Analytical Methods Order of Draw

Colorimetry Yellow Blood culture tubes (Sterile)

A. Spectrophotometry Light Blue Trisodium Citrate
- Light transmitted by a solution to determine the concentration of light-absorbing Red Serum tube, With or without clot activator or gel separator
substances in solution Green Heparin (Li, Na, NH₄)
B. Flame Emission Photometry (FEP) Lavender Ethylenediamine Tetraacetic Acid (EDTA)
- Light emitted by a single atom burned in a flame Gray NaF & K oxalate/iodoacetate & heparin
- Excitation of electrons from lower to higher energy state
C. Atomic Absorption Spectrophotometry (AAS)
- Light absorbed by atoms dissociated by heat
- Element is not excited by merely dissociated from its chemical bond and place in
Anticoagulants
an unionized, unexcited, ground state
Volumetry (Titrimetry) Combines with calcium to form an insoluble salt
- Unknown sample made to react with known solution in the presence of indicator Oxalate Interferes with Na, K, and most BUN (Urease) measurements
Turbidimetry Conc: 1-2 mg/mL
- Amount of light blocked by a particulate matter in a turbid solution Combines with calcium in a non-ionized form
Citrate
Nephelometry Conc: 3.2-3.8 g/dL
- Amount of scattered light by a particulate matter suspended in a turbid solution Combines with calcium in a process called chelation
Electrophoresis EDTA Used for CEA, TDM, lead poisoning & nucleic acid testing
- Migration of charged particles in an electric field Conc: 1-2 mg/mL
A. Densitometry Forms weakly dissociated calcium components
- Measures absorbance of stain-concentration of the dye and protein fraction Flouride Interferes with the measurement of Na, K, and BUN (Urease)
- Scans & quantitates electrophoretic pattern Conc: 10 mg/mL
B. Isoelectric Focusing Mucoitin Polysulfuric Acid
- Separates molecules by migration through a pH gradient Ideal universal anticoagulant
C. Capillary Electrophoresis Heparin Acts as antithrombin and antithromboplastin; anti-Factor X
- Sample molecules are separated by electro-osmotic flow (Li, Na) Accelerates the action of antithrombin III, neutralizing thrombin and
D. Western Blot preventing the formation of fibrin
- Separate, detect and identify 1 or more protein in a complex mixture Conc: 0.2 mg/mL
Chromatography
- Separation of soluble components in a solution by specific differences in physical
& chemical characteristics of the different constituents
A. Paper Chromatography Collection Tubes
- Fractionation of sugar and amino acid Stopper Color Code Additive Specimen Clinical Use
B. Thin Layer Chromatography (TLC) Red None Serum Gen Chemistry
- A semi-quantitative drug screening test Yellow & Red Polymer barrier (gel) Serum Gen Chemistry
- Sample components identified by comparison with standards on the same plate Gray & Red Polymer barrier Serum Gen Chemistry
C. Gas Chromatography (GC) Yellow/Gray/Orange Thrombin Serum Gen Chemistry
- Separation of steroids, barbiturates, blood, alcohol and lipids Gold Polymer barrier Serum Gen Chemistry
- Useful for compounds that are naturally volatile/easily converted to volatile form
Trace elements,
D. Mass Spectroscopy (MS)
toxicology,
- Fragmentation and ionization of molecules using a suitable source of energy None Serum
nutritional
E. Gas Chromatography-Mass Spectroscopy (GC-MS) Royal Blue (trace
studies, TDM
- Gold standard for drug testing element-free tube)
Toxicology,
- Uses electron beam to split drug emerging from the column to its component ions
Na heparin Plasma/WB nutritional
F. Tandem Mass Spectroscopy (MS/MS)
studies
- Detect 20 inborn errors of metabolism from a single blood spot
Na heparin (Glass
G. Liquid Chromatography (LC) Plasma/WB Lead testing
Tan tube)
- Distribution of solutes between a liquid mobile phase and a stationary phase
H. High Performance Liquid Chromatography (HPLC) K₂ EDTA (plastic tube) Plasma/WB Lead testing
- Uses pressure for fast separations, controlled temperature, in-line detectors and Green&Gray/Light Polymer barrier &
Plasma/WB Gen Chemistry
gradient elution technique Green/Black Lithium heparin
I. Liquid Chromatography-Mass Spectroscopy (LC_MS) Gen Chemistry &
None Serum
- For detecting nonvolatile substances in body fluids Serology
Fluorometry/Molecular Luminescence Spectrophotometry Pink K₂ EDTA-glass & plastic
ABO & Rh typing,
- Amount of light intensity present over a zero background (with cross-matched Plasma/WB
Ab screening (BB)
- Amount of light emitted by molecule after excitation by electromagnetic radiation label)
