Effects of Hypoglycin A and methylenecyclopropylglycine (MCPG) First, we need to understand how the body reacts when there is not enough glucose in circulation: this can be induced by the absence of meal in poor and young children (teenagers, adults and well fed child have more stock of glycogen). So when we are in hypoglycemia (when the body doesn’t have enough glucose to degrade to produce ATP) the body need to find another source of energy and glucose so the gluconeogenesis is triggered. This metabolic pathway results in the generation of glucose from certain non-carbohydrate carbon substrates. Figure 1: the gluconeogenesis One of the main step of the gluconeogenesis is the beta-oxidation of fatty acids. Figure 3 : Beta-oxidation cycle (occur in liver cell mitochondria) The beta-oxidation is a succession of chemical reactions which occur in the mitochondria of the liver cells and which produce ATP (approximately 14 ATP per cycle) and 1 Acetyl-CoA which are essential for the Krebs cycle (and we all know the importance of the Krebs cycle). If you want to have more details about all the reaction during beta-oxidation, I suggest you to read this (short and in French) course about the lipids degradation (http://calamar.univ-ag.fr/deugsv/Documents/Cours/Bioch-Zinsou/Lipidegrad.pdf) or if you prefer to just watch someting, this video (https://www.youtube.com/watch?v=WX8sLclDyvw ). So now that we have a better understanding of the beta-oxidation, we can focus on the interaction between those toxins and the beta-oxidation cycle. Figure 4 : Conversion of MCPG and hypoglycine into active metabolites and their sites of inhibition of beta-oxidation adapted from Metabolic consequences of methylenecyclopropylglycine poisoning in rats. As shown on the Figure 4, this two toxins inhibit several enzyme necessary to the beta-oxidation cycle. MCPG mainly inhibits the 3-Oxoacyl-CoA thiolase so the beta-oxidation cycle cannot produce Acetyl-CoA anymore. Hypoglycin A target mainly Acyl-CoA dehydrogenase and stop his action, so there is an accumulation of Branched-chain-oxo-acid dehydrogenase and that can be toxic. To conclude, those two toxins block the gluconeogenesis which are essential when there is not enough glucose supply to do the glycolysis who produce ATP and glycogen in normal case. And because of the absence of evening meal and glucose stocks in these children, they cannot perform the glycolysis and due to the litchi and toxin consumption they cannot perform gluconeogenesis. The main problem of this type of hypoglycemia is that the brain have a very small stock of glucose so very quickly (in a few minutes) after the “consumption” of all the glucose stocks, the ATP levels become depleted. More than the lost of cerebral energy, the main problem of the lack of glucose is the reduced of synthesis of neurotransmitter. Indeed, glucose oxidation in the brain provide several precursors for the synthesis of some neurotransmitter (acetylcholine, glutamate,...). And when the hypoglycemia is really acute, like here,there are neuronal cell death ensues, particularly in hippocampal and cortical structure. So this is why the children arrived why seizure and falled quickly in coma or death. The discovery of the two toxin in the litchi are new and not so well described in human and there is still some part a little bit vague. For example, there is a possibility that this toxin also impact carnitine which allow the way between mitochondria and cytosol for fatty acid, acetyl-CoA and other molecules. Also, there is a high possibility that this disease have some genetic factor, because some children didn’t become ill even if they have eat litchi only all day. So there is still a lot of things to discover around this two toxins. What you should bring home ? Litchi contain two toxin which disturb and block some part of ATP and glucose production. In “normal” case (adults, well feed, few litchi eaten), the other system of energy product will not be disturbed because we have enough glucose stock to avoid the hypoglycemia. But for Indian child who eat only litchi in an entire day, without a correct meal and without glycogen reserve, those toxin become dangerous and mortal. To avoid this, Indian Government has written some basics recommendation: don’t let the child eat litchi in orchard all day and give them a strong diner before let them all night without glucose intake. Figure 5 : Schema to summarize all our case
