Food Chemistry 141 (2013) 841–846

Contents lists available at SciVerse ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

The effect of holding live sea urchins (Evechinus chloroticus) in air priorto gonad removal on gonad adenine nucleotide profiles during storageat 4 �C

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.03.057

⇑ Corresponding author. Tel.: +64 3 479 5469; fax: +64 3 479 7567.E-mail addresses: phil.bremer@otago.ac.nz (P.J. Bremer).

Wasseela Verachia a, Giuseppe Lazzarino c, Brian Niven b, Philip J. Bremer a,⇑a Department of Food Science, University of Otago, P.O. Box 56, Dunedin 9054, New Zealandb Department of Mathematics and Statistics, University of Otago, P.O. Box 56, Dunedin 9054, New Zealandc Department of Biology, Geology and Environmental Sciences, Division of Biochemistry and Molecular Biology, Viale A. Doria 6, 95125 Catania, Italy

a r t i c l e i n f o

Article history:Received 17 September 2012Received in revised form 12 March 2013Accepted 15 March 2013Available online 26 March 2013

Keywords:Evechinus chloroticusSea urchinGonadK-ValueATPFreshness-indiceHPLCExtraction method

a b s t r a c t

Sea urchin gonads are usually sold as a fresh chilled product. Thus, to evaluate the effect of live urchin’spost-harvest conditions on gonad shelf-life, gonads were extracted either immediately after harvesting orafter holding urchins in air at either 4 or 15 �C for 144 and 72 h, respectively. Gonads were subsequentlywashed in brine and stored at 4 �C for 10 days prior to adenine nucleotide (nmol/g w/w) profile determi-nation. A decline in ATP (control: 376.16; urchins held in air: 231.58 and 245.16) and build-up of its deg-radation products, mainly inosine (control: 13.25; urchins held in air: 82.87 and 52.95), was observed ingonads recovered from urchins held in air. A faster increase in ATP degradation products was detectedduring storage of gonads recovered from urchins held in air, with final K-values (%) of 59.34 and 48.18being significantly higher than K-values obtained from the controls (29.69, p < 0.05), suggesting thatpost-harvest handling can negatively impact on gonad shelf-life.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Sea urchin gonads (also termed uni or roe) are an expensive andsought after delicacy whose quality and value in Asian markets areclosely linked to their freshness and shelf-life (McBride, Price, Tom,Lawrence, & Lawrence, 2004). Although New Zealand has goodstocks of sea urchins (Evechinus chloroticus, local name kina), deliv-ering gonads of suitable quality to distant high value Asian marketshas proven difficult due to a lack of understanding of the factorscontrolling shelf-life. Studies to improve shelf-life are complicatedby a reliance on experts to make assessments of freshness based onexperience.

Although many studies have reported on the evaluation offreshness for fish species, analytical techniques for measuring thefreshness of sea urchin gonads are limited. In searching for valu-able bio-indicators to assess product freshness and quality, studieswith marine products such as fish and seafood showed a positiverelationship between the loss of freshness and changes in concen-tration of the phosphorylated compound adenosine triphosphate

(ATP) into its breakdown products adenosine diphosphate (ADP),adenosine monophosphate (AMP), inosine monophosphate (IMP),inosine (Ino) and hypoxanthine (Hyp). Furthermore, it has beenreported that the nucleotide profile influences the taste and flavourof fishes and molluscs, with IMP in particular contributing to thesought after umami taste and fresh flavour of seafood which isassociated with excellent quality (Matsumoto & Yamanaka,1990). In contrast, Hyp has been reported to be responsible forthe progressive loss of desirable flavour (Fletcher & Statham,1988; Gill, 1990). By determining the absolute concentrations ofthe different adenine nucleotide derivatives (ATP, ADP, AMP, IMP,Ino and Hyp), calculation of the K-value (%) = [(Ino + Hyp)/(ATP + ADP + AMP + IMP + Ino + Hyp)] � 100 can be achieved. Thisparameter has been reported to be a valid biochemical index tomeasure fish freshness (Saito, Arai, & Matsuyoshi, 1959).

