Phytyl fatty acid esters in vegetables pose a risk for patients suffering from Refsum's disease.

Patients suffering from Refsum's disease show mutations in the enzyme necessary for the degradation of phytanic acid. Accumulation of this tetramethyl-branched fatty acid in inner organs leads to severe neurological and cardiac dysfunctions which can even result in death. Thus, patients with Refsum's disease have to follow a specific diet resigning foods with high levels of phytanic acid and trans-phytol like products from ruminant animals with a tolerable daily intake (TDI) of ≤ 10 mg/d. We recently reported the occurrence of phytyl fatty acid esters (PFAE, trans-phytol esterified with a fatty acid) in bell pepper with trans-phytol amounts of up to 5.4 mg/100 g fresh weight (FW). In this study we carried out in vitro-digestion experiments of PFAE with artificial digestion fluids. Our results demonstrate that PFAE actually are a source for bioavailable trans-phytol and thus add to the TDI. Eating only one portion of bell pepper (∼150 g) could therefore lead to exploitation of the TDI of up to 81%. Analysis of additional vegetable matrices showed that also rocket salad with up to 4.2 mg/100 g FW trans-phytol bound in PFAE represents a risk-relevant food for patients with Refsum's disease and should therefore be taken into account

Phytyl Fatty Acid Esters in the Pulp of Bell Pepper (Capsicum annuum)

Phytyl fatty acid esters (PFAE) are esters of fatty acids with the isoprenoid alcohol phytol (3,7<i>R</i>,11<i>R</i>,15-tetramethylhexadec-2<i>E</i>-enol). In this study, PFAE were identified and quantified in bell pepper using gas chromatography with mass spectrometry (GC-MS). All red (<i>n</i> = 14) and yellow (<i>n</i> = 6) samples contained six or seven PFAE at 0.9–11.2 mg/100 g fresh weight. By contrast, PFAE were not detected in green bell pepper samples (<i>n</i> = 3). PFAE might eventually be a source for bioavailable phytol, which can be transformed into phytanic acid by humans. Phytanic acid cannot be properly degraded by patients who suffer from Refsum’s disease (tolerable daily intake (TDI) ≤ 10 mg of phytanic acid). The phytol moiety of the PFAE (0.4–5.4 mg/100 g fresh weight) would contribute up to ∼50% to the TDI with the consumption of only one portion of bell pepper fruit pulp

Composition of the digestion fluids.

<p>Composition of the digestion fluids.</p

<i>trans</i>-Phytol contents derived from PFAE in different vegetables [mg/100 g FW] and max. tolerable portion size [kg] to fully exploit the TDI (10 mg/d).

<p><i>trans</i>-Phytol contents derived from PFAE in different vegetables [mg/100 g FW] and max. tolerable portion size [kg] to fully exploit the TDI (10 mg/d).</p

Phytyl fatty acid esters in vegetables pose a risk for patients suffering from Refsum’s disease - Fig 3

<p>Schematic metabolism of PFAE in the human body with cleavage of PFAE (a) into the free fatty acid and free <i>trans</i>-phytol (b), which is then further metabolized by oxidation into phytenic acid (c) and finally by reduction of the double bond into phytanic acid (d) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188035#pone.0188035.ref006" target="_blank">6</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188035#pone.0188035.ref030" target="_blank">30</a>].</p

Targeting of endogenous MID1 by miRNAs hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p leads to a reduction of AR protein.

<p>SH-SY5Y were transfected with a pool of miRNA mimics (hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p), or MID1-specific siRNAs as positive control or a non-specific control siRNA as negative control. Upper panel: Left: MID1 as well as AR protein levels were analyzed on a western blot using specific antibodies. Actin was detected on the same membranes as loading control. A representative blot of n = 3 is shown. Right: quantification of blots. Columns represent mean values +/- SEM (* p < 0.05).</p

MicroRNAs miR-19, miR-340, miR-374 and miR-542 regulate MID1 protein expression

<div><p>The MID1 ubiquitin ligase activates mTOR signaling and regulates mRNA translation. Misregulation of MID1 expression is associated with various diseases including midline malformation syndromes, cancer and neurodegenerative diseases. While this indicates that MID1 expression must be tightly regulated to prevent disease states specific mechanisms involved have not been identified. We examined miRNAs to determine mechanisms that regulate MID1 expression. MicroRNAs (miRNA) are small non-coding RNAs that recognize specific sequences in their target mRNAs. Upon binding, miRNAs typically downregulate expression of these targets. Here, we identified four miRNAs, miR-19, miR-340, miR-374 and miR-542 that bind to the 3’-UTR of the MID1 mRNA. These miRNAs not only regulate MID1 expression but also mTOR signaling and translation of disease associated mRNAs and could therefore serve as potential drugs for future therapy development.</p></div

Targeting of endogenous MID1 by miRNAs hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p leads to a reduction of HTT protein.

<p>HEK293T cells stably expressing HTT-exon 1 with 51 CAG-repeats were transfected with a pool of miRNA mimics (hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p), or a non-specific control siRNA. Upper panel: Left: HTT-exon 1 protein levels were analyzed on a western blot using HTT specific antibodies or Actin-specific antibodies. A representative blot of n = 7 is shown. Right: quantification of blots. Columns represent mean values +/- SEM (* p < 0.05). Lower panel: phospho-S6 (pS6) as well as total S6 protein levels were analyzed on a western blot using specific antibodies. Actin was detected on the same blots as a loading control. A representative blot of n = 3 is shown. Right: quantification of blots. Columns represent mean values +/- SEM (* p < 0.05).</p