Specific determination of free fatty acid in plasma

CLIXIC.4

CHIlLlIC.4 ACTA

SPECIFIC

DETERMINATION

B. J, M. REGOUW*t, J. H. F. SPIJKERS*,

187

OF FREE

FATTY

P. J. H. C. CORNELISSEN**, YVONNE M. M. WEEBER*

ACID

RIET

IN PLASMA

A. P. HELDER**,

*Clinical Chemical Labovatwy3 St. Lamb&us Ziekenhuis. Helmond, and **Clinical Chemical Labovatovy, R.K. Binnenziekenhuis, Eindhoven (The n;ethevlands) (Received

September

21, 1970)

SUMMARY

normal

In view of the diversity in methods and consequently the wide spread of the values mentioned in the literature, we searched for a reference method for

the determination of free fatty acids (FFA). In an attempt to eliminate factors interfering with titrimetric and calorimetric methods, separation with thin-layer chromatography was introduced. After detection with Nile blue and extraction of the FFA fraction, the FFA was determined as a copper complex. The method is described and explained. The normal values found are 200-800 ,umoles/l with a mean of 380 ,umolesjl.

INTRODUCTION

A number

of methods

have been described

for determining

the concentration

of free fatty acids group of the fatty mination by which the metal content

(FFA) in plasma. These methods are based on titration of the acid acids with an alkali in some form1-3 or on a calorimetric deterthe fatty acids are converted to copper or cobalt soaps, whereafter is determined colorimetrically4J. In the method according to Duncombe 4, FFA are extracted from plasma with chloroform; after this the copper salt is prepared with copper nitrate-triethanolamine-acetic acid (Cu-TEA) and finally copper is determined with sodium diethyldithiocarbamate (DDC). Laurel1 and Tibbli@ added methanol to the chloroform to obtain a better extraction and heptane to diminish the specific mass of the organic phase after extraction with Cu-TEA as described by Novaks. The co-extracted phospholipids are removed by shaking the plasma extract with silicic acid, after which the copper soap is prepared with Cu-TEA saturated with sodium chloride at pH 8.1. The copper is determined with diphenylcarbazide. The method described below has been developed, because the determination t

Present address:

Clinical Chemical Laboratory,

Van Dam Ziekenhuis

Rotterdam.

Clin. Chim. Acta, 31 (1971) 187-195

188

REGOUW

et d.

according to Duncombe* does not give reproducible results and the method of Laurel1 and Tibbling5 gives results which are too low, due to adsorption of FFA to silicic acid; however, this will be discussed later. Thus after extraction of FFA from plasma, we purified the extract by means of thin-layer chromatography (TLC) on silica gel. After this, the FFA were converted into copper soaps and the copper determined. REAGENTS

All reagents are p.a. quality (E. Merck, Darmstadt). liquid (CHM). Mix 280 ml chloroform with 2’10 ml heptane and IO ml methanol. SiEica gel Places with fluorescence indicator F 254 (Merck 5715/0025). Solaent (‘CM]. Add 20 ml methanol to 980 ml chloroform. C@$er reaged (Cu-TEA). Mix IO ml 0.5 M copper nitrate (-1 120.7 g Cu(NO,),+3 H,O/l) with IO ml I M triethanolamine (= 149 g (HOCH,CH,),N/l) with 3.5 ml I N NaOH and make up to TOOml with water. In this, dissolve 33 g sodium chloride and adjust the pH to 8.1. Prepare daily. S@ay reagent (NB). Dissolve 80 mg Nile blue chloride in IOOO ml chloroform, Colourin~ reagent (DDC). Dissolve IOO mg sodium diethyldithiocarbamate in TOOml butanol-z. Prepare daily. Stock standad. solution (2000 ,umoles/l). Dissolve 512 mg palmitic acid in IOOO ml chloroform. Working standard s~~ut~~~s (20 and 60 ~rno~esiL). Dilute I ml stock standard solution to 20 ml with CHM and dilute further 5 and 15 ml from this solution to 25 ml. Extraction

METHOD

Collection

of blood

Blood is taken from the fasting patient into a tube containing heparin. After separating the cells from the plasma, the FFA must be determined immediately. Procedwe

