Clinical Chemistry - 09 NPNs and Kidney Function Tests

CLINICAL CHEMISTRY NON-PROTEIN NITROGENOUS COMPOUNS & KIDNEY FUNCTION TESTS OUTLINE • The Kidney o Kidneys o Nephrons o Functions of Kidney • Non-Protein Nitrogenous Compounds (NPN) o Ammonia o Urea Nitrogen (Blood) o Creatinine o Creatine o Azotemia o Uric Acid • Renal Function Tests: Tests for Glomerular Filtration Rate o Glomerular Filtration Rate • Renal Function Tests: Tests for Renal Blood Flow o Urea Nitrogen Methodologies o Creatinine Methodologies o Uric Acid Methodologies • Renal Function Tests: Tests for Tubular Function o Excretion Tests o Concentration Test THE KIDNEY KIDNEYS • • • Pair of bean-shaped organs located retroperitoneally on either side of the spinal column. Has two regions: o Renal cortex (outer) o Renal medulla (inner) Each kidney contains 1-1.5 million nephrons (functional unit of kidneys) FUNCTIONS OF KIDNEY • • • • Main function is urine formation o Excretion of waste products (NPN) Maintenance of blood volume and electrolyte balance. o Aldosterone – is an adrenal hormone that regulates water and salt in the body ▪ Promotes reabsorption of sodium and excretion of potassium by the kidney ▪ When kidney detect increased sodium concentration, it will excrete sodium through urine reabsorbing potassium and vice versa o Vasopressin – aka anti-diuretic hormone; prevents water loss through urination Maintenance of acid-base balance o By excretion or reabsorption of bicarbonate o Bicarbonate - alkaline; basic component of acid-base balance/buffer system of blood Endocrine function – produces erythropoietin (RBC production) NON-PROTEIN NITROGENOUS (NPN) COMPOUNDS • NEPHRONS • • The functional unit of each kidney o Filters blood 5 basic parts: o Glomerulus ▪ Captures filtered blood from afferent & efferent arterioles o Proximal Convoluted Tubule ▪ Reabsorbs sodium, chloride, bicarbonate, glucose, amino acids, proteins, urea and uric acid o Loop of Henle ▪ Major exchange of water and salts ▪ Embedded in renal medulla o Distal Convoluted Tubule o Collecting duct ▪ Final site for either concentrating or diluting urine • • Products of metabolic processes (catabolic products/wastes) o Metabolic wastes - has no physiologic function ▪ From muscle, DNA, amino acid degradation o Normally excreted through the urine (normal response of body for substances with no physiologic function) Useful for assessing kidney function Plasma contains 20-35mg/dL of NPN compounds. o Urea (45%) o Amino acid (20%) o Uric acid (20%) o Creatinine (5%) o Creatine (1-2%) o Ammonia (0.2%) AMMONIA • • • Least abundant NPN Produced by the catabolism of amino acids and by bacterial metabolism in the intestine Excreted by the kidneys or consumed by the liver to produce Urea o Not easily excreted due to necessary conversion to urea before excretion • • • • • Toxic to the body o Due to increased ammonia concentration At normal blood pH: most exist as ammonium ion (NH 4+) Used for diagnosis: o Hepatic failure - decreased conversion in liver of ammonia to urea which increases ammonia levels o Inherited deficiencies of urea cycle enzymes o Reye’s syndrome – acute metabolic disorder of the liver (most common among children) More useful in liver function test Reference value: 19-60ug/dL (11-35 mmol/L) UREA NITROGEN (BLOOD) BUN • • • • • • • • • aka Urea Major end product of protein and amino acid catabolism Most abundant NPN (40%) Approximately 80% of the nitrogen excreted Easily removed by dialysis Formed through the Krebs-Henseleit (Urea) cycle in liver First metabolite to increase in kidney disease Reference value: 8-23 mg/dL (CF to mmol/L: 0.357) Normal Bun:Creatinine Ratio: 10-20:1 • Assays for urea were based on measurement of nitrogen in blood, hence the name blood urea nitrogen. Excreted