Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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cancer suggests that fatty acid synthesis helps pro-

mote tumor growth. This is in marked contrast to its

role as an anabolic energy storage pathway in liver

and adipose tissue, and fatty acid synthesis is now

associated with clinically aggressive tumor behavior

and tumor-cell growth and survival, and has become

a novel target pathway for chemotherapy develop-

ment.

See also: Adipose Tissue: Structure and Function of

White Adipose Tissue; Structure and Function of Brown

Adipose Tissue; Fats: Digestion, Absorption, and

Transport; Requirements; Classification; Fatty Acids:

Properties; Metabolism; Gamma-linolenic Acid; Analysis;

Dietary Importance; Hormones: Pancreatic Hormones;

Steroid Hormones; Triglycerides: Structures and

Properties; Characterization and Determination

Further Reading

Chirala SS, Jayakumar A, Gu A and Wakil SJ (2001)

Human fatty acid synthase: role of interdomain in the

formation of catalytically active synthase dimer. Pro-

ceedings of the National Academy of Sciences, USA 98:

3104–3108.

Coleman RA, Lewin T and Muoio DM (2000) Physiological

and nutritional regulation of enzymes of triacylglycerol

synthesis. Annual Review of Nutrition 20: 77–103.

Dutta-Roy AK (2000) Cellular uptake of long-chain fatty

acids: role of membrane-associated fatty-acid-binding/

transport proteins. Cellular and Molecular Life Science

57: 1360–1372.

Eaton S, Barlett K and Porfarzman M (1996) Mammalian

mitochondrial b-oxidation. Biochemical Journal 320:

345–357.

Girard J (1997) Mechanisms by which carbohydrates regu-

late expression of genes for glycolytic and lipogenic

enzymes. Annual Review of Nutrition 17: 325–352.

Holm C, Osterlund T, Laurell H and Contreras JA (2000)

Molecular mechanisms regulating hormone-sensitive

lipase and lipolysis. Annual Review of Nutrition 19:

365–393.

Hoppel CL (1976) Carnitine palmitoyltransferase and

transport of fatty acids. In: Martonosi A (ed.) The

Enzymes of Biological Membranes, vol. 2, pp. 119–

143. New York: Plenum.

Jump DB (1999) Regulation of gene expression by dietary

fat. Annual Review of Nutrition 19: 63–90.

Kerner J and Hoppel C (2000) Fatty acids import into

mitochondria. Biochimica et Biophysica Acta 1486:

1–17.

Kim K (1997) Regulation of mammalian acetyl-coenzyme A

carboxylase. Annual Review of Nutrition 17: 77–99.

Kuhajda FP (2000) Fatty-acid synthase and human cancer:

new perspectives on its role in tumor biology. Nutrition

16: 202–208.

Rasmussen BB and Wolfe RR (1999) Regulation of fatty

acid oxidation in skeletal muscle. Annual Review of

Nutrition 19: 463–484.

Shanklin J and Cahoon EB (1998) Desaturation and related

modifications of fatty acids. Annual Review of Plant

Molecular Biology 49: 611–641.

Smith S (1994) The animal fatty acid synthase: one gene,

one polypeptide, seven enzymes. FASEB Journal 8:

1248–1259.

Sprangers F, Romijn JA, Endert E, Ackermans MT and

Sauerwein HP (2001) The role of free fatty acids (FFA)

in the regulation of intrahepatic fluxes of glucose and

glycogen metabolism during short-term starvation in

healthy volunteers. Clinical Nutrition 20: 177–179.

Stanley H and Sherratt A (1994) Introduction: the regula-

tion of fatty acid oxidation in cells. Biochemical Society

Transactions 22: 421–422.

Storch J and Thumser AEA (2000) The fatty acid transport

function of fatty acid-binding proteins. Biochimica et

Biophysica Acta 1486: 28–44.

Subrahmanyamm SC, Jayakumar A, Gu A and Wakil SJ

(2001) Human fatty acid synthase: role of interdomain

in the formation of catalytically active synthase dimer.

Proceedings of the National Academy of Sciences, USA

98: 3104–3108.

Sul H and Wang D (1998) Nutritional and hormonal regu-

lation of enzymes in fat synthesis: studies of fatty acid

synthase and mitochondrial glycerol-3-phosphate acyl-

transferase gene transcription. Annual Review of Nutri-

tion 18: 331–351.

Towle HC and Kaytor EN (1997) Regulation of the expres-

sion of lipogenic enzyme genes by carbohydrate. Annual

Review of Nutrition 17: 405–433.

Wakil SJ (1989) Fatty acid synthase, a proficient multifunc-

tional enzyme. Biochemistry 28: 4523–4530.

Gamma-linolenic Acid

F D Gunstone, Scottish Crop Research Institute,

Invergowrie, Dundee, UK

Background

0001Linolenic acid (GLA) is an important member of the

n-6 family of polyunsaturated fatty acids, being an

intermediate in the bioconversion of linoleic acid to

arachidonic acid. Since the formation of GLA is rate-

determining in this sequence of changes there are

several circumstances when it might be desirable to

add it to the diet. GLA is available in seed oils of

evening primrose, borage, and blackcurrant, and

methods of raising the concentration of this acid in

these sources have been described.

Structure

0002g-Linolenic acid (GLA) is all-cis-6,9,12-octadecatri-

enoic acid and may also be designated as 18:3 (n-6).

2308 FATTY ACIDS/Gamma-linolenic Acid

This later term indicates an acid with 18 carbon

atoms and three double bonds starting on the sixth

carbon from the methyl group. It is understood that, in

the absence of other indicators, the double bonds have

a cis configuration and are methylene-interrupted, i.e.,

the unsaturated centers are separated from each other

by one CH

group.



