Food fortification and nutrient supplementation are important strategies to address micronutrient deficiencies. Mandatory folic acid fortification was implemented in Canada and the U.S. in 1998 to reduce the incidence of neural tube defects (NTD). However, the actual amount of folic acid added to foods has not been reported in Canada. We analyzed 95 fortified foods and found that there is 50% more folate in foods than that reported in food composition tables, which are primarily based on minimum mandated fortification levels. We then determined if these observed folate overages impacted the prevalence of dietary folate inadequacy or intakes above the Tolerable Upper Intake Level (UL). Using data from the 2004 nationally-representative Canadian Community Health Survey (CCHS) 2.2 (n = 35,107), adjusted for folate overages, we found a low prevalence of folate inadequacy in Canada post-fortification. However, few women 14-50y consumed 400µg/d of synthetic folic acid, an amount associated with maximal protection against an NTD. Conversely, we also showed that use of folic acid-containing supplements led to intakes >UL in the general population. To develop a tool that would help clinicians identify women with red blood cell (RBC) folate concentrations that were not maximally protective against an NTD (<906nmol/L), we used data from the nationally-representative U.S. National Health and Nutrition Examination Survey 2003-2006 to define risk factors of RBC folate <906nmol/L. We found that 35% of American women 19-45y had RBC folate <906nmol/L. Younger age, low dietary folate intake, not consuming supplemental folic acid, smoking, and being African-American were associated with increased risk of RBC folate <906nmol/L. Given our observations of a low prevalence of folate inadequacy and folic acid supplement use leading to intakes >UL, we used CCHS 2.2 data to compare the diets of supplement users and non-users in terms of inadequacy and intakes >UL for other nutrients. We showed that the prevalence of inadequacy was low for most nutrients, and from diet alone, supplement users were not at increased risk of inadequacy compared to non-users. Furthermore, inclusion of supplements led to intakes >UL above 10% for vitamins A, C, niacin, folic acid, and iron, zinc and magnesium.

Health in archaeological populations needs to be investigated using a holistic approach. Molecular techniques, particularly multiplex PCR, can be used with paleopathology and dietary analysis to understand aspects of population health. This thesis demonstrates how spina bifida, a multi-factorial disease, can be investigated using this paleoepidemiological approach. Based on skeletal evidence, spina bifida was present in a pre-Columbian Cuban population from the archaeological site of Canimar Abajo. Molecular techniques were employed to examine disease potential, examining individuals for five single nucleotide polymorphisms associated with spina bifida. It is postulated that the combined effect of these polymorphisms, as well as dietary factors, determines the risk of the population for spina bifida, and that these factors came together to create the observed high disease prevalence. Therefore, this thesis demonstrates how the methods of molecular paleopathology, corroborated by dietary analyses, can be used within a paleoepidemiological framework to understand population health and disease.

Food fortification and nutrient supplementation are important strategies to address micronutrient deficiencies. Mandatory folic acid fortification was implemented in Canada and the U.S. in 1998 to reduce the incidence of neural tube defects (NTD). However, the actual amount of folic acid added to foods has not been reported in Canada. We analyzed 95 fortified foods and found that there is 50% more folate in foods than that reported in food composition tables, which are primarily based on minimum mandated fortification levels. We then determined if these observed folate overages impacted the prevalence of dietary folate inadequacy or intakes above the Tolerable Upper Intake Level (UL). Using data from the 2004 nationally-representative Canadian Community Health Survey (CCHS) 2.2 (n = 35,107), adjusted for folate overages, we found a low prevalence of folate inadequacy in Canada post-fortification. However, few women 14-50y consumed 400µg/d of synthetic folic acid, an amount associated with maximal protection against an NTD. Conversely, we also showed that use of folic acid-containing supplements led to intakes >UL in the general population. To develop a tool that would help clinicians identify women with red blood cell (RBC) folate concentrations that were not maximally protective against an NTD (<906nmol/L), we used data from the nationally-representative U.S. National Health and Nutrition Examination Survey 2003-2006 to define risk factors of RBC folate <906nmol/L. We found that 35% of American women 19-45y had RBC folate <906nmol/L. Younger age, low dietary folate intake, not consuming supplemental folic acid, smoking, and being African-American were associated with increased risk of RBC folate <906nmol/L. Given our observations of a low prevalence of folate inadequacy and folic acid supplement use leading to intakes >UL, we used CCHS 2.2 data to compare the diets of supplement users and non-users in terms of inadequacy and intakes >UL for other nutrients. We showed that the prevalence of inadequacy was low for most nutrients, and from diet alone, supplement users were not at increased risk of inadequacy compared to non-users. Furthermore, inclusion of supplements led to intakes >UL above 10% for vitamins A, C, niacin, folic acid, and iron, zinc and magnesium.

