Sulfate production depicts fed-state adaptation to protein restriction in humans

Mazen J. Hamadeh, Alicia Schiffrin, and L. John Hoffer

Lady Davis Institute for Medical Research, and Division of Endocrinology, Jewish General Hospital, Montreal, H3T 1E2; and School of Dietetics and Human Nutrition, McGill University, Sainte Anne de Bellevue, Quebec, Canada H9X 3V9


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

One feature of the adaptation to dietary protein restriction is reduced urea production over the hours after consumption of a test meal of fixed composition. This adaptation is impaired in conventionally treated insulin-dependent diabetes mellitus (Hoffer LJ, Taveroff A, and Schiffrin A. Am J Physiol Endocrinol Metab 272: E59-E67, 1997). We have now tested the response to a test meal containing less protein and included as a main outcome variable the production of sulfate, a specific indicator of sulfur amino acid catabolism. Six normal men consumed a mixed test meal containing 0.25 g protein/kg and 10 kcal/kg while adapted to high (1.5 g · kg-1 · day-1) and low (0.3 g · kg-1 · day-1) protein intakes. They followed the identical protocol twice. Six subjects with insulin-dependent diabetes consumed the test meal while adapted to their customary high-protein diet. Adaptation to protein restriction reproducibly reduced 9-h cumulative postmeal urea N and S production by 22-29% and 49-52%, respectively (both P < 0.05). Similar results were obtained for a postmeal collection period of 6 h. The response of the diabetic subjects was normal. We conclude that reductions in postmeal urea and sulfate production after protein restriction are reproducible and are evident using a postmeal collection period as short as 6 h. Sulfate production effectively depicts fed-state adaptation to protein restriction.

urea; stable isotope; fed state; amino acid oxidation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

UREA PRODUCTION HAS OFTEN BEEN USED to measure short-term changes in amino acid catabolism (1, 28, 33, 34, 43, 51). We demonstrated that dietary protein restriction induces an adaptive reduction in postmeal urea production and showed this response to be impaired with conventional insulin therapy of insulin-dependent diabetes mellitus (IDDM) (24). This suggests that conventional insulin therapy of IDDM could increase the minimum protein requirement (22).

Despite its common use in clinical studies, the measurement of short-term urea production is subject to important confounding factors. Thus, even after correction for changes in the urea content in total body water (TBW), urinary urea excretion underestimates its true production by 15-30%, mainly due to urea hydrolysis (with partial recycling) in the gut (16, 32, 48, 51). As well, small analytical errors in the serum urea measurement are magnified when multiplied by TBW, a large volume. Tracer-determined urea rate of appearance has been used as an alternative (6, 12, 13, 50), but this underestimates short-term changes in urea production typical of the fed state (19).

In this study, we carefully examined the normal adaptive response to 3 days of protein restriction by measuring the reproducibility of cumulative urea and inorganic sulfate production after the consumption of a standard test meal. Sulfate is the predominant product of sulfur amino acid (SAA) catabolism and represents an attractive alternative to urea, because unlike urea, it distributes in a smaller body compartment (extracellular water), it is not subject to losses in the gut, and it is excreted almost entirely through the renal route (2, 7, 39, 42). Sulfate production can be measured accurately over several hours from its urinary excretion with a correction for changes in extracellular fluid (ECF) pool size (20).

We used a 0.5 g protein/kg test meal in our previous examinations of the effect of prior protein intake on the efficiency of fed-state protein retention (24, 45). However, a test meal containing less protein should be a more sensitive tool, because optimal retention of the amino acids in a low-protein meal calls for greater metabolic efficiency. The test meal in the present study contained 0.25 g protein/kg body wt. It was offered to normal research subjects before and after partial adaptation to protein restriction. Each subject underwent the protocol twice, the test meals in the replicate protocols differing only in that a tracer dose of [15N]alanine was included in the test meals for three subjects the first time they followed the protocol and a tracer dose of intrinsically labeled 15N-Spirulina platensis the second time, with the order reversed for the other three subjects. The effects of the different 15N-labeled amino acid tracers are described in a companion article (21). Finally, these measurements were made in healthy persons with IDDM receiving conventional insulin therapy while adapted to their customary, high protein intake, and the results were compared with those of the normal subjects.

