Serum 25-hydroxyvitamin D : determinants and associations with plasma lipids and bone mineral density in children

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DISSERTATIONS | SONJA SOININEN | SERUM 25-HYDROXYVITAMIN D: DETERMINANTS AND... | No 477

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THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences

ISBN 978-952-61-2847-4 ISSN 1798-5706

Dissertations in Health Sciences

THE UNIVERSITY OF EASTERN FINLAND

SONJA SOININEN

SERUM 25-HYDROXYVITAMIN D: DETERMINANTS AND ASSOCIATIONS WITH PLASMA LIPIDS AND BONE MINERAL DENSITY IN CHILDREN

This doctoral thesis provided data on dietary sources of vitamin D and the determinants of serum 25-hydroxyvitamin D (25[OH]D) and its associations with plasma lipids and bone mineral density (BMD) in a population sample

of Finnish children participating in the Physi- cal Activity and Nutrition in Children (PANIC)

study. Fortified milk products were the main source of vitamin D and the most important determinant of 25(OH)D. Higher 25(OH)D was associated with lower total cholesterol, low-den- sity lipoprotein cholesterol, high-density lipo-

protein cholesterol, and with higher BMD.

SONJA SOININEN

UEF_Vaitos_477_Soininen_kansi_18_07_20.indd 1 20.7.2018 8:34:47

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Serum 25-Hydroxyvitamin D:

Determinants and Associations with Plasma Lipids and Bone Mineral Density in

Children

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SONJA SOININEN

Serum 25-Hydroxyvitamin D:

Determinants and Associations with Plasma Lipids and Bone Mineral Density in

Children

To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Medistudia auditorium MS302, Kuopio,

on Friday, August 31^st 2018, at 12 noon

Publications of the University of Eastern Finland Dissertations in Health Sciences

Number 477

Institute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland

Kuopio 2018

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Grano Oy Jyväskylä, 2018

Series Editors:

Professor Tomi Laitinen, M.D., Ph.D.

Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences

Associate Professor Tarja Kvist, Ph.D.

Department of Nursing Science Faculty of Health Sciences

Professor Kai Kaarniranta, M.D., Ph.D.

Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences

Associate Professor (Tenure Track) Tarja Malm, Ph.D.

A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences

Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy

Faculty of Health Sciences

Distributor:

University of Eastern Finland Kuopio Campus Library

P.O.Box 1627 FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto

ISBN (print): 978-952-61-2847-4 ISBN (pdf): 978-952-61-2848-1

ISSN (print): 1798-5706 ISSN (pdf): 1798-5714

ISSN-L: 1798-5706

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Author’s address: Institute of Biomedicine/ School of Medicine University of Eastern Finland

KUOPIO FINLAND

Social and Health Center, City of Varkaus VARKAUS

FINLAND

Supervisors: Docent Virpi Lindi, Ph.D.

Institute of Biomedicine/School of Medicine University of Eastern Finland Library Kuopio University of Eastern Finland

KUOPIO FINLAND

Professor Timo Lakka, M.D., Ph.D.

Institute of Biomedicine/School of Medicine University of Eastern Finland

KUOPIO FINLAND

Virpi Sidoroff, M.D., Ph.D.

Department of Paediatrics North Karelia Central Hospital JOENSUU

FINLAND

Docent Anitta Mahonen, Ph.D.

Institute of Biomedicine/School of Medicine University of Eastern Finland

KUOPIO FINLAND

Reviewers: Professor Elina Hyppönen, Ph.D.

Centre for Population Health Research/School of Health Sciences University of South Australia

ADELAIDE AUSTRALIA

Dimitris Vlachopoulos, Ph.D.

Children’s Health and Exercise Research Centre Department of Sport and Health Sciences University of Exeter

EXETER

UNITED KINGDOM

Opponent: Professor Outi Mäkitie, M.D., Ph.D.

Children’s Hospital

University of Helsinki and Helsinki University Hospital HELSINKI

FINLAND

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Soininen, Sonja

Serum 25-Hydroxyvitamin D: Determinants and Associations with Plasma Lipids and Bone Mineral Density in Children

University of Eastern Finland, Faculty of Health Sciences

Publications of the University of Eastern Finland. Dissertations in Health Sciences Number 477. 2018. 74 p.

ISBN (print): 978-952-61-2847-4 ISBN (pdf): 978-952-61-2848-1 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

ABSTRACT:

Vitamin D is an essential factor in bone metabolism, and low serum 25-hydoxyvitamin D (25[OH]D) levels increase the risk of rickets, osteomalacia, and osteopenia. Vitamin D recommendations are mainly based on bone health, but there is still no consensus on an optimal serum 25(OH)D level. Moreover, knowledge on the associations of vitamin D with cardiovascular health and other health issues is still inconclusive, and there are few studies on these topics in children. Vitamin D deficiency is still common worldwide. Therefore, more information on the distribution and determinants of serum 25(OH)D concentration and its associations with various health issues in different age groups and geographic areas is needed.

The purpose of this thesis was 1) to investigate vitamin D intake, serum 25(OH)D levels, and the associations of dietary and other factors with serum 25(OH)D concentration; 2) to examine the associations of serum 25(OH)D with plasma lipids and whether gene variants previously associated with 25(OH)D modify these associations; and 3) to study the association of lean body mass (LM), body fat mass (FM), and related biomarkers, such as 25(OH)D, adipokines, myokines, and cytokines, with bone mineral density (BMD) in children. The analyses of this thesis are based on a population sample of 472 Finnish children aged 6–8 years who participated in the baseline measurements of the Physical Activity and Nutrition in Children (PANIC) study in 2007-2009.

The thesis showed that almost 20% of the children had serum 25(OH)D levels < 50 nmol/l, which is often used to indicate vitamin D insufficiency. However, mean serum 25(OH)D was > 65 nmol/l, and serum 25(OH)D < 25 nmol/l, indicating very low levels, were rare (0.5%). Milk, which is commonly fortified with vitamin D in Finland, was the main dietary source of vitamin D, providing about 50% of the daily vitamin D intake. Other important sources of vitamin D were fat products (27%) and fish products (10%). Milk products were also the most important determinant of 25(OH)D in both girls and boys. In girls, also younger age and a higher vitamin D intake from supplements were associated with higher 25(OH)D levels. About 40% of the children did not use vitamin D supplements, and 60% did not meet the total intake of 7.5 µg/d of vitamin D from food and supplements, which was the recommendation at the time of data collection. Children who drank ≥ 3 glasses (≥ 450 ml) of milk per day, spent ≥ 2.2 hours/day in physical activity, had ≥ 13.1 hours/day of daylight time, or were examined in autumn had a reduced risk of having serum 25(OH)D < 50 nmol/l.

Higher serum 25(OH)D was associated with lower plasma total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and lower plasma triglycerides. The inverse associations of 25(OH)D with total, LDL, and HDL cholesterol remained after controlling for body fat percentage, dietary factors, physical activity, sedentary behavior, and socioeconomic status. Gene variants in CYP2R1 (rs12794714 and rs10741657) and DBP (rs2282679) were associated with 25(OH)D. The same allele in CYP2R1 (rs12794714) that was associated with lower 25(OH)D was also associated with

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lower total and LDL cholesterol, and a gene variant in C10orf88 (rs6599638) was associated with HDL cholesterol when adjusted for 25(OH)D. However, these gene variants did not explain or modify the associations of 25(OH)D with lipids.