Health in archaeological populations needs to be investigated using a holistic approach. Molecular techniques, particularly multiplex PCR, can be used with paleopathology and dietary analysis to understand aspects of population health. This thesis demonstrates how spina bifida, a multi-factorial disease, can be investigated using this paleoepidemiological approach. Based on skeletal evidence, spina bifida was present in a pre-Columbian Cuban population from the archaeological site of Canimar Abajo. Molecular techniques were employed to examine disease potential, examining individuals for five single nucleotide polymorphisms associated with spina bifida. It is postulated that the combined effect of these polymorphisms, as well as dietary factors, determines the risk of the population for spina bifida, and that these factors came together to create the observed high disease prevalence. Therefore, this thesis demonstrates how the methods of molecular paleopathology, corroborated by dietary analyses, can be used within a paleoepidemiological framework to understand population health and disease.

Micronutrient poor diets around the globe and in particular in the developing world cause deficiencies in iron and folic acid. This may be rectified by the incorporation of these micronutrients into currently running salt iodization processes. The objective of this project was to develop folic acid and iodine spray solutions to be ready for pilot scale testing and to investigate the stability of triple fortified salt containing iodine, folic acid and microencapsulated ferrous fumarate. The optimal spray solutions were buffered to pH 9 with a carbonate/bicarbonate buffer to stabilize folic acid and contained 1%-2% w/v folic acid and 1%-3% w/v iodine (as KIO3). They remained in solution and retained ≥80% of both micronutrients after 5 months of storage at 25ºC and 45ºC. Double fortified salt produced using these spray solutions retained 100% of both folic acid and iodine over a 5 month period when stored at ambient conditions. Unfortunately triple fortified salt did not sufficiently retain the micronutrients due to excess moisture absorption and inadequate encapsulation of iron.

Micronutrient poor diets around the globe and in particular in the developing world cause deficiencies in iron and folic acid. This may be rectified by the incorporation of these micronutrients into currently running salt iodization processes. The objective of this project was to develop folic acid and iodine spray solutions to be ready for pilot scale testing and to investigate the stability of triple fortified salt containing iodine, folic acid and microencapsulated ferrous fumarate. The optimal spray solutions were buffered to pH 9 with a carbonate/bicarbonate buffer to stabilize folic acid and contained 1%-2% w/v folic acid and 1%-3% w/v iodine (as KIO3). They remained in solution and retained ≥80% of both micronutrients after 5 months of storage at 25ºC and 45ºC. Double fortified salt produced using these spray solutions retained 100% of both folic acid and iodine over a 5 month period when stored at ambient conditions. Unfortunately triple fortified salt did not sufficiently retain the micronutrients due to excess moisture absorption and inadequate encapsulation of iron.

