Clinical Chemistry Lecture: Finals Urea out of BUN = BUN x 2.

14
Nonprotein Nitrogen Compounds
BUN out of Urine = (Urine/BUN) /2.14
Nonprotein Nitrogen Compounds mg/dL -> mmol/L = mg/dL x 0.357
• Used for renal/kidney function
Specimen Requirements
• Considered as Protein Free Filtrate
• Does not require any fasting
• NPN are products of protein metabolism – e.g.
• Serum is the best specimen
Nitrogen, Free Ammonia, Amino Groups, Amino
acids • If plasma is used, avoid ammonia, sodium
citrate and sodium fluoride because it inhibits
• Measured through Spectrophotometry
urease
Urea Hemolysis – main interference of BUN
• Major excretory product of protein metabolism • Specimen should be non-hemolyzed
• Liver is the site of synthesis • Urea is susceptible to bacterial decomposition
• Nitrogen + Free Ammonia + Amino Groups = • Timed specimen (urine) should be refrigerated
Urea
Blood Urea Nitrogen Methods
• Urea is delivered through the kidney
(glomerulus) by blood then is filtered through 1. Chemical Methods (Direct Method)
the plasma – Diacetyl Monoxime Method
• Highest concentration in the blood (40%-50%) 2. Enzymatic Methods

Basis for Urea Measurement • Best/commonly used because it has values as

results, therefore it is accurate
• Renal Function
– Hydrolysis of Urea by Urease
• Protein diet/content
– Coupled Urease/ Glutamate
• Rate of Protein Catabolism
Dehydrogenase method
Clinical Applications – Isotope Dilution Mass
• Evaluate Renal Function Spectrophotometry

• Diagnose a Renal Disease Chemical Method

• Dialysis – check if process is effective through Diacetyl Monoxime Method
urea determination
• Just add the reagent to the specimen
• Determine Nitrogen Balance
𝑈𝑟𝑒𝑎 + 𝐷𝐴𝑀 → 𝑦𝑒𝑙𝑙𝑜𝑤 𝑐𝑜𝑙𝑜𝑟
Urea vs Blood Urea Nitrogen
• Urea is excreted through urine (first metabolite • Not used because high, low, normal results are
to elevate during kidney disease because if has only achieved
the highest concentration in the kidney).
Enzymatic Methods
• BUN is absorbed by blood circulated
Hydrolysis of Urea by Urease
throughout the body
• Ammonia is being measured
• Urine – Highest concentration of urea and
creatinine 𝑈𝑟𝑒𝑎 + 𝑢𝑟𝑒𝑎𝑠𝑒 → 𝑎𝑚𝑚𝑜𝑛𝑖𝑎 + 𝐶𝑂2
 Coupled Urease/ Diagnostic Criteria for Renal Azotemia
Glutamate Dehydrogenase (GLD)
 Blood Urea Nitrogen - >100 mg/dL
• Glutamate dehydrogenase is the coupling  Creatinine - >20 mg/dL
enzyme  Blood Uric Acid - >12 mg/dL

𝑈𝑟𝑒𝑎 + 𝑢𝑟𝑒𝑎𝑠𝑒 → 𝑎𝑚𝑚𝑜𝑛𝑖𝑎 + 𝐶𝑂2 Post Renal Azotemia

𝐴𝑚𝑚𝑜𝑛𝑖𝑎 + 2 − 𝑜𝑥𝑜𝑔𝑙𝑢𝑡𝑎𝑟𝑎𝑡𝑒 + 𝑁𝐴𝐷 • Result of urinary tract obstruction

𝐺𝐿𝐷
→ 𝑔𝑙𝑢𝑡𝑎𝑚𝑎𝑡𝑒 + 𝑁𝐴𝐷𝐻 + 𝐻2𝑂 Causes of Post Renal Azotemia
• Tumors of the bladder or prostate
• Measured the rate of disappearance of NAD =
• Renal calculi
340 nm
Azotemia Classification
Isotope Dilution Mass Spectrometry
• Proposed reference method • Azotemia are differentiated through
BUN : Creatinine Ratio
• Isotope is used as the quantifying agent in
determination of urea Normal Value BUN: Creatinine Ratio – 10:1 or 20:1

