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Quantitative determination of Zn protoporphyrin IX, heme and protoporphyrin IX in Parma ham by HPLC
Meat Science 82 (2009) 139–142
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Meat Science journal homepage: www.elsevier.com/locate/meatsci
Quantitative determination of Zn protoporphyrin IX, heme and protoporphyrin IX in Parma ham by HPLC Jun-ichi Wakamatsu a,b,*, Hiroko Odagiri b, Takanori Nishimura a,b, Akihito Hattori a,b a b
Meat Science Laboratory, Division of Bioresource and Product Science, Research Faculty of Agriculture, Hokkaido University, N-9 W-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan Department of Animal Science, Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan
a r t i c l e
i n f o
Article history: Received 3 July 2008 Received in revised form 27 November 2008 Accepted 22 December 2008
Keywords: Zn protoporphyrin IX Protoporphyrin IX Heme Parma ham HPLC determination
a b s t r a c t We measured the contents of Zn protoporphyrin IX (ZPP), heme and protoporphyrin IX (PPIX) in Parma ham by simultaneous analysis using high-performance liquid chromatography (HPLC). Extraction with ethyl acetate–acetic acid (4:1) was suitable for the quantitative analysis of ZPP. The contents of heme, ZPP and PPIX in Parma ham were 15.0–29.9, 27.7–47.0 and 0.4–1.1 lg/g, respectively, and total content of porphyrin was 43.7–76.6 lg/g. The amount of ZPP in Parma ham was larger than that of heme, and ZPP accounted for 60–70% of all porphyrins. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Parma ham has a bright and stable red color without the addition of nitrite and nitrate, and hence much interest has been shown in the origin of the red color. Wakamatsu et al. (Wakamatsu, Ito, Nishimura, & Hattori, 2007a; Wakamatsu, Nishimura, & Hattori, 2004a) revealed that Zn protoporphyrin IX (ZPP), in which Zn is coordinated instead of Fe in heme, contributes to the characteristic color of Parma ham. Results using a model experiment system showed that the formation of ZPP was inhibited by oxygen and was strongly dependent on pH, temperature and ionic strength suggesting that an enzyme was involved in the formation of ZPP (Wakamatsu, Okui, Ikeda, Nishimura, & Hattori, 2004b). It has been assumed that ZPP is formed by substitution of Fe with Zn in Parma ham (Wakamatsu et al., 2004a). It was later reported that ZPP is formed not from heme but from protoporphyrin IX (PPIX), which is formed independently (Wakamatsu, Okui, Hayashi, Nishimura, & Hattori, 2007b). On the other hand, it has recently been reported that ferrochelatase, which inserts Fe into PPIX in heme biosynthesis, catalyzes the removal of Fe from heme (Taketani et al., 2007). Iron-removing activity has also been reported in porcine heart tissue (Ishikawa et al., 2006). However, the mechanism by which ZPP is formed in meat products has not been fully elucidated. * Corresponding author. Address: Meat Science Laboratory, Division of Bioresource and Product Science, Research Faculty of Agriculture, Hokkaido University, N-9 W-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan. Tel./fax: +81 11 706 2547. E-mail address: [email protected] (J. Wakamatsu). 0309-1740/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2008.12.011
To elucidate the mechanism by which ZPP is formed in Parma ham, it is necessary to determine stoichiometrically the contents of heme, ZPP and PPIX, which is a precursor of both. However, there are only a few reports about the amounts of porphyrins in dry-cured hams like Parma ham. Although Laursen, Adamsen, Laursen, Olsen, and Møller (2008) determined the amounts of ZPP in Parma ham, Iberian ham and cured ham by using electron absorption, fluorescence and X-ray fluorescence (XRF) spectroscopy, the amounts of heme and PPIX have not been determined. Taketani et al. (2007) measured the amounts of ZPP and PPIX in Parma ham by using fluorescence spectroscopy and measured the amount of heme by the reduced-oxidized difference spectrum of pyridine hemochromogen. Moreover, the origin of the sample and sampling site were not stated in that report, and the contents were only shown as approximate values. Therefore, in this study, we examined the contents of ZPP, heme and PPIX in each muscle of Parma ham by simultaneous analysis using high-performance liquid chromatography (HPLC).
