Impact of folate absorption and transport for nutrition and drug targeting

In this thesis, interactions of folic acid with tea and tea components at the level of intestinal absorption have been investigated. Firstly, the interaction between folic acid and tea as well as tea catechins was studied in vitro, using Caco-2 cell monolayers and secondly, a clinical trial was designed and carried out. In addition, targeting of folic acid conjugated nanoparticles to FR expressing Caco-2 cells was studied in order to evaluate the principle of nutrient-receptor-coupled transport for drug targeting. In the first part of this work, it was shown that EGCG and ECG (gallated catechins) inhibit folic acid uptake (IC50 of 34.8 and 30.8 µmol/L) comparable to MTX (methotrexate) under these experimental conditions. Moreover, commercial green and black tea extracts inhibited folic acid uptake with IC50 values of approximately 7.5 and 3.6 mg/mL, respectively. These results clearly indicate an interaction between folic acid and green tea catechins at the level of intestinal uptake. The mechanism responsible for the inhibition process might be the inhibition of the influx transport routes for folates such as via RFC and/or PCFT. For understanding the in vivo relevance of this in vitro interaction, a phase one, open-labeled, randomized, cross-over clinical study in seven healthy volunteers was designed. For the 0.4 mg folic acid dose, the mean Cmax decreased by 39.2% and 38.6% and the mean AUC0 decreased by 26.6% and 17.9% by green tea and black tea, respectively. For the 5 mg folic acid dose, the mean Cmax decreased by 27.4% and mean AUC0 decreased by 39.9% when taken with green tea. The results of the clinical study confirm the interaction between tea and folic acid in vivo leading to lower bioavailabilities of folic acid. In the second part of the thesis, targeting studies using folic acid conjugated nanoparticles were conducted. Folic acid conjugated nanoparticles were shown to be internalized by the cell via FR (folate receptor) mediated endocytosis. DNA block copolymer micelles equipped with 2, 11 and 28 folic acid units respectively were applied on FR expressing Caco-2 cells. There was a direct proportion in the amount of internalized nanoparticle and the number of folic acid units on the periphery of the nanoparticle. To sum up, throughout this thesis, the importance of folic acid for nutrition and nutrient and drug related interactions of folic acid at intestinal level was shown. Furthermore, significance of FRs in targeting for cancer chemotherapy was demonstrated in in vitro cell culture experiments. Folic acid conjugated DNA block copolymer micelles were suggested as efficient nanoparticles for targeted drug delivery.

Data management and data analysis in the large European projects GEHA (GEnetics of Healthy Aging) and NU-AGE (NUtrition and AGEing): a bioinformatic approach

The aging process is characterized by the progressive fitness decline experienced at all the levels of physiological organization, from single molecules up to the whole organism. Studies confirmed inflammaging, a chronic low-level inflammation, as a deeply intertwined partner of the aging process, which may provide the “common soil” upon which age-related diseases develop and flourish. Thus, albeit inflammation per se represents a physiological process, it can rapidly become detrimental if it goes out of control causing an excess of local and systemic inflammatory response, a striking risk factor for the elderly population. Developing interventions to counteract the establishment of this state is thus a top priority. Diet, among other factors, represents a good candidate to regulate inflammation. Building on top of this consideration, the EU project NU-AGE is now trying to assess if a Mediterranean diet, fortified for the elderly population needs, may help in modulating inflammaging. To do so, NU-AGE enrolled a total of 1250 subjects, half of which followed a 1-year long diet, and characterized them by mean of the most advanced –omics and non –omics analyses. The aim of this thesis was the development of a solid data management pipeline able to efficiently cope with the results of these assays, which are now flowing inside a centralized database, ready to be used to test the most disparate scientific hypotheses. At the same time, the work hereby described encompasses the data analysis of the GEHA project, which was focused on identifying the genetic determinants of longevity, with a particular focus on developing and applying a method for detecting epistatic interactions in human mtDNA. Eventually, in an effort to propel the adoption of NGS technologies in everyday pipeline, we developed a NGS variant calling pipeline devoted to solve all the sequencing-related issues of the mtDNA.

Synthese und Evaluierung von 68Ga-Schiff'schen Basen zur Diagnostik der koronaren Herzerkrankung

