(M) Lec 3: Non-Nitrogenous Proteins Flashcards
- these have low molecular weight
- they contain nitrogen
- are distinguished from proteins based on their size
- are metabolic by-products/waste of proteins, nucleic acids, and muscle metabolism
Non-Protein Nitrogenous Compounds (NPNs)
A high NPN value is (advantageous or disadvantageous) to man
Disadvantageous
In what excretions (2) are a large number of NPNs found in?
Urine and Stool
In what body fluid are NPNs ideally found in small amounts/minimal concentrations?
Blood
What organ is responsible for properly excreting NPNs?
Kidney
NPNs are converted into proteins by bacteria found in the stomachs of (animals or humans)
Animals
Animals further convert NPNs into proteins while NPNs are the end products in humans (we lack the bacteria that convert it)
Where are NPNs incorporated in to increase its crude protein value which is measured based on nitrogen content?
Animal feeds
Crude protein values of NPNs in animal feeds
Protein: % N?
Urea: % N?
Melamine: % N?
Protein: 16%
Urea: 47%
Melamine: 66%
Order the NPNs from most significant to least (in %)
- Amino acids
- Ammonia
- Creatine
- Creatinine
- Urea
- Uric acids
I will give the percentages in the answer, just familiarize there
- Urea (45%)
- Amino Acids (20%)
- Uric Acids (20%)
- Creatinine (5%)
- Creatine (1-2%)
- Ammonia (0-2%)
What 4 NPNs are part of the parameters for a kidney function test?
- Urea
- Amino acids
- Creatinine
- Creatine
What NPN is part of the parameters for a liver function test?
Ammonia
What NPN will significantly increase if there is an increased result for NPNs in general?
Urea (highest concentration)
This NPN is technically not yet part of the “NPNs classified as waste products” as it is “not really” a protein yet
Amino acids
Note: From protein > amino acids > urea
Which among creatinine and creatine is the end product?
Creatinine (creatine signifies incomplete muscle metabolism)
Clinically Significant NPNs
This is the major excretory product of oxidative protein and amino acid metabolism
Urea or BUN
Clinically Significant NPNs
This is synthesized in the liver from CO2 and ammonia that arises from the deamination of amino acids
Urea or BUN
The synthesis of UREA in the liver from CO2 and ammonia that arises from the deamination of amino acids is known as what cycle?
Clue: It has 3 names
- Urea cycle
- Ornithine cycle
- Kreb’s Henseleit cycle
Urea is excreted in both urine and stool, but what percentage of it actually makes it to the urine?
90%
Consists of half of the urinary solids
Determination of Blood Concentration (UREA)
Abnormal levels of urea can give an idea about an individual’s renal function and perfusion, does it indicate acute or chronic disorders?
BOTH
Determination of Blood Concentration (UREA)
A diet rich in this component will result to high urea formation
High protein diet
Determination of Blood Concentration (UREA)
Urea is a good indicator for an individual’s state of what?
Hydration
Normal Urea = Hydrated
Abnormal Urea = Possible dehydration
Determination of Blood Concentration (UREA)
Refers to how FAST the body is able to catabolize proteins
Protein Catabolism Rate
Determination of Blood Concentration (UREA)
Urea is a good indicator for an individual’s intake of what?
Nitrogen
Urea has the HIGHEST nitrogen concentration among the NPNs
Urea Determination
This has been used to refer to a MEASUREMENT OF UREA, not the actual urea
Blood Urea Nitrogen (BUN)
Urea Determination
Actual Urea has a formula of what?
Actual urea is the level of urea minus the nitrogen
BUN x 2.14 mg/dL
The CF for urea is 2.14
Urea Determination
What is the conversion factor for actual urea when you have computed for the conventional unit (mg/dL) and would like to report it as an SI unit (mmol/L)?
Simply multiply the obtained mg/dL value with this CF to get mmol/L
0.357 or 0.36 mmol/L
Analytical Methods in Determining Urea
Which among enzymatic and chemical reactions are the direct and indirect methods for determining urea?
