Impact of Biotin Supplementation on Human Chorionic Gonadotropin Immunoassays Utilizing Biotin-Streptavidin Binding Methods in Urine.

BACKGROUND: Human chorionic gonadotropin (hCG) detection is indicative of pregnancy and can be indicative of some forms of cancerous tumors. The hCG drug itself, however, is a performance enhancing substance used by male athletes to increase testosterone production. Antidoping testing for hCG is conducted in urine, often on immunoanalyzer platforms, many of which utilize biotin-streptavidin dependent immunoassays in which the presence of biotin in samples is a known confounding factor. While biotin interference in serum has been well-studied, the extent of biotin interference in urine has not. METHODS: Ten active male individuals underwent a 2-week hCG administration protocol concurrent with supplementation with biotin (20 mg/day) or placebo. Urine and serum samples were collected throughout the study and analyzed for hCG and biotin concentrations. RESULTS: Urinary biotin levels in the hCG + biotin group increased 500-fold over baseline and 29-fold over corresponding serum biotin levels after biotin supplementation. When using a biotin-dependent immunoassay, the hCG + placebo group produced hCG-positive results (hCG ≥ 5 mIU/mL) in 71% of urine samples, while the hCG + biotin group produced positive results in only 19% of samples. Both groups had elevated hCG values in serum measurements by a biotin-dependent immunoassay and in urine when using a biotin-independent immunoassay. Urinary hCG measurements and biotin levels from the hCG + biotin group showed a negative correlation (Spearman r = -0.46, P < 0.0001) when measured using a biotin-dependent immunoassay. CONCLUSIONS: Biotin supplementation can severely suppress urinary hCG values in assays utilizing biotin-streptavidin binding methods and therefore these types of assays are not recommended for use in urine samples containing high levels of biotin. Clinicaltrials.gov Registration Number: NCT05450900.

Opioid-Sparing Anesthesia: Gabapentin and Postoperative Pain.

PURPOSE: Nonopioid analgesics are commonly used to augment or replace opioids in the perioperative setting. Perianesthesia nurses must consider timing and appropriateness when administering these medications to patients in the preoperative area or the postanesthesia care unit, particularly when other medications with sedative effects are being given. Gabapentin, originally proposed as an anticonvulsant medication, promotes analgesia and reduces risk for postoperative nausea and vomiting. This review examines the effect of gabapentin on postoperative pain. DESIGN: A systematic review. METHODS: CINAHL, PubMed, and Cochrane Review databases were searched to find a total of 93 sources that examined gabapentin and postoperative pain. After applying inclusion and exclusion criteria, four randomized controlled trials (RCT) were reviewed. Postoperative pain within the 24 hours of surgery was measured as the primary outcome using the visual analog scale in all sources FINDINGS: Three of the four reviewed RCTs determined gabapentin was both statistically and clinically significant in reducing postoperative pain, and all four sources showed a reduction in opioid consumption during the immediate postoperative period, which promoted patient satisfaction. CONCLUSIONS: Further study of gabapentin and postoperative pain is needed employing rigorous and robust methodology and diversity of the sample selections.

Perioperative Dexamethasone for Patients With Diabetes and Its Effect on Blood Glucose After Surgery.

PURPOSE: P Perioperative administration of single-dose dexamethasone helps reduce postoperative nausea and vomiting, inflammation, and pain. However, it is unclear which dose achieves these effects while minimizing the hyperglycemic impact in patients with diabetes. The purpose of this review was to elucidate the most appropriate perioperative dose of dexamethasone for diabetic patients, and whether it is necessary to withhold it in patients with poor glycemic control. DESIGN: A systematic review. METHODS: A literature search using PubMed and Cochrane Database of Systematic Reviews revealed 17 potential evidence sources. Eight sources met the inclusion criteria. Sources included one systematic review with meta-analysis, one randomized control trial, and six observational studies. FINDINGS: Evidence suggests diabetic patients who receive dexamethasone perioperatively are more likely to develop postoperative hyperglycemia, with a maximum blood glucose increase of 30 to 45 mg/dL in the first 24 hours following a single dose. One study described increased blood glucose levels with escalating doses, but no other sources have supported that finding. The available studies were markedly heterogeneous in both design and proportion of diabetic subjects included, and most were of low quality. CONCLUSIONS: There is not enough evidence to quantify the hyperglycemic effect of commonly used dexamethasone doses, and rigorous studies are needed to inform practice.

