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The quantification of methylenecyclopropyl compounds and acyl conjugates by UPLC-MS/MS in the study of the biochemical effects of the ingestion of canned ackee (Blighia sapida) and lychee (Litchi chinensis). Johannes Sander, Michael Terhardt, Stefanie Sander, and Nils Janzen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00224 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 14, 2017

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Journal of Agricultural and Food Chemistry

The quantification of methylenecyclopropyl compounds and acyl conjugates by UPLC-MS/MS in the study of the biochemical effects of the ingestion of canned ackee (Blighia sapida) and lychee (Litchi chinensis).

Johannes Sander* a, Michael Terhardt a, Stefanie Sander a, and Nils Janzen a,b

a

Screening-Labor Hannover, Postbox 91 10 09, 30430 Hannover, Germany

b

Department of Clinical Chemistry, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, Germany

*Corresponding author: E-mail address: [email protected] (J. Sander). Tel +49 5108 92163 31

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Abstract

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The consumption of ackee (Blighia sapida) and lychee (Litchi chinensis) fruit has led to

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severe poisoning. Considering their expanded agricultural production, toxicological

4

evaluation has become important. Therefore, the biochemical effects of eating 1 g/kg canned

5

ackee containing 99.2 µmol/kg hypoglycin A and 5 g/kg canned lychee containing 1.3

6

µmol/kg hypoglycin A were quantified in a self-experiment. Using ultra high performance

7

liquid chromatography/mass spectrometry, hypoglycin A, methylenecyclopropylacetyl-

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glycine and methylenecyclopropylformyl-glycine, as well as the respective carnitine

9

conjugates, were found in urine after ingesting ackee. Hypoglycin A and its glycine derivative

10

were also present in the urine after eating lychee. The excretion of physiological acyl

11

conjugates was significantly increased in the ackee experiment. The ingestion of ackee led to

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up to 15.1 nmol/L methylenecyclopropylacetyl-glycine and traces of

13

methylenecyclopropylformyl-carnitine in the serum. These compounds were not found in the

14

serum after eating lychee. Hypoglycin A accumulated in the serum in both experiments.

15 16

Keywords

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Ackee poisoning, Jamaican vomiting disease, Lychee, Litchi, Sapindaceae, Hypoglycin A,

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Methylenecyclopropylglycine, Methylenecyclopropylacetyl-glycine,

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Methylenecyclopropylacetyl-carnitine, Methylenecyclopropylformyl-glycine,

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Methylenecyclopropylformyl-carnitine, Inhibition of ß-oxidation, Acyl-CoA dehydrogenase

22

deficiency.

23 24


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Introduction

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Jamaican vomiting disease is a typical manifestation of ackee (Blighia sapida)

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poisoning. It affects both the digestive tract and the central nervous system. The disease has

28

been observed mainly in children eating the unripe fruits of ackee trees1-6. Encephalopathy in

29

children possibly caused by lychee (Litchi chinensis) has recently garnered great attention7-12.

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In a systematic analysis of a large outbrake of acute encephalopathy Shrivastava et al.13 very

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recently proved an association with lychee consumption. Many cases of acute poisoning have

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been described, but hardly anything is known about the toxicological reactions following the

33

ingestion of small quantities of fruits usually eaten with meals that normally cause no obvious

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clinical symptoms. Low doses of toxins may be of special interest when ingested by

35

individuals showing particular pre-existing disease conditions or certain genetic traits.

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Ackee and lychee trees belong to the botanical family Sapindaceae, which also

37

includes Acer species. The seeds of Acer species have recently been found to cause atypical

38

myopathy in horses14, 15. Blighia sapida is native to West Africa, but it is now also cultivated

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in other regions, such as the Caribbean and Florida. Ackee is a staple food in Jamaica and has

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become a heraldic plant of this country. Ackee and saltfish is a traditional Jamaican dish. The

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arils of the fruit are consumed boiled or fresh. Canned ackee is a major export product of

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several subtropical countries, especially Jamaica. Lychee originally comes from southern

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Asia, but it is now an important agricultural product in many subtropical countries. It is sold

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as fresh or canned fruit.

