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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 2020% 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
P Cr(mg/dL) X 1440 minutes/ 24 hours
1.73
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
Nomogram
1.
2.
3.
4.
Left side, find patient’s height( in feet or centimeters)
On right side, find patient’s weight (lbs or kg)
Using a straight edge draw a line through the points located
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
• Prerenal  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
• Prerenal  BUN ( Not related to renal function )
– Decreased dietary protein
– Increased protein synthesis ( Pregnant women , children )
Disease Correlations: BUN
• Renal causes of  BUN
• Renal disease with decreased glomerular filtration
– Glomerular nephritis
– Renal failure from Diabetes Mellitus
• Post renal causes of  BUN ( not related to renal function )
• Obstruction of urine flow
– Kidney stones
– Bladder or prostate tumors
– UTIs
BUN / Creatinine Ratio
– Normal BUN / Creatinine ratio is 10 – 20 to 1
– Pre-renal increased BUN / Creat ratio
– BUN is more susceptible to non-renal factors
– Post-renal increased ratio BUN / Creat ratio
– Both BUN and Creat are elevated
– Renal 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: Methodology
1. 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 protein
• Dipstick: Gives green color, does not check
for light chains
Negative – 10 mg/dl
Trace – 15-25 mg/dl
1-2+ – 30-100 mg/dl
3+ – 300 mg/dl
Sulfosalicylic 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
Microalbuminuria
• 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
• 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 azotemia
PMH: unremarkable
GPE: BP120/60 , LE edema
Labs: 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 gm
What other tests would you order to diagnose cause
of his renal dysfunction ?
UPEP,why?
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)
• 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
Creatinine
• 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
Creatinine
• Normal serum level 1–2 mg/dl
• 24 hour creatinine excretion
20 mg/kg/day for males
15 mg/kg/day for females
• Children, females, elderly, spinal cord injured
have low serum and urine creatinine
BUN/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
Volume
ADH
+
Renal Hypoperfusion
Diminished GFR
activation of RAS
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 ‹ 10:1
• Decreased urea load
Low protein diet
Liver failure
• Inhibition of creatinine secretion
Cimetidine
Trimethoprim
Probenecid
– Increased removal: Dialysis
BUN/Creatinine ratio ‹ 10:1
• Increased creatinine load
Ingestion of cooked meat
Rhabdomyolysis
• Interference with creatinine measurement
Ketosis
Cefoxitin
• Increased muscle mass
Anabolic steroids
Muscular 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
<350
Urine sodium (mmol/l)
<20
>40
Urine/plasma urea nitrogen
>8
<3
Urine/Plasma Creatinine
>40
<20
Fractional 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)
• 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 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)x70
72x2
*Applicable only when patient is in a steady state,
not edematous and not obese
GFR Estimation by Plasma
Creatinine(Pcr)
• In steady state
Creatinine excretion = creatinine production=constant
Creatinine excretion =Urine creatinine x Urine volume
Filtered creatinine =GFR x Plasma creatinine
As creatinine production is a function of muscle mass
and remains constant
Thus plasma creatinine values vary inversely with GFR
GFR1/2 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:1520mg/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)
0
1-10
15-30
40-100
150-350
>500
dipstick
0
trace
+1
+2
+3
+4
sulfosalicylic acid
no turbid
slight turbid
turbid
white without ppt
white with ppt
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 diseaseContrast 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 – HCO3excretion
• 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 voltagedependent 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-] >100150meq/L