EFFECTS OF INDUCED TEMPERATURE ON SOME BIOCHEMICAL

PARAMETERS IN HUMAN BLOOD SPECIMEN

BY

HASSAN, ABDULMALIK DIKKO

ADMISSION NO: 1311222096

A PROJECT SUBMITTED TO THE DEPARTMENT OF CHEMICAL

PATHOLOGY, SCHOOL OF MEDICAL LABORATORY SCIENCES, USMANU
DANFODIYO UNIVERSITY, SOKOTO. IN PARTIAL FULFILLMENT OF THE
CRITERIA FOR THE AWARD OF BACHELOR OF MEDICAL LABORATORY
SCIENCE (B. MLS).

OCTOBER, 2018.

i
 DEDICATION
This work is dedicated to my dear family. May Allah (S.W.T) continue to guide them.

ii
 CERTIFICATION

This project by Hassan, Abdulmalik Dikko (Adm. No.: 1311222096) has met the

requirements for the award of Bachelors’ degree of Medical Laboratory Sciences of

the Usmanu Danfodiyo University, Sokoto, and is approved for its contribution to

knowledge.

------------------------------ --------------------------
(External Examiner) Date

---------------------------
--------------------------
Prof. A.S. Mainasara Date
(Major Supervisor)

---------------------------
-------------------------
Mal. A.A.Ngaski Date
(Co- Supervisor)

------------------------- -------------------------
Dr. T. Oduola Date
(Head of Department)

iii
 AKNOWLEDGEMENTS

I am indeed grateful to Almighty Allah (SWT) for everything. I also wish to express my

profound gratitude to my Major supervisor and my mentor Prof. A. S. Mainasara for his

immense contribution, encouragement, guidance, and tolerance towards the actualization of

this research. Sir, my prayer is that may Allah (SWT) rewards you with paradise. I

acknowledge with gratitude my co-supervisor Mal. A.A. Ngaski for his assistance, guidance

and friendly advice to me towards the exploration of knowledge and completion of this

research work. Sir, I thank you once again. I also acknowledge with sincerity the non-stop

effort as well as kind gesture of the Ag. Head of the Department (Dr. T. Oduola). Sir, I thank

you a lot. Further gratitude goes to all staff of Chemical Pathology Department, School of

Medical Laboratory Sciences for their support, understanding, cooperation and prayers. I will

forever remain indebted to them. I must not forget to appreciate the support and cooperation

of my classmates, relatives and all my friends for their love and understanding. I also give

endless words of thanks my parents, my grandparent, beloved brothers and sisters, my Aunts,

uncles and my entire family for their moral support during this program. Finally I thank the

Management and staff of Usmanu Danfodiyo University Teaching Hospital Sokoto and

may Allah bless.

iv
 TABLE OF CONTENTS

COVER PAGE.....................................................................................................................i

DEDICATION.............................................................................................................ii

CERTIFICATION........................................................................................................iii

AKNOWLEDGEMENTS..............................................................................................iv

TABLE OF CONTENTS.............................................................................................-v

LIST OF TABLES.......................................................................................................x

LIST OF ABBREVIATIONS.........................................................................................xi

ABSTRACT.................................................................................................................xii

CHAPTER ONE

1.0INTRODUCTION -------------------------------------------------------------------------------1

1.1 STATEMENT OF THE RESEARCH PROBLEM-----------------------------------------2

1.2 RESEARCH QUESTION----------------------------------------------------------------------3

1.3 JUSTIFACATION OF THE STUDY---------------------------------------------------------3

1.4 AIM AND OBJECTIVES ---------------------------------------------------------------------4

1.4.1 Aim---------------------------------------------------------------------------------------------4

v
1.4.2 Objectives ---------------------------------------------------------------------------------------------------------------------------------5

1.5 RESEARCH HYPOTHESIS ----------------------------------------------------------------5

1.5.1 Null hypothesis -----------------------------------------------------------------------------5

1.5.2 Alternative Hypothesis --------------------------------------------------------------------5

CHAPTER TWO

2.0 LITERITURE REVIEW-----------------------------------------------------------------------------------------------------------.6

2.1 SODIUM --------------------------------------------------------------------------------------------------------------------------------------6

2.1.1 Regulation of Sodium -------------------------------------------------------------------------------------------------------------8

2.1.2Hyponatraemia ------------------------------------------------------------------------------------------------------------------------11

2.1.3 Hypernatraemia ---------------------------------------------------------------------------------------------------------------------12

2.2.0 POTASSIUM ------------------------------------------------------------------------------------------------------------------------13

2.2.1 Regulation of Potassium -----------------------------------------------------------------------------------------------------15

2.2.2Hypokalaemia-------------------------------------------------------------------------------------------------------------------------16

2.2.3Hyperkalaemia------------------------------------------------------------------------------------------------------------------------19

2.3.0 CHLORIDE --------------------------------------------------------------------------------------------------------------------------20

2.4.0 BICARBONATE -----------------------------------------------------------------------------------------------------------------21

2.4.1 Regulation of Bicarbonate -------------------------------------------------------------------------------------------------22

vi
2.5.0 GLUCOSE -----------------------------------------------------------------------------------------------------------------------------23

2.5.1 Regulation of Glucose ---------------------------------------------------------------------------------------------------------27

2.5.2Hyperglycaemia----------------------------------------------------------------------------------------------------------------------30

2.5.2.1 Diabetes Mellitus ---------------------------------------------------------------------------------------------------------------30

2.5.2.1.1 Pathophysiology of Diabetes Mellitus -----------------------------------------------------------------------31

2.5.3Hypoglycaemia------------------------------------------------------------------------------------------------------------------------33

2.6.0 UREA --------------------------------------------------------------------------------------------------------------------------------------35

2.6.1 Hyper-uraemia------------------------------------------------------------------------------------------------------------------------36

2.6.2 Hypo-uraemia-------------------------------------------------------------------------------------------------------------------------37

2.7.0 CREATININE -----------------------------------------------------------------------------------------------------------------------39

2.7.1 Serum Creatinine ------------------------------------------------------------------------------------------------------------------39

CHAPTER THREE

3.0 MATERIALS AND METHODS------------------------------------------------------------42

3.1 STUDY AREA --------------------------------------------------------------------------------42

3.2 STUDY POPULATION----------------------------------------------------------------------42

3.3 STUDY SUBJECTS---------------------------------------------------------------------------43

3.3.1 Criteria for inclusion -----------------------------------------------------------------------43

3.3.2 Criteria for exclusion -----------------------------------------------------------------------43

vii
3.4 ETHICAL CONSIDERATIONS -----------------------------------------------------------43

3.4.1 Informed Consent----------------------------------------------------------------------------43

3.5 SAMPLING TECHNIQUE ------------------------------------------------------------------44

3.5.1 Subjects Selection ---------------------------------------------------------------------------44

3.5.2 Samples Collection--------------------------------------------------------------------------44

3.5.3 Reagents and Equipment--------------------------------------------------------------------45

3.6 LABORATORY METHODS----------------------------------------------------------------45

3.6.1 Sodium, Potassium, Chloride and Bicarbonate Estimation-----------------------------45

3.6.2 Urea Estimation -----------------------------------------------------------------------------46

3.6.3 Creatinine Estimation -----------------------------------------------------------------------48

3.6.4 Random Glucose Estimation ---------------------------------------------------------------49

CAPTER FOUR

4.0 Results-----------------------------------------------------------------------------------------------------------------------------------------51

CHAPTER FIVE

5.0 Discussion-----------------------------------------------------------------------------------------------------------------------------------54

viii
 CHAPTER SIX

6.0 Conclusion ---------------------------------------------------------------------------------------------------------------------------------56

6.1 Recommendation ---------------------------------------------------------------------------56

References ---------------------------------------------------------------------------------------------------------------------------------------57

Appendix-------------------------------------------------------------------------------------------------------------------------------------------63

ix
 LIST OF TABLES

Table 1. Effect of temperature on some biochemical analytes (mean ± SEM) in

apparently healthy subjects...............................................................................................................................52

Table 2. Comparison of Effects of temperature and gender on some biochemical

analytes (mean ± SEM) in apparently healthy subjects…………………………………………53

x
 LIST OF ABBREVIATIONS USED

GI --------------------------------------------------Gastrointestinal
ECG -----------------------------------------------Electrocardiography
IDMS ---------------------------------------------Isotope Dilution Mass Spectroscopy
CDER --------------------------------------------Center for Drug Evaluation and Research
QRS ----------------------------------------------Quick Response Systems
CKD ----------------------------------------------Chronic Kidney Disease
AKI ---------------------------------------------- Acute Kidney Injury
eGFR -------------------------------------------- Estimated Glomerular Filtration Rate
GFR ---------------------------------------------- Glomerular Filtration Rate
pCO2 --------------------------------------------- Partial Pressure of Carbon dioxide
CA ----------------------------------------------- Carbonic Anhydrase
rpm ------------------------------------------------ revolution per minute
BUN ---------------------------------------------- Blood Urea Nitrogen
FDA ---------------------------------------------- Food Drug Administration
ECF ----------------------------------------------- Extracellular Fluid

IDDM ---------------------------------------------Insulin-dependent diabetes mellitus

