Biochemistry clinical significance

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1
Clinical Significance

2. 2
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Table of Contents
Page
Introduction .........................................................................5
Cancer Markers
Carcinoembryonic Antigen ...................................................9
Prostate Specific Antigen .................................................. 10
Cardiac Markers
Creatine Kinase-MB .......................................................... 15
Myoglobin ......................................................................... 16
Troponin-I ......................................................................... 18
Coagulation
General Information .......................................................... 23
Antithrombin III ................................................................ 26
Fibrin Degradation Products ............................................. 27
Fibrinogen ......................................................................... 28
Heparin ............................................................................. 29
Plasminogen ..................................................................... 31
Electrolytes
Carbon Dioxide ................................................................. 35
Chloride ............................................................................ 37
Potassium ......................................................................... 38
Sodium ............................................................................. 40
Endocrinology
Thyroid Function Tests: ..................................................... 45
Free Thyroxine Index ...................................................... 45
Thyroxine ........................................................................ 45
Thyronine Uptake ........................................................... 45
Thyroid Stimulating Hormone ........................................ 47
Enzymes
Acid Phosphatase ............................................................. 53
Alanine Aminotransferase ................................................. 54
Alkaline Phosphatase ........................................................ 55
Amylase ............................................................................ 57
Aspartate Aminotransferase .............................................. 58
Total Creatine Kinase ........................................................ 60
g-Glutamyl Transferase ..................................................... 61
Lactic Dehydrogenase ....................................................... 63
Lactate Dehydrogenase Isoenzyme 1 ............................... 63
Liver Lactic Dehydrogenase .............................................. 63
Lipase ............................................................................... 65
Pseudocholinesterase ....................................................... 66
Fertility Hormones
Follitropin .......................................................................... 69
Human Chorionic Gonadotropin (Choriogonadotropin) .... 72
Luteinizing Hormone ......................................................... 76
Prolactin ............................................................................ 80
Page
General Chemistry
Bilirubin (Conjugated and Total) ....................................... 85
Calcium ............................................................................. 87
Cerebrospinal Fluid Protein ............................................... 89
Cholesterol ........................................................................ 90
Creatinine .......................................................................... 92
Glucose ............................................................................. 93
High Density Lipoprotein Cholesterol ............................... 96
Iron ................................................................................... 98
Magnesium ..................................................................... 100
Neonatal Bilirubin ........................................................... 101
Phosphorus .................................................................... 103
Total Protein and Albumin ............................................... 105
Triglycerides .................................................................... 107
Urea (Blood Urea Nitrogen) ............................................ 109
Urinary Protein ............................................................... 110
Uric Acid ......................................................................... 113
Immunology/Serology
General Information ........................................................ 117
Hypergammaglobulinemias ............................................ 119
Hypogammaglobulinemias (Decreases in Immunoglobulins) ... 120
C-Reactive Protein .......................................................... 122
Therapeutic Drug Monitoring
Aminoglycoside Antibiotics ............................................ 127
Amikacin ....................................................................... 127
Gentamicin ................................................................... 127
Tobramycin ................................................................... 127
Antiarrhythmic Drugs ..................................................... 129
Digitoxin ....................................................................... 129
Digoxin ......................................................................... 129
Lidocaine ...................................................................... 130
N-Acetylprocainamide .................................................. 130
Procainamide................................................................ 130
Quinidine ...................................................................... 131
Antiasthmatic Drugs ....................................................... 132
Theophylline ................................................................. 132
Anti-convulsant Drugs: ................................................... 134
Carbamazepine ............................................................. 134
Ethosuximide ................................................................ 135
Phenobarbital ............................................................... 135
Primidone ..................................................................... 136
Phenytoin ..................................................................... 137
Valproic Acid ................................................................ 138
Chemotherapeutic Drug .................................................. 139
Methotrexate ................................................................ 139
Vancomycin .................................................................... 141

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Table of Contents (cont'd)
Page
Toxicology
Acetaminophen ............................................................... 145
Ethanol ("alcohol," ethyl alcohol) .................................... 146
Drug Screen Tests: .......................................................... 148
Barbiturate Screen ........................................................ 148
Benzodiazepine Screen ................................................. 148
Tricyclic Antidepressant Screen ................................... 148
Salicylate ......................................................................... 150
Urine Drugs of Abuse Screen Tests Introduction ............ 151
Urine Amphetamines Screen .......................................... 152
Urine Barbiturates Screen ............................................... 153
Urine Benzodiazepines Screen ........................................ 155
Urine Cannabinoids Screen ............................................. 156
Urine Cocaine Metabolite Screen .................................... 158
Urine Methadone Screen ................................................ 159
Urine Opiates Screen ...................................................... 160
Urine Phencyclidine Screen ............................................ 161
Specialty
Ammonia ........................................................................ 165
b2 Microglobulin ............................................................ 167
Ferritin ............................................................................ 168
Lactic Acid ...................................................................... 170
Urinary Albumin for the detection of microalbuminuria ................ 171

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Introduction
This collection of papers is intended to accompany the
technical descriptions of the procedures performed by the
Dade Behring aca® discrete clinical analyzer, Dimension®
clinical chemistry system, OPUS™/OPUS™ Plus/OPUS
Magnum® Immunoassay System and the Stratus® CS
STAT Fluorometric Analyzer. The purpose is to explain
in a very general way why the physician is interested in
the substances measured. The approach has been to de-scribe
briefly the normal physiology of the substance and
then to list some of the diseases in which abnormal val-ues
might be expected to occur. These are by no means
complete lists; only the most common diseases have
been included. Those wishing to know more about the
clinical relevance of these tests are referred to any one
of the standard textbooks of clinical pathology.
Some comments may be useful relative to the nature of
the specimen examined. Most of the tests are done on
serum. Blood, serum, and plasma are words which are
sometimes used interchangeably, yet they refer in fact to
different substances. Blood needs no explanation except
to say that it ordinarily refers to whole blood drawn from
the veins, that is, venous blood. There are differences
between arterial, capillary, and venous blood, but none
of these are of much importance with respect to the tests
now performed.
Plasma is the fluid part of the blood. It is obtained by
removing the cellular components (red cells, white cells,
and platelets) from whole blood by centrifugation. In order
to prevent clotting, an anticoagulant is added when the
blood is drawn. Plasma must be used when coagulation
factors are assayed, but it may also be used for the usual
chemistry procedures when a very rapid answer is re-quired.
For this purpose, heparin may be used as the
anticoagulant.
Serum is used in the measurement of most chemical
substances in blood. It is the fluid part of the blood which
remains after whole blood has been allowed to clot. Except
for the absence in serum of some of the coagulation fac-tors,
the differences between serum and plasma are of
little consequence. Serum has the advantage of remaining
fluid while plasma tends to clot eventually.
Many of the substances measured are referred to erro-neously
as though they were measured in a whole blood
sample when in fact it is serum (or plasma) which is as-sayed.
Thus physicians (and clinical chemists) refer to
“blood sugar”, “blood urea nitrogen”, etc., when the more
accurate reference would be to serum glucose or serum
urea nitrogen.
Frank C. Larson, M.D. Myrna Traver, MD
Professor Emeritus Pathologist
Department of Medicine Madison, WI
Medical School
University of Wisconsin-Madison

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7. Cancer Markers
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9. Carcinoembryonic Antigen
9
Structure and Function: CEA is one of approxi-mately
20 related complex glycoproteins that are mem-bers
of the immunoglobulin superfamily1. It’s molecular
mass varies between 150 and 300 kd2. The alteration in
molecular mass is due to extensive, but differing
glycosylation of the individual CEA molecules. CEA is
one of a number of proteins that are expressed during
normal differentiation of fetal tissue but are partially or
completely suppressed in the adult3. It resides on the
surface of epithelial cells of the gastrointestinal tract and
a variety of other tissues and functions to mediate inter-cellular
adhesion. In the normal adult, CEA is expressed
in only trace amounts and is attached to the cell surface
by way of a GPI (glycosylphosphatidylinositol) anchor4.
In cancerous states, surface CEA can be shed in larger
amounts and detected in the circulation and other body
fluids. No clear picture has emerged regarding the role
of CEA in cancer, but several studies suggest that over
expression in carcinomas interferes with cell differentiation.
Clinical Application: CEA is the most commonly
used tumor marker for gastrointestinal tract tumors. Once
thought to be specific for bowel cancer, elevated levels
are also detected in association with a number of other
cancers such as colorectal (70%), lung (45%), gastric
(50%), breast (40%), pancreatic (55%), ovarian (25%),
and uterine (40%). However, an abnormal serum level is
not specific for cancer or unique to malignancy. CEA lev-els
may also be elevated in some patients with benign
conditions such as cirrhosis (45%), pulmonary emphy-sema
(30%), rectal polyps (5%), benign breast disease
(15%), and ulcerative colitis (15%)2. Up to 19% of smok-ers
with no evidence of malignancy and 3% of a healthy
population may also demonstrate elevated CEA levels1.
Expected normal range values for nonsmokers are < 3.0
ng/ml and < 5.0 ng/ml for smokers. Age, sex, and race
have no significant effect on serum CEA levels.
Caution should be exercised when interpreting patient
CEA values. Quantitation is method dependent and there-fore
values should only be compared when using the
same method. Measurement of CEA is not recommended
as a screening tool given that false-positives arise from
non-cancerous conditions and false negatives may oc-cur
since many tumors do not produce CEA. It’s
quantitation can be used as an aid in the prognosis and
management of cancer patients in whom changing con-centrations
of CEA are observed. The American Society
of Clinical Oncology (ASCO) recommends the use of
CEA in colorectal cancer for preoperative evaluation,
postoperative follow-up, and monitoring of treatment for
metastatic disease.5
Preoperative Evaluation: CEA may be ordered pre-operatively
in patients with colorectal carcinoma if it would
assist in staging and surgical treatment planning. Al-though
elevated preoperative CEA (>5 ng/ml) may cor-relate
with poorer prognosis, data are insufficient to sup-port
the use of CEA to determine whether to treat a pa-tient
with adjunct therapy.5
Postoperative Follow-up: If resection of liver me-tastases
would be clinically indicated, it is recommended
that postoperative serum CEA testing may be performed
every 2 to 3 months in patients with stage II or III dis-ease
for 2 or more years after diagnosis. An elevated
CEA, if confirmed by retesting, warrants further evalua-tion
for metastatic disease, but does not justify the insti-tution
of adjunct therapy or systemic therapy for presumed
metastatic disease.5
Monitoring During Treatment for Metastatic
Disease: Present data are sufficient to recommend rou-tine
use of the serum CEA alone for monitoring response
to treatment. If no other test is available to indicate a
response, CEA should be measured at the start of treat-ment
for metastatic disease, and every 2 to 3 months
during active treatment. Two values above baseline are
adequate to document progressive disease, even in the
absence of corroborating radiographs. CEA is regarded
as the marker of choice for monitoring colorectal cancer.5
References:
1. E. P. Mitchell: Role of carcinoembryonic antigen in the manage-ment
of advanced colorectal cancer. Seminars in Oncol-ogy.
25:12-20, 1998.
2. D. W. Chan and S. Sell. Tumor Markers. In: C.A. Burtis and E.R.
Ashwood, eds. Textbook of Clinical Chemistry. Philadelphia: W.B.
Saunders, 1994:897-927.
3. J. Mendelsohn: Principles of Neoplasia. In: E. Braunwald, et al,
eds. Principles of Internal Medicine. New York: McGraw-Hill,
1987:412-431.
4. B. Obrink: CEA adhesion molecules: multifunctional proteins with
signal-regulatory properties. Current Opinion in Cell Biology.
9:616-626, 1997.
5. 1997 Update of recommendations for the use of tumor markers
in breast and colorectal cancer. J. Clinical Oncology. 16:793-795,
1998.

