J. Sci. Fd Agric. 1977, 28, 935-940
Histidine Metabolism in Fish. Urocanic Acid in Mackerel (Scomber scombrus)" Ian M. Mackie and Jose Fernandez-Salgdro* Torry Research Station, 135 Abbey Road, Aberdeen AB9 8DG, Scotland (Manuscript Received 14 March 1977)
Urocanic acid, isolated from perchloric extracts of the flesh of mackerel (Scomber scombrus) using an anion exchange resin Dowex-1 acetate, was identified by thin-layer chromatography, U.V. absorption and mass spectra. During storage of sterile and nonsterile tissue the concentration of urocanic acid increased at similar rates, indicating that L-histidine ammonia-lyase is not associated with bacterial spoilage. 1. Introduction
There is evidence that free histidine in fish muscle can be catabolised by two routes. The first, which is well documented, is decarboxylation brought about largely by contaminating bacteria,l and the second, about which there is little information, is deamination to urocanic acid. The latter route is the main pathway of histidine catabolism under normal physiological conditions.2 Sakaguchi and Kawai3 demonstrated for the first time that a histidine deaminase (histidine ammonia-lyase) existed in muscle and other organs of various species of fish including Japanese mackerel, Scomber japonicus, and showed that urocanic acid was produced from added histidine. Urocanic acid has been reported to be present in human sweat,4 epidermis5 and in urine.6 Its choline derivative is known to be pharmacologically active and to occur in molluscs.2 This paper describes a procedure for the determination of urocanic acid in fish and gives information on its production during storage of mackerel (Scomber scombrus) under sterile and nonsterile conditions. 2. Experimental 2.1. Material The mackerel (Scomber scombrus) were caught off Oban by hand-line and were transported by road to the laboratory in a container well mixed with ice. The sterile muscle portions and minces were prepared as described by Mackie and Fernandez-SalguCro7 and stored in sterile jars, while whole fish, fillets and non-sterile minces were sealed in polythene bags and stored at 0°C. Whole fish were also stored at 10 and 23°C respectively. 2.2. Preparation of perchloric acid extracts Tissue (100 g) either as whole muscle or as mince, was homogenised in 150 ml of 0.6 M perchloric acid (PCA) and centrifuged at 20 000 g for 15 min. This extraction procedure was repeated on the tissue residue and the combined extracts were filtered through Whatman No. 2V paper. The filtrates were neutralised with 4 M potassium hydroxide and cooled to precipitate potassium perchlorate which was removed by centrifugation. The precipitate was washed with cold distilled water and the combined washings were added to the supernatant solution. The total volume was then concentrated by evaporation under vacuum to approximately 15 ml and centrifuged to remove further amounts of potassium perchlorate. The precipitate was washed with cold distilled water and the washings a
Crown Copyright Reserved.
* Present address : Tecnologia y Bioquimica de 10s Alimentos, Facultad de Veterinaria, Cbrdoba, Spain. 975
I. M. Mackie and J. Fernandez-SalguBro
936
combined with the previous supernatant solution. This perchloric acid extract was diluted with distilled water to 25 ml in a volumetric flask. 2.3. Preparation of resin and column Dowex-1-chloride (200-400 mesh, 8 % cross-linked) was washed three times with 2 M sodium hydroxide followed by 6 M hydrochloric acid and, after washing with distilled water until chloridefree, it was eluted with a saturated solution of sodium acetate and washed finally with water. A chromatographic column (8 x 230 mm) was packed with resin to a height of 70 mm, and a flow-rate of 2ml/min was obtained using a Milton Roy pump. An Ultrograd (L.K.B. Instruments Ltd) gradient elution system was used to alter the concentration of eluant.
