Glucose 6 Phosphate Dehydrogenase

Deficiency

Section A2Group 5:

Capacio, Diane ChloeCardeno, Sheree Anne Rachille

Casem, Princess Joy P.Castaneda, Felix

Castro, John MichaelCeneta, Raymond

After the discussion, the class will be able to:• Explain the Hexose Monophosphate Shunt

• Discuss the importance of G6PD as to the reaction it catalyzes, and its role in the HMP shunt

• Define the classes of mutations involved in G6PD, the characteristics of each class, and to what class of mutation case 1 and case 2 belong

• Determine the role of Glutathione in the development of hemolytic anemia in G6PD deficiency

• Enumerate the drugs that can precipitate hemolytic anemia among patients with G6PD deficiency

• Know the reason why patients who have G6PD have a lesser tendency in contracting malaria

• Define what DNA analysis is best for identification of carriers and patient diagnosis

CASE 1J.R., a 26 y/o black male had been in a good condition until 1 wk before he was admitted he developed signs of Respiratory infection and low grade fever. Self medication with over the counter cold preparation did not alleviate his Sx. 1 day before admission, he experienced chills and hacking cough.

P.E.:-Icterus -T: 40 C

-Bronchial breathing with rales -PR: 120bpm

-Dyspneic

Lab Findings includes: -Hgb: 8.4 g/dL

WBC: 18,000/µL -Leukocyte & Bilirubin: 3.2 mg/dL Reticulocyte: 1.2% -Gram positive diplococcic in sputum

DX: Pneumococcus Pneumonia

TX: PENICILLIN

Lab findings after treatment:(+) Pneumoccoci in sputum and blood

-Less pronounced dyspnea

-afebrile

-Hgb: 13.8 g/dL

-Reticulocyte count: 12%

The doctor advised him never to take aspirin and a long list of drugs that should be avoided. The patient experienced no further hemolytic episode.

D.M. at 1 year:

1 day of Ultraviolet

light tx:

After several days:

Family Hx:

6 y/o: After 1 year:

14 y/o:

-Bilirubin: 14 mg/dL

Tx: ultraviolet light

-Bilirubin: 17 mg/dL

-Hgb: fell 13 g/dL

-Bilirubin: lower readings

-Brother: anemia and darkening of the urine during RTI and UTI

-Dark urine during respiratory infection-Hgb 5.4 g/dL

-Splenectomy was done bec. of spherocytosis-Hgb: no change

-RBC is profoundly deficient in G6PD.-During infxn D.M. sometimes exp. Hemolytic episodes.

-Reticulocyte count: 5-10%

-Maternal uncle: intermittent jaundice and anemia

-Transfusion of 1 U packed RBC

-Hgb: rose to his usual level

-Blood contained nucleated red cells and red cells with nuclear fragments (heinz bodies)

-Platelet count: 700 x 10^9/L

G6PD catalyzes the oxidation of Glucose 6 Phosphate to 6 Phosphogluconolactone, while reducing Nicotinamide adenine dinucleotide phosphate (NADP) to reduced form (NADPH)

Most common enzymatic disorder of RBC in humans. (Cpt Ali R. Elyassi etal., 2009)

Its activity is profoundly regulated by NADP/NADPH ratio.

NADPH then reduces oxidized glutathione into reduced glutathione which in turn counteracts the toxic hydrogen peroxide(H2O2).

OVERVIEW:

1st Reporter: Casem, Princess Joy P.

THE HEXOSE MONOPHOSPHATE SHUNT

• Also known as the Pentose Phosphate Pathway (PPP)

• Has 2 major functions:a) Generates Nicotinamide Adenine Diphosphate

(NADP) for reductive synthesis – Fatty acid and Steroid biosynthesis; reduction of glutathione

b) Synthesis of Ribose for nucleotide and nucleic acid formation.

•2 Phases:a)Oxidative phaseb)Nonoxidative phase

Glucose 6-Phosphate

6-Phosphogluconolactone

6-Phosphogluconate

Ribulose 5-Phosphate

Glucose 6-Phosphate dehydrogenase

Gluconolactone hydrolase

6-Phosphogluconate dehydrogenase

NADP+

NADPH

NADP+ NADPH

Importance of G6PD

• Enzyme that catalyzes G6P to 6 Phosphogluconolactone

• 1st enzyme in the PPP

•Rate limiting enzyme of the PPP

• Requires NADP as hydrogen acceptor thereby reducing NADP to NADPH

•The PPP produces NADPH in the RBC membrane

G6PD deficiency in Hemolytic Anemia

- Most common red cell enzymopathy associated with hemolysis.

