Clinical Chemistry 1: Non-Protein Nitrogen Substances    NPN in whole blood approximately 75% greater or 1.

76X higher than that of serum/plasma Total NPN in plasma 250-400 mg/L NPN waste product of metabolism

Biochemical process is: Ornithine + CO2 + NH3 -----------------> citrulline Citrulline + NH3 -------------> arginine arginase Arginine + H2O -------------> ornithine + urea is readily filtered from the plasma by the glomerulus excreted in the urine not good index of kidney function

Classification of nitrogenous compounds in blood: 1. 2. Protein nitrogen Non-protein nitrogen Approx. plasma conc. (of blood NPN; in %) 45 20 20 5 1-2 0.2 Example

NPN compounds 1. 2. 3. 4. 5. 6. Urea Amino acid Uric acid Creatinine Creatine Ammonia

Analytical Approaches: 1. Direct measurement of urea 1.1 diacetyl monoxine (DAM) method also known as the Fearon reaction R1-CO-NHR2 structure (R1-N; R2 is notnot an acyl radical) will react with diacetyl monoxime in the presence of a strong acid and oxidizing agent to produce a chromogen direct condensation assay (urea is condensed with diacetyl urea is not hydrolyzed to ammonia but is measured directly a colorimetric approach Principle: Steps 1. diacetyl monoxime + H2O -------------> diacetyl 2. Diacetyl + urea
H+ -------------> yellow diazine +

Biochemical functions of the kidney: Function 1. 2. 3. Synthesis Metabolism Excretion

erythropoietin, rennin, prostaglandins inactivation of vit. D, formation of creatinine ammonia, urea, uric acid, several minerals, toxic substances

+ hydroxylamine H 2O

3 major divisions of renal function test inn clinical chemistry: 1. Tests measuring glomerular filtration rate (GFR) 1.1 Creatinine clearance 1.2 Urea clearance 1.3 Inulin clearance Tests measuring renal blood flow 2.1 Kjeldah s digestion method 2.2 Dam Tests measuring function tests 3.1 Excretory function test 3.1.1 p-aminohipppurate (PA) or diodrast test 3.1.2 phenolsulfonaphthalein (PSP) dye excretion test 3.2 Concentration tests 3.2.1 Specific Gravity 3.2.2 Osmolality 3.2.3 Fishberg Concentration test

2. 3. 

   

yellow diazine unstable, photosensitive; so add thiosemicarbazide unknown and QCS undergo deproteinization to remove proteins clear and colorless ideal PFF blank check stability of the reagent; set spectrophotometer at 100%T (0 A) urea standard 10 mg%

other oxidizing agents: 1. 2. 3. K sulfate Perchloric acid Phenazone

Reagents used to intensify the color and minimize photosensitivity: 1. 2. 3. Thiosemicarbazide Phenylanthranilic acid Glucoronolactone

Specimen requirements: 1. 2. 3. 4. Serum Plasma Whole blood Other body fluids such as urine primary and most cost-effective test

Protein Urinalysis NPN 1. UREA -

2. Indirect approaches - uses urease enzyme to decompose/hydrolyze urea - oldest and most often used method - initial step: hydrolysis of urea (products: carbonate anion and ammonium cation) urease + Urea + H2O ------------> 2NH4 + HCO3  2NH4 - proportionate to amount of ammonia
+

Blood urea nitrogen (BUN) major NPN in blood major excretion product in protein metabolism synthesized in liver via the ornithine or Krebs-Hensleit urea cycle first metabolite

Quantitation of the 2NH4 2.1 NH4+ + Nessler s reagent -------------> yellow dimercuric ammonium iodide   Yellow dimercuric ammonium iodide double iodide of mercuric ammonium Category: enzymatic colorimetry (480-500 nm) Na + 2.2 NH4 + NAOGL + phenol ------------> indophenol (blue) nitroprusside NAOGL sodium hypochlorite Berthelot method (Chaney Marbach method or Urease-Berthelot reaction) Absorbance is read at 630 nm only in alkaline medium 2.3 coupled enzyme assay NH4
+

+ 

  

B.

urease Urea ------------>

Prerenal causes  Congestive heart failure  Shock  Hemorrhage  Starvation  Dehydration 2. Renal causes  Acute and chronic renal failure  Glomerular nephritis 3. Post renal causes  Urinary tract obstruction Decreased levels BUN 1. Decreased CHON intake  Severe liver disease decreased synthesis  Normal pregnancy increased glomerular function

1.

