BACKGROUND: A novel data warehouse based on automated retrieval from an institutional health care information system (HIS) was made available to be compared with a traditional prospectively maintained surgical database. METHODS: A newly established institutional data warehouse at a single-institution academic medical center autopopulated by HIS was queried for International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) diagnosis codes for pancreatic neoplasm. Patients with ICD-9-CM diagnosis codes for pancreatic neoplasm were captured. A parallel query was performed using a prospective database populated by manual entry. Duplicated patients and those unique to either data set were identified. All patients were manually reviewed to determine the accuracy of diagnosis. RESULTS: A total of 1107 patients were identified from the HIS-linked data set with pancreatic neoplasm from 1999-2009. Of these, 254 (22.9%) patients were also captured by the surgical database, whereas 853 (77.1%) patients were only in the HIS-linked data set. Manual review of the HIS-only group demonstrated that 45.0% of patients were without identifiable pancreatic pathology, suggesting erroneous capture, whereas 36.3% of patients were consistent with pancreatic neoplasm and 18.7% with other pancreatic pathology. Of the 394 patients identified by the surgical database, 254 (64.5%) patients were captured by HIS, whereas 140 (35.5%) patients were not. Manual review of patients only captured by the surgical database demonstrated 85.9% with pancreatic neoplasm and 14.1% with other pancreatic pathology. Finally, review of the 254 patient overlap demonstrated that 80.3% of patients had pancreatic neoplasm and 19.7% had other pancreatic pathology. CONCLUSIONS: These results suggest that cautious interpretation of administrative data rely only on ICD-9-CM diagnosis codes and clinical correlation through previously validated mechanisms.

The human erythrocyte glucose transporter is an allosteric complex of four GLUT1 proteins whose structure and substrate binding properties are stabilized by reductant-sensitive, noncovalent subunit interactions [Hebert, D. N., and Carruthers, A. (1992) J. Biol. Chem. 267, 23829-23838]. In the present study, we use biochemical and molecular approaches to isolate specific determinants of transporter oligomeric structure and transport function. When unfolded in denaturant, each subunit (GLUT1 protein) of the transporter complex exposes two sulfhydryl groups. Four additional thiol groups are accessible following subunit exposure to reductant. Assays of subunit disulfide bridge content suggest that two inaccessible sulfhydryl groups form an internal disulfide bridge. Differential alkylation/peptide mapping/N-terminal sequence analyses show that a GLUT1 carboxyl-terminal peptide (residues 232-492) contains three inaccessible sulfhydryl groups and that an N-terminal GLUT1 peptide (residues 147-261/299) contains two accessible thiols. The carboxyl-terminal peptide most likely contains the intramolecular disulfide bridge since neither its yield nor its electrophoretic mobility is altered by addition of reductant. Each GLUT1 cysteine was changed to serine by oligonucleotide-directed, in vitro mutagenesis. The resulting transport proteins were expressed in CHO cells and screened by immunofluorescence microscopy for their ability to expose tetrameric GLUT1-specific epitopes. Serine substitution at cysteine residues 133, 201, 207, and 429 does not inhibit exposure of tetrameric GLUT1-specific epitopes. Serine substitution at cysteines 347 or 421 prevents exposure of tetrameric GLUT1-specific epitopes. Hydrodynamic analysis of GLUT1/GLUT4 chimeras expressed in and subsequently solubilized from CHO cells indicates that GLUT1 residues 1-199 promote chimera dimerization and permit GLUT1/chimera heterotetramerization. This GLUT1 N-terminal domain is insufficient for chimera tetramerization which additionally requires GLUT1 residues 200-463. Extracellular reductants (dithiothreitol, beta-mercaptoethanol, or glutathione) reduce erythrocyte 3-O-methylglucose uptake by up to 15-fold. This noncompetitive inhibition of sugar uptake is reversed by the cell-impermeant, oxidized glutathione. Reductant is without effect on sugar exit from erythrocytes. Dithiothreitol doubles the cytochalasin B binding capacity of erythrocyte-resident glucose transporter, abolishes allosteric interactions between substrate binding sites on adjacent subunits, and occludes tetrameric GLUT1-specific GLUT1 epitopes in situ. CHO cell-resident GLUT1 structure and transport function are similarly affected by extracellular reductant. We conclude that each subunit of the glucose transporter contains an extracellular disulfide bridge (Cys347 and Cys421) that stabilizes transporter oligomeric structure and thereby accelerates transport function.

Hebert and Carruthers (1992) showed that the human erythrocyte glucose transporter is an allosteric complex of four GLUT1 proteins whose structure and substrate binding properties are stabilized by reductant-sensitive noncovalent subunit interactions. The GLUT1 tetramer dissociates into dimers upon exposure to reductant but subunits are not associated via disulfide bridges. Each subunit of SDS-denatured tetrameric GLUT1 exposes only two thiols while reduced denatured GLUT1 exposes all six sulfhydryl groups. They hypothesized that glucose transporter oligomeric structure and cooperative catalytic function resulted from noncovalent subunit interactions promoted or stabilized by intramolecular disulfide bridges. These interactions give rise to an antiparallel arrangement of substrate binding sites within the transporter complex. In the present studies, we tested aspects of this model. Specifically, we wanted 1) to understand why the native, noncovalent, homotetrameric GLUT1 complex is sensitive to reductant, 2) to determine whether the tetramer is more catalytically efficient than the dimer in situ, and 3) to test the hypothesis that it is the antiparallel arrangement of substrate binding sites between subunits that provides the transporter with its catalytic advantage. We used biochemical and molecular biological approaches to isolate specific determinants of transporter oligomeric structure and/or transport function in purified isolated transporter preparations, in intact red cells and in CHO cells. We have also examined the hypothesis that net sugar transport in the human erythrocyte is rate limited by reduced cytosolic diffusion of sugars and/or by reversible sugar association with intracellular macromolecules. Our findings support the hypothesis that each subunit of the parental glucose transporter contains a single intramolecular disulfide bridge located between cysteine residues 347 and 421. This disulfide seems to be necessary for GLUT1 tetramerization. Our findings suggest that GLUT1 N-terminal residues 1 through 199 provide contact surfaces for subunit dimerization but are insufficient for subunit tetramerization. Our studies also show that in situ disulfide disruption by cell impermeant reductants results in the loss of cooperative subunit interactions and a 3 to 15-fold reduction in the transport efficiency of the transporter. We further find that in situ GLUT1 is susceptible to exofacial proteolysis. Exofacial trypsin cleavage eliminates cooperativity between subunits but does not affect transporter oligomeric structure or transport activity. Thus catalytic efficiency does not derive directly from cooperative interactions between substrate binding sites on adjacent subunits. We have confirmed that 30MG transport in human erythrocytes is a diffusion limited process. We find that steady-state sugar uptake in red cells and K562 cells measures two processes - sugar translocation and intracellular sugar binding. We propose a model for native GLUT1 structure and function.