NIDDM ------------------------------------------ non-insulin-dependent diabetes mellitus
ACTH -------------------------------------------- Adrenocorticotropic hormone
HMP ---------------------------------------------- Hexose monophosphate shunt
NADP -------------------------------------------- Nicortinamide Adenine dinucleotide phosphate
NADH --------------------------------Reduced Nicortinamide Adenine dinucleotide phosphate
SIADH ------------------------------ Syndrome of Inappropriate antidiuretic hormone secretion
RBC ---------------------------------------------- Red blood cell
TCA ---------------------------------------------- Tri-carboxylic acid cycle

xi
ADH ---------------------------------------------- Antidiuretic hormone
ATPase ------------------------------------------- Adenosine tri-phosphatase

xii
 ABSTRACT

Biochemical anaytes have different level of tolerance to delays in sample

processing and temperature. Samples can be exposed to factors that necessitate
delay in analysis such as increased number of samples received for analysis that
can’t be met up either due to lack of advanced facilities or skilled personnel lead
to unavoidable delay in sample analysis which necessitates its exposure to
unfavorable atmospheric temperature. As temperature has effect on every
physiologic process of the body at certain degree, the aim of this study was to
determine the effect of temperature on some biochemical parameters of human
blood specimen. Blood samples were collected from 8 males and 7 females
subjects and analyzed for concentrations of; glucose, urea, creatinine, sodium,
potassium, chloride and bicarbonate. The mean concentration of urea
(3.6±0.02mmol/L) and glucose (3.7±0.08mmol/L) showed a significant
decrease (p≤0.05) when measured at 450C as compared to the levels measured
at 35oC. Glucose (4.7 ± 0.26mmol/L) increased significantly (P≤0.05) at 35oC
compared to the mean concentration (4.0±0.06mmol/L) at 25oC. At 35oC the
mean glucose concentration in females (4.01±0.06mmol/L) showed significant
(p≤0.05) increase compared to mean concentration in males at the same
temperature. At 450C, mean Na+ (132.14±0.69mmol/L) in females as well as
mean K+ (3.74±0.08) indicate significant (p≤0.05) decrease as compared to
levels in the corresponding male subjects at similar temperature. Almost all
analytes gave the most appropriate concentrations within normal reference
range at 350C with majority having marginally decreased level at 250C, though
not significant (p≥0.05). The result proved the existence of strong relationship
between temperature as well as gender and the integrity of the analytes in
question.

xiii
 CHAPTER ONE

1.0 INTRODUCTION

Each individual analyte has a different tolerance to delay in sample processing

and temperature. Since the laboratory receives the specimen mainly in the form

of whole blood and then separate the serum or plasma from clot and red cells, the

time interval and as well the temperature between the collection, separation, and

subsequent analysis must be controlled for reliable result to be obtained.

Hypothesis has shown that serum rather than plasma is more likely to be

contaminated by haemolysis (Heard and Whittier, 1997). As stated by World

Health Organization (WHO), 2 hours should be the maximum acceptable delay

time and as well room temperature for reliable sample processing. Other factors

affecting analyses include distance from the point of sample collection and point

of analysis, non-availability of advanced automations, power outage, as well

increase in number of sample received that usually makes laboratories unable to

process samples within short period of time which lead to unavoidable delays in

sample processing. These factors necessitate exposure of the samples to

atmospheric temperatures which may exceed the room temperature. A common

problem in clinical laboratories is maintaining the stability of serum analytes

during sample storage. Samples are usually stored at (4–8°C) of a refrigerator for

short durations or in a freezer (−20°C) for longer time periods. Thus, the

1
temperature at which the samples are stored constitutes an important pre-

analytical variable that may affect analytical results in the clinical chemistry

laboratory setting (Laessig et al., 1976).The stability of 72 analytes following

prolonged serum-cell contact has been previously reported (Laessig et al., 1976).

The effects of prolonged storage on the stability of 31 analytes in plasma and

serum separated from cells with a gel barrier have also been reported (Henis et

al., 1995). This situation needs to be addressed by establishing a standard local

ways to ensure the integrity of sample as well as reliable diagnostic laboratory

results. The present research therefore is motivated by the need to establish

maximum acceptable temperature to which sample can be exposed before

analysis in places like North Western part of Nigeria where the temperature is

harsh during certain time of the year. Storage of serum and other blood product is

often necessary in laboratories because of technical issues or to preserve samples

integrity for subsequent research purposes.

1.1 STATEMENT OF REASERCH PROBLEM

Tertiary care hospital laboratories receive over a thousand samples a day. These

laboratories face many challenges including equipment breakdown and the lack

of reagents, which can prevent daily/same-day processing of samples. In such

cases, sample can be exposed to atmospheric temperature (which can be as high

as 43°C in some northern part of this country e.g. Sokoto state). Therefore, the

2
only option is to preserve the samples in a freezer (−20°C). In addition, samples

are sometimes stored for an extended duration until subjected to routine batch

analysis for research purposes. Different temperatures affect some biochemical

analytes’ stability to varying degrees in the course of analysis, some varying from

normal range due to decrease in temperature while others become unstable at

higher temperature. Sokoto state is known for its harsh temperature at certain

periods of the year (up-to 43oC).

1.2 RESEARCH QUESTION

 Is there any significant effect of temperature on some blood biochemical

parameters?

 What temperature can human blood sample maximally tolerate if exposed

to prior to processing/analysis in North Western Nigeria?

 Which biochemical parameter(s) is/are likely to be affected due to

samples’ exposure to various temperatures in Sokoto (North Western

Nigeria)?

1.3 JUSTIFICATION OF STUDY

Previous studies have provided information regarding the stability of analytes in

serum using a number of methods that have since become obsolete (Ono et al.,

1981). Although many blood analytes have been shown to deteriorate within

3
hours in unseparated samples kept at ambient temperature, the few studies

examining unseparated samples stored at low temperatures involved prolonged

contact between serum and cells, which could have caused erroneous test results

(Boyanton et al., 2002). Moreover, most of these older studies were conducted

with animal samples (Kale et al., 2012).However, limited information is available

regarding the stability of commonly measured biochemical analytes in human

blood sample including the effect of atmospheric temperatures as high as 43°C on

blood-separated serum/plasma. Therefore, the present study shall examine the

stability of seven (7) routine chemistry analytes in spot serum/plasma separated

following exposure to varying temperatures (25ᵒC, 35ᵒC and 45ᵒC) for 30

minutes, using the standard guidelines for blood sample handling and separation

(Zhang et al., 1998).

1.4 AIM AND OBJECTIVES

1.4.1 Aim

The aim of the study is to determine the effects of induced temperature on some

biochemical parameters in human blood specimen.

4
 1.4.2 Specific Objectives

i. To measure the level of some biochemical parameters (urea, creatinine,

glucose, sodium, potassium, chloride and bicarbonate) in the samples at

different grades of induced temperatures (250C, 350C and 450C).

ii. To determine the differences in the values obtained of the biochemical

parameters at different temperatures.

iii. To determine the difference between the temperature variation with respect to

gender of the subjects.

iv. To establish the maximum acceptable temperature for biochemical analysis in

Sokoto with respect to these analytes.

1.5 RESEARCH HYPOTHESIS

1.5.1 Null Hypothesis

There is no significant effect of temperature on some biochemical parameters of

human blood serum.

1.5.2 Alternative Hypothesis

There is significant effect of temperature on some biochemical parameters of

human blood specimen.

5
 CHAPTER TWO
2.0 LITERITURE REVIEW
2.1 SODIUM

Normal plasma levels for sodium in adults range from 136 to 146 mEq/L, and

this balance is normally maintained by an average dietary intake of 90 to 250

mEq per day. Sodium excretion tends to reflect sodium intake, and on an average

diet, urine sodium excretion will range between 80 and 180 mEq per day. One of

the major roles of potassium/sodium balance in the body is that of the nerve

impulse. A differential in sodium and potassium concentration forms a polarity

across the nerve membrane that when stimulated (electrical, chemical,

mechanical, or thermal) leads to depolarization and propagation of the nerve

impulse along the cell membrane (Hole, 1978). In the nerve cell, active sodium-

potassium pumps create this differential by pumping two K+ atoms into the cell

for every three Na+ atoms pumped out of the cell. Active pumping, along with

negatively charged ions of other molecules inside the cell, leads to a voltage

potential across the cell membrane. The resulting voltage is approximately –

70mV (Barnett and Larkman, 2007). Following membrane stimulation, the

membrane becomes permeable to Na+ ions, allowing Na+ inside the cell, thus

eliminating the electrical potential across the membrane (depolarization).

Depolarization propagates in all directions from the initial point. For a very brief

6
time, the membrane is unable to depolarize again and remains unresponsive. Na +

and K+ play a key role in the depolarization of the muscle cell membrane.

Polarization, as with the neuron, requires an active ion pump and energy in the

form of ATP to create an ion gradient across the cell’s membrane. As with

neurons, the muscle cell membrane becomes impervious to Na+ while Na+ ions

are actively pumped out of the cell and K + ions into the cell; however, some K +

diffuse back out at a slower rate than Na + is pumped out. Homeostasis of Na+ and

K+ is critical to life, especially extracellular K+ levels. A number of homeostatic

mechanisms keep Na+ and K+ regulated. Normal extracellular and intracellular

Na+ Sodium is rapidly and actively taken up by the mucosal lining of the

gastrointestinal (GI) tract (Johnson, 2007). Unlike K+, however, it is not rapidly

sequestered into the cells. Only around 10% of Na+ body burdens are found in the

cells, 40% remains in extracellular fluid (Hole, 1978). Na+ is excreted through

urine, faeces, perspiration, and tears. It is also secreted back into the intestines at

the rate of 25 grams per day. To remain in homeostasis, the intestines must

absorb 25–35 of sodium every day (Guyton and Hall, 2000). This amount plus

the amount of Na+ lost from other routes (urine and perspiration) needs to be

reabsorbed every day for Na+ homeostasis to occur. It is easy to see why diseases

such as diarrhoea and intestinal influenza can easily upset the Na + maintenance in

the body and quickly lead to life threatening situations.

7
2.1.1 Regulation of Sodium

Some sodium is lost in faeces and sweat, but as was seen with potassium, the

majority of sodium regulation in the body occurs in the kidney. In the kidney,

sodium ions (approximately 70%) are reabsorbed into the proximal tubules and

loop of Henle after filtration through the glomerulus (Hole, 1978). However,

unlike K+, the driving force of Na+ homeostasis is the glomerular filtration rate

and tubule reabsorption. By the time the filtrate reaches the distal tubules almost

all the Na+ has been reabsorbed. As the filtrate formed at the glomerulus passes

through the proximal tubules, loop of Henle, and distal tubules, the solution

undergoes several transformations in tonicity that allows (along with active Na +

uptake throughout the loop) for reabsorption of water and Na +. The ascending

limb is impermeable to water yet still actively secretes Na + causing the interstitial

space around the ascending limb to become hypertonic. Since the interstitial

space around the ascending limb is immediately adjacent to the descending limb,

it creates an osmotic gradient between fluid inside the descending limb and the

interstitial fluid. This gradient drives the removal of water from the descending

limb, thereby increasing the fluid tonicity (forming a hypertonic solution). As the

fluid makes its way out of the descending limb into the ascending limb the tubule

becomes impermeable to water, yet Na+ continues to be actively pumped out.

