FISHERIES AND MARINE SERVICE 4WCHIVES!,

Translation Series No. 3230

Quantitative determination of total lipids

by Iwao Fukui, and Hideto Kushiro

Original title: (Ketsueki.Soshikichu no Shishitsu no Teiryoho) Soshishitsu

From: Rinsho-Byori: Rinji-Zokan-Tokushu Dai 19-Go, Showa 47-Nen, 1-Gatsu: Bessatsu (Special Supplement, Clinical Pathology), 19(special suppl.) : 38-51, 1972

Translated by the Translation Bureau(FRF/PS) Multilingual. Services Division

Department of the Secretary of State of Canada

Department of the Environment Fisheries and Marine Service

Halifax Laboratory Halifax, N.S.

1974

27, pages typescript

INTO - EN

English Fe.4 .gG23o

SECRÉTARIAT D'ÉTAT

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TRANSLATED FROM - TRADUCTION DE

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AUTFIOR - AUTEUR

Iwao FUKUI & Hideto KUSHIRO

TITLE IN ENGLISH - TITRE ANGLAIS

Quantitative Determination of Total Lipids

TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÈRE (TRANSCRIRE EN CARACTERES ROMAINS)

(Ketsueki.Soshikichu no Shishitsu no Teiryoho) Soshishitsu

REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS. RÉFÈRENCE EN LANGUE ÉTRANGÈRE (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTë.RES ROMAINS.

Rinsho-Byori: Rinji-Zokan-Tokushu Dai 19-Go, Showa 47-Nen, 1-Gatsu: Bessatsu, •

REFERENCE IN ENGLISH - RÉFiRENCE EN ANGLAIS

Special Supplement, Clinical Pathology, .19, January (1972)

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Quantitative Determination of Total Lipids

by Iwao Fukui and Hideto Kushiro *

I Introduction

Lipids are generally defined as organic substances which

(1) are soluble in organic solvents but more or less insoluble in

water, (2) form, or are able to form, fatty acids and esters, and

(3) are utilized by living systems. Like proteins or saccharides

they are not characterized by a definite chemical structure, but

include various substances of differing chemical structure.

Blood serum lipids include (1) cholesterol (ester form or

free acid form), (2) triglycerides (also called neutral lipids.

A small quantity of monoglyceride and diglyceride is also present.

(3) phosphatides, (4) free fatty acids, and (5) small amounts of

other fat soluble substances. The sum total of all these compon-

ent lipids is known as the total lipids.

* Central Laboratory for Clinical Investigation, Kyoto Prefectural University School of Medicine, Kyoto.

'S 0S-200-4 0.-31

) ,

2.

In the days when there were no precise methods of determ-

ining the individual component lipids, the measurement of blood

serum total lipids was carried out because of its appreciable

clinical significance. Also, the measurement of total lipids,

cholesterol, and phosphatide was used to calculate the triglycer-

ide values. However, since the sum total of the individual

component lipids encompasses not only varying chemical structure,

as stated above, but also varying physiological action and metab-

olism, the clinical significance of such determinations is obscure.

For example, it is sometimes observed that if one component

increases some other component may decrease, so that the total

lipids remains unchanged. The result is that at the present time,

when progress has been made in the determination of individual

component lipids, there is a gradual trend away from measuring

total lipids.

Nevertheless, a knowledge of blood serum total lipids is

not without value, a decrease in particular being quite signific-

ant clinically. Also, of late, there has been a revived develop-

ment of lipo-protein separation methods as a result of the simplic-

ity of the electrophoretic method. Such measurements are now

being combined with total lipid values in the determination of

the amount of each lipo-protein.

This paper will discuss the methods of measuring blood

serum total lipids and, in addition, will touch upon the measure-

ment of tissue total lipids.

