The Level of Zinc Serum after Oral Zinc on Mice with Escherichia

Coli LPS- Induced Diarrhea

Sulaiman Yusuf

, Yati Soenarto

, Muhammad Juffrie

, Wiryatun Lestariana

, Mudatsir

Department of Paediatric,

edical Faculty, Syiah

uala University, Banda Aceh/d

. ZainoelAbidin Hospital,

Aceh, Indonesia

Department of Paediatric, Medical Faculty, Gadjah Mada University,Yogyakarta, Indonesia

Department of Biochemistry, Medical Faculty, Gadjah Mada University,Yogyakarta, Indonesia

Department of Microbiology Medical Faculty Syiah Kuala University, Aceh, Indonesia

Keywords: Zinc, Escherichia Coli, Diarrhea

Abstract: The level of zinc requirements will increase due to infection, for formation of immune function and new

cells. The advantage of Zinc is maintain the integrity of the intestinal mucosa through its function in cell

regeneration and membrane stability. The aim of this study is to determine the level of zinc serum after oral

zinc on mice diarrheal induce E.Coli-LPS. This study used a controlled trial experimental design in the

laboratory. Sample of 20 mice were randomly divided into 4 groups:1). Control group was given standard

foods, 2). Trial group was given E.Coli-LPS 2.5 mg/kg/oral once on day-1, 3). Preventive group was given

E.Coli-LPS once 2.5 mg/kg/oral once on day-1 + 30 mg/kg/oral of zinc once daily for 12 days. 4). Therapy

group was given E.Coli-LPS 2.5 mg/kg/oral once on day-1, if diarrheal was given 30 mg/kg/oral of zinc

once daily for 12 days. Blood samples of mice were taken through the orbital sinus on the 0, 5

, 10

hour,

, 8

and 12

day. Data are presented in tables and graphic. We found that higher levels of zinc in the

preventive and therapy group especially on 4

, 8

and 12

day. I conclusion, oral administration of zinc

increase serum zinc levels, especially in control and experimental groups.

1 INTRODUCTION

Zinc deficiency is still common, especially in

developing countries. This can be related to lack of

intake, increasing needs, and the amount of zinc loss

from the body due to diseases, especially infections.

There is associated between infection and zinc

deficiency which influence each other. Zinc

requirements of the body will increase during

infection, formation of immune functions and new

cells. Zinc deficiency can cause suppression of

immune function, making it easier for infection to

occur (Baqui, 2006; King, 2003).

Zinc plays a role in maintaining the integrity of

the intestinal mucosa through its function in cell

regeneration and membrane stability. Zinc has a

direct impact on intestinal villi, disaccharidase brush

border activity and intestinal water and electrolyte

transport. Zinc also plays a role in T cell function

and enhances immunity thereby reducing the

severity of diarrhea (Roy, 1992).

The risk of zinc deficiency in Indonesia is

estimated being greater because of Indonesian

community menu, especially in the low socio-

economic group, with lower animal protein

consumption even though this type of protein

contains a lot of zinc. Moreover, the menu of

Indonesian society is relatively high in phytate and

fiber in inhibits zinc absorption, such as the habit of

drinking tea every day, even on certain social groups

that consume thick tea. Moreover, they also

consume lots of beans and cereal, including the

processed products. This food contains a lot of

phytate or tannin so that the potential lack of zinc in

Indonesian society is quite higher because the

absorption of zinc will be disrupted (Nona, 2010).

Escherichia coli is the main occupant of healthy

colon flora, but also cause various diseases such as

diarrhea by releasing endotoxins, which triggers the

release of pro-inflammatory mediators.

Lipopolysaccharide (LPS) is the main component of

the cell wall of gram-negative bacteria which is also

called endotoxin and is known to be the trigger of

Yusuf, S., Soenarto, Y., Juffrie, M., Lestariana, W. and Mudatsir, .

The Level of Zinc Serum after Oral Zinc on Mice with Escherichia Coli LPS- Induced Diarrhea.

DOI: 10.5220/0008788201630167

In Proceedings of the 2nd Syiah Kuala International Conference on Medicine and Health Sciences (SKIC-MHS 2018), pages 163-167

ISBN: 978-989-758-438-1

163

several types of inflammatory or infectious reactions

in macrophage cells and other cells that have CD14

receptors. In fact, endotoxin (LPS) of bacteria that

binds to TLR (Toll Like Receptor) in DC (Dendritic

Cells) can stimulate monocyte and macrophage cells

to secrete nitric oxide (NO) and inflammatory

substances (cytokines) such as Tumor Necrosis

Factor-alpha (TNF-α) and interleukin-1-beta (IL 1 -

β), IL-6 and IL-8 (Rahman, et al., 2007). As a result

of the release of excessive pro-inflammatory

cytokines can cause symptoms of decreased blood

pressure, fever and diarrhea (Baratawidjaya, 2009).

