Non-Protein Nitrogen Compounds (NPN)

Paired, bean-shaped organs located retroperitoneally on either side of the spinal column

Kidneys

Two regions of the kidneys

• cortex (outer)

• medulla (inner)

Functional unit of the kidney

Nephron

5 Basic Parts of Nephron

• glomerulus

• proximal convulated tubule

• loop of henle

• distal convulated tubule

• collecting duct

Proximal Convulated Tubule (PCT) is the site of reabsorption for:

• sodium

• chloride

• bicarbonate and other ions

• glucose

• amino acids and proteins

• urea

• uric acid

Anatomy of Kidney

Functions of the Kidneys

• Elimination of Waste Products

• Maintenance of Blood Volume

• Maintenance of Acid-Base Balance

• Endocrine Function > erythropoietin secretion

Renal Function Panel

• Glucose

• BUN

• Creatinine

• Sodium

• Potassium

• Chloride

• Phosphorous

• Calcium

• Albumin

• Carbon dioxide

Traditionally used to monitor renal function

Non-protein Nitrogen (NPN) Compounds

The term, Non-protein Nitrogen Compounds, originated when _

analytic methodology required removal of protein from sample before analysis

Concentration of nitrogen-containing compounds was quantified - by converting -

spectrophotometrically by converting nitrogen to ammonia

Non-Protein Nitrogen (NPN) Compounds

• Subsequent reaction with - produced a yellow color

Nessler's reagent (K2[HgI4])

NPN fraction comprises about - compunds of clinical interest

15

Majority of these compunds arise from _

catabolism of proteins and nucleic acids

The test for NPNs (specifically - and -) is considered -

• urea and creatinine

• kidney function test (KFT)

Clinically Significant NPNs

NPN compund present in highest concentration in blood

Urea

Urea is - formed in - from amino groups and free ammonia

• major excretory product of protein metabolism

• liver

• first to elevate in kidney diseases

• easily removed by dialysis

Urea

Urea - PHYSIOLOGY

• - is released as a result or protein metabolism converted to - and excreted as a waste product

• Nitrogen

• Urea (80%)

Urea - PHYSIOLOGY

• After synthesis in liver (through the - cycle), urea is carried in blood to the kidney and filtered out

• Krebs-Henseleit Cycle

Urea - PHYSIOLOGY

• Most urea in glomerular filtrate is excreted in - but some is reabsorbed in -

• urine (90%)

• renal tubules (10%)

Urea - CLINICAL APPLICATION

• Evaluates renal function

• Assess hydration status

• Determines nitrogen balance

• Aid in diagnosis of renal disease

• Verify adequacy of dialysis

Urea - Methods of Measurement

• Enzymatic > urease

• electrode

• isotope-dilution mass spectrometry > reference method

Urea - SPECIMEN REQUIREMENTS & INTERFERING SUBSTANCES

• may be measured in -

• in plasma > avoid -

• susceptible to -

• may be measured in plasma, serum, urine

• in plasma > avoid ammonium ions and high citrate and fluoride

• susceptible to bacterial decomposition > analyze quickly or refrigerate if urine

Enzymatic Methods for Urea use similar first step:

Enzymatic methods for urea

• GLDH coupled enzymatic

• Indicator dye

• Conductimetric

Other Methods

• Isotope dilution mass spectrometry

• used on many automated instruments

• best as kinetic measurement

GLDH coupled enzymatic

• used in automated systems, multilayer film reagents, and dry reagent strips

Indicator dye

• specific and rapid

• conversion of unionized urea to NH4+ and CO3 2- results in increased conductivity

Conductimetric

• proposed reference method

• detection of characteristic fragments following ionization, quantificationm using isotopically labeled compund

Isotope filution mass spectrometry

Indirect methods for measuring urea

• measure the nitrogen content of urea in blood urea nitrogen or BUN

Enzymatic assay for urea

Normal value for Urea Nitrogen in Adults

• In PLASMA/SERUM

  • 6 to 20 mg/dL (2.1 to 7.1 mmol/L)

• In URINE

  • 24-hour sample

  • 12 to 20 g/day (0.43 to 0.71 mol urea/day

Urea - PATHOPHYSIOLOGY

• elevated concentration of urea in blood

azotemia

Urea - PATHOPHYSIOLOGY

• Very high plasma urea concentration with renal failure > acideia and electrolyte imbalance

Uremia

Urea - PATHOPHYSIOLOGY

• reduced renal blood flow caused by:

  • high protein diet

  • congestive heart failure (CHF)

  • shock

  • hemorrhage

  • increased catabolism

  • corticosteroid therapy

Prerenal azotemia

Urea - PATHOPHYSIOLOGY

• decreased renal function caused by:

  • renal failure

  • glomerulonephritis

Urea - PATHOPHYSIOLOGY

• obstruction of urine flow caused by:

