or PFF.

The PFF contains Total Non-protein

NON-PROTEIN NITROGEN COMPOUNDS Nitrogens. We can't still identify which exact
Lecturer: Ms. Ms.Praise Selah G. Dagoc,RMT,MLS (ASPi)CM or what is the percentage of each individual
NPN because it is still just a filtrate. What
OUTLINE we have here is the Total NPN.
I. Introduction
I. NPNs, (Biochemistry, Methodologies) INTRODUCTION
a. Blood Urea Nitrogen ● NPN = Nitrogen compound that is not a protein.
b. Ammonia
c. Uric Acid The term Nonprotein nitrogen (NPN) originated in the early
d. Creatine Creatinine: this two may be connected by days of Clinical Chemistry when analytical methods required
they are not exactly the same. removal of CHONs before analysis.

LEARNING OBJECTIVES Based on the previous slide they added protein

At the end of the discussion the students must be able to: precipitants to get rid of protein, which we don’t do right
● Describe correctly the major NPN components and now kase modern napo tayo. Now we have developed
its corresponding concentration. methodologies that skip that process so that we can now
● Describe properly the biosynthesis and excretion of analyze NPNs without getting rid of proteins.
urea,creatinine,uric acid and ammonia.
● Discuss extensively the commonly used methods of Concentration of N-containing compounds in PFF was
determining its major components and the quantitated SPM by converting N to NH3 and subsequent
specimen collection,handling storage. reaction w/ Nessler's reagent, K2 (Hgl4), to produce a yellow
● Recognize the reference intervals for these NPN color. The majority of these compounds arise from the
substances. catabolism of proteins and nucleic acids.
● Relate some clinical conditions associated with
abnormal values. Total NPN is of value in the assessment of nitrogen balance in
nutritional management. More useful clinical information is
NON PROTEIN NITROGEN (NPN) obtained by analyzing individual NPN.
● What is NPN? - Non Protein Nitrogens
● What are proteins? (CHON) However clinically, it’s not really that requested. What is
It is made up of Carbon, Hydrogen, Oxygen, and more often requested by clinicians are the individual
Nitrogen, thus CHON. NPNs which is why we are discussing this in the next
slide.
Aside from the proteins, the building blocks and the
precursors to it which are the amino acids and CLINICALLY SIGNIFICANT NPN COMPOUNDS
peptides also has nitrogen. Here are the clinically significant NPN compounds that are
found in the blood. The values that we see are the percentage
● Blood Nitrogen Compounds (CHON + NPN) of the NPN.
o Composed of proteins and non-protein nitrogen
● Total Nitrogen (Review) ● Approx. plasma concentration of the individual NPNs (% of
o Total Protein + Non Protein Nitrogen compound Total NPN)
▪ which is Urea and creatine are measured.
NPN Percentag Notes
e
1. Urea 45-50 The highest. It composes
the largest plasma
concentration of NPN
2. Amino 25 Why amino acids if that is a
Acids protein?

Technically NO, it is still a

NPN, although it is a
building block of the
proteins. That is why in the
contents, we will not talk
about amino acids bc even
though technically it is a
● How to quantitate NPN? (WB + Protein Precipitants = NPN, it is a building block,
Protein-Free Filtrate (PFF)) still a protein and it has
o Historically and basically, it’s whole blood plus the been already been included
in the lecture.
addition of protein precipitants.
▪ Protein precipitants: To get rid of the 3. Uric Acid 10
protein, because it precipitates the protein. 4. Creatinin 5
And what is left is the Protein-Free Filtrate e
 LEC 10 – NON-PROTEIN NITROGEN COMPOUNDS

5. Creatine 1-2 2. Assess hydration status

6. Ammonia 0-2
● Remember the general ranking of the NPNs and deduce
why these are there amounts based on their biochemistry,
how they are metabolized and formed.
BLOOD UREA NITROGEN (BUN)
● Top 1 of the NPNs
● A Major excretory product of CHON metabolism.
o Why is it called BUN if it sounds redundant?
● BUN because historic assays for measuring urea were
based on the N content in urea. Urea N has been used to
refer to urea determination (indirect measurement).
They don’t measure urea itself, they measure the nitrogen.
Hence it is an indirect measurement because they are
measuring nitrogen instead of Urea itself.
HOW IS BLOOD UREA NITROGEN FORMED AND HOW ARE THEY
METABOLIZED?
In the small intestines, dietary CHONs are hydrolyzed by
proteases → AAs (amino acids).
● These amino acids go to 2 routes:
1. Large portion goes to the bloodstream for further
CHON synthesis or oxidized to produce energy or
stored as fat glycogen.
2. A significant portion undergoes deamination by
enzymes of bacteria in the glutamate lumen of S.I.
→ glutamate (dicarboxylic AA) is acted upon by the
enzyme 𝑔𝑙𝑢𝑡𝑎𝑚𝑎𝑡𝑒 𝑑𝑒ℎ𝑦𝑑𝑟𝑜𝑔𝑒𝑛𝑎𝑠𝑒→
α-ketoglutarate + NH3 (waste product) →
bloodstream (Take Note: Free NH3 is toxic,
therefore) → liver → NH3 - Krebs-Henseleit Cycle
→ less toxic UREA
● As mentioned earlier, some can be reabsorbed
depending on the hydration. This is a bit of a minor
purpose than the first one.
3. Determine Nitrogen balance
4. Aid in the Dx of Renal Disease
● Take note of “Aid”. If we take out the word aid, that
will be wrong because BUN only is not definitive in
the diagnosis of renal disease. You need to pair it
with other tests.
5. Verify adequacy of dialysis
● Dialysis is when the blood is filtered through an
external machine because the kidneys cannot do it
properly anymore. BUN can be used to check if the
dialysis procedure was adequate.
NORMAL VALUE OF UREA IN THE BLOOD
● NV of Urea in the blood: 6-20 mg/dL
● ↑ w/ age. Children have lower values (They don’t
eat much & Body mass).
UREMIA VS. AZOTEMIA
1. Azotemia - ↑ BUN, CREA & Other
Uremia or
prods of CHON metabolism
Uremic
2. Failure of excretory, regulatory, &
Syndrome
endocrine function of the kidney.
● If left untreated, can progress to stupor, coma, & death.
● Treatment: Dialysis and Kidney transplantation
● Uremia – presence of increased ↑ urea in the blood.
● Azotemia – a collective term to describe ↑ BUN, CREA &
Other prods of CHON metabolism
o Question: Do all azotemia also mean Uremia? Do
all uremia also mean azotemia? Based on the
table are both statements correct?