by the kidneys – 40% reabsorbed o Most are reabsorbed which indicate excretory function <10% of the total are excreted through the gastrointestinal tract and skin. Decreased: Low protein dietary intake, liver disease, severe vomiting or diarrhea, increase protein synthesis o Low protein diet - low protein synthesis decreasing urea formation from urea cycle o Liver disease - problem in conversion of ammonia to urea; decrease BUN and increased ammonia Concentration is determined by: o Renal function o Dietary intake o Protein catabolism rate • • • • • Clinical Application: o Evaluate renal function ▪ Large fraction of BUN is reabsorbed by kidney o To assess hydration status o To determine nitrogen balance o To aid in diagnosis of renal disease o To verify adequacy of dialysis CREATININE • • • • • • • End product of muscle metabolism derived from creatine and creatine phosphate Synthesized primarily by the liver from arginine, glycine and methionine Partially secreted by the proximal tubules via the organic cation transport pathway Not easily removed by dialysis Measure the completeness of 24 hour urine collection (urine creatinine). Synthesis of Creatinine o Creatinine is produced as waste product of creatine and creatine phosphate o Creatine is converted to creatinine by dehydration o Creatine phosphate is converted to creatinine by dephosphorylation. o Creatine may be phosphorylated to creatine phosphate by Creatine kinase Significance o Creatinine is released into circulation at stable rate proportional to muscle mass o Filtered by glomerulus o Excreted in urine o Plasma creatinine concentration is a function of: ▪ relative muscle mass, ▪ rate of creatine turnover ▪ renal function o Daily creatinine excretion is fairly stable. o Index of overall renal function ▪ Slight disturbance in excretion directly indicate impairment in renal function o Reference value (CF to umol/L: 88.4): ▪ Male: 0.9-1.3 mg/dL ▪ Female: 0.6-1.1 mg/dL ▪ Child: 0.3-0.7 mg/dL DISEASE CORRELATION • • • Elevated Creatinine is found is abnormal renal function. Measurement of creatinine concentration is used to determine: o sufficiency of kidney function o severity of kidney damage o monitor the progression of kidney disease. Acute Kidney Injury – functional or structural abnormalities or markers of kidney damage (seen in blood, urine, tissue test, imaging studies) present for less than three months. o Associated with retention of creatinine, urea, and other metabolic waste products that are normally excreted by the kidney ▪ Creatinine retention - inc. creatinine o Criteria: Stage 1 2 3 Serum Creatinine Criteria Urine Output Criteria >0.3 mg/dL or 150-200% <0.5 mL/Kg for >6hr >200-300% <0.5 mL/Kg for >12hr >300%, 4mg/dL, or acute <0.3 ml/Kg for >24 hr increase of >0.5 mg/dL or anuria >12h • • • POST RENAL AZOTEMIA • • • CREATINE • • • • • Elevated in plasma and urine in o Muscular dystrophy, hyperthyroidism, trauma. Plasma creatine levels usually normal, but urinary is elevated Specialized testing – not part of routine lab Analyzing the sample for creatinine before and after heating in acid solution using an endpoint Jaffe method. Heating converts creatine to creatinine and the difference between the two samples is the creatine concentration. o Creatine concentration - difference creatinine between heating and after heating • Elevated concentration of NPN (urea & creatinine) in blood Uremia/Uremic syndrome – elevated plasma urea concentration accompanied by renal failure Causes of urea plasma elevations are: o Prerenal o Renal o Postrenal Usually the result of Urinary Tract Obstruction Urea level is higher than creatinine due to back-diffusion/ reabsorption of urea into the circulation. Causes: nephrolithiasis, renal calculi, cancer/ tumors