MLS 403; Clinical Chemistry II

Disorders of Carbohydrates Metabolism

Dr. M Zachariah
Block 246/217
Email: zachariahm@ub.ac.bw
Tel: 3554651
 Objectives
• Describe the long-term sequelae of diabetes
mellitus
• Describe the principle of glycated hemoglobin
methods, sample management and commonly
encountered analytical interferences
• Interpret laboratory findings in diabetes
mellitus
• Selection and interpretation of laboratory
procedures
• Establishment and use of reference values
 Glucose metabolism
• Glucose is essential for life particularly the neurons
• Dependent on hypoglycemic and hyperglycemic
hormones. State the hormones and how he
regulate glucose?
• Glucose-induced insulin secretion from pancreatic
beta cells is in three phase;
– Transport and metabolism of glucose
– Metabolic changes in cellular ion influx
– Entry of calcium and calcium-dependent insulin release
Glucose metabolism; Insulin release
 Glucose metabolism
carbohydrates
digestion
absobtion

postprandial hyperglicem ia

insulin release insulin-independent cells

liver storage glucose m oves into protein synthesis inhibition:

glycogen insulin-dependent cells IN L IV E R lipolysis
(m uscle, adipose) glycogenolysis
gluconeogenesis
 Other Insulin secretagogues
• Leucin
Amino acid and amino acid by products;
• Ketoisocaporate mTOR dependent insulin secretion
• Methyl succinate
• Fatty acids
• Arginine Suppresses Glucagon release
• Ketones
• Incretins; Glucagon-like peptide 1 (GLP1) and Glucose-
dependent insulinotropic peptide (GIP); released from small
intestine in response to food/glucose to stimulate insulin
release and inhibit appetite.
• Sulfonylurea bind to the SUR1 to secrete more insulin to
cause influx which sensitizes the cells to uptake glucose and
feedback mechanism lowers insulin levels.
 Metabolic effects of Insulin
Liver Muscle Adipocytes
Glycogenolysis Glucose uptake Glucose uptake
Gluconeogenesis Ketone metabolism Lipolysis
Ketogenesis

What would be the effects of Glucagon, Growth hormone and Cortisol on the
insulin-sensitive organs?
Glucose homeostasis
Fasting state  Fed state  insulin
glucagon  insulin

peripheral uptake of
glucose
 peripheral uptake
of glucose hepatic
glycogenesis
 hepatic
glycogenesis glycogenolysis and
gluconeogenesis
 gluconeogenesis
lypolisis and
 lypolisis
ketogenesis
•
Case study 1
A 21-year-old was recently seen by her doctor and prescribed medication for
vaginal thrush. She had noted weight loss in the last month but was not dieting. In
the previous week, she was feeling tired, thirsty and drunk lots of water. She was
admitted to the hospital in a coma with rapid pulse and her breath smelled of
acetone. Her Clinical Chemistry results were as follows;
Blood glucose 35 mmol/L
Arterial blood pH 7.15
Urine Ketones positive 2+
Urine glucose positive ++
Serum potassium 5.9 mmol/L
a. What is the diagnosis?
b. Why does she have low pH, why her breath smell of acetone?
c. Why is serum K raised?
d. Why did she have thrush?
e. Why was she thirsty and drinking lots of water?
f. Why had she lost weight?
g. Why did she faint, have rapid pulse on admission?
h. How should she be managed?
 Case study 2
• A middle aged widow, who lived alone was admitted to hospital after her son
found her semi-conscious at home. He had not seen her for a week but she had
seemed well at their last meeting. On examination she was extremely dehydrated
but not ketotic. Her respiration was normal. Lab results are as follows;
Na 149mmol/L (135-145)
K 4.7 mmol/L (3.6-5.0)
HCO-3 18 mmol/L (22-30)
Urea 35 mmol/L (3.3-6.7)
Creatinine 180 umol/L (60-80)
Glucose 54 mmol/L
Total protein 90 g/L (60-80)

Calculate Osmolality. Comment on results. Diagnosis? Further test?

