A Primer on Fatty Acids and AnalysesMike Dugan

Meat Lipid ScientistAAFC-Lacombe

• Fat in beef is needed for–Flavour–Tenderness–Nutritional value

Meat Lipid studies try to understand and manipulate the feed to meat lipid conversion to

improve fatty acid profiles

• Fats in beef are composed of phospholipids and triglycerides.

• Phospholipids are found in membranes and serve structural roles.

• Triglycerides are found in marbling fat and basically store energy.

Cell Membrane

• Triglycerides have a glycerol backbone and 3 fatty acids

• Phospholipids have a glycerol backbone, a polar phosphate group and 2 fatty acids.

Fatty Acid

Fatty Acid

P ex. Choline

Fatty Acid

Fatty Acid

Fatty Acid

• Fatty acids are mostly made up of carbon and hydrogen

Hydrogen Carbon

Hydrogen can bond to CarbonCarbon can bond to Carbon

As a single or double bond

• Fatty acids are made up of a carbon chain with an acid group (carboxyl) attached at one end.

• when the rest of the bonds are taken up by hydrogen it a saturated fatty acid (SFA)

Acid

• With one double bond it’s a monounsaturated fatty acid (MUFA)

• With more than one double bond it’s a polyunsaturated fatty acid (PUFA)

• Double bonds can have 2 configs.

• hydrogen same side it’s cis

• hydrogen on opposite sides trans.

trans

cis

cis• A cis double bond bends the molecule,

• Fatty acids can’t pack together closely, decreases melting and boiling point

• Trans double bonds put a kink in the fatty acid

• FAs can pack together, has properties similar to saturated fatty acids.

trans

• Fatty acids are usually referred to by their trivial name or by chemical short-hand

• Common trivial names:

Saturated MUFA PUFA CLAStearic Oleic Linoleic (omega-6) RumenicPalmitic Vaccenic Linolenic, EPA, DHA

(all omega-3)

• There are 2 common shorthand systems• The Delta System – numbers carbons from the

acid (or delta end)• The Omega System – numbers carbons from

the methyl (or omega end).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

delta omega

Linoleic acid

c9,c12-18:2Delta System

c = cis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

delta omega

Linolenic acid

c9,c12,c15-18:3Delta System

Vaccenic Acid

t11-18:1Delta System

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

delta omega

t = trans

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

delta omega

Rumenic Acid

c9,t11-18:2Delta System

(main natural type or isomer of CLA)

2 double bonds

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

2 carbons

• CLA refers to a group of fatty acids• CLA’s have 18 carbons & 2 double bonds

separated by 2 carbons• Double bonds can be found at different places

along the carbons chain

• Besides Rumenic Acid (9c,11t-18:2), CLAs that can be found in beef include:

t7,c9-18:2t8,c10-18:2t10,c12-18:2t11,c13-18:2c12,t14-18:2

t7,t9-18:2t8,t10-18:2t9,t11-18:2t10,t12-18:2t11,t13-18:2t12,t14-18:2

c7,c9-18:2c8,c10-18:2c9,c11-18:2c10,c12-18:2c11,c13-18:2c12,c14-18:2

• Second shorthand system: The Omega System• Fatty acids named using this system:

– have all double bonds in cis configuration– adjacent double bonds are separated by 3 carbons

(methylene interrupted)– The system works for most fatty acids synthesized

by plants and animals– The system makes it easy to identify related series

of fatty acids (omega-6 and omega-3 fatty acids)

Linoleic acid

18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

delta omega

18:2n-6Omega System

c9,c12-18:2Delta System

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

delta omega

This stands for:18 carbons: 2 double bonds – first double bond at omega-6 carbon

Linolenic acid

18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

delta omega

18:3n-3Omega System

c9,c12,c15-18:3Delta System

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

delta omega

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

delta omega

• Plants and animals can add double bonds.

