ANEMIAS AND OTHER RED CELL DISORDERS - PART 9 ppt

䊏 CLASSIFICATION OF G6PD DEFICIENCY SYNDROMES 306 䊏 LABORATORY MANIFESTATIONS OF G6PD DEFICIENCY 311 Glucose-6-phosphate dehydrogenase G6PD deficiency is the most common throcyte enzyme d

of these patients.27 A former childhood disorder is increasingly a disease of adults.Widespread screening of individuals from populations whereβ-thalassemia trait is

prevalent has enabled prenatal diagnosis of affected fetuses In Greek American andItalian American communities, the number of affected infants has sharply decreased.The number of children with thalassemia in the United States is rising however

as a result of the increase in the number of Americans of Southeast Asian ancestry.These more recent arrivals to the country have not yet achieved the cohesive approach

to thalassemia that proved so successful in other American communities Until thisgoal is reached, thalassemia will remain an important issue in American medicine(Tables 14-3 and 14-4)

References

1 Cooley TB, Witwer ER, Lee P 1927 Anemia in children with splenomegaly and peculiarchanges in bones: Report of cases Am J Dis Child 34:347–355

2 Olivieri NF 1999 The beta-thalassemias N Engl J Med 341:99–109

3 Wasi P, Winichagoon P, Baramee T, Fucharoean S 1982 Globin chain synthesis in erozygous and homozygous hemoglobin E Hemoglobin 6:75–78

het-4 Fairbanks VF, Gilchrist GS, Brimhall B, Jereb JA, Goldston EC 1979 Hemoglobin E traitrevisited: A cause of microcytosis and erythrocytosis Blood 53:109–115

5 Fairbanks VF, Oliveros R, Brandabur JH, Willis RR, Fiester RF 1980 Homozygoushemoglobin E mimics b-thalassemia minor without anemia or hemolysis: Hematologic,functional and biosynthetic studies of first North American cases Am J Hematol 8:109–121

6 Rees DC, Styles L, Vichinsky EP, Clegg JB, Weatherall DJ 1998 The hemoglobin Esyndromes Ann N Y Acad Sci 850:334–343

7 Fucharoen S, Ketvichit P, Pootrakul P, Siritanaratkul N, Piankijagum A, Wasi P 2000.Clinical manifestation of beta thalassemia/hemoglobin E disease J Pediatr Hematol Oncol22:552–557

8 Chui DH, Fucharoen S, Chan V 2003 Hemoglobin H disease: Not necessarily a benigndisorder Blood 101:791–800

9 Chen FE, Ooi C, Ha SY, et al 2000 Genetic and clinical features of hemoglobin H disease

in Chinese patients N Engl J Med 343:544–550

10 Scopinaro F, Banci M, Vania A, et al 1993 Radioisotope assessment of heart damage inhypertransfused thalassaemic patients Eur J Nucl Med 20:603–608

11 Lattanzi F, Bellotti P, Picano E, et al 1993 Quantitative ultrasonic analysis of myocardium

in patients with thalassemia major and iron overload Circulation 87:748–754

12 Prennell DJ, Bland JM 2003 Deferiprone versus desferrioxamine in thalassemia, and T2∗validation and utility Lancet 361:182–184

13 Strickland GT, Elhefni H, Salman T, et al 2002 Role of hepatitis C infection in chronicliver disease in Egypt Am J Trop Med Hyg 67(4):436–442

14 Darwish MA, Faris R, Darwish N, et al 2001 Hepatitis C and cirrhotic liver disease in theNile delta of Egypt: A community-based study Am J Trop Med Hyg 64:147–153

15 Seeff LB, Hoofnagle JH 2003 Appendix: The National Institutes of Health ConsensusDevelopment Conference Management of Hepatitis C 2002 Clin Liver Dis 7:261–287

16 Gupta S, Bent S, Kohlwes J 2003 Test characteristics of alpha-fetoprotein for detectinghepatocellular carcinoma in patients with hepatitis C A systematic review and criticalanalysis Ann Intern Med 139:46–50.

