Clinical Biochemistry and
Molecular Biology
Anna Maria Eleuteri
Clinical Biochemistry
Clinical Biochemistry is an applied science which
studies the effect of a pathology or a drug on the
biochemical processes of organs, tissues and biological
fluids.
It examines all kind of biological samples to analyze
compounds or properties which can be interesting to
prevent, diagnose or treat a disease.
Specimen collection and processing
Collection of blood: it may be obtained from veins, arteries or
capillaries. Venipuncture is the method for obtaining this
specimen.
The medial cubital vein in the antecubital fossa, or crook of the
elbow, is the preferred site for collecting venous blood in adults,
because the vein is large and close to the surface of the skin.
Selection of a vein for venipuncture is facilitated by palpation.
Specimen collection and processing
Collection of blood
Procedure:
-the area around the site has to be cleansed
- a tourniquet is applied 10-15 cm above the site to obstruct the return of venous
blood to the heart and to distend the vein
- the blood volume to be drawn should be estimated
- the appropriate tube for plasma or serum has to be selected
- an appropriate needle must be selected
- when blood collection is complete, the tourniquet should be released and the
needle should be withdrawn
- the patient should hold a gauze pad over the puncture site with the arm raised
to avoid blood leakage.
Specimen collection and processing
Collection of blood
Evacuated blood tubes are considered to be more convenient to
use than syringes.
However syringes are invariably used for patients with difficult
veins.
Vigorous suction on a syringe during collection or forceful transfer
form the syringe to the receiving vessel may cause hemolysis.
Hemolysis is generally less likely to occur when blood is drawn
through a larger-bore needle because turbulence of the blood is
greater when a small-bore needle is used.
Specimen collection and processing
Collection of blood
Evacuated blood tubes are divided into two classes:
those that contain a serum separating material and
those that do not.
Various additives, including anticoagulants and
preservatives, may be used. Stopper color denotes the
type of additive as shown in the table.
Specimen collection and processing
Specimen collection and processing
Collection of blood
Serum separator tubes contain an inert, thixotropic, polymer gel
material with a specific gravity of ~ 1.04.
Silica or glass particles that accelerate clotting may be associated
with the gel.
Aspiration of blood into the tube and subsequent centrifugation
displace the gel, which settles like a disk between cells and
supernatant when the tube is centrifuged.
Release of intracellular components into the supernatant is prevented
by the barrier for several hours or, even, days.
Specimen collection and processing
Collection of blood
Venous occlusion. It is rarely necessary to leave the tourniquet in place
for longer than 1 min, but even within 1 min the composition of blood
changes.
When the blood flow in the vein is obstructed by the tourniquet, the
filtration pressure across the capillary walls is increased. This increase
causes fluid and low molecular weight compounds to pass through the
capillary wall.
The situation is analogous to that caused by a change of posture from
lying to standing.
Although the changes that occur in 1 min are slight, marked changes
have been observed after 3 min.
Specimen collection and processing
Specimen collection and processing
Anticoagulants
If whole blood or plasma is desired for testing an anticoagulant must
be added to the specimen during the collection procedure.
Whole blood is rarely required for clinical chemistry tests (blood gas,
ammonia, trace elements determinations).
Serum from coagulated blood is the specimen of choice for many assay
systems, but plasma obtained with an appropriate anticoagulant may be
an equally valid specimen.
In certain circumstances it may be preferable to serum.
Because harvest of serum requires a wait of 15 to 30 min for
completion of coagulation before centrifugation, use of plasma
expedites analysis in medical emergencies.
Specimen collection and processing
Anticoagulants
The formation of fibrin clots or fragments when
plasma is stored and the subsequent risk of clogging
samples probes of automated analytical instruments
is a disadvantage.
Plasma is also not suitable for electrophoretic
analyses because the presence of fibrinogen can
confound interpretation of electrophoretic patterns.
Specimen collection and processing
Anticoagulants – heparin
It is the most widely used anticoagulant for clinical chemical
analyses. It causes the least interference with tests.
It is a mucoitin polysulfuric acid and is available as
sodium,potassium, lithium, and ammonium salts.
This anticoagulant accelerates the action of antithrombin III,
which neutralizes thrombin and thus prevents the formation of
fibrin from fibrinogen.
About 20 units of heparin are usually required for anticoagulation
of 1 mL of blood; thus, most blood tubes are prepared with ~0.2 mg
heparin for each milliliter of blood to be collected.
The heparin is usually present as a dry powder. It is hygroscopic
and dissolves rapidly.
Specimen collection and processing
Anticoagulants – Ethylenediaminotetraacetic acid
(EDTA)
The chelating agent EDTA is useful for hematological
examinations because it preserves the cellular
components of blood.
It is used as the disodium or dipotassium salt, the latter
being more soluble.
It is effective at a concentration of 1 to 2 mg/ml of
blood.
EDTA prevents coagulation by binding calcium, which is
essential to the clotting mechanism.
Specimen collection and processing
Anticoagulants – Sodium fluoride
It is usually considered a preservative for blood glucose;
however it also acts as a weak anticoagulant.
As a preservative, together with another anticoagulant,
such as potassium oxalate, it is effective at a
concentration of 2 mg/ml of blood.
It inhibits the enzymes involved in glycolysis.
Specimen collection and processing
Anticoagulants – Sodium citrate
Sodium citrate solution, at a concentration of 3.4 or 3.8
g/dl in a ratio of 1 part to 9 parts of blood, is widely
used for coagulation studies, because its effect is easily
reversible by addition of calcium.
It inhibits the enzymes involved in glycolysis.
Specimen collection and processing
Anticoagulants – Oxalates.
Sodium, potassium, ammonium, and lithium oxalates inhibit
blood coagulation by forming rather insoluble complexes with
calcium ions. Potassium oxalate (K2C2O4. H20) at a
concentration of ~ 1 to 2 mg/mL of blood is the most widely
used oxalate.
Specimen collection and processing
Anticoagulants – Iodoacetate.
Sodium iodoacetate at a concentration of 2 g/L is an effective
antiglycolytic agent and a substitute for sodium fluoride.
Because it has no effect on urease, it can be used when glucose
and urea assays are perfomed on a single specimen.
It inhibits creatine kinase but appears to have no significant
effects in other clinical chemistry tests.
