Chapter 9:Nonprotein Nitrogen

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Chapter 9 Nonprotein Nitrogen
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Nonprotein Nitrogen (NPN)

• Monitored through renal (kidney) functions by
  measuring excretion or absorption of by products.
• Consist of 15 compounds that can be analyzed to
  monitor renal functions

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• Urea Blood Urea Nitrogen or BUN
• Constitutes ½ of the NPN substances in
  circulating blood.
• Synthesized in the liver from CO2 and NH4 arising
  from deamination of A.A. by the Kreb cycle.
• Transported by plasma to kidneys and filtered by
  the glomerulus.
• Most urea is excreted in urine
• 40 is reabsorbed by renal tubules. This is good
  indicator of renal function.

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• 4 Functions of the Kidneys
• Excrete waste products
• Preserves essential solutes
• Regulates hydration
• Regulates electrolyte balance
• Diseases associated with urea and normal function
  involve 3 major conditions.
• Prerenal
• Renal
• Post renal

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• Prerenal related to the renal circulation. The
  flow of blood to the kidneys.
• Ex Urea goes through the kidneys for excretion
  and reabsorption. If there is a prerenal
  problem, the urea doesnt make it to the kidneys
  to be filtered, so there is an increase or
  build-up of urea in the blood.
• Diseases Congested Heart Failure, Shock,
  Hemorrhage, dehydration
• Azotemia Increased in urea in the blood .

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• Renal involves the kidney there is a lack of
  ability to function correctly- decrease ability
  to excrete.
• Decrease renal function see an increase in blood
  urea levels.
• Diseases acute/chronic renal failure,
  glomerulonephritis, tubular necrosis, chronic
  nephritis, polycystic kidney.

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• Post renal obstruction of the flow of the urine
  from the kidneys. Kidney function impaired-
  unable to excrete normally.
• Diseases Kidney stone, tumor, UTI or other sever
  infection

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• To determine the kidney function and the cause of
  the increase in Urea in the body system, we use
  the BUN/Creatinine ratio (normal 101 to 151
  ratio)
• Analytical methods to determine urea in the blood
  involve 2 methods.
• Enzymatic
• Use urease
• Involves hydrolysis
• Quantitated by colorimetric method using Nessler
  reagent or Bertholot reaction, identifying NH4
  production.

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• 2. Coupled enzymatic method
• Good specificity and sensitivity, bad time
  consuming.
• Utilizes L-glutamate dehydrogenase 2-oxoglutarate
  and NADH.
• Quantitates NH4 production from urease hydrolysis
  by a series of coupled enzyme reactions that
  produce H2O2.
• Uses spectrophotometer

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• Instrumentation method
• Measures NH4 production by urease hydrolysis by
  detecting a color change and pH indicator.
• Specimen requirements and interference
• Serum or urine preferred, can use plasma (sodium
  citrate and sodium fluoride inhibit urease.)
• Stable at 4C, R.T. concerned with bacterial
  decomposition

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• Creatinine and creatine
• Closely related analytes
• Creatine synthesized in the liver from arginine,
  glycine and methionine. Transported to tissue to
  be converted to phosphocreatine- energy.

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• Creatinine is a anhydride of creatine after the
  loss of phosphoric acid or H2O.
• Valuable analyte for renal function- 100 is
  excreted.
• Insensitive monitor- see after gt50 of damage has
  occurred.
• Measure clearance ability
• Disease see increase creatinine levels in the
  blood- muscle diseases

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• Evaluate with BUN as a ratio to help
  differentiate between renal and prerenal disease
  of azotemia.
• Increase ratio prerenal (shock, CHF,
  dehydration)
• Decrease ratio renal

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• Creatine associated with muscle disorders.
  (M.D., M.S., hypothyroidism, crushing injury.)
• Clinical utilization ref. ranges for serum
  creatinine differ from men to women due to
  different muscle mass.
• Preferred analysis involves time clearance (24
  hr) and serum analysis.

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• Analytical methods
• Jaffe
• Colormetric method looks for a red to orange
  color change.
• Utilizes picric acid
• Disadvantage nonspecific and subject to
  interference from pyruvate glucose, ascorbate and
  acetone.

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• Fuller method
• More accurate
• Uses fullers earth or Lloyd reagent
• Use a protein free filter for the sample- less
  interference.

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• Kinetic Jaffe
• Various enzymatic methods
• Utilizes reagent picirate- measures rate of color
  change in absorbance _at_ 520nm _at_ 20 seconds and 80
  seconds.
• Disadvantage substance interference
• Advantage inexpensive, rapid and easy.

