Unit 4 Bilirubin and Non-Protein Nitrogen

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Unit 4 Bilirubin and Non-Protein Nitrogen
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2015-01-29 19:02:29
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Unit4 Clinical Chemistry
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Bilirubin, jaundice, and diseases. Methods for analysis. BUN and etc.
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  1. Three types of jaundice patterns (conjugated/unconjugated levels, and bilirubin/urobilinogen in urine) and examples of each:
    • Pre-hepatic Jaundice: excessive production of bilirubin
    • -Increased unconjugated bilirubin
    • -Normal conjugated bilirubin is normal
    • -Positive urine urobilinogen
    • -Negative urine bilirubin
    • -Examples - normal neonatal physiological jaundice, Gilbert's, Crigglar-Najjar, hemolytic disease of newborns, defects in RBCs, transfusion reactions

    • Hepatocelular Jaundice (Hepatitis): decreased ability to secrete into bile ducts
    • - Increased unconjugated and conjugated bilirubin
    • -Positive urine urobilinogen and bilirubin
    • -Examples - seen in any acute hepatitis (viral or chemical...ETOH, Acetaminophen, Chloroform, and other drugs).

    • Post-Hepatic (Hepatobiliary or Obstructive) Jaundice: a block somewhere after the liver
    • -Increased conjugated bilirubin
    • -Normal unconjugated bilirubin
    • -Low/negative urine urobilinogen
    • -Positive urine bilirubin
    • -Examples - primary biliary atresia and gallbladder disease.
  2. Describe the disease and the expected urine bilirubin and urobilinogen values:

    Hemolytic Disease of the Newborn
    Pre-hepatic jaundice.

    IgG antibodies of the mother cross in to the fetus and attack its RBCs.
  3. Describe the disease and the expected urine bilirubin and urobilinogen values:

    Neonatal Physiological Jaundice
    Pre-hepatic.

    • Normal jaundice that occurs in every newborn while their liver ramps up.
    • -Increased conjugated bilirubin.
    • -Positive urine urobilinogen.
  4. Describe the disease and the expected urine bilirubin and urobilinogen values:

    Gilbert's Syndrome (Disease)
    Pre-hepatic pattern.  

    Decreased uptake of bilirubin by the liber.  Genetic and fairly common.  

    Looks like pre-hepatic jaundice but all other tests will come back negative...whatever that means.
  5. Describe the disease and the expected urine bilirubin and urobilinogen values:

    Criggler-Najjar Syndrome (Disease)
    Pre-hepatic pattern

    • Type I - completely lacks enzyme to conjugate bilirubin.
    • Type II - Reduced enzyme.  Not much worse than Gilbert's.
  6. Describe the disease and the expected urine bilirubin and urobilinogen values:

    Dubin-Johnson Syndrome (Disease)
    Post-hepatic pattern. Obstructive physiological jaundice. Liver cells don't efficiently excrete conjugated bilirubin into the biliary track, so it looks like there is an obstruction.

    -Increased conjugated bilirubin.
  7. Collection, handling, and storage of specimens for bilirubin analysis:
    • Specimen of choice: fresh, unhemolyzed serum. (Unhemolyzed heparinized plasma is acceptable.)
    • Effects of hemolysis: causes increased values if method uses a wavelength below 600 nm.
    • Effects of light, temperature, and time: protect from light (light labile), keep cool, degrades with time so assay ASAP.
  8. Cite the principles of the reactions, the reagents, and the methods for bilirubin analysis and fractionation.
    • Evelyn-Malloy method:
    • -Uses the Diazo Reaction. Final product is red and follows Beer's Law, but it must be read below 600 nm where hemoglobin interferes.
    • -Reagents: Sulfanilic Acid which forms an N-triple bond with bilirubin.  
    • -Fractionation method: Direct bilirubin soluble in water vs. Total in ethanol or methanol.  (Only bilirubin that is dissolved will react with the reagent.)

