Motivation: The different coping styles used to respond to psychosocial stress have been linked to the development of cardiovascular disease (CVD). However, the manner in which the cardiovascular system is influenced differs between the coping styles. Of the different coping styles, defensive active coping (AC) has been shown to be the most detrimental to cardiovascular health. This is worsened by augmented α-adrenergic cardiac responses found in Africans. Furthermore, many studies have found that the prevalence of hypertension and other CVDs is much higher in urban Africans when compared to their Caucasian counterparts. This can be attributed to certain lifestyle changes implemented by Africans in the transition that occurs with urbanization, where they are forced to cope with an urban-dwelling lifestyle. One of these lifestyle factors, which also poses as a cardiovascular risk factor, is increased usage and in some cases abuse of alcohol. Certain discrepancies exist between ethnicities with regard to the metabolism of alcohol, which influences the effect of alcohol on the individual. Alcohol usage as a possible manner of coping has been supported in many instances, but the interdependent effects of alcohol usage and AC as cardiovascular risk factors has only been found in African men. Further investigation is needed to determine if coping and alcohol abuse act in tandem only in African men, or also in other ethnic or sex groups. What also needs to be discussed is whether the inconsistencies between ethnicities regarding alcohol metabolism, plays a part in the development of CVD in a bi-ethnic gender cohort. Objectives: The main aims of this study were to determine 1) receiver operated characteristic (ROC) ethnic specific cut points of alcohol usage in the prediction of ambulatory hypertension, and 2) to assess if these cut points in defensive active groups revealed increased cardiometabolic risk in a bi-ethnic sex cohort, and if so, whether the increased risk will be associated with a specific race or sex group? Methodology: This sub-study forms part of the SABPA (Sympathetic activity and Ambulatory Blood Pressure in Africans) study, conducted from 2008 to 2009. After exclusion criteria were applied, our bi-ethnic sex cohort consisted of 390 individuals. These participants were all from the Kenneth Kaunda Education District of the North-West province in South Africa, and they all signed informed consent prior to participation. The SABPA study was approved by the Ethics Review Board of the North-West University, with additional ethical approval for this sub-study. All procedures in this study complied with the guidelines of the Declaration of Helsinki. Each participant completed a psychosocial battery supervised by registered clinical psychologists, and information regarding their medication use and medical history was obtained. They also completed the Coping Style Indicator questionnaire which was developed by Amirkhan, to identify the coping style habitually used. Ambulatory blood pressure and ECG measurements were recorded for a 24h period with the Cardiotens CE120®. Anthropometric measurements were performed by ISAK (International Society for the Advancement of Kinanthropometry) level 2 accredited anthropometrists using calibrated instruments. Out of this, the body surface area were calculated. The physical activity of each participant was determined by use of the Actical® omnidirectional accelerometer. Resting blood samples were collected by a registered nurse. The following blood serum levels were determined: gamma-glutamyl transferase (γ-GT) as a marker for alcohol usage, C-reactive protein, cholesterol, high density lipoprotein, triglycerides, cotinine, reactive oxygen species and glycated haemoglobin levels. All statistical analyses were done using Statistica version 12.0. Descriptive statistics were conducted to state the baseline characteristics of the entire group, while Chi-square (X2) tests were used to determine prevalence for medications and pathology. ROC analyses were computed to establish a cut point for γ-GT predicting ambulatory hypertension in each ethnicity as well as in the entire group. Independent t-tests identified confounders, after which two-way analysis of covariance (ANCOVA) tests were computed to test a 2 x 2 main effects interaction (race x γ-GT cut points) for all cardiometabolic risk markers and to compare the different ethnic groups. ANCOVAs were then performed in the ethnic groups with high γ-GT as well as in above mean AC for the graphs that followed. Lastly, odds ratios (OR‟s) with 95% confidence intervals (CI‟s) were calculated in several models to highlight the odds of high alcohol intake to predict ambulatory hypertension in the ethnic-sex groups as well as in AC ethnic-sex groups. Significant values were noted