High-throughput ultra-high-performance liquid chromatography/tandem mass spectrometry quantitation of insulin-like growth factor-I and leucine-rich alpha-2-glycoprotein in serum as biomarkers of recombinant human growth hormone administration.

Insulin-like growth factor-I (IGF-I) is a known biomarker of recombinant human growth hormone (rhGH) abuse, and is also used clinically to confirm acromegaly. The protein leucine-rich alpha-2-glycoprotein (LRG) was recently identified as a putative biomarker of rhGH administration. The combination of an ACN depletion method and a 5-min ultra-high-performance liquid chromatography/tandem mass spectrometry (uHPLC/MS/MS)-based selected reaction monitoring (SRM) assay detected both IGF-I and LRG at endogenous concentrations. Four eight-point standard addition curves of IGF-I (16-2000 ng/mL) demonstrated good linearity (r(2) = 0.9991 and coefficients of variance (CVs) <13%). Serum samples from two rhGH administrations were extracted and their uHPLC/MS/MS-derived IGF-I concentrations correlated well against immunochemistry-derived values. Combining IGF-I and LRG data improved the separation of treated and placebo states compared with IGF-I alone, further strengthening the hypothesis that LRG is a biomarker of rhGH administration. Artificial neural networks (ANNs) analysis of the LRG and IGF-I data demonstrated an improved model over that developed using IGF-I alone, with a predictive accuracy of 97%, specificity of 96% and sensitivity of 100%. Receiver operator characteristic (ROC) analysis gave an AUC value of 0.98. This study demonstrates the first large scale and high throughput uHPLC/MS/MS-based quantitation of a medium abundance protein (IGF-I) in human serum. Furthermore, the data we have presented for the quantitative analysis of IGF-I suggest that, in this case, monitoring a single SRM transition to a trypsin peptide surrogate is a valid approach to protein quantitation by LC/MS/MS.

Multiple markers for lung cancer diagnosis: validation of models for localized lung cancer.

Sera from 71 patients with localized lung cancer, from 70 normal controls, and from 73 patients with benign lung diseases were analyzed for 10 substances to detect lung cancer: ferritin, lipid-bound sialic acid, total sialic acid, beta 2-microglobulin, lipotropin, the alpha and beta subunits of human chorionic gonadotropin, calcitonin (two assays), parathyroid hormone, and carcinoembryonic antigen (CEA). Individual markers were studied, and optimal combinations of markers were sought for discriminating patients with localized lung cancer from normal controls and from patients with benign lung disease. Both logistic regression and recursive partitioning methods for discrimination were tried. The best rules involved only CEA and ferritin for discriminating patients with lung cancer from normal controls, and CEA and age for discriminating patients with lung cancer from those with benign lung diseases. The performance of these rules was validated on an independent serum panel containing sera from 56 patients with localized lung cancer, 75 normal controls, and 75 patients with benign lung diseases. Three rules designed to achieve 95% specificity against normal controls attained 14%-36% sensitivity for localized lung cancer in the validation panels, whereas three rules designed to achieve 95% specificity against benign lung diseases attained 30%-39% sensitivity. Some aspects of potential clinical applications are discussed.

Multiple markers for lung cancer diagnosis: validation of models for advanced lung cancer.

Sera from 171 patients with advanced lung cancer, from 110 normals, and from 123 subjects with benign respiratory diseases were analyzed for 10 substances to detect lung cancer: ferritin, lipid-bound sialic acid, total sialic acid, beta 2-microglobulin, lipotropin, the alpha and beta subunits of human chorionic gonadotropin, calcitonin (two assays), parathyroid hormone, and carcinoembryonic antigen. Individual markers were studied, and optimal combinations of markers were sought for discriminating lung cancer patients from normals and from patients with benign lung disease. Numerous methods for combining the markers were examined, but the methods of logistic regression and recursive partitioning were finally adopted. The best discrimination rules we could find used only carcinoembryonic antigen (CEA) and total sialic acid (TSA). The performance of these rules was validated on an independent serum panel containing sera from 68 patients with advanced lung cancer, from 40 normals, and from 52 patients with benign respiratory disease. The combination rules based on TSA and CEA performed better than a rule based on CEA alone. Logistic discrimination rules with TSA and CEA that were designed to have 95% specificity achieved 54% sensitivity for discriminating advanced lung cancer from normal controls and 52% sensitivity for discriminating advanced lung cancer from controls with benign disease. Some aspects of clinical applicability are discussed, including planned studies for localized lung cancer and the requirement for further testing in specific clinical settings.