The concentration of ATP and its degradation products in thecalculation of the K-value, as an indicator of product quality, canbe measured quickly, reliably and with good reproducibility usingdifferent high performance liquid chromatographic (HPLC) meth-ods (Massa, Palacios, Paredi, & Crupkin, 2005). A critical issue inthis metabolite assay is represented by the type of sample process-ing adopted for efficient protein removal. Perchloric acid (PCA) is

Fig. 2. K-Value of gonads recovered from control urchins and gonads recoveredfrom urchins held in air at 15 �C for 72 h or 4 �C for 144 h. Histograms are the meanof 18 sea urchins (9 males and 9 females). Each vertical bar represents one standarddeviation ⁄ Significantly different attributes compared to the controls, p < 0.05. Kvalue (%) = [(Ino + Hyp)/(ATP + ADP + AMP + IMP + Ino + Hyp)] � 100. ATP = adeno-sine triphosphate, ADP = adenosine diphosphate, AMP = adenosine monophosphate,IMP = inosine monophosphate, Ino = inosine, Hyp = hypoxanthine.

842 W. Verachia et al. / Food Chemistry 141 (2013) 841–846

often used to deproteinise fish (Özogul, Özden, Özogul, & Erkan,2010), seafood (Massa, Paredi, & Crupkin, 2003) and sea urchingonadal tissues (Wang et al., 2011), even though concerns aboutits accuracy due to a significant loss (40–60%) of ATP have beenreported (Berne, 1963; Gebelein, Merdes, & Berger, 1992; Kimmich,Randles, & Brand, 1975; Williams & Forrester, 1976). Furthermore,perchloric acid and the inefficient precipitation of potassiumperchlorate have been reported to impact on the chromatographyof nucleotides by affecting either the retention times of analytes orthe column efficiency and life-span (Au, Su, & Wientjes, 1989; VanDoorn, Goormachtig, & De Haan, 1989).

To minimise changes in actual concentrations of adenine nucle-otides, an optimisation of organic solvent deproteinisation (Auet al., 1989) has been obtained by homogenising in the presenceof a mixture of acetonitrile (ACN)/10 mM potassium dihydrogenphosphate (KH2PO4) to remove proteins from samples of rat tissue(Lazzarino et al., 2003). Recently the protocol developed by Lazza-rino et al. (2003) for protein removal has been extended to measur-ing metabolites (ATP/ADP/AMP) accumulated by inter- andsubtidal Antarctic limpets (Nacella concinna) in response to aerialexposure (Weihe & Abele, 2008) and to determine sex-specificbiochemical and histological responses to the toxicant, phenan-threne, by the sea urchin Psammechinus miliaris gonads (Schäfer,Abele, Weihe, & Köhler, 2011).

In New Zealand, urchins are often harvested from remote loca-tions and it is a common practice to hold live urchins in air on thedeck of a fishing boat for several days before they are processed. Toevaluate the influence of holding live urchins in air prior to gonadrecovery on gonad energy metabolism, two different times andtemperatures of urchin holding conditions (15 �C for 72 h or 4 �Cfor 144 h) were chosen to simulate how live urchins in the South-ern region of New Zealand are handled. Once harvested, urchinsare commonly held out of water on the deck of the boat at ambienttemperatures of �4 �C during winter months and �15 �C duringsummer months. After urchins were held in air the recoveredgonads were subsequently washed in brine and stored in air at4 �C for 0, 3, 5, 7 and 10 days prior to the gonads being deprotein-ised and their adenine nucleotide profile, ATP and ATP-relatedcompounds (ADP, AMP, IMP, Ino and Hyp) being determined byHPLC and used as a valid biochemical index of freshness.