10. Prostate Specific Antigen
10
Structure and Function
Prostate specific antigen (PSA) is a single chain protein
consisting of 237 amino acids and one N-linked carbo-hydrate
chain. The molecular weight of 28.5 kDa includes
the 7% carbohydrate content. PSA is a serine protease
member of the kallikrein family and shares amino acid
sequence homology with at least two other family mem-bers,
human glandular kallikrein and pancreatic renal
kallikrein. PSA is produced mainly by the epithelial and
ductal cells of the prostate gland, but is also present in
the urethra, periurethral and anal glands of the male. Low
level PSA production has also been observed in the
female salivary glands and breast. PSA is a normal con-stituent
of seminal fluid where its role is liquefaction of
the coagulum to increase sperm motility. PSA may also
function as a growth factor modulator via proteolytic
cleavage of growth factor, as is the case with pro-EGF
(epidermal growth factor) or the transport protein for the
growth factor as is the case for the binding protein-3 for
insulin-like growth factor. PTH-rp (parathyroid hormone
related peptide) has also been shown to be a substrate
for PSA suggesting a role for PSA in intracellular cal-cium
regulation. The serum PSA test has become an
important aid in the clinical management of patients with
prostate cancer.
Serum PSA
Molecular Forms: In serum, PSA circulates as a “com-plex”
with proteolytic enzyme inhibitors and also in a “free”
form. The majority of measurable PSA consists of PSA
complexed in a 1:1 ratio with a-1 antichymotrypsin (ACT).
Smaller amounts of PSA are bound to a-1 antitrypsin
and protein C inhibitor. PSA complexed with a-2 macro-globulin
(2:1) ratio is not measurable with conventional
immunoassays, as the PSA is engulfed by the larger
a-2 macroglobulin. In some healthy subjects, the free
form of PSA in serum may constitute a significant frac-tion
of the total PSA (up to 40% for PSA values in the
range of 1.0 - 4.0 ng/mL). The free form may consist of
the proenzyme that is inappropriately released by the
prostate cell, or “nicked” PSA which is enzymatically in-activated
as a result of peptide bond cleavage at the
lysine-lysine 145-146 position and possibly at other sites
as well. The complexed form of PSA is cleared by the
liver with an apparent half-life of 2.7 days, while free PSA
has a half-life of approximately 1.5 hours.
Expected Ranges for Total Serum PSA: Normal
values for serum PSA are age related, reflecting the effects
of increasing prostate size as a man ages. The age-referenced
upper limits of normal (95th percentile) for
Caucasian males are: 40-49 years, 2.5 ng/mL; 50-59 years,
3.5 ng/mL; 60-69 years, 4.5 ng/mL and 70-79, 6.5 ng/mL.
Asian men may have lower PSA values and African
American men may have higher values than those given
for Caucasian males. The value of 4.0 ng/mL has become
the established cut-off value for diagnostic purposes. There
have been no reports of significant diurnal or circadian
variation for serum PSA, but some men may demonstrate
significant biological variation, showing coefficients of
variation as high as 50% for mean PSA concentrations
below 4.0 ng/mL.
Pre-Analytical Considerations: In some men, se-rum
PSA values may be increased by prostatic massage
as occurs with the digital rectal exam (DRE) or transrectal
ultrasonography (TRUS). Thus, it is recommended that
serum PSA values be determined prior to any of these
activities in order to minimize false positive PSA results.
Drug therapy with finasteride for benign prostatic hyper-trophic
(BPH) disease may cause serum PSA values to
decrease. Biopsy or surgery of the prostate will cause
significant false elevation of serum PSA values and it is
recommended that PSA measurements be delayed for
at least six weeks after these procedures are performed.
Human glandular kallikrein (HgK) is a potential source
of false positive error in PSA immunoassays due to its
high homology with PSA. However, careful selection of
immunoreagents and structural differences between PSA
and HgK appears to have minimized this potential problem.
Non-prostatic sources of PSA in males are secreted into
the urine tract and do not affect serum measurements.
Clinical Application
Patient Monitoring: Patients with cancer confined to
the prostate are usually treated by prostatectomy or ra-diation,
or may be offered “watchful waiting.” The “watch-ful
waiting” approach consists of close monitoring to as-certain
the rate of tumor growth. It is considered by many
to be most useful for older men who have less aggres-sive
tumors, that is, tumors with small volumes (less than
1.0 cc) of low Gleason grade.

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After radical prostatectomy, serum PSA values should
decline to values of less than 0.2 ng/mL after 2-3 days.
In many urologic practices, post-surgical PSA values
which rise above 0.5 ng/mL are strongly suggestive of
recumbinant tumor or metastasis and will signal additional
work-up or treatment.
The pattern of PSA change for patients undergoing ra-diation
therapy reflects the slower elimination of PSA
producing tumor cells compared to the more immediate
and complete removal of the tumor by surgery. Serum
PSA values may decrease for 6-12 months before stabi-lization.
The level to which the PSA must drop to reflect
effective radiation therapy is controversial. However, it is
apparent that the probability of relapse decreases with
the extent to which the PSA concentration drops. The
lowest relapse rate for radiation treated patients has been
achieved when the serum PSA value drops to levels below
0.2 ng/mL as demonstrated for patients treated by radical
prostatectomy. Rising PSA values in patients treated by
radiation therapy signals tumor recurrence and progres-sion
of disease. It is hoped that the early treatment of these
localized cancers will improve the overall cure-rate for pros-tate
cancer.
Hormonal treatments are commonly used to treat advanced
prostate cancer. The primary strategy is to reduce the con-centration
of testosterone by surgical castration (bilateral
orchiectomy), estrogen therapy, or androgen blockage with
the use of an LH-RH analogue and an anti-androgen. The
response to hormonal therapy and the resulting decline in
serum PSA concentration can be dramatic. However, the
response to hormone therapy as well as the PSA decline is
related to the composition of the tumor which consists of
both hormonally sensitive and insensitive cell clones. Ulti-mately,
the tumor will progress with the proliferation of the
androgen resistant clone. Serum PSA may not increase to
reflect the tumor burden in some of these cases, as the
hormonally insensitive clone is less well-differentiated and
may lack the ability to produce PSA to the same extent as
hormonally sensitive clones.
Staging and Prognosis: In routine practice, serum
PSA provides little discrimination between patients with
localized disease and those with extracapsular spread
of cancer. While serum PSA varies directly with tumor
volume, there is wide variation of PSA values and also
considerable overlap of PSA ranges for the various dis-ease
stages. Approximately 80% of prostate cancer pa-tients
have an elevated PSA value defined by the cut-off
value of 4.0 ng/mL. The majority of patients with PSA
values of less than 4.0 ng/mL will have disease confined
to the prostate, and most patients with PSA values greater
than 10.0 ng/mL will have metastatic disease to lymph
nodes or bone. Bone scans are used to confirm metasta-sis,
however, bone scans may not be necessary in the
routine workup of patients who present with no skeletal
symptoms and have PSA values of less than 20.0 ng/
mL. Since serum PSA cannot accurately identify patients
who have disease confined to the prostate, pretreatment
serum PSA measurement provides no prognostic infor-mation
for the individual patient.
Other Prostate Diseases: The primary cause of
false positive PSA values is BPH disease. While PSA
production in BPH tissue is estimated to be only one-tenth
that of prostate cancer tissue, the larger volume of
BPH tissue leads to serum PSA values greater than 4.0
ng/mL in 20-30% of patients. Most of these elevated BPH
values are in the range of 4.0-10.0 ng/mL. Acute urinary
retention, prostatic infarction and prostatitis may also
cause elevated serum PSA values.
Conclusion
In the management of prostatic carcinoma, serum PSA
values provide accurate assessment of the patient’s re-sponse
to therapy.
Prepared by:
Herbert A. Fritsche, Ph.D.
M. D. Anderson Cancer Center
Houston TX

