Clinical Chemistry

1
Clinical Chemistry

• Renal Assessment

2
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

3
Creatinine serum

• Increased

• Decreased

• Urinary tract obstruction
• Decreased glomerular filtration
• Chronic nephritis

• Muscular dustrophy

4
Creatinine Urine

• Increased

• Decreased

• Muscle Disease

• Kidney Disease

5
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

6
Reference Range Creatinine

• Serum

• Urine

• 0.5-1.5mg/dL

• 0.8-2.0gm/ 24 hour

7
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

8
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

9
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

10
Creatinine Clearance

• Instructions for urine collection

• Specimen requirements

• 24-hour urine
• Keep refrigerated
• Serum/Plasma
• Collected during 24-hour 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

11
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

12
Formula

• Ucr(mg/dL) X V Ur(mL/24 hour) X
  1.73
• P 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

13
Nomogram

1. Left side, find patients height( in feet or
   centimeters)
2. On right side, find patients weight (lbs or kg)
3. Using a straight edge draw a line through the
   points located
4. Read the surface area in square meters, on the
   middle line

14
Reference ranges

• Males
• 97 mL/min- 137 mL/min
• Females
• 88mL/miin-128 ml/min

15
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

16
Microalbumin

• Important in management of diabetes mellitus
• Perform an albumin/creatinine ratio

17
Urinalysis

• In-depth renal assessment
• Refer to UA notes for review of individual tests

18
Other Tests To Monitor Kidneys

• Measurement of the non-protein nitrogen
  substances
• BUN
• Uric Acid

19
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

20
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 kidneys fault, excessive hemorrhage,
  shock, and other reasons
• does not imply clinical illness, but can progress
  to symptomatic illness.

21
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

22
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 )

23
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

24

• 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

25
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

26

• Uric acid diseases
• Gout
• Increased plasma uric acid
• Painful uric acid crystals in joints
• Usually in older males ( gt 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 )

27
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 gt Allantoin
  H2O2 CO2
• (Absorbs at 293 nm)
  (Nonabsorbing at 293 nm)

28
Uric Acid

• Normal values
• Men 3.5 - 7.5 mg/dL
• Women 2.5 - 6.5 mg/dL

29
Laboratory Evaluation of Renal Function
30
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.
• GPEBP 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?

31
Case 1 continued

• Urine protein /Urine creatinine returns
  15mg/150mg ratio(lt0.1)
• 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?

32
Urine ProteinCategories of persistent
proteinuria

• Overflow Capacity to reabsorb normally filtered
  protein in proximal tubules over whelmed due to
  overproductione.g.light chains,hemoglobinuria
  and myoglobinuria
• Tubular proteinuria Decreased reabsorption of
  filtered proteins by tubules due to
  tubulointerstitial damage usually lt2 gm
• Glomerular proteinuria Microalbuminuria to overt
  proteinuria usuallygt3.5 gm

33
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

34
Urine protein Quantitative measurement

• 24 hour collection of urine for protein normal
  excretion is lt150 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 lt0.1 .
  A ratio gt3 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

35
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

36
Detection of Micro albuminuria 24 hour urine
collection

• Normal urine protein excretion lt150mg (20 of
  this is albumin)
• Therefore, normal urinary albumin excretion is lt
  30 mg/day
• Microalbuminuria urinary albumin excretion
  30-300 mg/day

37
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 lt30
  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?

38
Why and When to Screen Patients for
Microalbuminuria ?

• BP control with Ace_I and ARBs 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

39
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?

40
Clinical Assessment of Renal Function Glomerular
Filtration Rate(GFR)

• Parameters used
• Blood urea nitrogen
• Serum creatinine
• Endogenous creatinine clearance

41
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 lt
  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

42
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

43
Creatinine

• Formed at a constant rate by dehydration of
  muscle creatine
• Normally 12 of muscle creatine is broken into
  creatinine
• Mol. Wt. 113
• Creatinine is freely filtered by the glomerulii
  and is not reabsorbed
• 1015 is secreted into proximal tubule

44
(No Transcript)
45
Creatinine

• Normal serum level 12 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

46
BUN/Creatinine ratio 101

• Normal
• Chronic renal failure

47
D/D in Case 3 with BUN Creatinine ratio gt101

• Decreased perfusion
• Hypovolemia
• Congestive heart failure
• Increased urea load
• GI bleed
• Glucocorticoids
• -Tetracycline
• Hyper catabolic states
• High Protein diet
• Obstructive uropathy
• Decreased muscle mass

48
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

49
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 lt1
• Urine Sediment Hyaline casts
• What is the cause of lt 10 1 ,BUN to creat ratio
  now?

50
BUN/Creatinine ratio 101

• Decreased urea load
• Low protein diet
• Liver failure
• Inhibition of creatinine secretion
• Cimetidine
• Trimethoprim
• Probenecid
• Increased removal Dialysis

51
BUN/Creatinine ratio 101

• Increased creatinine load
• Ingestion of cooked meat
• Rhabdomyolysis
• Interference with creatinine measurement
• Ketosis
• Cefoxitin
• Increased muscle mass
• Anabolic steroids
• Muscular development

52
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?

