REVIEW NOTES IN CLINICAL CHEMISTRY Flashcards
: the act of obtaining a blood sample from a vein using a needle attached to a syringe or a stoppered evacuated tube; it is the most common way to collect blood specimens
VENIPUNCTURE
THE MAJOR VEINS FOR VENIPUNCTURE are in the antecubital fossa, the area of the arm in front of the elbow. The H pattern is displayed by approximately 70% of the population and includes the following veins:
Median
Cephalic
Basilic
Located near the center of the antecubital fossa
Median cubital vein
• Preferred vein because it is typically large, closer to the surface and the most stationary
Median cubital vein
Easiest and least painful to puncture
Median cubital vein
• Least likely to bruise
Median cubital vein
Second-choice vein
Cephalic vein
• Often harder to palpate than medial cubital vein
Cephalic vein
• Fairly well-anchored
Cephalic vein
• Often the only vein felt in obese patients
Cephalic vein
Last choice
Basilic vein
Not well anchored and rolls easily
Basilic vein
• Increased risk of puncturing a median cutaneous nerve branch or the brachial artery
Basilic vein
• Not recommended unless no other vein in either arm is more prominent
Basilic vein
OTHER VEINS
Although antecubital veins are used most frequently, veins on the [?] may also be used for venipuncture. Veins on the [?], however, should never be used for venipuncture. [?] are sometimes used but not without permission of the patient’s physician, due to a potential for significant medical complications.
back of the hand and wrist
underside of the wrist
Leg, ankle, and foot veins
• normally a clear, pale yellow fluid
Serum
• non-fasting serum can be cloudy due to lipids
Serum
• separated from clotted blood by centrifugation (approx. 10 minutes at an RCF of 1,000 to 2,000g)
Serum
• many chemistry tests are performed on serum
Serum
• normally a clear to slightly hazy, pale yellow fluid
Plasma
• separates from the cells when blood in an anticoagulant tube is centrifuged
Plasma
• contains fibrinogen (serum does not because it was used in clot formation)
Plasma
• Stat and other tests requiring a fast turnaround time (TAT) are often collected in tubes containing heparin anticoagulant because they can be centrifuged immediately to obtain plasma
Plasma
• contains both cells and plasma
Whole blood
• must be collected in an anticoagulant tube to keep it from clotting
Whole blood
• used for most hematology tests and many point-of-care tests (POCTs), especially in acute care and stat situations.
Whole blood
preferred method because blood is collected directly from the vein into a tube, minimizing the risk of specimen contamination and exposure to the blood.
Evacuated tube system (ETS)
• discouraged by CLSI due to safety and specimen quality issues
Needle and syringe
• sometimes used on small, fragile, or damaged veins
Needle and syringe
• can be used with the ETS or a syringe
Butterfly set
• often used to draw blood from:
Butterfly set
infants and children; hand veins; in other difficult-draw situations
Butterfly set
Fibrin degradation products
Light blue
Inhibits thrombin formation
Light green/black
Green
Tan (glass)
Royal blue (heparin)
Inhibits glycolysis
Gray
WBC preservative
Yellow
Clot activator
Yellow/gray and orange
Silica clot
Red/gray and gold
CAPILLARY PUNCTURE Length of lancet should be
less than 2.0 mm to avoid penetrating bone
CAPILLARY PUNCTURE Sites:
Palmar surface of 3rd and 4th fingers Lateral plantar heel surface (newborns)
Earlobes
Syringes are used instead of evacuated tubes because of the pressure in an arterial blood vessel.
ARTERIAL PUNCTURE
ARTERIAL PUNCTURE Preferred anticoagulant:
Heparin
Collect without a tourniquet
ARTERIAL PUNCTURE
ARTERIAL PUNCTURE Primary arterial sites (in order of preference):
radial, brachial and femoral arteries
ARTERIAL PUNCTURE Major complications of arterial puncture:
thrombosis, hemorrhage, and possible infection
done before the collecting arterial blood from radial artery
Modified Allen Test
to determine whether the ulnar artery can provide collateral circulation to the hand after the radial
arter puncture
Modified Allen Test
Albumin, ALP (↑older), phosphorus, cholesterol
Age
(↑older)
ALP
: Albumin, ALP, creatine, calcium, uric acid, CK, AST, PO4, BUN, magnesium, bilirubin, cholesterol
↑ males
: Fe, cholesterol, gamma-globulins, a-lipoproteins
↑ females
Peaks 4-6 AM; lowest 8 PM-12 AM; 50% lower at 8 PM than at 8 AM
Cortisol
Lower at night
ACTH, Plasma renin activity, Aldosterone, Insulin
Lower at night; higher standing than supine
Plasma renin activity
Higher in afternoon and evening
Acid phosphatase, Growth hormone
Higher levels at 4 and 8 AM and at 8 and 10 PM
Prolactin
Peaks early to late morning; decreases up to 30% during the day
Iron
≥20% for ALT, bilirubin, Fe, TSH, triglycerides
Day-to-day variation
↑ Glucose, insulin, triglycerides, gastrin, ionized calcium
Recent Food Ingestion
↓ chloride, phosphorus, potassium, amylase, ALP
Recent Food Ingestion
: albumin, cholesterol, aldosterone, calcium
↑ when standing
: CK
↑ in ambulatory patients
: lactic acid, creatine, protein, CK, AST, LD, thyroxine
↑ with exercise
: cholesterol and triglycerides
↓ with exercise
↑ACTH, cortisol, catecholamines, prolactin
Stress
: ↑TP, ↓ albumin
Black
: ↑CK/LD
Black males
: IgG ↑40% and IgA ↑20%
Black male vs white male
: ↑cholesterol and triglycerides
White & >40 years old
FBS, GTT, Triglycerides, lipid panel, gastrin, insulin, aldosterone/ renin
Require fasting
Lactic acid, ammonia, blood gas (if not cooled = ↓ pH and pO2)
Require Ice (Immediate Cooling)
↑ potassium, PO4, Fe, magnesium, ALT, AST, LD, ALP, catecholamines, CK
Hemolysis
CK
(marked hemolysis)
: method of determining the concentration of substance in solution by measuring the amount of light absorbed by that solution after appropriate treatment.
SPECTROPHOTOMETRY
- photons travelling in waves
Electromagnetic radiation
- distance between two peaks
Wavelength
- distance between peak and trough
Amplitude
Visible light:
400-700nm
states that the concentration of a substance is directly proportional to the amount of light absorbed or
inversely proportional to the logarithm of the transmitted light
BEER-LAMBERT LAW (BEER’S LAW)
Light Source
= most common source of light for work in the visible and near-infrared regions
a. Incandescent tungsten or tungsten-iodide lamp
= most commonly used for ultraviolet (UV) work
Deuterium - discharge lamp & Mercury - arc lamp
= most commonly used Monochromator
Diffraction gratings
= advantage over round cuvets in that there is less error from the lens effect, orientation in the spectrophotometer, and refraction.
Square
= used for applications in the visible range
Glass
= for applications requiring UV radiation.
Quartz
= least expensive
Barrier - layer cell or photo cell
= used in instruments designed to be extremely sensitive to very low light levels and light flashes of very short duration
Photomultiplier (PM) tube
PRINCIPLE: measures the quantity of light reflected by a liquid sample that has been dispensed onto a grainy or fibrous solid support
REFLECTOMETRY
COMPONENTS are very similar to those of a photometer
REFLECTOMETRY
APPLICATION:
urine dipstick analysis
dry slide chemical analysis
REFLECTOMETRY
PRINCIPLE: measurement of concentration is done by detecting the absorption of electromagnetic by atoms rather than molecules. When a ground-state atom absorbs light energy, an excited atom is produced. The excited atom ther returns to the ground state, emitting light of the same energy as it absorbed.
ATOMIC ABSORPTION SPECTROPHOTOMETRY
COMPONENTS
Hollow-cathode lamp
Flame
Monochromator
ATOMIC ABSORPTION SPECTROPHOTOMETRY
- usual light source
• Hollow-cathode lamp
- breaks chemical bonds and form free, unexcited atoms; serves as sample cells (instead of a cuvet)
Flame
- used to isolate the desired wavelength; also protects photodetector from excessive light emanating from flame emissions.
Monochromator
APPLICATION: measurement of unexcited trace metals e.g. calcium and magnesium
ATOMIC ABSORPTION SPECTROPHOTOMETRY
APPLICATION: measurement of unexcited trace metals e.g. calcium and magnesium
ATOMIC ABSORPTION SPECTROPHOTOMETRY
PRINCIPLE: measurement of light emitted by excited atoms
FLAME PHOTOMETRY
APPLICATION: Widely used before to determine the concentration of Na*, K+ or Lit
FLAME PHOTOMETRY
PRINCIPLE: measurement of the concentration of solutions that contain fluorescing molecules
FLUOROMETRY
COMPONENTS
Xenon lamp - most common light source
FLUOROMETRY
APPLICATION: is used to measure small particles, such as drugs.
FLUOROMETRY
PRINCIPLE: Chemical energy generated in a chemiluminiscent reaction produces excited intermediates that decay a ground state with the emission of photons; no excitation is required unlike in fluorometry
CHEMILUMINESCENCE
PRINCIPLE: measurements are made with a spectrophotometer to determine concentration of particulate matter in sample. The amount of light blocked by a suspension of particles depends not only on concentration but also on size.
TURBIDIMETRY
APPLICATIONS
1. Detection of bacterial growth and bacterial culture
2. Antibiotic sensitivity
3. Coagulation studies
4. Protein concentration in CSF and urine
TURBIDIMETRY
PRINCIPLE: light scattered by small particles is measured at an angle to the beam incident to the cuvet
NEPHELOMETRY
measure particles which are too large for spectrophotometry, such as antibody-antigen complexes formed in enzyme immunoassays.
NEPHELOMETRY
PRINCIPLE: involves measurement of the current or voltage generated by the activity of specific ions. techniques include potentiometry, coulometry, voltammetry, and amperometry.
ELECTROCHEMISTRY
Measurement of potential (voltage) between two electrodes in a solution to measure analyte concentration
Potentiometry
pH, pCO2, Nat, Ca?, K, NH4*
Potentiometry
measurement of the current flow produced by an oxidation-reduction reaction
Amperometry
pOz (Clark electrode), glucose, peroxidase
Amperometry
Electrochemical titration in which the titrant is electrochemically generated
Coulometry
Cl
Coulometry
Potential is applied to an electrochemical cell and the resulting current is measured
Voltammetry
Anodic stripping voltametry (for lead and iron)
Voltammetry
PRINCIPLE: separation of charged compounds based on their electrical charge
ELECTROPHORESIS
COMPONENTS
1. A driving force (electrical power)
2. Support more (electrical power)
a. Filter paper
b. Agarose
c. Cellulose acetate
d. Polyacrylamide
3. Buffer
4. Sample
5. Detecting system
ELECTROPHORESIS
: movement of buffer ions and solvent relative to the fixed support
ELECTROENDOSMOSIS
- most common and reliable way for quantitation of separated protein fractions
DENSITOMETRY
PRINCIPLE: separation of complex mixtures on the basis of different physical attractions between the individual compounds and the stationary phase of the system
CHROMATOGRAPHY
COMPONENTS
1. Mobile phase (gas or liquid)
2. Stationary phase (solid or liquid)
3. Column
4. Eluate
CHROMATOGRAPHY
: carries the complex mixture
- Mobile phase (gas or liquid)
: substance through which the mobile phase flows
- Stationary phase (solid or liquid)
: holds the stationary phase
- Column
: separated components
- Eluate
CHROMATOGRAPHIC PROCEDURES
- Thin-Layer Chromatography
- High-Performance Liquid Chromatography (HPLC)
- Gas Chromatography (GC)
- uses pressure for faster separations
- High-Performance Liquid Chromatography (HPLC)
- separate mixtures of compounds that are volatile or can be made volatile
- Gas Chromatography (GC)
PRINCIPLE: Sample in a MS is first volatilized and then ionized to form charged molecular ions and fragments that are separated according to their mass-to-charge (m/Z) ratio
MASS SPECTROMETRY
Allows definitive identification when used on samples eluting from GC or HPLC
MASS SPECTROMETRY
Gold standard for drug testing when coupled with GC
MASS SPECTROMETRY
PRINCIPLE
Two-step procedure: (1) MALDI, then (2) Time-of-Flight (TOF) Mass
MALDI-TOF MS (Matrix-Assisted Laser Desorption lonization Time-of-Flight) analysis
• A laser pulse irradiates the sample, causing desorption and ionization of both the matrix and the sample.
MALDI-TOF MS (Matrix-Assisted Laser Desorption lonization Time-of-Flight) analysis
• lons from the sample are focused into the mass spectrometer.
MALDI-TOF MS (Matrix-Assisted Laser Desorption lonization Time-of-Flight) analysis
The molecular weight of the proteins acquired by mass spectrometry is used to determine the identity of the sample and is helpful in determining posttranslational modifications that may have occurred.
MALDI-TOF MS (Matrix-Assisted Laser Desorption lonization Time-of-Flight) analysis
APPLICATION: used for the analysis of biomolecules, such as peptides and proteins
MALDI-TOF MS (Matrix-Assisted Laser Desorption lonization Time-of-Flight) analysis
- process by which lab ensures quality results by closely monitoring preanalytical, analytical, & postanalytical stages of testing.
QUALITY ASSURANCE
- everything that precedes test performance,
• Preanalytical
e.g., test ordering, patient preparation, patient ID, specimen collection, specimen transport, specimen processing.
• Preanalytical
- everything related to assay,
• Analytical
e.g., test analysis, quality control (QC), reagents, calibration,
preventive maintenance.
• Analytical
- everything that comes after test analysis, e.g., verification of calculations & reference ranges, review of results, notification of critical values, result reporting, test interpretation by physician, follow-up patient care.
• Postanalytical
- part of analytical phase of quality assurance
QUALITY CONTROL (QC)
process of monitoring results from control samples to verify accuracy of patient results.