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13. Cardiac Markers
13

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15. Creatine Kinase MB (CK Isoenzyme)
15
Physiology: Creatine phosphate and creatine kinase, the
enzyme which synthesizes and degrades it, are important
compounds in the storage and transfer of chemical energy
within cells. When food from the diet is oxidized, its poten-tial
energy is trapped as a high-energy compound which
may be utilized immediately or stored in the cell until it is
needed to perform mechanical work or to drive some en-ergy-
requiring metabolic reaction. The most common of the
“energy transfer” compounds is adenosine triphosphate,
or ATP. For example, the energy for muscle contraction (and
for many other metabolic reactions) is supplied by the hy-drolysis
of the terminal pyrophosphate bond of ATP to yield
adenosine diphosphate (ADP) and inorganic phosphate ion.
ATP is normally produced by the oxidation of foods through
a long series of reactions - a relatively slow process. There
is only a small amount of ATP in muscle cells - about 6
mmol/g. This is sufficient to sustain muscle contraction for
only a short period of time. Some additional ATP can be
produced fairly quickly by the partial degradation of glu-cose
liberated by hydrolysis of stored muscle glycogen.
However, at the onset of a sudden burst of intense muscle
contraction, muscle cells require some form of “stored”
energy which can be quickly converted to ATP. The high-energy
compound which serves to “store” energy in the
muscle cell is creatine phosphate (also called phosphocre-atine)
which has a high-energy guanidophosphate bond.
ATP can be quickly resynthesized by the one-step trans-fer
of the phosphate group of creatine phosphate to ADP.
This reaction is catalyzed by the enzyme creatine kinase
(CK - previously known as creatine phosphokinase, or
CPK).
All body cells probably contain some CK, but the largest
amounts are found in tissues in which a large amount of
energy is stored as creatine phosphate. Skeletal muscle
contains 2000-3000 units of this enzyme per gram of
tissue, heart muscle 400 U/g, and the smooth muscle of
the intestinal wall about 150 U/g. In contrast, there are
only 3 U/g in liver.
As is true of many other enzymes, several molecular
forms (isoenzymes) of CK exist. They differ in their
Michaelis constants and pH optima, but the physiologi-cal
significance of this is unknown. Each of the three
isoenzymes of CK is composed of two polypeptide
chains. In the case of skeletal muscle CK, the chains are
identical and are called M chains. The enzyme is called
type MM. Brain CK is also composed of two identical
chains, but these are different from the chains in muscle
CK. Brain CK is type BB, composed of two type B
chains. The third isoenzyme is a hybrid molecule,
called type MB, consisting of one M chain and one B
chain. Heart muscle, although it contains primarily type
MM CK, also contains some type MB. This is of major
clinical significance since CKMB is usually found only in
heart muscle.
Clinical Significance: Normal serum contains a small
amount of CKMM and practically no CKBB or CKMB.
When any tissue is damaged some of the intracellular
enzymes may leak into the blood. Thus injury to skeletal
muscle or heart muscle may be followed by a rise in serum
CK. Measurement of serum total CK has been widely
used in the diagnosis of acute myocardial infarction
(AMI).* This disease is the most common cause of death
in the western world. A portion of the heart muscle is de-prived
of its blood supply, usually because of occlusions
of a coronary artery by fatty plaques and/or by thrombus
formation. The heart muscle cells die, and CK is released
into the blood along with other enzymes such as AST
and LDH. Serum CK levels correlate with the volume of
damaged heart muscle and have been used as an index
of the extent of the infarction. The usefulness of serum to-tal
CK in MI has been compromised by the fact that some
injury to skeletal muscle may accompany AMI, and skel-etal
muscle contains 10 times as much CK as does heart
muscle. For example, injections of pain medication into
muscle (a common event in the treatment of a patient
with suspected AMI) may cause enough inflammation of
the muscle to give a spurious elevation in total serum
CK. This problem can be circumvented by measuring
only serum CKMB. This is a remarkable, sensitive and spe-cific
test for heart muscle damage. As with total CK, in-creases
in serum CKMB can usually be seen at 4 hours
after the onset of chest pain. The enzyme level peaks
soon after and returns to normal within 72 hours.
*CK is also used in the diagnosis of some diseases of muscle and
brain. These are discussed in the section on CK.

16. 16
Physiology
Structure and Function: Myoglobin is a 17,800 dalton
porphyrin-containing protein consisting of 153 amino acids.
Myoglobin is almost exclusively found in the cytoplasm of
skeletal and cardiac striated muscle. It constitutes 2% of
the total muscle protein, and there are approximately
2.8 mg of myoglobin per gram of heart tissue. No organ-specific
isoforms of myoglobin have been demonstrated
in humans thus far. One of the primary roles of myoglobin
in muscle tissue is to bind oxygen and release it when the
partial pressure (pO2) within the cells becomes very low.
Clinical Significance
Myoglobin Release from Cells: Clinical studies suggest
that myoglobin, owing to its small size and cytoplasmic
location, is one of the first tissue specific markers to
be released from the compromised skeletal or cardiac
myocyte. It is this characteristic on which its clinical sig-nificance
rests. Serum and plasma levels of myoglobin
increase within one hour of the onset of chest pain in
acute myocardial infarction (AMI), thus most clinical labo-ratories
today measure myoglobin levels.
Leakage by Molecular Size
When the myocyte experiences ischemia, one of the first
responses is swelling. This swelling results in a sequen-tial
loss of cytoplasmic constituents into the interstitial
spaces in a manner that is roughly dependent on the
molecular weight of each constituent. In this leakage
model, very small molecular weight molecules like elec-trolytes
are lost first. Then the macromolecules of increas-ing
size like myoglobin (17,800 daltons) and enzymes
like creatine kinase and its MB isoenzyme (CKMB) at
86,000 daltons are lost from the cytoplasm. Myoglobin
at 17,800 daltons is lost from the myocyte cytoplasm
before cell death in ischemic conditions such as unstable
angina; thus, circulating levels of myoglobin have been
found to increase in ischemia. In addition, myoglobin
seems to move directly to the coronary blood flow, while
CKMB and lactate dehydrogenase isoenzyme 1 (LD1)
are likely carried by lymphatic drainage from the injured
myocardium.
Myoglobin in Acute Myocardial Infarction
It has been known since the first reports by Kagen and
Stone in 1975 that serum and plasma myoglobin levels
increase with AMI. Information has begun to accumu-late
suggesting patients with AMI show a rise in myoglobin
levels before either mass CKMB or cardiac troponin I (cTnI).
The rapid clearance of myoglobin from the circulation in
uncomplicated AMI, makes this marker suitable for de-tection
of reinfarction in patients with recurrent chest pain.
Myoglobin in Emergency Department
Diagnosis of AMI
Until the early 1980’s most patients presenting with chest
pain were routinely being admitted to the hospital for
observation. However, two parallel events helped to
change how laboratory tests to detect AMI would be used
in the Emergency Department (ED) setting. The first of
these events was the adoption of the Diagnostic Related
Group (DRG) method of payment for in-patient treatment
of Medicare and Medicaid patients in 1983 and the rise
of managed care in the private sector. The payment
change introduced for the first time significant monetary
deincentives for inappropriate admission of patients who
turned out to not have myocardial events. Next, in 1985
the first reports began to appear on the use of tissue-type
plasminogen activator (TPA) and then subsequently
streptokinase as alternatives to angioplasty by their ability
to dissolve coronary occlusions. The common link be-tween
all the clot busting drugs was that the sooner they
were administered after the arrival in the ED, the better
the patient outcome. Therefore, the detection of
reperfusion by lab tests could significantly alter subse-quent
patient management. These two events contrib-uted
to the increased interest in finding better methods
to correctly identify patients actually having myocardial
events and to detect refusion as soon as possible after
administration of thrombolytic agents.
One of the difficulties in trying to make the diagnosis of
AMI in the ED is that patients present at widely variable
times after onset of chest pain depending on personal,
societal and logistical considerations. Some locations
may show 90% of patients presenting within 6 hours of
chest pain onset, while others in different parts of the
United States commonly see over 50% of patients pre-senting
more than 12 hours after onset of chest pain. A
further complication is that only 10% of those with chest
pain will have ST-segment elevations and AMI, the clas-sic
indicator of coronary artery occlusion. Another 10%
will have non-diagnostic ECG changes (e.g., ST-segment
decreases) and AMI. About 30% of those presenting with
chest pain will have acute coronary syndromes and no
AMI, while the remaining 50% of those presenting with
chest pain will have no cardiac etiology.
Myoglobin

17. 17
Laboratory testing is not involved in the diagnostic deci-sion
to use thrombolytic therapy since, with occlusion,
serum and plasma markers may not reflect the extent of
the myocardial damage due to their accumulation on the
other side of the blockage. The correct clinical diagno-sis
of the remaining AMI presenting population, which
have no ST-segment increase, relies increasingly on
laboratory tests. Since patients may present within less
than an hour to over 24 hours after onset of chest pain,
laboratory diagnostic markers of AMI need to be selected
based on when the levels in serum and plasma become
diagnostic. Myoglobin rises quickly after AMI and becomes
diagnostic within 2-4 hours post-infarct, peaking at 9-12
hours and returning to baseline within 24-36 hours. Myo-globin
tends to rise and fall sooner than other cardiac mark-ers
with AMI.
Myoglobin Utility on ED Presentation
The time of patient presentation in the Emergency De-partment
(ED) with possible AMI is nearly always more
reliably known than the time, based on patient recollec-tion,
of chest pain onset. Therefore, many reports relate
the clinical utility of the various markers for AMI to the
time of initial patient presentation in the ED. Generally
myoglobin is found to have a better sensitivity for AMI in
the first 4 hours after chest pain onset than mass CKMB,
cTnI and cardiac troponin-T (cTnT), but has a lower speci-ficity
than these other markers. A number of reports sug-gest
the measurement of myoglobin as a diagnostic aid
in “ruling-out” AMI with negative predictive values of up
to 100% reported at certain times after onset of symptoms.
A study in 1996 by Gornall and Levinott using serial sam-pling
at 1, 2, and 6 hours after presentation showed myo-globin
had the highest sensitivity, specificity, and nega-tive
predictive value for AMI in the first 2 hours of ED
presentation. They found myoglobin’s sensitivity to be
77% at 1 hour, and 93% at 2 hours, thus the serial pre-dictive
efficiency at presentation was 96% vs. Baseline
ECG at 70% and CKMB at 62%. Another study also
based on collecting blood at presentation and 1 and 2
hours later, using a criteria of myoglobin above 100 ng/
mL or a change (+/-) from baseline of more than 50%
found similar results. Sensitivity in the 0-2 hour period
was 93%, with a 95% confidence interval of 59-92%. The
literature suggests that by using the change from baseline
presentation, myoglobin may yield the most sensitive and
specific assessments of AMI detection if myoglobin mea-surements
alone must be employed.
Myoglobin with Perioperative Myocardial
Infarction
The same characteristics which make myoglobin useful
for the detection of AMI in the ED have been found in
perioperative MI in both cardiac and noncardiac surgical
patients. For example, one study of coronary artery by-pass
grafting (CABG) surgical patients found that myo-globin
levels peaked within 1 hour after aortic unclamping,
and returned to baseline within 4 hours. When a
perioperative MI occurred, myoglobin levels were elevated
1 hour after aortic unclamping, and continued to be
significantly elevated at least 3 hours post-surgery
compared with levels observed in non-MI patients. Myo-globin
levels in patients who suffered perioperative MIs
were found to be greater than 400 ng/mL 4 hours after
the procedure.
Cautions with Use of Myoglobin
There are several clinical situations where myoglobin
results should be used with caution in assessing AMI.
The most significant source of false myoglobin eleva-tions
is skeletal muscle injury. It has been found that
myoglobin and mass CKMB may be elevated in some
patients after electrical cardioversion while cardiac tropo-nin
I remains normal. Individuals running marathons
have also been shown to have elevations of myoglobin,
mass CKMB and CK isoforms but normal cTnI levels.
The specificity of myoglobin for AMI detection was found
to fall from 82% in non-cocaine users to 50% in individu-als
who recently used cocaine. Other clinical situations,
such as severe burn victims, Rhabdomyolysis patients
or other disease states where skeletal muscle injury is
involved could cause increased levels of myoglobin.

