Renal Function Tests (BLOCK3) PDF

Urinary and Genital system DR. ADEEB M. ALBADANI Renal Function Testing and Non-protein Nitrogen Substances Objectives Upon completion of this block the student will be able to: Define terminologies applied in the renal function tests Define the non protein nitrogenous (NPN) compounds Dis...

Urinary and Genital system DR. ADEEB M. ALBADANI Renal Function Testing and Non-protein Nitrogen Substances Objectives Upon completion of this block the student will be able to: Define terminologies applied in the renal function tests Define the non protein nitrogenous (NPN) compounds Discuss about, NPN compounds mainly of : 1. Urea 2. Creatinine, 3. Uric acid 4. Creatinine Clearance 5. Electrolytes source, metabolism, clinical significance ◘ Renal Stones  Outline of Introduction Definitions of important terminologies (concepts) Anatomy, Functions of the renal system Renal threshold Non- protein nitrogenous (NPN) compounds  Concepts Non protein nitrogenous (NPN) substances: are end products of metabolism that contains nitrogen Azotemia: An excess of urea or other nitrogenous compounds in the blood Anti diuretic hormone (ADH): is a posterior pituitary gland hormone, important for reabsorption of water from the kidneys. Diabetic insipidus: A disorder associated with secretion and metabolism of anti diuretic hormone (ADH), manifested by excessive urine production. Definitions, continued… Nephron: functional units of kidney Gout: Group of disorders of purine metabolism Renal failure: Acute or chronic decline in renal function Major Structures of Urinary System Kidney Ureters Bladder Urethra  Kidney Functions Filtration of small molecules Reabsorption of essential substances Secretion into urine from blood stream Excretion Hormonal regulation: erythropoietin, ADH, aldosterone Homeostasis Glomerular Filtration Non-selective filtration across semi-permeable membrane Low molecular weight substances will pass into the urine Filtrate lacks large molecular weight substances such as protein, protein-bound substances, cells GFR: ~120 mL/min Tubular Reabsorption Passive vs. active transport mechanisms  Conserves water and essential substances Water: always passively reabsorbed Glucose, HCO3: active transport Tubular Secretion Eliminates waste products not filtered by glomerulus  Drugs that are protein-bound (protein remains in bloodstream)  Organic waste: Blood urea nitrogen = BUN Creatinine Uric acid  Uromodulin protein secreted into tubules  Acid-base regulation: H+, HCO3 Renal Threshold Defined as the plasma concentration of a substance that when exceeded, the kidney tubules will not reabsorb any more into the bloodstream, resulting in the substance being excreted into the urine Example: glucose ~160-180 mg/dl  Non-Protein Nitrogen Compounds These are compounds that contain nitrogen, but are not proteins Often called NPN compounds Include end products of metabolism Kidneys act to excrete metabolic waste into urine Non-Protein Nitrogen Compounds Because the kidneys act to excrete these compounds into urine, measurement of NPN compounds in plasma is useful for assessment of kidney function Non-Protein Nitrogen Compounds Include >15 compounds  Amino acids  Ammonia  Blood urea nitrogen (BUN) Protein  amino acids  ammonia  urea  Creatinine Muscle breakdown product  Uric acid Nucleic acid catabolism Urea and BUN NPN compound present in highest concentration in blood and urine U= Urea Blood Urea Nitrogen = BUN Urea contains 2 nitrogen atoms: 28 g nitrogen/mole of urea BUN x 2.14 = urea  Out Line UREA - Introduction - Metabolism - clinical (Importance )Application - Normal Value - Clinical Significance - Determination  INTRODUCTION 1. Site Since ammonia is toxic to CNS even in traces liver rapidly removes ammonia from circulation and converts it to a non-toxic water soluble urea. Hence site of urea synthesis is liver. 2. The reactions leading to formation of urea from ammonia are proposed by Krebs and Henseleit. Hence, urea cycle is also called as Krebs-Henseleit cycle. 3. Formation of urea from ammonia in urea cycle occurs in five reactions. However the first reaction is not a part of the cycle but for the continuation of the cycle which consist of remaining four reactions product of the first reaction is essential. Further, the intermediates of the four reactions are amino acids. 