ࡱ>  kmhijtnprdq` ǯbjbjqPqP 4::5&zzz8>nLB"dddddd$h˴-:%dd:%:%˴dd222:%dd2:%22RM -d `oz(A0>_*b<--d 2 r"ddd˴˴x2Xddd>:%:%:%:%+ UNIVERSITY OF PAPUA NEW GUINEA SCHOOL OF MEDICINE AND HEALTH SCIENCES DIVISION OF BASIC MEDICAL SCIENCES DISCIPLINE OF BIOCHEMISTRY AND MOLECULAR BIOLOGY MBBS, BMLS BDS Year 3 WATER (FLUID) AND SODIUM BALANCE An Overview WATER (FLUID) BALANCE / WATER (FLUID) STEADY STATE: Amount of daily water intake varies among individuals Amount of daily water loss also varies among individuals, Water loss is normally seen as changes in volume of urine production (Urine Flow Rate can vary widely in a very short time) To maintain water balance: Amount of daily water intake must equal amount of daily water loss, Disruption of balance implies: Body will have either a net water gain or a net water loss Fig 1 shows schematic representation of Normal water balance What are the major sources of intake of water (fluid)? Water Drinking Water contained in Food Metabolic water (production of CO2 and H2O) What are some of the major route of water (fluid) loss? Urinary loss; Fecal loss Insensible H2O loss such as evaporation from Respiratory Tract and Skin surface (excluding sweat which is sensible, since it has a purpose) Sweat Losses at normal room temperature, sweating accounts for about 25% of heat losses In cold environments, H2O losses in sweat decrease In warm environments, or with exercise, sweat losses increase Pathological losses Include: Vascular Bleeding, Vomiting and Diarrhea What are the body fluid compartments? Water is the major body constituent Average person (70 kg) contains about 42 liters of Total Body Water Total body water is separated into 2 major compartments: Intracellular Fluid Compartment (ICF): Volume of fluid inside cells Extracellular Fluid Compartment (ECF): Volume of fluid outside cells Water Tank model can be used to illustrate body fluid compartments (Fig. 2) Does loss of fluid affect fluid compartments? Selective loss of fluid from ICF or ECF compartments gives rise to distinct signs and symptoms Loss of fluid from ICF (Intracellular fluid loss) can cause cellular dysfunction, which is most notably evident as Lethargy, Confusion and Coma Loss of fluid from ECF (Extracellular fluid loss), such as blood loss, can lead to Circulatory Collapse, Renal Shutdown and Shock Loss of Total Body Fluid usually produce similar effects as indicated above Signs of (substantial) fluid loss, is usually spread across both ECF and ICF compartments How is the state of hydration of a patient assessed? State of Hydration of a patient (i.e., Volume depletion or Volume expansion of body fluid compartments), is usually assessed on Clinical grounds: History taking to identify water intake and water loss Signs and Symptoms indicating Dehydration or Over hydration Figures 3a & 3b illustrate effect of Volume Depletion and Volume Expansion on water thank model of body fluid compartments How is water balance regulated by AVP? Fig. 4 Arginine Vasopressin (AVP; also called Anti-Diuretic Hormone, ADH) is a hormone produced by Posterior Pituitary Gland AVP tightly regulates water excretion by the kidneys Osmolality in Intracellular fluid is equal to that in Extracellular fluid Specialized cells in Hypothalamus play a role in maintaining the Osmolality between Intracellular and Extracellular fluid Specialized cells detect differences between their Intracellular Osmolality and that of the Extracellular fluid and adjust secretion of AVP from Posterior Pituitary gland Regulation is as follows: A rising Osmolality promotes the secretion of AVP A declining Osmolality switches off the secretion of AVP AVP causes water to be retained by the kidneys Fluid deprivation results in stimulation of endogenous AVP secretion causing reduction in Urine Flow Rate to as little as 0.5 ml/min in order to conserve body water Within ONE hour after drinking about 2 liters of water, the Urine Flow Rate may rise to about 15 ml/min as AVP secretion is Shut Down By regulating water Excretion or Retention, AVP maintains normal concentrations of Electrolytes within the body (Figure 4) SODIUM BALANCE: What is exchangeable and non-exchangeable sodium? (Fig. 5) Total Sodium in the body can be separated into Exchangeable and Non-Exchangeable About 25% of total Sodium is Non-Exchangeable Non-Exchangeable Sodium is incorporated into tissues such as bone and cartilage and has slow turnover rate About 75% of total Sodium is exchangeable Most exchangeable sodium is in ECF Sodium circulates in Plasma as Free Sodium Ions (Na+) Normal plasma or serum [Sodium] is between 135 and 145mmol/L Plasma or Serum [Sodium] does not reflect the state of Sodium balance Plasma or Serum [Sodium] primarily reflects body water content Sodium Intake: Intake of sodium is variable and depends on many factors: Habits, Taste In an health individual total body sodium does not change even if intake falls to as little as 5mmol/day or greater than 750mmol/day Sodium Loss: Loss of sodium is just as variable as sodium intake For health individuals: Urinary Sodium Excretion equals Sodium Intake Most sodium excretion is via the kidneys Some sodium is lost in sweat (on average about 5 mmol/day), and in the feces (about 5 mmol/day) In disease state GIT is often the major route of sodium loss This is a very important Clinical point, especially in Pediatric Cases, as Infantile Diarrhea may result in death from Salt and Water Depletion What factors regulate sodium excretion? Sodium excretion is controlled by: Intrinsic Renal Mechanisms, Suppression of Aldosterone Secretion and Stimulation of Secretion of Atrial Natriuretic Factor (ANF) What is the role of Aldosterone in regulation of sodium balance? Aldosterone decreases Urinary Sodium Excretion by Increasing Re-absorption of Na+ in the Renal Tubules in exchange for Tubule excretion of K+ and H+ Aldosterone does not concentrate the urine, because it exchanges one ion for another Aldosterone stimulates Sodium conservation by Sweat Glands and Mucosal Cells of the Colon, but in normal circumstances these effects are minimal How is the secretion of Aldosterone controlled? (Fig. 6) Volume of ECF is a major stimulus for secretion of Aldosterone Specialized cells in the Juxtaglomerular Apparatus of the Nephron sense decreases in Blood Pressure and Secrete Renin, Renin converts Angiotensinogen (produced in the Liver) to Angiotensin I Angiotensin I is converted to Angiotensin II by Angiotensin Converting Enzyme (ACP) Angiotensin II then act on Adrenal Cortex to produce Aldosterone What are the functions of Atrial Natriuretic Factor (ANF) or Atrial Natriuretic Peptide? Atrial Natriuretic factor is a polypeptide hormone that is predominantly secreted by the Cardiocytes of the Right Atrium of the Heart thus, it is referred to as Cardiac Hormone ANF increases urinary sodium excretion thus it is said to produce Natriuresis ANF is also play a role in regulation of ECF volume and sodium concentration How do AVP and Thirst indirectly regulates Sodium balance? AVP (ADH) and Thirst do not regulate Sodium directly, AVP and Thirst control fluid balance via the regulation of water absorption in the collecting duct of the Nephron Absorption and excretion of water alters the concentration of Sodium in the ECF When large intake of water lowers Serum Sodium concentration to less than 135 mmol/L, cell volume receptors in the Hypothalamus inhibit the secretion of AVP, excess water is excreted and circulating Sodium is returned to normal levels AVP secretion and Thirst are stimulated by: Hyper-Osmolality and Volume depletion by signals to the Baro-receptors and volume receptors in the great vessels and heart TAKE NOTE: Some important Physiological concepts: Water remains in Extracellular compartment by the Osmotic effect of ions Sodium ions (and Anions, mainly Chloride) are largely restricted to the extracellular compartment Amount of Sodium in the ECF determines the volume of the compartment Aldosterone and AVP interact to maintain normal volume and concentration of ECF How does AVP and Aldosterone interaction affect Osmolality? To understand this concept let us consider a patient who has been Vomiting and has Diarrhea from a GIT infection: With no intake the patient becomes Fluid depleted Consequently both water and Sodium have been lost Because the ECF volume is low, Aldosterone secretion is High Thus, as the patient begins to take fluids orally, any salt ingested is maximally retained As this raises the ECF Osmolality, AVP action then ensures that water is retained too Aldosterone and AVP interaction will continue until ECF fluid volume and composition return to normal Aldosterone regulating the Sodium, AVP regulating the water What are the Electrolytes in the ECF and ICF? Cations and Anions in solution in all body fluids Sodium ion (Na+) is the Principal Cation in ECF Potassium ion (K+) is the Principal Cation in ICF Proteins and Phosphates are the main Anions in the ICF Chloride ion (Cl-) and Bicarbonate ion (HCO3-) are the main Anions in ECF Na+ ions are present at highest concentration in ECF and make the largest contribution to the total plasma Osmolality Despite the low concentration of K+ ion in the ECF, changes in Plasma concentration of K+ ion is very important and may have life threatening consequences Urea and Creatinine concentrations are frequently measured with Serum or Plasma Electrolytes because they provide an indication of Renal Function Increase in concentrations of Urea and Creatinine usually indicates a decrease in the Glomerular Filtration Rate in the kidneys How are solute and solvent related to solution? Solution is made up of Solute and Solvent Concentration of solution is a ratio of two variables: Amount of Solute (e.g., Sodium ions) and amount of Solvent Concentration of solution can change if either or both variables change For example: Sodium ion concentration of 140mmol/l may becomes 130mmol/l because the amount of Sodium in the solution has fallen or the amount of solvent has increased HYPONATRAEMIA: Hyponatraemia is a significant fall in Serum Sodium concentration below the reference range of about 135 145 mmol/L (what reference range is used for serum sodium in PMGH?) Hypo-Osmolality is synonymous with Hyponatraemia because Sodium is the only ion in the ECF in sufficient amount such that a decrease in concentration would significantly affect the Osmolality What are the possible types of Hyponatraemia? (Figs. 7a & 7b) Hyponatraemia due to water retention: More water than normal is retained in the body compartments and dilutes the constituents of the extracellular space causing Hyponatraemia Hyponatraemia due to Sodium loss: When loss of sodium ions exceeds loss of water, hyponatraemia may result Example: Loss of body fluids that contain Sodium are replaced simply by water TAKE NOTE: Illustrations in Figs. 7a & 7b emphasizes that Biochemical observation of Hyponatraemia gives no information about the Volume of the ECF compartments Information on Volume of ECF compartments can only be obtained from Clinical Examination of the Patient Some patients with life-threatening Sodium depletion may present with a Normal Serum Sodium concentration (Figure 8a, b, c) Illustrations in Figs. 8a, b, c clearly indicates that the Clinician must always give greater emphasis and attention to the History, Signs, and Symptoms of the Patient than to the Laboratory results on Serum Sodium What is Osmolality (Osmolarity)? It is the concentration of osmotically active particles in a solution, i.e., particles that cannot cross the semi-permeable membrane Water moves easily through the cell membrane that separate ECF from the ICF Osmosis is the flow of solvent across a semi-permeable membrane from a low solute concentration to a higher solute concentration Osmotic pressure is the driving pressure for water to move the given concentration of osmotically active particles Osmotic pressure must always be the same on both sides of a cell membrane, In the living cell, water moves across the membrane so as to keep the Osmolality the same, even if this water movement causes cells to shrink or expand in volume Osmolality of the ICF is always the same as the Osmolality of the ECF The two compartments contain Isotonic solutions What is the unit for expressing Osmolality (Osmolarity)? Osmolality of a solution is expressed in mmol solute per kilogram (mmol/kg, or mOsmol/kg) of solvent (which is usually water) Osmolarity of a solution is expressed in mmol solute per liter (mmol/L or mOsmol/L) of solution In humans, the Osmolality of serum (and other body fluids except urine) is in the range 285 to 295 mmol/kg (285 to 295 mOsmol/L) How is Osmolality measured and calculated? Osmolality of serum or plasma sample can be measured directly, or it may be calculated if the concentrations of the major solutes are already known Osmolality can be calculated as follows: Serum Osmolality = 2 x molar concentration of serum Sodium ions This simple formula for calculating Osmolality can be used ONLY if the serum concentrations of Urea and Glucose are within the reference ranges If either or both are abnormally high, the concentration of either or both (in mmol/l) must be added in to give the calculated Osmolality value Normal Conditions: Estimating and Calculating