Effectiveness of Pilotage

The objective of the pilotage effectiveness study was to come up with a process descrip-tion of the pilotage procedure, to design performance indicators based on this process description, to be used by Finnpilot, and to work out a preliminary plan for the imple-mentation of the indicators within the Finnpilot organisation. The theoretical aspects of pilotage as well as the guidelines and standards used were determined through a literature review. Based on the literature review, a process flow model with the following phases was created: the planning of pilotage, the start of pilo-tage, the act of pilotage, the end of pilotage and the closing of pilotage. The model based on the literature review was tested through interviews and observation of pilotage. At the same time an e-mail survey directed at foreign pilotage organisations, which included a questionnaire concerning their standards and management systems, operations procedures, measurement tools and their attitude to the passage planning, was conducted. The main issues in the observations and interviews were the passage plan and the bridge team co-operation. The phases of the pilotage process model emerged in both the pilotage activities and the interviews whereas bridge team co-operation was relatively marginal. Most of the pilotage organisations, who responded to the query, also use some standard-based management system. All organisations who answered the survey use some sort of a pilotage process model. According to the query, the main measuring tools for pilotage are statistical information concerning pilotage and the organisations, the customer feedback surveys, and financial results. Attitudes to-wards passage planning were mostly positive among the organisations. A workshop with pilotage experts was arranged where the process model constructed on the basis of the literature review was tuned to match practical pilotage. In the workshop it was determined that certain phases and the corresponding tasks, through which pilo-tage can be described as a process, were identifiable in all pilotage. The result of the workshop was a complemented process model, which separates incoming and outgoing traffic, as well as the fairway pilotage and harbour pilotage from each other. Addition-ally indicators divided according to the data gathering method were defined. Data con-cerning safety and traffic flow is gathered in the form of customer feedback. The pilot's own perceptions of the pilotage process are gathered through self-assessment. The measurement data which is connected to the phases of the pilotage process is generated e.g. by gathering statistics of the success of the pilot dispatches, the accuracy of the pi-lotage and the incidents that occurred during the pilotage, near misses, deviations and accidents. The measurement data is collected via the PilotWeb at the closing of the pilo-tage. A separate project and a project group with pilots also participating will be established for the deployment of the performance indicators. The phases of the project are: the definition phase, the implementation phase and the deployment phase. The purpose of the definition phase is to prepare questions for ship commanders concerning the cus-tomer feedback questionnaire and also to work out the self-assessment queries and the queries concerning the process indicators.

Operointialueen huomioiminen risteilylaivan sähkötaseessa

Kiristyvät päästörajoitukset ja muuttuvat säädökset ovat muokanneet laivanrakennusalaa energiatehokkaampaan ja ympäristöystävällisempään suuntaan. Energiatehokkuutta tutkitaan yksittäisistä laiteratkaisuista laivan operointitasolle asti. Tämä työ on tehty osana STX Finland Oy:n laivojen energiatehokkuuden tutkimusta. Työn tarkoituksena oli uudistaa laivojen sähkötaseen laskennassa käytettyjä kuormitustaulukoita ja kehittää operointialueen vaikutuksia huomioiva laskentamenetelmä. Uudistuksen taustalla oli tieto operointialueiden ympäristöolosuhteiden vaikutuksista sähkönkulutukseen ja työn tarkoituksena oli mahdollistaa suunnittelun myöhäisemmässä vaiheessa tehtävä operointialuekohtainen sähkönkulutustarkastelu. Sähkötaseen laskenta haluttiin vastaamaan enemmän todellista sähkönkulutusta. Aikaisemmin käytetystä mitoitusperusteisesta laskennasta haluttiin siirtyä todellisempien kuormien arviointiin laitteiden ja muuntajien turhan ylimitoituksen poistamiseksi. Kuormitustaulukoiden toimintaa yksinkertaistettiin ja taulukoista tehtiin helpommin mukautuvia, jotta erilaisten operointitilanteiden vertailu olisi mahdollista. Perinteisen taselaskennan rinnalle kehitettiin operointialueiden vaikutuksia huomioivat laskentataulukot. Uudet taulukot huomioivat komponenttien sähkönkulutuksen prosentuaalisen muutoksen operointialueittain sekä yksilöllisesti että kulutusryhmittäin. Laskentataulukoiden yhteyteen tehtiin kuvaajat havainnollistamaan alueittaisia kulutuseroja.