Arg-188 and Trp-144 are implicated in the binding of urea and acetamide to the active site of the amidase from Pseudomonas aeruginosa.

Urea is a time-dependent active-site-directed inhibitor of Pseudomonas aeruginosa amidase. We found that 20 mM hydroxylamine caused bound urea to be released from the inactive urea:amidase complex with the restoration of enzyme activity. Bound urea restricts the titrability of the enzyme's -SH groups to 6 per hexameric molecule and protects it against thermal denaturation suggesting that urea binding provokes a conformational change in the enzyme. Mutations in the P. aeruginosa amidase gene that reduce the binding affinity of the enzyme for both urea and the substrate acetamide have been identified by direct sequencing of PCR-amplified mutant genes and confirmed by sequencing cloned PCR-amplified genes. The mutations were in two regions of the enzyme substituting either Arg-188 (or Gln-190, in one case) or Trp-144; one amidase that bound neither urea nor acetamide was doubly mutant with an amino-acid change at both sites.

Validating the GTP-cyclohydrolase 1-feedback regulatory complex as a therapeutic target using biophysical and in vivo approaches.

BACKGROUND AND PURPOSE: 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4 ) is an essential cofactor for nitric oxide biosynthesis. Substantial clinical evidence indicates that intravenous BH4 restores vascular function in patients. Unfortunately, oral BH4 has limited efficacy. Therefore, orally bioavailable pharmacological activators of endogenous BH4 biosynthesis hold significant therapeutic potential. GTP-cyclohydrolase 1 (GCH1), the rate limiting enzyme in BH4 synthesis, forms a protein complex with GCH1 feedback regulatory protein (GFRP). This complex is subject to allosteric feed-forward activation by L-phenylalanine (L-phe). We investigated the effects of L-phe on the biophysical interactions of GCH1 and GFRP and its potential to alter BH4 levels in vivo. EXPERIMENTAL APPROACH: Detailed characterization of GCH1-GFRP protein-protein interactions were performed using surface plasmon resonance (SPR) with or without L-phe. Effects on systemic and vascular BH4 biosynthesis in vivo were investigated following L-phe treatment (100 mg·kg(-1) , p.o.). KEY RESULTS: GCH1 and GFRP proteins interacted in the absence of known ligands or substrate but the presence of L-phe doubled maximal binding and enhanced binding affinity eightfold. Furthermore, the complex displayed very slow association and dissociation rates. In vivo, L-phe challenge induced a sustained elevation of aortic BH4 , an effect absent in GCH1(fl/fl)-Tie2Cre mice. CONCLUSIONS AND IMPLICATIONS: Biophysical data indicate that GCH1 and GFRP are constitutively bound. In vivo, data demonstrated that L-phe elevated vascular BH4 in an endothelial GCH1 dependent manner. Pharmacological agents which mimic the allosteric effects of L-phe on the GCH1-GFRP complex have the potential to elevate endothelial BH4 biosynthesis for numerous cardiovascular disorders.

Diabetic chronic hyperglycemia and neurologic outcome following global ischemia in dogs.

