CLINICAL CHEMISTRY III

3 MODULE 1
CARBOHYDRATE METABOLISM & DISORDERS

INTRODUCTION
1. Energy required by organisms are obtained from:

o Carbohydrates , Amino acids & Lipids

 All three are used for energy BUT Carbohydrates are the main energy source for the
brain, red cells and retinal cells in humans.

2. Carbohydrates are composed of the elements: C, H and O.

3. Classification of Carbohydrates = based on 4 different properties:

o Size of base Carbon Chain

o Location of CO functional group
o Number of sugar units
o Stereochemistry of the group
 Carbohydrates 2

4. Classification based on the number of sugar units in total chain:

o Monosaccharides = single sugar unit
o Disaccharides = two sugar units
o Oligosaccharides = 2 to 10 sugar units
o Polysaccharides = more than 10 sugar units

5. Monosaccharide:

Glucose

 Disaccharide:

6. This is called a condensation or dehydration reaction.

 Carbohydrates 3

7. Oligosaccharide:

 Polysaccharide:
 Carbohydrates 4

CARBOHYDRATE METABOLISM
& FUEL HOMEOSTASIS

• Glucose is the primary source of energy in humans.

WHY IS IT SO CRITICAL TO MAINTAIN [glucose] IN THE ECF?

• The nervous system & brain
• are totally dependent on glucose coming directly from the ECF as source of
=> energy.
• Nervous tissue does not concentrate or store glucose.
• Thus we need a steady supply to these tissues as needed
• [Glucose] must therefore be maintained within a narrow range.

FATE OF GLUCOSE
• Ingested polymers of carbohydrates are non-absorbable, eg.
=> starch
=> glycogen
• These polymers are digested by enzymes
=> salivary amylase
=> pancreatic amylase
• into => dextrins + disaccharides
• which are further hydrolyzed  monosaccharides (eg. sucrose and lactose)
• maltose  glucose + glucose
=> by the enzyme maltase (intestinal mucosa GIT)
• sucrose  glucose + fructose
=> by sucrase (GIT)
• lactose  glucose + galactose
=> by lactase (GIT)
 Carbohydrates 5

GLUCOSE METABOLISM
• Monosaccharides are absorbed in the gut / stomach, then
• transported to the liver
• via the hepatic portal venous blood.
• Glucose is the only carbohydrate that can be
=> directly utilized as energy or can be
=> stored as glycogen.
• Galactose & fructose are converted  glucose before it can be utilized.

• Glucose the enters the cell and is

• quickly shunted into a metabolic pathway (1) OR (2) OR (3),
• depending on the
=> availability of substrates, and the
=> nutritional status of the cell.
• The ultimate goal of the cell is to convert:
glucose  CO2 + H2O + ATP (high energy)

(ie. to oxidize glucose to obtain ENERGY in the form of ATP)

• The 1st step of all oxidizing pathways is the same:

glucose + phosphate  Glucose-6-phosphate
=> this step uses ATP
=> and is catalyzed by the enzyme hexokinase
• Then glucose-6-phosphate enters either the:
(1) Glycolysis / Embden-Meyerhof pathway Generate energy
(2) Hexose monophosphate pathway (glucose breakdown)

The opposite action can also take place, called glycogenesis:

where glucose is converted  glycogen Storage of glucose
Carbohydrates 6
Carbohydrates 7
 Carbohydrates 8

REGULATION OF GLUCOSE METABOLISM

• Blood glucose levels are kept within narrow range by the:
=> liver
=> pancreas
=> other endocrine glands.

• During a brief fast:

=> glucose is moved from the liver to the ECF
=> through glycogenolysis.

• When fasting lasts for >1 day:

=> glucose is synthesised from other sources (not coming from carbohydrates)
=> through gluconeogenesis.

WHICH HORMONES REGULATE THE GLUCOSE METABOLISM?

(1) Insulin
Two major hormones
(2) Glucagon
(3) Epinephrine
(4) Cortisol
(5) Growth hormone
(6) Somatostatin
(7) Thyroxine

(1) INSULIN
• peptide hormone
• released by the ß cells of the islets of Langerhans in the pancreas
• is the primary hormone responsible for facilitating the entry of glucose into cells,
promotes glycolysis, glycogenesis, and lipid & protein formation.
• thus there is an  insulin production & release after a meal as  [glucose] in the ECF
• The overall result is a reduction in blood glucose.
 Carbohydrates 9

• Insulin is synthesized & stored in vesicles in the ß cells in the pancreas until needed
• Insulin consists of a short leader peptide sequence attached to the rest of the
molecule, which is called
• pre-proinsulin, until the leader peptide is cleaved off (by a protease enzyme);
• After cleavage of the leader peptide, the molecule is known as
=> proinsulin, which is a peptide (82 amino acids long);
• This structure is further cleaved by specific proteases
• and another 31 amino acid part is removed in the middle, known as the
=> C peptide.
• The dipeptide that remains:
=>  chain (21 amino acids)
=> ß chain (30 amino acids)
• These 2 chains are linked via S-S (disulfide) bonds between Cysteine residues.

