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Lab Medicine Conference : Renal & Liver Function Tests Jim Holliman, M.D., F.A.C.E.P. Professor of Surgery and Emergency Medicine Director, Center for International Emergency Medicine M. S. Hershey Medicial Center Penn State University Hershey, Pennsylvania, U.S.A. Lab Medicine Conference : Renal and Liver Function Tests Lecture Objectives –Review renal & liver physiology as it relates to clinical testing –Review methodology for RFT's & LFT's –Discuss indications for obtaining RFT's & LFT's –Determine cost-effectiveness of RFT's & LFT's Physiology of Creatinine Is breakdown product of creatine (the storage source for high-energy phosphate in muscle cells) CPK acts to add high energy phosphate to creatine from ATP Creatine-phosphate transfers the phosphate to re-make ATP when energy is needed for metabolism Physiology of Creatinine (cont.) Synthesis of creatine –First step (guanidoacetate) occurs in kidney, small bowel, pancreas, liver –Second step (methylation of guanidoacetate) occurs in liver Distributed throughout body, mainly to muscle Total body content relatively constant & proportional to muscle mass Metabolic Breakdown of Creatine Creatine phosphate undergoes spontaneous & irreversible breakdown to creatinine Converted at constant rate : 2 % of total body stores per 24 hours Muscle mass is main determinant of amount produced Renal Handling of Creatinine Found in all body secretions, including CSF Has no metabolic "usefulness" Excreted almost entirely via kidneys Freely filtered at glomerulus No passive or active reabsorption along nephron So, major determinant of serum level is degree of renal function Rate of urine flow has no effect on serum level Renal handling of creatinine Urea Physiology Major end product of metabolism of nitrogen-containing substances (mainly protein) Generated mainly in liver –Small amount made in brain Freely diffusable across cell membranes except that of urinary bladder Renal Handling of Urea Excreted mainly renally –Small amounts lost in sweat or metabolized by gut bacteria Freely filtered at glomerulus 1/2 of filtered urea reabsorbed in proximal tubule Water reabsorption in distal tubule (via ADH) & collecting ducts increases tubular luminal concentration of urea While urea concentration in urine is high, only 40 to 80 % of filtered urea is excreted Renal handling of urea Urine Flow Effects on Urea Levels Major determinant of urea reabsorption is rate of urine flow –Depends on glomerular integrity & state of hydration At high urine flow rates (> 2 ml/min.) : 40 % of filtered urea is reabsorbed At lower flow rates, amount of reabsorbed urea proportionately increases Urea load filtered varies with degree of dietary protein intake & tissue breakdown General Measurement Methodology for Creatinine & BUN Done on serum Red top tube Should be run in 2 to 3 hours No problems related to sample collection Free hemoglobin may interfere with assays Measurement of Creatinine Jaffe reaction is standard method –Red solution results from reaction of creatinine & picric acid in alkaline medium –Color change is proportional to amount of creatinine, & follows Beer's Law –Reaction is sensitive to temp. & pH –Pre-Rx with aluminum silicate (Lloyd's Reagent) improves specificity Sakaguchi color reaction is alternate method Urea Analysis Quantification Enzyme urease added to specimen –Catalyzes hydrolysis of urea to carbonic acid & ammonia –Amount of ammonia produced is directly proportional to amount of urea Ammonia is then quantified –Automated analyzers used ƒ React ammonia with alphaketoglutaric acid ƒ Or, an ammonia - sensing electrode is used Urea Analysis : Alternate Method Diacetyl reaction with urea –Forms a measureable chromogen –Simple to perform –Disadvantages : ƒ Less specific ƒ The reagents stink ƒ Non-linear