High Serum osmolality, hyperglycemia which causes osmotic diuresis resulting in
decreased glomerulus filtration with retention in urea and creatinine. hypernatraemia
and high total protein due to loss of water from plasma. Bicarbonate low due to loss of
hydrogen ion at the kidneys
Insulin..9.5 mu/L (2-10)
Omolality ?? (275-295)
Non-ketotic hyperglacaemia due to T2DM.
 WHO diagnosis of Diabetes

• WHO estimates 529 million people will be

living with DM in 2035. Prevalence in
Botswana is 4-6%
• Fasting blood glucose ≥7.0 mmol/L
• 2 hour plasma glucose ≥11.1
mmol/l
• Impaired Glucose tolerance
2hour fasting plasma glucose ≥ 7.8 and
<11.1 mmol/L
• Impaired fasting glycaemia 6.1-6.9 mmol/L
 Causes of Diabetes
• Genetic; Type 1 DM
• Epigenetics; Type 2 DM
• Pregnancy; Gestational DM 4-5% pregnancies
• Secondary diabetes
 Secondary causes of impaired tolerance and DM
Type Cause Effect Severity
Malnutrition Childhood malnutrition with or without chronic pancreatitis Lack of protein Severe, Insulin
Cassava?/ requiring
Pancreatic disease Pancreatitis • Chronic damage to Mild
Pancreatic carcinoma endocrine pancreas
Total pancreatectomy • Local destruction of ß-
Haemochromatosis cell mass
• No endocrine pancreas
• Fibrosis due to iron
overload in pancreas

Drugs
Endocrine disorders
Pancreatic endocrine tumours
Mild-to-severe
Thiazide diuretics (Heart failure) • Impaired insulin Mild to severe
Beta blockers (Hypertension) secretion via low K
ß2-andrenergic agonists (asthma) • Insulin resistance
Immunosuppressants (transplants) • Increased
Corticosteroids (inflammation, overtreatment of Addison’s) gluconeogenesis
• Reduced glucose uptake
in muscle and adipose
Cushing’s syndrome (corticosteriods • Severe when due to Mild-severe
Acromegaly (growth hormone) ectopic ACTH
Phaechromocytoma (adrenaline) • Increased hepatic
Conn’s syndrome (hyperaldosteronism) glucose and reduced
glucose uptake
• Impaired insulin
secretion
Glucagonoma • Increased brekdown, Mild-to-severe
Somatostatinoma gluconeogenesis
• Increased Ketogenesis
• Reduced insulin secretion
and glucagon secretion
•
Case study 3
Lesedi Matlho, 18 years, was consulted by her doctor because of tiredness and weight loss.
She complains thirst and passing urine more than normal. Urine dipstick tested positive for
glucose. She was referred to diabetic clinic the following day. By then, however, she felt too ill
to get out of bed, drowsy, vomiting and cold extremities. She had Kussmaul respiration with
acetone breath. She was admitted and her results as follows;
Blood pressure 96/60mmHg
Pulse 112/min
Na 130 mmo/L (132-144)
K 5.8 mmol/L (3.5-5.0)
Urea 18 mmol/L (3.3-6.7)
Plasma Creatinine 140 umol/L (60-120)
Glucose 32 mmol/L (<10 )
pH 7.05 (7.35-7.45)
pCO2 2.0 kPA (4.7-6.0)
HCO3- 5 mmol/L (24-29)

Discuss the clinical presentation and results. Diagnosis? What other lab tests indicate?
Answer: low NA=cold extremities and tachycardia. Hyperventilation=low HCO -3, and high pH so
low pCo2 indicates non-respiratory acidosis with partial compensation. Renal impairment high
urea and creatinine and dehydration. Hyperkalemia due to lack/low insulin action as insulin leads
to K+ cellular uptake, acidosis and tissue catabolism or fluid from IC compartment. Ketonuria test.
Diagnosi
 Diabetic Ketoacidosis
• Functions of insulin??
• 6% mortality and occurs frequently in Type 1
diabetes
• Precipitating factors;
– UTI
– Pneumonia
– Heart attack
– Trauma
– Stroke
• <10% present with coma called Diabetic coma
• Obvious sign
– Pear drop smell
 Diabetic Ketoacidosis
Adipose tissue
Glycerol Acetyl Co A
Acettoaceta + Blood vessel
NEFA β-hydrobutyrate
Pancreas (ketone bodies)
Liver
Glucagon Glycogenolysis
Gluconeogensis
Or no Insulin Ketogenesis
Glucose
Ketones

