Měření hladin vitamínu E a saturace organismu vitamínem E

Association between genetic variants and response to long-term vitamin E supplementation

ATBC Study

• Randomized, placebo-controlled, double-blind intervention trial of alpha-tocopherol and ß-carotene supplementation that initially focused on the prevention of lung and other cancers [5]
• Genome-wide association study (GWAS)
• Common variants
• Circulating alpha-tocopherol concentrations
• Following 3 y of controlled supplementation
• 2112 middle-aged,
• Male smokers in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study cohort
• Alpha-tocopherol (50 mg/d)
• Fasting serum alpha-tocopherol concentrations measured after 3 y
• Models adjusted for age, BMI, serum total cholesterol, and cancer case status [5]

rs964184 on 11q23.3 (P = 2.6 × 10-12)

• (11q23.3:116648917, hg19, P = 2.6 × 10-12)
• Rs964184 in BUD13/ZNF259/APOA5
• Located between the BUD13 homolog (Saccharomyces cerevisiae) and zinc finger protein 259 (ZNF259) and only 359 bp 3' to ZNF259 [5]
• Subgroup analysis of men whose baseline concentrations of serum alpha-tocopherol were above the median value of 11.5 mg/L (26.7 µmol/L)
• Confirmed association of rs964184 with the 3-y supplemented ?-tocopherol concentrations (P = 4.8 × 10-10, ß = 0.08) [5]
• Minor allele in the same SNP, rs964184 associated with
• Lower HDL cholesterol
• Higher TG concentrations according to the results of recent meta-analyses [5]
• Associated with unsupplemented serum alpha-tocopherol in a population of women primarily comprosed of individuals who never smoked [5]

rs2108622 on 19pter-p13.11 (P = 2.2 × 10-7)

• Located between LOC100128347 and BUD13. [5]
• (19p13.12:15990431, hg19, P = 2.2 × 10-7)
• Is a missense mutation [V(Val) to M (Met)] in the region of CYP4F2 (cytochrome P450, family 4, subfamily F, polypeptide 2) [5]
• Associated with unsupplemented serum alpha-tocopherol in a population of women primarily comprosed of individuals who never smoked [5]

rs7834588 on 8q12.3 (P = 6.2 × 10-7)

• (8q12.3:63883593, hg19, P = 6.2 × 10-7)
• In an intron of Na+/K+ Transporting ATPase Interacting Protein 3 (NKAIN3) [5]

Výsledky

• Combined, these SNP explain 3.4% of the residual variance in serum alpha-tocopherol concentrations during controlled vitamin E supplementation - standard 50-mg daily dose [5]
• The mean serum concentration of alpha-tocopherol after 3 y of the standard dose-trial supplementation was higher than the baseline concentrations
• 18.1 vs. 11.9 mg/L (42.0 vs. 27.6 µmol/L) (P < 0.001)
• Wide range of response to 50 mg/d vitamin E supplementation
• Highly significant (P < 0.001) based on paired comparisons [mean change, 6.2 mg/L (14.4 µmol/L)]
• No significant changes between baseline and 3-y serum total and non-HDL cholesterol concentrations

Children

• Aged 9–17 years
• Mean/median serum alpha-tocopherol concentration was between about 15 and 30 µmol/L in seven European countries (Valtuena et al., 2011) [2]

• Plasma or serum alpha-tocopherol concentrations (after 12–14 hours of fasting) are commonly used to assess alpha-tocopherol status [2]

Diagnóza AVED

• Genetic testing finding two TTPA gene mutations may be useful to confirm the diagnosis [3]

Caused

• Mutation to the TTPA gene
• vitamin E cannot be distributed throughout the body
• Inherited in an autosomal recessive manner [3]

In vitro hydrogen peroxide–induced erythrocyte hemolysis test

• Chosen by the Institute of Medicine (IOM) in 2000 as a marker of vitamin E status
• Increased peroxide-induced erythrocyte hemolysis was correlated with increased erythrocyte fragility in vitamin E–deficient individuals.
• Anemia with increased erythrocyte turnover during vitamin E deficiency
• Observed in vitamin E–deficient children with cystic fibrosis [7]

• In 31 cystic fibrosis patients (males and females aged 1–42 years) with pancreatic insufficiency
• Not receiving alpha-tocopherol supplements or salicylates and not iron-deficient (Farrell et al., 1977)
• Mean (± standard error (SE)) RBC haemolysis (78 ± 4.5 %, range: 5–98 %)
• Was significantly higher than that of 32 adult controls (aged 18–40 years) (mean = 0.53 ± 0.12 %; range = 0–2 %, p < 0.001).
• Haemolysis
• 0 % ........... plasma alpha-tocopherol concentration above about 11.5–14 µmol/L
• Below 10 %..... about 9 µmol/L
• Nad 10%......... below cca 4.5 µmol/L [2]