• Insulin is released in response to an  [glucose] ECF.

• The release is biphasic:
(1) Initially a rapid small  in insulin;
(2) Then a prolonged release of insulin.
• The release of insulin is terminated when  [glucose] ECF until normal levels.

Please see attachment 1: "INSULIN SECRETION BY THE

PANCREATIC BETA CELLS"
 Carbohydrates 10

• GLUT4 = major transporter used for uptake of glucose

• GLUT4 glucose transporters are present in cytoplasmic vessels when [insulin] is
low.
MECHANISM OF GLUCOSE UPTAKE:
• Insulin binds to a receptor (R) on the surface of a cell where the
insulin-R complex initiates a chain of events (signal transduction).
• This leads rapidly to fusion of the cytoplasmic vessels (containing GLUT4) with
the plasma membrane and insertion of glucose transporters.
• This action results in an  number of glucose transporters to facilitate the
passage of glucose  cell);
• therefore an upregulation ( in glycogenesis.
• Glucose transporters are recycled back into cytoplasm when insulin ↓ and insulin
receptors not needed any longer
• Neurons, erythrocytes and retinal epithelium
obtain glucose without any endocrine control (directly).

(2) GLUCAGON
• Glucagon is a peptide hormone (H),
• synthesized by the  cells of the Islets of Langerhans in pancreas.
• This H indirectly 'antagonizes' insulin action.
 Carbohydrates 11

• So between glucagon & insulin these 2 hormones are responsible to maintain the
glucose range (by a 'push-and-pull' action).
• Glucagon is secreted if there is a  [glucose] in the ECF.
 Glucagon increases plasma glucose levels by (1)  hepatic glycogenolysis
(2)  gluconeogenesis
(3) inhibit glycolysis

(3) EPINEPHRINE
• secreted by the adrenal medulla, especially under physical or emotional stress
• stimulates glycogenolysis resulting in an increase in blood [glucose].

(4) CORTISOL
• secreted by the adrenal cortex
• helps to raise blood glucose levels by stimulating gluconeogenesis

(5) GROWTH HORMONE

• secreted by the anterior pituitary
• increases the blood glucose concentration by its antagonistic action to insulin.

(6) SOMATOSTATIN
• secreted by the delta cells of the Islets of Langerhans in the pancreas.
• increases plasma glucose levels by inhibiting insulin, glucagon and growth hormone
(7) THYROXINE
• secreted by the thyroid gland
• increase plasma glucose levels by increasing glycogenolysis, gluconeogenesis, and
intestinal absorption of glucose.
 Carbohydrates 12

ALTERATIONS IN CARBOHYDRATE METABOLISM

Hyperglycaemia
 Hyperglycaemia = An increase in plasma glucose levels.
 When hyperglycaemia is seen in a healthy patient, insulin is secreted by the -cells
of the pancreatic islets of Langerhans.
 Insulin increases membrane permeability of glucose in cells of the liver, muscle and
adipose tissue.
 Hyperglycaemia is caused by an imbalance of hormones.

DIABETES MELLITUS
• Diabetes mellitus is a group of metabolic diseases characterized by
hyperglycaemia resulting from defects in insulin secretion, insulin action, or both.
• The group of metabolic diseases has different aetiologies & clinical features.
• The common factor of all is
=> there is a relative / absolute deficiency insulin, because of a
(1) lack of insulin OR
(2) tissues have become insensitive to insulin.

• The serious medical complications of diabetes:

=> eyes
=> kidneys
=> nervous system
=> blood vessels

• CLASSIFICATION OF DIABETES MELLITUS (ADA/WHO)

1. Type 1 diabetes mellitus
2. Type 2 diabetes mellitus
3. Other specific types of diabetes mellitus
4. Gestational Diabetes Mellitus (GDM)
 Carbohydrates 13

Ketone bodies

 During starvation, and uncontrolled diabetes mellitus:

 glycogen reserves are rapidly depleted and
 the body begins to metabolize reserves of fat and protein.

