Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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A variety of spectrophotometric methods are also

available to determine protein content. These include

the biuret method (based on the binding of copper (II)

to a peptide bond in protein molecules at alkaline pH

values), the Lowry method (based on the reduction of

Folin–Ciocalteau reagent by oxidation of tyrosine

and tryptophan on the polypeptide side chain), and

the dye binding method (based on the measurement

of excess dye remaining in solution after removal

of the precipitated protein–dye complex). Most pro-

teins exhibit an absorption maximum at 280 nm,

because of the presence of a side chain of aromatic

amino acids, and so UV-spectrophotometry is also

used for protein identification and quantification. IR

spectrophotometry has become a popular method for

the determination of protein in cereal products,

because of its speed, simplicity of operation, safety,

and low running costs. Specific protein components

in food and food products can be quantified by chro-

matography, electrophoresis, immunology, or a

combination of these. Among the methods of chro-

matography, HPLC is becoming increasing important

in the analysis of specific protein components in foods

and food products. The separation of proteins in an

electric field on the basis of their charge density is a

powerful and frequently used technique for the analy-

sis of specific protein components in food. Methods

such as immunoelectrodiffusion and enzyme-linked

immunosorbent assay are immunological methods

(based on the interaction between an antigen and its

corresponding antibody) that are highly specific and

sensitive means to identify and quantify minor pro-

tein components in complex matrices. To improve

and optimize the accuracy of the protein quantifica-

tion in food and food products, further research for

new and alternative absolute protein quantification

methods is necessary. Amino acids are the building

blocks of proteins, and these can be determined

after hydrolysis of proteins by colorimetric, enzym-

atic, and chromatographic (ion-exchange and gas-

chromatography (GC)) methods.

0056 Lipids Most of the methods used in food lipid an-

alysis cannot be directly applied to foods themselves

because of their complexity. Lipids should first be

extracted either for total lipid estimation or for fur-

ther analysis. Neutral lipids (triacylglycerols) can be

easily extracted by nonpolar solvents such as petrol-

eum ether, hexane, or supercritical carbon dioxide. If

a sample contains phospho- or glycolipids, polar solv-

ents such as methanol must be used for quantitative

determination. Standard methods are used to deter-

mine the oil content (AOAC or AOCS) by refluxing

the sample in petroleum ether using Soxhlet appar-

atus. There are a number of procedures for oil

estimation in which a sample is not destroyed, such

as low-resolution NMR or high-resolution NMR.

Methods have been developed to separate different

classes of lipids such as neutral lipids, glycolipids and

phospholipids. The techniques commonly used are

TLC, column chromatography, and HPLC, especially

with mass detection.

0057General information on the constituent fatty acids

of fats can be obtained from different chemical ana-

lyses giving specific values such as the saponification

value, iodine value, hydroxyl value, etc. The presence

of free fatty acids in oil, which is an index of rancidity,

can be determined by titrating against a standard

alkali. Specific fatty acids in fat can be determined

by gas–liquid chromatography (GLC), which is still

the most informative technique in fatty acid analysis.

Mass spectrometry coupled to GC is the most power-

ful tool for identifying fatty acids separated by GLC.

IR spectroscopy and Raman spectroscopy are most

often used to detect trans-fatty acids and isomers of

cis-unsaturated fatty acids. UV-spectroscopy at 200–

400 nm is generally used to detect the presence of

conjugated double bonds. The processing and quality

of food oils are often determined by physical charac-

teristics such as their melting point, smoke point,

flash point, fire point, solid-fat content, and degree

of oxidation. These characteristics can be determined

by different analytical techniques.

Other Food Components

0058A large number of different analytical techniques

have been developed to analyze food components

such as bitter compounds like terpenoids, flavonoids,

phenols; pigments like carotenoids, chlorophyl,

anthocyanins; vitamins (water- and fat-soluble);

aroma compounds; dietary fibers; residues; alcohols;

organic acids; antioxidants; preservatives, etc. The

standard methods of the AOAC are generally adopted

for the analysis of these components. Apart from this,

often techniques that are commonly used for the

detection and quantification include colorimetric,

enzymatic, chromatography (paper chromatography

and TLC), UV-visible absorption spectroscopy, gas

chromatography, HPLC (reverse phase, normal

phase), capillary electrophoresis, etc. HPLC is the

most popular alternative to the standard methods

and is commonly used nowadays for the analysis of

almost all types of different food components.

See also: Chromatography: Principles; Thin-layer

Chromatography; Fatty Acids: Analysis;

Homogenization; Protein: Chemistry; Sensory

Evaluation: Sensory Characteristics of Human Foods;

Spectroscopy: Overview; Atomic Emission and

Absorption; Nuclear Magnetic Resonance

214 ANALYSIS OF FOOD

Further Reading

AACC (1983) Approved Methods of Analysis, 8th edn.

St. Paul, MN: American Association of Cereal Chemists.

AOAC (1995) Official Methods of Analysis, 16th edn. vols.

1–2. Arlington, Virginia USA: Association of Official

Analytical Chemists.

AOCS (1990) Official Methods of Recommended Prac-

tices. Chicago, IL: American Oil Chemists Society.

Birch GC and Parker KJ (1984) Control of Food Quality

and Analysis. London: Elsevier Applied Science.

Fennema OR (1996) Food Chemistry. New York: Marcel

Dekker.

Gruenwedel DW and Whitakar JR (eds) (1984) Principles

and Techniques, vols. 1–4. New York: Marcel Dekker.

James CS (1994) Analytical Chemistry of Foods. London:

Blackie Academic & Professional.

Kulp K and Ponte JG Jr. (2000) Handbook of Cereal Science

and Technology, 2nd edn. New York: Marcel Dekker.