Thus we tested, first, whether postmeal urea production before and after protein restriction is reproducible upon repeat testing; second, we explored the usefulness and reproducibility of sulfate production as an alternative or adjunct measure of postmeal amino acid catabolism; and finally, we compared postmeal urea N and sulfate production of normal and IDDM subjects after consumption of a test meal limited in its protein content.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects and protocols. Six healthy, nonsmoking men using no medications and with normal blood chemistries were admitted at 0700 to the clinical research unit, where they consumed a conventional, high-protein diet over that day (Table 1). All urine was collected for the duration of the protocol by means of serial 24-h collections beginning at 0700. The following morning (day 2) was the first test meal study, after which the subjects followed a low-protein diet for the rest of the day. Protein restriction continued on days 3 and 4. On the morning of day 5, the test meal procedure was repeated. The subjects returned 10 days later to repeat the same protocol, the replicate protocols differing only in that a tracer dose of [15N]alanine was included in the test meals for three subjects on the first occasion (protocol A), and a tracer dose of fully 15N-labeled whole protein, [15N]Spirulina platensis, was included on the second one (protocol S). The protocol order was reversed for the other three subjects. Four healthy men and two healthy women with IDDM presented to the research unit at 0700 for a study that they completed the same day. Their blood hemoglobin AIc concentrations were 6.2 ± 0.7% (mean ± SD; normal range 3.5-5.5%) and serum insulin C-peptide concentrations were 0.24 ± 0.04 nmol/l (normal range 0.46-0.72 nmol/l). Their diet was the typical Canadian diet, which provides ~1.5 g protein · kg-1 · day-1 (18); they did not change their dietary habit in the days before the test meal. To achieve a fasting blood glucose of 10-12 mmol/l typical of conventionally treated diabetes mellitus (10), they administered their usual short-acting insulin dose before the preceding evening meal and two-thirds of their usual intermediate-acting insulin dose. The next morning, they injected one-half of the estimated ideal short-acting and two-thirds of the estimated ideal intermediate-acting insulin dose 30 min before consuming the test meal. Fasting and postmeal capillary blood glucose concentrations were measured using the Accu-Chek III blood glucose monitor (Boehringer Mannheim, Laval, QC, Canada). All research volunteers gave their written consent to participate in the study, which was approved by the Research Ethics Committee of the Jewish General Hospital in Montreal.

                              
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Table 1.   Subject characteristics

Adaptation diet. Daily energy intake was 38 kcal/kg body wt, on the assumptions that resting energy expenditure was 24 kcal/kg (17) and that the energy cost of sedentary activity and diet-induced thermogenesis is 14 kcal/kg. On day 1, a high-protein breakfast (0830), lunch (1230), and dinner (1730) were consumed, providing 1.5 g/kg body wt (Table 2). On day 2, after completion of the test meal procedure, two low-protein meals were consumed, one at 1730 and and the other at 2130, for a total protein intake that day of 0.39 g/kg, while meeting the full energy requirement. This day was therefore considered the first day of protein restriction. On days 3 and 4, the diet provided 0.3 g protein/kg, and on day 5 (second test day), only the test meal was consumed. The food consisted of low-protein bread and wafers and standard low-protein foods (juice, butter, jam, mashed potatoes, green beans, carrots, tea, coffee, ginger ale, and sugar). A maximum of one cup of coffee (with coffee whitener) and two cups of tea were allowed per day; other foods and beverages were not permitted. While on the low-protein diet, subjects consumed one multiple vitamin-mineral tablet (Centrum Forte, Whitehall-Robins, Mississauga, ON, Canada) each day.