We tested the hypothesis that the neuropathologic outcome following recovery from incomplete ischemia is similar in normoglycemia and diabetes. Incomplete global ischemia was induced for 20 min in two groups of dogs: (a) normoglycemic, nondiabetic controls (n = 11) and (b) chronic (3 months), diabetic hyperglycemic subjects (n = 12). Animals were allowed to recover from surgery for 7 days after which they were perfusion-fixed for neuropathology. On paraffin processed tissue stained with hematoxylin and eosin (H&E), ischemic neurons were counted and the per cent of cell damage determined. All control animals survived for 7 days postischemia. Four of 12 diabetic animals survived for 7 days, with the remaining eight diabetic dogs dying within the first 3 days. On day 7, the percentage of neurons showing ischemic cell change in the four diabetic survivors and the 11 nondiabetic controls was similar in the cerebellum, CA1, superior temporal gyrus, and caudate. However, diabetic dogs that did not survive the 7-day recovery period showed cerebellar swelling, reduced Purkinje cell densities, and herniation. During the 3 months prior to ischemia, morning (10.7 +/- 4.4 versus 11.2 +/- 5.2 mM) and afternoon (8.8 +/- 5.0 versus 9.4 +/- 5.3 mM) blood glucose levels in the four surviving and eight nonsurviving diabetic animals, respectively, were similar. However, preischemic blood glucose was significantly elevated in animals that did not survive (7.8 +/- 2.8 versus 15.8 +/- 7.3 mM in survivors and nonsurvivors, respectively). This study shows that diabetic animals surviving 7 days postischemia and nondiabetic controls have similar neuropathology. However, diabetic animals in which glucose control deteriorated during the 24-h prior to ischemia did not survive, possibly due to severe hindbrain edema. These results show that in diabetes, blood glucose control immediately prior to incomplete global brain ischemia is an important determinant of morbidity and neuropathology.

Poor hemodynamic and metabolic recovery after global incomplete cerebral ischemia associated with short-term diabetes in dogs.

We determined the effect of 4-5 weeks of diabetes on ATP recovery following global incomplete cerebral ischemia. 31P magnetic resonance spectra of ATP, intracellular pH (pHi), and CBF (radiolabeled microspheres) were measured in three groups of anesthetized dogs (n = 8/group): chronic hyperglycemic diabetes (pancreatectomy followed by blood glucose of > 10 mM for 4-5 weeks); acute hyperglycemia (blood glucose of > 10 mM) during ischemia and reperfusion in nondiabetic dogs; and normoglycemic controls. Twenty minutes of incomplete ischemia was produced by ventricular fluid infusion to keep cerebral perfusion pressure (CPP) at 10 mm Hg during spontaneous variations in MABP. Intracranial pressure was increased initially to similar levels, resulting in a similar Cushing response among the groups. However, during the final 8 min of ischemia, MABP decreased to a greater extent in diabetic (86 +/- 42 mm Hg) than in hyperglycemic (162 +/- 30 mm Hg) and normoglycemic (135 +/- 54 mm Hg) groups and remained lower throughout 3 h of reperfusion. CPP was kept constant during ischemia, but was lower throughout reperfusion in diabetic dogs. During ischemia CBF was reduced similarly among groups: 5 +/- 3 ml.min-1 x 100 g-1 in hyperglycemic and normoglycemic and 4 +/- 3 ml.min-1 x 100 g-1 in diabetic dogs. During reperfusion early hyperemia was attenuated and delayed hypoperfusion was augmented (7 +/- 17 ml.min-1 x 100 g-1 by 180 min) as a result of low perfusion pressure in diabetics. However, medullary blood flow was similar among groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Pseudomonas aeruginosa aliphatic amidase is related to the nitrilase/cyanide hydratase enzyme family and Cys166 is predicted to be the active site nucleophile of the catalytic mechanism.

A database search indicated homology between some members of the nitrilase/cyanide hydratase family, Pseudomonas aeruginosa and Rhodococcus erythropolis amidases and several other proteins, some of unknown function. BLOCK and PROFILE searches confirmed these relationships and showed that four regions of the P. aeruginosa amidase had significant homology with corresponding regions of nitrilases. A phylogenetic tree placed the P. aeruginosa and R. erythropolis amidases in a group with nitrilases but separated other amidases into three groups. The active site cysteine in nitrilases is conserved in the P. aeruginosa amidase indicating that Cys166 is the active site nucleophile.