Normal Value Pre-renal ↓ Urea ↑ BUN Normal Creatinine

Creatinine
• Plasma and serum = 6 – 20 mg/dL
depends on the
• Urine = 12 – 20 g/day Renal ↑ or ↓ BUN
case of illness of
patient
Abnormalities
Post-renal ↑ Urea ↑ BUN ↑ Creatinine
• Azotemia – elevated concentration of urea in
blood (>20 mg/dL)
Low Ratio (BUN:Crea) <10:1 Causes:
Types: Pre-renal Azotemia
Low Protein Diet
Renal Azotemia
Acute Tubular
Post-renal Azotemia
Necrosis
Pre-renal Azotemia Repeated
Dialysis
• Reduced renal blood flow because less blood is
Hepatic Disease
delivered in the kidney, thus less urea is filtered
Causes of Pre-renal Azotemia: High Ration (BUN:Crea) >20:1 Causes:
with normal Pre-renal
• Dehydration
creatinine azotemia
• Shock
• Congestive Heart Failure Dehydration
Catabolic states
• Hemorrhage
GI hemorrhage
Renal Azotemia High protein diet
• The kidney is damaged High Ration (BUN:Crea) >20:1 Post renal
with increased azotemia
Causes of Renal Azotemia: creatinine Pre-renal
• Glomerulonephritis azotemia with
• Tubular Necrosis renal disease
Renal failure
• Acute/Chronic Renal Diseases
Uric Acid Specimen Requirements
• Product of catabolism of the purine nucleic • Fasting is not required but it is referred
acid
• Serum => best specimen
• Uric acid in nature is high in concentration in
• Plasma => use Heparin
the body
(do not use EDTA, Sodium Fluoride, and Sodium
• Insoluble in plasma citrate because it inhibits Uricase enzyme)
• Filtered out by the glomerulus and secreted by • Urine => buffered at pH 8.0
the distal tubules into the urine
• 3-5 days in Ref Temperature (4 C)
• Reabsorbed in the proximal tubules and
• 3 days in Room Temperature
reused
Main Interferences
• Stored in joints
• Bilirubin (Icteric)
𝑈𝑟𝑖𝑐 𝐴𝑐𝑖𝑑 + 𝑈𝑟𝑖𝑐𝑎𝑠𝑒
• Hemoglobin (Hemolyzed)
→ 5 − ℎ𝑦𝑑𝑟𝑜𝑥𝑦𝑖𝑠𝑜𝑢𝑟𝑎𝑡𝑒 (𝑎𝑙𝑙𝑎𝑛𝑡𝑜𝑖𝑛) + 𝐶𝑂2 + 𝐻2𝑂
• Triglyceride (Lipemic)
Uric Acid Metabolism • Glutathione (False Decrease)
• Salicylates
Purine • Thiazidine } (False Increase)
Synthesized

in the liver Normal Values
Uric Acid
• Male = 3.5 – 7.2 mg/dL
Blood • Female = 2.6 – 6.0 mg/dL
• Child = 2.0 – 5.5 mg/dL
Kidney
Uric Acid Methods
1. Chemical Methods (Direct Method)
PCT DCT – Oxidation-Reduction (Redox) Reaction
Oxidizing Agents:
Reabsorption Excretion
A. Caraway Method (Most common method)
98% - 100% 0% - 2%
– Sodium Carbonate (Na2CO3)
Purine Rich Foods B. Benedicts Fehling’s Newton
• Brain – Sodium Cyanide (NaCN)
• Kidney
2. Enzymatic Methods (Uricase Method)
• Meat
• Nuts • Uses enzyme uricase
• Bacon • More specific than Redox
• Beer => causes Gout
• Read absorbance: 293nm
• Liver
𝑈𝑟𝑖𝑐 𝐴𝑐𝑖𝑑 + 𝑈𝑟𝑖𝑐𝑎𝑠𝑒
→ 5 − ℎ𝑦𝑑𝑟𝑜𝑥𝑦𝑖𝑠𝑜𝑢𝑟𝑎𝑡𝑒 (𝑎𝑙𝑙𝑎𝑛𝑡𝑜𝑖𝑛) + 𝐶𝑂2 + 𝐻2𝑂

