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De industrie heeft een veelheid aan supplementen ontwikkeld die specifiek voor ouderen zijn. Sommige van deze supplementen bevatten hoge doseringen van bepaalde vitaminen en mineralen. Deze doseringen blijven onder de 'maximaal aanvaardbare bovengrens' (Upper Limit), en zijn meestal niet hoger dan drie keer de aanbevolen dagelijkse hoeveelheid (ADH). Uit onderzoek van het RIVM blijkt dat deze supplementen niet schadelijk zijn voor de gezondheid. Verder zijn er in supplementen voor ouderen hoge doseringen gevonden van stoffen waarvoor geen UL bestaat: B-vitamines, vitamine C en ijzer. Daardoor kunnen voor deze stoffen geen conclusies worden getrokken over de veiligheid van de doseringen. In algemene zin is daarom voorzichtigheid geboden als deze producten langere tijd worden gebruikt. Er zijn algemene supplementen op de markt, niet specifiek gericht op ouderen, die een dosering bevatten boven de Upper Limit. Ook ouderen kunnen deze producten gebruiken. Het gaat om bepaalde preparaten die hoge gehalten bevatten van de vitaminen foliumzuur, B6, D en E, en de mineralen zink en selenium. Het is niet uitgesloten dat een te hoge inname van de vitaminen B6 en E op de lange termijn schadelijk kan zijn voor de gezondheid en het welzijn van ouderen. Te hoge doseringen van vitamine B6 kan neurologische klachten veroorzaken, het gebruik van te veel vitamine E kan bloedstollingen veroorzaken. Samenvattend, supplementen die specifiek voor ouderen ontwikkeld zijn, kunnen beschouwd worden als niet gevaarlijk. Er zijn echter een aantal producten verkrijgbaar die niet specifiek op ouderen gericht zijn maar die wel door ouderen gebruikt kunnen worden, die de Upper Limit van toxiciteit overschrijden. Dagelijks gebruik van deze producten gedurende langere perioden is mogelijk schadelijk voor de gezondheid van ouderen. Voor dit onderzoek zijn alle supplementen die ouderen kunnen gebruiken geïnventariseerd.

The aim of this thesis was to evaluate micronutrient intake and status of socioeconomic disadvantaged populations, such as from Central and Eastern European (CEE) as compared to other European populations, and low socioeconomic status (SES) groups as compared to high SES groups within European countries. We addressed the micronutrients that have been prioritized because of their relevance for nutritional health by the EC-funded EURRECA Network of Excellence. Moreover, we assessed the association between folate intake and status which can be used in the process of setting folate DRVs. Micronutrient intake and status of CEE countries versus other European countries CEE countries have recently experienced rising income inequalities over a period of economic transition. There is some evidence that these reforms have been accompanied by health inequalities. Inadequacy in micronutrient intake and status may contribute to these inequalities. Because in more affluent Western European countries wide ranges in micronutrient intake and status are observed, we studied if low micronutrient intake and status levels are prevailing in CEE. The findings from this thesis signal no differences in micronutrient intake and status between CEE populations in comparison to those of other European regions with the exception of calcium intake in adults and iodine status in children that were lower in CEE than in other European regions. Since data from Western Europe indicate that inadequacies do exist among SES strata, more insight in the nutritional situation of lower SES populations in CEE and an understanding of both its determinants and consequences is needed. It is important to mention that evidence from grey literature added to that from open access sources. Fundamental to further studying of nutritional health in CEE, is suitable data. We underline the necessity for conducting nutritional surveillances on micronutrient intake and status in CEE as we have identified significant knowledge gaps for many life-stage groups. Differences in micronutrient intake between SES groups Considering that not enough studies have addressed the relationship between SES and micronutrient intake and status in their analyses of nutritional health, we performed a systematic review on this topic and we used data from the large European EPIC cohort to address that issue. To be able to conclude on socioeconomic, i.e. educational, occupational and income, inequalities associated with intake and status of prioritized micronutrients for all life stages in Europe, substantial knowledge gaps should be filled. Currently, data are mostly available for the intake of calcium, vitamin C and iron as collected from adults in Western European countries. When either of the above mentioned SES indicators was applied to estimate relative differences in micronutrient intake and status between the lowest and the highest SES category within one study, the results often, but not consistently, indicated a lower intake and/or status in low versus high SES groups. For example, in eight out of ten studies a lower intake for calcium intake was found with relative differences ranging from -2 to -14%. Similar patterns were found for vitamin C and iron: in eleven out of twelve studies relative differences ranged from -5 to -48% for vitamin C, whereas in nine of ten studies on iron relative differences went up to -14%. Studies on intake and/or status of folate, vitamin B12, zinc, iodine, and intake of vitamin D, selenium and copper were limited. Still, when differences were observed, it appeared that lower intake in low than in high SES groups was found except for vitamin B12 and zinc for which the findings were inconsistent. Furthermore, using education as a proxy for SES, we assessed differences in micronutrient intake between educational levels using the individual-participant data on European adults and elderly from the EPIC cohort. Based on data from 10 Western European countries it appeared that intake of calcium (except in France and a distinctive ‘health-conscious’ group in the UK), folate (except in Greece), and vitamin C was lower in the lowest than in the highest education groups: relative differences ranged up to 12, 13 and 23%, respectively. The intake of iron differed marginally, whereas the variation in intake of vitamins D and B12 was inconsistent. The observed association between educational level and intake of micronutrients was the same for men and women. Furthermore, differences in micronutrient intake were found to be larger between countries than between SES groups. With respect to SES differences in micronutrient intake and status, there are significant gaps in the open source literature for many life-stage groups in Europe, but particularly in CEE countries. There is a clear need for cross-country and within country comparative research and for the monitoring of trends in dietary intake across different SES groups and European countries. Relationship between folate intake and status to add complementary evidence for deriving folate dietary reference values (DRVs) DRVs are under continuous review and periodic revision as the cumulative evidence base and body of knowledge evolve. Folate is considered a public health priority micronutrient for which re-evaluation of DRVs is needed. For this micronutrient, a systematic review of observational studies on the relationship between intake and status was done followed by meta-analysis. The intake of folate was significantly associated with markers of folate status. The results of our meta-analysis showed that an average person with a folate intake of 100 µg/day has a serum/plasma folate status concentration that is 26% higher and a red blood cell folate status that is 21% higher than a person who has a folate intake of 50 µg/day; plasma homocysteine was found to be 16% lower. The difference between natural food folate and that from supplements and fortified foods (folic acid) significantly influenced the estimated relationship between folate intake and serum/plasma status. Associations were stronger when assessed as folate from the diet than as folate from diet and supplements. Dietary assessment method did not significantly influence the association, although pooled estimates were somewhat higher when FFQs were used as compared to 24-hour recalls combined with food records. To focus on the impact of poor intakes on related health outcomes, data modelling can be conducted to produce estimates for Average Nutrient Requirements. For this analysis datasets and statistical models developed within the EURRECA NoE are available and can be used. Overall, further research would benefit from methodologically comparable data on food intake in all age ranges, especially on so far understudied CEE populations. Both intakes obtained through diet and from supplements and fortified foods should be assessed. Monitoring of trends across SES strata should be done with standardized SES measurements that would also facilitate cross-country comparative research. The findings on the level and distribution of micronutrient intake and status could be used for development of food based dietary guidelines. To make them effective in meeting populations’ micronutrient needs, they should be created accounting for the country specific dietary patterns giving consideration to the socioeconomic context.