Chemiluminescence Buffered Na Citrate ESR
Black WB
- Emission of light is created from a chemical or electrochemical reaction (4:1 ratio) (Westergreen)
- Chemical reaction yields an electronically excited compound that emits light as it Na Polyanethol-
WB Blood culture
return to its ground state, or that transfers its energy to another compound, which Sulfonate (SPS)
then produces emission Yellow HLA
Acid citrate dextrose
Osmometry WB phenotyping,
(ACD)
- Measurement of osmolality of an aqueous solution (serum, plasma, urine) paternity testing
- Measuring changes in the colligative properties of solutions that occur owing to Molecular
White EDTA & gel Plasma
variations in particle concentration diagnostic
Electrochemistry Technique Trisodium citrate Plasma Coagulation tests
- Measurement of current and voltage generated by the activity of a specific ion Citrate, theophylline, Coagulation tests
A. Potentiometry adenosine & Plasma & heparin
Light Blue
- Measurement of electrical potential due to the activity of free ions – change in dipyridamole (CTAD) monitoring
voltage indicates activity of each analyte Thrombin & soybean
Plasma Coagulation tests
B. Ion Selective Electrode (ISE) trypsin inhibitor
- Electrical transducer capable of responding to one given ion
C. Coulometry
- Measurement of amount of electricity (in coulombs) at a fixed potential
D. Amperometry
- Measurement of the current flow produced by an oxidation-reaction
E. Polarography
- Measurement of difference in current at a constant voltage
F. Voltammetry
- Measurement of current after which a potential is applied to electrochemical cell

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Carbohydrates A. Chemical Methods
Carbohydrate 1. Oxidation Reduction Method
- Hydrates of aldehyde and ketone derivatives a. Alkaline Copper Reduction Method
- Monosaccharide, disaccharide, oligosaccharide, polysaccharide - Reduction of cupric ions to cuprous ions forming cuprous oxide in hot alkaline
Reducing Sugar solution by glucose
- Glucose (directly used for energy or stored as glycogen) a. Folin Wu Method – Blue
- Maltose, fructose, lactose, galactose b. Nelson Somogyi Method – Blue
Non-reducing Sugar c. Neocuproine Method – Yellow or Yellow orange
- Sucrose d. Benedict’s Method (Modified Folin Wu) – Brick Red
Pancreas - Used for detection and quantitation of reducing substances in body fluids
- Both an endocrine & exocrine organ in the control of carbohydrate metabolism b. Alkaline Ferric Reduction Method (Hagedorn Jensen)
A. Endocrine Gland - Reduction of a yellow ferricyanide to a colorless ferrocyanide by glucose
- Secretes hormones 2. Condensation Method
*Insulin (Synthesized by Beta Cells) a. Ortho-toluidine (Dubowski Method)
- Responsible for the entry of glucose into the cell B. Enzymatic Methods (acts only on glucose)
- Released when glucose levels are high 1. Glucose Oxidase Method
- Hypoglycemic agent (decrease glucose levels) - Measures beta-D glucose by CSF & urine glucose
*Glucagon (Synthesized by Alpha Cells) 2. Hexokinase Method
- Enhances catabolic function during fasting periods - Reference method/Gold standard for glucose analysis
- Released during stress and fasting states 3. Glucose Dehydrogenase Method
- Hyperglycemic agent (increase glucose levels) - Glucose is reduced to produce a chromophore
*Somatostatin (Synthesized by Delta Cells) 4. Dextrostics
- Inhibits the action of insulin, GH and glucagon - Important in establishing correct insulin amount for next dose
B. Exocrine Gland 5. Interstitial Glucose Measuring Device
- Produces and secretes an amylase responsible for the breakdown of ingested - For continuous monitoring of glucose levels in people with diabetes
complex carbohydrates Samples for Glucose Measurement
Clinical Conditions of Carbohydrate Metabolism A. Random Blood Sugar (RBS)
A. Hyperglycemia B. Fasting Blood Sugar (FBS)
- Increase blood glucose concentration - Overall glucose homeostasis
- FBS level: > 126 mg/dL C. 2-Hour Post Prandial Blood Sugar (PPBS)
- Lab findings: D. Glucose Tolerance Test (GTT)
↑ glucose in plasma & urine - For multiple blood sugar test
↑ urine specific gravity - Oral GTT
Ketones in serum & urine (Type 1 DM patients than Type 2) 1 Janney-Isaacson method / single dose method – most common
↓ blood & urine pH (acidosis) 2 Exton Rose method / double dose method / divided oral dose
Electrolyte imbalance (↓Na, ↑K, ↓HCO₃) - Intravenous GTT for DM patients with gastrointestinal disorders