The differences between the plasma and serum concentrations of
commonly ordered analytes are shown in the following Table .
Specimen collection and processing
Specimen collection and processing
Influence of Site of Collection on Blood Composition
Blood obtained from different sites differs in composition.
Skin puncture blood is more like arterial blood than venous
blood. Thus, no clinically significant differences exist
between freely flowing capillary blood and arterial blood in
pH, pCO2, p02, and oxygen saturation.
Specimen collection and processing
Influence of Site of Collection on Blood Composition
The pC02 of venous blood is up to 6 to 7 mm Hg (0.8-0.9
kPa) higher.
Venous blood glucose is as much as 7 mg/dL (0.39 mmol/L)
less than the capillary blood glucose as a result of tissue
utilization.
Blood obtained by skin puncture is contaminated to some
extent with interstitial and intracellular fluids.
The major differences between venous serum and capillary
serum are illustrated in Table 2 -4.
Specimen collection and processing
Specimen collection and processing
Hemolysis
Serum shows visual evidence of hemolysis when the hemoglobin
concentration exceeds 20 mg/dL.
Slight hemolysis has little effect on most test values.
Severe hemolysis causes a slight dilutional effect on those constituents
that are present at a lower concentration in the erythrocytes than in
plasma.
However, a marked effect may be observed on those constituents that are
present at a higher concentration in erythrocytes than in plasma.
Thus, plasma concentrations or activities of aldolase, total acid
phosphatase, lactate dehydrogenase, isocitrate dehydrogenase, potassium,
magnesium, and phosphate are particularly increased by hemolysis.
The inorganic phosphate in serum increases rapidly as the organic esters in
the cells are hydrolyzed.
Specimen collection and processing
Hemolysis
Aspartate aminotransferase activity is increased by 2% for each 10 mg/dL
increase in hemoglobin concentration.
Hemoglobin of 10 mg/dL increases serum lactate dehydrogenase by ~10%
and serum potassium by ~ 0.6%.
An additional band due to hemoglobin may be observed on serum protein
electrophoresis.
Although the amount of free hemoglobin could be measured and a
calculation made to correct test values affected by hemoglobin, this
practice is undesirable because factors other than hemoglobin could
contribute to the altered test values, and it would be impossible to assess
their impact.
Hemolysis may affect many unblanked or inadequately blanked analytical
methods.
Specimen collection and processing
HANDLING OF SPECIMENS FOR TESTING
MAINTENANCE OF SPECIMEN IDENTIFICATION
Valid test results require a representative. properly collected, and properly
preserved specimen.
Proper identification of the specimen must be maintained.
Every specimen container must be adequately labeled even if the specimen
must be placed in ice or if the container is so small that a label cannot be
placed along the tube, as might happen with a capillary blood tube. Direct
labeling of a capillary blood tube by folding the label like a flag around the
tube is preferred; a less satisfactory alternative is to label a larger container
into which the capillary tube can be placed.
Specimen collection and processing
HANDLING OF SPECIMENS FOR TESTING
MAINTENANCE OF SPECIMEN IDENTIFICATION
The minimum information on a label should include the patient's name,
location, and identifying number, as well as the date and time of collection.
No specific labeling should be attached to specimens from patients with
infectious diseases to suggest that these specimens should be handled with
special care.
All specimens should be treated as if they are potentially infectious!
However, labels suggesting special handling for other purposes are sometimes
indicated.
Specimen collection and processing
PRESERVATION OF SPECIMENS IN TRANSIT
The specimen must be properly treated both during its transport to the
laboratory and from the time the serum has been separated until it is
analyzed.
For some tests, specimens must be kept at 4°C from the time the blood is
drawn until the specimens are analyzed or until the serum or plasma is
separated from the cells. Transfer of these specimens to the laboratory
must be done by placing the specimen container in ice water.
For all test constituents that are thermally labile, serum and plasma should be
separated from cells in a refrigerated centrifuge. Specimens for bilirubin or
carotene must be protected from both daylight and fluorescent light to avoid
photodegradation.
Some special handling requirements are listed in Table 2 -6.
Specimen
collection and
processing
Specimen collection and processing
SEPARATION AND STORAGE OF SPECIMENS
Plasma or serum should be separated from cells as soon possible, and certainly
within 2 hours.
If it is impossible to centrifuge a blood specimen within 2 hours, the specimen
should be held at room temperature rather than at 4°C to decrease hemolysis.
If the specimens cannot be analyzed at once, the separated serum should
gently be stored in capped tubes at 4°C until analysis, to maintain stability of
the specimen and to reduce evaporation.
If a specimen for a particular test is sufficiently unstable at 4°C, the serum
specimen should be held at -20°C in a freezer capable of maintaining this
temperature.
Specimen collection and processing
SEPARATION AND STORAGE OF SPECIMENS
However, 4°C or -20°C is not the optimal storage temperature for all tests;
some lactate dehydrogenase isoenzymes, for instance, are more stable at
room temperature than at 4°C.
Specimen tubes should be centrifuged with stoppers in place.
Closure reduces evaporation and also prevents aerosolization of infectious
particles. Specimen tubes containing volatile substances such as ethanol must
be stoppered while they are spun.
Centrifuging specimens with the stopper in place maintains anaerobic
conditions that are important in the measurement of carbon dioxide and
ionized calcium.
Removal of the stopper before centrifugation allows loss of carbon dioxide
and an increase in blood pH.
Specimen collection and processing
PHYSIOLOGICAL FACTORS AFFECTING THE COMPOSITION OF
BODY FLUIDS
Standardization of specimen collection practices minimizes variables
that cause changes in test values within a certain day or from one day
to another and thereby reduces the difficulty in interpretation of
values.
However, in hospital practice, standardization is not always possible;
thus, it is important to understand the effects of both controllable
and uncontrollable variables on the composition of body fluids.
Specimen collection and processing
CONTROLLABLE BIOLOGICAL VARIABLES – Posture
The blood volume of an adult in an upright position is tipically 600 to 700
mL less than that of an adult in a recumbent position.
Change from a lying to an upright posture equates to a reduction of about
10% in the blood volume, but because only protein-free fluid passes through
the capillaries to the tissues, the reduction of the plasma volume is greater
than that of the blood volume.
