The non–protein nitrogen fraction of serum (and blood) is composed
of all nitrogenous compounds other than protein. The kidney plays an important
role in the elimination of this compound.
There are more than 15 NPN compounds in plasma. Urea nitrogen
comprises approximately of 45% of the total. Other compounds and there
percentages of the total NPN in plasma include amino acids (20%), uric acid
(20%), creatinine (5%), creatine (1–2 %) and ammonia (0.2%). NPN in blood is
approximately 75% greater than the plasma value owing to the glutathione
content of erythrocytes. NPN determination is most useful in connection with
kidney disease.
The test for NPN has been reduced by the more useful and convenient
test for urea nitrogen. The NPN does not offer any information in addition to
that provided by the urea nitrogen determination. An exception to this may be
the simultaneous determination of NPN and urea nitrogen in patient with hepatic
failure in the presence of renal disease. Under such conditions, the ration of
NPN to urea nitrogen may be higher than that normally found. This is due to the
decreased ability of the liver to synthesize urea and deaminate amino acid.
Increase in NPN fraction are mainly a reflection of an increased
in urea nitrogen. As the total NPN rises, the proportion of the urea also
increases.
Requirement common to all NPN compound
1.
Deproteinization
All proteins must be removed from
the samples before actual measurement. Somogyi type filtrates should not be
used since some NPN compounds are adsorbed on the precipitate.
2.
Digestion
This consists of breaking down
the nitrogenous compound after separating proteins and the conversion of
nitrogen into a common measurable form. This is done by using acid digestion
mixture and heat. There are many digestion mixtures which achieve the same
results. These are digestion mixtures consists of an oxidizing agent, a
catalyst and a chemical which increases the boiling point of the solution.
An example of an acid digestion
mixture contains the following:
a.
Sulfuric acid – oxidizing agent
b.
Copper sulfate or perchloric acid – catalyst
c.
Phosphoric acid or potassium sulfate – used
to raise the boiling point
During the digestion process, the
various nitrogenous substances are broken down and nitrogen is converted to
ammonia held in the form of ammonium sulfate by sulfuric acid.
3.
Measurement of ammonia by either
a.
Distillation
b.
Nesslerization
c.
Gasometrically
d.
Color reaction
Three common procedures in the determination
of NPN
1.
Folin Wu method
In this method, a protein free
filtrate is made. To the filtrate, an acid digestion mixture is added to help
liberate ammonia. This is heated to decompose nitrogenous substances in the
form of ammonium salts. Nessler’s solution is added to convert ammonium salts
to yellow simercuric ammonia iodide which is measured colorimetrically.
Reagents:
a.
Acid digestion mixture – a mixture of
concentrate sulfuric acid and 85% phosphoric acid. Concentrated sulfuric acid
is used to convert N2 to NH3 and to oxidize other organic
compounds while H3PO4 is used to raise the boiling point.
b.
Nessler’s reagent – alkaline potassium
mercuric iodide – used as color developer
c.
Gum ghatti – used to prevent cloudiness
d.
Glass beads – used to prevent bumping of the
solution
e.
Yellow – end color
2.
Koch–McMackin method
This is the same as the Folin–Wu
method except for the digestion mixture. Hydrogen peroxide is used instead of
phosphoric acid as catalyst. Sulfuric acid is used for stronger concentration.
3.
Berthelot’s color reaction
PFF is prepared. Sulfuric acid is
added to liberate ammonia. It is then heated to decompose nitrogen substance.
Phenol color reagent and alkali hypochlorite reagent is added and in the
presence of sodium nitroprusside as catalyst, blue color is produced which is
measured colorimetrically.
****** BLOOD UREA NITROGEN ******
It constitutes about 40–50% of the total NPN in the blood. It is
the chief end product of protein catabolism.
Urea is synthesized in the liver from NH3 produced as the result
of deamination of amino acids and CO2 and the process is known as
the ornithine cycle. From the liver, urea enters the blood to be distributed to
all intracellular and extracellular fluids, since urea is freely diffusible
across most cell membranes. Most of the urea is ultimately excreted by the
kidney by glomerular filtration but minimal amounts are also excreted in the
sweat and degraded by bacteria in the intestine.
