Identification and quantification of biomarkers to confirm the poisoning by Ginkgo biloba seeds in a 2-year-old boy

The seeds of Ginkgo biloba are commonly eaten in Japan, Korea and China, but it is important to know that they can be responsible for poisoning, especially in young children. Poisoning due to Ginkgo biloba seeds must also be considered as a possible clinical event in European countries, where Asian cuisine has recently become popular. This paper reports the case of a 23-months-old previously healthy male child who experienced two afebrile tonic-clonic seizures after the consumption of an unknown amount of Ginkgo seeds. The poisoning was identified by searching both in blood and urine for 4’-Omethylpyridoxine (MPN) as a specific biomarker for ginkgo poisoning, using an optimized and validated HPLC method with fluorimetric detection. The MPN concentrations in serum were 16.5 and 6.2 ng/mL 14 and 20 hours after the exposure, respectively, while the urine concentration was below the limit of detection (1.35 ng/mL). The involvement of ginkgo seeds in the described poisoning was confirmed by searching ginkgolide terpene lactones in urine, where the ratio between excreted ginkgolide A:B:C was 1:40:2. The analytical data obtained by looking for biomarkers in serum and urine demonstrate that even low values of MPN/ginkgolide concentration can be responsible for convulsions and other adverse effects. Introduction Ginkgo biloba (commonly known as ginkgo or Maindenhair tree) is an ancient tree with distinctive fan-shaped leaves, belonging to the family of Ginkgoaceae. Leaves and seeds are the parts most usually consumed, both as food (roasted or cooked seeds) and as extracts in food supplements and traditional medicines. The leaf extract is widely used in Europe as a traditional drug to improve the symptoms of early-stage Alzheimer's disease, vascular dementia, peripheral claudication and tinnitus of vascular origin. The adverse effects of Ginkgo biloba preparations containing leaves are numerous; approximately 30% of these cases are due to interaction with therapeutic drugs, including acetyl salicylic acid, ibuprofen and warfarin. The haemorrhagic complications, due to poisoning or interaction with prescription drugs, are probably due to the antiplatelet activity of ginkgosides, and in particular of Ginkgolide B. Ginkgo biloba seeds are commonly eaten in Japan, Korea and China for their nutritive value, but it is important to know that they can be responsible for poisoning, especially in young children. Even though they are infrequent, clinical cases of ginkgo seed poisoning show severe symptoms, including tonic or clonic convulsion, vomiting, irritability and loss of consciousness. Poisoning by ginkgo seeds is due to the neurotoxic compound 4'O-methylpyridoxine (MPN), which is chemically related to vitamin B6 and interfere with its biosynthesis, metabolism and function. The chemical structures of MPN and piridoxine are reported in Fig. 1. Ginkgo biloba seeds contain MPN at concentrations ranging between 170 and 400 mg/g of raw seeds. 10-13 The toxin is relatively heat-resistant so that cooking or other treatments only partially inactivate it. Kajiyama & Fujii (2002) described a case of poisoning in a 2-year-old girl who showed unexplained convulsions with vomiting, diarrhoea and irritability. The event was associated with the ingestion of 50 to 60 roasted ginkgo seeds, as reported by the parents. Similarly, a case of poisoning was described in another 2-year-old girl who had eaten about 50 ginkgo seeds 4 hours before the onset of symptoms 7 and a case of poisoning with ginkgo nuts was observed in a healthy 36-year-old woman, who consumed 70-80 seeds cooked in a microwave oven. The pathophysiologic mechanisms responsible for convulsions related to ginkgo poisoning are not fully understood. As known from in vivo studies, 4’-O-methylpyridoxine (MPN) competes with vitamin B6, which is a cofactor of glutamate decarboxylase; as a consequence, MPN indirectly inhibits the activity of this enzyme resulting in a decrease of the -aminobutyric acid (GABA) level in brain, which predisposes to convulsions. The study reported in this paper describes a case of ginkgo poisoning which occurred in Switzerland in a 2-year-old boy, where the association with the ingestion of ginkgo seeds was confirmed by measuring biomarkers both in blood and urine. Materials Clinical data A 23-months-old previously healthy male child of Asian origin experienced two afebrile tonic-clonic seizures for 15-30 seconds. He had had a 2-hour period of vomiting and somnolence. The parents reported that the child ingested an unknown amount of Ginkgo seeds 10 hours before the beginning of symptoms. The subsequent clinical course was uneventful with full recover. Blood samples were taken 14 and 20 hours after the ingestion of seeds, while urine was collected only 14 hours after intake. The patient’s family consented to the publication of this case report. Purified standards 4’-O-methylpyridoxine (MPN) The toxin 4'-O-methylpyridoxine was not commercially available at the time of the study, so that we synthesized it ourselves according to the method described by Harris (1940). A mixture of 2-methyl3-hydroxy-4,5-dihydroxymethylpyridine hydrochloride (10.25 g, 50.0 mmol) and sodium methoxide (2.71 g, 50.0 mmol) in methanol (80 mL) was heated in a bomb tube at 130 °C for 12 h. The solvent was evaporated off and the residue treated with acetone and filtered to remove the sodium chloride. The organic solution was concentrated to give an oily residue, which was chromatographed on silica gel. Elution with ethyl acetate/methanol (80:20, v/v) yielded 5.2 g (56.5%) of 2-methyl-3-hydroxy-4methoxymethyl-5-hydroxymethylpyridine as an orange oil. The compound was dissolved in ethanol (20 mL) and 2.6 M ethanolic HCl (15 mL) was added while stirring. The suspension was heated at reflux and, after cooling to 0°C, filtered to give 4.1 g (37.5%) of the compound as a white solid; the final product was chemically characterized to confirm the identity, as described in Results and Discussion.