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The Journal of Nutrition Vol. 128 No. 4 April 1998,
pp. 720-727
Institute of Human Nutrition, University of Southampton, Southampton SO16 7PX, UK
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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Urea kinetics were measured in normal women after 5 d consuming a low protein diet [LP, 67 mg N/(kg·d), 0.42 g protein/(kg·d)]. To determine whether the availability of methionine limits the utilization of nonessential nitrogen from low protein diets, the study was repeated on four further occasions with the addition of dietary supplements of L-methionine, 9 mg N/(kg·d) (LP-M); urea, 52 mg N/(kg·d) (LP-U); urea and methionine (LP-UM); or urea, 26 mg N/(kg·d), and glycine, 26 mg N/(kg·d), (LP-UG). Urea kinetics were derived after prime and intermittent oral doses of [15N15N]urea from the measurements of enrichment by isotope ratio mass spectrometry in urea isolated from urine. Nitrogen balance was significantly improved when the women consumed LP-U and LP-UG, but not LP-M or LP-UM. The urinary excretion of 5-L-oxoproline was measured as a marker of glycine availability and was significantly lower when women consumed LP-U and LP-UG compared with either LP or LP-M and LP-UM. There was a significant correlation between urinary 5-L-oxoproline and urinary sulfate excretion (r = 0.68, P = 0.00003). The availability of methionine was not limiting for nitrogen metabolism when women consumed these diets, whereas the response to supplementation with urea alone or urea with glycine showed that the availability of nonessential nitrogen was limiting. Glycine is consumed in the detoxification of excess methionine, and supplementation with methionine appeared to place a competitive demand on the availability of glycine for other metabolic processes.
KEY WORDS: glycine · serine · cysteine · sulfur · women
By varying the rate at which urea is excreted in urine, it is possible for humans to maintain nitrogen balance while consuming diets of different composition and across a wide range of protein intakes. The general perception has been that the change in urea excretion reflects a change in the rate of urea formation (Meijer et al. 1990 When the protein content of the diet is reduced acutely below the physiologic minimum level, 0.06 g N/(kg·d) (~30-35 g/d in adult men), nitrogen balance cannot be sustained (FAO/WHO/UNU 1985). Under this circumstance, the rate of urea production falls and the salvage of urea-nitrogen fails (Danielsen and Jackson 1992 Our goals in this study are to explore the possibility that, when diets that are low in protein are consumed, the availability of either methionine or glycine might be limiting, and to identify whether the efficiency with which urea-nitrogen is salvaged could be enhanced by dietary supplementation.
Subjects.
The studies were conducted in six normal women in good health, aged 22-28 y, who were staff and students of the Institute of Human Nutrition. They agreed to participate after an explanation of the nature of the study, which had been approved by the Southampton Hospitals and South West Hampshire Health Authority Ethical Subcommittee.
Diets.
The diets were consumed in the order LP,5 LP-U, LP-M, LP-UM, LP-UG; the only difference among the five study periods was the dietary supplement. During each period the subjects were provided with, and consumed, a diet designed to contain ~26 g protein/d, Table 1. All of the diets were provided as normal foods and the composition was calculated from food composition tables (Holland et al. 1991
Urea kinetics.
Urea kinetics were measured using prime and intermittent oral doses of [15N15N]urea (99% atoms 15N; Cambridge Isotope Laboratories, Cambridge, MA). An accurately measured amount of isotope was made up in sterile water and stored at 4°C until use. At 2400 h between d 4 and 5, a priming dose of isotopic urea equivalent to 15 h of intermittent infusion (28.5 mg) was given orally to shorten the time taken to achieve a plateau of enrichment in urinary urea. From 0600 until 1800 h, single doses of urea (5.5 mg) were administered at 3-h intervals. Urine was collected immediately before the administration of the prime dose of urea isotope and at 3-h intervals from 0600 until 2100 h to coincide with the taking of the intermittent doses of isotope. For the excretion of urea and nitrogen in urine, results were taken over the final 48 h period to reduce the within-person variability.
Analyses.
All specimens of urine were collected into acidified containers and were stored frozen until later analysis. The N concentration of the urine was measured by Kjeldahl analysis. The concentration of urea and ammonia in urine and plasma was determined by the Berthelot method (Kaplan 1965 Calculations.
