Quick Compendium of CP: Chemistry

1
Quick Compendium of CP Chemistry

• Enzymes, Serum Proteins, Acid-Base and
  Electrolytes
• ZW 7/7/08

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Enzymes Basics

• Michaelis-Menton kinetics
• The rate of enzyme activity varies linearly with
  substrate concentration up to the point that the
  enzyme is fully saturated with substrate
• At this point, enzyme working as fast as it can
  (Vmax)
• Rate of reaction at this point varies only with
  enzyme concentration

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Michaelis-Menton Kinetics
4
Enzymes

• Can measure enzyme concentration by
• Using excess of substrate
• Or, measure whether a reaction has taken place
• Measure reaction products
• NAD to NADH to NAD (NADH absorbs light at 340nm,
  NAD does not)

5
Coupled Enzyme Assay (NAD/NADH)

• Aspartate (Asp) a-ketoglutarate ? oxaloacetate
  (OAA) glutamate
• Does not utilize NADH
• Aspartate (Asp) a-ketoglutarate ? oxaloacetate
  (OAA) glutamate NADH ?malate NAD
• Add excess NADH and aKG, along with catalysts AST
  and MD
• The disappearance of NADH (absorbance at 340nm)
  can be used as a reflection of AST (enzyme at
  first arrow)

6
Measurement of Enzyme Antigen

• The quantity of enzyme determined by immunoassay
  corresponds to the enzyme ACTIVITY
• Discordance between concentration and ACTIVITY
  usually takes the form of the immuno assay
  overestimating the activity
• May be due to serum enzyme inhibitors
• Deficiency in necessary cofactor
• Defective enzyme
• Proteolytically inactivated enzymes

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Cofactors, Coenzymes

• Cofactors
• Substances that bind to an enzyme and enhance
  activity
• Include inorgantic cofactors like zinc, calcium,
  magnesium, iron
• Organic also called coenzymes
• Coenzymes
• Organic cofactors, include NAD, protein S,
  pyridoxine (vit B6)

8
Macroenzymes

• Ordinary enzymes bound to antibodies
• Has 2 effects
• Makes it incapable of functioning
• Prevents it from being cleared from blood

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Competitive INHIBITION
10
Non-competitive Inhibition
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Uncompetitive Inhibition
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Enzyme Units

• International Unit (IU)
• The amount of enzyme that catalyzes the
  conversion of 1 micromole of substrate per minute
• Katal
• 1 katal the amount of enzyme that catalyzes the
  conversion of 1 Mole of substrate per second
• 1IU 16.7 katals

13
Hepatic Enzymes

• Liver transaminases
• AST and ALT
• AST
• Cardiac muscle, liver, skeletal muscle, kidney,
  brain, lung, pancreas (in descending order)
• Found within cytoplasm (20)and mitochondria
  (80)
• ALT
• MORE SPECIFIC FOR LIVER, confined to liver and
  kidney
• Found entirely within cytoplasm

14
Hepatic Enzymes

• In children
• AST activity is slightly higher than ALT
• Pattern reverses at age 20
• In adults, AST activity a little lower than ALT
• May reverse with old age
• Both AST/ALT activities higher in adult males
  over females, and in African-Americans
• Hemolysis raises AST/ALT

15
Hepatic Enzymes

• Intra-individual variation more significant for
  ALT than AST
• Marked diurnal variation (highest in afternoon)
  and day-to-day variation up to 30
• Both AST/ALT elevated in heparin therapy to
  around 3X baseline
• In renal failure, both significantly lower than
  in healthy individuals

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Hepatic Enzymes

• Lactate dehydrogenase (LDH)
• Present in numerous tissues, traditionally
  separated into 5 isoenzymes by electrophoresis
• Fastest moving are LD1 and LD2
• Found in heart, RBC, kidney
• Slower moving are LD4 and LD5
• LIVER and skeletal muscle
• LD3 in lung, spleen, lymphocytes, and pancreas
• LD6- sixth LD is sometimes seen migrating
  cathodal to LD5
• PRESENCE THOUGHT TO BE A DIRE FINDING (hepatic
  insufficiency in setting of cardiovascular
  collapse)