B. Hypoglycemia E. Glycosylated Hemoglobin (HbA1c)
- Results from an imbalance between glucose utilization and production - The largest sub-fraction of normal Hgb A in both diabetic & non-diabetic individuals
- Decrease blood glucose concentration - Reliable method for monitoring long-term glucose control
- Whipple’s Triad - Indicative of DM: >6.5%
Low blood glucose concentration F. Fructosamine/Glycosylated Albumin
Typical symptoms - Reflection of short term glucose control (3-6 weeks)
Symptoms alleviated by glucose administration - Reference value: 205-285 umol/L
Diabetes Mellitus Inborn Errors of Carbohydrate Metabolism
- Group of metabolic disorders characterized by hyperglycemia resulting from defects A. Galactosemia
in insulin secretion, insulin receptors or both - Congenital deficiency of 1 of 3 enzymes involved in galactose metabolism
A. Type 1 Diabetes Mellitus 1 Galactose-1-phosphateuridyl transferase
- Formerly known as: 2 Galactokinase (GALK)
Insulin dependent diabetes mellitus 3 Uridine diphosphate galactose-4-epimerase (GALE)
Juvenile onset diabetes mellitus - Failure to thrive syndrome in infants
Brittle diabetes B. Essentail fructosuria
Ketosis-prone diabetes - Autosomal recessive disorder characterized by fructokinase deficiency
- Result of cellular-mediated autoimmune destruction of beta cells of the pancreas C. Hereditary fructose intolerance
- Insulinopenia (absolute insulin deficiency) due to loss of pancreatic beta cells, and - Defect of fructose-1,6-biphospahte aldolase B activity in liver, kidney and intestine
depend on insulin to sustain life and prevent ketosis D. Fructose-1,6-biphosphate deficiency
B. Idiopathic Type 1 Diabetes Mellitus - Defect of fructose-1,6-biphosphate results in failure of hepatic glucose generation
- A form of type 1 DM that has no etiology, strongly inherited, does not have beta cell E. Glycogen storage disease
autoantibodies and have episodic requirements for insulin replacement - Inherited autosomal recessive trait
C. Type 2 Diabetes Mellitus - A consequence of inherited deficiencies of enzymes that control the synthesis or
- Formerly known as: breakdown of glycogen
Non-insulin dependent diabetes mellitus - Most common: Von Gierke Disease
Adult type/maturity onset diabetes mellitus CSF Glucose
Stable diabetes - 40-60% of blood plasma glucose level
Ketosis-resistant diabetes - Increased DM,; Decreased bacterial meningitis, TB, fungal & amebic meningitis
Receptor-deficient diabetes mellitus - Reference value: adult = 40-70 mg/dL, child = 60-80 mg/dL
- Hyperglycemia due to an individual’s resistance to insulin; Relative insulin deficiency C-Peptide Test
- Described as geneticist’s nightmare - Formed during the conversion of pro-insulin to insulin
- Assoc. with strong genetic predisposition and not related to an autoimmune disease - Reliable indicator for pancreatic and insulin secretions (beta cell function)
D. Gestational Diabetes Mellitus (GDM) - Specimen: Fasting serum
- Impaired ability to metabolize carbohydrate usually caused by deficiency of insulin, - Reference value: 0.90-4.3 ng/mL
metabolic or hormonal changes Ketone Test
- Occurs during pregnancy and disappears after delivery - Normal ratio of beta-hydroxybutyrate and acetoacetic acid is 1:1
- Screening should be performed between 24&28 weeks of gestation - Beta-hydroxybutyrate levels are high in diabetic ketoacidosis (DKA) and fall with
- Screening & diagnosis by the performance of a 2-hour OGTT using 75g glucose load treatment whereas acetoacetic acid and acetone levels rose with treatment
- Revised diagnostic criteria FBS >92 mg/dL - Specimen: Fresh urine or serum
1-hour OGTT >180 mg/dL
2-hour OGTT >153 mg/dL
Glucose Methodologies
Venous Plasma Glucose
- Standard clinical specimen
Serum
- Appropriate for glucose analysis if serum is separated from the cells within 30
minutes, if longer than 30 minutes preserve with Sodium fluoride

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Lipids - Functions
Lipids Very important source of energy
- Referred to as fats, composed mostly of carbon-hydrogen bond Provide the substance for conversion to glucose
- Insoluble to blood and water but soluble in organic solvents Lipoproteins
Phospholipid (Conjugated Lipid) - Are large macromolecular complexes of lipids with special proteins known as
- Most abundant lipid derived from phosphatidic acid apolipoproteins (ALP)
- Originates in the liver and intestine - Main purpose is to transport TAG & Chol to sites of energy storage & utilization
