# Presentation: 077677

## Acrylamide:  Mechanism of Formation in Heated Foods

**David Zyzak, Ph.D.**
**Procter & Gamble**
**Snacks and Beverage Analytical and Microbiology **
**Cincinnati, Ohio**

**Notes:**
Thank you for giving me the opportunity to share our knowledge and understanding on the mechanism of acrylamide formation.  

## ACRYLAMIDE SHOCK  Press Release April 24, 2002

- Stockholm University/Swedish NFA revealed acrylamide presence in variety of cooked foods.

**Notes:**
April 24, 2002 – A shocking day for many in the food industry.  It was a surprise to hear about acrylamide being present at ppb/ppm levels in commonly consumed foods.  As many organizations began to implement/develop analytical capabilities for the analysis of acrylamide, it became evident that the data was accurate. 

## Sample Survey Results 

**Sample Survey Results **

**Notes:**
Here is a portion of our own product survey performed at P&G.  Indeed, we found acrylamide present in a variety of foods – such as: toasted bread products, roasted asparagus, potato chips and cooked taco shells.  

## What is Acrylamide?

- •  high boiling point

- •  very hydrophilic – water loving

**Notes:**
What is acrylamide? This molecule is a conjugated ene-amide thus displaying a high boiling point and is very hydrophilic.  Now, with confirmation of acrylamide in foods, the next question is how is it formed? 

## Initial Thoughts on the Mechanism of 

_**Acrylamide**_

**Initial Thoughts on the Mechanism of **
**Acrylamide Formation**

**Notes:**
Proposed mechanisms from JIFSAN, FRI, WHO and other groups focused on the possibility of oil degradation products such as acrylic acid and/or acrolein reacting with ammonia (formed in the Maillard reaction) to form acrylamide, however, these proposed mechanisms have not been successfully confirmed by experiments in various labs.  The difficulty lies in the formation of the amide bond.  However, there are two amino acids that have amide bonds already in their side chain – asparagine and glutamine.  And amino acids typically undergo decarboxylation (loss of CO2) and deamination (loss of NH2) reactions, at typically cooking conditions in the presence of reducing sugars, resulting in the formation of aroma molecules.  Thus it is reasonable to predict asparagine, and possible glutamine, would form acrylamide.  To test this, our laboratory developed a model food system similar to a potato chip.

## Effectiveness of Amino Acids and Dextrose to Form Acrylamide

- Acrylamide Formation
- Potato starch				<50 ppb
- Potato starch + dextrose			<50 ppb
- Potato starch + asparagine			117 ppb
- Potato starch + dextrose + asparagine		9270 ppb

- Other Amino Acids
- Alanine	<50 ppb		Arginine		<50 ppb
- Aspartic A.	<50 ppb		Cysteine		<50 ppb
- Lysine	<50 ppb		Methionine	<50 ppb
- Threonine	<50 ppb		Valine		<50 ppb
- Glutamine	156 ppb**	****	****Asparagine ****	****9270 ppb**

**Notes:**
This model system is composed of potato starch and water, and allows the addition of various reactants such as amino acids, reducing sugars, and a variety of other ingredients including possible inhibitors.  This model system is subjected to typical frying conditions and then analyzed for acrylamide.  The elegance of this model system is seen in its inability to form acrylamide thus providing an inert platform for studies.  Adding dextrose to this system did not result in acrylamide formation.  On the other hand, adding asparagine alone, did result in a small measurable amount of acrylamide.  In addition, the combination of asparagine with dextrose resulted in considerably more acrylamide formation.  Utilizing this system, our laboratory investigated other amino acids for acrylamide formation in combination with dextrose.  Only glutamine, the other amino acid with an amide side chain, formed detectable levels of acrylamide - ~1% the level of the asparagine.  In addition, with this model system, our laboratory investigated the potential impact of oil quality, oxidized oil vs. fresh oil – saw no difference.  
	These data suggest that asparagine is a critical factor in acrylamide formation.  And that other possible mechanisms such as starch and oil oxidation products (acrolein and/or acrylic acid) are not a factor.