II Blood Serum Lipids

As stated above, the lipids found in blood serum consist of

cholesterol (ester form and free acid form), phospholipids

(lecithin, cephalin, sphingomyelin, etc.), triglycerides, free

3.

fatty acids, and small amounts of fat soluble substances (vita-

mins A, D, E, and K, carotinoid, cerebroside, sterols, etc.). P39

The amounts of the components are: cholesterol 130-250 mg/di,

phospholipids 125-300 mg/di, triglycerides 29-134 mg/di, free

fatty acids 10-15 mg/di. Total lipids range from 450-1000 mg/di (1)

Although these lipids are insoluble in water, they combine

with proteins in the blood serum to exist in solution as

lipo-proteins. Cholesterol, phospholipids, and triglycerides

combine with globulin while free fatty acids combine with

albumin.

The lipids are reported to combine with the proteins in a

great number of ways, inc1uding covalent bonding, electrovalent

bonding, hydrogen bonding, and hydrophobic bonding (2) .

III Methods of Determining Serum Total Lipids

The methods presently being used to determine serum total

lipids can be classified as follows: (1) Gravimetric Method,

. (2) Oxidimetric Method, (3) Comparative Turbidity Method,

(4) Volumetric Method, (5) Dye Method, and (6) Colorimetric

Method based on the Sulfo-phospho-vanillin reaction. In addition,

numerous variations and improvements of the above methods are

being employed. A seventh, indirect method is also used in

which the total lipids are obtained from summing the individual

measurements of the total cholesterol, phospholipids, and

triglycerides.

A. Gravimetric Method

In this method the lipids are extracted from the serum

using an'organic solvent, refin -ed, and determined by weighing

with a chemical balance. This method is regarded as the standard

method of determining total lipids. Several variations in the

extracting and refining steps are employed.

As stated above, the serum lipids combine with proteins

to form lipo-proteins. It is reported that water molecules

participate in the bonding (3) . Polar solvents such as methanol,

ethanol, and acetone, which exert a dehydrating effect, are

employed in the extraction in order to sever these lipid-protein

bonds. Because serum also contains some lipids which are difficult

to extract, mixtures containing non-polar solvents such as

chloroform, ether, etc. are also used.

Some of the more common mixed extracting solvents are (1)

Bloor's solvent (ethanol:ether.-_-_, 3:1 v/v %) (4) , (2) Folch's

solvent (chloroform:methanol 2:1 v/v %) (5) , and (3) acetone:

ethanol ... 1:1 v/v % solution (6)

When the extraction is carried out using Bloor's solvent,

a solvent to serum ratio of 20:1 gives the greatest lipid

extracting power. However, this solvent will also extract non-

lipids such as grape sugar, urea, creatine, and glutathione,

as well as inorganic salts, and for this reason further refining

is necessary in order to remove these substances.

For the refining, Wilson (10) , Street (11) , Pernokis (9)

and Jacobs (12) used petroleum ether, while Kien et al(13) used

chloroform. When the total lipids are extracted using Folch's

solvent (chloroform.methanol), the non-lipids are removed by

washing with water (5 ' 14) . The lipid extraction is complete and

the lipids undergo no decomposition during refining. Further, the

removal of the non-lipids is also complete. This is said to be

a most superior extraction-refining method, and has been applied

to serum lipids by Sperry et al(15).

Sperry's method, however, lacks the speed necessary for

the everyday clinical examination of many patients. To overcome

this, the method has been simplified by Albrink(16), Carlson(17)

and Bragdon et al(18). Bragdon's method is particularlÿ simple:

Folch's solvent is added to the serum, then, without filtering,

dilute sulphuric acid is added: Upon standing the mixture

separates into two layers. The bottom layer is then taken and

quantitatively analyzed.

p409

B, Oxidime_tric Method

In the oxidimetric method the lipids are oxidized by bichrom-

ate. The customary procedure consists of the following four steps:

(1) extraction-refining, (2) saponification, (3) oxidation, and

(4) titration or colorimetric determination.

In this method the bichromate not consumed In the oxid-

ation is generally dealt with in one of two ways: (1) titration

with iodine, as employed.by Bang(19), Bloor(20), Katsura(2i),

2 )and Iwatsuru et a1(2 ,. or (2) colorimetric determination of

the chrome alum produced by regenerating the chromate with lipid,

(18)as employed by Bloor (23) and Bragdon et al

The colorimetric method is more convenient than the titri-

metric method. In Bragdon's method not only are the extraction

and refining steps simple, but the saponification step has been

omitted, so that this method is used in everyday testing.

