L-proline

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

other: Toxikokinetic assessment

Adequacy of study:

key study

Study period:

February 2012 to March 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Toxicokinetic assessment

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

2012

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Toxicokinetic assessment, based on
- Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance
- ECB EU Technical Guidance Document on Risk Assessment, 2003

GLP compliance:

no

Results and discussion

Main ADME results

Type:

absorption

Results:

Oral route: 100%. Dermal route: 10 %. Inhalation route: 100 %

Any other information on results incl. tables

L-proline is a natural occurring amino acid and a natural constituent of peptides and proteins. Its content in most proteins is ca. 4 – 7 %, high contents show e.g. gelatine (21.8 %), wheat protein (10.3 %) or casein (12.3 %) (Belitz et al, 2007). L-proline belongs to the group of amino acids with uncharged non-polar side chains. It is non-essential in humans.

 

L-proline biosynthesis begins with the ATP-driven phosphorylation and reduction of the carboxyl side chain of glutamate. The resulting glutamate gamma-semialdehyde then spontaneously cyclizes in a non-enzymatic reaction to produce delta1-pyrroline-5-carboxylate. This reduction of this intermediate creates the final pathway product, proline. In mammals, proline biosynthesis can occur through another route, through the urea cycle. In this pathway, arginine serves are the first intermediate that is converted to ornithine (Lehninger et al, 2000).

 

 

Adsorption

L-proline is absorbed from the gastrointestinal tract. Ingested dietary protein is denatured in the stomach due to low pH. Denaturing and unfolding of the protein makes the chain susceptible to proteolysis. Up to 15% of dietary protein may be cleaved to peptides and amino acids by pepsins in the stomach. In the duodenum and small intestine digestion continues through hydrolytic enzymes (e.g. trypsin, chymotrypsins, elastase, carboxypeptidase). The resultant mixture of peptides and amino acids is then transported into the mucosal cells by specific carrier systems for amino acids and for di- and tripeptides

 

The products of digestion are rapidly absorbed. Like other amino acids L-proline is absorbed from ileum and distal jejeunum.

 

 

Distribution

Absorbed peptides are further hydrolysed resulting in free amino acids which are secreted into the portal blood by specific carrier systems in the mucosal cell. Alternatively they are metabolised within the cell itself. Absorbed amino acids pass into the liver where a portion of the amino acids are used. The remainder pass through into the systemic circulation and are utilised by the peripheral tissue. L-proline is actively transported across the intestine from mucosa to serosal surface. The mechanism of absorption is that of the ion gradient. All L-amino acids are absorbed by Na⁺dependant, carrier mediated process. This transport is energy dependant by ATP. (All data from: Lehninger et al, 2000; Chatterjea and Shinde, 2012.)

 

Plasma L-proline concentrations in normal subjects are reported to be ca. 168 µM/L +/- 60 mM/L with plasma samples collected from healthy volunteers after an overnight fast (Cynober 2002). As with most nutrients, plasma concentration of L-proline is subject to homeostasis.

 

A number of hormones (e.g., thyroid hormone, catecholamines, and growth hormone) may affect plasma AA levels in diseases (Cynober et al., 1987). However, in the physiologic state, their influence is probably marginal. However, there is the counter-regulatory hormon system with cortisol and glucagon which influences the blood level of amino acids involved in gluconeogenesis, such as L-proline (Boden et al., 1984).

 

 

Metabolism

There is no storage form for amino acids in animals and human except in the biologically active protein of the cells.

 

L-proline exhibits the same metabolic pathway as several other amino acids do. Metabolism of L-proline is thus described by the entire pathway (Lehninger, 2010; Salway, 2004). This pathway (also known as “Ornithine and Proline Metabolism”) describes the co-metabolism of arginine, ornithine, proline, citrulline and glutamate in humans.

 

Arginine is synthesized from citrulline by the sequential action of the cytosolic enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Citrulline can be derived from ornithine via the catabolism of proline or glutamine/glutamate. Many of the reactions required to generate proline and glutamate from ornithine are located in the mitochondria. Proline is biosynthetically derived from glutamate and its immediate precursor, 1-pyrroline-5-carboxylate. The pathways linking arginine, glutamine, and proline are bidirectional. Thus, the net utilization or production of these amino acids is highly dependent on cell type and developmental stage.

 

On a whole-body basis, synthesis of arginine occurs principally via the intestinal–renal axis, wherein epithelial cells of the small intestine, which produce citrulline primarily from glutamine and glutamate, collaborate with the proximal tubule cells of the kidney, which extract citrulline from the circulation and convert it to arginine, which is returned to the circulation. Consequently, impairment of small bowel or renal function can reduce endogenous arginine synthesis, thereby increasing the dietary requirement.

 

Both proline and arginine are proteinogenic amino acids and are incorporated into proteins by prolyl-tRNA and arginyl-tRNA, which are synthesized by their respective tRNA synthetases. Arginine can also serve as a precursor for the synthesis of creatine and phopshocreatine through the intermediate guanidoacetic acid. A key component of the arginine/proline metabolic pathway is ornithine. In epithelial cells of the small intestine, ornithine is used primarily to synthesize citrulline and arginine, in liver cells surrounding the portal vein, ornithine functions primarily as an intermediate of the urea cycle, in liver cells surrounding the central vein, ornithine is used to synthesize glutamate and glutamine while in many peripheral tissues, ornithine is used for the synthesis of glutamate and proline.

 

 

Excretion

Body losses of amino acids are minimal because amino acids filtered by the kidneys are actively reabsorbed. Also cutaneous losses are negligible. Since there is no long term storage for amino acids in mammals, excess amino acids are degraded, mainly in the liver. Metabolism of amino acids involves removal of the amino group which is converted to urea and excreted in the urine. After removal of the amino group the rest of the acid is utilised as energy source or in anabolism of other endogenous substances.

