MTY1209 CLINICAL CHEMISTRY 1 (LECTURE)

W12
Nonprotein Nitrogen Compounds
Mr. Aaron Palmares | FEU BS Medical Technology 2023 L4
OUTLINE Creatine 1̶2 —
Nonprotein Nitrogen 1
Urea 1 Ammonia 0.2 2.8
Biochemistry 1 Table 12.1. Numerous compounds of clinical interest are
Clinical Application 1 included in the NPN fraction of plasma and urine. The most
Analytical Methods 2 abundant of these are listed
UREA
Specimen Requirements 2
● Urea
Reference Intervals 2 o The NPN compound present in highest
Pathophysiology 2 concentration in the blood
Uric Acid 3 o The major excretory product of protein
Biochemistry 3 metabolism.
Clinical Application 3 o It is formed in the liver from amino groups (−NH2)
and free ammonia generated during protein
Analytical Methods 3 catabolism.
Specimen Requirements 4 ▪ This enzymatically catalyzed process is termed
Reference Intervals 4 the urea cycle.
Pathophysiology 4 o The term blood urea nitrogen (BUN) has been used
Creatinine / Creatine 5 to refer to urea determination.
Biochemistry 5 ▪ Urea nitrogen (urea N) is a more appropriate
term.
Clinical Application 6
Analytical Methods 6
Creatinine 6
Specimen Requirements 6
Sources Of Error 7
Creatinine / Creatine 7 Figure 12.1. Structure of Urea.
Reference Intervals 7 BIOCHEMISTRY
Pathophysiology 7 ● Protein metabolism produces amino acids that can be
oxidized to produce energy or stored as fat and glycogen.
Creatinine 7 o These processes release nitrogen, which is
Creatine 7 converted to urea and excreted as a waste product.
Ammonia 7 ● Urea is carried in the blood to the kidney, where it is
Biochemistry 7 readily filtered from the plasma by the glomerulus.
Clinical Application 7 o Most of the urea in the glomerular filtrate is excreted
Analytical Methods 8 in the urine, although some urea is reabsorbed by
passive diffusion during passage of the filtrate
Specimen Requirements 8 through the renal tubules.
Sources Of Error 8 ● The amount reabsorbed depends on the urine flow rate
Reference Intervals 8 and extent of hydration.
Pathophysiology 8 ● Small quantities of urea (<10% of the total) are excreted
through the gastrointestinal (GI) tract and skin.
● The concentration of urea in the plasma is determined by
NONPROTEIN NITROGEN the protein content of the diet, the rate of protein
● Nonprotein Nitrogen (NPN) catabolism, and renal function and perfusion.
o The concentration of nitrogen-containing compounds
in this protein-free filtrate was quantified CLINICAL APPLICATION
spectrophotometrically by converting nitrogen to ● Measurement of urea is used to:
ammonia and subsequent reaction with Nessler's 1. Evaluate renal function
reagent (K2[HgI4]) to produce a yellow color. 2. Assess hydration status
3. Determine nitrogen balance
4. Aid in the diagnosis of renal disease
TABLE 12.1 Clinically Significant 5. Verify adequacy of dialysis.
Nonprotein Nitrogen Compounds o tapos pag may explanation
▪ pa
Approximate ● Measurements of urea were originally performed on a
Approximate protein-free filtrate of whole blood and based on
Plasma Urine
Compound Concentration measuring the amount of nitrogen.
Concentration ● Urea is often reported in terms of nitrogen concentration
(% of Total NPN) (% of Excreted
Nitrogen) rather than urea concentration.
● Urea N concentration can be converted to urea
Urea 45 ̶ 50 86.0 concentration by multiplying by 2.14, as follows:

Amino Acids 25 —
Uric Acid 10 1.7

Creatinine 5 4.5
(Eq. 12-1)

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 MTY1209 | LEC Nonprotein Nitrogen Compounds