The aim of this thesis was to evaluate micronutrient intake and status of socioeconomic disadvantaged populations, such as from Central and Eastern European (CEE) as compared to other European populations, and low socioeconomic status (SES) groups as compared to high SES groups within European countries. We addressed the micronutrients that have been prioritized because of their relevance for nutritional health by the EC-funded EURRECA Network of Excellence. Moreover, we assessed the association between folate intake and status which can be used in the process of setting folate DRVs. Micronutrient intake and status of CEE countries versus other European countries CEE countries have recently experienced rising income inequalities over a period of economic transition. There is some evidence that these reforms have been accompanied by health inequalities. Inadequacy in micronutrient intake and status may contribute to these inequalities. Because in more affluent Western European countries wide ranges in micronutrient intake and status are observed, we studied if low micronutrient intake and status levels are prevailing in CEE. The findings from this thesis signal no differences in micronutrient intake and status between CEE populations in comparison to those of other European regions with the exception of calcium intake in adults and iodine status in children that were lower in CEE than in other European regions. Since data from Western Europe indicate that inadequacies do exist among SES strata, more insight in the nutritional situation of lower SES populations in CEE and an understanding of both its determinants and consequences is needed. It is important to mention that evidence from grey literature added to that from open access sources. Fundamental to further studying of nutritional health in CEE, is suitable data. We underline the necessity for conducting nutritional surveillances on micronutrient intake and status in CEE as we have identified significant knowledge gaps for many life-stage groups. Differences in micronutrient intake between SES groups Considering that not enough studies have addressed the relationship between SES and micronutrient intake and status in their analyses of nutritional health, we performed a systematic review on this topic and we used data from the large European EPIC cohort to address that issue. To be able to conclude on socioeconomic, i.e. educational, occupational and income, inequalities associated with intake and status of prioritized micronutrients for all life stages in Europe, substantial knowledge gaps should be filled. Currently, data are mostly available for the intake of calcium, vitamin C and iron as collected from adults in Western European countries. When either of the above mentioned SES indicators was applied to estimate relative differences in micronutrient intake and status between the lowest and the highest SES category within one study, the results often, but not consistently, indicated a lower intake and/or status in low versus high SES groups. For example, in eight out of ten studies a lower intake for calcium intake was found with relative differences ranging from -2 to -14%. Similar patterns were found for vitamin C and iron: in eleven out of twelve studies relative differences ranged from -5 to -48% for vitamin C, whereas in nine of ten studies on iron relative differences went up to -14%. Studies on intake and/or status of folate, vitamin B12, zinc, iodine, and intake of vitamin D, selenium and copper were limited. Still, when differences were observed, it appeared that lower intake in low than in high SES groups was found except for vitamin B12 and zinc for which the findings were inconsistent. Furthermore, using education as a proxy for SES, we assessed differences in micronutrient intake between educational levels using the individual-participant data on European adults and elderly from the EPIC cohort. Based on data from 10 Western European countries it appeared that intake of calcium (except in France and a distinctive ‘health-conscious’ group in the UK), folate (except in Greece), and vitamin C was lower in the lowest than in the highest education groups: relative differences ranged up to 12, 13 and 23%, respectively. The intake of iron differed marginally, whereas the variation in intake of vitamins D and B12 was inconsistent. The observed association between educational level and intake of micronutrients was the same for men and women. Furthermore, differences in micronutrient intake were found to be larger between countries than between SES groups. With respect to SES differences in micronutrient intake and status, there are significant gaps in the open source literature for many life-stage groups in Europe, but particularly in CEE countries. There is a clear need for cross-country and within country comparative research and for the monitoring of trends in dietary intake across different SES groups and European countries. Relationship between folate intake and status to add complementary evidence for deriving folate dietary reference values (DRVs) DRVs are under continuous review and periodic revision as the cumulative evidence base and body of knowledge evolve. Folate is considered a public health priority micronutrient for which re-evaluation of DRVs is needed. For this micronutrient, a systematic review of observational studies on the relationship between intake and status was done followed by meta-analysis. The intake of folate was significantly associated with markers of folate status. The results of our meta-analysis showed that an average person with a folate intake of 100 µg/day has a serum/plasma folate status concentration that is 26% higher and a red blood cell folate status that is 21% higher than a person who has a folate intake of 50 µg/day; plasma homocysteine was found to be 16% lower. The difference between natural food folate and that from supplements and fortified foods (folic acid) significantly influenced the estimated relationship between folate intake and serum/plasma status. Associations were stronger when assessed as folate from the diet than as folate from diet and supplements. Dietary assessment method did not significantly influence the association, although pooled estimates were somewhat higher when FFQs were used as compared to 24-hour recalls combined with food records. To focus on the impact of poor intakes on related health outcomes, data modelling can be conducted to produce estimates for Average Nutrient Requirements. For this analysis datasets and statistical models developed within the EURRECA NoE are available and can be used. Overall, further research would benefit from methodologically comparable data on food intake in all age ranges, especially on so far understudied CEE populations. Both intakes obtained through diet and from supplements and fortified foods should be assessed. Monitoring of trends across SES strata should be done with standardized SES measurements that would also facilitate cross-country comparative research. The findings on the level and distribution of micronutrient intake and status could be used for development of food based dietary guidelines. To make them effective in meeting populations’ micronutrient needs, they should be created accounting for the country specific dietary patterns giving consideration to the socioeconomic context.

Dat vitaminen en mineralen een positieve invloed uit kunnen oefenen op specifieke lichaamsprocessen is bekend. Twee promovendi van Wageningen Universiteit richten zich op het mogelijke verband tussen vitamine B12, foliumzuur en vitamine D en verschillende leeftijdgerelateerde aandoeningen. De associatie tussen een verhoogd homocysteïnegehalte en een verhoogd risico op fracturen was aanleiding voor een groot trial onderzoek onder ouderen. Dit B-PROOF onderzoek loopt nog.

Dat vitaminen en mineralen een positieve invloed uit kunnen oefenen op specifieke lichaamsprocessen is bekend. Twee promovendi van Wageningen Universiteit richten zich op het mogelijke verband tussen vitamine B12, foliumzuur en vitamine D en verschillende leeftijdgerelateerde aandoeningen. De associatie tussen een verhoogd homocysteïnegehalte en een verhoogd risico op fracturen was aanleiding voor een groot trial onderzoek onder ouderen. Dit B-PROOF onderzoek loopt nog.

Introduction Adequate and safe micronutrient intake is important. Both insufficient and excessive intakes should be prevented as these can be associated with negative health effects. Therefore, the population intake distribution will ideally lay between insufficient and excessive intakes. For the development and evaluation of nutrition and food policy a good estimation of dietary micronutrient intake is of great importance. Aim Three challenges were addressed to improve the estimation of population micronutrient intake distributions: 1) how to estimate current habitual micronutrient intake when (detailed) data are lacking or data from different sources should be combined, 2) how to predict future intakes in order to support policy making, and 3) how to estimate a maximum safe fortification level per food item. The aim of this PhD-thesis is to further develop and apply statistical models which can cope with these challenges. Methods & Results Data from the Dutch National Food Consumption Surveys (DNFCSs) were used to develop and apply statistical models which can cope with the defined challenges. In addition, data from the Dutch food composition database (NEVO) and the Dutch dietary supplement database (NES) were used. Three main methodological improvements have been made. First, the combination of a deterministic approach with probabilistic approaches to be able to take into account uncertainty and variability were needed. This method was applied to estimate habitual iodine and salt intake distributions. From DNFCSs no detailed information was available on the discretionary use of (iodized) salt and no up to date information was available on the use of iodized salt in industrially processed foods. Estimates of the proportion of the population discretionarily using (iodized) salt and the proportion of industrially processed foods applying iodized salt were obtained from other data sources. The model accurately estimates habitual iodine and salt intake distributions when compared with studies measuring urinary iodine and sodium excretion. Additionally a framework was developed to simulate the habitual intake distribution for potential scenarios of future fortification strategies. Within this framework, deterministic and probabilistic approaches were combined when uncertainty or variability had to be taken into account. This framework was illustrated by the estimation of habitual folate-equivalent intake for different scenarios of mandatory or voluntary fortification with folic acid. Further this framework was applied to estimate the habitual iodine intake for several potential changes in the Dutch iodine policy and also for several scenarios of salt reduction strategies. A second methodological improvement was the development of a new statistical model to estimate habitual total micronutrient intake aggregated from food and dietary supplements. Within this 3-part model, habitual intake is estimated separately for a) intake from food for non-users of dietary supplements, b) intake from food for users of dietary supplements, and c) intake from dietary supplements for users only. Habitual total intake for the whole population was obtained by combination of the three separate habitual intake distributions (‘first shrink then add’). This 3-part model was illustrated by vitamin D intake for young children. With a more simple ‘first add then shrink’ approach the estimation of habitual total vitamin D intake distribution may give inconsistent results for the distribution of intake from foods and dietary supplements combined as compared to the intake from food only. In addition, this more simple approach may not be able to cope with multi modal distributions. With the newly developed model this inconsistency problem was solved and the multi-modal shape of the distribution as observed in the ‘raw’ data was preserved. Third, a model calculating the maximum safe fortification level per 100 kcal of a food was developed for the Dutch situation. By considering the tolerable upper intake level and reasonable high micronutrient intakes from food and dietary supplements, the ‘free space’ for voluntary fortification was calculated. This amount was divided over the amount of energy intake that can and may be fortified. The model was applied to derive safe maximum fortification levels for vitamin A, D, and folic acid. Based on these results the risk manager decided to legally allow voluntary fortification with vitamin D and folic acid up to a maximum level of 4.5 and 100 μg/100 kcal respectively. Conclusion The methodological improvements have resulted in higher accuracy for estimations of habitual intake distributions, which are essential for nutritional and food policy making. Furthermore, scenario analyses provide (under specific conditions) quantitative insight into proposed changes or areas such as maximum safe fortification levels. Several results and methods described are currently being used in research to assist Dutch and European food and nutrition policy making, which shows these methodologies are of immediate value to the practice of policy development and support.

