Sect 4 PPII - Non-Protein Nitrogen Compounds

Non-protein nitrogen compounds

the breakdown of proteins and nucleic acids results in the formation of:

Creatinine, Urea, Ammonia, Uric Acid

Non-protein compounds:

Creatine

Synthesized in the liver, pancreas, and kidneys

Transported through circulatory system to muscle, brain and other organs where it is converted to phosphocreatine and acts as energy source (like ATP)

arginine, glycine, methionine

Creatine is synthesized in the liver from:

**hint: amino acids

Creatinine

Produced in the muscle (amt in blood is proportional to pts lean muscle mass)

Produced as a waste product of creatine and phosphocreatine

NOT reabsorbed by kidneys; 100% excreted through the urine

serum and plasma

Specimen requirement for Creatinine methods

Fluoride and Ammonium heparin anticoagulant tubes

For Creatinine Measurement, what tube should NOT be used for specimens measured by the enzymatic rxn -- False neg

severe exercise and a high meat diet

For Creatinine Measurement, what can cause false pos

Jaffe Reaction (Chemcial)

Creatininase, Creatinase, Creatinine deaminase (Enzymatic)

What are the 2 methods used to measure Creatinine?

Jaffe reaction

Chemical method for Creatinine measurement

Creatinine reacts with picric acid in alkaline environment to generate an orange-red product

Most common reaction in the lab

picric acid

Chemical used in the Jaffe reaction

alkalkine environment

pH used in the Jaffee reaction

not specific for creatinine

Is the Jaffe reaction specific for creatinine?

interference with Jaffe reaction

False pos: elevated levels of bilirubin and hemolysis; protein, glucose, ascorbic acid, acetone, acetoacetate, pyruvate, guanidine and cephalosporins

Creatininase, Creatinase, Creatinine deaminase

3 enzymes utilized in the Enzymatic method for Creatinine measurement

Enzymatic method for creatinine measurement

Rxns are coupled with enzymatic reactions that can be measured spectrophotometrically

NADH to NAD+ and H2O2 to H2O → measuring colorless product

interference with Enzymatic method for Creatinine measurement

Less susceptible to interference from keto acids & cephalosporin antibiotics

False neg: fluoride and ammonia heparin anticoagulants inhibit enzyme activity

Inulin

gold standard for clearance tests

not typically used though because it is NOT endogenous (must be administered to paitent)

24hr urine and blood specimen

Specimen requirements for Creatinine clearance

Creatinine Clearance Formula

CC = (mL/min)

UC/PC = mg/dL

volume = mL

time = minutes

ASA/PSA = square meters

If you don't have PSA, remove PSA and ASA from formula

Schwarz formular

alternate method used to estimate the creatinine clearance in children

(k x Ht)/Creat

Cockcroft and Gault formula

alternate method to estimate creatinine clearance that adjusts for age difference (only used for 18 and older)

Cystatin C

Produced by all nucleated cells of the body at a constant rate

- releases when cell is broken down

- found in all body fluids EXCEPT urine → ALL is reabsorbed by proximal tubules

Unaffected by: muscle mass, sex, age, diet, ethnicity

Cystatin C increases

What happens to Cystatin C as GFR decreases?

**hint: all should be reabsorbed by proximal tubules

(compromised kidney = decreased reabsorption)

Blood Urea Nitrogen (BUN)

Urea is reported as what in the United States?

BUN

Breakdown of proteins

Synthesized in the liver and transported through blood to kidney, where it is filtered through the glomerulus, where almost HALF is reabsorbed back into the blood by passing though nephron tubule

protein intake and protein breakdown

The rate of urea synthesis depends on (2):

**hint: Amino acids → Ammonia + CO2 → Urea + water

75%

BUN accounts for more than _____% of non-protein nitrogen that is excreted from the body

**hint: the most abundant waste product in the urine

increase in BUN

- Disorders with excessive breakdown of proteins (febrile illnesses, dehydration, corticosteroid/tetracycline therapy, internal bleeding, and in presence of elevated thyroid hormones)

- High protein diets/Large ingestions of protein

- Pregnancy (due to increase GFR)

Cirrhosis of the liver

What causes decrease in BUN?

**hint: where urea cycle takes place -- can't breakdown ammonia to urea

10:1 to 20:1

Normal BUN:creatinine ratio

low BUN:creatinine ratio

5:1

Acute nephron tubular necrosis

Low protein intake (malnutrition)

Starvation

Severe liver disease

increased BUN with normal creatinine levels

30:1 (reabsorbing too much BUN)

Pre-renal uremia

High protein intake in diet

GI tract hemorrhage

Dehydration

increased BUN and creatinine

30:10

May indicate a post-renal problem (obstruction of urinary tract)

End-stage renal disease

Urease Method (Enzymatic)

Diacetyl monoxime (Colorimetric)

What are the 2 methods used to measure BUN?

