HEMOLYTIC ANEMIA Enzymopathies
Deficiencies in Hexose Monophosphate Shunt Glucose 6-Phosphate Dehydrogenase Deficiency Deficiencies in the EM Pathway Pyruvate Kinase Deficiency G6PD Five classes of G-6-PD deficiency exist based on enzyme activity levels, as follows:
Enzyme deficiency with chronic nonspherocytic hemolytic anemia
Severe enzyme deficiency (<10%)
Moderate-to-mild enzyme deficiency (10-60%)
Very mild-to-no enzyme deficiency (60%)
Increased enzyme activity G6PD
> 200 million people in the world Genetic heterogeneity (over 400 variants)
Severity of the problem can vary from hemolysis even in the absence of oxidative stress to hemolysis only on exposure to mild to marked oxidant stress Glucose-6-phosphate dehydrogenase
Gene symbol : G6PD
Location : Xq28 The G6PD gene is located on the X chromosome
Thus the deficiency state is an sex-linked trait seen only in hemizygote males
Most female carriers are asymptomatic Molecular data on human G6P
Size of gene (in kilobases) 18.5 DNA Number of exons 13 Number of introns 12
2269 mRNA Size (in nucleotides)
Number of amino acids 515 Protein Molecular weight (in Daltons) 59,265 Subunits per molecule of active enzyme 2 or 4 The G6PD gene is located on the X chromosome
Thus the deficiency state is an sex-linked trait seen only in hemizygote males
Most female carriers are asymptomatic Structure of G6PD
Glucose-6-Phosphate Dehydrogenase, is comprised of a dimer or tetramer of identical polypeptide chains
Each unit consists of 515 amino acids
The single G6PD locus in humans is located on the telomeric region of the long arm of the X-chromosome
Females have two X chromosomes, hence two copies of G6PD, while males have only one X chromosome and one copy of G6PD The normal G6PD is called as Type B
Type A- has two base substitutions and is seen in people from central Africa
A second variant is seen among the people of the Mediterranian and is more severe than Type A-
The third variant that is relatively more common but less severe is seen in southern China Familial Genetics of G6PD
Five genotypes can form from combinations of one normal (GdB) and one deficient form (e.g., GdA- or GdMed) of G6PD Females GdB/GdB, Homozygous Normal; "Normal" GdB /GdA-, Heterozygous; "Heterozygote" GdA-/GdA-, Homozygous Deficient; "G6PD Deficient" Males
GdB, Hemizygous Normal; "Normal" GdA-, Hemizygous Deficient; "G6PD Deficient Four most common variants out of 300+ known
GdB Normal Activity All World Populations
Normal Activity; Aspartic acid substituted for Africa GdA asparagine at position 126, Guanine for adenine at DNA position 376 (most common variant)
8 - 20% Normal Activity; Methionine for Valine at position 67 and Aspartic Acid for GdA- Asparagine at position 126, Adenine for Africa Guanine at position 202 and Guanine for Adenine at position 376
< 5% Normal Activity; Phenylalanine for Iran, Iraq, India, Pakistan, Med Serine at position 188; Thymine for Cytosine Gd Greece, Sardinia at position 563 Glucose 6-Phosphate Dehydrogenase Different Isozymes
1 0.8
0.6 Level needed for protection vs ordinary oxidative stress 0.4 0.2
G6PD Activity (%) Activity G6PD 0 0 20 40 60 80 100 120 RBC Age (Days)
Normal (GdB) Black Variant (GdA-) Mediterranean (Gd Med)
G6PD Heterozygotes
Because of the random inactivation of one X chromosome in each female body cell, heterozygotes have two kinds of Red Blood Cells G6PD Normal G6PD Deficient Depending on which X chromosome was inactivated in the stem cell giving rise to the particular RBC
The Pentose Phosphate Pathway. Note the importance of G6PD in the production of reduced G-SH, ribose, and NADPH
NADP+ = nicotinamide adenine dinucleotide phosphate NADPH = reduced nicotinamide adenine dinucleotide phosphate GS-SG = oxidized glutathione G-SH = reduced glutathione G6PD DEFICIENCY Function of G6PD
Infections Drugs Hgb 2 H2O H2O2
Sulf-Hgb GSSG 2 GSH
NADPH NADP Heinz bodies 6-PG G6P G6PD Hemolysis
Function of G6PD
present in the cytoplasm of all cells
In (RBC), which lack nuclei, mitochondria, and other organelles, G6PD is particularly significant
G6PD is involved in the first step of the Pentose Phosphate Shunt Catalyzes the oxidation of Glucose-6-Phosphate to 6-Phosphogluconolactone (Phosphogluconate) Only source of NADPH and GSH, necessary for the reduction of hydrogen peroxide Hydrogen Peroxide is a strong oxidant that will degrade the RBC and cause hemolysis if it is not reduced Thiol-ester (-SH) groups found in many cellular enzymes are maintained in a reduced state by NADPH and GSH through the action of G6PD in the hexose monophosphate shunt (pentose phosphate pathway)
