Cytoglobin (Cygb) and neuroglobin (Ngb) are among the newest members of vertebrate globin family characterized by a classical 3-over-3 α-helical fold and a heme prosthetic group capable of reversibly binding small ligands such as O2, CO and NO. The physiological functions of Cygb and Ngb remain to be determined; however, current data suggest that both proteins have a significant role in cytoprotection in hypoxic and genotoxic conditions. Cytoglobin and Ngb are distinct from their better-known counterparts, hemoglobin (Hb) and myoglobin (Mb), in several structural features. First, in the absence of an external ligand, the sixth coordination site of the heme iron in Cygb and Ngb is occupied by a distal histidine residue, leading to a complex ligand rebinding mechanism dependent on the rate of distal His dissociation from the heme iron. Although hexacoordination was observed before in plant and bacterial hemoglobins, the physiological role of this feature remains unknown. Second, both Ngb and Cygb are capable of forming an intraprotein disulfide bond, which has been shown to regulate ligand binding affinity, leading to a hypothesis that intracellular function of these proteins is redox-dependent. Lastly, Cygb contains 20 amino acid long extensions on both N- and C- termini, a unique feature among vertebrate globins with unknown physiological function. The work presented in the dissertation reveals that hexacoordinate heme reactivity is distinct from that of pentacoordinate heme and is strongly influenced by the distal histidine residue and the disulfide bond. In the case of human Cygb, experimental and computational approaches demonstrated that the disulfide bond regulates the flexibility of the N terminus and the accessibility of the 1,8-ANS binding site. Furthermore, molecular dynamics of the hexa- and pentacoordinate human Ngb were probed computationally to elucidate structural requirements that govern signal transmission between CD loop and the distal pocket. Lastly, Ngb and Cygb were reconstituted with a fluorescent analog of the native heme group to produce hexacoordinate variants with favorable photophysical properties that can be used to characterize protein-protein interactions.

A cor vermelha brilhante característica do presunto Parma é resultante, principalmente, do pigmento Zinco-protoporfirina IX (ZnPP). A ZnPP é formada a partir da mioglobina por uma reação de transmetalação, catalisada pela enzima ferroquelatase (FECH), em que o íon de Fe(II) coordenado ao grupo heme é substituído pelo íon Zn(II). O presunto Parma apresenta uma maior estabilidade oxidativa em relação aos demais produtos cárneos curados além de não conter nitrito e nitrato, portanto, são considerados mais saudáveis. A utilização da FECH no processamento de carnes curadas pode permitir a produção de produtos cárneos curados mais saudáveis e em menor tempo. No presente trabalho a proteína ferroquelatase de Bacillus subtilis (BsFECH) foi expressa em células de E. coli BL21(DE3), purificada por cromatografia de afinidade ao níquel e exclusão por tamanho e caracterizada por dicroísmo circular, emissão de fluorescência do triptofano e cromatografia de exclusão por tamanho analítico. Em termos de estabilidade foi encontrado que altas concentrações de sal aumentam a estabilidade da proteína frente aos agentes denaturantes ureia e temperatura. A BsFECH produzida é capaz de ligar-se ao substrato modelo de porfirina (TPPS), conforme verificado por espectroscopia de UV-Vis, com uma Ka = 3,8x105 M-1 e é capaz de se associar à metamioglobina, conforme verificado por reação de cross-linking com dissuccinimidil suberato e avaliado por SDS-PAGE. A BsFECH aumenta significativamente a taxa de inserção de íons de zinco na TPPS e mostra uma cinética de saturação com uma constante de ligação aparente de Zn(II) ao complexo [BsFECH-TPPS] de 1,3x104 M e uma constante de primeira ordem de 6,6x10-1 h-1 para a dissociação do complexo ternário. A reação de troca ferro/zinco na mioglobina catalisada pela BsFECH é facilitada pela proteólise limitada da mioglobina com pepsina que abre um caminho para a reação de troca metálica com base na interação proteína-proteína entre o fragmento globina da mioglobina e a BsFECH. / The bright red color, characteristic of the Parma ham, results mainly of the pigment Zinc-Protoporphyrin IX (ZnPP). The ZnPP is formed from myoglobin by the reaction, catalyzed by ferrochelatase enzyme (FECH), in which Fe(II) ions coordinated to the heme group is replaced by Zn(II) ions. Parma ham shows greater oxidative stability when compared to others cured meat products besides do not contain nitrite and nitrate and, therefore, is considered healthier. The use of FECH in the processing of cured meats may allow the production of healthier cured meat products in a shorter period of time. In this work, the ferrochelatase protein from Bacillus subtilis was expressed in E. coli BL21(DE3) cells, purified by nickel affinity chromatography and size exclusion, and characterized by circular dichroism, fluorescence emission of tryptophan and analytical size exclusion chromatography. In terms of stability, it was found that the high salt content enhances the protein stability against the denaturation agents urea and temperature. The BsFECH produced is able to bind to the porphyrin model substrate (TPPS), as verified by UV-Vis spectroscopy, with Ka = 3.8x105 M-1 and is capable to associate to metamyoglobin as verified by cross-linking reaction with dissuccinimidil suberato, as observed by SDS-PAGE. The BsFECH increases, significantly, the zinc ions insertion rate in TPPS and shows a saturation kinetics behavior with an apparent biding constant of Zn(II) to the [BsFECH-TPPS] complex of 1.3x104 M and a first order rate constant for the dissociation of ternary complex of 6.6x10-1 h-1. The Fe/Zn exchange reaction in the myoglobin as catalyzed by BsFECH is facilitated by myoglobin-limited proteolysis with pepsin that opens a reaction channel for the metallic exchange based on protein-protein interaction between the globin moiety of myoglobin and BsFECH.