17 McCord JM 1993 Human disease, free radicals, and the oxidant/antioxidant balance ClinBiochem 26:351–353

18 Farber JL 1994 Mechanisms of cell injury by activated oxygen species Environ HealthPerspect 102:17–24

19 Enright HU, Miller WJ, Hebbel RP 1992 Nucleosomal histone protein protects DNA fromiron-mediated damage Nucleic Acids Res 20:3341–3346

20 Bonkovsky, HL 1991 Iron and the liver Am J Med 301:32–43

21 Link G, Konijn AM, Hershko C 1999 Cardioprotective effect of alpha-tocopherol, bate, deferoxamine, and deferiprone: Mitochondrial function in cultured, iron-loaded heartcells J Lab Clin Med 133:179–188

ascor-22 Aldouri MA, Wonke B, Hoffbrand AV, et al 1990 High incidence of cardiomyopathy inbeta-thalassaemia patients receiving regular transfusion and iron chelation: Reversal byintensified chelation Acta Haematol 84:113–117

23 Olivieri NF, Nathan DG, MacMillan JH, et al 1994 Survival in medically treated patientswith homozygous beta-thalassemia N Engl J Med 331:574–578

24 Davis B, J Porter 2000 Long-term outcome of continuous 24-hour deferoxamine infusionvia indwelling intravenous catheters in high-risk beta-thalassemia Blood 95(4):1229–1236

25 Lucarelli G, Galimberti M, Polchi P, et al 1993 Marrow transplantation in patients withthalassemia responsive to iron chelation therapy N Engl J Med 329:840–844

26 Lucarelli G, Galimberti M, Giardini C, et al 1998 Bone marrow transplantation in lassemia The experience of Pesaro Ann N Y Acad Sci 850:270–275

tha-27 Pearson HA, Giardina P, Cohen A 1996 The changing profile of thalassemia major atrics 97:352–356

Enzymopathies

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䊏 CLASSIFICATION OF G6PD DEFICIENCY SYNDROMES 306

䊏 LABORATORY MANIFESTATIONS OF G6PD DEFICIENCY 311

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common throcyte enzyme defect, affecting over 400 million people The initial descriptions ofthe disorder arose in the wake of peculiar outbreaks of hemolytic anemia in militarypersonnel in the Pacific theater during World War II following prophylactic treatmentwith the antimalarial drug Primaquine.1A number of unusual attributes characterizedthe hemolytic episodes and whetted interest in the relationship between Primaquineand hemolysis Although the problem involved a significant number of people, only

ery-a minority of the group who used the drug were ery-affected The episodes of hemolysiswere self-limiting, with a sometimes explosive early phase followed by spontaneousrecovery After the initial hemolytic episode, susceptible people could continue Pri-maquine treatment without further problem A break of several months in Primaquineexposure saw a recrudescence in hemolytic sensitivity

The demographics of the problem also were unusual The hemolytic episodesoccurred almost exclusively in people of African heritage, pointing to an ethnic com-ponent of the susceptibility The familial pattern of drug sensitivity reinforced belief

305

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T A B L E 15 - 1 CLINICAL CLASSIFICATION OF G6PD SYNDROMES

Residual G6PD Class Activity (% normal) Clinical Characteristics

I <20 Chronic nonspherocytic hemolytic anemia (CNSHA)

Neonatal jaundice

II <10 Severe episodic hemolysis often related to drugs or other

oxidant exposure Fava bean mediated hemolysis.Neonatal jaundice

III 10–60 Episodic hemolysis often related to drug exposure

Hemolysis with infections Neonatal jaundice inpremature infants

IV 90–100 No clinical symptoms

in the genetic nature of the disorder Furthermore, men were the almost sole victims

of Primaquine-induced hemolysis, indicating an X-linked pattern of inheritance Inretrospect the episodes of hemolysis were a result of a form of G6PD deficiency thatoriginated in sub-Saharan Africa and spread to the New World as part of the Africandiaspora driven by the slave trade

Further investigation uncovered the existence of many other types of G6PD ciencies with varying manifestations and severity Over time, more than 400 descrip-tions of mutations in the gene encoding G6PD appeared in the literature.2While some

defi-of the mutations, such as those affecting African Americans, are common, most areextremely rare Also, the clinical characteristics of the G6PD deficiency varied sig-nificantly The WHO devised a classification that divided G6PD deficiency into fivegroups, depending on the clinical manifestations (Table 15-1) The strikingly differentpresentations, clinical characteristics, and manifestations seen within these subgroupsreflect the degree to which the G6PD enzyme levels fall below the normal range

䊏 CLASSIFICATION OF G6PD DEFICIENCY SYNDROMES

Table 15-1 outlines the five clinical classes of G6PD variant syndromes as categorized

by the WHO The Class I G6PD deficiency phenotype derives from a marked cyte enzyme deficiency The extreme sensitivity of these cells to oxidant stress pro-duces ongoing hemolysis with a moderately severe chronic nonspherocytic hemolyticanemia (CNSHA), with an associated marked reticulocytosis as the most common

erythro-manifestation Class II syndromes exhibit extreme enzyme deficiency in which G6PDvalues average less than 10% of the normal value Affected patients are healthy atbaseline but are susceptible to fulminant and sometimes life-threatening episodes

of hemolysis when exposed to certain oxidizing agents The Class III syndromeshave mild to moderate enzyme deficit This group was the primary subject of thePrimaquine studies The Class IV G6PD deficiency syndromes show at most onlymildly depressed enzyme levels and no clinical symptoms Class V, a phenotype alsowithout clinical manifestations, rounds out the categorization of the condition withG6PD enzymes whose activity exceeds the normal value

