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Serum vitamin B12 concentrations within reference values do not exclude functional vitamin B12 deficiency in PKU patients of various ages
Molecular Genetics and Metabolism 102 (2011) 13–17
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Molecular Genetics and Metabolism j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m g m e
Serum vitamin B12 concentrations within reference values do not exclude functional vitamin B12 deficiency in PKU patients of various ages Inge Vugteveen a,b, Marieke Hoeksma a,b,⁎, Anne-Lise Bjorke Monsen c, M. Rebecca Fokkema d, Dirk-Jan Reijngoud b,d, Margreet van Rijn a, Francjan J. van Spronsen a,b a
Section of Metabolic Diseases, Beatrix Children's Hospital, University Medical Centre of Groningen, University of Groningen, Groningen, The Netherlands GUIDE, Graduate School for Drug Exploration, Center for Liver, Digestive and Metabolic Diseases, University Medical Centre of Groningen, University of Groningen, Groningen, The Netherlands c Department of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway d Laboratory of Metabolic Diseases, Department of Laboratory Medicine, University Medical Centre of Groningen, University of Groningen, Groningen, The Netherlands b
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Article history: Received 22 April 2010 Received in revised form 2 July 2010 Accepted 3 July 2010 Available online 27 October 2010 Keywords: Phenylketonuria Vitamin B12 Methylmalonic acid Homocysteine Metabolic control
a b s t r a c t Homocysteine (Hcy) and in particular methylmalonic acid (MMA) are considered reliable parameters for vitamin B12 status in healthy individuals. Phenylketonuria (PKU) patients are at risk for functional vitamin B12 deficiency based on their diet. Objective: The aim of this study was to investigate the prevalence of functional vitamin B12 deficiency in continuously treated PKU patients and the association of parameters of vitamin B12 and metabolic control. Methods: In 75 continuously treated PKU patients of 1–37 years of age, serum vitamin B12 concentrations, plasma Hcy, MMA, and phenylalanine concentrations were studied. Results: Eight patients had vitamin B12 concentrations below normal. Out of these eight patients, two had elevated MMA and/or Hcy concentrations. Ten other patients with normal vitamin B12 concentrations had elevated concentrations of MMA and/or Hcy. Conclusions: A vitamin B12 concentration within the reference range does not automatically imply a sufficient vitamin B12 status. We recommend measuring serum MMA, or alternatively plasma Hcy, yearly in all PKU patients to diagnose functional vitamin B12 deficiency. © 2010 Elsevier Inc. All rights reserved.
Introduction Treatment of phenylketonuria (PKU; McKusick 261600) aims at lowering blood phenylalanine (Phe) concentrations by dietary Phe restriction [1]. Since Phe is part of natural protein, Phe restriction implies restriction of natural protein. Consequently, PKU patients consume individually tailored limited amounts of natural protein to maintain blood Phe concentrations within the target range for age. The requirement for the remaining amino acids and nitrogen is fulfilled with a protein substitute free of Phe. This substitute is enriched with micronutrients, normally provided for with the natural protein intake and with extra tyrosine being an essential amino acid in PKU patients. The minor intake of natural protein in PKU patients is mainly of low quality (from vegetable rather than animal origin), and a significant
Abbreviations: Hcy, homocysteine; MMA, methylmalonic acid; Phe, phenylalanine; PKU, phenylketonuria; Vit B12, vitamin B12. ⁎ Corresponding author. Beatrix Children's Hospital, University Medical Centre of Groningen, PO box 30.001, CA 80, 9700 RB Groningen, The Netherlands. Fax: +31 50 3614235. E-mail address: [email protected] (M. Hoeksma). 1096-7192/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2010.07.004
number of PKU patients, especially adolescents and young adults, do not take the protein substitute adequately [2,3]. As a result, PKU patients tend to be at risk for deficiencies of nutrients that are mainly found in protein of animal origin [4,5], e.g. carnitine [6], selenium [7], and vitamin B12 [4,8]. A vitamin B12 deficiency is often diagnosed based on a combination of clinical symptoms, morphological findings, and biochemical tests [9]. The clinical picture of vitamin B12 deficiency is diffuse and may comprise anemia, gastrointestinal and neurological symptoms including both low and brisk tendon reflexes. Brisk tendon reflexes are often seen in PKU patients and have been related to vitamin B12 deficiency [10–12]. Until now, vitamin B12 deficiencies in PKU patients have been investigated by especially studying serum vitamin B12 concentrations in blood in PKU patients [4,8,10,11,13,14]. Biochemically, there seems to be a discrepancy between vitamin B12 concentrations in serum and vitamin B12 activity [15]. Vitamin B12 is a cofactor of two enzymatic reactions converting methylmalonyl-CoA into succinyl-CoA, and homocysteine (Hcy) into methionine [16]. In vitamin B12 deficiency, methylmalonyl-coA is converted into methylmalonic acid (MMA) and released into the circulation. Elevated concentrations of MMA and/or Hcy in the blood are considered measures of impaired intracellular cobalamin status [17–19] and may
