Vol. 280, Issue 2, E214-E220, February 2001
Regulatory responses to an oral D-glutamate
load: formation of D-pyrrolidone carboxylic acid in
humans
Dominic
Raj,
Maryln
Langford,
Stephan
Krueger,
Martin
Shelton, and
Tomas
Welbourne
Departments of Medicine, Ophthalmology, Pediatrics, and Molecular
and Cellular Physiology, Louisiana State University Health Science
Center, Shreveport, Louisiana 71130
 |
ABSTRACT |
Previously published studies have shown D-glutamate to be
the most potent natural inhibitor of glutathione synthesis known, yet
how D-glutamate is handled in humans is unknown. Therefore, we administered an oral D-glutamate load to four healthy
volunteers and monitored the plasma D-glutamate
concentration and excretion over a 3-h postload period. Compared with
time controls, the plasma D-glutamate concentration
increased 10-fold in the 1st h and then reached a plateau over the
remaining time course. In contrast, plasma D-pyrrolidone
carboxylic acid increased progressively throughout the 3-h time course
to a level 10-fold higher than the D-glutamate plasma
concentration. Excretion of D-glutamate progressively
increased despite a constant filtered D-glutamate load
rising from only 5 to 95% of the filtered amount. Excretion of
D-pyrrolidone carboxylic acid increased with the rise in
filtered load without significant reabsorption. The amount of
D-pyrrolidone carboxylic acid excreted over the 3-h time
course was 10 times the amount excreted as D-glutamate and
accounted for almost 20% of the administered D-glutamate. These findings indicate that plasma D-glutamate
concentration is tightly regulated through two mechanisms:
1) the transport into cells and metabolic conversion to
D-pyrrolidone carboxylic acid and excretion, and
2) the enhancement of D-glutamate clearance by
the kidneys.
L-glutamate; D-glutamate cyclotransferase; renal handling; creatinine clearance; D-,L-glutamine synthetase; D-glutamine; glutamate transport
| |
INTRODUCTION |
D-GLUTAMATE LOADS are
normally presented to the intestine as the free amino acid present in
certain foods e.g., soybeans (5, 10) and from the turnover
of the intestinal tract microflora, whose cell walls contain
significant D-glutamate (19). Unlike other
D-amino acids, D-glutamate is not oxidized by
the D-amino acid oxidases (24), and therefore
this detoxification pathway is not available for handling
D-glutamate. Free D-glutamate is found in
mammalian tissue at surprisingly high levels, with
D-glutamate accounting for 9% of the total glutamate
present in liver (9). Noteworthy, significant accumulation
of D-glutamate in the body fluids would impair a number of
important physiological processes. For example, D-glutamate
is the most potent natural inhibitor of glutathione synthesis known
(21); in fact, the D-isomer is more reactive
[inhibitory constant (Ki) = 0.8 mM] than
the natural L-glutamate (Ki = 1.8 mM) with 
-glutamylcysteine synthetase (13). Given
the liver intracellular glutamate concentration of ~10 mM (9), D-glutamate concentration would approach
1 mM and thus might play a modulating role in the intraorgan
glutathione fluxes. Indeed, when D-glutamate at 1.6 mmol/kg
was injected into rats intraperitoneally, renal glutathione content
decreased 51% within an hour (21).
D-glutamate may also act as a false neurotransmitter in the
central nervous system (15) and may therefore contribute to excitotoxicity (18).
How D-glutamate is removed from the body fluids is not
clear. In the sole human study (16), radiolabeled
D-[14C]glutamate was infused intravenously
into a patient with multiple myeloma, and ~20% was recovered in the
urine unchanged as D-glutamate after 3 h
(16). Although taken as evidence for excretion without significant metabolic conversion, the low recovery suggests that D-glutamate could have undergone metabolic conversion. In
the rat, 75% of orally administered
D-[15N]glutamate was recovered in the urine
as D-[15N]pyrrolidone-carboxylic acid after
24 h (17). Conversion of 14C-labeled
D-glutamate to D-pyrrolidone-carboxylic acid
was observed when administered orally or intravenously to rats
(26), with >50% of the total administered radioactivity
recovered in the urine after 24 h as
D-pyrrolidone-carboxylic acid. The enzymatic activity of
D-glutamate cyclotransferase, responsible for converting D-glutamate to D-pyrrolidone-carboxylic acid,
was demonstrated to be highly expressed in mammalian kidney and livers
(12); kidney and liver obtained from humans had a
D-glutamate cyclotransferase activity approaching that of
the rat (12). These findings point to a potential
regulatory role for D-glutamate transport into cells and
metabolic conversion to D-pyrrolidone-carboxylic acid in
the handling of D-glutamate.
