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Clinical Chemistry
Renal Assessment
Creatinine
• Metabolic product cleared entirely by glomerular filtration
• Not reabsorped• In order to see increased creatinine in serum,
50% kidney function is lost• Correlates with muscle mass– Male values higher than females
Creatinine: serum
Increased• Urinary tract obstruction• Decreased glomerular
filtration– Chronic nephritis
Decreased• Muscular dustrophy
Creatinine: Urine
Increased• Muscle Disease
Decreased• Kidney Disease
Creatinine: Methodology• Jaffe reaction– basic reaction for creatinine– Kinetic• Principle: Protein-free filtrate(serum/urine) mixed with
alkaline picrate solution forms a red “tautomer” of creatinine picrate which absorbs light at 520 nm, proportional to the amount of creatinine present• Issues– Subject to interferences from cephalosporins and
alpha-keto acids– Enzymatic• New technology involving coupled reactions
Reference Range: Creatinine
Serum• 0.5-1.5mg/dL
Urine• 0.8-2.0gm/ 24 hour
Clearance Measurements
• Evaluation of renal function relies on waste product measurement, specifically the urea and creatinine
• Renal failure must be severe, where only 20-20% of the nephron is functioning before concentrations of the waste products increase in the blood
• The rate that creatinine and urea are cleared from the body is termed clearance
Clearance
• Definition– Volume of plasma from which a measured
amount of substance can be completely eliminated into urine per unit of time
– Expressed in milliliters per minute
• Function– Estimate the rate of glomerular filtration
Creatinine Clearance
• Used to determine GFR ( glomerular filtration rate)
• Most sensitive measure of kidney function• Mathematical derivation taking into effect the
serum creatinine concentration to the urine creatinine concentration over a 24- hour period
Creatinine Clearance
Specimen requirements• 24-hour urine
– Keep refrigerated
• Serum/Plasma– Collected during 24-hour
urine collection
Instructions for urine collection
• Empty bladder, discard urine, note exact time
• Collect, save and pool all urine produced in the next 24-hours.
• Exactly 24 hours from start time, empty bladder and add this sample to the collection
Creatinine clearance -
Procedure – Determine creatinine level on serum/plasma - in
mg/dL– Determine creatinine level on 24 hour urine• measure 24 hr. urine vol. in mL, take a aliquot• make a dilution (usually X 200)• run procedure as for serum• multiply results X dilution factor
– Plug results into formula
Formula
Ucr(mg/dL) X V Ur(mL/24 hour) X 1.73P Cr(mg/dL) X 1440 minutes/ 24 hours A
• U cr= urine creatinine• P cr= serum creatinine• 1.73= normalization factor for body surface
area in square meters• A= actual body surface area
Nomogram1. Left side, find patient’s height( in feet or centimeters)2. On right side, find patient’s weight (lbs or kg)3. Using a straight edge draw a line through the points located4. Read the surface area in square meters, on the middle line
Reference ranges
• Males– 97 mL/min- 137 mL/min
• Females– 88mL/miin-128 ml/min
Creatinine Clearance Exercise
• Female Patient: 5'6“ & 130 lbs.– Urine Creatinine – 98 mg/dL– Serum Creatinine – 0.9 mg/dL– 24 Hour Urine Volume – 1,200 mL
– Set up calculation
Microalbumin
• Important in management of diabetes mellitus
• Perform an albumin/creatinine ratio
Urinalysis
• In-depth renal assessment• Refer to UA notes for review of individual
tests
Other Tests To Monitor Kidneys
• Measurement of the non-protein nitrogen substances– BUN– Uric Acid
BUN
• Blood urea nitrogen – Urea is the nitrogenous end-produce of protein /
AA metabolism.– Urea is formed in the liver when ammonia (NH3) is
removed and combined with CO2.– Most widely used screening test of kidney
function
Blood urea nitrogen (BUN)
• Serum normal values – 5.0-20.0 mg/dL• Decreased concentration seen late in pregnancy
and in protein starvation.• If concentration exceeds 20.0 mg/dL, term
azotemia applies.– Azotemia – nitrogen in the blood
• not always kidney’s fault, excessive hemorrhage, shock, and other reasons
• does not imply clinical illness, but can progress to symptomatic illness.
