Determination of nucleotides in fish tissues using ... - ACS Publications

(17) Jorgenson, J. W.; Lukács, K. D. Anal. ... tions Science; John Wiley & Sons, Inc.: New York, 1973; Chapter. 17. ... (21) Jorgenson, J. W.; Rose, ...
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Anal. Chem. 1990, 62, 2490-2493

(17) Jorgenson, J. W.; Lukacs, K. D.Ana/. Chem. 1981, 53, 1298-1302. (18) Ewlng. A. G.; Wallingford, R. A.; Oleflrowicz, T. M. Anal. Chem. 1989, 61, 292A-303A. (19) Karger, B. L.; Snyder. L. R.; Horvath, C. An Introductbn to Separat&ns Science: John Wllev & Sons, Inc.: New York. 1973: ChaDter 17. (20) W h e m , D. H.; Loeb, A. L.; Overbeek, J. Th. G. J . colloid Interfece SCi. 1968, 22, 78-79. (21) Jorgenson,J. W.; Rose, D.J. Anal. Chem. 1988, 60, 642-648. (22) Giddings, J. C. Sep. S d . 1989, 4 , 181-189. (23) Karger. B. L.; Snyder. L. R.; tiorvath, c. An Intrcxiuctjm to separation Sclence: John Wlley 8 Sons, Inc.: New York, 1973: Chapter 5.

(24) Huang, X.; Coleman, W. F.; &re, R. N. J . C h r m t o g r . 1989, 480, 95-110.

RECEIVED for review March 21. 1990. AcceDted August 15. 1990. This research was supported by the U.S.Depirtment of Energy under Contract DE-AC06-76RLO 1830. Pacific Northwest Laboratory is operated for the US.Department of Energy by Battelle Memorial Institute under Contract DE-AC06-76RLO 1830.

Determination of Nucleotides in Fish Tissues Using Capillary Electrophoresis An-Lac Nguyen, John H. T. Luong,* and Claude Masson Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2

Capillary .kctrophore&s has been applled to quantltate nucleotide ckgrsdatlon In flrh tluues, to provlde a basis for detemrkrlng the K value, an lndicotor of f k h froshness. The three major comporndr,h o h e -te (IMP), Inoskre (HxR), and hypoxanthine (Hx) were dirtlncthrely wparated at 416 V/cm aQpUed potentlal, 100 mM CAPS buffer, pH 11. There was a good correlstbn betweem the peak area and the nueleotkk concentraton. By whrg a short dlstance (22 cm)from the same entrance to the detector, the Identtfkatkn and determlnatlon of these compounds fn each sampb were compkted wltMn 13 mln. The r d s OMalned correlated vsry w d l wtth how o b h d hy efum8tk assays. ThecapIhry wascompktdyregrrmwated with 1 N NaOH, to dlssoclate all bound materlak from the capwSry watl, mainly catlons In the Hsh extract. TMS provided the same d k a surface for repeated runs, resulting In reproducible electropherograms.

INTRODUCTION Capillary electrophoresis (CE) is conducted in an open capillary to resolve species according to their different migration rates resulting from an applied electric field. The electrophoreticmigration provides a high resolution while the capillary format confers high speed, accurate quantitation, and ease of automation. Various aspects of this new and promising technique have been reviewed and discussed recently (1-4). The impressive resolving power of CE is a great improvement over gel electrophoresis or high-pressure liquid chromatography (HPLC). This technique has been successfully used to separate organic and inorganic ions (5,6), amino acids (7),peptides (€0,and oligonucleotides (9,10). In the past, the separation of proteins preeented some difficulty due to protein adsorption onto the capillary wall, but the problem has now been overcome (11-13). The different separation principle of CE has enabled the detection of impurities in products which appeared homogeneous by HPLC analyses (13). Therefore CE has been proposed as a technique of crm-checking quality control. The speed of CE also suggests its applicability for process control and for analyses of biological samples. In the production of specialty biochemicals such as antibodies or synthetic poly0003-2700/90/0362-2490$02.50/0

