Det finns många biotekniska analys- och detekteringsmetoder. Metoderna används för identifiering och kvantifiering av biomarkörer. Denna studie har analyserat detekteringsmetoder i de fall där två hjärtspecifika biomarkörer används, troponin och kreatinkinas. Studien avsåg att först identifiera tillämpningsfrekvensen av detekteringsmetoder i Sverige samt internationellt. Vidare identifieras sambandet mellan avgörande parametrar i val av detekteringsmetod. Metoden gick ut på att först bestämma den mest frekventa detekteringsmetoden i Sverige med hjälp av en enkät som skickades till olika laboratorier, sedan studerades tidigare studier publicerade på olika internationella databaser. Studierna som tillämpades var på hjärtspecifika troponin och kreatinkinas för att identifiera val av detekteringsmetod, detekteringskaraktäristika och användarvänlighetsparametrar. Studiens resultat visade att nationellt finns det tre detekteringsmetoder som är de mest använda för identifiering av kreatinkinas: masspektrometri, elektrokemisk luminescence och spektrometri. Internationellt är den dominerande metoden däremot elektrokemisk luminescence. För troponin är den dominerande metoden nationellt: elektrokemisk luminescence och flödescytometri, medan internationellt: elektrokemisk luminescence. Elektrokemisk luminescence är i många fall en stark vinnare i tillämpningen. Ytterligare iakttogs korrelationskoefficienter mellan parametern för att identifiera det starkaste respektive svagaste sambandet. Avgörande parametrar i val av elektrokemisk luminescence, visar på flera samband. Elektrokemisk luminescence och kreatinkinas tilldelas en korrelationskoefficient nära ett för parametrar som volym och känslighet och en korrelationskoefficient nära minus ett för linjärt mätområde och volym, samt kostnad och minimummängd. Medan för troponin och elektrokemisk luminescence erhålls en korrelationskoefficient nära ett för parametrar som känslighet och kostnad och en koefficient nära minus ett för kostnad och tid. / There are many biotechnological analysis- and detection methods. The methods are used for identification and quantification of biomarkers. This study has analyzed detection methods incases where two heart-specific biomarkers are used, troponin and creatine kinase. The study was intended to first identify the application frequency of detection methods in Sweden and internationally. Then identify the relationship between crucial parameters in the choice of detection method. The method consisted of first determining the most frequent detection method in Sweden with the help of a questionnaire that was sent to different laboratories, then previous studies published on various international databases were observed. The studies applied were on topics regarding cardiac-specific troponin and creatine kinase to identify choice of detection method, detection characteristics, and ease of use parameters. The results of the study showed that nationally, the detection methods most used for creatine kinase are mass spectrometry, electrochemical luminescence, and spectrometry. Internationally, however, the dominant method is electrochemical luminescence. For troponin, on a national level the dominant methods are electrochemical luminescence and flow cytometry, while internationally: electrochemical luminescence. Electrochemical luminescence is in many cases a strong winner in application. In addition, correlation coefficients are observed between the decisive parameters for a detection method, to identify the strongest and weakest relationships. Electrochemical luminescence and creatine kinase are assigned a correlation coefficient close to one for parameters such as volume and sensitivity and a correlation coefficient when minus one for measurement range and volume, as well as cost and minimum amount. While for troponin and electrochemical luminescence, a correlation coefficient close to one is obtained for parameters such as sensitivity and cost and a coefficient close to minus one for cost and time.

Cardiac troponins are crucial for the diagnosis of acute myocardial infarction. Despite known differences in their diagnostic implication, there are no recommendations for only one of the two troponins, cardiac troponin I (cTnI) and troponin T (cTnT) so far. In an everyday routine diagnostic, cTnT (Roche) as well as cTnI (Abbott) were measured in 5667 samples from 3264 patient cases. We investigated the number of identical or discrepant troponin findings. Regarding cTnI, we considered both, sex-dependent and unisex cutoffs. In particular, the number of cTnT positive and cTnI negative results was strikingly high in 14.0% of cTnT positive samples and increases to 23.8% by using sex-specific cTnI cutoffs. This group was considerably greater than the group of cTnI positive and cTnT negative results, also after elimination of patients with an eGFR < 60 mL/min/1.73 m2. Comparing the troponin cases with a dynamic increase or decrease between two measurements, we saw a balanced number of discrepant cases (between cTnT+/cTnI− and cTnT−/cTnI+), which was, however, still present. Using ROC analysis, sex-dependent cutoffs improved sensitivity and specificity of cTnI. This study shows in a large cohort that comparing the two cardiac troponins does not amount to identical analytical results. Consideration of sex-dependent cutoffs may improve sensitivity and specificity.