Fig. 1. Concentrations of ATP and ATP degradation products (ADP, AMP, IMP, Inoand Hyp) extracted from gonads recovered from control urchins and from gonadsrecovered from urchins held in air at either 15 �C for 72 h or 4 �C for 144 h.Histograms are the mean of 18 sea urchins (9 males and 9 females). Each verticalbar represents one standard deviation. ⁄ Significantly different attributes comparedto the controls, p < 0.05. ATP = adenosine triphosphate, ADP = adenosine diphos-phate, AMP = adenosine monophosphate, IMP = inosine monophosphate, Ino = ino-sine, Hyp = hypoxanthine.

2. Methods

2.1. Sea urchin sampling and storage, gonad removal and incubation

Sea urchins (E. chloroticus) were obtained from waters aroundDoubtful Sound (45 160 S, 166 500 E) in summer (December) andtransported live to the Department of Food Science, University ofOtago, in 20 L bucket of seawater (approximately 1 urchin per 2 Lof seawater) within 24 h of being harvested.

Sea urchins [n = 18 (9 male and 9 female)] either had theirgonads removed immediately upon arrival (control group) or theywere held in the air at either 15 �C for 72 h or 4 �C for 144 h prior tothe gonads being recovered. On dissection, the weight and diame-ter of the whole urchin was recorded and gender determined bylight microscopy, to ensure an equal split of male to female urchinsin the study. The Aristotles lantern was removed, coelomic fluiddrained, and the test cut in half from the dorsal to ventral side,avoiding the gonads. The gonads were washed in serial solutionsof brine (3.2% NaCl, w/v in distilled water) at 4 �C, drained, weighedand the five gonads recovered from each urchin were stored indi-vidually in sealed petri dishes at 4 �C for up to 10 days. On days 0,3, 5, 7 and 10, one gonad from each set of five gonads recoveredfrom each urchin was frozen in liquid nitrogen and deproteinisedusing the Lazzarino et al. (2003) protocol described below. Pro-tein-free gonad extracts were stored at �80 �C until assayed byHPLC for the simultaneous determination of ATP and its degrada-tion products, which was then used to calculate the K-value, ameasure of gonad freshness.

2.2. Deproteinisation of sea urchin gonads

The deproteinisation protocol described by Lazzarino et al.(2003) was applied. A gonad was placed in a Corex centrifuge tube(3 mL) along with 7 mL ice cold, nitrogen-saturated precipitatingsolution comprising HPLC grade CH3CN (Merck, Darmstadt,Germany), 10 mM KH2PO4 (Merck, Darmstadt, Germany) (3:1;v:v, pH 7.40) and 80 lM dimethyladenine (internal standard ofpurine). The gonad was homogenised for 120 s using an Ultra-tur-rax T25 (Janke & Kunkel IKA�-Labortechnik) at 24,000 rpm (revolu-tions per minute). The precipitating solution was prepared fresheach week and its pH (7.40) checked immediately before use. Aftercentrifugation (21,000g, for 10 min at 4 �C), the clear supernatant

Fig. 3. Concentrations of ATP (Panel A) and ADP (Panel B) extracted from gonadsstored over 10 days of storage at 4 �C extracted from control sea urchins and seaurchins held in air at either 15 �C for 72 h or 4 �C for 144 h. Histograms are the meanof 18 sea urchins (9 males and 9 females). Each vertical bar represents one standarddeviation ⁄ Significantly different attributes compared to the controls, p < 0.05.⁄⁄ Significantly different attributes from day 0, p < 0.05. ATP = adenosine triphos-phate, ADP = adenosine diphosphate.

Fig. 4. Concentrations of AMP (Panel A) and IMP (Panel B) extracted from gonadsstored over 10 days of storage at 4 �C extracted from control sea urchins and seaurchins held at either 15 �C for 72 h or 4 �C for 144 h. Histograms are the mean of 18sea urchins (9 males and 9 females). Each vertical bar represents one standarddeviation ⁄ Significantly different attributes compared to the controls, p < 0.05.⁄⁄ Significantly different attributes from day 0, p < 0.05. AMP = adenosinemonophosphate, IMP = inosine monophosphate.

