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Aurelian Udristioiu¹, Radu G. Iliescu, ² Manole Cojoraru³ ¹Department of Hematology, Emergency County Hospital Târgu Jiu, Clinical Laboratory, Gorj, Romania, ²Department of Researches, Polytechnic Institute of New York University, Brooklyn, New York, ³Titu Maiorescu University, Medicine Faculty, Physiology, Bucharest, E-mail: ABSTRACT The aim of this work is to illustrate a difficult diagnostic in vitamin B12 deficiency because of using of multivitamin preparations to a patient with unknown etiology of anemia. History: a 70 year old engineer, following 8 years vegetable – dietary, was admitted to the county hospital, in neurology department, with diagnostic of poli-radiculonevrites, received in ward the treatment with milgama (containing 250 micro-gram multivitamin). After a lipotimia status the patient was transferred to cardiology department. Laboratory examinations showed in peripheral blood: Hb = 6 3 g/dl; Ht = 18.8%; RBC= 290.000/mm³; PLT. = 214000/mm³; WBC = 5300/³mm; Ret. = 3, 7%; Erythrocytes indices = normal values; ESR = 38 mm/h, moderate increase and serum iron decreased, 36 microgram/dl value. On blood smear in optic microscopy was registered: Band = 5% (with nucleus in ring!!!), differential count being normally with aspect flags on Coulter HMX Analyzer with 22 parameters: neutrophilia, lymphopenia, anemia. To microscopic examination of slide from bone morrow, have occurred the hyperplasic series of erythrocytes ~ 45%, deficiency of erythropoiesis, poly-cromathopil and acidophil erythroblasts with megaloblastic character, large metamielocytes and giant band forms. Macrocytes and ovalocytes where also presented. Biopsy results from gastric mucosa showed lesions of chronic gastritis, non-atrophic epithelium. Conclusions: Megaloblastosis appears in some time with vitamin B12 deficiently in bone morrow but no in peripheral blood because of administration of multivitamin drugs, deleting haematological shape of megaloblastic anemia. Keywords: Wait blood cells, Erythrocyte sedimentation ratio, haptocorin, transcobalamin. COVER LETTER FOR SUBMISSION OF MANUSCRIPT Open Access Pub – International Journal of Nutrition COVER LETTER FOR SUBMISSION OF MANUSCRIPT Date: 13/05/2016 I am enclosing herewith a manuscript entitled “ANEMIA BY VITAMIN B12 AND LATENT IRON DEFICIENCY ” in Journal International Journal of Nutrition for publication in for possible evaluation. The Corresponding author of this manuscript is Aurelian Udristioiu , ¹Department of Hematology, Emergency County Hospital Târgu Jiu, Clinical Laboratory, Gorj, and contribution of the authors as mentioned below with their responsibility in the research. 1. Radu G. Iliescu, ²Department of Researches, Polytechnic Institute of New York University, Brooklyn, New York 2. Manole Cojoraru, ³Titu Maiorescu University, Medicine Faculty, Physiology, Bucharest With the submission of this manuscript I would like to undertake that: • All authors of this research paper have directly participated in the planning, execution, or analysis of this study; • All authors of this paper have read and approved the final version submitted; • The contents of this manuscript have not been copyrighted or published previously; • The contents of this manuscript are not now under consideration for publication elsewhere; • The contents of this manuscript will not be copyrighted, submitted, or published elsewhere, while acceptance by the Journal is under consideration; • There are no directly related manuscripts or abstracts, published or unpublished, by any authors of this paper; • My Institute’s (Put name of your institute here) representative is fully aware of this submission. Submitted manuscript is a Research Article (Ex: Research Article, Review Article, Short Communication, Letters, Editorials) The research project was conducted under the supervision of: , ¹Department of Hematology, Emergency County Hospital Târgu Jiu, Clinical Laboratory, Gorj, Romania This research project was conducted from ___05/05/2016______________ to _13/05/2016_________________ Starting date Ending date From the same project I have already published the following manuscripts: If any Book:Hematological and