This results in a hypotonic fluid low in Na+ that leaves the ascending loop of

8
Henle. Following the reabsorption of Na+ in the ascending loop of Henle, Na+

reabsorption continues in the distal tubules. It is in this region of the kidney

where water retention occurs. The pituitary gland, in response to decreased water

concentration in the blood, releases stored antidiuretic hormone (ADH) into the

circulatory system. ADH causes the epithelial cells of the distal convoluted

tubules to become more permeable to water, thus concentrating urine and saving

water during times of water stress. Na + homeostasis is critical to life and thus

requires the amount of sodium intake to equal the amount of Na+ excretion. There

are numerous feedback loops and hormonal controls in play to regulate Na +

excretion such as blood pressure (pressure natriuresis and diuresis), blood

volume, antidiuretic hormone, angiotensin II, arterial baroreceptor, low pressure

stretch receptors reflexes, aldosterone, and natriuretic peptide. Regardless of the

mechanism (complex or simple), all these feedbacks work by altering either

glomerularfiltration rates or by Na+ reabsorption. Xenobiotics, disease, or even

fever can cause any of these mechanisms to alter Na+ balance. It is therefore

necessary to have a complex system of redundancy and rapid response to

maintain critical Na+ balance pressure natriuresis and diuresis: Blood pressure

drives both urinary volume and the amount of Na + filtered into the proximal

tubule. While increases and decreases in natriuresis pressure can help regulate

Na+ homeostasis when such pressure changes occur as a result of disease (e.g.,

9
hypertension) or other causes, the increase or decrease in pressure can cause

imbalances in sodium. Blood volume: Changes in blood volume quickly lead to

changes in cardiac output and blood pressure. Blood pressure changes can lead to

changes in Na+ excretion. Antidiuretic hormone: Pituitary gland, in response to

decreased water concentration in the blood, releases stored antidiuretic hormone

into the circulatory system. ADH causes the epithelial cells of the distal

convoluted tubules to become more permeable to water, thus concentrating urine

and saving water during times of water stress. Angiotensin II: Decreased levels of

angiotensin II result in decreased reabsorption of Na + in the renal tubules. Thus

decreases in angiotensin II are seen following increases in sodium intake.

Angiotensin II works by modifying the natriuresis pressure mechanism,

decreasing angiotensin II and increasing pressure when sodium needs to be

excreted (Crowley et al., 2006). It also indirectly stimulates aldosterone secretion

and constricts efferent arterioles. Angiotensin II is decreased by inhibiting renin,

an angiotensin II precursor. In some individuals, this renin-angiotensin system

(RAS) does not operate as efficiently, and greater increases in arteriole pressure

are needed to excrete sodium. This may lead to hypertension in some individuals

(Guyton and Hall, 2000). Arterial baroreceptor and low pressure stretch receptors

reflexes: Sympathetic activity can constrict renal arterioles, increase tubular

reabsorption, and stimulate renin release, all leading to increased retention of

10
sodium. This type of reflex is likely to occur from decreased blood volume, as in

following a large hemorrhage. Aldosterone: Na+ absorption in the kidney (the

ascending limb of the loop of Henle, the distal convoluted tubules, and collecting

ducts) is greatly influenced by the amount of aldosterone excreted by the adrenal

cortex (Hole, 1978). When Na+ levels drop, the adrenal cortex secretes

aldosterone, which results in an increase in the active reabsorption of Na+.

2.1.2 Hyponatraemia

Hyponatraemia represents a decrease in the serum sodium concentration below

the lower end of the normal range (136 mEq/L) (Berkow and Fletcher, 1992).

Clinical signs and symptoms associated with Hyponatraemia include

hypotension, and decreased extracellular fluid osmolarity resulting in intracellular

fluid increase (Chandrasoma and Taylor, 1991). Hyponatraemia is the most

common electrolyte disorder. In one study, the prevalence of Hyponatraemia was

28% in acute hospital care patients at the time of admission and 21% in

ambulatory patients (Hawkins, 2006). The risk factors for Hyponatraemia include

use of diuretics, liver failure, heart failure, myocardial infarction, and endocrine

changes which are mostly found in older patients. Hyponatraemia is associated

with various conditions that can be grouped into dilutional disorders

(characterized by water intake in excess of output; the condition implies impaired

water excretion) and depletional disorders (caused by sodium depletion in excess

11
of water depletion or replacement of fluid losses with water alone). Dilutional

disorders include primary causes such as renal failure and the syndrome of

inappropriate antidiuretic hormone secretion (SIADH). Other causes of dilutional

disorders include neuroendocrine dysfunction (adrenal and pituitary

insufficiency), diseases linked to sodium retention and edema (congestive heart

failure, cirrhosis, nephrotic syndrome), osmotic Hyponatraemia (severe

hyperglycemia in diabetes), and drug-induced disorders (mercurial diuretics,

chlorothiazide diuretics). Hyponatraemia with hypotonicity can also be induced

by diets with high water and low salt intake or by excessive beer drinking.

2.1.3 Hypernatraemia

Hypernatraemia represents an elevation in the serum sodium concentration above

the higher end of the normal range (145 mEq/L) (Berkow and Fletcher, 1992).

Clinical signs and symptoms associated with Hypernatraemia include

hypertension, increased extracellular fluid volume, and increased extracellular

fluid osmolarity resulting in intracellular fluid loss. Hypernatraemia is not as

common as Hyponatraemia. It is associated with abnormal renal excretion of

water with inadequate water intake disorders such as in pituitary ADH deficiency

(central diabetes insipidus) and nephrotic syndrome (nephrotic diabetes

insipidus), in which kidneys are ADH unresponsive, or with osmotic diuresis

such as severe glycosuria and manitol diuresis. Other diseases and states that may

12
be accompanied by Hypernatraemia are chronic renal failure, recovery phase of

acute renal failure, hypocalcemia, Hypokalaemia, and sickle cell anemia.Another

mechanism of Hypernatraemia is water depletion with normal renal conservation

of water but inadequate intake of water; causes include excessive sweating and

diarrhea (pronounced in children) (Bishop et al., 2010).

2.2.0 POTASSIUM

Potassium is essential for the proper function of all cells, tissues, and organs in

the human body. It is also crucial to heart function and plays a key role in skeletal

and smooth muscle contraction, making it important for normal digestive and

muscular function. Normal plasma levels for potassium in adults range from 3.5

to 5.0 mEq/L, and this balance is usually maintained in adults on an average

dietary intake of 80 to 200 mEq per day. It is noted that the normal intake,

minimal need, and maximum tolerance for potassium is almost the same as that

for sodium. Potassium ions are the major cations of the intracellular fluid

(Ganong, 1991).Potassium plays a key role in that potassium bicarbonate is the

primary intracellular inorganic buffer. Potassium enters the cell more readily than

sodium and initiates the brief sodium-potassium exchange across the cell

membranes. In the nerve cells, this sodium-potassium flux generates the electrical

potential that aids the conduction of nerve impulses. When potassium leaves the

cell, it changes the membrane potential and allows the nerve impulse to progress.

13
This electrical potential gradient, created by the “sodium-potassium pump”, helps

generate muscle contractions and regulates the heartbeat (Ganong, 1991).

Cellular uptake of potassium is regulated by the sodium-potassium pump, while

movement of potassium out of the cell is governed by passive forces (cell

membrane permeability and chemical and electrical gradients to the potassium

ions). Another of the pump’s most important functions is preventing the swelling

of cells. If sodium is not pumped out, water accumulates within the cell causing it

to swell and ultimately burst. (Bishop et al., 2010).

Potassium is very important in cellular biochemical reactions and energy

metabolism; it participates in the synthesis of proteins from amino acids in the

cell. Potassium also functions in carbohydrate metabolism; it is active in

glycogen and glucose metabolism, converting glucose to glycogen that can be

stored in the liver for future energy. Potassium is important for normal growth

and for building muscle. Though sodium is readily conserved by the body, there

is no effective method for potassium conservation. Even when a potassium

shortage exists, the kidneys continue to excrete it. Since the human body relies on

potassium balance for a regularly contracting heart and a healthy nervous system,

it is essential to strive for this electrolyte’s balance. The recommended intake of

potassium for adolescents and adults is 4700 mg/day (Karppanen et al., 2005).

Following ingestion, K+ is rapidly absorbed by active uptake in the mucosal

14
lining of the intestine. This rapid uptake could lead to severe K+ imbalance if it

was not for the rapid absorption of K+ into cells. Ninety-eight percent of gastro-

intestinally absorbed K+ is stored in cells, with 2% being found extracellularly

(Guyton and Hall, 2000). Even though cellular storage allows for the rapid

regulation of extracellular K+, long-term regulation is carried out in the kidney.

2.2.1 Regulation

A small percentage of excess K+ is excreted in the faeces, while the bulk of K+

excretion occurs in the urine following filtration, reabsorption, and secretion in

the kidneys. The kidney filters around 800 mg of K+ per day of which

approximately 65% and 27% is reabsorbed in the proximal tubule and loop of

Henle, respectively (Guyton and Hall, 2000). These percentages remain fairly

constant from day to day and do not significantly regulate daily variations from

changes in diet and absorption. The work of regulating daily variations occurs

mainly in the secretion of K+ in the distal tubules and cortical collecting tubules

(Giobiesch et al., 1996).Under normal potassium intake the amount of absorption

exceeds what the body needs, and secretion into the distal tubules and cortical

collecting tubules eliminates the excess through excretion in the urine. Under

extreme K+ deficiencies, reabsorption in the distal tubules can actually exceed

secretion and thus conserve K+. Aldosterone increases the Na+ /K+ ATPase pump

as Na+ is conserved, K+ is secreted into the urine. β -adrenergic stimulation:

15
Activation of β-2 -adrenergic receptors by stimulants such as epinephrine causes

K+ to move into cells. Drugs that block β-288receptors can prevent the uptake of

K+ into cells. Acid-base abnormalities: The activity of the sodium-potassium

ATPase pump is inhibited in the presence of increased hydrogen ion

concentration. Therefore disease or physiological states that affect acid-base

balance can affect K+ homeostasis as well Cell lysis: Necrosis or major cell death

can lead to the release of intracellular K+ causing a disruption in K+ homeostasis.

