ANALYSIS OF BIOGENIC AMINES IN FISH BY USING

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

NUR IDATUL DIANA BT AHMAD ZAKI (35120)

Bachelor of Science with Honours

(Resource Chemistry)

2015

ANALYSIS OF BIOGENIC AMINES IN FISH BY USING

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

NUR IDATUL DIANA BT AHMAD ZAKI

A Final Year Project Report is submitted in partial fulfillment of the requirements for the

award of the degree of Bachelor of Science with Honours

(Resource Chemistry)

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2015

i

ACKNOWLEDGEMENT

Firstly I would to express my special thanks to my research supervisor Professor Dr

Zaini Bin Asim for his guidance in helping me to finish my research project and this degree

thesis. Besides, the useful critiques, valuable suggestion during the development of this

research project and also willingness to spend his time has been much appreciated. I also

would like to dedicate special thanks to Mr Leo Bulin Unting, Science Officer Faculty

Resource Science and Technology whose guide and educating me in the usage of High

Performance Liquid Chromatography (HPLC), to my family, senior and my friends that give

moral support to me for completing this project and report.

ii

UNIVERSITI MALAYSIA SARAWAK

Grade:____________

Please tick (√)

Final Year Project Report

Masters

PhD

DECLARATION OF ORIGINAL WORK

This declaration is made on the ……………..day of……………..2015.

Student’s Declaration:

I NUR IDATUL DIANA BT AHMAD ZAKI, 35120, FACULTY OF RESOURCE SCIENCE

AND TECHNOLOGY hereby declare that the work entitled, ANALYSIS OF BIOGENIC

AMINES IN FISH BY USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY is

my original work. I have not copied from any other students’ work or from any other sources

except where due reference or acknowledgement is made explicitly in the text, nor has any

part been written for me by another person.

Date submitted NUR IDATUL DIANA BT AHMAD ZAKI (35120)

Supervisor’s Declaration:

I, PROF. DR.ZAINI BIN ASSIM, hereby certify that the work entitled, ANALYSIS OF

BIOGENIC AMINES IN FISH BY USING HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY was prepared by the above named student, and was submitted to the

“FACULTY OF RESOURCES SCIENCE AND TECHNOLOGY as a full fulfillment for the

conferment of BACHELOR DEGREE OF SCIENE (CHEMISTRY) WITH HONORS, and

the aforementioned work, to the best of my knowledge, is the said student’s work.

Received for examination by: _____________________ Date:___________________

PROF. DR. ZAINI BIN ASSIM

iii

I declare this Project/Thesis is classified as (Please tick (√)):

CONFIDENTIAL(Contains confidential information under the Official Secret Act 1972)*

RESTRICTED (Contains restricted information as specified by the organisation where research was done)*

OPEN ACCESS

Validation of Project/Thesis

I therefore duly affirmed with free consent and willingness declared that this said

Project/Thesis shall be placed officially in the Centre for Academic Information Services with

the abide interest and rights as follows:

This Project/Thesis is the sole legal property of Universiti Malaysia Sarawak

(UNIMAS).

The Centre for Academic Information Services has the lawful right to make copies for the purpose of academic and research only and not for other purpose.

The Centre for Academic Information Services has the lawful right to digitise the

content to for the Local Content Database.

The Centre for Academic Information Services has the lawful right to make copies of the Project/Thesis for academic exchange between Higher Learning Institute.

No dispute or any claim shall arise from the student himself / herself neither third party

on this Project/Thesis once it becomes sole property of UNIMAS.

This Project/Thesis or any material, data and information related to it shall not be

distributed, published or disclosed to any party by the student except with UNIMAS

permission.

Student’s signature ___________________ Supervisor’s signature: _____________ ( /5/2015 ) ( /5/2015)

Current Address:

21 JALAN SAGA 2 TAMAN SAGA 28400 MENTAKAB PAHANG Notes: * If the Project/Thesis is CONFIDENTIAL or RESTRICTED, please attach

together as annexure a letter from the organisation with the period and reasons of

confidentiality and restriction.

