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CONGRESSO NAZIONALE Società Italiana di Nutrizione Umana 20‐21 ottobre 2014 – ROMA
I MARCATORI DELLE VITAMINE IDROSOLUBILI
LUCIANA AVIGLIANO
DIPARTIMENTO DI MEDICINA SPERIMENTALE E CHIRURGIA
UNIVERSITA’ DI ROMA TOR VERGATA
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A balanced diet is one that provides adequate amounts of energy and
nutrients for health and well‐being.
Dietary Reference Values (DRVs) are the complete set of nutrient
recommendations and reference values, such as population reference
intakes /assunzione raccomandata per la popolazione (PRI), average
requirement/fabbisogno medio (AR), adequate intake/assunzione
adeguata (AI)
LARN 2014 (Livelli di Assunzione di Riferimento di Nutrienti ed energia pe r la popolazione Italiana) 2013‐2014 EFSA (European Food Safety Authority) DRVs for vitamin C Opinion published in November 2013
DRVs for biotin
Opinion published in February 2014.
DRVs for pantothenic acid Opinion published in February 2014.
DRVs for niacin
Opinion published in July 2014. DRVs for folate
Scheduled for public consultation from 22 July to 14 September 2014.
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based on published scientific literature
relationship between
nutrient status in the body
intake
of nutrient
suitable Indicators of nutrient requirement
Relation to human health
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INDICATORI
Livelli plasmatici: folati, vitamina C
Livelli urinari della vitamina e/o di suoi cataboliti: tiamina, niacina, biotina
Concentrazione cellulare: eritrociti ‐ folati
leucociti – vitamina C
Attivita enzimatica: attività transchetolasica eritrocitaria – tiamina
attività glutatione riduttasica eritrocitaria (EGRAC)‐ riboflavina
(Erythrocyte Glutathione Reductase Activity Coefficient)
Metaboliti: acido metilvalerico ‐ biotina
acido metilmalonico – vitamina B12
omocisteina – folati, vitamina B12 e B6, riboflavina
studi di deplezione‐replezione
VITAMINA
Prevenzione • malattia da carenza
• malattie croniche
• malformazioni fetali
STATO DI SALUTE
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CRITICITA’
 la misura di alcuni parametri può richiedere metodi analitici sofisticati, che devono essere validati;  indicatori diversi possono evidenziare situazioni diverse,
quali uno status recente (ore, giorni) dopo assunzione
alimentare oppure uno status vitaminico a lungo termine;
 i parametri possono variare in base all’età, al genere, allo
stato fisiologico, al genotipo del soggetto esaminato
 quasi tutte le vitamine, oltre alle funzioni coenzimatiche
su cui si basano i test, svolgono altri ruoli importanti (ad esempio epigenetico) non facilmente misurabili.
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VITAMIN C
WHAT IS THE OPTIMUN INTAKE of VITAMIN C IN HUMANS?
Frei et al. (2012) Critical reviews in Food Science and Nutrition 52:815‐
829
‐ Prevention of vitamin c deficiency disease
‐ Additional health benefits? Several health outcomes were considered
but data were found insufficient to establish DRV
Population reference intake/assunzione raccomandata per la popolazione (PRI) can be derived from indicators of vitamin status
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VITAMINA C
Studi di farmacocinetica mettono in correlazione l’assunzione della vitamina con i valori plasmatici, cellulari, urinari
Vitamin C pharmacokinetics in healthy volunteers: Evidence for a recommended dietary allowance
Levine M et al. Proc. Natl. Acad. Sci. USA 1996; 93, 3704‐9
Studio di deplezione (<5mg/die) –replezione in soggetti (n. 7 volontari sani ) ospedalizzati per 4‐6 settimane
A new recommended dietary allowance of vitamin C for healthy young women.
Levine M et al. Proc Natl Acad Sci U S A. 2001; 98:9842‐6.
