CHAPTER 2

REVIEW OF RELATED LITERATURE

This chapter provides the related literatures about the problem presented which

serves as its basis for its underlying principles on the study

.

Red Onions

Books sources:

According to Duke, 2003, Allium cepa, commonly termed as onion; in Filipino

terms—Sibuyas; in Bisayan—Cebuyas Bombay and; in Ilocano—Lasona. It belongs to

the family Mililiaceae and has been widely use for culinary and medicinal purposes. The

dried outer skin of the bulb reduces a bacteriocide and an excellent yellow dye. The

sscales outside the onion bulb are one of the richer sources of quercetin, a very useful

phytochemical also shared with evening primrose. Scales contain a heart stimulant that

increases pulse volume, affects the uterus, promotes bile production, and reduces blood

sugar. Onion bulbs are said to be aphrodisiac, diuretic, expectorant, emmenagogue,

hypoglycaemic, and stimulant (MPI). Onion juice and EO demonstrated antiaggregant

and hypocholesterolemic activities in humans subjects (MPI). Onions are alleged to

stimulate bile production, to speed healing of gunshot wounds, and to cure scorpion

bites, freckles, and the common cold.

Libster, 2000 stated that Asian Indians eat raw onions, spiced up with lemon,

pepper, and salt, for bronchitis, colic, edema, fever, and scurvy. Some people with

parasites macerate an onion in white wine and drink it on an empty stomach in the

morning. Or pediatric patients drink water in which onion has stepped overnight to kill

parasites. Cooked onions are consumed by Japanse macrobioticists to calm the

nervous system and alleviate irritability and sore muscles after heavy labor. A cut raw

onion is placed under the pillow to aid insomnia. Reputed to be hypotensive, onions

have recently been shown to contain the antihypertensive agent prostaglandin A1, but

only at ca. 1 ppm. With prostaglandins like this going at $10 per milligram, that means

that a kilogram of onions (now costing about $1) contains $10 worth of prostaglandin.

Add that to your chicken soup. Juice of the bulb is used for coughs and earache.

Macerated in ginm the bulbs are used for dropsy and gravel (GMH). In India, onions are

believed to be aphrodisiac, especially if retained in a cow dung year in a well-stoppered

pot for four months (DEP). Even wilder, in an Indian formula for acute dysentery, one

buries a grain of opium in an onion bulb and then roasts the onion (DEP). Most of the

real and folk medicinal attributes of onion are shared with garlic and other lesser known

members of the genus Allium. Garlic is popular with organic gardeners and naturopaths

for its biological activities. For millennia, onions have been famous for food, condiments,

and medicine. Green onions are eaten raw with meats, fish, cheese, or as a vegetable,

or chooped and added to cottage cheese, or cooked. Onions are eaten raw, boiled,

baked, creamed, broiled, fried, French-fried, roasted, or pickled, and in soups, stews,

dressings, or salads, but perhaps more importantly, added to other ingredients for

innumerable dishes. Dry onions may be served as a vegetable dish or to flavour meat,

fish, and poultry dishes and are also used in salt substitutes such as Spike, Mrs.Dash

and Vegit. A thick layer of cooked onion is used on the French dish pissaladiere,

sometimes called “Provencal pizza” (FAC). Onions are used in the Catalan sauces

sofregit and samfaina (FAC). In Tunisia, a fermented onion paste called “hrous” is used

to flavour couscous, soups, and stews (FAC). The papery outer skins, called “shuski” in

slavic Macedonia, are used as a dye for coloring Easter eggs, and in Egypt they are

used to color and flavour eggs called “hamine” (FAC). The leaves of some cultivars are

widely used as scallions. In Catalonia, the large shoots called “calcots” or “sprunzale”,

sprouted from bulbs planted in trenches, are blanched and eaten raw with bread, grilled,

or used for flavouring beans and sauces (FAC). Sprouted seeds used in salads and on

sandwiches.

Willey & Sons, 2002, stated that two flavonoid subgroups are found in the onion,

the anthocyanins, which impart a red/ purple color to some varieties and flavonols such

as quercitin and its derivatives responsible for the yellow and brown skins of many

varieties.

Journals sources:

According to Asis, 2001, onion is biennial plant which may persists vegetatively

as a perennial by means of bulbs. The root system is relative shallow and fibrous. The

underground stem is short and subconal; from it hallow cylindrical leaves are diverged

in one-half phyllotaxy (leaf arrangement). The sheathing base of each leaf completely

encircles the short stem, and it is this development of the fleshy leaf bases, together

with the absence of intermodal elongation, that results in the formation of the

commercial bulb. Onion is grown locally on a limited scale and gives good returns

although much labor is required in the cultivation. Crops produced in the Philippines are

relatively poor in quality and rot more quickly and storage. This may be related to the

short natural light period in the tropics during the time of bulbing. The water requirement

increases during the bulbing time. The bulbs need about two months storage at 4.4°-

10°C before planting. Red Bermuda or sibuyas Bombay is better adapted to our

conditions than the granex or excel variety but is off poorer keeping quality and more

pungent. Commercial production of onions is restricted to definite seasons. The cooler

months (Nov.-Jan.) are the planting months; the harvest takes place during the dry and

warm months (March- May).