Hypertrophic cardiomyopathy (HCM) is a primary disease of the myocardium. 4-11% of HCM is caused by mutations in cardiac troponin T (cTnT) and 65% of them are within the tropomyosin (TM)-binding TNT1 domain. Two of the known mutational hotspots within TNT1 are in the N and C-terminal domains. Unlike the N-terminal domain; no high-resolution structure exists for the highly conserved C-terminal domain limiting both our ability to understand the functional role of this extended domain in myofilament activation and molecular mechanism(s) of HCM. The Δ160E mutation is an in-frame deletion of a glutamic acid residue at position 160 of cTnT. This TNT1 C-terminal mutation is associated with an especially poor prognosis. The Δ160E mutation is located in a putative "hinge region" immediately adjacent to the unstructured flexible linker connecting the TM-binding TNT1 domain to the Ca²⁺-sensitive TNT2 domain. Unwinding of this α-helical hinge may provide the flexibility necessary for thin filament function. Previous regulated in vitro motility assay (R-IVM) data showed mutation-induced impairment of weak actomyosin binding. Thus, we hypothesized that the Δ160E mutation repositions the flexible linker which impairs weak electrostatic binding and ultimately leads to severe cardiac remodeling. The goal of our studies is two-fold: 1) to gain high-resolution insight into the position of the cTnT linker with respect to the C-terminus of TM, and 2) to identify Δ160E-induced positional changes using Fluorescence Resonance Energy Transfer (FRET) in a fully reconstituted thin filament. To this end, residues in the middle and distal regions of the cTnT linker were sequentially cysteine-substituted (A168C, A177C, A192C and S198C) and labeled with the energy donor IAEDANS. The energy acceptor, DABMI was attached to cysteine 190 (C190) in the C-terminal region of TM and FRET measurements were obtained in the presence and absence of Ca²⁺ and myosin subfragment 1 (S1). An all-atom thin filament model in the Ca²⁺–on state was employed to predict the pathogenic effects of the Δ160E mutation on the structure and the dynamics of the cTnT linker region. Our data suggest that the linker domain runs alongside the C-terminus of TM and is differentially repositioned by calcium, myosin and the Δ160E mutation. The Δ160E mutation moves the linker closer to the C-terminus of TM. The in silico model supported this finding and demonstrated a mutation-induced decrease in linker flexibility. Moreover, the model predicted a pathogenic change in the orientation of the middle region of the linker and in the position of the Ca²⁺-sensitive TNT2 domain and the TM-binding TNT1 domain in response to Δ160E mutation. Collectively, our findings suggest that the Δ160E mutation-induced changes in the structure, position and dynamics of the linker region cause steric blocking of weak myosin binding sites on actin and subsequent impairment of contraction and disruption of sarcomeric integrity. These studies, for the first time, provided information regarding the role of the extended linker in both myofilament activation and disease.

A novel luminescence resonance energy transfer (LRET) nanocircuit assay involving a donor and two acceptors in tandem was developed to study the dynamic interaction of skeletal muscle contraction proteins. The donor transmits energy relayed to the acceptors distinguishing myosin subfragment-1 (S1) lever arm orientations. The last acceptor allows the detection of S1's bound near or in between troponin complexes on the thin filament. Additionally, calcium related changes between troponin T and myosin were detected. Based on this data, the troponin complex situated every 7 actin monomers, hinders adjacently bound myosins to complete their power stroke; whereas myosins bound in between troponin complexes undergo complete power strokes.