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LDH

• Concentrations
• LD2gtLD1gtLD3gtLD4gtLD5
• LD 1 elevation (with flipped LD ratio LD1gtLD2)
• Acute MI
• Hemolysis
• Renal infarction
• Elevated LD4 and LD5
• LIVER DAMAGE or skeletal insult
• Elevated LD1 and LD5
• Acute MI with liver congestion
• Chronic alcoholism

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Alkaline phosphatase

• Two types of phosphatases
• Alkaline (optimum pH is 9)
• Bone, bile ducts, intestine, placenta
• Separate reference ranges for women and children
• Acid (optimum pH is 5)
• Found in prostate, RBC, and bone
• RBC acid phosphatase is susceptible to inhibition
  by 2 formaldehyde and resistance to inhibition
  by tartrate (this is also seen in hairy cell
  leukemia)

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Alkaline phosphatase

• 4 isoenzymes by electrophoresis
• Each displays characteristic degrees of
  inactivation by heating, urea incubation, and
  l-phenylalanine
• Heating produces significant inactivation of bone
  alk phos (bone burns), 50 inactivation of
  biliary alk phos, and NO inactivation of
  placental alk phos.

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Alkaline phosphatase

• Biliary alk phos
• Most sensitive marker of hepatic metastases
• Bone alk phos
• Produced by osteoBLASTS and reflects bone
  reforming activity
• Highest levels seen in Pagets disease of bone
• A specific immunoassay for bone alk phos available

21
Regan Isoenzyme

• Observed in about 5 of individuals with
  carcinoma
• Appears identical to placental alkaline
  phosphatase

22
Intestinal Alk Phos

• Elevation
• Can be factitious in non-fasting individuals,
  particularly in Lewis positive type B or O pts
• Ingesting a meal can elevate alk phos by 30 in
  2-12 hours
• Repeat fasting alk phos

23
Alk Phos

• Minor elevations are a common clinical problem
• Usually higher in men than women
• Higher in African-Americans
• Threshold of 1.5 times normal limit for further
  investigation (repeat in 6 months if borderline)
• Causes
• Pregnancy, CHF, hyperthyroidism, drugs

24
Alkaline phosphatase

• Sensitive indicator for hepatic metastases
• In women, investigation should include assay for
  anti-mitochondrial antibodies

25
Gamma-glutamyl transferase (GGT)

• GGT
• best test to confirm if elevate alk phos if of
  biliary tree origin
• Found in biliary epithelial cell, particularly
  those of the small interlobular bile ducts and
  ductules
• Exquisitely sensitive to biliary injury
• Also elevated in
• Steatosis, diabetes, hyperthyroidism, RA, acute
  MI, COPD

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GGT

• Present within the smooth endoplasmic reticulum
  of hepatocytes
• Whenever there is induction due to excess toxin,
  GGT levels increase
• This includes warfarin, barbiturates, dilantin,
  valproic acid, methotrexate, EtOH
• 2-3X normal limit in heavy drinkers
• Returns to normal after 3 weeks abstinence and
  can be followed as marker for alcohol consumption

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5 Nucleotidase

• Main source is biliary epithelium
• Levels highest in cholestatic conditions
• Another test to confirm if elevated alk phos is
  due to hepatobiliary disease
• Low sensitivity, best as confirmatory test,
  utility less than GGT

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Ammonia

• Hyperammonemia nearly always due to liver failure
• In children, it should raise the suspicion for
  an INBORN ERROR IN METABOLISM
• Sources of ammonia
• Skeletal muscle and gut
• Bacteria in GI tract produce ammonia
• Normally functioning liver removes this ammonia
  and discards it in the form of urea which is
  excreted in urine

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Ammonia

• Blood ammonia can become disastrously high when
• Too much collateral circulation
• Excess protein in gut (excess hemoglobin from
  variceal bleed)
• SIGNIFICANT HEPATOCYTE DISFUNCTION
• In cirrhotic patients, these conditions are often
  met, and neurotoxicity can result

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AMMONIA

• Measurement requires a FRESH specimen which has
  been CHILLED during transport and has undergone
  NO hemolysis
• Smoking patients must abstain for several hours
  before draw
• Anyone care to comment on the current state of
  the ammonia level as it can or cannot be ordered?