Plants

Animals Required

Linoleic acid18:2n-6

Linolenic acid18:3n-3

Used to make LCOmega-3 fatty acids

EPA 20:5n-3

DPA 22:5n-3

DHA 22:6n-3

Arachidonic acid

20:4n-6

Used to make LCOmega-6 fatty acids

• The two essential fatty acids are:

Beef Lipid AnalysesBeef Lipid Analyses

-2-Then homogenize

with organic solvent (2:1 CHCl3:CH3OH)

-1-First cut, grind and

mix to get a representative

sample

-3-Filter & add water

to separate out the pure lipids

+

Acid or Base

Methanol

Fatty Acid Methyl Ester (FAME)

Lipids

To analyze individual fatty acids…

GC and HPLC analysis

• Base catalyst works well for backfat because it contains mostly triglyceride.

• Base catalyst doesn’t work for all meat lipid classes (FFA, SM, DMA).

• Acid catalysts are a problem in meat when analyzing conjugated linoleic acid (CLA).

• For meat we combine results from analyses of separate acid and base methylations.

For GC Analysis

Inject on to column

Increase oven temperature

• On the column: separate based on boiling point and polarity– Short chains move faster than long chains– Saturated move faster than unsaturated– Trans move faster than cis

Flame Ionization Detector

• If you’d like to analyse the MAJOR fatty acids in beef, this can be done with

– Acid catalyzed methylation– One 15 minute GC analysis with automated peak

measurements

• To COMPREHENSIVELY analyze beef fatty acids– Acid and base methylation– 3 separate GC analyses– 1 HPLC analyses– 6 hours of machine time and time to make sure the

smaller peaks are identified and measured correctly

• In a feed sample we typically measure about 15 fatty acids

Saturates MUFA PUFA12:0 c9-16:1 18:2n-614:0 c9-18:1 18:3n-316:0 c11-18:1 20:2n-618:0 c11-20:120:0 c13-22:122:024:0

Intermediates of PUFA hydrogenation

• In a beef sample we analyse ~80 fatty acids

Feed or Animal

Saturates MUFA PUFAC10:0 c9-14:1 C18:2n-6C12:0 c9-16:1 C18:3n-6C14:0 c9-18:1 C18:3n-3C16:0 c9-20:1 C20:2n-6C18:0 c11-20:1 C20:3n-6C20:0 c13-22:1 C20:4n-6C22:0 t6-t7-16:1 C20:5n-3C24:0 t10-16:1 C22:3n-3C13:0 t11/t12-16:1 C22:4n-6C15:0 t6-t8-18:1 C22:5n-3C17:0 t9-18:1 C22:6n-3C19:0 t10-18:1 t9c11-CLAC14:0iso t11-18:1 c9,t11-CLAC15:0iso t12-18:1 t8,c10-CLAC15:0ai t13-t14-18:1t7,c9-CLAC16:0iso t15-18:1 t12,t14-CLAC17:0iso t16-18:1 t11,t13-CLAC17:0ai c9-15:1 t10,t12-CLAC18:0iso c10-16.1 t9,t11-CLA

c11-16:1 t8,t10-CLAc12-16:1 t7,t9-CLAc13-16:1 t11,c13-CLAc5-17:1 c11,t13-CLAc7-17:1 t10,c12-CLAc9-17:1 c9,c11-CLAc6-c8-18:1t9t12-18:2c11-18:1 c9t13-/t8c12-18:2c12-18:1 t8c13-18:2c13-18:1 t11c15-18:2c14-18:1 c9c15-18:2c15-18:1

Bacterial fatty acids

Odd chain

Branched chain

Trans-MUFA

Cis-MUFA

CLA isomers

Other dienes come from linolenic acid

Why do bacteria hydrogenate?• PUFA are toxic to bacteria• So bacteria rapidly hydrogenate linoleic (18:2n-6)

and linolenic acid (18:3n-3) to 18:0• This goes to completion unless PUFA somehow

protected or hydrogenation inhibited.• In most common feeds, >85-95% of the PUFA are

completely hydrogenated. • This presents a challenge or perhaps a

tremendous opportunity…

• So when measuring fatty acids:– You buy a standard that has the same fatty acids

your sample has.– You run your sample and standard on GC.– You use your standard to identify and measure

the fatty acids in your sample.– This works well for common fatty acids and when

fatty acids separate well on chromatograms.

