Identification and quantification of tocomonoenol isomers in plants and microalgae and investigation of their metabolism in liver cells

Tocopherols (T), tocomonoenols (T1), and tocotrienols (T3) are tocochromanols, a group of bioactive compounds composed of a chromanol ring and a 16-carbon sidechain with biological functions, such as the protection of lipid membranes from oxidation and the modulation of cellular signaling. T have saturated sidechains, while T1 and T3 have a single or three double bonds in theirs, respectively. The prefixes alpha-, beta-, gamma-, and delta- are assigned based on the number and positions of methyl groups on the chromanol ring. alpha-, beta-, gamma-, and delta-congeners of T1 have been reported, with alpha-T1 being the predominantly identified congener. Two different alpha-T1 isomers are known, 11-alpha-T1, which has been mainly found in land plants, and 12-alpha-T1, which has been mostly detected in marine organisms. However, little is known regarding the occurrence of T1 in photosynthetic organisms and their metabolism in the liver, a strong determinant of bioavailability and bioactivity. The aim of this thesis was to evaluate underutilized plant-based food sources, cyanobacteria, and microalgae as potential sources of T1 and to characterize the uptake and conversion into metabolites of T1 in cultured liver cells in comparison to T and T3. Acrocomia aculeata fruits were analyzed for alpha-T1 due to its phylogenetic relationship with Elaeis spp, the most common source of this congener. No alpha-T1 was detected in oils from endosperm and mesocarp of wild fruits of Acrocomia aculeata from Costa Rica. Aerial parts of the local underutilized leafy vegetable Urtica leptophylla were evaluated as source of T1 due to its agronomical potential and previous reports of T1 in leaves of plants. LC-MS analyses indicated that leaves and flowers of Costa Rican Urtica leptophylla contain minor amounts of alpha-T1 and gamma-T1. Cyanobacteria and microalgae from different species and origins were analyzed as source of alpha-T1 due to their role as primary producers in aquatic ecosystems and the reported presence of 12-alpha-T1 in marine phytoplankton. alpha-T1 in cyanobacteria and microalgae ranged from traces up to 17% of the total tocochromanol content. alpha-T1 concentrations alone were higher than the sum of all four T3. 11-alpha-T1 was the major alpha-T1 isomer in cyanobacteria and microalgae, as determined by GC-MS. Hence, 11-alpha-T1 is a product of biosynthetic pathways even in aquatic organisms. The effect of nitrogen depletion during the cultivation of microalgae on their alpha-T1 content was investigated. Nitrogen depletion did neither significantly affect the relative or absolute content of alpha-T1, despite an increase in tocochromanol content, nor the proportion of 11-alpha-T1/12-alpha-T1 in microalgae. The uptake and conversion into metabolites of 11-alpha-T1 in HepG2 liver cells was compared to those of alpha-T3 and alpha-T. Cellular uptake of alpha-T1 in liver cells was higher than that of alpha-T. 11-alpha-T1, similar to alpha-T, was converted mostly to alpha-carboxymethylhydroxychroman in a time dependent manner, but to lower extend than alpha-T3. The effect of both ring methylation and sidechain saturation on the uptake and metabolism of the alpha- and gamma-congeners of T1, T and T3 was studied in HepG2 cells. gamma-Congeners were metabolized at higher extent than alpha-congeners and metabolite production increased with increasing number of double bonds in the sidechain independently of chromanol ring methylation. In conclusion, alpha-T1 is present with up to 17% of total tocochromanols in cultured microalgae, which thus are an important new source of this congener. gamma-T1 is only a minor tocochromanol in U. leptophylla flowers. 11-alpha-T1, and not 12-alpha-T1, is the major alpha-T1 isomer in cyanobacteria and microalgae and nitrogen depletion of microalgae does not significantly affect alpha-T1 concentration. The metabolic conversion of alpha-T1 into alpha-carboxymethylhydroxychroman in HepG2 cells is similar to that of alpha-T and significantly lower than that of alpha-T3, suggesting that it may be handled by the organism similar to alpha-T. In conclusion, novel potential food sources of alpha-T1 have been identified and, because of similarities with alpha-T, its pharmacokinetics and biological activities warrant further investigation.Tocomonoenole (T1), Tocopherole (T) und Tocotrienole (T3) gehören