peptides, if the product level is quickly determined, corrective measures can be timely taken to optimize productivity. In the analyses of biological samples, the prerequisite is to establish conditions that provide good resolution of the substances of interest. In practice, application of CE to complex sample matrices is problematic. The interaction among the sample constituents and their interaction with the capillary wall is quite unpredictable. Reconditioning of the capillary after each run is still a trial-and-error procedure. Consequently, the reproducibility of the electropherogramcan only be obtained after a time-consuming series of trials. To date, there are only a few reports on the capillary electrophoresis of natural biological samples, to separate and quantitate the nucleotides in the organs of rats (14), and guinea pigs (15), and the polyamines in rat tissues (16). All other CE studies have been conducted on highly purified materials reconstituted in appropriate buffers. In this study, CE was applied to separate and quantitate the purine nucleotides in fiih tissue which are involved in the decomposition of adenosine 5’-triphosphate (ATP) following the death of a fish and during subsequent storage ATP ADP AMP IMP HxR Hx where ADP and AMP are adenosine diphosphate and adenosine monophosphate, IMP is inosine monophosphate, HxR is inosine, and Hx is hypoxanthine. In most fmh species, ATP degrades very quickly to IMP and this compound is reported to impart the pleasant flavor of fresh fish while the accumulation of Hx results in an off-taste. The concentrations of Hx, HxR, and IMP, and a freshness index derived from these concentrations (see definition later) have been used as indicators of fish freshness. The results obtained by enzymatic assays were also presented and compared with the CE results.

- - - - -

EXPERIMENTAL SECTION Materials. Xanthine, hypoxanthine, inosine, inosine 5’monophosphate, xanthine oxidase from butter milk (XO), nu-

cleoside phosphorylase from calf spleen (NP), and nucleotidase from Crotalus adamanteus venom (NT)were purchased from Sigma Chemical Co. (St. Louis,MO). Other reagents including 3-[cyclohexylamino]-l-propanesulfonic acid (CAPS)were products of Aldrich Chemical Co. (Milwaukee, WI). Fresh rainbow trout and frozen fillet of haddock were obtained from a local market. Preparation of Fish Extract. Tmue samples from fish fillet (5 g) were homogenized with 3 mL of 10% trichloroacetic acid. After centrifugation at 27000g,the supernatant was neutralized with 2 M sodium hydroxide. Phosphate buffer (10 mM, pH 7.8; 0 1990 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER

hereinafter referred to as buffer P7.8) was added to adjust the volume to 10 mL, designated as the neutralized extract. Determination of Concentrations by Enzymatic Assays. Following a published procedure (17), the analytes were enzymatically converted to uric acid. For Hx determination, only XO was required. For determining the s u m of Hx and HxR, XO was used together with NP. Combination of XO with both NP and NT was required to determine the sum of Hx, HxR, and IMP. The enzyme solutions were prepared separately, containing 1.52 IU of XO, 0.74 IU of NP,or 36 IU of NT per milliliter, respectively. The neutralized extract was diluted 2- to IO-fold with buffer P7.8, and 850 pL of the diluted solution was placed in quartz cuvette. Following the addition of one or more enzymes as required (50 p L each from the above-mentionedsolutions), buffer was added to attain a total assay volume of 1mL, and the absorbanceat 290 nm was immediately followed (Beckman spectrophotometer, Model DU-7). The increase in absorbance after 10 min indicated the amount of uric acid generated, which was then equated to the amount of analyte(s) in question. Capillary Electrophoresis. An automated d E instrument (Model 270A, Applied Biosystems, Foster City, CA) was used to obtain the electropherogram. For each electrophoresis run the capillary was first washed with a wash solution and then reconditioned with a selected buffer. A typical run consisted of a 2-min washing period followed by 2 min of capillary reconditioning. During these periods the solution was forced through the capillary by a vacuum. The sample was introduced by applying a precisely controlled 5 mmHg vacuum, required 1-5 s, resulting in 5-15 nL of sample being introduced to the capillary. After the sample introduction, voltage was applied and maintained at the preset value. The capillary was 72 cm long and the applied voltage was 30 kV, equivalent to 416 V/cm of capillary. The capillary could be installed so that the sample to detector distance, referred to as the effective capillary length, was either 50 or 22 cm. Both arrangements were used, the choice was indicated in each electropherogram. Electrophoresed components were detected with an W detector set at 250 nm. The detector signal was transmitted to an integrator (Model SP-4270,Spectra-Physics,San Jose, CA) which integrated and reported the area corresponding to each peak. Identification of the Electrophoretic Peaks. An electropherogram was first obtained for a sample of fish extract after appropriate dilution. An aliquot (10 pL) from stock solutions of 2 mM Hx in 10 mM NaOH, 2 mM HxR in 10 mM NaOH, or 2 mM IMP in water was added to the dilute extract and another electropherogramwas obtained. Each dilute extract streaked with Hx, HxR, or IMP showed a clearly intensified peak corresponding the added compound. By comparison of the migration time of the intensified peak with those present in the dilute extract, the peaks corresponding to Hx, HxR, or IMP were identified. Determination of Concentrations by Peak Areas. For each extract, a solution (designatedas unaltered extract) was prepared by adding 20 pL of 10 mM NaOH and 10 pL of water to a 30 pL extract aliquot, and an electropherogram was obtained. A streaked solution was prepared by adding 10 pL from each of the Hx, HxR, and IMP stock solutions, and another electropherogram was obtained at the same running conditions. Such a procedure assured the same level of constituents in the streaked and unaltered samples, except the analyte contents. Since the identity of each major peak is known, the concentration of each analyte is calculated as C = C,A/(A, - A ) , where C, is the increase in concentration due to the addition of stock solution, A is the peak area in the unaltered extract, and A , is the area of the corresponding peak in the streaked sample.