18. 18
Physiology
Structure and Function: Troponin C, I and T are polypep-tides
that combine to form a CIT complex on the thin fila-ment
(actin) portion of the contractile apparatus of cardiac
and skeletal striated muscle. This troponin complex is not
present in smooth muscle. Troponin C (TnC) can bind up to
four calcium ions and is responsible for regulating thin fila-ment
activation; troponin I (TnI) binds and inhibits acto-myosin
ATPase thereby blocking myosin movement, and
troponin T (TnT) binds TnI and TnC to the actin-tropomyo-sin
filament positioning the complex on the filament. The
size of the troponins vary from 23,500 daltons for TnI to
37,000 daltons for TnT. TnC has a variable molecular weight
depending on the number of calcium molecules bound.
The binding affinity of TnC for TnI is very high at about
108 L/mol if calcium is present, this enables TnI to wrap
around TnC contacting both N- and C-terminal globular
domain Ca2+ binding sites on TnC. Removal of Ca2+ with
EDTA decreases the TnC - TnI binding affinity 1,000 fold.
TnI has three striated muscle isoforms. Cardiac troponin
I (cTnI) is found only in heart striated muscle. Slow twitch
Troponin I (stTnI) is the isoform found in “red” striated
muscle such as in the soleus and diaphragm. Lastly, fast
twitch troponin I (ftTnI) is the isoform associated with
the “white” striated muscle in skeletal muscles such as
the biceps and triceps. Production of each isoform is
controlled by a different gene. Because the amino acid
sequence of each isoform is different, e. g., cTnI has 31
unique amino acids at its N-terminal end, antibodies can
be developed which can specifically detect cTnI, even in
the presence of high levels of the stTnI and ftTnI striated
muscle TnI isoforms. cTnI is not seen in fetal skeletal
muscle, in regenerating skeletal muscle or in chronic
muscle disease. TnT also shows differences between
cardiac and skeletal muscle isoforms; however, the pres-ence
of cardiac TnT in fetal skeletal muscle raises con-cern
that regenerating skeletal muscle may also produce
the cardiac TnT isoform. TnC has the identical amino acid
sequence in cardiac and skeletal muscle so it is not use-ful
as a marker for cardiac tissue damage.
Clinical Significance
Troponin Release from Cells: Most troponin is bound into
CIT complexes as part of the myofibril structure within the
muscle myocytes. cTnI and cTnT are present in stoichio-metrically
equal amounts in the contractile apparatus of
striated muscle. A smaller amount of cTnI, cTnT and TnC,
approximately 6% to 8% of cTnT and 2.5% of total cTnI,
resides in the myocyte’s cytoplasm. When the myocyte
experiences ischemia, one of the first responses is swelling.
The cell swelling is believed to result in a
sequential loss of cytoplasmic constituents into the in-terstitial
spaces. This cytoplasmic leakage occurs in a
manner that is roughly dependent on the molecular
weight of each constituent. In this leakage model, very
small molecular weight molecules such as electrolytes
are lost first. Then macromolecules of increasing size
such as myoglobin (17,800 daltons) and enzymes such
as creatine kinase (both MM and MB) at 86,000 daltons
and lactate dehydrogenase (LD) at 135,000 daltons are
lost from the cytoplasm. The macromolecule size, which
defines the threshold between leaking and ruptured cells,
is not well defined. However, it is likely that molecules
the size of CKMB and LD may not be able to escape
from an intact living cell. However, there is evidence that
cTnI, at approximately 23,500 daltons, is lost from myo-cyte
cytoplasm before cell death in conditions such as
unstable angina.
cTnI seems to be released from the cytoplasm of
myocytes in patients with acute myocardial infarction
(AMI) at about the same time as CKMB based on the
timing of cTnI’s appearance in serum and plasma. Both
mass CKMB and cTnI first attain optimal diagnostic effi-ciencies
for AMI in the range of 80% at about 6 hours
after onset of chest pain. However, cTnI is about 13 times
higher in concentration in the myocardium than CKMB,
offering the possibility of greater sensitivity to myocar-dial
damage than CKMB. If the ischemia is not reversed
the myocyte will die releasing structural elements, includ-ing
the myofibril troponin CIT complexes into the circula-tion.
Support for this sequential release model comes
from the well known sequence of appearance of bio-chemical
markers in serum and plasma. The bimodal rise
of serum and plasma cTnT is also believed to be due to
an initial cytoplasmic release followed by a second cTnT
rise due to the slower myofibrillar degradation occuring
with cell death.
Free vs Complexed cTnI
Little free cTnI is measurable in serum and plasma after
acute myocardial infarction. Because of the high binding
affinity of cTnI for TnC in the presence of Ca2+, nearly all
measurable serum and plasma cTnI exists as a cTnI-TnC
complex. The use of blood collection tubes containing
strong Ca2+ chelators like EDTA (purple top tubes) dra-matically
lowers the cTnI-TnC binding affinity, resulting
in more free cTnI in the sample. Studies using highly
selective antibodies for free and complexed cTnI show
that nearly all of the measurable cTnI in acute myocar-dial
infarction (AMI) is due to an increase of the cTnI-Troponin-
I

19. 19
TnC complexes. Peak concentrations of total cTnI lev-els
with AMI were found to be 5 to 12 times higher than
the corresponding free cTnI levels. This information sug-gests
that measurement of free cTnI should not contrib-ute
significantly to the diagnostic efficiency of serum and
plasma cTnI assays for myocardial infarction detection.
Cardiac Troponin I (cTnI) Measurement
cTnI’s unique 31 amino acid N-terminal sequence allows
antibodies to be developed to highly discrete epitope sites
on the polypeptide molecule. A properly selected anti-body
permits highly specific cTnI measurement in the
presence of severe skeletal muscle trauma and the high
levels of serum skeletal muscle troponin isoforms that
would result. Whether an immunoassay measures
complexed or free cTnI depends on the cTnI epitopes to
which the antibody is directed. AMI is generally associ-ated
with a serum rise of the cTnI-TnC complex; there-fore,
assays must measure this form of cTnI to be clini-cally
useful in AMI assessment. There is also evidence
that oxidation of cysteine-SH groups on cTnI may alter
the exposed epitopes and the ability of some assays to
detect cTnI. Since serum and plasma cTnI levels are
normally low to non-detectable, clinical laboratory mea-surement
of cTnI requires a highly specific antibody de-tection
system and use of a final measurement system
that can generate a detectable signal at low analyte con-centrations.
One of the biggest problems faced in the clinical appli-cation
of cTnI tests is that assays frequently employ an-tibodies
with different cTnI epitope specificities, and each
commercial method employs a different calibrator mate-rial.
The result is that commercial cTnI methods from dif-ferent
companies do not produce the same analytical
results and may vary as much as 30 fold on the same
sample. As a practical matter this means that diagnostic
cutoff values and clinical utility decision points presented
in the literature are highly method specific and should
not be exported to another cTnI method without repeating
the clinical studies needed to establish new numerical
decision points associated with, for example, unstable an-gina
and myocardial infarction. Therefore, the method used
(e. g., Dade Behring Stratus®) will be indicated as relevant
clinical literature findings are discussed in this article.
cTnI in Acute Myocardial Infarction
Several lines of evidence suggest cTnI may be a good
marker for myocardial injury. cTnI concentration is about
13 times higher in heart muscle than CKMB; therefore,
small amounts of injury should generate a large increase
from normal background levels. In practice, some clini-cal
decision points may be near the assay’s analytical
sensitivity since there is no measurable cTnI in serum or
plasma of individuals without cardiac involvement.
Indeed, cTnI (as measured by Dade Behring Stratus®)
has not been found to increase in serum or plasma in
conditions with acute skeletal muscle damage like trauma
or rhabdomyolysis, or with chronic skeletal muscle dam-age
such as polymyositis or Duchenne’s muscular
dystropy, or after running a marathon. Because those
same acute and chronic skeletal muscle pathologies can
result in increases in CKMB that are not associated with
AMI, the cTnI offers significantly improved specificity
for myocardial damage. Chronic renal failure with hemo-dialysis
or peritoneal dialysis also shows no increase of
cTnI. Elevations in cTnI that correlate closely with myo-cardial
injury have been verified by echocardiography.
These characteristics all suggest that cTnI should be a
better overall serum and plasma marker for routine as-sessment
of myocardial infarction than CKMB.
cTnI in Emergency Department Diagnosis of
AMI
One of the difficulties associated with making the diagno-sis
of acute myocardial infarction (AMI) in the emergency
department is that patients present at widely variable
times after the onset of chest pain depending on per-sonal,
societal and logistical considerations. Some loca-tions
may show 90% of patients presenting within 6 hours
of chest pain onset, while others (in different parts of the
United States) commonly see over 50% of patients pre-senting
more than 12 hours after the onset of chest pain.
To further complicate matters, currently only 50% of pa-tients
actually having AMI and 10% of individuals who
present with chest pain, show the ST-segment eleva-tions
associated with occlusion of coronary arteries and
require emergency thrombolytic therapy or angioplasty.
Another 10% of individuals who present with chest pain
will have nondiagnostic electrocardiogram (ECG)
changes (e. g., ST-segment decreases), and AMI. About
30% of those presenting with chest pain will have acute
coronary syndromes and no AMI, and in the remaining
50% of their chest pain will have no cardiac etiology.
Laboratory testing is often not involved in the diagnostic
decision to use thrombolytic therapy since, with occlusion,
cardiac markers may not reflect the extent of myocardial
damage due to their accumulation on the other side of a
blockage. The correct clinical diagnosis of patients pre-senting
with AMI who have no ST-segment increase
relies increasingly on laboratory tests. Since patients may
present within less than an hour to over 24 hours after
onset of chest pain, laboratory diagnostic markers of AMI
need to be selected based on when the levels in serum
and plasma become diagnostic. Myoglobin rises quickly
after AMI and becomes diagnostic at about 1 to 3 hours
after chest pain onset and remains elevated for 12 to 24
hours. It is a useful marker for “ruleout” AMI within 24
hours. CKMB begins to rise at 3 to 4 hours after onset of
chest pain, becomes diagnostic at about 6 hours, peaks
at 12 to 24 hours, and remains elevated about 24 to 36
hours. LD isoenzyme 1(LD1) becomes diagnostic at
about 10 hours after chest pain onset and remains el-

