Question

Case 8

Hemoglobin, the Oxygen Carrier   

Focus concept

            A mutation in the gene for hemoglobin results in an altered protein responsible for the disease sickle cell anemia. An understanding of the biochemistry of the disease may suggest possible treatments.

Prerequisite

Hemoglobin structure and function concepts.

Background

            Normal adult hemoglobin is called Hemoglobin A (Hb A). Ninety-eight percent of adult hemoglobin is Hb A and 2% is Hb A₂. There are other forms of hemoglobin. For example, the developing fetus has a different kind of hemoglobin than most normal adults. Fetal hemoglobin (or Hemoglobin F) consists of two α chains and two γ chains, whereas adult hemoglobin (Hemoglobin A) consists of two α chains and two β chains. Fetal hemoglobin is synthesized beginning at the third month of gestation and continues up through birth. After the neonate is born, hemoglobin F synthesis declines (because synthesis of the γ chain declines) and hemoglobin A is synthesized (because synthesis of β chains begins). By the time the baby is six months old, 98% of its hemoglobin is Hemoglobin A.

            There is also a mutant form of hemoglobin called Hemoglobin S which is found in persons with the disease sickle cell anemia. The disease sickle cell anemia is one of the major health problems facing the African-American community. The World Health Organization estimates that 250,000 babies world- wide are born with sickle cell anemia. Currently there is no cure. A person afflicted with sickle cell anemia has inherited a defective gene from each parent. (Parents who are carriers of the sickle cell gene are heterozygous AS, whereas the person afflicted with sickle cell anemia is SS; non-carriers are designated AA.) The defective gene is the one coding for the β-chain. The amino acid at position 6 on each β chain has been mutated from a glutamate to a valine. Normal α chains have a decreased affinity for the mutated β chains; thus assembly of the HbS tetramer is more difficult. Red blood cells containing HbS form a sickle shape because the Hb S molecules polymerize. Hb S molecules are more likely to polymerize when in the deoxygenated T form than in the oxygenated R form. The polymerized Hb deforms the normal discoid shape of the red blood cells, producing a sickle-shaped cell. The sickle shaped red blood cells become trapped in capillaries and organs, depriving the victim of adequate oxygen supply and causing chronic pain and organ damage.

            In this case we will consider our patient, a 10-year-old black male child named Michael B., who was admitted to the hospital because he was experiencing severe chest pain. He had been hospitalized on several previous occasions for vaso-occlusive episodes that caused him to experience severe pain that could not be managed with non-prescription drugs such as ibuprofen. He was slightly jaundiced, short of breath and easily tired, and feverish. A chest x-ray was taken and was abnormal. An arterial blood sample showed a p_O2 value of 6 kPa (normal is 10-13 kPa).

Questions

You suspect that Michael has sickle cell anemia and you have ordered an isoelectric focusing analysis of the child‟s lysed red blood cells. (Lysing the red blood cells releases the hemoglobin.) Draw a diagram of the predicted results. Why will this test allow you to diagnose this child‟s disease?

Why do you think that Hb S molecules would be likely to clump together whereas Hb A molecules do not?

In the emergency room, oxygen (100%) was administered to the patient. (Inspired air normally is about 20% oxygen.) Why was this an effective treatment?

You recall reading in the medical literature about a dramatic new drug treatment for sickle cell anemia, and you‟d like to try it on this patient. The drug is hydroxyurea, and is thought to function by stimulating the afflicted person‟s synthesis of fetal hemoglobin. Exactly how hydroxyurea stimulated fetal hemoglobin synthesis is unclear, but it is believed that hydroxyurea is metabolized to NO, which binds to a soluble guanylate cyclase enzyme which then catalyzes the synthesis of a second messenger, cyclic GMP (cGMP). The cGMP interacts with transcription factors in a manner that is not completely understood to induce the transcription (and then translation) of the fetal hemoglobin gene.

In a clinical study, patients who took hydroxyurea showed a 50% reduction in frequency of hospital admissions for severe pain, and there was also a decrease in the frequency of fever and abnormal chest x-rays. Why would increasing the synthesis of fetal hemoglobin result in alleviating the symptoms of sickle cell anemia?

Medical practioners who used hydroxyurea as a treatment for sickle cell anemia noted that their patients seemed to benefit from the administration of the drug long before the synthesis of fetal hemoglobin had time to take effect. It has recently been determined that hydroxyurea can react directly with the iron ion of oxy- and deoxyHb to form iron nitrosyl hemoglobin (HbNO). Why would this be of benefit to the sickle-cell anemic patient?