Enzymatic - indirect
Chemical - direct
Analytical Methods in Determining Urea (Enzymatic/Indirect Method)
- most common enzymatic method
- couples the urease with glutamate dehydrogenase (2 reactions)
- the amount of NAD produced corresponds to the amount of urea
Coupled Urease (Glutamate Dehydrogenase) Method
Analytical Methods in Determining Urea (Enzymatic/Indirect Method)
In Coupled Urease (Glutamate Dehydrogenase) Method, what are the end products [2] after the coupled reactions?
Glutamate and H2O
Analytical Methods in Determining Urea (Enzymatic/Indirect Method)
In Coupled Urease (Glutamate Dehydrogenase) Method, the amount of glutamate and H2O is not the main analyte being measured, instead what it is?
Nicotinamide Adenine Dinucleotide (NAD)
Analytical Methods in Determining Urea (Enzymatic/Indirect Method)
- ammonia will result in a color change with the help of a pH indicator
- thiosemicarbazide and ferric ions are added to enhance color development
- color intensity corresponds to the amount of urea
Note: W/o the reagents in the 2nd bullet, the color will be very light
Indicator Dye
Analytical Methods in Determining Urea (Enzymatic/Indirect Method)
- urea is broken down into ammonia and bicarbonate which produces conductive activity
- amount of conductivity produced is correspondent to the amount of urea
Note: This method has low sensitivity
Conductimetric
Analytical Methods in Determining Urea (Enzymatic/Indirect Method)
- this measures the production of indophenol blue when phenol is added as a reagent
- ammonia will result in a color change with the help of a pH indicator
Berthelot Reaction
Analytical Methods in Determining Urea (Enzymatic/Indirect Method)
- urea will produce ammonia and CO2 (the ammonia can be measured by treating it with Berthelot reagents)
- is commonly and routinely done
Hydrolysis of Urea (by urease)
Analytical Methods in Determining Urea (Enzymatic/Indirect Method)
- this is the REFERENCE METHOD for measuring urea
- the GOLD STANDARD
Isotope Dilution Mass Spectrometry (IDMS)
Note: This is not only for urea, it is also the reference method for other tests
Analytical Methods in Determining Urea (Chemical/Direct Method)
- most commonly used direct method
- diacetyl is added to urea to produce diazine
- it appears as a yellow-colored compound
- it is measured using a spectrophotometer and it has a higher sensitivity than other methods
Diacetyl Monoxime (DAM)
Analytical Methods in Determining Urea (Chemical/Direct Method)
In the Diacetyl Monoxime (DAM) Method, the production of diazine from the combination of urea and diacetyl is known as what reaction?
Fearon’s Reaction
Analytical Methods in Determining Urea (Chemical/Direct Method)
- the addition of o-phthaldehyde to urea will produce isoindoline which is acted upon by N-(1-naphthyl)-ethylenediamine resulting in a colored product
- the resulting color is dependent on the indicator added
O-phthaldehyde Method
Specimen Requirements (Urea)
What 3 bodily fluids can be used?
- Serum
- Plasma
- Urine
Specimen Requirements (Urea)
Urea should not be contamined with:
1. Fluoride
2. Citrate
3. Ammonia
Which among these results in false increased and false decreased values?
Note: I will explain the rationale in the results
Falsely decreased: Fluoride and Citrate
Falsely increased: Ammonia
Fluoride and Citrate - inhibits urease
Ammonia - a fellow NPN
Specimen Requirements (Urea)
TOF: You must fast before submitting a sample for urea testing
False (a non-fasting sample is needed)
Reference Values for Urea
- For serum?
- For 24H urine?
Serum: 6-20 mg/dL
Urine: 12-20 g/day
Disease Correlations of UREA
- elevated levels of all nitrogenous substances (urea has the most striking prominence)
- it has three types depending on the organ of origin
Azotemia
Disease Correlations of UREA
What are the 3 kinds of azotemia?