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be increased even when serum vitamin B12 concentrations are within reference values [20,21]. Therefore, in this study, we defined functional vitamin B12 deficiency as an increase of MMA and/or Hcy concentration in plasma. The present study aimed to investigate the prevalence of decreased functional vitamin B12 activity in continuously treated PKU patients of various ages, the prevalence of decreased vitamin B12 concentrations, and the association between serum vitamin B12 and functional vitamin B12 activity. Besides, the relation between vitamin B12 concentration in serum and biochemical vitamin B12 activity was investigated. We hypothesized that functional vitamin B12 deficiency should be considered more often than based on serum vitamin B12 concentrations alone. Patients and methods The PKU patients at the University Medical Centre of Groningen (n = 91) were investigated retrospectively. All PKU patients but three were diagnosed within 2 weeks after birth by neonatal screening. The three patients were diagnosed at 1 to 2 years of age, either before introduction of neonatal screening (n = 2) or missed by neonatal screening. From diagnosis onwards, patients were continuously treated with a Phe-restricted diet. None of the patients had hard clinical signs of functional vitamin B12 deficiency, although in most patients hyperreflexia can be found at least to some extent. From 2001 onwards, the aimed blood Phe concentrations were 120–360 μmol/L for patients below 12 years, 120–600 μmol/L for patients over 12 years, and 120– 240 μmol/L for women aiming to be pregnant and during pregnancy [22]. Whole blood spots for analysis of blood Phe are controlled with frequency depending on age, e.g., twice a week for young infants and once a month for adults. Of the 91 patients, 16 were excluded because of insufficient laboratory data (n=15) or pregnancy (n=1). No patients had severe comorbidity, leaving 75 patients (36 males and 39 females) aged 1–37 years, for statistical analysis of circulating concentrations of vitamin B12, MMA, and Hcy (Table 1). All patients were taken as one group. Starting in 2004, circulating concentrations of vitamin B12, MMA and Hcy were determined in all PKU patients on a routine basis at least yearly. Laboratory data were collected from the laboratory database. Measurements of serum vitamin B12, serum MMA, and plasma Hcy were performed in blood sampled taken at the same moment. Serum vitamin B12 concentration was measured by fluoroimmunoassay (AutoDelfia, PerkinElmer Life and Analytical Sciences, Wallac Oy, Finland). Plasma Hcy was measured by fluorescence polarization immunoassay (IMx; Abbott Laboratories, The Netherlands). Serum MMA was measured by gas chromatography-mass spectrometry. MMA and the internal standard MMA-D3 were derivatised with pentafluorobenzylbromide and detected in the selected ion monitoring mode at target ions of m/z 233.1 (MMA) and m/z 236.1 (MMA-D3). The method uses negative ion chemical ionisation with ammonia as the reagent gas. The local laboratory did not provide reference values for children for serum vitamin B12 and plasma Hcy. For the outer limits of the vitamin B12 and Hcy concentrations, we used the P2,5 and the P97,5 in the population of Monsen et al. [23]. To enable comparison of values between the laboratory of Monsen (Bergen method) and our laboratory (Groningen method), 36 samples from various age groups (1–32 years) were analyzed at both laboratories. Passing and Bablok regression analysis Table 1 Age dependent reference values for vitamin B12 and plasma Hcy. Age (years)
Vitamin B12 (pmol/L)
Plasma Hcy (μmol/L)
Blood Phe (μmol/L)
Plasma MMA (nmol/L)
1.6–11.9 12–19.9 N 20.0
194–1002 141–749 170–700
1.5–7.8 4.0–13.8 5.0–15.0
120–360 120–600 120–600
90–340 90–340 90–340
revealed a significant difference between methods with a slope of 1.15 and an intercept of −1.29 (r = 0.974). Corrected values were used for reference values of vitamin B12 in serum and Hcy in plasma (Table 1). The reference range (P2,5–P97,5) at the laboratory of the metabolic centre in Groningen of MMA is 90–340 nmol/L, and independent of age in children older than 1.5 years [23]. A functional vitamin B12 deficiency was defined as a MMA concentration above the P97,5. Blood spot Phe is analyzed by high-performance liquid chromatography with fluorescence detection. Blood spots are punched out and left to deproteinize and elute in trichloroacetic acid containing an internal standard. Primary and secondary amino acids subsequently react with 6aminoquinolyl-N-hydrozysuccinimidyl carbamate to obtain fluorescent derivatives. Metabolic control was defined as the average Phe concentration measured in the year preceding measurements of vitamin B12, MMA, and Hcy. Statistical analysis SPSS 14 was used for statistical analyses. Descriptive statistics of the different parameters were obtained. The median and interquartile ranges were used as the data were not evenly distributed. Spearman rank was used to study the relation between vitamin B12 concentrations and MMA and/or Hcy concentrations. Metabolic control (as derived from mean blood Phe concentrations) was taken into account as a possible confounding factor in Spearman correlation. Analyses were considered significant at P b 0.05. Results Clinical characteristics, blood Phe concentrations, and vitamin B12 status are presented in Table 2. Eight patients had serum vitamin B12 concentrations below reference ranges. One of these eight had an elevated serum MMA concentration and another had elevated concentrations of both Hcy and MMA. Ten other patients had biochemical evidence of functional vitamin