● In the International System of Units (SI), urea is reported

Other Methods
in units of millimoles per liter.
● Urea N concentration in milligrams per deciliter may be
converted to urea concentration in millimoles per liter by Isotope dilution Detection of Proposed
multiplying by 0.36. mass characteristic reference method
spectrometry fragments
ANALYTICAL METHODS following
● Enzymatic methods are used most frequently in clinical ionization;
laboratories. quantification
● The enzyme urease (urea amidohydrolase, EC 3.5.1.5) using isotopically
catalyzes hydrolysis of urea in the sample, and the labeled
ammonium ion ( ) produced in the reaction is quantified. compound
● The most common method couples the urease reaction
with glutamate dehydrogenase (GLDH, EC 1.4.1.3), and
the rate of disappearance of nicotinamide adenine SPECIMEN REQUIREMENTS
dinucleotide (reduced, NADH) at 340 nm is measured. ● If plasma is collected, ammonium ions and high
concentrations of sodium citrate and sodium fluoride
must be avoided; citrate and fluoride inhibit urease.
● Although the protein content of the diet influences urea
concentration, the effect of a single protein-containing
meal is minimal and a fasting sample is not usually
required.
● A non hemolyzed sample is recommended.
● Urea is susceptible to bacterial decomposition, so
specimens (particularly urine) that cannot be analyzed
within a few hours should be refrigerated.
● Timed urine specimens should be refrigerated during the
FIGURE 12.2 Enzymatic assay for urea.
collection period.
● Ammonium from the urease reaction can also be
● Methods for plasma or serum may require modification
measured by the color change associated with a pH
for use with urine specimens because of high urea
indicator.
concentration and the presence of endogenous
o This approach has been incorporated into
ammonia.
instruments using liquid reagents, a multilayer film
format, and reagent strips.
REFERENCE INTERVALS
● A method that uses an electrode to measure the rate of
increase in conductivity as ammonium ions are
produced from urea is in use in approximately 15% of
laboratories in the United States.
● A reference method using isotope dilution mass
spectrometry (IDMS) has been developed.

TABLE 12.1 Clinically Significant

PATHOPHYSIOLOGY
Nonprotein Nitrogen Compounds ● An elevated concentration of urea in the blood is called
azotemia.
Enzymatic Methods ● Very high plasma urea concentration accompanied by
renal failure is called uremia or the uremic syndrome.
This condition is eventually fatal if not treated by dialysis
Methods use a Enzymatic See Figure 12.2 or transplantation.
similar first step ̶ production of ● Conditions causing increased plasma urea are classified
catalyzed by ammonium ion according to the cause into three main categories:
urease (NH₄⁺) from urea 1. Prerenal
▪ Prerenal azotemia is a result of reduced renal
GLDH-coupled Enzymatic Used on many blood flow.
enzymatic reaction of NH₄⁺, automated ▪ Less blood is delivered to the kidney;
2-oxoglutarate, instruments; best consequently, less urea is filtered.
and NADH to as a kinetic ▪ Causative factors include congestive heart
form glutamate measurement failure, shock, hemorrhage, dehydration, and
and NAD⁺ other factors resulting in a significant decrease
in blood volume.
Indicator dye NH₄⁺ + pH Used in ▪ The amount of protein metabolism also induces
indicator → color automated prerenal changes in blood urea concentration.
change systems, ▪ A high-protein diet or increased protein
multilayer film catabolism, such as occurs in stress, fever,
reagents, and dry major illness, corticosteroid therapy, and GI
reagent strips hemorrhage, may increase the urea
concentration.
Conductometric Conversion of Specific and 2. Renal
unionized urea to rapid ▪ Decreased renal function causes an increase in
NH₄⁺ and CO²⁻ ₃ plasma urea concentration as a result of
results in compromised urea excretion.
increased ▪ Renal causes of elevated urea include acute
conductivity and chronic renal failure, glomerulonephritis,
tubular necrosis, and other intrinsic renal
disease

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 MTY1209 | LEC Nonprotein Nitrogen Compounds

