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The role of the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase also called the heme-regulated inhibitor (HRI) in regulating initiation of protein synthesis in reticulocytes is well established [1-5]. HRI is a cyclic AMP-independent protein kinase and coordinates the synthesis of globin chains in reticulocytes with heme availability . Upon HRI activation in response to heme-deficiency, it phosphorylates the 38 kDa α subunit of eIF-2 at ser-51 residue [7-10]. Phosphorylation of eIF2α causes the inhibition of polypeptide chain initiation and the arrest of protein synthesis. Translational arrest prevents the synthesis of globin chains in the absence of sufficient heme, which is required for the assembly of α- and β- globin chains into hemoglobin  and ultimately causes iron-deficiency anemia (IDA). The mechanism of inhibition of translation inhibition by the phosphorylation of eIF2 has been studied extensively [12,13]. In heme deficiency, which may lead to heme deficiency anemia, protein synthesis is inhibited, with a marked decrease in the formation of 40S-eukaryotic initiation factor 2 (eIF2)-Met-tRNAf GTP (43S preinitiation complex) and with increased phosphorylation of the α subunit of eIF2. The recycling of eIF2 in initiation requires the exchange of bound GDP for GTP [14- 17]. Under physiological conditions, eIF2 has a 400-fold-greater affinity for GDP than for GTP. The exchange of tightly bound GDP for GTP requires eIF2B, which is rate limiting and is present at 15 to 25% of the amount of eIF2. When eIF2α is Phosphorylated, the binding of eIF2(αP)-GDP to the regulatory subcomplex of eIF2B is much tighter than the binding of eIF2-GDP to eIF2B  This tighter interaction of phosphorylated eIF2 with eIF2B prevents the GDP-GTP exchange activity of eIF2B. Once the amount of phosphorylated eIF2 exceeds the amount of eIF2B, the shutoff of protein synthesis occurs .
HRI is a multidomain hemoprotein that appears to contain two distinct types of heme-binding sites . The unique N-terminal domain (NTD) of HRI is an autonomous heme-binding domain which has significant sequence similarity to mammalian α-globins . The binding of hemin to HRI regulates its maturation, transformation and activity . Mature-competent HRI is inactive, and its transformation into a stable, active kinase is inhibited by hemin. In the absence of hemin, mature-competent HRI is autophosphorylated and transformed into an active kinase which exhibits a slower electrophoretic mobility on SDS-PAGE. The binding of hemin to transformed HRI directly inhibits its autokinase and eIF2α kinase activities (repressed HRI) . Inactivation and reactivation of transformed HRI appears to be a direct effect of the binding and release of hemin [22, 23].
Among the various conditions and stresses that activate HRI, and inhibit globin synthesis and ultimately causes anemia in man, iron-deficiency anemia was our interest in this study. Anemia is the most common health problem in the world . Although some studies have shown a decline in the prevalence of anemia in the past three decades, anemia is still the most common nutritional problem due mainly to iron deficiency in infancy, childhood, and pregnancy [24, 25]. India has the highest prevalence of iron deficiency anemia among women in the world. Anemia among women can result in adverse pregnancy outcomes and severe anemia can lead to maternal deaths, reduced work productivity and impaired physical capabilities. Adolescence, as a period of growth and development, is considered the best time to intervene and assist in physical and mental development, and prevent later maternal anemia .
It has been shown that the quantity of HRI protein increases by 12-15-fold in the reticulocyte lysates of anemic rabbits as compared to that of normal, while the mRNA increases by two-fold only . Earlier we have reported that the pattern of expression of HRI shows a unique and gradual increase during drug (acetylphenylhydrazine) induced hemolytic anemia and its concomitant decrease upon recovery in rabbits .
