Bioaccumulation and Distribution of Indospicine and Its Foregut Metabolites in Camels Fed Indigofera Spicata
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toxins Article Bioaccumulation and Distribution of Indospicine and Its Foregut Metabolites in Camels Fed Indigofera spicata Gabriele Netzel 1, Eddie T. T. Tan 1,2, Mukan Yin 1, Cindy Giles 3, Ken W. L. Yong 3, Rafat Al Jassim 1 and Mary T. Fletcher 1,* 1 Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland, Health and Food Sciences Precinct, Coopers Plains, QLD 4108, Australia; [email protected] (G.N.); [email protected] (E.T.T.T.); [email protected] (M.Y.); [email protected] (R.A.J.) 2 Alliance of Research and Innovation for Food (ARIF), Faculty of Applied Sciences, Universiti Teknologi MARA, Cawangan Negeri Sembilan, Kuala Pilah Campus, Negeri Sembilan 72000, Malaysia 3 Department of Agriculture and Fisheries, Health and Food Sciences Precinct, Coopers Plains, QLD 4108, Australia; [email protected] (C.G.); [email protected] (K.W.L.Y.) * Correspondence: mary.fl[email protected]; Tel.: +61-7-3443-2479 Received: 20 February 2019; Accepted: 8 March 2019; Published: 19 March 2019 Abstract: In vitro experiments have demonstrated that camel foregut-fluid has the capacity to metabolize indospicine, a natural toxin which causes hepatotoxicosis, but such metabolism is in competition with absorption and outflow of indospicine from the different segments of the digestive system. Six young camels were fed Indigofera spicata (337 µg indospicine/kg BW/day) for 32 days, at which time three camels were euthanized. The remaining camels were monitored for a further 100 days after cessation of this indospicine diet. In a retrospective investigation, relative levels of indospicine foregut-metabolism products were examined by UHPLC-MS/MS in plasma, collected during both accumulation and depletion stages of this experiment. The metabolite 2-aminopimelamic acid could be detected at low levels in almost all plasma samples, whereas 2-aminopimelic acid could not be detected. In the euthanized camels, 2-aminopimelamic acid could be found in all tissues except muscle, whereas 2-aminopimelic acid was only found in the kidney, pancreas, and liver tissues. The clearance rate for these metabolites was considerably greater than for indospicine, which was still present in plasma of the remaining camels 100 days after cessation of Indigofera consumption. Keywords: indospicine; 2-aminopimelamic acid; 2-aminopimelic acid; in vivo; foregut metabolites; camel; food safety Key Contribution: The indospicine metabolites 2-aminopimelamic acid and 2-aminopimelic acid are formed during foregut microbial metabolism and can be detected in tissues of camels fed Indigofera spicata. These metabolites are not as persistent as indospicine in animal tissues after cessation of feeding, and could be used as markers for recent Indigofera consumption. 1. Introduction There are more than 60 Indigofera species distributed throughout the arid and semiarid regions of Australia [1–4]. Indigofera spp. are leguminous shrubs and herbs which are high in protein, as well as highly palatable for animals. These plants are considered a nutritious animal fodder, however, some species contain indospicine, a non-proteinogenic arginine analogue which causes hepatotoxicosis in Toxins 2019, 11, 169; doi:10.3390/toxins11030169 www.mdpi.com/journal/toxins Toxins 2019, 11, 169 2 of 8 Toxins 2019, 11, x FOR PEER REVIEW 2 of 8 sheep,hepatotoxicosis cows, rabbits, in sheep, and cows, dogs rabbits, [5–9]. The and introduced dogs [5–9]. species,The introducedIndigofera species, spicata Indigofera, has been spicata found, has to containbeen found high to levels contain of this high amino levels acid. of this Since amin thereo acid. is no Since known there mammalian is no known enzyme mammalian which is enzyme capable ofwhich degrading is capable indospicine of degrading [10], it can indospicine be toxic to simple-stomached[10], it can be toxic animals. to simple-stomached However, ruminants animals. seem toHowever, be less susceptible ruminants to seem certain to toxinsbe less due susceptible to the ability to ofcertain rumen toxins microflora due toto degradethe ability some of of rumen those foodbornemicroflora toxins.to degrade Feral some camels of those in Australia foodborne seem toxins to cope. Feral better camels in detoxification in Australia ofseem plant to toxinscope better from theirin detoxification fodder, compared of plant to toxins domesticated from their animals fodder, [11 compared]. to domesticated animals [11]. IndospicineIndospicine isis aa water-solublewater-soluble freefree aminoamino acidacid andand rumenrumen degradationdegradation processesprocesses competecompete withwith presumedpresumed rapidrapid passagepassage throughthrough thethe fermentationfermentation