Site-directed mutagenesis of predicted active site residues in glutamate carboxypeptidase II.

Glutamate carboxypeptidase II (GCP II) catalyzes the extracellular hydrolysis of the neuromodulator N-acetyl-aspartylglutamate to N-acetyl-aspartate and glutamate. GCP II also hydrolyzes gamma-glutamyl bonds in folylpolyglutamate. The predicted amino acid sequence of GCP II displays similarities to aminopeptidases from Streptomyces griseus and Vibrio proteolyticus, whose crystal structures have been determined. These aminopeptidases are cocatalytic zinc metallopeptidases belonging to the peptidase family M28. Specific zinc and substrate ligands have been proposed in GCP II based on the amino acid sequence alignment to these M28 family members. In the present study, site-directed mutagenesis has been used to test the assignment of these putative ligands in human GCP II. Substitutions to the five putative zinc ligands resulted in severely reduced enzyme activity, although mutant protein was expressed as demonstrated by immunoblot analysis. In addition, substitutions of amino acids near the putative zinc ligands have identified other specific residues important for enzyme structure and/or function. Substitutions to putative substrate ligands were less perturbing, and increases in Km were observed for substitutions that introduced a large charge perturbation (e.g., Lys to Glu). The results from substitutions at the proposed zinc and substrate ligands are consistent with the assignment of these residues and suggest that GCP II has a three-dimensional structure similar to other members of the peptidase family M28.

Lack of effect of olanzapine on the pharmacokinetics of a single aminophylline dose in healthy men.

STUDY OBJECTIVE: To test whether olanzapine, an atypical antipsychotic, is an inhibitor of cytochrome P450 (CYP) 1A2 activity, we conducted a drug interaction study with theophylline, a known CYP1A2 substrate. DESIGN: Two-way, randomized, crossover study. SETTING: Clinical research laboratory. SUBJECTS: Nineteen healthy males (16 smokers, 3 nonsmokers). INTERVENTIONS: Because the a priori expectation was no effect of olanzapine on theophylline pharmacokinetics, a parallel study using cimetidine was included as a positive control. In group 1, 12 healthy subjects received a 30-minute intravenous infusion of aminophylline 350 mg after 9 consecutive days of either olanzapine or placebo. In group 2, seven healthy subjects received a similar aminophylline infusion after 9 consecutive days of either cimetidine or placebo. MEASUREMENTS AND MAIN RESULTS: Concentrations of theophylline and its metabolites in serum and urine were measured for 24 and 72 hours, respectively. Plasma concentrations of olanzapine and its metabolites were measured for 24 hours after the next to last dose and 168 hours after the last olanzapine dose. Olanzapine did not affect theophylline pharmacokinetics. However, cimetidine significantly decreased theophylline clearance and the corresponding formation of its metabolites. Urinary excretion of theophylline and its metabolites was unaffected by olanzapine but was reduced significantly by cimetidine. Steady-state concentrations of olanzapine (15.3 ng/ml), 10-N-glucuronide (4.9 ng/ml), and 4'-N-desmethyl olanzapine (2.5 ng/ml) were observed after olanzapine 10 mg once/day and were unaffected by coadministration of theophylline. CONCLUSION: As predicted by in vitro studies, steady-state concentrations of olanzapine and its metabolites did not affect theophylline pharmacokinetics and should not affect the pharmacokinetics of other agents metabolized by the CYP1A2 isozyme.

Quantification of creatinine kinetic parameters in patients with acute renal failure.