CHCH

cis



CHCH

cis



CHðCH

cis

COOH:

There are two important families of polyunsaturated

fatty acids: the n-6 family based on linoleic acid, and

the n-3 family, based on linolenic acid. Since animals

– including humans – cannot produce linoleic or lino-

lenic acids themselves, these acids are essential parts

of the diet, obtainable from vegetable sources or from

animals that have already derived these acids from

a plant source. Once ingested, the two C18 acids can

be metabolized to important C20 and C22 acids

through a series of changes involving desaturation

and elongation. The same enzymes are required for

both families of acids, and there is competition for

these. The most important acids in these sequences

are arachidonic acid (20:4) in the n-6 series, and

eicosapentaenoic acid (n-3 20:5) and docosahexa-

enoic acid (22:6) in the n-3 series. These acids are

important components of phospholipid membranes,

and the two C20 acids are also precursors of prosta-

glandins and other eicosanoids.

0003 GLA is the first intermediate in the metabolic

conversion of linoleic acid to arachidonic acid. The

change involves introduction of a (third) double bond

at the D6 position under the catalytic influence of

the 6 desaturase enzyme. This step is believed to be

the rate-limiting stage in the metabolic pathway.

For humans in good health, this presents no problem,

but there are some conditions such as aging, smoking,

diabetes, high alcohol intake, stress-related hormones,

and viral infections that inhibit the rate-determining

step and where it is considered wise to supplement the

intake of GLA. Nutritional factors can also influence

this metabolic pathway.

0004 Table 1 shows the series of changes by which lino-

leic and linolenic acids are converted to the long-

chain polyunsaturated fatty acids.

Occurrence of GLA in Seed Oils and

Microorganisms

0005 As an intermediate in the conversion of linoleic acid

to arachidonic acid, it is not surprising that GLA

is found at low levels in many animal fats. It is present

in cow milk fat (*0.1%) and human milk fat

(0.35–1.0%), and its presence in the latter has often

been cited as an indication of its importance. How-

ever, richer sources of this acid are found in the seed

oils of some less common plants and are also pro-

duced by some microorganisms.

0006There are three commercial sources of GLA from

seed oils: evening primrose (Oenothera biennis and

O. lamarckiana), borage (Borago officinalis), and

blackcurrant (Ribes nigrum). Typical fatty acid pro-

files for these three oils are given in Table 2. Ucciani

found GLA in 164 higher plant families belonging to

10 botanical families.

0007Evening primrose oil was the first to be developed

and used as a source of GLA. It contains 6–14%

(generally 9–10%) of this acid and is accompanied

by high levels of linoleic acid. Borage (starflower) is a

richer source of GLA in terms of oil content of the

seed and level of GLA in the oil, but it has less linoleic

acid than evening primrose oil and also contains a

tbl0001Table 1 n-6 and n-3 families of polyunsaturated fatty acids

n-6 n-3

18:2 (9,12) Linoleic 18:3 (9,12,15) a-Linolenic

# a # a

18:3 (6,9,12) g-Linolenic 18:4 (6,9,12,15) Stearidonic

# b # b

20:3 (8,11,14) 20:4 (8,11,14,17)

# c # c

20:4 (5,8,11,14) Arachidonic 20:5 (5,8,11,14,17)

Eicosapentaenoic

# b

22:5 (7,10,13,16,19)

# b

24:5 (9,12,15,18,21)

# a

24:6 (6,9,12,15,18,21)

# d

22:6 (4,7,10,13,16,19)

Docosahexaenoic

a, 6-desaturase; b, elongase; c, 5-desaturase; d, b-oxidation.

tbl0002Table 2 Fatty acid composition of evening primrose oil, borage

oil, and blackcurrant oil and of oils from Mortierella isabellina and

Mucor javanicus

epo

bor

a,b

bck

Mortierella Mucor

Oil (%) 16–26 27–35 22–26

16:0 7–10 10 6–7 27 22–25

18:0 1.5–3.5 4 1.5 6 5–8

18:1 6–11 18 9–11 44 38–41

18:2 65–80 37 46–49 12 10–12

18:3 (n-6) 8–14

22 14–16 8 15–18

18:3 (n-3) 13–14

18:4 (n-3) 2.5–3

epo, evening primrose oil; bor, borage oil; bck, blackcurrant seed oil.

Also 20:1 4%, 22:1 2.5%, 24:1 1%.

Generally 9–10%.

FATTY ACIDS/Gamma-linolenic Acid 2309

series of n-9 higher monoene acids viz. 20:1, 22:1,

and 24:1 at a combined level of about 8%. Blackcur-

rant oil is intermediate between the other two sources

in its level of GLA, but it also contains the 18:3 and

18:4 n-3 acids.

0008 Molds belonging to the Mucorales family produce

GLA as the only 18:3 acid, and commercial processes

have been developed in Japan based on Mortierella

isabelina and in the UK based on Mucor javanicus.

UK production has been discontinued following a

change in ownership of the producer company.

These products differ from the seed oils in their higher

levels of saturated and monoene acids and their

lower level of linoleic acid. This makes the oil more

suitable as a source of pure GLA or of upgraded

triacylglycerols, since the necessary separations are

easier to effect.

0009 The blue–green algae Spirulina platensis (18–21%

GLA) and S. maxima (12% GLA) are also potential

sources of GLA. However, this is present in various

galactosyldiacylglycerols or phosphatidic acids rather

than in triacylglycerols.

0010 The distribution of these fatty acid chains between

the sn-1, 2, and 3 positions has been examined in a

number of ways. Results for GLA are summarized in

Table 3.