C. Comparative Turbidity Method

Lipids produce a turbidness in a. liqùid mixture of a hydro-

philic solvent and watcr. Based on this principle, De La Huerga(24)

W- V

6.

devised the comparative turbidity method. In this method the

serum lipids are extracted with Bloor's solvent, followed by

evaporation of the solvent. Turbidity is induced by dissolving

the residue in p-dioxane and adding 4% sulphuric acid. However,

,a weakness in this method has been pointed out by Davis et al(25)

who claim that differences in turbidity can be observed depending

on the type of lipid.

In addition, there is a simple phenol turbidity test(26) in

which phenol reagent ( an aqueous sodium chloride solution of

carbolic acid) is added directly to serum, giving an overall

value for total serum lipids.

D. Volumetric Method

This is a method in which the lipids are separated by the

use of a fat separating agent, and their volume measured.

Fat separating agents used in this method include: (1) a

fat soluble dye, 70% sulphuric acid, and isoamyl alcohol as

used by RLfckert(27) and Hermann et al(28), (2) sodium carbonate,

sodium hydroxide, sodium salicylate, and butanol as used by

Allen (29) , and (3) a surface active agent as used by Brandstein

et al(30). Generally speaking, this method is seldom used.

E. Dye Method

Lipo®proteins can be analyzed quantitatively by colouring

with fat soluble dyes such as Sudan Black B and Oil Red 0.

According to Swahn's method(31), the serum total lipids can be

determined from a 0.02 ml sample of blood serum.

tki

7.

F. Sulfo-phospho-vanillin Colorimetric Method

In this method sulphuric acid is added to the lipid and

heated, after which the mixture is reacted with phosphoric acid

and vanillin. The Sulfo-phospho-vanillin reaction (32,33•34)

develops a pink colour which is measured colorimetrically. This

is a two step method: (1) sulfonation of the lipid by sulphuric

acid, plus (2) generation of colour by the phosphoric acid-

vanillin reagent. It is a direct method, and does not require

an extraction step.

Frings (35) and Wada et al (36) have used this method. In

addition, Okuda (37) and Matsumiya et al (38) have "miniaturized"

and simplified the procedure. The usefulness of this method is

presently receiving attention.

IV Details of the Quantitative Methods of Analysis of Serum Total Lipids

This section will discuss the details of somè of the

methods of quantitatively determining serum total lipids.

Bragdon's method (18) will be chosen to represent the gravimetric

method, which is regarded as the standard method. Bragdon's

method of oxidimetric measurement (18) will also be discussed.

In addition, Frings' method using the Sulfe-phospho-vanillin

reaction (35) will be chosen as one of the more recent direct

methods requiring no extraction, which have lately been receiving

attention.

A. Braeon's Gravimetric Method (18)

1. Method

Blood serum lipids are treated with Folch's solvent and

p.41

8.

dilute sulphuric acid and extracted and refined according to

Bragdon's extraction method. The solvent is then evaporated and

the residue weighed.

2. Reagents* 1

a. Folch's Solvent

Mix 2 volumes of chloroform with 1 volume of methanol.

b. Dilute Sulphuric Acid

'Dilute 1.0 ml of concentrated sulphuric acid with water to

make 2 liters of solution.

3. Procedure

a. Extraction

To a stoppered centrifuge tube (50 ml capacity, level mark

at 25.0 ml) add 1.0 ml of serum. Vigorously add about 22 ml of

Folch's solvent*2 and, without shaking, let stand for 5 minutes.

After shaking and mixing for 30 seconds, add Folch's solvent up

to the marker line and again let stand for 5 minutes. Next, add-

ing 5 ml of dilute sulphuric acid, mix for 10 minutes by gently

inverting the tube, followed by 10 more minutes of standing.

Next, centrifuge the mixture for 15 minutes at 2000 rpm.