The G6PD deficiency syndromes show patterns of geographic and ethnic tering The Class III variety is the most prevalent form in people of African ethnicheritage, as noted earlier Class II G6PD deficiency occurs commonly, but not exclu-sively, in people of Mediterranean background.3 Both Class II and Class III G6PDdeficiency variants are common in China and Southeast Asia.4Knowledge of a pa-tient’s ethnic background greatly aids in the evaluation and treatment of hemolyticepisodes where G6PD deficiency is the possible culprit

clus-CLASS I G6PD DEFICIENCY

Patients who fall under this rubric typically have chronic hemolytic anemia that often

is moderate in severity, thanks to a robust compensatory reticulocyte response Class IG6PD deficiency manifests most commonly as congenital nonspherocytic hemolyticanemia The very low G6PD levels in the erythrocytes render these cells incapable

of withstanding the normal levels of oxidant stress visited upon all erythrocytes Thecompensating reticulocytosis commonly ranges between 10% and 40% Interestingly,splenomegaly is not a major manifestation of the condition Occasional patients withcongenital nonspherocytic hemolytic anemia due to G6PD deficiency develop recur-rent infections This problem apparently reflects dampening of neutrophil-generatedreactive oxygen species that are essential to the bacteriocidal capability of thesephagocytes.5,6

The marked elevation of bilirubin levels that sometimes occurs in the neonatalperiod raises the risk of kernicterus Although hemolysis contributes to the highbilirubin levels, an additional factor is a neonatal deficiency of the hepatic glucuronyltransferase enzyme with consequent impairment of bilirubin metabolism Neonatalhyperbilirubinemia often arises in association with Gilbert’s syndrome.7–9Supportivetreatment including phototherapy or even exchange transfusions may be necessary tocontrol hyperbilirubinemia

Most patients with CNSHA hemolytic anemia due to G6PD deficiency maintainhemoglobin levels in the range of 8–10 g/dL and compensate for anemia reasonablywell Epistatic factors may influence the degree of the anemia associated with thecondition, since siblings sometimes have very disparate courses Rarely, an anemiaarises whose severity necessitates chronic transfusion to maintain a reasonable level

of activity and comfort The lack of splenic enlargement associated with congenitalnonspherocytic hemolytic anemia due to Class I G6PD deficiency parallels the failure

of splenectomy to benefit most such patients

T A B L E 15 - 2 REPRESENTATIVE AGENTS CAUSING HEMOLYSIS IN

G6PD DEFICIENCY∗

Antibiotics Sulphonamides, Co-trimoxazole (Bactrim, Septrin), Dapsone,

Chloramphenicol, Nitrofurantoin, Nalidixic acidAntimalarials Chloroquine, Hydroxychloroquine, Primaquine, Quinine,

MepacrineOther drugs Aspirin, Phenacitin, Sulphasalazine, Methyldopa, pharmacological

doses of vitamin C, Hydralazine, Procainamide, Phenothiazine,vitamin K, penicillamine

Anthelmintics β-Naphthol, Stibophen, Niridazole

Chemicals Mothballs (naphthalene), Methylene blueFoods Raw fava beans (broad beans)

Infections Bacterial infection; viral infection, hepatitis

∗The drugs and agents listed are representative examples only Susceptibility to hemolytic episodes variesdepending on the subtype of G6PD deficiency Definitive information comes from the WHO Glucose-6- phosphate Working Group, Bull WHO 1989, 67:610.

The maintenance of a hemoglobin value that is both stable and substantive depends

on the brisk and ongoing production of new red cells Any phenomenon or process thatheightens hemolysis can precipitate life-threatening anemia Even small quantities ofoxidant drugs such as those listed in Table 15-2 can trigger massive hemolysis and adramatic fall in hematocrit Equally dangerous are events that dampen production ofnew erythrocytes Parvovirus B19 infection, for instance, shuts off erythropoiesis forseveral days, creating an anemia of life-threatening severity

CLASS II G6PD DEFICIENCY

Favism is a striking phenomenon that occurs in many people with Class II G6PD ficiency where spectacular and occasionally life-threatening hemolysis follows con-

de-sumption of raw, uncooked fava beans (Vicia fava) These innocent legumes of the

broad bean family are common throughout the world and are nutritional staples formillions of people The form of G6PD deficiency common in the Mediterranean ren-ders red cells particularly susceptible to hemolysis after consumption of raw favabeans The syndrome has been known for centuries without, of course, an under-standing of its basis In some Mediterranean countries, hospitals geared up each yearduring planting or harvesting of the beans for the expected influxes of people stricken

with favism In some regions, such as Sardinia, the economic and social burden offavism was enormous.