zur Familie der Tocochromanole, einer Gruppe bioaktiver Substanzen, die aus einem Chromanolring und einer Seitenkette aus 16 Kohlenstoffatomen bestehen. Während T eine gesättigte Seitenkette aufweisen, haben T1 eine einfach ungesättigte und T3 eine dreifach ungesättigte Seitenkette. Je nach Anzahl und Stellung von Methylgruppen am Chromanolring werden alpha-, beta-, gamma-, und delta-Kongenere unterschieden. Es wurden bereits alpha-, beta-, gamma- und delta-Kongenere der T1 beschrieben, wobei alpha-T1 das am Häufigsten gefundene T1-Kongener darstellt. Zwei verschiedene alpha-T1-Isomere sind bekannt: 11-alpha-T1, welches hauptsächlich in terrestrischen Pflanzen, und 12-alpha-T1, welches hauptsächlich in marinen Quellen gefunden wurde. Über mögliche Quellen für alpha-T1 und seinen Metabolismus in der Leber ist bislang nur wenig bekannt. Ziel der vorliegenden Arbeit war es daher, neue Quellen für T1 zu identifizieren sowie den Metabolismus von T1 in Leberzellen zu erforschen und mit dem von T und T3 zu vergleichen. Acrocomia aculeata wurde aufgrund seiner phylogenetischen Verwandtschaft mit Elaeis spp., einer Quelle für alpha-T1, auf den Gehalt dieses Kongeners hin untersucht. Im Endosperm- und Mesokarp von Früchten wilder Acrocomia aculeata aus Costa Rica konnte kein alpha-T1 nachgewiesen werden. Die oberirdischen Teile von Urtica leptophylla wurden aufgrund seines agronomischen Potenzials und vorangegangener Berichte über T1 in Blättern von Pflanzen als mögliche Quelle von T1 untersucht. Mittels LC-MS-Analyse wurden geringe Mengen an alpha-T1 und gamma-T1 in den Blättern und Blüten von Urtica leptophylla gefunden. Cyanobakterien und Mikroalgen unterschiedlicher Arten und Herkunft wurden aufgrund ihrer Rolle als Primärproduzenten in aquatischen Ökosystemen und des Vorkommens von 12-alpha-T1 in Phytoplankton als mögliche Quelle von alpha-T1 untersucht. Die Gehalte an alpha-T1 im Cyanobakterium Arthrospira platensis und in Mikroalgen der Gattungen Chlorella, Tetraselmis, Nannochloropsis und Monodopsis variierten vom Spurenbereich bis zu einem Anteil von bis zu 17% der Gesamt-Tocochromanolkonzentration. 11-alpha-T1 war das quantitativ wichtigste Isomer in allen Mikroalgen, unabhängig von Art und Herkunft, und in höheren Konzentrationen enthalten als T3. Der Einfluss eines Stickstoffmangels während der Kultivierung von Mikroalgen auf deren alpha-T1-Gehalte wurde erforscht. Die Kultivierung der Mikroalge Monodopsis subterranea unter Stickstoffmangel erhöhte den Tocochromanolgehalt, nicht aber die Konzentration von alpha-T1. Auch das Verhältnis von 11-alpha-T1 zu 12-alpha-T1 wurde nicht durch Stickstoffmangel beeinflusst. Weiterhin wurde die Aufnahme von 11-alpha-T1 in HepG2-Leberzellen und seine Umwandlung zu Metaboliten untersucht und mit denen von alpha-T und alpha-T3 verglichen. In mit Tocochromanolen inkubierten Leberzellen (HepG2) war die Aufnahme der T1 in die Zelle höher als die der T. 11-alpha-T1 wurde in einem ähnlichen Umfang wie alpha-T, aber einem geringeren als alpha-T3 vorwiegend in alpha-Carboxymethylhydroxychroman umgewandelt. Der Einfluss der Ringmethylierung sowie der Sättigung der Seitenkette auf die Aufnahme und Umwandlung der alpha- und gamma-Kongenere von T1, T und T3 wurde in HepG2 Zellen untersucht: Die metabolische Konversion der Tocochromanole nahm mit zunehmender Zahl an Doppelbindungen in der Seitenkette zu. gamma-Kongenere wurden stärker als alpha-Kongenere umgewandelt. Die Ergebnisse dieses Promotionsprojektes zeigen, dass alpha-T1 mit bis zu 17% des Gesamt-Tocochromanolgehalts in kultivierten Mikroalgen vorhanden und 11-alpha-T1, und nicht 12-alpha-T1, das wichtigste alpha-T1-Isomer in Mikroalgen ist. Weiterhin lässt sich schlussfolgern, dass ein Stickstoffmangel während der Kultivierung keinen Einfluss auf alpha-T1-Konzentrationen in Mikroalgen hat und gamma-T1 nur in geringen Mengen in Urtica leptophylla-Blüten vorhanden ist. Zum ersten Mal konnte in dieser Arbeit gezeigt werden, dass die Umwandlungsrate von alpha-T1 zu alpha-Carboxymethylhydroxychroman in HepG2-Zellen mit der von alpha-T vergleichbar und signifikant geringer als die von T3 ist, was darauf hindeutet, dass es im Organismus ähnlich wie alpha-T behandelt wird. Somit wurden im Rahmen der vorliegenden Arbeit neue Lebensmittelquellen für alpha-T1 identifiziert und Erkenntnisse über seinen Stoffwechsel gewonnen, die, aufgrund der Ähnlichkeit zu alpha-T, weitere Studien zu seiner Pharmakokinetik und seinen biologischen Aktivitäten rechtfertigen