Remove the upper liquid layer with a suction aspirator and place

15.0 ml of the lower, chloroform layer*3

into a 20 ml erlenmeyer

flask (accurately weighed at B mg). The lower liquid layer usually

amounts to about 18.0 ml.

*1 All reagents used were special grade JIS (Japan Industrial Standard). *2 Added with vigor to disperse the serum proteins as fine part-ides.

4" Since non-lipid bilirubin is also extracted in the lower liquid layer, in jaundiced blood sfrum the bilirubin value should, strict7y speakIng, be subtracted to give the serin total lipid ' val.

9.

b. Evaporative Dry_iiu

The erlenmeyer flask is immersed in a 700C hot water bath and

the solvent evaporo.tedy`4. The final remaining chloroform vapour

is completely removed using a pipette attached to an aspirator.

c. tiAleighing

The outer wall of the erlenmeyer flask is then rinsed with

distilled water and dried with clean gauze. The flask is next

placed in a CaC12 dessicator which is then evacuated with a vacuum

pump. After 2 hours the flask is weighed (A mg) using a chemical

balance.

4. Calculations

Serum total lipids (mg/dl) - (A-B)x^xi00

Since the 15.0 ml of extracted solvent contains the serum total

lipids in 15/18 ml of serum, the above equation gives the serum

total lipids in 100 ml of serum.

5. Discussion

a. Accuracv of -the Détermination

The gravimetric method involves extraction, refining, and

two weighings. Problems concerning the accuracy of the method

reside in the extraction and refining steps.

Although Bloor's solvent(4) yields good results(7'8) in

the extraction used to separate the lipo-protein-form serum

lipids from the proteins, non-lipid substances also present in

the serum are extracted at the same time(8'9), as stated above.

p42

Ideally, in order to avoid oxidative decomposition of the lipids,'the evaporation should be carried out under nitrogen or carbon

dioxide. However, no changes in measüred values have beenreported in the literature for failure to do this.

■-■•■•■-uà

10.

Therefore it is necessary to remove these non-lipid substances by

refining with petroleum.ether (9-12)

According to Jacobs' investigation (12) , refining with

petroleum ether does not exclude urea, uric acid, amino acids,

and sodium chloride; moreover, it has the adverse effect of causing

losses of the phosipho-lipids. This is influenced by the temperat-

ure (39) and the extent of evaporation (12) during the evaporation

of the Bloor's solvent.

For these reasons, it is difficult to equate values

obtained by extracting serum total lipids with Bloor's solvent

followed by refining with petroleum ether with the true value.

However, taking Jacobs' work as representative of this method, it

is found that the positive error in the non-lipid substances

cancels out the negative error in the phospho-lipids, so that it

may be said (12) that this method does in fact yield results in

close agreement with the values obtained using the superior

Sperry extraction method (15)

Bragdon's extraction method (18) , which has been chosen here,

is a simplification of Sperry's method (15) , which in turn is

based on Folch's method (5 ' 14) In Bragdon's method, dilute

sulphuric acid instead of water is used for the washing to ensure

the complete extraction of the fatty acids. However, according

to Albrink's investigations (16) , even when water is used there

is no difference in the extracted total lipids, so that both

Sperry's method and Bragdon's method may be said to yield the same

accuracy.

11.

b. Reproducibility of the Method

Using a Mettler direct-reading chemical balance (Model 1116,

weighing error + 0.01 mg) the authors carried out replicate

determinations (n=7) using this method. The average value was

7 823 mg/di, with standard deviation S.D. = + 31 mg/di, and

coefficient of variation C.V. = + 3.8%.

Comparing this with a coefficient of variation C.V. =± 11.0%

for Jacobs' method (15) it would appear that the superior

reproducibility of the present method is attributable to the

simplified and more stable extraction and refining steps.

B. BrmdmIsidirlietric Method (18)

1. Method

In this method, the refining-extraction is carried out

using Folch's solvent (CHC1 3 :CH3OH = 2:1 v/v %) and dilute sulph-

uric acid, as in the Bragdon extraction method, followed by evapor-

ation of the solvent .

