How the overlapping timescales for peptide binding and terrace exposure lead to non-linear step dynamics during growth of calcium oxalate monohydrate.

Using in situ atomic force microscopy (AFM), we investigate the inhibition of calcium oxalate monohydrate (COM) step growth by aspartic acid-rich peptides and find that the magnitude of the effect depends on terrace lifetime. We then derive a time dependent step-pinning model in which average impurity spacing depends on the terrace lifetime as given by the ratio of step spacing to step speed. We show that the measured variation in step speed is well fit by the model and allows us to extract the characteristic peptide adsorption time. The model also predicts that a crossover in the timescales for impurity adsorption and terrace exposure leads to bistable growth dynamics described mathematically by a catastrophe. We observe this behavior experimentally both through the sudden drop in step speed to zero upon decrease of supersaturation as well as through fluctuations in step speed between the two limiting values at the point where the catastrophe occurs. We discuss the model's general applicability to macromolecular modifiers and biomineral phases.

Subnanometer atomic force microscopy of peptide-mineral interactions links clustering and competition to acceleration and catastrophe.

In vitro observations have revealed major effects on the structure, growth, and composition of biomineral phases, including stabilization of amorphous precursors, acceleration and inhibition of kinetics, and alteration of impurity signatures. However, deciphering the mechanistic sources of these effects has been problematic due to a lack of tools to resolve molecular structures on mineral surfaces during growth. Here we report atomic force microscopy investigations using a system designed to maximize resolution while minimizing contact force. By imaging the growth of calcium-oxalate monohydrate under the influence of aspartic-rich peptides at single-molecule resolution, we reveal how the unique interactions of polypeptides with mineral surfaces lead to acceleration, inhibition, and switching of growth between two distinct states. Interaction with the positively charged face of calcium-oxalate monohydrate leads to formation of a peptide film, but the slow adsorption kinetics and gradual relaxation to a well-bound state result in time-dependent effects. These include a positive feedback between peptide adsorption and step inhibition described by a mathematical catastrophe that results in growth hysteresis, characterized by rapid switching from fast to near-zero growth rates for very small reductions in supersaturation. Interactions with the negatively charged face result in formation of peptide clusters that impede step advancement. The result is a competition between accelerated solute attachment and inhibition due to blocking of the steps by the clusters. The findings have implications for control of pathological mineralization and suggest artificial strategies for directing crystallization.

Pharmacokinetics and metabolism of nateglinide in humans.

The pharmacokinetics and metabolism of nateglinide were studied in six healthy male subjects receiving a single oral (120 mg) and intravenous (60 mg) dose of [14C]nateglinide in randomized order. Serial blood and complete urine and feces were collected for 120 h post dose. Nateglinide was rapidly (approximately 90%) absorbed, with peak blood and plasma concentrations at approximately 1 h post dose. The maximal plasma concentrations of radioactivity (6360 ngEq/ml) and nateglinide (5690 ng/ml) were comparable, and plasma radioactivity concentrations were about twice those of blood at all times. Oral bioavailability was 72%, indicating only a modest first-pass effect. After either dose, plasma nateglinide concentrations declined rapidly with elimination half-lives of 1.5 to 1.7 h and plasma clearance of 7.4 l/h. Plasma radioactivity was eliminated more slowly with half-lives of 52 and 35 h in plasma and blood, respectively, after the oral dose. The contribution of this more slowly eliminated component to the AUC(0-infinity) was minor. Nateglinide was extensively metabolized, with excretion predominantly (84-87%) in urine. Only approximately 16% of the dose was excreted unchanged in urine after either dosing route. The major metabolites were the result of oxidative modifications of the isopropyl group. Three of these were monohydroxylated, two of which appeared to be diastereoisomers. Additionally, one metabolite with an unsaturation in the isopropyl group and two diol-containing isomers were identified. Glucuronic acid conjugates resulting from direct glucuronidation of the carboxylic acid were also present. The major metabolite in plasma and urine was the result of hydroxylation of the methine carbon of the isopropyl group.

Treatment of colorectal liver metastases by cryotherapy.

