Non Protein Nitrogen compound

• NPN is more than 500 type the most important is

urea , creatinine ,uric acid, ammonia.
Estimation of NPN:-
 Nessler:-
Depend on converted nitrogen to ammonia
and subsequent reaction with nessler reagent
to produce yellow color .
Urea
 BUN
 urea is end product of protein metabolism ,it
formed in liver from ammonia and carbon dioxide .
 it carried in blood to kidney and filtrate by
glomerulus and excreted in urine small amount
<10%> excreted by GID and skin .
 Amount of urea is reabsorbed by passive diffusion
during passage of filtrate through renal tubules.
urea structure
Urea cycle
 Clinical application
 Evaluate renal function
 Asses hydration status
 Determine nitrogen balance
 Verify adequacy of dialysis
 pathological
• Increase concentration level in blood called azotemia
• Elevated the concentration accompanied by renal
failure called uremia
Increased concentration due to;-
1. Pre renal:-
 Congestive heart failure
 Shock , hemorrhage ,dehydration
 Increase protein catabolism
 high protein diet
2.Renal:-
 acute and chronic renal failure
 Glomerular nephritis
 Nephritic syndrome
 Tubular necrosis
3.Post renal:-
 Urinary tract obstruction
Decreased concentration :-
 Low protein intake
 Sever vomiting and diarrhea
 Liver disease
 pregnancy
 Estimation
 Sample:
Urine, serum ,plasma.
 Avoid:
Sodium citrate and sodium fluoride
anticoagulant
Avoid delayed of sample analyzed especial
urine sample
 Urea/ Creatinine ratio
• Normal:
 10:1 to20:1
• Abnormal
 Elevated urea +normal creatinine
Pre renal
 Elevated urea+ elevated creatinine
Post renal
 Low urea/ creatinine ratio
 BUN
• Amount of nitrogen in form of urea
• convert BUN to urea in mg/ dL by using
following formula:
• Urea [mg/dL]= BUN [mg/ dL] × 2.14
• factor derived by: MW of urea = 60, MW of
urea nitrogen = 14x2 => 60/28 = 2.14)
• Normal range of BUN=7-18mg/dl
 1)Chemical method
1-DAM method
Urea react with diacetyle monoxime at
High temperature, acid medium, and presence
of thiosemicarbazide
 Advantage:
estimate urea directly
 Disadvantage:
use strong acid and high temperature
Interference with protein and creatinine
2- o-phthaldehyde:-
 urea + o-phthaldehyde + H⁺ → isoindoline

 isoindoline+N(1naphthyl) ethylenediamine+H⁺
→ colored
2)Enzymatic method
• Same first step in all enzymatic method:
• By urease enzyme
urea +2H₂O→ ammonia
i. Method according to Berthelot
ammonia+ hypochlorite + phenol+ sodium
nitroprusside → indophenol
Reagent use:-
• Reagent 1:
Phenol + NaoH+ urease
• Reagent 2:
Hypo chloride +Nitro prusside
 Disadvantage:
• the method is rarely used, because it is quite
time-consuming and inadequately specific.

Advantage:
Not need deprotinization
 ii. GLDH coupled enzymatic;-
 ammonia + a-oxoglutaric acid + NADH →
glutamic acid+ NAD⁺
• The absorbance decrease due to NADH
reduction is proportional to the concentration
of urea
Advantage
it is rapid and very specific.
 3)Another method
 Conductimetric method
 Indicator dye
 H₂O₂ coupled
 Isotope dilution mass spectrometry
 Normal range:
In serum:
 10-50mg/dl
In urine<24h>
 12-20g/day
Creatinine & creatine
Creatinine :-
IS one of non protein nitrogenous substances
( wast product )formed from creatine &
creatine phosphate in muscle .

