2 Non-protein Nitrogen Compounds The determination of nonprotein nitrogenous substances in the blood has traditionally been used to monitor renal function.Nitrogen containing compounds that are not proteins or polypeptidesUseful clinical information is obtained from individual components of NPN fraction
3 Clinically Significant NPN The NPN fraction comprises about 15 compoundsMajority of these compounds arise from catabolism of proteins and nucleic acids
4 Urea Nitrogen (Blood) BUN Highest concentration of NPN in bloodMajor excretory product of protein metabolismThese processes release nitrogen, which is converted to ammoniaSynthesized in the liver from CO2 and Ammonia that arises from deamination of amino acidsOrganisms synthesize urea from ammonia because ammonia (a common metabolic waste product) raises pH in cells to toxic levels. Therefore, urea synthesis is necessary even though it costs energy to produce. Urea is neither acidic nor basic, so it is a perfect vehicle for getting rid of nitrogen waste
5 Urea Nitrogen (Blood) BUN Assays for urea were based on measurement of nitrogen, the term blood urea nitrogen (BUN) has been used to refer to urea determination.Excreted by the kidneys – 40% reabsorbed<10% of the total are excreted through the gastrointestinal tract and skin.Concentration is determined by:Renal functionDietary intakeProtein catabolism rate
6 Clinical Application Measurement of urea is used to: evaluate renal function,to assess hydration status,to determine nitrogen balance,to aid in the diagnosis of renal disease,and to verify adequacy of dialysis.
7 Disease Correlations Azotemia: elevated conc. of urea in blood Very high plasma urea concentration accompanied by renal failure is called uremia, or the uremic syndromeCauses of urea plasma elevations are:PrerenalRenaland postrenal
8 Pre-Renal AzotemiaReduced renal blood flow Less blood is delivered to the kidney less urea filteredAnything that produces a decrease in functional blood volume, include:Congestive heart failure,shock,hemorrhage,dehydrationHigh protein diet or increased catabolism (Fever, major illness, stress)
9 Renal AzotemiaDecreased renal function causes increased blood urea due to poor excretionAcute & Chronic renal failureGlomerular nephritisTubular necrosis& other Intrinsic renal diseaseGlomerulonephritis, also known as glomerular nephritis, abbreviated GN, is a renal disease characterized by inflammation of the glomeruli, or small blood vessels in the kidneys
10 Post-Renal Azotemia Obstruction of urine flow Renal calculiTumors of bladder or prostateSevere infections
11 Decreased Urea Nitrogen Low protein dietary intakeLiver disease (lack of synthesis)Severe vomiting and/or diarrhea (loss)Increase protein synthesis
12 Analytical methodsAssays for urea were based on measuring the amount of nitrogen in the sample (BUN)Current analytic methods have retained this custom and urea often is reported in terms of nitrogen concentration rather than urea concentration (urea nitrogen).Urea nitrogen concentration can be converted to urea concentration by multiplying by 2.14
13 Conversion of BUN to urea Atomic mass of nitrogen = 14 g/mol;Molecular mass of urea = g/mol.Urea contains two nitrogen atoms per molecule.Urea nitrogen (urea N) is 46.6% by weight of urea (28 divided by 60.06).Therefore: 10 mg/dL of BUN divided by = mg/dL of urea
14 Analytical methods Enzymatic Urease → hydrolysis of urea to ammonium ion , then detect ammonium ion (NH4+)EnzymaticThe most common method couples the urease reaction with glutamate dehydrogenaseglutamate dehydrogenase = GLDH
15 NH4+ + pH indicator → color change Analytical methodsIndicator dyeNH4+ + pH indicator → color changeConductimetricConversion of unionized urea to NH4+ and CO32- results in increased conductivityReference range of Urea N:Serum or plasma: mg/dl24 hours Urine: g/day:
16 Creatinine/ CreatineCreatine is synthesized in Liver from arginine, glycine & methionineConverted to Creatine Phosphate = high energy source for muscle tissueCreatinine is produced as a waste product of creatine and creatine phosphate.Creatine Phosphate – phosphoric acid = CreatinineCreatine – water = Creatinine
18 Creatinine/CreatineCreatinine is released into circulation at stable rate proportional to muscle massFiltered by glomerulusExcreted in urinePlasma creatinine concentration is a function of:relative muscle mass,rate of creatine turnoverand renal functionDaily creatinine excretion is fairly stable.It’s a very good test to evaluate renal function
19 Disease CorrelationsElevated Creatinine is found with abnormal renal function (i.e. GFR)Measurement of creatinine concentration is used to determine:sufficiency of kidney functionand the severity of kidney damageand to monitor the progression of kidney disease.