THREE (3) MAIN CATEGORIES OF INCREASED ↑ UREA

1. Pre-Renal – the processes before it arrives in the kidneys.
2. Renal - ↓Renal Function = ↓ Urea Excretion
3. Post-Renal – Urinary Obstruction. After the kidneys, there
are still several tubules before it becomes the final product
Then, from Urea, it goes to the blood stream. It then either – urine. Increased urea is determined if any of those
enters the: tubules are obstructed.

1. <10% reenters the G.I. Tract for microbial breakdown 1. PRE-RENAL

or excreted by the skin. Or; B. Decreased ↓ renal blood flow = less blood is delivered
2. Most of the urea enters the Kidney and is freely to the kidney = less urea is filtered out. Happens in:
filtered → urine excretion ● Congestive heart failure
● Some are reabsorbed by passive diffusion on ● Surgical shock
urine flow & extent of hydration ● Hemorrhage
● Dehydration
● Plasma urea is dependent on CHON content of diet, rate
of CHON metabolism and renal function & perfusion. B. Increased ↑ Amount of CHON Metabolism Stress
o If you are on a high protein diet, you would ● Fever
expect to have a higher plasma urea. ● Major illness
● Corticosteroid therapy

MEASUREMENT OF PLASMA UREA IS USED TO: ● G.I. hemorrhage
1. Evaluate renal function
● Because it is a majorly excreted through the 2. RENAL CAUSE
kidneys, and its presence or absence thereof can ● All of these are kidney diseases/disorders:
evaluate renal function. If you have more urea in 1. Acute & chronic renal failure
your blood than normal, then you would start to 2. Glomerular nephritis
question why is it not filtrated out. 3. Tubular necrosis

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 LEC 10 – NON-PROTEIN NITROGEN COMPOUNDS

● No = Normal

BUN METHODOLOGIES (INDIRECT)

3. POST-RENAL CAUSE ● Measures Ammonia instead of Urea.
● Renal calculi (Kidney Stones)
● Prostate or bladder tumor 1. COUPLED ENZYMATIC METHOD (@578NM)
● Severe infection ● Initial hydrolysis step with urease or urea
o UTI/Severe UTI can cause increased urea amidohydrolase
in the blood

THREE MAIN CATEGORIES OF DECREASED ↓ UREA

If you are asking if isn’t it a good thing since urea is a waste
product, well, not really. Lesser urea in the blood can still
indicate that there must be a problem.

What are the conditions that could lead to lesser urea in the
blood.
1. Increased CHON synthesis
● Late pregnancy & Infancy ● Urea with H2O and the action of Urease, it becomes
2. Decreased CHON Intake Ammonia and Carbon dioxide. Ammonia with
● Starvation & Malnutrition salicylate and the action of nitroprusside (catalyst)
3. Urea Formation turns into HCl and 2,2 dicarboxy indophenol.
● Liver Disease ● 2,2 dicarboxy indophenol is what’s being measured

1. INCREASED CHON SYNTHESIS: LATE PREGNANCY & 2. BERTHELOT METHOD (@340M)

INFANCY ● Indicator enzyme: GLDH (Glutamate dehydrogenase)
rd
● Within the 3 trimester, when the mother’s body
works overtime to make sure the fetus inside comes
out healthy.
● Infancy is the first few stages of human life so the
body is working overtime synthesizing CHON
● Protein is being used up and stored in cells, lesser
protein metabolize so lesser urea.