One hundred fifty-eight procedures were performed on 136 patients with unresectable hepatic metastases using hepatic cryotherapy to ablate the tumors. The median age was 62 years. Patients included 90 males and 46 females. Fifty-eight patients had synchronous metastases, 55 had bilobar lesions, and 90 had precryo chemotherapy. Median preoperative carcinoembryonic antigen (CEA) level was 14.4 ng/dl. The numbers of lesions treated, frozen, and resected were two and one. Median survival of all patients was 30 months. Survival for 39 patients was 37 months. Patients with a CEA level > 100 ng/dl had a statistically worse survival rate than those with a level < 100 ng/dl (P < .001). Twenty patients underwent recryotherapy with median survival of 34 months. Recurrent disease developed in 78% of patients--82% of the patients developed liver recurrence. Complication rates were comparable to liver resection. Operative mortality was 3.7%. Hepatic cryotherapy is effective and safe in treating colorectal hepatic metastases under ultrasound guidance.

Infectious complications of hepatic cryosurgery.

Hepatic cryosurgery is a novel procedure for patients with metastatic liver disease. To date, no reviews of the infectious complications of this procedure have been published. One hundred and fifty patients underwent 158 hepatic cryosurgical procedures at Allegheny General Hospital (Pittsburgh) from November 1987 through July 1995. Gastrointestinal malignancies accounted for 93% of the underlying diagnoses. The following 12 infections were directly related to the cryosurgical procedure: hepatic abscess (six), intraperitoneal abscess (three), ascending cholangitis (two), and an intrahepatic device (Infusaid; Strato/Infusoid, Norwood, MA) infection (one). Enterococcus was the most commonly isolated organism. Seven of the 12 infections were polymicrobial. The patients who developed infections had longer hospital stays (26 days vs. 13 days) and had more days of fever (6.5 days vs. 2.3 days). than those who did not develop infections. If perioperative manipulation of the biliary tree is avoided, the infection rate in patients who undergo hepatic cryosurgery may be decreased even further. Overall, cryoablation of the liver is not related to an increased risk of infection.

Pharmacokinetics of abecarnil in patients with renal insufficiency.

OBJECTIVE: To characterize the pharmacokinetics of a single 5 mg oral dose of abecarnil in subjects with varying degrees of renal impairment. METHODS: Twenty-six subjects were enrolled in this open-label parallel-group study. Ten subjects had normal renal function (NRF; creatinine clearance [CLCR] > or = 85 ml/min/1.73 m2), six subjects had mild to moderate renal insufficiency (MMRI; CLCR between 25 and 73 ml/min/1.73 m2), and 10 subjects had severe renal insufficiency (SRI; CLCR < or = 10 ml/min/1.73 m2). Abecarnil plasma concentrations were determined by means of HPLC, and plasma protein binding was determined by use of ultracentrifugation. Pharmacokinetic parameters were obtained with use of model-independent and model-dependent methods. RESULTS: In subjects with SRI, area under the concentration-time curve and maximum plasma concentration were reduced by 36% and 31%, respectively, compared with demographically matched subjects with NRF. The apparent total body clearance in the NRF, MMRI, and SRI groups was 13.0 +/- 6.89, 12.9 +/- 3.64, and 25.0 +/- 13 ml/min/kg, and the apparent volume of distribution was 14.0 +/- 3.78, 12.8 +/- 2.4, and 19.4 +/- 5.76 L/kg, respectively (mean +/- SD). The patients with SRI had a significantly lower protein bound fraction than subjects with NRF (0.850 +/- 0.077 versus 0.948 +/- 0.023). Despite an increase in the free fraction of abecarnil (f(u)), there was no significant change in the apparent unbound total body clearance and unbound volume of distribution between the SRI and NRF groups. The anticipated full effect of the increase in f(u) among the patients with SRI was not realized and suggests that the f(u) in tissue may be increased in patients with SRI. CONCLUSION: Dose adjustment will need to be made on the basis of titration to the desired clinical response and tolerability in patients with SRI just as in subjects with NRF.

Hepatic cryosurgery in treating colorectal metastases.