compartmentscompartments ofof thethe digestivedigestive tract.tract. ThisThis aminoamino acidacid has been detected detected in in meat, meat, which which indicates indicates that that at atleast least a portion a portion of indospicine of indospicine can can by-pass by-pass the thefermentation fermentation processes processes of the of theforegut foregut and and be beabsorb absorbeded as is as [12]. is [12 A]. number A number of ofdogs dogs have have died died of ofsecondary secondary hepatotoxicosis hepatotoxicosis after after consuming consuming indosp indospicine-contaminatedicine-contaminated horse horse meat meat [7] [7] and, moremore recently,recently, camelcamel meatmeat [[5],5], andand thisthis hashas raisedraised foodfood safetysafety concerns.concerns. In a recentrecent study,study, wewe havehave shownshown thatthat indospicineindospicine accumulatedaccumulated inin camelcamel meatmeat duringduring aa feedingfeeding trialtrial inin whichwhich sixsix camelscamels werewere fedfed aa dietdiet containingcontainingIndigofera Indigofera spicata spicatafor for 32 32 days, days, and and that that indospicine indospicine can can be detected be detected in plasma in plasma as long as aslong three as monthsthree months after removing after removingIndigofera Indigoferafrom the from diet the [13 diet]. [13]. WeWe alsoalso reportedreported previouslypreviously thatthat microfloramicroflora ofof bothboth thethe bovinebovine rumenrumen andand camelcamel foregutforegut fluidsfluids havehave thethe abilityability toto degradedegrade indospicine in vitro withinwithin anan incubationincubation period of 48 h [[14].14]. However, thethe inin vitrovitro degradability of indospicine is indicativeindicative of the potential degradability, andand notnot thethe actual degradability,degradability, thatthat occurs occurs in thein animalthe animal system. syst Factorsem. Factors including including the microbial the microbial community, community, residence timeresidence of the time solid of fractionthe solid offraction digesta, of digesta, and outflow and outflow rate of therate fluidof the phase fluid phase all play all anplay important an important role. Camelsrole. Camels are known are known to retain to retain low low quality quality fibre fibre diets diets longer longer in in the the foregut foregut compared compared with with ruminantruminant animals.animals. Retention time is always shorter when the diet is of higher quality, whichwhich shouldshould bebe thethe casecase withwith lushlush earlyearly seasonseason pasturepasture containingcontaining IndigoferaIndigofera spicataspicata atat thethe startstart ofof thethe wetwet season.season. ShiftShift toto suchsuch dietdiet increasesincreases thethe outflowoutflow raterate andand allowsallows moremore indospicineindospicine toto enterenter thethe intestinesintestines wherewhere itit thenthen getsgets absorbed.absorbed. Indospicine has been shown to be chemicallychemically stable and resistant toto bothboth acidicacidic andand basebase conditionsconditions [[15,16].15,16]. Since the camel foregut fluidfluid is only mildly acidic, it isis mostmost likelylikely thatthat rumenrumen bacteriabacteria areare responsibleresponsible forfor thethe observedobserved metabolismmetabolism ofof indospicineindospicine ((11)) intointo itsits degradationdegradation productproduct 2-aminopimelamic2-aminopimelamic acidacid ((22)) and,and, further,further, toto 2-aminopimelic2-aminopimelic acidacid ((33)) (Figure(Figure1 )[1)14 [14,17].,17]. FigureFigure 1.1. Chemical structures of of indospicine indospicine (1 (1) )and and its its metabolites metabolites 2-aminopimelamic 2-aminopimelamic acid acid (2) ( 2and) and 2- 2-aminopimelicaminopimelic acid acid (3 (),3 ),together together with with D D3-L-indospicine3-L-indospicine (4 (4) )which which is is used used as as an internal standardstandard inin LC-MS/MSLC-MS/MS analysis. analysis. AlthoughAlthough wewe could could show show previously previously that that indospicine indospicine accumulated accumulated as afree as aminoa free acidamino in variousacid in animalvarious tissues animalin tissues vivo [ 13in ],vivo it has [13], also it has been also demonstrated been demonstrated that indospicine that indospicine can be metabolized can be metabolizedin vitro byin foregutvitro by microbiota foregut microbiota [14]. These [14]. two These processes two ofprocesses removal of (outflow removal and (outflow absorption) and andabsorption) metabolism and metabolism could be considered to operate in competition, and there is nothing known about the extent of in vivo metabolism of indospicine and whether the metabolites are also transported and Toxins 2019, 11, 169 3 of 8 couldToxins 2019 be considered, 11, x FOR PEER to operateREVIEW in competition, and there is nothing known about the extent of in3 vivo of