Urea kinetics were calculated by using a modification of the model of Jackson et al. (1984)
The subjects were 1.68 ± 0.06 m tall and weighed 61.4 ± 7.2 kg, with a body mass index [BMI, weight (kg)/height(m2)] of 22 ± 2 kg/m2. Body fat was 26 ± 5% of body mass. When the amount of protein consumed in the diet is changed from one level to another, metabolic adjustments might continue to be made over several days, but the most marked changes in the excretion of N in urine take place within 2-3 d (Danielsen and Jackson 1992
sulfate = (23·5-L-oxoproline)
Despite a widespread perception that the availability of one or more of the indispensable amino acids will limit the effective utilization of a diet of marginal protein content, we have been impressed by the data that show that the generous addition of dispensable amino acids or other sources of "nonessential N" exerts a beneficial effect on N balance (Jackson 1995 Dietary supplements of methione and glycine were a gift from G. Hardy, Oxford Nutrition.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). However, when measured directly, urea production was found to vary by only 10% in groups of normal adults consuming diets that provide between 35 and 200 g protein/d (Child et al. 1997). Thus the variation in urea excretion associated with changes in the protein content of the diet are brought about by changes in the extent to which urea-nitrogen is salvaged and retained in the body after hydrolysis by the flora in the colon (Jackson 1995). The salvage of urea-nitrogen from the large bowel retains nitrogen for further metabolic interaction (Jackson 1995).
, Meakins and Jackson 1996). If an individual in negative nitrogen balance, consuming marginally inadequate dietary protein, is given a large dietary supplement of urea to simulate an increased rate of urea formation, urea salvage can be stimulated with an improvement in nitrogen balance (Meakins and Jackson 1996). However, the amount of urea required as a dietary supplement is large, raising the possibility that some other nutrient might limit the effective utilization of the additional urea. We have considered two possibilities. The first is that function is limited by the availability of dietarily indispensable amino acid. A comparison of the amino acid profile of the diet with the requirements (FAO/WHO/UNU 1985) shows that methionine (75% of the estimated requirement) is most likely to be limiting on the diets provided (Meakins and Jackson 1996). The second possibility is that, with the diets consumed during the study, the availability of nonessential nitrogen for the formation of dispensable (or nonessential) amino acids was limiting, and urea is being utilized as a source of nitrogen for their endogenous formation. The evidence suggests that although urea can be utilized as a source of nonessential nitrogen, it is less efficient than ammonia, other dispensable amino acids or indispensable amino acids (Jackson 1995, Kies 1972). There is direct evidence for a decrease in the formation of dispensable amino acids such as glycine, when the protein content of the diet is reduced (Gersovitz et al. 1980, Yu et al. 1985). We have found that in normal men and women consuming diets low in protein, the urinary excretion of 5-L-oxoproline, a marker for glycine sufficiency, is increased significantly, but this increased excretion is restored to normal levels when the diet is supplemented with generous amounts of nitrogen, as either protein, glycine or urea (Jackson et al. 1996, Persaud et al. 1996).
SUBJECTS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Complete collections of urine were made for the duration of the study; during the last 2 d of each study period, measurements of urea kinetics were made. A sample of blood was taken at 1500 h on d 5 of the study. The plasma was removed and stored at 
70°C. The studies were conducted at the same time of the menstrual cycle, wk 4, the luteal phase (McClelland and Jackson 1996), and hence at least 3 wk passed between consecutive studies. During the studies, the subjects engaged in their normal activities, but did not engage in vigorous exercise such as cycling or sports.
) with the use of a computerized data base (Comp-Eat, Nutrition Services, London, UK). Breakfast consisted of cereal with whole milk and orange juice. The main meals were either vegetable curry with rice, salad and dressing with peaches and cream, or vegetable lasagna, salad and dressing with pineapple and cream. Snacks were provided as jam sandwiches, potato snacks, cakes, sweets, apples and sweetened soft drinks. The nutrient composition of the diet is shown in Table 1. The low protein diet (LP) provided up to 67 mg N/(kg·d) [0.41 g protein/(kg·d) or 26 g mixed protein/d], and contained 0.55 mg N/(kg·d) as methionine (358 mg). It was estimated that a supplement of L-methionine, 0.9 mg N/(kg·d) (600 mg/d) would bring the dietary content to the recommended daily intake (FAO/WHO/UNU 1985, Raguso et al. 1997). The low protein diet supplemented with methionine, 0.55 mg N/(kg·d) (LP-M), provided 68 mg N/(kg·d), Table 2. A dietary supplement of urea, 6.9 g, has a nitrogen (N) content of 3.2 g, equivalent to about 20 g protein. The low protein diets supplemented with urea (LP-U), with urea and methionine (LP-UM) or with urea and glycine (LP-UG), provided about 120 mg N/(kg·d), Table 2.
