Investigation of functional vitamin B12 deficiency in healthy and phenylketonuria children with plasma methylmalonic acid and homocysteine levels

Introduction and aim of study: Phenylketonuria (PKU) patients are at risk for functional vitamin B12 deficiency, based on their diet restricted of natural protein rich from phenylalanine. Elevated concentrations of methylmalonic acid (MMA) and/or homocysteine (Hcy), which are more sensitive functional markers of deficiency than vitamin B12 concentrations in the blood, are considered as measures of impaired intracellular vitamin B12 status. However, the diagnostic value of these parameters are not studied extensively. The aims of this study are to investigate functional vitamin B12 deficiency in treated PKU patients and healthy subjects as control group and to compare the diagnostic value of methylmalonic acid and vitamin B12 measurements in vitamin B12 deficiency.Materials and methods: 31 PKU patients on phenylalanine-restricted diet and 26 healthy children aged 5-18 years were included in the study. Serum vitamin B12, ferritin and folate levels were determined by chemiluminescence immunoassay. Phenylalanine levels were measured by a fluorometric method. MCV values were studied with hemogram analyzer. HPLC was used for plasma Hcy assay. Plasma MMA was determined using a stable isotope dilution-multiple reaction monitoring liquid chromatography-tandem mass spectrometry (SID-MRM LC-MS/MS) method. Accuracy, linearity, intra-assay and inter-assay precision, limit of detection and quantification, and recovery were evaluated to validate this method. Statistical analyses of data were performed using SPSS 15.0. Analyses were considered significant at p<0.05.Results: Accuracy for MMA assay calculated with calibration standards in the range of 0.05 to 2.5 ?mol/L was within 100 ± 4%. The MMA assay was linear up to 100 ?mol/L. Intra-assay and inter-assay CVs% were smaller than 4.0 and 6.0, respectively. The limit of detection was 0.021 ?mol/L and limit of quantification was 0.085 ?mol/L. Recoveries of MMA added to plasma samples with low and high levels of MMA were 87.6% and 86.4%, respectively. When PKU and control groups were compared in terms of vitamin B12 deficiency, no significant differences were found for serum vitamin B12, plasma MMA and Hcy concentrations between the groups. 12.9% of patients with PKU were found with low serum vitamin B12 concentrations. 29% of patients with PKU had elevated plasma MMA and 9.7% had high Hcy levels. In the controls, 30.7% and 11.5% had elevated also plasma MMA and Hcy levels, respectively. PKU patients had significantly elevated folate and MCV values than controls (p<0.01). Regarding the correlations between the parameters investigated; in PKU patients, plasma Hcy were correlated with phenylalanine and MCV values and negatively correlated with folate (p<0.05). In control group, we have found a positive correlation between plasma MMA and Hcy concentrations (p<0.05). Taking data of PKU and control subjects together, plasma MMA and MCV were negatively related to serum vitamin B12 concentrations (p<0.01). We have additionally found that, in vitamin B12 deficiency plasma MMA measurement is significantly 5.33 times (95% CI=1.62-17.49) more diagnostic than serum vitamin B12 measurement.Conclusions: Our study clearly demonstrated that; even with serum vitamin B12 concentrations within reference ranges, functional vitamin B12 deficiency may be seen in both healthy subjects and PKU patients who are at risk for potential vitamin B12 deficiency. Additionally, the significantly higher diagnostic value of plasma MMA measurement compared to serum vitamin B12 in vitamin B12 deficiency, has been shown for the first time in PKU and healthy children.Key words: Vitamin B12, methylmalonic acid, homocysteine, functional vitamin B12 deficiency, phenylketonuria, multiple reaction monitoring-liquid chromatography-tandem mass spectrometry (MRM-LC-MS/MS)