• Eight children ( 1–17 years) with alpha-tocopherol deficiency
• Secondary to chronic severe liver disease
• Compared with five healthy controls ( 7–17 years) (Refat et al., 1991).
• Serum ‘vitamin E’ patients ranged from 1 - 4 mg/L ( equivalent to cca 2.3–9.3 µmol/L alpha-tocopherol)
• RBC haemolysis induced by peroxide was 100 % for five subjects
• 96, 41 and 0 % for the three others [2]
• Controls, serum ‘vitamin E’ concentrations were 10–13 mg/L (mean ± standard deviation (SD): 11 ± 1 mg/L)
• RBC haemolysis 0–2 %, for the three subjects [2]

• In vitro hydrogen peroxide-induced haemolysis is used to identify alpha-tocopherol deficiency
• It is not useful as a criterion for deriving the requirement for alpha-tocopherol [2]

V očních čočkách (intralentikulárně)

• Alfa-tokoferol 1,57-2,55 ng/g vlhké váhy
• Gamma-tokoferol 257-501 ng/g vlhké váhy
• Udržují v čočkách a sklivci hladinu reukovaného glutathionu jeho recyklací [1]

• Suplementace vit. E neovlivní hladinu vit. E v čočkách
• Hladiny alfa tokoferolu nad 20uM korelovaly s niží incid. katarakty, ale mnohé studie to neprokázaly [1]

Laboratorní chyby

• Oxidation of alpha-tocopherol by air
• May invalidate test results [4]

Opatření

• Centrifugation of the EDTA blood soon after venipuncture
• Quick separation of plasma from blood cells after centrifugation and subsequent flash freezing of the plasma in liquid nitrogen [4]
• Filling the space above the plasma with an inert gas (e.g., argon or nitrogen) [4]
• Protecting the sample from light by wrapping the container in aluminum foil, or using a black or light-shielded Eppendorf tube [4]
• Shipment of the sample to the test laboratory in dry ice [4]

Markers of oxidative damage

• Oxidative damage to DNA, proteins and lipids
• Plasma or preferably urinary F2-isoprostanes (EFSA NDA Panel, 2011) [2]

• Athlete runners consumed at dinner, before each trial, 75 mg each of D3-RRR and D6-all rac-alpha-tocopheryl acetates:
• Increased during a marathon race as compared with a rest period in the same subjects one month later (Mastaloudis et al., 2001)
• deuterated alpha-tocopherol disappearance rates
• plasma F2-isoprostane concentrations [2]

• All-rac-alpha-tocopheryl acetate supplementation was found to
• decrease urinary F2-isoprostanes in subjects
• With hypercholesterolaemia (Davi et al., 1997)
• In diabetics (Davi et al., 1999) [2]
• In subjects with polygenic hypercholesterolaemia supplemented with RRR-alpha-tocopherol (0–2 144 mg/day) for 16 weeks [2]

• Randomised controlled trial (RCT) in 30 healthy men and women
• Eight weeks either a placebo or alpha-tocopherol (at five different doses ranging from 134 to 1 340 mg/day, n = 5 in each group)
• Eight-week washout period
• Supplementation had no effect on two urinary isoprostanes measured in vivo at baseline and at 4, 8 and 16 weeks (Meagher et al., 2001)
• iPF(2alpha)-III
• iPF(2alpha)-VI [2]

• Markers of oxidative damage [2]
• Are not specific to the antioxidative effect of alpha-tocopherol [2]
• Relationship between alpha-tocopherol intake and these markers is ??? [2]
• Cannot be considered suitable biomarkers of function for alpha-tocopherol [2]

Insufficient data on markers of alpha-tocopherol intake/status/function

• Plasma/serum alpha-tocopherol concentration
• Hydrogen peroxide-induced haemolysis
• Urinary alpha-CEHC excretion
• Markers of oxidative damage
• To derive the requirement for alpha-tocopherol

Metabolický syndrom

• People with metabolic syndrome (MetS) may show half of the plasma VE found in the reference group (i.e., 12.5 µmol/L versus 25.5 µmol/L), despite a similar intake of VE (8.85 mg versus 9.33 mg of ?-tocopherol [8]

