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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was well documented and meets generally accepted scientific principles, but was not conducted in compliance with GLP.

Data source

Reference
Reference Type:
publication
Title:
Fatty alcohol metabolism in cultured human fibroblasts. Evidence for a fatty alcohol cycle.
Author:
Rizzo WB, Craft DA, Dammann AL, Phillips MW
Year:
1987
Bibliographic source:
J Biol Chem. 262:17412–17419

Materials and methods

Principles of method if other than guideline:
Cultured human fibroblasts were used to demonstrate the intracellular fatty alcohol cycle. The cells were incubated with radio-labelled palmitate (salts and esters of hexadecanoic acid) or with radio-labelled hexadecanol in the presence and absence of unlabelled hexadecanol, and the concentrations of labelled hexadecanol were measured.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Materials used:
[1-¹⁴C]Palmitic acid (58 miCi/mmmol) and [9,10-³H]palmitic acid (30 miCi/mmmol) were purified by thin layer chrmatography using hexane:diethyl ether:acetic acid (90:10:1).
[1-¹⁴C]hexadecanol was synthesised from [1-¹⁴C]palmitic acid by reduction with LiAlH followed by removal of excess hydride. The hexadecanol was then extracted into ether and then into hexane, followed by purification by thin layer chrmatography using benzene:ethyl ether:ethyl acetate:acetic acid (80:10:10:0.2). The identity of the hexadecanol was confirmed by TLC, acetate formation and comparison with a standard acetate derivative of hexadecanol . The overrall yield was 60-80%.
Hexadecanal was synthesised by oxidation of the alcohol.
Radiolabelling:
yes

Test animals

Species:
human
Strain:
other: skin fibroblasts
Details on test animals and environmental conditions:
Cell culture
Human cultured skin fibroblasts derived from normal subjects were grown from skin punch biopsies at 37°C in an atmosphere of 5% CO₂ 95% air in Dulbecco's minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/ml) and strptomycin (100 μg/ml). Lipid-free fetal bovine serum (LFFBS) was produced by solvent extraction and dialysis. Lipid-free medium used in experiments was DMEM supplemented with 10% LFFBS.

Administration / exposure

Details on study design:
1 MEASUREMENT OF INCORPORATION OF RADIOACTIVITY FROM FATTY ACID INTO FATTY ALCOHOL
CELL CULTURE AND LIPID EXTRACTION
Cells were grown to confluency in 100 mm culture dishes and medium changed to lipid-free DMEM 1 day prior to study.
[1-¹⁴C]Palmitic acid or [9,10-³H]palmitic acid bound to fatty acid-free bovine serum albumen at a ratio of 1:1 was added.
Cells were incubated for varying times at 37°C.
Radioactive medium was removed and monolayers were washed. Cells were collected and the lipids extracted overnight with 3ml chloroform:methanol (1:1). Insoluble cellular material was pelleted by centrifugation and protein content measured.

QUANTIFICATION OF FATTY ACIDS AND FATTY ALCOHOLS
The following method was used to quantify free and esterified fatty acids and fatty alcohols:
The supernatant from the pelleted cells (containing lipid) was dried and saponifiable lipids containing radioactive fatty acids were extracted into hexane and back extracted with water.
Radioactive fatty acids in the hexane layer containing the saponifiable lipid fraction were quantified by scintillation spectroscopy. Recovery of radioactive palmitate was 92 ± 3%.
The combined hexane extracts containing non-saponifiable lipids were dried under N₂. Carrier hexadecanol and palmitate were added to each sample. Fatty alcohol, palmitic acid and cholesterol were separated by thin layer chromatography (TLC), stained with rhodamine G and visualised under UV light. Areas containing hexadecanol were scraped off and transferred to scintillation vials for determination of radioactivity. (Silica gel and rhodamine G had negligible quenching effects). Recovery of radioactive hexadecanol was 85 ± 6%.

MEASUREMENT OF FATTY ALCOHOL METABOLISM IN INTACT FIBROBLASTS
Cells were grown to confluency in 100 mm culture dishes and medium changed to lipid-free DMEM 1 day prior to study.
[1-¹⁴C]hexadecanol in 95% ethanol was added to each dish (final concentration ethanol ≤ 0.1%).
Cells were incubated for varying times at 37°C.
In some experiments, palmitic acid bound to bovine serum albumen was added to some dishes.
Stock concentration of palmitate was determined by gas-liquid chromatography (GC).
Radioactive medium was removed and monolayers were washed. Cells were collected by scraping into 3ml methanol and the lipids extracted overnight after adding 3ml chloroform. Insoluble cellular material was pelleted by centrifugation and protein content measured.
Cell lipids were saponified for determination of radioactive fatty acid and fatty alcohol as described above.
In some experiments fibroblasts were incubated with 0.2 mM [1-¹⁴C]hexadecanol for 20 min, washed then placed in DMEM containing lipid-free serum. The cells were then incubated for varying periods of time prior to scraping into methanol.
Where radioactive lipids in the medium were measured, I ml of medium was added to 2 ml chloroform and 2 ml methanol containing 2% acetic acid. Samples were extracted and the lower phase removed for determination of fatty acid and fatty alcohol as described above.
To measure incorporation of [1-¹⁴C]hexadecanol into phospholipid alkyl ether linkages, monolayers were incubated in the presence of radioactive fatty alcohol and harvested, and cell lipids were extracted overnight in chloroform:methanol. Chloroform:methanol was removed by drying. Alkyl ether-linked radioactivity was measured after two-dimensional TLC and acid hydrolysis of the glycerol ethers

