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Following the current recommendations, most of the infant starting formulas on the market are whey protein-predominant (1). Related to the whey protein the threonine content of these formulas is approximately 20% higher than in human milk. The whey proteins which are used for infant formulas are sweet whey proteins. Sweet whey results from cheese production due to the enzymatic (rennin) clotting of milk casein by splitting of κ-casein into para-κ-casein and GMP (2). The GMP is soluble and remains in the sweet whey in a concentration up to 15 g/100 g of protein. The GMP is very rich in threonine (about 20 g of threonine/100 g GMP) and therefore causes the high threonine content of infant formulas.

Since the beginning of the 1980s, hyperthreoninemia has been a well known phenomenon in infants fed a whey protein-predominant formula (3). Particularly in preterm infants feeding whey protein-predominant formulas results in plasma threonine concentrations up to twice as high as in those preterm infants fed human milk fortified with human milk proteins (4–8). Nevertheless, also in term infants the plasma threonine levels are significantly higher when fed such formulas compared with breast-fed infants (9). In contrast, feeding a whey protein-dominant formula without GMP significantly reduces plasma threonine levels in preterm and term infants when compared with infants fed GMP containing whey protein-dominant formula (10,11).

In many rat experiments a significant correlation between threonine intake and plasma threonine concentration as well as between plasma and brain threonine concentration could be found (12–15). Based on the occurrence of growth retardation of weaning rats fed a low protein diet to which amino acids were gradually added, threonine was found to be one of the least toxic EAA (12) and, therefore, the physiologic relevance of high plasma threonine levels was underestimated. However, excess in dietary threonine results in increased levels of several nonEAAs, especially glycine and serine due to the close relationship of their metabolism to threonine (12,16). Thus, because of the extensive brain growth occurring during early infancy (17,18) hyperthreoninemia in plasma and subsequently in the brain is a potential risk factor for brain development. As a consequence, a recent review on the safety of amino acids recommended the testing of threonine in both animals and humans (19).

Most rat studies concerning the effects of dietary threonine are performed with bovine casein and/or corn protein as dietary protein. In the present study, the protein composition of the experimental diets based on bovine milk proteins with a whey protein/casein ratio of 60/40 which is similar to that commonly used in infant formula. The aim of the study was to clarify the consequences of high plasma threonine levels on the threonine metabolism in the brain and other peripheral tissues during feeding a whey protein dominant diet.

METHODS

Young male Wistar rats were kept in single cages on a 12-h light/12-h dark cycle, were pair-fed, and had a water supply ad libitum. In a first study, focused on the amino acid patterns in different compartments, 32 animals were included. Eight animals were fed chow pellets (Altromin C 1000; macronutrient composition: 17% protein as bovine caseins, 5.3% fat, 55% carbohydrates) as controls. Three groups of animals (n = 8 in each group) received experimental diets all based on a mixture of bovine proteins (whey protein/casein ratio 60/40), which is similar to the protein composition of a standard infant formula. The animals of group A were fed with a diet containing acid whey proteins which do not contain GMP. The threonine content of this diet was comparable to that of the typical rat diet with bovine caseins as protein (threonine content: 0.86 g/100 g of pellets and 0.77 g/100 g of pellets, respectively). The animals of group B were fed a GMP-containing sweet whey protein/casein diet (threonine content: 1.03 g/100 g of pellets) and the animals of group C were fed a sweet whey protein/casein diet similar to that used for the animals in group B, which was additionally supplemented with 92 mg of threonine/g of protein (threonine content: 2.21 g/100 g of pellets). The supplementation was calculated according to Yokogoshi et al. (20) to reach plasma threonine concentrations at least twice as high as in the animals fed the unsupplemented protein to meet the range of plasma threonine levels found in human infants fed either human milk or formula. The amino acid composition of the three experimental diets is given in Table 1.

Table 1 Amino acid composition (g of amino acids/100 g of pellets) of the diets used in the study
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Because the most dramatic changes in the tissue amino acids occurred in the liver and muscle, a second study was performed focusing on the effects of high plasma threonine on the liver and muscle protein metabolism. In this second study, only the two diets that resulted in the highest plasma threonine levels (i.e. diet of groups B and C) and normal chow diet as a control were used. Again, each group consisted of eight animals.

After a feeding period of 15 d (7-d setting down period, 8-d study period), the animals were killed without being fasted in the morning by decapitation, after an ether anesthesia. The study design was approved by the Veterinary Service, Office of Agriculture of the Canton Berne, Switzerland.

During the study period food intake, body weight, and urine excretion were measured daily. The consumption of food and water was calculated from the difference between the amount of food provided at the beginning and the amount not consumed after a 24-h period. At the end of the study amino acids were measured in plasma, liver, muscle, and brain (cerebral cortex, brain stem) in each of the animals of the first study.

In the animals of the second study, amino acids were measured similar to the first study. Additionally, urea and the activities of threonine dehydratase, choline esterase, and ornithine transcarbamylase were measured in the liver homogenate, and creatine concentrations were measured in the muscle homogenate.