BUN: Methodology• Kjeldahl – a classical method for determining urea
concentration by measuring the amount of nitrogen present
• Berthelot reaction - Good manual method - that measures ammonia– Uses an enzyme (urease – from Jack Bean meal) to
split off the ammonia• Diacetyl monoxide ( or monoxime)– Popular method but not well suited for manual
methods• because ➵ Uses strong acids and oxidizing
chemicals
Disease correlations: BUN
• PrerenalPrerenal BUN BUN ( Not related to renal function )
– Low Blood Pressure ( CHF, Shock, hemorrhage, dehydration )– Decreased blood flow to kidney = No filtration– Increased dietary protein or protein catabolism
• PrerenalPrerenal BUN BUN ( Not related to renal function )
– Decreased dietary protein– Increased protein synthesis ( Pregnant women , children )
Disease Correlations: BUN• RenalRenal causes of causes of BUN BUN
• Renal disease with decreased glomerular filtration
– Glomerular nephritis– Renal failure from Diabetes Mellitus
• Post renalPost renal causes of causes of BUN ( not related to renal function ) BUN ( not related to renal function )
• Obstruction of urine flow
– Kidney stones– Bladder or prostate tumors– UTIs
BUN / Creatinine RatioBUN / Creatinine Ratio– Normal BUN / Creatinine ratio is 10 – 20 to 1Normal BUN / Creatinine ratio is 10 – 20 to 1
– Pre-renal increased BUN / Creat ratio– BUN is more susceptible to non-renal factors
– Post-renalPost-renal increased ratio BUN / Creat ratio– Both BUN and Creat are elevated
– RenalRenal decreased BUN / Creat ratio– Low dietary protein or severe liver disease
Increased BUN
Normal Creat
Increased BUN
Increased Creat
Decreased BUN
Normal Creat
Uric acid
• Source– Final breakdown product of nucleic acid
catabolism - from both the food we eat, and breakdown of body cells.
– Uric acid is filtered by the glomerulus ( but 98 – 100 % reabsorbed )• Increased levels
– Not a primary test for kidney function - useful as a confirmatory or back - up test.
– * Most useful for diagnosis and monitoring gout– Also seen during toxemia of pregnancy
Uric acid diseases
• Gout
– Increased plasma uric acid– Painful uric acid crystals in joints– Usually in older males ( > 30 years-old )– Associated with alcohol consumption– Uric acid may also form kidney stones
• Other causes of increased uric acid
– Leukemias and lymphomas ( DNA catabolism )– Megaloblastic anemias ( DNA catabolism )– Renal disease ( but not very specific )
Uric Acid: Methodology1. Phosphotungstic Acid Reduction — This is the classical chemical method for uric
acid determination. In this reaction, urate reduces phosphotungstic acid to a blue phosphotungstate complex, which is measured spectrophotometrically.
2. Uricase Method — An added enzyme, uricase, catalyzes the oxidation of urate to allantoin, H2O2, and CO2. The serum urate / uric acid may be determined by measuring the absorbance before and after treatment with uricase. (Uricase breaks down uric acid.)
3. ACA — Uric acid, which absorbs light at 293 nm, is converted by uricase to allantoin, which is nonabsorbing at 293 nm.– Uric acid + 2H2O + O2 Uricase > Allantoin + H2O2 + CO2
(Absorbs at 293 nm) (Nonabsorbing at 293 nm)
Uric Acid
• Normal values– Men 3.5 - 7.5 mg/dL– Women 2.5 - 6.5 mg/dL
Laboratory Evaluation of Renal Function
Proteinuria Case 1• A 20 year old patient is referred to you for ,he has
been diabetic for 6 years ,he was told to have some kidney problem by his MD.He wants to know the cause of renal dysfunction.
• GPE:BP 145/90 ,otherwise exam is normal• How would you proceed ?• BUN 15mg/dl, creatinine 1.0mg/dl ,U/A shows SG
1.024 ,trace protein ,a few hyaline casts• What test would you order next ?• 24h protein collection , U protein/U creatinine ratio or
both?
