Running Head: TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL 1

FINCTION

Type 2 Diabetes and its Impact on Neutrophil Function

Alyaa Ali Abbas

Hager Nabil Abdul Aati

Maheen Abdeulrahim Yusuf

Maryam Abdulla Yusuf

Maryam Rashed A.Rahman

SBS 320

December 2018

College of Health Sciences, University of Bahrain

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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
Informed Consent Form

University of Bahrain

College of Health Sciences

Medical Laboratory Program

Project Title:

Type 2 Diabetes and its Impact on Neutrophil Function

Invitation:

This project is being carried out by a group of students from College of Health Sciences,

Alyaa Ali, Maryam Rashed, Maheen Abdelrahim, Hagar Nabil and Mariam Abdullah

supervised by Dr. Mohammed Yousef.

Purpose of Study:

Before you decide to participate in this study it is important that you understand why the

research is being done and what it will involve. Please read the following information

carefully. Please ask the researcher if there is anything that is not clear or if you need more

information.

The purpose of this study is to investigate the changes in neutrophil function among type 2

diabetic patients.

Study Procedure:

In this study, a 5ml blood sample will be collected from each participant for laboratory

examination.

Benefits and Risks:

There will be no financial imputation to you for your participation in this study. However, we

hope that the information obtained from this study may help you to increase the awareness of
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
the complications that you may face as a diabetic patient and how to avoid it. On the other

hand, there are no risks for your participation in this study.

Voluntary Participation:

Your participation in this study is voluntary. It is up to you to decide whether or not to take

part in this study. if you decide to take part in this study, you will be asked to sign this

consent form.

Confidentially:

Note that your personal information collected for this study will be anonymous. Participant

data will be kept confidential.

Note: If you have any questions you are free to ask anything regarding this study at any stage.

CONSENT

I have read and I understand the provided information and have had the opportunity to ask

questions. I understand that my participation is voluntary. I understand that I will be given a

copy of this consent form.

Participant's signature ______________________________ Date __________

Investigator's signature _____________________________ Date __________

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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
Diabetes mellitus (DM), commonly referred to as diabetes, is a serious, metabolic,

chronic disease characterized by high blood glucose levels. Diabetes occurs mainly either due

to deficiency of insulin production (a hormone produced in the pancreas gland and it is

primary function is to control blood glucose levels, which is achieved by transferring glucose

from the bloodstream to the body cells where it is converted to energy) or when there is an

impairment in the utilization of insulin by the body cells (WHO 2017). As a result, glucose

starts to build up in the blood vessels leading to hyperglycemia, which in turn may cause a

destruction of small blood vessels in the kidney, eye, heart or nerves.

There are three main types of diabetes, Type1 (Autoimmune), Type2, and gestational

diabetes. Type 1 diabetes is also known as juvenile diabetes, early onset diabetes, or insulin-

dependent diabetes and characterized by a lack of insulin. This type accounts for 10% of all

diabetes cases and mainly found among adolescents.

Type 2 diabetes is the most common form of diabetes and was previously called an adult-

onset or non-insulin dependent diabetes. However, this type of diabetes arising when body

cells are unable to utilize existing insulin properly hence the name insulin resistance. Usually

develops in adults aged over 45 years, but can also occur in other age groups such as

children, adolescents, and young adults. Nearly 90-95% of all diabetes cases worldwide are

of this type.

Gestational diabetes occurs during pregnancy and characterized by high blood glucose above

the normal range and below the diagnostic value of diabetes. This group at high risk to

develop type 2 diabetes (WHO 2017).

Several complications are associated with type 2 diabetes involves short-term complications

and long-term complications. Hypoglycemia and hyperosmolar hyperglycemic nonketotic

syndrome (HHNS) are the major short-term complications in diabetic patients. On the other

hand, long-term complications are subdivided into microvascular and macrovascular. As

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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
mentioned before, the damage caused in tiny blood vessels (microvascular) in kidneys, eyes,

or nerves due to hyperglycemia result in serious consequences such as nephropathy,

retinopathy, and lower limb amputation. In contrast, macrovascular complications are a heart

attack, stroke, and peripheral vascular disease. In addition, recurrent infections is another

significant complication among diabetic patients. According to Weintrob, A.C., and Sexton,

D.J. (2009) they were shown that diabetic patients are treated with infection medications

more than non-diabetic patients. Recently, several epidemiological studies have shown that

there are changes in neutrophil function occur in type 2 diabetes which contributes to

increased susceptibility and severity of infections. Neutrophils are the most abundant type of

white blood cells (~50-70%) in humans which have an essential role in the body. It is

considered as the first-line defense which migrates to the site of an infection to ingest the

microorganisms by releasing enzymes that will kill them (Phagocytosis).

The global prevalence of diabetes has been steadily increasing over the past few decades

from 4.7% in 1980 to 8.5% in 2014 (WHO 2017). In addition, the highest prevalence in

middle east and north Africa region are mainly found in Saudi Arabia (17.7%), UAE

(17.2%), Egypt (16.8%), Bahrain (16.3 %), and Qatar (16.3 %) (IDF 2017).

