Quick Compendium of CP:
Chemistry
Enzymes, Serum Proteins,
Acid-Base and Electrolytes
ZW 7/7/08
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
Michaelis-Menton Kinetics
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)
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)
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
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)
Macroenzymes
Ordinary enzymes bound to antibodies
Has 2 effects:
– Makes it incapable of functioning
– Prevents it from being cleared from blood
Competitive INHIBITION
Non-competitive Inhibition
Uncompetitive Inhibition
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
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
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 AfricanAmericans
Hemolysis raises AST/ALT
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
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)
LDH
Concentrations:
– LD2>LD1>LD3>LD4>LD5
– LD 1 elevation (with flipped LD ratio LD1>LD2):
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
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)
Alkaline phosphatase
4 isoenzymes by electrophoresis:
– Each displays characteristic degrees of
inactivation by heating, urea incubation, and lphenylalanine
– Heating produces significant inactivation of
bone alk phos (bone burns), 50% inactivation
of biliary alk phos, and NO inactivation of
placental alk phos.
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 Paget’s disease of
bone
– A specific immunoassay for bone alk phos
available
Regan Isoenzyme
Observed in about 5% of individuals with
carcinoma
– Appears identical to placental alkaline
phosphatase
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
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
Alkaline phosphatase
Sensitive indicator for hepatic metastases
In women, investigation should include
assay for anti-mitochondrial antibodies
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
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
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
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
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
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?
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
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)
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)
Causes of Hyperbilirubinemia
Unconjugated:
– Hemolysis (extravascular)
– Blood shunting (cirrhosis)
– Right heart failure
– Gilbert syndrome
– Drugs: rifampin
– Crigler-Najjar syndrome
– Hypothyroidism
Causes of Hyperbilirubinemia
Conjugated:
– Dubin-Johnson syndrome
– Hepatitis
– Endotoxin (sepsis)
– Pregnancy (estrogen)
– Drugs: estrogen, cyclosporine
– Mechanical obstruction:
PBC, PSC, tumor, stricture, stone
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
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
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)
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 >5 mg/dL
Conjugated bili (direct) that exceeds 2
mg/dL
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
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)
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)
Acute liver injury labs
Acute viral hepatitis due to HAV, HBV most often
leads to complete recovery
Acute HCV goes to chronic HCV in >80% of
cases
Serologic testing for HAV, HBV are very
dependable for diagnosing acute infx (IgM antiHAV, IgM anti-HBc, HBsAg)
Anti-HCV test only about 60% sensitive for acute
infx
HCV RNA testing 90-95% sensitive
Transaminases
Acute hepatic injury due to ischemic or toxic
injury produce PROFOUND elevations in
transaminases- often >100X upper limit of
normal (RARE in acute hepatitis)
AST > 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
AST:ALT ratio is over 2 in 80% of pts with toxic,
ischemic, and EtOH hepatitis (<1 in viral hep)
Amount of transam elevation poorly correlates
with LEVEL of injury
PT/BILIRUBIN
PT (protime)– Probably the best indicator of prognosis in acute
hepatic injury
– >4.0 secs indicates severe injury/unfav prog
BILI
– Jaundice in 70% of pts with EtOH, HAV
– Jaundice in <20% of pts with HBV, HCV
– Jaundice rare in kids with acute viral hep, rare in toxic
or ischemic injury
– >15 mg/dL indicates severe liver injury, bad prognosis
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
Serum Amylase
Rises within 2-24 hours of onset of acute
pancreatitis
Returns to normal in 2-3 days
Higher levels don’t correlate with severity
Higher levels are more specific for acute
pancreatitis
Persistance in elevation suggests
complication like pseudocyst
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
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
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)
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
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
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
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)
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.
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: 70%MM). 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
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
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
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
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)
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!
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
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)
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
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%
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
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
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
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)
Serum Protein Electrophoresis
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
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
IFE
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
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)
Prealbumin
Fastest migrating protein on SPEP
Sparse, not normally seen on traditional SPEP
Binds T4 and T3
Binds and carries retinal-binding protein:vitamin
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
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
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
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
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
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
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
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
C-reactive protein (CRP)
Produced in liver
Predictive value of low level elevations (>2-3
mg/L) for cardiac events
High sensitivity CRP (hsCRP) now available with
sensitivity of <0.5 mg/L
Distribution NOT Gaussian curve– Curve skewed significantly with dense cluster in very
lowest CRP levels and long tail extending into >10
range
Half of population has CRP >2
CRP
Three categories based on CRP:
– Normal CRP <3 mg/L
– High CRP >10 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
CRP
Low-level CRP elevation predicts poor
outcome following cardiovascular events
– Also correlates with mortality in non-cardiac
dzs
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
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
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
Monoclonal Gammopathy
SPEP shows a prominent discrete dark band (Mspike, 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
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
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)
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
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
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)
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
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
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 (antiIgG). This is the most common type of cryo
– Type III: mix of two polyclonal cryoglobulins
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
UPEP
In proteinuria, UPEP can determine source of
protein
– Glomerular proteinuria pattern:
Strong albumin, a1, B bands
Very large and very small proteins don’t 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 (a2macroglobulin, b2 microglobulin, light chains)
– Overflow proteinuria pattern:
Monoclonal light chain (Bence Jones)
Remember that these back up into SPEP in MYELOMA
KIDNEY
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)