LM was the strongest positive determinant of BMD, but also FM was positively and independently associated with BMD in this population sample of mainly normal-weight prepubertal children. Of biomarkers related to body composition, irisin had a positive association with BMD independently of LM and FM. Serum 25(OH)D was a positive correlate of BMD, but the association was weak and partly explained by LM.

This thesis provides data on the determinants of serum 25(OH)D and its associations with plasma lipids and BMD in children 6-8 years of age, a topic that has not been studied much in this age group in large population samples assessing several possible confounding factors. Higher serum 25(OH)D was independently associated with lower plasma total, LDL, and HDL cholesterol. This thesis showed new associations of SNPs related to 25(OH)D metabolism with plasma lipids, independently of 25(OH)D. However, the causality and mechanisms for the associations of serum 25(OH)D or the SNPs with plasma lipids need to be confirmed in larger population samples. Serum 25(OH)D was positively associated with BMD, but the association was much weaker than that of LM and FM. Even though the mean serum 25(OH)D was > 65 nmol/l, insufficient serum 25(OH)D levels and especially insufficient vitamin D intake were fairly common in Finnish children. Fortified milk products were the main source of vitamin D and the most important determinant of serum 25(OH)D levels. More attention should be paid to the sufficient intake of vitamin D from food and supplements, especially among children who do not use fortified milk products.

National Library of Medicine Classification: QT 250, QU 85, QU 95, QU 100, QU 145.5, QU 145.7, QU 173, QU 477, QW 541, QW 568, WA 105, WD 145, WE 202

Medical Subject Headings: Vitamin D; 25-Hydroxyvitamin D 2/blood; Calcifediol/blood; Vitamin D Deficiency; Risk Factors; Epidemiologic Factors; Food; Food, Fortified; Dairy Products; Milk; Dietary Fats;

Fish Products; Dietary Supplements; Recommended Dietary Allowances; Exercise; Sedentary Lifestyle;

Socioeconomic Factors; Sunlight; Cholesterol, LDL; Cholesterol, HDL; Triglycerides; Bone Density; Body Composition; Adipose Tissue; Biomarkers; Adipokines; Leptin; Cytokines; Polymorphism, Single Nucleotide;

Confounding Factors (Epidemiology); Child; Finland

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Soininen, Sonja

Seerumin D-vitamiinipitoisuus: selittävät tekijät ja yhteydet plasman rasva-arvoihin ja luuntiheyteen lapsilla Itä-Suomen yliopisto, terveystieteiden tiedekunta

Publications of the University of Eastern Finland. Dissertations in Health Sciences 477. 2018. 74 s.

ISBN (print): 978-952-61-2847-4 ISBN (pdf): 978-952-61-2848-1 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

TIIVISTELMÄ:

D-vitamiinin tiedetään olevan tärkeä luun aineenvaihduntaan liittyvä tekijä. D-vitamiinin saantisuositukset perustuvat pääasiassa sen luustovaikutuksiin ja matalan seerumin 25- hydroksi-D-vitamiinipitoisuuden (25[OH]D) tiedetäänkin olevan riisitaudin lisäksi yhteydessä myös osteomalasiaan ja osteopeniaan. Optimaalisesta seerumin 25(OH)D- pitoisuudesta ei kuitenkaan ole vielä päästy yhteisymmärrykseen. Tieto myös D-vitamiinin muista terveysvaikutuksista on lisääntynyt, mutta se on vielä riittämätöntä suositusten muodostamiseen. D-vitamiinin puute on edelleen yleistä maailmalla. Sen vuoksi tarvitaan lisää tietoa seerumin 25(OH)D-pitoisuudesta ja sitä selittävistä tekijöistä eri ikäryhmissä ja eri maantieteellisillä alueilla.

Tämän väitöskirjan tarkoituksena oli tutkia 1) D-vitamiinin saantia, seerumin 25(OH)D- pitoisuutta ja sitä selittäviä tekijöitä; 2) 25(OH)D-pitoisuuden yhteyttä plasman rasva- arvoihin ja muokkaavatko 25(OH)D-pitoisuuteen aiemmin yhteydessä olleet geenivariantit tätä yhteyttä; sekä 3) kehon lihas- ja rasvamassan sekä lihas- ja rasvakudokseen liittyvien biomarkkereiden, kuten 25(OH)D:n, myokiinien, adipokiinien ja sytokiinien yhteyttä luuntiheyteen lapsilla. Tutkimusaineistona oleva väestöotos koostuu 472 suomalaisesta 6−8-vuotiaasta lapsesta, jotka osallistuivat Lasten liikunta ja ravitsemus -tutkimuksen alkumittauksiin v.2007-2009.

Lapsista lähes 20 %:lla oli matala seerumin 25(OH)D-pitoisuus (< 50 nmol/l).

Keskimääräinen seerumin 25(OH)D-pitoisuus oli kuitenkin yli 65 nmol/l, ja 25(OH)D oli alle 25 nmol/l vain 0.5%:lla lapsista. Maito, joka on Suomessa pääosin D-vitaminoitua, oli tärkein D-vitamiinin lähde ja lapset saivat päivittäisestä D-vitamiiniannoksestaan noin 50 % maidosta. Muita tärkeitä D-vitamiinin lähteitä olivat ravintorasvat (27 % D-vitamiinin saannista) ja kalavalmisteet (10 % D-vitamiinin saannista). Maitotuotteet olivat myös tärkein 25(OH)D-pitoisuutta selittävä tekijä sekä tytöillä että pojilla. Tytöillä lisäksi nuorempi ikä ja D-vitamiinilisän käyttö olivat yhteydessä suurempaan 25(OH)D- pitoisuuteen. Lapsista noin 40 % ei käyttänyt D-vitamiinilisiä, ja 60 % ei saavuttanut tutkimusaineiston keruuvaiheessa voimassa ollutta D-vitamiinin kokonaissaantisuositusta, joka oli 7,5 µg päivässä. Vähintään kolme lasillista (≥ 450 ml) maitoa päivässä juovilla lapsilla ja yli 2,2 tuntia päivässä liikkuvilla lapsilla oli pienempi todennäköisuus matalaan 25(OH)D-pitoisuuteen (<50 nmol/l) kuin vähemmän maitoa juovilla tai alle 1,5 tuntia päivässä liikkuvilla. Myös suurempi päivän pituus (≥ 13,1 tuntia päivässä) ennen verinäytteenottoa ja syksyyn ajoittuva tutkimusajankohta olivat yhteydessä pienempään matalan 25(OH)D-pitoisuuden riskiin.