Introduction Adequate and safe micronutrient intake is important. Both insufficient and excessive intakes should be prevented as these can be associated with negative health effects. Therefore, the population intake distribution will ideally lay between insufficient and excessive intakes. For the development and evaluation of nutrition and food policy a good estimation of dietary micronutrient intake is of great importance. Aim Three challenges were addressed to improve the estimation of population micronutrient intake distributions: 1) how to estimate current habitual micronutrient intake when (detailed) data are lacking or data from different sources should be combined, 2) how to predict future intakes in order to support policy making, and 3) how to estimate a maximum safe fortification level per food item. The aim of this PhD-thesis is to further develop and apply statistical models which can cope with these challenges. Methods & Results Data from the Dutch National Food Consumption Surveys (DNFCSs) were used to develop and apply statistical models which can cope with the defined challenges. In addition, data from the Dutch food composition database (NEVO) and the Dutch dietary supplement database (NES) were used. Three main methodological improvements have been made. First, the combination of a deterministic approach with probabilistic approaches to be able to take into account uncertainty and variability were needed. This method was applied to estimate habitual iodine and salt intake distributions. From DNFCSs no detailed information was available on the discretionary use of (iodized) salt and no up to date information was available on the use of iodized salt in industrially processed foods. Estimates of the proportion of the population discretionarily using (iodized) salt and the proportion of industrially processed foods applying iodized salt were obtained from other data sources. The model accurately estimates habitual iodine and salt intake distributions when compared with studies measuring urinary iodine and sodium excretion. Additionally a framework was developed to simulate the habitual intake distribution for potential scenarios of future fortification strategies. Within this framework, deterministic and probabilistic approaches were combined when uncertainty or variability had to be taken into account. This framework was illustrated by the estimation of habitual folate-equivalent intake for different scenarios of mandatory or voluntary fortification with folic acid. Further this framework was applied to estimate the habitual iodine intake for several potential changes in the Dutch iodine policy and also for several scenarios of salt reduction strategies. A second methodological improvement was the development of a new statistical model to estimate habitual total micronutrient intake aggregated from food and dietary supplements. Within this 3-part model, habitual intake is estimated separately for a) intake from food for non-users of dietary supplements, b) intake from food for users of dietary supplements, and c) intake from dietary supplements for users only. Habitual total intake for the whole population was obtained by combination of the three separate habitual intake distributions (‘first shrink then add’). This 3-part model was illustrated by vitamin D intake for young children. With a more simple ‘first add then shrink’ approach the estimation of habitual total vitamin D intake distribution may give inconsistent results for the distribution of intake from foods and dietary supplements combined as compared to the intake from food only. In addition, this more simple approach may not be able to cope with multi modal distributions. With the newly developed model this inconsistency problem was solved and the multi-modal shape of the distribution as observed in the ‘raw’ data was preserved. Third, a model calculating the maximum safe fortification level per 100 kcal of a food was developed for the Dutch situation. By considering the tolerable upper intake level and reasonable high micronutrient intakes from food and dietary supplements, the ‘free space’ for voluntary fortification was calculated. This amount was divided over the amount of energy intake that can and may be fortified. The model was applied to derive safe maximum fortification levels for vitamin A, D, and folic acid. Based on these results the risk manager decided to legally allow voluntary fortification with vitamin D and folic acid up to a maximum level of 4.5 and 100 μg/100 kcal respectively. Conclusion The methodological improvements have resulted in higher accuracy for estimations of habitual intake distributions, which are essential for nutritional and food policy making. Furthermore, scenario analyses provide (under specific conditions) quantitative insight into proposed changes or areas such as maximum safe fortification levels. Several results and methods described are currently being used in research to assist Dutch and European food and nutrition policy making, which shows these methodologies are of immediate value to the practice of policy development and support.

Flavoprotein oxidases represent an interesting class of enzymes because of their increasing interest to the fine-chemical and pharmaceutical industries. Owing to their high regio- and enantioselectivity and low costs, the use of oxidases is preferred over classical organic chemistry. A specific class of oxidases is formed by carbohydrate oxidases. Many flavin-harboring carbohydrate oxidases contain a vocalently linked FAD corfactor. Therefore, the approach used in this thesis to find carbohydrate oxidases was based on searching the genome databases for putative covalent FAD-containing proteins, thereby increasing the chance of finding an oxidase. We focused the search for novel carbohydrate oxidases on the vanillyl-alcohol oxidase family of flavoproteins. This family harbors a large number of putative covalent flavoproteins, and most known carbohydrate oxidases belong to this family.

Flavoprotein oxidases represent an interesting class of enzymes because of their increasing interest to the fine-chemical and pharmaceutical industries. Owing to their high regio- and enantioselectivity and low costs, the use of oxidases is preferred over classical organic chemistry. A specific class of oxidases is formed by carbohydrate oxidases. Many flavin-harboring carbohydrate oxidases contain a vocalently linked FAD corfactor. Therefore, the approach used in this thesis to find carbohydrate oxidases was based on searching the genome databases for putative covalent FAD-containing proteins, thereby increasing the chance of finding an oxidase. We focused the search for novel carbohydrate oxidases on the vanillyl-alcohol oxidase family of flavoproteins. This family harbors a large number of putative covalent flavoproteins, and most known carbohydrate oxidases belong to this family.

Tijdens dit onderzoek zijn een aantal nieuwe methoden ontwikkeld voor het maken van enantiomeerzuivere haloalcoholen, epoxiden en andere verbindingen. Deze stoffen vinden onder andere toepassing in het onderzoek naar en de productie van nieuwe medicijnen, kunststoffen, harsen e.d. Een van de belangrijkste speerpunten van de methoden ontwikkeld tijdens dit promotieonderzoek was het gebruik van enzymen om reacties te versnellen, eventueel in combinatie met katalysatoren op basis van metalen.

Tijdens dit onderzoek zijn een aantal nieuwe methoden ontwikkeld voor het maken van enantiomeerzuivere haloalcoholen, epoxiden en andere verbindingen. Deze stoffen vinden onder andere toepassing in het onderzoek naar en de productie van nieuwe medicijnen, kunststoffen, harsen e.d. Een van de belangrijkste speerpunten van de methoden ontwikkeld tijdens dit promotieonderzoek was het gebruik van enzymen om reacties te versnellen, eventueel in combinatie met katalysatoren op basis van metalen.

Background: A number of modifiable predictors for osteoporosis and fractures have been identified, including nutritional factors, such as vitamin D deficiency and low calcium intake. Cobalamin deficiency has been suggested to affect bone metabolism. Pernicious anaemia, which can result in cobalamin deficiency, has been identified as a risk factor for osteoporosis. Also, it is known that homocystinuria patients are of ten diagnosed with osteoporosis. Both moderate hyperhomocysteinemia and vitamin B12 deficiency are highly prevalent in old age and may play a role in diseases characteristic for old age.Objectives: 1) To show the associations of vitamin B12 and homocysteine with measures of bone health in three different populations. 2) To assess the effect of supplementation with 1000 mg crystalline cobalamin, carried either by a milk product or a capsule, on cobalamin status in mildly cobalamin deficient Dutch elderly people .Results: Data analyses in three different populations supplied the following information. Macrobiotic-fed adolescents: adolescents (9-15 y) with a low BMD had a significantly less favorable vitamin B12 status (adjusted mean, SD: 344 :t 24 pmol/L) and MMA status (adjusted mean, P5, P95: 0.31 [0.26, 0.35] f-lIDol/L) than adolescents with a normal BMD, with levels of respectively 442 (18) and 0.20 (0.16, 0.23) f-lmol/L. Free-living elderly: An increased Hcy level appeared to be a strong and independent risk factor for osteoporotic fractures in elderly men and women of the Longitudinal Aging Study Amsterdam (n=1267, mean age: 76 yrs). Relative risks (95% CI) for the highest Hcy quartile versus lowest three homocysteine quartiles were 4.6 (1.4-14.5) in men, and 1.8 (0.8-3.7) in women. Frail elderly people: Osteoporosis (defined by BMD T-score< -2.5) occurred almost five times more of ten in frail elderly women with a marginal vitamin B12 status and seven times more of ten in women with a deficient vitamin B12 status than in women with a normal status in the VFit-study. Intervention study with vitamin B12: Crystalline cobalamin added to milk is an effective alternative for cobalamin capsules in improving cobalamin status.Conclusions: We found a relevant association between vitamin B12 metabolites and bone health in various studies with different study designs and diverse populations. Since these observed associations broaden the scope for randomized clinical trials, we conducted an intervention study in which we showed that milk enriched with vitamin B12 is an as effective treatment as cobalamin capsules.

