MT6317 – CLINICAL CHEMISTRY (Lecture/ Laboratory)

Unit _ – Unit Title

SECOND SHIFTING PERIOD
SEPTEMBER - OCTOBER 2021

OUTLINE o Body makes uric acid when it breaks down

purines which are mainly found in the body
REFERENCES
 Lecture PPT – Blood Uric Acid Determination by Ms. Anna Lissa V. and the foods we eat; purines are organic
Magcaling, RMT compounds that are not necessarily harmful,
 Asynchronous video of Ms. Anna Lissa V. Magcaling, RMT
 Asynchronous video of Sir Ron
however when purines break down into a
waste substance called uric acid and if it builds
up, this causes health issues
GENERAL INSTRUCTIONS o 📖 Purines, such as adenine and guanine from
 Please don’t fill up the outline part. the breakdown of ingested nucleic acids or
 All highlighted yellow parts should be replaced. from tissue destruction, are converted into uric
 Font Sizes: acid, primarily in the liver.
o Heading 1: Caps Lock, Helvetica, 10  Transformation mainly occurs in the liver.
o Heading 2, 3: Helvetica, 9 o Before it is transported to the kidneys where it
o Text: Helvetica, 9, 1.15 spacing is filtered by the glomerulus
 Lecture PPT: Black  📖 Filtered by the glomerulus and secreted by the
 Info from the Lecture: Same color with the distal tubules into the urine
Trans Subject  Most uric acids are reabsorbed and 1% is excreted.
o Green for Clinical Chemistry o 📖 Most uric acid is reabsorbed in the
 Follow the bullet format, if di mahanap copy proximal tubules and reused.
paste from the previous list (PLEASE o 📖 Reabsorption of 98% to 100% of the uric
FOLLOW THIS) acid from the glomerular filtrate occurs in the
 📖 ß Copy this and place it before the text if proximal tubules.
you’re going to add info from the book. So its o 📖 Renal excretion accounts for about 70% of
easier to identify. uric acid elimination; the remainder passes
into the GI tract and is degraded by bacterial
NONPROTEIN NITROGEN COMPOUNDS enzymes.
 One characteristic of uric acid is its insolubility in
Approximate Approximate plasma that when present in high concentrations,
Plasma Urine they may be deposited in the joints and tissue as
Compound Concentration Concentration tophi which induce inflammatory response known
(% of Total (% of as gout
NPN) Excreted o Urate crystals can occur in urines when uric
Nitrogen) acid concentration is abnormally high
Uric Acid 10 1.7  Usually requested to confirm diagnosis and
monitor treatment of gout, assess inherited
BLOOD URIC ACID DETERMINATION disorders of purine metabolism and assist in the
 Uric acid is one of the non-protein nitrogenous diagnosis of renal calculi.
compounds. o Also, to prevent acid nephropathy during
 Main product of the catabolism of exogenous and chemotherapeutic treatment and to detect
endogenous purine nucleosides. (Adenine and kidney dysfunction
Guanine)

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 MT6317Lec/Lab - Topic

Method Catalyzed by of 12.3

s uricase allantoin Very
from uric specific
acid
Coupled H2O2 + Readily
enzymatic – indicator automate
Sharp crystals may form in the big toe or joints such Peroxidase dye à d;
as the knees and the hands which causes episodes of colored reducing
swelling and pain called gout attacks compound agents
interfere
METHOD OF DETERMINATION
Spectrophotom Decrease Hemoglo
etric in bin and
Uric Acid +O2 +2 H 2 O uricase allantoin+C O2 + H 2 O2
→ absorbanc xanthine
e at 293 interfere
2 H 2 O2 +4− AAP+ EHSPT peroxidase quinoneimine+ HCl+ 4 H 2 nm
→
measured
 Enzymatic method
Other Isotope Detection Proposed
o In the presence of uricase, uric acid is
methods dilution mass of reference
converted to allantoin
spectrometry characteris method
 Uricase acts to speed up the
tic
reaction
fragments
o Hydrogen peroxide reacts under the
following
catalysis of peroxidase with amino-4-
ionization;
antipyrine and N-ethyl-2-hydroxy-3-
quantificat
sulfopropyl toluidine
ion using
o (Sir Ron) 4-AAP and EHSPT: coupled
isotopicall
chromogen y labeled
o Quinoneimine: red, violet dye compound
 Uric acid is readily oxidized to allantoin, so it
SUMMARY OF ANALYTIC METHODS – functions as the reducing agent in the chemical
URIC ACID reactions
Chemic Phosphotungsti In Non-  Most common: Caraway method
al c Acid carbonate specific; o Based on oxidation of uric acid in a PFF
Method solution requires
with subsequent reduction of
s (Na2CO3/ protein
phosphotungstic acid in alkaline solution
OH-), uric removal
à tungsten blue
acid +
o Lacks specificity
H3PW12O40
 More specific: enzymatic method with uricase
+ O2 à
o Used almost exclusively in clinical
allantoin +
laboratories
tungsten
blue +  Coupled enzyme method (Peroxidase/Catalase): it
CO2 measures hydrogen peroxide produced from uric
Enzyma Similar first Enzymatic See acid à allantoin
tic step – production Figure
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o Peroxidase catalyzes chemical indicator o Uricase can also be referred as urate

