*3.1.1.2 Clinical indicators*

 Signs of chronic, long-standing VAD of xerophthalmia are conjunctival xerosis (X1A) and Bitot's spots with conjunctival xerosis (X1B). In general, a very bright torchlight in natural light is used to examine the eyes [29]. Conjunctival xerosis or drying can occur in both eyes where eyes turn dry and non-wettable with wrinkle presence at the temporal conjunctiva [31, 32]. Bitot's spots are the accumulation of fine white foamy cheesy material comprising keratin, on the conjunctival surface [28].

 Signs of acute, sudden onset of VAD are corneal xerosis (X2), corneal ulceration with xerosis (X3A), keratomalacia (X3B) and xerophthalmia fundus (XF). Corneal xerosis (X2) is drying of the cornea due to the lack of mucus and tears (wetting agent) because glands in the conjunctiva are no more functioning normally [33]. Lesions on the cornea become denser, and stromal oedema starts to develop during corneal xerosis. The cornea appears to be granular, rough and blurry when examined using a hand light [32]. At this stage, treatment with vitamin A will heal the eyes within 1 to 2 weeks without leaving any scars. Corneal ulceration with xerosis (X3A) is permanent destruction of all or some parts of the corneal stroma which are prominent. Ulcers may be shallow but usually become deep if it penetrates into the cornea. Vitamin A therapy can cure superficial ulcer, leaving small scars, while deeper ulcers and perforations form dense scars [28].

 Keratomalacia (X3B) means softening of the cornea, and it is a rare stage of xerophthalmia. The cornea may become thickened and melt away due to a progression of necrosis or death of tissue, affecting the collagen in the cornea [32]. Blindness is usually inevitable, although other eyes and the lives of children can be instantly saved by vitamin A therapy. Keratomalacia is also usually associated with secondary eye infections but can be treated with an antibiotic [28]. Xerophthalmia fundus (XF) is the appearance of small yellowish lesions on the fundus of the eye, which occurs due to the loss of pigment from the retinal pigment epithelium caused by VAD. The lesions are sometimes accompanied by blind spots or scotomas, congruent with their distribution on the retina [34]. The healing or end result of corneal ulceration and keratomalacia is corneal scars (XS). Scars are left on the cornea with varying densities, known as staphyloma (permanent bulging of the damaged cornea) or phthisis bulbi (shrunken globe), whereby the contents of the intraocular are gone and can lead to blindness [28].

#### *3.1.1.3 Histological indicators*

The morphological changes of epithelial cells from the conjunctiva surface can be assessed using a piece of filter paper. Normal conjunctiva cells show an abundance of mucin-secreting goblet cells and small epithelial cells. However, if there is a deficiency in vitamin A, the goblet cells and mucin droplets will reduce, and the epithelial cells become enlarged, separated and flattened [29]. Histological indicators include conjunctival impression cytology (CIC) and impression cytology with transfer (ICT). Assessing VAD using both techniques requires standard pore size filter paper, slides and a simple light microscope. The method involves gently applying a filter paper on the surface of the conjunctiva for 2–3 seconds, and after removal, it is placed in fixative and stained to differentiate the goblet cells from the endothelial cells. The eye is classified as normal or abnormal based on the number of goblet cells, which is counted under a microscope [35]. The differences between the two techniques are ICT only require single staining while CIC include extra processing steps for fixing, staining and mounting specimens. Comparatively, the CIC technique is more efficient in transferring cells of high quality from filter paper to slide [29].

## *3.1.2 Biochemical indicators*

Biochemical indicators include serum and breast milk retinol concentrations, relative dose response (RDR) test, modified relative dose response (MRDR) test and isotope dilution (ID) assay.