photometric curve Normal Reference Ranges for BUN & Creatinine BUN : –8 to 26 mg/dl –2.9 to 9.3 mmol/liter (International Units) Creatinine –0.7 to 1.5 mg/dl –0.062 to 0.113 mmol/liter Normal BUN : Creat. ratio : –8 to 15 : 1 Azotemia Represents abnormal condition in which the non-protein nitrogenous (NPN) compounds of urea & creatinine are elevated Classed as : –Prerenal –Renal –Postrenal General Causes of Hyperuremia (BUN > 26) Prerenal azotmia Postrenal azotemia Renal dysfunction Increased protein load to liver –Endogenous –Exogenous Prerenal Azotemia Functional integrity of nephrons maintained Due to : –Inadequate renal perfusion ƒ Dehydration ƒ Shock ƒ Blood loss ƒ Congestive heart failure ƒ Renal artery stenosis –Or, increased NPN production Prerenal Azotemia : Causes of Increased NPN Production Endogenous –GI hemorrhage –Catabolic states –Antianabolic medications (steroids, tetracycline) –Cancer chemoRx Exogenous –Increased protein intake Causes of Postrenal Azotemia Generally due to urinary tract obstruction & stasis of urine flow –Renal vein thrombosis –Bilateral ureteral stricture, calculi, or compression –Prostatic hypertrophy or tumor –Bladder obstruction ƒ Tumor ƒ Trauma ƒ Stone or foreign body ƒ Autonomic dysfunction (spinal cord dysfunction) Causes of Renal Azotemia Due to renal insufficiency or failure due to intrinsic renal disease Not reversed by correcting pre- or postrenal problems Etiologies : –Acute tubular necrosis –Acute interstitial nephritis –Nephrotic syndrome –Collagen vascular diseases –Malignancy –Metabolic diseases (esp. diabetes) Mechanisms Causing Increased NPN Compounds with Renal Disease Renal vasoconstriction / decreased renal blood flow Urine stasis from tubular obstruction by debris Back leakage of filtrate into blood Decreased glomerular permeability & GFR Shunting or redistribution of renal blood flow resulting in decreased GFR Causes of Hypouremia (BUN < 6 mg/dl) Physiologic –Newborn –Pregnancy (increased GFR & urine flow) –Overhydration –Decreased protein intake Pathologic –Acute or chronic liver disease General Factors Affecting the BUN Level BUN is dependent on : –Protein intake –Functional integrity of kidneys –Functional integrity of liver –Urine flow rate Causes of Elevated Creatinine Levels (> 1.5 mg %) Intrinsic renal disease Mild elevations from pre- or post- renal azotemia (Transiently) from ingestion of large amounts of meat Extensive muscle trauma Muscle wasting diseases (MD, ALS, myasthenia gravis) Factitious (lab assay interference) Causes of Factitious Elevations of Creatinine Levels Ketone bodies Hyperglycemia Other proteins Barbiturates Penicillins Cephalosporins Methanol Causes of Low Creatinine Levels (< 0.7 mg %) Basically due to decreased muscle mass : –Children –Females –Pathologic : later stages of muscle-wasting diseases Indications to Check BUN & Creatinine Assess dehydration not obvious by physical exam Differentiate renal vs. pre- or post- renal azotemia as cause for decreased urine output Indicate presence of "occult" blood in upper GI tract Verify renal function O.K. prior to dye studies, surgery, or nephrotoxic Rx Evaluate for transplant rejection Monitor for ongoing nephrotoxic drug effect Situations NOT Requiring Checking BUN & Creatinine Dehydration in healthy adults from gastroenteritis Preop in healthy adults for simple abdominal or orthopedic surgery Uncomplicated UTI's Uncomplicated respiratory tract and head & neck infections Mild to moderate back trauma without hematuria Clinical Situations Requiring Periodic BUN & Creatinine Monitoring Aminoglycosides Amphotericin ACE inhibitors Moderate to severe hypertension Diabetes Structural renal disease (polycystic, etc.) Renal transplant Rhabdomyolysis Lab Charges at Hershey Med Center for BUN / Creatinine Both together ("renal profile") : $11.00 Either separate : $ 11.00 "SMA-7" : $ 12.00 Stat fee (for E.D. or inpatients) : 11/2 Times the above $ Laboratory Evaluation of Liver Disease : Topics Covered Enzymes –Alkaline phosphatase –Aminotransferases (transaminases) –Lactate dehydrogenase (LDH) Bilirubin –Direct –Indirect Serologies for viral hepatitis Alkaline Phosphatase (ALP) Physiology Is heterogeneous group of enzymes catalyzing same reaction using different substrates Hydrolyze phosphomonoesters to alcohol & inorganic phosphate at alkaline pH Play role in transport of sugars & phosphates : –Intestinal mucosa –Renal tubules –Bone –Placenta Isoenzymes exist but difficult to separate Causes of Increased ALP Activity in Serum Physiologic –Rapid growth periods in children (ages 5 to 14 years) ƒ Value is 2 to 3 X normal –Pregnancy –Aging –Post fatty meal Pathologic Causes of Increased ALP Activity in Serum Hepatic lesions –Acute hepatitis, mononucleosis, cirrhosis, cholestasis Osteoblastic lesions –Hyperparathyroidism, Rickets, Paget's, fractures, tumors Tumors –Ectopic production Gastrointestinal lesions –Stomach, duodenal, or colon ulcerations Infarcts –Cardiac, pulmonary, renal, spleen Physiology of Aminotransferases (Transaminases) Catalyze reversible transfer of amino group from an alpha amino acid to an alpha keto acid Results in formation of oxaloacetic & pyruvic acids 2 main ones in serum : –Aspartate aminotransferase (AST) ƒ Formerly glutamate oxaloacetic transaminase (GOT) –Alanine aminotransferase (ALT) ƒ Formerly glutamate pyruvate transaminase (GPT) Aspartate Aminotransferase (AST) Found in heart, liver, skeletal muscle, brain, kidney Catalyzes transfer of amino group from aspartate to alpha ketoglutarate Present in both mitochondria & cytosol Present in serum as both apoenzyme & holoenzyme (i.e., with & without cofactor pyridoxal-5-phosphate; same as for ALT) Currently measurement of AST isoenzymes not clinically useful Causes of Elevated AST Levels Myocardial infarction Acute hepatic necrosis Pulmonary infarction (mild elevations in 30 %) Congestive heart failure (passive liver congestion) Pericarditis Rheumatic fever Skeletal muscle injury Alanine Aminotransferase (ALT) Localized primarily in liver Catalyzes transfer of amino group from alanine to alphaketoglutarate Is specific marker for hepatic disease or injury Only in cytosol (not in mitochondria) Lactate Dehydrogenase (LDH) Physiology Catalyzes the reversible reaction : –lactate + NAD pyruvate + NADH Maintains balance between anabolism & catabolism of carbohydrates In liver is involved in gluconeogenesis & glycogen synthesis from lactate In heart enables lactate to enter citric acid cycle & be used as fuel to generate ATP & NAD Most tissues have high quantities of LDH Isoenzymes of LDH Are tetramers made of 4 subunits containing one of 2 tissue types : H (heart) or M (skeletal muscle) There are 5 isozymes of LDH which consist of combos of the monomers Normal LDH activity in serum is mainly of erythrocyte origin Isoenzymes of LDH TYPE MONOMERS ORGAN LOCATION LDH 1 HHHH Myocardium, erythrocytes LDH 2 HHHM Myocardium, erythrocytes LDH 3 HHMM Brain, kidney, less in liver & muscle LDH 4 HMMM Liver, brain, kidney, muscle LDH 5 MMMM Liver, muscle, less in kidney Conditions with Increased LDH Levels Cardiac –Myocardial infarction –CHF –Pulmonary infarction Hematologic –Megaloblastic anemia –Sickle cell disease –Hemolytic anemia –Leukemias –Lymphoma –Infectious mononucleosis Conditions with Increased LDH Levels (cont.) Hepatic –Hepatitis –Obstructive jaundice –Cirrhosis –Metastatic tumors Skeletal –Muscular dystrophy –Delerium tremens LDH Isoenzyme Patterns in Different Conditions DISEASE Acute MI Meg.anemia Hem.anemia Mus.dystro. Leukemia Pancreatitis Ca. mets Pulm infarct C.H.F. Hepatitis Cirrhosis LDH-1 LDH-2 + + + + + + + + LDH-3 + + + LDH-4 LDH-5 + + + + + + + Measurement Methodology for Liver