Nutrient Metabolism

Nutrient Metabolism

Urinary Sulfur Excretion and the Nitrogen/Sulfur Balance Ratio Reveal Nonprotein Sulfur Amino Acid Retention in Piglets1,2 Chunsheng Hou,*† Linda J. Wykes* and L. John Hoffer*†3 *School of Dietetics and Human Nutrition, McGill University, Montreal, Quebec, Canada H9X 3V9 and † Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2 ABSTRACT We evaluated the use of urinary sulfur (S) excretion as a measure of sulfur amino acid (SAA) catabolism and the nitrogen/sulfur (N/S) molar balance ratio as an indicator of nonprotein SAA storage in growing piglets. After confirming that an intravenous dose of sulfate is fully recovered in urinary sulfate, we measured urinary S recovery after an intravenous dose of methionine in 6 piglets fed an adequate protein (AP) diet and 6 piglets fed a low protein (LP) diet with normal energy provision. As measured over 48 h, recoveries of the methionine load as urinary total S was 106% in the AP group but only 69% in the LP group (P ⬍ 0.05). On the baseline diets the N/S balance ratio in the AP group was 36, whereas that in the LP group was 30 (P ⬍ 0.05); immediately after the methionine load, this ratio remained constant in the AP group but decreased further, to 26 (P ⬍ 0.05) in the LP group. These results indicate that protein-deficient piglets accumulate relatively more S than N from their diet, and under these conditions a significant portion of the S derived from a methionine load is retained in nonprotein compounds. Urinary S excretion, a simple nontracer measurement, can provide an accurate measure of SAA catabolism, and the N/S balance ratio is a potentially useful indicator of changes in nonprotein SAA stores of growing piglets. J. Nutr. 133: 766 –772, 2003. KEY WORDS:

●

glutathione

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methionine

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protein restriction

Because the partitioning of amino acids between protein synthesis and catabolism is mainly decided in the fed state (1– 5), the identification of simple and accurate ways to measure amino acid catabolism in the fed state could lead to better understanding of the factors regulating whole body protein economy. Current methods for measuring fed-state amino acid catabolism are tracer-determined amino acid oxidation and urea production. The former is expensive and laborious, and both methods have problems of accuracy and precision in the nonsteady state that follows normal food consumption (6 – 8). Sulfate production has been proposed as an alternative (9). End-product sulfur (S) arises predominantly from the catabolism of the sulfur amino acids (SAA) methionine and cysteine. Sulfate and taurine are the two main S-containing end products and, once formed, they are excreted almost entirely in the urine (10 – 14). The measurement of urinary S or urinary inorganic sulfate and taurine represents a simple, inexpensive, nonisotopic means to measure SAA catabolism and, in some situations, whole body amino acid catabolism. An important feature that distinguishes SAA catabolism from amino acid catabolism in general is the considerable storage of cysteine in a nonprotein reservoir, glutathione

●

sulfate

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taurine

(GSH) (15– 18). This raises the opportunity of using SAA catabolism in relation to total amino acid catabolism to infer information about whole body GSH stores. Because the N/S molar ratio of GSH is much lower than in whole proteins, changes in the whole body N/S balance ratio could indicate increases or decreases in the whole body GSH pool size (9,19). A better understanding of SAA metabolism is important in part because more information is needed about the SAA requirements of infants and children, including their metabolic response to inadequate protein intakes (20 – 22). The metabolic similarity of amino acid metabolism in piglets and human infants makes it a suitable animal model for such research (23,24). The present study was undertaken to validate the method of using urinary S excretion to measure whole body SAA catabolism, and to explore the use of the whole body N/S balance ratio as a noninvasive indicator of nonprotein SAA storage under different nutritional conditions in growing piglets. We first studied newborn piglets fed adequate protein and surfeit SAA with the aim of producing a state with normally filled GSH stores. We then used this model in a moderately protein-deficient state. Our aim was to create a dietary situation in which tissue GSH depletion is likely to occur, then use urinary S recovery to measure SAA retention and the N/S molar balance ratio as an indicator of nonprotein S storage.

1 Supported by Grant MOP-8725 from the Canadian Institutes of Health Research. 2 Presented in part in abstract form at the meeting of the Canadian Federation of Biological Societies, June 2002, Montreal, Quebec, Canada (Canadian Federation of Biological Societies Programme/Proceedings 2000, abstract T023, p. 82). 3 To whom correspondence should be addressed. E-mail: [email protected] 4 Abbreviations used: AP, adequate protein; GSH, glutathionine; LP low protein; N, nitrogen; NRC, National Research Council; PEG, polyethylene glycol; S, sulfur; SAA, sulfur amino acid.