BACKGROUND: Urea kinetic modeling (UKM) and creatinine (Cr) kinetic modeling (CKM) are used in the nutritional evaluation of end-stage renal disease (ESRD) patients. Both the UKM-derived normalized protein catabolic rate (nPCR) and the CKM-derived estimate of lean body mass (LBM) may also provide important information in critically ill acute renal failure (ARF) patients. Estimation of LBM may be particularly useful as previous data demonstrate that malnutrition adversely influences outcome in ARF patients. METHODS: Eleven critically ill ARF patients (age 52 +/- 21 years; mean +/- SD) treated with continuous venovenous hemofiltration (CVVH) were the study group. They were analyzed at steady state with a single-pool variable-volume model that determined the creatinine generation rate (GCr) by a methodology that we have previously described. RESULTS: The CVVH ultrafiltrate production rate was 913 +/- 49 ml/hr, yielding a blood Cr clearance of 15.2 +/- 0.9 ml/min and a steady state serum Cr of 3.4 +/- 1.7 mg/dl. Daily creatinine generation normalized to body wt (creatinine index: CI) was 6.3 +/- 0.8 and 10.6 +/- 3.0 mg/kg/day for females (N = 4) and males (N = 7), respectively (P < 0.05). Estimated mean LBM was 30.0 +/- 2.0 and 41.2 +/- 7.0 kg in females and males, respectively (P < 0.05), while the same parameter normalized to body wt was 0.50 +/- 0.05 and 0.52 +/- 0.10, respectively. These values are substantially lower than those previously reported for both normal and ESRD patients. Regression analysis demonstrated both GCr (r2 = 0.96; P < 0.001) and LBM (r2 = 0.96; P < 0.001) were significantly correlated with steady state serum Cr in a linear manner. However, no significant correlation (r2 = 0.06; P = 0.24) between nPCR and CI was observed. CONCLUSIONS: These data suggest critically ill ARF patients have severe somatic protein depletion. This malnourished state is likely due to deficits established prior to the development of ARF, such as those secondary to underlying chronic illnesses or prolonged hospitalization, and deficits related to acute hypercatabolism. Quantitative assessment of malnutrition in ARF patients with this CKM-based methodology may permit a better understanding of predisposing factors and, consequently, facilitate the development of interventions designed to prevent malnutrition in these patients.

Renal replacement therapy quantification in acute renal failure.

Recent results suggest that RRT delivery affects outcome in critically ill ARF patients. These data have generated interest in the use of RRT quantification methods, originally developed for ESRD patients, in ARF. However, the fundamental differences between ARF and ESRD, with respect to both patient and therapy characteristics, must be fully appreciated before making this extrapolation. These differences may render many of the simplified ESRD quantification formulae of little use in ARF. As is the case in ESRD, the use of clearance-based methods to compare disparate therapies is problematic in ARF. Although the optimal technique for RRT quantification in ARF remains to be defined, dialysate-side quantification may be the most rational approach for the future, as has been suggested for ESRD patients [43].

The role of renal replacement therapy quantification in acute renal failure.

The recognition that both morbidity and mortality are inversely related to delivered hemodialysis (HD) dose in end-stage renal disease (ESRD) patients has substantially changed clinical practices in the United States. A number of quantification techniques, which differ greatly in complexity and sophistication, are now used in ESRD patients. Investigators recently have attempted to extrapolate some of these ESRD quantification methods to the acute renal failure (ARF) setting. This review focuses on these recent attempts. Both patient-related and renal replacement therapy (RRT)-related differences in ESRD and ARF are discussed. In addition, the potential pitfalls of extrapolating certain ESRD quantification methods to RRT in ARF are discussed. Prescription considerations for both intermittent HD (IHD) and continuous RRT (CRRT) are presented. The optimal technique for RRT quantification in ARF remains to be determined.

Extracorporeal therapy requirements for patients with acute renal failure.