In the absence of an antioxidative mechanism, oxidant stress results in conversion of reactive thiol groups in haemoglobin to disulphides and sulphates, with ferriheme dissociation
sulfhaemoglobin accumulation, and finally large aggregates of precipitated haemoglobin (Heinz bodies) G6PD Activity
Declines with age of RBC
GdB has 62 day half-life for decay of activity Sustains GSH levels for 100 to 120 day RBC life span GdA- has normal activity when new, but the activity half-life is only 13 days Deficiency is due to instability of the enzyme GdMed has greater instability with 8 day half-life New cells already have reduced activity, and mature RBC have enzyme levels < 1% normal activity Glucose 6-Phosphate Dehydrogenase Functions
Regenerates NADPH, allowing regeneration of glutathione Protects against oxidative stress Lack of G6PD leads to hemolysis during oxidative stress Infection Medications Fava beans Oxidative stress leads to Heinz body formation The diagnosis can be confirmed by G6PD assay
The test may be negative during a hemolytic crisis when the older and defective RBCs are replaced by younger cells
In such cases, the test has to be repeated Diagnosis . Female heterozygotes may be difficult to detect as they have G6PD levels intermediate between those of hemizygous deficient males and normal individuals
G6PD screening during an acute attack may result in a false-normal result as reticulocytes have higher G6PD content than mature red cells
Heinz bodies may be detected during the early phase of haemolysis Symptoms of G6PD deficiency
G6PD deficiency is manifested as anemia, with RBCs being prematurely destroyed
RBCs are also extremely susceptible to oxidative stress
Neonatal jaundice is a yellowish discoloration sclera, skin, and mucous membranes caused by deposition of bile salts in these tissues The hemolytic crisis may manifest within hours of exposure to oxidant stress In severe cases, hemoglobinuria and peripheral circulatory collapse can occur Since only older red cells are affected most, the problem is usually self-limiting There will be a rapid drop in hematocrit, rise in plasma hemoglobin and unconjugated bilirubin.
Heinz bodies can be seen on crystal violet staining. These are removed in the spleen in a day or two and 'bite cells', with loss of a portion of the periphery of the red cell, may be seen Hemolytic episodes triggered by : viral or bacterial infections drugs or toxins that have an oxidating potential :
Antimalarials like primaquine, pamaquine, dapsone, sulfonamides like sulfamethoxazole, nitrofurantoin, vitamin K, doxorubicin, methylene blue, nalidixic acid, furazolidone etc. and naphthalene balls can cause hemolysis in defective individuals. severe form of this is a direct result of insufficient activity of the G6PD enzyme in the liver
In some cases, the neonatal jaundice is severe enough to cause death or permanent neurologic damage Acute haemolysis may occur
1. Drugs : aspirin, sulfonamides, antimalarials, nitrofurantoin, para-aminosalicylic acid, chloramphenicol, naphthalene, methylene blue and vitamin K analogues. 2. Following infections 3. After ingestion of broad beans (favism) 4. In the neonatal period (Icterus Neonatorum) chloroquine phenylhydrazine hydroxychloroquine pyridium mepacrine (quinacrine) quinine pamaquine toluidine blue pentaquine trinitrotoluene primaquine urate oxidase quinine vitamin K (water soluble) quinocide CYTOTOXIC/ANTIBACTERIAL CARDIOVASCULAR DRUGS chloramphenicol procainamide co-trimoxazole quinidine furazolidone SULFONAMIDES/SULFONES furmethonol dapsone nalidixic acid sulfacetamide neoarsphenamine sulfamethoxypyrimidine nitrofurantoin sulfanilamide nitrofurazone sulfapyridine PAS sulfasalazine para-aminosalicylic acid sulfisoxazole PLEASE READ DISCLAIMER Infection trigger
Oxidative metabolites produced by bacterial, viral, and rickettsial cause an anemic response
Viral hepatitis, pneumonia, and typhoid fever are particularly likely to precipitate a hemolytic episode in G6PD deficient individuals
Bite Cells Heinz Bodies
G6PD Deficiency: Acute haemolysis Reticulocytosis and "blister" or "bite" Blood film shows anisocytosis with a single blister cell. Heinz Bodies Haemoglobin denaturation leads to its precipitation as Heinz bodies. Heinz bodies bind to the red cell membrane and alter its rigidity, resulting in premature destruction in the spleen. The spleen also removes membrane-bound Heinz bodies from red cells resulting in "blister" or "bite" cells.