- Produced from the conjugation of 2 fatty acids and a phosphorylated glycerol - Cholesterol & TAG travel in plasma not as free-floating molecules but as part of the
- Functions water-soluble complexes known as lipoproteins
Surfactant to lower the surface tension at the air/liquid interface within the alveoli - Major Lipoproteins
of the lung A. Chylomicrons (CM)
Participates in cellular metabolism and blood coagulation - Largest and least dense of the LP particles
- Reference value: 150-380 mg/dL (serum) - Produced in the intestine and transports exogenous/dietary TAG to liver,
- Forms muscles and fat depot
Lecithin/Phosphatidyl choline 70% - Apolipoproteins: Apo B-48, Apo A-1, Apo C, Apo E
Spingomyelin 20% - Density: <0.95 kg/L
Cephalin 10% B. Very Low Density Lipoprotein/Pre-Beta Lipoprotein (VLDL)
- Spingomyelin – Only phospholipid in membrane that is not derived from glycerol but - Secreted in the liver and transports endogenous TAG from the liver to muscle,
from an amino alcohol called sphigosine; Accumulates in the liver & spleen of fat depots and peripheral tissues
patients suffering from Niemann-Pick Disease (Lipid storage disorder) - Apolipoproteins: Apo B-100, Apo C, Apo E
- Methods - Density: 0.95-1.006 kg/L
Estimation of serum lipid phosphorous C. High Density Lipoprotein/Alpha Lipoprotein (HDL)
Status of fetal lung maturation (Lecithin/Spingomyelin Ratio) - Smallest LP but the most dense (3-12 nm)
Cholesterol (3-hydroxy-5,6-cholestene) - Produced in the liver & intestine and transports excess cholesterol from the
- Unsaturated steroid alcohol containing 4 rings, & it has a single C-H side chain tail tissues and return it to the liver (which maintains the equilibrium of cholesterol
- Synthesized in the liver; Found on the surface of lipid layers in peripheral cells)
- Transport and excretion promoted by estrogen - Apolipoproteins: Apo A-I, Apo A-II, Apo C
- Functions - Reference value: 40 mg/dL
Precursor of 5 major classes of steroids: progestin, glucocorticoids, - Density: 1.063-1.21 kg/L
mineralocorticoids, androgen & estrogen D. Low Density Lipoprotein/Beta Lipoprotein (LDL)
Important constituent in the assembly of cell membranes and bile acids - Major end-product from the catabolism of VLDL
- Reference values - Synthesized in the liver and is the major source of cholesterol for tissues
<200 mg/dL desirable - Important in assessing patients with or without coronary heart disease (CHD)
200-239 mg/dL borderline - Apolipoproteins: Apo B-100, Apo E
>240 mg/dL high cholesterol - Reference value: <100 mg/dL
- Forms - Density: 1.019-1.063 kg/L
Cholesterol ester 70% - Minor Lipoproteins
Free cholesterol 30% A. Intermediate Density Lipoprotein (IDL)
- Lecithin-Cholesterol Acyl Transferase (LCAT) – In human plasma; Catalyzes the - Product of VLDL catabolism (VLDL remnant)
esterification of cholesterol (HDL) by promoting the transfer of fatty acids from - Migrates either in the pre-beta or beta region (electrophoresis)
lecithin to cholesterol which results in the formation of lysolecithin and cholesterol - ALP: Apo B-100
ester; Activated by Apo A-1 - Density: 1.006-1.019 kg/L
- Methods B. Lipoprotein (a)/ Lp(a)
A. Chemical Method - Variable migration = pre-beta, or sometimes between LDL & albumin
- Dehydration and oxidation of cholesterol to form a colored compound - Known as “Sinking pre-beta LP” due to electrophoretic mobility same as VLDL but
a. Liebermann Burchardt Reaction – Green density like LDL
b. Salkowski Reaction – Red - ALP: Apo B-100, Apo (a)
B. Enzymatic Method - Abnormal Lipoproteins
a. Cholesterol Oxidase Reaction – Measure amount of hydrogen H₂O₂ produced A. Lipoprotein X
C. Reference Method - Abnormal LP found in obstructive jaundice & LCAT deficiency
a. Abell, Levy and Brodie Method – Hexane extraction after hydrolysis with - Specific and sensitive indicator of cholestasis
alcoholic KOH followed by reaction with Liebermann Burchardt color reagent - Lipid content mostly phospholipid and free cholesterol and contains Apo C & Alb
Triglyceride/Triacylglycerol (TAG) or Neutral Fat B. Beta-VLDL (Floating Beta LP)
- Contains 3 molecules of fatty acids and 1 molecule of glycerol by ester bonds - Known as “Abnormally migrating Beta-VLDL” & “VLDL rich in cholesterol” due to
- Main storage lipid in man (adipose tissue) defective catabolism of VLDL
- Function: When TAG are metabolized, their fatty acids are released to the cells and - Density: <1.006 kg/L
