Editor-in-Chief Hatice Kübra Elçioğlu Vice Editors Levent Kabasakal Esra Tatar Online ISSN 2630-6344 Publisher Marmara University Frequency Bimonthly (Six issues / year) Abbreviation J.Res.Pharm. Former Name Marmara Pharmaceutical Journal
Journal of Research in Pharmacy 2010 , Vol 14 , Num 3
Detection of vitamin B12 levels with the aid of some hematological and biochemical parameters that are more sensitive
Süheyl Asma, Ferit Erdoğan, Aydan Ünsal, Can Boğa, Hakan Özdoğu, Çiğdem Gereklioğlu, İlknur Kozanoğlu, Erkan Maytalman
Başkent Üniversitesi, Tıp Fakültesi, Adana, Türkiye DOI : 10.12991/201014447

Summary

Vitamin B12 eksikliği genellikle yaşlı hastalarda karşılaşılan, hematolojik ve nörolojik rahatsızlara sebep olan önemli bir sağlık sorunudur. Bazı hastalarda serum vitamin B12 düzeylerinin gerçek düzeyi tam olarak yansıtmadığı düşünülmektedir. Bu çalışmamızda vitamin B12 serum düzeylerinin, bazı hematolojik ve biyokimyasal parametreleri dikkate alarak, hangi düzeylerin gerçek doku düzeylerini yansıtabileceğini amaçladık. Gereç ve Yöntem: Vitamin B12 düzeyi düşük saptanan yaşları 16 ile 90 arasında, 91'i kadın 138 hasta incelendi. Hastaların tam kan sayımı, anti parietal antikor (APA), folik asit, homosistein ve periferik yayma değerlendirmesi ile gastroduodenoskopi bulguları değerlendirildi. Bulgular: Vitamin B12 düzeyi 60 pg/mL'ın altında saptanan hasta grubunda (Grup 1), ortalama alyuvar hacmi (OEH) 105 Fl olarak bulundu. Yüksek homosistein değerleri homosistein % 87 olguda, nötrofillerde hipersegmentasyon % 80, atrofik gastrit % 52 ve APA pozitifliği % 45 oranında saptandı. Bu gruptaki hastaların yaş ortalamaları 65.1 olarak belirlendi. Vitamin B12 seviyesi 61-100 pg/mL olan grupta (Grup 2) homosistein yüksekliği % 45, OEH 79Fl olduğu tespit edildi. Vitamin düzeyleri 101-140 pg/mL'ın arasındaki grupta ise (Grup 3) homosistein yüksekliği % 21, OEH 80 ve 141-178 pg/mL arasında olan grupta ise (Grup 4) homosistein yüksekliği % 16 ve OEH 76 olduğu belirlendi. Grup 1 dışındaki gruplarda homosistein, APA pozitifliği, OEH ve hipersegmentasyon değerlerinin anlamlı şekilde azaldığı belirlendi. Sonuç: Bu çalışma sonucunda referans değerlerinin altındaki serum vitamin B12 düzeyinin gerçek doku düzeyini yansıtmayabileceği, vitamin B12 düzeyi 60 pg/mL ve altındaki değerlerin daha özgül olduğu saptandı.

Introduction

The diagnosis and treatment of the megaloblastic anemia regarding vitamin B12 or folic acid deficiency are important. The vitamin B12 and folic acid deficiency are a group of diseases whose their diagnosis are easy and the cost of their treatments are fairly lower. The delays in the treatment could lead to series complications such as anemia, irreversible neurological impairment neurological tube defects and thromboembolism [1]-[3]. The deficiency of vitamin B12 is more prevalent than the previously known in the population [4], [5]. The prevalence in elderly people is approximately between the 10 and 20 %.

However about 5–10 % of the patients with vitamin B12 deficiency is asymptomatic. The classical clinical findings and signs are the neuro-psychiatric symptoms, glossitis, macrocytic anemia and other cytopenias. For the diagnosis, the levels of serum vitamin B12 should be measured and in addition, homocysteine and methyl malonic acid levels should also be detected for correct interpretation of vitamin B12 levels [6].

The serum level of vitamin B12, which is one of the current methods of diagnosis, is known not to represent the real tissue level [7]. In the serum of patients with vitamin B12 deficiency, the anti parietal cell antibodies (APA) in 90% of cases, anti intrinsic factor antibodies in 60% of cases and anti thyroid antibodies in 40% of cases are present in the serum of patients [8]. The measurement of the methyl malonic acid and homocysteine levels that have been elevated in the early period of vitamine B12 deficiency is a more sensitive method for screening the vitamin B12 deficiency. Since the elevation of at least one metabolite is 94% sensitive and 99% specific.