ECF Osmolarity: Plasma Osmolarity is representative of ECF Osmolarity and is clinically accessible ECF Osmolarity is dominated by [Na+] and the associated Anions which are necessary to maintain Electro-neutrality Under Normal conditions, ECF Osmolarity can be roughly estimated as: EMBED Unknown (Where Posm is Plasma Osmolarity) Since Intracellular Osmolarity is the same as Extracellular Osmolarity under normal conditions, this also provides an estimate of Intracellular Osmolarity.) Clinical Laboratory Measurement: Plasma Osmolarity measured in Clinical Laboratory includes contributions from Glucose and Urea In healthy individuals contribution from Glucose and Urea is small Under certain Pathological conditions, concentrations of Glucose and Urea may increase, Thus the measured Plasma Osmolarity measured by Clinical laboratory can be calculated as: Posm (measured) = 2[Na]P = [glucose]P + [BUN]P BUN = Blood Urea Nitrogen, Normal [glucose]p is 60 100 mg/dl, Normal [BUN]p is 10 20 mg/dl Glucose and BUN normally contribute about 5mOsm each (i.e., about 2% of the Plasma Osmolarity measured in the clinical lab What is effective and ineffective Osmolarity? Effective Osmolarity: Glucose, Na+ and Anions associated with Na+ have concentration gradients across the cell membrane and are therefore Effective Osmoles in the sense that they determine the distribution of water between ECF and ICF Ineffective Osmolarity: Urea (BUN) crosses cell membranes just as easily as water, so it does not contribute to redistribution of water between ECF and ICF Urea is therefore called an Ineffective Osmole Effective Osmolarity is given as: Posm (effective) = 2[Na]P + [glucose]P or, Posm (effective) = Posm (measured) [BUN]P What is Osmolal Gap (OG)? Osmolal gap is the difference between Measured and Calculated Osmolality (Osmolarity) Osmolal gap occurred when the Clinically measured Osmolality (Osmolarity) is higher than the Calculated Osmolality Osmolal gap suggests the presence of an unmeasured osmotically active substance in the blood Knowledge of the serum concentration of Sodium ions, Glucose and Urea or blood urea nitrogen (BUN) allows calculation of the Serum Osmolality (Osmolarity) to a degree that compares quite well to measured Osmolality (Osmolarity) How is Osmolal gap (OG) calculated? To calculate the OG, serum determination of MO, Na+, Glucose and Urea are necessary Determination of MO and for CO should be performed on the same serum sample Difference between Measured Osmolality (MO) and Calculated Osmolality (CO) is known as the Osmolality Gap or Osmolal Gap (OG) Osmolality (Osmolarity) Gap (OG) = MO CO How is OG interpreted? A large positive OG can help identify the presence in Serum of substances such as Ethanol, Methanol, Isopropanol, Ethylene Glycol, and Acetone Proper interpretation of OG requires knowledge of Anion Gap (AG), blood pH, and qualitative testing of the serum Ketone Bodies Anion Gap = [Na+] {[HCO3-] + [Cl-]} In Summary: Water is lost from the body as Urine and as obligatory Insensible losses from skin and lungs Na+ ions may be lost from the body in prolonged vomiting, diarrhea and intestinal fistulae AVP regulates renal water loss and thus causes changes in the Osmolality of body fluid compartments Aldosterone regulates renal Na+ ion loss and controls Na+ content of the ECF Changes in Na+ ion content of the ECF cause changes in volume of this compartment because of the combined actions of AVP and Aldosterone Hyponatraemia because of water retention is the commonest biochemical disturbance encountered in clinical practice In many patients the non-osmotic regulation of AVP overrides the osmotic regulatory mechanism and this results in water retention, which is a non-specific features of illness Patients with Hyponatraemia without oedema, but have normal serum urea and creatinine and blood pressure, have water overload Patients with Hyponatraemia and with Oedema are likely to have both water and sodium overload Hyponatraemia may occur in the patient with gastrointestinal or renal fluid losses, which have caused sodium depletion The low sodium concentration in serum occurs because water retention is stimulated by increased AVP secretion Patients with hyponatraemia because of sodium depletion show clinical signs of fluid loss such as Hypotension, such patients usually do not have Oedema SCHOOL OF MEDICINE AND HEALTH SCIENCES DIVISION OF BASIC MEDICAL SCIENCES DISCIPLINE OF BIOCHEMISTRY AND MOLECULAR BIOLOGY CLINICAL BIOCHEMISTRY: BMLS & BDS Yr. 3 INVESTIGATION OF RENAL FUNCTION An Overview The kidneys are the major excretory system in humans and other ureotelic organisms. The functional unit of the kidney is the Nephron (Fig 1a and 1b)) What are the major components of the Nephron? Major components of the nephron include the following: Glomerulus where filtration occurs Proximal tubule where main reabsorption occurs; Loop of Henle where concentration of filtrate occurs; Distal tubule where secretion occurs and Collecting duct where water reabsorption occurs What are some of the functions of the Kidneys? Kidneys regulate Extracellular Fluid (ECF) Volume and Electrolyte composition to compensate for wide daily variations in Water and Electrolyte intake. Kidneys form urine in which the potentially toxic waste products of metabolism are excreted. What are the Regulatory Functions of kidneys? Regulation of Water / Fluid Balance: Arginine Vasopressin (AVP) {Anti-Diuretic Hormone} produced by Posterior Pituitary acts on Kidney Tubules causing Reabsorption of Water from Glomerular filtrate. AVP stimulates incorporation of Aqua-porins into cell membranes in the collection duct where reabsorption of water occurs. Regulation of Electrolyte Balance: Aldosterone acts on Kidney Tubules causing Reabsorption of Na+ ions in exchange for secretion of K+ ions and H+ ions Regulation of Acid-base balance: Kidneys involve in maintaining pH (acidity/alkalinity) in blood and other body fluids Parathyroid Hormone (PTH): Promotes Tubular Re-absorption of Calcium; Promotes Phosphate Excretion Promotes Synthesis of 1, 25-Dihydroxy-Cholecalciferol, which regulates Calcium Absorption in Gastrointestinal Tract (GIT) Renin: An enzyme produced by Juxtaglomerular cells in kidneys Renin catalyzes conversion of Angiotensinogen to Angiotensin-1 Angiotensin Converting Enzyme (ACE) then converts Angiotensin-1 to Angiotensin II Angiotensin II stimulates biosynthesis of Aldosterone in Adrenal Cortex Erythropoietin: Peptide hormone that promotes biosynthesis of Hemoglobin Production of Erythropoietin is partly regulated by Kidneys Endocrine effects of the kidneys remain intact until End Stage Renal Failure How are the functions of the kidneys assessed? Functions of the kidneys can be assessed by a collection of tests sometimes referred to as Renal Function Tests. What are the Renal Function Tests? Collective term for a variety of individual tests and procedures used to evaluate the Functional State of the kidney: Tests for Glomerular Functions Tests for Tubular Functions Renal Function Tests Include the following: Urinalysis: First line test for Renal Function Creatinine Clearance (CC): used to measure Glomerular Filtration Rate (GFR) Inulin Clearance: used to measure GFR Para-Amino-Hippuric Acid (PAH): used to measure Renal Plasma Flow (RPF) Urine Osmolality, Plasma Creatinine, Plasma Urea, Plasma Electrolyte What tests are involved in Urinalysis? Randomly collected Urine sample is examined: Physically for: Color, Odor, Appearance, and Concentration (Specific Gravity) or Osmolarity Chemically for: Protein, Glucose, and Urine pH (acidity/ alkalinity); Microscopically for the presence of: Cellular elements (RBC, WBC, Epithelial cells), Bacteria, Crystals, Casts (structures formed by deposit of protein, cells, and other substances in the kidneys' tubules) How is the Creatinine Clearance calculated? Creatinine clearance is a measure of the GFR (i.e., the number of milliliters of filtrate made by the kidneys per minute.) of the kidney It measures the rate at which the kidneys can clear a compound from the blood Maximum rate that the plasma can be Cleared of any substance is equal to the GFR. GFR can be calculated from the Clearance of some plasma constituent, which is freely filtered at the Glomerulus, and is neither reabsorbed nor secreted by the kidney tubules In clinical practice Creatinine, which is already present in blood as a normal product of muscle metabolism, comes close to fulfilling the above requirements An estimate of the GFR can be calculated from the Creatinine content of a 24-hour urine collection, and the Plasma concentration of Creatinine within this period. Clearance of Creatinine from plasma is directly related to GFR provided that: Urine volume is collected accurately. There are no Ketone and Heavy Proteinuria present to interfere with the Creatinine determination. What is Glomerular Filtration Rate (GFR)? GFR is a useful index of the numbers of functioning Glomeruli GFR in the number of milliliters of filtrate made by the kidneys per minute GFR measures the rate at which the kidneys can clear a compound from the blood Normal GFR depends on normal Renal Blood Flow and Pressure GFR is directly related to body size, thus it is higher in men than in women GFR is also affected by age, with a reduced rate in elderly Reduction in GFR can be caused by: Restriction of Renal blood supply, Low Cardiac Output, Destruction of Nephrons by Renal Diseases, etc Reduction in GFR results in Retention of Waste Products of Metabolism in blood How can GFR be determined / calculated? GFR is directly related to Clearance, GFR can be calculated from the Clearance of some Plasma Constituent, which is freely Filtered at the Glomerulus, and is neither Reabsorbed nor Secreted by Kidney Tubules For Clinical Practice Creatinine, present in blood as a normal product of muscle metabolism, comes close to fulfilling the above requirements GFR can be calculated from the Creatinine content of a 24-hour urine collection, and the Plasma concentration of Creatinine within this period Inulin can be used to measure GFR because it is filtered but not re-absorbed or secreted by Renal Tubules GFR is Calculated as follows: (Creatinine Clearance or Inulin Clearance) GFR = CC = (U x V)/P Where U = Urine concentration of Creatinine (mmol/L) P = Plasma or Serum concentration of Creatinine (mmol/L or as (mol/L) V = Urine Flow Rate (ml/minute) {Urine Flow Rate = Total Volume of Urine collected in 24-hours divided by 24 x 60} GFR value must be corrected for body surface area of patients, this is calculated from the Age and Height of the patient in relation to the Standard Average Body Surface Area of normal individual Standard average body surface area = 1.73m2 EXAMPLE: QUESTION: Your are requested to determine the Creatinine Clearance in a male patient age 45 years admitted with loin pain in the Clinical ward in PMG. The volume of urine collected in 24-hour was 2160ml. The concentration of creatinine in the urine was 7.5mmol/L. The concentration of Creatinine in plasma was 150 (mol/L. Calculate the Creatinine Clearance of the patient and comment on the results. (Assume that the correction factor for body surface area is 0.8). The nursing staff that was helping you to collect the urine came to see you later and told you that the 2160ml of urine was collected in 17hours not 24-hours. How does this affect the result and its interpretation? ANSWER: The creatinine clearance (CC) adjusted for body surface area is calculated using the formula: CC = 1.73(U x V)/ (PA) Where U is the concentration of Creatinine in urine; V is the Urine Flow Rate; P is the concentration creatinine in plasma; 1.73 is the standard average body surface area of normal individual; A is the body surface area of the patient obtained from his height and age. As there are 1440 minutes in a day (24 x 60 = 1440), the Urine Flow Rate of this patient is V = 2160/1440 = 1.5 ml/minute. His urinary Creatinine concentration must be in the same units as his Plasma Creatinine concentration. Therefore his urinary Creatinine concentration U = 7.5 mmol/L. His plasma Creatinine concentration P = 150/1000 = 0.15 mmol/L. The correction factor for body surface area 1.73/A is given as 0.8. Thus CC = 0.8(7.5 x 1.5)/0.15 = 60.0 ml/minute. This value is low for a young male. (The normal range for male is 80 130 ml/minute). When it was discovered that the urine collection was for 17 hours and not 24 hours his Urine Flow Rate was recalculated. V = 2160/1020 = 2.1 ml/minute. Recalculating the CC value gives: CC = 0.8(7.5 x 2.1)/0.15 = 84.0 ml/minute Comments: This is the range expected in a young male. One can see, therefore, how errors in the timing and collection of urine significantly influence the calculation of the creatinine clearance. Errors in collection are by far the most common and serious errors encountered when estimating the creatinine clearance. Low Clearance values for Creatinine and Urea indicate diminished ability of the kidneys to filter these waste products from the blood and excrete them in the urine. As Clearance levels decrease, blood levels of Creatinine and Urea Nitrogen increase. Factors affecting CC values: The CC value can be increased, because of the following: Exercise, Pregnancy (this is due in part to the increased load placed on the kidney by the growing fetus), High cardiac output syndromes (note that as blood flow increases to the kidney, GFR and CC increase) The CC value can be reduced, because of the following: Impaired kidney function Conditions causing decreased GFR (e.g., congestive heart failure, cirrhosis with ascites, shock, and dehydration) conditions that are associated with decreased blood flow to the kidney will decrease GFR. How can the Creatinine Clearance be calculated using the Cockcroft and Gault equation? Cockcroft and Gault equation using the Ideal Body Weight (IBW) of a patient: For males: CC = {(140 A) x IBW} / (S x 72) For females: CC = {(140 A) x IBW} / (S x 0.85) A = age in years; S = concentration of creatinine in serum (umol/L) Actual body weight (ABW) should be used if it is less that the IBW If serum creatinine concentration is less than 1.0umol/L for patients over 65years, then use serum creatinine concentration of 1.0umol/L to calculate the CC. Estimation of IBW (kg): For male patients: IBW is 50.0kg plus 2.3kg for every 2.5cm over 1.5meter For female patients: IBW is 45.5kg plus 2.3kg for every 2.5cm over 1.5meter Cockcroft and Gault equation using adjusted body weight: This equation recommended for elderly obese patients with Actual Body Weight 25% over the IBW For males: CC = {(140 A) x Adjusted Body Weight} / (S x 72) For females: CC = {(140 A) x Adjusted Body Weight} / (S x 0.85) A = age in years; S = concentration of creatinine in serum (umol/L) Estimation of Adjusted Body Weight (ABW) = IBW plus 0.3(ABW IBW) Another method using Cockcroft and Gault formula to calculate Creatinine Clearance Cockcroft and Gault formula can be used to estimate the Creatinine Clearance of patients with the following exceptions: Patients should not be severely malnourished Patients should not be very obese Renal Function should not be severely impaired (GFR < 20 ml/min) (140 Age in yrs) x Wt in Kg x (0.85 for a female) CC (ml/min) -------------------------------------------------------- 0.814 x Serum Creatinine (micromoles / L) NB: To correct for muscle mass in Male patients: Multiple results by 1.22 What is Proteinuria? Glomerular basement membrane does not usually allow passage of albumin and large molecular weight proteins Small amount of protein, usually less than 25mg/24h is found in urine Positive screening test for protein (included in a routine urinalysis) on a random urine sample is usually followed-up with a test on a 24-hour urine sample that more precisely measures the quantity of protein Protein, in excess of 250mg/24h urine sample indicates significant damage to Glomerular membrane Persistent presence of significant amounts of protein in the urine, is an indicator of kidney disease What are the different types and causes of Proteinuria? Glomerular Proteinuria: Abnormal leaking of large and small molecular weight proteins into Glomerular filtrate resulting from damaged of Glomerular membrane May be due to Exercise, Fever (Febrile Proteinuria), Congestive Cardiac Failure, Glomerulonephritis, Renal Stenosis Glomerulonephritis: Common cause of persistent Proteinuria Amount of protein in urine depends on: Extent of Glomerular damage, Molecular mass of protein, Capacity of Tubule to reabsorb or metabolize the proteins May be mild, moderate or Severe Proteinuria Severe Proteinuria: Protein loss in urine exceeds synthetic capacity of body to replace protein, thus resulting in HypoProteinemia (low protein in blood) Severe persistent Proteinuria is one of the feature of Nephrotic Syndrome Nephrotic Syndrome: Large amount of protein loss in urine Leads to Hypo-Proteinemia and Edema Edema may be caused by low albumin and secondary Hyper-Aldosteronism Patients may also develop Hyper-Lipidemia Causes of Nephrotic Syndrome: Glomerulonephritis, Systemic Lupus Erythematosus, Diabetes Nephropathy Tubular Proteinuria: Failure of Tubules to reabsorb filtered plasma proteins Abnormal secretion of protein into urinary tract May be due to Tubular or Interstitial damage Low with low molecular mass are excreted by Tubules Loss of protein usually mild about 2.0g/24hours Sensitive test for assessment of Renal Tubular damage: Measure Urinary 2-Microglobulin (> 0.4mg/24hours indicates damage) Overflow Proteinuria: Large amount of low molecular weight proteins accumulate the urine. Proteins are filtered at the Glomerulus, but are not reabsorbed or catabolised completely by the Tubules Causes of Overflow Proteinuria: Acute Pancreatitis, Multiple Myeloma, Intravascular Hemolysis, Myelomonocytic Leukemia, Crush Injuries Orthostatic (Postural) Proteinuria: Proteinuria occurs after standing for a long time Protein absent in early morning urine samples Plasma Creatinine: Creatinine is a by-product of muscle energy metabolism Creatinine is cleared from the blood by the kidneys and excreted in urine Production of Creatinine depends on muscle mass, which fluctuates very little Normally Creatinine in blood remains relatively constant Plasma Creatinine concentration is inversely proportional to Creatinine Clearance Plasma Creatinine concentration is affected very little by Liver function, Elevated Plasma Creatinine is a sensitive indication of impaired Renal function Normal Plasma Creatinine concentration of a patient does not always indicate normal Renal function Progressive rise in serial Plasma Creatinine concentration indicates impaired Renal function Blood Urea Nitrogen (BUN) Urea is a by-product of protein metabolism Urea is formed in the Liver, released in the blood then filtered by the Glomerulus and excreted in the urine BUN test measures the amount of Nitrogen contained in Urea High BUN levels can indicate kidney dysfunction, but because BUN is also affected by protein intake and liver function, the test is usually done in conjunction with blood creatinine, a more specific indicator of kidney function Urea can be affected by other factors, thus elevated BUN, by itself, is suggestive, but not diagnostic, of kidney dysfunction What other parameters in blood that can be measured: Measurement of the blood levels of other elements regulated in part by the kidneys can also be useful in evaluating kidney function. These include: Electrolytes (Sodium, Potassium, Chloride, Bicarbonate), Calcium, Magnesium, Phosphorus, Protein, Uric Acid, and Glucose Urine Osmolality: Osmolality is the measurement of Urine concentration that depends on the number of particles dissolved in the urine Osmolality is expressed as mOsm/kg (milli-Osmols per kilogram) of water For apparently healthy person the Urine is usually more concentrated than Plasma Urine Osmolality / Plasma Osmolality > 1 Measurement of Osmolality can be carried out on early morning urine samples, on multiple timed urine samples, or on a cumulative sample collected over a 24-hour period Inability of the kidney to concentrate the urine in response to restricted fluid intake, or to dilute the urine in response to increased fluid intake may indicate decreased Renal Function Renal Tubular Function: Tubular reabsorption must be efficient to ensure effective reabsorption of: Water, Sodium, Glucose, Bicarbonate, etc are not lost from the body About 180 litres of fluid pass into the Glomerular filtrate each day, and about 99% is reabsorbed via the Tubules Tubular function can be assess by comparing Osmolality of Urine and Osmolality of Plasma Urine / Plasma Osmolality Ratio is usually between 1.0 and 3.0, because Urine is normally more concentrated than Plasma Urine / Plasma ratio < 1.0, indicates that Renal Tubules are not reabsorbing water Some disorders of Tubular function are inherited: Example, Some patients are unable to reduce their urine pH below 6.5, because of a specific failure of Hydrogen ion secretion How does the kidney regulate Acid-Base balance? Kidney regulates Acid-Base Balance by controlling: Re-absorption of Bicarbonate ions (HCO3-) Secretion of Hydrogen ions (H+) Both processes depend on formation of HCO3- ions H+ ions from CO2 and H2O within Renal Tubular cells: Carbonic Anhydrase CO2 + H2O (======( H2CO3 (=====( H+ + HCO3- H+ ions formed are actively secreted into Tubule fluid in exchange for Na+ What mechanisms are used in the kidney for elimination of Acids? Mechanisms used for elimination of Acids include: Re-absorption of Sodium Bicarbonate (NaHCO3) by Proximal Renal Tubules, (Fig. 1) Regeneration of HCO-3 by Distal Renal Tubules (Fig. 2) Production of Ammonia (NH3) by Distal Renal Tubules, which secrete H+ ions and maintain a gradient of H+ ions between cell and lumen (Figs. 3 & 4) Secretion of H+ ions by the Tubular cells serves initially to reabsorb HCO3- ions from the Glomerular filtrate so that they are not lost from the body When all the HCO3- ions have been recovered (reabsorbed), any deficit due to the buffering process is regenerated How are the HCO3- ions in the Glomerular Filtrate Reabsorbed (Reclaimed or Recovered)? (Interpretation of Fig. 1) HCO3- ions are freely filtered by the Glomerulus Amount of HCO3- ions in filtrate is equivalent to that in Plasma If HCO3- ions were not reabsorbed in Renal Tubules the Buffering Capacity of Blood Plasma would be depleted rapidly Reabsorption of HCO3- ions occur mainly in Proximal Tubule HCO3- ions filtered through the Glomerulus combine with H+ ions secreted from the Tubular cell forming Carbonic Acid (H2CO3) H2CO3 is then converted to CO2 and H2O by Carbonic Anhydrase II, which is present in the brush border of Renal tubular cells CO2 produced readily crosses into Tubular cell Inside Tubular cell the CO2 interacts with H2O again, to form H2CO3 catalyzed by Carbonic Anhydrase II H2CO3 then dissociates to form HCO3- ions and H+ ions HCO3- ions formed diffuse into the blood stream whilst the H+ ions are transported into the Tubular Lumen in exchange for Na+ ions About 80 90% of HCO3- ions in the Glomerular Filtrate are Reabsorbed or Reclaimed in Proximal Tubule How are the HCO3- ions Regenerated? After reabsorption of HCO3- ions is completed, the process of regeneration compensates for any deficit in the amount of HCO3- reabsorbed Mechanisms for reabsorption of HCO3- ions and for the regeneration of HCO3- ions are completely different (Compare Fig. 1 and Fig. 2) How are Hydrogen ions (H+) excreted by the Renal Tubules? H+ ions are secreted in exchange for Na+ ions Energy for this exchange comes from Na+ - K+ -ATPase (Sodium-Potassium Pump) that maintains the concentration gradient for Na+ ions H+ ions are secreted by two major buffers: Phosphate buffer (H2 PO4- / HPO42-) Ammonium buffer (NH4+ / NH3) Phosphate (HPO42-) is freely filtered by Glomerulus and passes down the Tubule where it combines with H+ ions to form H2 PO4- (Fig. 3) Ammonia (NH3) is produced in Renal Tubular cells by the action of the enzyme Glutaminase on the Amino acid Glutamine Glutamine + H2O =======( Glutamate + NH3 Glutaminase functions optimally at a lower (more acidic) than normal pH More Ammonia is produced during Acidosis thus improving the buffering capacity of the Urine Ammonia is un-ionised and so rapidly crosses into the Renal Tubular Lumen down its concentration gradient NH3 combines with H+ ions to form NH4+ ions (Ammonium ions), which being ionised does not pass back into the tubular cell NH4+ ions are lost in urine, along with H+ ions (Fig. 4) What is anion gap? Anion Gap (AG) calculation is the sum of routinely measured Cations minus routinely measured Anions: (Na+ + K+) (Cl- + HCO3-). However, because K+ is a small value numerically, it is usually omitted from the AG equation so that, the most commonly use equation is AG = Na+ - (Cl- + HCO3-) It is the venous value of HCO3- that should be used in the calculation. Venous value of CO2 can be used in place of venous Bicarbonate value. Equation will then be AG = Na+ - (Cl- + CO2) The normal AG calculated in this manner (without K+) is about 12.4mEq/L. Anion Gap exists simply because not all Electrolytes are routinely measured. Normally there is electrochemical balance, so that the sum of all negatively charged electrolytes (Anions) equals the sum of all positively charged electrolytes (Cations). However, several anions are not measured routinely, leading to the anion gap. Anion Gap is thus an artifact of measurement, and not a Physiologic reality. UNIVERSITY OF PNG SCHOOL OF MEDICINE AND HEALTH SCIENCES DIVISON OF BASIC MEDICAL SCIENCES DISCIPLINE OF BIOCHEMISTRY AND MOLECULAR BIOLOGY BMLS & BDS Yr 3 RENAL FAILURE: OLIGURIA AND ANURIA An Overview What are some of the major functions of the kidneys? Regulation of Water (Fluid), Regulation of Electrolyte Regulation of Acid-Base Balance Excretion of products of Protein and Nucleic Acid metabolism such as Urea, Creatinine, Creatine, Uric Acid, Sulfate, Phosphate, etc. Kidneys are also Endocrine Organs, producing a number of Hormones, and are under the control of other hormones How can the functional state of the kidneys be assessed? To answer this question check your lecture on Renal function for details RENAL FAULURE: Renal failure is the cessation of kidney function Acute Renal Failure (ARF) is when the kidneys suddenly fail to carryout major functions, this can occur over a period of hours or days. ARF is usually reversible and normal Renal Function can be