20. 20
evated for at least 72 hours. cTnI begins to rise 4 to 6
hours after chest pain onset, becomes diagnostic at about
6 hours, and will remain elevated for from 4 to 14 days.
Consequently, cTnI tends to rise like CKMB in serum
but stays increased for as long as LD1. This release
pattern enables the use of cTnI to detect AMI over a
longer period of time from the onset of chest pain than
the other three traditional markers of AMI.
Well controlled studies wherein serum or plasma levels
of multiple markers of myocardial injury were examined
in the same sample and related to time of chest pain
onset show cTnI rises in serum at or before the time of
CKMB. These studies consistently show that CKMB and
cTnI have poor diagnostic sensitivity (e. g., less than
50%) for myocardial injury before 6 hours after onset of
chest pain. In one typical study, in the 7 to 36 hour pe-riod
after chest pain onset cTnI sensitivity was 88 - 100%
compared to CKMB sensitivity of 61% to 81%.
Studies using serial testing of patients with possible acute
myocardial events, but no ST-segment elevation, have
consistently shown that absence of cTnI rise indicates a
low risk of near term coronary death or infarction. In one
study, 773 consecutive patients presenting with chest
pain of less than 12 hours duration and no ST-segment
elevation were tested on admission and again 4 hours
later, or at a second time corresponding to at least 6
hours after chest pain onset. The risk of coronary death
or infarct in those with negative cTnI levels on both tests
was only 0.3%. In another study, cTnI values less than
0.6 ng/mL showed negative predictive values (NPV-%)
results, to be 85% at presentation to the ED, 85.5 at 1
hour later, 87.4 at 2 hours, 96.7 at 6 hours, and 98% at
12-24 hours. The positive predictive value (NPV+%) at
time of ED presentation was 25%, 40% 1 hour later,
60% at 2 hours, 88% at 6 hours, and 88.9% at 12-24
hours. This and other studies strongly suggest serial cTnI
testing assists in the clinical decision to discharge or ad-mit
a patient presenting at the ED with possible AMI.
Available information suggests that cTnI or CKMB results
from serum or plasma collected less than 4 hours after
onset of chest pain may yield results still within the
normal range that could change to positive in samples
collected later.
cTnI in Acute Coronary Syndromes
Acute coronary syndromes (ACS) share a common
pathophysiologic mechanism which is initiated when a
coronary atherosclerotic plaque ruptures and a throm-bus
forms that interrupts perfusion of myocardial tissue.
Patients presenting with resting chest pain, but with no
ST-segment elevation on the electrocardiogram (ECG),
may have an ACS such as unstable angina or non-Q
wave AMI. Detection of ACS frequently cannot be done
clinically or by angiography. Consequently, analysis of
serum markers of myocardial damage such as CKMB
and cTnI have been routinely ordered to assist in detection
of ACS. Making the diagnosis at the time the patient
presents with the first ACS chest pain event is critical
since, without intervention, 40% of those patients will
have an AMI within 3 months. About 17% of those pre-senting
with ACS will die within 3 months. Early inter-vention
can reduce the risk of AMI to 8% and death to
3% within the same 3 month period.
Evidence now suggests that increased serum and
plasma cTnI level in the first 24 hours from onset of chest
pain is a much more sensitive and specific marker for
ACS than CKMB. The prognostic value (predictive value
of positive results) of cTnI increase is highest in patients
presenting more than six hours after onset of chest pain.
One study used samples collected from 1,404 patients
presenting with unstable angina or non-Q wave AMI as
part of the Thrombolysis in Myocardial Ischemia Phase
IIIB (TIMI IIIB) study. It was found that 2.6% of patients
presenting within 6 to 24 hours after chest pain onset,
having cTnI (Dade Behring Stratus®) levels above 0.4
ng/mL but negative mass CKMB levels, died from car-diac
causes within 42 days. If cTnI was less than 0.4 ng/
mL, mortality within the 42 days was 5 fold less, at 0.5%.
This and other studies suggest that cTnI is a useful di-agnostic
marker for ACS, the utility of which will likely
improve as detection methods for cTnI evolve.
cTnI to Assess Cardiac Contusion
Detection of cardiac injury in patients with chest trauma
can be difficult because the serum and plasma level of mass
CKMB could be elevated due to collateral skeletal muscle
injury. Studies using transthoracic echocardiograms as the
criteria for myocardial damage show that serum and plasma
cTnI levels are much more specific than mass CKMB lev-els
for detecting cardiac contusion. In one study (using Dade
Behring Stratus® II) of 44 patients without cardiac contu-sion,
none had serum cTnI levels above 0.35 ng/mL, sug-gesting
no cardiac damage. In contrast, 26 patients had
serum CKMB mass levels above 6.7 ng/mL, suggesting
cardiac damage was present. That the commonly employed
CKMB/Total CK ratio percent does not correct for all skel-etal
muscle damage is suggested by the finding that 3% of
the patients in this study had levels above 2.5%, a range
generally attributed to cardiac damage. Several studies also
suggest that cTnI may be useful for detection of unsus-pected
perioperative myocardial injury or infarction.
cTnI to Assess Myocardial Injury in Critically
Ill Patients
Critically ill patients are exposed to stresses that increase
tissue oxygen demand at a time when oxygen supply to
the myocardium could decrease due to a combination of
variables such as hypoxia, fall in blood pressure, tachycar-dia,
anemia and septicemia. Available evidence suggests
that myocardial injury may go undetected in about 66% of
critically ill patients in the absence of cTnI surveillance.

21. Coagulation
21

22. 22

23. 23
General Information
Hemostasis, the process which prevents the loss of blood
from a blood vessel, is accomplished through three inter-related
events: vascular constriction, platelet aggregation,
and coagulation. The initial vasoconstriction is short-lived.
It enables the second event, platelet aggregation, to pro-duce
a fragile, unstable platelet plug. Prolonged hemosta-sis
depends upon the third phenomenon, the formation
of a blood clot. To be effective a clot must form promptly
and yet be closely confined to the damaged portion of
the vessel. Coagulation is a highly complicated process
in which the factors which prevent clotting are delicately
balanced against those which promote it. Understanding
the complex coagulation process is made more difficult
by the lack of standard nomenclature. In some cases,
the clotting factors are given their chemical names (i.e.
calcium, prothrombin, fibrinogen). Other factors have also
been named for their functions (proaccelerin, proconver-tin,
antihemophilic factor). In still other cases factors were
given the surname of the person in whom hereditary
deficiency of that factor was first detected (Hageman,
Fletcher, and Stuart). In an attempt to reduce confusion,
Roman numerals have been assigned to most of the clot-ting
factors. The numbers reflect the order of their dis-covery,
and not the sequence in which they participate
in the clotting reactions. Factors may exist in plasma as
the inactive protein or precursor which is converted to
the active form during the clotting process. The active
form is identified by appending a lower case ‘a’ to the
Roman numeral, e.g. factor Xa. Even in normal plasma
the stage is constantly set for clotting. It can be triggered
by events which take place either within the blood itself,
in the blood vessel wall, or in the surrounding tissues.
Regardless of how clotting is initiated, the last several
steps are identical. These final steps result in the formation
of a three-dimensional fibrin lattice in which platelets,
red cells, and white cells are trapped.
Fibrin is an insoluble protein which polymerizes spontane-ously.
The initial polymers aggregate due to electrostatic
and hydrophobic bonds. The immature fibrin aggregate
is therefore gelatinous and easily broken up. As the clot
matures, covalent (peptide) bonds are formed between
the fibrin strands, strengthening and contracting the clot.
This process is catalyzed by a transglutaminase, factor XIIIa.
Fibrin cannot exist per se in plasma. It must neverthe-less
be readily available in abundant quantity. Plasma
therefore contains large amounts of a fibrin precursor,
fibrinogen. Fibrinogen is converted to fibrin by the catalytic
action of the enzyme thrombin.
Figure 1
Xa
Prothrombin Thrombin
Fibrinogen
Fibrin Monomer
Soluble Fibrin
Stabilized Fibrin
(Fibrin Clot)
Ca++, PF3
Va
Ca++
XIII XIIIa
Thrombin also is responsible for the activation of factor
XIII (i.e. the conversion of protransglutaminase to trans-glutaminase)
in the final step of clot formation.
Thrombin is a key enzyme in coagulation. If thrombin
existed in the active form in plasma, uncontrolled clotting
would occur. It, too, circulates as an inactive precursor,
prothrombin. The conversion of prothrombin to thrombin
is catalyzed by factor Xa. This has proved to be a com-plex
enzymatic reaction requiring the presence of factor
Va, calcium, and a phospholipid-Platelet Factor 3 (PF3)
(See figure 1).
The activation of factor X is pivotal in clotting. It can come
about in two different ways, referred to as the intrinsic
and extrinsic pathways; the distinction is based upon the
source of the activators. The intrinsic pathway is acti-vated
by trauma within the vascular system such as ex-posed
endothelium. In contrast, the extrinsic pathway is
activated by external trauma such as a cut. Of the two
systems, the intrinsic is slower but more important.
The intrinsic system involves a series of reactions in
which the product of one acts as a catalyst or cofactor
for the succeeding reaction. This reaction chain is often
referred to as a “cascade” (See figure 2). The intrinsic
pathway is triggered when blood comes in contact with
an unfamiliar surface such as a damaged blood vessel
wall, causing a conformational change in factor XII and
converting it to factor XIIa. Factor XIIa is a weak initiator
of several reactions, including the activation of factor XI
to XIa and the conversion of prekallikrein (Fletcher factor)
to kallikrein. Factor XIa activates factor IX. Factor IXa is
an endopeptidase which, in the presence of factor VIIIa
converts factor X to factor Xa. The concomitant presence
of factor VIIIa, platelet phospholipids (PF3), and calcium
is required for the activation of factor X to proceed rapidly.