3. Postrenal ● At the pH of plasma (pH ~ 7), urate is relatively insoluble;

▪ Postrenal azotemia can be due to obstruction of at concentrations greater than 6.8 mg/dL, the plasma is
urine flow anywhere in the urinary tract by renal saturated.
calculi, tumors of the bladder or prostate, or ● As a result, urate crystals may form and precipitate in the
severe infection. tissues.
● The major causes of decreased plasma urea ● In acidic urine (pH < 5.75), uric acid is the predominant
concentration include low protein intake and severe species and uric acid crystals may form.
liver disease.
● Plasma urea concentration is decreased during late CLINICAL APPLICATION
pregnancy and in infancy as a result of increased protein ● Uric acid is measured to confirm diagnosis and monitor
synthesis. treatment of gout, to prevent uric acid nephropathy
● Differentiation of the cause of abnormal urea during chemotherapeutic treatment, to assess inherited
concentration is aided by calculation of the urea disorders of purine metabolism, to detect kidney
nitrogen/creatinine (urea N/creatinine) ratio, which is dysfunction, and to assist in the diagnosis of renal calculi.
normally 10:1 to 20:1.
● Prerenal conditions tend to elevate plasma urea, whereas ANALYTICAL METHODS
plasma creatinine remains normal, causing a high urea ● Uric acid is readily oxidized to allantoin and, therefore,
N/creatinine ratio. can function as a reducing agent in chemical reactions.
● A high urea N/creatinine ratio with an elevated creatinine ● The most common method of this type is the Caraway
is usually seen in postrenal conditions. method, which is based on the oxidation of uric acid in a
● A low urea N/creatinine ratio is observed in conditions protein-free filtrate, with subsequent reduction of
associated with decreased urea production, such as low phosphotungstic acid in alkaline solution to tungsten blue.
protein intake, acute tubular necrosis, and severe liver o The method lacks specificity.
disease. ● Methods using uricase (urate oxidase, EC 1.7.3.3), the
enzyme that catalyzes the oxidation of uric acid to
allantoin, are more specific and are used almost
TABLE 12.3 Causes of Abnormal exclusively in clinical laboratories.
Plasma Urea Concentration o The simplest of these methods measures the
differential absorption of uric acid and allantoin
Increased Concentration at 293 nm.
o The difference in absorbance before and after
incubation with uricase is proportional to the uric
Prerenal Congestive heart failure acid concentration.
Shock, hemorrhage o Proteins can cause high background absorbance,
Dehydration reducing sensitivity; hemoglobin and xanthine can
Increased protein catabolism cause negative interference.
High-protein diet ● Peroxidase or catalase (EC 1.11.1.6) is used to catalyze
a chemical indicator reaction.
Renal Acute and chronic renal failure o The color produced is proportional to the quantity of
Renal disease, including uric acid in the specimen.
glomerular nephritis and o Enzymatic methods of this kind have been adapted
tubular necrosis for use on traditional wet chemistry analyzers and
for dry chemistry slide analyzers.
Postrenal Urinary tract obstruction o Bilirubin and ascorbic acid, which destroy peroxide,
if present in sufficient quantity, can interfere.
Decreased Concentration o Commercial reagent preparations often include
potassium ferricyanide and ascorbate oxidase to
minimize these interferences.
Low protein intake ● HPLC (high-performance liquid chromatography)
Severe vomiting and diarrhea methods, typically using UV detection, have been
Liver disease developed.
Pregnancy ● IDMS has been proposed as a candidate reference
method.
URIC ACID
● Uric acid
o The product of catabolism of the purine nucleic acids.
o Filtered by the glomerulus and secreted by the distal
tubules into the urine.
o Most uric acid is reabsorbed in the proximal tubules
and reused.
o Relatively insoluble in plasma and, at high
concentrations, can be deposited in the joints and
tissue, causing painful inflammation.

BIOCHEMISTRY
● Reabsorption of 98% to 100% of the uric acid from the
glomerular filtrate occurs in the proximal tubules.
● Small amounts of uric acid are secreted by the distal
tubules into the urine.
● Renal excretion accounts for about 70% of uric acid
elimination; the remainder passes into the GI tract and is
degraded by bacterial enzymes.
● All of the uric acid in plasma is present as monosodium
urate.

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 MTY1209 | LEC Nonprotein Nitrogen Compounds

fluoride additives should not be used for specimens that

will be tested by a uricase method.
● Urine collections must be alkaline (pH 8).

REFERENCE INTERVALS
● Results expressed in conventional units of milligrams per
deciliter can be converted to SI units using the molecular
mass of uric acid (168 g/mol).

Uric Acid (Uricase Method)

FIGURE 12.3. Conversion of uric acid to allantoin.

Adult Plasma or
TABLE 12.4 Summary of Analytic Serum
Methods—Uric Acid
Chemical Methods Male 3.5-7.2 mg/dL 0.21-
0.43 mmol/L

Phosphotungst In carbonate Nonspecific;

ic acid solution(𝑁𝑎2 𝐶𝑂3 /𝑂𝐻− ), requires Female 2.6-6.0 mg/dL 0.16-
uric acid + protein
0.36 mmol/L
𝐻3 𝑃𝑊12 𝑂40 +𝑂2 → removal
allantoin + tungsten
blue + 𝐶𝑂2
Child 2.0-5.5 mg/dL 0.12-
Enzymatic Methods 0.33 mmol/L

Similar first Enzymatic production See Figure Adult Urine, 24 h 250-750 mg/d 1.5-
step ̶ catalyzed of allantoin from uric 12.3. 4.4 mmol/d
by uricase acid Very specific

