NON-PROTEIN NITROGEN

COMPOUNDS
Prepared by: Winona Mei A. Reyes, RMT
 OBJECTIVES:
1. List the nonprotein nitrogen components of the blood
2. Describe the biosynthesis and excretion of urea, uric acid, creatinine, creatine, and
ammonia
3. Describe the major pathological conditions associated with increase and decrease
plasma concentrations of each NPN.
4. State the specimen collection, transport, and storage requirements necessary for
determinations of NPN
5. Discuss methods and interferences used to determine NPN
6. Describe the toxic effects of increased ammonia concentration
7. Suggest possible clinical conditions associated with test results
 NONPROTEIN NITROGEN
COMPOUNDS (NPN)
• Consists of nitrogen, but are not proteins
• Measurement has been used to monitor renal function
• Originated when methodology required removal of protein
before analysis
 NONPROTEIN NITROGEN
COMPOUNDS
1. Urea  45-50%
2. Amino acids  20%
3. Uric acid  20%
4. Creatinine  5%
5. Creatine  1-2%
6. Ammonia  0.5%
PROTEIN STRUCTURE
METABOLISM OF PROTEIN
ORNITHINE OR KREB’S HENSELEIT
CYCLE
 AMMONIA
• From deamination of amino acids

• Consumed by parenchymal cells of liver to produce urea

• Neurotoxic

• Monitors progress of severe clinical conditions

 AMMONIA - VALUES

Plasma: 19-60 microgram/dL

Urine 24hr: 140 – 1500 mg N/day
 AMMONIA – DISEASE CONDITIONS
• Liver failure
• Reye’s syndrome
children, viral infection, aspirin
• Deficiencies of urea cycle enzymes
 AMMONIA – SPECIMEN
CONSIDERATIONS
• EDTA or heparin
• ICED
• Stable for 3-5 hours on ice bath
• Affected by smoking
 AMMONIA – ANALYTICAL MTDS
1. Colorimetric mtd
2. Coupled enzyme  GLDH  glutamate+NADP (340 nm)
3. Conway  titrated
4. Spectrophotometric  bromphenol blue
5. Ion selective  potentiometry
6. Ion Exchange  Dowex 50 resin
 AMMONIA – ANALYTICAL MTDS

Colorimetric methods:
• Nessler’s
• Berthelot’s
• Highest plasma concentration among other NPN UREA
• Major excretory product of protein metabolism
• Synthesized in the liver from ammonia and CO2
• Ornithine or Kreb’s Henseleit cycle
• Readily filtered by glomerulus
• Governed by renal fxn, protein content of diet, and protein
catabolism
• First metabolite to rise in kidney diseases
• Good indicator of nitrogen intake
 UREA

• 25g is excreted, in stable nitrogen balance

• Expressed as BUN
• BUN x 2.14 = urea (mg%)
• BUN:Creatinine; 10:1 – 20:1
 UREA - VALUES

• BUN (serum/plasma) = 6 – 20 mg/dL

• BUN x 2.14 = urea
• Conversion factor = 0.357
 UREA – DISEASE CONDITIONS

Increased?
1. Pre-renal
2. Renal
3. Post-renal

Decreased?
 1. PRE-RENAL CONDITIONS

a. Decreased blood flow to kidneys

• CHF
• Shock
• Dehydration
b. Level of protein metabolism
c. Increased protein catabolism
• Fever
• Burns
• Stress
•
2. RENAL CONDITIONS
 3. POST-RENAL CONDITIONS
• Obstruction, urinary tract disease
 DECREASED UREA PLASMA
• Decreased protein intake
• Poor nutrition
• Overhydration
• Severe liver disease
• Increased protein synthesis  pregnancy, infancy
• Impaired absorption  celiac disease
 UREA – SPECIMEN
CONSIDERATIONS
• Non hemolyzed serum, plasma, urine
• Urine should be refrigerated or added with thymol
• Freeze samples if not processed right away
• Na Fluoride and Na citrate will inhibit urease
• Thiosemicarbazide and Fe+++ will enhance color dev.
 UREA – ANALYTICAL METHODS

1. Chemical or Direct or nonspecific

DAM
2. Enzymatic or Indirect or specific
Urease
GLDH
3. Colorimetric assay
Nessler’s
Berthelot’s
4. Isotope Dilution Mass Spectrometry (IDMS)
 UREA – ANALYTICAL MTDS