The particulars of the syndrome are puzzling and suggest that factors other thanexposure to the bean contribute to or modify the degree of hemolysis Perhaps the mostcurious aspect of favism is that not every exposure to the beans produces hemolysis

in people with the Mediterranean variety of G6PD deficiency Some people consumefava beans for years without difficulty and then are felled by a hemolytic episode.Others are so sensitive to the active agent in fava beans that hemolysis can developafter mere exposure to pollen from the plant Interestingly, hemolysis is more common

in children than in adults, with as many as three-quarters of episodes occurring inchildren between the ages of 2 and 10 years

As many as 48 unremarkable hours commonly pass following ingestion of thebeans The child then develops lethargy, sometimes in conjunction with confusion and

a mild fever Nausea, abdominal pain, and diarrhea are other nonspecific tions A more telling feature (in retrospect) is back pain, which commonly accompa-nies acute hemolytic episodes of any cause The alarming and illuminating aspect ofthe illness that usually brings the child to medical attention is the passage of dark red

manifesta-or brown urine Physical examination at this point reveals frank jaundice, pallmanifesta-or, andtachycardia Moderately severe anemia quickly follows, often with the hemoglobinfalling to range of 4–7 g/dL Although extremely severe cases that threaten circulatorycollapse sometimes demand transfusion therapy,10most children recover without thisintervention The hemoglobin level usually returns to normal in 4–6 weeks

The precise basis of hemolysis induced by fava beans in people with the ranean variety of G6PD deficiency is unclear The likely mediators are vicine andconvicine, constituents in the beans that are metabolized to compounds capable ofgenerating reactive oxygen species The extreme deficiency of G6PD leaves the redcells compromised and very susceptible to oxidant damage and consequent hemol-ysis The delay in the onset of manifestations following consumption of fava beansprobably represents the time required to convert the latent compounds to their activemetabolites Favism is only one clinical manifestation of Class II G6PD deficiency.People with the condition are also susceptible to oxidizing compounds and drugs such

Mediter-as those listed in Table 15-2 Neonatal jaundice with possible kernicterus is anotherissue in Class II G6PD deficiency The problem of hyperbilirubinemia in the newbornreflects both decreased hepatic Conjugation of bilirubin as well as hemolysis

An interesting neonatal syndrome sometimes designated “Greek Baby Jaundice”

is common in newborns from Greece and the islands of the Aegean Sea, characterized

by extreme hyperbilirubin and a risk of kernicterus Affected babies can be shown tohave Type II G6PD deficiency; however, unknown environmental factors must also beoperative, because the syndrome is not seen with any frequency in children of Greekfamilies who have emigrated to North America or Australia

CLASS III G6PD DEFICIENCY

Class III G6PD deficiency is associated with only a modest depression of the throcyte enzyme level (Table 15-1) The most commonly encountered variety of

ery-condition in the United States is that seen in African Americans, which is designated

as the A−subtype of G6PD deficiency People with the condition ordinarily have anormal hematological profile and most are unaware of any issue with respect to theirred cells Problems arise only with stressful conditions such as exposure to drugs(Table 15-2) that can produce significant red cell oxidant damage

Most drugs and chemicals that produce problems do so after an initial sion to metabolites that then generate oxidant species The result is a delay of 1–2days between drug exposure and the manifestations of hemolysis An investigation

conver-of hemolysis possibly related to G6PD deficiency must include potential exposuresoccurring in the days preceding the attack The severity of hemolytic reactions variessubstantially between patients Furthermore, separate episodes of hemolysis experi-enced by a single patient can vary in course and severity Other as yet poorly under-stood modifying factors likely contribute to the inconsistent hemolytic reactions inpatients with G6PD deficiency of the A−variety

On the first day of the hemolytic reaction, patients commonly experience jaundiceand dark urine Some develop back pain and a few experience low-grade fever Ageneral malaise afflicts the patient often without specific localizing features Pallor

is a prominent aspect of the clinical complex, but detection often requires morethan a glancing observation in patients with dark skin Pale conjunctivae, mucousmembranes, and nail beds are the features most easily assessed The low hemoglobinvalue often produces prominent retinal pallor in which the eye grounds take on asalmon color