B12 deficiency. All of these 10 patients had vitamin B12 concentrations within reference range. Five out of these ten patients had elevated serum MMA concentrations, and two patients had elevated concentrations of both MMA and Hcy. Three other patients had elevated plasma Hcy concentrations (Fig. 1; Table 3). Table 2 shows that the number of patients with an increased MMA and/or Hcy was equal or higher than the number of patients with decreased serum vitamin B12 concentrations. Fig. 1 shows the relationship between Hcy/MMA and vitamin B12 concentrations in patients with increased Hcy and/or MMA concentrations. Table 3 shows the absolute concentrations of the 18 patients with abnormal concentrations of vitamin B12, MMA, and/or Hcy. Correlations of serum vitamin B12 concentration with serum MMA and plasma Hcy for various age groups did not show consistently Table 2 Patients characteristics and vitamin B12 markers. n Male (%) Serum vitamin B12, pmol/L Median Interquartile range Deficient (number of patients) Serum MMA, nmol/L Median Interquartile range Elevated MMA (number of patients) Plasma Hcy, μmol/L Median Interquartile range Elevated Hcy (number of patients) Blood Phe, μmol/L Median Interquartile range
75 48.0 288.0 (n = 69) 202.0–386.0 8 229.5 (n = 68) 175.6–271.3 9 6.0 (n = 67) 4.9–8.5 6 380.0 (n = 62) 289.8–513.8
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Fig. 1. Normal concentrations of Hcy and MMA, elevated concentrations of Hcy and/or MMA, and vitamin B12 concentrations (number of patients).
significant correlations between these variables. Taking data of all ages together, MMA concentration, plasma Hcy concentration, and metabolic control were negatively related to serum vitamin B12 concentrations (r = −0.452, P = 0.000, r = −0.690, P = 0.000, r = −0.391, P = 0.002, respectively). The blood Phe concentration did not correlate significant with serum MMA and plasma Hcy concentrations. This was corrected for age. Discussion The most important findings of this study were that in 12 of these 75 continuously treated PKU patients, a functional vitamin B12 deficiency, defined as an increase of MMA and/or Hcy, was found and that there was no consistent relationship between circulating MMA and Hcy concentrations and the metabolic control. Significant correlations were shown between serum vitamin B12 and circulating Hcy and MMA and between serum vitamin B12 and metabolic control. Before discussing the results in more detail, a few methodological issues will be dealt with. Table 3 Absolute concentrations of patients with a functional vitamin B12 deficiency.a Age
Vitamin B12 concentration (pmol/L)
Serum MMA (nmol/L)
Plasma Hcy (μmol/L)
1.5 7 8 9 9 13 14 15 16 18 20 21 26 28 28 29 30 37
254 216 203 113 181 143 185 227 301 109 109 186 186 151 104 130 364 134
257 245 378(↑) 251 258 682 (↑) 1280 (↑) 437 (↑) 285 935 (↑) 226 430 (↑) 342 (↑) 177 160 790 (↑) 359 (↑) 255
9(↑) 8 (↑) 6 6 7 12 (↑) 17 (↑) 9 14 (↑) 8 9 14 12 14 10 35 (↑) 7 11
a
(↓) (↓)
(↓) (↓)
(↓) (↓) (↓) (↓)
Vitamin B12 and Hcy have age-related values in contrast to MMA.
First, it was difficult to obtain reference values of Hcy and vitamin B12 in children, since the laboratory of the University Medical Centre of Groningen did not have reference values for children for these measures. We obtained reference values from Monsen et al. [23]. To enable comparison between the data of the laboratory of Monsen and our laboratory, samples from various age groups were analyzed at both the laboratory of Dr. Monsen and our laboratory, so that our values could be adequately compared to the reference values. Reference values for MMA are not considered age-dependent in children older than 1.5 years [23], and we used 340 nmol/L as the upper reference concentration for all ages. From external quality control programs, it is known that interlaboratory variation of serum vitamin B12 is large. This is mainly dependent on differences in the antibodies that are used. Correction of the constant bias of 57 pmol/L between the two laboratories was not considered to be a problem given the good agreement between the method of Monsen et al. and ours (r= 0.974). Second, analyses were based on one rather than a series of measurements of Hcy, MMA and vitamin B12 in all individuals. We decided this was acceptable as intraindividual variation was rather small in our population (results not shown). Third, there is a lack of consensus about the definition of vitamin B12 deficiency. Definitions vary between ‘impaired cobalamin status’, ‘cobalamin deficiency’, and ‘vitamin B12 deficiency’ [4,11,23–25], while Savage et al. [17] also used hematologic and neurologic findings and responses to therapy. We prefer to use the term ‘functional vitamin B12 deficiency’ defined by an increase of MMA. We also measured Hcy, although it is known that an increase of this functional marker is not specific for vitamin B12 deficiency. MMA (and Hcy) are considered to be at the start of a continuum that ultimately results in clinically evident vitamin B12 deficiency. Moreover, clinical vitamin B12 deficiency is difficult to recognize, since many patients never develop macrocytic anemia and neurological symptoms are nonspecific and eventually irreversible. For example, the hyperreflexia observed in almost all our patients after some years of treatment could be due to some degree of functional vitamin B12 deficiency. This is in line with Pietz et al. [26]. With regard to the results of the present study, 10 of the 67 patients with serum vitamin B12 concentrations within reference values had elevated MMA and/or Hcy. Only 2 of the 12 patients with elevated MMA and/or Hcy showed vitamin B12 concentrations below reference range. Elevated Hcy and MMA concentrations are sensitive markers for reduced vitamin B12 activity [19]. These results indicate that the vitamin B12 concentration does not prove or exclude functional vitamin B12
