Clinical Chemistry part 2 Flashcards
Organic molecules that are water insoluble, fat-soluble
Lipids and lipoproteins
Building blocks of lipids; hydrocarbon chains with a terminal COO group
Fatty acids
3 fatty acid molecules attached to one molecule of glycerol by Ester bonds; 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; contains polar and non-polar end; consitutent of cell membranes
Phospholipids
Serves as part of cell membranes and as parent chain for cholesterol-based hormones
Cholesterol
2 forms of cholesterol
Cholesterol esters and free cholesterol
approximately 70% of total cholesterol
Cholesterol esters
approximately 30% of total cholesterol
free cholesterol
Typically spherical in shape with sizes ranging from 10 to 1200 nm
Lipoprotein
Lipoproteins are composed of lipids and proteins called
apolipoproteins
The size of lipoproteins particle correlates with its lipid content
T
The various lipoproteins were separated through
Ultra centrifugation
Located on the surface of lipoproteins particles; maintain structural integrity of lipoproteins; serve as ligands for cell receptors
Apolipoproteins
Function of lipoproteins
Transport lipids
Apo A-I location
HDL
Apo A-II
HDL
Apo A-IV
Chylos, VLDL, HDL
Apo B-100
LDL, VLDL
Apo B-48
Chylos
Apo C-I
Chylos, VLDL, HDL
Apo C-II
Chylos, VLDL, HDL
Apo C-III
Chylos, VLDL, HDL
Apo-E
VLDL, HDL
Apo (a)
Lp(a)
Largest lipoprotein; least dense; highest TAG content; can block light: causes post-prandial turbidity
Chylomicrons
Transports exogenous or dietary TAG
Chylomicrons
2nd largest, 2nd least dense, 2nd highest TAG content
VLDL
Causes FASTING HYPERLIPIDEMIC TURBIDITY
VLDL
Transports endogenous or hepatic TAG
VLDL
Small; highest cholesterol content; transports cholesterol To peripheral tissues
LDLQ\
Target of cholesterol lowering therapy (statins)
LDL
Deposition of cholesterol
Atherosclerosis
Smallest, densest, highest protein content
HDL
Reverse transport of cholesterol
HDL
Triglycerides rich lipoproteins
CM, VLDL
Protein-rich lipoproteins
LDL, HDL
Highest TAG content
CM
Highest cholesterol content
LDL
Highest protein content
HDL
Apo B containing
CM, VLDL, LDL
Alpha lipoprotein
HDL
Pre-beta lipoprotein
VLDL
Beta lipoprotein
LDL
Target for station therapy
LDL
Floating beta lipoprotein
Beta-VLDL
Increased in familial dysbetalipoproteinemia
Beta-VLDL
Sinking Pre-beta lipoprotein
Lp(a)
LDL-like particule; increased risk of premature coronary heart disease and stroke
Lp(a)
Seen in patients with biliary cirrhosis or cholestasis and in patients with mutations in the enzyme LCAT
LpX
Triglyceride content of CM
85-95%
Triglyceride content of VLDL
45-65%
cholesterol content of LDL
6-8%
Protein content of HDL
45-55%
Optimal LDL cholesterol Reference range
<100
Near optimal LDL cholesterol Reference range
100-129
Borderline high LDL cholesterol Reference range
130-159
High LDL cholesterol Reference range
160-189
Very high LDL cholesterol Reference range
> /= 190
Desirable total cholesterol Reference range
<200
Borderline high total cholesterol Reference range
200-239
High total cholesterol Reference range
> /= 240
High HDL cholesterol Reference range
> /= 60
Low HLD cholesterol Reference range
< 40
Normal TAG reference range
< 150
Borderline high TAG reference range
150-199
HIigh TAG reference range
200-499
Very high TAG reference range
> /= 500
Used to characterize lipid disorders
Fredrickson Classification
The Fredrickson Classification used ___ and ___ for CM to correlate clinical disease syndromes with laboratory phenotypes.
Electrophoresis and standing plasma test
Type 1 Hyperchylomicronemia (Familiar LPL deficieny)
Increased TG and CM
Type 2a (Familial Hypercholeseterolemia
Increased cholesterol and LDL
Type 2b (Familial Combined Hyperlipidemia)
Increased TG, Cholesterol, LDL, VLDL
Type 3 (Familial Dysbetalipoproteinemia)
Increased TG, Cholesterol, and VLDL
Type 4 (Familial Hypertriglyceridemia)
Increased TG, VLDL
Type 5
Increased all except LDL
Fasting requirement for Lipid profile
12 hours
2 parameters that can be measured nonfasting
TC and HDL-C
Prolonged tourniquet application can cause ___
Hemoconcentration
Reclined patients causes ___ values
Decreased
Preferred sample for lipid profile
Serum or plasma
Preferred sample in electrophoresis and ultracentrifugation (lipid profile)
Plasma
Capillary blood samples have generally __ values
Lower
Lipemic samples are seen when triglyceride levels exceed ___
4.6 mmol/L (400 mg/dl)
Cholesterol measurement (3)
- Abell-Kendall method
- Enzymatic methods
- IDMS
Cholesterol measurement wherein the initial extraction with zeolite is used to remove sterols redissolving of cholesterol hydrolysis of cholesterol esters to cholesterol
Abell-Kendall method
3 components of the Liebermann-Burchard reagent
Glacial acetic acid, sulfuric acid, acetic anhydride
Absorbance of the Abell-Kendall method
410 nm
Reference method for Cholesterol measurement
GC-MS method
Gold standard for drug testing
GC-MS method
In the enzymatic method of cholesterol measurement, what is the first enzyme in the reaction
Cholesteryl esterase
Definitive method of cholesterol measurement
Isotope Dilution Mass Spectrometry (IDMS)
Triglyceride measurement (3)
- Enzymatic method
- Chemical methods
- GC-MS method
In the enzymatic method of triglyceride measurement, what is the first enzyme in the reaction?