• Problems arise with measuring beef fatty acids because: – trans-18:1 isomers are difficult to separate

and can overlap with cis-18:1 isomers.– Many CLA isomers cannot be separated using

GC and you have to use HPLC. – Standards for most of the hydro. products

are not commercially available.– You have to use literature reports,

experience, and complementary analyses to piece together which peaks are which.

• Early studies using comprehensive trans and CLA analysis of beef indicated most trans-18:1 was vaccenic acid (t11-18:1) and most CLA was rumenic acid (c9,t11-18:2)

• This created some problems:– Diets fed during these studies were forage based.– People assumed results would be similar when any diets

were fed.– In many instances, people used and still use methods that

don’t separate individual isomers and assume all trans is vaccenic and all CLA is rumenic acid.

• Cattle get essential fatty acids from the diet• In general:

– forages are a source of linolenic acid (omega-3)– grains are a souce of linoleic acid (omega-6)– oilseeds including sunflower and safflower have

higher levels of linoleic (omega-6)– Flax has a high level of linolenic acid (omega-3).– algae, fish oils and fish meals have high levels of

long chain omega-3’s.

P Choline

• When cattle eat, rumen bacteria rapidly hydrolyse lipids to release free fatty acids.

• In a few steps bacteria shift double bonds and then add hydrogen

Hydrogen

CLA

Trans-18:1

Stearic acid (18:0)

Hydrogen

• For years intermediates in hydrogenation like CLA were ignored.

• In the late 1970’s Mike Pariza’s group from the University of Wisconsin found:– CLA from beef protected against cancer– synthetic CLA reduced body fat

• This led to a number of research projects studying the effects of CLA and how to increase levels in beef and dairy products.

• From 1995-2003 my research focused on pork• AAFC already had people working with beef

lipids and I was happy to work with pork.• We did some of the first work feeding CLA to

pigs to show it reduces body fat and increases lean.

• I was a post doc with John Kramer in 1995 and we’ve worked on methods for trans and CLA isomer analyses over the past 10-15 years.

• From 2002 to 2004, pork research was interrupted at Lacombe due to barn renovations.

• In 2003 I had the opportunity to analyse some muskox and compared these to conventionally finished beef.

• From the literature we expected both cattle and muskox would have mostly rumenic and vaccenic acid as hydrogenation products:

• But found all trans isn’t vaccenic and all CLA isn’t rumenic acid.

Vaccenic acid (t11-18:1)

Rumenic acid (c9,t11-18:2)

PUFA

• For our current analyses, we use the techniques developed when working with pork, dairy and muskox/beef samples.

• First we do one GC analysis with a 175C plateau which gets most of the fatty acids.

But trans 18:1’s don’t separate well

• We then do a 150C run to further separate trans-18:1’s and 18:3 hydrogentation products.