Projektin suunnittelu ja johtaminen julkishallinnon organisaatiossa

Työn tavoitteena on tutkia projektisuunnittelun ja projektijohtamisen perusteiden toteutu-mista projektityöskentelyssä julkishallinnon organisaatiossa. Tutkimuksen ydin on tunnistaa projektisuunnitelman, muutoshallinnan ja projektipäällikön osaamisen sekä johtamis-käyttäytymisen merkitys projektin onnistumisen näkökulmasta. Tutkimus käsittää projektihallinnan kokonaisuutena, jota toteutetaan suunnittelun, toimin-nan ja johtamisen vuoropuhelussa. Tutkimuksessa verrataan kirjallisuudesta esitettyä pro-jektisuunnittelun ja johtamisen teoriaa Liikenneviraston projektihallinnan ohjeistukseen, mallipohjiin ja kokemukseen projektipäällikön johtamiskäyttäytymisestä. Johtopäätöksinä esitetään Liikenneviraston projektiohjeistuksen kehittämiskohteet sekä Liikenneviraston projektijohtamisen kulttuurissa hyviksi johtajaominaisuuksiksi koetut projektipäällikön ominaisuudet. Keskeisenä havaintona tunnistetaan projektityöskentelyn kehämäinen luonne ja siitä aiheutuva jatkuvan suunnittelun, arvioinnin ja johtamisen tarve.

Russian metals for arctic offshore structures

With an increasingly growing demand for natural resources, the Arctic region has become an attractive area, holding about 15% of world oil. Ice shrinkage caused by global warming encourages the development of offshore and ship-building sectors. Russia, as one of the leading oil and gas production countries is participating actively in cold resistant materials research, since half of its territory belongs to the Arctic environment, which held considerable stores of oil. Nowadays most Russian offshore platforms are located in the Sakhalin Island area, which geographically does not belong to the Arctic, but has com-parable environmental conditions. Russia recently has manufactured several offshore platforms. It became clear that further development of the Arctic off-shore structures with necessary reliability is highly depending on the materials employed. This work pursues the following objectives:  to provide a comprehensive review on Russian metals used for Arctic offshore structures on the base of standards, books, journal articles and companies reports  to overview various Arctic offshore structures and its structural characteristics  briefly discuss materials testing methods for low temperatures Master`s thesis focuses on specifications and description of Russian metals which are already in use and can be used for Arctic offshore structures. Work overviews several groups of steel, such as low carbon, low alloy, chromium containing steels, stainless steels, aluminium and nanostructured steels. Materials under discussion are grouped based on the standards, for instance the work covers shipbuilding and structural steels at the different sections. This paper provides an overview of important Russian Arctic offshore projects built for use in Russia and ordered by foreign countries. Future trends in development of the Arctic materials are discussed. Based on the information provided in this Master`s thesis it is possible to learn about Russian metals used for ships and offshore platforms operated in the Arctic region. Paper can be used as the comprehensive review of current materials, such as various steels, aluminium and nanomaterials.

Applications and Benefits of Adaptive Pulsed GMAW

In ship and offshore terminal construction, welded cross sections are thick and the number of welds very high. Consequently, there are two aspects of great importance; cost and heat input. Reduction in the welding operation time decreases the costs of the work force and avoids excessive heat, preventing distortion and other weld defects. The need to increase productivity while using a single wire in the GMAW process has led to the use of a high current and voltage to improve the melting rate. Unfortunately, this also increases the heat input. Innovative GMAW processes, mostly implemented for sheet plate sections, have shown significant reduction in heat input (Q), low distortion and increase in welding speed. The aim of this study is to investigate adaptive pulsed GMAW processes and assess relevant applications in the high power range, considering possible benefits when welding thicker sections and high yield strength steel. The study experimentally tests the usability of adaptive welding processes and evaluates their effects on weld properties, penetration and shapes of the weld bead.The study first briefly reviews adaptive GMAW to evaluate different approaches and their applications and to identify benefits in adaptive pulsed. Experiments are then performed using Synergic Pulsed GMAW, WiseFusionTM and Synergic GMAW processes to weld a T-joint in a horizontal position (PB). The air gap between the parts ranges from 0 to 2.5 mm. The base materials are structural steel grade S355MC and filler material G3Si1. The experiment investigates heat input, mechanical properties and microstructure of the welded joint. Analysis of the literature reveals that different approaches have been suggested using advanced digital power sources with accurate waveform, current, voltage, and feedback control. In addition, studies have clearly indicated the efficiency of lower energy welding processes. Interest in the high power range is growing and a number of different approaches have been suggested. The welding experiments in this study reveal a significant reduction of heat input and a weld microstructure with the presence of acicular ferrite (AF) beneficial for resistance to crack propagation. The WiseFusion bead had higher dilution, due to the weld bead shape, and low defects. Adaptive pulse GMAW processes can be a favoured choice when welding structures with many welded joints. The total heat reduction mitigates residual stresses and the bead shape allows a higher amperage limit. The stability of the arc during the process is virtually spatter free and allows an increase in welding speed.

