Spectrophotometric Determination of Histamine in Fisheries Using an Enzyme Immunoassay Method

Tânia L. P. Pessatti, José D. Fontana, and Marcos L. Pessatti

1. Introduction

The biogenic amines are low-weight organic bases that exhibit variable biological activity. Approximately 30 vasoactive and psycoactive amines have been found in foodstuffs (1). Histamine, tyramine, phenylethylamine, tryptamine, cadaverine, putrescine, spermine, and spermidine are examples of biogenic amines detectable in meat and its derivatives (2). These amines occur naturally in animals, plants, and microorganisms owing to their natural metabolic processes, and are usually formed by decarboxylation of amino acids.

Histamine, 5-imidazol-ethylamine (MW 111), is produced by oxidative decarboxy-lation of histidine, and decarboxylase-positive microorganisms are able to produce it as a byproduct of their action on host tissues. This process, associated with enterobac-teria such as Escherichia, Salmonella, Clostridium, and Bacillus, can be fast if the meat storage conditions are inadequate (3).

Additionally, because of its nonvolatile behavior, histamine may bestow toxicity on the product even before it is considered decayed or organoleptically unacceptable. Secondary amines such as piperidine and pyrrolidine can be produced in the catabo-lism of histamine, and they, in turn, are precursors of nitrosamines, substances with known carcinogenic activity (4). Some cases of histamine toxicity have been reported (5-7).

Therefore, the histamine content of fishery products has been proposed as an index for microbial deterioration, caused by low-quality fish or inadequate processing. The evaluation of histamine levels has been demonstrated to be an important indicator in the quality control of both fresh and processed fish (8,9), particularly fish of the Scombridae family, which exhibit elevated levels of histidine in their tissues.

For these reasons, legal regulations were implemented to monitor the histamine content of fish, with the maximum limit being set from 50 to 200 mg histamine/kg fish (50-200 ppm) of sample, varying by country (10). The values reported for fish in an advanced stage of decomposition varied from 100 to > 4000 mg/kg (11).

Some analytical methods for the determination of histamine have been developed employing two main techniques: spectrofluorometry (9,12) and/or chromatography (liquid [13], liquid-gas [1], or in thin layer [14]).

Fluorometric determination, based on histamine derivation with o-phthalaldehyde (OPA) (11,15), has no specificity since histidine can also react with the fluorogenic reagent, which thus interferes with the reaction. Even though these methods have been modified and evolved, they are still time-consuming and expensive.

In chromatographic determination, the reagents also do not exhibit specific reactivity and therefore require a laborious extraction step to eliminate possible interfering substances, specially histidine itself, peptides with histidine, and other amines (16,17). This can make this methodology less practical, particularly when a fast, trustworthy result is needed and when combining for a large number of samples is required.

More recently, a method of histamine determination by immunoassay has been developed. It consists of competitive binding between a monoclonal antibody coated on the wells in a microplate and modified histamine (acylated) and a conjugate (histamine bound to alkaline phosphatase). Upon alkaline phosphatase catalysis, the p-nitro-phenyl phosphate substrate (p-NPP) releases free phenol, which develops a yellow coloration of an intensity inversely related to the histamine concentration in the sample. This immunoassay method has been shown to be more efficient, less expensive, faster, and sensitive (1 mg histamine/kg fishery = 1 ppm).

This chapter deals with the use of an immunoassay method (the Histamarine® kit) to evaluate the levels of histamine in skipjack tuna samples (Katsuwonuspelamis), in order to monitor the effect of storage temperature on histamine levels, color determination being carried out by spectrophotometry.

2. Materials

1. The object of this study was a marine fish species of economic interest and widespread commercialization, the skipjack tuna (Katsuwonus pelamis). The fish were caught by a local fishery in the Atlantic Ocean in the zone of Itajai Port, Santa Catarina State, Southern Brazil and maintained on crushed ice for 5 d.

2. Once transported to the laboratory, tuna samples were immediately cut longitudinally to get only the dorsal muscle. Each piece was transversely sectioned in three slices of approx 2 cm each and then separately stored in sterile plastic bags at -20°C or 0°C or 8°C, respectively. The procedures for manipulating the samples were aseptic to avoid cross-contamination.

3. All reagents used for sample acylation and immunoassay were from the Histamarine® kit (Immunotech, France) (see Note 1).

4. Other materials required for the immunoassays but not provided by the kit:

a. Precision balance (e.g., AG204 from Mettler Toledo, Switzerland).

b. Homogenizer (see Note 2).

c. Spectrophotometer (e.g., CE1020 from CECIL, Cambridge) or microplate reader (Xmax = 405-414 nm).

d. Plastic tubes (see Note 3).

e. Variable volume micropipets (20, 200, and 1000 ^L).

g. 25-mL and 1-L Graduated cylinders.

3. Methods

3.1. Sample Preparation and Histamine Extraction

1. Take a 10-g aliquot of each slice from fish dorsal muscle for analysis at time zero, and store the remainder at -20°C, 0°C, and 8°C, respectively.

