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【摘要】 Ureagenesis in the liver consumes up to 1,000 mmol of /day in humans as a result of + urea + CO 2 + 3H 2 O. Whether the liver contributes to the regulation of acid-base equilibrium by controlling the rate of ureagenesis and, therefore, consumption in response to changes in plasma acidity has not been adequately evaluated in humans. Rates of ureagenesis were measured in eight healthy volunteers during control, chronic metabolic acidosis (induced by oral administration of CaCl 2 3.2 mmol·kg body wt -1 ·day -1 for 11 days), and recovery as well as during bicarbonate infusion (200 mmol over 240 min; acute metabolic alkalosis). Rates of ureagenesis were correlated negatively with plasma concentration both during adaption to metabolic acidosis and during the chronic, steady-state phase. Thus ureagenesis, an acidifying process, increased rather than decreased in metabolic acidosis. During bicarbonate infusion, rates of ureagenesis decreased significantly. Thus ureagenesis did not appear to be involved in the regulated elimination of excess. The finding of a negative correlation between ureagenesis and plasma concentration over a wide range of concentrations, altered both chronically and acutely, suggests that the ureagenic process per se is maladaptive for acid-base regulation and that ureagenesis has no discernible homeostatic effect on acid-base equilibrium.
【关键词】 acidosis net acid excretion alkalosis
UREAGENESIS IN THE LIVER CONSUMES up to 1,000 mmol of bicarbonate/day in humans as a result of + urea + CO 2 + 3H 2 O.
The liver is believed to contribute to regulation of acid-base equilibrium by controlling the rate of ureagenesis and, therefore, consumption in response to changes in plasma acidity. Accordingly, present teaching attributes increased rates of ureagenesis to the defense against overload (metabolic alkalosis) and decreased rates to conservation of in the defense against metabolic acidosis ( 9 ). This clinicopathophysiological concept challenges the traditional view that regulation of acid excretion by the kidney is the principal, if not exclusive, mechanism regulating acid-base balance ( 18, 24 ).
Two pathways compete for hepatic elimination: ureagenesis (an -consuming process, metabolically equivalent to proton generation) and net glutamine synthesis, which does not consume directly. 1 Based on extensive in vitro and in vivo measurements of ureagenesis in acidotic and normal rats, it has been argued that the regulation of acid-base composition is accomplished via control of the rate of ureagenesis ( 3, 9 ). In acute metabolic acidosis in rats induced by HCl administration, results have been modestly supportive of the hepatic regulator theory, as both decreases ( 3, 5 ) and no change ( 8, 13 ) in ureagenesis were reported.
In chronic metabolic acidosis (CMA) in rats, however, the results have been reasonably uniform in support of the hepatic acid-base control theory inasmuch as significant reductions in urea production have been observed consistently ( 6, 19, 23, 27 ), although one of the reports found only a transient reduction ( 19 ). Hepatic control of acid-base homeostasis may also operate in chronic metabolic alkalosis, as chronic alkali exposure in the walking catfish has been recently reported to greatly increase ureagenesis ( 25 ). The in vivo data favoring hepatic control of plasma acid-base composition as mediated by acidbase effects on ureagenesis have been confirmed consistently in both in vitro and perfused liver studies in rats (reviewed in Ref. 19 ). In four normal human subjects administered 200 mmol/day of HCl for 4 days, a significant reduction in urinary urea excretion was observed on day 4, but interpretation is hampered by a lack of standard metabolic control ( 10 ).
A regulated shift from ureagenesis to increased hepatic glutamine release during an acid load would thus ensure continued elimination of toxic while simultaneously decreasing consumption and, thereby, conserving /base stores. Additional support for this thesis comes from perfused liver studies in rats showing that flux through glutamine synthetase is stimulated by a decrease in ambient concentration and that further increases in hepatic glutamine release in acidosis may be related to an observed diminished nitrogen flux through the hepatic phosphate-dependent glutaminase pathway (decreased ureagenesis) ( 1, 11, 12 ). The findings of both increased flux in the glutamine synthetase pathway and decreased flux in the glutaminase pathway in perfused liver have been confirmed with detailed in vitro flux measurements in incubated liver cells from rats with CMA ( 22 ). In vivo data in rats with either acute metabolic acidosis or CMA, however, have not confirmed acidosis-induced increases in hepatic glutamine release ( 5, 6 ).