How the kidneys handle D-glutamate has more recently been
studied using microperfusion (6, 22) and extraction
procedures (4). In the rat proximal convoluted tubule,
luminal D-glutamate is neither taken up nor does it inhibit
the avid uptake of L-glutamate (22). In
contrast, both D-glutamate and L-glutamate are
taken up in the proximal straight and loop segments (6).
In extraction studies, the rat kidney removes almost 70% of the
D-glutamate arterial load, indicating both luminal and
antiluminal uptake sites for the D-glutamate
(4); in addition, virtually all of the filtered
D-glutamate was reabsorbed (4), indicating a
significant tubular reabsorptive capacity apparently in the proximal
straight and loop segments (6). Furthermore, the uptake of
D-glutamate could be largely, if not entirely, accounted
for as D-pyrrolidone-carboxylic acid formation in the
functioning rat kidney (20).
D-pyrrolidone-carboxylic acid, in turn, is filtered and
excreted in contrast to the D-glutamate (20).
The purpose of the present study was to determine the response in
humans to an oral D-glutamate load. Specifically we wished to know whether D-glutamate would be taken up from the
intestine and appear in the systemic circulation. If so, then the
handling of the D-isomer becomes subject to physiological
regulation, potentially involving both transport into cells and
metabolic conversion. Our second objective was to determine whether
D-glutamate entering the plasma would be excreted as
D-glutamate or metabolically converted to
D-pyrrolidone-carboxylic acid. Finally, we wished to know
how the D-glutamate and, if formed,
D-pyrrolidone-carboxylic acid, would be handled by the
kidneys. The results to follow show that D-glutamate
appears in the plasma after an oral load, confirming its uptake from
the intestine. Whereas the rise in plasma D-glutamate plateaus, plasma D-pyrrolidone-carboxylic acid
concentration rises to a level 10-fold higher than plasma
D-glutamate. The kidneys effectively excrete the filtered
D-pyrrolidone-carboxylic acid without reabsorption, whereas
D-glutamate excretion progressively increases despite a
constant filtered load.
| |
MATERIALS AND METHODS |
Studies were carried out on four healthy male volunteers. The
authors were the volunteers and gave their consent to a protocol approved by the Louisiana State University Medical Center Institutional Review Board for Human Research. Vehicle, 500 ml water, vehicle plus
2 g (13.3 mmol) D-glutamate (D-Glu), or
vehicle plus 2 g D-glutamine (D-Gln;
Sigma, St. Louis, MO) were ingested within a 20-min period 1 h
after a light breakfast (coffee, toast, and juice). This large dose of
D-Glu was chosen to challenge the putative systems and to
reveal the underlying mechanisms. Time controls and D-Glu
or D-Gln loading were performed over 2 wk, one paired test
per week. The D-Gln loading provided an index of renal
D-Glu formation from this potential metabolite of
D-Glu. After subjects emptied their bladders and an initial
forearm venous blood sample was obtained (t = 0), either
vehicle or vehicle plus D-Glu was ingested. Blood samples
were then obtained at hourly intervals for 3 h (t = 1, 2, and 3), with urine collected at the end of each hourly period; water
was consumed at a rate matching the urine volumes to maintain the state
of hydration.
Plasma and urine samples were placed on ice immediately and processed
for same day analysis. Total glutamate concentration of plasma and
urine was determined on aliquots of protein-cleared samples by HPLC as
previously described (20). Replicate analysis of 100 µM
L-Glu standards was made with a coefficient of variation of
1.6% (n = 3). Recovery of L-Glu (50 nmol)
added to 1 ml plasma was 103 ± 4% (n = 3). To
determine the D-Glu concentration, 15 µl of untreated
plasma and urine samples or standards were incubated at 37°C with
0.02 U of L-glutamate oxidase (1 unit converts 1 µmol
L-Glu/min at 30°C, Sigma) for 2 h in 10 mM phosphate
buffer, pH 7.4. The samples and standards were then processed as
described above and analyzed for glutamate by HPLC (Fig.