All glassware is purified with a dichromate-sulphuric acid mixture. In a test tube with polyethylene cap, 300 ~1 plasma is extracted with 6 ml CHM by shaking during 30 set on a Vortex mixer. After 15 min the tubes are shaken again IO sec. After cent~fugi~lg (5 min, 4000 rev./min) the water and protein layer is sucked off. Five ml from the organic layer is transferred to another tube. To construct the standard curve, hereafter 5 ml of the two working standard solutions are subjected to the same procedure. The tubes with standard, respectively plasma extract are placed in a waterbath of 35” and the contents are dried by a flow of air. The residue is collected at the bottom of the tubes by pouring IOO~1 CM against the wall of the tube and dried by air in the same manner. The residue is taken up in 150 ~1 CM and xoo ~1 are applied on a plate. A few hours before use of the chamber, 50 ml of solvent is poured into the chamber. After migration of the front to 15 cm from the start, chromatography is terminated, the plates are air-dried and sprayed with NB. The plates are examined under illumination with light of 350 nm. By this illumination the plate gives a red fluorescence and the fatty acids are visible by fluorescence C&z. Chim. .4cta, 31 (x97x) 157-195

FFA IN PLASMA

I89

quenching. The FFA extracted from the plasma are identified by the corresponding spot of the standard. The FFA show a RF-value of 0.3. The spots are eluted with 5 ml of CHM by shaking 30 set on a Vortex mixer. After centrifuging (5 min, 4000 rev./min) 4 ml supernatant is transferred to another tube containing 2. ml Cu-TEA. During 5 min the tubes are vigorously shaken horizontally. After centrifuging (5 min, 4000 rev./min) 3 ml of the supernatant is mixed with 0.5 ml DDC and the absorbancy is determined at 437 nm using chloroform as a blank. The flow cell should be rinsed and dried with acetone. RESULTS AND DISCUSSION

A number of extraction liquids have been tested. For the extraction, chloroform and methanol are necessary; addition of methanol gives a better extraction. Duncombed uses only chloroform for the extraction. Addition of petroleum ether (b.p. 40-60” and 60-80”), dichloroethane, benzene, cyclohexane, pyridine, dioxan or dimethylformamide in various amounts to 2% methanol in chloroform gave either a poor extraction or no separation in two layers. We can confirm that for a complete extraction of FFA from plasma the mixture of chloroform and methanol gives the best results, as observed by Laurel1 and Tibbling5. They used for the extraction 2% methanol in chloroform-heptane (4:3 v/v). Laurel1 and Tibblings tried to avoid the adhering disadvantage, the co-extraction of a part of the phospholipids, by absorbing these to silicic acid. By the determination as described by Laurel1 and Tibblings more than 999/o of the phospholipids present was adsorbed to 250 mg silicic acid. However, we have found that a part of the FFA is co-adsorbed, as shown in Fig. I.

Fig. I. Influence of the addition of silicic acid on the standard solutions: O-O acid: A---A with silicic acid.

without silicic

The first curve (I) shows the relation between the concentration of FFA in the standard solution after formation of copper soap and copper-diphenylcarbazide complex and the absorbancy according to Laurel1 and Tibbling6, however without shaking with silicic acid. The second curve (2) shows the results of shaking with 250 mg silicic acid (Sil-R-roe mesh, Sigma). In order to prevent the effect of co-adsorption we Clin.

Chim.

Ada,

31 (1971)

187-195

tried to separate chromatographically the phospholipids from the FFA. During chromatography of the copper salts, the copper complex decomposes, the copper remaining at start, while the FFA migrate. Because of this decomposition it is necessar! to visualize the FFA with another method. Spraying with Sudan III indicates clearly the FFA, but on elution of the FFA, the Sudan III goes over into the eluent. Spraying the plate with NB appeared suitable to detect the FFA. NH is an alkaline dye used as an indicator for fats with an acid character. Influence

of NB

The amount of NB sprayed on the plate has no effect on the determination; it does not show any absorbance at 437 nm (Fig. a), besides XI3 remains ad$orbrd on the silica gel. Some experiments, the results of which are given in Table I, show that spraying the plate with NB has no influence. Both CH and CHM were used as clution liquids. Table I demonstrates that CHM gives a better yield.

