89

Technical

A Simple and Rapid Colorimetric Method for Determination

of Free Fatty Acids for Lipase Assay
Dae Y. Kwon and Joon S. Rhee*
Department of Biological Science and Engineering, Korea Advanced Instituteof Science and Technology,
Seoul, Korea

A simple and rapid colorimetric method was developed replacing benzene with hexane for determination of free
to determine the lipase activity for fat splitting. Free fatty acids formed in the enzymatic hydrolysis of olive
fatty acids produced by lipase from triacylglycerols oil in the solvent system {6).
were determined by observing the color developed The purpose of this paper is to report a further
using cupric acetate-pyridine as a color developing simplification of the method of Lowry and Tinsley (15)
reagent. This modified method requires only a few for determination of free fatty acids by eliminating the
minutes to determine the free fatty acids, whereas it solvent evaporation and centrifugation steps for lipase
takes over 20 min by the conventional methods which assay.
require solvent evaporation and centrifugation steps.
The sensitivity and reproducibility of the method were
good for caproic, caprylic, capric, lauric, myristic, EXPERIMENTAL
palmitic, stearic and oleic acids. Materials. Oleic, stearic, palmitic, myristic, lauric,
capric, caprylic, caproic and butyric acids, specially
manufactured as fatty acid standards, were purchased
Lipases or acylglycerol-acylhydrolases (EC 3.1.1.3) are from Sigma Chemical Co. (St. Louis, Missouri).
the enzymes which hydrolyze the esters of long chain Benzene, isooctane and other solvents were purchased
aliphatic acids from glycerol at oil/water interface (1). from Tokyo Kasei Chemical Co., Ltd. {Tokyo, Japan).
Recently, lipases have been used extensively in the All other chemicals and r e a g e n t s used were of
hydrolysis of lipid under mild conditions (2) and analytical grade.
synthesis of new triacylglycerols by interesterification Copper reagent was prepared according to Lowry
(3). Many research papers on fat splitting using and Tinsley {15}. A 5% (w/v) aqueous solution of cupric
microbial lipases in the substrate emulsion system {4), acetate was prepared and filtered, pH being adjusted
or in the two-phase system (5) have been published. to 6.1 using pyridine. The solution does not require any
We reported the effect of organic solvents on the other color reagent.
stability and catalytic activity of the lipases from Standard Curves of Free Fatty Acids. Samples
Candida rugosa for fat splitting, and isooctane was containing 2.0-50.0 ~mole free f a t t y acids, butyric,
recommended as the most suitable solvent in the caproic, caprylic, capric, lauric, myristic, palmitic,
two-phase system (6). stearic and oleic acids each, were prepared by dissolv-
There is a need to determine the lipase activity by ing them in test tubes with 5 ml of isooctane. Slight
measuring the f a t t y acids produced. F a t t y acids warming was necessary to make solution for solid
produced by lipase can be determined by titrimetry, stearic, palmitic and myristic acids. Then 1.0 ml of
copper soap colorimetry, chromophore spectropho- cupric acetate-pyridine reagent was added and the two
tometry, isotopic methods, gas liquid chromatography, phases thus formed were mixed vigorously for 90 sec
enzymatic methods and immunological methods (7). using a vortex mixer. The mixture was allowed to
Jensen (7) reported that the most practical methods are stand still for about 10-20 sec until the aqueous phase
titrimetry and copper soap colorimetry for the study of was sedimented clearly from the solution of isooctane
enzymatic fat splitting and that copper soap colori- and fatty acid. The standard curves of free fatty acids
metry is better than titrimetry. vs. absorbancy were determined by measuring the
Copper soap colorimetry measures color after fatty absorbance of isooctane solution at 715 nm against the
acids are converted to copper soaps with color control which contains no free fatty acids.
reagents. This colorimetric method, originally devel- Determination of Lipase Activity. The enzyme reac-
oped by Duncombe (8) using Cu(NO3)23H20 and tion in the emulsion system (4) was stopped by adding
triethanolamine as a copper reagent and a color 6N HC1 and isooctane, followed by mixing and boiling
reagent, respectively, was later modified and improved the reaction mixture for 5 min. The 5 ml of upper
by many workers for their specific research purposes isooctane layer containing the fatty acids was drawn off
(9-14}. Lowry and Tinsley (15) developed a rapid to a test tube for analysis. The lipase activity in the
colorimetric determination of free fatty acids with a two-phase system {16) was determined by adding 6N
good sensitivity and reproducibility using cupric HC1 at the very end of the reaction, and the mixture was
acetate-pyridine. This method is again modified by agitated vigorously for 30 sec. after which 5 ml of the
*To whom correspondence should be addressed at Department of upper layer was taken in a test tube. Free fatty acids
Biological Science and Engineering, Korea Advanced Institute of dissolved in isooctane were determined by spectros-
Science and Technology, P.O. Box 150, Chongyang, Seoul 131, copy according to the method described above. Lipase
Korea. activity was determined by measuring the amount of

