Nutrition and bone metabolism : In vivo effects of inorganic phosphate on rats and in vitro effects of bioactive tripeptides on human osteoblasts

Nutrition affects bone health throughout life. To optimize peak bone mass development and maintenance, it is important to pay attention to the dietary factors that enhance and impair bone metabolism. In this study, the in vivo effects of inorganic dietary phosphate and the in vitro effects of bioactive tripeptides, IPP, VPP and LKP were investigated. Dietary phosphate intake is increased through the use of convenience foods and soft drinks rich in phosphate-containing food additives. Our results show that increased dietary phosphate intake hinders mineral deposition in cortical bone and diminishes bone mineral density (BMD) in the aged skeleton in a rodent model (Study I). In the growing skeleton (Study II), increased phosphate intake was observed to reduce bone material and structural properties, leading to diminished bone strength. Studies I and II revealed that a low Ca:P ratio has negative effects on the mature and growing rat skeleton even when calcium intake is sufficient. High dietary protein intake is beneficial for bone health. Protein is essential for bone turnover and matrix formation. In addition, hydrolysis of proteins in the gastrointestinal tract produces short peptides that possess a biological function beyond that of being tissue building blocks. The effects of three bioactive tripeptides, IPP, VPP and LKP, were assessed in short- and long-term in vitro experiments. Short-term treatment (24 h) with tripeptide IPP, VPP or LKP influenced osteoblast gene expression (Study III). IPP in particular, regulates genes associated with cell differentiation, cell growth and cell signal transduction. The upregulation of these genes indicates that IPP enhances osteoblast proliferation and differentiation. Long-term treatment with IPP enhanced osteoblast gene expression in favour of bone formation and increased mineralization (Study IV). The in vivo effects of IPP on osteoblast differentiation might differ since eating frequency drives food consumption, and protein degradation products, such as bioactive peptides, are available periodically, not continuously as in this study. To sum up, Studies I and II raise concern about the appropriate amount of dietary phosphate to support bone health as excess is harmful. Studies III and IV in turn, support findings of the beneficial effects of dietary protein on bone and provide a mechanistic explanation since cell proliferation and osteoblast function were improved by treatment with bioactive tripeptide IPP.

Enzyme molecular choreography : studies on soluble inorganic pyrophosphatases

Inorganic pyrophosphatases (PPases, EC 3.6.1.1) hydrolyse pyrophosphate in a reaction that provides the thermodynamic 'push' for many reactions in the cell, including DNA and protein synthesis. Soluble PPases can be classified into two families that differ completely in both sequence and structure. While Family I PPases are found in all kingdoms, family II PPases occur only in certain prokaryotes. The enzyme from baker's yeast (Saccharomyces cerevisiae) is very well characterised both kinetically and structurally, but the exact mechanism has remained elusive. The enzyme uses divalent cations as cofactors; in vivo the metal is magnesium. Two metals are permanently bound to the enzyme, while two come with the substrate. The reaction cycle involves the activation of the nucleophilic oxygen and allows different pathways for product release. In this thesis I have solved the crystal structures of wild type yeast PPase and seven active site variants in the presence of the native cofactor magnesium. These structures explain the effects of the mutations and have allowed me to describe each intermediate along the catalytic pathway with a structure. Although establishing the ʻchoreographyʼ of the heavy atoms is an important step in understanding the mechanism, hydrogen atoms are crucial for the mechanism. The most unambiguous method to determine the positions of these hydrogen atoms is neutron crystallography. In order to determine the neutron structure of yeast PPase I perdeuterated the enzyme and grew large crystals of it. Since the crystals were not stable at ambient temperature, a cooling device was developed to allow neutron data collection. In order to investigate the structural changes during the reaction in real time by time-resolved crystallography a photolysable substrate precursor is needed. I synthesised a candidate molecule and characterised its photolysis kinetics, but unfortunately it is hydrolysed by both yeast and Thermotoga maritima PPases. The mechanism of Family II PPases is subtly different from Family I. The native metal cofactor is manganese instead of magnesium, but the metal activation is more complex because the metal ions that arrive with the substrate are magnesium different from those permanently bound to the enzyme. I determined the crystal structures of wild type Bacillus subtilis PPase with the inhibitor imidodiphosphate and an inactive H98Q variant with the substrate pyrophosphate. These structures revealed a new trimetal site that activates the nucleophile. I also determined that the metal ion sites were partially occupied by manganese and iron using anomalous X- ray scattering.