The hemoglobin often falls to levels that are 3–4 g/dL below baseline Palpitationsand dyspnea are common complaints and tachycardia accompanies these symptoms.The precipitous decline in the hemoglobin value produces lightheadedness and dizzi-ness in many patients Older people can experience serious secondary side effectsincluding angina and peripheral edema Nausea and sometimes vomiting are lessfrequent clinical features

The focus on drugs as agents of hemolysis in G6PD deficiency is an able result of the key role played by drug exposure in bringing the disorder to themedical spotlight Worldwide, however, infection likely is the most common cause of

understand-hemolysis due to deficiency of the enzyme Bacterial processes, such as Streptococcus

pneumoniae infections, are quite prominent in this regard Viral hepatitis often is at

the root of episodes of hemolysis in people with G6PD deficiency The variability

in the association between infection and hemolysis exceeds that seen with the druginsult The precise mechanism by which infection produces hemolysis in people withG6PD deficiency is unknown Many factors undoubtedly contribute in small ways tothe final effect One speculation on mechanism posits that reactive oxygen speciesgenerated by phagocytes in the battle with bacteria somehow set off a chain reactionwith inadvertent oxidant injury to susceptible erythrocytes.11

Hemolytic episodes in people with Class III G6PD deficiency rarely produce thelife-threatening scenarios that can arise with Class I or Class II deficiency The pro-portion of cells in the circulation that are susceptible to oxidant-mediated hemolysis

is much lower than in the latter two conditions Recovery from the acute hemolyticreaction proceeds due to replenishment of the circulation with young erythrocytes

containing sufficient G6PD to resist further oxidant assault A recovery phase occurswithin about 10 days of the initial insult even with continued administration of theoffending drug By 3–4 weeks of chronic exposure to the oxidant drug, a new steadystate ensues wherein a higher reticulocyte count (usually about twofold greater than

baseline) maintains a normal hemoglobin value Interestingly, the K m of the G6PD

A−variant for G6P is normal The problem for the red cell is poor enzyme stabilitythat leads to depletion in older erythrocytes (see below) The fresh reticulocytes haveabundant and active G6PD with which to resist oxidant assault Neonatal jaundiceassociate with Class III G6PD may occur in babies whose mothers ingested mothballsprior to delivery; and hyperbilirubinemia may occur in premature African Americanbabies, even without known drug exposures

CLASS IV AND V G6PD VARIANTS

Class IV and V G6PD variants are rare anomalies that do not produce clinical lems People with Class IV variants have G6PD activity that is either normal or onlyslightly below normal The very unusual Class V variants have G6PD activity that ex-ceeds the normal value The lack of clinical symptoms associated with these variantsmeans that serendipity commonly underlies their discovery

prob-䊏 LABORATORY MANIFESTATIONS OF G6PD DEFICIENCY

Hemolysis due to G6PD deficiency invariably lowers the hemoglobin level Thedegree of decline depends on the class of the deficiency state and the magnitude ofthe oxidant insult Mild episodes might register a fall in hemoglobin of only 1 or

2 g/dL to values of 9 or 10 g/dL In contrast, the hemoglobin might plunge to values

as low as 3 or 4 g/dL in people with severe G6PD deficiency

The intravascular red cell destruction produces prominent hemoglobinemia andhemoglobinuria Massive release of hemoglobin into the circulation reduces hap-toglobin levels to zero Methemalbumin is prominent when looked for Urinaryhemosiderin appears within several days of the hemolytic episode and can persistfor weeks following the insult While assay for hemopexin is usually a research tool,the protein is invariably absent in the aftermath of hemolysis due to G6PD deficiency.Hemolysis due to G6PD deficiency dramatically alters erythrocyte morphology.Some deformed red cells appear to have bites taken out of them, producing thecharacteristic “bite cells” that mark this type of hemolysis (Figure 15-1) Occasionalspherocytes and other nonspecifically deformed red cells also appear in the peripheralblood Prominent polychromasia reflects the large number of reticulocytes that pourout in the wake of the hemolytic episode

Clumps of denatured hemoglobin, called Heinz bodies, form within the raddledred cells Visualization of Heinz bodies by ordinary microscopy is difficult due to thesubtle nature of these inclusions Wright-Geimsa staining shows dark, irregular bulges

or blisters at the edges of some cells presumably due to clusters of Heinz bodies in