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deficiency. By this, our results are in line with the results in various nonPKU studied populations [17,19,27,28]. We found only inconsistent relations between serum vitamin B12 concentrations, serum MMA and plasma Hcy concentrations in different age groups. Because of the relatively small groups when divided into different age groups, we treated the data of the total population as one group. In the whole group, we found significant relations between the serum vitamin B12 concentration and the plasma Hcy concentration and serum MMA concentration and between vitamin B12 concentrations and metabolic control. Our data do not substantiate the results of some studies in PKU patients that did not show a significant correlation between plasma Hcy concentrations and vitamin B12 concentrations [14,26,29]. Part of these differences can be explained by the fact that (part of the) patients studied by Colome et al. [26] and Hvas et al. [29] received extra supplementation of vitamins in addition to the protein substitute. At this moment, we cannot explain the difference in the study of Huemer et al. [14] that showed no relation between Hcy and vitamin B12 concentrations. Theoretically, discrepancies between our results and results of Huemer et al. [14] and Hvas et al. [29] may be caused by differences in folate or vitamin B6 concentration that also influence Hcy concentration in plasma [4,16,17,23,26,30]. Folate has an important role in vitamin B12 metabolism and can also be an important confounder of vitamin B12 metabolism [31]. Unfortunately, in our study, neither folate nor vitamin B6 concentrations were measured. In PKU, deficiencies of vitamin B12 have always been attributed to deficient intake [8,11,13]. Intake of vitamin B12, however, may only be one of the factors. In this study, the intake of protein was not studied. Another important factor may be the bioavailability of vitamin B12 [32]. In general, PKU patients do not have malabsorption, but the possibility of lower bioavailability of vitamin B12 within the protein substitute has not been studied and may result in lower intracellular concentrations. Such mechanisms have been reported in PKU for the intake of calcium [33,34] and amino acids [35]. Another reason of the functional vitamin B12 deficiency may be the timing of the intake rather than the amount and content of the protein substitute. A study about vitamin B12 sources and bioavailability of Watanabe [32] showed a significant decrease of the availability of vitamin B12 with increasing intake of vitamin B12 per meal. It is recommended to take the protein substitute as evenly distributed over the day as possible [3,36] and lifelong [37,38]. PKU patients in our population reported to take their main source of vitamin B12 (the protein substitute) three times a day. However, a less frequent intake of the protein substitute cannot be excluded. The combination of a possibly lower availability and taking the protein substitute in less than three times a day may be a reason for finding more biochemical vitamin B12 deficiencies in PKU patients than in the healthy population. In this way, increased MMA and/or Hcy may be considered not only indicative of functional vitamin B12 deficiency, but also as a marker of possible inadequate intake of the protein substitute as a whole. When functional vitamin B12 deficiency is due to factors such as inadequate (timing of) intake or nonoptimal bioavailability, the risk of vitamin B12 deficiency may also apply for other nutrients. In conclusion, a relation between concentrations of vitamin B12 and MMA and Hcy, functional vitamin B12 deficiency, defined as elevated serum MMA or plasma Hcy concentrations, is seen relatively often in PKU patients even with a serum vitamin B12 concentration within reference values. An elevated serum MMA or plasma Hcy concentration rather than low serum vitamin B12 concentrations is a reliable indicator of lack of active vitamin B12. It, therefore, is important to evaluate serum MMA or plasma Hcy concentrations regularly in PKU patients. Hcy may be measured if MMA measurements are not readily available, although it has to be considered that Hcy may also be increased with deficiencies of folate and vitamin B6. When MMA or Hcy concentrations in PKU patients are increased, it is necessary to optimize the intake of the protein substitute to ensure
adequate intake of both vitamin B12 and other micronutrients. When increased MMA or Hcy persists, knowledge on the response (clinical or biochemical) on vitamin B12 supplementation is indicated. Not only inadequate intake but perhaps also less optimal bioavailability from the protein supplement may be a cause of deficient vitamin B12 status. The latter possibility needs to be studied further. Acknowledgment We kindly acknowledge the laboratory workers of the department of laboratory medicine in Groningen and the laboratory workers at the laboratory in Bergen, Norway, for their kind help. References [1] C.R. Scriver, S. Kaufman, Hyperphenylalaninemia: phenylalanine hydroxylase deficiency, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic & Molecular Bases of Inherited Disease, McGraw-Hill, Inc., New York, 2001, pp. 1667–1724. [2] A.P. Prince, M.P. McMurray, N.R. 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Molecular Genetics and Metabolism j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m g m e