reaction oxidase because it catalyzes the oxidation
o Have been adapted for use in traditional of uric acid to allantoin
 In the presence of uricase, uric acid is converted to
wet chemistry and dry chemistry slide
allantoin and there is a formation of hydrogen
analyzer peroxide
o Bilirubin and ascorbic acid destroy  More specific than Caraway
peroxide, and, in sufficient quantity, can  Almost exclusively used in clinical laboratories
interfere  Measures different absorbance of uric acid at 293
o Commercial reagent preparation includes nm
potassium ferricyanide and ascorbate o The difference of before and after is
oxidase to minimize interferences proportional to the uric acid concentration
 Negative interference: hemoglobin, xanthine
(Sir Ron) ANALYTIC METHODS
 Reduce sensitivity: proteins
o 📖 Proteins can cause high background
 Uric acid is readily oxidized to allantoin and,
absorbance, reducing sensitivity
therefore, can function as a reducing agent in
chemical reactions
Coupled Enzyme (Peroxidase/Catalase)
 Conversion of uric acid to allantoin
 Principle: measures hydrogen peroxide produced
 📖 Allantoin: a more water-soluble end product. as uric acid is converted to allantoin
 📖 Peroxidase or catalase is used to catalyze a
Chemical Methods chemical indicator reaction
 Color produced is proportional to the quantity of
Phosphotungstic Acid (Caraway Method) uric acid
 Principle: Reduction-Oxidation (Redox) Reaction  Adapted for use in traditional wet chemistry and
dry slide analyzers
 Most common method of analyzing uric acid
 Interferences: bilirubin, ascorbic acid
 Based on the oxidation of the uric acid o Consequently, destroys peroxide
 First rule of caraway method is that it should be o To minimize, commercial reagent
protein free preparation includes potassium
 Specimen: Protein free filtrate ferricyanide or ascorbate oxidase
Na2CO3/OH-
Spectrophotometric (Blauch and Koch)
Uric acid + H3PW12O40 + O2 allantoin + tungsten blue + CO2
 Interferences: hemoglobin and xanthine
 Sodium carbonate (Na2CO3/OH-): what makes the
reaction alkaline  Uric acid has a UV absorbance peak of 293 nm
 Lacks specificity  Allantoin does not have a UV peak at 293 nm,
therefore, the decrease in absorbance in
 Sodium cyanide (NaCN): another type of similar
proportional to the concentration of uric acid
method
present in the sample
o Sodium cyanide is used instead of sodium
carbonate
Other Methods
 📖 HPLC (high-performance liquid
Enzymatic Methods
chromatography) methods, typically using UV
detection, have been developed.
First Step (Uricase)
 Principle: enzymatic production of allantoin from  📖 IDMS has been proposed as a candidate
uric acid crystals reference method.