Three-dimensional reconstruction of leukocyte internalisation in the luminal uterine epithelium following mating.

Following mating, leukocytes are recruited to the uterine epithelium where they phagocytose spermatozoa and mediate maternal immune tolerance as well as a mild inflammatory response. In this ultrastructural study we utilised array tomography, a high-resolution volume scanning electron microscopy approach to 3D reconstruct the cellular relationships formed by leukocytes recruited to the luminal uterine epithelium 12 h post-mating in the rat. We report that following mating, neutrophils and macrophages are internalised by the luminal uterine epithelium, with multiple leukocytes internalised via contortion through a small tunnel in the apical membrane into a large membrane-bound vacuole within the cytoplasm of luminal uterine epithelial cells (UECs). Once internalised within the UECs, recruited leukocytes appear to phagocytose material within the membrane-bound vacuole and most ultimately undergo a specialised cell death, including vacuolisation and loss of membrane integrity. As these observations involve ultrastructurally normal leukocytic cells internalised within non-phagocytic epithelial cells, these observations are consistent with the formation of cell-in-cell structures via entosis, rather than phagocytic engulfment by UECs. Although cell-in-cell structures have been reported in normal and pathological conditions elsewhere, the data collected herein represents the first evidence of the formation of cell-in-cell structures within the uterine epithelium as a novel component of the maternal inflammatory response to mating.

Resuming Breastfeeding After Surgery: Influencing Practice Recommendations.

PURPOSE: Mothers often request guidance on when it is safe to resume breastfeeding after surgery. At our institution, this guidance was inconsistent and not well-grounded in current research. This project sought to bring recommendations to patients in line with current evidence about when to recommend resumption of breastfeeding after surgery. DESIGN: A local practice guideline was developed based on our systematic review, then staff were educated about the guideline. METHODS: Transfer to clinical practice was measured by reported practice recommendations. A repeated measures design measured change in provider knowledge, recommendations, and confidence in these recommendations. A follow-up assessment was conducted at 2 years to measure long-term impact. FINDINGS: After the educational session, there was a two-fold increase in the number of perianesthesia staff who recommended resumption of breastfeeding as soon as the mother had recovered from anesthesia. CONCLUSIONS: This evidence-based practice project standardized delivery of breastfeeding recommendations by perioperative staff.

Identification and Management of Ischemic Stroke in the Postanesthesia Care Unit.

A thromboembolic stroke is a debilitating event that can occur with little or no warning. This report details the case of a 63-year-old male experiencing a stroke in the immediate postoperative period after total knee arthroplasty. Risk for perioperative stroke is influenced by age, sex, ethnicity, comorbidities, and some medications. The depressed neurocognitive state of patients recovering from anesthesia warrants special consideration for the identification and management of perioperative stroke.

Increasing Access to Auricular Acupuncture for Postoperative Nausea and Vomiting.

PURPOSE: Postoperative nausea and vomiting (PONV) affects more than 30% of surgical patients. Auricular acupuncture (AA) has been shown to decrease the incidence of PONV in select populations. DESIGN: An evidence-based quality improvement project made AA available to all adult surgical patients, and the results were recorded in a database. A retrospective between-groups analysis of 210 database entries was conducted, of those 25 receiving AA. FINDINGS: More risk factors for PONV were present in the AA group (P < .001). Both groups experienced a less-than-expected rate of PONV. Similar rates were shown between groups for PONV, postanesthesia care unit length of stay, and opioid consumption. Patient satisfaction was 96% with AA. The AA group was treated with less antiemetic medication (P < .001), yet PONV rates remained similar. CONCLUSIONS: A multimodal approach treating patients at risk for PONV is recommended. Administration of multiple antiemetics may result in unnecessary cost or unfavorable side effects when effective and less costly alternatives exist. AA is a viable treatment for PONV, considering cost and patient satisfaction.