The reduction in plasma is associated with a comparable increase in the
plasma protein concentration.
The concentrations of all proteins, including enzymes and protein hormones,
and of such compounds as drugs, calcium and bilirubin, which circulate partly
bound to proteins are also affected.
Normally, the alteration of blood volume that takes place with a change
from standing to lying is complete in 10 min.
The decrease with the change from lying to standing is complete in 10 min.
Specimen collection and processing
CONTROLLABLE BIOLOGICAL VARIABLES – Posture
Changes in concentration of proteins and protein-bound constituents in
serum is greater in hypertensive persons, in persons with a low plasma
protein concentration, and in elderly persons.
Most of the plasma oncotic pressure is attributable to albumin because
of its high concentration, so protein malnutrition and its associated
reduction of plasma albumin concentration reduces the retention of the
fluid within capillaries.
Conversely, the impact of posture is less in individuals with abnormally
high concentrations of protein, such as those with a monoclonal
gammopathy (multiple myeloma).
Specimen collection and processing
CONTROLLABLE BIOLOGICAL VARIABLES – Posture
In general, the concentrations of freely diffusible constituents with
molecular weights of less than 5000 are unaffected by postural changes.
However, a significant increase in potassium (about 0.2 -0.3 mmol/L)
occurs with 30 min of standing.
This increase in K+ has been attributed to the release of intracellular
potassium from muscle.
Although postural changes affect urinary sodium excretion, sodium
concentration in plasma is only slightly affected.
Changes in the concentration of some major serum costituents with
change in posture are listed in Table 2-7.
Specimen collection and processing
Specimen collection and processing
Hospitalization and Immobilization
With prolonged bed rest fluid retention occurs, and serum protein and
albumin concentrations may be decreased by an average of 5 and 3 g/L,
respectively.
The concentrations of protein-bound constituents are also reduced,
although mobilization of calcium from bones with an increased ionized
fraction compensates for the reduced protein-bound calcium, so total
serum calcium is less affected.
Prolonged bed rest is associated with increased urinary nitrogen
excretion.
Calcium, sodium, potassium, phosphate, and sulfate excretions are
increased; hydrogen ion excretion is reduced, presumably due to
decreased metabolism of skeletal muscles.
When an individua1 becomes active after a period of bed rest, more than
3 weeks are required before calcium excretion reverts to normal, and
another 3 weeks must pass before positive calcium balance is achieved.
Several weeks are required before positive nitrogen balance is restored.
Specimen collection and processing
Exercise
The influence of exercise on the composition of body fluids is
related to the duration and intensity of the activity.
The provoked stress-response causes an increase in the blood
glucose, which stimulates insulin secretion.
Plasma pyruvate and lactate are increased by the increased
metabolic activity of skeletal muscle.
Even mild exercise may increase the plasma lactate two-fold.
Specimen collection and processing
Exercise
Arterial pH and pCO2 are reduced by exercise.
Reduced renal blood flow causes a slight increase in the
serum creatinine concentration.
Competition between uric acid and lactate and products of
increased tissue catabolism for renal excretion cause the
serum urate concentration to increase.
Exercise causes a reduction of cellular ATP, which increases
cellular permeability.
The increased permeability causes slight increases in the
serum activities of enzymes originating from skeletal muscle.
such as aspartate aminotransferase, lactate dehydrogenase,
creatine kinase, and aldolase.
As little as 5 min of walking increases the activity of these
enzymes in plasma.
Specimen collection and processing
Mild exercise produces a slight decrease in the serum
cholesterol and triglyceride concentrations that may persist for
several days.
Generally, the effects of strenuous exercise are exaggerations
of those that occur with mild exercise.
Thus,hypoglycemia and increased glucose tolerance may occur.
The plasma lactate rnay be increased ten-fold.
Severe exercise increases the concentration of plasma proteins
owing to an influx of protein from interstitial spaces, which
occurs after an initial loss of both fluid and protein through the
capillaries.
Specimen collection and processing
Glycoproteins, transferrin and 2-macroglobulin concentrations are
typically increased. Fibrinolytic activity is also increased.
Strenuous exercise may more than double creatine kinase activity,
but the activity of enzymes with primarily liver or kidney origins is
little changed, although both hepatic and renal blood flow are
reduced.
Some representative changes in concentration or activity of serum
constituents induced by strenuous exercise are listed in Table 2 - 8.
These are typically exaggerations of the effects of moderate
exercise.
Specimen
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processing
Specimen collection and processing
Physical Training
Athletes generally have a higher serum activity of enzymes of
skeletal muscular origin at rest than do non-athletes.
However, the response of these enzymes to exercise is less in
athletes than in other individuals.
Serum concentrations of urea, urate, creatinine, and thyroxine
are higher in athletes than in comparable untrained individuals.
This is probably related to the increased muscle mass and a good
turnover of muscle mass in athletes.
Specimen collection and processing
Physical Training
The total serum lipid concentration is reduced by physical
conditioning; serum cholesterol may be lowered by as much as
25%. High-density lipoprotein (HDL)-colesterol,however, is
increased.
Thus, the decrease is mostly due to a reduction in low-density
lipoprotein (LDL)-cholesterol.
The serum triglyceride concentration may be reduced by up to
20 mg/dL (0.23 mmol/L) but the free fatty acid concentration
is higher in fit individuals than in others.
Generally, the same exercise produces a less marked
biochemical response in the fit person than in the unfit person.
Specimen collection and processing
Circadian Variation
Many constituents of body fluids exhibit cyclical variations
throughout the day.
Factors contributing to such variations include posture,
activity, food ingestion, stress, presence of daylight or
darkness, and state of sleep or wakefulness.
These cyclical variations may be quite large, and therefore the
drawing of the specimen must be strictly controlled.
The concentration of serum iron, for example, may change by
as much as 50% from 8.00 to 14.00 hours, and that of cortisol
may change by a similar amount between 8.00 and 16.00 hours.
The typical total variation of several commonly measured serum
constituents over 6 hours is illustrated in Table 2-9.
The total variation is listed together with analytical error.
Specimen
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Specimen collection and processing
Circadian Variation
Hormones are secreted in bursts and this, together with the
cyclical variation to which most hormones are subject, may
make it very difficult to properly interpret their serum
concentration.