Studies of new inorganic species using relativistic quantum chemistry

In the present work the methods of relativistic quantum chemistry have been applied to a number of small systems containing heavy elements, for which relativistic effects are important. First, a thorough introduction of the methods used is presented. This includes some of the general methods of computational chemistry and a special section dealing with how to include the effects of relativity in quantum chemical calculations. Second, after this introduction the results obtained are presented. Investigations on high-valent mercury compounds are presented and new ways to synthesise such compounds are proposed. The methods described were applied to certain systems containing short Pt-Tl contacts. It was possible to explain the interesting bonding situation in these compounds. One of the most common actinide compounds, uranium hexafluoride was investigated and a new picture of the bonding was presented. Furthermore the rareness of uranium-cyanide compounds was discussed. In a foray into the chemistry of gold, well known for its strong relativistic effects, investigations on different gold systems were performed. Analogies between Au$^+$ and platinum on one hand and oxygen on the other were found. New systems with multiple bonds to gold were proposed to experimentalists. One of the proposed systems was spectroscopically observed shortly afterwards. A very interesting molecule, which was theoretically predicted a few years ago is WAu$_{12}$. Some of its properties were calculated and the bonding situation was discussed. In a further study on gold compounds it was possible to explain the substitution pattern in bis[phosphane-gold(I)] thiocyanate complexes. This is of some help to experimentalists as the systems could not be crystallised and the structure was therefore unknown. Finally, computations on one of the heaviest elements in the periodic table were performed. Calculation on compounds containing element 110, darmstadtium, showed that it behaves similarly as its lighter homologue platinum. The extreme importance of relativistic effects for these systems was also shown.

Marking one-summer old whitefish with fluorescent pigment spraying method and results of whitefish stockings in the Gulf of Bothnia

Anadromous whitefish is one of the most important fish species in the Finnish coastal fisheries in the Gulf fo Bothnia. To compensate the lost reproduction due to river damming and to support the fisheries, several million one-summer old whitefish are released yearly into the Gulf of Bothnia. Since there are naturally reproducing whitefish in the Gulf as well, and the wild and stocked fish can not be separated in the catch, stocking impact can only be estimated by marking the stocked fish. Due to the small size and large number of released whitefish, the scattered fishery and large area where the whitefish migrate, most of the traditionally used fish marking methods were either unsuitable (e.g. Carlin-tags) or proved to be too expensive (e.g. coded wire tags). Fluorescent pigment spraying method offers a fast and cost-effective method to mass-mark young fish. However, the results are not always satisfactory due to low long-time retention of the marks in some species. The method has to be tested and proper marking conditions and methods determined for each species. This thesis is based on work that was accomplished while developing the fluorescent pigment spraying method for marking one-summer old whitefish fingerlings, and it draws together the results of mass-marking whitefish fingerlings that were released in the Gulf of Bothnia. Fluorescent pigment spraying method is suitable for one-summer old whitefish larger than 8 cm total length. The water temperature during the marking should not exceed 10o C. Suitable spraying pressure is 6 bars measured in the compressor outlet, and the distance of the spraying gun nozzle should be ca 20 cm from the fish. Under such conditions, the marking results in long-term retention of the mark with low or no mortality. The stress level of the fish (measured as muscle water content) rises during the marking procedure, but if the fish are allowed to recover after marking, the overall stress level remains within the limits observed in normal fish handling during the capture-loading-transport-stocking procedure. The marked whitefish fingerlings are released into the sea at larger size and later in the season than the wild whitefish. However, the stocked individuals migrate to the southern feeding grounds in a similar pattern to the wild ones. The catch produced by whitefish stocking in the Gulf of Bothnia varied between released fingerling groups, but was within the limits reported elsewhere in Finland. The releases in the southern Bothnian Bay resulted in a larger catch than those made in the northern Bothnian Bay. The size of the released fingerlings seemed to have some effect on survival of the fish during the first winter in the sea. However, when the different marking groups were compared, the mean fingerling size was not related to stocking success.

