Concentraciones Objetivo para Porciones comestibles de Cultivos Biofortificables Una aproximación metodológica para fines de nutrición publica Erick Boy CIAT, Cali, Colombia (Febrero 25, 2013) Provitamin A (strongest story) Sweetpotato, efficacy has been demonstrated, even effectiveness (Low-Mozambique; REU-Uganda have evidence on serum retinol) Orange maize, a complex efficacy trial is about to begin in Zambia with JHU as the lead institution Yellow cassava, an efficacy is about to begin in Kenya with Wageningen as the lead institution (say also something on plans for Nigeria) Evidence on bioavailability in maize and cassava.   Iron (shaping up to be a strong story relatively soon) Efficacy has been demonstrated in beans in Mexico, an upcoming bean trial in Rwanda is planned. Something about the preliminary results from bioavailability and efficacy of pearl millet in India, timetable for having final results I wouldn’t go into all the bioavailability studies that ETH has done, but have a summary slide ready in case there is a question about them – the bottom line is that measuring bioavailability in test meals does not take account of possible adaptation to high levels of phytates over time; efficacy trials do take account of such adaptation. Even though iron in rice is no longer a target, as a proof of concept, you might have a slide on the nun efficacy trial in the Philippines. Zinc (not yet much evidence) Start with bioavailability of the wheat – old Hambidge study Not sure what evidence is now available from pearl millet, but could present something on this Talk about plans for rice and wheat efficacy trials; talk about new methodologies being tested for new markers of improved zinc status Similar to beans, I would have a summary slide on the rice bioavailability study (IR68-144) ready in case there are questions; I would characterize this as testing bioavailability percentages from a “fast-track” variety developed/discovered in the 1990s and used also in the nun feeding trial to test iron; the study is useful in that it demonstrates the importance of meeting zinc targets holding phytate levels constant;  there is no analogous presumption (as with iron) that there will be adaptation to high levels of phytate over time in an efficacy trial. After this, you could present a couple of slides on the breeding targets, how they are calculated – where we are in terms of thinking that levels can be adjusted and vis-à-vis the breeding achievements that Wolf has projected – first, second, and third waves: Provitamin A (again the strongest story) Targets can probably be lowered; in any event, third wave releases will meet original targets for cassava and maize in high-yielding backgrounds Iron (also a  strong story) Targets should remain where they are; third wave releases will meet these targets for beans and pearl millet in high-yielding backgrounds Zinc (again our weakest story) Targets will likely need to be increased by 60% due to new evidence/thinking in the zinc nutrition community Third wave releases will  hit xx% of new target levels; we still have to confirm that phytate levels are held constant in new varieties with high zinc levels; nevertheless, target levels assume that 40% of EAR will be achieved -- which is an arbitrary assumption; for example, in third wave varieties we are adding +4 mg (??) of zinc per day to women’s diets in Bangladesh, and +2.0 mg (??) of zinc per day to preschooler diets in Bangladesh – which increases current zinc intakes by an estimated 50% (??); the zinc experts will be consulted if they think such levels are useful, that is, can these absolute increases be expected to be significant for improving public health? You might end with a slide about plans in general for measuring effectiveness during Phase 3. Again, I would concentrate on the story and conclusions and not so much methodology; you can answer questions that come up about methodology.

Guía Marco de Referencia Principio y Métodos Ejemplos Conclusiones Reflexiones

Objetivo estimado para la concentración del nutriente Marco conceptual para el Impacto de la biofortificación como intervención nutricional Objetivo estimado para la concentración del nutriente Desarrollo Retención Desafíos: Variación de perdidas de MNs Consumo de Alimentos Desafios: disponibilidad de datos representativos Biodisponibilidad baja absorción de Hierro; Cuantificación de efectos de inhibidores y promotores de la dieta completa; Efecto de enfermedades Evaluación Eficacia Identificación de biomarcadores sensitivos del estado nutricional de los MNs Efectividad Infección; Permanencia de rasgos nutricionales y agronómicos; demanda sostenida; Sistemas efectivos/eficientes de distribución de semilla; Aceptación x consumidores Too much detail on boxes—impossible to read. Nutritionists should know definitions..better that slides show challenges. Hotz & McClafferty, 2007, FNB, 28 (2), S271-79

