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Nutrition and Disease

Dietary Glycomacropeptide Supports Growth and Reduces the Concentrations of Phenylalanine in Plasma and Brain in a Murine Model of Phenylketonuria^1,2

³ Department of Nutritional Sciences and ⁴ Department of Food Science, University of Wisconsin, Madison, WI 53706 and ⁵ Biochemical Genetics Program, Waisman Center, University of Wisconsin, Madison, WI 53705

^* To whom correspondence should be addressed: ney{at}nutrisci.wisc.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Phenylketonuria (PKU) is a genetic disorder caused by deficiency of phenylalanine hydroxylase (PAH) that requires life-long adherence to a low-phenylalanine (Phe) diet. Glycomacropeptide (GMP) is uniquely suited to the nutritional management of PKU, because pure GMP contains no Phe. Our aim was to assess how ingestion of diets containing GMP support growth and affect the concentrations of amino acids in plasma and brains of mice with a deficiency of PAH, the Pah^enu2 mouse (PKU mouse). Experiments were conducted in 4- to 6-wk-old wild-type (WT) (C57Bl/6) and PKU mice fed diets containing 20% protein from casein, amino acids, or GMP supplemented with limiting indispensable amino acids (IAA). PKU mice fed the GMP diet showed gains in body weight, feed efficiency, and a protein efficiency ratio that did not differ from the amino acid diet. The concentrations of isoleucine and threonine in plasma showed a significant 2- to 3-fold increase for WT and PKU mice fed GMP compared with casein or amino acid diets, respectively. PKU mice fed the GMP diet had decreased concentrations of Phe in plasma (11% decrease) and in 5 regions of the brain (20% decrease) compared with the amino acid diet. The concentration of Phe in the brain was inversely correlated with the concentrations of isoleucine, threonine, and valine in plasma (R² = 0.74; P < 0.0001), suggesting competitive inhibition of Phe transport into the brain. In summary, PKU mice fed GMP showed comparable growth and reduced concentrations of Phe in plasma and the brain compared with an amino acid diet. These data support the use of GMP supplemented with IAA as an alternative source of dietary protein for individuals with PKU.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Glycomacropeptide (GMP) is ideally suited to the nutritional management of PKU, because it is abundant and in its pure form contains no Phe (4). GMP is a 64-amino acid glycophosphopeptide produced during cheese making when bovine -casein is cleaved by chymosin into para--casein, which remains with the cheese curd, and GMP, which remains with the whey (4). GMP comprises 15–20% of the protein in bovine milk whey. The unique amino acid profile of GMP is associated with an absence of aromatic amino acids, Phe, tryptophan, and tyrosine, as well as arginine, cysteine, and histidine, and concentrations of isoleucine and threonine that are 2- to 3-fold greater, respectively, than those found in other dietary proteins (4). GMP contains limiting amounts of 4 indispensable amino acids (IAA) for individuals with PKU (5): histidine, leucine, tryptophan, and tyrosine. GMP supplemented with limiting IAA could provide an alternative source of protein, instead of amino acids, for individuals with PKU as supported by our report describing the development of a variety of palatable foods and beverages made with GMP (6). Moreover, GMP has an excellent safety record as part of the human diet based on widespread supplementation of foods with whey protein and the use of whey-predominant infant formulas (7).