Bacterial lipase
Hydrolysis of glycerol is accomplished using alcoholic KOH Oxidation of glycerol by periodic acid, forming formaldehyde and formic acid formaldehyde combines with a variety of reagents forming colored products and fluorescence
Chemical methods of triglyceride measurement
Colorimetric method of triglyceride measurement
Van Handel and Zilversmit
Fluorometric method of triglyceride measurement
Hantzsch
Reagent in Van Handel and Zilversmit (TG)
Chromotropic acid
Product formed in Van Handel and Zilversmit
Blue colored compound
Reagent in Hantzsch method (TG)
Acetylacetone (diacetyl acetone; reactant of choice)
Produce formed in Hantzsch method
Strong absorption maximum at 412 nm and has a good fluorescence
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
Takes advantage of differences in size and charge
Electrophoresis
___ can be performed using unfractionated plasma or in plasma fractions that contain other serum proteins.
Electrophoresis
Most anodal lipoprotein
HDL
Lipoprotein electrophoretograms are usually visualized with a lipid-staining dye such as ___
Oil Red O
Fat Red 7B
Sudan Black B
“OFS or Only Find Sun”
Depends on particle size, charge, and differences in the apolipoprotein content; primarily used in research labs only. It uses polyanions (heparin sulfate, dextran sulfate and phosphotungstate) and divalent cations such as magnesium, calcium and manganese)
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
Chromatographic methods
Recall the Friedewald calculation
[Plasma TG/5] = mg/dl
[Plasma TG/2.175] = mmol/l
[Plasma TG/2.825] = gives more accurate estimate of VLDL-C , which is equivalent to [Plasma TG/6.5]
Involves large and medium sized arteries
Atherosclerosis
Increased risk of atherosclerosis
LDL
Decreased risk of atherosclerosis
HDL
Narrowing of blood vessels result in impaired blood flow and ischemia leading to
Peripheral vascular disease
Angina
Ischemic bowel disease
Complications of atherosclerosis
Plaque rupture thrombosis ___
Myocardial infarction and stroke
Plaque rupture embolization ___
Atherosclerotic emboli
Weakening of blood vessel wall results in ___
Aneurysm
An extreme form of hypoalphalipoproteinemia (isolated decrease in circulating HDL)
Tangier Disease
Tangier disease is associated with HDL cholesterol concentrations as low as ____ in homozygotes, accompanied by total cholesterol concentrations of ___
1-2 mg/dl (0.03 -0.05 mmol/l;
50 – 80 mg/dl (1.3 – 2.1 mmol/L)
Tangier disease is associated with increased risk of premature ____
Coronary heart disease (CHD)
Linear polymers of amino acids
Proteins
Regulate metabolism
Facilitate contraction in the muscle
Provide structural framework
Shuttle molecules in the bloodstream
Component of the immune system
Functions of proteins
Number and types of amino acids in the specific amino acid sequence
Primary
Regularly repeating structures stabilized by hydrogen bonds between the amino acid within the protein (local folding; geometrically structured levels)
Secondary
Overall shape, or conformation of the protein molecule
Tertiary
Shape or structure that results from the interaction or more than one protein molecule, or protein subunits held together by noncovalent forces
Quaternary
Number and types of polypeptide units of oligometric proteins and their spatial arrangement
Quaternary
Most plasma proteins are synthesized in the ___ and secreted by the hepatocyte into the circulation
Liver
___ are synthesized in plasma cells
Immunoglobulins
___ are produced by endothelial cells and megakaryocytes
vWF
In liver failure, all proteins are decreased except
Immunoglobulin and VWF
The nitrogen content of serum protein is approximately ___
16%
Site of protein synthesis within the cell
Ribosomes
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
Aplha-1-fetoprotein
May be related to immune response
Alpha-1-acid glycoprotein
Binds to hemoglobin
Haptoglobin
Transports copper; peroxidase activity
Ceruloplasmin
Binds heme
Hemopexin
Immune response
Complement
Precursor of fibrin
Fibrinogen
Opsonin
C-reactive protein
Migrates before albumin in the serum protein electrophoresis
Prealbumin
Transport protein for thyroid hormones; transports Vitamin A by forming a complex with retinol-binding protein
Prealbumin (Transthyretin)
Decreased in hepatic damage, acute-phase inflammatory response and tissue necrosis
Prealbumin
Sensitive marker of poor nutritional status
Low prealbumin
Increased in patients receiving steroids, in alcoholism, and in chronic renal failure
Prealbumin
Negative Acute Phase Reactant (decreased in inflammation)
Prealbumin, Albumin, Transferrin
Protein present in the highest concentration in serum (2/3 of total protein)
Albumin
Provide nearly 80% of colloid osmotic pressure (COP) of intravascular fluid
Albumin
Buffers pH