Coupled 𝐻2 𝑂2 + indicator dye → Readily

enzymatic ̶ colored compound automated;
peroxidase reducing PATHOPHYSIOLOGY
agents ● Abnormally increased plasma uric acid concentration is
interfere found in gout, increased catabolism of nucleic acids, and
renal disease
Spectrophoto Decrease in Hemoglobin o Gout is a disease found primarily in men and is
metric absorbance at 293 nm and xanthine usually first diagnosed between 30 and 50 years of
measured interfere age. Affected individuals have pain and inflammation
of the joints caused by precipitation of sodium urates.
Other Methods o In 25% to 30% of these patients, hyperuricemia is a
result of overproduction of uric acid, although
hyperuricemia may be exacerbated by a purine-rich
Isotope Detection of Proposed diet, drugs, and alcohol.
dilution mass characteristic reference o Plasma uric acid concentration in affected
spectrometry fragments following method individuals is usually greater than 6.0 mg/dL.
ionization; Patients with gout are susceptible to the formation
quantification using of renal calculi, although not all persons with
isotopically labelled abnormally high serum urate concentrations develop
compound this complication.
▪ In women, urate concentration rises after
SPECIMEN REQUIREMENTS menopause. Postmenopausal women may
● Uric acid may be measured in heparinized plasma, develop hyperuricemia and gout. In severe
serum, or urine. Serum should be removed from cells as cases, deposits of crystalline uric acid and
quickly as possible to prevent dilution by intracellular urates called tophi form in tissue, causing
contents. deformities.
● Diet may affect uric acid concentration overall, but a ● Another common cause of elevated plasma uric acid
recent meal has no significant effect and a fasting concentration is increased metabolism of cell nuclei,
specimen is unnecessary. Gross lipemia should be as occurs in patients on chemotherapy for such
avoided. proliferative diseases as leukemia, lymphoma, multiple
● High bilirubin concentration may falsely decrease
results obtained by peroxidase methods. myeloma, and polycythemia.
● Significant hemolysis, with concomitant glutathione o Monitoring uric acid concentration in these patients
release, may result in low values. is important to avoid nephrotoxicity. Allopurinol,
● Drugs such as salicylates and thiazides have been which inhibits xanthine oxidase (EC 1.1.3.22), an
shown to increase values for uric acid enzyme in the uric acid synthesis pathway, is used
● Uric acid is stable in plasma or serum after red blood for treatment.
cells have been removed. ● Patients with hemolytic or megaloblastic anemia may
● Serum samples may be stored refrigerated for 3 to 5 exhibit elevated uric acid concentration.
days. Ethylenediaminetetraacetic acid (EDTA) or

