Lab Medicine Conference :

Renal & Liver FunctionTests

Jim Holliman, M.D., F.A.C.E.P.Professor of Surgery and Emergency Medicine

Director, Center for International Emergency MedicineM. S. Hershey Medicial Center

Penn State UniversityHershey, 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 CSFHas no metabolic "usefulness"Excreted almost entirely via kidneysFreely filtered at glomerulusNo passive or active reabsorption along

nephronSo, major determinant of serum level is degree

of renal functionRate 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 UreaExcreted mainly renally

–Small amounts lost in sweat or metabolized by gut bacteria

Freely filtered at glomerulus1/2 of filtered urea reabsorbed in proximal

tubuleWater 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 &

BUNDone on serumRed top tubeShould be run in 2 to 3 hoursNo problems related to sample

collectionFree 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 acidƒ 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 specificƒ The reagents stinkƒ Non-linear photometric curve

Normal Reference Rangesfor 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 azotmiaPostrenal azotemiaRenal dysfunctionIncreased protein load to liver

–Endogenous–Exogenous

Prerenal Azotemia

Functional integrity of nephrons maintained

Due to :–Inadequate renal perfusion

ƒ Dehydrationƒ Shockƒ Blood lossƒ Congestive heart failureƒ Renal artery stenosis

–Or, increased NPN production

Prerenal Azotemia :Causes of Increased NPN

ProductionEndogenous

–GI hemorrhage–Catabolic states–Antianabolic medications (steroids, tetracycline)–Cancer chemoRx

Exogenous–Increased protein intake

Causes of Postrenal AzotemiaGenerally 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 Tumorƒ Traumaƒ Stone or foreign bodyƒ Autonomic dysfunction (spinal cord dysfunction)

Causes of Renal AzotemiaDue to renal insufficiency or failure due

to intrinsic renal diseaseNot reversed by correcting pre- or post-

renal problemsEtiologies :

–Acute tubular necrosis–Acute interstitial nephritis–Nephrotic syndrome–Collagen vascular diseases–Malignancy–Metabolic diseases (esp. diabetes)

Mechanisms Causing Increased NPN

Compounds with Renal DiseaseRenal vasoconstriction / decreased

renal blood flowUrine stasis from tubular obstruction by

debrisBack leakage of filtrate into bloodDecreased glomerular permeability &

GFRShunting 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 Affectingthe 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 diseaseMild elevations from pre- or post- renal

azotemia(Transiently) from ingestion of large

amounts of meatExtensive muscle traumaMuscle wasting diseases (MD, ALS,

myasthenia gravis)Factitious (lab assay interference)

Causes of Factitious Elevations

of Creatinine Levels

Ketone bodiesHyperglycemiaOther proteinsBarbituratesPenicillinsCephalosporinsMethanol

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 CheckBUN & 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 rejectionMonitor for ongoing nephrotoxic drug effect

Situations NOT RequiringChecking BUN & Creatinine

Dehydration in healthy adults from gastroenteritis

Preop in healthy adults for simple abdominal or orthopedic surgery

Uncomplicated UTI'sUncomplicated respiratory tract and

head & neck infectionsMild to moderate back trauma without

hematuria

Clinical Situations Requiring Periodic

BUN & Creatinine MonitoringAminoglycosidesAmphotericinACE inhibitorsModerate to severe hypertensionDiabetesStructural renal disease (polycystic, etc.)Renal transplantRhabdomyolysis

Lab Charges at Hershey Med Center for BUN / Creatinine

Both together ("renal profile") : $11.00Either separate : $ 11.00"SMA-7" : $ 12.00Stat fee (for E.D. or inpatients) : 11/2

Times the above $

Laboratory Evaluation of Liver Disease :

Topics CoveredEnzymes

–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 IncreasedALP 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 & cytosolPresent 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 infarctionAcute hepatic necrosisPulmonary infarction (mild elevations in