converted into energy; Provides excellent insulation - Methodologies
- Fasting requirement: 12-14 hours A. Ultracentrifugation (Density Gradient)
- Reference values - Reference method for quantitation of LP; Expressed in svedverg (s) unit
<150 mg/dL normal - Based on protein (1.4 mg/L) and TAG (1.0 g/mL) contents of LP
150-199 mg/dL borderline - Reagent: Potassium bromide solution with 1.063 density
200-499 mg/dL high B. Electrophoresis
>500 mg/dL very high - Electrophoretic pattern: HDL, VLDL, LDL, chylomicrons
- Methods - Preferred supporting medium: Agarose gel (speed, sensitive, resolves LP classes)
A. Chemical Method - Lipid-staining dyes: Oil red O, Fat red 7B, Sudan black B (react primarily with the
a. Colorimetric Method (Van Handel & Zilversmith) – Blue ester bonds in TAG and cholesterol esters
b. Fluorometric Method (Hantzsch Condensation) C. Chemical Precipitation
B. Enzymatic Method (Relatively specific, rapid & easy to use) - Uses polyanions and divalent cations
a. Glycerol Kinase Method – Hydrolysis of TAG to free fatty acids and glycerol, - Enzymatic with coupled detergent precipitation is the most useful LP test
followed by the phosphorylation of glycerol to glycerophosphate D. Chromatographic Methods
C. Reference Method - Utilizes either gel chromatography or affinity chromatography
a. Modified Van Handel & Zilversmith E. Immunochemical Methos
- Time-consuming manual method which cannot be automated - Uses antibodies specific to epitopes on the ALP
- Involves alkaline hydrolysis (saponification) using alcoholic KOH, solvent F. Immunoassay or Immunonephelometry (ALP assay)
extraction with chloroform and the extract is treated with silicic acid - Turbidity created by ALP-anitibody complexes
(chromatography) to isolate TAG, and a color reaction with chromotropic acid,
giving rise to a pink end color Main
ALP Function Comments
Fatty Acids Distribution
Activates LCAT that
- Linear chains of C-H bonds that terminate with a carboxyl group
esterifies cholesterol in Synthesized in liver & intestine;
- Mostly found as constituents of phospholipid and TAG A-I HDL
plasma; HDL biosynthesis
- Derived from hydrolysis of TAG in adipose tissue Ligand for ABCA1
- Small amount present in plasma (free unsterified form), most is bound to albumin Inhibit LP & hepatic lipases;
A-II HDL
- Reference value: 9-15 mg/dL Increases plasma TAG

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 HDL, CM, & Cofactor for LCAT;
A-IV free in Increased during fat absorption;
plasma HDL biosynthesis
Carboxy-terminal
Very large structural protein,
B- recognition signal targets
VLDL, LDL synthesized in liver with lipids of
100 LDL to the LDL (apoB,E)
endogenous origin
receptor
Synthesized in intestine, encoded by
Not recognized by LDL
B-48 CM same gene and same amino terminus
receptor
as apoB-100
Inhibit hepatic uptake of VLDL and
C-I CM, VLDL
CETP
Activates LP lipase; Deficiency causes reduced clearance
C-II CM, VLDL
Stimulates hydrolysis of TAG of TAG-rich LP
Inh. lipolysis of TAG-rich LP;
Deficiency causes reduced clearance
C-III VLDL, HDL Decreases clearance rate of
of TAG-rich LP
remnant particles
D HDL Activates LCAT
Recognition factor that
E-2 (associated with type 3
targets CM and VLDL
CM, VLDL, hyperlipoproteinemia), E-3, & E-4
remnants to hepatic
E IDL, remnants (associated with high LDL-C, higher
receptor;
of HDL risk of CHD and Alzheimer’s disease)
Binds to cell surface LDL
isoforms
receptors & proteoglycans
HDL, LDL,
F Regulates CETP function
VLDL
Abs against apoH or B₂-glycoprotein-I
Related to activation of LPL are a subset of antiphospholipid Abs,
H VLDL
TAG metabolism and may be associated with
hyperthrombosis and stroke
Cell-aggregating factor in
Sertoli cells;
Inhibitor of the C5b*7 Involved in apoptosis;
complement complex; Linked to neurologic diseases like
J
Beta-amyloid clearance in Pick’s and Alzheimer’s;
glial cells; Also known as clusterin
Cholesterol trafficking in
brain
May be linked to reverse cholesterol
L HDL
transport; Nor present in plasma
HDL, CM,
M May be linked to HDL remodeling
LDL, VLDL
Homologous to plasminogen;
Apo May be prothrombotic;
Lp(a)
(a) Bound to apoB-100 by disulfide
linkage

Disorders Associated with Lipids and Lipoproteins

A. Familial Hypercholesterolemia (Type 2a)
- Defective or deficient LDL receptors
B. Familial Dysbetalipoproteinemia (Type 3)
- Accumulation of plasma VLDL rich in cholesterol and chylomicron remnants
C. Abetalipoproteinemia (Bassen-Kornzweig Syndrome)
