Determination of 25-hydroxyvitamin D [25(OH)D] represents a unique challenge, considering its lipophilic na - ture. Considering the widespread prevalence of vitamin D deficiency, which leads to increasing number of requests for 25(OH)D determination, immunoassay measurements adjusted to automated analyzers are being developed. Because of the variability among assays, it is often difficult to monitor vitamin D status and supplementation. The aim of this study was to compare the results of two immunoassays with high performance liquid chromatography with ultraviolet detection (HPLC-UV). Also, the aim was to estimate vitamin D status, since up to date the prevalence of vitamin D deficiency in Serbia was not examined. We have evaluated analytical characteristics of two automated immunoassays for 25(OH)D determination, from Roche (CobasR e601) and Abbott (Architect). For comparison studies we used HPLC analysis of 25-(OH)-Vitamin D3/D2 from Chromsystems (Waters isocratic system). In order to estimate vitamin D status in general population, we have searched the database of the laboratory information system and analyzed the data from 533 patients whose 25(OH)D was determined together with intact parathyroid hormone (iPTH). For imprecision assessment, four serum pools were prepared with following 25(OH)D concentrations: 35 nmol/L, ∼50 nmol/L, ∼75 nmol/L and ∼125 nmol/L. Obtai ned CVs for Roche method were 1.5-2.8% for within-run and 4.0-6.7% for between-run imprecision. For Abbott method, CVs were 0.7-4.4% for within- run and 3.8-7.2% for between-run imprecision. Inaccuracy was analyzed with commercial control sera. Obtained deviations from target value were 2.1% for Roche assay and 1.3-1.5% for Abbott method, and were not statistically significant (P>0.05). Comparison of Roche and HPLC-UV methods using Passing-Bablok regression analysis gave the following equation for the regression line y=0.937x+9.518 (r=0.739; n=97) and the regression line equation from the comparison of Abbott and HPLC-UV methods was y=0.745x+10.343 (r=0.793; n=97). Mean difference and SD for Bland-Altman plot were -4.5 nmol/L and 21.75 nmo/L, respectively for Roche method and 6.4 nmol/L and 18.8 nmol/L, respectively for Abbott. Statistical analysis (Chi-square test) of frequency distribution among different vitamin D status categories (<25 nmol/L severe deficiency, 25-50 nmol/L deficiency, 50-75 nmol/L insufficiency and >75 nmol/L sufficiency) showed that the frequency distribution obtained with Abbott method was significantly different from the distribution of the HPLC results, in contrast to Roche results frequency distribution which did not differ significantly. Also, statistical analysis of the agreement between the three methods for each vitamin D status category showed that results of both Roche and Abbott methods were significantly higher than HPLC in the two deficiency categories (P=0.005 for Roche, P=0.0407 for Abbott), and in the sufficiency category Abbott method significantly underestimated concentration of 25(OH)D compared to HPLC results (P<0.0001). Median population values of 25(OH)D and iPTH were 41.8 nmol/L and 76.6 ng/L, respectively. ANOVA analyses showed significant (P<0.05) decrease in iPTH and Ca2+ concentrations across the 25(OH)D concentration categories. Stepwise multiple linear regression analysis indicated independent correlation of iPTH with 25(OH)D concentration (b=-0.290, P=0.0008). Also, one-way ANOVA with Student-Newman-Keuls test demonstrated that 25(OH)D concentrations measured in summer and autumn were significantly (P<0.001) higher compared to those determined in winter and spring. Despite acceptable imprecision and inaccuracy of both examined methods, results obtained with them did not correlate well with HPLC-UV (r<0.9), which was used as a reference. However, methods showed satisfactory ability to classify patients into vitamin D status categories, which is important for diagnosis of vitamin D deficiency and therapy follow-up. About two thirds (68.5%) of the examined po pulation had vitamin D deficiency (25(OH)D<50 nmol/L) and only 8% had sufficient 25(OH)D concentration (>75 nmol/L).
Anđelo Beletić, Duško Mirković, Aleksandra Dudvarski-Ilić, Branislava Milenković, Ljudmila Nagorni-Obradović, Valentina Đorđević, Svetlana Ignjatović and Nada Majkić-Singh
Background: An increased homocysteine (Hcy) concentration may represent a metabolic marker of folate and vitamin B12 deficiency, both significant public health problems. For different reasons, patients with chronic obstructive pulmonary disease (COPD) are prone to these deficiencies. The study evaluates the reliability of Hcy concentration in predicting folate or vitamin B12 deficiency in these patients.
Methods: A group of 50 COPD patients (28 males/22 females, age
years was enrolled. A chemiluminescent microparticle immunoassay was applied for homocysteine, folate and vitamin B12 concentration. Kolmogorov-Smirnov, Mann-Whitney U and χ2 tests, Spearman’s correlation and ROC analysis were included in the statistical analysis, with the level of significance set at 0.05.
