**Abstract**

Folic acid is a B complex water-soluble vitamin that is essential to humans, and its deficiency can cause problems including neural tube defects as well as heart-related diseases. An important feature of such vitamins is that they are generally not synthesized by mammalian cells and therefore must be supplied in sufficient amounts in the diet. Folate is a generic term for compounds, possessing vitamin activity similar to that of pteroylglutamic acid, and is the form of the vitamin, which is naturally present in foods. The main dietary sources of folic acid are dark green and leafy vegetables such as spinach, asparagus, romaine lettuce, broccoli, bok choy, turnip green, beet, dried or fresh beans, and peas. The amount of folate that is absorbed and utilized physiologically varies among different food sources and different chemical forms of the vitamin. About 85% of folic acid is estimated to be bioavailable; however, the bioavailability of food folate is estimated at about 50% of folic acid. Several national health authorities have introduced mandatory food fortification with synthetic folic acid, which is considered a convenient fortificant, being cost efficient in production, more stable than natural food folate, and superior in terms of bioavailability and bio-efficacy. Presently, many countries affected by diseases associated with a lack of folic acid have made it mandatory to supplement foods with the vitamin. Considering the need, several analytical procedures were standardized to determine the presence of folic acid in different food matrices. The reported methods are simple, selective, robust, and reproducible and can be used in routine analyses.

**Keywords:** folic acid, vitamin B9, green leafy vegetables, bio-fortification, bio-availabilty

#### **1. Introduction**

Vitamin B9 also called folate or folic acid (FA) or pteroyl-L-glutamic acid is one of the eight water-soluble B vitamins. The chemical name of folic acid is *N*-[4-[[(2 amino-3,4-dihydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-l-glutamic acid. The chemical formula of folic acid is C19H19N7O6 and its molecular weight is 441.4. The IUPAC name is (2*S*)-2-[[4-[(2-amino-4-oxo-3*H*-pteridin-6-yl) methyl amino] benzoyl] amino] pentanedioic acid. Folic acid appears as odorless orange-yellow needles. Folic acid is a synthetic form of folate, found in vitamin supplements and fortified foods. Its structure has been shown in **Figure 1**.

#### **Figure 1.** *Chemical structure of the folic acid molecule.*

Folate, vitamin B12, and riboflavin have attracted scientific as well as health interest in recent years. Folate has a well-established role in preventing neural tube defects (NTDs); however, there are several other reports highlighting the potential role of folate and B-vitamins in protecting against several lifestyle diseases including heartrelated cardiovascular diseases (especially strokes), certain types of cancers, cognitive impairment, and bone-related osteoporosis. Folic acid is involved in carbon transfer reactions of amino acid metabolism, in addition to purine and pyrimidine synthesis, and is essential for hematopoiesis and red blood cell production. It is found in many foods and particularly in leafy green vegetables that are essential for the critical biosynthetic pathways involving the transfer of methyl groups to organic compounds. Folate is important for a range of functions in the body. Folic acid (vitamin B9) works with vitamin B12 and vitamin C to help the body break down, use, and make new proteins. Folate is required in the synthesis of nucleic acids *viz*., DNA and RNA and is also part of the protein metabolism. It helps in the degradation of homocysteine, which is a risk factor for heart disease. Folic acid is required for growth, reproduction (during gestation and lactation), and antibody formation. As a coenzyme, it is involved in glycine metabolism and is essential for the synthesis of purines, as well as pyrimidines. It plays a major role in cell division and protein synthesis. Its deficiency induces chromosomal abnormalities [1]. It is essential for the formation of RBCs and prevention of folate deficiency anemia [2]. Folic acid deficiency can lead to congenital malformations in the fetus (spina bifida, encephalocele, cleft palate, and hydrocephalus), as well as heart disease [3–5]. Deficiency of folate leads to megaloblastic anemia (a condition where there is a reduction in RBCs, and the red blood cells are larger in size than normal). Other symptoms include weakness, fatigue; irregular heartbeat, shortness of breath, hair loss, pale skin, mouth sores, etc. The nutrient is very important during early pregnancy to reduce the risk of birth defects of the brain and spine [1]. An important feature of this vitamin is that they are generally not synthesized by mammalian cells and therefore must be supplied in sufficient amounts in the diet [6, 7].

#### **2. Recommended dietary allowance (RDA)**

The RDA for males and females aged 15 years and older is 400 μg DFE day−1 (**Table 1**), whereas the RDA ranges from 65 to 300 μg DFE day−1 for ages between *Vitamin B9 in Dark Green Vegetables: Deficiency Disorders, Bio-Availability, and Fortification… DOI: http://dx.doi.org/10.5772/intechopen.100318*


#### **Table 1.**

*Recommended dietary allowances (RDA) for folate.*

0 and 14 years. The requirement of folate for pregnant and lactating women is comparatively higher, respectively, 600 and 500 μg DFE day−1. The upper tolerable limit (UL) for folic acid has been reported as 1000 μg day−1. Men and women of age 19 years and older should aim for 400 μg DFE. Those who are breastfeeding should aim to take around 500 μg per day. People who regularly drink alcohol should aim for at least 600 mcg DFE of folate daily since alcohol can impair its absorption. Higher daily doses (up to 4 mg) are recommended for women who have had a baby with a neural tube defect. Folate is essentially nontoxic, although there is some concern that high doses may mask pernicious anemia. The body absorbs folic acid from supplements and fortified foods better than the folate from naturally occurring foods.