+

2HCO3 

source of urease

soybeans/jackbeans

*azotemia increased level of urea as well as creatinine in the blood largely related to decreased GFR *uremia very high levels of plasma urea and creatinine Signs and Symptoms of Renal Failure: 1. 2. 3. Metabolic acidosis due to failure of kidney to eliminate acidic products of metabolism. Hyperkalemia (increased K) due to failure of K excretion. Generalized edema due to water retention.

Indicator reaction: NH4+   
GLDH + alphaketoglutarate + NADH -------------> Glutamic acid + NAD+ + H2O
+ 

   

GLDH glutamate dehydrogenase + unique characteristic use of coenzyme which is NADH or nicotinamide adenine dinucleotide coenzyme not an enzyme can be altered or reduced attached tightly to an enzyme added as a reagent high peak of absorbance at 340 nm uses multiple readings/analysis of absorbance + + decreased absorbance reading as NAD is formed from NADH + principle: rate of decrease of absorbance of NADH at 340 nm is proportionate to amount of urea category of estimation/quantitation: enzymatic UV spectrophotometry

Increased CHON diet:   Fever Major illness: may increase urea levels because of increased CHON breakdown

Used for renal function test:    BUN Creatinine test Uric acid test

Other enzymatic methods: 4.1 4.2 4.3 acid titration of ammonium Van Slyke-Cullen method urograph urostrat? (paper chromatography method) Conway micro diffusion method

*Creatine and ammonia level determinations are not good renal function tests *ammonia det. is more on liver function test Creatine and Creatinine Physiologic Chemistry/Biochemistry Creatine:     From glycine, arginie and methionine Synthesized mainly in the liver Transported as phosphorylated (muscle cells) creatine kinase/creatine phosphate (high energy source in muscle tissue) Creatine phosphate/creatine, under physiologic conditions -> lose phosphoric acid and H2O

General considerations in BUN method: 1. The determination is affected by increase CHON diet, hydration and other physiologic functions. 2. Whole blood should be deproteinized to eliminate interference of hemoglobin. 3. NH4-containing anticoagulants are contraindicated in enzymatic methods. 4. Increase concentration of NaF and sodium citrate must be avoided since they will inhibit the urease. 5. Urea is quite susceptible to bacterial decomposition; samples (esp. urine) that can t be analyzed within a few hours should be refrigerated. A few crystals of thymol can be added to the urine sample as preservative. 6. Bilirubin at a concentration of 10mg/100ml increases the urea nitrogen value by about 3mg/100ml of serum. 7. Hemolysis and lipemia does not interfere with the coupled enzyme assay but a nonhemolyzed sample is ideal. Clinical Significance A. Increased level BUN decreased renal blood flow

Creatinine:        Anhydride form of creatine as a result of non-enzymatic dehydration of muscle creatine (degradation product of creatine) Excreted into the circulation at a relatively constant rate Removed from the plasma almost entirely by GF and excreted in urine, therefore it is used to check the completeness of a 24hr urine sample Measurement of creatinine clearance (CC) is used as a measurement of GF and thus plasma creatinine maybe used as an index of GF/index of renal function More reliable than BUN Free creatinine is not reutilized in the body s metabolism, thus function solely as a waste product of creatine Increased in the latter part of renal diseases because it increases slowly than BUN but also decreases more slowly with hemodialysis 



Is not routinely affected by diet (CHON intake), however, large quantities of creatine are present in: sterilized canned meat (e.g. sardines), cooked meat It is among the most frequently performed clinical assay ranking second only with glucose (K+) and BUN *EHSPT

Analytical Assays: 1. Jaffe s rxn (direct approach)  Classical assay for serum and urine creatinine Principle: Creatinine (PFF) + alk. Picric acid ----------> amber yellow/bright orange-red alkaline creatinine picrate complex

sarcosine O2 + H2O --------------> glycine + HCHO + H2O2 oxidase H2O2 + EHSPT + 4-aminoantipyrine ------> red quinine (quinonimine) (A@600-700 nm) n-ethyl-n-2-hydroxy-3-sulfopropyl-n-toluidine