BACKGROUND: The purpose of this study was to determine the effectiveness of cryosurgery as an adjunct to resection in treating patients with hepatic metastases from colorectal adenocarcinoma. METHODS: Forty-seven patients with documented metastases limited to the liver from colorectal adenocarcinoma were treated with cryosurgery with or without resection from November 1987 to February 1992 and were followed until February 1994. Intraoperative ultrasound was used to map the lesions and place the cryoprobes. Each lesion was frozen to -196 degrees centigrade for 15 minutes, thawed for 10 minutes, and frozen again for 15 minutes. Follow-up computed tomographic scans were obtained before hospital discharge and 6 months and 1 year after cryosurgery. Carcinoembryonic antigen levels were obtained monthly. RESULTS: Thirty-one males and 16 females, with a median age of 63 years, were treated. The median hospital stay was 10 days, and follow-up ranged from 24 to 57 months, with a median follow-up of 26 months. The actual survival at 24 months was 62%. Eleven percent of these patients had no evidence of disease at a median follow-up of 30 months. Complications included myoglobinuria, coagulopathy, pleural effusions, and bile duct injuries. Two patients (4%) died because of multisystem organ failure with irreversible coagulopathies. CONCLUSIONS: Cryosurgical ablation increases the number of patients with liver metastases who potentially can become disease free. However, the effect on overall survival will require a longer follow-up period.

Hepatic cryosurgery in the treatment of unresectable metastases.

The treatment of unresectable hepatic metastases has generally been limited to systemic or intra-arterial chemotherapy. Cryosurgery has the advantage of potentially ablating such unresectable tumours. From November 1987 to August 1994, 140 patients underwent 155 procedures using hepatic cryosurgery with and without resection for documented metastatic disease. Intra-operative ultrasound was used for monitoring the freezing zone. The tumours were frozen using liquid nitrogen cooled to -196 degrees C for 15 min. The median number of lesions treated was three. Median hospital stay was 10 days. The operative mortality was 4%. Complications included coagulopathy, hypothermia, myoglobinuria, pleural effusions, ATN and infection. The median survival for all patients was 22 months. Of those patients followed for more than 2 years, the median survival was 25 months. Of the 65 patients that are still alive, the median follow-up is 27 months.

The effect of oral dose volume on the absorption of a highly and a poorly water-soluble drug in the rat.

The influence of dose volume on drug absorption following oral administration of a highly and a poorly water soluble drug was examined in male Sprague-Dawley rats. A constant mass of each 14C-labeled compound was given via gavage in dose volumes of 1, 5, 10, and 20 mL kg-1. Blood levels, as well as the quantitative excretion of radioactivity, were measured following each treatment. No significant changes in either the rate or extent of absorption of the water soluble drug were detected. In contrast, the absorption rate of the poorly water soluble drug increased with higher dose volumes, whereas no changes in the extent of absorption were observed. Drug solubility and gastric emptying appeared to be important factors affecting the rate of absorption of the poorly water soluble drug. Since changes in dose volume may affect the absorption characteristics of orally administered compounds, and the extent of such changes may be dependent upon the physicochemical properties of the drug, it is apparent that dose volume is an important experimental variable to be considered in studies comparing absorption data.

Cryosurgery of liver cancer.

Cryosurgery, the in situ destruction of tissue using subzero temperatures, has been used to treat hepatic metastases. Because it is a focal treatment, cryosurgery can be used in patients with unresectable lesions due to location (next to major blood vessels) or multiplicity. In this study, 57 patients with unresectable hepatic metastases were treated with cryosurgery (with at least a 6-month follow-up). The number of lesions treated ranged from 1-16 with a mean of 4.6. Forty-three patients (73%) had bilobar disease, while 25 patients (42%) were treated with a combination of resection and cryosurgery. The disease-free survival rate (patients with normal computed tomography [CT] scans and carcinoembryonic antigen [CEA] levels) was 27% with a mean follow-up of 21 months. This is comparable to other hepatic cryosurgery studies that have found survival rates of 25-37.5%. Although the results are still short-term, this study indicates that hepatic cryosurgery offers the hope of long-term survival in patients with unresectable hepatic metastases.