4. Synthesis of urea from ammonia is a energy dependent process. 5. Enzymes of urea cycle are present in mitochondria and cytosol. 6. First two reactions of urea formation occurs in mitochondria and remaining reactions occur in cytosol.  MEDICAL IMPORTANCE 1. Urea formation is impaired in several inherited diseases. 2. They are due to deficiency of enzymes of urea cycle. 3. The rate of incidence of urea cycle disorders is 1 in 2500. 4. Most of these inherited diseases are due to defective genes and are fatal. 5. Since the urea cycle converts ammonia to urea these disorders of urea cycle cause ammonia intoxication. 6. Some common clinical symptoms seen in these diseases are vomiting, irritability, lethargy, seizures, mental retardation, coma and early death Urea (BUN) Formation and excretion  Synthesized in the liver: ammonia  urea Protein  amino acids  ammonia [LIVER]  urea  Conversion of ammonia to urea is last liver function to fail in end stage liver disease Plasma ammonia levels rise The urea cycle Fig. 12.7  Reactions Of Urea Cycle - For the synthesis of urea only one ammonia molecule is used as such. aspartate serves as donor of another molecule of ammonia. HCO3 – serves as source of co2 required for urea formation. 1. Formation of carbamoyl phosphate - First reaction leading to urea formation is condensation of ammonia and hco3 – at the expense of two high-energy bonds to form carbamoyl phosphate. the reaction is catalyzed by mitochondrial carbamoyl phosphate synthetase-I. - The enzyme requires n-acetyl glutamate and mg 2+. n-acetyl - glutamate is synthesized from acetyl-coa and glutamate in the liver. - 2ATP molecules are hydrolyzed to 2 ADP and 2Pi in the first reaction. - Of the 2PI one pi is consumed in the reaction. - Since the product of the reaction carbamoyl phosphate is high energy compound its formation thermodynamically pulls subsequent reactions of urea cycle towards urea formation.  2. Now the first reaction of urea cycle is catalyzed by ornithine transcarbamoylase. - It condenses carbamoyl phosphate and ornithine to form citrulline. - This enzyme is present in mitochondria. - Since the subsequent reactions of urea cycle occurs in cytosol, citrulline formed eners cytosol through specific transporter present in inner mitochondrial membrane. 3. Arginino - succinate synthetase present in cytosol catalyzes second reaction of urea cycle. - It condenses citrulline and aspartate at the expense of two high energy bonds to form argininosuccinate. One high energy bond is consumed by the hydrolys is of ATP to AMP and PPi. Further hydrolysis of PPi to 2Pi by pyrophosphatase leads to consumption of another high energy bond. 4. In the third reaction of urea cycle argininosuccinate is cleaved by argininosuccinase to arginine and fumarate. 5. Regeneration of ornithine and formation of urea from arginine is the final reaction of urea cycle. - This reaction is catalyzed by arginase. - The ornithine so formed enters mitochondria through specific transporter present in inner mitochondrial membrane to start reactions of urea cycle once again. Reaction of urea formation are shown in Fig. 12.7 Overall equation for urea formation NH3 + HCO–3 + ASPARTATE + 4ATP UREA + FUMARATE + 4ADP + 4PI  Fate of urea - Urea has no physiological function. Hence it is transported to kidneys where it is excreted in urine. - It is major end product of protein catabolism in humans. About 10-25 gm of urea is excreted in urine per day which makes up to 80-90% of total nitrogen excreted per day. - However, blood also contains some urea. REGULATION OF UREA FORMATION - Formation of urea is regulated by activity of carbamoyl phosphate synthetase-I. - This enzyme catalyzes committed step in urea synthesis. - N-acetylglutamate regulates this enzyme activity. - It is an allosteric activator. - High protein in take leads to more N- acetylglutamate formation. - Thus high protein in take influences urea formation. - In starvation also urea synthesis is more mostly due to increased protein breakdown. Blood Urea N (BUN) Reference Range For adults (Serum/plasma)……………….. 