The murine model of PAH deficiency, the Pah^enu2 mouse (PKU mouse) is a suitable model to study the nutritional management of PKU as it exhibits hyperphenylalaninemia and cognitive defects similar to humans with PKU (8). Moreover, parallel to the human low-Phe diet in which the majority of dietary protein is provided by amino acids, studies in the PKU mouse utilize an amino acid-based diet often free of Phe with provision of Phe in drinking water (9). Our objective was to assess how ingestion of diets containing GMP as the sole protein source support growth and impact the concentrations of amino acids, in particular Phe, in plasma and brain of wild-type (WT) and PKU mice. Our positive findings of suitable growth and significantly reduced concentrations of Phe in plasma and the brain of PKU mice fed GMP compared with an amino acid diet provide support for our metabolic study to evaluate the safety and efficacy of GMP in the diet of individuals with PKU.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Mice. The animal facilities and protocols reported were approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee. Male and female 4- to 6-wk-old WT mice weighing 18–22 g were bred on the same background as the PKU mice (C57Bl/6, Jackson Laboratories). PKU mice were homozygous for the Pah mutation but were bred and backcrossed onto the C57Bl/6 background to increase breeding facility (10). Breeding pairs of PKU mice were generously provided by Cary O. Harding, Oregon Health and Science University, Portland, OR. Genotyping for the presence of the Pah^enu2 mutation was performed by PCR analysis of tail biopsy DNA on an amplified region of exon 7. Mice were individually housed in stainless steel, wire-bottom cages in a room maintained at 22°C on a 12-:12-h light:dark cycle and were given free access to water. The mice were weighed every day at 1000 and food intake was determined daily. At the conclusion of each experiment, mice were anesthetized using isoflurane via an anesthesia machine (IsoFlo, Abbott Laboratories) and killed by cardiac puncture/exsanguination between 0800 and 1000 with removal of food 1 h before being killed.

    Diets. Purified diets were designed in collaboration with Dr. Barbara Mickelson (Harlan Teklad) to provide similar amounts of vitamins, minerals, energy, and macronutrients (Table 1) (11). The protein source in the diets was provided by casein, free amino acids (9), GMP (BioPURE GMP, Davisco Foods), and GMP processed to reduce residual Phe content (4). The GMP diets were supplemented with 1.5 times the NRC suggested requirement for the following limiting IAA to compensate for faster absorption and degradation of amino acids compared with intact protein (12): arginine, histidine, leucine, methionine, tryptophan, and tyrosine. The nitrogen content of the amino acid and GMP low-Phe diets was similar, 24.1 and 22.9 g nitrogen/kg diet, respectively, and both diets provided 175 g amino acids/kg of diet. Complete amino acid analysis of the diets was conducted in the Experiment Station Chemical Laboratories, University of Missouri-Columbia (Columbia, MO) (Table 2) (13).

Expt. 2 tested the ability of diets containing amino acids and GMP to support growth in male and female PKU mice (5–8 wk old) when Phe was provided in the drinking water (1 g Phe/L) for 21 d. Three dietary treatment groups were included (n = 10/group): PKU mice fed the GMP Phe-deficient diet, PKU mice fed an amino acid Phe-deficient diet, and WT mice fed the GMP-adequate diet. We measured drinking water intake daily in PKU mice and adjusted for evaporation to determine the amount of Phe consumed.

Expt. 3 evaluated the ability of diets containing amino acids and GMP that were supplemented with a minimum amount of Phe (determined from Expt. 2) to support growth and affect the concentrations of amino acids in plasma and the brain of male and female PKU mice fed for 47 d. Four dietary treatment groups were included: 6-wk-old WT mice fed casein (n = 8) or the GMP-adequate diet (n = 7) and 8- to 10-wk-old PKU mice fed the amino acid, low-Phe (n = 10) or the GMP, low-Phe diet (n = 11). There were similar numbers of male and female mice in each treatment group. Blood samples were obtained by orbital bleeding for amino acid analysis using heparinized capillary tubes after 21 d of feeding (n = 5/group). Mice were anesthetized, killed by cardiac exsanguination, and decapitated after 47 d. The brains were quickly removed and placed on a glass plate cooled by dry ice. Using visual landmarks, samples were taken from the following 5 regions; cerebellum, brain stem, hypothalamus, parietal cortex, and the anterior piriform cortex (14). The samples were placed in preweighed polystyrene tubes, weighed to determine sample mass, and stored at –80°C until processing.