2. Materials and methods 2.1. Sampling Whole Parma ham (deboned) was purchased from Ferrarini S.p.A. (Rivaltella, Reggio Emilia, Italy). Sampling was carried out from 3 sections (arrows; A, B and C) of Parma ham as shown in Fig. 1, in accordance with our previous study (Wakamatsu, Odagiri,
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A stock ZPP standard was prepared by dissolving 10 mg of ZPP (Aldrich Chem. Co., Milwaukee, WI) in 50 ml of dimethylformamide (Kanto Chemical Co., Inc., Tokyo, Japan). After dissolving 10 mg of PPIX disodium salt (Aldrich Chem. Co., Milwaukee, WI) with a few drops of 1 M HCl, a stock PPIX standard was prepared by diluting it in 50 ml of N,N-dimethylformamide (DMF). After dissolving 10 mg of hemin (Sigma–Aldrich Co., St Louis, MO) with a few drops of 5% ammonia, a heme standard was prepared by diluting it in 50 ml of DMF. Working solutions of ZPP, PPIX and hemin were prepared in methanol/ammonium acetate (86:14, v/v, pH 5.16). 2.4. Statistical analysis Results are expressed as mean values of two independent trials. Data were analyzed using one-way analysis of variance (ANOVA) with Scheffe´s test. Statistics were calculated using Microsoft Excel 2002 and a statistic add-in software (Excel Toukei 2002 for Windows, Social Survey Research Information Co., Ltd., Tokyo, Japan). A probability of p < 0.05 was considered statistically significant. Fig. 1. Sampling from Parma ham. Three major muscles (semimembranosus (SM), semitendinosus (ST) and biceps femoris (BT)) were obtained from three sections (arrows: A, B and C) of Parma ham.
Nishimura, & Hattori, 2006). Semimembranosus (SM), semitendinosus (ST) and biceps femoris (BT) from sections A and B, and SM and BF from section C were used in this study. As soon as possible after sampling, samples were wrapped in aluminum foil and stored at 80 °C. 2.2. Extraction of porphyrin Porphyrins were extracted with 0.7% HCl–75% acetone (Okayama & Nagata, 1979), ethyl acetate–acetic acid (4:1) (Smith, Doran, Mazur, & Bush, 1980), tetrahydrofuran (THF) (Parolari, Gabba, & Saccani, 2003) and disodium phosphate–methanol (1:4) (Oshima, Yamada, Saito, & Hayakawa, 1996) with a minor modification. Parma ham homogenate (40% in water) was homogenized on ice with 10 volumes of each extraction solvent (15,000 rpm, 1 min). After extraction on ice for 15 min and centrifugation (3000 rpm, 15 min, 4 °C), the supernatant was collected into a volumetric flask and diluted with each extraction solvent. Extractions with the solvent were performed once or twice in darkness as much as possible.
3. Results Extraction of ZPP has been performed mainly by the 75% acetone method (Adamsen, Møller, Laursen, Olsen, & Skibsted, 2006; Adamsen, Møller, Parolari, Gabba, & Skibsted, 2006; Laursen et al., 2008; Morita, Niu, Sakata, & Nagata, 1996; Møller, Adamsen, Catharino, Skibsted, & Eberlin, 2007; Parolari et al., 2003; Wakamatsu et al., 2004a; Wakamatsu et al., 2004b). Since only nitrosylheme in heme derivatives was extracted with 75% acetone, we investigated the effectiveness of 0.7% HCl–75% acetone, ethyl acetate–acetic acid (4:1), THF and disodium phosphate–methanol (1:4) as extraction solvents for all porphyrins in Parma ham. First, we extracted porphyrins from Parma ham by using 0.7% HCl–75% acetone, which has been used to extract total heme pigments in meat products (Okayama & Nagata, 1979). When total porphyrins were extracted with 0.7% HCl–75% acetone, the fluorescence spectrum of the extract was consistent not with that of ZPP but PPIX (data not shown). It was shown that Zn was released from ZPP and extraction with 0.7% HCl–75% acetone was not suitable for the measurement of ZPP. Next, ZPP was extracted from Parma ham by using ethyl acetate–acetic acid, THF and disodium
2.3. Quantitative determination of ZPP, heme and PPIX Quantitative determination of ZPP, heme and PPIX was carried out as described by Wakamatsu, Okui, Hayashi, Nishimura, and Hattori (2007b) with a minor modification. The above-mentioned ethyl acetate–acetic acid extract was mixed with an equal volume of methanol/ammonium acetate (86:14, v/v, pH 5.16). The sample was filtered through a 0.45-lm filter (Minisart RC4, Sartorius AG, Goettingen, Germany). An STR ODS-II column (4.6 150 mm, Shinwa Chemical Industries Ltd., Kyoto, Japan) was used for separation of porphyrins, and the separation was carried out by isocratic elution using methanol/ammonium acetate (86:14, v/v, pH 5.16) at a flow rate of 0.6 ml/min at 35 °C. Forty microliters of each sample was injected. Detection of ZPP and PPIX was carried out at excitation and emission wavelengths of 420/400 and 590/630 nm, respectively. Heme was monitored by absorption at 400 nm. All extraction oparations were carried out in darkness as much as possible.
Fig. 2. Amount of ZPP in each solution after extraction with various solvents. After one extraction (h) or two extractions (j) with a 10-times volume of ethyl acetate– acetic acid (EtOAc-AcOH; 4:1), THF or disodium phosphate–methanol (DSP-MetOH; 1:4) from Parma ham homogenate, the extracts were diluted in equal amounts. Amounts of ZPP in the solutions were measured with a fluorescence photometer. Bars represent SD. Data with different characters differ significantly (p < 0.05).