MATERIALS AND METHODS Experimental design Six newborn piglets were assigned to an adequate protein (AP) group and six to a low protein (LP) group. Piglets in the AP group were

0022-3166/03 $3.00 © 2003 American Society for Nutritional Sciences. Manuscript received 10 September 2002. Initial review completed 28 September 2002. Revision accepted 8 December 2002. 766

SULFUR AMINO ACID CATABOLISM IN PIGLETS

continuously fed an elemental diet providing adequate protein and surfeit SAA with sufficient energy for normal growth; and those in the LP group, a low protein, low SAA diet with sufficient energy. The method of using urinary sulfate or total S excretion as an indicator of sulfate production was validated in the AP piglets by infusing a known amount of sulfate intravenously and measuring its urinary recovery. In both groups a methionine infusion study was performed to determine the relationship between urinary S end products and SAA catabolism under conditions of surfeit and deficient SAA intake. Urinary S recovery was used to determine SAA retention, and the N/S molar balance ratio to indicate nonprotein S storage.

Animals and surgical procedures Twelve male piglets (Landrace ⫻ Yorkshire) were obtained from Macdonald Farm, McGill University. The six piglets in the AP group were removed from the sow at 3 d of age for surgery to implant catheters. The six piglets in the LP group were removed from the sow at 6 d of age for the same procedure. All procedures were in accordance with the Canadian Council on Animal Care Guidelines and were approved by the Animal Care Committee of McGill University. Piglets were premedicated with atropine (0.05 mg/kg intramuscularly [i.m.]; atropine sulfate; MTC Pharmaceuticals, Cambridge, ON, Canada), Baytril (2.5 mg/kg i.m.; enrofloxacin; Bayer, Etobicoke, ON, Canada) and Buprenorphine (7.5 ␮g/kg i.m.; buprenorphine hydrochloride; Reckitt & Colman Pharmaceutical, Richmond, VA) followed by the induction of anesthesia with 5% isoflurane (MTC Pharmaceuticals). Anesthesia was maintained with 2% isoflurane in 50% oxygen by a tightly secured mask. Using a method modified from one described earlier (25), the following silicone catheters were aseptically inserted: a venous sampling catheter (1.0 mm i.d. ⫻ 2.2 mm o.d.) into the right external jugular; a femoral catheter (0.8 mm i.d. ⫻ 2.0 mm o.d.) into the right femoral vein for magnesium sulfate and methionine administration; a feeding catheter (1.57 mm i.d. ⫻ 3.18 mm o.d.) through a hole in the stomach; and a urine sampling catheter (1.02 mm i.d. ⫻ 2.16 mm o.d.) through a hole in the bladder wall. The catheters were filled with saline solution, capped and secured in the pocket of a mesh jacket worn by piglets, which were housed in metabolic cages. Each piglet’s urinary bladder was fully emptied every morning, after which piglets were weighed to the nearest 5 g on an electronic balance. Collected urine was pooled over 24 h to measure N and S balance.

Diets Elemental diets were used to permit the intake of the individual amino acids to be manipulated and precisely known. The AP diet was closely similar to one previously administered parenterally (25); it supplied nutrients at or above National Research Council (NRC) requirement levels for neonatal piglets (26), including those of the vitamins, trace elements and minerals (MULTI-12K1 Pediatric multiple vitamins for infusion and MICRO⫹6 Pediatric trace elements; Sabex, Boucherville, QC, Canada) except for calcium and phosphorus, which could be supplied only at 30% of the requirement because of their limited solubility. This diet provided 15.8 g 䡠 kg⫺1 䡠 d⫺1 of crystalline amino acids (Ajinimoto USA, Raleigh, NC). The amino acid profile was similar to that of milk protein, with tyrosine supplied as glycyl-tyrosine. All indispensable amino acids were supplied in excess of their requirement with the SAA supplied at 120% of requirement (see Table 1). This diet supplied 1.1 MJ 䡠 kg⫺1 䡠 d⫺1 of metabolizable energy, with amino acids, glucose polymer (Polycose; Ross Laboratories, Columbus, OH) and lipid (Intralipid 30%; Pharmacia, Mississauga, ON, Canada) supplying 24, 38 and 38%, respectively, of the total energy. The LP diet supplied 6.45 g 䡠 kg⫺1 䡠 d⫺1 total amino acids. The lower amino acid content of this diet was achieved by reducing the level of each indispensable amino acid (including methionine and cysteine) to 50% of the NRC requirement and reducing the dispensable amino acid content to 50% of the level in the AP diet. The LP diet was identical to the AP diet in all other respects, with the missing amino acid energy being replaced isoenergetically with glucose polymer; thus, amino acids, glucose polymer and lipid supplied 10, 52 and 38% of the total energy, respectively.