1). Figure 1, A and
B, shows the complete removal (>99%; Sigma
L-Glu appears to have 0.8% D-Glu) of 50 nmol
L-Glu/ml (Fig. 1A) after the 2-h incubation with
L-glutamate oxidase (Fig. 1B). In contrast, with
25 nmol/ml of the D-Glu standard (Fig. 1C),
virtually all (>95%) remains after treatment with
L-glutamate oxidase (Fig. 1D). Plasma sampled 3 h after D-Glu loading was analyzed before (Fig.
1E) and 2 h after (Fig. 1F) treatment with
L-glutamate oxidase. Of the 60 nmol/ml total glutamate
(Fig. 1E), 20% (12 nmol/ml) was in the D-Glu
form (Fig. 1F). In urine collected at the 3-h postload
D-Glu period, 89% of the total glutamate present (Fig.
1G) was present in the D-form (Fig.
1H). Recoveries of L-Glu (50 nmol/ml) added to
plasma and urine in the presence of L-glutamate oxidase
were <1%. Note that under these conditions the
L-glutamate oxidase activity was sufficient to completely
remove 
300 nmol/ml of the L-Glu. Recovery of 50 µM
D-Glu as glutamate added to plasma was 94 ± 4%
(n = 3) in the absence of L-glutamate
oxidase and 97 ± 3% (n = 3) when added in the
presence of L-glutamate oxidase. Thus there does not appear
to be an inhibitor of L-glutamate oxidase present in human
plasma.
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
The enzyme L-glutamate oxidase ( E or +E)
effectively removes L-glutamate from plasma and urine
samples. A and B show that 50 nmol/ml of
L-glutamate (L-Glu; A) are
completely oxidized after 2 h (B). C and
D show that 20 nmol/ml of D-glutamate
(D-Glu) are resistant to the oxidase. E and
F show that plasma glutamate (E) contains
oxidase-resistant D-Glu (F). G and
H show that urine glutamate (G) contains mostly
D-Glu 3 h after the D-Glu load. Glutamate
and homoserine (internal standard) had retention times of 6.8 and 13.5 min, respectively.
|
|
Total pyrrolidone carboxylic acid was determined by mild acid
hydrolysis of plasma and urine samples, as previously described (20). Aliquots of the protein-cleared samples and
standards, together with equal volumes of 3 N HCl, were incubated for
2 h at 95°C, cooled, and then neutralized with 3 N NaOH. The
samples were then analyzed for total glutamate by HPLC and for
L-Glu by fluorometric enzymatic analysis by use of
L-glutamate dehydrogenase (L-GDH), as
previously described (20). Total pyrrolidone carboxylic acid (analyzed by HPLC as for glutamate) was taken as the increase in
glutamate after acid hydrolysis after the plasma glutamine and
glutamate concentrations were subtracted; the D-isomer was obtained from the difference between the total glutamate present measured by HPLC and the L-form as measured with
L-GDH.
As an index of potential liver and renal damage, the plasma and urine
-glutamyltransferase (GGt) activities were assayed during the time
course; GGt activity is expressed in units/ml plasma or as units per
period excreted.
Excreted amounts of D- and L-Glu as well as
D-pyrridoline carboxylic acid (D-PCA) were
calculated from the hourly urine volume times the urine concentration.
Filtered amounts were determined as the average (midpoint) plasma
concentration for each 1-h period times the glomerular filtration rate
estimated from the endogenous creatinine clearance (calculated from the
standard formula U/P × V, where U and P are the plasma and urine
creatinine concentrations, respectively, and V is the urine flow).
Fractional excretion (FE) rates (as percentages) are the amount
excreted divided by the amount filtered times 100. Statistical analyses
were performed using ANOVA for differences as the result of treatments
(time control and D-glutamate load) or the Student's
t-test for differences between groups. The null hypothesis
was rejected with P < 0.05.
| |
RESULTS |
The plasma concentrations of D-Glu and
D-PCA before and for 3 h after the oral
D-Glu load are shown in Fig.
2. The corresponding time control is
shown in the inset; note the difference in y-axis scales.
The circulating D-Glu concentration increases 10-fold (14 ± 1 vs. 1.1 ± 0.1 µM for the time control,
P < 0.0001) over the 1st h and then plateaus at this
concentration for the remainder of the 3-h time course (14 ± 1 and 12 ± 1 µM at 2 and 3 h, all P < 0.01 vs. time controls). The plasma D-PCA concentration before the 18 ± 7 µM load, Fig. 2, rose 5-, 13-, and 18-fold above the time controls at 1, 2 and 3 h postload (70 ± 14 vs. 14 ± 3, 176 ± 24 vs. 14 ± 5, and 182 ± 21 vs. 10 ± 1 µM, respectively, all P < 0.005 vs. time
controls). Whereas the plasma D-Glu concentration increases
to ~14 µM and plateaus, the increase in plasma D-PCA concentration is far greater and progressive.