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Bilirubin

• Unconjugated (indirect) bilirubin
• Water-insoluble form produced by breakdown of
  heme
• Taken to liver tightly bound to albumin where it
  under goes glucuronidation to produce
  water-soluble (as in bile) conjugated (direct)
  bilirubin
• Conjugated bili excreted in bile where intestinal
  bacteria convert to urobilinogen

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Bilirubin

• Urobilinogen ends up in feces, some of which is
  reabsorbed and excreted in urine
• Some urobilinogen is converted by colonic
  bacteria into brown pigments (complete biliary
  obstruction leads to yellow-white stool- the
  Silver Stool of Thompson)

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Bilirubin

• Unconjugated bilirubin, even when it is quite
  high, does NOT appear in urine
• Thus, bilirubinuria indicates CONJUGATED
  hyperbilirubinemia
• 2 test methods
• Diazo-colorimetric methods
• Rely on formation of colored dye through reaction
  of bili with diazo compound
• Without the addition of an accelerator (alcohol),
  only conjugated bilirubin is measured
• Addition of accelerators measures combined unconj
  and conjugated (total) bilirubin
• Direct spectrophotometry
• Bilirubin concentration measured by absorbance
  (455nm)

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Causes of Hyperbilirubinemia

• Unconjugated
• Hemolysis (extravascular)
• Blood shunting (cirrhosis)
• Right heart failure
• Gilbert syndrome
• Drugs rifampin
• Crigler-Najjar syndrome
• Hypothyroidism

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Causes of Hyperbilirubinemia

• Conjugated
• Dubin-Johnson syndrome
• Hepatitis
• Endotoxin (sepsis)
• Pregnancy (estrogen)
• Drugs estrogen, cyclosporine
• Mechanical obstruction
• PBC, PSC, tumor, stricture, stone

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Additional Hepatic Function Tests

• PT
• Factor VII has half life of 12 hours (ON RISE
  EXAM)
• Sensitive marker for impaired hepatic synthetic
  function
• Impaired bile secretion can lead to Vit K
  deficiency (bile salts required for absorption)
• How do you distinguish between a prolonged PT
  because of cholestasis/impaired Vit K absorption
  and hepatocyte injury?
• Add parental vit K
• Gammaglobulins
• Serum gammaglobulins elevated in liver injury,
  especially autoimmune

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Neonatal Jaundice

• Most cases of neonatal jaundice are entirely
  benign (physiologic jaundice)
• Hepatic enzymes not yet at full capacity leading
  to build-up of unconjugated bilirubin
• Usually noted between days 2-3 of neonatal life
• Usually peaks at 4-5 days rarely exceeds 5-6
  mg/dL

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Severe hyperbilirubinemia in neonates

• Most common causes
• Hemolytic disease of the newborn (HDN)
• Sepsis
• Poorly developed blood-brain barrier causes
  unconjugated bili to pass to CNS and cause damage
  (kernicterus)

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When to worry about neonatal jaundice?

• Appearance in first 24 hours of life
• Rising bili beyond 1 week
• Persistance of jaundice past 10 days
• Total bili that exceeds 12 mg/dL
• Single-day increase of gt5 mg/dL
• Conjugated bili (direct) that exceeds 2 mg/dL

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Therapy for neonatal jaundice

• Phototherapy
• Consider when bili exceeds 10 mg/dL before 12
  hours of age 12 mg/dL before 18 hours of age 14
  mg/dL before 24 hours of age
• Phototherapy converts unconj bili to a molecule
  that can be excreted WITHOUT conj
• Not useful for conj hyperbili
• Exchange transfusion
• When bili exceeds 20 mg/dL

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DDX of neonate hyperbili

• Jaundice in 1st 24 hours
• Erythroblastosis fetalis
• Concealed hemorrhage
• Sepsis
• TORCH infection
• Jaundice between 3rd and 7th day
• Bacterial sepsis (usually UTI origin)
• Arising after 1st week
• Breast milk jaundice, sepsis, extrahepatic
  biliary atresia, cystic fibrosis, congenital
  paucity of bile ducts (Alagille syndrome),
  neonatal hepatitis, glactosemia, inherited
  hemolytic anemia (PK def, hered. Spherocytosis,
  G6PD def)

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Lab Eval of Acute Liver Injury

• May be symptomatic, Jaundice, Elevated
  transaminases
• May be due to viral hepatitis (HAV, HBV, HCV),
  autoimmune hep, toxin, drug, ischemia, or Wilson
  disease
• Labs
• Hepatitis serologies, ANA, ceruloplasmin,
  clinical history (new drugs usually cause damage
  within 4 months of starting)