Sarcosine +

2.1.2 reagent strip method/Sakaguchi reaction Creatinine + DNBA ------> purple chromogens *DNBA 3,5-dinitrobenzenoic acid/dinitrobenzene or its derivative

y

2.2 UV-spectrophotometric method
creatininase 2.2.1 creatinine + H2O --------------->

y

Composition of alkaline picrate sol n: 1 part 10% NaOH 5 parts saturated picric acid (2,4,6-trinitrophenol) Major drawbacks: 1. Lacks specificity and sensitivity (because of pseudocreatinine like glucose, ascorbic acid, and alpha-keto acids such as acetone and acetoacetate) 2. Temperature sensitive (should not exceed 30°C) 3. Affected by length of time of incubation (10 min) 4. Affected by incomplete removal of CHON To improve Jaffe s method, modifications were made: Addition of Lloyd s rgt. Employing ion-exchange resins

creatine creatine phosphate + ADP ATP + pyruvate

Creatine kinase Creatine + ATP ------------------------> phosphokinase ADP + PEP ----------------------->

y

Phosphoenolpyruvate Indicator reaction: + + Pyruvate + NADH + H ----------> L-lactate + NAD *the concentration of creatinine is proportionate to the rate of decrease in absorbance at 340nm *NADH characteristics: reduced form co-enzyme high absorbance at 340nm + *NADH oxidized to NAD *advantages: precise and accurate not subject to interference Specimen Requirement: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Specimen: serum, plasma, urine Fasting not required Serum/plasma is preferred over whole blood Hemolyzed sample should be avoided esp. when using Jaffe method Bilirubin, elevated amounts in icteric serum samples causes low creatinine result in the Kinetic Jaffe and enzymatic methods Lipemic samples falsely low results in kinetic jaffe Fresh specimens are recommended as creatinine and creatine are labile, stable when frozen and ph of specimen should be maintained at ph7 during storage to minimize conversion Separation of serum and cells is required Levels of alphaketo acids at levels that maybe found in diabetic patients will give falsely high values Ascorbate, pyruvate, acetone and glucose will also form a color in the presence of the reagent significant (+) error in Jaffe method using PFF Drugs: methyldopa, cefoxitin, dopamine false high Antibiotics: cephalosporin false high with Jaffe s reaction

y 1. 2.

Lloyd s reagent  purified fuller s earth which is a hydrated aluminum silicate powder as a kaolin is added for the system to improve sensitivity and specificity of the Jaffe s method

Purposes of the reagent: 1. 2. used to absorb, therefore separate the creatinine from the other chromogens a trapping agent which removes reactive impurities before the alkaline picrate sol n is added

Oxalic acid   Added to PFF which results to the formation of creatinine oxalate which is then absorbed and held by Lloyd s reagent in the acid sol n Color of true creatinine is less resistant to acidification than color of pseudocreatinine substances

Advantages of the use of Lloyd s reagent: 1. Kinetic Jaffe method by Romeo Principle: Serum/plasma + alkaline picrate ------> rate of change in A is measured bet 2 time points Advantages: inexpensive, rapid and easy to do still subject to interferences by acids and cephalosporins Disadvantages: Bilirubin and hemoglobin give A(-) bias probably due to their destruction in strong base used --------------------------------------------------------------------------------------------------------------------------------

11.

2. Indirect Analytical Approaches 2.1 Enzymatic spectrophotometric 2.1.1 creatinine + Creatine +
creatininase ---------------> creatine creatinase H2O -------------> sarcosine + urea

H2O

Clinical Significance: A. High creatinine levels 1. Renal diseases (decrease renal blood flow) 2. Shock 3. Congestive heart failure 4. Muscular dystrophies 5. Decreased urine excretion B. Low creatinine levels  Not associated with any disease  Due to drug interference

Reference values:   Male: 0.6-1.2mg/dl (53-106 umol/L) Female: 0.5-1.0mg/dl (44-99 umol/L)