ZusammenfassungrnDie häufigsten Todesfälle weltweit sind auf Herzerkrankungen zurückzuführen. Bei der koronaren Herzkrankheit (KHK) sammeln sich über Jahre arteriosklerotische Ablagerungen in den Herzkranzgefäßen an und führen so zu einer verminderten Durchblutung und Versorgung des Herzmuskelgewebes mit Sauerstoff und Nährstoffen. Zur nuklearmedizinischen Bildgebung finden am häufigsten das SPECT-Nuklid 201Tl sowie die beiden 99mTc-Radiopharmaka Sestamibi und Tetrofosmin Anwendung. Die PET-Technik ist der SPECT-Technik in Bezug auf absolute Quantifizierung sowie Auflösung überlegen. Ziel der vorliegenden Arbeit war es, ein mögliches PET-Radiopharmakon zur Diagnostik der KHK zu entwickeln. Um eine dem 99mTc-Nuklid vergleichbare Verfügbarkeit im klinischen Alltag zu erreichen, sollte als Basis des neuen Radiopharmakons das mittels Radionuklid-Generator verfügbare 68Ga dienen. Schiff’sche Basen-Verbindungen zeigten nach Komplexierung mit 67/68Ga eine deutliche Aufnahme in die Herzmuskelzellen. Auf dieser Grundlage wurden verschiedene Schiff’sche Basen-Strukturen synthetisiert. Diese unterscheiden sich einerseits durch das Substitutionsmuster der verwendeten Aldehyde und andererseits durch das verwendete Rückgrat. Alle synthetisierten Chelatoren wurden erfolgreich mit 68Ga radioaktiv markiert und konnten anschließend aufgereinigt werden. Die Evaluierung dieser Substanzen in vitro zeigte, dass sie in unterschiedlichen Medien stabil ist. Die Lipophilie der 68Ga-Verbindungen (log D) lag zwischen 0,87±0,24 und 2,72±0,14. Die Ladung der Verbindungen wurde mittels Papierelektrophorese bei pH= 7 als kationisch bestimmt. Zusätzlich fanden in vitro-Untersuchungen zur Bestimmung der Aufnahme der Komplexe in HL-1 Herzzellen statt. Um den Einfluss des Zellmembranpotentials bzw. des Mitochondrienmembranpotentials zu untersuchen, wurde ein Teil der Zellen dafür mit Valinomycin (Ionophor, zerstört das Potential) behandelt. Mittels ex vivo-Biodistributionen wurde die Organverteilung von zwei Schiff’schen Basen (68Ga-BADED-2 und 68Ga-BAPDMEN-2) mit dem routinemäßig in der Klinik eingesetzten Derivat 99mTc-Sestamibi sowie dem 18F-Flurpiridaz in Ratten verglichen. Alle Verbindungen zeigten dabei eine deutliche Herzaufnahme von mehr als 2 % der injizierten Dosis pro Gramm Gewebe. Durch in vivo-PET-Aufnahmen wurden die Zeit-Aktivitätskurven der 68Ga-Verbindungen sowie zum Vergleich des 18F-Flurpiridaz bestimmt. Die Aufnahmen lagen im Bereich von 0,63±0,15 für 68Ga-BAPEN-3 bis 2,72±0,86 für 68Ga-BADED-8.In dem zweiten Teil der Arbeit wurden die Vorteile des hochaffinen Herztracers Flurpiridaz mit dem lipophilen, positiv-geladenen Ga-Schiff’sche Base-Chelator kombiniert. Hierzu wurde zunächst das Insektizid Flurpiridaz synthetisiert und mit dem BAPEN-Rückgrat gekoppelt. Die entstandene Verbindung wurde erstmals mit 68Ga radioaktiv markiert und muss in weiterführenden Arbeiten evaluiert werden.

Synthesis, binding and cellular uptake properties of oligodeoxynucleotides containing cationic bicyclo-thymidine residues

The synthesis and incorporation into oligodeoxynucleotides of two novel derivatives of bicyclothymidine carrying a cationic diaminopropyl or lysine unit in the C(6′)-β position is described. Compared to unmodified DNA these oligonucleotides show Tm-neutral behavior when paired against complementary DNA and are destabilizing when paired against RNA. Unaided uptake experiments of a decamer containing five lys-bcT units into HeLa and HEK293T cells showed substantial internalization with mostly cytosolic distribution which was not observed in the case of an unmodified control oligonucleotide.

The adaptive potential of a survival artist: characterization of the in vitro interactions of Toxoplasma gondii tachyzoites with di-cationic compounds in human fibroblast cell cultures

The impact of di-cationic pentamidine-analogues against Toxoplama gondii (Rh- and Me49-background) was investigated. The 72 h-growth assays showed that the arylimidamide DB750 inhibited the proliferation of tachyzoites of T. gondii Rh and T. gondii Me49 with an IC(50) of 0.11 and 0.13 muM, respectively. Pre-incubation of fibroblast monolayers with 1 muM DB750 for 12 h and subsequent culture in the absence of the drug also resulted in a pronounced inhibiton of parasite proliferation. However, upon 5-6 days of drug exposure, T. gondii tachyzoites adapted to the compound and resumed proliferation up to a concentration of 1.2 muM. Out of a set of 32 di-cationic compounds screened for in vitro activity against T. gondii, the arylimidamide DB745, exhibiting an IC(50) of 0.03 muM and favourable selective toxicity was chosen for further studies. DB745 also inhibited the proliferation of DB750-adapted T. gondii (IC(50)=0.07 muM). In contrast to DB750, DB745 also had a profound negative impact on extracellular non-adapted T. gondii tachyzoites, but not on DB750-adapted T. gondii. Adaptation of T. gondii to DB745 (up to a concentration of 0.46 muM) was much more difficult to achieve and feasible only over a period of 110 days. In cultures infected with DB750-adapted T. gondii seemingly intact parasites could occasionally be detected by TEM. This illustrates the astonishing capacity of T. gondii tachyzoites to adapt to environmental changes, at least under in vitro conditions, and suggests that DB745 could be an interesting drug candidate for further assessments in appropriate in vivo models.