A Test Bed Applied Mobile Robot Navigation

It is presented a test bed applied to studies on dynamics, control, and navigation of mobile robots. A cargo ship scale model was chosen, which can be radio-controlled or operated autonomously through an embedded control system. A control program, which manages on board mission execution, is implemented on a microcontroller. Navigation is based on an electronic compass, which includes automatic compensation for pitch and roll motions. Heading control loop is based on this sensor, and on a rudder positioning system. A propulsion control system is also implemented. Typical manoeuvres as the turning test and "zig-zag", were implemented and tested. They are included on a manoeuvre library, and can be accessed independently or in combined modes. The embedded system is also in charge of signal acquisition and storing during the missions. It is possible to analyse experiments on identification of ship dynamics, control, and navigation, through the data transferred to a PC by serial communication. Navigation is going to be improved by including inertial sensors on board, and a DGPS. Preliminary tests are aimed to ship identification, and manoeuvrability, using free model tests. Future steps include extending this system for developing other mobile robots as, ROVs, AUVs, and aerial vehicles.

CS discourses of co-operatives structuring the food industry

Tämä tutkimus osallistuu tuottaja-osuuskuntiin liittyviin kestävän kehityksen keskusteluihin ja tarjoaa esimerkin pohjoismaisesta ruoantuotannon toimialasta. Tämä tutkimus seuraa kvalitatiivisen tutkimuksen suuntaviivoja ja Fairclough’n kriittinen diskurssianalyysi tarjoaa metodin yritystekstien analyysille. Kieli ymmärretään poststrukturalistisesta näkökulmasta, joka luo kielellisiä identiteettejä ja rakentaa vastuullisuuskeskustelua. Tutkimuksen tavoitteena on kuvata diskurssit, jotka rakentavat vastuullisuuskeskustelua kontekstissaan. Lisäksi tavoitteena on kuvata ja ymmärtää kuinka nämä diskurssit rakentavat rooleja ja identiteettejä ympärilleen sekä kuinka yritystekstit institutionalisoituvat ja vaikuttavat kontekstiinsa. Analyysi määrittää kolme vastuullisuusdiskurssia, jotka kuvaavat yrityskansalaisuutta, liiketoimintalähtöisyyttä ja integriteettiä. Diskurssien tavoitteiden, roolien, ja identiteettien ymmärrys ja vaikutus ympäristöön auttaa laajentamaan tietämystä kestävän kehityksen moniulotteisesta luonteesta. Lisäksi tutkimus muodostaa itsessään äänen osuuskuntatutkimukselle ja kestävän kehityksen ymmärryksen laajentamiselle.

Design and testing of an armature-reaction-compensated permanent magnet synchronous generator for island operation