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TABLE 1 Amino acid composition of adequate protein (AP) and low protein (LP) diets in comparison to the National Research Council (NRC) amino acid requirements of piglets Amino acid

AP

LP

NRC1

g 䡠 kg⫺1 䡠 d⫺1 Arginine Glycine Alanine Serine Glutamate Proline Methionine Cysteine Phenylalanine Tyrosine Threonine Tryptophan Histidine Aspartate Isoleucine Leucine Valine Lysine Taurine

0.92 0.56 1.47 0.87 1.60 1.26 0.36 0.31 0.61 0.39 0.87 0.39 0.47 0.99 0.79 1.77 0.87 1.26 0.07

0.19 0.30 0.75 0.45 0.80 0.64 0.13 0.15 0.29 0.15 0.32 0.09 0.15 0.50 0.27 0.48 0.33 0.48 0.04

0.38

0.25 0.30 0.58 0.30 0.63 0.18 0.30 0.53 0.95 0.65 0.95

1 Based on the National Research Council requirement for 3- to 5-kg piglets, assuming average weight of 4 kg (26). Cysteine and tyrosine requirements were calculated by subtracting methionine and phenylalanine from total sulfur and aromatic amino acid requirements, respectively.

The diets were administered by gastric catheter at a constant rate using a COMPAT 199235 enteral feeding pump (Novartis Nutrition, Minneapolis, MN), with the feeding rates adjusted every morning according to body weight. The target infusion rates were 275 mL 䡠 kg⫺1 䡠 d⫺1 of undiluted elemental diet, which was coinfused with lipid at a rate of 34 mL 䡠 kg⫺1 䡠 d⫺1. To avoid hyperosmolar diarrhea or fluid overload it was necessary to use glucose polymer rather than glucose, and the diets were diluted according to a graded schedule, in which the diet infusion began ⬃3 h postsurgery at a submaximal rate, and gradually increased during d 0 and d 1. The complete elemental diet was diluted 4-fold on the day of surgery (d 0), then 3-fold for 12 h followed by 2.5-fold for 12 h on d 1; 2-fold for 12 h followed by 1.75-fold for the remainder of d 2 and 3; then 1.5-fold for d 4 to d 6. From d 7 to the end of the study, the solution was diluted 1.2-fold. A full rate of diet provision was reached from the beginning of d 3 postsurgery in the AP group, and from the beginning of d 4 postsurgery in the LP group.

Study protocol Sulfate infusion. In the AP piglets, urinary sulfate and total S excretion were measured over two 24-h basal periods on d 4 and d 5 after surgery. On the beginning of d 6, 1.2 mmol 䡠 kg⫺1 of MgSO4 䡠 7H2O (Sabex, Boucherville, QC, Canada) was infused intravenously over 2 h. Two 24-h urine samples were collected before the MgSO4 infusion to measure baseline urinary sulfur excretion, after which a 24-h urine sample was obtained to measure urinary sulfate and total S excretion. Methionine infusion. In the AP piglets, 24-h urine collections were obtained on d 8 and d 9 postsurgery to measure baseline urinary excretions. At the beginning of d 10, L-methionine (1.2 mmol 䡠 kg⫺1) was infused intravenously at a constant rate over 2 h and urine was collected over the following 2 d to measure urinary S and N excretion. Venous blood samples were obtained just before and 24 and 48 h after the methionine infusion. Piglets were then killed by an intravenous injection of 750 mg sodium pentobarbital (Euthansol; Scher-

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HOU ET AL.

ing Canada, Pointe Claire, QC, Canada). In the LP piglets, a methionine infusion study was performed when they were the same age as the AP piglets (13 d) using the same infusion and measurement procedure.