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The fruits of Sapindaceae, especially ackee and lychee, are known to contain

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hypoglycin A (HGA)16-19. There are several chemical names for the compound, but the

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designation methylenecyclopropylalanine underlines its amino acid structure. The amino acid

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may also form a dipeptide with glutamate, and this compound is called hypoglycin B (HGB).

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Both compounds are toxic18, 19. In addition, another homolog of HGA,


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methylenecyclopropylglycine (MCPG), has been detected in Sapindaceae fruit20-22. MCPG

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also may form a dipeptide with glutamate21.

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With the aim to protect people against poisoning with HGA, which is not destroyed by

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heating, the Food and Drug Administration (FDA, USA) has defined concentration limits for

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HGA in canned ackee. Import to the USA is only permitted when the level of HGA has been

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shown not to exceed 100 mg/kg of canned ackee23. There are no comparable restrictions based

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on toxicological limits for exporting or importing lychee or fruits of other Sapindaceae

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species.

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The clinical symptomatology and pathogenesis of ackee poisoning have been

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described repeatedly in cases of severe accidental poisoning24-27. Neither a relation between

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quantitiy of ingested fruit and toxic effect nor differences in toxin availability in fresh as

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compared to canned fruit have been examined. There are some data on experimental HGA

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and MCPG poisoning in laboratory animals using high doses19, 30-32. The biochemical

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background of HGA poisoning in horses has recently been analyzed and reviewed33, 34.

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HGA and MCPG themselves are not toxic. They first have to be metabolized over

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several steps to MCPA-CoA and MCPF-CoA. While MCPA-CoA reacts with acyl-CoA

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dehydrogenases, MCPF-CoA reacts with enoyl-CoA hydratases35-38. The CoA esters are

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bound but not metabolized. In this way, MCPA-CoA interrupts the first step of the ß-

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oxidation of fatty acids. As acyl-CoA dehydrogenases responsible for the degradation of

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branched-chain amino acids are part of the spectrum of inhibited enzymes, the metabolism of

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leucine, isoleucine and valine is also affected. MCPF-CoA mainly inhibits the second step of

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the ß-oxidation spiral. The consequences of inhibition of ß-oxidation by both toxins are

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similar. Acyl residues which are not further degraded are excreted as organic acids into the

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urine. Acyl residues may also be conjugated to amino acids, especially to glycine , or to

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carnitine. Toxicokinetic differences may exist between MCPG and HGA. Nothing, however,

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is known about the effects caused by the simultaneous action of the two homologs. ACS Paragon Plus Environment

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Sensitive and specific laboratory methods for detecting HGA and MCPG have been

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developed; some methods quantify the metabolites of HGA or MCPG conjugated to glycine

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or carnitine10, 14, 15, 39-42. We measured the levels of toxins and their metabolites

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simultaneously with the products of fatty acid ß-oxidation43, 44. In this way, it is possible not

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only to prove the ingestion of HGA and MCPG but also to assess the toxicological

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consequences in a single analytical procedure. In this study, we apply a variation of this

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method that made it sensitive enough to quantify low concentrations of the relevant poison

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and typical metabolites.

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Our objective was to use a self-experiment as a model for future research to assess

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whether eating a small quantity of canned ackee or lychee that did not induce clinical

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symptoms might be sufficient to lead to measurable concentrations of HGA in the serum and

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the significant excretion of metabolites of HGA and MCPG. Additionally, the ability to

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produce biochemical effects indicating toxicological reactions was probed. For demonstrating

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an inhibitory effect on fatty acid metabolism we considered a spectrum of 8 acyl conjugates

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of short and medium chain length sufficient, for future testing the spectrum of acyl

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compounds, however, may be extended.

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Materials and methods

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Experimental design

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Canned ackee and lychee were eaten in two separate experiments. A healthy adult

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male volunteer, non-smoker, not taking any medication, consumed the test material early in

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the morning without additional food or drink. Regular meals consisting of potatoes,

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vegetables and some meat and bread were than taken in at 1 pm and 7 pm.