W. Verachia et al. / Food Chemistry 141 (2013) 841–846 843

was saved and 3 mL of the precipitating solution were added to thepellet which was homogenised again, as above. The resulting sus-pension was again centrifuged as above, the pellet saved and thesupernatant combined with that obtained from the first centrifuga-tion step. The combined supernatant sample was extracted byvigorous agitation with a double volume of HPLC-grade chloroform(CHCl3) (Mallinckrodt Chemicals, Phillipsburg, New Jersey, USA)and centrifuged as above. The upper aqueous phase (containingwater-soluble, low molecular-weight compounds) was collected,subjected to chloroform washings two more times (this procedureallowed the removal of all the organic solvent and any lipid solublecompound from the buffered gonad extracts) and finally adjustedin volume with 10 mM KH2PO4, pH 7.40, to achieve an aqueous10% weight/volume gonad homogenate which was stored at�80 �C until HPLC assay of adenine nucleotide profile.

2.3. HPLC analysis of adenosine triphosphate and its degradationcompounds

The simultaneous separation of ATP, ADP, AMP, IMP, Ino andHyp was carried out using an ion pairing HPLC method (Lazzarinoet al., 2003), which utilizes tetrabutylammonium hydroxide(Sigma Chemical Co., St. Louis, Missouri, USA) as the pairing

reagent. The HPLC system consisted of a quaternary pumpequipped with a diode array detector set at 267 nm (AgilentTechnologies, 120 Series, Victoria, Australia). Separation wascarried out on 200 lL of each ACN/10 mM KH2PO4 deproteinisedgonad sample using a 250 � 4.6 mm, 5 lm particle size, reversedphase C-18 Gemini column fitted with a guard column (Phenome-nex, Auckland, NZ), using a step gradient with two buffers: buffer A(10 mM tetrabutylammonium hydroxide, 10 mM KH2PO4 and0.125% methanol, pH 7.00) and buffer B (2.8 mM tetrabutylammo-nium hydroxide, 100 mM KH2PO4 and 30% methanol, pH 5.50).Runs were carried out at a flow rate of 1.2 mL/min and a constantcolumn temperature of 18 �C. Peaks in the chromatographic runs ofthe extracts were identified by comparing retention times andabsorption spectra with those of freshly prepared ultrapurestandard mixtures, containing ATP, ADP, AMP, IMP, Hyp (SigmaChemical Co., St. Louis, Missouri, USA) and Ino (Fluka BioChemika,Germany). Concentrations of the diverse compounds were calcu-lated by comparing peak areas with those of standard mixturesof known concentrations.

2.4. Statistical analysis

Data from each experiment were analysed using the statisticalpackage SPSS 17.0 (SPSS Inc, Chicago, IL, USA) at a significance level

Fig. 5. Concentrations of Ino (Panel A) and Hyp (Panel B) extracted from gonadsstored over 10 days of storage at 4 �C extracted from control sea urchins and seaurchins held in air at either 15 �C for 72 h or 4 �C for 144 h. Histograms are the meanof 18 sea urchins (9 males and 9 females). Each vertical bar represents one standarddeviation ⁄ Significantly different attributes compared to the controls, p < 0.05.⁄⁄ Significantly different attributes from day 0, p < 0.05. Ino = inosine, Hyp =hypoxanthine.

Fig. 6. K-Value of gonads stored for 10 days at 4 �C extracted from control seaurchins and sea urchins held in air at either 15 �C for 72 h or 4 �C for 144 h.Histograms are the mean of 18 sea urchins (9 males and 9 females). Standarddeviations are represented by vertical bars. ⁄ Significantly different attributescompared to the controls, p < 0.05. ⁄⁄ Significantly different attributes from day 0,p < 0.05. K value (%) = [(Ino + Hyp)/(ATP + ADP + AMP + IMP + Ino + Hyp)] � 100.ATP = adenosine triphosphate, ADP = adenosine diphosphate, AMP = adenosinemonophosphate, IMP = inosine monophosphate, Ino = inosine, Hyp = hypoxanthine.