Linden G (ed.) (1996) Analytical Techniques for Foods and

Agricultural Products. New York: VCH.

Multon JL (ed.) (1997) Analysis of Food Constituents. New

York: Wiley-VCH.

Nollet LML (ed.) (2000) Food Analysis by HPLC, 2nd edn.

New York: Marcel Dekker.

Nollet LML (ed.) (1996) Handbook of Food Analysis Vols.

1–2. New York: Marcel Dekker.

Poumeranz Y and Meloan CE (1994) Food Analysis, Theory

and Practice, 3rd edn. New York: Chapman & Hall.

Sorensen (2000) Chromatography and Capillary Electro-

phoresis in Food Analysis. New York: Springer.

Wilson RH (ed.) (1994) Spectroscopic Techniques for Food

Analysis. New York: Wiley-VCH.

Analytical Techniques See Chromatography: Principles; Thin-layer Chromatography; High-

performance Liquid Chromatography; Gas Chromatography; Supercritical Fluid Chromatography; Combined

Chromatography and Mass Spectrometry; Immunoassays: Principles; Radioimmunoassay and Enzyme

Immunoassay; Mass Spectrometry: Principles and Instrumentation; Applications; Spectroscopy: Overview;

Infrared and Raman; Near-infrared; Fluorescence; Atomic Emission and Absorption; Nuclear Magnetic Resonance;

Visible Spectroscopy and Colorimetry

ANEMIA (ANAEMIA)

Contents

Iron-deficiency Anemia

Megaloblastic Anemias

Other Nutritional Causes

Iron-deficiency Anemia

S R Lynch, Eastern Virginia Medical School, Norfolk,

VA, USA

Introduction

0001 A lack of iron is the commonest nutritional deficiency

disorder in human beings. Anemia occurs when

body iron stores have been exhausted and the rate of

delivery of iron to developing red blood cells in the

bone marrow is insufficient to support the level of

heme synthesis required to maintain a normal circu-

lating red cell mass and hemoglobin concentration.

It is estimated that more than 500 million people

throughout the world suffer from iron-deficiency

anemia. This article will review the causes, methods

of detection, physiological consequences, as well as

treatment and prevention of iron-deficiency anemia.

(See Iron: Physiology.)

Iron Balance

0002Iron balance in human beings is uniquely dependent

on the body’s ability to match the rate of iron absorp-

tion from the proximal small intestine to iron require-

ments. During childhood, iron absorption normally

exceeds immediate requirements, ensuring a positive

balance and the gradual establishment of an iron

store. In the adult, the level of absorption is approxi-

mately equal to a relatively fixed rate of loss that is

ANEMIA (ANAEMIA)/Iron-deficiency Anemia 215

governed by factors unrelated to iron balance. When

requirements are increased, e.g., during periods of

rapid growth and in pregnancy, absorption increases

to replace the storage iron that is used up. (See Chil-

dren: Nutritional Requirements; Lactation: Human

Milk: Composition and Nutritional Value.)

0003 Once iron stores are exhausted by increased

demand, anemia supervenes if adaptive changes in

the rate of absorption are insufficient to restore iron

balance. Dietary iron intake almost always exceeds

the body’s requirements by a large margin, but the

ability of absorptive mechanisms to extract the iron

from food may be limited. Iron-deficiency anemia is

therefore most commonly encountered when the

relationship between requirement and dietary intake

of bioavailable iron is least favorable. Periods of

increased vulnerability include infancy, adolescence

(particularly among girls after the onset of menstru-

ation), and the child-bearing years for women.

0004 After the first 4–6 months of age, stores are no

longer the primary source of iron for growth. By the

age of 12 months total body iron should have

increased by about 70%. Iron-deficiency anemia is

therefore commonly encountered in infancy. The

risk tends to decrease after about 18 months of age

because the rate of growth slows and the diet becomes

richer in bioavailable sources of iron. The adolescent

growth spurt and the onset of menstruation in girls

constitute the second high-risk period. In adulthood,

increased menstrual bleeding or blood loss from the

gastrointestinal tract may contribute to an unfavor-

able balance. For example, the monthly menstrual

loss in 10% of healthy women is more than 80 ml

(average for most women is 30 ml). This increases the

daily iron requirement by about 0.8 mg or 6% of the

dietary intake. It would be necessary for such an

individual to increase her absorption of dietary iron

from about 10% to 16%. Hookworm infestation,

which is prevalent in many developing countries,

may induce sufficient blood loss to increase iron

requirements by 3–4 mg day

1

. Iron balance would

only be maintained if percentage absorption from

the diet were increased by 20–30%. Many of these

individuals will become anemic because maximal

total absorption from a normal diet is usually only

4–5 mg day

1

. Pregnancy presents the greatest

physiological challenge to the body’s adaptive mech-

anisms. The daily iron requirement during the last

trimester is between 4 and 6 mg. (See Premenstrual

Syndrome: Nutritional Aspects.)

0005 The diets of most western adults contain large

quantities of meat, which is rich in highly available

heme iron, as well as fresh fruit and other sources of

vitamin C, that increase the absorption of inorganic

iron. This diet provides sufficient bioavailable iron to

prevent anemia even in the presence of higher require-

ments. For example, a recent study in which dietary

iron absorption was measured over a 2-week period

indicated that a menstruating woman eating a mixed

diet containing meat would absorb about 10% of the

iron and maintain an iron store of approximately

350 mg. The composite 10% absorption value is

achieved by assimilation of about 30% of the heme

iron and 6% of the nonheme iron. A woman with

relatively high menstrual losses would also be

expected to maintain iron balance by increasing her

nonheme iron absorption to 12%, although her iron

store would be estimated at only 200 mg.