Metabolical Aspects of Laboratory Medicine (3-th Edition/2016). Publisher: Aurelian Udristioiu. My Research Project was partially or fully sponsorised (n/a) with grant number (n/a). If you have undergone your research project without any financial assistance, then you must provide information who was the sponsor of the project? Please suggest potential three reviewers for this submission. (OPTIONAL) Use the fields below to give us contact information for each suggested reviewer, and please provide specific reasons for your suggestion in the comments box for each person. Please note that the journal may not use your suggestions, but your help is appreciated and may speed up the selection of appropriate reviewers. Reviewers' field: No. 1: First Name Dana Middle Initial Last Name Panait Academic Degree(s) PhD Position Researcher Department Microbiology Institution National Agency of Drugs, Bucharest E-mail Address Reason Scientific No. 2: First Name Dali Middle Initial Last Name Vlad Academic Degree(s) MD Position Lab Director Department Clinical Trial Institution Central Laboratory Synevo, Bucharest E-mail Address Reason Scientific No. 3: First Name Sorin Middle Initial Last Name Giju Academic Degree(s) PhD Position Chief in Biochemistry Department Department Biochemistry Institution Emergency Hospital University Timisoara, Romania E-mail Address Reason Scientific Date: 13/05/2016 ___________Aurelian Udristioiu______________________________________ (Signature of corresponding author on behalf of all authors) (A scanned signature is valid) Introduction Deficiency of vitamin B12 was a disease that has intrigued numerous investigators for many years. In fact, the history of pernicious anaemia was an “illustrative case” of the development and progress of medicine. The aim of this work was to illustrate, by one difficult case patient, admitted in hospital, the diagnosis of B12 vitamin deficiency, intrigued with iron deficiency, because of multivitamin drugs, used as an individual treatment of patient, without a previous medical consult.. History: A 70 year old engineer, since 8 years vegetable – diet, was admitted to the county hospital in month august 20 – 2011, because of weakness, 10 kilo in last two months], dizziness, exertional dyspneea, ataxia and numbress of the hands, with lung disease in evolution. After screening investigations without a concluded diagnosis, in 10 - 20 September/2012, was transferred to department of neurology because of symptoms came to include a loss of sensitivity in extremities, paresthesia, an unsteady gait [ataxia] and loss of balance. Psychiatric symptoms such as memory disturbance, depression and cognitive decline have been also reported. For a presumptive diagnosis of poly-radiculonevrites, the patient followed treatment with milgama, contained ciancobalamin vitamin 250 microgr /vial, prednisone, levomepromazin, antibiotics. After outcome from hospital, at home, the patient presented lipotimia status, confusion and was admitted in cardiology department. Laboratory blood – CBC – showed: HGB = 6.3 g/dl; HTC = 18.8%; RBC = 290.000/³mm, Thrombocyte = 214000/.mm³, WBC = 5300/³mm; Reticulocyte = 3.7%; Erythrocyte indices = normal values. On blood smear in optic microscopy: Band = 5% [nucleus in ring!?], Segmented = 75%, Eosinophile = 1%; Basophile = 1%, Lymphocyte = 11%, Monocyte = 3%, ESR = 38 mm/h.; Suspect flags on coulter HMX. Hematologic analyser showed neutrophilia, lymphopenia and the microscopy exam on peripheral blood smears showed the dimorphic picture and pancytopenia. Macropolicytes and ovlaocytes were also presented, Other laboratory tests relieved an increased level LDH = 710 u/l [N = 313 – 618], glucose = 143 mg/dl [N = 75 – 110], urea = 111 mg/dl, creatinine = 1,3 mg/dl, triglyceride = 251 mg/dl [20 – 150], iron = 35 ug/dl [N = 49 – 181], serum ferritn = 15 ng/ m l, Total bilirubin < 0,2 mg/dl and Indirect bilirubin = 0,1 mg/dl, ALT and AST with normal values. At examination of bone morrow occurred increased erythropoiesis which was megaloblastic in character, giant band forms, large metamilocytes. In conclusion, bone morrow with hyperplasic series erythrocytes ~ 45%, deficiency of erythropoiesis [predominant acidophil and