A cor vermelha brilhante característica do presunto Parma é resultante, principalmente, do pigmento Zinco-protoporfirina IX (ZnPP). A ZnPP é formada a partir da mioglobina por uma reação de transmetalação, catalisada pela enzima ferroquelatase (FECH), em que o íon de Fe(II) coordenado ao grupo heme é substituído pelo íon Zn(II). O presunto Parma apresenta uma maior estabilidade oxidativa em relação aos demais produtos cárneos curados além de não conter nitrito e nitrato, portanto, são considerados mais saudáveis. A utilização da FECH no processamento de carnes curadas pode permitir a produção de produtos cárneos curados mais saudáveis e em menor tempo. No presente trabalho a proteína ferroquelatase de Bacillus subtilis (BsFECH) foi expressa em células de E. coli BL21(DE3), purificada por cromatografia de afinidade ao níquel e exclusão por tamanho e caracterizada por dicroísmo circular, emissão de fluorescência do triptofano e cromatografia de exclusão por tamanho analítico. Em termos de estabilidade foi encontrado que altas concentrações de sal aumentam a estabilidade da proteína frente aos agentes denaturantes ureia e temperatura. A BsFECH produzida é capaz de ligar-se ao substrato modelo de porfirina (TPPS), conforme verificado por espectroscopia de UV-Vis, com uma Ka = 3,8x105 M-1 e é capaz de se associar à metamioglobina, conforme verificado por reação de cross-linking com dissuccinimidil suberato e avaliado por SDS-PAGE. A BsFECH aumenta significativamente a taxa de inserção de íons de zinco na TPPS e mostra uma cinética de saturação com uma constante de ligação aparente de Zn(II) ao complexo [BsFECH-TPPS] de 1,3x104 M e uma constante de primeira ordem de 6,6x10-1 h-1 para a dissociação do complexo ternário. A reação de troca ferro/zinco na mioglobina catalisada pela BsFECH é facilitada pela proteólise limitada da mioglobina com pepsina que abre um caminho para a reação de troca metálica com base na interação proteína-proteína entre o fragmento globina da mioglobina e a BsFECH. / The bright red color, characteristic of the Parma ham, results mainly of the pigment Zinc-Protoporphyrin IX (ZnPP). The ZnPP is formed from myoglobin by the reaction, catalyzed by ferrochelatase enzyme (FECH), in which Fe(II) ions coordinated to the heme group is replaced by Zn(II) ions. Parma ham shows greater oxidative stability when compared to others cured meat products besides do not contain nitrite and nitrate and, therefore, is considered healthier. The use of FECH in the processing of cured meats may allow the production of healthier cured meat products in a shorter period of time. In this work, the ferrochelatase protein from Bacillus subtilis was expressed in E. coli BL21(DE3) cells, purified by nickel affinity chromatography and size exclusion, and characterized by circular dichroism, fluorescence emission of tryptophan and analytical size exclusion chromatography. In terms of stability, it was found that the high salt content enhances the protein stability against the denaturation agents urea and temperature. The BsFECH produced is able to bind to the porphyrin model substrate (TPPS), as verified by UV-Vis spectroscopy, with Ka = 3.8x105 M-1 and is capable to associate to metamyoglobin as verified by cross-linking reaction with dissuccinimidil suberato, as observed by SDS-PAGE. The BsFECH increases, significantly, the zinc ions insertion rate in TPPS and shows a saturation kinetics behavior with an apparent biding constant of Zn(II) to the [BsFECH-TPPS] complex of 1.3x104 M and a first order rate constant for the dissociation of ternary complex of 6.6x10-1 h-1. The Fe/Zn exchange reaction in the myoglobin as catalyzed by BsFECH is facilitated by myoglobin-limited proteolysis with pepsin that opens a reaction channel for the metallic exchange based on protein-protein interaction between the globin moiety of myoglobin and BsFECH.

