NON-PROTEIN NITROGENS

NPNs

• substances that are not considered as proteins but contains nitrogen

NPNs

• waste products

• include urea, uric acid, creatinine and ammonia, and amino acids

Urea

Uric Acid

Creatinine

• NPNs that are used to assess renal function

Ammonia

• NPN that is used to assess liver function

Creatinine

• a non-protein nitrogenous compound that is produced by

  the breakdown of creatine in muscle.

Creatinine

• found in serum, plasma, and urine and is excreted by glomerular filtration at a constant rate and in the same concentration as in plasma.

Creatinine

• a more reliable indicator of renal function than BUN because it is less influenced by other factors such as diet and hydration.

Creatinine

• synthesized in the kidneys, liver, and pancreas by two emzymatically mediated reactions

1. Kidneys

2. Liver

3. Pancreas

• organs that synthesize creatine

1. Transamidation

2. Methylation

• steps of creatine synthesis

Phosphocreatine

• high-energy compound formed in the muscle and brain through phosphorylation

Creatine Phosphate

• phospocreatine

• phosphorylated derivative of creatine found in muscle, is a high-energy compound that provides a small but rapidly mobilized reserve of high-energy phosphates that can be reversibly transferred to adenosine diphosphate to maintain the intracellular level of adenosine triphosphate (ATP) during the first few minutes of intense muscular

  contraction.

Phosphocreatine

• muscle cells used this to store energy

Creatinine

• Interconversion of phosphocreatine and creatine is a particular feature of the metabolic processes of muscle contraction.

Creatinine

• proportion of the free creatine in muscle (thought tobe between 1% and 2%/d) spontaneously and irreversibly converts to its anhydride waste product

True

T or F

The amount of creatinine produced each day is relatively constant and is related to the muscle mass.

Creatinine

• present in all body fluids and secretions and freely filtered by the glomerulus

  • not reabsorbed to any great extent by the renal tubules

1-2 g

• amount of creatinine excreted by male adults in a day

Creatinine

• break-down product of creatine phosphate and creatine in muscles, and is usually produced at a fairly constant rate by the body depending on muscle mass

Creatinine

• anhydride of creatine

• produced when creatine loses water

Creatinine

• produced when creatine phosphate loses phosphoric acid

Creatinine

• end product of muscle metabolism

• not reabsorbed by the renal tubules in significant amounts

Creatinine

• assess the completeness of a 24-hr urine specimen as it is excreted at a constant rate

10:1 - 20:1

• normal BUN to creatinine ratio

Creatinine

• its measurement is used to determine the sufficiency of kidney function, to determine the severity of kidney damage, and to monitor the progression of kidney disease.

True

T or F

Strenuous exercise and fist clenching can affect creatinine measurement.

1. Glomerular Filtration Rate

2. Plasma Creatinine

3. Creatinine Clearance

• clinical significance of creatinine

Glomeruli

• filters water and low molecular weight components of the blood while retaining cells and high molecular weight components.

True

False (baliktad)

T or F

GFR declines with age.

GFR declines to a greater extent in females than in males.

GFR

• considered to be a reliable measure of the functional capacity of the kidneys and is often thought of as indicative of the number of functioning nephrons.

GFR

• has proved to be a useful marker of changes in overall

  renal function.

• depends upon the balance between hydrostatic and oncotic forces along the afferent arteriole and across the glomerular filter.

True

T or F

A decrease in GFR precedes kidney failure in all forms of progressive disease.

1. End Stage Renal Disease

2. Dialysis Dependency

• pathological kidney conditions due to decreased GFR

Plasma Creatinine

• is a function of relative muscle mass, the rate of creatine

  turnover, and renal function.

True

T or F

Protein content in diet affect plasma creatinine concentration.

Urinary Creatinine Excretion

• has been used as a measure of the completeness of 24-hour urine collections in a given individual, although the uncertainty associated with this practice may exceed that introduced by use of the urine volume and collection time for standardization.

Creatinine Clearance

• An estimate of the GFR can be made by measuring the urinary excretion of a substance that is completely filtered from the blood by the glomeruli and is not secreted, reabsorbed or metabolized by the renal tubules.