RESULTS AND DISCUSSION Reproducibility of the Electropherogram. The neutralized extract contained a significant amount of cations as indicated by its high conductivity. Such cations may bind to the capillary wall and would not be completely dissociated with weak washing solution. As experimentally confirmed, when the capillary was washed with 0.1 N NaOH after each run, the retention time was observed to differ significantly from one run to the next. The retention time of the hypoxanthine peak increased from 4.3 to 7.0 min between the first

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2.17

lb I

.-

78

f-

Figure 1.

Effect of sample dilution. E f f i of a neutralized extract without dilution (a)and with 1:l dlkrtbn (b): voltage, 416 V/cm; current, 18 pA; buffer, 100 mM CAPS, pH 10.5; effective capillary length, 50 cm; capillary diameter, 50 pm; temperature 30 'C.

and the fifth run. Prolonged washing up to 10 min with the same solution did not lead to any improvement. It was further noted that if the capillary was washed with 0.1 N NaOH, flushed and filled with the running buffer, and left overnight, the f i s t electropherogram obtained in the next day displayed the same retention time as that in the very first run. Apparently, this treatment effectively removed the cations bound to the capillary wall. However, when washing was made with 1 N NaOH, the electropherogram was reproducible for successive repetitions. Evidently, such a strong alkaline solution was able to dissociate all cations from the capillary wall to provide the same silica surface for each run. Therefore washing with 1N NaOH was performed after each analysis. Influence of Sample Constituents. The electropherogram of a neutralized extract is presented in Figure la. The doublet a t 6.97 and 7.44 was attributed to IMP by a streaking procedure (Experimental Section). For 2-fold diluted extracts, the doublet merged to a single peak at 6.78. In a previous study, this effect of sample dilution was also observed with the extract prepared from whole mussels (18). Conceivably the cations present in the extract could form salt with the phosphate group of IMP and thus influence its migration. The cations could also bind to the negatively charged wall and affect the mobility. It was also probable that after dilution the sample's ionic strength was decreased, creating a higher potential gradient across the sample zone, and thereby resulted in a concentrating effect on the sample constituents. More importantly, the buffers used in CE are often of low molarity, the extract constituents could easily upset the pH of the buffer, at least in the capillary spaces immediately preceeding and followingthe sample. Such a pH upset would also disturb the migration of the analytes. Further studies are required to understand these effects. However, at present, it is important to note that some complex samples which displayed widely distorted peaks resulted in good peak characteristics after dilution. Buffer Selection. Since CE depends on electrophoretic migration, the pH of the running buffer is the most important operating parameter. At pH 9.5, 10 mM borate (figure not

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ANALYTICAL CHEMISTRY, VOL. 62, NO. 22,

NOVEMBER 15, 1990 Table I. Peak AreaConcentration Relationship added

i.

mM

migration time, min

peak area, arb unit

additional peak area, arb unit

total [Hxl, mM

0.0 0.1 0.2 0.4 0.6 0.8 1.0

4.25 4.25 4.30 4.27 4.28 4.29 4.33

323 366 400 488 543 628 707

43 77 164 220 305 383

0.82 0.92 1.02 1.22 1.42 1.62 1.82

Table 11. Peak Areas of Unaltered and Streaked Extracts and Calculated Concentrations 2b

migration peak area, arb unit time, min analyte unaltered streaked 3.82 6.05 11.11

13.48

I

WxI,

HxR

Hx IMP

284 123 224

concn in extract, m M CE enzyme 1.36 0.49 0.41

423 289 589

1.40 0.51 0.44

I

Figure 2. Effect of buffer. Electropherogram of the same neutralized extract run with 100 mM CAPS at pH 10.0, current 10 pA (a),and pH 10.6, current 22 pA (b). Other conditions and the same as in Figure 1.

