Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 51
Filter
1.
J Perinatol ; 37(6): 723-727, 2017 06.
Article in English | MEDLINE | ID: mdl-28181997

ABSTRACT

OBJECTIVE: To estimate the presence and sources of inter-center variation (ICV) in the risk of death or tracheostomy placement (D/T) among infants with severe bronchopulmonary dysplasia (sBPD)Study design:We analyzed the Children's Hospitals Neonatal Database between 2010 and 2013 to identify referred infants born <32 weeks' gestation with sBPD. The association between center and the primary outcome of D/T was analyzed by multivariable modeling. Hypothesized diagnoses/practices were included to determine if these explained any observed ICV in D/T. RESULTS: D/T occurred in 280 (20%) of 1383 eligible infants from 21 centers. ICV was significant for D/T (range 2-46% by center, P<0.001) and tracheostomy placement (n=187, range 2-37%, P<0.001), but not death (n=93, range 0-19%, P=0.08). This association persisted in multivariable analysis (adjusted center-specific odds ratios for D/T varied 5.5-fold, P=0.009). CONCLUSIONS: ICV in D/T is apparent among infants with sBPD. These results highlight that the indications for tracheostomy (and subsequent chronic ventilation) remain uncertain.


Subject(s)
Bronchopulmonary Dysplasia/mortality , Bronchopulmonary Dysplasia/surgery , Infant, Extremely Premature , Intensive Care Units, Neonatal/statistics & numerical data , Tracheostomy/statistics & numerical data , Databases, Factual , Female , Gestational Age , Humans , Infant , Infant, Newborn , Male , Multivariate Analysis , Severity of Illness Index , United States/epidemiology
2.
J Perinatol ; 37(2): 172-176, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27787506

ABSTRACT

OBJECTIVE: Infants requiring resuscitation at birth are resuscitated using neonatal resuscitation guidelines. Sometime after birth, resuscitation practice must transition to pediatric guidelines. There is no evidence on when this transition should occur. The objective of this study was to describe infant resuscitation practices in Neonatal Intensive Care Units (NICUs), Pediatric Intensive Care Units (PICUs) and Cardiac Intensive Care Units (CICUs). STUDY DESIGN: An electronic survey was sent to medical directors of NICUs, PICUs and CICUs in the U.S. The survey examined resuscitation practices, and preference for use of neonatal or pediatric guidelines, for different postnatal ages, clinical scenarios and etiologies of arrest. RESULTS: A total of 152 responses were received, including 118 NICUs, 19 PICUs and 15 CICUs. The majority of NICU responders used greater than 28 days as the time to change from neonatal to pediatric guidelines. The majority of PICU and CICU transitioned to pediatric guidelines immediately after birth. Pediatric guidelines were preferred in the PICU and CICU regardless of the arrest etiology. NICU responders favored pediatric guidelines only if the arrest was cardiac. CONCLUSIONS: Our results suggest that infants are resuscitated using neonatal guidelines in the NICU and pediatric guidelines in the PICU and CICU, even if they are the same age and have the same etiology of arrest. There is no agreement on the time to change from neonatal to pediatric guidelines. Further research comparing the outcomes of infants resuscitated in these different units could inform future guideline refinement.

3.
J Perinatol ; 36(8): 654-9, 2016 08.
Article in English | MEDLINE | ID: mdl-26963428

ABSTRACT

OBJECTIVE: To predict mortality or length of stay (LOS) >109 days (90th percentile) among infants with congenital diaphragmatic hernia (CDH). STUDY DESIGN: We conducted a retrospective analysis using the Children's Hospital Neonatal Database during 2010 to 2014. Infants born >34 weeks gestation with CDH admitted at 22 participating regional neonatal intensive care units were included; patients who were repaired or were at home before admission were excluded. The primary outcome was death before discharge or LOS >109 days. Factors associated with this outcome were used to develop a multivariable equation using 80% of the cohort. Validation was performed in the remaining 20% of infants. RESULTS: The median gestation and age at referral in this cohort (n=677) were 38 weeks and 6 h, respectively. The primary outcome occurred in 242 (35.7%) infants, and was distributed between mortality (n=180, 27%) and LOS >109 days (n=66, 10%). Regression analyses showed that small for gestational age (odds ratio (OR) 2.5, P=0.008), presence of major birth anomalies (OR 5.9, P<0.0001), 5- min Apgar score ⩽3 (OR 7.0, P=0.0002), gradient of acidosis at the time of referral (P<0.001), the receipt of extracorporeal support (OR 8.4, P<0.0001) and bloodstream infections (OR 2.2, P=0.004) were independently associated with death or LOS >109 days. This model performed well in the validation cohort (area under curve (AUC)=0.856, goodness-of-fit (GF) χ(2), P=0.16) and acted similarly even after omitting extracorporeal support (AUC=0.82, GF χ(2), P=0.05). CONCLUSIONS: Six variables predicted death or LOS ⩾109 days in this large, contemporary cohort with CDH. These results can assist in risk adjustment for comparative benchmarking and for counseling affected families.