2.4. Isolation and determination of urocanic acid An aliquot (5.0 ml) of the above perchloric extract was applied to the column which was then washed with 200 ml of distilled water followed by 50 ml 0.001 M acetic acid. The urocanic acid adsorbed on the resin was then eluted by increasing the concentration of acetic acid to 0.06 M. The transmission of the eluate was monitored continuously at 254 nm with a LKB Uvichord (Figure 1). The urocanic acid was collected in approximately 30 ml, and, after adjustment of the pH to 7.4 with 2 M dipotassium hydrogen phosphate, the volume was made up to 50 ml. The concentration of urocanic acid was determined from a standard plot of the absorbance at 277 nm of authentic urocanic acid over the range of concentration (0-10-4 M) at pH 7.4 in 2 M dipotassium hydrogen phosphate solutions using a Unicam SP 1700 Ultraviolet spectrophotometer. For the blank, 300 ml of perchloric acid was taken through the same procedure of neutralisation and elution through the column.
c
100
200
Time ( m i n )
Figure 1. Elution profile of urocanic acid from column of Dowex-1 acetate. --, nni; ------, concentration of acetic acid in eluant.
percent transmission at 254
931
Histidine metabolism in fish
2.5. Thin-layer chromatography A layer of Silica Gel CT, 250 pm thick, was spread on glass plates (200 x 200 mm), allowed to dry and then activated at 100°C for 30 min. Ascending one-dimensional chromatography was carried out using n-butanol : acetic acid : water (12 : 3 : 5 by vol) as developing agent. Pauly’s reagentg was used to detect urocanic acid and amines. 2.6. Mass spectrometry Mass spectra of the non-derivatised acids were determined by Dr A. McGill of Torry Research Station on anAEI-902 mass spectrometer, the sample being introducedvia the direct insertion probe. The ion source at a temperature of 180°C above ambient was operated at an electron voltage of 70 eV and a filament current of 100 PA.
3. Results and discussion
The isolation of urocanic acid from the perchloric acid extract of tissue was based on the procedure described by Tabor,s using the anion-exchange resin Dowex-1, in the acetate form. Prior to elution of urocanic acid from the column with 0 . 0 6 ~acetic acid, weakly absorbing anions and other contaminants were removed by washing with water and 0.01 M acetic acid in his procedure. In our experience elution with 0.01 M acetic acid released some urocanic acid and satisfactory recoveries of added urocanic acid were only obtained if the concentration of the acetic acid in this preliminary wash did not exceed 0.001 M. Using this modified procedure recoveries of added urocanic acid
Figure 2. Thin-layer chromatographic separation of urocanic acid in crude extracts and after purification on a Dowex-1 acetate column. Lanes 1 and 9, authentic urocanic acid; lanes 7 and 8, eluates from column; lanes 2-6, crude perchloric acid extracts of muscle of whole fish stored for 4,7, 10, 14 and 18 days respectively. Chromatogram developed with Pauly’s reagent for imidazole compound^.^
938
I. M. Mackie and J. Fernandez-Salguero
were consistently around 96.9% on further elution with 0.06 M acetic acid. Figure 1 is a typical elution profile of a perchloric acid extract of muscle from the column of Dowex-1. The fraction corresponding to the peak, which was well separated from the other U.V. absorbing material in the eluate, was identified as urocanic acid by the following three criteria. The spectrum obtained at pH 7.4 for the eluate from the column coincided with that of the authentic sample, with a maximum absorption between 276 and 278 nm. Examination of the thin-layer chromatogram for imidazole compounds (Figure 2) showed that the eluate from the column gave an orange-red spot on spraying with Pauly’s reagent, with the same colour and mobility (RF0.59) as authentic urocanic acid. The extent of purification obtained
Standard
I 3 8 (Mt)
I Eluate from column
1
L I40 mie
Figure 3. Mass spectra of authentic urocanic acid (mol.wt 138) and of urocanic acid isolated from mackerel.
