Uric Acid Determination

C5H4N4O3

What is the organic compound in Uric Acid?

(Uric acid) (adenine & guanine) (DNA, RNA, ATP)

(_____) is the final oxidation product of purine metabolism in humans and primates.

-catobolic products

purines are? (__________&_________)

Purines are nitrogen containing bases that are found in a

(_____, ______, ______) and other nuclceotides

(adinosin monophosphate and guanosine monophosphate) (Diet)

(__________________&_________________)

Nucleic acids are degraded into nucleotides such as (____)

plays a major role in the formation of uric acid

(inosine) (hypoxanthine) (guanosine) (guanine)

Adenosine monophosphate is converted into → (_____) and then → (________)

Guanosine monophosphate is broken into → (________) and then → (_________)

(xanthine) (guanine diaminise)

Guanine is converted into (_______) by enzyme (_______) and

(xanthine oxidase)

both hypoxanthine and xanthine are oxidase by the enzyme (_______) to produce uric acid

(molybdenum, iron-sulfur, Flavin adenine dinucleotide)

This enzyme is mainly active in the liver and small intestines and requires co-factors such as (_________, ______________, ___________)

Uricase

Unlike most mammals, they possess a certain enzyme which is used to convert uric acid into a more soluble compound.

What do you call that enzyme?

allantoin

Uricase will convert uric acid into a more soluble compound. What do you call this soluble compound?

(monosodium urate)

Humans lack the enzyme uricase due to an evolutionary gene mutation. As a result, uric acid levels in humans are naturally higher. Once formed, uric acid is then transported in the bloodstream primarily as (_____________)

(7.4) (urate crystals)

Depending on the blood pH. At a normal physiological pH of about (___) it is largely ionized and soluble. But when levels become too high (_________) can form, especially in the joints in the kidneys.

(70%) (30%) (filtration) (reabsorption) (secretion) (post-secretory reabsorption) (10%)

The excretion of uric acid is handled mainly by the kidneys that’s around (____) and also in the excretion in the intestines, about around (____). in the kidneys, uric acid undergoes (______) and then (_______), (______) and then there is another (________________________) meaning only around (___) of the filtered uric acid is finally excreted in our urines, The intestines also play a role in excretion where certain bacteria can further break it down.

(strong antioxidant) (neutralizing free radicals) (protecting cells) (scavenge reactive molecules) (hydroxyl radicals) (peroxy nitrite)

(maintining cellular redox balance)

Although uric acid is often associated with gout and kidney stones, it also serves important. Physiological functions. Number one, it acts as a

1. (__________),

2. (____________), and

3. (________) from oxidative stress.

4. It can also (________________) such as (__________) and (_________),

5. helping (__________________).

Furthermore, uric acid may have neuroprotective effects, such as studies that suggest that moderate levels helps protect our central nervous system from oxidative damage, possibly reducing the risk of neurodegenerative diseases like Parkinson’s and Alzheimer’s.

(enhance antioxidant defenses) (support longer lifespan)

(improve brain function.)

Evolutionarily, the loss of uricase in humans may have been beneficial because the resulting higher uric acid levels could (__________), (______________), and (______________)

(hyperasemia) (purines) (kidneys) (red meat and seafood) (diuretics)

most common condition related to uric acid is (___________)

• this is a state where uric acid levels exceed the normal values and it can be by a whole lot of reasons. Can be cause by overproduction of (______) can be decreased excretion by the (________)

High consumption of purine rich foods such as (_____&______) also high alcohol intake which lead to hyper uricimia,

Another reason is the use of certain medication like (__________)

(Hyperuricemia)

(_____________) can lead to gout, which involves painful inflammation of joints due to uric acid crystal deposition as well as kidney stones and a higher risk of metabolic and cardiovascular diseases.

conversely, low uric acid levels or hypothurismia may result from liver dysfunction, which can also be due to excessive kidney excretion or there is xanthine oxidase deficiency or can be wilson’s diseases.