Analytical methods Total S, inorganic sulfate and ester sulfate. These measurements were based on the turbidimetry of barium sulfate in the presence of a small amount of preformed barium sulfate and the stabilizing agent polyethylene glycol (PEG), as described by Lundquist et al. (27) with modifications described below. Total S was determined after wet oxidation of urine samples with nitric acid and perchloric acid in the presence of catalytic vanadate (28). All water used was distilled and deionized. A 3-mL urine sample was transferred to a 150 ⫻ 25 mm Kimax digestion tube (VWR International, Ville-Montre´ al, QC, Canada) and dried under plain airflow at 120°C. To this tube was added 5 mL of nitric acid/perchloric acid/potassium dichromate/ ammonium metavanadate digestion mixture (27) and the sample heated in a Tecator Digestion System 12:1009 Digester (Mandel Scientific, Montreal, QC, Canada) at 140°C for 2 h, leaving the end of the tube uncovered to allow evaporation of all nitric acid. A funnel was then placed over the mouth of the tube and the temperature raised to 220°C for 3 h to oxidize all S-containing molecules to inorganic sulfate. Complete oxidation was indicated by a change in color of the sample from green to orange as chromic ion (green) was oxidized to dichromate (orange). After the sample had cooled, 20 mL of acetic acid/hydrochloric acid/phosphoric acid diluent (27) was added and carefully mixed to dissolve precipitates. The contents of the digestion tube (with water washes) were transferred to a long 100-mL Kjeldahl tube and the volume made up to the 50-mL mark with deionized water. For the sulfate analysis, 1 mL of the diluted digestate was mixed with 3 mL of deionized water and 1 mL of freshly prepared barium/PEG/sulfate reagent (27) and left at room temperature for precisely 23 min, when it was mixed and transferred to a 2-mL quartz cuvette and the absorbance was read precisely at 25 min using a Perkin Elmer Lambda 3A UV/VIS spectrophotometer (Perkin Elmer, Shelton, CT) set at 600 nm. To measure inorganic sulfate, a 0.1-mL urine sample was diluted to 3 mL with deionized water. To this was added 1 mL of 0.5 mol/L HCl and 1 mL of barium/PEG/sulfate reagent, followed by thorough mixing. The sample was then treated as described above. Ester sulfate was measured after first eliminating inorganic sulfate from the urine by barium chloride precipitation. An acid barium chloride solution (3 mL) (27) was mixed with 1.5 mL of urine. The mixture was centrifuged at 1400 ⫻ g for 10 min at room temperature, and 1.5 mL of the supernatant applied to a 1-mL column of cation exchange resin (AG 50W-X8, 100 – 200 mesh hydrogen form; Bio-Rad Laboratories, Richmond, CA). Water (2.5 mL) was added, and the eluate collected in a 15-mL Kimax screw-cap glass tube. The sealed tube was kept in a boiling water bath for 30 min to hydrolyze sulfate esters. After cooling to room temperature, 2 mL of the resulting solution was mixed with 1 mL of barium/PEG/sulfate reagent and mixed well. The other 2 mL was mixed with 1 mL of deionized water and served as a control, given that the hydrolyzed urine samples may develop a faint blue color with boiling alone. The sulfate measurement was then made as described above. The difference between the absorbance of the control and deionized water was subtracted from the absorbance obtained in the complete assay. Methionine, taurine and homocysteine. Plasma methionine and taurine and urinary taurine were derivatized with phenylisothiocyanate and measured by ultraviolet detection HPLC, as described previously (29). Plasma total homocysteine was measured by fluorescence HPLC after reduction of all disulfides using tris-(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl; Pierce Chemicals, Rockford, IL; 100 mg dissolved in 0.9 mL of water), and conversion to a fluorescent derivative with monobromobimane (Calbiochem, San Diego, CA) using a method modified from that of Hum et al. (30). Total N, urea and creatinine. Total urinary N was measured by high temperature combustion followed by chemiluminescent detection of nitric oxide (ANTEK model 7000B total N analyzer; ANTEK Instruments, Houston, TX). Serum and urine urea and creatinine

were analyzed using a Hitachi 917 automated analyzer (Laval, QC, Canada).

Calculations N balance was calculated as Nbal (mmol䡠kg⫺1䡠d⫺1) ⫽ Nin ⫺ Nout. N retention (Nret) was calculated as Nret (%) ⫽ [Nbal/Nin] ⫻ 100. Nout was equal to urinary N only, given that stool output was negligible. S balance (Sbal) was calculated as Sbal (mmol䡠kg⫺1䡠d⫺1) ⫽ Sin ⫺ Sout. SAA retention (SAAret) was calculated as SAAret (%) ⫽ [Sbal/SAAin] ⫻ 100. Sout was equal to urinary total S only, given that stool output was negligible. The N/S molar balance ratio was calculated as Nbal/Sbal. The recovery of infused magnesium sulfate or methionine as urinary sulfate or S was calculated as the increase in urinary sulfate or S excretion above its stable baseline excretion. Urinary sulfate or S production attributed to the infused substrate was equal to total sulfate or S production minus baseline sulfate or S production over the 2 d before the infusion, which was assumed to remain constant over the subsequent 2 d. The absolute dose of methionine infused was determined by the body weight on the morning of the infusion. Because excretions were expressed per kg body weight, it was necessary to take account of the slightly greater body weight on the 2nd d of the two 24-h collection periods. To obtain a value for metabolite excretion above basal on d 2, the extrapolated basal excretion/kg of body weight on d 2 was subtracted from the total excretion/kg of body weight. The result was then multiplied by (body weight on d 2)/(body weight on d 1).

Statistical analysis After homogeneous variance and normality were verified, two-way repeated-measures ANOVA was used to determine differences in N balance, S balance, N/S balance ratio, N retention and S retention, the two factors being diet (AP vs. LP) and different study states (before and after methionine infusion). Two-way repeated-measures ANOVA was used to determined differences in body weight, circulating metabolite concentrations and S recoveries in the methionine infusion study, the two factors being diet (AP vs. LP) and time. Within the same group, circulating metabolite concentrations and urinary S excretion over time were analyzed by one-way repeated ANOVA. When ANOVA results showed significance, the Newman–Keuls test was used post hoc to determine the source of difference. A paired t test was used to determine significance in urinary S and sulfate recoveries with 100%. All statistical analyses were performed using the Sigmastat program (Sigmastat for Windows version 2.03, SPSS, Chicago, IL). All results are presented as means ⫾ SEM.