Korkeampi seerumin 25(OH)D-pitoisuus oli yhteydessä matalampaan plasman kokonais-, LDL-, ja HDL-kolesterolipitoisuuteen sekä matalampaan triglyseridipitoisuuteen. Yhteys kokonais-, LDL- ja HDL-kolesteroliin säilyi, vaikka analyyseissä vakioitiin kehon koostumus, ravitsemustekijät, fyysinen aktiivisuus ja perheen sosioekonominen tilanne. D-vitamiinin aineenvaihduntaan liittyvistä geenivarianteista CYP2R1 (rs12794714 ja rs10741657) sekä DBP (rs2282679) olivat yhteydessä seerumin 25(OH)D-pitoisuuteen. Lisäksi CYP2R1 (rs12794714):n alleeli, joka oli yhteydessä

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matalampaan 25(OH)D-pitoisuuteen, oli yhteydessä myös matalampaan kokonais- ja LDL- kolesteroliin, ja C10orf88 (rs6599638) oli yhteydessä HDL-kolesteroliin, riippumatta seerumin 25(OH)D-pitoisuudesta. Geenivariantit eivät kuitenkaan selittäneet tai muokanneet 25(OH)D:n yhteyttä veren rasva-arvoihin.

Kehon lihasmassa oli tärkein luuntiheyttä selittävä tekijä. Myös kehon suurempi rasvamassa oli yhteydessä suurempaan luuntiheyteen riippumatta lihasmassasta. Irisiini, jota erittyy lihaksista liikunnan vaikutuksesta, oli tutkimuksen perusteella yhteydessä suurempaan luuntiheyteen. Suuremmalla seerumin 25(OH)D-pitoisuudella havaittiin yhteys suurempaan luuntiheyteen, mutta tämä yhteys selittyi osittain lihasmassalla.

Tämä väitöskirja tuo tietoa seerumin 25(OH)D-pitoisuutta selittävistä tekijöistä ja sen yhteydestä plasman rasva-arvoihin ja luuntiheyteen ikäryhmässä, jossa näitä asioita on tutkittu vähän. Tutkimuksen vahvuus on kohtalaisen suuri väestöotos, jossa on mitattu ja pystytty huomioimaan lukuisia mahdollisia sekoittavia tekijöitä. Suurempi seerumin 25(OH)D-pitoisuus oli yhteydessä matalampiin plasman rasva-arvoihin. Seerumin 25(OH)D-pitoisuuteen aiemmin yhteydessä olleiden geenivarianttien 25(OH)D:sta riippumaton yhteys veren rasva-arvoihin on uusi havainto, joka vaatii varmentamista suuremmissa väestöotoksissa. Myös 25(OH)D-pitoisuuden ja veren rasva-arvojen yhteyden syy-seuraussuhteen selvittäminen sekä mekanismien löytäminen vaatii vielä lisää tutkimuksia. Korkeamman seerumin 25(OH)D-pitoisuuden yhteys suurempaan luuntiheyteen oli verrattain heikko ja kehon koostumus oli merkittävämpi luuntiheyttä selittävä tekijä. Vaikka keskimääräinen seerumin 25(OH)D-pitoisuus oli yli 65 nmol/l, matalat 25(OH)D-pitoisuudet ja erityisesti riittämätön D-vitamiinin saanti oli lapsilla melko yleistä. D-vitaminoidut maitotuotteet olivat tärkein ravinnon D-vitamiinin lähde, ja myös tärkein seerumin 25(OH)D-pitoisuutta selittävä tekijä. Riittävään D-vitamiinin saantiin ja D-vitamiinilisien käyttöön tulisikin kiinnittää huomiota erityisesti niillä lapsilla, jotka eivät käytä D-vitaminoituja maitovalmisteita tai muita D-vitaminoituja tuotteita.

Luokitus: QT 250, QU 85, QU 95, QU 100, QU 145.5, QU 145.7, QU 173, QU 477, QW 541, QW 568, WA 105, WD 145, WE 202

Yleinen suomalainen asiasanasto: D-vitamiini; maitovalmisteet; maito; ravintorasvat; kalavalmisteet;

vitaminointi; ravintoainevalmisteet; ravitsemussuositukset; fyysinen aktiivisuus; liikkumattomuus;

sosioekonomiset tekijät; päivänpituus; lipoproteiinit; LDL-kolesteroli; HDL-kolesteroli; triglyseridit;

luuntiheys; kehonkoostumus; rasvakudokset; lihasmassa; leptiini; sytokiinit; markkerit; geenitutkimus;

riskitekijät; puutostilat; kouluikäiset; Suomi

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Acknowledgements

This study was carried out at the Institute of Biomedicine, University of Eastern Finland, Kuopio, as a part of the Physical Activity and Nutrition in Children (PANIC) study. I warmly thank all children and families participating the PANIC study. The doctoral studies were carried out in the Doctoral Programme of Clinical Research.

I have had a privilege to get Virpi Lindi as my principal supervisor. Virpi, you led me to the world of science and I have learnt a lot from you. I admire your optimism and warmth, you have always found time for my questions and you have been supporting me so much during these years. I am grateful to the principal investigator of the PANIC study and my supervisor Timo Lakka. Timo, you introduced me to the PANIC study already at the pilot phase when I conducted my Master’s thesis. I have had an honor to be part of the team since then, even though I have been performing first my medical studies and then clinical work. You encouraged me to doctoral studies and challenged me to do my best in writing the manuscripts. I own my deepest thanks to my other supervisors, Anitta Mahonen and Virpi Sidoroff. Anitta, you taught me biochemistry already at my principal studies, and I want to thank you for sharing your expertise and experience during this project. Virpi, thank you for all the conversations and support especially on topics related to bone metabolism and on clinical aspects. I am grateful you have found time for me along with clinical work.

I sincerely thank the pre-examiners Professor Elina Hyppönen and Dr. Dimitris Vlachopoulos for the valuable comments and constructive criticism that helped me to find still some new aspects and to improve the thesis. I am also honoured to Professor Outi Mäkitie, who has agreed to act as opponent during public examination.

I want to thank all my co-authors Aino-Maija Eloranta, Taisa Venäläinen, Anna Viitasalo, Jarmo Jääskeläinen, Mustafa Atalay, David Laaksonen, Tomi Laitinen, Nina Zaproudina, Liisa Kröger, Heikki Kröger, Arja Erkkilä, and Geneviève Dion for their contribution to this work. David, I also want to thank you for the language editing of my thesis. I owe my warmest thanks to the whole PANIC study group who have been helping with collecting the data. It has been so nice to work in the “zoo team” with a great team spirit, humor, and friendship. Particularly, thanks to Juuso Väistö who has kindly helped me in the problems with information technology, and Niina Lintu who helped me a lot with my first grant applications.

In appreciation of the financial support of this thesis, I thank the Institute of Biomedicine and Institute of Dentistry in University of Eastern Finland, Finnish Cultural Foundation, Orion Research Foundation sr, and Kuopio University Hospital State Research Funding. I also thank all the other funders of the whole PANIC study.

I warmly thank my employer City of Varkaus and my managers in the Social and Health Center for the positive attitude and the opportunity for conducting research. I also thank my colleagues for their support.

I owe my warmest thanks to my friends and family for the support. I thank my mother- in-law and father-in-law Irma and Seppo for taking care of our children. Matias, Katja and Julia: my dearest siblings, I always miss you, thank you for being there for me. Katja and Anna, remember to seek your motivation dresses! Mum and dad, I am so grateful for all your help and love, you have always supported us children in all ways. Touko and Silja, my sweethearts, you have given us so much happiness and love, and you always remind me what is really important in life. Mum always loves you! I owe my deepest gratitude to my beloved husband Janne. Even though you may not have always understood my enthusiasm towards research, you have supported me with patience and love.