as p ≤ 0.05. Results: The Africans revealed higher cardiometabolic risk markers, above mean defensive active coping, seeking social support with less avoidance coping scores. ROC analyses revealed that ambulatory hypertension commences at a much higher level of γ-GT in the Africans [55.7U/l (AUC=0.69; 95% CI: 0.61; 0.76)] with sensitivity /specificity of 47%/83% compared to the Caucasians [19.5U/l (AUC=0.747; 95% CI: 0.68; 0.82)] with sensitivity/specificity of 70%/73%. The Caucasians thus reveal an increased sensitivity for alcohol ingestion at a much lower γ-GT cut point compared to the Africans. When comparing ethnic specific ROC cut point groups, we found that certain levels of cardiometabolic risk factors such as C-reactive protein, systolic blood pressure, waist circumference and silent ischemic events, were significantly higher in the African group, especially in above mean AC groups. Out of the Africans with high γ-GT levels, 73% used the AC style, suggesting hypervigilant AC coping and increased CVD risk in Africans. Clinical significance was determined by OR‟s, which demonstrated that high γ-GT levels in AC African men predicted ambulatory hypertension with an OR of 7.37 (95% CI: 6.71 – 8.05). Higher alcohol intake predicted ambulatory hypertension in AC Caucasians with an OR of 2.77 (95% CI: 2.31 – 3.23) in men and 6.42 (95% CI: 5.85 – 7.0) in women respectively. Conclusion: γ-GT cut-points in defensive active groups revealed increased cardiometabolic risk markers in a bi-ethnic sex cohort. A possible hypermetabolic state in African men may initially protect them against CVD morbidity but if chronically challenged with no forthcoming social support, CVD risk is imminent. / MSc (Physiology), North-West University, Potchefstroom Campus, 2015

Motivation: The different coping styles used to respond to psychosocial stress have been linked to the development of cardiovascular disease (CVD). However, the manner in which the cardiovascular system is influenced differs between the coping styles. Of the different coping styles, defensive active coping (AC) has been shown to be the most detrimental to cardiovascular health. This is worsened by augmented α-adrenergic cardiac responses found in Africans. Furthermore, many studies have found that the prevalence of hypertension and other CVDs is much higher in urban Africans when compared to their Caucasian counterparts. This can be attributed to certain lifestyle changes implemented by Africans in the transition that occurs with urbanization, where they are forced to cope with an urban-dwelling lifestyle. One of these lifestyle factors, which also poses as a cardiovascular risk factor, is increased usage and in some cases abuse of alcohol. Certain discrepancies exist between ethnicities with regard to the metabolism of alcohol, which influences the effect of alcohol on the individual. Alcohol usage as a possible manner of coping has been supported in many instances, but the interdependent effects of alcohol usage and AC as cardiovascular risk factors has only been found in African men. Further investigation is needed to determine if coping and alcohol abuse act in tandem only in African men, or also in other ethnic or sex groups. What also needs to be discussed is whether the inconsistencies between ethnicities regarding alcohol metabolism, plays a part in the development of CVD in a bi-ethnic gender cohort. Objectives: The main aims of this study were to determine 1) receiver operated characteristic (ROC) ethnic specific cut points of alcohol usage in the prediction of ambulatory hypertension, and 2) to assess if these cut points in defensive active groups revealed increased cardiometabolic risk in a bi-ethnic sex cohort, and if so, whether the increased risk will be associated with a specific race or sex group? Methodology: This sub-study forms part of the SABPA (Sympathetic activity and Ambulatory Blood Pressure in Africans) study, conducted from 2008 to 2009. After exclusion criteria were applied, our bi-ethnic sex cohort consisted of 390 individuals. These participants were all from the Kenneth Kaunda Education District of the North-West province in South Africa, and they all signed informed consent prior to participation. The SABPA study was approved by the Ethics Review Board of the North-West University, with additional ethical approval for this sub-study. All procedures in this study complied with the guidelines of the Declaration of Helsinki. Each participant completed a psychosocial battery supervised by registered clinical psychologists, and information regarding their medication use and medical history was obtained. They also completed the Coping Style Indicator questionnaire which was developed by Amirkhan, to identify the coping style habitually used. Ambulatory blood pressure and ECG