Urine Analysis I: Chemical Examination: Lenka Fialová & Martin Vejražka
Urine Analysis I: Chemical Examination: Lenka Fialová & Martin Vejražka
Urine Analysis I: Chemical Examination: Lenka Fialová & Martin Vejražka
LF UK
Urine analysis I:
Chemical examination
General Medicine
2018/2019
Chemical analysis of urine
Urine is a biological fluid whose analysis provides valuable information about condition of the human
body and its metabolic state. Examination of urine is one of the basic procedures in clinical chemistry
that significantly contributes to diagnostic process, as well as monitoring of disease course and effects
of therapy. Analysis of urine employs a wide array of techniques ranging from the simplest colored
and precipitating test-tube reactions to very sophisticated and automated ones such as flow cytometry
and computer analysis of urinary sediment. Test strips enable basic examination of urine not only in
the laboratory, but also directly in the consulting rooms or at patient’s beds in the hospitals.
This material describes the usual procedures of routine analysis of urine.
1. Collection of urine
In order to get valid results from urine analysis, an appropriate and correct technique of urine sample
collection is essential.
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Chemical analysis of urine
The routine chemical examination of urine involves qualitative tests for protein, glucose, hemoglobin,
ketone bodies and bile pigments. These components are mostly present in urine from healthy
individuals as well, but in tiny amounts undetectable by the routine tests. Various pathological
conditions increase their concentration in urine.
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Chemical analysis of urine
In the past, colored/precipitating reactions performed as ‘wet chemistry’, i.e. in test-tubes, were used
for detection of the pathologic components of urine. Some of these classical reactions have been
already introduced in the previous practical lessons (which see), and their principles are summarized
in table 1.
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Chemical analysis of urine
The test strips (diagnostic strips) enable detection of pathological urine components directly at
patient’s bed or in physician’s consulting room. The strips consist of a plastic support onto which one
or more indication zones are attached. In manufacture of the indication zones, a liquid analytic reagent
is applied on a suitable absorbent (e.g. special filtration paper), and then gently dried. In Czech
Republic the strips PHAN made by Lachema/Pliva a.s. are the most widely used.
There are various types of the diagnostic strips: they come either as monofunctional, or polyfunctional,
or strips for specialised examinations.
Monofunctional strips contain basic indication zones for semiquantitative estimation of one urine
component. Polyfunctional strips bear several indication zones and hence enable examination of
several biochemical parameters simultaneously. They are designed for situations where it is useful to
obtain as much information on the patient’s health condition as possible, such as in various screening
examinations.
Strips for specialized examinations include two or more indication zones chosen with respect to
screening for or examination of a specific disease. For example strips for diabetes mellitus screening
contain zones for ketone bodies and glucose, strips for renal diseases have zones for blood, protein, pH
and nitrite, and strips for hepatic diseases examine bilirubin and urobilinogen.
The strips enable estimation of the following parameters in urine:
protein ascorbic acid
glucose leukocytes
ketone bodies nitrites
bilirubin pH
urobilinogen specific gravity
hemoglobin, red blood cells
The reactions involved in test strips detection are largely based on similar principles as the classical
test-tube ones (table 1, table 2). Further we discuss in detail only the reactions not introduced in the
previous practical lessons.
Ascorbic acid
Ascorbic acid (vitamin C) is examined because of its strong reducing properties that affect estimation
of other analytes in urine. In particular, it interferes with reactions employing hydrogen peroxide,
which reduces directly; and it also decomposes diazonium salts.
The principle of ascorbic acid detection utilises phosphomolybdenic acid, which is reduced with
ascorbic acid to molybdenum blue. The test is not specific for ascorbic acid as other strong reducing
agents would react in a similar manner.
Leukocytes
Chemical detection of leukocytes with the diagnostic strip is based on demonstration of enzymes
esterases present in granulocytes. The esterase hydrolyses ester of indoxylcarbonic acid to indoxyl,
which reacts with a stable diazonium salt yielding the corresponding azo dye (Fig. 1). In normal
(negative) reaction the strip zone is colored creamy yellow; in case of positive reaction it turns pink or
violet. The chemical demonstration of leukocytes cannot substitute the microscopic examination of
urinary sediment. On the other hand, however, the biochemical examination can also find lysed
leukocytes (e.g. in hypotonic urine), which is not possible with microscopy.
Leukocyturia comes generally as a sign of kidney or urinary tract inflammation. Majority of positive
findings are caused by a bacterial infection of the urinary tract. In case of a positive leukocyte test it is
recommended to perform also examination of proteinuria, hematuria, nitrituria, examination of the
urinary sediment and microbiological examination.
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Chemical analysis of urine
Leukocytic esterase
Ester of indoxyl
Azo dye
Diazonium salt
Violet color
Bacteria:
NO3 → NO2
Nitrite (bacteria)
Sulfanilamide Diazonium salt
Diazonium salt
Azo dye
Coupling reagent
Pink to violet color
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Chemical analysis of urine
Table 2: Principles of detection of pathological components in urine with the test strips, and
common causes of interference
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Chemical analysis of urine
In some inborn errors urine has a typical smell caused by the abnormal metabolite accumulation.
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Chemical analysis of urine
Reaction of sodium nitroprusside with sulfhydryl compounds (e.g. cysteine, homocysteine) yields
rose or purple-red complex products:
[FeII(CN)5NO]2 + S2 → [FeII(CN)5NOS]4
Disulfides (cystine, homocystine) do not give the reaction and therefore must be first reduced to
free sulfhydryls by alkaline sodium cyanide.
Cystinuria is one of inborn errors caused by a disorder in the transport of amino acids in the kidney and gut.
It is caused by a defect in the reverse transport of cystine, lysine, arginine and ornithine in the epithelial cells
of renal tubuli and digestive tract. Biochemical tests reveal increased urinary excretion of all the mentioned
amino acids. Cystine urolithiasis is the major clinical sign; it is caused by a low water solubility of cystine.
The presence of cystine in urine can be demonstrated by the Brand’s test with sodium nitroprusside.
2,4-dinitrophenylhydrazine in acidic medium reacts with 2-oxo acids (-keto acids) and forms
precipitates of hydrazones. Positive test indicates elevated excretion of keto acids or other ketone
bodies in urine.
Leucinosis (maple syrup urine disease) develops due to defect in the enzyme system performing oxidative
decarboxylation of branched-chain α-ketoacids. High levels of leucine, valine and isoleucine are found in the
serum. Oxo-acids that are formed by transamination of these amino acids (2-oxoisovalerate, 2-oxo-3-
methylvalerate and 2-oxoisocapronate) are excreted in urine in amounts 10-100 higher than normal. The oxo-
acids in urine can be demonstrated by the reaction with 2,4-dinitrophenylhydrazine.