Non-Protein Nitrogen (Laboratory) PDF

Non-Protein Nitrogen (Laboratory) COURSE LEARNING OUTCOMES: ✓ Determine the requirements regarding patient preparation, specimen collection, transport, processing and handling ✓ Demonstrate and compare methods of analysis used for Non- Protein Nitrogen ✓ Interpret laboratory test results...

Non-Protein Nitrogen (Laboratory) COURSE LEARNING OUTCOMES: ✓ Determine the requirements regarding patient preparation, specimen collection, transport, processing and handling ✓ Demonstrate and compare methods of analysis used for Non- Protein Nitrogen ✓ Interpret laboratory test results and correlate with normal and pathologic conditions ✓ Determine the significance of Non-Protein Nitrogen as a marker for Kidney Function Test ✓ Determine other laboratory Methodology in the assessment of kidney function CONTENT I. Urea a. Lab Methodologies & Analytical Techniques b. Precautions & Specimen Considerations c. Reference Interval II. Creatinine a. Lab Methodologies & Analytical Techniques b. Precautions & Specimen Considerations c. Reference Interval III. Uric Acid a. Lab Methodologies & Analytical Techniques b. Precautions & Specimen Considerations c. Reference Interval IV. Ammonia a. Lab Methodologies & Analytical Techniques b. Precautions & Specimen Considerations c. Reference Interval I. UREA LABORATORY METHODOLOGIES & ANALYTICAL TECHNIQUES A. Direct Methods- measure urea levels directly B. Indirect Methods (Blood Urea Nitrogen) - assess the nitrogen content in urea BUN x 2.14 = UREA LABORATORY METHODOLOGIES & ANALYTICAL TECHNIQUES 1. Micro - Kjeldahl (Indirect) 2. DAM: Diacetyl Monoxime (Direct) 3. Enzymatic Method (Indirect) 4. ISOTOPE DILUTION/ MASS SPECTROMETRY 1. MICRO - KJELDAHL (INDIRECT) Classical reference method Indirect method Digests nitrogen to become ammonia Principle: Nitrogen in PFF is converted to ammonia using hot conc. sulfuric acid in the presence of a catalyst 1. MICRO - KJELDAHL (INDIRECT) Measurement of ammonia 1. Micro-Kjeldahl Nessler Nessler’s Reaction (Nesslerization) di mercuric potassium iodide (Nessler’s reagent) gum ghatti NH3 + K2Hg2I2 NH2Hg2I2 (yellow) BUN x 2.14 = UREA 1. MICRO - KJELDAHL (INDIRECT) 2. Micro-Kjeldahl Berthelot Berthelot Reaction NH3 + phenol + Na nitroprusside indophenol hypochlorite blue BUN x 2.14 = UREA 2. DAM: DIACETYL MONOXIME (DIRECT) EMPLOYED IN AUTOANALYZERS strong acid yellow diazine UREA + DAM derivative Reaction is intensified by: ✓ ferric ions and thiosemicarbazide ✓ intense red color formed is measured at 540 nm Utilizes the FEARON Reaction 2. DAM: DIACETYL MONOXIME (DIRECT) ADVANTAGES: Simple Direct measurement of UREA Shows no interference by ammonia LABORATORY METHODOLOGIES & ANALYTICAL TECHNIQUES Berthelot Reaction NH3 Color Reaction Nessler’s Reaction N Color Reaction Fearon Reaction UREA Color Reaction 3. ENZYMATIC METHOD (INDIRECT) a. Urease-Nessler Urea Urease CO2 + NH3 + K2Hg2I2 (yellow) b. Urease-Berthelot Urease CO2 + NH3 Urea + phenol hypochlorite (indophenol blue @ 630 nm) BUN x 2.14 = UREA 3. ENZYMATIC METHOD (INDIRECT) c. Coupled Urease/Glutamate Dehydrogenase (GLDH) Method CANDIDATE REFERENCE METHOD 4. ISOTOPE DILUTION/ MASS SPECTROMETRY GOLD STANDARD used only by REFERENCE LABORATORIES due to its high cost PRECAUTIONS & SPECIMEN CONSIDERATIONS ✓Specimens: Plasma, serum, or urine. ✓Non-hemolyzed samples are recommended. ✓Highly influenced by