QUALITY CONTROL (QC)
- most frequently used measure of variation
b. Standard deviation (SD)
- an index of precision used to compare the dispersion of two or more groups of data with different units / concentrations
c. Coefficient of variation (CV)
- used to determine if there is a significant difference between the MEANS of two groups of data
T-test
- used to determine if there is a significant difference between the SD of two groups of data
F-test
describes many continuous laboratory variables and
deviation shares several unique characteristics
The Gaussian Distribution (Normal Distribution)
the mean, median, and mode are [?];
the distribution is [?] — meaning half the values fall to the left of the mean, and the other half fall to the right (the symmetrical shape is often referred to as a “bell curve.”)
The total area under the gaussian curve is [?].
identical
symmetric
1.0, or 100%
summarizes the above relationships between the area under a Gaussian distribution and the SD.
“68-95-99 Rule”
“68-95-99 Rule”
In other words, given any Gaussian distributed data
of the data fall between ‡1 SD from the mean
of the data fall between +2 SDs from the mean
fall between ‡3 SDs from the mean
68%
295%
99%
- nearness or closeness of assayed values to the true value
- Accuracy
- nearness or closeness of assayed values to each other
- Precision (Reproducibility)
- ability of an analytical method to maintain accuracy and precision over an extended period of time
- Reliability
- degree by which a method can easily be repeated
- Practicability
- ability to measure the smallest concentration of the analyte of interest
- Analytical sensitivity
- ability to measure only the analyte of interest
- Analytical specificity
- also known as linearity; range of values over which lab can verify accuracy of test system
- Reportable range
Formerly called normal value.
- Reference interval
Can vary for different patient populations (age, gender, race).
- Reference interval
Established by testing minimum of 120 healthy subjects & determining range in which 95% fall.
- Reference interval
Verifying a reference interval (transference) can required as few as 20 study individuals
- Reference interval
Reporting a positive result in a patient who has the disease
True positive (TP)
Reporting a positive result in a patient who doesn’t have the disease
False positive (FP)
Reporting a negative result in a patient who doesn’t have the disease
True negative (TN)
Reporting a negative result in a patient who has the disease
False negative (FN)
% of population with the disease that test positive
Diagnostic sensitivity
ability of the analytical method to detect the proportion of individuals with the disease
Diagnostic sensitivity
% of population without the disease that test negative
Diagnostic specificity
ability of the analytical method to detect the proportion of individuals without the disease
Diagnostic specificity
Number individuals without the disease with a negative test × 100%
Diagnostic Specificity (%)
% of time that a positive result is correct
Positive predictive value (PPV)
% of time that a negative result is correct
Negative predictive value (NPV)
• Assayed on a regular schedule to verify that a laboratory procedure is performing correctly
QC SAMPLES
• Generally, two different concentrations are necessary for adequate statistical QC
QC SAMPLES
• Chemically & physically similar to unknown specimen & is tested in exactly the same manner
QC SAMPLES
• New instrument or new lot of reagents: analyze QC materials for 20 days
QC SAMPLES
CHARACTERISTICS OF IDEAL QC MATERIALS
- Must resemble human samples
- Inexpensive and stable for long periods
- No communicable disease
- No known matrix effects
- With known analyte concentrations (for assayed controls)
- Convenient packaging for easy dispensing and storage
Also called a Shewart plot
LEVEY-JENNINGS CONTROL CHART
• Most common presentation for evaluating QC results
LEVEY-JENNINGS CONTROL CHART
shows each QC result sequentially over time
LEVEY-JENNINGS CONTROL CHART
- control values increasing or decreasing for six consecutive runs
Trend
Trend Main cause:
DETERIORATION OF REAGENTS
- six consecutive control values on the same side of the mean
Shift
Shift Main cause:
IMPROPER CALIBRATION OF INSTRUMENT
- highly deviating values
Outliers
control result outside established limits
Outliers
1 control >‡ 2s from mean.
1(2S)
1 control >‡ 2s from mean.
1(2S)
Warning flag of possible change in accuracy or precision.
1(2S)
Initiates testing of other rules (warning rule). If no violation of other rules, run is considered in control.
1(2S)
1 control >‡ 3s from mean
1(3S)
2 consecutive controls >2s from mean on same side
2(2S)
Random; Rejection rule
Systematic; Rejection rule
2 consecutive controls differ by >4s
R(4S)
4 consecutive controls > 1s from mean on same side
4(1S)
10 consecutive controls on same side of mean
10x
Present in all measurements; due to chance; no means of predicting it
- Random error
Error that doesn’t recur in regular pattern
- Random error
Associated with violations of the 12s, 13s and R4s Westgard rules
- Random error
• Error that influences ALL observations consistently in one direction
- Systematic error
Recurring error inherent in test procedure
- Systematic error
• Associated with violations of the 22s and 41s Westgard rules
- Systematic error
• Also known as external quality assessment
PROFICIENCY TESTING
• consists of evaluation of method performance by comparison of results versus those of other
PROFICIENCY TESTING
• laboratories for the same set of samples
PROFICIENCY TESTING
PROFICIENCY TESTING • Basic procedure:
PT providers circulate a set of samples among a group of
laboratories.
Each laboratory includes the PT samples along with patient samples in the usual assay process.
Results for the PT samples are reported to the PT provider for evaluation.
Error due to dirty glassware
RANDOM ERRORS
Dirty photometer
SYSTEMATIC ERRORS
Use of wrong pipet
RANDOM ERRORS
Faulty ISE
SYSTEMATIC ERRORS
Voltage fluctuation
RANDOM ERRORS
Evaporation or contamination of standards or reagents
SYSTEMATIC ERRORS
Sampling error
RANDOM ERRORS
Anticoagulant or drug interference
RANDOM ERRORS
• Comparison of patient data with previous results.
Delta checks
• Detects specimen mix-up & other errors.
Delta checks
• When limit is exceeded, must determine if due to medical change in patient or lab error.
Delta checks
Test results that indicate a potentially life-threatening situation.
Critical values
List typically includes glucose, Na+, K+, total CO2, Ca2+, Mg2+, phosphorus, total billrubin (neonates), blood gases
Critical values
• Patient care personnel must be notified immediately.
Critical values
Critical values• Example:
Serum glucose
<40 mg/dL
>500 mg/dL
Schedule of maintenance to keep equipment in peak operating condition; must be documented & must follow manufacturer’ s specifications & frequencies.
Preventive maintenance
CARBOHYDRATES
1. Contain C, H and 0; Empiric formula: (CH20)n
3. Can be reducing or non-reducing sugars ; Can be classified according to the number of sugar units
CARBOHYDRATES
CARBOHYDRATES Functions:
Major energy source (?)
Storage form of energy e.g. [?]
Components of cell membranes e.g. [?]
Structural component in plants, bacteria, insects (e.g. ?)
glucose
glycogen
glycoproteins
chitin, cellulose
- one sugar unit e.g. glucose, fructose, galactose
- 2 sugar units linked together by a glycosidic bond e.g. sucrose, lactose, maltose
- 3 to 10 sugar units
- more than 10 sugar units (e.g. starch, glycogen, cellulose)
a. Monosaccharides
b. Disaccharides
Oligosaccharides
d. Polysaccharides
Glucose + fructose
Sucrose-Sucrase
Glucose + galactose
Lactose-Lactase
Glucose + glucose
Maltose-Maltase
Metabolism of glucose molecule to pyruvate or lactate for production of energy
Glycolysis
Formation of glucose-6-phosphate from non-carbohydrate sources
Gluconeogenesis
Breakdown of glycogen to glucose for use as energy
Glycogenolysis
Conversion of glucose to glycogen for storage
Glycogenesis
Conversion of carbohydrates to fatty acids
Lipogenesis
Decomposition of fat
Lipolysis
Carbohydrates in the diet constitute about 50% of the calories in the average diet:
- 60%
- 30% C.
- 5%
- 5%
(part of dietary fiber)
a. Starch and dextrins
b. Sucrose
C. Lactose
d. Other sugars
e. Cellulose
breakdown polymers to dextrins and disaccharides.
Salivary amylase (ptyalin) and pancreatic amylase (amylopsin)
are further hydrolyzed into monosaccharides by specific enzymes (disaccharidases)
Disaccharides
are absorbed by the gut via active transport (glucose and galactose) or facilitated diffusion (fructose).
Monosaccharides
They are then transported into the liver through the portal circulation.
Monosaccharides
is the only carbohydrate to be used directly for energy.
Glucose
After glucose enters the cell, it undergoes phosphorylation into glucose-6-phosphate through the action of
hexokinase or glucokinase.
Glucose-6-phosphate is then shunted into the following metabolic pathways:
a. Glycolysis (Embden-Meyerhof pathway)
b. Glycogenesis
c. Hexose-Monophosphate shunt
• Produced by the beta cells of the islets of Langerhans (pancreas)
Insulin
→ insulin
• Preproinsulin → proinsulin
• Target: most cells of the body
Insulin
Increases utilization of glucose by the cells by increasing cellular uptake and hepatic glycolysis
Insulin
Increases glycogenesis and inhibits glycogenolysis; Inhibits gluconeogenesis
Insulin
Stimulates lipogenesis while inhibiting lipolysis
Insulin
Stimulates protein synthesis and stimulates uptake of amino acids into muscles
Insulin
• Produced by the alpha cells of the islets of Langerhans
GLUCAGON
• Target: liver
GLUCAGON
• Target: liver
GLUCAGON
Promotes liver glycogenolysis
GLUCAGON
Increases gluconeogenesis
GLUCAGON
Inhibits glycolysis
GLUCAGON
Increases gluconeogenesis
Cortisol (Glucocorticoids)
Decreases glucose uptake and utilization by extrahepatic tissues
Cortisol (Glucocorticoids)
Stimulates glycogenolysis
Catecholamines
Increases glucose absorption in the small intestines
Thyroid hormone
Inhibit glucagon and insulin secretion
Somatostatin
Increases liver gluconeogenesis
Growth hormone
Inhibits glucose transport
Growth hormone
- heterogeneous group of multifactorial, polygenic syndromes characterized by an elevated fasting blood glucose caused by a relative or absolute deficiency in insulin
DIABETES MELLITUS
- characterized by an absolute deficiency of insulin caused by an autoimmune attack on the beta cells of the pancreas
- Type 1 DM
- characterized by a combination of insulin resistance and dysfunctional beta cells
- Type 2 DM
Juvenile Onset DM; Insulin Dependent DM; Most common in children and young adults
TYPE 1 DIABETES
Adult Onset DM; Non-insulin Dependent DM; Most common with advancing age
TYPE 2 DIABETES
5-10%
TYPE 1 DIABETES
90-95%
TYPE 2 DIABETES
Genetic, autoimmune, environmental (e.g. viral infection)
TYPE 1 DIABETES
HLA DR3/4 ; Autoantibodies
-Anti-islet cell cytoplasmic antibody
-Insulin autoantibodies
-Anti-GAD (glutamic acid decarboxylase)
TYPE 1 DIABETES
Genetic, obesity, sedentary lifestyle, race/ethnicity
TYPE 2 DIABETES
Destruction of pancreatic beta cells, usually autoimmune
TYPE 1 DIABETES
No autoimmunity; Insulin resistance and progressive insulin deficiency
TYPE 2 DIABETES
Very low or undetectable c-peptide
TYPE 1 DIABETES
Detectable c-peptide
TYPE 2 DIABETES
Low to absent plasma insulin
TYPE 1 DIABETES
High in early disease; low to absent in disease of long duration plasma insulin
TYPE 2 DIABETES
Prone to ketoacidosis and diabetic complications
TYPE 1 DIABETES
Not prone to ketoacidosis
TYPE 2 DIABETES
Insulin absolutely necessary; muliple
TYPE 1 DIABETES
Oral agents (insulin sometimes daily injections or insulin pump indicated)
TYPE 2 DIABETES
None known therapy
TYPE 1 DIABETES
Lifestyle, oral medicines
TYPE 2 DIABETES
- associated with secondary conditions
E.g. genetic defects of beta cell function; pancreatic disease; endocrine disease; drug or chemical induced; insulin receptor abnormalities; other genetic syndromes
Other specific types of DM
Glucose intolerance with onset or first recognition during pregnancy
Gestational Diabetes Mellitus (GDM)
• Due to metabolic and hormonal changes
Gestational Diabetes Mellitus (GDM)
• Large % of patients develop DM Within 5 to 10 years
Gestational Diabetes Mellitus (GDM)
• Infants born to mothers with diabetes are at increased risk for RDS, hypocalcemia, hyperbilirubinemia and other complications
Gestational Diabetes Mellitus (GDM)
• Screening: 2-hour OGTT using a 75 g glucose load
Gestational Diabetes Mellitus (GDM)
Random plasma glucose
≥200 mg/dL (211.1 mmol/L), +symptoms of DM
Fasting plasma glucose
≥126 mg/dL (27.0 mmol/L)
Two-h plasma glucose
≥200 mg/dL (≥11.1 mmol/L)
N.B. In absence of unequivocal hyperglycemia, these criteria should be confirmed by repeat testing on a different day. The third measure (OGTT) is not recommended for routine clinical use.
Normal Fasting plasma glucose
<100 mg/dL
<5.6 mmol/L
Normal 2-hour plasma glucose level (after 75 g load)
<140 mg/dL
<7.8 mmol/L
Pre-diabetes HbA1c
5.7-6.4 %
Impaired fasting glucose Fasting plasma glucose
100-125 mg/dL
5.6-6.9 mmol/L
Impaired glucose tolerance 2-hour plasma glucose level (after 75 g load)
140-199 mg/dL
7.8-11.0 mmol/L
Diabetes mellitus Fasting plasma glucose
≥126 mg/dL
≥7.0 mmol/L
Diabetes mellitus 2-hour plasma glucose level (after 75 g load)
≥200 mg/dL
≥11.1 mmol/L
Diabetes mellitus HbA1c
≥6.5%
Use: detection of GDM
ORAL GLUCOSE TOLERANCE TEST (OGTT)
Before an OGTT is performed, individuals should ingest [?]
preceding the test.