20 Disease CorrelationsGFR is the volume of plasma filtered (V) by the glomerulus per unit of timeGFR is used to estimate renal functionCreatinine ClearanceA measure of the amount of creatinine eliminated from the blood by the kidneys per unit timePlasma concentration of creatinine is inversely proportional to clearanceTherefore increased plasma levels mean decreased GFR
21 Analytic Methods Jaffe reaction Kinetic Jaffe Reaction Most frequently used, was first described in 1886Creatinine reacts with picric acid in alkaline solution → red-orange chromogenKinetic Jaffe ReactionRate of change in absorbance is measuredEnzymatic MethodUsing creatininase, creatine kinase, pyruvate kinase and lactate dehydrogenase
22 Analytic MethodscreatininasePhosphoenolpyruvate= PEP
23 Creatine Elevated in plasma and urine in Muscular dystrophy, hyperthyroidism, trauma,Plasma creatinine levels usually normal, but urinary is elevatedSpecialized testing – not part of routine lab
24 Assay of creatineAnalyzing 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.
25 Uric AcidUric acid is a final breakdown product of purine metabolism (adenosine/guanine) in liverMost other mammals degrade it further to allantoinUric acid is transported to kidney and filtered (70%)98% reabsorbed in PCTSome secreted by DCTNet amount 6-12% of filtered amountRemaining 30% by GITPCT= proximal convoluted tubuleDCT= distal
26 Uric Acid Present in plasma as monosodium urate At plasma pH → relatively insolubleConc. > 6.8 mg/dl → plasma saturated → urate crystals may form & precipitate in tissueUric acid is measured to:assess inherited disorders of purine metabolism,to confirm diagnosis and monitor treatment of gout,to assist in the diagnosis of renal calculi,to prevent uric acid nephropathy during chemotherapeutic treatment,and to detect kidney dysfunction
27 Disease Correlations Gout Primarily in men Onset 30-50 years UA greater than 6.0 mg/dLPain & inflammation of joints by precipitation of sodium urates in tissuesIncreased risk of renal calculihyperuricemia due to overproduction of uric acid in 25-30%
28 Disease Correlations Increased catabolism Chronic renal disease occurs in patients on chemotherapy for diseases such as leukemia & multiple myeloma.Allopurinol inhibits xanthine oxidase, an enzyme in the uric acid synthesis pathway, is used to treat these patients.Chronic renal diseasecauses elevated levels of uric acid because filtration and secretion are hindered.
30 Disease Correlations Hypouricemia Secondary to severe liver disease Defective renal tubular reabsorptionFanconi’s SyndromeChemotherapy with 6-mercaptopurine or azathioprine – inhibit purine synthesisOver treatment with allopurinolFanconi Syndrome (also known as Fanconi's syndrome) is a disorder in which the proximal tubular function of the kidney is impaired, resulting in decreased reabsorption of electrolytes and nutrients back into the bloodstream
31 Analytic MethodsPrimary method uses enzyme uricase (urate oxidase) to convert uric acid to allantoinDifferential absorption at 293 nmuric acid has a uv absorpance peak at 293 nm. Whereas allantoin does notProteins also absorb near this wavelengthuric acid has a uv absorpance peak at 293 nm. Whereas allantoin does not
32 Reference range: Males 0.5-7.2, Females: 2.6-6.0 mg/dl Analytic MethodsNewer methods couple uricase with catalase or peroxidase action on hydrogen peroxide product from allantoin productionSome interferences from reducing agentsReference range: Males , Females: mg/dl
33 Ammonia Comes from deamination of amino acids Digestive & bacterial enzymes in intestineAlso released from muscle during exerciseConsumed by parenchymal cells of liver and converted to ureaFree ammonia is toxic;however, ammonia is present in the plasma in low concentrations
34 Disease Correlations Severe liver disease Most common cause of abnormal ammonia levelsAmmonia is not removed from circulation & not converted to ureaElevated ammonia levels are neurotoxic and are often associated with encephalopathy.
35 Disease Correlations Reye’s Syndrome Most commonly seen in children Often preceded by viral infection treated with aspirinSevere fatty infiltration of liverMay be fatal if ammonia levels remain high100% survival if ammonia stays below 5x normalReye's syndrome is a potentially fatal disease that causes numerous detrimental effects to many organs, especially the brain and liver. It is associated with aspirin consumption by children with viral diseases such as chickenpox.The disease causes fatty liver with minimal inflammation, and severe encephalopathy (with swelling of the brain). The liver may become slightly enlarged and firm, and there is a change in the appearance of the kidneys. Jaundice is not usually present
36 Disease CorrelationsAmmonia is of use in the diagnosis of inherited deficiencies of urea cycle enzymesMeasurement of ammonia used to diagnose and monitor treatment
38 Analytic Methods Glutamate dehydrogenase Direct ISE Decrease in absorbance at 340 as NADPH is consumed (oxidized)Direct ISEChange in pH of solution as ammonia diffuses through semi-permeable membraneReference Interval: Adult Plasma 19 – 60 μg / dl