regained. Chronic Renal Failure (CRF) develops over months or years and leads eventually to End Stage Renal Failure (ESRF). CRF usually cause irreversible damage to the kidneys. ARF can arise from a variety of problems affecting the kidneys and/or their circulation. ARF usually presents as a sudden deterioration of Renal function indicated by rapidly rising: Serum urea concentration and Serum creatinine concentrations. Severely ill patients may develop ARF thus monitoring of kidney function is important in these groups of patients. Usually in the first 24 hours of ARF: Serum and Urine tests for renal function might not reveal any abnormality. If the Urine Output of the patient falls to less than 400ml in 24 hours, the patient is said to be Oliguric (Oliguria). Some patients may have Normal Urine Output, with reduced Glomerular Filtration and Tubular Dysfunction (this is referred as Non-Oliguric ARF) Some patient may not pass any urine, such patients are said to be Anuric (Anuria). What are the different phases of ARF? Oliguric Phases, Diuretic Phase and Recovery Phase OLIGURIC PHASE - in brief: When the urine output falls to less than 400 ml in 24 hours, the patient is said to be Oliguric. Some patients may not pass any urine, such patients are said to be Anuric Some patients may have normal urine flow, low GFR, when tubular dysfunction predominates (Non-Oliguric ARF) Oliguria is mainly due to a fall in the GFR In Oliguric phase the urine formed usually has Osmolality similar to Plasma and a relatively high Na+ concentration, since the composition of the small amount of Glomerular Filtrate produced is only slightly altered by the damaged Tubules. Plasma Na+ ion concentration is usually low due to a combination of factors: Intake of water in excess of the amount able to be excreted, Increase in metabolic water from increased tissue catabolism, and A shift of Na+ ions from ECF to (ICF) Plasma K+ ion concentration, on the other hand, is usually increased due to: Impaired Renal output and Increased tissue catabolism, Aggravated by the shift of K+ ions out of cells that accompanies the metabolic acidosis which develops due to failure to excrete H+ ions and to the increased formation of H+ ions from tissue catabolism Retention of: Urea, Creatinine, Phosphate, Sulfate and other waste products occurs Rate of increase of urea concentration in plasma depends on the rate of tissue catabolism; this, in turn, depends on the cause of the ARF. How can the low urinary output in ARF be differentiated from Hypovolaemia? To differentiate the low urinary output of suspected ARF from that due to severe circulatory impairment with reduced blood volume the tests in the table below may be helpful. Note that none of these tests can be completely relied upon to make the important and urgent distinction between Renal Failure and Hypovolaemia. Careful assessment of fluid status of the patient, possibly including measurement of the central venous pressure, is also required. INVESTIGATION OF LOW URINARY OUTPUTInvestigationSimple HypovolaemiaAcute Renal FailureUrine Osmolality usually > 500mmol/kgusually < 400mmol/kgUrine [urea]:Plasma [urea] usually > 10usually < 5Urine [Na+] usually < 20mmol/LUsually > 40mmol/L What laboratory parameters are used to monitor the Oliguric phase? For monitoring patients in the Oliguric phase of ARF: The following are very important and must be determined at least once daily Plasma Creatinine, Plasma Urea and K+ ion concentrations are particularly important, In order to determine fluid and Electrolyte replacement requirement, the following must be regularly assessed: Volume of urine and its Electrolyte composition Volume and composition of any other measurable sources of fluid loss DIURETIC PHASE: With the onset of the diuretic phase: Urine volume increases, Clearance of Urea, Creatinine and other waste products may not improve to the same extent Plasma Urea and Creatinine concentrations may therefore continue to rise, at least at the start of the Diuretic phase. Large losses of Electrolytes may occur in urine, this should be monitored and replaced as appropriate. Plasma K+ ion concentration tends to fall as the diuretic phase continues, due to the shift of K+ ions back into the cells and to marked losses in the urine resulting from impaired conservation of K+ ions by the still-damaged tubules. Usually, Na+ deficiency occurs also, due to failure of renal conservation. Throughout the diuretic phase therefore, it is important to measure: Plasma creatinine, Plasma urea, Na+ and K+ concentrations at least once daily, and Monitor the urine flow rate and electrolyte output. How can Acute Renal Failure be classified? ARF is usually caused by problems that affect the kidneys. A simplified classification of ARF or uraemia is as follows: Pre-Renal: When blood supply to the kidneys is affected, this may be due to vascular obstruction or to reduced perfusion Renal: When the problem is within the kidneys (damage kidneys). May be due to a variety of diseases, or the Renal damage may be a consequence of prolonged Pre-renal or Post-renal problems. Post-renal: When the urinary drainage of the kidneys is impaired because of an obstruction, May be due to either Renal Stones, Carcinoma of cervix, Prostate, or Bladder. What are some of the factors that can cause Pre-renal ARF? Some of the factors that can precipitate Pre-renal ARF are usually associated with a reduced effective ECF volume and may include some of the following: Decreased plasma volume because of: Blood loss, Burns, Prolonged Vomiting or Diarrhea Diminished Cardiac Output Local factors such as an Occlusion of Renal Artery. Pre-renal factors usually lead to decreased renal perfusion and reduction in glomerular filtration rate (GFR). Both Arginine Vasopressin (AVP act to influence water balance) and Aldosterone (affects Na+ reabsorption in the nephron) are secreted maximally and a small volume of concentrated urine is produced. What are some of the Biochemical finding in a patient with Pre-renal ARF? Some of the Biochemical finding in Pre-renal ARF include the following: Serum Urea and Creatinine are increased: Urea is increased proportionally more than Creatinine because of its reabsorption by the Tubular cells, particularly at low urine flow-rates. This leads to a relatively higher serum Urea concentration than Creatinine, which is not so reabsorbed. Hyperkalaemia due to decreased GFR and Acidosis. Metabolic Acidosis due to the inability of the kidney to excrete H+ ions High urine Osmolality. What are some of the factors that can cause Post-renal ARF? Post-renal factors usually cause decreased Renal function, because: Effective Filtration pressure of the Glomeruli is reduced due to the backpressure caused by the blockage. Some of the causes of this include the following: Renal Stones, Carcinoma of Cervix, Prostate or Occasionally Bladder. Failure to correct the Pre-renal or Post-renal factors in a patient can lead to Intrinsic Renal damage (Acute Tubular Necrosis). Patients in the early stages of Acute Tubular Necrosis may have only a modestly increased Serum Urea and Creatinine, which then rises rapidly over a period of days, in contrast to the slow increase over months and years seen in chronic renal failure. It may be difficult to decide the reason for a patients oliguria. The biochemical features that distinguish pre-renal ARF from Intrinsic Renal damage are shown below. Biochemical features in the differential diagnosis of Oliguric patient Biochemical featurePre-renal failureIntrinsic Renal DamageUrine sodium< 20 mmol/L> 40 mmol/LUrine [urea] : Serum [urea]> 10:1< 3:1Urine/Plasma Osmolality > 1.5:1< 1.1:1 Bicarbonate buffer system: The main buffer in the blood is the Bicarbonate buffer, which is regulated by the enzyme called Carbonic Anhydrase. The buffer can be represented as follows: CO2 + H2O (==( H2CO3 (==( H+ + HCO3- pH = pKa + Log {[HCO3-] /(PCO2)} This equation shows that the pH (or H+ ion concentration) in blood varies as the Bicarbonate ion concentration (i.e., [HCO3-]) and Partial Pressure of CO2 (i.e., PCO2) change. What are the factors that affect the Bicarbonate buffer? Factors that can affect the Bicarbonate buffer are as follows: Following factors cause increase in [H+] ion, which implies decrease in pH. Adding H+ ions, Removing HCO3- ion or Increasing PCO2 The following factors will cause the [H+] ion to fall, which implies an increase in pH. Removing H+ ions, Adding HCO3- or Lowering PCO2 What do you understand by Metabolic and Respiratory Acid Base disorders? Metabolic Acid Base disorders are those, which directly cause a change in the Bicarbonate concentration (i.e., [HCO3-]). Examples include: Loss of Bicarbonate ions from the Extracellular Fluid or Build up of H+ ions from the Ionization of Ketone bodies. Respiratory Acid Base disorders are those that directly affect the PCO2. Impaired Respiratory function causes a build up of CO2 in blood, whereas, less commonly, Hyperventilation can cause a decrease of PCO2. The definitions are as follows: Metabolic acidosis The primary disorder is a decrease in [HCO3-] Metabolic alkalosis The primary disorder is an increased [HCO3-] Respiratory acidosis The primary disorder is an increased PCO2 Respiratory alkalosis The primary disorder is a decreased PCO2 What are the compensatory responses for Primary Acid Base disorders? In general the predicted Compensatory response in [HCO3-] or PCO2 when [H+] changes as a result of Primary Acid Base disorders are show in the table below: PRIMARY DISORDERCOMPENSATORY RESPONES( PCO2 (Respiratory acidosis)( HCO3-( PCO2 (Respiratory alkalosis)( HCO3-( HCO3- (Metabolic acidosis)( PCO2( HCO3- (Metabolic alkalosis)( PCO2  UNIVERSITY OF PNG SCHOOL OF MEDICINE AND HEALTH SCIENCES DISCIPLINE OF BIOCHEMISTRY AND MOLECULAR BIOLOGY DIVISION OF BASIC MEDICAL SCIENCES CLINICAL BIOCHEMISTRY LECTURE: BMLS and BDS YEAR 3 CARBOHYDRATE HOMEOSTASIS PART 1 WHAT IS HOMEOSTASIS (give an example)? All organisms have mechanisms in place for maintaining a relatively constant internal environment for the cell Homeostasis. These mechanisms also provide for appropriate responses to changes in the external environment and the associated demands those changes place on maintaining homeostasis in other words: Changes that potentially produce a stress on the system, A means of sensing the changes, A graded response, and A correction of the stress put on the system, That is, a complete feedback-loop resulting in the return to the homeostatic set-point. Endocrine, nervous and immune systems etc. are responsible for mediating both detection and response from the level of the cell to that of the whole organism. An example of homeostatic control is the maintenance of Glucose level in the blood. Maintenance of blood glucose level is significant to humans and normally this is under control of numerous exquisitely sensitive Homeostatic mechanisms. The major system involves the cells of the Endocrine pancreas, their detection of blood glucose levels, and the hormones that they secrete. Defects in this system are responsible for one of the major challenges to human health Diabetes Mellitus. Why does the body need adequate amount of Glucose? Under normal Physiological conditions the Brain and the rest of the nervous tissue utilize Glucose as a major substrate for energy production. Even in long-term fasting the Brain still requires significant amount of glucose. Red blood cells do not contain mitochondria therefore they can only obtain energy by Anaerobic Glycolysis. Skeletal muscle at rest uses predominantly Lipid as the energy source but in heavy exercise also draws upon muscle Glycogen and blood glucose for energy production. However, because Brain and Red Blood Cells depend almost exclusively upon Glucose as their major source of energy, it is essential that Glucose is always available in adequate amount in the blood. How does High Intake of Glucose relate to Insulin level in the blood? Digestion of Carbohydrate is very rapid and glucose levels increase in the blood shortly after dietary intake (following a meal), but this increase is only temporary and, within two to three hours, blood glucose levels are restored to the Pre-prandial level. Increase in blood glucose level after a meal is immediately followed by increase in Blood Insulin level (See Fig. 1.) Insulin secretion is stimulated by several events that are associated with Glucose intake. These events include the following: Elevated blood glucose directly stimulates Pancreatic Insulin release from the beta cells (Islets of Langahan) in the Pancreas. Insulin release is also stimulated by other components of the typical diet, notably Leucine and Arginine derived from the digestive hydrolysis of protein in the diet. Digestive process stimulates the release from the GIT of Gastrin, Pancreozymin, Cholecystokinin, and Glucagon-like gastrointestinal peptide Glicentin. These hormones appear to feed forward to the Pancreatic (-cell and stimulate insulin release in an anticipatory manner. Insulin release is also under neural control, possibly also as an anticipatory event. How does the composition of the diet affect blood levels of Insulin and Glucagon? Blood levels of both Insulin and Glucagon are changed after consumption