It is customary in most laboratories to express urea as urea
nitrogen. The same is through the desire to compare the quantity of nitrogen in
urea with that of other components included in the non–protein category. Since
its molecular mass is 60 daltons, and it contains 2 nitrogen atoms with a
combined weight of 28, a urea nitrogen value can be converted to urea by 60 ÷
28 or 2.14
Urea = BUN x 2.14
Example:
BUN = 15mg%
Urea = 15 mg% x 2.14
=
32.10 mg%
Methods of
BUN determination
1. Colorimetric determination by its
reaction with diacetyl monoxime
Fearon reaction and Diacetyl monoxime (DAM)
In 1939, Fearon found that reaction of ammonia with diacetyl
monoxime followed by oxidation gives a color.
In 1942, Ormsby applied this reaction to the determination of
urea. The sample is heated with diacetyl monoxime in acid solution and the
resultant color is intensified by oxidation with potassium persulfate of the
hydroxylamine formed in the reaction.
Disadvantages of this method:
a.
Color develops rapidly and fades rapidly
b.
Color is photosensitive
c.
Color does not follow Beer’s law.
d.
Unpleasant odor and irritant fumes of the
reagent.
e.
The time of heating for maximal color
development is dependent on the urea concentration.
f.
The reaction is not completely specific.
2. Enzymatic method by the action of
urease on urea
a.
Karr method
To protein free filtrate, a buffer solution is added to control
the pH. Enzyme urease is then added and the mixture is incubated to decompose
urea and form ammonium carbonate. Gum ghatti is added as protective colloid.
Lastly, Nessler’s is added to yellow dimercuric ammonium iodide. This is then
measured photometrically.
b.
Van Slyke Cullen method
A buffer solution is added to oxalated blood to control the pH.
Enzyme urease is added to decompose urea and form ammonium carbonate. Potassium
carbonate is added to liberate ammonia which is collected in boric acid
solution. This is then titrated with a standard acid solution and urea nitrogen
is determined by calculation.
c.
Gentzkow Massen method
Oxalated blood is diluted and enzyme urease is added to decompose
urea and form ammonium carbonate. Then, the proteins are precipitated.
Nessler’s reagent is then added to the filtrate to convert ammonium carbons to
yellow dimercuric ammonium iodide which is measured photometrically.
d.
Folin–Svedberg method
A PFF is made and a buffer solution is added to control the pH. An
ezyme is added to decompose urea and form ammonium carbonate. Sodium borate is
added to liberate ammonia which is then collected in dilute hydrochloride acid.
Nessler’s solution is added to convert ammonium salts to yellow dimercuric
ammonium iodide which is then measured colorimetrically.
e.
Berthelot color reaction
Buffered urease is added to serum or plasma to decompose urea and
form ammonia. The mixture is incubated to enhance the reaction. Phenol color
reagent and alkali hypocholorite reagent are added which is measured
colorimetrically.
f.
Leiboff method
The principle is similar to the Folin method except that a special
Leiboff pressure tube is used and the sample and sulfuric acid are placed in
Leiboff and immersed in an oil both heated at 150oC for about 10 minutes. After
cooling, it is nesslerized and the yellow color formed is measured
colorimetrically.
g.
Urograph (Urastrat) method
This consists of chromatography paper bonded by controlled amount
of reagent (buffered urease, potassium carbonate and bromcresol green acidified
with quantitative titrated tartaric acid). The method will measure directly in
30 minutes the BUN concentration from 10–75 mg%. The urograph chemical reaction
parallels closely those of the Conway microdiffusion method:
(1) Digestion
with urease
(2) Release of
ammonia by K2CO3
(3) Titration
with acid
When the serum or plasma in the bottom of the test tube travels up
through the urograph, it passes through a series of chemical adventures. The
sample first encounters a highly potent urease which produces ammonia from the
urea present in the sample.
