PRACTICAL PART Flashcards
Estimation of Hemoglobin in blood
The photometric assay is based on oxidation of ferrous iron in hemoglobin to ferric iron
with potassium ferricyanide. The resulting methemoglobin is converted in the next
reaction with potassium cyanide to a very stable cyanmethemoglobin with a broad
absorption maximum at 540 nm
1. Oxidation of hemoglobin to methemoglobin:
HbFe2+ + [Fe3+(CN)6]3− ® HbFe3+ + [Fe2+(CN)6]4−
Hemoglobin methemoglobin
2. Conversion of methemoglobin to cyanmethemoglobin:
HbFe3+ + CN− ® HbFe3+ CN
methemoglobin cyanmethemoglobin
Evaluation:
Estimation of hemoglobin in the blood is one of the most essential laboratory
examinations. When hemoglobin and/or number of erythrocytes below the lower limit of
reference values are found, the term anemia is used. It is a common finding in clinical practice. Anemia leads to low transport capacity for oxygen and impairment of tissue
respiration.
Anemia in principle results from any condition in which erythropoiesis does not match the
body demands for red blood cells. It develops due to:
• Blood loss (acute or chronic bleeding)
• Increased destruction of red blood cells (autoimmune, enzymes, membrane or shape)
• Insufficient production of red blood cells(any factors needed, infectious inflammations chemical physical irritations). See causes of anemia.
Reference:
Reference values for hemoglobin in blood:
Healthy adult man: 130-180 g/l
Healthy adult woman, children: 120-160 g/l
Estimation of total serum protein.
The biuret reaction is used. Proteins form a violet complex with cupric salts in alkali,
suitable for photometric estimation. The resulting complex of Cu2+ ions with peptidic
bonds strongly absorbs light at 540-560 nm. The color intensity is proportional to the
concentration of protein. In general, substances containing at least two groups –CO-NH2 or
Proteins in serum and urine
7
–CO-NH- give the biuret reaction, i.e. the reaction is not specific for proteins. The simplest
compound giving the reaction with cupric salts in alkaline medium is biuret NH2-CO-NHCO-
NH2 (dimer of urea), hence the name of the reaction. Amino acids and dipeptides do
not react.
Reference values (S-protein): 65 – 85 g/l
Hyper or hypo proteinemia,
The basic examination of proteins in serum or plasma is an estimation of their total
concentration, called ‘total serum protein’. If a pathologic value is found, or in other
indicated cases, further detailed examination follows, encompassing electrophoresis of
serum proteins, immunofixation and targeted estimation of concentration of selected
proteins.
Estimation of total protein is a common and affordable test providing basic information on
protein synthesis, utilization and excretion. Many diseases alter the spectrum of serum
proteins, but only some of them alter also the total protein level.
Changes in total serum protein:
Hypoproteinemia (serum protein is low):
Absolute: amount of serum protein is decreased due to
a) high loss by:
kidney
gastrointestinal tract (e.g. intestinal inflammation)
skin (burns)
bleeding
to the ‘third space’ (e.g. abdominal cavity in ascites)
b) low protein biosynthesis (chronic liver diseases)
c) insufficient intake of protein (malnutrition)
Relative: in hyperhydration state the actual amount of serum protein is actually
unchanged, but the protein is diluted due to retention of water and salts. Concentrations of
all particular proteins are decreased proportionally.
Hyperproteinemia (serum protein is high):
Absolute: usually caused by an increased production of certain specific proteins,
such as immunoglobulins in plasmocytoma.
Relative: comes in dehydration state due to insufficient intake or excessive loss of
fluids (diarrhoe, vomiting). The total amount of protein is preserved, but apparent
concentrations of all particular proteins are proportionally increased.
Estimation of total bilirubin in serum.
Almost all tests for bilirubin are based on the azo coupling reaction of bilirubin with some
diazonium salt. Diazotized sulfanilic acid, produced by reacting sulfanilic acid with
sodium nitrite in excess of hydrochloric acid (diazo reagent)1, is used most often – the Van
den Bergh diazoreaction. In the presence of diazotized sulfanilic acid bilirubin splits to
yield two molecules of azobilirubin (Fig. 1). The azobilirubin behaves as an acid-base
indicator with several color transitions: in weakly acidic medium it is red, whereas in
strong alkali it has blue color.