For amino acid analysis the tissues were homogenized (1:20 wt/vol) with perchloric acid (0.5 M) containing 0.08 mM norvaline as an internal standard as described previously (21). An aliquot was used to determine amino acids using automated ion exchange chromatography (Biotronic LC 3000, Munich, FRG).

In the liver, the activity of ornithine transcarbamylase was measured according to Campbell et al. (22), the activity of threonine dehydratase according to Moundras et al. (23), and urea by using the urease/glutamate dehydrogenase reaction. In the muscle creatine was measured according to Cramer et al. (24). Values are related to gram wet weight of tissue.

Statistical values are given as the mean ± SD. Group comparison of means were calculated by ANOVA followed by Fisher's post hoc test in case of significance. Correlation between variables were evaluated by linear regression analysis. A p value of <0.05 was considered as statistically significant.

RESULTS

In the first study, there was a significant correlation between threonine intake and plasma threonine levels (r = 0.687, p < 0.001). The mean plasma levels reflected the different intakes of the feeding groups (Table 2).

Table 2 Mean (M ± SD) concentration of essential amino acids, sum of essential (Σ EAA) and total amino acids (Σ TAA), as well as glycine and serine (mmol/L) in the plasma of rats fed three experimental diets different in their threonine content and a normal rat diet as controls
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Plasma threonine concentrations correlated significantly with the threonine concentration in the cortex (r = 0.821, p < 0.01) and the brain stem (r = 0.882, p < 0.01) resulting in highest threonine concentrations in group C (Tables 3 and 4).

Table 3 Mean (M ± SD) concentration of essential amino acids, sum of essential (Σ EAA) and total amino acids (Σ TAA), as well as glycine and serine (nmol/g) in the brain stem of rats fed three experimental diets different in their threonine content and a normal rat diet as controls
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Table 4 Mean (M ± SD) concentration of essential amino acids, sum of essential (Σ EAA) and total amino acids (Σ TAA), as well as glycine and serine (nmol/g) in the brain stem of rats fed three experimental diets different in their threonine content and a normal rat diet as controls
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There were also significant correlations between plasma threonine concentration and threonine concentration in the muscle (r = 0.632, p < 0.01) and the liver (r = 0.547, p < 0.01). However, in both tissues the threonine concentrations in group C did not exceed those of group B (Tables 5 and 6).

Table 5 Mean (M ± SD) concentration of essential amino acids, sum of essential (Σ EAA) and total amino acids (Σ TAA), as well as glycine and serine (nmol/g) in the liver of rats fed three experimental diets different in their threonine content and a normal rat diet as controls
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Table 6 Mean (M ± SD) concentration of essential amino acids, sum of essential (Σ EAA) and total amino acids (Σ TAA), as well as glycine and serine (nmol/g) in the muscle of rats fed three experimental diets different in their threonine content and a normal rat diet as controls
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There was a significant positive correlation between threonine and glycine concentrations in the cortex (r = 0.673, p < 0.01) (Fig. 1) and the brain stem (r = 0.542, p < 0.01) (Fig. 2). As consequence, the glycine concentrations in the cortex and the brain stem were significantly higher in the group fed the threonine supplemented diet compared with the other three feeding groups (Tables 3 and 4).

Figure 1
figure 1

Relationship between concentration of threonine (nmol/g) and glycine (nmol/g) in the cortex in rats fed a normal rat diet based on bovine milk casein (□) or experimental diets based on a whey protein/casein mixture (60/40) without the threonine rich GMP (▴), with GMP (▪), or with GMP plus free threonine (•).

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Figure 2
figure 2

Relationship between concentration of threonine (nmol/g) and glycine (nmol/g) in the brain stem in rats fed a normal rat diet based on bovine milk casein (□) or experimental diets based on a whey protein/casein mixture (60/40) without the threonine-rich GMP (▴), with GMP (▪), or with GMP plus free threonine (•).

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In the brain stem, the correlation between threonine and serine concentrations was weaker (r = 0.373; p < 0.05) than the correlation between threonine and glycine concentrations. However, the serine concentrations in group C were significantly higher than in the other three feeding groups (Table 4). There was no significant correlation between threonine and serine concentrations in the cortex (r = 0.329; p = 0.066) and the serine concentrations in the cortex were not significantly different among the groups (Table 3).

There was no correlation between threonine and glycine concentrations in the liver and muscle. In the liver and the muscle, but also in the cortex the sum of the EAAs except threonine (Val, Met, Ile, Leu, Phe, His, Lys) was lowest in the group with the highest threonine intake (Tables 3, 5, and 6).

In the second study, the amino acid pattern were similar to those found in the first study. The liver weight was not different among the feeding groups (data not shown). In the liver, there were no significant differences in the enzymes activities between the groups, and also the urea concentrations were not different (Table 7). In the muscle, the creatine concentration was significantly higher in group C than in group B (11.7 ± 1.3 versus 7.9 ± 2.7 µmol/g of protein; p < 0.01).