Serum vitamin B12 concentrations within reference values do not exclude functional vitamin B12 deficiency in PKU patients of various ages Inge Vugteveen a,b, Marieke Hoeksma a,b,⁎, Anne-Lise Bjorke Monsen c, M. Rebecca Fokkema d, Dirk-Jan Reijngoud b,d, Margreet van Rijn a, Francjan J. van Spronsen a,b a
Section of Metabolic Diseases, Beatrix Children's Hospital, University Medical Centre of Groningen, University of Groningen, Groningen, The Netherlands GUIDE, Graduate School for Drug Exploration, Center for Liver, Digestive and Metabolic Diseases, University Medical Centre of Groningen, University of Groningen, Groningen, The Netherlands c Department of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway d Laboratory of Metabolic Diseases, Department of Laboratory Medicine, University Medical Centre of Groningen, University of Groningen, Groningen, The Netherlands b
a r t i c l e
i n f o
Article history: Received 22 April 2010 Received in revised form 2 July 2010 Accepted 3 July 2010 Available online 27 October 2010 Keywords: Phenylketonuria Vitamin B12 Methylmalonic acid Homocysteine Metabolic control
a b s t r a c t Homocysteine (Hcy) and in particular methylmalonic acid (MMA) are considered reliable parameters for vitamin B12 status in healthy individuals. Phenylketonuria (PKU) patients are at risk for functional vitamin B12 deficiency based on their diet. Objective: The aim of this study was to investigate the prevalence of functional vitamin B12 deficiency in continuously treated PKU patients and the association of parameters of vitamin B12 and metabolic control. Methods: In 75 continuously treated PKU patients of 1–37 years of age, serum vitamin B12 concentrations, plasma Hcy, MMA, and phenylalanine concentrations were studied. Results: Eight patients had vitamin B12 concentrations below normal. Out of these eight patients, two had elevated MMA and/or Hcy concentrations. Ten other patients with normal vitamin B12 concentrations had elevated concentrations of MMA and/or Hcy. Conclusions: A vitamin B12 concentration within the reference range does not automatically imply a sufficient vitamin B12 status. We recommend measuring serum MMA, or alternatively plasma Hcy, yearly in all PKU patients to diagnose functional vitamin B12 deficiency. © 2010 Elsevier Inc. All rights reserved.
Introduction Treatment of phenylketonuria (PKU; McKusick 261600) aims at lowering blood phenylalanine (Phe) concentrations by dietary Phe restriction [1]. Since Phe is part of natural protein, Phe restriction implies restriction of natural protein. Consequently, PKU patients consume individually tailored limited amounts of natural protein to maintain blood Phe concentrations within the target range for age. The requirement for the remaining amino acids and nitrogen is fulfilled with a protein substitute free of Phe. This substitute is enriched with micronutrients, normally provided for with the natural protein intake and with extra tyrosine being an essential amino acid in PKU patients. The minor intake of natural protein in PKU patients is mainly of low quality (from vegetable rather than animal origin), and a significant
Abbreviations: Hcy, homocysteine; MMA, methylmalonic acid; Phe, phenylalanine; PKU, phenylketonuria; Vit B12, vitamin B12. ⁎ Corresponding author. Beatrix Children's Hospital, University Medical Centre of Groningen, PO box 30.001, CA 80, 9700 RB Groningen, The Netherlands. Fax: +31 50 3614235. E-mail address: [email protected] (M. Hoeksma). 1096-7192/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2010.07.004
number of PKU patients, especially adolescents and young adults, do not take the protein substitute adequately [2,3]. As a result, PKU patients tend to be at risk for deficiencies of nutrients that are mainly found in protein of animal origin [4,5], e.g. carnitine [6], selenium [7], and vitamin B12 [4,8]. A vitamin B12 deficiency is often diagnosed based on a combination of clinical symptoms, morphological findings, and biochemical tests [9]. The clinical picture of vitamin B12 deficiency is diffuse and may comprise anemia, gastrointestinal and neurological symptoms including both low and brisk tendon reflexes. Brisk tendon reflexes are often seen in PKU patients and have been related to vitamin B12 deficiency [10–12]. Until now, vitamin B12 deficiencies in PKU patients have been investigated by especially studying serum vitamin B12 concentrations in blood in PKU patients [4,8,10,11,13,14]. Biochemically, there seems to be a discrepancy between vitamin B12 concentrations in serum and vitamin B12 activity [15]. Vitamin B12 is a cofactor of two enzymatic reactions converting methylmalonyl-CoA into succinyl-CoA, and homocysteine (Hcy) into methionine [16]. In vitamin B12 deficiency, methylmalonyl-coA is converted into methylmalonic acid (MMA) and released into the circulation. Elevated concentrations of MMA and/or Hcy in the blood are considered measures of impaired intracellular cobalamin status [17–19] and may