Albumin
Binds to various substances in blood (hormones, drugs, electrolytes, unconjugated bilirubin)
Albumin
50% of calcium is transported by ___
Albumin
Decreased in liver disease, malnutrition, malabsorption, kidney loss (nephrotic syndrome), hemodilution
Increased in hydration
Albumin
Most important function is the 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 is a major component of a1-globulin band deficiency of AAt seen as lack of an a1-globulin band on SPE
True
Excessive elastase breaks down elastin in alveoli leading to ___
Emphysema
Normal albumin-globulin ration
2:1
Hypoalbuminemia leads to
Edema (H20 goes out of the BV and enters the tissue)
One of the COPDs (chronic obstructive pulmonary diseases). Most common cause is smoking
Emphysema
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
“Pink puffers” “barrel-chest”, hypoxemia
Emphysema
Synthesized by the developing embryo and fetus; thought to protect the fetus from immunologic attack by the mother. No known function in normal adults
Alpha-1-fetoprotein
Elevated AFP
Neural tube defects, presence of twins
Low AFP
Increased risk for Down syndrome
Tumor marker for hepatocellular carcinoma, some testicular carcinomas
Alpha-1-fetoprotein
Cooper-containing (contains >90% of total serum copper)
Ceruloplasmin
Used in diagnosis of Wilson’s disease
Ceruloplasmin (decreased)
Decreased levels of ceruloplasmin. Excess storage of copper in various organs.
Liver hepatic cirrhosis
Brain neurologic damage
Cornea Kayser-Fleischer rings
Wilson’s disease
Total serum copper decreased
Free serum copper increased
Urinary copper increased
Wilson’s disease
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)
Disruption of the electrical charges that produce the tightly fitting podocyte barrier resulting in massive loss of protein and lipids
Nephrotic syndrome
Manifestations of nephrotic syndrome
Hypoalbuminemia – pitting edemia
Hypogammaglobulinemia – increased risk of infection
Hypercoagulable state – due to loss of anti-thrombin III
Hyperlipidemia and hypercholesterolemia – may result in fatty casts in the urine
Marker for intravascular hemolysis
Haptoglobin
Bind free hemoglobin to prevent loss of hemoglobin and its constituent iron into the urine
Haptoglobin
Haptoglobin-hemoglobin complex is removed by
Reticuloendothelial system (mainly in the spleen)
Transports two molecules of ferric iron
Transferrin
Major component of the beta-globulin fraction
Transferrin
Tested to determine cause of anemia
Transferrin
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
Diagnostic of hemolytic anemia
Low hemopexin levels
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
___ is the same protein but is named for the newer, monoclonal antibody-based test methodologies that can detect CRP at levels below 1 mg/L
High-sensitivity CRP
The ___ test determines risk of cardiovascular disease
High-sensitivity CRP
Glycoprotein produced by fetal membranes responsible for the cellular adhesiveness of placenta and membranes to the decidua
Fibronectin
Fetal ___ 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
Fibronectin test for assessment of the risk for ___ in women between 24 to 35 weeks gestational age
Preterm delivery
Proteolytic fragments of collagen I formed during bone resorption
Cross-Linked C-Telopeptides
Biochemical marker of bone resorption that can be detected in serum and urine
Cross-linked C-telopeptides
Gold standard for the detection of myocardial infarction
Troponin
Govern excitation-contraction coupling in muscle
Troponin
Used as an AMI indicator because of specificity and early rise in serum concentration
Troponin T or Troponin I
Troponin T rises within ___
Peaks in ___
Returns to normal in ___
3-4 hours
10-24 hours
10-24 days
Troponin I rises within ____
Peaks in ___
Returns to normal in ____
3-6 hours
14-20 hours
5-10 days
Earliest protein marker for Myocardial infarction
Myoglobin
Heme-containing protein that binds oxygen with cardiac and skeletal muscle. Levels are related to muscle mass and activity (reasonable sensitivity but poor specificity)
Myoglobin
Increased in skeletal muscle injuries, muscular dystrophy, and AMI
Myoglobin
Myoglobin rises in ___
Peaks in ___
Returns to normal in ___
1-3 hours
5-12 hours
18-30 hours
Myoglobin is not tissue specific. It is better used as a negative predictor in the first 2-4 hours following chest pain
True
Neurohormones that affect body fluid homeostasis (through natriuresis and diuresis) and blood pressure
Natriuretic peptides
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 BNP
BNP has become a popular marker for ___
Congestive Heart failure
A1- globulins
A1-antitrypsin
A1-fetoprotein
A1-acid glycoprotein
A1-lipoprotein
A1-antichymotrypsin
Inter-a-trypsin inhibitor
Gc globulin
A2 globulins