measurements were recorded for a 24h period with the Cardiotens CE120®. Anthropometric measurements were performed by ISAK (International Society for the Advancement of Kinanthropometry) level 2 accredited anthropometrists using calibrated instruments. Out of this, the body surface area were calculated. The physical activity of each participant was determined by use of the Actical® omnidirectional accelerometer. Resting blood samples were collected by a registered nurse. The following blood serum levels were determined: gamma-glutamyl transferase (γ-GT) as a marker for alcohol usage, C-reactive protein, cholesterol, high density lipoprotein, triglycerides, cotinine, reactive oxygen species and glycated haemoglobin levels. All statistical analyses were done using Statistica version 12.0. Descriptive statistics were conducted to state the baseline characteristics of the entire group, while Chi-square (X2) tests were used to determine prevalence for medications and pathology. ROC analyses were computed to establish a cut point for γ-GT predicting ambulatory hypertension in each ethnicity as well as in the entire group. Independent t-tests identified confounders, after which two-way analysis of covariance (ANCOVA) tests were computed to test a 2 x 2 main effects interaction (race x γ-GT cut points) for all cardiometabolic risk markers and to compare the different ethnic groups. ANCOVAs were then performed in the ethnic groups with high γ-GT as well as in above mean AC for the graphs that followed. Lastly, odds ratios (OR‟s) with 95% confidence intervals (CI‟s) were calculated in several models to highlight the odds of high alcohol intake to predict ambulatory hypertension in the ethnic-sex groups as well as in AC ethnic-sex groups. Significant values were noted as p ≤ 0.05. Results: The Africans revealed higher cardiometabolic risk markers, above mean defensive active coping, seeking social support with less avoidance coping scores. ROC analyses revealed that ambulatory hypertension commences at a much higher level of γ-GT in the Africans [55.7U/l (AUC=0.69; 95% CI: 0.61; 0.76)] with sensitivity /specificity of 47%/83% compared to the Caucasians [19.5U/l (AUC=0.747; 95% CI: 0.68; 0.82)] with sensitivity/specificity of 70%/73%. The Caucasians thus reveal an increased sensitivity for alcohol ingestion at a much lower γ-GT cut point compared to the Africans. When comparing ethnic specific ROC cut point groups, we found that certain levels of cardiometabolic risk factors such as C-reactive protein, systolic blood pressure, waist circumference and silent ischemic events, were significantly higher in the African group, especially in above mean AC groups. Out of the Africans with high γ-GT levels, 73% used the AC style, suggesting hypervigilant AC coping and increased CVD risk in Africans. Clinical significance was determined by OR‟s, which demonstrated that high γ-GT levels in AC African men predicted ambulatory hypertension with an OR of 7.37 (95% CI: 6.71 – 8.05). Higher alcohol intake predicted ambulatory hypertension in AC Caucasians with an OR of 2.77 (95% CI: 2.31 – 3.23) in men and 6.42 (95% CI: 5.85 – 7.0) in women respectively. Conclusion: γ-GT cut-points in defensive active groups revealed increased cardiometabolic risk markers in a bi-ethnic sex cohort. A possible hypermetabolic state in African men may initially protect them against CVD morbidity but if chronically challenged with no forthcoming social support, CVD risk is imminent. / MSc (Physiology), North-West University, Potchefstroom Campus, 2015

The enzyme gamma-glutamyl transferase (GGT) is a multifunctional enzyme that participates in a number of metabolic processes, including the conversion of leukotriene C4 (LTC4) to leukotriene D4 (LTD4). LTD4 is necessary for normal myeloid proliferation and differentiation. We have examined the ability of hematopoietic growth factors (HGF) to induce GGT enzyme activity and mRNA content in a HGF-responsive cell line (KG-1). Incubation of KG-1 with recombinant human cytokines interleukin-1β (IL- 1β), interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF), but not interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF) or monocyte colony-stimulating factor (M-CSF), results in significant increases in GGT enzyme activity. The increases in GGT activity are both dose- and time-dependent. In response to IL-1. Increases in enzyme activity are seen by 6 hours and activity is maximal by 24 hours. GGT mRNA increases also occur and peak by 3 to 6 hours. These results indicate that induction of increases in GGT mRNA levels and enzyme activity occur in myeloid cells in response to HGFs. This induction, together with the requirement for LTD4 for normal granulopoiesis, supports a role for GGT in the cellular events occurring in myeloid cells in response to HGFs.