Background: A number of modifiable predictors for osteoporosis and fractures have been identified, including nutritional factors, such as vitamin D deficiency and low calcium intake. Cobalamin deficiency has been suggested to affect bone metabolism. Pernicious anaemia, which can result in cobalamin deficiency, has been identified as a risk factor for osteoporosis. Also, it is known that homocystinuria patients are of ten diagnosed with osteoporosis. Both moderate hyperhomocysteinemia and vitamin B12 deficiency are highly prevalent in old age and may play a role in diseases characteristic for old age.Objectives: 1) To show the associations of vitamin B12 and homocysteine with measures of bone health in three different populations. 2) To assess the effect of supplementation with 1000 mg crystalline cobalamin, carried either by a milk product or a capsule, on cobalamin status in mildly cobalamin deficient Dutch elderly people .Results: Data analyses in three different populations supplied the following information. Macrobiotic-fed adolescents: adolescents (9-15 y) with a low BMD had a significantly less favorable vitamin B12 status (adjusted mean, SD: 344 :t 24 pmol/L) and MMA status (adjusted mean, P5, P95: 0.31 [0.26, 0.35] f-lIDol/L) than adolescents with a normal BMD, with levels of respectively 442 (18) and 0.20 (0.16, 0.23) f-lmol/L. Free-living elderly: An increased Hcy level appeared to be a strong and independent risk factor for osteoporotic fractures in elderly men and women of the Longitudinal Aging Study Amsterdam (n=1267, mean age: 76 yrs). Relative risks (95% CI) for the highest Hcy quartile versus lowest three homocysteine quartiles were 4.6 (1.4-14.5) in men, and 1.8 (0.8-3.7) in women. Frail elderly people: Osteoporosis (defined by BMD T-score< -2.5) occurred almost five times more of ten in frail elderly women with a marginal vitamin B12 status and seven times more of ten in women with a deficient vitamin B12 status than in women with a normal status in the VFit-study. Intervention study with vitamin B12: Crystalline cobalamin added to milk is an effective alternative for cobalamin capsules in improving cobalamin status.Conclusions: We found a relevant association between vitamin B12 metabolites and bone health in various studies with different study designs and diverse populations. Since these observed associations broaden the scope for randomized clinical trials, we conducted an intervention study in which we showed that milk enriched with vitamin B12 is an as effective treatment as cobalamin capsules.

Acute respiratory infections are the most frequent of all infectious diseases. In popular speech common cold, flu (influenza), and pneumonia all denote acute respiratory infections. Elderly people show an increased risk of these infections and their complications. In The Netherlands about 2.000 elderly people die annually from influenza and influenza-related illness. Because the number of elderly people is growing rapidly worldwide, factors that could diminish the risk of theinfections,may have great public health importance.This thesis focuses on the effects of micronutrients and lifestyle on acute respiratory infections in 652 apparently healthy well-nourished elderly people of 60-95 years. Our 15-months randomized controlled trial showed that neither multivitamins-minerals in doses near the recommended dietary allowance nor 200mg of vitamin E had beneficial effects on the incidence and severity of acute respiratory infections. Incidence rate ratio (95% confidence interval) was 0.95 (0.75-1.15) in groups that received multivitamins-minerals and 1.12 (0.88-1.25) in groups that received vitamin E. Instead, we observed unfavorable effects of vitamin E on illness-severity. In the vitamin E compared to the no vitamin E group, illness-duration was 19 versus 14 days (p=0.02); no. of symptoms was six versus four (p=0.03); presence of fever was 37% versus 25% (p=0.009); and presence of activity-restriction was 52% versus 41% (p=0.02). Episodes of respiratory infection were self-assessed by means of a diary and substantiated by nurse telephone check, home-visits, and microbiology and serology testing in a subset. Infections turned out to be laboratory confirmed in 58% of the patients, whereas in only four percent of persons without symptoms of infection a pathogen was identified. Major pathogens in patients suffering from acute respiratory infection were rhinoviruses (32%), coronaviruses (17%), and influenzaviruses(7%).In addition, observational analyses were performed using the data of the trial. High plasma beta-carotene concentrations may reduce the incidence of acute respiratory infections, whereas alcohol consumption may increase the risk. Observational studies are however sensitive to bias and the influence of plasma carotenoids and lifestyle factors, i.e. alcohol consumption, smoking, and physical activity, on acute respiratory infections remain therefore subject to debate. Our main conclusion is that relatively healthy, well-nourished elderly people will not benefit from multivitamin-mineral and vitamin E supplementation in reducing acute respiratory infections.

Acute respiratory infections are the most frequent of all infectious diseases. In popular speech common cold, flu (influenza), and pneumonia all denote acute respiratory infections. Elderly people show an increased risk of these infections and their complications. In The Netherlands about 2.000 elderly people die annually from influenza and influenza-related illness. Because the number of elderly people is growing rapidly worldwide, factors that could diminish the risk of theinfections,may have great public health importance.This thesis focuses on the effects of micronutrients and lifestyle on acute respiratory infections in 652 apparently healthy well-nourished elderly people of 60-95 years. Our 15-months randomized controlled trial showed that neither multivitamins-minerals in doses near the recommended dietary allowance nor 200mg of vitamin E had beneficial effects on the incidence and severity of acute respiratory infections. Incidence rate ratio (95% confidence interval) was 0.95 (0.75-1.15) in groups that received multivitamins-minerals and 1.12 (0.88-1.25) in groups that received vitamin E. Instead, we observed unfavorable effects of vitamin E on illness-severity. In the vitamin E compared to the no vitamin E group, illness-duration was 19 versus 14 days (p=0.02); no. of symptoms was six versus four (p=0.03); presence of fever was 37% versus 25% (p=0.009); and presence of activity-restriction was 52% versus 41% (p=0.02). Episodes of respiratory infection were self-assessed by means of a diary and substantiated by nurse telephone check, home-visits, and microbiology and serology testing in a subset. Infections turned out to be laboratory confirmed in 58% of the patients, whereas in only four percent of persons without symptoms of infection a pathogen was identified. Major pathogens in patients suffering from acute respiratory infection were rhinoviruses (32%), coronaviruses (17%), and influenzaviruses(7%).In addition, observational analyses were performed using the data of the trial. High plasma beta-carotene concentrations may reduce the incidence of acute respiratory infections, whereas alcohol consumption may increase the risk. Observational studies are however sensitive to bias and the influence of plasma carotenoids and lifestyle factors, i.e. alcohol consumption, smoking, and physical activity, on acute respiratory infections remain therefore subject to debate. Our main conclusion is that relatively healthy, well-nourished elderly people will not benefit from multivitamin-mineral and vitamin E supplementation in reducing acute respiratory infections.

Dat vitaminen en mineralen een positieve invloed uit kunnen oefenen op specifieke lichaamsprocessen is bekend. Twee promovendi van Wageningen Universiteit richten zich op het mogelijke verband tussen vitamine B12, foliumzuur en vitamine D en verschillende leeftijdgerelateerde aandoeningen. De associatie tussen een verhoogd homocysteïnegehalte en een verhoogd risico op fracturen was aanleiding voor een groot trial onderzoek onder ouderen. Dit B-PROOF onderzoek loopt nog.