Strenuous exercise: Muscle cells release K+ during long-duration exercise.

Usually this is not a problem except in individuals that may already be sensitive

to K+ disturbances (diabetics, people taking beta blockers) (Guyton and Hall,

2000).

2.2.2 Hypokalaemia

Hypokalaemia represents the low potassium levels. In adults, potassium blood

levels drop below 3.5 mEq/L, which is the lower range of normal values. Clinical

signs and symptoms associated with Hypokalaemia include neuromuscular

(weakness, paralysis, fasciculation and tetany), gastrointestinal (ileus, nausea,

vomiting, abdominal distention), and renal effects (polyuria). Cardiac effects

present themselves as dysrhythmias and conduction defects. ECG manifestations

include decreased amplitude and broadening of the T waves, prominent U waves,

ST segment depression, increased QRS duration, and increase in P wave

16
amplitude and duration. The changes may lead to atrioventricular block and

cardiac arrest (Unwin et al., 2011). With Hypokalaemia, cardiac arrest occurs

during systole (Krupp, 1990). Potassium homeostasis depends on external

balance (i.e., dietary intake and absorption versus excretion) and internal

balance (i.e., the distribution of potassium between intracellular and extracellular

fluids (Chandrasoma and Taylor, 1991)). External losses include those through

the gastrointestinal tract (e.g., diarrhea, villous adenoma of recto-sigmoid colon,

inadequate intake) or through the skin (e.g., profuse sweating). Urine potassium

is usually <20 mEq/24 hours. In external losses through the kidneys, urine

potassium is usually >20 mEq/24 hours. Eating disorders and starvation:

Anorexia nervosa and bulimia are psychological eating disorders. Medical

consequences of these eating disorders include heart damage, failure of the

endocrine system, perforation of the stomach or esophagus, aspiration of vomit,

erosions of teeth enamel, and depression (Brom, 1985). Death by starvation has

been reported in up to 24% of the patients with anorexia. Biochemical changes

are also pronounced (Winston, 2012). Hypokalaemia is the most common

electrolyte disturbance. It is often reflected by changes on the electrocardiograms.

Metabolic alkalosis is found in patients who vomit or abuse diuretics, whereas

acidosis is found in those abusing laxatives. In laxative abuse, potassium is lost

directly from the intestines. In contrast, the loss of potassium in those who vomit

17
is largely due to metabolic alkalosis, which is secondary to loss of hydrogen ions

in the vomitus. This results in increased availability of bicarbonate from blood

and increased renal excretion of potassium (Weinstein, 2003). Hypokalemic

nephropathy is also associated with laxative abuse. Severe chronic Hypokalaemia

in these patients was found to result in a progressive decrease in renal function

and histological changes suggestive of chronic glomerular damage. Chronic

tubule-interstitial nephropathy has been also reported (Abdel-Rahman and

Moorthy, 1997). Hypokalaemia is also associated with starvation related to other

causes. For example, Hypokalaemia was reported in malnourished children on

poor protein-calorie diets all over the world. In these children, decrease in total

body potassium was correlated with decreased muscle potassium established by

analysis of biopsy samples (Nichols et al., 1969). This result correlated with loss

of total muscle mass. In contrast, muscle water was increased. Wasting is one

aspect of the muscle loss; however, a contributing factor may be a decreased

muscle build-up. Several laboratory studies showed the importance of potassium

in protein synthesis. Causes for potassium renal losses are complex (Pepin and

Shields, 2012). Contributing clinical factors are increased mineralocorticoid-

receptor stimulation (primary hyper-reninism distinguished by increased renin

and aldosterone levels that cannot be suppressed by saline); primary

aldosteronism (e.g., Conn syndrome); a primary increase in the effectiveness

18
and/or amount of non-aldosterone mineralocorticoid-receptor agonist (e.g.,

Cushing syndrome, congenital adrenal hyperplasia); and increased distal sodium

delivery and/or non-reabsorbable ions in the distal nephron (e.g., magnesium

deficit, Bartter syndrome) ( Unwin et al., 2011 ).

2.2.3 Hyperkalaemia

In adults, Hyperkalaemia refers to blood values of potassium >5 mEq/L. Clinical

manifestations of Hyperkalaemia include neuromuscular effects (weakness,

ascending paralysis, and respiratory failure) and ECG changes (Chandrasoma and

Taylor, 1991). The changes in heart conductivity can lead to sinus arrest,

ventricular tachycardia, and brillation at >10 mEq/L (El-Sherif and Turitto,

2011). With Hyperkalaemia, cardiac arrest occurs during diastole (Krupp, 1990).

Hyperkalaemia is less common than Hypokalaemia. However, there are two

major mechanisms for Hyperkalaemia development. Redistribution

Hyperkalaemia is caused by potassium shifting from the intracellular space into

the extracellular space, thus raising serum potassium concentration. Potassium is

forced out of cells in exchange for hydrogen ion in both metabolic and respiratory

acidosis. Similarly, potassium leaks out of cells in hypertonic states, in burns and

injuries, and in massive digitalis overdose. Hyperkalaemia secondary to impaired

potassium excretion is the major cause of this electrolyte disorder. It may be due

to aldosterone deficiency (e.g., primary adrenal failure, Addison’s disease) or

19
tubular unresponsiveness to aldosterone (e.g., chronic renal diseases, some

pharmaceuticals). Hyperaldosteronism is a disease caused by an excess

production of adrenal hormone aldosterone. This hormone is responsible for

sodium and potassium balance, which then directly controls water balance to

maintain appropriate blood pressure and blood volume. With adrenal

insufficiency, there is inappropriate sodium excretion. When adrenal aldosterone

production is increased (as in shock, heart failure, or cirrhosis) sodium excretion

is decreased. People with a deficiency of aldosterone, especially found in

association with cortisol deficiency in Addison’s disease, have low blood volume

and therefore low blood pressure, low sodium and high potassium. Just the

opposite is seen in hyperaldosteronism. There are several drugs that affect the

renin-angiotensin-aldosterone system and thus may impact potassium levels.

Metabolic acidosis is caused by the failure of hydrogen ion excretion.

Hyperkalaemia is one of the later signs of the disease; so is the development of

secondary hyperparathyroidism and renal osteodystrophy. Black patients seemed

to better tolerate Hyperkalaemia than whites. Hypokalaemia was associated with

faster chronic kidney disease progression in both races. (Guyton and Hall, 2000).

2.3.0 CHLORIDE

Chloride (Cl-) is the principal extracellular anion and is involved in the

maintenance of extracellular fluid balance. It is readily filtered by the glomerulus

20
and is passively reabsorbed as a counter ion when sodium is reabsorbed in the

proximal convoluted tubule. In the ascending limb of the loop of Henle,

potassium is actively reabsorbed by a distinct chloride “pump,” which also

reabsorbs sodium. This pump can be inhibited by loop diuretics, such as

furosemide. As expected, the regulation of chloride is controlled by the same

forces that regulate sodium (Agoreyo and Nwanzi, 2010).

2.4.0 BICARBONATE

This is the second most abundant anion in the ECF. Total CO 2 comprises the

bicarbonate ion (HCO3-), carbonic acid (H2CO3), and dissolved CO2, with HCO3-

accounting for more than 90% of the total CO 2 at physiologic pH. Because HCO3-

composes the largest fraction of total CO2, total CO2 measurement is indicative of

HCO3- measurement. HCO3 is the major component of the buffering system in the

blood. Carbonic anhydrase in RBCs converts CO2 and H2O to carbonic acid,

which dissociates into H+ and HCO3-. CO2+ H2O ←⎯CA→ H2CO3 ←⎯CA→ H+ +

HCO3-+ CA, carbonic anhydrase. HCO3 diffuses out of the cell in exchange for

Cl- to maintain ionic charge neutrality within the cell (chloride shift). This

process converts potentially toxic CO2 in the plasma to an effective buffer: HCO 3.

HCO3- buffers excess H+ by combining with acid, then eventually dissociating

into H2O and CO2 in the lungs where the acidic gas CO2 is eliminated (Burtis et

al., 2008).

21
2.4.1 Regulation of Bicarbonate

Most of the HCO3- in the kidneys (85%) is reabsorbed by the proximal tubules,

with 15% being reabsorbed by the distal tubules. Because tubules are only

slightly permeable to HCO3-, it is usually reabsorbed as CO 2. This happens as

HCO3-, after filtering into the tubules, combines with H to form carbonic acid,

which then dissociates into H2O and CO2. The CO2 readily diffuses back into the

ECF. Normally, nearly all the HCO3- is reabsorbed from the tubules, with little

lost in the urine. When HCO3- is filtered in excess of H+ available, almost all

excess HCO3- flows into the urine. In alkalosis, with a relative increase in HCO3-

compared to CO2, the kidneys increase excretion of HCO3- into the urine,

carrying along a cation such as Na+. This loss of HCO3- from the body helps

correct pH. Among the responses of the body to acidosis is an increased excretion

of H+ into the urine. In addition, HCO3- reabsorption is virtually complete; with

90% of the filtered HCO3 reabsorb the filtered HCO3- reabsorbed in the proximal

tubule and the remainder in the distal tubule. Acid-base imbalances cause

changes in HCO3- and CO2 levels. A decreased HCO3- may occur from metabolic

acidosis as HCO3- combines with H to produce CO 2, which is exhaled by the

lungs. The typical response to metabolic acidosis is compensation by

hyperventilation, which lowers pCO2. Elevated total CO2 concentrations occur in

metabolic alkalosis as HCO3 is retained, often with increased pCO 2 as a result of

22
compensation by hypoventilation. Typical causes of metabolic alkalosis include

severe vomiting, Hypokalaemia, and excessive alkali intake (Bishop et al., 2010).