844 W. Verachia et al. / Food Chemistry 141 (2013) 841–846

of 0.05. GLM two-way ANOVA was used to test for differences inconcentrations of metabolites and K-value of the gonad with thefixed effects being the type of post-harvest storage conditions ofsea urchins prior to gonad recovery and day of storage after gonadrecovery, and the interaction effect being the type of post-harveststorage of sea urchins prior to gonad recovery and day of storageafter gonad recovery, with Tukey’s post hoc analysis being usedto determine if type of post-harvest storage of sea urchins priorto gonad recovery were significantly different to each other.

3. Results and discussion

3.1. Impact of sea urchin post-harvest handling on the adeninenucleotides and K-value of recovered gonads

Gonads recovered from urchins either immediately after theirremoval from seawater (control) or from urchins held in air ateither15 �C for 72 h or 4 �C for 144 h were evaluated for their con-centrations of ATP and ATP-related compounds (ADP, AMP, IMP,Ino and Hyp) (Fig. 1).

Gonads recovered from urchins held in air (15 �C for 72 h or 4 �Cfor 144 h) had lower values of energy metabolites compared tothose in gonads recovered from control urchins due to a changein the energy balance caused by a decreased ability to supply anadequate rate of ATP production (Fig. 1). On day 0, the concentra-tion of ATP in gonads recovered from control urchins was376.16 ± 52.74 nmol/g w/w while the ATP extracted from thegonads recovered from urchins held at 15 �C for 72 h or 4 �C for144 h was 245.16 ± 50.62 and 231.58 ± 46.09 nmol/g w/w, respec-tively (p < 0.05). Consequently, gonads recovered from urchinsheld in air had higher levels of the ATP degradation productsADP, AMP and Ino, compared to the levels in gonads recoveredfrom control urchins. ATP concentrations decreased between 35%and 38% whilst inosine concentrations increased 4- and 6-fold inthe gonads recovered from urchins held in air (Fig. 1). The concur-rent decreases in ATP concentrations and increases in inosineresulted in a significantly (p < 0.05) higher K-value (day 0) forgonads recovered from urchins held in air (Fig. 2). The K-valuesof gonads recovered from urchins held in air at 4 �C for 144 h or15 �C for 72 h were 60% or 116% higher, respectively, than theK-value of gonads recovered from the control urchins(8.01 ± 1.40). This finding confirms that post-harvest storage con-ditions of urchins have a significant (p < 0.05) negative effect onenergy metabolism and potentially the shelf-life of recoveredgonads. The use of the K-value has been established to assesschemically the freshness of fish during post mortem storage since1959 (Saito et al., 1959) and it has successfully been used toevaluate sea urchin gonad freshness and quality in a study byWang et al. (2011), who stressed the importance of maintaininga high oxygen saturation in water during the storage of gonadsrecovered from recently harvested sea urchins. This researchshowed a clear relationship between the length of gonad storageand changes in their K-values (Wang et al., 2011). However, inthe market place, gonads are generally arranged on small woodentrays with clear pastic covers that are held in air at 2–4 �C, withonly a small percentage of gonads being stored in sea water.

3.2. Change in gonad adenine nucleotide pattern and K-value during10 days of storage at 4 �C

The conditions under which the live urchins were held prior toprocessing also affected the concentration of ATP and its degrada-

Fig. 7. Chromatogram of sea urchin gonad extract deproteinised (20 ll injected) with ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4, pH 7.40 (3:1; v:v), showing theidentification of the 6 compounds forming the standard mixture illustrated above in (A). Concentrations of ATP and ATP degradation products (ADP, AMP, IMP, Ino and Hyp)extracted from gonads recovered from control urchins Day 0 (B) and from gonads recovered from urchins held in air at either 15 �C for 72 h Day 0 (C) or 4 �C for 144 h Day 0(D). Sample processing and chromatographic conditions are fully described under materials and methods. ATP = adenosine triphosphate, ADP = adenosine diphosphate,AMP = adenosine monophosphate, IMP = inosine monophosphate, Ino = inosine, Hyp = hypoxanthine.