Case 1 continued
• Urine protein /Urine creatinine returns 15mg/150mg ratio(<0.1)
• Does this patient have abnormal proteinuria ?• Patient wants to know if he has
microalbuminuria ,you order urine micro albumin result is :60mg micro albumin /gm creatinine .
• Is this abnormal, does this patient have diabetic nephropathy?
Urine Protein:Categories of persistent proteinuria
• Overflow: Capacity to reabsorb normally filtered protein in proximal tubules over whelmed due to overproduction:e.g.light chains,hemoglobinuria and myoglobinuria
• Tubular proteinuria: Decreased reabsorption of filtered proteins by tubules due to tubulointerstitial damage ; usually <2 gm
• Glomerular proteinuria: Microalbuminuria to overt proteinuria usually>3.5 gm
Screening for Urine proteinScreening for Urine protein
• Dipstick: Gives green color, does not check for light chainsNegative – 10 mg/dlTrace – 15-25 mg/dl
1-2+ – 30-100 mg/dl3+ – 300 mg/dlSulfosalicylic acid: white precipitate
Urine protein :Quantitative measurement
24 hour collection of urine for protein normal excretion is <150 mg/24 hour
Spot urine protein/urine creatinine ratio : (as 24 h urine creatinine excretion is a function of muscle mass i.e. 15 mg/kg for females and 20mg/kg for males ) a normal ratio is 150/1500 or <0.1 . A ratio >3 indicates nephrotic range proteinuria
Case 1 has normal urine protein excretion, trace protein on u/a is due to highly concentrated urine ,pt may still have microalbuminuria
MicroalbuminuriaMicroalbuminuria
• Urine albumin excretion below detection by regular dipstick
• First clinical sign of diabetic nephropathy• Incidence increases with the duration of
diabetes and may be present at the diagnosis of NIDDM
• Transient albuminuria may occur with fever,infection,exercise,decompensated CHF
• Associated with poor glycemic control and elevated BP
Detection of Micro albuminuria: 24 hour urine collection
Detection of Micro albuminuria: 24 hour urine collection
• Normal urine protein excretion : <150mg (20% of this is albumin)
• Therefore, normal urinary albumin excretion is < 30 mg/day
• Microalbuminuria :urinary albumin excretion 30-300 mg/day
Microalbuminuria :Detection by Spot Urine Albumin to Urine Creatinine ratio
• Easier than cumbersome 24 hr.collection• If we assume daily creatinine excretion to be 1000
mg and normal urine albumin excretion <30 mg; albumin / creatinine ratio should be less than 0.03 or 30mg/g creatinine
• Thus case 1 has micro albuminuria which is likely due to diabetic nephropathy.How would you manage him now?
Why and When to Screen Patients for
Microalbuminuria ? • BP control with Ace_I and ARB’s have been
known to reduce microalbuminuria and delay the progression of kidney disease in diabetics
• IDDM patients should be screened yearly,beginning 5 years after the onset of disease
• Patients with NIDDM should be screened at presentation
Proteinuria Case 2
A70 year- old male is referred for chronic azotemiaPMH: unremarkableGPE: BP120/60 , LE edemaLabs: U/A SG 1.010 pH 6.0 , protein neg, glucose 2+,
Uprotein /U creatinine ratio 4 BUN 30mg/dl creat.3.0, Blood Sugar 78mg/dl albumin
2.8, Hb 10 gmWhat other tests would you order to diagnose cause
of his renal dysfunction ? UPEP,why?