HarvestPlus: Fases de Investigación en Nutrición Consumo del Alimento Objetivo Inicial Mínimo Desarrollo Retención del nutriente Biodisponibilidad Evaluación Eficacia (ECC) Efectividad De ensayo De programa FNB 2007;28 (2), S271-79

Principio Las poblaciones objetivo tienen ingestas insuficientes de vitaminas y minerales esenciales que determinan la deficiencia nutricional y sus efectos (contexto) El aporte adicional de nutrientes a la dieta tradicional disminuye el déficit y mejora el estado nutricional de forma medible (limites de biomarcadores actuales)

Aporte nutricional (% del requerimiento estimado promedio, REP) Nutriente % mínimo SUPUESTO del requerimiento diario con impacto biológico (REP) Mujer NE NL Niños (4-6 años) Vitamina A (μg RAE) 50% (500 μg RAE/d) 50% (275 μg RAE/d) Zinc (mg) 40% ( 1.86 mg/d)* 40% ( 0.83 mg/d)* Hierro (mg) 30% (1.46 mg/d) 30% (0.50 mg/d)

Método Definido el % del requerimiento que producirá un efecto biológico medible (50/40/30) Supuestos (o evidencia) sobre: Ingesta promedio del alimento Pérdidas de nutrientes: del campo a la mesa Almacenamiento Procesamiento Preparación en el punto de consumo Biodisponibilidad (absorción y utilización)

¿Cómo se calcula el objetivo? Consumo (g/dia) % retenido después de almacenamiento y preparación % absorbido OBJETIVO % Requerimiento Promedio Estimado

Ejemplo para Fe en frijol para mujeres Consumo 200 g/d % retenido = 90-98% 5% absorbido 44 μg/g 0.30 x 1460 mcg/d

50% Pérdidas por procesamiento y almacenamiento Nutriente en alimento Crudo Absorción 8% 50% 4%

Original Beans and Pearl Millet Iron Breeding Targets for Non Pregnant Non lactating women Nutrient FOOD Consumed (g/day) X "+Nutrient Concentration (mcg/g)" Nutrient Retained (%) Absorbed proportion (%) = Nutrient absorbed (μg/day)   EAR NP NL Woman (mcg) Iron Beans 200 x 44.0 x  90  x 5.0  = 396 1460 Iron Pearl Millet 300 30.0 406 Iron absorbed % EAR 27% Beans   Iron Required 28% Pearl Millet

Objetivos Iniciales para VA, Fe, Zn: Mujer no embarazada / no en lactancia Nutrient FOOD Consumed (g/day) X "+Nutrient Concentration (mcg/g)" Nutrient Retained (%) Absorbed proportion (%) = Nutrient absorbed (μg/day)   EAR NP NL Woman (mcg) Vit. A maize 400 15.0 50 8.0  = 240 500 Iron Beans 200 44.0 90 5.0 396 1460 Zinc Rice 25.0 720 1860 Nutrient absorbed % EAR 48 VA  Nutrient Required 27 Fe 39 Zn

Proof of Concept Framework Nutrient retained harvest (storage, processing, cooking) Initial Nutrient Concentration (raw food) Amount of Food Consumed % nutrient absorbed  utilized (bioavailable) Contribution to nutritional sufficiency

Summary of crops intake – HarvestPlus Background Surveys (2008-2012) Grupo Etario Arroz Trigo Mijo Perla Frijol Maíz Yuca (2 aldeas) (1 distrito) Gujarat Maharashtra Rwanda (2 provincias) Zambia (2 distritos) Nigeria (Estado de Akwa Ibom) Mujeres 19 – 50 años 422 ± 135 208 ± 98 302 ± 135 244 ± 104 123 ± 112 287 ± 112 940 ± 777 Niños 0.5 – 2 años 41 ± 33 24 ± 33 86 ± 84 42 ± 43 47 ± 56 84 ± 67 167± 236 3 – 5 años 146 ± 78 72 ± 57 141± 113 72 ± 63 65 ± 63 172 ± 82 348± 317