protein intake, but the impact of a recent protein meal is minimal. Fasting is generally not required ✓Gray and blue-top tubes are unsuitable for BUN determination Fluoride and citrate inhibit the urease enzyme. PRECAUTIONS & SPECIMEN CONSIDERATIONS ✓Avoid contamination with ammonium salts ✓Prevent prolonged standing, as urea can convert to ammonia. ✓Urea is prone to bacterial breakdown. Specimens, especially urine and timed urine samples, should be refrigerated if not analyzed within a few hours. UREA: REFERENCE INTERVAL / RANGE CONVERSION FACTOR: (Conventional to SI unit) 0.357 Conventional unit: 8 – 23 mg/dL SI unit: 2.9 – 8.2 mmol/L ATTENDANCE CHECK II. CREATININE LABORATORY METHODOLOGIES & ANALYTICAL TECHNIQUES 1. Direct JAFFE Reaction Method 2. Kinetic Jaffe Method 3. Enzymatic Methods 4. Isotope Dilution/ Mass Spectrometry 1. DIRECT JAFFE REACTION METHOD Sample must first undergo deproteinization Creatinine in a protein Alkaline picrate free filtrate Red-orange tautomer of creatinine picrate 1. DIRECT JAFFE REACTION METHOD Reagent: ALKALINE PICRATE Picric acid (trinitrophenol) mixed with 10% NaOH should be freshly made, as it can turn into picramic acid if left standing for too long. 1. DIRECT JAFFE REACTION METHOD DISADVANTAGES: Not specific for creatinine FALSE INCREASE FALSE DECREASE Ketones Bilirubin Glucose Hemoglobin Fructose Protein Urea Ascorbic acid Cephalosporins 1. DIRECT JAFFE REACTION METHOD To eliminate interferences, use: Lloyd’s Reagent (Method of Hare): Sodium aluminum silicate Fuller’s Earth Reagent: Aluminum magnesium silicate These reagents remove interferences, enhancing the method’s specificity and sensitivity. 2. KINETIC JAFFE METHOD Measures the varying rates at which color develops from non-creatinine chromogens, helping to eliminate interferences. It is widely used, cost-effective, quick, and straightforward. Requires automated equipment. No need for protein-free filtrate; serum can be used directly. Product: Red-orange tautomer of creatinine picrate 3. ENZYMATIC METHODS Free from interference by glucose and other Jaffe chromogens. a. Creatinine Iminohydrolase Method A reduction in absorbance at 340 nm reveals the level of creatinine. 3. ENZYMATIC METHODS b. Creatinine Amidohydrolase Method 3. ENZYMATIC METHODS Different coupled enzymatic approaches, designed for use with a dry slide analyzer, include: Creatininase (creatinine amidohydrolase) Creatinase (creatine amidinohydrolase) Sarcosine oxidase Peroxidase 4. ISOTOPE DILUTION/ MASS SPECTROMETRY ULTIMATE REFERENCE STANDARD for creatinine measurement PRECAUTIONS & SPECIMEN CONSIDERATIONS ✓Specimens: Plasma, serum, or urine. ✓Avoid hemolyzed samples, as red blood cells contain significant non-creatinine chromogens. ✓Lipemic and icteric samples can cause incorrect results. ✓Ensure that specimens are stored correctly and maintain a pH of 7.0. PRECAUTIONS & SPECIMEN CONSIDERATIONS ✓Fasting is not required, but a high-protein diet can temporarily boost serum creatinine levels. ✓Urine: Refrigerate it right after collection. ✓If you need to store urine for more than 4 days, freeze it. ✓Effects of Specific Medications: ✓Dopamine can affect the accuracy of Jaffe and enzymatic assays. ✓Lidocaine may introduce a false increase in results for some enzymatic assays  CREATININE: REFERENCE INTERVAL / RANGE CONVERSION FACTOR: (Conventional to SI unit) 88.40 ADULT CHILDREN ( Plasma