On the study conducted by Lanzotti, 2006, onion is characterized by polar

compounds of phenolic and steroidal origin, often glycosilated, showing interesting

pharmacological properties. The flavonoids in onion tend to be more concentrated in the

outer layers of the flesh.

Agric, 2007, mentioned that the qualitative anthocyanin content of red onion

cultivars includes a wide structural assortment including several unique flavonoid

structure. Red onion is a rich source of anthocyanin. The index onion cultivars

according to their content of flavonoids measured as quercetin. The only compound

belonging to flavonols, anthocyanins, and the dihydroflavonols have been report to

occur in onion bulb. Red onion contains 415-1917 mg of flavonols per kilogram of FW.

Flavonols are the predominant pigments of onion. The anthocyanin of red onion is

mainly cyaniding glycosides acylated with malonic acid or non-acylated. The quantities

content of anthocyanin in some red onion cultivars has been reported to be

approximately 10% of the total flavonoid content or 39-240 mg kg -1.

Internet sources:

Onion is an olfactory indicator. The onion odor isn't detectable in strongly basic

solutions. Red onion can act as a visual indicator at the same time. It changes from pale

red in acid solution to green in basic solution (antoine.frostburg.edu retrieved: 2/10/13).

On the other hand, World Health Foods states that onions are members of the

Allium family that are rich in sulfur-containing compounds that are responsible for their

pungent odor and for many of their health-promoting effects. Onions are an outstanding

source of polyphenols, including the flavonoid polyphenols which makes it a standout

source of quercetin. The flavonoids in onion tend to be more concentrated in the outer

layers of the flesh. In animal studies, there is evidence that onion’s sulfur compound

may work in an anti-clotting capacity and help prevent the unwanted clumping together

of blood platelet cells. It can also lower blood levels of cholesterol and triglycerides and

improve cell membrane function in red blood cells. In human studies, onion provides

protection for the heart and blood vessels when consumed in a diet that is rich in other

vegetables and fruits – especially flavonoid-containing vegetables and fruits. It can also

increase our bone density and provide direct benefits to our connective tissue due to its

high sulfur content (whfoods.com retrieved Jan.2013).

Anthocyanins

Books sources:

Harborne, 2000, mentioned that lycopene, saponins, tannins, anthocyanin, and

flavonoids are the usual components commonly seen in the of leaves, fruits, trunks,

peels, roots of plants like guava, tomato, garlic, onions, atis and cabbage. Anthocyanins

are all based chemically on a single aromatic structure, that of cyaniding, and all are

derived from this pigment by addition or subtraction of hydroxyl groups or by

methylation or by glycosylation. Orange-red colours are due to pelargonidin with one

less hydroxyl group than cyaniding, while mauve, purple and blue colours are generally

due to delphinidin, which has one more hydroxyl group than cyaniding.

According to Willstatter, 2003, nearly any fruit or flower that is bright red, blue or

purple contains pigment molecules that are based on cyanidin. The molecular structure

is responsible for all these colors. like phenolphthalein, cyanidin’s structure changes

with pH. In acidic solution, there is a high formal charge on the oxygen in the structure

which makes it bright red. In basic solution, removal of hydrogen from the OH group on

the right outmost ring and forms a blue or violet color. In natural forms of the molecule,

the hydrogens on at least one of the -OH groups are replaced with more sugar

molecules. A cyanidin with attached sugars is called an anthocyan or anthocyanin.

Guevarra, 2005 added that anthocyanins also make up the most important and

widespread group of coloring matter in plants. It is one of the subclass of the phenolic

compound named flavonoids.

Journals sources:

In a research article entitled, “Use Of Anthocyanin Extracted From Natural Plant

Materials To Develop A Ph Test Kit For Measuring Effluent From Animal Farms”,

Suppadit et al., 2011, Anthocyanins (from the Greek anthos = flower and kianos = blue)

are the most important pigments of the vascular plants; they are harmless and can be

easily incorporated into aqueous media which makes them interesting for use as natural

watersoluble colorants. These pigments are responsible for the shiny orange, pink, red,

violet and blue colors in the flowers and fruits of some plants. Anthocyanins can be

found in different chemical forms depending on the pH of the solution. In the research,

two factors were considered in optimal extraction of anthocyanin. First, the type of

natural plant materials is considered. The research utilized butterfly pea (Clitoria

ternatea L.) flower roselle red (Hibiscus sabdariffa L.) flower and dragon fruit

(Hylocereus undatus (Haw) Britt. &Rose.) peel. Second is the type of solvent to be

used. This includes consisting of distilled water, 1% HCl/95% ethanol, 0.1 N acetic

acid, 0.5% vinegar and 20% white liquor. The butterfly pea flowe yielded the highest

amount of anthocyanins at 541mg/100 g dry weight followed by roselle red flower (280

mg/100 g) and dragon fruit peel. For solvents, the distilled water used for the extraction

yielded the highest amount of anthocyanins (394 mg/100 g dry weight) followed by

white liquor (388 mg/100 g), 1% HCl/95% ethanol (303 mg/100g), acetic acid (288

mg/100 g) and vinegar (282 mg/100 g) (P<0.05).

From a journal written by Frosburg, 2001 he mentioned that any substance that

undergoes a reversible chemical change when pH changes can be used as an acid-

base indicator. In practice, a sharp change in some easily detectable property of the

substance is required. Usually, the property is color; but other properties such as odor

can also change with pH. Almost any flower, fruit, or plant part that is red, blue, or

purple contains a class of chemical compounds called anthocyanins that change color

with pH. The color of a flower or fruit depends on which anthocyanins are present, the

pH of the pigment-bearing tissues, and the presence of other pigments, like yellow

flavones. Red cabbage contains a mixture of anthocyanins and other pigments that

indicate a wide range of pH.

Accorrding to Medwell Journals, 2010, despite their advantages with respect to

heat, light, ph ability and purity, compared with natural colorants such as anthocyanins,

synthetic pigments are increasingly rejected by consumers owing to health concerns,

thus there is worldwide interest in additional use of anthocyanins as consequence of

perceived consumer preferences as well as legialative action which has continued the

delisting of approved artificial dyes. The onion solid wastes was frozen with liquid

nitrogen and ground with a pestle and a mortar. An amount of approximately 500

milligrams of ground tissue was place in a 30 ml glass vials and 10 ml of solvent was

added. Extraction was carried under magnetic stirring at 400 rpm at room temperature

for predetermined time periods. Upon completion of extraction, the extract were filtered

through paper filter and stored at negative 20°C until analyzed. All extracts were also

filtered through 0.45 micrometer syringe filters prior to determinations. Briefly an aliquot

of extract was combined with ethanolic HCL solution (0.25M) to give a dilution 1:10. The

solution was mixed thoroughly and the absorbance at 520 nm was read after 5 mins

using the ethanolic HCL solution as blank. Total anthocyanin content was determined

as cyanin per 100 g fresh tissue using as ɛ= 26900 and MW=449.2.

Anthocyanin was also extracted from rose petals to be used as acid-base

indicators as describe by Vankar & Majpai, 2010, anthocyanin extraction were carried

out by three methods. By 1) HCl, 2) Citric acid 3) Tatrtaric acid. 1) Anthocyanins were

extracted from flowers with 0.1% HCL in methanol for two to three hours at room

temperature in darkness. The mixture was filtered on a Buchner funnel and the

remaining solids were washed with 0.1% HCl in methanol until a clear solution was

obtained. The combined filtrates were dried using a rotary evaporator at 30°C. The

concentrate was dissolved in 0.01% HCL in DW and in the solution was used as

indicator. 2) Anthocyanins were extracted from flowers with 4.0% citric acid in methanol

for 2-3 hours at room temperature in darkness. The mixture was filtered in a Buchner

funnel and the remaining solids were washed with 4.0% citric acid in methanol until a

clear solution was obtained. The combined filtrates were dried using a rotary evaporator

at 30°C. The concentrate was dissolved in 4.0% citric acid in DW and in the solution

was used as indicator. 3) Anthocyanins were extracted from flowers with 4.0% tartaric

acid in methanol for 2-3 hours at room temperature in darkness. The mixture was

filtered in a Buchner funnel and the remaining solids were washed with 4.0% tartaric

acid in methanol until a clear solution was obtained. The combined filtrates were dried

using a rotary evaporator at 30°C. The concentrate was dissolved in 4.0% tartaric acid

in DW and in the solution was used as indicator.

Electronic Journal of Environmental, Agriculture and Food Chemistry, 2010,

added that anthocyanin changes color when the ph is 2-9 and the color will be from dark

pink to mehdi green. Phenolphthalein is an organic compound (C20H14O4) used as an

acid-base indicator.

Internet sources:

Anthocyanins are water soluble pigment that provides color to plants. The cyan

part of the name comes from the Greek word for blue, and cyan is also a

complementary color of red. Some good food sources of anthocyanins are eggplant,

checkberries, cherries, elderberries, red grapes, blueberries, oranges, red onion, red

wine, strawberries, radishes and purple cabbage (thirdplanetfood.com , r etrieved 2013).