At present, permanent magnet synchronous generators (PMSGs) are of great interest. Since they do not have electrical excitation losses, the highly efficient, lightweight and compact PMSGs equipped with damper windings work perfectly when connected to a network. However, in island operation, the generator (or parallel generators) alone is responsible for the building up of the network and maintaining its voltage and reactive power level. Thus, in island operation, a PMSG faces very tight constraints, which are difficult to meet, because the flux produced by the permanent magnets (PMs) is constant and the voltage of the generator cannot be controlled. Traditional electrically excited synchronous generators (EESGs) can easily meet these constraints, because the field winding current is controllable. The main drawback of the conventional EESG is the relatively high excitation loss. This doctoral thesis presents a study of an alternative solution termed as a hybrid excitation synchronous generator (HESG). HESGs are a special class of electrical machines, where the total rotor current linkage is produced by the simultaneous action of two different excitation sources: the electrical and permanent magnet (PM) excitation. An overview of the existing HESGs is given. Several HESGs are introduced and compared with the conventional EESG from technical and economic points of view. In the study, the armature-reaction-compensated permanent magnet synchronous generator with alternated current linkages (ARC-PMSG with ACL) showed a better performance than the other options. Therefore, this machine type is studied in more detail. An electromagnetic design and a thermal analysis are presented. To verify the operation principle and the electromagnetic design, a down-sized prototype of 69 kVA apparent power was built. The experimental results are demonstrated and compared with the predicted ones. A prerequisite for an ARC-PMSG with ACL is an even number of pole pairs (p = 2, 4, 6, …) in the machine. Naturally, the HESG technology is not limited to even-pole-pair machines. However, the analysis of machines with p = 3, 5, 7, … becomes more complicated, especially if analytical tools are used, and is outside the scope of this thesis. The contribution of this study is to propose a solution where an ARC-PMSG replaces an EESG in electrical power generation while meeting all the requirements set for generators given for instance by ship classification societies, particularly as regards island operation. The maximum power level when applying the technology studied here is mainly limited by the economy of the machine. The larger the machine is, the smaller is the efficiency benefit. However, it seems that machines up to ten megawatts of power could benefit from the technology. However, in low-power applications, for instance in the 500 kW range, the efficiency increase can be significant.

Overview on the Cost-effectiveness of Maritime Safety Policy Instruments

Maritime safety is an issue that has gained a lot of attention in the Baltic Sea area due to the dense maritime traffic and transportation of oil in the area. Lots of effort has been paid to enhance maritime safety in the area. The risk exists that excessive legislation and other requirements mean more costs for limited benefit. In order to utilize both public and private resources efficiently, awareness is required of what kind of costs maritime safety policy instruments cause and whether the costs are in relation to benefits. The aim of this report is to present an overview of the cost-effectiveness of maritime safety policy instruments focusing on the cost aspect: what kind of costs maritime safety policy causes, to whom, what affects the cost-effectiveness and how cost-effectiveness is studied. The study is based on a literature review and on the interviews of Finnish maritime experts. The results of this study imply that cost-effectiveness is a complicated issue to evaluate. There are no uniform practices for which costs and benefits should be included in the evaluation and how they should be valued. One of the challenges is how to measure costs and benefits during the course of a longer time period. Often a lack of data erodes the reliability of evaluation. In the prevention of maritime accidents, costs typically include investments in ship structures or equipment, as well as maintenance and labor costs. Also large investments may be justifiable if they respectively provide significant improvements to maritime safety. Measures are cost-effective only if they are implemented properly. Costeffectiveness is decreased if a measure causes overlapping or repetitious work. Costeffectiveness is also decreased if the technology isn’t user-friendly or if it is soon replaced with a new technology or another new appliance. In future studies on the cost-effectiveness of maritime safety policy, it is important to acknowledge the dependency between different policy instruments and the uncertainty of the factors affecting cost-effectiveness. The costs of a single measure are rarely relatively significant and the effect of each measure on safety tends to be positive. The challenge is to rank the measures and to find the most effective combination of different policy instruments. The greatest potential offered for the analysis of cost-effectiveness of individual measures is their implementation in clearly defined risk situations, in which different measures are truly alternative to each other. Overall, maritime safety measures do not seem to be considered burdening for the shipping industry in Finland at the moment. Generally actors in the Finnish shipping industry seem to find maintaining a high safety level important and act accordingly.

Corporate Social Responsibility and Shipping Views of Baltic Sea Shipping Companies on the Benefits of Responsibility