View this table:
Table 1.
The composition of a low protein diet analyzed with a computerized database and designed to provide ~0.4 g protein/(kg·d) for women who consumed the diet, or consumed the diet with supplements of urea, glycine or methionine1
View this table:
Table 2.
The protein, nitrogen content of five diets consumed
for periods of 5 d by women in whom
urea kinetics were measured1
). Urea-N was isolated from urine for mass spectrometry using short ion-exchange column chromatography (Jackson et al. 1980). Nitrogen gas was liberated from urea by reaction with alkaline hypobromite. In this reaction, N2 is released from urea in a monomolecular reaction (Walser et al. 1954); hence the relative proportions of [15N15N]urea, [15N14N]urea and [14N14N]urea can be determined. Measurements were made in a triple collector isotope ratio mass spectrometer (SIRA 10, VG Isogas, Cheshire, UK). For each study, the plateau level of enrichment was taken as the average enrichment in either [15N15N]urea or [15N14N]urea during the final 12-h period, the last four specimens of urine.
).
).
, shown in Figure 1. Catabolism of amino acids in the body gives rise to urea (Pu), which mixes with the general body pool. Urea also appears in the pool directly from that ingested from the dietary supplement (Iu). Once an isotopic steady state has been achieved, the dilution of an intermittent dose of [15N15N]urea gives a measure of the rate of urea appearance in the body (Au) as follows:
where d is the rate of administration of [15N15N]urea in mg N/(kg·d) and E is the ratio of [15N15N]urea to [14N14N]urea in urine once an isotopic plateau has been achieved. The difference between the appearance of urea in the body and that taken orally gives a measure of the endogenous production of urea (Pu). A proportion of the urea appearing in the urea pool is excreted in the urine (Eu). The difference between the urea that appears in the pool and that excreted is presumed to have been transferred to the bowel (T), where it is hydrolyzed with the N being returned to the general metabolic pool. Although the size of the urea pool shows a diurnal variation, on a standard diet, the urea pool size is similar at the same time each day (Quevedo et al. 1994
). In this study, because urea excretion was measured for either 24 or 48 h, it can be presumed that urea not excreted was hydrolyzed in the bowel, not retained within the urea pool. Thus
A part of the N derived from urea hydrolysis is resynthesized to urea (Pr), whereas the remainder is available for synthesis into amino acids and proteins (S).
The total urea produced is comprised of two inputs, a component derived from the catabolism of dietary and endogenous amino acids and a component (Pr) that comes from urea hydrolysis in the bowel. The urea entering the bowel is doubly labeled, [15N15N]urea. The recycled urea (Pr) that is synthesized in the liver from 15NH3, generated from N derived from urea hydrolyzed in the bowel, will be singly labeled, [14N15N]urea. Pr is determined from measurements of singly labeled urea in the urine (Jackson et al. 1984
, Meakins and Jackson, 1996).
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Fig 1.
Model of urea kinetics used in this study, consisting of a single pool of urea with two sources of entry and two of loss. The rate at which urea appears in the pool, Au, is measured by isotopic dilution. Au is derived from two sources, endogenous urea production, Pu, and the dietary intake, lu. In a metabolic steady state, the rate of appearance of urea in the pool is equal to the rate of loss of urea from the pool. The loss from the pool is through two forms, loss as excretion to urine, Eu, or loss to hydrolysis in the bowel, T. Hydrolyzed urea nitrogen undergoes further metabolic interaction, either by returning to the formation of urea, Pr, or entering other metabolic pathways, S.
). The percentage of the urea pool excreted daily to the colon was calculated, assuming that colonic excretion was equivalent to T.