2. DECREASED CHON INTAKE

● Starvation & malnutrition
o Low protein diet because no protein input,
there is low urea Urea plus water with the action of Urease becomes
ammonium ions and bicarbonate. Ammonium ions plus
3. UREA FORMATION 2-oxoglutarate with the action of GLDH becomes glutamate
and water. With that process, NADH plus hydrogen ions
● Liver disease
turns into NAD plus.
o If Liver not functioning properly so less urea
will be formed because less protein is
● α-ketoglutarate or 2-oxoglutarate (same thing)
metabolized
● Best as kinetic measurement
● MoM: rate of decrease ↓ in abs @ 340m as NADH is
UREA N/CREATININE RATIO oxidized to NAD+ which does not absorb light at 340nm
● This ratio normally at 10:1 to 20:1 (Urea:Crea) aids
differentiation of the cause of abnormal urea
concentration. ● Why is there a decrease in absorbance?
o Because while NADH absorbs light, NAD+ does not
absorb light at 340 nm
o The more there is action, from this to this (NH4+ +
2-oxoglutarate → glutamate + H2O), the more the
NADH is consumed to become NAD+.
o The more NAD+, the smaller the absorbance of
light because this NAD+ does not absorb light.

● To sum it up:
o The more urea, the more ammonium ions
o The more ammonium ions, the more reactions with
NADH
o The more NADH, the more NAD+ products
o The more NAD+ products, the greater the decrease
in the absorbance

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 LEC 10 – NON-PROTEIN NITROGEN COMPOUNDS
o So here, since this is a kinetic measurement, we
would call it the rate of decrease in absorbance.
(Kinetic means action, you would expect the value
to be a rate)
3. CONDUCTOMETRIC METHOD
● In this method, urea with water and with the action of the
urease enzyme, turns into ammonium ions and
bicarbonate.
● In water, the products are carbonate anion & ammonium
cation
● Conductivity of the solution is increased, then the
ammonium is measured by ISE.
● Concentration is determined from the measured potential
using the Nernst equation.
● Advantage:
o Rapid & specific
o The ammonium contamination is not a problem
▪ Why? Because ammonium is not part of
the substrate. It’s part of the product. If you
compare it with the previous method dba
duha toh cya ka parts. Although yes, the
ammonium is the product in the first part.
In the second part, the ammonium
becomes the substrate. And so, if your
sample is contaminated with ammonium,
that contamination would react together
with the ammonium that you derived from
the urea in the blood. Therefore, it would
lead to what? False increase or decrease?
K, think about it.
To infer whether a certain compound is
significant in terms of contamination or
not, check and see where it is in the
formula.
If it becomes part of the substrate, then
yes that can affect. If it’s in the end
product, okay ra cya.
4. NH4+ MEASUREMENT BY COLOR CHANGE ASSOCIATED WITH
PH INDICATOR
● So here, ammonium + pH indicator would lead to color
change
● Would ammonium contamination affect your results? Yes
or No
● This method is Incorporated into instruments using liquid
reagents, a multilayer film format and reagent strips
5. ISOTOPE DILUTION MASS SPECTROMETRY (IDMS)
● Proposed reference method
● Capable of detection of characteristic fragments ff.
Ionization
● Quantitation using isotropically labelled compound
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BUN METHODOLOGIES (DIRECT METHODS)
1. DIACETYLMONOXIME (DAM) METHOD
● In this case, BUN with diacetyl and the action of heat and
hydrogen ions, becomes diazine + water
● End product: Yellow chromogen diazine at 540 nm
o In some laboratory kits, it’s 480nm but the
recommended standard is 540nm (refer to the
instructions and the manuals or leaflet that you
find in your laboratory kits.
o Even though yes, we do study these things in
school but remember laboratory subjects,
especially clinical chemistry, is constantly
evolving. What you may be studying now may not
be exactly the same as the one you will be
practicing later on as licensed medical
technologists or when you order for tests when
you become doctors. Does that mean na useless
inyu gtun an karun? No, because these are the
foundations.
● Inexpensive and CHONs don’t interfere
● Lacks specificity
● Asterisk in Diacetyl means. Diacetyl is in solution: DAM +
acid → unstable diacetyl
1. DAM Method – is based on Fearon’s Reaction
● FEARON’s reaction - It is where urea and other
compounds with R1-CO-NHR2 structure react with
DAM in the presence of a strong acid and an
oxidizing agent to produce a chromogen.
● Principle: Urea is condensed with diacetyl produced
by the oxidation of DAM in arsenic/nitric acid
(provides strong acid environment) plus heat (boiling
water) to form the YELLOW DIAZINE
derivatives/YELLOW condensation product.
o In this case, oxidising agent is the
arsenic/nitric acid
o Do you remember the previous formula? It’s
heat + H+ so the source of your hydrogen
ions is your acid.
● Urea is not hydrolyzed to ammonia but is measured
directly
● Category of estimation: COLORIMETRIC approach
● Schematic Reaction Principle (2 Steps):
1. Diacetylmonoxime + H2O ℎ𝑒𝑎𝑡 (𝑎𝑟𝑠𝑒𝑛𝑖𝑐 𝑎𝑐𝑖𝑑) → Diacetyl
+ Hydroxylamine
2.
+
Diacetyl + Urea 𝐻 → Diazine (yellow) + 2H2O
o Diazine has a strong absorbance at 540 nm
o Diacetyl - an unstable compound, has been used
to eliminate hydroxylamine thus obviating the
need for an oxidizing agent.
o Hydroxylamine - interferes with urea
quantitatively, but is also eliminated by arsenic
acid, the oxidizing agent.
o Nganong wala nay arsenic acid in the 2nd
formula? Because you already have diacetyl.
Diacetyl can eliminate the hydroxylamine
o Other oxidizing agents:
4
 LEC 10 – NON-PROTEIN NITROGEN COMPOUNDS