### *3.1.2.1 Serum retinol concentrations*

 Serum retinol concentrations are among the most common method used to identify populations at risk of VAD [36]. They are determined using highperformance liquid chromatography (HPLC). The current cut-offs for VAD are <0.70 μmol/L, while severe VAD is classified below 0.35 μmol/L [21]. However, this indicator is affected by infections [37], inflammation and inadequate intakes of protein, zinc or energy, which are needed for retinol-binding synthesis [38]. Therefore, before using serum retinol concentration to assess VAD in a population, these factors should also be taken into consideration. In addition, serum retinol concentrations are homeostatically controlled over a broad range of body store and only decline when the liver reserves are very low [39]. Serum retinol concentrations should be used in conjunction with another biological indicator or when four or more of the following risk factors are detected in the population being assessed [40]. These risk factors include:


#### *3.1.2.2 Breast milk retinol concentrations*

 Breast milk retinol concentration is a unique indicator in lactating women. It has also been proposed as a measure of the population status of vitamin A, since the probability of infant and children at risk of VAD is very high if the lactating women are of a community with marginal vitamin A status [41]. Vitamin A deficiency is considered a moderate public health problem if the prevalence of inadequate milk retinol concentrations (≤1.05 mmol/L or ≤8 mg/g milk fat) is ≥10–<25% [29]. The breast milk samples are easier to obtain, and the concentration of retinol in milk can be determined after saponification by HPLC, similar to those used to determine serum retinol [42].

### *3.1.2.3 Relative dose response test (RDR)*

 The test principle of the RDR is on the basis that when vitamin A undergoes depletion, apo-retinol-binding protein (apo-RBP) accumulates in the liver. In this test, a challenge dose of retinyl ester is given to the subject, and blood samples are withdrawn prior to dosing (baseline) and 5 hours after dosing. The retinol from retinyl ester will bind to the excess RBP and is released into serum as holo-retinol/retinol-binding protein complex (holo-RBP-retinol complex). A percentage change is measured as per Eq. 1 where RDR ≥ 20% indicates VAD [35].

$$RDR\,\{\text{\(\%\)}\,\,=\frac{[A5]-[A0]}{[A5]}\times\text{100}\,\tag{1}$$

where, [A5] is the serum retinol concentration at 5-hr post-dosing; and [A0] is the serum retinol concentration just before dosing (baseline).

## *3.1.2.4 Modified relative dose response test (MRDR)*

 MRDR is a modified test of RDR using 3, 4-didehydroretinyl acetate (DRA) as the challenge dose, followed by a high-fat snack to ensure adequate absorption. In this method, a single blood sample is taken after 4 to 7 hours dosing [43]. In parallel to retinyl esters, DRA is hydrolysed to 3, 4-didehydroretinol (DR) within small intestine, taken up by enterocytes and esterified to form various didehydroretinyl esters. The esters are de-esterified to form DR in the liver, which can bind to apo-RBP and be released into serum or can be re-esterified and stored in stellate cells. The only difference between DR and retinol is the presence of a double bond located in the 3–4 position on β-ionone ring of DR. This structural difference can be separated using HPLC due to their difference in polarity. The MRDR value, which is used to indicate liver reserves, is the ratio of DR to retinol in serum [27]. The ratio of 3, 4-didehydroretinol (DR) to retinol is calculated, and the value of ≥0.06 indicates VAD in children [44]. The MRDR test has been widely used to diagnose a subclinical vitamin A status.

#### *3.1.2.5 Isotope dilution (ID) assay*

 Of all the indicators available, the most accurate method to indirectly measure the vitamin A storage in the liver known till now is the isotope dilution assay [45–47]. Isotope dilution assay could detect a full range of vitamin A content in the body from deficient state up to the toxic level [48]. This test involves blood sample collection before and after the administration of a stable isotope tracer (deuterated or 13C-labelled retinyl acetate) at an appropriate equilibration period. The variations in the equation and assumptions used in the calculation are dependent on the study design based on the population assessed. The method of mass spectrometry used, the dosage size given to the subjects and the time allowed for equilibration were also taken into consideration when calculating the total body reserve in the ID test [41]. The ID assay is determined as shown in Eq. 2.