RESULTS Growth Piglets in both groups were healthy and active throughout the study. Figure 1 shows their growth curves. After a full dietary intake was achieved, the rate of weight gain of AP piglets (79.4 ⫾ 2.8 g 䡠 kg⫺1 䡠 d⫺1) was similar to that previously reported for sow-raised piglets (79 g 䡠 kg⫺1 䡠 d⫺1) (25). The rate of weight gain by the LP piglets was 70.5 ⫾ 5.4 g䡠kg⫺1䡠d⫺1 (P ⫽ 0.11). Daily urinary S excretion Daily urinary total S, inorganic sulfate, taurine and ester sulfate excretion by the AP piglets were constant during the baseline periods (d 7, 8, 11, 12) with values of 2.51 ⫾ 0.08, 1.69 ⫾ 0.06, 0.67 ⫾ 0.03 and 0.036 ⫾ 0.002 mmol 䡠 kg⫺1 䡠 d⫺1, respectively. Excretion of these metabolites by the LP piglets was also constant during the baseline period (d 11, 12), and less than by the AP piglets (see Table 2). Ester sulfate accounted for only 3.0 ⫾ 0.2% of total S excretion; it was unaffected by diet, sulfate or methionine administration.


769

over the subsequent 24 h, returning to the baseline level after 24 h. Taurine excretion was unaffected. Urinary total S and inorganic sulfate excretion above baseline amounted to 95.9 ⫾ 3.1 and 98.6 ⫾ 2.7%, respectively, of the total sulfate infused; neither was different from 100%. When the same molar dose of methionine was administrated to the AP piglets, 70% of the S load was excreted within 24 h as urinary total S or the sum of inorganic sulfate and taurine (44% as inorganic sulfate and 18% as taurine). Inorganic sulfate, but not taurine excretion, had returned to baseline after 48 h, by which time total S recovery was 106% (not different from 100%). When this dose of methionine was administrated to the LP piglets, urinary total S recovery was 32% after 24 h and 69% after 48 h, both significantly less (P ⬍ 0.001) than in the AP group. Table 3 shows that plasma methionine, taurine and homocysteine concentrations in the AP piglets were not affected by the methionine infusion. Baseline plasma methionine, taurine and homocysteine concentrations in the LP group were significantly reduced (P ⬍ 0.05), and unlike the AP piglets, their plasma methionine and homocysteine concentrations remained substantially increased (P ⬍ 0.05) after 24 h, returning to close to normal by 48 h (Table 3). Before methionine administration, serum urea concentrations and endogenous creatinine clearance of the LP piglets was lower than that of the AP piglets (both P ⬍ 0.05). In both groups N balance was slightly less positive during the 48 h after methionine administration (see Table 4). In the AP group, 48-h S balance and the N/S balance ratio after methionine administration were not different from baseline, but in the LP group, the 48-h S balance became more positive after the methionine infusion (P ⬍ 0.05). Over the 48 h before the methionine infusion, the N/S balance ratio of LP piglets was 30, lower than the value of 36 observed for the AP piglets (P ⬍ 0.001). After the methionine infusion, the 48-h N/S balance ratio in the protein-deprived piglets decreased further, to 26 (P ⫽ 0.001).

FIGURE 1 Weight (mean ⫾ SEM) of six piglets fed adequate protein (AP) and six piglets fed low protein (LP) diets. MgSO4 indicates magnesium sulfate infusion in the AP piglets; MET indicates L-methionine infusion in both groups.

Because the excretion of all S-containing products was constant over the baseline periods, these values were used to predict their basal excretion/kg on the days sulfate and methionine were administered. Sulfate and methionine infusions When 1.2 mmol 䡠 kg⫺1 of magnesium sulfate was administrated to the AP piglets, urinary total S and inorganic sulfate excretion increased from baseline levels of 2.50 ⫾ 0.13 and 1.86 ⫾ 0.19 to 3.65 ⫾ 0.13 and 3.04 ⫾ 0.17 mmol 䡠 kg⫺1 䡠 d⫺1

TABLE 2 Urinary sulfate, taurine and total S excretion over 24 and 48 h after L-methionine administration to piglets fed adequate protein (AP) or low protein (LP) diets1 AP

LP

24 h

48 h

24 h

48 h

␮mol 䡠 kg⫺1 Basal excretion Sulfate Taurine Total S Excretion following methionine infusion Sulfate Taurine Total S Excretion above basal Sulfate Taurine Total S