Is blood blood? Comparing quantitation of endogenous steroids and luteinizing hormone in concurrently collected venous serum and Tasso+ SST capillary serum samples.

The monitoring of endogenous steroids in urine has been an important component of the Athlete Biological Passport (ABP) for the last decade. Recently, the quantitation of endogenous steroids in blood has been incorporated into the ABP to increase sensitivity in circumstances where the excretion of urinary ABP biomarkers is low. Current ABP guidelines mandate the use of venous blood draws for blood steroid sample collections, however, recent efforts have focused on investigating the use of less invasive sample collection methods, such as capillary blood collected from the upper arm. The focus of this study was to compare the analytical results of venous and capillary blood collected weekly from 20 individuals, 10 males and 10 females, over six weeks. The two primary biomarkers of the blood steroid ABP module, testosterone (T) and the testosterone/androstenedione (T/A4) ratio, were compared, as well as luteinizing hormone (LH) and the T/LH ratio in male participants, two biomarkers known to be responsive to T use. All biomarkers showed excellent agreement between venous and capillary blood. Longitudinal stability between sample types within individuals was also comparable for all biomarkers. Finally, storage of simultaneously collected capillary samples at room temperature and frozen conditions was compared with evaluate the potential impact of non-cold chain shipping conditions. Most biomarkers showed excellent agreement between frozen and room temperature storage conditions. These results indicate capillary blood collections represent a promising alternative to venous blood collections for the blood steroid module of the ABP.

Metabolism of beclomethasone dipropionate by cytochrome P450 3A enzymes.

Inhaled glucocorticoids, such as beclomethasone dipropionate (BDP), are the mainstay treatment of asthma. However, ≈ 30% of patients exhibit little to no benefit from treatment. It has been postulated that glucocorticoid resistance, or insensitivity, is attributable to individual differences in glucocorticoid receptor-mediated processes. It is possible that variations in cytochrome P450 3A enzyme-mediated metabolism of BDP may contribute to this phenomenon. This hypothesis was explored by evaluating the contributions of CYP3A4, 3A5, 3A7, and esterase enzymes in the metabolism of BDP in vitro and relating metabolism to changes in CYP3A enzyme mRNA expression via the glucocorticoid receptor in lung and liver cells. CYP3A4 and CYP3A5 metabolized BDP via hydroxylation ([M4] and [M6]) and dehydrogenation ([M5]) at similar rates; CYP3A7 did not metabolize BDP. A new metabolite [M6], formed by the combined action of esterases and CYP3A4 hydroxylation, was also characterized. To validate the results observed using microsomes and recombinant enzymes, studies were also conducted using A549 lung and DPX2 liver cells. Both liver and lung cells produced esterase-dependent metabolites [M1-M3], with [M1] correlating with CYP3A5 mRNA induction in A549 cells. Liver cells produced both hydroxylated and dehydrogenated metabolites [M4, M5, and M6], but lung cells produced only the dehydrogenated metabolite [M5]. These studies show that CYP3A4 and CYP3A5 metabolize BDP to inactive metabolites and suggest that differences in the expression or function of these enzymes in the lung and/or liver could influence BDP disposition in humans.

Metabolic pathways of inhaled glucocorticoids by the CYP3A enzymes.

Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6ß-hydroxylation and Δ(6)-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of Δ(6)-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography-mass spectrometry and NMR analysis.

Influence of multiple human chorionic gonadotropin administrations on serum and urinary steroid Athlete Biological Passport profiles in males.