Subject(s)
Hernias, Diaphragmatic, Congenital/mortality , Length of Stay/statistics & numerical data , Databases, Factual , Female , Gestational Age , Humans , Infant , Infant, Newborn , Intensive Care Units, Neonatal , Logistic Models , Male , Multivariate Analysis , Odds Ratio , Retrospective Studies , Risk Adjustment/methods , United States/epidemiology
4.
J Perinatol ; 34(7): 543-8, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24651732

ABSTRACT

OBJECTIVE: To estimate the risk of death or tracheostomy placement (D/T) in infants with severe bronchopulmonary dysplasia (sBPD) born < 32 weeks' gestation referred to regional neonatal intensive care units. STUDY DESIGN: We conducted a retrospective cohort study in infants born < 32 weeks' gestation with sBPD in 2010-2011, using the Children's Hospital Neonatal Database. sBPD was defined as the need for FiO2 ⩾ 0.3, nasal cannula support >2 l min(-1) or positive pressure at 36 weeks' post menstrual age. The primary outcome was D/T before discharge. Predictors associated with D/T in bivariable analyses (P < 0.2) were used to develop a multivariable logistic regression equation using 80% of the cohort. This equation was validated in the remaining 20% of infants. RESULT: Of 793 eligible patients, the mean gestational age was 26 weeks' and the median age at referral was 6.4 weeks. D/T occurred in 20% of infants. Multivariable analysis showed that later gestational age at birth, later age at referral along with pulmonary management as the primary reason for referral, mechanical ventilation at the time of referral, clinically diagnosed pulmonary hypertension, systemic corticosteroids after referral and occurrence of a bloodstream infection after referral were each associated with D/T. The model performed well with validation (area under curve 0.86, goodness-of-fit χ(2), P = 0.66). CONCLUSION: Seven clinical variables predicted D/T in this large, contemporary cohort with sBPD. These results can be used to inform clinicians who counsel families of affected infants and to assist in the design of future prospective trials.


Subject(s)
Bronchopulmonary Dysplasia/mortality , Tracheostomy/statistics & numerical data , Bronchopulmonary Dysplasia/surgery , Cohort Studies , Female , Gestational Age , Humans , Infant , Infant, Newborn , Infant, Premature , Intensive Care Units, Neonatal , Length of Stay/statistics & numerical data , Logistic Models , Male , Respiration, Artificial , Retrospective Studies , Risk Assessment
5.
J Perinatol ; 33(11): 877-81, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23828204

ABSTRACT

OBJECTIVE: To characterize the treatments and short-term outcomes in infants with severe bronchopulmonary dysplasia (sBPD) referred to regional neonatal intensive care units. STUDY DESIGN: Infants born <32 weeks' gestation with sBPD were identified using the Children's Hospital Neonatal Database. Descriptive outcomes are reported. RESULT: A total of 867 patients were eligible. On average, infants were born at 26 weeks' gestation and referred 43 days after birth. Infants frequently experienced lung injury (pneumonia: 24.1%; air leak: 9%) and received systemic corticosteroids (61%) and mechanical ventilation (median duration 37 days). Although 91% survived to discharge, the mean post-menstrual age was 47 weeks. Ongoing care such as supplemental oxygen (66%) and tracheostomy (5%) were frequently needed. CONCLUSION: Referred infants with sBPD sustain multiple insults to lung function and development. Because affected infants have no proven, safe or efficacious therapy and endure an exceptional burden of care even after referral, urgent work is required to observe and improve their outcomes.


Subject(s)
Bronchopulmonary Dysplasia/therapy , Infant, Premature , Adrenal Cortex Hormones/therapeutic use , Humans , Infant, Newborn , Intensive Care Units, Neonatal , Respiration, Artificial , Treatment Outcome
6.
Clin Appl Thromb Hemost ; 12(3): 369-72, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16959693

ABSTRACT

Pulmonary embolism (PE) and Deep vein thrombosis (DVT) are separate but related aspects of the same dynamic process termed as venous thrombembolism (VTE). The existing Asian literature has shown a wide variation in the prevalence of VTE, with very limited data from the Indian subcontinent. Between January 2001 and July 2004, 1,552 patients with clinically suspected lower limb DVT underwent a combined ascending radionuclide venogram and lung perfusion scan for assessment of the total thrombus burden. Of 744 patients with radionuclide venography proven DVT, 294 (40%) had a high probability lung scan. Nearly half of these patients were asymptomatic for pulmonary embolism. The high prevalence of PE in patients with DVT suggests the need for evaluation of thrombus load in the venous as well as pulmonary circulation. A combination radionuclide ascending venography with lung perfusion scan is a useful and reliable single test for this purpose.