View larger version (12K):
[in this window]
[in a new window]
|
Fig. 2.
Plasma D-Glu and
D-pyrrolidone-carboxylic acid (D-PCA)
concentrations before and for 3 h after a D-Glu load.
Results are means ± SE from 4 subjects. The increase in both
D-Glu and D-PCA concentrations at 1, 2, and
3 h was significant compared with the time 0 by ANOVA,
P < 0.001. Inset: time-control value for 4 subjects; note the difference in y-axis scales.
|
|
The corresponding excretion of D-Glu and D-PCA
over the 3-h time course is shown in Fig.
3. The corresponding time controls are
shown in the inset; note the difference in the y-axis
scales. The D-Glu excretion is 33-fold greater
(P < 0.005) than the time control for the 1st h
(198 ± 33 vs. 6 ± 4 nmol/min) and then increases to 130- and 351-fold higher (650 ± 147 vs. 5 ± 3 and 700 ± 124 vs. 2 ± 1 nmol D-Glu/min) than their respective
time controls. This progressively higher D-Glu excretion
occurs despite an unchanged plasma D-Glu concentration, as
shown in Fig. 2. Excretion of D-PCA before the
D-Glu load, 891 ± 128 nmol/min (Fig. 3), increased 2- (P < 0.09), 6- (P < 0.002), and 12 (P < 0.01)-fold above the time controls at 1, 2, and
3 h, respectively (2,726 ± 651 vs. 1,383 ± 361, 6,992 ± 919 vs. 1,200 ± 213, and 11,435 ± 2,355 vs. 983 ± 84 nmol/min). Note that the excretion of D-PCA
is 11 and 17 times higher than that of the D-Glu over the
2nd and 3rd h. The excretion of D-PCA was 13,569 ± 2,379 nmol/min at the 4th h postloading, indicating that the excretion
of D-PCA reaches a plateau. These results show that
metabolic conversion of D-Glu to D-PCA is the
major pathway for eliminating the D-Glu load in humans and
that D-PCA excretion is significant in the absence of an
exogenous D-Glu load.
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Excretion of D-Glu and D-PCA
before and for 3 h after an oral D-Glu load. Results
are means ± SE from 4 subjects. The increase in D-Glu
excretion at 1, 2, and 3 h was significant compared with
time 0 by ANOVA, P < 0.001. Inset: time-control value for 4 subjects; note the
difference in y-axis scales.
|
|
How the kidneys handle the filtered D-Glu and
D-PCA as well as L-Glu is shown in Fig.
4 and Table
1. Figure 4 shows the FE of
L- and D-Glu as well as D-PCA
before and for 3 h after the D-Glu load. Table 1
presents the filtered load and excreted amounts for each, as well as
the corresponding time controls. D-Glu shows a progressive
increase in the FE (Fig. 4) from 3 ± 3% at t = 0 to
40 ± 11 (P < 0.02) to 68 ± 12 (P < 0.004) and 95 ± 15% (P < 0.002) at 1, 2, and 3 h, respectively, compared with time controls (5 ± 3, 4 ± 3, and 1 ± 1%, respectively). The
increase in FE occurs despite an unchanged filtered D-Glu
load (Table 1). The L-Glu FE tends to increase from 3 ± 1 to 19 ± 13 and 23 ± 12% (P < 0.09) compared with the time controls (2 ± 1, 1 ± 0, and 1.5 ± 0% of the filtered L-Glu is excreted at 1, 2, and
3 h, respectively). On the other hand, the FE of D-PCA
is not different from 100% of the filtered load in both the
D-Glu and time controls, although the filtered amounts are
11-fold higher over the last hour. The renal handling of the neutral
amino acid glutamine was not changed over the postload period.
View larger version (29K):
[in this window]
[in a new window]
|
Fig. 4.
Fractional excretion (FE) of L-Glu and
D-Glu and D-PCA before and for 3 h after a
D-Glu load. Results are means ± SE from 4 subjects.