JAOCS, Vol. 63, no. 1 (January 1986)

90
D.Y. KWON AND J.S. RHEE

free fatty acids from the standard curves of free fatty

acids.

RESULTS A N D D I S C U S S I O N
To determine the lipase activity according to the
conventional colorimetric methods (15) for an emulsion / S ~~
system (4), we must selectively extract the free fatty
acids from the reaction mixture with the solvent,
evaporate the solvent, replace that with benzene or
other solvents, and then add the color reagents
l i
followed by mixing the color reagent mixture vigor-
ously. This mixture must be centrifuged in order to
separate the solvent phase from the aqueous phase. I I I
C ~ 6 7 8
Lipase activity was determined by observing the color
development of free fatty acids in the solvent phase. pH
For a two-phase s y s t e m (6}, lipase a c t i v i t y was
determined by taking the supernatant containing free Fig. 1. pH dependency of absorption cupric acetate-oleic acid
soaps in 5 ml isooctane at 715 nm. 5% (w/v) aqueous solution of
fatty acids from the reaction mixture and evaporating cupric acetate w a s used as a color reagent. Relative absorbancy
the solvent which was replaced by benzene or other of color development using 30 ~eq oleic acid was represented by
solvents, followed by centrifugation of the color varying the pH of cupric acetate solution (1 ml) with pyridine.
reagent mixture. Then the lipase activity was deter- Curve A, isooctane; curve B, benzene. Curve B represents the
data reported by Lowry and Tins|ey (15). Tests were run in
mined by assaying the f a t t y acids. All of these duplicate.
colorimetric methods required extraction, evaporation
and centrifugation.

/
This modification of the colorimetric method of free 100 ~ - - "
fatty acid determination is based on the fact that the
density and water miscibility of benzene are greater
than those of isooctane, which was selected as the most
suitable solvent for fat splitting in the two-phase
system (6). The density of isooctane and benzene is
>,
0.69 and 0.88 at 20 C, respectively, and water
immiscibility of isooctane is greater than t h a t of 5o
benzene (Table 1). When isooctane was used we were
able to determine the free fatty acids for assaying the
lipase activity rapidly while elimating the centrifuga-
tion step.
The spectrum of the color developed by reaction of
free fatty acids with cupric acetate-pyridine at various
wavelengths of spectrophotometer was investigated as 12 14 1 18
a preliminary step. The result showed t h a t the
Carbon Number of Fatty acid
absorbance in the range of 710 nm and 720 nm was
maximum, as reported by Lowry and Tinsley (15). They Fig. 2. Effect of carbon numbers of fatty acid chain on color
reported that the optimum wavelength was 715 nm. development. Relative absorbancy at 715 nm of color developed
The effect of the pH of cupric acetate solution on the for each 30 ~eq butyric (C,), eaproic (C~), caprylic (C8), capric (C,o),
color development of cupric acetate-oleic acids soaps lauric ( C , } and oleic acids (CI8:1} in 5 ml i s o o c t a n e were
represented. 10 peq stearic (CiJ, paimitic (C1~) and myristic acids
in isooctane was investigated by varying the pH with (C~3 in 5 mi isooctane were used for calibration. Tests were run in
pyridine and comparing the pH dependency of absorp- duplicate.
tion in this s o l v e n t with t h a t in benzene. The
absorbance of this color in isooctane was maximum at
pH 5.8-6.4 (Fig. 1), whereas the absorbance in benzene estimation of the fatty acids produced when triacyl-
was maximum at pH 6.0-6.2 (15). This broad optimum glycerol having fatty acids of carbon numbers larger
pH range for an isooctane system is considered to be than C,o was used as a substrate. However, each
due to the higher water immiscibility of isooctane. caproic and caprylic acid also could be determined with
Broad spectrum of the pH dependency in isooctane is good reproducibility.
one of the advantages of this method for reproducibili- Standard curves for various free fatty acids were
ty. prepared using caproic, caprylic, capric, lauric, myristic,
To study the effect of carbon numbers of fatty acids palmitic, stearic and oleic acids. The regression
on their color development, the absorbance was equation of the standard curves for capric, lauric,
measured at 715 nm with 30 peq free fatty acid/5ml of myristic, palmitic, stearic and oleic acids were almost
isooctane, unless otherwise specified. The color devel- the same. Figure 3 shows the standard curves for oleic
opments of caproic, caprylic, capric, lauric, myristic, acid (line A) and caproic acid (line B). The data show
palmitic, stearic and oleic acids are shown in Figure 2. good reproducibility and sensitivity up to 50 ~mole of
The results suggest that this method was suitable for free fatty acid/5 mI of isooctane without centrifugation

