Jo Bergholte

Oxidation of 15-hydroxyeicosatetraenoic acid and other hydroxy fatty acids by lung prostaglandin dehydrogenase

The oxidation of the 15-hydroxy group of prostaglandins of the A, E, and F series by the NAD+-dependent prostaglandin dehydrogenase (PGDH) has been well documented. In addition to prostaglandins, we have observed that the purified lung PGDH also will oxidize 15-HETE to a novel metabolite that was isolated by reverse-phase HPLC and identified by gas chromatography-mass spectrometry as the 15-keto-5,8,11-cis-13-trans-eicosatetraenoic acid (15-KETE). The Km for 15-HETE was 16 microM, which was 2.5 times lower than the value obtained for PGE1. In addition to 15-HETE, 5,15-diHETE and 8,15-diHETE also were substrates for the lung PGDH with Km values of 138 and 178 microM, respectively. Other hydroxy derivatives of eicosatetraenoic acid that did not have a hydroxy group at carbon atom 15 did not support the PGDH-mediated reduction of NAD+. In addition to the 15-hydroxy derivatives of eicosatetraenoic acid, 12-HHT also was a substrate for the lung enzyme with a Km of 12 microM. These data indicate that omega 6-hydroxy fatty acids, in addition to prostaglandins, are also substrates of the lung NAD+-dependent PGDH and that the enzyme does not require the cyclopentane ring of prostaglandins.

The anesthetic effectiveness of lidocaine-adrenaline-tetracaine gel on finger lacerations

Objectives: To determine the effectiveness of lidocaine-adrenaline-tetracaine (LAT) in providing adequate anesthesia for the repair of finger lacerations and to monitor the risk of digital ischemia following application of LAT gel to finger lacerations. Methods: A prospective case series. Children aged 5 to 18 years with a simple finger laceration-requiring repair were eligible for enrollment. The primary outcome measure was LAT success/failure. Failure was defined as any sharp sensation reported by the patient either before or during suturing. Enrolled patients had LAT gel applied to their laceration for 45 minutes, followed by an examination for signs of digital ischemia and standard laceration repair. Infiltration anesthesia (local subcutaneous injection/digital block) was provided for all LAT failures. Patients were followed up by phone within 3 to 5 days from discharge. Results: Sixty-seven patients were analyzed in the study. The mean age was 11.9 years. Forty-four (65.7%) of 67 patients were male and 46 (68.7%) were white. Locations of the lacerations were equally distributed on the dorsal and ventral surfaces. The overall LAT success rate was 53.7% (95% confidence interval [CI], 41.1% to 66.0%; 36/67). The success rate for dorsal surface lacerations was 68.6% (95% CI, 50.7% to 83.1%; 24/35) versus 37.5% (95% CI, 21.1% to 56.3%; 12/32) for ventral surface lacerations. The difference in success rates between dorsal and ventral surface lacerations was significant (Δ 31.1% [95% CI, 8.3% to 53.8%]). There were no differences in success rates for age, sex, or race. No signs of digital ischemia were noted in any of the 67 cases (0% [95% CI, 0.0% to 5.4%]). Conclusions: LAT gel appears to be a safe and effective means of providing anesthesia for the repair of simple finger lacerations in children. It was most effective on the dorsal surface of the finger.

Isolation and properties of lung 15-hydroxyprostaglandin dehydrogenase from pregnant rabbits.

A NAD-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was purified to a specific activity of over 25,000 nmol NADH formed/min/mg protein with 50 microM prostaglandin E1 as substrate from the lungs of 28-day-old pregnant rabbits. This represented a 2600-fold purification of the enzyme with a recovery of 6% of the starting enzyme activity. The lungs of pregnant rabbits were used because a 42- to 55-fold induction of the PGDH activity was observed after 20 days of gestation. The enzyme was purified by CM-cellulose, DEAE-cellulose, Sephadex G-75, octylamino-agarose, and hydroxylapatite chromatography. The enzyme could not be purified by affinity chromatography using NAD- or blue dextran-bound resins. The purified enzyme was specific for NAD and had a subunit molecular weight of 29,000. The optimal pH range for the oxidation of prostaglandin E1 was between 10.0 and 10.4 using 3-(cyclohexylamino)propanesulfonic acid as the buffer. The Km and Vmax values for prostaglandin E1 were 33 microM and 40,260 nmol/min/mg protein, respectively, while the Km and Vmax values for prostaglandin E2 were 59 microM and 43,319 nmol/min/mg protein, respectively. The Km for prostaglandin F2 alpha was four times the value for prostaglandin E1. The PGDH activity was inhibited by p-chloromercuriphenylsulfonic acid but the enzymatic activity was restored by the addition of dithiothreitol. n-Ethylmaleimide also produced a rapid decline in enzymatic activity but when NAD was included in the incubation system, no inhibition was observed.

Office preparedness for pediatric emergencies: baseline preparedness and the impact of guideline distribution.

Objective: To determine the baseline level of preparedness of Wisconsin primary care physician offices for pediatric emergencies and to assess the impact of mail distribution of guidelines on this level of preparedness. Method: Preintervention surveys were mailed to all Wisconsin physicians requesting information about pediatric equipment, medications, training, and policies. Guidelines were distributed by mail to all physicians who returned completed surveys. An identical postintervention survey was mailed to these physicians, and an analysis of the paired surveys for each respondent was performed. Results: Baseline preparedness of 1051 Wisconsin physician offices ranged from 37% with intraosseous needles to 96% with albuterol solution for inhalation. Physician certification in pediatric advanced life support was required in 26% of offices. A total of 568 paired preintervention and postintervention surveys were analyzed. Improvements were identified for the availability of equipment and medications, transport policies, and reference guides. Conclusions: Mail distribution of guidelines was minimally effective in improving the preparedness of Wisconsin offices for pediatric emergencies.