Time-control FE values were 1.8 ± 0.5, 1.8 ± 0.4, 1 ± 0.3, and 1.5 ± 6% for L-Glu and 2 ± 0.6, 5 ± 5, 4 ± 3, and 1 ± 1% for D-Glu.
|
|
Because D-Glu may be converted to D-Gln by the
liver and subsequently hydrolyzed intraluminally in the kidney to
D-Glu, D-Gln was orally administered (2 g), and
the filtered and excreted D-Glu was measured as shown in
Fig. 5. Before the D-Gln
load, FE of the filtered D-Glu was only 4 ± 3%,
indicating near complete reabsorption of the filtered
D-Glu. Subsequent to the D-Gln load, the
apparent FE for filtered D-Glu increased from 4 ± 3 to 143 ± 17, 411 ± 103, and 366 ± 195% as the result
of intraluminal D-Gln hydrolysis (25). These
results show that the D-PCA is filtered and excreted without significant reabsorption, whereas L-Glu is
reabsorbed far more efficiently than D-Glu throughout the
postload period. However, there is a progressive increase in the
excretion of both isomers, and most notably D-Glu, which
may reflect impaired reabsorption or intrarenal synthesis, or both.
View larger version (12K):
[in this window]
[in a new window]
|
Fig. 5.
Filtered and excreted D-Glu after a
D-Glu load. Results are means ± SE from 3 subjects.
Excreted D-Glu significantly greater than filtered amount,
*P < 0.05.
|
|
Figure 6 shows the glomerular filtration
rate measured as the endogenous creatinine clearance rate for the time
controls and after D-Glu loading. The estimated filtration
rate drops 27% (P < 0.02) and 33% (P < 0.004) over the second and third clearance periods (70 ± 4 and
66 ± 6 vs. 96 ± 15 and 98 ± 6 ml/min for time controls, respectively). Neither the GGt activity in plasma (517 ± 37, 509 ± 42, 458 ± 21, and 535 ± 76 units/ml
plasma, respectively) nor that in urine (69 ± 1, 46 ± 1, 46 ± 6, and 76 ± 12 × 103 U/period)
increased over the 3 h after the D-Glu load.
View larger version (12K):
[in this window]
[in a new window]
|
Fig. 6.
Creatinine clearance from 4 subjects before and for
3 h after either D-Glu load or vehicle (time control).
Results are means ± SE from 4 subjects. Significantly different
from time control, *P < 0.05.
|
|
| |
DISCUSSION |
The biological significance of D-glutamate uptake and
conversion to D-pyrrolidone-carboxylic acid and excretion
is suggested by the presence of the small, but detectable, plasma
D-glutamate (Fig. 2) and the considerable excretion of
D-pyrrolidone-carboxylic acid (Fig. 3), even in the absence
of the exogenous D-glutamate load. Based on the minute
excretion rate (Table 1) of D-pyrrolidone-carboxylic acid,
and extrapolated to 24 h, the daily D-glutamate load
approximates 0.5-1 mmol/day. In addition, a large exogenous
D-glutamate load greatly enhances the
D-pyrrolidone-carboxylic acid formation, consistent with a
precursor-product relationship and the demonstration in rats that
15N-labeled D-glutamate appears in the urine as
D-pyrrolidone-carboxylic acid (17). There has
been one other report of a detectable D-glutamate concentration in normal human plasma by use of chiral chromatography (3), but this determination encompasses plasma
D-glutamine, which, according to the authors, is hydrolyzed
to D-glutamate under their conditions. Whether
D-glutamine is normally present in plasma, and how much,
remains to be determined. Thus the present paper is the first report of
D-glutamate in human plasma and the first demonstration
that D-pyrrolidone-carboxylic acid formation is indeed a
significant, if not the sole, detoxification pathway for
D-glutamate in humans.
The accumulation of the D-pyrrolidone-carboxylic acid in
plasma after the oral administration of D-glutamate (Fig.
2) and the 13-fold increase in D-pyrrolidone-carboxylic
acid excretion (Fig. 3) are consistent with the absorption of
D-glutamate from the intestine and conversion via
D-glutamate cyclotransferase (12). The
cumulative excretion of D-pyrrolidone-carboxylic acid over
this 3-h time course accounted for ~20% of the administered D-glutamate. If the excretion rate had continued at the 3-h
rate, virtually all of the load would have been excreted in 
15 h. The fact that a significant fraction appears as
D-pyrrolidone-carboxylic acid confirms that humans utilize
the same pathway as that present in the rat to detoxify this
potentially harmful metabolite.