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phosphate–methanol (Fig. 2). The amounts of ZPP extracted with ethyl acetate–acetic acid and THF were significantly larger than that of ZPP extracted with disodium phosphate–methanol (p < 0.05). The amount of ZPP extracted with ethyl acetate–acetic acid was equivalent to that of ZPP extracted with THF, while the amounts of ZPP varied widely between one extraction and two extractions with ethyl acetate–acetic acid. Although ZPP was completely extracted after two extractions with ethyl acetate–acetic acid, a minute amount of ZPP still remained after two extractions with THF (Fig. 3). Therefore, these results indicated that two extractions with ethyl acetate–acetic acid are suitable for the determination of ZPP. Since most porphyrins are extracted with ethyl acetate–acetic acid and the content of each porphyrin cannot be determined by spectroscopy or by fluorescent spectroscopy, we determined the contents of heme, ZPP and PPIX in Parma ham by using HPLC. The contents of heme, ZPP and PPIX in Parma ham were 15.0– 29.9, 27.7–47.0 and 0.4–1.1 lg/g, respectively, and total content of porphyrin was 43.7–76.6 lg/g (Table 1). The amount of ZPP in Parma ham was larger than that of heme, and ZPP accounted for 60–70% of all porphyrins. There was only a small amount of PPIX in Parma ham compared with the amounts of heme and ZPP. The amounts of porphyrins did not show any obvious relationships among muscles or sections, but those of heme and ZPP in section C of SM were large. Since the contents of heme, ZPP and PPIX per gram of dry matter (DM) were almost the same for different sampling parts (data not shown), the high contents of heme and ZPP
Fig. 3. Autofluorescence spectra of residues after solvent extraction from Parma ham. Autofluorescence spectra of residues were measured (excitation wavelength: ) or two extractions ( ) with 420 nm) after one extraction ( ethyl acetate–acetic acid (4:1) and after one extraction ( ) or two ) with THF. Arrow indicates the fluorescence peak extractions ( originating from ZPP.
Table 1 Concentrations and composition ratio of heme, ZPP and PPIX in each section of three muscles (lg/g). Heme BF
SM ST
A B C A B C A B
23.1 ± 2.3 17.1 ± 1.7 18.0 ± 0.5 20.4 ± 1.2 15.0 ± 3.9 28.9 ± 1.5 19.8 ± 0.4 15.7 ± 0.7
ZPP (43.4%) (29.7%) (34.7%) (36.8%) (34.3%) (37.8%) (34.3%) (27.7%)
29.7 ± 1.0 39.6 ± 2.7 33.5 ± 2.0 34.5 ± 0.5 27.7 ± 6.8 47.0 ± 3.1 37.0 ± 1.3 39.8 ± 5.0
Data are expressed as means ±SD (n=2).
PPIX (55.8%) (68.7%) (64.5%) (62.3%) (63.4%) (61.4%) (64.1%) (70.3%)
0.4 ± 0.3 0.9 ± 0.1 0.4 ± 0.1 0.5 ± 0.1 1.0 ± 0.3 0.7 ± 0.2 0.9 ± 0.3 1.1 ± 0.3
Total (0.8%) (1.6%) (0.8%) (0.8%) (2.3%) (0.9%) (1.6%) (1.9%)
53.2 ± 1.0 57.6 ± 4.4 51.9 ± 1.6 55.5 ± 1.9 43.8 ± 11.0 76.5 ± 4.5 57.7 ± 1.2 56.6 ± 5.9
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seemed to be caused by excessive drying during processing. On the other hand, the content of total porphyrin in section B of SM was lower than that in other parts. In section B of SM, the standard deviations of the contents of ZPP and heme were higher than those in other parts. Since the contents of ZPP and heme varied considerably in section B of SM, the amounts of ZPP and heme were estimated to be small.
