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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics, other
Remarks:
review
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting 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:
review article or handbook
Title:
Branched Chain Fatty Acids (BCFA) in the neonatal gut, and estimated dietary intake ininfancy and adulthood
Author:
R Ran-Ressler, R Glahn, S Bae, J Brenna
Year:
2013
Bibliographic source:
Nestle Nutr Inst Workshop Ser. 2013, 77, 133–143.

Materials and methods

Objective of study:
bioaccessibility (or bioavailability)
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
12-Methyltridecanoic acid
EC Number:
608-066-8
Cas Number:
2724-57-4
Molecular formula:
C14H28O2
IUPAC Name:
12-Methyltridecanoic acid
Constituent 2
Chemical structure
Reference substance name:
14-Methylhexadecanoic acid
Cas Number:
5918-29-6
Molecular formula:
C17H34O2
IUPAC Name:
14-Methylhexadecanoic acid
Constituent 3
Chemical structure
Reference substance name:
Isooctadecanoic acid
EC Number:
250-178-0
EC Name:
Isooctadecanoic acid
Cas Number:
30399-84-9
Molecular formula:
C18H36O2
IUPAC Name:
16-methylheptadecanoic acid
Constituent 4
Chemical structure
Reference substance name:
18-Methylnonadecanoic acid
Cas Number:
6250-72-2
Molecular formula:
C20H40O2
IUPAC Name:
18-Methylnonadecanoic acid
Test material form:
liquid
Radiolabelling:
not specified

Results and discussion

Metabolite characterisation studies

Metabolites identified:
not specified

Any other information on results incl. tables

Metabolism of BCFA


 


BCFA are normal constituents of the human neonatal gut and are metabolized by enterocytes.


To investigate human enterocyte metabolism of BCFA, we studied Caco-2 human colon carcinoma cells that differentiate into enterocyte cells [9], which, have characteristics common to fetal intestinal cells [10] and are used as a model for FA uptake [11]. Graded levels (0.1-0.5 mM) of a mixture of four pure BCFA with chain lengths of C14-C20 (iso-14:0, anteiso-17:0, iso-18:0 and iso-20:0) were delivered with bile salts in a micellar solution to simulate the postprandial intestine. Table 1 demonstrates that BCFA are taken up by Caco-2 in a dose responsive manner until they reached a plateau at around 0.50 mM. The evidence for both saturable and diffusive mechanisms for FA uptake by Caco-2 were described previously [11], and the saturable mechanism is consistent with the uptake pattern observed here. In addition, BCFA were incorporated into cell lipid classes in a selective manner (Table 2): anteiso-17:0 was unfavored in diacylglycerol (DAG) fraction compared to phospholipids (PL) and triacylglycerol (TAG). iso-18:0 was favored in PL and iso-20:0 was favored in DAG.


 


The relative uptake of dietary BCFA was calculated as the percent of a BCFA in the cells (%w/w) divided by the percent of that BCFA in the dietary mixture (%w/w), × 100. Cells were incubated for 4 hours with graded levels (0.1-0.5 mM) of a mixture of 4 BCFA (iso-14:0, anteiso-17:0, iso-18:0, and iso-20:0). BCFA were delivered to the cells in fat-free media with 10 mM Taurocholate salt and 0.2 mM mono-olein, as described by Ho et al. [11]. Control cells were treated with 10 mM Taurocholate salt and 0.2 mM mono-olein, with and without graded levels of oleic acid corresponding to doses of BCFA. BCFA were negligible in Control cells (not shown). Comparisons were made among the relative uptake of individual BCFA across treatment groups and confirm dose-dependent uptake (different superscripts (a,b,c,d) indicate a statistically significant difference, p<0.05). Cells took up BCFA from the medium in a dose responsive manner until they reached a plateau at around 0.50 mM of BCFA in the dietary mixture.


 


Table 1: Caco-2 cell relative uptake of BCFA (%; mean ± SEM) dose-response















































  Amount of total BCFA in the diet   
 BCFA type 0.1mM 0.25mM 0.4mM 0.5mM
 iso-14:0 12.6 ± 0.6 a 15.9 ± 0.2 b 22.1 ± 0.6 c20.9 ± 0.4 c
 anteiso-17:0 15.9 ± 0.2 a 18.7 ± 0.5 b 25.6 ± 0.3 c26.1 ± 0.7 b
 iso-18:019.2 ± 0.6 a  20.9 ± 0.5 a 26.9 ± 0.3 b  26.4 ± 0.7 b
 iso-20:0 9.4 ± 0.3 a  10.7 ± 0.3 ab 17.3 ± 0.2 bc  22.4 ± 2.8 c