Haptoglobin
Ceruloplasmin
A2-macroglobulin
“CHA”
Beta globulins
Pre-b-lipoprotein
b-lipoprotein
Transferrin
Hemopexin
B2-microglobulin
Complement
Fibrinogen
CRP
y-Globulins
IgA
IgD
IgE
IgG
IgM
CRP
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 reactants
CRP response time
Normal concentration (mg/dl)
Increase
Function
6-10 hours (preferred marker for inflammation)
0.5
1000x
Opsonization, complement activation
Serum amyloid A response time
Normal concentration (mg/dl)
Increase
Function
3.0
1000x
Removal of cholesterol
A-1-antitrypsin response time
Normal concentration (mg/dl)
Increase
Function
24 hours
200-400
2-5x
Protease inhibitor
Fibrinogen response time
Normal concentration (mg/dl)
Increase
Function
24 hours
110-400
2-5x
Clot formation
Haptoglobin response time
Normal concentration (mg/dl)
Increase
Function
24 hours
40-200
2-10x
Binds hemoglobin
Ceruloplasmin response time
Normal concentration (mg/dl)
Increase
Function
48-72 hours
20-40
2x
Binds coppere, oxidizes iron
C3 response time
Normal concentration (mg/dl)
Increase
Function
48-72 hours
60-140
2x
Opsonization, lysis
Mannose-binding protein response time
Normal concentration (mg/dl)
Increase
Function
?
0.15-1.0
?
Complement activation
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
Increased in total plasma proteins
Hyperproteinemia
Not an actual disease state, but is the result of dehydration
Hyperproteinemia
Abnormal proliferation of plasma cells produce many Ig (which are abnormal)
Multiple myeloma
Total Protein analysis (4 methods)
Kjeldahl
Refractometry
Biuret
Dye binding
Digestion of protein; measurement of nitrogen content
Reference method of total protein analysis; assume average nitrogen content of 16%
Kjeldahl
Conversion factor in Kjeldahl method
6.25 or 6.54
Measurement of refractive index due to solutes in serum
Rapid and simple; assume nonprotein solids are present in same concentration as in the calibrating serum
Refractometry
Formation of violet-colored chelate between Cuprous ions and peptide bonds
Routine method; requires at least 2 peptide bonds and an alkaline medium
Biuret
Protein binds to dye and causes a spectral shift in the absorbance maximum of the dye
For research use
Dye binding
Globulins are precipitated in high salt concentrations; albumin in supernatant is quantitated by biuret reaction
Labor intensive
Salt-precipitation (Historical method)
Albumin binds to dye; causes shift in absorption maximum;
Nonspecific for albumin
Methyl orange
Albumin binds to dye; causes shift in absorption maximum;
Many interferences (salicylates, bilirubin)
HABA
Albumin binds to dye; causes shift in absorption maximum;
Sensitive; overestimates low albumin levels; most commonly used dye
Bromcresol green
Albumin binds to dye; causes shift in absorption maximum;
Specific, sensitive, precise
Bromcresol purple
Protein separated based on electric charge; accurate, gives overview of relative changes in different protein fractions
Electrophoresis
Performed when an abnormality in the total protein or albumin is found. Separation of proteins based on their charge density
Protein electrophoresis
Regions in protein electrophoresis are stained using:
Coomassie Blue, Amido Black, Ponceau S
Beta-gamma bridging electrophoretic pattern
Cirrhosis
Monoclonal spike electrophoretic pattern
Multiple myeloma
Increased a2, increased b2, decreased albumin electrophoretic pattern
Nephrotic syndrome
Decreased a1-antitrypsin electrophoretic pattern
Emphysema
Increased beta region electrophoretic pattern
Use of plasma instead of serum (fibrinogen)
The most significant finding from an electrophoretic pattern is ___
Monoclonal immunoglobulin disease
Non-protein nitrogen compounds
Urea
Amino acids
Uric acid
Creatinine
Creatine
Ammonia
NPN present in the highest concentration in blood
Urea
Major excretory product of protein metabolism
Urea
Concentration of urea in the plasma is determined by
Renal function and perfusion,
Protein content of the diet
Rate of protein catabolism
Most common cause of death or liver failure
Increased ammonia
Evaluate renal function
Assess hydration status
Aid in diagnosis of renal disease
Verify frequency of dialysis
Clinical applications of urea
Urea nitrogen concentration is converted to urea concentration by multiplying
2.14
Most used method in determining urea concentration
Enzymatic methods
Proposed reference method for the urea determination
IDMS
Elevated concentration of urea in the blood
Azotemia
Very high plasma urea concentration accompanied by renal failure is called
Uremia or the uremic syndrome
Prerenal azotemia
Congestive heart failure
Shock, hemorrhage
Dehydration
Increased protein catabolism
High-protein diet
Renal azotemia
Acute and chronic renal failure
Renal disease (glomerular nephritis, tubular necrosis)
Postrenal azotemia
