KASHIN-BECK DISEASE: EVALUATION OF MINERAL INTAKE IN YOUNG TIBETAN CHILDREN FROM ENDEMIC AREAS GEORGES LOGNAY, FRANÇOISE MATHIEU MICHAËL DERMIENCE ANNÉE ACADÉMIQUE

I. Introduction 1. Objectives 2. Tibet Autonomous Region, P.R. China 3. Kashin-Beck disease 4. Trace elements and their roles in relation to bone metabolism

II. Material and methods 1. Choice of families and sampling 2. Nutritional survey 3. Mineral elements analyses 4. Validation of methods 5. Evaluation of daily intakes

III. Results and discussion 1. Choice of families and sampling 2. Nutritional survey 3. Analyses of foods mineral contents 4. Evaluation of daily intakes IV. Conclusion and perspectives

I. Introduction

 Main objective:  to measure the mineral content of most consumed Tibetan foods  Secondary objectives:  to compare the measured food contents with food reference tables  to develop a food sample procedure matching field conditions  to establish and validate an analytical method of measurement  to estimate mineral daily intakes by the means of a nutritional program: Kidmenu®

 Located in the South-Western border area of Popular Republic of China  About 1,228,400 km², and over 2.62 millions peoples  Physionomy is diversified (mountains, deserts, grasslands and forests)  Many rivers have their sources in Tibetan Plateau (Yangtze, Yellow River, Brahmaputra, Ganges, etc)  Farming and animal husbandry are the major agricultural activities but productivity is very low

 According to Haubruge and al.(2000), Malaisse and al.(2008) populations share out 4 macro- ecosytems: ◦ The urban zone ◦ The suburban zone which is mechanized and has communication media ◦ The agricultural zone with subsistence farming ◦ The pastoral zone over 4500 m with nomads and yaks The Kashin-Beck disease is only encountered in the agricultural group. Diet is a major difference between this group and the others.

 Kashin-Beck disease is an endemic and chronic osteochondropathy  This disease is characterized by short stature and skeletal deformities especially in long bones and joints  Symptoms appear during childhood (3-5 years) and become progressively worse  Clinical and radiological diagnosis exist, distinguishing several stages

 Although many studies have already been conducted and many others are still underway, its ethiology remains unknown  A multifactorial hypothesis has been proposed:  selenium deficiency  high concentration of organic matters in drinking water  mycotoxin in cereals

 Calcium  Phosphorus  Magnesium  Iron  Zinc  Copper  Manganese  Selenium  Nickel  Molybdenum  Arsenic  Strontium  Fluorine  Cadmium

Daily reference intakes (DRIs), West Suitor et al., 2006 : Life Stage Group P (mg/d)Ca (mg/d)Mg (mg/d)Fe (mg/d) RDA/AI*ULRDA/AI*ULRDA/AI*ULRDA/AI*UL 1-3 years * years * Zn (mg/d)Cu (µg/d)Mn (mg/d)Se (µg/d) RDA/AI*ULRDA/AI*ULRDA/AI*ULRDA/AI*UL 1-3 years * years * Ni (mg/d)As (µg/d)Mo (µg/d) RDA/AI*ULRDA/AI*ULRDA/AI*UL 1-3 yearsND0.2ND yearsND0.3ND 22600

II. Material and methods

10 families from two distinct areas:  Living in endemic areas  Including a 3 to 5 year-old child  This child having a KBD brother or sister

 A sampling campaign split up into two periods  Most eaten foods collected in each family:  Barley flour  Wheat flour  Rice  Potato  Black tea  Yak butter  Two foods collected in Lhasa:  Chinese cabbage  Instant noodles

24-Hour food recall of the 3-5 years old child

 Mineralization: microwave-assisted wet process  Mineral measurements:  Flame Atomic Absorption Spectrometry (FAAS)  Electrothermal Atomic Absorption Spectrometry (ETAAS)  Hydride generation  Cold vapour generation  Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES)  Molecular Absorption (UV-Vis)