Conjugated bilirubin reacts with diazotized sulfanilic acid directly and rapidly – direct
Van den Bergh reaction. This fraction of bilirubin is therefore called “direct” bilirubin.
Unconjugated bilirubin, which is poorly soluble in water and non-covalently bound to
albumin, reacts with diazo reagent much more slowly and the color develops only after
addition of an accelerator – indirect Van den Bergh reaction. As the accelerator,
alcohols (methanol, ethanol) or other substances (sodium benzoate, caffeine, urea) are
used. The accelerator facilitates reaction by releasing bilirubin from bond to albumin and
disrupts intramolecular hydrogen bridges, rendering bilirubin more soluble. Fraction of
bilirubin that requires an accelerator to give the reaction is called “indirect” bilirubin.
In the presence of accelerator all forms of bilirubin react; hence, in this way total bilirubin
is estimated. The actual amount of unconjugated (indirect) bilirubin can be calculated as a
difference between total and direct bilirubin (Fig. 2).
Photometric estimation of total bilirubin is best performed in alkaline medium, where it is
the most sensitive, and interference of other compounds in serum is minimal. The basic pH
is provided by strongly alkaline tartrate. Nowadays, the recommended approach for routine
estimation is the Jendrassik-Grof modification, utilizing caffeine and sodium benzoate as
the accelerator.
1 The Ehrlich diazo reagent: Diazo I: sulfanilic acid in HCl, diazo II: sodium
Reference values:
fS-Total bilirubin: up to 17 μmol/l
fS-Direct bilirubin: up to 5 μmol/l
If concentration of serum bilirubin exceeds 17 μmol/l, we talk about hyperbilirubinemia.
It can in principle arise either from overproduction of bilirubin, or from defects in its
metabolism and secretion. Bilirubin passes from blood stream to the tissues and if
bilirubinemia exceeds about 35 μmol/l, it brings about a yellow color of the eye white and
the skin – condition known as jaundice, icterus. Because bilirubin is degraded by light,
the yellowish color is first visible only on mucosal membranes and on the parts of sclera
covered by eyelids (subicterus). Color of skin in moderate hyperbilirubinemias need not be
explicitly yellow; rather it can resemble a sun tan.
We can distinguish three main types of hyperbilirubinemias:
• unconjugated: pre-hepatic (hemolytic) icterus
- increased bilirubin production, e.g. hemolytic anemia
- decreased uptake and/or conjugation in the liver, e.g. jaundice of the newborn,
some inborn errors such as Gilbert’s disease, Criegler-Najjar’s syndrome
• conjugated: post-hepatic (obstructive) icterus
- biliary obstruction, e.g. due to cholelithiasis (stones in gall bladder or biliary
ducts) or tumors of biliary tract or pancreas
- impairment of the secretion of conjugated bilirubin, e.g. some inborn errors
such as Dubin-Johnson’s and Rotor’s syndromes
• mixed: hepatocellular (hepatic) icterus
- e.g. viral hepatitis, toxic liver damage
Estimation of triacylglycerols in serum.
The recommended methods for the estimation of serum triacylglycerols include several enzymatic
reactions: Triacylglycerols are first hydrolyzed by lipoprotein lipase to produce glycerol and free
fatty acids. Glycerol is converted to glycerol-3 phosphate by glycerol kinase in the presence of
ATP. In the next enzymatic reaction catalysed by glycerol-3-phosphate oxidase glycerol-3
phosphate is oxidised to produce dihydroxyacetone phosphate and H2O2. Finally, horseradish
peroxidase uses the hydrogen peroxide for oxidation of a chromogen to yield a colour product,
measurable spectrophotometrically (Fig. 2)
Draw diagram page 8 of lipoproteins
Reference values of serum triacylglycerols: 0.45 – 1.7 mmol/l
Triacylglycerol values depend on the prandial state. Higher levels occur after meal, therefore, blood
for triacylglycerol determination should be taken after a 14-hour fasting.
Evaluation of serum triacylglycerol concentration with respect to the risk of atherosclerosis:
Elevated triacylglycerols > 1.7 mmol/l (fasting) are an independent risk factor for atherosclerosis.
Increased triacylglycerol concentration tends to be associated with decreased HDL cholesterol.
Estimation of total cholesterol in serum.