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 MTY1209 | LEC Nonprotein Nitrogen Compounds
o Increased urate concentrations may be found
following ingestion of a diet rich in purines (e.g., liver,
kidney, sweetbreads, and shellfish) or as a result of
increased tissue catabolism due to inadequate
dietary intake (starvation)
● Inherited disorders of purine metabolism are
associated with significant increases in physiological uric
acid concentrations
o Lesch-Nyhan syndrome is an X-linked genetic
disorder (seen only in males) caused by the
complete deficiency of hypoxanthine–guanine
phosphoribosyltransferase (EC 2.4.2.8),
important enzyme in the biosynthesis of purines.
▪ Lack of this enzyme prevents the reutilization
of purine bases in the nucleotide salvage
pathway and results in increased de novo
synthesis of purine nucleotides and high
plasma and urine concentrations of uric
acid.
▪ Neurologic symptoms, mental retardation,
and selfmutilation characterize this extremely
rare disease. Mutations in the first enzyme in the
purine synthesis pathway,
phosphoribosylpyrophosphate synthetase (EC
2.7.6.1), also cause elevated uric acid
concentration.
▪ Increased uric acid is found secondary to
glycogen storage disease (deficiency of
glucose-6-phosphatase, EC 3.1.3.9) and
fructose intolerance (deficiency of fructose-1-
phosphate aldolase, EC 2.1.2.13).
▪ Metabolites such as lactate and triglycerides
are produced in excess and compete with urate
for renal excretion in these diseases.
● Hyperuricemia as a result of decreased uric acid
excretion is a common feature of toxemia of pregnancy
(preeclampsia) and lactic acidosis presumably as a result
of competition for binding sites in the renal tubules.
o Chronic renal disease causes elevated uric acid
concentration because filtration and secretion are
impaired.
● Uric acid nephrolithiasis, the formation of kidney stones
(renal calculi), may occur due to a variety of predisposing
factors and conditions.
o In acidic urine, the relatively insoluble uric acid
precipitates to form calculi, which can cause intense
flank pain.
o The stones may be dissolved by alkalinization of the
urine or treated by increased fluid intake and
administration of xanthine oxidase inhibitors to
reduce uric acid production.
● Hypouricemia is less common than hyperuricemia and
is usually secondary to severe liver disease or defective
tubular reabsorption, as in Fanconi syndrome (a disorder
of reabsorption in the proximal convoluted tubules of the
kidney).
o Decreased plasma uric acid can be caused by
chemotherapy with 6- mercaptopurine
azathioprine, inhibitors of de novo purine synthesis,
and as a result of overtreatment with allopurinol.
o Some studies have shown an association between
low uric acid concentrations and neurodegenerative
conditions such as Alzheimer's and Parkinson's
diseases.
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Table 12.5 Causes of Abnormal Plasma Uric Acid
Concentration
Increased Concentration
Gout
Treatment of myeloproliferative disease with cytotoxic
drugs
Hemolytic and proliferative processes
an Purine-rich diet
Increased tissue catabolism or starvation
Enzyme deficiencies
Lesch-Nyhan syndrome (hypoxanthine guanine
phosphoribosyl transferase deficiency)
Phosphoribosylpyrophosphate synthetase deficiency
Glycogen storage disease type I (glucose-6-
phoosphatase deficiency)
Fructose intolerance (fructose-1-phosphate aldolase
deficiency)
Toxemia of pregnancy
Lactic acidosis
Chronic renal disease
Drugs and poisons
Decreased Concentration
Liver disease
Defective tubular reabsorption (Fanconi syndrome)
Chemotherapy with azathioprine or 6-mercaptopurine
Overtreatment with allopurinol
CREATININE / CREATINE
● Creatinine is formed from creatine and creatine
phosphate in muscle and is excreted into the plasma at a
constant rate related to muscle mass.
o Plasma creatinine is inversely related to glomerular
filtration rate (GFR) and, although an imperfect
measure, it is commonly used to assess renal
filtration function.
BIOCHEMISTRY
● Creatine is synthesized primarily in the liver from
arginine, glycine, and methionine.
o It is then transported to other tissues, such as
muscle, where it is converted to creatine
phosphate, which serves as a high-energy source.
o Creatine phosphate loses phosphoric acid and
creatine loses water to form the cyclic compound,
creatinine, which diffuses into the plasma and is
excreted in the urine.
Figure 12.4 Interconversion of creatine, creatine
phosphate, and creatinine.
or
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 MTY1209 | LEC Nonprotein Nitrogen Compounds
● Creatinine is released into the circulation at a relatively
constant rate that has been shown to be proportional to
an individual's muscle mass. It is removed from the
circulation by glomerular filtration and excreted in the
urine.
o Small amounts of creatinine are secreted by the
proximal tubule and reabsorbed by the renal tubules
o Daily creatinine excretion is reasonably stable.
CLINICAL APPLICATION
● Measurement of creatinine concentration is used to
determine the sufficiency of kidney function, to
determine the severity of kidney damage, and to
monitor the progression of kidney disease