Although there are some data available from animal model studies, not much is known on the usage of HRI as a molecular marker for diagnosis or even prognosis of anemia in man. In this investigation, therefore, we have addressed the question that whether HRI could be used as a possible indicator of anemia by designing experiments using human blood. We have used human blood for RNA extraction and RT-PCR analysis, protein extraction and western blot analysis, ferritin estimation and also for measuring routine hematological tests which are commonly done at hospitals. Our results indicate that firstly, HRI is over expressed in anemic individuals who were also recognized anemic by hematological tests; secondly, HRI was over expressed in number of individuals who had normal hematological measures but very low ferritin. Thus, our results, for the first time, suggest that expression of HRI can be used possibly as a marker for anemia even before signs and symptoms of anemia could be diagnosed or routine hematological tests could show anemia. These results underscore the usage of HRI as a marker for anemia, particularly iron deficiency anemia.
All the general laboratory chemicals were purchased from Sigma Chemical Co., USA and Life Technologies (GIBCO BRL), USA. First strand cDNA synthesis kit for RT-PCR was purchased from Roche Molecular Biochemicals, Germany. All the chemicals and instruments for hematology tests were purchased from Beacon Diagnostics, India. DNA ladder (100 bp) and protein molecular weight marker (medium range) were purchased from Bangalore Genei Pvt Ltd, India. HRI and β- actin primers were purchased from Sigma chemical Co., USA. TRIZOL LS reagent was purchased from Life Technologies (Gibco BRL), USA. Microplate 96 wells coated with anti-ferritin antibody was purchased from Omega Diagnostics (Pathozyme) kit, London. Nitro cellulose membrane was purchased from Sigma chemical Co., USA and BM Chemiluminescence Western Blotting kit (Mouse/Rabbit) was purchased from Roche Molecular Biochemicals, Germany. Hemoglobin standard, New Methylen Blue, Drabkin solution, PCV capillaries, sealant and EDTA coated 5ml blood containers were purchased from Beacon Diagnostics, India. Phospho-eIF2α (Ser51) and eIF2α polyclonal antibodies were procured from Cell Signaling Technology, USA. Other routinely used chemicals were purchased from MERCK India Ltd. Human blood samples were drawn from randomly selected young college going individuals.
Keeping in mind the ethical issues of drawing blood from anemic individuals, the study employed a cross-sectional study design. 141 volunteers of the age group of 20 to 28 years were invited to participate in this study.
20 μl of blood was taken in to 5 ml of Drabkin solution and incubated for 10 minutes, and then the OD of mixture was measured at 546nm wave length. The calculation was done by dividing the OD of Test by OD of Standard multiplied into 15.06 (dilution factor).
Two third of specific PCV capillaries were filled with blood and sealed with sealant and centrifuged by hematocrit centrifuge for 5 minutes at 5000 rpm. The value was read by PCV reader. The value is normally between 0.42 and 0.52 for males and between 0.36 and 0.48 for women
Two volume of well-mixed EDTA anticoagulated blood was incubated with one volumes of New Methylene Blue (10g/l) for 20 minutes, and then a thin blood film was prepared on a clean glass slide and air-dried. Reticulocytes were identified and counted by microscope and blood cell counter. Counting was done systematically in consecutive fields and percentage of reticulocytes was calculated. The normal range is 0.5-2%.
Blood serum was collected by allowing the blood to coagulate, and then serum was stored in -20 oC for ferritin estimation. 20μl of test serum was dispensed and in microtitration wells which were coated with specific anti-ferritin antibody followed by addition of 100μl of antiferitin HRP conjugate and incubated for 55 minutes at room tempareture. Contents of the wells were discarded and washed 5 times with distilled water. 100μl of substrate solution was added to each well, gently shaked and incubated in dark for 20 minutes at room temperature. 100μl of stop solution was added to each well, shaked gently for 30 seconds. OD of the solution was obtained immediately (less than 10 minutes) at 450nm.
Total RNA was extracted from blood cell samples as per manufacturer's (Life Technologies, USA) protocol using TRIZOL® reagent. In brief, 150μl of blood was diluted by DEPC treated water to increase lysis efficiency followed by addition of 750 μl of TRIZOL® reagent and passing the cell lysate several times through a pipette and incubated for 15 minutes at room temperature. The cell lysate was added with 200μl chloroform and centrifuged at 12,000g for 20 minutes at 4 oC. RNA was precipitated and pelleted from the aqueous phase by mixing with isopropyl alcohol followed by centrifugation at 12,000g for 20 minutes at 4 oC. The RNA pellet was washed with 75% ethanol and subsequently redissolved in RNase-free distilled water. The total RNA was quantified by routine spectrophotometric method.
