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.

Novel Organo-Noble-Gas Hydrides

Noble gases are mostly known as inert monatomic gases due to their limited reactivity with other elements. However, the first predictions of noble-gas compounds were suggested by Kossel in 1916, by von Antropoff in 1924, and by Pauling in 1930. It took many decades until the first noble-gas compound, XePtF6, was synthesized by Neil Bartlett in 1962. This was followed by gradual development of the field and many noble-gas compounds have been prepared. In 1995, a family of noble-gas hydride molecules was discovered at the University of Helsinki. These molecules have the general formula of HNgY, where H is a hydrogen atom, Ng is a noble-gas atom (Ar, Kr, or Xe), and Y is an electronegative fragment. The first molecular species made include HXeI, HXeBr, HXeCl, HKrCl and HXeH. Nowadays the total number of prepared HNgY molecules is 23 including both inorganic and organic compounds. The first and only neutral ground-state argon compound, HArF, was synthetized in 2000. Helium and neon are the only elements in the periodic table that do not form neutral, ground-state molecules. In this Thesis, experimental preparation of eight novel xenon- and krypton-containing organo-noble-gas hydrides made from acetylene (HCCH), diacetylene (HCCCCH) and cyanoacetylene (HCCCN) are presented. These novel species include the first organic krypton compound, HKrCCH, as well as the first noble-gas hydride molecule containing two Xe atoms, HXeCCXeH. Other new compounds are HXeCCH, HXeCC, HXeC4H, HKrC4H, HXeC3N, and HKrC3N. These molecules are prepared in noble-gas matrices (krypton or xenon) using ultraviolet photolysis of the precursor molecule and thermal mobilization of the photogenerated H atoms. The molecules were identified using infrared spectroscopy and ab initio calculations. The formation mechanisms of the organo-noble-gas molecules are studied and discussed in this context. The focus is to evidence experimentally the neutral formation mechanisms of HNgY molecules upon global mobility of H atoms. The formation of HXeCCXeH from another noble-gas compound (HXeCC) is demonstrated and discussed. Interactions with the surrounding matrix and molecular complexes of the HXeCCH molecule are studied. HXeCCH was prepared in argon and krypton solids in addition to a Xe matrix. The weak HXeCCH∙∙∙CO2 complex is prepared and identified. Preparation of the HXeCCH∙∙∙CO2 complex demonstrates an advanced approach to studies of HNgY complexes where the precursor complex (HCCH∙∙∙CO2) is obtained using photolysis of a larger molecule (propiolic acid).

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.