1735 702 2608

⫾ 120 ⫾ 71 ⫾ 153

3470 1405 5216

⫾ 24 ⫾ 14 ⫾ 306

1099 167 1398

⫾ 17 ⫾ 29 ⫾ 41

2199 333 2797

⫾ 33 ⫾ 58 ⫾ 82

2259 920 3450

⫾ 110 ⫾ 68 ⫾ 130

4051 1953 6462

⫾ 208 ⫾ 169 ⫾ 311

1394 300 1782

⫾ 18 ⫾ 17 ⫾ 37

2730 696 3609

⫾ 33 ⫾ 38 ⫾ 55

524 218 842

⫾ 43 ⫾ 46 ⫾ 88

585 567 1269

⫾ 48 ⫾ 75 ⫾ 32

295 134 383

⫾ 15 ⫾ 16 ⫾ 30

541 373 831

⫾ 35 ⫾ 37 ⫾ 57

% of infused dose Recovery Sulfate Taurine Total S

43.6 ⫾ 18.0 ⫾ 70.1 ⫾

3.6 3.8 7.3

48.7 ⫾ 48.8 ⫾ 105.5 ⫾

4.0 6.6† 2.9†

23.9 ⫾ 0.9* 11.1 ⫾ 1.4 32.0 ⫾ 2.5*

45.4 ⫾ 3.3† 31.0 ⫾ 3.1*† 69.2 ⫾ 4.8*†

1 L-Methionine (1200 ␮mol 䡠 kg⫺1) was infused at the beginning of the first day. Basal excretion refers to excretion over the 2 days prior to methionine infusion. 48 h excretion refers to the entire 48-h period. Values are means ⫾ SEM, n ⫽ 6. * Significantly different from AP group, P ⬍ 0.05; † significantly different from 24 h, P ⬍ 0.05.

HOU ET AL.

770

TABLE 3 Circulating metabolite concentrations and endogenous creatinine clearance before and after L-methionine administration to piglets fed adequate protein (AP) or low protein (LP) diets1

AP diet Plasma methionine, ␮mol 䡠 L⫺1 Plasma taurine, ␮mol 䡠 L⫺1 Plasma homocysteine, ␮mol 䡠 L⫺1 Serum urea, mmol 䡠 L⫺1 Creatinine clearance, mL 䡠 min⫺1 䡠 kg⫺1 LP diet Plasma methionine, ␮mol 䡠 L⫺1 Plasma taurine, ␮mol 䡠 L⫺1 Plasma homocysteine, ␮mol 䡠 L⫺1 Serum urea, mmol 䡠 L⫺1 Creatinine clearance, mL 䡠 min⫺1 䡠 kg⫺1

0h

24 h

48 h

119 ⫾ 14 137 ⫾ 19 29 ⫾ 3 2.22 ⫾ 0.28 5.7 ⫾ 0.6

112 ⫾ 19 139 ⫾ 22 37 ⫾ 6 2.58 ⫾ 0.41 4.4 ⫾ 0.3*

112 ⫾ 13 140 ⫾ 19 39 ⫾ 8 3.08 ⫾ 0.50* 4.2 ⫾ 0.4*

61 ⫾ 7† 47 ⫾ 3† 9 ⫾ 1† 0.30 ⫾ 0.04† 3.6 ⫾ 0.2†

251 ⫾ 36* 63 ⫾ 3* 23 ⫾ 5* 0.35 ⫾ 0.07† 3.5 ⫾ 0.2

84 ⫾ 6 68 ⫾ 7* 12 ⫾ 1 0.50 ⫾ 0.16† 4.4 ⫾ 0.2

1 Value are means ⫾ SEM, n ⫽ 6. * Significantly different from 0 h, P ⬍ 0.05; † significantly different from AP group, P ⬍ 0.05.

DISCUSSION We have established a model in growing piglets to study SAA metabolism by using urinary S excretion as an indicator of whole body SAA catabolism. We confirmed that urinary sulfate is a valid indicator of sulfate production in this setting by administering sulfate intravenously in a physiologic dose that corresponded to the amount of S in the methionine used subsequently. Cumulative urinary total S and sulfate recovery were 96 and 99% in the 24 h after the magnesium sulfate infusion, respectively. This agrees with the 90– 100% recovery in urine as inorganic sulfate over 24 h after intravenous administration of a large dose of sodium sulfate (31), and 98% recovery as inorganic sulfate in urine and extracellular water over 9 h after an intravenous administration of magnesium sulfate in adult humans (9). We then determined what fraction of the S in an intravenous dose of methionine can be accounted for as increased urinary excretion of S end products 24 and 48 h after its administration in wellnourished (AP) and protein-depleted (LP) piglets. Piglets were fed continuously at a rate adjusted every morning according to their body weight, and hence received the same amount of diet per kg body weight every day. It was therefore expected that