In the next band containing potassium carbonate, the ammonia is
liberated as free gas, the quantity of which is proportional to the original
urea nitrogen concentration of the sample. The upward migration of the serum or
plasm is eventually stopped by the plastic barrier. The indicator band of the
bromcresol green tartaric acid located above the plastic layer quantitatively traps
and reads the ammonia released. The urea nitrogen is quantitatively indicated
by the height of the portion of the indicator band which has changed color at
the end of 30 minutes incubation period.
The height of the indicator band in which color change is noted is
measured in terms of millimeters which is then multiplied by 5 and 10 is added.
This will be mg%.
3.
Xanthydral reaction (direct
determination of urea)
Urea is precipitated with xanthydrol and then the resulting
dixanthydryl urea is estimated. The method requires a special apparatus, time
consuming and expensive to manipulate.
Reference
range for BUN is 8 – 26 mg / dl:
A value
within this range, however, does not imply that renal function is unimpaired. A
patient with baseline of 12 mg/dl whose level increases to 24 mg/dl in a steady
state of hydration and protein intake may well have significant reduction of
renal function regardless of the fact that the level is within the reference
range.
Precautions in BUN determination:
1.
BUN determination is affected by high protein
diet.
2.
Whole blood should be deproteinized to
eliminate interferences of hemoglobin.
3.
Ammonium–containing anticoagulants are
contraindicated in enzymatic method.
4.
Sodium fluoride inhibits the action of
urease.
5.
Upon prolonged standing, ammonium
concentration in the sample raises 2 – 3 times the original value due to
enzymatic deamination of labile amides like glutathione.
Clinical significance
The determination of serum urea nitrogen is presently the most
popular screening test for the evaluation of kidney function. The test is
frequently requested along the serum creatinine test since simultaneous
determination of these two compounds appears to aid in different diagnosis of
pre–renal, renal and post–renal hyperuremia.
1.
Pre–renal causes – conditions in which
circulation through the kidney is less efficient than usual.
a.
Cardiac decompensation
b.
Water depletion due to decreased intake or
excessive loss.
2.
Renal causes – with lesions of the renal
parenchyma
a.
Glomerulonephritis
b.
Chronic nephritis
c.
Polycystic kidney
d.
Nephrosclerosis
e.
Tubular necrosis
3.
Post–renal – due to obstruction of the
urinary tract
a.
Stone
b.
Enlarged prostate gland
c.
Tumors
Azotemia – biochemical abnormality that
refers to an increase in BUN and creatinine levels which is largely related to
decreased GFR.
Uremia – increase in urea and creatinine
values with accompanying clinical signs and symptoms of renal failure like:
a.
Metabolic acidosis due to failure of the
kidneys to eliminate acidic products of metabolism
b.
Hyperkalemia due to failure of potassium
excretion.
c.
Generalized edema due to water retention.
****** CREATINE AND CREATININE ******
CREATINE
Creatine is synthesized in the liver and pancreas from three amino
acids: arginine, glycine and methionine. After synthesis, creatine diffuses
into the vascular system and is supplied to many kinds of cells, particularly
the muscle, where it is phosphorylated. Creatine phosphate serves as a
reservoir of high energy and is readily convertible to adenosine triphosphate
in muscles and other tissues.
Creatine and creatine phosphate total approximately 400 mg per 100
gram of fresh muscles. Both creatine and creatine phosphate are spontaneously
converted into creatine at a rate of approximately 2% per day.
Method of
determination:
It is determined as the
difference between the preform creatinine and the total creatinine that results
after the creatine present has been converted to creatinine by heating at an
acid pH.
Total
creatinine – Preformed creatinine = Creatine
CREATININE
Creatinine is a wasted product derived from creatine and creatine
phosphate. It is an anhydride formed when creatine loses a water molecule and
creatine phosphate loses a phosphoric acid molecule. The reaction occurs
spontaneously.
Approximately 2% of creatine is transformed into creatine every 24
hours. The body content of creatinine is proportional to muscle mass; therefore,
the levels of creatinine in the body are also proportional to muscle mass. The
conversion of creatine to creatinine occurs at an accelerated rate in acid or
alkaline solutions.