 Triglycerides are liberated from adipose tissue  [free fatty acids (FFA’s)] in
blood

 FFA’s  fatty acyl carnitine (within the liver cells), and this molecule is
converted to acetyl CoA, which in turn reach the mitochondria.

 The entry of acetyl CoA into the citric acid cycle depends on:
 availability of oxaloacetic acid for the formation of citric acid.

 In starvation or uncontrolled diabetes situations:

 oxaloacetic acid is not available for use with acetyl CoA (it is used instead
to synthesize glucose).
 acetyl CoA now produced more rapidly than it is consumed

 as [acetyl CoA] , its molecules have increasing opportunities to react with

each other.

 In three steps, two acetyl CoA react to form acetoacetic acid.

 The acetoacetic acid may be changed into either acetone or -hydroxybutyric

acid.

 Acetoacetic acid and -hydroxybutyric acid leave liver and enter the general
circulation.

 Both are normally present in blood in low concentrations, can be used by body.

 in starvation or uncontrolled diabetes situations both can be used by the heart,

kidneys, and brain for metabolism to produce energy.

 Ketosis = overall accumulation of ketone bodies in blood and urine.

 Carbohydrates 14

Acetyl CoA Acetyl CoA

Acetoacetic acid

-hydroxybutyric acid acetone

Formation of ketone bodies

 Carbohydrates 15

1. Type 1 Diabetes mellitus

• Approximately 5% to 10% of all cases of diabetes mellitus.

• Type 1 diabetes mellitus is a result of cellular-mediated autoimmune destruction of
the -cells of the pancreas, causing an absolute deficiency of insulin secretion.
• Occurs in childhood and adolescence
• Usually initiated by an environmental factor ( a chemical) or infection (usually a virus)
in individuals with a genetic pre-disposition and causes the immune destruction of the
β-cells of the pancreas and, therefore a decreased production of insulin.
• Abrupt onset, insulin dependence and a ketosis tendency characterize Type diabetes
mellitus.
• This type of diabetes is genetically related.
• One or more of the following autoantibody markers of immune destruction are
present in 85% to 90% of individuals with immune-mediated diabetes when fasting
hyperglycaemia is initially detected:
 Islet cell autoantibodies
 Insulin autoantibodies
 Glutamic acid decarboxylase autoantibodies (GAD 65) and
 autoantibodies to the tyrosine phosphatases IA-2 and IA-2B.

 Signs and symptoms:

=> polydipsia
=> polyphagia
=> polyuria
=> rapid weight loss
=> hyperventilation
=> mental confusion
=> possible loss of consciousness ( due to  glucose to the brain)
=> ketonaemia
=> ketonuria may be present
* These symptoms usually occur abruptly
 Carbohydrates 16

 Complications
=> nephropathy
=> neuropathy
=> retinopathy
=> increased heart disease may also be found

 Idiopathic Type 1 diabetes mellitus

• A form of Type 1 diabetes mellitus with no known aetiology, is strongly
inherited and does not have β-cell autoimmunity.

TREATMENT?
• Episodic requirements for insulin replacement.

2.Type 2 Diabetes mellitus

• Comprises approximately 90% of all cases of diabetes

• Includes individuals who have insulin resistance and usually have relative (rather
than absolute) insulin deficiency.

• Most patients are obese or have an increased % of body fat distribution in the
abdominal region.

• This diabetes is associated with a strong genetic predisposition.

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

• Patients are more likely to go into a hyperosmolar coma and are at an increased risk
of developing macrovascular and microvascular complications.

CAUSES?
• Genetic factors play a larger role here than in Type 1 diabetes mellitus.
but the genetic marker(s) have not been discovered yet
 Carbohydrates 17

• The aetiology is still unknown

• Environmental factors:
=> Diet (obesity is common in individuals having Type 2 diabetes mellitus)
=> Changes in the normal concentrations of [substrate] delivered to liver,
adipose tissue, skeletal muscle may affect normal function insulin-receptor.

TREATMENT?
• Restriction of a person’s caloric intake & weight loss ALONE may control Type 2
diabetes mellitus.
• Drugs, such as sulfonylureas / other oral hypoglycaemic drugs.
=> These drugs help to release insulin from the ß cells;
=> or it may  the sensitivity of the target tissues to insulin;
=> insulin administration in very few cases.

3. Other specific types of diabetes

• This subclass includes uncommon patients in whom hyperglycaemia is due to a

specific underlying disorder eg:
1. genetic defects of β-cell function
2. genetic defects in insulin action
3. disease of the exocrine pancreas
4. endocrinopathies eg Cushing’s Disease and
5. infections

4. Gestational Diabetes Mellitus (GDM)

• GDM is “any degree of glucose intolerance with onset of first recognition during
pregnancy.
• Causes include metabolic and hormonal changes.
• Patients with GDM frequently return to normal postpartum.
• However GDM is associated with increased perinatal complications and an
 Carbohydrates 18

increased risk for development of diabetes in later life.

