Non-Protein Nitrogen(NPN) Compounds Lecture 7

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

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

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

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.

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 

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

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)

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

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

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

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

Conversion of BUN to urea Atomic mass of nitrogen = 14 g/mol; Molecular mass of urea = 60.06 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 0.466 = 21.46 mg/dL of urea

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

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: 6-20 mg/dl 24 hours Urine: 12-20 g/day :

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

Creatinine production

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

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.

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

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

Analytic Methods creatininase Phosphoenolpyruvate= PEP

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

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.

BUN-to-Creatinine ratio Test Units BUN (Urea) 7–20 mg/dL Urea 20-40 mg/dL Creatinine 0.7-1.2 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).

7–20 mg/dL 0.7-1.2 mg/dL

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.

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.

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

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

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

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.

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

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

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 0.5-7.2, Females: 2.6-6.0 mg/dl

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

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.

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

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

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)

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

occurring within or administered into the spinal theca.

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.

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