Reaction products resulting from the esterification of C18 unsaturated fatty acid with glycerol and glycerol oligomers

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-09-25 to 2014-10-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented scientific experiment, performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Objective of study:

metabolism

toxicokinetics

Test guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

Lipases (EC number 3.1.1.3) are esterases that catalyze the hydrolytic cleavage of triglycerides, fats and oils into glycerol and free fatty acids. In this study it was elucidated if and to what extent the potential substrate Emulsogen OG acts as a substrate for PPL. In order to compare the extent of this catalysis a proven substrate for the enzyme (here: olive oil) was treated in the same way as the potential substrate. As a consequence of in vitro lipase catalysed hydrolysis the concentration of free fatty acids increased during incubation time. The post-reaction concentration of the liberated fatty acids was analysed using a commercial test kit.
The study aims to obtain valuable hints concerning the metabolic fate of Emulsogen OG following ingestion.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products resulting from esterification of C18 unsaturated fatty acid with glycerol oligomers

IUPAC Name:

Reaction products resulting from esterification of C18 unsaturated fatty acid with glycerol oligomers

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Emulsogen OG
- chemical Name: 9-Octadecenoic acid (Z)-, ester with oxybis [propanediol]
- Physical state: yellow liquid
- Purity: 99.1 % (w/w)
- Expiry date: 2015-08-21
- Lot/batch No.: ESD0017954
- Storage condition: at room temperature

Radiolabelling:

no

Test animals

Species:

other: in vitro assay with porcine pancreas lipase

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

- Characterisation of PPL:
Lipase from procine pancreas 100-400 units/mg protein
Product no.: L3126
Lot No. SLBJ1166V
Brand: Sigma
CAS no. 9001-62-1
MDL Number: MFCD00131509
Starage temperature: 2-8 °C

- Characterisation of Test Kit:
Name: Fee Fatty Acid Quantification Kit
Product Number: ab65341
Lot Number: GR186460-1
Brand: abcam
Storage Temperature: < -20 °C

- Further Equipment:
Microplate reader:TECAN Infinite M200
Software: Magellan Tracker V 6.4
All reagents were freshly prepared on each assay day according to the manufacturer’s specifications

Administration / exposure

Route of administration:

other: in vitro

Details on study design:

Study Design
Triplicate reaction mixes of both Emulsogen OG and olive oil, serving as a reaction positive control were incubated under vigorous shaking at 37 °C for 10 minutes in Tris/HCl-buffer (pH 7.7) solution containing 50 U of PPL. The reaction was then stopped by adding ethanol. For both substrates there was one negative control where enzymatic reaction was excluded by adding the PPL only after incubation and stopping of the reaction. After cooling down and subsequent centrifugation of the reaction mixes the supernatant were transferred into another vial and later analyzed for fatty acid content using the Free Fatty Acid Quantification Kit.
Analysis
All samples were analyzed after overnight storage at <-20°C. Fatty acids are carboxylic acids with long hydrocarbon chains. The hydrocarbon chain length may vary from 8-30 carbons. The Free Fatty Acid Quantification Kit employed here provides a sensitive enzyme-based method for detecting the long-chain free fatty acids (FA) in various biological samples, such as serum, plasma and other body fluids, food, growth media, etc. In this assay, fatty acids are converted to their CoA derivatives, which are subsequently oxidized, leading to formation of color. Fatty acids can then be easily quantified colorimetrically (spectrophotometry at λ= 570 nm).

Evaluation
By using the regression equation obtained from plotting the blank corrected standard curve values, the fatty acid concentration (nmol/well) in each sample was calculated. By subtracting the negative control value for each substrate and taking into account the different volumes of the reaction mix that were employed in the assay, the total fatty acid content per reaction vial (µmol/vial) was calculated. In a second step the substrate turnover per minute (%/min) was calculated under consideration of the substrate amount in mole and under the hypothesis that for each mole of formed fatty acid one mole of substrate is consumed. Both substrates were put into relation in order to display the relative efficiency of turnover by PPL (%).

Results and discussion

Main ADME results

Type:

metabolism

Results:

Emulsogen OG is subject to fatty acid releasing hydrolysis by PPL. The mean turnover rate for Emulsogen OG being approximately 149.8 % of the rate obtained for olive oil.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
It could be shown that Emulsogen OG is subject to fatty acid releasing hydrolysis by PPL, the mean turnover rate for Emulsogen OG being approximately 149.8 % of the rate obtained for olive oil. Under the chosen experimental conditions, Emulsogen OG is broken down at a comparable rate as the representative substrate olive oil. These in vitro experimental results support the hypothesis that accumulation of Emulsogen OG in the gut is very unlikely due to its property of being a substrate for PPL.

Executive summary:

The metabolism and kinetic of Emulsogen OG was evaluated by a Lipase Assay. Lipases (EC number 3.1.1.3) are esterases that catalyze the hydrolytic cleavage of triglycerides, fats and oils into glycerol and free fatty acids. In this study it was elucidated if and to what extent the potential substrate Emulsogen OG acts as a substrate for porcine pancreas lipase (PPL). In order to compare the extent of this catalysis a proven substrate for the enzyme (here: olive oil) was treated in the same way as the potential substrate. As a consequence of in vitro lipase catalyzed hydrolysis the concentration of free fatty acids increased during incubation time. The post-reaction concentration of the liberated fatty acids was analyzed using a commercial test kit.

In this lipase assay two substrates, olive oil and Emulsogen OG were incubated with PPL in triplicate for 10 minutes. It could be shown that the fatty acid concentration in all reaction vials with the substrate Emulsogen OG and the reference substrate olive oil increased in a time dependent manner. The speed of fatty acid formation was comparable for both substrates. The substrate turnover rate was 0.723 %/min for Emulsogen OG and 0.483 %/min for olive oil. Under the chosen experimental conditions, the enzyme PPL causes the hydrolytic release of fatty acids approximately 1.5 times faster with the substrate Emulsogen OG as compared to the reference substrate olive oil.

These in vitro experimental results support the hypothesis that accumulation of Emulsogen OG in the gut is very unlikely due to its property of being a substrate for PPL.