Der Folsäureantagonist Methotrexat (MTX) wird zur Behandlung onkologischer und rheumatoider Erkrankungen eingesetzt. Die Aufnahme des Methotrexats in die Zielzelle ist dabei Vorraussetzung für die Bindung an seine intrazellulären Zielstrukturen und erfolgt über verschiedene Transportsysteme. In diesem Zusammenhang ist bei entsprechenden Plasma-konzentrationen von MTX der Reduced Folate Carrier (RFC1) von besonderer Bedeutung. 1994 konnte erstmals die cDNA dieses Transporters aus Maus- und Hamstergewebe isoliert werden. Die cDNA für einen mit dem RFC1 identischen hepatozellulären MTX-Transporter der Ratte wurde 2000 kloniert. Vorhergehende Gen-Expressionsstudien zeigten, dass die RFC1-mRNA ubiquitär gebildet wird. Die Proteinexpression wurde jedoch bisher nur in ausgewählten Geweben der Maus untersucht. Systematische Arbeiten, in denen in vergleichender Weise sowohl die RFC1 Gen- als auch die Proteinexpression in allen Geweben mit einer möglichen Relevanz für die Folat- und Antifolataufnahme, Speicherung und Eliminierung untersucht werden, fehlten bisher. Insbesondere die Expression des RFC1-Proteins der Ratte (rRFC1) mittels immunologischer Verfahren ist bisher nicht beschrieben worden. Ziel dieser Arbeit war es daher, die Gen- und Proteinexpression des rRFC1 in ausgewählten Geweben der Ratte darzustellen. Dieses schließt die Generierung spezifischer Antiseren gegen den rRFC1 als ersten Schritt mit ein. Es wurden geeignete antigene Aminosäuresequenzen des rRFC1 bestimmt und die entsprechenden cDNA Sequenzen wurden amplifiziert und in einen geeigneten Expressionsvektor kloniert. Rekombinante rRFC1 Fusionsproteine konnten mittels E. coli Zellen hergestellt und anschließend aufgereinigt werden. Nachfolgend wurden entweder die rRFC1 Fusionsproteine oder die rRFC1 spezifischen Peptide, welche von dem Affinitätspeptid separiert worden waren, für die Immunisierung von Kaninchen verwendet Drei Antiseren mit ausreichender Reaktivität und Spezifität konnten gewonnen und mittels Affinitätschromatographie aufgereinigt werden. Die erhaltenen Antiseren sind gegen die intrazellulären N- und C-terminalen Regionen (ID1, ID7) bzw. gegen die erste extrazelluläre Schleife (OD1) gerichtet. In Western-Blot Studien konnte mittels dieser Antiseren für den rRFC1, der in transfizierten Nierenepithelzellen (MDCK-rRFC-HA) stabil exprimiert wurde, ein Molekulargewicht von 71 kD für die glykosylierte Form und von 53 kD für die unglykosylierte Form ermittelt werden. Weiter konnte belegt werden, dass das Protein in MDCK-rRFC1-HA Zellen überwiegend in der glycosylierten Form vorliegt. Mittels RT-PCR Analysen wurde die Genexpression des rRFC1 in allen untersuchten Geweben nachgewiesen. Besonders hohe mRNA-Gehalte waren in Thymus, Niere und Milz vorhanden, während in Herz- und Muskelgewebe sowie in Leukozyten nur ein Signal nahe der Nachweisgrenze detektierbar war. Durch immunhistologische Untersuchungen konnten die rRFC1 Proteinexpression und beträchtliche Unterschiede in der Signalintensität bestätigt werden. Zusätzlich konnten neue Informationen über die unterschiedliche subzelluläre Lokalisation gewonnen werden: so konnte eine starke Expression des Transporters in der apikalen Membran von Dünn- und Dickdarmmukosa dargestellt werden, während die ebenfalls starke Färbung in der Niere auf den Bereich der basolateralen Membran der Tubuli beschränkt war. In der Leber war eine Expression mittlerer Intensität im Bereich der Lebertrias erkennbar. Während in der Milz nur in der roten Pulpa das RFC1-Protein detektiert wurde, konnten im Thymus sowohl in der Rinde als auch im Mark positive Zellen nachgewiesen werden. Im Hoden konnte der Transporter in den Sertoli-Zellen dargestellt werden. Eine starke Expression des Transporters wurde im Gehirn im Bereich der apikalen Membran der Ependymzellen des Plexus choroideus nachgewiesen. In der Skelettmuskulatur und im Herzgewebe beschränkte sich die Expression des rRFC1 auf das Perimysium des Muskelgewebes und auf kleinere Gefäße des Muskel- und Herzgewebes. In dieser Arbeit konnte somit gezeigt werden, dass der RFC1 der Ratte ubiquitär exprimiert wird, wobei die Expressionsstärke jedoch stark variiert. Die beobachtete Gewebslokalisation des RFC1 belegt sowohl dessen zentrale Rolle in der Folathomöostase als auch in der MTX vermittelten Organtoxizität und Pharmakokinetik, insbesondere bei der intestinalen Resorption sowie der hepatischen und renalen Exkretion.

Univ., Diss.--Erlangen, 2004.

Universiẗat, Diss.--Kiel, 1997.

Foram estudadas 363 gestantes de primeira consulta, que não faziam uso de medicamentos que continham ferro, ácido fólico, vitamina B12 ou associação destes, na ocasião da coleta do material, em Centros de Saúde do Estado, no Subdistrito de Paz do Butantã, município de São Paulo. A prevalência de anemia (concentração de hemoglobina inferior a 11,6 g/dl) foi de 12,4%. As médias das concentrações de hemoglobina e as prevalências de anemia, segundo o trimestre de gestação, foram respectivamente: 13,47 g/dl e 3,57% no primeiro, 12,47 g/dl e 20,86% no segundo e 12,25 g/dl e 32,14% no terceiro trimestre. Não encontramos diferença. estatisticamente significativa entre as médias das concentrações de hemoglobina e entre as prevalências de anemia nas gestantes primigestas e multigestas. O mesmo aconteceu com as médias e as prevalências das gestantes multigestas com intervalo do último parto até dois anos e maior que dois anos. Na amostra estudada, encontramos maior prevalência de anemia naquelas gestantes que pertenciam às famílias que tinham renda per capita até 0,5 SMPC (salário mínimo per capita). As prevalências de verminose e de ancilostomídeos nas 300 gestantes foram respectivamente: 64,7% e 14,0%. Nas gestantes anêmicas foram respectivamente: 73,7% e 7,9%. Nas gestantes anêmicas, a prevalência de deficiência de ferro foi de 42,2% (concentração de ferro sérico <50 &#181;g/dl) ou 46,7% (saturação da transferrina <15% ou 40,0% (concentração de ferro sérico <50 &#181;g/dl e saturação de transferrina <15%). A prevalência de deficiência de ácido fólico foi 44,4%. A prevalência de deficiência de ferro e ácido fólico foi de 17,8%. Não encontramos deficiência de vitamina B<SUB<12 nas gestantes anêmicas. / Abstract not available