24. 24
Figure 2
XII
Activator
Chemicals
Collagen
Exposed
Endothelium
Platelets
XIIa
kallikrein
prekallikrein
Intrinsic Pathway
XI XIa
IX IXa
Ca++, PF3
VIIIa
X Xa
Clotting is initiated through the extrinsic system when
blood escapes from the vascular system and contacts
damaged tissues. A poorly understood tissue factor called
thromboplastin is released from the damaged cells. It
forms a complex with calcium and factor VII which is able
to activate factor X directly (See figure 3). Thus several
of the early steps which must occur in the intrinsic system
are bypassed in the extrinsic system. Clotting occurs
much more quickly by this route.
Figure 3
Extrinsic Pathway
VII
Tissue Factor
VIIa
X Xa
Control of Clotting
Once clotting has started it is important to control its rate
and, when hemostasis is achieved, to stop it altogether.
This is accomplished by several mechanisms. The flow
of blood past the site of the developing clot washes away
the activated clotting factors and delivers them to the
liver where they are removed from the blood. In addition
there are several circulating clotting inhibitors, including
antithrombin III (AT III), alpha-1-antitrypsin, C1 inhibitor,
and alpha-2-macroglobulin. Of these, AT III is the most
important. As its name implies, its primary role is to com-bine
with and thereby inactivate thrombin. This inhibition
proceeds relatively slowly but is greatly accelerated in
the presence of heparin. AT III also inactivates factors
Xa, and to a lesser extent, inactivates IXa, XIa, and XIIa.
Inhibition of these factors is also accelerated by heparin
(See figure 4).
Figure 4
Antithrombin III
+ Heparin
Inactive
Xa
Complex
Inactive
Thrombin
Complex
Xa Thrombin

25. Inactive Xa
Complex
Activators
Kallikrein (Intrinsic)
Tissue Activator (Extrinsic)
Urokinase
Streptokinase
Antithrombin III
+
Heparin
Prothrombin Thrombin
25
Clot Dissolution
Blood clots are not intended to be permanent. Their pur-pose
is to stop the flow of blood until the damaged blood
vessel can be repaired. Normally all but the largest clots
will be completely dissolved. Dissolution, which begins
almost as soon as the clot is formed, is accomplished by
the hydrolytic action of the enzyme plasmin. Plasmin also
hydrolyzes fibrinogen and other clotting factors. Therefore,
it cannot exist in its active form in plasma. An elaborate
control system exists which allows clot lysis to proceed while
protecting clotting factors against destructive hydrolysis.
Normal plasma contains an inactive precursor of plasmin
called plasminogen. Plasminogen has an affinity for fibrin
molecules and is incorporated within the fibrin strands
as the clot forms. Plasminogen remains dormant inside
the clot until activated by specific enzymes called plas-minogen
activators. Such activators have been found in
tissues, in urine, in plasma, and, most importantly, in the
endothelial cells which line blood vessels. Factor XIIa
also accelerates the activation of plasminogen. Thus, the
factor which triggers the clotting process also contributes
to clot dissolution. The fact that a clot forms at all is ex-plained
by the relative rates of the two processes. Clot for-mation
occurs rapidly until it is slowed by specific limiting
mechanisms. Clot dissolution, on the other hand, is slow in
starting, but is a continuous process (See figure 5).
Hemostasis
XII XIIa
Activator
Chemicals
Collagen
Exposed
Endothelium
Platelets
Kallikrein
Prekallikrein
Kallikrein
Extrinsic – Intrinsic Interaction
Extrinsic System
Intrinsic System
Common Pathway
Fibrinolytic Pathway
Inhibition
VIIa
XIa
Tissue Factor
XI
VII
IX IXa
X Xa
XIII XIIIa
Ca++, PF3
VIIIa
Ca++, PF3
Va
Activators
Kallikrein (Intrinsic)
Tissue Activator
(Extrinsic)
Urokinase
Streptokinase
Inactive
Thrombin
Complex
inactive
plasmin
complex
Fibrin (ogen)
Degradation
Product
Stabilized Fibrin
Plasminogen
a2antiplasmin
Plasmin
Fibrinogen
FpA
FpB
Fibrin Monomer
Soluble Fibrin
Figure 5
inactive
plasmin
complex
Fibrin (ogen)
Degradation
Product
Fibrinogen
Fibrin Monomer
Soluble Fibrin
Stabilized Fibrin
Plasminogen
a2antiplasmin
Plasmin
Active plasmin sometimes finds its way into the plasma;
it may escape from a dissolving clot or be formed if plas-minogen
activators gain access to the blood stream.
When this happens, fibrinogen and other essential clotting
proteins, including factors V and VIII, are destroyed. To
prevent this, plasmin inhibitors, the most important
of which is alpha-2-antiplasmin, quickly inactivate any
plasmin in the circulation.

26. 26
Antithrombin III
Physiology: Antithrombin III (AT III) is a plasma pro-tein
which is an inhibitor of serine proteases. As the name
implies, its main physiologic function is the inactivation
of the key clotting enzyme, thrombin. It also inactivates
other clotting and clot-lysing enzymes, including factors
Xa, IXa, XIa, XIIa, kallikrein, urokinase, and plasmin, all
of which are serine proteases. AT III is a glycoprotein
with a molecular weight of 65,000. It migrates as an al-pha-
globulin on electrophoresis. It is synthesized by the
liver and is widely distributed in body fluids. Its physi-ologic
half-life is about four days. As with other coagulation
factors, the exact catabolic pathway is not known.
Furthermore, normal plasma contains more AT III than
prothrombin. The fact that clots are able to form at all is
due to the fact that the inhibitory reaction is much slower
than the thrombin-catalyzed fibrinogen to fibrin reaction.
This situation is altered in the presence of heparin, which
greatly increases the inhibitory activity of AT III through
a conformational change in the molecule. Heparin is
therefore an accelerator of AT III. This appears to be the
mode of action for this important anticoagulant.
Clinical Significance: AT III is normally present in
plasma. In disease, its concentration may be either
increased or decreased.
Increases: Damage to body tissue may produce a rise
in plasma AT III which could result in an increased ten-dency
to bleed.
Decreases: Decreased AT III is of much greater clinical
importance. A reduction in AT III leads to an increased
tendency for blood to clot within the blood vessels (throm-bosis).
Once formed thrombi tend to enlarge or progagate.
Rapidly propagating thrombi may break away from the
site where they are formed and lodge elsewhere in the
vascular system (embolization). Serious consequences,
even death, may follow embolization to the heart, lung
or brain. AT III levels less than 60% of normal are asso-ciated
with increased risk of thromboembolism.
An inherited inability to synthesize normal AT III accounts
for only a very small percentage of patients (2%) with
thromboembolism. Several families with this disorder
have been discovered. These individuals have repeated
episodes of thromboembolism. Thromboembolism is very
rare among young persons; when it does occur, hereditary
AT III deficiency should be considered as a possible cause.
Decreased AT III is more commonly an acquired condition.
Impaired synthesis or increased utilization, or a combi-nation
of the two, may be the basic problem. Since AT III
is synthesized in the liver, diseases of this organ may be
responsible for decreases in plasma AT III levels.
AT III is a molecule about the same size as albumin and,
consequently, may be easily lost in the urine in kidney dis-ease.
Nephrosis, which is characterized by massive albu-minuria,
may also be accompanied by AT III deficiency.
AT III deficiency may result from (as well as cause) exten-sive
intravascular coagulation (DIC). In DIC, thrombin, plas-min,
and other serine proteases may be produced at such
a rate as to deplete plasma AT III.
Prolonged use of oral contraceptives is accompanied by
some decrease in AT III levels. The exact significance of
this is not known, but it may contribute to the slightly
increased incidence of thromboembolism observed
among women takin anovulatory medication. The assay
of plasma AT III is helpful in monitoring heparin therapy.
Patients with low AT III levels have a reduced response
to heparin. Heparin may itself lower AT III levels. When
this occurs, the patient has an increased tendency toward
thrombosis after the heparin therapy is discontinued.
Regardless of cause, low AT III signifies a hypercoagulable
state which can be dangerous and which, with proper
treatment, can be avoided.

27. Fibrin Degradation Products
27
Physiology: The proteolytic enzyme, plasmin, cleaves
fibrin (and fibrinogen), producing variable-sized, soluble
fragments called fibrin split products, or fibrin degrada-tion
products. In normal circulating plasma there is very
little active plasmin, hence only small amounts of FDP
are formed and are quickly removed from the body by
the liver.
Plasmin activity may be increased secondary to a variety
of disease processes. The level of FDP will increase if the
rate of their formation exceeds the rate of removal. They
have an inhibitory effect on the conversion of fibrinogen
to fibrin, and may also impede platelet aggregation. Fi-brin
degradation products are measured in serum, using
an antibody against fibrinogen and FDP. The fragmenta-tion
of fibrin/fibrinogen by plasmin destroys the ability of
fibrinogen to form an insoluble clot, but only slightly
changes the antigenic sites. Fibrinogen, which will cross-react
with the FDP antibody, is removed from the sample
by allowing it to clot.
Clinical Significance: Fibrin degradation products
are present in plasma in small amounts normally. In-creases
indicate clot lysis is proceeding at a faster than
normal rate.
The FDP assay may also be clinically useful in monitoring
patients receiving streptokinase, a plasminogen activator
used in treatment of thromboembolic disorders such as
deep vein thrombosis, pulmonary embolism, and myo-cardial
infarction. Patients receiving streptokinase should
have elevated levels of FDP.
Increases: FDP levels are increased in a number of
diseases such as liver disease, malignant tumors, renal
failure, transplant rejection, and disseminated intravascu-lar
coagulation (DIC). The highest levels occur in DIC (de-scribed
more fully in the section dealing with fibrinogen).
In DIC, the delicate balance between clot formation and
clot dissolution is disturbed and widespread intravascular
clotting occurs. In malignancy or certain obstetrical
complications large amounts of procoagulants may be
released into the blood stream. In massive injury or burns,
the extrinsic clotting pathway is activated. If there is
significant damage to vascular endothelium, plasma is
exposed to collagen and collagen-like substances which
can activate the intrinsic pathway. Whatever the cause
of DIC, there is a compensatory increase in the fibrin-olytic
activity, and an increase in levels of FDP.

