Iron and Zinc Bioavailability From Biofortified Potatoes
Condition: Biofortification; Potato
Study type: Interventional

Summary: 
      Two of the most common nutritional deficiencies are anemia and zinc deficiency. One strategy
      for combating nutritional deficiencies is biofortification. The primary objective of the
      proposed research is to determine in humans the bioavailability of iron and zinc from
      biofortified potatoes as compared to a non-fortified local variety. The secondary objective
      is to use the data obtained from the human studies to model the potential impact of the
      introduction of biofortified potatoes to the alleviation of iron and zinc deficiencies in the
      Andean Highlands and low and middle-income countries elsewhere.

      Iron bioavailability studies: The investigators will compare bioavailability of iron from a
      non-fortified potato variety with a biofortified potato that has significantly higher iron
      content though the use of a randomized cross-over intervention study. Female volunteers will
      consume both biofortified potato extrinsically labelled with 58FeSO4 and a nonfortified
      potato labelled with 57FeSO4. Thirty women with marginal iron status (plasma ferritin < 25
      ng/ml) will be selected from an initial screening of 180 women. Every woman will receive 2
      different types of test meals in a series of 20 servings for 10 days each. Blood samples will
      be collected during screening and on days 1, 15, 26 and 40 and the amount of 58Fe and 57Fe
      incorporated into hemoglobin and serum ferritin quantified.

      Zinc bioavailability study: The investigators will compare bioavailability of zinc from a
      non-fortified potato variety with that of a biofortified potato that has significantly higher
      zinc content (p<0.001) with a crossover study. Forty volunteers will be randomly assigned to
      receive first either the biofortified or the non-fortified potato and receive the second meal
      30 days later. Zinc absorption from two meals will be measured using the double stable
      isotope technique. Every volunteer will be given an i.v. infusion of 70zinc and two test
      meals of 250 g cooked potato labelled extrinsically with 67zinc separated by 3-4 hr. A spot
      urine sample will be collected 96 hours after each set of test meal and the ratio of the two
      isotopes measured to calculate absorption of zinc from the test meal.

      Modelling and Impact assessment: Data on bioavailability will be used in a Disability
      Adjusted Life Year model to assess the potential impact of biofortified potatoes to alleviate
      iron and zinc deficiencies in the Andean Highlands and low and middle-income countries
      elsewhere.
    

Detailed description: 
      Type of Study and population Double blind randomized trial of naturally biofortified potatoes
      to be carried out in Huancavelica, Perú. It will be recruited about 100 women and 80 men aged
      18-45 years living in the city of Huancavelica.

      ETHICS Applications was submitted and approved by Research Ethics Committee (REC) of the
      University of East Anglia Faculty of Medicine and Health (FMH) in Norwich (UK) and a REC of
      the Instituto de Investigacion Nutricional (IIN) in Peru.

      Every participant will be fully informed of all the procedures and will be given the
      opportunity to ask questions and receive any further details participants require.

      The procedures (blood samples, urine samples, anthropometry, dietary recall) will be
      explained to the individuals who agree to take part in the initial screening, and
      participants will be invited to sign the first consent form.

      SPECIFIC METHODS FOR IRON STUDY (Phase 1) Sample size The study requires an enrollment of a
      total of 30 women to allow for 33% who do not adhere to the protocol, to reach a final sample
      size of 20 women. The investigators estimate that the investigators may need to screen about
      100 women.

      Nutritional Status As part of the screening all the potential candidates will be weighed and
      measured using standardized equipment.

      Measurement of physical activity All the participants will be asked to use an actigraph (a
      tri-axial accelerometer) which measures physical activity (movement) in three dimensions.

      Blood sample An appropriately trained member of the study team will collect a venous blood
      sample (approximately 6ml in a test tube with heparin) to measure ferritin, hemoglobin, and
      CRP (screening and 3 more times: baseline, 16 and 40 days after first day of potato intake).

      Urine sample Participants will be asked to provide a urine sample (30ml) to carry out a
      pregnancy test using a test strip.

      Sociodemographic and Food security survey The investigators will include a socioeconomic
      survey and the ELCSA food security questionnaire both will be applied to all enrolled
      participants.

      Study Procedures According to the results of the screening and revision of the criteria for
      inclusion and exclusion and following information and verification that participants
      understand the iron study consent form, subjects will be invited to participate. Consented
      participants will be randomly assigned to consume one or other of the two types of potato
      (biofortified-A or not-B). The first potato variety will be given during the first 2 weeks
      (day 1-12) and the second sequence between days 15-26, (in both cases excluding weekend days
      Saturday and Sunday). The potatoes will be cooked in water and offered mashed.