Subject(s)
Pulmonary Embolism/diagnosis , Severity of Illness Index , Venous Thrombosis/diagnosis , Adult , Aged , Female , Humans , Male , Middle Aged , Phlebography , Practice Guidelines as Topic , Prevalence , Radionuclide Imaging , Venous Thrombosis/diagnostic imaging , Ventilation-Perfusion Ratio
7.
Am J Physiol Lung Cell Mol Physiol ; 281(5): L1157-63, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11597907

ABSTRACT

Nitric oxide (NO) and prostacyclin (PGI(2)) are potent fetal pulmonary vasodilators, but their relative roles and interactions in the regulation of the perinatal pulmonary circulation are poorly understood. We compared the separate and combined effects of nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibition during acute hemodynamic stress caused by brief mechanical compression of the ductus arteriosus (DA) in chronically prepared fetal lambs. Nitro-L-arginine (L-NNA; NOS antagonist), meclofenamate (Mec; COX inhibitor), combined drugs (L-NNA-Mec), or saline (control) was infused into the left pulmonary artery (LPA) before DA compression. In controls, DA compression decreased pulmonary vascular resistance (PVR) by 43% (P < 0.01). L-NNA, but not Mec, treatment completely blocked vasodilation and caused a paradoxical increase in PVR (+31%; P < 0.05). The effects of L-NNA-Mec and L-NNA on PVR were similar. To determine if the vasodilator effect of PGI(2) is partly mediated by NO release, we studied PGI(2)-induced vasodilation before and after NOS inhibition. L-NNA treatment blocked the PGI(2)-induced rise in LPA blood flow by 73% (P < 0.001). We conclude that NO has a greater role than PGs in fetal pulmonary vasoregulation during acute hemodynamic stress and that PGI(2)-induced pulmonary vasodilation is largely mediated by NO release in the fetal lung.


Subject(s)
Cyclic GMP/analogs & derivatives , Hemodynamics/physiology , Lung/blood supply , Nitric Oxide/metabolism , Prostaglandins/metabolism , Pulmonary Circulation/physiology , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Animals , Blood Pressure/drug effects , Cyclic GMP/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Drug Interactions , Ductus Arteriosus/physiology , Female , Lung/embryology , Meclofenamic Acid/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Nitroarginine/chemistry , Nitroarginine/pharmacology , Pregnancy , Prostaglandin-Endoperoxide Synthases/metabolism , Pulmonary Circulation/drug effects , Sheep , Stress, Mechanical , Vascular Resistance , Vasodilation
8.
Cancer ; 92(6 Suppl): 1708-13, 2001 Sep 15.
Article in English | MEDLINE | ID: mdl-11598891

ABSTRACT

Cancer-related fatigue is now the most prevalent symptom of cancer, occurring in 60-90% of patients. Fatigue has been identified by cancer patients as a factor influencing functionality and quality of life. Our objectives in developing a fatigue specialty clinic at The University of Texas M. D. Anderson Cancer Center were to improve our patients' quality of life by decreasing fatigue; educate health care providers, patients, and patients' families about cancer-related fatigue; develop an appropriate clinical and diagnostic evaluation for this symptom; correlate objective measures of fatigue with its clinical evaluation; and develop innovative treatment plans for cancer-related fatigue. This article describes the general clinic design and operations and the preliminary analysis of the first 40 patients evaluated in the fatigue clinic.


Subject(s)
Fatigue/therapy , Neoplasms/complications , Outpatient Clinics, Hospital , Adult , Aged , Fatigue/diagnosis , Fatigue/etiology , Female , Humans , Male , Middle Aged , Outpatient Clinics, Hospital/organization & administration
9.
Am J Physiol Heart Circ Physiol ; 281(3): H1005-14, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11514265

ABSTRACT

Prolonged infusions of 17beta-estradiol reduce fetal pulmonary vascular resistance (PVR), but the effects of endogenous estrogens in the fetal pulmonary circulation are unknown. To test the hypothesis that endogenous estrogen promotes pulmonary vasodilation at birth, we studied the hemodynamic effects of prolonged estrogen-receptor blockade during late gestation and at birth in fetal lambs. We treated chronically prepared fetal lambs with ICI-182,780 (ICI, a specific estrogen-receptor blocker, n = 5) or 1% DMSO (CTRL, n = 5) for 7 days and then measured pulmonary hemodynamic responses to ventilation with low- and high-fraction inspired oxygen (FI(O(2))). Treatment with ICI did not change basal fetal PVR or arterial blood gas tensions. However, treatment with ICI abolished the vasodilator response to ventilation with low FI(O(2)) [change in PVR -30 +/- 6% (CTRL) vs. +10 +/- 13%, (ICI), P < 0.05] without reducing the vasodilator response to ventilation with high FI(O(2)) [change in PVR, -73 +/- 3% (CTRL) vs. -77 +/- 4%, (ICI); P = not significant]. ICI treatment reduced prostacyclin synthase (PGIS) expression by 33% (P < 0.05) without altering expression of endothelial nitric oxide synthase or cyclooxygenase-1 and -2. In situ hybridization and immunohistochemistry revealed that PGIS is predominantly expressed in the airway epithelium of late gestation fetal lambs. We conclude that prolonged estrogen-receptor blockade inhibits the pulmonary vasodilator response at birth and that this effect may be mediated by downregulation of PGIS. We speculate that estrogen exposure during late gestation prepares the pulmonary circulation for postnatal adaptation.