The commonest methods for estimation of cholesterol are enzymatic assays. The first step in the
enzymatic methods is hydrolysis of cholesterol esters by cholesterol esterase to produce free
cholesterol and fatty acids. In the presence of oxygen the released free cholesterol together with
the one initially present in the sample is then oxidised by cholesterol oxidase to 4-cholesten-3-one
and H2O2.
The final step again uses the ability of produced H2O2 to oxidise various compounds to coloured
products in the presence of peroxidase. The approach most widely used for routine cholesterol
testing is based on the reaction of phenol with 4-aminoantipyrine forming a quinonimine dye (red
colour). The intensity of a red dye is proportional to the cholesterol concentration in the sample
Draw diagram page 9 of lipoproteins
Reference values: 2.9 – 5.0 mmol/l
Evaluation of total cholesterol concentration with respect to the risk of atherosclerosis:
Cholesterol values in serum < 5.0 mmol/l indicate the risk of coronary heart disease is low. The
determination of total cholesterol is sufficient for screening purposes. If the concentration of total
cholesterol is above 5.0 mmol/l, it is necessary to measure LDL and HDL cholesterol.
Fasting is not required if only total cholesterol is measured.
Estimation of calcium in serum.
Estimation of serum calcium represents a basic screening examination for assessment of calcium
homeostasis.
Calcium in serum exists in several forms (Fig. 3):
• 60 % of total calcium is diffusible – filtered by renal glomeruli. From this fraction:
- 50 % of total calcium is in free (ionized) form (denoted as Ca2+). This is the
biologically active form of calcium.
- 10 % of total calcium occurs in low-molecular-weight complexes with citrate,
phosphate or hydrogen carbonate
• 40 % of total calcium is not diffusible (does not pass the glomerular membrane) as it is
bound to plasma proteins (90 % to albumin, 10 % to globulins). The protein-bound calcium is
not biologically active, but rather it represents a readily accessible reserve from which calcium
can be quickly released during hypocalcemia.
In hypoalbuminemia the calcium fraction bound to albumin decreases. Drop of plasmatic albumin
of 10 g/l makes the total serum calcium level 0.2 mmol/l lower without any effect on the plasma
concentration of ionized calcium. On the other hand, hyperproteinemia (e.g. in malign myeloma)
may lead to a high increase in total calcemia, again without change in ionized calcium level.
Therefore, both parameters, i.e. serum calcium and albumin, should be considered together. The
amount of Ca2+ depends on pH: it decreases in alkalosis and increases in acidosis, due to mutual
competition of Ca2+ and H+ ions for the binding sites on albumin.
Reference values:
Normally, concentration of total calcium in the serum (S-Ca, calcemia) is kept within a rather
narrow range of 2.25 – 2.75 mmol/l. Calcemia around 4 – 5 mmol/l is considered as the upper limit
of serum calcium concentration compatible with life. Death in hypercalcemia is due to heart arrest.
On the other hand, the lower limit of calcemia still compatible with life lies around 1 mmol/l.
Severe hypocalcemia leads to convulsions (tetania).
One of the methods recommended for routine
measurements of total calcium in serum and
urine is based on spectrophotometry of
colored complexes, formed in a reaction of
calcium with a suitable chelating substance.
One such complex-forming substance in use is
o-cresolphthalexone: with calcium ions in
alkaline medium it produces a violet complex;
the color of the solution is proportional to
concentration of calcium ions. Another such
complex-forming agent in use is a metallochromogene
arsenazo III.
Estimation of total calcium in serum is one of the tests suffering from relatively big errors of
measurement. The complex-forming agent does not bind all the calcium present in the blood
sample because it has to compete with plasmatic proteins and other calcium chelating substances in
blood. Significant deviations from normal composition of blood (e.g. pronounced dysproteinemia)
may therefore produce false high or low values of calcemia.
The free (ionized) fraction of calcium can be measured by means of potentiometry using ionselective
electrodes. The major advantage of this technique is its rapidity. Another important fact
is that this measurement provides information on the biologically active form of calcium regardless
of the various factors influencing the levels of total calcium (e.g. severe hypoproteinemia, acidbase
balance disorders etc.). On the other hand, the main difficulty is brought about by necessity to
process only very fresh blood samples – unless analyzed immediately, the pH in blood sample
quickly deteriorates which obviously affects also the availability of ionized calcium.
