Table V.
Comparison of Methods Blood Alcohol
Sample Subject I Venous Finger tip, drawn at same time Subject I1 Venous Finger tip, drawn at same time
for
Alcohol Found, % Chemical Enzyme method method 0.049 0.050
0,050 0.047 0,047 0.048
...
0.136 0.131 0.134
0.129 0.124
...
0.114 0.120 0.121
Venous, drawn 30 minutes later 0,146 0.143 Standard samples, analyzed by 16 operating laboratories I, mean, 0.055 0 . 050a
...
0.143 0.146
0.056 0.056 11, mean, 0.210 0 . 1 9 8 ~ 0.208 0.213 0.305 111, mean, 0.293 0.293a 0.305 Mean of triplicate determinations.
$1ith methanol, isopropyl alcohol, acetone, paraldehyde, formaldehyde, lactic acid, and formamide. The first four were added to the sample a t approximately 0.15 %. The last three were analyzed directly without ethyl alcohol being present. Only isopropyl alcohol produced any significant alteration in results. amounting to about 15 % of the alcohol added, expressed as ethyl alcohol. Small negative variations were
found n ith the aldehydes and ketones, their magnitude being within the limits of error of the method and, therefore, not significant. The most important result of these tests )vas confirmation of earlier findings that methanol does not interfere. This is one of the more troublesome interferences with chemical methods, especially in cases of death, in which intoxication may be involved. Discussion. By virtue of its superiority in specificity, small amount of analyst time, small amount of equipment, and small blood sample required, t h e enzymatic method of alcohol determination in blood is considered superior to all others so far described. I n the clinic, and particularly as a method for rapid checking of t h e chronic alcoholic, it should prove superior. It also is directly applicable to other situations in which a knodedge of blood alcohol is desired, uncomplicated by possible error due to the presence of other similar materials. I t s utilization requires certain precautions. as do the more complex chemical methods. The reagent must be relatively fresh and kept refrigerated a t all times when not in use. Frequent checks on calibration are desirable. A well preserved alcohol sample in water may be used for this purpose by inserting a sample or two along with a series of analyses. If finger blood is utilized, special care must be applied in drawing it. and improvements in the methods of minimizing evaporation of alcohol, while drawing and measuring the sample, are still needed. The enzymatic method is most convenient when large numbers of determinations are required, because all
ma? be set up serially and read in rotation as the reaction becomes complete. This situation is especially economical of reagent, all of which may be made a t one time for a series, with little to be discarded at the end of the series. The chemical oxidative method described is more suitable when small numbers of analyses or occasional analysis are required. The reagents are relatively more stable, and the time requirement for the occasional analysis is very small. LITERATURE CITED
(1) Bonnichsen, R. K., Lundgren, G., Acta Pharmacol. Toxicol. 13, 256 (1957). Bonnichsen, R. K., Theorell, H., Scand. J . Clin. Lab. Invest 3, 58 (1951). Biicher, T., Redetzki, H., Klin Wochschr. 29, 615 (1951). Cavett. J. W.. J . Lab. Clin. Med. 23, 543 (1939). Dotzauer. G.. Redetzki. H.. Johannsmeier, ’ K.’, Bucher, T., Deut. Z. gerichtl. Med. 41, 15 (1952). Feldstein, M., Klendshoj, N. C., Can. J . M e d . Technol. 16, 48 11954). ~
T. E., Ibid., 115, 47 Harger, R. K., I Baker. R. S.. Quart. - J . Studies Alcohdl 17, 1 (1956). (10) Kozelka, F. L., Hine, C. H , 1x0. ESG. CHERI.,ANAL.ED. 13, 905
Friedemann,
(1941). (11) Widmark, E. M. P., Biochem. 7. 131, 473 (1922); 218, 465 (1930). RECEIVEDfor review September 17, 1957. Accepted March 26, 1958: .lided by grants from the California State Alcohol Rehabilitation Commission and Research Committee, University of California.
Determination of Polyunsaturated Acids in Lipides of Plasma and Tissue RALPH T. HOLMAN and HERBERT HAYES The Hormel Institute and Department o f Physiological Chemistry, University o f Minnesota, Austin, Minn.
A micromethod for determination of polyunsaturated acids in 1 gram of tissue or a few milliliters of plasma has been developed. The optimum conditions of isomerization were determined upon pure polyunsaturated acids, and constants were established for the calculation of the various polyene types. Reproducibility of isomerization varies between 2 and 10% for the various polyenes. Nonacid polyenes have been isomerized to determine possible interference by
1422
ANALYTICAL CHEMISTRY
unsaponifiable matter containing polyunsaturated materials.