Retinol Equivalency of provitamin A rich foods: human studies In our study population, the vitamin A equivalence of the b-carotene and other provitamin A carotenoids in the maize was 6.2 to 1 (by weight) and 2.8 to 1 (by weight) in cassava. The average bioefficacy of the provitamin A carotenoids was therefore 15.0% and 35%, respectively The b-carotene in b-carotene–biofortified maize and cassva is more bioavailable than the b-carotene in conventional plant sources (~12:1). 12

Nutrition Research to Date (January 2013) Dietary intake & nutritional status Nutrient Retention Absorption/ Bioavailability Efficacy Effective-ness OFSP  MAIZE 2  2015 CASSAVA BEANS 3 2013? PEARL MILLET RICE 2013-14 2016 WHEAT 2012-13 (2) Complete:  Ongoing: 

YUCA

YUCA - Nigeria retention in 2012 HH practices gari production (60%)* Maize consumption & VA status Survey 2011 Akwa Ibom: Women: 840g/dy Children: 165 - 358g/day; VAD of Moderate PH significance, 14% Retention, 2009-10 70% after boiling 15-30% in gari retention in 2012 HH practices gari production (60%)* Bioavailability: 2009 & 2011 2.8:1 (range: 1.15-6.6) 4.3:1 & 4.7:1 (w/oil); (range: 1.9 – 7.7) Efficacy 2012-2013: Kenya (WAU) 2013-14 Nigeria 2nd wave releases *Freshly made gari (losses during storage of up to 4 months being studied in Nigeria)

Yuca – Nigeria (gari 1 mes Akwa Ibom rural) Δ Nutriente retenido: Gari fresco: 40% 1 mes almacen: 50%  20% Δ Concentration Inicial (crudo): 15 μg/g Consumo promedio diario: 800 g/day % nutriente biodisponible  17% (6:1) Contribucion al Requerimiento Promedio Diario (Mujer NE NL: 81%

Yuca – Nigeria, Akwa Ibom (Vit. A de gari 1 mes - niños 4-6 años) Δ Nutriente retenido: Gari fresco: 40% 1 mes almacen: 50%  20% Δ Concentration Inicial (crudo): 15 μg/g Consumo promedio diario: 350 g/day % nutriente biodisponible  17% (6:1) Contribucion al Requerimiento Promedio Diario (3-5 años): 97%

Yuca – Nigeria, (Vit. A de gari 1 mes para niños 1-2 años- Akwa Ibom) Δ Nutriente retenido: Gari fresco: 40% 1 mes almacen: 50%  20% Δ Concentration Inicial (crudo): 15 μg/g Consumo promedio diario: 150 g/day % nutriente biodisponible  17% (6:1) Contribucion al Requerimiento Promedio Diario (1-2 años): 54%

MineralEs: HIERRO Y ZINC

Original Beans and Pearl Millet Iron Breeding Targets for Non Pregnant Non lactating women Nutrient FOOD Consumed (g/day) X "+Nutrient Concentration (mcg/g)" Nutrient Retained (%) Absorbed proportion (%) = Nutrient absorbed (μg/day)   EAR NP NL Woman (mcg) Iron Beans 200 x 44.0 x  90  x 5.0  = 396 1460 Iron Pearl Millet 300 30.0 406 Iron absorbed % EAR 27% Beans   Iron Required 28% Pearl Millet

Updated Fe Breeding Targets for Beans & Pearl Millet Non Pregnant Non lactating women Nutrient FOOD Consumed (g/day) X "+Nutrient Concentration (mcg/g)" Nutrient Retained (%) Absorbed proportion (%) = Nutrient absorbed (μg/day)   EAR NP NL Woman (mcg) Iron Beans 150 x 44.0 x  95  x 5.0  = 313 1460 Iron Pearl Millet 167 30.0 7.0 333 Iron absorbed % EAR 21% Beans Rwanda   Iron Required 23% Pearl Millet   Maharashtra