Einleitung: Vitamin B12 hat wichtige Funktionen im Energiestoffwechsel sowie bei der Erythropoese. Beide Funktionskreise werden bei Hochleistungskühen besonders beansprucht und können bei Belastungen und ungenügender Vitamin-B12-Versorgung Ausgangspunkt für klinische Störungen werden. Ziele der Untersuchungen: In den vorliegenden Studien wurde der Fragestellung nachgegangen, wie sich die Vitamin-B12-Konzentration im Blutserum von Milchkühen in der Frühlaktation verhält und welche Zusammenhänge zu Stoffwechselparametern, dem Erythrogramm sowie dem Gesundheitsstatus der Kühe bestehen. Des Weiteren wurde geprüft, inwieweit der postpartale Stoffwechsel und der Gesundheitsstatus durch orale Vitamin-B12-Substitutionen stabilisiert werden können. Material und Methoden: Die Untersuchungen zur Vitamin-B12-Statuserhebung erfolgten an 157 Kühen der Rasse Holstein Friesian. Blutproben zur Stoffwechselanalytik wurden 2-6 Tage p.p. sowie 4-5 Wochen p.p. entnommen. Parallel dazu wurden klinische Daten zu Leistung und Gesundheitsstatus bis 3 Monate p.p. erhoben. In einem zweiten Versuch wurden die Kühe in 2 Gruppen eingeteilt, wobei die Versuchsgruppe (65 Kühe) eine orale Vitamin-B12-Substitution in Höhe von 0,5 g Cyanocobalamin/Kuh/Tag 4-6 Wochen a.p. beginnend bis zur Kalbung erhielt. 71 Kühe, die das stallübliche Mineralfutter erhielten, dienten als Kontrollgruppe. Auch hier erfolgten die Blutkontrollen 2-6 Tage p.p. sowie 4-5 Wochen p.p. Es wurden die Milchleistung sowie auftretende Erkrankungen dokumentiert. Die Blutentnahme erfolgte aus der Vena caudalis mediana. Neben den hämatologischen Untersuchungen wurden folgende Parameter aus dem Serum bestimmt: Freie Fettsäuren (FFS), Betahydroxybutyrat (BHB), Glukose, Bilirubin, Cholesterol, Gamma-Glutamyltransferase (GGT), Creatinkinase (CK), Harnstoff, Calcium, Eisen, anorganisches Phosphat (Pi), Cyanocobalamin und Cobalt. Ergebnisse: Die Vitamin-B12-Konzentration zeigt eine signifikante Laktationsdynamik. Alle untersuchten Kühe hatten 4 Wochen p. p. gegenüber 2–6 Tage p. p. erniedrigte Vitamin-B12-Konzentrationen (p ≤ 0,05). Bei den p.p. kranken Kühen sank gegenüber den gesunden Kühen die Vitamin-B12-Konzentration weniger stark ab (p ≤ 0,05), d.h., höhere Vitamin-B12-Konzentrationen können auf klinische Probleme hinweisen. Gesunde sowie auch p.p. kranke Kühe wiesen 2–6 Tage p. p. höhere Werte für die Parameter Erythrozytenzahl, Hämatokrit und Hämoglobinkonzentration auf als 4 Wochen p. p. Die BHB-, FFS- und Bilirubin-Konzentrationen waren bei allen Kühen 2–6 Tage p. p. infolge der partusbedingten Lipolysesteigerung erhöht (p ≤ 0,05). Bei allen Kühen korrelierte die Aktivität der cholestaseanzeigenden GGT eng mit der Vitamin-B12-Konzentration (p ≤ 0,01). Aufgrund dieser engen Korrelation mit der GGT-Aktivität sowie der Bilirubinkonzentration kann Vitamin B12 bei einer Serumkonzentration ≥ 227 ng/l bei Kühen cholestatische Stoffwechselbelastungen anzeigen. Nach Vitamin-B12-Substitution blieben in der Versuchsgruppe 60 % und in der Kontrollgruppe 47,9 % der Kühe in der Frühlaktation gesund. In der Kontrollgruppe hatten 12,7 % eine Nachgeburtsverhaltung und 40,8 % eine Mastitis, in der Versuchsgruppe betrugen die Anteile 21,5 % sowie 26,2 %. Mit x̃ = 320 pg/ml war die Vitamin-B12-Konzentration 2-6 Tage p.p. in der Vitamin-B12-substituierten Gruppe gegenüber x̃ = 224 pg/ml in der Kontrollgruppe gesichert höher. Auch vier Wochen p.p. war die Differenz noch signifikant. Cobalt, die Parameter des Leber- und Energiestoffwechsels sowie Harnstoff und CK unterschieden sich zwischen der Vitamin-B12-substituierten Gruppe und der Kontrollgruppe nicht gesichert. Die Erythrozytenzahlen sowie die Hämoglobin-Konzentrationen waren in der Vitamin-B12-substituierten Gruppe gesichert höher. Der Milchfettgehalt (%) war bei den Vitamin-B12-substituierten Kühen gegenüber den Kontrollkühen signifikant erhöht (p = 0,022), die Milchleistung unterschied sich unwesentlich. Schlussfolgerungen: Signifikant höhere Vitamin-B12- und