Search

Malnutrisi merupakan masalah penting pada pasien sepsis karena memperburuk outcome akibat penyembuhan luka terganggu, menurunkan fungsi imun, risiko infeksi tinggi, kelemahan otot sehingga weaning ventilator mekanik lebih sulit, meningkatkan lama tinggal dan biaya tinggi di unit perawatan intensif serta peningkatan mortalitas. Skrining risiko nutrisi saat pasien masuk unit perawatan intensif dengan modified NUTRIC score dapat mengidentifikasi kelompok risiko nutrisi tinggi atau risiko rendah. Penelitian kohort retrospektif untuk mengetahui korelasi antara modified NUTRIC score dengan mortalitas pada pasien sepsis yang dirawat dari Januari‒Desember 2018 di RSUP Dr. Mohammad Hoesin Palembang. Jumlah sampel 100 orang, dibagi 2 kelompok berdasarkan mortalitas, kemudian dilakukan pengukuran modified NUTRIC score yang terdiri dari usia, skor APACHE II, skor SOFA, lama rawat sebelum masuk perawatan intensif, dan jumlah komorbid. Modified NUTRIC score memiliki nilai 0‒9. Hasil analisis ROC curve modified NUTRIC score dengan mortalitas didapatkan sensitiftas sebesar 96%, spesifisitas 71%, nilai area under the curve (AUC) 0,916 dengan nilai cut off >4. Terdapat korelasi antara modified NUTRIC score dengan mortalitas dalam 28 hari pasien sepsis dengan koefisien korelasi (r)=0,716 dan nilai p=0,0001.

Objective: Central nervous system (CNS) damage from HIV infection or treatment can lead to developmental delays and poor educational outcomes in children living with HIV (CLWH). Early markers of central nervous system dysfunction are needed to target interventions and prevent life-long disability. The frequency following response (FFR) is an auditory electrophysiology test that can reflect the health of the central nervous system. In this study, we explore whether the FFR reveals auditory central nervous system dysfunction in CLWH., Study Design: Cross-sectional analysis of an ongoing cohort study. Data were from the child's first visit in the study., Setting: The infectious disease center in Dar es Salaam, Tanzania., Methods: We collected the FFR from 151 CLWH and 151 HIV-negative children. To evoke the FFR, three speech syllabi (/da/, /ba/, /ga/) were played monaurally to the child's right ear. Response measures included neural timing (peak latencies), strength of frequency encoding (fundamental frequency and first formant amplitude), encoding consistency (inter-response consistency), and encoding precision (stimulus-to-response correlation)., Results: CLWH showed smaller first formant amplitudes ( P  < 0.0001), weaker inter-response consistencies ( P  < 0.0001) and smaller stimulus to response correlations ( P  < 0.0001) than FFRs from HIV-negative children. These findings generalized across the three speech stimuli with moderately strong effect sizes (partial η2 ranged from 0.061 to 0.094)., Conclusion: The FFR shows auditory central nervous system dysfunction in CLWH. Neural encoding of auditory stimuli was less robust, more variable, and less accurate. As the FFR is a passive and objective test, it may offer an effective way to assess and detect central nervous system function in CLWH., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: Evaluate whether a portable, tablet-based central auditory processing (CAP) test system using native language training videos and administered by minimally trained community health workers can produce CAP results comparable to previously published norms. Our secondary aim was to determine subject parameters that influence test results., Study Design: Cross-sectional study., Setting: Community-based settings in Chontales, Nicaragua, New Hampshire, and Florida., Patients: English- and/or Spanish-speaking children and adolescents (n = 245; average age, 12.20 yr; range, 6-18 yr)., Main Outcome Measures: Completion of the following tests with responses comparable to published norms: Pure-tone average (PTA), gap detection threshold (GDT), fixed-level frequency threshold, masking level difference (MLD), Hearing in Noise Test (HINT), Dichotic Digits Test (DDT), and Frequency Pattern Recognition (FPR) test., Results: GDT, HINT, and DDT had comparable results to previously published normative values. MLD and FPR results differed compared with previously published normative values. Most CAP tests (MLD, GDT, HINT) results were independent of age and PTA (p = 0.1-0.9). However, DDT was associated with age and PTA (p < 0.0001)., Conclusions: Pediatric CAP testing can be successfully completed in remote low- and middle- income country environments using a tablet-based platform without the presence of an audiologist. Performance on DDT improved with age but deteriorated with hearing loss. Further investigation is warranted to assess the variability of FPR., (Copyright © 2024, Otology & Neurotology, Inc.)