FIGURE 15–1 Peripheral blood with drug-induced G6PD hemolysis The smear is 3 days into the acute hemolytic episode Several bite cells appear on the smear along with other nonspecific shape anomalies Small bulges exist along the edge of some of the cells (From Kapff CT and Jandl JH 1991 Blood: Atlas and Sourcebook of Hematology, 2nd edn Boston: Little, Brown and Company Figure 23-1, p 53 Reproduced with permission of the publisher.)

the region Although phase contrast microscopes easily show these anomalous ies within erythrocytes, most clinical laboratories lack these relatively sophisticatedinstruments A more readily available means of visualizing Heinz bodies involvesstaining the red cells with crystal violet or methyl violet in supravital preparations(Figure 15-2) However, Heinz bodies are not evident after 24–48 hours

bod-Heinz bodies cause a number of problems for red cells Methemoglobin mulates due to the dearth of NADPH to support methemoglobin reductase activity.Hemoglobin denatures and the oxidized heme slips out of its usual site in the heme-binding pocket The resulting amalgam forms hemichromes that can themselves fa-cilitate further formation of reactive oxygen species This downward spiral due tooxidant injury dooms the G6PD-deficient erythrocytes

accu-Heinz bodies also adhere to the red cell membrane where they disrupt ture, promote oxidation and cross-linking of lipids and proteins, and impair function

architec-of membrane-associated enzymes including ion channels Cross-links architec-of membranestructures produce rigid red cells with impaired ability to pass through capillaries

FIGURE 15–2 Heinz body prep of G6PD deficient cells The red cells are ghostly outlines with the Heinz bodies appearing as dark inclusions.

The spleen briskly clears the circulation of these decrepit red cells Disturbed ionchannel activity produces erythrocyte swelling and rupture Some cells show bizarredistributions of hemoglobin with protein confined to one portion of the cell and aghostly appearance to the remainder

Reticuloendothelial cells rapidly remove these spavined erythrocytes from thecirculation Sometimes an erythrocyte rips free of the reticuloendothelial cell, leaving

a large rent in its membrane The cells appear to have bites taken out of them, givingrise to the common moniker “bite cells.” Many such cells are scalloped and somehave thin peduncles reflecting narrow escapes from reticuloendothelial cell assault.The spleen eventually clears the circulation of these aberrant erythrocytes

Levels of unconjugated bilirubin rise during the hemolytic acute attack, mirroringthe jaundice on physical examination Small rises in the level of conjugated bilirubinoccur occasionally, particularly with massive hemolysis Transient, small elevations

in BUN occur most often in the wake of marked hemolysis

The high RDW in the condition reflects the tremendous size variation of red cellsthat include reticulocytes, cell fragments, and distorted cells The augmented marrowactivity that produces the reticulocytosis also raises production of neutrophils This

at times confuses the distinction between hemolysis due to infection and that due tooxidant agents The platelet count typically varies little from baseline

䊏 G6PD AND RED CELL METABOLISM

The monomer subunit of G6PD is a 59-kDa polypeptide Two subunits join to create

a protein dimer that includes a tightly bound NADP molecule The enzymatically

Auto-oxidant drugs; Natural oxidant reactions

Methemoglobin reductase;

other reduction reactions

G6P Dehydrogenase

2 O 2

H 2 O GSH

NADPH 6PG

GSSG Reductase

GSH Peroxidase

FIGURE 15–3 Schematic representation of the hexose monophospate (HMP) shunt G6PD dehydrogenase converts glucose-6-phosphate (G6PD), the initial product of glycolysis, into 6-phosphogluconate (6PG) with the coupled conversion of NADP to NADPH This reaction is the sole source of NADPH in the red cell, which is needed for a number of important metabolic functions including the rescue of hemoglobin from constant oxidation to methemoglobin A host of normal metabolic processes generate hydrogen peroxide (H2O2) as a usual byproduct This compound, along with other pernicious reactive oxygen species that it produces, has the potential of wrecking widespread cell injury including the cross-linking of lipids and proteins.