Subject(s)
Estradiol/administration & dosage , Estrogen Antagonists/administration & dosage , Prenatal Exposure Delayed Effects , Pulmonary Circulation/drug effects , Receptors, Estrogen/antagonists & inhibitors , Animals , Animals, Newborn , Blotting, Western , Cyclooxygenase 1 , Cyclooxygenase 2 , Cytochrome P-450 Enzyme System/metabolism , Drug Administration Schedule , Estradiol/analogs & derivatives , Female , Fetus/blood supply , Fetus/drug effects , Fetus/physiology , Fulvestrant , Hemodynamics/drug effects , Infusions, Intra-Arterial/methods , Intramolecular Oxidoreductases/metabolism , Isoenzymes/metabolism , Lung/drug effects , Lung/embryology , Lung/enzymology , Nitric Oxide/metabolism , Nitric Oxide/pharmacology , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type III , Organ Specificity , Pregnancy , Prostaglandin-Endoperoxide Synthases/metabolism , Pulmonary Artery/embryology , Pulmonary Artery/physiology , Pulmonary Circulation/physiology , Pulmonary Ventilation/drug effects , Sheep , Vasodilation/drug effects , Vasodilation/physiology
10.
Pediatr Res ; 48(1): 75-83, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10879803

ABSTRACT

Antenatal administration of glucocorticoids has been shown to improve postnatal lung function after preterm birth in the ovine fetus. Mechanisms of steroid-induced lung maturation include increased surfactant production and altered parenchymal lung structure. Whether steroid treatment also affects lung vascular function is unclear. Because nitric oxide contributes to the fall in pulmonary vascular resistance at birth, we hypothesized that the improvement of postnatal lung function of preterm lambs after treatment with prenatal glucocorticoids may be in part caused by an increase in endothelial nitric oxide synthase (eNOS) activity. To determine whether glucocorticoid treatment increases lung eNOS expression, we measured eNOS protein content by Western blot analysis of distal lung homogenates and immunostaining of formalin-fixed lungs from ovine fetuses delivered at preterm and term gestation after prenatal administration of glucocorticoids. Treatment protocols were followed in which ewes were treated with intramuscular betamethasone (0.5 mg/kg) at single or multiple doses at weekly intervals, and fetuses were delivered at 125, 135, or 145 d gestation. All groups were compared with saline-treated controls. Western blot analysis of whole lung homogenates demonstrated a 4-fold increase in eNOS protein content in lambs treated with repetitive doses of glucocorticoids and delivery at term (145 d; p < 0.002). In addition, a small increase in lung eNOS protein content was seen in lambs treated with a single dose of betamethasone at 128 d gestation with delivery at 135 d gestation. In comparison with control animals, there were no differences in lung eNOS content from the remaining lambs treated with glucocorticoids when delivery occurred at preterm ages (125 and 135 d). Immunostaining showed eNOS predominantly in the vascular endothelium in all vessel sizes. Pattern of staining was not altered by treatment with antenatal glucocorticoids. We conclude that maternal treatment with glucocorticoids increases lung eNOS content after multiple doses and delivery at term gestation. We speculate that antenatal glucocorticoids may up-regulate eNOS but that the timing and duration of steroid administration appears to be critical to this response.


Subject(s)
Betamethasone/pharmacology , Glucocorticoids/pharmacology , Lung/embryology , Lung/enzymology , Nitric Oxide Synthase/metabolism , Animals , Animals, Newborn , Betamethasone/administration & dosage , Cesarean Section , Drug Administration Schedule , Female , Gestational Age , Immunohistochemistry , Injections, Intramuscular , Lung/drug effects , Nitric Oxide Synthase Type III , Pregnancy , Reference Values , Sheep
11.
Am J Physiol Lung Cell Mol Physiol ; 278(4): L779-84, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10749755