by the ion-exchange process was evident on comparing the lanes of the eluates from the column with those of crude perchloric acid extracts. The spot corresponding to urocanic acid was detected in crude extracts of fish stored for 10 days or more at 0°C but only as a minor constituent. For mass spectrometric analysis, the eluate from the column was repeatedly evaporated to dryness and redissolved in distilled water in an attempt to crystallise urocanic acid. Only a small quantity of crystals was obtained, but the weak spectrum (Figure 3) was non the less consistent with that for authentic urocanic acid; significant amounts of impurities were, however, present. Using this anion-exchange procedure for the determination of urocanic acid in perchloric acid extracts of fish muscle, a study was made of the formation of urocanic acid in various preparations of mackerel muscle during storage under sterile and non-sterile conditions. The sterile and non-sterile minces and muscle portions were prepared and stored at 0, 10 and 23°C respectively. Whole fish
939
Histidine metabolism in fish Table 1. Urocanic acid development in various preparations of mackerel heId at 0°C Concentrations of urocanic acid in samples (pg/g) (means of duplicate analyses) ~~
Sterile muscle
Time of storage (days)
Sterile mince
Fillet
Mince
Muscle (whole fish)
4.1 7.6 6.1 13.7 21.8 52.5
3.6 5.0 7.3 17.4 41.2 47.4
1 4 7 10 14 18
7.7 5.7 10.4 14.3 17.6 38.9
9.9 11.8 10.2 14.7 21.3 34.5
9.3 14.9 13.2 17.4 29.4 43.5
Table 2. Urocanic acid development (pglg) in muscle from whole fish held at 10°C and 23°C Temperature of storage (“C) Time of storage
(h) 6 15 24 36 72 120
10
23
-
5.2 7.4 14.0 33.3
-
6.7 -
10.1 28.8
-
were included for comparison as mackerel is normally stored ungutted under commercial conditions. The initial concentrations of urocanic acid in the samples were in the range 3.9-9.9 pg/g of tissue (Tables 1 and 2) and during storage they increased steadily at all temperatures. Some of the scatter in the results must be due to variations between fish as each sample analysed was from a different fish. At 0°C there was no marked difference in the build-up of this acid among the preparations. As expected, the rate of production increased with temperature of storage (at 10°C the rate was about three times higher than at 0 and at 23°C about six times higher). It is of significance that there was no apparent difference in the amount of urocanic acid produced in sterile and nonsterile muscle. This provides good evidence that the catabolism of histidine in mackerel to urocanic acid was entirely an autolytic process. Although the major pathway of histidine catabolism in mammals and in bacteria is initially to urocanic acid and then to imidazolonepropionic acid by histidine ammonia-lyase and urocanase respectively, it would appear that any such enzyme produced by bacteria in the non-sterile samples must be insignificant and that the major bacterial-induced breakdown of histidine is by the histidine decarboxylase route. 0 CH -N
I1
C
c< I NH,-CH
‘d
CH-N
I COOH L-Histidine
59
/I
I1
CH
II I
C-N
I1 +
CHz
I
It NH
I
CH
I COOH
+NH,
Urocanic acid
CH,
I
COOH Imidazolonepropionic acid
940
I. M. Mackie and J. Fernandez-Salgu6ro
These results were taken from an experiment which included the measurement of h’istamine formation during storage of mackerel.7 Free histidine was present at a level of 4000 pg/g of tissue and histamine at less than 0.5 pg/g in whole muscle and minced muscle held under sterile conditions. The concentrations of urocanic acid were much higher than those of histamine even after storage for 1 day at 0°C. However, considering the large amount of free histidine present in the muscle, the activity of histidine ammonia lyase was not high. Since the concentration of urocanic acid did not decrease towards the later stages of storage it would appear that, if urocanase was present in either sterile or non-sterile preparations of mackerel muscle, its activity must have been very weak. Acknowledgement The authors are grateful to Dr J. Gordon of the Dunstaffnage Marine Research Laboratory of the Scottish Marine Biological Association, Oban, for arrangements made to catch the mackerel. The work was carried out by J.F.S. during the tenure of a grant from the Ministry of Education and Science of Spain. References 1. Ienistea, C. In The Microbiological Safety of Food 1973, p. 327 (Hobbs, B. C.; Christian, J. H. B., eds) New York and London, Academic Press. 2. Meister, A. In Biochemistry of the Amino Acids 1965, Vol. 11, p. 825, New York and London, Academic Press. 3. Sakaguchi, M.; Kawai, A. Bull. Jap. SOC.Sci. Fish. 1968, 34, 507. 4. Zenisek, A.; Kral, J. A. Biochim. biophys. Acta 1953, 12, 479. 5. Everett, M. A.; Anglin, J. H.; Bever, A. T. Archs. Derm. 1959, 84, 717. 6. Kerr, J. W. Lancet 1963, 2, 709. 7. Mackie, 1. M.; Fernandez-SalguBro, J. (in preparation). 8. Tabor, H. In Methods in Enzymology 1963, Vol. VI, p. 581 (Colowick, Sidney P.; Kaplan, Nathan O., eds) New York and London, Academic Press. 9. Smith, I. In Chromatographic and Electrophoretic Techniques 1960, Vol. I, p. 218, London, Heinemann.