JAOCS, Vol. 63, no, 1 ( J a n u a r y 1986)

 91
RAPID DETERMINATION OF FREE FATTY ACIDS

1.2 TABLE 1
Comparison of Density and Water Miscibility tWater Solubility
in Solvent, Solvent Solubility in Water} of Solvent (17}

1.(]
Density at 20 Water solubiU- Solvent solu-
Solvent C g/ml ty of solvent, bility of water,
%(w/w) a t 2 5 C %(w/w) a t 2 5 C

.8
Iso-octane 0.692 0.0055a 0.00024
Benzene 0.879 0.063 0.1780
0
" .6 awater solubility at 20 C.

TABLE 2
The Concentration R a n g e for Determination of Absorbancy of
Various Free F a t t y Acids

Free fatty acids b Concentration (~mole FFA/5 ml)

Lower range Upper range

Butyric >50c
10 20 30 40 Caproic 10 >50d
Myristic 0 30
Fattyacid Concentration, pmole/5mL Palmitic 0 22
Stearic 0 18
Fig. 3. Standard curves of fatty acids in isooctane. Absorbancy
at 715 nm w i t h the pyridine-cupric a c e t a t e r e a g e n t w a s
represented, line A, oleic acid; line B, caproic acid. The regression aExperiment was carried out up to 50 ~mole of FFA in 5 ml of
equation for line A is y = 0.0227x + 0.002, and for line B is y = isooctane.
0.0180x - 0.203, where y = absorbancy at 715 nm and x = ~eq
free fatty acid/5 ml of isooctane. Each point represents the mean bThe data for caprylic, capric, lauric and oleic acids are excluded
value of three determinations. in this table, because the color of copper soap of these fatty acids
develops well over 50 ~mole FFA with good sensitivity.
CColor does not develop uo to 50 umole of FFA.
and solvent evaporation steps. In case of caproic acid dNo limitation was detected up to 50 ~mole of FFA.
{line B), color did not develop up to 10 ~mole caproic
acid/5 ml of i s o o c t a n e , a l t h o u g h t h e c o l o r was
d e v e l o p e d in linear p r o p o r t i o n to t h e f a t t y acid
concentration at above 10 ~mole/5 ml. Because the free associated with an ionized free f a t t y acid (RCOO-) to
caproic acid is slightly soluble in water, some part of form a nonionized free fatty acid (RCOOH), and the
the caproic acid which is not e x t r a c t e d readily by the nonionized form (RCOO-). In the mixture of isooctane-
solvent m a y remain in aqueous phase so t h a t the color cupric acetate-pyridine, only free f a t t y acids form a
does not develop up to 10 ~mole of caproic acid/5 ml of cage-like complex with cupric acetate and the color
solvent. As shown in Table 2, the data for the butyric development is not interfered by monoacylglycerols,
acid seem to be in agreement with this fact. For diacylglycerols, triacylglycerols, or another lipid {15}.
butyric acid, which is relatively soluble in water, copper In fact, we c a l c u l a t e d the lipase a c t i v i t y b y this
soap color did n o t d e v e l o p up to 50 ~mole of method to investigate the solvent effects on the lipase
b u t y r i c acid/5 ml of s o l v e n t b e c a u s e of its low stability and lipase activity {19}, and compared the
e x t r a c t a b i l i t y to the solvent. T a b l e 2 shows the m e t h o d with the conventional methods 16, 15). Prev-
s t a n d a r d c u r v e s of such s a t u r a t e d f a t t y acids as ious r e p o r t s (6) d e t e c t e d t h e l i p a s e s t a b i l i t y b y
stearic, palmitic and myristic acids were constructed determining the residual activity after incubating in
only up to 18 ~mole of stearic, 22 ~mole of palmitic and organic solvents, and the lipase activity in organic
30 ~mole of myristic acids/5 ml of solvent. Over these solvent. The present m e t h o d has the a d v a n t a g e of
concentrations, soap-like emulsion developed probably eliminating evaporation and centrifugation, which are
due to the limited solubility of s a t u r a t e d f a t t y acids in required in conventional methods. As a result, the
solvent {18}. present method takes only a few minutes to determine
For the s t u d y of lipase assay for both the emulsion the free f a t t y acids in contrast to the conventional
system and the two-phase system, using the isooctane methods of 20 .min. The result of the method agreed
as an extraction solvent excludes laborious solvent with results of the conventional methods in determin-
e v a p o r a t i o n and c e n t r i f u g a t i o n steps. A d d i n g the ing the solvent stability of lipase; it also shows better
c o n c e n t r a t e d hydrochloric acid helps not only for reproducibility. On the other hand, in determining the
stopping the lipase reaction b u t also for increasing the lipase activity the two phase system was recommend-
extractability of free f a t t y acids to the solvent. HC1 able, b e c a u s e t h e r e p r o d u c i b i l i t y of t h e d a t a in