Hämoglobin-Konzentrationen, höhere Erythrozytenzahlen sowie geringere Morbidität sprechen für positive Effekte der Vitamin-B12-Substitution. Anhand der Parameter des Leber-Energiestoffwechsels sowie der Milchleistung ließ sich dies nicht bestätigen. Cholestase stört die Vitamin-B12-Bewertung im Blut bzw. ist bei der Interpretation zu beachten. / Introduction: Vitamin B12 has important functions in energy metabolism and erythropoiesis. Both functional groups are particularly stressed in high-yielding dairy cows and can be a starting point for clinical disorders under stress and insufficient vitamin B12 supply. Objective: The studies presented were designed to ascertain the characteristics of the serum vitamin B12 concentration of dairy cows in early lactation and to check the relations with metabolic parameters, the erythrogram as well as the health status of the cows. Furthermore, it was examined to what extent the postpartum metabolism can be stabilized by oral vitamin B12 substitutions. Material and methods: The investigations on vitamin B12 status survey were carried out on 157 cows of the Holstein Friesian breed. Blood samples were taken for metabolic analysis at 2-6 days p.p. and at 4-5 weeks p.p. In parallel, clinical findings on the milk yield and the health status were compiled up to 3 months p.p. In a second trial the cows were divided into 2 groups in which the experimental group (65 cows) received an oral vitamin B12 substitution in the amount of 0.5 g cyanocobalamin/cow/day starting 4-6 weeks a.p. up to calving. 71 cows, which recieved the common mineral feed, served as control group. Again the blood tests were performed at 2-6 days p.p. and at 4-5 weeks p.p. The milk yield and emerging diseases were documented. The blood samples werde taken from the Vena caudalis mediana. In addition to haematological investigations the following parameteres were measured: Non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHB), glucose, bilirubin, cholesterol, gamma-glutamyl transferase (GGT), creatinkinase (CK), urea, calcium, ferric, anorganic phosphor, cyanocobalamin and cobalt. Results: The vitamin B12 concentration shows a significant dynamic in lactation. All examined cows had decreased vitamin B12 concentrations at 4 weeks p. p. compared to 2-6 days p. p. (p ≤ 0.05). In the p.p. morbid cows the vitamin B12 concentration fell less than in the healthy cows (p ≤ 0.05), which means higher vitamin B12 concentrations may indicate clinical problems. Healthy as well as p.p. morbid cows showed higher values for the parameters erythrocyte count, hematocrit and hemoglobin concentration at 2-6 days p.p. than 4 weeks p.p. BHB, FFS, and bilirubin levels were increased in all cows at 2-6 days p.p. as a result of partus related rise in lipolysis (p ≤ 0.05). In all cows, the activity of the GGT, which indicates cholestasis, was closely correlated with the vitamin B12 concentration p ≤ 0.01). Because of this close correlation with GGT activity and bilirubin concentration, vitamin B12 may show cholestatic metabolic stress in cows at a serum concentration ≥ 227 ng/l. After vitamin B12 substitution 60 % of the cows in the experimental group and 47.9 % of the cows in the control group remained healthy during early lactation. In the control group 12.7 % had a Retentio secundinarum and 40.8 % had a mastitis, in the experimental group the proportions were 21.5 % and 26.2 %. At two to six days p.p. the vitamin B12 concentration in the vitamin B12 substituted group was significant higher (x̃ = 320 pg/ml) than in the control group (x̃ = 224 pg/ml). This difference was still significant at four weeks p.p. Cobalt, parameters of liver and energy metabolism as well as urea and CK did not differ significantly between the vitamin B12 substituted group and the control group. Erythrocyte counts and hemoglobin concentrations were significant higher in the vitamin B12 substituted group. Milk fat content (%) was significant higher in the vitamin B12 substituted group compared to the control group (p = 0.022), the milk yield did not differ significantly. Conclusions: Significant higher vitamin B12 and hemoglobin concentrations, higher erythrocyte counts as well as lower morbidity speak for positive effects of vitamin B12 substitution. Based on the parameters of hepatic and energy metabolism as well as milk yield this could not be confirmed. Cholestasis interferes with the evaluation of vitamin B12 in the blood respectively should be considered in the interpretation.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