## Amino Acid Composition in Potatoes

**Approximately 50% of amino acids are in the free state **
**	****	****(not incorporated into protein).**

**Asparagine is roughly half of the free amino acid content.**

**Notes:**
	In potatoes, ~50% of all amino acids are in the free state, i.e. non-protein bound.  Out of that 50%, half is asparagine.  

## Free vs. Bound Asparagine

- Asparagine occurring as component of protein does not have an accessible primary amine group for Schiff base formation, and would not be expected to participate in the production of acrylamide.  Blocking the amine group in asparagine, N-acetyl asparagine, is an effective analogue to test.

- Result:	No acrylamide formation observed

**Notes:**
Next we were interested in understanding more about the mechanism of acrylamide formation from asparagine and dextrose.  A question to answer was:  Can protein bound asparagine form acrylamide?  To test this we used N-acetyl-asparagine as a protein-bound analogue.  In this experiment, no acrylamide was formed; therefore, asparagine must be in the free state allowing the alpha-amine group to react with dextrose as part of the reaction mechanism.  

## Dose/Response:  Dextrose

**Notes:**
Next we looked at acrylamide formation with a fixed asparagine concentration and increasing dextrose (10g asparagine is equivalent to 1.25% in the system).  As you can see, there is a linear response to dextrose up to a 1:1 ratio with asparagine.  Thus, controlling both asparagine and dextrose (reducing sugars) will affect acrylamide formation. While most of our studies focused on dextrose, we next looked at the ability of other carbonyl sources to form acrylamide.

## Other Carbonyl Sources Which Produce Acrylamide

- Also:  ribose

- All of these carbonyl sources produce significant acrylamide in the model system with asparagine.

**Notes:**
We chose sugars typically formed during the Maillard reaction – glyoxal, glyceraldehyde, ribose.  The Maillard reaction is the browning reaction that occurs during the heating of amino acids with reducing sugars.  This reaction results in the formation of sugar degradation products such as glyoxal, glyceraldehyde, and ribose.  These sugars are more reactive than glucose and are considered to be responsible for the browning reactions.  All of these carbonyl compounds were effective in reacting with asparagine and forming acrylamide.  In one of our experiments, we used 2-deoxyglucose as the carbonyl source and showed that it too formed comparable levels of acrylamide.  Since 2-deoxyglucose does not have a hydroxyl group adjacent to the carbonyl, it can only form the Schiff base adduct and cannot undergo the Amadori rearrangement which leads to the formation of dicarbonyl compounds e.g. 3-deoxyglucosone.  In addition, Becalaski, at Health Canada, reported that octanal was able to react with asparagine to form acrylamide.  Our studies, using decanal, produced similar results.  Combined, these observations suggest the necessity of carbonyls in the formation of acrylamide and that dicarbonyls are not required for the formation of acrylamide from asparagine.  Next, our laboratories’ focus was to identify the source of C’s and N in acrylamide formation from asparagine utilizing labeled isotopes.

## Use of Isotopes to Understand the Mechanism of Acrylamide Formation from the Reaction of Asparagine and Dextrose 

**Notes:**
We believe the side chain of asparagine is the source of the atoms in acrylamide formation.  To test this idea, we used three different isotopes: amide label; amine label; and uniformly C and N labeled asparagine.  The reactions were carried out using our model system (potato starch and water) as the matrix and adding the labeled asparagine with dextrose.  Utilizing LC/MS, we could monitor the shift in mass units for acrylamide and therefore determine the location of C’s and N.  Acrylamide has a molecule weight of 71 daltons and its analysis by ESI-MS would produce the m/z = 72 ion.  

## Label Expt #1: Amide15N-Labeled Asparagine

- m/z 73

**Notes:**
In the first experiment using amide labeled asparagine, The detection of acrylamide was greater than 97% at m/z = 73.  Indicating that the amide nitrogen of asparagine is being incorporated into acrylamide.  This was verified by the 2nd experiment using amine labeled asparagine.