I
study of biological phenomena involving polyunsaturated acids, it is often necessary to determine the content of these substances in very small samples. The methods now in common use require the isolation and weighing of lipides prior to isomerization (4, Is), and in many cases this may complicate or preclude an adeN THE
quate analysis. Some of the methods use conditions of isomerization far from the optimum for the more highly unsaturated acids (I, I S ) , and some are not standardized against pure acids or all of the polyene acid types. A detailed discussion of methods appeared in a recent review (6). A modification of the method of alkaline isomerization is based upon a systematic study of isomerization and uses highly purified polyunsaturated acids as standards.
ing a t 2 ml. per second, prevents entry of oxygen. After being heated for 20 minutes, the vessels are chilled in ice water and allowed t o come to room temperature The contents are made up t o volume (5.0 ml.). Absorbance of these solutions is measured a t 375, 346, and 315 mp against a blank consisting of reagent and ethyl alcohol which has been treated the same as the samples. A tenfold dilution usually allows the measurement of densities at 268 and 233 nip.
EXPERIMENTAL
Equipment and Supplies. Constant-temperature apparatus may consist 01 a hot oil bath (preferably Don Corning Silicone KO.710) or a metal block (6, 8), and it is maintained a t 180" rt 0.5" C. In the work reported here, a thermostated aluminum block was found t o be reliable and corn-enient. The holes for reacels are 2.5 cni. in diameter and 20 em. deep, and contain sufficirnt silicone fluid to immerse the lower 5 c m of the vessel. The reaction vessels (Figure 1) are made by sealing the tops of 5.0-nil. volumetric flasks a t the bottom of 16 X 150 mm. test tubes n i t h lips. The vessels are provided n-ith nitrogen inlet tubes which cover the tops and introduce an atmosphere of inert gas over the reagent and sample. An ultraviolet spectrophotometer is nrcded for the measurement of the spectra of tlie samples before and after isomerization. It may be a manually operated or a self-recording instrument, equipped n i t h quartz or fused eilica cells having a light path of 1.0 em. Potassium hydroxide in ethylene glycol (4) is used to isomerize the fatty acids. Ethylene glycol is heated t o 190" C for 10 minutes to drive off water, and then cooled to 150" C. TI\ enty-eight grams of potassium hydroxide (85% analytical reagent grade) is added t o each 100 grams of glycol. The solution is reheated to 190" C. for 10 minutrs and cooled. The content of potassium hydroxide is determined titrimetrically and adjusted to 21.0 i0.1y0 KOH. All manipulations must be performed under nitrogen, and the reagent should be stored under nitrogen in glass-stoppered bottles below 5" C. Solvents required for the extraction of lipides and in the analysis include 95% ethyl alcohol, light petroleum ether (Shelly F), methanol (reagent grade), and ethyl ether (reagent grade, stored in contact, with iron). The methanol should have a density of less than 0.4 at 220 i i i u when measured against water. Acidic ethyl alcohol-ether is prepared by adding 5 parts of concentrated hydrochloric acid to 95 parts of freshly prepared ethyl alcohol-ether, 3 to 1. Extraction of Lipides. The extraction of lipides from serum, blood, or tissue may be made by any standard procedure TT hich does not leave traces of halogenated solvents in the lipide samples These solvents, if present, impart strong extraneous ultraviolet absorption in isomerized samples. The lipide content of the extracts need not be known if a n aliquot of the extract can be related t o a known 11-eight of tissue or volume of fluid.
PROPOSED PROCEDURE. One volume of serum, plasma, or other fluid (usually 1 to 10 ml.) is added with rapid mixing t o 20 volumes of ethyl alcoholether, mixed thoroughly a t room temperature, and filtered on a Riichner
Calculations. If the weight of the lipide is knoTm the extinction coeffiD D cients, ka = SlL - and lzc = QIL,
Figure 1.