PREGNANCY & INFANTS OF DIABETIC MOTHERS

• There is an  insulin resistance in the mother as a pregnancy progresses
=> considered normal.
WHY?
• In order to  substrates delivered to fetus.
• Mother's lipolysis pathway is upregulated
=> ketosis may occur if only 1 meal is missed.
• Because of an of substrates to the fetus
• The fetus receives more glucose, lipids, amino acids
• This triggers the fetus to produce its own ß cells (hyperplastic ß cells)
• Thus the fetus overproduces insulin
• This in turn leads to an  in fetal growth, and these big babies are known as
=> macrosomic infants.
• The infant has:
=>  weight & length
=>  internal organs, except for the brain
=>  adipose tissue
• It is important to monitor the infant for hyperglycaemia
• Complications may be
=> hyperglycaemia
=> respiratory distress syndrome
=> hypocalcaemia
=> polycythaemia
=> hyperbilirubinaemia
 Carbohydrates 19

PATHOPHYSIOLOGY OF DIABETES MELLITUS

• Both Type 1 and Type 2 diabetes mellitus are characterised by:

=> hyperglycaemia (which may be severe), and
=> glucosuria (because the renal tubular transporter system for glucose becomes
saturated)
• Glucosuria happens when the plasma [glucose] >9.9 mmol/L
• BUT when the hepatic glucose overproduction continues
• Plasma [glucose] reaches a plateau between 17 – 28 mmol/L,
• and the excess is excreted via urine, provided that the renal output is maintained.

• Type 1 diabetes mellitus is more likely to produce ketones

=> diabetic ketoacidosis
• Type 2 diabetes mellitus seldom produces ketones BUT is more likely to develop a
=> nonketotic hyperosmolar state.

WHY ARE KETONES PRODUCED IN Type 1 diabetes mellitus ?

• There is a difference in the ratios of [insulin] & [glucagon] between Type 2
diabetes mellitus and Type 1 diabetes mellitus
• In Type 1 diabetes mellitus :
=> insulin is absent
=> there is an excess glucagon
=> THUS gluconeogenesis & lipolysis is stimulated
=> therefore ketones are produced => ketonaemia & ketonuria
• In Type 2 diabetes mellitus:
=> insulin is present (sometimes even hyperinsulinaemia)
=> thus glucagon is suppressed & fatty acid oxidation is inhibited
=> THUS there is hypertriglyceridaemia
(where excess fatty acids are incorporated into triglycerides as VLDL)
 Carbohydrates 20

LABORATORY FINDINGS
• Diabetic ketoacidosis
=> dehydration
=> electrolyte disturbances
=> acidosis
Production of ketone bodies
(1) acetoacetate
(2) acetone
(3) ß-hydroxybutyrate
-
=> HCO3 & PCO2  because of Kussmaul breathing (deep respiration)

=> anion gap may be increased >16 mmol/l

=>  serum osmolality (because of the hyperglycaemia)
+
=>  Na (polyuria & water shift from cells because  glucose)
+
=> hyperkalaemia (displacement of K from cells in acidosis)

• Diabetic nonketotic hyperosmolar state

FEATURES
=> more likely caused by Type 2 diabetes mellitus
=> overproduction of glucose
(imbalance between the production & elimination via urine)
=> often precipitated by heart disease / stroke / pancreatitis
=> [glucose] usually in excess of >17 – 28 mmol/L
=> severe dehydration (inability to excrete glucose in urine)
=> high mortality
=> no ketones are produced
(because the hyperosmolar state inhibits glucagon to stimulate lipolysis)
 Carbohydrates 21

LABORATORY FINDINGS
=> *plasma [glucose] raises extremely high >55 mmol/L
+ +
=> N /  plasma Na & K
-
=> slightly  HCO3

=> * BUN & creatinine

=> * osmolality
=> *ketones absent
(* These features clearly distinguishes this conditions from diabetic ketoacidosis).

Impaired Glucose Tolerance (IGT)

• IGT is diagnosed in people who have fasting blood [glucose] less than that those
required for a diagnosis of diabetes mellitus , but have a plasma glucose response
during the Oral Glucose Tolerance Test (OGTT) between normal and diabetic states.
• An OGTT is needed to assign a patient to this class.
• Development of overt diabetes occurs at a rate of 1% - 5% per year, but
• A large proportion spontaneously revert to normal glucose tolerance.
• Microvascular disease in this group is quite rare
• Increased prevalence of atherosclerosis and mortality from CVD.