                              
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Table 2.   Diet composition

Test meal. The test meal was Glucerna (Ross Laboratories, St. Laurent, QC, Canada), to which beet sugar (Rogers Sugar, Winnipeg, Manitoba, Canada) was added to provide 0.25 g of protein and 10 kcal of energy/kg body wt (10% protein, 30% fat, and 60% carbohydrate). As in our previous research (24), 200 mg [13C]urea (99%; MSD Isotopes, Montreal, QC, Canada) were added to each test meal with the aim of using the total recovery of this tracer in serum and urine as a measure of extraurinary urea losses. A preliminary study indicated that, unlike cane sugar, beet sugar consumption does not increase breath 13CO2 enrichment. The protein N and amino acid S contents of Glucerna were 6.6 and 0.31 mg/ml, respectively, on the basis of data supplied by Ross Laboratories.

Test meal procedure. On the test days (days 2 and 5), urine was collected in 3 pools: 1) before test meal consumption, 2) over the first 6 h after test meal consumption, and 3) over the last 3 h of the study. NaBr (30 mg/kg body wt) was taken by mouth at hour 6 to determine the corrected bromide space (CBS), a measure of the ECF (36, 46). Blood samples were drawn hourly from an arterialized arm vein kept patent with 77 mmol/l NaCl infused at 80 ml/h. The normal subjects drank 207 ± 23 ml water/h (mean ± SD) and had a urine flow of 279 ± 36 ml/h during the study. The IDDM subjects drank 355 ± 37 ml/h; their urine flow was 432 ± 32 ml/h. Body weight (which was also measured daily) and TBW (by bioimpedance analysis; RJL Systems BIA-101A, Mt. Clemens, MI) (29) were measured just before and after completion of the 9-h study.

Analytical methods. Clotted arterialized venous blood was centrifuged at 1,400 g for 30 min at room temperature, and the serum was stored at -30°C until analysis. Urea was analyzed using a Hitachi 917 automated analyzer (Laval, QC, Canada) with reagents from Roche-Boehringer Mannheim (Laval, QC, Canada). Although capillary blood glucose levels were measured on site with the Accu-Chek III blood glucose monitor, serum was subsequently analyzed for glucose using a Hitachi 917 automated analyzer. Capillary blood glucose levels, as measured with the Accu-Chek III blood glucose monitor, strongly correlated with serum glucose (r = 0.98, slope = 0.90, P < 0.0001).

Serum and urine were analyzed for inorganic sulfate and bromide by ion exchange chromatography with conductivity detection (IEC-CD; Dionex 2110i; Dionex, Sunnyvale, CA), as previously reported (5). [13C]urea enrichment was measured by gas chromatography-mass spectrometry (GC-MS), as previously described (19). Serum C-peptide was analyzed by radioimmunoassay in the laboratory of Dr. John Dupré, University of Western Ontario (London, ON, Canada).

Calculations. Urea production was calculated as its urinary excretion corrected for changes in TBW content. Sulfate production was calculated as inorganic sulfate urinary excretion, corrected for changes in ECF content (20). ECF was estimated from the CBS as described by Bell et al. (4). Net protein utilization (NPU) or SAA retention was calculated as
<FR><NU>(total N or S intake<IT>−</IT>urea N or sulfate produced)<IT>×</IT>100</NU><DE>total N or S intake</DE></FR>
Because [M+1]urea arises both from the [13C]urea tracer provided in the test meal and from [15N]urea synthesized in the body, [M+1]urea specifically due to [13C]urea was calculated by subtracting the contribution due to [15N]urea, as determined by isotope ratio-mass spectrometry (IRMS) from the total GC-MS-determined [M+1]urea enrichment in serum and urine. Details about the IRMS analysis are given in the companion article (21). We determined that the [13C]urea tracer, although indeed 99% 13C, was also 8% [18O,13C]urea (and hence of mass M+3). This was accounted for in the mole ratio standard curve.