    • Jendrassic-Grof method:
    • -Same as Evelyn-Mally method except...
    • -Fractionation method: Direct bilirubin soluble in HCl (pH 1.3) vs. Total in Acetate-Caffeine Reagents (pH 8.0).
    • -Fehling II reagent (pH 13.0) is added to shift the product to Blue Azobilirubin (instead of red) and is read at 600 nm where hemoglobin does not interfere.

    • Direct reading bilirubinometer method:
    • -Colorimetric measuring of the yellow chromophore of the original bilirubin. The specimen is diluted serum or plasma.

    • Vitros Dry Chemistry Slide Analyzers
    • -dry slide
    • -TBIL - a diazo reaction reading color using 1 wavelength.
    • -Bu/Bc - diazo reaction reading color using 2 wavelengths. Bu and Bc reflect at slightly different wavelengths.
    • -Adults: DBili = TBIL - Bu  
    • *The Bc may differ from DBili due to Delta-Bilirubin
    • -Neonates: NBili = total of Bu/Bc slide.
    • *During the first few days of life, neonates only have unconjugated bilirubin. TBili is not FDA approved for babies under 3 weeks.
  9. Define Delta-bilirubin.
    Conjugated bilirubin covalently bound to albumin.

    This accounts for the discrepancy between TBili and Bu/Bc totals. First recognized for neonates but then noticed for some adults (those with Hepatitis or Choleostasis).
  10. Explain the biochemistry and disease correlations for Ammonia:
    It is the strongest naturally occuring base, ripping H+ off water molecules. Liver clears it out by converting it to harmless Urea. Ammonia is, therefore, a Hepatic marker.

    High = Metabolic Alkalosis (Liver Disease). Toxic to nerves, shaking, spasms. Measured in µmol/L because blood concentration is so low.

    Specimen of choice: Heparinized Plasma (not NH4 Heparin and not serum because deaminase enzymes will break urea into ammonia over time), keep on ice, tightly capped, and assay immediately.
  11. Explain the biochemistry and disease correlations for Urea:
    End product of protein catabolism. Synthesized by the liver from bicarbonate and ammonia. Harmless. Water soluble. Renal marker because it is cleared by the Nephrons of the kidneys.

    Low = Hepatic disease (not being produced)

    High = Renal or Pre-Renal condition (not being eliminated)

    Usually reported as BUN (blood urea nitrogen). Conversion factor is 2.14.

    Specimen of choice: unhemolyzed serum or heparinized plasma. Usually part of a BMP when other tests may require fasting or anticoagulants.
  12. Explain the biochemistry and disease correlations for Creatinine:
    • -Originates in muscle cells. Creatine → Creatine-PO4 (for storage) 
    • -Some Creatine escapes cell → Creatinine (can’t reenter cell) → eliminated by Kidneys

    • -Higher blood concentrations with ↑ muscle mass, ↓ renal function, ↑ muscle activity. 
    • -Assuming muscle mass and muscle activity are constant, concentration is influence by renal function. (Renal marker.)
    • -Shock → pre-renal azotemia → elevates blood creatinine 

    • Specimen of choice:
    • 1. Serum or heparinized plasma. If not assayed immediately, separate from cells and freeze.
    • 2. 24 hour urine collection: refrigerated during collection, avoid collecting with pH extremes. 
    • If not assayed immediately then freeze.
  13. Explain the biochemistry and disease correlations for Uric Acid:
    It is the end product of Purine catabolism in humans and is eliminated by the nephrons of the kidney. However, it is not a good independent renal marker because it can rise in the blood from many things.

    Adenine or Guanine (Purines) → Hypoxanthine or Xanthine → Uric Acid (same ring structure)

    • Barely soluble.
    • High synovial fluid levels leads to crystals = Gout.
    • High Urine levels leads to crystals = Kidney Stones

    • Specimen of choice:
    • 1. Non-hemolyzed serum. (Heparinized plasma is acceptable.) Be sure to be accurate. High uric acid means an expensive drug to stop its catabolism at Xanthine.
    • 2. 24-hour urine. Jug must contain around 10 mL of 6.0 Molar NaOH to keep urine at a pH greater than 8.0 so Uric Acid won’t crystalize.
  14. Interconvert Urea and BUN values.
    • Urea MW = 60
    • Nitrogen functional groups MW = 28 60/28 = 2.14

    Urea is the larger number. BUN is the smaller number. (To help you remember whether you divide or multiply by 2.14.)