Impaired Fasting Glucose (IFG)

• Diagnosed by a fasting glucose value between that of normal and diabetic

individuals.
• It is a metabolic stage between normal glucose homeostasis and diabetes.
• Adipose tissue also play important roles in glucose usage
• Individuals are at an increased risk for the development of diabetes and CVD.
 Carbohydrates 22

DISEASES & CONDITIONS THAT CAUSE HYPOGLYCAEMIA

• Hypoglycaemia has many causes and could either be:
• transient & insignificant, OR
• life-threatening.
• Glucagon & other glycaemic factors are released when blood glucose is:
3.6 – 3.9 mmol/L
• Observable symptoms of hypoglycaemia present when blood glucose is:
2.8 - 3.0 mmol/L
• Warning signs and symptoms all related to CNS.
• Epinephrine & norepinephrine & glucagon are all released in an attempt to
=>  blood glucose

HYPOGLYCAEMIA
? Diagnosis
Whipple’s Triad
Three conditions :
 Symptoms of hypoglycaemia
 Low blood glucose <2.2 mmol/L
 Immediate reversal of symptoms on glucose administration.

SYMPTOMS
• anxiety / anxiousness
•  heart rate
• dizzy
• cold
• sweaty
• tremors (hands)

• There would also be a sustained release of epinephrine.

• If the condition is not corrected, there would be further symptoms involving loss of
 Carbohydrates 23

neuronal function:
=> slurred speech
=> loss motor coordination
=> unconsciousness
=> coma, death...

CLASSIFICATION OF HYPOGLYCAEMIA
(1) Induced by exogenous agent

(2) Occur spontaneously during fasting

DRUGS
• Insulin
• Missed meals in diabetics on insulin, undertake strenuous exercise, overdose
inadvertently.
• Deliberate self-administration of insulin.
• Alcohol - Hypoglycaemia observed after excessive alcohol consumption without food
intake.

INSULINOMA
• Pancreatic β-cell tumour secreting insulin independently.
• Generally benign.
• Diagnosis: Inappropriately high insulin levels (>10mU/L) in the presence of
hypoglycaemia (<2.2 mmol/L).

NON-PANCREATIC NEOPLASMS
• Hypoglycaemic attacks may occur in patients with malignant hepatomas.
• Tumours secrete insulin-like growth factor 2 (IGF-2), which, at high concentrations,
cross-reacts with the insulin receptor.
 Carbohydrates 24

HYPOGLYCAEMIA : Induced
ENDOCRINE DISEASE
• A deficiency in any of the hormones that antagonize insulin can cause fasting
hypoglycaemia.

HYPOGLYCAEMIA IN CHILDHOOD
• May be due to any of the above causes (eg. insulinoma).
• However, certain types of hypoglycaemia are specific to the younger age group.
• Neonatal hypoglycaemia: Transient hypoglycaemia may occur in otherwise healthy
neonates. Blood glucose is maintained In utero by placental glucose transfer from
the mother, but, after delivery, depends on hepatic glycogen reserves until feeding
commences.

Genetic defects in carbohydrate metabolism

(1) Glycogen storage diseases

• a result of the deficiency of a specific enzyme that causes an alteration of glycogen
metabolism
• Glucose-6-phosphatase deficiency type 1(von Gierke disease) - most common
• Disease characterized by severe hypoglycaemia that coincides with metabolic
acidosis, ketonemia and  lactate and alanine.

(2) Galactosaemia
• a congenital deficiency of one of three enzymes involved in galactose metabolism,
leading to an  galactose in plasma.
• Galactose-1-phosphate uridyl transferase = most common enzyme deficiency
• Galactosaemia occurs because of inhibition of glycogenolysis and is accompanied
by diarrhoea and vomiting.
 Carbohydrates 25

LAB DIAGNOSIS & MANAGEMENT OF PATIENTS WITH GLUCOSE METABOLIC

ALTERATIONS

GLUCOSE MEASUREMENT

SPECIMENS
• plasma
• serum
• whole blood (11% lower than serum / plasma)

• serum / plasma separated from cells

• refrigerated within 1 hr
• sodium fluoride
=> inhibits glycolytic enzymes Grey top vacutainer tube
• potassium oxalate
=> anticoagulant

METHODS
• mostly enzymatic methods
(1) glucose oxidase (Trinder reaction)