The rise in plasma D-glutamate during the postload period
demonstrates that the D-glutamate is taken up into the
blood and reaches the systemic circulation. Rather than continue to
rise, the blood concentration plateaus, despite continuous absorption (based on the D-pyrrolidone-carboxylic acid formation). In
contrast to the plasma D-glutamate concentration that is
defended at 14 µM, the plasma D-pyrrolidone-carboxylic
acid concentration progressively increases to levels that are more than
fivefold higher than the D-glutamate plasma concentration
in the 1st h. It may be noteworthy that this plasma concentration of
D-pyrrolidone-carboxylic acid (70 µM) corresponds to, or
is slightly higher than, that reported circulating in the plasma of
patients with chronic renal failure [total pyrrolidone-carboxylic
acid = 86 µM, with 33 µM as the D-isomer
(14)].
Although the detoxification of D-glutamate requires at a
minimum a transporter capable of delivering D-glutamate
into cells expressing D-glutamate cyclotransferase activity
and functioning kidneys for effective clearance of the
D-pyrrolidone-carboxylic acid, there may be other pathways
involved in the transit of D-glutamate. In our previous
studies in the rat with intravenously infused D-glutamate,
the combination of renal extraction and conversion to
D-pyrrolidone-carboxylic acid was the major organ-metabolic pathway (4, 20). In contrast, an oral
D-glutamate load would be delivered to the liver, which
expresses both D-glutamate cyclotransferase activity
(12) and D-,L-glutamine synthetase
(11). The D-pyrrolidone-carboxylic acid formed
in the liver is effectively cleared through the kidney, as demonstrated
in Fig. 4. On the other hand, D-glutamate incorporated into
D-glutamine by hepatic
D-,L-glutamine synthetase (11)
would present another problem, because it can be readily hydrolyzed to
D-glutamate at specific sites expressing GGt activity. In
the present study this was demonstrated for the kidneys by the large excretion of D-glutamate after an oral
D-glutamine load (Fig. 5). If D-glutamine was
indeed formed after the D-glutamate load, then intraluminal
hydrolysis might explain the increase in D-glutamate's FE
(Fig. 4). In addition, the antiluminal D-glutamine
hydrolysis (25) and D-glutamate uptake
(20), coupled to renal D-glutamate cyclotransferase activity (20), would result in release of
D-pyrrolidone-carboxylic acid into the renal vein.
Consequently, any D-pyrrolidone-carboxylic acid formed in
the kidney from D-glutamine would join that produced by the
liver for renal filtration and excretion. Regardless of the relative
contributions of the liver and kidneys, over the 3 h ~20% of
the D-glutamate load could be accounted for as
D-pyrrolidone-carboxylic acid and <5% as
D-glutamate.
How the kidney handles D-glutamate was another objective of
our study. In the time controls, <5% of the filtered
D-glutamate was excreted, consistent with previous studies
in the rat demonstrating that filtered D-glutamate is
largely, if not entirely, reabsorbed (4). Although the
anionic amino acid transport system XAG
displays a
stereospecific anomoly for D-aspartate (8),
the affinity of XAG subtypes for
D-glutamate is far below that for L-glutamate
(1). Nevertheless, there appears to be a transporter
present in the kidney capable of high-affinity D-glutamate
transport, possibly xc, that accounts for the
reabsorption; it is also possible that this transporter is inhibited by
D-pyrrolidone-carboxylic acid. After the
D-glutamate load, there was an apparent progressive diminution of reabsorption from 95, 60, and 32, to only 5% of the
filtered D-glutamate. Because the amount filtered did not change, the reduced reabsorption does not reflect saturation of this
carrier and points to either a reduction in the reabsorption of the
filtered D-glutamate or de novo formation of
D-glutamate within the kidney (25). These
observations may explain why some studies observed filtration and
excretion without reabsorption (2, 16), whereas others
found a significant renal reabsorptive capacity (4, 6).
Suppressing reabsorption of D-glutamate could account for
the decreasing reabsorption over the course of the study. For example, if D-pyrrolidone-carboxylic acid inhibits glutamate
transport via the high-affinity transporter, then the progressively
increasing D-pyrrolidone-carboxylic acid concentration (mM
concentrations in the urine) in the tubule lumen could become more
effective in inhibiting glutamate reabsorption; this would explain the
progressive increase in FE with time, as actually observed in Fig. 4.
