MLS2001 sem2 Lecture Notes

Objectives

• Key words: ketones, ketoacidosis, polyuria, glycosuria, polydipsia, post-prandial, C-peptide, insulin receptors, static and dynamic tests, etc.

• Know the hormones and organs that are involved in the regulation of extracellular glucose.

• Hyperglycaemia VERSUS Hypoglycaemia.

• Assays to analyse and assist in the diagnosis of hyperglycaemia & hypoglycaemia:

  • FBG, RBG, post-prandial glucose

  • OGTT

  • HbA1c

  • urinary (albumin, glucose & ketones)

  • C-peptide, Insulin

Definitions

• Hormone regulation of Blood Glucose

• Regulation of blood glucose is largely done through the endocrine hormones of the pancreas

• Balance of hormones achieved through a negative feedback loop

• Glucagon and insulin are key hormones.

Glucagon

• 29 A.A polypeptide secreted by the α-cells of the pancreatic islets.

• Increase secretion when plasma glucose concentration decreases.

• Stimulates hepatic glycogenolysis & gluconeogenesis.

Insulin

• Secreted by the β cells of the pancreatic islets of Langerhans in response to an increase in plasma glucose.

• Insulin allows glucose to move into muscles and adipose cells through insulin receptors on cell membranes, facilitating energy and storage.

• Insulin stimulates:

  • Glycolysis

  • Glycogenesis

  • Lipogenesis

• Insulin inhibits:

  • Glycogenolysis

  • Gluconeogenesis

• Insulin is synthesized in pancreatic β cells in response to hyperglycaemia.

• It binds to insulin receptors in target cells (skeletal muscle cells, hepatocytes, and adipocytes), inducing signalling pathways that promote glucose uptake, catabolism, or storage.

• This process reduces plasma glucose levels.

Extracellular Glucose

• Glucose is especially important for brain cells to maintain normal cerebral function; hyperglycaemia can affect cerebral dysfunction.

• Blood glucose levels must be maintained at a constant level, even during intermittent load.

• The level is maintained between 4.5 to 11 mmol/L.

• A constant level must be maintained (including in the brain) to minimize loss from the body.

• The kidney reabsorbs glucose via renal tubular cells, so glucose should be very low in urine, even after a carbohydrate meal or high sugar load if everything is working as it should.

• Glycosuria: glucose passed in urine.

• Occurs when plasma glucose is very high (>11 mmol/L) and the renal threshold is exceeded.

Assessment of Hyperglycaemia and Hypoglycaemia

• Hypoglycaemia

  • Causes: Too much insulin, little or no food, unexpected exercise, gastrointestinal disturbances.

  • Onset: Usually rapid with 'good health' previously.

  • Blood glucose level: Usually less than 3 mmol/L.

  • Signs and symptoms: Pale, cold, clammy skin, bounding, rapid pulse, normal or high blood pressure, normal or shallow breathing, irritable, confused, uncoordinated, aggressive and anti-social behaviour, headache, dizziness, hunger, rapid loss of consciousness, possible tremors and convulsions.

• Hyperglycaemia

  • Causes: Omission or insufficient insulin, excessive food consumption, infection or digestive disturbance.

  • Onset: Usually slow with ill health a few days before.

  • Blood glucose level: Usually greater than 20 mmol/L.

  • Signs and symptoms: Flushed, hot, dry skin, weak, rapid pulse, low blood pressure, laboured breathing, sweet, fruity (pear drops) odour on breath, abdominal pain, nausea and vomiting, polydipsia (excessive thirst), polyuria (excessive urine output), polyphagia (excessive intake of food), restless, drowsy or lethargic behaviour, unconsciousness.

Complications of Hyperglycaemia

• Stroke, transient ischemic attack, cognitive impairment

• Hypertension, coronary artery disease, increased carotid intima-media thickness, oxidative stress, endothelial dysfunction

• Diabetic retinopathy, glaucoma, cataracts

• Peripheral vascular diseases of extremities (diabetic foot)

• Diabetic kidney disease, nephropathy

• Diabetic neuropathy

Diabetes Mellitus

• Type 1 DM

  • Autoimmune disease: Rapid, T-cell mediated autoimmune destruction of the β-cells of the pancreas.

  • Absolute deficiency of insulin secretion.

  • Increase Ketones, decrease Insulin, increase Glucagon = increase Gluconeogenesis & Lipolysis.

  • Insulin required to prevent ketoacidosis.

• Type 2 DM

  • Characterized by hyperglycaemia caused by individual resistance to insulin combined with an insulin secretory defect.