Literature Review

PubMed, Google Scholar, and MEDLINE were searched to identify studies published

between 2005 and 2018 reporting the relationship between type 2 diabetes and neutrophil

function. Neutrophils play an essential role to protect the host from infectious agents and it is

important cells because their ability to migrate to varies areas in the body. Once the foreign

antigens (such as bacteria) enter the body tissue it will stimulate the chemotaxis process

which results in the release of chemokines that attract circulating neutrophils to the

endothelium. Then, several receptors expressed on the endothelial surface (E-selectin) to bind
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
with neutrophil receptors (L-selectin) to allow rolling of neutrophil over the endothelial

surface. After rolling, neutrophil activates their integrin receptors allowing them to adhere to

endothelial I-CAM receptors which initiate the migration of neutrophil from circulation into

the affected tissue (diapedesis) with the help of PECAM-1 receptor ( a receptor present on the

endothelial surface ). After neutrophil enter the tissue, the most important neutrophil function

will begin by killing the organisms through the phagocytosis process. First of all, PRRs

receptor (pattern recognition receptors) which is present on neutrophils surface will

specifically recognize PAMPs receptor (pathogen-associated molecular pattern) which found

on many microorganisms. Then, the plasma membrane of the neutrophil extended around the

foreign body and start to engulf it. After that, the engulfed particles are surrounded by a

membrane-bound vacuole called phagosome followed by fusion of lysozyme with

phagosome to form a phagolysosome. As a result of this fusion, the lysozyme will release

hydrolytic enzymes which caused an acidic environment in the sac that lead to a destruction

of the microbes. Finally, the cellular waste products will be discharged from the cell through

exocytosis process.

One of the most significant components of neutrophils is NADPH which is scattered in

plasma membrane and cytoplasm during resting state. During phagocytosis, NADPH moves

to phagosome membrane and reduced to NADP and H +¿¿ by an oxidation process. At the

same time, this step will lead to oxidation of O2 into O2 (superoxide anion). After that,
−¿ ¿

superoxide anion will react with H +¿¿ to produce H 2 O2 (Hydrogen peroxide) but this

molecule is not very efficient as bactericidal agent, so in the presence of MPO

(myeloperoxidase enzyme that found naturally in azurophilic granules of neutrophil) and

Cl−¿¿, hydrogen peroxide will be converted to hypochlorite and this product is more effective

as bactericidal agent .
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL

Prevalence

Globally

The global prevalence of diabetes shown that the number of people with diabetes has

risen from 108 million in 1980 to 422 million in 2014, 422M are mostly adults over 18 years

old.

The report of WHO (2016) shown that the largest population with Diabetes disease are

present in South-East Asia and Western Pacific Regions. In few decades the same report has

shown that the numbers of Diabetes are raises rapidly due to same reasons, for example,

increase the age average of the population, population growth and increase in the prevalence

of diabetes at each age.

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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
The mortality of people with diabetes was 1.5 million in 2012, it was conceded the eighth

leading cause of death during that years. Almost most of the death cases in diabetes are due to

high blood glucose level. The great mortality case due to the high glucose level occurs in

upper-middle income countries (1.5 million) and the lowest number in low-income countries

(0.3million).

Although Type 2 Diabetes seen mostly in adult and elderly people , now its arise in young

and children. There are no data on the true incident about the type 2 patient because most of

the time it is undiagnosed. The report from seven countries found that 24%-62% of people

with diabetes are not diagnosed and not treated. (WHO 2016).

Regionally

Recently, the prevalence of diabetes has been increased significantly in the Middle East

and North Africa (MENA) region. According to the International Diabetes Federation (IDF)

in 2017, almost 38.7 million adults aged 20-79 years were found in the MENA region. Nearly

to half (49.1%) of these cases were undiagnosed. More than two-thirds (67.3%) of adults with

diabetes live in urban regions.

MENA region becomes one of the highest rates of diabetes in the world. In addition, the

prevalence of diabetes has demonstrated a growing burden, and among the three types of

diabetes, type 2 diabetes is predominating while type 1 diabetes and gestational diabetes are

less common. However, the Gulf region has the highest prevalence among the MENA region,

four of the Gulf countries (Saudi Arabia, Bahrain, Kuwait, and United Arabic Emirate)

reporting the prevalence of equal to or more than 15% while Oman and Qatar with a

prevalence of less than 15% (IDF, 2017). The reason of increasing the prevalence in the Gulf

region is their recent societal changes with an elevation of economic development and growth

associated with decreased infant mortality and increasing life expectancy, resulting in a huge

change in the lifestyles, diet and physical activity in their population. However, Saudi Arabia
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
ranked first in the MENA region with a prevalence of 18.5% in 2017, while Yemen ranked

last with a prevalence of 3.8% in 2017 (IDF 2017).