The short-term outcome of seizure management by prehospital personnel: A comparison of two protocols

Objective To evaluate the impact of an emergency medical service protocol with reduced diazepam dose on the intubation rate of children with seizure activity treated by emergency medical service personnel and to evaluate the short-term outcome comparing 2 emergency medical service treatment protocols. Methods Retrospective review of the emergency medical service and hospital databases of children 0-18 years with seizure activity. Prior to January 1996, the county emergency medical service protocol recommended a diazepam dose of 0.2-0.5 mg/kg iv or pr for termination of seizure activity (group 1). As of January 1996, the diazepam dose was reduced to 0.05-0.1 mg/kg iv or pr (group 2). Demographics, emergency medical service and emergency department interventions, and disposition data were abstracted. Results 1516 subjects met the enrollment criteria: 1003 (66%) in group 1 and 513 (34%) in group 2. Emergency medical service administered diazepam to 288 subjects: 189 (19%) in group 1 and 99 (19%) in group 2. Twenty (7%) of all treated subjects required intubation: 19 in group 1 and 1 in group 2 (relative risk 9.7, 95% CI 1.30-72.5). Mean diazepam dose was 0.17 mg/kg in group 1 and 0.13 mg/kg in group 2 (mean difference 0.04, 95% CI 0.02-0.06). No significant difference in the requirement for repeated anticonvulsant dose, complications, or emergency department interventions was noted. However, hospital admission rate was lower in group 2 (rate difference 0.06, 95% CI 0.01-0.11). Conclusions Our study demonstrated a reduction in intubation rate and a need for hospitalization in the reduced diazepam dose emergency medical service protocol. The reduction in the diazepam dose was effective in terminating the seizure activity and did not increase the risk of adverse events.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase: Immunochemical characterization of the lung enzyme from pregnant rabbits☆

A polyclonal antibody was produced in guinea pig against the lung NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) purified from pregnant rabbits. Western blot analysis demonstrated that the protein identified by this antibody in the 105,000g supernatant fraction of lung tissue from pregnant rabbits had a molecular mass of 30 kDa and comigrated with the purified PGDH. The specific activity of the lung PGDH in pregnant rabbits (25- to 28-day gestations) was 36.7 nmol NADH formed/min/mg protein compared to 0.3 nmol NADH formed/min/mg protein in nonpregnant rabbits. Although the PGDH activity in the lung cytosol of nonpregnant rabbits was inhibited by the anti-lung PGDH antibody, the 30-kDa protein was not detected by Western blot analysis. An examination of this 30-kDa protein during the gestational period indicated that the protein was present after 10 days and the amount of the protein increased from Day 10 to Day 28. This increase in the immunochemically reactive protein correlated with the marked increase in PGDH specific activity between 10 and 28 days. An immunochemically reactive protein also was observed in the ovary of 25- to 28-day pregnant rabbits and the specific activity of the ovary PGDH was 19.3 nmol NADH formed/min/mg protein. Only trace levels of the PGDH activity were detected in the ovaries of nonpregnant rabbits. A 30-kDa protein was not detected by the anti-rabbit lung PGDH in brain, kidney, bladder, uterus, liver, and heart tissue of pregnant or nonpregnant rabbits. When rabbit or human placental cytosol was examined with the anti-rabbit lung PGDH only faint 30-kDa bands were observed by Western blot analysis. A monoclonal antibody prepared against human placental PGDH did not recognize the 30-kDa band in the pregnant rabbit lung. Localization studies indicated a marked increase in immunochemical staining in pulmonary epithelial cells of pregnant rabbits as compared to nonpregnant rabbits. Lung epithelial cells but not endothelial cells were identified as containing the PGDH.

Characterization of an endogenous inhibitor of lung 15-hydroxyprostaglandin dehydrogenase

An endogenous inhibitor of the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase was isolated from the 105,000 X g supernatant fraction of lungs of pregnant rabbits following DEAE chromatography. The material was heat stable and was resistant to pronase treatment. The inhibitor contained a mixture of saturated and mono-unsaturated fatty acids and cholesterol with palmitate and oleate representing the major fatty acids in the inhibitory factor. The factor inhibited prostaglandin dehydrogenase activity but had only minor effects on the activity of NAD+-dependent alcohol and lactate dehydrogenases or the NADP+-dependent isocitrate dehydrogenase. In an attempt to develop a greater understanding of the inhibitory action of fatty acids on prostaglandin dehydrogenase activity, a variety of standard fatty acids were examined for their ability to decrease enzymic activity. Oleate and palmitate inhibited enzymic activity by 70% at 10 microM, whereas arachidonate and myristate were only 30% inhibitory at this concentration. A comparison among the 18-carbon-containing fatty acids demonstrated that oleate was more potent than linoleate and linolenate in inhibiting prostaglandin dehydrogenase activity. The coenzyme A derivatives of oleate, linoleate and linolenate were less inhibitory than the free fatty acids.