$$\mathbf{(F\_a \times a) + (F\_b \times b) = (F\_c \times c)}\tag{2}$$

where:

a is the amount of dose absorbed and stored (dose × absorption rate). b is the baseline total body reserves of vitamin A.

c is the total body reserve in μmol after the dose (c = a + b).

$$\mathbf{F} = \frac{\mathbf{R}}{\mathbf{R} \star \mathbf{1}} \quad \text{and} \, \mathbf{R} \, \mathbf{i} \, \mathbf{s}^{13} \mathbf{C} / {}^{12} \mathbf{C} \tag{3}$$

where Fa, Fb and Fc are the abundance of isotopes [13C/total C; At %/100; R/(R + 1)] from dose, baseline serum and serum after the dose.

## **4. Effects of vitamin A deficiency or toxicity on human health**

 Routine monitoring of vitamin A status serves as an important measure in the determination of toxicity due to excessive intake or deficiency in a population. Under circumstances where dietary consumption does not meet the recommended criteria, this could lead to vitamin deficiency or toxicity depending on whether the vitamin consumption is insufficient or in excess, respectively. Various health implications have been reported as a consequence of both vitamin deficiency and excess, as discussed below.

#### **4.1 Vitamin A deficiency (VAD)**

 Dietary source of vitamin A is generally available in various forms, of which the preformed retinol from animal-based source (eggs, liver, dairy) is the most bioavailable form of vitamin A. Plant-based food sources are rich in pro-vitamin A; however, populations that are dependent solely on these sources are at higher risk of VAD since its absorption is reliant on various factors [49, 50]. VAD is commonly associated with decreased immunity and higher risk of night blindness [51]. It is worthwhile to note that this deficiency is highly prevalent in countries with an alarming increase of diabetes especially among those of lower income group in United States as well as Asian developing countries [51, 52].

 Vulnerability to VAD differs according to specific life stages that include infancy, childhood and pregnancy. VAD in neonates is highly related to insufficient vitamin A in breast milk or formula milk. Apart from dietary shortage, VAD could have also been triggered by reduced intestinal absorption of vitamin A. Prolonged deprivation of body requirements for vitamin A leads to vitamin A deficiency disorders (VADDs) that affects gastrointestinal, renal, musco-skeletal organ systems as well as harming growth and development [53]. Xerophthalmia and anaemia are two most common examples of VADDs. In line with vitamin A roles as immunity enhancer, its deficiency is often associated with an increased risk of infections [54, 55]. Respiratory tract infections and diarrhoeal diseases are the most common form of infections with high incidence of mortality along with marked susceptibility to severe measles infection [55–57]. The representation of VADD association to risk of mortality is presented in **Figure 2** below.

#### *4.1.1 Vitamin A deficiency and xerophthalmia*

 Xerophthalmia refers to a spectrum of ocular manifestations due to VAD and varies according to its severity and age. It is characterised by pathological dryness of the conjunctiva and cornea that turns out as a leading cause of childhood corneal blindness, especially in nutritionally deprived populations [58]. All of such signs encompass those involving impaired retinal sensitivity to light (night blindness) and epithelial disruptions of the cornea and conjunctiva (conjunctival xerosis, Bitot's spot, corneal xerosis and keratomalacia) [59, 60]. The classifications of xerophthalmia stages in order of severity based on WHO criteria are shown in **Table 2** (Section 3.1.1).

#### **Figure 2.**

*Representation of VADDs in relation to risk of mortality (adapted from [53]).* 

 Xerophthalmia can occur in any age group with higher possibilities in preschoolage children, adolescents and pregnant women. In line with greater requirements for growth, children are more prone to VAD and xerophthalmia [61]. The initial symptoms of VAD are characterised by impaired adaptation to dark, which starts when the serum retinol concentration falls below 1.0 μmol/L and becomes more often when it falls lower than 0.7 μmol/L. A further drop in serum retinol concentration level below 0.35 μmol/L leads to more frequent and severe xerophthalmia condition [62, 63]. The incidence of xerophthalmia is often associated with higher risk of mortality [57].