0011 Because of the high level of linoleic acid in evening

primrose oil the major triacylglycerols contain two

(or three) linoleic acyl chains: LLL 54.3 mol%, LLG

17.6%, LLO 13.7%, LLP 7.9%, and other 6.5% (L ¼

linoleic, G ¼ g-linolenic, O ¼ oleic, and P ¼ palmitic

acyl group). The triacylglycerol composition of

borage oil is more complex, with 10 molecular

species each exceeding 4 mol%: LLG 15.4, OLG

12.3, LLL 10.1, LGG 9.8, PLG 9.5, PLL/OOG 8.9,

OLL 8.2, SLG/POG 5.0, LLE 4.1, OOL 4.1, and

other 12.6 (symbols as above and E ¼ 20:1, S ¼

stearic and). No sterochemistry is implied in these

symbols, and PLG, for example, represents the sum

of six isomers.

0012 Evening primrose oil grows in many countries in

Europe and also in New Zealand, but for commercial

reasons, the crop is now grown mainly in China (75–

80%) and Eastern Europe. Seed is produced at a level

of 1000–1200 kg ha

1

. Annual production is esti-

mated to be about 10 000–12 000 tonnes of seed

yielding 1300–1500 tonnes of oil. Over 10 years to

1999, the price of the oil fell from $45 to $17.5.

0013Britain is the main source of borage (starflower),

but it is also grown in Holland, Canada, New Zea-

land, and Poland. Annual production of seed is esti-

mated at 3000–4000 tonnes, which provides about

800 tonnes of oil. Borage seed is $3000–4000 per

tonne. The price of the oil fell from $60 to $35 per

kilogram over the 10 years to 1999.

0014Blackcurrant seed oil is a byproduct of the produc-

tion of blackcurrant juice and jelly, and production is

estimated to be perhaps 50–100 tonnes per annum.

0015These oils are most often supplied in capsules with

added tocopherol and ascorbyl palmitate to serve as

antioxidants. The products have a shelf-life of about

3 years.

Sources with Higher Levels of GLA

0016It is possible to raise the level of GLA in these oils by

selective enzymic reaction, and enhanced evening

primrose and borage oils, with twice the normal

level of GLA, have been produced and offered for

sale. These materials are mainly triacylglycerols but

may contain up to 20% of diacylglycerols. Proced-

ures are based on the fact that some enzymes discrim-

inate against acids having a double bond close to the

acyl function and GLA with D6 unsaturation can be

distinguished from the more common unsaturated

acids with D9 unsaturation (oleic and linoleic). This

differentiation is apparent in hydrolysis, esterifica-

tion, acidolysis, and alcoholysis reactions promoted

by appropriate lipases. The following examples are

typical.

0017Borage oil (22% GLA) is hydrolyzed completely

(Pseudomonas species, 35



C, 24 h), and the acids

are selectively esterified with lauric alcohol in

the presence of Rhizopus delemar lipase at 30



for 20 h. The latter enzyme discriminates against

GLA, and this acid is concentrated in the unester-

ified acids, which finally contain 70% GLA (74%

recovery). If the esterification is repeated, 94%

GLA is obtained with 68% recovery (C. rugosa,



C, 15 h).

0018Selective hydrolysis of borage oil (22% GLA) with

C. rugosa lipase at 35



C for 15 h gives glycerol

esters with 46% of this acid that can hardly be

raised above this value. After removal of free

acids, a second hydrolysis raises the concentration

to 54% with 76% recovery. On a pilot-plant scale,

7 kg of borage oil gives 1.5 kg of upgraded oil (56%

tbl0003 Table 3 Distribution of GLA between the sn-1, 2, and 3

positions in evening primrose, borage, and blackcurrant seed

oils

epo

bor

bck

Tag 9.6 9.3 24.8 15.9

sn-1 7.2 3.6 4.0 4.1

sn-2 10.7 10.7 40.4 17.4

sn-3 10.9 13.5 30.1 25.8

Forabbreviations,see Table 2.

From Laakso P and Christie WW (1990) Lipids 25: 349–353.

From Lawson LD and Hughes BG (1988) Lipids 23: 313–317.

2310 FATTY ACIDS/Gamma-linolenic Acid

GLA) after removal of free acid by molecular dis-

tillation. Starting with an already upgraded borage

oil (10 kg, 45% GLA), high-quality GLA (2.1 kg,

98% pure, 49% recovery) is obtained by hydrolysis

and two separate partial esterifications.

There are several reports of plants such as rapeseed/

canola genetically modified to produce GLA, but

commercial products are not yet available. One

example that has been cited has the following fatty

acid composition: 16:0 4.2%, 18:0 3.7%, 9–18:1

21.6%, 11–18:1 3.4%, 18:2 26.0%, 6,9,12–18:3

37.0%, 9,12,15–18:3 1.3%, and other 2.8%.

Nutritional and Medical Uses

0019 The major applications for the oils are in the area of

healthfood supplements, but markets have also been

developed in infant nutrition, pet food, and cosmet-

ics, and the total market for these oils is around $50

million per year.

0020 Available evidence indicates that only around

5–10% of the daily intake of linoleic acid can be

converted to GLA and beyond. For a 60-kg adult

with a dietary intake of 5–20 g day

1

of linoleic acid,

the endogenous rate of formation of GLA will be

250–1000 mg day

1

or around 4–17 mg kg

1

day

1

0021 Human breast milk contains 100–400 mg l

1

GLA þ DGLA (20:3 n-6). A 5 kg baby consuming 1 l

of milk per day receives 20–80 mg kg

1

day

1

of these

two acids combined. DGLA is converted to two main

eicosanoids: PGE

and 15-hydroxy DGLA. PGE

(prostaglandin E

) is antiinflammatory, antithrombo-

tic, a vasodilator, and stimulates the formation of

cyclic adenosine monophosphate, which inhibits

phospholipase A

, whereas 5-hydroxy DGLA is a

potent natural antiinflammatory agent inhibiting the

conversion of AA (arachidonic acid) to its 5- and 12-

lipoxygenase metabolites. GLA and DGLA are con-

sidered to be useful in the treatment of diabetes,

atopic eczema, inflammation, stress, cardiovascular

disease, and cancer.