Renal replacement therapy (RRT) requirements for critically ill patients with acute renal failure (ARF) depend on numerous factors, including the degree of hypercatabolism, patient size, and desired level of metabolic control. However, the current practice at many institutions is to prescribe generally similar amounts of RRT to ARF patients essentially without regard for the above factors. In this study, a computer-based model designed to permit individualized RRT prescription to ARF patients was developed. The critical input parameter is the desired level of metabolic control, which is the time-averaged BUN (BUNa) or steady-state BUN (BUNs) for intermittent hemodialysis (IHD) or continuous RRT (CRRT), respectively. The basis for the model was a group of 20 patients who received uninterrupted CRRT for at least 5 days. In these patients, the normalized protein catabolic rate (nPCR) increased linearly (r = 0.974) from 1.55 +/- 0.14 g/kg per day (mean +/- SEM) on day 1 to 1.95 +/- 0.15 g/kg per day on day 6. The daily urea generation rate (G), determined from the above linear relationship, was utilized to produce BUN versus time curves by the direct quantification method for simulated patients of varying dry weights (50 to 100 kg) who received variable CRRT urea clearances (500 to 2000 ml/h). Steady-state BUN versus time profiles for the same simulated patient population treated with IHD regimens (K = 180 ml/min, T = 4 h) of variable frequency were generated by use of a variable-volume, single-pool kinetic model. From these profiles, regression lines of required IHD frequency (per week) versus patient weight for desired BUNa values of 60, 80, and 100 mg/dl were obtained. Regression lines of required CRRT urea K (ml/h) versus patient weight for desired BUNs values of 60, 80, and 100 mg/dl were also generated. For the attainment of intensive IHD metabolic control (BUNa = 60 mg/dl) at steady state, a required treatment frequency of 4.4 dialyses per week is predicted for a 50-kg patient. However, the model predicts that the same degree of metabolic control cannot be achieved even with daily IHD therapy in patients > or = 90 kg. On the other hand, for the attainment of intensive CRRT metabolic control (BUNs = 60 mg/dl), required urea clearance rates of approximately 900 ml/h and 1900 ml/h are predicted for 50- and 100-kg patients, respectively. This model suggests that, for many patients, rigorous azotemia control equivalent to that readily attainable with most CRRT can only be achieved with intensive IHD regimens. Following prospective clinical validation, this methodology may be a useful RRT prescription tool for critically ill ARF patients.

Dialysis prescription and kinetics in acute renal failure.

The recognition that both morbidity and mortality are inversely related to delivered hemodialysis (HD) dose in end-stage renal disease (ESRD) patients has substantially changed clinical practices in the United States. A number of quantification techniques, which differ greatly in complexity and sophistication, are now used in ESRD patients. Investigators recently have attempted to extrapolate some of these ESRD quantification methods to the acute renal failure (ARF) setting. This review focuses on these recent attempts. Both patient-related and renal replacement therapy (RRT)-related differences in ESRD and ARF are discussed. In addition, the potential pitfalls of extrapolating certain ESRD quantification methods to RRT in ARF are discussed. Prescription considerations for both intermittent HD (IHD) and continuous RRT (CRRT) are presented. Finally, recent data suggesting survival in critically ill ARF patients is directly correlated with delivered therapy dose are reviewed. The optimal technique for RRT quantification in ARF remains to be determined.

Effects of dialysis membrane on intradialytic vancomycin administration.

STUDY OBJECTIVE: To quantify the influence of hemodialyzers on vancomycin removal when the drug was infused during hemodialysis. DESIGN: Prospective, controlled, crossover study with three arms. SETTING: A university-affiliated medical center. PATIENTS: Eight subjects receiving outpatient hemodialysis. INTERVENTIONS: The three treatment arms were vancomycin 1000 mg infused after dialysis was completed (control), and the same dosages infused during the last hour of hemodialysis with a cellulose triacetate (CT) and a cellulose acetate (CA) hemodialyzer. MEASUREMENTS AND MAIN RESULTS: The areas under the curve from time zero to 44 hours (AUC0-44 hrs) for the three study arms were significantly different (p < 0.05), with the mean vancomycin AUC0-44 hrs being significantly lower when administered during CT and CA dialysis (73.7% and 87.2% of control; p < 0.05 vs control). The mean vancomycin peak concentration achieved during CT dialysis was significantly lower than for the CA and control arms (20.5, 23.9, 27.0 mg/L, respectively). Forty-four-hour postinfusion concentrations were similarly lower. CONCLUSION: Clinicians should recognize that the composition of the hemodialyzer significantly influences vancomycin serum concentrations when the drug is administered during hemodialysis.

Impact of the nutritional regimen on protein catabolism and nitrogen balance in patients with acute renal failure.