    (2.14 x mg/dL BUN = 1 mg/dL UREA)
  15. Define and distinguish between Azotemia and Uremia.
    Azotemia: elevated blood levels of NPN waste products (BUN, creatinine, Uric Acid) which may be caused by many things.

    Uremia (renal failure): high potassium, low bicarbonate, low calcium, high sodium, high phosphates, anemia, bone resorption, and azotemia.
  16. Explain what is meant by Pre-renal, Renal, and Post-renal causes of elevated blood NPN compounds.
    • Pre-renal:
    • -Not perfusing the kidneys with blood
    • -Causes: Diet Heavy metal poisoning

    • Renal (Intra-Renal) – intrinsic damage to nephrons of kineys
    • Acute Glomeruloephritis – damage to basement membrane of glomerulus due to Immune Complexes. (Such as Gropu A Beta Strep or Viral Hepatitis B and C)
    • -Proteinuria with tons of RBCs and RBC casts.
    • -Elivated blood K+
    • Acute Pyelonephritis – Almost always due to complication of urinary tract infection (E. coli, Proteus Vulgaris, Mirabilis, Pseudomonas, etc.) --WBCs, WBC casts, bacteria, renal ipithelial cells.
    • Interstitial Nephritis (Nephrosis) – indirect damage to tubules of nephrons by inflammation of parenchymal kidney cells due to aspirin, anti-freeze, yellow fever toxins, etc.
    • Acute Tubular Necrosis (Intra-Renal Azotemia) – similar to Interstitial Nephritis, except that toxins directly damage tubules of the nephrons. Caused by hypoxia (lack of oxygen) or shock.

    • Post-renal - urethral obstructions or abnormalities – back up pressure within bladder impedes glomerular filtration.
    • -Kidney stones – stone attemps to pass but blocks ureters or urethra.
    • -Usually severe enough that medical help is sought before permanent damage occus.
  17. Explain how BUN/Creatinine ratios can distinguish between Pre-renal azotemia and intra-renal disease.


    • Intra-renal disease: Creatinine rises faster than BUN. 
    • ≤ 15 correlates to actual renal disease, either glomerular, tubular, or both. 

    • Pre-renal Azotemia: BUN rises faster than Creatinine. 
    • ≥ 24 correlates to prerenal Azotemia 
    • *Shock is a common cause - a biggie for ER trauma and surgery patients.There is no normal range.

    Do not calculate this for normal people. These ratios are only useful when one or both BUN and/or Creatinine are elevated. There really is no meaningful normal range for the ratio.
  18. Discuss why serum Creatinine assays are more sensitive for renal function disorders than are BUN or Uric Acid assays.
    • -0.1 mg/dL increase in serum Creatinine corresponds to 5% – 7% renal function loss. It works this way from 1.0 to 4.0 mg/dL.
    • -Uric Acid levels are affected by many other factors. In actual renal disease, Urea and BUN do not elevate until 60%-70% renal function is lost
  19. Explain the principle of the Creatinine Clearance test, how the test is performed, and how the test is used to assess renal function.
    Measures Glomerular Filtration Rate as mL of plasma cleared of Creatinine per minute. This will decrease in both Glomerular and Tumular Diseases.

    • For this test:
    • 1. Serum or plasma sample, and
    • 2. 24-hour urine collection
    • 3. Patient’s height and weight to calculate BSA (Body Surface Area)
    • -Huge people w/ more muscle mass will have more creatinine. Teeny people will have less, which is why 1.73 BSA ratio is added in, to normalize with the standard male.