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List of the original publications

This thesis is based on the following original publications:

I Soininen S, Eloranta AM, Lindi V, Venäläinen T, Zaproudina N, Mahonen A, and Lakka TA. Determinants of Serum 25-Hydroxyvitamin D Concentration in Finnish Children: The Physical Activity and Nutrition in Children (PANIC) study. Br J Nutr 2016;115:1080-91.

II Soininen S, Eloranta AM, Viitasalo A, Dion G, Erkkilä A, Sidoroff V, Lindi V, Mahonen A, and Lakka TA. Serum 25-hydroxyvitamin D, Plasma Lipids, and Associated Gene Variants in Prepubertal Children. J Clin Endocrinol Metab 2018;

103:2670-2679.

III Soininen S, Sidoroff V, Lindi V, Mahonen A, Kröger L, Kröger H, Jääskeläinen J, Atalay M, Laaksonen DE, Laitinen T, Lakka TA. Body fat mass, lean body mass and associated biomarkers as determinants of bone mineral density in children 6- 8 years of age - The Physical Activity and Nutrition in Children (PANIC) study.

Bone 2018;108:106-114.

The publications were adapted with the permission of the copyright owners. In addition, some previously unpublished data are presented.

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Abbreviations

1,25(OH)2D, 1,25-dihydroxyvitamin D, calcitriol

25(OH)D, 25-hydroxyvitamin D, calcidiol BF%, body fat percentage

BMC, bone mineral content BMD, bone mineral density BMI, body mass index Ca, calcium

C10orf88, chromosome 10 open reading frame 88

CI, confidence interval

CYP2R1, cytochrome P450 family 2 subfamily R member 1, vitamin D-25- hydroxylase

CYP24A1, cytochrome P450 family 24 subfamily A member 1, vitamin D-24- hydroxylase

CYP27B1, cytochrome P450 family 27 subfamily B member 1, 25(OH)D-1α- hydroxylase

DBP, vitamin D binding protein

DHCR7, 7-dehydrocholesterol reductase DHEAS, dehydroepiandrosterone

sulphate

DXA, dual-energy x-ray absorptiometry ELISA, enzyme linked immunosorbent

assay

FM, body fat mass

GC, vitamin D binding protein

GWAS, Genome-wide association study HDL, high-density lipoprotein

HOMA-IR, homeostatic model assessment for insulin resistance

hs-CRP, high-sensitivity C-reactive protein

IGF-1, insulin-like growth factor 1 IL-6, interleukin 6

IOM, Institute of Medicine of the USA LDL, low-density lipoprotein

LM, lean body mass

MAF, minor allele frequency MUFA, monounsaturated fatty acid NADSYN1, NAD synthetase 1

NNR, Nordic nutrition recommendations OR, odds ratio

PANIC study, Physical Activity and Nutrition in Children study Pi, phosphate

PTH, parathyroid hormone PUFA, polyunsaturated fatty acid SD, standard deviation

SDS, standard deviation score SFA, saturated fatty acid

SNP, single nucleotide polymorphism TNF-α, tumor necrosis factor α UV, ultraviolet

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1 Introduction

Vitamin D is one of the oldest hormone precursors existed on Earth, because it was found in phytoplanktons that have survived for over 750 million years [1]. Along with evolution, vitamin D became essential for calcium metabolism. As a result of industrialization, children living in sunless conditions developed clinical signs of rickets, which was recognized as a major health problem in children in the 1600s [1,2]. In the 1800s some researchers thought that rickets may be somehow related to sunlight. In 1919, exposure to ultraviolet (UV) light from a mercury lamp was reported to cure rickets [1]. Cod liver oil was recognized to cure rickets in Germany and France already in the 1800s [2]. However, cod liver oil became more commonly used in the 1900s, when it was reported to contain an anti-rachitic factor, which was named as “vitamin D”. The sensible exposure of the children to sunlight and the ultraviolet irradiation of foods were used to cure rickets, and later on, vitamin D supplementation was provided for children. The uncontrolled fortification of foods led to the outbreak of vitamin D intoxication in Great Britain and some other countries [2]. After that, the fortification of food products with vitamin D became more controlled in Europe. In Finland, the appreciated archiater Arvo Ylppö recognized rickets as a major problem in 1925 [3]. The recommendation for vitamin D supplementation among infants was 100 µg/d in the beginning of the 1900s, was first halved to 50 µg/d in the 1960s [4] and was further gradually reduced ending up to the current recommendation of 10 µg/d for infants and 7,5 µg/d for older children [5,6].

Since the days of recognizing vitamin D, knowledge on its health effects has been increasing. A recent study found a total of 25 992 research articles on vitamin D published in 1900−2014 [7]. Even though it is known that vitamin D is essential for bone metabolism and that low serum 25-hydroxyvitamin D (25[OH]D) levels increase the risk of rickets, osteomalacia, and osteopenia [8], there is still no consensus on the optimal serum 25(OH)D levels. Moreover, vitamin D has been suggested to have multiple extraskeletal and mostly beneficial health effects on for instance cardiometabolic risk factors, but evidence for such effects has been insufficient to make recommendations [8,9]. Vitamin D deficiency is still common worldwide and also in Europe [10,11]. Therefore, more information on the distribution and determinants of serum 25(OH)D concentration and its associations with various health issues in different age groups and geographic areas is needed.

There are a few studies on serum 25(OH)D concentration and its determinants, including the intake of vitamin D from food and supplements, among children from Nordic countries and other countries located at the same latitude. These children are at increased risk of vitamin D deficiency due to long and dark winters. Plasma lipid abnormalities, especially high low-density lipoprotein (LDL) cholesterol, are risk factors for atherosclerosis already in childhood [12]. Moreover, cardiometabolic risk factors tend to track from childhood to adulthood [13]. It is therefore important to understand the associations, mechanisms, and potential confounding factors between serum 25(OH)D and plasma lipids.

The associations of 25(OH)D with plasma lipid levels in children and adolescents have been conflicting [14–20], and very few studies have been carried out in prepubertal children [18]. Increased serum 25(OH)D may be due to a leaner body composition and healthy lifestyle, including a healthy diet, and spending plenty of time on physical activities outdoors resulting in increased vitamin D production in the skin, all of which may also be associated with a more favorable plasma lipid profile. Many studies in children lack data on these potential confounding factors. Several gene variants related to vitamin D metabolism are recognized to be associated with serum 25(OH)D levels [21,22]. However, there are few studies on the associations of these gene variants with plasma lipids [23–25].

Vitamin D and cholesterol have a common precursor, 7-dehydrocholesterol and vitamin D

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receptor complexes have been suggested to regulate cholesterol metabolism [26]. We therefore hypothesized that genetic factors related to vitamin D metabolism may partly explain or modify the association between serum 25(OH)D and plasma lipids.

Lean body mass (LM) has been positively associated with bone mineral density (BMD) in children and adolescents, but the relationship between body fat mass (FM) and BMD remains controversial. Serum 25(OH)D is important for bone metabolism, and it has been suggested to be associated with body adiposity. In addition to the mechanical load caused by LM, FM, and physical activity, several biomarkers secreted by adipose tissue, skeletal muscle, or bone may affect bone metabolism and BMD. However, there are still little data on these issues especially in children.