 


The relative uptake of dietary BCFA was calculated as the percent of a BCFA in the cells (%w/w) divided by the percent of that BCFA in the dietary mixture (%w/w), × 100. Cells were incubated for 4 hours with graded levels (0.1-0.5 mM) of a mixture of 4 BCFA (iso-14:0, anteiso-17:0, iso-18:0, and iso-20:0). BCFA were delivered to the cells in fat-free media with 10 mM Taurocholate salt and 0.2 mM mono-olein, as described by Ho et al.. Control cells were treated with 10 mM Taurocholate salt and 0.2 mM mono-olein, with and without graded levels of oleic acid corresponding to doses of BCFA. BCFA were negligible in Control cells (not shown). Comparisons were made among the relative uptake of individual BCFA across treatment groups and confirm dose-dependent uptake (different superscripts (a,b,c,d) indicate a statistically significant difference, p<0.05). Cells took up BCFA from the medium in a dose responsive manner until they reached a plateau at around 0.50 mM of BCFA in the dietary mixture.


 


Table 2: BCFA uptake (%BCFA; mean ± SEM) into Caco-2 cells’ lipid classes








































Fatty acid DietPhospholipids (PL)  Diacylglycerols (DAG) Triacylglycerols (TAG)
 iso-14:0 16.8

 14.2 ± 1.6 a



 14.2 ± 2.7 a



 20.3 ± 1.5 a



 anteiso-17:0



 23.7



 27.5 ± 0.4 a



 21.6 ± 0.6 b



 28.5 ± 1.0 a



 iso-18:0



 27.2



 37.2 ± 0.4 a



 23.4 ± 0.7 b



 30.0 ± 1.2 c



 iso-20:0



 32.2



 37.2 ± 0.4 a



 23.4 ± 0.7 b



 20.5 ± 1.0 a



 


Cells were incubated as described in Table 1. Comparisons were made among individual BCFA across lipid classes (different superscripts (a,b,c,d) indicate a statistically significant difference between lipid classes, p<0.05). For example: anteiso-17:0 was unfavored in diacylglycerol (DAG) fraction compared to phospholipids (PL) and triacylglycerol (TAG). iso-18:0 was favored in PL and iso-20:0 was favored in DAG.


 


BCFA intake in infants and adults


 


The ingestion of BCFA is not limited to the perinatal period. The breastfed infant consumes BCFA since they are found in colostrum and mature milk at concentrations up to 1.5% w/w [23]. Taking 0.6% as a conservative estimate of mean BCFA concentration in American human milk [24] with 3.2% fat, we estimate that consumption of BCFA is about 19 mg BCFA per 100 mL human milk. Taking 600 mL as the mean intake of human milk by a one month-old infant, then a typical male infant weighing 4.5 kg (http://www.cdc.gov/growthcharts/data/who/GrChrt_Boys_24LW_100611.pdf, (Table 15-7)) consumes about 114 mg total BCFA per day, or about 25 mg BCFA per kg body weight. A three-month-old infant consumes, on average, 800 mL human milk per day, ingesting about 152 mg BCFA/day or about 23 mg BCFA/kg body weight for a male infant weighing the median of 6.5kg (http://www.cdc.gov/growthcharts/data/who/GrChrt_Boys_24LW_100611.pdf). For comparison, the mean human milk concentrations of the bioactive long chain polyun-saturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) is 0.32%w/w [25] and comparable total intakes for 1 and 3 month old infants would be 13 and 12 mg DHA per kg body weight per day, respectively. Thus, BCFA are consumed at levels that exceed DHA, a bioactive FA of great interest in infant nutrition. BCFA are not, however, a component of powdered milk formulas that use vegetable oils as their fat source, and in this sense these formula lipids differ from human milk.


BCFA are prominent in ruminant meat and milk, constituting 2% w/w of fat in the US retail cow’s milk supply [26]. Our preliminary analyses of foods from an Ithaca, New York, USA market show BCFA to be about 0.9-1.8% in various cheese and ground beef types (Ran-Ressler, et al., unpublished data). Based on these initial figures, and taking 1.3% and 0.9% as conservative estimates of the mean BCFA concentration in cheeses and in ground beef, respectively, we can estimate BCFA consumption in children. For children ages 2-11 years, the mean intake of beef is 43.5 g/d beef [27]. Children who consume a total of 28 g (1 oz) of cheese with an average of 27% fat, and 43.5 g/d of cooked beef with an average of 18% fat, consume about 168 mg BCFA per day. Addition of 1 cup of whole milk (155 mg BCFA) increases their daily BCFA intake to 323 mg. For comparison, the mean intake of DHA and arachidonic acid (ARA) in Canadian children, ages 4-7 years, was recently estimated to be 37 mg and 57mg per day [28]; both combined are only one third of the daily BCFA consumption from milk, cheese and beef.