Enzymes ALP –Rate of conversion of p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) in presence of buffer AMP –Change in absorbance at 405 nm due to formation of p-NP is proportional to ALP activity AST, ALT –Change in absorbance at 340 nm due to disappearance of NADH is proportional to AST & ALT activity LDH –Change in absorbance at 340 nm due to appearance of NADH is proportional to total LDH activity –LDH isozymes separated electrophoretically & stained General Diagnostic Interpretation of AST & ALT Levels ALT is most specific measure of hepatocellular damage (necrosis) Highest AST & ALT levels occur with : –Acute viral hepatitis –Toxin - induced hepatic necrosis –Circulatory shock Damage to as little as 1% of liver cells raises ALT AST & ALT rise 7 to 14 days before jaundice AST elevation can be screen for Reye's Syndrome General Interpretation of AST & ALT Levels (cont.) Degree of elevation not necessarily related to severity of disease process Levels < 500 U/liter usually mean mild illness Ratio of AST : ALT > 2 highly suggestive of alcoholic hepatitis (unless ALT > 300, then this does not apply) LDH usually normal or only slightly elevated with hepatitis or obstructive jaundice Patterns of Enzyme Elevations in Liver and Biliary Diseases DISEASE ALP AST ALT LDH Acute Liver Injury 4 - 10 X >20 X >20 X +/- Alcoholic hepatitis 2-4X 4 - 10 X 2-4X +/- Infectious Mononucleosis Cholestatic jaundice 2 - 10 X 10 - 20 X 10 - 20 X 10 - 20 X 4 - 10 X 4 - 10 X Primary or Secon. cancer 10 - 20 X 4 - 10 X 4 - 10 X 4 - 20 X Primary biliary cirrhosis 10 - >20 X 4 - 10 X 4 - 10 X 2-4X Alcoholic fatty liver 2-4X 2-4X +/- Cirrhosis 2-4X 2-4X 2-4X 2-4X Chronic active hepatitis 2-4X 10 - 20 X 4 - 10 X 2-4X 2 - 10 X +/- +/- Time pattern of serum transaminases Bilirubin Metabolism Originates from breakdown of heme (from hemoglobin, myoglobin, & cytochromes) into biliverdin which is reduced to form bilirubin Can be produced by most cells Free (unconjugated) bilirubin enters plasma from sites of production –Is tightly bound to albumin –Not filtered at glomerulus (not excreted in urine) –Taken up by hepatocytes –Conjugated in microsomes by enzyme bilirubin glucuronyl transferase –Bilirubin diglucuronide (water soluble) then excreted via bile Disposition of Conjugated Bilirubin Enters intestine via bile Further reduced by colonic bacteria to stercobilinogen / urobilinogen, which is spontaneously oxidized to brown bilin pigment (accounts for normal stool color) Some of this pigment undergoes enterohepatic cycling Trace amounts excreted in urine as urobilinogen, which autooxidizes to urobilin "Direct" versus "Indirect" Bilirubin "Indirect" = unconjugated (non- liver metabolized) –Is nonmiscible with aqueous diazonium salts –So solvent such as methyl alcohol is needed to render it water soluble, permitting a color reaction "Direct" = conjugated (acted upon by liver cells) –Reacts directly with diazo reagents to make a measureable color change Normal serum total bilirubin is 0.5 to 1.2 mg/dl (< 20 % unconjugated) Bilirubin metabolism Bilirubin metabolism in hepatic disease Bilirubin metabolism in extra- hepatic obstruction Causes of Jaundice from Unconjugated Hyperbilirubinemia Pigment loading –Hemolytic anemia –Extravascular blood (surgery or trauma) –Liver disease (unable to conjugate) Gilbert's Syndrome –Usually benign –Bilirubin levels elevate with fasting Crigler-Najjar Syndrome –If homozygous is severe & needs liver transplantation Jaundice from Conjugated Hyperbilirubiinemia Usually reflects cholestasis –Retention of bilirubin & bile salts –Can be intra- or extra- hepatic cause –Urine is dark brown (from conjugated bilirubin) –Urine froths if shaken (from detergent action of bile acids) –Patients often have pruritis from bile acids Intrahepatic Causes of Conjugated Hyperbilirubinemia Hepatocellular injury