Objective: Automated threshold audiometry (ATA) could increase access to paediatric hearing assessment in low- and middle-income countries, but few studies have evaluated test-retest repeatability of ATA in children. This study aims to analyse test-retest repeatability of ATA and to identify factors that affect the reliability of this method., Design: ATA was performed twice in a cohort of Nicaraguan schoolchildren. During testing, the proportion of responses occurring in the absence of a stimulus was measured by calculating a stimulus response false positive rate (SRFP). Absolute test-retest repeatability was determined between the two trials, as well as the impact of age, gender, ambient noise, head circumference, and SRFP on these results., Study Sample: 807 children were randomly selected from 35 schools in northern Nicaragua., Results: Across all frequencies, the absolute value of the difference between measurements was 5.5 ± 7.8 dB. 89.6% of test-retest differences were within 10 dB. Intra-class correlation coefficients between the two measurements showed that lower SRFP was associated with improved repeatability. No effect of age, gender, or ambient noise was found., Conclusions: ATA produced moderate test-retest repeatability in Nicaraguan schoolchildren. Participant testing behaviours, such as delayed or otherwise inappropriate response patterns, significantly impacts the repeatability of these measurements.

Importance: Despite normal audiometry, adults living with HIV have lower distortion product otoacoustic emissions (DPOAEs) compared with HIV-negative controls, but the degree of these differences in children living with HIV is unknown. If subclinical auditory deficits are present, results could affect developmental outcomes in children living with HIV (CLWH)., Objective: To compare DPOAEs and auditory brainstem responses (ABR) between 2 age- and sex-matched groups of younger children with normal audiometry, 1 infected with HIV and the other uninfected., Design, Setting, and Participants: Cohort study in an infectious disease center in Dar es Salaam, Tanzania. Participants included 340 Tanzanian children aged 3 to 9 years with clinically normal hearing, type A tympanograms bilaterally, and air-conduction thresholds of 20 dB HL or less from 0.5 to 8 kHz. Participants in the cohort repeated testing approximately every 6 months (approximately 2.2 sessions per participant) for a total of 744 total observations. Data were analyzed from March 2020 to January 2022., Main Outcomes and Measures: DPOAE amplitudes from 1.5 to 8 kHz using an f2 to f1 ratio of 1.2 and L1/L2 values of 65/55 dB sound pressure level and click-evoked ABR using a slow (21.1/s) and fast (61.1/s) click rate., Results: A total of 141 CLWH (70 female participants [49.3%]; mean [SD] age, 7.24 [1.67] years) and 199 HIV-negative individuals (99 female participants [49.7%]; mean [SD] age, 7.26 [1.44] years) participated in the study. The groups did not differ significantly in age, static immittance, or air-conduction thresholds. HIV status was independently associated with approximately 1.4 dB (95% CI, -3.28 to 0.30 dB) to 3.8 dB (95% CI, 6.03 to -1.99 dB) lower DPOAE amplitudes at 6 and 8 kHz bilaterally and 0.28 μV (95% CI, 0.01 to 0.33 μV) lower ABR wave V amplitudes in the right ear., Conclusions and Relevance: Consistent with previous findings in young adults, CLWH had slightly, but reliably, lower DPOAEs and ABR wave V amplitudes than HIV-negative controls. The magnitude of these differences was small, but results suggest an early and consistent association between HIV infection or treatment and outer hair cell and auditory brainstem responses in children as young as 3 years. These subclinical changes suggest tracking both auditory function and development outcomes in CLWH is warranted.