policromatophil erythroblasts]. Biopsy results from gastric mucosa showed lesions of chronic gastritis, non-atrophic. Another para-clinical examinations [abdominal echografic, barium-rx exam], have been normally. B12 vitamin dosage on the Analyzer Chemi Immulite 2000 in peripheral blood showed 150 pg/m L (biological reference interval =193-982 pg/m L) and folic acid dosage into erythrocytes was in normal value, 1632 nmol/L(biological reference interval =995-2499). Diagnosis: anemia by cobalamin deficiency and iron deficiency The patient followed therapeutic sample [with B12 vitamin, 1000 gamma, im./per day, 2 weeks and than 1000 gamma per week, Omeran 1 tb./day, in morning 10 o’clock, Ferumgradumet 1 tablet/day, protection of diet. Evolution After therapeutic sample, in 10 days, Hb became 9,8 g/dl, Ht = 29%, Reticulocyte = 11% with IP = 3,48, Leucocyte = 5000/cu-mm, Thrombocyte = 281000/, ESR = 18 mm/h. The peripheral blood picture showed: Neutrophiles = 73%, Eosinophile = 2%, Basophile = 2%, Monocyte = 15%, Lymphocyte = 8%. The blood smear shows anisocytosis: normocytosis with ease macrocytosis. Urea was normalised = 48 mg/dl, plasma iron = 46 ug/dl, TIBC = 382mg/100 ml, sample of coagulation with normal values [aPTT, fibr, At3, PDF (-), TELCE [-]. In present time the patient there is in treatment with B12 vitamin, 50 gamma/week with the normal level serum ferritin, 122 ng/m L((biological reference interval =25-285 ng/m L for man and 5-148 ng/m L for women. Efficiency of treatment in periodically will be monitored and by performing of metilmalonic acid test (heparnied plasma) and homocystein level (EDTA plasma). Comment: In first time the neurogical manifestation occurred to patients in absence of blood picture in peripheral blood of typical megaloblastic anemia. To the patient with vegetable diet anemia by B12 vitamin is accompanied of neurological syndrome with frequently iron deficiency, specialty to elder men. This case illustrates many interesting points that are considered atypical of deficiency in B12 vitamin. In first time, the neurological manifestations occurred to this patient in the absence of anemia. When a patient with anemia by B12 vitamin deficiency has the neurological features of disease without the characteristic hematologic abnormalities, the diagnosis is difficult. On such circumstance, the patient has received B12 vitamin and folic acid in an amount that is sufficient to convert the megaloblastic anemia type of erythropoiesis to the normoblastic type in peripheral blood and raise the erythrocyte count. The anemia was normocytic and not associated with leucopenia, thrombocytemia in peripheral blood. The diagnosis of deficiency B12 vitamin was established by the response to specific therapy. When and iron deficiency is severe the anemia is hypochromic and microcytic but in milder degrees of iron deficiency the anemia is normocytic. A combined deficiency of iron B12 vitamin might result in a normocytic anemia that required both iron and vitamin B12 in treatment [1]. Impaired absorption of vitamin B12 occurs and in other conditions different of diet deficiencies associated with diarrhea or other evidence of the mal-absorption syndrome. Such impairment has been demonstrated to patients with: - Blind intestinal loops, fistulae, strictures, and diverticula of the small bowel, - Gastrectomy, because absence of the gastric intrinsic factor, altered gastric secretion, gastric atrophy with degenerative lesions, mal-absorption syndrome, gastro duodenal disease and pancreatic, abnormal bacterial proliferations in bowel, infestation with parasites, failure pancreatic, liver, chronic hepatitis cirrhosis, - Disease of lymphoma group, myeloproliferative syndrome (MSD) by elevated consummations in neoplasia and hyperthyroid – but a study of the bone morrow usually enable one to establish the diagnosis [2]. In some patient particularly since the advent of folic acid and the widespread use of multivitamin preparations the aematological abnormalities are mild or absent when neurological manifestations are predominant. The bone morrow is usually hyper-cellular. The characteristic feature is the occurrence of the megaloblastic type of erythropoiesis. If anemia is