The purpose of this thesis was to investigate vitamin D intake and determinants of serum 25(OH)D concentration in a population sample of Finnish children aged 6 – 8 years.

The second aim was to examine the associations of serum 25(OH)D and potential confounding lifestyle factors with plasma lipids, and whether gene variants related to vitamin D metabolism modify this association. The third aim was to study the associations of LM, FM, and related biomarkers such as 25(OH)D, adipokines, myokines, and other cytokines with BMD.

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2 Review of the Literature

2.1 VITAMIN D METABOLISM

Vitamin D is a fat-soluble vitamin that can be obtained from foods and supplements [10,27].

It has two forms, ergocalciferol (Vitamin D²), which is plant origin and may only be obtained from food, and cholecalciferol (Vitamin D3), which is animal origin and is also synthetized endogenously in the skin (Figure 1). Cholesterol and vitamin D³ are synthesized from a common precursor, 7-dehydrocholesterol from the Kandutsch-Russel pathway of cholesterol synthesis. The 7-dehydrocholesterol reductase (DHCR7) converts 7- dehydrocholesterol to cholesterol. However, in the presence of ultraviolet B radiation (wavelength 290 to 315 nm) in the skin, 7-dehydrocholesterol is converted to previtamin D3, and further in the skin to vitamin D³. Prolonged exposure to sunlight causes the isomerization of previtamin D3 into its inactive metabolite lumisterol or tachysterol, preventing sun-induced vitamin D intoxication.

Vitamin D³ synthesized in the skin, or vitamin D² or D³ derived from food, are transported by vitamin D binding proteins (DBP) through blood into the liver [10,27]. There it is converted by 25-hydroxylase (CYP2R1) into 25(OH)D, the major circulating vitamin D metabolite, which is used as an indicator of vitamin D status. This biologically inactive metabolite is converted mainly in the kidney by 25(OH)D-1α-hydroxylase (CYP27B1) to the active metabolite 1,25-dihydroxyvitamin D (1,25[OH]²D). However, this activating process may to lesser extent happen also in tissues other than kidney [10,27].

The active metabolite 1,25(OH)²D regulates calcium, phosphorus, and bone metabolism [10,27]. On the other hand, the activating process from 25(OH)D to 1,25(OH)2D is tightly regulated by parathyroid hormone (PTH), circulating calcium and phosphate, fibroblast growth factor 23, and 1,25(OH)²D itself. Moreover, 1,25(OH)²D increases the expression of 25(OH)D-24-hydroxylase (CYP24A1) to catabolize 1,25(OH)2D to the water-soluble, biologically inactive calcitroic acid, which is excreted in the bile. The biological effects of 1,25(OH)2D are mediated by vitamin D receptor (VDR), which binds with retinoic acid x- receptor, forming a complex with VDR. This complex binds to specific nucleotide sequences in DNA, vitamin D response elements, and through transcription factors finally causes up- or downregulation of the target gene’s activity [10]. The main physiological function of vitamin D is to maintain normal circulating calcium levels. In small intestine, 1,25(OH)2D enhances calcium and phosphorus absorption. In osteoblasts, 1,25(OH)2D causes increase in the expression of the receptor activator of nuclear-factor-κB ligand, which in turn interacts with its receptor in preosteoclasts [10,27]. This induces preosteoclasts to become mature osteoclasts, which remove calcium and phosphorus from bone, maintaining their normal circulating levels. In addition to small intestine and bone, VDRs are found from most tissues of the body, and vitamin D is suggested to regulate hundreds or even thousands of genes [10]. This provides a basis for a variety of possible health effects of vitamin D.

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Figure 1. Vitamin D metabolism.

Abbreviations: UV-B, ultraviolet B; DBP, vitamin D binding protein; CYP2R1, cytochrome P450 family 2 subfamily R member 1=vitamin D-25-hydroxylase; CYP27B1, cytochrome P450 family 24 subfamily B member 1=25(OH)D-1α-hydroxylase; Ca, calcium; Pi, phosphate; PTH, parathyroid hormone

2.2 MEASUREMENT OF SERUM 25(OH)D

Total serum 25(OH)D concentration has been used as a biomarker of vitamin D status, and the recommendations for vitamin D intakes and definitions of vitamin D deficiency are based on 25(OH)D levels [28]. Total 25(OH)D is a sum of 25(OH)D2 and 25(OH)D3 levels.

There are several methods for measuring serum 25(OH)D, including chromatographic methods, protein-binding methods, and immunochemical methods, which may give different results on 25(OH)D [29,30]. The 25(OH)D has been described as a difficult analyte due to its hydrophobic nature and its binding to the DBP and the impact of sample matrix on assay performance. In addition, some of the assays measure total 25(OH)D, and some can differentiate 25(OH)D² and 25(OH)D³levels. The non-equimolar detection of 25(OH)D² and 25(OH)D3 or a presence of a molecule called 3-epimer of 25(OH)D can also be sources of bias. The methods used vary across the studies and it has been difficult to compare 25(OH)D levels from different studies.

Vitamin D External Quality Assessment Scheme was incorporated in 1989 to ensure the analytical reliability of 25(OH)D and 1,25(OH)²D assays [31]. In November 2010, the National Institutes of Health Office of Dietary Supplements in United States of America (USA) in collaboration with Centers for Disease Control and Prevention National Center for Environmental Health, National Institute of Standards and Technology, and Ghent University, established the Vitamin D Standardization Program [28,32]. It is an international collaborative effort to standardize the laboratory measurement of vitamin D status so that the results will be accurate and comparable over time, location, and laboratory procedure. A standardized laboratory measurement of 25(OH)D is comparable to the National Institute of Standards and Technology [33] and Ghent [34] Reference Measurement Procedures. National Institute of Standards and Technology has provided a certified standard reference serum (SRM972) with assigned values for the content of 25OHD2, 25OHD3, and 3-epimer of 25OHD3 [35]. One of the targets of the Vitamin D

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Standardization Program is to standardize national health survey 25(OH)D measurements across the world, which will be valuable when developing clinical guidelines for vitamin D intake and recommendations on 25(OH)D levels [28]. Total 25(OH)D concentration can be reported in units of nanograms per millilitre (ng/ml) or nanomoles per litre (nmol/l); ng/ml can be converted approximately into nmol/l using the formula: ng/ml x 2.5 ~ nmol/l. Nmol/l is used as the unit of measure in this thesis.

2.3 RECOMMENDATIONS FOR VITAMIN D INTAKE AND SERUM 25(OH)D CONCENTRATION IN CHILDREN

2.3.1 Recommendations for vitamin D intake in children

The dietary reference intakes provide values for the estimated average requirement, recommended dietary allowance or recommended intake, lower intake level, and upper tolerable level for vitamin D. The average requirement reflects the estimated median requirement and the value is to be primarily used to assess the risk for inadequate intake of micronutrients in a certain group of individuals [8,9]. Recommended intake or recommended dietary allowance for vitamin D are targeted so that intake meets or exceeds the needs of > 97.5% of the population [8]. Long-term intakes below the lower intake level are associated with an increased risk of developing deficiency symptoms, but due to substantial uncertainty, they should be applied with caution. Therefore, a lower intake level is provided only for adults in Nordic Nutrition Recommendations (NNR) [9]. One international unit (IU) corresponds to 0.025 μg vitamin D, and μg is used as the unit of measure in this thesis.