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Acute liver injury labs

• Acute viral hepatitis due to HAV, HBV most often
  leads to complete recovery
• Acute HCV goes to chronic HCV in gt80 of cases
• Serologic testing for HAV, HBV are very
  dependable for diagnosing acute infx (IgM
  anti-HAV, IgM anti-HBc, HBsAg)
• Anti-HCV test only about 60 sensitive for acute
  infx
• HCV RNA testing 90-95 sensitive

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Transaminases

• Acute hepatic injury due to ischemic or toxic
  injury produce PROFOUND elevations in
  transaminases- often gt100X upper limit of normal
  (RARE in acute hepatitis)
• AST gt 3,000 U/L toxin in 90 of cases
• AST 10X upper limit of normal in acute viral
  hepatitis, but reaches this level RARELY in
  alcoholic hepatitis
• ASTALT ratio is over 2 in 80 of pts with toxic,
  ischemic, and EtOH hepatitis (lt1 in viral hep)
• Amount of transam elevation poorly correlates
  with LEVEL of injury

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PT/BILIRUBIN

• PT (protime)-
• Probably the best indicator of prognosis in acute
  hepatic injury
• gt4.0 secs indicates severe injury/unfav prog
• BILI
• Jaundice in 70 of pts with EtOH, HAV
• Jaundice in lt20 of pts with HBV, HCV
• Jaundice rare in kids with acute viral hep, rare
  in toxic or ischemic injury
• gt15 mg/dL indicates severe liver injury, bad
  prognosis

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Pancreatic Enzymes

• AMYLASE
• Serum amylase salivary and pancreatic
  isoenzymes
• On electrophoresis? 6 bands result
• 1st three are salivary
• Slower 3 are pancreatic
• Can be separated by inhibition as well
• Salivary amylase sensitive to inhibition by wheat
  germ lectin (treticum vulgaris)
• Assays based on monoclonal Abs directed against
  specific isoenzymes are very accurate

47
Serum Amylase

• Rises within 2-24 hours of onset of acute
  pancreatitis
• Returns to normal in 2-3 days
• Higher levels dont correlate with severity
• Higher levels are more specific for acute
  pancreatitis
• Persistance in elevation suggests complication
  like pseudocyst

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Urine amylase

• Nearly all pts have concomitant increase in urine
  amylase
• Amylase primarily cleared by glomeruli
• Renal insufficiency spurious amylase elevation
• Fractional excretion of compound (x) FEx

49
Amylase

• Sensitivity of serum amylase for acute
  pancreatitis is 90-98
• Specificity is only around 70-75
• Specificity of urine amylase and FEamylase is
  higher

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Additional causes of increased amylase

• Diabetic ketoacidosis, peptic ulcer dz, acute
  cholecystitis, ectopic pregnancy, salpingitis,
  bowel ischemia, intestinal obstruction,
  macroamylasemia, and renal insufficiency, opioid
  analgesics (contraction of sphincter of oddi)

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Pancreatic Enzymes

• LIPASE
• Unlike amylase, essentially specific for pancreas
• Rise parallels rise in amylase, but remains
  elevated for 14 days
• Less reliant on renal clearance than amylase
• Often considered superior to amylase in dx of
  acute pancreatitis

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Lab Eval for Acute Pancreatitis

• Amylase limited in sensitivity and specificity
• Hypertriglyceridemia (common cause of acute
  pancreatitis) interferes with amylase assay
  (false negative)
• Lipase remains elevated longer giving greater
  sensitivity
• Others trypsinogen-2 and elastase-1 (not used
  often) but have excellent negative predictive
  value

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Acute Pancreatitis Prognosis

• Ranson Criteria
• Aggressive management
• ICU admission
• Parenteral feeding
• Systemic antibiotics
• Provides specificity of 90
• Cannot be assigned until 48 hours after admission
• Serum amylase/lipase are poor predictors of
  outcome

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Etiology of Acute Pancreatitis

• Stones
• EtOH
• Viruses
• Inherited diseases
• Mutations in
• Cationic trypsinogen (PRSS-1)
• Pancreatic secretory trypsin inhibitor (PSTI)
• Cystic fibrosis transmembrane conductance
  regulator (CFTR)

55
Pancreatic EXOCRINE function

• Tests include
• Secretin-cholecystokinin (secretin CCK, secretin
  pancreozymin), fecal elastase-1, fecal fat
• FECAL FAT
• Oil-red O stain and 72 hour fecal fat quant
• Stain has sensitivity of 70
• Fecal fat quant quite sensitive for panc. Insuff.

