CENTRAL TEXAS COLLEGE 2401-CLINICAL CHEMISTRY Non-Protein Nitrogen Compounds I.

Renal System: Organs (kidneys, ureters, bladder, and urethra) that produce, collect, and eliminate urine A. Gross Anatomy of Kidneys 1. 2. External: 3 layers of tissue surround kidneys that protect against trauma and infection and anchors kidney to abdominal wall Internal anatomy a. Cortex: outer peripheral portion of kidney that contains the glomeruli, proximal and distal convoluted tubules b. Medulla: inner portion of the kidney that contains loops of HenIe, vasa recta, and collecting ducts c. Nephrons end with collecting ducts located in the renal pyramids, which drain into calyces, which drains into the renal pelvis, which drains into a ureter d. Renal pelvis: large cavity that drains urine from major calyces into the ureter

B.

Reservoir components 1. Ureters: Pair of fibromuscular tubes which transport urine from renal pelvis to the bladder 2. Bladder: Elastic, hollow muscular organ that consists of transitional epithelial cells that can stretch 3. Urethra: Muscular tubular passageway from bladder to the outside of the body

II.

Microscopic Structures A. Nephron: Functional unit of the kidney 1. Number: Each kidney contains 1 million nephrons Components of Nephrons 1. Glomerulus a. Dense capillary mass b. Filtration begins here 2. Bowmans capsule - Double-walled cup that surrounds glomerulus and collects the filtrate (filtered blood) 3. Proximal convoluted tubule - Consists of cuboidal epithelial cells containing numerous mitochondria needed for active transport 4. Loop of HenIe a. Thin (descending) segment: squamous epithelial cells b. Thick (ascending) segment: cuboidal cells containing numerous mitochondria 5. Distal convoluted tubule: consists of cuboidal cells and few microvilli 6. Collecting ducts: cuboidal and columnar cells

B.

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C.

Blood supply 1. Afferent arteriole: supplies blood to glomerulus 2. Efferent arteriole 3. Vasa recta a. Thin walled vessels along the Loop of HenIe b. Important in forming concentrated urine

III.

Nephron Physiology (Formation of urine) A. Glomerular filtration 1. High blood pressure in glomerulus forces fluid and dissolved substances from plasma through permeable membrane into Bowmans capsule 2. Ultra filtrate formed: essentially same as plasma without large molecules (Large proteins (MW > 15,000) and cells) 3. Filtrate - - in composition to interstitial fluid 4. - 180 liters of filtrate produced per day Tubular reabsorption 1. Movement from filtrate in tubule back into blood 2. Specific amounts of certain substances are reabsorbed depending on bodys need at the time 3. Reabsorption mechanisms a. Active transport: energy requiring; may require carrier molecules (primarily Na and Cl) b. Passive transport: equalization of concentrations across a membrane (primarily water) 4. Areas of reabsorption a. Proximal convoluted tubule 1) 80% of tubular reabsorption 2) Electrolytes (Na, Cl, HC03, Ca), phosphate, glucose, and amino acids b. Loop of HenIe 1) Electrolytes (Na and Cl) and some water c. Distal convoluted tubule: mostly electrolytes (primary function is to secrete) d. Collecting ducts 1) Water is reabsorbed 2) Dilute or concentrated urine formation under antidiuretic hormone (ADH) control 3) ADH renders collecting ducts permeable to water Tubular secretion 1. Movement of substances from the blood into the tubule. Many of these substances were not filtered by the glomerulus 2. Secretion rids the body of certain substances and helps control blood pH a. Distal tubule secretes H ions and reabsorbs Na and HC03 ions to aide acidbase regulation 3. Substances secreted a. Potassium and hydrogen ions b. Ammonia c. Creatinine Page 2 of 7

B.

C.

4. 5.

6.

Passive or active transport Areas of secretion a. Primarily the distal convoluted tubule b. Collecting duct Countercurrent System: Wastes must be eliminated regardless of bodys state of hydration. Countercurrent system allows the body to concentrate urine with very little water loss

IV.

Non-protein Nitrogens - Role of glomerular filtration and tubular secretor function is to eliminate waste products of metabolism (NPNs) A. Non- protein nitrogens: 1. Amino acids (from proteins) 2. Ammonia (NH3 (from amino acids) 3. Urea (from ammonia) 4. Creatinine (from creatine) 5. Uric acid (from purine nucleosides, ex. adenine, and guanine) Urea 1. 2.