The enzyme glucose oxidase catalyzes the oxidation of glucose to gluconic

acid and hydrogen peroxide.
Glucose Oxidase
Glucose + H2O + O2 Gluconic acid + H2O2
The hydrogen peroxide formed reacts, under catalysis of peroxidase, with
phenol and 4-aminoantipyrine, to form a red-violet quinoneimine dye as
indicator, the intensity of the colour which is directly proportional to the
concentration of glucose in the sample @ 500nm.
Peroxidase
2H2O2 + phenol + 4-aminoantipyrine Red quinone + 4H2O
 Carbohydrates 26

(2) hexokinase
Glucose is phosphorylated by ATP in the presence of hexokinase
and Mg++. The glucose-6-phosphate formed is oxidized by glucose-
6-phosphate dehydrogenase (G6PD) to phosphogluconate in the
presence of nicotinamide-adenine dinucleotide phosphate
(NADP+). The amount of NADPH produced is directly proportional
to the amount of glucose in the sample and is measured by
absorbance at 340nm.
Hexokinase, Mg++
Glucose + ATP Glucose-6-phosphate + ADP
G-6-PD
Glucose-6-phosphate + NADP+ 6-Phosphogluconate + NADPH + H+

NB!! The hexokinase method is considered more accurate than the glucose oxidase
methods because the coupling reaction using glucose-6-phosphate dehydrogenase is
highly specific; therefore, it has less interference than the coupled glucose oxidase
procedure. Please see attachment 2: "SEMDSA 2017 Recommendations"
 Carbohydrates 27

CRITERIA FOR THE DIAGNOSIS OF DIABETES MELLITUS

CATEGORIES OF FASTING PLASMA GLUCOSE

• Normal fasting glucose FPG  5.6 mmol/L
• Impaired fasting glucose FPG  6.0
5.6 mmol/L and 7.0 mmol/L
- 6.9
• Provisional diabetes diagnosis FPG  7.0 mmol/L

ORAL GLUCOSE TOLERANCE TEST

• a person must have had a normal diet of 150 g carbohydrates per day for the
preceding 3 days before the test
• the person the fasts at least 10h overnight, not more than 16h
• the next morning a fasting blood sample is collected
• the person then consumes a glucose load containing the equivalent of 75g glucose
anhydrous dissolved in 250ml water over 5 minutes (for children only 1.75 g
glucose/kg body weight)

• a blood sample for glucose analysis is collected after 2 hrs.

 Carbohydrates 28

CATEGORIES OF ORAL GLUCOSE TOLERANCE

• Normal glucose tolerance 2-hour PG  7.8 mmol/L
• Impaired glucose tolerance 2-hour PG  7.8 mmol/L and- 11.0
11.1 mmol/L
• Provisional diabetes diagnosis 2-hour PG  11.1 mmol/L

URINARY GLUCOSE
• This test is usually used as a screening test for glucose
• specific test strips / dipsticks are used

TEST PRINCIPLE
• These test strips / dipsticks contain:
(1) glucose oxidase +
(2) peroxidase +
(3) chromogen
INTERFERENCES
• reducing agents interfere with the oxidation of the chromogen
thus can cause false negative results
eg. reducing agents:
=> uric acid
=> ascorbic acid
=> salicylates

URINARY KETONES
TM TM
• Acetest / Ketostix tablets or strips
TEST PRINCIPLE
• contains nitroprusside
• that will detect one of the following ketone groups:
=> acetone
=> acetoacetic acid
 Carbohydrates 29

• ß-hydroxybutyric acid (the 3rd ketone body) lacks a ketone group

• thus will not be detected
• BUT all 3 fatty acid breakdown products are produced at the same time
• thus the presence of acetone and acetoacetic acid will show up positive for ketones
although
• ß-hydroxybutyric acid actually is the predominant ketone
• therefore a weak (+) result may be misleading of the extent of ketosis
and this is only a semi-quantitative screening test

REDUCING SUBSTANCES

Copper Reduction Test

• Measurement of glucose by the copper reduction method was one of the earliest
chemical tests performed on urine.
• Test relies on the ability of glucose and other substances to reduce copper sulfate to
cuprous oxide in the presence of alkali and heat.
• A color change progressing from a negative blue (CuSO4) through green, yellow,
and orange/red (Cu2O) occurs when the reaction takes place.