Fasting is NOT required for the specimen. Lipemia can impact the accuracy of results. Hemolysis can cause a false increase in total protein levels. Total protein levels are generally higher in ambulatory patients compared to those in a recumbent position due to fluid redistribution into extracellular compartments. At birth, total protein levels are lower and typically reach adult levels by age 3. With aging, there is a gradual decline in albumin levels. Pregnancy can also result in lower total protein levels. LAB METHODOLOGIES 1. Kjeldahl Method 2. Biuret Method 3. Folin-Ciocalteau (Lowry) Method 4. Ultraviolet Absorption Method 5. Salt Fractionation 6. Refractometry 7. Turbidimetric and Nephelometric Methods 8. Serum Protein Electrophoresis KJELDAHL METHOD (INDIRECT METHOD) widely accepted standard for protein measurement. Determines protein concentration based on the nitrogen content of proteins Proteins contain an average of 16% nitrogen, and protein content is calculated by multiplying the nitrogen amount by 6.25 The process includes converting nitrogen into ammonia through digestion with an acid mixture KJELDAHL METHOD (INDIRECT METHOD) Process: Serum is treated with tungstic acid, which separates proteins from the solution, leaving a protein-free filtrate. Reagents: Sulfuric Acid (H₂SO₄): Used to digest the sample and convert nitrogen into a measurable form. End Product: Ammonia: The amount of ammonia produced during the digestion process is quantified to estimate protein content. BIURET METHOD (DIRECT METHOD) Most used method for protein quantification. Also referred to as Piotrowski's Test Endorsed by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) BIURET METHOD (DIRECT METHOD) Principle: Cupric ions react with peptide bonds in an alkaline environment to form a violet-colored complex. The intensity of the violet color is proportional to the number of peptide bonds and indicates the total protein concentration. The measurement is taken at a wavelength of 545 nm. This method requires a minimum of two peptide bonds and an alkaline medium to produce a detectable reaction. BIURET METHOD (DIRECT METHOD) Reagents: 1. Copper Sulfate: Primary Reagent: Cupric ions from copper sulfate form complexes with peptide bonds. 2. Potassium Sodium Tartrate (Rochelle's Salt): Stabilizes Copper: Helps keep copper in solution. 3. Potassium Iodide: Prevents Auto reduction: Stops copper from reducing itself during the reaction. 4. Sodium Hydroxide: Alkalinizes the Solution: Creates the alkaline environment necessary for the reaction. Note: Avoid Hemolysis: Hemoglobin absorbs light at the same wavelength as the biuret reagent, which can interfere with accurate measurement. FOLIN-CIOCALTEAU (LOWRY) METHOD Highest Analytical Sensitivity among protein determination methods. Principle: Oxidation of phenolic compounds, including tyrosine, tryptophan, and histidine, results in a deep blue color. Reagents: Phenol Reagent or Phosphotungstic-Molybdic Acid: Used to induce the color reaction. Color Enhancer: Biuret Reagent: Enhances the color intensity of the reaction, aiding in the measurement. ULTRAVIOLET ABSORPTION METHOD Principle: Proteins absorb ultraviolet light due to specific components: 210 nm: Absorption is attributed to peptide bonds in the protein. 