(n. 26 volontari sani)
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Vitamin C plasma concentration
Intracellular vitamin C concentrations (millimolar) in circulating cells Bioavailability of vitamin C at steady state for each dose:
Dose, mg
15 30
50
100 200
500
1250
Vitamin C excretion over 24 h Median bioavailability, %
89
87.3 58 80 72
63 46
Vitamin C: a concentration‐function approach yields
pharmacology and therapeutic discoveries. Levine M et
al
Adv Nutr 2011
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lo stato nutrizionale viene valutato sulla base della  concentrazione di vitamina C plasmatica:
riflette un’assunzione a breve termine e non indica le effettive riserve
 concentrazione nei leucociti: è considerato il più sensibile indicatore dello stato in vit C,
ma la misura è tecnicamente complessa e l’interpretazione dei dati è complicata dalla variabilità del contenuto di vitamina nelle diverse frazioni leucocitarie e dalla mancanza di procedure standard.
Di conseguenza, la concentrazione plasmatica della vitamina rimane il test più ampiamente utilizzato.
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LARN, EFSA
I criteri fanno riferimento alle concentrazioni plasmatiche di vitamina C necessarie per la quasi saturazione tissutale, in modo da consentire un’ottimale funzione antiossidante e coenzimatica della vitamina.
Il fabbisogno per l’adulto corrisponde alla dose di vitamina C necessaria affinchè la concentrazione nei granulociti neutrofili sia vicina alla saturazione (1 mmol/L) con la minima escrezione urinaria. Tali criteri corrispondono ad una concentrazione plasmatica di 50‐ 60 μmol/L.
LARN Assunzione raccomandata per la popolazione (PRI) Maschio adulto Femmina adulta
105 mg/die
85 mg/die EFSA Assunzione raccomandata per la popolazione (PRI) Maschio adulto 110 mg/die
Femmina adulta
95 mg/die
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BIOTIN  CHARACTERIZE THE EFFECTS OF MARGINAL BIOTIN DEFICIENCY
‐ Marginal biotin deficiency during normal pregnancy
Mock DM et al. American Journal of Clinical Nutrition, 75, 295‐29, 2002
 Accelerated biotin catabolism
 Fetal accumulation by biotin. Fetal to maternal concetrations ratio = 6:1 (might contribute to maternal depletion)
Marginal biotin deficiency causes birth defects in humans?
‐ Protein‐energy malnutrition (children)
‐ Long‐term therapy with certain anticonvulsant
 DECIPHER THE ROLE OF HISTONE BIOTINYLATION IN GENE REGULATION AND GENOME STABILITY
Biotin requirements for DNA damage prevention. Zempleni J et al. Mutat Res. 733:58‐60, 2012 .
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DIRECT MEASURES
• Urinary excretion
of biotin and catabolites
18‐79 nmol/die (4,4‐35 µg/die) An early and sensitive indicator of biotin
deficiency
( The 24 h urinary excretion decreases
significantly over the depletion period )
• Serum concentration
Is not significantly affected
Zempleni J et al., 2013. Identification and assessment of markers of biotin status in healthy adults. British Journal of Nutrition, 110, 321‐329.
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INDIRECT MEASURES
Activity of PCC
24 h urinary excretion
CH3‐CH2‐CHOH‐CH2‐COOH (3HIA)
77‐195 μmol/die
3HIA‐ carnitine
Leucina 
Level of biotynilated PCC or MCC in lymphocytes
Increased urinary excretion of 3‐ hydroxyisovaleric acid and decreased urinary excretion of biotin are sensitive early indicators of decreased biotin status in experimental biotin deficiency. Mock DM et al , American Journal of Clinical Nutrition, 65, 951‐958, 1997. Urinary excretion of 3‐hydroxyisovaleryl carnitine is an early and sensitive indicator of marginal biotin deficiency in humans. Stratton SL et al , J Nut. 141: 353‐358, 2011.
Identification and assessment of markers of biotin status in healthy adults. Br J Nutr. Eng WK et al, 110, 321‐329, 2013.