0006The iron requirements during the second and third

trimesters of pregnancy usually exceed the body’s

capacity to extract sufficient iron from the diet even

if the bioavailability is high. Most women require a

small iron supplement to prevent iron imbalance.

0007As indicated above, relative iron requirements are

also high in infants between the ages of 6 and 18

months. Foods given to young children during and

immediately after the weaning period usually have

low iron bioavailability. However some countries

have been very successful in preventing iron defi-

ciency during this vulnerable period by fortifying

infant foods with iron.

0008Iron balance is much less satisfactory in many

people living in developing countries. Heme iron is

often absent from the diet since meat is not eaten in

significant quantities. The bioavailability of the non-

heme iron is usually low because the staple foods

tend to have high levels of phytate and polyphenols.

Iron-deficient individuals may be able to absorb only

5–10% of the nonheme dietary iron. Under these

circumstances even modestly increased requirements

may not be met. Poor dietary iron availability is often

accompanied by high requirements due to factors

such as multiple pregnancies and hookworm infest-

ation. As a result, iron-deficiency anemia is wide-

spread.

Prevalence

0009On the basis of data obtained during the third

National Health and Nutrition Examination Survey

(NHANES III, 1988–1994) the highest prevalence

rates for iron-deficiency anemia in the USA occur

in toddlers (3%), and adolescent girls and men-

struating women (2–5%). Iron-deficiency anemia is

rare in men and postmenopausal women. Among the

elderly, inflammatory disease, not iron deficiency, is

the commonest cause of anemia. Iron deficiency

is much more frequently encountered in developing

countries. At least 500 million people are affected

globally.

216 ANEMIA (ANAEMIA)/Iron-deficiency Anemia

Laboratory Diagnosis

0010 Iron-deficiency anemia may be suspected on the basis

of the clinical symptoms and the setting in which it

occurs, but precise identification depends on the

results of laboratory tests designed to confirm the pres-

ence of anemia and to evaluate iron status (Table 1).

Identification of Anemia

0011 Anemia is identified by the finding of a hemoglobin

concentration or hematocrit that is below the normal

range. This usually presents little difficulty in the

clinical situation since symptoms only occur in indi-

viduals with moderate or severe anemia. On the other

hand, prevalence studies of nutritional anemia

depend on the identification of mild anemia in an

otherwise healthy population. The World Health

Organization (WHO) criteria established in 1975

are the most widely accepted. Hemoglobin concen-

trations below 13 g dl

1

for adult males, 12 g dl

1

for

menstruating women, and 11 g dl

1

in pregnancy are

considered indicative of anemia.

0012 In mild iron deficiency, the diagnostic value of a

low hemoglobin concentration or hematocrit is

limited by the significant overlap in the frequency

distribution curves for anemic and normal popula-

tions. In one study using a single value criterion,

17% of women responded to oral iron despite the

fact that they were classified as normal, while 35%

of ‘anemic’ women failed to show any change in

hemoglobin after being treated. The problem is even

more complex in children, where developmental

changes affect the normal hemoglobin concentration.

The accuracy of diagnosis can be improved by using a

criterion that employs an abnormal hemoglobin or

hematocrit value in combination with one or more

laboratory markers of iron deficiency.

Evaluation of Iron Status

0013 Laboratory tests of iron status are used both to dis-

tinguish iron deficiency from other causes of anemia

and to facilitate the recognition of mild iron-

deficiency anemia in nutritional surveys. They supply

specific information about the adequacy of iron stores

and the supply of iron to the bone marrow and other

organs.

Iron Stores

0014Bone marrow iron Histological evaluation of stor-

age iron by using the Prussian-blue method to stain

macrophage hemosiderin in needle aspirate or biopsy

specimens of bone marrow provides a semiquantita-

tive estimate of storage iron. Iron-deficiency anemia

is characterized by an absence of stainable bone

marrow iron. This procedure remains the gold stand-

ard in patients suffering from complicated illnesses,

but is rarely necessary under most other circum-

stances and impractical in nutritional surveys.

0015Serum ferritin Body iron not immediately required

for hemoglobin synthesis or other metabolic pro-

cesses is stored in cells as ferritin and hemosiderin.

Ferritin is also present in very small amounts in the

plasma. The development of sensitive immunological

methods has made it possible to measure serum ferri-

tin (SF) accurately. This has become the most useful

indirect estimate of body iron stores. SF concentra-

tion is directly proportional to the size of the storage

pool in health and in uncomplicated iron deficiency:

1 mgl

1

is equivalent to 8–10 mg storage iron in

adults (SF 1 mgl

1

is equivalent to 140 mgkg

1

stor-

age iron). A serum ferritin below 12 mgl

1

is diagnos-

tic of iron deficiency. Unfortunately, higher SF levels

do not exclude iron deficiency when disorders such as

infection, chronic inflammatory diseases, malignant

disorders, and liver disease are present because they

elevate SF values independently.

Internal Iron Supply

0016Serum transferrin receptor concentration Transfer-

rin receptors are expressed on the surfaces of all cells

in concert with their need for iron. A truncated form

of the extracellular domain of the receptor is pro-

duced by proteolytic cleavage and is present in the

plasma in direct proportion to the total number of

receptors expressed in all body tissues. When func-

tional iron depletion occurs more transferrin recep-

tors appear on cell surfaces and the concentration

of the proteolytically cleaved extracellular domain

(serum transferrin receptor or STfR) in the plasma

increases concomitantly. The magnitude of the

increase is proportional to the size of the functional

iron deficit. This assay has only recently become

available, but may prove to be a very important

measure of functional iron deficiency. The transferrin

saturation and free erythrocyte protoporphyrin levels

tbl0001 Table 1 Laboratory evaluation of iron-deficiency anemia

Purpose of test Laboratory test

Confirm presence of anemia Hemoglobin

Hematocrit

Evaluate iron status

Iron stores Bone marrow iron

Serum ferritin

Iron supply Serum transferrin receptor

concentration

Transferrin saturation

Free erythrocyte protoporphyrin

Red blood cell indices

ANEMIA (ANAEMIA)/Iron-deficiency Anemia 217

are both abnormal in the anemia of chronic disease

even if iron stores are adequate because the iron

supply is reduced by impaired release from stores.