Country/territory Prevalence(%), 2017

Saudi Arabia 18.5
Kuwait 15.1
Qatar 14.1
Bahrain 16.2
United Arab Emirates 15,6
Egypt 15.1
Lebanon 14.6
Oman 10.7
Jordan 9.5
Iran 8.9
Afghanistan 6.7
Algeria 6.9
Libya 11.2
morocco 7.3
Pakistan 6.9
Palestine 7.0
Sudan 10.9
Syria 7.5
Yemen 3.8
Table1: the prevalence in the adult of the MENA region in 2017 (IDF)

Nationally

Bahrain has experienced a marked and rapid development in its socio-economic status,

leading to significant changes in lifestyle and patterns of health and disease. There has been a

sharp decline in infectious diseases and a rise in genetic diseases, including diabetes, which

has become one of the most common health problems in the kingdom.

According to the International Diabetes Federation, 5 out of 19 countries in the Middle East

and North Africa (MENA) with the highest prevalence of type 2 diabetes are the Gulf States,

including Bahrain, which ranks second where the prevalence rate reached 16.2 %. In 2016,

the number of diabetes cases among Bahrainis was 52,806 (Ministry of Health 2016). In

2017, this number has increased sharply, making the total number of diabetes cases become

165,300 (International Diabetes Federation 2017).

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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
Bahrain has witnessed a significant increase in diabetes mortality. Diabetes mortality rate was

10% in 2012, but this percent gradually rose to become 15% by the end of 2013 (Ministry of

Health 2012,2013). The probability of premature death from one of the non-communicable

diseases (NCDs), including diabetes is 13%, which means nearly one out of five adults die

from NCDs before age 70 (UN Task Force on NCDs 2017).

Obesity, physical inactivity, tobacco use, and an unhealthy diet have played an important role

in increasing diabetes among Bahrainis. As a result, recent estimates suggest that diabetes

prevalence will increase by more than two-fold in Bahrain in the coming two decades,

making it a national issue that needs to be taken with high importance.

Lab procedures

One of the most important tests to detect neutrophils function is nitroblue tetrazolium test

(NBT). NBT is a *qualitative /quantitative* test that used to measure the reactive oxygen

species (ROS) production particularly superoxide in neutrophil and the ability of neutrophil

to perform phagocytosis. A colorless chemical called nitro blue tetrazolium is added to white

blood cells in the lab to check whether there is change on its color or no. Normally,

neutrophil changes the colorless compound of NBT into dark blue aggregation and this

indicates that cells should be able to kill bacteria and protect the person from infections. But

If there is no change in color when NBT is added this indicates a reduced in ROS production

due to NADPH oxidase defect and thus dysfunctional neutrophils.

Literature Read & literature rejected:

summary of literature review:

methodology:
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL

References

Alba-Loureiro, T. C., Hirabara, S. M., Mendonca, J. R., Curi, R., & Pithon-Curi, T. C.

(2006). Diabetes causes marked changes in function and metabolism of rat

neutrophils. Journal of endocrinology, 188(2), 295-303.

Alba-Loureiro, T. C., Munhoz, C. D., Martins, J. O., Cerchiaro, G. A., Scavone, C., Curi, R.,

& Sannomiya, P. (2007). Neutrophil function and metabolism in individuals with

diabetes mellitus. Brazilian Journal of Medical and Biological Research, 40(8), 1037-

1044.

Gorudko, I. V., Kostevich, V. A., Sokolov, A. V., Shamova, E. V., Buko, I. V.,

Konstantinova, E. E., ... & Panasenko, O. M. (2012). Functional activity of

neutrophils in diabetes mellitus and coronary heart disease: role of myeloperoxidase

in the development of oxidative stress. Bulletin of experimental biology and

medicine, 154(1), 23-26.

Hou, H. W., Tay, H. M., Dalan, R., & Boehm, B. O. (2017). Advances in Neutrophil Testing

In Type 2 Diabetes Mellitus.

International Diabetes Federation. (2017). The IDF Middle East and North Africa (MENA)

Region. International Diabetes Federation.

Khan, S., Raghuram, G. V., Pathak, N., Jain, S. K., Chandra, D. H., & Mishra, P. K. (2014).

Impairment of mitochondrial–nuclear cross talk in neutrophils of patients with type 2

diabetes mellitus. Indian Journal of Clinical Biochemistry, 29(1), 38-44.

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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
Muller, L. M. A. J., Gorter, K. J., Hak, E., Goudzwaard, W. L., Schellevis, F. G., Hoepelman,

A. I. M., & Rutten, G. E. H. M. (2005). Increased risk of common infections in

patients with type 1 and type 2 diabetes mellitus. Clinical infectious diseases, 41(3),

281-288.

Tshikongo, A. K., Songela, R. K., Koke, J. K., Mawazo, S. S., Kalala, Z. L., & Otshudi, A. L.

(2018). Polynuclear Neutrophil Variation and Oxidative Stress in Type 2

Diabetics. JMR, 4(1), 16-19.

Weintrob, A. C., & Sexton, D. J. (2009). Susceptibility to infections in persons with diabetes

mellitus. Waltham (MA): Uptodate.

World Health Organization. (2016). Global report on diabetes. World Health Organization.