Chapter 7

Carbohydrate

1
Objectives
Upon completion of this chapter the student will be able to:
1. Describe carbohydrates and their classification based on
different properties
2. Discuss about carbohydrate metabolism and regulation
3. Explain the clinical significance of glucose measurement,
specifically the role of measuring serum or plasma glucose in
diabetes screening and diagnosis
4. Describe the reactions of the glucose oxidase and hexokinase
methods, including the reactants, enzymes, products and
indicator (signal) measured
5. List the advantages, limitations and specimen requirements of
the glucose oxidase and hexokinase methods
6. Describe the utility and limitations of glucose measurement in
urine using the glucose oxidase; and of reducing sugars by the
copper-reduction method
2
Objectives…
Upon completion of this chapter the student will be able to:
7. Describe the clinical utility of quantitative cerebrospinal fluid
(CSF) glucose
8. Describe the laboratory definition of diabetes mellitus using
the following techniques: random glucose, fasting plasma
glucose specimens, and oral glucose tolerance test
9. Describe a generic procedure for a manual photometer used
for glucose oxidase and hexokinase assays
10. Determine the glucose concentration of patient samples and
quality control samples by calculating their absorbance from
a single calibrator or standard
11. List common causes of pre-analytical, analytical and post-
analytical error for glucose analysis
12. Describe typical quality assurance procedures required for
glucose analysis

3
Outline
• Introduction to carbohydrate

• Classification of carbohydrates

• Functions of carbohydrates

• Carbohydrate metabolism

• Hormonal regulation of glucose

• Disorders of carbohydrate metabolism

• Why glucose analysis is performed?

• Measurement of glucose
4
Introduction
• Organisms rely on the oxidation of complex organic
compounds to obtain energy
 carbohydrates, amino acids, and lipids

• carbohydrates are the primary source for brain, erythrocytes,

and retinal cells in humans.

• Carbohydrates are the major food source and energy supply of

the body and are stored primarily as liver and muscle glycogen.

5
 Intro…
• Carbohydrates are a class of organic compounds that includes
sugars, starches, cellulose…..

• Carbohydrates are the most abundant compounds found in nature

(cellulose: 100 billion tons annually)

• All carbohydrate molecules contain the elements carbon,

hydrogen, and oxygen

• The hydrogen to oxygen ratio in a carbohydrate molecule is the

same as in the water molecule—H2O (2:1)
Chemical composition of carbohydrates

• The empirical formula for a carbohydrate is:

Cx(H2O)y
The "x": the number of carbon atoms; "y“: the number of water molecules

• The "x" and the "y" will be the same number only for
monosaccharides
• All carbohydrates contain C = O and O - H functional groups
• Not all carbohydrates have this empirical formula: deoxysugars,
aminosugars
The functional groups of glucose are shown in the
figure below.

8
Chemical composition of carbohydrates…
• There are some derivatives from this basic formula because
carbohydrate derivatives can be formed by the addition of other
chemical groups, such as phosphates, sulfates, and amines. E.g.
alpha-D-Glucose-6-Phosphate, deoxysugars, aminosugars

Alpha-D-Glucose-6-Phosphate
9
•What are the functions of carbohydrates?

10
Function of carbohydrates
• Energy source (4 kilocalories per gram )
• Cell structure (plants-cellulose & animals- chitin)
• Recognition markers
 e.g. A,B,O blood types
• Structural component of nucleic acids
• Part of plasma membrane
• Intermediates in the biosynthesis of other basic biochemical
entities (fats and proteins)
• Participate in biological transport, cell-cell recognition,
activation of growth factors, modulation of the immune system

11
Classification of carbohydrates
• The classification of carbohydrates is based on four different
properties:
1. The size of the base carbon chain
2. The location of the CO function group
3. The number of sugar units and
4. The stereochemistry of the compound

12
Classification of carbohydrates…

Carbohydrates can be grouped into generic classifications

based on the number of carbons in the molecule

For example, trioses contain three carbons, tetroses contain

four, pentoses contain five, and hexoses contain six

In actual practice, the smallest carbohydrate is

glyceraldehyde, a three-carbon compound

13
Classification of carbohydrates…

Carbohydrates are hydrates of aldehyde or ketone derivatives

based on the location of the CO functional group

The two forms of carbohydrates are aldose and ketose

The aldose form has a terminal carbonyl group (O= CH-)

called an aldehyde group

whereas the ketose form has a carbonyl group (O=C) in the

middle linked to two other carbon atoms called a ketone
group
14
Classification of carbohydrates…

15
Classification of carbohydrates…
• Another classification of carbohydrates is based on number of
sugar units in the chain:
Monosaccharides
Disaccharides
Oligosaccharides
Polysaccharides

• This chaining of sugars relies on the formation of glycoside

bonds that are bridges of oxygen atoms
• When two carbohydrate molecules join, a water molecule is
produced
• When they split, one molecule of water is used to form the
individual compounds
16
Classification of carbohydrates…

17
Monosaccharides

• Monosaccharides are simple sugars that cannot be hydrolyzed

to a simpler form

• These sugars can contain three, four, five, and six or more
carbon atoms (known as trioses, tetroses, pentoses, and
hexoses, respectively)