Anthocyanins are contained in red, purple, and blue colored flowers, fruits,

leaves, and roots of higher plants. They mainly exist as glycosides in plants and their

aglycon. Anthocyanidin is a chromophore in pigments. It change thie color with pH like

litmus form flavylium ions strongly acidic solutions resulting in a very stable orange to

red. In weakly acidic or neutral solutions they first begin to form anhydrobases, so that

the color is reddish violet to violet. The blue they produce in alkaline media is the

predominance of anhydrobase anions. However, anhydrobases and anhydrobase

anions are unstable and are easily hydrated at the 2-position of the anthocyanidin

nucleus, resulting in a rapid change to the colorless pseudobase (crcnetbase.com,

retrieved 2011).

According to math.ufl.edu, retrieved 2010, Anthocyanins have played a

prominent role in the enrichment of human lives for thousands of years. Historians and

scientists believe that cave paintings from as far back as 15,000 B.C.E. were colored

using various plant pigments, and in Egypt and China, dyed fabrics have been found

and dated back to 2,000 B.C.E. The ancient Britons used a blue plant dye to color their

bodies in an attempt to frighten enemies in battle, and more recently in history, the

famous “red coats,” worn by British soldiers in the American Revolutionary War, were

dyed with a plant called ‘madder root’. Richard Martin Willstätter was the first scientist to

identify anthocyanins as the primary red/blue pigmentation in some plants and fruits. He

received the Nobel Prize in Chemistry in 1915 for his work with chlorophyll in connection

to anthocyanins and plant coloring. Specifically, he isolated the characteristic pigment in

cornflowers, roses, pelargonias, larkspurs, and hollyhock, and showed that

anthocyanidins attached to glucoses produced an anthocyanin. Willstätter also

explained how the same anthocyanin can have blue or red color properties, and

proposed that in roses the anthocyanin is bonded to a plant acid, which makes it red.

Conversely, he claimed, in cornflower, the anthocyanin is bonded to a plant alkali, which

is why it is distinctly blue.The word anthocyanin is derived from two Greek words,

anthos, which means ‘flower’, and kyáneos, which means ‘purple’. Nearly three

hundred different anthocyanins have been discovered, and different fruits and

vegetables have their own signature mix of pigments. Red wine, for example, contains

over fifteen different anthocyanin compounds, depending on the amount and type of

grapes with which it is made. The differing concentrations and types of compounds are

what give wine its different color shades. Anthocyanins are also thought to play an

important role in the high antioxidant levels in fruits and vegetables. Blueberries, for

example, contain a very high concentration of antioxidant compounds, which guard the

cell walls of the berry from harmful free radicals existing inside the plant. When people

ingest blueberries, they obtain the same protection from free radicals, which can be just

as harmful to cell membranes as cell walls. Blueberries, cranberries, and cherries can

contain up to 400mg of antioxidants per 100g of berry, and concord grapes—used in

many red wines—can contain up to 750mg per 100g of grape (Sriram, 2004). In the

twelfth century, bilberry (Vaccinium myrtillus) was used as an herbal medicine to induce

menstruation, and during World War II, British pilots took the same drug before

nighttime missions to enhance their night-vision. Now, researchers know that although

anthocyanins probably cannot increase nighttime awareness, nor encourage

menstruation, they can prevent oxidation damage in both large and small blood vessels

because of their anti-oxidant properties. Anthocyanins are also believed to inhibit

degenerative nerve damage, and in laboratory conditions, delphinidin and cyanidin

compounds have been found to inhibit the epidermal growth factor receptor in cancer

cells, which could potentially stunt the growth of tumor cells in humans. Also under

study are anthocyanins’ abilities to reduce LDL (the “bad”) cholesterol, and prevent

blood clotting.

In organic compounds, conjugated (alternating double) bonds primarily affect the

color the compound absorbs. In phenolphthalein, every carbon except for the central

carbon has overlapping p-orbitals, whichcreate pi bonds between these carbon atoms.

The light absorbed by this structure is actually in the ultraviolet range, reflecting in the

infrared range, which is why phenolphthalein in a pH below 8.2 appears clear. When

Phenolphthalein is in the presence of an alkali, the hydrogen atoms in phenolphthalein’s

Hydroxide ions are removed first. In a solution with a pH higher than 8.2, the structure

opens up, and the central carbon acquires a pi bond. Because electrons are less

confined in pi bonds than sigma bonds, the absorption for this molecule shifts

bathochromically to the blue-green range of the visible spectrum (redder than ultra-

violet), which makes the light it reflects pink. Anthocyanins, because of their hydroxide

groups, can act much the same way as phenolphthalein and other weak-acid indicators.

