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Diss Factsheets

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Remarks:
performed with a group of esterified alcohols
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Hydrolysis of fully esterified alcohols containing from one to eight hydroxyl groups by the lipolytic enzymes of rat pancreatic juice.
Author:
Mattson, F.H. and Volpenhain, R.A.
Year:
1972
Bibliographic source:
J Lipid Res.; 13(3):325-8.

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The enzymatic hydrolysis in vitro of the esters of methanol, ethylene glycol, glycerol, erythritol, pentaerythritol, adonitol, sorbitol, and sucrose in which all alcohols groups were esterified with oleic acid was studied. Various preparations of rat pancreatic juice, including pure lipase, were used as the sources of enzymes. To distinguish lipase form non-specific lipase activity, incubations containing sodium taurocholate and pancreatic juice treated with proteolytic enzymes were included.
GLP compliance:
not specified

Test material

Specific details on test material used for the study:
- Name of test material (as cited in study report):methanol, ethylene glycol, glycerol, erythritol, pentaerythritol, adonitol, sorbitol, sucrose and oleic acid.
- Analytical purity: Oleic acid - 99% pure by Gas-liquid chromatography.
- Other: The alcohols, except ethylene glycol, were transesterified with an excess of methyl oleate to form the complete esters. Ethylene glycol dioleate was pre- pared by acylation of the alcohol with oleoyl chloride

Test animals

Details on test animals or test system and environmental conditions:
Preparation of enzymes:
- Combination fluid - The common bile-pancreatic duct was cannulated at a point near its entrance into the duodenum. In the 24 -hr period following the cannulation, the combination of bile and pancreatic fluid was collected at 4°C. The lipolytic enzymes in this preparation retained their activity for at least 48 hr.
- Untreated pancreatic juice solids - Pancreatic juice, free of bile, was obtained by cannulating separately the bile and pancreatic ducts. The pancreatic fluid was collected at 4°C and freezed-dried. On the day of use, the solids were reconstituted, 3 mg/ mL, in 0.01 M histidine, pH 7.0.
- Treated pancreatic juice solids - To inactivate nonspecific lipase in the study, 150 mg of crude pancreatic juice solids and 1 mg of α-chymotrypsin was dissolved in 80 mL of 0.01 M histidine, pH 9.0.
- Purified Lipase - Lipase (EC 3.1.1.3) was isolated from the untreated pancreatic juice solids using the method of Vandermeers, A and Christophe, J (Biochim. Biophy. Acta. 154:110 -129).

Administration / exposure

Route of administration:
other: In incubation medium
Details on exposure:
- The esters were digested in a cylindrical, flat bottomed glass tube 30 mm (I.D.) x 90 mm. Four 3-mm indentations in the side wall prevented vortexing during stirring.
- Each digest contained 100 mg of substrates, 85 µmoles of CaCl2, 3.5 mg of histidine (final concentration 0.002 M), 152 mg of NaCl (final concentration 0.15 M), and the enzyme in a total volume of 10 mL. For incubations with untreated pancreatic juice, treated pancreatic juice and purified lipase incubations with and without 200 mg of sodium taurocholate (final concentration 37 mM) were included. Prior to the addition of the enzyme, all the components of the digest were stirred for 5 min.
- The digestions were carried out at pH 9.0 and at 27°C. Under these conditions all substrates were liquids.
- The pH was maintained with the aid of a pH stat by the addition of 0.02 N KOH.
Doses / concentrations
Dose / conc.:
100 other: mg
Control animals:
other: sodium taurocholate
Details on dosing and sampling:
The rate of addition of alkali (0.02 N KOH) was linear during the first several minutes of digestion and was reported as the rate of hydrolysis: µmoles of free fatty acid released per minute per milligrams or per millilitre of enzyme preparation.

Results and discussion

Metabolite characterisation studies

Metabolites identified:
no
Details on metabolites:
- The combination of bile-pancreatic fluid digested all substrates with the exception of sorbital hexaoleate and sucrose octaoleate. This failure of hydrolysis was obtained in spite of using 400 times as much combination bile-pancreatic fluid as was used when triolein was the substrate.
- When pancreatic juice without bile was used as a source of the enzymes, the esters that were hydrolysed depended on the presence or absence of added sodium taurocholate. In the absence of sodium taurocholate, only those substrates that contained less than four ester groups were hydrolysed. The addition of sodium taurocholate to the digest permitted the hydrolysis also of the substrates containing four and five ester groups. There were marked differences in the rates of hydrolysis of the oleate esters of methanol, ethylene glycol, and glycerol if taurocholate was not present, but these differences disappeared if this bile salt was added to the digest.
- In the absence of sodium taurocholate, the pattern of digestion by treated pancreatic juice was similar to that seen with the untreated pancreateic juice. However, in the presence of added sodium taurocholate, pancreatic juice that had been treated with the proteolytic enzyme could not digest any of the substrates.
- The final set of results was obtained with purified pancreatic lipase. If sodium taurocholate was not present, this enzyme hydrolysed methyl oleate, ethylene glycol dioleate, and triolein, but did not hydrolyse the substrates that contained more than three ester groups. The additions of sodium taurocholate blocked completely the hydrolytic activity of this enzyme.
- The observation that four and five ester groups were hydrolysed by certain preparations of pancreatic juice is attributed to the enzyme nonspecific lipase. This enzyme also hydrolysed esters of primary alcohols.
- Details are provided in 'any other information on results incl. tables'.

Any other information on results incl. tables

The rate of hydrolysis of the esters of the eight different alcohols by the various preparations of rat pancreatic juice is given in the table below. The numbers in parentheses are the volume or weight of enzyme preparation that was used in that particular digest.

Table 1. Relative rates of hydrolysis by rat pancreatic juice enzymes of the complete oleate esters of the listed alcohols.

 

 

 

 

 

 

 

 

Pancreatic-Bile Juice

Untreated Pancreatic Juice

Treated Pancreatic Juicea

Purified Lipase

No TCb

No TC

TC added

No TC

TC Added

No TC

TC added

 

µmoles FFA min/ mL

µmoles FFA min/ mg

µmoles FFA min/ mg

µmoles FFA min/ mg

Methanol, 1c

54    (0.05)d

2.6 (1.2)

4.0 (1.2)

2.5 (1.2)

0 (1.2)

63 (0.02)

0 (0.3)

Ethylene glycol, 2

160 (0.025)

10  (0.3)

4.3 (0.3)

7.7 (0.3)

0 (0.3)

200 (0.01)

0 (0.1)

Glycerol, 3

2100 (0.005)

73 (0.075)

6.0 (0.15)

70 (0.06)

0 (0.3)

1900 (0.002)

0 (0.02)

Erythritol, 4

1.9   (1)

0   (6)

1.4 (3)

0 (6)

0 (6)

0 (0.1)

0 (0.1)

Pentaerythritol, 4

1.1   (2)

0   (6)

1.1 (3)

0 (6)

0 (6)

0 (0.1)

0 (0.1)

Adonitol, 5

0.53 (2)

0   (6)

0.25 (3)

0 (6)

 

0 (0.1)

0 (0.1)

Sorbitol, 6

0      (2)

0   (6)

0   (12)

0 (6)

0   (12)

0 (0.1)

0 (0.1)

Sucrose, 8

0      (2)

0   (6)

0   (12)

0 (6)

0   (12)

0 (0.1)

0 (0.1)

a Nonspecific lipase was inactivated by treatment with α-chymotrypsin.

b TC, Sodium taurocholate.

c Number of ester groups.

d The number in parentheses is the volume or weight of the enzyme preparation that was used.

Applicant's summary and conclusion