(diagnosing gout) (monitoring renal function) (assessing metabolic conditions.) (monitoring patient undergoing chemotherapy) (enzymatic) (lifestyle)

Uric acid testing is important, especially in

(_____________), (____________), and also (__________). it is aslo use to (________________) because there is rapid cellular breakdown when the patient is undergoing chemotherapy and in doing so there will be the release of large amounts of purines causing dangerous uric acid accumulation in our patients. Regulation of uric acid levels can be achieved through both (__________) and (_________) means

-xanthine oxidase controls the rate of uric acid productions.

(-Gout)

(Treatment of myeloproliferative disease with cytotoxic drugs)

(Hemolytic and proliferative disease)

(Purine-rich diet)

(Increased tissue catabolism or starvation)

(Lesch-Nyhan syndrome (Hypoxanthine guanine Phosphoribosyl transferase deficiency)

(Phosphoribosyl pyrophosphate synthetase deficiency)

(Glycogen storage disease Type 1 (G6PD deficiency)

(Fructose intolerance (Fructose-1-phosphate aldolase deficiency)

(Toxemia of pregnancy)

(Lactic acidosis)

(Chronic renal disease)

(Drugs and poison)

Increased Uric Acid Concentration

-
-
-

-
-

Enzyme deficiencies

-
-
-

-
-
-

-
-

(Liver disease)

(Defective tubular reabsorption (Fanconi syndrome)

(Chemotherapy with azathioprine or 6-mercaptopurine)

(Overtreatment with allopurinol)

Decreased Uric Acid Concentration

• 
  

• 
  

• 
  

• 
  

Caraway method

ANALYTIC METHODS

● Most common early method

● It is based on oxidation of uric acid in a protein-free filtrate with a subsequent reduction of phosphotungstic acid in an alkaline solution, it produces tungsten blue.

● It is a common method but non-specific and tedious because it requires protein removal

(Enzymatic method) (Enzyme Commission (EC) (1.7.3.3)

(_________________)

● Uses uricase/urate oxidase

● Uricase catalyzes uric acid → allantoin

● The enzyme is identified with

(___________________)

number (_____)

● More specific and used

almost exclusively in clinical

laboratories

(Spectophotometric method) (Hemoglobin and xanthine)

(______________________)

● The simplest uricase method

measures the differential

absorbance of uric acid and

allantoin at 293 nm

● The difference in absorbance

before and after incubation

with uricase is proportional to

uric acid concentration

● Proteins can cause high

background absorbance

which reduces sensitivity

● (___________&__________)

can also cause negative

interference

(Coupled enzymatic method) (1.11.1.6.) (Traditional wet chemistry analyzers )

(Dry chemistry slide analyzers) (bilirubin) (ascorbic acid)

(potassium ferricyanide) (ascorbate oxidase)

(_____________________)

● This measures the hydrogen peroxide produced when uric acid is converted to allantoin

● Peroxidase or catalase has the enzyme commission number (_______)

● The enzyme catalyzes a chemical indicator reaction.

● The color produced is proportional to quantity of uric acid in the specimen

● These enzymatic methods have been adapted for:

○ (_______________________)

○ (___________________)

● If the specimen contains sufficient amounts of (_______) and (__________). These substances can interfere with the reaction.

● (6) (____________) can destroy peroxide

● Commercial reagent preparations may include (___________) and (_____________) to minimize these interferences.

(High-performance liquid chromatography) (blood and urine) (260-285 nm)

(_______________)

(___) can accurately

determine uric acid levels in

biological samples such as:

(______&______)

● Advantages include good linearity, good precision, and more reliable determination

● It involves reversed-phase column, mobile phase containing water, acetonitrile, and buffer

● Detection can be done by UV and mass spectrometry at (_________)

(Isotope dilution mass spectrometry) (endogenous uric acid) (chromatography) (mass spectrometry) (labeled) (unlabeled)

(accurately calculated)

● (__________________________) can also be used for uric acid determination.