The Institute of Medicine (IOM) in the USA has recommended the intake of vitamin D from food and supplements of 10 μg/d for infants and 15 μg/d for older children and adults [8]. In 2012, the Nordic and Finnish experts recommended the intake of vitamin D from food and supplements of 10 μg/d for all children [6,9], raising the recommended intake from 7.5 μg/d for children over 2-year-old given in previous NNR [36]. Moreover, in Finland, infants under 2 years of age are recommended to use 10 μg/d and older children 7.5 μg/d of vitamin D supplements year-round, regardless of their dietary intake of vitamin D [6]. This recommendation for vitamin D supplementation [6] was slightly increased and is simpler to follow than the previous one [37,38]. In other Nordic countries, the use of supplements is generally recommended for infants but not for older children, unless the dietary intake of vitamin D is insufficient [9]. The upper tolerable level is the highest average daily intake that is likely to pose no risk of adverse effects to almost all individuals in the general population [8]. According to the NNR, the upper tolerable level for vitamin D is 100 μg/d for adults and adolescents 11-17 years of age, 50 μg/d for children under 11 years of age and 25 μg/d for infants younger than one year [9]. IOM has used slightly different values and set upper tolerable level of 100 μg/d for adults and children over 9 years old, 75 μg/d for children 4−9 years old, and 63 μg/d for children 1-3 years old, 38 μg/d for infants 6-12 months old and 25 μg/d for infants younger than 6 months [8]. In the United Kingdom (UK), the recommendation for vitamin D intake for groups other than children under 4 years old and elderly over 65 years old was not given until 2016, because cutaneous synthesis was supposed to cover the needs of other age groups [39]. The current recommendation on vitamin D intake in the UK is 10 μg/d for children over 4 years and adults throughout the year, and safe intake was set from 8.5 to 10 μg/d for infants and 10 μg/d for children from 1 to 4 years of age. Despite recommended upper tolerable levels, higher doses of vitamin D may be needed when treating vitamin D deficiency [40–42].

2.3.2 Recommendations for serum 25(OH)D concentration

Both the NNR and IOM ended up defining serum levels of 25(OH)D below 30 nmol/l as deficiency, 30 nmol/l−50 nmol/l as insufficiency, and concentrations above 50 nmol/l as a

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sufficient status in both children and adults (Table 1) [8,9]. IOM concluded that a serum level of 50 nmol/l is consistent with a dietary allowance-type reference value and appears to cover the needs of 97.5% of the population [8]. More recently, international experts ended up to the same limits for deficiency and insufficiency in their consensus statement for preventing and treating rickets in children [42]. However, different definitions for vitamin D deficiency and for recommended 25(OH)D levels have also been used. Also 50 nmol/l has been suggested as cut-off for deficiency in children and in adults [27,40,43]. Moreover, some experts have suggested that 25(OH)D levels 50−75 nmol/l will still be insufficient, and over 75 nmol/l may be an optimal level [40,43,44].

The differences in definitions for deficiency and sufficiency seem to be related with different aspects and purposes of the recommendations. The recommendations of IOM and NNR [8,9] are targeted for the general population and are the basis for national nutrition policies. They demand very strong evidence for health benefits and are therefore based on bone outcomes, concluding that the evidence of extraskeletal benefits of vitamin D is not strong enough to inform recommendations. The recommendation for prevention and management of nutritional rickets [42] is also based on bone outcomes. The recommendations for higher cut-offs for deficiency and sufficiency, such as the those by Endocrine Society [40] and the Society of Adolescent Health and Medicine [43], are targeted not only for general population, but also for risk groups and patients. These recommendations include expert opinions and take into account also the increasing evidence on extraskeletal benefits of vitamin D. Therefore, they conclude that many of their target groups probably benefit from the higher recommended 25(OH)D levels [40,43].

A rare condition of vitamin D intoxication with hypercalcemia, hypercalciuria and hyperphosphatemia is observed at 25(OH)D levels over 375 nmol/l [27,40], but may occur also at lower concentrations [45]. Endocrine Society and international paediatric experts have considered 250 nmol/l as a safety margin [40,42]. However, IOM concluded that serum levels over 75 nmol/l may not provide additional benefit, and there may be a reason for concern at serum levels over 125 nmol/l [8].

Table 1. Recommendations for serum 25(OH)D concentration, nmol/l.

Definition

All age groups Children and adolescents IOM [8]

NNR [9] and

Endocrine society [40]

Global Consensus Recommendations on

Prevention and Management of Nutritional Rickets [42]

Society of Adolescent Health

and Medicine [43]

Deficiency < 30 < 50 < 30 < 50

Insufficiency 30-50 50-75 30-50 50-75

Sufficiency > 50 > 75 > 50 75-125

Excess > 125 > 250 > 250 > 250

Abbreviations: 25(OH)D, 25-hydroxyvitamin D; IOM, Institute of Medicine; NNR, Nordic Nutrition Recommendations

2.4 SERUM 25(OH)D CONCENTRATION AMONG CHILDREN IN FINLAND AND OTHER COUNTRIES

Low serum 25(OH)D levels have been common among children and adolescents. Over 70%

of 195 school children from Helsinki, Finland, were vitamin D insufficient with serum 25(OH)D below 50 nmol/l [46]. In a small Finnish study, 20% of the 1-year old infants had 25(OH)D levels below 50 nmol/l [47], and in a small Swedish study, 25% of the preschool- age children had serum 25(OH)D levels below 50 nmol/l in summer and 40% in the winter [48]. In a study including 199 adolescents girls from Denmark, Finland, Ireland and Poland in 2002, 37% of the girls had 25(OH)D below 25 nmol/l and 92% below 50 nmol/l in winter

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[49]. In a British study, 29% of 7560 children had 25(OH)D levels below 50 nmol/l [50].

Large National Health and Nutrition Examination Survey 2001-2004 in USA found 9% of the 1-21-year-old children and adolescents being vitamin D deficient with 25(OH)D below 37.5 nmol/l (< 15 ng/ml) and 61% were classified as vitamin D insufficient with 25(OH)D 37.5−72.5 nmol/l (15−29 ng/ml) [51]. In a recent large Canadian population-based study, 5.6% of the children 3−18 years of age were determined as vitamin D deficient, having 25(OH)D levels below 30 nmol/l, and 71% were vitamin D sufficient, having 25(OH)D levels above 50 nmol/l [52].

However, the prevalence of vitamin D deficiency depends on the cut-off value of 25(OH)D used, and there may also be differences across different analytical methods, as described in Section 2.2. A study applying Vitamin D Standardization Program protocols from several European population studies investigated the prevalence of vitamin D deficiency in Europe [11]. The prevalence of vitamin D deficiency, defined as serum 25(OH)D below 30 nmol/l using standardized values of 25(OH)D, was 4−7% in studies including children 1−14 years of age, and 12−40% in teenage populations.

