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1H-NMR Study of monocrotaline and its metabolites
in human blood
Yi-Cheng Yanga, John Crowdera, Nick J. Wardlea, Li Yangb,...
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Yicheng Yang Poster

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Yicheng Yang Poster

  1. 1. 1H-NMR Study of monocrotaline and its metabolites in human blood Yi-Cheng Yanga, John Crowdera, Nick J. Wardlea, Li Yangb, Kenneth N. Whitea, Zheng-Tao Wanga,b, S. W. Annie Bligha* aInstitute for Health Research and Policy, London Metropolitan University, 166 - 220 Holloway Road, London N7 8DB, UK bKey Laboratory of Standardization of Chinese Medicines of Ministry of Education, Shanghai University of Traditional Chinese Medicine, 1200 Cai Lun Road, Zhangjiang Hi-Tech Park, Shanghai 201203, PR ChinaRoad, Zhangjiang Hi-Tech Park, Shanghai 201203, PR China Overview Monocrotaline (MCT) is a phytotoxin and belongs to a class of pyrrolizidine alkaloids obtained from Crotalaria spectabilis and Crotalaria retusa (Figure 1). Metabolites of MCT, dehydromonocrotaline (DHM) and dehydroretronecine (DHR), are reported to produce pathological injury not only to liver, but also to other organs, such as lung, kidney and heart, and the question arises how toxic metabolites being transported from the liver to other organs. This study is aimed at further understanding the Figure 1. Plants of Crotalaria spectabilis and Crotalaria retusa from the liver to other organs. This study is aimed at further understanding the transportation of MCT, DHM and DHR in blood in mediating its toxicity. For the first time, It is confirmed that both human plasma and Red Blood Cells (RBCs) can be the carriers for the transportation of MCT and its metabolites, DHM and DHR, from the liver to other organs by using 1H nuclear magnetic resonance spectroscopy (1H-NMR). It is found that the transport of MCT and RET by RBCs provides them a chance to develop toxic metabolites in other organs; and DHM and DHR are stabilized by interacting with plasma and RBCs during the transport and exert toxicities in the Aims and Objectives MCT and RET were found to enter RBCs and stable in the cell, while DHM could be hydrolyzed to give hydrolyzed necic acid and To investigate the state of MCT and its metabolites DHM, RET and DHR (Figure 2) in blood plasma and red blood cells by using 1H-NMR. To semi-quantified the uptake of MCT and DHM by RBCs. by interacting with plasma and RBCs during the transport and exert toxicities in the destined organs. lactone acid (H-17”, H-18” and H-19”) and DHR bind to the cell contents once being mixed with RBCs (Figure 4). Figure 4. NMR spectra of MCT and its metabolites in RBCs being recorded after 30 min incubation. Table 1. The uptake of MCT by RBCs after 0.5, 1, 1.5 and 2h incubation at 37°°°°C. MCT DHM RET DHR Figure 2 Chemical structures of MCT and its metabolites. The uptake of MCT and DHM was performed by measuring the amount of Incubation time (h) 0.5 1.0 1.5 2.0 Concentration of MCT in the 1st washing (mM) 0.7902 0.7327 0.7071 0.7104 MCT outside RBCs (%) 53.9 50.1 48.3 48.5 MCT entered RBCs (%) 46.1 49.9 51.7 51.5 Materials and Methods NMR study of plasma with MCT, DHM, RET and DHR MCT, DHM, RET and DHR were added into plasma and recorded by 1H-NMR immediately as well as after being incubated at 37°C overnight . NMR study of RBCs with MCT, DHM, RET and DHR RBCs were incubated with MCT, DHM, RET and DHR at 37°C for 30 min and then washed five times with PBS. The first, fifth washings and cells measuring the amount of MCT and DHM outside RBCs, which was mainly in the 1st washing. The uptake of MCT was summarized in Table 1. As the uptake of DHM could be contributed by its hydrolyzed ester products from outside cells, the RBCs enter rate of those products was obtained. The actual uptake of DHM by RBCs were showed in Table 2. Incubation time (h) 0.5 1.0 1.5 2.0 DHM hydrolyzed ester products in the buffer without the presence of RBCs (%) 71.2 71.3 72.9 74.4 DHM hydrolyzed ester products in the buffer with the presence of RBCs (%) 65.5 62.9 64.5 64.2 DHM hydrolyzed ester products entered RBCs (%) 5.7 8.4 8.4 10.2 Actual uptake of DHM by RBCs (%) 48.9 49.7 50.5 52.1 then washed five times with PBS. The first, fifth washings and cells contents were taken for 1H-NMR analysis. Time study of the uptake of MCT and DHM by RBCs Time studies of MCT and DHM internalised by RBCs were carried out at timed intervals of 0.5 h, 1 h, 1.5 h and 2 h. Results Discussion MCT and RET remained intact and weakly associated with plasma proteins; and DHM was rapidly hydrolysed releasing the Table 2. The uptake of DHM by RBCs after 0.5, 1, 1.5 and 2h incubation at 37°°°°C. Table 2. In this study we have demonstrated that by using 1H-NMR Discussion Conclusion Our findings confirm that MCT, a hepatotoxic retronecine pyrrolizidine alkaloid, is stable in blood plasma and can enter human red blood cells and stay intact. Reactions of DHM and DHR in both plasma and RBCs can prevent them from losing their alkylating abilities in other organs, such as lungs, to cause further damage. plasma proteins; and DHM was rapidly hydrolysed releasing the hydrolyzed necic acid (H-17’, H-18’ and H-19’). All proton signals of DHR disappeared immediately after mixing with plasma (Figure 3). Acknowledgements Yicheng acknowledges the financial support of Institute for Health Research and Policy at London Metropolitan University. The authors are grateful to Prof. Zheng Tao Wang (SUTCM) for his kind donation of PAs reference standards used in this study. In this study we have demonstrated that by using 1H-NMR spectroscopy, MCT and metabolites can be monitored for reaction in human blood. It confirmed that both human plasma and RBCs can be the carriers for the transportation of MCT and its metabolites, DHM and DHR, from the liver to other organs. Figure 3. NMR spectra of MCT and its metabolites in plasma being recorded immediately after mixing.

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