Urease method

Enzymatic method for Urea (BUN) measurement

Ammonia (NH4+) is liberated in this rxn and can be measured using the Berthelot reaction

Urease

Enzyme utilized in the Enzymatic method for Urea (BUN) measurement

Berthelot's reaction

Coupled reaction utilized with the Urease method

Ammonia (NH4+) ion that is liberated in the urease rxn is most commonly measured utilizing this rxn

Diacetyl monoxime method

Colorimetric method for Urea (BUN) measurement

conversion of urea to BUN

Urea (mg/dL) = BUN (mg/dL) x 2.14

BUN (mg/dL) = Urea (mg/dL) / 2.14

interference with Colorimetric method for BUN measurement

False neg: anti-coagulants containing fluoride or citrate

(inhibits rxn form occurring = no color formed)

Urinary Protein

used to monitor kidney function not usually present in urine

more sensitive

Urine concentration of protein is less than the concentration of blood; so urine methodologies have to be ____________ than blood protein levels

Turbidimetric

Dye-Binding/Colorimetric

What are the 2 methods used to measure total protein in urine?

Turbidimetric

Protein is precipitated out with Sulfosalicylic acid (SSA), then turbidity of the precipitated is measured

Semi-Quantitative test

other: Trichloroacetic acid or Benzethonium chloride

Sulfosalicyclic acid

Trichloracetic acid

Benzethonium chloride

Chemicals for turbidimetric method for urinary protein measurement

Dye-Binding/Colorimetric

Dye binds to albumin and causes a shift in the max absorption; resulting color change is measured

Methyl orange, Bromcresol green (BCG), or Bromcresol purple (BCP) may be used

Methyl Orange

Bromcremsol Green

Bromcresol Purple

Dye-binding chemicals used to measure urinary albumin

BCG most common

Uric Acid

Produced from the cellular breakdown products of the purine nucleosides

Filtered through the renal glomerulus and is almost completely reabsorbed in the proximal tubules, but some end up in urine (6-12%)

adenosine and guanine

purine nucleosides that uric acid is derived from

Urate crystals

At acidic urine pH levels above 5.7, what type of crystals are formed?

chemical form that is soluble in urine

Uric acid crystals

At acidic urine pH levels below 5.7, what type of crystals are formed?

painful accumulation in joints or present in kidney stones

leukemia and gout

Increased levels of uric acid are seen in (2):

Hyperuricemia

Increased uric acid in the blood; > 7.0 mg/dL

may be asymptomatic, but are at increased risk for associated renal problems

Gout

a disease in which deposits of urates are deposited in body fluids (especially in synovial fluid - joints)

caused by: over-production of purines or defects in the metabolic pathways of purine metabolism

big toe

Where is the most common place we see gout in the body?

gout arthritis

Classic symptom of gout

**hint: uric acid is deposited in joints causing pain & inflammation

causes of Hyperuricemia

over-production of purines

renal retention of uric acid

acidemia (low pH = uric crystals)

defects in the enzymes of the purine metabolic pathway (Lesch-Nyhan syndrome)

Lesch-Nyhan syndrome

a deficiency of a transferase enzyme in the purine metabolic pathway, leading to tophi deposits

hyperuricemia

can be:

minor - depending on location

major - accumulation in brain tissue which can lead to intellectual disability, signs of cerebral palsy, and self-injurious behavior

Hypouricemia

decreased uric acid

causes of Hypocuricemia

reduced purine synthesis

defective reabsorption of uric acid in the kidney (Fanconi's syndrome)

Fanconi's syndrome

a disorder of the proximal tubule of the nephron, resulting in poor transport

hypouricemia

Uricase Method (Enzymatic)

Phosphotungstate Method (Colorimetric)

What are the 2 methods used to measure Uric acid?

Uricase Method

Enzymatic method for Uric Acid measurement

Rxn measured in 2 ways:

1. Allantoin is produced and measured as a decrease in absorbance at 293nm due to a loss of urate

2. Hydrogen peroxide may be converted to a chromogen using 4-aminophenazone & peroxidase in a couple reaction

Uricase

enzyme that converts uric acid to allantoin

interference with Uricase method

False pos: excessive amounts of protein and lipids; hemoglobin and bilirubin

False neg: Xanthine (competitive inhibitor -- keeps uric acid from binding & reacting) & anti-coagulants containing fluoride or citrate

Phosphotungstate Method

Colorimetric method for Uric Acid measurement

protein-free filtrate for serum or plasma

Phosphotungstate method for uric acid requires:

**hint: proteins are interfering substances

interference of Phosphotungstate Method

False pos: Glucose, Ascorbic acid, Glutathione, Hemoglobin and drugs (Acetaminophen)

False neg: Caffeine (competitive inhibitor -- keeps uric acid from binding & reacting) & anti-coagulants containing fluoride or citrate

serum, heparinized plasma and urine

Specimen requirements for Uricase Method & Phosphotungstate Method

Anticoagulants containing fluoride or citrate should not be used