• The most common include glucose, fructose, and galactose

18
Classification of monosaccharides
• Monosaccharides are classified according to:

1. The number of carbon atoms present in the molecule

2. Whether they contain an aldehyde or keto group

C Ketone Aldehyde Examples

4 Tetrose Tetrulose Erythrose,Threose
5 Pentose Pentulose Ribose, Ribulose
6 Hexose Hexulose Fructose, Glucose
7 Heptose Heptulose Sedoheptose, Sedoheptulose
Monosaccharides

• Two of the hexoses, glucose and galactose, are aldose sugars,

whereas fructose is a ketose

• Has two functional groups

1. hydroxyl (- OH)

2. Carbonyl

20
Aldose Sugars

H H H H H

C O C O C O C O C O

(H C OH)n H C OH H C OH H C OH H C OH

CH2OH CH2OH H C OH H C OH H C OH

Aldose Aldotriose CH2OH H C OH H C OH

n=1
Aldotetrose CH2OH
n=2 H C OH
Aldopentose
CH2OH
n=3
Aldohexose
n=4

21
 Ketose Sugars

CH2OH CH2OH
CH2OH
CH2OH
C O C O CH2OH
C O
C O
(H C OH)n H C OH C O
H C OH
CH2OH
CH2OH H C OH H C OH
CH2OH
CH2OH H OH
Ketose Ketotriose Ketotetrose
n=1 Ketopentose H C OH
n=0
n=2
CH2OH
Ketohexose
n=3

22
Structural Representation of Sugars

 Fisher projection: straight chain representation

 Haworth projection: simple ring in perspective

 Conformational representation: chair and boat configurations

23
Fischer Projections
• According to the conventions proposed by Fischer
D-monosaccharide: when written as a Fischer projection,
has the -OH on its one before the last carbon on the right
L-monosaccharide: when written as a Fischer projection,
has the -OH on its one before the last carbon on the left
Haworth projection

• Is a common way of writing a structural formula to represent the

cyclic structure of monosaccharides with a simple three-
dimensional perspective

25
• D-glucose can cyclize in two ways
forming either furanose or
pyranose structures
26
• D-ribose and other five-carbon
saccharides can form either furanose
or pyranose structures

27
Disaccharides
• Disaccharides are formed when two monosaccharide units are
joined by a glycosidic linkage

28
Disaccharides…

• On hydrolysis, disaccharides will be split into two

monosaccharides by disaccharide enzymes (e.g., lactase)
located on the microvilli of the intestine

• These monosaccharides are then actively absorbed

• The most common disaccharides are maltose, lactose, and

sucrose

29
Disaccharides…
• Maltose (malt-sugar)= glucose + glucose (blood sugar)

• Lactose (milk sugar)= 1 glucose + 1 galactose

• Sucrose (table sugar)= 1 glucose + 1 fructose (fruit sugar)

30
Maltose

• Composed of two D-glucose molecules

• Obtained from the hydrolysis of starch

• Linked by an -1,4-glycosidic bond formed from the −OH on

C1 of the first glucose and −OH on C4 of the second glucose

• Used in cereals, candies, and brewing

• Found in both the - and β - forms

31
Maltose, Continued

• The glycosidic bond is α(1→4)

32
Lactose
• Is a disaccharide of β-D-galactose and α- or β-D-glucose

• Contains a β -1,4-glycosidic bond

• Is found in milk and milk products

• Used in infant formulations, medium for penicillin production

and as a diluent in pharmaceuticals

33
Lactose…

34
Milk Lactose and Baby Food

 Lactose is the most suitable sugar as a milk sweetener for baby

feeding because;
i) it has the lowest degree of sweetness that delays loss of the
baby appetite;
ii) it is non-fermentable and so no colic-causing gases are
produced by the large intestine bacteria;
iii) it is a mild laxative and helps preventing constipation;
iv) it is not an irritant to the stomach and helps preventing
vomiting;
v) the unabsorbed sugar is used as a fuel for large intestinal
bacteria that produces some vitamins; and,
vi) it facilitates absorption of milk minerals

35
Sucrose
Sucrose or table sugar
 Is obtained from sugar cane and sugar beets

 Consists of α-D-glucose and β-D-fructose

 Has an α,β-1,2-glycosidic bond

 Used pharmaceutically to make syrups, troches

α-D-glucose

β-D-fructose
36
Sucrose is not the sweetest sugar
• Although used as sweetener in most of the food preparations,
sucrose is not the sweetest of them all
• Fructose is almost twice as sweet as sucrose
• With sucrose as a reference (with 100 degree sweetness), the
degree of sweetness of some sugars is as follows: 173 for
fructose; 74 for glucose; 32 for maltose and galactose and 16
for lactose.
• Diabetes mellitus patients and people on weight reduction
protocols avoid sucrose as sweetener
• Most of the artificial sweeteners commonly known as ‘Sugar
Free’ contain aspartame, which is a dipeptide L-aspartyl-L-
phenylalanine methyl ester
• Aspartame is also added to the beverages marketed as ‘low
caloric’ or ‘Diet drinks’. 37
Polysaccharides
• They are polymers of monosaccharides

• They contain more than ten monosaccharide units

• The monosaccharides are joined together by glycosidic linkage

• The two classes of polysaccharides are

Homo-polysaccharides

Hetero-polysaccharides

• Important homopolysaccharides are starch, glycogen, cellulose,

dextran, inulin and chitin
All these contain glucose as repeating unit
38
Example of a Homopolysaccharide:
Cellulose
• It is composed of multiple D-glucose residues (only),
bonded to each other by β-(1⟶4) glycosidic bonds