Creatinine is removed from the plasma almost entirely by
glomerular filtration with a small contribution from tubular secretion. No
reabsorption of creatinine occurs in the renal tubules. Urine contains
significantly more creatinine than creatine because of the differences in renal
handling.
Method of determination
1.
Jaffe Reaction
PFF is treated with alkaline
sodium picrate solution to produce a red – orange tautomer of creatine picrate
(Jaffe reaction) and this is measured phtometrically.
The Jaffe reaction is not
specific for creatine since there are many substances in the red cells which
give the same reaction as creatinine. This is the reason why plasma and serum
are preferred to whole blood since considerable amount of non – creatinine
chromogens are present in the red cells.
Lloyd’s reagent is used for
isolation of creatinine from interfering substances. After deproteinization of
the specimen, creatinine is absorbed from an acid medium on Lloyd’s reagent, an
aluminum silicate, and subsequently described in an alkaline solution. This
method is highly specific and gives true creatinine level.
Notes on creatinine
determination:
a.
Alkaline sodium picrate solution is unstable
and should therefore be prepared and stored for 30 minutes before use.
b.
Solution should be thoroughly mixed after the
addition of alkaline picrate.
c.
The end color in the Jaffe reaction slowly
fades and should be read within ½ hour.
Chief source of difficulty of the
Jaffe reaction
a.
Lack of specificity
b.
Sensitivity to certain variable like ascorbic
acid, pyruvate, acetone, glucocyanidine, amino–hippurate, diacetic acid,
glucose and protein.
Variables involved in Jaffe
reaction:
a.
Picric acid – commercial preparations must be
purified.
b.
Temperature – color development between 15 –
25oC is not much important but the temperature of solution while it
is being read is important.
c.
Protein precipitation – 85 – 90% recovery at
pH 3 – 4.5; complete recovery at pH below 2.
d.
pH – color intensity of alkaline picrate
decreases with decreasing pH, conversely, color intensity of alkaline picrate
increases as pH increases.
2.
Enzymatic method
3.
Development of a purple rose
color formed between creatinine and 3,5– dinitrobenzoic acid in alkaline
solution.
4.
Turbidimetric method employing a
modified Nessler’s reagent.
5.
Reaction of creatinine with
potassium mercury thiocyanate
6.
Degradation to methyl guanidine
followed by Sakaguchi color reaction.
7.
Isolation of creatinine by
absorption of an ion exchange resin and quantitation by measurement of
absorbance at its peak.
Clinical significance
Creatinine is considered as the best index for prognosis of kidney
impairment. As renal function diminishes, serum creatinine rises but the rise
is less than the change in BUN. An elevated serum creatinine level indicates
severe, long standing renal impairment.
By virtue of its relative independence from such factors as diet
(protein intake), degree of hydration and protein metabolism, the plasma
creatinine is a significantly more reliable screening test or index of renal
function than BUN.
Most experts advocate the establishment of a baseline GFR with
creatinine clearance test, and then the serum creatinine can be used to
establish changes in GFR.
Creatinine in serum represents a small part of the non – protein
nitrogen function. Its determination has little clinical value in kidney
diseases. It is used principally in evaluating muscle disorders. It is a
product of endogenous muscle breakdown and when this breakdown is accelerated,
as in muscular dystrophy, large amount of creatine maybe excreted in urine.
****** BLOOD URIC ACID ******
Uric acid is a waste product derived from purine of the diet and
those synthesized in the body. It has been shown that the healthy adult human
body contains about 1.1 grams of uric acid and that about one sixth of this is
present in the blood, the remainder being in other tissues. Normally, about one
half of the total uric acid is eliminated and replaced each day, partly by way
of urinary excretion and partly through destruction in the intestinal tract by
microorganisms. Uric acid is one of the components of the NPN fraction of
plasma.
