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Automated Colorimetric Determ ination of FreeFatty Acids in B io logic F luidsC . D alto n and C. Kowaiski

F or th e ro utin e d ete rm in atio n o f fre e fa tty acids (FFA ) the authors recommend amodific atio n o f th e a utomate d c olo rim e tric m e th od o f A nto nis (1 ) u sin g th e e xtra c-tion procedu re of ltaya and U i (2). T his d ire ct e xtra ctio n a nd a utomate d a na ly siss how s g oo d c orre la tio n w ith T ro uts (3 ) modificatio n of D oles (4) titrimetricprocedure.

HE MOST FREQUENTLY USED procedure for the determ ination of freefatty acids (FFA) in body fluids or bio logic m edia is that of Dole (4).This method is based on the ex traction of fatty acids w ith organic sol-vents fo llowed by titration w ith d ilu te alkali. D espite improvements inspecificity (3, 5 , 6), th is method is subject to error resulting from dis-solved atm ospheric carbon dioxide. W hen applied to large numbers ofsamples, the difficulty of endpoint detection in a two-phase systemm akes it laborious.

Colorimetric methods for long-chain FFA determination stem fromt ho se d ev is ed by Ayers (7 ) and Iw aya ma (8), who measured th e coppersoaps formed in chloroform solution upon shaking a solution of potas-sium soaps with copper nitrate. Although Duncombe (9 , 10 ) improvedth e sensitivity of th e colorimetric procedure by complexing th e coppersoap with sodium diethyldithiocarbamate, th e method was still no t sat-isfactory for the determ ination of plasma FF1 due to interference byphospholipids.

A number of extraction procedures have been proposed to elim inateth is phospholipid in terference. In Anstall and Trujillo s modification(11), an acetone-magnesium chloride precipitation was used; A .ntonis(1 ) used a silicic acid-d iisopropyl ether slurry in h is automated pro-cedure; and Kyam et al. (12) used a slight m odification (hexane rather

From the Department of Pharmacology, Research D ivision, Hoffmann-La Roche, Inc., Nutley,N . J. 07110.

The authors wish to thank D r. D . E. M aynard , Department of C linical Pharmacology,Hoffman-La Roche Inc. for his help in preparing this manuscript.Received for publication Nov. 10 , ]966; accepted for publication Jan. 23 , 1967.

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Vol. 13, N o. 9, 1967 FREE FA1T( AC IDS 745than heptane), of Doles (4) extraction technic. In all these procedures,t he p hos pho lip id- fre e ext rac t must he evaporated and redissolved inchloroform , which becom es cumbersome w ith large numbers of samples.Recently, the extraction procedure of itaya and U i (2 ) u sing pho sphatebuffer (pH 6-7) was adapted to I)uncombes procedure (9, 10 ) to elim -inate phospholipids. This approach is direct and allows the easiesthandling of routine colorim etric FFA analysis. This paper w ill showthe utility of combining the extraction procedure of Itaya and U i (2 )w ith tile automated colorim etric method of A lltoflis (1).

ReagentsChloroform A .R .. grade, from freshly opened or tightly sealed bot-

tles so that evaporation is prevented . This m inim izes the concentrationof contam inating ethanol which would increase blank values. W ashingw ith distilled water and drying over CaC l0 before use as suggested byAntonis (1) was not found necessary.

Copper reagent Prepared as suggested by Antonis (1 ) and usuallymade up in 1-L. quantities containing 450 m l. of 2 NI triethanolam ine,500 m l. 10% (w /v) Cu(N03)0, and 50 m l. of 2 N acetic acid . This rea-gent w as stored in a refrigerator and was stab le for at least 3 weeks at4#{176}.t was found simpler to prepare large quantities of the triethanol-am ine, cupric nitrate, and acetic acid which could he kept indefinitelyat room temperature and m ixed when needed.

Color reagent D iethyld ith iocarbam ate, 0 .2% (w /v), in secondarybutanol was used. This w as found to be as satisfactory a solvent asisobutanol or butanol, as suggested by Antonis (1), if the copper con-centration of a given lot w as low enough to prevent a high base line.