280 nm: Absorption is due to aromatic amino acids, specifically tryptophan, tyrosine, and phenylalanine. SALT FRACTIONATION Purpose: Separates globulins from albumin using salting-out techniques with sodium salts. Salt Used: Sodium sulfate is employed for the salting-out process. Process: Albumin that remains in the supernatant after salting-out can be quantified using any of the total protein measurement methods. SALT FRACTIONATION REFRACTOMETRY Relies on measuring the refractive index caused by solutes in the serum. TURBIDIMETRIC AND NEPHELOMETRICMETHODS Reagents: Sulfosalicylic acid (SSA) Trichloroacetic acid (TCA) Principle: Nephelometry: Measures the scattering of light by a fine, uniform precipitate in suspension. Turbidimetry: Measures the reduction of light transmission caused by precipitates that block light. TURBIDIMETRIC AND NEPHELOMETRICMETHODS Factors that affects the nephelometry and turbidity: Size and number of particles Depth of the tube Cross sectional areas of each particle SERUM PROTEIN ELECTROPHORESIS (SPE) Introduced By: Tiselius Principle: Charged particles migrate in an electric field. Proteins are separated based on their electrical charge. Applications: Identifies monoclonal spikes of immunoglobulins Differentiates monoclonal gammopathies from polyclonal hypergammaglobulinemia SERUM PROTEIN ELECTROPHORESIS (SPE) Cations: Positively charged ions Anions: Negatively charged ions At pH 8.6: Proteins acquire a negative charge Positive Electrode: Anode Negative Electrode: Cathode Migration of Serum Proteins: From the cathode to the anode SERUM PROTEIN ELECTROPHORESIS (SPE) Fastest to slowest: SERUM PROTEIN ELECTROPHORESIS (SPE) ALBUMIN ALPHA 1 ALPHA 2 BETA GAMMA alpha1-antitrypsin (90%) haptoglobin transferrin (90%) Immunoglobulins (Igs) alpha1-fetoprotein ceruloplasmin hemopexin C-reactive protein (CRP) alpha1-acidglycoprotein alpha2- IgA (orosomucoid) macroglobulin alpha1-antichymotrypsin beta2- microglobulin thyroxine-binding complements(C3, globulin(TBG) C4, C5) antithrombin III SERUM PROTEIN ELECTROPHORESIS (SPE) Staining Agents: Bromphenol blue Ponceau S Amido Black 10B Lissamine green Coomassie Brilliant Blue Quantification: After staining, the bands are scanned with a DENSITOMETER to measure the amount of protein in each band. SERUM PROTEIN ELECTROPHORESIS (SPE) SERUM PROTEIN ELECTROPHORESIS (SPE) OTHER METHODS Coomassie Brilliant Blue Dye: Sensitive enough to detect as little as 1 µg of protein. Used as a stain for cerebrospinal fluid (CSF) electrophoresis. Ninhydrin: Produces a violet color when reacting with primary amines. Commonly used for detecting peptides and amino acids after paper chromatography. REMEMBER! Protein Profile – evaluates the synthetic function of the liver. Total Serum Protein (TSP) = albumin + globulin Globulin = TSP – albumin Total Plasma Protein (TPP) = albumin + globulin + fibrinogen TPP - TSP = fibrinogen B. Albumin Determination ALBUMIN DETERMINATION Albumin Synthesis: Produced in the liver. Normal Synthesis Rate: Approximately 120 mg/kg/day. Major Serum Protein. ALBUMIN DETERMINATION Relationship with Liver Disease: Albumin concentration decreases as liver disease severity increases. Measurement Methods: Direct methods utilize the dye-binding property of albumin. Binding Characteristics: Albumin binds many small molecules, including specific dyes that do not interact with other serum proteins  ALBUMIN DETERMINATION Isolation and Measurement: Isolation Process: Mix serum with Na2SO4 or (NH4)2SO4 and centrifuge. Residue: Precipitated globulins. Filtrate: Contains albumin. Measurement Methods: Micro-Kjeldahl Method: Multiply the result by 6.25. Biuret Method: Identifies albumin by producing a violet color. Dye-Binding Methods: Measures albumin based on dye binding. ALBUMIN DETERMINATION DYE-BINDING METHODS Dyes Used for Measurement: Bromcresol Purple: The most specific dye (603 nm) Bromcresol Green: The most commonly used dye (628 nm) Methyl Orange Hydroxyaminobenzoic Acid (HABA) ALBUMIN DETERMINATION INCREASED IN DECREASED IN severe dehydration liver disease prolonged tourniquet application nephrotic syndrome malabsorption syndrome muscle wasting disease thyrotoxicosis malnutrition burns Low total protein + Low albumin = Hepatic cirrhosis & Nephrotic syndrome C. Albumin/Globulin Ratio ALBUMIN/GLOBULIN RATIO Purpose: Determines if globulin levels are higher than albumin in the serum. Globulin: Analysis: Not directly measured; computed as TSP - albumin. Reference Range: 1.3-3:1 (Typically, albumin > globulin). Increased Globulin Indications: (Inverted A/G Ratio) Chronic inflammation Multiple myeloma Waldenstrom macroglobulinemia Inverted Ratio: Occurs when globulin > albumin, known as an inverted A/G ratio. REFERENCE INTERVAL / RANGE ALBUMIN CONVERSION FACTOR: (Conventional to SI unit) 10 Conventional unit: 3.5-5 g/dL (gm %) SI unit: 35-50 g/L REFERENCE VALUESFOR OTHER PROTEIN FRACTIONS Alpha1-globulin 0.1-0.3 g/dL Alpha2-globulin 0.6-1.0 g/dL Beta-globulin 0.7-1.1 g/dL Gamma globulin 0.8-1.6 g/dL MUST KNOW! Analbuminemia hereditary absence of albumin Bisalbuminemia associated with the presence of therapeutic drugs in serum (↑penicillin = bisalbuminemia) D. Prothrombin Time/ Vitamin K Response Test PROTHROMBIN TIMETEST/ VITAMIN K RESPONSE TEST Liver Function Responsible for synthesizing Vitamin K-dependent clotting factors Intrahepatic vs. Extrahepatic Obstructive Liver Disease Intrahepatic Disorder: PT (Prothrombin Time) is prolonged Extrahepatic Disorder: PT (Prothrombin Time) is normal Vitamin K Administration Route: Intramuscular (IM) Dosage: 10 mg daily for 1-3 days PROTHROMBIN TIMETEST/ VITAMIN K RESPONSE TEST Assessment of Liver Disease Severity Serum Albumin Vitamin K-Dependent Clotting Factors These are the most useful indicators for evaluating the severity of liver disease PART 2 III. Tests that evaluate hepatic conjugation and excretion function  B1 B2 Unconjugated bilirubin Conjugated bilirubin Water Insoluble Water Soluble Non-polar bilirubin Polar Bilirubin Indirect reacting Direct reacting Hemobilirubin Cholebilirubin Slow reacting One-minute/promt bilirubin Prehepatic bilirubin Post hepatic/obstructive and regurgitative bilirubin  I. BILIRUBIN ASSAYS ✓end product of hemoglobin metabolism ✓Function: Principal pigment in bile ✓Formation: Results from the breakdown of heme- containing substances, including: Myoglobin Cytochrome oxidase Catalase I. BILIRUBIN ASSAYS Specimen Considerations for Bilirubin Assay ✓Avoid: Hemolysis Lipemia ✓Sample Requirements: Fasting sample preferred to prevent increased bilirubin concentration from lipemia Hemolyzed samples can reduce bilirubin reaction with diazo reagent I. BILIRUBIN ASSAYS Specimen Considerations for Bilirubin Assay ✓Processing: Protect specimen from light Process as soon as possible (preferably within 2-3 hours) ✓Note: Photoisomerization converts B1 to B2, potentially