Plasma uric acid is filtered by the glomeruli and is subsequently
reabsorbed to about 90% by the tubules. It is the end product of purine
metabolism in man. Other mammals are able to metabolize the uric acid molecule
to a more soluble end product, allantoin. It is greatly affected by extra–renal
as well as renal factors.
Methods of determination
1.
Colorimetric method
Most methods of uric acid
determination are based on the reducing property of this substance. The end
result of most of the procedure is the production of a blue color by the action
of uric acid on phosphotungstic acid.
Phosphotungstic acid serves both
as protein precipitating agent and color agent.
A glycerine–silicate reagent
increases the sensitivity and also provides alkalinity for the reduction of
phosphotungstic acid to tungsten blue.
Sodium polyanethol sulfonate is
added to prevent turbidity
a.
Benedict’s method
The disadvantage of this method
is that 5% NaCN, which is added to intensify the color is poisonous.
Uric acid is reacted with
phosphotungstic acid solution in an alkaline media. The phosphotungstate
complex is reduced to phosphotungstite (blue) in which intensity of color is
directly related to the concentration of uric acid in the sample.
b.
Folin method
A PFF is treated with urea
cyanide and phosphotungstic acid to form a phosphotungstate complex. This is
reduced by the uric acid present to form blue phosphotungstite complex. This then
measured colorimetrically with a standard.
c.
Brown method
A PFF is treated with sodium
cyanide, urea and phosphotungstic acid to form a phosphotungstate complex. The uric
acid present reduces the phosphotungstate to the blue phosphotungstite complex.
The depth of the color is measured in a colorimeter and compared with a
standard.
d.
Newton method
A special PFF is made and treated
with urea–cyanide solution and lithium arsenotungstate to form arsenotungstate
complex. The depth of the color is measured in a colorimeter and compared with
a standard.
e.
Archibald method
A special PFF is made and
glycerol silicate polyanethol sodium sulfonate and phosphotungstic acid are
added to form phosphotungstic complex which reduced to blue phosphotungstite
complex. This is measured in a colorimeter and compared with a standard.
The specificity of the method is
enhanced by pre–treatment of the serum with sodium hydroxide which causes an
oxidative destruction of ascorbic acid and sulfhydryl compound which would lead
to false high values.
f.
Henry method
Uric acid in serum, plasma and
urine reduce an alkaline phosphotungstate solution to a “tungsten blue.” The alkali
used is sodium carbonate. The depth of the color is measured photometrically
and compared with a standard.
g.
Caraway method
Similar and identical with Henry
method. The recent methods of Henry and Caraway returned to the older methods
of providing alkalinity due to the disadvantages and hazards when one is using
cyanide.
h.
Kern–Stransky method
There are several disadvantages
with the cyanide technique:
a.
Reagent blanks with cyanide usually have
appreciable absorbance which varies with age of the reagent and brand.
b.
Standard curves with cyanide are not continuously
reproducible.
c.
Cyanide is undesirable because of its highly poisonous
character.
d.
Extra care in the storing of the reagent is
needed as it requires refrigeration.
One of the problems involved in
the colorimetric method of uric acid determination is the appearance of
turbidity in the final colored solution.
Methods or reagents used to avoid
the formation of turbidities are:
a.
Purification of the cyanide reagent because
of their carbonate contents.
b.
Others add lithium salts to the
phosphotungstic acid reagent. This reagent helps to avoid turbidity by
inhibiting the formation of sodium or potassium phosphotungstate.
c.
Addition of urea to the cyanide technique.
d.
Kern and Stransky introduced a method
employing sodium silicate as the source of the alkali.
e.
Archibald, unable to avoid turbidities by the
Kern – Stransky method introduced a technique retaining glycerine silicate but
added sodium polyanethol sulfonate to prevent turbidity formation.
f.
Henry, et.al. included lithium sulfate in the
phosphotungstic acid reagent. He found that the same color intensity could be
obtained with sodium carbonate, sodium hydroxide and sodium metasilicate.
With regards to the problem of
specificity of the reaction, the following techniques have been employed:
a.
Isolation of uric acid on ion exchange resin.
b.