 

L-proline is completely used by the organism after oral intake but rapidly converted and metabolised (Jaksic et al, 1987). Increase in plasma L-proline concentration is associated with a rapid decrease in L-proline endogenous biosynthesis with total inhibition at high rates of administered L-proline. Excess L-proline is shunted to other amino acids. Experiments show that there is a homeostatic regulation of L-proline metabolism in healthy human affecting the elimination via the urine to steady state level (Jaksic et al., 1984).

 

After uptake (orally, intravenously) of L-proline the concentration in blood of L-proline reaches steady state levels.

Intestinal absorption rates of amino acids were found to be > 50 % for most amino acids (Adibi et al, 1967) whereby the absorption rate of individual amino acids may increase depending of the administered concentration.These findings were made with amino acid concentrations far below the saturation plateau of the transport system. The absorption rate expressed as g/h of amino acids from different dietary proteins is high (Bilsborough and Mann, 2006) and indicates absorption rates > 50 % for individual amino acids. An oral absorption rate of 100 % is more realistic than a rate of 50 %.

The amounts of protein and, therefore, of amino acids consumed by humans vary over a wide range.When dietary nitrogen and essential amino acid intakes are above the requirement levels, healthy individuals appear to adapt well to highly variable dietary protein intakes, because frank signs or symptoms of amino acid excess are observed rarely, if at all, under usual dietary conditions (Bier, 2003). When considering the fact that DNELs based upon 100 % absorption rate are by factors well lower than the daily dietary intake of humans, an absorption rate of 100 % is not only realistic for the purpose of safety assessment but sufficiently safe.

For risk assessment purposes oral absorption of L-proline is set at 100%.

 

Other routes of excretion than that following to dietary intake are not relevant (e.g. exhalation).

 

L-proline is of low volatility due to a very low vapour pressure (0.00000403 Pa). From this and from the particle size it is not expected that L-proline reaches the nasopharyncheal region or subsequently the tracheobronchial or pulmonary region in significant amounts.

 

However, being a very hydrophilic substance with a molecular mass of only 115.13, any L-proline reaching the lungs might be absorbed through aqueous pores (ECHA, 2008). For risk assessment purposes, although it is unlikely that L-proline will be available to a high extent after inhalation via the lungs due to the low vapour pressure and high MMAD, the inhalation absorption of l-proline is set at 100%.

 

L-proline with high water solubility (162 g/L) and the log P value below 0 (-2.54) may be too hydrophilic to cross the lipid rich environment of the stratum corneum. Therefore, 10% dermal absorption of L-proline is proposed for risk assessment purposes.

 

Setting the dermal absorption rate tof 10 % considers derivations in ECHA (2008). Initially, basic physico-chemical information should be taken into account, i.e. molecular mass and lipophilicity (log P). Following, a default value of 100% skin absorption is generally used unless molecular mass is above 500 and log P is outside the range [-1, 4], in which case a value of 10% skin absorption is chosen (de Heer et al, 1999). The lower limit of 10% was chosen, because there is evidence in the literature that substances with molecular weight and/or log P values at these extremes can to a limited extent cross the skin.

For substances with log P values <0, poor lipophilicity will limit penetration into the stratum corneum and hence dermal absorption. Values <–1 suggest that a substance is not likely to be sufficiently lipophilic to cross the stratum corneum, therefore dermal absorption is likely to be low.

 

 

Citations:

Adibi SA,Gray SJ,Menden E. (1967). The kinetics of amino acid absorption and alteration of plasma composition of free amino acids after intestinal perfusion of amino acid mixtures.Am J Clin Nutr.1967 Jan;20(1):24-33.

Belitz H-D, Grosch W und Schieberle P. (2007): Lehrbuch der Lebensmittelchemie. 6. Auflage. Springer-Verlag, Berlin and Heidelberg

 

Bier, D.M. (2003): Amino acid pharmacokinetics and safety assessment. J. Nutr. 133:2034S-2039S.

Bilsborough, S and Mann, N. (2006): A review of issues of dietary protein intake in humans.International Journal of Sport nutrition and Excercise Metabolism, 16, 129-152

Boden G, Rezvani I, Owen OE (1984):. Efects of glucagon on plasma amino acids. J Clin Invest ,73:785

 

Chatterjea M and Shinde R (2012): Textbook of Medical Biochemistry. Jaypee Brothers Medical Publishers, New Delhi

 

Cynober L (2002): Plasma Amino Acid Levels With a Note on Membrane Transport: Characteristics, Regulation, and Metabolic Significance. Nutrition 18 (9), 761-766

 

Cynober L, Coudray-Lucas C, Ziegler F, et al. (1987): Metabolisme azote´ chez le sujet sain. Nutr Clin Metabol;3:87

 

De Heer, C., Wilschut, A., Stevenson, H., and Hakkert, B. C. Guidance document on the estimation of dermal absorption according to a tiered approach: an update.V98.1237. 1999. Zeist, NL, TNO.

ECHA (2008): Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance

 

Jaksic T, Wagner D, Burke E, Young, V (1987): Plasma Proline Kinetics and Regulation of Proline Synthesis in Man. Metabolism 36 (11), 1040-1045

 

Lehninger A, Nelson D, Cox M (2000), Principles of Biochemistry (3rd ed.), New York: W. H. Freeman

 

Salway J.G. (2004): Metabolism at a glance (3 rd ed.). Alden, Mass. : Blackwell Pub.

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: The absorption factors for risk assessment purposes have been set as follows: absorption oral 100%, absorption dermal 10% and absorption inhalation 100%
For risk assessment purposes:
Absorption oral: 100%,
Absorption dermal: 10%
Absorption inhalation: 100%