shown) and pH 10.0,100 mM CAPS (Figure 2a) the analytes could not be resolved; Hx and HxR comigrated while IMP exhibited very poor peak characteristics. On the contrary, a t pH 10.6, 100 mM CAPS the three analytes were distinctively separated (Figure 2b). Although the IMP peak was not sharp, likely due to the interaction between IMP and the cations, it was reproducible. Such a good resolution was obtained with CAPS buffers of pH 10.5 to 11.0, a better resolution was provided by higher pH. Most of the electropherograms were obtained in the presence of mesityl oxide, a neutral compound, which reached the detector before all the analytes of interest. In other words, at pH 9.5 and higher, all analytes were negatively charged, HxR being the least and IMP being the most negative. It was thus not surprising that higher pH provided better resolution. However, due to the amount of NaOH required to adjust the pH, CAPS solutions of higher pH exhibited higher conductivity, resulting in high current when a constant voltage was applied. The pH value of 11.0 was thus the maximum that could be used. At this pH the most negative analyte, IMP, exhibited a migration time of about 25 min when the effective capillary length was 50 cm. In order to take advantage of the good resolution at pH 11.0 and shorten the running time, the capillary was inverted. With capillary inversion, the total length of the capillary remained the same as before (72 cm), but the effective capillary length was only 22 cm. All the electropherograms presented hereinafter were obtained with this arrangement. Peak Area-Concentration Relationship. If the electropherogram is to be used for quantitation of an analyte, a relationship between the concentrationand the peak area must be established a priori. The peak areas corresponding to Hx in a series of electropherograms obtained by adding varying amounts of Hx to the same fish extract, originally containing 0.82 mM of Hx, are given in Table I. Theoretically, the Hx content of the extract could be assewed by using the peak area shown by the unaltered extract and that obtained with any of the streaked samples. In reality, the values obtained by all different combinations gave a value of 820 f 60 pM. From the data of Table I, it can be seen that a linear relationship existed between the additional peak area and the added concentration of Hx (slope = 377 and correlation coefficient

3a

3b

Figwe 3. Electropherogram of an extract unaltered (a), and streaked (b): voltage, 416 V/cm; current, 40 pA; buffer, 100 mM CAPS, pH 11; effective capillary length, 22 cm; capillary diameter, 50 pm; temperature, 30 ' C . = 1.00). Similarly, a plot of the total Hx concentration versus the peak area also yielded a linear relationship. Assays of Extracts. The electropherogram of a typical extract, obtained at optimal running conditions, is presented in Figure 3a. Addition of Hx, HxR, or IMP individually to the sample would intensify the peaks at 6.04,3.78, and 11.14 min correspondingly. Figure 3b presents the electropherogram of the same extract spiked with HxR, Hx, and IMP. Table I1 summarizes the data for the peaks of interest, and the calculated concentrations of the analytes in question. For comparison, the concentrations in the extracts BB determined by enzymatic assays are also presented in this table.

ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990

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Table 111. Comparison of the K Values Obtained by CE and by Enzymatic Assays enzyme

"

c

0

100

200

300

400

500

600

"t, .

,

500

0

0

500

1000

1500

2000

I ' I

"0

1000

2000

3000

Concrntrrtlon by CE ( c Y) e 4. Conc8ntratbnsdetermined by CE and by enzymatic assays: (A)IMP (0)HxR; (0)Hx. m

Figure 4 presents results determined by CE and enzymatic assays for a set of tissue extracts. A line representing the perfect correspondence is drawn in each graph for ease of observation. In general, there was good agreement between the values obtained by CE and those determined by enzymatic assays. In particular, the Hx results obtained by the two methods correlated very well, since the Hx concentrations in the streaked extracts were well within the limit of the linear relationship between the peak area and concentration, as presented in the previous section. In the determination of H a , some results at high contents did not compare well. The streaked extracts corresponding to those results had HxR contents above 1.5 mM, likely those concentrations were beyond the linear range of the peak area-concentration relationship. The IMP concentrations determined by two methods showed a significant difference. The presence of the phosphate group rendered IMP highly negatively charged, resulting in high electrophoretic mobility and a long retention time. The variety of cations in the sample matrix likely would interfere with the IMP migration as indicated by the broadness of the IMP peak. In fact, the reproducibility of the IMP peak area was not satisfactory, at times there was 10% difference between two consecutive runs of the same sample. Consequently, the IMP concentration as determined by CE could involve an error of about 5%. In CE assays of the extracts, each unaltered extract was run in duplicate. Each extract was then streaked with all the analytes and the resulting sample was also analyzed in duplicate. Each concentration presented in Figure 4 was determined by the formula presented in the Experimental Section, using the average value for A and A,. The concentrations could also be calculated by using the EPG of the unaltered extracts and that of only one streaked extract. For extracts obtained from the same fillet, this simplified procedure gave results close to those reported above. However, the concentration calculated by comparing the EPG of an unaltered extract of one fish species (e.g. trout) with an EPG of

sample

description

CE

assays

1 2 3 4 5

trout freshly killed sample 1 after 2 ha sample 1 after 4.5 h sample 1 after being frozen and thawed sample 4 after 2 h frozen haddock after thawing sample 6 after 4 h sample 6 after 6 h

