20 June 2016

Lecture #9: Non-protein Nitrogenous Compounds


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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