J Inherit Metab Dis (2006) 29: 47–53 DOI 10.1007/s10545-006-0108-3
O R I G I NA L A RT I C L E
Vitamin B12 and vitamin B6 supplementation is needed among adults with phenylketonuria (PKU) A. M. Hvas · E. Nexo · J. B. Nielsen
Received: 11 February 2005 / Accepted: 7 June 2005 C SSIEM and Springer 2006
Summary Phenylketonuria (PKU) is caused by an autosomal recessive deficiency of the enzyme phelnylalanine hydroxylase leading to a failure to convert phenylalanine to tyrosine. To avoid irreversible neurological damage because of increased phenylalanine, treatment is instituted rapidly after birth. We examined 31 adult PKU patients living on a less protein-restricted diet. Theoretically, these PKU patients had an increased risk of developing vitamin B12 and B6 deficiency because of a limited intake of animal products. Besides laboratory tests (n = 31) we obtained clinical information (n = 30) and detailed information on food consumption (n = 28). Three-quarters of the patients had early biochemical signs of vitamin B12 deficiency. In spite of a normal folate status, 9 (29%) had a plasma homocysteine above 12 μmol/L. In accord with these findings, the food questionnaires indicated that 11 (39%) patients received less than the recommended daily vitamin B12 , and 20 (71%) received less vitamin B6 than recommended. A significant association was found between reduced vitamin B12 intake and both reduced serum cobalamins ( p = 0.04) and reduced serum transcobalCommunicating editor: Georg Hoffmann Competing interests: None declared A. M. Hvas () Department of Clinical Biochemistry, SKS, Aarhus University Hospital, Aarhus, Denmark e-mail:
[email protected] E. Nexo Department of Clinical Biochemistry, AS, Aarhus University Hospital, Aarhus Denmark J. B. Nielsen Centre for PKU Treatment, John F. Kennedy Institute, Glostrup, Denmark
amin saturation ( p = 0.03). Eleven patients took a vitamin pill daily, and these patients had a significantly lower plasma homocysteine compared to the rest. The present study suggests that adult PKU patients were at increased risk of developing vitamin B12 deficiency, and their intake of vitamin B6 was below the recommended daily intake. In conclusion PKU patients need continuing dietary guidance throughout adult life, and considering the risks, costs and potential benefits, daily vitamin supplementation seems justified in these patients.
Introduction Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism in Europeans (Zschocke 2003). The incidence of treatment requiring PKU in Denmark is 1 in 12 000 newborns per year, which amounts to about six children per year. It is caused by an autosomal recessive deficiency of the hepatic enzyme phenylalanine hydroxylase. Failure to convert phenylalanine to tyrosine leads to an increase of phenylalanine, causing irreversible neurological damage including mental retardation in almost all cases if the patients are not treated with diet instituted rapidly after birth. In Denmark all newborns are systematically screened for PKU, and every child identified with increased phenylalanine is referred to the John F. Kennedy Institute. All patients are screened for mutations in the phenylalanine hydroxylase gene and classified as having classical, moderate or mild PKU according to Guldberg and colleagues (1998). During their childhood, patients with PKU get a diet very restricted in protein and they therefore need supplements containing
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all amino acids except phenylalanine as well as vitamins and minerals. Generally the diet becomes less restricted as the children grow up, as the mature brain is less sensitive to phenylalanine. From adolescence some of the patients are treated according to a different principle, with more natural protein in the diet and a supplement consisting only of the essential large, neutral amino acids without vitamins and minerals added (Moats et al 2003). Although the patients are now allowed to eat foods containing proteins, they might not do so because they find that these nutrients have an unpleasant taste. The main sources of vitamin B12 are meat, poultry and seafood and to a lesser degree dairy products, while vitamin B6 is found in a wide variety of foods including beans, meat, poultry, fish and some fruits and vegetables. It has been reported that patients with PKU on an unrestricted diet have an increased risk of vitamin B12 deficiency, probably because of too low a vitamin B12 intake (Robinson et al 2000; Hanley et al 1996; Aung et al 1997). A previous study described the food and nutrient intake in adolescents and young adults (Schulz and Bremer 1995); however, to our knowledge no reports have commented on the intake of vitamin B6 among a pure population of adult patients with PKU on an unrestricted diet. If the intrinsic factor-based mechanism of vitamin B12 absorption is intact, 60–70% of the dietary vitamin B12 is absorbed (Carmel 2001). If not treated in time, vitamin B12 deficiency may lead to macrocytic anaemia and/or irreversible neurological damage (Healton et al 1991). Serum cobalamins and the metabolites plasma methylmalonic acid and