Corticotropin secretion is influenced by cortisol-like steroids,
but it is also affected by posture, light and darkness and
stress.
Its secretion is increased three- to five-fold from its minimum
between afternoon and midnight to its maximum around waking.
Cortisol concentrations are greatest around 6.00 to 8.00 hours.
Specimen collection and processing
Circadian Variation
Maximum renin activity normally occurs early in the morning
during sleep; its minimum occurs late in the afternoon.
Glomerular filtration rate (GFR) varies inversely with the
secretion of renin; GFR is least at the time of maximum renin
secretion and -20% greater in the afternoon when renin
activity is at a minimum.
No circadian variation exists in the plasma concentrations of
follicle-stimulating hormone and luteinizing hormone in men, but
a 20% to 40% increase of plasma testosterone occurs during
the night.
Serum thyroid-stimulating hormone is at a maximum
concentration between 2.00 and 4.00 hours and at a minimum
between 18.00 and 22.00 hours.
The variation is of the order of 50%.
Specimen collection and processing
Circadian Variation
Growth hormone secretion is greatest shortly after sleep
commences.
Conversely, basal plasma insulin concentration is higher in the
morning than later in the day, and its response to glucose is
greatest in the morning and least about midnight.
When a glucose tolerance test is performed in the afternoon,
higher glucose values are obtained than when the test is given
early in the day.
Specimen collection and processing
INFLUENCE OF FOOD AND STIMULANTS
Recent Food Ingestion
The concentration of certain plasma constituents is affected
by the ingestion of a meal.
The biggest increases in serum concentrations occur for
glucose, iron, total lipids and alkaline phosphatase.
The increase in alkaline phosphatase (mainly intestinal
isoenzyme) is greater when a fatty meal is ingested and is
influenced by the blood group of the individual and the
substrate used for the enzyme assay.
Lipemia may affect some analytical methods used to measure
serum constituents.
Ultracentrifugation or the use of serum blanks can reduce the
adverse analytical effects of lipemia.
Specimen collection and processing
INFLUENCE OF FOOD AND STIMULANTS
Recent Food Ingestion
Because the effects of a meal may be long lasting, ingestion of a
protein-rich meal in the evening may cause increases in the serum
urea nitrogen, phosphorus, and urate concentrations that are still
apparent 12 hours later.
Nevertheless, these changes may be less than the typical
intraindividual variability.
Large protein meals at lunch or in the evening also increase the
serum cholesterol and growth hormone concentrations for at
least 1 hour after a meal.
Specimen collection and processing
INFLUENCE OF FOOD AND STIMULANTS
Recent Food Ingestion
The effect of carbohydrate meals on blood composition is less
than that of protein meals.
No change in the cortisol concentration is noted when breakfast
is taken, probably because cortisol occupies all cortisol-binding
sites on its binding protein in the early morning.
Glucagon and insulin secretions are stimulated by a protein meal
and insulin is also stimulated by carbohydrate meals.
Specimen collection and processing
INFLUENCE OF FOOD AND STIMULANTS
Ingestion of Specific Foods and Beverages
Caffeine, which is contained in many beverages including
coffee, tea, and colas, has considerable effect on the
concentration of blood constituents.
Caffeine stimulates the adrenal medulla, causing an increased
excretion of the catecholamines and their metabolites and a
slight increase in the plasma glucose concentration with
impairment of glucose tolerance.
The adrenal cortex is also affected; plasma cortisol is
increased, accompanied by increased excretion of free cortisol,
11-hydroxycorticoids and 5-hydroxyindoleacetic acid.
Specimen collection and processing
INFLUENCE OF FOOD AND STIMULANTS
Ingestion of Specific Foods and Beverages
The effect of caffeine may be so marked that the normal diurnal
variation of plasma cortisol may be eliminated.
Caffeine has a marked effect on lipid metabolism.
Ingestion of two cups of coffee may increase the plasma free
fatty acid concentration by as much as 30%.
Caffeine is also a potent stimulant of gastric juice, hydrochloric
acid and pepsin secretion.
Coffee has a diuretic effect and also increases the excretion of
erythrocytes and renal tubular cells in the urine.
Specimen collection and processing
Smoking
Smoking, through the action of nicotine, may affect several
laboratory tests.
The extent of the effect is related to the number of cigarettes
smoked and to the amount of smoke inhaled.
Through stimulation of the adrenal medulla, nicotine increases the
concentration of epinephrine in the plasma and the urinary
excretion of catecholamines and their metabolites.
Glucose concentration rnay be increased by 10 mg/dL (0.56
mmol/L) within 1O min of smoking a cigarette.
The increase may persist for 1 hour.
Plasma insulin concentration shows a delayed response to the
increased blood glucose, rising about 1 hour after a cigarette is
smoked.
Specimen collection and processing
Smoking
Typically, the plasma glucose concentration is higher in smokers than in
non-smokers, and glucose tolerance is mildly impaired in smokers.
The plasma growth hormone concentration is particularly sensitive to
smoking.
It may increase ten-fold within 30 min after an individual has smoked a
cigarette.
Plasma -lipoprotein, cholesterol, and triglyceride concentrations are
higher and HDL-cholesterol levels lower in smokers than in non
smokers.
Free fatty acid concentration tends to be variable, but inhalation
during smoking produces an immediate increase of free fatty acids of
about 30%.
Some of the effects of smoking on serum constituents are listed in
Table 2- 10.
Specimen collection and processing
Specimen collection and processing
Smoking
Smoking affects the adrenal cortex as well as the medulla; plasma 11hydroxycorticosteroids may be increased by 75% with heavy smoking.
In addition, the plasma cortisol concentration may increase by as much as
40% within 5 min of the start of smoking, although the normal diurnal
rhythmicity of cortisol is unaffected.
Smokers excrete more 5-hydroxyindoleacetic acid than do non smokers.
The blood erythrocyte count is increased in smokers.
The amount of carboxyhemoglobin may exceed 10% of the total hemoglobin
in heavy smokers, and the increased number of cells compensates for
impaired ability of the erythrocytes to transport oxygen. The blood p02 of
the habitual smoker is usually about 5 mm Hg (0.7 kPa) less than in the nonsmoker, whereas the pC02 is unaffected.
