DETECTION AND QUANTIFICATION OF HISTAMINE AND IRON (Fe) IN THREE VARIETIES OF TUNA

Janne Paulyne P. Libunao, Kristoffer John M. Nerona & Janel R. Cacuyog

Introduction

Fish is widely consumed in many parts of the world by humans because it has high protein content, low saturated fat and also contains omega 3 fatty acids known

to support good health. Histamine fish poisoning, an entirely preventable condition, is among the most common toxicities related to fish ingestion. Histamine fish poisoning,

which had been previously termed scombroid fish poisoning, pseudo allergic fish poisoning, histamine overdose, or mahi-mahi flush, accounts for less than 0.5% of all food-

borne outbreaks reported to the Centers for Disease Control and Prevention (CDC) and 37% of all seafood-related food-borne illnesses. It presents as a possible allergic

reaction after consuming certain fish but is actually caused by ingesting toxins within the fish's tissues .Biologically active substance found in a great variety of living

organisms is histamine. It is distributed widely, albeit unevenly, throughout the animal kingdom and is present in many plants and bacteria and in insect venom. It is formed

by the decarboxylation (the removal of a carboxyl group) of the amino acid histidine. Iron is a trace element of considerable concern in public health. A complete, accurate

and quantitative knowledge of the levels and forms (heme or nonheme) of iron in foods is important since the bioavailability of each type of iron differs. Fish is a major

source of iron for adults and children. Iron deficiency causes anemia. Meat is the main source of heme iron in human diets, and meat also makes a large contribution to the

nonheme iron content of human diets. Levels of total, nonheme and heme iron are often determined in meats, but little effort has been spent on validating the methods used

to analyze iron. Most types of fish contain iron, providing up to about 10 percent of the RDA in an average serving. For example, a 6-ounce serving of canned salmon or

tuna contains about 1.5 milligrams of iron. Other varieties of fish also provide iron, including cod, with 0.8 milligrams in 6 ounces; flounder, with 0.6 milligrams in 6 ounces;

and haddock, with 0.4 milligrams in a similar-sized serving. Both wild-caught and farmed fish supply iron. Our body uses iron in many ways. One of the most important uses

is for manufacturing heme, an iron-containing compound that binds oxygen. As part of hemoglobin, heme carries iron in your blood to all parts of your body. Iron is also part

of myoglobin, another oxygen-carrying compound found in your muscles. Besides its function in oxygen transport, iron is also necessary for the action of many enzymes.

Our body needs the mineral iron for many extremely important functions, including helping your red blood cells carry oxygen to all your cells and tissues. A lack of iron is the

most prevalent nutrient deficiency in America, according to the Linus Pauling Institute. You can increase your intake of this vital micronutrient by adding iron-rich seafood to

your diet.

Statement of the Problem

The study aimed to detect and quantify

Histamine and Iron in Three Varieties of Tuna.

Specifically, it answered the following questions:

1.) Which of the following varieties has

the most percentage of Histamine

and Iron?

2.1) Blue fin Tuna

2.2) Yellow fin Tuna

2.3) Big eye Tuna

2.) What are the factors affecting

Histamine and Iron level in the three

varieties of Tuna?

3.) What percentage of the Tuna is

positive with Iron and Histamine?

What percentage of Tuna is negative

with Iron and Histamine?

4.) Is there a significant difference in the

levels of Histmine and Iron among

the three varieties of Tuna?

Methodology

Preparation of Materials The fresh raw

tuna and histamine dihydrochloride will be bought

at the Microbiology Laboratory of Notre Dame of

Dadiangas University – Main Campus. The

Histamine Dihydrochloride of 1mM acetonitrile

solution of 2, 3- naphathalene dicarboxylaldehyde

will also be prepared. Fresh raw tuna will be

weighed 5 g. The laboratory equipments to be

used include the following: filter paper discs, test

tubes, petri dishes, injection syringe. Gathering

and Preparation of Samples The experiment wil

be performed by addition of a histamine standard

preparation to a real sample of fresh fish meat.

The histamine standard preparation used was

histamine dihydrochloride, and the histamine

detection reagent used was a 1mM acetonitrile

solution of 2, 3- naphathalene dicarboxylaldehyde.

Preparation of Fresh Raw Tuna The procedure

weighed 5g of fresh raw tuna in a homogenizer,

added 25 ppm, 50 ppm, and 100 ppm aqueous

solutions of the histamine standard preparation

and 30 ml of a 5% aqueous TCA solution, and

homogenized the respective mixtures for 1 minute.

After centrifugation of each of the homogenized

solutions at 4º C the respective mixtures for 1

minute. After centrifugation of each of the

homogenized solutions at 4° C., 1000 rpm, 1000 μl

of the supernatant was mixed with 250 μl of a 1 M

sodium hydroxide solution with stirring to give a

TCA-extracted sample solution. The TCA-extracted

sample solutions had pH of 8. Methanol-extracted

sample solutions were prepared by the similar

procedure with replacement of the aqueous TCA

solution by methanol and with addition of 20 μl of

the 1 M sodium hydroxide solution in place of 250

μl. The methanol-extracted sample solutions had

pH of 7. A. Histamine detection method The

procedure introduced 500 μl of each sample

solution to the histamine detection cartridge and

applied pressure to the carrier with a piston of the

injection syringe to make the sample solution pass

through the carrier. The procedure then similarly

introduced 500 μl of each sample solution to the

cartridge under pressure to make the sample

solution pass through the carrier, sequentially

introduced one 200 μl aliquot of a 0.2 M phosphate

buffer (pH 6.0) and four 200 μl aliquots of water to

the cartridge to wash the carrier, and subsequently

introduced 200 μl of the histamine detection

reagent to make the reagent pass through the

carrier in the similar manner. B. Total Iron Method

Tuna samples will be accurately weighed into 125

mL Erlenmeyer flasks and 15 mL of concentrated

nitric acid will be added. Each flask will be left to

predigest at room temperature for 4-6 hours or

overnight. The flask will be placed on a hot plate

set at 100°C until dry. Hydrogen peroxide sulfuric

reagent containing peroxymonosulfuric acid, will be

added in 1 mL aliquots to each sample until they all

become clear, typically after three or four additions.

The flask will be left on the hot plate until all

peroxide will be expelled and the white vapors of

sulfuric acid become evident. The clear digest will

allow to cool and quantitatively transferred to 10

mL volumetric flask using 0.01 N HCl as the rinse.

Quantitative Analysis In order to determine the

absorbance value of histamine and the iron in tuna,

the data will be subjected to quantitative analysis.

Method Histamine

T1

T1 – Histamine Dihydrocholride

T2 – Total Iron Method

R1- Big Eye Tuna

R2- Yellow Fin Tuna

R3- Blue Fin Tuna

Figure 1. Research Design

Absorbance Value

R1

Detection and QuantificationR2

R3

T2

R1

R2

R3

Iron

Detection and Quantification

Absorbance Value