28. Fibrinogen (Clotting Factor I)
28
Physiology: Fibrinogen is the soluble precursor of in-soluble
fibrin, the major component of a blood clot. It is a
long, rod-like, moderately large (340,000 daltons) glyco-protein.
The molecule is constructed of polypeptide chains
of three different types designated alpha, beta and gamma,
The alpha, beta and gamma chains are joined together by
disulfide bonds near their N-terminal segment, forming a
monomer. Two such monomers are joined at their heads
again by disulfide bonds to form a dimer of six polypeptide
chains. When fibrinogen is attacked by the hydrolytic en-zyme
thrombin, four segments, two alpha and two beta,
are split off and released as fibrinopeptides A and B (FpA,
FpB). Assay of fibrinopeptides may be used to estimate the
presence and extent of thrombosis. Once thrombin has split
off these terminal segments, the remaining molecules are
free to polymerize into fibrin strands which ultimately form
the basic structure of the blood clot. Most of the total body
fibrinogen is intravascular. The liver synthesizes 2-5 grams
of fibrinogen per day. Its physiologic half-life is about 4
days. The normal catabolic path way for fibrinogen is not
well understood.
Fibrinogen
FpA
FpB
Fibrin
Clinical Significance: Fibrinogen is measured in
plasma. Its concentration may be increased or decreased.
In general, decreases are of more clinical importance
than increases.
Decreases: Decreases in plasma fibrinogen levels
might be expected in liver disease since that organ is
the site of synthesis. Although hypofibrinogenemia does
occur in liver disease, it is not common.
The most common cause of low plasma fibrinogen levels
is disseminated intravascular coagulation (DIC), a condi-tion
in which blood clots form throughout the microvascular
system. The process may be either acute or chronic with a
mild or fatal outcome. There are many causes of DIC. It
accompanies many serious complications of childbirth. Pre-mature
separation of the placenta (abruptio placenta) and
amniotic fluid embolism (the forced injection of amniotic
fluid into the maternal circulation during labor) are the two
most common obstetric incidents, in which DIC occurs.
Other problems which may produce DIC are retention of a
dead fetus and abortion induced by the intra-amniotic in-jection
of hypertonic saline.
DIC can have two serious consequences: The clots may
occlude important blood vessels and the consumption
of fibrinogen (and factors VIII and V) may outstrip pro-duction.
When fibrinogen levels fall to the point where
blood is unable to clot, dangerous bleeding ensues.
Fibrinogen levels below 100 mg/dl are associated with
an increased risk of bleeding.
Increases: Fibrinogen is one of several proteins which
increase in the plasma whenever tissue is damaged. Fi-brinogen
is not usually assayed directly for the purpose
of assessing tissue damage. On the other hand, mea-surement
of the rate at which red cells in anticoagulated
blood aggregate and settle to the bottom of the tube, the
red cell sedimentation rate, is commonly used to detect
and monitor inflammation. Fibrinogen is more potent than
any other plasma protein in increasing the erythrocyte
sedimentation rate.
A large number of inherited disorders of fibrinogen synthe-sis
have been discovered. A total inability to synthesize this
protein (afibrinogenemia) is known, but it is extremely rare.
There are many genetic disorders which presumably are
due to errors in the polypeptide structure although their exact
nature remains to be defined. These disorders are referred
to as dysfibrinogenemias. The quantity of plasma fibrino-gen
is normal in such persons, but the ability to form clots
(functional activity) is impaired, usually due to defective
polymerization. For this reason, assays whose end point is
clot formation are abnormal, and are interpreted as a
decreased fibrinogen level. From a functional point of view,
this interpretation is correct.
Low fibrinogen levels occasionally result from the presence
in plasma of plasmin which has not been inactivated. Plas-min
degrades fibrinogen as it does fibrin. This process is
known as fibrinogenolysis. It may result from the use of
plasminogen activators such as streptokinase in the treat-ment
of clotting disorders, or the release of activators from
malignant tumors. It may also be due to failure of synthesis
of adequate amounts of plasmin inhibitors.

29. 29
Heparin: Heparin was discovered in 1916 by a medi-cal
student, Jay McLean, who was attempting to isolate,
from various tissues, a substance (thromboplastin) which
promotes coagulation. McLean found that certain extracts
of tissues contained a substance which prevented instead
of promoted coagulation. He and his mentor, Professor
W. H. Howell, named this substance “heparin” because
of its abundance in the liver (hepar). High concentrations
were also found to be present in other tissues notably
lung and the walls of small intestines, the two most com-mon
sources of commercial heparin today.
Chemistry: Heparin is a mucopolysaccharide bearing
chemical resemblance to hyaluronic acid, chondroitin
sulfate and keratan sulfate. It is a polymer made up of
repeating units of sulfated and non-sulfated D-glucosamin,
L-iduronic acid, and D-gluronic acid. Phar-macologic
preparations of heparin are heterogeneous
with a distribution of molecular weights between 5,000
and 30,000 daltons. There are a large number of O- and
N- sulfate linkages and carboxyl groups, making it the
strongest organic acid in the body.
Physiology: Mast cells or endothelial cells are the
source of heparin in the body. Mast cells are medium
sized mononuclear cells, the cytoplasm of which is stuffed
with granules which stain intensely with basic stains.
These granules have been found to be comprised mainly
of heparin. Mast cells are found throughout loose con-nective
tissue in the body but are in greatest numbers in
the organs known to be rich in heparin, i.e., lung and
liver. Two main functions have been ascribed to heparin.
The first is the prevention of intravascular clotting of blood
and the second, the clearing of fat from the blood plasma
after the ingestion of a heavy fat meal. The anticoagulation
function of heparin is generally believed to be the most
important one. Heparin has no known effects except those
on blood.
Effects On Coagulation: Heparin inhibits coagulation
of blood in vivo (in the body) and in vitro (in glass). This
inhibitory action requires the presence in blood plasma of
a naturally occurring alpha-globulin, antithrombin III. Anti-thrombin
III (discussed elsewhere in this booklet) is syn-thesized
in the liver. Its presence in plasma prevents the
spontaneous intravascular coagulation of blood, i.e., it helps
maintain blood in the fluid state. Antithrombin III is only slowly
active in the absence of heparin but becomes very active
in its presence. The apparent mechanism of action involves
first the interaction of heparin with the lysine group of anti-thrombin
III which exposes a reactive arginine group which
in turn combines with and inactivates serine proteases.
Coagulation occurs as a result of a series of enzyme
reactions. At each step, an inactive proenzyme is converted
to an active protease which in turn converts a subse-quent
proenzyme to another active protease enzyme.
There are several proteins involved in the coagulation of
blood. Of these, some are activated by serine proteases
(e.g., Prothrombin, and factors VII, IX, X, XI, and XII).
Others are co-factors in these reactions (factors Va, VIIIa,
kallikrein, and kinin) and one is converted to a transami-dating
enzyme (factor XIII). The antithrombin III-heparin
complex appears to neutralize all of the serine proteases.
(The complex will also neutralize plasmin which is any
enzyme active in dissolving clots). Heparin, then, acts at
many stages of clot formation but the net effect is the
inhibition of the formation of thrombin from prothrombin.
Heparin, unlike the anticoagulant, Coumadin®, does not
interfere with the synthesis of coagulation-related proteins.
Heparin has a weak inhibitory effect on thrombin itself.
This is probably not physiologically or pharmacologically
important because it requires 30 to 40 times as much
heparin to inhibit the action of thrombin than to prevents
its formation.
Lipoprotein Lipase Activation: Heparin appears
to have a specific effect on the release of an enzyme,
which catalyzes the hydrolysis of triglycerides in chylo-microns,
releasing free fatty acids which rapidly enter
tissues. This enzyme, lipoprotein lipase, has the effect of
recuing the turbidity of serum. Because of this effect, it is
also known as the “plasma clearing factor.” Uncertainty
remains regarding the physiologic importance of heparin
activation on this lipase.
Absorption and Metabolism: Heparin is inactive
when given orally; it must be administered parenterally.
It may be given intravenously, intramuscularly or subcu-taneously.
The biologic half-life is a function of dosage.
High dosages are associated with relatively long half-lives.
With standard dosage the half-life is in the range of
one to two hours. At these dose levels, it is metabolized
in the liver to a weakly active form which is excreted by
the kidneys. A discovery of this derivative in urine led to its
name - uroheparin. Following very large doses of heparin,
especially when given intravenously, heparin itself does
not cross the placental barrier nor does it enter the milk
of lactating women.
Heparin
Coumadin® is a registered trademark of E.I. du Pont de Nemours &
Company, Wilmington, DE 19898