Histidine Metabolism in Fish. Urocanic Acid in Mackerel (Scomber scombrus)" Ian M. Mackie and Jose Fernandez-Salgdro* Torry Research Station, 135 Abbey Road, Aberdeen AB9 8DG, Scotland (Manuscript Received 14 March 1977)
Urocanic acid, isolated from perchloric extracts of the flesh of mackerel (Scomber scombrus) using an anion exchange resin Dowex-1 acetate, was identified by thin-layer chromatography, U.V. absorption and mass spectra. During storage of sterile and nonsterile tissue the concentration of urocanic acid increased at similar rates, indicating that L-histidine ammonia-lyase is not associated with bacterial spoilage. 1. Introduction
There is evidence that free histidine in fish muscle can be catabolised by two routes. The first, which is well documented, is decarboxylation brought about largely by contaminating bacteria,l and the second, about which there is little information, is deamination to urocanic acid. The latter route is the main pathway of histidine catabolism under normal physiological conditions.2 Sakaguchi and Kawai3 demonstrated for the first time that a histidine deaminase (histidine ammonia-lyase) existed in muscle and other organs of various species of fish including Japanese mackerel, Scomber japonicus, and showed that urocanic acid was produced from added histidine. Urocanic acid has been reported to be present in human sweat,4 epidermis5 and in urine.6 Its choline derivative is known to be pharmacologically active and to occur in molluscs.2 This paper describes a procedure for the determination of urocanic acid in fish and gives information on its production during storage of mackerel (Scomber scombrus) under sterile and nonsterile conditions. 2. Experimental 2.1. Material The mackerel (Scomber scombrus) were caught off Oban by hand-line and were transported by road to the laboratory in a container well mixed with ice. The sterile muscle portions and minces were prepared as described by Mackie and Fernandez-SalguCro7 and stored in sterile jars, while whole fish, fillets and non-sterile minces were sealed in polythene bags and stored at 0°C. Whole fish were also stored at 10 and 23°C respectively. 2.2. Preparation of perchloric acid extracts Tissue (100 g) either as whole muscle or as mince, was homogenised in 150 ml of 0.6 M perchloric acid (PCA) and centrifuged at 20 000 g for 15 min. This extraction procedure was repeated on the tissue residue and the combined extracts were filtered through Whatman No. 2V paper. The filtrates were neutralised with 4 M potassium hydroxide and cooled to precipitate potassium perchlorate which was removed by centrifugation. The precipitate was washed with cold distilled water and the combined washings were added to the supernatant solution. The total volume was then concentrated by evaporation under vacuum to approximately 15 ml and centrifuged to remove further amounts of potassium perchlorate. The precipitate was washed with cold distilled water and the washings a
Crown Copyright Reserved.
* Present address : Tecnologia y Bioquimica de 10s Alimentos, Facultad de Veterinaria, Cbrdoba, Spain. 975
I. M. Mackie and J. Fernandez-SalguBro
936
combined with the previous supernatant solution. This perchloric acid extract was diluted with distilled water to 25 ml in a volumetric flask. 2.3. Preparation of resin and column Dowex-1-chloride (200-400 mesh, 8 % cross-linked) was washed three times with 2 M sodium hydroxide followed by 6 M hydrochloric acid and, after washing with distilled water until chloridefree, it was eluted with a saturated solution of sodium acetate and washed finally with water. A chromatographic column (8 x 230 mm) was packed with resin to a height of 70 mm, and a flow-rate of 2ml/min was obtained using a Milton Roy pump. An Ultrograd (L.K.B. Instruments Ltd) gradient elution system was used to alter the concentration of eluant.
