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Nutrient Metabolism
Research Communication

The Balance of Dietary Sulfur Amino Acids and the Route of Feeding Affect Plasma Homocysteine Concentrations in Neonatal Piglets¹

Anna K. Shoveller^*, James D. House, Janet A. Brunton^**, Paul B. Pencharz^*,,, and Ronald O. Ball^*,,,2

^* Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada T6G 2P5; Department of Animal Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2; ^** The Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9; Research Institute, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8; and Department of Paediatrics and Department of Nutritional Sciences, University of Toronto, Toronto, ON, Canada M5G 1X8

²To whom correspondence should be addressed. E-mail: ron.ball{at}ualberta.ca.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Plasma total homocysteine (tHcy) concentrations are associated with atherogenesis in adults and increased risk of stroke in infants and children. After a series of experiments to compare the methionine (Met) requirement and cysteine (Cys)-sparing capacity in piglets that were parenterally or enterally fed, we examined the effects of route of feeding and dietary Cys on plasma tHcy concentrations. Piglets (n = 60; 6–8 d old) were fed elemental diets, intragastrically (n = 28) or intravenously (n = 32), with 0.55 g · kg^-1 · d^-1 dietary Cys (n = 28) or without dietary Cys (n = 32). Dietary Met ranged from deficient to excess. Increasing Met intake increased (P < 0.01) plasma tHcy in all treatment groups. Plasma tHcy concentrations were higher (P < 0.05) in the enterally fed piglets that did not receive dietary Cys than in all other groups, which did not differ from each other. Therefore, both route of feeding and dietary supply of Met and Cys significantly affected the concentrations of plasma tHcy. These dramatic and rapid alterations in plasma tHcy warrant further studies of sulfur amino acid metabolism in neonatal animals.

KEY WORDS: • homocysteine • methionine • cysteine • total parenteral nutrition • enteral feeding

Hyperhomocysteinemia is an independent risk factor for cardiovascular and atherosclerotic disease in adults (1). It is also associated with an increased risk of ischemic and hemorrhagic stroke in newborn infants and children (2,3). Reference values for plasma total homocysteine (tHcy)³ in human infants range from 5 to 8 µmol/L (2,4,5). There is substantial evidence that blood values of tHcy are often greater when vitamin B-6, vitamin B-12, and folate are deficient in adults and infants (6,7). Ambrosi and colleagues (8) showed that 4.5-mo-old pigs fed a methionine (Met)-rich, casein-based diet for 1 mo developed hyperhomocysteinemia, which led to arterial lesions and thrombotic events. To our knowledge, no one has previously investigated the relation between plasma tHcy concentration and dietary Met intake in neonatal animals. The neonatal pig may be a useful model to study the relation between hyperhomocysteinemia and stroke in infants and children.

There is currently great variation in the absolute concentration and ratio of sulfur amino acids (SAAs) in enteral infant formulas (9) and breast milk (10,11). Whether these differences affect plasma tHcy concentrations is not known. The dietary recommendation for adults, children, and infants with hyperhomocysteinemia is to supplement folate and vitamin B-12 (2). However, there are few data on how the dietary supply of SAAs affects plasma tHcy concentrations in neonates with adequate intake of vitamin B-6, vitamin B-12, and folate. The potential effects of the differences in SAA supply and ratio on plasma tHcy concentrations and also on long-term health need to be determined.

Little information exists on how total parenteral nutrition (TPN) and the dietary concentration and ratio of SAAs in TPN affect plasma tHcy concentrations. Neonates fed TPN have lower plasma cysteine (Cys) concentrations than do enterally fed neonates (12–15). Much like enteral formulas, there is great variation in the absolute concentration of SAAs and the Met:Cys ratio in currently available TPN solutions (16). The clinical goal in both TPN and enteral feeding of neonates is to provide sufficient nutrients to support rapid growth while avoiding potentially harmful excesses. One such deleterious effect that may occur with an inappropriate Met:Cys ratio is the alteration of plasma tHcy and Cys concentrations. Rabbits that were fed either by TPN or enterally with a standard diet and that received supplemental i.v. Met had higher plasma tHcy concentrations than did control rabbits fed a standard diet (15). In addition, rabbits fed either by TPN or with a standard diet that received i.v. Met treatment had decreased bile flow and hepatobiliary function; these events precede cholestasis, a common complication in TPN-fed patients (15). Altering the Met:Cys ratio may alleviate the potentially harmful effects of high Met concentrations in TPN. Therefore, it is necessary to define the SAA requirements of TPN-fed neonates and understand how the intake and ratio of these amino acids affect plasma tHcy concentrations.