pH

Muscle
Amino acid
 Hyperosmolar hyperglycemic syndrome (HHS)
• A.K.A Hyperosmolar non-ketotic state
• Marked hyperglycaemia; often >50 mmol/L without presence of
ketones/ketoacidosis
• High Osmolality (320-340 mOsm/kg.
• Occurs often in T2m patients with some endogenous insulin release which
reduces the ketogenesis and lipolysis seen in DKA but insufficient to suppress
hepatic glucogeneosis and promote glucose uptake.
• Associated with high sugary intake and polyuria and occurs over a period of
time
• Sign and symptoms include; seizures, severe dehydration, progressive
confusion or altered consciousness and serious problem might be cerebral
oedema during fluid replacement
• In African American, Afro-Caribbean and older patients mostly affected and
often present to the doctor as T2DM
• Mortality is 30%
• Tx; treat precipitating cause (infections, MI drugs like antipyschotic), normal
saline 0.9% not too quickly, insulin infusion 3-4 mmol/L
 DKA vs HHS
Diabetic Ketoacidosis
Short onset (few days)
Osmolality rarely > 320
mOsm/kg
Significant hyperketonaemia
and acidosis
Hyperglycaemia relatively
modest/rarely absent
Occurs mostly in Type 1 DM
Mortality <6%
Hyperosmolar
Hyperglycaemic syndrome
Long onset (many days)
Osmolality frequently >320
mOsm/kg
None or low hyperketonaemia
and acidosis
Severe Hyperglycaemia (>50
mmol/L)
Occurs mostly in T2DM
Mortality up to 30%
 Case study 2 continues
• A year later after good follow ups, 18 year old Lesedi’s,
diabetic patient, lab results after out patient check up came
as follows;
Urine glucose (early morning) 2%
Blood fasting Glucose 18 mmol/L (<10 )
HbA1c 6.1%

Discuss the clinical presentation and results.

Answer: Hba1c=good diabetic control. High glucosuria and
hyperglycemia. It transpired that Lesedi had been to a party the
night before and had considerably eaten more than usual and
admitted to have consumed “1-2-3 beers.”
Would you include AGEs measurement as a monitoring test in
diabetic?
 Case study 4
• A 59 year old man, was consulted at Hukuntsi Primary Hospital
feeling tired and lethargic. He used to be physically active and
involved in several sports. He stopped playing 6 years ago and had
since gained 18kg in weight, mostly around the abdomen, with
BMI of 32kg/m2 (18.5-24.5). His clinical work-up is as follows;
Blood pressure 200/160 mmHg
Blood random glucose 7.5 mmol/L
OGTT blood glucose 12.5 mmol/L
Total cholesterol 6.2 mmol/L
HDL cholesterol 1.0 mmol/L
Fasting triacylglycerols 3.5 mmo/L (0.8-2.2)

Discuss the results. What is the diagnosis? What is the cause of the
results. How should he be treated? What other test should be done?
 Lactic acidosis
• Uncommon complication of DM normally seen
in patients taking phenformin
• Also seen in decreased perfusion, reduced
arteral pCO2, high intake of fructose, sorbitol
and congenital diseases such as G6PD and
defective gluconeogenesis and pyruvate
oxidation.
• Characterised by high anion gap and high
chloride levels
 Hypoglycaemia
• Glucose levels <2.8 mmol/L but
symptoms begin to develop at levels ~3.0
mmol/L
• Treated with catecholamines. Explain
why?
• List the causes of the following
conditions.
– Reactive hypoglycaemia
– Fasting hypoglycaemia
 Metabolic Syndrome (Syndrome X)
International Diabetes Federation Definition:
Abdominal obesity plus two other components: elevated BP, low
HDL, elevated TG, or impaired fasting glucose, oxidative stress and
inflammation

Prevalence increase with age and greater industrialization and

urbanization
 Metabolic Syndrome (Syndrome X)