Background The potential dire consequences of iron deficiency (ID) and iron deficiency anaemia (IDA) on childhood development are of major public health concern. Many factors contribute to anaemia, ID being only one progressive factor. The prevalence of ID and IDA must be accurately determined before iron intervention strategies can be safely prescribed. There is continued uncertainty regarding the optimal approach to identifying and measuring ID, as indicators have different roles, explore different aspects of iron metabolism and cannot be directly compared. Furthermore, inflammation and infection have a confounding effect on the commonly applied indicator and acute phase reactant, serum ferritin (SF). In the public health setting, a suitable method to assess iron status in developing countries has to be inexpensive, standardised, established, easy to measure and its applications specific to identifying ID. Aim We conducted secondary analysis of screening data from three independent iron intervention studies to critically evaluate the indicators used to determine iron status in 6-11-year-old primary school children from three South African provinces. Study design and methods A cross-sectional descriptive analysis was performed on the screening data collected in 2009 and 2010 during iron intervention studies in KwaZulu-Natal (n=736), Northern Cape (n= 1045), and North West (n=546). The three distinct study sites were analysed independently and collectively. Children’s haemoglobin (Hb), SF, transferrin receptor (TfR), zinc protoporphyrin (ZPP), and C-reactive protein (CRP) concentrations were measured and body iron calculated. ID prevalence was compared using different methods (namely the single indicators SF, TfR and ZPP, body iron and the multiple criteria model), and the influence of inflammation on SF was considered. Literature suggests that the multiple criteria model provides a more complete assessment of iron status. The performance of single and body iron indicators were compared to the multiple criteria model (by assessing sensitivity, specificity and predictive values). Results Significant positive correlations between CRP (indicator of inflammation) and SF existed in all study sites and the combined sample (p < 0.01). The mean SF concentration was substantially higher in subjects with inflammation than those without. A different SF cut-off to identify ID was applied to subjects with inflammation. The percentage of ID subjects varied using different indicators (4.2 – 26.5% in KwaZulu-Natal; 4.1 – 13.4% in Northern Cape; 7.0 – 24.4% in North West; and 5.4 – 15.2% in the combined sample). The sensitivity, specificity and predictive values of alternate ID indicators varied within and between study sites, compared to the multiple criteria model. Conclusion Simply using Hb as an ID indicator is inaccurate. The vast differences between percentages identified as ID by different indicators is reason for concern. No consistent agreement appeared between single ID indicators, body iron and the multiple criteria model for ID identification after correcting for inflammation in primary school children. The global view of the multiple criteria model as the gold standard for estimating ID is debatable and potentially impractical at a public health level. Current evidence cautions against overestimating the prevalence of ID, as there is more associated harm than deficiency underestimation. This critical analysis has confirmed a need for research to identify a suitable, accurate and precise alternative to Hb as a tool in the South African public health setting. Furthermore, the impact of inflammation on iron status indicators, in particular SF, should be assessed in context to clearly set parameters for its use in nationally-representative nutrition surveys, the cornerstone of iron intervention strategies. / MSc (Nutrition), North-West University, Potchefstroom Campus, 2015

Background The potential dire consequences of iron deficiency (ID) and iron deficiency anaemia (IDA) on childhood development are of major public health concern. Many factors contribute to anaemia, ID being only one progressive factor. The prevalence of ID and IDA must be accurately determined before iron intervention strategies can be safely prescribed. There is continued uncertainty regarding the optimal approach to identifying and measuring ID, as indicators have different roles, explore different aspects of iron metabolism and cannot be directly compared. Furthermore, inflammation and infection have a confounding effect on the commonly applied indicator and acute phase reactant, serum ferritin (SF). In the public health setting, a suitable method to assess iron status in developing countries has to be inexpensive, standardised, established, easy to measure and its applications specific to identifying ID. Aim We conducted secondary analysis of screening data from three independent iron intervention studies to critically evaluate the indicators used to determine iron status in 6-11-year-old primary school children from three South African provinces. Study