British Journal of Nutrition, 2016; 116: 126–131.

The naturally occurring α-tocopherol stereoisomer RRRα-tocopherol is predominant in the human infant brain.

Matthew J. Kuchan1, Søren K. Jensen2, Elizabeth J. Johnson3 and Jacqueline C. Lieblein-Boff1

1 Research, Scientific and Medical Affairs, Abbott Nutrition, Columbus, OH 43219, USA

2 Department of Animal Sciences, Aarhus University, Blichers Allé 20, DK-8830 Tjele, Denmark

3 Tufts University, Boston, MA 20111, USA

 

Abstract

Objectives:  α-Tocopherol is the principal source of vitamin E, an essential nutrient that plays a crucial role in maintaining healthy brain function. Infant formula is routinely supplemented with synthetic α-tocopherol, a racaemic mixture of eight stereoisomers with less bioactivity than the natural stereoisomer RRR-α-tocopherol. α-Tocopherol stereoisomer profiles have not been previously reported in the human brain.

Methods:  In the present study, we analysed total α-tocopherol and α-tocopherol stereoisomers in the frontal cortex (FC), hippocampus (HPC) and visual cortex (VC) of infants (n 36) who died of sudden infant death syndrome or other conditions.

Results:  RRR-α-tocopherol was the predominant stereoisomer in all brain regions (P <0·0001) and samples, despite a large intra-decedent range in total α-tocopherol (5–17 μg/g). Mean RRR-α-tocopherol concentrations in FC, HPC and VC were 10·5, 6·8 and 5·5 μg/g, respectively. In contrast, mean levels of the synthetic stereoisomers were RRS, 1–1·5; RSR, 0·8–1·0; RSS, 0·7–0·9; and Σ2S 0·2–0·3 μg/g. Samples from all but two decedents contained measurable levels of the synthetic stereoisomers, but the intra-decedent variation was large. The ratio of RRR:the sum of the synthetic 2R stereoisomers (RRS + RSR + RSS) averaged 2·5, 2·3 and 2·4 in FC, HPC and VC, respectively, and ranged from 1 to at least 4·7, indicating that infant brain discriminates against synthetic 2R stereoisomers in favour of RRR.

Conclusions:  These findings reveal that RRR-α-tocopherol is the predominant stereoisomer in infant brain. These data also indicate that the infant brain discriminates against the synthetic 2R stereoisomers, but is unable to do so completely. On the basis of these findings, investigation into the impact of α-tocopherol stereoisomers on neurodevelopment is warranted.

 

Supplementary

Key points:

Human beings, and in fact all vertebrates, must consume vitamin E since animals cannot synthesize this essential nutrient.  Consequently the newborn infant must receive vitamin E from breast milk and/or infant formula, which is necessarily fortified.  While vitamin E deficiency is rare in human beings, it leads to neuromuscular abnormalities with ataxia and myopathy thus emphasizing the role of vitamin E in normal functioning and development of the nervous system (1-3).

Vitamin E activity can be derived from four tocopherols (α-, β-, γ-, and δ-) and their corresponding four tocotrienols (α-, β-, γ-, and δ-).  However, α-tocopherol (a-T) is the most biologically active form of vitamin E (4).  The three chiral carbon atoms in the structure of a-T result in significant structural complexity as eight a-T stereoisomers are possible.  However, naturally occurring a-T only exists as the RRR stereoisomer (2R, 4’R, 8’R-a-T), commonly referred to as Natural Vitamin E.  In contrast, synthetic a-T (all-rac-a-T) is a racemic mixture of the eight possible stereoisomers.  Based on these differences in stereochemistry, extensive research has established that RRR-a-T has 1.36 to 2 times the biologic value as all-rac-a-T (5,6).  Discrimination amongst stereoisomers is known to occur in the liver through the action of α-Tocopherol Transfer Protein (7), which preferentially binds 2R stereoisomers resulting in degradation of the majority of 2S stereoisomers in all-rac-α-T (8).

RRR-α-T was the sole source of α-T during vertebrate evolution during which all vertebrates became dependent on exogenous/dietary sources.  Synthetic vitamin E entered the human food chain more recently.  Consistent with this, the traditional rat fetal resorption assay for vitamin E activity has revealed that RRR-a-T and the synthetic stereoisomers have different anti-fetal resorption potencies, with RRR-α-T being the most efficacious (9).