At least 150g/day of carbohydrates for the 3 days (no restriction of diet)
No limitation in physical activity
ORAL GLUCOSE TOLERANCE TEST (OGTT)
Test should be performed after an overnight 8- to 14-hour fast.
ORAL GLUCOSE TOLERANCE TEST (OGTT)
The individual should not eat food, drink tea, coffee, or alcohol, or smoke cigarettes during the test, and should be seated.
ORAL GLUCOSE TOLERANCE TEST (OGTT)
Venous glucose samples are preferably collected in gray-top tubes containing fluoride and an anticoagulant (Henry’s, 23rd ed)
ORAL GLUCOSE TOLERANCE TEST (OGTT)
FBG is measured right before the administration of the glucose load. A FBG of greater than 140 mg/dL necessitates that the test be stopped immediately. Proceed with the glucose load if FBG is less than 140 mg/dL.
ORAL GLUCOSE TOLERANCE TEST (OGTT)
ORAL GLUCOSE TOLERANCE TEST (OGTT)
• Glucose load for adults
• Glucose load for children
• Pregnant women
75 g
1.75g/kg bw (max: 75 g)
75 g or 100 g
Patient should finish glucose load within 5 - 15 minutes.
ORAL GLUCOSE TOLERANCE TEST (OGTT)
ORAL GLUCOSE TOLERANCE TEST (OGTT)
Patient should NOT vomit. If patient vomits, [?]
Discontinue the test
DIAGNOSIS OF GESTATIONAL DIABETES MELLITUS
Gestational diabetes mellitus is diagnosed if [?] plasma glucose levels are exceeded (American Diabetes Association, 2004)
≥2
• Rate of formation of Hbac is proportional to the average blood glucose concentration over the previous 3 months
GLYCOSYLATED HEMOGLOBIN (HbA1c)
• For every 1% increase in Hba1c, there is a corresponding 35mg/dL change in plasma glucose
GLYCOSYLATED HEMOGLOBIN (HbA1c)
• The ADA also recommends that it be tested at least twice a year to monitor long-term glycemic control.
GLYCOSYLATED HEMOGLOBIN (HbA1c)
• Spn: EDTA-WB → hemolysate
GLYCOSYLATED HEMOGLOBIN (HbA1c)
• False decrease: decreased RBC lifespan
GLYCOSYLATED HEMOGLOBIN (HbA1c)
• Monitoring glucose control over past 2-3 weeks
FRUCTOSAMINE (Glycated Albumin)
• Albumin has a life span of 20 days in circulation
FRUCTOSAMINE (Glycated Albumin)
• Affected by albumin levels; false decrease in patients with hypoalbuminemia
FRUCTOSAMINE (Glycated Albumin)
100 g OGTT plasma glucose/75 g OGTT plasma glucose
Fasting
≥95 mg/dL
≥5.3 mmol/L
100 g OGTT plasma glucose/75 g OGTT plasma glucose
1 hour
≥180 mg/dL
≥10.0 mmol/L
100 g OGTT plasma glucose/75 g OGTT plasma glucose
2 hour
≥ 155 mg/dL
≥8.6 mmol/L
100 g OGTT plasma glucose
3 hour
≥140 mg/dL
≥7.8 mmol/L
HYPOGLYCEMIA
The plasma glucose concentration at which glucagon and other glycemic factors are released is between [?]. At about [?], observable symptoms of hypoglycemia appear. Warning signs and symptoms are all related to the central nervous system
65 and 70 mg/dL
50 to 55 mg/dL
HYPOGLYCEMIA
Most causes are secondary to other illnesses and resolve themselves when the primary disorder is treated.
Examples:
Insulinoma
Various liver disorders
Gastrointestinal disorders and surgery
HYPOGLYCEMIA
Possible specimens =
WB, serum, plasma, urine, CSF, serous fluid, synovial fluid
HYPOGLYCEMIA Standard clinical specimen
Fasting venous plasma
HYPOGLYCEMIA Fasting blood sugar should be obtained after
8 - 10 hours of fasting
HYPOGLYCEMIA Whole blood glucose levels
10-15% lower vs plasma levels
HYPOGLYCEMIA Glucose is metabolized at room temperature at a rate of
7 mg/dl/hour
HYPOGLYCEMIA At 4°C, glucose decreases by approximately
2 mg/dl/hour
HYPOGLYCEMIA Evacuated tube
Gray top (NaF)
HYPOGLYCEMIA CSF glucose levels
60-70% of plasma levels (decreased in bacterial meningitis)
HYPOGLYCEMIA As little as 10% contamination with 5% dextrose (D5W) will elevate glucose in a sample by
500 mg/dL or more
Principle: Glucose and other carbohydrates are capable of converting cupric ions in alkaline solution to cuprous ions.
Chemical Methods
GLUCOSE: Chemical Methods
a. Oxidation-reduction method
i. Alkaline Copper Reduction Method
Folin-Wu
Nelson-Somogyi
Neocuproine method
Benedict’s method
ii. Alkaline Ferric Reduction Method (Hagedorn-Jensen)
a. Condensation method (Dubowski)
GLUCOSE: Enzymatic Methods
a. Glucose oxidase method
i. Colorimetric method
ii. Polarographic method
b. Hexokinase method
is the most specific enzyme reacting with only B-D-glucose
Glucose oxidase
- uses a side reaction that consumes H202
i. Colorimetric method
- measure the rate of disappearance of oxygen using an oxygen electrode
ii. Polarographic method
• More accurate than glucose oxidase methods because the coupling reaction using G6PDH is highly specific; therefore it has less interference than the coupled glucose oxidase procedure
b. Hexokinase method
• NADPH has a strong absorbance at 340 nm
b. Hexokinase method
Generally accepted as the reference method
b. Hexokinase method
• Not affected by ascorbic acid or uric acid
b. Hexokinase method
Most common cause of lactose intolerance
Lactase deficiency
Lactose is not digested at a normal rate and accumulates in the gut, where it is metabolized by bacteria. Bloating, abdominal cramps, and watery diarrhea result
Lactase deficiency
result of the deficiency of a specific enzyme that causes an alternation of glycogen metabolism
Glycogen storage diseases
Glycogen storage diseases Most common congenital form:
Von Gierke Disease
i. Autosomal recessive disease
Von Gierke Disease
ii. Characterized by hepatomegaly, severe hypoglycemia, metabolic acidosis, ketonemia, and elevated lactate and alanine
Von Gierke Disease
• Building blocks of lipids
FATTY ACIDS
• Hydrocarbon chains with a terminal COO- group
FATTY ACIDS
• 3 fatty acid molecules attached to one molecule of glycerol by ester bonds
TRIGLYCERIDES
• Serves as main storage form of energy, insulator, shock absorber and integral part of cell membrane
TRIGLYCERIDES
• Similar to triglycerides except that the third position on the glycerol backbone contains a phospholipid head group
PHOSPHOLIPIDS
• Contains polar and non-polar end
PHOSPHOLIPIDS
• Constituent of cell membranes
PHOSPHOLIPIDS
• Serves as part of cell membranes and as parent chain for cholesterol-based hormones, e.g. aldosterone, cortisol and the sex hormones
CHOLESTEROL
CHOLESTEROL• Exists in two forms
- approximately 70% of total cholesterol
- approximately 30% of total cholesterol
• Cholesterol esters
• Free cholesterol
Typically spherical in shape with sizes ranging from 10 to 1200 nm
LIPOPROTEIN
Composed of lipids and proteins, called apolipoproteins
LIPOPROTEIN
Size particle correlates with its lipid content
LIPOPROTEIN
originally separated through ultracentrifugation
LIPOPROTEIN
• located on the surface of lipoprotein particles maintain structural integrity of lipoproteins
Apolipoproteins
• serve as ligands for cell receptors
Apolipoproteins
Apolipoproteins• Important types:
- largest
-least dense - highest TG content
- postprandial turbidity
- fxn: transports exogenous / dietary triglycerides
CHYLOMICRONS
- 2nd largest
-2nd least dense - 2nd highest TG content
- fasting hyperlipidemic turbidity
- fxn: transports endogenous/hepatic triglycerides
VERY LOW DENSITY LIPOPROTEIN (VLDL)
- small –> can cross BV walls
–> deposition of lipid - highest cholesterol content
- fxn: transports cholesterol to peripheral tissues
→inc LDL –> in atherosclerosis - target for cholesterol lowering therapy
LOW DENSITY LIPOPROTEIN (LDL)
- smallest but densest
- highest protein content
- fxns: reverse transport cholesterol (peripheral tissues –> liver)
- inc HDL –> dec atherosclerosis
HIGH DENSITY LIPOPROTEIN (HDL)
• Chylomicrons accumulate as a floating “cream” layer and can be detected visually. The presence of chylomicrons in fasting plasma is considered to be abnormal.
Standing Plasma Test
• A plasma sample that remains turbid after standing overnight contains excessive amounts of VLDL; if a floating
“cream” layer also forms, chylomicrons are present as well. (Henry, 23rd ed)
Standing Plasma Test
Floating Beta-lipoprotein
- Beta-VLDL
Increased in familial dysbetalipoproteinema
- Beta-VLDL
• Sinking pre-beta lipoprotein
- Lp(a)
• LDL - like particle
- Lp(a)
Increased risk of premature coronary heart disease and stroke
- Lp(a)
Seen in patient with biliary cirrhosis or cholestasis and in patients with mutations in the enzyme lecithin: cholesterol acyltransferase (LCAT)
- Lpx
happens in the intestines
Absorption Pathway
CM transports exogenous TG
Exogenous Pathway
VLDL and hDL transports endogenous TG
Endogenous Pathway
LDL transports Cholesterol
Reverse Cholesterol Transport Pathway
was used to characterize lipid disorders; used electrophoresis and a standing plasma test for CM to correlate clinical disease syndromes with laboratory phenotypes. Note that each phenotype is not a specific disease but rather a variety of disorders that affect the same lipoproteins and therefore express the same lipid pattern.
FREDRICKSON CLASSIFICATION OF LIPID DISORDERS
-Hyperchylomicronemia
-Familial LPL deficiency
Type 1
-Familial Hypercholesterolemia
Type 2a
-Familial Combined Hyperlipidemia
Type 2b
Familial Dysbetalipoproteinemia
Type 3
Familial Hypertriglyceridemia
Type 4
Low cardiac risk; eruptive xanthoma; recurrent pancreatitis
Type 1
High cardiac risk; xanthelasma; tendon xanthoma; corneal arcus; hypothyroidism and nephrotic syndrome
Type 2a
High cardiac risk
Type 2b
Eruptive and palmar xanthomas
Type 3
Low cardiac risk
Type 4
Low cardiac risk; eruptive xanthoma, may be associated
with pancreatitis
Type 5
Fasting = 12 hours before venipuncture
LIPIDS AND LIPOPROTEINS
• Can be measured non-fasting = TC and HDL-C
LIPIDS AND LIPOPROTEINS
• Prolonged tourniquet application = causes hemoconcentration
LIPIDS AND LIPOPROTEINS
Reclined patients = decreased values
LIPIDS AND LIPOPROTEINS
Preferred sample = Serum or plasma but plasma preferred in electrophoresis and ultracentrifugation
LIPIDS AND LIPOPROTEINS
Capillary blood samples = generally lower values
LIPIDS AND LIPOPROTEINS
Lipemic samples = seen when triglyceride levels exceed 4.6 mmol/L (400 mg/dL).
LIPIDS AND LIPOPROTEINS
Initial extraction with zeolite to remove sterols
- Abell-Kendall method
Redissolving of cholesterol
- Abell-Kendall method
Hydrolysis of cholesterol esters to cholesteriol
- Abell-Kendall method
Liebermann-Burchard reagent
glacial acetic acid
sulfuric acid
acetic anhydride
(+) formation of product which strongly absorbs at 410 nm
- Abell-Kendall method
Recently, the reference method has changed to a [?] that now specifically measures cholesterol and does not detect related sterols. (Bishop 7h ed.)
GC-MS method
- definitive method
- Isotope Dilution Mass Spectrometry (IDMS)
Hydrolysis of glycerol is accomplished using alcoholic KOH
- Chemical methods (triglyceride)
Oxidation of glycerol by periodic acid, forming formaldehyde and formic acid
- Chemical methods (triglyceride)
Formaldehyde combines with a variety of reagents:
- Chemical methods (triglyceride)
Reagent: Chromotropic acid → blue colored compound
a. Van Handel & Zilversmit (Colorimetric method)
Reagent: acetylacetone (aka diacetyl acetone; reactant of choice)
Hantzsch (Fluorometric method)
Product has a strong absorption maximum at 412 nm and also has good fluorescence
Hantzsch (Fluorometric method)
- new reference method for triglyceride measurements; involve the hydrolysis of fatty acids on triglycerides and the measurement of glycerol.
GC-MS method
: range in density observed among lipoprotein classes is a function of lipid and protein content and enables fractionation by density using ultracentrifugation
- Ultracentrifugation
: takes advantage of differences in size and charge
- Electrophoresis
: depends on particle size, charge and differences in the
apolipoprotein content; primarily used in research labs only
- Chemical precipitation
Uses polyanions (heparin sulfate, dextran sulfate and phosphotungstate) and divalent cations such as magnesium, calcium andmanganese e.g. HDL - dextran sulfate + magnesium
- Chemical precipitation
: uses antibodies specific to apolipoproteins to bind and separate lipoprotein classes
- Immunoassays
: takes advantage of size differences in molecular sieving methods or composition in affinity methods e.g. gel chromatography or affinity chromatography
- Chromatographic methods
• The term [Plasma TG]/5 is used when concentrations are expressed in mg/dL.