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In the team in which I do my internship, there is a habit: every Friday we meet all together to explain what we have done during the week and each week a student presents an article or a particular subject. Last Friday, it was my turn to present something and I thought that it could maybe be interesting for you too. It’s not really in relation to my internship subject but it’s a very actual problematic. What happened ? Figure 1: Map of the Bihar region, India, the point is Muzaffarpur (from Google map) As you may know, since now many years (1995 approximately) in Bihar area in India, every spring a lot of children are affected by a mysterious disease and each year many child die (30% of the affected children). In the figure 2, we can see that during June more than 15 children arrived each day in the hospital and in pink we can see that a lot of kids died during this period. There is no time between the first cases and the first deaths. They arrive in the hospital with different symptoms: seizure, fever, coma caused by acute low blood glucose level which leads to encephalopathy. (1) Figure 2: Acute neurological illness by date of hospital admission in Muzaffarpur, May 29–July 17, 2014 (2) This mysterious disease made the headline in the past 2 months thanks to The Lancet Global Health. Indeed the journal published a global alert with the explanation of the origin of this disease. First hypothesis Since 1995, scientists and doctors have made several hypothesis about this illness. First, they thought of Japanese encephalitis, heat stroke or unusual medication, but the precise and restrict location, as well as the precise period of the disease development eliminated these possibilities. During the research, scientists have established an interesting correlation. Bihar is one of the poorest areas in India and the largest litchi fruit (Litchi chinensis) cultivation region in the country. And as a random part, the season of litchi harvesting is during spring precisely at the same time as the emergence of the disease each year. With these pics of information scientist started to search for causality between the disease and the cultivation. They investigated different ideas: the pesticide, herbicides and insecticides used in the cultivation, the exposure to heavy metals in the earth of the cultivation and a possible zoonotic origin, due to bats bacteria (the bats bit the fruit and let saliva in fruit, eaten by the children afterwards). And the last hypothesis was the presence of an environmental toxin. (3) Method To determine which hypothesis is the good one, a case control study was performed by Indian and American scientists in Muzaffarpur (see map above) and published in The Lancet Global Health accompanied by the commentary coming as a warning on January 30, 2017. To carry out this survey, scientists went to two hospitals in Muzaffarpur and studied 390 patients under 15 years with new-onset of seizure or altered sensorium. The control subjects were age-matched children admitted to the same hospitals for non-neurologic illness. Clinical specimen (blood, cerebrospinal, urine) were collected from each patient. (4) The patient profils As we already saw, this disease affects only the children (median age around 4 years and nobody above 15 years), almost all of them come to the hospital with generalized seizure and altered mental status and their level of glucose in blood or cerebrospinal fluid (CSF) glucose are very low. All the tables are extract from Association of acute toxic encephalopathy with litchi consumption in an outbreak in Muzaffarpur, India, 2014: a case-control study (Shrivastava, Aakash et al. The Lancet Global Health , Volume 0 , Issue 0) After interview with the parents, scientifics also established several similar behaviors, 65% of the child have ate litchi and the index of confidence for this fact is very high (red frame on Table 2) and the absence of evening meal for 78% of the subjects. Figure 3: picture of litchi (Litchi chinensis, Sapindacea) So now after that they try to find if there is a causality between death and litchi in addition to the correlation. Results Tests for several viruses (Japanese encephalitis, West Nile and enterovirus has been performed. As well as the examination of blood and urine to find pesticide or toxins. And litchi were also examined for pesticide residues. All the samples were negative for all the assays tested. But when they analysed urine in search for some toxins, they found some interesting things. Table 3: Analysis of acylcarnitine, organic urinary acids, and metabolites of hypoglycin A and MCPG in cases and controls in Muzaffarpur, 2013–14 Data are n/N (%). NA=not available. MCPG=methylenecyclopropylglycine. We can see in the Table 3 that there are some toxins in the children urine. Indeed, hypoglycin A and MCPG are toxins who disturb the fatty oxidation cycle, they are metabolite related to the common amino acid lysine. The abnormal acylcarnitine profile and the abnormal urine organic acid profile are direct consequences of the two toxins effects. Acylcarnitine is an ester of fatty acid and carnitine who allow the fatty acids to cross the mitochondrial membrane. Hypoglycin A was already known in another fruit, the unripe ackee (Blighia sapida, Sapindacées). This fruit is at the origin of an acute encephalopathy disease called Jamaican vomiting sickness which have many similarities with the Muzaffarpur children onset. Figure 4: picture of an ackee (Blighia