  • renal calculi

  • tumors of bladder and prostate

  • severe infection

Postrenal azotemia

Urea - DISEASE CORRELATIONS

• The following increases urea levels

• Chronic renal disease

• Stress

• Burns

• High protein diet

• Dehydration

Urea - DISEASE CORRELATION

• The following decreases urea levels:

• poor nutrition

• high fluid intake / excessive IV fluids

• pregnancy

• severe liver diseases

• effects of some hormones

• severe vomiting or diarrhea

Differentiation of the cause of abnormal urea concentration:

Urea Nitrogen / Creatinine ration

Norma Urea Nitrogen / Creatinine ratio

10:1 to 20:1

Urea Nitrogen / Creatinine ratio in Prerenal Azotemia

• high urea

• normal creatinine

• HIGH UREA NITROGEN/CREATININE RATION

Urea Nitrogen / Creatinine ratio in Postrenal Azotemia

• high urea

• high creatinine

• HIGH UREA NITROGEN / CREATININE RATIO

There will be LOW UREA NITROGEn / CREATININE RATIO in:

• Decreased urea production

  • low protein intake

  • acute tubular necrossi

  • severe liver disease

product of catabolism or purine nucleic acids

Uric acid

Uric Acid

• most is reabsorbed in - and reused

proximal convulated tubules (98% to 100%)

Uric Acid

• at pH of - , more than -% of uric acid in body exists as -

• pH of 7.4

• 95%

• monosodium urate

Uric Acid

• relatively insoluble in -

• in high concentrations, can be deposited in -

• relatively insoluble in plasma

• in high concentrations, can be deposited in joints and tissues > painful inflammation

Uric Acid - Physiology

• - are converted to uric acid in -

• Uric acid is transprted in plasma from - to - and filtered by glomerulus

• purine

• liver to kidney

Uric Acid - Physiology

• -% is eliminated by -, remainder passes into - and is degraded by bacterial enzymes

• 70%

• renal excretion

• GI tract

Uric Acid - CLINICAL APPLICATION

• Assessment of inherited disorders of purine metabolism

• Confirmation of diagnosis and monitoring of treatment of gout

• Assistance in diagnosis of renal calculi

• Prevention of uric acid nephropathy in chemotheraphy

• Detection of kidney dysfunction

Uric Acid - Methods of Measurement

• Caraway method

• Uricase method

• Coupled enzyme methods

• Isotope dilution mass spectrometry (proposed)

Uric Acid - SPECIMEN REQUIREMENTS and INTERFERING SUBSTANCES

• measured in -

• remove - from cells quickly to prevent -

• Avoid gross -,-,-

• Diet may affect concentration overall but - not necessary

• measured in heparinized plasma, serum, or urine

• remove serum from cells quickly to prevent dilution by intracellular contents

• Avoid gross lipemia, bilirubinemia, hemolysis

• Diet may affect concentration overall but fasting not necessary

Uric Acid - CHEMICAL METHODS

• Nonspecific

• Requires protein removal

Phosphotungstic acid

Uric Acid - ENZYMATIC METHODS

• Spectrophotometric

• Coupled enzymatic (I)

• Coupled enzymatic (II)

Uric Acid - ENZYMATIC METHODS

• have similar first step:

• very specific

Uric Acid - Enzymatic Methods

• decrease in absorbance at 293 nm measured

• hemoglobin and xanthin interfere

Spectrophotometric

Uric Acid - Enzymatic Methods

• commonly automated method

• negative bias with reducing agents

Coupled Enzymatic (I)

Uric Acid - Enzymatic Methods

• readili automated

• reducing agents interfere

Coupled enzymatic (II)

Uric Acid - OTHER METHODS

• proposed reference method

• detection of characteristic fragments following ionization

• quantification using isotopically labeled compound

- Isotope dilution mass spectrometry (IDMS)

Uric Acid - NORMAL VALUE

• ADULT

  • plasma/serum

    →MALE = 3.5 to 7.2 mg/dL

    →FEMALE = 2.6 to 6.0 mg/dL

• CHILD

  • plasma/serum

    → 2.0 to 5.5 mg/dL

Uric Acid - Pathophysiology

• elevated levels of uric acid

hyperuricemia

Uric Acid - Pathophysiology

• Hyperuricemia is found in:

• inherited disorders of purine metabolism

• Glucose-6-phosphate deficiency

• Fructose intolerance > Fructose-1-phosphate aldolase deficiency

• Gout

• Treatment of myeloproliferative disorders with cytotoxic drugs

• Chronic renal disease

• Toxemia of pregnancy

• Lactic acidosis

• Drugs and poisons

• Purine-rich diest

• Increased tissue catabolism or starvation

Uric Acid - Pathophysiology

• decreased levels of uric acid

hypouricemia

Uric Acid - Pathophysiology

• Hypouricemia is found in:

• Liver disease

• Defective tubular resorption > Fanconi syndrome

• Chemotherapy > azathioprine or 6-mercaptopurine

• Overtreatment with allopurinol

Uric Acid - Disease Correlations

• Gout

• Lesch-Nyhan Syndrome

Uric Acid - Disease Correlations

• degenerative disorder

• commonly found in males where there is deposition of uric acid crystals in joints or TOPHI

Gout

Uric Acid - Disease Correlations

• in-born errors of metabolism

• deficiency of hypoxanthine guanine phosphoribosyl transferase (HGPRT) > important in biosynthesis of purines

Lesch-Nyhan Syndrome

Uric Acid - Disease Correlations

• Lesch-Nyhan Syndrome

  • Clue:

Orange sand in diapers

Uric Acid - Disease Correlations

• Lesch-Nyhan Syndrome

  • Complication:

can lead to mental retardation

• Major end-product of metabolism

• best index of kidney function

Creatinine / Creatine

Creatinine is formed from - and - in muscle and is excreted into plasma at rate related to -

• creatine and creatine phosphate

• muscle mass

Plasma creatinine is inversely related to - and is commonly used to assess -

• glomerular filtration rate (GFR)

• renal filtration function

Creatinine / Creatine - PHYSIOLOGY

• Creatine is synthesized mainly in - from -,-, and -

• liver

• arginine, glycine, methionine

Creatinine / Creatine - PHYSIOLOGY

• It is then transported to other tissues and converted to - which serves as high-energy source

creatine phosphate

Creatinine / Creatine - PHYSIOLOGY

• Creatine phosphate and creatine for creatinine which difuses into plasma and is excreted

• - % excreted

• maximum of -% reabsorbed

• 100% excreted

• 1% reabsorbed

Creatinine / Creatine - CLINICAL APPLICATION

• determine sufficiency of kidney function and severity of kidney damage

• monitor progression of kidney disease

Creatinine / Creatine - CLINICAL APPLICATION

• measure of amount of creatinine eliminated from the blood by the kidneys

Creatinine Clearance

Creatinine / Creatine - CLINICAL APPLICATION

• volume of plasma filtered by glomerulus per unit of time

Glomerular Filtration Rate (GFR)

Creatinine / Creatine - CLINICAL APPLICATION

• Abbreviated Modification of Diet in Renal Disease (MDRD) equation include 4 variables:

• serum creatinine concentration

• age

• gender

• ethnicity

Creatinine - Methods of Measurement

• Jaffe Reaction

• Kinetic Jaffe Method

• Coupled Enzymatic Methods

• Isotope Dilution Mass Spectrometry

Creatinine - Methods of Measurement

• time-consuming, not readily automated

Jaffe Reaction

Creatinine - Methods of Measurement

• Jaffe Reaction

  • Creatinine reacts with - in alkaline solution to form -

• picric acid

• red-orange chromogen

Creatinine - Methods of Measurement

• rapid

• inexpensive

• easy to perform

Kinetic Jaffe Method

Creatinine - Methods of Measurement

• Kinetic Jaffe Method

  • Serum is mixed with - and - is measured

• alkaline picrate

• rate of change in absorbance

Creatinine - Methods of Measurement

• improves specificity

Coupled enzymatic methods

Creatinine - Methods of Measurement

• reference method

Isotope dilution mass spectrometry

Creatinine - CHEMICAL METHODS BASED ON JAFFE REACTION

• Jaffe-Kinetic

• Jaffe with adsorbent

• Jaffe witount adsorbent

Creatinine - CHEMICAL METHODS BASED ON JAFFE REACTION

• Jaffe reaction performed direactly on sample

• Detection of color formation timed to avoid interference of noncreatinine chromogens

Jaffe-Kinetic

Creatinine - CHEMICAL METHODS BASED ON JAFFE REACTION

• Jaffe-Kinetic

  • positive bias from:

• alpha-ketoacids and cephalosporins

• requires automated equipment for precision

Creatinine - CHEMICAL METHODS BASED ON JAFFE REACTION

• Creatinine in protein-free filtrate adsorbed onto Fuller's earth (aluminum magnesium silicate)

• Then, reacted with alkaline picrate to form colored complex

Jaffe with adsorbent

Creatinine - CHEMICAL METHODS BASED ON JAFFE REACTION

• Jaffe with adsorbent

  • Adsorbent improves -

specificity

Creatinine - CHEMICAL METHODS BASED ON JAFFE REACTION

• previously considered reference method

Jaffe with adsorbent

Creatinine - CHEMICAL METHODS BASED ON JAFFE REACTION

• Creatinine in protein-free filtrate reacts with - to form colored complex

• alkaline picrate

Creatinine - ENZYMATIC METHODS

• requires large sample

• not used widely

Creatininase-CK

Creatinine - ENZYMATIC METHODS

• adapted for use as dry slide method

• potential to replace Jaffe

Creatininase-H2O2

Creatinine - ENZYMATIC METHODS

• No interference from -

• Some positive bias due to -

• acetoacetate or cephalosporins

• lidocaine