Statistical analysis. Three-way repeated-measures ANOVA was used to determine significant differences in serum sulfate and urea concentrations for the normal group, the factors being diet (high vs. low protein), protocol (A vs. S), and time. Two-way repeated-measures ANOVA was used to determine significant differences in body weight and 24-h urea and sulfate excretion, the factors being protocol and day. Two-way repeated-measures ANOVA was used to determine significant differences in postmeal urea N and sulfate metabolism, postmeal N/S ratio, serum postabsorptive urea and sulfate concentrations, and [13C]urea recovery, the two factors being diet and protocol. Within the same protocol and diet, serum urea and sulfate concentrations over time were analyzed by one-way repeated-measures ANOVA. When significance occurred, a Newman-Keuls test was used post hoc to determine the source of difference. Student's unpaired t-test and two-way repeated-measures ANOVA were used to determine significant differences between the normal and IDDM groups, also with the Newman-Keuls test to determine the source of difference. Differences were considered significant at P <=  0.05. Results are presented as means ± SE unless otherwise indicated.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All subjects tolerated both study protocols without difficulty and without any change in body weight. A pronounced and closely reproducible reduction in daily urinary urea and sulfate excretion was evident by the 1st day of protein restriction (Fig. 1). Figure 2 illustrates the close relationship between urinary urea and sulfate excretion over the 3 days of protein restriction.


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Fig. 1.   Daily urea and sulfate excretion and N-to-S molar ratio of 6 men before (day 1) and during (days 2-4) protein restriction. Filled and open bars indicate values from replicate executions of the protocol (filled bars, protocol A; open bars, protocol S). Different letters indicate significant differences (P < 0.05).



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Fig. 2.   Relationship between 24-h urinary urea N and sulfate excretion (r = 0.95, P < 0.0001). Sulfate excretion = (0.024 ± 0.001) × urea N excretion + (1.68 ± 0.701). The inverse of the slope is the molar N/S ratio, 41.

Pre- and postmeal serum urea and sulfate concentrations and their urinary excretion were measured before and after protein restriction. Protein restriction reduced postabsorptive serum urea concentrations by 60-62%. When the subjects were adapted to a high protein intake, the test meal decreased their serum urea concentrations, but when the same test meal was consumed after protein restriction, serum urea concentrations increased (Fig. 3). Postmeal urea N production was 22-29% lower after protein restriction, when measured over either 6 or 9 h after the meal (P < 0.05; Table 3). There was an associated rise in NPU. These results are indicated in Table 3.


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Fig. 3.   Serum urea and sulfate concentrations (mmol/l) before and after test meal consumption, with the test meal consumed immediately after the zero time measurement. The protocol was executed twice. Left: protocol A; right: protocol S. (), concentrations before protein restriction; (), concentrations after protein restriction.


                              
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Table 3.   Postmeal urea and sulfate metabolism

Protein restriction reproducibly reduced postabsorptive and postmeal serum sulfate concentrations (P < 0.05; Fig. 3). Unlike with urea, serum sulfate remained constant after the test meals. Postmeal sulfate excretion was reduced by ~50% after protein restriction (P < 0.05), with the result that SAA retention, which was negative when the diet was high in protein, was positive after protein restriction. The direction, magnitude, and variability of these responses were similar whether the observation period was over 6 or 9 h after the test meal (Table 3). Protein restriction significantly increased postmeal N/S production, implying a higher proportional retention of SAA than total amino acids over the 6 and 9 h after the test meal.