• Benedict solution contains copper sulfate, sodium carbonate, and sodium citrate
buffer.
• Urine is added to the solution, heat applied, and the resulting precipitate observed
for colour.
• More convenient method that employs Benedict’s principle is the Clinitest tablet.
• Upon addition of the tablet to water and urine, heat is produced by the hydrolysis of
sodium hydroxide and its reaction with sodium citrate, and carbon dioxide is
 Carbohydrates 30

released from the sodium carbonate to prevent room air from interfering with the
reduction reaction.
• Tubes should be placed in a rack and not held in the hand because the reaction heat
could cause a burn.
• At the conclusion of the effervescent reaction, the tube is gently shaken, and the
colour ranging from blue to orange/red can be compared with the manufacturer’s
color chart to determine the approximate amount of reducing substance.

KETONES QUANTITATIVE ASSAYS: Blood/Plasma/Serum

• Serum/plasma ketones can be measured with the tablets or dipsticks routinely used
for urine ketone measurements, but predominant ketone body in DKA (ß-
hydroxybutyric acid), is not detected, as with urine ketone testing
• Blood samples can be collected into tubes containing heparin, EDTA,
fluoride, citrate, or oxalate.
• Sample stability differs among methods, but whole-blood samples are generally
stable at 4 °C for up to 24 h. Serum/plasma samples are stable for up to 1 week at 4
°C and for at least several weeks at -20 °C
• Several different assay methods, but enzyme method is preferred.
• The principle of the enzymatic methods is that ß-hydroxybutyrate dehydrogenase
in the presence of NAD+ converts ß-HBA to AcAc and NADH.
Under alkaline conditions (pH 8.5–9.5), the reaction favors the
formation of AcAc from ß-HBA. The NADH produced can be
quantified spectrophotometrically (usually kinetically) with
the use of a peroxidase reagent.
 Carbohydrates 31

GLYCATED HAEMOGLOBIN (HbA1c)

Hb : Glycated and Non-glycated fractions

(by ion-exchange)

Hb

Normal Adult Hb HbA HbA2 HbF Fetal Hb

(ßß) () ()

97% 2.5% 0.5%

Non-
HbA0 HbA1 Glycated
glycated
94%
5%
- main glycohemoglobin
A1a and A1b:
- used since > 20 years
concentration HbA1a HbA1b HbA1c for glycemic control with
very low
HPLC

 In humans, haemoglobin consists of several fractions: HbA, HbA2 and HbF.

 The predominant fraction is HbA, which itself is divided into HbAo and HbA1, while
HbA1 is further divided into several minor components HbA1a, HbA1b and
HbA1c .

 They are collectively known as glycated haemoglobins. The main fraction is Hba1c.

 Glycated haemoglobins are formed by the glycation of haemoglobin.

 Specifically, glucose reacts with valine which is the terminal amino acid of each β-
 Carbohydrates 32

Chain of the haemoglobin molecule.

 This reaction is non-enzymatic. The initial product is an unstable Schiff base

which undergoes a re-arrangement to form a stable keto-amine Amadori product ,
called glycosylated haemoglobin. The reaction resulting in the formation of the Schiff
base is reversible, whereas the second one in the formation of the Amadori product
is not.

The AIM of diabetic management is to:

=> maintain blood [glucose] in the non-diabetic range, with
=> minimal fluctuations (thus minimal tissue damage)

SHORT-TERM MANAGEMENT
• serum / plasma [glucose] measured in the lab
• OR self-monitoring of whole blood [glucose] by the patient at home / work

LONG-TERM MANAGEMENT
• measure glycosylated haemoglobin (Hb)
• this gives a time-averaged picture of the patient’s blood [glucose]
• N-terminus of a protein (Hb) + carbohydrate (glucose) => glycosylated product
• glycosylated Hb (stable ketoamine)
 Carbohydrates 33

• ketoamine HbA1C is the largest subfraction of HbA (in normal & diabetic

individuals)
• thus HbA1C measurement reflects the [glucose] over 60 days (½-life of erythrocytes)

Specimen

• EDTA blood sample.

REFERENCE RANGE
*• <Non
6.5% = Inconclusive
diabetic range: HbA1c = 4 - 6%
* > 6.5% = Diabetes mellitus in a patient with symptoms of hyperglycaemia.
*• >HbA1c
6.5% =target for glycaemic
Diabetes mellitus incontrol: < 7% (most
an asymptomatic methods)
individual repeated on separate days
within a 2 wk period.

METHODOLOGY
Methods are grouped into two major categories:
1. based on charge differences between glycosylated and non-glycosylated
haemoglobin ( cation-exchange chromatography, electrophoresis and isoelectric
focusing) , and

2. structural characteristics of glycogroups on haemoglobin (affinity chromatography

and immunoassay).