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MT6317Lec/Lab - Topic

REAGENTS/ MATERIALS/ INSTRUMENTATION

NEEDED
 Serological pipet 1 ml or 2 ml, 0.1 ml
 Rubber aspirator
 Tissue
 Parafilm
 Specimen (serum or plasma) SPECIMEN REQUIREMENTS
 Ellitrol I (Normal Control)  Heparinized plasma, serum, or urine
o Serum should be removed immediately from
the cells to prevent dilution by intracellular
contents
o Uric acid is stable in plasma or serum after
RBCs are removed
o Serum samples can be stored or refrigerated
 Ellitrol II (Pathologic Control)
for 3-5 days
 Gross lipemia should be avoided
o Diet may affect uric acid concentration, but a
recent meal has no significant effect and
fasting specimen is unnecessary
 High bilirubin concentration can interfere
 Spectrophotometer o High bilirubin concentration may falsely
 Kahn test tube (or cuvette) decrease results obtained by peroxidase
 Centrifuge method
 Elical 2 (Calibrator) not included in the reagent kit  Hemolysis may result in low values
o Significant hemolysis with concomitant
glutathione release may result in low values of
uric acid
 Salicylates and thiazides increase values of uric
acid
 EDTA or Fluoride additives should not be used
 Uric Acid Reagent Kit containing:
o Especially for uricase method
o Phosphate buffer (pH 7.0)
 Urine collections must be alkaline
o EHSPT
o Should have a pH of at least 8
o Amino-4-antipyrine
o Uricase PROCEDURES
o Peroxidase  Prepare and label the tubes properly as follows: B,
o Ferrocyanide S, CN, CP, U
o Sodium azide  In labelling, make sure that the specimen number
o Standard: Uric acid 6 mg/dl or 357umol/L; is legibly written on the unknown tube.
may vary  Then follow the pipetting scheme summarized in
 May vary depending on the manufacturer. the table below. Practice correct manual pipetting
technique.

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MT6317Lec/Lab - Topic

 Since the volume of the sample is very small, Patholo ml

deliver the desired volume of the sample first, into gic
the tube by touching the tip of the pipet directly to Serum
the bottom of the tube. Unknow - - - - 0.025
o Since the sample is too small, do not let the n Serum ml
pipette touch the sides but pipet it directly on Workin 1.0 1.0ml 1.0ml 1.0ml 1.0ml
the bottom of the tube g ml
 Then add the required volume of the reagent by Reagent
touching the tip of the pipet to the side of the Notes: Mix, incubate for 5 minutes at 37ºC.
tube. Measure the absorbance of standard and the sample
 Mix the solution by covering the mouth of the test against the blank within 15 minutes.
tube with a parafilm and inverting the tubes 2-3 1.0 mL contains uricase – enzyme that will react to
times. Uric Acid
o Make sure to put parafilm on the mouth of the B – Blank; S – Standard; CN – Control Normal; CP –
tube before mixing Control Pathological; U - Unknown
 Start the incubation time.
 Read the absorbance following the assay CALCULATION
requirements.  Use the formula below to calculate for Uric Acid
concentration.
ASSAY REQUIREMENTS  Serum or Plasma:
 Wavelength  Top: Concentration of Uric Acid In
o 546 nm Serum/Plasma (SI Unit)
 Optical path  Bottom: Concentration of Uric Acid in
o 1 cm Serum/Plasma (Conventional Unit)
 Temperature 
o 37°C  Urine:
 Measurement
o Read against reagent blank (only one reagent
blank per series is required)

PIPETTING SCHEME: SEMI-MICRO

Pipette B S CN CP U
into
Cuvette
s
Standar - 0.025 - - -
d ml
Solution
Control - - 0.025 - -
Normal ml
Serum
Control - - - 0.025 -

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 MT6317Lec/Lab - Topic

 Top: Concentration of Uric Acid In Urine (SI o 📖 Affected individuals have pain and
Unit) inflammation of the joints caused by
 Bottom: Concentration of Uric Acid in Urine precipitation of sodium urates.
o In 25-30% of patients, hyperuricemia
Notes: is a result of overproduction of uric
acid.
Legend:  May be exacerbated by a purine
B = Blank rich diet, drugs, and alcohol
S = Standard o 📖 Plasma uric acid concentration in
CN = Control Normal affected individuals is usually greater
CP = Control Pathologic than 6.0 mg/dL.
U = Unknown o 📖 Patients with gout are susceptible to
C = Concentration the formation of renal calculi, although
A = Absorbance not all persons with abnormally high
(Conventional Unit) serum urate concentrations develop
this complication.
REFERENCE RANGE
o 📖 In women, urate concentration rises
 SERUM
after menopause.
o SI UNIT
o 📖 Postmenopausal women may
 Males:3.5-7.2 mg/dL
develop hyperuricemia and gout.
 Females: 2.6-6.0 mg/dL
o 📖 In severe cases, deposits of
o CONVENTIONAL UNIT
crystalline uric acid and urates called
 Males: 206-428 umol/ /L tophi form in tissue, causing
 Females: 155-357 umol/ /L
deformities.
 