    Amino acid analysis. Blood was collected by cardiac puncture into syringes containing a final concentration of 2.7 mmol/L EDTA and plasma was isolated by centrifugation at 1700 x g; 15 min at 4°C. The profile of free amino acids in plasma was determined using a Beckmann 6300 amino acid analyzer equipped with an ion chromatography system using post column ninhydrin derivatization (15). The samples were deproteinized with sulfosalicylic acid, centrifuged (14,000 x g; 5 min, and passed through a 0.2-µm syringe filter before adding an internal standard and injecting into the column.

The profile of free amino acids in the brain was determined in the Amino Acid Analysis Laboratory, University of California-Davis, School of Veterinary Medicine (Davis, CA) using a Biochrom 30 amino acid analyzer (Biochrom). The procedure for extraction of amino acids from the brain samples included the addition of 3% sulfosalicylic acid containing 100 µmol/L Norleucine as an internal standard (Sigma Chemicals) in a ratio of 1:10 (wt:v), homogenization with an ultrasonic needle for 2 min, centrifugation at 14,000 x g; 20 min at 4°C, and filtration of supernatant through a 0.45-µm syringe drive filter. The filtrate was adjusted to pH 2.2 with 0.4 mol/L LiOH and 0.05 mL was injected into the column. Values are expressed as nmol amino acid/g wet tissue weight.

    Statistics. Statistical analyses were conducted using SAS version 8.2 (SAS Institute) and R (Universitat Wien, Vienna, Austria). Data were analyzed using general linear models. The differences between dietary treatment groups were determined by the protected least significant difference technique. Statistics were performed on log-transformed data when residual plots indicated unequal variance among groups as occurred for some of the data. Where appropriate, sex was included as a covariate to adjust for its potential influence. Changes in body weight (BW) among treatment groups were assessed with repeated measures analysis in Expt. 1. Among PKU mice, simple linear regression was used to examine the correlations between dietary intakes of amino acids 48 h prior to death and the concentrations of amino acids in plasma and brain. All values are presented as means ± SE; P 0.05 was considered significant.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Expt. 1. Initial and final BW did not differ among the 3 dietary treatment groups (Fig. 1). Food intake and BW did not differ when comparing the casein and GMP adequate groups throughout the 42-d study. Mice stopped eating the GMP Phe-deficient diet after 3 d, at which time Phe was added to the drinking water and food intake resumed. The 3 dietary groups did not differ in changes in daily BW from d 14 to d 42.

View larger version (13K): [in this window] [in a new window]   FIGURE 1  Changes in BW in weanling WT mice fed diets containing casein, GMP supplemented with limiting IAA (GMP adequate), or GMP supplemented with limiting IAA except Phe (GMP Phe deficient) for 42 d in Expt. 1. Values are means ± SEM; n = 10. Phe was added to the drinking water for the GMP Phe-deficient group (1 g Phe/L) on d 4 through the end of the study. There were no significant differences for changes in daily BW from d 14 to d 42.

    Expt. 2. Initial (16–18 ± 1.4 g) and final (19–21 ± 1.3 g) BW and food intake (3.3 to 4.1 ± 0.3 g/d) did not significantly differ among the 3 treatment groups for 21 d. Mean Phe intake in PKU mice was 6.5 ± 0.5 mg Phe/d with ingestion of the amino acid Phe-deficient diet and 5.9 ± 0.3 mg Phe/d with ingestion of the GMP Phe-deficient diet (P > 0.10). John and Bell (12,16) reported that growing mice fed an adequate level of tyrosine have a minimum Phe requirement of >2.5 g Phe/kg diet but 4.0 g Phe/kg diet. Considering our observations from Expt. 1 and 2 that growth may be limited with provision of Phe in the drinking water, we decided to supplement the low-Phe amino acid and GMP diets for Expt. 3 to contain 2.5 g Phe/kg diet. This provided a daily Phe intake for growing PKU mice of 7.5–10 mg Phe.