Clearance

• the removal of the substance from plasma into urine over a fixed time

Inversely Prop

• relationship of plasma creatinine concentration and creatinine clearance

• formula of creatinine clearance

Creatinine Clearance

• measured as a rate; therefore, the test must be timed.

• The test measures the movement of the substance from blood to urine; therefore, both blood and urine concentrations of the chemical must be measured.

Male: 97-137 mL/min

Female: 88-128 mL/min

• reference ranges for creatinine clearance in males and females

1. High cardiac output

2. Pregnancy

3. Burns

4. Carbon monoxide poisoning

• causes of increased creatinine clearance

1. Impaired kidney function

2. Shock, dehydration

3. Hemorrhage

4. Congestive heart failure

• causes of decreased creatinine clearance

Creatinine

• associated with abnormal renal function

Inversely prop

• relationship of plasma creatinine concentration and GFR

  • indicates renal damage

Plasma Creatinine

• a relatively insensitive marker and may not be measurably

  increased until renal function has deteriorated more than 50%.

Creatine

• increased in muscle diseases

  a. muscle dystrophy

  b. poliomyelitis

  c. hyperthyroidism

  d. trauma

Creatine Kinase

• its measurement is used typically for the diagnosis of muscle disease because analytic methods for creatine are not readily available in most clinical laboratories.

Plasma Creatine

• not elevated in renal disease

Jaffe Principle

• most common methods for creatinine determination

7 days at 4C

• stability of creatinine in serum or urine

1. Chemical Methods (Jaffe reaction)

   a. Folin-Wu

   b. O’ Leary Method

   c. Lloyd or Fuller’s Earth method

   d. Hare Method

   e. Kinetic Jaffe Method

2. Enzymatic Methods

   a. Creatininase - Creatine Kinase Method

   b. Creatininase - Hydrogen Peroxide Method

   c. Creatinine Deaminase

3. Isotope Dilution Mass Spectrometry (IDMS)

• methods for creatinine determination

Jaffe reaction (Alk Picric Method)

• Creatinine reacts with picric acid (trinitrophenol) in an

  alkaline solution to form a red-orange chromogen

Red-Orange

490-500 nm

• color for Jaffe Reaction and its absorbance

lacks specificity to creatinine

• problem with Jaffe Reaction

1. Ketones

2. Glucose

3. Fructose

4. Protein

5. Urea

6. Uric Acid

7. Ascorbic Acid

8. Blood-Substitute products

9. Cephalosporin

10. Protein

11. Pyruvate

12. Guanidine

• non-creatinine chromogens that reacts with picrate

• falsely increases creatinine concentration in Jaffe Reaction

1. Bilirubin

2. Hemoglobin

• causes falsely decreased creatinine concentration in Jaffe reaction

Buffering Ions (borate, phosphate with surfactant)

• minimizes interferences from bilirubin and hemoglobin in Jaffe Reaction

Folin-Wu Method

• Adopted Jaffe reaction for the measurement of blood creatinine in 1919.

O’Leary Method

• Uses ferricyanide to oxidize bilirubin to biliverdin hence reducing its interference.

Lloyd or Fuller’s Earth Method

• Adsorbent improves specificity: adsorbent removes interferences present in the specimen.

• Time-consuming and not readily automated, therefore not routinely performed.

Fuller’s Earth

• uses aluminum magnesium silicate

LLoyd’s Method

• uses sodium aluminum silicate

Hare Method

• Involves isolating creatinine by absorption into Lloyd’s reagent and discarding the plasma containing interfering chromogens.

Kinetic Jaffe Method

• Serum is mixed with alkaline picrate and the rate of change in absorbance is measured.

• The detection of color formation is timed (thus, termed as kinetic) to avoid interference of noncreatine chromogens.

Kinetic Jaffe Method

• Improvement of specificity in the kinetic assays was achieved by selecting times or rate measurements 20 to 80 seconds after initiation of the reaction (mixing).

• This approach has been implemented with various automated instruments, and kinetic assays are now widely used to measure creatinine concentrations in body fluids.

Kinetic Jaffe Method

• Rapid, inexpensive, and easy to perform.

1. (+) Alpha - keto (2-oxo) acids and cephalosporins

2. (-) Bilirubin and hemoglobin

• positive and negative interference in Kinetic Jaffe Method

Creatininase (Creatinine Aminohydrolase) - Creatine Kinase (CK) Method

• Creatininase (creatinine amidohydrolase) catayzes the conversion of creatinine to creatine.