1. Potassium persulfate o The use of blue and gray top tubes should be
2. Perchloric acid avoided since these contain Na Citrate and
NaF which may interfere with the test.
Advantages Disadvantages
2. URINE
● Measures urea directly ● Color develops & fades
● Ammonia does not rapidly ● Susceptible to bacterial decomposition
interfere ● Color is photosensitive ● Remedy if test is not run immediately: refrigerate
● Color is not linear ● Methods for plasma and serum may require
● Unpleasant /Irritating modifications with urine specimen because of the
fumes of reagents increase ↑ in urea content and presence of
endogenous NH3
● Other Factors to Consider:
1. Condensation Process 3. SERUM
● Entails boiling and cooling ● Non-hemolyzed
2. Boiling Water Bath ● In most clinical laboratories, serum is used for the
● Initiate condensation of urea w/ DAM measurement of BUN.
3. Thiosemicarbazide & ferric Ions (If added) ● You may use the red top tube or gold top tube.
● Stabilizes the color, minimizes the Orange top tubes are rarely used.
photosensitivity of the yellow diazine-end
product & excludes protein interferences
● Can somehow override the disadvantages of the REFERENCE RANGE FOR BUN
method but not much
Plasma/Seru 6-20 mg/dL or 2.1 - 7.1 mmol/L
● Measurement of urea were originally performed on PFF m
(protein free filtrate) and based on measure nitrogen.
Urine (24 h) 12-20 g/d or 0.43 - 0.71 mol urea/d

PIT STOP
1. TRUE about the DAM Method:

2. O-PHTHALALDEHYDE METHOD
● Condensation product of urea and o-phthalaldehyde
reacts with N-(l-naphthyl)ethylenediamine to yield a
colored product measurable at 505 nm.
o N-(l-naphthyl)ethylenediamine can also be
replaced by;
● 8-(4amino-l-methylbutylamino)-6-methoxyquinoline is
a popular alternative to N(l-naphthyl)ethylenediamine, in
which case the chromophore is measured at 510nm.
● Unlike the Fearon methods, these reactions do not require
incubation at high temperatures. The reaction has been
adapted for use in continuous flow, manual, end-point and
kinetic methods.
● Method remained unpopular because of the caustic (can
irritate and burn the skin) nature of some of the reagents,
despite the stability, cheapness and speed of the reaction.
● Subject to interference.
● Due to this, it is not used as often anymore.
AMMONIA (NH3)
● Still a byproduct of protein metabolism.
● Primary site of NH3 formation: Small Intestine
● Endogenous NH3 has three 3 routes of formation:
1. By-product of CHON metabolism
2. Urea re-enters S.I. for microbial breakdown
3. Some results from anaerobic metabolism in
skeletal muscle during exercise
We say endogenous ammonia because we have external
sources of ammonia e.g. poisoning. In this case we are
talking about endogenous ammonia derived from body
functions.
AMMMONIA BIOCHEMISTRY

SPECIMEN REQUIREMENTS FOR BUN

1. PLASMA
● Non-hemolyzed
● NH4+ must be avoided
● Increase ↑ in Na citrate and NaF inhibits urease Urea in the small intestine becomes ammonia which then
o Tubes with Lithium heparin (light green top goes to the bloodstream. There, you find an equilibrium of
tubes) are recommended. ammonia and ammonium ions. The ammonium ions go to