In dieser Arbeit wurden drei verschiedene Gruppen von humanen Myokardproben aus dem interventrikulären Septum mittels elektrophoretischer Verfahren auf Veränderungen in der Zusammensetzung der kontraktilen Proteine untersucht. 6 der insgesamt 38 Proben stammten von gesunden Herzen, die aus technischen Gründen nicht transplantiert werden konnten. 19 der Proben stammten von Patienten, die an einer hypertrophischen-obstruktiven Kardiomyopathie (HOCM) litten und die restlichen 13 Proben von Patienten mit einer valvulären Aortenstenose (AS). Die 32 kranken Herzen befanden sich allesamt im Stadium der kompensierten Hypertrophie, an klinischen Daten waren von diesen Patienten die Ejektionsfraktion (EF), der Durchmesser des interventrikulären Septums (IVS) sowie die linksventrikuläre enddiastolische Füllungsdruck (LVEDP). Die Ejektionsfraktion lag bei allen diesen Patienten mit Werten zwischen 62% und 88% (Mittelwert 73 ± 7%) im Normbereich, zwischen der HOCM- und der Aortenstenosegruppe bestand kein signifikanter Unterschied. Die insgesamt 38 Gewebeproben wurden mittels 3 verschiedener elektrophoretischer Verfahren auf das Vorliegen von 3 verschiedener Veränderungen in der Proteinzusammensetzung untersucht: 1. Mittels 2-dimensionaler Polyacrylamidgel-Elektrophorese (2D-PAGE) wurde der Phosphorylierungsgrad des kardialen Troponin I (cTnI) bestimmt. 2. Mittels 2-dimensionaler Polyacrylamidgel-Elektrophorese (2D-PAGE) wurde eine Analyse der leichten Myosinketten (MLC) durchgeführt, vor allem im Hinblick auf die Frage, ob und inwieweit es zu einer Expression der atrialen leichten Kette vom Typ I (ALC-1) kommt . 3. Mittels Natriumdodecylsulfat-Polyacrylamidgel-Elektrophorese (SDS-PAGE) wurde eine Bestimmung der schweren Myosinketten (MHC) vorgenommen, vor allem im Hinblick auf die Frage, ob es im hypertrophierten Myokard zu einer Expression der &#945;-Isoform der schweren Myosinkette (&#945;-MHC) kommt. Für alle dieser drei oben genannten Veränderungen finden sich Hinweise in der Literatur, dass sie möglicherweise eine Rolle bei der Myokardhypertrophie spielen könnten ohne dass bislang eine abschließende Klärung möglich war. In dieser Arbeit wurde zum ersten Mal ein derartig großes, klinisch gut evaluiertes Probenkollektiv von menschlichen Herzen im Stadium der kompensierten Hypertrophie auf das Vorliegen der o.g. Veränderungen untersucht. Ein weiterer wichtiger Aspekt ist das Vorliegen von zwei verschiedenen Ursachen (Aortenstenose und hypertrophisch-obstruktive Kardiomyopathie) für die Herzhypertrophie im Probenkollektiv dieser Arbeit. In der Zusammensetzung der schweren Myosinketten (MHC) sowie im Phosphorylierungsgrad des kardialen Troponin I (cTnI) konnten in dieser Arbeit keine signifikanten Unterschiede zwischen dem hypertrophiertem und dem gesunden Myokard gefunden werden. Im Bereich der leichten Myosinketten (MLC) konnte jedoch nachgewiesen werden, dass es in den hypertrophierten Herzen zu einer deutlichen, signifikanten Expression der atrialen leichten Myosinkette (ALC-1) in der Größenordnung von 10,8 ± 1,5 % an der Gesamtmenge der leichten Myosinketten vom Typ 1 (MLC-1) gekommen war. Im Gegensatz hierzu konnte die atriale leichte Kette vom Typ 1 (ALC-1) in keinem der gesunden Herzen nachgewiesen werden. Zudem konnte eine statistische hochsignifikante positive Korrelation (Koeffizient 0,56 nach Pearson) zwischen der Höhe der Ejektionsfraktion und dem Anteil der ALC-1 an der Gesamtmenge der leichten Myosinketten ermittelt werden. Diese Ergebnisse legen nahe, dass der Expression der ALC-1 ein hoher Stellenwert bei der Anpassung an erhöhte hämodynamische Anforderungen zukommt. Die positive Korrelation zwischen der Höhe der ALC-1-Expression und der Ejektionsfraktion weisen daraufhin, dass der ALC-1-Expression zumindest im Rahmen der kompensierten Hypertrophie ein positiver Effekt auf das Myokard zukommt. Dieser Effekt lässt sich anhand von früheren Veröffentlichungen erklären, die z.B. zeigten, dass die ALC-1 über eine Erhöhung der Ablösungsgeschwindigkeit zu einer Beschleunigung des Querbrückenzyklus und zu einer Erhöhung der Verkürzungsgeschwindigkeit und der isometrischen Kraftentwicklung führt. / To assess changes in the composition of contractile proteins we examined human myocardial samples from three different groups by means of electrophoretic analysis. 6 of 38 samples in total have been taken from healthy hearts which could not be transplanted due to technical reasons. 