The blood leukocyte concentration is increased by as much as 30% in
smokers, but the leukocyte concentration of ascorbic acid is greatly
reduced.
Specimen collection and processing
Alcohol Ingestion
A single moderate dose of alcohol has few effects on laboratory tests.
Ingestion of enough alcohol to produce mild inebriation may increase the
blood glucose concentration by 20% to 50%.
The increase may be even more marked in diabetics.
More commonly, inhibition of gluconeogenesis occurs and becomes apparent
as hypoglycemia.
Marked hypertriglyceridemia following alcohol ingestion is due to a
combination of increased triglyceride formation in the liver and impaired
removal of chylomicrons and very-low-density lipoproteins (VLDL) from the
circulation.
The effect is most noticeable when alcohol is ingested with a fatty meal.
The effect may persist for longer than 12 hours.
When moderate amounts of alcohol are ingested for 1 week, the serum
triglyceride concentration is increased by more than 20 mg/dL
(0.23mmol/L).
Intoxicating amounts of alcohol stimulate the release of cortisol, although
the effect is more related to the intoxication than to the alcohol per se.
Specimen collection and processing
Alcohol Ingestion
Sympathicomedullary activity is increased by acute alcohol ingestion but
without detectable effect on the plasma epinephrine concentration and
with only a mild effect on norepinephrine.
With intoxication, plasma concentrations of catecholamines are markedly
increased.
Chronic alcohol ingestion affects the activity of many serum enzymes.
-glutamyltransferase activity has been most studied and increased activity
of the enzyme is used as a marker of persistent drinking.
Chronic alcoholism is associated with many characteristic biochemical
abnormalities, including abnormal pituitary, adrenal cortical and medullary
function.
Acute alcohol ingestion has been reported to increase the activity of
several serum enzymes, including -glutamyltransferase, isocitrate
dehydrogenase, and ornithine carbamoyl transferase.
Specimen collection and processing
DRUG ADMINISTRATION
It is rare for a patient to be hospitalized without receiving some
drugs. For certain medical conditions, more than 10 drugs may be
administered at one time.
Individuals with chronic diseases often ingest drugs on a continuing
basis. Drugs may have both in vivo and in vitro effects on laboratory
tests.
The in vivo effects arise from the therapeutic intent of drugs, their
side effects, and patient idiosyncrasies.
Effects on the composition of body fluids are likely to be more
apparent when large doses of a drug are administered for a long time
than when administration of a single dose occurs on an isolated
occasion.
Specimen collection and processing
DRUG ADMINISTRATION
When administered intramuscularly, many drugs cause sufficient muscle
irritation to increase amounts of enzyme released into the serum.
The activities of creatine kinase, aldolase, and the skeletal muscle
component of lactate dehydrogenase are increased in the serum.
The increased activities may persist for several days after a single
injection, and consistently high values may be observed during a course of
treatment.
Penicillin derivatives given intramuscularly are particularly likely to increase
the activity of these enzymes, although any drug given intramuscularly
appears capable of increasing enzyme activity.
Opiates such as morphine or meperidine can cause spasm of the sphincter
of Oddi. The spasm transmits pressure back to the liver, causing release of
liver and pancreatic enzymes into the serum. Increases in aspartate
aminotransferase activity may be so large that they may be suggestive of a
myocardial infarction.
Specimen collection and processing
DRUG ADMINISTRATION
Oral contraceptives affect many different constituents measured in the
clinical laboratory. Tests are affected by both the progestin and estrogen
components. The overall effect depends on the proportion of the two
components.
Diuretic drugs often cause a mild reduction of the plasma potassium
concentration; hyponatremia may be observed. Hypercalcemia may occur
with hemoconcentration, but occasionally the ionized as well as the proteinbound fraction is increased.
Thiazides cause hyperglycemia and reduce glucose tolerance, especially in
diabetics. Thiazides may cause prerenal azotemia with hyperuricemia due to
decreased renal blood flow and glomerular filtration rate as a result of
reduced blood volume.
Specimen collection and processing
UNDERLYING MEDICAL CONDITIONS
Some general clinical conditions have an effect per se on the composition of
body fluids. These conditions may exist in addition to the primary complaint
that prompted a patient's admission to the hospital.
Fever
Fever provokes many hormone responses. Hyperglycemia occurs early and
stimulates the secretion of insulin, which improves glucose tolerance; but
insulin secretion does not necessarily reduce the blood glucose
concentration, as increased secretion of growth hormone and glucagon
also occurs.
Fever appears to reduce the secretion of thyroxine, as do acute illnesses
without fever.
Specimen collection and processing
Fever
In response to increased corticotropin secretion, the plasma cortisol
concentration is increased and its normal diurnal variation may be abolished.
The urinary excretion of free cortisol, 17-hydroxycorticosteroids. and 17ketosteroids is increased. If acute fever lessens but persists for a
prolonged period, or if it subsides, the hormone responses diminish.
Glycogenolysis and a negative nitrogen balance occur with the onset of
fever. These are prompted by the typically decreased food intake and
wastage of skeletal muscle that accompany fever.
Although there is usually an increase in the blood volume with fever, the
serum concentrations of creatinine and uric acid are usually increased.
Aldosterone secretion is increased with retention of sodium and chloride.
Secretion of antidiuretic hormone also contributes to the retention of
water by the kidneys.
Specimen collection and processing
Fever
Increased synthesis of protein occurs in the liver, and the plasma
concentrations of acute-phase reactants and glycoproteins are increased.
Fever accelerates lipid metabolism.
The serum concentrations of cholesterol, nonesterified fatty acids and the
other lipids may decrease initially, but within a few days the free fatty
acid concentration may increase markedly.
Specimen collection and processing
Shock and Trauma
Regardless of the cause of shock or trauma certain characteristic
biochemical changes ensue.
Corticotropin secretion is stimulated to produce a three- to five-fold
increase in the serum cortisol concentration.
Aldosterone secretion is stimulated.
Plasma renin activity is increased, as are the secretions of growth hormone,
glucagon and insulin.
Anxiety and stress increase the excretion of catecholamines.
The stress of surgery has been shown to reduce the serum
triiodothyronine concentration by 50% in patients without thyroid disease.