Turnover of alpha-tocopherol

Mean half-life of the dose

• Was 44 days in plasma
• 96 days in red blood cells (RBC)
• High individual differences were observed [2]

Plasmatické hladiny

• Multi-compartmental model of alpha-tocopherol metabolism was developed
• To determine mean transfer rates among body compartments
• Alpha-tocopherol concentrations in 12 healthy subjects
• (mean (range): 23 (19–27) µmol/L) [2]

Intake of RRR-alpha-tocopherol necessary to maintain stable plasmatic values

• Estimated 4 mg/day

Plasmatické hladiny

• Plasma/serum alpha-tocopherol concentration is not a sensitive marker of dietary alpha-tocopherol intake
• Lack of data on the relationship between plasma/serum alpha-tocopherol concentrations and alpha-tocopherol concentrations in peripheral tissues
• Plasma/serum alpha-tocopherol concentrations below about 12 µmol/L may be indicative of alpha-tocopherol deficiency
• Lack of data to set a precise cut-off value above which alpha-tocopherol status may be considered as adequate [2]
• vitamin E deficiency is suggested if the alpha-tocopherol level is
• Pod 5 mcg/mL
• Pod 11.6 mcmol/L
• Because abnormal lipid levels can affect vitamin E status, a
• Low ratio of serum alpha-tocopherol to lipids is the most accurate indicator in adults with hyperlipidemia
• Pod 0.8 mg/g total lipid [9]

Individuals with AVED

• vitamin E levels 0.00 - 3.76 µmol/L
• Mean 0.95 µmol/L, SD 1.79 µmol/L; n=132
• Plasma vitamin E concentration is generally lower than 4.0 µmol/L (<1.7 mg/L) [Cavalier et al 1998, Mariotti et al 2004] [4]

Normální hladiny vit. E v krvi

• 9.0 and 29.8 µmol/L (mean ± 2 SD)
• 16.3-34.9 µmol/L (El Euch-Fayache et al [2014])

Plazmatické koncentrace

• 15-40 uM
• Normally, the plasma alpha-tocopherol level is 5 to 20 mcg/mL (11.6 to 46.4 mcmol/L) [9]
• Suplementací zvýšení plazm. hladin cca 2-3x [1]

Correction of plasma alpha-tocopherol concentrations for lipids

• Is not appropriate in cases in which circulating lipids are below normal concentrations [7]
• Both malnutrition and infectious diseases can lower circulating cholesterol and its lipoprotein carriers [7]

Serum alpha-tocopherol concentration

• Dietary alpha-tocopherol intake significantly correlated with plasma alpha-tocopherol in 233 adults (men and women)
• Plasma alpha-tocopherol concentrations do 33 µmol/L14 (p = 0.001, n = 200) of both groups
• Median alpha-tocopherol intake
• 8.6 mg/day non-supplement users
• 17.8 mg/day supplement users [2]

• Dietary alpha-tocopherol intake adjusted for energy intake
• Correlated weakly with plasma alpha-tocopherol concentration
• Non-supplement users (n = 458), alpha-tocopherol intake (mean ± standard error of the mean (SEM))
• 8.7 ± 0.2 mg/day for men
• 9.7 ± 0.6 mg/day for women adjusted for energy intake [2]
• Alpha-Tocopherol intake (11.4, 7.7–15.5 mg/day including supplements)
• Serum alpha-tocopherol concentration were not associated with intake (IOM, 2000) [2]

• Controlled diet (alpha-tocopherol content: 2.1 ± 1.9 mg/day), and supplemented with 50 (week 2), 150 (week 3), 350 (week 4) and 800 (week 5) mg/day RRR-alpha-tocopherol [2]
• Average plasma alpha-tocopherol concentration increased with supplementation dose (from 24.6 ± 3.6 to 61.8 ± 18.1 µmol/L) (Schultz et al., 1995) [2]

V tukové tkáni

• Estimated that the adipose tissue contains cca 90 % of the total body alpha-tocopherol pool
• 99 % of alpha-tocopherol of the adipose tissue is in the bulk lipid (Traber and Kayden,1987) [2]
• 90 to 99 % of the total body RRR-alpha-tocopherol pool are contained in the adipose tissue

• Mean total body RRR-alpha-tocopherol pool of about 11 g (about 26 mmol)
• About 99 % was associated with a slowly turning-over compartment - adipose tissue (compartmental model of Novotny et al.; 2012) [2]