Next, a potassium bichromate.sulphuric acid solution is

added . Upon heating, the lipids reduce the potassium bichromate

(K2Cr20 7 , reddish brown) to chrome alum (K2Cr2 (SO4)4, dark brown),

which is analyzed colorimetrically to yield indirectly the amount

of lipid. The reaction is as follows:

3 C 1 e31 00011 + 184 H2s04 + 46 K2 Cr207 (palmitic acid) (reddish brown)

46 K2cr2 (So- )1. 4 + 232 1120 + 48 C O2 t (dark brown)

*5 2. Reagents ' •

a. Folch's Solvent (discussed above)

*5 All reagents used were special grade JIS.

>es

p43

12.

t

b. Dilute Sulphuric Acid (discussed above)

c. Potassium Bichromate

Twenty grains of potassium bichromate is dissolved in 1

liter of concentrated sulphuric acid*6 . The reagent is stored in

a brown bottle to exclude light, and will keep for one month at

room temperature.

d. Standard Solution

Dissolve 50 mg of palmitic acid in chloroform and dilute

with chloroform to 100 ml.

3. Procedure

a. Extraction

The same procedure as in the extraction step of Bragdon's

gravimetric method is followed.

b. Ev:corative Drying

Two ml aliquots each of extracted solution, of standard solution and

of chloroform "to be used for testing blindness"* are placed in hardened

test tubes, which are then immersed in a 70°C hot water bath to evaporate

the solvent. The remaining chloroform vapour is completely removed

using a pipette attached to an aspirator*7

.

• c. Oxidation

Add 4.0 ml of potassium bichromate solution to each test

tube, stopper and heat in a 1000 0 boiling water bath for 30 minutes.

d. Colorimetry

After cooling with running water, add 6.0 ml of water and

stir with a glass stirring rod. After again cooling with

*6 Dissolve by heating in water.

*7 If the solvent is not completely removed, K 2Cr207 will be con-sumed in its oxidation, leading to a positive ert.or.

* T.M.: literal translation; "blindness" could also be translated as "ignorance"

13.

running water, analyze colorimetrically at a wavelength of 580 mil.

. 4. Calcula.tions

la Serum total lipids (mg/di) = absorbance of sample x1x7 x 100 absorb , of stand. sol'n

absorbance of sample x 900 absorbance of standard sol'n

Two ml of the standard solution contains 1.0 mg of palmitic

acid, while the 2.0 ml of extract solution contains the total

lipids in 2/18 ml of serum, hence, using the above equation, the

total lipids in 100 ml of blood serum can be determined.

5. Discussion

a. Abq.P..E.P.ILM_P.21EY!

The abSorption curve for this method is shown in Figure 1 (a).

It can be seen that the maximum absorption ( X max ) occurs at

600mp.

b. Calibration Curve

As seen from Figure 1(b), the calibration curve for this

method is a straight line passing through the origin.

c. Reproducibility of the Method

Replicate determinations (n = 6) carried out by the authors

for this method gave an average value of 7 = 745 mg/dl, standard

deviation S.D. = + 31 mg/di, and coefficient of variation C.V. =

+ 4.2%.

d. Standard Solution

The accuracy of the values obtained using the oxidimetric

method has the same limitations ab found for the extraction and

refining steps of the previously discussed Bragdon gravimetric

method, except that now, in addition, an oxidation step must

14.

O.D.

.200

.150

.100

.050

450 900 (mg/dl)

Seruni Total Lipides

also be taken into consideration.

In the oxidation step, a different amount of K2Cr2 07 is

reduced by a given amount of each lipid contained in the serum.

However, according to Bragdon (40 ,) , the -mount of K2Cr2 07

reduced by 1.0 mg of the various lipids in the serum is:

cholesterol 19.1 mg, triglyceride 17.7 mg, phospholipid 15.2 mg,

and palmitic acid 17.6 mg. Thus 1 mg of serum total lipids

reduces an amount of K2Cr207 which is extremely close to the

amount reduced by 1 mg of palmitic acid. Accordingly, it is

claimed that the use of palmitic acid as the standard substance

does not impair the accuracy of the determination.