The serum transferrin receptor concentration may

help to distinguish these two common causes of

anemia, since serum transferrin receptor levels are

normal in the anemia of chronic disease.

0017 Transferrin saturation As body iron stores become

depleted, the concentration of the iron transport pro-

tein, transferrin, in the plasma increases, leading to a

rise in the total iron-binding capacity (TIBC). Once

iron stores are exhausted and the tissue demands

exceed the rate of supply, the serum iron (SI) concen-

tration falls. Since an impaired iron supply in an iron-

deficient individual is characterized by a rise in TIBC

and a fall in SI, a reduction in the transferrin satur-

ation (TS ¼ SI  100/TIBC) from the normal value of

approximately 35% to less than 15% is the best

single criterion of a suboptimal supply.

0018 Free erythrocyte protoporphyrin An impaired iron

supply can also be recognized by measuring the pro-

toporphyrin IX (free erythrocyte protoporphyrin or

FEP) in peripheral red blood cells. Insufficient iron

limits the final step in heme synthesis, leading to the

accumulation of FEP in red cell precursors. The cellu-

lar content remains unchanged throughout the life

span of the erythrocyte. Peripheral blood FEP values

therefore reflect iron supply during red cell produc-

tion. FEP levels are also raised in lead poisoning. If

exposure to lead can be excluded, a raised FEP pro-

vides the same information as a reduced TS, but it is

more stable. FEP is raised only after several weeks of

iron-deficient erythropoiesis and requires a similar

period for normalization once an adequate supply of

iron is reestablished.

0019 Red blood cell indices As the severity of iron

deficiency progresses, morphological abnormalities

(microcytosis and hypochromia), resulting from re-

duced hemoglobin synthesis, appear in the peripheral

blood. The sensitivity with which these changes can

be recognized has improved dramatically with the

introduction of electronic counting and sizing equip-

ment. Both the mean corpuscular volume (MCV) and

the mean corpuscular hemoglobin (MCH) are reliable

measures of impaired hemoglobin synthesis, although

not specific for iron deficiency. The MCV is the more

widely used.

0020 The best approach to diagnosing iron-deficiency

anemia depends on the setting in which it is being

evaluated. The accuracy of prevalence studies of

nutritional anemia is improved by the concurrent

measurement of hemoglobin or hematocrit and one

or more specific tests for iron status, such as SF, STfR,

FEP, or TS. The SF concentration is currently the most

widely used measure of iron status. A similar

approach or a therapeutic trial of oral iron may be

satisfactory for clinic or office patients in whom iron

deficiency is considered likely. In hospitalized pa-

tients, anemia complicated by other illnesses may

occasionally require the direct histological evaluation

of bone marrow iron stores. (See Nutritional Assess-

ment: Anthropometry and Clinical Examination;

Biochemical Tests for Vitamins and Minerals; Func-

tional Tests.)

Physiological Consequences of Iron-

deficiency Anemia

0021Mild anemia usually causes few symptoms in individ-

uals with sedentary occupations because of compen-

satory physiological adjustments, including increased

cardiac output and a shift in the oxygen dissociation

curve with improved oxygen delivery to tissues.

Aerobic work capacity in human beings is however

reduced when the hemoglobin concentration falls

below 12 g dl

1

0022Patients with moderate or severe anemia are pale

and have symptoms such as reduced exercise capacity,

shortness of breath, and palpitations that are indica-

tive of a decreased cardiopulmonary reserve.

0023Iron deficiency usually develops gradually. Com-

pensatory adjustments to oxygen transport insure ad-

equate oxygen delivery at rest. However the ability to

carry out sustained physical activity is significantly

impaired, even in relatively mild iron-deficiency

anemia, because of a reduction in available oxygen

and the associated tissue iron deficiency that directly

affects skeletal muscle function. Manual laborers

who suffer from iron-deficiency anemia are less pro-

ductive and may have a reduced earning capacity.

0024A study carried out among Indonesian rubber plan-

tation latex tappers, who were paid a daily wage

based on the weight of latex they collected, demon-

strated a direct correlation between hemoglobin con-

centration and income. Anemic men were 18.7% less

productive than their nonanemic fellow workers;

when they were given 100 mg elemental iron per day

for 60 days their income increased by 37%. It has

been estimated that for every 1% increase in hemo-

globin, work output will increase by 1–2%.

0025Iron-deficiency anemia also has insidious effects in

children. Mental and motor development is impaired

in infants. Cognitive function and school perform-

ance are affected in older children. The observed

abnormalities are thought to be due primarily to

direct effects of iron deficiency on cellular and neuro-

transmitter function. They are discussed in the chap-

ter on physiology.

218 ANEMIA (ANAEMIA)/Iron-deficiency Anemia

0026 During pregnancy iron-deficiency anemia has sig-

nificant consequences for the mother and the infant.

Maternal anemia is associated with low infant birth

weight, premature delivery, and increased perinatal

mortality. Furthermore, although fetal requirements

tend to be met at the expense of the mother’s needs,

several studies indicate that infants born to

iron-deficient women have lower iron stores at birth

and lower SF levels at 3–6 months of age. The risk of

maternal death is also increased in women with

severe anemia.

0027 Finally iron-deficiency anemia may be associated

with several abnormalities that are considered to be

specific consequences of a tissue iron lack. They

include impaired immunity, mucosal and epithelial

changes, the eating of nonfood material (pica), and

impaired temperature regulation.