Urinary tract obstruction
Decreased urea concentration
Low protein intake
Severe vomiting and diarrhea
Liver disease
Pregnancy
Product of 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 proximal tubules and reused
Uric acid
Relatively insoluble in plasma, and a high concentrations, can be deposited in the joints and tissues, causing painful inflammation
Uric acid
Uric acid is mostly present as ___ in plasma, wherein it is insoluble at around pH 7
Monosodium urate
Increased uric acid in urine
“Orange sand” in diapers
UA crystals
Lesch-Nyhan syndrome
Uric acid determination (phosphotungstic acid and tungsten blue)
Caraway method
Proposed reference method for uric acid
IDMS
Uric acid determination or method where there are problems with turbidity and several common drugs interference
Colorimetric
Preferred marker for kidney function (glomerular filtration)
Creatinine or creatine
Creatinine is formed from ___ (synthesized primarily in the liver from arginine, glycine, and methionine) and creatine phosphate in muscle
Creatine
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
Monitor the progression of kidney disease
Measure of completeness of 24 hour collections
Creatinine
Increased creatinine concentration
Abnormal renal function
Muscle disease
Normal BUN/Creatinine ratio
10:1 – 20:1
The BUN/Creatinine ratio rises in prerenal disease to
> 20:1
In true renal disease, both BUN and creatinine rise together, maintaining BUN/Creatinine ratio at __
10-20:1
__ performed directly on sample; detection of color formation times to avoid interference of noncreatinine chromogens
Jaffe-kinetic
Creatinine in protein-free filtrate adsorbed onto Fuller’s earth (aluminum magnesium silicate); then reacted with alkaline picrate to form colored complex
Lloyd’s reagent (sodium aluminum silicate)
Jaffe with adsorbent
Highly specific, accepted reference method for creatinine
IDMS
Formed through the deamination of amino acids during protein metabolism
Ammonia
Ammonia is removed from the circulation and converted to __ in the liver
Urea
Provide useful information on clinical conditions such as hepatic failure, Reye’s syndrome and inherited deficiencies of the urea cycle enzymes
Ammonia
Indicator in the spectrophotometric method of ammonia determination
Bromphenol blue
Enzymatic method of ammonia determination
Glutamate dehydrogenase
Specimen consideration for ammonia
Should be iced,
Avoid smoking prior to collection
Increased BUN, Normal creatinine, Increased BUN/Crea ratio
Prerenal azotemia
Increased BUN, increased creatinine, Normal Ratio
Renal, postrenal azotemia
Largest and heaviest internal organ
Liver
Weight of the liver
1.2 – 1.5 kg
Ligament the divides the right and left lobe of the liver
Falciform ligament
Functional units of the liver
Lobules/ acini
Supplies oxygen-rick blood in the liver (main blood vessel)
Hepatic artery
Supplies deoxygenated blood to the liver; delivers blood from the gallbladder, spleen, GIT to the liver
Hepatic portal vein
Capillary-like blood vessel where blood from hepatic artery and hepatic vein miX
Sinusoids
Small passages in the liver
Canaliculi
Perform most of the liver function; contribute to the regenerative properties of the liver
Hepatocytes
Derived from macrophages; found in sinusoids
Kupffer cells
Synthesize nitric oxide (regulate blood flow)
Stellate/Ito cells
Liver stem cells (regeneration of hepatocytes and bile ducts)
Oval cells
Carries bile
Common bile duct
Layer of loose connective tissue in the liver
Glisson’s capsule
Covers the exterior portion of the liver
Peritoneum
Synthetic function of the liver
Produce substances such as proteins, bile salts, lipids, and carbohydrates
Storage function of the liver
Glycogen, iron, amino acids, and some lipids
Excretes bilirubin, bile acids, and ammonia
Excretes bilirubin, bile acids, and ammonia
Detoxification function of the liver and drug metabolism
Convert toxic substances
Bile acids are conjugated with amino acids (taurine, glycine) to form
Bile salts
Aids in absorption and digestion of vitamins
Bile salts
Major metabolite of heme; orange-yellow pigment derived from hemoglobin
Bilirubin
Daily bilirubin production
250-300 mg
Bilirubin is mainly transported by __
Albumin
Bilirubin production is contributed by __
85% liver, spleen, BM (reticuloendothelial)
15% ineffective erythropoiesis, heme-containing proteins/hemoproteins
3 heme-containing proteins/hemoproteins
Cytochromes
Peroxidases
Myoglobin
How many grams of albumin is produced a day by the liver
12 g
One of the synthetic functions of the liver is the metabolism of cholesterol into ___
Primary bile acids
Promote fat absorption and emulsification
Primary bile acids
2 types of primary bile acids
Deoxycholic acid and chenodeoxxycholic acid