Specimen collection and processing
Shock and Trauma
Loss of fluid to extravascular tissues takes place immediately following an
injury, with a resultant decrease in plasma volume.
If the decrease is enough to impair circulation glomerular filtration is
diminished. Diminished renal function leads to the accumulation of urea and
other end products of protein metabolism in the circulation.
In burned patients, serum total protein concentration falls by as much as 0.8
g/dL because of both loss to extravascular spaces and catabolism of protein.
Serum 1, 2 and -globulin concentrations increase but not enough to
compensate for the reduced albumin concentration.
The plasma fibrinogen concentration responds dramatically to trauma and
may double in 2 to 8 days following surgery.
With tissue destruction, urinary excretion of the major components of
skeletal muscle increases.
The muscle damage associated with the trauma of surgery can markedly
increase the serum activity of enzymes originating in skeletal muscle, and this
increased activity may persist for several days.
Specimen collection and processing
LONG-TERM BIOLOGICAL INFLUENCES ON BODY FLUIDS
Published reference intervals usually are broad; however
intraindividual variability is significantly less than group or
population variability.
Indeed, a situation can arise in which the values obtained in one
individual are very different from those found in another
individual, yet both sets of values could fall within a predetermined
reference interval.
Not only does the set-point for different individuals appear to be
different, but the extent to which values change from one occasion
to another, even when specimens are obtained under standardized
conditions, appears to differ among individuals.
Specimen collection and processing
LONG-TERM BIOLOGICAL INFLUENCES ON BODY FLUIDS
Both of these factors are probably determined genetically, and
the difference in set-points underscores the fallibility of using a
population reference interval to associate small changes in
laboratory data with a deviation from health for a given
individual.
Age, second only to gender, influences reference values, but the
most important influences that determine the overall effect of
age are the degree of sexual maturity and the amount of skeletal
muscle mass of the individual. Generally, individuals can be
considered in four groups- the newborn, the older child to
adolescent, the sexually mature adult, and the elderly adult.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Newborn
The body fluids of the newborn infant reflect both the trauma
of birth and the changes related to the infant's adaptation to an
independent existence.
The composition of the blood is affected by the maturity of the
infant at birth. In the mature infant, most of the hemoglobin is
the adult form, hemoglobin A, whereas in the immature infant,
much of the hemoglobin may be the fetal form, hemoglobin F.
In infants, even in the absence of disease, the concentration of
bilirubin rises following birth and peaks about the third to fifth
day of life.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Newborn
The physiological jaundice of the newborn rarely produces serum
bilirubin values greater than 5 mg/dL (85 pmol/L). Distinguishing
this naturally occurring phenomenon from other conditions that
produce neonatal hyperbilirubinemia may be difficult and the
chronological course of the hyperbilirubinemia is important.
The blood glucose concentration is low in newborns because of
their small glycogen reserves, although some attribute the low
glucose level to adrenal immaturity.
Blood lipid concentrations are low but reach -80% of the adult
values after 2 weeks.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Newborn
The plasma urea nitrogen concentration decreases following birth
as the infant synthesizes new protein, and the concentration
does not begin to rise until tissue catabolism becomes prominent.
The serum thyroxine concentration of the healthy newborn, like
that in the pregnant woman, is considerably higher than in the
nonpregnant adult.
Following its birth, an infant secretes thyroid-stimulating
hormone, which causes a further increase in the serum thyroxine
concentration.
The physiological hyperthyroidism gradually declines over the
first year of life.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Older Child to Adolescent
Plasma protein concentrations increase after infancy and adult
concentration values are attained by the age of 10 years.
Serum IgG increases slightly out of proportion to the increase in
concentration of 2-globulin.
The serum activity of most enzymes decreases during childhood
to adult values by puberty or earlier, although the activity of
alanine aminotransferase may continue to rise, at least in men,
until middle age.
Serum alkaline phosphatase activity is high in infancy, but it
decreases during childhood and rises again with growth before
puberty.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Older Child to Adolescent
The activity of the enzyme is better correlated with skeletal
growth and sexual maturity than with chronological age; it is
greatest at the time of maximum osteoblastic activity occurring
with bone growth. The activity decreases rapidly after puberty,
especially in girls.
The serum creatinine concentration increases steadily from
infancy to puberty, parallel to development of skeletal muscle;
until puberty, there is little difference in the concentration
between sexes.
The serum uric acid concentration decreases from its high at
birth until age 7 to 10 years, at which time it begins to increase,
especially in boys, until about age 16 years.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Sexually Mature Adult
The concentrations of most test constituents remain quite
constant between puberty and menopause in women and between
puberty and middle age in men.
During the midlife years serum total protein and albumin
concentrations decrease slightly. There may be a slight decrease
in the serum calcium concentration in both sexes.
In men, the serum phosphorus decreases markedly after age 20
years; in women, the phosphorus also decreases until menopause,
when a marked increase takes place. The serum alkaline
phosphatase begins to rise in women at menopause so that in
elderly women activity of this enzyme may actually be higher
than that in men.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Sexually Mature Adult
Serum uric acid concentrations peak in men in their 20s and in
women during middle age.
Urea concentration increases in both sexes in middle age.
Age does not affect the serum creatinine concentration in men,
but it does increase the concentration in women. The serum
total cholesterol and triglyceride concentrations increase in both
men and women at a rate of -2 mg/dL (0.02 mmol/L) per year to a
maximum between ages 50 and 60 years.
The activity of most enzymes in serum is less during adult life
than during adolescence. This increased enzyme activity
presumably reflects the greater physical activity of adolescents.
The concentration of glucose in plasma 1 hour after a loading
dose rises ~8 mg/dL (0.44 mmol/L) per decade.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Elderly Adult
Significant increases in the plasma concentrations of many
constituents occur in women after menopause.
Estrogen secretion in women begins to decrease before the
menopause and continues at a greater rate after the menopause,
whereas gonadotropins show a feedback-mediated reciprocal
rise.
Serum concentrations of estrogens decrease by 70% or more and
urinary excretion of estrogens is decreased comparably.
The decreased estrogen secretion may be responsible for the
increase of serum cholesterol that occurs up to age 60 years
in women.
Estrogen secretion in men, although always less than in women,
declines with age.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Elderly Adult
Renal concentrating ability is reduced in the elderly adult, so that
creatinine clearance may decline by as much as 50% between the
third and ninth decades.