Curiously, there are no studies as to whether
D-pyrrolidone-carboxylic acid can inhibit glutamate
transport. However, it has been shown that micromolar concentrations of
the L-isomer of pyrrolidone-carboxylic acid competitively
inhibit the high-affinity uptake of radiolabeled L-glutamate into rat striatal synaptosomes and,
surprisingly, as effectively as L-glutamate
(7). If the D-pyrrolidone-carboxylic acid has
a similar, or, more likely, lesser reactivity with one or more
glutamate transporters, then inhibition of glutamate uptake could occur
and might explain reductions in both D- and
L-glutamate reabsorption. Further studies into whether
D-pyrrolidone-carboxylic acid can inhibit either
D- or L-glutamate transport would seem warranted.
The reduction in apparent reabsorption might also reflect the
intraluminal hydrolysis of D-glutamine, leading to the
formation of D-glutamate, as previously demonstrated in the
functioning rat kidney (25). Because
D-glutamine is a substrate for GGt's glutaminase activity
(23, 25), the formation of D-glutamate as the
result of filtered D-glutamine hydrolysis might also
contribute to the rising D-glutamate excretion. Regardless
of the actual mechanism(s), the rise in D-glutamate
excretion is obviously another mechanism for eliminating
D-glutamate and maintaining a low plasma concentration.
The fact that plasma D-glutamate concentration is so
tightly regulated after an oral D-glutamate load points to
an intricate regulatory mechanism for maintaining the normal low plasma
concentration. This regulation is more significant because the
D-glutamate is not metabolized by D-amino acid
oxidase (2, 24), as are other D-amino acids.
The present study shows that this regulation involves both
D-glutamate transport and metabolism and enhanced renal
clearance. The availability of an active metabolic pathway(s), coupled
to a high-affinity transporter, clearly would provide a tighter
regulation of the plasma D-glutamate concentration. A
second mechanism serving to prevent the rise in plasma
D-glutamate is the enhanced renal clearance, which although
quantitatively less significant than conversion to
D-pyrrolidone-carboxylic acid, would nevertheless contribute at the critical time that the metabolic disposal reaches a
maximum. This of course might be fortuitous, or might be by design if
the tubular interactions proposed actually occur. Nevertheless, together the two regulatory mechanisms would be reinforcing and guarantee effective removal of the D-glutamate, thereby
defending the low plasma D-glutamate concentrations.
| |
ACKNOWLEDGEMENTS |
We thank the Biomedical Research Foundation of Northwest Lousiana;
Dr. Neil Granger, Chairman of the Molecular and Cellular Physiology Department at LSUHSC; and the Southern Arizona Foundation for their support.
| |
FOOTNOTES |
Address for reprint requests and other correspondence: T. C. Welbourne, Dept. of Molecular and Cellular Physiology, LSUHSC, Shreveport, LA 71130 (E-mail twelbo{at}lsumc.edu).
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 28 April 2000; accepted in final form 4 October 2000.
| |
REFERENCES |
1.
Arriza, JL,
Fairman WA,
Wadiche JI,
Murdoch GH,
Kavanaugh MP,
and
Amara SG.
Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex.
J Neurosci
14:
5559-5569,
1994[Abstract].
2.
Berg, CP.
Physiology of the D-amino acids.
Physiol Rev
33:
145-188,
1953[Free Full Text].
3.
Bruckner, H,
and
Hausch M.
Gas chromatographic characterization of free D-amino acids in the blood serum of patients with renal disorders and of healthy volunteers.
J Chromatogr
614:
7-17,
1993[ISI][Medline].
4.
Carter, P,
and
Welbourne TC.
Glutamate transport asymmetry in renal glutamine metabolism.
Am J Physiol Endocrinol Metab
274:
E877-E884,
1998[Abstract/Free Full Text].
5.
Christensen, HN.
What is the physiological origin of free D-amino acids in mammals?
Nutr Rev
50:
294-295,
1992[ISI][Medline].
6.
Dantzler, WH,
and
Silbernagl S.
Specificity of amino acid transport in renal papilla: microperfusion of Henle's loops and vasa recta.
Am J Physiol Renal Fluid Electrolyte Physiol
261:
F495-F504,
1991[Abstract/Free Full Text].
7.
Dusticer, N,
Kerkerian L,
Errami M,
and
Nieoullon A.
