Non Protein Nitrogen (NPN) Urea & Ammonia Presentation PDF

Non protein nitrogen (NPN) Urea &Ammonia Fatima Yousuf Mohamed MSc Clinical Chemistry Objectives To identify NPN compounds, and the clinically significant NPN. To discuss the physiology and clinical significance of urea and ammonia measurement...

Non protein nitrogen (NPN) Urea &Ammonia Fatima Yousuf Mohamed MSc Clinical Chemistry Objectives To identify NPN compounds, and the clinically significant NPN. To discuss the physiology and clinical significance of urea and ammonia measurement. To understand Blood Urea Nitrogen (BUN)&BUN: Creatinine ratio. To study the analytical methods of urea &ammonia measurement. To understand pathophysiology (disease correlations) of urea and ammonia. Introduction Non protein nitrogen (NPN) are nitrogen containing compounds that are not proteins or polypeptides. Include 15 compounds, the majority of them rise from the catabolism of proteins and nucleic acids. Their determination in the blood has been used to assess renal function, because the kidneys act to excrete these compounds in the urine. Clinically Significant NPN Compounds Compound Approximate plasma conc (% of Total NPN) Urea 45–50 Amino acids 25 Uric acid 10 Creatinine 5 Creatine 1–2 Ammonia 0.2 Urea physiology Urea is the major excretory product of protein metabolism. Synthesized in the liver from carbon dioxide and ammonia arising from the deamination of amino acids. Urea physiology Cont… Following synthesis in the liver, urea is carried in the blood to the kidney, where it is filtered from the plasma by the glomerulus. Most of the urea in the glomerular filtrate is excreted in the urine, some urea is reabsorbed by passive diffusion through the renal tubule. Small quantities of urea (10% of the total) are excreted through the gastrointestinal tract and skin. Clinical significance of urea measurement Used to evaluate renal function Assess hydration status Determine nitrogen balance Aid in the diagnosis of renal disease Verify adequacy of dialysis Pathophysiology (Disease correlations) Elevated concentration of urea in the blood is called azotemia. Very high plasma urea concentration accompanied by renal failure is called uremia, or the uremic syndrome. Causes of increased blood urea are classified in to : Pre renal causes Renal causes Post renal causes Pre renal causes caused by reduced renal blood flow due to:  Hemorrhage  Dehydration  Congestive heart failure  Shock High-protein diet or increased protein catabolism Renal causes Acute and chronic renal failure Renal disease, including glomerular nephritis, tubular necrosis Post renal causes Urinary tract obstruction due to renal calculi, tumors of the bladder or prostate. Causes of low urea concentration Decreased protein intake Severe liver disease Pregnancy Blood Urea Nitrogen (BUN) Calculation of correlated nitrogen content to urea. Nitrogen gram molecular weight: 14 g/mole Urea contains 2 nitrogens: 28 g /mole of urea Molecular weight of urea: 60 g/mole 60/ 28 = 2.14 BUN= urea/2.14 BUN: Creatinine ratio Used for differentiation the cause of abnormal urea concentration. Normally 10:1 to 20:1. A high ratio with normal creatinine seen in pre renal conditions. A high ratio with an elevated creatinine is usually seen in renal, and post renal conditions. A low ratio is observed in conditions associated with decreased urea production. Analytical Methods Enzymatic methods (in direct):  Involves the hydrolysis of urea by the enzyme urease and quantitation of the NH +4 by: 1. Colorimetric Berthelot reaction to detect the NH+4 produced. 2. The coupled enzymatic assay: Is a kinetic assay that involves a second enzyme glutamate dehydrogenase (GLDH), in addition to urease enzyme. the rate of decreased nicotinamide adenine dinucleotide (reduced, NADH) is measured at 340 nm. Analytical Methods Cont… Chemical method diacetyl monoxime (DAM) (direct):  It measures urea directly.  Urea reacts with DAM in an acidic medium to produce a colored complex. The color is intensified by using thiosemicarbazide and cadmium salt. Assignment Other analytical methods used for urea measurement Specimen Requirements and Interfering Substances Non hemolyzed plasma, serum, or urine can be used. Avoid citrated plasma and sample collected in fluoride, that will inhibit the urease enzyme. Samples (especially urine) that cannot be analyzed within a few hours should be refrigerated, because urea is susceptible to bacterial decomposition. For urine specimen, the methods used require modification, because of high urea concentration and the presence of endogenous ammonia. Reference Interval of urea nitrogen Plasma or serum 6–20 mg/dL Urine, 24-h 12–20 g/day Ammonia Physiology Is formed by the deamination of amino acids and by bacterial metabolism in the lumen of the intestine. Some ammonia results from anaerobic metabolic reactions that occur in skeletal muscle during exercise. It is removed from the circulation and converted to urea in the liver. Free ammonia is toxic, it present in the plasma in low concentrations. Clinical significance of Ammonia measurement Blood ammonia concentration can be correlated with both the severity and prognosis of the disease such as:  Severe liver disease.  Hepatic failure  Reye’s syndrome  Inherited deficiencies of urea cycle enzymes. Analytical Methods 1) Enzymatic method: Ammonium reacts with α- ketoglutarate in the presence of glutamate dehydrogenase enzyme, and reduced nicotinamide adenine dinucleotide phosphate (NADPH), to form L-glutamate and nicotinamide adenine dinucleotide phosphate (NADP), with changes in absorbance measured spectrophotometrically. Analytical Methods Cont 2) Chemical methods: The ammonia electrode: Based on diffusion of NH3 through a selective membrane into NH4Cl solution causing pH change.  Ammonia reacts with an indicator bromophenol blue to produce a colored compound that is detected spectrophotometrically. Specimen Requirements Heparin and EDTA should be used. Venous blood should be obtained without trauma and placed on ice immediately, because of in vitro amino acid deamination. Samples should be centrifuged at 0°C to 4°C within 20 minutes of collection. Hemolysis should be avoided ( RBCs contain two to three times as much ammonia as plasma). Patients do not smoke for several hours before the sample (smoking is a significant source of ammonia contamination). Interfering Substances Ammonium salts, barbiturates, diuretics, ethanol, narcotic, analgesics, and some other drugs may increase ammonia in plasma. Lactobacillus acidophilus, lactulose, levodopa, and several antibiotics decrease values. Reference Interval Adult plasma 19–60 µg/dL Child ((10 days–2 years) plasma 68–136 µg/dL Pathophysiology Hyperammonemia is neurotoxic and often associated with encephalopathy, can be caused by :  Severe liver disease (impaired function of parenchymal liver cell)  Inherited deficiency of enzymes of the urea cycle. References Clinical Chemistry principles, procedures, correlations (Bishop)