[The instrument was duly prepared by The Centre for Academic Information Services]

iv

TABLE OF CONTENTS

Content Page

ACKNOWLEDGEMENT i

DECLARATION ii-iii

TABLE OF CONTENT iv-vi

LIST OF ABBREVIATIONS vii

LIST OF TABLES viii

LIST OF FIGURES ix

ABSTRACT x

ABSTRAK Xi

1. INTRODUCTION

1.1 General Introduction 1-2

1.2 Problem Statement 3

1.3 Objectives of the Project 3

2. LITERATURE REVIEW

2.1 Biogenic Amine 4-5

2.2 Hazard Characteristics

2.2.1 Histamine 6

2.2.2 Cadaverine and Putriscine 6-7

2.3 Microorganism Involved in Biogenic Amine Production 7

2.4 Condition Supporting the Formation of Biogenic Amines

v

2.4.1 Substrate availability 7-8

2.4.2 Temperature 8

2.4.3 Effect of pH 8

2.4.4 Others factors 9

2.5 Toxicology Aspects of Biogenic Amine in Response of Human

2.5.1 Histamine 9-10

2.5.2 Cadaverine and Putrescine 10

2.5.3 Tyramine and Phenylethylamine and Tryptamine 10-11

2.6 Biogenic Amine in Fish 11-12

2.7 Biogenic Amine as Quality Index in Meat and Meat Products 12-13

2.8 Analytical Methods for Biogenic Amine 13-14

3. MATERIALS AND METHODS

3.1 Chemical and Reagent 15

3.2 Chromatographic Condition 15

3.3 Application of Validated Method for Biogenic Amines in Fish

3.3.1 Sample of Raw Fish 16

3.3.2 Analysis of Biogenic Amines in Fish 16

3.4 Method Validation for Analysis of Biogenic Amines

3.4.1 Preparation of Biogenic Amine Standards 16

3.4.2 HPLC Analysis of Biogenic Amine Standards 17

3.4.3 Validation Parameters 17

vi

3.5 Statistical Analysis using One Way ANOVA 18

4. RESULTS AND DISCUSSION

4.1 Method Validation 19-23

4.2 Linearity and Working Range 24-27

4.3 Limit of Detection(LOD) and Limit of Quantification (LOQ) 27-28

4.4 Accuracy

4.4.1 Repeatability Precision 29-30

4.4.2 Reproducibility Precision 31-32

4.5 Precision 33-35

5. CONCLUSION 36

REFERENCES 37-44

APPENDICES 45-47

vii

LIST OF ABBREVIATIONS

Cad Cadaverine

CE Capillary Electrophoresis

DAO Diamine oxides

HMT Histamine-N-methyltransferase

GC Gas Chromatography

Him Histamine

HOC Histidine decarboxylase

HPLC High Performance Liquid Chromatography

LOD Limit of detection

LOQ Limit of quantification

MAO Monoamine oxidase

ppm Part per million

Put Putrescine

RT Retention time

RSD Relative standard deviation

Spm Spermine

SD Standard deviation

Spd Spermidine

SDr Standard deviation repeatability

SDR Standard deviation reproducibility

TLC Thin Layer Chromatography

viii

LIST OF TABLES

Table Page

Table 2.1 Structure and molecular weight and boiling point of six standard biogenic

amines

4

Table 2.2 Concentration of histamine in fish related to histamine fish poisoning 12

Table 4.1 Parameters in method validation 19

Table 4.2 Comparison of different columns used in the identification of biogenic

amines

20

Table 4.3 Determined retention time of Biogenic Amines on Purospher® STAR

column

22

Table 4.4 Result of SD & RSD of area and retention time of biogenic amines 23

Table 4.5 Correlation coefficients of calibration curves of biogenic amines 27

Table 4.6 LOD and LOQ values for each investigation of biogenic amines 28

Table 4.7 SDr and RSD value of biogenic amines in repeatability condition in muscle

fish.