B.

3. C.

Renal disease can cause increase of plasma urea concentrations (measured as BUN) known as uremia Other factors cause increased BUN such as high protein diet or muscle wasting with starvation, so must look at BUN to creatinine ratio to determine prerenal vs postrenal source Normal BUN:Creatinine ratio =12-20

Creatinine 1. Creatine is produced in kidneys, liver, and pancreas then transported to muscle and brain 2. Creatine is energy used for muscle contraction 3. Creatine converts to creatinine (1-2% daily in muscle); amount produced is proportional to muscle mass 4. Production is very constant, therefore clearance is measured as indicator of glomerular filtration rate (GFR)

V.

Functions of the Renal System A. Endocrine function Hormones and/ or enzymes regulate volume and composition of urine and blood 1. Primary - produces hormones a. Renin and prostaglandin: affect vascular system (renin-angiotensin system regulates BP; regulates aldosterones effect on Na and K exchange) b. Erythropoietin (EPO): renal insufficiency can lead to anemia 2. Secondary - receptor of hormones a. Degradation of insulin, glucagon, and aldosterone b. Site of most active form ofVit D: chronic renal failure associated with osteomalacia (due to inadequate bone mineralization) 3. ADH: Makes distal tubule and collecting ducts permeable to water, thereby concentrating urine. Absence of ADH 6 dilute urine (high HOH content) is excreted Page 3 of 7

4. 5. 6.

Aldosterone: Allows Na+ to be reabsorbed from distal tubule and collecting ducts, decreasing amount ofNa+ excreted in urine Renin: Stimulates adrenal cortex to release aldosterone, which increases Na+ reabsorption; which increases water reabsorption Renal erythropoietic factor (REF) a. Release is commonly due to hypoxia b. Converts plasma proteins to erythropoietin c. Erythropoietin stimulates erythropoiesis

B.

Regulatory function 1. Maintenance of the internal fluid environment (homeostasis) a. Water and electrolyte balance b. By absorption and secretion the optimal chemical composition of interstitial and intracellular fluids are maintained throughout the body 2. Secondary regulation of the bodys acid-base balance (in conjunction with respiratory system) a. In acidosis (blood pH < 7.35) 1. Increased excretion of acids (tf) 2. Conservation of base (tubular reabsorption ofHC03) b. In alkalosis (blood pH > 7.45) 1. Increased excretion of base (HC03) 2. Conservation of acids (decreased tubular 1-T secretion) Excretory function: concentration and elimination of the waste products of metabolism 1. Urea: 25 - 30 gms excreted each day 2. Acid: Excreted or retained based on bodys need 3. Creatinine: End product ofcreatine metabolism Is not reabsorbed 4. Also, ammonia and HC03, a.a. in small quantities, and excess from diet (Na, K, Cl, Ca, Phos, Mg, and sulfate)

C.

VI.

Abnormal renal function A. Renal blood flow 1. Increase in rate of blood flow thru nephrons greatly increases glomerular filtration rate 2. Tubules are unable to reabsorb those substances that need to be conserved in the body 3. Decrease in rate of blood flow thru nephrons [ glomerular filtration rate 4. Tubules reabsorb waste products, which should be eliminated B. Glomerular 1. Acute Glomerulonephritis: Caused by an abnormal immune reaction a. 1-3 weeks following grp A beta streptococci infection elsewhere in body (streptococcal sore throat) b. Insoluble Ag-Ab immune complex becomes entrapped in the glomerulus c. Glomeruli become blocked by this inflammatory reaction 2. Chronic Glomerulonephritis a. Glomerular membrane becomes progressively thickened and is eventually invaded by fibrous tissue b. Glomeruli completely replaced by fibrous tissue in final disease stages

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C.

Tubular 1. Pyelonephritis a. Bacterial infection of renal pelvis and renal parenchyma b. Usually results from fecal contamination of urinary tract Inflammation/Obstruction 1. Renal calculi: (kidney stones) 2. Sexually transmitted diseases: (gonorrhea)

D.

VII.