Ionized calcium (fS-Ca2+): 0.9 – 1.3 mmol/l
Estimation of iron in serum.
Principle of estimation:
For estimation of serum iron colorimetric methods, atomic absorption spectrophotometry,
and other specialized techniques can be used. The photometric methods, based on reaction
of iron with a complex-forming reagent, are used most widely.
All these assays encompass the following steps (Fig. 5):
• Release of Fe3+ from complex with transferrin by action of acids (such as HCl) or
detergents
• Reduction of Fe3+ to Fe2+, necessary for the
next reaction with the chelating agent. As a
reductant, e.g. ascorbic acid can be used.
• Chelation of Fe2+ by the complex-forming
agent containing –N=C–C=N– groups, yielding a
colored complex. Two chelators are nowadays
used: bathophenanthroline and ferrozine [3-(2-
pyridyl)-5,6-bis-(4-sulfophenyl)-1,2,4-triazine,
registered name FerroZine]. The latter has higher
absorption coefficient and is more soluble in
water.
) Transferrin (Fe3+)2 + HCl ® 2 Fe3+ + transferrin
2) Fe3+ + reducing agent ® Fe2+
(ascorbic acid)
3) Fe2+ + ferrozine ® colored complex
Evaluation:
Levels of serum iron fluctuate with circadian rhythms and are affected by many other
factors. Accordingly, diagnostic significance of this marker is limited. Serum iron poorly
reflects status of tissue iron stores and must be always assessed together with serum
transferrin and iron-binding capacity.
Low levels of iron in serum are observed in iron deficiency due to e.g. big or repeated
bleeding, low iron intake or impaired iron absorption; but serum iron is low also in acute
infections and chronic inflammatory diseases (sequestration of iron in tissues). On the
other hand, high serum iron is found in hemochromatosis, iron overload or iron
intoxication, increased destruction of erythrocytes and some liver diseases.
Reference values (S-iron):
Men: 9 – 29 μmol/l
Women: 7 – 28 μmol/l
Spectrophotometry of cerebrospinal fluid.
Spectrophotometry of CSF is used in diagnostics of brain stroke, especially in cases of suspect
bleeding to subarachnoid space. It is important mainly in the types of brain hemorrhage that are
difficult to see by imaging methods. In particular it is valuable in early stages of the disease. It
provides information on time period since the bleeding episode, as well as whether the bleeding has
been protracted or recurrent. Spectrophotometric examination of CSF makes use of different
maxims in the visible region for oxyhemoglobin (at 415 nm), methemoglobin (at 405 nm) and
bilirubin (at 420 − 460 nm).
At the beginning of brain hemorrhage the CSF contains mostly oxyhemoglobin, in the later stages
spectrophotometry yields a summation spectra of oxyhemoglobin/methemoglobin and bilirubin.
The rate of hemoglobin degradation to bilirubin displays a high individual variability, but isolated
bilirubin xanthochromia in general does not appear earlier than 5 days after bleeding.
Spectrophotometry of CSF is performed on a scanning spectrophotometer in the wavelength range
370 – 600 nm. It is recommended to centrifuge the CSF sample within one hour since its collection.
Evaluation:
Physiological trace
Spectrophotometric trace of normal CSF is flat or slightly rising in the direction from 600 nm to
370 nm. The absorbance values in the visible region are less than 0.02.
Read up about detection of oxyhemoglobin, methemoglobin and bilirubin and the graphs
Estimation of ALT activity in serum.
Aminotransferases are enzymes that catalyze reversible transfer of the amino group from
an amino acid to a keto acid. Pyridoxal-5’-phosphate serves as a cofactor. In clinical
biochemistry, alanine aminotransferase and aspartate aminotransferase are the most
significant.
2.1. Alanine aminotransferase (ALT):
ALT catalyzes a reversible transfer of amino group from alanine to 2-oxoglutarate:
COO- COO-
C=O NH2 -C- H
COO-
CH2 ALT COO-
CH2
®
NH2 -C- H + CH2 ¬ C=O + CH2
P-5´-P
CH3 COO- CH3 COO-
L-alanine 2-oxoglutarate pyruvate L-glutamate (Page 3 of liver doc)
Alanine to pyruvate. Then pyruvate plus NADH +H to yield NAD and Lactate, determination of ALT depends on the reduction in absorbance of reduced NADH. Waburg optical test.