19 of the samples are from patients suffering from hypertophic obstructive cardiomyopathy and the remaining 13 samples from patients with a valvular aortic stenosis. The 32 impaired hearts have all been in the stadium of compensated hypertrophy, the ejection fraction, the diameter of the interventricular septum and the leftventricular enddiastolic pressure were kown. In all patients the ejection fraction was between 62% and 88% (73 ± 7% in the mean), thus in the normal range, there was no significant difference between the hypertrophic obstructive group and the valvular aortic stenosis group. All of the 38 samples have been examined by means of 3 different electrophoretical procedures. 1. 2-dimensional polyacrylamide gelelectrophoresis (2D-PAGE) for assessing the level of phosphorylation of the cardial troponin I(cTnI) 2. 2-dimensional polyacrylamidegelelectrophoresis (2D-PAGE) for analysing the composition of the myosin light chains (MLC)to answer the question whether there is an reexpression of the atrial light chain 1 (ALC-1) and if, to which extent. 3. Sodiumdodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE) to assess the composition of the myosin heavy chains to answer the question whether there is an expression of the &#945;-Isoform of the myosin heavy chain (&#945;-MHC)in the hypertrophic myocardium. There are hints in literature that all 3 changes mentioned above could play a role in myocardial hypertrophy but it has not been possible to definitely clarify the role. We have analysed for the first time a great number of clinically well evaluated samples from hypertrophic human hearts in the stadium of compensated hypertrophy regarding those changes. Another important aspect of our work is that in our samples the cause of the hypertrophy have been two pathogenetically different diseases (valvular aortic stenosis and hypertrophic obstructive cardiomyopathy). We could not find any significant differences between the hypertrophic and the nonhypertrophic hearts regarding the level of phosphorylation of the cardial troponin I (cTnI). We proved that in the hypertrophic heart there is a significant expression of the atrial light chain 1 (ALC-1) of about 10,8 ± 1,5 % of the total amount of myosin light chain 1 (MLC-1). In contrast to that there was no atrial light chain 1 (ALC-1) found in the nonhypertrophic hearts. Statistically there is a highly significant correlation (coefficient 0,56 after Pearson) between the level of the ejection fraction and the amount of the atrial light chain 1 (ALC-1) compared to the myosin light chain 1 (MLC-1) in total. These results suggest a highly important role of the ALC-1 expression in the adjustment of the heart to increased hemodynamic demands. The positive correlation between the level of the ALC-1 expression and the level of the ejection fraction suggests a positive effect of the ALC-1 expression on the myocardium during compensated hypertrophy. This effect can be explained by former publications which have shown that the expression of the ALC-1 can lead to an increased speed of displacement hence to an increased shortening velocity and an increased isometric force generation.

O diagnóstico da tromboembolia pulmonar aguda (EPA) e a avaliação da gravidade desta condição é desafiador. Enquanto as concentrações de troponina I cardíaca (TI) já estão bem estabelecidas quanto ao risco de estratificação, não há estudos prévios que tenham examinado se há alguma relação linear entre as concentrações de TI cardíaca e a gravidade da EPA. Além disso, as metaloproteinases (MMPs) da matriz extracelular estão envolvidas na fisiopatologia da EPA. Entretanto, é desconhecido se o aumento da atividade gelatinolítica das MMPs após a EPA reflete a gravidade desta condição. Nós examinamos se as concentrações circulantes destes biomarcadores aumentam em proporção à gravidade da EPA experimental induzida em cães anestesiados. A EPA foi induzida com coágulos de sangue autólogo (salina, 1, 3 ou 5 mL/Kg) injetados no átrio direito. As avaliações hemodinâmicas foram realizadas no momento basal e 120 minutos após a EPA. Da mesma forma, foram realizadas as quantificações de troponina I no soro e a zimografia das MMPs 2 e 9 no plasma. Nossos resultados sugerem não haver aumento significativo da atividade gelatinolítica da pró-MMP-2 no plasma após a EPA, enquanto que a atividade da pró-MMP-9 aumenta em 80% apenas no grupo que recebeu 5 mL/Kg de coágulos. A TI cardíaca no soro e a atividade da pró-MMP-9 no plasma tiveram uma correlação positiva com o índice de resistência vascular pulmonar (p=0,007 e rs=0,833 para a TI, e p=0,034 e rs=0,684 para a pró-MMP-9) e com a pressão média na artéria pulmonar (p=0,005 e rs=0,610 para a TI, e p=0,022 e rs=0,720 para a pró-MMP-9). Concluímos que a TI cardíaca e a pró-MMP-9 circulantes aumentam em proporção à gravidade da EPA, embora o aumento da pró-MMP-9 não seja muito evidente em graus menos severos da EPA. Estes achados podem ser relevantes para a clínica da EPA. / Making