Non-Protein Nitrogen (CC1 Lab)

Page 1: Non-Protein Nitrogen(NPN) Compounds

  • Introduction to non-protein nitrogenous substances in the blood

  • Used to monitor renal function

Page 2: Non-protein Nitrogen Compounds

  • Definition of non-protein nitrogen compounds

  • Useful clinical information obtained from individual components of NPN fraction

Page 3: Clinically Significant NPN

  • NPN fraction comprises about 15 compounds

  • Majority of compounds arise from catabolism of proteins and nucleic acids

Page 4: Urea Nitrogen (Blood) BUN

  • Nitrogen released during processes converted to ammonia

  • Urea synthesized in the liver from CO2 and ammonia

  • Urea is the major excretory product of protein metabolism

Page 5: Urea Nitrogen (Blood) BUN

  • Assays for urea based on measurement of nitrogen

  • Urea concentration determined by renal function, dietary intake, and protein catabolism rate

  • Urea excreted by the kidneys, with 40% reabsorbed and <10% excreted through the gastrointestinal tract and skin

Page 6: Clinical Application

  • Measurement of urea used to evaluate renal function, assess hydration status, determine nitrogen balance, aid in the diagnosis of renal disease, and verify adequacy of dialysis

Page 7: Disease Correlations

  • Azotemia: elevated concentration of urea in blood

  • Uremia or uremic syndrome: very high plasma urea concentration accompanied by renal failure

  • Causes of urea plasma elevations: prerenal, renal, and postrenal

Page 8: Pre-Renal Azotemia

  • Reduced renal blood flow leads to less urea filtered

  • Factors causing decrease in functional blood volume include congestive heart failure, shock, hemorrhage, and dehydration

  • High protein diet or increased catabolism can also contribute to pre-renal azotemia

Page 9: Renal Azotemia

  • Decreased renal function leads to increased blood urea due to poor excretion

  • Causes of renal azotemia include acute and chronic renal failure, glomerular nephritis, tubular necrosis, and other intrinsic renal diseases

Page 10: Post-Renal Azotemia

  • Obstruction of urine flow can cause post-renal azotemia

  • Causes include renal calculi, tumors of the bladder or prostate, and severe infections

Page 11: Decreased Urea Nitrogen

  • Low protein dietary intake can lead to decreased urea nitrogen

  • Liver disease can cause lack of urea synthesis

  • Severe vomiting and/or diarrhea can result in loss of urea

  • Increase in protein synthesis can also decrease urea nitrogen

Page 12: Analytical methods

  • Assays for urea based on measuring the amount of nitrogen in the sample

  • Urea nitrogen concentration can be converted to urea concentration by multiplying by 2.14

Page 13: Analytical methods

  • Urease hydrolyzes urea to ammonium ion, which is then detected

  • Enzymatic methods commonly used, coupling the urease reaction with glutamate dehydrogenase

Page 14: Analytical methods

  • Indicator dye used to detect ammonium ion

  • Conductimetric method measures increased conductivity resulting from conversion of unionized urea to ammonium ion and carbonate ion

  • Reference range of urea nitrogen: serum or plasma: 6-20 mg/dl, 24 hours urine: 12-20 g/day

Page 15: Creatinine/ Creatine

  • Creatine synthesized in the liver from arginine, glycine, and methionine

  • Converted to creatine phosphate, a high energy source for muscle tissue

  • Creatinine produced as a waste product of creatine and creatine phosphate

Page 16: Creatinine production

  • Diagram showing the production of creatinine from creatine and creatine phosphate

Page 17: Creatinine/Creatine

  • Creatinine released into circulation at a stable rate proportional to muscle mass

  • Filtered by glomerulus and excreted in urine

  • Plasma creatinine concentration is a function of relative muscle mass, rate of creatine turnover, and renal function

  • Daily creatinine excretion is stable and used to evaluate renal function

Page 18: Disease Correlations

  • Elevated creatinine found with abnormal renal function

  • Measurement of creatinine concentration used to determine sufficiency of kidney function, severity of kidney damage, and monitor the progression of kidney disease

Page 19: Disease Correlations

  • GFR (glomerular filtration rate) used to estimate renal function

  • Creatinine clearance measures the amount of creatinine eliminated from the blood by the kidneys per unit time

  • Plasma concentration of creatinine inversely proportional to clearance, increased plasma levels indicate decreased GFR

Page 20: Analytic Methods

  • Jaffe reaction most frequently used for creatinine measurement

  • Kinetic Jaffe reaction measures the rate of change in absorbance

  • Enzymatic method using creatininase, creatine kinase, pyruvate kinase, and lactate dehydrogenase

Page 21: Analytic Methods

  • Diagram showing the enzymatic conversion of creatinine to lactate

  • Reference intervals for creatinine in plasma or serum

Page 22: Creatine

  • Elevated in plasma and urine in conditions such as muscular dystrophy, hyperthyroidism, and trauma