  • Less likely to have ketoacidosis because insulin is present at times, thus glucagon is not elevated.

  • Decrease FA oxidation (muscle / liver) = increase Triglycerides (VLDL).

  • Early & late Adulthood due to: decrease insulin secretion, insulin deficiency, receptor defects.

• DM due to secondary conditions

  1. Absolute insulin deficiency: pancreas problems (check serum Amylase).

  2. Relative insulin deficiency: check for 2° problems [GH (acromegaly), glucocorticoids (Cushing’s)].

  3. Drugs: Thiazides, steroids (get patient’s history).

  4. Lifestyle factors.

Impaired Glucose Tolerance (IGT)

• FBG (WHO criteria): Venous = 5.5 – 7.8 mmol/L.

• Post-prandial Plasma: = 7.8 – 11.1 mmol/L.

• Only a small proportion of subjects with IGT will develop DM.

• Thus, not diagnosed as diabetics!

• Obtain patient history, test patient, and treat accordingly (diet first).

Ketoacidosis

• Diabetic ketoacidosis (DKA) is a life-threatening problem that affects people with diabetes.

• Occurs when the body starts breaking down fat at a rate that is much too fast. The liver processes the fat into a fuel called ketones, which causes the blood to become acidic.

• Diabetic ketoacidosis (DKA) may be a presenting feature of type 1 DM or may develop in a patient who omits to take insulin or whose insulin dosage becomes inadequate because of an increased requirement (infection, trauma, or acute illness).

Ketones

• Byproducts of fat metabolism.

• Why does it happen?

  • Fasting states: mild ketosis = 12-hour fast.

  • Reduced nutrient absorption & Vomiting.

  • Diabetes: T1 / T2 (resistance vs no production).

• What does it reflect? Intracellular glucose deficiency and low insulin activity.

Types of Ketones

• Acetoacetate

  • First ketone body produced.

  • Created from the breakdown of fatty acids (and some amino acids).

  • Either used as energy, converted into BHB, or broken down into acetone.

  • Can be detected in the urine and serve as a proxy measure for BHB levels.

• Beta-Hydroxybutyrate (BHB)

  • Most prevalent ketone body.

  • The most energy-efficient ketone body.

  • Produced in higher quantities after sustained carb restriction.

  • Used as energy in the muscles, heart, brain, and other tissues.

  • Can be measured directly with a blood ketone meter.

• Acetone

  • May be spontaneously formed after acetoacetate is produced.

  • Virtually useless to the body.

  • Most of it is excreted through the breath.

  • Can be measured with an acetone breath meter and serve as a proxy measure for BHB levels.

Different Types of Ketosis

Prolonged Fasting/Ketogenic Diet

• Ketones: 0.5-10 mmol

• Blood glucose: 60-120

Starvation vs. DKA

Uncontrolled Diabetic Ketoacidosis

• Ketones: > 15 mmol

• Blood glucose: > 200

Laboratory Investigations - Sample Collection

• Blood

  • Type of Blood collection tube:

    1. Serum: ONLY if sample centrifuged within 1hr of collection – RBCs continue to metabolise glucose.

    2. Plasma: INHIBITOR = Sodium fluoride (grey vacutainer) – inhibits glycolytic enzymes.
       *NOTE: Serum or plasma refrigerated and separated within 1 hour to prevent substantial losses of glucose by cellular fraction unless collected in potassium oxalate/sodium fluoride anticoagulant.

  • TESTED: Enzymatic test (glucose oxidase or hexokinase) or colorimeter can be used.

• Urine

  • URINE specimen: collected ~1 hr after a meal (Void).

  • Double void technique may be applied: empty bladder & discard urine; recollect 10-15mins later & test!

  • Post-meal & NOT stored in bladder for some time (false negative = enough time to clear).

  • Fasting: Glycosuria detected if patient diabetic or has renal Glycosuria.

Oral Glucose Tolerance Test (OGTT)

• How to investigate Abnormal Carbohydrate Metabolism?

  • Patient diagnosed with plasma glucose = REPEAT!

  • How to investigate? Symptoms = glycosuria, polydipsia, familial, etc.

• Blood collection:

  • FBG = At least 8-10 hours fast.

  • Post-prandial = 2 hours after a meal (often used in conjunction with FBG).

  • RBG = at random (no fast!).

  • OGTT (dynamic) = Part of a glucose LOAD test.

• DM Confirmed: FBG (x2) = >7.8 mmol/L; RBG (x2) = >11.1 mmol/L.