of genitourinary tract, severe infection o Nephrolithiasis - presence of stones in the nephrons URIC ACID (BUA) • • • • AZOTEMIA • • Lab Results: o BUN – abrupt elevation (>100 mg/dL) o Creatinine – slow elevation (20 mg/dL) o BUA – 12 mg/dL o Anemia (due to impairment in EPO production for RBC synthesis) o Electrolyte Imbalance (due to electrolyte balance function) Causes: o Acute/chronic renal disease o Glomerulonephritis o Tubular necrosis Complications: o Coma o Neuropsychiatric changes • Major product of purine (adenine and guanine) catabolism The final breakdown of nucleic acids catabolism in humans o Ingested nucleoproteins o Endogenous nucleoproteins (nucleic acids in body) o Direct transformation of endogenous purine nucleotides Formed from xanthine by the action of xanthine oxidase in the liver and intestine. Uric acid is transported to kidney and filtered (70%) o About 1 gram of uric acid is excreted normal o 98% reabsorbed in PCT o Some secreted by DCT o Net amount 6-12% of filtered amount Remaining 30% by GIT PRE-RENAL AZOTEMIA • • Reduced renal blood flow → less blood delivered to the kidney → less urea filtered Causes: o Anything that produces a decrease in functional blood volume, include: ▪ Congestive heart failure ▪ Shock ▪ Hemorrhage ▪ Dehydration o High protein diet or increased catabolism (Fever, major illness, stress) RENAL AZOTEMIA • • • True renal disease Characterized by damage within the kidney Damaged kidneys → Poor excretion → Increased Urea • Clinical Significance o Present in plasma as monosodium urate (95%) o At plasma pH → relatively insoluble o Conc. > 6.8 mg/dl → plasma saturated → urate crystals may form & precipitate in tissue o Reference value (CF to mmol/L: 0.059): ▪ Male: 3.5-7.2 mg/dL ▪ Female: 2.6-6.0 mg/dL ▪ Child: 2.0-5.5 mg/dL o Uric acid is measured to: ▪ assess inherited disorders of purine metabolism ▪ confirm diagnosis and monitor treatment of gout ▪ assist in the diagnosis of renal calculi ▪ prevent uric acid nephropathy during chemotherapeutic treatment ▪ detect kidney dysfunction HYPERURICEMIA • • • • • • Increased uric acid concentration in blood Gout o Most common cause of hyperuricemia o Primarily in men and first diagnosed between 3rd and 5th decade of life (30-50 years old) o Pain & inflammation of joints by precipitation of sodium urates in tissues ▪ Definitive diagnosis: presence of “birefringent crystal in synovial fluid” o UA greater than 6.0 mg/dL o Increased risk of renal calculi/nephrolithiasis o Hyperuricemia due to overproduction of uric acid in 25-30% Increased nuclear metabolism o Occurs in patients on chemotherapy for diseases such as leukemia & multiple myeloma. ▪ Chemotherapeutic drugs increase metabolism of nucleic acids which increases uric acid o Allopurinol inhibits xanthine oxidase, an enzyme in uric acid synthesis pathway, is used to treat these patients. Chronic Renal Disease o Causes elevated levels of uric acid because filtration and secretion are hindered. o BUA: >10mg/dL (can cause production of urinary tract calculi) Lesch-Nyhan Syndrome (inborn errors of purine metabolism) o It is deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) Other Causes: 1. Secondary to glycogen storage disease 2. Toxemia of pregnancy and lactic acidosis 3. Increased dietary intake (purine-rich food) 4. Ethanol consumption • • • • • CLEARANCE • • • • • • • • • • • Increased uric acid concentration in blood Fanconi’s syndrome o Disorder of reabsorption in the PCT of the kidney Wilson’s disease Hodgkin’s disease Overtreatment with allopurinol Chemotherapy (6-mercaptopurine, azathioprine) Alzheimer’s disease Parkinson’s disease RENAL FUNCTION TESTS: TESTS FOR GLOMERULAR FILTRATION RATE GLOMERULAR FILTRATION RATE (GFR) • • Best overall indicator of the level