Non-Protein Nitrogen(NPN) Compounds

1. Non-Protein Nitrogen(NPN)Compounds

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 polypeptides • Useful clinical information is obtained from individual components of NPN fraction

3. Clinically Significant NPN • The NPN fraction comprises about 15 compounds • Majority of these compounds arise from catabolism of proteins and nucleic acids

4. Urea Nitrogen (Blood) BUN • These processes release nitrogen, which is converted to ammonia • Synthesized in the liver from CO2 and Ammonia that arises from deamination of amino acids • Highest concentration of NPN in blood • Major excretory product of protein metabolism

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 function • Dietary intake • Protein 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 syndrome • Causes of urea plasma elevations are: • Prerenal • Renal • and postrenal

8. Pre-Renal Azotemia • Reduced renal blood flow Less blood is delivered to the kidney less urea filtered • Anything that produces a decrease in functional blood volume, include: • Congestive heart failure, • shock, • hemorrhage, • dehydration • High protein diet or increased catabolism (Fever, major illness, stress)

9. Renal Azotemia • Decreased renal function causes increased blood urea due to poor excretion • Acute & Chronic renal failure • Glomerular nephritis • Tubular necrosis • & other Intrinsic renal disease

10. Post-Renal Azotemia • Obstruction of urine flow • Renal calculi • Tumors of bladder or prostate • Severe infections

11. Decreased Urea Nitrogen • Low protein dietary intake • Liver disease (lack of synthesis) • Severe vomiting and/or diarrhea (loss) • Increase protein synthesis

12. Analytical methods • Assays 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. Analytical methods • Urease → hydrolysis of urea to ammonium ion , then detect ammonium ion (NH4+) • Enzymatic • The most common method couples the urease reaction with glutamate dehydrogenase

14. Analytical methods • Indicator dye NH4+ + pH indicator → color change • Conductimetric • Conversion of unionized urea to NH4+ and CO32- results in increased conductivity Reference range of Urea N: Serum or plasma: 6-20 mg/dl 24 hours Urine: 12-20 g/day

15. Creatinine/ Creatine • Creatine is synthesized in Liver from arginine, glycine & methionine • Converted to Creatine Phosphate = high energy source for muscle tissue • Creatinine is produced as a waste product of creatine and creatine phosphate. Creatine Phosphate – phosphoric acid = Creatinine Creatine – water = Creatinine

16. Creatinine production

17. Creatinine/Creatine • Creatinine is released into circulation at stable rate proportional to muscle mass • Filtered by glomerulus • Excreted in urine • Plasma creatinine concentration is a function of: • relative muscle mass, • rate of creatine turnover • and renal function • Daily creatinine excretion is fairly stable. • It’s a very good test to evaluate renal function

18. Disease Correlations • Elevated Creatinine is found with abnormal renal function (i.e. GFR) • Measurement of creatinine concentration is used to determine: • sufficiency of kidney function • and the severity of kidney damage • and to monitor the progression of kidney disease.

19. Disease Correlations • GFR is the volume of plasma filtered (V) by the glomerulus per unit of time • GFR is used to estimate renal function • Creatinine Clearance • A measure of the amount of creatinine eliminated from the blood by the kidneys per unit time • Plasma concentration of creatinine is inversely proportional to clearance • Therefore increased plasma levels mean decreased GFR

20. Analytic Methods • Jaffe reaction • Most frequently used, was first described in 1886 Creatininereacts with picric acid in alkaline solution → red-orange chromogen • Kinetic Jaffe Reaction • Rate of change in absorbance is measured • Enzymatic Method • Using creatininase, creatinekinase, pyruvatekinase and lactate dehydrogenase

21. Analytic Methods creatininase

22. Creatine • Elevated in plasma and urine in • Muscular dystrophy, hyperthyroidism, trauma, • Plasma creatinine levels usually normal, but urinary is elevated • Specialized testing – not part of routine lab

23. Assay of creatine • Analyzing 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.

24. Uric Acid • Uric acid is a final breakdown product of purine metabolism (adenosine/guanine) in liver • Most other mammals degrade it further to allantoin • Uric acid is transported to kidney and filtered (70%) • 98% reabsorbed in PCT • Some secreted by DCT • Net amount 6-12% of filtered amount • Remaining 30% by GIT

25. Uric Acid • Present in plasma as monosodium urate • At plasma pH → relatively insoluble • Conc. > 6.8 mg/dl → plasma saturated → urate crystals may form & precipitate in tissue • Uric 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

26. Disease Correlations • Gout • Primarily in men • Onset 30-50 years • UA greater than 6.0 mg/dL • Pain & inflammation of joints by precipitation of sodium urates in tissues • Increased risk of renal calculi • hyperuricemia due to overproduction of uric acid in 25-30%

27. Disease Correlations • Increased catabolism • occurs in patients on chemotherapy for diseases such as leukemia & multiple myeloma. • Allopurinol inhibits xanthineoxidase, an enzyme in the uric acid synthesis pathway, is used to treat these patients. • Chronic renal disease • causes elevated levels of uric acid because filtration and secretion are hindered.

29. Disease Correlations • Hypouricemia • Secondary to severe liver disease • Defective renal tubular reabsorption • Fanconi’s Syndrome • Chemotherapy with 6-mercaptopurine or azathioprine – inhibit purine synthesis • Over treatment with allopurinol

30. Analytic Methods • Primary method uses enzyme uricase (urate oxidase) to convert uric acid to allantoin • Differential absorption at 293 nm • uric acid has a uv absorpance peak at 293 nm. Whereas allantoin does not • Proteins also absorb near this wavelength

31. Analytic Methods • Newer methods couple uricase with catalase or peroxidase action on hydrogen peroxide product from allantoin production • Some interferences from reducing agents Reference range: Males 0.5-7.2, Females: 2.6-6.0 mg/dl

32. Ammonia • Comes from deamination of amino acids • Digestive & bacterial enzymes in intestine • Also released from muscle during exercise • Consumed by parenchymal cells of liver and converted to urea • Free ammonia is toxic; • however, ammonia is present in the plasma in low concentrations

33. Disease Correlations • Severe liver disease • Most common cause of abnormal ammonia levels • Ammonia is not removed from circulation & not converted to urea • Elevated ammonia levels are neurotoxic and are often associated with encephalopathy.

34. Disease Correlations • Reye’s Syndrome • Most commonly seen in children • Often preceded by viral infection treated with aspirin • Severe fatty infiltration of liver • May be fatal if ammonia levels remain high • 100% survival if ammonia stays below 5x normal

35. Disease Correlations • Ammonia is of use in the diagnosis of inherited deficiencies of urea cycle enzymes • Measurement of ammonia used to diagnose and monitor treatment

36. Analytic Methods • Low concentration, volatile nature, instability, easy contamination – testing difficult • Historical Methods • Conway 1935 – volatilize, absorbed then titrated • Dowex 50 cation-exchange column + Berthelot reaction

37. Analytic Methods • Glutamate dehydrogenase • Decrease in absorbance at 340 as NADPH is consumed (oxidized) • Direct ISE • Change in pH of solution as ammonia diffuses through semi-permeable membrane • Reference Interval: Adult Plasma 19 – 60 μg / dl