Propanoic acid, 2-hydroxy-, propyl ester, (2S)-

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-11-16 to 1999-01-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

S9 homogenates of liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium, prepared from healthy male Wistar rats, were used. The rates of hydrolysis of the lactate esters by these homogenates were determined, and the enzyme kinetic parameters Km and Vmax were established, where possible. The S9 homogenates used were similar to those used in a previous in vitro hydrolysis study with L-lactic acid esters (TNO-report V97.552). The esterase acitivities of the various homogenates towards the model substrate p-nitrophenyl-butyrate, were determined according to the method described by Bogdanffy et al. (1987): 100 µM p-nitrophenyl-butyrate and a suitable amount of homogenate were incubated in 0.1 M phosphate buffer pH 7.8 at 25°C in a total volume of 1 ml. The rate of hydrolysis was measured spectrophotometrically at 400 nm.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

propyl (2S)-2-hydroxypropanoate

EC Number:

611-025-7

Cas Number:

53651-69-7

Molecular formula:

C6H12O3

IUPAC Name:

propyl (2S)-2-hydroxypropanoate

Specific details on test material used for the study:

- Name of test material used in the report: n-Propyl-(L)-lactate
- Batch no.: MJE-8
- Purity: >99%
- Storage: ambient temperature

Radiolabelling:

no

Test animals

Species:

other: incubation with homogenate of rat blood, skin, liver, small intestinal mucosa and nasal olfactory epithelium.

Strain:

Wistar

Sex:

male

Administration / exposure

Route of administration:

other: incubation with homogenate of rat blood, skin, liver, small intestinal mucosa and nasal olfactory epithelium

Vehicle:

unchanged (no vehicle)

Details on exposure:

General incubation conditions:
The lactate esters were incubated at 37°C in 1 ml incubation mixtures containing 0.10 M potassium phosphate buffer pH 7.4. The chemical hydrolysis was determined in incubation experiments without homogenates. Blanks were homogenates without test substances. Incubations were performed in open tubes. The reaction was terminated by addition of 3 mL of ice-cold ethanol. After placing the samples in the freezer for at least 20 minutes, the tubes were centrifuged for 10 min at 4300 x g (room temperature) and decanted into new tubes. The samples were evaporated to dryness with nitrogen and stored at < -15°C until analysis.

Times and concentrations:
The lactate esters were incubated with the various homogenates for 5, 10, 20, 40 and 120 minutes. Chemical hydrolysis was measured by incubating the substrates without homogenates during 120 min. The lactate ester concentrations used were 50, 100, 250, 500 and 1250 µM.

Analysis:
The liberated amount of L-lactic acid was determined by using the Boehringer test for the determination of L-lactic acid in foodstuffs. The liberated amount of D-lactic acid was determined by using the Boehringer test for the determination of D- and L-lactic acid in foodstuffs and other materials. Instead of the glycylglycine buffer (pH 10) included in the kit, a 0.1 M potassium phosphate buffer pH 7.4 (assay buffer) was used, in order to minimize hydrolysis of the lactate esters during the measurement of the liberated amounts of L- and D-lactic acid.

Duration and frequency of treatment / exposure:

Single application for 5, 10, 20, 40 and 120 minutes.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 250 other: µM

Doses / concentrations 2

Dose / conc.:

500 other: µM

Doses / concentrations 3

Dose / conc.:

250 other: µM

Doses / concentrations 4

Dose / conc.:

100 other: µM

Doses / concentrations 5

Dose / conc.:

50 other: µM

No. of animals per sex per dose / concentration:

The amounts of tissue protein used in the incubation experiments, are: 2.38 µg of nasal olfactory epithelium protein, 192.4 µg of small intestinal mucosal protein, 7.43 µg of liver protein, 50.4 µg of skin protein and 832 µg of blood protein.

Control animals:

other: Chemical hydrolysis was determined in incubation experiments without homogenates. Blanks were homogenates without test substances.

Positive control reference chemical:

The esterase acitivities of the various homogenates towards the model substrate p-nitrophenylbutyrate were determined.

Details on dosing and sampling:

Analysis:
determination of L-lactic acid
The liberated amount of L-lactic acid was determined by using the Boehringer test for the determination of L-lactic acid in foodstuffs and other materials. The liberated amount of D-lactic acid was determined by using the Boehringher test for the determination of D- and L-lactic acid in foodstuffs and other materials. Instead of the glycylglycine buffer (pH 10), included in the kit, a 0.1 M potassium phosphate buffer pH 7.4 (assay buffer) was used, in order to minimize hydrolysis of the lactate esters during the measurement of the liberated amount of L- and D-lactic acid.
The detection limit of the method was arbitrarily fixed at 10 nmol (absorption ~ 0.02).

Statistics:

Calculations:
The amounts L-lactic acid or D-lactic acid formed during the incubations were calculated from the respective standard curves.
The rates of hydrolysis were corrected for the chemical hydrolysis, which was assumed to be a linear chemical proces.
The initital rates of hydrolysis were calculated from the amounts of liberated L-lactic acid/D-lactic acid (corrected for chemical hydrolysis) with the regression model:
liberated lactic acid (nmol) = a . (time) + b . (time)²,
or with the regression model
liberated lactic acid (nmol) = a.(time),
with a = regression coefficient of the linear component and b = regression coefficient of the quadratic component.
The regression coefficient 'a' respresents the initital rate of hydrolysis expressed as nmol/min.

After calculating the initial rates of hydrolysis expressed as nmol/min/mg S9 protein, the enzyme kinetic parameters Km and Vmax were determined by the curve-fitting program "EZ-FIT" (version 4.13 for MS Windows).