Six persons with conventionally treated IDDM consumed the test meal while adapted to their customary, high-protein diet. Before the test meal was consumed, serum glucose concentrations were 9.8 ± 0.9 mmol/l, rising to a maximum of 14.9 ± 2.0 mmol/l 2 h after the test meal (P < 0.05; postabsorptive serum glucose was 5.3 ± 0.1 mmol/l in the nondiabetic subjects and did not change after the test meal). Postabsorptive serum urea (4.1 ± 0.7 mmol/l) and sulfate (316 ± 36 µmol/l) concentrations and their postmeal time profiles were similar to those of the normal subjects (data not shown). Urea N and S metabolism over the 6 and 9 h after the test meal are shown in Table 3. Despite greater variability, there was no indication of excessive fed-state N or sulfate production.

A trace amount (2.98 mmol) of [13C]urea was added to each test meal with the aim of using its recovery in the serum and urine to indicate the effects of diet or diabetes mellitus on unmeasured extraurinary urea losses or altered distribution of urea between excretion and retention in body water. When [15N]alanine was in the test meal (protocol A), postmeal recovery of the urea tracer was 97 ± 6 and 99 ± 10% for the normal subjects (high and low previous protein intakes, respectively) and 108 ± 3% for the IDDM subjects. Recovery was significantly greater (P < 0.05) when the 15N-labeled amino acid tracer in the meal was the intrinsically labeled protein (protocol S; 111 ± 6 and 121 ± 6% for high and low previous protein intakes, respectively). [13C]urea tracer recoveries did not differ significantly with regard to the adaptation diet or between the normal and IDDM subjects.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We determined several parameters related to the use of urea and sulfate production as measures of amino acid catabolism in the fed state. Urea and sulfate production were measured before and after 3 days of protein restriction by use of a dietary protocol that was completed two times, once when the test meals contained a trace amount of [15N]alanine (protocol A) and once when it contained a trace amount of [15N]Spirulina platensis (protocol S). The resulting patterns of 15N-labeled amino acid and [15N]urea enrichment are described in the accompanying article (21). The present results support the following conclusions. 1) Postmeal urea and sulfate production were closely reproducible. 2) When measured using a relatively low protein (0.25 g/kg) test meal, adaptation to protein restriction reduced postmeal urea production by ~25% and sulfate production by a considerably greater ~50%, a conclusion that is unaltered whether the observation period is over 6 or 9 h from the time the meal was consumed. 3) Postmeal urea and sulfate production was measured in persons with conventionally treated IDDM who consumed the test meal while adapted to their customary, high-protein diet. Although their response was more variable, there was no indication that the IDDM subjects had higher postmeal urea or sulfate productions than normal.

An important question is whether 3 days of protein restriction are adequate for full metabolic adaptation to a change in the dietary protein level. The answer is no. It is customary, in protein metabolic studies, to allow a minimum of 4 days for full metabolic adjustment to a dietary change (14, 23, 41). In a previous study, which demonstrated increased urinary obligatory N excretion in intensively treated IDDM, we allowed 10 days (31). Notwithstanding this caveat, adaptation is substantially underway within 3 days of a change in protein intake (37). Figure 1 shows that, even by the 2nd day of protein restriction, daily urea and sulfate excretion had fallen by ~70% to a low and nearly constant level. Lakshmanan et al. (30) observed a similarly prompt ~85% reduction in urinary sulfate excretion upon consumption of an SAA-free diet. Thus, although 3 days of low protein intake are insufficient for complete adaptation, the adaptation that occurs is large and reproducible.

We found postmeal urea production to be reproducible despite the potential for problems with this measurement. One problem is the need to adjust urinary excretion for any change in the body's large urea pool. In the present study, serum urea concentrations fell after consumption of the test meal when the previous protein intake had been high, a phenomenon also observed by Owen et al. (40). We attribute this to the relatively low protein content of the test meal in the face of continuing turnover of a large urea pool. Postmeal serum urea rose when the test meal followed protein restriction, for the protein in the test meal was now more than twice what the subjects consumed for breakfast on their protein-restricted diet and, hence, sufficient to increase the size of their diminished body urea pool.