K. FRIEDEWALD CALCULATION
It has been reported that the factor [Plasma TG]/2.825 gives a more accurate estimate of VLDL-C (DeLong, 1986). This is equivalent to Plasma TG/6.5, when concentrations are expressed in mg/dL.
K. FRIEDEWALD CALCULATION
a. Involves large- and medium-sized arteries (e.g. abdominal aorta, coronary artery, popliteal artery, internal carotid artery)
- Atherosclerosis
b. LDL - increased risk ; HDL - decreased risk
- Atherosclerosis
c. Complications
Narrowing of blood vessels result in impaired blood flow and ischemia leading to:
(i) Peripheral vascular disease
(ii) Angina
(iii) Ischemic bowel disease
- Atherosclerosis
Plaque rupture → thrombosis → myocardial infarction and stroke
Plaque rupture → embolization atherosclerotic embolism
Weakening of blood vessel wall results in aneurysm
- Atherosclerosis
An extreme form of hypoalphalipoproteinemia (isolated decrease in circulating HDL)
- Tangier Disease
Associated with HDL cholesterol concentrations as low as 1-2 mg/dL (0.03-0.05 mmol/L) in homozygotes, accompanied by total cholesterol concentrations of 50 to 80 mg/dL (1.3-2.1 mmol/L).
- Tangier Disease
Associated with increased risk of premature coronary heart disease (CHD).
- Tangier Disease
Linear polymers of amino acids; Perform diverse functions
PROTEIN
Regulate metabolism
PROTEIN
• Facilitate contraction in the muscle
PROTEIN
Provide structural framework
PROTEIN
Shuttle molecules in the bloodstream
PROTEIN
• Component of the immune system
PROTEIN
: determined by amino acid sequence
: folding of short segments of polypeptide into geometrically ordered units (e.g. alpha-helix, beta-sheet)
: overall 3-dimensional shape of the protein (globular vs fibrous)
: number and types of polypeptide units of oligomeric proteins and their spatial arrangement
Primary
Secondary
Tertiary
Quaternary
Indicator of malnutrition; binds thyroid hormones and retinol-binding protein
Prealbumin (Transthyretin)
Binds bilirubin, steroids, fatty acids; major contributor to oncotic pressure
Albumin
Protease inhibitor
Alpha-1-antitrypsin
Principal fetal protein
Alpha-1-fetoprotein
May be related to immune response
Alpha-1- acid glycoprotein
Binds hemoglobin
Transports copper; peroxidase activity
Haptoglobin
Ceruloplasmin
Inhibits thrombin, trypsin and pepsin
Alpha-2-macroglobulin
Transports iron
Binds heme
Immune response
Precursor of fibrin
Opsonin
Transferrin
Hemopexin
Complement
Fibrinogen
C-reactive protein
- Most plasma proteins are synthesized in the [?]
and secreted by the hepatocyte into the circulation.
The immunoglobulins are exceptions because they are synthesized in plasma cells.
Liver (hepatocytes)
- The nitrogen content of serum proteins is, on average,
16%
: site of protein synthesis within the cell
- Ribosomes
• Aka Transthyretin
• Migrates before albumin in the serum protein electrophoresis
- PREALBUMIN
• Function: Transport protein for thyroid hormones; transports vitamin A by forming a complex with retinol-binding protein
- PREALBUMIN
Decreased in hepatic damage, acute-phase inflammatory response, and tissue necrosis
- PREALBUMIN
- A low prealbumin level is a sensitive marker of poor nutritional status
- PREALBUMIN
Increased in patients receiving steroids, in alcoholism, and in chronic renal failure
- PREALBUMIN
• Protein present in the highest concentration in serum
- ALBUMIN
Provide nearly 80% of colloid osmotic pressure (COP) of intravascular fluid
- ALBUMIN
Buffers pH
- ALBUMIN
Binds to various substances in blood (e.g. some hormones, drugs, electrolytes, unconjugated bilirubin)
- ALBUMIN
Negative acute-phase reactant
- ALBUMIN
Decreased: liver disease, malnutrition, malabsorption, kidney loss, hemodilution
- ALBUMIN
Increased: dehydration
- ALBUMIN
• Most important function: inhibition of the protease neutrophil elastase
- ALPHA-1-ANTITRYPSIN
• Abnormal form of AAT can also accumulate in the liver and cause cirrhosis
- ALPHA-1-ANTITRYPSIN
• Major component of a1-globulin band → deficiency of AAT seen as lack of an a1-globulin band on SPE
- ALPHA-1-ANTITRYPSIN
One of the COPs (chronic obstructive pulmonary diseases)
Emphysema
Most common cause: smoking
Emphysema
• Pathophysiology: excessive inflammation or lack of AAT leads to destruction of alveolar air sacs → loss of elastic recoil and collapse of airways during exhalation → obstruction and air trapping
Emphysema
• Dyspnea, cough with minimal sputum
Emphysema
“Pink puffers”, “barrel-chest”, hypoxemia
Emphysema
Synthesized by the developing embryo and fetus; thought to protect the fetus from immunologic attack by
the mother
ALPHA-1-FETOPROTEIN
• No known function in normal adults
ALPHA-1-FETOPROTEIN
: neural tube defects (e.g. spina bifida), presence of twins
: increased risk for Down syndrome (trisomy 21)
Elevated AFP
Low AFP
- Tumor marker for hepatocellular carcinoma, some testicular carcinomas
AFP
• Copper-containing (contains >90% of total serum copper)
CERULOPLASMIN
• Used in the diagnosis of Wilson’s disease
CERULOPLASMIN
• Autosomal recessive
• Decreased levels of ceruloplasmin
Wilson’s disease
Wilson’s disease
• Excess storage of copper in various organs
Liver →
- Brain →
- Cornea →
hepatic cirrhosis
neurologic damage
Kayser-Fleischer rings
• Large protein that inhibits proteases such as trypsin, thrombin, kallikrein, and plasmin
ALPHA-2-MACROGLOBULIN
• Increased in nephrotic syndrome (large size aids in its retention)
ALPHA-2-MACROGLOBULIN
• Glomerular disorder characterized by proteinuria (>3.5 g/day)
Nephrotic syndrome
• Pathophysiology: Disruption of the electrical charges that produce the tightly fitting podocyte barrier resulting in massive loss of protein and lipids
Nephrotic syndrome
Nephrotic syndrome
• Manifestations
- pitting edema
- increased risk of infection
- due to the loss of anti-thrombin III
- may result in fatty casts in the urine
- Hypoalbuminemia
- Hypogammaglobulinemia
- Hypercoagulable state
- Hyperlipidemia and hypercholesterolemia
Function: bind free hemoglobin to prevent loss of hemoglobin and its constituent, iron, into the urine
HAPTOGLOBIN
Used primarl to holp detect and evaluate hemolylic anemia
HAPTOGLOBIN
Transports two molecules of ferric iron
TRANSFERRIN
• Negative acute-phase reactant
TRANSFERRIN
• Major component of the beta-globulin fraction
TRANSFERRIN
• Tested to determine cause of anemia (e.g. increased levels in IDA)
TRANSFERRIN
• Function: scavenge heme released or lost by the turnover of heme proteins such as hemoglobin → protect body from oxidative damage that free heme can cause
HEMOPEXIN
• Low levels are diagnostic of hemolytic anemia
HEMOPEXIN
• Precipitates with C substance, a polysaccharide of pneumococci
C-REACTIVE PROTEIN
• Functions in opsonization
C-REACTIVE PROTEIN
One of the first acute-phase proteins to rise in inflammatory disease
C-REACTIVE PROTEIN
• High or increasing amount of CRP suggests an acute infection or inflammation
C-REACTIVE PROTEIN
• Glycoprotein produced by fetal membranes responsible for the cellular adhesiveness of placenta and membranes to the decidua.
FIBRONECTIN
• Fetal fibronectin is produced at the boundary between the amniotic sac and the decidua (the lining of the uterus) and functions to maintain the adherence of the placenta to the uterus.
FIBRONECTIN
• Test for assessment of the risk for [PRE-TERM DELIVERY] in women between 24 to 35 weeks gestational age.
FIBRONECTIN
• Proteolytic fragments of collagen I formed during bone resorption
CROSS-LINKED C-TELOPEPTIDES
• CTX is a biochemical marker of bone resorption that can be detected in serum and urine.
CROSS-LINKED C-TELOPEPTIDES
Govern excitation-contraction coupling in muscle
TROPONIN• Three subunits: Troponin T (cTnT), Troponin C (TnC), Troponin I (cTnl)
TROPONIN
is used as an AMI indicator because of specificity and early rise in serum concentration following AMI
rises within 3-4 hours, peaks in 10-24 hours, returns to normal in 10-24 days.
rises within 3-6 hours, peaks in 14-20 hours, and returns to normal in 5-10 days.
cTnT or cTnI
cTnT
cTnl
• Now known as the “gold standard” for diagnosis of MI
TROPONIN
• Heme-containing protein that binds oxygen with cardiac and skeletal muscle
MYOGLOBIN
• Levels are related to muscle mass and activity (reasonable sensitivity but poor specificity)
MYOGLOBIN
Increased in skeletal injuries, muscular dystrophy, and AMI
MYOGLOBIN
is released early in cases of AMI, rising in 1-3 hours and peaks in 5-12 hours, and returns to normal in 18-30 hours. However, it is not tissue specific. It is better used as a negative predictor in the first 2-4 hours following chest pain.
MYOGLOBIN
are neurohormones that affect body fluid homeostasis (through natriuresis and diuresis) and blood pressure
BRAIN NATRIURETIC PEPTIDE (BNP) AND N-TERMINAL-BRAIN NATRIURETIC PEPTIDE (NT-BNP)
BNP has become a popular marker for
CONGESTIVE EART FAILURE
are found in largest concentration in the left ventricular myocardium but are also detectable in atrial tissue as well as in the myocardium of the right ventricle.
NT-proBNP and BP
ELECTROPHORETIC PATTERNS OF SERUM PROTEINS
share the property of showing elevations in concentrations in response to stressful or inflammatory states that occur with infection, injury, surgery, trauma, or other tissue necrosis.
acute phase reactant proteins
They include AAT, orosomucoid, haptoglobin, ceruloplasmin, fibrinogen, serum amyloid A protein, and CRP. Others are Factor VIII, ferritin, lipoproteins, complement proteins, and immunoglobulins.
acute phase reactant proteins
Opsonization, complement activation
CRP
Removal of cholesterol
Serum amyloid A
Protease inhibitor
Alpha1-antitrypsin
Binds hemoglobin
Haptoglobin
Clot formation
Fibrinogen
Binds copper, oxidizes iron
Ceruloplasmin
Opsonization, lysis
С3
Complement activation
Mannose-binding protein
• Total protein level less than the reference interval
Hypoproteinemia
• occurs in any condition where a negative nitrogen balance exists (excessive loss, decreased intake, decreased synthesis, accelerated catabolism)
Hypoproteinemia
• Increase in total plasma proteins
Hyperproteinemia
• Not an actual disease state but is the result of dehydration
Hyperproteinemia
When excess water is lost from the vascular system, the proteins, because of their size, remain within the blood vessels
Hyperproteinemia
May also be cause by excessive production, primarily of gamma-globulins e.g. multiple myeloma
Hyperproteinemia
Digestion of protein; measurement of nitrogen content
Kjeldahl
Reference method; assume average nitrogen content of 16%
Kjeldahl
Measurement of refractive index due to solutes in serum
Refractometry
Rapid and simple; assume nonprotein solids are present in same concentration as in the calibrating serum
Refractometry
Formation of violet-colored chelate between Cu?+ ions and peptide bonds
Biuret
Routine method; requires at least two peptide bonds and an alkaline medium
Biuret
Protein binds to dye and causes a spectral shift in the absorbance maximum of the dye
Dye binding
Research use
Dye binding
Globulins are precipitated in high salt concentrations; albumin in supernatant is quantitated by biuret reaction
Salt precipitation
Labor intensive
Salt precipitation
Albumin binds to dye; causes shift in absorption maximum
Methyl orange
HABA [2,4’-hydroxyazobenzene)-benzoic acid]
BCG (bromcresol green)
BCP (bromcresol purple)
Nonspecific for albumin
Methyl orange
Many interferences (salicylates, bilirubin)
HABA [2,4’-hydroxyazobenzene)-benzoic acid]
Sensitive; overestimates low albumin levels; most commonly used dye
BCG (bromcresol green)
Specific, sensitive, precise
BCP (bromcresol purple)
Proteins separated based on electric charge
Electrophoresis
Accurate; gives overview of relative changes in different protein fractions
Electrophoresis
Performed when an abnormality in the total protein or albumin is found
Protein Electrophoresis
Principle: separation of proteins based on their charge density
Protein Electrophoresis
Regions are stained using: Coomassie Blue, Amido Black, Ponceau S
Protein Electrophoresis
Electrophoretic patterns:
Protein Electrophoresis
Probably the most significant finding from an electrophoretic pattern is
(Bishop, 6th ed)
monoclonal immunoglobulin disease
Beta-gamma bridging:
Monoclonal spike:
↑a2, ↑B, ↓albumin:
↓a1-antitrypsin:
↑B:
Cirrhosis
Multiple myeloma
Nephrotic
Emphysema
Inflammation
NPN present in the highest corientation in blood
UREA
Major excretory product of protein metabolism
UREA
• Following synthesis in the liver, urea is carried in the blood to the kidney, where it is readily filtered from the plasma by the glomerulus. Most of the urea in the glomerular filtrate is excreted in the urine.
UREA
• The concentration of urea in the plasma is determined by:
i. renal function and perfusion
ii. the protein content of the diet
iii. rate of protein catabolism
UREA
• evaluate renal function
UREA
• assess hydration status aid in the diagnosis of renal disease
UREA
• verify frequency of dialysis
UREA
• Urea nitrogen concentration is converted to urea concentration by multiplying [?]
• Enzymatic methods are used most frequently in clinical laboratories.