BACKGROUND: Patients with acute renal failure are in substantial negative nitrogen balance as a result of their extremely high protein catabolic rates. We prospectively evaluated a series of patients with acute renal failure managed with continuous venovenous hemofiltration to determine which nutritional and nonnutritional variables might influence protein catabolism and nitrogen balance. METHODS: Forty consecutive patients (aged 52 +/- 20 years; mean +/- SD) were monitored for 357 treatment days (average treatment duration 8.9 +/- 8.6 days). All data (including nutritional regimen, laboratory values, APACHE II score, administered blood products, hemofiltration parameters, and medications) were collected daily. RESULTS: For all patients, the mean normalized protein catabolic rate was 1.4 +/- 0.5 g/kg per day. The rate did not differ between those who received nutrition support and those who did not. The net nitrogen deficit was less in those patients receiving nutrition support (-6.0 +/- 5.2 vs -14.0 +/- 5.6 g N/d; p = .02). Using regression techniques (adjusted for the within-person correlation and the previous day's normalized protein catabolic rate), the level of protein and energy provision and the interaction between protein and energy provision were predictive of the normalized protein catabolic rate. Predicted values, using this equation, suggest that at low protein administration rates (< 1 g/kg per day), increasing energy provision may reduce the protein catabolism. However, at this level of protein provision, patients remain in negative nitrogen balance. At protein administration rates necessary to achieve nitrogen balance (approximately 1.5 to 1.8 g/kg per day), protein catabolism may increase. Providing relatively low levels of energy may diminish the magnitude of this increase. CONCLUSION: These results suggest that the optimal nutritional regimen for patients with acute renal failure may require a high-protein (approximately 1.5 to 1.8 g/kg per day) and a relatively low-energy (approximately 25 to 35 kcal/kg per day) content.

Azotemia control by extracorporeal therapy in patients with acute renal failure.

The mortality rate for patients with acute renal failure (ARF) requiring renal replacement therapy remains unacceptably high. The cause of death in these patients has been thought to relate primarily to the nature of the condition that precipitated renal failure. However, recent investigations challenge that notion and suggest that the characteristics of the renal replacement procedure itself may influence outcome. The major considerations for the clinician prescribing renal replacement therapy to the patient with ARF are the therapy mode, the type of membrane used, and the dose of delivered therapy. The first two considerations have been discussed extensively in the medical literature and are reviewed elsewhere in this issue. However, the determination of the amount of delivered therapy, although standard practice in patients with end-stage renal disease, has not been assessed routinely in patients with ARF. Furthermore, the influence on patient outcome of the level of azotemia control achieved by the delivered therapy is unknown. The purpose of this review is to provide some insight into quantifying the amount of renal replacement therapy delivered to patients with ARF treated with either continuous or intermittent therapies. The expected level of azotemia control that can be achieved with each of these therapies is discussed. We suggest that quantification of the amount of delivered therapy and the level of azotemia control are important variables to be obtained and evaluated in future investigations seeking to understand the high mortality rate of patients with ARF.

Plasma protein adsorption to highly permeable hemodialysis membranes.

Although membrane adsorption of plasma proteins is one of several factors determining the biocompatibility and mass transfer characteristics of a hemodialyzer, this process has not been evaluated rigorously. We performed an equilibrium and kinetic analysis of the binding of proteins of differing molecular weight to highly permeable membranes of differing hydrophobicity and surface change. Hydrophobic, anionic polyacrylonitrile (PAN) and hydrophilic, uncharged cellulose triacetate (CT) membrane fragments were incubated in buffer containing radioiodinated beta 2-microglobulin (beta 2m) or bovine serum albumin (BSA). From an initial solution concentration of 50 mg/liter, both membranes adsorbed significantly more beta 2m than BSA at equilibrium (PAN, 352 +/- 30 vs. 32.1 +/- 2.4 ng; CT, 87.0 +/- 0.6 vs. 30.8 +/- 1.7 ng). These results were consistent with membrane pore exclusion of BSA. Comparison of the slopes of the equilibrium isotherm lines (concentration range, 0 to 220 mg/liter) showed the PAN binding affinity for beta 2m and BSA was 28 and 1.4 times that of CT, respectively. In kinetic studies, the approach to equilibrium versus (time)1/2 was assessed. For all protein-membrane combinations, this relationship was linear, consistent with a diffusion-controlled process. This latter characteristic permitted the determination of beta 2m membrane diffusivity values for both PAN and CT, which were found to be 0.30 and 3.25 x 10(-7) cm2/sec, respectively. These data suggest membrane hydrophobicity more significantly influences the binding of low-molecular weight proteins than that of pore-excluded proteins. In addition, these results demonstrate electrostatic membrane-protein interactions may influence the kinetics of both the adsorption and transmembrane mass transfer of plasma proteins.