First strand cDNA was synthesized from RNA samples using the cDNA synthesis kit for RT-PCR (AMV) from Roche Molecular Biochemicals, Germany, as per specifications provided in the kit. PCR amplification of HRI cDNA was carried out using HRI-specific primers (1 μM each) as described earlier . The HRI specific primers used for HRI cDNA amplification were as follows: 5'-AAAATAGGAGACTTTGGTCTGGCCTGCGCCGACATC-3' and 5'- CTCCATCTCTGTCCCGAAGGGCTGGAA-3'. Simultaneous amplification of human β-actin sequence was done using actin-specific primers: 5'-GTGGGGCGCC CCAGGCACCA-3' and 5'-CTCCTTAATGTCACGCACGA -3', as an internal control. PCR products were analyzed on 1.5% Agarose gel followed by ethidium bromide staining. The results were analyzed and saved using gel documentation system (Bio-Rad).
Samples containing equal quantity of proteins were denatured in sample buffer for 3-5 min at 100°C and analyzed by SDS PAGE. Electrophoresis was carried out at a constant current of 25mA at room temperature. Following SDS PAGE, proteins were electrophoretically transferred to a nitrocellulose membrane. Blots were then processed for immuno-reaction using Phospho-eIF2α (Ser51) and eIF2α polyclonal antibodies. In brief, blots were saturated with 2% Blocking reagent (provided by the kit) for 1h, and incubated overnight with primary antibody in Trisbuffered saline containing 0.1% (v/v) Tween-20 (TBST, pH 7.5) and then with antimouse/ rabbit IgG HRP-conjugated secondary antibody for 1h at room temperature. Following each antibody incubation, blots were washed thrice (5 min each) in TBST. Blots were developed using the chemiluminescence detection kit. The results were analysed using Biorad-gel documentation system (USA).
Analysis of association with Fisher's Exact test was performed to reveal statistical significances. P values less than 0.05 were considered statistically significant.
Consent of the 141 participants was taken. The participants had the choice to participate or withdraw from the study. Assurance was given to the participants for the confidentiality. Purpose and benefits of the study was explained to the participants.
In order to differentiate the expression of HRI in normal versus anemic individuals, and compare the expression profile with the hematological results namely, hemoglobin, PCV, reticulocyte count and ferritin estimation, blood samples were drawn from randomly selected individuals aged between 21 to 28. Total RNA was extracted and used for RT-PCR experiments. RT-PCR analysis using HRIspecific primers revealed an increased expression of HRI in anemic individuals (Fig. 1A, lanes 3 and 11). As seen in figure 1A the 230 bp HRI amplificate was 2-2.5-fold more in the anemic individuals as compared to that in the normal individuals. β-actin, which was used as an internal control, did not show changes (600 bp amplificate).
All the volunteers who were determined anemic showed a strong HRI expression in RT-PCR analysis. Hb of 2 volunteers was below 12 g/dL which one of them was severely anemic with the Hb of 8.4 g/dL. Expression of HRI in anemic volunteers showed a drastic increase when it was compared with the normal volunteers (Fig. 2A, lanes 10-11). Two individuals had Hb and PCV in the normal range but unexpectedly they had HRI expression more than other normal volunteers (Fig. 2A, lanes 6-7), but very same individuals had a very low ferritin and they were detected as iron deficient individuals (Fig. 2B, lanes 6-7).
Association of HRI expression, anemia and ferritin was determined by comparing the pattern of HRI expression, hematological tests and ferritin level. The results indicated that all the volunteers who showed over-expression of HRI (Fig. 1A, lanes 3 and 11; Fig. 2A, lanes 10-11) were determined anemic considering their hematological parameters, and also had very low amount of ferritin (less than 20 ng/ml) in their blood (Fig. 1B, lanes 2-4; Fig. 2B, lanes 10-11), which is the indication of iron deficiency anemia (P value < 0.007).
