NPNs Flashcards
non-protein nitrogen compounds
compounds that contain nitrogen but are not proteins
ex. urea, creatinine, creatine, amino acids, uric acid, ammonia
Urea
***of all the NPNs, urea is present in highest concentration the blood
- *Synthesized in the liver from amino groups (-NH2) & free ammonia from protein catabolism
- catalyzed by enzymes in the urea cycle
protein–> amino acids–>ammonia–> urea
urea- biochemistry
urea is the major nitrogen-containing product of protein catabolism
more than 90% of urea is excreted through the kidneys
- minor losses through GI tract
Most urea in the glomerular filtrate is excreted in urine, but some is reabsorbed in the renal tubules
patients with kidney disease will have an increased level of urea in their blood ( Azotemia)
Urea vs BUN
historically, whole blood samples were used to measure nitrogen content & testing was called Blood Urea Nitrogen (BUN)
this is now an obsolete term, although it is still used in the United States
- Urea Nitrogen is a more appropriate term
( bc we use plasma not whole blood)
To convert between BUN & Urea :
BUNx 2.14 = Urea
Urea/2.14= BUN
Urea - specimen requirements
Can be measured on serum, plasma or urine
if plasma is collected:
- avoid ammonium ions
-do not use sodium citrate ( blue top) or sodium fluoride ( grey top) tubes
- citrate & fluoride inhibit urease ( enzyme we use
in measurement of urea)
avoid hemolysis
specimen are susceptible to bacterial decomposition ( particularly urine)
- refrigerate specimen if not analyzed within a few
hours ( 24hrs)
- evaluate renal function w/urea
Urea- Analytical Methods
enzymatic ( urease)
Electrode
Isotope Dilution Mass Spectrometry (IDMS)
-Reference Method
Urea- Coupled Enzymatic Assay
urea is hydrolyzed with urease to produce ammonium ions (NH4+)
-ammonium ions ( what we quantify @ the end)
Urease Urea + 2H2O ------> 2NH4^+ + CO3^2-
GLDH NH4^+ +2-oxoglutarate ------->gluatamate + H2O
NADH + H+ NAD+
a decrease in absorbance of NAD+ is measured at 340nm
Can be measured as an endpoint or kinetic reaction
** more common on automated analyzers
Urea- Colorimetric method
**Urea in an acidic medium condenses with diacetly monoxime at 100 degrees to form a red colored complex
urea + diacetyl monoxime——> red colored complex
the intensity of color formed is directly proportional to the amount of urea present in the sample
Urea- Berthelot Method
Urease hydrolyses urea to ammonia & CO2
urease
Urea + H2O ———-> ammonia + CO2
ammonia reacts with a phenolic chromagen & hypochlorite to form a green colored complex
ammonia + Phenolic chromagen + hypochlorite (green colored complex )
the intensity of color produced is proportional to the amounnt of urea in the sample
Electrochemical Analysis of Urea
Urea is hydrolyzed by urease to ammoinum ions (NH4+) ***common step
2 methods to measure NH4+
1. Conductimetric:
measures the rate of change of conductivity ( inc. conductivity ) as the ion is formed
- Potentiometric :
used in some point of care devices
uses an ammonium ion-selective electrode with a membrane containing immobilized urease
Urea - reference ranges
plasma/serum : 2.1-7.1 mmol/L ( based on normal protein intake )
Urine : 12-20 g/day or 430-710 mmol/day
Urea levels gradually increase with age & are typically higher in men than women ( bc of muscle mass)
Clinical significance of urea
increase in urea in the blood is called Azotemia **
Very high levels with renal failure us called uremia or uremic syndrome ***( dialysis or transplant required)
Azotemia is classified into 3 main categories based on cause:
prerenal azotemia
renal azotemia
postrenal azotemia
Prerenal Azotemia
caused by : Decreased renal blood flow - congestive heart failure - shock -hemorrhage - dehydration ( less blood is delivered to the kidney & therefore, less urea is filtered) **
Increased protein catabolism
- stress, fever, major illness
- corticosteroid therapy
- GI hemorrhage
Renal Azotemia
caused by decreased renal function & compromised renal excretion of urea
- acute or chronic renal failure
- Glomerulornephritis
- Tubular necrosis
very high plasma urea along with renal failure = uremia or uremic syndrome
- can be fatal if not treated by dialysis or kidney transplant
Postrenal Azotemia
caused by an obstruction of urine flow anywhere in the urinary tract
- renal calculi
- bladder or prostate tumors
- severe infection
decreased urea
low protein diet
severe liver disease
late pregnancy or infancy
severe vomiting & diarrhea
Urea nitrogen /creatinine ratio
ratio can help differentiate the cause of abnormal urea concentrations
***normal ratio 10:1 to 20:1
prerenal conditions :
elevated urea; normal creatinine - causes high ration
renal conditions :
both urea & creatinine elevated - normal ratio
postrenal conditions:
both elevated, urea elevated more - high ratio
low ratio is associated with low protein intake, acute tubular necrosis & severe liver disease
urea as a diagnostic tool
blood urea can vary greatly with non- renal factors
not a good independent indicator of renal function
mostly used together with creatinine determination
Creatine
**creatine is synthesized in the kidney, liver & pancreas from amino acids
**after synthesis, it is transported in the blood to other organs such as muscle & brain
**in muscle, creatine is phosphorylated to phosphocreatine, a high-energy compound
a small proportion of creatine spontaneously & irreversibly converts to creatinine ( its anhydride waste product)