0022 Soft gelatin capsules containing evening primrose

oil have been sold as nutritional supplements for

more than 20 years and of borage oil (also marketed

as starflower oil) for more than 10 years. Samples

may be blended with other oils (fish, linseed) or

have added vitamins, minerals, or herbal extracts.

Nutritional supplements probably account for 80%

of the total usage of GLA oils.

0023 GLA has been used to treat a wide range of condi-

tions including: atopic eczema, dermatitis and other

inflammatory skin conditions, diabetic neuropathy,

breast pain, premenstrual syndrome, high blood pres-

sure, and cancer, but not all of these claims have been

confirmed.

0024Doses have not been fully defined, but the

following levels have been recommended (levels

refer to GLA and not to GLA-containing oils): nutri-

tional purposes (25–50 mg day

1

), therapeutic use

(100–500 mg day

1

), and pharmacological effect

(500–2000 mg day

1

0025Two products have been licensed as pharmaceut-

icals Efamast

for relief of cyclical mastalgia and

Epogam

for treatment of atopic eczema. There are

also a range of preterm and term infant formulations

on the market (particularly in Europe) containing

GLA from various sources. Levels of GLA in infant

formula range from 0 to 0.9%. Many skin-care

products (creams, lotions, soaps) contain GLA oils

at levels between 0.1 and 2.0%.

See also: Essential Fatty Acids; Fatty Acids:

Metabolism; Dietary Importance; Vegetable Oils: Types

and Properties; Oil Production and Processing;

Composition and Analysis; Dietary Importance

Further Reading

Christie WW (1999) The analysis of evening primrose oil.

Industrial Crops and Products 10: 73–83.

Clough P (2001) Specialty vegetable oils containing gamma

linolenic acid and stearidonic acid. In: Gunstone FD

(ed.) Structured and Modified Lipids. New York: Marcel

Dekker.

Gunstone FD (1992) Gamma linolenic acid – occurrence

and physical and chemical properties. Progress in Lipid

Research 31: 145–161.

Horrobin DF (1992) Nutritional and medical importance of

gamma linolenic acid. I. Progress in Lipid Research 31:

163–194.

Huang Y-S and Mills DE (1996) g-Linolenic Acid Metabol-

ism and its Roles in Nutrition and Medicine. Cham-

paign, IL: AOCS Press.

Huang Y-S and Ziboh A (2001) Recent Advances in Bio-

technology and Clinical Applications of Gamma Lino-

lenic Acid. Champaign, IL: AOCS Press.

Ucciani E (1995) Sources potentielles d’acide gamma-

linolenique: une revue. Oleagineux Corps gras Lipides

2: 319–322.

Analysis

E W Hammond, Greenisle-Consulting, Warkton,

Kettering, UK

Background

0001Fatty acids occur in food lipids mainly as esters

of glycerol. Fatty acids are represented in food

lipids (typical ranges shown in brackets) by

FATTY ACIDS/Analysis 2311

triacylglycerides (> 90%), diacylglycerides and mono-

acyglycerides (in the range 0.5–4%), other polar

lipids such as lecithin (0.5–5%), phytosterol esters

(up to 1%), and free fatty acids (up to 1%).

Extraction of Lipids from Foods

0002 The extraction of lipids from foods in order to ana-

lyze their fatty acids is often difficult and specialized.

For example, foods may be essentially anhydrous

(e.g., biscuits, wheat flour) or wet (e.g., fresh meats,

fish). Each food type may require very different

handling to obtain the total lipids present. The fat

in biscuits can be obtained just by suspending the

crushed samples in light petroleum. However, to

obtain the total lipids from flour requires rigorous

extraction with solvents such as n-butanol. Samples

containing water in any quantity require dehydrating

first, which process might change the lipid materials,

or extraction with alcohol based solvent mixtures.

The references included in the bibliography cover a

range of techniques for specific materials.

Determination of Total Free Fatty Acids

0003 Free fatty acids (FFA) occur in refined edible oils at

less than 0.1% (w/w) and in crude oils typically from

1 to 15%. FFA at such levels are present because of

lipid hydrolysis and not as a normal natural com-

ponent of vegetable or animal lipids. The high levels

sometimes found in crude oils are as a consequence of

tissue damage leading to the release of natural lipases.

Tropical fruits such as palm are particularly prone to

high FFA levels in crude oils. (See Phospholipids:

Determination; Triglycerides: Characterization and

Determination; Vegetable Oils: Composition and

Analysis.)

0004 The normal method of determination is titration

(AOAC28:029–28:034) with a solution of potassium

hydroxide [approximately 0.1 M in 95% (v/v) etha-

nol]. This procedure is applicable to all oils and fats

that are soluble in the solvent mixture 1/1 (v/v) of

ethanol 95% (v/v) and diethyl ether. The titrimetric

procedure uses an indicator solution which is phenol-

phthalein [10 g l

1

in ethanol 95% (v/v)], for deter-

mining the end point. In the case of fats, which

give colored solutions, a potentiometric technique

should be used to determine the end point. The result

of the titration is the average of two duplicate

determinations and is expressed as one of the

following:

0005 Acid value (AV): the number of milligrams of

potassium hydroxide required to neutralize 1 g

of the fat. This is given by the formula:

A:V: ¼ð56:1PVÞ=m,

where V ¼ number of milliliters of potassium

hydroxide; P ¼ exact molarity of potassium hy-

droxide; and m ¼ mass (g) of the test portion.