1. loading phase
Day ‐ 14  ‐7 supplement 300 µg/d
15 subjects (10 woman) consumed an egg white beverage (22‐32 % of the diet dried weight) with each meal to produce marginal biotin deficiency. 3. Repletion phase
2. Depletion phase
3‐hydroxyisovaleric acid (3HIA) bisnorbiotin (BNB)
biotin
biotin sulfoxides (BSO) Indicators of marginal biotin deficiency and repletion in humans: validation of 3‐hydroxyisovaleric acid excretion and a leucine challenge. Mock DM et al. Am J Clin Nutr. 2002; 76; 1061‐8 14
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Biomarkers sensitive to biotin depletion have been identified in
adults
HOWEVER
the variability characteristics of these biomarkers and their ability
to discriminate between biotin insufficiency and adequacy are not
well known.
dose‐response relationships between biotin intakes and these
biomarkers have not been established.
the data are not sufficient to derive Average Requirement (AR) and
Population reference intake (PRI) PRI for biotin from the use of
available biomarkers of intake or status for any population.
an adequate Intake/assunzione adeguata (AI) is proposed for all population groups
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Biotin intake
Germania, Irlanda, UK, USA (LARN) Germania, Irlanda, Austria, Ungheria, Lettonia (EFSA)
mean/median intakes ranged from 26 (F) to 50 (M) μg/day: 18‐65 years
In older adult men and women (> 65 y), mean/median intakes ranged from 24 to 43 μg/day: LARN AI = 30 µg/die (≥ 18 years M, F) AI = 35 µg/die (pregnancy) EFSA AI = 40 µg/die (≥ 18 years M, F) (midpoint of the observed
median intake)
AI = 40 µg/die (pregnancy)
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FOLATI (e metabolismo dell’unita monocarboniosa)
Acido folico
MTHFR
Genotipo 677CT
Folati naturali
(metile –CH3)
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Biomarkers of intake and status (LARN, EFSA)
Serum/plasma folate concentration is a marker of recent dietary intake; as indicated by supplementation studies (100 – 4.000 µg/die of folic acid or 113 ‐
416 µg/die of 5‐ methyl THF )
Concentrations < 10 nmol/L, confirmed on consecutive occasions, indicate folate deficiency.
Red blood cell folate concentration. It is the most reliable biomarker of folate
status as it reflects tissue folate stores (amount available in bone marrow at the time of erythropoiesis)
Linear relationship within the folic acid intake range 50‐400 416 µg/die Concentrations < 340 nmol/L are indicative of folate deficiency.
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24‐hour urinary folate excretion reflects differences in dietary folate
intake within the range of 300‐1600 μg DFE/day). However, folate continues to be excreted in the urine even in advanced stages of folate suggesting that it is not a useful indicator of low dietary intake and status. Mean cell volume is of limited use since can be detected only at an advanced stage of folate deficiency
macrocytic cells appear in the bone marrow shortly after initiation of folate
depletion and before the fall in red blood cell folate concentration. However, given the long life span of the circulating red blood cells (i.e. 120 days) in the peripheral blood, macrocytosis can be detected only at an advanced stage of folate deficiency
Biomarkers of function Plasma homocysteine < 12 μmol/L
Plasma Hcy is a sensitive but not a specific biomarkers of folate status as it is closely dependent on genetic and nutritional factors: folate, vitamin B12 and vitamin B6
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Effects of genotypes
Some polymorphisms of genes encoding enzymes and transport proteins involved in folate metabolism are reported to have an impact on folate status and health
677CT polymorphism of the gene encoding the MTHFR enzyme. Homozygosity for the T allele is associated with reduced enzyme activity (up to 70 % lower) and around 20‐25 % lower serum folate and higher plasma total homocysteine
concentrations compared with the 677CC genotype
Biochemical abnormalities in the 677TT genotype are more pronounced in the face of a low folate status.
High prevalence in some European countries 12 % in the Northern and up to 24 % in Southern Europe
This polymorphim should be considered in determining the requirements for folate.
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Dietary Folate Equivalent – DFE
Institute of Medicine (IOM) 2000 ‐ Dietary Reference Intake for vitamins B and choline
Folate requirement and metabolism in nonpregnant women. Sauberlich HE et al. Am J Clin Nutr. 1987 Dec;46(6):1016‐28.
