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Non-Protein Nitrogen(NPN) Compounds

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Non-Protein Nitrogen(NPN) Compounds Disease Correlations Severe liver disease Most common cause of abnormal ammonia levels Ammonia is not removed from circulation ... – PowerPoint PPT presentation

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Title: 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
  • 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

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
  • lt10 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
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

14
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

15
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

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.
  • Its 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
  • 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
Analytic Methods
  • Jaffe reaction
  • Most frequently used, was first described in 1886
  • Creatinine reacts with picric acid in alkaline
    solution ? red-orange chromogen
  • 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
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
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

26
Uric Acid
  • Present in plasma as monosodium urate
  • At plasma pH ? relatively insoluble
  • Conc. gt 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

27
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

28
Disease Correlations
  • Increased catabolism
  • occurs in patients on chemotherapy for diseases
    such as leukemia multiple myeloma.
  • 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.

29
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30
Disease Correlations
  • Hypouricemia
  • Secondary to severe liver disease
  • Defective renal tubular reabsorption
  • Fanconis Syndrome
  • Chemotherapy with 6-mercaptopurine or
    azathioprine inhibit purine synthesis
  • Over treatment with allopurinol

31
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

32
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
33
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

34
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.

35
Disease Correlations
  • Reyes 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

36
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

37
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

38
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
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