4. Discussion Although ZPP is abundant in meat products, such as Parma ham, without addition of nitrite and nitrate, there has been only a few studies in which the amount of ZPP was determined (Laursen et al., 2008; Taketani et al., 2007). In this study, the contents of heme, ZPP and PPIX in Parma ham were determined simultaneously by using HPLC. The amount of total porphyrin around the part that was cut off from the body was slightly high due to dehydration during processing, but there was no obvious difference among the amounts of heme, ZPP and PPIX. ZPP was the most abundant porphyrin in Parma ham in our study (27.7–47.0 lg/g) as was reported by Taketani et al. (2007) (25–29 lg/g, approximately 67% of total porphyrins (60–70 nmol/g)). Laursen et al. (2008) reported that contents of ZPP in Parma ham and Iberian ham were 12.1–15.6 ppm (12.1– 15.6 lg/g) and 12.5–14.6 ppm (12.5–14.6 lg/g), respectively. Although our results were similar to the results of Taketani et al. (2007), the content of ZPP determined in this study was higher than the contents reported by Taketani et al. (2007) and Laursen et al. (2008). The total content of the three porphyrins was 43.7– 76.6 lg/g in this study, whereas Taketani et al. (2007) reported the total content to be 60–70 nmol/g (37–43 lg/g). The content was lower than that in our study (43.7–76.6 lg/g). The content of heme in our study (15.7–28.9 lg/g) was much higher than that reported by Taketani et al. (2007) (approximately 4 lg/g). In contrast, the content of PPIX in our study (0.4–1.1 lg/g) was much lower than that reported by Taketani et al. (approximately 8–9 lg/g). The ratio of PPIX to total porphyrins in the study by Taketani et al. (2007) (23%) was therefore much higher than that in this study (0.8–2.3%). PPIX is unstable to light. However, extraction from Parma ham and preparation of samples in this study were carried out in darkness as much as possible, and there have been many studies in which PPIX was analyzed by HPLC (Lim, 2002); hence, the breakdown of PPIX did not seem to be the main cause of the decrease. The difference in reported porphyrin contents might be due to different extraction solvents used. We used ethyl acetate/acetic acid (4:1) as an extraction solvent, and Taketani et al. (2007) used ethyl acetate/acetic acid (3:1) for the extraction of heme and acetone/ethanol (2:1) for the extraction of ZPP and PPIX. On the other hand, Laursen et al. (2008) used 75% acetone for the extraction of ZPP. Although the 75% acetone method is suitable for the extraction of ZPP, the efficiency of this method and the amount of ZPP extracted by this method have not been validated. This might be the reason for the lower concentration of ZPP in the study by Laursen et al. (2008) than the concentrations in the study by Taketani et al. (2007) and in our study. Taketani et al. (2007) measured the content of ZPP extracted from Parma ham with acetone/ethanol (2:1, v/v). Laursen et al. (2008) measured the content of ZPP that was extracted from Parma ham with 75% acetone and then isolated. Since the sampling procedure and the manufacturers from which Parma ham was obtained might be different in the three studies, further investigations are needed. ZPP and heme accounted for two-thirds and one-third of all porphyrins, respectively. Data indicated that ZPP contributes mainly to the characteristic color of Parma ham and that heme does not contribute greatly to the color of Parma ham. Our results suggest
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that PPIX does not contribute to the color, in contrast to the results of a previous report (Taketani et al., 2007), and our results agrees with those of a previous study in which PPIX was not detected from the autofluorescence spectrum of Parma ham (Wakamatsu et al., 2006). Since there was almost no difference in the amount of ZPP and the composition ratio of the three measured porphyrins among muscles or among parts, our findings indicate that the red pigments in fully matured Parma ham in the marketplace are almost uniform throughout the ham. References Adamsen, C. E., Møller, J. K. S., Laursen, K., Olsen, K., & Skibsted, L. H. (2006). Zn– porphyrin formation in cured meat products: Effect of added salt and nitrite. Meat Science, 72, 672–679. Adamsen, C. E., Møller, J. K. S., Parolari, G., Gabba, L., & Skibsted, L. H. (2006). Changes in Zn–porphyrin and proteinous pigments in Italian dry-cured ham during processing and maturation. Meat Science, 74, 373–379. Ishikawa, H., Yoshihara, M., Baba, A., Kawabuchi, T., Sato, M., Numata, M., et al. (2006). Formation of zinc protoporphyrin IX from myoglobin in porcine heart extract. Food Science and Technology Research, 12, 125–130. Laursen, K., Adamsen, C. E., Laursen, J., Olsen, K., & Møller, J. K. S. (2008). Quantification of zinc–porphyrin in dry-cured ham products by spectroscopic methods: Comparison of absorption, fluorescence and X-ray fluorescence spectroscopy. Meat Science, 78, 336–341. Lim, C. K. (2002). Analysis of biosynthetic intermediates, 5-aminolevulinic acid to heme. In A. G. Smith & M. Witty (Eds.), Heme, chlorophyll, and bilins: Methods and protocols (pp. 95–110). Totowa, NJ: Humana Press. Møller, J. K. S., Adamsen, C. E., Catharino, R. R., Skibsted, L. H., & Eberlin, M. N. (2007). Mass spectrometric evidence for a zinc–porphyrin complex as the red pigment in dry-cured Iberian and Parma ham. Meat Science, 75, 203–210.