o Treatment of myeloproliferative disease with
📖 Reference Intervals
cytotoxic drugs
Uric Acid (Uricase Method) o Increased metabolism of cell nuclei
Adult  Plasma or Serum 
 📖 Another common cause of
Male  3.5 – 7.2 mg/dL 0.21 – 0.43 mmol/L  elevated plasma uric acid
concentration
Female 2.6 – 6.0 mg/dL  0.16 – 0.36 mmol/L   Occurs in patients on
Child 2.0 – 5.5 mg/dL  0.12 – 0.33 mmol/L 
  Urine, 24 h  chemotherapy for such
Adult 250 – 750 mg/dL 1.5 – 4.4 mmol/L  proliferative diseases such as
leukemia, lymphoma, multiple
CLINICAL SIGNIFICANCE myeloma, and polycythemia.
 Increased Concentration  📖 Monitoring uric acid
o Gout concentration in these patients
o Abnormally increased plasma uric acid is important to avoid
concentration nephrotoxicity.
o Increased catabolism of nucleic acids  📖 Allopurinol, which inhibits
in renal disease xanthine oxidase, an enzyme in
o Found primarily in men and is usually the uric acid synthesis pathway,
first diagnosed between 30 and 50 is used for treatment.
years of age. o Hemolytic and proliferative processes
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MT6317Lec/Lab - Topic

o Patients with hemolytic or bases in the nucleotide

megaloblastic anemia may exhibit salvage pathway and
elevated uric acid concentration. results in increased de
o Purine-rich diet novo synthesis of purine
o 📖 Increased urate concentrations may nucleotides and high
be found following ingestion of a diet plasma and urine
rich in purines concentrations of uric
 Liver, kidney, sweetbreads, acid.
shellfish  📖 Characterized by
o Increased tissue catabolism or starvation neurologic symptoms,
o Due to inadequate dietary intake mental retardation, and
o 📖 as a result, increases urate self-mutilation
concentrations o Phosphoribosylphyrophosphate
o Enzyme deficiencies like Lesch-Nyhan synthetase deficiency
syndrome, phosphoribosylpyrophosphate  Mutations in the first enzyme in
synthetase deficiency, glycogen storage the purine synthesis pathway
disease type 1, fructose intolerance o Glycogen storage disease type 1
o Enzyme deficiencies  Deficiency of glucose-6-
 Cannot metabolize purine/uric phosphatase
acid, increase in purine o Fructose intolerance
o Inherited disorders of purine  Deficiency of fructose 1-
metabolism which are associated with phosphate aldolase
significant increases in physiological o 📖 Metabolites such as lactate and
uric acid concentration. triglycerides are produced in excess
o Lesch-Nyhan syndrome and compete with urate for renal
 X-linked genetic disorder excretion in these diseases.
which is seen only in males  Increased Concentration REMOVE NA
 Metabolic disorder most likely o Toxemia of pregnancy
seen in babies o Also known as Preeclampsia
 “Orange sand diapers”  Serious condition in late
 Too much uric acid in pregnancy characterized by a
blood can lead to uric sudden rise in blood pressure,
acid crystals in urine excessive weight gain,
 Sandy diaper = crystals generalized edema, proteinuria,
which are orange in severe headache, and visual
appearance disturbances
 Caused by complete deficiency o Hyperuricemia as a result of decreased
of hypoxanthine-guanine uric acid excretion
phosophoribosyltransferase o Lactic acidosis
 Important enzyme in the o Presumably as a result of competition
biosynthesis of purines for binding sites in the renal tubules
 📖 Lack of this enzyme o Chronic renal disease
prevents the o Filtration and secretion are impaired
reutilization of purine
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 MT6317Lec/Lab - Topic

