Bishop

• NPN


  1. Concentration of nitrogen-containing compounds in a protein-free filtrate was quantified spectrophotometrically by converting nitrogen to ammonia

  2. Followed by a subsequent reaction with Nessler's reagent (K2 [HgI4]) to produce a yellow color
• Though it was technically difficult to perform, the method provided an accurate determination of total NPN concentration
• Urea


  • Highest concentration in the blood
  • The major excretory product of protein metabolism
  • Formed in the liver from amino groups (-NH2) and free ammonia generated during protein catabolism
  • Enzymatically catalyzed process termed the urea cycle to reduce the levels of ammonia in the blood, as ammonia is toxic to cells
• Urea cycle


  In the liver, ammonia is bound with CO2 to form carbamoyl phosphate, which enters the urea cycle and ultimately becomes urea
• Blood urea nitrogen (BUN)


  Historic assays for urea were based on the measurement of nitrogen via blood samples, but urea nitrogen (urea N) is a more appropriate term
• Protein metabolism


  • Produces amino acids that can be oxidized to produce energy or stored as fat and glycogen
  • During protein metabolism, nitrogen is released, converted to urea, and excreted as a waste product
• Urea excretion


  1. Following synthesis in the liver, urea is carried in the blood to the kidney and readily filtered from the plasma by the glomerulus

  2. Most of the urea in the glomerular filtrate is excreted in the urine, although some urea is reabsorbed by passive diffusion during passage of the filtrate through the renal tubules

  3. The amount reabsorbed depends on the urine flow rate and extent of hydration

  4. Small quantities of urea (<10% of the total) are excreted through the gastrointestinal (GI) tract and skin
• Urea concentration in plasma

  Determined by the protein content of the diet, the rate of protein catabolism, and renal function and perfusion
• Measurement of urea


  • Used to evaluate renal function, to assess hydration status, to determine nitrogen balance, to aid in the diagnosis of renal disease, and to verify adequacy of dialysis
  • Originally performed on a protein-free filtrate of whole blood and based on measuring the amount of nitrogen
  • Current analytic methods have retained this custom, and urea is often reported in terms of nitrogen concentration rather than urea concentration
• Urea concentration conversion


  • Urea concentration can be converted to urea concentration by multiplying by 2.14
  • In the International System of Units (SI), urea is reported in units of millimoles per liter
  • Urea concentration in milligrams per deciliter may be converted to urea concentration in millimoles per liter by multiplying by 0.36
• Enzymatic methods for urea analysis

  1. The enzyme urease (urea amidohydrolase) catalyzes hydrolysis of urea in the sample, and the ammonium ion produced in the reaction is quantified

  2. The most common method couples the urease reaction with glutamate dehydrogenase (GLDH), where the conversion of nicotinamide adenine dinucleotide (reduced, NADH) at 340 nm is measured

  3. Ammonium from the urease reaction can also be measured by the color change associated with a pH indicator

  4. A method that uses an electrode to measure the rate of increase in conductivity as ammonium ions are produced from urea is in use in approximately 15% of laboratories in the United States
• A reference method using isotope dilution mass spectrometry (IDMS) has been developed
• Specimen requirements for urea analysis

  • Measured in plasma, serum, or urine
  • If plasma is collected, ammonium ions and high concentrations of sodium citrate and sodium fluoride must be avoided, as citrate and fluoride inhibit urease
  • Although the protein content of the diet influences urea production, the effect of a single protein-containing meal on urea concentration is minimal and a fasting sample is not generally required
  • Hemolyzed samples should not be used
  • Urea is susceptible to bacterial decomposition, so specimens (particularly urine) that cannot be analyzed within a few hours should be refrigerated
  • Timed urine specimens should be refrigerated during the collection period
  • Methods for plasma or serum may require modification for use with urine specimens because of high urea concentration and the presence of endogenous ammonia
• Azotemia


  An elevated concentration of urea in the blood
• Uremia or Uremic Syndrome


  Very high plasma urea concentration accompanied by renal failure
• Symptoms of Uremia or Uremic Syndrome

  • Fatigue, nausea or vomiting, and generalized confusion and can eventually become fatal if not treated by dialysis or renal transplantation
• Conditions resulting in increased plasma urea concentration

  • Prerenal
  • Renal
  • Postrenal
• Prerenal azotemia


  Occurs as a result of reduced renal blood flow, less blood is delivered to the kidney and so consequently, less urea is filtered
• Causative factors of prerenal azotemia

  • Congestive heart failure
  • Shock
  • Hemorrhage
  • Dehydration
  • Other factors resulting in a significant decrease in blood volume
• Protein metabolism


  Induces prerenal changes in blood urea concentration
• Factors that may increase urea concentration

  • Regular high-protein diet
  • Increased protein catabolism (stress, fever, major illness, corticosteroid therapy, GI hemorrhage)
• Renal causes of elevated urea

  Acute and chronic renal failure, glomerular nephritis, tubular necrosis, and other intrinsic renal disease
• Postrenal azotemia


  Due to obstruction of urine flow anywhere in the urinary tract by renal calculi, tumors of the bladder or prostate, or severe infection
• Decreased plasma urea concentration