Detection of renal dysfunction during and after anaesthesia and surgery - evaluation of the influence of inorganic fluoride, ketorolac and clonidine

Drugs and surgical techniques may have harmful renal effects during the perioperative period. Traditional biomarkers are often insensitive to minor renal changes, but novel biomarkers may more accurately detect disturbances in glomerular and tubular function and integrity. The purpose of this study was first, to evaluate the renal effects of ketorolac and clonidine during inhalation anesthesia with sevoflurane and isoflurane, and secondly, to evaluate the effect of tobacco smoking on the production of inorganic fluoride (F-) following enflurane and sevoflurane anesthesia as well as to determine the effect of F- on renal function and cellular integrity in surgical patients. A total of 143 patients undergoing either conventional (n = 75) or endoscopic (n = 68) inpatient surgery were enrolled in four studies. The ketorolac and clonidine studies were prospective, randomized, placebo controlled and double-blinded, while the cigarette smoking studies were prospective cohort studies with two parallel groups. As a sign of proximal tubular deterioration, a similar transient increase in urine N-acetyl-beta-D-glucosaminidase/creatinine (U-NAG/crea) was noted in both the ketorolac group and in the controls (baseline vs. at two hours of anesthesia, p = 0.015) with a 3.3 minimum alveolar concentration hour sevoflurane anesthesia. Uncorrelated U-NAG increased above the maximum concentration measured from healthy volunteers (6.1 units/l) in 5/15 patients with ketorolac and in none of the controls (p = 0.042). As a sign of proximal tubular deterioration, U-glutathione transferase-alpha/crea (U-GST-alpha/crea) increased in both groups at two hours after anesthesia but a more significant increase was noted in the patients with ketorolac. U-GST-alpha/crea increased above the maximum ratio measured from healthy volunteers in 7/15 patients with ketorolac and in 3/15 controls. Clonidine diminished the activation of the renin-angiotensin aldosterone system during pneumoperitoneum; urine output was better preserved in the patients treated with clonidine (1/15 patients developed oliguria) than in the controls (8/15 developed oliguria (p=0.005)). Most patients with pneumoperitoneum and isoflurane anesthesia developed a transient proximal tubular deterioration, as U-NAG increased above 6.1 units/L in 11/15 patients with clonidine and in 7/15 controls. In the patients receiving clonidine treatment, the median of U-NAG/crea was higher than in the controls at 60 minutes of pneumoperitoneum (p = 0.01), suggesting that clonidine seems to worsen proximal tubular deterioration. Smoking induced the metabolism of enflurane, but the renal function remained intact in both the smokers and the non-smokers with enflurane anesthesia. On the contrary, smoking did not induce sevoflurane metabolism, but glomerular function decreased in 4/25 non-smokers and in 7/25 smokers with sevoflurane anesthesia. All five patients with S-F- ≥ 40 micromol/L, but only 6/45 with S-F- less than 40 micromol/L (p = 0.001), developed a S-tumor associated trypsin inhibitor concentration above 3 nmol/L as a sign of glomerular dysfunction. As a sign of proximal tubulus deterioration, U-beta 2-microglobulin increased in 2/5 patients with S-F- over 40 micromol/L compared to 2/45 patients with the highest S-F- less than 40 micromol/L (p = 0.005). To conclude, sevoflurane anesthesia may cause a transient proximal tubular deterioration which may be worsened by a co-administration of ketorolac. Clonidine premedication prevents the activation of the renin-angiotensin aldosterone system and preserves normal urine output, but may be harmful for proximal tubules during pneumoperitoneum. Smoking induces the metabolism of enflurane but not that of sevoflurane. Serum F- of 40 micromol/L or higher may induce glomerular dysfunction and proximal tubulus deterioration in patients with sevoflurane anesthesia. The novel renal biomarkers warrant further studies in order to establish reference values for surgical patients having inhalation anesthesia.

A metabolomic approach to studying mechanisms of polymeric gene delivery to retinal pigment epithelial cells

Tiivistelmä ReferatAbstract Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.