39
Polysaccharides…
• An example is the cellulose molecule which contains about
10,000 glucose units per molecule

• Humans cannot digest cellulose, since an appropriate enzyme is

not secreted by the intestinal tract

• The polysaccharide of prime interest is starch, which contains

350 to 1300 glucose units per molecule

40
Polysaccharides…
• Starch
Most common storage polysaccharide in plants
Composed of 10 – 30% α-amylose and 70-90% amylopectin
depending on the source
The chains are of varying length, having molecular weights
from several thousands to half a million
Provides 80% of dietary calories in humans worldwide

41
 Polysaccharides…
• Amylose is composed of multiple D-glucose residues
(only), bonded to each other by α-(1⟶4) glycosidic bonds.
• It is long straight glucose chains

42
• Amylopectin is composed of multiple D-glucose residues (only), bonded
to each other by α-(1⟶4) glycosidic bonds (as in amylose) with other
chains of D-glucose that branch from carbon number 6.
Branching usually occurs every 24 to 30 glucose residues

43
Polysaccharides….
• Glycogen
Also known as animal starch and stored in muscle and liver
Present in cells as granules (high MW)
Contains both α(1,4) links and α(1,6) branches at every 8 to 12
glucose unit i.e. more branched than starch
identical to amylopectin, the only difference is that it branches
more frequently.
Complete hydrolysis yields glucose
Glycogen and iodine gives a red-violet color
Hydrolyzed by both α and β-amylases and by glycogen
phosphorylase

44
Structural polysaccharides

Cellulose
 Polymers of glucose
 Most abundant of all carbohydrates
 Major component of plant cell walls
 Very few organisms produce cellulose, enzyme that
hydrolyze cellulose
 Gives no color with iodine

45
Structural polysaccharides…
Chitin
• Made of glucose with a nitrogen containing group
• Major component of arthropod exoskeleton & fungal cell walls
• Chitin is used commercially in coatings (extends the shelf life of fruits
and meats)

46
Example of a Heteropolysaccharide: Hyaluronic acid

• Hyaluronic acid contains D-glucuronic acid and N-acetyl-D-

glucosamine residues, connected to each other in the bonding
pattern shown below.

47
Chemical Properties of Carbohydrates
• Some carbohydrates are reducing substances; these
carbohydrates can reduce other compounds

• To be a reducing substance, the carbohydrate must contain a

ketone or an aldehyde group

• This property was used in many laboratory methods in the past

in the determination of carbohydrates

• Examples of reducing substances include glucose, galactose,

fructose, lactose, and maltose
48
Chemical Properties of Carbohydrates…
• All monosaccharides and many disaccharides are reducing
agents

• This is because a free aldehyde or ketone (the open chain form)

can be oxidized under the proper conditions

• Disaccharide remains a reducing agent when the ketal hydroxyl

group is not linked to another molecule

• Both maltose and lactose are reducing agents, whereas sucrose

is not

49
Digestion of carbohydrates
 About sixty percent of the human diet consists of carbohydrates

 Most of the carbohydrates in the food stuffs are complexed with

proteins, lipids or even nucleic acids

 The separation and then breakdown of these food stuffs into

simpler assimilable forms constitutes the process of digestion

50
 Digestion of carbohydrates…
The dietary carbohydrates can be divided into three groups

A) Ready-to-absorb carbohydrates: which do not require

digestion and are absorbed as such, e.g., monosaccharides:
glucose, mannose, galactose, fructose and pentoses

B) Digestible carbohydrates: These include starch, glycogen,

maltose, sucrose, and lactose (oligosaccharides and
polysaccharides)

• They are completely digested into their respective monosaccharides

51
 Digestion of carbohydrates

C) Non-digestible carbohydrates:

 The indigestiblility of these dietary fibers is primarily due to the

absence of specific digestive enzymes

 Most of these indigestible carbohydrates are plant polysaccharides

like cellulose, hemicellulose, lignin, gums and pectins

52
 Carbohydrate Metabolism
• Amylase
• Lactase
• Sucrase
• Maltase
Carbohydrate Metabolism
• Polysaccharides (starch and glycogen) are hydrolyzed (digested)
to the disaccharide maltose by the enzyme amylase

• The body has two sources of amylase, the salivary glands and
the pancreas

• Salivary amylase is of minor importance in the hydrolysis of

these two polysaccharides, since this enzyme has its greatest
activity around pH 7

54
Carbohydrate Metabolism…
• The pH of the stomach is between 1 and 4; consequently,
salivary amylase activity is markedly inhibited when the food
reaches the stomach

• The pH of the duodenum is approximately 7; hence, pancreatic

amylase is very active

55
Carbohydrate Metabolism…
• The three most important disaccharides are maltose, from starch
and glycogen hydrolysis; sucrose, or table sugar (cane sugar);
and lactose, or milk sugar