Another problem is urea hydrolysis in the gut (16, 32, 47, 51). Our calculation of urea production ignored this and therefore underestimated actual urea synthesis. We added a trace amount of [13C]urea to each test meal with the intention of using the recovery of this isotopic tracer as a whole body "internal standard" that could reveal effects of diet or IDDM on extraurinary urea losses. [13C]urea concentrations in serum and urine were calculated as the product of total urea concentration and the fraction of it that was mass M+1, while subtracting the contribution to this mass of the [15N]urea synthesized in the body from the 15N-labeled amino acid tracer in the test meals (see METHODS). Recovery of the [13C]urea tracer in serum and urine was ~98% for protocol A (in which [15N]alanine was added to the test meal) whether the adaptation diet was high or low in protein, and ~116% for Protocol S (in which [15N]Spirulina platensis was added to the test meal). Both recoveries are substantially greater than the ~80% that was anticipated with a nonrecycling urea tracer (32). More [15N]urea was produced in protocol S, which was associated with a much greater apparent [13C]urea recovery. This suggests that our mathematical correction for the [15N]urea contribution to total [M+1]urea was inadequate, probably owing to the inequality of enrichment values measured by GC-MS (for total [M+1]urea) and IRMS (for [15N]urea). In principle, the technique of using the recovery of a nonrecycling urea tracer to correct for hydrolysis and other nonurinary losses should be sound. We are undertaking experiments to elucidate the reasons for these unexpectedly high apparent [13C]urea recoveries.

Despite these concerns, the physiologically important observation is that there was no significant difference in recovery of the urea tracer added to the test meal after high- or low-protein diets or between the normal and IDDM subjects. This permits the conclusion that postmeal changes in urea production ascribed to protein restriction or IDDM were not confounded by alterations in nonrenal urea elimination or renal clearance. In a previous study using this method with [15N]alanine, we reported [13C]urea recoveries of 71-75% (24). When reviewing those results in light of the present findings, we discovered a calculation error in the earlier data. The correct values are 91 ± 4% (first test meal) and 96 ± 2% (second test meal) for the normal subjects and 92 ± 3% (first test meal) and 94 ± 3% (second test meal) for the subjects with diabetes mellitus.

Sulfate production showed a pattern generally similar to that of urea. When measured over 24 h, urea excretion and sulfate excretion were closely related (Fig. 2), in agreement with previous comparisons of urinary sulfate with urinary urea N (27) or total N excretion (27, 30). The slope of the regression line indicates a molar N/S ratio of ~40, consistent with the N/S ratio in mixed-body proteins and in high-quality dietary proteins (3, 8, 49).

There are theoretical advantages to using sulfate production to study the factors governing metabolic adaptation and short-term amino acid catabolism (8). First, from the strictly technical point of view, the sulfate production measurement must be more reliable than urea production in short-term metabolic studies. Unlike urea, which distributes throughout TBW, sulfate distributes in the considerably smaller ECF; moreover, unlike urea, it is not subject to important losses in the gut (20). An additional technical advantage in the present study was that, unlike urea, serum concentrations remained constant in the postprandial state, entirely obviating the need for a body pool correction with its attendant potential for error (Fig. 3). An important feature that distinguishes SAA from total amino acid catabolism is the considerable storage of cysteine in a nonprotein reservoir, glutathione (GSH), which occurs under certain circumstances (8, 9, 15, 44). To the extent that this occurs, sulfate production will not simply indicate whole body net proteolysis; rather, in conjunction with an appropriate measure of whole body urea production, it could provide additional insight into the mechanisms governing metabolic adaptation (20).

This is illustrated in Table 3, which shows that adaptation to protein restriction was associated with a ~25% reduction in postmeal urea production but a far greater, ~50% reduction in postmeal sulfate production and a corresponding, marked increase in the postmeal N/S production ratio. This simple observation suggests that an important feature of the adaptation to protein restriction is specific replenishment of the nonprotein SAA pool (presumably GSH), in addition to new protein synthesis, after consumption of a protein-containing meal.