Isomerization reaction vessel
funnel. For the extraction of tissue, 1 part (0.5 to 2.0 grams) is homogenized in 30 parts of acid ethyl alcohol-ether using a Potter homogenizer equipped with a Teflon pestle. The precipitate from either plasma or tissue is washed once with ethyl ether and once m-ith light petroleum ether on the funnel. The combined extracts are reduced in volume in suction flasks to about one fourth of the original volume and transferred \yith 7 5 ml. of light petroleum ether to a separatory funnel. Fifty milliliters of water is added, and the contents are shaken. The aqueous layer is extracted twice more with 75nil. portions of light petroleum ether. The petroleum ether extracts are combined and washed twice with 75-m1. portions of distilled water and dried with anhydrous sodium sulfate. After filtration, the dried extract is evaporated down to 2 or 3 nil. in a pear-shaped flask, and the lipide is transferred to a volumetric flask quantitatively with light petroleum ether. The size of the flask is chosen such t h a t lipide from 1 ml. of serum or 1 gram of tissue is contained in about 1 ml. of petroleum ether. If desired, the lipide may be saponified, and the unsaponifiable matter may be removed prior to isomerization. Analysis. The background absorption is determined upon the petroleum ether solution a t 375, 346, 315, 268, and 233 mp. One milliliter of the solution is transferred to the vessel used for isomerization. The petroleum ether is evaporated under nitrogen, 1 ml. of 95% ethyl alcohol is added, 0.85 ml. (1.1 gram) of ethylene glycol-potassium hydroxide reagent is measured into the vessel using a syringe, and the nitrogen cap is replaced. The contents are mixed by shaking, and the tube is placed in the hot block and left there for exactly 20 minutes. The boiling of the ethyl alcohol during the first few minutes provides thorough mixing, facilitates saponification, and sweeps out any dissolved oxygen. The nitrogen. flow-
calculated for the sample before and after isomerization, respectively. for all five wave lengths measured. The differences, k = k , - kb, are substituted into the following equations for the calculation of polyenoic acid contents expressed as per cent of total lipide :
% docosahexaenoic acid = 4.1861237, -
0.1778kade 1.559 k 3 ~ 1.628km % arachidonic acid = 1.4561031s 1.344kws - 0.4128kax 5 linolenic acid = 1.26612~~ 0.8028k31j 0 3172kj16 - 1.778k375 5 linoleic acid = 1.087k2~- 0.615km 0,13541281, - 0.1072k346 - 0.412ks-, eicosapentaenoic acid
=
+
If the weight of lipide is not known, the content of polyunsaturated acids may be calculated and expressed as milligrams per 100 ml. of fluid or milligrams per 100 grams of tissue. In this case, the relative densities are calculated-that is, the densities derived from 1 gram of tissue or 1 ml. of fluid. This is equivalent t o multiplying the observed densities by the dilution factor, When the relative densities corrected for background absorption are substituted for the absorptivities, IC, the results are expressed as milligrams of fatty acids per 100 ml. of fluid or as milligrams per 100 grams of tissue. In the derivation of the equations, the spectral constants of linoleic, linolenic, eicosatetraenoic, eicosapentaenoic, and docosahexaenoic acids were used. The results, although expressed in terms of these acids, do not necessarily indicate the presence of these specific acids in the sample. DISCUSSION
To develop a method for the quantitative measurement of polyunsaturated acids, it was necessary first to obtain highly purified fatty acids for use as primary standards. Methyl linoleate (Hormel Foundation) was isolated from saflower oil fatty acids by means of low temperature crystallization of the acid, and fractional distillation of the methyl esters. It had an iodine value of 171 (theory 172.5), less than 0.2% conjugated double bonds, no trans unsaturaVOL. 30, NO. 8, AUGUST 1958
1423
Table I.
Extinction Coefficients Developed b y Pure Polyunsaturated Acids after Isomerization 11p
375
ilcid
346
315
268
Linoleic5 Linolenic5 Eicosatrienoic Octadecatetraenoic EicosatetraenoicG Eicosapentaenoica Docosahexaenoica 25.0 26.1 31.2 48.6 Rccommended slit width, nirn. 0.2 0.2 0.3 0.5 a Constants from these acids were used for derivation of equations given in text. Table II.