53
Dirty Brown Sediment in ATN
54
Urinary Indices in Diagnosis of Acute Renal
Failure

• 
  Pre renal ATN
• Uosm(mosm/kgH20) gt500 lt350
• Urine sodium (mmol/l) lt20
  gt40
• Urine/plasma urea nitrogen gt8 lt3
• Urine/Plasma Creatinine gt40
  lt20
• Fractional Excretion of Sodiumlt1 gt1
• Sediment normal
  dirty brown

55
Fractional Excretion of filtered Sodium(FeNa)

• FeNa Amount of Na excreted
  Amount of Na filtered
• FeNaUNa 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

56
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 ?

57
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

58
GFR Estimation by Plasma Creatinine

• Cockcroft and Gault Formula
• Calculated creatinine clearance
• (140age) 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

59
GFR Estimation by Plasma Creatinine(Pcr)

• In steady state
• Creatinine excretion creatinine
  productionconstant
• 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

60
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

61
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

62
Reading of renal function
63
Glomerular filtration rate

• Clearance of inulin
• Clearance of creatininenormal range
• Male12025 mL/min
• Female9520mL/min
• Infant17 mL/min/1.73M2

64
PInulin GFR UInulin urine volume
65

• Difference between inulin and creatinine
• Age effect age gt40y/o -gt Ccr decrease 1mL/min/yr
• Urine Cr collection
• Age?60y/omale 20-25mg/kg female15-20mg/kg
• Agegt60y/o10mg/kg

66
Plasma Cr
67
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

68
Estimate Ccr

• Cockcroft and Gault equation CCr(140-age(yr))
  BW(kg) 72Pcr(mg/dl)
• Female above data0.85
• 1/Pcr
• EsGFR(ml/min/1.73M2)KL(body length, cm) Pcr
• K
• LBW0.33
• NB-1yr0.45
• 2yr-adolescent girls 0.55
• 2yr-adolescent boys0.77

69
(No Transcript)
70
BUN

• Reverse relationship with GFR, but many
  confounding factors
• Urea nitrogen can reabsorb paralleling with Na
  and H2O resorption
• BUNPcr 15-201

71
Urinalysis

• Urine sample fresh (30-60min)
• 3000rpm, 3-5min -gt suspension with pellet
• Color

72
(No Transcript)
73
Urine protein

• Daily urinary protein150mg/day
• Microalbuminuria
• Detection dipstick
• Tetrabromophenol blue dye albumin
• Sulfosalicylic acid

74

• Protein(mg/dL) dipstick sulfosalicylic acid
• 0 0 no turbid
• 1-10 trace slight turbid
• 15-30 1 turbid
• 40-100 2 white without ppt
• 150-350 3 white with ppt
• gt500 4 coarse ppt

75
Urine protein

• 24 hr daily protein loss
• Spot UTP/UCr

76
Urine pH and osmolality

• Normal range4.5-8.0
• How about alkalization urine?
• Urine sp. Gr. To estimate urine osmolality
• Plasma osmolality urine osmolality

77
(No Transcript)
78
Urine Na excretion

• Urine excretion intake Na amount
• Urine Nalt20meq/L
• Urine Nagt40meq/L
• Significance of FENa

79
ARF with FENa lt1

• Prerenal factor
• ATN
• Non-oliguric ATN (10)
• Chronic prerenal disease-
• Contrast media
• Sepsis
• Myoglobulinuria or hemoglobulinuria
• AGN or vasculitis
• Obstructive nephropathy

80
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

81
Renal acidification evaluation

• Urinary pH
• Net acid excretion
• Urinary anion gap
• Acidification loading test

82
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.

83
Net acid excretion

• Total acid excretiontitratable acid NH4
• Net acid excretiontotal acid excretion HCO3-
  excretion
• Titratable acid buffer solution of H3PO4 with
  urea nitrogen
• Def. of titratable acid excretionthe amount of
  NaOH(meq) to elevate UpH to 7.4

84
Urinary anion gap

• Total conc. Of anions total conc. Of cations
• NaKNH4Ca2Mg2Cl-H2PO4-SO4-organic
  anions
• NaKNH4Cl-80
• Urinary anion gapNaK-Cl-

85
Urinary acid loading tests

• Acid loading test
• Sodium sulfate infusion test or furosemide test
• Buffer loading test

86
Acid loading test

• NH4Cl 0.1g(1.9meq)/kg, po -gt collection urine pH
  and net acid excretion for 2-8hr.(normal
  UpHlt5.5)
• CaCl2
• Arginine HCL
• Diamox test

• Normal urine CO2gt80mmHg
• U-BPCO2gt30mmHg

87
Increase distal tubule Na conc. Test for proton
pump or voltage-dependent defect

• Furosemide test 1mg/kg, collect urine pH, net
  acid excretion and Uk, po 5hr or iv 3hr
• ReadingUpH increase in 1hr and then UpH down to
  5.5 in future 2-4hrs Uk and acid increase 2
  fold
• Sodium sulfate

88
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
• gt15
• U-BPCO2 gt20-30mmHg, when UHCO3- gt100-150meq/L