design and methods A cross-sectional descriptive analysis was performed on the screening data collected in 2009 and 2010 during iron intervention studies in KwaZulu-Natal (n=736), Northern Cape (n= 1045), and North West (n=546). The three distinct study sites were analysed independently and collectively. Children’s haemoglobin (Hb), SF, transferrin receptor (TfR), zinc protoporphyrin (ZPP), and C-reactive protein (CRP) concentrations were measured and body iron calculated. ID prevalence was compared using different methods (namely the single indicators SF, TfR and ZPP, body iron and the multiple criteria model), and the influence of inflammation on SF was considered. Literature suggests that the multiple criteria model provides a more complete assessment of iron status. The performance of single and body iron indicators were compared to the multiple criteria model (by assessing sensitivity, specificity and predictive values). Results Significant positive correlations between CRP (indicator of inflammation) and SF existed in all study sites and the combined sample (p < 0.01). The mean SF concentration was substantially higher in subjects with inflammation than those without. A different SF cut-off to identify ID was applied to subjects with inflammation. The percentage of ID subjects varied using different indicators (4.2 – 26.5% in KwaZulu-Natal; 4.1 – 13.4% in Northern Cape; 7.0 – 24.4% in North West; and 5.4 – 15.2% in the combined sample). The sensitivity, specificity and predictive values of alternate ID indicators varied within and between study sites, compared to the multiple criteria model. Conclusion Simply using Hb as an ID indicator is inaccurate. The vast differences between percentages identified as ID by different indicators is reason for concern. No consistent agreement appeared between single ID indicators, body iron and the multiple criteria model for ID identification after correcting for inflammation in primary school children. The global view of the multiple criteria model as the gold standard for estimating ID is debatable and potentially impractical at a public health level. Current evidence cautions against overestimating the prevalence of ID, as there is more associated harm than deficiency underestimation. This critical analysis has confirmed a need for research to identify a suitable, accurate and precise alternative to Hb as a tool in the South African public health setting. Furthermore, the impact of inflammation on iron status indicators, in particular SF, should be assessed in context to clearly set parameters for its use in nationally-representative nutrition surveys, the cornerstone of iron intervention strategies. / MSc (Nutrition), North-West University, Potchefstroom Campus, 2015

A zinco protoporfirina (ZPP) eritrocitária é um metabólito formado pela adição do zinco no sítio do ferro durante a formação da molécula de hemoglobina, quando este último está total ou parcialmente indisponível. O objetivo deste trabalho foi realizar a padronização dos valores da ZPP eritrocitária em bezerros sadios com até um mês de vida, assim como a validade da ZPP como previsora da ocorrência de anemia ferropriva em bezerros neonatos, em comparação com outros métodos. Para tanto foram utilizados 134 bezerros da raça Holandesa, com idades variando do nascimento até 30 dias de vida, provenientes de fazendas produtoras de leite localizadas no Estado de São Paulo, classificados como sadios (67 animais) e anêmicos (67 animais). Os animais foram monitorados por meio de exames físico e complementares (hemograma e reticulocitometria, teores de ferro sérico (FT) e capacidade total de ligação do ferro (CTLF), teores de bilirrubinas e de uréia séricas, e concentrações da ZPP). Durante a padronização do exame, os valores médios encontrados foram: concentração de ZPP eritrocitária das amostras de hemácias não lavadas em até 3 horas após a colheita de sangue de 30 animais sadios - 80,90 &#181;mol ZPP/mol heme; concentração de ZPP eritrocitária, após a lavagem de hemácias, determinadas até 3 horas e 12 horas após colheita de sangue do mesmo grupo - 61,40 &#181;mol ZPP/mol heme e 61,03 &#181;mol ZPP/mol heme, respectivamente. Com base nos resultados obtidos, foi possível concluir que as amostras de sangue colhidas para a mensuração da ZPP podem ser armazenadas, sob refrigeração a 4°C, por até 12 horas após a colheita, sem alterações significativas dos seus valores, sendo recomendável a lavagem das hemácias antes da mensuração dos valores da ZPP eritrocitária devido à presença de substâncias interferentes no plasma do animal. Foi encontrada diferença significativa nas concentrações da ZPP e de todos os componentes do eritrograma, assim como nos teores do metabolismo de ferro entre os animais anêmicos e sadios. As concentrações de bilirrubinas e ureia séricas apresentaram-se no intervalo fisiológico de variação, não interferindo na mensuração da ZPP nos eritrócitos dos bezerros com ou sem anemia. A correlação entre os valores encontrados dos teores de ferro sérico e da capacidade total de ligação do ferro com as concentrações de