The Athlete Biological Passport (ABP) is a longitudinal tool used in anti-doping to monitor biological parameters known to change with performance-enhancing drug use. The ABP consists of multiple modules, including two aimed at detecting the use of endogenous anabolic androgenic steroids: the urinary and serum steroid modules. Human chorionic gonadotropin (hCG) is a protein hormone potentially abused by male athletes to increase the production of endogenous testosterone. To date, no studies have investigated the impact of extended hCG administration on the urinary and serum steroid modules of the ABP. The goal of this study was to identify the impact of multiple hCG administrations on the parameters tracked as part of the urinary and serum steroid modules of the ABP. Ten recreationally active, healthy male individuals self-administered seven 250 µg hCG injections over 3 weeks. Serum and urine samples were collected before, during, and 2 weeks following the final injection. All ABP parameters were quantified in the respective matrix, and steroid profiles were created with Anti-Doping Administration and Management System adaptive model upper and lower limits for both matrices. In both serum and urine profiles, testosterone increased; however, the testosterone/epitestosterone ratio in urine and the testosterone/androstenedione ratio in serum showed minimal changes. Additionally, serum luteinizing hormone (LH) was quantified using an immunoassay, and a serum testosterone/LH ratio was generated. Serum LH values decreased during administration causing large increases in the serum T/LH ratio, indicating this ratio may be a more sensitive parameter for detecting hCG abuse than urinary testosterone/epitestosterone or serum testosterone/androstenedione.

Simulation in Nurse Anesthesia Educational Programs: A Report by the AANA Simulation Subcommittee.

Simulation is an integral part of the healthcare educational landscape and a key element in the future of graduate professional education. For the past three decades, simulation-based educational methodology has been gaining popularity in nurse anesthesia educational programs (NAEP). There is currently limited objective evidence documenting modalities used or educational outcomes addressed through simulation in NAEPs. In 2018, the American Association of Nurse Anesthesiology (AANA) established a Simulation Subcommittee of the AANA Education Committee and tasked the group with two primary goals: 1) to gain a better understanding of the current state of simulation education and 2) to review responses with regard to how NAEPs could best incorporate simulation elements within their curriculum to meet requirements while adhering to the guidelines of the Council on Accreditation of Nurse Anesthesia Educational Programs. A survey tool was developed and distributed to all programs to assess the utilization of simulation, available simulation resources, ongoing faculty development efforts, and barriers to use of this educational approach. Survey results indicated that simulation is valued as an effective method within NAEPs for a variety of teaching and learning activities and is utilized to support achievement of both technical and nontechnical learning outcomes for student registered nurse anesthetists.

Respective roles of CYP2A5 and CYP2F2 in the bioactivation of 3-methylindole in mouse olfactory mucosa and lung: studies using Cyp2a5-null and Cyp2f2-null mouse models.

The aim of this study was to determine whether mouse CYP2A5 and CYP2F2 play critical roles in the bioactivation of 3-methylindole (3MI), a tissue-selective toxicant, in the target tissues, the nasal olfactory mucosa (OM) and lung. Five metabolites of 3MI were identified in NADPH- and GSH-fortified microsomal reactions, including 3-glutathionyl-S-methylindole (GS-A1), 3-methyl-2-glutathionyl-S-indole (GS-A2), 3-hydroxy-3-methyleneindolenine (HMI), indole-3-carbinol (I-3-C), and 3-methyloxindole (MOI). The metabolite profiles and enzyme kinetics of the reactions were compared between OM and lung, and among wild-type, Cyp2a5-null, and Cyp2f2-null mice. In lung reactions, GS-A1, GS-A2, and HMI were detected as major products, and I-3-C and MOI, as minor metabolites. In OM reactions, all five metabolites were detected in ample amounts. The loss of CYP2F2 affected formation of all 3MI metabolites in the lung and formation of HMI, GS-A1, and GS-A2 in the OM. In contrast, loss of CYP2A5 did not affect formation of 3MI metabolites in the lung but caused substantial decreases in I-3-C and MOI formation in the OM. Thus, whereas CYP2F2 plays a critical role in the 3MI metabolism in the lung, both CYP2A5 and CYP2F2 play important roles in 3MI metabolism in the OM. Furthermore, the fate of the reactive metabolites produced by the two enzymes through common dehydrogenation and epoxidation pathways seemed to differ with CYP2A5 supporting direct conversion to stable metabolites and CYP2F2 supporting further formation of reactive iminium ions. These results provide the basis for understanding the respective roles of CYP2A5 and CYP2F2 in 3MI's toxicity in the respiratory tract.