30

Table 4.8 Result of accuracy (intermediate precision) for the chromatographic method

evaluated with the same HPLC in three consecutive days

31

Table 4.9 Recovery values in % of spiked fish 34

ix

LIST OF FIGURES

Figure Page

Figure 2.1 Structure of histamine, tyramine, 2-phenylethylamine, putrescine and

cadaverine

5

Figure 2.2 Formation of biogenic amine 9

Figure 4.1 Chromatogram for 100 ppm biogenic amines analyzed using

Purospher® STAR HPLC column

20

Figure 4.2 Chromatogram for 100 ppm biogenic amines analyzed using μ

Porasil HPLC column

21

Figure 4.3 The calibration curve of the biogenic amines standards of tyramine 25

Figure 4.4 The calibration curve of the biogenic amines standards of cadaverine 25

Figure 4.5 The calibration curve of the biogenic amines standards of histamine 26

Figure 4.6 Components of accuracy 29

Figure 4.7 HPLC chromatogram of spiked sample 35

Figure 4.8 HPLC chromatogram of unspiked sample 35

x

Analysis of Biogenic Amines in Fish by using

High Performance Liquid Chromatography

ABSTRACT

Biogenic amines (BAs) are organic basis formed in food by microorganisms through

enzymatic decarboxylation of amino acid, non volatile and heat stable. Biogenic amine can be

found in several of food especially food that inappropriate handle and stored scombroid fish

like tuna. The aim of this study was for the verification of an high performance liquid

chromatography (HPLC) method for the determination of biogenic amines in fish. The

implementation of the method included the optimization of sample preparation, HPLC

analysis and data evaluation. Thereafter, the method performance was verified to demonstrate

that the results obtained were consistent, correct and satisfactory for analysis of biogenic

amines in fish. Separation of biogenic amine was achieved on a Purospher® STAR column

with gradient elution system coupled with a UV detector (198 nm). A mixture of standard

biogenic amines consist of tyramine, cadaverine and histamine were used for the verification

test such as linearity, limit of detection, limit of quantification, accuracy, precison and

recovery. The calibration curves for biogenic amines are linear from the concentration of 10

ppm until 75 ppm and linearity coefficients (r2) >0.900 for tyramine and histamine. Relative

standard deviation (RSD) for biogenic amines <10% which are 4.74%, 5.15% and 5.53% for

tyramine, cadaverine and histamine. The recovery in the concentration of tyramine and

cadaverine are 86% and 95%, respectively.

Keywords: biogenic amine, HPLC, validation, fish

xi

ABSTRAK

Amina biogenik merupakan organik asas yang terbentuk di dalam makanan oleh bakteria

melalui pendekarboksilan enzim asid amino, tidak mudah meruap dan tahan kepada suhu yang

tinggi. Amina biogenik boleh dijumpai di dalam makanan terutamanya makanan yang

dikendalikan dengan cara yang tidak sesuai dan kumpulan ikan scrombroid seperti tuna yang

telah disimpan. Tujuan utama kajian ini adalah bagi menentukan kandungan amina biogenik

di dalam ikan dengan menggunakan kromatografi cecair prestasi tinggi (KCPT). Perlaksanaan

kaedah ini adalah termasuk pengoptimuman penyediaan sampel, analisis KCPT dan proses

pengolahan data. Seterusnya kaedah yang terhasil disahkan bagi menunjukkan keputusan yang

diperolehi konsisten, betul dan memuaskan bagi analisis amina biogenik di dalam ikan.

Pemisahan amina biogenik terhasil baik dengan menggunakan turus Purospher@ STAR dan

UV sebagai pengesan (198 nm). Amina biogenik seperti tiramina. kadaverina dan histamina

digunakan dalam pengesahan analisis seperti kelinearan, had pengesanan, had kuantifikasi,

ketetapan, dan kepresisan. Graf kalibrasi bagi amina biogenik adalah linear pada julat

kepekatan antara 10 hingga 75 ppm dan menunjukkan nilai pekali kelinearan >0.900 bagi

tiramina dan histamina. Sisihan piawai relatif untuk amina biogenik <10% iaitu sebanyak

4.74%, 5.15% and 5.53% untuk tiramina, kadaverina dan histamina. Nilai perolehan semula

bagi amina biogenik tiramina dan kadaverina adalah 86% dan 95%, masing-masingnya.