Renal Function Tests A. Urea Nitrogen (Diacetyl monoxime Thiosemicarbazide) 1. Principle: Urea in serum or plasma reacts directly with diacetyl monoxime in a hot acid environment to form a yellow colored complex (diazine) 2. Catalyst - thiosemicarbazide 6 intensifies and stabilizes color 3. Intensity of the color read at 520 nm is % to cone. of urea in the specimen 4. Oldest and most often used: hydrolysis of urea by urease, quantitated NH4+ produced using coupled enzymatic method, kinetic assay 5. Manual method: used Nesslers reagent 6. Specimens a. Serum b. Urine c. Plasma 7. Reference range for Blood Urea Nitrogen (BUN) 7-18 mg/dL 8. Clinical significance of increased values a. Pre-renal 1) Dehydration or any condition resulting in excess water loss 2) Heart failure (decreased blood flow) 3) Excessive protein destruction b. Renal Kidney disease c. Post-renal urinary obstruction 9. Decreased values are of little clinical significance, except in ending stage of liver disease (pregnancy and infants normally have lower values)

B.

Uric Acid (Enzymatic/Colorimetric) 1. Principle: Uric acid, an end point ofpurine metabolism, reacts with 02 and H20 to form allantoin and hydrogen peroxide (H202). 1 mole of H202 is produced for every mole of uric acid oxidized. Reaction is catalyzed by enzyme uricase 2. Peroxide produced is utilized in an oxidative coupling of 4aminoantipyrine (4AAP) and 3,5- dichloro-2-hydroxybenzene sulfonate (DHBS) to produce red quinoneimine dye (Trinders reaction). Reaction is catalyzed by enzyme peroxidase 3. Intensity of color produced is % to uric acid concentration and can be measured at 520 nm. Procedure is linear to 25.0 mg/dL 4. Caraway method: oxidation reaction with reduction of phosphotungstic acid to tungsten blue, Na Carbonate gives alkaline pH for color change. 5. Reference Values for serum / Heparinized plasma a. Males: 3.5-7.2 mg/dL b. Females: 2.6 - 6.0 mg/dL

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PRECAUTION: In blood samples, hemolysis should be avoided (decreases values)

C.

Creatinine and Creatinine Clearance test (Jaffe reaction) 1. Principle: Creatinine in diluted urine or a protein free supernatant of plasma or serum, is reacted with alkaline picrate to form orange-red color complex whose intensity is measured at 520 nm. Amount of red color produced is % to amount ofcreatinine present. Procedure is linear to 15.0 mg/dL 2. Creatinine clearance: measures amount of plasma, which is theoretically cleared of creatinine per minute by both kidneys. Procedure relies on a comparison of plasma/serum and 24 hr urine creatinine concentrations calculated against normal body surface area 3. Creatinine clearance calculations a. Clearance: 24 hr creatinine output (used to assess completeness of 24-hour collection) mg creatinine = mg urine creatinine/dL X Total vol (in mL) 24 hr 100 b. Creatinine Clearance Formula 1) (U) (V) x 1.73 = ml plasma cleared P A min

c.

Where U = urine concentration (mg/dL) P = serum creatinine cone.(mg/dL) V = urine excreted mL / min* * (TV / 1440) A = body surface area in m2 Body Surface Area - use height and weight nomogram (p. 615) [patient height and weight required] 1.) 1.73 factor normalizes clearance for average body surface area 2.) Use 1.73 by itself if patient height and weight not provided

2)

Calculate: Urine creat =110 mg/dL Plasma creat =1.0 mg/dL 24 hour urine volume = 1600 mL Patient height = 6 ft 0 in Patient weight = 200 Ibs _____ X 1.73 = 4. mL/min

Reference values: a. Serum 1) Males: 0.6-1.2 mg/dL 2) Females: 0.5-1.1 mg/dL b. Urine 1) Males: 14-26 mg/kg/24 h 2) Females: 11 - 20 mg/kg/24 h c. Clearance (age < 40 yrs.) 1) males: 97 - 137 mL/min/1.73 m 2) females: 88-128 mL/min/1.73 m

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Specimen: serum, plasma, or urine. Avoid hemolysis, affected by lipemia and icterus, fasting not required. Jaffe reaction decreased by glucose, uric acid, alpha ketones, and ascorbic acid; measuring kinetically instead of end point can reduce affects; cephalosporins may increase results 5. Clinical significance: a. Serum creatinine 1) Elevated concentrations a) Moderate to severe kidney damage b) Urinary tract obstruction c) Severe muscle damage or disease d) Hyperthyroidism 2) Decreased concentrations a) Muscular dystrophy b) Debilitation b. Urine creatinine 1) Increased conc.s mimics serum creatinine 2) Decreased concentrations a) Anemias b) Leukemia

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