Five cases of missed cystic fibrosis heterozygous mutations identified after a positive newborn screen on a sibling.

In Michigan (MI), NBS for CF was started in October 2007 using the IRT/DNA protocol. In 2016, a component of the Hologic molecular test kit used by the MI NBS lab was recalled (40 CF mutation 2nd tier test). This recall had a major impact on states using the Hologic test kits in their NBS programs. Michigan specimens were sent to another state's NBS Lab for 2nd tier testing using the Luminex 60 mutation test kit until the Luminex kit could be procured and validated in MI. In this report, we present five cases born during this time period. These cases were initially reported out as having normal NBS results for CF but had heterozygous F508 del (c.1521_1523delCTT) mutations later identified. Of the five cases, one was diagnosed with CF (Case1), one with CF related metabolic syndrome (CRMS), and the other three were carriers.

Allergic Broncho-Pulmonary Aspergillosis.

Allergic broncho-pulmonary aspergillosis (ABPA) is an immunologically mediated lung disease that usually occurs in people with a diagnosis of asthma or cystic fibrosis. It is a noninvasive lung disease caused by colonization of the airways with Aspergillus fumigatus. In people who are susceptible, Aspergillus leads to an exaggerated immune response and ultimately pulmonary inflammation and lung damage. Patients with ABPA typically present with poorly controlled asthma, recurrent pulmonary infiltrates, and bronchiectasis. Diagnosis of ABPA is established based on a combination of clinical manifestations as well as laboratory and radiological evaluations. Delay in diagnosis can result in airway destruction and pulmonary fibrosis, which may result in significant morbidity and mortality. This article discusses the clinical characteristics, diagnosis, and management of patients with ABPA. It aims to serve as a tool for pediatricians to aid in early recognition of this debilitating disease and consider referral, facilitating early diagnosis and treatment. [Pediatr Ann. 2021;50(5):e214-e221.].

Health Effects Reported by Adolescent Water Pipe and/or Cigarette Smokers Compared to Nonsmokers.

PURPOSE: The purpose of this study was to determine the prevalence of respiratory and/or physical fitness health problems in adolescent (ages 18-21) water pipe (WP) smokers (with or without cigarette smoking), cigarette-only smokers, and nonsmokers. METHODS: A comparative four-group study design was used to recruit a non-probability sample of 153 WP smokers only, 103 cigarette smokers only, and 102 cigarette+WP smokers along with 296 nonsmokers. Our hypothesis was that youth who smoked WPs and/or cigarettes would report more respiratory problems and/or poorer physical fitness than those who did not smoke. RESULTS: The results showed that coughs were significantly associated with smoking in all three of the smoking groups (p < .05). Cigarette-only smokers reported the most adverse outcomes with more wheezing, difficulty breathing, and less ability to exercise without shortness of breath. A dose-response analysis showed similar patterns of adverse health effects for both WP and cigarette smokers. The combined use of both products was not appreciably worse than smoking one product alone. This could be due to cigarette+WP smokers' reporting using less of the respective products when only one product was smoked. CONCLUSIONS: Even during the adolescent years, WP and/or cigarette smoking youth experienced reportable negative health effects.

Effects of an Antioxidant-enriched Multivitamin in Cystic Fibrosis. A Randomized, Controlled, Multicenter Clinical Trial.

RATIONALE: Cystic fibrosis (CF) is characterized by dietary antioxidant deficiencies, which may contribute to an oxidant-antioxidant imbalance and oxidative stress. OBJECTIVES: Evaluate the effects of an oral antioxidant-enriched multivitamin supplement on antioxidant concentrations, markers of inflammation and oxidative stress, and clinical outcomes. METHODS: In this investigator-initiated, multicenter, randomized, double-blind, controlled trial, 73 pancreatic-insufficient subjects with CF 10 years of age and older with an FEV1 between 40% and 100% predicted were randomized to 16 weeks of an antioxidant-enriched multivitamin or control multivitamin without antioxidant enrichment. Endpoints included systemic antioxidant concentrations, markers of inflammation and oxidative stress, clinical outcomes (pulmonary exacerbations, anthropometric measures, pulmonary function), safety, and tolerability. MEASUREMENTS AND MAIN RESULTS: Change in sputum myeloperoxidase concentration over 16 weeks, the primary efficacy endpoint, was not significantly different between the treated and control groups. Systemic antioxidant (ß-carotene, coenzyme Q10, γ-tocopherol, and lutein) concentrations significantly increased in the antioxidant-treated group (P < 0.001 for each), whereas circulating calprotectin and myeloperoxidase decreased in the treated group compared with the control group at Week 4. The treated group had a lower risk of first pulmonary exacerbation requiring antibiotics than the control group (adjusted hazard ratio, 0.50; P = 0.04). Lung function and growth endpoints did not differ between groups. Adverse events and tolerability were similar between groups. CONCLUSIONS: Antioxidant supplementation was safe and well tolerated, resulting in increased systemic antioxidant concentrations and modest reductions in systemic inflammation after 4 weeks. Antioxidant treatment was also associated with a lower risk of first pulmonary exacerbation. Clinical trial registered with www.clinicaltrials.gov (NCT01859390).