Kata kunci : amina biogenik, kromatografi cecair prestasi tinggi (KPTC), ikan

1

CHAPTER 1

INTRODUCTION

1.1 General Introduction

High Performance Liquid Chromatography (HLPC) is the instrument that is widely

applicable techniques leading the field of analytical chemistry and the most versatile (McNair

and Miller, 1998). The advantage by using HPLC is the sample does not need to derivatise like

the Gas Chromatography (GC). In HPLC, the mobile phase will carried the analyte through

column or stationary phase. Water, methanol, acetonitrile are the example for the mobile

phase. The analyte will be separate by the stationary phase and the elute enter the detector

which produces an electrical signal. The data system generates an image called

chromatography.

Biogenic amines (BAs) are result of removal the carboxylic acid group on the amino

by enzymic reactions from corresponding amino acid. The bacterial actions cause the

formation of biogenic amine in raw food. Biogenic amine can be found in diverse foods like

meat, fermented foods, fish and fish product and cheese (Eerola et al., 1993). Mishandled

during the storage and processing, can causes breakdown of protein to free amino. Amino acid

can also be naturally found within the food. According to Den Brinker et al. (1995) food that

was contaminated with bacteria that contain the decarboxylase enzymes, produce biogenic

amine because the free amino acids undergo decarboxylation. Biogenic amines may cause

vasoactive or psychoactive effects if too large quantity of biogenic amine is consumed orally

(El-Habib, 2011). Microbial growth, availability of free amino acids, temperature, and high the

presence of decarboxylase enzymes are factor influenced the amount of biogenic amines are

2

conditions (Halasz et al.,1994). Histamine poisoning, which results from intake of foods that

contain significant amounts of histamine, has been reported to be one of the major illness

among foodbrone disease. Other amines like putrescine and cadaverine have been described as

potentiators that enhance the toxicity of histamine (El-Habib, 2011). Histidine decarboxylase

is the enzyme that involved in the production of histamine. Temperaute greater than 15 ºC and

30 ºC as the optimum temperature is needed for the production of histamine. Fish are usually

caught in temperatures exceeding 20ºC in the tropical areas of the world and if the fish are not

refrigerated immediately, conditions are favorable for biogenic amine production, providing

bacteria containing decarboxylase enzymes. According to Ahmed (1991), when the

temperature is around 0-5ºC, bacterial growth will stop, but enzymic activity will still continue

resulting in further amine production. Taylor (1985) reported, histamine is toxic in larger

doses, but in small doses it has little effect. Excess quantity of histamine in the body can cause

dilating blood cells and can result in hypotension. The incubation period of histamine

poisoning is short. According to Taylor et al. (1989), eating of a meal that contain high level

of histamine can cause the poisoning effects occur within several minute to a few hours.

Diamine oxides (DAO) and histamine-N-methyl transferase (HMT) is enzyme in human body,

which convert histamine to harmless degradation products (Den Brinker et al., 1995). These

enzymic reactions will be inhibited by putrescine and cadaverine and therefore potentiate of

histamine toxicity. There are several fish suspected of causing histamine but only one type of

fish will be used as samples which are tenualosa toli (terubuk).

3

1.2 Problem Statements

Biogenic amines are basic nitrogenous compounds found in a variety of foods like fish,

meat, fish product and many others. The formations of biogenic amines were mainly due to the

amino acid decarboxylase activity of certain microorganisms. Availability of free amino acid,

microbial growth and elevated temperature conditions are the factor that influences the

biogenic amines production. They can cause different toxicological effects to humans,

depending on biogenic amines concentration, individual sensitivity and the specific biogenic

amines. Some of them have powerful physiological effect and have an important biological

activity. This study will focus on validation of analytical method using HPLC for detection of

biogenic amine. Information regarding the biogenic amine contains in raw fish and fish

product in Sarawak is still rather scanty and inadequate. Hence, attention should be given to

ensure the safety of this product.