• The hydrolysis of these disaccharides takes place in the

duodenum, which contains the enzymes needed for the
hydrolysis of these sugars

• These enzymes are sucrase, maltase, and lactase

56
Carbohydrate Metabolism…

• They are secreted from the mucosal lining of the duodenum

• The enzyme sucrase hydrolyzes sucrose to the monosaccharides

glucose and fructose

• maltase hydrolyzes maltose to two molecules of glucose; and

• lactase hydrolyzes lactose to galactose and glucose

57
Carbohydrate Metabolism…
• The monosaccharides glucose, fructose, and galactose, which
are produced by the hydrolysis of the disaccharides, are all
absorbed in the duodenum

• The absorption of these monosaccharides depends on the

following conditions:
Amylase must be present to hydrolyze the polysaccharides, so there must be
normal pancreatic function;

The mucosal lining of the duodenum must possess the enzymes sucrase, lactase,
and maltase to hydrolyze the disaccharides;

The intestinal cells must be functioning normally for absorption of the

monosaccharides
58
Carbohydrate Metabolism…
Fate of the Absorbed Hexoses

• After the three monosaccharides (glucose, fructose, and

galactose) are absorbed from the duodenum, they are carried by
the portal circulation to the liver

• Fructose and galactose are phosphorylated by the liver enzymes

and either converted to glucose or follow similar metabolic
pathways

59
Fates of Glucose

 Fed state Synthesis and breakdown occur

at all times regardless of state...
 Storage as glycogen

 Liver The relative rates of synthesis

 Skeletal muscle and breakdown change
 Storage as lipids

 Adipose tissue

 Fasted state
 Metabolized for energy

 New glucose synthesized

60
 High Blood Glucose

Pancreas

Insulin:
Muscle Liver

Glucose absorbed Glucose absorbed

Adipose
Cells

Glucose absorbed

Immediately after eating a meal…

61
Fate of Absorbed Glucose

• 1st Priority: glycogen storage

Stored in muscle and liver

• 2nd Priority: provide energy

Oxidized to ATP

• 3rd Priority: stored as fat

Only excess glucose
Stored as triglycerides in adipose

62
Glucose Utilization

Energy
Adipose Stores Glycogen

Glucose
Pentose Glycolysis
Phosphate
Pathway

Ribose-5-phosphate Pyruvate
63
64
 Glycogenesis

• Liver
• 7–10% of wet weight
• Use glycogen to export glucose to the bloodstream when
blood sugar is low
• Glycogen stores are depleted after approximately 24hrs
of fasting (in humans)
• De novo synthesis of glucose from glycogen

65
Glycogenesis
• Skeletal muscle
• 1% of wet weight
• More muscle than liver, therefore more glycogen in
muscle, overall
• Use glycogen (i.e., glucose) for energy only (no export
of glucose to blood)
• Use already-made glucose for synthesis of glycogen

66
67
• Glucose 1-phosphate produced by glycogen phosphorylase is
converted to glucose 6-phosphate by phosphoglucomutase,
which catalyzes the reversible reaction

Glycolysis Pentose phosphate pathway

68
 Glycolysis

Glucose + 2 ADP + 2 Pi
2 Lactate + 2 ATP + 2 H2O

69
Carbohydrate Metabolism…

70
• Assignment 7: Mechanism of glucose absorption

71
Hormonal control of blood glucose levels

72
Hormonal regulation
1. Hormone that decrease plasma glucose
 Insulin

2. Hormones that increase blood glucose

 Glucagon

 Epinephrine/adrenalin
 Growth hormone & ACTH(adrenocorticotropic hormone)
 Glucocortico steroids
 Thyroid hormone
 Somatostatin

73
Insulin
• Insulin is a hormone produced in the beta-cells of the pancreatic
islets of Langerhans

• The amount of insulin secreted by the pancreas is controlled by

the level of blood glucose

• Insulin functions in the following manner:

It increases the entry of glucose into cells

It increases the rate of glycolysis and glycogenesis

Reduces the level of blood glucose

74
 Insulin……..
• Overall insulin promotes storage of glucose as a fuel and a
reduction of blood glucose when elevated

75
 Glucagon
• Peptide hormone that is synthesized by the alpha cells of the islets
cells of the pancreas and released during stress and fasting states

• Released in response to decreased body glucose

• Main function is to increase hepatic glycogenolysis, inhibit

glycolysis and increase gluconeogenesis

• Hyperglycemic agent
Major effects of glucagon:
• Stimulates breakdown of glycogen stored
in the liver

• Activates hepatic gluconeogenesis (using

amino acids and other non-carbohydrate
precursors)

• Overall the effects of glucagon are to

increase blood glucose when it is low

77
Pathways regulated by the release of:
 Glucagon (in response to a lowering of blood glucose levels)
 Insulin (in response to an elevation of blood glucose levels)

78
Epinephrine
• Hormone produced by the adrenal gland

• Increases plasma glucose by inhibiting insulin secretion,

increasing glycogenolysis and promotes lipolysis

• Release during times of stress

Glucocorticoids

• Cortisol is released when stimulated by ACTH

• Cortisol increases plasma glucose by decreasing entry into the

cells and increasing gluconeogenesis, liver glycogenolysis and
lipolysis

• Insulin antagonist
Thyroxin

• The thyroid gland is stimulated by TSH to release thyroxin

• Its effect on glucose metabolism and control is minimal

• Increases glucose levels by increasing glycogenolysis,

glucogenesis and intestinal absorption of glucose
Growth hormone

• Its principal actions include blocking glucose uptake into cells

and stimulating lipolysis

• Its action is antagonizes insulin action

Somatostatin

• Produced by the delta cells of the islets of langerhans of the

pancreas

• Increases plasma glucose levels by the inhibition of insulin

Carbohydrate metabolism disorders
• Hyperglycemia

• Hypoglycemia

• Galactosemia

• Glycogen storage disorders

• Fructosuria

84
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

• Hyperglycemia, or increased plasma glucose levels, is caused

by an imbalance of hormone( insulin)