• cluster of disorders of your body’s

metabolism, including:
High blood
pressure

CVD High insulin

Abnormal
Stroke levels
cholesterol
Diabetes
levels

Excess body
weight

• a.k.a Insulin Resistance Syndrome

• Increase the risk of Polycystic ovarian syndrome (40-50%) and and alcoholic
fatty liver disorder (NFALD)
 Metabolic Syndrome (Syndrome X)
• High glucose levels, NEFA, inflammatory
cytokines and interactions with oxidized LDL
induce oxidative stress in endothelium.
 Metabolic Syndrome
(Syndrome X)

• 34% of 33-54 year old workers at Kanye SDA Hospital,Botswana

had MS (Garrido R, et al., 2009)

Garrido, Roberto A., et al. "Metabolic syndrome and obesity among workers at Kanye Seventh-day Adventist
Hospital, Botswana." SAMJ: South African Medical Journal 99.5 (2009): 331-334.
Other long term complications of diabetes
• Hyperglycemia and excess glucose within target
tissue leads to non-enzymatic glycation of molecules
e.g proteins
• Glucose binds to Hemoglobin A; HbA1c
• Other proteins and lipoproteins can be glycated and
further transformed into advanced glycation end
products (AGEs)
• AGEs are pro-inflammatory, bind to target cells
especially endothelium triggering adhesion
molecules; CVD risk
• Lipedemia leads to atherosclerosis
• Atherosclerosis hallmark of CVD and hypertension
• How does uncontrolled diabetes result in CKD?
 …cont’

• Macrovascular disease; atherosclerosis affecting peripheral vessels.

– Clotting and limping due to insufficient O2 to lower extremities (claudication)
– Increased risks of CVD, stroke and death
• Microvascular diseases; damage to the smaller blood vessels impair
function to the kidney, eyes and nervous system.
– Nephropathy; common in people with long-term DM
– Retinopathy; blocked/leaky blood vessels in the retina
– Neuropathy; nerve damage in the brain, spinal cord, muscles, skin, penis. Hence Erectile
Dysfunction in T2DM in males.
– Hyperglycaemia and neuropathy combined causes delayed wound-healing in DM
promoting diabetic ulcers especially in the lower extremities (major cause of disability
in DM)
• Musculoskeletal damage; Hyperglycaemia might cause damage to
connective tissue esp. collagen
– Dupuytren’s contracture
– Carpal Tunnel Syndrome
– Tenosynovitis
– Frozen shoulder
– Mastopathy
– Skin rashes (necrobiosis lipoidica)
 HbA1c measurement
• Not subject to fluctuations
• Measurement; based as percentage of Hb
– Charge difference
– Structural difference
– Chemical analysis
– m/z
• Common, current
– Immunoassays
– Ion-exchange chromatography; resin has affinity
for Hb
– HPLC
 HbA1c measurement
Type Ion-exchange Immunoasay HPLC Affinity
chromatography chromatography
Principle Separates Hb Antibodies Separate Hb Affinity gel columns
variants based on against fractions on separate glycated Hb
charge Amadoru cation-exchange which binds to m-
product of columns aminophenylboronic
glucose Beta acid. Sorbitol elute the
chain in Hb GHb measured at
amino acids. 415nm
agglutination

Sample A haemolysed Whole blood 5 uL whole blood Whole blood

Venous blood aliquot (20-100uL) haemolysed with
on borate @415 and
EDTA/oxalate/fl 690nm
uoride

Interferences Temperature None specific none None from non-

4 degree C Reagents to HbA1c glycated Hb but
pH negligence from labile
Ionic strength HbA1c (Schiff base)
Ketoamines
Uremia

Electrophoresis?? Reading assignment

 Case study 5
• 28 year old male was admitted at ER when a
room mate found him to confused and
disorientated. The attending doctor noted a
scar on the abdomen and excess skin on the
limps and abdomen. His blood glucose was 1.2
mmol/L.
• What could have happened?
Reactive hypoglycaemia common on patient
gastric/gastric bypass patient 90-150 minutes
after high sugar food.
Small intestines release incretins leading to
excessive insulin response.