    Expt. 3. Gain in BW, feed utilization based on the ratio of feed intake to gain in BW, and the protein efficiency ratio did not differ among the 4 dietary treatment groups (Table 3). PKU mice were 2 g heavier than WT mice (P < 0.05), consistent with the former being 2 wk older. Female mice of both genotypes weighed less than male mice at the end of the study (20 ± 1 g vs. 25 ± 1 g; n = 17–18; P < 0.0001).

The profile of amino acids in plasma was affected by diet and sex (Table 4). PKU mice fed either the amino acid or GMP low-Phe diet showed 15-fold greater plasma concentrations of Phe and a 60–70% decrease in plasma concentrations of tyrosine and proline compared with WT mice fed either the casein or GMP diets. Both WT and PKU mice fed GMP diets showed plasma concentrations of threonine and isoleucine that were 2 times the values in WT and PKU mice fed the casein or amino acid diets (P < 0.002). Decreased plasma concentrations of lysine were noted in WT and PKU mice fed GMP diets (272 ± 11 µmol/L) compared with WT and PKU mice fed the casein or amino acid diets (443 ± 31 µmol/L; P < 0.0001; n = 17–18). Female mice of both genotypes showed greater plasma concentrations of tyrosine (74 ± 6 vs. 53 ± 8 µmol/L) and tryptophan (113 ± 5 vs. 81 ± 5 µmol/L) compared with male mice (P < 0.01; n = 17–18).

The profile of amino acids in the cerebellum differed significantly due to diet but not sex (Table 5). The concentration of Phe in cerebellum of PKU mice was 3 to 4 times the value in WT mice (P < 0.0001). The concentrations of tyrosine and the sum of the branched chain amino acids in cerebellum of PKU mice were 50% of that in WT mice regardless of diet (P < 0.0001) as previously noted (17). PKU mice fed the GMP diet had a 20% decrease in the concentration of Phe in cerebellum compared with PKU mice fed the amino acid diet. Moreover, this response of a 20% decrease in Phe concentration was noted in each of 5 sections of brain sampled: cerebellum, brain stem, hypothalamus, parietal cortex, and anterior piriform cortex (Fig. 2). The concentrations of threonine and isoleucine in the cerebellum increased 70–100% in PKU mice fed the GMP diet compared with the amino acid diet (P < 0.0001). A similar trend was noted for higher valine concentration in the cerebellum of PKU mice fed the GMP diet compared with the amino acid diet (P < 0.10). The concentration of Phe in the cerebellum of PKU mice was inversely correlated with the concentrations of threonine, isoleucine, and valine in plasma as well as the sum of the concentrations of threonine, isoleucine, and valine in plasma, R² = 0.65–0.77 (P < 0.0001) (Fig. 3).

View larger version (14K): [in this window] [in a new window]   FIGURE 2  The concentration of Phe in 5 sections of brain, cerebellum, brain stem, hypothalamus, parietal cortex, and anterior piriform cortex, of PKU mice fed the GMP or amino acid (AA) diet for 47 d in Expt. 3. Values are means ± SEM; n = 8. *Different from AA, P 0.001.

View larger version (9K): [in this window] [in a new window]   FIGURE 3  Inverse correlation between plasma threonine (Thr) + isoleucine (Iso) + valine (Val) and cerebellum Phe for PKU mice fed the GMP or amino acid (AA) diet for 47 d in Expt. 3.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
The primary therapy for PKU is lifelong adherence to a highly restrictive, low-Phe diet in which the majority of dietary protein is provided by amino acids. GMP is an abundant protein in cheese whey that has a unique amino acid profile, including the absence of Phe (4). Thus, GMP supplemented with IAA provides a potential alternative protein source in the PKU diet. This study assesses for the first time, to our knowledge, the ability of diets containing GMP supplemented with IAA as the sole protein source to support growth and affect the concentrations of amino acids in the plasma and brains of PKU mice. In support of utilization of GMP as a source of low-Phe protein in the PKU diet, we observed similar growth with significantly lower concentrations of Phe in the plasma and brains of PKU mice fed GMP compared with an amino acid diet.