99 100

The ackee experiment was performed by ingesting 1 g per kg body weight of canned ackee fruit (Caribbean Food Centre, Grace Foods UK). The concentration of HGA in the


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drained fruit, measured using our established method43, was 99.2 µmol/kg (14 mg/kg).

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Because of the lack of original MCPG, we were not able to quantify this compound.

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For the lychee experiment, the same volunteer ate 5 g per kg body weight of canned lychee (Bonasia, China). The concentration of HGA was 1.3 µmol/L (0.18 mg/kg). Spontaneously voided urine was sampled in intervals, as shown in table 4. Ten

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minutes before sampling, the bladder was emptied. The urine that was produced within the

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following 10 minutes was then collected. The serum was obtained after clotting and

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centrifugation of periodically collected blood (table 6). Subjectively no adverse effects after

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the ingestion of the test meals were observed.

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Chemicals General laboratory reagents were of the same analytical grade as stated in the method

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published earlier44. The chromatographic chemicals used were commercial products of the

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highest quality made for liquid chromatography and mass spectrometry (Biosolve BV,

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Valkenswaard, The Netherlands). Butanolic HCl (3 N) was the highest analytical quality

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available (Fluka, Deisenhofen, Germany). HGA was purchased with a purity of 85% (Toronto

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Research Chemicals, Toronto, Canada). Labeled and unlabeled MCPA-G and MCPF-G were

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supplied from IsoSciences (King of Prussia, Pennsylvania, USA) as > 97% pure. Mass

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transitions and concentrations of the internal standards used are listed in table 1. They were

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dissolved in methanol.

121 122 123

Analysis In our study, we applied a modification of a previously published method43. To be

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sensitive enough to measure subtoxic concentrations, the sample volume was increased, and

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the dilution of the extracts was reduced. Briefly, a methanolic internal standard solution (300

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µL) was added to 25 µL of serum or urine for extraction. The mixture was vortexed for 20 ACS Paragon Plus Environment

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seconds and centrifuged for 10 minutes at 17,000 RCF. A total of 250 µL of the cleared

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supernatant was dried in a microtiter plate at 65 °C for approximately 30 minutes under a

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gentle stream of nitrogen. The residue was treated with 50 µL of 3 N butanolic HCl for 15

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minutes at 65 °C and dried again at 65 °C under nitrogen. The dry material was dissolved in

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70 µL methanol/water (80:20 vol/vol) and further diluted 1:2 with water. From this solution,

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90 µL was transferred to a 384-well microtiter plate, centrifuged at 17,000 RCF in order to

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sediment any particles and then used for ultra high performance chromatography-tandem

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mass spectrometry (UPLC-MS/MS) analysis. From the solution, 5 µL was injected onto an

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ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 x 50 mm, Waters, Eschborn, Germany) for

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gradient chromatography44. Tandem mass spectrometric analysis was conducted with single-

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point calibration on a Xevo TQ-MS UPLC-MS/MS system (Waters). The mass transitions are

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listed in table 2. The concentrations of creatinine in the urine were measured by the Jaffee

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kinetic method using a commercial test kit (Bioanalytic GmbH, D79224 Umkirch, Germany).

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Quality criteria Quality criteria were established using spiked serum and urine from a volunteer. A full

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evaluation of the analytical quality was conducted for HGA, MCPA-G and MCPF-G. As

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quantification of the levels of medium chain acyl conjugates has been a standard procedure in

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pediatric laboratories for many years, we confined the evaluation to intra-assay CV values.

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For MCPA-C and MCPF-C no authentic material was available. Therefore we deducted

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concentrations from the peak height of the internal standards reported in table 2. Values

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obtained in these cases do not represent absolute quantifications but rather a relative

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quantification. We established CV values for these compounds using the serum of a horse

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available from our earlier studies43. The horse had ingested seeds of Acer pseudoplatanus.