2.4. Isolation and determination of urocanic acid An aliquot (5.0 ml) of the above perchloric extract was applied to the column which was then washed with 200 ml of distilled water followed by 50 ml 0.001 M acetic acid. The urocanic acid adsorbed on the resin was then eluted by increasing the concentration of acetic acid to 0.06 M. The transmission of the eluate was monitored continuously at 254 nm with a LKB Uvichord (Figure 1). The urocanic acid was collected in approximately 30 ml, and, after adjustment of the pH to 7.4 with 2 M dipotassium hydrogen phosphate, the volume was made up to 50 ml. The concentration of urocanic acid was determined from a standard plot of the absorbance at 277 nm of authentic urocanic acid over the range of concentration (0-10-4 M) at pH 7.4 in 2 M dipotassium hydrogen phosphate solutions using a Unicam SP 1700 Ultraviolet spectrophotometer. For the blank, 300 ml of perchloric acid was taken through the same procedure of neutralisation and elution through the column.
c
100
200
Time ( m i n )
Figure 1. Elution profile of urocanic acid from column of Dowex-1 acetate. --, nni; ------, concentration of acetic acid in eluant.
percent transmission at 254
931
Histidine metabolism in fish
2.5. Thin-layer chromatography A layer of Silica Gel CT, 250 pm thick, was spread on glass plates (200 x 200 mm), allowed to dry and then activated at 100°C for 30 min. Ascending one-dimensional chromatography was carried out using n-butanol : acetic acid : water (12 : 3 : 5 by vol) as developing agent. Pauly’s reagentg was used to detect urocanic acid and amines. 2.6. Mass spectrometry Mass spectra of the non-derivatised acids were determined by Dr A. McGill of Torry Research Station on anAEI-902 mass spectrometer, the sample being introducedvia the direct insertion probe. The ion source at a temperature of 180°C above ambient was operated at an electron voltage of 70 eV and a filament current of 100 PA.
3. Results and discussion
The isolation of urocanic acid from the perchloric acid extract of tissue was based on the procedure described by Tabor,s using the anion-exchange resin Dowex-1, in the acetate form. Prior to elution of urocanic acid from the column with 0 . 0 6 ~acetic acid, weakly absorbing anions and other contaminants were removed by washing with water and 0.01 M acetic acid in his procedure. In our experience elution with 0.01 M acetic acid released some urocanic acid and satisfactory recoveries of added urocanic acid were only obtained if the concentration of the acetic acid in this preliminary wash did not exceed 0.001 M. Using this modified procedure recoveries of added urocanic acid
Figure 2. Thin-layer chromatographic separation of urocanic acid in crude extracts and after purification on a Dowex-1 acetate column. Lanes 1 and 9, authentic urocanic acid; lanes 7 and 8, eluates from column; lanes 2-6, crude perchloric acid extracts of muscle of whole fish stored for 4,7, 10, 14 and 18 days respectively. Chromatogram developed with Pauly’s reagent for imidazole compound^.^
938
I. M. Mackie and J. Fernandez-Salguero
were consistently around 96.9% on further elution with 0.06 M acetic acid. Figure 1 is a typical elution profile of a perchloric acid extract of muscle from the column of Dowex-1. The fraction corresponding to the peak, which was well separated from the other U.V. absorbing material in the eluate, was identified as urocanic acid by the following three criteria. The spectrum obtained at pH 7.4 for the eluate from the column coincided with that of the authentic sample, with a maximum absorption between 276 and 278 nm. Examination of the thin-layer chromatogram for imidazole compounds (Figure 2) showed that the eluate from the column gave an orange-red spot on spraying with Pauly’s reagent, with the same colour and mobility (RF0.59) as authentic urocanic acid. The extent of purification obtained
Standard
I 3 8 (Mt)
I Eluate from column
1
L I40 mie
Figure 3. Mass spectra of authentic urocanic acid (mol.wt 138) and of urocanic acid isolated from mackerel.