Unconjugated, nonpolar, water insoluble, indirect bilirubin, hemobilirubin
B1
Conjugated bilirubin, polar, water soluble, direct bilirubin, cholebilirubin
B2
First type of bilirubin formed
B1
Type of bilirubin not normally present in the urine
B1
Type of bilirubin that slowly reacts with diazo reagent
B1
Bilirubin that is normally in the circulation
B1
Type of bilirubin that needs an accelerator or solubilizer
B1
Bilirubin that is freely filtered in plasma and present in the urine
B2
Bilirubin that directly reacts with diazo reagent
B2
Aka biliprotein; a bilirubin that is covalently bound to albumin thus contributes to the direct bilirubin
Delta bilirubin
Formula for total bilirubin
Total bilirubin = unconjugated + conjugated + delta bilirubin
“Jaune” = yellow
Yellow discoloration of the skin, eyes, and mucous membranes due to bilirubin retention
Jaundice
Serum bilirubin in adults and neonates
2-3 mg/dl (adults)
5 mg/dl (neonates)
Amount of bilirubin that becomes noticeable to the naked eye
> 3 mg/dl
Unconjugated hyperbilirubinemia;
Mild type of jaundice that occurs prior to liver metabolism
Pre-hepatic jaundice
Most common cause of pre-hepatic jaundice
Hemolytic anemia
Ineffective erythropoiesis
Results from intrinsic liver disease due to defects in bilirubin metabolism and transport; due to diseases resulting to hepatocellular injury
Hepatic jaundice
Inherited, autosomal recessive mild form of unconjugated hyperbilirubinemia
Serum bilirubin: 1.5 – 3 mg/dl
The gene responsible for the expression of the enzyme UDPGT is mutated or due to transport deficit across the hepatocyte membrane
Gilbert syndrome
Problem in conjugation deficit
Criggler-Najjar syndrome
UDPGT absent; serum bilirubin >20 mg/dl; kernicterus; therapy is liver transplant
Type 1 Crigler-Najjar syndrome
Partial UDPGT deficiency (25% activity); serum bilirubin 5-20 mg/dl; no kernicterus; patients responds to UV therapy
Type 2 Crigler-Najjar syndrome
Transporter gene deficit
Dubin-Johnson syndrome
Predominantly elevated conjugated bilirubin; excretion of bilirubin is impaired; liver biopsy shows dark brown pigment in hepatocytes due to accumulation of lipofuscin-like pigment; B2 cannot be transported out of the hepatocyte
Dubin-Johnson syndrome
Conjugated hyperbilirubinemia similar to Dubin-Johnson syndrome but without liver pigmentation
Rotor syndrome
Transient familiar neonatal hyperbilirubinemia; presence of UDPGT inhibitor (antibody); mild hyperbilirubinemia that lasts for the first 2-3 weeks of life; increased B2
Lucey-Driscoll syndrome
3 types of unconjugated hyperbilirubinemia in the jaundice of the newborn
Physiologic jaundice of the newborn
Hemolytic disease (HDFN)
Breastmilk hyperbilirubinemia
In breastmilk hyperbilirubinemia what inhibits the conjugation
Alpha glucoronidase
Conjugated bilirubin is >1.5 mg/dl; most important causes are idiopathic neonatal hepatitis and biliary atresia
Conjugated hyperbilirubinemia in the jaundice of the newborn
Results from biliary obstructive disease
Post hepatic jaundice
Most common causes of post hepatic jaundice
Gall stones or tumors in the biliary tree
Common cause of gallstones
Ketodiets (intermittent fasting)
High fat diet and steatorrhea
Increased serum B1, Normal serum B2, increased serum total bilirubin
Pre-hepatic jaundice
Increased serum B1, decreased serum B2, increased serum total bilirubin
Gilbert syndrome, Crigler-Najjar syndrome, jaundice of the newborn, Lucey-Driscoll syndrome
Normal serum B1, increased serum B2, Increased serum total bilirubin
Dubin-Johnson syndrome, Rotor syndrome, Post-hepatic jaundice
Viral hepatitis that causes hepatocellular injury
Hepatitis A,B,C,D,E,G
Acute injury of hepatocytes caused by toxic drugs, ischemia, or immunologically mediated injury
Acute hepatitis
Accumulation of fats as a consequence of insulin resistance
Non-alcoholic fatty liver disease and non-alcoholic steatohepatitis
Chronic liver inflammation that persists for at least 6 months
Chronic hepatitis
Diffused fibrosis with nodular regeneration (end stage of scar formation)
Liver cirrhosis
Causes mild hyperbilirubinemia with three fold rise in transaminases (AST and ALT); has a strong association with intake of aspirin; characterized by non-inflammatory encephalopathy and fatty degeneration of the liver; usually affects children 2-14 years old after a viral infection – flu and chickenpox
(most harmful in affecting brain and liver)
Reye Syndrome
Methods of bilirubin determination
Ehrlich reaction
Van den Berg reaction
Jendrassik-Grof method
Evelyn-Malloy method
Bilirubinometry
Bilirubin + diazotized sulfanilic acid azobilirubin
Initially done on urine and stool samples (freshly collected 24 hour samples)
Ehrlich reaction
Bilirubin + diazotized sulfanilic acid azobilirubin
Done using serum samples;