t6-t

8-1

8:1

t9-1

8:1

t10

-18

:1t1

1-1

8:1

c9-1

8:1

c11

-/t1

5-1

8:1

c12

-18

:1 c13

-18

:1

c14

-18

:1t1

6-1

8:1

c15

-18

:1

C1

9:0

c9t1

3-1

8:2

t8c1

3-1

8:2

t8c1

2-1

8:2

c9t1

2-1

8:2

c16

-18

:1

t11

c15

-18

:2C

18

:2n-6

unkn-d

iene

c9,c

15

-18

:2

trans-18:1

18:3 hydro products

• We do silver-ion HPLC to separate the CLA isomers not separating by GC

GC

reagent

bla

nk

t12

,t1

4t1

1,t

13

t10

,t1

2t9

,t1

1t8

,t1

0t7

,t9

t6,t

8

unkn-t

12

,c1

4unkn-c

12

,t1

4unkn-a

fter

c12

,t1

4t1

1,c

13

c11

,t1

3t1

0,c

12

9c,

11

t

t8,c

10

7t9

cunkn a

fter

tole

ic a

cid

HPLC

• In the muskox/beef study:– Beef diet – barley/barley silage with linoleic acid

(18:2n-6) as the most concentrated PUFA.– Muskox diet – sedges from the arctic tundra with

equal amounts of linoleic and linolenic acid (18:3n-3).

Figure 2: Conjugated Linoleic Acid (CLA) Composition

0

0.1

0.2

0.3

0.4

0.5

0.6

9c11t-CLA 9t11c-CLA 11t13c-CLA 7t9c-CLA 10t12c-CLA Total tt-CLA

CLA-Isomer

% o

f tot

al fa

tty

acid

s

MuskoxBeef

*

**

*

**

n = 16; * P< 0.05

Most concentrated inBeef and Muskox

BeefMuskox

of Backfat

• Presently no one knows what effects t7,c9-18:2 or t11,c13-18:2 are in humans BUT levels are important to know for future ref.

• Vaccenic acid (t11-18:1) was the most concentrated trans fatty acid in muskox but…

• In beef, we found t10-18:1 was the most concentrated, and it was quite variable

min36 37 38 39

6-8t 13t/

14t

/ 6

-8c

9t

10t

11t12t

9c

11c

12c13c 14c

16t 15c

10c15t

min65 66

10t

11t9t6-8t 12t

/ 6-

8c

9c

Trans Fatty Acids

• A high level of vaccenic acid (t11-18:1) is good as animals use this to make rumenic acid (c9,t11-18:2) but….

• Increased levels of t10-18:1 are not positive– t10-18:1 has properties similar to industrially

produced trans fats which negatively effect blood cholesterol levels in animal models.

• First we wanted to see what the extent of the problem was:–We took samples from a study comparing A

(youthful) vs D (cow) grades (from commercial packing plant).

–We also conducted a retail survey and analysed striploin, backfat, hamburger from Calgary and Guelph&Ohio.

• Second we wanted to figure out how to limit t10-18:1 and reverse this to t11 and c9,t11-18:2 if possible.

• Our current understanding:– Grain diets rich in starch are rapidly

fermented in the rumen.– Rapid fermentation leads to reduce rumen

pH.– Lack of fibre, high starch and low rumen pH

shift the rumen bacterial population from t11-18:1 to t10-18:1 producing species.

Low pHHigh GrainLow Roughage

• The D vs A-Grade Study – confirmed results of Muskox study

0.0

0.5

1.0

1.5

2.0

2.5

3.0

D1 D2 D3 D4 Y1OTM Y1UTM

Maturity/Grade

% o

f T

ota

l F

att

y A

cid

s A

CID

S t6- to t8-18:1

t9-18:1

t10-18:1

t11-18:1

t12-18:1

a aa a a

babc

ab aabc c

ab

a aab

b

c

b b

bb

b

a

a a ab aab b

Trans Fatty Acids

• Cows (D grades) likely had more forage than than concentrate in the diet, yielding more vaccenic acid than t10-18:1.

• Youthful over 30 months of age, likely summered on pasture before a short stay in the feedlot. Still more vaccenic than t10-18:1.

• Youthful under 30 months, definite “shift” from vaccenic to t10-18:1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

D1 D2 D3 D4 Y1OTM Y1UTM

Maturity/Grade

% o

f T

ota

l F

att

y A

cid

s

t8,c10-18:2

t7,c9-18:2

c9,t11-18:2

t11,c13-18:2

t10,c12-18:2

a a b a

c

aab

a a ab

ba

bb

b

b

b

a

b b b b baa a a a a b

CLA – rumenic acid main isomer for all

• To try and reverse the 10t shift:– We checked to see if some common

antibiotics might shift the balance back to t11-18:1.