Objective: Little is known about peripheral auditory function in young adults with HIV, who might be expected to show early evidence of hearing loss if HIV infection or treatment does affect peripheral function. The goal of this study was to compare peripheral auditory function in 2 age- and gender-matched groups of young adults with clinically normal hearing with and without HIV., Study Design: Matched cohort study with repeated measures., Setting: Infectious disease center in Dar es Salaam, Tanzania., Methods: Participants included HIV-positive (n = 38) and HIV-negative (n = 38) adults aged 20 to 30 years who had clinically normal hearing, defined as type A tympanograms, air conduction thresholds ≤25 dB HL bilaterally from 0.5 to 8 kHz, and distortion product otoacoustic emissions (DPOAEs) >6 dB above the noise floor bilaterally from 1.5 to 8 kHz. Participants were tested multiple times over 6-month intervals (average, 2.7 sessions/participant) for a total of 208 observations. Primary outcome measures included tympanograms, air conduction audiograms, DPOAEs, and click-evoked auditory brainstem responses., Results: HIV groups did not significantly differ in age, static immittance, or air conduction thresholds. HIV-positive status was independently associated with approximately 3.7-dB lower DPOAE amplitudes from 2 to 8 kHz (95% CI, 1.01-6.82) in both ears and 0.04-µV lower (95% CI, 0.003-0.076) auditory brainstem response wave I amplitudes in the right ear., Conclusion: Young adults living with HIV have slightly but reliably smaller DPOAEs and auditory brainstem response wave I amplitudes than matched HIV-negative controls. The magnitude of these differences is small, but these results support measuring peripheral auditory function in HIV-positive individuals as they age.

Background: Hospital readmissions are a key quality metric, which has been tied to reimbursement. One strategy to reduce readmissions is to direct resources to patients at the highest risk of readmission. This strategy necessitates a robust predictive model coupled with effective, patient-centered interventions., Objective: The aim of this study was to reduce unplanned hospital readmissions through the use of artificial intelligence-based clinical decision support., Methods: A commercially vended artificial intelligence tool was implemented at a regional hospital in La Crosse, Wisconsin between November 2018 and April 2019. The tool assessed all patients admitted to general care units for risk of readmission and generated recommendations for interventions intended to decrease readmission risk. Similar hospitals were used as controls. Change in readmission rate was assessed by comparing the 6-month intervention period to the same months of the previous calendar year in exposure and control hospitals., Results: Among 2,460 hospitalizations assessed using the tool, 611 were designated by the tool as high risk. Sensitivity and specificity for risk assignment were 65% and 89%, respectively. Over 6 months following implementation, readmission rates decreased from 11.4% during the comparison period to 8.1% ( p  < 0.001). After accounting for the 0.5% decrease in readmission rates (from 9.3 to 8.8%) at control hospitals, the relative reduction in readmission rate was 25% ( p  < 0.001). Among patients designated as high risk, the number needed to treat to avoid one readmission was 11., Conclusion: We observed a decrease in hospital readmission after implementing artificial intelligence-based clinical decision support. Our experience suggests that use of artificial intelligence to identify patients at the highest risk for readmission can reduce quality gaps when coupled with patient-centered interventions., Competing Interests: Mayo Clinic and the third-party AI-based CDS software tool developer (Jvion; Johns Creek, Georgia) have existing licensing agreements for unrelated technology. Jvion was not involved in the production of this manuscript except to answer queries made during the peer review process regarding details of the software. Jvion otherwise played no role in the conception, drafting, or revision of the manuscript nor did they play a role in the decision to submit it for publication. The pilot project was funded with internal Mayo Clinic funds., (Georg Thieme Verlag KG Stuttgart · New York.)

Background: "Artificial intelligence" (AI) is often referred to as "augmented human intelligence" (AHI). The latter term implies that computers support-rather than replace-human decision-making. It is unclear whether the terminology used affects attitudes and perceptions in practice., Methods: In the context of a quality improvement project implementing AI/AHI-based decision support in a regional health system, we surveyed staff's attitudes about AI/AHI, randomizing question prompts to refer to either AI or AHI., Results: Ninety-three staff completed surveys. With a power of 0.95 to detect a difference larger than 0.8 points on a 5-point scale, we did not detect a significant difference in responses to six questions regarding attitudes when respondents were alternatively asked about AI versus AHI (mean difference range: 0.04-0.22 points; p > 0.05)., Conclusion: Although findings may be setting-specific, we observed that use of the terms "AI" and "AHI" in a survey on attitudes of clinical staff elicited similar responses.