56
Myocardial Enzymes

• CK (creatine kinase)
• Three isoenzymes distinguishable by
  electrophoresis
• CK-MM, CK-MB, CK-BB
• Fastest migrating is BB (CK1), then MB (CK2),
  then MM (CK3)
• CK-BB found primarily in brain, with lesser
  amounts in bladder, stomach, and prostate.
• CK-MM (CK3) found in skeletal muscle and cardiac
  muscle (Skel.muscle 99 MM, cardiac 70MM). In
  normal subjects, serum CK is 100 MM
• CK-MB (CK2) found in cardiac and skeletal muscle
  (card 30, skel 1), skeletal muscle is source
  of nearly all MB in circulation in serum

57
CK

• Now use immunoassays to measure CK
• Much faster and more accurate than
  electrophoresis (particularly in low/clinical
  range)
• Total CK? enzymatic assay
• Ratio of CK-MB to total CK (RI relative index)
  adds to ability to distinguish MI
• RI of 2 is usually cut-off

58
Troponin I

• Group of enzymes consisting of
• Troponin T (TnT)
• Troponin I (TnI)
• Troponin C (TnC)
• Involved in mediating the actin-myosin
  interactions that result in muscle contraction
• Immunoassays distinguish cardiac troponins (cTnI
  and cTnT) from skeletal muscle troponins

59
Troponin

• Vast majority of cardiac muscle troponin is bound
  to actin/mysosin
• Very small amount free in cytoplasm
• So
• Immediate release of cytoplasmic troponin in MI
  (4-8 hours)
• And sustained release of bound troponin over next
  10-14 days

60
Cardiac Troponin I

• Only cTnI is currently widely available for use
  in clinical diagnosis
• cTnT marginally less cardiospecific
• cTnI NOT elevated in skeletal muscle injury
• cTnI may be elevated in other forms of cardiac
  muscle injury(contusion, myocarditis)

61
Myoglobin

• Mgb is THE MOST SENSITIVE of the cardiac markers
• Earliest marker of acute MI
• Myoglobin should be elevated as soon as an
  infarcting patient present to ED
• LEAST CARDIOSPECIFIC of cardiac markers!

62
Ischemia-modified albumin (IMA)

• When albumin circulates through harsh
  environments (acidosis, hypoxemia, free radicals,
  altered calcium- all seen in ischemia), its
  ability to rapidly and tightly bind cobalt is
  altered
• Altered Cobalt Binding (ACB) assay
• Add cobalt, measure unbound amount
• Measurement reflects ischemia modified albumin

63
B-type Natriuretic Peptide (BNP)

• Natriuretic peptides
• Cause vasodilation and sodium excretion
• A-type stored in granules in atrial myocytes,
  affected by atrial filling pressure, ventrilar
  wall tension
• B-type synthesized in ventricular myocytes
• Correlates directly with ventricular wall tension
• N-terminal peptide fragment (N-terminal pro-BNP)
  is cleaved from pro-BNP to make active hormone
  BNP
• N-terminal pro-BNP is more stable, provides more
  longitudinal info
• Elevated in heart failure
• Distinguishes between cardiac/non-cardiac dyspnea
• Provides prognostic info for pts with CHF and
  acute coronary syndrome (ACS)

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ACS

• Acute coronary syndrome
• Encompasses many clinical situations with
  myocardial ischemic damage
• Stable angina, unstable angina, acute MI, sudden
  cardiac death
• Lab assays good at diagnosing MI, bad at
  diagnosing the remainder
• Usual biomarkers for necrosis (MB, myoglobin,
  troponin) are overall poor markers for non-AMI
  ACS
• BNP is predictive for both recurrence and higher
  likelihood of sudden cardiac death in non-AMI ACS
• C-reactive protein? good predictor of development
  of ACS in healthy individuals