the diagnosis of acute pulmonary thromboembolism (APT) and assessing its severity is very challenging. While cardiac troponin I (CTI) levels are promising in risk stratification, no previous study has examined whether there is a linear relation between CTI levels and the severity of APT. Moreover, matrix metalloproteinases (MMPs) are involved in the pathophysiology of APT. However, it is unknown whether the increases in MMP levels after APT reflect the severity of this condition. We examined whether the circulating levels of these biomarkers increase in proportion to the severity of experimental APT induced in anesthetized dogs. APT was induced with autologous blood clots (saline, 1, 3, or 5 mL/kg) injected into the right atrium. Hemodynamic evaluations were carried out for 120 min. Gelatin zymography of MMP-2 and MMP-9 from plasma samples were performed and serum CTI levels were determined at baseline and 120 min after APT. Our results sugest that while no significant increases in pro-MMP-2 levels were found after APT, pro-MMP-9 levels increased by 80% only after 5 mL/kg of clot embolization. Serum CTI and plasma pro-MMP-9 levels correlated positively with pulmonary vascular resistance (p=0.007 and rs=0.833 for troponin I, and p=0.034 and rs=0.684 for pro-MMP-9) and with pulmonary artery pressure (p=0.005 and rs=0.610 for troponin I, and p=0.022 and rs=0.720 for pro-MMP-9). We conclude that circulating CTI and pro-MMP-9 increase in proportion to the severity of APT, although the increases in plasma pro-MMP-9 are less clear with less severe APT. These findings may be relevant for clinical APT.

Com o objetivo de avaliar se o exercício físico prolongado causa alterações miocárdicas em eqüinos de enduro e se estas alterações cardíacas têm influência no desempenho dos animais durante as provas, avaliaram-se 30 cavalos, divididos em três grupos de dez animais cada, sendo G1 composto por animais que percorreram distâncias acima de 100 km, G2 por animais que percorreram distâncias menores de 100 km e G3 por animais desqualificados por alterações metabólicas. Os animais foram avaliados em três momentos distintos, T0 (pré-exercício, em repouso), T1 (entre 30 e 60 minutos após o exercício) e T2 (entre 90 e 120 minutos pós-exercício). Realizaram-se o exame físico e o exame ecocardiográfico, além de colheita de amostras de sangue para determinação de troponina I cardíaca sérica e outras provas bioquímicas. Não houve diferença nos valores de troponina I cardíaca entre os diversos grupos nem nos diferentes tempos. Observou-se diminuição dos valores do diâmetro interno do ventrículo esquerdo em diástole e aumento de espessura de septo interventricular pós-exercício. Não houve diferença nos índices funcionais cardíacos e houve manutenção do débito cardíaco por aumento da freqüência cardíaca. Estas alterações ecocardiográficas de pequena magnitude foram mais evidentes nos animais desqualificados por alterações metabólicas e não parecem estar relacionadas a injúria miocárdica e sim secundárias a outras condições orgânicas. Conclui-se que o exercício físico prolongado não leva a injúrias cardíacas severas em cavalos de enduro. / With the purpose of evaluating myocardial alterations caused by prolonged physical exercise and whether these alterations influence the endurance horses\' performance during the races, 30 horses were evaluated, divided into three groups of ten horses each, being G1 composed by animals that performed distances of more than 100 km, G2 by animals that performed distances of less than 100 km and G3 by animals disqualified by metabolic alterations. The horses were evaluated in three distinct moments, T0 (pre-exercise, at rest), T1 (between 30 and 60 minutes post-exercise) and T2 (between 90 and 120 minutes post-exercise). Physical and echocardiographic examinations and else blood sample collection for the determination of cardiac troponin I and other biochemical tests were done. There was no difference in cardiac troponin I values neither between the various groups, nor between the moments. There was a decrease in the post-exercise values of the diastolic left ventricle internal diameter and an increase in the post-exercise values of the interventricular septal thickness. There was no difference in the cardiac functional indexes and the cardiac output was maintainded by augmentation of the heart rate. These minor echocardiographic alterations were more evident in the animals that were disqualified by metabolic alterations, and they don\'t seem to be related to myocardial injury, but secondary to other organic conditions. Based on these results, prolonged physical exercise doesn\'t seem to cause severe cardiac injuries in endurance horses.