In experimental studies, zinc deficiency has a

direct effect on the digestive tract, in the form of

villous atrophy, a decrease of disaccharidase enzyme

activity in brush borders, and impaired intestinal

transport. However, the exact mechanism that

connects the pathophysiology of diarrhea with zinc

deficiency has not been agreed yet. However, the

incidence of persistent diarrhea has been reduced by

zinc supplementation, and administration of ORS

(oral rehydration solutions) with zinc has

substantially reduced the duration and severity of

diarrhea in children with acute and persistent

diarrhea (Bhandari et al., 2002). Although the

mechanism of zinc supplementation reduces diarrhea

is unknown, zinc therapy in diarrhea patients has

shown better absorption of water and electrolytes by

the intestine, faster regeneration of intestinal

epithelial cells, increased levels of enzymes from

brush border, and increased immune response so that

it can eliminate pathogens in the intestine (Fenwick

et al., 2004).

The purpose of this study was to determine

serum zinc levels after administration of oral zinc in

mice diarrheal induced E. coli-LPS.

2 METHODS

This study is designed with Controlled Trial Design,

and done in The Laboratory of Nutrition and Food

Centre (PSPG) in GadjahMada University

Yogyakarta. Sample consist of 20 white mice

Sprague Dawleywhich is randomly chosen and

divided into 4 groups randomly and each group

consist of 5 mice. 1). Control Group (G1), was

given standard nutrition, 2). Trial Group (G2) was

given LPS E.Coli 2,5 mg/kg/oral in day 1-3, 3).

Preventive Group (G3) was given LPS E.Coli 2,5

mg/kg/oral in day 1 + zinc 30 mg/kg/oral since day 1

until 12 days, 4). Therapy Group (G4)was given

LPS E.Coli 2,5 mg/kg/oral in day 1, if diarrhea

occur, this group will be given zinc 30 mg/kg/oral

since day 1 until 12 days. Sample of the mice blood

was taken from sinus orbitalin 0, 5

, 10

hour, 4

, and 12

day to measure zinc level in serum

(Madiono, et, al., 2002).

3 RESULTS

The characteristics of subjects in this study include

weight and Hemoglobin level (Hb) presented in

Table 1.

Table 1. The Characteristics of subjects.

Group Mean of

Weight(gram)

Standard

Deviation

value*

73,4

88,4

93,8

89,6

8,02

10,92

15,06

7,335

0,04

Group Mean of

Hb(g%)

Standard

Deviation

value*

11,88

11,76

11,36

11,57

0,362

0,295

0,268

0,441

0,138

* ANOVA, p < 0,05

G1= Control group G3=Preventive groupG2= Trial

groupG4= Therapy group

3.1 The Level of Zinc based on

Treatment Time

The results of zinc level can be seen in Table 2 and

Picture 1. In control group (G1) there is no

difference of zinc level based on treatment time in 5

mice that has been evaluated, the level range is 1,14

until 1,2. However, zinc level in trial group (G2)

seems to be lower compared to group G1, G3 and

G4 which can be seen obviously in Picture 1, since

treatment time in 4

and 12

day in group G2,

mice 3

and 5

experience diarrhea.

The difference also can bee seen in preventive

group (G3), zinc level in 5

hour and 10

hour after

treatment with LPS is 0,94 µg/dl and 0,77 µg/dl,

meanwhile in 4

, 8

and 12

day, zinc level is

higher compared to group G2 and can be seen

obviously in Picture 1, this event occur in group G3

mice 3

and 5

. In preventive group all mice

were given zinc everyday.

SKIC-MHS 2018 - The 2nd Syiah Kuala International Conference on Medicine and Health Sciences

164

Table 2: The level of zinc (µg/dl) based on treatment time.

Treatment

time

(µg/dl)

hour 1,14 0,95 0,94 0,93

hour 1,19 0,83 0,77 0,52

day 1,16 0,51 1,17 1,05

day 1,18 0,5 1,18 1,09

day 1,2 0,6 1,2 1,07

It is the same in therapy group (G4) zinc level is

higher in mice 3

, 4

and 5

compared to mice in

group G2 in 4

, 8

and 12

day. Actually zinc level

is higher in mice 3 in the group G3 3 days after

receiving zinc, it is also the same for mice 4 in group

G4, zinc level is higher in day 6 after receiving zinc,

mean while in mice 5 in group G4, zinc level is

higher after 10 days receiving zinc, can be seen in

Table 2 and Figure 1.

Figure 1. Diagram of zinc level in serum on control group

(G1), Trial group (G2), Preventive Group (G3) and

Therapy group (G4).

The result of zinc level in trial group (G2) is lower

than control group (G1) since 5

hour after

treatment time of LPS and lower in 10

hour, 4

, 8

and 12

day can be seen in Table 2 and Picture 1.