Directed evolution reveals requisite sequence elements in the functional expression of P450 2F1 in Escherichia coli.

Cytochrome P450 2F1 (P450 2F1) is expressed exclusively in the human respiratory tract and is implicated in 3-methylindole (3MI)-induced pneumotoxicity via dehydrogenation of 3MI to a reactive electrophilic intermediate, 3-methyleneindolenine (3-MEI). Studies of P450 2F1 to date have been limited by the failure to express this enzyme in Escherichia coli. By contrast, P450 2F3, a caprine homologue that shares 84% sequence identity with P450 2F1 (86 amino acid differences), has been expressed in E. coli at yields greater than 250 nmol/L culture. We hypothesized that a limited number of sequence differences between P450s 2F1 and 2F3 could limit P450 2F1 expression in E. coli and that problematic P450 2F1 sequence elements could be identified by directed evolution. A library of P450 2F1/2F3 mutants was created by DNA family shuffling and screened for expression in E. coli. Three generations of DNA shuffling revealed a mutant (named JH_2F_F3_1_007) with 96.5% nucleotide sequence identity to P450 2F1 and which expressed 119 ± 40 pmol (n = 3, mean ± SD) hemoprotein in 1 mL microaerobic cultures. Across all three generations, two regions were observed where P450 2F3-derived sequence was consistently substituted for P450 2F1 sequence in expressing mutants, encoding nine amino acid differences between P450s 2F1 and 2F3: nucleotides 191-278 (amino acids 65-92) and 794-924 (amino acids 265-305). Chimeras constructed to specifically test the importance of these two regions confirmed that P450 2F3 sequence is essential in both regions for expression in E. coli but that other non-P450 2F1 sequence elements outside of these regions also improved the expression of mutant JH_2F_F3_1_007. Mutant JH_2F_F3_1_007 catalyzed the dehydrogenation of 3MI to 3-MEI as indicated by the observation of glutathione adducts after incubation in the presence of glutathione. The JH_2F_F3_1_007 protein differs from P450 2F1 at only 20 amino acids and should facilitate further studies of the structure-activity relationships of P450s of the 2F subfamily.

Detection of insulin analogues and large peptides >2 kDa in urine.

Insulin analogues and large bioactive peptides may be used by athletes to enhance performance and are banned by the World Anti-Doping Agency (WADA). In addition to insulin analogues, the large peptides include a structurally diverse set of peptides including analogues of growth hormone releasing hormone (GHRH), insulin-like growth factor-1 (IGF-1), and mechano-growth factor (MGF). Detection of this class of peptides is difficult due to their absorptive losses and presence at very low concentrations in urine. In this report, a high throughput method is described that allows sensitive detection of four classes of large peptides in one assay. Sample extraction is performed by ultrafiltration to concentrate the urine followed by solid phase extraction in a 96-well micro-elution plate. Peptides in the urine samples are detected on a triple quadrupole mass spectrometer coupled to standard flow liquid chromatography. The method was validated and evaluated for limit of detection, limit of identification, specificity, precision, carryover, recovery, matrix interference, and post-extraction stability. The limit of detection for insulin analogues is between 5 and 25 pg/ml and between 5 and 50 pg/ml for the other peptide classes. Specificity was good with no detection of interfering peaks in blank urine samples. Carryover from a high concentration sample was not observed and the post-extraction stability was between 77% and 107%. The method was able to detect insulin analogues in three diabetic urine samples. Increased screening for this class of peptides will improve detection and deterrence.

MAPK signaling pathways regulate IL-8 mRNA stability and IL-8 protein expression in cystic fibrosis lung epithelial cell lines.