Background: To explore attitudes about artificial intelligence (AI) among staff who utilized AI-based clinical decision support (CDS)., Methods: A survey was designed to assess staff attitudes about AI-based CDS tools. The survey was anonymously and voluntarily completed by clinical staff in three primary care outpatient clinics before and after implementation of an AI-based CDS system aimed to improve glycemic control in patients with diabetes as part of a quality improvement project. The CDS identified patients at risk for poor glycemic control and generated intervention recommendations intended to reduce patients' risk., Results: Staff completed 45 surveys pre-intervention and 38 post-intervention. Following implementation, staff felt that care was better coordinated (11 favorable responses, 14 unfavorable responses pre-intervention; 21 favorable responses, 3 unfavorable responses post-intervention; p < 0.01). However, only 14 % of users would recommend the AI-based CDS. Staff feedback revealed that the most favorable aspect of the CDS was that it promoted team dialog about patient needs (N = 14, 52 %), and the least favorable aspect was inadequacy of the interventions recommended by the CDS., Conclusions: AI-based CDS tools that are perceived negatively by staff may reduce staff excitement about AI technology, and hands-on experience with AI may lead to more realistic expectations about the technology's capabilities. In our setting, although AI-based CDS prompted an interdisciplinary discussion about the needs of patients at high risk for poor glycemic control, the interventions recommended by the CDS were often perceived to be poorly tailored, inappropriate, or not useful. Developers should carefully consider tasks that are best performed by AI and those best performed by the patient's care team., (Copyright © 2019. Published by Elsevier B.V.)

Objective: Determine whether an electronic tablet-based Wireless Automated Hearing-Test System can perform high-quality audiometry to assess schoolchildren for hearing loss in the field in Nicaragua., Study Design: Cross-sectional., Setting: A school and hospital-based audiology clinic in Jinotega, Nicaragua., Subjects and Methods: Second and third graders (n = 120) were randomly selected for hearing testing in a school. Air conduction hearing thresholds were obtained bilaterally using a Wireless Automated Hearing-Test System at 1000, 2000, and 4000 Hz. Referral criteria were set at more than 25 dBHL at one or more frequencies. A cohort of children was retested with conventional audiometry in a hospital-based sound booth. Factors influencing false-positive examinations, including ambient noise and behavior, were examined., Results: All children with hearing loss were detected using an automated, manual, or two-step (those referred from automated testing were tested manually) protocol in the school (sensitivity = 100%). Specificity was 76% for automated testing, 97% for manual testing, and 99% for the two-step protocol. The variability between thresholds obtained with automated testing was greater than manual testing when compared with conventional audiometry. The percentage of participant responses when no stimulus tone was presented during automated testing was higher in children with false-positive examinations., Conclusion: A Wireless Automated Hearing-Test System identified all children with hearing loss in a challenging field setting. A two-step protocol (those referred from automated testing are tested manually) reduced false-positive examinations and unnecessary referrals. Children who respond frequently when no tone is presented are more likely to have false-positive automated examinations and should be tested manually.

Story is an intimate human resource to describe our being within the world. This article demonstrates the use of this human resource within a patient- and family-centered care initiative developed with and for the pediatric patient population. It describes the purpose, objectives, and design of the MyStory initiative as well as its impact on patient satisfaction scores. Finally, this article challenges current methods of data collection and assessment to include patient stories in order to involve children in care planning and decision making to ensure whole-person care.