Vitamin A deficiency is a serious health problem in many developing countries. Improved vitamin A status can reduce morbidity and mortality by one-third in developing countries. Vitamin A occurs in food as preformed vitamin A (retinol), present in animal foods and breastmilk, and as provitamin A carotenoids - the major source of vitamin A for a large proportion of the world's population. However, the contribution of plant foods can only be substantial when not only the consumption and provitamin A content of foods but also the bioefficacy of provitamin A carotenoids in such foods is high. With respect to provitamin A carotenoids, bioefficacy is the product of the fraction of the ingested amount which is absorbed (bioavailability) and the fraction of that which is converted to retinol in the body (bioconversion). Isotopic tracer techniques can supply accurate and precise data on bioavailability, bioconversion and bioefficacy of carotenoids in humans.A new stable isotope technique - based on reaching plateau isotopic enrichment ofβ-carotene and retinol in serum during prolonged intake of multiple low doses ofβ-carotene and retinol, each specifically labeled with 10 13C atoms - has been developed. In 2 studies, for periods≤10 wk, > 100 Indonesian children (aged 7-13 y), consumed daily 2-3 doses of specifically labeled [ 13C 10 ]β-carotene (≤80μg/dose) and [ 13C 10 ]retinyl palmitate (≤90μg/dose). In the second study, half of the subjects ( n = 41) also received spinach while the others ( n = 36) received pumpkin. Either in serum or in feces, the degree of isotopic enrichment of retinol with [ 13C 5 ]retinol - derived from administered [ 13C 10 ]β-carotene - and with [ 13C 10 ]retinol, and ofβ-carotene with [ 13C 10 ]β-carotene was measured by high performance liquid chromatography with atmospheric pressure chemical ionization liquid chromatography-mass spectrometry (APCI LC-MS). These techniques have emerged as the most effective and convenient for studying the bioavailability, bioconversion and bioefficacy of provitamin A carotenoids. For interpretation of data, a mathematical model has been developed based on assumptions that can be readily justified in the light of present knowledge on carotenoid metabolism.From the studies presented, it can be concluded that 2.6μgβ-carotene in oil has the same vitamin A activity as 1μg retinol (bioefficacy ofβ-carotene in oil, 36%). The bioavailability ofβ-carotene in oil was found to be 86%. The bioavailability and bioefficacy ofβ-carotene in pumpkin ranged from 1.7 to 1.8 times those ofβ-carotene in spinach. Results obtained using data from feces confirmed results obtained using data from serum.This plateau isotopic enrichment technique can be applied for studying the effect of individual SLAMENGHI factors which affect the bioavailability and bioefficacy of carotenoids in humans (SLAMENGHI is a mnemonic for such factors). The studies presented have provided the most reliable estimates of the bioefficacy ofβ-carotene in oil available to date. The estimates are most probably applicable to children in other developing countries. Results from recent studies and from those presented in this thesis indicate that the current guidelines that the vitamin A activity of 1μ>g retinol can be supplied by 6μg (FAO/WHO) and 12μg (US Institute of Medicine) ofβ-carotene in a mixed diet overestimateβ-carotene bioefficacy about 2 to 3 times. This means that approaches other than the promotion of fruit and vegetables are required for eliminating vitamin A deficiency.

Vitamin A deficiency is a serious health problem in many developing countries. Improved vitamin A status can reduce morbidity and mortality by one-third in developing countries. Vitamin A occurs in food as preformed vitamin A (retinol), present in animal foods and breastmilk, and as provitamin A carotenoids - the major source of vitamin A for a large proportion of the world's population. However, the contribution of plant foods can only be substantial when not only the consumption and provitamin A content of foods but also the bioefficacy of provitamin A carotenoids in such foods is high. With respect to provitamin A carotenoids, bioefficacy is the product of the fraction of the ingested amount which is absorbed (bioavailability) and the fraction of that which is converted to retinol in the body (bioconversion). Isotopic tracer techniques can supply accurate and precise data on bioavailability, bioconversion and bioefficacy of carotenoids in humans.A new stable isotope technique - based on reaching plateau isotopic enrichment ofβ-carotene and retinol in serum during prolonged intake of multiple low doses ofβ-carotene and retinol, each specifically labeled with 10 13C atoms - has been developed. In 2 studies, for periods≤10 wk, > 100 Indonesian children (aged 7-13 y), consumed daily 2-3 doses of specifically labeled [ 13C 10 ]β-carotene (≤80μg/dose) and [ 13C 10 ]retinyl palmitate (≤90μg/dose). In the second study, half of the subjects ( n = 41) also received spinach while the others ( n = 36) received pumpkin. Either in serum or in feces, the degree of isotopic enrichment of retinol with [ 13C 5 ]retinol - derived from administered [ 13C 10 ]β-carotene - and with [ 13C 10 ]retinol, and ofβ-carotene with [ 13C 10 ]β-carotene was measured by high performance liquid chromatography with atmospheric pressure chemical ionization liquid chromatography-mass spectrometry (APCI LC-MS). These techniques have emerged as the most effective and convenient for studying the bioavailability, bioconversion and bioefficacy of provitamin A carotenoids. For interpretation of data, a mathematical model has been developed based on assumptions that can be readily justified in the light of present knowledge on carotenoid metabolism.From the studies presented, it can be concluded that 2.6μgβ-carotene in oil has the same vitamin A activity as 1μg retinol (bioefficacy ofβ-carotene in oil, 36%). The bioavailability ofβ-carotene in oil was found to be 86%. The bioavailability and bioefficacy ofβ-carotene in pumpkin ranged from 1.7 to 1.8 times those ofβ-carotene in spinach. Results obtained using data from feces confirmed results obtained using data from serum.This plateau isotopic enrichment technique can be applied for studying the effect of individual SLAMENGHI factors which affect the bioavailability and bioefficacy of carotenoids in humans (SLAMENGHI is a mnemonic for such factors). The studies presented have provided the most reliable estimates of the bioefficacy ofβ-carotene in oil available to date. The estimates are most probably applicable to children in other developing countries. Results from recent studies and from those presented in this thesis indicate that the current guidelines that the vitamin A activity of 1μ>g retinol can be supplied by 6μg (FAO/WHO) and 12μg (US Institute of Medicine) ofβ-carotene in a mixed diet overestimateβ-carotene bioefficacy about 2 to 3 times. This means that approaches other than the promotion of fruit and vegetables are required for eliminating vitamin A deficiency.

The experiments presented in this thesis were carried out to simulate the interrelationship between vitamin A and iron metabolism, which has been illustrated in human studies in third world countries and to elucidate underlying mechanisms.Effects of manipulation with dietary vitamin A on vitamin A and iron status were studied in rats. Recurrent effects on iron metabolism with moderate vitamin A deficiency were: iron accumulation in spleen and bone; reduction in total ironbinding capacity, a measure of transferrin and reduced total liver iron, caused by a reduction in liver weight. These effects were reversed by vitamin A supplementation for 10-12 days. Mild vitamin A deficiency produced anaemia followed by increased iron absorption. When vitamin A status further deteriorated, haemoglobin concentrations rose, due to haemoconcentration. Rats with chronic vitamin A deficiency may be less affected by haemoconcentration. But there was no anaemia in mature female rats with stable low plasma retinol levels.As to the mechanism by which vitamin A might influence iron metabolism, we have speculated that with vitamin A deficiency, blood cell synthesis is impaired, leading to increased destruction of malformed red blood cells and increased iron stores in the macrophages of bone marrow and spleen. In addition synthesis of transferrin might be impaired, possibly at the level of transferrin glycosylation. These hypotheses could not be confirmed by measuring transferrin glycosylation and indicators of red blood cell synthesis and breakdown in rats fed diets deficient in vitamin A.Alternatively, transferrin synthesis might be regulated by vitamin A at the level of gene transcription. With the use of a gene bank, candidate retinoic acid response elements for transferrin and erythropoietin were located. However, it was not possible to confirm a role for vitamin A in transferrin gene transcription by using cultured liver cells incubated with all- trans and 9- cis retinoic acid.Finally, evaluating the rat as animal model it can be deducted from these studies that for optimal standardization of experiments with young growing rats involving nutrients such as vitamin A and iron, diets fed to their dams should also be defined and controlled.