plasma homocysteine, which increase with lack of vitamin B12 , are usually employed for diagnosing vitamin B12 deficiency. In plasma, cobalamin is bound to the two proteins haptocorrin and transcobalamin. Cobalamin attached to transcobalamin, holotranscobalamin, represents the biologically active fraction that can be delivered into all DNA-synthesizing cells. Recently, new methods for measurement of holotranscobalamin have been introduced (Nexo et al 2002; Ulleland et al 2002). Vitamin B6 is involved in the metabolism of amino acids, carbohydrates, neurotransmitters and lipids (Leklem 1996). The major forms of vitamin B6 in mammalian tissues are pyridoxine, pyridoxal and pyridoxamine as well as their phosphorylated derivatives and the end product 4-pyridoxic acid. After passive intestinal absorption, the major part of vitamin B6 is delivered to the liver and converted to pyridoxal 5 -phosphate (Leklem 1992). Currently, there is no agreement concerning which form of vitamin B6 should be used for routine assessment of vitamin B6 status (Bor et al 2003). The aim of the present study was to examine the vitamin B status among Danish adult for PKU patients and to consider the need of vitamin supplementation among these patients. Springer
Methods Study population All PKU patients in Denmark are registered at the John F. Kennedy Institute. We contacted all PKU patients above 17 years of age who as adults ceased to follow the prescribed diet without any supplements of amino acids (26 patients) or received large neutral amino acids in tablets, PreKUnil (67 patients). The patients were included from March 2004 to September 2004. Besides a blood test we conducted an interview on neurological disability and the patients filled in two questionnaires on food consumption. Laboratory investigations The blood tests were obtained at the general practitioner or at a laboratory near to the patient. The patients were not fasting. The blood tests were sent by post over one night to the Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark, where all the laboratory investigations were performed. Serum total transcobalamin (totalTC) and cobalamin saturated transcobalamin (holoTC) were measured by ELISA (Nexo et al 2000, 2002) employing an automated ELISA analyser (BEP-2000, Dade Behring, Germany), and serum TC saturation was calculated as holoTC/totalTC. Analytical imprecision was 5% for totalTC and 8% for holoTC. Serum cobalamins and erythrocyte folate were determined on the ACS:Centaur Automated Chemiluminescence System (Bayer A/S) by a competitive protein-binding assay, with analytical imprecision 12 μmol/L
Mil (n = 12) Moderate (n = 2) Classical (n = 17) Total
4 1 2 7
8 1 15 24
3 1 7 11
5a 1 3a 9
distribution of the use of daily vitamin pills is shown in Table 3. Table 4 shows the median energy intake and the estimated percentage content of protein, fat and carbohydrates. More than half of the patients received less protein than recommended, and 11 patients (39%) received less than the recommended daily vitamin B12 intake from the diet (Table 4). Among these 11 patients, 6 patients did not take any kind of vitamin pills, and all but one had a serum holoTC below the reference interval. A reduced vitamin B12 intake estimated from the sevenday food registrations was significantly associated with both reduced serum cobalamins and reduced TC saturation (Fig. 2). This association was also found between serum
Table 4 Intake among PKU patients (n = 28; female, n = 14; males, n = 14) and recommended intake of protein, fat, carbohydrate, vitamin B12 , and vitamin B6 as expressed as average daily intakes over time. Intakes of protein, fat and carbohydrate are indicated percentages of the total energy intake (excluding energy from alcohol) PKU patients
Total energy (kJ/day) Females Males Protein (%) Females Males Fat (%) Females Males Carbohydrate (%) Females Males Vitamin B12 (μg/day) Females Males Vitamin B6 (mg/day) Females Males a
Becker et al (2004)
Median
Range
Recommendeda
7037 8615
3242–10661 4138–14099
5800 7550
13 13
6–18 8–16
15 30
30 32
18–46 20–39
30 30
58 52
45–65 46–59
55 55
1.2 2.9
0.5–8.0 1.7–7.2
2.0 2.0
1.0 1.4
0.5–2.6 0.5–2.1
1.2 1.6
Fig. 2 (A) The association between serum cobalamins and the vitamin B12 intake estimated from the seven-day food registrations (n = 28). (B) The association between serum transcobalamin saturation and the vitamin B12 intake estimated from the seven-day food registrations (n = 28)
holoTC and vitamin B12 intake, though not significant ( p = 0.06). Remarkably, 9 out of 14 men and 10 out of 14 women received less than the recommended daily vitamin B6 intake (Table 4). Overall, no association was found between the level of plasma homocysteine and the estimated vitamin B6 intake ( p = 0.86, linear regression). The 11 patients taking one vitamin pill daily had a significantly lower plasma homocysteine ( p = 0.006, means Springer
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compared by two-sample t-test) as compared to the rest. The level of serum cobalamins, serum holoTC, and plasma methylmalonic acid did not differ between the vitamin-group and the rest.