● Plasma creatinine concentration is a function of
relative muscle mass, the rate of creatine turnover, and
renal function.
o Small amounts of creatinine are secreted by the
proximal tubule and reabsorbed by the renal
tubules
o The amount of creatinine in the bloodstream is
reasonably stable, although the protein content of
the diet does influence the plasma concentration.
o Because of the constancy of endogenous
production, urinary creatinine excretion has been
used as a measure of the completeness of 24-hour
urine collections in a given individual, although the
uncertainty associated with this practice may exceed
that introduced by use of the urine volume and
collection time for standardization.
▪ Urinary constituents may be expressed as a
ratio to creatinine quantity rather than as mass
excreted per day.
● Creatinine clearance (CrCl), a measure of the amount
of creatinine eliminated from the blood by the kidneys,
and GFR are used to gauge renal function.
● The GFR is the volume of plasma filtered (V) by the
glomerulus per unit of time (t):
v
GFR =
t (Eq.12-2)
● Assuming a substance, S, can be measured and is freely
filtered at the glomerulus and neither secreted nor
reabsorbed by the tubules, the volume of plasma filtered
would be equal to the mass of S filtered (MS) divided by
its plasma concentration (PS):
𝑀𝑠
𝑉 = 𝑃𝑠 (Eq. 12-3)
● The mass of S filtered is equal to the product of its urine
concentration (US) and the urine volume (VU):
𝑀𝑠 = 𝑈𝑠𝑉𝑠 (Eq. 12-4)
● If the urine and plasma concentrations of S, the volume
of urine collected, and the time over which the sample
was collected are known, the GFR can be calculated:
𝑈𝑠𝑉𝑢
𝐺𝐹𝑅 = 𝑃𝑠𝑡
(Eq. 12-5)
● The clearance of a substance is the volume of plasma
from which thatsubstance is removed per unit time. The
formula for CrCl is given as follows, where UCr is urine
creatinine concentration and PCr is plasma creatinine
concentration:
𝑈𝑐𝑟 𝑉𝑢
𝐶𝑟𝐶𝑙 =
𝑃𝑐𝑟𝑡 (Eq. 12-6)
o CrCl is usually reported in units of mL/min and can
be corrected for body surface area (see Chapter 27).
CrCl overestimates GFR because a small amount of
creatinine is reabsorbed by the renal tubules and up
to 10% of urine creatinine is secreted by the tubules.
However, CrCl provides a reasonable approximation
of GFR.
o The observed relationship between plasma
creatinine and GFR and relatively constant plasma
creatinine concentrations should make the analyte a
good endogenous filtration marker. However,
measurement of plasma creatinine does not provide
CGM NIEVES, MKR SIBUG, RJKM REYES | 2021
sufficient sensitivity for the detection of mild renal
dysfunction.
● Clinical laboratories have been strongly encouraged to
report an estimated GFR when serum creatinine is
ordered as a means to increase identification of kidney
disease and improve patient care. Initially, the
abbreviated Modification of Diet in Renal Disease
(MDRD) equation was advocated.
o When serum creatinine is measured using an IDMS-
traceable method, the MDRD equation for
estimated glomerular filtration rate (eGFR) is
(Eq. 12-7)
● The CKDEPI equation is used to report higher values
(>60 mL/min/1.73 m2) for adults 18 years and older. A
modified Schwartz equation has been developed to
calculate eGFR in the pediatric population. The equation
is
(Eq. 12-8)
ANALYTICAL METHODS
CREATININE
● The methods most frequently used to measure creatinine
are based on the Jaffe reaction first described in 1886
● In this reaction, creatinine reacts with picric acid in
alkaline solution to form a red-orange chromogen. The
reaction was adopted for the measurement of blood
creatinine by Folin and Wu in 1919.
● The reaction is nonspecific and subject to positive
interference by a large number of compounds, including
acetoacetate, acetone, ascorbate, glucose, and
pyruvate.
● More accurate results are obtained when creatinine in a
protein-free filtrate is adsorbed onto Fuller's earth
(aluminum magnesium silicate) or Lloyd's reagent
(sodium aluminum silicate), then eluted and reacted
with alkaline picrate
● This method is time consuming and not readily
automated; it is not routinely used.
● In the kinetic Jaffe method, serum is mixed with alkaline
picrate and the rate of change in absorbance is measured
● The kinetic Jaffe method is used routinely despite these
problems because it is inexpensive, rapid, and easy to
perform.
● In an effort to enhance the specificity of the Jaffe reaction,
several coupled enzymatic methods have been
developed. The method using creatininase (creatinine
amidohydrolase, EC 3.5.2.10), creatinase (creatine
amidinohydrolase,EC 3.5.3.3), sarcosine oxidase (EC
1.5.3.1), and peroxidase (EC 1.11.1.7) was adapted for
use on a dry slide analyzer
● IDMS is used as a reference method. Assays used on
automated analyzers are designated as “traceable”
(calibrated) to an IDMS method.
Summary of Analytic Methods for creatinine is summarized on
Table 12.6 (last page)
SPECIMEN REQUIREMENTS
● Creatinine may be measured in plasma, serum, or urine.
● Hemolyzed and icteric samples should be avoided,
particularly if a Jaffe method is used.
● Lipemic samples may produce erroneous results in
some methods.
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 MTY1209 | LEC Nonprotein Nitrogen Compounds

