Molybdenum is part of the molybdenum cofactor in the enzymes sulfite oxidase, xanthine oxidase, and aldehyde oxidase (1) and has been acknowledged as an essential trace element since 1953 (2,3). Clinical symptoms of molybdenum deficiency have been described in an adult patient receiving prolonged parenteral nutrition: irritability leading to coma, tachycardia, tachypnea, and night blindness accompanied by biochemical findings of low tissue sulfite oxidase activity, increased thiosulfate excretion, reduced sulfate output, and increased plasma methionine (4). Molybdenum excess results in defects in osteogenesis, skeletal and joint deformities, spontaneous subepiphyseal fractures, and mandibular exostoses (5). Molybdenosis, which may result in a secondary copper deficiency, was initially described in ruminants (6).
The unimpaired early development of infants with phenylketonuria (PKU) requires dietetic restrictions of phenylalanine. Special dietetic products are fortified with essential trace elements and minerals, and the present national directive in Germany for molybdenum demands an daily intake of 30 to 80 μg (7,8), whereas an intake of 15 to 40 μg is recommended for healthy infants (9,10). The latest directive of the Commission of the European Communities set a molybdenum content of less than 10 μg/100 kcal (11).
Although the molybdenum content of human colostrum is 15 μg/l, a decline is observed during the first weeks of lactation, and mean values (2 μg/l in mature milk) have been reported by several investigators (12–15). Excessive concentrations of more than 100 μg/l were measured in formulas (16–18). Little is known about the molybdenum balance in infancy. Alexander et al. (19) reported a mean absorption of higher than 50% of the intake in healthy and dietetically treated infants and children. However, the groups were rather heterogeneous. This study was therefore undertaken to investigate whether molybdenum supplementation in diets results in a retention equivalent to that of healthy breast-fed term infants. In addition, plasma concentrations were longitudinally assessed.
MATERIALS AND METHODS
Four infants with PKU that had been diagnosed and treated within the first 2 to 3 weeks of life were studied. The infants received the usual diet with synthetic amino acid mixtures in addition to human milk and infant formula (Table 1). They were investigated initially in a balance study and longitudinally in plasma investigations. Their results were compared with those of breast-fed infants (n = 17). A number of participants missed investigation due to acute disease, limited plasma volume, or technical reasons in the course of the study (Table 1). Standardized nutrition enabled participation later.
Conduction of Balance Studies and Collection of Plasma Specimens
The intake was measured by bottle weighing (accuracy ±1 g) or test weighing in breast-fed infants using scales with an accuracy of ±5 g (Seca & Halke, Hamburg, Germany). Specimens were collected daily or at each feeding in infants with PKU. Human milk was collected with electric breast pumps (Mini Electric; Medela, Eching, Germany) or manual expression pumps (Ruska, Hanover, Germany) at each breast-feeding (n = 248). Samples were pooled from fore milk (milk pumped before breast feeding) and hind milk (milk pumped after breast feeding) of the left or right side, respectively. The families were provided with molybdenum-free water for the cleaning of materials.
To avoid inaccuracies due to molybdenum intake through other nutritive sources, the families were supplied with standardized formulas, complementary foods, and mineral water (Table 1). The breast-fed group was instructed to follow the recommendations of the Institute of Child Nutrition, Dortmund, Germany (20). These recommendations were also applied to the dietetically treated patients, as far as possible. Intakes through tea and drugs were analyzed and added, although amounts were marginal (Table 2).
Infant or pediatric-sized 24-hour collection bags (Hollister Inc., Ballina, Ireland) were used for urinary collection and changed every 24 hours. Feces were collected with diaper inserts (Blümia, Moltex Baby Hygiene GmbH, Mayen, Germany). The stool collection was defined by the appearance of two doses of carmine red fed the infants 72 hours apart. All specimens were stored at −20°C until analysis.
Blood samples were obtained by routine methods of pediatric venipuncture and were transported in heparinized tubes (Microtainer; Becton Dickinson GmbH, Heidelberg, Germany). They were centrifuged at arrival in the clinic, and the plasma was frozen at −20°C until analysis.
The materials of the balance studies were analyzed in the department of pediatrics with atomic absorption spectroscopy. Urinary samples and tea were directly measured; milk, most of the drugs, and the fecal samples were lyophilized and ashed with nitric acid according to Kotz et al. (21). Standard materials were used as precision controls, Table 2.