This decreased clearance is caused more by a decrease in urinary
creatinine excretion as a result of decreased lean body mass
than by renal problems.
The tubular maximum capacity for glucose is reduced.
The plasma urea concentration rises with age, as does the urinary
excretion of protein.
Hormone concentrations are also affected by aging.
However, changes in concentration are much less pronounced
than an endocrine organ's response to stimuli.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Elderly Adult
Triiodothyronine concentration decreases by up to 40% in persons older
than 40 years. Although thyroxine secretion is reduced, the thyroxine
concentration is not changed, because its degradation is also reduced.
Plasma parathyroid hormone concentration decreases with age.
Cortisol secretion is reduced, although the serum concentration
may not be affected.
The secretion and metabolic clearance of aldosterone are decreased with
a reduction of -50% in the plasma concentration.
The aldosterone response to sodium restriction is diminished.
Basal insulin concentration is unaffected by aging, but its response to
glucose is reduced. In the male, the secretion rate and concentration of
testosterone are reduced after age 50 years.
In women, the concentration of pituitary gonadotropins, especially
follicle-stimulating hormone, is increased in the blood and urine.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Gender
Until puberty, few differences in laboratory data are seen between boys
and girls.
After puberty, the serum activities of alkaline phosphatase, the
aminotransferases, creatine kinase, and aldolase are greater in men than
in women. The higher activity of enzymes originating from skeletal muscle
in men is related to their greater muscle mass.
The concentrations of albumin, calcium and magnesium are higher in men
than in women, but the concentration of -globulin is less.
Blood hemoglobin concentrations are lower in women; thus, the serum
bilirubin concentrations are also slightly lower.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Gender
Serum iron is low during woman's fertile years, and her plasma ferritin
may be only one third the concentration of that in males.
The reduced iron concentration in women is attributable to menstrual
blood loss.
Cholesterol concentration is typically higher in men than in women, while
the -lipoprotein concentration is less.
The plasma amino acid concentrations as well as the concentrations of
creatinine, urea and uric acid are higher in men than in women.
The effect of age on the difference in concentrations of serum
constituents between men and women is illustrated in Table 2 – 11.
Specimen collection and processing
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Race
Differentiation of the effects of race from those of socioeconomic
conditions is often difficult.
Nevertheless, the total serum protein concentration is known to be
higher in blacks than in whites.
This is largely attributable to a much higher -globulin concentration,
although usually the concentrations of 1- and -globulins are also
increased.
The serum albumin concentration is typically lower in blacks than in
whites. In black men, serum IgG is often 40% higher, and serum IgA may
be as much as 20% higher than in white men.
The activity of creatine kinase and lactate dehydrogenase is usually much
higher in both black men and women than in white men and women.
Specimen collection and processing
INFLUENCES OF AGE, GENDER, AND RACE
Race
This is presumed to be related to the amount of skeletal muscle, which
tends to be greater in blacks than in whites.
After age 40 years the serum cholesterol and triglyceride concentrations
are consistently higher in both white men and women than in black men
and women.
These may be dietary rather than racial factors, because the
concentration of plasma lipids has been shown to be different for the
same racial group in different parts of the world.
The blood hemoglobin concentration is as much as 1.0 g/dL higher in
whites than in blacks.
Specimen collection and processing
EFFECT OF ENVIRONMENTAL FACTORS
In individuals living at a high altitude the blood hemoglobin is
markedly increased due to reduced atmospheric pO2.
Erythrocyte 2,3-diphosphoglycerate is also increased, and the oxygen
dissociation curve is shifted to the right.
The increased erythrocyte concentration leads to an increased
turnover of nucleoproteins and excretion of urate.
The fasting, basal concentration of growth hormone concentration is
high in individuals living at a high altitude.
Specimen collection and processing
LONG-TERM CYCLICAL CHANGES
Seasonal lnfluences
Seasonal influences on the composition of body fluids are small in
comparison with those related to changes in posture or effected by
misuse of a tourniquet.
Probable factors are dietary changes as different foods come into
season and altered physical activity as more or different forms of
exercise become feasible.
In summer in the northern hemisphere, the -globulin may increase by
as much as 50%.
Serum urate concentration appears to be about 5% to 7% higher in
summer than in winter.
Serum triglyceride concentration is up to 10% higher in summer,
whereas the serum cholesterol has been reported to be up to 50
mg/dL ( 1.3 mmol/L) higher in men and 30 mg/dL (0.7 mmol/L) higher
in women in winter than in summer.
Specimen collection and processing
LONG-TERM CYCLICAL CHANGES
Seasonal lnfluences
Activities of serum enzymes arising from skeletal muscle are higher
in summer than in winter, presumably as a result of increased physical
activity. The increase of serum lactate dehydrogenase may be as
much as 20%.
Calcium metabolism is affected by an individual's exposure to
sunlight. Dehydrocholecalciferol in the skin is converted by
ultraviolet irradiation to cholecalciferol, which is further
metabolized in the liver and kidney to 1,25-dihydroxycholecalciferol.
The calcium concentration in serum is increased, as is its elimination
in urine.
Exposure to sunshine for a weekend during summer may cause enough
photodegradation of bilirubin to reduce the serum concentration by
20%. Some seasonal effects on the composition of body fluids are
listed in Table 2- 12.
Specimen collection and processing
Specimen collection and processing
LONG-TERM CYCLICAL CHANGES
Influence of Menstrual Cycle
The plasma concentrations of many female sex hormones, as well as
other hormones, are affected by the menstrual cycle.
Thus, the plasma corticosterone concentration is as much as 50%
higher in the luteal phase than in the follicular phase.
The urinary excretion of 17-hydroxycorticosteroids reaches a peak
at midcycle.
Plasma androstenedione concentration and plasma aldosterone
concentration increase from the follicular phase to the luteal phase
of the menstrual cycle.
On the pre-ovulatory day the aldosterone concentration may actually
be twice that of the early part of the follicular phase.
The change in renin activity is almost as great.
These changes are usually more marked in women who retain fluid
prior to menstruation.
Specimen collection and processing
LONG-TERM CYCLICAL CHANGES
Influence of Menstrual Cycle
Urinary catecholamine excretion increases at midcycle and remains
high throughout the luteal phase.