UREA
UREA: Used on many automated instruments; best as kinetic measurement
GLDH coupled enzymatic
UREA: Used in automated systems, multilayer film reagents, and dry reagent strips
Indicator dye
UREA: Specific and rapid
Conductimetric
UREA: Proposed reference method
Isotope dilution mass spectrometry
An elevated concentration in the blood is called azotemia. Very high plasma concentration accompanied by renal failure is called uremia, or the uremic syndrome.
urea
This condition is eventually fatal if not treated by dialysis or transplantation.
uremia, or the uremic syndrome
Low protein intake
Severe vomiting and diarrhea
Liver disease
Pregnancy
DECREASED UREA CONCENTRATION
Congestive heart failure
Shock, hemorrhage
Dehydration
Increased protein catabolism
High-protein diet
INCREASED UREA CONCENTRATION: Prerenal
Acute and chronic renal failure
Renal disease, including glomerular nephritis, tubular necrosis
INCREASED UREA CONCENTRATION: Renal
Urinary tract obstruction
INCREASED UREA CONCENTRATION: Postrenal
- Product of the catabolism of purines (guanine and adenosine)
URIC ACID
- Filtered by the glomerulus and secreted by the distal tubules into the urine, but mostly reabsorbed in the proxima tubules and reused
URIC ACID
- Relatively insoluble in plasma, and at high concentrations, can be deposited in the joints and tissue, causing painful inflammation; mostly present as monosodium urate in plasma, wherein it is insoluble at around pH 7
URIC ACID
• Assess inherited disorders of purine metabolism
URIC ACID
Confirm diagnosis and monitor the treatment of gout
URIC ACID
Assist in the diagnosis of renal calculi
URIC ACID
• Prevent uric acid nephropathy during chemotherapeutic treatment
URIC ACID
• Detect kidney dysfunction
URIC ACID
Liver disease
DECREASED URIC ACID CONCENTRATION
Defective tubular reabsorption (Fanconi syndrome)
DECREASED URIC ACID CONCENTRATION
Chemotherapy with azathioprine or 6-mercaptopurine
DECREASED URIC ACID CONCENTRATION
Overtreatment with allopurinol
DECREASED URIC ACID CONCENTRATION
Enzyme deficiencies
Lesch - Nyhan Syndrome (hypoxanthine guanine phosphoribosyltransferase deficiency)
Phosphoribosy|pyrophosphate synthetase deficiency
Glycogen storage disease type I (glucose-6-phosphatase deficiency)
Fructose intolerance (fructose-1-phosphate aldolase deficiency)
INCREASED URIC ACID CONCENTRATION
Gout
Treatment of myeloproliferative disease with cytotoxic drugs
Hemolytic and proliferative processes
Chronic renal disease
Toxemia of pregnancy
Lactic acidosis
Drugs and poisons
Purine-rich diet
Increased tissue catabolism or starvation
INCREASED URIC ACID CONCENTRATION
is formed from creatine (synthesized primarily in the liver from arginine, glycine and methionine) and creatine phosphate in muscle
Creatinine
Excreted in plasma at a constant rate related to muscle mass
CREATININE
Daily excretion is fairly stable thus it is commonly used to assess renal filtration function
CREATININE
Determine sufficiency of kidney function and severity of disease
CREATININE
Monitor the progression of kidney disease
CREATININE
Measure of completeness of 24 hour collections
CREATININE
Abnormal renal function
INCREASED CREATININE CONCENTRATION
Muscle disease:
Muscular dystrophy
Poliomyelitis
Hyperthyroidism
Trauma
INCREASED CREATININE CONCENTRATION
The normal BUN/CREATININE RATIO is [?]
In prerenal disease, it rises to well [?]
In true renal disease, both BUN and creatinine rise together, maintaining BUN/Creatinine ratio at [?]
(Bishop, 6th ed.)
10:1 to 20:1.
10:1 to 20:1.
10-20:1.
Formed through the deamination of amino acids during protein metabolism
AMMONIA
Removed from the circulation and converted to urea in the liver
AMMONIA
Free ammonia is toxic; however ammonia is present in the plasma in low concentrations
AMMONIA
• Provide useful information on clinical conditions such as hepatic failure, Reye’s syndrome and inherited deficiencies of the urea cycle enzymes
AMMONIA
Measurement of urine ammonia can be used to confirm the ability of the kidneys to produce ammonia
AMMONIA
Severe liver disease
Inherited deficiencies of enzymes of the urea cycle
INCREASED AMMONIA CONCENTRATION
- protein catalysts; increase velocity of a chemical reaction without being consumed during the reaction they catalyze. This is achieved by decreasing the energy of activation (Ea) of a chemical reaction.
Enzymes
- protein catalysts; increase velocity of a chemical reaction without being consumed during the reaction they catalyze. This is achieved by decreasing the energy of activation (Ea) of a chemical reaction.
Enzymes
Majority are proteins so they can be denatured by certain agents (acids, strong bases, detergents, etc.)
Enzymes
Majority are proteins so they can be denatured by certain agents (acids, strong bases, detergents, etc.)
Active site - site where substrate interacts with enzymes
Allosteric site- site other than the active site
Isoenzyme - multiple forms of an enzyme with different genetic origin
Isoform - results when an enzyme is subject to post-translational modifications
Cofactors - nonprotein molecule that must bind to particular enzymes for enzyme reactions to occur
a. Activators - inorganic cofactors
b. Coenzymes - organic cofactor (e.g. vitamins)
Holoenzyme = apoenzyme + prosthetic group (a coenzyme tightly bound to its enzyme)
Proenzyme / zymogen - inactive form
IZYME CLASSIFICATION AND NOMENCLATURE
The International Union of Biochemistry (IUB) Enzyme Commission categorized all enzymes into six (6) classes based on the catalytic activity of an enzyme:
Enzymes
are highly specific for their substrates and products. Many recognize only a single compound as a substrate.
Enzymes
- site where substrate interacts with enzymes
- site other than the active site
Active site
Allosteric site
- multiple forms of an enzyme with different genetic origin
Isoenzyme
- results when an enzyme is subject to post-translational modifications
Isoform
- nonprotein molecule that must bind to particular enzymes for enzyme reactions to occur
Cofactors
- inorganic cofactors
- organic cofactor (e.g. vitamins)
a. Activators
b. Coenzymes
= apoenzyme + prosthetic group (a coenzyme tightly bound to its enzyme)
Holoenzyme
- inactive form
Proenzyme / zymogen
categorized all enzymes into six (6)
classes based on the catalytic activity of an enzyme
The International Union of Biochemistry (IUB) Enzyme Commission
- catalyze an oxidation-reduction reaction between two substrates
- Oxidoreductases
- catalyze the transfer of a group other than hydrogen from one substrate to another
- Transferases
- catalyze hydrolysis of various bonds
- Hydrolases
- catalyze removal of groups from substrates without hydrolysis; the product contains double bonds
- Lyases
- catalyze the interconversion of geometric, optical or positional isomers
- Isomerases
- catalyze the joining of two substrate molecules, coupled with the breaking of the pyrophosphate bond in ATP or a similar compound
Ligases
- rate of reaction is almost directly proportional to substrate concentration at low level
FIRST-ORDER KINETICS
- rate of reaction is almost directly proportional to substrate concentration at low level
FIRST-ORDER KINETICS
• substrate is high enough to saturate all available enzymes
reaction rate is unaffected by increase in substrate concentration
ZERO-ORDER KINETICS
• dependent on enzyme concentration only
ZERO-ORDER KINETICS
• when maximum velocity is reached, the rate of increase in velocity is “zero”
ZERO-ORDER KINETICS
pH
a. most enzymes react at pH
b. controlled through
7.0 - 8.0
buffer solutions
Temperature
a. Increased temperatures increase
b. For each 10°C, rate of reaction is
c. Temperatures which are too high can
rate of reaction
doubled
denature proteins
- required for enzymatic activity but are not consumed in the process (unlike substrates)
Cofactors
- the presence of inhibitors can affect the reaction velocity
Inhibitors
- compete for the substrate at the active site of the enzyme and form an enzyme - inhibitor complex; competitive inhibition can be reversed by increasing substrate [S]
a. Competitive inhibitors
- bind to the enzyme or enzyme-substrate at a site distinct from the active site, decreasing the activity of the enzyme. Inhibition cannot be overcome by increasing substrate.
b. Non-competitive inhibitors
- inhibitor binds to the enzyme-substrate complex, preventing the formation of a product. Increasing substrate concentration further increases inhibition.
Uncompetitive inhibitors
: reaction is stopped then measurement is done
: multiple measurements are done at different intervals
Fixed time
Continuous monitoring or kinetic
1 International Unit: amount of enzyme that will catalyze the reaction of [?]
1 Katal: amount of enzyme that will catalyze the reaction of [?]
1 IU = [?]
1 umol of substrate in 1 minute
1 mole of substrate in 1 second
17nkat
HIGH SPECIFICITY
- RBC & Prostate
- Liver
- Pancreas & Salivary glands
- Pancreas
MODERATE SPECIFICITY
- Liver, Heart, Skeletal muscle
- Heart, Skeletal muscle, brain
- Liver, Bone, Kidney, Placenta
LOW SPECIFICITY
- all tissues
ACP
ALT
AMS
LIPASE
AST
CK
ALP
LDH
HEART
CK-MB, AST, LD1>LD2
BONE
ALP
LIVER
Hepatocellular disorders:
Biliary tract obstruction:
AST, ALT, LD5
ALP, GGT
PANCREAS
Amylase Lipase
SKELETAL MUSCLE
CK-MM, AST, LD, Aldolase
BRAIN
СК-BB
PROSTATE
ACP
Widely distributed in skeletal muscle, brain and cardiac muscle
CREATINE KINASE
May be detected in nerve tissue, testicular tissue, amniotic fluid and in certain malignant tissues
CREATINE KINASE
exists as a dimer (subunits: M or B)
CREATINE KINASE
• Major form in sera of healthy people and in striated muscle
СК-ММ
• Increased in hypothyroidism, IM injections, mild to strenuous activity
СК-ММ
• Cardiac tissue contains significant amounts
СК-МВ
• Value in detection of AMI
СК-МВ
Rise within 4-8 hours, peak at 12-24 hours, return to normal within 48 to 72 hours
СК-МВ
Highest concentration in CNS
СК-ВВ
• Increased with extensive damage to the brain and carcinoma of various organs
СК-ВВ
Frequently elevated in disorders of cardiac and skeletal muscles
CREATINE KINASE
Sensitive indicator of AMI and Duchenne-type muscular dystrophy
CREATINE KINASE
Separation of total CK into its various isoenzyme fractions is considered a more specific indicator of various disorders than total levels:
CREATINE KINASE
• Optimum pH: 9.0
• Coupled with pyruvate kinase and lactate dehydrogenase (NADH → NAD+)
• Absorbance at 340 nm is determined
CK: Forward Reaction (Tanzer-Gilvarg)
• More commonly performed method in the laboratory
• Reverse reaction is two to six times faster and has less interferences
• Optimum pH: 6.8
CK: Reverse Reaction (Oliver-Rosalki)
Sources of Error
Anticoagulants inhibit enzyme activity
Avoid hemolysis
Serum should be stored in the dark
Muscular activity and muscle mass have effects on levels
CREATINE KINASE
Widely distributed in the tissues of the body
LACTATE DEHYDROGENASE
High concentrations in the heart and liver
LACTATE DEHYDROGENASE
Five major isoenzyme fractions ; Each isoenzyme is composed of four subunits. The subunits are of two different structures: (heart) and M (muscle) polypeptide
LACTATE DEHYDROGENASE
LACTATE DEHYDROGENASE Serum concentration:
LD flipped pattern:
LD-2 > LD-1 > LD-3 > LD-4 > LD-5
Heart, RBCs
LD-1
LD-2
Myocardial infarction
Hemolytic anemia
LD-1
Megaloblastic anemia
Acute renal infarct
Hemolyzed specimen
LD-2
Lung, lymphocytes, spleen and pancreas
LD-3
Pulmonary embolism
Extensive pulmonary pneumonia
Lymphocytosis
Acute pancreatitis
Carcinoma
LD-3
Liver
Hepatic injury or inflammation
LD-4
Skeletal muscles
Skeletal muscle injury
LD-5
Because of its widespread activity in numerous body tissue, [?] is elevated in a variety of disorders.
LDH
Highest levels are seen in pernicious anemia and hemolytic disorders
LDH
In AMI: rises 12-24 hours after onset, peaks 48-72 hours, remains elevated for 10 days An elevated total value is a nonspecific finding. Assays therefore assume more clinical significance when separated into isoenzyme fractions.
LDH
Optimum pH: 8.3 to 8.9
LDH: Forward reaction (Wacker et al)
Optimum pH: 7.1 to 7.4
Rate of reaction is three times faster → smaller sample volumes and shorter reaction times
More susceptible to substrate exhaustion and loss of linearity
LDH: Reverse reaction (Wrobleuski & La Due)
Sources of Error
Avoid hemolysis
Do not freeze specimen
Serum is the sample of choice since some anticoagulants (e.g. oxalate) inhibit the enzyme
LDH
increases within 4-8 hours of MI, peaks at 12-24 hours, elevated 3-4 days
increases within 12-24 hours, peaks at 72 hours, elevated 10 days
CK
LD
[?] are heat stable; [?] labile
[?]↑with liver and skeletal muscle disease.