Effects of pyroglutamic acid on corticostriatal glutamatergic transmission.
Neuropharmacology
24:
903-908,
1985[ISI][Medline].
8.
Gazzola, GC,
Dall'Asta V,
Bussolati O,
Makowske M,
and
Christensen HN.
A stereospecific anomaly in dicarboxylic amino acid transport.
J Biol Chem
256:
6054-6059,
1981[Free Full Text].
9.
Kera Aoyama, YH,
Matsumura H,
Hasegawa A,
Nagasaki H,
and
Yamada RH.
Presence of free D-glutamate and D-aspartate in rat tissues.
Biochem Biophys Acta
1243:
282-286,
1995.
10.
Man, EH,
and
Bada JL.
Dietary D-amino acids.
Ann Rev Nutr
7:
209-225,
1987[ISI][Medline].
11.
Meister, A.
Catalytic mechanism of glutamine synthetase; overview of glutamine metabolism.
In: Glutamine: Metabolism, Enzymology, and Regulation, edited by Palacios R,
and Mora J.. New York: Academic, 1980, p. 1-40.
12.
Meister, A,
and
Bukenberger MW.
Enzymatic conversion of D-glutamic acid to D-pyrrolidone carboxylic acid by mammalian tissues.
Nature
194:
557-559,
1962.
13.
Orlowski, M,
and
Meister A.
Partial reactions catalyzed by -glutamylcysteine synthetase and evidence for an activated glutamate intermediate.
J Biol Chem
246:
7095-7105,
1971[Abstract/Free Full Text].
14.
Palekar, AG,
Tate SS,
Sullivan JF,
and
Meister A.
Accumulation of 5-oxo-L-proline and 5-oxo-D-proline in the blood plasma in end stage renal disease.
Biochem Med
14:
339-345,
1975[ISI][Medline].
15.
Pan, ZZ,
Tong G,
and
Jahr CE.
A false transmitter at excitatory synapses.
Neuron
11:
85-91,
1993[ISI][Medline].
16.
Putnam, FW,
Miyake A,
and
Meyer F.
The metabolism of DL-glutamic acid-1-14C in man.
J Biol Chem
231:
657-669,
1958[Free Full Text].
17.
Ratner, S.
Conversion of D-glutamic acid to pyrrolidone carboxylic acid by the rat.
J Biol Chem
152:
559-564,
1944[Free Full Text].
18.
Rosenberg, PA,
Amin S,
and
Leitner M.
Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture.
J Neurosci
12:
56-61,
1992[Abstract].
19.
Schleifer, KH,
and
Kandler O.
Peptidoglycan types of bacterial cell walls and their taxomomic implications.
Bacteriol Rev
36:
407-477,
1972[Free Full Text].
20.
Schuldt, S,
Carter P,
and
Welbourne T.
Glutamate transport asymmetry and metabolism in the functioning kidney.
Am J Physiol Endocrinol Metab
277:
E439-E466,
1999[Abstract/Free Full Text].
21.
Sekura, R,
van der Werf P,
and
Meister A.
Mechanism and significance of the mammalian pathway for elimination of D-glutamate inhibition of glutathione synthesis by D-glutamate.
Biochem Biophys Res Comm
71:
11-18,
1976[ISI][Medline].
22.
Silbernagl, S.
Kinetics and localization of tubular reabsorption of "acidic" amino acids. A microperfusion and free flow micropuncture study in rat kidney.
Pfluegers Arch
396:
218-224,
1983[ISI][Medline].
23.
Thompson, G,
and
Meister A.
Modulation of -glutamyltranspeptidase activities by hippurate and related compounds.
J Biol Chem
255:
2109-2113,
1980[Free Full Text].
24.
Van der Werf, P,
and
Meister A.
The metabolic formation and utilization of 5-oxo-L-proline (L-pyroglutamate, pyrrolidone carboxylic acid).
Adv Enzymol
43:
519-556,
1975.
25.
Welbourne, T,
and
Dass P.
Mechanism of the acidosis induced adaptation in renal -glutamyltransferase.
Life Sci
28:
1219-1224,
1981[ISI][Medline].
26.
Wilson, WE,
and
Koeppe RE.
The metabolism of D- and L-glutamic acid in the rat.
J Biol Chem
236:
365-369,
1961[Free Full Text].
Am J Physiol Endocrinol Metab 280(2):E214-E220
0193-1849/01 $5.00
Copyright © 2001 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
