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 4 C the respective mixtures for 1
minute. After centrifugation of each of the
homogenized solutions at 4 C., 1000 rpm, 1000 l
of the supernatant was mixed with 250 l 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
l of the 1 M sodium hydroxide solution in place of
250 l. The methanol-extracted sample solutions
had pH of 7. A. Histamine detection method
The procedure introduced 500 l 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 l of each sample solution to the
cartridge under pressure to make the sample
solution pass through the carrier, sequentially
introduced one 200 l aliquot of a 0.2 M phosphate
buffer (pH 6.0) and four 200 l aliquots of water to
the cartridge to wash the carrier, and subsequently
introduced 200 l 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 100C 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
Quantification R2
R3
T2
R1
R2
R3
Iron
Detection and
Quantification
Absorbance
Value