## Label Expt #2: Amine 15N-Labeled Asparagine

- m/z 72

**Notes:**
Here, only the unlabeled acrylamide is detected – thus proving that the amide nitrogen is the source of N in acrylamide formation from asparagine.  Asparagine and glutamine are known to undergo deamination at the side chain during heating, resulting in the release of ammonia.  Is this occurring here, is ammonia reacting with acrolein/acrylic acid or some sugar degradation product and forming acrylamide?  Probably not, because if that was occurring, asparagine and glutamine would form similar levels of acrylamide.  We suspected that the amide bond in asparagine was remaining intact.  Next, we wanted to understand where the carbons are coming from.  

## Label Expt #3:  Uniformly Labeled Asparagine

- m/z 76

**Notes:**
We addressed this question using uniformly labeled 13C6, 15N2-asparagine.  As you see, we detected only the tetralabeled acrylamide – the amide nitrogen being one of the labels and the other three coming from the carbons in asparagine.  We confirmed these results utilizing uniformly labeled 13C glucose reacting with asparagine and found that none of the 13C were incorporated into acrylamide.  Thus confirming that asparagine provides the total source of C’s and N in the formation of acrylamide.  From these results, the following mechanism was derived.

## Acrylamide Formation Mechanism

**Notes:**
The free amine group of asparagine reacts with a carbonyl source resulting in the loss of water and the formation of a Schiff Base.  This reaction is favored during typically cooking conditions as water is evaporated.  Under heat, the Schiff base de-carboxylates (facilitated by de-localization of negative charge which Schiff base formation allows), forming a product that can react one of two ways.  It can hydrolyze to form -alanine amide that can further degrade via the elimination of ammonia to form acrylamide when heated.  Alternatively, the Schiff base can decompose directly to form acrylamide via elimination of an imine. 
In our model heated food system, we determined that –alanine amide heated without dextrose formed five times the level of acrylamide as compared to an asparagine/dextrose reaction mixture.

## Monitoring Intermediates in Acrylamide Formation 

- Monitoring Intermediates in Acrylamide Formation

_**A**_

_**B**_

**Notes:**
Additional evidence to support this mechanism was developed using an aqueous reaction system comprising dextrose and asparagine.  Figure A displays chromatograms that monitor the disappearance of asparagine and dextrose, and the formation of Schiff base, -alanine amide, and acrylamide.  Before heating (t=0 s), only asparagine and dextrose are detected.  At t=180 s, the Schiff base appears. At t=270 s, dextrose and Schiff base are depleted, and –alanine amide and acrylamide appear.                        
We used several techniques to identify the intermediates and products indicated in Figure A.  The molecular formulae of the asparagine, dextrose, Schiff base, and acrylamide peaks were confirmed by high resolution mass spectrometry.  The -alanine amide peak was confirmed by injecting a standard and comparing its retention time and +ESI mass spectrum with that of the indicated peak in Figure A, t=270 s.  Finally, all of these identifications are confirmed in Figure B:  the same experiment as A but with uniformly labeled asparagine substituted for asparagine.  The ions displayed are for the same compounds as in the unlabeled experiment, but adjusted in mass to account for incorporation of the number of labels was expected from the mechanism.  For example, -alanine amide retains five of the six labels of the precursor asparagine, and m/z 94 behaves in near-identical fashion to m/z 89 in the unlabeled experiment.  The appropriate mass shift has occurred for each component, confirming the identifications of intermediates and products generated in the mechanism.  Data from the high resolution system, when plotted, yielded similar profiles.  

Dr. Richard Stadler, at Nestle, has also shown that the Schiff base formed from asparagine and dextrose is more efficient in forming acrylamide than the reactants alone.