_L. 650

Fig. 1(a) Absorption Curve for Bragdon Oxidimetric Method

Fig. 1(b) Calibration Curve for Bragdon Oxidimetric Method (Shimatsu SP-20, 580 m/.4 )

C. Colorimetrï.c Method Using the Sulfo-phospho-vanillinReact_i_cin ( T{`rings I Method) (35

1. Me th od

)

Serum lipids are sulphonated by adding sulphuric acid and

heating. Next, phosphoric acid.vanillin reagent is added, where-

upon the so-called Su1fo--phospho-vanillin reaction occurs. The

total lipids are obtained by colorimetrically analyzing the piri.k

colour so produced.

2. Reagents"

a. Concentrated Sulphuric Acid

b. Aqueous Vanillin Solution (0.6 w/v /),F9

Dissolve 1.2 g vanillin in water and dilute to 200 ml.

c. Phosphoric Acid.Va.nillin Reagent*10^_ - _.._._....__ .^..._...-.

Add 200 ml of aqueous vanillin solution to a 2-liter erlen-

meyer flask. Add 800 ml of 85% phosphoric acid with.stirring. If

kept in a brown bottle, this reagent will keep for six weeks at

room temperature.

d. Standard Solution (500 mg/dl)

Weigh out 500 mg of olive oil and dilute to 100 ml with

ethanol.

Standard serum (i.e. serum in which the total lipids is

known) can also be used.

*8 All reagents used were special grade JIS.

*9 0.6 w/v % is the optimum concentration,

*10 The more concentrated the phosphoric acid, the more intensethe colour.

15.

p45

1 6. J

3. Procedure

11 a. Sulphonation*

Place 0.1 ml each of serum, of standard solution and of ethanol

to be used for testing blindness* in hardened test tubes. To each add

2.0 ml of concentrated sulphuric acid and heat for 10 minutes in a 100 0 0

boiling water bath. After cooling under running water for 5 minutes,

transfer 0.1 ml of each mixture to a hardened test tube.

b. Sulfo-phomhafnnillin Reaction*12

Add 5.0 ml of phosphoric acid.vanillin reagent to each

test tube, mix well, and react at room temperature for 15 minutes.

c. Colorimetry

Analyze colorimetrically at a wavelength of 540 mp.

4. Calculations

absorbance of Samp l e Serum total lipids (mg/di) e2» absorbance of standard sol'n x 500

5. Discussion

a. Sulfo-phospho-vanillin Reaction

Although the details of the mechanism of this reaction are

still uncertain, the reaction is believed,to originate in the

unsaturated bonds of the lipid components (33) . The colour may

be produced by reactions between ketonized lipid fragments and

aromatic aldehydes, formed as decomposition products or perhaps

through a peroxide formation process, etc.

*11 Under the conditions of this method, the sulphonation is complete after 10 minutes of heating at 100'C.

*12 The colour is stable at room temperature for 15-30 minutes, after which it slowly fades.

see T.N.'S p. 12

O.D. O.D.

b. Absorption Curve

Figure 2(a) shows the absorption curve for the coloured

solution produced in this method. It can be seen that the max-

imum absorbanee (>\ max ) occurs at 530 mp, but, since the absorb-

ance of the blank reagent was low at 540 mp, this latter is the

wavelength employed.

c. Lirit

Figure 2(h) shows the calibration curve obtained for this

method. It can be seen that the line is linear up to serum total

lipids = 1000 mg/di, and passes through the origin.

17.