- G6PD enzyme is necessary for subsequent production of NADPH and glutathione through HMP shunt pathway.

-Reduced Glutathione protects enzymes and hemoglobin against oxidation by reducing hydrogen peroxide and free radicals.

- Heme is liberated from globin and globin denatures forming Heinz bodies.2nd Reporter: Castaneda, Felix II

Heinz bodies

- consist of denatured hemoglobin -best identified by staining with basic

dyes such as cresyl violet. - Occurs in individuals with a deficiency

of G6PD - causes of Heinz bodies formation:

Oxidative stress results from fava beans and primaquine consumption

leads to the formation of “Bite cell”

Heinz bodies

Bite cells

When RBC with heinz bodies passes the spleen, the spleenic macrophages will attack the hb removing along with it a part of RBC membrane while leaving the red cell intact. (pitting)

Bite cell

Mutation into the gene for G6PD

Activity of G6PD

Levels of NADPH

Regeneration of GSH (Reduced Glutathione) from GSSG (Oxidized) by Glutathione Reductase (which uses NADPH)

Oxidation due to decreased level of GSH and Increase level of Intracellular oxidants (e.g H2O2) of SH groups of Hb (forming heinz bodies) , and of membrane proteins, altering membrane structure and increasing susceptibility to ingestion by macrophages.

Hemolysis

Class 1—enzyme deficiency with chronic hemolytic anemia (<10% of normal activity)

Class 2—Class II variants also have severe enzyme deficiency, but there is usually only intermittent hemolysis.

Class 3—Class III variants have moderate enzyme deficiency (10 to 60% of normal) with intermittent hemolysis, usually associated with infection or drugs.

Class 4—have no enzyme deficiency nor hemolysis

Class 5—increased enzyme activity

Fortunately, only a small number of people fall into Class 1

Classes IV and V are of no clinical significance.

5 Classes of G6PD:

Drugs that can precipitate hemolytic anemia in patients with G6PD

3rd Reporter: Capacio, Diane Chloe

Definite Risk of Hemolysis Possible Risk of Hemolysis

Class Drugs Class Drugs

Antihelmintic Niridazole Analgesic AcetanilideAspirin

Antibiotic NitrofurantoinNalidixic acid

Sulfamethoxazole Sulfacetamide

Antibiotic Furazolidone

Antimalarial Primaquine Dx. Agent for CA

detection

Toluidine Blue

Antimethemoglobinemic

Methylene Blue Others NaphthaleneTrinitrotolueneUrate Oxide

Antimycobacterium

Thiazosulfone

Genitourinary analgesic

Phenazipyridine

This drugs may induce hemolysis in G6PD deficiency (Koda-Kimble, 1978)

In G6PD deficient individuals, oxidative stress may result in the denaturation, or unfolding, of the hemoglobin molecule, the principal oxygen carrying molecule inside the red blood cells.

This results in the loss of biological function with respect to hemoglobin and leads to the inability of the red blood cell to effectively transport oxygen throughout the body (Yoshida & Beutler, 1986). 

Overview about Malaria

Malaria is a febrile illness caused by sporozoa of genus Plasmodium, with four species which infects humans: P. falciparum, P. vivax, P. ovale, and P. malariae

Undergo developmental stages in the female Anopheles, which is a vector.

G6PD have lesser tendency to contact malaria

a deficiency in G6PD has been shown to sometimes confer a resistance to the malaria-causing parasite, Plasmodium falciparum  (Scriver et al., 1995).

P. falciparum – most severe form of malaria

Found in warm, moist climates and distributed over most tropical places like Africa, South East Asia etc.

This resistance is due to the fact that the parasite selectively infects red blood cells.

This renders mature erythrocytes more vulnerable to oxidative stress and the mechanism of protection produced by the G6PD deficiency condition.

In G6PD deficient red blood cells, an essential metabolite for the survival of the parasite is present in insufficient quantities.

A reduction in the amount of functional glucose-6-dehydrogenase appears to make it more difficult for this parasite to invade red blood cells.

This is due to decreased activity of G6PD within these cells which ultimately leads to the death of the parasite (Farid, personal interview).

Method in identifying patients and carriers with G6PD

Genetic analysis of DNA provides accurate results in patients without symptoms and those undergoing hemolysis.

