DAVAO DOCTORS COLLEGE

MEDICAL LABORATORY SCIENCE DEPARTMENT

STUDENT NOTES: CLINCHEM1
Prepared by: Merryl Louise C. Matus, RMT

MIDTERM 1: LIPIDS AND LIPOPROTEINS  Very Low Density Lipoprotein (VLDL)

- Lipoprotein made in the liver
 Lipids are basically fats. - Second hightest TAG content
 Biological function of lipids: - function: Endogenous transport of TAG; body's internal
- Fuel transport mechanism for lipids
- Insulation - Responsible for fasting hyperlipidemic turbidity of
- Protect or cushion internal organs sample
- Building blocks of other substances in the body  Intermediate Density Lipoprotein (IDL)
- Constituent of cell walls - VLDL remnant
- similar to LDL wherein it transports TAG, fats and
Lipid Chemistry cholesterol and can also promote the growth of atheroma
 Triglycerides - function: enables fats and cholesterol to move within
- major class of dietary lipids
water-based solution of the bloodstream
- comprises 95% of lipids in food and human body
 Low Density Lipoprotein (LDL)
- FATS: lipids that are solid at RT - products of VLDL and IDL metabolism
- OILS: lipids that are liquid at RT - most cholesterol-rich
 Phospholipids - function: principal cholesterol and fat transport in blood
- constituent of cell membranes
- known as the “bad cholesterol” because it is involved in
FORMS:
the progression of CVD like atherosclerosis or stroke
- 70% Lecithin/Phosphatidyl choline - metabolism: 40-60% cleared by hepatic LDL receptors;
- 20% Sphingomyelin the rest are taken up by non-hepatic (scavenger) receptors
- 10% Cephalin such as macrophages
 Cholesterol
 High Density Lipoprotein (HDL)
- precursor of steroid hormones
- smallest and densest
- not used for energy - most abundant in apolipoproteins
 Fatty Acids - phospholipid is the main lipidic content
- building blocks of lipids - function: reverse cholesterol transport; transport of excess
cholesterol from tissues and cells back to the liver
Lipoproteins
- known as the “good cholesterol” because it protects
 bound to the proteins which allow fats to move through the
against heart disease
water in and out of cells
- metabolism: synthesized in the liver and intestine
 Function: emulsify lipid molecules
 Lipoproteins
Apolipoproteins
- Chylomicrons  Apolipoproteins are proteins that bind lipids to form
- Very Low Density Lipoprotein lipoprotein
- Intermediate Density Lipoprotein
 They serve as enzyme cofactors and receptor ligands and
- Low Density Lipoprotein
they also regulate the metabolism of LP and their uptake in
- High Density Lipoprotein tissues
 Most dense to the least (based on density):
HDL > LDL > VLDL > Chylomicron ApoLP LP CARRIED/FUNCTION
 Heaviest to lightest (based on Molecular Weight): Apo A-1 Main protein in HDL; Activator of LCAT for
Chylomicron > VLDL > LDL > HDL esterification
 Chylomicron Apo B-100 VLDL, LDL
- Contains:
Apo B-48 Chylomicrons
85-92% TAG
Apo C-II Lipemia clearing factor
6-12% phospholipids
1-3% cholesterol Activates LPL, targets TAG and removes CM
1-2% proteins after meal
- function: transport TAG absorbed from the intestine to Apo E VLDL, LDL, HDL
adipose, cardiac & skeletal muscle Increased in Alzheimer’s Dx
- Responsible for post-prandial turbidity of sample
 Lipid Storage Diseases Friedwald Equation
NIEMANN-PICK Accumulation of sphingomyelin in the TC = HDL + LDL + VLDL
DISEASE bone marrow, spleen and lymph LDL = TC – HDL – TAG/5 (mg/dL)
nodes LDL = TC – HDL – TAG/2.175 (mmol/L)
TANGIER’S Complete absence of HDL (HDL: 1-2
DISEASE mg/dL) De Long Equation
TC = HDL + LDL + VLDL
Clin cx: orange or yellow discoloration
LDL = TC – HDL – TAG/ 6.5 (mmol/L)
of tonsils and pharynx
LDL = TC – HDL – TAG/2.825 (mmol/L)
TAY-SACH’S Deficiency of hexosaminidase A
DISEASE Accumulation of sphingolipids in the Cholesterol Measurement
brain  Abell-Kendall Method (OLD reference method)
ANDERSON’S Chylomicron-retention disease - 3-step method: Saponification -> Extraction -> Colorimetry
DISEASE - Libermann-Burchard rgt: gHAc, acetic anhydride,
SITOSTEROLEMIA Plant sterols absorbed and concentrated H2SO4
accumulated in the blood and tissues - End Product: cholestadienyl monosulfuric acid
- End Color: GREEN
Lipid Disorders
TAG CH CM LDL VLD  Abell, Levey, Brody Method (Current Reference Method)
OLE L - Reagents: Libermann-Burchard rgt + KOH and alcoholic
1:
Hyperchylo
LPL deficiency (Low
cardiac risk, eruptive
↑ N ↑ N N hexane
- End Color: GREEN
micronemia xanthoma, recurrent
pancreatitis)
 Sperry Method
2a: Familial
hypercholes
Defective or deficient
LDL receptors