Buffer solution Na phosphate buffer, 0.1 N I, pH 6.2.Palm .itic acid standards A stock solution containing 2 mEq./L .

(2000 Eq./L .) w as prepared by dissolving 128 tug. of palm itic acid in250 m l. of chloroform . This stock was diluted w ith chloroform to giveworking palm itic acid standards ranging from 10-100 pYq./L . Thiscorresponded to 200-2000 SEq. of FFA per liter of plasma which isroutinely diluted 1 :20 when extracted as described below . These work-ing and stock solutions are stable indefinitely if refrigerated and wellsealed.

AutoAnalyzerAn Autolnalyzer consisting of Sampler IT , proportion ing pump,

co lorim eter, and recorder was used. The arral1ement of ind iv idualmodules and tubing was as shown in Fj. 1. Special materials, precau-tions, and modifications from the Antonis (1) arrangement w ere as fol-

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COL OR I M ET ER

74 6 DALTON & KOW ALSKI C Ii& ce l C h em is tr ylow s. Polyethylelle sample cups were used instead of the regular poly-styrene. The polythylene cups were resistant to chloroform and elim -inated the use of specially prepared glass sample cups. They were usedonce and then discarded. An alum inum cover p late replaced the spe-

SAMPLER30/HR 1:2

TUBINGO .090 AO.065TO.0451O.035SO .056 IO.073 A0.065A0. 100A

Fig. 1. Flow diagram for free fatty acid determ ination. Pump tubing w ith I.D . as indicated:A, acidflex; T, tygon; 8, solvaflex. A ll connections m ade w ith acidflex transm ission tubing andp oly eth yle ne n ip ples .

cially-prepared Teflon-protected cover plate.* W e found the normalreservoir on the Sampler II to be resistan t to chloroform , but we suggestw ash ing it w ith chloroform to remove any interfering substances. Reg-ular acidflex transm ission tubing was used whenever lines came intocontact w ith ch loroform ; i.e ., the waste line, the separator to proportion-ing pump line, and the connecting line to the colorim eter flow cell. Theuse of two .005-in. (I.D .) standard pump tubing pulse suppressorsw as not necessary for a stable base line. Tile sample-line pump tubingused was .090 in. I.D ., which does not necessitate adjusting the lengthsof the two delivery lines from sampler to pump and reservoir to pump.The use of an .090-in. acidflex sample line, while using a reservoir line*Both the polyethylene cups and alum inum cover p late were supplied by Tecljiiieo ii Corpora.

tion , A rdsley (Chauncey), N . V .

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Vol. 13 , No. 9 , 1 967 FREE FATTY ACIDS 747

of .100-in. acidflex gave lower sensitiv ity but guards against dry ing ofthe reservo ir on the Sampler II. D rying or decreased flow from tilereservo ir would allow copper so lutions to pass through the separatorand cause m ixing w ith the diethyld ith iocarbamate solution .

MethodsExt rac t ion Procedure

The procedure used was essentially that of Itaya and U i (2). Plasmaor serum was separated in a refrigerated centrifuge at 100 at 1500 rpmand extracted as rapidly as possible. To 2.0 m l. of phosphate buffer and6.0 m l. of chloroform was added 0 .3 m l. of sample. This m ix ture wasshaken m anually for 90 sec., allow ed to separate, and then the upperphosphate layer was aspirated off w ith most of the protein precipitate.The chloroform was dried and the remainder of the protein was re-moved by filtering through filter paper (any paper not prev iouslywashed w ith acid is suitable). The 6.0 m l. of chloroform extracted isadequate for duplicate free fatty acid determ inations.Automa ted Ana lys is P rocedu re