0006FFA%: the acidity in per cent given by the

formula:

Acidity ¼ðPVM

Þ=ð10mÞ,

where V ¼ number of milliliters of potassium

hydroxide; P ¼ exact molarity of potassium

hydroxide; M

¼ relative molecular mass (see

Table 1); and m ¼ mass (g) of the test portion.

0007When determining FFA%, it is conventional to

express the value as ‘oleic acid.’ However, where the

type of fat is known, the relative molecular mass

should be used for improved accuracy. This can be

calculated from the average molecular weight of fatty

acids, determined from the analysis of fatty acids by

gas chromatography. Otherwise, the values for M

,as

shown in Table 1, should be used.

0008To maintain precision, the size of sample to be used

in the determination will depend on the expected level

of FFA and should follow the data in Table 2.(See

Chromatography: Gas Chromatography.)

Method: Total FFA by Titration

1. 0009A quantity of the solvent mixture (1/1 ethanol/

diethyl ether) should be neutralized, just prior to

use, by dropwise addition of 0.1 M potassium hy-

droxide solution after adding the phenolphthalein

indicator at the rate of 0.5 ml per liter of solvent.

0010The quantity of sample required is determined

by reference to Table 2. Duplicate samples are

weighed out accordingly, paying attention to

accuracy. The weights are recorded. The samples

are dissolved in 50–150 ml of the solvent, each.

tbl0001Table 1 Total free fatty acids

Type of fat Expressedas M

Coconut, palm kernel Lauric acid 200

Palm oil Palmitic acid 256

All other oils Oleic acid 282

tbl0002Table 2 Sample mass required for determination of FFA

Expected

acid value

PercentageFFA Mass of test

portion (g)

Accuracy of

weighing (g)

<1 (<1) 20 0.05

1–4 (<2) 10 0.02

4–70 (2–10) 2.0 0.01

20–70 (10–40) 0.5 0.001

>70 (>40) 0.1 0.0002

2312 FATTY ACIDS/Analysis

3.0011 While the solution is stirred continuously, titration

with 0.1 M potassium hydroxide should proceed

to the end point. The end point is a pink color,

which should persist for at least 10 s. The volume

of titrant is recorded.

Notes:

0012 If the FFA is very low (< 0.2%), atmospheric

carbon dioxide may interfere significantly. It is

useful to replace the air in the titration flask with

nitrogen.

0013 If the solution becomes cloudy during titration,

the volume of neutralized solvent may be in-

creased. Warming should be avoided.

0014 If the quantity of hydroxide required for titration

exceeds 10 ml, a solution of 0.5 M may be used.

Determination of Individual Free Fatty

Acids

0015 Certain situations arise where it may be advantageous

to determine a single fatty acid or the distribution of

fatty acids in the FFA fraction of a fat or oil. In this

case, titration is of little use, since it does not discrim-

inate between the different fatty acids. The quickest

method is to use gas chromatography with an added

internal standard. In this method, quantitation of

both individual and total FFA is obtained and can be

measured down to 0.001% with confidence.

0016 It is possible to analyze free fatty acids directly by

capillary gas–liquid chromatography (GLC) using

special deactivated acidic phases, such as Restek

Stabilwax-DA (Stabilwax-DA crossbond fused silica

capillary column; Thames Restek UK Ltd., Berkshire,

UK). These reduce the tailing effects and nonpropor-

tional losses that are a consequence of hydrogen

bonding of FFA on the column. Analysis of deriva-

tives is advised to reduce quantitative errors that can

result.

0017 Where FFA is to be analyzed directly, it should first

be isolated by thin-layer chromatography (TLC) or

clean-up column procedures (see below). Prior to this,

an internal standard of heptadecanoic acid (C17:0)

should be added to the fat at a level consistent with

the expected level of FFA present in the fat. (See

Chromatography: Thin-layer Chromatography.)

0018 The FFA, dissolved in toluene (up to 5 mg ml

1

total FFA), is injected (0.5 ml) using the direct on-

column technique. The column can be a 30-m length

of 0.53-mm ID fused silica, with a 0.25-mm, cross-

bonded film suitable for the analysis of underivatized

fatty acids (see Figure 1). A 1.0-m length of fully

deactivated, blank fused silica should be fitted as

a ‘retention gap.’ Typical conditions for analysis

are shown in Figure 1. A full profile, including

unsaturated fatty acids, from butyric acid (C4:0) to

arachidic (C20:0) acid can be achieved within 30 min.

0019Where the chain-length distribution of the FFA is

wider than C14 to C20, it may be necessary to apply

correction factors to the detector response. The flame

ionization detector responds to nonoxidized carbon

in a linear relationship. However, the carboxyl carbon

is oxidized and does not respond; therefore, formic

acid gives no response, while acetic shows the re-

sponse of one carbon, and so on through the series.

Since an internal standard is used in this method, it is

a simple job to determine the response factors for the

FFA of interest relative to this standard.

Analysis of Derivatized FFA

0020Methods There are often problems in the measure-

ment of FFA by GLC. It is advisable to derivatize for

improved accuracy. Again, it will be necessary to use

an internal standard of C17:0 and relative response

factors. Two approaches may be used: methyl esters

of FFA or silyl ethers of FFA.