#### **3. Common symptoms of folate deficiency**

A diet lacking folate or folic acid can lead to a folate deficiency. Inadequate levels of folate (vitamin B9) and vitamin B12 during pregnancy have been reported to lead to an increased risk of neural tube defects (NTDs) [9]. Although both are part of the same biopathway, folate deficiency is much more common and therefore it is of much concern [10]. NTDs are birth defects of the brain, spine, or spinal cord and Spina bifida as well as anencephaly are the most common ones. The spinal column of the fetus does not close completely in spina bifida, whereas in anencephaly, most of the brain and skull do not develop properly due to which babies with anencephaly are either stillborn or may die shortly after birth. The peri-conceptional folate supplementation can reduce the risk of neural tube defects (NTDs) and other congenital abnormalities such as cardiovascular malformations (CVMs), cleft lip and palate [4], urogenital abnormalities, and limb reductions [11]. Supplementation with folic acid reduces the prevalence of NTDs by approximately 70% indicating that 30% of these defects are not folate-dependent and are due to some other reasons, rather than alterations of methylation patterns. Many other genes related to NTDs exist, which may be responsible for folate insensitive NTDs. However, folate deficiency can also occur in people who is suffering from celiac disease that prevents the small intestine from absorbing nutrients from foods (malabsorption syndromes). Deficiency of folic acid can cause a wide range of problems in the human body, which may include tiredness, fatigue, and lethargy, besides muscle weakness and other neurological signs, such as tingling, burning, or peripheral neuropathy leading to numbness. It may also cause psychological problems, such as depression and memory problems, and gastrointestinal symptoms, such as nausea,

vomiting, abdominal pain, weight loss and diarrhea, headache and dizziness, and shortness of breath. Anemia, particularly megaloblastic anemia, is often the first sign that there is an underlying folate deficiency, and doctors will usually test for folate deficiencies when they encounter anemia. In pernicious anemia, our immune system attacks healthy cells in our stomach, which prevents the absorption of vitamin B12 from the food we eat, and this is the most common cause of vitamin B12 deficiency. Other factors include the lack of these vitamins in our diet. Besides this, certain medicines, including anticonvulsants and proton pump inhibitors (PPIs), can affect how much of these vitamins our body absorbs both. Vitamin B12 deficiency and folate deficiency are more common in older people. The folates are hydrolyzed to monoglutamate in the gut before absorption by active transport across the intestinal mucosa. Sometimes, passive diffusion also occurs when pharmacological doses of folic acid are consumed. Before it enters the bloodstream, the enzyme dihydrofolate reductase reduces the monoglutamate to tetrahydrofolate [12]. The major folate in plasma is 5-methyl-THF. The activity of dihydrofolate reductase varies among individuals [13]. It is yet not known whether the unmetabolized folic acid has any biological activity or it can be used as a biomarker of folate status [14]. Folate is also synthesized by colonic microbiota and can be absorbed across the colon, although the extent to which colonic folate contributes to folate status is unclear [15]. The folate content of the body is estimated to be around 15–30 mg. Half of this amount is stored in the liver and the rest amount is found in blood and body tissues [16]. Normally, the serum folate concentration is used to assess the folate status of the body. A value higher than 3 ng/mL indicates adequacy [17]. The erythrocyte folate concentration provides a longer-term measure of folate intake; a concentration above 140 ng/mL indicates adequate folate status [13, 17]. A combination of serum or erythrocyte folate concentration can also be utilized to assess folate status. Sometimes plasma homocysteine concentration is also used as a functional indicator of folate status because homocysteine levels rise when the body is unable to convert homocysteine to methionine due to deficiency of 5-methyl tetra hydrafolate [17]. Homocysteine levels, however, are not a highly specific indicator of folate status because they can be influenced by other factors, including kidney

**Figure 2.** *Neural tube defect—Spina bifida.*

*Vitamin B9 in Dark Green Vegetables: Deficiency Disorders, Bio-Availability, and Fortification… DOI: http://dx.doi.org/10.5772/intechopen.100318*

dysfunction and deficiencies of vitamin B12 and other micronutrients [17, 18]. The most commonly used cutoff value for elevated homocysteine levels is 16 μmol/L. A homocysteine cutoff of 10 μmol/L has been proposed for assessing folate status in populations (**Figure 2**) [13].
