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Non-Protein Nitrogen (NPN)
Clinical Chemistry 1
BS Medical Technology BSMT 3-3 | Prof. Eric E. Carpo
Compound Approximate Plasma Concentration (% of Total NPN) Approximate Urine Concentration (% of Excreted N) Urea 45-50 86. Amino Acids 25 — Uric Acid 10 1. Creatinine 5 4. Creatine 1-2 — Ammonia 0.2 2. A. Blood Urea Nitrogen (BUN) ● It is the major end product of protein & amino acid catabolism ● Highest NPN compound in the blood 45-50% ● It is synthesized in the liver from CO₂ & ammonia from the deamination of amino acids via the Kreb's-Henseleit cycle or Ornithine cycle ● Following its synthesis in the liver, it is transported to the kidneys for excretion ● It is freely filtered at the glomerulus but reabsorbed substantially at the PCT & the inner collecting duct ● The concentration of urea in the plasma is determined by renal function, protein content of the diet & the rate of protein catabolism ● The concentration of urea is expressed only by the nitrogen content of urea so to get the concentration of urea from BUN, urea= BUN x 2. ● Clinical significance: ○ to evaluate renal function ○ to assess hydration status ○ to determine nitrogen balance ○ to aid in the diagnosis of renal disease (first metabolite to increase in renal disease) ○ to determine adequacy of dialysis (easily removed by dialysis) ● Azotemia means elevated concentration of NPNs in the blood. ● Uremia is a clinical term that describes the patient's signs & symptoms when symptomatic end- stage renal failure is present. ● Decreased BUN is due: ○ to low protein intake ○ severe vomiting & diarrhea ○ severe liver disease ○ pregnancy ● BUN: Creatinine ratio is 10:1 - 20: Table 2. Classification & causes of azotemia Classification Causes Pre-renal ● refers to conditions with reduced blood flow to the kidneys thereby reducing urine output & causing retention of waste product ● High BUN: Crea ratio with normal plasma creatinine ● congestive heart failure (CHF) ● hemorrhage ● shock ● dehydration ● high protein diet ● increased protein catabolism Renal ● indicates that the kidneys itself are dysfunctional ● diseases of the renal vessels, glomerulus, tubules & renal interstitium ● acute & chronic renal failure ● acute glomerulonephritis
Post-renal ● results from anatomic obstruction to urine flow out of the kidney ● high BUN: Crea ratio with high plasma creatinine ● kidney stones (nephrolithiasis) ● congenital anomaly ● inflammatory lesion ● neoplasm Blood Urea Nitrogen (BUN) Specimen requirements: ● non-fasting sample (serum, plasma & urine) ● citrate & fluoride inhibit urease ● avoid hemolyzed sample ● refrigeration is important for urine specimens to prevent bacterial decomposition I. Chemical Method (Direct Method) A. Diacetyl Monoxime Method ● uses ferrithiocyanate as color developer ● This reaction is known as Fearon's Reaction. II. Enzymatic Method (Kinetic/Indirect Method) A. Hydrolysis of urea by urease ● the ammonia produced is measured by various methods to calculate the concentration of urea in the original sample. ● NH, Measurement Methods:
1. by use of coupled enzyme method to measure the rate of disappearance of NADH to NAD read at 340 nm. ● used by many automated instruments, best as a kinetic measurement 2. by using the Berthelot reaction- Nessler **Reagent
- by using an indicator dye** ● used in automated systems, multilayer film reagents & dry reagent strips 4. by measuring the conductivity of ammonium ion-Conductometric ● conversion of unionized urea to NH+ 4 , & CO²- 3 , resulting in increased conductivity III. Reference Method ● Isotope Dilution Mass Spectrometry ○ Not used in clinical laboratory B. Uric Acid (Blood Uric Acid/BUA) ● The final breakdown of purine (adenine & guanine) metabolism in humans. ● It is formed from xanthine by the action of xanthine oxidase in the liver & intestine. ● It is freely filtered, partially reabsorbed & secreted in the renal tubules. ● It is a weak acid; at pH 7.4 & is relatively insoluble