• DM Excluded: FBG (x2) = <5.5 mmol/L; RBG (x2) = <9.0 mmol/L

• When to do OGTT: FBG = 5.5 – 7.8mmol/L.

• RBG = 7.8 – 11.1 mmol/L.

• Important points:

  • During test, patient should be resting and NOT smoke/eat/drink.

  • Fast overnight (>8hrs but not >16hrs); water allowed.

  • Withdraw blood (finger prick or phlebotomy). Collect urine at baseline.

  • 75g *of glucose given in 300ml water (75g = average amount used by the body in 2hrs).

• Factors affecting test:

  • Previous diet (no carbohydrate restrictions).

  • Time of day (normally morning).

  • Drugs (e.g. steroids, oral contra, thiazides).

OGTT Results

• Resorption of Glucose from intestine & release in plasma

  • Glucose is absorbed through the intestine and enters the bloodstream

  • Scenarios:

    • A patient with hyperthyroidism has a rapid intestinal absorption and a rapid combustion of glucose.

    • The myxoedematous patient (hypothyroidism) has a slow absorption and a slow combustion of glucose.

    • Malabsorption = flat curve.

• Insulin secretion = Glycogen synthesis

  • Dependent on good liver function (glycogen synthesis) and on insulin action in the liver.

  • 80% of glucose is converted to glycogen in the liver under normal conditions.

  • Blood glucose concentration in the portal vein is up to 22.2 mmol/L, while the concentration of Glucose is maximum 11.1 mmol/L in peripheral tissues.

  • Increasing glycaemia stimulates insulin secretion into the blood.

  • The peak of glycaemia is reached in 45 - 60 minutes

• Utilisation of Glucose

  • The descending part of the curve is dependent on insulin action.

  • Diabetes: Curve: slowed-down & insufficient return to normal values - A classical manifestation of diabetes mellitus due to absolute or relative lack of insulin.

Diabetic Curve

1. Fasting blood glucose is higher than normal

2. The highest value is attained at 1 hr to 1hr 30 mins

3. The highest value exceeds the renal threshold

4. Glucose is found in almost all the urine samples

5. The blood glucose level does not return to the fasting level even within 2hr 30 minutes

Lag Curve

1. Fasting blood glucose is normal

2. Sharp rise within 30 mins to 1 hr

3. The blood glucose levels exceed the renal threshold

4. The decline is rapid and the normal levels are attained back

5. Some of the urine samples contain glucose, where the blood glucose is above the renal threshold

• Could be due to: Hyperthyroidism, Pregnancy, after gastrectomy, early DM

Gestational Diabetes

• Also known as: Diabetes in Pregnancy

• Pregestational diabetes

  • Type 1 diabetes

  • Type 2 diabetes

• What is it?

  • Increasing glucose during pregnancy (intolerance), occurring in women without previous DM diagnosis.

  • Done between 22-24 weeks of pregnancy (when it peaks).

Risk factors for Gestational Diabetes

• OR = odds ratio; RR = relative risk.

Consequences of Gestational Diabetes

• For the mother

  • High blood pressure

    • Preeclampsia, can affect mother and baby

  • Type 2 diabetes (50% develop T2D within 10 years)

• For the baby

  • Premature birth and malformations

  • Respiratory problems

  • Fetal death

  • Macrosomia (excessive birth weight)

  • Cesarean birth

  • Hypoglycemia and hyperbilirubinemia

  • Obesity and type 2 diabetes in adulthood

Why does gestational diabetes occur?

• Insulin receptors do not function properly since pregnancy hormones block the effect of insulin (e.g., human placental lactogen).

• Usually occurs around the 24 – 28th week of pregnancy.

• GDM (mothers & babies) should be monitored even after birth

• Pregnant women should be tested for GDM at the initial visit & around 24 weeks when GDM is most prevalent

Glycosylated Proteins & HbA1c

• Purpose: Diabetic monitoring & management, also diagnosis now

• HbA1c reflects the concentration of glucose present in the body over a prolonged period of time (120-day RBC lifespan), indicating a patient’s glucose level over 3 months.

• Principle: Glucose can rapidly react with Hb to form a labile aldamine, which can undergo rearrangement to form glycosylated Hb (ketoamine). Ketoamine is stable & cannot revert back to Hb & glucose. HbA1c = Hb with glucose attached to it.

• HbA1c = largest fraction of HbA, formed by reaction of β-chain of HbA with glucose

• Normal level: 4-6% (depending on analyser & kit used)

  • >6.5% = DM, uncontrolled

  • 5.7 – 6.4% = pre-diabetes

• Detection methods: HPLC; Low-pressure cation exchange chromatography + gradient elution

HbA1c Interferences

• If an individual has a haemoglobin variant, such as sickle cell haemoglobin (haemoglobin S), that person will have a decreased amount of haemoglobin A. This may limit the usefulness of the A1c test in diagnosing and/or monitoring this person's diabetes, depending on the method used.