of kidney function Measures of the clearance of substances not bound to protein. o Freely filtered by glomerulus o Not secreted not reabsorbed by the tubules. o Substances totally excreted. Removal of the substance from plasma into urine over a fixed time. It represents the volume of plasma that would contribute all the solute excreted. Plasma concentration is inversely proportional to clearance Decreased clearance of substance = increased plasma concentration Formula: U – conc. of analyte (urine) P – conc. of analyte (Plasma) Volume – Urine volume in mL (24 hours) Minutes – time required to collect urine (1440 minutes) o 1.73 – ave. body surface of adult individual (0.717 for pediatric) o A – body surface of patient (nomogram: height and weight are taken) Inulin Clearance Test o Reference method o Inulin: ▪ Exogenous substance (never produced by body) ▪ Introduced via intravenously (500 mL of 1.5% inulin solution) ▪ Urine collection are times over many hours o Amount of inulin administered should be near the amount excreted o Not routinely done due to the necessity for continuous IV infusion o Alternatives to Inulin ▪ Radioactive marker: 125I-iothalamate & 99mTc-DTPA ▪ Iohexol and Chromium 51-labelled EDTA ▪ Nonradiolabeled iothalamate o Reference value: ▪ Male: 127 mL/min ▪ Female: 118 mL/min Creatinine Clearance o Best alternative method to Inulin o Creatinine – endogenous substance freely filtered by the glomeruli but not reabsorbed (excreted in urine) ▪ Production and excretion of creatinine are directly related to muscle mass. ▪ Excretion is not routinely affected by diet – 1.21.5 g creatinine excreted/day. o Excellent measure of renal function ▪ Kidney issues may affect excretion of creatinine o Measure of completeness of 24 hour urine collection o Serum creatinine should be collected within 24 hour urine collection (for comparison) o Major limitation: accurate urine collection ▪ w/in 24 hours, all urine voided should be collected o Reference value: ▪ Male: 85-125 mL/min ▪ Female: 75-112 mL/min o o o o • HYPOURICEMIA • • 150 liters of glomerular filtrate is produced daily. GFR decreased by 1.0 mL/minute/year after age 20-30 years About 180 liters of water if filtered daily 150 liters is reabsorbed in the PCT 5 liters in the descending limb of henle of cortical nephrons. • o Creatinine Clearance Formula: • GFR formula using β Trace Protein o White Formula ▪ Affected by gender ▪ o Affected by body surface area and correction for body mass should be included in the formula • 1.73/A = correction factor (std. body surface area for adults) Cockcrooft-Gault Formula: o Page Formula ▪ Unaffected by gender ▪ ▪ o Results are not corrected for body surface area Assumes that women will have a 15% lower creatinine clearance than men at the same level of serum creatinine Modification of Diet in Renal Disease Formula (MDRD) RENAL FUNCTION TESTS: TESTS FOR RENAL BLOOD FLOW ▪ ▪ ▪ • Multiply result by 1.212 if black Multiply result by 0.742 if female Provide more accurate assessment of GFR than Cockcroft-Gault formula ▪ Does not require patient weight ▪ Variables: age, serum creatinine, race, gender Urea Clearance o Demonstrate progression of renal disease or response to therapy o Not reliable GFR – urea is freely filtered but variably reabsorbed by the tubules UREA NITROGEN METHODOLOGIES • • • CYSTATIN C • • • • • • • • • Test for renal blood flow Low molecular weight protease inhibitor and produced at a constant rate by all nucleated cells. Indirect estimate of GFR o Freely filtered by the glomerulus but completely reabsorbed by the tubules (not secreted) o Healthy urine = “0” Cystatin C o Presence in urine denotes damage in the PCT Not affected by muscle mass, age, diet and gender. Only kidney function test never measured in urine. Specimen: serum or plasma Increased (↓ GFR): acute and chronic renal failure, diabetic nephropathy Reference value: o Adult: 0.5-1.0 mg/L o >65 years old: 0.9-3.4 mg/L GFR is computed using Modified Cystatin C Equation: Sample: serum, plasma, urine o Fluoride and Citrate: inhibits urease ▪ Not to be used as anticoagulants o Refrigerate sample if delay in testing is expected ▪ To prevent decomposition of urea by ureasepositive bacteria Assays for urea were based on measurement of nitrogen, hence the name blood urea nitrogen. 