0.09 0.36

0.08

0.55 0.77 0.81

0.52 0.77

0.12

0.13

0.27

0.28 0.36

6

7 9

Storage a t 20

0.38

0.34

0.82

OC.

the streaked extract of another species (e.g. haddock) was very erroneous. This observation thus reconfirmed the influence of the sample constituents. Indicators of Freshness. Although the hypoxanthine concentration in fish tissues can be used as an indicator of fish freshness, other indicators have been found to be applicable to a wider variety of fish species. One composite indicator that was found to correlate well with sensory results (i.e. panel tasting) is the K value, as defined by K = ([Hx] + [HxR])/([Hx] + [HxR] + [IMP]) (19).Since IMP imparts the pleasant flavor and Hx creates the off-taste, a K value close to 0 indicates freshness while a value close to 1 indicates advanced degree of degradation. Table I11 clearly confirms this trend and also shows that the K values determined by CE corresponded very well with those determined by enzymatic assays. It should be noted that xanthine oxidase catalyzes both hypoxanthineand xanthine, therefore the K values determined by enzymatic assays could be different from those determined by CE if the fish extracts contained significant amounts of xanthine. The experimentalresults obtained during this study showed that the xanthine contents in all extracts were insignificant. Such a result was not unexpected since xanthine oxidase is not present in the fish tissues and hypoxanthine is the final product resulting from the autodegradation of ATP. Registry No. IMP, 131-99-7; HxR, 58-63-9; Hx, 68-94-0.

LITERATURE CITED Ewing, A. G.; Wallingford. R. A.; Oiefirowicz, T. M. Anal. Chem. 1989, 6 1 , 292A-303A. Eiby, M. J. Bio/Technology 1989, 7 , 903-911. Karger, B. L. Nature 1989, 339, 641-642. Banks, P. J . NIH Res. 1990, 2 , 87-90. Hjertbn, S.; Elenbring, K.; Kilar, F.; Llao, J.I.; Chen, A. J. C.; Slebert, C. J.: ZhU. M.-D. J . ChrOmetW. 1987. 403. 47-61. Gross, L.; Yeung, E. S. Anal. & e m . 1990. 62, 427-431. Grossman, P. D.; Wilson, K. J.; Petrie, G.; L a w , H. H. Anal. Blochem. 1988, 173, 265-270. Cobb, K. A.; Novotny, M. Anal. Chem. 1989, 6 1 , 2226-2231. Cohen, A. S.; Terabe, S.; Smith, J. A,; Karger, B. L. Anal. Chem. 1987. .... 59. .. 1021-1029 Cohen, A.' S.;Naja;in, D.; Smith, J. A,; Karger, 8. L. J . Chromatogr. 1988. 458. 323-333. Hjertin, S . ' J . Chromatogr. 1985, 347, 191-198. McCormick, R. M. Anal. Chem. 1988, 60, 2322-2328. Grossman, P. D.; Colburn, J. C.; Lauer, H. H.; Nieisen. R. G.; Riggin, R. M.; Sittampalam, G. S.; Rickard, E. C. Anal. Cham. 1989, 61 1 , 1188- 1194. Tsuda, T.; Nakagawa, G.; Sato, M.; Yagi, K. J . Appl. Biochem. 1983, 5 , 330-336. Tsuda, T.; Takagi, K.; Watanabe, T.; Satake. T. HRC CC, J . High Resolut. Chromatogr. Chromtatogr. Commun. 1988. I f , 721-723. Tsuda, T.; Kabayashi, Y.; Horl. A.; Matsumoto, T.; Suzuki, 0. J . Mcrocolumn Sep. 1690, 2 , 21-25. Mulchandani, A.; Male, K. B.; Luong, J. H. T. Biotechnol. Bioeng. 1990. 35. 739-745. Nguyen, A.-L.; Luong, J. H. T.; Masson. C. Anal. Lett. 1990, 23,9. Karube, I.; Matsuoka, H.; Suzukl. S.; Watanabe, E.: Toyama, K. J . Agric. Food Chem. 1984, 32, 314-319.

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RECEIVED for review May 30,1990. Accepted August 9,1990. NRC Canada publication number 31459.