6-20 mg/dl New borne upto one week( Serum/plasma)……… 3- 25mg/dl Adult over 60 (Serum/plasma) ……………………..8-23mg/dl Urine, 12-20 g/24hrs Convert 22 mg/dL BUN to urea mg/dL BUN 22 x 2.14 = Urea 47 mg/dL Urea and BUN Clinical Significance Plasma levels are dependent upon  Diet  Liver function  Kidney function  State of hydration Urea (BUN) Clinical Significance Increased urea (BUN):  Azotemia or uremia Increased protein intake (leads to increased urea formation) Decreased kidney function (decreased excretion into urine results in elevated plasma levels) Dehydration (lack of body water results in increased levels) Urea (BUN) Clinical Significance: Decreased urea (BUN): Decreased protein intake (leads to decreased urea formation) Decreased liver function (decreased conversion of ammonia to urea) Not a good test for GFR: Influenced by diet, liver function Urea laboratory diagnostic techniques Enzymatic (indirect) method Chemical (direct) method Ammonia Toxicity and Metabolism  AMMONIA TOXICITY - Since ammonia is toxic to central nervous system particularly to glial cells blood ammonia level must be within normal range. - If blood ammonia level raises due to any reason symptoms of ammonia intoxication appears. -They are slurred speech, blurred vision and tremors. - Coma and death can occur in severe cases. MECHANISM OF AMMONIA TOXICITY Mechanism of toxic effect of ammonia on brain is not clearly understood. However ammonia can cause brain toxicity by three ways 1. The entry of ammonia into brain leads to formation of glutamate by the reversal of glutamate dehydrogenase reaction. This depletes available -keto glutarate in the brain. As a result citric acid cycle operation is impaired and ATP production diminishes. This leads to brain cell dysfunction. 2. Since the brain is rich in glutamine synthetase the ammonia which enters brain is used for glutamine synthesis. This leads to depletion of cellular ATP and cell dysfunction. 3. Since glutamate is considered as neurotransmitter the toxice effect of ammW2onia may be due to over stimulation of nerve cells by glutamate formed from ammonia and -keto glutarate by the action of glutamate dehydrogenase. CAUSES FOR AMMONIA TOXICITY 1. If hepatic function is impaired plasma ammonia rises to toxic level. Liver function can impair in cases of poisoning due to carbon tetra chloride, heavy metals and viral infections. 2. If collateral communication is developed between portal vein and systematic blood plasma ammonia rises to toxic level. In cirrhosis collateral communication develops between portal vein and systematic blood. 3. Consumption of protein rich diet after gastro intestinal haemorrhage can cause ammonia toxicity.  Ammonia metabolism is shown in Fig. 12.6.  Out Line - Introduction CREATININE - Metabolism - Produce - Clinical Application - Normal Value - Clinical Significance - Determination Creatinine Introduction Formation and Excretion  Spontaneously derived from creatine in muscle High energy ATP storage and use in muscle  Produced at a constant rate day to day  Excreted into urine through glomerular filtration; not significantly reabsorbed or secreted by tubules METABOLISM OF CEATININE Creatinine Reference Range Serum Adult male: 0.6-1.1 mg/dl Adult female: 0.5-0.8 mg/dl Child: 0-0.6 mg/dl Urine Male: 800-2000 mg/24hr Female: 600-1800 mg/24hr Amniotic fluid: 1-2 mg/dl Creatinine Clinical Significance  Endogenous substance  Amount produced is constant day to day: levels vary 59 ml/min Creatinine Clearance Clinical Significance  Used to monitor GFR  As renal function fails, CrCl decreases  Dialysis indicated when CrCl critically low (GFR ~ 10-20 mL/min) Correlates with Increased BUN/ Creatinine ratio with increased BUN, increased plasma creatinine and decreased ur. creatinine  Out Line - Introduction URIC ACID - Metabolism - Produce - Clinical Application - Normal Value - Clinical Significance - Determination Normal values of Internal Chemical Environment controlled by the Kidneys  SODIUM 135 to 145 mEq/L POTASSIUM 3.5 to 5.5 mEq/L CHLORIDES 100 to 110 mEq/L BICARBONATE 24 to 26 mEq/L CALCIUM 8.6 to 10 mg/dl MAGNESIUM 1.6 to 2.4 mg/dl PHOSPHORUS 3.0 to 5.0 mg/dl URIC ACID 2.5 to 6.0 mg/dl pH 7.4 CREATININE 0.8 to 1.4 mg/dl BUN (Blood Urea Nitrogen) 15 to 20 mg/dl Renal Stones  Kidney Stones Affects 12% of men & Formation is 5% of women promoted by: Crystalline mass in  Reduced urine volume urinary tract  Blocked urine flow  