Minerales Arroz con hierro marginalmente eficaz(Haas J et al, AJCN 2005). Frijol con hierro: Eficacia demostrada en México (sTfRs) Ruanda: múltiples estudios de absorción (3.5-9%) y un 2o estudio de eficacia activo en Ruanda c/indicadores de efectos funcionales). Mijo Perla: Fe > biodisponible (7%) que lo previsto (mujeres y niños); Estudio de eficacia concluido en India, 2012. 65% reducción en probabilidad de def. De hierro en escolares.

Zinc Requirements & Breeding Target Int. J. Vitam. Nutr. Res. 81 (1) c 2011

Absorbed Zinc Requirements: IZiNCG and IOM comparison showing effect of adjustment to original IZiNCG estimates

Conclusión: Requerimiento para mujeres debe incrementarse de 1.86 a ~2.5-3.0 mg/día “The estimates of zinc physiological requirements by International Zinc Nutrition Consultative Group (IZiNCG) in 2004 were conspicuously low in comparison with those estimated by the Institute of Medicine (IOM) in 2001.” Physiological requirements for zinc. Hambidge M et. al. Int. J. Vitam. Nutr. Res. 2011, 81(1):72–78 Abstract : The estimates of zinc physiological requirements by the International Zinc Nutrition Consultative Group (IZiNCG) in 2004 were conspicuously low in comparison with those estimated by the Institute of Medicine (IOM) in 2001. The objective of this review is to explore the reasons for this gap and to refl ect on lessons learned. All estimates of inevitable losses of endogenous zinc, especially intestinal excretion of endogenous zinc, were reviewed. An error in zinc menstrual losses, as well as a minor error in the linear regression of endogenous fecal zinc (EFZ) vs. total daily zinc absorption (TAZ) by IOM, were corrected. The review revealed an error by IZiNCG in selecting two data points for the linear regression of EFZ on TAZ. A second major reason for the “gap” is attributable to weighting of the data in the regression analysis by number of subjects per study by IZiNCG. Adjusting for these factors, together with use of the same reference data for body weights, resulted in satisfactory agreement between the two estimates of physiological requirements. The lessons to be learned from this review are discussed together with suggestions for future action by IOM as well as a constructive role for IZiNCG. Implicacion para fito-mejoramiento: objetivo en trigo para contribuir 40% REP para mujeres debe incrementarse de +8 a ~ +13 ppm (en trigo entero/harina integral)

Investigación y Objetivos Iniciales Provitamina A: objetivos exceden las metas nutricionales en algunos contextos donde la ingesta es muy alta; la 2a (yuca) y 3a olas (maíz) de variedades liberadas cumplirán con el objetivo inicial en un fondo de alta productividad. Hierro: objetivos sin cambio; en el frijol es necesario disminuir el contenido de fitato. Zinc: objetivos deben incrementar debido a ajuste en requerimiento fisiológico (Consulta con Expertos, Julio 24,2012,WDC).

CONCLUSIONES Los objetivos para los cultivos deben apuntar al máximo posible sin menoscabar rasgos agronómicos (rendimiento). Las metas nutricionales están condicionadas por cada contexto y su estimación requiere datos locales (grupo poblacional, prácticas culinarias y alimentarias, estado de salud, etc.)