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Results ACS Paragon Plus Environment

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Analytical performance Chromatographic separation was achieved for all compounds of interest within a run

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time of 14 minutes. The intra-assay and inter-assay imprecision values for HGA, MCPA-G

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and MCPF-G are summarized in table 3. The linearity measurements of the quantitative

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results for these compounds were found to be in the range of 1 to 100 nmol/L, with the r2

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values being > 0.98 in serum and urine. The lines, however, do not run through the origin,

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indicating a low nonspecific signal. A CV below 20% was used as a basis for calculating the

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lower limit of quantification (LLOQ). Thus, we defined 10 nmol/L for MCPF-G, MCPA-G

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and HGA in the urine and serum as the LLOQ. With this concentration we found CV values

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for spiked serum of 12.2, 17.0 and, 14.1 and for spiked urine of 14.6, 19.9 and 5.8 for

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MCPFG, MCPAG and HGA respectively. We found an intra-assay CV for MCPF-C of 8.0%

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at a concentration of 70.0 nmol/L in the serum. This value for MCPA-C was 6.5% at a

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concentration of 170 nmol/L. The intra-assay CV values in the blank serum of the volunteer

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for acyl-carnitines were below 5% at the concentrations given. The concentrations of

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hexanoyl-glycine in the serum were too low to be quantified. In blank urine, the CV values

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were also below 5%, except for that of butyryl-carnitine (7.5%).

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Urine samples: Following the ackee meal, the excretion of HGA and the metabolites of HGA and

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MCPG was observed beginning with the first sample collected 1 hour after ingestion (table 4).

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At this point, the highest concentrations were found for MCPA-G. The corresponding

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carnitine derivative was excreted only in trace amount. The excretion of the MCPF conjugates

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proved the presence of MCPG in the ackee fruit. The excretion of HGA and specific

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metabolites lasted for at least 24 hours. As shown in table 5, elevated concentrations of 7 acyl

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conjugates were found in the urine after ingestion of ackee fruit.


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In the lychee experiment, the urine concentration of MCPA-G reached 114 nmol/L,

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while MCPF-G was found in trace amounts. Significant amounts of each compound (61

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nmol/L and 14 nmol/L, respectively), however, were still excreted after 24 hours. The

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corresponding carnitine derivatives did not accumulate to quantifiable levels. HGA was

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excreted in trace amounts during the 24 hours of the experiment. Unlike the ackee meal, the

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lychee meal did not induce elevated excretion of physiological acyl conjugates.

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Serum samples:

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Corresponding to much higher concentrations of HGA in the ackee material, the

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maximal concentrations of HGA found in the ackee experiment exceeded those measured

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after ingestion of lychee by a factor of more than twenty. The concentration of HGA remained

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high for 10 hours (table 6). The compound was still present in trace amounts in a serum

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sample collected 24 hours after ingesting lychee. Unfortunately, serum was not collected after

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24 hours in the ackee experiment. The ingestion of ackee led to levels of MCPA-G up to 15.1

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nmol/L and traces of MCPF-C. MCPA-C and MCPF-G did not accumulate to quantifiable

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levels. The metabolites of HGA and MCPG were not found in the serum collected after eating

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lychee.

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Following ackee ingestion, no significant alterations in 4 out of the 6 acyl conjugates

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measured were found in the serum samples. However, isobutyryl carnitine and isovaleryl

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carnitine showed a steady increase in concentration from 320 to 678 nmol/L (p < 0.05) and 54

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to 286 nmol/L (p < 0.05), respectively, within 10 hours. Elevated concentrations of medium-

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chain acyl conjugates were not seen in the serum after the ingestion of canned lychee.

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Discussion Inhibition of enzymes responsible for the stepwise degradation of CoA activated fatty acids is the basis of toxicity of HGA and MCPG, as mentioned above. These enzymes are ACS Paragon Plus Environment

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involved in energy production by fatty acid oxidation and are active in the degradation of

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branched-chain amino acids. At the present state of research, it is not possible to define a safe

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concentration of HGA or MCPG or their metabolites in serum.