o Uric acid nephrolithiasis or formation

of kidney stones/renal calculi which
may occur due to a variety of
predisposing factors and conditions
 📖 In acidic urine, the relatively
insoluble uric acid precipitates
to form calculi, which can
cause intense flank pain.
 📖 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.
o Drugs and poison
 Decreased Concentration (Hypouricemia, less
common than hyperuricemia) secondary to:
o Liver disease
o Defective tubular reabsorption (Fanconi
Syndrome)
o Disorder of reabsorption in the
proximal convoluted tubules of the PRESENTATION
kidney.  Show the following:
o Chemotherapy with azathioprine or 6- o Request form
mercaptopurine o Test tube Set up
o Overtreatment with allopurinol o Computation
o 📖 Decreased plasma uric acid can be caused o Result form
by chemotherapy with 6-mercaptopurine or o Evaluation of Results: Compute for the values
azathioprine, inhibitors of de novo purine of CN, CP, and U. And evaluate the results by
synthesis, and as a result of overtreatment with answering the following questions:
allopurinol.  Is the test valid? Explain your answer. Can
o 📖 Some studies have shown an association you release the result?
between low uric acid concentrations and  What can you say about the obtained Uric
neurodegenerative conditions such as Acid values of the patient? Are they within
Alzheimer's and Parkinson's diseases. the normal range? Explain.

Table 12.5 Causes of Abnormal Plasma Uric Acid (Sir Ron) BASIC PHYSIOLOGY
Concentration  Major end-product of purine catabolism
primarily in the liver
o (Sir Ron) We don’t say that the food we eat is
high in uric acid, rather we say that it is
purine-rich; once purine is metabolized, that’s
the time it becomes uric acid

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MT6317Lec/Lab - Topic

o Final breakdown of nucleic acids, catabolism  Confirm diagnosis and monitor treatment of gout
in humans is found by the action of xanthine o Pag nagmemeasure ng uric acid sa blood,
oxidase in the liver and intestines either to establish diagnosis of arthritis related
 After the blood is filtered at the glomerulus, the to uric acid
resulting fluid enters the tubules of the kidneys to o If may arthritis na, minomonitor natin yung
be secreted as urine Uric acid to manage formation of gouty
 Uric acid is considered a weak acid at pH 7.4 and arthritis
more than 95% of it will be existing as a  Diagnosis of renal calculi
monosodium urate o Pag nalabas na kidney stones, stone analysis
o There are many types of uric acid, but majority
ang test
is monosodium urate o What type of stone does this patient have?
o 📖 Nearly all of the uric acid in plasma is
o Most of the time is uric acid
present as monosodium urate.
o Kahit walang stone analysis, naprepredict
 The concentrations of uric acid is greater than 6.8
naman
mg/dL and if the plasma is saturated at that level
 Prevent uric acid nephropathy during
as a result, when there is too much uric acid in the
circulation, it will crystalize/precipitate in tissues chemotherapy
 📖 At the pH of plasma (pH ~ 7), urate is  Detect kidney dysfunction
relatively insoluble; at concentrations greater than o When it comes to kidney function, urea and
6.8 mg/dL, the plasma is saturated. creatinine have more bearing and better
 📖 As a result, urate crystals may form and assessment of kidney performance among
precipitate in the tissues. In acidic urine (pH <
NPNs unlike uric acid.
5.75), uric acid is the predominant species and uric
acid crystals may form.
o May sometimes flock/accumulate in our joints
o Either ilalabas sa urine or kung saan siya may
affinity sa mga joints
o Pag nasa blood, most likely lalabas din sa
urine
o Pag mataas uric acid sa blood, lalabas siya sa
urine, most likely mag crycrystalize
o Stone forming kaya nagkakakidney stone in
the form of uric acid crystals
o Purine rich foods: lamang loob

(Sir Ron) CLINICAL APPLICATION

 Assess inherited disorders of purine metabolism

o Purine will form into uric acid
o Pag nakain saka magiging uric acid
o Metabolic disorders
 Di mametabolize ng tao yung purine so
tataas purine and uric acid
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