95% exists as monosodium urate (MSU) but at concentrations >6.8 mg/dL the plasma is saturated thus precipitates in the tissues, MSU precipitates In acidic urine as uric acid crystals ● Derived from 3 sources: ○ catabolism of ingested nucleoproteins (dietary) ○ catabolism of endogenous nucleoproteins ○ direct transformation of endogenous purine nucleotides ● Clinical significance: ○ Hyperuricemia is found in the following conditions: 1. Gout ● disease found primarily in men 30-50 y/o, post-menopausal women ● pain & inflammation of the joints cause by precipitation of MSU ● persons with gout are highly susceptible to nephrolithiasis ● in severe cases deposition of uric acid crystals & urates in tissues occur known as tophi, causing deformities 2. Increased nuclear metabolism ● occurs in patients on chemotherapy for proliferative diseases such as leukemia, lymphoma, multiple myeloma, polycythemia, hemolytic & megaloblastic anemia ● monitoring of blood uric acid levels is important to avoid nephrotoxicity 3. Chronic renal disease ● causes hyperuricemia because filtration & secretion are impaired 4. Enzyme deficiencies ● inherited disorders of
of creatine kinase (CK). Phosphocreatine provides a ready, rapid-source of energy. ● Creatinine is not reused in the body's metabolism, solely as a waste product & not easily removed by dialysis. ● It is commonly used to monitor renal function, an index of overall renal function. ● It is a measure of the completeness of 24-hour urine collection. ● It is used to evaluate fetal kidney maturity- as gestation progresses, more creatinine is excreted by the fetus into the amniotic fluid (2mg/dL). ● Clinical significance: ○ Increased in impaired renal function- severity & monitor progression of disease ○ used to measure sufficiency of renal function ○ chronic nephritis ○ congestive heart failure ● decreased in: ○ decreased muscle mass ○ severe liver diseases ○ pregnancy ○ Inadequate dietary protein Creatinine Specimen requirements: ● non-lasting sample (serum, plasma & urine) ● hemolyzed & icteric samples should be avoided ● lipemia yields erroneous results ● Urine should be refrigerated after collection or frozen if longer than 4 days is required I. Chemical Methods A. Jaffe reaction ● classic assay for serum & urine creatinine where creatinine reacts with picric acid in alkaline solution (NaOH) to form a red-orange complex with an absorbance at approximately 490-505 nm ● non-specific method ● ascorbic acid, glucose, protein & acetoacetate (a-ketoacids) give falsely elevated values ● bilirubin & hemoglobin give falsely decreased values. ● 2 general chemical Jaffe methods:
1. Jaffe reaction without adsorbent-Folin Wu Method ● sensitive but non-specific method ● uses protein-free filtrate 2. Jalle reaction with adsorbent- Lloyd or Fuller's Earth Method ● sensitive & specific method uses adsorbent: ○ Lloyd's - sodium aluminum silicate ○ Fuller's Eartn - aluminum magnesium silicate ● to remove interferences present in the specimen & elution techniques are then utilized to separate the creatinine from the adsorbent which is then made to react with the freshly prepared Jaffle reagent ● It is time consuming ● not routinely used B. Kinetic Jaffe Method ● serum is mixed with Jalfe reagent & the rate of change in absorbance is measured between 2 points (ex. after 20 seconds & after 80 seconds incubation) ● By measuring the rate of color formation in the "window" between these 2 sets of reactions, the color contributed by creatinine can be more accurately assessed. ● Although this method eliminates many interferences, a-ketoacids & cephalosphorins are still present to cause falsely increased levels. II. Enzymatic Methods A. Creatinase-Creatine Kinase Method ● read at 340 nm ● this requires large sample & lacks sensitivity thus not widely used B. Creatinase-H ₂ O, Method ● adapted for dry-chemistry ● potential to replace Jaffe method because it is more specific ● no interferences from acetoacetate or cephalosphorins