65
ACUTE MI

• Typical rise and fall of CK-MB or troponin with
  ischemic symptoms
• ECG changes
• Or interventionally demonstrated coronary artery
  abnormality
• TROPONINS single positive troponin is highly
  specific for AMI
• Single low troponin has low sensitivity for
  negative predictor
• CK-MB increase detectable within 3-6 hours of
  AMI, peaks at 20-24 hours, returns to normal in
  72 hours sensitivity using serial measurements
  approaches 100
• Myoglobin rapidly released from damaged muscle,
  early indicator, negative predictive value 2
  hours post symptoms approaches 100

66
SERUM PROTEINS

• Protein quantitation
• Nitrogen count (Kjeldahl technique) is gold
  standard involves acid digestion of protein to
  release ammonium ions which are quantified
• Assumption is serum proteins are 16 nitrogen by
  mass
• Colorimetry (Biuret technique) is recommended
  routine method for measuring total protein
• Absorbance from chelate with copper at 540 nm is
  proportional to total protein

67
Protein Separation

• Use PRECIPITATION
• Electrophoresis
• Movement of proteins due to electrical potential
• Charge applied across a medium composed of solid
  support (gel) and fluid buffer. Charge creates
  electromotive force
• Solid support has slight neg charge and is drawn
  towards the anode ( pole), but being solid,
  cannot move
• Compensatory flow of fluid buffer towards
  negative pole (cathode)
• This flow is called endosmosis- has capacity to
  carry substances suspended in medium

68
Protein Separation

• If proteins added, two forces are on the
  proteins
• Electromotive force
• Endosmotic force
• Most proteins have negative charge
• Electromotive force pulls towards anode
• Endosmosis pulls towards cathode
• Gammaglobulins weak net negative charge
• Electromotive force exceeds endosmotic force
• Move to variable extent towards anode

69
Serum Protein Electrophoresis

• SPEP
• When electrophoresis is carried out on serum at
  pH 8.6 on agarose gel, then fixed and stained
• Five distinct bands can be seen
• Fastest moving band is albumin
• Next fastest- two a bands (a1 and a2)
• Then the b band
• Then the g band (move very slowly)

70
Serum Protein Electrophoresis
71
Protein Electrophoresis

• Increasing endosmosis is used to separate gamma
  globulins into oligoclonal bands in CSF
  electrophorsis
• Capillary electrophoresis can also be used if
  there is
• Small sample size
• Needed automation
• Need for speed

72
Immunofixation Electrophoresis

• IFE
• Method for characterizing a suspected monoclonal
  band observed in SPEP or UPEP
• Much simpler to interpret than IEP
• Place pt serum into six wells in agarose gel
• Five different monospecific antisera are applied
• Anti-IgG, IgA, IgM, Kappa, and lambda
• Entire gel is stained
• IEP (immunoelectrophoresis)- not commonly used
  anymore

73
IFE
74
Serum Proteins

• Albumin
• Most abundant protein in human plasma
• 2/3 of total plasma protein
• Many functions
• Maintains serum osmotic pressure, carries
  multiple substances
• Congenital absence NOT a serious problem (mild
  anemia and hyperlipidemia)
• Several allotypes
• Most common is Albumin A
• When variant is present, might get 2 peaks

75
Albumin

• Clinical utility
• Nutritional status (half-life is 17 days)
• Hepatic synthetic function (ESLD)
• Diabetic control
• In normal pts, up to 8 of albumin is
  glycosylated
• In diabetics with poor control, up to 25 may be
  glycosylated
• Negative acute phase reactant (decreases in
  inflammatory conditions)

76
Prealbumin

• Fastest migrating protein on SPEP
• Sparse, not normally seen on traditional SPEP
• Binds T4 and T3
• Binds and carries retinal-binding proteinvitamin
  A complex
• Also, prealbumin is the precursor protein in
  senile cardiac amyloidosis
• Short ½ life of 48 hours
• True elevations seen in chronic EtOH, steroids
• Negative acute phase reactant

77
a1 antitrypsin (AAT)

• Major component of the a1 band
• Main function is to inactivate various proteases
  like tyrpsin and elastase
• SPEP can be used to screen for AAT deficiency
• Markedly positive acute phase reactant

78
a1-acid glycoprotein (orosomucoid)

• Briskly positive acute phase reactant
• Minor component of a1 band
• Major component of increased a1 band seen in
  inflammation
• May be used to monitor chronic inflammatory
  conditions like ulcerative colitis