The purpose of the present studies was, therefore, to evaluate the direction and magnitude of changes in the rates of ureagenesis in response to decreases (acutely and chronically) and increases in plasma concentration. If ureagenesis contributes to regulation of acid-base equilibrium in humans, a direct correlation among rates of ureagenesis and plasma concentration (or body base content in the steady state) would be expected.
METHODS
Eight healthy male volunteers (71.1 ± 7.9 kg, aged 24-26 yr) were studied under metabolic balance conditions. The metabolic diet provided [per kilogram body weight (BW) and day] 1.8 mmol sodium, 1.05 mmol potassium, 45 ml water, 0.84 g protein, and 36 kcal. CMA was induced by oral administration of 3.2 mmol of CaCl 2 ·kg BW -1 ·day -1 dissolved in water (flavored with strawberry syrup) and ingested in six divided doses. The metabolic diet was supplemented with neutral oral sodium phosphate (0.53 mmol·kg -1 ·day phosphate -1 ) during control and the first acidosis period ( A1 ), yielding a total phosphate content of 0.65 mmol·kg -1 ·day -1 (20.2 mg·kg -1 ·day -1 ). During the second acidosis period ( A2 ) and during recovery, the sodium phosphate supplement was removed to provide a low-normal daily phosphate content of 0.12 mmol·kg -1 ·day -1. The oral phosphate supplement was administered between CaCl 2 doses. Oral administration of CaCl 2 induces metabolic acidosis by generation of poorly absorbable CaCO 3 complexes in the intestine, effectively interrupting reabsorption of luminal of pancreatic origin ( 15 ).
Arterialized venous blood T CO 2 and pH analysis, determination of plasma and urine electrolytes, and analysis of renal acid excretion and creatinine were performed as previously described ( 17 ). Plasma ammonia/um, glutamine, glutamate, and urea analyses were performed by HPLC after automated precolumn separation ( 26 ). Determination of nitrogen in stool was performed with the Kjeldahl method ( 14 ). Metabolic period demarcation in stool production was by radiographic identification of orally ingested pellets present in stool ( 7 ).
Urea was assumed to be distributed uniformly in total body water. Water space was assumed to be 65% of BW (kg). The "urea production rate" was calculated as the 24-h urinary excretion rate plus the change in urea pool content. The change in urea pool was computed as plasma urea concentration x 0.65 BW (kg) at the beginning - plasma urea concentration x 0.65 BW (kg) at the end of the 24-h sampling periods. The term urea production rate reflects net production or appearance of urea in extracellular fluid and, when used to compute values on a daily basis, is arithmetically similar to urinary excretion, being adjusted only for daily variation in total body water urea pool size.
Values are given as means ± SE. Statistical analysis was performed by ANOVA for repeated measurements.
RESULTS
Tables 1 and 2 illustrate that in the eight healthy human subjects studied, a chronic exogenous acid load administered by CaCl 2 ingestion (3.2 mmol·kg BW -1 ·day -1 ) resulted in chronic, hyperchloremic metabolic acidosis of moderate severity, a mean decrease in plasma concentration () by 6.8 ± 0.8 mmol/l ( P < 0.001). A large increase in renal net acid excretion (NAE) was observed (increase from 59 ± 10 to 186 ± 12 mmol/day, P = 0.005). Reduction of phosphate intake during the second phase of acidosis (decrease in phosphate intake from 0.65 in A1 to 0.12 mmol·kg BW -1 ·day -1 in A2, Table 1 ) did not result in significant changes in the severity of acidosis or magnitude of NAE. However, the components of NAE were quantitatively altered. The anticipated significant decrease in renal titratable acid excretion during A2 was offset by a significant increase in renal excretion.