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 LEC 10 – NON-PROTEIN NITROGEN COMPOUNDS
the kidney for secretion. Whereas the ammonia
goes to the liver parenchymal cells where it goes
through the Krebs Henseleit Cycle.
The equilibrium depends on the blood pH. The
lesser the pH, the higher the hydrogen ions, the
more the ammonium ions. In normal pH, you
have an abundance of ammonium ions. So,
ammonia plus hydrogen turns into ammonium
ions but this is a reversible reaction meaning
the ammonium ions can also turn into ammonia
and hydrogen ions.
2. LIVER PARENCHYMAL CELLS - KREBS HENSELEIT CYCLE
● Simplified version of the Krebs Cycle.
● Ammonia plus carbon dioxide with ATP becomes
carbamoyl phosphate. The carbamoyl phosphate plus
ornithine with further transformation then regenerates
ornithine, which is recycled, and UREA, which is the
by-product.
PIT STOP
MEASUREMENT OF AMMONIA
Measurement of Ammonia is used for:
1. Severe liver diseases - Used to determine prognosis,
however, not consistent; Arterial NH3 is a better indicator.
● prognosis = when you are trying to see or determine
the outcome of a condition.
● Remember that the blood we take for CC is venous,
so if you want to use it as a prognosis of severe liver
diseases then arterial ammonia might be better.
● Parenchymal cells function is severely impaired, NH3
is not removed in the circulation.
● ↑ NH3 is neurotoxic & often associated with
encephalopathy (diffuse disease of the brain that
alters brain function or structure)
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2. Reye’s Syndrome - Correlation with both severity of the
disease and prognosis
● Acute metabolic liver disorder
● Occurs most commonly in children
● Serious disease that can be fatal
● Preceded by viral infection & AD of aspirin
o Right now aspirin can’t be bought over the
counter, it is already a prescription-required
medicine because it has been found to have
so many side effects in so many people
● Autopsy reveals severe fatty infiltration
o There really is a liver dysfunction.
● Survival reaches 100% if NH3 remains below 5x
normal.
o Ammonia needs to be maintained lower than
the normal
3. Inherited Deficiency of Urea Cycle Enzymes - for
diagnosis; testing should be considered for any neonate
with unexplained nausea, vomiting or neurological
deterioration assoc. with feeding
4. Hyperalimentation Therapy
5. Urine Ammonia used to confirm ability of kidneys to
product NH3 (Recall: Since ammonia has one route going
to the kidneys)
CLINICAL SIGNIFICANCE: PRIMARY CAUSE OF ↑NH3 IN ADULTS
Primary cause of increased NH3 in adults (Clinical
Significance):
1. Hepatic encephalopathy
● Venous blood doesn’t pass thru the liver in large
enough quantities for detoxification
● Personality changes develop, neurological
alterations appear & coma as end result of liver
damage
● May involve liver necrosis or cirrhosis
2. Renal failure - inability to excrete sufficient urea & NH3
3. Pulmonary problems - 2° to altered blood pH, which
distorts equilibrium between NH3 & NH4+
● It is associated with pulmonary problems because
remember that the equilibrium is affected by the blood
pH and that is affected by pulmonary problems
4. Reye’s Syndrome
5. Any abnormality in Krebs Henseleit Cycle - impaired
metabolism (which can be attributed to hereditary
disorders in urea cycle enzymes)
AMMONIA METHODOLOGIES
SPECIMEN REQUIREMENTS
● Plasma is preferred specimen for rapid separation
because test must be done ASAP to eliminate problems
with endogenous NH3 formation.
o Do take note that for this test, it must be ASAP
because if it cannot be done ASAP as in cases
wherein that hospital does not have an test
available in their laboratory, it needs to be sent
out placed…
● Place specimen in ice bath or ref to slow down this
process
6
 LEC 10 – NON-PROTEIN NITROGEN COMPOUNDS

o If it cannot be done ASAP as in cases where the composed of 400mg from endogenous purines + 300mg from
hospital doesn’t have an ammonia NH3 test is not dietary purines and fed into the Bloodstream
available and it needs to be sent out
● Smoking and caffeine cases marked ↑ in NH3
o It is not common to have ammonia measurement
which is why a lot of labs don’t have ammonia as
part of their test roster
o Moderation and balance is key
o Do you notice when you’ve had too much coffee,
when you urinate there is a certain smell? That’s
actually ammonia. This tells us that your renal or
kidneys are functioning well because it is able to
excrete the extra ammonia from your system
which you got from your coffee.

1. COLORIMETRIC ASSAY
● NH3 binds to cation exchange resin, isolated from the ● Two 2 routes in the bloodstream:
remainder of the material by elution. 1. Kidney
● Following elution from the resin, 2. Intestines
o 50% of total BUA is secreted

1. Kidney
● Most are filtered
● 98-100% are reabsorbed in the PCT (proximal
convoluted tubule)
Ammonium ions plus NaOCl plus phenol hypochlorite with the ● 40% is reabsorbed in the distal convoluted tubule
action of OH- turns into indophenol which can be read at through active transport processes
630nm ● Only 6-12% Uric Acid is excreted (400-500mg/d)
● Renal excretion accounts for 70% BUA elimination
2. ENZYMATIC ASSAY
● Produces lower values than colorimetric 2. Intestines
● Using the reverse of the metabolic process leading to ● Degradation by bacterial enzymes
NH3 formation in the body: ● 200-500mg eliminated in the stool
o Kidney → Urine
o Intestine → Stool
● Nearly all uric acid in plasma is present as
monosodium urate
● NADPH absorbs light at 340 nm; MoM: ff decrease ↓ ● Uric acid has limited solubility
in absorbance at 340 nm. Why? ● At pH 7.0 urate is relatively insoluble and at >6.8
o There is a decrease in absorbance at 340 mg/dL, plasma is saturated
nm because the product is NADP not ● Urate crystals precipitate and can deposit in joints
NADPH such as in gout and tissues causing painful
o NADPH will absorb while the NADP will not inflammation
● Reference Range: 20-80 ug/dl o Gouty arthritis is caused by urate crystals
● At urine pH of <5.75, uric acid is predominant and
URIC ACID uric crystals can form
● End product of purine nucleoside catabolism
o Purines: Adenine and Guanine BUA | MEASUREMENT
o Pyrimidine: Cytosine and Thymine o How or why is BUA measured?
o How can we use measurement of Blood Uric Acid?
URIC ACID (BUA) BIOCHEMISTRY. ▪ BUA - Blood Uric Acid
▪ UA - Urinalysis
● Measuremnent of BUA is useful in:
1. Gout
● It aids in the diagnosis of gout
● Confirming diagnosis and monitoring
2. Chemotherapy Patients
● Monitoring to prevent uric acid nephropathy or
nephrotoxicity
3. Inherited disorders of purine metabolism
● For assessment
4. Kidney Dysfunction
The Endogenous Purines are the adenosine and guanosine ● Detect
nucleotides from tissue destructions plus the Dietary Purines ● Expected filtrate of the kidney
which are the breakdown of ingested nucleic acids is then 5. Renal Calculi
delivered to the liver where is become Uric Acid which is ● Or kidney stones
● Assist Dx

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 LEC 10 – NON-PROTEIN NITROGEN COMPOUNDS

okay to eat this but in moderation. Also cut down alcohol

REFERENCE RANGE OF URIC ACID: intake. As early as now, eat healthy!