  • Major causes include low protein intake and severe liver disease
  • Additionally, plasma urea concentration can decrease during late pregnancy and in infancy as a result of increased protein synthesis
• Urea nitrogen/creatinine (BUN/creatinine) ratio

  • Normally 10:1 to 20:1
  • Prerenal conditions tend to elevate the BUN/creatinine ratio
  • Prerenal conditions that result in a lowered BUN/creatinine ratio are associated with decreased urea production as seen in low protein intake and severe liver disease
  • An increase in both plasma BUN and creatinine, which in turn shows a "normal" BUN/creatinine ratio, is usually seen in renal conditions
  • A high BUN/creatinine ratio with an elevated creatinine is usually seen in postrenal conditions involving urinary flow obstruction
• Uric acid


  • The final product of catabolism of purine nucleic acids
  • Filtered by the glomerulus and secreted by the distal tubules into the urine, but most uric acid is reabsorbed in the proximal tubules, maintaining a steady physiological concentration
  • Relatively insoluble in plasma and, at high concentrations, can be deposited in the joints and tissue, causing painful inflammation
  • The inability of humans and other higher primates to further metabolize uric acid to the more soluble allantoin is attributed to a series of progressive evolutionary events ultimately resulting in the prevention of uricase production, the enzyme responsible for further uric acid degradation
• Uric acid formation and excretion

  1. Primarily formed in the liver as the end product of purine metabolism

  2. Transported in the plasma from the liver to the kidney, where it is filtered by the glomerulus

  3. Reabsorption of 98% to 100% of the uric acid from the glomerular filtrate occurs in the proximal tubules

  4. Small amounts of uric acid are secreted by the distal tubules into the urine

  5. Renal excretion accounts for about 70% of uric acid elimination; the remainder passes into the GI tract and is degraded by bacterial enzymes
• Increase in affinity for uric acid in renal transport proteins has been identified as occurring concurrently with the cessation of enzymatic uricase production in humans, adding to the theory that increased uric acid levels provide evolutionary advantage to humans and other higher primates
• Uric acid in plasma

  • Nearly all of the uric acid in plasma is present as monosodium urate
  • At the pH of plasma (pH ~ 7), urate is relatively insoluble; at concentrations greater than 6.8 mg/dL, the plasma is saturated, and urate crystals may form and precipitate in the tissues
  • In acidic urine (pH < 5.75), uric acid is the predominant species, and uric acid crystals may form
• Clinical applications of uric acid measurement

  • Confirm diagnosis and monitor treatment of gout
  • Assess and prevent uric acid nephropathy during chemotherapeutic treatment
  • Assess inherited disorders of purine metabolism
  • Detect kidney dysfunction
  • Assist in the diagnosis of renal calculi
• Caraway method for uric acid analysis

  The most common method of this type; which is based on the oxidation of uric acid in a protein-free filtrate, with subsequent reduction of phosphotungstic acid in alkaline solution to tungsten blue
• The Caraway method lacks specificity
• Uricase (urate oxidase)


  The enzyme that catalyzes the oxidation of uric acid to allantoin
• Uricase-based methods for uric acid analysis

  1. More specific and are used almost exclusively in clinical laboratories

  2. Measure the differential absorption of uric acid and allantoin at 293 nm, where the difference in absorbance before and after incubation with uricase is proportional to the uric acid concentration

  3. Coupled enzymatic methods measure uric acids levels by measuring the hydrogen peroxide produced as uric acid is converted to allantoin, with peroxidase or catalase used to catalyze a chemical indicator reaction
• Interferences in uric acid analysis

  • Proteins can cause high background absorbance, reducing sensitivity
  • Hemoglobin and xanthine can cause negative interference
  • Bilirubin and ascorbic acid destroy peroxide if present in sufficient quantity and can interfere
• Commercial reagent preparations often include potassium ferricyanide and ascorbate oxidase to minimize these interferences
• HPLC (high-performance liquid chromatography) methods, typically using UV detection, have been developed for uric acid analysis
• IDMS has been proposed as a candidate reference method for uric acid analysis
• Specimen requirements for uric acid analysis

  • Uric acid may be measured in heparinized plasma, serum, or urine
  • Serum should be removed from cells as quickly as possible to prevent dilution by intracellular contents
  • Diet can affect uric acid concentration overall, but a recent meal has no significant effect; therefore, a fasting specimen is unnecessary
  • Gross lipemia (seen in individuals with elevated triglyceride levels) should be avoided
  • High bilirubin concentration may falsely decrease results obtained by peroxidase methods
  • Significant hemolysis, with concomitant glutathione release, may result in low values
  • Drugs such as salicylates and thiazides have been shown to increase values for uric acid
  • Uric acid is stable in plasma or serum after red blood cells have been removed
  • Serum samples may be stored refrigerated for 3 to 5 days
  • EDTA or fluoride additives should not be used for specimens that will be tested by a uricase method
  • Urine collections must be alkaline (pH 8)
• Abnormally increased plasma uric acid concentration is seen in certain conditions