2.5 DETERMINANTS OF SERUM 25(OH)D

2.5.1 Sunlight

A seasonal variation in 25(OH)D levels has been described in several studies in children and adolescents, with lower 25(OH)D levels in winter [50,53,54]. As described in section 2.1, vitamin D is synthesized in the skin in the presence of UV-radiation from the sun. Sunlight has been stated to be the main source of vitamin D on a global level, but this may not hold true at higher latitudes [55]. The solar zenith angle, which varies according to the season and latitude, strongly affects solar radiation. According to the Holick’s rule, sun exposure 25% of a minimal erythemal dose over 25% of a body is equivalent to 1000 IU, corresponding to 25 µg, of oral vitamin D3 [56]. An estimated time for achieving this dose at latitude 62.5° N (close to the latitude 62.89° N of Kuopio) with exposure of the face, arms, and hands (25% of body surface) for the sun from clear sky at noon, varies from 7 to 36 min according to the skin type in June and from 89 to 276 min in March [57]. At latitudes over 50° N, the sunlight is inadequate for vitamin D production part of the year, which is called as vitamin D winter [58]. For example, in Helsinki, at a latitude of 60° N, this period lasts from mid-October to mid-March [9]. In addition, the effect of UV-radiation can be diminished due to clouds, thickness of ozone layer, surface reflection from for example snow, and also altitude has an effect on radiation dose [58]. The time spent outdoors, clothing or sunscreen use also affect the amount of UV radiation reaching the skin [55], and the time spent outdoors and UV-protection score have been associated with 25(OH)D3

concentrations in children [50].

The skin type affects vitamin D production: dark skin produces less vitamin D than pale skin [57], and children and adolescents with dark skin or non-Caucasian race have been at risk for low 25(OH)D levels [50,51,53,54]. The capacity to produce vitamin D in the skin reduces with aging [59]. Also in children and adolescents, advancing age has been associated with lower 25(OH)D levels [51], but this may be due to factors other than cutaneous production of vitamin D. The increase in serum vitamin D³concentration has been lower after exposure to UV-B irradiation in obese than in non-obese adults [60].

However, the difference was suggested to be rather due to attenuated release of vitamin D³ from the skin into the circulation than differences in skin synthesis. Moreover, when the initial 25(OH)D level is low, the effect of vitamin D synthesis in the skin and the effect of vitamin D intake on serum 25(OH)D levels may be enhanced [55,61,62]. Even though exposure to sunlight is beneficial for vitamin D production in the skin, excess exposure to sunlight should be avoided and sun-protection strategies are recommended because exposure to UV radiation elevates the risk of developing skin cancer [63].

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2.5.2 Diet and supplements

Higher vitamin D intake has been associated with higher 25(OH)D levels in children and adolescents [46,50]. However, natural dietary sources of vitamin D are limited. Vitamin D3

can be obtained from fish, egg yolk, and offals such as liver, and to a small amount from meat [9,64]. Meat and eggs also contain 25(OH)D which is suggested to have higher biopotency[65,66]. In addition, some mushrooms and yeast contain vitamin D², which has slightly lower biopotency compared with vitamin D3 [9,65]. Fish has been the most important natural source of vitamin D [67].

To increase the intake of vitamin D at the population level, some foods such as milk, margarine, or juices are fortified with vitamin D2 or increasingly with vitamin D3

[9,64,68,69]. The fortification policies vary between countries. In Finland, most fluid milk products, spreads, and some other single food products have been fortified with vitamin D after the recommendation of the Ministry of Social Affairs and Health in 2003. A Finnish study assessing the impacts of the initiation of fortification of fluid milk products and margarines with vitamin D found a higher vitamin D intake and a higher mean serum 25(OH)D concentration after fortification in 4-year-old children [70]. In adolescent females, the effect of fortification on vitamin D status was modest [71]. The amount of fortification was regarded insufficient, and in 2010, the Finnish recommendation for vitamin D fortification was increased from 0.5 to 1 μg/100 g for fluid milk products and from 10 to 20 μg/100 g for spreads [38]. A recent study on the effect of vitamin D fortification policy on vitamin D status concluded that between 2000 and 2011, the mean increase in 25(OH)D in adults not consuming vitamin D supplements was 20 nmol/l in those consuming fluid milk products, whereas in non-consumers of fluid milk products, the increase in 25(OH)D levels was 6 nmol/l less [72]. Another study in Finnish adults observed an increase in vitamin D intake from 5 µg/d to 17 µg/d in men and from 3 µg/d to 18 µg/d in women between 2002 and 2012, the intake of milk products, fat spreads, and fish being the main sources of vitamin D [73]. Studies on the effect of the increase in fortification in children are not yet available.

Vitamin D-fortified foods have been a major source of vitamin D also in North America [69]. A higher intake of milk fortified with vitamin D has been associated with a higher serum 25(OH)D concentration [74] and with a lower risk of having vitamin D deficiency [75] in Canadian and US studies among children. In another recent large Canadian study, children who consumed vitamin D-fortified milk daily and children who had used vitamin D supplements during previous month, were less likely to be vitamin D deficient [52].

However, only 9% of the children had used vitamin D supplements. Because milk and margarine are the only mandatorily fortified foods in Canada and there is no recommendation on supplement use for children, the authors suggested that fortification of more food items, promoting the consumption of vitamin D rich products and providing recommendations on vitamin D supplementation may be beneficial in improving vitamin D status. A study from UK, where only infant formula milk and margarine are mandatorily fortified with vitamin D, also concluded that the fortification of food may be the most effective way to improve vitamin D status in children [76].

In addition to classical fortification where exogenous vitamin D is added to foodstuff, fortification strategies include biofortification. Biofortification means enrichment of a food staple with another food rich in vitamin D [69]. Biofortification can be performed for example by adding vitamin D to animal food, theoretically leading to higher vitamin D³ contents of the animal produce (such as eggs, chicken, beef, or milk), or by using UV- irradiation of mushrooms or yeast, which may increase their vitamin D2 content [68,69].

Vitamin D supplement use has been associated with higher serum 25(OH)D levels [51,77], and lower risk for vitamin D insufficiency [78] in children and adolescents.

Supplement use has also been reported to blunt seasonal variation in 25(OH)D levels [79].

The systematic literature review for NNR concluded that according to dose-response studies relating vitamin D intake (fortification and supplementation), an intake of 1-2.5

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µg/day will increase the serum concentration of 25(OH)D by 1-2 nmol/l but this is dependent on solar exposure and the basal concentration, with a greater response when the basal concentration is low [80].

During pregnancy, the mother is the only source for vitamin D for the fetus, and adequate vitamin D intake with sufficient 25(OH)D levels are important for fetal development [10,40]. The 25(OH)D levels of the new-born thus depend on the vitamin D status of the mother, and vitamin D deficiency can occur in neonates born to vitamin D deficient mothers [10,41]. In case of vitamin D sufficiency of the mother, an infant can maintain sufficient 25(OH)D levels for several weeks after birth [40]. However, breast milk includes little vitamin D, and therefore vitamin D supplementation is recommended for infants [6,8].