Male up to 7.0 mg/dL

Female up to 6.0 mg/dL
● Increase synthesis of purine precursor lead to increase ↑
uric acid (uricemia)

BUA | CAUSES OF HYPERURICEMIA

● There is an increase levels of uric acid in the blood
This is sweetbread where the meat comes from the thymus
1. Alcohol and patient’s dietary purine intake - (Liver, It is in the internal organ so it is high in purine, therefore eating
kidney, sweetbreads and shellfish). Both could lead to too much of this will lead to higher uric acid because uric acid
gout comes from metabolized purines
● Sweetbreads that is mentioned is not croissant or
any bread BUT a culinary term used to describe 3. Tissue hypoxia
meat that comes from the thymus or pancreas
● Meat from the thymus is sweeter than the regular 4. ↑ NA (nitric acid) turnover when there is ↑ metabolism
meat and bread for the morsel of cell nuclei
● Alcohol and patient’s dietary purine intake are ● Chemotherapy for proliferative diseases (leukemia,
AVOIDABLE by balanced diet. Unless it is genetic lymphoma, multiple myeloma (MM), polycythemia)
hereditary disorder then it is uncontrollable ● In leukemia, ↑ cellular breakdown → ↑ levels of
purine → ↑ purine turnover → ↑ uric acid production
2. Gout (as high as 18,000-30,000 mg for Gouty Arthritis) ● Radiotherapy
● With that amount of uric acid in the blood, where the ● Trauma
kidney and intestine cannot excrete anymore
therefore, it will go to the joints. 5. Inherited disorders of purine metabolism
● Patients usu. >6.0 mg/dL ● Out of your control because it is inherited; you got it
● Disease primarily found in 30-50 years old men from your genes
o It still does happen to women but it is more
often associated with men I. Lesch-Nyhan Syndrome
o Why men? Estrogen which is the hormone o X-linked disorder (seen only in ♂) - Remember,
that is found abundantly in women helps in in the male, you have XY, so if this disease is in
getting rid in uric acid X, there is no other X to neutralize. Thus, the
o That is why uric acid is higher in men Lesch-Nyhan syndrome manifests. In females
because men have lesser estrogen levels (XX), it can be neutralized by the other X
● Joint pain and inflammation caused by precipitation chromosome, thus the Lesch-Nyhan does not
of sodium urates. manifest.
o Uric acid forms urate crystals that get o Complete deficiency of hypoxanthine-guanine
lodged in the joints. phosphoribosyl transferase (important in the
● In 25-30% of patients hyperuricemia is caused by biosynthesis of purines)
overproduction of uric acid which may be
exacerbated by a purine-rich diet, drugs and alcohol II. Mutations in phosphoribosyl pyrophosphate
● Patients are susceptible to the formation of renal synthetase
calculi although not all patients with abnormally high
uric acid can develop this complication III. Competition w/ uric acid for renal excretion when
● In females, uric acid can rise after menopause. there is excess lactate or triglyceride
Postmenopausal women may develop hyperuricemia o Glycogen storage disease type 1
and gout (glucose-6-phosphatase deficiency)
o Because estrogen in decreased o Fructose intolerance (fructose-1-phosphate
● In severe cases deposits of crystalline uric acid and aldolase deficiency)
urates called tophi form in tissue, causing
deformities 6. ↑ Renal Absorption

7. ↓ Excretion

I. Preeclampsia or (toxemia of pregnancy)

II. Impaired secretion due to diabetic
ketoacidosis (DKA) and lactic acidosis
(presumably as a result of competition for binding
sites in the tubules. Lactic acidosis is a common
finding in DKA)
III. Rapid weight loss (impaired secretion)
Ginabot is a rich source of uric acid along with other internal IV. Chronic renal disease (impaired filtration &
organs such as liver, batikon, shellfish, and many more. It’s secretion)

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 LEC 10 – NON-PROTEIN NITROGEN COMPOUNDS

8. Thiazide diuretics - either dehydration as more body H2O

is lost or because of blockage of tubular secretion of uric ● Very specific
acid ● Method: Differential absorption of uric acid & allantoin
9. Lead poisoning at 293nm before & after incubation w/ uricase
10. Starvation - ↑ tissue catabolism ● MoM: rate of ↓ in Abs at 293nm
11. Hemolytic or Megaloblastic anemia ● Use of special quartz cuvette (glass does not transmit
12. Bone disease - cause of ↑ uric acid is not known light <340nm)
(Calbreath) ● CHONs cause high background Abs reducing
sensitivity
HYPERURICEMIA + HYPERURICARIA ● Hemoglobin & Xanthine → negative interferences
● Leads to: ● Reference Ranges:
1. Kidney Disease
2. Gout
3. Renal Calculi