Creatine :-
Is one of non protein nitrogenous substances
formed from amino acids arginine , glycine &
methionine in the liver .
 biosynthesis of creatine
 Creatine is synthesized primary in the liver
(endogenous source) from amino acid arginine
,glycin and methionine
 in present of arginine glycin amidinotransferase
arginine + glycin → guanidinoacetate
 In present of guanidinoacetate N.methyltransferar
guanidinoacetate →creatine
both enzyme found in the liver and kidney.
Also Found exogenous source from diet mainly
meat
 biosynthesis of creatinine
 Creatine come from the liver and
transport mainly through circulation to
muscle with Creatine kinase convert to
creatine phosphate as battery that store the
energy .
 creatine phosphate loss phosphoric acid and
creatine loss water to form cyclic compound
,creatinine which diffuse in to the plasma at
relatively constant rate that proportional to
an individuals muscle mass and excreted in
the urine
small amount of creatinine are secreted by
PCT and reabsorbed by renal tubule.
 Pathophysiology
 Creatinine
Increasing in:
 Abnormal renal function
 Temporarily as result of muscle injuring
 Increase muscle catabolism
 Diabetic ketoacidosis
• Decrease :
Are not common
 they can seen with condition that result in decrease
muscle mass
 Slightly lower seen during pregnancy and with ageing
Creatine:
• Elevate in muscle diseases
• Plasma creatine is not elevate in renal disease
 analytical method
• specimen require :
creatinine may be measured in plasma,
serum ,or urine....
• Interfering substance:
ascorbate , glucose , a. ketoacid….
creatinine:-
A. chemical method:
base on jaffe reaction

1.classical jaffe method

-principle
creatinine reacts with picric acid in alkaline solution to form
a red-orange chromogen can be read
spectrophotometrically(520nm)
-disadvantage
1.posative interference by large number of compound
include …
• 2.modification of classical jaffe reaction:

i. follin and Wu method:

-principle
10%Na2wo4, 2/3H2SO4
-disadvantage
non specific ,subject to positive bias from ....
jaffe with adsorbent
principle
 ii- jaffe with adsorbent
principle
• Creatinine (in a protein free filtrate is adsorbed on fuller’s
earth (aluminum magnesium silicate) or Lloyd’s reagent
(sodium aluminum silicate) then eluted and reacted with
alkaline picrate to form colored complex can measure by SPM

• o
• Advantage :
More accurate results &adsorbent improve
specificity .
Disadvantage
-time consuming
-not readily automated
-not used routinely
iii. Kinetic jaffe method:
-Principle
Serum is mixed with alkaline picrate & the rate of change in
absorbance is measured between to points.
-Advantages:
-rapid, inexpensive .
-Easily to perform
-Eliminate some of non specific reactions
-:Disadvantage
-Subject to interference by a.keto acid & cephalosporine .
-bilirubin & Hb may cause(-ve) bias.
iv-Acidification
V-Dialysis
 Enzymatic methods
1- creatininase –CK :
Principle:
Creatinine + H₂O +creatinase →creatine
Creatine + ATP +CK →creatine phosphate + ADP
PEP + ADP +PK→ pyruvate + ATP
Pyruvate NADH + H⁺+LD →Lactate +NAD⁺
2- creatinase - H₂O₂
Principle
Creatinine + H₂O₂ + creatininase →creatine
Creatine+ H₂O₂ +creatininase →sarcosine +Urea
Sarcosine +O₂+H₂O₂+ sarcosine oxidase →
glycine + CH₂O+ H₂O₂
H₂O₂+ phonel+4-aminophenazone+ peroxidase =
→colored product +H₂O
 Other methods

1- 3.5dinitrobezonic acid method

2- IDMS
3- HPLC.
Creatine:-
1-end point jaffe method
2- coupled enzyme (creatininase CK)
3-HPLC
 Normal range
• Adult male:0.9-1.3 mg/dl
• Adult female: 0.6-1.1 mg/dl
• Child:0.3-0.7mgldl
 24h urine:
• Adult male:800-2000mg/dl
• Adult female:600-1800mg/dl
 GFR
• Volume of plasma filtrate from renal
glomeular capillary in to the Bowman capsule
per unit (ml/min)
• GFR=
urine con (mg/dl )×urine volume (ml)
plasma con (mg/dl)× time (min)
 Important of GFR
• 1-assessment of renal function
• 2-grading of chronic renal insufficiency
• Dosage of drugs that are excreted primarily
via urine also based on GFR
 Can calculate by measure any chemical substance
which has certain characteristics :
1- should be filtered freely in the glomeruli neither
reabsorbed nor secreted by renal tubular
2- not metabolized or store in the body
3- not bind with plasma protein
4-not toxic
5- not affect by died
6-produce by body
 factor affecting GFR
1-surface area( number of glomeruli)
2-hydrostatic pressure of glomeruli
3-intracellular pressure
4.Plasma protein osmotic pressure
 Type of clearance (GFR) Test
• 1- creatinine clearance test
• 2-inulin clearance test
• 3-urea clearance test
• 4-cysteine clearance test
• 5- radioactive substance
 5-Equation for estimated GFR
 CrCl (ml/min/1.73m)=
 Schwartz
(length<cm>×K)/Scr
 Shulletal
(0.035×age)+(0.236)×100/S cr
 Cockcroft – Gault formula
 (140-age)×mass(kg)×0.85 if femal
/ 72 ×SCr mg/dl
 Normal range
• Male: 97-137ml/min/1.73m
• Femle:88-128 ml/min/1.73m
Uric acid
Nitrogenus bases :