.100

.400

.300 .050

.200

520 540 560 (mp

Fig. 2(a) Absorption Curve for SPV Method (Frings Method)

Fig. 2(h) Calibration Curve for SPV Method (Frings' Method) (Shimatsu SP-20, 540 rnAt )

500 560 600

.100

I

100 300 500 1000 (mg/d/) Serum Total Lipides

d. Reproducibility of the Method

Simultaneous determinations (n = 7) using this method galie

the following values: average value 7 . 485 mg/di, standard

deviation S.D. = + 18 mg/di, and coefficient of variation

C.V. = +

18.

e. Standard Solution

For each lipid, the strength of the absorbance (molar

absorption coefficient) produced through the Sulfo-phospho-van-

illin reaction is different: cholesterol 5.63 x 10 3 , cholesterol

oleate 1.14 x 10 lecithin 7.75 X 103 , triolein 1.65 x 104 ,

oleic acid 5.27 x 10 3 , and palmitic acid 1.08 x 10 3 . The colour

intensity with the saturated fatty acids is particularly low,

and the choice of standard solution becomes a problem. Olive

oil has been chosen as standard substance because gas chromatog-

raphy has revealed that its fatty acid structure is analogous

to that of human blood serum (38) .

The use of control serum of known concentration as

standard solution instead of olive oil is said to improve the

method even further (37)

V Comparison of Serum Total Lipid Values Obtained bm Bragdon Gravimetric Me_tholl_llragdon Oxidimetric Method, and Sulfo-phos ho-vanillin Colorimetric Method (Frings' Method)

Bragdon's Gravimetric Method (18) , Bragdon's Oxidimetric

Method (18), and Frings' Sulfo-phospho-vanillin(SPV) Method (35)

have been selected as quantitative methods of analyzing serum

total lipids, and have been discussed in detail above. These

three methods were used concurrently to determine, in duplicate,

the serum total lipids values for 33 samples of blood serum.

Figures 3(a), (b), and (c) compare the results obtained.

As already discussed, the reproducibility of the three

methods was as follows: Bragdon Gravimetric Method C.V. = + 3.8%,

Bragdon Oxidimetric Method C.V. + 4.2%, Sulfo-phospho-vanillin

Colorimetric Method C.V. = + 3.7%. -

S

•

Oxidimetric 'Method

( B ragdon;

(mg/dl)1200

I

19.

Gravimetric Method Gravi:netric Method

( Sragdon ) (Bragdon)t mg/dl )l (mg/dl i

1200

1000

800

600

400

200

1000

800

.600

400

200

n=33

'x=657 mg/d!

37730 mg/d!

r=0.96

12001-

1000

S00

600

400

200

I I I i i i200 400 600 800 1000 1200 (mg%dl)

Oxidimetric Method (Bragdon)

e

n=33

z=.517mg/d1

V=657mg/dl

r=0.97

I I ^ f

200 400 600 800 1000 1200 (m g•'d/)SPV•Ntethod 'Frings• ,

Figure 3(c)

.n=33z=517mg/df

j'=730mg/dl

r=0.90

I I L LL

200 .400 600 800 500 1200 (m g!dl)

SPV-Tvrethod ( FrinV)

Figure 3(b)

The correlation coefficient between the Bragdon gravimetric

method and the Bragdon oxidimetric method was ^- 0.96, and that

between the Bragdon gravimetric method and the SPV method was

'K _ 0.90, while that between the Bragdon oxidimetric method and

the SPV method was ^ = 0.97.

Although the correlation coefficients among the three

methods were in good agreement, disparities were evident in the

average values: Bragdon gra virlletric method - 730 mg/dl, Bragdon

Figure 3(a)

20.

oxidimetric method .- 657 mg/dl, SPV method = 517 mg/dl.

If the serum total lipid values obtained using the gravi-

metric method are taken as the standard, then, owing to the

variety of lipids present in serum, it will be a problem choosing

a standard substance which gives the same values when the oxidi-

metric or SPV method are employed.

VI Normal Values for. Serum Total. Lipids

The more important data published in the literature(41-45)

for the normal values of serum total lipids are shown below in

Table 1:

Table 1 Normal Values of SerumTotal Lipids

Researcher Normal Values mg/dl

1. Shuko NIT0411•93 - 841

2. Susumu SHTBATA(42) 320 - 620

3 . Tadao YASUGI et a1(43) 500 - 600*

4. Henry, R. J.(1) 450 - 1000

5. Fredrickson, D. S.(44) 400 - 1000

6. Cantarow, A. (45) 385 - 675

* over 40 years of age

It can be seen that although there is not a great deal of

variation in the lower limits of these values, the upper limits

reported by Henry() and Fredrickson(44) have a tendency to be

high, giving rise to a rather large range of values.