The experiments presented in this thesis were carried out to simulate the interrelationship between vitamin A and iron metabolism, which has been illustrated in human studies in third world countries and to elucidate underlying mechanisms.Effects of manipulation with dietary vitamin A on vitamin A and iron status were studied in rats. Recurrent effects on iron metabolism with moderate vitamin A deficiency were: iron accumulation in spleen and bone; reduction in total ironbinding capacity, a measure of transferrin and reduced total liver iron, caused by a reduction in liver weight. These effects were reversed by vitamin A supplementation for 10-12 days. Mild vitamin A deficiency produced anaemia followed by increased iron absorption. When vitamin A status further deteriorated, haemoglobin concentrations rose, due to haemoconcentration. Rats with chronic vitamin A deficiency may be less affected by haemoconcentration. But there was no anaemia in mature female rats with stable low plasma retinol levels.As to the mechanism by which vitamin A might influence iron metabolism, we have speculated that with vitamin A deficiency, blood cell synthesis is impaired, leading to increased destruction of malformed red blood cells and increased iron stores in the macrophages of bone marrow and spleen. In addition synthesis of transferrin might be impaired, possibly at the level of transferrin glycosylation. These hypotheses could not be confirmed by measuring transferrin glycosylation and indicators of red blood cell synthesis and breakdown in rats fed diets deficient in vitamin A.Alternatively, transferrin synthesis might be regulated by vitamin A at the level of gene transcription. With the use of a gene bank, candidate retinoic acid response elements for transferrin and erythropoietin were located. However, it was not possible to confirm a role for vitamin A in transferrin gene transcription by using cultured liver cells incubated with all- trans and 9- cis retinoic acid.Finally, evaluating the rat as animal model it can be deducted from these studies that for optimal standardization of experiments with young growing rats involving nutrients such as vitamin A and iron, diets fed to their dams should also be defined and controlled.

Micronutrient deficiencies seriously hinder mental and physical development and are still an important cause of death in developing countries. Therefore, goals have been set worldwide for the year 2000: to eliminate deficiencies of vitamin A and iodine and to reduce prevalence of iron deficiency anemia in women by onethird of 1990 levels. Food-based approaches for the control of micronutrient deficiencies, using foods naturally rich in micronutrients and/or fortified foods, are preferable, because a pharmaceutical approach can only reach a selective group and is less sustainable. This thesis describes research on the role of foods for improving vitamin A status of breastfeeding women in Indonesia.In developing countries, people derive 80-85% of vitamin A from plant sources in the form of provitamin A carotenoids, which the body converts to vitamin A. Preformed vitamin A, retinol, exists only in animal foods and accounts for 15-20% of vitamin A intake. Our questionnaire confirmed that dark-green leafy vegetables are the most important source of vitamin A. However, feeding a portion of dark-green leafy vegetables daily for 12 weeks did not improve vitamin A status, while it improved after feeding a wafer enriched with the same amount ofβ-carotene. This, as well as results from other carefully controlled studies, contradicts the assumption that vegetables rich in provitamin A carotenoids can play an important role in the control of vitamin A deficiency. Causes of poor bioavailability of vegetable carotenoids could include: complex matrix of leaves; absorption inhibitors, such as fibre which entraps carotenoids, or other carotenoids which may compete for absorption; parasitic infestation; and genetic and/or dietary factors.A very limited effect on vitamin A status should not lead to the conclusion that promoting the consumption of dark-green leafy vegetables is no longer necessary, but food approaches should be based on a variety of foods. Also, factors to calculate vitamin A activity of carotenoids should be reconsidered. In addition, we conclude that vitamin A intake questionnaires, which could be very simple, should only be used to estimate the risk of poor vitamin A status at community level. Vitamin A status and changes in status are best assessed by measuring serum retinol. The measurement of serumβ-carotene is recommended for evaluating food-based interventions, because it is very responsive.

Micronutrient deficiencies seriously hinder mental and physical development and are still an important cause of death in developing countries. Therefore, goals have been set worldwide for the year 2000: to eliminate deficiencies of vitamin A and iodine and to reduce prevalence of iron deficiency anemia in women by onethird of 1990 levels. Food-based approaches for the control of micronutrient deficiencies, using foods naturally rich in micronutrients and/or fortified foods, are preferable, because a pharmaceutical approach can only reach a selective group and is less sustainable. This thesis describes research on the role of foods for improving vitamin A status of breastfeeding women in Indonesia.In developing countries, people derive 80-85% of vitamin A from plant sources in the form of provitamin A carotenoids, which the body converts to vitamin A. Preformed vitamin A, retinol, exists only in animal foods and accounts for 15-20% of vitamin A intake. Our questionnaire confirmed that dark-green leafy vegetables are the most important source of vitamin A. However, feeding a portion of dark-green leafy vegetables daily for 12 weeks did not improve vitamin A status, while it improved after feeding a wafer enriched with the same amount ofβ-carotene. This, as well as results from other carefully controlled studies, contradicts the assumption that vegetables rich in provitamin A carotenoids can play an important role in the control of vitamin A deficiency. Causes of poor bioavailability of vegetable carotenoids could include: complex matrix of leaves; absorption inhibitors, such as fibre which entraps carotenoids, or other carotenoids which may compete for absorption; parasitic infestation; and genetic and/or dietary factors.A very limited effect on vitamin A status should not lead to the conclusion that promoting the consumption of dark-green leafy vegetables is no longer necessary, but food approaches should be based on a variety of foods. Also, factors to calculate vitamin A activity of carotenoids should be reconsidered. In addition, we conclude that vitamin A intake questionnaires, which could be very simple, should only be used to estimate the risk of poor vitamin A status at community level. Vitamin A status and changes in status are best assessed by measuring serum retinol. The measurement of serumβ-carotene is recommended for evaluating food-based interventions, because it is very responsive.

Application of antibacterial drugs plays an important role in the control of bacterial diseases in intensive fish culture. However, since the scientific basis for the use of these drugs is yet inadequate, there is a growing concern about the safety of fish medication for the consumer, the environment and the target animals. This led to the research presented in this thesis, which is focused on the influence of the antibacterial drug flumequine on the defence system of European eel (Anguilla anguilla L., 1758). Eel appeared to be an interesting model because the initial, pharmacokinetic investigations demonstrated that flumequine is very slowly eliminated from both plasma and tissues in this species. Treatment of eels with flumequine resulted in an enhanced proliferation of (probably surface immunoglobulin negative) lymphoid cells as assessed by means of an in vivo lymphocyte stimulation assay and monoclonal antibodies to eel immunoglobulin, respectively. The effect of the drug on the integral functioning of the defence system was subsequently examined by using a ,challenge with the parasitic nematode Anguillicola crassus. The resulting parasite recoveries pointed to an improved protection in flumequine-treated eels, which seemed to be related to a modulation of the cellular response.

Application of antibacterial drugs plays an important role in the control of bacterial diseases in intensive fish culture. However, since the scientific basis for the use of these drugs is yet inadequate, there is a growing concern about the safety of fish medication for the consumer, the environment and the target animals. This led to the research presented in this thesis, which is focused on the influence of the antibacterial drug flumequine on the defence system of European eel (Anguilla anguilla L., 1758). Eel appeared to be an interesting model because the initial, pharmacokinetic investigations demonstrated that flumequine is very slowly eliminated from both plasma and tissues in this species. Treatment of eels with flumequine resulted in an enhanced proliferation of (probably surface immunoglobulin negative) lymphoid cells as assessed by means of an in vivo lymphocyte stimulation assay and monoclonal antibodies to eel immunoglobulin, respectively. The effect of the drug on the integral functioning of the defence system was subsequently examined by using a ,challenge with the parasitic nematode Anguillicola crassus. The resulting parasite recoveries pointed to an improved protection in flumequine-treated eels, which seemed to be related to a modulation of the cellular response.

In order to assess the state of the art of vitamin analysis in foods, an intercomparison of methods was set up. Eighteen laboratmies in Europe specialised in vitamin analysis participated in this trial. Each Iabaratory received samples of dry foods in the farm of homogeneaus powders, milk powder, freeze-dried park muscle and haricots verts beans. The food samples presently to be certified for major nutrients, had proved to be homogeneaus with respect to these compounds.

As part of a Community Bureau of Reference (BCR) certification project on food reference materials for nutrients, homogeneity and stability of 5 candidate reference materials (RMs), whole milk powder, pork muscle, wheat and rye flourand harkots verts beans were studiecl. Homogeneity was stuclied by camparing the within-sachet ancl between-sachets variation of the nutrients of interest using the F-test

As part of a Community Bureau of Reference (BCR) certification project on food reference materials for nutrients, homogeneity and stability of 5 candidate reference materials (RMs), whole milk powder, pork muscle, wheat and rye flourand harkots verts beans were studiecl. Homogeneity was stuclied by camparing the within-sachet ancl between-sachets variation of the nutrients of interest using the F-test

In order to assess the state of the art of vitamin analysis in foods, an intercomparison of methods was set up. Eighteen laboratmies in Europe specialised in vitamin analysis participated in this trial. Each Iabaratory received samples of dry foods in the farm of homogeneaus powders, milk powder, freeze-dried park muscle and haricots verts beans. The food samples presently to be certified for major nutrients, had proved to be homogeneaus with respect to these compounds.