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be increased even when serum vitamin B12 concentrations are within reference values [20,21]. Therefore, in this study, we defined functional vitamin B12 deficiency as an increase of MMA and/or Hcy concentration in plasma. The present study aimed to investigate the prevalence of decreased functional vitamin B12 activity in continuously treated PKU patients of various ages, the prevalence of decreased vitamin B12 concentrations, and the association between serum vitamin B12 and functional vitamin B12 activity. Besides, the relation between vitamin B12 concentration in serum and biochemical vitamin B12 activity was investigated. We hypothesized that functional vitamin B12 deficiency should be considered more often than based on serum vitamin B12 concentrations alone. Patients and methods The PKU patients at the University Medical Centre of Groningen (n = 91) were investigated retrospectively. All PKU patients but three were diagnosed within 2 weeks after birth by neonatal screening. The three patients were diagnosed at 1 to 2 years of age, either before introduction of neonatal screening (n = 2) or missed by neonatal screening. From diagnosis onwards, patients were continuously treated with a Phe-restricted diet. None of the patients had hard clinical signs of functional vitamin B12 deficiency, although in most patients hyperreflexia can be found at least to some extent. From 2001 onwards, the aimed blood Phe concentrations were 120–360 μmol/L for patients below 12 years, 120–600 μmol/L for patients over 12 years, and 120– 240 μmol/L for women aiming to be pregnant and during pregnancy [22]. Whole blood spots for analysis of blood Phe are controlled with frequency depending on age, e.g., twice a week for young infants and once a month for adults. Of the 91 patients, 16 were excluded because of insufficient laboratory data (n=15) or pregnancy (n=1). No patients had severe comorbidity, leaving 75 patients (36 males and 39 females) aged 1–37 years, for statistical analysis of circulating concentrations of vitamin B12, MMA, and Hcy (Table 1). All patients were taken as one group. Starting in 2004, circulating concentrations of vitamin B12, MMA and Hcy were determined in all PKU patients on a routine basis at least yearly. Laboratory data were collected from the laboratory database. Measurements of serum vitamin B12, serum MMA, and plasma Hcy were performed in blood sampled taken at the same moment. Serum vitamin B12 concentration was measured by fluoroimmunoassay (AutoDelfia, PerkinElmer Life and Analytical Sciences, Wallac Oy, Finland). Plasma Hcy was measured by fluorescence polarization immunoassay (IMx; Abbott Laboratories, The Netherlands). Serum MMA was measured by gas chromatography-mass spectrometry. MMA and the internal standard MMA-D3 were derivatised with pentafluorobenzylbromide and detected in the selected ion monitoring mode at target ions of m/z 233.1 (MMA) and m/z 236.1 (MMA-D3). The method uses negative ion chemical ionisation with ammonia as the reagent gas. The local laboratory did not provide reference values for children for serum vitamin B12 and plasma Hcy. For the outer limits of the vitamin B12 and Hcy concentrations, we used the P2,5 and the P97,5 in the population of Monsen et al. [23]. To enable comparison of values between the laboratory of Monsen (Bergen method) and our laboratory (Groningen method), 36 samples from various age groups (1–32 years) were analyzed at both laboratories. Passing and Bablok regression analysis Table 1 Age dependent reference values for vitamin B12 and plasma Hcy. Age (years)
Vitamin B12 (pmol/L)
Plasma Hcy (μmol/L)
Blood Phe (μmol/L)
Plasma MMA (nmol/L)
1.6–11.9 12–19.9 N 20.0
194–1002 141–749 170–700
1.5–7.8 4.0–13.8 5.0–15.0
120–360 120–600 120–600
90–340 90–340 90–340
revealed a significant difference between methods with a slope of 1.15 and an intercept of −1.29 (r = 0.974). Corrected values were used for reference values of vitamin B12 in serum and Hcy in plasma (Table 1). The reference range (P2,5–P97,5) at the laboratory of the metabolic centre in Groningen of MMA is 90–340 nmol/L, and independent of age in children older than 1.5 years [23]. A functional vitamin B12 deficiency was defined as a MMA concentration above the P97,5. Blood spot Phe is analyzed by high-performance liquid chromatography with fluorescence detection. Blood spots are punched out and left to deproteinize and elute in trichloroacetic acid containing an internal standard. Primary and secondary amino acids subsequently react with 6aminoquinolyl-N-hydrozysuccinimidyl carbamate to obtain fluorescent derivatives. Metabolic control was defined as the average Phe concentration measured in the year preceding measurements of vitamin B12, MMA, and Hcy. Statistical analysis SPSS 14 was used for statistical analyses. Descriptive statistics of the different parameters were obtained. The median and interquartile ranges were used as the data were not evenly distributed. Spearman rank was used to study the relation between vitamin B12 concentrations and MMA and/or Hcy concentrations. Metabolic control (as derived from mean blood Phe concentrations) was taken into account as a possible confounding factor in Spearman correlation. Analyses were considered significant at P b 0.05. Results Clinical characteristics, blood Phe concentrations, and vitamin B12 status are presented in Table 2. Eight patients had serum vitamin B12 concentrations below reference ranges. One of these eight had an elevated serum MMA concentration and another had elevated concentrations of both Hcy and MMA. Ten other patients had biochemical evidence of functional vitamin B12 deficiency. All of these 10 patients had vitamin B12 concentrations within reference range. Five out of these ten patients had elevated serum MMA