Parenteral nutrition does not improve postoperative recovery from radical cystectomy: results of a prospective randomised trial

After radical cystectomy, patients are in a catabolic state because of postoperative stress response, extensive wound healing, and ileus.

ESPEN Guidelines on Enteral Nutrition: Geriatrics

Nutritional intake is often compromised in elderly, multimorbid patients. Enteral nutrition (EN) by means of oral nutritional supplements (ONS) and tube feeding (TF) offers the possibility to increase or to insure nutrient intake in case of insufficient oral food intake. The present guideline is intended to give evidence-based recommendations for the use of ONS and TF in geriatric patients. It was developed by an interdisciplinary expert group in accordance with officially accepted standards and is based on all relevant publications since 1985. The guideline was discussed and accepted in a consensus conference. EN by means of ONS is recommended for geriatric patients at nutritional risk, in case of multimorbidity and frailty, and following orthopaedic-surgical procedures. In elderly people at risk of undernutrition ONS improve nutritional status and reduce mortality. After orthopaedic-surgery ONS reduce unfavourable outcome. TF is clearly indicated in patients with neurologic dysphagia. In contrast, TF is not indicated in final disease states, including final dementia, and in order to facilitate patient care. Altogether, it is strongly recommended not to wait until severe undernutrition has developed, but to start EN therapy early, as soon as a nutritional risk becomes apparent.

[Seasonal fluctuations and influence of nutrition on macular pigment density]

PURPOSE: We have previously reported on measuring macular pigment density (MPD) with a scanning laser ophthalmoscope (HRA, Heidelberg Engineering, Heidelberg, Germany). This study war undertaken to evaluate the variation of MPD over a period of 1 year in healthy subjects. METHOD: We used autofluorescence images recorded with a HRA to evaluate MPD with a 2 degrees circle centered on the fovea. Healthy subjects were included in the study and MPD measurements were repeated every 2 months over a period of 1 year. RESULTS: We included a total of 30 healthy subjects aged 19-34 years (mean: 23+/-2 years). Mean MPD at time point 1 was 0.215+/-0.056 density units (DU), at time point 2 0.235+/-0.051 DU, at time point 3 0.218+/-0.055 DU, at time point 4 0.228+/-0.057 DU, at time point 5 0.225+/-0.053 DU, and at time point 6 0.203+/-0.050 DU. The statistical analysis revealed no significant variation of MPD over the follow-up period of 1 year. CONCLUSION: This study demonstrates that MPD shows no variation over a period of 1 year in healthy subjects.

Nutrition in clinical practice-the refeeding syndrome: illustrative cases and guidelines for prevention and treatment

The refeeding syndrome is a potentially lethal complication of refeeding in patients who are severely malnourished from whatever cause. Too rapid refeeding, particularly with carbohydrate may precipitate a number of metabolic and pathophysiological complications, which may adversely affect the cardiac, respiratory, haematological, hepatic and neuromuscular systems leading to clinical complications and even death. We aimed to review the development of the refeeding syndrome in a variety of situations and, from this and the literature, devise guidelines to prevent and treat the condition. We report seven cases illustrating different aspects of the refeeding syndrome and the measures used to treat it. The specific complications encountered, their physiological mechanisms, identification of patients at risk, and prevention and treatment are discussed. Each case developed one or more of the features of the refeeding syndrome including deficiencies and low plasma levels of potassium, phosphate, magnesium and thiamine combined with salt and water retention. These responded to specific interventions. In most cases, these abnormalities could have been anticipated and prevented. The main features of the refeeding syndrome are described with a protocol to anticipate, prevent and treat the condition in adults.

Nutrition with 'light and water'? In strict isolation for 10 days without food - a critical case study

In her book 'Living on Light', Jasmuheen tries to animate people worldwide to follow her drastic nutrition rules in order to boost their quality of life. Several deaths have been reported as a fatal consequence. A doctor of chemistry who believably claimed to have been 'living on light' for 2 years, except for the daily intake of up to 1.5 l of fluid containing no or almost no calories was interested in a scientific study on this phenomenon. PARTICIPANT AND METHODS: The 54-year-old man was subjected to a rigorous 10-day isolation study with complete absence of nutrition. During the study he obtained an unlimited amount of tea and mineral water but had no caloric intake. Parameters to monitor his metabolic and psychological state and vital parameters were measured regularly and the safety of the individual was ensured throughout the study. The subject agreed on these terms and the study was approved by the local ethics committee.