And finally, hydroxyurea has been shown to produce NO directly, in less than an hour after hydroxyurea administration. It‟s also possible that HbNO could produce NO, either directly or indirectly. NO is an important second messenger which, even in nanomolar amounts, stimulates vasodilation. How could NO production help the sickle-cell anemic patient?

A year ago, at a conference, one of your colleagues told you that she had “cured” a patient of sickle cell anemia by performing a bone marrow transplant. Why would this procedure “cure” sickle cell anemia?

The patient‟s parents tell you that they are planning on having another child and that they are confident that subsequent children will not have sickle cell anemia, since they already have a child with the disease. What will you tell them?

References

Glew, R. H., and Ninomiya, Y. (1997) Clinical Studies in Medical Biochemistry, Oxford University Press, pp. 78-90.

Glover, R. E., Ivy, E. D., Orringer, E. P., Maeda, H., and Mason, R. (1999) Molecular Pharmacology 55, pp. 1006-1010.

Huang, J., Hadimani, S. B., Rupon, J. W., Ballas, S. K., Kim-Shapiro, D. B., and S. Bruce King (2002) Biochemistry 41, pp. 2466-2474.

S. Bruce King (2003) J.Clin. Invest., 111, pp. 171-172.

Answer 1

Ans

1] Isoelectric focusing analysis is a process of testing the presence of abnormal red blood cells in a person's blood. In this ,a pH gradient is established by carrier ampholytes exposed to an electric current. The Hb molecules migrate across the gradient until they reach a position where their net charge is zero.

So,the molecules arrange themselves in sharp bands. Hb S molecules form a band at the far end which is clearly demarked from the other Hb bands.

Ans 2] We already know that HbS molecules are different from Hb A molecules in their biochemical and physical structure. In Hb S , a single amino acid , beta Glu6 is substituted by a valine.

When the Hb S loses oxygen, another hydrophobic patch becomes exposed on it. Valine interacts with this and and large fibrous aggregates of Hb form. Because of the fibers within the cell , the cells become hard and less flexible resulting in their clumping together. These clumps block the narrow blood vessels.

On the other hand, the normal Hb A cells are soft and flexible and squeeze easily through the narrowest of the capillaries, carrying oxygen and collecting CO2.

Ans 3] Treating the patient of sickle cell anaemia with 100% oxygen will be effective because with such a high concentration of O2 , there will be more absorption of oxygen by the plasma [ normally a small amount of oxygen is carried in the dissolved form by the blood plasma]. This will help to make up for some amount of oxygen reaching the O2 starved cells.

Ans 4.a] Hb F starts to be synthesized by the third month of geststion and continues till the baby is birn. Now, the Hb F starts to convert into Hb A because the synthesis of Y chain stops and B chain begins. In my view, if the synthesis of fetal haemoglobin is increased then there are chances that they will change to Hb A. This means, the more the synthesis of Hb F, the more will be the conversion to Hb A . This will definitely help in diminishing the symptoms of the sickle cell anaemia in the patient.

Ans b] The presence of nitrosyl in the haemoglobin might help in dilating the blood vessels and make the movement of O2 carrying cells easier .

c] Vasodilation means the broadening of the blood vessels for easy passage of blood through them . If NO causes vasodilation then even the Hb S will be able to pass through the blood vessels. And as we are aware, the main problem of sickle cell patients is the inability of the Hb S molecules to pass through, which ends up in starving the cells of oxygen. So, vasodilation will definitely help the patients of sickle cell anaemia.

Ans 5] A person with sickle cell disease has bone marrow that produces red blood cells with defective haemoglobin S. If the person's bone marrow is replaced by healthy bone marrow, then it might start producing healthy red blood cells. Some children have been successfully treated with blood stem cell or bone marrow transplant. High doses of chemotherapy is used to destroy all the child's bone marrow and then it is replaced with marrow from a donor. But in many cases the disease was reversed as one can do nothing with the defective gene !

Ans 6] First the child's parents will have to be told that sickle cell anaemia is an inheritable disease i.e. it passes on from parents to children. It is a genetic disease which is caused due to the presence of a defective gene in both of them which Michael has inherited. Since their first child is showing symptoms of the disease, it is very likely that their other children will also suffer from it because both the parents are carriers for that defective gene.

So, I would tell them to be mentally prepared for the occurance of sickle cell anaemia in their other children too.