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Considering the inhibition of fatty acid ß-oxidation by the toxins of Sapindaceae,

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clinical experience with genetically based alterations in fatty acid metabolism may be cause

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for concern. Heterozygous carriers, as well as some compound heterozygous individuals, may

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have reduced activities of the enzymes of the ß-oxidation system but appear healthy as long as

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certain stress situations do not occur. Muscular activity, fasting or febrile illness has

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repeatedly been shown to be sufficient to induce metabolic decompensation, including severe

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hypoglycemia and organic aciduria. Neuropathy, myopathy and retinopathy may follow45. In

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the pregnancies of heterozygous women, severe pre-eclampsia, liver disease and intrauterine

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growth retardation of infants have been observed46. No information is available on whether

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the otherwise tolerable ingestion of Sapindaceae fruits may have similar consequences in the

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case of febrile infections or other metabolic stress factors. It is not known to what extent a

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genetically based reduced capacity for ß-oxidation in heterozygous carriers may be

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dangerously affected by toxic compounds of Sapindaceae at concentrations irrelevant to

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healthy individuals. No studies have been conducted to determine whether eating the fruits of

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Sapindaceae might pose a special risk for children or adults heterozygous for any genetic

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deficiency in the ß-oxidation of fatty acids or branched-chain amino acid metabolism.

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In a study on cases of acute encephalitis syndrome, Isenberg et al.10 found

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concentrations of MCPA-G up to 54,900 nmol/L in urine samples. This value is

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approximately 5 times higher than the highest concentration measured in our ackee

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experiment, which did not induce any symptoms. The lowest concentration reported by

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Isenberg et al.10 was 680 nmol/L. In the same samples, the concentrations of MCPF-G,

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however, ranged between 677 and 16,903 nmol/L. These values exceeded ours by several


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orders of magnitude. Whether these children had ingested not only the flesh but also other

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parts of the fruit is not mentioned.

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A major route of elimination of HGA and MCPG is detoxification by the conjugation

232

of metabolites to glycine and other compounds for urinary excretion. Conjugates of MCPA

233

and MCPF have been shown here to be excreted for many hours after the consumption of

234

HGA- and MCPG-containing material. However, within 24 hours, the levels had fallen

235

considerably. The concentration profile might have been different if instead of canned

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material fresh fruit had been ingested. In contrast to the conjugates of MCPG and HGA, the

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excretion of acyl conjugates of medium chain fatty acids showed a tendency to increase

238

within the course of time; in serum the concentrations of isobutyryl and isovaleryl carnitines

239

increased during the test period. In ackee poisoning, symptoms of the central nervous system

240

usually appear several hours after vomiting has occurred. Whether the accumulation of

241

metabolites plays a role has not been examined.

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When looking at the variability of the concentrations over the time course, it becomes

243

apparent that for toxicological evaluations, it is important to obtain samples under controlled

244

conditions. In addition, as shown here, positive test results for HGA, MCPG and their

245

metabolites may serve as indicators of the ingestion of fruits of Sapindaceae, but they do not

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justify the conclusion that the fruit toxins are the cause of an observed case of severe disease.

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Only simultaneous demonstration of the toxins, along with evidence of severe inhibition of

248

enzymes in the ß-oxidation spiral, such as elevated excretion of organic acids or - as used in

249

this study - acyl conjugates, is diagnostic. However, to this day, no fixed limits have been

250

described for any of the metabolites which could be used for defining a degree of inhibition to

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be called severe. As described here partial inhibition may not cause any clinical symptoms.

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The experimental setup as described here will allow for studies to broaden the

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knowledge regarding ackee and lychee poisoning, which will contribute to the better

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assessment of the risks linked to the consumption of these fruits. ACS Paragon Plus Environment

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255 256

Abbreviations

257 258

HGA

hypoglycin A

259

MCPG

methylenecyclopropylglycine

260

CoA

Coenzyme A

261

MCPA-CoA

methylenecyclopropylacetyl-CoA

262

MCPF-CoA

methylenecyclopropylformyl-CoA

263

MCPA-G

methylenecyclopropylacetyl-glycine

264

MCPA-C

methylenecyclopropylacetyl-carnitine

265

MCPF-G

methylenecyclopropylformyl-glycine

266

MCPF-C

methylenecyclopropylformyl-carnitine

267

UPLC-MS/MS

ultra high performance liquid chromatography-tandem mass

268

spectrometry


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References 1. Scott H. On the vomiting sickness of Jamaica. Annals of tropical medicine and parasitology 1916, 10, 1-78