2.3 enzymatic UV-spectrophotometric assay uricase u.a. + 2H2O <-------------> allantoin + CO2 + H2O2
catalase H2O2 + ethanol <------------> oxidized acetaldehyde + 2 H2O2 AIDH Acetaldehyde + NADP+ <----------> acetate + NADPH + H+

SI unit = conventional unit x 88.4 2.   URIC ACID Major end product of purine metabolism Final breakdown product in purine metabolism in humans   

*NAPD oxidized form *kung naay H reduced acetaldehyde is oxidized to acetate by aldehyde dehydrogenase and the simultaneous reduction of NADP+ to NADPH, the increase in A is proportional to the u.a. concentration nd (initial A is lower than the 2 reading) @340nm u.a. has a maximum absorption peak at 285-293nm while allantoin and H2O2 has no absorption at this wavelength the disappearance of u.a. is indicated by the decrease in light A and is proportional to the u.a. initially present in the sample

*urinary excretion depends upon: purine, metabolism, renal function *purine rich foods: legumes, visceral organs, sweet breads, shellfish

y y y y

96.8% of uric acid in blood plasma is present as sodium urate or monosodium urate, relatively insoluble UA level above 6.4mg/dl, the plasma is saturated, urate crystals may form and be deposited in the joint-tophi Or deposited in the gut as UA stones Not a primary evaluation test for kidney function but serves as a confirmatory check on creatinine and BUN analyses

Specifications: 1. 2. 3. 4. 5. 6. 7. diet specimen: serum/heparinized plasma and urine, potassium oxalate can t be used since it combines with phosphotungstate promoting turbidity gross lipemia/grossly turbid sample: erroneously high result refrigerate serum sample to avoid bacterial contamination fluoride and thymol are added as preservatives elevated bilirubin (3mg/dk) erroneously low results with the enzymatic assay due to destruction of peroxidase ascorbate (vit. C) usually causes low results due to competition of ascorbate with chromogen as a hydrogen donor in the reaction *competition is eliminated by including K ferricyanide and ascorbic acid oxidase in the reagent to oxidize ascorbate. This occurs in the uricase/H2O2 coupled reaction. Hemolysis releases nonspecific reducing substance such as ergothionine, glutathione, tyrosine, ascorbic acid and glucose, which, like u.a., reduce phosphotungstic acid

Analytical Assays: 1. Caraway method  Direct redox method  Based on the alkalinizing agents such as Na cyanide, Na carbonate

Principle: NaCN u.a. (serum) + phosphotungstic acid <-----------> (blue) tungsten + allantoin + CO2 Na2CO3 u.a. is oxidized by phosphotungstic acid to allantoin in the presence of sodium carbonate phosphotungstic acid reduced to tungsten by u.a.; color developer and oxidizing agent sodium carbonate provides the alkaline (buffer) medium necessary for adequate color development 2. Coupled Enzymatic Assay 2.1 Blaunch and Koch Method  An enzymatic method  Utilizes the uricase enzyme  Enzymatic-colorimetric analysis  Aka uricase method  A coupled enzymatic reduction assay Principle: uricase u.a --------> allantoin + CO2 + H2O2    H2O2 + o-aminobenzenoic acid + MBTH ------> blue chromogen (w/ an A at 520nm) *MBTH 2.2 H 2O2 3-methyl-2-benzothiazolinone hydrazone

8.

Reference values:   Male: 3.5-7.2mg/dl Female: 2.6-6.0mg/dl

*SI unit in mmol/L Causes of increased plasma uric acid: 1. 2. 3. 4. Increased dietary intake Increased urate production: gout, treatment of myeloproliferative disease with cytotoxic drugs Decreased excretion: lactic acidosis, toxemia of pregnancy, glycogen storage disease type 1 B (glucose-6-phosphate deficiency), drug therapy, poisons (lead, alcohol), renal disease Catabolic pathway enzyme defects: Lesch-Nyhan Syndrome

Coupled Enzymatic-Spectrophotometric Assay peroxidase + 3,5 DCHBS + 4-aminophenazone ------------------>

red dye complex

*3,5- DCHBS 3,5-dichloro-2-hydroxybenzene sulfonic acid Indicator compounds: 1. 2. O-aminobenzoic acid MBTH produces color with absorbance in the visible region

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