[?] with hemolysis
LD1 and LD2; LD5
LD4 and LD5
LD1 > LD2
Widely distributed in human tissue
Aspartate aminotransferase (SGOT)
Highest concentration: cardiac tissue, liver & skeletal muscle
Aspartate aminotransferase (SGOT)
Smaller amounts: kidney, pancreas & RBCs
Aspartate aminotransferase (SGOT)
Bilocular enzyme: cytoplasmic & mitochondrial isoenzyme
Aspartate aminotransferase (SGOT)
Distributed in many tissues
Alanine aminotransferase (SGPT)
Comparatively high concentrations in the liver (more liver-specific enzyme of the aminotransferases)
Alanine aminotransferase (SGPT)
catalyze the interconversion of amino acids & alpha-ketoacids by transfer of amino groups
AMINOTRANSFERASES
acts as the coenzyme for both AST and ALT
Pyridoxal phosphate
AMI: rises within 6-8 hours, peaks 24 hours, returns to normal in 5 days
AST
measurements are mainly confined to evaluation of hepatic disorders
• ALT
• With most forms of acute hepatocellular injury (e.g. hepatitis) [?] will be higher than ALT initially, because of the higher activity of [?] in hepatocytes. Within 24 - 48 hours, particularly if ongoing damage occurs, ALT will become higher than AST, based on its longer half-life
AST
AST
In alcoholic hepatitis, the elevations in ALT are comparatively lower than AST, resulting in an AST/ALT ratio (De Ritis ratio) [?]. (Bishop)
greater than 2
Oxaloacetate from AST activity reacts with Malate dehydrogenase as the indicator enzyme
Monitors the change in absorbance at 340 nm
continuously as NADH is oxidized to NAD
Optimal pH: 7.3 - 7.8
Karmen Method (for AST)
Pyruvate from ALT activity reacts with lactate dehydrogenase as the indicator enzyme
Walker et al Method (for ALT)
Sources of Error
[?] can be dramatically increased in hemolyzed specimens while [?] is relatively unaffected.
AST
ALT
Involved in the cleavage of phosphate-containing compounds in alkaline pH
ALKALINE PHOSPHATASE
facilitates the transfer of metabolites across cell membranes associated with lipid transport and the calcification process in osseous tissues
ALKALINE PHOSPHATASE
Located in a wide variety of tissues
None in erythrocytes
ALKALINE PHOSPHATASE
ALKALINE PHOSPHATASE: Separation is done through the following methods
[?]- reduces activity of intestinal and placental isoenzymes
[?]- Reduces activity of bone and liver enzyme
Heat fractionation
Electrophoretic fractionation
Inhibition with phenylalanine
Inhibition with levamisole
Often used in evaluation of hepatobiliary (obstructive conditions) and bone disorders (osteoblast involvement)
ALKALINE PHOSPHATASE
Highest elevation of ALP is seen in [?]
Physiologic elevation of ALP can be seen in [?]
Sources of Error
Use serum or heparinized plasma only
Increased activity in specimens left standing on the clot for a long time due to a gradual development of a more basic pH in the system as CO2 is lost
ALKALINE PHOSPHATASE
ACID PHOSPHATASE: Tissue distribution and isoenzymes
- band 1; inhibited by tartrate
- bands 2 & 4
- band 3; major form in plasma
- band 5; osteoclasts
Prostatic ACP
Granulocytic ACP
Platelets, RBs & Monocytes
Bone isoenzyme
Separation is done through the following methods
Electrophoretic separation
Chemical inhibition - pACP inhibited by L-tartrate
Immunoassays
ACID PHOSPHATASE
Prostatic cancer: ACP is inferior to PSA
↑ Prostatic ACP
Prostatic hyperplasia and prostatic infarction
↑ Prostatic ACP
Urinary tract obstruction, carcinoid tumors of rectum and prostatic massage
↑ Prostatic ACP
assays have proven useful in forensic clinical chemistry, particularly in the investigation of rape. Vaginal washings are examined for seminal fluid (presumptive evidence of rape in such cases).
ACID PHOSPHATASE
Sources of Error
Store at appropriate pH and temperature
Sample of choice: plasma (to minimize contamination by platelets)
Preferred anticoagulant citrate buffered to a pH of 6.2 to 6.6
Avoid hemolysis
ACID PHOSPHATASE
Catalyze the hydrolytic cleavage of peptides to form amino acids or smaller peptides
GAMMA-GLUTAMYLTRANSFERASE
Plasma membrane-bound on cells
GAMMA-GLUTAMYLTRANSFERASE
Widely distributed in a number of tissues
GAMMA-GLUTAMYLTRANSFERASE
Highest amounts of GGT in the kidneys / Significant amounts in pancreas and liver
GAMMA-GLUTAMYLTRANSFERASE
Useful marker for liver damage
GAMMA-GLUTAMYLTRANSFERASE
Increased in obstructive liver disease, inflammation of the liver, obstruction of the biliary tract
GAMMA-GLUTAMYLTRANSFERASE
Increase over time in patients on long-term medications
GAMMA-GLUTAMYLTRANSFERASE
Most useful application for GGT measurements:
GAMMA-GLUTAMYLTRANSFERASE
Reference method:
Product of the reaction: 5-amino-2-nitrobenzoate (410 nm)
Szasz Assay
Sources of Error
Preferred specimen: serum
Some anticoagulants inhibit activity
GAMMA-GLUTAMYLTRANSFERASE
Primarily responsible for starch digestion
Splits complex CHO made up of a-D glucose units
AMYLASE
Two major sources:
AMYLASE
Major clinical reason for increased serum amylase:
cancers
kidney impairment (e.g. renal failure)
conditions affecting salivary glands
↑ AMYLASE
: number of milligrams of glucose released in 30 minutes at 37 deg C under specific assay conditions
Somogyi unit
: inhibits salivary amylase
Wheat germ lectin
Electrophoresis: salivary amylase is more
anodal
Measures the rate of disappearance of the starch substrate
Amyloclastic (lodometric)
Measures the amount of reducing sugars produced by the hydrolysis of starch
Saccharogenic (Nelson Somogyi mod. By Henry & Chiamon)
Measures the increasing color from production of product coupled with a chromogenic dye
Chromogenic (Klein, Foreman, Searcy)
This measures the change in turbidity of starch solution over a short reaction period. This is used for stat analysis to rule out acute upper abdominal pain
Turbidimetry and Nephelometry (Peralta
& Reinhart)
AMYLASE Classic reference method:
Enzyme that hydrolyzes glycerol esters of long chain fatty acids (e.g. triglycerides)
LIPASE
Full activity in the presence of bile salts and colipase
LIPASE
Highest concentrations found in pancreatic tissue and secretions
Assessment of acute pancreatitis
LIPASE
• Rise: 2 - 12 hours
• Peak value within 48 - 72 hours
• Remains elevated for 10 - 14 days
LIPASE
LPS: Cherry-Crandall method
Substrate:
Liberated fatty acids were measured after a 24-h incubation
LPS: Cherry-Crandall method
Modifications - used triolein as substrate
LPS: Cherry-Crandall method
Simpler and more rapid
LPS: Turbidimetric method
Based on coupled reactions with enzymes such as peroxidase or glycerol kinase
LPS: Colorimetric method
Sources of Error
Bacterial contamination - false elevation
Avoid hemolysis - hemoglobin inhibits lipase thus causing falsely lower values
LPS
Sources include the adrenal cortex, spleen, thymus, lymph nodes, lactating mammary gland and RBCs
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
Little activity found in normal serum
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
Specimen
RBC hemolysate: to detect enzyme deficiencies
Serum: to detect enzyme evaluations
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
Inherited sex-linked trait
Drug-induced hemolytic anemia
Increased levels in MI and megaloblastic anemias (but not routinely tested in these conditions)
G6PD deficiency
Reaction catalyzed: hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid, a reaction necessary to allow a cholinergic neuron to return to its resting state after activation
CHOLINESTERASE
Uses only acetylcholine as a substrate (high substrate specificity)
“TRUE” CHOLINESTERASE
Hydrolyzes a variety of choline esters
“PSEUDO” CHOLINESTERASE
Also known as acetylcholinesterase
“TRUE” CHOLINESTERASE
Referred to as “cholinesterase”
“PSEUDO” CHOLINESTERASE
Primary location are synapses of nerve cells.
Also seen in RBCs, lung, brain, spleen
“TRUE” CHOLINESTERASE
Found in serum and in the white matter of the central and peripheral nervous system Also in the heart, liver and pancrease
“PSEUDO” CHOLINESTERASE
Important part of the process for the transmission of nerve impulses
“TRUE” CHOLINESTERASE
Protective function in the body; hydrolyzes choline esters (other than acetylcholine) which can inhibit acetylcholinesterase
“PSEUDO” CHOLINESTERASE
CHOLINESTERASE
Pathological values are
Important roles in the diagnosis and management of
Richest source in prostate. Also in bone, liver, spleen, kidneys, RBCs and platelets.
Acid phosphatase (ACP)
↑in prostatic carcinoma, bone disease
Acid phosphatase (ACP)
Catalyze hydrolysis of phosphomonoesters = alcohol + phosphate ion
Acid phosphatase (ACP)
Alkaline phosphatase (ALP)
Reacts optimally at pH 5.0
Acid phosphatase (ACP)
Differentiate prostatic portion using tartrate as inhibitor (inhibited by tartrate).
Acid phosphatase (ACP)
Also used in rape investigation.
Acid phosphatase (ACP)
Activity associated with osteoclasts.
Acid phosphatase (ACP)
Separate serum from RBCs ASAP
Acid phosphatase (ACP)
Freeze or acidify to <pH 6.5
Acid phosphatase (ACP)
Avoid hemolysis.
Acid phosphatase (ACP)
Alkaline phosphatase (ALP)
AST
Most human tissues. Highest concentrations in intestines, liver, bone, spleen, placenta and kidney.
Alkaline phosphatase (ALP)
↑in hepatobiliary disease and bone disorders; highest ↑with Paget’s disease
Alkaline phosphatase (ALP)
Reacts optimally at pH 9.0 to 10.0
Alkaline phosphatase (ALP)
Requires Mg2+
Alkaline phosphatase (ALP)
Associated with osteoblast activity
Alkaline phosphatase (ALP)
Diet may induce elevations in ALP activity of blood group B and O individuals who are secretors.
Alkaline phosphatase (ALP)
Primarily in kidney, brain, prostate, pancreas and liver.
Gamma-glutamyltransferase (GGT)
↑ with biliary tract obstruction, chronic alcoholism
Gamma-glutamyltransferase (GGT)
Glutathione serves as gamma-glutamyl donor in most biologic systems.
Gamma-glutamyltransferase (GGT)
Lower values in females.
Gamma-glutamyltransferase (GGT)
Hemolysis does not interefere.
Gamma-glutamyltransferase (GGT)
Many tissues; Considered the liver-specific enzyme of the transferases
Alanine aminotransferase (ALT)
↑ with liver disease
Alanine aminotransferase (ALT)
Formerly SGPT. Pyridoxal phosphate as coenzyme.
Alanine aminotransferase (ALT)
Relatively unaffected by hemolysis.
Alanine aminotransferase (ALT)
Many. Highest in liver, heart, skeletal muscle.
Alanine aminotransferase (ALT)
↑ with liver disease, myocardial infarction (MI, muscular dystrophy
Alanine aminotransferase (ALT)
Formerly SGOT. Pyridoxal phosphate as coenzyme.
Alanine aminotransferase (ALT)
Marked elevation with viral hepatitis.
Alanine aminotransferase (ALT)
Don’t use ammonium heparin
Alanine aminotransferase (ALT)
All. Highest in liver, heart, skeletal muscle, RBCs.
Lactate dehydrogenase (LD)
↑ with MI, liver disease, pernicious anemia
Lactate dehydrogenase (LD)
Catalyzes lactic acid › pyruvic acid
Lactate dehydrogenase (LD)
Serum should be separated in clot immediately to prevent ↑in LD1 and LD2.
Lactate dehydrogenase (LD)
Unstable; Store at 25 °C
Lactate dehydrogenase (LD)
Highest levels with pernicious anemia.
Lactate dehydrogenase (LD)
Enzyme that stays elevated longest with MI.
Lactate dehydrogenase (LD)
Some anticoagulants interfere.
Lactate dehydrogenase (LD)
Cardiac muscle, skeletal muscle, brain.
Creatine kinase (CK)
↑ with MI and muscular dystrophy
Creatine kinase (CK)
Catalyzes phosphocreatinine + ADP → creatine +ATP.
Creatine kinase (CK)
Most sensitive enzyme for skeletal muscle disease.
Creatine kinase (CK)
Highest levels with muscular dystrophy.
Creatine kinase (CK)
First enzyme to increase with MI.
Creatine kinase (CK)
Inhibited by all anticoagulants except heparin.
Creatine kinase (CK)
Salivary glands, pancreas
Amylase (AMS)
↑ with acute pancreatitis
Amylase (AMS)
Breaks down starch to simple sugars.
Amylase (AMS)
Saccharogenic method measures sugar produced. lodometric or amyloclastic measures starch remaining. Chromogenic method measures dye released from the breakdown of polysaccharide. Kinetic method measures change of NAD to NADH at 340 nm.
Amylase (AMS)
Ca2+ and Cl required.
Amylase (AMS)
Don’t use EDTA, citrate or oxalate.
Amylase (AMS)
Urine levels stay elevated longer than serum.
Amylase (AMS)
Pancreas;↑ with acute pancreatitis
Lipase (LPS)
Breaks down triglycerides into fatty acids and glycerol.
Lipase (LPS)
Olive oil substrate.
Lipase (LPS)
Levels usually parallel amylase, but may peak a little later and stay elevated longer.
Lipase (LPS)
RBCs;↓ with hereditary predisposition to hemolytic crises after ingestion of oxidant drugs such as primaquine, infection or diabetic ketoacidosis
Glucose-6-phosphate dehydrogenase (G6PD)
Involved in first step of glucose metabolism.
Glucose-6-phosphate dehydrogenase (G6PD)
Measure in hemolysate of whole blood.