by the ion-exchange process was evident on comparing the lanes of the eluates from the column with those of crude perchloric acid extracts. The spot corresponding to urocanic acid was detected in crude extracts of fish stored for 10 days or more at 0°C but only as a minor constituent. For mass spectrometric analysis, the eluate from the column was repeatedly evaporated to dryness and redissolved in distilled water in an attempt to crystallise urocanic acid. Only a small quantity of crystals was obtained, but the weak spectrum (Figure 3) was non the less consistent with that for authentic urocanic acid; significant amounts of impurities were, however, present. Using this anion-exchange procedure for the determination of urocanic acid in perchloric acid extracts of fish muscle, a study was made of the formation of urocanic acid in various preparations of mackerel muscle during storage under sterile and non-sterile conditions. The sterile and non-sterile minces and muscle portions were prepared and stored at 0, 10 and 23°C respectively. Whole fish
939
Histidine metabolism in fish Table 1. Urocanic acid development in various preparations of mackerel heId at 0°C Concentrations of urocanic acid in samples (pg/g) (means of duplicate analyses) ~~
Sterile muscle
Time of storage (days)
Sterile mince
Fillet
Mince
Muscle (whole fish)
4.1 7.6 6.1 13.7 21.8 52.5
3.6 5.0 7.3 17.4 41.2 47.4
1 4 7 10 14 18
7.7 5.7 10.4 14.3 17.6 38.9
9.9 11.8 10.2 14.7 21.3 34.5
9.3 14.9 13.2 17.4 29.4 43.5
Table 2. Urocanic acid development (pglg) in muscle from whole fish held at 10°C and 23°C Temperature of storage (“C) Time of storage
(h) 6 15 24 36 72 120
10
23
-
5.2 7.4 14.0 33.3
-
6.7 -
10.1 28.8
-
were included for comparison as mackerel is normally stored ungutted under commercial conditions. The initial concentrations of urocanic acid in the samples were in the range 3.9-9.9 pg/g of tissue (Tables 1 and 2) and during storage they increased steadily at all temperatures. Some of the scatter in the results must be due to variations between fish as each sample analysed was from a different fish. At 0°C there was no marked difference in the build-up of this acid among the preparations. As expected, the rate of production increased with temperature of storage (at 10°C the rate was about three times higher than at 0 and at 23°C about six times higher). It is of significance that there was no apparent difference in the amount of urocanic acid produced in sterile and nonsterile muscle. This provides good evidence that the catabolism of histidine in mackerel to urocanic acid was entirely an autolytic process. Although the major pathway of histidine catabolism in mammals and in bacteria is initially to urocanic acid and then to imidazolonepropionic acid by histidine ammonia-lyase and urocanase respectively, it would appear that any such enzyme produced by bacteria in the non-sterile samples must be insignificant and that the major bacterial-induced breakdown of histidine is by the histidine decarboxylase route. 0 CH -N
I1
C
c< I NH,-CH
‘d
CH-N
I COOH L-Histidine
59
/I
I1
CH
II I
C-N
I1 +
CHz
I
It NH
I
CH
I COOH
+NH,
Urocanic acid
CH,
I
COOH Imidazolonepropionic acid
940
I. M. Mackie and J. Fernandez-Salgu6ro
These results were taken from an experiment which included the measurement of h’istamine formation during storage of mackerel.7 Free histidine was present at a level of 4000 pg/g of tissue and histamine at less than 0.5 pg/g in whole muscle and minced muscle held under sterile conditions. The concentrations of urocanic acid were much higher than those of histamine even after storage for 1 day at 0°C. However, considering the large amount of free histidine present in the muscle, the activity of histidine ammonia lyase was not high. Since the concentration of urocanic acid did not decrease towards the later stages of storage it would appear that, if urocanase was present in either sterile or non-sterile preparations of mackerel muscle, its activity must have been very weak. Acknowledgement The authors are grateful to Dr J. Gordon of the Dunstaffnage Marine Research Laboratory of the Scottish Marine Biological Association, Oban, for arrangements made to catch the mackerel. The work was carried out by J.F.S. during the tenure of a grant from the Ministry of Education and Science of Spain. References 1. Ienistea, C. In The Microbiological Safety of Food 1973, p. 327 (Hobbs, B. C.; Christian, J. H. B., eds) New York and London, Academic Press. 2. Meister, A. In Biochemistry of the Amino Acids 1965, Vol. 11, p. 825, New York and London, Academic Press. 3. Sakaguchi, M.; Kawai, A. Bull. Jap. SOC.Sci. Fish. 1968, 34, 507. 4. Zenisek, A.; Kral, J. A. Biochim. biophys. Acta 1953, 12, 479. 5. Everett, M. A.; Anglin, J. H.; Bever, A. T. Archs. Derm. 1959, 84, 717. 6. Kerr, J. W. Lancet 1963, 2, 709. 7. Mackie, 1. M.; Fernandez-SalguBro, J. (in preparation). 8. Tabor, H. In Methods in Enzymology 1963, Vol. VI, p. 581 (Colowick, Sidney P.; Kaplan, Nathan O., eds) New York and London, Academic Press. 9. Smith, I. In Chromatographic and Electrophoretic Techniques 1960, Vol. I, p. 218, London, Heinemann.