Clinical Assessment of Renal Function: Glomerular Filtration Rate(GFR)
Clinical Assessment of Renal Function: Glomerular Filtration Rate(GFR)
• Parameters used Blood urea nitrogen Serum creatinine Endogenous creatinine clearance
Case 3 Azotemia • A 55 year old diabetic female is admitted with intractable
vomiting and low urine output• Exam: BP 120/60 with postural hypotension• Labs: BUN 60, Creat. 2.0 mg/dl ( baseline 1.0mg/dl), Hb
16gm• ,U/A: SG 1.020, sediment: hyaline casts,UNa: 10
mmol/L,UOsm: 600 mosm/kg,Ucreat.150mg/dl ,Fe Na < 0.5
• Q.What is the cause of her high BUN to creatinine ratio and her renal failure? What are the other causes of high BUN to creatinine ratio
Blood Urea Nitrogen (BUN)Blood Urea Nitrogen (BUN)• Catabolism of aminoacids generates NH3
NH2
2 NH3 + CO2 = C = 0 + H2O NH2
• Urea Mol wt : 60• BUN Mol wt. : 28• Normal BUN 10-20 mg/dl• After filtration › 50% is reabsorbed by the tubule• BUN level is related to: Renal function, protein
intake, and liver function
CreatinineCreatinine
• Formed at a constant rate by dehydration of muscle creatine
• Normally 1–2% of muscle creatine is broken into creatinine
• Mol. Wt. 113• Creatinine is freely filtered by the glomerulii
and is not reabsorbed 10–15% is secreted into proximal tubule
CreatinineCreatinine
• Normal serum level 1–2 mg/dl• 24 hour creatinine excretion
20 mg/kg/day for males15 mg/kg/day for females
• Children, females, elderly, spinal cord injured have low serum and urine creatinine
BUN/Creatinine ratio 10:1BUN/Creatinine ratio 10:1
• Normal• Chronic renal failure
D/D in Case 3 with BUN Creatinine ratio >10:1
• Decreased perfusion»Hypovolemia»Congestive heart failure
• Increased urea load–GI bleed–Glucocorticoids
-Tetracycline–Hyper catabolic states–High Protein diet
• Obstructive uropathy• Decreased muscle mass
Pathophysiology of Pre-renal Azotemia in Case 3
Decreased “Effective” Intravascular ADH
Volume +
Renal Hypoperfusion activation of RAS Diminished GFR aldosterone
Low urine volume and U sodium and high Uosmolality
Case 3 :Diabetic patient continued..
• Vomiting stopped ,BP improved and BUN/creat lowered to 35/1.8mg/dl. 24 hours later she developed UTI, trimethaprim/sulfamethoxazole was started
• Next day 24 hr urine output 800 mL• Exam: Unremarkable• BUN: 20 mg/dl Creat: 3.0 mg/dl • Uosm: 600 mosm/kg ,UNa: 10 mom/l, FeNa: <1%• Urine Sediment: Hyaline casts• What is the cause of < 10: 1 ,BUN to creat ratio now?
BUN/Creatinine ratio ‹ 10:1BUN/Creatinine ratio ‹ 10:1
• Decreased urea loadLow protein dietLiver failure
• Inhibition of creatinine secretionCimetidineTrimethoprim Probenecid– Increased removal: Dialysis
BUN/Creatinine ratio ‹ 10:1BUN/Creatinine ratio ‹ 10:1
• Increased creatinine loadIngestion of cooked meatRhabdomyolysis
• Interference with creatinine measurementKetosisCefoxitin
• Increased muscle massAnabolic steroidsMuscular development
Case 3 continued… 6 months later
• Pt was discharged with normal BUN and creatinine,6 months later she is admitted with vague abdominal pain, an US done shows 6 cm abdominal aortic aneurysm, she undergoes resection with cross-clamping of aorta for 2 hours.
• Post surgery she is oliguric (u/o less than 70ml in 8 hours).On exam well hydrated.
• U/A: SG 1.015 ,”Dirty brown sediment “U Na 40 mEq /L U osmolality 350 mOsm/l ,Fe Na 2%
• What is your diagnosis after reviewing the lab data ? How would you manage?