ZPP foram: rs = - 0,45, p < 0,001 (ZPP x FT) e rs = 0,51, p < 0,001 (ZPP x CTLF), respectivamente, demonstrando que quanto menores os teores de ferro, maiores serão as concentrações de ZPP. Portanto, a utilização da ZPP como previsora dos quadros de anemia ferropriva em bezerros neonatos com até um mês de vida se mostrou válida e, considerando que a hematofluorometria é um método rápido e não oneroso, pode ser recomendado como exame complementar de rotina. / The zinc protoporphyrin (ZPP) is a metabolic originated by zinc addition in the iron site during the synthesis of hemoglobin molecule, when iron is total or partially unavailable. The aim of this study was to standardize the values of zinc protoporphyrin (ZPP) in erythrocyte of healthy calves until one month of life, and to evaluate this determination as a predictor of the occurrence of iron deficiency anemia in newborn calves, compared with other methods. Therefore, 134 Holstein calves were used, aged from birth to 30 days of life, from dairy farms located in the São Paulo State, splitted into two groups, classified as healthies (67 animals), and anemics (67 animals). The animals were monitored by physical examination and laboratory assessments (complete blood cell count and reticulocytes, serum iron (SI) and total iron binding capacity (TIBC), levels of serum bilirubin and urea, and ZPP levels). During the exam standardization, the mean values found were: erythrocyte ZPP of unwashed erythrocytes within 3 hours after blood sampling of 30 healthy animals - 80.9 &#181;mol ZPP/mol heme; erythrocyte ZPP after washing of red blood cells, determined by 3 hours and 12 hours after blood collection, from the same group were 61.40 &#181;mol ZPP/mol heme and 61.03 &#181;mol ZPP/mol heme, respectively. Based on these results, it was concluded that blood samples taken for measurement of ZPP may be stored under refrigeration at 4 °C for up to 12 hours, without significant changes of the values of erythrocyte ZPP, and it is also advisable to wash the red cells before erythrocyte ZPP values measuring, due to the presence of interfering substances in the animal plasma. A significant difference was found in the ZPP levels and in all erythrogram components, as well as on the levels of iron metabolism between anemic and healthy animals. The bilirubin and serum urea levels remained within physiological variation, does not interfering with the measurement of erythrocyte ZPP of calves with or without anemia. The correlation values between SI and TIBC with ZPP levels were: rs = - 0.45, p < 0.001 (ZPP x SI) and rs = 0.51, p < 0.001 (ZPP x TIBC), respectively, i.e. decreasing biochemical levels of iron metabolism will increase levels of ZPP. Therefore, the use of ZPP as an iron deficiency anemia predictor in newborn calves up to one month of life has proven its validity, and considering hematofluorometry as a quick and inexpensive method, it might be recommended as a routine exam.

A zinco protoporfirina (ZPP) eritrocitária é um metabólito formado pela adição do zinco no sítio do ferro durante a formação da molécula de hemoglobina, quando este último está total ou parcialmente indisponível. O objetivo deste trabalho foi realizar a padronização dos valores da ZPP eritrocitária em bezerros sadios com até um mês de vida, assim como a validade da ZPP como previsora da ocorrência de anemia ferropriva em bezerros neonatos, em comparação com outros métodos. Para tanto foram utilizados 134 bezerros da raça Holandesa, com idades variando do nascimento até 30 dias de vida, provenientes de fazendas produtoras de leite localizadas no Estado de São Paulo, classificados como sadios (67 animais) e anêmicos (67 animais). Os animais foram monitorados por meio de exames físico e complementares (hemograma e reticulocitometria, teores de ferro sérico (FT) e capacidade total de ligação do ferro (CTLF), teores de bilirrubinas e de uréia séricas, e concentrações da ZPP). Durante a padronização do exame, os valores médios encontrados foram: concentração de ZPP eritrocitária das amostras de hemácias não lavadas em até 3 horas após a colheita de sangue de 30 animais sadios - 80,90 &#181;mol ZPP/mol heme; concentração de ZPP eritrocitária, após a lavagem de hemácias, determinadas até 3 horas e 12 horas após colheita de sangue do mesmo grupo - 61,40 &#181;mol ZPP/mol heme e 61,03 &#181;mol ZPP/mol heme, respectivamente. Com base nos resultados obtidos, foi possível concluir que as amostras de sangue colhidas para a mensuração da ZPP podem ser armazenadas, sob refrigeração a 4°C, por até 12 horas após a colheita, sem alterações significativas dos seus valores, sendo recomendável a lavagem das hemácias antes da mensuração dos valores da ZPP eritrocitária devido à presença de substâncias interferentes no plasma do animal. Foi encontrada diferença significativa nas concentrações da ZPP e de todos os componentes do eritrograma, assim como nos teores do metabolismo de ferro entre os animais anêmicos e sadios. As concentrações de bilirrubinas e ureia séricas apresentaram-se no intervalo fisiológico de variação, não interferindo na