233 92 0
43.3 1.0
Standard Deviations of Analyses of Polyunsaturated Acids
Hexaene
Pentaene
Total Fat, c& Tetraene
Triene Diene Hg plasma lipide 0 . i 8 Z!Z 0.07 0.83 i 0 . 0 2 2.89 f 0.06 1 . 9 7 =t0 . 0 4 15.0 i 0 . 8 6 Herring oil 5.29 i 0.54 14.05 f 0 . 5 9 6.76 i 0 . 2 % 5.28 f 0.62 3 . 6 5 i 0 . 1 7
tion detectable in the infrared spectruni, arid was found uniform by paper chromatography ( I O , 1 2 ) . Yethyllinolenate (Hormel Foundation) was preparrd by broniination of linseed oil acids, debromination of the hesabromostearic acid, and fractional distillation of the niethvl ester. It had an iodine value of 260 (theory 260.5), contained about 0.5% esters n ith conjugated double bonds, and 10% esters with trans isolated double bonds. This preparation was used for a triene standard because all-cis linolenic acid was not available. Octadecatetraenoic acid n-as isolated from cod liver oil by fractional distillation of the methyl esters, followed by displacement chromatography of the free acids (6). It was contaminated by a trace of an acid having 20 carbon atoms, which mas detected in a hydrogenated sample by paper chromatography. Eicosatrienoie, eicosatetraeiioic, and eicosapentaenoic acids n ere isolated by Yontag and Klenk ( 1 1 ) from beef liver phosphatide fatty acids by fractional distillation and countercurrent distribution. They n ere free of higher and lorn-er unsaturated acids as judged by ultraviolet spectra after isomerization and by paper ehromatography ( I O , I @ , Docosahesaenoic acid n a s isolated froni a concentrate of methyl docosahexaenoate by displacement chromatography, and was found uniform bv paper chromatography. The concentrate was prepared from hog brain phosphatides by low temperatur: crystallization and fractional distillation ( 3 ) . T o adapt the method of alkaline isomerization to a micro scale, the amount of reagent was rpduced. The lipide was added to the reagent in or with ethyl alcohol to facilitate mixing and saponification of the sample, and to swerp out oxygen from the solution. To elinii1424 *
ANALYTICAL CHEMISTRY
c3,
F-
e
LINOLEIC ACID
a
EICOSATF ENOIC AC-Ex-
Figure 2. Course of isomerization of polyunsaturated fatty acids under standardized conditions
nxte one transfer, tlie rmction vessel ivas adapted from a 5.0-ml. volurnetris flask. K i t h these inodifications in procedure and equipment, the rates of 1-omerization of mixed fatty acids from natural sources n ere studied. Imiierization proceeded to approxiniately tlie same degree in 20 minutes under these conditions as in 15 minutes under the conditions of Herb and Rienienschneider ( 4 ) . The temperature within the reaction vessel was measured throughout an isomerization reaction; the temperature rose to 170' C. nithin 5 minutes and increased sloivly there after. It thus appeared that the 1.0 nil. of ethyl alcohol had the effect of delaying the isomerization approximately 5 minutes. With the conditions thus established, the rates of isomerization were determined on the highly purified acids and esters, and their extinction coefficients TI ere measured a t least in triplicate after isomerization. The limited amounts of these pure substances precluded niorr replication. The developnient of the distinctive conjugated polyene from each of the nonconjugated po1)enoic acids is slioivn in Figure 2. Thc optimum time for nia\-imum con-
jugation varies from acid to acid. Thus, eicosatrienoic acid requires 20 minutes to develop maximuin conjugated triene, if-hereas masimuni liesaene conjugation is developed froni docosnhesaenoic acid within 8 minutes. S o n chosen n-liicli is optione time ~ n be mal for d l acids. The 20-minute reaction tinic is a reasonable conil)romise, bxause the conjugation of most acids remains high. The extinction coefficients of tlie isomerized pure' polyenoic nritls under the st,anclartlizcd conditions are given in Table I. So regular pattern can be sern in the extinction corfficients of acids having the same niunbcr of double l~onds but differing in chain length. This call scarcely be expected because the constants are measured under conditions which are not optimal for all ueitli. and a t 20 minutes the development of a given chromopliore may be increasing or decreasing. Tlie eyuations for the calciilat'ions of pentuenoie and hesaeiioic acids \vert' ticrivd by simultaneous equations n-1iic.h expross the contribution of each : i d to t81ie absorptions a t 375 and 3-16 nip. The equations for tetraenoic, trienoic, and dienoic acid contcnts were then clerivtd by successive substitutions. 111 iiieasurenients of spectral ~ b s o r p tioii were m:itle in a Beckninn DL spectrophotometer using a hydrogen arc lanip. This n-as done 1~ce:iuse even a t 373 nip tlip required slit n-itlth using tlit. tungsten lanip was not significwntly sniall