      Blood samples will be taken on 5 occasions, 1) at screening to verify compliance with the
      exclusion and inclusion criteria; then on day 1 before participants eat the first type of
      potato, day 15 before eating the second type of potato, day 26 after eating all both potato
      types and on day 40 two weeks after eating all the potato.

      Consumption of the potato Each participant will be served 500 g of potato divided into two
      portions of 250 g each. each participant will take, depending on their allocation, 0.3 mg of
      57FeSO4 (Peruanita potato) or 0.3 mg of 58FeSO4 (biofortified potato), diluted in 50 ml of
      distilled water, which should be drunk while participants are eating the potato - halfway
      through the meal. In addition, each participant will receive 200 ml of distilled water to
      drink. The isotopes will be provided by ETH (Swiss Federal Institute of Technology in
      Zurich). In addition, the isotopes that the investigators will use are stable isotopes, that
      is participants exist naturally in the environment and as such do not pose any risk.

      Collection of biological samples for the measurement of iron using stable isotopes Blood
      samples will be collected throughout the intervention period as previously described and will
      be sent to ETH, Zurich, following standard operating procedures. Participants will be
      informed and will provide consent for the transfer and use of their samples outside Peru' for
      the purpose of this research study. Samples will be anonymized and will not be transported
      with personal identifiable data.

      In terms of processing, samples will be mixed with NO3 and H2O2 and digested in a microwave,
      followed by separation of the iron from the blood matrix by way of exchange-anion
      chromatography and a solvent (diluent) or extraction from the solvent using distilled.

      The isotopes will be analyzed using a plasma coupled inductive multicollector mass
      spectrometer (Neptune; Thermo Finnigan). The amount of the labelled iron isotopes 57Fe and
      58Fe will be measured 14 days after consumption of both types of potato has been completed.
      The calculation (of absorption) will be done considering the subjects estimated blood volume
      based on weight, height, and the subject's measured hemoglobin concentration (considering the
      average of the initial and final blood sample). The calculation is based on the principle of
      isotopic dilution considering as the factor for the iron isotope marker rather than the
      monoisotopic marker8. For the calculation of the fraction absorbed the investigators will
      assume 80% incorporation of the absorbed iron.

      SPECIFIC METHODS FOR ZINC STUDY (Phase 2) Sample size The study requires a samples size of 40
      women. This allows for a 40% loss to reach a final total of 24 women.

      Procedures According to the results of screening, and after checking that participants comply
      with all the inclusion criteria and none of the exclusion criteria participants will be
      invited to participate in the zinc part of the study. For this participant will be given the
      second consent form to read and those who accept and sign the form will be invited to
      participate in the study.

      The people who accept will be randomly allocated to eat one or other of the types of potato
      (biofortified-A or no-B). All the participants will be given both types of potato but in
      different order, in this way each participant will be their own control. The first variety of
      potato will be given on day 1, after acceptance of participating in the study, and after
      accepting and signing the consent form for this part: (consumption protocol and second
      variety will be eaten 29 days after (the order of potato varieties will be randomly
      assigned).

      The investigators will take blood samples on three occasions: enrolment and days 1 and 29
      (the days when participants eat the potato) after fasting for 8 hours (food) and 6 hours
      (liquids).

      Study Measurements Anthropometry As part of the screening all the potential candidates will
      be weighed and measured using standardized equipment. According to their weight and BMI the
      investigators will decide whether participants are eligible.

      Consented participants will be asked to provide a venous blood sample (approximately 10ml in
      a test tube with heparin). Plasma and packed red cells will be obtained from the blood
      samples and stored at <18oC.

      Urine sample Consented participants will provide a urine sample (30ml) as part of the
      screening phase to carry out a pregnancy test. On days 1, 3, 4, 5, 6, 7, 29, 32, 33, 34, 35
      and 36 the investigators will collect urine samples to analyze zinc content. On each day the
      investigators need 100ml of urine, collected in the morning (between 7-12 o'clock in the
      morning).

      Consumption of the potato The subjects will be given a total of 1000 g of potato in two 500 g
      portions. The first portion will be given while participants are fasted, (at least 8 hours
      for food and 6 hours for drink) and in two separate servings (each one 250 g). When
      participants have eaten the first 250 g serving, the subject will receive 0.25 mg of 67Zn
      diluted in 50 ml of bottled water and then participants will receive the second serving of
      potato. Two hours later, participants will receive an intravenous infusion of 70Zn (200 ug)
      diluted in 50 ml of bottled water. After a further 2 hours participants will receive the
      second two servings of potato, and, as before, after the first serving participants will
      receive 0.25 mg of 67Zn diluted in 50 ml of bottled water. During this period and for 3 hours
      after the second portion of potato the subject will not consume any food or liquid, except
      for water which participants will be provided with. Each subject will receive 200 ml of water
      with each meal and 200 ml of water between meals. Currently, using isotopes is the best
      method for measuring the absorption of zinc. In addition, the isotopes that will be used are
      stable isotopes, that is participants exist naturally in the environment and pose no risk. No
      food or drink can be consumed for at least 3 hours after eating the final portion of potato.