mensuração da ZPP nos eritrócitos dos bezerros com ou sem anemia. A correlação entre os valores encontrados dos teores de ferro sérico e da capacidade total de ligação do ferro com as concentrações de ZPP foram: rs = - 0,45, p < 0,001 (ZPP x FT) e rs = 0,51, p < 0,001 (ZPP x CTLF), respectivamente, demonstrando que quanto menores os teores de ferro, maiores serão as concentrações de ZPP. Portanto, a utilização da ZPP como previsora dos quadros de anemia ferropriva em bezerros neonatos com até um mês de vida se mostrou válida e, considerando que a hematofluorometria é um método rápido e não oneroso, pode ser recomendado como exame complementar de rotina. / The zinc protoporphyrin (ZPP) is a metabolic originated by zinc addition in the iron site during the synthesis of hemoglobin molecule, when iron is total or partially unavailable. The aim of this study was to standardize the values of zinc protoporphyrin (ZPP) in erythrocyte of healthy calves until one month of life, and to evaluate this determination as a predictor of the occurrence of iron deficiency anemia in newborn calves, compared with other methods. Therefore, 134 Holstein calves were used, aged from birth to 30 days of life, from dairy farms located in the São Paulo State, splitted into two groups, classified as healthies (67 animals), and anemics (67 animals). The animals were monitored by physical examination and laboratory assessments (complete blood cell count and reticulocytes, serum iron (SI) and total iron binding capacity (TIBC), levels of serum bilirubin and urea, and ZPP levels). During the exam standardization, the mean values found were: erythrocyte ZPP of unwashed erythrocytes within 3 hours after blood sampling of 30 healthy animals - 80.9 &#181;mol ZPP/mol heme; erythrocyte ZPP after washing of red blood cells, determined by 3 hours and 12 hours after blood collection, from the same group were 61.40 &#181;mol ZPP/mol heme and 61.03 &#181;mol ZPP/mol heme, respectively. Based on these results, it was concluded that blood samples taken for measurement of ZPP may be stored under refrigeration at 4 °C for up to 12 hours, without significant changes of the values of erythrocyte ZPP, and it is also advisable to wash the red cells before erythrocyte ZPP values measuring, due to the presence of interfering substances in the animal plasma. A significant difference was found in the ZPP levels and in all erythrogram components, as well as on the levels of iron metabolism between anemic and healthy animals. The bilirubin and serum urea levels remained within physiological variation, does not interfering with the measurement of erythrocyte ZPP of calves with or without anemia. The correlation values between SI and TIBC with ZPP levels were: rs = - 0.45, p < 0.001 (ZPP x SI) and rs = 0.51, p < 0.001 (ZPP x TIBC), respectively, i.e. decreasing biochemical levels of iron metabolism will increase levels of ZPP. Therefore, the use of ZPP as an iron deficiency anemia predictor in newborn calves up to one month of life has proven its validity, and considering hematofluorometry as a quick and inexpensive method, it might be recommended as a routine exam.

Tesis por compendio / [ES] El hígado de cerdo es un coproducto de origen animal que contiene una elevada cantidad de proteínas, así como otros compuestos de interés, entre los que destacan las enzimas como la ferroquelatasa (FeQ). La FeQ actúa como catalizadora en la formación del pigmento zinc protoporfirina (ZnPP), responsable del color característico del jamón de Parma y que puede resultar relevante en la elaboración de productos cárnicos crudo-curados. Así, en la presente Tesis Doctoral se va a estudiar la aplicación de tecnologías emergentes, como los ultrasonidos (US), para favorecer la extracción de la enzima FeQ y para la mejora de la formación del pigmento ZnPP. Asimismo, con el objetivo de la valorización del hígado como fuente de proteínas, en primer lugar se estudiará su estabilización mediante el secado convectivo, a un amplio rango de temperaturas, para optimizar el producto (fracción proteica). Posteriormente, el hígado deshidratado se desgrasará y desodorizará utilizando técnicas convencionales como el arrastre por vapor con aplicación de vacío (AVV) y CO2 supercríticos (CO2-SC), para lograr un producto rico en proteínas que sea aceptable organolépticamente por el consumidor. Los resultados experimentales mostraron que la aplicación de US intensificó en gran medida la extracción de la enzima FeQ del hígado de cerdo. Así, la aplicación de US durante la extracción de FeQ condujo a una mejora de la actividad enzimática y consecuentemente a un aumento de la formación de ZnPP. En cuanto a la formación de ZnPP asistida por US a partir hígado homogeneizado (Hhc) e hígado homogeneizado, con adición de oxihemoglobina (Hhc+OxiHb), el máximo de ZnPP formado cuando se aplicaron US a baja potencia (7.05 W/L) fue de 0.405 mmol ZnPP/L en Hhc y 0.449 mmol ZnPP/L en Hhc+OxiHb, alcanzándose este valor a las 12 h, mientras que sin aplicación de US el máximo se alcanzó a las 24 h, siendo éste de 0.322 mmol ZnPP/L en Hhc y 