● Adding a stable-isotope-labeled uric acid to a sample like serum

○ It is allowed to equilibrate with (___________________).

○ The mixture is separated, often by (_______________).

○ It is derivatized to make it suitable for (_____________).

○ The isotope ratio of (______) and (_________) uric acid is measured.

○ The original uric acid concentration is (___________________).

(ERBA) (hydrogen peroxide) (4-aminoantipyrine (4-AAP) and DHBS (3,5- dichloro-2-hydroxybenzenesulfonate) (colorimetric uric acid test) (505 nm) (liquid form)

For the lab activity: Used: (_____)

● Uric acid is oxidized to allantoin by uricase with the production of (____________)

● The hydrogen peroxide then reacts with (______________________________) with the presence of peroxidase → Red quinoneimine dye

○ DHBS is chemically used in (____________)

● Red quinoneimine dye

○ Measured at (______)

○ The absorbance is directly proportional to uric acid concentration in the sample.

○ More uric acid → more dye produced → greater absorbance at 505 nm

○ The rgt are already in (_____) and are ready to use

(Pipes buffer (7.0)) (50 mmol/L) (DHBS) (0.50 mmol/L) (Uricase) (≥ 0.32 kU/L) (Peroxidase) (≥ 1.0 kU/L) (4-aminoantipyrine) (0.31 mmol/L)

(Uric acid) (357 umol/L)

RGT COMPOSITION

R1	- - - - - -	- - - - - -
R2	-	-

(non-hemolytic serum, heparinized plasma, EDTA plasma, 24-hour urine sample) (24 hrs) (24-hour urine) (15) (5) (sodium hydroxide) (> 8) (10)

(discard the sample) () () ()

SPECIMEN COLLECTION AND HANDLING

● Used in lab activity: (_________, __________, _____________, ______________)

● Urine (_____)

○ When performing a (_______) uric

acid determination, (_) mL of (_) mol/L

(_______________) is added to the urine

collector to prevent precipitation of uric

acid.

■ The purpose is to maintain urine

pH (___)

○ Dilute using 1 (urine):9 (distilled water) and

multiply the result by (__)

○ Contaminated urine: (__________)

(3-4 days) (20-25°C ) (7 days) (4-8°C) (6 months) (-20°C)

Stability in serum/plasma	At temperatures
-	-
-	-

-	-

(505 nm) (37°C) (5)

Wavelength: (____)

Temp: (____)

Mix and incubate for (_) mins at 37°C.

𝑈𝑟𝑖𝑐 𝑎𝑐𝑖𝑑 (𝑚𝑔/𝑑𝑙) =

Δ𝐴 𝑠𝑎𝑚𝑝𝑙𝑒

____________×𝐶𝑠t

Δ𝐴 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑

standard (calibrator) concentration

Calculation:

Cst = (_____________________)

(3.5 - 7.2 mg/dl)

(2.6 - 6.0 mg/dl)

(2.0 - 5.5 mg/dl)

(250 - 750 mg/dl)

(< 1000 mg/dl)

(< 480 mg/dl)

(< 420 mg/dl)

REFERENCE VALUES

Serum	Adult male	(__________)
	Adult female	(_________)
	Child	(__________)

---

Urine (24 hrs)	Average diet	(________)
	High-purine diet	(_________)
	Low-purine diet	(_________)

	Purine-free diet	(_________)

𝑚𝑔/𝑑𝑙 × 60 = 𝑢mol/L

Unit conversion

(Hemoglobin) (Bilirubin) (Triglycerides) (N-acetylcysteine (NAC), acetaminophen, and metamizole) (N-acetyl-p-benzoquinone)

ANALYTES THAT DO NOT INTERFERE

(_________)	Up to 10 g/l
(_________)	Up to 40 mg/dl
(_________)	Up to 2000 mg/dl

(_________)	False low results
(_________)	False low results