The pattern of HRI expression, hematological tests and ferritin level of individuals were compared in order to determine the association between HRI expression and iron deficiency. Among individuals with normal hemoglobin level, we found few number of individuals who had very low ferritin level but higher expression of HRI. HRI profile (Fig. 2A, lanes 6-7) of these individuals are shown to indicate the association of HRI expression and iron deficiency (P value 0.00). This result shows that HRI expression is much more sensitive and accurate than hemoglobin estimation to show iron deficiency and iron deficiency anemia which might not be shown by routine hematological tests.
Since over-expression of HRI was conformed by RT-PCR at mRNA level in anemic individuals, it was of interest to determine the HRI activity in blood samples by measuring eIF-2α and eIF-2α(P) level in anemic and normal individuals. Equal quantity of protein extracts were electrophoresed by SDS PAGE, and transferred to NC membrane for Western blot analysis. Three antibodies namely, anti-actin, antieIF- 2α and anti-eIF-2α(P) were used for Western blotting of the total blood cell protein extracts. The results obtained from Western blot experiments clearly indicated that amount of eIF-2α(P) increased significantly (2-2.5 fold) in anemic individuals (Fig. 3C). However, there was no variation in actin protein, which was used as the loading control (Fig. 3A). Total eIF-2α showed a marginal variation in different individuals (Fig. 3B).
Fisher's Exact test was used to indicate the association of HRI expression and hemoglobin content and ferritin level of all volunteers. Statistical analysis ensured that those subjects who had low hemoglobin, also showed HRI over-expression (P value < 0.007) which indicated a significant association between HRI expression and hemoglobin content (Fig. 1B, lanes 2-4; Fig. 2B, lanes 10-11). On the other hand, those volunteers with low ferritin (iron-deficient) also expressed HRI more than normal volunteers (P value 0.00) which is the indication of a great association between low ferritin level and HRI over-expression (Fig. 2B, lanes 6-7).
In previous investigation on animal models in our laboratory we have reported that HRI is almost undetectable in blood samples of normal rabbits and it increases by 12- 15-fold in the reticulocytes of drug-induced anemic rabbits . It was determined that this increase in the quantity of HRI is gradual and it could be an indicator of anemia. Furthermore, when rabbits recovered from anemia due to individual response to the drug, quantity of HRI decreased significantly. These observations were novel and hence prompted us to undertake further studies on the usage of HRI expression as a molecular marker for anemia in human. In the present study, we determined HRI expression in normal and anemic adolescent volunteers which the pattern of expression was compared to routine hematological tests and ferritin level. The following are noteworthy regarding sensitivity and specificity of HRI expression and activity in anemic and normal conditions, and validity of usage of HRI expression for early detection of iron-deficiency and iron-deficiency anemia in man prior to appearance of all signs and symptoms.
In our study, we demonstrated for the first time that determination of HRI expression in the erythroid cells might be used as a prognostic/diagnostic approach for anemia in man. Iron deficiency anemia is one of the most common nutritional disorders world-wide, especially in India and other developing countries. Young children and women in the reproductive age group are the most vulnerable group of the society to iron deficiency anemia. Surveys in different parts of India revealed that 87% of pregnant women suffer from anemia and about 10% show severe anemia (Hb less than 8 g/dl) . A study on the prevalence and etiology of nutritional anemia in early childhood in an urban slum area of east Delhi indicated a high prevalence (76%) of anemia and iron deficiency in 41% of children . Earlier studies from National Institute of Nutrition (NIN) in India prior to 1985 showed an average anemia prevalence rate of 68% in preschool children . This is partly due to the fact that anemia is a gradual process, and iron deficiency commonly remains unrecognised.