When fed as the sole source of dietary protein, GMP contains limiting amounts of several IAA for growing mice including: arginine, histidine, leucine, methionine, Phe, tryptophan, and tyrosine. Our results showed adequate growth of mice that are fed GMP supplemented with these limiting IAA. In Expt. 1, weanling WT mice fed casein or the GMP-adequate diet had virtually identical growth over 6 wk. In Expt. 3, PKU mice fed GMP or amino acid diets with similar Phe intake showed gains in BW, feed efficiency and a protein efficiency ratio that were not significantly different. These data demonstrate that GMP supplemented with limiting IAA provides a nutritionally adequate source of dietary protein for growing mice.

Consumption of a diet that is deficient in an IAA rapidly depresses the concentration of the limiting IAA in plasma and brain with reduced food intake in rats (18). Thus, it was not surprising that in Expt. 1, mice stopped eating the GMP Phe-deficient diet and lost BW after only 3 d of this diet and that addition of Phe to the drinking water normalized food intake and gain in BW. The plasma concentrations of isoleucine and threonine in WT mice fed the GMP-adequate diet were 2 to 3 times those in WT mice fed the casein diet. However, these alterations in plasma amino acid concentrations did not impair food intake in mice fed GMP once the deficiency of Phe was corrected (19). Thus, in contrast to evidence that GMP may suppress appetite (20), we conclude that ingestion of GMP supplemented with all limiting IAA alters the plasma amino acid profile without reducing food intake in growing mice.

In contrast to other IAA, hepatic uptake of threonine is low and oxidation of threonine to CO₂ via liver threonine dehydratase activity (EC 4.2.1.16) is limited in both humans (21) and rats (18). Thus, an increase in dietary threonine without an increase in total protein intake results in expansion of the plasma threonine pool without toxicity if diets provide <15 times the normal level of threonine (18,22,23). The concentration of threonine in plasma showed the largest increase with ingestion of GMP compared with the casein and amino acid diet in all 3 experiments. Similarly, an increase in plasma threonine concentration is observed in response to increased threonine intake in the human infant fed whey predominant formulas (24) and in human adults (25) as well as rats (19,23). Degradation of threonine to glycine via threonine dehydrogenase (EC 1.1.1.103) is a major catabolic pathway in rats but not in humans (21). Elevated glycine levels are potentially neurotoxic in the brain due to the glycinergic neurotransmitter system that can inhibit or stimulate transmission of nervous impulses (26). However, our demonstration of no increase in the concentration of glycine in both plasma and brain suggests that feeding a GMP diet that provides 3 times the normal intake of threonine is not sufficient to modify the concentration of glycine in brain (23). Taken together, these findings support the safety of dietary GMP.

The 11% decrease in the concentration of Phe in plasma of PKU mice fed GMP compared with the amino acid diet for 47 d is a positive finding for the nutritional management of PKU. Similarly, a 26–36% decrease in the concentration of Phe in plasma was noted in rats fed whey-supplemented diets with a threonine content similar to the current study (19) and in individuals with hyperphenylalaninemia given an oral daily supplement of 50 mg threonine/kg BW for 8 wk (27). There is no known direct link between threonine and Phe intermediary metabolism. Two explanations may account for decreased circulating concentrations of Phe with ingestion of GMP. GMP may have stimulated whole-body protein synthesis, resulting in lower circulating concentrations of Phe compared with the amino acid diet. However, greater protein synthesis would be expected to lower concentrations of other IAA and this was not observed in the PKU mice fed GMP compared with the amino acid diet.