2 FATTY ALCOHOL:NAD⁺ OXIDOREDUCTASE ACTIVITY
Cells were collected by tryptinisation and washed. The cell pellet was homogenised. Assay of fatty alcohol:NAD⁺ oxidoreductase activity was performed by modification of method described by Lee, T (1979). After the reaction was stopped the homogenate was extracted into 1ml water and 1 ml chloroform, centrifuged at 3000 g for 5 minutes and the aqueous phase removed. The chloroform phase was dried. Carrier palmitate and hexadecanol were added to the dried lipids which were then dissolved in chloroform:methanol (1:1) and separated by TLC.

3 MEASUREMENT OF FIBROBLAST HEXADECANOL CONTENT
Confluent cells were incubated overnight in DMEM containing lipid-free serum. To some cultures, palmitate bound to fatty acid-free bovine serum albumin was added at a final concentration of 50 μM for 2 hours then washed. Cells were harvested by scraping into methanol then lipids extracted overnight with chloroform:methanol 1:1. 2 μg pentadecanol was added as internal standard. Cells were centrifuged and solvent removed and the pellet was assayed for protein. Chloroform:methanol was dried under nitrogen. Dried lipids were extracted into hexane and dried, then acetate derivatives of fatty alcohols made by reaction with acetic anhydride. The acetate derivatives were extracted and purified by TLC, stained with rhodamine G and visualised under UV light. Appropriate areas were scraped off and the alcohol acetates extracted with hexane:benzene (3:2) and dried prior to analysis by GC calibrated with appropriate standards.
Lee, T (1979). J Biol Chem. 254, 2892 - 2896.

Results and discussion

Main ADME results
Type:
metabolism
Results:
the interconversion of hexadecanol and hexadecanoic acid was demonstrated.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
The metabolic pathway for interconversion of hexadecanol and hexadecanoic acid was demonstrated and characterised.

Any other information on results incl. tables

In brief: the study demonstrated that the synthesis and oxidation of fatty alcohols takes place within cultured skin fibroblasts. Fatty alcohol is oxidised to the corresponding aldehyde, which is followed by further oxidation to fatty acid. In turn, fatty acids can be converted via the corresponding aldehyde to fatty alcohol. This interconversion of fatty acids and fatty alchols is known as the fatty alcohol cycle.

Cells incubated in radioactive palmitate (salts or esters of hexadecanoic acid, C16) showed increased levels of labelled hexadecanol. In the presence of non-labelled hexadecanol, and labelled palmitate, cellular levels of labelled hexadecanol increased up to 10-fold, which suggests that rapid metabolism of palmitate to hexadecanol is occurring.

When incubated in the absence of fatty acid and the presence of labelled hexadecanol, cells rapidly oxidised hexadecanol to palmitic acid (hexadecanoic acid).

Double labelling demonstrated simultaneous interconversion of hexdecanol and palmitic acid.

METABOLISM OF RADIOACTIVE PALMITATE TO FATTY ALCOHOL

Cells incubated with [¹⁴C]Palmitic acid accumulated radioactive material in the non-saponifiable lipid extract. This material was demonstrated to be fatty alcohol by 1) co-migration with known hexadecanol in TLC with three different solvent systems; 2) formation of acetate derivative and co-migration with known hexadecanol acetate derivative in TLC; 3) double-label experiments involving incubation of cells with [¹⁴C]Palmitic and [³H]acetate which demonstrated that the material identified as hexadecanol had a ¹⁴C to ³H ratio much higher than cholesterol; and 4) after separation with reverse-phase TLC with methanol as the solvent, >90% of radioactivity was found in hexadecanol.

When cells were incubated with [¹⁴C]palmitate, radioactive hexadecanol accumulated in a concentration dependent manner. Hexadecanol appeared to plateau at palmitate concentrations > 30 μM, but uptake of [¹⁴C]palmitate increased in parallel with the amount of cellular fatty alcohol suggesting that alcohol accumulation was limited by fatty acid uptake.

In presence of 30 μM [¹⁴C]palmitate, hexadecanol accumulated with time, reaching a plateau after 1 hour even though additional [¹⁴C]palmitate was taken up for at least 4 hours. This is consistent with feedback inhibition by hexadecanol of further reduction of [¹⁴C]palmitate, the metabolism of radioactive hexadecanol through other pathways, or loss of labelled hexadecanol into the medium. The latter was not supported by measurement.