Morita, H., Niu, J., Sakata, R., & Nagata, Y. (1996). Red pigment of Parma ham and bacterial influence on its formation. Journal of Food Science, 61, 1021–1023. Okayama, T., & Nagata, Y. (1979). A method for the determination of total heme pigments in meat products. Japanese Journal of Zootechnical Science, 50, 15–21. Oshima, H., Yamada, S., Saito, I., & Hayakawa, J. (1996). Heme, zinc protoporphyrin (IX) and heavy metals in edible tissues of livestock and poultry. Porphyrin, 5, 301–306. Parolari, G., Gabba, L., & Saccani, G. (2003). Extraction properties and absorption spectra of dry cured hams made with and without nitrate. Meat Science, 64, 483–490. Smith, R. M., Doran, D., Mazur, M., & Bush, B. (1980). High-performance liquid chromatographic determination of protoporphyrin and zinc protoporphyrin in blood. Journal of Chromatography B: Biomedical Sciences and Applications, 181, 319–327. Taketani, S., Ishigaki, M., Mizutani, A., Uebayashi, M., Numata, M., Ohgari, Y., et al. (2007). Heme synthase (ferrochelatase) catalyzes the removal of iron from heme and demetalation of metalloporphyrins. Biochemistry, 46, 15054–15061. Wakamatsu, J., Ito, T., Nishimura, T., & Hattori, A. (2007a). Direct demonstration of the presence of zinc in the acetone-extractable red pigment from Parma ham. Meat Science, 76, 385–387. Wakamatsu, J., Nishimura, T., & Hattori, A. (2004a). A Zn–porphyrin complex contributes to bright red color in Parma ham. Meat Science, 67, 95–100. Wakamatsu, J., Odagiri, H., Nishimura, T., & Hattori, A. (2006). Observation of the distribution of Zn protoporphyrin IX (ZPP) in Parma ham by using purple LED and image analysis. Meat Science, 74, 594–599. Wakamatsu, J., Okui, J., Hayashi, N., Nishimura, T., & Hattori, A. (2007b). Zn protoporphyrin IX is formed not from heme but from protoporphyrin IX. Meat Science, 77, 580–586. Wakamatsu, J., Okui, J., Ikeda, Y., Nishimura, T., & Hattori, A. (2004b). Establishment of a model experiment system to elucidate the mechanism by which Znprotoporphyrin IX is formed in nitrite-free dry-cured ham. Meat Science, 68, 313–317.
Contents lists available at ScienceDirect
Meat Science journal homepage: www.elsevier.com/locate/meatsci
Quantitative determination of Zn protoporphyrin IX, heme and protoporphyrin IX in Parma ham by HPLC Jun-ichi Wakamatsu a,b,*, Hiroko Odagiri b, Takanori Nishimura a,b, Akihito Hattori a,b a b
Meat Science Laboratory, Division of Bioresource and Product Science, Research Faculty of Agriculture, Hokkaido University, N-9 W-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan Department of Animal Science, Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan
a r t i c l e
i n f o
Article history: Received 3 July 2008 Received in revised form 27 November 2008 Accepted 22 December 2008
Keywords: Zn protoporphyrin IX Protoporphyrin IX Heme Parma ham HPLC determination
a b s t r a c t We measured the contents of Zn protoporphyrin IX (ZPP), heme and protoporphyrin IX (PPIX) in Parma ham by simultaneous analysis using high-performance liquid chromatography (HPLC). Extraction with ethyl acetate–acetic acid (4:1) was suitable for the quantitative analysis of ZPP. The contents of heme, ZPP and PPIX in Parma ham were 15.0–29.9, 27.7–47.0 and 0.4–1.1 lg/g, respectively, and total content of porphyrin was 43.7–76.6 lg/g. The amount of ZPP in Parma ham was larger than that of heme, and ZPP accounted for 60–70% of all porphyrins. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Parma ham has a bright and stable red color without the addition of nitrite and nitrate, and hence much interest has been shown in the origin of the red color. Wakamatsu et al. (Wakamatsu, Ito, Nishimura, & Hattori, 2007a; Wakamatsu, Nishimura, & Hattori, 2004a) revealed that Zn protoporphyrin IX (ZPP), in which Zn is coordinated instead of Fe in heme, contributes to the characteristic color of Parma ham. Results using a model experiment system showed that the formation of ZPP was inhibited by oxygen and was strongly dependent on pH, temperature and ionic strength suggesting that an enzyme was involved in the formation of ZPP (Wakamatsu, Okui, Ikeda, Nishimura, & Hattori, 2004b). It has been assumed that ZPP is formed by substitution of Fe with Zn in Parma ham (Wakamatsu et al., 2004a). It was later reported that ZPP is formed not from heme but from protoporphyrin IX (PPIX), which is formed independently (Wakamatsu, Okui, Hayashi, Nishimura, & Hattori, 2007b). On the other hand, it has recently been reported that ferrochelatase, which inserts Fe into PPIX in heme biosynthesis, catalyzes the removal of Fe from heme (Taketani et al., 2007). Iron-removing activity has also been reported in porcine heart tissue (Ishikawa et al., 2006). However, the mechanism by which ZPP is formed in meat products has not been fully elucidated. * Corresponding author. Address: Meat Science Laboratory, Division of Bioresource and Product Science, Research Faculty of Agriculture, Hokkaido University, N-9 W-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan. Tel./fax: +81 11 706 2547. E-mail address: [email protected] (J. Wakamatsu). 0309-1740/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2008.12.011
To elucidate the mechanism by which ZPP is formed in Parma ham, it is necessary to determine stoichiometrically the contents of heme, ZPP and PPIX, which is a precursor of both. However, there are only a few reports about the amounts of porphyrins in dry-cured hams like Parma ham. Although Laursen, Adamsen, Laursen, Olsen, and Møller (2008) determined the amounts of ZPP in Parma ham, Iberian ham and cured ham by using electron absorption, fluorescence and X-ray fluorescence (XRF) spectroscopy, the amounts of heme and PPIX have not been determined. Taketani et al. (2007) measured the amounts of ZPP and PPIX in Parma ham by using fluorescence spectroscopy and measured the amount of heme by the reduced-oxidized difference spectrum of pyridine hemochromogen. Moreover, the origin of the sample and sampling site were not stated in that report, and the contents were only shown as approximate values. Therefore, in this study, we examined the contents of ZPP, heme and PPIX in each muscle of Parma ham by simultaneous analysis using high-performance liquid chromatography (HPLC).