• The most important cause of inappropriate hyperglycemia seen

in the clinical laboratory is diabetes mellitus

85
Diabetes Mellitus (DM)
• DM is a group of metabolic disorders characterized by
hyperglycemia

• The hyperglycemia results from defects in insulin secretion,

insulin action or both

• The chronic hyperglycemia of diabetes is associated with

specific chronic complications resulting in damage to or failure
of various organs, notably the eyes, kidneys, nerves, heart and
blood vessels

86
Classification of Diabetes mellitus
• The ADA/World Health Organization (WHO) guidelines
recommend the following categories of diabetes:

1. Type 1 diabetes

2. Type 2 diabetes

3. Gestational diabetes mellitus (GDM)

4. Other specific types of diabetes

87
 Type I or IDDM
• Type 1 diabetes is characterized by inappropriate hyperglycemia
primarily a result of pancreatic islet β-cell destruction and a
tendency to ketoacidosis

• It is a result of cellular-mediated autoimmune destruction of the β-

cells of the pancreas, causing an absolute deficiency of insulin
secretion

• Upper limit of 110 mg/dL on the fasting plasma glucose is

designated as the upper limit of normal blood glucose

88
Type I or IDDM…

• Type 1 constitutes only 10% to 20% of all cases of diabetes and

commonly occurs in childhood and adolescence

• Characteristics of type 1 diabetes include abrupt onset, insulin

dependence, and ketosis tendency

89
Lab findings Type 1 DM
• Ketoacidosis

• Bicarbonate and total carbon dioxide are decreased due to deep

respiration- body trying to compensate for acidosis by blowing
off CO2 and removing H ions

• Anion gap greater than 16 mmol/L

• Serum osmoality is increased

• Sodium decreased due to polyuria and shift in water from cells

• Hyperkalemia is almost always present due to displacement of

potassium in cells that occurs in acidosis
•Assignment 8 : Diabetic Ketoacidosis

91
Type 2 diabetes (NIDDM)

• Type 2 diabetes is characterized by hyperglycemia as a result of an

individual’s resistance to insulin with an insulin secretory defect

• This resistance results in a relative, not an absolute, insulin

deficiency

• It constitutes the majority of the diabetes cases

• Most patients in this type are obese or have an increased

percentage of body fat distribution in the abdominal region

92
Type 2 diabetes…
• Characteristics usually include adult onset of the disease and
milder symptoms than in type 1, with ketoacidosis seldom
occurring

• However, these patients are more likely to go into a

hyperosmolar coma and are at an increased risk of developing
macro vascular and micro vascular complications

93
Type 2 diabetes…
• Insulin resistance associated with
• Obesity
• Physical inactivity
• Malnutrition in early life, pregnancy, drugs
• Hyperglycaemia – ‘glucose toxicity’
• Having Cushing’s disease or polycystic ovary disease
• Taking high doses of steroids over an extended period of time
• Having chronic stress
• Having a high-calorie DIET, high-carbohydrate or high-sugar diet

94
Lab findings with Type 2
• Over production of glucose: > 300-500 mg/dl

• Dehydration due to the inability to excrete glucose in urine

• No ketones bodies formed because of the lack of lipolysis

• Can lead to coma if glucose levels reach > 1000 mg/dl, in

addition to elevated sodium and potassium, slight decrease in
bicarbonate and increase in BUN: Creatinine ratio, increased
osmolality
Type 1 and Type 2 Diabetes Mellitus

• Type 1 and type 2 diabetes mellitus differ in their clinical

presentation as well as their etiology

• Type 1 diabetics are usually younger and thinner than type 2

diabetics

• Type 1 diabetics present with acute symptoms, while type 2

diabetes develops more slowly over time

• Type 1 diabetics are more prone to develop ketoacidosis than

type 2 diabetics

96
Type 1 and Type 2 Diabetes Mellitus

• Type 1 and Type 2 diabetes:

• There is an increase in blood glucose levels (hyperglycemic)

• There is also elevation of glucose in urine (glucosuria) if

glucose levels in blood exceed 180 mg/dl
Gestational diabetes mellitus (GDM)
• GDM is any degree of glucose intolerance with onset or first
recognition during pregnancy

• Causes of GDM include metabolic and hormonal changes

• Patients with GDM frequently return to normal post-partum

• However, this disease is associated with increased perinatal

complications and an increased risk for development of diabetes
in later years

98
Other specific types of diabetes
• Other specific types of diabetes are associated with certain
conditions (secondary), including:
Genetic defects of β-cell function or insulin action
Pancreatic disease
Diseases of endocrine origin
Drug- or chemical-induced insulin receptor abnormalities
Certain genetic syndromes

• The characteristics and prognosis of this form of diabetes

depend on the primary disorder
• Maturity-onset diabetes of youth (MODY) is a rare form of
diabetes that is inherited in an autosomal dominant fashion

