Lipid Methodologies 1.

ANALYTICAL PROCEDURES FOR LIPIDS Lipid profiles are important diagnostic tool for categorizing the various disorders of lipids, commonly known as hyperlipidemias 1975: total Ce, TGs, serum appearance after standing the in the refrigerator for several hours Today: specific tests for Ce, HDL, LDL, apoA, apoB and TGs along with family history and patient’s nutritional and lifestyle habit

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

1.2 Serum or Plasma Appearance • • After it has remained undisturbed for 4-6 hours at 4-8C may prove to be helpful in classifying andultimately treating hypolipidemic pateients A creamy layer at the surface of serum contains chylomicrons (indication of either nonfasting specimen or a serious defect in LPL production or function)  Varying degrees of cloudiness (lipemia) are usually associated with high TG levels  Dilution of the specimen may be required to bring the TG levels within linear range of the analytical method 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)

2.1 Chemical Methods 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

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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) Chromotrpoic acid (disodium salt with 4,5-hydroxy-2,7-naphthalene disulfonic acid) Phenylhydrazone Acetyl acetone o o In the Hartzeh reaction, acetylacetone forms a cyclic derivative with formaldehyde Product formed exhibits a strong Abs maximum at 412nm and also has good fluorescence giving 2 methods of detection for quantitating TG

Reagents   

E. Methods a. Neri and Frings

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After the removal of PLs, glycerol and glucose by absorption of albumins (Al+3 oxides), TGs are saponified (KOH and isopropyl) Release glycerol → (oxidized by metaperiodate) → formaldehyde → (condensed by acetyl acetone; NH+4) → 3,5 – diacetyl – 1,4 – dihydrolutidine **Yellow, 405nm

b. Kessler and Lederer

Isopropanol extracts of serum are treated with Zeolite Llyod’s reagent for removal of PLs, glucose and other chromogenic material. The isopropanol, PL-free extract is mixed with KOH to saponify TGs. The hydrated glycerol is oxidized with periodic acid to form formaldehyde. Formaldehyde + Diacetylacetone & Ammonia to give a product Fluorometrically measured

 c.

Van Handel and Zilversmit    TGs extracts from serum with chloroform-methanol PLs are removed by adsorption of silicic acid TGs → glycerol → (periodic acid) → formaldehyde Formaldehyde determined photmetrically by reaction with chromotrophic acid **Blue end product 564nm

2.2 Enzymatic Methods Extraction is eliminated Hydrolysis accomplished enzymatically using LIPASE

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Used in excess to ensure complete breakdown of all ester linkages Uses chymotrypsin as part of hydrolysis step (cleavage of CHON portion of any LP complex containing TG may be necessary for complete reaction of TG) Correlation for free glycerol is easily accomplished by using a 2nd sample through analysis but eliminates the lipase hydrolysis step

Glycerol formed reacts directly with an indicator system in a series of enzyme reaction


Actual end product removed is NAD or NADH Basic reaction: • • • TG → (lipase) → glycerol + FFAs Glycerol + ATP → (glycerol kinase) → glycerol-3-phosphate + ADP Approaches to subsequent analysis: a. Bucolo and David  ADP + PEP → (PK) → ATP + pyruvate Pyruvate + NADH + H → (LD) → lactate + NAD


Follows the rate of decrease in absorbance at 340nm (because NAD does not absorb UV light)

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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



3.1 Chemical Methods Called for production of colored products (chromogen) by reacting with Ce with strongly acidic reagents Essentially oxidative - - step by step procedures with successive introduction of double bonds into the steroid ring A. Color reactions used for Cholesterol determination a. Liebermann-Burchardt method

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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 o (free) Ce → strong acids → 3,5 cholestadiene 3,5 cholestadiene → cholestahexaene sulfonic acid (Abs at 410nm) Oxidation step:

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


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.

3.2 Enzymatic Methods

A. The earliest method carried out analysis in 3 enzymatic steps
 Hydrolysis of the Ce esters by Ce esterase (CE): Ce esters → (CE) → Ce + FAs Oxidation of Ce by Ce oxidase form H2O2 and cholestone: Ce + O2 → (CO) → Cholest-4-3-one + 2H2O2

Catalysis of H2O2 by horseradish peroxidase (HRP) to produce free O2 radical that oxidizes Ce reduced dye to the colored end product: H2O2 + aminophenazone (reduced) + phenol → (HRP) → quinoneimine (oxidized) + 2H2O (Abs 500nm Coupled enzyme method is the major method used of analysis for total Ce

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

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3.3 Cholesterol Fractions Will include Ce of HDL, LDL Separation technique employed • • Chemical precipitation using salts and organic solutions Ultracentrifigation (Gold Standard Reference Method)  Took advantage of 2 properties of LPs:      •  by virtue of their lipid content, they have lower densities than other plasma molecules each class of LPs has different density

Chylomicrons and VLDL float, LDL and HDL are sedimented at 1.006kg/L (density) LDL and VLDL both float at 1.063kg/L All the LPs and HDL float at 1.21kg/L The ability to separate LP classes by electrophoresis led to the development of a LP phenotyping classification by D.S. Fredrickson and colleagues   Used in the classification of hyperlipoproteinemia Blood LP patterns in patients with hyperlipoproteinemia TY PE I IIa IIb III IV V LP PATTERN Extremely high TG due to presence of chylomicrons High LDL High LDL and VLDL High Ce, TG; presence of β-VLDL; VLDL-Ce/plasma TG ratio > 0.3 High VLDL High VLDL and presence of chylomicrons

LP electrophoresis

 High resolution gel electrophoresis methods are becoming available providing greater
reliability in determining all LP fractions 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

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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 New methods (more specific methods) Use of antibodies to apoB Ab will remove those fractions (LDL, VLDL, Chylomicrons) that contain apoB

a. Immunoprecipitation Assays

b. Homogeneous Enzymatic Assays     Use Ce oxidase and Ce esterase that are modified by polyethylene glycol These enzymes under specific condition of cation and dextran concentration will react with HDLs only without having to pretreat sample to separate HDL Comparable wit hold method, but has greater susceptibility to interference 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: Assumes that TG/5 is a relatively accurate estimate of VLDL

LDL = Total Ce – (HDL + [TG/5])  Moderately elevated TG levels or the appearance of abnormal LPs will invalidate the results of the calculation resulting in a falsely decreased LDL  New methods to fractionate and directly measure LPL:

a. Immunoprecipitation methods 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)

3.4 Fractions Affecting Plasma Cholesterol Levels
A. Concentration of Ce transporting LP since a high Ce level reflects high LP concentration B. Rate of metabolism of Ce transporting LP C. Biosynthesis capacity and integrity of Ce synthesizing organs and tissues D. Rate of FFA catabolism E. F. Feeding state Endocrine influence a. b. Thyroid hormone    Lowers Ce levels by increasing the number of hepatic LDL receptors Lowers Ce levels by decreasing hepatic catabolism of LPL Men are more prone to atherosclerosis since testosterone has the opposite effect Estrogen

3.5 Outpatient Testing for Cholesterol Variant of enzyme based quantitation A. Dipstick Method B. Cartridge system for separating plasma from cells, followed by measurement of plasma cholesterol

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


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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

4.1 Apo A-I Has been proposed as an alternative to HDL • • Provided that the specimen is fasting, trace mounts of VLDL and chylomicrons are present in this type of sample Reference values:  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 Female: 1.20 – 1.52 g/L  Male: 1.21 – 1.38 g/L

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