Treatment and Prevention

Treatment

0028 The recognition of iron-deficiency anemia is never a

complete diagnosis. Iron administration may produce

a rapid and gratifying correction of the anemia, but

a satisfactory long-term solution to the problem

depends on a clear understanding of the reasons for

increased requirements and/or reduced absorption.

For example, iron-deficiency anemia in western men

is usually a symptom of unsuspected blood loss from

the gastrointestinal tract. Identification of the under-

lying disorder is essential (Table 2).

0029 Oral iron therapy using a soluble ferrous salt, such

as sulfate, gluconate, or fumarate, that provides a

daily dose of 60–120 mg elemental iron in adults, or

2–3mgkg

1

in children, is usually the most appropri-

ate method both to repair the anemia and restore a

normal body iron store in patients with moderate or

severe anemia. Once this has been accomplished,

continued iron balance can only be assured if the

cause of excessive requirements is eliminated or if

more available dietary iron is supplied (in situations

in which iron-deficiency anemia is the result of an

inadequate intake of bioavailable iron).

Prevention

0030 The prevalence of nutritional iron-deficiency anemia

in western societies has fallen markedly in recent

years. The many factors that have been suggested to

contribute to this improvement in adults include diet-

ary fortification, a general improvement in income

level and diet, a large increase in the sale of medicinal

iron preparations, routine iron supplementation in

pregnancy, and reduced menstrual blood loss as a

result of the use of oral contraceptives.

0031Similarly, increasing awareness of the importance

of adequate iron nutrition in early infancy has led to a

reduction in the prevalence of iron deficiency in

young children. As with adults, improved iron nutri-

tion reflects the effect of several different factors,

including the use of iron-fortified infant foods and

the provision of supplemental iron when appropriate.

0032The situation in developing countries is far less

satisfactory. Attempts to reduce iron losses or im-

prove the intake of bioavailable iron have had only

limited success. Iron supplementation (usually the

provision of iron tablets) has been effective in pilot

studies, but the inadequacy and cost of distribution

systems, as well as poor compliance, have limited the

value of this approach as a long-term solution. Never-

theless, supplementation is the preferred method

of intervention in severely anemic individuals and

during pregnancy.

0033Increasing the population’s intake of bioavailable

dietary iron is the only feasible means of improving

iron nutrition at the national level. Despite years of

research and numerous field trials, this has proved to

be extremely difficult to achieve. The traditional diet

of people in most developing countries tends to

inhibit iron absorption. Dietary customs are not

easily modified. In addition, foods that promote

iron bioavailability tend to be expensive. Iron

tbl0002Table 2 Causes of iron-deficiency anemia

Underlyingdisorder Site of action Causal factors

Nutritional deficiency Low dietary

bioavailability

Unrefined cereal diet

Malabsorption Gastric surgery

Clay eating

Gluten-induced

enteropathy

Blood loss Uterine Menorrhagia

Gastrointestinal Hookworm,

schistosomiasis

Peptic ulcer disease,

esophagitis

Stomach and colorectal

cancer

Angiodysplasia,

hereditary

telangiectasia

Aspirin, NSAIDs

Inflammatory bowel

disease

Diverticulosis,

hemorrhoids

Renal tract Hemoglobinuria

Severe hematuria

Lung Idiopathic pulmonary

hemosiderosis

NSAIDs, nonsteroidal antiinflammatory drugs.

ANEMIA (ANAEMIA)/Iron-deficiency Anemia 219

fortification of the diet has also encountered several

obstacles. The introduction of iron into the food

chain in developing countries is complicated by the

difficulty of identifying a suitable vehicle. Most foods

are produced at the local level and not processed or

distributed through a limited number of centers

where the fortificant can be introduced into the

food. In addition, the inhibitory effect of vegetable

diets on bioavailability tends to reduce the effective-

ness of fortification iron. Finally it is often difficult to

add iron to dietary items without affecting consumer

acceptability. Iron salts that do not alter the appear-

ance or organoleptic and storage properties of foods

tend to be poorly absorbed. Despite these formidable

obstacles, field trials and targeted national programs

have been successful in Chile, South Africa and, more

recently, Venezuela. (See Food Fortification.)

See also: Children: Nutritional Requirements; Folic Acid:

Properties and Determination; Physiology; Food

Fortification; Iron: Physiology; Lactation: Human Milk:

Composition and Nutritional Value; Physiology;

Nutritional Assessment: Anthropometry and Clinical

Examination; Biochemical Tests for Vitamins and

Minerals; Functional Tests; Premenstrual Syndrome:

Nutritional Aspects

Further Reading

Brock JH, Halliday JW, Pippard MJ and Powell LW (1994).

Iron Metabolism in Health and Disease. London: WB

Saunders.

Cook JD (1982) Clinical evaluation of iron deficiency.

Seminars in Hematology 19: 6–18.

Cook JD (1990) Adaptation in iron metabolism. American

Journal of Clinical Nutrition 51: 301–308.

Cook JD, Skikne BS and Baynes RD (1993) Serum transfer-

rin receptor. Annual Review of Medicine 44: 63–74.

Looker AC, Dallman PR, Carroll MD, Gunter EW and

Johnson CL (1997) Prevalence of iron deficiency in the

United States. Journal of the American Medical Associ-

ation 277: 973–976.