“Dirty Brown” Sediment in ATN
Urinary Indices in Diagnosis of Acute Renal Failure
Pre renal ATN Uosm(mosm/kgH20) >500 <350Urine sodium (mmol/l) <20 >40Urine/plasma urea nitrogen >8 <3Urine/Plasma Creatinine >40 <20Fractional Excretion of Sodium<1% >1%Sediment normal “dirty brown”
Fractional Excretion of filtered Sodium(FeNa)
• FeNa= Amount of Na excreted Amount of Na filtered
• FeNa=UNa x Urine volume PNa x GFR
• FeNa = UNa x V PNa x[(UCr x V) /PCr]
• FeNa % =UNa x PCr X 100 PNa x UCr
Case 4
• 20 y/o male is seen at West point ,on admission physical : wt 70Kg , BUN 10mg/dl, serum creatinine 1.0mg/dl, GFR was 100ml/min and he excreted 1500mg creatinine /day in the urine. 2 months later he develops acute glomerulonephritis with RBC and fatty casts.His serum creatinine increases to 2mg/dl and remains at 2mg/dl at 1 year follow up .Wt is 72kg
• What is his estimated GFR by Cockcroft and Gault formula and by serum creatinine?
• What would be the creatinine excretion now at 1 year ?
Concept of Clearance ? Measurement of GFR by Creatinine
Clearance(Ccr)
Concept of Clearance ? Measurement of GFR by Creatinine
Clearance(Ccr)
• Urine is collected for 24 hours and plasma creatinine is measured the next day
• 1. Filtered creatinine = Excreted creatinine• 2. GFR x Pcr = Ucr x Volume• 3. GFR = Ucr. mg/dl x V ml Pcr.mg/dl• Normal GFR = 100 ml/min• GFR declines by 1 ml/min/year after age 40
GFR Estimation by Plasma CreatinineGFR Estimation by Plasma Creatinine
Cockcroft and Gault Formula*Calculated creatinine clearance = (140–age) x wt (kg)72 X serum creatinine(mg/dl)
For females, subtract 15% (or multiply by 0.85); for paraplegics multiply by 0.8, for quadriplegics, multiply by 0.6
Est GFR for this pt is ..(140-20)x7072x2
*Applicable only when patient is in a steady state, not edematous and not obese
GFR Estimation by Plasma Creatinine(Pcr)
GFR Estimation by Plasma Creatinine(Pcr)
• In steady stateCreatinine excretion = creatinine production=constantCreatinine excretion =Urine creatinine x Urine volume
Filtered creatinine =GFR x Plasma creatinineAs creatinine production is a function of muscle mass
and remains constantThus plasma creatinine values vary inversely with GFRGFR1/2 X 2 Pcr = GFR x Pcr = constant
• A rise in Pcr almost always represents a fall in GFR
In case 4 ,serum creatinine increased from from 1 to 2 mg/dl and remained at that
level, his 24urine creatinine will remain the same
• Another example :70 kg man with serum creat. of 1 mg/dl and GFR of 100 ml/min was excreting 1500 mg creatinine/day,if you remove his one kidney , next day his GFR will be 50ml/min,urine creatinine excretion will be 750 mg /day.Over the next few days creatinine will accumulate in the blood and level will increase to 2 mg /dl and thus filtered and excreted amount will be the same
Summary
• How to evaluate a patient with renal disease• How to interpret u/a,urine protein to creatinine
ratios• Interpretation of urea nitrogen and creatinine ratios• Estimation and measurement of GFR& to see when
a patient would need renal replacement therapy• Interpret urine indices in evaluation of various
causes of ARF
Reading of renal function
Glomerular filtration rate
• Clearance of inulin• Clearance of creatinine:normal range– Male:120±25 mL/min– Female:95±20mL/min– Infant:17 mL/min/1.73M2
P[Inulin] × GFR = U[Inulin] × urine volume
• Difference between inulin and creatinine• Age effect: age >40y/o -> Ccr decrease
1mL/min/yr• Urine Cr collection:– Age 60y/o:male: 20-25mg/kg; ≦
female:15-20mg/kg– Age>60y/o:10mg/kg