ALT is present mostly in the liver; its activity in other organs (skeletal muscle,
myocardium, etc.) is much lower. Unlike AST it localizes only to the cytosol.
Estimation of ALT is a sensitive and relatively specific test for hepatocyte damage. Its
activity in serum rises even in a small damage of the liver cell, caused by increased
permeability of the cell membrane. In inflammation of the liver (viral hepatitis), for
instance, elevation of ALT is the earliest indicator that hepatocyte cell membrane integrity
is compromised. Repeated ALT estimation is suitable for monitoring course of the disease.
Reference values:
Catalytic concentration of serum ALT (S-ALT):
Men up to 0.80 μkat/l
Women up to 0.60 μkat/l
Evaluation page 5
Estimation of AST activity in serum.
AST catalyzes a reversible transfer of amino group from aspartate to 2-oxoglutarate: Learn reaction: Aspartate to OAA then OAA plus NADH +H to yield NAD and Malate, determination of AST depends on the reduction in absorbance of reduced NADH. Waburg optical test.
AST occurs in numerous organs: liver, heart, skeletal muscle, kidney, pancreas, and red
blood cells.
It exists in two isoenzymes: mitochondrial (about 70 %), and cytosolic (about 30 %).
Cytosolic fraction is readily released into circulation due to mild alterations of hepatocyte
cell membrane permeability. In contrast, the mitochondrial fraction is released only after
destruction (necrosis) of the hepatocyte. Therefore, a high increase of serum AST is a
marker of hepatocyte destruction, because both isoenzymes are likely to participate in the
increase.
Since AST is not specific for the liver tissue, it can be elevated also in damage of skeletal
muscle and myocardium. AST in blood rises in acute myocardial infarction (heart stroke)
and following heart surgery, but also due to a long lasting strenuous physical exercise.
Hemolysis of the sample can cause false positive results of AST estimation, since quite
high levels of the enzyme are present in the erythrocytes.
Reference values:
Catalytic concentration of serum AST (S-AST):
Men up to 0.85 μkat/l
Women up to 0.60 /l
Read evaluation page 5
Estimation of γ-glutamyl transferase activity in serum.
g-glutamyl transferase (GGT, formerly also GMT) is a key enzyme of the g-glutamyl cycle
that provides transport of some amino acids and peptides through the cell membrane from
extracellular fluid to the cells. GGT catalyzes transfer of g-glutamyl moiety from g-
glutamyl-peptides to other peptides, amino acids, or water. The physiologic donor of g-
glutamyl is the tripeptide glutathione in the cytosol, and the acceptor is an amino acid
localized extracellularly. Cysteinyl-glycine remaining from glutathione following cleavage
of g-glutamyl is converted by other reactions back to glutathione.
The basic GGT-catalyzed reaction:
GGT occurs in membranes of cells with high secretion or absorption capacity. In the
liver it is present in the microsomal fraction and in the membranes of biliary tract endothelium
g-glutamyl-cysteinyl-glycine (GSH) GGT g-glutamyl amino acid
+ ® +
amino acid or peptide ¬ cysteinyl-glycine
. High activity of GGT is also found in proximal tubules of the kidney, in
enterocytes, and in the pancreas. Synthesis of GGT can be induced by some drugs
(barbiturates, antidepressants, alcohol). It is possible to release GGT from membranes by
detergent action of bile acids or alcohol.
Increase in GGT is especially characteristic for damage of the hepatobiliary tract:
• intrahepatic or extrahepatic cholestasis – in these cases alkaline phosphatase is
increased as well
• hepatocellular damage – acute and chronic liver diseases
• chronic alcohol abuse – a high isolated elevation of GGT is characteristic. Its
increase in alcoholics precedes the liver damage (induction of GGT synthesis)
• tumors of the liver and pancreas
Reference values:
Catalytic concentration of serum GGT (fS-GGT):
Men: 0.14-0.84 μkat/l
Women: 0.14-0.68 μkat/l
GGT is higher in men since the enzyme is relatively abundant in prostate gland.
Estimation of alkaline phosphatase activity in serum.
Phosphatases can catalyze hydrolysis of various organic phosphomonoesters to phosphate anion
and the corresponding alcohol or phenol. In addition, the ALP mediates also a
transphosphorylation reaction, in which the phosphate group is transferred to a suitable acceptor.