0.430 mmol ZnPP/L en Hhc+OxiHb. Sin embargo, cuando se aplicaron US a moderada potencia (36.53 W/L) fue solo de 0.037 mmol ZnPP/L a 24 h de incubación, debido a que el sistema de control de temperatura no pudo ser controlado. En relación al secado de hígado de cerdo a diferentes temperaturas (de -10 a 70ºC), los resultados revelaron que el aumento de la temperatura de secado influyó tanto en la actividad enzimática como en la concentración de FeQ aparente. Así, la condición óptima se encontró en el rango de 10 a 20ºC. Además, la temperatura de secado y el desgrasado también influyó en las propiedades fisicoquímicas y tecno-funcionales del hígado de cerdo, siendo la temperatura de 40ºC la que conllevó una menor degradación de proteínas en comparación con el deshidratado a 70ºC. En cuanto a la desodorización, los resultados mostraron que ambas técnicas (AVV y CO2-SC) fueron eficaces en la reducción y eliminación compuestos volátiles (COVs) característicos del hígado del cerdo. A través del AVV se redujo el contenido de COVs se redujo en su conjunto un 67.5%, mientras que mediante CO2-SC se redujo un 81.2%, con respecto al hígado de cerdo deshidratado sin desodorizar. Además, tras la aplicación de CO2-SC se eliminaron COVs característicos del olor desagradable del hígado de cerdo. Asimismo, mediante la extracción con CO2-SC se redujo el contenido de grasa un 24.9%, en comparación con la muestra sin desodorizar. Así pues, se puede concluir que las tecnologías emergentes como los US mejoraron el proceso de extracción de FeQ, así como su actividad enzimática en la formación de ZnPP. Además, mediante el secado y desgrasado del hígado, es posible alargar la vida útil del hígado. Así, a partir del hígado deshidratado se extrajo FeQ con actividad catalítica para la formación del pigmento ZnPP, y a partir del hígado deshidratado-desgrasado se recuperó la fracción proteica, la cual presentaba excelentes propiedades fisicoquímicas y tecno-funcionales. La extracción mediante CO2-SC resultó ser una alternativa a la técnica AVV para la desodorización. / [CA] El fetge de porc és un coproducte d'origen animal que conté una elevada quantitat de proteïnes, així com altres compostos d'interès, entre els quals destaquen els enzims com la ferroquelatasa (FeQ). La FeQ actua com a catalitzadora en la formació del pigment zinc protoporfirina (ZnPP), responsable del color característic del pernil de Parma i que pot resultar rellevant en l'elaboració de productes carnis crucurats. Així, en aquesta Tesi Doctoral s'estudiarà l'aplicació de tecnologies emergents, com els ultrasons (US), per afavorir l'extracció de l'enzim FeQ i per a la millora de la formació del pigment ZnPP. Així mateix, amb l'objectiu de la valorització del fetge com a font de proteïnes, en primer lloc se n'estudiarà l'estabilització mitjançant l'assecatge convectiu, a un ampli rang de temperatures, per optimitzar el producte (fracció proteica). Posteriorment, el fetge deshidratat es desgreixarà i desodoritzarà utilitzant tècniques convencionals com l'arrossegament per vapor amb aplicació de buit (AVV) i CO2 supercrítics (CO2-SC), per aconseguir un producte ric en proteïnes que sigui acceptable organolèpticament pel consumidor. Els resultats experimentals van mostrar que l'aplicació US va intensificar en gran mesura l'extracció de l'enzim FeQ del fetge de porc. Així, l'aplicació US durant l'extracció de FeQ va conduir a una millora de l'activitat enzimàtica i conseqüentment a un augment de la formació de ZnPP. Quant a la formació de ZnPP assistida per US a partir fetge homogeneïtzat (Hfp) i fetge homogeneïtzat, amb addició de oxihemoglobina (Hfp+OxiHb) , el màxim de ZnPP format quan es van aplicar US a baixa potència (7.05 W/L) va ser de 0.405 mmol ZnPP/L a Hhc i 0.449 mmol ZnPP /L a Hhc+OxiHb, assolint-se aquest valor a les 12 h, mentre que sense aplicació d'US el màxim es va assolir a les 24 h, sent aquest de 0.322 mmol ZnPP/L a Hhc i 0.430 mmol ZnPP/L a Hhc+OxiHb . Tot i això, quan es van aplicar US a moderada potència (36.53 W/L) va ser només de 0.037 mmol ZnPP/L a 24 h d'incubació, pel fet que el sistema de control de temperatura no va poder ser controlat. Pel que fa a l'assecatge de fetge de porc a diferents temperatures (de -10 a 70ºC), els resultats van revelar que l'augment de la temperatura d'assecatge va influir tant en l'activitat enzimàtica com en la concentració de FeQ aparent. Així, la condició òptima es va trobar al rang de 10 a 20ºC. A més, la temperatura d'assecatge i el desgreixat també va influir en les propietats fisicoquímiques i tecnofuncionals del fetge de porc, sent la temperatura de 40ºC la que va comportar una menor degradació de proteïnes en comparació del deshidratat a 70ºC. Pel que fa a la desodorització, els resultats van mostrar que ambdues tècniques (AVV i CO2-SC) van ser eficaces en la reducció i eliminació compostos volàtils (COVs) característics del fetge del porc. A través de l'AVV es va reduir el contingut de COVs es va reduir en conjunt un 67.5%, mentre que mitjançant CO2-SC es va reduir un 81.2%, respecte al fetge de porc deshidratat sense