We previously established the Met requirements of parenterally and enterally fed piglets with (12) and without dietary Cys (17). These data demonstrated that the gut utilizes 30% of the SAA, that dietary Cys can spare the Met requirement by 40% in both routes of feeding, and that Cys is not an indispensable dietary amino acid in either enterally or parenterally fed neonatal piglets. We also measured plasma tHcy concentrations in the samples obtained during these studies. The objective of the present study was to examine the effect of dietary Met and Cys intake on plasma tHcy concentrations in parenterally and enterally fed neonatal piglets. Dietary Cys decreases transmethylation and increases remethylation of Met (18,19), and thereby decreases plasma tHcy concentrations overall. We hypothesized that plasma tHcy levels would be positively associated with Met intake in both parenterally and enterally fed piglets, but would be lower in piglets fed diets that provide Cys.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and study protocol. The Faculty of Agriculture, Forestry, and Home Economics Animal Policy and Welfare Committee at the University of Alberta approved all procedures used in this study. Landrace/Large White piglets (Genex Swine Group; 53 male, 7 female; age = 1–2 d; body wt = 1.4–1.7 kg) were obtained from the University of Alberta, Swine Unit. Venous catheters were surgically implanted in the piglets as previously described (17).

    Animal housing. The piglets were housed and cared for as described in Shoveller et al. (17).

    Diets. The piglets received 15 g amino acids · kg^–1 · d^-1 and 1.1 MJ metabolizable energy · kg^–1 · d^-1, with glucose and lipid (Intralipid 20%; Fresenius-Kabi) each supplying 50% of the nonprotein energy intake. The base amino acid profile of the complete elemental diet was as previously described (17). Vitamins were supplied in a commercial solution (3 mL per 900 mL of complete diet; Multi-12K₁ Pediatric; Sabex) that was added to the diet prior to feeding. The multivitamin solution provided 115% of the required levels of cofactors involved in Met metabolism, vitamin B-12, vitamin B-6, and folate (20). Trace minerals were supplied in a solution that provided 200% of required levels (20), as previously described (17).

TPN was initiated immediately after surgery, and both enteral and parenteral feeding groups were increased to full infusion rates (13.5 mL · kg^-1 · h^-1 of TPN and lipid) by the end of d 1. Complete TPN was continued until 1800 h on d 5. Piglets were then randomly allocated to one of the Met test levels, with either no dietary Cys or excess dietary Cys (0.55 g · kg^-1 · d^-1). This concentration of dietary Cys is 10% above the NRC recommendation for total SAA (20), and the increments of dietary Met ranged from 5 to 200% of the total recommended intake for SAA (20). The dietary Met intake ranged from 0.025 to 1.0 g · kg^-1 · d^-1. All test diet solutions were made isonitrogenous by balancing the test concentration of L-Met with an increased or decreased L-alanine concentration. Due to the highly unstable nature of L-Cys in aqueous solutions, the test diet solutions were prepared immediately prior to infusion. The piglets were fed the test diet from 1800 h on d 5 until 1800 h on d 6. Blood was sampled at 1400 h on d 6 (20 h after initiation of the test diet). Subsequently, the piglets were again fed the complete diet for 24 h. At 1800 h on d 7, the piglets were randomly assigned to a second test diet, and blood was again sampled at 1400 h on d 8 (20 h after initiation of the second test diet). The piglets were then killed by injection of 1000 mg of sodium pentobarbital through a venous catheter.

    Blood collection. Blood samples were collected with heparinized syringes via the femoral catheter. Whole blood samples were centrifuged at 4000 x g for 5 min, and the plasma was removed, frozen with liquid nitrogen, and stored at -80°C until further analysis.

    Determination of plasma homocysteine concentrations. Plasma tHcy concentrations were analyzed using the reverse-phase HPLC method of Araki and Sako (21), with modifications as suggested by Gilfix et al. (22). Briefly, plasma samples were incubated with tris-carboxyethylphosphine (Pierce Chemicals) to reduce protein-bound and oxidized forms of homocysteine, followed by derivatization with 7-fluorobenzofurazan-4-sulfonic acid ammonium salt (SBD-F; Sigma). The fluorescent thiol derivatives were separated on a Waters C-18 column (5 µmol/L, 4.5 x 250 mm; Waters Canada), using isocratic elution (98% 0.1 mol/L acetate, pH 5.5; 2% methanol) by means of a Shimadzu HPLC system (Man-Tech Associates) complete with autoinjector and fluorescence detector (excitation = 385 nm; emission = 515 nm). Concentrations of tHcy were determined using an external standard curve (inter- and intra-assay CV < 0.02). This is a commonly used and well-supported method of assaying tHcy concentration (23).

