Transcript Non-protein Nitrogen Compounds

Non-Protein
Nitrogen(NPN)
Compounds
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

2
Clinically Significant NPN
The NPN fraction comprises about 15
compounds
 Majority of these compounds arise from
catabolism of proteins and nucleic acids

3
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
4
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
5
Clinical Application
 Measurement of urea
◦ Evaluate renal function,
◦ Assess hydration status,
is used to:
 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.
6
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
7
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)
8
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
9
Post-Renal Azotemia
 Obstruction
of urine flow
◦ Renal calculi
 Tumors
of bladder or prostate
 Severe infections
10
Decreased Urea Nitrogen
 Low
protein dietary intake
 Liver disease (lack of synthesis)
 Severe vomiting and/or diarrhea (loss)
 Increase protein synthesis
11
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

12
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

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 (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
20
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

Enzymatic Method
◦ Rate of change in absorbance is measured
◦ Using creatininase, creatine kinase, pyruvate kinase and
lactate dehydrogenase
21
Analytic Methods
creatininase
Phosphoenolpyruvate= PEP
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
BUN-to-Creatinine ratio
Test
Units
BUN (Urea)
7–20 mg/dL
Urea
20-40 mg/dL
Creatinine
0.7-1.2 mg/dL
25
26


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.
27

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.
28
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 PCT
 Some secreted by DCT
 Net amount 6-12% of filtered amount

Remaining 30% by GIT
29
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
30
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%
31
Disease Correlations
Increased catabolism

◦ 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.
32
33
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
34
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
35
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
36
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
37
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.
38
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
39
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

40
Analytic Methods
Low concentration, volatile nature, instability,
easy contamination – testing difficult
 Historical Methods

◦ Conway 1935 – volatilize, absorbed then titrated
41
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
 Measured potentiometrically

Reference Interval: Adult Plasma 19 – 60 μg / dl
42
43

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.
44

It is probably due to decreased intake (patient is unable to
eat); a determination of total serum protein and albumin
would be helpful.
45