In an alkaline solution, H+ ions from the anthocyanin are removed by excess hydroxide

ions. This allows electrons in the anthocyanin to spread out in oxygen’s p-orbitals,

causing a hypsochromic shift, but also leaving a bonding site open. Metal ions such as

Mg2+ , Fe2+ , Fe3+, Ca2+,and Al3+ are known to bond with anthocyanin compounds,

and the addition of these metal ions could cause a change in color as well. Some of

these metals will chelate with multiple anthocyanins, which can produce a very different

color than is typically exhibited by the metal ion, or the anthocyanin itself. This also

emphasizes the idea that chelation requires a pH above the pKa of the phenolic group,

because the H+ ions need to be removed for the metal ion(s) to have an open bonding

site. Because the acidified anthocyanins are generally accepted as red in color,

deprotonated anthocyanins must be present either alone, or chelated with certain metal

ions to change the color.

Fecal Occult Blood

Books sources:

Strasinger & Di Lorenzo, 2008 mentioned that by far the most frequently

peroformed fecal analysis is the chemical screening test for the detection of occult

(hidden) blood. Since, any bleeding in excess of 2.5 ml/150 g of stool is considered

pathologically significant, and no visible signs of bleeding may be present with this

amount of blood, fecal occut blood testing (FOBT) is necessary. Originally used

primarily to test suspected cases of gastrointestinal diseases, FOBT has currently

becaome widely used as a mass screening procedure for the early detection of

colorectal cancer. Annual testing for occult blood has high positive predictive value for

detection of colorectal cancer in the early stages and is recommended by the American

Cancer Society, particularly for persons older than age 50. The most frequently

encounterd screening test for occult blood are based on detection of the

pseudoperoxidase activity of hemoglobin. This is the same principle as the reagent strip

test for urinary blood, but uses a different indicator chromogen. The reaction uses the

pseudoperoxidase activity of hemoglobin reacting with hydrogen peroxide to oxidize a

colorless compound to a colored compound.

Hemoglobin H2O2 (pseudo peroxidase) Guaiac Oxidized guaiac + H2O (blue color)

However, blood presumptive test can rule out the possibility if the fluid studied is

blood. These tests relies on the use of chemicals that will change color when in the

presence of blood like phenolphthalein which turn from colorless to pink when added to

feces (Lerner & Lerner, 2006).

Journals sources:

According to National Cancer Institute, fecalysis is a major test that is least

invasive and more affordable screening test for colorectal cancer. The presence of fecal

occult blood is what should be detected because presence of blood in stool may be the

only symptom of colorectal cancer (webMD.com, retrieved January 27, 2011). There is

now strong evidence that screening for colorectal cancer with fecal occult blood tests

(FOBTs) is effective in reducing the incidence and mortality of this disease (Department

of Medicine (Gastroenterology), 2002).

According to Higaki & Philip, 2001 it has been known for many years that fecal

occult blood test shows considerable peroxidise like enzymatic activity and therefore in

the presence of hydrogen peroxide will give highly coloured products with certain

substrates and in particular, with amines such as benzidine. On their study shows the

relative merits of using benzidine and phenolphthalein as reagents in indicator test for

blood centers primarily on the sensitivity, stability and specificity of two substrates.

Results indicate that the enzyme peroxidise, which is widely distributed in plants, does

not contribute to false positive results in the three-stage phenolphthalein indicator test

for blood stain. The phenolphthalein reagent was prepared. Serial dilutions of fresh

human, neat to 0.2 x 10-6 were prepared and tested and directly in solution form and in

stain form. The direct test in stain form consisted of application of reagents to a stained

thread on filter paper. The stains have been air-dried for 24 hours. Regardless of the

means of application, phenolphthalein compared quite favorable with benzidine for

detecting blood in solution. The peroxidase based test were sensitive to diltuons of 10 -4

to 10-5 approximately equivalent to benzidine, while tests using sodium perborate all

surpassed this sensitivity of benzidine. For detecting blood in stain form, the peroxide

based test had a maximum sensitivity of 10-3. The use of perborate did not measurably

increase this sensitivity when directly testing dried stains. Benzidine was100 times more

sensitive with an end point of 10-5.

In the article of Allison, 2007, it stated that fecal occult blood testing remains an

important screening option for colorectal cancer. Furthermore, it also states that it was

no surprise to the gastroenterology community that a one-time test—colonoscopy—

would be better than a one-time sigmoidoscopy or one-time FOBT, but the press and

public interpreted this information as “If a person is screened with a test other than

colonoscopy there is a good chance he or she will die from a missed colorectal cancer.”