In starting a sample run, chloroform was usually allowed to runthrough the sam ple and color-reagent lines until the extractor w as filled .This removed any remaining isopropanol or m ethanol in the coils andflow cell. The copper and diethyldithiocarbamate reagents were thenintroduced and pumped for a maximum of 20-30 mm. to estab lish anorm al base line. Sample analysis was begun when the base line ha dbeen stab le for at least 5 mm . Upon completion of a series of deter-m ination, methanol or isopropanol w ere equally effective as washingagents due to their m iscibility w ith water and chloroform . The wash-outprocedure was as fo llows: the copper line was removed from the rea-gen t and water w as pumped for at least 1 mm . to remove the excesscopper; then air w as allowed to pass through the copper line. Isopro-panol or methanol were then introduced through both the sample andcolor-reagent lines. W hen the extractor showed clear methanol or iso-propanol present, indicating that all the water and remain ing copperwere washed out, the sample line was removed. A m inimum of 2 mm .was required for the g lass-beaded special m ixing coil to dry. The color-reagent line was then rem oved and all tubing was allowed to dry . Thisprocedure involved a 10-12-mm . wash-out period.

To compensate for possible downward drift the base line was usuallyset between 97-98% transm ission. This allowed better results than thoseo f .A n ato nis (i ) who recommended 98-100% setting. W e found less drift

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U4

748 DALTON & KOWALSKI C lin ical Chem istrythan described by Antonis so standards were run hourly-after 30 sam -ples rather than after 10 . This allowed more samples to be assayed ina given tim e.

ResultsA typical tracing obtained by the above procedure is shown in Fig. 2.

A linear relationsh ip exists for the standard solutions and fair agree-

IiE;41 I-i_-i,- : L. -F lE E F AT TY A CID co NcE NT RA TIo N (MIcr..q,Iv.Ieafs p.r uSsr)

Fig . 2. AntoAnalyzer chart recordings of relationship between free fatty acid concentrationand per cen t transm ission . Tracing from left to righ t: palm itic acid standards. 10-100 Eq./L .; and trip licate album in ex tractions of the fo llowing concen trations in Eq./L .: a 14.0 , b23.6, c 34.3, d 52.8, e 63.8, f 73.8,g 93.3,and h 106.8.ment is obtained betw een trip licate album in sam ples. The extraction ofplasma FFA was tested by comparison w ith T routs (3 ) modificationof Dole technic (4). The colorim etric method gave a slightly lowervalue than tile titration procedure for the 39 samples assayed by thetwo methods (Table 1). A graphic representation of these data (F ig. 3)shows, linearity, w ith a correlation coefficient of 0.997. The recovery ofpalm itic acid standards from a 5% album in-Krehs R inger phosphatebuffer w as determ ined . An extraction m ixture consisted of 0 .3 m l. al-bum in-buffer, 2 .0 m l. of the phosphate buffer, and 3.0 m l. of standardchloroform solution. Recovery was considered to he virtually completew ith a range from 95 to 108% and a mean of 99.0 0.7 (Table 2).

DiscussionThe method described, an adaptation of the ex traction procedure of

Itaya and U i (2 ) combined w ith the autom ated colorim etric procedureof Antonis (1), is an improvement on previously described technics.

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Vol. 13, N o. 9, 1967 FREE F ATTY ACIDS 749

The rapid ity of the manual extraction procedure, compared w ith otherextraction methods reported , has proved to he a real advantage whenapplied to a large series of samples. Under our conditions, 180 plasmasam ples have been extracted and analyzed by one technician in a sing le

Table 1. CONCENTRATION OF PLASMA FFA I)ErsRM1NED flYCOLORIM ETRIC AND TITRIM ETRIC M ETH ODS

Method,

Semi -at4o,nated colorim etric (A ) M anual titrim etric (B )Sample no . (pEq./L.) (jEq./L.) Ratio (A:B)