- Pre-renal
- Renal
- Post-renal
Disease Correlations of UREA
- accumulation of NPNs in the blood due to the inability of urea to enter the kidneys (before it even arrives at the kidneys)
- caused by conditions already existing in the blood (specifically a decrease in renal perfusion)
- caused by dehydration, shock, and congestive heart failure
Pre-renal Azotemia
Disease Correlations of UREA
In Pre-renal Azotemia, there is a decreased rate of what but with normal renal function?
Glomerular Filtration Rate (GFR)
Disease Correlations of UREA
In Pre-renal Azotemia, increased urea comes with (decreased, normal, or increased) blood creatinine
Normal
Disease Correlations of UREA
- the TRUE kidney disease
- there is increased BUN due to poor excretion
- caused by either acute or chronic kidney disease and glomerulonephritis
- can cause coma and neuropsychiatric changes
Renal Azotemia
Disease Correlations of UREA
In Renal Azotemia, striking urea levels come with (decreased, normal, or increased) blood creatinine
Increased (slowly rising)
Disease Correlations of UREA
Renal Azotemia Criteria (give the values for the ff. analytes:)
1. BUN
2. Creatinine
3. Uric Acid
- BUN - ≥100 mg/dL
- Creatinine - ≥20 mg/dL
- Uric Acid - ≥12 mg/dL
Disease Correlations of UREA
- there is marked elevation in plasma urea accompanied by acidemia and electrolyte imbalance (potassium is elevated)
- the kidneys fail to eliminate waste products
- characterized by anemia (normocytic normochromic), uremic frost (dirty skin of metallic color), edema, foul breath, and urine in sweat
Uremia
Note: Only BUN is high in this disease (because it is found in plasma)
Disease Correlations of UREA
What are the normal values of the BUN to CREATININE (B:C) ratio which differentiates the causes of abnormal urea in the blood?
10:1 to 20:1
Disease Correlations of UREA
Indicate if:
A. Low B:C Ratio (< 10:1)
B. Increased B:C w/ normal creatinine
C. Increased B:C w/ high creatinine
- decreased urea production
- low-protein diet
- acute tubular necrosis
- repeated dialysis
- hepatic disease
A.
Disease Correlations of UREA
Indicate if:
A. Low B:C Ratio (< 10:1)
B. Increased B:C w/ normal creatinine
C. Increased B:C w/ high creatinine
- pre-renal azotemia
- high-protein intake
- thyrotoxicosis
- GI hemorhhage
- dehydration
- catabolic states
B.
Disease Correlations of UREA
Indicate if:
A. Low B:C Ratio (< 10:1)
B. Increased B:C w/ normal creatinine
C. Increased B:C with high creatinine
- post-renal azotemia
- pre-renal azotemia with renal disease
- uremia
- renal failure
C.
TOF: Urea is the hardest waste to remove in dialysis due to its abundance
False (the easiest)
Answer if increased or decreased BUN
- azotemia (all three types)
- fever
- stress
- burns
- high-protein diet
- dehydration
- chronic renal disease
Increased
Answer if increased or decreased BUN
- poor nutrition
- high fluid intake (overhydration)
- pregnancy
- severe liver disease
- hormonal effects
- malnutrition
- hepatic diseases
Decreased
- end product of muscle metabolism
- is not reabsorbed and minimally secreted in the kidneys
- amount generated is proportional to the muscle mass
- is not affected by protein intake
- a marker for glomerular filtration rate which makes it an index for overall renal function
- measures the completeness of 24H urine
Creatinine
Once creatine is produced via muscle metabolism, it will be (dehydrated, hydrolyzed, methylated, or deaminated) to form creatinine
Dehydrated
Creatinine is also produced by 3 amino acids, what are those?
Methionine, Arginine, and Lysine
TOF: Creatinine is not easily removed via dialysis
True
Increased or Decreased Creatinine?
- impaired renal function
- chronic nephritis
- muscular diseases
- congestive heart failure
Increased
Increased or Decreased Creatinine?