Ao contrário do homem, as cardiopatias de natureza hipóxica/isquêmica são pouco relatadas nos cães. Raros são os relatos de infarto agudo do miocárdio (IAM) nesses espécimes; entretanto, existem achados eletrocardiográficos que indicam hipóxia/isquemia miocárdica, como os desníveis (infra e supra) do segmento ST. Com o intuito de constatar algum dano nas células do miocárdio em condições de má perfusão, utilizaram-se 38 cães, dos quais 20 com traçados eletrocardiográficos normais e 18 com desníveis (infra e supra) do segmento ST, na derivação DII, velocidade de 50 mm/s e sensibilidade N. Nos animais normais (grupo 1), a dosagem sérica da enzima creatinafosfoquinase isoenzima MB (CK-MB) e da troponina I (cTnI) destinou-se à obtenção dos valores de referência (em ng/mL). Estes valores de referência foram confrontados com os obtidos de cães portadores de desnível (grupo 2), permitindo confirmar ou não a injúria miocárdica. Em relação à CK-MB, os cães do grupo 1 apresentaram média de 0,54 ng/mL e desvio-padrão de 0,89 ng/mL e os do grupo 2 apresentaram média de 0,44 ng/mL e desvio-padrão de 1,106 ng/mL. A média e o desvio-padrão foram, respectivamente, de 0,16 ng/mL e 0,110 ng/mL e de 0,20 ng/mL e 0,111 ng/mL, nos grupos 1 e 2. Houve 18 valores nulos de CK-MB, igualmente distribuídos entre ambos os grupos. O grupo 1 apresentou três valores nulos para cTnI. Houve diferença marcante em relação à idade, sendo o grupo 1 constituído por animais, preponderantemente, abaixo de 7 anos; o contrário ocorreu no grupo 2. São significativas, ao nível de significância de 5%, as associações da variável CK-MB com as variáveis idade, massa e CK-T (creatinafosfoquinase total) no grupo 1, e com a variável CK-T no grupo 2. A variável cTnI não apresentou evidências de associação, ao nível de significância de 5% , com as variáveis idade, massa, CK-T e nível sérico de potássio, para cada um dos níveis da variável grupo. Tanto para a variável CK-MB quanto para a cTnI, não houve diferenças significativas, ao nível de 5%, entre os grupos 1 e 2. Conclui-se que é possível a utilização do \"kit\" de ensaio imunométrico quimioluminescente humano para a espécie canina e que a hipóxia/isquemia, revelada pelo desnível do segmento ST, não acarreta dano miocárdico, ou que este é mínimo. / Although very often in men, hypoxic and ischemic heart diseases are poorly documented in dogs. There are few reports of acute myocardial infarction (AMI) in this species. However, some electrocardiographic findings may suggest myocardium hypoxia/ischemia, like ST segment elevation or depression. In order to investigate myocardial cells injury in poor perfusion conditions, 38 dogs, being 20 with normal electrocardiogram and 18 with ST segment elevation or depression in lead II, at a paper speed of 50 mm/sec and N sensibility (1 mV = 1cm), were included. Serum measurement of creatinephosphokinase isoenzyme MB (CK-MB) enzym and troponin I (cTnI) in normal dogs (group 1) determined reference values (in ng/ml). These values were compared to those obtained in dogs with deviation (group 2), which allowed confirmation or not of myocardial injury. CK-MB mean values obtained from dogs in groups 1 and 2 was 0,54 ng/mL (SD±0,54 ng/mL) and 0,44 ng/mL (SD±1,106), respectively. Mean cTnI values in groups 1 and 2 was 0,16 ng/mL (SD±0,110 ng/mL) and 0,20 ng/mL (SD±0,111 ng/mL) respectively. Three cTnI null values were found in group 1. There was a marked difference concerning to age, being group 1 composed, mainly, by animals ageing under 7 years, on the contrary of group 2. At a significance level of 5%, was significant the relation of CK-MB with age, mass and total creatinephosphokinase (CK-T) in group 1 and with CK-T in group 2. There is no relation, at a significance level of 5%, of cTnI with age, mass, CK-T or serum potassium concentration, for each level of group variable. Both CK-MB and cTnI variables showed no difference, at 5% level, between groups 1 and 2. In conclusion, it is possible to use the human chemiluminescent immunometric assay kit in canine species and that hypoxia/ischemia revealed by ST segment deviation does not mean significant myocardium injury.