Belang van vitamine E voor leghennen wordt uitvoerig uiteengezet. Behoeftenormen worden weergegeven

Belang van vitamine E voor leghennen wordt uitvoerig uiteengezet. Behoeftenormen worden weergegeven

The experiments described in this thesis were undertaken to get a better understanding of the kinetics and biotransformation ofthe nitrofuran furazolidone upon oral administration to swine. Such information forms an essential prerequisite for making anappropriate assessment of the consumer hazards of edible products originating from animals treated with this compound. The experiments were conducted according to two different approaches:- kinetic studies in the target animal to determine the elimination kinetics of furazolidone and its metabolites from plasma andtissues upon oral administration of furazolidone- in vitro biotransformation studies using swine and rat liver microsomes to elucidate biotransformation routes and to identifyreactive intermediates responsible for the interaction with biological macromolecules.Part I of this thesis starts with a description of some developments in animal husbandry during the last decades. This is followed by a review of literature data on physical/chemical and antimicrobial characteristics, toxicity, elimination kinetics and biotransformation of furazolidone (chapter 1).Part II of this thesis deals with the present study. In chapter 2 a fast, sensitive method has been described for the determination of furazolidone in swine plasma, muscle, liver, kidney, fat and urine based on high-performance liquid chromatographic separation after solid-phase extraction on Extrelut R1. The sensitivity of the method was 1-2 ng/ml (g) for plasma and tissues and 25 ng/ml for urine.From the kinetic studies the conclusion can be drawn that no accumulation of furazolidone occurs in blood after oral administration of furazolidone to both piglets (chapter 3) and adult swine (chapter 4); the half life time was respectively 45 and 60 minutes. Furazolidone was rapidly and almost completely metabolized and urine proved to be the major excretion pathway of formed metabolites: 61% of the radioactive dose administered to piglets had been exreted via the urine and 18% via faeces, while in urine of adult swine only traces of total dosed furazolidone could be recovered. In tissues of piglets and adult swine no residues of furazolidone could be detected at all. However, the experiments with piglets showed that relatively high levels of radioactivity were present in all tissues studied. After a withdrawal period of 14 days the concentrations varied from 0.9-4.3 pg-equivalents per gram tissue. Up to 56% of the radioactivity detected in organs and tissues appeared to be non-extractable and was partly associated with DNA. This may either be indicative for endogenous incorporation in physiologically occurring compounds or for covalent binding of reactive intermediate metabolites of furazolidone to biological macromolecules (chapter 3).Studies with the adult swine further revealed the formation of a cyano-derivative from furazolidone, namely 3-(4-cyano-2- oxobutylidene amino)-2-oxazolidone. In studies in other animal species, it was claimed that this cyano-derivative was a major metabolite in urine. However, this compound proved to be a minor metabolite in plasma (half life time: 4 hours) and tissues of adult swine. This may be explained by species differences in biotransformation of furazolidone, by a very fast elimination of the cyano-derivative via urine or by an effective trapping of reactive intermediate metabolites of furazolidoneby biological macromolecules or agents like glutathione (chapter 4).Two major ethyl acetate extractable metabolites of furazolidone could be observed upon incubation in rat liver microsomes: the cyano-derivative referred to before and a reaction product of furazolidone with its open-chain acrylonitrile derivative, namely 2,3-dihydro-3cyanomethyl-2-hydroxy-5-nitro-lα,2-di-(2-oxo-oxazolidin-3-yl)iminomethylfuro[2,3-b]furan. Approximately 2-7% of the totally formed metabolites proved to be covalently bound to microsomal protein. This covalent binding could be inhibited by addition of glutathione, which also resulted in an almost complete shift from non-polar to watersoluble metabolites. No binding to DNA was detected when calf thymus DNA was added to a microsomal incubation mixture with furazolidone in contrast to the in vivo DNA interaction observed in the piglets. This may be explained by differences between the in vitro and in vivo situation in the availability of reactive intermediate metabolites of furazolidone for reaction with DNA (chapter 5).The above mentioned ethyl acetate extractable metabolites of furazolidone proved to be only minor conversion products of furazolidone upon incubation in swine liver microsomes, while the percentage of the protein bound metabolites formed was much higher, namely 12-22%. The site of attack on microsomal protein is probably formed by the thiol-group of cysteine as indicated by results of experiments performed with amino acids. Using mercapto-ethanol as a trapping agent for reactive intermediates, a new metabolite could be isolated upon incubation of furazolidone in swine liver microsomes. This compound was identified as a conjugation product of the open-chain acrylonitrile derivative of furazolidone with mercapto-ethanol, namely 3-(4-cyano-3-β-hydroxyethyl-mercapto2-oxobutylidene amino)-2-oxazolidone. This conjugate accounted for approximately 50% of total amount of metabolites formed. In addition, neither the above mentioned ethyl acetate extractable metabolites nor covalent binding to microsomal protein could be observed when mercaptoethanol was added to the incubation mixture, indicating that the openchain acrylonitrile derivative plays a central role in reductive biotransformation of furazolidone by swine liver microsomes (chapter 6).The open-chain acrylonitrile derivative of furazolidone binds reversibly with thiolgroup containing agents such as glutathione and mercapto-ethanol or with microsomal protein. The reversibility of the exchange reaction is dependent on pH as is demonstrated for the mercapto-ethanol conjugate. Below pH 2 this conjugate is stable; optimal exchange to microsomal protein is found between pH 7 and 10. The mercapto-ethanol conjugate gives a distinct direct positive response in the Salmonella/microsome test using tester strain TA 100. This is probably due to an interaction of the acrylonitrile moiety with DNA (chapter 7).It can be concluded that furazolidone is rapidly and almost completely metabolized upon oral administration to piglets. A major part of the formed metabolites proved to be non-extractable from the tissues. It can not be excluded that these non-extractable metabolites are the result of covalent binding of a reactive intermediate metabolite of furazolidone to biological macromolecules such as protein or DNA. From in vitro studies using swine liver microsomes evidence has been obtained showing that the open-chain acrylonitrile derivative plays a central role in the biotransformation of furazolidone.It has been shown that this reactive intermediate can bind reversibly to microsomal protein. Whether this non-enzymatic reaction also occurs in vivo is not yet known. As the possible presence of such covalently bound residues in edible tissues could have serious consequences for the acceptibility of the drug for massmedication of food producing animals, the identity of the non-extractable radioactive material in tissue of piglets should be further investigated, together with its bioavailability and toxicity. Efforts should also be made to identify the extractable radioactivity in tissues of piglets as only a small part of this fraction as so far has been identified.