Colonization of the gastric epithelium by Helicobacter pylori induces the transcription factor nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) and the innate immune response. Virulent strains of H. pylori carry a cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS). Recent publications have shown controversial data regarding the role of the T4SS and the effector protein cytotoxin associated gene A (CagA), which becomes translocated by the T4SS into the eukaryotic epithelial cell, in H. pylori-induced NF-κB activation. Thus, this study analyses by using three different H. pylori strains (P1, B128 and G27) whether CagA is required to initiate activation of different molecules of inhibitors of kappa B (IκB) and the NF-κB transcription factor RelA. We provide experimental evidence that H. pylori induces phosphorylation of NF-κB inhibitors IκBα, IκBβ and IκBɛ, and degradation of IκBα. Further, H. pylori stimulates phosphorylation of RelA at amino acids S536, S468 and S276, promotes DNA binding of RelA, and interleukin 8 (IL-8) gene expression in a T4SS-, but CagA-independent manner at early time points., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Much of the work in quality and patient safety has been based in hospitals and other acute care settings, yet it has become increasingly apparent that there are errors in delivery of care in other settings, all of which require attention. This article reviews sources of errors in 3 nonacute settings: the home, children's day care, and ambulatory surgery centers. Also described are standards that have been developed to guide practice, as well as specific regulatory agencies that are involved and some of the safeguards that have been instituted in each of the settings to protect against errors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Helicobacter pylori, the etiological agent of several human gastric diseases, induces the transcription factor nuclear factor-κB (NF-κB) in colonized epithelial cells leading to the release of proinflammatory mediators. Activation of NF-κB involves the IκB kinase (IKK)-complex composed of two catalytic subunits, IKKα and IKKβ, and a regulatory scaffold protein, IKKγ. IKKβ was shown to be essential for NF-κB activation in response to a variety of stimuli including H. pylori. In addition to the phosphorylation of serine residues, tyrosine phosphorylation could be crucial for IKKβ activation. Here we provide evidence that IKKβ phosphorylation is induced in lipid rafts (DRM fractions) of H. pylori-infected cells, but not TNFα-stimulated cells. Furthermore, H. pylori transiently induces binding of IKKβ to c-Src kinase. Inhibition of c-Src by specific inhibitors as well as knockdown of c-Src by small interfering RNA reduced phosphorylation of IκBα as well as of p65. Thus, tyrosine-phosphorylated IKKβ contributes at least in part to NF-κB activation in response to H. pylori infection.

It is supposed that human pathogens, e.g. Helicobacter pylori abuse lipid raft domains on the host cell plasma membrane to infect the cell. Investigating DRM-associated molecules we identified the transmembrane adapter proteins (TRAPs), non-T cell activation linker (NTAL) and lymphocyte-specific protein tyrosine kinase (Lck)-interacting membrane protein (LIME) to be regulated by H. pylori in the human epithelial cell line HCA-7. Up to now, raft-associated TRAPs were exclusively described to mediate signal propagation downstream of antigen receptors. Our results posed the question whether these proteins adopt a role in H. pylori-infected epithelial cells too. Our studies revealed that H. pylori induces tyrosine phosphorylation of NTAL as well as LIME within 15 min of infection. We observed that activated NTAL and LIME bind to the Src homology 2 (SH2)-domain of growth factor receptor-bound protein 2 (Grb2) within 15 to 30 min of infection and associate with the c-Met receptor. Further, NTAL has a contributory role in regulating H. pylori-induced extracellular signal-regulated kinase (ERK) activation. After suppression of NTAL protein levels by siRNA, ERK phosphorylation was reduced to approximately 50%. Additionally, the knockdown of NTAL suppressed the phosphorylation of cytosolic phospholipase A2 (cPLA2). Activated cPLA2 catalyzes the release of arachidonic acid (AA), whose metabolites are pivotal mediators in the H. pylori-induced inflammatory response. Thus, we propose that NTAL participates in the activation of the c-Met-Grb2-ERK-cPLA2 signalling cascade at early stages of H. pylori infection.

