NPN (CC1 Lec)

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  • Nonprotein nitrogen compounds (NPNs) are excreted by the kidneys and are useful for diagnosing kidney function.

    • Urea is the major NPN in plasma and urine, with a concentration of 45-50% in plasma and 86% in urine.

    • Amino acids are seen in the plasma but not normally seen in urine.

    • Uric acid has a concentration of 10% in plasma and 1.7% in urine.

    • Creatinine has a concentration of 5% in plasma and 4.5% in urine.

    • Creatine is mainly present in muscle and liver, with a concentration of 1-2% in plasma.

    • Ammonia has a very low concentration in plasma (0.2%) due to its toxicity.

  • Urea is a major waste product of protein catabolism and is excreted by the kidneys.

  • The amount of urea is dependent on urine flow rate and extent of hydration.

  • The concentration of urea in the plasma is determined by protein content in the diet, rate of protein catabolism, and renal function and perfusion.

  • Urea is used for evaluating renal function, assessing hydration status, determining nitrogen balance, diagnosing renal disease, and verifying adequacy of dialysis.

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  • Urea is the first metabolite to increase in renal disease and can be used to evaluate renal function.

  • Urea is a good indicator of hydration status and contributes to plasma osmolality.

  • Urea can be used to determine nitrogen balance and diagnose renal disease.

  • Urea is easily removed in dialysis, making it useful for verifying adequacy of dialysis.

  • Glucose, sodium, and chloride are major contributors to plasma osmolality levels.

  • Enzymatic methods, such as the GLDH coupled enzymatic method, can be used to measure urea.

  • Chemical methods, such as Fearon's reaction, can also be used to directly measure urea.

  • Isotope Dilution Mass Spectrometry (IDMS) is a proposed reference method for measuring urea.

  • The reference intervals for urea in adults are 6-20 mg/dl in plasma/serum and 12-20 g/day in urine/24 hours.

  • A 24-hour urine sample should be collected in a wide-mouth sterile container, refrigerated, and transported to the laboratory immediately.

  • Fasting blood samples are preferred for urea analysis, and fluoride or citrate anticoagulants should be avoided.

  • Azotemia refers to an increase in urea in the blood, while uremia refers to a very high urea concentration in the blood.

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  • Pre-renal azotemia is characterized by a high BUN creatinine ratio with normal creatinine and is caused by reduced blood flow.

  • Renal azotemia is characterized by a high BUN creatinine ratio and high creatinine, indicating damage to the filtering structures of the kidney.

  • Post-renal azotemia is characterized by a high BUN creatinine ratio and high creatinine, and is caused by urinary tract obstruction.

  • Uric acid is the major end-product of purine catabolism and is primarily produced in the liver.

  • Uric acid can precipitate as urate crystals in tissues when its concentration exceeds 6.8 mg/dL.

  • Uric acid is filtered in the glomerulus and secreted by the distal tubules into the urine.

  • Xanthine oxidase (XO) is the enzyme responsible for the conversion of purine bases to uric acid.

  • Uric acid can cause gout when urate crystals precipitate in tissues, forming tophi.

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  • An increase in urea concentration can be caused by pre-renal azotemia, renal azotemia, and post-renal azotemia.

  • Pre-renal azotemia is caused by reduced blood flow, while renal azotemia is caused by damage to the filtering structures of the kidney.

  • Post-renal azotemia is caused by urinary tract obstruction.

  • A decrease in urea concentration can be caused by low protein intake, severe vomiting and diarrhea, liver disease, and pregnancy.

  • Uric acid is the major end-product of purine catabolism and is primarily produced in the liver.

  • Uric acid can form urate crystals in tissues when its concentration exceeds 6.8 mg/dL, leading to conditions like gout.

  • Uric acid is filtered in the glomerulus and secreted by the distal tubules into the urine.

  • Xanthine oxidase (XO) is the enzyme responsible for the conversion of purine bases to uric acid.