1.3 Objectives of the Project

The objectives of this study are as following:

a. to validate the analytical method for biogenic amines analysis using high

performance liquid chromatography (HPLC),

b. to evaluate the recovery efficiency of extract method,

c. to isolate biogenic amines from fish.

4

CHAPTER 2

LITERATURE REVIEWS

2.1 Biogenic Amines

Biogenic amines such as spermine, cadaverine, tryptamine, histamine, putrescine,

heptylamine, and tyramine (Table 2.1) are organic bases formed mainly by the

decarboxylation of amino acid by enzymic reaction.

Table 2.1: Structure and molecular weight and boiling point of six standard biogenic amines

(Nakovich, 2003)

Amines Molecular

Weight

Boiling Point

( ˚C)

Structure Precursor

Histamine 111.15 167

Histidine

putrescine 88.15 158-160

Orthine,

agmatine

cadaverine 102.18 178-180

Lysine

Tyramine 137.18 175-181

Tyrosine

Spermidine 145.25 128-130

Arginine

5

According to Fathi et al. (2013), biogenic amine may be present in various foods that

rich in protein like fish and meat. The chemical structure of biogenic amines are heterocyclic

(tryptamine and histamine) or aliphatic (cadaverin, putrescine, espermine and espermidine) or

as aromatic (phenylethylamine and tyramine) (Figure 2.1).

Heterocyclic amines Aromatic amines

Aliphatic amine

Figure 2.1: Structure of putrescine, tyramine, cadaverine, histamine and 2-phenylethylamine

(EFSA, 2011).

Various bacteria will be produce decarboxylase enzyme in high temperature and kept its

activity to slightly lower 5°C (Fathi et al., 2013). Improper storage of fish can cause quality

degradation and the accumulation of biogenic amine in a short period. Technological

processes for example fermentation, ripening, salting or marination can increase the possibility

formation of biogenic amine (Pons-Sanchez-Cascado et al. 2005a). A study conducted by

Fathi et al. (2013), at low pH around 4.0–5.5, is favorable for enhanced amino acid

decarboxylase activity which can be achieved in salted anchovies. Presence of histamine in

raw fish or fish products is the most important quality indicator as chemical indicators for

spoilage of fish and is considered as a threat to public health bodies.

6

2.2 Hazard Characteristics

2.2.1 Histamine

L-histidine that was contained in Pyridoxal phosphate decarboxylase from the amino

acid histidine in enterochromaffine cells, histaminergic neurons, basophils, mast cells, and

platelets, synthesized the histamine (Maintz and Novak, 2007). Histidine to histamine is the

decarboxylation of the amino acid that is naturally occurring substance in the human body.

Foods that contain free histidine can be present with histamine. Histamine is generated by

certain bacteria during spoilage and fermentation of fish. Endogenous histamine has vital

physiological functions related to local neuromodulation, immune responses and gastric acid

secretion. Sensitive individuals cannot tolerance with histamine-rich foods and food poisoning

may cause because of histamine contamination in fish and fish products (Taylor, 1985).

2.2.2 Putrescine and Cadaverine

Cadaverine and putrescine are example of biogenic amines that may found in fish.

Gram-negative bacteria has mainly been to related cadaverine and putrescine production,

especially in the Pseudomonadaceae, Shewanellaceae and Enterobacteriaceae,family’s

normally related with spoilage (Lopez-Caballero et al., 2001). In the fermented product,

putrescine is one of the most common biogenic amine that can be found. Bacteria like

lactobacilli mainly, staphylococci and lactic acid bacteria have also been reported to be able to

generate cadaverine or putrescine (Arena and Manca de Nadra, 2001; Beneduce et al.,2010;

Coton et al., 2010b). Like histamine, they are produced from amino acids by bacteria during

spoilage and fermentation. The precursors of putrescine and cadaverine are ornithine and

lysine respectively. Putrescine and cadaverine are both found frequently in improperly handled

7

fish and have been studied as spoilage indicators. In some fish spoilage studies, cadaverine

appeared to be formed and increased earlier than histamine (Pons-Sanchez-Cascado et al.,

2005b; Rossi et al., 2002).