30. 30
Clinical Uses of Heparin: Heparin is used to pre-vent
intravascular clotting or to stop further progression
of intravascular clotting (clot propagation) once it has
begun. Heparin has the advantage over other antico-agulant
drugs in that it is immediate in its action. In gen-eral,
less heparin is required to prevent the initiation of
clotting (prophylaxis) than to halt active clot formation
(thrombosis).
Physicians use heparin for clot prophylaxis in certain high
risk patients. Among these are those of advanced age,
with fractures of the leg or hip, with pelvic surgery, with
congestive heart disease, with cancer, and with acute
myocardial infarction. Common to this group of patients
is that they may require long periods of bed rest during
which time the blood flow in the large veins of the legs
and pelvis is slowed, increasing the tendency of clot for-mation.
These clots (thrombi) propagate forming fragile
extensions which are not firmly bound to the vessel wall.
Fragments of these clots tend to break off and move in
the blood stream (embolize). A common and serious
event is the movement of a large clot fragment (an em-bolus)
from one of the deep veins of the legs or pelvis to
the lung. The embolus is caught up in one of the major
arteries of the lung abruptly stopping the flow of blood to
that part of the lung. The disorder that follows, pulmonary
embolization, is a very common cause of death in these
patients. The use of low dose heparin in preventing deep
vein thrombosis and high dosages in treating it once it
has occurred has greatly reduced the mortality rate
among these high risk patients.
Heparin is believed to be of value in the treatment of a
phenomenon known as disseminated intravascular coagu-lation.
Normally blood does not clot in the blood vessels.
There are at least two reasons for this. Circulating blood
does not come in contact with substances which induce
clotting and whatever activated clotting factors do appear
are rapidly removed by body cells, particularly those of
the liver. In many diseases, sufficient clot activation factors
may enter the blood stream and initiate clotting throughout
the vascular system. This state is referred to as dissemi-nated
intravascular coagulation (DIC). DIC has many
causes. Among these are serious infections, complications
of pregnancy, malignant disease (cancer), and massive
physical trauma. The successful treatment of DIC requires
the control or elimination of the cause but until that
can be achieved, heparin is useful in preventing further
intravascular clot formations.
Because of the quickness of action and the ease of
neutralization of its anticoagulation effect by the use of
such substances as protamine sulfate, heparin is used
to prevent coagulation during the use of extracorporeal
apparatus such as the heart-lung machine, which is em-ployed
to maintain aeration and circulation of blood while
patients are undergoing heart surgery or hemodialysis
apparatus which are used to clear the blood of unwanted
metabolites in patients without functioning kidneys.
In all of these uses, it is important to monitor the plasma in
order to make appropriate adjustments in heparin dosage.
Tests Used to Monitor Heparin Treatment: In
general, heparin treatment is monitored by the measure-ment
of its effect on coagulation. The first such test was
the measurement of the length of time required for whole
blood to clot in a test tube (the Lee White clotting test).
Today the most commonly used test is the activated partial
thromboplastin test (APTT). This test is performed on
citrated plasma in which the clotting factors have been
activated by a surface contact activator such as kaolin.
The APTT is the time required for clot formations after
the plasma has been treated with the activator, and phos-pholipid
and calcium chloride have been added. The
APTT is sensitive to small amounts of heparin. Unfortu-nately,
commercially available thromboplastin preparations
vary in their sensitivity to heparin. Some are so insensitive
as to fail to detect therapeutic levels of heparin at all.
This variation in sensitivity means that each batch of re-agents
should be tested and standards established. Each
hospital laboratory, therefore, must determine its own
therapeutic range.
An assay system which measures heparin directly and
depends upon the inhibition of factor Xa by heparin-activated
antithrombin III has been introduced. In this
procedure, a synthetic peptide which has a chromogenic
group attached is employed as a substrate for factor Xa.
Factor Xa, a serine protease, causes the release of the
chromogen producing a color change. The assay is sen-sitive
to very small quantities of heparin but even more
important, it is reproducible, accurate, and can be stan-dardized
over several reagent lots and heparin sources.

31. 31
Physiology: Plasminogen is the precursor of the fibrin-splitting
enzyme, plasmin. It is a single chain glycoprotein
with a molecular weight of about 90,000 daltons. It circu-lates
in normal plasma in concentration of about 20 mg/dl.
Plasminogen is synthesized by the liver and has a biologic
half-life of about 2 days. The conversion of inactive plasmi-nogen
to the active, trypsin-like protease, plasmin is brought
about by several activators. The most important of these is
found in the endothelial cells which line blood vessels.
Clinical Significance: Plasminogen is measured in
plasma. Its concentration may be increased or decreased.
In general, only decreases are of clinical importance.
Increases: Physiologic increases in plasminogen con-centrations
occur in pregnancy. Certain contraceptives
may also increase circulating plasminogen levels. In-creases
can occur in the acute phase of inflammation.
Decreases: Low plasminogen levels are usually due
to liver disease with impaired synthesis, or to increased
utilization associated with DIC (this disorder is discussed
in detail in the section on fibrinogen). Low levels may
occur in severe nephrosis due to loss of this relatively
small protein molecule in the urine.
Primary fibrinolysis is a rare cause of low plasminogen
levels. In this disorder abnormally large quantities of plas-minogen
activators are released into the circulation. There
are also rare genetic disorders in which individuals syn-thesize
a defective, inactive plasminogen molecule (dys-functional
plasminogen).
Plasminogen

32. 32

33. Electrolytes
33

34. 34

35. 35
Carbon Dioxide
Physiology: The major end products of the metabolism
of most foodstuffs are water and carbon dioxide (CO2).
Approximately 20 moles of CO2 are produced each day
(the exact amount varies with body size and physical
activity) and excreted by the lungs. The CO2 formed body
cells promptly dissolve in water and combine with it to
form carbonic acid. The latter process is a slow one in a
simple aqueous solution in a test tube, but in the body it
is markedly accelerated by the ubiquitous enzyme, car-bonic
anhydrase. The carbonic acid partially dissociates
into hydrogen ion (H+) and bicarbonate ion (HCO3).
These reactions are summarized in the following equations.
K1 K CO 2 + H2O
H2CO3
2 H+ + HCO3 Thus carbon dioxide is transported from the tissues to the
lungs in three forms: dissolved CO2, undissociated or
molecular H2CO3, and bicarbonate ion (HCO3). At normal
plasma pH (7.4), the amounts of each of these are as
follows: hydrogen ion, 4 x 10–8 M; dissolved CO2, 1 x 10–3 M;
molecular carbonic acid, 5 x 10–6 M; bicarbonate ion,
25 x 10–3 M. These relative amounts are determined by
the equilibrium constants for the equations above.
The role of bicarbonate ion as the major transport form
of CO2 is ordinarily of only passing interest to the physician;
of much greater importance is its role in control of plasma
pH, which normally lies within the narrow range of 7.35
to 7.45. The concentration of bicarbonate ion can and
does vary more with alteration in acid-base balance than
do chloride, sodium, or potassium ion concentrations.
The body has control of the excretion of CO2 through
changes in the rate and depth of respiration. Because
the various forms of CO2 in plasma are in equilibrium, a
change in CO2 concentration will result in a concomitant
change in hydrogen ion and bicarbonate ion concentra-tions.
The lungs are therefore able to compensate to some
degree for abnormal production or loss of hydrogen ion.
The importance of CO2 derives also from that bicarbonate
ion buffering capacity, that is, its effectiveness in limiting
changes in pH when acid or base enters the plasma. At
first glance, the bicarbonate-carbonic acid buffer system
would not appear to be particularly effective at the usual
plasma pH of 7.4 since the effective pK1* of this system
in plasma is 6.1 (buffers work best at pH values close to
their pK). Buffering is in fact remarkably effective because
the concentration of carbonic acid can be changed by
changes in respiration. In brief, the addition of a strong
acid to plasma (the usual direction of pH change in the
body) results in excretion of the lungs of enough carbonic
acid (in the form of CO2 and water) to partially correct the
fall in pH. At the same time, the bicarbonate-carbonic acid
buffer system becomes more effective at this lower pH.
Clinical Significance: Knowledge of the CO2 con-centration
permits a first approximation of the acid-base
balance and goes at least part of the way toward eluci-dating
the cause when an abnormality exists. Full ap-preciation
of the acid-base status also requires knowledge
of plasma pH, buffering capacity, hemoglobin concentra-tion,
pO2 and pCO2. Nevertheless, at the present time,
the determination of CO2 concentration remains the most
commonly performed initial test in the evaluation of the
body’s ability to control pH.
Decreased Bicarbonate With Elevated pH -
Respiratory Alkalosis: If the respiratory rate is in-creased,
there is an increase in excretion of carbon diox-ide,
and levels of plasma carbonic acid and dissolved CO2
fall. The plasma pH rises, hence the name respiratory alka-losis.
The causes of this condition are varied, but they have
in common the over-stimulation of that part of the brain
which controls breathing (the “respiratory center”). The most
common cause is simple anxiety with increased rate and
depth of breathing - the so called “hyperventilation syn-drome”.
Other causes include toxic substances (e.g.,
salicylates, as in aspirin overdosage) which stimulate the
respiratory center, and central nervous system lesions such
as tumors located in this part of the brain.
Decreased Bicarbonate With Decreased pH -
Metabolic Acidosis: The condition usually results
from the addition of excess amounts of acid to the plasma.
Usually these acids are products of normal metabolic
reactions, but they are being formed at a faster rate that
they can be degraded or excreted. For example, in dia-betic
coma, increased amounts of acetoacetic acid are
produced from the oxidation of fatty acids. It accumu-lates
in the plasma because of the decreased ability to
further metabolize it via the citric acid cycle. Most of the
acetoacetic acid is reduced to beta-hydroxybutyric acid.
Both of these acids are increased in the plasma in dia-betic
coma. If insulin is not given to correct the metabolic
abnormalities, coma and death occur. Another example
of metabolic acidosis is the accumulation of lactic acid
in the clinical condition called “shock”. The blood pres-sure
is low and the blood supply to muscles therefore
decreased. The muscles received insufficient oxygen to
completely burn glucose; it is metabolized only to the point
The pK1 value for the net reaction [CO2 + H2O H+ + HCO3
– ]
in plasma (rather than in simple aqueous solution) is 6.1.

36. 36
of formation of pyruvic and then lactic acids. Lactic acid
accumulates in the plasma. In both diabetic coma and shock
the bicarbonate level may be very low, and the pH may fall
below 7.0. Some of the hydrogen ions from these acids
combine with bicarbonate ion to form carbonic acid and
then CO2 and water. The CO2 is excreted by the lungs. Thus
the level of plasma bicarbonate is decreased. There is still
an excess of hydrogen ion in the plasma, so the pH is low.
In fact the changes in this condition can be summarized as
the substitution of a strong acid (lactic or betahydroxybutyric)
for a weak acid (carbonic). Metabolic acidosis also occurs
in patients with kidney disease: phosphoric and sulfuric acids
(produced from the breakdown of phosphate and sulfate-containing
proteins) can not be removed from the plasma
at the normal rate because the kidneys are damaged.
Increase Bicarbonate With Decreased pH-Res-piratory
Acidosis: The condition usually results from
diseases of the lungs which impede the excretion of
carbon dioxide. A typical example is emphysema, a dis-ease
in which there is widespread destruction of lung
tissue. Carbon dioxide, carbonic acid, and hydrogen and
bicarbonate ions accumulate in the plasma.
Increased Bicarbonate With Elevated pH-Metabolic
Alkalosis: This condition can result from
excess intake of sodium bicarbonate (common baking
soda), a commonly used remedy for abdominal pain such
as “heartburn” or pain of peptic ulcer. Metabolic alkalosis
can also result from prolonged vomiting or, in the hospi-talized
patient, over-use of gastric suction with loss of
the acid stomach contents. There is a net loss of a strong
acid, HCl, and a rise in plasma pH. The body attempts to
replace the lost HCl by decreasing the excretion of CO2
by the lungs. Retention of CO2 will produce an increase
in concentrations of hydrogen and bicarbonate ions, thus
correcting the pH change to some extent. The net effect
is to replace a strong acid, HCl, with a weak acid, car-bonic
acid; therefore the pH remains abnormally high.