N ↑ N ↑ N - 4-step method: Saponification -> Extraction -> Colorimetry
terolemia (High cardiac risk, -> Purification with digitonide
xanthelasma, tendon
xanthoma, corneal  Salkowski Method
arcus, hypothyroidism,
nephritic syndrome)
- End Product: cholestadienyl disulfuric acid
- End Color: RED
2b: Familial
combined
Most common
(High cardiac risk)
↑ ↑ N ↑ ↑
hyperlipide  Enzymatic Method (most commonly used)
mia - interferences: ascorbate, hemoglobin, bilirubin
3: Familial
dysbetalipop
Accumulated VLDL
(Eruptive and palmar
↑ ↑ N N ↑
 Isotope Dilution/Mass Spectrometry (IDMS)
roteinemia xanthomas)
- Definitive method
4: Familial
hypertriglyc
Low cardiac risk
↑ N N ↑
eridemia Triglyceride Measurement
 Van Handel & Zilversmit Method
5: Low cardiac risk,
eruptive xanthoma
↑ ↑ N ↑ - Colorimetric Method
- Reagent: chromotropic acid
Abetalipopro
teinemia/
Defective apo β
synthesis
↓ ↓ Not found in
- End Color: BLUE
plasma
Bassen- (Cerebral ataxia,
Kornzweig acanthocytosis, fat
 Modified Van Handel & Zilversmit Method
Syndrome malabsorption)
- Colorimetric Method and Reference Method
- Reagent: sulphuric acid, salicylic acid
Specimen Considerations
- End Color: PINK
 Requires at least 10-16 hours of fasting
 Not affected by fasting: CHOLESTEROL determination
 Hantzsch Method
 Hemolyzed and icteric samples are not accepted
- Fluorometric method
 Specimen: Serum or plasma EDTA
- Reagent: acetylacetone (diacetyl acetone)
- End Color: YELLOW
Lipid Profile
 Triglyceride determination
 Total Cholesterol determination Lipoprotein Analysis
 Ultracentrifugation
 HDL determination
- Reference method; Measured in Svedverg units
 LDL determination (computed)
- Reagent: potassium bromide w/ 1.063 density
 VLDL determination (computed)
- Fluorometric method
 - Floating layer: Chylo, VLDL  Albumin
- Sinking layer: LDL, HDL - most abundant protein generally used for transport
- maintains fluid balance in tissues
 Electrophoresis Clinical Significance:
- Preferred gel: agarose gel - negative acute phase reactant
- From origin to the most anodal: Chylo, LDL, VLDL, HDL - decreased levels: malnutrition, malabsorption, liver
disease, renal disease, skin loss, dilution
 α-1 antitrypsin
MIDTERM 2: PROTEINS - major component of α-1 globulins
 The word PROTEINS come from the Greek word “proteis” - inactivate proteases
which means “first rank of importance” Clinical Significance:
 Most proteins are synthesized in the liver except: - acute phase reactant
immunoglobulins, adult hgb, factor VIII - deficiency: pulmonary emphysema, hepatic cirrhosis
 Proteins are amphoteric which means that they are able to  α-1 antichymotrypsin
react both as a base and as an acid - serine proteinase inhibitor
 Biological function of proteins: Clinical Significance:
- Structural support for the tissue (collagen and keratin) - acute phase reactant
- Contraction and relaxation of muscles - associated with: Alzheimer disease and Parkinson’s
- Coagulation and immunologic function disease
- Transport of metabolic substances  α-1 fetoprotein
- pH buffer - protects fetus from immunologic attack by the mother
- Precursor of hormones - levels decreases gradually after birth
- Maintenance of osmotic pressure Clinical Significance:
- Biochemical catalysts - increased levels: Spina Bifida, Neural Tube Defects,
Anencephaly
Protein Classification - decreased levels: Down Syndrome and Trisomy 18
 Primary Structure - tumor marker: hepatocellular CA
- linear sequence of amino acids  α-1 glycoprotein
- it determines the identity of protein, molecular structure, - only protein that is negatively charged even in acidic state
function and binding capacity Clinical Significance:
 Secondary Structure - Acute phase reactant
- refers to specific regular three-dimensional formations into - increased levels: stress, CA, RA, inflammation, AMI,
which portions of the polypeptide chain fold pregnancy, pneumonia, surgery
 Tertiary Structure  α-2 macroglobulin
- actual 3-dimentional structure or folding pattern - largest major non-immunoglobulin
- responsible for physical and chemical properties of - inactivate proteases
proteins Clinical Significance:
 Quaternary Structure - increased levels: nephrotic syndrome
- association of 2 or more polypeptide chains  Lipoprotein
- transports cholesterol, triglycerides and phospholipids
Structural Classification of Proteins  Ceruloplasmin
 Simple Proteins - transport protein for 90% of copper (other 10% is bound to
- contain peptide chains which on hydrolysis will yield only albumin)
amino acids Clinical Significance:
 Conjugated Proteins - Acute phase reactant