Cystic fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung, caused by mutations in the CFTR gene. IL-8 and other proinflammatory mediators are elevated in the CF airway, and the immediate mechanism may depend on disease-specific stabilization of IL-8 mRNA in CF lung epithelial cells. MAPK signaling pathways impact directly on IL-8 protein expression in CF cells, and we have hypothesized that the mechanism may also involve stabilization of the IL-8 mRNA. To test this hypothesis, we have examined the effects of pharmacological and molecular inhibitors of p38, and downstream MK2, ERK1/2, and JNK, on stability of IL-8 mRNA in CF lung epithelial cells. We previously showed that tristetraprolin (TTP) was constitutively low in CF and that raising TTP destabilized the IL-8 mRNA. We therefore also tested these effects on CF lung epithelial cells stably expressing TTP. TTP binds to AU-rich elements in the 3'-UTR of the IL-8 mRNA. We find that inhibition of p38 and ERK1/2 reduces the stability of IL-8 mRNA in parental CF cells. However, neither intervention further lowers TTP-dependent destabilization of IL-8 mRNA. By contrast, inhibition of the JNK-2 pathway has no effect on IL-8 mRNA stability in parental CF cell, but rather increases the stability of the message in cells expressing high levels of TTP. However, we find that inhibition of ERK1/2 or p38 leads to suppression of the effect of JNK-2 inhibition on IL-8 mRNA stability. These data thus lend support to our hypothesis that constitutive MAPK signaling and proteasomal activity might also contribute, along with aberrantly lower TTP, to the proinflammatory phenotype in CF lung epithelial cells by increasing IL-8 mRNA stability and IL-8 protein expression.

Training Program for Ultrasound-Guided Intravenous Catheter Insertion.

INTRODUCTION: Ultrasound guidance is now widely available in military treatment facilities and civilian hospitals alike, both in the USA and in forward-deployed military environments. Technical mastery of ultrasound-guided peripheral intravenous (USGPIV) catheter insertion can be easily achieved through a short training course. Mastery can be achieved even when trainees have a limited medical background before course attendance. An evidence-based practice project team sought to improve the knowledge, confidence, and skills in the placement of USGPIV catheters by clinicians at Naval Hospital Jacksonville. Completion of an USGPIV training program can equip healthcare providers with knowledge and confidence for placement of peripheral access necessary in critical situations such as those requiring medications or blood products. MATERIALS AND METHODS: The project team conducted a literature review to evaluate the appropriateness of USGPIV training for the nurses and military medical technicians in this setting. The team developed and delivered a USGPIV training program based on adaptations from the literature. During the training period, knowledge and confidence scores were reported by each trainee to evaluate the perceptions of the quality of training. The number of attempted catheter placements and ultrasound utilization was recorded in the pre- and post-implementation periods to evaluate the project's effect on the delivery of patient care. Statistical analysis was conducted to evaluate project outcomes. RESULTS: In the pre-intervention period, none of the 252 intravenous catheters were placed with the USGPIV technique, compared to 50 of 267 in the post-intervention period. These results demonstrate an 18.7% increase in the USGPIV access approach by nursing staff. Mean knowledge scores significantly increased following the delivery of the training, 60% versus 80% in the pre- and post-training assessments, respectively (P < .001). Mean self-reported skill confidence scores also significantly improved (P < .001). CONCLUSION: Knowledge and self-reported confidence in USGPIV access improved for the trainees. Mean knowledge improved from 60% to 80%, while mean confidence scores increased from 2.74 to 3.79 for corpsman and from 3.0 to 3.88 for nurses. Utilization of the USGPIV technique increased by 18.7% in the post-intervention period. These results demonstrate that implementing this training program can improve knowledge, confidence, and use of ultrasound during the placement of PIV catheters.

Prevention of Pruritis following Spinal Morphine for Scheduled Cesarean Birth.