2.5.0 GLUCOSE

Glucose is a primary source of energy for humans. The nervous system, including

the brain, totally depends on glucose from the surrounding extracellular fluid

(ECF) for energy. Nervous tissue cannot concentrate or store carbohydrates;

therefore, it is critical to maintain a steady supply of glucose to the tissue. For this

reason, the concentration of glucose in the ECF must be maintained in a narrow

range. When the concentration falls below a certain level, the nervous tissues lose

the primary energy source and are incapable of maintaining normal function

(Burtis et al., 2008).

By virtue of the fate of Glucose, most of our ingested carbohydrates are

polymers, such as starch and glycogen. Salivary amylase and pancreatic amylase

are responsible for the digestion of these non-absorbable polymers to dextrins and

disaccharides, which are further hydrolyzed to monosaccharides by maltase, an

enzyme released by the intestinal mucosa. Sucrase and lactase are two other

important gut-derived enzymes that hydrolyze sucrose to glucose and fructose

and lactose to glucose and galactose. When disaccharides are converted to

monosaccharides, they are absorbed by the gut and transported to the liver by the

hepatic portal venous blood supply. Glucose is the only carbohydrate to be

23
directly used for energy or stored as glycogen. Galactose and fructose must be

converted to glucose before they can be used. After glucose enters the cell, it is

quickly shunted into one of three possible metabolic pathways, depending on the

availability of substrates or the nutritional status of the cell. The ultimate goal of

the cell is to convert glucose to carbon dioxide and water. During this process,

the cell obtains the high-energy molecule adenosine triphosphate (ATP) from

inorganic phosphate and adenosine diphosphate (ADP). The cell requires oxygen

for the final steps in the electron transport chain (ETC). Nicotinamide adenine

dinucleotide (NAD) in its reduced form (NADH) will act as an intermediate to

couple glucose oxidation to the ETC in the mitochondria where much of the ATP

is gained. The first step for all three pathways requires glucose to be converted to

glucose-6-phosphate using the high energy molecule, ATP. This reaction is

catalyzed by the enzyme hexokinase. Glucose-6-phosphate can enter the

Embden-Myerhof pathway or the hexose monophosphate pathway or can be

converted to glycogen. The first two pathways are important for the generation of

energy from glucose; the conversion to glycogen pathway is important for the

storage of glucose. In the Embden-Myerhof pathway, glucose is broken down

into two, three-carbon molecules of pyruvic acid that can enter the tricarboxylic

acid cycle (TCA cycle) on conversion to acetyl-coenzyme A (acetyl-CoA). This

pathway requires oxygen and is called the aerobic pathway. Other substrates have

24
the opportunity to enter the pathway at several points. Glycerol released from the

hydrolysis of triglycerides can enter at 3-phosphoglycerate, and fatty acids and

ketones and some amino acids are converted or catabolized to acetyl-CoA, which

is part of the TCA cycle. Other amino acids enter the pathway as pyruvate or as

deaminated-ketoacids and -oxoacids. The conversion of amino acids by the liver

and other specialized tissue, such as the kidney, to substrates that can be

converted to glucose is called gluconeogenesis. Gluconeogenesis also

encompasses the conversion of glycerol, lactate, and pyruvate to glucose.

Anaerobic glycolysis is important for tissue such as muscle, which often have

important energy requirements without an adequate oxygen supply. These tissues

can derive ATP from glucose in an oxygen-deficient environment by converting

pyruvic acid into lactic acid. The lactic acid diffuses from the muscle cell, enters

the systemic circulation, and is then taken up and used by the liver. For anaerobic

glycolysis to occur, 2 moles of ATP must be consumed for each mole of glucose;

however, 4 moles of ATP are directly produced, resulting in a net gain of 2 moles

of ATP. Further gains of ATP result from the introduction of pyruvate into the

TCA cycle and NADH into the ETC. The second energy pathway is the hexose

monophosphate shunt (HMP shunt), which is actually a detour of glucose-6-

phosphate from the glycolytic pathway to become 6-phosphogluconic acid. This

oxidized product permits the formation of ribose-5-phosphate and NADP in its

25
reduced form (NADPH). NADPH is important to erythrocytes that lack

mitochondria and are therefore incapable of the TCA cycle. The reducing power

of NADPH is required for the protection of the cell from oxidative and free

radical damage. Without NADPH, the lipid bilayer membrane of the cell and

critical enzymes would eventually be destroyed, resulting in cell death. The HMP

shunt also permits pentoses, such as ribose, to enter the glycolytic pathway.

When the cell’s energy requirements are being met, glucose can be stored as

glycogen. This third pathway, which is called glycogenesis, is relatively

straightforward. Glucose-6-phosphate is converted to glucose-1-phosphate, which

is then converted to uridinediphosphoglucose and then to glycogen by glycogen

synthase. Several tissues are capable of the synthesis of glycogen, especially the

liver and muscles. Hepatocytes are capable of releasing glucose from glycogen or

other sources to maintain the blood glucose concentration. This is because the

liver synthesizes the enzyme glucose-6-phosphatase. Without this enzyme,

glucose is trapped in the glycolytic pathway. Muscle cells do not synthesize

glucose-6-phosphatase and, therefore, they are incapable of dephosphorylating

glucose. Once glucose enters a muscle cell, it remains as glycogen unless it is

catabolized. Glycogenolysis is the process by which glycogen is converted back

to glucose 6-phosphate for entry into the glycolytic pathway. Overall, dietary

glucose and other carbohydrates either can be used by the liver and other cells for

26
energy or can be stored as glycogen for later use. When the supply of glucose is

low, the liver will use glycogen and other substrates to elevate the blood glucose

concentration. These substrates include glycerol from triglycerides, lactic acid

from skin and muscles, and amino acids. If the lipolysis of triglycerides is

unregulated, it results in the formation of ketone bodies, which the brain can use

as a source of energy through the TCA cycle. The synthesis of glucose from

amino acids is gluconeogenesis. This process is used in conjunction with the

formation of ketone bodies when glycogen stores are depleted, conditions

normally associated with starvation. The principal pathway for glucose oxidation

is through the Embden-Myerhof pathway. NADPH can be synthesized through

the HMP shunt, which is a side pathway from the anaerobic glycolytic pathway

(Burtis et al., 2008).

2.5.1Regulation of Glucose

The liver, pancreas, and other endocrine glands are all involved in controlling the

blood glucose concentrations within a narrow range. During a brief fast, glucose

is supplied to the ECF from the liver through glycogenolysis. When the fasting

period is longer than 1 day, glucose is synthesized from other sources through

gluconeogenesis. Control of blood glucose is under two major hormones: insulin

and glucagon, both produced by the pancreas. Their actions oppose each other.

Other hormones and neuroendocrine substances also exert some control over

27
blood glucose concentrations, permitting the body to respond to increased

demands for glucose or to survive prolonged fasts. It also permits the

conservation of energy as lipids when excess substrates are ingested. Insulin is

the primary hormone responsible for the entry of glucose into the cell. It is

synthesized by the cells of islets of Langerhans in the pancreas. When these cells

detect an increase in body glucose, they release insulin. The release of insulin

causes an increased movement of glucose into the cells and increased glucose

metabolism. Insulin is normally released when glucose levels are high and is not

released when glucose levels are decreased. It decreases plasma glucose levels by

increasing the transport entry of glucose in muscle and adipose tissue by way of

nonspecific receptors. It also regulates glucose by increasing glycogenesis,

lipogenesis, and glycolysis and inhibiting glycogenolysis. Insulin is the only

hormone that decreases glucose levels and can be referred to as a hypoglycemic

agent. Glucagon is the primary hormone responsible for increasing glucose

levels. It is synthesized by the cells of islets of Langerhans in the pancreas and

released during stress and fasting states. When these cells detect a decrease in

body glucose, they release glucagon. Glucagon acts by increasing plasma glucose

levels by glycogenolysis in the liver and an increase in gluconeogenesis. It can be

referred to as a hyperglycemic agent. Two hormones produced by the adrenal

gland affect carbohydrate metabolism. Epinephrine, produced by the adrenal

28
medulla, increases plasma glucose by inhibiting insulin secretion, increasing

glycogenolysis, and promoting lipolysis. Epinephrine is released during times of

stress. Glucocorticoids, primarily cortisol, are released from the adrenal cortex on

stimulation by adrenocorticotropic hormone (ACTH). Cortisol increases plasma

glucose by decreasing intestinal entry into the cell and increasing

gluconeogenesis, liver glycogen, and lipolysis. Two anterior pituitary hormones,

growth hormone and ACTH, promote increased plasma glucose. Growth

hormone increases plasma glucose by decreasing the entry of glucose into the

cells and increasing glycolysis. Its release from the pituitary is stimulated by

decreased glucose levels and inhibited by increased glucose. Decreased levels of

cortisol stimulate the anterior pituitary to release ACTH. ACTH, in turn,

stimulates the adrenal cortex to release cortisol and increases plasma glucose

levels by converting liver glycogen to glucose and promoting gluconeogenesis.

Two other hormones affect glucose levels: thyroxine and somatostatin. The

thyroid gland is stimulated by the production of thyroid-stimulating hormone

(TSH) to release thyroxine that increases plasma glucose levels by increasing

glycogenolysis, gluconeogenesis, and intestinal absorption of glucose.

Somatostatin, produced by the cells of the islets of Langerhans of the pancreas,

increases plasma glucose levels by the inhibition of insulin, glucagon, growth

hormone, and other endocrine hormones (Bishop et al., 2010).

29
2.5.2 Hyperglycemia

Hyperglycemia is an increase in plasma glucose levels. In healthy patients, during

a hyperglycemia state, insulin is secreted by the cells of the pancreatic islets of

Langerhans. Insulin enhances membrane permeability to cells in the liver,

muscle, and adipose tissue. It also alters the glucose metabolic pathways.

Hyperglycemia, or increased plasma glucose levels, is caused by an imbalance of

hormones (Jeppsson et al., 2002).

2.5.2.1 Diabetes Mellitus

Diabetes mellitus is actually a group of metabolic diseases characterized by

hyperglycemia resulting from defects in insulin secretion, insulin action, or both.

