1. Non-Protein Nitrogen(NPN) Compounds
Lecture 7

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
Highest concentration of NPN in blood
Major excretory product of protein metabolism
These processes release nitrogen, which is converted to ammonia
Synthesized in the liver from CO2 and Ammonia that arises from deamination of amino acids
Organisms 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 function
Dietary intake
Protein catabolism rate
Some urea is reabsorbed by passive diffusion during passage of the filtrate through the renal tubules.

6. Clinical Application Measurement of urea is used to:
Evaluate renal function,
Assess hydration status,
The amount of urea reabsorbed depends on urine flow rate and extent of hydration
Determine nitrogen balance,
Aid in the diagnosis of renal disease,
And to verify adequacy of dialysis.
In dehydration there is increased reabsorption of urea by the kidneys urea transporter UT-A1 in the inner medullary collecting duct is directly sensitive to vasopressin and upregulates its expression 

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
caused by a lack of oxygen to the kidney tissues (ischemia of the kidneys).
& other Intrinsic renal disease
Glomerulonephritis, 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 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. 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+)
Enzymatic
The most common method couples the urease reaction with glutamate dehydrogenase
NADH ( a common co-enzyme ) ( the reduced form ) absorbs light at 340 NM but not NAD
Glutamate dehydrogenase = GLDH

15. NH4+ + pH indicator → color change
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: mg/dl
24 hours Urine: g/day :

16. 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

17. Creatinine production

18. 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

19. 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.

20. Disease Correlations
GFR is the volume of plasma filtered (V) by the glomerulus per unit of time (t) [GFR=V/t]
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

21. Creatinine reacts with picric acid in alkaline solution →
Analytic Methods
Jaffe reaction
Most frequently used, was first described in 1886
Creatinine reacts with picric acid in alkaline solution →
red-orange chromogen
Glucose, -ketoacids, and uric acid may increase creatinine concentration measured by the Jaffe reaction
Kinetic Jaffe Reaction
Rate of change in absorbance is measured
Enzymatic Method
Using creatininase, creatine kinase, pyruvate kinase and lactate dehydrogenase

22. Analytic Methods creatininase Phosphoenolpyruvate= PEP

23. 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

24. 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.

25. BUN-to-Creatinine ratio
Test
Units
BUN (Urea)
7–20 mg/dL
Urea
20-40 mg/dL
Creatinine
mg/dL
The principle behind this ratio is the fact that both urea (BUN) and creatinine are freely filtered by the glomerulus, however urea reabsorbed by the tubules can be regulated (increased or decreased) whereas creatinine reabsorption remains the same (minimal reabsorption).

26. 7–20 mg/dL
mg/dL

27. The most likely cause is chronic renal disease
The most likely cause is chronic renal disease. Supporting data are the essentially BUN/creatinine ratio and the significant elevation of all nonprotein nitrogen (NPN) values.
There was no significant improvement when cardiac function improved, further eliminating congestive heart failure as a cause of elevated BUN.

28. If the levels of acetone and other α-ketoacids were elevated, as might be found in diabetes, the patient’s elevated creatinine levels could have been an erroneous result. α- Ketoacids cause a positive bias when creatinine is measured by a kinetic Jaffe reaction, the most commonly used assay method. However, the normal glucose level and abnormal values for other NPN substances make this unlikely.

29. 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 (Renal excretion accounts for about 70% of uric acid elimination)
98% reabsorbed in proximal convoluted tubule (PCT)
Some secreted by distal convoluted tubule (DCT)
Net amount 6-12% of filtered amount
Remaining 30% by GIT
PCT= proximal convoluted tubule
DCT= distal

30. 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

31. 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%
Hyperuricemia is typically asymptomatic and does not lead to gout, but gout is preceded by hyperuricemia

32. Disease Correlations Increased catabolism Chronic renal disease
Occurs in patients on chemotherapy for diseases such as leukemia & multiple myeloma.
Treatment: Allopurinol inhibits xanthine oxidase, 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.

34. Disease Correlations Hypouricemia Secondary to severe liver disease
Defective renal tubular reabsorption
Fanconi’s Syndrome (proximal tubular function of the kidney is impaired)
Chemotherapy with 6-mercaptopurine or azathioprine – inhibit purine synthesis
Over treatment with allopurinol
Fanconi Syndrome (also known as Fanconi's syndrome) is a disorder in which the proximal tubular function of the kidney is impaired,[1] resulting in decreased reabsorption of electrolytes and nutrients back into the bloodstream

35. 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
uric acid has a uv absorpance peak at 293 nm. Whereas allantoin does not

36. Reference range: Males 0.5-7.2, Females: 2.6-6.0 mg/dl
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 , Females: mg/dl

37. 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

38. 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.

39. 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
Reye'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

40. 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

41. Analytic Methods
Low concentration, volatile nature, instability, easy contamination – testing difficult
Historical Methods
Conway 1935 – volatilize, absorbed then titrated
Dowex-50 (cation exchanger, sodium form).
Berthelot reaction NH3 + NaOCl → H2NCl (chloramine) + NaOH
[Fe(CN)5H2O]-3 + H2NCl → ‘Complex’
‘Complex’ + Phenol _OH→ Colored indophenol blue (560 nm)

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

44. occurring within or administered into the spinal theca.

45. Increased uric acid is a result of the significant increase in nuclear breakdown in the presence of a high WBC. The increase is not from renal disease, because BUN and creatinine are normal.
Chemotherapy has reduced the WBC to below normal levels, and the patient is taking allopurinol.

46. It is probably due to decreased intake (patient is unable to eat); a determination of total serum protein and albumin would be helpful.