One-step affinity purification of amidase from mutant strains of Pseudomonas aeruginosa.

Amidases (acylamide amidohydrolase EC 3.5.1.4) from mutant strains (i.e., B6, AI3, AIU1N, OUCH 4 and L10) of Pseudomonas aeruginosa were purified in one-step by ligand affinity chromatography using Epoxy-activated Sepharose 4B-acetamide. The yields of the purified enzymes were about 90% for all mutant strains with purification factors of about 10 and were apparently homogeneous when analysed by SDS-PAGE and native PAGE. The protein bands on native PAGE coincided with the stained band of enzyme activity for all amidase preparations. Affinity columns had a maximum binding capacity of 0.5 mg amidase protein/ml of sedimented gel and could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either semicarbazide or urea were also found useful for the isolation of amidase. The differences in substrate specificity of these amidases reported previously were also observed in the elution behaviour of these enzymes from the affinity columns.

Cell-to-cell contact not soluble factors mediate suppression of lymphocyte proliferation by bovine parainfluenza virus type 3.

We have previously characterized the ability of parainfluenza virus type 3-infected (PIV-3) and noninfected bovine alveolar macrophages (BAM) to support lymphocyte proliferation. While uninfected macrophages support proliferation of lymphocytes stimulated with concanavalin A (Con A), ovalbumin, and interleukin 2 (IL-2), lymphocyte [3H]thymidine incorporation was suppressed in the presence of PIV-3-infected BAM. Since viral infection of macrophages has been shown to alter arachidonic acid metabolism and cytokine secretion, we have determined if arachidonate metabolism or the lack of IL-1 and IL-2 mediated the suppression of lymphocyte proliferation by PIV-3. Inhibition of arachidonic acid metabolism failed to reverse the suppressive effect of viral infection as did supplementation of cultures with bovine recombinant IL-1 beta, IL-2, or lymphocyte-conditioned medium. Further, lymphocytes proliferated normally when physically separated from virus infected BAM by a semipermeable membrane. Stimulation of lymphocytes in contact with infected BAM resulted in marked suppression of lymphocyte [3H]thymidine incorporation. Interactions between stimulated lymphocytes and PIV-3-infected BAM resulted in PIV-3 infection of lymphocytes. Virus infection of lymphocytes was confirmed ultrastructurally by the presence of characteristic parainfluenza virus inclusions and virus budding from lymphocyte plasma membranes. It was concluded that suppression of lymphocyte proliferation by PIV-3 is mediated in part by infection of stimulated lymphocytes during cell-to-cell contact with BAM.

Localization of CAI and CAII isoenzymes in normal term human placenta by immunofluorescence techniques.

The carbonic anhydrase isoenzymes, low activity CAI and high activity CAII, were localized in normal term human placenta by immunocytochemical techniques. Both CAI and CAII isoenzymes were present in the syncytial trophoblasts. Fetal erythrocytes in the placental capillaries also showed positive staining for both CAI and CAII isoenzymes. The possible physiological roles of CA in human placenta are also discussed.

The platelet defect in acute myeloid leukaemia.

In a study of platelets from 13 patients with acute myeloid leukaemia abnormal aggregation and release reactions were found. A previously unrecognised quantitative defect of thromboxane B2 production may, at least in part, explain these findings. In contrast to a previous report, we were unable to show a convincing storage pool defect in these platelets. The platelet membrane glycoproteins were largely normal.

NAD-dependent glutamate dehydrogenase from Pseudomonas aeruginosa is a membrane-bound enzyme.

Measurements of the deaminating activity of NAD-dependent glutamate dehydrogenase (NAD-GDH) in Pseudomonas aeruginosa strain 8602 (PAC 1) showed an initially constant rate that gave way to a 3.5-fold increased rate on prolonged incubation. Only the faster rate was observed when assay mixtures were preflushed with nitrogen or were treated with the detergent Triton X-100. Comparison of the intracellular distribution of NAD-GDH with marker enzymes showed it to be associated with the cytoplasmic membrane. The results suggest that NAD-GDH may be linked to oxygen through an electron-transport system.