350

400

450

500 Wavelength

550 in nm

Fig. 2. Absorbancy curve of Nile blue. TABLE

I

INFLUENCE

OFNILE

BLUE

ON

ABSORBANCY

.mution

FFA

liquid

without NB

with ‘VB

CH CHM

0.136 0.169

0.162

InfEuence

Blank

0.139

mthout IVB

wzth NB

0.062

0.065

0.061

0.065

of plasma

Applying this method on a plasma extract and illuminating the plate with light of 350 nm (Camag lamp) the plate gives a red fluorescence, which is quenched at 4 distinct areas: I. Phospholipids at the start (cf. lecithin). 2. FFA with RF about 0.3 identified with a standard migrating simultaneously. 3. A faint spot with RF about 0.7 probably deriving from di- and triglycerides and cholesterol. 4. A faint spot just behind the front probably deriving from steroids and steroid esters. C&z. Chim.

Acta,

31

(1971)

187-195

FFA IN PLASMA

191

After spraying with NB all spots visible upon illuminating the plate with UV light and the parts of the chromatogram between these spots were removed individually from the plate and eluted with CHM. In each eluate, FFA determination was carried out as described. The results of two of these tests are summarized in Table II. TABLE REVIEW ___-

11 OF THE ____-

VARIOUS

PARTS

OF

THE

CHROMATOGRAM -

0.85 0.95

Abs.

pm&s/l

405

o.ozx 0.096 0.035

330 _

0.033

0.03’

-

0.030 0.032

0.033 0.034 0.040

-

0.119

4oo

0.03’

Phospholipids FFA Intersection Di- and triglycerides and cholesterol Intersection Steroid(esters) Blank Standard 20 ,umoles/l _ 400 pmoles/l plasma

0

0.3 0.5 0.7

fimoles /l

Abs.

spot

RF

0.118

0.116

400

From Table II it appears that the FFA migrate with RF 0.3 and the other substances from the plasma extract have no influence or cannot be eluted from the spot. For better separation between FFA and phospholipids, we preferred CM to CHM as solvent. InJuence of phospholipids There is no loss in the yield of FFA by the TLC separation of FFA and phospholipids. We demonstrated this with a mixture of lecithin and palmitic acid, the concentrations of which correspond to normal and elevated values respectively. The sample of lecithin we used appeared to be chromatographically impure. Beside a heavy spot at the start, there appeared a very faint spot at RF 0.3 which, we assumed, was caused by FFA in the lecithin sample. We prepared the following mixture in CHM : I: zo ,umoles palmitic acid/l N 400 pmoles FFAjl plasma g6 ,umoles lecithin/l N rgzo pmoles phospholipids/l plasma; II : 60 ,umoles palmitic acid/l N IZOO pmoles FFAjl plasma 480 ,umoles lecithin/l N 9600 pmoles phospholipidsj plasma. Table III shows the absorbance of the FFA spots from lecithin alone, palmitic acid alone and a mixture of lecithin and palmitic acid together. All determinations were carried out in triplicate. TABLE SEPARATION

FFA

III OF

spot from

LECITHIN

PALMITIC

ACID

II

I A

Lecithin Palmitic acid Mixture

FROM

&pot

0.056 0.152 O.ilIZ

A blank 0.036 0.036 0.036

A,-Ab 0.020

0.114 0.172

pmoh?S/l AbsBPOt 170 4oo 550

0.072 0.381 0.460

AbSblank

A,-Ab

pmoles/l

0.032 0.032 0.032

0.040 0.349 0.428

270 1200 ‘440

CZin. Chim.

Acta,

31 (1971) 187-195

It is recommended to warm up tlje solution to 25-30” WIWII dissolving chloride to prevent chilling beneatll room temperature, and to prepare freshly. The stability of the Cu-DDC complex appeared to be constant 20 min. measured on a Unicam SP 800.

the sodium the reagent for at least

Standard curve

The relation Clin.

Chim.