- Defective apo B synthesis
D. Hypobetalipoproteinemia
- Apo-B deficiency resulting from point mutation in apo B
E. Niemann-Pick Disease (Lipid Storage Disease)
- Inherited disorder of lipid metabolism in which there are accumulations of
spingomyelin in the bone marrow, spleen and lymph nodes
F. Tangler’s Disease
- Complete absence of HDL due to mutation in the ABCA1 gene on chromosome 9
G. Lipoprotein Lipase (LPL) Deficiency
- Childhood with abdominal pain and pancreatitis
- Results to inability to clear CM particles
H. LCAT Deficiency
- Due to mutation in the LCAT gene
- Milder form of LCAT deficiency is Fish-eye disease
I. Tay-Sachs Disease
- Inherited neurodegenerative disorder of lipid metabolism characterized by a
deficiency of the enzyme hexosaminidase A
- Results in the accumulation of spingolipids in the brain
J. Chylomicron Retention Disease (Anderson’s Disease)
- Is distinct from abetalipoproteinemia, as only apoB-48 appears to be affected
K. Sitosterolemia
- Plant sterols are absorbed and accumulate in plasma and peripheral tissues
Friedrickson Classification
A. Type1 Hyperchylomicronemia/Familial LPL Deficiency
- Block in the progression from CM to CM remnants results in the accumulation of
CM in type 1 and 5
B. Type2 Hyperlipoproteinemia
- Block in LDL metabolism and defective apo B that does not bind to LDL receptor
C. Type3 Dysbetalipoproteinemia
- Due to failure to convert VLDL to LDL causing IDL to accumulate
D. Type4 Hyperlipoproteinemia/Hypertriglyceridemia
- Block in the conversion of VLDL to IDL and LDL results to elevated TAG and VLDL,
but LDL is normal
E. Type5 Hyperlipoproteinemia
- LPL deficiency
- Inability to breakdown TAG

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Proteins - Found on the surface of most nucleated cells
Proteins - Needed in the production of CD8 cells
- Synthesized in the liver and secreted by the hepatocyte to the circulation except Igs - RV: 0.2-2.8 ug/dL
- Are macromolecules composed of polymers of covalently lined amino acids that are k. Transferrin (Siderophilin)
involved in every cellular processes - Major component of the ß₂-globulin fraction (electrophoresis)
Functions - Sythesized in liver which transports iron to its storage sites
Repair body tissues - RV: Male 215-365 mg/dL; Female 250-380 mg/dL
Important in blood coagulation and immunologic function l. Immunoglobulins
Transport of metabolic substances - Synthesized in plasma cells
Maintenance of osmotic pressure - IgG (most abundant antibody found in plasma and lymph);
Maintenance of blood pH (buffers) IgA (main antibody found in mucous secretions);
Biocatalysts IgM (first antibody that appears in response to antigenic stimulation;
Structures IgD (present mostly on the presence of B cells);
A. Primary Structure IgE (antibody associated with allergic and anaphylactic reactions)
- Linear sequence of the amino acid m. Lipoprotein
B. Secondary Structure - Binds with proteins and lipids forming VLDL, HDL, LDL, & CM
- Winding of the polypeptide chain - Transport cholesterol, TAG and phospholipids
C. Tertiary Structure n. Fibrinogen
- Responsible for the many of the physical and chemical properties of the proteins - One of the largest proteins in the blood
D. Quaternary Structure - Synthesized in liver and the most abundant of the coagulation factor (forms a
- Association of 2 or more polypeptide chains to form a functional protein molecule fibrin clot when activated by thrombin)
Classification - Serve as a marker for long-term prognosis of cardiovascular disease
A. Simple Proteins o. Complement
- Contain peptide chains which on hydrolysis yield only amino acids - One of the natural defense mechanisms that protects the human body from
- May be fibrous or globular infection
B. Conjugated Proteins - Participates in immune reaction and serve as a link to inflammatory response
- Composed on a protein (apoprotein) and a non-protein moiety (prosthetic group) - C3 is the most abundant form in serum (in the pathogenesis of age-related
- These proteins impart certain characteristics to the proteins: metalloprotein, macular degeneration
lipoprotein, glycoprotein, mucoprotein or proteoglycans, nucleoprotein p. C-Reactive Protein (CRP)
Plasma Proteins - Binds to the C-polysaccharide of the pneumococcus
A. Prealbumin (Transthyretin) - General scavenger molecule; Gamma-migrating protein
- Migrates ahead of albumin - Cardiac and inflammatory marker
- Half-life of 2 days; Rich in tryptophan - RV: <1.0 mg/dL