Membrane adsorption of beta 2-microglobulin: equilibrium and kinetic characterization.

Enhanced extracorporeal removal of beta 2-microglobulin (beta 2m) may prevent the development of dialysis-related amyloidosis (DRA). One mechanism of beta 2m removal is membrane adsorption. Therefore, we fundamentally characterized beta 2m adsorption to the highly permeable polyacrylonitrile (PAN) membrane. Porous and nonporous PAN fragments were incubated in buffer containing 125I-beta 2m. Over a concentration range of 8 to 60 mg/liter, the equilibrium adsorption isotherm was linear (r = 0.99) for porous PAN while the isotherm for nonporous PAN suggested either multilayer binding or adsorption of proteins with differing orientations. In kinetic analyses, the approach to equilibrium versus (time)1/2 was evaluated. For both porous and nonporous PAN, this relationship was linear (r = 0.99), consistent with a diffusion-controlled process. Adsorption reversibility was assessed by comparing the amount bound at varying residence times (0 to 4 hr) to the amount remaining adsorbed after a subsequent incubation in buffer. The fractions remaining bound at 60, 120, and 240 minutes (0.34 +/- 0.02, 0.36 +/- 0.06, and 0.44 +/- 0.03; mean +/- SEM) were significantly greater (P < 0.05) than the value at five minutes (0.23 +/- 0.01). This suggests membrane-induced conformational changes in adsorbed beta 2m. This investigation permits the comparison of beta 2m adsorptive properties of PAN to those of other membrane-based and nonmembrane-based therapies designed to prevent DRA.

A comparison of metabolic control by continuous and intermittent therapies in acute renal failure.

Azotemia control provided by blood pump-assisted continuous hemofiltration has not been rigorously compared with that provided by intermittent hemodialysis (IHD) for critically ill patients with acute renal failure (ARF). The metabolic control achieved by continuous venovenous hemofiltration (CVVH) and IHD was compared. In ARF patients treated with CVVH (N = 11), the normalized daily dose of therapy was 0.59 +/- 0.23 (mean +/- SD) and the normalized protein catabolic rate was 1.82 +/- 0.95 g/kg per day. The serum urea nitrogen concentration (SUN) declined with CVVH from an initial value of 114 +/- 32 to 79 +/- 17 mg/dL at steady state (SUNs). The initial analysis was a theoretical comparison between CVVH azotemia control and the control that would have been provided by IHD. Simulated IHD data were generated by conventional urea kinetic methods. The peak concentration hypothesis was invoked to compare CVVH SUNs and the peak IHD SUN (SUNp). A simulated IHD frequency of five times or more weekly was required to achieve a SUNp that did not differ from the CVVH SUNs. A similar comparison between the CVVH group and a separate group of ARF patients (N = 11) who received IHD was also performed. In the latter group, the normalized protein catabolic rate and the normalized daily dose of therapy were similar to those of the CVVH group. The SUNp (101 +/- 12 mg/dL) in the IHD group was significantly higher than the mean CVVH SUNs (P < 0.05). These data suggest that intensive hemodialysis is required to provide azotemia control similar to that provided by CVVH.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of imipenem pharmacokinetics in patients with acute or chronic renal failure treated with continuous hemofiltration.