Biliary atresia Primary biliary cirrhosis Steroids (especially estrogens) Space - occupying hepatic lesions Dubin-Johnson Syndrome Extrahepatic Causes of Conjugated Hyperbilirubinemia Choledocholithiasis Caroli's Disease Postoperative biliary tract strictures Sclerosing cholangitis Cholangiocarcinoma Pancreatitis Ampullary or pancreatic cancer Compression from adjacent cysts Parasites Other Tests to Consider for Evaluation of Liver Disease Protime –Measures presence of liver-synthesized vitamin K dependent factors II, VII, X –Factor VII has half life < 12 hours –Indicates significant liver dysfunction if prolonged > 2 seconds Serum albumin –Normal level 3.5 to 5 g/dl –Synthesized exclusively in liverMax. synthesis is 25 g/day (half life is up to 20 days) Serum globulin –Levels often > 2 g/dl (normal < 1.1 g/dl) with chronic liver disease Gamma glutamyl transpeptidase Gamma Glutamyl Transpeptidase (GGTP) Found in liver, kidney, pancreas, heart, brain Elevates in cholestatic disorders Inducible by many drugs Half life 26 days If GGTP level is normal, it suggests a concurrent ALP elevation is from bone or placenta Can be elevated in non-liver disorders (other LFT's are then normal) Additional Tests to Consider To Rule Out Specific Liver Disorders Alpha-1-antitrypsin level –Rule out alpha-1-antitrypsin deficiency –These patients can have CAH & COPD –Is autosomal recessive ; relatives should be screened Serum ceruloplasmin –Rule out Wilson's Disease –Should confirm with liver biopsy –Is treatable Serum iron / TIBC –Rule out hemochromatosis ; also treatable Elevated AST Algorithm for evaluation of elevated alkaline phosphatase Hepatitis A Serologies Hepatitis A IgM antibody (IgM anti-HAV) –If positive, represents current or recent acute hepatitis A –Persists typically 4 to 6 months (but up to 12) post infection Hepatitis A total antibody (total antiHAV) –Tests IgM & IgA early, and mainly IgG later Time course of hepatitis A serologies Hepatitis B Serologies Antibody to hepatitis B surface antigen (anti-HBs) –If present indicates : ƒ Prior hepatitis B, now immune ƒ Or prior hepatitis B vaccination ƒ Or recent hepatitis B immune globulin prophylaxis –If HBsAg also present, indicates chronic hepatitis B (carrier) Hepatitis B surface antigen (HBsAg) –If present, indicates acute or chronic hepatitis B and patient is infectious Hepatitis B Serologies (cont.) Hepatitis B e antigen (HBeAg) –If present, indicates acute or chronic hepatitis B with active viral replication Antibody to hepatitis B e antigen (antiHBe) –Indicates suppression of hepatitis B viral replication Hepatitis B Serologies (cont.) Hepatitis B core IgM antibody (IgM antiHBc) –Indicates current or recent hepatitis B (in past 4 to 6 months), or chronic hepatitis B with active viral replication (less common) Hepatitis B core total antibody (total antiHBc) –Just indicates prior hepatitis B infection, but does not indicate infectivity or chronicity Time course of acute hepatitis B serologies Time course of chronic hepatitis B serologies Serologies with hepatitis D superinfection Serologies with acute hepatitis D coinfection Hepatitis C Serologies Hepatitis C antibody by enzyme immunoassay (antiHCV by EIA) –Indicates chronic hepatitis C ( rarely detectable for acute hepatitis C) Hepatitis C antibody by recombinant immunoblot assay (anti-HCV by RIBA) –Indicates chronic hepatitis C (useful for evaluating suspected false positive anti-HCV by EIA) Hepatitis C RNA by polymerase chain reaction (HCV RNA by PCR) –Indicates acute or chronic hepatitis C These antibodies do not confer protection against infection Lab Charges for Liver Function Tests at Hershey Med Center "LFT panel" (ALP, AST, total bili) : $18 ALT alone : $10 AST alone : $10 Hepatitis serologies : –HCV antibody : $42 –HAV antibody : $37 –HBc antibody : $30 –HBsAg : $30 –anti-HBs : $43