– We tried adding buffer to the diet (partly funded by BCRC).

– We tried adding distillers’ grains (i.e. grain without starch but higher oil content)(partly funded by BCRC)

– We analyzed grass versus 1-2mo grain finishing to see when the trans and CLA profiles would be affected.

– More recently we have unreported results on the effects of adding vitamin E.

• From the D versus A Grade study– We were also interested in enriching omega-3’s in

beef.– We calculated hamburger from 1 in 20 animals

had the potential to be labelled omega-3 enriched (300 mg/100g serving).

• From the 2008 Beef Fatty Acid Workshop in Lacombe:– We prepared a proposal looking at ways to

increase omega-3’s in mature and youthful beef.

– We knew pasture/forage feeding could play a key role in enriching omega-3s.

• This was based on literature reports on the effects of forage versus concentrate finishing.

• We wanted to start by feeding flax combined with forage (Red Clover) to protect linolenic acid in the rumen.

• Positive results were reported from Kansas (LaBrune et al., 2008) finishing cattle with flax in the diet:– 10% flax was fed in a corn based diet for 85 d

increased linolenic acid (18:3n-3) in longissimus muscle from 0.2% to 2%.

– Fatty acids reported included:

Saturates MUFA PUFAC10:0 C14:1 C18:2n-6C12:0 C16:1 C18:3n-3C14:0 C18:1 C20:3C16:0 C24:1 C20:4C18:0 C15:1 C20:5C20:0 C17:1C24:0C11:0C13:0C15:0C17:0

-No hydrogenation products-No trans-No CLA-No other 18:3 hydrogenation products

-No DHA or DPA

• With this critical information missing we felt a baseline finishing trial feeding flax in a barley based diet was needed.– Fed 0 vs 10% flax in a Barley/Hay diet over 90d

Control (0% flax)

c15-18:1

t11c15-18:2

c9t12-18:2

t8c13-18:2c9t13-18:2t8c12-18:2

t11

-18

:1

t6-t

8-1

8:1

t9-1

8:1

t10

-18

:1t1

2-1

8:1 t1

3-t

14

--1

8:1

c9-c

10

-/t1

5-1

8:1

c11

-18

:1c1

2-1

8:1 c1

3-1

8:1

t16

/c1

4-1

8:1 control CLA18:3n-3

18:2n-618:3 hydrogenation

products

cis and trans-18:1

10% flax

Mostly not reported byothersSome negative but mostly unknown effects

t5-1

8:1

t6-t

8-1

8:1

t9-1

8:1

t10-1

8:1 t1

1-1

8:1

t12-1

8:1

t13-+

t14--

18:1

c9--

18:1

c11-1

8:1

c12-1

8:1

c13-1

8:1

t16/c

14-1

8:1

c15-1

8:1

Also a different transFA profile was found

18:2n-6 Linoleic

t10,c12-18:2

t10-18:1

(t7,c9-18:2)+

Grain

oil, monensin

c9,t11-18:2

t11-18:1

FO

RA

GE

18:3n-3 Linolenic

t13-t14-18:1

Grain +

Flax

CLAs + Other Dienes

c15-18:1

c9,t11-18:2

t11-18:1

FO

RA

GE

(t11,c13-18:2)+

Major Points• If we want to increase or decrease 1-2 fatty

acids in beef, we have to:– Be able to comprehensively analyse the fatty acids.– Know what happens to the rest of the fatty acid.

• If you don’t do this and you’ve developed a product:– You’ll have troubles if negative health effects are

found later.– You’ll have repeat all your studies and analyses to

see what’s in your product and how to modify it.