Specimen collection and processing
BODY HABITUS
The serum concentrations of cholesterol, triglycerides and
- lipoproteins are positively correlated with obesity.
The serum urate concentration is also correlated with body weight,
especially in individuals weighing more than 80 kg.
Serum lactate dehydrogenase activity serum and glucose
concentration increase in both sexes with increasing body weight.
In men, serum aspartate amino-transferase, creatinine, and total
protein increase with increasing body weight, as does the blood
hemoglobin concentration.
In women, serum calcium increases with increasing body weight.
In both sexes, serum phosphate decreases with increased body mass.
Cortisol production is increased in obese individuals.
However, increased metabolism maintains the serum concentration
unchanged so that urinary excretion of 17-hydroxycorticosteroids
and 17-ketosteroids is increased.
Specimen collection and processing
BODY HABITUS
Because growth hormone concentration is reduced in obese
individuals, it responds poorly to the normal challenges.
Plasma insulin concentration is increased, but glucose tolerance is
impaired in the obese.
Although the serum thyroxine concentration is unaffected by
obesity, the serum triiodothyronine concentration correlates
significantly with body weight and increases further with overeating.
In obese men, the serum testosterone concentration is reduced.
Specimen collection and processing
INFLUENCE OF DIET
An individual's typical diet has considerable influence on the
composition of his or her plasma.
Studies with synthetic diets have shown that day-to-day changes in
the amount of protein are reflected within a few days in the
composition of the plasma and in the excretion of end products of
protein metabolism.
Specimen collection and processing
INFLUENCE OF DIET
Vegetarianism
In long-time vegetarians, the concentrations of low-density and verylow-density lipoproteins are low.
The total lipid and phospholipid concentrations are reduced, and the
concentrations of cholesterol and triglyceride may be only two-thirds
of those in persons on a mixed diet. The effects are less marked in
individuals who have been on a vegetarian diet for only a short time.
The lipid concentrations are also less in individuals who eat only a
vegetable diet than in those who consume eggs and milk as well.
When individuals previously on a mixed diet begin a vegetarian diet,
their serum albumin concentration may fall by 10% and their urea
concentration by 50%.
However, there is little difference in the concentration of protein or
of activities of enzymes in the serum of long-time vegetarians and
individuals on a mixed diet.
Specimen collection and processing
INFLUENCE OF DIET
Malnutrition
In malnutntion, total serum protein, albumin, and -globulin
concentrations are reduced. The increased concentration of globulin does not fully compensate for the decrease in other proteins.
The concentrations of complement C3, retinol-binding globulin,
transferrin and prealbumin decrease rapidly with the onset of
malnutrition and are measured to define the severity of the
condition.
The plasma concentrations of lipoproteins are reduced and serum
cholesterol and triglycerides may be only 50% of the concentrations
in healthy individuals.
In spite of severe malnutrition, glucose concentration is maintained
at a level close to that in healthy individuals.
Specimen collection and processing
INFLUENCE OF DIET
Malnutrition
However, the concentrations of serum urea and creatinine are
greatly reduced as a result of decreased skeletal mass and creatinine
clearance is also decreased.
Plasma cortisol concentration is increased due to decreased
metabolic clearance.
The plasma concentration of total triiodothyronine, thyroxine, and
thyroid-stimulating hormone are considerably reduced, with the
thyroxine concentration being most affected.
This is partly due to reduced concentrations of thyroxine-binding
globulin and prealbumin.
Specimen collection and processing
INFLUENCE OF DIET
Malnutrition
Erythrocyte and plasma folate concentrations are reduced in proteincalorie malnutrition, but the serum vitamin B12 concentration is
unaffected or may even be slightly increased.
The plasma concentrations of vitamins A and E are much reduced.
Although the blood hemoglobin concentration is reduced, the serum
iron concentration is initially little affected by malnutrition.
The activity of most of the commonly measured enzymes is reduced,
but it increases with restoration of good nutrition.
Specimen collection and processing
INFLUENCE OF DIET
Fasting and Starvation
Withdrawal of most or all caloric intake has been used to treat
certain cases of obesity. Such withdrawal provokes many metabolic
responses. The body attempts to conserve protein at the expense of
other sources of energy such as fat. The blood glucose concentration
decreases by as much as 18 mg/dL ( 1 mmol/L) within the first 3 days
of the start of a fast despite the body's attempts to maintain
glucose production.
Specimen collection and processing
INFLUENCE OF DIET
Fasting and Starvation
Insulin secretion is markedly reduced, whereas glucagon secretion
may double in an attempt to maintain normal glucose concentration.
Lipolysis and hepatic ketogenesis are stimulated.
Ketoacids and fatty acids become the principal sources of energy for
muscle. In addition, the concentrations of ketone bodies, fatty acids,
and glycerol in serum rise considerably.
Serum triglycerides increase by 20% after 48 hours of fasting but
decline thereafter; the cholesterol concentration also decreases.
Amino acids are released from skeletal muscle and the plasma
concentration of the branched-chain amino acids may increase by as
much as 100% with 1 day of fasting.
Specimen collection and processing
INFLUENCE OF DIET
Fasting and Starvation
Despite the catabolism of tissue induced by starvation, the serum
protein concentration is little affected initially; ultimately, a
reduction occurs.
However, from the beginning, the catabolism of nucleoproteins
causes an increased serum urate level. Rise in serum urate is
exacerbated by the reduced glomerular filtration rate and the
competition for excretion from lactate and ketoacids.
With the onset of starvation, aldosterone secretion increases with
the results of increased urinary excretion and decreased plasma
concentration of potassium.
Specimen collection and processing
INFLUENCE OF DIET
Fasting and Starvation
Magnesium, calcium, and phosphate are affected similarly, although
the urinary excretion of phosphate gradually declines.
Plasma growth hormone concentration may rise by as much as 15
times at the start of a fast but may return to normal after 3 days.
Levels of free and total triodothyronine decrease by up to 50%
within 3 days of the start of a fast.
Free thyroxine concentration is also affected, but to a lesser extent;
total thyroxine is little changed. Urinary free cortisol is decreased
by fasting and the plasma cortisol concentration (free and total)
shows a slight increase along with loss of the normal diurnal variation.