Creatinine
creatinine is a spontaneous decomposition product of creatine
the amount of creatinine produced is relatively constant & related to muscle mass
it is revived firm plasma via glomerular filtration & excreted in the urine
Creatinine - specimen requirements
can be measured on serum, plasma or urine
hemolyzed & icteric samples should be avoided
high protein ingestion may temporarily elevate serum concentrationa
serum & urine creatinine is stable for 7 days at 4 degrees ( or freeze)
creatinine - analytical methods
can be measured using:
chemical methods based on Jaffe reaction
enzymatic methods
- **isotope dilution mass spectrometry
- reference method
Jaffe reaction for creatinine
creatinine reacts with picric acid in an alkaline solution to form a red-orange chromogen
NaOH
creatinine + picric acid——–> Creatinine picrate
main disadvantage: not specific for creatinine many non- creatinine Jaffe- like chromagens interfere - ascorbic acid -glucose -ketone bodies - protein -cephalsporins ( antibiotics ) -pyruvate
Approaches to increase specificity of Jaffe Reaction
increased accuracy can be obtained by using a protein- free filtrate absorbed onto Fuller’s earth ( aluminum magnesium silicate) or Lloyd’s reagent ( sodium aluminum silicate )
Increased specificity can be obtained by using:
- a kinetic method ( Jaffe method) ***
- an enzyme method ( Jaffe Method)
Creatinine - kinetic methods
2 types of non creatinine chromogens hsve been identified in rate reactions
- those that interfere within the first 20 seconds have been identified in rate reactions
- those that interfere 80-100 seconds after mixing
the window between 20-80 seconds will be more specific for creatinine
- picrate reaction
used with automated instruments
Creatinine - Enzymatic methods
coupled enzymatic reactions involving : creatininase creatinase sarcosine oxidase peroxidase
adapted to use on dry-slide analyzers ( like vitros)
Sources of error
at temps over 30 degrees the following can interfere: ascorbate glucose ⍺-ketoacids uric acid
bilirubin causes a negative bias
patients taking cephalosporin antibiotics may have falsely elevated results ( creatinine )
Creatinine - Reference Ranges
serum/plasma : 50-110 µmol/L
urine 5-18 mmol/day
men will have higher creatinine levels than women (due to increased muscle mass)
creatinine - clinical significance
elevated serum creatinine is associated with abnormal renal function
if serum creatinine is inc. glomerular filtration rate (GFR) is decreased.
increased with :
abnormal renal function
normal with:
muscle diseases
insensitive marker
- may not be noticably increased until kidney function is <50%
Creatine- clinical significance
increased in : muscle diseases - muscle dystrophy - poliomyelitis - hyperthyroidism -trauma
not increased in :
renal disease
we measure CK –> creatine kinase ( bc there is no good method to measure creatine directly )
Kidney function test
serum/plasma urea
serum/plasma creatinine
creatinine clearance test
glomerular filtration rate
creatinine has the advantage over urea in that it is not affected by protein intake
creatinine clearance is the best test fro kidney/glomerular function
Glomerular filtration rate (GFR)
a reliable measure of functional capacity of the kidneys
indicates the # of functioning nephrons
a decrease in GFR precedes all forms of progressive kidney disease
measuring GFR is useful to : target treatment monitor progression predict when renal replacement therapy (RRT) will be needed - dialysis, transplants
Glomerular filtration rate
GFR= [Us] x V
————
[Ps]
Us= urinary concentration pf the substance V= volumetric flow rate of urine in mL/min Ps= plasma concentration of the substance
labs are encouraged to report an estimated GFR (eGFR) when serum creatinine is ordered to increase identification of kidney disease
eGFR accounts for a patients body surface area, age, gender & ethnicity
Creatinine clearance
renal clearance is “ the volume of plasma from which the substance is completely cleared by the kidneys per unit of time “
clearance depends on:
- the net result of glomerular filtration & tubular reabsorption & secretion
- is proportional to a BSA( body surface area)of 1.73 m^2
creatinine :
- eliminated mainly by glomerular filtration
- only a small amount is reabsorbed by the tubules
- creatinine clearance is a good measure of glomerular filtration
Creatinine clearance - specimen
precisely timed urine specimen
- 24 hr is most common
serum or plasma sample collected within 24 hrs of urine sample ***
creatinine is measured on both urine & serum
creatinine clearance -calculation
creatinine clearance = UV/P x 1.73/A
U = urine creatinine (µmol/L)
V= urine volume ( mL/s or mL/min)
P= plasma creatinine (µmol/L)
A= body surface area ( m^2)
- height & weight on requisition neededCreatinine clearance - sources of error
incorrect timing of urine collection
loss of urine during collection
vigorous excercise
patient must be properly hydrated
- improves accuracy
- eliminates retention of urine in the bladder as a source of energy
Creatinine clearance - reference ranges
75-125 mL/min
1.24-2.08 mL/s
plasma creatinine level is inversely related to creatinine clearance
if patient has an incr. plasma creatinine, they will have a decr. creatinine clearance