The serum total lipids in new-born infants is about

100-250 mg/dl. The value doubles within a few days, and within

a year is the same as the value found in adults(46'47) .

p47

r ...._

■ • 21.

The serum total lipids increases with the age of the

individual (48 ' 69) ; the rate of increase is especially rapid above

the age of 40 (35) . After the age of about 65-70, however, the

change reverses and there is a decrease in serum total lipids (50) .

Furthermore, it is reported that there are no differences

based on sex distinction (49 ' 51)

The value increases if lipid-containing foods are eaten (52) .

This phenomenon is due to the increase in triglycerides. Accord-

ingly, as a general rule, when determining the serum total lipids

the blood sample is taken when the patient has not eaten for

12-14 hours, usually in the early morning,

VII Determination of Tissue Total Lipids

Tissue total lipids are classified according to the

difficulty of extraction with a fat solvent, into (i) lipids

which are easily dissolved by solvent and extracted, and (ii) lipid

substances (bonded lipids) which are first dissolved in aqueous

acids or alkalis, then extracted with solvents.

In most tissue, 96-97% of the lipids are of the first type,

while the remaining 3-4% are of the second type. The value of

the first type is often considered acceptable as representing

the total lipids.

In the extraction of tissue total lipids, usually 3-5

times as much Folch's solvent as tissue is used (for enzyme

solutions, 20 times the volume of homogenate).

The extraction of tissue lipids is best carried out at

room temperature (25-2eC) or, at the very highest, at less than 35 ° C

When a chloroform.methanol mixture is used, a sufficiently

effective extraction can be carried ou-qat these temperatures, with

22.

relatively little destruction of the lipid components (2 ' 55)

The extract will still contain a fair amount of non-lipid

impurities. The non-lipids present differ with the tissue and

with the individual lipids in the tissue. Accordingly, researchers

have devised various means to remove these impurities, but the

most common methods are Folch's washing method (14,54,55) and

the chromatographic method (56 sic )

In Folch's washing method (2155) 0,2 volumes of water (or

commonly, a dilute salt solution) is added to each volume of the

mixed (2:1) chloroform:methanol extract and shaken well. After

standing for some time, the mixture separates into two layers

(more accurately, three layers if the "fluff" layer at the boundary

is included). The upper layer, which contains the non-lipids,

is discarded, and the total lipids are collected from the lower,

chloroform layer.

Chromatography (using a column or paper chromatography)

is also employed for the same purpose.

After refining, the total lipids are determined using the

gravimetric method.

Concerning the lipids in tissue, the main objective after

the extraction and refining is usually not so much to obtain the

total lipids as it is to re-use the extracting solution to its

fullest capacity in separating out lipids, so that quantitative

total lipid determination methods are much less frequently

applied to tissue than to serum.

For want of space here, the reader is referred to the

literature (14 ' 54-56 sic ) for more details.

-)

fr 2 3.

VIII Conclusions

The above discussion centred primarily around the quantit-

ative analysis of serum total lipids. It was explained that the

serum total lipids consists of the sum total of the component

lipids, and that because the information and clinical significance

gained therefrom is not clearly defined, the determination of total

lipids is gradually falling into disuse.

The extraction and refining differ, in the strict sense

of the word, with the method of determination. The extent of

potassium bichromate reduction depends on which lipids are present:

the extent of the Sulfo-phospho-vanillin reaction is not exactly

identical; differences occur depending on the standard substance

used in the determination. For these and similar reasons, great

care must be exercised when using these methods. It is, none-

theless, believed that the total lipids is more meaningful than

the sum of the component lipids. Accordingly, it is necessary

. to read the measured values of these things with a considerable

amount of careful thought.

(The authors wish to thank Yoko Mizuguchi, B. Pharmacol., of

the Clinical Investigation Section of the Biochemistry Department

of this university, who supervised the experiments mentioned near

the end of the manuscript.)

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