- decreased muscle mass
- advanced and severe lung disease
- pregnancy
- inadequate dietary protein
Decreased
Analytical Methods in Determining Creatinine
- most frequently used and first described in 1886
- creatinine reacts with picric acid in an alkaline solution to form a red-orange tautomer of creatinine picrate
- has many interferences (ascorbate, glucose, uric acid, and a-ketoacids)
Direct Jaffe Reaction
Analytical Methods in Determining Creatinine
This is a saturated reagent comprised of picric acid and 10% NaOH
Jaffe Reagent
Analytical Methods in Determining Creatinine
- requires automated equipment to measure the rate of change of absorbance
- it takes more than one measurement
- rate of color production is measured (spectrophotometer)
Kinetic Jaffe Method or Janovsky-like Reaction
Analytical Methods in Determining Creatinine
- uses creatininase or creatinine amidohydrolase, creatine kinase, pyruvate kinase, and lactate dehydrogenase
- peroxidase may also be used in a dry slide technique
- this eliminates the non-specificity of the Jaffe reaction as it is more specific
Enzymatic Method
Analytical Methods in Determining Creatinine (Enzymatic Methods)
- produces lactate with NAD (which are measured) as these are always directly proportional to creatinine
Creatininase-Creatine Kinase
Analytical Methods in Determining Creatinine (Enzymatic Methods)
- using peroxidase, the end product exhibits a color through the addition of an indicator to measure the color intensity
Creatininase-Hydrogen Peroxide
Analytical Methods in Determining Creatinine
- modified Jaffe reaction with adsorbent (done via elution)
- the picric acid with adsorbent now functions to remove interferences
Chemical Method
Analytical Methods in Determining Creatinine
What adsorbent is composed of sodium aluminum silicate?
Lloyd’s reagent
Analytical Methods in Determining Creatinine
What adsorbent is composed of aluminum magnesium silicate?
Fuller’s earth
Analytical Methods in Determining Creatinine
The reference method for creatinine measurement
Isotope Dilution Mass Spectrometry (IDMS)
Specimen Requirements for Creatinine
What 3 body fluids can be used?
- Plasma
- Serum
- Urine
Specimen Requirements for Creatinine
Creatinine requires a (fasting/non-fasting) sample
Non-fasting
TOF: Protein ingestion cannot elevate creatinine levels
False (it can transiently elevate but only minimally)
Interferences of Creatinine (falsely increased or decreased)
- a-ketoacids
- glucose
- ascorbic acids
- uric acid
- cephalosporin
- high ketones
False increase
Interferences of Creatinine (falsely increased or decreased)
- bilirubin
- hemoglobin (hemolyzed sample)
- contamination
False decrease
Creatinine Interferences
Bilirubin and hemoglobin cause what resulting in a false decrease in results?
Dilution
Creatinine Interferences
This is an anesthesia that can cause interferences in enzymatic methods
Lidocaine
Creatinine Interferences
This can cause interferences in BOTH enzymatic and Jaffe reactions
Dopamine
Creatinine Interferences
This causes false increased levels using Jaffe’s reaction
Cephalosporin
Reference Values for Creatinine
Choices:
A. Female adult (Jaffe reaction)
B. Female adult (Enzymatic method)
C. Male adult (Jaffe reaction)
D. Male adult (Enzymatic method)
E. Children (Jaffe reaction)
F. Children (Enzymatic method)
Given: 0.6 - 1.1
A and D
Reference Values for Creatinine
Choices:
A. Female adult (Jaffe reaction)
B. Female adult (Enzymatic method)
C. Male adult (Jaffe reaction)
D. Male adult (Enzymatic method)
E. Children (Jaffe reaction)
F. Children (Enzymatic method)
Given: 0.9 - 1.3
C.
Reference Values for Creatinine
Choices:
A. Female adult (Jaffe reaction)
B. Female adult (Enzymatic method)
C. Male adult (Jaffe reaction)
D. Male adult (Enzymatic method)
E. Children (Jaffe reaction)
F. Children (Enzymatic method)
Given: 0.5 - 0.8
B.
Reference Values for Creatinine
Choices:
A. Female adult (Jaffe reaction)
B. Female adult (Enzymatic method)
C. Male adult (Jaffe reaction)
D. Male adult (Enzymatic method)
E. Children (Jaffe reaction)
F. Children (Enzymatic method)
Given: 0.3 - 0.7
E.