Discussion This is the first study including PKU patients receiving only the large neutral amino acids, and in which examination of the vitamin B12 status also included measurement of serum holoTC and the metabolites plasma methylmalonic acid and plasma homocysteine. We studied 31 adult PKU patients who have been on an unrestricted diet since they were teenagers. The majority had received PreKUnil, which contained only the large neutral amino acids. One limitation of the study was that only 31/93 PKU patients participated in the study. The participating patients did not differ from the nonparticipating patients as regards age and sex. Vitamin B12 deficiency has earlier been reported in patients with PKU as case reports (Hanley et al 1993, 1996; Aung et al 1997; Robinson et al 2000), and low serum cobalamins have been found in PKU populations consisting of both children and young adults (Hanley et al 1993; Robinson et al 2000). Our main finding was that about three quarters of the patients showed biochemical signs of beginning vitamin B12 deficiency as judged from low serum cobalamins and serum holoTC as well as increased erythrocyte mean cell volume. None of the patients had overt vitamin B12 deficiency, but patients with at least one neurological symptom tended to have lower serum cobalamins compared to the rest. The finding that less than half of the patients received as much protein as recommended supported the suspicion that a considerable proportion the PKU patients did not eat enough food of animal origin. According to the food frequency questionnaire, the patients ate meat and/or poultry rather frequently; however, according to the seven-day registration they did not eat much of it. As a consequence only 61% of the PKU patients received the recommended amount of vitamin B12 daily from the diet. This insufficient intake of vitamin B12 was reflected in the reduced serum cobalamins and reduced TC saturation. In addition we found that 29% of the patients had increased plasma homocysteine (above 12 μmol/L). This finding might be alarming in the light of the discussion of mild hyperhomocysteinemia as a possible risk factor for cardiovascular disease (Refsum et al 2004; Weiss et al 2004). Apart from vitamin B12 deficiency, plasma homocysteine increases in lack of folate and vitamin B6 and in the case of decreased renal function (Refsum et al 2004). The present PKU patients Springer
had increased plasma homocysteine in spite of a normal folate status and normal renal function, but we found that 71% of the patients received less vitamin B6 than recommended. Although we could not determine the exact reason for the increased concentration of homocysteine, patients who took a vitamin pill daily had a significantly lower homocysteine than the rest. In conclusion, our study suggests that adult PKU patients were at increased risk of developing vitamin B12 deficiency. Further, we report that the majority had a vitamin B6 intake below the recommended daily intake. These findings indicate that PKU patients need continuing dietary guidance throughout adult life, and considering the risks, costs and potential benefits, daily vitamin supplementation seems justified in these patients. Acknowledgements The excellent technical assistance of Anna-Lisa Christensen and Jette Fisker Pedersen is warmly acknowledged. The study was financially supported by EU Biomed (QLK3-CT-200201775) and Institute of Clinical Medicine, Aarhus University, Denmark.
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53 Schulz B, Bremer HJ (1995) Nutrient intake and food comsumption of adolescents and young adults with phenylketonuria. Acta Paediatr 84: 743–748. Ulleland M, Eilertsen I, Quadros EV, et al (2002) Direct assay for cobalamin bound to transcobalamin (holotranscobalamin) in serum. Clin Chem 48: 526–532. Weiss N, Hilge R, Hoffmann U, et al (2004) Mild hyperhomocysteinemia: risk factor or just risk predictor for cardiovascular diseases? Vasa 33: 191–203. Zschocke J (2003) Phenylketonuria mutations in Europe. Hum Mutat 21: 345–356.
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