The total clearance of imipenem, a carbapenem antibiotic, is reduced from approximately 230 mL/min in patients with normal renal function to approximately 50 mL/min in patients with chronic renal failure. This decline in clearance results not only from the loss of renal clearance, but also from a reduction in the nonrenal clearance from 130 to 50 mL/min. Current dosing recommendations for the administration of imipenem to patients with acute or chronic renal failure are based on this reduced clearance rate. We investigated the pharmacokinetics of imipenem in critically ill patients with acute or chronic renal failure to determine whether published dosing guidelines were applicable to both patient populations. Imipenem pharmacokinetic parameters were determined in 10 anuric patients with renal failure managed by continuous venovenous hemofiltration (CVVH). Seven patients had acute renal failure, while the other three had preexisting chronic renal failure. Imipenem serum concentration data were incorporated into a first-order, single-compartment pharmacokinetic model. Determinations of the area under the serum concentration-time curve were made by the trapezoidal rule. Dosing regimens were calculated from clearance data to achieve a mid-dose imipenem serum concentration of 12 mg/L. The total clearance of imipenem in patients with acute renal failure (108.3 +/- 13.8 mL/min; mean +/- SD) was significantly greater than the total clearance measured in patients with chronic renal failure (64.4 +/- 10.5 mL/min; P < 0.02). This increased clearance resulted from a greater nonrenal clearance of the drug in patients with acute renal failure (95.0 +/- 13.8 v 51.1 +/- 10.5 mL/min; P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

NaCl transport by Madin Darby canine kidney cyst epithelial cells.

The mechanism of NaCl transport across the epithelium of intact MDCK cysts grown in a collagen gel matrix was investigated. Double-barreled microelectrodes were used to measure basolateral membrane PD (Vbl), transepithelial PD (Vt), and intracellular (Cli) and intralumenal (Clcy) Cl- activities in cysts under different conditions. In a control Ringer's solution (RS), Cli (60 +/- 1 mM) and Clcy (107 +/- 2 mM) exceeded the values corresponding to electrochemical equilibrium across the basolateral membrane and epithelium, respectively. Cli was reduced by superfusing the cysts with a low Cl- RS (Cli, 20 +/- 3 mM), a low Na+ RS (Cli, 40 +/- 4 mM), or by adding amiloride to the control RS (Cli, 46 +/- 1 mM). Cli was unaffected by removal of either K+ or HCO3- from the RS or by adding furosemide or SITS to the control RS. Vbl in the control RS was -50 +/- 2 mV and was affected only by removal from the RS of K+ (Vbl, -31 +/- 3 mV) or HCO3- (Vbl, -29 +/- 4 mV) or by the addition of SITS to the control RS (Vbl, -59 +/- 5 mV). Vt in control RS was -2 +/- 0.2 mV (lumen negative), and was increased by reducing bath Na+ (Vt, -37 +/- 2 mV) but not by reducing bath Cl-. These data indicate that Cl- is secreted in a basolateral to apical direction by the cyst epithelium. Basolateral Cl- transport probably occurs mainly by an electroneutral Cl-/HCO3- exchanger. Transepithelial Na+ transport seems to occur via a paracellular route which appears to be cation selective. These experiments also support the existence, in the basolateral membrane, of a Na+/K+ ATPase, a Na+/H+ exchanger, and possibly a Na+/HCO3-/CO3(2-) transporter.

Urea kinetics during continuous hemofiltration.

Urea kinetic analysis allows for the calculation of the urea distribution volume and urea generation rate. This method was employed in patients with acute renal failure managed by continuous venovenous hemofiltration (CVVH). Based on serial serum urea nitrogen concentration measurements, each patient's treatment course consisted of both steady state and non-steady state periods. Thirteen data sets were obtained from 11 critically ill patients treated with CVVH. The duration of therapy was 9.5 +/- 7.5 days (mean +/- SD). Serum urea nitrogen concentration fell from 114 +/- 32 mg/dl to a steady state value of 79 +/- 17 mg/dl (p < 0.0005). The urea distribution volume was 0.55 +/- 0.11 L/kg (range 0.29-0.73), and the urea generation rate 11.7 +/- 3.1 mg urea N/min (range 7.1-17.3). The steady state serum urea nitrogen concentration had a linear relationship to the rate of urea generation (r = 0.92). Urea kinetic analysis permitted the simultaneous determination of the urea generation rate and distribution volume, on an individualized basis, in patients with acute renal failure treated with CVVH.