Megaloblastic Anemias

N M F Trugo, Universidade Federal do Rio de Janeiro,

Instituto de Quimica, Rio de Janeiro, Brazil

Background

0001 Megaloblastic anemia is characterized by morpho-

logical abnormalities of hemopoietic cells that in-

clude the formation of abnormally large erythrocyte

precursors (megaloblasts) and giant metamyelocytes

in the bone marrow, and abnormally large (macro-

cytic) erythrocytes and hypersegmented neutrophils

in the blood. Megaloblastic anemia is caused by a

reduction in the rate of DNA biosynthesis, resulting

in abnormal nuclear maturation and ineffective ery-

thropoiesis. Folate and cobalamin deficiencies are the

most common causes of the impaired DNA synthesis

that leads to megaloblastic hemopoiesis; other less

common factors are the use of drugs that interrupt

DNA biosynthesis and inherited conditions present-

ing defective enzymes of DNA biosynthesis. Megalo-

blastic anemia caused by folate and/or cobalamin

deficiencies present indistinguishable morphological

abnormalities, and will be the main focus in this

chapter.

Etiology

Consequences of Folate and Cobalamin

Deficiencies

0002Folate and cobalamin deficiencies are primary causes

of megaloblastic anemia. Folate is an essential cofac-

tor involved in many single-carbon transfer reactions

in intermediary metabolism, including the synthesis

of purine bases, thymidylate and methionine. Folate

deficiency thus causes impairment in the DNA

synthesis and in the methylation reactions that are

dependent on S-adenosylmethionine.

0003Both folate and cobalamin are required for the

methyl transfer reaction catalyzed by methionine

synthase. In this reaction methionine is recycled by

remethylation of homocysteine and simultaneous

demethylation of 5-methyltetrahydrofolate to tetra-

hydrofolate, a source of other folate coenzymes.

Methionine synthase requires methylcobalamin as a

cofactor. Impairment of this reaction by cobalamin

deficiency thus causes a decrease in the availability of

10-formyl-tetrahydrofolate for purine synthesis and

of 5,10-methylene-tetrahydrofolate for conversion of

deoxyuridylate into deoxythymidylate, which might

be explained by ‘trapping’ of methyltetrahydrofolate

that cannot be converted back to the other folate

forms, resulting in a decrease of folate coenzymes.

An alternative hypothesis is that the impairment of

the methionine synthase reaction decreases the supply

of formate that would be required for generation

of 10-formyl-, 5,10-methenyl- and 5,10-methylene

tetrahydrofolate, due to the failure of methionine

synthesis and resulting low levels of S-adenosyl-

methionine.

0004Therefore, deficiencies of both or either of these

vitamins affect DNA replication and consequently

cell division, especially in rapidly proliferating

220 ANEMIA (ANAEMIA)/Megaloblastic Anemias

tissues. In the bone marrow, the impaired DNA repli-

cation causes asynchronous maturation of the

nucleus and cytoplasm of cells in the erythroid and

myeloid series, with cytoplasmic components being

synthesized in excessive amounts during the delay

between cell divisions. This results in the enlargement

of the precursor cells and the presence of larger than

normal circulating erythrocytes and leukocytes, which

are characteristic features of megaloblastic anemia.

0005 Besides megaloblastic anemia, cobalamin defi-

ciency also causes a neuropathy characterized by an

early demyelination of peripheral nerves, as a result of

the impaired methylation of the myelin basic protein,

a major constituent of myelin. This is currently

explained by the inadequate activity of methionine

synthase in cobalamin deficiency, which leads to

a reduction in the methylation cycle due to the

impaired recycling of S-adenosylmethionine. Several

evidences indicate that the other enzymatic reaction

requiring cobalamin as a coenzyme (5

-deoxyadeno-

sylcobalamin), which is the conversion of methyl-

malonyl-coenzyme A (CoA) to succinyl-CoA by

methylmalonyl-CoA mutase, seems not to be impli-

cated in the neuropathy of cobalamin deficiency.

Folate deficiency usually does not lead to this neur-

opathy, unless the deficiency is prolonged and severe,

possibly because the neural tissue concentrates more

folate than other tissues and may also have alternative

pathways to preserve the supply of S-adenosylmethio-

nine when there is folate deprivation. (See Cobala-

mins: Physiology; Folic Acid: Physiology.)

Causes of Cobalamin and Folate Deficiencies

0006 Deficiencies of cobalamin and folate may result

from inadequate intake, increased requirements and

impaired utilization, due to several underlying factors

that are presented in Tables 1 and 2.

0007 Cobalamin deficiency due to insufficient dietary

intake is rare, but may occur in strict vegetarians,

since cobalamin is supplied primarily by foods of

animal origin. Recommended daily intakes of dietary

cobalamin are in the range of 1–2 mg for adults. A

normal western diet usually provides an excess of the

body requirements for cobalamin, and cobalamin

body stores represent a further protection, with an

efficient conservation through the enterohepatic

circulation.

0008 Among the causes of cobalamin deficiency de-

scribed in Table 1, the most common is intestinal

malabsorption, which can be the result of a decreased

production or lack of gastric intrinsic factor, a protein

required for cobalamin absorption, as occurs in

atrophic gastritis, gastrectomy, and pernicious

anemia, an autoimmune disease in which there is

production of autoantibodies against intrinsic factor

and of autoantibodies that cause destruction of

gastric parietal cells. Moreover, hypochlorydria or

achlorydria and low pepsin secretion in the stomach

affect release of cobalamin from its bound state in

food. When this happens, but secretion of intrinsic

factor is normal, there is protein-bound cobalamin

tbl0001Table 1 Causes of cobalamin deficiency

Reduced intake

Strict vegetarianism

Breast-feeding by severely cobalamin-deficient mothers

Malabsorption

Pernicious anemia

Gastric dysfunction caused by gastritis due to Helicobacter

pylori infection or alcohol consumption

Gastrectomy or gastric bypass

Protein-bound cobalamin malabsorption

Enteropathy: Crohn’s disease, celiac disease

Ileal resection

Pancreatic insufficiency

Microbial overgrowth in intestinal stasis and parasitic

(fish tapeworm) infestation

Use of drugs such as colchicine, neomycine, omeprazole, or

p-aminosalicylic acid

Congenital absence or dysfunction of intrinsic factor

Lack of ileal receptor for intrinsic factor

Impaired utilization

Congenital transcobalamin II deficiency

Inactivation of cobalamin by prolonged exposure to

nitrous oxide

Decreased conversion to active coenzyme forms of

cobalamin due to enzyme defect

tbl0002Table 2 Causes of folate deficiency

Reduced intake

Malnutrition

Alcoholism

Intake of foods poor in folate (raw; processed)