no / Glutathione depletion is a consistent feature of the progression of mild to severe acute pancreatitis. In this study, we examined the temporal relationship between cysteine, homocysteine, and cysteinyl-glycine levels; total reduced erythrocyte glutathione; gamma-glutamyl transpeptidase activity; and disease severity. Initially, cysteine concentration was low, at levels similar to those of healthy controls. However, glutathione was reduced whilst cysteinyl glycine and gamma-glutamyl transpeptidase activity were increased in both mild and severe attacks. As the disease progressed, glutathione and cysteinyl glycine were further increased in mild attacks and cysteine levels correlated with homocysteine (r = 0.8, P < 0.001) and gamma-glutamyl transpeptidase activity (r = 0.75, P < 0.001). The progress of severe attacks was associated with glutathione depletion, reduced gamma-glutamyl transpeptidase activity, and increased cysteinyl glycine that correlated with glutathione depletion (r = 0.99, P = 0.01). These results show that glutathione depletion associated with severe acute pancreatitis occurs despite an adequate cysteine supply and could be attributed to heightened oxidative stress coupled to impaired downstream biosynthesis.

No description available.

Einleitung: Vitamin B12 hat wichtige Funktionen im Energiestoffwechsel sowie bei der Erythropoese. Beide Funktionskreise werden bei Hochleistungskühen besonders beansprucht und können bei Belastungen und ungenügender Vitamin-B12-Versorgung Ausgangspunkt für klinische Störungen werden. Ziele der Untersuchungen: In den vorliegenden Studien wurde der Fragestellung nachgegangen, wie sich die Vitamin-B12-Konzentration im Blutserum von Milchkühen in der Frühlaktation verhält und welche Zusammenhänge zu Stoffwechselparametern, dem Erythrogramm sowie dem Gesundheitsstatus der Kühe bestehen. Des Weiteren wurde geprüft, inwieweit der postpartale Stoffwechsel und der Gesundheitsstatus durch orale Vitamin-B12-Substitutionen stabilisiert werden können. Material und Methoden: Die Untersuchungen zur Vitamin-B12-Statuserhebung erfolgten an 157 Kühen der Rasse Holstein Friesian. Blutproben zur Stoffwechselanalytik wurden 2-6 Tage p.p. sowie 4-5 Wochen p.p. entnommen. Parallel dazu wurden klinische Daten zu Leistung und Gesundheitsstatus bis 3 Monate p.p. erhoben. In einem zweiten Versuch wurden die Kühe in 2 Gruppen eingeteilt, wobei die Versuchsgruppe (65 Kühe) eine orale Vitamin-B12-Substitution in Höhe von 0,5 g Cyanocobalamin/Kuh/Tag 4-6 Wochen a.p. beginnend bis zur Kalbung erhielt. 71 Kühe, die das stallübliche Mineralfutter erhielten, dienten als Kontrollgruppe. Auch hier erfolgten die Blutkontrollen 2-6 Tage p.p. sowie 4-5 Wochen p.p. Es wurden die Milchleistung sowie auftretende Erkrankungen dokumentiert. Die Blutentnahme erfolgte aus der Vena caudalis mediana. Neben den hämatologischen Untersuchungen wurden folgende Parameter aus dem Serum bestimmt: Freie Fettsäuren (FFS), Betahydroxybutyrat (BHB), Glukose, Bilirubin, Cholesterol, Gamma-Glutamyltransferase (GGT), Creatinkinase (CK), Harnstoff, Calcium, Eisen, anorganisches Phosphat (Pi), Cyanocobalamin und Cobalt. Ergebnisse: Die Vitamin-B12-Konzentration zeigt eine signifikante Laktationsdynamik. Alle untersuchten Kühe hatten 4 Wochen p. p. gegenüber 2–6 Tage p. p. erniedrigte Vitamin-B12-Konzentrationen (p ≤ 0,05). Bei den p.p. kranken Kühen sank gegenüber den gesunden Kühen die Vitamin-B12-Konzentration weniger stark ab (p ≤ 0,05), d.h., höhere Vitamin-B12-Konzentrationen können auf klinische Probleme hinweisen. Gesunde sowie auch p.p. kranke Kühe wiesen 2–6 Tage p. p. höhere Werte für die Parameter Erythrozytenzahl, Hämatokrit und Hämoglobinkonzentration auf als 4 Wochen p. p. Die BHB-, FFS- und Bilirubin-Konzentrationen waren bei allen Kühen 2–6 Tage p. p. infolge der partusbedingten Lipolysesteigerung erhöht (p ≤ 0,05). Bei allen Kühen korrelierte die Aktivität der cholestaseanzeigenden GGT eng mit der Vitamin-B12-Konzentration (p ≤ 0,01). Aufgrund dieser engen Korrelation mit der GGT-Aktivität sowie der Bilirubinkonzentration kann Vitamin B12 bei einer Serumkonzentration ≥ 227 ng/l bei Kühen cholestatische Stoffwechselbelastungen anzeigen. Nach Vitamin-B12-Substitution blieben in der Versuchsgruppe 60 % und in der Kontrollgruppe 47,9 % der Kühe in der Frühlaktation gesund. In der Kontrollgruppe hatten 12,7 % eine Nachgeburtsverhaltung und 40,8 % eine Mastitis, in der Versuchsgruppe betrugen die Anteile 21,5 % sowie 26,2 %. Mit x̃ = 320 pg/ml war die Vitamin-B12-Konzentration 2-6 Tage p.p. in der Vitamin-B12-substituierten Gruppe gegenüber x̃ = 224 pg/ml in der Kontrollgruppe gesichert höher. Auch vier Wochen p.p. war die Differenz noch signifikant. Cobalt, die Parameter des Leber- und Energiestoffwechsels sowie Harnstoff und CK unterschieden sich zwischen der Vitamin-B12-substituierten Gruppe und der Kontrollgruppe nicht gesichert. Die Erythrozytenzahlen sowie die Hämoglobin-Konzentrationen waren in der Vitamin-B12-substituierten Gruppe gesichert höher. Der Milchfettgehalt (%) war bei den Vitamin-B12-substituierten Kühen gegenüber den Kontrollkühen signifikant erhöht (p = 0,022), die Milchleistung unterschied sich unwesentlich. Schlussfolgerungen: Signifikant höhere Vitamin-B12- und Hämoglobin-Konzentrationen, höhere Erythrozytenzahlen sowie geringere Morbidität sprechen für positive Effekte der Vitamin-B12-Substitution. Anhand der Parameter des Leber-Energiestoffwechsels sowie der Milchleistung ließ sich dies nicht bestätigen. Cholestase stört die Vitamin-B12-Bewertung im Blut bzw. ist bei der Interpretation zu beachten. / Introduction: Vitamin B12 has important functions in energy metabolism and erythropoiesis. Both functional groups are particularly stressed in high-yielding dairy cows and can be a starting point for clinical disorders under stress and insufficient vitamin B12 supply. Objective: The studies presented were designed to ascertain the characteristics of the serum vitamin B12 concentration of dairy cows in early lactation and to check the relations with metabolic parameters, the erythrogram as well as the health status of the cows. Furthermore, it was examined to what extent the postpartum metabolism can be stabilized by oral vitamin B12 substitutions. Material and methods: The investigations on vitamin B12 status survey were carried out on 157 cows of the Holstein Friesian breed. Blood samples were taken for metabolic analysis at 2-6 days p.p. and at 4-5 weeks p.p. In parallel, clinical findings on the milk yield and the health status were compiled up to 3 months p.p. In a second trial the cows were divided into 2 groups in which the experimental group (65 cows) received an oral vitamin B12 substitution in the amount of 0.5 g cyanocobalamin/cow/day starting 4-6 weeks a.p. up to calving. 71 cows, which recieved the common mineral feed, served as control group. Again the blood tests were performed at 2-6 days p.p. and at 4-5 weeks p.p. The milk yield and emerging diseases were documented. The blood samples werde taken from the Vena caudalis mediana. In addition to haematological investigations the following parameteres were measured: Non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHB), glucose, bilirubin, cholesterol, gamma-glutamyl transferase (GGT), creatinkinase (CK), urea, calcium, ferric, anorganic phosphor, cyanocobalamin and cobalt. Results: The vitamin B12 concentration shows a significant dynamic in lactation. All examined cows