0021In the case of derivatives of FFA, one must consider

whether they are to be analyzed free (isolated) or in

the presence of other lipid classes such as triacylgly-

ceride (TAG) and partial glycerides. If short-chain,

volatile FFA is present (e.g., in butterfat), it is best

not to have any concentration step. By far the most

specific reagent is diazomethane, but care must be

exercised in its use, as the reagent is highly toxic and

potentially explosive.

0022The preparation of methyl esters in this way will

provide good quantitative data on the FFA portion of

the fat only. If silyl ethers are prepared, and the whole

fat mixture is analyzed by GLC, total fat information

is obtained.

0023Methyl Esters via Diazomethane An appropriate

amount (up to 50 mg) of the isolated FFA is dissolved

in diethyl ether (2 ml) containing a few drops of

methanol. The diazomethane in diethyl ether solution

is prepared, with all manipulations carried out in a

fume cupboard. Sufficient diazomethane solution is

added to the sample to leave a slight excess of yellow

color remaining. The mixture is left for no longer

than 5 min; otherwise, artificially high results may

occur. Formic acid in methanol (10%) solution is

added dropwise to remove the excess reagent. This

solution is now ready for analysis without concen-

tration. The type of GLC column used is typically a

30 m 0.32 mm i.d. fused silica, with a 1.0-m reten-

tion gap of fully deactivated fused silica. A polar

phase of bonded FFAP at 1.0-mm film thickness is

suitable. The sample is injected using the on-column

technique and not split injection. The carrier gas may

be hydrogen (linear velocity 40 cm s

1

) or helium

FATTY ACIDS/Analysis 2313

(linear velocity 20 cm s

1

). The column initial tem-

perature will depend upon the type of sample but

typically is 100



C, held for 5 min. The temperature

is then programmed to 200



Cat5



Cmin

1

. The

elution order and profile will appear very similar to

that for FFA above (Figure 1), with the advantage that

peak width and retention time will be improved.

0024 O-Trimethylsilyl Ethers (OTMSi) The most useful

reagents for the derivatization of fats are bis-

trimethylsilyl acetamide (BSA) and trimethylsilyl imi-

dazole (TSIM) (Pierce Chemical Co.), with the latter

being the stronger reagent, particularly when diacyl-

glycerides (DAG) and monoacylglycerides (MAG)

are present. Derivatization is achieved in the neat

reagent or in a solution of the lipid in chloroform or

tetrahydrofuran (10 mg ml

1

). During the procedure,

the anhydrous solution is warmed at 50



C for 5 min

in a closed vial. In this reaction, all free hydroxyls and

carboxyls are derivatized. Normally, 100% volume

excess of the reagent over the fat is sufficient, where

the level of FFA and partial glycerides is not abnor-

mally high. The minimum level of reagent will be 50–

100-fold molar excess over total free hydroxyls and

carboxyls. When using chloroform, allowance must

be made for the 2% of ethanol stabilizer. The

resulting solution is stable for up to 5 h if maintained

anhydrous in a closed vial. After this time, it should

be discarded. The solution is injected directly ‘on

column’ and not via a split technique. For GLC, use

a column of fused silica, 0.53 mm i.d. by 10 m in

length with a bonded phase of OV1, OV101 (or

min. 4 8 12 16 20 24

c-gram#162

fig0001 Figure 1 Organic acids and free fatty acids [Stabilwax DA, direct injection; 30 m, 0.53 mm i.d., 0.25 mm Stabilwax-DA (cat. #11025),

0.5 ml direct injection of a 5 mg ml

1

standard]. Oven temperature: 100



C (hold for 2 min) to 250



Cat8



C min

1

. Injection and detection

temperature: 280



C. Carrier gas: helium. Linear velocity: 40 cm s

1

(flow rate: 5.2 cm

min

1

). FID sensitivity: 8  10

11

AFS. 1: C2:0,

acetic acid; 2: C3:0, propionic acid; 3: C4:0, butyric acid; 4: C5:0, valeric acid; 5: C6:0, caproic acid; 6: C7:0, enanthic acid; 7: C8:0,

caprylic acid; 8: C9:0, capric acid; 9: C10:0, lauric acid; 10: C11:0, myristic acid; 11: C12:0, pentadecanoic acid; 12: C13:0, palmitic acid;

13: C14:0, palmitoleic acid; 14: C15:0, stearic acid; 15: C16:0, oleic acid; 16: C17:0, linoleic acid; 17: C18:1, linolenic acid. Reproduced by

permission of Restek Corporation.

2314 FATTY ACIDS/Analysis

equivalent) of film thickness 0.1–0.2 mm. A 1.0-m

length of fully deactivated, blank fused silica is fitted

as a ‘retention gap.’ The helium carrier gas is set high

at about 30 ml min

1

and a start temperature of 50



held for 5 min. The temperature is then programed at



C min

1

up to 320



C in a single ramp. The elution

order is fatty acids, MAG, DAG, and TAG in order of

molecular weight. The method is suitable for neutral

fat extracts. In some fats, the level of free sterols is

significant, and OTMSi ethers of sterols will appear

on the chromatogram.

Fatty Acid Methyl Esters from

Triacylglycerides

0025 There are a number of methods used to prepare fatty

acid methyl esters (FAME) from TAG to enable their

analysis by GC. The boron trifluoride/methanol re-

agent is widely used (AOAC -28:057). The reagent is

toxic and unstable during storage, and has been

found to be prone to form artefacts from some oxy-

genated, cyclic, and some polyunsaturated fatty acids.

0026 Transmethylation techniques are very rapid, but

they produce FAME only from glyceride esters and

not from any FFA component. It is important, also,

that the reagent and fat solutions are anhydrous;

otherwise, significant (and even large) amounts of

unesterified FFA can result. Of this group, 0.5 M

sodium methoxide/methanol is widely used. This

reagent is hazardous to prepare and dispose of.