Table 1. Effect of CaCl 2 administration on steady-state blood and urinary acid-base composition
Table 2. Effect of CaCl 2 on steady-state plasma electrolytes, creatinine clearance, and body weight
Tables 3 and 4 show the changes in urine and stool electrolyte excretion. Tables 5 and 6 show that plasma concentrations of the nitrogen-containing substances glutamine, glutamate,, and urea did not differ significantly from control during acidosis. As expected, due to enhanced renal ammoniagenesis, urinary glutamine and glutamate excretion rates decreased, whereas urinary excretion increased in response to CaCl 2 administration.
Table 3. Effect of CaCl 2 on steady-state urinary electrolyte excretion
Table 4. Effect of CaCl 2 on stool electrolyte and nitrogen excretion
Table 5. Effect of CaCl 2 administration on nitrogen-containing plasma metabolites
Table 6. Effect of CaCl 2 administration on nitrogen-containing metabolites in urine and stool
The subjects were in approximately neutral nitrogen balance during the control period (Tables 3, 4, 5, 6 ): mean daily nitrogen excretion for urine was computed as 44 mmol/day as NH4, 639 mmol/day as urea, 46 mmol/day as creatinine, and 14 mmol/day as uric acid, totaling 743 mmol of urinary nitrogen/day. When mean stool nitrogen (117 mmol/day) is added, total nitrogen excretion averaged 860 mmol/day. Because measured dietary protein intake (0.84 g·kg -1 ·day -1 ) comprised 9.556 g nitrogen/day in our subjects (6.25 g protein/g nitrogen), total nitrogen intake averaged 683 mmol/day for a nominal mean nitrogen balance that approached neutral (-60 ± 22 mmol/day).
Induction of CMA (decrease in plasma ) resulted in an immediate and significant increase in urea production that persisted for the duration of CMA ( Fig. 1, depicting daily urea production rates and daily mean plasma ). During the recovery phase, CaCl 2 administration was discontinued, resulting in an increase in plasma ( Fig. 1 ) with a rapid and sustained, significant decrease in urea production. During a separate infusion protocol performed on day 4 of recovery (200 mmol of NaHCO 3 /study subject infused over 240 min), plasma increased further from 27.5 ± 0.9 to 32.5 ± 1.4 mmol/l. As illustrated in Fig. 2, the increase in plasma into the frankly alkalotic range was associated with a further and significant decline in the urea production rate.
Fig. 1. Daily urea production rates in control, acidosis, and recovery periods. X P < 0.05 compared with the mean of the last 3 days during control.
Fig. 2. Effect of acute metabolic alkalosis (bicarbonate infusion; Inf) on urea production rate. X P < 0.05.
Figure 3 depicts the daily changes in urea production rate compared with the original control period in the case of the two acidosis periods and compared with the last 2 acidosis days in the case of the recovery period. Induction of acidosis increased the urea production rate, resulting in a significant cumulative increase in production by 500 mmol urea over the 11 days of acidosis (CaCl 2 ingestion). By the end of period A2, daily nitrogen balance had become significantly negative vs. that of the control period (-123 ± 48 mmol/day, P < 0.025).
Fig. 3. Daily changes in urea production (open bars) and cumulative sum of the daily changes in urea production for acidosis and recovery periods, respectively (line and ). X P < 0.05 compared with the mean in the control period. X P < 0.05 recovery compared with the mean of the last 2 days of acidosis. For the cumulative change in urea production, comparison is made with 0.
DISCUSSION
The novel results of these studies are 1 ) CMA in humans leads to stimulated rates of ureagenesis and 2 ) ureagenesis is inhibited during acute metabolic alkalosis (bicarbonate infusion); thus 3 ) rates of ureagenesis in humans are inversely related to plasma concentration in both chronic and acute metabolic disturbances of acid-base equilibrium.