Although it has been shown that iron deficiency causes a notable increase in HRI activity and its higher expression at mRNA level and subsequently inhibition of protein synthesis , nothing was known on the usage of HRI for prognosis/diagnosis of anemia in human. Our results indicated that the level of HRI expression was hemoglobin concentration-independent. Interestingly, results of hematological tests (Hb, PCV, reticulocyte count and ferritin) were consistent with expression of HRI; Hb, PCV and ferritin showed inverse co-relation, but reticulocyte count showed direct co-relation in anemic cases. In anemic condition when hemoglobin, PCV and ferritin tests were below the normal range (Hb less than 12 g/dl and PCV less than 0.36 % and ferritin less than 20 ng/ml), HRI expression detected by RT-PCR analysis was 2-2.5-fold more and in non-anemic volunteers who had their hemoglobin and PCV in the normal range, HRI expression was negligible.
Earlier reports in the literature on expression of HRI in iron deficiency shows that, when the concentration of iron is very low, due to nonavailibility of heme, globin synthesis in immature nucleated erythriod cells has to be regulated according to heme deficiency condition which is present as a kind of stress in the cells . In erythroid cells, HRI is the most predominant eIF2α kinase  and is responsible for the phosphorylation of eIF2α by heme deficeincy and most other cytoplasmic stresses . It may therefore be postulated that RT-PCR based analysis of HRI expression in man has the potentiality to be valid as an accurate and very sensitive technique for diagnosis of iron deficiency and iron deficiency anemia in human.
It has been reported that in iron-deficient HRI -/- mice, globins devoid of heme aggregated within the RBC and its precursors, resulting in a hyperchromic, normocytic anemia with decreased RBC counts, compensatory erythriod hyperplasia and accelerated apoptosis in bone marrow and spleen . It is also proved that HRI is a physiological regulator of gene expression and cell survival in erythriod lineage . A report from our laboratory indicated that as anemia in rabbit advances, HRI expression also increases gradually . These data taken together strengthen our earlier hypothesis that RT-PCR analysis for HRI detection can be a promising tool for diagnosis of anemia in man.
Our further studies revealed, iron-deficient cases that had normal hemoglobin and PCV test results, showed HRI expression at higher level than other normal volunteers. Ferritin test was performed for all the samples, and iron-deficiency of these cases was confirmed. Iron-deficient cases had their ferritin level below normal range (less than 20ng/ml), which could be a possible physiological explanation for over-expression and activation of HRI. Amount of iron absorbed in intestine is only 10% of the total iron intake in daily diet (only 1-2 mg of iron out of 10-20 mg of total intake) . If there was not enough iron to be stored in ferritin molecules, the physiological homeostasis strategy would be to keep hemoglobin synthesis and erythropoiesis in the bone marrow in its normal condition, and avoid storing iron in ferritin molecules, which results in producing very less ferritin in the body .
In correlation with this data, our ferritin estimation and HRI expression revealed that analysis of RT-PCR based HRI detection in human blood can indicate early decrease of iron stores in the body. This perhaps indicates the onset of iron depletion in the body which is reflected by increase in HRI expression. This is the time that signs and symptoms of iron-deficiency have not yet been diagnosed. Therefore, HRI could be used as a unique molecular marker for iron-deficiency anemia.
In conclusion, (1) we showed for the first time that the expression of HRI in human (as expressed by the HRI/β-actin ratio) was sensitive to extremely small changes in routine hematological measures. (2) Statistical analysis showed no anemia risk factor significantly associated with HRI expression other than anemia (low Hb) or iron deficiency (low ferritin). (3) Our results also demonstrated that expression pattern of HRI (by RT-PCR) at mRNA level is a sensitive indicator of changes in hemoglobin concentration and more precisely ferritin status of volunteers, and thereby could be used as a molecular marker for diagnosis of iron-deficiency anemia (IDA) in humans. (4) Western blot analysis indicated that phosphorylation of eIF-2α increased in anemic individuals by 2- to 2.5 fold which is tallying with the over-expression of HRI in the same anemic individuals. (5) HRI could be used as an indicator to show the onset of depletion of iron stores of the body (iron deficiency) prior to any hematological test positive for anemia, which is of a great prognostic value and it provides an upper hand chance for early treatment.
We would like to thank Dr. Padmashastry for allowing us to use lab equipments at NCCS, University of Pune. Financial help in the form of research grants from Dr. JKP is duly acknowledged.