99
Diagnosis of Diabetes mellitus
 Depends on the demonstration of hyperglycemia

Diagnostic criteria

1. History

2. Classic symptoms of diabetes

3. Demonstration of significant hyperglycemia

100
Criteria for the diagnosis diabetes mellitus

101
Complications of Diabetes mellitus
 Stroke

 Peripheral arterial disease

 Coronary disease

 Ketoacidosis

 Neuropathy

 Retinopathy

 Nephropathy

 Infection

102
Organs affected in Diabetes Complications

103
Hypoglycemia
• Hypoglycemia is characterized by blood glucose levels that are
less than normal

• Exact definition of the glucose level in hypoglycemia is under

debate; symptoms of hypoglycemia usually occur when blood
glucose has fallen below 50 mg/dL

• Two types of hypoglycemia occur, reactive (postprandial, or

after meals) and fasting (post-absorptive)

104
Hypoglycemia
• Hypoglycemia that is caused by a stimulus such as excessive
insulin administration, ethanol ingestion, or over-regulation of
diabetes is termed reactive hypoglycemia

• Fasting hypoglycemia may occur

As a response to insulin-producing tumors of the pancreas (insulinomas)
or other tissues
Hepatic dysfunction
Glucocorticoid deficiency
Sepsis or
Low glycogen stores

105
Insulinoma
• An insulinoma is a tumor of the pancreas that is derived from
beta cells and secretes insulin

• The secretion of insulin by insulinomas is not properly regulated

by glucose and the tumors will continue to secrete insulin
causing glucose levels to fall further than normal

• Tumor that secretes insulin- no counter measure use for

treatment

• Extremely elevated insulin levels with decreased glucose levels

 Genetic Defects

• Glycogen storage defect is due to deficiency of specific enzyme that

cause an alternation of glycogen metabolism

• Most common form is glucose-6-phosphatase deficiency type 1 –

von Gierke disease

• Hypoglycemic state is due to the inability of glycogen to be

converted back to glucose by hepatic glycogenolysis
 Galactosemia
• It is a rare genetic metabolic disorder that affects an individual's
ability to metabolize the sugar galactose properly

• Defect in enzyme needed to metabolize galactose- results in an

increase in galactose in plasma

Enzyme that is most commonly deficient : galatose-1phosphate uridyl transferase

• Due to inhibition of glycogenolysis accompanied by diarrhea and

vomiting

• Must remove galactose from diet, if not will build up in the system
cause retardation and cataracts
 Fructosuria
• Fructosuria caused by a deficiency of hepatic fructokinase is a
clinically benign condition characterized by the incomplete
metabolism of fructose in the liver, leading to its excretion in
urine
• Essential fructosuria
• Hereditary fructose intolerance

• Hereditary fructose intolerance, or the presence of fructose in the

blood (fructosemia), is caused by a deficiency of aldolase B, the
second enzyme involved in the metabolism of fructose
Lactose intolerance
• Lactose intolerance, also called lactase deficiency and
hypolactasia, is the inability to digest lactose, a sugar found in
milk (and hence some dairy products)

• Lactose intolerant individuals have insufficient levels of lactase,

the enzyme that metabolizes lactose into glucose and galactose,
in their digestive system

• In most cases, such individuals will experience symptoms such

as abdominal bloating and cramps, flatulence, diarrhea, nausea,
borborygmi (rumbling stomach) and/or vomiting
Diabetic Profile Tests:
1. Blood Glucose
 FBG (= FBS= PG=FPG)….Fasting blood glucose

 PP glucose = Post prandial glucose

 GGT= Glucose tolerance test

2. C-peptide (Differentiates between type I and type II)

3. HbA1c = Glycosylated hemoglobin

4. Ketones

5. Insulin

6. ICA = islet cell antibodies

111
Samples for Glucose Assays
• Serum and plasma are the most common specimens for
quantitative glucose analysis

• Glucose is also measured in CSF and urine

• Whole blood capillary glucose limited to patient administered

(self) or point-of-care glucose monitoring

• Not acceptable for diagnosis of DM

Blood Collection for Plasma and Serum
• Serum (red top or serum separator tube)

• Plasma (heparin, citrate, EDTA)

• Fluoride-oxalate (grey-top) will inhibit

glycolysis

• Serum/ plasma should be separated from

cells after centrifugation

• Glucose is reduced 5-7% per hour in

uncentrifuged/ unseparated sample
Blood Collection for Plasma and Serum…

• Specimen collection time options for blood glucose

measurements
– Random blood sugar (RBS)
– FBS (fasting blood sugar )
– Two- hours post prandial (2h.pp)
Measuring Glucose in Plasma/ Serum
• Glucose can be measured by 3 types of methods:
• Oxidation-reduction [Redox ]
• Condensation
• Enzymatic
 Oxidation-reduction [Redox ] method
1. Alkaline ferric cyanide method

Glucose + ferric cyanide alkaline ferrous cyanide + oxidized glucose

(yellow) 1000C (colorless)
 Decrease in yellow color is directly proportional to glucose
concentration & can be read at 420 nm
Oxidation-reduction [Redox ] method
2. Copper reduction method