● In acidic urine, uric acid precipitates to form calculi

(stones), causing intense flank pain
● Dissolved by alkalinization of urine or ↑ fluid intake
● If urine has become quite acidic basing on AUBF / too 3. COUPLED ENZYME ASSAY
dark in physical examination, neutralize with fluid intake ● Measures H2O2 by peroxidase or catalase used to
catalyze chemical indicator reaction
BUA | CAUSES OF HYPOURICEMIA
1. 2° to liver disease

2. Fanconi Syndrome - defective reabsorption in PCT

(proximal convoluted tubule) ● Readily automated, adapted in traditional wet chem
analyzers (e.g., Abbott Architect) & for dry chem slide
3. Chemotherapy w/ 6-mercaptopurine or azathioprine analyzers (e.g., Vitros) commonly seen in hospital
laboratories
4. Overtreatment w/ allopurinol (medication for gout) ● Reducing substances react w/ H2O2 → false ↑ in uric
acid levels
5. Exposure to toxic agents ● Bilirubin & ascorbic acid destroy peroxide if present in
sufficient quantity
● Addition of K ferricyanide & ascorbate oxidase
minimize these interferences.
BUA | METHODOLOGIES
1. COLORIMETRIC: PHOSPHOTUNGSTIC ACID 4. ELECTROCHEMICAL & BIOSENSOR SYSTEMS
● Caraway Method
● Uses uricase in oxygen electrode
● Requires CHON removal by dialysis or precipitation
● In carbonate solution,
5. HPLC
● Use ion-exchange resins or reverse-phase columns
6. IDMS
● Proposed reference method

● Turbidity can be a problem particularly if the pH is not CREATININE (AND CREATINE)

strongly alkaline
● Aspirin & its metabolites, acetaminophen, caffeine,
CREATININE (AND CREATINE) | BIOCHEMISTRY
and theophylline produce color → false ↑ (false
First, it starts with Arginine, Glycine, and Methionine which
contamination)
are amino acids. They go to the liver and deposit creatine.
● Basing from the info, it is not a very recommendable
method bc of a quite number of interferences.
● Reference Range:
o Male (♂) - 4.5 - 8.2 mg/dL,
o Female (♀) - 3.0 - 6.5 mg/dL

2. ENZYMATIC: URICASE
● Source of uricase or urate oxidase: mammals other
than humans & apes (otherwise can lead to false or
erroneous results)
● Result: 1 mg/dL lower than colorimetric
● Uric acid can act as reducing agent in “Uricase
Reaction” (Calbreath) Creatine goes to the bloodstream and reaches the Brain &
Muscle and is then metabolized into Creatine Phosphate
(aka Phosphocreatine). The Creatine Phosphate while inside
the muscle is used up in a way that the Phosphocreatine which

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 LEC 10 – NON-PROTEIN NITROGEN COMPOUNDS
donates the phosphate to the ADP through creatine kinase to
become ATP which fuels muscle contraction. Now, the
Phosphocreatine lost its phosphate group to go back being a
Creatine then 1-2% loses water in irreversible reaction to
become Creatinine which is the waste product or the anhydride
form of creatine.
CREATININE MEASUREMENT
● Constant production of creatinine, related to muscle mass.
o Why? The muscle still contracts even though
you’re lying in bed and when the muscles
contract, it produces creatinine.
● Diet, age, sex & exercise DO NOT affect blood CREA
levels although high CHON ingestion may transiently
elevate conc. of creatinine
● Creatinine is a function of:
1. Relative muscle mass
o Obese individuals have lesser muscle mass
because what they have is more fat.
Meaning, lesser muscle mass in obese than
in non-obese individuals. Thus there is
lesser creatinine in obese individuals and
non-obese individuals despite having the
same body mass because non-obese indivs
have leaner muscle mass.
2. Rate of creatine turnover
3. Renal function
● Fasting sample is NOT required
Creatinine goes to the Kidneys where it is excreted to the
Urine. Small amounts can be reabsorbed by renal tubules but
6-8% are eliminated by PCT secretion.
The CREA is inversely proportional to the Glomerular
Filtration Rate (GRP) and although an imperfect
measurement, it is commonly used to assess renal filtration
function.
● Reference Range:
● Measurement of CREA provides useful info for:
1. Detection of sufficiency of kidney function
o Creatinine clearance & GFR are used to
gauge renal fn.
2. Monitor organ function after transplantation
o Signal dss progression/rejection
o In kidney transplant, higher crea is expected
if recipient’s body rejects the organ since the
kidney cannot do its function well thus lead
to higher crea in the blood because it is not
filtered out by the transplanted kidney.
3. Detection of severity of kidney DSS
4. Monitor progression of kidney DSS
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CREATINE MEASUREMENT
1. Muscle Disease = increase ↑ creatine leads to increase ↑
urinary CREA, however normal blood CREA.
● Muscular dystrophy
● Poliomyelitis
● Hyperthyroidism
● Trauma
Measurement of CK (creatine kinase) is better for dx because
creatine measurement is usually N/A in the clinical lab.
URINE CREATININE MEASUREMENT
● A measure of completeness of 24 hr urine
● Urinary constituents may be expressed as a ratio to
creatinine quantity rather as mass excreted per day.
o Why? Because there is constant production of
creatinine
CREATININE METHODOLOGIES
1. JAFFE RNX
● First and most famous
● First described in 1886, was adopted for
measurement of blood CREA by Folin and Wu in
1919. It involves:
o Creatinine + pictrate → orange-red complex
@ 490-505 nm
o CHON should be removed by ppt’n or
dialysis
o Temp. sensitive; @ >30 C, other compounds
react w/ pictrate
o Extended incubation even @RT →
nonspecific colored cmpds (Calbreath)
o Not specific or creatinine & positive
interfering substances from CHON, glucose,
acetoacetate, ascorbic acid, pyruvate,
acetone, ketones falsely elevating conc. Up
to 0.3-0.4 g/dL
o Classical assay for serum/urine creatinine.
● Principle:
Creatinine (PFF) + Alk. Picric Acid → Alk. Creatinine Picrate complex
amber-yellow/bright orange red
o Composition of Alkaline Picrate Solution:
▪ 1 part 10% NaOH
▪ 5 parts saturated picric
acid (2,4,6 Trinitrophenol)
● Drawbacks of the classical Jaffe
Method:
o Lacks specificity and sensitivity
o Temp. sensitive
o Affected by length of time of incubation
o Affected by incomplete removal of protein
● Modified Jaffe Rxn - Colorimetric Endpoint
10
 LEC 10 – NON-PROTEIN NITROGEN COMPOUNDS