• The creatine is then detected with a series of enzyme-

  mediated reactions involving (1)creatine kinase (CK), (2) pyruvate kinase (PK), and (3) lactate dehydrogenase (LDH), with monitoring of the decrease in absorbance at 340 nm.

Creatininase (Creatinine Aminohydrolase) - Creatine Kinase (CK) Method

• Requires large sample; not widely used.

Creatininase (Creatinine Aminohydrolase) - Hydrogen Peroxide (H202) Method

• Creatinase is used to yield sarcosine and urea. Sarcosine is then measured by further enzyme-mediated steps using sarcosine oxidase to produce:

  (1) glycine,

  (2) formaldehyde, and

  (3) hydrogen peroxide.

Creatininase (Creatinine Aminohydrolase) - Hydrogen Peroxide (H202) Method

• In the presence of peroxidase (e.g.horseradish peroxidase), an indicator (e.g. 2,4-dichlorophenolsulfonate) is converted toa colorless polymer by hydrogen peroxide, and the concentration of the polymer is thenmeasured at 510 nm.

1. Bilirubin

2. Ascorbic Acid

• potential interferences of Creatininase (Creatinine Aminohydrolase) - Hydrogen Peroxide (H202) Method

K Ferricyanide or Bilirubin Oxidase

• how is bilirubin minimized in the Creatininase (Creatinine Aminohydrolase) - Hydrogen Peroxide (H202) Method

Ascorbate Oxidase

• how is ascorbate minimized in the Creatininase (Creatinine Aminohydrolase) - Hydrogen Peroxide (H202) Method

Creatininase (Creatinine Aminohydrolase) - Hydrogen Peroxide (H202) Method

• adapted for use as dry slide method

Creatininase (Creatinine Aminohydrolase) - Hydrogen Peroxide (H202) Method

• has the potential to replace Jaffe

False

T or F

Acetoacetate and cephalosporins interferes with Creatininase (Creatinine Aminohydrolase) - Hydrogen Peroxide (H202) Method

Lidocaine

• Creatininase (Creatinine Aminohydrolase) - Hydrogen Peroxide (H202) Method has a positive bias because of this

Creatinine deaminase (Creatinine iminohydrolase)

• Creatinine is hydrolyzed by creatinine iminohydrolase to ammonia and N- methylhydantoin.

• The ammonia then combines with 2-oxoglutarate and

  nicotinamide adenine dinucleotide (NADH) in the presence of glutamate dehydrogenase to produce glutamate and NAD+.

• Consumption of NADH measured as a decrease in absorbance at 340 nm is used to measure the concentration of creatinine.

Creatinine deaminase (Creatinine iminohydrolase)

• Adapted for use as a POCT testing device

Isotope Dilution Mass Spectrometry (IDMS)

• reference method for creatinine determination

Gas Chromatography - IDMS (GC-IDMS)

• method of choice for establishing the true concentration of creatinine in serum; excellent specificity and low imprecision.

• reference intervals for creatinine determination (table from module)

88.4

• conversion factor for creatinine determination

True

T or F

Creatinine is the main storage component of high energy phosphate needed for muscle metabolism.

False

T or F

Serum or plasma creatine concentration and urine creatine are decreased by skeletal muscle necrosis or atrophy.

Urea

• synthesized in the liver, primarily as a by-product of the deamination of amino acids.

Urea

• its elimination in the urine represents themajor route for nitrogen excretion.

Urea

• filtered from the blood by the glomeruli but significant tubular reabsorption occurs through passive diffusion.

Urea

• major nitrogen-containing metabolic product of protein catabolism in humans

Kidney

• the only significant route of excretion for urea

Urea

• readily filtered by the glomerulus

Urea

• produced by the liver through the Krebs-Henseleit Cycle, excreted by the kidneys but partially reabsorbed

Collecting Ducts

• 40-60% of urea is reabsorbed in this

Blood Urea Nitrogen

• refer to the nitrogen content only of urea

• obtained by indirect methods

Urea Concentration

• concentration of urea as a whole molecule

• obtained by direct methods

BUN x 2.14

• formula for Urea Concentration via BUN

• flow of urea synthesis