In 1979, the National Diabetes Data Group developed a classification and

diagnosis scheme for diabetes mellitus. This scheme included dividing diabetes

into two broad categories: type 1, insulin-dependent diabetes mellitus (IDDM);

and type 2, non–insulin-dependent diabetes mellitus (NIDDM). Established in

1995, the International Expert Committee on the Diagnosis and Classification of

Diabetes Mellitus, working under the sponsorship of the American Diabetes

Association, was given the task of updating the 1979 classification system. The

proposed changes included eliminating the older terms of IDDM and NIDDM.

The categories of type 1 and type 2 were retained, with the adoption of Arabic

numerals instead of Roman numerals (Bishop et al., 2010).

30
2.5.2.1.1 Pathophysiology of Diabetes Mellitus

In both type 1 and type 2 diabetes,the individual will be hyperglycemic, which

can be severe. Glucosuria can also occur after the renal tubular transporter system

for glucose becomes saturated. This happens when the glucose concentration of

plasma exceeds roughly 180 mg/dL in an individual with normal renal function

and urine output. As hepatic glucose overproduction continues, the plasma

glucose concentration reaches a plateau around 300 to 500 mg/dL (17–28

mmol/L). Provided renal output is maintained, glucose excretion will match the

overproduction, causing the plateau. The individual with type 1 diabetes has a

higher tendency to produce ketones. Patients with type 2 diabetes seldom

generate ketones but instead have a greater tendency to develop hyperosmolar

non-ketotic states. The difference in glucagon and insulin concentrations in these

two groups appears to be responsible for the generation of ketones through

increased -oxidation. In type 1, there is an absence of insulin with an excess of

glucagon. This permits gluconeogenesis and lipolysis to occur. In type 2, insulin

is present, as is (at times) hyper-insulinaemia; therefore, glucagon is attenuated.

Fatty acid oxidation is inhibited in type 2. This causes fatty acids to be

incorporated into triglycerides for release as very low density lipoproteins

(VLDL). The laboratory findings of a patient with diabetes with ketoacidosis tend

to reflect dehydration, electrolyte disturbances, and acidosis. Acetoacetate, -

31
hydroxybutyrate, and acetone are produced from the oxidation of fatty acids. The

two former ketone bodies contribute to the acidosis. Lactate, fatty acids, and

other organic acids can also contribute to a lesser degree. Bicarbonate and total

carbon dioxide are usually decreased due to Kussmaul-Kien respiration (deep

respirations). This is a compensatory mechanism to blow off carbon dioxide and

remove hydrogen ions in the process. The anion gap in this acidosis can exceed

16 mmol/L. Serum osmolality is high as a result of hyperglycemia; sodium

concentrations tend to be lower due in part to losses (polyuria) and in part to a

shift of water from cells because of the hyperglycemia. The sodium value should

not be falsely underestimated because of hypertriglyceridemia. Grossly elevated

triglycerides will displace plasma volume and give the appearance of decreased

electrolytes when flame photometry or prediluted, ion-specific electrodes are

used for sodium determinations. Hyperkalaemia is almost always present as a

result of the displacement of potassium from cells in acidosis. This is somewhat

misleading because the patient’s total body potassium is usually decreased. More

typical of the untreated patient with type 2 diabetes is the non-ketotic

hyperosmolar state. The individual presenting with this syndrome has an

overproduction of glucose; however, there appears to be an imbalance between

production and elimination in urine. Often, this state is precipitated by heart

disease, stroke, or pancreatitis. Glucose concentrations exceed 300 to 500 mg/dL

32
(17–28 mmol/L) and severe dehydration is present. The severe dehydration

contributes to the inability to excrete glucose in the urine. Mortality is high with

this condition. Ketones are not observed because the severe hyperosmolar state

inhibits the ability of glucagon to stimulate lipolysis. The laboratoryfindings of

non-ketotic hyperosmolar coma include plasma glucose values exceeding 1,000

mg/dL (55 mmol/L), normal or elevated plasma sodium and potassium, slightly

decreased bicarbonate, elevated blood urea nitrogen (BUN) and creatinine, and

an elevated osmolality (greater than 320 mOsm/dL). The gross elevation in

glucose and osmolality, the elevation in BUN, and the absence of ketones

distinguish this condition from diabetic ketoacidosis. Other forms of impaired

glucose metabolism that do not meet the criteria for diabetes mellitus include

impaired fasting glucose and impaired glucose tolerance. These forms are

discussed in the following section (Rohlfing et al., 2002).

2.5.3 Hypoglycaemia

Hypoglycaemia involves decreased plasma glucose levels and can have many

causes; some are transient and relatively insignificant, but others can be life

threatening. The plasma glucose concentration at which glucagon and other

glycemic factors are released is between 65 and 70 mg/dL (3.6–3.9 mmol/L); at

about 50 to 55 mg/dL (2.8–3.0 mmol/L), observable symptoms of

Hypoglycaemia appear. The warning signs and symptoms of Hypoglycaemia are

33
all related to the central nervous system. The release of epinephrine into the

systemic circulation and of norepinephrine at nerve endings of specific neurons

acts in unison with glucagon to increase plasma glucose. Glucagon is released

from the islet cells of the pancreas and inhibits insulin. Epinephrine is released

from the adrenal gland and increases glucose metabolism and inhibits insulin. In

addition, cortisol and growth hormone are released and increase glucose

metabolism. Historically, Hypoglycaemia was classified as post-absorptive

(fasting) and postprandial (reactive) Hypoglycaemia. However, the reactive

Hypoglycaemia only described the timing of Hypoglycaemia, not the etiology.

Current approaches suggest classification based on clinical characteristics. This

classification separates patients into those who appear healthy and those who are

sick. Among healthy-appearing patients are those with and without a

compensated coexistent disease. This category includes individuals in whom

medications may be the cause of Hypoglycaemia through accidental ingestion by

dispensing error. Sick persons may have an illness that predisposes to

Hypoglycaemia or may experience drug and illness interaction leading to

Hypoglycaemia. Hypoglycaemia in hospitalized patients can often be ascribed to

iatrogenic factors. Symptoms of Hypoglycaemia are increased hunger, sweating,

nausea and vomiting, dizziness, nervousness and shaking, blurring of speech and

sight, and mental confusion. Laboratory findings include decreased plasma

34
glucose levels during hypoglycemic episode and extremely elevated insulin levels

in patients with pancreatic -cell tumors (insulinoma). To investigate an

insulinoma, the patient is required to fast under controlled conditions. Men and

women have different metabolic patterns in prolonged fasts. The healthy male

will maintain plasma glucose of 55 to 60 mg/dL (3.1–3.3 mmol/L) for several

days. Healthy females will produce ketones more readily and permit plasma

glucose to decrease to 40 mg/dL (2.2 mmol/L) or lower. Diagnostic criteria for an

insulinoma include a change in glucose level of 25 mg/dL (1.4 mmol/L)

coincident with an insulin level of 6 U/mL (36 pmol/L), C-peptide levels of 0.2

nmol/L, proinsulin levels of 5 pmol/L, and/or -hydroxybutyrate levels of 2.7

mmol/L (Bishop et al., 2010).

2.6.0 UREA

Urea is eliminated via sweat and the gut, but most of the urea produced in the

liver is transported in blood to the kidneys where it is eliminated from the body in

urine. This process of renal elimination, which is detailed in a recent review

(Weiner, et al., 2015), begins with filtration of blood at the glomeruli of the

approximately 1 million nephrons contained within each kidney. During

glomerular filtration, urea passes from blood to the glomerular filtrate, the fluid

that is the precursor of urine. The concentration of urea in the filtrate as it is

formed is similar to that in plasma so the amount of urea entering the proximal

35
tube of the nephron from the glomerulus is determined by the glomerular

filtration rate (GFR). Urea is both reabsorbed and secreted (recycled back into the

filtrate) during passage of the filtrate through the rest of the tubule of the

nephron; the net effect of these two processes results in around 30-50 % of the

filtered urea appearing in urine. The facility of the kidney to adjust urea

reabsorption and secretion as the filtrate passes through the tubule determines an

important role for urea in the production of maximally concentrated urine, when

this becomes necessary. The mechanism of this water-conserving action of urea

within the nephron is well detailed (Weiner et al., 2015). Although often

considered simply a metabolic waste product, urea has two important

physiological functions outlined above: detoxification of ammonia and water

conservation.

2.6.1 Hyper-Uraemia

Serum/plasma urea concentration reflects the balance between urea production in

the liver and urea elimination by the kidneys, in urine; so increased plasma/serum

urea can be caused by increased urea production, decreased urea elimination, or a

combination of the two. By far the highest levels occur in the context of reduced

urinary elimination of urea due to advanced renal disease and associated marked

reduction in glomerular filtration rate (GFR). GFR is a parameter of prime

clinical significance because it defines kidney function. All those with reduced

36
kidney function, whatever its cause have reduced GFR and there is good

correlation between GFR and severity of kidney disease. The rate of decline in

GFR distinguishes chronic kidney disease (CKD) and acute kidney injury (AKI).

CKD is associated with irreversible slow decline in GFR over a period of many

months, years or even decades; whereas AKI is associated with precipitous

decline in GFR over a period of hours or days; AKI is potentially reversible. The

value of urea as a test of renal function depends on the observation that

serum/plasma urea concentration reflects GFR: as GFR declines, plasma/serum

urea rises. The limitation of urea as a test of renal function is that in some

circumstances plasma urea is not a sufficiently accurate reflection of GFR. For

example, urea is an insensitive indicator of reduced GFR; GFR must be reduced

by around 50 % before serum/plasma urea increases above the upper limit of the

reference range (Baum et al., 1975). Furthermore, urea may be raised despite a

normal GFR (i.e. normal renal function) so as a test of renal function, urea lacks

specificity (McWilliam and Macnab, 2009).

2.6.2 Hypo-Uremia

Reduced plasma/serum urea is less common (Lum and Leal-Khouri, 1989) and

usually of less clinical significance than increased plasma/serum urea. Since urea

concentration in plasma or serum reflects the balance between urea production

and urea elimination in urine, reduced plasma/serum urea can be caused by

37
decreased urea production, increased urinary urea excretion, or a combination of

the two. There are two physiological causes of reduced concentration: low-

protein diet, and pregnancy. Low-protein diet is associated with reduced urea

production and consequent tendency to reduced plasma/serum urea concentration.