Molecular techniques: Polymerase chain reaction amplification Restriction enzyme digestion

Polymerase chain reaction amplification

is a laboratory technique for "amplifying" a specific DNA sequence. PCR is extremely efficient and sensitive; it can make millions or billions of copies of any specific sequence of DNA.

Restriction enzyme digestion

are enzymes isolated from bacteria that recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments. Restriction enzymes play a very important role in the construction of recombinant DNA molecules.

Laboratory Diagnostic Tests

Serum Direct Bilirubin Level Complete blood count Haptoglobin Level Reticulocyte count

Serum Direct Bilirubin Test

an important part of routine newborn (neonatal) diagnostic screening tests.

The level of bilirubin in a newborn's blood serum is measured to determine if the circulating level of bilirubin is normal or abnormal.

Case 1 Lab findings:

bilirubin test = 3.2 mg/dL

Normal: 0.1-0.4mg/dL

Complete Blood Count (CBC)

It is a screening test used to diagnose and manage numerous diseases. The results can reflect problems with fluid volume (such as dehydration) or loss of blood.

The test can reveal problems with red blood cell production and destruction, or help diagnose infection, allergies, and problems with blood clotting.

Case 1 Lab findings:

Hemoglobin count = 8.4 g/dL

(N= above 14 g/dL)

WBC = 18,000/uL

(N= 5,000 – 10,000/uL)

Case 2 Lab findings:

Hemoglobin count = 13.8 g/dL

Haptoglobin Test

is a blood protein made by the liver. The haptoglobin levels decrease in hemolytic anemia. Hemolytic anemias include a variety of conditions that result in hemolyzed, or burst of the red blood cells.

Normal: 27-139mg/dL

Reticulocyte Count The test is done to determine if red blood

cells are being created in the bone marrow at an appropriate rate.

The number of reticulocytes in the blood is a sign of how quickly they are being produced and released by the bone marrow.

Normal: 1 – 2% adult1 - 3% infant

Case 1 Lab findings:

Reticulocyte count = 1.2%

(N= 1 – 2%)

Case 2 Lab findings:

Reticulocyte count = 10%

NORMAL VALUES: RBC (varies with altitude):

Male: 4.7 to 6.1 million cells/mcL Female: 4.2 to 5.4 million cells/mcL

WBC: 4,500 to 10,000 cells/mcL Hematocrit (varies with altitude):

Male: 40.7 to 50.3 % Female: 36.1 to 44.3 %

Hemoglobin (varies with altitude): Male: 13.8 to 17.2 gm/dL Female: 12.1 to 15.1 gm/dL

REFERENCES:Elyassi, Rowsh 2009. Perioperative Management of the Glucose-6-Phosphate Dehydrogenase

Deficient Patient:A Review of Literature

Murray, Bender, Botham, Kennely, Rodwell, Weil. Harper’s illustrated Biochemistry 28th ed.

Miles 2003. Pentose Phosphate Pathway Aka the Hexose Monophosphate Shunt

Lucio Luzzatto and Vincenzo Poggi 2008. Glucose-6-Phosphate Dehydrogenase DeficiencyGregg XT, Prchal JT. Red blood cell enzymopathies. In: Hoffman R, Benz Jr. EJ, Shattil SJ, et al., eds.

Hematology: Basic Principles and Practice. 5th ed. Philadelphia, Pa: Churchill Livingston; 2008:chap 45.

Philippine Pharmaceutical Directory Review 5th Edition. Jocelyn J. Yambao et al. Ed. Medicom Pacific Inc. 2005.

MIMS Philippines. Volume 31, Number 3, 2002. Medi Media. 2002.Yearbook of Physical Anthropology Vol. 36. Lawrence S. Greene et al. 1993.Principles of Pharmacology: A Tropical Approach. D.T. Okpako et al. 1991.

Cappellini MD, Fiorelli G. Glucose-6-phosphate dehydrogenasedeficiency. Lancet. 2008;371 :64^74.

World Health Organization.Working group glucose-6-phosphate dehydrogenase deficiency. Bull WHO. 1989;67: 601^611.

Beutler E. The Molecular biology of enzymes of erythrocyte metabolism. In: Stamatoyannopoulos G, Nienhus AW, Majerus PW, et al, eds:The Molecular Basis of Blood Disease. Philadelphia:WB Saunders; 1993.

Harper’s illustrated biochemistry, 28 editionhttp://rialto.com/g6pd

http://themedicalbiochemistrypage.org/g6pdhdeficiency.html

http://www.answers.com/topic/polymerase-chain-reaction