The safety of shipping operations in the Baltic Sea is an extensively studied issue due to the density of traffic and the ecological sensitivity of the area. The focus has, however, mainly been on ship technology or on traffic control measures and the operative safety aspect of shipping is in a minor position in maritime safety studies and is lacking in terms of solutions. Self-regulatory and voluntary measures could be effective ways to improve the operational safety of shipping. Corporate social responsibility, or CSR, is one example of a voluntary measure that the shipping companies can take. CSR can enhance maritime safety and improve the shipping companies’ competitiveness. The aim of this study is to increase the knowledge of CSR in the maritime sector and study its applicability and benefits to the shipping companies. The research comprises of a theory part and a questionnaire study, which examine the significance of corporate social responsibility in shipping companies’ maritime safety and competitiveness. The aim of the questionnaire study is to find out how corporate social responsibility is implemented in the shipping companies. According to the literature review, responsible actions can produce financial and time costs, but due to these actions cost savings in the form of learning and increased efficiency can be achieved. Responsible actions can also produce concrete improvements and a reputation of responsibility that can lead to both cost savings and increase in the company’s income. CSR is recognised as having real business benefits in terms of attracting customers and high-quality employees. In shipping, CSR usually focuses on environmental issues. Environmental social responsibility in shipping is mainly motivated by the need to comply with existing and forthcoming regulation. Shipping companies engage in CSR to gain competitive advantage and to increase maritime safety. The social aspects of CSR take into account the well-being and skills of the employees, corporation and other stakeholders of the company. The questionnaire study revealed that the most common CSR measures in shipping companies are environmental measures, and that environmental concerns are considered to be the most important reason to engage in CSR. From the preliminary question about the concept of CSR it can also be seen that safety issues are commonly considered to be a part of CSR and safety gains are the second most important reason to engage in CSR. From the questionnaire, it can also be extrapolated that gaining a better reputation is one of the most important reasons to engage in CSR in the first place. For example, the main economic benefit was seen to be the increase of customer numbers as a result of a better reputation. Based on the study, it would seem that companies are starting to realise that they might gain competitive advantage and be favoured as shippers if they engage in sustainable, responsible operations and present themselves as “green”.

Modeling and Parameter Estimation of Double-Star Permanent Magnet Synchronous Machines

The power rating of wind turbines is constantly increasing; however, keeping the voltage rating at the low-voltage level results in high kilo-ampere currents. An alternative for increasing the power levels without raising the voltage level is provided by multiphase machines. Multiphase machines are used for instance in ship propulsion systems, aerospace applications, electric vehicles, and in other high-power applications including wind energy conversion systems. A machine model in an appropriate reference frame is required in order to design an efficient control for the electric drive. Modeling of multiphase machines poses a challenge because of the mutual couplings between the phases. Mutual couplings degrade the drive performance unless they are properly considered. In certain multiphase machines there is also a problem of high current harmonics, which are easily generated because of the small current path impedance of the harmonic components. However, multiphase machines provide special characteristics compared with the three-phase counterparts: Multiphase machines have a better fault tolerance, and are thus more robust. In addition, the controlled power can be divided among more inverter legs by increasing the number of phases. Moreover, the torque pulsation can be decreased and the harmonic frequency of the torque ripple increased by an appropriate multiphase configuration. By increasing the number of phases it is also possible to obtain more torque per RMS ampere for the same volume, and thus, increase the power density. In this doctoral thesis, a decoupled d–q model of double-star permanent-magnet (PM) synchronous machines is derived based on the inductance matrix diagonalization. The double-star machine is a special type of multiphase machines. Its armature consists of two three-phase winding sets, which are commonly displaced by 30 electrical degrees. In this study, the displacement angle between the sets is considered a parameter. The diagonalization of the inductance matrix results in a simplified model structure, in which the mutual couplings between the reference frames are eliminated. Moreover, the current harmonics are mapped into a reference frame, in which they can be easily controlled. The work also presents methods to determine the machine inductances by a finite-element analysis and by voltage-source inverters on-site. The derived model is validated by experimental results obtained with an example double-star interior PM (IPM) synchronous machine having the sets displaced by 30 electrical degrees. The derived transformation, and consequently, the decoupled d–q machine model, are shown to model the behavior of an actual machine with an acceptable accuracy. Thus, the proposed model is suitable to be used for the model-based control design of electric drives consisting of double-star IPM synchronous machines.