- Serves as transport protein for T₄ and retinol (Vitamin A) Miscellaneous Proteins
- Detect malnutrition and individual’s response to dietary supplementation A. Myoglobin
- Landmark to confirm that the specimen is really CSF - Small heme protein found in skeletal and cardiac muscle
- Reference value: 18-45 mg/dL - Transports and stores oxygen from hemoglobin to intracellular respiratory
B. Albumin enzymes of contractile cells
- Synthesized in liver; Present in highest concentration in plasma - Marker for chest pain (angina) and acute myocardial infarction (AMI)
- General transport protein (binds to various substances in the blood) B. Troponins (Tn)
- Serves as circulating reservoir of amino acids - Complex of 3 proteins that bind to the thin filaments of cardiac muscles
- Reference value: 3.5-5.0 g/dL - Regulators of actin and myosin
C. Globulin - Most important marker for cardiac injury (AMI)
- A group of protein consisting of alpha1, 2, beta and gamma fractions - Cardiac troponins
- Measurement: Total protein – Albumin = Globulin a. Troponin T (TnT)/Tropomyosin-binding subunit
- Reference value: 2.3-3.5 g/dL - Valuable tool in diagnosis of AMI
a. ἀ₁-Antitrypsin (AAT) - Sensitive marker for the diagnosis of unstable angina (angina at rest)
- Neutralizes trypsin-like enzymes which is released from WBCs combat infection b. Troponin I (TnI)/Inhibitory subunit or Actin-binding unit
but it can also destroy alveoli which can lead to emphysema - Only found in the myocardium (greater cardiac specificity that TnT
- Major inhibitor of protease activity (prevents self-destruction of tissues) - Highly specific for AMI
- RV: 145-270 mg/dL - Very sensitive indicator of even minor amount of cardiac necrosis
b. ἀ₁-Fetoprotein (AFP) - RV: <0.1 ng/mL
- Most abundant protein in fetal serum C. B-Type Natriuretic Peptide (BNP)
- Synthesized initially by fetal yolk sac, then by fetal parenchymal cells in the liver - Cardiac maker
- For hepatic & gonodal cancer - Increases in response to peptide ventricular systolic and diastolic dysfunction and
- RV: 5 ng/mL is diagnostic of congestive heart failure
c. ἀ₁- Acid Glycoprotein/Orosomucoid (AAG) D. Cystatin C
- Useful diagnostic tool in neonates with bacterial infections - Low molecular weight protein and a cysteine proteinase inhibitor
- RV: 55-140 mg/dL - Endogenous renal marker (sensitivity for determining GFR)
d. ἀ₁- Antichymotrypsin (ἀ₁-x) Proteins in Body Fluids
- Synthesized in the liver which binds and inactivates Prostate-Specific Antigen A. Urinary Proteins
- RV: 30-60 mg/dL - Majority of proteins found in the urine arise from the blood
e. Hemopexin - Presence of urine albumin is generally considered abnormal even in trace amount
- Binds heme released by degradation of hemoglobin (has the strongest affinity Microalbuminuria – Early indicator of glomerular dysfunction and precedes
for heme) nephropathy associated with type 1 diabetes; RV: 0-29 ug/mg creatinine
- RV: 50-115 mg/dL B. CSF Proteins
f. Group Specific Component (Gc)-Globulin - Ultrafiltrate of plasma formed in the choroids plexus of the ventricles of the brain
- Exhibits affinity with vitamin D and actin (vitamin D binding protein) - CSF albumin is 10-30 mg/dL
- RV: 20-55 mg/dL - RV: 15-45 mg/dL
g. Haptoglobin CSF Oligoclonal Banding
- Synthesized by the hepatocytes which binds free hemoglobin by its ἀ chain - Indication of antibody production within the CNS
- RV: 26-185 mg/dL - Primary purpose of CSF protein electrophoresis: To observe this type of banding
h. Ceruloplasmin representing inflammation within the CNS
- Copper-binding ἀ₂ glycoprotein that has enzymatic activities Aminoacidopathies
- Synthesized in liver which imparts a blue color to protein - Inherited disorders of amino acid metabolism
- Marker for Wilson’s Disease (0.1 g/L of ceruloplasmin) - Exist in either the activity of a specific enzyme in the metabolic pathway or in the
- RV: 18-45 mg/dL membrane transport system for amino acids
i. ἀ₂- Macroglobulin (AMG) a. Alkaptonuria – Inborn error of metabolism characterized by the absence of
- Largest non-immunoglobulin protein in plasma homogentisate oxidase in the tyrosine pathway
- Found in the intravascular spaces and inhibits proteases b. Homocystinuria – Impaired activity of cystathionine ß-synthetase
- RV: 15-420 mg/dL c. Maple Syrup Urine Disease – Markedly reduced or absence of ἀ-ketoacid decarboxylase
j. ß₂-Microglobulin d. Phenylketonuria (PKU) – Deficiency of enzyme phenylalanine hydrolase which catalyzes the