sapida, Sapindacées) After the discovery of this type of toxin in children urine, scientists searched in litchi fruits if the toxin comes from him or from another environmental source. As we can see in the table 4, there is MCPG and hypoglycin A in the litchi, even if they are ripe. So now we can assume the correlation between the disease and the litchi consumption. Table 4 : Analysis of hypoglycin A and MCPG in litchi fruit arils in Muzaffarpur, 2014 MCPG=methylenecyclopropylglycine. Both the ripe and unripe groups contained 6 homogenates. Once again something need to be noticed, in the table 2 we saw that almost all the kids didn’t have an evening meal (78%), children who have eat an evening meal didn’t have or much less morning onset. This is due to the effect of the toxin on the glucose metabolism. Now that you know the history and the culprit of those mysterious deaths, it's time to know how the toxins can affect the children and kill them. But this will be for the next episode ;) Sources:
- (1) http://edition.cnn.com/2017/02/01/health/lychee-fatal-illness-india/ - (2) The enigma of litchi toxicity: an emerging health concern in southern Asia, Spencer, Peter S et al. The Lancet Global Health , Volume 0 , Issue 0 , - (3) John, TJ and Das, M. Acute encephalitis syndrome in children in Muzaffarpur: hypothesis of toxic origin. Curr Sci. 2014; 106: 1184–1185 - (4) Association of acute toxic encephalopathy with litchi consumption in an outbreak in Muzaffarpur, India, 2014: a case-control study, Shrivastava, Aakash et al. The Lancet Global Health , Volume 0 , Issue 0 , During my internship I will work on a project in collaboration between BioCIS laboratory (Laboratoire Pharmacognosie et Substance naturelles, CNRS UMR 8076, Faculté de Pharmacie, Châtenay Malabry), the Faculté de Pharmacie de Paris Descartes (Chimie des Substances Naturelles, Electrochimie UMR CNRS 8638 COMETE) and the Conservatoire National des Plantes à Parfum, Médicinales et Aromatiques de Milly la Forêt. What are the research areas at BioCIS ? In BioCIS laboratory, there is several fields of research: extractive and analytical chemistry to find new active components in different plants and other natural sources (sponges, insects,... ) from Africa, New Caledonia or Guyane. And also synthesis and methodology to synthesis natural products and different analysis (RMN, GC/FID, biologic activity, toxicologic,...) on natural products which can influence the central nervous system or have an antiparasitic action. (1) However, the natural product are more than ever a big part of the medicine world. Indeed, in some poor country they don’t have access to modern drugs and still use knowledge of the elders and wild plants for curing diseases. And even in the western world most of the drugs come from plants or were based on them, as you can see below. (2) Figure 1: The different type of new approved drug between 1981 and 2014 (2) And also more and more people turns to more traditional medicine, full of plants, for their health. So one of the big issues, in this research fields, are to explore the possible toxicity of the plants, the dosage, often unknowns, and the quality control of the natural products.(3) What is the project for which I am going to work ? The conservatory of Milly is a semi-public, professional association who tries to save our natural heritage and make everyone discover the amazing world of useful plants. For example, they select and product seeds for aromatic and medicinal plants cultivation. (4) They have a new project to share the benefit of their plants: to produce essential oil from 6 different plants, lemon balm, hyssop, thyme, sage, everlasting and mint (Melissa officinalis, Hyssopus procumbens L. var. decumbens, Thymus vulgaris L. (CT * thuyanol), Salvia sclarea L., Helichrysum italicum and Mentha x piperita L.). (5) I will write another blog post to describe essentials oils in general and the benefits and toxicity of the essential oil of each of this species. ( * CT = chemotype = “plants that are practically indistinguishable from one another in appearance but are nevertheless unique in their composition and therefore are used to treat distinct diseases”. (6)) For the moment they have tried different crops condition:
Figure 2: Various location of plantation with different conditions of soil, sun,...
What is the goal of this internship ? The aim of my internship is to analyze all the samples with GC/MS and then with GC/FID, to compare them, using a metabolomic approach when needed, to identify each components, to check if it’s relevant with the French and European standard (Pharmacopée, AFNOR ) (7) and to determine which combination of conditions is the best in terms of composition. Sources:
Annex: Timeline of all the project, realized by ADéPAM For the moment, I analyze the essential oil from the 2a) rectangle. I added this timeline thanks to Noémie to give an overview of the project timeline.
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