79
a2 macroglobulin

• Protease inhibitor
• Serum concentration elevated in liver and renal
  disease
• Large size prevents its loss in nephrotic
  syndrome, leading to a 10-fold increase in
  concentration

80
Ceruloplasmin

• a2 protein
• Functions in copper transport
• Decreased serum ceruloplasmin important marker
  for Wilson disease, ddx includes
• Hepatic failure
• malnutrition
• Menke syndrome
• Acute phase reactant
• Elevated in inflammation and pregnancy

81
Haptoglobin

• Third major component of a2
• Binds free hemoglobin
• Decreased or absent in acute intravascular
  hemolysis
• Very sensitive marker for hemolysis
• Haplotypes I and II
• Phenotypes 1-1, 1-2, 2-2
• 2-2 phenotype is independent risk factor for CAD
  in diabetes
• Acute phase reactant

82
Transferrin

• Major b globulin
• Functions to transport ferric iron (Fe3)
  normally 30 saturated
• Marked increase in Fe def. abnormally
  masquerades as an M-protein
• Increased in pregnancy and estrogen tx
• Decreases in acute phase but rises is
  inflammation persists
• Blood brain barrier transports transferrin to CSF
  in a modified form (Tau-protein)
• Carbohydrate-deficient transferrin superior to
  GGT as marker for EtOH abuse

83
Fibrinogen

• Also called b globulin
• In normal course of events, no fibrinogen in
  serum as it is consumed by formation of clot
• If specimen clots incompletely (heparinized pt)
  fibrinogen may be seen
• Can straddle the b-g interface
• When present in serum, may be misinterpreted as
  an M-protein
• May be seen in serum in
• Dysfibrinogenemia, APL syndrome, liver dz,
  vitamin K def, or heparin

84
C-reactive protein (CRP)

• Produced in liver
• Predictive value of low level elevations (gt2-3
  mg/L) for cardiac events
• High sensitivity CRP (hsCRP) now available with
  sensitivity of lt0.5 mg/L
• Distribution NOT Gaussian curve-
• Curve skewed significantly with dense cluster in
  very lowest CRP levels and long tail extending
  into gt10 range
• Half of population has CRP gt2

85
CRP

• Three categories based on CRP
• Normal CRP lt3 mg/L
• High CRP gt10 mg/L (active inflammation)
• Low-level elevations 3-10 mg/L (cellular stress)
• Low level elevation may indicate
• Minor disease states
• Genetic factors
• Demographic variables
• Behavioral patters
• Individuals normal set point is inherited

86
CRP

• Low-level CRP elevation predicts poor outcome
  following cardiovascular events
• Also correlates with mortality in non-cardiac dzs

87
SPEP Patterns

• Normal serum
• Invisible prealbumin band
• Very large albumin band
• Then, small peaked a1, a2, a bimodal B, and a
  broad gamma band
• Bisalbuminemia
• Seen in heterozygotes for albumin allotypes
• Double albumin spike
• No clinical consequence

88
SPEP Patterns

• a1-antitrypsin (AAT) deficiency
• AAT is the major component of a1 band
• Genotype PiZZ individuals have a visibly and
  quantitatively decreased band
• Nephrotic syndrome
• Massive loss of small proteins, particularly
  albumin
• Minimal change disease especially high loss of
  albumin
• With other forms of nephrotic syndrome, gamma
  globulins also lost
• LARGE PROTEIN MOLECULES RETAINED
• Result dimming of all electrophoretic bands,
  with exception of a2 which contains
  a2-MACROglobulin

89
SPEP Patterns

• Acute inflammation
• Acute phase reactants account for increases in
  a1, a2 bands
• Albumin slightly increased
• Prolonged inflammation shows polyclonal gamma
  globulin increase
• Beta-gamma bridging (BODOR noted this has been on
  several Board Exams)
• Hallmark of CIRRHOSIS
• Also, hypoalbuminemia, blunted a1 and a2
• Beta-gamma bridging mainly due to increase serum
  IgA

90
Monoclonal Gammopathy

• SPEP shows a prominent discrete dark band
  (M-spike, m-protein) usually within the gamma
  region (sometimes in B or a2)
• A monoclonal gammopathy (paraprotein) shows
  immunochemically homogeneous immunoglobulin
  (M-protein) in serum
• May be result of
• Multiple myeloma
• Neoplastic proliferations like solitary
  plasmactyoma, MGUS, Waldenstroms
  macroglobulinemia (lymphoplasmacytic lymphoma),
  and CLL/SLL