The reduced plasma/serum urea that commonly occurs during pregnancy is due to

the combined effect of reduced urea production and increased urea excretion

(Kalhan, 2000). The increased urea excretion is consequent on increased GFR, a

well-documented physiological adaptation to pregnancy. Pathological cause of

reduced urea concentration is largely confined to advanced liver disease (Kalhan,

2000). This reflects the central role that the liver plays in urea production via the

urea cycle. Inherited deficiency of any one of the five enzymes of the urea cycle

describes a rare group of conditions (called the urea cycle defects) that can give

rise to reduced urea synthesis and consequent reduced plasma/serum urea

concentration. A recently published case history (Salek et al., 2010) exemplifies

this very rare cause of decreased plasma/serum urea. Over-hydration induces

increased GFR and consequent increased excretion of urea. For this reason over-

hydration, as might occur, for example, in the syndrome of inappropriate

antidiuretic hormone (SIADH), is often associated with decreased plasma/serum

urea.

38
2.7.0 CREATININE

Creatinine is formed from creatine and creatine phosphate in muscle and is

secreted into the plasma at a constant rate related to muscle mass (Rose, 2001).

Serum creatinine is the most commonly used indicator (but not direct measure) of

renal function. Elevated creatinine is not always representative of a true reduction

in GFR. A high reading may be due to increased production of creatinine not due

to decreased kidney function, to interference with the assay, or to decreased

tubular secretion of creatinine. An increase in serum creatinine can be due to

increased ingestion of cooked meat (which contains creatinine converted from

creatine by the heat from cooking) or excessive intake of protein and creatine

supplements, taken to enhance athletic performance. Intense exercise can increase

creatinine by increasing muscle breakdown. Dehydration secondary to an

inflammatory process with fever may cause a false increase in creatinine levels

not related to an actual kidney injury, as in some cases with cholecystitis. Several

medications and chromogens can interfere with the assay. Creatinine secretion by

the tubules can be blocked by some medications, again increasing measured

creatinine (Samra and Abcar, 2012).

2.7.1 Serum Creatinine

Measuring serum creatinine is a simple test, and it is the most commonly used

indicator of renal function (Taylor, 1989). A rise in blood creatinine level is a late

39
marker, observed only with marked damage to functioning nephrons. Therefore,

this test is unsuitable for detecting early-stage kidney disease. A better estimation

of kidney function is given by calculating the estimated glomerular filtration rate

(eGFR). eGFR can be accurately calculated without a 24-hour urine collection

using serum creatinine concentration and some or all of the following variables:

sex, age, weight, and race, as suggested by the American Diabetes Association

(Gross et al., 2005). Many laboratories will automatically calculate eGFR when a

creatinine test is requested. Algorithms to estimate GFR from creatinine level and

other parameters are discussed in the renal function article. A concern as of late

2010 relates to the adoption of a new analytical methodology, and a possible

impact this may have in clinical medicine. Most clinical laboratories now align

their creatinine measurements against a new standardized isotope dilution mass

spectrometry (IDMS) method to measure serum creatinine. IDMS appears to give

lower values than older methods when the serum creatinine values are relatively

low, for example 0.7 mg/dL. The IDMS method would result in a comparative

overestimation of the corresponding calculated GFR in some patients with

normal renal function. A few medicines are dosed even in normal renal function

on that derived GFR. The dose, unless further modified, could now be higher

than desired, potentially causing increased drug-related toxicity. To counter the

40
effect of changing to IDMS, new FDA guidelines have suggested limiting doses

to specified maxima with carboplatin, a chemotherapy drug (CDER, 2011).

41
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 STUDY AREA

This prospective cross-sectional study was carried out in the Department of

Chemical Pathology, Usmanu Danfodiyo University Teaching Hospital

(UDUTH), Sokoto, North Western Nigeria. Sokoto State metropolis is located in

the extreme North West of Nigeria, near to the confluence of Sokoto River and

Rima River. The state is located between longitude 11’ 30ᵒ, 13’ 50ᵒ east and

latitude 4’ to 6’ 0ᵒ north. It is bordered to the north by Niger Republic and

Zamfara state to the east while Kebbi State borders most of the south and western

parts with peak temperature attainable of about 43oC during certain periods of the

year (Mamman, 2000).

3.2 STUDY POPULATION

This research study was carried out on apparently healthy individuals residing

within Sokotometropolis, Sokoto state.

42
3.3 STUDY SUBJECTS

Fifteen (15) apparently healthy subjects were selected for this study comprising

both gender; 8 males and 7 females. Informed consent was sought prior to the

commencement of the study as per the work of Abdoljalal (2008).

3.3.1 Criteria for inclusion of subjects

 Apparently healthy individuals

 Adult of legal age (≥18years).

 Subjects that gave their informed consent

3.3.2 Criteria for Exclusion of Subject

 Subject with any known disease condition(s)

 Subject outside the range of the legal age

3.4 ETHICAL CONSIDERATION

The ethical approval for this research was obtained from the Ethics and Research

Committee of State Ministry of Health Sokoto.

3.4.1 Informed Consent

In line with the ethical provision of medical research procedure, informed

consent was sought from each individual who participated in this research work

prior to their recruitment.

43
3.5 SAMPLING TECHNIQUE

3.5.1 Subject Selection

Apparently healthy and consenting adults within the hospital community were

recruited for the study. The subjects that physically satisfy the inclusion criteria

were contacted through good manner of approach. Both the physical assessment

as well as the laboratory examination process was explained to the subjects in the

language they understand most and hence, their informed consent for

participation was sought.

3.5.2 Blood Sample Collection and Processing

From each selected subject, a total of 10 millilitres (10mls) of venous blood

specimen was collected using a sterile vacutainer blood specimen bottle (plain

and fluoride oxalate containing), holder and needle. The sample collected in plain

container was then allowed to clot at 25oC. Both the anticoagulated blood

samples as well as the non-anticoagulated blood samples were then immediately

centrifuged at 4000 rpm for duration of 10 minutes after which a clear and non-

haemolyzed plasma and serum were obtained, respectively. The plasma (from the

anti-coagulated blood) and serum was harvested into 6 discrete sterile cryovail

bottle for each subject’s sample (each 3 from the plain and anticoagulated sample

44
container). Two (2) discrete cryovails containing serum and Plasma (1 each from

the plain and the other from the anticoagulant containing bottle) of the aliquoted

sample serum was then analyzed at baseline 25oC while the other 2 parts; each of

plain and anti-coagulated samples was exposed to varying temperature before

analysis.

3.5.3 REAGENTS AND EQUIPMENT

All the reagent and equipment used were of Analytical grade.

3.6.0 LABORATORY METHODS

As most commonly routine analytes in clinical chemistry laboratory analysis,

biochemical parameters investigated were 7 in number. These include

Electrolytes (sodium, potassium, chloride and bicarbonate), Urea, Creatinine and

glucose (Random glucose).

3.6.1 Serum Electrolyte Estimation

The level of serum electrolytes (sodium, potassium, bicarbonate and chloride)

was determined by using the following method:

By the use of Ion Selective Electrode System (Automated Machine), the above

mentioned electrolytes were measured by method of Automation using ISE

(Pirkle, 2016).

45
3.6.1.1 Principle
An Ion-Selective Electrode (ISE) makes use of the unique properties of certain

membrane materials to develop an electrical potential (electromotive force, EMF)

for the measurements of ions in solution. The complete measurement system for a

particular ion includes the ISE, a reference electrode, and electronic circuits to

measure and process the EMF to give the test ion concentration.

3.6.1.2 Procedure;

The machine was firstly calibrated using the standard solutions recommended by

the manufacturer and then set for analysis of sample, about 250microlitre of the

sample was brought in close contact with the aspirating component of machine to

feed in the sample by pressing the button "measure" on the display screen after

feeding the machine with the sample via the aspirator, the sample container was

immediately withdrawn from the tip of the aspirating tube after few seconds, the

sample got analyzed and result was displayed on the machine's screen in mmol/L.

3.6.2 Urea Estimation

Serum urea was estimated using the method; Diacetylmonoxime-Fearon’s of the

version 2.0 (Wybenga et al., 1971).

46
3.6.2.1 Principle

Urea reacts with diacetylmonoxime at high temperature in an acidic medium in

the presence of cadmium ions and thiosemicarbazide. The absorbance of the

reddish color produced is measured in a colorimeter using a green filter of

wavelength 520 nm or a spectrophotometer at 530 nm wavelengths.

3.6.2.2 Procedure

Three clean, dry and detergent traces free test tubes for each one of the samples

to be analyzed were place in a test tube rack subsequent to labeling them as; test,

standard and blank.1 ml of distilled water was dispensed into each of the labeled

test tubes.1 ml of mixed acid reagent was added into the test tubes. Also, 1 ml of

mixed color reagent was also dispensed into the test tubes.10μl of sample was

added unto the test tube except to the one labeled “standard”. The content of each

test tube was mixed thoroughly and then incubated in boiling water bath for 15

minutes. Hence the absorbance was read at wavelength 520 nm using a

spectrophotometer.

3.6.2.3 Calculation

The level of serum urea was determined by the formular;

ODTEST
XCONC . OFSTD (8.3mmol/L).
ODSTD

47
3.6.3.0 Creatinine Estimation

Creatinine level was determined using the Jaffe-Slots’ method (Max Jaffe,

1886).

3.6.3.1 Principle

Creatinine reacts with picric acid in an alkaline medium. The absorbance of the

yellow-red color produced is measured in a colorimeter using a blue-green filter

at 490 nm or in a spectrophotometer at wavelength of 490 nm.

3.6.3.2 Procedure

The procedure is as follows:

Three clean, dry and detergent traces free test tubes were labeled as; test, standard

and blank for each sample to be analyzed and then placed in a test tube rack. 2.0

ml of distilled water was added to the tube labeled blank, and then 1.5 ml to that

labeled as test. 500 μl of the serum was dispensed into the tube labeled “test”. 0.5

2
ml of N H 2 SO 4was added to the test tube labeled “Blank” and “Test”. 0.5 ml
3

of 10% sodium tungstate was added to the tube labeled “Blank” and “Test”.