## Understanding Acrylamide Formation in Food Products

**Selective removal of asparagine with asparaginase to address these questions.**

**What about other potential sources of acrylamide?        **
**methionine, glutamine, cysteine, acrolein, etc...**

** ****Is asparagine the only precursor to acrylamide in heated foods?**

**Notes:**
Next, we wanted to understand how well our model system related to real food systems.
	Is asparagine the only precursor to acrylamide formation in heated foods?
	What about other precursors which have been postulated such as:  methionine, glutamine, cysteine, and acrolein?
We decided we could address these questions by selectively removing asparagine, using the enzyme asparaginase, in a food product, heating that food product, and then measuring acrylamide.  Because, removal of asparagine with no resulting acrylamide formation would indicate that other components are not significant factors in the formation of acrylamide.

## Asparaginase:  Mode of Action 

**Notes:**
Asparaginase selectively hydrolyzes the amide bond of asparagine resulting in the formation of aspartic acid and ammonia.  Recall from one of my initial slides that we looked at aspartic acid and it did not form acrylamide when reacted with dextrose.  Thus, this process should convert asparagine into a non-acrylamide forming molecule.

## Asparaginase Experiment on Potato Product 

- Boil for 1 hour

- Blend flesh 1:3 with distilled water

- Asparaginase-treated

- Microwave @ 2 min intervals for total of 10 min.
*Highly Cooked to Maximize Acrylamide Formation*
-    (both control and asparaginase-treated products were dry and brown)

- Control

- 45 min @ RT

- Washed Russet Burbank Potatoes

**Notes:**
Our system was as follows:  Russet Burbank Potatoes, purchased from the local grocery store, were washed and then boiled for 1 hour.  The flesh was blended with distilled water in a 1:3 ratio (potato:water).  The sample was split – one aliquot was treated with asparaginase and the other was untreated.  This treatment was carried out with stirring for 45 min at room temperature.  Next, the samples were microwaved at 2-min intervals for a total of 10 min.  Both samples were of similar brown color and fairly dry following the microwaving step.  These products were highly cooked in order to maximize acrylamide formation.  To check the progress of the experiment, we analyzed for both asparagine and aspartic acid in both samples.

## Asparagine Analysis of Enzyme-Treated 

**Potato Product **

**Asparaginase treated**

**Control**

**Asparagine**

**Asparatic acid**

**Asparagine**

**Unreacted **
**FMOC**

**ISTD**

**Notes:**
This is an HPLC amino acid analysis chromatogram using fluorescence detection.  As expected, the asparaginase-treated sample has reduced the asparagine content and resulted in the formation of aspartic acid.  In this experiment, we degraded ~88% of the asparagine.  Next, we analyzed the microwaved products for acrylamide.

## Asparaginase Reduces Acrylamide in Cooked Potato Products 

**Asparaginase Reduces Acrylamide in Cooked Potato Products **

**Notes:**
These samples were highly cooked in order to maximize acrylamide formation and this is shown in the level of acrylamide in the control sample which produced 20,500 ppb acrylamide; however, the asparaginase treated sample produced only 164 ppb acrylamide – a 99% reduction.  Since we only removed asparagine during this experiment, these data suggest that asparagine is the major source of acrylamide formation in potato products.  In this experiment, we did not affect the level of any other amino acids or precursors that could be formed during the cooking procedure

## Acrylamide Precursors – Where to Intervene

-  Factors affecting asparagine and reducing sugars
- 	- Variety of potato
- 	- Storage conditions

**Notes:**
Based on this work, there are two factors that contribute to acrylamide formation in potatoes – Asparagine and reducing sugars (glucose, fructose, sucrose hydrolysis during cooking).  And these two factors can be affected by the variety of the potato and the storage conditions. 

## Conclusions

-  Asparagine is the major source of acrylamide formation in foods.

-  Carbonyl source (reducing sugars) is required in the reaction.

-  Oil oxidation products and starch do not appear to be significant factors in acrylamide formation.

**Notes:**
We conclude that Asparagine is the major source of acrylamide formation in foods and that a carbonyl source, usually in the form of a reducing sugar such as glucose or fructose, is required in the reaction.