W. Verachia et al. / Food Chemistry 141 (2013) 841–846 845

tion products (ADP, AMP, IMP, Ino and Hyp) present in the gonadsand their K-value over a subsequent 10 days storage period. Asshown in Fig. 3 (Panel A), a time dependent decrease in ATP wasrecorded in gonads recovered from both control urchins and urch-ins held in air. After 7 days of storage at 4 �C for gonads recoveredfrom control urchins, the percentage of ATP was at 30%, the K-valuewas still below 20% and the sum of adenylates (ATP + ADP + AM-P + IMP + Ino + Hyp) was 567.29 ± 87.17 nmol/g w/w (value at day0 = 744.56 ± 69.85 nmol/g w/w, �23.8% of the value at day 0;p < 0.05). By day 10 of storage, the ATP concentration was108.09 ± 16.84 nmol/g w/w (�71.3%; p < 0.05 with respect to day0). For urchins held in air at either 15 or 4 �C prior to gonad recov-ery and storage at 4 �C, the ATP concentration at day 0 of storagewas comparable to that of gonads recovered from control urchinsafter day 3 of storage at 4 �C (Fig. 3, Panel A). The concentrationof ATP recorded thereafter decreased significantly (p < 0.05) to aminimal concentration of 14.60 ± 5.82 (6% of ATP at day 0 ofcontrol urchins; p < 0.05) and 13.83 ± 2.41 nmol/g w/w (6% of ATPat day 0 of control urchins; p < 0.05) which was recorded at day10 of storage at 4 �C. ADP in control gonads increased to133.95 ± 28.00 nmol/g w/w (p < 0.05 in comparison to day 0)during the initial 5 days of storage, after which its concentrationdecreased, returning to values not significantly different those ofday 0. For the urchins held in air prior to processing, the concentra-tion of ADP progressively decreased over the storage period (Fig. 3,Panel B) with minimal concentrations of ADP measured at day 10of storage. Gonads recovered from urchins held at either 15 �Cfor 72 h or 4 �C for 144 h had values of 6.25 ± 2.09 and 9.27 ±1.76 nmol/g w/w, respectively (p < 0.05). Changes in the AMP

concentration of gonads recovered from both the control urchinsand urchins held in air (Fig. 4, Panel A) mirrored those recordedfor ADP, with an initial increase and subsequent decrease in thegonads recovered from the control urchins and a steady declinefor gonads recovered from urchins held in the air. Concentrationsof IMP in gonads recovered from control urchins also showed asignificant (p < 0.05) increase by day 3 of storage at 4 �C (+26%, incomparison to day 0). However, the IMP concentrations decreasedafter day 3, returning to concentration levels recorded at day 0(Fig. 4, Panel B). In comparison with their respective day 0 values,IMP values in gonads recovered from urchins held in air increasedslightly by day 5 of storage (+5% for urchins held at 15 �C for72 h,+3% for urchins held at 4 �C for 144 h) but then decreased tovalues below that recorded at day 0 of storage at 4 �C (�29% forurchins held at 15 �C for 72 h, �15% for urchins held at 4 �C for144 h; in comparison to day 0). Furthermore, IMP levels in gonadsrecovered from urchins held in air were lower than that recordedin gonads recovered from control urchins throughout storage.IMP has been reported to be indispensable for producing thecharacteristic sought after umami fresh and fresh seafood flavourassociated with excellent quality of sea urchin gonads (Fuke &Konosu, 1991; Matsumoto & Yamanaka, 1990) and when the IMPcomponent was removed from a synthetic sea urchin gonad extractthere was a notable decrease in the umami flavour (Fuke & Ueda,1996). Inosine, the first dephosphorylated ATP catabolite mea-sured, slowly increased in gonads recovered from control urchinsduring the first 7 days of storage at 4 �C (Fig. 5, Panel A), with asignificant (p < 0.05) increase by day 10. On day 10 of storage theIno concentration was 8.5-fold higher than on day 0. In gonads