Mixing was then done separately and the centrifugation at 4000 rpm for 10

minutes. To the fresh empty test tubes labeled “blank” and “test”, 1.5 ml of

supernatant fluid was added. To the test tubes labeled; blank, standard and test,

48
0.5 ml of 0.75N NaOH was then dispensed. 0.5 ml of picric acid was added to all

the test tubes. The content of each tube was rocked gently to mix and then

incubated at room temperature for 15 minutes. At the wavelength of 520 nm (for

spectrophotometer), using reagent blank to zero the instrument, absorbance was

read and recorded for both the test and standard test tube content.

3.6.3.3 Calculation

OD TEST
Hence, creatinine concentration was determined in mg/dl by; X Conc.
OD STD

STD

3.6.4 Random Glucose Estimation

Glucose level was determined by using a laboratory method of Glucose Oxidase-

Peroxidase.

3.6.4.1 Principle

Glucose in serum sample reacts with the enzyme glucose oxidase to give rise to

gluconic acid and hydrogen peroxide. The hydrogen peroxide formed is then

acted upon by the enzyme peroxidase to form oxygen and water. The formed

oxygen then react with phenol and 4-aminophenazone (4-aminoantipyrine) to

form a pink color, the absorbance of which is read at 520 nm using a green filter

for colorimeter or at 500 nm in spectrophotometer.

49
3.6.4.2 Procedure

The level of serum glucose was estimated by the following procedure:

Three separate test tubes were firstly be labeled as; Test, Standard and blank for

each sample analyzed, which were then placed in a test tube rack. To all the test

tubes, 1 ml of glucose reagent were placed into each test tube.10 μl of the serum

sample was added unto the content of the tube labeled; “test” and “standard”.

Mixing was done and then incubation at 370C for 10 minutes. At a

spectrophotometric wavelength of 500 nm, the absorbance of the test sample was

read against that of reagent blank.

3.6.4.3 Calculation

Serum glucose concentration (in mmol/L) was then be calculated by using the

OD TEST
formula: XConc . Std .
OD STD

50
 CHAPTER FOUR
RESULT 4.0

Table 4.1Showed the effects of temperature on some biochemical analytes (mean ±

SEM). The result indicated significant (P≤0.05) decrease in concentration of Urea

between group B(350C)and C(450C) with significant (P≤0.05) increase in

concentration of Glucose between group A(250C) and B(350C) which then

significantly (p≤0.05) decrease in concentration from group B (350C) to group C

(450C). All the rest five analytes showed insignificant (p ≥0.05) change in

concentration at all temperature of analysis.

Table 4.2 Showed comparison of the effect of temperature and gender on some

biochemical analytes (mean ± SEM) of the subjects. The result obtained indicated

significant (p ≤ 0.05) increase in level of glucose in females at 35 0C with

corresponding significant (p ≤ 0.05) decrease in concentration of Na+ and K+ in

females than in males at 450C. All the other analytes showed insignificant (p ≥0.05)

change in concentrations between males and females subject at all the three

temperature of analysis.

51
GROUP N UREA Creat. Glu Na+ K+ Cl- HCO3-
(mmol/L) (mg/dL) (mmol/L) (mmol/L) (mmol/L) (mmol/L) (mmol/L)
A 15 4.0±0.28 0.9±0.07 4.0±0.06 135.9±1.21 3.8±0.07 104.3±1.05 21.5±1.08
B 15 4.7±0.26 1.1±0.09 4.2±0.05* 137.3±1.64 3.8±0.10 102.3±0.95 25.6±1.67
C 15 3.6±0.20* 1.1±0.08 3.7±0.08* 135.1±1.53 3.9±0.11 101.1±1.29 24.1±1.51
P Value 0.018 0.068 0.000 0.581 0.418 0.131 0.137
Post-hoc analysis, Bonferroni
Group A Vs B P= 0.196 P= 0.085 P= 0.041 P= 1.000 P= 1.000 P= 0.651 P= 0.150
Group A Vs C P= 0.898 P= 0.235 P= 0.029 P= 1.000 P= 0.955 P= 0.140 P= 0.634
Group B Vs C P= 0.016 P= 1.000 P= 0.000 P= 0.921 P= 0.642 P= 1.000 P= 1.000
Table 4.1 Effect of Temperature on Biochemical Analytes (mean + SEM) in
Serum/Plasma of apparently healthy adult individuals.

Values are mean ± SEM; Creat. = creatinine concentration, Glu = Glucose

concentration, Na+ = Sodium ion concentration, K+ = Potassium ion concentration, Cl-
= Chloride ion concentration, HCO3- = Bicarbonate ion concentration.

Key:
Group A = 25oC
Group B = 35o
Group C = 45oC
Vs = Versus.
P = p-values.
Level of statistical significance is considered at p ≤ 0.05

52
 Table 4.2 Comparison of the Effect of Temperature and Gender on some Biochemical
Analytes (mean ± SEM) of the Subjects.

GROUP G N Glu. Urea Creat. Na+ K+ Cl- HCO3-

(mmol/L) (mmol/L) (mg/dL) (mmol/L) (mmol/L) (mmol/L) (mmol/L)
A2 X 8 3.93±0.10 4.21±0.35 0.86±0.07 135.88±1.81 3.81±0.10 104.55±1.64 21.16±1.70
Y 7 4.06±0.80 3.77±0.45 0.86±0.14 135.84±1.71 3.70±0.10 104.00±1.38 21.83±1.49
P-value 0.25 0.27 0.06 0.81 0.54 0.68 0.78

B2 X 8 3.96±0.11 4.00±0.41 0.88±0.07 135.79±1.80 3.79±0.11 105.76±1.71 19.86±1.43

Y 7 4.01±0.06 4.01±0.40 0.84±0.41 135.94±1.76 3.73±0.09 102.61±0.88 23.31±1.43
P-value 0.01 0.65 0.06 0.73 0.24 0.30 0.96

C2 X 8 3.71±0.13 3.66±0.25 1.04±0.11 137.67±2.52 4.03±0.19 101.63±1.60 26.19±2.08

Y 7 3.74±0.12 3.61±0.34 1.10±0.12 132.14±0.69 3.74±0.08 100.47±2.20 21.63±1.96
P-value 0.75 0.32 0.95 0.04 0.01 0.91 0.43
Values are in Mean ± SEM; Creat. = creatinine concentration, Glu = Glucose concentration,
Na+ = Sodium ion concentration, K+ = Potassium ion concentration, Cl- = Chloride ion
concentration, HCO3- = Bicarbonate ion concentration.
Key:
A2 = 25OC

B2 = 35OC
C2 = 45OC
X = Male
Y = Female
G = Gender
N = Number of subjects.
Level of statistical significance is considered when p ≤ 0.05

53
 CHAPTER FIVE

5.0 DISCUSSION

The effect of temperature was examined on 7 biochemical analytes (urea, creatinine,

glucose, sodium, potassium, chloride and bicarbonate) in spot samples separated

human blood specimen. Mean Urea concentration decreased significantly (p ≤ 0.016)

when measured at 45oC. Glucose concentration (4.2 ± 0.05) was found to have

increased significantly (p ≤ 0.029) when analyzed after exposure of the sample to

35oC as compared to the mean concentration (4.0 ± 0.041) at 25 oC which then

decreased (3.7 ± 0.08) significantly (p ≤ 0.01) when analyzed at 45oC. This may be

due to enzymatic cleavage of precursor molecules and later partial denaturation of

enzymes. However, all other 5 parameters had variations in concentration which were

not significant (p ≥ 0.05) when analyzed at all the three different grades of

temperatures. Arshad Khan et al (2002) in their study on the effect of induced heat

stress (in vivo) on some biochemical values in broiler chicks found out that

parameters like urea, creatinine, glucose and sodium have increased in value between

groups (280C-350C and 400C-450C). This difference in findings could be due to mode

of heat inducement (in-vivo or in-vitro) or differences in the subjects used for the two

studies. Feiza et al (2012) in their study on the Effect of heat stress on some

biochemical values and humoral immunity in broiler chickens also reported a

significant increase in glucose concentration when comparison was made between

54
240C and 330C temperature grade. This is in close agreement with the findings of this

current study.

In respect to gender groups (male and female) as well as temperature, the mean

concentration of glucose in females at 35 oC increased significantly (p ≤ 0.05) as

compared to males at same temperature. This may be due to the fact that most females

undertake less of physical exercise. Mean Sodium ion and Potassium ion

concentrations were significantly (p ≤ 0.05) decreased in the female subjects relative

to male subjects when analyzed after exposing the sample to a temperature of 45 oC.

With the exception glucose, sodium ion and potassium ion, all the other four analytes

showed no significant (p ≤0.05) variation in mean concentrations between male and

female groups at all the three grades of temperature. Some analyte behaving nearly

stable could be due to the fact that all the subject were adults of same age group

(nether infant nor elderly) Musch et al (2006).

55
 CHAPTER SIX

6.0 CONCLUSION

This study has shown that there was a significant decrease in mean glucose and urea

levels at 450C with corresponding increase in mean glucose concentration at 35oC.

This goes to show that varying temperatures has effect on the integrity of some

biochemical parameters. It can also be deduced from the study that, the integrity of

the measured analytes is maintained best within the range of 350C. Temperature

relative to gender was also found to have significant (p ≤ 0.05) effect on some of the

measured analytes (glucose, sodium and potassium).

6.1 RECOMMENDATION

Blood samples for the analysis of biochemical parameters should not be exposed to

harsh atmospheric temperature as in the case of Sokoto where the temperature reaches

up to 43oC during certain period of the year prior to or during the cause of analysis.

This study was carried out on human blood samples with limited number of

parameters measured. It is therefore recommended that similar study should be

conducted to incorporate additional parameters beyond temperature and gender using

lager sample size.

56
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 Appendix

Materials Manufacturer

Test tubes Pyrex, France

Pasteur pipette DHT, UK

Automatic micropipette Mbi, Great Britain

Anticoagulatedplasma container Bectton Dickson, USA

Plain serum container Bectton Dickson, USA

Centrifuge machine Alc, Italy

Water bath Assistant, Germany

Spectrophotometer LabomedInc, USA

Ion Selective Electrode (ISE) RocheCobas, USA

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