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