o To remove reactive impurities before Alk. Pic. 6. REACTION 2

sol’n is added o More popular, usually for POCT instruments
o Adapted for use in dry slide analyzer
2. TRAPPING OF OTHER MATERIALS W/ ALUMINUM-SILICATE
● First method that uses the Modified Jaffe rxn principle
● CREA in PFF is adsorbed onto fuller’s earth
(aluminum magnesium silicate) or Lloyd’s rgt (sodium
aluminum silicate) then eluted & reacted w/ alk picrate
o This method is no longer used now but it did
set the stepping stones for the modern
creatinine methods. This is one that has
modified the original Jaffe Reaction
● Adsorbent improves specificity
● Previously considered reference method, but since;
● Method is time consuming & not readily automated, it
o In the reaction, the end product reacts with the H2O2
is;
(product of the 3rd rexn), and then reacts with the
● Not routinely used
indicator. What is measured is the oxidized form of
the indicator.
3. ION-EXCHANGE RESINS o The reason why this coupled enzyme method is more
● Simple, BUT nonspecific popular even though the process is long, is because
in the modern clinical chemistry is now done through
4. MODIFIED JAFFE REACTION – KINETIC automation, so dili na siya manomanohon ug add ang
o Rapid, increased ↑ specificity mga reagents..It’s the analyzers that do this. With the
o Measured rate of color formation is between 25 - 60s series of reactions, it gets rid of possible
after initiating the reaction interferences.
o Interfering substances take time before they o Here, the enzymes are provided in the reagent kit
contribute significant Absorbance and CREA can be along with the water.
more accurately measured.
o Even then, some interferences are still present: 7. REACTION 3
α-keto acids & cephalosporins give positive bias
o Bilirubin & Hemoglobin may cause a negative bias, o Used in dry chem systems and employs a two-slide
probably a result of their destruction in the strong system.
base used
o Kinetic Jaffe is used routinely because it is
inexpensive, rapid & easy to perform
o May require automated equipment for precision.

OTHER APPROACHES TO MINIMIZE INTERFERENCES INCLUDE

MULTISTEP ENZYMATIC OR COUPLED ENZYME ASSAYS

5. REACTION 1: CREATININE –CK

o Initial reaction w/ creatininase/ craetine amidino
hydrolase
o There are a series of 4 reactions:

o 5 Enzymes are involved here.

o The last products that react with the indicator as with
the other reaction is hydrogen peroxide which react
with the red. Indicator and becomes oxidized with the
action of peroxidase.

8. OTHER ENZYMATIC METHOD (NOT A COUPLE):

o Measured either colorimetrically or w/ ISE

o Creatinine 𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑎𝑠𝑒 𝑖𝑚𝑖𝑛𝑜ℎ𝑦𝑑𝑟𝑜𝑙𝑎𝑠𝑒 → NH4+

o Creatinine is in the sample
o Creatininase enzyme is taken from the kit/reagent 9. IDMS – ACCEPTED REFERENCE METHOD
o With this, it can override the interferences present in o Assays used on automated analyzers are designated
the previous method. as “traceable’ (calibrated) to an IDMS method.
o Not popular, suffers poor sensitivity, poor precision &
relatively expensive reagents

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 LEC 10 – NON-PROTEIN NITROGEN COMPOUNDS

CREATININE (CREA) | REFERENCE RANGE

● Values in children are lower, but begin to rise during

adolescence

PRACTICE QUESTION
● The following catalysts are involved in the indicator
reaction for these methods, EXCEPT;

● Odd one Out

REFERENCES
● Bishop,M.L.,Fody,E.P.,Schoeff,L.E.(2013).ClinicalChemi
stry:Principles,Techniques and Correlations.7th ed.
● Larson,D.L.(2017). Clinical Chemistry:Fundamentals
andLaboratory Techniques.
● Calbreath,D.F.(1998).Clinical Chemistry:A
FundamentalTextbook

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