of a diet. Magnitude and direction of the change depends on the composition of the diet consumed. If the diet contains only Carbohydrate, then, there will be a fall in the blood level of Glucagon as a result of direct Glucose inhibition of the alpha-cells in the Islets and due to the released of Insulin from the beta-cells in the Islets. If the diet is high in protein and low in Carbohydrate then Glucagon secretion will be stimulated as a consequence of Amino Acid Influx. In a typical diet that contains both Carbohydrate and Protein, plasma Glucagon levels often may not change noticeably, while Insulin level may increase. HOW DOES THE BODY NORMALLY DISPOSE OF HIGH INTAKE OF GLUCOSE? What is the role of the Liver in the disposal of high intake of glucose? After periods of fasting, such as overnight fasting, a substantial amount of carbohydrate consumed in the diet is converted to hepatic Glycogen. The first important site of ingested Glucose metabolism is the Liver. The liver, which is freely permeable to glucose, quite typically extracts about 50% of the Carbohydrate load. In the liver glucose is first converted to G-6-P (by either Glucokinase or Hexokinase) and then to Glycogen. The major signals for this include: Insulin, which promotes the activation of Glycogen Synthase. Low level of plasma Glucagon that causes reduction in the level of hepatic cyclic-AMP; thus blocking the cyclic-AMP-dependent protein kinase-catalyzed inactivation of Glycogen Synthase. High levels of plasma glucose that promote high levels of G-6-P, which serves as a feed-forward mechanism to allosterically stimulate the Glycogen Synthase. These signals result in hepatic glucose storage. Thus, available glucose will be converted into hepatic glycogen until the liver has restored its optimal level of glycogen. The signal, which terminates the synthesis of Glycogen, is the level of Glycogen itself, which acts by inhibiting Glycogen Synthase Phosphatase. Thus, the glucose, which is not taken up by the liver is distributed to other tissues, for metabolism in those tissues. What is the role of the Muscle tissue in the disposal of high intake of glucose after the action of the liver? Insulin directly stimulates the uptake of glucose into muscle. Glucose taken up by the muscle is normally used to replenish the muscle stores of Glycogen. Excess glucose can also be used for protein synthesis, so as to replenish those proteins that might have been degraded for Gluconeogenesis during the period of fasting. The signal for elevated protein synthesis from glucose is insulin. What happens to the excess glucose remaining in the blood after the Liver and Muscle tissues have extracted and stored enough glucose as Glycogen? With the exception of the Brain, Liver, and Blood cells, insulin directly stimulates both the entrance of glucose into most cells of the body and its use as substrate for anabolic processes. In humans, the live plays a major role in converting excess glucose into Triacylglycerols (Triglycerides), packaging them into VLDL and exporting the VLDL to Adipose tissue. As a consequence, much of the glucose in excess of that needed to restore Glycogen levels ends up as Triacylglycerols stored in the Adipocytes. In other words, most of the glucose in excess of that used by Liver and Muscle will be taken up by Adipocytes. The primary signal need for conversion of excess glucose to Triacylglycerols for storage in Adipocytes is again the Insulin-stimulated transport of glucose into the cell. Take Note: A general summary of the disposition of high glucose intake is presented in Fig. 2. The general concept is as follows: As much glucose as is practical is stored in the Liver and in Muscle. When this level is optimal, the bulk of the remaining glucose is used for other biosynthetic purposes and any excess is then converted into Fatty acids and stored as Triacylglycerols. Although the glucose levels in systemic blood are elevated, they do not exceed the renal threshold (200mg %) therefore, glucose does not spill over into the urine. Thus, in the disposal of high blood glucose, Insulin plays a pivotal role in stimulating a range of anabolic processes in addition to those of Glycogen, Protein and Triacylglycerol synthesis. Insulin does so by a variety of Regulatory mechanisms, which may involve either the modification of key regulatory enzymes and/or regulation of their synthesis. In addition to the role of insulin is the absence of the insulin Counter-regulatory hormones Glucagon, Glucocorticoids and Catecholamines. How is the glucose level in the blood regulated during fasting? As already discussed above after meal, glucose is rapidly absorbed and dispersed into the various tissues. For the rest of the day, for the person with a typical intermittent eating schedule, blood glucose level remains constant. In a healthy individual, the blood glucose level usually remains constant, even if no food is consumed within a 24-hour period. In a healthy individual, during prolonged fasting the blood glucose level usually decreases only slightly, but is within normal range. During the period of prolong fasting, glucose is still being actively metabolized by tissues such as brain and the red blood cells therefore, the blood glucose that is utilized must be replenished. The primary source of the glucose that keeps the blood glucose level within the normal range during the period of fasting is the liver. This is done initially by bread down of Glycogen stored in the liver (Hepatic Glycogenolysis), and later by contribution from Gluconeogenesis (the synthesis of Glucose from Non-carbohydrate sources) in the liver. What is the role of the liver in regulating blood glucose level during fasting? The role of the live in the regulation of blood glucose during fasting involves two major pathways: Utilization of Glycogen stored in the liver (Glycogenolysis) and formation of Glucose from non-carbohydrate sources (Gluconeogenesis) Glycogenolysis: Amount of Glycogen stored in liver is about 5% to 10% of wet weight of the liver. Most of the Glycogen stored in the liver is usually mobilized and used up within the first 24 to 36 hours of fasting. The First positive signal for stimulation of Glycogen breakdown (Glycogenolysis) in the liver is increase plasma level of the hormone Glucagon, which is secreted in response to Hypoglycemia. The second positive signal is the absence of insulin. During Hypoglycemia, plasma level of Insulin is usually low. During hepatic Glycogenolysis, Glucose-1-Phosphate is produced from Glycogen. The Glucose-1-Phosphate is then converted to Glucose-6-Phosphate The G-6-Phosphate is then converted to Glucose by Glucose-6-Phosphatase, which is an enzyme that is very active in the Liver. The Glucose is then released into the blood to keep the blood level normal. Thus, through the hormones Glucagon and Insulin there is a tight regulation of Glycogen breakdown to Glucose that directly maintains the level of Glucose in the blood. In the initial phases of starvation/fasting this is the major Glucose-producing mechanism. Hepatic Glycogenolysis is also regulated by Catecholamines (Adrenaline and Noradrenaline). Catecholamine release is a less sensitive hypoglycemic signal compared to Glucagon, but it does play a significant role in stimulating hepatic Glycogenolysis in circumstances of additional stress and marked Hypoglycemia. Gluconeogenesis: As hepatic Glycogen stores become depleted during fasting (or starvation) the only other significant source of Glucose is Gluconeogenesis. The site of Gluconeogenesis and the sources of precursors depend upon the duration of Caloric deprivation. Although the Kidney assumes importance as a source of new glucose during protracted starvation, during brief fasting at least 90% of total Gluconeogenesis occurs in the Liver. How does skeletal muscle regulating blood glucose level during fasting? Glycogen content in Skeletal muscle (about 1% wet weight) is lower that in the liver, however because the total mass of skeletal muscle is much higher than that of liver, the Total Glycogen content in skeletal muscle is much higher than Glycogen content in the Liver. Glycogen in skeletal muscle is not readily available to maintain blood glucose concentration. This is because muscle tissue does not contain the enzyme Glucose-6-Phosphatase, and thus cannot convert Glucose-6-Phosphose to Glucose. Under anaerobic conditions the muscle converts Glucose to Lactate, which via the Cori cycle can be converted to Glucose in the Liver. How is Gluconeogenesis regulated? Gluconeogenesis is regulated by multiple factors. The primary signals that attune the body to the status of Gluconeogenesis are as follows: Glucagon as an Acute Modulator: The actions of Glucagon are directed towards the production of Glucose. Glucagon stimulates Glycogen break down, Glucagon stimulates formation of Glucose from Gluconeogenic intermediates, Glucagon stimulates Triacylglycerol and Fatty acid breakdown and their Oxidation. The absence of Insulin: The actions of Insulin are directly opposite to those of Glucagon. Insulin stimulates Glycogen synthesis, Insulin stimulates Glycolysis, and Insulin stimulates the biosynthesis of fatty acids. Glucocorticoids (e.g. Cortisol) as Chronic Modulators: Glucocorticoid actions are more complex than either Insulin or Glucagon. In simple terms: Glucocorticoids stimulate Fatty acid breakdown and stimulate Gluconeogenesis, but they also increase the rate of hepatic Glycogen synthesis. Glucocorticoids are one of the major signals for the degradation of muscle proteins, with the amino acids serving as precursors for Gluconeogenesis. Effects of Glucocorticoids are long term, effects of Glucagon are moment to moment. Take Note: The three primary tissues involved in Glucose conservation are the Liver, Skeletal muscle and Adipose tissue. Glucagon actions are essentially restricted to the Liver and Adipose tissue. WHY?? In the Liver, Glucagon stimulates Glycogen break down and also stimulates Gluconeogenesis. Glucocorticoids activate hepatic Gluconeogenesis synergistically with Glucagon. A major site of Glucocorticoids actions is on Skeletal Muscle; The presence of Glucocorticoids and the absence of Insulin are the Primary signals for enhanced Protein degradation. Five Phases of Glucose Homeostasis: Glucose Homeostasis in Humans can be divided into Five Phases as shown in Fig. 3. SUMMARY: Understanding Glucose Homeostasis: The balancing acts: Hypoglycemia and Hyperglycemia: Glucose Homeostasis involves extensive contributions from various metabolic tissues (Liver, Skeletal muscle, Adipose tissue) tightly regulated and balanced by metabolic Endocrines. Hypoglycemia and Hyperglycemia refers to circumstances when this balance is disturbed, giving uncharacteristically low or high blood glucose concentrations, respectively. The circumstances that give rise to Hypoglycemia or Hyperglycemia can generally be divided in three categories, namely: Factors related to effective Insulin concentration, Insulin Counter-Regulatory Hormones Sources of Fuel for the tissues. Insulin Counter-Regulatory Hormones: The Insulin Counter-Regulatory Hormones, (Glucagon, Catecholamines, Glucocorticoids and Growth hormones) antagonizes the actions of Insulin. Each is elevated in plasma in response to hypoglycemia. Study Questions for Glucose Homeostasis: Why does the body need adequate amount of glucose? How is the body able to dispose of high glucose level in the blood after a meal? How is the blood glucose level regulated during period of fasting? What is the role of the liver in the regulation of blood glucose during fasting? What is the role of the muscle in the regulation of blood glucose during fasting? CLINICAL BIOCHEMISTRY BMLS-3 & BDS CARBOHYDRATE HOMEOSTASIS PART 2 DIABETES MELLITUS DIAGNOSIS AND MONITORING What are some of the metabolic functions affected by Insulin? Metabolic functions enhanced are: Glucose uptake in muscle and adipose tissue Glycogenesis Glycolysis, Protein synthesis, Cellular uptake of ions, especially Potassium and Phosphate ions Insulin stimulates biosynthesis of: Glycogen, Fats, Proteins, Insulin inhibits degradation of: Glycogen, Fat, Proteins Insulin affects the uptake of Glucose into: Muscle cells, Adipose tissue, Connective tissues, White blood cells Insulin DOES NOT affects uptake of Glucose into: Brain, Liver, Kidneys Insulin counter regulatory hormones, such as Glucagon, Epinephrine, Glucocorticoids, and Growth hormone oppose the actions of Insulin What is the Insulin feedback loop? Insulin feedback loop is: Action of Insulin and Insulin Counter Regulatory Hormones in regulating blood glucose level Homeostatic regulation of blood glucose is the result of balance between action of Insulin and Insulin Counter-regulatory Hormones: INSULIN FEEDBACK LOOP Failure of Insulin feedback loop affects homeostatic regulation of blood glucose Failure of part of the loop can cause increase in blood glucose level, Glucose cannot get into cells that use or store it Excess Glucose may be dumped into the urine resulting in Sweet Urine (Diabetes Mellitus!!!!) DIABETES MELLITUS (DM) What is the definition of Diabetes Mellitus? Precise definition of Diabetes Mellitus is very difficult DM: a disease characterized by derangements