Schilling test which has been utilized for the detection of pernicious anemia has usually been replaced with the serological detection of antibodies to intrinsic factor and parietal cells [9].

Many unnecessary treatments and interventions might be performed in patients without clinical signs of findings when their serum levels of vitamin B12 have been found to be lower in the clinics. In the present study, we aimed to find the more significant vitamin B12 levels with hematological and biochemical tests in terms of detecting the deficiencies in the cases whose serum vitamin B12 levels are measured lower than reference values.

Methods

Selection of the cases and design of the study
Of the patients that have been applied to our center for anemia between April 2005 and November 2007 or are referred to hematology consultation for having lower vitamin B12 levels, a total of one hundred fifty eight cases whose ages are between 16 and 90 and fifty three of those are female; have been retrieved retrospectively for the information regarding headings of diagnosis/referring diagnosis, cause of application, physical examination findings, assessment/plan laboratory data anti parietal antibodies result, radiology/endoscopy/ pathology consultation, etiology, treatments and advises heading from computer software (Avicenna HBSY, Ankara, Turkey) for the nutritional anemias. The patients with the combined iron deficiency, folic acid deficiencies or thalessemia carriers have been excluded from the study.

Patients have been categorized into four groups based on their vitamin B12 levels. The 34 patients whose vitamin B12 levels are lower than < 60 pg/mL, their ages ranged from 32 to 90 and 13 of them are females constituted the group 1 one; the 32 patients, whose vitamin B12 levels are between 61 and 100 pg /mL, their ages ranged from 19 to 87 and 14 of them are females constituted the group 2, 29 patients one, whose vitamin B12 levels are between 101 and 140 pg/mL, their ages ranged from 21 to 88 and 12 of them are females constituted the group 3, 28 patients, whose vitamin B12 levels are higher than 141 to 178 pg/ mL, their ages ranged from 16 to 62 and 14 of them are females constituted the group 4. A total of 128 patients have been included into study. Demographic information and clinical findings of the patients have been recorded.

Laboratory tests
The complete blood count, thyroid function tests, folic acid and homocysteine levels and the peripheral blood smear evaluations have been assessed in the venous samples of the peripheral blood that have been drawn from the fasting patients. The preparations have been used to detect the auto-anti bodies against to parietal cells commercially (Euroimmun, AG, Germany). They have been incubated with 1/10 patient serum for initial dilution. Then they have been incubated with IgG – FITC and the parietal antibodies have been detected with immune fluorescent microscope (Euro star, Euroimmune, AG Germany). The reference range has been detected as the 179 – 883 pg/mL, with the chemiluminescent microparticle immune assay (Abbott Laboratories, Abbott Park, IL, USA) in the Arcitect device. The homocysteine levels have been measured in the AXYM device (Abbott park, IL, USA) with the fluorescence polarization immune assay method and reference range has been taken as 5.9 – 16 μmol/L in males, 3.36–20.44 μmol/ L in females. The blood counts have been analyzed with the Cell Dyn 37000 automatic blood count analyzer (Abbott park, IL USA) . Thyroid function tests have been measured with Chemiluminescent Microparticle Immunoassay in the Arcitect device (Abbott Park, IL, USA). The patients with combined folic acid and Vitamin B12 deficiencies have been excluded from the study.

The helicobacter pylori positivity, has been shown either with the fast urease test (Pronto Dry, Medical Instr, Solothurn, Switzerland) or the histopathological assessment in the patients who underwent endoscopy. The quality control measurements have been performed with Biorad External Quality Assay System 3517 in the laboratory.

The data that were gathered have been transferred to SPSS 11.01 (15 th November 2001) statistical software package and analyzed with one way ANOVA and Pearson Chi square. P values that are less than 0 .05 were considered as statistically significant.

Results

The correlations between the vitamin B12 levels and the age hemoglobuline, homocysteine, APA, hyper segmentation finding in peripheral blood smear, MCV, thyroid function tests and patients with clinically diagnosed neurological problems have been depicted in table 1. The age, MCV, hemoglobine homocysteine, hypersegmentation in neutrophils, atrophic gastritis, positivity of helicobacter pylori, APA positivity and neurological problems are obviously higher in the group 1, in which the vitamin B12 level has been detected lower than the 60/pg/mL. This group has been found as the eldest group with the mean age of 65.1±14.8. When The group 1 has been compared individually to other groups, the age, hemoglobin value, MCV, level of homocysteine, APA positivity, hypersegmentation gastric atrophy and neurological problems have been significantly different in statistical analysis (p<0.05). There have been no statistically significant differences of thyroid function tests and helicobacter pylori positivity status between group 1 and other study groups (p >0. 05).

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