The evidence suggests, however, that if the other available screening tests are

employed as recommended, the incremental benefit of colonoscopy in decreasing

patient mortality from colorectal cancer is small. The concern about missed “advanced

neoplasms” in once-only testing with methods other than colonoscopy may not be as

important as it has been portrayed. Annual FOBT testing and flexible sigmoidoscopy

every 5 years are the current recommendations, leaving the potential for discovery of a

missed advanced neoplasm on subsequent screens before it has become malignant or

lethal.

Paimela et al., 2010 stated that Faecal occult blood test (FOBT) screening has

been shown to decrease the incidence and mortality from colorectal cancer. This study

compared the stage profile of patients with colorectal cancer diagnosed at the first

FOBT screening round with that of an unscreened control group.

Internet sources:

Fecal occult blood indicates the blood found in the feces that is apparently not

visible with the naked eye. Fecal occult blood test is used to detect the hidden blood in

the stool and a positive result suggests a blood loss or bleeding in the gastrointestinal

tract. Blood may appear in the stool because of one or more conditions, such as benign

or malignant growths or polyps of the colon, hemorrhoids (swollen blood vessels near

the anus and lower rectum that can rupture causing bleeding), anal fissures, intestinal

infections that cause inflammation, ulcers, ulcerative colitis, Crohn’s disease,

diverticular disease caused by outpouchings of the colon wall, abnormalities of the

blood vessels in the large intestine, and Meckel’s diverticulum. (webmd.com, retrieved

02/10/13).

Detection of blood is not only done on the diagnosis of diseases but can also be

performed in solving crimes. One the classic tools in analyzing evidence at a crime

scene is the Kastle-Meyer test for the presence of blood. A spot that might be blood is

wiped with a cotton swab to collect some of the substance. A drop of phenolphthalein

reagent is added to the sample, then a drop of hydrogen is applied to the swab. If the

swab turns pink rapidly, it is said to test positive for blood. The test result is actually

presumptive positive, meaning it is not a conclusive test for blood, and other analyses

would typically be carried out to confirm the presence of blood. Fecal occult blood test

relies on the iron in the hemoglobin, which is the iron-containing portion of a red blood

cell, to promote the oxidation of phenolphthalein to phenolphthalein. Phenolphthalein is

colorless, but in the presence of blood and hydrogen peroxide, it changes to

phenolphthalein, which makes the solution pink (sciencebuddies.org, retrieved

2/8/2013).

Guaiac’s Test

Books sources:

According to Starsinger & Di Lorenzo, 2008 many commercial testing kits are

available for occult blood testing with guaiac reagent. The kits contain guaiac-

impregnated filter paper, to which the fecal specimen and hydrogen peroxide are added.

Two or three filter paper areas are provided for application of material taken from

different areas of stool, and positive and negative controls are also included. Obtaining

samples from the center of the stool avoids false-positive from external contamination.

Addition of hydrogen peroxide to the back of the filter paper that contains the stool

produces a blue color with guaiac reagent when pseudoperoxidase activity is present.

However, Turgeon, 2008 state that various interfering substances may give false

positive results. These are mostly enzyme peroxidase from hemoglobin and myoglobin

found in red meat, vegetables like horseradish, turnips and brocoli, and fruits like

bananas, black grapes, pears, plums and melons. In addition, WBCs and bateria also

have peroxidase activity.

According to Harborne, 2000 most test for occult blood in feces is gum guiac, a

phenolic compound that produces a blue color when oxidized. The test requires the

presence of hydrogen peroxide or a suitable precursor. The peroxidase activity of the

hemoglobin molecule results in the liberation of oxygen.

Journals sources:

According to Dr. Winchester and Wansbrough 2003, guiac’s tests rely on the fact

that heme can catalyze the breakdown of hydrogen peroxide. As the H2O2 breaks

down, another substance in the reaction mixture is oxidized, producing a color change.

It is important to note that a positive test does not mean that a given stain is blood, let

alone that it is human blood, as various enzymes and certain metals can also give

positive results.

However, heme is present in red meat and peroxidase activity is present in fresh

fruits and vegetables such as radishes, turnips and broccoli. These foods, therefore,

have the potential to produce false-positive results (Alliso, 2004).

In an article by Allison, 2007, it was mentioned that in 1985, Simon published an

excellent review of FOBT testing for colorectal cancer. He stated that the concept of

occult blood detection is generally credited to Van Deen, who in 1864 used gum guaiac,

as an indicator reagent. The guaiac FOBTs (GT) detects the peroxidase activity of

heme either as intact hemoglobin or free heme. In the presence of heme and a

developer (hydrogen peroxide) guaiac acid is oxidized producing a blue color. In

screening for colorectal neoplasms a true positive GT is one which indicates bleeding

from a colon cancer or polyp. All other positive results are considered to be false

positive. Heme is present in red meat and peroxidase activity is present in fresh fruits

and vegetables such as radishes, turnips and broccoli. These foods, therefore, have the

potential to produce false-positive results. Although there are several available GTs only

three, Hemoccult II, Hemoccult Sensa (Beckman Coulter Inc.; PrimaryCare Diagnostics,

Los Angeles, CA), and hema-screen (Immunostics, Ocean, NJ), have been extensively

evaluated in large screening populations. The Hemoccult test first became available

around 1970 and was in use until modifications in 1977 led to the Hemoccult II test.