Glucose-6-phosphate dehydrogenase (G6PD)
Liver, brain
Pseudocholinesterase (PChE)
↑ after exposure to organophosphorus compounds found in insecticides and nerve gases, with hypersensitivity to succinylcholine and liver disease
Pseudocholinesterase (PChE)
1.2 to 1.5 kg
Extremely vascular - blood supply comes from the hepatic artery and portal vein
1500 mL blood/min
Functional unit: LOBULE
LIVER
-Six-sided with one portal triad (comprised of a hepatic artery, a portal vein and a bile duct) on each side
-Two major cell types:
• Kupffer cells
• Hepatocytes (80%)
LOBULE
major waste product of heme catabolism
Approximately 200-300 mg produced per day
BILIRUBIN
Synthesis:
Proteins - albumin, cholinesterase, coagulation proteins, cholesterol, bile salts, glycogen
Cholesterol
Bile salts
Glycogen
BILIRUBIN
Metabolism:
Glucose to acetyl-CoA, gluconeogenesis, amino acid conversions, fatty acids
BILIRUBIN
Detoxification:
Bilirubin, drugs, ammonia
BILIRUBIN
Excretion:
Bile acids
BILIRUBIN
Jaundice becomes noticeable to the naked eye once bilirubin levels reach
> 3.0 mg/dL.
: increased amounts of bilirubin are being presented to the liver
a. Prehepatic Jaundice
: primary problem is within the liver
b. Hepatic Jaundice
: lower production of UDPGT due to a genetic lesion and overall lower enzymatic activity; an additional defect related to a transport deficit in the sinusoidal membrane of the hepatocyte may be present
Gilbert Disease
: more serious disorder; multiple mutations in the gene coding for UDPGT results in the production of mildly dysfunctional to completely nonfunctional UDPGT.
Crigler- Najjar Syndrome
: more serious form; homozygously nonfunctioning proteins
• Crigler- Najjar Syndrome Type I
: severe deficiency
• Crigler- Najjar Syndrome Туре Il
Infants who are affected with Crigler-Najjar syndrome, especially the more severe form, develop severe unconjugated hyperbilirubinemia, which typically leads to kernicterus, the deposition of bilirubin in the brain. Motor dysfunction and retardation can result. The danger of kernicterus is a certainty at levels exceeding
20 mg/dL.
: removal of conjugated bilirubin from the liver cell & the excretion into the bile are defective
Dubin-Johnson
• Caused by deficiency of the canalicular transporter protein (MDR2/cMOAT)
Dubin-Johnson
• Appearance of dark-stained granules on a liver biopsy
Dubin-Johnson
: hypothesized to be due to a reduction in the concentration or activity of intracellular binding proteins; Liver biopsy does NOT show dark pigmented granules
iv. Rotor syndrome
v. Physiologic jaundice:
: biliary obstructive disease; stool loses color (clay-colored)
c. Posthepatic jaundice
: biliary obstructive disease; stool loses color (clay-colored)
c. Posthepatic jaundice
• Clinical condition in which tissue scar replaces normal, healthy liver tissue
Cirrhosis
• Most common causes include chronic alcoholism and chronic hepatitis C infection
Cirrhosis
are more common than primary liver cancers
a. Metastatic liver cancers
is the most common malignant tumor of the liver
b. Hepatocellular carcinoma
Reye Syndrome
• Almost exclusively seen in
• Often preceded by a
• Strong association with intake of
• Acute illness characterized by non-inflammatory encephalopathy and fatty degeneration of the liver
Reye Syndrome
• Mild hyperbilirubinemia; threefold changes in ammonia and aminotransferases (AST and ALT)
Reye Syndrome
Drug and Alcohol-related Disorders
Drugs cause injury most commonly via immune-mediated injury to the [?]
[?]: most important drug which can cause liver damage; can lead to alcoholic cirrhosis
[?] can cause fatal hepatic necrosis
hepatocytes
Acetaminophen
• Bilirubin + diazotized sulfanilic acid → azobilirubin (pink-purple)
Classic Diazo Reaction
Reaction Initially done before on urine samples
Classic Diazo
• Can be done on serum samples, but only in the presence of accelerators (solubilizer)
Classic Diazo Reaction
Classic Diazo Reaction
• Evelyn-Malloy reaction:
• Jendrassik-Grof :
Total Bilirubin =
unconjugated bilirubin + conjugated bilirubin + delta bilirubin
Attached to one or two glucuronic acid molecules
CONJUGATED BILIRUBIN
Reacts DIRECTLY with the color reagent
CONJUGATED BILIRUBIN
Noncovalently attached to albumin
UNCONJUGATED BILIRUBIN
Does not react with the color reagent until the bilirubin is first dissociated from albumin using an accelerator (INDIRECT)
UNCONJUGATED BILIRUBIN
Non-polar; water insoluble
UNCONJUGATED BILIRUBIN
AKA hemobilirubin / slow-reacting bilirubin
UNCONJUGATED BILIRUBIN
Polar; water soluble
CONJUGATED BILIRUBIN
AKA cholebilirubin / one-minute / prompt bilirubin
CONJUGATED BILIRUBIN
• Third fraction of bilirubin
Delta bilirubin
• Conjugated bilirubin that is covalently bound to albumin.
Delta bilirubin
Seen only when there is significant hepatic obstruction.
Delta bilirubin
• When present, will react in most laboratory methods as conjugated bilirubin.
Delta bilirubin
Bilirubin is very sensitive to and is destroyed by light; therefore, specimens should be protected from
light. If left unprotected from light, bilirubin values may reduce by
30% to 50% per hour.
WATER BALANCE
• Average water content of the human body ranges from [?]
• Located within the [?] compartments.
• Concentrations of ions within cells are maintained both by [?]
40% to 75% of total body weight.
intracellular (2/3) and extracellular (1/3)
active and passive transport.
Physical property based on the concentration of solutes (colligative property)
OSMOLALITY
The sensation of thirst and arginine vasopressin hormone (AVP formerly ADH; posterior pituitary gland) secretion are stimulated by the hypothalamus in response to an increased osmolality of blood
OSMOLALITY
Normal plasma osmolality =
275 - 295 mOsm/kg of plasma H20
Indirectly indicates the presence of osmotically active substances other than Na+, urea or glucose, such as:
Ethanol
Lactate
Methanol
Betanydroxybutyrate
Ethylene glycol
OSMOLAL GAP
Volume and osmotic regulation
Sodium, potassium, chloride
Myocardial rhythm and contractility
Potassium, magnesium, calcium
Cofactors in enzyme activation
Magnesium, calcium, zinc
Regulation of ATPase ion pumps
Magnesium
Acid-base balance
Bicarbonate, potassium, chloride
Blood coagulation
Calcium, magnesium
Neuromuscular excitability
Potassium, calcium, magnesium
Production and use of ATP from glucose
Magnesium, phosphate
Greatly depends on intake and excretion of water and on renal regulation of Na*; AVP, aldosterone, angiotensin Il and ANP
Sodium
Kidneys are important in regulation; aldosterone stimulates secretion into urine
Potassium
Aldosterone secretion conserves Cl; changes usually parallel Na*
Chloride
Most reabsorbed in kidneys as CO2
Bicarbonate
Regulation controlled largely by kidneys; regulation can be related to Ca?+ and Na*; PTH increases renal reabsorption and intestinal absorption; aldosterone and thyroxine increase renal excretion
Magnesium
↑: PTH, vitamin D
↓: calcitonin
Calcium
Kidneys play major role in regulation
↓: PTH
↑: Vitamin D and Growth hormone
Phosphate
Not specifically regulated; liver is the major organ for removing lactate
Lactate
- Difference between unmeasured anions and unmeasured cations
ANION GAP
- Useful in indicating an increase in one or more of the unmeasured anions in the serum and also as a form of QC for the analyzer used to measure these electrolytes
ANION GAP
Hypoalbuminemia
LOW ANION GAP
Severe hypercalcemia
LOW ANION GAP
uremia/ renal failure
ketoacidosis
methanol, ethanol, ethylene glycol, salicylate poisoning
lactic acidosis
hypernatremia
instrument error
ELEVATED ANION GAP
is amperometric (Clarke electrode).
• p02 measurement
are potentiometric, using the Severinghaus and glass membrane electrodes respectively.
pCO2 and pH measurements
• Several acid-base parameters can be calculated from measured pH and pCO2 values:
1. calculation is based on the Henderson Hasselbach equation.
2. can be calculated using the solubility coefficient of CO2 in plasma at 37°C
3. is the bicarbonate plus the dissolved CO2 (carbonic acid) plus the associated CO2 with proteins (carbamates).
HCO3
Carbonic acid concentration
Total CO2 content
Mixture of a weak acid and its salt with the capability of combining with protons and releasing protons in response to external shifts in pH
BUFFERS
Purpose: maintain a defined pH
BUFFERS
BUFFER SYSTEMS IN THE BODY
A. Plasma bicarbonate buffer
B. RBC hemoglobin/oxyhemoglobin buffer
C. Organic and inorganic phosphate buffer
D. Plasma proteins
Carbon dioxide = driving force in the bicarbonate carbonic acid system
TRH, CRF, GnRH, others
Hypothalamus
TSH, ACTH, FSH, LH, prolactin, GH
Anterior Pituitary
Vasopressin, oxytocin
Posterior Pituitary
Epinephrine, norepinephrine
Adrenal Medulla
Cortisol, 11-deoxycortisol, aldosterone
Adrenal Cortex
T3, T4, calcitonin
Thyroid
Parathyroid hormone (PTH)
Parathyroid
Insulin, glucagon
Pancreas
Estrogens
Ovaries
Testosterone, other androgens
Testes
Released directly from the tissue into the bloodstream and carried to the specific site of action.
HORMONES
Acts at a specific site or sites (target cells) to induce certain characteristic biochemical changes.
HORMONES
Adrenal glands, gonads, and placenta
STEROID HORMONES
Anterior pituitary, placenta, and parathyroid glands
PROTEIN HORMONES
Thyroid and adrenal glands
AMINE HORMONES
Cholesterol
STEROID HORMONES
Protein
PROTEIN HORMONES
Amino acids
AMINE HORMONES
Synthesized as needed, not stored
STEROID HORMONES
Synthesized, then stored in cell as secretory granules until needed
PROTEIN HORMONES
Synthesized, then stored in the cell as secretory granules until needed
AMINE HORMONES
Lipid
Water
STEROID HORMONES
PROTEIN HORMONES ; AMINE HORMONES
Lipid
Water
STEROID HORMONES
PROTEIN HORMONES ; AMINE HORMONES
Protein
Do not need protein
Require a carrier protein and others do not
STEROID HORMONES
PROTEIN HORMONES
AMINE HORMONES
Cortisol Aldosterone Testosterone Estrogen, and Progesterone
STEROID HORMONES
FSH*
LH*
TSH*
hCG*
Glucagon
Parathyroid hormone
Growth hormone
Prolactin
PROTEIN HORMONES
Epinephrine
Norepinephrine
Thyroxine
Triiodothyronine
AMINE HORMONES
- Composed of two polypeptide chains containing carbohydrate. Alpha chains are the same in all. Beta chains determine specificity
*Glycoproteins
Gonad (tropic)
Luteinizing Hormone (LH)
Follicle-stimulating hormone (FSH)
Maturation of follicles, ovulation, production of estrogen progesterone, testosterone
Luteinizing Hormone (LH)
• Regulated by GnRH from hypothalamus.
Luteinizing Hormone (LH)
• Sharp ↑ just before ovulation.
Luteinizing Hormone (LH)
↑ FSH & LH in
ovarian failure, menopause
Sperm and egg production
Follicle-stimulating hormone (FSH)
• Regulated by gonadotropin-releasing hormone (GnRH) from hypothalamus
Follicle-stimulating hormone (FSH)
Thyroid (tropic)
Thyroid-stimulating hormone (TSH)
Production of T3 and T4 by thyroid
Thyroid-stimulating hormone (TSH)
• Thyrotropin-releasing hormone (TRH) from hypothalamus.
Thyroid-stimulating hormone (TSH)
Adrenal (tropic)
Adrenocorticotropic hormone (ACTH)
Production of adrenocortical hormones by adrenal cortex
Adrenocorticotropic hormone (ACTH)
• Regulated by corticotropin-releasing hormone (CRH) from hypothalamus.
Adrenocorticotropic hormone (ACTH)
• Diurnal variation: highest levels in early am, lowest in late afternoon.
Adrenocorticotropic hormone (ACTH)
↑ ACTH in
Cushing’s disease.
(GH; aka somatotropin)
Growth hormone
Multiple (direct effector)
Growth hormone
Allows an individual to transition from a fed state to a fasting state
Growth hormone
↑ protein synthesis in skeletal muscle and other tissues
Growth hormone
Antagonizes the effects of insulin
Growth hormone
• Regulated by growth-hormone releasing hormone (GHRH) & somatostatin from hypothalamus.
Growth hormone
↑ GH in
↓ GH in
Gigantism & Acromegaly
Dwarfism.
Breasts (direct effector)
Prolactin
Lactation
Prolactin
Regulated by prolactin-releasing factor (PRF) & prolactin inhibiting factor (PIF) from hypothalamus.
Prolactin
Breasts and uterus (direct effector)
Oxytocin
Critical role in lactation
Oxytocin
Major role in labor and parturition
Oxytocin
Produced in hypothalamus. Stored in posterior pituitary
Oxytocin
Kidneys (direct effector)
Vasopressin (Antidiuretic hormone)
Regulation of renal free water excretion
Vasopressin (Antidiuretic hormone)
Produced in hypothalamus. Stored in posterior pituitary.
Vasopressin (Antidiuretic hormone)
Release stimulated by ↑ osmolality, ↓ blood volume or blood pressure.
Vasopressin (Antidiuretic hormone)
↓ in diabetes insipidus.