1.Purine {adenine (A) ,guanine (G)}.

2.pyrimidine {thymine (T), cytosine (C) ,
Uracil (U)} .

 Sources of purine :
1.Synthesis in the body .
2.Diet .
3.Endogenous catabolism of nucleic acids .
Fate of purine:

Purine may be oxidized to urate or reused for

nucleic acid synthesis .
a.Purine reused for nucleic acid synthesis :

 Some xanthine ,hypoxanthine & guanine can be

resynthesized to purine nucleotides by pathway
involving among other enzyme .
-Hypoxanthine guanine phosphoribosyl
transferase (HGPRT) .
-Adenine phosphoribosyl transferase (APRT) .
b. purine oxidized to urate :-
Excretion of urate :-
 Uric acid transported by the plasma from the liver
to the kidneys where it is filtered by the
glomerulus .
 Reabsorption of 98% to 100% of the uric acid in the
glomerular filtrates' occur in the proximal tubules .
 Small amount of uric acid is secreted by the distal
tubules into the urine .
 This represent about 70% of daily uric acid
excretion .
 The remainder amount is excreted into GIT
and degraded by bacterial enzymes
(uricolysis).
 TAG , ketone bodies & lactic acid have been
shown to compete with urate for excretory
sites in renal tubules .
 Nearly all of the uric acid in plasma is present
as monosodium urate .
 In the urine at pH level <5.75 uric acid is
predominant form of frequently appear as
uric acid crystals and may form renal calculi .
Disease correlation with hyperuricaemia :

1-Gout:-
 Is disease found primarily in males & usually is first
diagnosed between the age of 30 &50 years .
 Patients have pain and inflammation of the joints
caused by precipitation of sodium urate.
 In 25% to 30% of these patients hyperuricemia is
due to over production of uric acid , although other
common causes are drugs and alcohol .
 Patient with gout are highly susceptible to develop
renal calculi .
Pseudogout :-
 Is not a disorder of purine metabolism
although the clinical presentation is similar to
that of gout .
 It is caused by calcium pyrophosphate
precipitation in joints .
 Associated with ↑ PTH,↓ TH,
hemochromatosis and acromegaly .
2- Increased turn over of nucleic acid :-

a. Patients on chemotherapy for such

proliferative diseases .
- monitoring uric acid level to avoid
nephrotoxicity by allopurinol .
b. Patients with hemolytic & megaloblastic
anemia .
c. Trauma as in surgery .
3-Reduced excretion of urate :-

a. Chronic renal diseases lead defect in

filtration
& secretion .
urate : creatinine conc. ratio
- Ratio less than 0.7 →renal dysfunction is
primary .
- Ratio greater than 0.7 → urate nephropathy .
b. Chronic lead intoxication .
c. Lactic acidosis .
4-combined (both Î production & ↓excretion):
a. Alcohol.
b. During starvation .
c. Type 2 diabetes mellitus
d. Glycogen storage disease type (2) :
is due to glucose 6 phosphotase
deficiency .
 This deficiency impairs the ability of the liver to
produce free glucose from glycogen and
glyconeogenesis .
 More G6P available for metabolism, through
-pentose phosphate pathway → increase purine
synthesis (over production of urate ) .
-Glycolysis increase lactic acid production .
4-Toxemia of pregnancy (ketosis).
5-lesch –nyhan syndrome :-
 Is X linked genetic disorder (only seen in males ) .