). Differences were accepted as significant if P < 0.05. Results are reported as means ± SEM and associations between variables were explored by linear regression analysis.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
, Quevedo et al. 1994). There was a significant reduction in urinary urea when the subjects consumed the experimental diets compared with their usual diets (Fig. 2). Most of the change was seen in the first 2 d; the much smaller changes in excretion after d 3 were within the range of day-to-day variation. Estimates of N balance were made over d 4 and 5 of the experimental period for each of the diets as shown in Table 3. Urinary N varied with the diets consumed and was significantly greater during the periods when urea was added as a supplement, LP-U, LP-UM and LP-UG diets, compared with the LP and LP-M diets. Apparent N balance was calculated as the difference between N intake and urinary N. Based on an estimate for fecal and other miscellaneous losses of N, 24 mg/(kg·d) (FAO/WHO/UNU 1985), an estimated of balance was calculated as Intake [urine N + 24 mg N/(kg·d)]. The addition of the methionine supplement, LP-M, to the low protein diet, LP, did not affect N balance. However, the addition of a dietary urea supplement, LP-U, to the LP diet significantly improved N balance. The benefit afforded by the dietary supplement of urea was not maintained when urea and methionine were supplemented together; when subjects consumed the LP-UM diet, N balance was not different from when they consumed the LP or the LP-M diets. When a part of the urea supplement was replaced on an isonitrogenous basis with glycine, LP-UG, there was no difference in N balance compared with the LP-U diet period; N balance was significantly improved compared with the LP, LP-M and LP-UM diet periods.
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Fig 2.
The daily excretion of urea in urine in women consuming low protein diets of different composition for periods of 5 d. Values are means ± SEM, n = 6 for each point; closed circle LP, low protein diet, 0.42 g protein/(kg·d); open circle, LP-M, low protein diet with a supplement of L-methionine; star, LP-U, low protein diet with supplement of urea; closed square, LP-UM, low protein diet with a supplement of urea and methionine; open square, LP-UG, low protein diet with a supplement of urea and glycine.
View this table:
Table 3.
Nitrogen balance in six normal women in whom urea kinetics were measured on five occasions while consuming diets
of different nitrogen content, over d 4 and 5 of each dietary period1,2
View this table:
Table 4.
Urea kinetics in six normal women measured on five occasions while consuming low protein diets of different composition1,2
View this table:
Table 5.
The urea pool size and clearance of urea into the urine or bowel, and the excretion in urine of sulfate and 5-L-oxoproline in six normal women in whom urea kinetics were measured on five occasions while consuming diets of different nitrogen content1,2
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Fig 3.
Urinary excretion of sulfate and 5-L-oxoproline measured in young women consuming either their habitual diet, a low protein diet for 5 d or a low protein diet supplemented with methionine, urea, methionine and urea or glycine and urea. Based on linear regression analysis:
63 (r = 0.68, P = 0.00003).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Kies 1972). In a previous study, we found improvements in N balance after the addition of generous supplements of urea [83 mg N/(kg·d)] to the diet of young men consuming a diet that was too low in protein to maintain N balance [50-60 mg N/(kg·d)] (Meakins and Jackson 1996). Of special interest was the observation that the response to urea was seen only at high levels of supplementation; we wondered whether that reflected a limitation in methionine, the first limiting indispensable amino acid in the diet. This hypothesis has been tested in a group of healthy young women consuming a diet in which the protein content was similar in relation to body weight [~60 mg N/(kg·d)] and too low to support N balance. In these women, a dietary supplement of urea significantly improved N balance, confirming earlier studies that the addition of nonessential N to a diet low in protein can promote improvements in N balance (Kies 1972). The demonstration that N balance was influenced at a lower level of urea supplementation than that seen in men indicates the need to determine whether the difference is real and related to a different sensitivity to dietary protein and supplements of nonessential N in women, or is simply a reflection of the form of expression (whether expressed as an absolute amount, or relative to body weight or to lean body mass). We found no evidence to support the suggestion that methionine was limiting when women consumed the LP diet. Indeed, not only did supplementation with methionine fail to confer any benefit, but the supplement of methionine removed the beneficial effect of a supplement of urea when given at the same time as the urea. In this study, we did not identify any benefit in providing nonessential nitrogen as an amino acid, glycine, compared with urea.