• If a person has anaemia, haemolysis, or heavy bleeding, A1c test results may be falsely low.

• If someone is iron-deficient, the A1c level may be increased.

• If a person receives erythropoietin therapy or has had a recent blood transfusion, the A1c may be inaccurate and may not accurately reflect glucose control for 2-3 months.

Urinary Glucose (Glycosuria)

• When Glycosuria detected: Renal threshold exceeded (increasing plasma glucose & increasing Glomerular filtrate ( >11mmol/L)

• Renal glycosuria: Decreasing re-absorptive capacity, decreasing filtration. Pregnancy = reversible

• TESTING: using enzyme reagent strips; screening test. Test depends on the action of the enzyme glucose oxidase, which is incorporated into the strip.

  • Insensitive but good for screening & monitoring

• Why insensitive? Increasing Plasma glucose But Decreasing Kidney GFR = (ve) glucose in urine → no use for monitoring DM

• FALSE POSITIVES: Other substances that may interfere (e.g. creatinine, lactose)

• FALSE NEGATIVES: Due to ascorbic acid in urine

Microalbumin

• Increase Urinary albumin

• Principle: assess damage to kidneys due to diabetic renal nephropathy

• Purpose: useful to assist in the diagnosis of DM at an early stage and prior to the development of proteinuria. No urinary albumin → Microalbuminuria → Proteinuria

• Normal level: 20 – 300 mg/d (depending on analyser & kit used)

• Measurement: strips, spectrophotometry, Immunoassay

• Specimen: urine

Other Tests

• Monitor Kidney function: Creatinine clearance - eGFR - Urea, Creatinine, Electrolytes – Cystatin C - Acid-base balance (Blood gases)

• Monitor cholesterol: Lipid profile

• Hormones: Cortisol, GH, epinephrine, FT4, etc.

Common features of DM

• Hyperglycaemia

• Glycosuria (exceeded renal threshold) & increased specific gravity

• Polyuria (high passage of urine: Increase urine glucose = osmotic diuresis; Decrease Na), increased serum and urine osmolality

• Ketones in serum & urine (ketonemia & ketouria)

• Decreased blood and urine pH (acidosis)

• Polydipsia (dehydration = thirst)

• Electrolyte imbalance due to loss in urine

• Hyperlipidaemia: Decrease Insulin = increase lipolysis = increasing FFA → Triglycerides + VLDL → LDL

• Hyperchylomicronaemia: Decrease decrease Insulin (rem effect of insulin of chylomicrons → LPL)

• Increase Proteolysis = muscle wasting

Hypoglycaemia in Adults

• Definition: Plasma glucose = <2.5 mmol/L. Consider Time of collection & analysis!

• Get HISTORY

  • Fasting hypoglycaemia or strenuous exercise: abnormality of glucose-sparing or forming mechanisms (e.g. liver disease, pancreas tumours, glucocorticoid def).

  • Non-fasting hypoglycaemia: 5-6hrs after meal due to ingestion of certain foods (e.g. fructose) or drugs (insulin).

Provoked hypoglycaemia: Examples

• Insulin-induced hypoglycaemia or other drugs (e.g. sulphonylureas)

• Insulinoma: benign primary tumour of the islet cells of the pancreas

• Alcohol-induced hypoglycaemia: gluconeogenesis as a result of alcohol metabolism

• Prolonged fasting

Differentiation of Hypoglycaemia in Adults

• Measure: Plasma Insulin and C-peptide assay

Keep in mind

1. Time of attack in relation to meal

2. Drugs (e.g. hyperglycaemic drugs)

3. Possible hypopituitarism or adrenocortical hypofunction

4. Non-pancreatic tumours

5. In case of elevated insulin & C-peptide (e.g.Insulinoma) – INSULIN SUPPRESSION test (fails to lower Insulin!).

• C-peptide = endogenous production

C-Peptide test

• When & Why are C-peptide tested in diabetics?

  • Differentiate between type 1 and type 2 diabetes:

    • Type 1 DM: the pancreas produces little or no insulin, thus there is little or no C- peptide

    • Type 2 DM: C-peptide levels normal or high, since pancreas produces more insulin (to overcome resistance by receptors)

  • Help Determine cause of Hypoglycaemia: Insulinoma vs Insulin administration