Postmeal urea and sulfate production by the IDDM subjects was normal. This supports our earlier conclusion that IDDM subjects previously adapted to a high protein intake have normal postmeal urea production (24). In fact, we predicted that our low-protein test meal might unmask a state of inefficient amino acid conservation created by the mildly insulin-deficient state characteristic of conventionally treated IDDM but concealed by the high protein content of the customary diet (22). The customary daily protein intake of ~1.5 g/kg corresponds roughly to three 0.5 g/kg protein meals, about twice what was in the test meal. A person adapted to such a high protein intake would be expected to catabolize an excessive amount of the protein in the first of a series of low-protein meals until adaptive mechanisms come into play to reduce amino acid catabolism to a lower level compatible with zero N balance (23).

This is illustrated in Table 4, which incorporates data from an earlier study using a 0.5 g protein/kg test meal (24). When a conventional amount of protein was consumed by persons adapted to a customarily high protein intake, their metabolic N balance and NPU were positive, as would be anticipated under such conditions. When similarly conditioned subjects were presented with a test meal that contained one-half the amount of protein they were habituated to (present study), their postmeal metabolic N balance and NPU were negative. We predicted that IDDM subjects would have an even more profoundly negative N (or S) balance on account of their relative insulin deficiency, but despite basal hyperglycemia and a postmeal rise in plasma glucose consistent with conventional insulin therapy (11), this proved not to be the case.

                              
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Table 4.   Effects of protein content of a test meal and previous protein intake on postmeal N metabolism

We further predicted that postmeal sulfate production would be even more markedly abnormal in IDDM. Insulin withdrawal in IDDM increases both splanchnic proteolysis and splanchnic protein synthesis, the latter stimulated in part by an influx of amino acids resulting from increased muscle proteolysis (38). Such increased splanchnic amino acid turnover should yield abundant cysteine for GSH synthesis, and indeed, unlike the fasting (26) or protein-deficient rat (25), the IDDM rat has a normal hepatic GSH store (35). As a consequence of their mildly increased visceral protein turnover, persons with imperfectly regulated IDDM may have better filled GSH stores before consuming a protein meal and, hence, promptly convert a greater fraction of incoming dietary SAA to sulfate. We consider in the accompanying article (21), in the context of the 15N tracer results, possible reasons why this result was not obtained.

In summary, normal adaptation to dietary protein restriction includes a pronounced reduction in postmeal urea and sulfate production that is highly reproducible on repeat testing. A protein content of 0.25 g/kg in the test meal usefully detects this adaptation and, in principle, could prove to be more sensitive than a test meal with surfeit protein at detecting states of inefficient dietary amino acid conservation. Sulfate production effectively depicts fed-state adaptation to protein restriction and has theoretical advantages (and some disadvantages) compared with the traditional urea production measurement. Postmeal adaptive changes in urea and sulfate production can be depicted accurately over observation periods as short as 6 h. When adapted to a high protein intake, persons with conventionally treated IDDM manifest normal post-test-meal urea and sulfate productions.


    ACKNOWLEDGEMENTS

We thank Line Robitaille for technical assistance and Chantal Bellerose for dietetic expertise and assistance. We thank Margaret Behme and Dr. John Dupré for the insulin C-peptide measurements and Ross Laboratories for providing the product used in the test meals.


    FOOTNOTES

M. J. Hamadeh was recipient of the McGill University 1999-2000 Standard Life Dissertation Fellowship. Support for the Clinical Research Unit is provided by the Fonds de la Recherche en Santé du Québec. This study was supported by Grants MT8725 and MME6712 from the Canadian Institutes for Health Research.

Address for reprint requests and other correspondence: L. J. Hoffer, Lady Davis Inst. for Medical Research, Jewish General Hospital, 3755 Cote-Ste-Catherine Rd., Montreal, QC H3T 1E2, Canada (E-mail:mi90{at}musica.mcgill.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 September 2000; accepted in final form 21 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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