Plasma Cr
Condition associated with PCr increased and not changed GFR
• Increased Cr production– Rhabdomyolysis– Meat
• Decreased Cr excretion– Cimetidine, triamterene, probenecid, amiloride,
trimethoprim, spironolactone• Measured bias– Endogeneous: ketone, ketoacids, glucose, bilirubin,
urate, urea, fatty acid– Exogeneous: cephalosporines, 5-FU, phenylacetyl urea,
acetoheximide
Estimate Ccr
• Cockcroft and Gault equation: CCr=[(140-age(yr)) ×BW(kg)] ÷[72×Pcr(mg/dl)]
• Female: above data×0.85• 1/Pcr• EsGFR(ml/min/1.73M2)=KL(body length, cm) ÷ Pcr– K
• LBW:0.33• NB-1yr:0.45• 2yr-adolescent girls: 0.55• 2yr-adolescent boys:0.77
BUN
• Reverse relationship with GFR, but many confounding factors
• Urea nitrogen can reabsorb paralleling with Na and H2O resorption
• BUN: Pcr = 15-20:1
Urinalysis
• Urine sample: fresh (30-60min)• 3000rpm, 3-5min -> suspension with pellet• Color
Urine protein
• Daily urinary protein:150mg/day• Microalbuminuria• Detection: dipstick– Tetrabromophenol blue dye –albumin– Sulfosalicylic acid
Protein(mg/dL) dipstick sulfosalicylic acid0 0 no turbid1-10 trace slight turbid15-30 +1 turbid40-100 +2 white without ppt150-350 +3 white with ppt>500 +4 coarse ppt
Urine protein
• 24 hr daily protein loss• Spot UTP/UCr
Urine pH and osmolality
• Normal range:4.5-8.0• How about alkalization urine?• Urine sp. Gr. To estimate urine osmolality• Plasma osmolality & urine osmolality
Urine Na excretion
• Urine excretion = intake Na amount• Urine [Na]<20meq/L• Urine [Na]>40meq/L• Significance of %FENa
ARF with %FENa <1%
• Prerenal factor• ATN– Non-oliguric ATN (10%)– Chronic prerenal disease-– Contrast media– Sepsis– Myoglobulinuria or hemoglobulinuria
• AGN or vasculitis• Obstructive nephropathy
Urinary cast
Hyaline cast conc. Urine or diuretics
Red cell cast GN or vasculitis
WBC cast TIN, APN, GN
Epithelial cast ATN, GN
Fatty cast GN with proteinuria, NS
Granular cast proteinuria, degenerative cells
Waxy cast CRF
Renal acidification evaluation
• Urinary pH: • Net acid excretion:• Urinary anion gap:• Acidification loading test:
Urine pH
• Fresh urine• Collect in the morning• Must rule out UTI• Many confounding factors- proton pump,
electro-gradient of membrane, buffer conc., diet, et. al.
Net acid excretion
• Total acid excretion=titratable acid + NH4+
• Net acid excretion=total acid excretion – HCO3-
excretion• Titratable acid= buffer solution of H3PO4 with urea
nitrogen• Def. of titratable acid excretion:the amount of
NaOH(meq) to elevate UpH to 7.4
Urinary anion gap
• Total conc. Of anions = total conc. Of cations
• Na++K++NH4++Ca+2+Mg+2=Cl-+H2PO4
-+SO4-
+organic anions• Na++K++NH4
+=Cl-+80
• Urinary anion gap:Na++K+-Cl-
Urinary acid loading tests
• Acid loading test• Sodium sulfate infusion test or furosemide
test• Buffer loading test
Acid loading test
• NH4Cl 0.1g(1.9meq)/kg, po -> collection urine pH and net acid excretion for 2-8hr.(normal: UpH<5.5)
• CaCl2
• Arginine HCL• Diamox test
Normal urine CO2>80mmHg
U-B[PCO2]>30mmHg
Increase distal tubule Na conc. Test – for proton pump or voltage-
dependent defect• Furosemide test: 1mg/kg, collect urine pH, net
acid excretion and U[k], po 5hr or iv 3hr– Reading:UpH increase in 1hr and then UpH down
to 5.5 in future 2-4hrs; U[k] and acid increase 2 fold
• Sodium sulfate
Buffer loading test
• IV drip or 2-3ml/min NaHCO3 100-150mEq(total) till plasma NaHCO3 30meq/L≧– Then check blood and urine pH, [HCO3
-], CO2
– Calculate %FEHCO3-
• 3-5%
• >15%
– U-B[PCO2] >20-30mmHg, when U[HCO3-] >100-150meq/L