ABSTRACT

As observed with nitric oxide (NO), carbon monoxide (CO) binds and may activate soluble guanylate cyclase and increase cGMP levels in smooth muscle cells in vitro. Because inhaled NO (I(NO)) causes potent and sustained pulmonary vasodilation, we hypothesized that inhaled CO (I(CO)) may have similar effects on the perinatal lung. To determine whether I(CO) can lower pulmonary vascular resistance (PVR) during the perinatal period, we studied the effects of I(CO) on late-gestation fetal lambs. Catheters were placed in the main pulmonary artery, left pulmonary artery (LPA), aorta, and left atrium to measure pressure. An ultrasonic flow transducer was placed on the LPA to measure blood flow to the left lung. After baseline measurements, fetal lambs were mechanically ventilated with a hypoxic gas mixture (inspired O(2) fraction < 0.10) to maintain a constant fetal arterial PO(2). After 60 min (baseline), the lambs were treated with I(CO) [5-2,500 parts/million (ppm)]. Comparisons were made with I(NO) (5 and 20 ppm) and combined I(NO) (5 ppm) and I(CO) (100 and 2,500 ppm). We found that I(CO) did not alter left lung blood flow or PVR at any of the study doses. In contrast, low-dose I(NO) decreased PVR by 47% (P < 0.005). The combination of I(NO) and I(CO) did not enhance the vasodilator response to I(NO). To determine whether endogenous CO contributes to vascular tone in the fetal lung, zinc protoporphyrin IX, an inhibitor of heme oxygenase, was infused into the LPA in three lambs. Zinc protoporphyrin IX had no effect on baseline PVR, aortic pressure, or the pressure gradient across the ductus arteriosus. We conclude that I(CO) does not cause vasodilation in the near-term ovine transitional circulation, and endogenous CO does not contribute significantly to baseline pulmonary vascular tone or ductus arteriosus tone in the late-gestation ovine fetus.


Subject(s)
Carbon Monoxide/pharmacology , Fetus/physiology , Pulmonary Circulation/drug effects , Vasodilation , Administration, Inhalation , Animals , Blood Pressure/drug effects , Gestational Age , Hemodynamics/drug effects , Injections, Intra-Arterial , Protoporphyrins/pharmacology , Pulmonary Artery/drug effects , Sheep/embryology , Vascular Resistance/drug effects
12.
Am J Physiol Lung Cell Mol Physiol ; 278(2): L374-81, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10666122

ABSTRACT

Partial ligation of the ductus arteriosus (DA) in the fetal lamb causes sustained elevation of pulmonary vascular resistance (PVR) and hypertensive structural changes in small pulmonary arteries, providing an animal model for persistent pulmonary hypertension of the newborn. Based on its vasodilator and antimitogenic properties in other experimental studies, we hypothesized that estradiol (E(2)) would attenuate the pulmonary vascular structural and hemodynamic changes caused by pulmonary hypertension in utero. To test our hypothesis, we treated chronically instrumented fetal lambs (128 days, term = 147 days) with daily infusions of E(2) (10 microg; E(2) group, n = 6) or saline (control group, n = 5) after partial ligation of the DA. We measured intrauterine pulmonary and systemic artery pressures in both groups throughout the study period. After 8 days, we delivered the study animals by cesarean section to measure their hemodynamic responses to birth-related stimuli. Although pulmonary and systemic arterial pressures were not different in utero, fetal PVR immediately before ventilation was reduced in the E(2)-treated group (2.43 +/- 0.79 vs. 1.48 +/- 0.26 mmHg. ml(-1). min, control vs. E(2), P < 0.05). During the subsequent delivery study, PVR was lower in the E(2)-treated group in response to ventilation with hypoxic gas but was not different between groups with ventilation with 100% O(2). During mechanical ventilation after delivery, arterial partial O(2) pressure was higher in E(2) animals than controls (41 +/- 11 vs. 80 +/- 35 Torr, control vs. E(2), P < 0. 05). Morphometric studies of hypertensive vascular changes revealed that E(2) treatment decreased wall thickness of small pulmonary arteries (59 +/- 1 vs. 48 +/- 1%, control vs. E(2), P < 0.01). We conclude that chronic E(2) treatment in utero attenuates the pulmonary hemodynamic and histological changes caused by DA ligation in fetal lambs.


Subject(s)
Animals, Newborn/physiology , Blood Vessels/physiopathology , Estradiol/pharmacology , Fetus/physiology , Hypertension, Pulmonary/physiopathology , Pulmonary Circulation/drug effects , Animals , Ductus Arteriosus , Female , Hemodynamics/drug effects , Hypertension, Pulmonary/complications , Hypertension, Pulmonary/pathology , Hypertrophy, Right Ventricular/etiology , Ligation , Muscle, Smooth/drug effects , Physical Stimulation , Pregnancy , Pulmonary Artery/drug effects , Pulmonary Artery/pathology , Sheep/embryology
13.
Anal Chem ; 70(4): 673-83, 1998 Feb 15.
Article in English | MEDLINE | ID: mdl-9491752