2. Materials and methods 2.1. Sampling Whole Parma ham (deboned) was purchased from Ferrarini S.p.A. (Rivaltella, Reggio Emilia, Italy). Sampling was carried out from 3 sections (arrows; A, B and C) of Parma ham as shown in Fig. 1, in accordance with our previous study (Wakamatsu, Odagiri,
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A stock ZPP standard was prepared by dissolving 10 mg of ZPP (Aldrich Chem. Co., Milwaukee, WI) in 50 ml of dimethylformamide (Kanto Chemical Co., Inc., Tokyo, Japan). After dissolving 10 mg of PPIX disodium salt (Aldrich Chem. Co., Milwaukee, WI) with a few drops of 1 M HCl, a stock PPIX standard was prepared by diluting it in 50 ml of N,N-dimethylformamide (DMF). After dissolving 10 mg of hemin (Sigma–Aldrich Co., St Louis, MO) with a few drops of 5% ammonia, a heme standard was prepared by diluting it in 50 ml of DMF. Working solutions of ZPP, PPIX and hemin were prepared in methanol/ammonium acetate (86:14, v/v, pH 5.16). 2.4. Statistical analysis Results are expressed as mean values of two independent trials. Data were analyzed using one-way analysis of variance (ANOVA) with Scheffe´s test. Statistics were calculated using Microsoft Excel 2002 and a statistic add-in software (Excel Toukei 2002 for Windows, Social Survey Research Information Co., Ltd., Tokyo, Japan). A probability of p < 0.05 was considered statistically significant. Fig. 1. Sampling from Parma ham. Three major muscles (semimembranosus (SM), semitendinosus (ST) and biceps femoris (BT)) were obtained from three sections (arrows: A, B and C) of Parma ham.
Nishimura, & Hattori, 2006). Semimembranosus (SM), semitendinosus (ST) and biceps femoris (BT) from sections A and B, and SM and BF from section C were used in this study. As soon as possible after sampling, samples were wrapped in aluminum foil and stored at 80 °C. 2.2. Extraction of porphyrin Porphyrins were extracted with 0.7% HCl–75% acetone (Okayama & Nagata, 1979), ethyl acetate–acetic acid (4:1) (Smith, Doran, Mazur, & Bush, 1980), tetrahydrofuran (THF) (Parolari, Gabba, & Saccani, 2003) and disodium phosphate–methanol (1:4) (Oshima, Yamada, Saito, & Hayakawa, 1996) with a minor modification. Parma ham homogenate (40% in water) was homogenized on ice with 10 volumes of each extraction solvent (15,000 rpm, 1 min). After extraction on ice for 15 min and centrifugation (3000 rpm, 15 min, 4 °C), the supernatant was collected into a volumetric flask and diluted with each extraction solvent. Extractions with the solvent were performed once or twice in darkness as much as possible.
3. Results Extraction of ZPP has been performed mainly by the 75% acetone method (Adamsen, Møller, Laursen, Olsen, & Skibsted, 2006; Adamsen, Møller, Parolari, Gabba, & Skibsted, 2006; Laursen et al., 2008; Morita, Niu, Sakata, & Nagata, 1996; Møller, Adamsen, Catharino, Skibsted, & Eberlin, 2007; Parolari et al., 2003; Wakamatsu et al., 2004a; Wakamatsu et al., 2004b). Since only nitrosylheme in heme derivatives was extracted with 75% acetone, we investigated the effectiveness of 0.7% HCl–75% acetone, ethyl acetate–acetic acid (4:1), THF and disodium phosphate–methanol (1:4) as extraction solvents for all porphyrins in Parma ham. First, we extracted porphyrins from Parma ham by using 0.7% HCl–75% acetone, which has been used to extract total heme pigments in meat products (Okayama & Nagata, 1979). When total porphyrins were extracted with 0.7% HCl–75% acetone, the fluorescence spectrum of the extract was consistent not with that of ZPP but PPIX (data not shown). It was shown that Zn was released from ZPP and extraction with 0.7% HCl–75% acetone was not suitable for the measurement of ZPP. Next, ZPP was extracted from Parma ham by using ethyl acetate–acetic acid, THF and disodium
2.3. Quantitative determination of ZPP, heme and PPIX Quantitative determination of ZPP, heme and PPIX was carried out as described by Wakamatsu, Okui, Hayashi, Nishimura, and Hattori (2007b) with a minor modification. The above-mentioned ethyl acetate–acetic acid extract was mixed with an equal volume of methanol/ammonium acetate (86:14, v/v, pH 5.16). The sample was filtered through a 0.45-lm filter (Minisart RC4, Sartorius AG, Goettingen, Germany). An STR ODS-II column (4.6 150 mm, Shinwa Chemical Industries Ltd., Kyoto, Japan) was used for separation of porphyrins, and the separation was carried out by isocratic elution using methanol/ammonium acetate (86:14, v/v, pH 5.16) at a flow rate of 0.6 ml/min at 35 °C. Forty microliters of each sample was injected. Detection of ZPP and PPIX was carried out at excitation and emission wavelengths of 420/400 and 590/630 nm, respectively. Heme was monitored by absorption at 400 nm. All extraction oparations were carried out in darkness as much as possible.