Intermolecular Interactions of Noble-Gas-Containing Species

The importance of intermolecular interactions to chemistry, physics, and biology is difficult to overestimate. Without intermolecular forces, condensed phase matter could not form. The simplest way to categorize different types of intermolecular interactions is to describe them using van der Waals and hydrogen bonded (H-bonded) interactions. In the H-bond, the intermolecular interaction appears between a positively charged hydrogen atom and electronegative fragments and it originates from strong electrostatic interactions. H-bonding is important when considering the properties of condensed phase water and in many biological systems including the structure of DNA and proteins. Vibrational spectroscopy is a useful tool for studying complexes and the solvation of molecules. Vibrational frequency shift has been used to characterize complex formation. In an H-bonded system A∙∙∙H-X (A and X are acceptor and donor species, respectively), the vibrational frequency of the H-X stretching vibration usually decreases from its value in free H-X (red-shift). This frequency shift has been used as evidence for H-bond formation and the magnitude of the shift has been used as an indicator of the H-bonding strength. In contrast to this normal behavior are the blue-shifting H-bonds, in which the H-X vibrational frequency increases upon complex formation. In the last decade, there has been active discussion regarding these blue-shifting H-bonds. Noble-gases have been considered inert due to their limited reactivity with other elements. In the early 1930 s, Pauling predicted the stable noble-gas compounds XeF6 and KrF6. It was not until three decades later Neil Bartlett synthesized the first noble-gas compound, XePtF6, in 1962. A renaissance of noble-gas chemistry began in 1995 with the discovery of noble-gas hydride molecules at the University of Helsinki. The first hydrides were HXeCl, HXeBr, HXeI, HKrCl, and HXeH. These molecules have the general formula of HNgY, where H is a hydrogen atom, Ng is a noble-gas atom (Ar, Kr, or Xe), and Y is an electronegative fragment. At present, this class of molecules comprises 23 members including both inorganic and organic compounds. The first and only argon-containing neutral chemical compound HArF was synthesized in 2000 and its properties have since been investigated in a number of studies. A helium-containing chemical compound, HHeF, was predicted computationally, but its lifetime has been predicted to be severely limited by hydrogen tunneling. Helium and neon are the only elements in the periodic table that do not form neutral, ground state molecules. A noble-gas matrix is a useful medium in which to study unstable and reactive species including ions. A solvated proton forms a centrosymmetric NgHNg+ (Ng = Ar, Kr, and Xe) structure in a noble-gas matrix and this is probably the simplest example of a solvated proton. Interestingly, the hypothetical NeHNe+ cation is isoelectronic with the water-solvated proton H5O2+ (Zundel-ion). In addition to the NgHNg+ cations, the isoelectronic YHY- (Y = halogen atom or pseudohalogen fragment) anions have been studied with the matrix-isolation technique. These species have been known to exist in alkali metal salts (YHY)-M+ (M = alkali metal e.g. K or Na) for more than 80 years. Hydrated HF forms the FHF- structure in aqueous solutions, and these ions participate in several important chemical processes. In this thesis, studies of the intermolecular interactions of HNgY molecules and centrosymmetric ions with various species are presented. The HNgY complexes show unusual spectral features, e.g. large blue-shifts of the H-Ng stretching vibration upon complexation. It is suggested that the blue-shift is a normal effect for these molecules, and that originates from the enhanced (HNg)+Y- ion-pair character upon complexation. It is also found that the HNgY molecules are energetically stabilized in the complexed form, and this effect is computationally demonstrated for the HHeF molecule. The NgHNg+ and YHY- ions also show blue-shifts in their asymmetric stretching vibration upon complexation with nitrogen. Additionally, the matrix site structure and hindered rotation (libration) of the HNgY molecules were studied. The librational motion is a much-discussed solid state phenomenon, and the HNgY molecules embedded in noble-gas matrices are good model systems to study this effect. The formation mechanisms of the HNgY molecules and the decay mechanism of NgHNg+ cations are discussed. A new electron tunneling model for the decay of NgHNg+ absorptions in noble-gas matrices is proposed. Studies of the NgHNg+∙∙∙N2 complexes support this electron tunneling mechanism.

A New Rare-Gas Compound: HXeOXeH

Rare-gas chemistry is of growing interest, and the recent advances include the "insertion" of a Xe atom into OH and water in the rare-gas hydrides HXeO and HXeOH. The insertion of Xe atoms into the H-C bonds of hydrocarbons was also demonstrated for HXeCC, HXeCCH and HXeCCXeH, the last of which was the first rare-gas hydride containing two rare-gas atoms. We describe the preparation and characterization of a new rare-gas compound, HXeOXeH. HXeOXeH was prepared in solid xenon by photolysis of a suitable precursor, for example water, and subsequent mobilization of the photoproducts. The experimental identification was carried out by FTIR spectroscopy, isotopic substitution and by use of various precursors. The photolytical and thermal stability of the new rare-gas hydride was also studied. The experimental work was supported by extensive quantum chemical calculations provided by our co-workers. HXeOXeH forms in a cryogenic xenon matrix from neutral O and H atoms in a two-step diffusion-controlled process involving HXeO as an intermediate [reactions (1) and (2)]. This formation mechanism is unique in that a rare-gas hydride is formed from another rare-gas hydride. H + Xe + O → HXeO (1) HXeO + Xe + H → HXeOXeH (2) Similarly to other rare-gas hydrides, HXeOXeH has a strongly IR-active H-Xe stretching vibration, allowing its spectral detection at 1379.3 cm-1. HXeOXeH is a very high-energy metastable species, yet thermally more stable than many other rare-gas hydrides. The calculated bending barrier of 0.57 eV, is not enough to explain the observed stability, and HXeOXeH might be affected by additional stabilization from the solid xenon environment. Chemical bonding between xenon and environmentally abundant species like water is of particular importance due to the “missing-xenon” problem. The relatively high thermal stability of HXeOXeH compared to other oxygen containing rare-gas compounds is relevant in this respect. Our work also raises the possibility of polymeric (–Xe–O)n networks, similarly to the computationally studied (XeCC)n polymers.