If 4-nitrophenyl phosphate is used as the substrate; the alkaline phosphatase catalyses the following
reactions:
1) hydrolysis:
4-nitrophenyl phosphate + H2O ® 4-nitrophenol + phosphate (Fig. 6)
2) transphosphorylation:
4-nitrophenyl phosphate + N-methyl-D-glucamine ®
® 4-nitrophenol + N-methyl-D-glucamine phosphate
In the first reaction the phosphate is hydrolytically cleaved from the substrate (Fig. 6). In the
second transphosphorylation reaction the N-methyl-D-glucamine acts simultaneously as a buffer
and as an acceptor for phosphate; in this way the reaction is accelerated.
The substrate 4-nitrophenyl phosphate is colorless in the alkaline medium, while product of its
ALP-dependent hydrolysis has a quinone-like form (Fig. 6) at alkaline pH, which is intensely
yellow. The released 4-nitrophenol, then, is a measure of ALP activity and is estimated
photometrically using either kinetics or end-point approach (following termination of the enzyme
reaction by an inhibitor). ALP is activated by sodium chloride.
Isoenzymes of alkaline phosphatase can be distinguished on a basis of their different physical,
chemical, immunological and electrophoretic properties.
Methods for estimation of ALP isoenzymes utilize selective inactivation of some isoenzymes by
heat, phenylalanine, leucine, or urea; or differences in electrophoretic mobility. Good separation of
liver and bone isoenzymes is sometimes difficult. Recently, new methods for estimation of bone
isoenzyme have appeared, based on immunochemistry, or binding of the bone ALP to a specific
lectin followed by electrophoresis.
Reference values:
Total catalytic concentration of ALP (fS-ALP, μkat/l):
Adults 0.66 – 2.2 μkat/l
Children (1 – 10 years) 1.12 – 6.2 μkat/l
Children (10 – 15 years) 1.35 – 7.5 μkat/l
Bone isoenzyme of ALP:
Adults 0.26 – 0.40 μkat/l
Estimation of urea in biological sample
In general, urea in biological fluids can be estimated either directly, or indirectly as ammonia. In the indirect assay, first an enzyme urease cleaves urea to carbon dioxide and ammonia that in aqueous medium exists as ammonium ion. Next, the amount of ammonium is estimated by the Berthelot’s reaction: ammonium ion with sodium hypochlorite and phenol or salicylate form a colored product. The reaction is catalyzed by sodium nitroprusside.
Nowadays, the recommended routine method for measurement of the ammonium ions generated by the urease reaction utilizes conversion of α-ketoglutarate to glutamate. The reaction is catalyzed by glutamate dehydrogenase, and coupled to oxidation of NADH to NAD+ (Warburg’s optical test).
Urea + H2O + 2H+
NH4+ + 2-oxoglutarate + NADH
Urease
Glutamate dehydrogenase
2NH4+ + CO2 L-glutamate + NAD+ + H2O
Reference values (fS-Urea): Women: 2.0 – 6.7 mmol/l Men: 2.8 – 8.0 mmol/l
Increased serum Urea: Impaired kidney function, high protein diet, high protein catabolism, dehydration
Decreased serum Urea: Impaired liver function, late pregnancy (increased foetus demand), low protein diet
Estimation of uric acid in biological sample
Most state-of-art techniques for measuring uric acid concentration employ uricase, an enzyme converting uric acid into allantoin, hydrogen peroxide and carbon dioxide (Fig. 9). Decrease in uric acid concentration in the reaction mixture may be determined by direct photometry at 290 – 293 nm, because uric acid and allantoin differ in their absorption spectra. Uric acid has an absorption peak at this wavelength while allantoin does not absorb light in this range.
Another choice is indirect measurement in which hydrogen peroxide formed in the uricase reaction is used in a coupled reaction catalyzed by peroxidase. A quinonimine dye is formed by oxidative coupling of (usually) 4-aminoantipyrine and a suitable derivative of phenol (e.g. N-ethyl-N-(2-hydroxy-2-sulphopropyl)-m-toluidine)). Intensity of the resulting color is proportional to the concentration of uric acid in solution. Ascorbic acid interferes with this method; therefore ascorbate oxidase is added to eliminate such an influence.