846 W. Verachia et al. / Food Chemistry 141 (2013) 841–846

recovered from urchins held in air, the Ino concentration was sig-nificantly (p < 0.05) higher than that detected in gonads recoveredfrom control urchins during the first 7 days of storage. Significantchanges in the Hyp concentration were observed only on day 7and 10 of storage for all recovered gonads (Fig. 5, Panel B). Thus,for gonads recovered from control urchins, a notable depletion ofcompounds related to ATP metabolism and energy state occurredonly after 10 days of storage at 4 �C. While for gonads recoveredfrom urchins held in air, after minimal changes occurred duringthe initial 3 days of storage at 4 �C, significant (p < 0.05) changesin the ATP content and in most of its catabolites (particularlyADP, AMP and Ino) were noted.

As a consequence of the relatively slow rate of imbalance ofenergy metabolism of gonads recovered from control urchin, theK-value (Fig. 6) increased exponentially (r2 = 0.948; p < 0.05)remaining below 20% by day 7 of storage and finally reaching avalue of 29.69% ± 3.15 by day 10 of storage at 4 �C (2.7-fold higherthan the K-value at day 0; p < 0.05). In the gonads recovered fromurchins held in air (Fig. 6), a steady increase in the K-value wasnoted, which was also linearly related to the length of storage at4 �C (15 �C for 72 h: r2 = 0.939, p < 0.05; 4 �C for 144 h: r2 = 0.947,p < 0.05). By day 5 of storage at 4 �C, gonads recovered fromurchins held in air exhibited K-values well above 20% (p < 0.05;compared to control urchins). Final K-values after 10 days ofstorage at 4 �C of gonads recovered from urchins previously heldat 15 �C for 72 h or 4 �C for 144 h were 59.34 ± 6.27 and 48.18 ±5.71, respectively (p < 0.05), indicating a progressive decline inshelf-life and quality of these gonads. The K-value therefore clearlyindicated that holding the urchins in air prior to recovery compro-mised cell energy metabolism at day 0 of storage, thereby reducingthe subsequent shelf-life at 4 �C which may be explained byassuming that the progressive dephosphorylation of nucleotides(ATP, ADP and IMP) causes a concomitant increase in the dephos-phorylated compounds Ino and Hyp (Saito et al., 1959) (see Fig. 7).

4. Conclusion

In conclusion, the results of this study demonstrate that seaurchin post-harvest holding regimes can negatively affect cellenergy metabolism, thereby reducing the time of subsequent go-nad shelf-life as evidenced by dramatic changes in ATP, ATPcatabolites and K-value. Since the K-value, calculated from theactual concentration of ATP and its degradation products in thetissue, has been indicated to correlate strictly with freshness ofsea foods (Massa et al., 2003, 2005; Özogul et al., 2010), includingsea urchin gonads (Wang et al., 2011), it is appropriate that theanalysis of adenylates used to calculate the K-value is applied toevaluate freshness of sea urchin gonads as part of routine qualitycontrol. However, this analysis should be performed using a properprotocol of tissue deproteinisation (aimed at minimising metabo-lite loss with the highest efficiency of extraction) as well as areliable HPLC method for their determination.

Acknowledgements

Funding from the Commonwealth Scholarship Scheme as wellas the New Zealand Foundation for Research, Science and Technol-

ogy is gratefully acknowledged. The authors also wish to thank Dr.Miles Lamare for the procurement of E. chloroticus. The authorswould also like to thank Dr. Alaa El-Din Behkit, Dr. Jackie Woodsand Mrs. Yuko Matzuno for their unwavering support and all theirassistance in making this project possible.

References

Au, J., Su, M., & Wientjes, M. (1989). Extraction of intracellular nucleosides andnucleotides with acetonitrile. Clinical Chemistry, 35(1), 48–51.

Berne, R. M. (1963). Cardiac nucleotides in hypoxia: Possible role in regulation ofcoronary blood flow. American Journal of Physiology – Cell Physiology, 204(2),317–322.