2.3 Microorganism Involved in Biogenic Amine Production

Amino acids and amino acid decarboxylases, synthesized by bacteria required in the

formation of biogenic amine (EFSA, 2011). Some microorganisms that have enzyme histidine

decarboxylase (HDC) can lead the formation of histamine in fish. At the highest level of

temperature, the naturally present of free histidine in the muscle of some fish will be converted

to histamine by HDC. HDC can be form by both Gram-negative and Gram-positive bacteria

but, the forms of the enzymes differ (EFSA, 2011; Bjornsdottir-Butler et al.,2010).

FAO/WHO (2013), reported that in fish, biogenic amine-producing bacteria can be likely to be

found on the gills, skin, or in the gastrointestinal tract. Transfer of these bacteria to the flesh of

the fish, where free amino acids may be present, leads to development of biogenic amines.

Transfer can occur from the gastrointestinal tract after harvest through migration, or via

rupture or spillage of gastric contents during gutting.

2.4 Conditions Supporting the Formation of Biogenic Amines

2.4.1 Substrate availability

The amino acids substrate is one example of the prerequisites for biogenic amines

synthesis. Proteolysis is one of the factors that are directly related to availability of free amino

8

acids which provide a substrate for biogenic amines formation. Biogenic amines formation

increases based on conditions of enhanced proteolysis or accelerated (Leuschner et al.,1998a;

Innocente and D`Agostin, 2002; Fernandez et al., 2007a; Komprda et al., 2008).

2.4.2 Temperature

Temperature is the major factor of the quantity production of biogenic amines (Zaman

et al. 2009). As the temperature increase, the production of amine also increases. On the

contrary, at low temperatures microbial growth and the reduction of enzyme activity is

inhibited, thus the accumulation of the biogenic amine is minimized. 20 to 37 °C is the

optimum temperature for the formation of biogenic amine but at the temperature of 5 °C or

above 40 °C the production of biogenic amine is decrease (EFSA, 2011). In seafood

Morganella morganii is a powerful histamine producer, although at temperatures above 7-10

ºC (Kim et al. 2002; Lehane and Olley, 2000). At 20ºC rather than 10ºC, Klebsiella

pneumoniae was produce cadaverine while an Enterobacter cloaca was able to produce

putrescine (Halaz et al., 1994). Very low temperature is needed for refrigerated foods because

even at 5 ºC, psychro tolerant bacteria can give to the amine accumulation. To reduce the

growth of bacteria and proteolytic and decarboxylase activities, low temperatures should be

applied during the storage (Hernandez-Orte et al. 2008; Rezaei et al., 2007)

2.4.3 Effect of pH

Other factors that influence the amino acid decarboxylase activity are pH (Silla Santos,

1996). In low pH environment, bacteria are more stimulated to produce decarboxylase as a

part of their defence mechanisms (Fernandez et al. 2007a; Bover-Cid et al. 2006b; Molenaar

et al. 1993).

9

2.4.4 Other Factors

Biogenic amines are produces by enzymatic decarboxylation of certain free amino acid

(Figure 2.2). According to Bardocz (1995), bacterial decarboxylases are not very specific, but

their activity varies according to the bacterial species and strain. Thus, the presence of free

amino acids, the decarboxylase enzyme and suitable environmental conditions are required for

biogenic amine formation (Ruiz-Capillas and Jimenez-Colmenero, 2004).