ABSTRACT

Metastable ion decay in matrix-assisted laser desorption/ionization (MALDI) has become a routine method for obtaining primary structures of peptides. Significant fragmentation occurs in the MALDI ion source and can be observed via delayed ion extraction TOF-MS. In-source decay (ISD) can provide C- and N-terminal primary sequence data for even moderate-sized peptides (< 5000 Da). The unique cn series fragmentation that occurs in ISD has been exploited to obtain partial C-terminal sequences for proteins as large as human apotransferrin (75 kDa). Two approaches for combining this ISD MALDI-generated partial sequence information with protein database searching techniques are presented. In one approach, cyanogen bromide is used to cleave relatively large peptide fragments from a sample of human apotransferrin. One of the larger cleavage products (6034.84 Da) was isolated by HPLC and subjected to ISD MALDI analysis. An easily identified cn fragment ion series allowed two noncontiguous segments of the peptide's sequence to be determined (about 55% of the total sequence). This partial sequence information was used to search protein and oligonucleotide sequence databases. In addition to uniquely identifying human apotransferrin in a protein sequence database, an example of the use of this ISD MALDI-determined partial sequence information to search expressed sequence tag databases is presented. Such searches have the potential for rapidly identifying new genes that code for target proteins. An alternate approach for obtaining partial sequence information on proteins is also demonstrated that utilizes ISD MALDI fragmentation of the intact protein to generate partial sequence information. This approach is shown to generate about 5-7% of a protein's sequence, usually near the C-terminus of the protein. Examples of the ISD MALDI fragmentation data obtained from intact (reduced) human apotransferrin and intact (nonreduced) bovine serum albumin (66 kDa) proteins are presented.


Subject(s)
Proteins/analysis , Animals , Cattle , Databases, Factual , Humans , Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
14.
Biochemistry ; 36(1): 139-47, 1997 Jan 07.
Article in English | MEDLINE | ID: mdl-8993327

ABSTRACT

Efficient peroxidase substrates may have a critical role in the oxidation of secondary compounds by peroxidases. Hydrazines are often oxidized slowly by peroxidases due, in part, to hydrazine-dependent inactivation of these enzymes. Peroxidase-catalyzed oxidation of hydrazines may be dramatically affected by an efficient peroxidase substrate. We investigated this hypothesis in a model system using the well-known peroxidase substrate chlorpromazine (CPZ) and the hydrazine derivative isoniazid. CPZ stimulated isoniazid oxidation as measured by nitroblue tetrazolium (NBT) reduction and O2 consumption. The kinetics of isoniazid and CPZ oxidation by horseradish peroxidase (HRP) in the presence of both compounds suggested CPZ was acting as an electron transfer mediator between HRP and isoniazid. Indeed, CPZ.+, the product of CPZ oxidation by HRP, was able to oxidize isoniazid. The rate constant for this pH-dependent reaction was (2.6 +/- 0.1) x 10(4) M-1 s-1 at pH 4.5. In the absence of CPZ, isoniazid-dependent irreversible inactivation of HRP was observed. The inactivation process involved the formation of compound III followed by accumulation of irreversibly inactivated HRP. CPZ completely inhibited inactivation. Thus, by acting as a redox mediator and preventing HRP inactivation, CPZ stimulated isoniazid oxidation by several orders of magnitude. Similarly, other efficient peroxidase substrates, such as phenol and tyrosine, were also able to dramatically stimulate isoniazid oxidation by HRP. We suggest that the presence of efficient peroxidase substrates may potentiate the activation of isoniazid and other hydrazines. As such, these substrates may have a vital role in the pharmacological and toxicological properties of hydrazines and other compounds.


Subject(s)
Enzyme Activation/physiology , Horseradish Peroxidase/metabolism , Hydrazines/metabolism , Carbon Monoxide/metabolism , Chemical Phenomena , Chemistry , Chlorpromazine/metabolism , Chlorpromazine/pharmacology , Enzyme Inhibitors/pharmacology , Hydrogen-Ion Concentration , Isoniazid/metabolism , Isoniazid/pharmacology , Models, Chemical , Oxidation-Reduction , Oxygen/metabolism , Phenol , Phenols/metabolism , Spectrophotometry , Substrate Specificity , Tyrosine/metabolism
15.
Chem Res Toxicol ; 9(8): 1333-9, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8951237

ABSTRACT

Hypochlorous acid (HOCl) derived from activated neutrophils and monocytes has been implicated in the activation of hydrazine-containing drugs to toxic intermediates. However, reactive intermediates formed during the reaction between HOCl and these drugs have not been identified. We investigated the oxidation of the hydrazine derivatives isoniazid, iproniazid, and hydralazine by HOCl. The reaction between HOCl and all three hydrazines resulted in O2 consumption, indicating that free radicals were produced, but the rate and extent of O2 consumption were different for each hydrazine. Moreover, reduction of nitroblue tetrazolium (NBT) was observed only during the reaction between HOCl and isoniazid, suggesting that different radical species may be produced from HOCl reaction with each hydrazine. The oxidation of iproniazid by HOCl in the presence of the radical trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) resulted in the formation of a carbon-centered radical adduct. In contrast, the reaction between HOCl and hydralazine resulted in the formation of a nitrogen-centered DMPO radical adduct. The oxidation of isoniazid by HOCl resulted in the formation of two oxygen-centered radical adducts, DMPO-OOH and DMPO-OH. Myeloperoxidase-catalyzed oxidation of these hydrazines in the presence of Cl- and H2O2 produced radical species that were identical to those observed with HOCl. Thus, some of the toxic side effects of these drugs may be the result of the production of free-radical intermediates from reaction with neutrophil-derived oxidants, such as HOCl. The types of radicals produced and the consequences of generating these reactive species are discussed.