2. Hassall, CH.; Reyle, K. The toxicity of the Ackee (Blighia sapida) and its relationship to the vomiting sickness of Jamaica. West Indian Med J. 1955, 4, 83-90

3. Gaillard, Y.; Carlier, J.; Berscht, M.; Mazoyer, C.; Bevalot, F.; Guitton, J.; Fanton, L. Fatal intoxication due to ackee (Blighia sapida) in Suriname and French Guyana. GCMS detection and quantification of hypoglycin-A. Forensic Sci Int. 2011, 206, 1-3

4. Barceloux, DG. Akee fruit and Jamaican vomiting sickness (Blighia sapida Köenig). Dis Mon. 2009, 55, 318-326

5. Joskow, R.; Belson, M.; Vesper, H.; Backer L.; Rubin C. Ackee fruit poisoning: an outbreak investigation in Haiti 2000-2001, and review of the literature. Clin Toxicol (Phila). 2006, 44, 267-273

6. Katibi, O.S.; Olaosebikan, R.; Abdulkadir, M.B.; Ogunkunle, T.O.; Ibraheem, R.M.; Murtala, R. Ackee Fruit Poisoning in Eight Siblings: Implications for Public Health Awareness. Am J Trop Med Hyg. 2015, 93, 1122-1123

7. Shrivastava, A.; Srikantiah.; P, Kumar, A.; Bhushan, G.; Goel, K.; Kumar, S.; Kumar, T.; Mohankumar, R.; Pandey, R.; Pathan, P.; Tulsian, Y.; Pappanna, M.; Pasi, A.; Pradhan, A.; Singh, P.; Somashekar, D.; Velayudhan, A.; Yadav, R.; Chhabra, M.; Mittal, V.; Khare, S.; Sejvar, J.J.; Dwivedi, M.; Laserson, K, .; Earhart, K.C.; ACS Paragon Plus Environment

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Sivaperumal, P.; Kumar, A.R.; Chakrabarti, A.; Thomas, J.; Schier, J.; Singh, R, .; Singh, R.S.; Dhariwal, A.C.; Chauhan, L.S.; Centers for Disease Control and Prevention (CDC) Outbreaks of unexplained neurologic illness - Muzaffarpur, India, 2013-2014. Morb Mortal Wkly Rep. 2015, 64, 49-53.

8. Spencer P.S.; Palmer V.S.; Mazumder, R. Probable toxic cause for suspected lycheelinked viral encephalitis. Emerg Infect Dis. 2015, 21, 904-905.

9. Paireau, J.; Tuan, N.H.; Lefrançois, R.; Buckwalter, M.R, .; Nghia, N.D.; Hien N,T.; Lortholary, O.; Poirée, S.; Manuguerra, J.C.; Gessain, A.; Albert, M.L.; Brey, P.T.; Nga, P.T.; Fontanet, A. Litchi-associated acute encephalitis in children, Northern Vietnam, 2004-2009. Emerg Infect Dis. 2012, 18, 1817-1824.

10. Isenberg, S.L.; Carter, M.D.; Graham, L.A.; Mathews, T.P.; Johnson, D.; Thomas, J.D.; Pirkle, J.L.; Johnson, R.C. Quantification of metabolites for assessing human exposure to soapberry toxins hypoglycin A and methylenecyclopropylglycine. Chem Res Toxicol. 2015, 28, 1753-1759.

11. Vashishtha, V.M. Outbreaks of Hypoglycemic Encephalopathy in Muzaffarpur, India: Are These Caused by Toxins in Litchi Fruit?: The Counterpoint. Indian Pediatr. 2016, 53, 399-402.