      Blood samples for measurement of zinc The investigators will take a total of three blood
      samples in total. Absorption using stable isotopes (Day 29) The blood samples that have been
      collected will be sent to ETH, Zurich. ETH will undertake the analysis of CRP for Zinc. The
      CRP will be measured using an ELISA kit. The plasma zinc concentration will be measured by
      induction plasma coupled mass spectrometry.

      Urine samples for measurement of zinc using stable isotopes Samples of urine will be
      concentrated using freeze drying and then mineralized by digestion in a microwave oven
      (MLS-Turbowave; MLS GmbH) using a mixture of HNO3 and zinc isolated from the master matrix by
      ion exchange chromatography. The investigators will use ultrapure acids for the preparation
      of the samples.

      Determination of zinc absorption Blood and zinc isotopic composition will be analyzed in
      duplicate at the ETH Human Nutrition Laboratory. Urine samples will be mineralized by
      microwave digestion (MLS-ETHOS plus; MLS GmbH) using a mixture of HNO3 and H2O2 and zinc
      isolated from the sample matrix by ion-exchange chromatography (Wegmuller et al 2014). All
      acids used for the preparation of samples will be ultrapure. 70Zn:66Zn and 67Zn:66Zn isotopic
      ratios will be measured to determine 70Zn and 67Zn enrichment in the urine samples by using a
      high-resolution double-focusing magnetic sector field multi-collector inductively coupled
      plasma mass spectrometer (Finnigan Neptune; Thermo Electron).12 Fractional absorption of the
      67Zn dose from the test meals will be calculated by using the oral to i.v. tracers ratio
      method applied to spot urine samples, according to principles described by Friel et al.

      7-day food record Using the method of registering all foods eaten over a 7-day period the
      investigators can obtain information on all foods eaten in a week (including Saturday and
      Sunday). To achieve this, the investigators will ask the subjects to make a record of all
      foods consumed, details of food preparation, and all drinks (including water) that
      participants have eaten or drunk. Participants should note the meal at which the food and
      drink were consumed, and the time taken to eat it (breakfast, lunch, supper and between meals
      (snacks) and the amount eaten at each time of the day. To do this the investigators will give
      the subjects a tablet in which to register the consumption/meals and as well take a photo of
      all the food eaten.

      METHODS SPECIFIC FOR IMPACT ASSESSMENT The data on dietary intake, serum ferritin and iron
      bioavailability will be used for two modelling approaches. Firstly, the investigators will
      apply the Disability Adjusted Life Year (DALY) method, which is an appropriate method to
      estimate nutritional and health impacts of biofortified crops. This will build upon models
      that have been developed jointly by CIP and IIN that has made assumptions concerning iron and
      zinc bioavailability from potatoes. Secondly, the investigators will use data on habitual
      iron intake, serum ferritin concentrations and calculated physiological requirements for iron
      to adapt a model estimate dietary iron bioavailability in Europe, the Andean highland region
      and, more generally, to low- and middle-income countries.
    

Intervention name(s): Iron biofortified potato; Zinc biofortified potato
Intervention type(s): Other; Other
Intervention description(s): A non-blinded randomized cross-over design with multiple meal feedings will be used to quantify iron bioavailability from iron biofortified and non-biofortified potatoes. Women volunteers will consume biofortified ('A') and a local non-fortified Peruanita potato variety ('B') in a randomized order (either A-B or B-A). The potatoes have significantly different (p<0.001) content of iron or zinc, but no significant differences in vitamin C or phenolics (p>0.05). Based on this study design, every woman is her own control.; A crossover study will be used to compare zinc bioavailability of biofortified and non-fortified potatoes. Nutritional pre-screening is not required as there are no major systemic influences on zinc absorption.Volunteers will be randomly assigned to consume first either the biofortified or the non-biofortified potato, and consume the second potato-type 30 days later. Zinc absorption from two meals of biofortified or non-fortified potato will be measured using the double stable isotope technique.
Allocation: Randomized
Intervention model: Crossover Assignment
Intervention model description: Double blind randomized trial of naturally biofortified with iron or zinc potatoes to be carried out in Huancavelica.
Masking: None (Open Label)
Primary purpose: Prevention
Keywords: Biofortification; Zinc; Iron; Potato