Table 7 Mean (M ± SD) activity of liver enzymes and liver urea concentration of rats fed two experimental diets different in their threonine content
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In both studies, the food intake and weight gain were not different among the feeding groups A-C and were not different when compared with the group fed the typical rat diet (Table 8).

Table 8 Mean (m ± SD) food intake, water intake, and weight gain during the study period of rats fed three experimental diets different in their threonine content and a normal rat diet as controls
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DISCUSSION

In the present study, increasing the threonine intake led to elevated plasma and organ threonine concentrations. In the brain this was accompanied by increased glycine concentrations. With the exception of the brain stem, the elevated concentrations of threonine were accompanied by a decrease of the other EAAs.

We found a significant correlation between the plasma and brain threonine concentrations indicating an efficient transfer of threonine across the blood brain barrier which is in agreement with other studies (25,26). Theoretically, high threonine plasma concentrations may diminish the entry of other neutral amino acids through competition at the level of the transport systems in the blood brain barrier (27,28). In the cortex, the sum of the EAAs except threonine was significantly lower in the group with the highest threonine intake if compared with the other feeding groups. This may indicate a transport competition between threonine and the other EAAs at this high plasma threonine level. However, because only amino acid concentrations were measured, the data do not allow speculations concerning amino acid fluxes.

Threonine catabolism in mammals appears to be due primarily (70-80%) to the activity of threonine dehydrogenase (EC 1.1.1.103) that oxidizes threonine to 2-amino-3-oxobutyrate, which forms glycine and acetyl CoA, whereas threonine dehydratase (EC 4.2.1.16) that catabolizes threonine into 2-oxobutyrate and ammonia, is significantly less active (29).

The positive significant correlation between threonine and glycine concentrations in the brain indicates a conversion of threonine to glycine. Elevated plasma threonine concentrations must therefore exert some effects on the glycinergic neurotransmitter system of the brain. There was no correlation between threonine and glycine concentrations in the liver, muscle, or plasma, which may indicate a different threonine metabolism and/or a different glycine utilization in the brain in comparison with the other peripheral tissues.

Glycine has been identified as important postsynaptic inhibitory transmitter predominantly in the spinal cord and brain stem (18) and exhibits also an excitatory action as co-agonist of the N-methyl-D-aspartate-stimulated glutamate receptor modulating its activity particularly in the cortex and forebrain (30). Glycine is involved in locomotion (31), audition (32), and cognitive functions (33), which may be of importance during the rapid growth period of the brain.

The human brain undergoes two periods of changes during intrauterine and postnatal life. The first period takes place in early pregnancy and is characterized by considerable proliferation of neuroblasts. The second period starts during the last period of gestation and remains up to the postnatal period. This period is characterized by expansion of glial tissue, the dendritic network and synapses (17). There is evidence that glycinergic transmission modulates the maturation of dendrites during development (34). A second aspect is that neurotransmitter receptors undergo a functional development during fetal and postnatal life which makes the immature brain selectively vulnerable to neurotransmitter receptor overstimulation (35). Thus, an increase in glycine of the brain may influence the brain development during the perinatal period.

In the present study, the difference between group A and B represents the nutritional situation in human newborn infants.

With the exception of plasma threonine, no other parameters were significantly different. The threonine effect on brain glycine concentration could be demonstrated if group C with the highest threonine intake was included in the analysis. Apart from species differences, the brain of weaned rats in much more developed than the brain of the human newborn infant (36). Thus, the data obtained in the present study do not allow direct conclusions concerning the threonine effect in human newborn infants. This is particularly true for the question at which plasma threonine level the conversion of threonine to glycine in the brain becomes relevant. However, the experience with threonine treatment of spasticity in which threonine is used to increase the glycine concentration in spinal cord and brain (18,37) demonstrates that the findings in rats are also relevant for the human brain and spinal cord. Thus, even if the toxicity of threonine per se is assumed to be low, hyperthreoninemia may affect the brain development due to its possible influence of the glycinergic neurotransmission.

Another surprising result is the finding that increasing threonine concentrations in liver and muscle were accompanied by decreasing concentrations of the other EAAs which again may indicate a transport competition between threonine and the other EAAs. The similar urea concentration and the similar activity of ornithine transcarbamylase may suggest that the nitrogen metabolism was not significantly affected by the different threonine intakes which is in agreement with other studies (16,26,27).

In our experiment the addition of threonine of the diet did not increase the threonine dehydratase activity in the liver. Similar observations about the lack of induction of threonine dehydrogenase and/or threonine dehydratase activity by excess of threonine intake were previously made by Sawar et al. (12). If this is also true for the human infant, it could explain the increased threonine plasma levels seen particularly in preterm infants fed GMP-containing formulas (3–8).

In conclusion, the presented data indicate that increasing the threonine plasma concentrations leads to accumulation of threonine and glycine in the brain. Such accumulation affects the neurotransmitter balance which may have consequences for the brain development during early postnatal life. Thus, excessive threonine intake during infant feeding should be avoided.