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  • Concentration of carbohydrates, lipids, proteins, and NPNs is determined by:

    • Catabolism of dietary nucleoprotein (exogenous)

    • Catabolism of endogenous nucleoproteins (derived from tissue destruction)

    • Direct transformation of endogenous purine nucleotides

  • Clinical applications of measuring concentration:

    • Assess inherited disorders of purine metabolism

    • Confirm diagnosis and monitor treatment of gout

    • Diagnosis of renal calculi

    • Prevent uric acid nephropathy during chemotherapy

    • Detect kidney dysfunction

  • Chemical method:

    • Phosphotungstic acid (Caraway method) - measure tungsten blue as the product

    • Principle: based on the oxidation of uric acid in a protein-free filtrate

    • Characteristic: nonspecific and requires removal of protein

  • Enzymatic method:

    • First step: Uric Acid + O2 +2 H2O - Uricaseà allantoin + CO2 + H2O2

    • Spectrophotometric method (Blauch and Koch) measures the decrease in absorbance at 293 nm (uric acid v. allantoin)

    • Coupled enzyme system using catalase and peroxidase to produce colored compounds

    • Colorimetric method, specific and recommended for routine testing

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  • Reference intervals for uric acid concentration:

    • Adult Male Plasma/Serum: 3.5 - 7.2 mg/dL (0.21-0.43 mmol/L)

    • Adult Female Plasma/Serum: 2.6 - 6.0 mg/dL (0.16-0.36 mmol/L)

    • Child Plasma/Serum: 2.0-5.5 mg /dL (0.12-0.33 mmol/L)

    • Adult Urine/24Hour: 250-750 mg/day (1.5-4.4 mmol/day)

  • Specimen consideration:

    • Uric acid can be measured using heparinized plasma, serum, or urine

    • Avoid gross lipemia, high bilirubin concentration, and hemolysis

    • Avoid EDTA or fluoride additives (affects uricase method)

    • Salicylates and thiazides can increase values for uric acid

  • Pathophysiology of hyperuricemia (increased concentration):

    • Enzyme deficiencies: Lesch-Nyhan syndrome, phosphoribosylpyrophosphate synthetase deficiency, glycogen storage disease type 1, fructose intolerance

    • Hemolytic and proliferative processes

    • Chronic renal disease

    • Toxemia of pregnancy and lactic acidosis

    • Drugs and poisons

    • Purine-rich diet or increase in tissue catabolism or starvation

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  • Pathophysiology of hypouricemia (decreased concentration):

    • Liver disease

    • Defective tubular reabsorption (Fanconi syndrome)

    • Chemotherapy with azathioprine or 6-mercaptopurine

    • Overtreatment with allopurinol

  • Creatinine physiology:

    • Chief product of muscle metabolism

    • Not affected by protein diet

    • Level is directly influenced by the muscle mass and activity of the patient

    • Excretion in the kidney is at a constant rate

  • Creatine:

    • Formed primarily in the liver from the amino acids arginine, glycine, and methionine

    • Converted to creatine phosphate in other tissues, such as muscle, which serves as a high-energy source

    • During muscle activity, creatine phosphate is utilized as an energy source and converted to cyclic creatinine

    • Creatinine is formed in the muscle and liver, released into the plasma, and excreted at a constant rate

    • Increased creatine level in the plasma indicates muscle damage

  • Clinical applications of measuring creatinine:

    • Determine sufficiency of kidney function

    • Determine severity of kidney damage

    • Monitor the progression of kidney disease

    • Measure completeness of 24-hour urine

  • Renal clearance and glomerular filtration rate:

    • Glomerular filtration rate (creatinine clearance) is inversely proportional to the concentration of plasma creatinine

    • Calculation of creatinine clearance requires serum creatinine, urine creatinine, urine volume, and surface area

  • Method of analysis: chemical method

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  • Chemical method:

    • Principle: Direct Jaffe Reaction

    • Creatinine + picrate → red-orange complex

    • Jaffe-kinetic method detects the rate of change of absorbance to avoid interference of non-creatinine chromogens