30 %)Congestive heart failure (passive liver

congestion)PericarditisRheumatic feverSkeletal muscle injury

Alanine Aminotransferase (ALT)

Localized primarily in liverCatalyzes transfer of amino group from

alanine to alphaketoglutarateIs specific marker for hepatic disease or

injuryOnly 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 IncreasedLDH Levels

Cardiac–Myocardial infarction–CHF–Pulmonary infarction

Hematologic–Megaloblastic anemia–Sickle cell disease–Hemolytic anemia–Leukemias–Lymphoma–Infectious mononucleosis

Conditions with IncreasedLDH Levels (cont.)

Hepatic–Hepatitis–Obstructive jaundice–Cirrhosis–Metastatic tumors

Skeletal–Muscular dystrophy–Delerium tremens

LDH Isoenzyme Patternsin Different Conditions

DISEASE LDH-1 LDH-2 LDH-3 LDH-4 LDH-5

Acute MI + + Meg.anemia + + Hem.anemia + + Mus.dystro. + + Leukemia + + Pancreatitis + + Ca. mets + + Pulm infarct + C.H.F. + Hepatitis + Cirrhosis +

Measurement Methodologyfor 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

LevelsALT 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 jaundiceAST elevation can be screen for Reye's Syndrome

General Interpretation ofAST & ALT Levels (cont.)

Degree of elevation not necessarily related to severity of disease process

Levels < 500 U/liter usually mean mild illnessRatio 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 inLiver and Biliary Diseases

DISEASE ALP AST ALT LDH

Acute Liver Injury 4 - 10 X >20 X >20 X +/-

Alcoholic hepatitis 2 - 4 X 4 - 10 X 2 - 4 X +/-

InfectiousMononucleosis

2 - 10 X 10 - 20 X 10 - 20 X 2 - 10 X

Cholestaticjaundice

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 biliarycirrhosis

10 - >20 X 4 - 10 X 4 - 10 X 2 - 4 X

Alcoholic fattyliver

2 - 4 X 2 - 4 X +/- +/-

Cirrhosis 2 - 4 X 2 - 4 X 2 - 4 X 2 - 4 X

Chronic activehepatitis

2 - 4 X 10 - 20 X 4 - 10 X 2 - 4 X

Time pattern of serum transaminases

Bilirubin MetabolismOriginates from breakdown of heme (from

hemoglobin, myoglobin, & cytochromes) into biliverdin which is reduced to form bilirubin

Can be produced by most cellsFree (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 ConjugatedBilirubin

Enters intestine via bileFurther 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 fromUnconjugated

HyperbilirubinemiaPigment 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 ConjugatedHyperbilirubiinemia

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 ofConjugated

Hyperbilirubinemia

Hepatocellular injuryBiliary atresiaPrimary biliary cirrhosisSteroids (especially estrogens)Space - occupying hepatic lesionsDubin-Johnson Syndrome

Extrahepatic Causes ofConjugated Hyperbilirubinemia

CholedocholithiasisCaroli's DiseasePostoperative biliary tract stricturesSclerosing cholangitisCholangiocarcinomaPancreatitisAmpullary or pancreatic cancerCompression from adjacent cystsParasites

Other Tests to Consider forEvaluation 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 disordersInducible by many drugsHalf life 26 daysIf 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 ToRule Out Specific Liver

DisordersAlpha-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 anti-HAV)–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 immuneƒ Or prior hepatitis B vaccinationƒ 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 (anti-HBe)–Indicates suppression of hepatitis B viral replication

Hepatitis B Serologies (cont.)

Hepatitis B core IgM antibody (IgM anti-HBc)–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 anti-HBc)–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 SerologiesHepatitis C antibody by enzyme immunoassay (anti-

HCV 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) : $18ALT alone : $10AST alone : $10Hepatitis serologies :

–HCV antibody : $42–HAV antibody : $37–HBc antibody : $30–HBsAg : $30–anti-HBs : $43