α-T is widely accepted to play an important role in the protection of membrane-bound unsaturated fatty acids from oxidation through its potent lipid soluble, chain-breaking antioxidant activity.  The antioxidant activities of the α-T stereoisomers do not differ in vitro.  Extending on its antioxidant activity, α-T is also believed to play roles in diverse biological processes, including regulation of enzymatic activity (10), gene expression (11), and cell signaling (11,12).  More recent evidence indicates that dietary RRR-α-T or all-rac-α-T have differential impact on gene expression [13].  It is generally believed that differences in bioavailability between RRR-α-T and all-rac-α-T can be overcome by supplementing with a higher level of all-rac.  However, if α-T stereoisomers have differential impact on gene expression, it is important to understand if, and to what extent, the human infant brain discriminates amongst them.  Available evidence in animals indicates that the brain and other tissues discriminate for RRR-α-T at the expense of all-rac-α-T (14-16).  All but one of these studies was unable to determine if the animal brain is able to discriminate amongst RRR-α-T and the synthetic 2R stereoisomers; RRS-, RSR-, and RSS-a-T.  For obvious reasons, access to human infant brain tissue for nutrient analyses is inherently difficult.  For these reasons, we chose to analyze the α-T stereoisomer profile in autopsied infant brain samples.

 

The Study:

Voluntarily donated, frozen brain samples were obtained from the National Institute of Child Health and Human Development Brain and Tissue Bank for Developmental Disorders at the University of Maryland.  Samples for 33 of the decedents were collected between 1995 and 2005, and between 2007 and 2008 for the remaining 3 decedents.  Tissues were voluntarily donated by the parents or legal guardians and were identified using a unique numerical identifier which obscured the identity of the decedent.  Identity obscured autopsy reports were reviewed to confirm decedents were otherwise healthy and did not suffer from brain abnormalities or other systemic pathologies. A total of 79 samples were obtained from frontal cortex (n=28), hippocampus (n=25) and occipital cortex (n=26) and it is noteworthy that these regions of the brain are associated with memory, executive function, and vision, respectively.

 

Findings:

We discovered that RRR-a-T was the predominant a-T stereoisomer in each region tested; occipital cortex, hippocampus, and frontal cortex.  Even more dramatic was the finding that RRR-a-T was the predominant stereoisomer in each of the 78 samples tested.  Surprisingly, total a-T concentration varied widely amongst decedents and tended to increase with the age of the decedent – this variability was not related to the death-preservation interval.  This high degree of variability in total a-T complicates the comparison of stereoisomer proportions amongst decedents.  For this reason, we chose to express stereoisomer proportions as % of total a-T in this summary (Figure 1).   On average the predominance was dramatic with approximately 70% of total a-T found as RRR-a-T.  The highest mean % of total for a synthetic stereoisomer was RRS-a-T at approximately 14%.

 

 

Figure 1.  Mean a-tocopherol stereoisomer profile in occipital cortex (A), hippocampus (B) and frontal cortex (C) from infant brain.  Profiles are the mean % of total α-tocopherol for each α-tocopherol stereoisomer in each brain region.  Percent stereoisomer profile totals 100% in each brain region.  Decedents (n = 36) were less than 365 days of age.  Samples were donated by the decedent’s legal guardian.

 

 

 

However, important information about these valuable samples is uncovered by additional analyses.  For this reason, we present individual a-T stereoisomer profiles for 14 of the 33 decedents we studied.  For these 14 decedents, we were able to acquire and analyze each of the three brain regions (Figure 2).  Examining these data in this way allows us to compare the % stereoisomer profile for the 14 decedents within each brain region, and also to compare the pattern for each decedent in each of the brain regions tested.    In the occipital cortex, all fourteen decedents had 48% or more of total a-T as RRR-a-T, and this stereoisomer was clearly the most common in each of the decedents.  It is also apparent that the order of stereoisomer predominance generally decreased as presented from left to right with the 2S stereoisomers being the least common.   Similar patterns were found for both hippocampus and frontal cortex.  Comparing across brain regions by decedent, we see that decedents 14 and 12 together with a grouping of decedents 10, 7, 6 and 3 had relatively low proportions of RRR-a-T in each region.  Likewise, decedents 13, 5 and 4 had relatively high proportions of RRR-a-T in each region.  Taken together, these data indicate that the a-T stereoisomer proportions were relatively consistent within an individual.  Decedent 5 was high RRR-a-T in each region, but was 100% RRR-a-T in hippocampus.  It is not clear if this reflects analytical error, natural variation or a real phenomenon.