● A fasting sample is not required, although high-protein

ingestion may transiently elevate serum concentrations.
● Urine should be refrigerated after collection or frozen if
longer storage than 4 days is required
SOURCES OF ERROR
● Ascorbate, glucose, α-keto acids, and uric acid may
increase creatinine concentration measured by the Jaffe
reaction, especially at temperatures above 30°C.
● Bilirubin causes a negative bias in both Jaffe and
enzymatic methods. Ascorbate will interfere in
enzymatic methods that use peroxidase as a reagent.
● Patients taking cephalosporin antibiotics may have
falsely elevated results when the Jaffe reaction is used.
Other drugs have been shown to increase creatinine
results.
● Dopamine, in particular, is known to affect both
enzymatic and Jaffe methods. Lidocaine causes a
positive bias in some enzymatic methods
CREATININE / CREATINE
● The traditional method for creatine measurement relies
on the analysis of the sample using an endpoint Jaffe
method for creatinine before and after it is heated in acid
solution.
● Heating converts creatine to creatinine and the
difference between the two sample measurements is the
creatine concentration.
● High temperatures may result in the formation of
additional chromogens and the precision of this method
is poor.
o Several enzymatic methods have been developed;
one is the creatininase assay. The initial enzyme is
omitted and creatine kinase (EC 2.7.3.2), pyruvate
kinase (EC 2.1.7.40), and lactate dehydrogenase
(EC 1.1.1.27) are coupled to produce a measurable
colored product.
o Creatine can be measured by HPLC
REFERENCE INTERVALS
● Reference intervals vary with assay type, age, and
gender. Creatinine concentration decreases with age
beginning in the 5th decade of life.
CREATININE
ADULT PLASMA JAFFE ENZYMATIC
OR METHOD METHOD
SERUM
PATHOPHYSIOLOGY
CREATININE
● Elevated creatinine concentration is associated with
abnormal renal function, especially as it relates to
glomerular function.
● Plasma concentration of creatinine is inversely
proportional to the clearance of creatinine
o Therefore, when plasma creatinine concentration is
elevated, GFR is decreased, indicating renal
damage.
o Plasma creatinine is a relatively insensitive marker
and may not be measurably increased until renal
function has deteriorated more than 50%.
CREATINE
● In muscle disease such as muscular dystrophy,
poliomyelitis, hyperthyroidism, and trauma, both
plasma creatine and urinary creatinine are often
elevated.
● Measurement of creatine kinase is used typically for the
diagnosis of muscle disease because analytic methods
for creatine are not readily available in most clinical
laboratories.
● Plasma creatine concentration is not elevated in renal
disease
AMMONIA
● Ammonia is produced in the deamination of amino acids
during protein metabolism
● It is removed from the circulation and converted to
urea in the liver. Free ammonia is toxic; however,
ammonia is present in the plasma in low concentrations.
BIOCHEMISTRY
● Ammonia (NH3) is produced in the catabolism of amino
acids and by bacterial metabolism in the lumen of the
intestine
o Some endogenous ammonia results from anaerobic
metabolic reactions that occur in skeletal muscle
during exercise.
o Ammonia is consumed by the parenchymal cells of
the liver in the Krebs- Henseleit or urea cycle to
produce urea, a nontoxic compound that is excreted
in the urine.
o At normal physiologic pH, most ammonia in the
blood exists as ammonium ion (NH4+).
o Ammonia is excreted as ammonium ion by the
kidney and acts to buffer urine.

Male 0.9-1.3 0.6-1.1

mg/dL mg/dL (53-
(80- 97μmol/L) FIGURE 12.5 Interconversion of ammonium ion and
115μmol/L) ammonia

Female 0.6-1.1 0.5-0.8 CLINICAL APPLICATION

mg/dL mg/dL ● Clinical conditions in which blood ammonia concentration
(53- (44-71 provides useful information are hepatic failure, Reye's
97μmol/L) μmol/L) syndrome, and inherited deficiencies of urea cycle
enzymes
Child 0.3-0.7 0.0-0.6 ● Severe liver disease is the most common cause of
mg/dL mg/dL disturbed ammonia metabolism
(27-62 (0-53 μmol/L) o The monitoring of blood ammonia may be used to
μmol/L) determine prognosis, although correlation between
the extent of hepatic encephalopathy and plasma
Adult Urine, 24 ammonia concentration is not always consistent.
h ● Reye's syndrome, occurring most commonly in children,
is a serious disease that can be fatal. Frequently, the
Male 800- disease is preceded by a viral infection and the
2000,mg/d administration of aspirin.
(7.1-17.7 o Reye's syndrome is an acute metabolic disorder of
mmol/d) the liver, and autopsy findings show severe fatty
infiltration of that organ
Female 600-1,800 o Blood ammonia concentration can be correlated
mg/d (5.3- with both the severity of the disease and prognosis.
15.9 mmol/d) Survival reaches 100% if plasma NH3 concentration
remains below five times normal

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 MTY1209 | LEC Nonprotein Nitrogen Compounds