    • *100% renal function equals 130 mL/minute. Anything higher than this represents an error (BSA correction, not a true 24 hour urine, urine measuring error, dilution error)
    • *GFR of 56 mL/min means 43% renal function. (56/130 = 43%)
  20. Explain why elevated levels of Uric Acid may be found in the blood of patients having:
       -Primary Gout
       -Secondary Gout
       -Renal failure
       -Malignant tumors
       -Polycythemias, Leukemias  Cell proliferation
       -Pernicious Anemia
       -Diets rich in nucleoproteins
    • a. Primary Gout (rare): Occurs if you are unable to recycle purines you’ve made yourself, so you have to make them all from scratch. This is only 40% efficient. All the left over scraps (the other 60%) increase uric acid.
    • b. Secondary Gout (heavy metal induced): heavy metals make synthesis of purines less efficient.
    • c. Renal failure: filters out Uric Acid
    • d. Malignant tumors – cell proliferation: making more DNA and RNA means more waste products.
    • e. Polycythemias, Leukemias Cell proliferation: making more DNA and RNA.
    • f. Pernicious Anemia: cells are dying prematurely, leaving waste to break down.
    • g. Diets rich in nucleoproteins: can’t reuse these. You have to break them down into uric acid so you can excrete them.
  21. Explain why urine Uric Acid specimens must be collected at a pH greater than pH 8.0.
    Jug must contain around 10 mL of 6.0 Molar NaOH to keep urine at a pH greater than 8.0 so Uric Acid won’t crystalize.
  22. Describe the test methods for Ammonia:
    • Glutamic Dehydrogenase/NADPH method (enzymatic) 
    • • 2-Oxo-glutarate + NH4 + NADPH → Glutamate + NAD 
    • • NADPH absorbs at 340 nm. Measure drop in absorbance. 

    Ion-Selective Electrode (ISE). The best Ammonia assay.
  23. Describe the test methods for Urea or BUN:
    • Diacetylmonoxime-Thiosemicarbazide method
    • • Older test. Great but clumsy. High heat. Reproducible.
    • • Form unstable Diacetyl and react with urea → diazine (red) 

    • Urease methods (enzymatic beginning)
    • • Urea → split off Ammonia; then… 
    • a. Berthelot: Add Bleach + Phenol → Indophenol which is blue 
    • b. Nessler: add HgI + KI → form Orange chromagen 
    • c. GluDH: add 2-Oxo-glutarate + NADPH → Glutamate + NAD
  24. Describe the test methods for Creatinine:
    • Jaffe method – been the gold standard for years
    • • Creatinine + trinitrophenol (picoric acid) → red (Beer’s law) 
    • • Not 100% specific for creatinine but reacts w/ creatinine first
    • • Picoric acid is more explosive than TNT.

    • Creatinine Hydrolase + CK
    • • A bunch of steps, but specific for creatinine.
    •     o Creatinine → Creatine
    •     o Creatine + ATP → Creatine-PO4 + ADP
    •     o ADP + PEP → ATP + Pyruvic Acid
    •     o Pyruvic Acid + NADH → Lactic Acid + NAD

    • Creatininase + NH4 ISE
    • • Creatinine → Boring stuff + NH4
    • • Use ISE to quantitate NH4. ISE coated with monactin and nonactin (antibiotics)
  25. Describe the test methods for Uric Acid:
    • Phosphotungstic reduction (Henry-Caraway)
    • • Not entirely specific for Uric Acid
    • • Not used much anymore.
    • • Uric Acid + Phosphotungstic Acid → Allentoin + tungsten blue

    • Uricase method (most common) (Trinder Method)
    • • Uric Acid → Allantoin + H2O2
    • • Oxidize peroxide to a chromophore.
    • o Similar to the glucose oxidase method. Same colors. Same wavelength.
  26. Name four NPN compounds:
    • 1. Ammonia
    • 2. Urea or BUN
    • 3. Creatinine
    • 4. Uric Acid

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