 It caused by complete deficiency of hypoxanthine

guanine phosphoribosyl transferase (HGPRT) , a
major enzyme in biosynthesis of purine .

 Hypoxanthine and other purine can not be recycled

to form nucleotides and produce more urate .

 This syndrome characterized by neurologic

symptoms ,mental retardation .
 Partial deficiency of HGPRT is called kelley
seegmiller .
6- Excessive intake of diet in purines ( liver ,
kidney ,……).
Hypouricaemia :-

Causes :
1-severe liver disease .
2- Over treatment of hyperuricaemia with
allopurinol .
3-Defective tubular reabsorption as in fanconi
syndrome .
4- xanthinuria:
Xanthinuria :-
 This is a very rare inborn error of purine
metabolism inherited as an autosomal
recessive disorder in which there is deficiency
of xanthine oxidase in the liver .
 purine break down stops at xanthine and
hypoxanthine stage .
 plasma and urinary urate conc. are very low .
 increased xanthine excretion may lead to the
formation of xanthine stones .
 Analytical methods :-
Specimen requirement :
 uric acid may be determined in serum ,urine
or heparinized plasma .

 Serum should be removed from cells rapidly.

 Gross lipemia should be avoided .

 Fasting is not necessary .

Methods of estimation :-
A. Chemical methods (reduction methods):
1.Caraway method .
2.Reduction of ferric irons.
 1-Caraway method :-
Principle:-
PTA is reduced by uric acid in alkaline medium
resulting formation of tungestic blue which can be
read by spectrophotometry .

 Alkaline medium obtained by;

1.Sodium carbonate .
2.Sodium cynide.

 Sample should be deproteinized .

2- Reduction of ferric ions :-

principle:
Fe³ reduced by uric acid forming colored
compound which can be measured by SPM .
Disadvantages :
- Fe⁺² can be oxidized by O₂ .
B-Enzymatic method:-
Uric acid + uricase → allantoin +H₂O₂
1.Direct method :-
 Principle :
Is based on the fact that uric acid has UV
absorbance peak at 293nm . Where as allantoin
does not have .
The difference in absorbance before and after
incubation with uricase is proportional to the
uric acid conc. .
Disadvantages:
(-ve) interference with xanthine &
hypoxanthine.
2-Indirect method :-
Principle:
Uric acid + uricase → allantoin +H₂O₂
H₂O₂ catalase +(peroxidase)→ H₂O +O₂
O₂ + O₂ acceptor → colored compound

 Absorbance of color measured by SPM .

Disadvantages:
Interferences can occur with bilirubin and
ascorbic acid which destroy peroxidase .
C-Other method :
1- HPLC .
2- NIST .

Reference range :
Male :3.6 -7.0 mg/dl .
Female : 2.5 -6.0 mg/dl .
Ammonia
 Ammonia is a compound produce by
damnation of amino acid by intestinal
bacteria & body cells .
 It transport to liver and use in the production
of urea.
Ammonia toxicity :
 High concentration of ammonia are
neurotoxic and associated with
encephalopathy.
Cause of high ammonia concentration :

1.sever liver disease.

2.reyes syndrome .
3.Inherited defects in urea cycle enzymes
except argininosuccinase .
Specimen requirement :
• 1.sample,serm & plasma .

• 2.anticoagulants (e.g. heparin ,EDTA ) .

• 3 .sample examined immediately or frozen .
Analytical method
1-first method depend on the volatility of
ammonia to separate the compound in
microdiffusion chamber .
ammonia gas from the sample diffuses into
separate compartment and is absorbed in
solution containing a pH indicator .
the amount of ammonia was determined by
titration.
2.Cation exchange resin :
Principle:
Use the cation exchange resin followed by
elution of ammonia with sodium chloride and
quantification by Berthelot reaction .
3. Enzymatic Assay :

 Using glutamate dehydrogenase .

NH₄⁺ + 2- oxoglutarate + NADPH + GLDH →

glutamate + H₂O + NADP⁺
4.Ion selective electrode :

Principle :
potentiometric method of analysis
involve the direct measurment of electrical
potential due to the activity of free ions.
5.Spectrometric method :

Principle :
In this method ammonia reacts with an
indicator to produce a color compound that Is
spectrophotometrically detected .