  • Plasma creatinine levels usually normal, but urinary creatine is elevated

  • Specialized testing required, not part of routine lab

Page 23: Assay of creatine

  • Analyzing the sample for creatinine before and after heating in acid solution using an endpoint Jaffe method

  • Heating converts creatine to creatinine, and the difference between the two samples is the creatine concentration

Page 24: Uric Acid

  • Uric acid is a final breakdown product of purine metabolism in the liver

  • Most other mammals degrade it further to allantoin

  • Uric acid is measured to assess inherited disorders of purine metabolism, confirm diagnosis and monitor treatment of gout, assist in the diagnosis of renal calculi, prevent uric acid nephropathy during chemotherapeutic treatment, and detect kidney dysfunction

Page 25: Uric Acid

  • Uric acid is transported to the kidney and filtered, with 98% reabsorbed in the proximal convoluted tubule and some secreted by the distal convoluted tubule

  • Uric acid measured to assess inherited disorders of purine metabolism, confirm diagnosis and monitor treatment of gout, assist in the diagnosis of renal calculi, prevent uric acid nephropathy during chemotherapeutic treatment, and detect kidney dysfunction

Page 26:

  • Disease Correlations:

    • Gout:

      • Primarily affects men

      • Onset between 30-50 years

      • Elevated levels of uric acid (UA) greater than 6.0 mg/dL

      • Symptoms include pain and inflammation of joints due to the precipitation of sodium urates in tissues

      • Increased risk of renal calculi

      • Hyperuricemia in 25-30% of cases due to overproduction of uric acid

Page 27:

  • Disease Correlations:

    • Increased catabolism:

      • Occurs in patients on chemotherapy for diseases such as leukemia and multiple myeloma

      • Allopurinol is used to inhibit xanthine oxidase, an enzyme in the uric acid synthesis pathway, in these patients

    • Chronic renal disease:

      • Causes elevated levels of uric acid due to hindered filtration and secretion

Page 28:

  • Xanthine oxidase pathway:

    • Diagram of the pathway

    • Shows the conversion of hypoxanthine to xanthine to uric acid

    • Xanthine oxidase is the enzyme involved in this pathway

Page 29:

  • Disease Correlations:

    • Hypouricemia:

      • Secondary to severe liver disease

      • Defective renal tubular reabsorption, such as in Fanconi's Syndrome

      • Chemotherapy with 6-mercaptopurine or azathioprine, which inhibit purine synthesis

      • Over-treatment with allopurinol

Page 30:

  • Analytic Methods:

    • Primary method uses enzyme uricase (urate oxidase) to convert uric acid to allantoin

    • Differential absorption at 293 nm:

      • Uric acid has a UV absorpance peak at 293 nm, while allantoin does not

      • Proteins also absorb near this wavelength

Page 31:

  • Analytic Methods:

    • Newer methods couple uricase with catalase or peroxidase action on hydrogen peroxide product from allantoin production

    • Some interferences from reducing agents

    • Reference range: Males 0.5-7.2 mg/dL, Females: 2.6-6.0 mg/dL

Page 32:

  • Ammonia:

    • Comes from deamination of amino acids

    • Digestive and bacterial enzymes in the intestine also contribute to its production

    • Released from muscle during exercise

    • Consumed by liver cells and converted to urea

    • Free ammonia is toxic, but it is present in low concentrations in the plasma

Page 33:

  • Disease Correlations:

    • Severe liver disease:

      • Most common cause of abnormal ammonia levels

      • Ammonia is not removed from circulation and not converted to urea

    • Elevated ammonia levels are neurotoxic and often associated with encephalopathy

Page 34:

  • Disease Correlations:

    • Reye's Syndrome:

      • Most commonly seen in children

      • Often preceded by viral infection treated with aspirin

      • Severe fatty infiltration of the liver

      • High ammonia levels can be fatal

      • 100% survival if ammonia stays below 5x normal

Page 35:

  • Disease Correlations:

    • Ammonia is useful in the diagnosis of inherited deficiencies of urea cycle enzymes

    • Measurement of ammonia is used to diagnose and monitor treatment

Page 36:

  • Analytic Methods:

    • Low concentration, volatile nature, instability, and easy contamination make testing difficult

    • Historical Methods:

      • Conway 1935: volatilize, absorbed, then titrated

      • Dowex 50 cation-exchange column + Berthelot reaction

Page 37:

  • Analytic Methods:

    • Glutamate dehydrogenase:

      • Decrease in absorbance at 340 nm as NADPH is consumed (oxidized)

    • Direct ISE:

      • Change in pH of solution as ammonia diffuses through a semi-permeable membrane

    • Reference Interval: Adult Plasma 19-60