2. For histamine extraction, put each sample into a sterile bag with 80 mL of iced distilled water (the amount of muscle sample may vary between 1 and 10 g, but the relationship sample(g): distilled water [mL] must be 1:8) and homogenize with a BagMixer® 400 (Interscience, France) for 10 min. Filter the homogenate with a paper filter (2x), which results in a translucent liquid (filtrate) that is then used in the histamine analysis. Alternatively, the double-filtration step may be replaced by centrifugation (10,000g for 5 min).

3.2. Sample Acylation

Acylate the filtrate or supernatant using the reagent supplied in the commercial kit, as follows: Add 180 ^L of the acylation buffer to a sterile (or sufficiently clean) plastic tube, followed by 20 ^L of the extracted sample and 50 ^L of the acylation reagent. This reaction is instantaneous, and the product is quite stable (for 48 h at 4°C).

3.3. Enzyme Immunoassay

1. Wash procedure.

a. Put 50 ^L of either the acylated sample or the standard in the microplate wells coated with the high-affinity specific monoclonal antibody (antihistamine), followed by 200 ^L of the alkaline phosphatase-histamine conjugate.

b. After 30 min, wash the wells with buffered water wash solution. For this washing step, turn the microplates upside down and shake vigorously over the sink; use a micropipet (or a squeeze bottle) to fill the wells carefully with wash solution. Repeat this procedure three times (see Note 5).

c. Finally, tap the wells firmly on clean absorbent paper, still in the upside-down position.

d. The wash procedure may also be performed with an automated microplate washer machine.

2. After the washing step, add 200 ^L of the _p-NPP substrate and incubate the solution for 30 min at room temperature (18-25°C), covering the microplate to avoid evaporation. Stop the reaction with 50 ^L of 1 N sodium hydroxide.

3. To determine the absorbance of the solutions in the wells, siphon the respective volumes individually using a micropipet and then read them in a spectrophotometer at 405 nm using a microcuvette of silica (quartz) with a maximum capacity of 200 ^L. Ideally, the solution absorbances must be determined in a microplate reader at 405-414 nm. Then compare each absorbance reading with the calibration curve using a semi-log scale (Fig. 1) to obtain the histamine concentration directly from the graph and/or by the equation y = 2.3609 x~0-3101 (R2 = 0.9949), derived from the data. Reading the microplates can be delayed up to a maximum of 2 h.

Fig. 1. Calibration curve of histamine at 405 nm. The exponential equation calculated was y = 2.3609 x-03101, with R2 = 0.9949.

4. Analysis of results.

a. Figure 1 shows that the sensitivity limit of the calibration curve is 1 ppm (1 mg histamine/kg fish), even though the detection limit of the method is supposed to be 0.5 ppm.

b. As the histamine levels must be below 50-200 ppm (mg/kg), depending on local legislation, the calibration curve demonstrates that this immunotechnique is an efficient methodological tool to control fishery quality in terms of onset of putrefaction.

c. To provide examples, histamine analyses were done on fish samples (Katsuwonus pelamis) stored at temperatures of -20°C, 0°C, and 8°C (Fig. 2).

d. The results show that temperature is a determining factor in the quality of fishery products, especially those that exhibit a flora positive for histidine decarboxylase, such as the Scombridae (tuna).

e. Furthermore, the results show that at freezing temperatures (-20°C and 0°C) histamine levels were almost constant during the 50-d storage. In the fish kept at 8°C, the histamine levels increased quickly. From the 10th d onwards the histamine level exceeded the legal limit and reached nearly 1000 ppm, meaning that fish stored at 8°C could not be considered safe for human consumption.

4. Notes

1. The Histamarine kit contains the following: microplate, standards (1, 10, 50, 100, and 500 mg/kg [ppm], dimethyl sulfoxide [DMSO]), acylation reagent (to be dissolved in DMSO), acylation buffer (stored at 2-8°C), conjugate (dissolved in distilled water), wash solution (diluted in distilled water), substrate buffer, substrate tablets of p-NPP (to be diluted in substrate buffer; the tablets must be handled with forceps, since contact with skin may affect the product adversely), and stop solution (1 N NaOH). All solvents, reagents, resulting solutions, and microplates are stored at 2-8°C or frozen at -20°C until further use, at which time they are carefully thawed to room temperature. All reagents in the unopened kit are stable at 2-8°C until the expiration date, and it is recommended to allow the components of the kit to reach room temperature before use.

Fig. 2. Determination of histamine levels in samples of skipjack tuna (Katsuwonuspelamis) stored at -20°C (A). 0°C (□), and 8°C (•).

2. Homogenization is carried out in a solid sample blender. e.g.. BagMixer 400. but a common blender may be substituted.

3. Care should be taken to avoid glass tubes since histamine adsorbs to glass.

4. Direct contact of samples with the naked hands should be avoided since histamine may be present in human secretions such as sweat.

5. Careful washing is important since incomplete removal of unbound conjugate will lead to high background levels.


The authors thank Central de Produtos Enzimaticos e Imunologicos Ltda. (CPEI)

for support with the Histamarine kit. CCTMAR-UNIVALI-SC for funding. and Prof.

A. Barreto for reading the manuscript.


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