JAOCS, Vol. 63, no. 1 (January 1986)

 92
D.Y. KWON AND J.S. RHEE

the two phase s y s t e m was better t h a n t h a t of the 10. Sahasrabudhe, M.R., JAOCS 59:354 (1982}.
emulsion system. 11. Bowyer, D.E., J.S. Cridland and J.P.King, J. Lipid Res.
The results indicate t h a t the p r e s e n t m e t h o d is a 19:274 (1978}.
v e r y simple and rapid one to determine free f a t t y acids, 12. Radding, W., G.G. Mayer and J.W. CorreU, J. Lipid Res.
24:100 (1983).
and is suitable for the determination of lipase activity. 13. Shipe, W.F., G.F. Senyk and K.B. Fountain, J. Dairy Sci.
63:193 (1980).
REFERENCES 14. Bains, G.S., S.V. Rao and D.S. Bhatia, JAOCS 41:831
(1964).
1. Brockman, H. L., in Lipases, edited by B. Borgstrbm and H. 15. Lowry, R.R., and I.J. Tinsley,JAOCS 53:470 (1976).
L. Brockman, Elsevier Sci. Pub., Amsterdam, 1984, pp. 3-46. 16. Leuenherger, H.G.W., in Biotechnology, edited by H.-J.
2. Linfield, W.M., R.A. Barauskas, L. Sivieri, S. Scrota and Rehm and G. Reed, Verlag Chemie, Weinheim,1984, Vol. 6a
R.W. Stevensor Sr., JAOCS 61:191 (1984}. pp. 5-29.
3. Macrae, A.R., JAOCS 60:291 (1983}. 17. Riddick, J.A., and W.B. Bunger, in Organic Solvents, edited
4. Kwon, D.Y., and J.S. Rhee, Korean J. Chem. Eng. 1:153 by A. Weissberger, 3rd edn., John Wiley & Sons, New York,
(1984). 1970, pp. 95-108.
5. Blain, J.A., M.W. Akhtar and J.D.E. Patterson, Pak. J. 18. Singleton, W.S., in Fatty Acids, edited by K.S. Markley,
Biochem. 10:41 (1976). Interscience Pub. Inc., New York 1960, pp. 609-678.
6. Kim, K.H., D.Y. Kwon and J.S. Rhee, Lipids 19:975 (1984). 19. Kwon, D.Y., and J.S. Rhee, Korean J. Food Scs Technol.
7. Jensen, R.G., Lipids 18:650 (1983}. 17:490 (1985).
8. Duncombe, W.G., Biochem. J. 88:7 (1963}.
9. Hron, W.T., and L.A. Menahan, J. Lipid Res. 22:377 (1981). [Received J u n e 28, 1985]