Severepain  Increased  Can obstruct tract concentrations of stone-forming substances Types of Stones Calcium oxalate Uric acid stones stones  Abnormally acidic urine  Most common  Associated with gout  Reduce intake of  Low-purine diet oxalate Cystine stones  Avoid vitamin C  Inherited disorder cystinuria supplements Struvite stones  Form in alkaline urine Calcium Oxalate Stone © 2007 Thomson - Wadsworth Consequences Renal colic Urinary tract complications  Severe, continuous  Urgency pain  Frequency  Inabilityto urinate  Begins in the back &  Obstruction travels toward bladder  Infection  Nausea & vomiting Prevention & Treatment Drink 12-16 cups of fluids/day Tea, coffee, wine, beer No apple or grapefruit juices ELECTROLYTES  Chapter KEY TERMS Hyper / Hypo … natremia , Electrolyte kalemia, calcemia Osmolality Parathyroid Hormone ( PTH ) Acidosis / Alkalosis Osmolality Calcitonin Polydipsia Ion Selective Electrode ADH Na = Sodium Hypothalamus Gland K = Potassium Renin - Angiotensin - Aldosterone Cl = Chloride System CO2 = Carbon Dioxide Ca = Calcium Mg = Magnesium PO4 = Phosphate 95 Types of dysfunction for renal  Out Line - Introduction - Metabolism - Clinical Application - Normal Value - Clinical Significance - Determination  Electrolytes Electrolytes  Substances whose molecules dissociate into ions when they are placed in water.  CATIONS (+) ANIONS (-) Medically significant (panel Diagnosis ) include:  sodium (Na)  potassium (K)  chloride (Cl)  CO2 (in its ion form = HCO3- ) 98  Classifications of ions : - by their charge 1- Cations – have a positive charge - in an electrical field, (move toward the cathode) Na+ = most abundant extracellular cation K+ = most abundant intracellular cation 2- Anions – have a negative charge - move toward the anode Cl– (1st) most abundant extracellular anion HCO–3 – (bicarbonate) second most abundant extracellular anion 99 Electrolyte Functions Volume and osmotic regulation Myocardial rhythm and contractility Cofactors in enzyme activation Regulation of ATPase ion pumps Acid-base balance Blood coagulation Neuromuscular excitability Production of ATP from glucose 100 Electrolytes Water (the diluent for all electrolytes) constitutes 40-70% of total body and is distributed:  Intracellular – inside cells 2/3 of body water (ICf)  Extracellular – outside cells 1/3 of body water  Intravascular – plasma 93% water  Intrastitial - surrounds the cells in tissue (ISF) 101 Electrolytes 102  Transport of ions : 1- active(energy requirement). 2-passive(diffusion) transport. principles to keep water and ion concentration in place 103  Out Line Osmolality Electrolytes Osmolality Physical property of a solution based on solute concentration  Water concentration is regulated by thirst and urine output  Thirst and urine production are regulated by plasma osmolality 105 Osmolality -  osmolality stimulates two responses that regulate water  Hypothalamus stimulates the sensation of thirst  Posterior pituitary secrets ADH ( ADH increases H2O re-absorption by renal collection ducts ) In both cases, plasma water increases Osmolality – concentration of solute / kg – reported as mOsm / kg another term: – Osmolarity - mOsm / L - not often use 106  Determination 2 methods or principles to determine osmolality Freezing point depression  (the preferred method) Vapor pressure depression  Also called ‘dewpoint’ -Specimen Collection Serum Urine Plasma not recommended due to osmotically active substances that can be introduced into sample Samples should be free of particulate matter..no turbid samples, must centrifuge 107  Electrolytes Calculated osmolality  uses glucose, BUN, & Na values  (Plasma Sodium accounts for 90 % of plasma osmolality) Formula:  1.86 (Na) + glucose∕18 + BUN∕2.8 = calculated osmolality 108  Electrolytes Increase in the difference between measured and calculated  would indicate presence of osmo active substances such as possibly alcohol - ethanol, methanol, or ethylene glycol or other substance.  