conversion of phenylalanine to tyrosine
- Light chain component of the major human leukocyte antigen (HLA)
e. Tyrosinemia

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KIDNEY FUNCTION TESTS
Kidneys
- Are paired, bean-shaped organs located retroperitoneally on either side of the
spinal column
- Nephrons: The functional unit of each kidney, are composed of 5 basic parts
namely, glomerulus, proximal convoluted tubule (PCT), loop of Henle, distal
convoluted tubule (DCT) and collecting duct
- Functions:
Elimination of waste products
Maintenance of blood volume
Maintenance of electrolyte balance
Maintenance of acid-base balance
Endocrine function (erythropoietin secretion)
- Renal function panel: Glucose, BUN, creatinine, sodium, chloride, phosphorus,
calcium, albumin and CO₂
Tests for the Glomerular Filtration Rate (GFR)
- Is a measure of the clearance of normal molecules that are not bound to protein and
are freely filtered by the glomeruli neither reabsorbed nor secreted by the tubules
- Considered the best overall indicator of the level of kidney function
Clearance (mL/min)
- The removal of the substance from plasma into urine over a fixed period of time
𝑈 𝑉𝑜𝑙𝑢𝑚𝑒 (𝑚𝐿) 1.73
𝑥 𝑥
𝑃 1440 (𝑚𝑖𝑛𝑢𝑡𝑒𝑠) 𝐴
a. Inulin Clearance (Reference Method)
- Not routinely done because of the necessity for continuous IV infusion –
requires an intravenous infusion and timed urine collections over many hours
- RV: Male (127 mL/min); Female (118 mL/min)
b. Creatinine Clearance
- Provides an estimate of the amount of plasma that must flowed through the
kidney glomeruli/minute
- Excellent measure of renal function – creatinine is freely filtered by the
glomeruli but not reabsorbed
- Measure of the completeness of a 24-hour urine collection
- RV: Male (85-125 mL/min); Female (75-112 mL/min)
c. Urea Clearance
- Demonstrate progression of renal disease or response to therapy

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Enzymes
Enzyme
- Proteins produced by living cells that hastens chemical reactions in organic matter
Factors Affecting Enzymatic Reactions
A. Enzyme Concentration
B. Substrate Concentration
C. Cofactors
- Non-protein entities that must bind to particular enzymes before a reaction occur
a. Coenzymes – Organic compound (second substrate)
b. Activators – Inorganic ions which alters the spatial configuration of the enzyme
for proper substrate binding
c. Metalloenzymes – Inorganic ion attached to a molecule
D. Inhibitors
a. Competitive – Physically binds to the active site of an enzyme; Both substrate
and inhibitor compete for the same active site of the enzyme
b. Non-Competitive – Does not compete with the substrate but look for areas other
than the active site; The substrate and inhibitor may bind an enzyme
simultaneously
c. Uncompetitive – Inhibitor binds to the enzyme-substrate (ES) complex
E. Isoenzymes – Are enzymes having the same catalytic reactions but slightly different
molecular structures
F. Temperature
G. Hiydrogen Ion Concentration (pH)
H. Storage
I. Hemolysis
J. Lactescense or Milky Specimen
Enzyme Nomenclature
A. First Digit Classification
B. Second & Third Digits Subclass
C. Final & Fourth Numbers Serial Number
Classification of Enzymes
A. Oxidoreductase - Removal/addition of electrons
B. Transferase - Transfer of a chem. group other than H from 1 substrate to another
C. Hydrolase - Hydrolysis or splitting of a bond by the addition of water
D. Lyase - Removal of groups from substrates w/o hydrolysis
E. Isomerase - Intramolecular arrangement of the substrate compound
F. Ligase - Joining of 2 substrate molecules
Enzyme Activity
A. Change in the substrate concentration
B. Change in the product concentration
C. Change in coenzyme concentration
Causes of Elevated Plasma Enzyme Levels
A. Impaired removal of enzyme from plasma
B. Increased permeability of cell membrane
C. Increased in the number of cells or the production of cells
D. Increased in the normal cell turnover
E. Decreased clearance of enzymes from the circulation
F. Tissue necrosis and degeneration – death of enzyme-containing cells
Major Clinical Enzymes
A. Phosphatase
B. Transferase/Transaminase
C. Amylase/Alpha-1,4-glucan-4-glucohydrolase
D. Lipase/Triacylglycerol Acylhydrolase
E. Lactate Dehydrogenase
F. Creatine Kinase/ATP-creatine-N-phosphotransferase
G. Aldolase/Fructose-1,6-diphosphate aldolase
Phosphatase
A. Alkaline Phosphatase (ALP)
B. Acid Phosphatase (ACP)

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