Alternatively, the high concentration of threonine in GMP may have reduced plasma Phe concentration by competing with Phe for absorption from the intestinal mucosa. The large neutral amino acids (LNAA), Phe, tyrosine, tryptophan, threonine, isoleucine, leucine, valine, and methionine, share a similar common transporter across the blood brain barrier and intestinal mucosa and compete with one another for transport in the brain and gut (28,29). Individuals with PKU given daily supplements of LNAA (250–500 mg/kg BW) show significant decreases in the concentration of Phe in plasma consistent with competitive inhibition of Phe absorption from the gut (30,31). An in vitro study in human intestinal epithelial Caco-2 cells showed a 50% inhibition of Phe transport with 100-fold concentrations of tyrosine or leucine compared with Phe (29). In the current study, the GMP diet provided a 30-fold greater concentration of the LNAA, threonine, isoleucine, and valine, compared with Phe concentration. Additional studies are needed to determine whether the concentrations of LNAA in GMP are sufficient to competitively inhibit Phe transport across the intestinal mucosa.

The finding of greatest relevance to the management of PKU and the known neurotoxic effects of Phe is our observation that PKU mice fed GMP compared with the amino acid diet had a 20% decrease in the concentrations of Phe in 5 sections of brain. The concentration of Phe in brain is the best correlate of mental impairment in individuals with PKU (1). For example, there are intellectually normal persons with PKU who have never been treated with a low-Phe diet and show very high blood levels of Phe but low levels of Phe in the brain due to reduced transport (32). The most likely explanation for the reduced concentration of Phe in the brain of PKU mice fed GMP is that elevated plasma levels of LNAA due to ingestion of GMP competitively inhibit Phe transport across the blood brain barrier via the LNAA carrier protein that has a significantly lower Km in the brain compared with the gut (30). This conclusion is supported by a significant inverse correlation between higher plasma concentrations of threonine, isoleucine, and valine and lower brain concentration of Phe. Interestingly, previous research demonstrates that isoleucine, but not threonine, competitively inhibits Phe transport in rat brain (33). In support of our finding that ingestion of GMP lowers the concentration of Phe in brain, oral supplementation with LNAA reduces the concentration of Phe in brain of PKU mice (30) and in adults with PKU as measured by magnetic resonance spectroscopy (34). In contrast to other IAA, threonine and the branched chain amino acids, isoleucine, leucine, and valine, tend to accumulate in plasma and the brain in proportion to their concentrations in the diet (22).

In summary, we demonstrate for the first time to our knowledge that PKU mice fed a diet with 20% GMP supplemented with IAA compared with an amino acid diet show similar growth and lower concentrations of Phe in plasma and the brain. These data establish that GMP can be formulated into a nutritionally adequate complete protein for growing mice and suggest that long-term feeding studies may provide further insight into the metabolism of GMP. Our findings support continued research to establish the efficacy of foods and beverages made with GMP in the nutritional management of PKU in humans (6).

	ACKNOWLEDGMENTS

 
We thank the following individuals for their helpful input regarding this research: Quinton Rogers, University of California-Davis; Jean Tews, University of Wisconsin-Madison; and Sally Gleason and Albee Messing, Waisman Center, University of Wisconsin-Madison.

	FOOTNOTES

 
¹ Supported by Robert Draper Technology Innovation Funding, Graduate School and the USDA Cooperative State Research, Education and Extension Service project WISO1084, University of Wisconsin-Madison; and NIH P30 HD03352.

² Author disclosures: D. M. Ney, A. K. Hull, S. C. van Calcar, X. Liu, and M. R. Etzel, no conflicts of interest.

⁶ Abbreviations used: BW, body weight; GMP, glycomacropeptide; LNAA, large neutral amino acids; PAH, phenylalanine hydroxylase; Pah^enu2 mouse, PKU mouse; PKU, phenylketonuria; IAA, indispensable amino acid; WT, wild type.

Manuscript received 21 September 2007. Initial review completed 13 November 2007. Revision accepted 6 December 2007.

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