Registration Dossier

Toxicological information

Basic toxicokinetics

Currently viewing:

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication meeting basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1971

Materials and methods

Objective of study:
metabolism
Principles of method if other than guideline:
The lipolytic activity of human gastric and duodenal juice against medium chain and long chain triglycerides was compared.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Triglycerides and fatty acids were obtained from the Hormel Institute (Austin, Minn., USA) and were found to be 99% class pure by TLC.
- Glyceryl 2-oleyl monoether was synthesized from oleyl alcohol and 1,3-benzylidene glycerol.
- A triglyceride analogue, 2-octanoyl-1,3-diethoxyglycerol, was prepared from 1,3-diethoxy-2-propanol (Aldrich Chemical Comp)
- Name of test material (as cited in study report): Triolein (CAS No. 122-32-7), Trioctanoin (CAS No. 538-23-8), Trihexanoin (CAS No. 621-70-5), Tributyrin (CAS No. 60-01-5)
Radiolabelling:
yes
Remarks:
Glyceryl trioctanoate-1-14C

Test animals

Species:
human

Administration / exposure

Route of administration:
other: in vitro testing

Results and discussion

Any other information on results incl. tables

Enzymatic Lipolysis by Gastric and Duodenal Juice:

All samples of gastric juice showed lipolytic activity against trioctanoin and triolein. Hydrolysis of emulsified trioctanoin was greater than of emulsified triolein. Hydrolysis of unemulsified trioctanoin was less and more variable.

Duodenal juice was more active, even against unemulsified trioctanoin and triolein. Duodenal juice was more active against unemulsified substrate than gastric juice against emulsified substrate.

Table 1: Hydrolysis of trioctanoin and triolein*

 

Substrate and form

(μmoles)

Hydrolysis (%)

 

Trioctanoin

Triolein

Gastric juice

30, unemulsified

21

1

 

60, emulsified

33

16

Duodenal juice

30, unemulsified

40

34

 

45, emulsified

42

35

 

105, emulsified

45

36

*Gastric or duodenal juice (1 mL) was incubated (1 hour, continuous shaking, 37ºC) with 1 mL of buffer and unemulsified substrate or 1 mL of substrate emulsified in 10 mM sodium taurodeoxycholate, pH6.

pH Optimum

In the presence of bile acids, gastric lipolytic activity against trioctanoin had a broad pH optimum, between 4 and 7. The lipolytic activity of duodenal juice had a sharper pH optimum, between 6 and 8. The pH optimum was lower for short chain triglycerides, indicating that pH optimum values for lipases must be defined for a particular substrate.

Chain Length Specificity

Lipolysis rates increased with decreasing chain lengths for pure triglycerides.

Tributyrin was cleaved more rapidly than trihexanoin which in turn was cleaved more rapidly than trioctanoin (ratio of rates, 100:69:53). Because the pH optimum of gastric lipase is lower for short chain triglycerides than for MCT, trihexanoin and tributyrin were cleaved much more rapidly than, for example, trioctanoin at pH5.

Esterification and Fatty Acid Acceptors by Gastric and Duodenal Lipases

Gastric and duodenal lipases did not induce esterification of the fatty acid acceptor, glyceryl 2 -monooleyl ester, by octanoic acid over the pH range of 2 to 6. However, it was esterified by oleic acid in the presence of gastric juice, duodenal juice, or pancreatic fistula juice when bile acids were added. Esterification, calculated by disappearance of titratable fatty acid, was confirmed by TLC which showed the formation of compounds having the mobilities of a monoether monoester and a monoether diester. Control incubations without enzyme showed no loss of oleic acid or appearance of new lipids by TLC. To determine the amount of disubstituted and trisubstituted glyceryl derivatives which were formed, 14C-labeled glyceryl 2 -monooleyl ether was used and the products of the reaction were examined by zonal scanning. The glyceryl 2 -monooleyl ether was not cleaved during the incubation procedure. The amounts of ester bonds formed estimated by titration and by zonal scanning were in good agreement.

Products of Lipolysis and Positional Specificity

The specificity of pancreatic lipase for the 1 -ester bond in LCT has been demonstrated previously by establishing the formation of 2 -monoglycerides and fatty acid as end products of lipolysis. This procedure cannot be used for MCT because medium chain 2 -monoglycerides are either cleaved by pancreatic lipase or rapidly isomerized to the 1 -isomer which is rapidly hydrolyzed or both. Indeed, chromatographic examination of the products of hydrolysis of trioctanoin-14C showed only a small fraction of monoglyceride present.

Table 2: Products of hydrolysis of trioctanoin by gastric juice*

 

Radioactivity distribution** (%)

Lipolysis

(%)

 

Monoglyceride

Diglyceride

Fatty acid

Triglyceride

Buffer (control)

0

0

0

100

0

Gastric juice

1 mL

3

26

26

44

34

3

28

24

43

33

4

28

25

43

36

4

28

25

43

36

Duodenal juice

 

 

 

 

 

0.4 mL

4

9

15

72

26

0.5 mL

4

14

20

62

40

*Glyceryl trioctanoate-1-14C was added to 1 mL of emulsified trioctanoin (60 μmoles) and incubated for 1 hour at 37ºC with buffer (blank) or gastric or duodenal juice. The reaction mixture was extracted and a 50 μL aliquot was analyzed by TLC and zonal scanning. A 3 mL aliquot was titrated to quantify fatty acids liberated.

Discussion:

The work confirmed extensive literature showing that gastric juice contains lipolytic activity, that ingested triglyceride is hydrolyzed in the stomach, even after pancreatic diversion, that lipase may be demonstrated histochemically in gastric mucosa, and that gastric mucosal homogenates have lipolytic activity. Pancreatic lipase has some activity at the pH of gastric content, which is between pH6 and pH3 in normal subjects.

Applicant's summary and conclusion