Objective: An important issue in epileptology is the question whether information extracted from the EEG of epilepsy patients can be used for the prediction of seizures. Several studies have claimed evidence for the existence of a pre-seizure state that can be detected using different characterizing measures. In this paper, we evaluate the predictability of seizures by comparing the predictive performance of a variety of univariate and bivariate measures comprising both linear and non-linear approaches., Methods: We compared 30 measures in terms of their ability to distinguish between the interictal period and the pre-seizure period. After completely analyzing continuous inctracranial multi-channel recordings from five patients lasting over days, we used ROC curves to distinguish between the amplitude distributions of interictal and preictal time profiles calculated for the respective measures. We compared different evaluation schemes including channelwise and seizurewise analysis plus constant and adaptive reference levels. Particular emphasis was placed on statistical validity and significance., Results: Univariate measures showed statistically significant performance only in a channelwise, seizurewise analysis using an adaptive baseline. Preictal changes for these measures occurred 5-30 min before seizures. Bivariate measures exhibited high performance values reaching statistical significance for a channelwise analysis using a constant baseline. Preictal changes were found at least 240 min before seizures. Linear measures were found to perform similar or better than non-linear measures., Conclusions: Results provide statistically significant evidence for the existence of a preictal state. Based on our findings, the most promising approach for prospective seizure anticipation could be a combination of bivariate and univariate measures., Significance: Many measures reported capable of seizure prediction in earlier studies are found to be insignificant in performance, which underlines the need for statistical validation in this field.

We have recently introduced a measure for nonstationarity using a recurrence time statistic to assess stationarity. In this paper we propose an extension of this method based on a detailed study of the statistics for the case of stationary systems. We derive a simple scheme that allows us to estimate the effective number of degrees of freedom relevant for this statistic. This substantially improves the statistical significance of the method and can be used to improve the significance of various other nonlinear statistics.

The question whether information extracted from the electroencephalogram (EEG) of epilepsy patients can be used for the prediction of seizures has recently attracted much attention. Several studies have reported evidence for the existence of a preseizure state that can be detected using different measures derived from the theory of dynamical systems. Most of these studies, however, have neglected to sufficiently investigate the specificity of the observed effects or suffer from other methodological shortcomings. In this paper we present an automated technique for the detection of a preseizure state from EEG recordings using two different measures for synchronization between recording sites, namely, the mean phase coherence as a measure for phase synchronization and the maximum linear cross correlation as a measure for lag synchronization. Based on the observation of characteristic drops in synchronization prior to seizure onset, we used this phenomenon for the characterization of a preseizure state and its distinction from the remaining seizure-free interval. After optimizing our technique on a group of 10 patients with temporal lobe epilepsy we obtained a successful detection of a preseizure state prior to 12 out of 14 analyzed seizures for both measures at a very high specificity as tested on recordings from the seizure-free interval. After checking for in-sample overtraining via cross validation, we applied a surrogate test to validate the observed predictability. Based on our results, we discuss the differences of the two synchronization measures in terms of the dynamics underlying seizure generation in focal epilepsies.

A rapidly growing number of studies deals with the prediction of epileptic seizures. For this purpose, various techniques derived from linear and nonlinear time series analysis have been applied to the electroencephalogram of epilepsy patients. In none of these works, however, the performance of the seizure prediction statistics is tested against a null hypothesis, an otherwise ubiquitous concept in science. In consequence, the evaluation of the reported performance values is problematic. Here, we propose the technique of seizure time surrogates based on a Monte Carlo simulation to remedy this deficit.

We propose a measure for nonstationarity which is based on the analysis of distributions of temporal distances of neighboring vectors in state space. As an extension of previous techniques our method does not require a partitioning of the time series. Moreover, the deviation of mean recurrence times from frequency distributions that would be expected under stationary conditions allows us to estimate the statistical significance of the method.