Depletion period of four weeks , n=10 donne (food folate = 50% folic acid)
Absorption of folate from fortified cereal‐ grain products and of supplemental folate
consumed with or without food determined by using a dual‐label stable‐isotope protocol. Pfeiffer CM et al. Am J Clin Nutr 1997;66:1388‐1397.
1 µg DFE = 1 µg food folate = 0,6 µg folic acid from a fortified food or as a supplement consumed with food
= 0,5 µg as a folic acid supplement taken on an empty stomach
µg DFE = µg food folate + (µg folic acid x 1,7)
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EFSA 2014
A long‐term controlled folate feeding study in young women supports the validity of the 1.7 multiplier in the dietary folate equivalency equation. Yang TL. Journal of Nutrition, et al. 2005. 135, 1139‐1145
Determining bioavailability of food folates in a controlled intervention study. Hannon‐Fletcher MP et al American Journal of Clinical Nutrition, 2004, 80, 911‐918.
Bioavailability of food folates is 80% of that of folic acid. Winkels RM, American Journal of Clinical Nutrition, 2007, 85, 465‐473.
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EFSA The Panel considers that two of three long‐term investigations using whole diets indicate that the bioavailability of food folate relative to folic acid may be higher than previously assumed.
The Panel also considers that results for folate bioavailability in these studies vary and that there is wide variation around estimates.
The Panel also considers that 5‐methyl‐THF has a bioavailability that is similar to that of folic acid.
The Panel considers that the difference in bioavailability between food folate
and folic acid needs to be accounted for.
In the absence of better data, agrees with the previous definition of the DFE ‐ bioavailability of food folate is around 50 %, half that of folic acid taken on an empty stomach
‐ bioavailability of folic acid from fortified foods or from a supplement ingested with food is about 85 %.
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EFSA 2014
DFE (Dietary Folate Equivalent)
average requirement (AR) adults (M, F) = 250 µg DFE
coefficient of variation (CV) of 15 % in order to account for the additional variability associated with the higher requirement for folate in individuals with the MTHFR 677TT genotype
population reference intake (PRI) = 320 µg DFE
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LARN 2014
Dietary folate from vegetables and citrus fruit decreases plasma homocysteine concentrations in humans in a dietary controlled trial. Brouwer et al., J Nutr 1999;129:1135–1139
The maternal Mediterranean dietary pattern is associated with a reduced risk of spina bifida in the offspring.
Vujkovic M et al. British J. Obstetrics and Gynaecology,116:408‐15, 2009.
Homocysteine lowering by folate‐
rich diet or pharmacological supplementations in subjects with moderat
e hyperhomocysteinemia.
Zappacosta, Mastroiacovo, Ruggeri, et al., Nutrients 5, 1531‐43, 2013
(Univ Cattolica; Ospedale Bambin Gesù; INRAN ‐ ROMA)
Supplementation with natural folate rich foods, folic acid and 5‐MTHF in a free‐
living mediterranean population reached a similar reduction in Hcy concentr
ations (also in C667T MTHFR) . However, it could be advisable to prefer a pharmacological supplementation in particular conditions such as pregnancy. 26
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LARN 2014
(a cura di Giulia Cairella, Stefania Ruggeri, Domenica Taruscio)
una dieta ricca di folati naturali ha lo stesso effetto dell’acido folico
sintetico nell’aumentare la folatemia plasmatica e nel ridurre
l’omocisteinemia.
I LARN esprimono le raccomandazioni in folati totali e non in folati
equivalenti, promuovendo un’alimentazione basata sul consumo di alimenti ricchi in folati.
Fabbisogno medio (AR) adulto (M e F) = 320 µg folati
Assunzione raccomandata per la popolazione (PRI) = 400 µg folati
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GRUPPO DI LAVORO LARN per le VITAMINE IDROSOLUBILI
Luciana Avigliano, Giulia Cairella, Francesca Garbagnati, Stefania Ruggeri, Isabella Savini, Paolo Simonetti, Domenica Taruscio
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