Glucose + Cu2+ Alkaline Cu+ + oxidized glucose

100oc
Cu+ + arsenomolybidic acid Cu2+ + reduced arsenomolybidate
• Intensity of blue color is directly proportional to glucose
concentration

• Absorbance read at 680 nm

Sources of Errors
Draw backs

1. Lacks specificity – due to reducing ability of uric acids & others

2. Protein, Requires protein precipitation- like 20% Trichloroacetic acid
(TCA) or tungstic acid

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 Condensation methods
• Glucose can undergo condensation with a variety of aromatic
compounds in hot acid solution to yield colored products
• Condensation with aromatic amines:
O-toluidine
P-amino-benzoic acid
M-P-aminosalicylic acid
aminophenol
O-aminodiphenyl

• Condensation with o-toluidine is the most widely used aromatic

amine method
• The O-toluidine method is more sensitive than all non-
enzymatic glucose assay methods
 O-toluidine method
{in hot acetic acid solution}

Glucose + O-toluidine  Green colored glycosyl amine

• Increase in Absorbance is measured

Additional Reagents:

• TCA PFF is used to increase specificity

• Thio-urea is added to stabilize reagent

The O-toluidine method is the most specific of the non-enzymatic

glucose assay methods
Sources of Error
• Other hexoses can react; e.g. galactose

• Bilirubin, lipemia interfere

• Protein

TCA [trichloroacetic acid PFF is used to remove interference due to

proteins]

• Reagent is toxic and carcinogenic

Enzymatic Methods
• Common enzymatic methods for glucose measurement in serum
or plasma:
Glucose oxidase

Glucose hexokinase
Glucose dehydrogenase

• These methods will not measure other mono- or disaccharides,

i.e., fructose, galactose, sucrose or lactose
Glucose oxidase method

D-Glucose + O2 Glucose Oxidase Gluconic Acid + 2H2O2

H2O2 + 0 – Dianisidine Peroxidase 2 H2O + Oxidized 0-Dianisidine

 Absorbance is measured at 546 nm

• Intensity of the color is proportional to the amount of glucose

• Dyes used in various systems:

 O-dianisidine, 4-aminoantipyrine, or tetramethylbenzidine

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Hexokinase Method

Glucose + ATP Hexokinase G - 6 - P + ADP

G - 6 – P + NADP+ G6- P DH 6 – phosphogluconate +NADPH+

• The amount NADPH+ produced is directly proportional to

glucose amount

• Absorbance measured at 340nm

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 Glucose Dehydrogenase method
Glucose + NAD+ GDH D- glucono-δ-lactone +NADH + H+
• The increase in absorbance at 340 nm is measured
• The enzyme glucose dehydrogenase [GDH] catalyzes the oxidation
of glucose to gluconolactone
• The amount of NADH generated is proportional to the glucose
concentration
• Mutarotase is added to shorten the time necessary to reach the end
point
Sources of Error
• Hemolysis

• Lipemic

• Time of collection

• Time of serum separation

• Inappropriate anticoagulant use

Glucose tolerance test (GTT)
• GTT is a test used to diagnose mild or hidden cases of
diabetes

• Non-preferred method of diabetes diagnosis

• GTT can be

– Oral glucose tolerance

– Intravenous glucose tolerance test

• A solution containing 75 g of glucose is administered, and a

specimen for plasma glucose measurement is drawn 2 hours
later
Oral glucose tolerance test (OGTT)
• Measures the body's ability to use glucose

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Factors affecting CHO tolerances

• Quantity of glucose administered

• Rate of absorption
 low-in malabsorption
 High-in Hyperthyroidism

• Age
 Elderly people have decreased CHO tolerance
C-peptide test
• Concentration of C-peptide reflects the production of
endogenous insulin by the pancreas

• Insulin is stored in the pancreas as the biologically inactive

protein proinsulin

• Proinsulin is cleaved into the active hormone, insulin, and an

inactive peptide, C-peptide
C-peptide…..

• Therefore, the concentration of C-peptide represents the

concentration of the endogenous insulin that is produced in the
pancreas

• It is better indicator of B-cell function than peripheral insulin

• A C-peptide test can be done when diabetes has just been found
and it is not clear whether type 1 diabetes or type 2 diabetes is
present

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 C-peptide…..

• A person whose pancreas does not make any insulin (type 1

diabetes) has a low level of insulin and C-peptide

• A person with type 2 diabetes has a normal or high level of

C-peptide

• Normal value: Fasting 0.51-2.72ng/ml

• However, the difficulty in measuring this hormone

accurately diminishes its usefulness for diagnosis

132
C-peptide…..
• A C-peptide test can also help find the cause of low blood
sugar(hypoglycemia)
excessive use of medicine to treat diabetes
a noncancerous growth (tumor) in the pancreas
(insulinoma)

133
Glycosylated Hemoglobin/Hemoglobin A1c

• Glycosylated hemoglobin is the term used to describe the

formation of a hemoglobin compound produced when glucose
(a reducing sugar) reacts with the amino group of hemoglobin (a
protein)

• The glucose molecule attaches non-enzymatically to the

hemoglobin molecule to form a ketoamine
Glycosylated hemoglobin (HbA1c)

• The rate of formation is directly proportional to the plasma

glucose concentrations

• Because the average red blood cell lives approximately 120

days, the glycosylated hemoglobin level at any one time
reflects the average blood glucose level over the previous 2
to 3 months