Plasma molybdenum analysis was performed at the Institute of Geosciences, Department of Geology, by high-resolution inductively coupled plasma mass spectrometry (PlasmaTrace 2, HR-ICP-MS; Micromass, Ltd., Wythenshaw, United Kingdom). Results of precision controls and accuracy are shown in Table 3.
The nonparametric test (Wilcoxon, Mann–Whitney) was used for statistical evaluation, which was performed by computer (Statistica 5.0 software; StatSoft, Inc. Tulsa, OK, U.S.A.). The threshold for significance was set to P < 0.05. A descriptive presentation based on median and range was chosen for the balance trials and plasma concentrations (Figs. 1 and 2).
Informed consent was obtained from the parents of all participants before their inclusion in the study. The investigation was approved by the institutional ethics committee and performed in accordance with the Declaration of Helsinki.
The molybdenum concentration in the PKU diet exceeded that of human milk by far. This was reflected in urinary molybdenum concentrations but not in the fecal concentration (P > 0.05, Table 4). The molybdenum intake from diet or human milk in the balance studies contributed almost exclusively to the molybdenum intake; the content in tea and drugs was negligible. The percentage of urinary excretion in infants with PKU was 50.9% of the intake, the fecal excretion 9.7%. The lower quantitative intake of molybdenum in breast-fed infants resulted in an increased percentage of molybdenum in feces (44.8% of the intake;Table 5).
In the balance studies, daily intakes of 0.23 μg/kg resulted in a balanced molybdenum metabolism in breast-fed infants. The dietetically treated patients receiving supplemented diets retained 3.2 μg/kg. This exceeded the complete intake of breast-fed infants more than 13-fold. Although the ranges within the groups are considerable, there was no correlation of total molybdenum intake and excretion between breast-fed infants and infants with PKU (Fig. 1).
The plasma concentrations reflected the results obtained in the balance trials. With the exception of one extreme value registered in one breast-fed participant at 4 months, the results in infants with PKU were above those of the healthy infants. The initial median plasma concentration was 0.04 μg/l in breast-fed infants compared with 2.5 μg/l in the infants with PKU. The values were 1.48 μg/l (range, 1.22–3.65 μg/l) in infants with PKU and 1.41 μg/l (range, 0.47–2.09 μg/l) in breast-fed infants at 1 year of age. With a molybdenum supply predominantly from supplementary food, the differences in plasma concentrations were negligible (Fig. 2).
Human milk is regarded as the preferred feeding in the first 4 to 6 months of infancy (20,22). Many dietetically treated infants with inborn errors of metabolism cannot be nourished in accordance with these recommendations. Their supply of essential nutrients, however, should be equivalent to that of healthy breast-fed infants, when evaluated by comparative studies.
In conservative trace element balance studies, human milk supplied the breast-fed infant daily with 0.23 μg molybdenum/kg (Fig. 1). This was sufficient to maintain balanced results. This intake was similar to the results of other intake studies (13). In infants with PKU, however, not only the daily molybdenum intake of 6.6 μg/kg, but also the retention of 3.2 μg/kg was considerably higher. Alexander et al. (19) reported a daily retention of 2 μg/kg in two infants with PKU, which was comparable to the retention in infants in our study. Neither the nature of the disease nor knowledge gained from previous studies suggests that the high retention is due to special needs of this group. Unnecessary storage is more likely. Because it has been shown in adults that molybdenum retention is dependent on urinary excretion (23), supplementation leading to substantial quantitative retention of this trace element may be specific to early infancy, related to the physiological development of renal function.
Molybdenum intake in infancy is predominantly dependent on four aspects of nutrition:
1. The route of application (enteral or parenteral). Parenteral products may contain considerable amounts of molybdenum by contamination (24). Supplementation in infancy is recommended only in long-term total parenteral nutrition (0.25 μg/kg per day) (25).
2. The choice of nutrition modifies the intake, in that there is a low concentration in human milk (14), a wide range of concentrations in infant formulas (17), and a high concentration in special foods for medical purposes, supplemented in accordance with dietetic directions (17,18).
3. The time of the introduction of supplementary foods outlined by the nutritional concept pursued (e.g., 20) limits the importance of formulas or human milk as exclusive nutritive sources.