Protein

Proteinases

Endopeptidases

Peptides

Tyrosine Histidine Trytophan Lysine Glutamine Ornithine Arginine

Amino acid

decarboxylases

Tyramine Histamine Tryptamine Cadaverine Spermine Putrescine Spermidine

Figure 2.2 Biogenic amine Formation (Ruiz-Capillas and Jimenez-Colmenero, 2004)

2.5 Toxicological Aspects of Biogenic Amine in Responses of Human

2.5.1 Histamine

Intestinal problem, headaches, pseudo-allergic, gastric and migraine are several effect

if consume foods that contain high concentration of biogenic amine which is brought by the

toxic action of tyramine and histamine, known as cheese reaction and histamine poisoning

(Stratton et al., 1991; Taylor, 1985; Smit, 1980). Even though endogenous concentrations of

histamine are necessary and are required for normal physiological function, histamine is toxic

10

when large doses enter the circulatory system. This results in symptoms of poisoning, which

involve a wide range of organs include vascular, neurological, heart and gastrointestinal

(Taylor, 1985).

2.5.2 Cadaverine and Putrescine

Putrescine and cadaverine, can enhance the effect of tyramine and histamine by

interacting with the amino oxidase and interfering with the detoxifying mechanism and also

know as histamine potentiators and both are not considered toxic individually (Sattler et al.,

1988; Taylor, 1985; Rice et al., 1976). The minimum concentration of putrescine or

cadaverine that potentiates histamine toxicity is unknown. The ratio of putrescine or

cadaverine to histamine may need to be high to produce an effect, and it is not clear whether

the levels present in spoiled fish are sufficient to enhance the toxicity of histamine in humans

(FAO/WHO, 2013).

2.5.3 Tyramine, Phenylethylamine and Tryptamine

According to EFSA (2011), phenylethylamine, tryptamine and tyramine are

synthesised in humans from their corresponding amino acids (phenylalanine, tryptophan, and

tyrosine respectively) by decarboxylation and also known as ‘trace amines’. Monoamine

oxidase (MAO) is their main catabolism there are two MAO enzyme exist (A and B), with

different substrate specificity and locations. MAO-A predominates in the placenta, stomach

and intestine has polar aromatic amines (as octopamine and noradrenalin) as preferred

substrates. In the brain MAO-B was predominates and selectively deaminates non-polar

aromatic amines (as dopamine and phenylethylamine) (EFSA, 2011). In humans, tyramine

acts as a catecholamine (including norepinephrine [noradrenaline], dopamine, epinephrine

11

[adrenaline]) releasing agent, resulting in increased blood pressure (FAO/WHO, 2013).

According to Joosten (1987), headaches, increased blood pressure, and migraines are typical

symptoms of tyramine poisoning. Tyramine also causes the release of noradrenaline from the

sympathetic nervous system (Bardocz, 1995).

2.6 Biogenic Amine in Fish

A majority incident of histamine poisoning was related with the fish like tuna,

skipjack, and mackerel (Taylor, 1985). Scombroid fish poisoning, also known as histamine

fish poisoning is because of the histamine as the main toxin involved in. That poisoning

generally associated with high levels of histamine that is higher or equal to 500 mg/kg in fish.

There is not a straightforward dose-response relationship even though there is compelling

evidence to implicate histamine as the causative agent in histamine fish poisoning, because

spoiled fish containing histamine tends to be more toxic than the equivalent amount of pure

histamine dosed orally (Lehane and Olley, 2000). The main evidences supporting the

involvement of histamine in histamine fish poisoning are the identical symptoms of histamine

poisoning to those of intravenous histamine administration and the efficacy of antihistamines

in histamine fish poisoning therapy (Koutsoumanis et al. 2010). Concentration of histamine

that is lower than 500 mg/kg, was also reported as histamine poisoning. For example

scrombroid poisoning was linked to canned anchovies containing 365 mg/kg histamine (NSW

Food Authority, 2010) and tuna burgers with histamine levels of 213 mg/kg were implicated

(Becker et al. 2001). Various histamine concentrations in fish with different range sub-

samples of the same sample, 102 – 180 mg/kg, 27 – 152 mg/kg, 50 – 500 mg/kg of 147 mg/kg,

below 10 to 1000 mg/kg and 5 to 208 were recorder in the European Rapid Alert system for