Fig. 2. Amount of ZPP in each solution after extraction with various solvents. After one extraction (h) or two extractions (j) with a 10-times volume of ethyl acetate– acetic acid (EtOAc-AcOH; 4:1), THF or disodium phosphate–methanol (DSP-MetOH; 1:4) from Parma ham homogenate, the extracts were diluted in equal amounts. Amounts of ZPP in the solutions were measured with a fluorescence photometer. Bars represent SD. Data with different characters differ significantly (p < 0.05).
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phosphate–methanol (Fig. 2). The amounts of ZPP extracted with ethyl acetate–acetic acid and THF were significantly larger than that of ZPP extracted with disodium phosphate–methanol (p < 0.05). The amount of ZPP extracted with ethyl acetate–acetic acid was equivalent to that of ZPP extracted with THF, while the amounts of ZPP varied widely between one extraction and two extractions with ethyl acetate–acetic acid. Although ZPP was completely extracted after two extractions with ethyl acetate–acetic acid, a minute amount of ZPP still remained after two extractions with THF (Fig. 3). Therefore, these results indicated that two extractions with ethyl acetate–acetic acid are suitable for the determination of ZPP. Since most porphyrins are extracted with ethyl acetate–acetic acid and the content of each porphyrin cannot be determined by spectroscopy or by fluorescent spectroscopy, we determined the contents of heme, ZPP and PPIX in Parma ham by using HPLC. The contents of heme, ZPP and PPIX in Parma ham were 15.0– 29.9, 27.7–47.0 and 0.4–1.1 lg/g, respectively, and total content of porphyrin was 43.7–76.6 lg/g (Table 1). The amount of ZPP in Parma ham was larger than that of heme, and ZPP accounted for 60–70% of all porphyrins. There was only a small amount of PPIX in Parma ham compared with the amounts of heme and ZPP. The amounts of porphyrins did not show any obvious relationships among muscles or sections, but those of heme and ZPP in section C of SM were large. Since the contents of heme, ZPP and PPIX per gram of dry matter (DM) were almost the same for different sampling parts (data not shown), the high contents of heme and ZPP
Fig. 3. Autofluorescence spectra of residues after solvent extraction from Parma ham. Autofluorescence spectra of residues were measured (excitation wavelength: ) or two extractions ( ) with 420 nm) after one extraction ( ethyl acetate–acetic acid (4:1) and after one extraction ( ) or two ) with THF. Arrow indicates the fluorescence peak extractions ( originating from ZPP.
Table 1 Concentrations and composition ratio of heme, ZPP and PPIX in each section of three muscles (lg/g). Heme BF
SM ST
A B C A B C A B
23.1 ± 2.3 17.1 ± 1.7 18.0 ± 0.5 20.4 ± 1.2 15.0 ± 3.9 28.9 ± 1.5 19.8 ± 0.4 15.7 ± 0.7
ZPP (43.4%) (29.7%) (34.7%) (36.8%) (34.3%) (37.8%) (34.3%) (27.7%)
29.7 ± 1.0 39.6 ± 2.7 33.5 ± 2.0 34.5 ± 0.5 27.7 ± 6.8 47.0 ± 3.1 37.0 ± 1.3 39.8 ± 5.0
Data are expressed as means ±SD (n=2).
PPIX (55.8%) (68.7%) (64.5%) (62.3%) (63.4%) (61.4%) (64.1%) (70.3%)
0.4 ± 0.3 0.9 ± 0.1 0.4 ± 0.1 0.5 ± 0.1 1.0 ± 0.3 0.7 ± 0.2 0.9 ± 0.3 1.1 ± 0.3
Total (0.8%) (1.6%) (0.8%) (0.8%) (2.3%) (0.9%) (1.6%) (1.9%)
53.2 ± 1.0 57.6 ± 4.4 51.9 ± 1.6 55.5 ± 1.9 43.8 ± 11.0 76.5 ± 4.5 57.7 ± 1.2 56.6 ± 5.9
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seemed to be caused by excessive drying during processing. On the other hand, the content of total porphyrin in section B of SM was lower than that in other parts. In section B of SM, the standard deviations of the contents of ZPP and heme were higher than those in other parts. Since the contents of ZPP and heme varied considerably in section B of SM, the amounts of ZPP and heme were estimated to be small.