III. Reference Method ● Isotope Dilution Mass Spectrometry ○ not used in clinical laboratory D. Ammonia ● Formed by the deamination of amino acids during protein metabolism. ● It is removed from the circulation & converted to urea in the liver. ● Its production is not dependent on renal function but on liver function. ● Ammonia is neurotoxic & is associated with encephalopathy. ● Clinical significance: ○ prognostic indicator for hepatic failure ○ severe liver disease ○ Reye's syndrome (acute metabolic disorder of the liver) Methods
1. lon Selective Electrode ● diffusion of NH 3 , through selective membrane in NH 4 CI causes a pH change which is measured potentiometrically ● good accuracy & precision 2. Enzymatic method using Glutamate Dehydrogenase (GLDH) ● most common on automated instruments ● read at 340 nm 3. Spectrophotometic Table 1. Reference Ranges Analyte Male Female BUN Serum/Plasma 24-hr Urine 6 - 20 mg/dL (2.1 - 7.1 mmol/L) 12 - 20 mg/dL 6 - 20 mg/dL (2.1 - 7.1 mmol/L) BUA 3.5 - 7.2 mg/dL (0.21 - 0.43 mmol/L) 2.6 - 6.0 mg/dL (0.16 - 0.36 mmol/L) Creatinine Jaffe: 0.9 - 1.3 mg/dL (80-115 umol/L) Enzymatic: Jaffe: 0.6 - 1.1 mg/dL (53 - 97 umol/L) Enzymatic: 0.6 - 1.1 mg/dL (53 - 97 umol/L) 0.5 - 0.8 mg/dL (44 - 71 umol/L)
Beta 2-Macroglo bulin Plasma Proteins:
1. Prealbumin (Transthyretin) ● transthyretin & retinol-binding protein are transport proteins that migrate together ● acts as a transport mechanism for thyroid hormones ● migrates ahead of albumin on high resolution electrophoresis ● has half-life of only 2 days ● used as a marker for CSF ● DECREASE : indicates poor nutritional status ● INCREASE : alcoholism, chronic renal failure, steroid treatment, NSAID therapy, Hodgkin disease ● Reference value : 18-45 mg/dL (0.1-0.4 g/L) 2. Albumin ● small, globular protein that is present in highest concentration in plasma ● major protein component of most extravascular body fluids ● maintains colloidal osmotic pressure ● serves as indicator of nutritional status ● serves as reservoir of amino acids ● major transport protein carrier for free fatty acids, phospholipids, metallic ions, drugs, hormones & bilirubin ● INCREASE : Dehydration (Hemoconcentration) ● DECREASE : Acute Inflammation, Malnutrition, Liver disease- decreased synthesis, Renal disease- increased urinary loss; ex. nephrotic syndrome, GI disorders- peptic ulcer or colitis ● Reference value : 3.5-5.0 g/dL (35-55 g/L) 3. Alpha-1 antitrypsin (AAT) ● is a serpin (serine protease inhibitor); it neutralizes trypsin-like enzymes ● other serpins are alpha- antichymotrypsin, alpha-2 antiplasmin, anti-thrombin III, C1 inhibitor ● acute phase reactant ● it neutralizes the enzyme neutrophil elastase released by WBCs ● present in highest concentration of alpha-1 plasma proteins ● INCREASE : Inflammation, Pregnancy, Contraceptive Use ● DECREASE : Emphysema, Juvenile hepatic cirrhosis/AAT deficiency ● Reference valu e: 145-270 mg/dL 4. Alpha-1 Fetoprotein (AFP) ● one of the first α-2 globulins to appear in mammalian sera during development of the embryo & is the dominant serum protein in early embryonic life ● major protein in fetal serum, synthesized primarily by the yolk sac & liver ● AFP reappears in the adult serum during certain pathological states ● migrates between albumin & AAT on electrophoresis of fetal or newborn serum ● INCREASE : Spina bifida/Neural tube defects, Multiple fetuses, Fetal demise, Feto-maternal bleed, Incorrect estimation of fetal age, Hepatocellular carcinoma ● DECREASE : Fetal trisomy 18; Trisomy 21 5. Alpha-1 acid glycoprotein/orosmucoid (AAG) ● implicated in the formation of cell membrane ● negative charge in acid solutions ● binds & inactivates basic & lipophilic hormones, including progesterone & progesterone antagonist RU486; binds & reduces bioavailability of many drugs ● INCREASE : inflammatory disease, malignant