). The supplement of urea was selected to bring the total N intake to ~120 mg/(kg·d) [equivalent to 0.76 g protein/(kg·d) or 47 g protein/d], an intake that would have been adequate to maintain N balance, if the pattern of amino acids was suitable (FAO/WHO/UNU 1985). The supplement of methionine was generous, so as to ensure that there would be no limitation of sulfur amino acids, and was set at the recommended level of intake (FAO/WHO/UNU 1985, Raguso et al. 1997). The glycine supplement was selected as an isonitrogenous replacement for half of the urea supplement. For none of the diets was 5 d adequate to achieve full accommodation, but on each diet the subjects achieved a metabolic state that we judged to approximate a steady state within the recognized range of day-to-day variability, Figure 2 (Quevedo et al. 1994). Under these circumstances, there were significant improvements in N balance when subjects consumed the LP-U and LP-UG diets. In contrast, there was no improvement in N balance with the addition of methionine to the low protein diet, LP-M, and methionine exerted an adverse effect when added to the low protein diet with supplemental urea, LP-UM. We conclude that methionine was not limiting for these diets and that supplementation of methionine at this level exerted an adverse effect.
) indicates that the movement of urea within the body may be subject to closer control than had been appreciated and raises interesting questions about the possible importance of urea movement in relation to interactions among protein intake on the one hand and salt and water homeostasis on the other. The demonstration that the same transporter is located in the colon allows the possibility of coordinate control between renal retention and colonic secretion of urea (You et al. 1993). There is now provisional evidence that the flora of the colon utilize the salvaged urea-N for the synthesis of indispensable amino acids, such as lysine, which are available to the host in functionally important amounts (Gibson et al. 1997a and 1997b, Metges et al. 1997). This evidence raises fundamental questions about our perceptions of the physiologic and dietary requirements for indispensable and dispensable amino acid and the extent to which they might be satisfied from the diet, from endogenous formation or as the result of the metabolic activity of the flora in the colon (Jackson 1995).
, Kies and Fox 1972, Young et al. 1984, Zezulka and Calloway 1976). Excessive supplements of methionine have adverse effects and it has been suggested that a supplement >1.1 g methionine/d to individuals consuming diets that provided ~4 g N/d abolished any benefit seen with lower levels of supplementation in men (Kies et al. 1975, Young et al. 1984); similar observations have been made in women (Reynolds et al. 1958). The results of this study indicate that the margin of safety may be less than this and requires further consideration. There are theoretical reasons why adding methionine to the diet might exert an influence either directly or indirectly on the behavior of the flora in the colon (Christl et al. 1992), and although there is much information on the effect of dietary sulfate on gastro-intestinal function (Hill 1995), knowledge of the effect of sulfur amino acids is sparse. Early studies suggest that losses of sulfur in the stool are constant, even with dietary sulfur supplementation (Wright et al. 1960). However, the observation in this study that increasing the dietary methionine three times resulted in a much more modest 50% increase in urinary losses suggests that there are other important routes for sulfur disposal, which may include increased fecal losses, Table 5.
). We used urinary excretion of 5-L-oxoproline as a marker for glycine status and showed that in normal people, there is a significant increase in the excretion of 5-L-oxoproline after 5 d consuming diets low in protein. This high level of excretion is brought back to normal when the protein content of the diet is increased, or when the diet is supplemented with other sources of N, such as urea (Jackson et al. 1996). This study shows that the change in 5-L-oxoproline excretion when the diet is supplemented with urea is completely blocked by the addition of methionine to diet. The excretion of 5-L-oxoproline was highest when subjects consumed the LP diet and when supplements of methionine were provided, LP-M and LP-UM. Methionine is considered to be among the most toxic amino acids; any excess beyond that that can be used in metabolism must be removed. It is well established that there are close interactions between the metabolism of methionine and the metabolism of glycine and that toxic levels of methionine in the diet can be corrected by the addition of glycine (Harper et al. 1970). The pathways through which this protective effect operates are not clear but are thought to include enhanced degradation of methionine through the transulfuration pathway, with glycine acting as a receptor for methyl groups and as the precursor for serine, and hence cysteine formation (Bagley and Stipanuk 1995). This implies that a methionine load presents a competitive metabolic pathway for the available glycine and, in this study, would explain why urinary 5-L-oxoproline was increased when supplements of methionine were provided. This interpretation is supported by the demonstration that for all of the study periods, there was a close relationship between the excretion of sulfate in urine and the urinary excretion of 5-L-oxoproline, Figure 3, leading to the conclusion that a methionine challenge may exert its toxic effect in part through competing for available glycine and thereby limiting the availability of glycine for other metabolic interactions, such as the formation of glutathione.
ACKNOWLEDGMENT
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FOOTNOTES |
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Manuscript received 3 September 1997. Initial reviews completed 13 October 1997. Revision accepted 21 December 1997.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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