1 30 0 317 .952 250 25 4 .9 83 240 231 1.084 220 216 1.025 210 20 5 1.036 240 243 .997 670 731 .928 70 0 728 .969 430 481 .8910 800 813 .9911 665 687 .9712 695 832 .8413 750 690 1 .0914 650 743 .8815 705 701 1.0116 68 0 646 1.0517 685 854 8018 310 317 .9819 28 0 261 1.0720 225 239 .9421 340 317 1.0722 29 0 280 1.0423 255 239 1.0724 755 75 0 1.0123 715 713 1.0026 760 754 1.0127 65 0 657 .9928 66 0 728 .9129 743 713 1.0530 855 936 .9231 740 69 0 1.0732 700 709 .9933 790 784 1.0134 600 646 .9335 730 75 4 .9 736 243 216 1.1337 250 228 1 .1038 295 302 .9839 283 328 1.17

Mean S .E . .012

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S.I

IV

I8

Fig. 3. C orrelation betw eenmanual titrim etric method andautomated colorimetric method.

Table 2. RECOVERY OF PALMITIC ACID FROM 5% ALBUMIN INK REI3S RINGER PHO SPHATE BUFFER (pH 7.4)

TITIIMIT1IC MITHOC ( u. Iq P t. .. .. E V Al ilt ., )

20 0

Pal m iS c a ci dAdded (pEq./L .) Found (jEq./L .) Recovery (% )

40 0

80 0

1000

1200

1600

750 DALTON & KOWALSKI C li ni ca l Ch em i st ry

Mean SE.

day. The m ethod also has tile desired versatility, being readily appliedto FFA determ inations iii biologic media used ill-vitro experim entsutilizing epididymal fat pads or isolated adipose tissue cells. It is alsouseful for assaying hormone-sensitive lipase.

19 020 019 039 040 043 077 077 079 098 099 01020117012201200156016301570

95.0100.095 .0r97_100.0107.596.396.398.898.099.0102.097.5101 .7

100.097.5101.998.1

99.0 0.7

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Vol. 13 , No. 9 , 1967 FREE F ATTY ACIDS 751

References1. Antonis, A ., Sein inu tom ated m ethod for the colorimetric determ ination of plasm a free

fatty acids. J. L ipid . .Res. 6 , 3 07 (19 65 ).2 . Itaya, K ., and Iji, M ., Colorimetric determ ination of free fatty acids in b iological fluids.

J. L ipid Res. 6, 16 (19 65 ).3. T rou t, D . L ., E stes, E . H ., Jr., and Friedberg, S . J., T itration of free fatty acids of

p lasma: A study of curren t methods and a new modification. J. Lipid Res. 1, 199 (1960).4. Dole, V . P ., A relation between non-esterified fatty acids in p lasma and the metabolism

o f g lu co se . J. C lin. Invest. 35, 1 50 (19 56 ).5. Gordon, B . S ., Jr., Cherkes, A ., and Gates, H ., IJnesterified fatty acid in hum an blood

plasma. II. The transport function of unesterified fatty acid. J. G un .. I nve st. 36, 81 0(1957).

6. Dole, V . P ., and M einertz, H ., M icrodeterm ination of long-chain fatty acids in plasm aa nd tis sue s. J. Biol. Chem. 235, 2595 (1960).

7 . Ayers, C . w ., Estimation of the h igher fatty acids C7-C1,. A nal. C huin. A da 15 , 77 (1 95 6).8. Iw ayam a, V ., N ew colorim etric determ ination of higher fatty acids. J. Ph arm . Soc. Japaa

79, 552 (1959).9. D uncom be, w. G ., The co lorim etric determ ination of long .chain fatty acids in the 0.05-0.5

m ole range. Biochem . J. 83, 6 (1962).1 0. D un co mb e, w. G.,The colorimetrie m icrodeterm ination of long-chain fatty acids. Bioche-m.J. 88 , 7 (1963).11. Anstall, H . B ., and Tru jillo , J. M., Determ ination of free fatty acids in plasma by a

co lorimetric procedure: An appraisal of the method and comparison w ith o ther techn ics.G un. Chem . 11, 7 41 (19 65 ).

12. Kvam , D . C ., Schm idt, J. G ., R igg ilo, P . A ., and Gallo, D . G ., C oloritnetric ,nicrodeterm ina-tion of plasm a free fatty acids. J. Pharm. Sci. 53, 9 88 ( 19 64 ).