To investigate feedback inhibition, cells were incubated with radioactive palmitate in the presence and absence of unlabelled hexadecanol. In the presence of a large excess of unlabelled fatty alcohol, fibroblasts accumulated about 10-fold more radioactive hexadecanol than in the absence of unlabelled fatty alcohol; uptake of radioactive palmitate was unaffected. No evidence for feedback inhibition of palmitate reduction was found, suggesting excess unlabelled alcohol prevented further metabolism or loss into the medium.

METABOLISM OF FATTY ALCOHOL

Cells incubated with 5 μM [¹⁴C]hexadecanol accumulated radioactivity with time. Radiolabelled hexadecanol taken up by cells was rapidly oxidised to fatty acid; by 4 h, 98% of cell-associated radioactivity was present as fatty acid. The rate was dependent on fatty alcohol concentration (see table 1).

The effect of exogenous palmitate on hexadecanol oxidation was investigated, and found to inhibit [¹⁴C]hexadecanol oxidation in concentration-dependent manner. Cell uptake of [¹⁴C]hexadecanol was also inhibited.

The effect of exogenous palmitate on hexadecanol oxidation was investigated, and found to inhibit [¹⁴C]hexadecanol oxidation in concentration-dependent manner. Cell uptake of [¹⁴C]hexadecanol was also inhibited. The half-life of radioactive hexadecanol loaded into fibroblasts was 15 mins in the absence of exogenous palmitate and 80 minutes in the presence of 50 μM exogenous palmitate. The presence of exogenous did not affect the increase the relative incorporation of [¹⁴C]hexadecanol into phosphatidylethanolamine ether linkages, but did increase the loss of labelled alcohol into the medium.

Intracellular fibroblast hexadecanol content

Intracellular content of hexadecanol fibroblasts was 137 ± pmol/mg protein in the absence of exogenous palmitate. After 2 hours exposure to 50 μM exogenous palmitate intracellular hexadecanol content was 288 ± 64 pmol/mg protein.

Fatty aldehyde intermediate

Cells were incubated with [¹⁴C]palmitate or [¹⁴C]hexadecanol in the presence or absence of a large excess of unlabelled hexadecanal, and radioactive fatty aldehyde was measured. The results are shown in Table 2. [¹⁴C]Palmitate in presence of excess hexadecanol resulted in a slight increase in radioactive fatty aldehyde and a slight (10%) decrease in radioactive fatty alcohol. [¹⁴C]hexadecanol in presence of excess hexadecanol did not result in trapping of radioactive fatty aldehyde, though incorporation of radioactivity into fatty acid was decreased by 62%.

Simultaneous synthesis and oxidation of hexadecanol

The metabolism of [³H]palmitate and [¹⁴C]hexadecanol to hexadecanol and palmitate respectively was measured, and it was shown that synthesis and oxidation proceeded simultaneously. Conversion of [³H]palmitate to [³H]hexadecanol appeared to plateau, possibly due to re-oxidation of newly synthesised [³H]hexadecanol to [³H]palmitate. Conversion of [¹⁴C]hexadecanol to [¹⁴C]palmitate was linear with time. The presence of the much larger intracellular fatty acid pool and resulting dilution of [¹⁴C] radioactivity would mean that re-conversion of [¹⁴C]palmitate to [¹⁴C]hexadecanol would not cause a similar plateau. The cellular hexadecanol pool was measured as 670 pmol/mg protein, and the rate of hexadecanol oxidation (in conditions with 20 μM palmitate and 16 μM hexadecanol in medium) was 12.6 pmol/min/mg protein. It would take 53 minutes to renew the cellular hexadecanol pool at this rate.

These results demonstrated simultaneous synthesis and oxidation of hexadecanol. Incorporation of radioactivity from [1-¹⁴C]hexadecanol into lipids was measured (Table 3); the distribution was similar to that seen when cells were incubated with [1-¹⁴C]palmitate under identical conditions, showing that palmitate derived from hexadecanol apparently entered the general intracellular fatty acid pool.

FATTY ALCOHOL:NAD⁺ OXIDOREDUCTASE ACTIVITY

Fatty alcohol:NAD⁺ oxidoreductase catalyses oxidation of long chain alcohols to fatty acids. [1‑¹⁴C]hexadecanol was oxidised to free palmitate by fibroblast homogenates, dependent on NAD⁺. ATP and CoA had no effect on activity. The effect of palmitate and palmitoyl-CoA was investigated: palmitoyl-CoA inhibited activity; palmitate was less inhibitory. NADH was innhibitory (Table 4). The inhibitory effect of palmitoyl-CoA appeared to be synergistic with that of NADH at lower concentrations of palmitoyl-CoA (10-20 μM); at 80 μM the effects were additive. In the presence of 80 μM palmitoyl-CoA or 0.4 mM NADH, the amount of radioactive aldehyde increased. Hexadecanal did not affect reaction rate.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: the interconversion of fatty alcohols and fatty acids was demonstrated.
In a study using radio-labelled hexadecanoic acid and hexadecanol, the interconversion of fatty acid and fatty alcohol was demonstrated. The cellular concentration of hexadecanol was reported to be 137 ± 58 pmol/mg protein. A fatty alcohol cycle was proposed.