Osmolality : are concerns for  Infants  Unconscious patients  Elderly 109  Decreased osmolality  Diabetes insipidus ADH deficiency Because they have little / no water reabsorption, produce 10 – 20 liters of urine per day Osmolality normal values – Serum – 275-295 mOsm/Kgm – 24 hour urine – 300-900 mOsm/Kgm – urine/serum ratio – 1.0-3.0 110 Out Line Sodium Routinely measured electrolytes Sodium  the major cation of extracellular fluid outside cells  Most abundant (90 %) extracellular cation  Functions - recall influence on regulation of body water Osmotic activity - sodium determines osmotic activity (Main contributor to plasma osmolality) Neuromuscular excitability - extremes in concentration can result in neuromuscular symptoms 112 Regulation of Sodium Concentration depends on:  intake of water in response to thirst  excretion of water due to blood volume or osmolality changes Renal regulation of sodium  Kidneys can conserve or excrete Na+ depending on ECF and blood volume by aldosterone and the renin-angiotensin system  this system will stimulate the adrenal cortex to secrete aldosterone. 113  Sodium (Na) Aldosterone  From the (adrenal cortex)  Functions promote excretion of K in exchange for reabsorption of Na 114 Clinical Features: Sodium 1- Hyponatremia: < 135 mmol/L  Increased Na+ loss Aldosterone deficiency  Addison’s disease (hypo-adrenalism, result in ➷ aldosterone) Diabetes mellitus  In acidosis of diabetes, Na is excreted with ketones Potassium depletion  K normally excreted , if none, then Na Loss of gastric contents 115  Hyponatremia Increased water retention  Dilution of serum/plasma Na+  excretion of > 20 mmol /mEq urine sodium) Renal failure Nephrotic syndrome Water imbalance  Excess water intake  Chronic condition 116 2- Hypernatremia Excess water loss resulting in dehydration (relative increase)  Sweating  Diarrhea  Burns  Dehydration from inadequate water intake, including thirst mechanism problems  Diabetes insipidus (ADH deficiency … H2O loss ) Excessive IV therapy comatose diabetics following treatment with insulin. Some Na in the cells is kicked out as it is replaced with potassium. – Cushing's syndrome - opposite of Addison’s 117  Specimen Collection: Sodium (Na) serum (sl hemolysis is OK, but not gross) heparinized plasma timed urine sweat GI fluids liquid feces (would be only time of excessive loss) Note: Increased lipids or proteins may cause false decrease in results. artifactual/pseudo- hyponatremia 118  Sodium determination - Ion-selective (specific) electrode Membrane composition = lithium aluminum silicate glass Semi-permeable membrane allows sodium ions to cross 300X faster than potassium and is insensitive to hydrogen ions. 119  Out Line Potassium (K) Routinely measured electrolytes Potassium (K)  the major cation of intracellular fluid Only 2 % of potassium is in the plasma Potassium concentration inside cells is 20 X greater than it is outside. This is maintained by the Na pump, (exchanges 3 Na for 1 K) INSIDE 20  OUTSIDE 1 121  Potassium (K) Function – critically important to the functions of neuromuscular cells  Critical for the control of heart muscle contraction! ↑ potassium promotes muscular excitability ↓ potassium decreases excitability (paralysis and arrhythmias) 122  Regulation of Potassium (K)  Diet easily consumed (bananas etc.)  Kidneys Kidneys - responsible for regulation. Potassium is readily excreted, but gets reabsorbed in the proximal tubule - under the control of ALDOSTERONE Potassium normal values – Serum (adults) – 3.5 - 5.3 mEq/L – Newborns slightly higher – 3.7 - 5.9 mEq/L – Urine (24 hour collection) – 25 - 125 mEq/L 123 - Decrease in K concentration Effects neuromuscular weakness cardiac arrhythmia - Causes of hypokalemia  Excessive fluid loss ( diarrhea, vomiting, diuretics )  ↑ Aldosterone promote Na reabsorption …  K is excreted in its place (Cushing’s syndrome = hyper aldosterone)  Insulin IVs promote rapid cellular potassium uptake 124 Causes of hypokalemia Increased plasma pH ( decreased Hydrogen ion ) RBC H+ K+ K+ moves into RBCs to preserve electrical balance, causing plasma potassium to decrease. ( Sodium also shows a slight decrease ) 125  Hyperkalemia Increased K concentration Causes  IV’S or other increased intake  Renal disease – impaired excretion  Acidosis (Diabetes mellitus ) H+ competes with K+ to get into cells & to be excreted by kidneys Decreased insulin promotes cellular K loss Hyperosomolar plasma (from ↑ glucose) pulls H2O and potassium into the plasma 126  Hyperkalemia Causes  Tissuebreakdown ( RBC hemolysis )  Addison’s - hypo- adrenal; hypo- aldosterone Specimen Collection : Potassium Non-hemolyzed serum heparinized plasma 24 hr urine. 