concentrations, and two patients had elevated concentrations of both MMA and Hcy. Three other patients had elevated plasma Hcy concentrations (Fig. 1; Table 3). Table 2 shows that the number of patients with an increased MMA and/or Hcy was equal or higher than the number of patients with decreased serum vitamin B12 concentrations. Fig. 1 shows the relationship between Hcy/MMA and vitamin B12 concentrations in patients with increased Hcy and/or MMA concentrations. Table 3 shows the absolute concentrations of the 18 patients with abnormal concentrations of vitamin B12, MMA, and/or Hcy. Correlations of serum vitamin B12 concentration with serum MMA and plasma Hcy for various age groups did not show consistently Table 2 Patients characteristics and vitamin B12 markers. n Male (%) Serum vitamin B12, pmol/L Median Interquartile range Deficient (number of patients) Serum MMA, nmol/L Median Interquartile range Elevated MMA (number of patients) Plasma Hcy, μmol/L Median Interquartile range Elevated Hcy (number of patients) Blood Phe, μmol/L Median Interquartile range
75 48.0 288.0 (n = 69) 202.0–386.0 8 229.5 (n = 68) 175.6–271.3 9 6.0 (n = 67) 4.9–8.5 6 380.0 (n = 62) 289.8–513.8
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Fig. 1. Normal concentrations of Hcy and MMA, elevated concentrations of Hcy and/or MMA, and vitamin B12 concentrations (number of patients).
significant correlations between these variables. Taking data of all ages together, MMA concentration, plasma Hcy concentration, and metabolic control were negatively related to serum vitamin B12 concentrations (r = −0.452, P = 0.000, r = −0.690, P = 0.000, r = −0.391, P = 0.002, respectively). The blood Phe concentration did not correlate significant with serum MMA and plasma Hcy concentrations. This was corrected for age. Discussion The most important findings of this study were that in 12 of these 75 continuously treated PKU patients, a functional vitamin B12 deficiency, defined as an increase of MMA and/or Hcy, was found and that there was no consistent relationship between circulating MMA and Hcy concentrations and the metabolic control. Significant correlations were shown between serum vitamin B12 and circulating Hcy and MMA and between serum vitamin B12 and metabolic control. Before discussing the results in more detail, a few methodological issues will be dealt with. Table 3 Absolute concentrations of patients with a functional vitamin B12 deficiency.a Age
Vitamin B12 concentration (pmol/L)
Serum MMA (nmol/L)
Plasma Hcy (μmol/L)
1.5 7 8 9 9 13 14 15 16 18 20 21 26 28 28 29 30 37
254 216 203 113 181 143 185 227 301 109 109 186 186 151 104 130 364 134
257 245 378(↑) 251 258 682 (↑) 1280 (↑) 437 (↑) 285 935 (↑) 226 430 (↑) 342 (↑) 177 160 790 (↑) 359 (↑) 255
9(↑) 8 (↑) 6 6 7 12 (↑) 17 (↑) 9 14 (↑) 8 9 14 12 14 10 35 (↑) 7 11
a
(↓) (↓)
(↓) (↓)
(↓) (↓) (↓) (↓)
Vitamin B12 and Hcy have age-related values in contrast to MMA.
First, it was difficult to obtain reference values of Hcy and vitamin B12 in children, since the laboratory of the University Medical Centre of Groningen did not have reference values for children for these measures. We obtained reference values from Monsen et al. [23]. To enable comparison between the data of the laboratory of Monsen and our laboratory, samples from various age groups were analyzed at both the laboratory of Dr. Monsen and our laboratory, so that our values could be adequately compared to the reference values. Reference values for MMA are not considered age-dependent in children older than 1.5 years [23], and we used 340 nmol/L as the upper reference concentration for all ages. From external quality control programs, it is known that interlaboratory variation of serum vitamin B12 is large. This is mainly dependent on differences in the antibodies that are used. Correction of the constant bias of 57 pmol/L between the two laboratories was not considered to be a problem given the good agreement between the method of Monsen et al. and ours (r= 0.974). Second, analyses were based on one rather than a series of measurements of Hcy, MMA and vitamin B12 in all individuals. We decided this was acceptable as intraindividual variation was rather small in our population (results not shown). Third, there is a lack of consensus about the definition of vitamin B12 deficiency. Definitions vary between ‘impaired cobalamin status’, ‘cobalamin deficiency’, and ‘vitamin B12 deficiency’ [4,11,23–25], while Savage et al. [17] also used hematologic and neurologic findings and responses to therapy. We prefer to use the term ‘functional vitamin B12 deficiency’ defined by an increase of MMA. We also measured Hcy, although it is known that an increase of this functional marker is not specific for vitamin B12 deficiency. MMA (and Hcy) are considered to be at the start of a continuum that ultimately results in clinically evident vitamin B12 deficiency. Moreover, clinical vitamin B12 deficiency is difficult to recognize, since many patients never develop macrocytic anemia and neurological symptoms are nonspecific and eventually irreversible. For example, the hyperreflexia observed in almost all our patients after some years of treatment could be due to some degree of functional vitamin B12 deficiency. This is in line with Pietz et al. [26]. With regard to the results of the present study, 10 of the 67 patients with serum vitamin B12 concentrations within reference values had elevated MMA and/or Hcy. Only 2 of the 12 patients with elevated MMA and/or Hcy showed vitamin B12 concentrations below reference range. Elevated Hcy and MMA concentrations are sensitive markers for reduced vitamin B12 activity [19]. These results indicate that the vitamin B12 concentration does not prove or exclude functional vitamin B12