135
Glycosylated hemoglobin (HbA1c)

• A glycohemoglobin test indicates how well diabetes has been

controlled in the 2 to 3 months before the test

• The HbA1C level is directly related to complications from

diabetes

(The lower the A1C level, the lower the risk for
complications)

Glycemic status is categorized as poor glycemic control if

HbA1c ≥7% (ADA, 2007)

136
Glycosylated Hemoglobin/Hemoglobin A1c…
• The specimen requirement for HbA1c measurement is an EDTA whole
blood sample

• The methods of measurement are grouped into two major categories:

1. Based on charge differences between glycosylated and non-

glycosylated hemoglobin
– Cation-exchange chromatography
– Electrophoresis
– Isoelectric focusing

2. Structural characteristics of glycogroups on hemoglobin

– Affinity chromatography
– Immunoassay
Urine Glucose Testing
• Urine reagent strips (“dipsticks”) use
the glucose oxidase method

• Chromogens vary by manufacturer

• Semi-quantitative and susceptible to

interference

• Glucose in urine occurs when serum

level is >9.7 mmol/L(180 mg/dl
therefore not a reliable screening test
Copper Reduction Test for Reducing Sugars
Benedict’s test
• Detects reducing sugars and other
reducing substances

• Screen urine from infants for

galactosemia and other congenital
carbohydrate disorders

• Examples of reducing substances:

ascorbic acid, uric acid, mono and Bayer “Clinitest” color chart

disaccharides (i.e., glucose, galactose,

fructose), not sucrose
Principle of the copper reduction test
• Reducing substances lower the valence of an ion by lending
electrons (e.g., Cu3+ + e- → Cu2+)

• In the reaction - an alkaline solution w/ cupric ions (Cu3+) is

blue; when a reducing substance is present - cuprous ions Cu2+
are formed and the solution changes to green, yellow, or orange,
depending upon amount of reducing substance

• Reagent is caustic (contains NaOH) and generates significant

heat

• Follow manufacturer directions carefully!

Cerebrospinal Fluid (CSF) Glucose
• Glucose enters the CSF by selective transport across the blood-
brain barrier, which results in a normal value that is
approximately 60% to 70% that of the plasma glucose.

• For an accurate evaluation of CSF glucose, a blood glucose test

must be run for comparison

• CSF glucose is analyzed using the same procedures employed

for blood glucose

(Glucose oxidase method )

Cerebrospinal Fluid (CSF) Glucose…

• The diagnostic significance of CSF glucose is confined to the

findings of values that are decreased in relation to plasma values

• Low CSF glucose values can be of considerable diagnostic value

in determining the causative agents in meningitis

• Elevated CSF glucose values are always a result of plasma

elevations
Cerebrospinal Fluid (CSF) Glucose…

• A high CSF glucose concentration has no specific diagnostic

importance and is related to an elevated blood glucose
concentration, for example, in diabetics

• Glucose oxidase method used to measure glucose in CSF

• Bacteria and other cells may contaminate CSF, so it should be

analyzed immediately or centrifuged and stored at 4°C

• Specimens should be tested immediately because glycolysis

occurs rapidly in the CSF.
 Self-monitoring test
• Glucose Oxidase
Typically used in POCT/self-monitoring and dedicated
instrumentation.
Glucose Oxidase
Glucose + 2H2O + O2  gluconic acid +2H2O2

Peroxidase
Chromogenic oxygen receptor +H2O2  Coloured oxidised product + H2O

• Glucose oxidase is specific for ß D-glucose, so mutarotase is

required to convert all to ß form.
Interpretation of Results
Reference range

Fasting blood glucose

 Serum---------------------------70-110 mg/dl

 Whole blood------------------- 65 -95 mg/dl

 CSF----------------------------- 40-70 mg/dl

2-hour postprandial: less than 140 mg/dl

Random: less than 126 mg/dl

145
Cont’……

Criteria for diagnosis of DM are met when any of the following

results have been repeated at least two different days

FBS is 126 mg/dl or higher

2hr oral glucose tolerance test result is 200 mg/dl or higher

Symptoms of diabetes are present and a random blood

glucose test is 200 mg/dl or higher

146
Cont’…..
• If the patient FBS is between 110-126 mg/dl consider to have
pre-diabetes (impaired fasting glucose)

• FBS level below 40 mg/dl in women or below 50 mg/dl in

men: Hypoglycemia:
 Insulinoma may be caused by tumor

147
Documentation of Glucose Results
• Record patient results in result logbook
• Record QC results in QC logbook
• Retain records for recommended time
Quality Control

• A normal & abnormal quality control sample should be

analyzed along with patient samples, using Westgard or other
quality control rules for acceptance or rejection of the analytical
run.

• Assayed known samples

• Commercially manufactured
Next Chapter: Chapter 8
Renal Function Tests

150
Reference
1. Burtis, Carl A., and Ashwood, Edward R.. Tietz: Fundamentals of
Clinical Chemistry. Philadelphia, 2001.

2. Arneson, W and J Brickell: Clinical Chemistry: A Laboratory

Perspective 1st ed. 2007 FA Davis

3. Burtis, Carl A., and Ashwood, Edward R.. Tietz: textbook of Clinical
Chemistry. Philadelphia, 1999.

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