Reference Values for Creatinine
Choices:
A. Female adult (Jaffe reaction)
B. Female adult (Enzymatic method)
C. Male adult (Jaffe reaction)
D. Male adult (Enzymatic method)
E. Children (Jaffe reaction)
F. Children (Enzymatic method)
Given: 0 to 0.6
F.
Creatinine has different reference values because it depends on the what of the individual?
Muscle Mass
What is the conversion factor of creatinine to its SI unit?
88.7 (to umol/L)
Creatinine 24H Urine Values
Male or Female:
800-2000 mg/dL
Male
Creatinine 24H Urine Values
Male or Female:
600-800 mg/dL
Female
Disease Correlations of Creatinine
Elevated creatinine in the blood is correlated to what kidney disease?
Renal Failure
Disease Correlations of Creatinine
Creatinine is used in what test to measure glomerular filtration rate and how effective the kidneys are in filtering wastes in the blood
Clearance Test
- major end product of purines (adenine and guanine) metabolism
- the final breakdown of nucleic acid catabolism in humans
- is freely filtered but significantly reabsorbed and secreted in the tubules
Uric Acid
For higher primates, uric acid is the end product of purine metabolism but in some animals, uric acid is further converted into what?
Allantoin
Uric Acid
More than (>) 95% of uric acid exists as what kind of urate at a pH of 7.4 (slightly alkaline)?
Monosodium Urate
Uric Acid
Uric acid at a pH of 5.4 and below exists as?
Uric acid (lol)
Note: It has a different form when in an alkaline environment
Uric Acid
A concentration of > 6.4 mg/dL in plasma is considered (saturated, normal, unsaturated)
Saturated
Hyperuricemia or Hypouricemia
- leukemia
- lymphoma
- multiple myeloma
- drugs, diet, and obesity
- hypertriglyceridemia
- renal disease
- gout
- Lesch Nyhan syndrome
- ethanol consumption
Hyperuricemia
Hyperuricemia or Hypouricemia
- Fanconi’s
- Wilson’s
- Hodgkin’s
- Bronchogenic CA
- Xanthinuria
Hypouricemia
Analytical Methods in Determining Uric Acid
- the simplest and most specific method
- is acted upon by uricase to produce allantoin
Enzymatic Method
Analytical Methods in Determining Uric Acid (Enzymatic Methods)
- measures the difference in absorbance before and after adding uricase
- a decrease in absorbance is proportional to the uric acid content
Spectrophotometric
Analytical Methods in Determining Uric Acid (Enzymatic Methods)
Uric acid is measured at a peak absorbance of what using the spectrophotometric method?
293nm
Analytical Methods in Determining Uric Acid (Enzymatic Methods)
Which among the two is the Coupled Enzymatic Reaction I and II?
- is coupled with catalase to produce a colored compound
- is coupled with peroxidase to produce a colored compound
- CATALASE = enzymatic reaction I
- PEROXIDASE = enzymatic reaction II
Analytical Methods in Determining Uric Acid (Chemical Methods)
- involves a reaction with uric acid and alkaline phosphogluconate with tungsten blue as the end product
- the principle is a redox reaction upon incubation
Phosphotungstic Method
Analytical Methods in Determining Uric Acid (Chemical Methods)
In the spectrophotometric method, this additive is used in Brown’s, Benedict’s, Folin’s, and Newton’s methods
Sodium Bicarbonate
Analytical Methods in Determining Uric Acid (Chemical Methods)
In the spectrophotometric method, this additive is used in Archibal’s, Caraway’s, and Henry’s methods
Sodium Cyanide
Analytical Methods in Determining Uric Acid (Chemical Methods)
In spectrophotometry, this will occur after the addition of the additives to inactivate non-uric acid reactants to isolate uric acid for measurement
Lag Phase
Analytical Methods in Determining Uric Acid (Chemical Methods)
The reference method for uric acid measurement
Isotope Dilution Mass Spectrometry (IDMS)
Specimen Requirements for Uric Acid
TOF: Uric acid is stable in both serum and plasma for 3 days at room temperature
False (serum and URINE, not plasma)
The other parameters are correct
Specimen Requirements for Uric Acid
This substance cannot be used as an anticoagulant as it can interfere with results
Potassium oxalate
Specimen Requirements for Uric Acid
What are the 2 major interferences that falsely decrease results?