neoplasms ● DECREASE : estrogens (from pregnancy & OCP) , nephrotic syndromes ● Reference value : 55-140 mg/dL 6. Alpha-1 anti-chymotrypsin ● migrates between the α1 & α2 zones ● serine protease inhibitor– inhibits cathepsin G, pancreatic elastase, mast cell chymase & chymotrypsin ● binds & inactivates PSA ● associated with the pathogenesis of Alzeimer’s disease- it is a vital component of the amyloid deposits found in persons with this disorder ● INCREASE : infection, malignancy, burns, AMI & Alzheimer’s disease ● DECREASE : liver disease ● Reference value : 30-60 mg/dL 7. Haptoglobin ● binds free hemoglobin ● it prevents loss of hemoglobin in the urine ● natural bacteriostatic agent for iron-requiring bacteria such as E.coli ● evaluates the degree of intravascular hemolysis ● INCREASE : corticosteroid hormones & many NSAIDs ● DECREASE : hemolysis, estrogens ● Reference value : 26-185 mg/dL 8. Ceruloplasmin ● copper-containing protein which also has peroxidase activity ● marker for Wilson’s disease ● INCREASE : inflammation, cancer, pregnancy
● DECREASE : Wilson’s disease, malnutrition, malabsorption, Menke’s kinky hair syndrome ● Reference value : 150-240 mg/dL
9. Alpha-2 macroglobulin (AMG) ● major component of the alpha-2 band in the serum protein electrophoresis (SPE) ● inhibits proteases such as trypsin, pepsin & plasmin ● forms a complex with prostate-specific antigen (PSA) ● INCREASE : nephrotic syndrome, diabetes & liver disease, estrogen, children ● DECREASE : severe acute pancreatitis, advanced prostate carcinoma ● Reference value : 150-420 mg/dL 10. Hemopexin ● it binds the heme released by degradation of hemoglobin ● helps in early dx of hemolysis ● INCREASE : inflammation, diabetes mellitus, Duchenne Muscular Dystrophy & some malignancies ● DECREASE : hemolytic anemias ● Reference value : 50-115 mg/dL 11. Transferrin (Siderophilin) ● transports iron ● used for differential diagnosis of anemias ● INCREASED : Iron Deficiency Anemia (IDA) ● DECREASED : inflammation, liver disease, malnutrition ● Reference value: ○ Male ■ 215-365 mg/dL ○ Female ■ 250-380 mg/dL 12. Fibrinogen ● most abundant coagulation factor ● acute phase reactant- markedly increased in inflammatory process ● high levels in plasma may cause elevated ESR- by coating the cells & allowing them to sediment in faster clumps ● INCREASE : inflammatory disorders, pregnancy, Oral Contraceptive Pill ● DECREASE : coagulation ● Reference value : 200-400 mg/dL 13. Complement ● acts as an opsonin, facilitating phagocytosis & cytolysis ● acute phase reactant produced by the liver ● INCREASE : inflammation ● DECREASE : DIC, hemolysis, malnutrition 14. C-Reactive Protein (CRP) ● cardiac marker- used as an early warning test for persons at risk with coronary artery disease ● opsonin ● an inflammatory marker that appears to reflect the severity of CHD & may contribute to its pathogenesis ● used as a rapid test for presumptive diagnosis between bacterial infection vs. infection **15. Lipoproteins
- Immunoglobulins Miscellaneous Proteins:
- Myoglobin** ● found in skeletal & cardiac muscles ● transports & stores oxygen from Hgb to intracellular respiratory enzymes of contractile cells ● higher affinity for oxygen than does Hgb ● has small MW (18 kDa) thus leaks from damaged cells more easily ● potential nephrotoxin ● one of the early protein markers for MI: onset 1-3 hours, peak 5-12 hours, normalizes in 18-30 hours; marker for angina 2. Troponins- Troponin T , Troponin I & Troponin C ● complex of three proteins that bind to the thin filaments of cardiac muscles ● regulators of actin & myosin ● TnT, Tn I & TnC are found in cardiac & skeletal muscles ● Tn T & Tn I are undetectable in many healthy individuals ● they serve as the most marker for AMI ● their levels may elevate after AMI attack in the absence of CK-MB elevations a. Troponin T ● valuable for AMI diagnosis ● useful for the assessment of early & late AMI, also in renal & muscle diseases ● sensitive marker for unstable angina ● useful in monitoring effectiveness of thrombolytics ● it rises in 3-4 hours after MI, peak level is at 10-24 hours, return to normal in 7 days (but may remain elevated for 10-14 days) ● ≥1.5 ng/mL is suggestive of AMI b. Troponin I ● only found in the myocardium ● highly specific for AMI ● 13x more abundant in the myocardium than CK-MB on a weight basis
