250

mg/dl

Total cholesterol Triglyceride LDL-cholesterol HDL-cholesterol LDL/HDL ratio

Control

Patients

100

150

200

250

300

350

400

nm
ol

Malondialdehyde

0.5

1.5

2.5

nm
ol/

Glutathione

Iraqi J Pharm Sci , Vol.18 (Suppl.), 2009 Brucellosis on lipid and oxidant-antioxidants

intake. Glutathione is tripeptide helps to

detoxify free radicals, peroxides and

electrophilic compounds of endogenous and

exogenous origin
(31,32)

.More ever ,serum

glutathione level was higher in our patients

than in control groups. Additional

investigation is needed to verify whether the

high values of glutathione constitute an

adaptive to extrapolate from an experimental

model
(33)

. These high concentrations of

glutathione may help prevent oxidation of α-

tocopherol, and preserve ascorbic acid

concentration
(34)

.Free radicals are known to

occur as natural byproducts under physiologic

conditions. However, their over production has

been implicated in the pathogenesis of gut

inflammation and intestinal injury in

inflammatory bowel disease
(28)

.Oxyradical

induced cytotoxicity gives rise to lipid

peroxidation through the reaction of free

radicals and peroxides with fats in cellular

membranes, resulting in malondialdehyde

formation
(35)

. Our patients had significantly

higher circulating malondialdehyde

concentrations than did control groups. It is

unclear whether the excess serum

malondialdehyde was generated in the patient's

blood or was produced by the inflamed

intestine and translocated in to the circulation.

The presence of malondialdehyde in the

circulation may explain the increased

formation of glutathione as a mean of

preventing oxidative damage. Moreover, was

showed that proinflamatory cytokines can

adversely affect lipoprotein metabolism. For

example, tumor necrosis factor α and

interlukin-6 were shown to affect intestinal fat

handling and lipid metabolism
(32,

35)
.Nevertheless, our data failed to show an

effect of disease activity on these indexes,

likely because of the relatively small number

of patients, further studies are required in

which these indexes are analyzed serially in

brucellosis patients over time.In conclusion we

found substantial abnormalities in the

concentrations of plasma lipids,

malondialdehyde, and antioxidants of

brucellosis patients. Moreover, the presence of

excessive lipid peroxidation strategies should

be elaborated to bolster the antioxidants

system in patients with brucellosis. From our

results in this study we suggested that

brucellosis patients had high risk to

atherosclerosis due to the significant increase

in plasma total cholesterol, LDL-cholesterol

and high level of LDL/HDL ratio, the three

indexes of high risk cholesterol mortality.

According to the result of this study we can

concluded that patients with brucellosis may

have an increasing incidence of metabolic and

cardiovascular events.Further investigation is

required to elucidate the mechanisms involved

in aforementioned abnormalities.

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