4. Discussion Although ZPP is abundant in meat products, such as Parma ham, without addition of nitrite and nitrate, there has been only a few studies in which the amount of ZPP was determined (Laursen et al., 2008; Taketani et al., 2007). In this study, the contents of heme, ZPP and PPIX in Parma ham were determined simultaneously by using HPLC. The amount of total porphyrin around the part that was cut off from the body was slightly high due to dehydration during processing, but there was no obvious difference among the amounts of heme, ZPP and PPIX. ZPP was the most abundant porphyrin in Parma ham in our study (27.7–47.0 lg/g) as was reported by Taketani et al. (2007) (25–29 lg/g, approximately 67% of total porphyrins (60–70 nmol/g)). Laursen et al. (2008) reported that contents of ZPP in Parma ham and Iberian ham were 12.1–15.6 ppm (12.1– 15.6 lg/g) and 12.5–14.6 ppm (12.5–14.6 lg/g), respectively. Although our results were similar to the results of Taketani et al. (2007), the content of ZPP determined in this study was higher than the contents reported by Taketani et al. (2007) and Laursen et al. (2008). The total content of the three porphyrins was 43.7– 76.6 lg/g in this study, whereas Taketani et al. (2007) reported the total content to be 60–70 nmol/g (37–43 lg/g). The content was lower than that in our study (43.7–76.6 lg/g). The content of heme in our study (15.7–28.9 lg/g) was much higher than that reported by Taketani et al. (2007) (approximately 4 lg/g). In contrast, the content of PPIX in our study (0.4–1.1 lg/g) was much lower than that reported by Taketani et al. (approximately 8–9 lg/g). The ratio of PPIX to total porphyrins in the study by Taketani et al. (2007) (23%) was therefore much higher than that in this study (0.8–2.3%). PPIX is unstable to light. However, extraction from Parma ham and preparation of samples in this study were carried out in darkness as much as possible, and there have been many studies in which PPIX was analyzed by HPLC (Lim, 2002); hence, the breakdown of PPIX did not seem to be the main cause of the decrease. The difference in reported porphyrin contents might be due to different extraction solvents used. We used ethyl acetate/acetic acid (4:1) as an extraction solvent, and Taketani et al. (2007) used ethyl acetate/acetic acid (3:1) for the extraction of heme and acetone/ethanol (2:1) for the extraction of ZPP and PPIX. On the other hand, Laursen et al. (2008) used 75% acetone for the extraction of ZPP. Although the 75% acetone method is suitable for the extraction of ZPP, the efficiency of this method and the amount of ZPP extracted by this method have not been validated. This might be the reason for the lower concentration of ZPP in the study by Laursen et al. (2008) than the concentrations in the study by Taketani et al. (2007) and in our study. Taketani et al. (2007) measured the content of ZPP extracted from Parma ham with acetone/ethanol (2:1, v/v). Laursen et al. (2008) measured the content of ZPP that was extracted from Parma ham with 75% acetone and then isolated. Since the sampling procedure and the manufacturers from which Parma ham was obtained might be different in the three studies, further investigations are needed. ZPP and heme accounted for two-thirds and one-third of all porphyrins, respectively. Data indicated that ZPP contributes mainly to the characteristic color of Parma ham and that heme does not contribute greatly to the color of Parma ham. Our results suggest
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that PPIX does not contribute to the color, in contrast to the results of a previous report (Taketani et al., 2007), and our results agrees with those of a previous study in which PPIX was not detected from the autofluorescence spectrum of Parma ham (Wakamatsu et al., 2006). Since there was almost no difference in the amount of ZPP and the composition ratio of the three measured porphyrins among muscles or among parts, our findings indicate that the red pigments in fully matured Parma ham in the marketplace are almost uniform throughout the ham. References Adamsen, C. E., Møller, J. K. S., Laursen, K., Olsen, K., & Skibsted, L. H. (2006). Zn– porphyrin formation in cured meat products: Effect of added salt and nitrite. Meat Science, 72, 672–679. Adamsen, C. E., Møller, J. K. S., Parolari, G., Gabba, L., & Skibsted, L. H. (2006). Changes in Zn–porphyrin and proteinous pigments in Italian dry-cured ham during processing and maturation. Meat Science, 74, 373–379. Ishikawa, H., Yoshihara, M., Baba, A., Kawabuchi, T., Sato, M., Numata, M., et al. (2006). Formation of zinc protoporphyrin IX from myoglobin in porcine heart extract. Food Science and Technology Research, 12, 125–130. Laursen, K., Adamsen, C. E., Laursen, J., Olsen, K., & Møller, J. K. S. (2008). Quantification of zinc–porphyrin in dry-cured ham products by spectroscopic methods: Comparison of absorption, fluorescence and X-ray fluorescence spectroscopy. Meat Science, 78, 336–341. Lim, C. K. (2002). Analysis of biosynthetic intermediates, 5-aminolevulinic acid to heme. In A. G. Smith & M. Witty (Eds.), Heme, chlorophyll, and bilins: Methods and protocols (pp. 95–110). Totowa, NJ: Humana Press. Møller, J. K. S., Adamsen, C. E., Catharino, R. R., Skibsted, L. H., & Eberlin, M. N. (2007). Mass spectrometric evidence for a zinc–porphyrin complex as the red pigment in dry-cured Iberian and Parma ham. Meat Science, 75, 203–210.
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