 


Estimated BCFA content by representative serving size














































   

BCFA intake in common use serving sizes   



Food



 Fat content (% wt)



 BCFA (% w/w)



Amount consumed, g



BCFA content, mg



 Cow’s milk



 3.2



2.0 



 240.0



 155.0



Cooked beef



 18.0



 0.9 *



 100.0



 162.0



 Cheese



 27.0 **



 1.3 **



 28.0



 98.0



 Total BCFA



 



 



 



 415.0



* Preliminary determination of BCFA concentrations in beef, purchased from a local retail store.


** Preliminary determination of the mean fat and BCFA concentrations in different cheeses, purchased from a local retail store.


 


Based on the same preliminary food data, the BCFA intake of an adult who consumes 28g (1oz) of cheese, 100 g of cooked beef and 1 cup of whole milk will total approximately 415 mg/day (Table 4) (Ran-Ressler, et al., unpublished data). BCFA intake is greater than the 100 mg average daily consumption of the DHA and eicosapentaenoic acid (EPA) reported in a survey of 8604 Americans between 1999 and 2000 and by women of child-bearing age, based on NHANES III data [29,30]. Thus, BCFA are being consumed in substantial amounts by most non-vegans, during all life stages.


In conclusion, BCFA are normal constituents in the gut from very early age, and they are present in the gut throughout the human life cycle. Studies in human cell and animal models show that BCFA are not inert components of the GI tract but are metabolized by enterocytes.


 


References


9. Pinto M, Robine-Leon S, Maric-Dominique A, et al. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line caco-2 in culture. Biol Cell. 1983;47:323–330.


10. Blais A, Bissonnette P, Berteloot A. Common characteristics for na+-dependent sugar-transport in caco-2 cells and human-fetal colon. Journal of Membrane Biology. 1987;99:113–125.


11. Ho SY, Storch J. Common mechanisms of monoacylglycerol and fatty acid uptake by human intestinal caco-2 cells. Am J Physiol Cell Physiol. 2001;281:C1106–1117.


24. Aitchison JM, Dunkley WL, Canolty NL, et al. Influence of diet on trans fatty acids in human milk. Am J Clin Nutr. 1977;30:2006–2015.


25. Brenna JT, Varamini B, Jensen RG, et al. Docosahexaenoic and arachidonic acid concentrations in human breast milk worldwide. Am J Clin Nutr. 2007;85:1457–1464.


26. Ran-Ressler RR, Sim D, O’Donnell-Megaro AM, et al. Branched chain fatty acid content of united states retail cow’s milk and implications for dietary intake. Lipids. 2011;46:569–576.


27. Daniel CR, Cross AJ, Koebnick C, et al. Trends in meat consumption in the USA. Public Health Nutr. 2011;14:575–583.


28. Lien VW, Clandinin MT. Dietary assessment of arachidonic acid and docosahexaenoic acid intake in 4-7 year-old children. J Am Coll Nutr. 2009;28:7–15.


29. Ervin RB, Wright JD, Wang CY, et al. Dietary intake of fats and fatty acids for the united states population: 1999-2000. Adv Data. 2004:1–6.


30. Brenna JT, Lapillonne A. Background paper on fat and fatty acid requirements during pregnancy and lactation. Ann Nutr Metab. 2009;55:97–122.

Applicant's summary and conclusion

Conclusions:
Branched chain fatty acids (BCFA) are primarily saturated fatty acids (FA) with a methyl branch, usually near the terminal methyl group. BCFA are abundant in bacteria, skin, and vernix caseosa but have seldom been studied with respect to human nutrition. They are constituents of the term newborn infant gut lumen, being swallowed as vernix particulate components of amniotic fluid in the last trimester of normal pregnancy. The few published reports of BCFA in human milk enable an estimate that breast fed infants consume 19 mg BCFA per 100ml milk. Dietary BCFA consumption from milkfat and other ruminant products, the main sources of dietary BCFA, is more than 400 mg BCFA per day in adult Americans. This estimate exceeds by several fold the average dietary intake of bioactive FA, such as docosahexaenoic acid. In conclusion, BCFA are normal constituents in the gut from very early age, and they are present in the gut throughout the human life cycle. Studies in human cell and animal models show that BCFA are not inert components of the GI tract but are metabolized by enterocytes.

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