3 methods: o Chemical Method ▪ Diacetyl Monoxime (DAM) Method – non-specific method ▪ Yellow diazine derivative - measured spectrophotometrically o o Enzymatic Method Isotope Dilution Mass Spectrometry (Reference Method) UREA TO BUN • • • • • Atomic mass of Nitrogen = 14 g/mol Molecular mass of urea = 60 g/mol Urea = 2 Nitrogen atoms (46.6% of the total weight of urea) Hence, there is 28 g/mol of Nitrogen per 60g/mol of Urea Therefore, o 1 gram BUN = 0.4666 g/mol of Urea (28/60) Urea = BUN x 2.14 ENZYMATIC METHODS (BUN) β TRACE PROTEIN • • • • A low molecular weight glycoprotein Belongs to the lipocalin protein family and functions as prostaglandin D synthase Isolated primarily from CSF (indirect measure of GFR) o Freely filtered by the glomerulus but completely reabsorbed and catabolized by the proximal tubule. Increased: Renal disease (because of reduced filtration in the presence if constant production) • Urease Method o Hydrolysis of Urea to ammonia by the enzyme urease o Urease – prepared from jack beans o Ammonia may be subjected to Berthelot reagents to form a chromogenic end o • Ammonia and CO2 can be measured by different method to calculate concentration of urea in sample. ▪ Measurement of ammonia is commonly used Coupled Urease/Glutamate dehydrogenase (GLD) method o UV Enzymatic Method o Measures consumption of NADH ▪ More consumed NADH = more NAD ▪ NAD inversely proportional to urea KINETIC JAFFE METHOD • • • Requires automated analyzers for precision Principle: Serum is mixed with alkaline picrate solution and the rate of change in absorbance is measured between two points. o When reagent is added to sample, it will read the intitial absorbance (1st pt.) o Read another absorbance after another time (2nd pt.) Interferences : o False increased: α-ketoglutarate, cephalosporins CREATININE METHODOLOGIES • • • • Sample: serum, plasma, urine Interference: hemolysis, ictericia, lipemia 24 hr urine sample with <0.8 g/day of creatinine indicates that some of the urine was probably discarded o Overall indicator of complete 24hr urine specimen 4 Methods: 1. Direct Jaffe Method (Chemical Method) 2. Kinetic Jaffe Method 3. Enzymatic Method 4. Isotope Dilution Mass Spectrometry (IDMS) – reference method ENZYMATIC METHOD • • • • • Routinely used method; more commonly used method Used to eliminate non-specificity of the Jaffe reaction Specific than Jaffe test Creatinase – creatinine aminohydrolase Creatinine Aminohydrolase-CK Method o Requires a large volume of pre-incubated sample o Not widely used • Creatinase-Hydrogen Peroxide Method o Has potential to replace Jaffe method (more specific) o W/o interference from acetoacetate & cephalosporins DIRECT JAFFE METHOD • Principle: a red-orange tautomer of creatinine picrate is formed when creatinine is mixed with alkaline picrate reagent (Jaffe reagent) • Jaffe reagent: o Saturated picric acid o 10% NaOH o Unstable reagent: prolonged storage may form picramic acid + methyl guanidine (orange color) ▪ Discard when orange color is observed Interferences: o False increase: ▪ Creatinine-like analytes: Uric acid, ascorbic acid, glucose, α-keto acids ▪ Medications: cephalosporins, dopamine, lidocaine o False decrease: bilirubin, hemoglobin Folin Wu Method