Results and discussion

Metabolite characterisation studies

Metabolites identified:

yes

Any other information on results incl. tables

The protein concentrations, and the esterase activities towards the model substrate p-nitrophenylbutyrate of the various tissue homogenates are presented in Table 1. These results show that esterase activity was present in the various homogenates and thus could be used for the measurement of esterase activity towards the various lactate esters.

 

Table 1:  Mean protein concentrations, and esterase activities towards p-nitrophenylbutyrate (mean ± sd) of the various rat tissue homogenates.

Homogenate	Protein concentration homogenate (mg/ml)	Esterase activity towards p-nitrophenylbutyrate
		Amount of protein used in assay (µg)	Activity (µmol/min/mg protein)
Nasal olfactory	7.14	11.9	1.013 ± 0.028
Small intestinal mucosa	9.62	2.41	7.836 ± 0.064
Liver	22.3	11.2	0.852 ± 0.010
Skin	2.52	63.0	0.113 ± 0.003
Blood	41.6	104	0.0134 ± 0.0001

 

The amounts of lactic acid formed by chemical hydrolysis after 2 hour incubations are presented in Table 2. From the results it can be concluded that the rate of chemical hydrolysis of n-propyl-L-lactate at pH 7.4 and 37°C is very low.

Table 2: Chemical hydrolysis of n-propyl-L-lactate to L-lactic acid.

Lactate ester	nmol of L-lactic acid formed at the various concentrations
	50 µM	100 µM	250 µM	500 µM	1250 µM
n-propyl-(L)-lactate	< 10	< 10	< 10	< 10	15.3

 

The initial rates of hydrolysis obtained for the various incubation time periods are presented in Table 3. The hydrolyses of n-propyl-L-lactate was most efficiently catalyzed by nasal olfactory epithelium and liver homogenates. The lowest activities were found for blood.

Table 3. Initial rates of hydrolysis of n-propyl-L-lactate by various rat tissue homogenates to L-lactic acid. Enzyme activities are expressed as nmol/min/mg protein.

	Concentration (µM)	Initial rates of hydrolysis
		Nasal olfactory epithelium	Small intestinal mucosa	Liver	Skin	Blood
n-propyl-(L)-lactate	50	260	3.7	165	23.6	0.7
	100	559	7.0	283	40.0	1.3
	250	982	13.0	516	75.0	2.8
	500	1128	21.4	674	104.3	4.9
	1250	1216	56.9	795	127.8	8.9

 

The enzyme kinetic parameters Km and Vmax, presented in Table 4, were calculated from the initial rates of hydrolysis of n-propyl-L-lactate. The hydrolysis of n-propyl-L-lactate by the homogenates of nasal epithelium, liver and skin showed Km values in the same order of magnitude (range 150-278 µM), while blood and small intestinal mucosa showed a high Km value, or first order kinetics in the tested concentration range. With respect to the obtained Vmax values it is observed that n-propyl-L-lactate was most efficiently hydrolyzed by nasal olfactory epithelium and liver homogenate.

 

Table 4. Enzyme kinetic parameters (mean ± sd) of the hydrolysis of n-propyl-L-lactate. V_max is expressed as nmol/min/mg protein.

Homogenate	Km (µM)	Vmax
Nasal olfactory epithelium	150 ± 37	1420 ± 109
Small intestinal mucosa	first order¹ v = 0.0455xS
Liver	220 ± 17	949 ± 25
Skin	278 ± 18	158 ± 4
Blood	1450 ± 62	19.2 ± 0.5

* v = rate expressed as nmol/min/mg protein; S = ester concentration

In order to extrapolate the obtained kinetic parameters in terms of disappearance rates of n-propyl-L-lactate in the organs/tissues, the obtained kinetic parameters were scaled up to hydrolysis rates expressed per weight of tissue, by using the total amount of protein/gram of tissue. Subsequently, the disappearance in time of the compound in the organs/tissues was calculated by the Michaelis-Menten or first order equation and the data presented in Table 4. A starting concentration of 500 µM was used. However, it had to be assumed that the equilibrium of the reactions are completely on the side of the hydrolyzed compounds. The calculated disappearance rates would be higher in vivo. The times were calculated in which at least 99% of the ester would be hydrolyzed (Table 5). Table 5 shows that n-propyl-L-lactate were hydrolysed very rapidly by nasal olfactory epithelium and liver homogenate, while blood and small intestinal mucosa were much less efficient. Table 5 also shows that chemical hydrolysis, compared to the enzymatical hydrolysis by nasal olfactory epithelium and liver, is not detectable.

 

Table 5. Calculated times (seconds) in which at least 99% of n-propyl-L-lactate would be hydrolyzed. The starting concentration was 500 µM. The reactions were assumed to be completely oriented towards the hydrolyzed compounds.

Homogenate	Time (s)
Nasal olfactory epithelium	0.9
Small intestinal mucosa	136
Liver	1.0
Skin	31
Blood	132
Chemical hydrolysis	-

- = no detectable rate of hydrolysis

Applicant's summary and conclusion

Conclusions:

In the present study, the hydrolysis of propyl (S)-lactate was studied by conducting incubation experiments with various rat tissue homogenates. It was found that the propyl (S)-lactate is hydrolysed very rapidely by the different tissue homgenates.

Executive summary:

The rates of hydrolysis of propyl (S)-lactate to L-lactic acid by homogenates of liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium, prepared from healthy mal Wistar rats, was studied. Enzym kinetic parameters Km and Vmax were established, where possible.

All homogenates showed esterase activity to propyl (S)-lactate. Nasal olfactory epithelium, liver and skin were, in this order, the most efficient tissues with repect to the hydrolysis of propyl (S)-lactate. Enzymatic hydrolysis of n-propyl-L-lactate in vivo would be much faster than chemical hydrolysis.