 Certified reference materials (CRM): ◦ Trueness ◦ Fidelity  Repeatability study: ◦ CRM ◦ Sample

Nutritional surveyFood contentsP. De Voghel’study, 2008 Kidmenu® Daily intakes: Ca, P, Mg, Fe, Zn, Cu & Mn Comparison with DRIs table

III. Results and discussion

 Food sampling  Dry matters and comparison with reference tables (USDA, Souci):  ± equals to references, slightly different for barley flour

 Extremely monotonous cereal-based diet Poor diversity of dishes  Most common beverages:  Butter tea  Chang  Consumption of :  Meat is low  Fruits and dairy products is rare

 Limit of quantification:  Outliers set aside via the Grubb’s test  All determination coefficient (R²) of calibration curves > Method successfully validated (except for Se, Sr and As) Repeatability: RSD < 10%

 Food mineral contents expressed in mg/100g FM  Compared to reference tables (USDA, Souci):  Iron content is markedly high  Selenium is clearly deficient  Black tea is rich in elements Brewed black tea is drunk so much diluted that mineral contents is finally really low

 Creation of anamnesis, edition of children’s menus and coding of food contents in Kidmenu®  Some restrictions: ◦ Omission of some foods (milk, yak meat, chang, fruits, etc.) ◦ Standardization of dish (cooking pot) ◦ Relative density approximation

Mineral intakes compared to daily reference intakes:  We confirm a marked deficiency in Ca

Mineral intakes compared to daily reference intakes:  We confirm a marked deficiency in Ca  Ca/P ratio rarely exceeds 0.1

Mineral intakes compared to daily reference intakes:  We confirm a marked deficiency in Ca  Ca/P ratio rarely exceeds 0.1  Fe and Cu are in excess and sometimes reach upper limits

Mineral intakes compared to daily reference intakes:  We confirm a marked deficiency in Ca  Ca/P ratio rarely exceeds 0.1  Fe and Cu are in excess and sometimes reach upper limits  Zn is around 100 % of RDA but absorbability is probably weak

Mineral intakes compared to daily reference intakes:  We confirm a marked deficiency in Ca  Ca/P ratio rarely exceeds 0.1  Fe and Cu are in excess and sometimes reach upper limits  Zn is around 100 % of RDA but absorbability is probably weak  Selenium approximation suggests a deficiency

Mineral intakes compared to daily reference intakes:  We confirm a marked deficiency in Ca  Ca/P ratio rarely exceeds 0.1  Fe and Cu are in excess and sometimes reach upper limits  Zn is around 100 % of RDA but absorbability is probably weak  Selenium approximation suggests a deficiency  Manganese often exceeds till two or three times upper limits!

IV. Conclusion and perspectives

 Original nutritional study in the Kashin- Beck ethiology context  Main objective and secondary objectives were reached

 This work presents limits: Omission of some foods Representativeness ? Extrapolation to every endemic areas ? Bioavailability of minerals is a critical point Bioavailability largely influences DRIs DRIs are established for a standard population with a quite balanced diet  Tibetans may not be compared to a standard population and their diet is not balanced at all

Antinutritional factors could be measured (phytate,...) Bioavailability could be evaluated by experimentations (e.g. rats fed with a Tibetan diet) A larger study over a longer term covering both endemic and non-endemic regions is required for definite conclusions to be reached

Acknowledgements Pr. Georges Lognay Ph.D. Françoise Mathieu William Claus and the KBD fund asbl-vzw Pr. Jean-Paul Barthélémy Ir. Philippe Maesen Pr. Philippe Goyens Ms. Martine Robert and M.D. Shancy Rooze The members of the BEAGx and especially Laurent Lessire The Tibet Center for Disease Control and Prevention The KBD fund team in T.A.R. The members of the analytical chemistry unit Ph.D. Jean-Michel Romnee The Tibetan families My family and my friends Thanks for your attention