Approximately 30% of people admitted to ICU in the UK have co-existing cardiovascular disease (CVD), and this may rise as life-expectancy increases. Patients with CVD have impaired compensatory mechanisms to enable maximum oxygen delivery to the tissues in the event of critical illness, which itself increases global oxygen demand, further stressing the heart. This is exacerbated by tachycardia and hypotension, which may relatively reduce blood flow to the coronary arteries, and catecholamines which increase myocardial oxygen demand. The myocardium extracts 75% of the oxygen supplied by the coronary arteries at rest, and atheroma-related flow limitation further compromises myocardial oxygen delivery. However, the diagnosis of acute coronary syndrome in critical illness is not straightforward, due to patient inability to communicate symptoms, non-specific ECG changes, and poorly understood cardiac biomarker troponin elevation. My overall hypothesis is that patients with CVD benefit from increased oxygen delivery to the myocardium during critical illness. A focus is the importance of anaemia. The aims of the studies presented in this thesis are (i) to systematically review the literature regarding blood transfusion thresholds specifically in patients with CVD; (ii) to explore the association between Troponin I (TnI) within 24 hours of ICU admission and hospital mortality (iii) to describe and quantify the dynamics of TnI in patients with CVD during the first ten days after ICU admission; and (iv) to define myocardial infarction in the context of critical illness. I have performed a systematic review and meta-analysis of randomised controlled trials comparing a restrictive with liberal transfusion threshold and that included patients with CVD. In total, 11 trials enrolling patients with CVD (n=3033) were included for meta-analysis (restrictive n=1514, liberal=1519). The pooled risk ratio for the association between a restrictive transfusion threshold and 30 day mortality was 1.15 (95% CI 0.88 to 1.50, p=0.50, I2=14%). The risk of acute coronary syndrome in patients managed with restrictive compared with liberal transfusion was increased (nine trials, risk ratio 1.78, 95% CI 1.18 to 2.70, p=0.0, I2=0%). In contrast to broader literature supporting restrictive thresholds, our systematic review shows that a restrictive transfusion threshold of less than 80g/l may not be safe in patients with co-existing CVD, and highlights the variability in diagnostic definitions of ACS and the potential for ascertainment bias in transfusion trials. I undertook a retrospective cohort study in two independently collected cohorts of general ICU patients who had TnI measured within 24 hours of ICU admission. Importantly, the majority of TnI samples were collected routinely rather than for clinical indications. We used the Abbott ARCHITECT Stat assay (limit of detection 0.01mcg/l. We performed multivariable regression, adjusting for components of the APACHE II model. We derived the risk prediction score from the multivariable model with TnI. TnI was associated with all cause hospital mortality (OR per doubling TnI 1.16, 95% CI 1.13 to 1.20, p < 0.001) which persisted after adjustment for APACHE II model components (OR TnI 1.05, 95% CI 1.01 to 1.09, p=0.003). TnI correlated highly with the Acute Physiological Score component of APACHE II (r=0.39), suggesting that TnI release may be largely explained by acute physiological stress. Addition of TnI to the APACHE II model did not improve the performance of the risk prediction model and we would not advocate the adoption of a routine single troponin sample at admission. I designed, set up, and recruited 279 patients to a prospective cohort study TROPonin I in Cardiovascular patients in CriticAL care (TROPICCAL, UKCRN 19253) in 11 UK centres. The aims were to (i) determine the incidence of Myocardial Injury and Infarction, defined by the Third Universal Definition of Myocardial Infarction; (ii) explore factors associated with Injury and Infarction from multivariable analyses; and (iii) explore the relationship between Injury/Infarction and outcome in unadjusted and adjusted analyses. We recorded baseline characteristics, and took daily hs-TnI for ten days after ICU admission, severity of illness measures and ECGs for 5 days. There was a wide range of peak TnI (med 114ng/l (min 3, Q1 27, Q3 412, max 58820ng/l)) and a high prevalence of myocardial injury on systematic screening: 71% of patients had peak TnI greater than the sex-specific diagnostic threshold ('Injury'), and 24% had peak TnI greater than the sex-specific diagnostic threshold and dynamic changes on ECG consistent with ischaemia ('Infarction'). TnI consistently showed a rise-and-fall pattern consistent with an acute myocardial 'hit' rather than persisting injury, which peaked early during ICU stay. Importantly, only 12 (4.4%) patients were diagnosed with MI by the clinicians looking after the patients. Independent predictors of peak TnI in the preceding 24 hours were SOFA score, dynamic ECG ischaemia, lactate, haemoglobin, and age. The lack of association with CRP (representing systemic inflammation), with stronger association with lactate (representing inadequate perfusion/oxygen supply), Hb and ECG ischaemia support the conjecture that injury results in part from an acute ischaemic hit in this population. Patients with Infarction had similar baseline demographics to patients with Injury, but had higher peak TnI concentrations, and higher hospital and six month mortality (Figure 2). This supports the importance of including systematic assessment of dynamic ECG changes in the myocardial injury 'construct' in ICU. My work has shown an increased risk of ACS in patients with CVD randomised to restrictive transfusion thresholds. TnI elevation is prevalent in general ICU patients, and is independently associated with hospital mortality. A systematic approach to the detection of myocardial injury in critically ill patients with co-existing CVD who are unable to communicate symptoms, can identify a high risk population who have poorer survival than patients with no injury. Markers of ischaemia are more associated with TnI rise than markers of inflammation, supporting the hypothesis that myocardial injury in this population is at least in part due to oxygen supply-demand imbalance 'myocardial infarction'. From this work, I would recommend (i) a more liberal transfusion threshold of at least 80g/l in patients with coexisting CVD; (ii) systematic use of sequential ECGs in ICU to screen for myocardial injury in 'at risk' patients; and (iii) manipulation of physiological parameters such as anaemia, hypotension and tachycardia should be considered for patients with dynamic ECG changes plus troponin increase consistent with Infarction. Future research should include 'precision medicine' trials in the substantial cohort of ICU patients with co-existing CVD to explore whether interventions that increase myocardial oxygen supply and/or treat infarction alter outcomes.