Each Hemoccult II and Hemoccult Sensa slide has two windows of guaiac impregnated

paper, on which a small amount of stool is smeared. This is repeated with two

subsequent bowel movements. The three-slide package is then returned to the

laboratory or physician’s office for development. It is important to remember that

screening for colorectal cancer with FOBT should not be done with stool samples

obtained at a digital rectal examination (DRE). FOBT results of a single stool sample

obtained by DRE should be considered inadequate screening as there is a possibility of

an increased false positivity rate and a decreased sensitivity when compared to the

standard three specimen requirement (16,17).

Internet sources:

Guaiac Test is a test for blood in urine or feces using a reagent containing

guaiacum that yields a blue color when blood is present (http://en.wikipedia.org, ret.

2013). But the fecal occult blood test results are largely affected by how an individual

prepare oneself for the test. Do not perform the test if one has diarrhea, colitis,

constipation, diverticulitis, ulcers, hemorrhoids, flare-ups, and menstrual period.

Because certain foods can alter the test results, a special diet is often recommended for

48 to 72 hours before the test. The following foods should not be eaten 48 to 72 hours

before taking the test: Beets, Broccoli, Cantaloupe, Carrots, Cauliflower, Cucumbers,

Grapefruit, Horseradish, Mushrooms, Radishes, Red meat (especially meat that is

cooked rare), Turnips, and Vitamin C-enriched foods or beverages

(http://www.webmd.com/colorectal-cancer/guide/fecal-occult-blood-test retrieved:

2/8/13).

Colorectal Cancer

Book source:

Colorectal cancer (CRC) is the second leading cause of cancer death among

men and women combined in the United States and is the third most common cause of

cancer, separately, in men and in women. The lifetime risk if developing CRC is 1:18.

Surgery will cure almost 50% of all diagnosed patients, although almost 80,000 people

develop metastatic CRC each year. The incidence of colon cancer is higher in the more

economically developed regions, such as the United States or Western Europe, than in

Asia, Africa, or South America. Although certain conditions predispose patients to

develop colon cancer, up to 70% of patients have no identifiable risk factors. Such are

age, ethnicity, personal history and tobacco use. Obesity also is a risk factor

considering dietary factors, calcium deficiency, micronutrient deficiency and such. The

pathophysiology of the disease reveals that more than 90% of CRC is adenocarcinoma.

Colon carcinogenesis involves progression from hyperproliferative mucosa to polyp

formation, with dysplasia, and transformation to non-invasive lesions and subsequent

tumor cells, with invasive and metastaic capabilities. The screening tests for early

detection of colorectal cancer have been developed by several professional societies.

Any positive or abnormal screening test should be followed up with colonoscopy

( Abraham, 2010)

According to McPherson & Pincus, 2011 the idea is that if blood is discovered in

the stool, there is the chance that a colonic tumor is the cause, and further workup, such

as colonoscopy, is therefore strongly recommended.

Journal source:

According to World Health Organization, 2008 statistics, there are many factors

contributing to colorectal cancer, the main only are tobacco use, alcohol use, unhealthy

diet and physical inactivity.

Internet source:

A stool test is one of many tests used to look for colorectal cancer. These tests

may find cancer early, when treatment works better. Colorectal cancer affects the large

intestine (colon ) and the rectum. There are three kinds of stool tests: Fecal

occult blood test (FOBT). Fecal immunochemical test (FIT). Stool DNA test

(sDNA). Blood in the stool may be the only symptom of colorectal cancer, but not all

blood in the stool is caused by cancer. Other conditions that can cause blood in the

stool include: 1) Hemorrhoids. These are enlarged, swollen veins in

the anus. Hemorrhoids can form inside the anus (internal hemorrhoids) or outside of the

anus (external hemorrhoids). 2) Anal fissures. These are thin tears in the tissue that

lines the anus (anal sphincter) up into the anal canal. 3) Colon polyps. These growths of

tissue are attached to the colon and often look like a stem or stalk with a round top. 4)

Peptic ulcers. These sores form when the digestive juices made in the stomacheat

away at the lining of the digestive tract. 5) Ulcerative colitis. This type of inflammatory

bowel disease (IBD) causes inflammation and sores (ulcers) in the inner lining of the

colon and rectum. 6) Gastroesophageal reflux disease (GERD). This is the abnormal

backflow (reflux) of food, stomach acid, and other digestive juices into the esophagus.