BACKGROUND: Intrathecal morphine provides effective analgesia after cesarean birth, yet up to 90% of women who receive it experience excessive itching, an undesirable dose-dependent effect. Pruritis may increase nursing workload, delay breastfeeding, and decrease patient satisfaction. When 0.1 mg spinal morphine is given, pruritis is markedly reduced while analgesia is preserved. PURPOSE: The purpose of this project was to determine possible causes and solutions for pruritus after cesarean birth. METHODS: Anesthesia providers were educated and encouraged to limit spinal morphine to 0.1 mg as a strategy to prevent pruritus. In a repeated measures design, the rate of treatment-required pruritus and opioid consumption were measured 24 hours after surgery. The project included an evaluation of 30 medical records before and 30 medical records after the project intervention. RESULTS: Preintervention rate of treatment-required pruritis was 37%, all received spinal morphine ≥ 1.5 mg. Postintervention rate of treatment-required pruritis was 13% and 57% after spinal morphine 0.1 mg and 0.2 mg, respectively. Opioid consumption was similar between groups. CLINICAL IMPLICATIONS: Mother-baby nurses can have an impact on the practice of anesthesia providers by advocating for evidence-based dosing of intrathecal morphine to reduce the incidence of pruritis while maintaining effective analgesia for women after cesarean birth.

CYP3A4-Mediated oxygenation versus dehydrogenation of raloxifene.

Raloxifene was approved in 2007 by the FDA for the chemoprevention of breast cancer in postmenopausal women at high risk for invasive breast cancer. Approval was based in part on the improved safety profile for raloxifene relative to the standard treatment of tamoxifen. However, recent studies have demonstrated the ability of raloxifene to form reactive intermediates and act as a mechanism-based inhibitor of cytochrome P450 3A4 (CYP3A4) by forming adducts with the apoprotein. However, previous studies could not differentiate between dehydrogenation to a diquinone methide and the more common oxygenation pathway to an arene oxide as the most likely intermediate to inactivate CYP3A4. In the current work, (18)O-incorporation studies were utilized to carefully elucidate CYP3A4-mediated oxygenation versus dehydrogenation of raloxifene. These studies established that 3'-hydroxyraloxifene is produced exclusively via CYP3A4-mediated oxygenation and provide convincing evidence for the mechanism of CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide, while excluding the alternative arene oxide pathway. Furthermore, it was demonstrated that 7-hydroxyraloxifene, which was previously believed to be a typical O(2)-derived metabolite of CYP3A4, is in fact produced by a highly unusual hydrolysis pathway from a putative ester, formed by the conjugation of raloxifene diquinone methide with a carboxylic acid moiety of CYP3A4, or other proteins in the reconstituted system. These findings not only confirm CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide but also suggest a novel route of raloxifene toxicity.

Improved cytochrome P450 3A4 molecular models accurately predict the Phe215 requirement for raloxifene dehydrogenation selectivity.

The use of molecular modeling in conjunction with site-directed mutagenesis has been extensively used to study substrate orientation within cytochrome P450 active sites and to identify potential residues involved in the positioning and catalytic mechanisms of these substrates. However, because docking studies utilize static models to simulate dynamic P450 enzymes, the effectiveness of these studies is strongly dependent on accurate enzyme models. This study employed a cytochrome P450 3A4 (CYP3A4) crystal structure (Protein Data Bank entry 1W0E) to predict the sites of metabolism of the known CYP3A4 substrate raloxifene. In addition, partial charges were incorporated into the P450 heme moiety to investigate the effect of the modified CYP3A4 model on metabolite prediction with the ligand docking program Autodock. Dehydrogenation of raloxifene to an electrophilic diquinone methide intermediate has been linked to the potent inactivation of CYP3A4. Active site residues involved in the positioning and/or catalysis of raloxifene supporting dehydrogenation were identified with the two models, and site-directed mutagenesis studies were conducted to validate the models. The addition of partial charges to the heme moiety improved the accuracy of the docking studies, increasing the number of conformations predicting dehydrogenation and facilitating the identification of substrate-active site residue interactions. On the basis of the improved model, the Phe215 residue was hypothesized to play an important role in orienting raloxifene for dehydrogenation through a combination of electrostatic and steric interactions. Substitution of this residue with glycine or glutamine significantly decreased dehydrogenation rates without concurrent changes in the rates of raloxifene oxygenation. Thus, the improved structural model predicted novel enzyme-substrate interactions that control the selective dehydrogenation of raloxifene to its protein-binding intermediate.