    Statistical analyses. The day of sampling (d 6 or d 8) and piglet weight at sampling had no significant effect (P > 0.05) on plasma tHcy level, as determined by ANOVA (SAS/STAT, version 8.1; SAS Institute). The regression analysis used dietary Met intake as the independent variable and plasma tHcy concentration as the dependent variable. Slopes were tested for differences among groups (GraphPad Prism, version 3.00 for Windows; GraphPad Software). The slope of the regression line did not differ among groups; therefore, differences between the y-intercepts of the regression lines for route of infusion and dietary treatment (with or without dietary Cys) were determined by pdiff, analyzed as a 2 x 2 factorial using route of infusion and Cys inclusion as the independent variables (SAS/STAT, version 8.1; SAS Institute). The y-intercept represents the plasma tHcy concentration.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
All piglets were healthy and active during the course of the study. Piglet weight on arrival (1645 g; age = 2 d; pooled SD = 149) and at the time of sampling (2661 g; age = 9 d; pooled SD = 297) did not differ among dietary treatment levels or routes of feeding. Mean daily weight gain prior to initiation of the test diet (160 g/d; pooled SD = 34) did not differ among dietary treatment levels or routes of feeding.

Plasma homocysteine concentration.

Plasma tHcy concentration increased linearly with Met intake within each treatment group (P < 0.01; Fig. 1; Table 1). The slope did not differ among treatment groups (P > 0.05); however, the intercept was significantly higher for enterally fed piglets that did not receive Cys (IG group) than for all other treatment groups (Table 1; P < 0.05). The linear response of the plasma tHcy concentration was similar both below and above the dietary requirement for the total SAAs (Fig. 1; 12,17).

View larger version (20K): [in this window] [in a new window]   FIGURE 1 The relation between plasma tHcy concentration and Met intake in intravenously fed neonatal piglets (A) receiving no Cys (IV group; y = 25.39x + 4.29; R2 = 0.55, P < 0.0001) or excess Cys (IV+C group; y = 26.99x + 3.86; R2 = 0.84, P < 0.0001) and in intragastrically fed neonatal piglets (B) receiving no Cys (IG group; y = 30.42x + 9.94; R2 = 0.37, P = 0.0006) or excess Cys (IG+C group; y = 17.64x + 5.14; R2 = 0.25, P = 0.009).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

These data have implications for the provision of SAAs in enteral infant formulas because of the health risks associated with elevated plasma tHcy concentrations in newborns (2) and children (3). Currently, the Met:Cys ratio in infant formulas varies depending on the protein source. Casein-based formulas have a greater proportion of Met, whereas both soy- and whey-based products provide less Met and more taurine, and few formulas contain additional Cys (9). Considering our findings in a model that is relevant to nutrition in human infants (16), SAA intake and the SAA ratio may have a profound effect on plasma tHcy concentrations. The questions of the optimal SAA intake and ratio for formula-fed infants and the effects on plasma tHcy concentrations should be pursued further. When the majority of the SAAs in a formula are supplied by Met, the addition of Cys should be considered as a means of decreasing the plasma tHcy concentrations in the infants fed the formula.

In the present study, all piglets received similar and more than adequate intakes of vitamin B-12, vitamin B-6, and folate and similar intakes of all other amino acids (20). Therefore, the increases in plasma tHcy concentrations we observed must be due solely to the Met intake and not to differences in protein source or intakes of vitamins, nitrogen, amino acids, or minerals. Other than Met, the only dietary amino acid that was altered was alanine, which was used to make the diets isonitrogenous. The increase in plasma tHcy levels as dietary Met increased suggests that the proportion of Met that was transmethylated increased relative to the proportion that was transsulfurated and/or remethylated.

In the present study, the IG group had higher plasma tHcy concentrations at every level of Met intake than did the IG+C group. In adult men, the replacement of dietary Met with Cys (18) or glutathione (19) decreases transmethylation of Met to homocysteine and increases the remethylation of homocysteine to Met. A reduction in transmethylation causes less homocysteine to be synthesized, whereas an increase in remethylation causes more homocysteine to be remethylated to Met. In total, the addition of dietary Cys decreases the homocysteine balance (appearance–disappearance) and therefore decreases the plasma tHcy concentration, as observed in the present study. Regulatory processes similar to those in adult humans appear to be present in piglets, as shown by the lower plasma tHcy concentrations in the IG+C group, compared to the IG group. The regulatory effect of dietary Cys on plasma tHcy does not exist in parenterally fed subjects, when the gut is bypassed. This is evidenced by the fact that plasma tHcy concentrations did not differ between the IV+C and IV groups. The transmethylation/remethylation cycle is present in all cells (24), so it is possible that the gut exports large amounts of homocysteine. However, given that oral dietary Cys intake decreases transmethylation (18) and increases remethylation (18,19), the addition of dietary Cys may have partly mediated these effects within the gut.