1. Chemical (Direct, Non specific, Fearon’s rxn)

DAM + H20  diacetyl + HONH2

Urea + diacteyl  diazine derivative (YELLOW) + 2H20

• Thiosemecarbazide and Fe+++ will stabilize color

 UREA – ANALYTICAL MTDS
2. Enzymatic (Indirect, kinetic, more specific)

a) UREASE
- jack beans

b) GLDH
- UV enzymatic method
- measures disappearance of NAD (reduced, NADH) @340nm
 UREA – ANALYTICAL MTDS
3. Colorimetric (Non specific, Indirect)

Nessler’s
NH3 + HgI2KI  iodide salt of mercury and potassium; orange

Berthelot’s
NH4 + 5NaOCl + 2phenol  (NaOH or Na nitroprusside)
indophenol blue +5NaCl + 5 H20
 UREA – ANALYTICAL MTDS

4. Isotope Dilution Mass Spectrometry (IDMS)

definitive method
reference method
 CREATINE
• From arginine, glycine, methionine
• Produced by liver and pancreas
 CREATINE
Increased in?
• Muscular dystrophy
• Poliomyelitis
• Hyperthyroidism
• Trauma

Plasma creatine concentration is not elevated in renal disease

 CREATININE
• Waste product of creatine
(insert equation here)
• Produced by 3 amino acids: methionine, arginine, lysine
• Released in to the circulation at a constant rate that is
proportional to the muscle mass
• Readily filtered, not reabsorbed
• Governed by muscle mass, rate of creatinine turnover, and renal
fxn
 CREATININE
Renal marker:

• More specific
• Insensitive and not measured until renal has deteriorated more than
50%
• Index of overall function
 CREATININE - VALUES

Male: 0.9 – 1.3 mg/dL

80-115 micromole/L

Female: 0.6 – 1.1 mg/dL

53-97 micromole/L
 CREATININE – DISEASE
CONDITIONS
Increased?
• Impaired renal function
• Chronic nephritis
• CHF

Decreased?
• Decreased muscle mass
• Liver disease
• Pregnancy
•
 CREATININE – SPECIMEN
CONSIDERATIONS
• Avoid hemolyzed and icteric samples
• 24hr urine specimen collection; creatinine <0.8 g/day 
• Urine samples should be maintained at 7.0 pH
• Cephalosporin may cause false increased results in Jaffe
reaction
 CREATININE – ANALYTICAL MTDS
1. Jaffe chemical
methods
2. Jaffe with adsorbent
3. Kinetic Jaffe
enzymatic
4. Creatininase-CK methods
5. Creatininase-H2O2

6. IDMS
 CREATININE – ANALYTICAL MTDS
CHEMICAL METHOD
Alkaline picrate
Red orange tautomer

ENZYMATIC METHOD
more specific than Jaffe
Requires large volume specimen
Pre-incubate sample
Creatininase also known as “creatinine aminohydrolase”
 CREATININE – ANALYTICAL MTDS
1. Jaffe reaction

Creatine + NaOH,picric acid  red orange tautomer of creatinine

picrate

Absorbs at 520 nm
F(I): ascorbate, glucose, uric acid, cephalosporins
F(D): bilirubin, hemoglobin
 CREATININE – ANALYTICAL MTDS

2. Jaffe with adsorbent

sensitive and specific

Lloyd’s reagent – sodium aluminum silicate

Fuller’s earth – aluminum magnesium silicate

Removes interferences by isolating creatinine from

noncreatinine chromogen
 CREATININE – ANALYTICAL MTDS

3. Kinetic Jaffe
Janovsky like reaction
automatic, inexpensive, rapid, easy

serum + alkaline picrate

rate of change in absorbance is measured
 CREATININE – ANALYTICAL MTDS

4. Creatininase – Creatine Kinase

Requires a large volume of pre-incubated sample

 CREATININE – ANALYTICAL MTDS
5. Creatininase – Hydrogen Peroxide

Potential to replace Jaffe

1) Creatinine  creatine
2) Creatine + H20  sarcosine + urea
3) Sarcosine + O2  glycine, formaldehyde, H202
4) H202 + indicator  oxidized indicator + 2H202
 CREATININE – ANALYTICAL MTDS