12. John, T.J.; Das, M. Acute encephalitis syndrome in children in Muzaffarpur: hypothesis of toxic origin. Curr Sci 2014, 106, 1184-1185


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13. Shrivastava A, Kumar A, Thomas JD, Laserson KF, Bhushan G, Carter MD, Chhabra M, Mittal V, Khare S, Sejvar JJ, Dwivedi M, Isenberg SL, Johnson R, Pirkle JL, Sharer JD, Hall PL, Yadav R, Velayudhan A, Papanna M, Singh P, Somashekar D, Pradhan A, Goel K, Pandey R, Kumar M, Kumar S, Chakrabarti A, Sivaperumal P, Kumar AR, Schier JG, Chang A, Graham LA, Mathews TP, Johnson D, Valentin L, Caldwell KL, Jarrett JM, Harden LA, Takeoka GR, Tong S, Queen K, Paden C, Whitney A, Haberling DL, Singh R, Singh RS, Earhart KC, Dhariwal AC, Chauhan LS, Venkatesh S, Srikantiah P. Association of acute toxic encephalopathy with litchi consumption in an outbreak in Muzaffarpur, India, 2014: a case-control study. Lancet Glob Health. 2017 Jan 30. pii: S2214-109X(17)30035-9. doi: 10.1016/S2214109X(17)30035-9.

14. Valberg, S.J.; Sponseller, B.T.; Hegeman, A.D.; Earing, J.; Bender, J.B.; Martinson, K.L.; Patterson, S.E; Sweetman, L. Seasonal pasture myopathy/atypical myopathy in North America associated with ingestion of hypoglycin A within seeds of the box elder tree. Equine Vet J. 2013, 45, 419-426.

15. Votion, D.M.; van Galen, G.; Sweetman, L.; Boemer, F.; de Tullio, P.; Dopagne, C.; Lefère, L.; Mouithys-Mickalad, A.; Patarin, F.; Rouxhet, S.; van Loon, G.; Serteyn, D.; Sponseller, B.T.; Valberg, S.J. Identification of methylenecyclopropyl acetic acid in serum of European horses with atypical myopathy. Equine Vet J. 2014, 46, 146149.

16. Feng, P.C. Hypoglycin - from ackee: a review. West Indian Med J. 1969, 18, 238-243.


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18. Hassall, C.H.; Reyle, K. Hypoglycin A,B. Biologically active polypeptides from Blighia sapida. Nature. 1954, 173, 356-357.

19. Chen, K.K.; Anderson, R.C.; McCowen, M.C.; Harris, P.N. Pharmacologic action of hypoglycin A and B. J Pharmacol Exp Ther 1957, 121, 272-285

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23. Food and Drug Administration, Division of Import Operations (HFC-170), Rockville, USA: Import Alert 2016, 21-11 ACS Paragon Plus Environment

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24. Melde, K.; Buettner, H.; Boschert, W.; Wolf, H.P.; Ghisla, S. Mechanism of hypoglycaemic action of methylenecyclopropylglycine. Biochem J. 1989, 259, 921924.

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47. Tables

Table 1. The Mass Transitions, Concentrations and Commercial Sources of the Internal Standards. Mass Transition Concentration Internal Standard of the Butylated Supplier nmol/L Compounds ten Brink, Academic Medisch D3-octanoyl carnitine 347.3 85.0 25.4 Center, Amsterdam, NL D9-isovaleryl carnitine 311.3 85.0 26.8 ten Brink D7-butyryl carnitine 295.3 85.0 50.5 ten Brink D3-hexanoyl glycine 233.2 132.0 324.6 ten Brink Cambridge Isotope Laboratories, D3-leucine 191.0 89 5.78 Andover, MA, USA [13C2, 15N] MCPA-G 229.1 75.9 38.7 IsoSciences [13C2, 15N] MCPF-G 215.1 80.9 42.2 IsoSciences


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Table 2. Mass Transitions Used in the UPLC-MS/MS Analysis for Quantifying the Toxins and Metabolites in Serum and Urine and the Respective Internal Standards Used for Quantification.