Given that plasma tHcy concentrations did not differ between the IV and IV+C groups, we speculate that the gut is a larger site of homocysteine synthesis and release than the liver in neonatal piglets. However, adult rat hepatocytes incubated in Met exported more homocysteine than did hepatocytes incubated in Met and Cys (25). Further research to elucidate possible differences in homocysteine metabolism among different species and tissues is needed. Alternatively, if Cys utilization is greater in parenteral feeding, the homocysteine balance may be reduced by the upregulation of the transsulfuration pathway in an attempt to maintain the plasma Cys concentration. Indeed, plasma Cys concentrations were lowest in the IV group, suggesting increased utilization or catabolism of Cys (12). Therefore, we speculate that the luminal presence of Cys causes less homocysteine to be exported from the gut, compared to when no dietary Cys is provided, and that parenterally fed piglets may require more Cys than enterally fed piglets.

Given that the IG group had significantly higher plasma tHcy concentrations than the IG+C group, the whole body availability of Cys may also play a role in homocysteine balance. Ventura et al. (26) found that i.v. infusion of N-acetylcysteine, a Cys precursor, for 60 min increased urinary excretion of homocysteine in adults (22.4 basal vs. 13.1 nmol/mL^-1). Therefore, dietary Cys may increase the renal clearance and/or metabolism of homocysteine by displacing homocysteine from albumin binding sites and enabling it to be readily filtered by the kidney. This would result in a decrease in plasma tHcy concentration; however, urinalysis was not conducted in the present experiment. Clearly, both the route of diet administration and the Met:Cys ratio play a regulatory role in SAA metabolism, and further investigation is needed to elucidate the regulation of this phenomenon.

Although meeting the total SAA requirement for growth is important, supplying the correct Met:Cys ratio while avoiding excessive dietary Met intake is also important. The results of the present study agree with those reported by Rolland et al. (27), who demonstrated that minipigs fed a Met-rich caseinate-based diet developed hyperhomocysteinemia, establishing that pigs are a suitable model for the study of hyperhomocysteinemia. In addition, given the markedly higher plasma tHcy concentrations in the IG group piglets compared to those in the IG+C group, supplying Met as the sole SAA source—even at or below the dietary requirement level—may result in dangerously high plasma tHcy concentrations. If plasma tHcy is directly related to an increased risk of ischemic and hemorrhagic stroke (2,3) our research implies that Met intake by infants should be restricted. However, if Met intake is restricted, Cys supplementation is critical for the maintenance of growth. Cysteine is unstable in aqueous solution; therefore, a Cys precursor, such as N-acetylcysteine, could be added to both enteral and parenteral diets to lower plasma tHcy levels in enterally fed infants and improve the Cys status of parenterally fed neonates. N-acetylcysteine may be an effective precursor of Cys because it is stable in aqueous solution, highly bioavailable (28), and resistant to Maillard reactions (29).

The present data clearly demonstrate that Met intake, route of feeding, and Cys inclusion affect plasma tHcy concentrations in neonatal piglets. The higher concentration of plasma tHcy in enterally fed piglets that did not receive dietary Cys has important implications regarding the composition of infant formulas. Research into the role of splanchnic metabolism on homocysteine metabolism must be pursued using more complex in vivo techniques to measure SAA kinetics under different dietary conditions and routes of feeding. Finally, this study shows that the neonatal piglet model is a sensitive model for the investigation of homocysteine metabolism in both parenterally and enterally fed neonates.

	FOOTNOTES

 
¹ Supported by grants from Alberta Pork, Alberta Agricultural Research Institute, CIHR Fund no.12928, and Natural Science and Engineering Research Council (J.D.H.).

³ Abbreviations used: Cys, cysteine; IG, intragastric feeding without cysteine; IG+C, intragastric feeding with cysteine; IV, intravenous feeding without cysteine; IV+C, intravenous feeding with cysteine; Met, methionine; SAA, sulfur amino acids; tHcy, total homocysteine; TPN, total parenteral nutrition.

Manuscript received 19 October 2003. Initial review completed 5 November 2003. Revision accepted 2 December 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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