    • Jaffe with adsorbent uses Fuller's earth or Lloyd's reagent to adsorb creatinine in protein-free filtrate before reacting with alkaline picrate

    • Jaffe without adsorbent directly reacts creatinine in protein-free filtrate with alkaline picrate to form a colored complex

  • Enzymatic method:

    • Principle: Creatininase-CK and Creatininase-H2O2 reactions

    • Creatininase-CK converts creatinine to creatine, which is further metabolized to produce colored compounds

    • Creatininase-H2O2 reaction produces sarcosine, which is then oxidized to glycine, producing H2O2 and a colored product

  • Reference value: not mentioned in the transcript

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Notes in CARBOHYDRATES, LIPIDS, PROTEINS, AND NPNs

  • Specimen reference values for different categories:

    • Adult Male:

      • Plasma/Serum:

        • 0.9 – 1.3 mg/dL (80-115 µmol/L)

      • Urine/24 Hour:

        • 800 – 2,000 mg/day

    • Adult Female:

      • Plasma/Serum:

        • 0.6 – 1.1 mg/dL (55-96 µmol/L)

      • Urine/24 Hour:

        • 600 – 1,800 mg/day

    • Child:

      • Plasma/Serum:

        • 0.3 – 0.7 mg /dL (27-62 µmol/L)

      • Urine/24 Hour:

        • 0.0 – 0.6 mg/dL (052 µmol/L)

  • Specimen considerations:

    • Falsely increase results due to positive bias:

      • Glucose

      • α-ketoacids

      • Ascorbate

      • Uric Acid

      • Cephalosporins

      • Dopamine

    • Falsely decrease results due to negative bias:

      • Bilirubin

      • Hemoglobin

      • Lipemic specimens

  • BUN/CREATININE RATIO:

    • Comparison of BUN and creatinine levels is a better indicator of the source of elevation of either substance

    • The normal BUN-Creatinine ratio is 10:1 to 20:1

  • Pathophysiology:

    • Increase concentration:

      • Renal failure (glomerular function)

      • ↑ Plasma Concentration = ↓ GFR

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Notes in CARBOHYDRATES, LIPIDS, PROTEINS, AND NPNs

  • Ammonia physiology:

    • Byproduct of amino acid deamination

    • Removed from the circulation and converted to urea in the liver

    • Further converted to urea in the urea cycle; toxic

    • Most forms are in ammonium hydroxide (when water binds with ammonia)

    • Neurotoxic

  • Clinical application:

    • Diagnosis of hepatic failure and hepatic coma

      • Free ammonia is no longer converted to urea

      • Can cause irreversible damage to the tissue and could lead to comatose

    • Reye's syndrome – acute metabolic disorder of the liver

    • Inherited deficiencies of urea cycle

  • Methods of analysis:

    • Chemical method:

      • GLDH:

        • Decrease in absorbance is measured at 340 nm

        • NH4 + 2-oxoglutarate + NADPH + H+ → Glutamate + NADP+ + H2O

      • Ion-selective electrode:

        • Diffusion of NH3 through selective membrane into NH4Cl causing pH change, which is measured potentiometrically

    • Enzymatic method:

      • Spectrophotometric:

        • NH3 + bromphenol blue → blue dye

        • Measured spectrophotometrically

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Notes in CARBOHYDRATES, LIPIDS, PROTEINS, AND NPNs

  • Specimen reference values:

    • Adult:

      • Plasma:

        • 19-60 ug/dL

        • 11-35 umol/L

    • Child (10 days to 2 years):

      • Plasma:

        • 68-136 ug/dL

        • 40-80 umol/L

  • Specimen requirements/considerations:

    • May be measured using heparinized and EDTA tubes

    • Samples should be centrifuged at 0°C to 4°C within 20 minutes of collection and the plasma or serum removed

    • Avoid cigarette smoking for several hours (smoke can cause false increase level of ammonia in