Platelet aggregation by oral streptococci.

One proposed mechanism in the pathogenesis of infective endocarditis is the direct aggregation of platelets by the bacteria causing the disease. Some, but not all, strains of Streptococcus sanguis have been reported to aggregate platelets but the taxonomy of this and related taxa has changed recently. The ability to aggregate platelets by 24 genetically grouped laboratory stock strains was studied along with 8 recent isolates from cases of endocarditis. Strains belonging to S. sanguis could aggregate platelets, but not S. gordonii, "S. parasanguis", S. mitis, S. oralis or related taxa. Also, preliminary data indicate that certain biotypes of S. sanguis lack the ability to aggregate platelets. Of the recent clinical isolates, only 4 aggregated platelets and each of these showed phenotypes typical of S. sanguis. These data suggest that the ability to aggregate platelets is not essential for an organism to be able to cause endocarditis, although it may be a significant virulence factor.

Substitution of Glu-59 by Val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme.

A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase. The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene. The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography. The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method. The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70-80%. The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles. The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography. On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a M(r) of 38,000 and 78,000 Dalton, respectively. These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme. Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase.

Human erythrocyte CAI and CAII isoenzymes in hypoxemic and anemic fetuses.

OBJECTIVES: The aims of the present study are (1) to determine the erythrocyte CAI and CAII concentrations in SGA and anemic fetuses and to compare them with normal levels, and (2) to examine whether there is any correlation between fetal hypoxia or anemia and isoenzyme concentrations. METHODS: Human erythrocyte CAI and CAII concentrations were measured in SGA (n = 25) and anemic (n = 32) fetuses (20-36 weeks' gestation), using enzyme-linked immunosorbent assays (ELISA). The blood gases and pH were measured with an ABL-2c blood gas analyzer. RESULTS AND CONCLUSION: In the group of SGA fetuses, there was a tendency for CAI and CAII to be higher than for normal fetuses. The CAI/CAII ratio was also significantly higher than the normal ratio. There were significant correlations between blood Delta pH and Delta CAI, Delta CAII, or Delta CAI/CAII ratio in this group of fetuses. The levels of CAI, CAII, and CAI/CAII ratio were the same as for normal fetuses in the group of anemic fetuses from red cell isoimmunized pregnancies before their first intrauterine blood transfusion. However, in the group of anemic fetuses after their first blood transfusion, the levels of both isoenzymes were significantly higher than the normal blood levels. Within the latter group there was a significant negative correlation between the Delta CAI and the percentage of fetal erythrocytes in the circulation.

Measurement of human erythrocyte CAI and CAII in adult, newborn, and fetal blood.

OBJECTIVE: The aim of the present study was to determine the erythrocyte CAI and CAII concentrations in fetal blood over a wide gestational range, and compare levels to those found in neonates and adults. METHODS: Human erythrocyte CAI and CAII concentrations were measured in fetal (n=38), neonatal (n=10) and adult (n=30) blood, using enzyme-linked immunosorbent assays (ELISA). For the measurement of CAII, a new ELISA method was developed. RESULTS AND CONCLUSIONS: The ELISA method was found to be simple, sensitive, economical, and precise. The normal mean levels of erythrocyte CAI and CAII in adults with standard deviation were 13.68 +/- 2.79, and 1.59 +/- 0.21 mg/g Hb, respectively. The corresponding values in cord blood obtained at delivery at 38-40 weeks gestation were 1.20 +/- 0.68 and 0.46 +/- 0.13 mg/g Hb. The mean CAI/CAII ratio in adults was 8.8 and, in newborns, it was 2.5. The normal mean fetal erythrocyte CAI concentration increased significantly with gestation from 39 microgram/g Hb at 20 weeks to 380 microgram/g Hb at 38 weeks of gestation. Similarly, the CAII concentration increased from 53 microgram/g Hb at 20 weeks to 120 microgram/g Hb at 38 weeks of gestation. The CAI to CAII ratio also increased with gestation from 0.9 at 20 weeks to 2.5 at 38 weeks.