Vasopressin (Antidiuretic hormone)
• Located near larynx; two lobes connected by a thin piece of tissue
THYROID GLAND
Exerts significant control over the rate of metabolism in humans
THYROID GLAND
THYROID GLAND Hormones produced:
T3, T4, calcitonin
Stimulated by TSH (from the anterior pituitary to produce and secrete T3, T4
THYROID GLAND
THYROID GLAND Functional unit:
thyroid follicle
Comprised of follicular cells surrounding a central colloid
thyroid follicle
Colloid contains thyroglobulin, which is rich in tyrosine (the amino acid that forms the backbone for the thyroid hormone molecules)
thyroid follicle
- contains tyrosine which forms the backbone for the thyroid hormone molecules
•Thyroglobulin
- contains tyrosine which forms the backbone for the thyroid hormone molecules
•Thyroglobulin
= T3 (3, 5, 3’-triiodothyronine)
• Monoiodotyrosine + dodotyrosine
= T4 (3, 5, 3’, 5’-tetraiodothyronine)
• Diiodotyrosine + diodotyrosine
is more potent (T4 is converted to T3 suggesting that T3 is more important)
• T3
Major transport protein for T3 and T4
Thyroxine-binding globulin (TBG)
: 0.03-0.05% is unbound (almost completely bound to proteins)
: 0.5% free (weaker attachment to proteins)
T4
T3
RIA, fluorometric enzyme immunoassay, fluorescence polarization immunoassay (FPIA)
Thyroxine
RIA, microparticle enzyme immunoassay, fluorometric
enzyme immunoassay
Triiodothyronine
Resin uptake, FPIA
Thyroid hormone binding ratio, T3 uptake, T uptake
Equilibrium dialysis, immunometric assay (chemiluminescence)
Free T4
RIA
Free T3
Calculation from T4 and THBR
Free T4 index, Free thyroxine index (FTI), T7
Calculation from T3 and THBR
Free T3 index
RIA, Immunometric assay (IMA)
Thyroid-stimulating hormone
Cretinism
Hashimoto’s thyroiditis
Hypothyroidism
• Hypothyroidism in neonates and infants
Cretinism
• Characterized by mental retardation, short stature with skeletal abnormalities, coarse facial features, enlarged tongue and umbilical hernia
Cretinism
• Causes:
Maternal hypothyroidism during early pregnancy
Thyroid agenesis lodine deficiency
Dyshormonogenetic goiter (congenital defect in thyroid hormone production; most commonly involves thyroid peroxidase)
Cretinism
• Most common cause of hypothyroidism in regions where iodine levels are adequate
Hashimoto’s thyroiditis
• Autoimmune destruction of thyroid gland
Hashimoto’s thyroiditis
• Anti-thyroglobulin and anti-thyroid peroxidase antibodies are often present
Hashimoto’s thyroiditis
• Associated with HLA-DR5
Hashimoto’s thyroiditis
Subclinical hypothyroidism =
Subclinical hyperthyroidism =
↑ TSH, normal FT4
↓ TSH, normal FT4
Grave’s disease
Hyperthyroidism
• Most common cause of hyperthyroidism
Grave’s disease
• Autoantibody (IgG) stimulates TSH receptor → increased synthesis and release of thyroid hormones
Grave’s disease
• Clinical features:
- Hyperthyroidism
- Diffuse goiter
- Exophthalmos
- Pretibial myxedema
Grave’s disease
• Composed of three layers; each secretes predominantly one class of hormones.
ADRENAL CORTEX
produces mineralocorticoids (e.g. aldosterone)
Zona glomerulosa
produces glucocorticoids (e.g. cortisol)
Zona fasciculata
produces androgens (e.g. DHEA, dehydroepiandrosterone)
Zona reticularis
are derived from cholesterol.
Cortical hormones
= Fluid and electrolyte balance
a. Mineralocorticoids
= Fluid and electrolyte balance
a. Mineralocorticoids
= Glucose production and protein metabolism
b. Glucocorticoids
= Regulate sexual development and control many aspects of pregnancy
c. Sex steroids
All adrenal steroids are derived by sequential enzymatic conversion of a common substrate, cholesterol.
STEROIDOGENESIS
Non-specific; carries many steroids
Albumin
Cortisol and derivatives; progesterone
Cortisol-binding globulin
Testosterone and estradiol
Sex hormone- binding globulin
• critical for sodium retention, (volume), potassium, and acid-base homeostasis
MINERALOCORTICOIDS (ALDOSTERONE)
Most commonly due to bilateral adrenal hyperplasia (60%) or adrenal adenoma (40%, Conn syndrome)
Primary Hyperaldosteronism
Arises with activation of renin-angiotensin system (e.g. renovascular hypertension)
Secondary Hyperaldosteronism
Muscle weakness with thin extremities - cortisol increases muscle break down to produce amino acids for gluconeogenesis
Hypercortisolism (Cushing Syndrome)
Moon facies, buffalo hump, and truncal obesity - high glucose → high insulin increased storage of fat centrally
Hypercortisolism (Cushing Syndrome)
- Abdominal striae - impaired collagen synthesis results in thinning of skin
Hypercortisolism (Cushing Syndrome)
Hypertension often with hypokalemia and metabolic alkalosis
Hypercortisolism (Cushing Syndrome)
Osteoporosis
Hypercortisolism (Cushing Syndrome)
Immunosuppression
Hypercortisolism (Cushing Syndrome)
- 24-hour urine cortisol level (increased)
Hypercortisolism (Cushing Syndrome)
- Late night salivary cortisol level (increased)
Hypercortisolism (Cushing Syndrome)
- Low-dose dexamethasone suppression test
• Low dose dexamethasone suppresses cortisol in normal individuals but fails to suppress cortisol in all cases of Cushing syndrome - High dose dexamethasone suppression test
• High dose dexamethasone suppresses ACTH production by a pituitary adenoma (serum cortisol is lowered) but does not suppress ectopic ACTH production (serum cortisol remains high)
Hypercortisolism (Cushing Syndrome)
• Due to enzymatic defects in cortisol production
Congenital Adrenal Hyperplasia
- High ACTH (decreased negative feedback) leads to bilateral adrenal hyperplasia
Congenital Adrenal Hyperplasia
- Mineralocorticoids and androgens may be increased or decreased depending on the enzyme defect
Congenital Adrenal Hyperplasia
- Most common cause: 21-hydroxylase deficiency (90% of cases)
Congenital Adrenal Hyperplasia
- Aldosterone and cortisol are decreased; steroidogenesis is shunted towards androgens
21-hydroxylase deficiency
- Classic form presents in neonates as:
• Hyponatremia and hyperkalemia with life-threatening hypotension (salt-wasting type);
• Females have clitoral enlargement (genital ambiguity)
21-hydroxylase deficiency
- Newborn screening for CAH: measurement of serum 17-hydroxyprogesterone
21-hydroxylase deficiency
Presents as weakness and shock
Acute ADRENAL INSUFFICIENCY
Abrupt withdrawal of glucocorticoids
Acute ADRENAL INSUFFICIENCY
Waterhouse-Friderichsen syndrome (hemorrhagic necrosis of adrenal glands, usually due to sepsis and DIC in young children with N. meningitidis infection
Acute ADRENAL INSUFFICIENCY
Presents with vague, progressive symptoms such as hypotension, weakness, fatigue, nausea, vomiting and weight loss
Chronic ADRENAL INSUFFICIENCY (Addison disease)
Caused by progressive renal damage:
Autoimmune destruction
Tuberculosis
Metastatic carcinoma
Chronic ADRENAL INSUFFICIENCY (Addison disease)
- Formed by the conversion of tyrosine
CATECHOLAMINES
- Best known catecholamines: epinephrine (adrenaline) and norepinephrine (noradrenaline)
CATECHOLAMINES
- Synthesized and stored by the chromaffin cells of the adrenal medulla
CATECHOLAMINES
- End products of catecholamine metabolism:
a. Homovanillic acid
b. Vanillylmandelic acid
CATECHOLAMINES
- Increased breakdown of triglycerides
CATECHOLAMINES
- Enhanced synthesis of glucose from amino acids
CATECHOLAMINES
- Enhanced breakdown of liver glycogen
CATECHOLAMINES
- Decrease in protein synthesis
CATECHOLAMINES
- Increase in blood glucose levels
CATECHOLAMINES
Colorimetric assay for total metanephrines
Pisano Method
Does not distinguish between metanephrine and normetanephrine but gives the total of the two components
Pisano Method
Involves extraction followed by colorimetric reaction
Pisano Method
Conversion to vanillin (absorbance maximum 360 nm) is accomplished through periodate oxidation
Pisano Method
Hyperthyroidism
DECREASED CATECHOLAMINE
Diabetes: long-term
DECREASED CATECHOLAMINE
Pheochromocytoma
Neuroblastoma
Essential hypertension
Biabric adesis
Cardiac disease
Burns
Septicemia
Depression
INCREASED CATECHOLAMINE
• Catecholamine-producing tumor arising from chromaffin tissue; rare (<0.1% of hypertensive patients)
PHEOCHROMOCYTOMA
Clinical features are due to episodic release of catecholamines (episodic hypertension, headaches, palpitations, tachycardia, sweating)
PHEOCHROMOCYTOMA
Most sensitive screening test: measuring both total plasma catecholamines and urine metanephrines
PHEOCHROMOCYTOMA
• Treatment is adrenalectomy
PHEOCHROMOCYTOMA
Development of female reproductive organs & secondary sex characteristics.
Estrogens
Regulation of menstrual cycle.
Estrogens
Maintenance of pregnancy
Estrogens
is major estrogen produced by ovaries; most potent estrogen.
Estradiol (E2)
Also produced in adrenal cortex.
Estrogens
Preparation of uterus for ovum implantation, maintenance of pregnancy
Progesterone
Also produced by placenta. Metabolite is pregnanediol
Progesterone
Useful in infertility studies & to assess placental function.
Progesterone
No hormonal activity
Estrogen (estriol)
Used to monitor fetal growth & development.
Estrogen (estriol)
Progesterone production by corpus luteum during early pregnancy.
HCG
Development of fetal gonads
HCG
Used to detect pregnancy, gestational trophoblastic disease (e.g., hydatidiform mole), testicular tumor, & other HCG-producing tumors.
HCG
Estrogen & progesterone production by corpus luteum.
Human placental lactogen (HPL)
Development of mammary glands
Human placental lactogen (HPL)
Used to assess placental function.
Human placental lactogen (HPL)
Development of male reproductive organs & secondary sex characteristics
Testosterone
Also produced in adrenal cortex.
Testosterone
: Most abundant estrogen in post-menopausal women
Estrone (E1)
: Most potent; most abundant in pre-menopausal women
Estradiol (E2)
: metabolite of estradiol; estrogen found in maternal women; major estrogen secreted by placenta
Estriol (E3)
: metabolite of estradiol; estrogen found in maternal women; major estrogen secreted by placenta
Estriol (E3)
: metabolite of estradiol; estrogen found in maternal women; major estrogen secreted by placenta
Estriol (E3)
Visual pigments in the retina; regulation of gene expression and cell differentiation (B-carotene is an antioxidant)
Retinol, ß-carotene
Night blindness, xerophthalmia; keratinization of skin
Retinol, ß-carotene
Maintenance of calcium balance; enhances intestinal absorption of Ca and mobilizes bone mineral; regulation of gene expression and cell differentiation
Calciferol
Rickets = poor mineralization of bone; osteomalacia = bone demineralization
Calciferol
Antioxidant, especially in cell membranes; roles in cell signaling
Tocopherols, tocotrienols
Extremely rare-serious neurologic dysfunction
Tocopherols, tocotrienols
Coenzyme in formation of y-carboxyglutamate in enzymes of blood clotting and bone matrix
Phylloquinone; menaquinones
Impaired blood clotting, hemorrhagic disease
Phylloquinone; menaquinones
Coenzyme in pyruvate and a-ketoglutarate dehydrogenases, and transketolase; regulates Cl channel in nerve conduction
Thiamin
Peripheral nerve damage (beriberi) or central nervous system lesions (Wernicke-Korsakoff syndrome)
Thiamin
Coenzyme in oxidation and reduction reactions (FAD and
FMN); prosthetic group of flavoproteins
Riboflavin
Lesions of corner of mouth, lips, and tongue, seborrheic dermatitis
Riboflavin
Coenzyme in oxidation and reduction reactions, functional part of NAD and NADP; role in intracellular calcium regulation and cell signaling
Nicotinic acid, nicotinamide
Pellagra —photosensitive dermatitis, depressive psychosis
Nicotinic acid, nicotinamide
Coenzyme in transamination and decarboxylation of amino acids and glycogen phosphorylase; modulation of steroid hormone action
Pyridoxine, pyridoxal, pyridoxamine
Disorders of amino acid metabolism, convulsions
Pyridoxine, pyridoxal, pyridoxamine
Coenzyme in transfer of one-carbon fragments
Folic acid
Megaloblastic anemia
Folic acid
Coenzyme in transfer of one-carbon fragments and metabolism of folic acid
Cobalamin
Pernicious anemia = megaloblastic anemia
with degeneration of the spinal cord
Cobalamin
Functional part of CoA and acyl carrier protein: fatty acid synthesis and metabolism
Pantothenic acid
Peripheral nerve damage (nutritional melalgia or “burning foot syndrome”)
Pantothenic acid
Coenzyme in carboxylation reactions in gluconeogenesis and fatty acid synthesis; role in regulation of cell cycle
Biotin
Impaired fat and carbohydrate metabolism, dermatitis
Biotin
Coenzyme in hydroxylation of proline and lysine in collagen synthesis; antioxidant; enhances absorption of iron
Ascorbic acid
Scurvy—impaired wound healing, loss of dental cement, subcutaneo
Ascorbic acid
Structural function
Calcium, magnesium, phosphate
Involved in membrane function: principal cations of extracellular-and intracellular fluids, respectively
Sodium, potassium
Function as prosthetic groups in enzymes
Cobalt, copper, iron, molybde-num, selenium, zinc
Regulatory role or role in hormone action
Calcium, chromium, iodine, magnesium, manganese, sodium, potassium
Known to be essential, but function unknown
Silicon, vanadium, nickel, tin
Have effects in the body, but essentiality is not established
Fluoride, lithium
Without known nutritional function but toxic in excess
Aluminum, arsenic, antimony, boron, bromine, cadmium, ce-sium, germanium, lead, mercury, silver, strontium