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.

Diffusion of Be, Co and Mn impurities in compound semiconductors and glassy carbon studied by the modified radiotracer technique

The main method of modifying properties of semiconductors is to introduce small amount of impurities inside the material. This is used to control magnetic and optical properties of materials and to realize p- and n-type semiconductors out of intrinsic material in order to manufacture fundamental components such as diodes. As diffusion can be described as random mixing of material due to thermal movement of atoms, it is essential to know the diffusion behavior of the impurities in order to manufacture working components. In modified radiotracer technique diffusion is studied using radioactive isotopes of elements as tracers. The technique is called modified as atoms are deployed inside the material by ion beam implantation. With ion implantation, a distinct distribution of impurities can be deployed inside the sample surface with good con- trol over the amount of implanted atoms. As electromagnetic radiation and other nuclear decay products emitted by radioactive materials can be easily detected, only very low amount of impurities can be used. This makes it possible to study diffusion in pure materials without essentially modifying the initial properties by doping. In this thesis a modified radiotracer technique is used to study the diffusion of beryllium in GaN, ZnO, SiGe and glassy carbon. GaN, ZnO and SiGe are of great interest to the semiconductor industry and beryllium as a small and possibly rapid dopant hasn t been studied previously using the technique. Glassy carbon has been added to demonstrate the feasibility of the technique. In addition, the diffusion of magnetic impurities, Mn and Co, has been studied in GaAs and ZnO (respectively) with spintronic applications in mind.

Disjunct eddy covariance measurements of volatile organic compound fluxes using proton transfer reaction mass spectrometry

Volatile organic compounds (VOCs) are emitted into the atmosphere from natural and anthropogenic sources, vegetation being the dominant source on a global scale. Some of these reactive compounds are deemed major contributors or inhibitors to aerosol particle formation and growth, thus making VOC measurements essential for current climate change research. This thesis discusses ecosystem scale VOC fluxes measured above a boreal Scots pine dominated forest in southern Finland. The flux measurements were performed using the micrometeorological disjunct eddy covariance (DEC) method combined with proton transfer reaction mass spectrometry (PTR-MS), which is an online technique for measuring VOC concentrations. The measurement, calibration, and calculation procedures developed in this work proved to be well suited to long-term VOC concentration and flux measurements with PTR-MS. A new averaging approach based on running averaged covariance functions improved the determination of the lag time between wind and concentration measurements, which is a common challenge in DEC when measuring fluxes near the detection limit. The ecosystem scale emissions of methanol, acetaldehyde, and acetone were substantial. These three oxygenated VOCs made up about half of the total emissions, with the rest comprised of monoterpenes. Contrary to the traditional assumption that monoterpene emissions from Scots pine originate mainly as evaporation from specialized storage pools, the DEC measurements indicated a significant contribution from de novo biosynthesis to the ecosystem scale monoterpene emissions. This thesis offers practical guidelines for long-term DEC measurements with PTR-MS. In particular, the new averaging approach to the lag time determination seems useful in the automation of DEC flux calculations. Seasonal variation in the monoterpene biosynthesis and the detailed structure of a revised hybrid algorithm, describing both de novo and pool emissions, should be determined in further studies to improve biological realism in the modelling of monoterpene emissions from Scots pine forests. The increasing number of DEC measurements of oxygenated VOCs will probably enable better estimates of the role of these compounds in plant physiology and tropospheric chemistry. Keywords: disjunct eddy covariance, lag time determination, long-term flux measurements, proton transfer reaction mass spectrometry, Scots pine forests, volatile organic compounds