Acidosis was induced by oral administration of CaCl 2, an agent that interrupts reabsorption of intestinal luminal of pancreatic origin ( 15 ). This model of CMA was chosen to avoid alterations in nitrogen intake attendant to NH 4 Cl acidosis and its potential as an ureagenic stimulus. The use of disparate phosphate intakes to modulate urinary phosphate excretion enabled the experimental partitioning of NAE among and titratable acid (comprised of ) and thus afforded an examination of ureagenesis during experimentally controlled differences in urinary excretion as it might impact the partitioning of total nitrogen excretion among and urea. As indicated by Figs. 1 and 3, changes in dietary phosphate did not result in significant changes in ureagenesis, despite significant changes in and titratable acid (and phosphate) excretion.
During the acidosis periods, the maximum amount of consumed (or, equivalently, protons produced) via ingestion of CaCl 2 in a 70-kg subject is estimated as 4,928 mmol (assuming complete complexing of by the 3.2 mmol CaCl 2 ·kg BW -1 ·day -1 over 11 days). The surplus 500 mmol of urea generated during this time ( Fig. 3 ) necessitated the consumption of an additional 1,000 mmol of during that period, a process directionally maldapative for acid-base homeostasis.
While the directional differences observed in the ureagenic response to CMA in the humans of the present study and those reported previously in rats ( 6, 19, 23, 27 ) remain unresolved, the degree of acidosis reported in the rat studies was modest and of lesser magnitude than that in the human data. Future studies exploring CMA in multiple species and over a range of magnitudes will be of great interest.
Thus our results indicate that the rate of ureagenesis in humans is not geared to subserve acid-base homeostasis but is directionally controlled by the need to remove or retain nitrogen (e.g., ) as results from the net effect of ongoing protein anabolism or catabolism dictated by the metabolic state of the organism and independent of metabolic acid-base status. CMA has been reported to result in negative nitrogen balance in humans ( 4 ) and confirmed in the current study due to net catabolism of endogenous protein, a process attributable in rats to glucocorticoid-dependent proteolysis via the ubiquitin/proteasome pathway ( 20 ). Thus the changes in catabolic nitrogen waste load during changes in acid-base equilibirum exert a dominant role in the regulation of ureagenesis and override any possible counterbalancing effects of increases and decreases in systemic content, even under ambient acid-base conditions (acidosis) that have been demonstrated in vitro and in vivo in rats to inhibit ureagenesis ( 6, 19, 23, 27 ) and to stimulate nitrogen flux through hepatic glutamine synthetase and increase hepatic glutamine release during liver perfusion ( 1, 11, 12 ) and in vitro incubation ( 22 ).
In summary, urea production rates in humans are correlated inversely, and not directly as reported in rats, with base content (plasma ) both during acute and chronic alterations. There is thus no discernible role for ureagenesis in the regulation of acid-base equilibrium in humans. The demonstrated increased urea production in response to acid loads in humans, in itself, consumes and thus would potentially aggravate acidosis by causing further depletion of the body's diminished content. It is thus likely that other quantitatively important metabolic processes (e.g., oxidation of glutamate or other alkali-generating reactions) operate in CMA to offset the accelerated proton production of ureagenesis. The nature and regulation of these pathways with respect to maintenance or defense of acid-base equilibrium require further investigation.
The finding that the rate of ureagenesis decreased when body and plasma content increased (recovery from metabolic acidosis and induction of metabolic alkalosis) provides additional evidence against a role for ureagenesis in the regulated elimination of base and, therefore, in the prevention or attenuation of metabolic alkalosis (primary elevation of base content).
ACKNOWLEDGMENTS
The authors thank Dr. C. Rothen for performing stool nitrogen and electrolyte analysis and Isabelle Grilli and René Groell for laboratory assistance.
GRANTS
This study was supported by institutional financial resources (Medizinische Universitaetsklinik Kantonsspital Bruderholz) including professional fees.
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作者单位：1 Medizinische Unversitaetsklinik, Kantonsspital Bruderholz, CH-4101 Bruderholz/Switzerland; and 2 Genentech, Incorporated, South San Francisco, California 94080