The zinc level is lower in control group (G2)

compared to preventive group (G3) and therapy

group (G4) especially in 4

, 8

and 12

day. Mice in

preventive and therapy group (G4) is given zinc

everyday, meanwhile in trial group (G2) is not given

zinc.

4 DISCUSSION

In Figure 1, generally we can see zinc level in

control group (G2) is lower in mice experienced

diarrhea and zinc level is higher in group received

zinc, preventive group (G3) and therapy group (G4).

In this study, mice in trial group (G2) were not given

zinc and experienced diarrhea since 3

day until

day, in which zinc level is the lowest since 4

and 12

day. Zinc associated with gut villi

regeneration and function, therefore it will influence

formation of disaccharide enzyme such as lactase,

sucrose and maltase. Therefore, zinc can influence

process of osmotic diarrhea, most of which caused

by malabsorption andmaldigestion. In diarrhea, there

is excessive loss of zinc. Prolonged diarhhea cause

decreased the serum level of zink. There is a

circulation between diarrhea, zinc deficiency,

duration of diarrhea, and malnutrition.

Administration of micro zinc in oral can replace loss

of zinc in diarrhea (Artana et al., 2005).

Some studies show the role of zinc associated

with red blood cell formation. In biosynthesis of

heme, enzyme δ ALA dehydratase which is depend

on zinc plays an important role. There are lots of

studies show lower level of zinc can distract

synthesis of Heme. Meanwhile, the higher

supplement of zinc can also distract the absorption

of copper and iron. This can distract the immune

system response which lead to anemia (Olivares et

al., 2007). Another mechanism which shows by

another researchers were animal study with zinc

deficiency shows that decrease of erythrocyte

precursor in bone marrow and erythropoietin level in

plasma in which experiment were mouse and rat

(King et al., 2001; King et al., 2005; Konomi, 2005).

Another opinion shows that deficiency of

mineral can reduce the red blood cell lifetime

because zinc is a cofactor of Superoxide Dismutase

(SOD) in red blood cell (RBC-SOD) which

contribute in protecting from oxidative stress and

cell integrity (Powell, 2000; O’Dell, 2000).

According to the theory which developed by

Shankar & Prasad that zinc deficiency make less

productivity and activity of SOD enzyme and can

reduced free radical activity, which lead to excessive

fat peroxidation. The free radical in intestinal

mucosa cause atrophy of intestinal mucosa through

cell apoptosis. The atrophy intestinal mucosa that

caused by zinc deficiency can make decreasing

productivity and activity of SOD enzyme in

intestinal mucosa cell, therefore fre radical activity

increase which can lead to fragmentation of DNS

and also can lead to cell apoptosis. The apoptosis of

0,5

1,5

5th

hour

10th

hour

4thday8thday 12th

day

Zinc(µg/dl)

TreatmentTime

The level of zinc serum

Controlgroup(G1)

Trialgroup(G2)

Preventivegroup(G3)

Therapygroup(G4)

The Level of Zinc Serum after Oral Zinc on Mice with Escherichia Coli LPS- Induced Diarrhea

165

cell make atrophy of gut villus. Cumulative

effect of intestinal atrophy and broken of tight

junction caused the permeability of membrane

increase and disturb intestinal absorption and lead to

diarrhea (Shankar, 1998).

Patel et al in their meta-analysis found that zinc

supplementation has a modest beneficial association

(9% reduction) with incidence of diarrhea, a stronger

beneficial association (19% reduction) with

prevalence of diarrhoea and occurrence of multiple

diarrheal episodes (28% reduction) (Kalavakuri,

2017).

Zinc is usually given as zinc sulphate, zinc

acetate, or zinc gluconate, which are all water-

soluble compounds. The World Health Organization

(WHO) and the United Nations Children’s Fund

(UNICEF) recommend 10 mg to 20 mg of zinc per

day for children with diarrhoea. There are several

mechanism of action of zinc on acute diarrhoea,

some of which are speciﬁc to the gastrointestinal

system: zinc restores mucosal barrier integrity and

enterocyte brush-border enzyme activity, it promotes

the production of antibodies and circulating

lymphocytes against intestinal pathogens, and has a

direct effect on ion channels, acting as a potassium

channel blocker of adenosine 3-5-cyclic

monophosphate-mediated chlorine secretion

(Lazzerini, 2016).

The conclusion of this study are the level serum

of zinc can increase in preventive group (G3) and

therapy group (G4) after receiving oral zinc and

lower in trial control (G2) which did not received

oral zinc in mice with diarrhea induced by LPS

from E. coli.

ACKNOWLEDGEMENTS

Our thank you note is given to Mr.Yulianto as a staff

of Centre of Biotechnology Laboratory Among

University (PAU) Gadjah Mada University,

Yogyakarta which has been helpful in doing

research in trial animals and Mr.Munawar, M. App.

Stats. As a Lecturer in Faculty of Mathematics and

Science in Syiah Kuala University who has been

helping analysing the study data in statistics.

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