● Ammonia is of use in the diagnosis of inherited

deficiency of urea cycle enzymes. and NADPH accurate
to form and precise
● Assay of blood ammonia can be used to monitor
glutamate and
hyperalimentation therapy and measurement of urine
NADP-, which
ammonia can be used to confirm the ability of the kidneys
is detected
to produce ammonia.
spectrophoto
ANALYTICAL METHODS
metrically
● The accurate laboratory measurement of ammonia in
plasma is complicated by its low concentration,
instability, and pervasive contamination
● Two approaches have been used for the measurement of SPECIMEN REQUIREMENTS
plasma ammonia. ● Whole blood ammonia concentration increases rapidly
o One is a two-step approach in which ammonia is following specimen collection because of in vitro amino
isolated from the sample and then assayed. acid deamination.
o The second involves direct measurement of ● Venous blood should be obtained without trauma and
ammonia by an enzymatic method or ion-selective placed on ice immediately.
electrode. Assays detect NH3 or NH4+ ● Heparin and EDTA are suitable anticoagulants.
● One of the first analytic methods for ammonia, developed Commercial collection containers should be evaluated for
by Conway in 1935, exploited the volatility of ammonia ammonia interference before a new lot is put into use
to separate the compound in a microdiffusion chamber ● Samples should be centrifuged at 0 to 4°C within 20
o Ammonia gas from the sample diffuses into a minutes of collection and the plasma removed.
separate compartment and is absorbed in a solution ● Specimens should be assayed as soon as possible or
containing a pH indicator. The amount of ammonia is frozen. Frozen plasma is stable for several days at
determined by titration −20°C. Erythrocytes contain two to three times as much
● Ammonia can be measured by an enzymatic method ammonia as plasma; hemolysis should be avoided.
using GLDH. This method is convenient and the most ● Cigarette smoking by the patient is a significant source
common technique used currently. of ammonia contamination. It is recommended that
1. NADPH is the preferred coenzyme patients do not smoke for several hours before a
because it is used specifically by GLDH; specimen is collected
NADH will participate in reactions of other ● Many substances influence the in vivo ammonia
endogenous substrates, such as pyruvate. concentration. Ammonium salts, asparaginase,
2. Adenosine diphosphate is added to the barbiturates, diuretics, ethanol, hyperalimentation,
reaction mixture to increase the rate of the narcotic analgesics, and some other drugs may
reaction and to stabilize GLDH.61 This increase ammonia in plasma.
method is used on many automated ● Diphenhydramine, Lactobacillus acidophilus,
systems and is available as a prepared kit lactulose, levodopa, and several antibiotics decrease
from numerous manufacturers concentrations.
● A dry slide automated system uses a thin-film ● Glucose at concentrations greater than 600 mg/dL
colorimetric assay.62 In this method, ammonia reacts (33 mmol/L) interferes in dry slide methods.
with an indicator to produce a colored compound that is SOURCES OF ERROR
detected spectrophotometrically ● Ammonia contamination is a potential problem in the
● Direct measurement using an ion-selective electrode laboratory measurement of ammonia.
has been developed. The electrode measures the o Precautions must be taken to minimize
change in pH of a solution of ammonium chloride as contamination in the laboratory in which the assay is
ammonia diffuses across a semipermeable membrane. performed
o Elimination of sources of ammonia
Summary of the analytical methods for ammonia are contamination can significantly improve the
summarized in Table 12.7 (last page) accuracy of ammonia assay results
o Sources of contamination include tobacco smoke,
TABLE 12.7 SUMMARY OF ANALYTIC METHODS- urine, and ammonia in detergents, glassware,
AMMONIA reagents, and water.
● The ammonia content of serum-based control material is
unstable.
Chemical Methods Diffusion of Good
o Frozen aliquots of human serum albumin
Ion-selective electrode NH3 through accuracy
containing known amounts of ammonium chloride or
selective and
ammonium sulfate may be used.
membrane precision;
REFERENCE INTERVALS
into NH4Cl membrane
causes a pH stability may ● Values obtained vary somewhat with the method used.
change, be a protein Higher concentrations are seen in newborns
which is
measured AMMONIA
potentiometric
ally Adult Plasma 19-60μg/dL 11-35
μmol/L
Spectrophotometric NH3+
bromophenol Urine, 24 h 140-1,500 10-107
blue -> blue mg N/d mmol N/d
color
Child (10 d Plasma 68-136 40-80
Enzymatic Methods to 2 y) μg/dL μmol/L
Catalyzed by GLDH Enzymatic Most
reaction of common on
NH4+ 2- automated PATHOPHYSIOLOGY
oxoglutarate, instruments; ● In severe liver disease in which there is significant
collateral circulation (as in cirrhosis) or if parenchymal
CGM NIEVES, MKR SIBUG, RJKM REYES | 2021 Page 8 of 10
 MTY1209 | LEC Nonprotein Nitrogen Compounds

liver cell function is severely impaired, ammonia is not

removed from the circulation and blood concentration
increases.
o High concentrations of NH3 are neurotoxic and
often associated with encephalopathy.
o Ammonia alters metabolic processes in the brain,
which results in accumulation of toxic species, and
impairs astrocyte function
● Hyperammonemia is associated with inherited
deficiency of urea cycle enzymes
● Measurement of plasma ammonia is important in the
diagnosis and monitoring of these inherited
metabolic disorders

References

Bishop, M.L.; Fody E.P. & Schoeff L.E. (2018). Total Protein Abnormalities
& Methods of Analysis. Clinical Chemistry: Principles, Techniques, and
Correlations (8th edition). Page 638-666

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