in Carbohydrate, Fat and Protein metabolism DM: a syndrome characterized by Hyperglycemia due to: An absolute or relative lack of Insulin and/or Insulin Resistance What are the major types of Diabetes Mellitus? Primary DM is generally sub-classified into: Type I DM {Insulin Dependent Diabetes Mellitus (IDDM or)} Type 2 {Non-Insulin Dependent Diabetes Mellitus (NIDDM)} Secondary DM: may result from a number of causes including: Pancreatic disease, Endocrine disease such as Cushings syndrome, Drug therapy, Insulin receptor abnormalities, Gestational diabetes What are some of the possible causes of Type 1 DM? Type 1 DM, also called Juvenile-Onset Diabetes because it usually appears in childhood or in teenagers Type 1 DM is not only limited to juvenile patients Causes of Type 1 DM include: Inability to produce Insulin, due to either: Defective Beta cells in Pancreatic Islets, or Absent of Beta cells in Pancreatic Islets Autoimmune process causing destruction of Beta cells in Pancreatic Islets Presence of Islet cell antibodies in serum predicts development of Type 1 DM Islet-cell antibodies act against Glutamic Acid Decarboxylase (GAD) Environmental precipitating factors: Viral infection, Dietary factors (Anti-metabolites) What are some of the characteristics of Type 1 DM? Type 1 DM is usually characterized by: Deficiency in Insulin and the consequent Hyperglycemia Hyperglycemia causes blood glucose level to exceed Renal Threshold of 200mg/dl or 11mmol/L, Resulting in Glucosuria Following sequence of events occur: Sugar is excreted in urine (Glucosuria) Water follows the sugar due to osmosis (Osmotic diuresis) Large volume of urine is passed out (Polyuria) Patient becomes thirsty, thus drinks a lot of water (Polydipsia) Lack of Insulin: Muscles, Adipose tissue, Connective tissues and White Blood Cells cannot utilize Glucose in blood (Starvation in the midst of plenty), Patient become hungry and eats a lot (Polyphagia) Due to continuous lack of Insulin, glucose cannot enter Muscle and other tissues, thus patient may start to loose weight (Wasting) Patient may develop Ketoacidosis (Why?) What are some of the consequences if Type 1 DM is not controlled? Hyperglycemia: Partly due to inability of Insulin-dependent tissues to take up glucose from blood (Starvation in the midst of plenty!! (Why?) Increased Hepatic Gluconeogenesis, and Depressed Glycolysis resulting from low glucose levels in cells Hyper-Lipoproteinemia (Chylomicrons and VLDL): May be due to low Lipoprotein Lipase activity in Adipose tissue, Insulin is required for biosynthesis of Lipoprotein Lipase Ketoacidosis: Increased production of Ketone bodies: Acetone, Acetoacetic acid, and (-Hydroxybutyric acid Why is insulin used to control Type 1 DM? Administration of insulin does not cure Type 1 DM, it alters clinical cause of the disease Insulin promotes Glucose uptake and restoration of normal metabolism When the hypoglycemia is corrected: Loss of water and electrolytes ceases Formation of Ketone bodies ceases, and Acid-Base balance returns to normal Metabolism of Glucose via Glycolysis and TCA Cycle also allows the Acid-Base balance to return to normal Changes in plasma Bicarbonate levels during treatment serve as a guide to monitor the success of treatment What are some of the consequences of DKA? Decreased Glucose transport into tissues leads to Hyperglycemia, which gives rise to Glucosuria Increased Lipolysis leads to formation of Ketone bodies, Resulting in Ketonemia, and Ketonuria Acetone is exhaled in Lungs and manifests itself in breath Acetoacetic acid and (-Hydroxybutyric acid causes acidosis HCO3- concentration in blood falls (Metabolic acidosis) and more Carbonic acid (H2CO3) is formed, (================= CO2 + H2O (==(H2CO3 (==( H+ + HCO3- Carbonic acid is converted to CO2, which then stimulates respiratory center to remove excess CO2 Increased removal of CO2 causes rapid deep breathing (Hyperventilation) observed in patients with DKA Hyperventilation (Kussmaul breathing) is a response by the lungs to compensate for Metabolic Acidosis, by removing excess CO2 Glycosuria causes Osmotic Diuresis, which leads to: Loss of water, Loss of Electrolytes, Loss of Calcium, Magnesium, and Phosphate Dehydration if severe produces Pre-renal Uremia and leads to Hypovolaemic Shock Frequent vomiting is usually present and accentuates the loss of water and electrolytes Take Note: Development of DKA is a series of interlocking vicious circles all of which must be broken to aid restoration of normal Carbohydrate, Lipid and Protein metabolism Correction of DKA requires rapid treatment dictated by severity of the metabolic abnormalities and the associated tissue water and electrolyte imbalance Why is Insulin essential in the control of DKA? Insulin lowers plasma Glucagon level, Insulin stimulates Glucose uptake into target tissues Insulin antagonizes Catabolic effects of Glucagon on the Liver, Insulin inhibits flow of Ketogenic and Gluconeogenic substrates (free fatty acids and amino acids) from the periphery General occurrence of Type 2 DM: Type 2 DM accounts for about 85% of diagnosed cases of DM in PNG Type 2 DM: Formally called: Non-Insulin Dependent Diabetes Mellitus (NIDDM) Maturity-onset diabetes mellitus (most common in middle-age obese individuals, can occur in non-obese middle-age individuals, can occur in any age group) What are some of the possible causes of Type 2 DM? May be due to any of the following: Resistance of peripheral tissues to Insulin, despite normal or high Insulin level in circulation Deficiency or defect in Insulin Receptors in target tissues (Relative Insulin deficiency) Obesity, (most commonly associated clinical feature of Type 2 DM) Defect in Insulin Receptors is related to increased levels of Tumor Necrosis Factor-( (TNF-() in Adipocytes Increase in adipose tissue mass causes increase in production of TNF-(, which then blocks Insulin Receptors Diet can often be used to control Type 2 DM in Obese patient Obese patients that are motivated to lose weight: Insulin receptors will increase in number, and the Post-receptor abnormalities will improve, which may result in increased tissue sensitivity to insulin and Glucose tolerance Defects occurring within Insulin-responsive cells at sites beyond Insulin receptors Non-obese individuals: Type 2 DM may be cause not only by Insulin Resistance, but also by Impaired Pancreatic (-cell function resulting in Relative Insulin Deficiency What are the consequences of uncontrolled Type 2 DM? Uncontrolled Type 2 DM is characterized by: Hyperglycemia, Hyper-Triglyceridemia Hyperglycemia causes accumulation of glucose in: Eyes (Lens epithelium, Retinal capillaries), Peripheral Nerve cells (Schwann cells), Kidneys (Papillae, Glomerulus) Aldose Reductase and Sorbitol Dehydrogenase in these tissues converts: Glucose to: Fructose, Dulcitol and Sorbitol Sorbitol accumulates, crystallizes causing damage to tissues by causing them to swell Resulting in conditions such as: Cataract formation in the eyes (diabetic cataract), Diabetic Neuropathy including loss of sensation Retinopathy (damage to retina) Damage to blood vessels (Vascular disease) Damage to kidneys leading to renal failure Damage to Cardiac tissue (Ischemic heart disease) Ketoacidosis is not PRESENT in patients Type 2 DM (WHY?) DIAGNOSIS OF DIABETES MELLITUS: Is the diagnosis of DM the same as monitoring of DM? Diagnosis of DM is not the same as monitoring of DM Diagnosis: to clinically establish a condition in a patient Monitor: to follow progress on a condition that has been diagnosed Specific biochemical tests and guidelines are used for diagnosis of DM Specific biochemical tests and guideline are used for monitoring DM Some Biochemical tests for diagnosis of DM Glucosuria (Glycosuria): Glucosuria is a good first-line screening test for DM Glucose usually appears in urine when plasma glucose concentration rises above renal threshold (11mmol/L or 200mg/dL) Glucosuria may occur in some individuals with low renal threshold for glucose; Individuals have Glucosuria without DM Conversely, renal glucose threshold increases with age, thus some diabetics may have DM without Glucosuria Glucosuria indicates Hyperglycemia over the period of formation of the urine, it does not reflect the exact level of blood glucose at the time of testing Ketone in Urine (Ketonuria) or in Blood plasma (Ketonemia) Ketone bodies (Acetone, Acetoacetate, and Beta-Hydroxybutyrate) may accumulate in plasma and appear in urine in Type 1 DM Ketonuria or Ketonemia is not an automatic diagnosis of ketoacidosis, which is a serious condition Ketonuria or Ketonemia may occur during prolonged fasting Dry reagent strips, which detect Acetoacetate but not Beta-Hydroxybutyrate usually provides an underestimation of Ketonemia or Ketonuria Fasting blood glucose (FBG): FBG is measured after an overnight fast (about 8 to 10 hours) FBG is better than RBG for diagnostic purposes FBG above 8.0mmol/L on two occasions may be diagnostic of DM FBG between 6.0 to 8.0mmol/L may be interpreted as borderline Measurement of FBG on Whole blood, Plasma or Capillary blood have different cut-off points (see Table above) Random blood glucose (RBG) RBG is one of the major tests required in an emergency RBG of less than 8.0mmol/L is usually expected in non-diabetics RBG higher than 11.0mmol/L in more than one occasion indicates that the individual be investigated more thoroughly for DM Two Hours Post-Prandial blood glucose: Measure blood glucose level 2-hours after consumption of a meal It is a better indicator of DM that RBG and FBG Individuals with blood glucose above 11.0mmol/L should be investigated more thoroughly for DM Briefly explain how to perform oral glucose tolerance test (OGTT)? OGTT is recommended only if results from RBG and FBG tests cannot be interpreted clearly to justify DM in a patient OGTT must be carried out under proper clinical supervision Patient must be properly briefed before starting the procedure Measure FBG and urine glucose of patient after an overnight fast: Record both results Prepare a solution containing 75.0g glucose in about 300ml water Patient is requested to drink the solution within 5 minutes Measure blood glucose level of patient every 30 minutes for 2 hours Measure glucose in urine after 2 hours Patient should be sitting comfortably throughout the test, should not smoke or exercise and should have been on a normal diet for at least 3 days prior to the test WHO recommended guidelines for diagnosis of DM World Health Organization (WHO) published guidelines for diagnosis of DM on the basis of blood glucose results and the response to an Oral Glucose Load Table shows the WHO criteria for diagnosis of DM and Impaired Glucose Tolerance (IGT) RANDOM GLUCOSE SAMPLE (mmol/L)Diabetes likelyDiabetes uncertainDiabetes unlikelyVenous plasma( 11.1 5.5 - < 11.1< 5.5 (99.0 mg/dl)Venous blood( 10.04.4 - < 10.0< 4.4 (79.2 mg/dl)Capillary plasma( 12.25.5 - < 12.2< 5.5Capillary blood( 11.14.4 - < 11.1< 4.4STANDADIZED ORAG GLUCOSE TOLLERANCE TEST (mmol/L)DiabetesIGTVenous plasmaFasting 2hours( 7.8 ( 11.1< 7.8 7.8 - < 11.1Venous bloodFasting 2hours( 6.7 ( 10.0< 6.7 6.7 - < 10.0Capillary plasmaFasting 2hours ( 7.8 ( 12.2< 7.8 8.9 - < 12.2Capillary bloodFasting 2hours( 6.7 ( 11.1< 6.7 7.8 - < 11.1(Note: to convert mmol/L to mg/dl multiply by 18.0) How do you interpret the OGTT result? Use the data obtained to draw a graph of Time vs. Blood glucose level In Asymptomatic patients, OGTT should be interpreted as diagnostic of DM only when: There is an increased 2-house glucose level, and Blood glucose was equal to or greater than 11.0mmol/L (200.0 mg/dL) at some other point during the test If patient has normal fasting plasma glucose and only the 2-Hour value is in the diabetic range, the test should be repeated after approximately 6 weeks Impaired Glucose Tolerance (IGT) should be regarded as abnormal because it signals that the patient is at an intermediate stage between normality and DM and is at an increased risk of developing DM Such patients should be followed up yearly, and dietary treatment may be used MONITORING OF DIABETES MELLITUS: How can a patient with DM be monitored? (Long-term indices of diabetic control) Glycation of ECF proteins A high concentration of glucose in the extracellular fluid (ECF) leads to its non-enzymatic attachment to the Lysine residues of a variety of proteins. This is called Glycation. The extent of this process depends on the Blod glucose level. It is virtually irreversible at physiological pH concentration and therefore the glucose molecule will remain attached until the protein molecule is degraded. The concentration of Glycated protein is therefore a reflection of a mean blood glucose level prevailing in the ECF for the duration of that protein. Glycosylated Hemoglobin (HbA1c) In adults about 98% of Hb in the RBC is Hb A1. About 7% of Hb A consists of a type of Hb (HbA1) that can combine strongly with Glucose in a process called Glycosylation. Once Glycosylation occurs, it is not easily reversible. HbA1 is made up of three components (HbA1a, HbA1b, and HbA1c). Of these HbA1c is the highest in concentration and it is also the component that most strongly undergoes Glycosylation with Glucose. Thus, as the RBC circulates its HbA1 