leading to falsely elevated B2 levels Visible icterus occurs at bilirubin levels >25 mg/L I. BILIRUBIN ASSAYS Notes to Remember ✓Unconjugated bilirubin reacts SLOWLY Therefore, accelerants such as CAFFEINE or METHANOL are used to measure both direct and indirect bili (total bilirubin) Without accelerants, only direct-acting bilirubin will be measured ✓Conjugated bilirubin: the fraction that produced a color in aqueous solution ✓Unconjugated bilirubin: the fraction that produce a color only after addition of alcohol. I. BILIRUBIN ASSAYS Notes to Remember ✓Total bilirubin is measured 15 minutes after adding the accelerant/coupling accelerator. ✓Caffeine benzoate is preferred over methanol Because methanol causes protein ppt’n and turbidity I. BILIRUBIN ASSAYS MUST KNOW!!! ✓BILIRUBIN absorbs light maximally at 450nm ✓It imparts yellow color to serum, urine and amniotic fluid (body fluids) I. BILIRUBIN ASSAYS PRINCIPLE OF BILIRUBIN ASSAYS: Van den Bergh Reaction ✓diazotization of bilirubin to produce azobilirubin I. BILIRUBIN ASSAYS DIAZO REACTIONS Principle: Reaction of bilirubin with diazotized sulfanilic acid forms a colored product, azobilirubin Reactions: Bilirubin + diazotized sulfanilic acid → azobilirubin (conjugated bilirubin) Bilirubin + diazotized sulfanilic acid + accelerator → total bilirubin Calculating Unconjugated Bilirubin: Total bilirubin - conjugated bilirubin = unconjugated bilirubin  I. BILIRUBIN ASSAYS DIAZO REACTIONS ✓Historically first performed on urine samples only, but Van den Bergh found that diazo reaction may be applied on serum samples but only in the presence of accelerators (solubilizers) ✓The third fraction, delta bilirubin will react as conjugated bilirubin Delta bilirubin is conjugated bilirubin tightly bound to ALBUMIN. It is elevated in obstructive jaundice I. BILIRUBIN ASSAYS 1. Diazo Reaction A. EVELYN MALLOY B. JENDRASSIK AND GROF C. MODIFIED JENDRASSIK AND GROF I. BILIRUBIN ASSAYS 1. Diazo Reaction A. EVELYN AND MALLOY METHOD Coupling accelerator: 50% Methanol Final reaction: (+) pink to purple azobilirubin (560 nm) Uses pH 1.2 I. BILIRUBIN ASSAYS 1. Diazo Reaction B. JENDRASSIK AND GROF METHOD MOST SENSITIVE AND MOST WIDELY USED More sensitive than Evelyn Malloy Popular technique for the discreet analyzers Not falsely elevated by hemolysis  I. BILIRUBIN ASSAYS 1. Diazo Reaction B. JENDRASSIK AND GROF METHOD ✓Coupling accelerator: CAFFEINE SODIUM BENZOATE ✓Buffer: Sodium Acetate less sensitive to pH change ✓Ascorbic acid: terminates the initial reaction and destroys the excess diazo reagent ✓Final reaction: (+) pink to blue azobilirubin (600 nm)  I. BILIRUBIN ASSAYS 1. Diazo Reaction C. MODIFIED JENDRASSIK AND GROF METHOD CANDIDATE REFERENCE METHOD FOR TOTAL BILIRUBIN Coupling accelerator: caffeine-benzoate  I. BILIRUBIN ASSAYS MUST KNOW! Obstruction in the biliary tract also causes elevated total cholesterol due to block in the normal excretion of cholesterol in the bile.  I. BILIRUBIN ASSAYS MUST KNOW! If there is obstruction, 1. Conjugated bilirubin will re-enter the circulation 2. It will accumulate 3. Then spontaneously form a covalent bond with albumin forming delta bilirubin or biliprotein with a half-life of 17 days thereby prolonging its half-life in the circulation. Note: (B1 = 55 or premature Low LDL-c menopause for women Family history of premature CHD Current cigarrete smoking High LDL-c Low HDL-c (