Increased demands

Pregnancy

Rapid growth rate (prematurity; early infancy; adolescence)

Diseases associated with increased cell turnover (hemolytic

anemias; leukemia; chronic diseases; exfoliative

dermatitis; severe infection)

Excessive losses (hemodialysis; liver disease)

Malabsorption

Enteropathy: Crohn’s disease, celiac disease, tropical sprue

Intestinal resection

Chronic giardiasis

Bacterial overgrowth

Use of sulfasalazine and of anticonvulsive drugs (phenytoin,

phenobarbital)

Impaired utilization

Congenital abnormalities in enzymes of folate metabolism

(5,10-methylene tetrahydrofolate reductase; methionine

synthase; di-hydrofolate reductase)

Use of drugs such as methotrexate and oral contraceptives

Alcoholism

ANEMIA (ANAEMIA)/Megaloblastic Anemias 221

malabsorption, because cobalamin from food is

poorly absorbed whereas absorption of ingested

synthetic cobalamin is not affected.

0009 Differently from cobalamin, dietary inadequacy is

a common cause of folate deficiency (Table 2), since

folate intake is not usually in great excess of nutri-

tional requirements and the body reserves are meager.

Recommended daily intakes of folate are in the range

of 0.15–0.20 mg for adults, 0.4 mg for women of

reproductive age, and 0.6 mg for pregnant women.

Diets poor in folate are characterized by a predomin-

ance of starches and grains (white bread, white maize

meal, polished rice, etc.) and overcooked vegetables,

and insufficient amount or lack of foods of animal

origin, dark green vegetables, fresh or lightly cooked,

and fruits, as occurs in some parts of Africa and India.

In developed countries, nutritional folate deficiency

has been found in elderly persons living on a marginal

subsistence diet, and in people on very low incomes.

Alcoholism may cause folate deficiency, not only

because of the reduced folate intake, but also due

to altered folate metabolism and interference with

the folate enterohepatic cycle.

0010 Intestinal malabsorption can also be a cause of

folate deficiency and may be associated with ineffect-

ive food folate deconjugation prior to absorption as

occurs in subtotal gastrectomy and with the use of

sulfasalazine, as well as with ineffective absorption

due to extensive intestinal resection, tropical sprue,

gluten-sensitive enteropathy, chronic giardiasis, and

the use of drugs such as anticonvulsants (phenytoin

and phenobarbital).

0011 Due to the high folate requirements, periods of

intense cell proliferation such as pregnancy, early

infancy, and adolescence predispose to folate defi-

ciency. Increased requirements of folate may also

occur in diseases such as hemolytic anemia, malig-

nant metastatic tumors, and leukemias. Folate defi-

ciency may also occur with the use of drugs that are

folate antagonists and impair folate utilization, such

as those used for cancer chemotherapy (methotrex-

ate) and immunosuppression, as well as antiprotozoal

(pyrimethamine) and antibacterial (trimethroprim,

pentamidine) drugs.

0012 A genetically carried impairment in the folate util-

ization, due to one variant of 5,10-methylenetetra-

hydrofolate reductase that has a lower affinity for

folate, and which is apparently common in many

populations, increases folate requirements. Another

genetic problem related to 5,10-methylenetetra-

hydrofolate reductase is its deficient production in

some patients, leading to hyperhomocysteinemia,

homocystinuria, and neurological disturbances, but

without any hematological abnormality. (See Coba-

lamins: Physiology; Folic Acid: Physiology.)

Morphological and Clinical Aspects

0013The reduction in DNA biosynthesis in the bone

marrow delays the development of the erythrocytes,

leading to megaloblastosis of erythocyte precursors

and macrocytosis of erythrocytes, and reduces their

number, leading to anemia. The megaloblastic

anemia is morphologically indistinguishable in the

deficiency of either vitamin. The changes in the eryth-

roid and myeloid cells result in pallor and weakness.

0014In the bone marrow, there is an overt megalo-

blastosis and a reduction in the ratio of myeloid to

erythroid cells. Megaloblasts present a characteristic

chromatin pattern with a lacy appearance. There are

more immature erythroblasts than mature ones, due

to selective death of the latter. In the myeloid series,

giant and abnormally shaped metamyelocytes

(granulocyte precursors) are found. Megakaryocytes

(platelet precursors) may also be enlarged, with

increased numbers of nuclear lobes.

0015In blood, erythrocytes are large (macrocytic) and

often oval in shape, and some may be fragmented

and irregularly shaped. Neutrophilic leukocytes

often have abnormal nuclei, with an increase in the

number of nuclear segments (hypersegmentation).

There is an increased mean corpuscular volume of

red cells, and decreased erythrocyte, reticulocyte,

leukocyte, and platelet counts in peripheral blood,

due to ineffective erythropoiesis that results in cell

destruction before maturity. The decrease in erythro-

cyte counts leads to low hemoglobin values, as the

deficiency becomes more severe.

0016Besides the hemopoietic system, the epithelial cells

of gastrointestinal, respiratory, urinary, and female

genital tracts, and the gonads are also affected. The

decreased replication of enterocytes in the small in-

testine leads to villus atrophy that, if severe enough,

can cause malabsorption. Other symptoms, such as

glossitis (sore tongue), cheilitis (sore lips), diarrhea,

and weight loss may thus result from impaired prolif-

eration of epithelial cells. Infertility and impotence

may also be present.