LIMITATIONS
 HbA1C assays may not be useful, or more specific assays may be
necessary, in a variety of circumstances:
- Haemoglobinopathies (HbS, HbC, HbF, HbE, thalassaemia traits)
- States of increased or reduced red cell turnover (haemolytic anaemia,
chronic malaria, blood loss, blood transfusions).
- Age (elevates HbA1C)
- Different race/ethnic groups
- Rapid onset type 1 diabetes
 Carbohydrates 34

FRUCTOSAMINE

 Fructosamine = a compound that can be considered the result of a reaction between

fructose and ammonia or an amine.
 In humans, a fructosamine is also formed when the carbonyl group of glucose reacts
with an amino group of a protein.
 Since albumin is the most abundant serum protein, it accounts for 80% of the
glycated serum proteins, and thus, a high proportion of the fructosamine.

 Fructosamines formed from serum albumin are known as glycated albumin and can
be used to identify the plasma glucose concentration over time.
 The circulating half-life for albumin is  20 days.
 Therefore the concentration of glycated albumin reflects glucose control over a
period of 2 – 3 weeks.
Specimen

• Serum

• Plasma in heparin or EDTA.

Storage: 3 days at 20-25° C

2 weeks at 2-8° C
2 months at -20° C

REFERENCE RANGE
• Fructosamine = 205 – 285 umol/L
METHODOLOGY
 Methods include: Affinity chromatography, HPLC & Spectrophotometry.
 Spectrophotometry
Colorimetric assay based on the ability of ketoamines to reduce nitrotetrazolium
blue (NBT) in an alkaline environment.
The speed of formazan formation is directly proportional to the fructosamine
concentration.
The reaction speed is measured by photometry at 546 nm.
 Carbohydrates 35

ALBUMINURIA (formerly microalbuminuria)

• diabetes mellitus

progressive kidney changes

diabetic renal nephropathy
• this damage may take years and may be delayed if proper glycaemic control is
achieved
• a very early sign of nephropathy =>  urinary albumin (before kidney damage is
evident)
• measurement of microalbumin = useful to assist in diagnosis of kidney disease at an
early stage and before proteinuria develops.
Urinary albumin excretion (UAE) Urine Albumin: creatinine ratio (UACR) Urine dipstick
Normal < 30 mg/24hr < 2.5 mg/mmol Negative
High Albuminuria 30 to 299 mg/24hr 2.5 - 30 mg/mmol Negative
Albuminuria  300 mg/24hr > 30 mg/mmol +/++
(clinical proteinuria)

Specimen

• 24hr urine specimen preferred, random collection acceptable.

After sampling, the test should be performed without delay..

METHODOLOGY
 Semi-quantitative assays are available
 Quantitative assays
 RIA, ELISA & Immunoturbidimetry
 Immunoturbidimetry
When an antigen-antibody reaction occurs between μ-ALB in a
sample and anti-ALB antibody, agglutination results. This agglutination is
detected as an absorbance change, with the magnitude of the change
being proportional to the quantity of μ-ALB in the sample. The actual
concentration is then determined by interpolation from a calibration
curve prepared from calibrators of known concentration.
 Carbohydrates 36

INSULIN

 Useful in diagnosis of insulinoma (during prolonged fasting, when the

patient's glucose level is <2.2 mmol/L,  insulin +  of proinsulin and C-
peptide suggest insulinoma).

 Used in management of diabetes mellitus.

Specimen

• Serum

Reference range
• Insulin = 6 – 27 mU/L

METHODOLOGY
 Many assays are commercially available.
 Standardization is a problem.
 Immunoassays most widely used.

C-PEPTIDE

 C-peptide (connecting peptide), a 31-amino-acid polypeptide, represents

the midportion of the proinsulin molecule.
 Carbohydrates 37

 Useful in diagnostic work-up of hypoglycemia:

-Diagnosis of factitious hypoglycemia due to surreptitious administration of
insulin
-Evaluation of possible insulinoma
 Assessing insulin secretory reserve in selected diabetic patients
 Serum C-peptide & Insulin both  in eg renal failure, insulinoma
 Serum C-peptide & Insulin both  Type 1 Diabetes mellitus & Type 2 Diabetes
mellitus (long-standing).
 Factitious hypoglycaemia due to surreptitious administration of insulin
suppresses endogenous insulin and C-peptide secretion.

Simultaneously, the peripherally administered insulin bypasses the hepatic

first-pass metabolism, and leads to  insulin and  C-peptide levels.

Specimen

• Serum (Fasting)

Reference range
C-peptide = 297-1,419 pmol/L

METHODOLOGY
Several Immunometric methods are available.