• Average body weight (67 kg)
• Estimated percentage of body fat (25 %) of the participants, Novotny et al. (2012)
• Calculated alpha-tocopherol concentration in adipose tissue 657 µg/g (1.53 µmol/g)
• Measurements provide variable results
• Alpha-tocopherol concentrations ranged from 61 - 811 µg/g (0.14–1.89 µmol/g) (Parker, 1988)
• Means varied from 73 - 245 µg/g (four groups studied post mortem) (0.17–0.57 µmol/g) (Dju et al., 1958)
• 83 - 268 µg/g in men (0.19–0.62 µmol/g)
• 123 - 355 µg/g in women (0.29–0.82 µmol/g) (biopsies) (Kardinaal et al., 1995; Su et al., 1998; El-Sohemy et al., 2001) [2]

• Changes in adipose tissue alpha-tocopherol concentrations take years (Schaefer et al., 1983; Handelman et al., 1994)
• Adipose tissue alpha-tocopherol concentration increased (10 - 60 % according to subjects) with 800 mg/day all-rac-alpha-tocopherol supplementation for one year
• Did not decrease after one year of discontinuation of the supplement [2]

• Efflux of alpha-tocopherol from adipocytes may be tightly regulated
• During weight loss, the triglyceride content of adipocytes and their size significantly decreased (three subjects)
• Without any change in ‘tocopherol’ content per cell (one subject) (Schaefer et al., 1983) [2]

• Flux of alpha-tocopherol from the adipose tissue to plasma lipoproteins is very low (close to 0 mg/day). [2]

Adipose tissue alpha-tocopherol concentration

• 85 healthy Dutch adults (men and women, aged 50–70 years) not taking vitamin supplements (Kardinaal et al., 1995)
• Intake, assessed by FFQ
• Significantly correlated with alpha-tocopherol concentrations in adipose tissue from biopsies of the buttock
• Costa Rican men (mean age ± SD: 56 ± 11 years) and women ( mean age ± SD: 60 ± 10 years) (El-Sohemy et al., 2001)
• Alpha-tocopherol intake adjusted for energy intake (assessed by FFQ)
• Significantly correlated with alpha-tocopherol concentrations in adipose tissue from biopsies of the buttock
• Correlations were low either for the whole sample or when vitamin supplement users were excluded
• Healthy men (aged 20–55 years) from Norway
• No association between alpha-tocopherol intake, assessed by FFQ (median, P25–P75: 11.4, 7.7–15.5 mg/day, including supplements)
• Adjustments for total plasma cholesterol, plasma triglycerides, BMI, age, sex, ethnicity and energy intake.

• Alpha-tocopherol in adipose tissue (microg/g total fatty acid methyl esters, n = 119 biopsies from the buttock) (Andersen et al., 1999).
• Changes in adipose tissue alpha-tocopherol concentrations take years (Schaefer et al., 1983; Handelman et al., 1994)
• Is not a good marker of either alpha-tocopherol intake or alpha-tocopherol status.

Typical levels of individual tocopherols and tocotrienols

• 115 mg/g alpha-tocopherol (101 mg/g minimum)
• 5 mg/g beta-tocopherol (pod 1 mg/g minimum)
• 45 mg/g gamma-tocopherol (25 mg/g minimum)
• 12 mg/g delta-tocopherol (3 mg/g minimum)
• 67 mg/g alpha-tocotrienol (30 mg/g minimum)
• Pod 1 mg/g beta-tocotrienol (pod 1 mg/g minimum)
• 82 mg/g gamma-tocotrienol (45 mg/g minimum)
• 5 mg/g delta-tocotrienol (pod 1 mg/g minimum)
• According to Directive 2002/46/EC [2]

High-Plasma Vitamin E Associated with Impaired Lipoprotein Transport

Vyjimky z jednoduchosti

• Plasma alpha-tocopherol has been used as a proxy to assess VE status
• Although an association between dietary intake and plasma alpha-tocopherol concentration has not been found consistently [8]
• Impaired VE bioavailability
• Main cause of low VE plasma levels
• In most cases, higher VE intake
• Would be expected to compensate for the dysfunction
• But unlike in variants related to lipoprotein malfunctioning
• Greater amount of VE in plasma
• Decreased VE availability in the liver and other tissues

Apolipoprotein synthesis and metabolism

• Play a crucial role in tocopherol and tocotrienol transport
• VE and its derivatives can be actively exchanged between the different circulating lipoproteins
• Positive correlations between VE isoforms and different circulating apolipoproteins have been observed
• Plasma proteome to characterize VE deficiency in children [8]
• Main apolipoproteins involved in alpha-tocopherol circulation