Draw reactions: uricase and oxidative coupling of 4 aminoantipyrine and a phenolic compound
Reference values:
Uric acid excreted per day: Clearance of uric acid
Fractional excretion of uric acid:
1.5 – 4.5 mmol/day 0.07 – 0.22 ml/s 0.04 – 0.12
Reference values (fS-UA): Women 140 – 340 μmol/l Men 220 – 420 μmol/l
Overproduction of uric acid
• Excessive de novo synthesis of purines associated with hyperuricemia is found in
some genetically determined impairments of purine metabolism. Partial or
complete defect of hypoxanthine-guanine phosphoribosyltransferase (Lesch-
Nyhan’s syndrome) may serve as an example. Re-utilization of purine nucleotides
is impaired and purines are therefore degraded to uric acid. Another genetic
disorder leading to increased production of uric acid is increased activity of
phosphoribosylphosphate synthetase.
• Anticancer therapy (cytostatic chemotherapy, radiotherapy) induces extensive cell
lysis and leads to increased formation of uric acid. Purine nucleotides are released
from degraded nucleic acids and metabolized to uric acid. Some hematological
diseases associated with excessive production (polycythemia vera) or degradation
of cells (leukemia, hemolytic anemia) are, in a similar way, joined with
hyperuricemia.
• Increased intake of foodstuffs rich on purines (offal, meat, legume, in smaller
extent also chocolate, cocoa and coffee) also leads to overproduction of uric acid.
Kidneys may fail to compensate the overload with uric acid and uricemia grows up.
• Alcohol increases the level of uric acid. Increased production of lactate inhibits
renal secretion of uric acid. The decreased excretion of uric acid is later replaced
with high uricosuria
Decreased excretion of uric acid
Decreased excretion of uric acid is one of the most frequent causes of hyperuricemia.
• Decreased tubular secretion of uric acid is common in patients with
hyperuricemia. The cause of this disorder is unknown.
• Excretion of uric acid is lowered in states associated with decreased glomerular
filtration and impaired tubular function.
Estimation of glucose in serum
Measurement of glucose concentration can be based on different principles; but enzyme methods are the ones most widespread. In general, any enzyme that metabolizes glucose can be employed for glucose estimation. The recommended routine technique utilizes coupled enzyme reactions of glucose oxidase (GOD) and peroxidase (POD) (Fig. 3a).
In the first reaction the enzyme glucose oxidase catalyzes glucose oxidation with air oxygen producing gluconic acid in the form of its inner ester - gluconolactone. The glucose
Diabetes mellitus
6
solutions normally consist of 36 % of α-anomer and 64 % of β-anomer. GOD is highly specific for β-D-glucopyranose. Therefore, in order to achieve oxidation of both anomers, conversion of α to β-anomer is necessary, which, however, occurs spontaneously during sufficiently long incubation. The other product of the glucose oxidase reaction is hydrogen peroxide in amount equivalent to that of glucose.
In the next reaction catalyzed by peroxidase the hydrogen peroxide reacts with a suitable chromogen, e.g. derivative of phenol that is oxidized to a reactive intermediate, which, in turn, reacts with another compound, such as 4-aminoantipyrine, yielding a stable soluble dye (Fig. 3a), whose absorbance is measured.
Alternatively, it is possible to measure decay of oxygen consumed in the glucose oxidase reaction, by means of electrochemical techniques (oxygen electrode or an enzyme electrode).
The hexokinase method (Fig. 3b) is also a highly specific one. Glucose is first phosphorylated with ATP to glucose 6-phosphate, which is then oxidized with NADP+ to 6-phosphogluconolactone in the reaction catalyzed by glucose-6-phosphate dehydro- genase. Reduction of NADP+ to NADPH can be followed directly as an increase of absorbance in the UV region (the Warburg optical test).
Fasting glycemia < 5.6 mmol/l, and in OGTT glycemia in 120 minutes < 7.8 mmol/l are considered normal.
In contrast, fasting glycemia ≥ 7.0 mmol/l, or glycemia ≥ 11.1 mmol/l in random sample or in 120 minutes of OGTT together with classical clinical signs (thirst, polyuria, unexplained weight loss) point to diagnosis of diabetes mellitus. For confirmation of diagnosis of DM the examination must be repeated (Fig. 5, Table 2)
Draw reactions (diabetes protocol)