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deficiency. By this, our results are in line with the results in various nonPKU studied populations [17,19,27,28]. We found only inconsistent relations between serum vitamin B12 concentrations, serum MMA and plasma Hcy concentrations in different age groups. Because of the relatively small groups when divided into different age groups, we treated the data of the total population as one group. In the whole group, we found significant relations between the serum vitamin B12 concentration and the plasma Hcy concentration and serum MMA concentration and between vitamin B12 concentrations and metabolic control. Our data do not substantiate the results of some studies in PKU patients that did not show a significant correlation between plasma Hcy concentrations and vitamin B12 concentrations [14,26,29]. Part of these differences can be explained by the fact that (part of the) patients studied by Colome et al. [26] and Hvas et al. [29] received extra supplementation of vitamins in addition to the protein substitute. At this moment, we cannot explain the difference in the study of Huemer et al. [14] that showed no relation between Hcy and vitamin B12 concentrations. Theoretically, discrepancies between our results and results of Huemer et al. [14] and Hvas et al. [29] may be caused by differences in folate or vitamin B6 concentration that also influence Hcy concentration in plasma [4,16,17,23,26,30]. Folate has an important role in vitamin B12 metabolism and can also be an important confounder of vitamin B12 metabolism [31]. Unfortunately, in our study, neither folate nor vitamin B6 concentrations were measured. In PKU, deficiencies of vitamin B12 have always been attributed to deficient intake [8,11,13]. Intake of vitamin B12, however, may only be one of the factors. In this study, the intake of protein was not studied. Another important factor may be the bioavailability of vitamin B12 [32]. In general, PKU patients do not have malabsorption, but the possibility of lower bioavailability of vitamin B12 within the protein substitute has not been studied and may result in lower intracellular concentrations. Such mechanisms have been reported in PKU for the intake of calcium [33,34] and amino acids [35]. Another reason of the functional vitamin B12 deficiency may be the timing of the intake rather than the amount and content of the protein substitute. A study about vitamin B12 sources and bioavailability of Watanabe [32] showed a significant decrease of the availability of vitamin B12 with increasing intake of vitamin B12 per meal. It is recommended to take the protein substitute as evenly distributed over the day as possible [3,36] and lifelong [37,38]. PKU patients in our population reported to take their main source of vitamin B12 (the protein substitute) three times a day. However, a less frequent intake of the protein substitute cannot be excluded. The combination of a possibly lower availability and taking the protein substitute in less than three times a day may be a reason for finding more biochemical vitamin B12 deficiencies in PKU patients than in the healthy population. In this way, increased MMA and/or Hcy may be considered not only indicative of functional vitamin B12 deficiency, but also as a marker of possible inadequate intake of the protein substitute as a whole. When functional vitamin B12 deficiency is due to factors such as inadequate (timing of) intake or nonoptimal bioavailability, the risk of vitamin B12 deficiency may also apply for other nutrients. In conclusion, a relation between concentrations of vitamin B12 and MMA and Hcy, functional vitamin B12 deficiency, defined as elevated serum MMA or plasma Hcy concentrations, is seen relatively often in PKU patients even with a serum vitamin B12 concentration within reference values. An elevated serum MMA or plasma Hcy concentration rather than low serum vitamin B12 concentrations is a reliable indicator of lack of active vitamin B12. It, therefore, is important to evaluate serum MMA or plasma Hcy concentrations regularly in PKU patients. Hcy may be measured if MMA measurements are not readily available, although it has to be considered that Hcy may also be increased with deficiencies of folate and vitamin B6. When MMA or Hcy concentrations in PKU patients are increased, it is necessary to optimize the intake of the protein substitute to ensure
adequate intake of both vitamin B12 and other micronutrients. When increased MMA or Hcy persists, knowledge on the response (clinical or biochemical) on vitamin B12 supplementation is indicated. Not only inadequate intake but perhaps also less optimal bioavailability from the protein supplement may be a cause of deficient vitamin B12 status. The latter possibility needs to be studied further. Acknowledgment We kindly acknowledge the laboratory workers of the department of laboratory medicine in Groningen and the laboratory workers at the laboratory in Bergen, Norway, for their kind help. References [1] C.R. Scriver, S. Kaufman, Hyperphenylalaninemia: phenylalanine hydroxylase deficiency, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic & Molecular Bases of Inherited Disease, McGraw-Hill, Inc., New York, 2001, pp. 1667–1724. [2] A.P. Prince, M.P. McMurray, N.R. 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