GRACIAS OBRIGADO Thank you

Dosis y Efecto (Zinc) X mg Zn/dia X # dias Cambio en zinc serico Y mg Zn/dia X # dias Incremento en crecimiento Y mg Zn/dia X # dias Disminucion de diarrea Brown et al. FNB 2009;

Mexico Bean Trial: Morbidity and Inflammation Prevalence in High Iron and Control Schools After statistical control for school level variation in prevalence of morbidity and APP there is no significant difference in log Ferritin between High Fe and control subjects (p=0.72) Suggests twice the prevalence of infections in the control schools * High CRP = CRP>3.0 mg/L **High APR = AGP>1.0 g/L # Reported fever, diarrhea, productive cough in previous 2 weeks

Rice: Zinc content and sources of diets Dietary component CR ZnBfR CR+Zn Composite diet (mg/d) 2.64 3.63 Zinc tracer + non-enriched zinc (mg/d) 1.01 1.00 3.18 Other food (banana), (mg/d) 0.16 0.20 0.21 Total measured zinc intake (mg/d) 3.81 4.83 6.03 Total estimated phytate intake (mg/d) ‡ 767 1083 Estimated phytate : zinc molar ratio 20 22 13 Table 3 – Measured zinc intakes and estimated phytate intakes by dietary component, study group and dietary period *Composite diet included cooked rice and lentils and papaya recipes. Zinc concentration of composite diets and zinc concentration of zinc solutions were measured in the laboratory. †Amount of zinc intake from banana was determined from the measured amount of banana consumption and estimated zinc content of bananas, as derived from a food composition table (WorldFood Dietary Assessment System, 1996). ‡ Phytate content of the diet was estimated as the sum of the measured phytate content of milled, washed, uncooked rice (319 mg/100 g or 479 mg/d for BR-28, and 530 mg/100 or 795 mg/d for IR-68144) and the estimated phytate content of 30 g lentils (288 mg/d), as derived from a food composition table (WorldFood Dietary Assessment System, 1996).

Total dietary zinc intake: fractional and total zinc absorption CR ZnBfR CR +Zn p-value* Total dietary zinc intake (mg/d) 3.81 4.83 6.03 Fractional zinc absorption (%) 25.1 ± 4.1a 19.5 ± 3.1b 18.8 ± 3.4c <0.001 Total absorbed zinc (mg/d) 0.96 ±0.16a 0.95 ± 0.16a 1.13 ± 0.20b *p-value for ANOVA comparing three dietary periods (two ZnBfR periods combined). Dietary periods with different superscripts are significantly different, p<0.05.

Akwa Ibom - Red palm oil intake (preliminary result, grams/day) Mean All ( SD) Mean among consumers only ( SD) % consumption Children 18  14 19  14 91% Women 39  29 40  28 97% Children 18 g of red palm oil= 338 µg RAE of vitamin A (EAR=275) Women 39 g of red palm oil= 731 µg RAE of vitamin A (EAR=500)

Baseline ferritin p<.0001 Baseline body iron p<.0001 Difference in final ferritin and body iron between rice groups1 Non-anemic subjects (n=138) Control Rice (n=69) Mean+SE2 High Iron Rice (n=68) Rice Group Effect t-value Covariates Ferritin, logn,mg/L (mg/L) 3.21+0.06 (24.8) 3.45+0.06 (31.5) 2.58 p=.011 Baseline ferritin p<.0001 Rice intake p<.10 Body Iron, mg/kg 4.99+0.36 6.03+0.36 2.27 p=. 025 Baseline body iron p<.0001 Rice intake p<.05 1Mixed model ANOVA, random effect=convent. Philippines Iron rich rice trial. Haas et al 2005 2Least square means + SE after controlling for covariates and convent

Evidence-based Nutrition & Evidence-based Medicine: the same guidelines apply Question Level 1 Level 2 Level 3 Level 4 Level 5 Does this Intervention help? Systematic review of randomized trails or n-of-1 trails Randomized trial or observational study with dramatic effect Non-randomized controlled cohort/follow -up study** Case-series, case-control studies, or historically controlled studies** Mechanism-based reasoning *Level may be graded down on the basis of study quality, imprecision, because of inconsistency between studies, or because the absolute effect size is very small; Level may be graded up if there is a large or very large effect size. ** A systematic review is generally better than an individual study. Reference: OCEBM Levels of Evidence Working Group. “The Oxford 2011 Levels of Evidence”. Oxford Centre for Evidence-Based Medicine. h