91
Biclonal Gammopathy

• 2 M-proteins
• Occurs in 3-4 of cases
• If biclonal gammopathy has IgA spikes having a
  single light chain (by IFE)
• Most likely due to appearance of both monomers
  and dimers in SPEP
• Should be considered monoclonal

92
SPEP Hypogammaglobulinemia

• 10 of SPEPs
• When not due to myeloma
• May be due to congenital deficiency, lymphoma,
  nephrotic syndrome, or corticosteroids
• Pts with myeloma and hypogammaglobulinemia are
  likely to have free-light chains in urine
  (Bence-Jones proteins)

93
IFE

• Indicated to characterize M-protein on SPEP
• Even if no m-spike is seen, do IFE if
• Strong suspicion of myeloma (lytic bone lesions
  in 80 year old male)
• Systemic AL amyloidosis
• Hypogammaglobulinemia
• In pts with negative serum screens with high
  suspicion, do UPEP (for light chain disease)
• May be losing all of light chain in urine

94
M-protein

• Usually an intact immunoglobulin composed of 2
  heavy and 2 light chains
• Sometimes, it is light chain only or very rarely,
  heavy chain only
• Systemic amyloidosis may result from monoclonal
  gammopathy, when M protein produced has unusual
  (amyloidogenic) properties

95
After diagnosis

• Protein electrophoresis is used to follow disease
  progression and efficacy of treatment
• 3 quantitive results are important
• M-protein concentration
• Degree of suppression of other immunoglobulins
  (IgG, IgA, IgM, whichever is not involved)
• Quantity of free serum light chain, especially in
  light chain only myelomas (extremely sensitive to
  myeloma recurrence after tx)

96
Hyperviscosity syndrome

• Normally, viscosity of serum is 1.5-1.8
  centipoise (cp)
• Hyperviscosity syndrome exceeds 3.0 cp
• Symptoms
• Nasal bleeding, blurred vision, retinal vein
  dilation, neurologic symptoms

97
Cryoglobulins

• May be looked for in pts with M-proteins
• Precipitate reversibly at low temps
• Blood is drawn and kept at 37 degrees C until
  clotted
• Centrifuged at 37 degrees
• Remaining serum stored at 4 deg C for at least 3
  days, then centrifuged at 4 deg C
• Any precipitate that is formed is CRYOPRECIPITATE
  and can be subject to electrophoresis

98
Cryoglobulins

• 3 types
• Type I monoclonal immunoglobulins associated
  with MM or Waldenstroms
• Type II mix of monoclonal IgM and polyclonal
  IgG. IgM has Rheumatoid factor activity
  (anti-IgG). This is the most common type of cryo
• Type III mix of two polyclonal cryoglobulins

99
Mixed Cryoglobulinemia

• Types II III
• Found in variety of conditions
• Lymphoproliferative d/o, chronic infx, chronic
  liver dz, autoimmune dz (SLE)
• Most common in women in 4th-5th decades of life
• 30-50 of cases have underlying HepC virus (most
  common cause)
• Clinical manifestation palpable purpura,
  arthralgias, hepatoslenomegaly, lymphadenopathy,
  anemia, sensoineural defects, and
  glomerulonephritis
• Tx corticosteroids, plasmapheresis, a-interferon

100
UPEP

• In proteinuria, UPEP can determine source of
  protein
• Glomerular proteinuria pattern
• Strong albumin, a1, B bands
• Very large and very small proteins dont make it
  into urine, leaving albumin, AAT, and transferrin
• Tubular proteinuria pattern
• Weak albumin band, strong a1 and B bands
• Impaired tubular reabsorption of LMW proteins
  normally filtered freely by glomerulus
  (a2-macroglobulin, b2 microglobulin, light
  chains)
• Overflow proteinuria pattern
• Monoclonal light chain (Bence Jones)
• Remember that these back up into SPEP in MYELOMA
  KIDNEY

101
CSF Protein electrophoresis

• Different than serum
• All proteins in serum, but smaller quantities
• Prominent prealbumin band and double beta
  (transferrin) band (transferrin conjugated to
  Tau-protein on way into CSF)