.%Addition of Phthalimidonitrene to Acetylenic Fatty Acid Esters:

Synthesis of Long-Chain 2-Phthalimido-2H-Azirines
M.H. Ansari, F. A h m a d and M. A h m a d "
Section of Oils and Fats, Department of Chemistry, Aligarh Muslim University,Aligarh 202001, India

Lead tetraacetate (LTA) oxidations of N-aminophthalim- addition of aminonitrene to acetylenes and r e p o r t e d the
ide in the presence of acetylenic fatty acid esters have formation of B, which probably occurred b y the rearrange-
resulted in the formation of corresponding 1H-azirines m e n t of A. We r e p o r t here the s y n t h e s i s of chain-
that spontaneously rearranged to give 2H-azirines in s u b s t i t u t e d 2 H - f a t t y azirine b y the reaction of acetylenic
moderate yields. 2H-Azirine derivatives (IV, V and VI) f a t t y acid esters (terminal, penultimate, internal) with the
of acetylenic fatty acid esters, methyl l(~undecynoate (I), nitrene intermediate generated in situ b y the L T A oxida-
methyl 9-undecynoate (II) and methyl 9-octadecynoate tion of N-aminophthalimide.
(III), respectively, have been prepared and characterized
with the help of spectral and micro analyses.
RESULTS AND DISCUSSION
The oxidation of N - a m i n o p h t h a l i m i d e in the presence of
To continue our studies on the synthesis of long chain
m e t h y l 10-undecynoate (I) {Scheme 1), using L T A as an
N-aminoaziridines (12) b y the addition of aminonitrene
oxidant at r o o m t e m p e r a t u r e on final work up and col-
intermediate to olefins, we focused on m o n o u n s a t u r a t e d
u m n c h r o m a t o g r a p h i c fractionation, g a v e an inseparable
analogs of aziridine, i.e., 1H-azirine CA) and 2H-azirine (B).
\ isomeric B {IV). I t s infrared {IR) s p e c t r u m showed a
characteristic s h a r p b a n d at 1775 cm-', which has been
--C~---C-- C--C--
\ / / \ // assigned to the highly strained carbon nitrogen double
bond vibration of the azirine ring (10). A b r o a d b a n d in
N N
the region of 1740-1680 revealed the presence of carbonyl
functions of ester and phthalimido groups. B a n d s at 1600
(A) (B)
and 1455 cm -1 accounted for C - - - C s t r e t c h i n g of the
benzene ring, a b a n d at 1070 cm -' accounted for C - - H
No azirine CA) h a s been i s o l a t e d y e t or e v e n
bending and one at 705 cm -1 accounted for an out-of-plane
d e m o n s t r a t e d clearly to be a reaction intermediate. How-
ring b y s e x t a n t s of the benzene ring. I t s N M R s p e c t r u m
ever, it is believed t h a t A is formed first and t h e n rear-
g a v e a s h a r p multiplet at 68.57 showing long r a n g e cou-
ranges very rapidly to B. The r e a r r a n g e m e n t m a y be due
to the high a n t i a r o m a t i c C3,4) nature of A. A n u m b e r of I
pling ( H C - - C - - ) and a multiplet at 67.82 for four protons
m e t h o d s {5-8) to prepare azirines are described in the \\/
literature, b u t the addition of nitrene to acetylenes is a
N
relatively new m e t h o d t h a t g a v e a fairly good yield of
of the benzene ring along with usual signals of f a t t y
azirine in one step. A n d e r s o n et al. (9) first described the
methyl ester. These d a t a confirmed the structure of prod-
*To whom correspondence should be addressed. uct IV as 2-{8-carbomethoxyoctyl)-2-phthalimido-2H-

JAOCS, Vol. 63, no. 1 (January 1986)