Diazotized sulfanilic acid is formed by reacting sulfanilic acid with sodium nitrite and hydrochloric acid
Van den Berg reaction (and Muller reaction)
Results of the Van den Berg reaction at neural pH and at high or low pH
At neutral pH reddish purple
At low or high pH blue
Diazotized sulfanilic acid + accelerator/solubilizer (caffeine sodium benzoate) ;
Buffer: sodium acetate
Strong alkaline tartrate is added to convert original purple color into blue (measured spectrophotometrically at 600nm)
Jendrassik-Grof method
Advantages of Jendrassik Grof method:
Insensitive to sample pH changes and variation in protein concentration of sample
Not affected by hemoglobin up to 750 mg/dl
Has adequate optical sensitivity even for low bilirubin concentrations
True
Accelerator in Evelyn-Malloy method
50% methanol
Azobilirubin: red to reddish purple color in acid pH (measured at 560 nm)
Evelyn-Malloy method
Done in neonates
Transcutaneous bilirubin concentration device;
Measurement of reflected light from the skin using 2 wavelengths, providing a numerical index based on spectral reflectance;
Has a positive interference with carotenoid compounds thus cannot be done in adults
Bilirubinometry
Diazo reagents
Diazo A: 0.1% Sulfanilic acid + HCl
Diazo B: 0.5% Sodium nitrite
Diazo Blank: 1.5% HCl
Tests Measuring Hepatic Synthetic Ability
Total Protein determination
Prothrombin time – Vitamin K response test
Test for albumin
Albumin/Globulin ratio
Total bilirubin
Up to 1.0 mg/dl
Direct bilirubin
<0.5 mg/dl (other references 0 – 0.2 mg/dl)
Indirect bilirubin
0.2 – 0.8 mg/dl (other references 0 – 1 mg/dl)
Conversion factor of bilirubin
17.1
Critical value of bilirubin
> 18 mg/dl (may cause kernicterus)
Intamuscular injection of 10 mg Vitamin K everyday for 1 – 3 days
Vitamin K response test
Results in vitamin K response test
Prolonged: Intrahepatic disorder
Normal: Extrahepatic disorder
Consistently prolonged: Loss of hepatic synthetic ability of liver
Tests measuring conjugation and excretion function
Bilirubin assay
Bromosulfophthalein dye exretion test
Urobilinogen test
2 methods under Bromosulfophthalein dye exretion test
Rosenthal White method
MacDonald
Rosenthal White Method
Double collection method
After 5 mins: 50% dye retention
After 30 mins: 0%
MacDonald (Reference method)
Single collection method
NV: After 45 mins +/- 5% retention of dye
Ehrlich’s reagent
p-dimethyl aminobenzaldehyde
Uses Ehrlich’s reagent
Specimen: 2-hour freshly collected urine or stool sample
Positive reaction: red color production
Urobilinogen Test
Consists of binding site and catalytic site; a site where substrate interacts with the enzyme
Active site
Site other than the active site; binds to regulator molecules
Allosteric site
Non-protein substances needed for maximal activity of the enzyme
Cofactors
Inorganic cofactors
Activators (anions and cations)
Organic cofactors
Coenzymes (acts as co-substrate in enzyme reactions)
Complete active enzyme system with full catalytic activity; apoenzyme + prosthetic group
Holoenzyme
Inactive form of an enzyme
Proenzyme/ Zymogen
Multiple forms of serum proteins that are functionally related; results from post translational modifications
Isoform
Multiple forms of an enzyme that catalyzes the same biochemical reaction
Isoenzyme
6 classes of enzymes
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
Catalyzes an electron transfer or oxidation-reduction reaction between 2 substrates
Oxidoreductasses
Catalyzes the transfer of various chemical groups from one molecule to another
Transferases
Catalyzes the hydrolysis of various bonds with the addition of water; involves splitting of molecules
Hydrolases
Catalyzes the removal of groups from substrates without hydrolysis; formation of double bonds
Lyases
Catalyzes the interconversion of geometric, optical or positional isomers within a molecule
Isomerases
Catalyzes the joining of two substrate molecules coupled with breaking of the pyrophosphate bond in ATP
Ligases (synthetases)
Rate of reaction is almost directly proportional to substrate concentration at a low level
First order kinetics
Substrate concentration is high enough to saturate all available active sites of the enzymes;
Reaction is independent of substrate concentration
Reaction rate depends only on enzyme concentration
Zero-order kinetics
Enzymes are affected by changes in ___ as it is a factor in their stability
pH
Most favorable pH value at which enzyme is most active
Optimum pH (7.0 – 8.0)
ALP optimum pH
9.0 – 10.0
ACP optimum pH
5.0
pH are controlled using ___
Buffer solutions
Optimum temperature for most enzymes is ___
37 deg C
Increased temperature = increased rate of ___
Chemical reaction
For every ___ increase in temperature, rate of reaction is doubled until it is denatured
10 deg C
Absence means enzyme activity may not occur.