Asiakastoimialojen vaikutukset yrityksen myynnin kehitykseen

Työn tavoitteena oli tutkia asiakkaiden toimialojen muutoksen, esimerkiksi taantuman, vaikutuksia Mikpolis Oy:n puutekniikan myynnin kehitykseen aikajaksolla 2007–2014. Työssä tutustuttiin markkinoinnin ja talouden osa-alueisiin. Tutkimuksessa yrityksen asiakastiedoista muodostettiin useita arvoanalyysejä, joita tehtiin päätuoteryhmistä, tuotesegmenteistä, asiakkaista ja asiakkaiden toimialoista. Yrityksen sisäisiä tekijöitä tutkittiin SWOT -analyysillä ja havainnoimalla aiempia selvityksiä. Lisäksi haastateltiin asiakastoimialojen edustajia. Päätuoteryhmät myynnin määrän perusteella ovat NC-koneistuspalvelut, erityisesti lasikuitu- ja huonekaluteollisuuden segmenteissä, sekä puun testauspalvelut. Asiakkaat jaoteltiin A-, B- ja C-asiakkaisiin myyntimäärän suhteen. Huolestuttavan suuri osa avainasiakkaista on konkurssissa. Tavoiteltavat toimialat tulevaisuudessa ovat laivarakennus-, ajoneuvo- ja koneenrakennusteollisuus sekä koulutus ja testaustoiminta. Mikpolis Oy:llä parantamisen varaa on erityisesti markkinoinnissa ja investoinneissa. Asiakastoimialojen muutokset vaikuttavat yrityksen myyntiin, mutta yritys voi vaikuttaa asiaan sisäisillä tekijöillä, joita ovat markkinointi ja tuotetarjonta.

The effect of environmentally differentiated port fees to shipping companies’ environmental investments

The pressure has grown to develop cost-effective emission reduction strategies in the Baltic Sea. The forthcoming stringent regulations of the International Maritime Organization for reducing harmful emissions of shipping in the Baltic Sea are causing increasing expenses for the operators. A market-based attitude towards pricing of economic incentives could be seen as a new approach for a successful application for the additional emission reduction of nitrogen oxides (NOx). In this study the aim is to understand the phenomenon of environmentally differentiated port fees and its effects on shipping companies’ emission reduction investments. The goal is to examine empirically the real-life effects of the possible environmental differentiated port fee system and the effect of environmentally differentiated port fees on NOx reduction investments in the Baltic Sea. The research approach of this study is nomothetical. In this study research questions are answered by analyzing the broad database of the Baltic Sea fleet. Also the framework of theory is confirmed and plays an important role in analyzing the research problem. Existing investment costs of NOx emission reduction technology to ship owners are estimated and compared to investment costs with granted discounts added to the cash flows. The statistical analysis in this study is descriptive. The major statistic examination of this study is the calculation of the net present values of investments with different port fee scenarios. This is done to investigate if the NOx technology investments could be economically reasonable. Based on calculations it is clear that the effect of environmentally differentiated port fees is not adequate to compensate the total investment costs for NOx reduction. If the investment decision is made only with profitability considerations, sources will prefer to emission abatement as long as incomes from the given subsidy exceeds their abatement costs. Despite of the results, evidence was found that shipping companies are nevertheless willing to invest on voluntary emission abatement technology. In that case, investment decision could be made with criteria of, for example, sustainable strategy or brand image. Combined fairway and port fee system or governmental regulations and recommendation could also function as additional incentives to compensate the investment costs. Also, the results imply that the use of NPV is not necessarily the best method to evaluate environmental investments. If the calculations would be done with more environmental methods the results would probably be different.

A chromatographic method for the production of a human immunoglobulin G solution for intravenous use

Immunoglobulin G (IgG) of excellent quality for intravenous use was obtained from the cryosupernatant of human plasma by a chromatographic method based on a mixture of ion-exchange, DEAE-Sepharose FF and arginine Sepharose 4B affinity chromatography and a final purification step by Sephacryl S-300 HR gel filtration. The yield of 10 experimental batches produced was 3.5 g IgG per liter of plasma. A solvent/detergent combination of 1% Tri (n-butyl) phosphate and 1% Triton X-100 was used to inactivate lipid-coated viruses. Analysis of the final product (5% liquid IgG) based on the mean for 10 batches showed 94% monomers, 5.5% dimers and 0.5% polymers and aggregates. Anticomplementary activity was 0.3 CH50/mg IgG and prekallikrein activator levels were less than 5 IU/ml. Stability at 37ºC for 30 days in the liquid state was satisfactory. IgG was stored in flasks (2.5 g/flask) at 4 to 8ºC. All the characteristics of the product were consistent with the requirements of the 1997 Pharmacopée Européenne.

Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.