severe, promegaloblastic and basophilic megaloblasts are increased in number and mitotic divisions are numerous [3]. The polychromatophilic megaloblasts which are easiest cells to identify in this serious persist even after the anemia in the peripheral blood has been abolished by transfusion. Deficiency of vitamin B12 or folic acid also leads to the production of giant metamielocytes and multi-segmented macropolicites. The abnormalities in the granulocytic series do not disappear as promptly as then megaloblasts after specific therapy and their present may be helpful in diagnosis. There are the conversion of the megaloblastic type of erythropoiesis to the normoblastic type within two day, a rise in reticolocytes during the first week, and a return of the haemoglobin and red count to normal levels in succeeding weeks. The absorption of the vitamin may be affected by atrophic gastritis [around 20% individuals over 60 years this conditions [4]. The recent years the picture has become more complex. For example: 24 – 28% of patients do not have anemia, 17 – 33% have a normal mean corpuscular volume [MCV], there is an apparent normal cobalamin levels to patients with clinical evidence of deficiency in B12 vitamin [5]. In last time, attention has recently been focused on measuring metabolic levels methylmalonic acid [- MMA -], and homocystein [- Hcy -], as these should accumulate when a deficiency stare exists. However, both of these methabolits can be affected by the renal function of the patient and Hcy can be elevated when there is an underling folate deficiency [6]. The situation becomes more warring when abnormal levels of metabolites are detected but the total Hcy are apparently well within normal range. The notion that metabolic levels change before that total Cbl levels decreases this fact not proven. Current made techniques for measuring plasma Cbl that is bond to both haptocorin [HC] and transcobalamin [TC], complex describing Holo-Transcobalamin. Hololotranscobalamin has been suggested as a better marker for cobalamin deficiency because of: - measures the physiologically relevant fraction of circulating cobalamin, -It has a short-life and so may be a good early good marker. It is hoped that the new methods [antibodies and physico-chemical methods] will be enable to the investigators to address the many questions surrounding holo-trancobalamin and also enable carefully designed clinical surveys to be undertaken [7]. Conclusions: To the vegetarian elder men, anemia by deficiency of B12 vitamin is accompanied of neurological syndrome. There are frequent and deficiency of iron also to the patient which eats vegetal reached in folic acid, cofactor of B12 vitamin, but poor in B12 vitamin. Megaloblastoza appears in same time with B12 vitamin deficiency, in bone morrow but not in peripheral blood. Neuropathy was results from accumulations of a metabolised metylmalonic acid [from propionic acid] with decrease of acetyl-coA, ATP and energy cells. Drugs with poli-vitamins administrated without corresponding investigations can delete haematological shape of megaloblastic anemia. References [1] William S. Beck, the book: Haematology – Fourth Editions, Lecture 4, “Megaloblastic Anemias”, WH, USA 1990. [2] Carmel R. Current Measuring and Interpreting Holo – Transcobalamin (Holo-Transcobalamin 11). (Editorial), Clinical Chemistry 2002; (48); 3: 407-408. [3] Ulleland M. Direct Assay for Cobalamin Bound to Transcobalamin (Holo-Transcobalamin) in Seum, Clinical Chemistry 2002; (8); 3: 526 – 532. [4] Nexo E. Quantification of Transcobalamin, a Marker of Vitamin B 12 Deficiency. Clinical Chemistry 2004; (8); 3: 561-562. [5] Association of Cognitive Impairment with Combination of Vitamin B12-Related Parameters. Clinical Chemistry 2011; (57); 10: 1436-1443. [6] Bennett J M. Practical Diagnosis of Hematological Disorders.2th ed. Lett. Anemia of Chronic Diseases. Chicago: ASCP Press 2006; p. 615 – 671 [7] Steensma DP, Tefferi A. Anemia in the elderly: how should we define it, when does it matter, and what can be done?. Mayo Clin Proc 2007; 82(8): 958-66. ANEMIA BY VITAMIN B12 AND LATENT IRON DEFICIENCY. International Journal of Nutrition.

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