It is clear that with the exception of decedent 5 in hippocampus, the remaining decedents, and decedent 5 in the remaining brain regions, all had measurable proportions of synthetic a-T stereoisomers.  The majority of decedents had at least 25% of total a-T as synthetic stereoisomers, and some had 40% – 52% of total a-T as synthetic stereoisomers.

Further examination of the ratio of RRR-a-T to the sum of RRS– + RSR– + RSS-a-T revealed that the lowest value for this ratio was 1 with average values of 2.3 -2.5 for each brain region.  These data are consistent with supra-hepatic selection against the synthetic 2R stereoisomers, and for RRR-a-T.

Importance of the study: This study is the first to describe the stereoisomer profile of a-T in human brain.  a-T is an essential nutrient important for development and function of neural tissue.  We chose to study the infant brain because synthetic a-T (all-rac-a-T) is widely used in infant formula and maternal supplements, but is known to have less vitamin E activity than RRR-a-T.  The predominance of RRR-a-T in all samples tested emphasized its importance in infant brain.  This finding is particularly surprising given the predominant use of all-rac-a-T.   The presence of significant quantities of synthetic stereoisomers in the brain of many of the decedents studied raises new and important questions.  Recent research has indicated that dietary RRR-a-T and all-rac-a-T have differential effects on gene expression in murine lymphocytes.  What if this is also true in brain tissue?  We believe this is an extremely important question for future research to address.

 

 

References:

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(3)       Traber MG, Sokol RJ, Ringel SP, et al. (1987) Lack of tocopherol in peripheral nerves of vitamin E-deficient patients with peripheral neuropathy N. Engl. J. Med. 317, 262–265.

(4)      Food and Nutrition Board, Institute of Medicine. 2000. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. Washington, DC: Natl. Acad. Press. 529 pp.

(5)       Brigelius-Flohe R, Traber MG (1999) Vitamin E: function and metabolism. FASEB J 13, 1145-1155.

(6)      Traber MG (2007) Vitamin E regulatory mechanisms. Ann Rev Nutr  27, 347–362.

(7)       Ferslew KE, Acuff RV, Daigneault EA, et al. (1993) Pharmacokinetics and bioavailability of the RRR and all racemic stereoisomers of alpha-tocopherol in humans after single oral administration.  J Clin Pharmacol 33, 84–88.

(8)      Traber MG, Burton GW, Ingold KU, et al. (1990) RRR– and SRR-alpha-tocopherols are secreted without discrimination in human chylomicrons, but RRR-alpha-tocopherol is preferentially secreted in very low density lipoproteins. J Lipid Res 31, 675–85.

(9)      Steele CE, Jeffery EH, Diplock AT (1974) The effect of vitamin E and synthetic antioxidants on the growth in vitro of explanted rat embryos. J Reprod Fertil  38, 115-123.

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(11)      Azzi A, Gysin R, Kempna P, et al. (2004) Vitamin E mediates cell signaling and regulation of gene expression. Ann N Y Acad Sci 1031:86-95.

(12)     Azzi A. (2007) Molecular mechanism of alpha-tocopherol action. Free Radic Biol Med 43, 16-21.

(13)    Han SN, Pang E, Zingg JM et al. (2010)  Differential effects of natural and synthetic vitamin E on gene transcription in murine T lymphocytes.  Arch Biochem Biophys 495, 49–55.

(14)    Leonard SW, Terasawa Y, Farese RV, Jr., et al. (2002) Incorporation of deuterated RRR- or all-rac-alpha-tocopherol in plasma and tissues of alpha-tocopherol transfer protein–null mice. Am J Clin Nutr 75:555-560

(15)    Weiser H, Riss G, Kormann AW (1996) Biodiscrimination of the eight alpha-tocopherol stereoisomers results in preferential accumulation of the four 2R forms in tissues and plasma of rats. J Nutr 126:2539-2549.

(16)    Lauridsen C, Engel H, Jensen SK, et al. (2002) Lactating sows and suckling piglets preferentially incorporate RRR– over all-rac-a-tocopherol into Milk, Plasma and Tissues. J. Nutr. 132, 1258–1264.

 

Contact:

Matthew J Kuchan, Ph.D.

Abbott Nutrition, Discovery Research

3300 Stelzer Road, Dept 105500/RP4-2,

Columbus, OH 43219-3034         

matthew.kuchan@abbott.com

 

 

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