Favism
The Fava Bean (Vicia faba) is a favored cultigen in areas where the GdMed allele is common Vicine and convicine make up approximately 0.5% of the wet weight of the Fava bean These compounds metabolize to divicine and isouramil in the intestine These metabolites decrease RBC reduced glutathione (GSH) Produce hydrogen peroxide and free radicals Creates a severe oxidant stress in G6PD deficient cells The defect is known to provide partial protection against malaria, by providing defective environment in the affected red cells Plasmodium in the RBC
Plasmodium preferentially attack immature RBC but P. falciparum can invade RBC of all ages Plasmodium oxidizes RBC NADPH from the Pentose Phosphate pathway for its metabolism This results in a deficiency of RBC GSH, most severe in G6PD deficient individuals, leading to peroxide- induced hemolysis which curtails the development of Plasmodium After several cell cycles the Plasmodium can adapt to produce its own G6PD, reducing the adaptive benefit of G6PD deficiency Fava Beans and Malaria
Recall that fava beans contain compounds that metabolize to powerful oxidants In a cell that is oxidant-stressed by Plasmodium infection, the addition of another strong oxidant can lead a rapid build-up of peroxide In vitro and in vivo (mouse) studies indicate a suppressant effect of divicine and isouramil on Plasmodium in G6PD normals This effect would be expected to be even greater in G6PD deficient individuals Fava bean cultivation is widespread, especially throughout the circum-Mediterranean region
There is substantial overlap between the cultivation of fava beans and the GdMed allele
About 1 in 12 cases of favism results in mortality
Mostly affects children (up to 95% of cases) Responses to Favism
Mediterranean populations have developed several responses including food taboos, preparation techniques, and folk remedies
Highly susceptible groups including children and pregnant women are frequently forbidden to consume fava beans
Drying, soaking, and removing the skins appear to reduce toxicity
Increasing sugar consumption reduces the severity of an impending hemolytic crisis World distribution of G6PD deficiency. "The values shown by the different shadings are gene frequencies in the different populations"
No specific treatment is needed since the condition is usually self limiting
Rarely blood transfusions are indicated
Adequate urine output should be ensured MALARIA and the RED CELL A remarkable example of Darwinism in action. All elements -- blood groups on the envelope (e.g.Duffy), the hemoglobin, and the presence of G6PD deficiency are all involved in guarding against malaria. PYROVATE KINAZE Next most common enzymopathy after G6PD deficiency, but is rare
It is inherited in a autosomal recessive pattern and is the commonest cause of the so-called :
"congenital non-spherocytic haemolytic anaemias" (CNSHA) Pathophysiology
PK catalyses the conversion of phosphoenolpyruvate to pyruvate with the generation of ATP
Inadequate ATP generation leads to premature red cell death There is considerable variation in the severity of haemolysis
Most patients are anaemic or jaundiced in childhood
Gallstones, splenomegaly and skeletal deformities due to marrow expansion may occur
Aplastic crises due to parvovirus have been described. Since the erythrocyte has no mitochondria, it has no Krebs cycle. Its only source of ATP is through the Embden-Myerhof pathway (the hexose monophosphate shunt does not generate any high-energy phosphate bonds, thus no ATP)
ATP is needed primarily for the maintenance of the ATP-dependent potassium/sodium pump pyruvate kinase deficiency PK deficiency differs from G-6-PD deficiency
Mode of inheritance is autosomal recessive
Hemolysis is chronic and ongoing, unlike G-6-PD deficiency, which episodic and related to environmental exposures Blood film: PK deficiency: Characteristic "prickle cells" may be seen
Other red cell enzyme defects causing haemolysis Glycolytic pathway enzymes e.g. hexokinase, glucose phosphate isomerase, aldolase, phosphoglycerate kinase etc
Glutathione metabolism pathway enzymes e.g. glutathione synthetase, glutathione reductase etc.
Nucleotide metabolism pathway enzymes e.g. adenylate kinase, adenosine deaminase, pyrimidine 5' nucleotidase (associated with marked basophilic stippling) etc.
All are extremely rare