desodoritzar. A més, després de l'aplicació de CO2-SC es van eliminar COV característics de l'olor desagradable del fetge de porc. Així mateix, mitjançant l'extracció amb CO2-SC es va reduir el contingut de greix un 24.9%, en comparació de la mostra sense desodoritzar. Així doncs, es pot concloure que les tecnologies emergents com els US van millorar el procés d'extracció de FeQ, així com la seva activitat enzimàtica a la formació de ZnPP. A més, mitjançant l'assecatge i desgreixatge del fetge, és possible allargar la vida útil del fetge. Així, a partir del fetge deshidratat es va extreure FeQ amb activitat catalítica per a la formació del pigment ZnPP, ia partir del fetge deshidratat-desgreixat es va recuperar la fracció proteica del fetge, la qual presentava excel·lents propietats fisicoquímiques i tecnofuncionals. L'extracció mitjançant CO2-SC va resultar ser una alternativa a la tècnica de AVV per a la desodorització. / [EN] Pork liver is a co-product of animal origin that contains a high amount of protein, as well as other compounds of interest, among which enzymes such as ferrochelatase (FeCH). FeCH acts as a catalyst in the formation of the pigment zinc protoporphyrin (ZnPP), responsible for the characteristic color of Parma ham and which may be relevant in the production of raw-cured meat products. Thus, in this Doctoral Thesis, the application of emerging technologies, such as ultrasound (US), will be studied to favor the extraction of the FeCH enzyme and to improve the formation of the ZnPP pigment. Likewise, with the aim of valorizing the liver as a source of protein, its stabilization by means of convective drying will first be studied, at a wide range of temperatures, to optimize the product (protein fraction). Subsequently, the dehydrated liver will be defatted and deodorized using conventional techniques such asvacuum steam distillation (VSD) and supercritical CO2 (SC-CO2), to achieve a protein-rich product that is organoleptically acceptable to the consumer. The experimental results showed that the application of US greatly enhanced the extraction of FeCH enzyme from pork liver. Thus, the application of US during the FeCH extraction led to an improvement in the enzymatic activity and consequently to an increase in the ZnPP formation. Regarding US-assisted ZnPP formation from homogenized pork liver (HLi) and homogenized pork liver, with the addition of oxyhemoglobin (HLi+OxyHb, the maximum ZnPP formed when US was applied at low power (7.05 W/L) was 0.405 mmol ZnPP/L in Hhc and 0.449 mmol ZnPP /L in Hhc+OxyHb, reaching this value at 12 h, while without application of US the maximum was reached at 24 h, being 0.322 mmol ZnPP/L in Hhc and 0.430 mmol ZnPP/L in Hhc+OxyHb. However, when US was applied at moderate power (36.53 W/L) it was only 0.037 mmol ZnPP/L at 24 h of incubation, because the temperature control system could not be controlled. Regarding the drying of pig liver at different temperatures (from -10 to 70ºC), the results revealed that the increase in the drying temperature influenced both the enzymatic activity and the apparent FeCH concentration. Thus, the optimal condition was found in the range of 10 to 20ºC. In addition, the drying temperature and defatting also influenced the physicochemical and techno-functional properties of pork liver, being the temperature of 40ºC the one that led to less protein degradation compared to that dehydrated at 70ºC. Regarding deodorization, the results showed that both techniques (VSD and SC-CO2) were effective in reducing and eliminating volatile compounds (VOCs) characteristic of pig liver. Through AVV the content of VOCs was reduced as a whole by 67.5%, while through SC-CO2 towas reduced by 81.2%, with respect to dried pork liver without deodorizing. In addition, after the application of SC-CO2, VOCs characteristic of the unpleasant odor of pig liver were eliminated. Likewise, by extraction with SC-CO2, the fat content was reduced by 24.9%, compared to the sample without deodorization. Thus, it can be concluded that emerging technologies such as US improved the FeCH extraction process, as well as its enzymatic activity in the ZnPP formation. Furthermore, by drying and defatting the liver, it is possible to extend the lifespan of the liver. Thus, from the dehydrated liver, FeCH was extracted with catalytic activity for the formation of the ZnPP pigment, and from the dried-defatted liver, the protein fraction of the liver was recovered, which presented excellent physicochemical and techno-functional properties. The SC-CO2 extraction turned out to be an alternative to the VSD technique for the deodorization. / The authors acknowledge the financial support from the “Ministerio de Economía y Competitividad (MINECO)” and “Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)” in Spain (Projects RTA2017-00024-C04-03 and RTA2017-00024-C04-02). / Abril Gisbert, B. (2023). Aplicación de tecnologías emergentes para la obtención de Zinc-protoporfirina y proteínas funcionales a partir de co-productos cárnicos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/192244 / Compendio