Metabolite MCPF-G MCPA-G MCPF-C MCPA-C HGA Isobutyryl/butyryl carnitine Isovaleryl/valeryl carnitine Hexanoyl carnitine Hexanoyl glycine Octanoyl carnitine Decenoyl carnitine

Mass Transition of the Butylated Compounds 212.1 81.0 226.1 74.0 298.2 85.0 312.2 85.0 198.1 73.9 288.2 85.0 302.2 85.0 316.2 85.0 230.2 132.0 344.2 85.0 370.2 85.0

Internal Standard Used [13C2, 15N] MCPF-G [13C2, 15N] MCPA-G D7-butyryl carnitine D3-octanoyl carnitine D3-leucine D7-butyryl carnitine D9-isovaleryl carnitine D3-octanoyl carnitine D3-hexanoyl glycine D3-octanoyl carnitine D3-octanoyl carnitine


22

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Table 3. The Coefficients of Variation (6 Injections of the Same Sample for Intra-Assay CV, 6 Separate Preparations for Inter-Assay CV) for Methylenecyclopropylformyl-Glycine (MCPF-G), Methylenecyclopropylacetyl-Glycine (MCPA-G) and Hypoglycin A (HGA).

Intra-assay CV MCPF-G MCPA-G HGA Inter-assay CV MCPF-G MCPA-G HGA a n.d.: not done

10 nmol/L Urine Serum




14.9 19.9 7.3

18.1 17.6 6.8

10.0 12.7 6.3

13.2 11.5 3.6

n.d.a n.d. 2.8

n.d. n.d. 2.8

3.7 10.1 -

4.6 8.1 -

n.d. n.d. 10.8

n.d. n.d. 9.7

11.2 16.7 n.d.

9.7 15.9 n.d.

n.d. n.d. 5.6

n.d. n.d. 3.9

5.7 12.5 n.d.

5.5 8.6 n.d.


23


Page 24 of 27

Table 4. Ackee Experiment: The Measurement of the Levels of Hypoglycin A (HGA), Methylenecyclopropylformyl-Glycine (MCPF-G), Methylenecyclopropylacetyl-Glycine (MCPA-G), Methylenecyclopropylformyl-Carnitine (MCPF-C), and Methylenecyclopropylacetyl-Carnitine (MCPA-C) in the Urine After the Ingestion of 1 g Canned Ackee per kg Body Weight Time After Ingestion Hours

HGA nmol/mmol Creatinine

MCPF-G nmol/mmol Creatinine

MCPA-G nmol/mmol Creatinine

MCPF-C nmol/mmol Creatinine

MCPA-C nmol/mmol Creatinine

0

< LLODa

< LLOD

< LLOD

< LLOD

< LLOD

1

9.1

11.4

707.9

22.8

trace

3

5.8

11.3

332.1

15.3

< LLOD

10

4.3

5.0

391.1

7,6

trace

24

2.1

4.9

45.6

3.3

trace

a

< LLOD: Below Lower Limit of Detection


24

Page 25 of 27


Table 5. Ackee Experiment: The Renal Excretion of Acyl Conjugates at different Times During the Experiment Time After Ingestion Hours 0 1 3 10 24 a

Decenoyl carnitinea

Octanoyl carnitinea

Hexanoyl glycinea

Hexanoyl carnitinea

Isovaleryl carnitinea

Isobutyryl carnitinea

Butyryl carnitinea

4.9 38.8 30.7 34.3 45.8

1.2 12.2 6.8 23.0 30.1

14.4 72.5 30.0 103 170

0.5 6.0 2.6 9.9 14.7

18.9 25.2 19.6 39.3 52.8

146 1,012 1,284 1,608 1,543

2.7 25.5 25.1 63.3 81.5

nmol/mmol Creatinine


25


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Table 6. The concentrations of hypoglycin A in the serum after the ingestion of canned ackee (1 g per kg body weight) and canned lychee (5 g per kg body weight). Time After Test Meal Hours 0 2 5 7 10

Ackee Lychee Experiment Experiment HGA HGA nmol/L nmol/L

Quantification of Methylenecyclopropyl ... - ACS Publications

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