 Night blindness is generally the earliest manifestation, and it is indicated by vision limitation under dim light and is both a sensitive and specific indicators for low serum retinol levels [63, 64]. Vitamin A in the form of retinal within the eyes combines with opsin to form rhodopsin, which is the photosensitive visual pigment of rods. Rhodopsin level decreases when vitamin A is deficient, and this impairs the rod function causing night blindness [61]. Bitot's spot is the representation of opaque whitish deposits on the scleral conjunctiva, which is the most characteristic sign of problems related to VAD. Conjunctival xerosis is already present at this stage, with the conjunctiva appearing dry and dull. Under conditions where VAD persists, corneal xerosis (hazy cornea) occurs, followed by keratomalacia (liquefaction of part or all cornea) [61].

Several risk factors have been associated with onset of VAD and xerophthalmia based on epidemiological findings. These include demographic, geographic, childhood, parents and household factors. The mechanism of these factor effects on the prevalence of xerophthalmia is summarised in **Table 3**.

#### **4.2 Vitamin A toxicity**

On another note, the increase in supply and consumption of fortified foods and supplements led to intake of preformed vitamin A at higher than the recommended level [75]. The side effects of vitamin A excess could occur in two forms, known as hypervitaminosis A and hypercarotenemia [76].

#### *4.2.1 Hypervitaminosis A*

Hypervitaminosis A can occur due to both acute and chronic intoxications that generally result from excessive intake of vitamin A from nutritional supplements


#### **Table 3.**

*Risk factors associated with prevalence of VAD and xerophthalmia.* 

and foods rich in vitamin A [76]. Acute toxicity occurs when adults and children ingest more than their respective recommended dietary allowance within few hours or days, while chronic toxicity results from prolonged consumption of preformed vitamin A over the months or years. However, acute conditions create minimal consequences to human health compared to those under chronic toxicity [77, 78]. Vitamin A, being an essential fat-soluble micronutrient, is quickly absorbed upon ingestion, although it is only cleared slowly from the body. Under such conditions, toxicity could arise either from high-dose exposure or low intake over short or prolonged duration, respectively [79]. Chronic hypervitaminosis A leads to various clinical manifestations that include xerosis, epistaxis, alopecia, weakness and fatigue, bone and joint pain, insomnia, drowsiness, anorexia, bulging fontanelle in infants as well as psychiatric symptoms [76].

Previous research findings have shown that elevated serum concentrations of vitamin A is highly associated with risk of hip fracture [80, 81]. This association is supported by evidence of rat-based experimental studies that demonstrated excessive intake of vitamin A leads to increased bone resorption and less formation at the outer surface that results in bone narrowing [82, 83]. In contrast, the mechanism takes place in opposing effect on the bone marrow surface, where an increase in vitamin A intake reduces bone resorption while increasing its formation. This contradictory effect takes place by the action of vitamin A or its metabolites on osteoblasts at the outer surface together with indirect effect on bone marrow surface [83, 84].

#### *4.2.2 Hypercarotenemia*

Hypercarotenemia, which is also referred to as carotenemia or carotenodermia, is a benign phenomenon characterised by pigmentation of the skin. The yelloworange pigmentation is a result of carotene deposition at the stratum corneum, which is the outermost layer of epidermis [76]. Hyperlipidaemia, consumption of excessive carotene or failure of converting carotenes into vitamin A are conditions that lead to the onset of carotenemia. In view that there is a direct relationship between β-carotene and β-lipoprotein, other medical conditions that are associated with hyperlipidaemia also could lead to this pigmentation. Those conditions include diabetes mellitus, nephrotic syndrome and hypothyroidism. Apart from these, patients suffering from liver disease are also at higher risk of carotenemia due to the

#### *Vitamin A in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.84460*

 impaired conversion of β-carotene into vitamin A [76]. In contrast to hypervitaminosis A, there are no clear indications of carotenemia to health, and the pigmentation could disappear within weeks to months along with a steady decrease in β-carotene concentration [76, 85].