4. The molybdenum content of supplementary foods depends on the choice and origin of products (26).
While pastry and fruits do not contain much molybdenum, not only animal products but also vegetables have higher molybdenum concentrations (26). The range of molybdenum concentrations in water is considerable; mineral waters contain 0.22 to 21.95 μg/l (27). The upper limit in drinking water was set at 70 μg/l by the World Health Organization (28,29). Diets in infants with PKU are thus not necessarily prone to render an insufficient supply of molybdenum.
In accordance with the results of calculations and conservative balance studies (30) and based on stable isotope investigations in adults (23,31), a minimum intake of 25 μg molybdenum is regarded as sufficient (32). The upper limit is 500 μg/day. Absorption of the trace element is higher than 84%(23,31,33,34). Its retention is regulated by urinary excretion. Repetitive biokinetic studies in the same adult showed that individual characteristics of intestinal absorption and plasma clearance are the main processes of molybdenum metabolism (33–35). In addition, the binding pattern of molybdenum may be of importance for resorption and excretion of the trace metal. Using quantitative and qualitative speciation analyses it is possible today to specify the differences between formulas and human milk. The relationships between trace elements in the maternal dietary intake and breast milk can also be determined (36).
Longitudinal plasma studies underline the importance of the nutritional supply in early infancy (Fig. 2). The different ranges assessed for the groups were dependent on nutrition rather than age. The initial results of infants with PKU exceeded those of breast-fed infants by far during a period of more than 4 months and reflected the molybdenum retention observed in parallel balance trials (Fig. 1). The plasma concentration in breast-fed infants decreased with age until the introduction of supplementary foods. There was no difference between the groups at the age of 1 year.
Systematic data on molybdenum plasma concentrations in relation to nutrition in infancy are scarce. Cord blood and blood samples drawn from the respective mothers rendered a significant correlation between the molybdenum concentrations (37). The mean result of 1.44 μg/l, however, was not confirmed by other investigators who found 0.7 μg/l (range, 0.4–1.6 μg/l) (38). Values of 0.6 μg/l in breast-fed infants and 1.9 μg/l in infants fed breast milk and formula were observed (39). Concentrations assessed at the end of the first year in the present study are equivalent to those in patients with PKU (1.33 ± 0.5 μg/l) and healthy children aged 2 to 12 years (1.75 ± 0.8 μg/l) (40). Recent studies revealed a mean molybdenum concentration of 1.6 and 1.9 μg/l in infants with PKU at the ages of 3 and 6 months, respectively (41). The molybdenum intake in formula-fed preterm infants correlated with urinary excretion as well as with plasma and urine concentrations (16).
Molybdenum was not completely excreted in long-term ammonium tetrathiomolybdate-treated sheep but was widely distributed in many organs, including the brain and pituitary (42). The response of nonruminants on molybdenum excess is partly suggestive of an effect on copper use (43,44). The reaction of molybdate with sulfide generated by bacterial reduction of sulfate within the gastrointestinal tract may lead to the formation of thiomolybdates reacting with copper. Based on the fact that large amounts of sulfide can be generated within the human colon on high sulfate intake (45), high human intakes of molybdenum (46) may be sufficient for thiomolybdate formation and effects on copper utilization. Therapeutic applications of ammonium tetrathiomolybdate in adults have been recommended to enhance copper excretion in the treatment of Wilson's disease (120–240 mg molybdenum per day) (47,48)). Though molybdenum intakes provided by formulas are considerably lower, copper metabolism should be monitored in recipients of long-term molybdenum-supplemented diets.
In conclusion, molybdenum supplementation of diets in PKU leads to a substantial retention, exceeding the respective results of healthy breast-fed infants by far. This is reflected by increased plasma concentrations in the first months of life. There remains a deficit in data to support the necessity of molybdenum supplementation and exclusion of interactions with other trace metals. The present practice of molybdenum fortification of diets used in early infancy should be revised in light of the actual findings.
The authors thank the patients' families for their participation and cooperation. The study was funded by the Deutsche Forschungsgemeinschaft (Si 514-1). The authors thank Milupa Co. (Friedrichsdorf, Germany), Hipp AG (Pfaffenhofen, Germany), Fürst Bismarck Quelle (Aumühle, Germany), and SHS GmbH (Heilbronn, Germany) for providing a continuous supply of defined nutritional products.
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