Results: Average (SD) concentrations of folate and vitamin B12 were 4.13 (2.16) μg/L and 463.6 (271.0) ng/L, whereas only vitamin B12 correlated with the Hcy level (P=−0.310 (R=0.029)). Gender related differences were not significant and only a borderline significant correlation between age and folate was confirmed (R=0.279 (P=0.047)). The incidence of folate and vitamin B12 deficiency differed significantly (P=0.000 and P<0.000 for folate and vitamin B12 respectively), depending on the cut-off used for classification (4.4, 6.6 and 8.0 μg/L – folate; 203 and 473 ng/L – vitamin B12). ROC analyses failed to show any significance of hyperhomocysteinemia as a predictor of folate or vitamin B12 deficiency.
Conclusion: Reliability of the Hcy concentration as a biomarker of folate or vitamin B12 depletion in COPD patients is not satisfactory, so their deficiency cannot be predicted by the occurrence of HHcy.
Anđelo Beletić, Aleksandra Dudvarski-Ilić, Branislava Milenković, Ljudmila Nagorni-Obradović, Mila Ljujić, Valentina Đorđević, Dragica Radojković and Nada Majkić-Singh
The primary role of alpha-1-antitrypsin (AAT), encoded by the highly polymorphic SERPINA1 gene, is to protect the lung parenchyma from proteolysis by neutrophil elastase. AAT deficiency (AATD) is an autosomal recessive disease, considered as the most important genetic cause of liver disease in children and emphysema in adults. According to frequency, deficient alleles can be classified as »common« (Z and S) and »rare« (Mmalton, Mheerlen, Mprocida etc). Type, intensity and onset of clinical disease associated with AATD occur as a result of interaction between AATD and additional genetic and acquired factors (tobacco smoking, air pollution exposure etc). The most frequent clinical manifestations include premature emphysema, chronic hepatitis, cirrhosis and hepatocellular carcinoma. Epidemiological studies highlight the need for improvement in diagnostic efficiency for AATD. It is recommended for a diagnostic approach to integrate precise, internationally recognized clinical criteria and a standardized laboratory protocol, based on a combination of biochemical and molecular methods. The predilection site of cli nical manifestations guides the therapeutic approach. Augmen tation therapy is possible in lung disease, while currently the only specific measure in patients with severe liver failure due to AATD is transplantation. In all patients, preventive measures, ammeliorating the deleterious effects of habits and environmental factors are recommended. Introduction of gene therapy is expected to additionally improve health outcomes in affected persons. Current results with an integrative AATD diagnostic strategy in the Serbian population are highly encouraging, prompting towards its further implementation in common medical practice with the ultimate goal to establish a national register of affected individuals.
Anđelo Beletić, Valentina Đorđević, Aleksandra Dudvarski-Ilić, Ivana Obradović, Duško Mirković, Mirka Ilić, Dragica Radojković and Nada Majkić-Singh
Isoelectrofocusing and PCR Amplification-Reverse Hybridization Assay in Evaluation of Alpha-1-Antitrypsin Deficiency
Alpha-1-antitrypsin deficiency is a potentially lethal genetic disorder, which has pulmonary and liver manifestations. The standardized biochemical and molecular diagnostic protocol for detection of clinically relevant alleles is needed. The paper summarizes current concepts about AATD, describes the potentials of isoelectric focusing and PCR amplification-reverse allele specific oligonucleotide hybridization assay in the detection of affected individuals and shortly presents our experiences in the evaluation of AATD. We conclude that the systematic clinical laboratory approach to AATD might be based on the combination of mentioned methods, coordinated by alpha-1-antritrypsin quantification. Additionally, its complete medical implementation is achieved through teamwork between clinical chemists, molecular biologists and clinicians.
Anđelo Beletić, Duško Mirković, Nebojša Antonijević, Valentina Đorđević, Violeta Šango, Branko Jakovljević, Jovan Peruničić, Mirka Ilić, Zorana Vasiljević and Nada Majkić-Singh
Incidence of Hyperhomocysteinemia and Mthfr C677T Polymorphism Among Young Patients with Acute Myocardial Infarction
Hyperhomocysteinemia is considered an independent risk factor for premature cardiovascular disease. Mutation MTHFR C677T reduces the activity of methylenetetra-hydrofolatereductase and may cause hyperhomocysteinemia. Incidence of hyperhomocysteinemia (homocysteine above 12 μmol/L), homocysteine level, and distribution of MTHFR C677T genotypes (C/C, C/T and T/T) are compared between young patients with acute myocardial infarction and healthy persons, matched by age. Study involved 86 patients younger than 45 years (77 men and 9 women) and 35 controls. Homocysteine was measured by an HPLC method and the MTHFR C677T genotype determined using PCR amplification and digestion with Hinf I. Statistical analyses included chisquare and Mann-Whitney U tests. Hyperhomocysteinemia was present in 32.6% patients and 14.3% controls, revealing a significant difference (P= 0.038). Median homocysteine levels in patients (10.4 μmol/L) and controls (9.6 μmol/L) were significantly different (P=0.035). Among patients, 50.0% had C/C, 41.9% C/T and 8.1% T/T genotype, and the genotype had no influence on hyperhomocysteinemia incidence and homocysteine level. Genotype distribution in patients was not significantly different from that observed in controls. The conclusion is that young patients with acute myocardial infarction have higher incidence of hyperhomocysteinemia and higher homocysteine levels than healthy young adults, while there is no significant difference in the distribution of MTHFR C677T genotypes.