In the last step of the HMP shunt, glutathione peroxidase converts this destructive chemical to water.

inactive dimer is in equilibrium with the fully functional tetramer form of the molecule.Structural studies show that the NADP lodges near the dimer contact point and isessential to molecular stability.12

G6PD provides red cells with crucial protection against oxidant damage 6-phosphate is the first metabolite produced in glycolysis The Embden-Meyerhofpathway is the primary metabolic route for the compound, producing ATP along theway and generating lactate in the final step A portion of the glucose-6-phosphate takes

Glucose-an alternate route involving the hexose monophosphate (HMP) shunt The metabolicsteps of the HMP eventually swing back to merge with the Embden-Meyerhoff path-way leading to lactate Along the way, however, the HMP shunt produces severalcompounds important to the health of the cell

Figure 15-3 is a simplified representation of the HMP shunt G6PD catalyzesthe first step that converts glucose-6-phosphate to 6-phosphogluconate The NADPH

generated from NADP in the process provides the cell with a metabolite that iscentral to many reductive biochemical reactions Hydrogen peroxide (H2O2) produced

by natural processes in the cell oxidizes heme iron from the Fe(II) valance state

to Fe(III), thereby daily converting as much as 3% of hemoglobin to functionallyinactive methemoglobin The NADPH-dependent enzyme, methemoglobin reductase,regenerates hemoglobin from methemoglobin allowing continued normal oxygendelivery

H2O2transforms into other reactive oxygen species including the highly volatilehydroxyl radical (•OH) These destructive molecules attack carbon–carbon doublebonds of unsaturated fatty acids, producing fatty acid hydroperoxides that promotefurther cleavage and cross-linking of carbon–carbon bonds in membrane lipids Thedamaged regions of membrane loose their fluidity and accumulate aggregates of pro-teins that normally are integral to or closely associated with the membrane Thesechanges alter both the biophysical and biochemical properties of red cell mem-branes and promote hemolysis As Figure 15-2 shows, glutathione peroxidase defusesH2O2 before it can wreck havoc on the cell, a job shared with catalase Glutathioneperoxidase performs the additional valuable function of decomposing fatty acidperoxides

G6PD deficiency blunts the efficiency of this entire cascade of events The degree

of enzyme deficiency markedly influences the clinical character of the condition.Syndromes in which the G6PD deficit is only mild or moderate, such as occurs withClass III deficiency, show hemolysis only with major insults that rain vast quantities

of H2O2 on the cell People with Class I G6PD deficiency in contrast are in suchdelicate balance with the forces producing oxidant cell stress that a relatively minorupswing in cellular oxidant activity can mean disaster

Red cell G6PD levels normally decline slowly with age, due to among other thingsprotein inactivation by oxidant damage Reticulocyte levels of G6PD in fact exceed

by nearly fivefold those of the oldest erythrocytes Figure 15-4 schematically showsthat the normal decay of G6PD maintains sufficient levels of the enzyme to protecterythrocytes from exorbitant oxidant damage out to the end of the cell life span The

enzyme produced by the G6PD A−gene is unstable and decays much more rapidlythan normal Consequently, older cells fall below the protective threshold and arevulnerable to the type of oxidant stress produced by Primaquine, for instance Afterthe initial bout of hemolysis following exposure to an oxidant drug, the compensatoryreticulocytosis produces young cells containing sufficient enzyme to defend the cellintegrity A new steady state maintains the hemoglobin in the normal range even withcontinued exposure to the oxidant drug

The G6PD Mediterranean deficiency, in contrast, results from an extremely ble enzyme whose half-life in the cell is exceedingly short As indicated in Figure 15-3,most of the cells fall into the oxidant danger zone soon after their production Themassive hemolysis seen with this form of G6PD deficiency reflects the large number

unsta-of vulnerable erythrocytes in the circulation Maximally augmented erythropoiesis unsta-ten fails to keep pace with the rate of hemolysis creating a potentially life-threateningsituation

of-Red Cell G6PD Level

100 %

0 %

Red Cell Life Span

is very unstable and falls quickly to low levels that leave a large fraction of circulating red cells susceptible to destruction by oxidant assault.

䊏 THE MOLECULAR BIOLOGY OF G6PD DEFICIENCY

The gene encoding G6PD located on the X-chromosome, X(q28), is over 20 Kb inlength and contains 13 exons.13Most of the hundreds of known mutations are pointmutations that produce some degree of reduced enzymatic activity A few mutants havenormal or even enhanced enzymatic activity The absence of a mutant that producescomplete enzyme loss suggests that complete G6PD deficiency is incompatible withlife

Table 15-3 provides information on some common and representative examples ofG6PD deficiency The designation “G6PD B” attached to the normal enzyme derivesfrom its mobility on gel electrophoresis relative to the more rapidly migrating bandseen commonly in African Americans, which is designated “G6PD A+.” The alteredmobility of G6PD A+ derives from a nucleotide and amino acid difference relative

to G6PD B, but the protein has full enzymatic activity A mutation on the G6PD

A+background that lowers G6PD enzymatic activity is the basis of the G6PD A−deficiency seen in African Americans.14