- contain protein and non-protein group - increased levels: Menke’s Kinky Hair Syndrome
Ex. Metalloproteins, lipoproteins, glycoproteins, - decreased levels: Wilson’s disease, malnutrition,
nucleoproteins malabsorption
 Haptoglobin
Types of Plasma Proteins - binds w hgb released by lysis of RBC
 Prealbumin - removed from the circulation by reticuloendothelial system
- transport protein bound to thyroxine (T3) and Retinol(Vit A) - does not bind with myoglobin
- best quantified by immunologic measurements since it is Clinical Significance:
below the level of detection by electrophoresis - evaluates degree of intravascular hemolysis (HTR and
Clinical Significance: HDN)
- Marker for nutritional status - increased levels: Myoglobinuria
- Confirms if specimen is CSF - decreased levels: Intravascular Hemolysis,
Hemoglobinuria
 Β2-microglobulin  GC globulin
- found on the surface of nucleated cells - migrates between alpha 1 and 2 region exhibits affinity to
- present in high concentration on lymphocytes vitamin D
- needed in the production of CD8 cells
 Transferrin Total Protein Abnormalities
- major component of beta2 region  Hypoproteinemia
- transport protein of iron Nephrotic syndrome Protein loss through urine
- prevents loss of iron through the kidney Extensive burns Protein loss through extrusion of plasma
Clinical Significance: Hepatocellular disease Protein not synthesized
- negative acute phase reactant
- increased levels: Iron Deficiency Anemia  Hyperproteinemia
TP ALB GLOB DISEASE
 Complement
↑ ↑ ↑ Dehydration
- participates in immune reaction
↑ N ↑ Multiple myeloma; Polyclonal and monoclonal
- linked to inflammatory response gammopathies
 Hemopexin ↓ ↓ ↓ Hyperaldoseteronemia/Salt Retention
- binds w heme released from hgb  destroyed in the liver Syndrome
 preserve body’s iron ↓ ↓ N Malabsorption, inadequate diet; Nephrotic
Clinical Significance: syndrome
↓ N ↓ Immunodeficiency syndromes
- acute phase reactant
↓/N ↓ ↑/N Hepatic damage such as cirrhosis, hepatitis
- decreased levels: Hemolytic Anemia and obstructive jaundice; burns, trauma
 Fibrinogen
- one of the largest plasma protein Specimen Considerations
- thrombin cleaves fibrinogen to form fibrin  Specimen: Serum is preferred; 24-hr urine and serous fluid
- most abundant coagulation factor which forms the fibrin clot can be used
Clinical Significance:  Hemolyzed and lipemic samples are not accepted
- negative acute phase reactant
 C-Reactive Protein Total Protein Measurement
- named because it binds with the C-polysaccharide of the  Kjeldahl Method (reference method)
pneumococcus - Indirect method since it measures the nitrogen content
- “general scavenger” - Average Nitrogen Content: 16%
Clinical Significance: - 3 step method
- highly sensitive acute phase reactant (1) precipitation by trichloroacetic acid (TCA) or tungstic
- first inflammatory marker to appear acid
- rapid presumptive test of bacterial vs viral infection (2) acid digestion by H2SO4 + heat + cupric sulfate (as
- increased levels: inflammatory dses catalyst) = nitrogen is converted to ammonium ions
- acute rheumatic fever, AMI, RA, gout (3) quantitation by back titration with HCl or Nesslerization
 Immunoglobulins with HgI2 or KI
- synthesized by plasma cells
- immune response Nessler’s reaction Gum ghatti Yellow
- IgG, IgA, IgM, IgE, IgD Berthelot’s reaction Hypochlorite Blue
Clinical Significance:
- increased levels: multiple myeloma, infections, allergic  Biuret Method (routine method)
reactions, hepatic dse - cupric ions complex with peptide bond = formation of violet
 Myoglobin colored chelate
- primary oxygen-carrying protein in striated skeletal and - read at 540nm
cardiac muscle - requires at least 2 peptide bonds to be positive
- nephrotoxic Reagents:
Clinical Significance: - Rochelle salt (sodium potassium tartrate)
- Cardiac marker - NaOH
- increased levels: AMI, muscular diseases - Alkaline copper sulfate
 Troponin - Potassium iodide
- Troponin I, Troponin T, Troponin C  Dye Binding
Clinical Significance: - based on the ability of most proteins to bind dyes (read at
- Gold standard marker for AMI 465nm – 595nm)
 Brain Natriuretic Peptide - dyes: Bromphenol Blue, Ponceau S, Amido Black,
- released in response to volume expansion and the Limassine Green, Coomassie Brilliant Blue
increased wall stress of cardiac myocytes  Lowry/Folin Ciocaltau (highest analytical sensitivity)
- cardiac marker - Oxidation of phenolic compounds to a DEEP BLUE color
 Refractometry
- rapid and simples test
- measures refractive index of solutes in serum