Hepatic cryosurgery with and without the Bair Hugger.

Hypothermia is a significant clinical problem during hepatic cryosurgery, which at times causes the procedure to be halted until the patient's body temperature can be raised. This study examines the effects of the Bair Hugger (a warming device) on body temperature during hepatic cryosurgery. Twenty-eight cases of hepatic cryosurgery were performed without the Bair Hugger, while 44 cases included the Bair Hugger. The lowest mean temperature was significantly lower in the group without the Bair Hugger (34.2 degrees C vs. 35.3 degrees C; P < 0.0001). In addition, this group showed a significantly greater mean change in temperature during the procedure (1.81 degrees C vs. 0.73 degrees C; P < 0.0001). No patient in the Bair Hugger group reached the point of clinically significant hypothermia. In conclusion, the Bair Hugger is safe and very effective in regulating body temperature and it is an essential piece of equipment performing hepatic cryosurgery.

Salivary flow induction by buccal permucosal pilocarpine in anesthetized beagle dogs.

We tested whether permucosal delivery of pilocarpine nitrate could be used to elicit significant salivary secretion. Pilocarpine (pKa 6.6 at 37 degrees C) was applied as solutions (pHs 5.6, 6.6, 7.6; 15 mg/mL) to the buccal mucosa (2.8 cm2) of 6 anesthetized dogs. Saliva was collected continuously from cannulated submandibular and parotid ducts and blood sampled during and after drug administration. Plasma pilocarpine levels were determined by reversed-phase HPLC. Absorption rates were determined by use of data from separate zero-order intravenous infusions to the same dogs. Pilocarpine was buccally absorbed at a constant rate of 72.9 +/- 38.5 micrograms/kg/h following its application at pH 7.6. At this pH of the drug solution, the time to appearance of pilocarpine in blood plasma was 0.31 +/- 0.08 h, and the time to appearance of salivary flow was 0.86 +/- 0.32 h. A threshold dose of 32.9 +/- 7.5 micrograms/kg was required to induce secretion with the pH 7.6 drug, the steady-state submandibular flow rate was 0.14 +/- 0.11 mL/min/gland pair. Salivary flow induction was symmetrical and reached levels as high as 0.35 mL/min/submandibular gland pair without apparent tachyphylaxis. Results at pHs 5.6, 6.6, and 7.6 were consistent with the hypothesis that pilocarpine is primarily absorbed as un-ionized drug. The data indicate that transmucosal delivery of pilocarpine, avoiding "first pass" hepatic loss, may hold promise for the treatment of xerostomia.

High-performance liquid chromatographic determination of pilocarpine in plasma.

A high-performance liquid chromatographic procedure requiring neither derivatization nor complex sample work-up is reported for reproducibly and sensitively determining pilocarpine in plasma. Following stabilization of pilocarpine against in vitro hydrolysis using sodium fluoride, plasma samples were extracted and the extracts chromatographed on a 5-microns, low-carbon-load (6%) C18 reversed-phase column. The assay was linear between 10 and 300 ng/ml (r = 0.998). It had sufficient sensitivity to quantitate pilocarpine at concentrations as low as 10 ng/ml (signal-to-noise ratio > or = 4) using a 500-microliters sample. The assay appears to be the first published specifically for plasma determinations and has proven capable of supporting pharmacokinetics studies of pilocarpine disposition in the anesthetized dog.

Pilocarpine disposition and salivary flow responses following intravenous administration to dogs.

Oral doses of pilocarpine increase salivary flow rates in patients afflicted with xerostomia (dry mouth). This study examined the pharmacokinetics of and a pharmacodynamic response (salivation) to intravenous pilocarpine nitrate administration in dogs. Disposition was linear over a dose range of 225-600 micrograms/kg; plasma concentrations were 10-120 micrograms/L. Elimination was rapid and generally biphasic, with a terminal elimination half-life of approximately 1.3 hr. The systemic clearance of pilocarpine was high (2.22 +/- 0.49 L/kg/hr) and its steady-state volume of distribution (2.30 +/- 0.64 L/kg) was comparable to that of many other basic drugs. All doses of pilocarpine induced measurable submaxillary and parotid salivary flow rates which could be maintained constant over time. Cumulative submaxillary saliva flow was linearly related to total pilocarpine dose. Plasma pilocarpine concentration was linearly related to both steady-state and postinfusion submaxillary salivary flow rates.

Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells.

We have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment (3.9 or 1.3 microM) for 24 hr virtually abolished Cl- transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br- uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatment, but not after treatment for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl- transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl- permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl- permeability.

Correlations on radioimmunoassay, fluorescence polarization immunoassay, and enzyme immunoassay of cannabis metabolites with gas chromatography/mass spectrometry analysis of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in urine specimens.

Results obtained from three commercial immunoassay kits, Abuscreen, TDx, and EMIT, commonly used for the initial test of urine cannabinoids (and metabolites) were correlated with the 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (9-THC-COOH) concentration as determined by GC/MS. Correlation coefficients obtained based on 26 (out of 1359 total sample population) highly relevant samples, are 0.601 and 0.438 for Abuscreen and TDx. Correlation coefficients obtained from a parallel study on a different set of 47 (out of 5070 total sample population) highly relevant specimens are 0.658 and 0.575 for Abuscreen and Emit. The immunoassay concentration levels, that correspond to the commonly used 15 ng/ml GC/MS cutoff value for 9-THC-COOH, as calculated from the regression equations are 82 ng/ml and 75 ng/ml for TDx and EMIT and 120 ng/ml and 72 ng/ml for Abuscreen manufactured at two different time periods. The difference of these calculated corresponding concentrations provides quantitative evidence of the reagent specificity differences.

Regulation of Cl- permeability in normal and cystic fibrosis sweat duct cells.

Reabsorptive cells of the human sweat gland normally exhibit a high basal Cl- permeability but are markedly impermeable to Cl- in cystic fibrosis (CF). We examined the possibility that the reduced basal Cl- permeability of CF sweat duct cells in primary culture is due to a defective regulation of plasma membrane Cl- permeability by prostaglandin E2 (PGE2), which is endogenously produced by cultured sweat duct cells. The macroscopic Cl- permeabilities of normal and CF sweat duct cells were assessed using a halide-specific fluorescent dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, in combination with fluorescence digital-imaging microscopy. The Cl- and Br- permeabilities of normal sweat duct cells were markedly reduced by inhibiting endogenous PGE2 production with indomethacin. This inhibition of Cl- permeability by indomethacin was largely reversed by the addition of PGE2 (10 nM to 1 microM), but not forskolin. Conversely, PGE2 failed to stimulate the low Cl- permeabilities of sweat duct cells cultured from CF subjects. Our results support the following conclusions: 1) a defective regulation of Cl- permeability in CF is a feature of reabsorptive as well as secretory epithelial cells, and 2) the nature of this regulatory defect extends beyond altered Cl- permeability regulation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase.

Thoracic spine injuries in victims of motorcycle accidents.

Spinal cord injury can be devastating. Cervical spine roentgenography has been recommended in all severe multisystem trauma patients but little attention has been given to the thoracic spine. In a series of 266 motorcycle accident victims, seen over a 42-month period, 13 cases of thoracic spine injury were identified. During this same time interval four cases of cervical spine injury were identified. Eleven of the 13 cases involved a catapulting ejection from the motorcycle and resultant axial loading to the thoracic spine. Thoracic spine injuries are more common in these patients and therefore the thoracic spine should be immobilized until full thoracic spine roentgenography can be carried out.

Aluminum ingestion and behavior in the Long-Evans rat.

A variety of behaviors was measured in adult Long-Evans male rats fed ground rat chow containing either no added aluminum, low aluminum (1500 mg/kg), moderate aluminum (2500 mg/kg), or high aluminum (3500 mg/kg). There were no effects of aluminum on either body weight or mouse killing. There was an inverse relationship between brain aluminum and open-field activity. Elevated brain aluminum was correlated with relatively poor performance on a single-trial passive-avoidance task and on a visual discrimination with reversal task.