baseline S excretion/kg body weight would be constant, as indeed occurred. The growth rate of the AP piglets was similar to that of sow-fed piglets (25), whereas that of the LP piglets was slightly lower; however, the composition of the tissue gain was dramatically different. Assuming that 1 g positive N balance is equivalent to the gain of 6.25 g protein and 33 g lean tissue (32,33), it can be calculated from the data in Table 4 that 79% of the weight gain of the AP piglets was lean tissue and 14.9% was protein, similar to the 14.5% protein gain previously observed in parenterally fed piglets (25). By contrast, only 36% of the weight gain of the LP piglets was lean tissue. Their only slightly slower weight gain can be explained by the fact that they were supplied generously with energy. This, in the context of their lower lean tissue mass (and consequently lower metabolic rate), resulted in a weight gain attributed to fat that largely offset their slower lean tissue gain. In the well-nourished state with surfeit SAA intake, the pattern of urinary total S, inorganic sulfate and taurine excretion after intravenous methionine indicated that the entire methionine load was catabolized and excreted in the urine as

TABLE 4 S and N balance and N/S molar balance ratio 48 h before and after L-methionine administration to piglets fed adequate protein (AP) or low protein (LP) diets1 AP Group study period N metabolism, mmol 䡠 kg⫺1 N intake N output N balance N retention, % S metabolism, mmol 䡠 kg⫺1 S intake SAA intake S output S balance SAA retention, % N/S balance ratio2

Before methionine

315.2 44.2 271.0 86.0

⫾ 1.0 ⫾ 4.7 ⫾ 5.0 ⫾ 1.5

12.87 ⫾ 0.06 10.96 ⫾ 0.05 5.22 ⫾ 0.31 7.65 ⫾ 0.28 69.8 ⫾ 2.7 35.6 ⫾ 1.1

LP After methionine

317.5 53.5 264.4 83.2

⫾ 0.6 ⫾ 6.1 ⫾ 6.0* ⫾ 1.9*

14.13 ⫾ 0.03 12.22 ⫾ 0.02 6.46 ⫾ 0.31 7.67 ⫾ 0.32 62.7 ⫾ 2.6* 34.7 ⫾ 1.3

Before methionine

121.3 8.7 112.7 92.9

⫾ 0.3 ⫾ 0.8 ⫾ 1.0† ⫾ 0.6†

6.60 ⫾ 0.02 4.69 ⫾ 0.01 2.80 ⫾ 0.08 3.81 ⫾ 0.09† 81.1 ⫾ 1.8† 29.7 ⫾ 0.5†

After methionine

121.2 12.3 108.9 89.9

⫾ 0.7 ⫾ 1.0 ⫾ 1.2*† ⫾ 0.8*†

7.73 ⫾ 0.05 5.82 ⫾ 0.05 3.61 ⫾ 0.06 4.12 ⫾ 0.06*† 70.8 ⫾ 0.8*† 26.4 ⫾ 0.1*†

1 Values are means ⫾ SEM, n ⫽ 6. * Significantly different from before methionine, P ⬍ 0.05; † significantly different from AP group, P ⬍ 0.05. 2 The N/S ratio is the N balance divided by S balance (see Methods).


S end products within 48 h, half as inorganic sulfate (which was almost entirely excreted within the first 24 h), and the other half as taurine (which was largely excreted in the second 24 h). Taurine is actively transported into cells (mainly into muscle), where it has a large and slowly exchanged body pool (11,34,35). We therefore presume that most of the recovered taurine was formed in the first 24 h after the methionine infusion, although some of it was retained in the muscle taurine pool and released over the subsequent 24 h. This is the first report in piglets that by 48 h after a methionine load all of the dose is fully catabolized and excreted as urinary S end products. It agrees with the 95% recovery of L-cysteine in urine as inorganic sulfate and taurine over a few days after its administration to well-nourished rats (36). These observations in well-nourished piglets validate the concept of using urinary S excretion as an indicator of SAA catabolism in this setting. The same method was used to determine the fate of a methionine load during mild protein depletion. When methionine was administered to the LP piglets, urinary total S recovery was 32% over 24 h and 69% over 48 h, both significantly lower than in the well-nourished state. Also unlike the AP group, plasma methionine, homocysteine and taurine concentrations of the LP piglets remained substantially above their baseline level 24 h after the methionine infusion. Free methionine could account for ⬃12% of the infused methionine dose, if it is assumed that intracellular and extracellular methionine concentrations are approximately equal (37) and total body water is 75% of body weight (25). Even after accounting for this, 56% of the administered methionine S was unaccounted for (and hence retained in some form in the body) 24 h after its administration. By 48 h, plasma methionine and homocysteine had returned to close to their baseline concentrations. Although 12% of the infused methionine was catabolized in the second 24 h, and some part of it may have been catabolized to taurine and retained in the muscle taurine pool, 31% of the methionine load remained unaccounted for 48 h after its administration. This S retention was not due to increased methionine uptake for new protein synthesis because there was no increase in N balance, even as the S balance increased considerably. This indicates that the methionine S must have been retained in a nonprotein form such as GSH or taurine. The N/S molar balance ratio provides insight into this phenomenon. The basal N/S balance ratio in the AP piglets was 36, and remained at this level after methionine administration. This is close to the N/S molar ratio of 38 reported for pork protein (38,39), and suggests that these piglets accumulated SAA normally in their lean tissues, and did not increase their nonprotein S store to a higher level than normal after the methionine load. This is consistent with reports that excess SAA provision does not increase the liver GSH concentration above the level obtained with adequate protein feeding (16,40– 42). The N/S balance ratio was quite different in the LP piglets. Before the methionine infusion, their basal N and S balances were considerably less positive than those of the AP piglets and their N and SAA retention rates were higher, consistent with the increased efficiency of dietary amino acid utilization characteristic of the adaptation to protein restriction (8). Their N/S balance ratio of 30 was significantly lower than in the AP piglets. We suggest this was attributable to a markedly reduced muscle protein accretion rate in the context of a less severely slowed accretion of splanchnic tissues rich in GSH (N/S ratio 3). After methionine administration, the 48-h N/S balance ratio of the LP piglets decreased even further, to 26. Protein-deficient diets decrease hepatic GSH by 20– 50% in rats (41– 44), and decrease intestinal mucosal GSH by ⬎ 50% in