A&z,

31

between (197’)

the absorbance

187-195

and the 1;I;A concentration

of the working

FFA IN

PLASMA

standard ,umolesjl

193

solutions

appeared

(corresponding

not

entire]:”

linear.

to zoo ,.umolt~s/l 111plasma)

I’or concentrations tile relation&ip

exceeding IO will tw linear.

1’ and i;ig. 3. This appties for tlhe standard curve, determined directly as weI as after TLC. For low FFA concentrations in plasma, below the normal limit, the determinations will be inaccurate. The extrapolation of this curl.e does not go had similar experiences. I”rolxtbl,P, through the origin. Iwa\~ama’ and I)uncombes the cause of the Nan-iinearit~ is that the formation of the Cu--TEA complex is not completed. Increasing the copper: fatty acid ratio--by raising the volume of the See Table

0.6

Fig. 4. Concentration-absorbance determination with DDC: e---e

curve of palmitic acid after Cu complex formation and Cu direct determination; -;----+ determination by TLC.

INFLUEKCE OF

CU-‘rE.&

AND

TBX

on

VARIOUS

AMOUSTS

the standard

REAGENT

CUYW, tkavacterized

.%I A,

Blank abs. A% A,

Blank abs. Al ‘4

0.0.27 -O.IOl.$ o.orji( 2 ml z-fold COMC.

0.07’ -0.0280 0.0178 2 nd .?$oId cant.

AND

I-3-FOLD

COSCENTRATIOSS

OF COPPER

I’ = .4,~r A,X. 6 ~1 i-foid cm1c.

? ml r-fold cont.

Blank abs.

by

0.053 -0.0120 O.OI.$l

4 vzi z-fold

6 n?E r-fold

EOIG.

cnnc.

0.067 -0.0225

0.092 -0.0216 0.0182

O.OI&

o.rr3 -0.0210 O.OZO‘& _-~___-

__I

-.-__-_.. Ch.

&him.

ACta,

31 (1971)

187-195

REGOCTW et

194

(11.

copper-TEA reagent or by increasing the copper-TEA concentration in a constant volume-shifts the standard curve in the direction of the origin. Table VI shows these influences. The curves are characterized by the formula: Y = ,4.+11,X. From Table \‘I it appears that the composition of the Cu-TEA reagent ac-

l;ig. 5. Absorbance of the Cu-UDC Cu-FFA complex formation.

complex

after extraction

of various amounts of plasma and

IO i

pmoles FFAjl Fig. 6. Values qf the FIT:\ concentration Clin. Chim. Acta, jr (1971) 187-195

in plasma of healthy adults.

FFA

IN PLASMA

I95

cording to Laurel1 and Tibbli@, used in this study, is not quite optimal. The greater part of these experiments were already concluded however, when we came to this conclusion. The relationship between various amounts of extracted plasma and the absorbance is demonstrated in Table VII and Fig. 5. Remarkable is the complete linearity of the curve in Fig. 5, and in this respect it is different from the standard curve.The reason of this discrepancy might be caused by the difference in handling the standard and the sample and the character of these liquids. Because position and slope of the standard curves are not always exactly the same in spite of all precautions, it is recommendable to plot these standard curves each time separately.

The FFA value of 40 normal adults varied from zoo to 800 pmoles/l, average value of 410,umoles/l and a mean value of 380 @moles/l (Fig. 6). The determinations were carried out in triplicate in plasma.

with an

REFERENCES

I V. P. DOLE, J. C/in. Invest., 35 (1956) 150. L D. L. TROUT, E. H. ESTES AND S. J. FRIEDBERG, J. Lipid&s., I (1960) 199. 3 J. KEUL, N. LINSET AND E. ESCHENBRUCH, Z. Klzn. Chem. Klin.Biochem.. 5 (1968) 394. 4 U’. G. DUXCOMBE, Clin. Chim. Acta, 9 (1964) 122. 5 S. LAURELL ASD G. TIBBLING, Clin.Chim. Acta. 16 (1967) 57. 6 ill. NOVAK. J. Lipid Res., 6 (1965) 431. 7 ‘I’. IWAYAMA. J. Pharm. Sot. Japan. 79 (1959) 552. C2in. Chim. Acta, 31 (1971) 187-195