Insufficient vitamin D intake has been common among European children [81–84], but recommendations have been met more often in infants [85]. In Finland, the average intake of vitamin D was above the previous recommendation of 7.5 μg/d in 1-year-old children in 2003−2005 [86]. The proportion of vitamin D supplement users was 86% at the age of 1 years, 70% at the age of 2 years, 47% at the age of 3 years, 31% at the age of 4 years, and only 21% at the age of 6 years [86]. In older children and adolescents in 2007−2008, the average intake was lower than the recommended levels, and only 16% of the girls and 13%

of the boys used vitamin D supplements [87]. In another study, the intake of vitamin D was below the recommendation in 34% of the children and adolescents, but still 71% of children and adolescents had serum 25(OH)D concentrations below 50 nmol/l [46]. Noteworthy is that after conduction of these studies, the Finnish recommendation for vitamin D intake has been increased [9].

2.5.3 Physical activity and sedentary behavior

In large National Health and Nutrition Survey in US children and adolescents, those who spent > 4 hours per day watching television and videos or playing computer had increased risk for vitamin D deficiency [51]. Another study from the same survey found vigorous activity to be associated with higher 25(OH)D [54]. In Dutch study, children watching television ≥ 2h/d and children playing outside < 1h/d were at increased risk for vitamin D deficiency [88]. In large British study in children, lower amount of physical activity was a risk factor for low 25(OH)D levels, but the association was explained by other factors, which included time spent outdoors [50].

2.5.4 Body composition

Higher BMI [50,51], waist circumference, visceral fat, and body fat percentage [54] have been associated with vitamin D deficiency or insufficiency in children and adolescents, but not all studies have found an association between fat mass or lean mass and 25(OH)D [53].

Serum 25(OH)D concentration has been inversely associated with the development of adiposity in school-aged children [89]. However, a bi-directional Mendelian randomization analysis in adults concluded that higher BMI leads to lower 25(OH)D, but the effect of lower 25(OH)D to increase BMI is likely small [90]. Based on some small studies, obese children and adolescents [91] and adults [60] may require higher vitamin D doses to normalize serum 25(OH)D levels, but the evidence has been insufficient, and separate recommendations on vitamin D intake for obese individuals have not been considered necessary in IOM and NNR [8,9]. However, the Endocrine Society has suggested that obese children and adults need at least two to three times more vitamin D than recommended for the age group to satisfy their body’s vitamin D requirement [40].

The response of serum vitamin D³to UV-B irradiation has been lower in obese than in non-obese adults probably due to attenuated release of vitamin D3 from the skin into the circulation [60]. Vitamin D is also stored in the adipose tissue, even though the mechanisms of the regulation of accumulation and release of vitamin D from adipose tissue are not yet clear [8,60,92,93]. In some studies, weight loss has led to increase in 25(OH)D

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concentrations in children and adolescents [94], which may be a result from the release of 25(OH)D from fat. Another reason for the association between obesity and lower 25(OH)D has been suggested to be volumetric dilution to the higher fat mass [95]. Also differences on ability to activate vitamin D and several physiological functions in adipose tissue have been suggested [92]. It is also possible that obese children and adolescents spend less time outdoors, and the association may be related to decreased sun exposure.

2.5.5 Sociodemographic factors

An older age has been associated with lower 25(OH)D levels in children and adolescents in several [50,51,53,88], but not all [54] studies. Also a positive association between age and 25(OH)D has been found in girls [96]. In several studies among children and adolescents, girls have had lower serum 25(OH)D concentrations than boys [50,51,54]. Lower socioeconomic status has been associated with vitamin D deficiency [50,52,88]. As described in section 2.5.1, non-white ethnicity has been associated with lower 25(OH)D levels [50,51,53,54].

2.5.6 Genetic factors

Genome-wide association studies (GWAS) have identified several single nucleotide polymorphisms (SNPs) in the genes related to vitamin D pathway to be associated with 25(OH)D levels [21,22]. In a GWAS by Wang et al., variants near gene coding vitamin D binding protein DBP, also called as GC (rs2282679), near nicotinamide adenine dinucleotide synthetase-1 / 7-dehydrocolesterol reductase (NADSYN1/DHCR7, rs12785878), near CYP2R1 (rs10741657), and additionally in a pooled sample, a variant near CYP24A1 (rs6013897) were associated with 25(OH)D levels [22]. Those in the top quartile of genotype score calculated by three confirmed variants had a 2−2.5-fold odds for being vitamin D deficient, and in discovery cohorts, those identified SNPs explained 1−4% of the variation in 25(OH)D. Another GWAS of Ahn et al, also found a SNP in DBP/GC (rs2282679) to be associated with 25(OH)D, and suggestive signals for SNPs near NADSYN1/DHCR7 (rs3829251), near the region harbouring chromosome 10 open reading frame 88 area (C10orf88, rs6599638), and near CYP2R1 (rs2060793). Other SNPs except the one near C10orf88, were confirmed to be genome-wide significant in additional samples. A recent GWAS published in 2018 found two loci of genom-wide significant associations with 25(OH)D in addition to the previously known GC, NADSYN1/DHCR7 and CYPR1 [97].

The new loci were in Sec23 Homolog A, coat protein complex II component (rs8018720) and in aminohydrolase domain containing 1 (rs10745742).

In addition to the genes identified in GWASs [21,22,97], genetic association studies on candidate genes have found associations with 25(OH)D in SNPs located in genes CYP27B1, VDR, and retinoid acid x receptor alpha [98]. The association of SNPs near these genes with 25(OH)D levels is reasonable, because most of these genes code enzymes that are involved in vitamin D metabolism, as described in section 2.1.

Rare conditions causing vitamin D deficiency include heritable genetic disorders in vitamin D pathway [27,41,99]. In contrast to SNPs that cause mild differences on the function of certain genes, also more severe mutations causing total loss or reduced function of the gene in producing e.g. certain enzymes have been recognized. Mutations in CYP27B1 cause 1α-hydroxylase deficiency, leading to reduced or complete loss of activation of 25(OH)D to 1,25(OH)2D in the kidney, a condition known as vitamin D-dependent rickets type 1A or hereditary pseudo-vitamin D deficient rickets [27,41,99]. Mutations in CYP2R1 are rare, but can cause 25-hydroxylase deficiency, affecting the conversion of vitamin D to 25(OH)D in liver, a condition called vitamin D-dependent rickets type 1B [41,99]. Mutations in VDR cause partial or total resistance to the action of 1,25(OH)2D where 1,25(OH)2D are increased due to this resistance, a condition known as vitamin D resistant rickets or vitamin D-dependent rickets type 2A [27,41,99]. Also vitamin D dependent rickets type 2B or type 3 due to defects in the gene coding heterogeneous nuclear ribonucleoprotein C has been

Serum 25-hydroxyvitamin D : determinants and associations with plasma lipids and bone mineral density in children

uef.fi

Dissertations in Health Sciences

SONJA SOININEN

SERUM 25-HYDROXYVITAMIN D: DETERMINANTS AND ASSOCIATIONS WITH PLASMA LIPIDS AND BONE MINERAL DENSITY IN CHILDREN

SONJA SOININEN

Serum 25-Hydroxyvitamin D:

Determinants and Associations with Plasma Lipids and Bone Mineral Density in

Children

Serum 25-Hydroxyvitamin D:

Determinants and Associations with Plasma Lipids and Bone Mineral Density in

Children

Acknowledgements

List of the original publications

Contents

Abbreviations

1 Introduction

2 Review of the Literature