Fletcher, G. C., & Statham, J. A. (1988). Shell-life of sterile yellow eyed mullet(Aldrichetta forsteri) at 4 �C. Journal of Food Science, 53(5), 1030.

Fuke, S., & Konosu, S. (1991). Taste-active components in some foods: A review ofJapanese research. Physiology and Behavior, 49(5), 863–868.

Fuke, S., & Ueda, Y. (1996). Interactions between umami and other flavorcharacteristics. Trends in Food Science and Technology, 7(12), 407–411.

Gebelein, M., Merdes, G., & Berger, M. R. (1992). Nucleotide preparation from cellsand determination of nucleotides by ion-pair high-performance liquidchromatography. Journal of Chromatography B Biomedical Sciences andApplications, 577(1), 146–150.

Gill, T. A. (1990). Objective analysis of seafood quality. Food Reviews International, 6,681–714.

Kimmich, G. A., Randles, J., & Brand, J. S. (1975). Assay of picomole amounts of ATP,ADP, and AMP using the luciferase enzyme system. Analytical Biochemistry,69(1), 187–206.

Lazzarino, G., Amorini, A. M., Fazzina, G., Vagnozzi, R., Signoretti, S., Donzelli, S.,et al. (2003). Single-sample preparation for simultaneous cellular redox andenergy state determination. Analytical Biochemistry, 322(1), 51–59.

Massa, A. E., Palacios, D. L., Paredi, M. E., & Crupkin, M. (2005). Postmortem changesin quality indices of ice-stored flounder (Paralichthys patagonicus). Journal ofFood Biochemistry, 29, 570–590.

Massa, A. E., Paredi, M. E., & Crupkin, M. (2003). A chemical assessment of freshnessin stored adductor muscle from scallops. Brazilian Journal of ChemicalEngineering, 20, 147–152.

Matsumoto, M., & Yamanaka, H. (1990). Postmortem biochemical change in themuscle kuruma prawn during storage and evaluation of the freshness. NipponSuisan Gakkaishi, 56, 1145.

McBride, S. C., Price, R. J., Tom, P. D., Lawrence, J. M., & Lawrence, A. L. (2004).Comparison of gonad quality factors: Color, hardness and resilience, ofStrongylocentrotus franciscanus between sea urchins fed prepared feed or algaldiets and sea urchins harvested from the Northern California fishery.Aquaculture, 233(1–4), 405–422.

Özogul, F., Özden, Ö., Özogul, Y., & Erkan, N. (2010). Nucleotide degradationproducts of gamma-irradiated sea bream (Sparus aurata) stored in ice.International Journal of Food Science and Technology, 45(11), 2290–2296.

Saito, T., Arai, K., & Matsuyoshi, M. (1959). A new method for estimating thefreshness of fish. Bulletin of the Japanese Society of Scientific Fisheries, 24,749–750.

Schäfer, S., Abele, D., Weihe, E., & Köhler, A. (2011). Sex-specific biochemical andhistological differences in gonads of sea urchins (Psammechinus miliaris) andtheir response to phenanthrene exposure. CORD Conference Proceedings, 71(1),70–78.

Van Doorn, J. E., Goormachtig, P. F. J., & De Haan, A. (1989). Influence of perchloricacid on ion-pair high-performance liquid chromatography of nucleotides.Journal of Chromatography B Biomedical Sciences and Applications, 496, 441–449.

Wang, C., Xue, C., Xue, Y., Li, Z., Lv, Y., & Zhang, H. (2011). Quality changes in seaurchin (Strongylocentrotus nudus) during storage in artificial seawater saturatedwith oxygen, nitrogen and air. Journal of the Science of Food and Agriculture.

Weihe, E., & Abele, D. (2008). Differences in the physiological response of inter- andsubtidal Antarctic limpets Nacella concinna to aerial exposure. Aquatic Biology,4(2), 155–166.

Williams, C., & Forrester, T. (1976). Loss of ATP in micromolar amounts afterperchloric acid treatment. Pflügers Archives European Journal of Physiology,366(2), 281–283.