Human erythrocyte superoxide dismutase in adults, neonates, and normal, hypoxaemic, anaemic, and chromosomally abnormal fetuses.

An enzyme-linked immunosorbent method is described for the assay of human erythrocyte superoxide dismutase (SOD). The method was specific and precise as well as simple, economical, and reliable. The mean levels of adult SOD with SD were 652 +/- 122 mg/kg hemoglobin (Hb) for males and 635 +/- 100 mg/kg Hb for females. In neonates the levels were 528 +/- 92 mg/kg Hb. Normal fetal levels at 20-36 weeks gestation were 421 +/- 90 mg/kg Hb. Mean SOD levels in hypoxemic growth-retarded fetuses and in anemic fetuses from red cell iso-immunised pregnancies (before their first intrauterine blood transfusion) were the same as those in normal fetuses. However, in a case of trisomy 21 the fetal level of SOD was significantly increased.

Contaminated fractures of the tibia: a comparison of treatment modalities in an animal model.

External fixation is the current standard treatment for skeletal stabilization of open tibial fractures, but intramedullary fixation techniques have become increasingly popular. The aim of this study was to compare, in an animal model, the susceptibility to infection of contaminated fractures stabilized with external fixation with that of contaminated fractures fixed with intramedullary locking nails with or without reaming. A unilateral osteotomy of the tibia was performed in 15 goats under general anesthesia. Each osteotomy was stabilized with either (a) a unilateral biplanar external fixator, (b) an 8 mm diameter intramedullary rod inserted without reaming of the medullary cavity, or (c) a 10 mm diameter rod inserted after reaming. A standardized inoculum of Staphylococcus aureus, 10(3) colony forming units per milliliter, was placed at each osteotomy site on a piece of absorbable gelatin sponge, to simulate contamination of an open fracture. Antibiotics were not administered. The animals were allowed full activity after the procedure. Fourteen days postoperatively, the animals were killed, radiographs of the tibiae were taken, and the tibiae were harvested in a sterile manner. Multiple specimens for quantitative microbiological analysis were taken from the fracture site and from sites 3 cm distal and 6 cm proximal to the fracture. Additional specimens of bone were taken for histological study. Clinical, radiographic, and microbiological analysis demonstrated that, in this animal model, there were significantly fewer and less severe infections in fractures fixed with external fixation than in those fixed with an intramedullary nail with or without reaming. There was marked cortical necrosis in tibiae that had been fixed with nailing and reaming.

High performance liquid chromatographic determination of serum UV profiles of normal subjects and patients with breast cancer and benign fibrocystic changes.

High performance liquid chromatography (HPLC) was used to determine the UV profiles of serum samples taken postoperatively from 22 patients with histologically documented breast cancer, 8 patients with benign breast fibrocystic changes and 10 normal subjects. The analyses were performed on coded serum samples and after they were completed, the code was broken and the results correlated with the clinical data. Only one ml of serum was required for the HPLC analysis and identification. Detection limits for the nucleosides and bases were in the 10--20 pmol range and the injection volume of the deproteinated serum was 75 mul. The UV profiles of the normal subjects were very reproducible and similar to those of the patients with benign fibrocystic changes. The profiles of some of the cancer patients were distinctly different from the two other groups, 1-methylinosine and N2-methylguanosine, which were not detected in sera from normal subjects and patients with benign fibrocystic changes, were found in 45.5% and 22.7% of the cancer patients, respectively. Patients with the metastatic disease also showed elevated levels of guanosine and uridine. Only one false positive was found in the normal population. At present, it is not clear whether this indicates a subclinical manifestation of the disease and it must await further follow-up.