- Ascorbic Acid
- Bilirubin
Reference Values for Uric Acid
For females?
2.6 - 6.0 mg/dL
Reference Values for Uric Acid
For males?
0.5 - 7.2 mg/dL
Reference Values for Uric Acid
What is the conversion factor to convert mg/dL to mmol/L?
0.0595
Disease Correlations for Uric Acid (hyperuricemia)
- pain and inflammation in the joints
- crystals are found in the synovial fluid
- can be treated with allopurinol
Gout
Disease Correlations for Uric Acid (hyperuricemia)
- leukemia, lymphoma, multiple myeloma, and polycythemia vera
- can be treated with allopurinol
Increased nuclear metabolism
Disease Correlations for Uric Acid (hyperuricemia)
- due to increased GFR and tubular secretion
Chronic renal disease
Disease Correlations for Uric Acid (hyperuricemia)
- an inborn error of purine metabolism (X-linked)
- decreased hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
Lesch-Nyhan Syndrome (aka Juvenile Gout)
Disease Correlations for Uric Acid (hyperuricemia)
- glycogen storage diseases
- toxemia of pregnancy and lactic acidosis
- due to increased dietary intake and ethanol consumption
Other causes of hyperuricemia (secondary to the disease)
Disease Correlations for Uric Acid
- Fanconi’s syndrome (congenital tubular necrosis)
- Wilson’s disease
- Hodgkin’s disease
Hypouricemia
What NPNs can be measured with the IDMS method?
- Urea
- Creatinine
- Uric Acid
- a byproduct of protein catabolism that arises from the deamination of amino acids due to the action of intestinal flora
- also released due to skeletal muscle metabolism
- is useful for liver function tests
Ammonia
The liver removes ammonia (which is toxic) through the circulation in what vein before it is converted to urea?
Portal Vein
Ammonia is converted to what in cases of severe liver disorders?
Glutamine
TOF: Ammonia exists as an ion in highly acidic and alkaline pH levels
False (it is an ammonium ion in PHYSIOLOGICAL pH levels)
Analytical Methods in Determining Ammonia
- uses glutamate dehydrogenase, same in urea measurement
Enzymatic Method
Analytical Methods in Determining Ammonia
- ion-exchange
- ion selective electrode
- spectrophotometric
Chemical
Specimen Requirements for Ammonia
- whole blood ammonia (rises/drops) rapidly following specimen collection
Rises
Specimen Requirements for Ammonia
Ammonia samples must be placed on (ice/heated blocks) to prevent the loss of ammonia
Ice
Specimen Requirements for Ammonia
Air exposure can cause ammonia to (falsely increase/decrease)
Increase
Specimen Requirements for Ammonia
What anticoagulants (2) are used to measure ammonia?
EDTA and Heparin
Specimen Requirements for Ammonia
Centrifuge the sample in what temperature range?
0-4ºC
Specimen Requirements for Ammonia
Ammonia is assayed ASAP or if frozen, it can stay stable for several days at what temperature?
-20ºC
Specimen Requirements for Ammonia
(RBCs, WBCs, or platelets) contain 2-3 times as much ammonia as plasma
RBCs
Specimen Requirements for Ammonia
Cigarette smoking is a significant contaminant and should be stopped how many hours prior to testing?
24-48 hours
Specimen Requirements for Ammonia
If sample is hemolyzed, reject immediately as this can cause a rapid (increase/decrease) of the result upon collection
Increase
Specimen Requirements for Ammonia
What is the conversion factor from ug/dL to umol/L?
0.587
Please study the images on the transes for each enzymatic/chemical reaction as these may show up on the tests, and the mere descriptions in these cards may not be enough.
Thank you.