had decreased vitamin B12 concentrations at 4 weeks p. p. compared to 2-6 days p. p. (p ≤ 0.05). In the p.p. morbid cows the vitamin B12 concentration fell less than in the healthy cows (p ≤ 0.05), which means higher vitamin B12 concentrations may indicate clinical problems. Healthy as well as p.p. morbid cows showed higher values for the parameters erythrocyte count, hematocrit and hemoglobin concentration at 2-6 days p.p. than 4 weeks p.p. BHB, FFS, and bilirubin levels were increased in all cows at 2-6 days p.p. as a result of partus related rise in lipolysis (p ≤ 0.05). In all cows, the activity of the GGT, which indicates cholestasis, was closely correlated with the vitamin B12 concentration p ≤ 0.01). Because of this close correlation with GGT activity and bilirubin concentration, vitamin B12 may show cholestatic metabolic stress in cows at a serum concentration ≥ 227 ng/l. After vitamin B12 substitution 60 % of the cows in the experimental group and 47.9 % of the cows in the control group remained healthy during early lactation. In the control group 12.7 % had a Retentio secundinarum and 40.8 % had a mastitis, in the experimental group the proportions were 21.5 % and 26.2 %. At two to six days p.p. the vitamin B12 concentration in the vitamin B12 substituted group was significant higher (x̃ = 320 pg/ml) than in the control group (x̃ = 224 pg/ml). This difference was still significant at four weeks p.p. Cobalt, parameters of liver and energy metabolism as well as urea and CK did not differ significantly between the vitamin B12 substituted group and the control group. Erythrocyte counts and hemoglobin concentrations were significant higher in the vitamin B12 substituted group. Milk fat content (%) was significant higher in the vitamin B12 substituted group compared to the control group (p = 0.022), the milk yield did not differ significantly. Conclusions: Significant higher vitamin B12 and hemoglobin concentrations, higher erythrocyte counts as well as lower morbidity speak for positive effects of vitamin B12 substitution. Based on the parameters of hepatic and energy metabolism as well as milk yield this could not be confirmed. Cholestasis interferes with the evaluation of vitamin B12 in the blood respectively should be considered in the interpretation.

Electron crystallography of two-dimensional crystals is a structure-determination method well suited to the study of membrane protein structure-function. Two-dimensional crystals consist of ordered arrays of protein within reconstituted lipid bilayers, an arrangement that mimics the natural membrane environment. In this work we describe our recent progress in the use of this method with three different proteins, each providing a window into a separate paradigm in the electron crystallographic pipeline. Specific crystallization conditions for human leukotriene C₄ synthase (LTC₄S) have previously been determined, but our continued refinement of purification and crystallization has identified a number of additional parameters that greatly affect crystal size and quality, and we have developed a protocol to rapidly and reproducibly grow large, non-mosaic crystals of LTC₄S. The human gamma-glutamyl carboxylase (GGCX) has also been crystallized, but is sensitive to cryo-EM sample preparation conditions and we present here the successful reproduction of crystallization and refinement of cryo-EM sample preparation conditions. Lastly, we describe our crystallization screens with the Vibrio cholerae sodium-pumping NADH:ubiquinone reductase complex (Na⁺-NQR), and identify the factors critical to membrane reconstitution of the complex, a necessary first step towards crystallization. We also describe a semi-quantitative crystal screening protocol we have developed that provides quick and accurate method to assess two- dimensional crystallization trials, and discuss some general observations in optimization of membrane protein purification and two-dimensional crystallization for electron crystallography.