The mutations that produce the Class II G6PD variants tend to cluster in the region

of the NADP binding site or the interface between the two dimers Protein structuralalterations in these regions appear to be particularly detrimental to enzymatic activity.NADP binds tightly to the G6PD dimer behaving more as a structural component than

T A B L E 15 - 3 REPRESENTATIVE G6PD VARIANTS

Common Distribution Classification Designation Substitution

Worldwide IV Normal; G6PD B NoneAfrican ethnicity IV G6PD A+ 68 Val→ MetSporadic I G6PD Marion 213 Val→ LeuMediterranean II G6PD Mediterranean 188 Ser→ PheSoutheast Asia II G6PD Canton 463 Arg→ LeuAfrican Ethnicity III G6PD A− 68 Val→ Met

126 Asn→ Asp

as a simple enzyme substrate Mutations that interrupt NADP binding severely dampenenzyme activity The fact that G6PD activity is confined to the tetramer means thatmutants disrupting the interface between dimers are particularly deleterious

䊏 DIAGNOSIS OF G6PD DEFICIENCY

A number of enzyme assays exist that assess G6PD activity Such tests have limitations

in certain clinical settings, but are very useful and reliable within the parameters set

by those limitations The most discriminating assay detects the conversion of NADP

to NADPH wherein a resonating structure forms in one of the molecule’s rings andproduces a characteristic UV absorption band The assay is precise and provides aquantitative readout of G6PD activity, but is relatively complex A simplified variant

of that is particularly suitable for population screening is the fluorescent spot test.The test takes advantage of the change in UV absorption profile by an approach thatrequires a simple UV lamp rather than a sophisticated spectrophotometer

A number of other simple screening tests for G6PD rely on biochemical changesproduced by the active enzyme, including dye decolorization and reduction of methe-moglobin These semiquantitative tests are best at providing a “positive” or “negative”answer, which requires setting proper cutoffs for the reading Used correctly, thesetests are very useful in the detection of G6PD deficiency A number of caveats exist,however

The key drawback to all these approaches when applied to patients suspected ofhaving hemolysis due to G6PD deficiency is that an acute hemolytic episode destroysthe cohort of cells that are deficient in G6PD (Figure 15-3) Patients with the G6PD

A−deficiency, for instance, often show normal testing readouts for more than 3 weeks

after drug exposure Patients with the more severe Class II defects are less of a problemsince even very young red cells have substantial enzyme deficits Physicians who ordertesting for G6PD deficiency must be aware of the laboratory procedure employed andalert to the possibility that it will elide a Class III G6PD deficiency in particular.The ascorbate-cyanide test provides a broad screen for red cells that are sensitive

to oxidant stress The test involves inactivating catalase with cyanide followed bytreatment with sodium ascorbate The ascorbate produces reactive oxygen interme-diates that convert hemoglobin to methemoglobin, seen as a brown pigment that isreadily visible to the unaided eye The ascorbate-cyanide test is a sensitive but notspecific screen with respect to G6PD deficiency Positive results occur in a variety ofsettings with red cells that are sensitive to oxidant stress, including pyruvate kinasedeficiency The test can be very useful in screening for Class III G6PD deficiency

in the immediate aftermath of a hemolytic episode, however The extreme oxidantstress of the ascorbate-cyanide test overwhelms red cells with low levels of G6PD thatmanaged to survive the in vivo hemolysis The positive test supports the existence of

a red cell metabolic defect whose nature later can be definitely documented.Depending on the urgency of obtaining a diagnosis of G6PD deficiency, one pos-sible solution to the dilemma of testing in the aftermath of hemolysis is to wait for afew weeks and perform the assay after the patient has returned to steady state Whentime is an issue, another approach is to test the patient’s mother who is an obligate het-erozygote for this X-linked disorder However, heterozygotes sometimes have normalenzyme levels due to selective lyonization of the affected X-chromosome Testing ofthe patient’s brothers, who have a 50% chance of also having G6PD deficiency, canalso provide indirect support for the diagnosis The size and availability of the familydictates the usefulness of this approach to the issue

T A B L E 15 - 4 KEY DIAGNOSTIC POINTS WITH G6PD DEFICIENCY

Congenital hemolysis Anemia, reticulocytosis,

elevated bilirubin

r Assess G6PD level

r Heinz body prep

r Osmotic fragility test

Acute hemolysisfollowing drug exposure

Falling hematocrit,reticulocytosis, elevatedbilirubin, hematuria

r Obtain history of drugexposure over precedingseveral days

r Urine hemosiderin ifhemolytic episode occurredseveral weeks beforeevaluation

r G6PD level Low in Class Iand II Speciously normal inClass III