combines with blood glucose in a non-enzymatic reaction to form Glycosylated Hb (HbA1c). The amount of HbA1c formed is dependent on the concentration of Glucose in the blood over the 120-day life span of the RBC. Therefore determination of the HbA1c value reflects the average blood sugar level for the 100- to 120-day period before the test. The more glucose the RBC is exposed to, the greater the amount of HbA1c formed. One important advantage of this test is that the blood sample can be drawn at any time, because it is not affected by short-term variations (e.g., food intake, exercise, stress, hypoglycemic agents, and patient cooperation). Very high short-term blood glucose levels can cause elevation of HbA1c. Elevation of HbA1c occurs about 3 weeks after sustained elevation in blood glucose. Needs 4 weeks for HbA1c to decrease after a sustained reduction in blood glucose. HbA1c is a good Clinical indicator of the time-average control of blood glucose. In normo-glycemic individuals HbA1c represents 4% to 6% of the total HbA. In prolonged Hyperglycemia con of HbA1c may rise to as much 12% of the Total Hb. Patients with DM have high conc. of blood glucose, thus high amounts of HbA1c. This test is accepted as a good index of diabetic control and is used routinely in most diabetic clinics to complement the information from single blood glucose levels, or indeed a patients log of his or her own blood glucose measurements. What are some of the major uses of the Hb A1c test? Some of the major uses of HbA1c test include: Evaluating the success of diabetic treatment and patient compliance. Comparing and contrasting the success of past and new forms of therapy. Determining the duration of hyperglycemia in patients with newly diagnosed DM. Providing a sensitive estimate of glucose imbalance in patients with mild diabetes. Individualizing diabetic control regimens. Providing a sense of reward for many patients when the test shows achievement of good diabetic control. Evaluating diabetics whose glucose levels change significantly day to day (brittle diabetes). Differentiating short-term hyperglycemia in non-diabetics (e.g., recent stress or myocardial infarction) and diabetics (in whom the glucose has been persistently elevated). Fructosamine: Many other proteins in addition to Hb are Glycated when exposed to glucose in the blood. An indication of the extent of this glycation can be obtained by measuring Fructosamine, the Ketoamine product of non-enzymatic glycation. As albumin is the most abundant plasma protein, Glycated Albumin is the major contributor to serum Fructosamine measurements. As this protein has a shorter half-life than Hb, Fructosamine measurements are complementary to Hb A1c providing an index of glucose control over the 3 weeks prior to its measurement. Microalbuminuria: Microalbuminuria may be defined as an albumin excretion rate intermediate between normality (2.5 to 25 mg/day) and Macroalbuminuria (> 250 mg/day). The small increase in urinary albumin excretion is not detected by simple albumin stick tests and requires confirmation by careful quantization in a 24hour urine specimen. The importance of Microalbuminuria in the diabetic patient is that it is a signal of early, reversible renal damage. SUMMARY: Diagnosis and monitoring of Diabetes Mellitus: The diagnosis of DM is made on the basis of blood glucose concentrations either alone or in response to an oral glucose load. In Asymptomatic patients the results of an OGTT should be interpreted as diagnostic of DM only when there is an increased 2 h glucose concentration, and the blood glucose is also equal to or greater than 11.0mmol/L (198.0 g/dl) at some other point during the test. The HbA1c and Fructosamine are measures of protein glycation and serve as indices of long-term glucose control. Microalbuminuria is a measure of early, reversible, diabetic nephropathy. Hypoglycemia: Hypoglycemia is a laboratory diagnosis which is usually taken to mean a blood glucose level less than 2.2mmol/L (45.0 g/dl). Hypoglycemia may be due to a number of underlying conditions including endocrine disorders, liver disease, inborn errors of metabolism and gastrointestinal surgery. The cause (or biochemical basis) is an imbalance between glucose intake, endogenous glucose production and glucose utilization. A low blood glucose level normally leads to the stimulation of Catecholamine secretion and correction of hypoglycemia through suppression of Insulin secretion and stimulation of Glucagon, Cortisol and Growth Hormone. The Catecholamine surge accounts for the signs and symptoms most commonly seen in Hypoglycemia, i.e., Sweating, Shaking, Tachycardia, Nausea and Weakness. Hypoglycemia decreases the glucose fuel supply to the brain and may lead to brain damage particularly in infants. Laboratory Investigation: The Biochemistry laboratory can confirm hypoglycemia and may also provide some useful clues to the underlying cause. Blood glucose: The detection of hypoglycemia is by blood glucose testing. Urine testing cannot detect hypoglycemia. WHY??? Plasma Insulin: Insulin measurements can lead to the diagnosis or exclusion of Insulinoma. They play no part in the diagnosis of diabetes mellitus. Insulin/Glucose ratio: In order to make better diagnostic use of Insulin measurements, the ratio of Insulin and Glucose concentrations, measured on the same sample, should be reported. Plasma C-peptide: Insulin secretion in Insulin-treated diabetics cannot be assessed by the measurement of plasma insulin since the insulin given therapeutically will also be measured in the assay. However, insulin and its associated Connecting-peptide (or C-peptide) are secreted by the Islet cells in equimolar amounts and thus measurement of C-peptide levels together with insulin can differentiate between hypoglycemia due to Insulinoma (high C-peptide) and that due to exogenous insulin (low C-peptide). Study Questions: What are some of the characteristics of IDDM (Type 1 DM)? Briefly explain why insulin can be used to control IDDM? What are some of the factors that can cause DKO in patients with IDDM? Briefly outline the consequences of DKO? Briefly explain possible causes of NIDDM (Type 2 DM). List the Biochemical tests that can be used for the diagnosis of DM List the Biochemical tests that can be used for monitoring of DM Explain the procedures for carrying out OGTT. Give an overview of the interpretation of an OGTT. Briefly explain why Hb A1C can be used to monitor DM in a patient. Reference: Textbook of Biochemistry, with clinical correlations, Ed. By T. M. Devlin, 4th Edition. Pages: 62, 287, 390, 537, 548 551; 536 540 Harpers Biochemistry 24th Edition. Ed. By R. K. Murray et. al. Page: 203, 204, 586, 587, and 826, 827. Biochemistry, By V. L. Davidson & D. B. Sittman. 3rd Edition. Pages: 517 519.  HYPERLINK "http://WWW.zonehome.com/met/metglucose.html" WWW.zonehome.com/met/metglucose.html  HYPERLINK "http://WWW.niko.unl.edu/bs101/notes/lecture12.html" WWW.niko.unl.edu/bs101/notes/lecture12.html  HYPERLINK "http://WWW.mun.ca/biochem/courses/1430/diabetes.html" WWW.mun.ca/biochem/courses/1430/diabetes.html SCHOOL OF MEDICINE AND HEALTH SCIENCES DIVISION OF BASIC MEDICAL SCIENCES DISCIPLINE OF BIOCHEMISTRY AND MOLECULAR BIOLOGY PBL SEMINAR; BMLS & BDS Year 3 THYROID HORMONES: An Overview What are the Thyroid Hormones? Thyroid Hormones are: Thyroxine (T4, also called 3,5,3,5 Tetra-Iodothyronine) and Tri-Iodothyronine (T3 also called 3,5,3 Tri-Iodothyronine) T4 contains Four Iodine atoms while T3 contains Three Iodine atoms. T3 is the biological active form of the Thyroid hormone, because it is the hormone that binds to receptors and trigger the end-organ effects Thyroid hormones are unique in that they require the trace element Iodine for Biological activity. Biological inactive form of the thyroid hormone is called Reverse T3 (rT3, 3,3,5-Tri-Iodothyronine) (See Fig. 1). Outline the biosynthesis of the Thyroid Hormones (Fig. 2): Thyroid hormones are synthesized in the Thyroid gland The biosynthesis of Thyroid involves the Iodination and Coupling of Tyrosine molecules attached to a Complex Protein called Thyroglobulin (TG). Biosynthesis process can be separated into two stages as follows: Stage one: Iodination reactions (or Organification): The process begins by Trapping of Iodide from plasma by the Thyroid gland The Iodination reactions (Organification) of the Tyrosine residues in Thyroglobulin are carried out by the enzyme Thyroid peroxidase, which catalyses the oxidation of Iodide (I-) to Iodine using locally generated Hydrogen Peroxide (H2 O2). The Thyroid Peroxidase then uses the Iodine to Iodinate Tyrosine residues in Thyroglobulin forming 3-Monoiodotyrosine (MIT) residues. The 3-Monoiodotyrosine can then be Iodinated a Second time to form 3,5-Diiodotyrosine (DIT) in a reaction catalysed by Thyroid Peroxidase. Both MIT and DIT still remain attached to Thyroglobulin. Stage two: Coupling reactions: At this stage of the process Thyroid Peroxidase cleaves off a molecule of MIT or DIT and Couples it to an Acceptor DIT molecule. Three combinations are possible: DIT + DIT coupling gives T4; MIT + DIT coupling gives T3 DIT + MIT coupling gives reverse T3 (which is not active) The major coupling process that occurs is the formation of T4. Finally, T4 and T3 are released into the plasma, (note that the thyroid gland secretes mostly T4 into the plasma). How is T4 utilized in peripheral tissues? (Production of T3 in peripheral tissues) T4 is a Pro-hormone and it is produced exclusively by the Thyroid gland. The biologically active Thyroid hormone is T3. The liver and kidneys have a lower affinity, higher capacity enzyme system (De-Iodinase) that catalyzes the de-iodination of T4. The Liver and Kidney, De-iodinate T4 to produce approximately two-thirds of the T3 present in plasma. The De-Iodinase enzyme that catalyzes the conversion of T4 to T3 requires the trace element Selenium, because it contains a specific Amino Acid called Seleno-Cysteine. There is also an enzyme called 5-De-Iodinase that does not require Selenium. This enzyme catalyzes the conversion of T4 to Reverse T3 Deficiency of the trace element Selenium can result in a decrease in the conversion of T4 to T3, thus causing at the same time an increase in the conversion of T4 to reverse T3 (rT3), by the enzyme 5-Deiodinase that does not contain the amino acid Seleno-Cysteine. Other body cells are capable of taking up T4 and De-iodinating it to form the more biologically active T3. Alternatively, T4 can be metabolised to Reverse T3 (rT3), which is biologically inactive. By modulating the relative production of T3 and rT3, tissues can Fine Tune their local Thyroid Status. What are some of the factors that can affect the conversion of T4 to T3? A number of factors can affect the conversion of T4 to T3 in body cells. Some of these factors act by decreasing the activity of the enzyme 5- De-Iodinase, resulting in increased rT3/ T3 ratio, less T4 to T3 conversion. Some of these factors include the following: Pregnancy or oral contraceptive pills, Fasting, Stress, High plasma Cortisol, Catabolic diseases, Hepatic and Renal diseases Thiouracil drugs (blocks thyroid peroxidase activity) How are the Thyroid hormones transported in plasma? (Thyroid Hormone Binding in Plasma): Both T4 and T3 circulate in plasma bound to specific binding proteins: Thyroxine-Binding Globulin (TBG), Transthyretin (also called Thyroxine-binding pre-albumin or TBPA) Some of the Thyroid hormones are also bound to plasma Albumin. In the plasma TBG is quantitatively the most important binding protein for the Thyroid hormones. TBG binds about 70% of plasma T4 and about 80% plasma T3. Approximately 0.05% of plasma T4 and 0.2% of plasma T3 are Free (i.e., unbound to protein in plasma). TBG is synthesized in the Liver; Estrogens (pregnancy and birth control pills) increase the synthesis of TBG. TAKE NOTE: The plasma contains both Bound and unbound (Free) Thyroid hormones It is the amount of unbound or Free T4 and T3 (i.e., FT4 and FT3) that are important for the biological effects of the Thyroid hormones, including the feedback to the Pituitary and Hypothalamus. This is because only the Free Fractions can cross the cell membrane and affect intracellular metabolism. How is the secretion of Thyroid hormones regulated? (See Figs. 3 & 4): Feedback regulation of Thyroid hormones occurs via the Hypothalamic-Pituitary-Thyroid axis (HPT axis). The Hypothalamus secretes a tripeptide called Thyrotropin-Releasing Hormone (TRH, also called Thyroliberin). TRH then stimulates the Anterior Pituitary to synthesize and release a large Glycoprotein Hormone, called Thyroid-Stimulating Hormone (TSH, also called Thyrotropin). The TSH then stimulates the Thyroid glands to produce the Thyroid hormones. Excess circulating unbound Thyroid hormones (FT4 and FT3) act via the long loop feedback mechanism to block the production of TSH and TRH. TSH can also act via the short loop feedback mechanism to block the production of TRH by the Hypothalamus. Knowledge of the regulation of Thyroid hormones via the feedback loops is essential for interpretation of results in the investigation of thyroid status. TAKE NOTE: It is import to note the following: If the Thyroid gland of a patient