127  Determination Potassium (K)  Ion-selective electrode (valinomycin membrane) insensitive to H+, & prefers K+ 1000 X over Na+  Flame photometry - K λ 766 nm 128  Out Line Chloride (Cl ) -  Chloride ( Cl - ) Chloride - the major anion of extracellular fluid  Chloride moves passively with Na+ or against HCO3- to maintain neutral electrical charge  Chloride usually follows Na (if one is abnormal, so is the other) Function - not completely known body hydration osmotic pressure electrical neutrality & other functions 130  Regulation via diet and kidneys  In the kidney, Cl is reabsorbed in the renal proximal tubules, along with sodium.  Deficiencies of either one limits the reabsorption of the other. Normal values – Serum – 100 -110 mEq/L – 24 hour urine – 110-250 mEq/L varies with intake – CSF – 120-132 mEq/L 131  1- Hypochloremia Decreased serum Cl  loss of gastric HCl  salt loosing renal diseases  metabolic alkalosis;  increased HCO3- & decreased Cl- 2-Hyperchloremia Increased serum Cl – dehydration (relative increase) – excessive intake (IV) – congestive heart failure – renal tubular disease – metabolic acidosis – decreased HCO3- & increased Cl- 132 Specimen Collection: Chloride Serum heparinized plasma 24 hr urine sweat Determination – Amperometric/Coulometric titration – involves titration with silver ions. Mercurimetric titration of Schales and Schales – Precipitate protein out - 1 st step – Titrate using solution of mercury Hg +2 + 2 Cl- = HgCl2 – When all chloride is removed, next drop of mercury will complex with diphenylcarbazone indicator to produce violet color = endpoint a calculation required to determine amt of Cl present by 133 the amt of Hg used  Colorimetric  Procedure suitable for automation  Chloride complexes with mercuric thiocyanate  forms a reddish color proportional to amt of Cl in the specimen. Sweat chloride – – Remember, need fresh sweat to accurately measure true Cl concentration. – Testing purpose - to ID cystic fibrosis patients by the increased salt concentration in their sweat. Pilocarpine iontophoresis – Pilocarpine = the chemical used to stimulate the sweat production – Iontophoresis = mild electrical current that simulates sweat production 134 Chloride ( Cl - ) CSF chloride  NV = 120 - 132 mEq/L (higher than serum)  Often CSF Cl is decreased when CSF protein is increased, as often occurs in bacterial meningitis. 135  Out Line Bicarbonate ion (HCO3- )  bicarbonate ion (HCO3- ) Carbon dioxide/bicarbonate –  * the major anion of intracellular fluid  2nd most important anion (2nd to Cl) Note: most abundant intra-cellular anion 2nd most abundant extra-cellular anion Total plasma CO2 = HCO3- + H2CO3- + CO2 – HCO3- (carbonate ion) accounts for 90% of total plasma CO2 – H2CO3- carbonic acid (bicarbonate) Regulation: – Bicarbonate is regulated by secretion / reabsorption of the renal tubules – Acidosis : ↓ renal excretion 137 – Alkalosis : ↑ renal excretion  Kidney regulation requires the enzyme carbonic anhydrase - which is present in renal tubular cells & RBCs carbonic anhydrase carbonic anhydrase Reaction: CO2 + H2O ⇋ H2CO3 → H+ + HCO–3 CO2 Transport forms – 8% dissolved in plasma dissolved CO2 – 27% carbamino compounds C02 bound to hemoglobin – 65% bicarbonate ion HCO3- - carbonate ion 138  Normal values :  Total Carbon dioxide (venous) – 22-30 mmol/L includes bicarb, dissolved & undissociated H2CO3 - carbonic acid (bicarbonate)  Bicarbonate ion (HCO3–) – 22-26 mEq/L Function : – CO2 is a waste product – continuously produced as a result of cell metabolism, – the ability of the bicarbonate ion to accept a hydrogen ion makes it an efficient and effective means of buffering body pH – dominant buffering system of plasma – makes up 95% of the buffering capacity of 139 plasma bicarbonate ion (HCO3- ) Clinical Significance :  Thebicarbonate ion (HCO–3) is the body's major base substance  Determiningits concentration provides information concerning metabolic acid/base 140  CO2 /bicarb Determination :  Specimen can be heparinized plasma, arterial whole blood or fresh serum. Anaerobic collection preferred. methods Ion selective electrodes Colorimetric Calculated from pH and PCO2 values Measurement of liberated gas 141