Anhydrous potassium hydroxide/methanol (0.2 M)

is perhaps a more preferable reagent, being easy to

prepare as required, although storage is acceptable

provided it is kept dry. It should be noted that if

conjugated unsaturated fatty acids are known to be

present, they might be unstable in alkaline reagents.

These reagents should be stored in bottles with poly-

carbonate stoppers; glass stoppers will fuse to glass

bottles.

0027 For up to 50 mg of sample in 3–5 ml of reagent

(with 1.0 ml of toluene added as solubilizer), heated

to 50



C, the reaction time is normally 15 min. At the

end of reaction, the sample is allowed to cool, and

5 ml of aqueous acetic acid (5%) is added carefully,

followed by up to 10 ml of hexane (dependent upon

sample size) such that the ester concentration is about

5mgml

1

(suitable for direct sampling on to GLC).

This mixture needs to be shaken thoroughly and

allowed to separate. The lower layer is removed by

aspiration and discarded. A 5-ml aliquot of water is

added, and the mixture shaken and separated again.

Most of the top layer is then transferred to a vial

containing about 2 g of anhydrous sodium sulfate,

which dehydrates the solution ready for GLC. No

concentration step should normally be necessary.

0028Acid methanolysis reagents methylate most lipid

classes including FFA. They require relatively long

reaction times but very short processing times,

which means that technicians are not tied to one

technique for long periods. Since the reagents are

acidic, they are not suitable for epoxy fatty acids or

other acid-labile fatty acids. Anhydrous hydrochloric

acid/methanol (typically 5% or saturated) is suitable

and popular. This produces methyl chloride during

storage. In addition, while it is easy to use and pre-

pare by bubbling hydrogen chloride gas from a cylin-

der into anhydrous methanol, this can be hazardous.

Alternatively, acetyl chloride (50 ml) can be added to

cold (5



C) anhydrous methanol (450 ml); methyl

acetate is formed as a byproduct. However, several

workers, when using this latter reagent, have reported

yield problems.

0029Sulfuric acid/methanol with toluene as solubilizer

(1:10:20 by volume of sulfuric acid–toluene–metha-

nol) is a convenient reagent. It is easy and safe to

prepare, is stable at ambient for long periods, and is

easy to use. Sulfuric acid is added to cooled and

stirred methanol, followed by the toluene. The solu-

tion is stored in a brown stoppered bottle and must be

kept dry. Acid-resistant gloves and a face shield

should be worn while mixing in the acid. For use,

5 ml of reagent is added to up to 50 mg of sample,

which is refluxed for 60 min in a tube. The sample is

then diluted with 5 ml of water. A volume of hexane is

added (to give an approximate sample concentration

of 5 mg ml

1

), and the mixture is shaken thoroughly.

On separation, the bottom layer is aspirated to waste.

A further 5 ml of water is added and the mixture

shaken. The top layer is transferred to a vial contain-

ing 2 g of anhydrous sodium sulfate. This solution is

then ready for analysis by GLC. If short-chain (< C10)

fatty acids are present, the toluene may interfere with

the GLC analysis. In this case, a reagent made up

without the toluene solubilizer should be used, but

note that an extended reflux (90 min) may be neces-

sary, unless the fat dissolves rapidly.

Chromatographic Methods

0030Some of the specific points have been covered above

and should be considered in combination with this

information.

Thin-layer Chromatography (TLC)

0031FFA is easily separated from other lipid classes on

silicic acid TLC plates. This technique is excellent

for preparative work. Plates coated with Kieselgel

with (‘G’) or without (‘H’) calcium sulfate binder can

be used. The plates should be activated before use.

For speed, individual plates may be placed flat on the

FATTY ACIDS/Analysis 2315

turntable in a microwave oven for 2 min at full power

and then cooled. Multiple samples may be spotted in

lanes or, for preparative work, the sample may be

applied as a continuous streak 1 cm above the base

of the plate. The sample area is focused before the

main chromatography by developing the plate to just

above the sample application line twice in a solvent of

diethyl ether. The plate is air-dried between each

focus development, and then developed in the main

solvent, which, for FFA, is a mixture of diethyl ether/

light petroleum 40–60



with added formic acid (pro-

portions 18/82/1 by volume). After development to

within 1 cm of the top of the plate, the plate is air-

dried and sprayed lightly with a methanolic solution

of dichlorofluorescein (0.1%). After drying, the plate

is viewed under ultraviolet (UV) light (254–320 nm),

which causes most lipid components to fluoresce.

Lipids that absorb in the UV (e.g., conjugated unsa-

turates) appear as violet spots. FFA lies between TAG

and DAG with an Rf of approximately 0.6. If there is

some confusion over the position of the FFA, a stand-

ard can be run in 1-cm lanes at the sides of the plate.

Samples that contain FFA with a wide range of chain

lengths (C4 to C20, for instance) often give a broad

band or even double bands. The longer-chain FFA is

less polar than the short-chain FFA and therefore runs

slightly higher up the plate. The FFA band should be

marked, scraped off into a sinter glass disc filter stick,

and eluted off with diethyl ether. Careful concentra-

tion yields the dry FFA fraction. If small or volatile

fractions are suspected (this is particularly useful for

radioactive labeled fractions), they may be stabilized

prior to concentration by the addition of a known,

small amount of 10% potassium hydroxide in metha-

nol (enough to form the salts). After concentration,

the salts are dissolved in a small volume of 1:1 diethyl

ether:methanol containing sufficient formic acid to

re-form the acids prior to methylation.