is producing too much Thyroid hormone, then the circulating TSH will be suppressed. (Why?) If the Thyroid gland of a patient is not secreting enough Thyroid hormone, the TSH level will be very high in an attempt to stimulate the Thyroid gland to secrete more Thyroid hormone. In Non-Thyroidal illness (NTI) a number of hormones and other agents have been shown to be responsible for inhibiting the release of TSH. These include the following: Dopamine, Somatostatin, Glucocorticoids, Interleukins What are some of the cellular actions of the Thyroid hormones? Plasma FT3 is the biologically active form of Thyroid hormone. After binding to high affinity binding sites on the membranes of target cells, the Thyroid hormones are actively transported into the cell by an ATP-dependent mechanism. In the cell, T3 acts mainly at the Nucleus, where it binds to specific receptors (Hormone Response Elements, HRE) that in turn activate T3-responsive Genes. These genes appear to exert a number of effects on cell metabolism, which include: Stimulation of Basal Metabolic Rate Metabolism of Lipids, Carbohydrates and Proteins Regulation of Gene Expression, Regulation of Tissue Differentiation and General Development, which are essential for the normal maturation and metabolism of all the tissues in the body. High Thyroid hormone concentration causes increased Metabolic State by: Increasing the Mobilization of Endogenous Protein, Fat and Carbohydrate for the production of substrates needed for Energy Production The effects of Thyroid hormones on tissue maturation are most dramatically seen in Congenital Hypothyroidism, a condition, which unless treated within a short time after birth, may results in permanent brain damage. Hypothyroid children have delayed skeletal maturation, short stature and delayed puberty. An example of the effect of Thyroid hormones on lipid metabolism is the observation of High Serum Cholesterol in some Hypothyroid Patients. This is a consequence of the reduction in cholesterol metabolism, due to down regulation of Low-Density Lipoprotein (LDL) receptors on Liver cell membranes, with a subsequent failure of Sterol excretion via the gut. Briefly state the actions of Thyroid hormones Thyroid hormones: Increase Basal Metabolic Rate (BMR) Increase Oxygen consumption Increase Thermogenesis (heat production in the body) Activate Na+-K+-ATPase in cells Increase number of Mitochondria in cells Increase mobilization of endogenous Carbohydrate, Fat and Protein to produce substrates for energy metabolism Increase Glycolysis, Glycogenolysis, Gluconeogenesis, Increase Lipolysis and Protein degradation Decrease Muscle mass Decrease Adipose Tissue Increase Beta-Adrenergic receptors, which leads to increase Cardiac Output Increase Systolic blood pressure only Increase Ventilation Rate Required for maturation of Ovary and Testis Required for Actions of Growth Hormone (GH) to promote linear growth / bone formation Required for development of the CNS in the Foetus Study Questions: Give a brief outline of the biosynthesis of thyroid hormones List some factors that can affect the conversion of T4 to T3 How are Thyroid hormones transported in plasma? How is the secretion of Thyroid hormones regulated? Briefly state the cellular functions of Thyroid hormones  SCHOOL OF MEDICINE AND HEALTH SCIENCES DIVISION OF BASIC MEDICAL SCIENCES DISCIPLINE OF BIOCHEMISTRY AND MOLECUALR BIOLOGY PBL SEMINAR and BMLS Lecture THYROID FUNCTION TEST An Overview How can Thyroid function be investigated? Test for investigation of Thyroid dysfunction can be separated into Two categories: Groups of Tests to established Thyroid status: Measurement of [TSH] in Plasma or Serum Measurements of [Thyroid Hormones] {T4 and T3} in Plasma or Serum Groups of Test to elucidate cause of Thyroid dysfunction: Thyroid Auto-antibody, Serum [Thyroglobulin], Thyroid Peroxidase, Biopsy of the Thyroid, Ultrasound and Isotopic Thyroid Scanning TAKE NOTE: Thyroid status MUST be determined before tests to elucidate cause of dysfunction What are the tests used to determine Thyroid status? Thyroid-Stimulating Hormone (TSH): Single most sensitive, specific and reliable test of Thyroid status in both overt and subclinical thyroid disease, Can be used to diagnose Primary Hypothyroidism and to differentiate it from Secondary Hypothyroidism Thyroid-Releasing Hormone (TRH): Test assists in evaluation of patients with Hyperthyroidism and Hypothyroidism Especially helpful in differential diagnosis of Hypothyroidism Thyroid-Binding Globulin (TBG): Measurement of TBG, the major thyroid hormone protein carrier Used in evaluation of patients who have abnormal Total T4 and T3 levels Can be done concurrently with Total T4 and Total T3 test, for proper interpretation of Total T4 and Total T3 Total Thyroxine (Total T4): Used in assessing Thyroid Function Used to monitor Replacement and Suppressive Therapy Total Triiodothyronine (Total T3): Used to evaluate Thyroid Function Mainly used to diagnose Hyperthyroidism Used to monitor Thyroid Replacement and Suppressive therapy Free Thyroxine (FT4): Used to evaluate Thyroid Function Used to diagnose Hyperthyroidism and Hypothyroidism Free Triiodothyronine (F T3): Used to diagnose Thyroid Function and to monitor replacement and suppressive therapy How significant is TSH test (TSH, Thyrotropin)? {Reference range: 0.4 to 4.5mU/L} TSH release is very sensitive to alterations in plasma [Thyroid Hormones] Decrease in Plasma [Thyroid Hormones] causes Increase secretion of TSH Increase in Plasma [Thyroid hormones] suppresses secretion of TSH Feedback control mechanism in HPT axis Measurement of Plasma [TSH] in basal blood sample by Immuno-metric Assay provides one of the single most sensitive, specific and reliable test of Thyroid status in both Overt and Subclinical Thyroid Disease In Primary Hypothyroidism, Plasma [TSH] is increased above Normal reference range {Why?} In Primary Hyperthyroidism (e.g., Thyrotoxicosis) Plasma [TSH] is reduced below Normal reference range {Why?} Take note of following questions and answers Thyrotoxicosis {Low [TSH], Why?}: Thyroid automatically manufactures too much T4 and T3, which suppresses production of TSH via Feedback Mechanism When laboratory result shows raised TSH level, then FT4 should be measured When laboratory result shows a suppressed TSH level then FT4 and FT3 should be measured Why should both FT4 and FT3 be measured in the second case? In some patients Thyroid over secrete only T3, a condition called T3 Toxicosis and both hormones need to be measured to detect this form of Thyrotoxicosis Condition is usually seen in patients who previously had Thyroidectomy or had been treated with Radioactive Iodine for Thyrotoxicosis in the past Exceptions: both raised and undetected plasma TSH concentration may be seen in some Euthyroid patients How are the results of TSH tests interpreted? High sensitivity TSH assay measures the [TSH] in Serum Healthy individuals: Serum [TSH] is usually between 0.4 and 4.5m U/L TSH is under: Negative Feedback Control of FT4 and FT3 in circulation Positive Control of TRH in Hypothalamic Thyroid hormone deficiency should cause elevated Serum [TSH] Serum [TSH] greater than 20m U/L is a good indicator of Primary Failure of Thyroid Gland Serum [TSH] between 4.5 and 15m U/L is borderline thyroid dysfunction, which requires more careful evaluation In Secondary Hypothyroid status, [TSH] may be low, normal or occasionally borderline range Serum [TSH] above 15m U/L is very good evidence for Primary Hypothyroidism Serum [TSH] below 5 is very good evidence against Primary Hypothyroidism Presence of Low [FT4] with [TSH] less than 10m U/L strongly suggests a Secondary Hypothyroidism High [FT4] and [FT3] will suppress TSH levels, in almost all case of Hyperthyroidism, thus, [TSH] is usually below 0.3m U/L and may be less than 0.1m U/L Interpreting the use of Serum [TSH] for monitoring Serum [TSH] can be effectively used to follow patients being treated with Thyroid Hormone High [TSH] indicates under-treatment, Low [TSH] usually indicates over-treatment Abnormal [TSH] should be interpreted with [FT4] or [FT3] before modifying therapy because Serum Thyroid Hormone levels change more quickly than TSH levels Patients who recently started using Thyroid Hormone, or who have been non-compliant until shortly before a visit to the doctor may have normal [FT4] and [FT3], though their TSH levels are still elevated Serum [TSH] may be affected by acute illness and several medications, including Dopamine and Glucocorticoids (Non-Thyroidal Illness, NTI) TAKE NOTE: Serum [TSH] and [FT4] are frequently measured to differentiate between Secondary and Primary Thyroid dysfunction Decrease [FT4] and Normal or Elevated [TSH] indicates Primary Thyroid disorder {Why?} Decrease [FT4] with a decreased [TSH] indicates Secondary Thyroid disorder {Why?} What is the Thyroxine Binding Globulin (TBG) test? Determination of Plasma [TBG] Determination of Plasma [Total T4] Determination of Plasma [FT4] Used in evaluation of patients with abnormal Plasma [Total T4] Conditions that causes increase in Plasma [TBG] include: Pregnancy, Hormone Replacement Therapy, Oral Contraceptives, Infections, and Hepatitis, Conditions that causes decrease in Plasma [TBG]: Hypoproteinemia, Nephrotic syndrome, and Malnutrition Fig. 1: Strategy for investigation of Low TSH  Fig. 2: Strategy for investigating High TSH  Plasma [FT4] and [FT3] are independent of changes in Plasma [TBG] Provide a more reliable means of diagnosing Thyroid Dysfunction than measurement of Plasma [Total T4] alone (Fig. 3) Fig. 3: TBG Test and Interpretation of results  How significant is the Thyroxine (T4) screen? Plasma [FT4] (Reference range: 10 to 27 pmol/L) Plasma [Total T4] (Reference range: 70 to 150 nmol/L) Plasma [Total T4] or [FT4] can be determined by several methods: Radioimmunoassay (RIA), Enzyme-Linked Immunosorbent Assay (ELISA), Enzyme Immunoassay (EIA), Microplate Enzyme Immunoassay (MEIA) All laboratories should be encouraged to measure Plasma or Serum [FT4] What factors can affect Interpretation of Plasma [Total T4] results? Some laboratories still measure Plasma [Total T4], Results of Plasma [Total T4] depends on Plasma [TBG], thus results should be interpreted with care Plasma [TBG] may be Low in some patients with Inherited but harmless deficiency Plasma [Total T4] is Low in these patients, but [FT4] may be Normal Plasma [TBG] may be elevated in Pregnant women and in Women using Oestrogen-containing Oral Contraceptive Pill, Plasma [Total T4] may be elevated well above the Reference range, but [FT4] may be normal Determination of Plasma [FT4] is recommended in conditions where [TBG] may be altered, e.g., Pregnancy, users of Oral Contraceptive Pill and patients with Nephrotic Syndrome How significant is the Tri-Iodothyronine (T3) test? Plasma [FT3] (Reference range: 3 to 9 pmol/L) Plasma [Total T3] (Reference range: 1.2 to 2.8 nmol/L) Test is primarily used to diagnose Hyperthyroidism and to monitor thyroid replacement and suppressive therapy Plasma [Total T3] or [FT43] can be determined by several methods: Radioimmunoassay (RIA), Enzyme-Linked Immunosorbent Assay (ELISA), Enzyme Immunoassay (EIA), Microplate Enzyme Immunoassay (MEIA) FT3 comprises about 0.3% of Total circulating T3 in blood Gradually laboratories are moving over to FT3 measurements as more reliable FT3 assays become available TAKE NOTE: Conversion of T4 to T3 depends on a number of situations such as Chronic illness or Surgical stress, which cause a fall in T4 to T3 conversion (called low T3 syndrome) Starvation can alter T4 to T3 conversion with a fall in T3 as the body tries to reduce its metabolism to conserve energy Plasma [Total T3] provides a useful test for Hyperthyroidism, as values are often raised proportionately more than Plasma [Total T4] Determination of Plasma [Total T3] is of no value in investigating patients with suspected Hypothyroidism, as normal results are often found How reliable is Thyroid function test for assessing Thyroid status during Pregnancy? Plasma [TSH] is reliable indicator of Thyroid status in 2nd and 3rd Trimester of pregnancy Plasma [TSH] is not a reliable indicator during 1st Trimester of pregnancy (Why?) Plasma [TSH] is usually low, may be due to weak Thyrotrophic effect of Placental hCG (Human Chorionic Gonadotropin), which is high during 1st Trimester Plasma [Free Thyroid hormone] increases during 1st Trimester, then decline later Plasma [TBG] increase during pregnancy, causing elevated in Plasma [Total T4] and [Total T3]  In Summary: Plasma [TSH] is the single best test for assessing Thyroid Status Plasma [TSH] is elevated in Primary Hypothyroidism Plasma [TSH] is suppressed in Primary Hyperthyroidism Normal Plasma [TSH] usually excludes Primary Thyroid Disorder Plasma [FT4] and [TSH] can be used to assess severity of Thyroid disease and distinguish Subclinical from Overt disease Plasma [FT3] and [TSH] can be used to determine severity of Hyperthyroidism and to identify patients with T3 Hyperthyroidism Plasma [Free Thyroid Hormones] correlates more closely with Thyroid Status than Plasma [Total Thyroid hormones], which are heavily influenced by changes in Plasma [TBG] Thyroid 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