We compare dynamical properties of brain electrical activity from different recording regions and from different physiological and pathological brain states. Using the nonlinear prediction error and an estimate of an effective correlation dimension in combination with the method of iterative amplitude adjusted surrogate data, we analyze sets of electroencephalographic (EEG) time series: surface EEG recordings from healthy volunteers with eyes closed and eyes open, and intracranial EEG recordings from epilepsy patients during the seizure free interval from within and from outside the seizure generating area as well as intracranial EEG recordings of epileptic seizures. As a preanalysis step an inclusion criterion of weak stationarity was applied. Surface EEG recordings with eyes open were compatible with the surrogates' null hypothesis of a Gaussian linear stochastic process. Strongest indications of nonlinear deterministic dynamics were found for seizure activity. Results of the other sets were found to be inbetween these two extremes.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) can oxygenate 18-22 carbon polyunsaturated fatty acids, albeit with varying efficiencies. Here we report the crystal structures of eicosapentaenoic acid (EPA, 20:5 n-3) and linoleic acid (LA, 18:2 n-6) bound in the cyclooxygenase active site of Co(3+) protoporphyrin IX-reconstituted ovine PGHS-1 (Co(3+)-oPGHS-1) and compare the effects of active site substitutions on the rates of oxygenation of EPA, LA, and arachidonic acid (AA). Both EPA and LA bind in the active site with orientations similar to those seen previously with AA and dihomo-gamma-linolenic acid (DHLA). For EPA, the presence of an additional double bond (C-17/C-18) causes this substrate to bind in a "strained" conformation in which C-13 is misaligned with respect to Tyr-385, the residue that abstracts hydrogen from substrate fatty acids. Presumably, this misalignment is responsible for the low rate of EPA oxygenation. For LA, the carboxyl half binds in a more extended configuration than AA, which results in positioning C-11 next to Tyr-385. Val-349 and Ser-530, recently identified as important determinants for efficient oxygenation of DHLA by PGHS-1, play similar roles in the oxygenation of EPA and LA. Approximately 750- and 175-fold reductions in the oxygenation efficiency of EPA and LA were observed with V349A oPGHS-1, compared with a 2-fold change for AA. Val-349 contacts C-2 and C-3 of EPA and C-4 of LA orienting the carboxyl halves of these substrates so that the omega-ends are aligned properly for hydrogen abstraction. An S530T substitution decreases the V(max)/K(m) of EPA and LA by 375- and 140-fold. Ser-530 makes six contacts with EPA and four with LA involving C-8 through C-16; these interactions influence the alignment of the substrate for hydrogen abstraction. Interestingly, replacement of Phe-205 increases the volume of the cyclooxygenase site allowing EPA to be oxygenated more efficiently than with native oPGHS-1.

Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O(2), and two electrons to prostaglandin endoperoxide H(2) (PGH(2)). Formation of PGH(2) involves an initial oxygenation of arachidonate to yield PGG(2) catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG(2) to form PGH(2) catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co(3+)-heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG(2) versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually make less but measurable contributions to optimal catalytic efficiency.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) catalyze the committed step in prostaglandin biosynthesis. Both isozymes can oxygenate a variety of related polyunsaturated fatty acids. We report here the x-ray crystal structure of dihomo-gamma-linolenic acid (DHLA) in the cyclooxygenase site of PGHS-1 and the effects of active site substitutions on the oxygenation of DHLA, and we compare these results to those obtained previously with arachidonic acid (AA). DHLA is bound within the cyclooxygenase site in the same overall L-shaped conformation as AA. C-1 and C-11 through C-20 are in the same positions for both substrates, but the positions of C-2 through C-10 differ by up to 1.74 A. In general, substitutions of active site residues caused parallel changes in the oxygenation of both AA and DHLA. Two significant exceptions were Val-349 and Ser-530. A V349A substitution caused an 800-fold decrease in the V(max)/K(m) for DHLA but less than a 2-fold change with AA; kinetic evidence indicates that C-13 of DHLA is improperly positioned with respect to Tyr-385 in the V349A mutant thereby preventing efficient hydrogen abstraction. Val-349 contacts C-5 of DHLA and appears to serve as a structural bumper positioning the carboxyl half of DHLA, which, in turn, positions properly the omega-half of this substrate. A V349A substitution in PGHS-2 has similar, minor effects on the rates of oxygenation of AA and DHLA. Thus, Val-349 is a major determinant of substrate specificity for PGHS-1 but not for PGHS-2. Ser-530 also influences the substrate specificity of PGHS-1; an S530T substitution causes 40- and 750-fold decreases in oxygenation efficiencies for AA and DHLA, respectively.