7) Crohn's disease. This type of inflammatory bowel disease causes inflammation and

ulcers that may affect the deep layers of the lining of the digestive tract. 8) Use

of aspirin or nonsteroidal anti-inflammatory drugs (NSAIDs). Stool tests may be used to

check for colorectal cancer, but they are never used to diagnose it. Other tests for

colorectal cancer include flexible sigmoidoscopy, colonoscopy, and CT

scan (virtual colonoscopy) (http://www.webmd.com/colorectal-cancer/fecal-occult-blood-

test-fobt retrieved: 2/9/13).

Peroxidase and Hydrogen peroxide

Books sources

Peroxidases are enzymes that belong to class I of enzymes—the

oxidoreductases. These enzymes catalyze oxidation-reduction reactions. Oxidation

means the loss of electrons, and reduction means the addition of electrons. Many

different electron acceptors are used in biological systems. Similar mechanism is seen

in the cell’s peroxisomes which have catalase, a heme enzyme, that catalyzes the

conversion of hydrogen peroxide to water and oxygen (Delvin, 2011).

As defined by Funk and Wagnalls New World Encyclopedia (2002), hydrogen

peroxide is a chemical compound of hydrogen and oxygen with the formula H2O2. Pure,

anhydrous hydrogen peroxide is a colorless, syrupy liquid with a sp.gr. of 1.44. It blisters

the skin and has a metallic taste. The liquid solidifies at –0.41° C (31.4° F).

Concentrated solutions are unstable, and the pure liquid may explode violently if heated

to a temperature above 100° C (302.4° F). It is soluble in water in all proportions, and

the usual commercial forms are a 3% and a 30% aqueous solution. To retard the

decomposition of the peroxide into water and oxygen, organic substances, such as

acetanilide, are added to the solutions, and they are kept in dark bottles at low

temperature. Hydrogen peroxide is manufactured in large amounts by the electrolysis of

aqueous solutions of sulfuric acid or of potassium bisulfate or ammonium bisulfate. It is

also prepared by the action of acid on other peroxides, such as those of sodium and

barium. Hydrogen peroxide acts as both an oxidizing and a reducing agent. Its oxidizing

properties are used in the bleaching of substances, such as hair, ivory, feathers, and

delicate fabrics, which would be destroyed by other agents. It is also used medicinally,

in the form of a 3% aqueous solution, as an antiseptic and throat wash. Hydrogen

peroxide is used in restoring the original colors to paintings that have darkened through

the conversion of the white lead used in the paintings to lead sulfide. The hydrogen

peroxide oxidizes the black lead sulfide to white lead sulfate. It is also used as a source

of oxygen in the fuel mixture for many rockets and torpedoes. As a reducing agent it

reacts only with such easily reduced chemicals as silver oxide and potassium

permanganate.

Journal source

In an article entitled, “The Decomposition of Hydrogen Peroxide by Blood.

George Senter’s Discovery of the Enzyme Involved” (Stock & Stuart, 2005), various

experiments were done to determine the enzyme that catalyze the oxidation of

hydrogen peroxide. Enzymes in substances that caused the decomposition of hydrogen

peroxide were provisionally termed superoxidases. Preliminary experiments on the rate

of decomposition, carried out with diluted blood, implied that the rate of reaction was of

first order with respect to the concentration of hydrogen peroxide. The research was

conducted by George Senter by the 1900s. Senter concluded that, in dilute solutions,

the rate of decomposition of hydrogen peroxide was proportional to the product of the

respective concentrations of peroxide and of Hämase (now termed catalase) (Stock &

Stuart, 2005).

Internet Source

The enzyme catalase in blood speeds up the decomposition of hydrogen

peroxide. Catalase is very efficient at decomposing hydrogen peroxide; one molecule of

the enzyme can catalyse the conversion of over 6000,000 hydrogen peroxide molecules

into water and oxygen every second. The enzyme occurs widely in tissues such as the

liver and prevents accumulation of, and tissue damage by, hydrogen peroxide that is

produced during metabolism. Catalase found in human red blood cells is a complicated

chemical consisting of four polypeptide chains with 500 amino acids in each chain. Each

peptide chain includes a porphyrin haem group. These are the active components which

allow the enzyme to catalyze the decomposition of hydrogen peroxide. Hydrogen

peroxide is a powerful oxidising agent but is unusual in that it can act as reducing agent

under certain conditions. The oxidation number of oxygen in hydrogen peroxide is -1,

intermediate between 0 in oxygen and -2 in water, and this allows the oxygen to act as

both a reductant and oxidant in either acid (H2O2) or alkali (HO2-) solution.

(www.eic.com, ret, February 2013)