A Primer on Fatty Acids and Analyses Mike Dugan Meat Lipid Scientist AAFC-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. Glycerol Fatty Acid Pex. Choline Glycerol 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

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: SaturatedMUFAPUFACLA StearicOleicLinoleic (omega-6)Rumenic PalmiticVaccenicLinolenic, 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).

deltaomega Linoleic acid c9,c12-18:2 Delta System c = cis

deltaomega Linolenic acid c9,c12,c15-18:3 Delta System

Vaccenic Acid t11-18:1 Delta System deltaomega t = trans

deltaomega Rumenic Acid c9,t11-18:2 Delta System (main natural type or isomer of CLA)

2 double bonds 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,c9-18:2 t8,c10-18:2 t10,c12-18:2 t11,c13-18:2 c12,t14-18:2 t7,t9-18:2 t8,t10-18:2 t9,t11-18:2 t10,t12-18:2 t11,t13-18:2 t12,t14-18:2 c7,c9-18:2 c8,c10-18:2 c9,c11-18:2 c10,c12-18:2 c11,c13-18:2 c12,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 deltaomega 18:2n-6 Omega System c9,c12-18:2 Delta System deltaomega This stands for: 18 carbons: 2 double bonds – first double bond at omega-6 carbon

Linolenic acid deltaomega 18:3n-3 Omega System c9,c12,c15-18:3 Delta System deltaomega

deltaomega Plants and animals can add double bonds. Plants AnimalsRequired

Linoleic acid 18:2n-6 Linolenic acid 18:3n-3 Used to make LC Omega-3 fatty acids EPA 20:5n-3 DPA 22:5n-3 DHA 22:6n-3 Arachidonic acid 20:4n-6 Used to make LC Omega-6 fatty acids The two essential fatty acids are:

Beef Lipid Analyses

-2- Then homogenize with organic solvent (2:1 CHCl 3 :CH 3 OH) -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 SaturatesMUFAPUFA 12:0c9-16:118:2n-6 14:0c9-18:118:3n-3 16:0c11-18:120:2n-6 18:0c11-20:1 20:0c13-22:1 22:0 24:0

Intermediates of PUFA hydrogenation In a beef sample we analyse ~80 fatty acids Feed or Animal 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 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 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-t8-18:1 t9-18:1 t10-18:1 t11-18:1 c9-18:1 c11-/t15-18:1 c12-18:1 c13-18:1 c14-18:1 t16-18:1 c15-18:1 C19:0 c9t13-18:2 t8c13-18:2 t8c12-18:2 c9t12-18:2 c16-18:1 t11c15-18:2 C18:2n-6 unkn-diene c9,c15-18:2 trans-18:1 18:3 hydro products

We do silver-ion HPLC to separate the CLA isomers not separating by GC GC reagent blank t12,t14 t11,t13 t10,t12 t9,t11 t8,t10 t7,t9 t6,t8 unkn-t12,c14 unkn-c12,t14 unkn-after c12,t14 t11,c13 c11,t13 t10,c12 9c,11t t8,c10 7t9c unkn after t oleic acid 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).

Most concentrated in Beef 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 min t 13t/14t / 6-8c 9t9t 10t 11t 12t 9c9c 11c 12c 13c 14c 16t 15c 10c 15t min t 11t 9t9t 6-8t 12t / 6-8c 9c9c 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 pH High Grain Low Roughage

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

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:

-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 t8c13-18:2 c9t13-18:2 t8c12-18:2 t11-18:1 t6-t8-18:1 t9-18:1 t10-18:1 t12-18:1 t13-t14--18:1 c9-c10-/t15-18:1 c11-18:1 c12-18:1 c13-18:1 t16/c14-18:1 control CLA18:3n-3 18:2n-6 18:3 hydrogenation products cis and trans-18:1 10% flax Mostly not reported by others Some negative but mostly unknown effects

t5-18:1 t6-t8-18:1 t9-18:1 t10-18:1 t11-18:1 t12-18:1 t13-+ t14--18:1 c9--18:1 c11-18:1 c12-18:1 c13-18:1 t16/c14-18:1 c15-18:1 Also a different trans FA 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 FORAGE

18:3n-3 Linolenic t13-t14-18:1 Grain + Flax CLAs + Other Dienes c15-18:1 c9,t11-18:2 t11-18:1 FORAGE (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.