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• Specimen Serum, plasma or urine
• Avoid hemolysis, lipemic, and icteric
• Urine needs to refrigerated.
• Errors in Jaffe reaction
• Glucose, uric acid, alpha-keto acid and ascorbate
  increase _at_ 30C.
• Timing
• Antibiotic interfere

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Uric Acid UA

• Final breakdown product of purine metabolism in
  the liver. (adonosine and gluamine)
• Transported by plasma from liver to kidneys
• Filterd by glomerulus then 98-100 is reabsorbed
  by the proximal tubules- small amount is secreted
  in the distal tubules and found in urine.
• 96-98 of UA is present in the form of monosodium
  urates- at levels above 6.4mg/dl there is the
  formation of urate crystals.
• Disease Gout, Chemotherapy, Chronic renal
  failure.
• Misc. Disease Lesch-Nyhan Syndrome

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• Decreased levels are rare disorders associated
  with secondary liver disease and tubular
  reabsorbtion.
• Analysis involves measuring the oxidization of
  uric acid to allontonin- a reducing agent.
• Methods
• Caraway
• Measures oxidation of uric acid in a protein
  free-filtrate
• Reduction of phosphotungstic acid to tungsten
  blue in the presence of uric acid.
• Simple method, measure UV absorbance before and
  after incubation.

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• Disadvantage uses low frequency 293nm- not
  feasible on most instrumentation.
• Interference seen in high levels of protein.
• 2. Coupled enzyme
• Specific for uricase- no UV measurement used.
• Measures the amount of peroxide formed in primary
  reaction.
• Uses 4 aminoantipyrine and phenol or 3-methyl-2
  benzothiazolinone hydrosone and dimethylaniline.

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Specimen

• Serum preferred, urine and heparinized plasma
• Diet will effect values
• Avoid gross lipemia
• Increased bilirubin- decrease levels
• Hemolysis should be avoided- decreases levels
• Drugs interfere

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Ammonia

• NPN in low levels
• Derived from the deamination of A.A through
  digestive and bacterial enzymes on proteins in
  the intestinal tracts.
• A.A. involved ornithine, citrulline and arginine
• Released from the metabolic reaction that occurs
  in skeletal muscle.
• Liver disease ammonia is not remove sufficiently
  therefore there is a build-up in the blood- leads
  to brain damage.

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• Diseases of increased levels liver failure and
  Reyes syndrome
• Decreased levels very rare and are not a concern.
• Analysis
• 2 step method- isolate the ammonia then measure-
  not utilized
• Direct measurement-

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Direct measurement

• Conway micro diffusion method
• 1st methods, measures the amount ammonia
  liberated from a sample by the addition of
  alkali then ammonia is absorbed into an acid to
  be quantitated by titration or colorimetry
• Not very accurate

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Cation-exchange resin

• Use to isolate ammonia followed by elution of
  ammonia with NaCl- quantitate by Bertholot
  reaction- time consuming

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Coupled Enzyme Assay

• Use absorbance _at_340 nm- measure decrease in
  absorbance- proportional to NH3 concentration -
  measure coupled conversion of NAD to NADH.
• Ammonia electrode
• Measure pH of solution of ammonium chloride as
  ammonia diffuses across semi-permeable membrane.

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Specimen

• Rapidly increase upon collection
• Prevent by collecting on Ice.
• EDTA preferred specimen Heparin may give false
  result.
• Glass ware used needs to be free of ammonia
• Errors occur if not iced, wrong anticoagulant,
  contamination

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Renal Function-Chap 24

• Renal Anatomy
• Kidneys
• Bilateral ureters
• Nephrons
• Glomerulus
• Proximal convuluting tubules
• Loop of Henle
• Ascending Loop of Henle
• Distal convuluting tubules
• Collecting ducts

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• 3 major NPN eliminated by the renal system
• Urea
• Creatinine
• Uric acid
• Creatinine clearance test 24 hour urine test to
  analyze renal clearance ability.
• Formula in lab math book

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• Disease process
• Acute glomerulonephritis increase in BUN, CREAT,
  ALB (quick onset)
• Chronic glomerulonephritis increase in BUN,
  Creat, Alb ( slow onset)
• Nephrotic syndrome increase in BUN, Creat and
  Alb (gt 3.5 g/day)

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Chapter 22 Liver Function

• Liver is one of the largest organs of the body.
• Two main lobes
• Red-brown in color- located under the diaphram
• Abundant blood supply-filters 15ml/minute of
  blood from the Hepatic artery and portal vein
• Structural units include lobules, portal tract,
  sinusoids, kupffers cells, and primary bile
  canalculi.

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Excretory and Secretory Function

• Major function secrete bile and bile salts.
• Fasting or between meal major portion of bile
  acid pool concentrates up to 10X in the
  gallbladder.
• Bile acids reach the intestines when the
  gallbladder contracts after a meal.