Subject(s)
Free Radicals/chemistry , Hydrazines/chemistry , Hypochlorous Acid/chemistry , Electron Spin Resonance Spectroscopy , Indicators and Reagents/chemistry , Isoniazid/chemistry , Nitroblue Tetrazolium/chemistry , Oxidants/chemistry , Oxidation-Reduction , Oxygen Consumption/drug effects , Oxygen Consumption/physiology , Reactive Oxygen Species , Spin Trapping
16.
Chem Res Toxicol ; 9(2): 476-83, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8839052

ABSTRACT

Many drugs, industrial pollutants, and other xenobiotics are known to be oxidized by peroxidases to potentially harmful free-radical intermediates. We have examined the possibility that certain compounds, acting as efficient peroxidase substrates, may stimulate the formation of reactive free radicals by acting as mediators of electron transfer reactions (redox mediators). To explore this hypothesis, we have investigated the interaction of two well-known peroxidase substrates, chlorpromazine and aminopyrine. As shown by ESR and UV-visible spectroscopy, chlorpromazine radical was able to oxidize aminopyrine to aminopyrine cation radical. The rate constant for this rapid, pH-dependent, reaction was estimated to be 1 x 10(7) M-1 s-1 at pH 4.5. Transient-state and steady-state kinetic studies both showed that rate constants for chlorpromazine oxidation to its cation radical by horseradish peroxidase (HRP) were about 100-fold greater than for the corresponding HRP-catalyzed oxidation of aminopyrine to its cation radical. When both aminopyrine and chlorpromazine were present with HRP and H2O2, aminopyrine cation radical formation was stimulated 100-fold. Concomitantly, the accumulation of chlorpromazine cation radical was completely inhibited in the presence of aminopyrine. Similar results were obtained when lactoperoxidase, myeloperoxidase, or the myeloperoxidase mimic HOCl were substituted for HRP. These data suggest that chlorpromazine can act as a redox mediator for peroxidase-catalyzed oxidation of aminopyrine and other chemicals. We suggest that some peroxidase substrates, acting as redox mediators, may stimulate the production of toxic free-radical intermediates from various drugs and other xenobiotics. As such, this may have implications for a number of adverse effects caused by these xenobiotic chemicals.


Subject(s)
Aminopyrine/metabolism , Drug Interactions/physiology , Horseradish Peroxidase/metabolism , Chlorpromazine/metabolism , Kinetics , Oxidation-Reduction/drug effects , Spectrum Analysis
17.
Anal Biochem ; 231(2): 333-8, 1995 Nov 01.
Article in English | MEDLINE | ID: mdl-8594981

ABSTRACT

We describe an improved enzyme-monitored stopped-flow method for determining rate constants for peroxidase reactions that are too rapid to measure by conventional pseudo-first-order methods. Ascorbate will reduce many substrate radicals as rapidly as they are generated by a peroxidase. This ensures a constant substrate concentration during the peroxidase reaction. We investigated the reactions of horseradish peroxidase compound I (HRP-I) with three standard substrates (chlorpromazine (CPZ), 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), and p-methoxyphenol) in the presence and absence of ascorbate. The rate of reaction of CPZ with HRP-I is slow enough that it can be measured using pseudo-first-order conditions maintained by a minimum 10-fold excess of CPZ, or by the addition of ascorbate at very low CPZ concentrations. The same rate constant was obtained with either method. The rate of reaction of ABTS with HRP-I at lower pH (5.0-3.3) is rapid; consequently, we were unable to obtain rate constants using concentrations of ABTS which constitute pseudo-first-order conditions. However, using much lower ABTS concentrations with the addition of ascorbate, we obtained rate constants that ranged from 5 x 10(7) to 8 x 10(8) M-1 s-1. Hence, ascorbate provides a simplified way to maintain pseudo-first-order conditions for fast peroxidase reactions even at low substrate concentrations.