High-performance Liquid Chromatography (HPLC)

0032 It is recommended that FFA be analyzed by GLC, but

for various reasons, the use of HPLC may be dictated.

To overcome the lack of a chromaphore, most workers

use UV-absorbing derivatives of FFA. The most popu-

lar and successful has been the phenacyl ester. The

dansyl piperazide derivative has been applied success-

fully for fluorescence. (See Chromatography: High-

performance Liquid Chromatography.)

0033 Typical chromatography conditions for phenacyl

esters include a 25-cm 4-mm i.d. column packed

with C18 reversed phase material of 5-mm particle

size. A solvent of acetonitrile–water (80:20, v/v) is

run isocratically at 2.0 ml min

1

for the first 30 min.

The solvent is then linearly programed to 85:15 (v/v)

for another 15 min. There is a considerable overlap of

peak compositions, and it is essential that a full set of

standard materials is chromatographed to determine

retention behavior and times. For instance, myristic

acid (C14:0) has an elution time of about 16 min,

whereas arachidonic acid (C20:4) elutes around

18 min. Stearic acid (C18:0) has an elution time of

about 50 min.

Gas–liquid Chromatography (GLC)

0034This item has been covered earlier. However, the ap-

plications described are designed for capillary GLC.

Modern instruments together with capillary or wide

bore columns are relatively easy to use and produce

high-quality results. However, the methods do not

preclude the use of packed columns, although the

chromatography of unesterified FFA is less successful.

For esterified FFA, packed columns of either 2 or

4 mm i.d. by about 2 m in length should be used,

with a carrier gas of nitrogen at 30 ml min

1

for a

2-mm column and 60 ml/min for a 4-mm column. For

the column packing, a phase of 10% SP2330 on 100/

120 mesh Supelcoport should be used, and this should

be operated isothermally at 180



C or programed

from 50 to 200



Cat5



Cmin

1

after holding for

5 min. The sample solution is then injected directly

on-column into a heated injection area set isother-

mally at 200



Determination of

Trans

Fatty Acids

0035Isolated, methylene-interrupted trans double bonds

show an infrared (IR) band absorbing at about

967 cm

1

. This absorption may be used to estimate

the trans content in edible fats. The AOAC publishes

a typical method AOAC 28.052–28.067 for trans

values (TV). There are some drawbacks with the IR

method, which can lead to some inaccuracies. Essen-

tially, when TV is measured on triacylglyceride, the

value obtained is some 2 units higher than when the

same sample is measured as methyl esters. This prob-

lem is worse for samples below 15% trans. Also,

isomerized or oxidized fats (mainly) exhibit conju-

gated species that show absorption close to the isol-

ated trans bond. This interferes with the correct

allocation of baseline and makes low values less easy

to define accurately. The following method takes into

account these problems and introduces the use of

standards and references, although it must be remem-

bered that problems still exist with the determination

of absolute TVs.

0036The method measures the TV on triacylglyceride,

but we recommend conversion to methyl esters for

values of less than 5% trans. The values are expressed

as the percentage of trielaidin compared with a stand-

ard curve of trielaidin in tristearin. Confusions in

2316 FATTY ACIDS/Analysis

interpretation exist where there are compounds pre-

sent that show absorption in the region 970 cm

1

Pure trielaidin and tristearin (99%) are required as a

standard and reference, together with a recording

double-beam IR spectrometer suitable for quantita-

tion between 1100 and 900 cm

1

and a matched pair

of cuvettes of path length 1 mm (deviation < 1%) with

windows of sodium chloride or potassium bromide.

The solvent is carbon disulfide and solutions and

measurements are all at 20



C. The solvent is toxic,

and therefore, all manipulations of open solutions

should be done in a fume cupboard and appropriate

solvent-resistant gloves must be worn. Alternatively,

the use of Fourier transform infrared (FTIR) greatly

improves the methodology and accuracy.

Method: Determination of

Trans

Value by IR

Spectroscopy

1.0037 The spectrometer is set to record in the range

1050–900 cm

1

, with a narrow slit and slow

recording speed. (See Spectroscopy: Infrared and

Raman.)

0038 Solutions are prepared according to Table 3 and

made up to exactly 10 ml in volumetric flasks.

0039 A cuvette is filled with solution 1 and placed in

the reference beam. The absorbances of the other

solutions are recorded against this.

0040 For each spectral record, a straight baseline is

drawn joining the minima at 1000 and 925 cm

1

The absorbance of the peak is calculated. If

transmission is recorded, the absorbance can be

determined by the formula:

Absorbance A ¼ log

ðBD=BCÞ,

where B is the zero/? chart point, D is the center

point of the baseline, and C is the peak apex.

0041 The calibration line is constructed (this should be

a straight line).

0042 If necessary, all samples are liquefied and hom-

ogenized. Solutions in carbon disulfide of 200 mg

(to the nearest 0.1 mg) in 10-ml volumetric flasks

are made up to volume.

0043 All sample solutions are read against the same

reference solution 1 (tristearin). The baselines are

constructed, and the peaks are measured as above.

0044The values (equivalent to trielaidin) for samples

are read off the standard line, and the percentage

trans from the sample weight is calculated.

0045If there is a significant degree of interference to the

construction of the baseline, the samples should be

converted to methyl esters. This should also be

done where the TV is below 5%. In this method,

the standard is methyl elaidate, and the reference

becomes methyl stearate. All other manipulations

are the same.

See also: Chromatography: Thin-layer Chromatography;

High-performance Liquid Chromatography; Fats:

Classification; Phospholipids: Determination;

Spectroscopy: Infrared and Raman; Triglycerides:

Characterization and Determination; Vegetable Oils:

Composition and Analysis