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Bilirubin

• Principle pigment in bile
• Formed from the breakdown of hemaglobulin when
  RBCs are phagocytized by the retuculoendothelial
  system.
• Heme iron released
• Porphyrins is a result of release of iron and
  globulin being released when porphyrin ring
  splits and forms biliverdin-precursor if
  bilirubin.

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Bilirubin-cont.

• Transported by the liver in the blood stream
  bound to albumin- picked up by the hepatic cells
  after split from albumin.

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Conjugated Bilirubin

• A.K.A Direct Bilirubin
• Formed by the combination of enzyme
  uridyldiphosphate glucuronyl transferase.
• Bilirubin Diglucuronide is conjugated bilirubin.
• Conjugation occurs in the endoplasmic reticulum.
• Water soluble
• Secreted from hepatic cells into bile canalculi
  into large bile ducts into the intestine.

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Mesobilirubin

• Formed from the bile pigment that is acted on by
  enzymes present in the intestinal bacteria.
• Mesobilirubinogen reduced form of mesobilirubin

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Urobilinogen

• Reduced product of mesobilirubinogen.
• Colorless product
• When oxidized produces red-brown color-pigment
  for urobilin.
• Urobilin is the pigment for stool
• Filtered by the kidneys-excreted in the Urine.

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Total Bilirubin

• Is a waste product of Hgb. Metabolism (iron,
  protein and bilirubin)
• Consist of two types
• Conjugated bilirubin (direct)
• Bilirubin that has been processed with the
  addition of glucoronic acid.
• Water soluble.
• Excreted by the kidneys and liver.

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• 2. Unconjugated bilirubin
• Bilirubin molecule that has albumin attached and
  is going to be processed in the liver.
• A build-up of this product causes a change in
  skin color, whites of the eyes, etc. (jaundice)

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Specimen for analysis of Bilirubin

• Free if hemolysis and lipemia- causes false
  elevation
• Protect from light light sensitive.
• Store in the dark _at_ 4ºC for a week
• 200-300 mg produced daily
• 200-300 mg eliminated on a daily bases.
• Excreted in the conjugated form- stool
• Circulating form unconjugated form

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Jaundice

• Three forms
• Prehepatic
• Hepatic
• Posthepatic

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Prehepatic

• Increase amounts of bilirubin is sent to the
  liver for metabolism. (Hemolytic anemia)
• Characterized by unconjugated hybilirubinemia.
• Unconjugated bilirubin is not H2O soluble and is
  bound to albumin for transport and cannot be
  filtered by the kidneys- nor bilirubin found in
  the urine.

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Hepatic

• Results form impaired cellular uptake from
  defective conjugation in the intestines or
  abnormal secretion by the liver cells.
• Gilberts Syndrome
• Crigler-Najjar Syndrome
• Dublin-Johnson
• Rotors Syndrome

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Post Hepatic

• Results from impaired excretion of bilirubin
  usually caused by an obstruction.
• Increase in serum conjugated bilirubin- less
  pigment in stool.
• Conjugated bilirubin seen in the urine with a
  decrease in urine urobilinogen.

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Infant jaundice

• Hyperbilirubinemia
• Due liver not functioning normally at birth.
• Level greater than 15-20 mg/dl can cause CNS
  damage.
• Treat with exposure to U.V. light and lots of
  fluids.

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Cirrhosis

• Scarring process of the liver of the nodules.
• Caused by various diseases that involve over
  worked liver.

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Tumors

• Heptocellular carcinoma
• Heptoma
• Heptocarcinoma
• Related to previous infection of Hepatitis virus.
• Has poor prognosis.

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Reyes Syndrome

• That evolves from some form of viral infection
  can cause hepatic destruction.

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Role of the Liver

• Plasma protein production
• Production and regulation of coagulation factors
• Conversion of glucose to glycogen
• Fat metabolism
• Forms ketone bodies
• Storage site for fat soluble vitamins.

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Liver Enzymes

• AST/SGOT
• ALT/SGPT
• ALP
• 5NT
• GGT
• T. Bilirubin (direct and indirect)

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Bilirubin Analysis

• Ehrilich 1st method, measured by a color
  formation of red or blue in presence of bilirubin
  coupled with diazotized sulfanilic acid.
• Mallory and Evelyn 1st quantitative methods.
  Uses 50 methanol solution to make bilirubin
  soluble in an acid pH.
• Jendrassik Groff uses caffeine
  bensoate-actetate to accelerate reaction,
  alkaline tartrate to shift to correct pH.

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Urobilinogen analysis

• Quantitative method based on reaction of
  urobilinogen with para-dimethyl-aminobenzaldehyde
  to form a red color (Ehrlich)
• Terwen improved method by use of alkaline
  ferrous hydroxide to reduce bilirubin to
  urobilinogen.