Week 7 Non Protein Nitrogenous Substances Part 1 PDF

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Taught by Connie Thurber...

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MLSC-2111U, Clinical Biochemistry Urea -

Chief nitrogen containing waste product of protein and amino acid metabolism. Has no known function in humans. The rate of synthesis depends on both the amount protein intake and breakdown. Urine is not only in the blood but also excreted in urine. It is water soluble but 40-70% is reabsorbed at the renal tubules. 75%-85% for NPNs in the urine are urea molecules. Urea Nitrogen, a measurement of only the nitrogen portion of urea in plasma. Blood Urea Nitrogen (BUN), the measurement of the nitrogen part of the urea using a whole blood sample. Urea nitrogen is different than urea itself. BUN can be converted to urea multiplying BUN by 2.14. The BUN remains in use in the US and the Vitros analyzers. In Canada, urea is used instead of BUN.

Clinical Significance of Urea -

Elevated levels are indicative of renal disease. Used as a screening tool and it is a kidney marker. The typical reference interval for urea is 3.0 – 7.0 mmol/L. Azotemia, clinical term used for increased urea in the blood. Hyperuremia, laboratory term for increased plasma urea levels. Uremia, clinical term for very high plasma urea accompanied by acute kidney injury. Urine in the blood.

Causes of Increased Urea -

Pre-Renal, reduced blow flow or increased urea synthesis from protein catabolism or muscle wasting. Renal, reduced renal function leading to compromised urea excretion (nephritis, tubular necrosis, renal failure etc.) Post-Renal, blockage of urine flow (renal stones, urinary tract obstruction).

Causes of Decreased Urea -

Urea is also a liver marker and problems in the liver can cause hypouremia due to impaired urea synthesis in the liver.

MLSC-2111U, Clinical Biochemistry Urea Testing -

Methods for measuring urea typically use the urease enzyme to catalyze the hydrolysis of urea into carbon dioxide and ammonia. This method requires initial hydrolysis of urea using the specific enzyme urease to produce ammonium ions. Ammonium ions can then be used to measure urea.

Diacetyl Monoxime Method -

A direct estimation of urea. Using a condensation reaction between urea and diacetyl monoxime. No longer in use in the clinical lab and not clinically relevant.

Berthelot Method -

In this test, ammonium is reacted with reagents to produce coloured end products for spectrophotometry (Indophenol, blue and measured at 560 nm). Additional ammonia in the sample from other sources is an interference.

Glutamate Dehydrogenase Method (GLDH) -

Urease is used to convert urea to ammonia and GLDH to convert NADH to NAD in proportion the amount of ammonia produced by urease. This is a coupled reaction. Since NADH is being turned into NAD, we are measuring a decrease in absorbance at a wavelength of 340 nm.

Pre-Analytical Factors of Urea Results -

Urea can be done on most bodily fluids. Urease is used in almost all tests. Ammonia from detergents, smoke and urine can contaminate samples and falsely increase urea levels. Na Fluoride and Sodium citrate tubes will inhibit urease. Na Fluoride tubes are more problematic since they are more commonly used than the blue Sodium citrate tubes. Ammonium heparin tubes also can false increases in urea levels. These tubes cannot be used for urea tests. Urine samples must be refrigerated to avoid bacterial decomposition of urea to ammonia. Thymol can be added as a growth inhibitor. Causes falsely low results.

Creatinine -

Nitrogenous waste product of muscle metabolism. Synthesized in the liver from slow but spontaneously degradation of creatinine phosphate in muscle. Diffuses into the plasma at a constant rate that is proportional to the persons muscle mass.

MLSC-2111U, Clinical Biochemistry -

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Creatinine is completely filtered by the kidneys. There is slight secretion in the tubules but not enough to be significant. It is not reabsorbed at all by the kidney. Almost all creatinine is found in the urine. Primarily used to evaluate kidney function. Plasma levels of creatinine are influenced by muscle mass and the rate of creatinine turn over and kidney filtration. Male and female reference levels are 50 – 130 and 45 – 100 umol/L respectively. In normal healthy individuals creatinine levels in the body are low and constant.

Clinical Significance of Creatinine -

Elevated serum creatinine is often indicative of renal disease. As GFR decreases, creatinine in the blood increases. Act as an indirect measure of GFR. A reduction of 50% of normal renal function results in only a doubling of SCr. Creatinine levels can therefor still within the reference range despite renal disease. Creatinine is not always sensitive to early or mild to moderate renal disease.

Causes of Abnormal Creatinine Levels -

Pre-Renal, CHF, dehydration, shock, hemorrhage, strenuous exercise and muscular dystrophy can cause high creatinine results. Post-Renal, urinary tract obstruction due to GI tumours, renal stones or infection can cause high creatinine. Non-Renal, drugs that compete with tubular secretion will increase the amount of blood creatinine. Decreased creatinine can be caused by pregnancy and decreased muscle mass.

Creatinine Testing Jaffe Reaction -

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The oldest clinical chemistry reaction still in use today. In this test, creatinine is oxidized by saturated picric acid in an alkaline solution to form creatinine picrate. Read at 510-520 nm. Not specific since it has a positive interference from any other reducing agents that react with picric acid or red coloured substances. Negative interferences include hemoglobin and bilirubin (hemolysis). Absorbance is read at 20-80 seconds. The kinetic Jaffe reaction uses the rate of change in reaction to measure creatinine levels. This method is better but still is interfered with by hemoglobin and bilirubin. Commonly used in automated procedures. The Vitros does not used the Jaffe reaction and instead uses the Creatininase method. Has very few interferences. Do not add water or handle dry picric acid to avoid exploding.

MLSC-2111U, Clinical Biochemistry Urine Creatinine -

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Creatinine levels in urine provide additional information regarding kidney function. A single random urine creatinine test is not as diagnostically strong as timed urine creatinine tests. Timed urine collection is the amount of urine that is produced by the patient over a specific collection time period.

Timed Urine Collection -

To begin, the patient is given a collection vessel for urine. Their urine is voided that next day and discarded. All other voidings afterwards are collected and documented. The container must be refrigerated. Can be measured using a graduated cylinder or by weight. If weight is used, you must account for the weight of the container by subtracting it from the total weight.

Creatinine Clearance Test -

Measures the movement of creatinine from blood to urine. The elimination of a substance as related to its removal from the plasma by the kidney. It measures the rate at which the kidneys are able to remove a filterable substance from the plasma. Can be used to indirectly measure the GFR by measuring an analyte filtered by the kidneys. Substances that work for clearance tests must be freely filtered through the glomerulus, not reabsorbed or secreted and is physiologically inert. Stability, consistency of the analyte and the availability of its test methods should also be considered. Inulin is the best substance but no tests exist for measuring it, so creatinine is used....