Em todo o mundo, milhões de pacientes são atendidos em emergências por apresentarem dor torácica de início aguda, mas apenas uma parcela deve-se a síndrome coronariana aguda (SCA). Em situações como essa é de extrema importância distinguir quando a dor torácica é devido à isquemia do miocárdio, pois esta é de alto risco e o início do tratamento deve ser imediato. Novos biomarcadores são necessários para auxiliar no diagnóstico e conduta clínica a ser tomada diante de situações de emergência como esta. Recentemente a quantificação de troponinas através de ensaios ultrassensíveis tem sido amplamente utilizado para diagnósticos e prognóstico de isquemia cardíaca, porém esses ensaios não tiveram seus valores de referências estabelecidos e validados para diversas situações clínicas. O presente estudo identificou a troponina I cardíaca nitrada como um novo biomarcador para isquemia cardíaca. Através de experimentos de imunoluorecência, foi possível colocalizar a marcação de troponina I cardíaca e nitrotirosina em modelos celulares e murinos de isquemia cardíaca, sugerindo assim que a troponina I cardíaca é nitrada. A partir do soro de modelos porcinos de isquemia, foi realizado o enriquecimento de proteínas nitradas por imunoprecipitação seguido da identificação da troponina I cardíaca por western blot. Dessa maneira foi possível identificar a troponina I cardíaca nitrada no soro poucos minutos após o evento x isquêmico, a qual permaneceu circulante por até 24 horas. Nessas mesmas amostras outros biomarcadores de isquemia como CKMB, Troponina I e Troponina T ultrassensível foram dosados e nenhum marcador de elevou após a isquemia cardíaca seguida de reperfusão. A troponina I cardíaca nitrada foi caracterizada por espectrometria de massas. Esse proteína é um potencial marcador circulante sensível para o diagnóstico e prognóstico precoce de isquemia cardíaca com ou sem necrose do miocárdio / Worldwide, millions of patients are treated in emergencies because they had acute-onset chest pain, but only a portion is due to coronary syndrome. In situations like this is extremely important to distinguish when the chest pain is due to myocardial ischemia, as this is high risk and initiation of treatment should be immediate. New biomarkers are needed to assist clinical decision-making in ACS. Recently, the quantification of ultra-sensitive tests for troponins has been widely used for diagnosis and prognosis of myocardial ischemia, however the reference values was not well validated and established for different subjects groups. The present study identified the nitrated cardiac troponin I as a novel biomarker of cardiac ischemia. We performed immunofluorescence colocalization marking of cardiac troponin I and nitrotyrosine in cell and rat model of cardiac ischemia, suggesting that cardiac troponin I is a nitrated protein. From serum of porcine models cardiac ischemia was made enrichment of nitrated proteins by immunoprecipitation with anti-nitrotyrosine followed by detection of cardiac troponin I by western blot. It was possible to identify the cardiac troponin I in serum nitrated few minutes after the ischemic event, which remains current for up to 24 hours. In these samples, other markers of cardiac ischemia such as CK-MB, troponin I and ultra-sensitive troponin T did not increase after ischemia followed by reperfusion. Nitrated cardiac troponin I was characterized by MS/MS. The xii nitrated cardiac troponin I is a potential circulating marker sensitive for the diagnosis and prognosis for early cardiac ischemia with or without myocardial necrosis