0017Cobalamin deficiency may also result in a complex

neurologic syndrome, manifested with or without

megaloblastic anemia, including peripheral neur-

opathy, subacute combined degeneration of the spinal

cord, and optic atrophy. Common neurological symp-

toms are paresthesias, ataxia, numbness, impairment

of memory, depression, dementia, and psychosis.

Folate deficiency can occasionally cause mental

changes, such as mental slowing, depression, demen-

tia, and other neurologic syndromes.

0018There are, however, variations in the hematologic

and neurologic expressions of cobalamin and folate

deficiencies among patients. In children, for instance,

222 ANEMIA (ANAEMIA)/Megaloblastic Anemias

hereditary and acquired deficiencies result more often

in developmental delay and neurological dysfunc-

tions than in megaloblastic anemia. Also, genetic

and acquired characteristics of ethnic and racial

groups seem to impose variations related to cobala-

min deficiency, which could have implications in

diagnosis and issues of public health.

0019 During pregnancy, risks from folate deficiency in-

clude poor pregnancy outcome, such as prematurity

and low birth weight. In addition, there is consider-

able evidence that folate is a key factor for prevention

of neural tube defects (NTD; congenital birth defects

such as spina bifida and anencephalus). Although

NTD have multiple etiologies, maternal folate sup-

plementation during the periconceptional period can

reduce both their occurrence and recurrence. The first

3 weeks after conception represent a crucial period

because closure of the neural tube occurs about the

23rd day. Although the underlying mechanism for the

folate role in the prevention of NTD is not known,

there are indications that in the vast majority of cases

the problem is not of maternal dietary deficiency, and

may be due to a maternal and/or fetal defect in folate

metabolism or transport into the cells, that can be

overcome by maternal supplementation. An impair-

ment in homocysteine metabolism seems to be

involved in NTD manifestation. Although NTD

may affect pregnancies in women who present a

wide range of folate erythrocyte levels, from low to

normal, the risk is strongly reduced when status is

improved, even in those women who do not present

low erythrocyte folate levels. Cobalamin status, not

necessarily in the deficient range, is also an independ-

ent risk factor for NTD. (See Cobalamins: Physi-

ology; Folic Acid: Physiology; Pregnancy: Maternal

Diet, Vitamins, and Neural Tube Defects.)

Laboratory Diagnosis

Hematology

0020 An elevated blood mean corpuscular volume (MCV)

due to macrocytosis is suggestive of megaloblastosis.

In cobalamin and folate deficiencies the MCV tends

to increase before the decrease in the hemoglobin

levels. The increase in MCV may not be present

when there is concurrent microcytosis due to iron

deficiency and thalassemia. Examination of a bone

marrow aspirate may be useful for the diagnosis of

megaloblastic anemia, since macrocytosis can be due

to other conditions (chronic alcoholism, liver disease,

etc.) that do not cause changes in the bone marrow.

However, megaloblastosis can also be due to the use of

anticancer drugs, mainly methotrexate, which impair

folate metabolism. The presence of hypersegmented

neutrophils, with five or more nuclear lobes, in the

peripheral blood smear is a sensitive indicator of

megaloblastic anemia, and is useful for the diagnosis

of folate and cobalamin deficiencies when erythro-

cyte changes are masked by coexistent iron deficiency

and when folate and cobalamin deficiencies are still

subclinical, because hypersegmentation occurs earlier

than the anemia and macrocytosis.

Biochemical Tests

0021A range of biochemical tests are used to investigate

whether megaloblastic anemia is due to folate or

cobalamin deficiency. Biochemical indicators of

either vitamin status corresponding to different

degrees of depletion are used for this purpose, despite

the problems in establishing ‘cut-off’ levels.

0022Serum (or plasma) cobalamin may be a poor indi-

cator of tissue levels, but levels lower than 200 pg

1

probably indicate a deficiency state of cobala-

min. About one-third of patients with folate defi-

ciency present low serum cobalamin levels, which

return to normal with folate therapy. Low serum

folate (less than 3 ng ml

1

) is an indicator of negative

balance that, if persistent, can result in tissue deple-

tion, which is assessed by determination of erythro-

cyte folate. Values of erythrocyte folate of less than

150 ng ml

1

are indicative of an initial folate defi-

ciency (marginal status) and those less than 120 ng

1

indicate established deficiency. These three indi-

cators are the most widely used to assess cobalamin

and folate status. They can be measured by micro-

biological assays or by competitive protein-binding

radioassays, but since values vary between assay pro-

cedures, the range of normal values should be estab-

lished for each procedure.

0023Once established whether megaloblastic anemia is

due to folate or cobalamin deficiency, or to other

causes, the underlying causes of these deficiencies

should also be investigated. For instance, dietary

assessment is useful to detect whether dietary insuffi-

ciency of either vitamin is an underlying factor.

0024Other biochemical and functional tests provide

valuable information regarding the nutritional status

of folate and cobalamin and the underlying causes of

deficiency. Serum homocysteine is a sensitive indica-

tor of intracellular folate and cobalamin, and it is

increased when depletion of either vitamin occurs

because they are required for conversion of homo-

cysteine to methionine.

0025The deoxyuridine suppression test in bone marrow

cells or in lymphocytes assesses the thymidylate

synthesis from deoxyuridylate, which requires 5,10-

methylene tetrahydrofolate as a coenzyme and is

impaired in folate and cobalamin deficiencies.

Exogenous deoxyuridine added to normal cells

ANEMIA (ANAEMIA)/Megaloblastic Anemias 223