o A sensitive but not specific method Lloyd or Fuller’s Earth Method o A sensitive and specific method o Removes interferences present in specimen by an adsorbent (creatinine-like analytes) o Adsorbents: ▪ Lloyd’s reagent • Sodium aluminum silicate ▪ Fuller’s Earth Reagent • Aluminum Mg2+ silicate o Disadvatanges: ▪ Time consuming ▪ Not employed to automated analyzers • • • URIC ACID METHODOLOGIES • • • • • Sample: serum, heparinized plasma, urine o Serum may be stored for 3-5 days (refrigerator 4C) o For uricase method: EDTA nor fluoride should not be used ▪ Heparinized - preferred anticoagulant o Urine sample (pH 8.0) Recent meal does not affect the uric acid concentration Fasting is not required 3 Methods: 1. Chemical Methods 2. Enzymatic Methods 3. Isotope Dilution Mass Spectrometry – reference method Interferences: o False increased: lipemia, salicylates, thiazides o False decreased: high bilirubin, hemolysis CHEMICAL METHOD • Principle: Reduction-oxidation reaction (redox reaction) • • • Sodium cyanide (NaCN): Folin, Newton, Brown, Benedict Sodium carbonate (NaCO3): Caraway, Archibald, Henry Lag phase – the incubation period after the addition of NaCN/NaCO3 to inactivate non-uric acid reactants • • • ENZYMATIC METHOD • • • Uricase method Specific method Principle: Uric acid is oxidized to form allantoin by uricase enzyme. • • • Uric acid: peak absorbance at 293 nm. Allantoin: not absorbed at 293 nm. The decrease in absorbance is proportional to the concentration of uric acid • INTERPRETATION OF RESULTS RENAL FUNCTION TESTS: TESTS FOR TUBULAR FUNCTION • EXCRETION TESTS • PARA-AMINO HIPPURATE TEST • • • • Diodrast test Measures renal plasma flow This method requires clearance of dye (para-amino Hippurate) Reference value: 600-700 mL/min • PHENOLSULFONTHALEIN (PSP) DYE TEST • • • Measures excretion of dye proportional to renal tubular mass Dose: 6 mg of PSP (IV) Reference value: 1200 mL blood flow/minute CONCENTRATION TEST URINE SPECIFIC GRAVITY • • • • • • Simplest test Compares the weight of fluid with that of distilled water at a reference temperature Affected by solute number and mass o Glucose, urea, protein o SG: 0.003 unit increase = 1% change in protein Fixed Urine SG = 1.010 o Loss of concentrating ability of kidneys o 1.010 SG is equal to the SG of protein-free plasma Specimen: first morning urine Reference value: 1.005-1.030 OSMOLALITY • • • • Expression of concentration in terms of total number of solute particles present per kilogram of solvent (moles/Kg solvent) More reliable that urine specific gravity Only affected by the number of solutes present o Urine Osmolality – primarily due to urea o Serum Osmolality – primarily due to Na+ & Clo Protein & Lipids do not affect osmolality Useful for assessing water deficit or excess Sample: Serum or 24 hr Urine Reference value: o Serum: 275-295 mOsm/Kg o 24 hr Urine: 300-900 mOsm/Kg Direct Method o Directly measure osmolality of sample o Methods: ▪ Freezing Point Osmometry (popular method) ▪ Vapor Pressure Osmometry (Seebeck Effect) o An increase in osmolality (solute) decreases freezing point and vapor pressure Indirect Method o Formula for serum osmolality o To use glucose or urea in osmolality, calculations must be converted from milligram units to molar units o Glucose – not requires fasting • Concentrated urine: o Specific gravity: 1.025 o Osmolality: >800 mOsm/Kg Chronic renal disease: o Specific gravity: fixed 1.010 on multiple sample o Osmolality: 290 mOsm/Kg Serum-to-urine osmolality ratio: o 1:1 - Normal o >1:1 – glomerular disease, increased solutes in the urinary filtrate o <1:1 – Distal tubular disease (diabetes insipidus – severe polyuria) ▪ Decreased ADU - reduce suppression of urine which increases urine output causing concentration of serum