● Principle : migration of charged particles in an electrical field (Proteins separated based on electric charge densities) ● The single most important clinical application is for the identification of monoclonal spike of immunoglobulins & differentiating them from polyclonal hypergammaglobulinemia ● Cellulose acetate/agarose gel (support media) ● After separation, protein fractions are immersed in acid solution then stained by dyes (e.g. Coomassie blue) ● The medium is placed in scanning densitometer which compute the area under the absorbance ● Albumin (fastest), alpha-1 (2nd), alpha-2 (3rd), beta (4th) & gamma (5th) ● Reference values for each fraction: ○ Albumin ■ 53-65% (3.5-5.0 g/dL) ○ Alpha- ■ 2.5-5% (0.1-0.3 g/dL) ○ Alpha- ■ 7-13% (0.6-1.0 g/dL) ○ Beta ■ 8-14% (0.7-1.1 g/dL) ○ Gamma ■ 12-22% (0.8-1.6 g/dL) ● Abnormal patterns: ○ Gamma spike ■ multiple myeloma ○ Beta-Gamma bridge ■ hepatic cirrhosis (“tau”) ○ Alpha-2 spike ■ nephrotic syndrome ○ Alpha-1 flat curve ■ juvenile cirrhosis (AAT deficiency) ○ Spikes at Alpha-1, Alpha-2 & Beta regions ■ inflammation ● Dyes used: ○ Bromphenol Blue ■ (for paper medium) ○ Ponceau S ■ (gel & acetate) ○ Amido Black ■ (gel & acetate) ○ Coomasie Brilliant Blue Fig. 1. Electrophoretic pattern of protein fractions.
6. Refractometry ● Based on the refractive index of solutes in serum ● Is an alternative to chemical analyses ● Not useful in the clinical laboratory 7. Turbidimetric or Nephelometric Methods ● Uses SSA or Trichloroacetic Acid ● Measurement depends on the formation of a uniform fine precipitate which scatters incident light in suspension (nephelometry) or block light (turbidimetry) 8. Salt Fractionation ● Globulins can be separated from ablumin by salting-out procedures using sodium salts ● Reagents : Sodium Sulfate Salt Albumin ● Concentration is inversely proportional to the severity of liver disease ● Plasma levels decline when severe hepatocellular disease lasts for more than 3 weeks ● Decreased serum albumin concentration may be due to decreased synthesis **Methods:
- Salt Precipitation** ● Globulins are precipitated, albumin in supernatant is quantitated by biuret reaction ● time consuming & labor intensive 2. Dye Binding Technique ● widely used method for albumin determination ● Uses only dyes or indicators that bind very tightly to albumin ● Ideally 100% of dye will be bound to albumin none to other fractions ● It is a dissociation-association technique influenced by temperature ● Serum is the sample of choice ● Heparin yields falsely high results bec. it enhances dye binding to albumin ● Binding should not be affected by small changes in ionic strength pH a. Bromcresol Green (BCG) ● most commonly used method ● sensitive; overestimates low albumin levels ● used extensively in automatic analyzers in parallel with Biuret reagent for TP b. Methyl Orange ● non-specific for albumin c. Hydroxyazobenzene Benzoic Acid (HABA)
● many interferences (salicylates, bilirubin) d. Bromcresol Purple (BCP) ● most sensitive & specific method, accurate ● gives overview of relative changes in different protein fractions Evaluation of Total Protein Concentration: Albumin-Globulin Ratio (A/G): Reference range : 1.1-1. ● clinically useful in evaluating TP concentrations ● Globulins are not measured but calculated from TP & albumin ● A/G ratio= Albumin TP-Albumin ● Decreased A/G ○ decreased albumin or increased globulins ● Increased A/G ○ decreased globulin synthesis & increased albumin (dehydration) Fig. 2. Sample abnormal serum protein electrophoresis patterns.