Albumin Measurement
 Salt Fractionation
- Globulins are precipitated in high salt concentrations
(ammonium sulfate)
- Albumin is quantified using biuret reaction Beta-Gamma Bridging: Liver Cirrhosis
- Reference range:
Total Protein = 6.5-8.3 g/dL
Albumin = 3.5-5.5 g/dL
- Conversion factor for both (g/dL to g/L) = 10
 Dye Binding
- Albumin binds to dye causing shift in absorption maximum

Methyl orange Non specific for albumin

HABA Many interferences such as
2-Hydroxyazobenzen- salicylates & bilirubin
4'-Carboxylic Acid Monoclonal Spike: Monoclonal Gammopathy
Bromcresol green Most commonly used (Multiple Myeloma and Waldenstrom’s
Bromcresol purple Most specific, sensitive and Macroglobulinemia)
precise

Protein Electrophoresis
 Proteins are negatively charged at pH 8.6 (anion) and they
move towards the anode
 After electrophoresis  fixative  stained 
densitometry (measures the absorbance of stain –
concentration of the dye and protein band)
Components
1. Support Media decreased α-1 Antitrypsin: Emphysema
 Agarose- most commonly used for lipoproteins
 Cellulose acetate- most commonly used for SPE
 Polyacrilamide- most commonly used for isoenzymes
 Nitrocellulose- most commonly used for western blot
2. Buffer- maintain a constant pH (pH 8.6)
3. Electric current
 Proteins are (-) charged, so sample is placed on the negative
side
 Smaller particles and more negatively charged particles will
migrate farther towards the anode

Electrophoretic Patterns
decreased Albumin and increased α-2 region:
Nephrotic Syndrome

Normal serum protein electrophoretic pattern

Increased α1 and α2, slightly decreased albumin:

acute inflammation