C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-03-30 to 2013-03-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented scientific experiment, performed under GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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 Hostacerin DGI 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.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

EC Number:

701-479-5

Molecular formula:

C42H82O7

IUPAC Name:

C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

Test material form:

other: colorless to light yellow viscous liquid

Details on test material:

- Name of test material (as cited in study report): Hostacerin DGI
- Physical state: colorless to light yellow viscous liquid
- Purity: 100 %
- Purity test date: n.a.
- Lot/batch No.: ESD0012304
- Expiration date of the lot/batch: 2013-06-09
- Storage condition of test material: at room temperature

Radiolabelling:

no

Test animals

Species:

other: in virto 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. 090M1393V
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: GR84554-2
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 test

Details on study design:

Study Design
Triplicate reaction mixes of both Hostacerin DGI 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 (see table 3). 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 with the Free Fatty Acid Quantification Kit using an isostearic acid standard curve for quantification.

Analysis
All samples were analyzed immediately. 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

Preliminary studies:

It could be shown that under the experimental conditions chosen here olive oil serves as substrate for PPL, resulting in significant absorption values after incubation with the enzyme. Hostacerin DGI seems to be a similarly preferred substrate as judged by the amount of fatty acids released over time (µmol/reaction vial).
Under consideration of the specific weight (Hostacerin DGI: 0.941 g/ml; olive oil: 0.9165 g/ml) and molecular mass (Hostacerin DGI: 832.38 g/mol; olive oil: 874 g/mol) of both substrates, the substrate amount per reaction vial in mole was calculated. In a second step, the substrate turnover per minute (%/min) was calculated under consideration of the substrate amount in mole and the hypothesis that for each mole of formed fatty acid one mole of substrate was consumed.
By dividing the turnover value obtained for Hostacerin DGI with the turnover value for olive oil the relative turnover rate can be calculated. The speed of fatty acid formation was comparable for both substrates. The substrate turnover rate was 0.119 %/min for Hostacerin DGI and 0.176 %/min for olive oil. Relative substrate turnover rate of Hostcerin DGI was estimated to be 67.6% compared to the hydrolysis turnover of the reference substance olive oil. With other words the enzyme PPL causes the hydrolytic release of fatty acids approximately 1.5 times slower with the substrate Hostacerin DGI as compared to the reference substrate olive oil. These in vitro experimental results support the hypothesis that accumulation of Hostacerin DGI in the gut is rather unlikely due to its property of being a substrate for PPL.

Main ADME results

Type:

metabolism

Results:

The speed of fatty acid formation was comparable for both substrates. Under the chosen experimental conditions, the relative turnover of Hostacerin DGI was estimated to be 67.6 % as compared to olive oil.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results
Under the chosen experimental conditions, the relative turnover of Hostacerin DGI into fatty acids was estimated to be 67.6 % as compared to the hydrolysis turnover of the reference substance olive oil. These in vitro experimental results support the hypothesis that accumulation of Hostacerin DGI in the gut is rather unlikely due to its property of being a substrate for PPL.

Executive summary:

The metabolism and kinetic of Hostacerin 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 Hostcerin DGI 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 in vitro Lipase Assay it could be shown that the fatty acid concentration in all reaction vials with the substrate Hostacerin DGI as well as 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 0.119 %/min for Hostacerin DGI and 0.176 %/min for olive oil. Under the chosen experimental conditions, the relative turnover of Hostacerin DGI was estimated to be 67.6 % as compared to the hydrolysis turnover of the reference substance olive oil. With other words the enzyme PPL causes the hydrolytic release of fatty acids approximately 1.5 times slower with the substrate Hostacerin DGI as compared to the reference substrate olive oil.

Thesein vitroexperimental results support the hypothesis that accumulation of Hostacerin DGI in the gut is rather unlikely due to its property of being a substrate for PPL.