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growing piglets (45). The observed postmethionine decrease in the N/S balance ratio in these piglets is therefore consistent with the retention of a part of the cysteine derived from it to increase the tissue GSH pool, as has been reported when either methionine or cysteine is administered to protein-restricted rats (41,46). The proportion of the S in the administered methionine retained as GSH vs. taurine in the LP piglets cannot be determined from the present data. Studies to test this prediction by measuring tissue GSH and taurine concentrations are planned. Taurine makes a minor contribution to urinary S excretion in humans, (13,47– 49), but the present results clearly show that piglets catabolize a large fraction of administered methionine to taurine. Taurine is abundant in pig milk (50), and for that reason is commonly added to piglet elemental diets as a semi-indispensable amino acid (51). However, urinary taurine excretion was 125% of dietary taurine in the AP piglets, suggesting that when methionine and cysteine intake are adequate, newborn piglets do not require taurine supplementation. In human neonates and adults, ester sulfate accounts for 17 and 6 – 10% of urinary total S (27,48,52,53), respectively, but it accounted for 3% at most of urinary total S excretion in piglets in this study, and it was not increased by methionine administration. Because ester sulfate excretion was constant and made only a very minor contribution to their total S excretion, it appears to be unimportant in SAA catabolism in elemental-diet– fed piglets. These results indicate that, despite many similarities in amino acid metabolism, piglets and humans differ in certain respects with regard to SAA. Unlike humans, piglets convert cysteine to GSH and taurine, both of which may represent a form of nonprotein S storage. A S balance determination in piglets therefore requires measuring either total S or the sum of sulfate and taurine. In humans, the measurement of sulfate alone may suffice, unless taurine is administered in quantitatively important amounts. It is interesting that before the methionine load, plasma methionine and homocysteine concentrations were substantially reduced in the LP group (P ⬍ 0.05), whereas 24 h after the methionine load plasma methionine and homocysteine concentrations were still well above baseline. This exaggerated response to a methionine load suggests that protein-depleted piglets downregulate enzyme activity in the transsulfuration pathway. In humans, higher protein or methionine intakes either do not affect, or even decrease, fasting plasma homocysteine (54,55). This is presumed to occur because a high amino acid intake increases glomerular filtration (56,57), which in turn increases renal homocysteine catabolism (58). In this study, the LP diet was associated with a slightly lower endogenous creatinine clearance but a substantial reduction in plasma homocysteine. It appears that the reduced methionine provision in the LP diet reduced de novo homocysteine synthesis and its plasma concentration, despite a possible small reduction in the glomerular filtration rate. In summary, this report describes a growing piglet model for studying SAA metabolism. We validated the method of using urinary S excretion as an accurate indicator of whole body SAA catabolism. We found that piglets, unlike humans, metabolize a substantial proportion of SAA to taurine. We showed that in the protein-depleted state, a significant fraction of the S in a dose of intravenous methionine is stored in the body in nonprotein forms, presumably as GSH and taurine. The noninvasive, nontracer approach described here can potentially provide valuable information about whole body SAA metabolism and nonprotein S storage in animals and humans.

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ACKNOWLEDGMENTS We thank Farhad Saboohi for technical assistance, and Mazen J. Hamadeh and Mark Warren for helpful scientific discussions.

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