The experiments described in this thesis were undertaken to get a better understanding of the kinetics and biotransformation ofthe nitrofuran furazolidone upon oral administration to swine. Such information forms an essential prerequisite for making anappropriate assessment of the consumer hazards of edible products originating from animals treated with this compound. The experiments were conducted according to two different approaches:- kinetic studies in the target animal to determine the elimination kinetics of furazolidone and its metabolites from plasma andtissues upon oral administration of furazolidone- in vitro biotransformation studies using swine and rat liver microsomes to elucidate biotransformation routes and to identifyreactive intermediates responsible for the interaction with biological macromolecules.Part I of this thesis starts with a description of some developments in animal husbandry during the last decades. This is followed by a review of literature data on physical/chemical and antimicrobial characteristics, toxicity, elimination kinetics and biotransformation of furazolidone (chapter 1).Part II of this thesis deals with the present study. In chapter 2 a fast, sensitive method has been described for the determination of furazolidone in swine plasma, muscle, liver, kidney, fat and urine based on high-performance liquid chromatographic separation after solid-phase extraction on Extrelut R1. The sensitivity of the method was 1-2 ng/ml (g) for plasma and tissues and 25 ng/ml for urine.From the kinetic studies the conclusion can be drawn that no accumulation of furazolidone occurs in blood after oral administration of furazolidone to both piglets (chapter 3) and adult swine (chapter 4); the half life time was respectively 45 and 60 minutes. Furazolidone was rapidly and almost completely metabolized and urine proved to be the major excretion pathway of formed metabolites: 61% of the radioactive dose administered to piglets had been exreted via the urine and 18% via faeces, while in urine of adult swine only traces of total dosed furazolidone could be recovered. In tissues of piglets and adult swine no residues of furazolidone could be detected at all. However, the experiments with piglets showed that relatively high levels of radioactivity were present in all tissues studied. After a withdrawal period of 14 days the concentrations varied from 0.9-4.3 pg-equivalents per gram tissue. Up to 56% of the radioactivity detected in organs and tissues appeared to be non-extractable and was partly associated with DNA. This may either be indicative for endogenous incorporation in physiologically occurring compounds or for covalent binding of reactive intermediate metabolites of furazolidone to biological macromolecules (chapter 3).Studies with the adult swine further revealed the formation of a cyano-derivative from furazolidone, namely 3-(4-cyano-2- oxobutylidene amino)-2-oxazolidone. In studies in other animal species, it was claimed that this cyano-derivative was a major metabolite in urine. However, this compound proved to be a minor metabolite in plasma (half life time: 4 hours) and tissues of adult swine. This may be explained by species differences in biotransformation of furazolidone, by a very fast elimination of the cyano-derivative via urine or by an effective trapping of reactive intermediate metabolites of furazolidoneby biological macromolecules or agents like glutathione (chapter 4).Two major ethyl acetate extractable metabolites of furazolidone could be observed upon incubation in rat liver microsomes: the cyano-derivative referred to before and a reaction product of furazolidone with its open-chain acrylonitrile derivative, namely 2,3-dihydro-3cyanomethyl-2-hydroxy-5-nitro-lα,2-di-(2-oxo-oxazolidin-3-yl)iminomethylfuro[2,3-b]furan. Approximately 2-7% of the totally formed metabolites proved to be covalently bound to microsomal protein. This covalent binding could be inhibited by addition of glutathione, which also resulted in an almost complete shift from non-polar to watersoluble metabolites. No binding to DNA was detected when calf thymus DNA was added to a microsomal incubation mixture with furazolidone in contrast to the in vivo DNA interaction observed in the piglets. This may be explained by differences between the in vitro and in vivo situation in the availability of reactive intermediate metabolites of furazolidone for reaction with DNA (chapter 5).The above mentioned ethyl acetate extractable metabolites of furazolidone proved to be only minor conversion products of furazolidone upon incubation in swine liver microsomes, while the percentage of the protein bound metabolites formed was much higher, namely 12-22%. The site of attack on microsomal protein is probably formed by the thiol-group of cysteine as indicated by results of experiments performed with amino acids. Using mercapto-ethanol as a trapping agent for reactive intermediates, a new metabolite could be isolated upon incubation of furazolidone in swine liver microsomes. This compound was identified as a conjugation product of the open-chain acrylonitrile derivative of furazolidone with mercapto-ethanol, namely 3-(4-cyano-3-β-hydroxyethyl-mercapto2-oxobutylidene amino)-2-oxazolidone. This conjugate accounted for approximately 50% of total amount of metabolites formed. In addition, neither the above mentioned ethyl acetate extractable metabolites nor covalent binding to microsomal protein could be observed when mercaptoethanol was added to the incubation mixture, indicating that the openchain acrylonitrile derivative plays a central role in reductive biotransformation of furazolidone by swine liver microsomes (chapter 6).The open-chain acrylonitrile derivative of furazolidone binds reversibly with thiolgroup containing agents such as glutathione and mercapto-ethanol or with microsomal protein. The reversibility of the exchange reaction is dependent on pH as is demonstrated for the mercapto-ethanol conjugate. Below pH 2 this conjugate is stable; optimal exchange to microsomal protein is found between pH 7 and 10. The mercapto-ethanol conjugate gives a distinct direct positive response in the Salmonella/microsome test using tester strain TA 100. This is probably due to an interaction of the acrylonitrile moiety with DNA (chapter 7).It can be concluded that furazolidone is rapidly and almost completely metabolized upon oral administration to piglets. A major part of the formed metabolites proved to be non-extractable from the tissues. It can not be excluded that these non-extractable metabolites are the result of covalent binding of a reactive intermediate metabolite of furazolidone to biological macromolecules such as protein or DNA. From in vitro studies using swine liver microsomes evidence has been obtained showing that the open-chain acrylonitrile derivative plays a central role in the biotransformation of furazolidone.It has been shown that this reactive intermediate can bind reversibly to microsomal protein. Whether this non-enzymatic reaction also occurs in vivo is not yet known. As the possible presence of such covalently bound residues in edible tissues could have serious consequences for the acceptibility of the drug for massmedication of food producing animals, the identity of the non-extractable radioactive material in tissue of piglets should be further investigated, together with its bioavailability and toxicity. Efforts should also be made to identify the extractable radioactivity in tissues of piglets as only a small part of this fraction as so far has been identified.

The application of quantitative thin-layer chromatography (TLC) involving in situ spectrodensitometric measurements with a flying-spot densitometer is described in this study for the analysis of aflatoxin B 1 , quinine hydrochloride and L(+)-ascorbic acid (vitamin C) in various food and foodstuffs.Several aspects dealing with the quantitative analysis of aflatoxin B 1 in peanuts and peanut products were investigated: isolation, thin-layer chromatographic conditions, visual estimations and fluorodensitometric: measurements. The results of this study contributed to the development of the Official method of analysis for aflatoxin B 1 in the Netherlands. A new spotting procedure for aflatoxin assay was introduced ("anti-diagonal" technique) improving the accuracy for visual B 1 estiminations for samples with low contamination levels (0-10μg/kg).An in situ fluorodensitometric procedure was developed for the quantitative determination of quinine hydrochloride in soft drinks containing quinine whereas in situ densitometric transmittance measurements were applied for the quantitative determination of L(+)-ascorbic acid (vitamin C) in buttermilk.It was generally concluded that in situ spectrodensitometric procedures were suitable for quantitative routine determinations in food control work, for specific purposes.

The occurrence and heat inactivation of native and bacterial lipases and proteinases in milk were studied.Production of these enzymes by Gram-negative psychrotrophic bacteria in milk was found to take place towards the end of exponential growth and in the stationary growth phase.Kinetics of heat inactivation in milk of milk lipoprotein lipase, alkaline milk proteinase and lipases and proteinases of some Gram-negative bacteria are given.The effects of residual lipolytic and proteolytic activity on the keeping quality of milk products were studied. In order to prevent activity of native lipases and proteinases, the milk should be heated sufficiently. The only way of preventing activity of bacterial lipases and proteinases in milk is to take measures against the growth of Gram-negative bacteria in the milk to be processed. In het onderzoek zijn het voorkomen en de hitte-inactivering bestudeerd van zowel de lipasen en proteinasen die van nature in de melk aanwezig zijn als van die welke door bacterien in de melk zijn geproduceerd. Ook wordt besproken welke gevolgen de verschillende hittebehandelingen, die in de zuivelindustrie worden toegepast, hebben voor de houdbaarheid van melk en melkprodukten

The application of quantitative thin-layer chromatography (TLC) involving in situ spectrodensitometric measurements with a flying-spot densitometer is described in this study for the analysis of aflatoxin B 1 , quinine hydrochloride and L(+)-ascorbic acid (vitamin C) in various food and foodstuffs.Several aspects dealing with the quantitative analysis of aflatoxin B 1 in peanuts and peanut products were investigated: isolation, thin-layer chromatographic conditions, visual estimations and fluorodensitometric: measurements. The results of this study contributed to the development of the Official method of analysis for aflatoxin B 1 in the Netherlands. A new spotting procedure for aflatoxin assay was introduced ("anti-diagonal" technique) improving the accuracy for visual B 1 estiminations for samples with low contamination levels (0-10μg/kg).An in situ fluorodensitometric procedure was developed for the quantitative determination of quinine hydrochloride in soft drinks containing quinine whereas in situ densitometric transmittance measurements were applied for the quantitative determination of L(+)-ascorbic acid (vitamin C) in buttermilk.It was generally concluded that in situ spectrodensitometric procedures were suitable for quantitative routine determinations in food control work, for specific purposes.

The occurrence and heat inactivation of native and bacterial lipases and proteinases in milk were studied.Production of these enzymes by Gram-negative psychrotrophic bacteria in milk was found to take place towards the end of exponential growth and in the stationary growth phase.Kinetics of heat inactivation in milk of milk lipoprotein lipase, alkaline milk proteinase and lipases and proteinases of some Gram-negative bacteria are given.The effects of residual lipolytic and proteolytic activity on the keeping quality of milk products were studied. In order to prevent activity of native lipases and proteinases, the milk should be heated sufficiently. The only way of preventing activity of bacterial lipases and proteinases in milk is to take measures against the growth of Gram-negative bacteria in the milk to be processed.