In excess, may inhibit enzyme activity
Cofactors
Substrate competes with active site
Competitive inhibitor
Substrate competes other than the active site
Non-competitive inhibitor
Substrate competes with the substrate-enzyme complex
Uncompetitive inhibitor
Enzyme concentration is measured in fixed period of time; the reaction is stopped and a measurement is made
Fixed time assay (End point assay)
Multiple measurements (absorbance change) are made at specific time interval
Continuous monitoring assay
Amount of enzyme that will catalyze the reaction of 1 umol of substrate in 1 minute
1 International Unit
Amount of enzyme that would catalyze the reaction of 1 mole substrate in one second
1 Katal
EC numerical code of AST
2.6.1.1
Tissue source of AST
Cardiac tissue, liver, skeletal muscle
AST is previously known as
SGOT
Aspartate + alpha-ketoglutarate ____ + ____
Oxaloacetate, glutamate
Coenzyme of AST
Pyridoxal phosphate
2 enzyme fractions of AST
Cytoplasmic AST
Mitochondrial AST
Most predominant AST in the serum
Cytoplasmic AST
Mitochondrial AST becomes highest than cytoplasmic AST in serum in ____
Cellular necrosis
Normal cAST > mAST
Necrosis
cAST < mAST
AST highest elevation: 5 or more x ULN
Acute hepatocellular disorder
Myocardial infarction
Circulatory collapse
Acute pancreatitis
Infectious mononucleosis
AST 3 or more x ULN
Stop the medication (potential hepatotoxic drug)
AST in AMI
Rise within 6-8 hours
Peaks at 24 hours
Returns to normal within 3-5 days
Marked elevation of AST is observed in __
Viral hepatitis and AMI
Hemolysis increases AST concentration (T/F)
T
AST activity is stable in serum for ___ at refrigerated temperature
3-4 days
Methodologies for AST
Karmen method
Reitman-Frankel method
Diazonium salt reaction
Coupled enzymatic reaction
Basis for IFCC
Malate dehydrogenase is added: oxaloacetate + NADH Malate + NAD
Karmen Method (AST)
In the Karmen method, the rate of decrease in absorbance at __ due to the formation of NAD is directly proportional to the activity of AST in the sample
340 nm
Optimal pH of Karmen method for AST determination
pH 7.3 – 7.8 (7.5)
Colorimetric method of AST determination
Substrate: aspartic alpha-ketohlutarate
Color developer: 2,4-dinitrophenylhydrazone
Reitman-Frankel method
Final reaction of Reitman-Frankel method of AST determination
Blue-colored complex, measured at 505 nm
The Reitman-Frankel method lacks specificity because it reacts with any ___
Keto-compound
Colorimetric method of AST determination
Stabilized diazonium salt + oxaloacetic acid red colored compound
Formation of diazonium derivatives
Diazonium salt reaction
EC numerical code for ALT
2.6.1.2
Tissue sources of ALT
Highest concentration in liver and kidney, smaller amount in cardiac tissue and skeletal muscle
ALT is formerly known as
SGPT
More liver specific than AST
ALT
(ALT>AST)
Involved in the reversible transfer of an amino group between alanine and alpha-ketoglutarate
Alanine + alpha-ketoglutarate ___+ _____
Pyruvate; glutamate
ALT has the highest elevation in __
Acute viral hepatitis
De Ritis ration (AST/ALT quotient)
>1
Liver cirrhosis, metastatic carcinoma
De Ritis ration (AST/ALT quotient)
3-4:1
Alcohol-induced liver disease
ALT is stable in serum for ___ days at 4 deg C
3-4
ALT is unaffected by hemolysis
True
In liver disease, ALT > AST, except in __
Hepatic cirrhosis and liver neoplasia
ALT is increased, HCV (-), accept or reject donor?
Defer (consider window period)
Acute hepatocellular injury (AST/ALT ratio)
AST>ALT
Methodologies for ALT determination (4)
Wroblewski and La Due
Reiman-Frankel method
Diazonium salt reaction
Coupled enzymatic reaction (ALT determination)
Basis for the IFCC recommended method
Lactate dehydrogenase is added to convert endogenous pyruvate in the sample into lactate (excess pyruvate is inhibitory to ALT activity)
Pyruvate + NADH lactate + NAD (340 nm)
Wroblewski and La Due
Optimal pH in Wroblewski and La Due (ALT determination)
pH 7.3 – 7.8 (7.5)
Substrate in Reiman-Frankel method (ALT determination)
Alpha-ketoglutarate
Anticoagulant of choice for TDM
Heparin
