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1.1 Samples
• Serum is the sample of choice for all lipid determinations
• 12-14 hours fasting in order to minimize dietary impact on the TG levels
• Ce levels do not change to any significant degree in response to daily dietary intake
2. TRIGLYCERIDES
- General procedure or process:
• Hydrolysis of TGs to form glycerol + FFAs
• Measurement of glycerol present (either as glycerol or after conversion to another product)
A. Extraction
Requires pretreatment of sample to remove lipids from CHON and other constituents
Organic solvents are employed to extract lipids
Solvent of choice: Diethyl Ether
Use of alcohols results in only one solvent layer formed requiring less manipulation
Use of solid absorbing material such as Florisil or Zoolite removes interfering substances which would
react to the color reagent employed later in the analysis
B. Hydrolysis/Saponification
Accomplished in basic solution at elevated temperatures
KOH in ethanol is the preferred reagent (OH- ion promotes hydrolysis and ethanol allows material to
remain in the solution)
C. Oxidation
All glycerol present is oxidized by periodate anion forming formaldehyde and formic acid
Needs careful control, only formaldehyde will be assayed by the color reagent
Formaldehyde can undergo further oxidation to produce formic acid which does not react in
the system
D. Quantitation
Formaldehyde combines with a variety of reagent to form a colored end product
Some material employed give products with absorption maximum in the range of 500-600nk
(Bb, Hgb influences are eliminated)
Reagents
Chromotrpoic acid (disodium salt with 4,5-hydroxy-2,7-naphthalene disulfonic acid)
Phenylhydrazone
Acetyl acetone
o In the Hartzeh reaction, acetylacetone forms a cyclic derivative with formaldehyde
o Product formed exhibits a strong Abs maximum at 412nm and also has good
fluorescence giving 2 methods of detection for quantitating TG
E. Methods
a.
Glycerol-3-phosphate + NAD → (G-6-PD) → dihydroxyacetone PO4 → NADH
Trapping agent (hydrazine) for dihydroxyacetone phosphate is used to pull the 3rd reaction formed
NADH can be quantitated directly at 340nm
NADH may be made to react with a reagent forming a colored end product with strong light
absorbance in the visible region or a highly fluorescent product
3. TOTAL CHOLESTEROL
a. Liebermann-Burchardt method
Measures Ce extracted into cold chloroform then treated with acetic anhydride, acetic acid,
concentrated H2SO4 to form a green complex
Bloor; Schoenheiner & Sparry; Carr & Drekter; Abell & Kendell (reference method)
Dehydration step:
o (free) Ce → strong acids → 3,5 cholestadiene
Oxidation step:
o 3,5 cholestadiene → cholestahexaene sulfonic acid (Abs at 410nm)
b. Other classic color reaction of Ce incorporated the use of iron salts and H2SO4 to produce a
reddish purple complex (Salkowski reaction)
c. Methods
Wybenga et al
o Ce in serum is oxidized to a tetraene derivative by Fe+3 perchlorate and Abs of mixture is
compared with that of a pure solution fo Ce
Abell et al
o After conversion of esterified Ce to free Ce by treatment with alcoholic KOH (saponification),
the free Ce produced together with that already present in the serum is extracted into
petroleum ether. After removal of the solvent (evaporation), the Ce is determined by the
green color produced with the LB reaction.
B. Alternative enzymatic method is the polagraphic O2 electrode method that measures the decrease
in O2 tension during the reaction as Ce is oxidized
Since O2 consumption is actually being used up and measures, H2O2 interactions with other reducing
substances will not interfere
Requires the use of a different type of instrument (non-spectrophotometric) and the additional burden
of maintaining the electrode and its membrane
Expected reference range for total Ce is based on the values of the NCEP (Nat’l Cholesterol Education
Program… WOW! Naa juy naghimo ug ing-ani sa??)
<200 mg/dL (<5.18mmol/L) is desirable
200-239 mg/dL (5.18 – 6.19 mmol/L) is the borderline
>= 240mg/dL (>6.22mmol/L) high
A. HDL
May be separated by selective precipitation methods that use divalent cations such as Ca, Mg or Mn in
solutions of buffer, heparin dextran sulfates or polyethylene glycol
LDL, IDL and VLDL will tend to float on top of the serum where they may be aspirated away; the
infranatant will contain only HDLs
HDL determination will follow using the methods employed for total Ce
Interferences are minimized by manipulating the concentration of the cations and salts in the
precipitation reagents
Excessive TG levels may not be fully precipitated and may remain in the supernatant
Females tend to have a slightly lower HDL levels than a similarly aged male
NCEP reference range has been established at a minimal value of 40mg/dL
(1.02mmol/L)
o Values lower than this minimum are considered to be at a greater risk
developing CAD
B. LDL Cholesterol
May be separated by ultracentrifugation or electrophoresis
NCEP has recommended the use of Friedwald Calculation method
Requires prior determination of total Ce, TG and HDL
Formula:
LDL = Total Ce – (HDL + [TG/5])
Assumes that TG/5 is a relatively accurate estimate of VLDL
Moderately elevated TG levels or the appearance of abnormal LPs will invalidate the
results of the calculation resulting in a falsely decreased LDL
b. Use of Ab to apoA and apoE will remove HDL & VLDL fractions leaving the LDL (to be directly
assayed using the Ce oxidase method)
c. Chemical Precipitation method
LDL is precipitated
Use of buffered heparin solution
Remaining HDL and VLDL are measured and subtracted from total Ce
Less susceptible to hypertriglyceridemia than the Friedwald method
d. Homogeneous LDL Ce
LDL selectively reacts with precipiants
Eliminates pretreatment step reducing analytical time and costs while maintaining the reliability of
the assay even in the presence of high TGs
NCP recommended values is <= 100mg/dL (2.60mmol/L)
4. APOLIPOPROTEINS
- Play a critical role in lipid metabolism and are now recognized as important indicators of disease
- Measurement provides data to evaluate a patient’s risk for cardiovascular disease
- Most common method employed is Immunoprecipitation
• Specific Ab + ALP (in the sample) result to partially insoluble immune complexes
• Radial Immunodiffusion methods were among the first who employed the above principle
• RID was replaced by Rate Nephelometry which measures light scattering by large membrane complexes
Scattering of light by complexes is directly proportional to their concentration
4.2 Apo B
- Found primarily on LDL & VLDL
- May be used as an alternative to the calculated LDL
- Reference values:
Female: 0.92 – 1.38 g/L
Male: 0.99 – 1.36 g/L