Subject(s)
Ascorbic Acid/chemistry , Horseradish Peroxidase/chemistry , Computer Simulation , Hydrogen-Ion Concentration , Kinetics , Spectrophotometry , Time Factors
18.
Arch Biochem Biophys ; 320(2): 243-9, 1995 Jul 10.
Article in English | MEDLINE | ID: mdl-7625830

ABSTRACT

A quinone produced from veratryl alcohol by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was tested for its ability to mediate reduction. The quinone (2-hydroxymethyl-5-methoxy-1,4-benzoquinone), reduced chemically or by cellobiose:quinone reductase isolated from cultures of the fungus, mediated the reduction of cytochrome c in reactions containing either Mn(III), a manganese-dependent peroxidase, Mn(II) and H2O2, or lignin peroxidase and H2O2. Formation of the semiquinone, the species responsible for reducing cytochrome c, was observed by electron spin resonance spectroscopy in these reactions. The production of the quinone was observed in the extracellular fraction of cultures grown under nutrient nitrogen-deficient conditions (2.4 mM ammonium tartrate) for over 10 days, starting on Day 2, but not under nutrient nitrogen-sufficient conditions. These results suggest that a quinone produced by lignin peroxidase can serve as a physiological mediator of reductive reactions catalyzed by the fungal peroxidases.


Subject(s)
Benzoquinones/metabolism , Cytochrome c Group/metabolism , Fungi/metabolism , Peroxidases/metabolism , Benzoquinones/chemical synthesis , Electron Spin Resonance Spectroscopy , Oxidation-Reduction
19.
Biochemistry ; 34(18): 6020-5, 1995 May 09.
Article in English | MEDLINE | ID: mdl-7742304

ABSTRACT

Lignin peroxidases (LiP) catalyze the H2O2-dependent two-electron oxidation of veratryl alcohol (VA) to veratryl aldehyde. We present here, electron spin resonance (ESR) evidence for the formation of the one-electron oxidized intermediate, the veratryl alcohol cation radical (VA.+). The ESR spectrum of VA.+ was first obtained in a fast-flow system with Ce(IV) as an oxidant and 10% HNO3 to stabilize the radical. This ESR signal was deconvoluted, and the hyperfine splitting constants were determined. The identity of the radical was confirmed by computer simulation of the ESR spectrum and calculation of spin and charge densities on the radical. An identical radical signal was observed with LiP, also in a fast-flow incubation containing 10 microM LiP, 2 mM VA, and 500 microM H2O2 at pH 3.5. The Fourier transforms of the ESR signals further confirmed that the spectra obtained with both Ce(IV) and LiP were due to the same radical species. The VA.+ had a distinct visible spectrum in 98% H2SO4 with an absorbance maximum at 529 nm. The extinction coefficient of the VA.+ spectral band at 529 nm was calculated to be 11,000 M-1 cm-1. The VA.+ was found to be a strong acid, as are other cation radicals, with the pKa at -1.0 pH. This value was determined by quantitating both the concentration of VA.+ by visual and ESR spectrometry and the g-value of the ESR signal at various pH values.


Subject(s)
Benzyl Alcohols/metabolism , Peroxidases/metabolism , Cations/metabolism , Electron Spin Resonance Spectroscopy , Free Radicals/metabolism , Oxidation-Reduction
20.
Biochemistry ; 34(15): 5060-5, 1995 Apr 18.
Article in English | MEDLINE | ID: mdl-7711026

ABSTRACT

We have examined the hypothesis that veratryl alcohol (VA) may act as a redox mediator in lignin peroxidase (LiP)-catalyzed oxidations. The oxidation of chlorpromazine (CPZ) by this system was used to evaluate this hypothesis. Chlorpromazine can be oxidized by one electron to form a stable cation radical (CPZ.+). This cation radical can be oxidized by another electron to the sulfoxide (CPZSO). These oxidation steps are easily monitored, making CPZ a useful chemical to investigate redox mediation by VA. Lignin peroxidase oxidized CPZ to CPZ.+ whether or not VA was present. The inclusion of VA, however, stimulated CPZ oxidation to CPZ.+ and subsequent oxidation of CPZ.+ to CPZSO. In the absence of VA, the initial rates of CPZ oxidation by LiP were CPZ concentration-dependent. However, when saturating concentrations of VA were added, the oxidation of CPZ and CPZ.+ became independent of CPZ concentration. When the oxidation of VA to veratryl aldehyde was examined, increasing concentrations of CPZ produced a lag in veratryl aldehyde appearance proportional to the concentration of CPZ. Conversely, increasing concentrations of VA never inhibited CPZ oxidation. Transient-state kinetic studies indicated that both VA and CPZ reduced the compound I and compound II forms of LiP. However, when saturating concentrations of VA were utilized, LiP turnover was independent of CPZ concentration. We suggest these data demonstrate that VA may act as a redox mediator for the indirect oxidation of compounds by LiP.


Subject(s)
Benzyl Alcohols/metabolism , Peroxidases/metabolism , Catalysis , Chlorpromazine/metabolism , Electrons , Kinetics , Models, Chemical , Oxidation-Reduction
SELECTION OF CITATIONS
SEARCH DETAIL
...