3. Isotope Dilution Mass Spectrometry

– Proposed Reference Method
*Chemical Method is not accurate because the Creatinine Methods
reporting is color or high/low.
1. Chemical Method
*Enzymatic method is more specific.
Jaffe Reaction (Rate of change of absorbance before
Clinical Applications and after heat is applied) 𝑆𝑒𝑟𝑢𝑚 +
Δ
• Diagnosis of Gout (Hyperuricemia) 𝑆𝑎𝑡𝑢𝑟𝑎𝑡𝑒𝑑 𝑃𝑖𝑐𝑟𝑖𝑐 𝐴𝑐𝑖𝑑 → 𝑟𝑒𝑑 − 𝑜𝑟𝑎𝑛𝑔𝑒 𝑐𝑜𝑚𝑝𝑙𝑒𝑥
= Most commonly found in males *Rate of change of absorbance before and
= 3rd to 5th decade of life after heat is applied
= Inflammation of joints
Formula: Abs 1 – Abs 2
• Prevent nephropathy during chemotherapy
2 Classifications:
= radiation melts UA, making it soluble in
plasma Kinetic Jaffe
• To assess kidney dysfunction Jaffe with adsorbent
• Diagnosis of Renal calculi
Kinetic Jaffe
• Detection of Lesch-Nyhan Syndrome
• Measures rate of absorbance
= Inborn errors of purine metabolism
= Deficiency of hypoxanthine-guanine- • This is routinely used
phosphoribosyl transferase • Has + and – biases
= SYMPTOM: Mental Retardation
• Negative Bias – When specimen is icteric and
Creatinine hemolyzed (Makes creatinine abnormal)
• Formed from creatine and creatine • Positive Bias – If cephalosporin and Alpha-
phosphatase ketoacid is applied (Makes creatinine normal)
• Found in muscle (muscle metabolism) Advantages of Kinetic Jaffe
• Generally used to assess kidney damage, • It is inexpensive
determine the sufficiency of kidney function,
• It is rapid
severity of kidney damage and progression of
kidney disease • Easy to perform

Specimen Requirements --------------------------------------

• Does not require fasting Jaffe with Adsorbent
(If the person has eaten protein rich food then
did exercise, the creatinine will increase) • Adsorbent is used because it improves its
specificity by deleting positive and negative
• Serum, plasma, urine (Serum is best) biases/interferences
• Hemolyzed, icteric and lipemic specimens • Previous reference method
should be avoided
Disadvantages:
• Urine = Stable up to 4 days (ref temp)
• Expensive
• Not readily available

Absorbents
 • Fuller’s Earth 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 + 𝐻2𝑂 →
𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑎𝑠𝑒
𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑒
– Aluminum Magnesium Silicate 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑎𝑠𝑒
𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑒 + 𝐻2𝑂 → 𝑆𝑎𝑟𝑐𝑜𝑠𝑖𝑛𝑒 + 𝑢𝑟𝑒𝑎
• Lloyd’s Reagent
𝑆𝑎𝑟𝑐𝑜𝑠𝑖𝑛𝑒 𝑂𝑥𝑖𝑑𝑎𝑠𝑒
– Sodium Aluminum Silicate 𝑆𝑎𝑟𝑐𝑜𝑠𝑖𝑛𝑒 + 𝐻2𝑂 + 𝑂2 → 𝐺𝑙𝑦𝑐𝑖𝑛𝑒
+ 𝐻𝐶𝐻𝑂 + 𝐻2𝑂2
Isotope Dilution Mass Spectrometry
𝐻2𝑂2 + 𝑃ℎ𝑒𝑛𝑜𝑙 + 4 𝑎𝑚𝑖𝑛𝑜𝑝ℎ𝑒𝑛𝑎𝑧𝑜𝑛𝑒
• Proposed reference method 𝑃𝑒𝑟𝑜𝑥𝑖𝑑𝑎𝑠𝑒
→ 𝐵𝑒𝑛𝑧𝑜𝑞𝑢𝑖𝑛𝑜𝑛𝑒𝑚𝑖𝑛𝑒 𝑑𝑦𝑒 (𝑟𝑒𝑑)

Complications
2. Enzymatic Method • Muscle dystrophy
• Creatininase – CK Method • Poliomyelitis
– Requires a large volume of pre- • Hyperthyroidism
incubated sample; not widely used
• Trauma
• Creatininase – Hydrogen Peroxide Method
– Has the potential to replace Jaffe
method Normal Values: Creatinine
– Without interference from Jaffe Enzymatic Urine
acetoacetate and cephalosphorins
Male 0.9 – 1.3 0.6 – 1.1 800 – 2000
– Creatininase is also known as Creatinine
mg/dL mg/dL mg/day
Aminohydrolase
Female 0.6 – 1.1 0.5 – 0.8 600 – 1800
Creatininase – CK Method
mg/dL mg/dL mg/day
𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑎𝑠𝑒
𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 + 𝐻2𝑂 → Creatine Child 0.3 – 0.7 0 – 0.6 mg/dL
𝐶𝐾 mg/dL
𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑒 + 𝐴𝑇𝑃 → Creatine PO4 + ADP
𝑃𝐾
𝐴𝐷𝑃 + 𝑃ℎ𝑜𝑠𝑝ℎ𝑜𝑒𝑛𝑜𝑙 𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒 → 𝐴𝑇𝑃 + 𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒
𝐿𝐷𝐻
𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝑁𝐴𝐷𝐻 → 𝐿𝑎𝑐𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷 +

Creatinase-Hydrogen Peroxide Method