Bulger and Johns (1941) used the specific enzyme
uricase. The end products resulting from the oxidation of uric acid by uricase
are dependent on pH and specific buffer employed.
(1) Tris buffer
or phosphate buffer at pH 7.2 – 8.5; uric acid is oxidized mainly to allantoin
(2) Presence of
borate buffer and at pH 7.2, the main products are urea and allaxonic acid
(3) Borate buffer
at pH 9, the products of uric acid oxidation are urea, allaxonic acid and
allantoin.
c.
Kalokar introduced the technique of
differential spectrophotometry for the determination of uric acid. Uric acid
has an absorption peak in the region of 290 –293 mu, whereas, the end product
after destruction by uricase has no absorption at this wavelength. Thus, the
decrease in absorbance in this wavelength resulting from uricase action is
proportional to the uric acid originally present.
2.
Spectrophotometric method
Uricase is an enzyme found in
most mammals except man, which catalyzes the complicated transformation of uric
acid into allantoin. Uric acid has a maximum absorption peak at about 203 – 290
mu. When uricase destroys uric acid, the resultant products have no absorption
at this wavelength. The decrease in optical density of the specimen, after
incubating it with uricase, is proportional to the amount of the uric acid
present. This method has great specificity because uricase acts on uric acid alone.
It is more time consuming and requires the use of an instrument capable of
reading in the ultraviolet region; therefore, it is seldom used routinely.
Normal values: Female = 2 – 6 mg%
Male = 3 – 7 mg%
Clinical significance:
Determination of serum uric acid levels is most helpful in the
diagnosis of gout. It is also increased whenever there is increased metabolism
of nucleoproteins, such as leukemia and polycythemia or after the intake of
food rich in nucleoproteins, e.g. liver, kidney or sweetbread. It is also a
constant finding in familial idiopathic hyperuricemia, of which there are two
types. In one type, there is an overproduction of uric acid in the presence of
normal excretion and in the other; there is a decreased rate of excretion in
the presence of normal uric acid production. Uric acid levels are elevated in
decreased renal function and are valuable for early diagnosis of kidney
impairment. It is excreted least daily.
Gout – a chronic disturbance of metabolism in which there is a
noted accumulation of uric acid in the blood as a result of the disturbance of the
exogenous and endogenous uric acid formation. In gout, the blood uric acid
level is elevated and there is often deposition of crystalline uric acid around
various joints called “tophi.”
****** AMMONIA
******
The ammonia concentration in blood is low (approximately, 40 – 75 mg%).
Ammonia is increased in: (1) Bleeding esophageal varices (2) Hepatic coma.
A rapid quantitative screening procedure for blood ammonia has
been proposed which is carried out in a Conway disk.
Blood is alkalinized with potassium carbonate and ammonia thus
released is absorbed in a boric acid solution. A mixed indicator is used to
estimate the degree of neutralization of the boric acid by ammonia.
Seligson and Katsugi described a quantitative procedure in which
blood is alkalinized in a close bottle containing a suspended red coated with
acid. The released ammonia is absorbed by the acid and the red is the dipped
into Nessler’s solution for the subsequent determination of ammonia.
Because of this low concentration and its relative obiquity, ammonia methods are
difficult to set up and control.
****** AMINO ACID NITROGEN ******
Normal value: 4 –
8 mg / 100 ml
Elevated valued may be seen in the following diseases:
1.
Uremia
2.
Leukemia
3.
Yellow atrophy of the liver
Determination is not widely used.
Classical procedure for amino acids determination utilizes a
specific reaction in which ninhydrin liberates carbon dioxide from the carboxyl
group of the amino acids and the carbon dioxide measured in a gas apparatus.
Less precise methods employ a color reaction between the amino
acids and beta–napthoquinone–4–sulfonate. In this procedure, a portion of PFF
is alkalinized, the boiled with napthoquinone. The excess napthoquinone is
decolorized with formaldehyde and thiosulfate, and the remaining brown color is
proportional to the amino acid nitrogen concentration.
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