Monodopsis subterranea is a source of α‐tocomonoenol, and its concentration, in contrast to α‐tocopherol, is not affected by nitrogen depletion

α‐Tomonoenols (αT1) are tocochromanols structurally related to tocopherols (T) and tocotrienols (T3), the bioactive members of the vitamin E family. However, limited evidence exists regarding the sources and biosynthesis of tocomonoenols. Nitrogen depletion increases the content of α‐tocopherol (αT), the main vitamin E congener, in microalgae, but little is known regarding its effect on other tocochromanols, such as tocomonoenols and tocotrienols. We therefore quantified the concentrations of T, T1, and T3, in freeze‐dried biomass from nitrogen‐sufficient, and nitrogen‐depleted Monodopsis subterranea (Eustigmatophyceae). The identities of isomers of αT1 were confirmed by LC-MS and GC-MS. αT was the predominant tocochromanol (82% of total tocochromanols). αT1 was present in higher quantities than the sum of all T3 (6% vs. 1% of total tocochromanols). 11′‐αT1 was the main αT1 isomer. Nitrogen depletion increased αT, but not αT1 or T3 in M. subterranea. In conclusion, nitrogen depletion increased the content of αT, the biologically most active form of vitamin E, in M. subterranea without affecting αT1 and T3 and could potentially be used as a strategy to enhance its nutritional value but not to increase αT1 content, indicating that αT1 accumulation is independent of that of αT in microalgae.Baden‐Württemberg StiftungFood Security Center (University of Hohenheim)German Academic Exchange Service (DAAD)German Federal Ministry for Economic Cooperation and Development (BMZ

Effect of sitagliptin on cardiovascular outcomes in type 2 diabetes

BACKGROUND: Data are lacking on the long-term effect on cardiovascular events of adding sitagliptin, a dipeptidyl peptidase 4 inhibitor, to usual care in patients with type 2 diabetes and cardiovascular disease. METHODS: In this randomized, double-blind study, we assigned 14,671 patients to add either sitagliptin or placebo to their existing therapy. Open-label use of antihyperglycemic therapy was encouraged as required, aimed at reaching individually appropriate glycemic targets in all patients. To determine whether sitagliptin was noninferior to placebo, we used a relative risk of 1.3 as the marginal upper boundary. The primary cardiovascular outcome was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for unstable angina. RESULTS: During a median follow-up of 3.0 years, there was a small difference in glycated hemoglobin levels (least-squares mean difference for sitagliptin vs. placebo, -0.29 percentage points; 95% confidence interval [CI], -0.32 to -0.27). Overall, the primary outcome occurred in 839 patients in the sitagliptin group (11.4%; 4.06 per 100 person-years) and 851 patients in the placebo group (11.6%; 4.17 per 100 person-years). Sitagliptin was noninferior to placebo for the primary composite cardiovascular outcome (hazard ratio, 0.98; 95% CI, 0.88 to 1.09; P<0.001). Rates of hospitalization for heart failure did not differ between the two groups (hazard ratio, 1.00; 95% CI, 0.83 to 1.20; P = 0.98). There were no significant between-group differences in rates of acute pancreatitis (P = 0.07) or pancreatic cancer (P = 0.32). CONCLUSIONS: Among patients with type 2 diabetes and established cardiovascular disease, adding sitagliptin to usual care did not appear to increase the risk of major adverse cardiovascular events, hospitalization for heart failure, or other adverse events

Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy

Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe

Tocochromanol Profiles in Chlorella sorokiniana, Nannochloropsis limnetica and Tetraselmis suecica Confirm the Presence of 11&prime;-&alpha;-Tocomonoenol in Cultured Microalgae Independently of Species and Origin