6. Isotope Dilution Mass Spectrometry

IDMS
reference method
 CREATININE CLEARANCE

C (ml/min) = UV/P x 1.73

m2/BSA
 CREATININE CLEARANCE

• Males: 105 +- 20 ml/min/1.73 m2

• Females: 95 +- 20 ml/min/1.73 m2
 ACUTE KIDNEY INJURY
• Used to classify Acute Kidney Injury (AKI)
• Functional or structural abnormalities or markers of kidney damage
including abnormalities in blood, urine, or tissue tests or imaging
studies present for less than 3 months
• AKI = retention of metabolic waste products that are usually
excreted
• Anuria, oliguria, normal, increased urine volume
BUN : CREATININE
10-20 : 1
 AFFECTE
METABOLI AFFECTE
SM BUN:CRE D BY
RENAL D BY
PRODUCT A RATIO MUSCLE
OF? DIET?
MASS
FIRST TO
UREA Protein 10-20 YES NO
RISE
MORE
SPECIFIC
CREATININ
E Muscle , 1 NO YES
OVERAL
L INDEX
 DISEASE CORRELATION
• Azotemia: increase of NPN in blood, particularly urea and
creatinine

• Uremia: increase of urea in blood with renal failure

acidemia, K+ elevation, anemia, burr cells +, uremic
frost, foul breath, urine like sweat
 AZOTEMIA

a. PRE-RENAL
b. AZOTEMIA
c. POST-RENAL

Pg. 104, Rodriguez

 URIC ACID
• Major product of purine (adenine, guanine) catabolism

• From xanthine by xanthine oxidase in liver and intestine

• Weak acid

• pH 7.4 = monosodium urate

• pH <5-7.5 = uric acid
 URIC ACID - VALUES

Male = 3.5 – 7.2 mg/dL

0.21 – 0.43 mmol/L

Female = 2.6 – 6.0 mg/dL

0.16 – 0.36 mmol/L

>7 mg/dL = gout, monosodium urate

>10 mg/dL = renal calculi
 URIC ACID – DISEASE CONDITIONS
Hyperuricemia
1. Gout
2. Increased nuclear metabolism
3. Chronic renal disease
4. Lesch-Nyhan syndrome – hypoxanthone guanine phosphoribosyltransferase

Hypouricemia
5. Fanconi’s syndrome
6. Wilson’s disease
7. Hodgkin’s disease
 URIC ACID – SPECIMEN
CONSIDERATIONS
• Fasting sample is preferred
• Free from hemolysis and lipemia
• Stable in serum and urine for 3 days
• K+ oxalate and fluoride can not be used
• Heparin  susceptible to destruction be bacteria and thymol
• Ascorbic acid and bilirubin = interferences
 URIC ACID – ANALYTICAL MTDS

1. Chemical/Colorimetric
2. Enzymatic - Uricase
3. IDMS

all methods rely that uric acid is oxidized into allantoin,

which acts as a reducing agent
 URIC ACID – ANALYTICAL MTDS

1. Chemical/Colorimetric
Caraway method

uric acid + phosphotungstic acid  Tungsten Blue

+ Allantoin + CO2

Provides alkaline pH which is

Na cyanine (NaCN)
needed for color development;
Na carbonate (Na2CO3) inhibits reducing substances
 URIC ACID – ANALYTICAL MTDS

1. Chemical/Colorimetric

Intereferences:
turbidity, aspirin, acetaminophen, caffeine, theophylline
 URIC ACID – ANALYTICAL MTDS

2. Enzymatic
uses URICASE
specific
UV absorbance at 293 nm

Coupled enzymes: catalase, peroxidase, ethanol

 URIC ACID – ANALYTICAL MTDS

REMEMBER!!!

Allantoin has no absorbance at 293 nm, only uric acid

Uric acid concentration is inversely proportional to the

absorbance
 URIC ACID – ANALYTICAL MTDS
• Isotope Dilution Mass Spectrometry

IDMS
reference method
 METABOLISM OF…
Urea Protein
Ammonia Protein
Uric Acid Purine
Creatinine Muscle/creatine
Arginine, Glycine,
Creatine
Methionine