11&prime;-&alpha;-Tocomonoenol (11&prime;-&alpha;T1) is structurally related to vitamin E and has been quantified in the microalgae Tetraselmis sp. and Nannochloropsis oceanica. However, it is not known whether 11&prime;-&alpha;T1 is present in other microalgae independent of species and origin. The aim of this study was to analyze the tocochromanol profiles of Chlorella sorokiniana, Nannochloropsis limnetica, and Tetraselmis suecica and to determine if 11&prime;-&alpha;T1 is present in these microalgae. Cultured microalgae were freeze-dried and the presence and identity of &alpha;-tocomonoenols were confirmed by LC-MSn (liquid chromatography coupled to mass spectroscopy) and GC-MS (gas chromatography coupled to mass spectroscopy). Tocochromanol profiles were determined by HPLC-FLD (liquid chromatography with fluorescence detection) and fatty acid profiles (as fatty acid methyl esters; FAME) by GC-MS. As confirmed by LC-MSn and GC-MS, 11&prime;-&alpha;T1 was the dominant &alpha;T1 isomer in cultured microalgae instead of 12&prime;-&alpha;T1, the isomer also known as marine-derived tocopherol. &alpha;T1 represented less than 1% of total tocochromanols in all analyzed samples and tended to be more abundant in microalgae with higher proportions of polyunsaturated fatty acids. In conclusion, our findings confirm that &alpha;T1 is not restricted to terrestrial photosynthetic organisms, but can also accumulate in microalgae of different species, with 11&prime;-&alpha;T1&mdash;and not the marine-derived tocopherol (12&prime;-&alpha;T1)&mdash;as the predominant &alpha;T1 isomer

Tocochromanol Profiles in <i>Chlorella sorokiniana</i>, <i>Nannochloropsis limnetica</i> and <i>Tetraselmis suecica</i> Confirm the Presence of 11′-α-Tocomonoenol in Cultured Microalgae Independently of Species and Origin

11′-α-Tocomonoenol (11′-αT1) is structurally related to vitamin E and has been quantified in the microalgae Tetraselmis sp. and Nannochloropsis oceanica. However, it is not known whether 11′-αT1 is present in other microalgae independent of species and origin. The aim of this study was to analyze the tocochromanol profiles of Chlorella sorokiniana, Nannochloropsis limnetica, and Tetraselmis suecica and to determine if 11′-αT1 is present in these microalgae. Cultured microalgae were freeze-dried and the presence and identity of α-tocomonoenols were confirmed by LC-MSn (liquid chromatography coupled to mass spectroscopy) and GC-MS (gas chromatography coupled to mass spectroscopy). Tocochromanol profiles were determined by HPLC-FLD (liquid chromatography with fluorescence detection) and fatty acid profiles (as fatty acid methyl esters; FAME) by GC-MS. As confirmed by LC-MSn and GC-MS, 11′-αT1 was the dominant αT1 isomer in cultured microalgae instead of 12′-αT1, the isomer also known as marine-derived tocopherol. αT1 represented less than 1% of total tocochromanols in all analyzed samples and tended to be more abundant in microalgae with higher proportions of polyunsaturated fatty acids. In conclusion, our findings confirm that αT1 is not restricted to terrestrial photosynthetic organisms, but can also accumulate in microalgae of different species, with 11′-αT1—and not the marine-derived tocopherol (12′-αT1)—as the predominant αT1 isomer

Monodopsis subterranea is a source of α-tocomonoenol, and its concentration, in contrast to α-tocopherol, is not affected by nitrogen depletion

α-Tomonoenols (αT1) are tocochromanols structurally related to tocopherols (T) and tocotrienols (T3), the bioactive members of the vitamin E family. However, limited evidence exists regarding the sources and biosynthesis of tocomonoenols. Nitrogen depletion increases the content of α-tocopherol (αT), the main vitamin E congener, in microalgae, but little is known regarding its effect on other tocochromanols, such as tocomonoenols and tocotrienols. We therefore quantified the concentrations of T, T1, and T3, in freeze-dried biomass from nitrogen-sufficient, and nitrogen-depleted Monodopsis subterranea (Eustigmatophyceae). The identities of isomers of αT1 were confirmed by LC–MS and GC–MS. αT was the predominant tocochromanol (82% of total tocochromanols). αT1 was present in higher quantities than the sum of all T3 (6% vs. 1% of total tocochromanols). 11′-αT1 was the main αT1 isomer. Nitrogen depletion increased αT, but not αT1 or T3 in M. subterranea. In conclusion, nitrogen depletion increased the content of αT, the biologically most active form of vitamin E, in M. subterranea without affecting αT1 and T3 and could potentially be used as a strategy to enhance its nutritional value but not to increase αT1 content, indicating that αT1 accumulation is independent of that of αT in microalgae