Adenotonsillectomy Normalizes Hormones and Urinary Electrolytes in Children With Nocturnal Enuresis and Sleep-Disordered Breathing.

OBJECTIVE: To assess (1) plasma levels of antidiuretic hormone (ADH) and brain natriuretic peptide (BNP) and urinary levels of electrolytes in children with sleep disordered breathing (SDB), with or without nocturnal enuresis (NE), and (2) the effect of adenotonsillectomy (T&A) on urinary electrolytes and the secretion of ADH and BNP in children with NE and SDB. We previously reported post-T&A improvements in plasma levels of BNP and ADH in children with SDB and NE. However, the differences in plasma concentration of these hormones in SDB children with and without NE, and their relationships with urinary electrolytes, have not yet been addressed. METHODS: This prospective study compared concentrations of urinary electrolytes and plasma ADH and BNP in (1) children with SDB and NE (study group) and an age- and sex-matched control group of children with SDB without NE, and (2) the study group before and 1-month after T&A. RESULTS: Compared with the control group (n = 31), the study group (n = 37) exhibited significantly lower ADH (P = .04) and higher BNP (P = .009) plasma levels. The differences in urinary electrolytes were not significant. Post-T&A, the study group showed significantly decreased BNP (P = .018), urinary sodium-to-creatinine ratio (P = .02), and urinary calcium-to-creatinine ratio (P = .007) compared with the pre-T&A values. Post-T&A changes in urinary calcium were significantly correlated with changes in sodium excretion (P = .002) and in plasma levels of BNP (P <.001). CONCLUSION: The presence of NE is associated with altered ADH and BNP levels in children with SDB. T&A led to normalization of ADH and BNP, probably through a calcium- and sodium-dependent mechanism.

Adenotonsillectomy improves quality of life in children with sleep-disordered breathing regardless of nocturnal enuresis outcome.

BACKGROUND: Nocturnal enuresis (NE) and sleep-disordered breathing (SDB) have both been associated with impaired health-related quality of life (HRQoL). The following were investigated: (1) whether tonsillectomy and/or adenoidectomy (T&A) significantly affect the HRQoL in children with NE and SDB, and 2) differences in HRQoL between children with NE persistence versus resolution post-T&A. METHODS: This was a prospective study comparing the HRQoL of children with SDB and NE (study group) pre- and 4 weeks post-T&A, and the HRQoL of children with SDB without NE (control group) (independent t-tests). HRQol was assessed using the Obstructive Sleep Apnea Quality of Life 18 questionnaire (OSAS-18), a validated measure containing five subscales that combine to create a total score. Individual items were scored on a Likert-type scale ranging from 1 (none of the time) to 7 (all of the time). Symptoms of SDB were evaluated using the validated Pediatric Sleep Questionnaire (PSQ). Mixed ANOVA was conducted to evaluate changes in the measures between the wet and dry children post-T&A. Pre- and post-T&A change scores were calculated for both the PSQ and the OSAS-18. RESULTS: There were 30 children in the study group (18 male, mean age 9.07 years, SD 2.19), and 30 age-matched controls (16 male). There were no statistically significant differences between the two groups in regards to OSAS-18 total, PSQ total, BMI, diagnosis of snoring or OSAS on sleep study, or race. Overall, OSAS-18 and PSQ scores significantly improved in all children post-surgery (p < 0.001; p < 0.001, respectively), with no significant differences between dry and wet children post-T&A. The correlation between the pre- and post-T&A change scores on the OSAS-18 and PSQ was significant (r(29) = 0.58, p = 0.001), suggesting that a reduction in SDB symptoms post T&A is related to improved HRQoL. CONCLUSIONS: T&A significantly improved HRQoL in all children with SDB and NE, regardless of NE outcomes. These findings support recommendations for T&A in children with SDB with or without NE.

Effect of salt supplementation on the rate of inadequate sweat collection for infants less than 3 months of age referred for the sweat test.

BACKGROUND: Sweat testing in young infants (≤ 3 months) with a positive newborn screen for Cystic Fibrosis (CF) can yield higher rates of inadequate sweat collection. The role of salt supplements in improving sweat collection has not been studied before. METHODS: All young infants referred to our CF center for sweat testing were randomized to either receive salt supplements {1/8th teaspoon salt (750 mg)} mixed in formula feeds 1 day prior to sweat testing (study group) or no salt supplement (controls). RESULTS: Of the 151 young infants that underwent sweat testing over 18 months, 75 received salt supplements, while 76 did not. A total of 9 (11.8%) infants in the salt supplement group had inadequate sweat collection, as compared to 4 (5.2%) infants in the control group (p = 0.16, Fisher's Exact Test). CONCLUSIONS: Oral salt supplementation for young infants prior to sweat testing does not help to reduce the rates of inadequate sweat collection.

Improving screening for diabetes in cystic fibrosis.

PURPOSE: Annual screening for cystic fibrosis-related-diabetes (CFRD) using oral glucose tolerance test (OGTT) is recommended, but national testing rates are low. The purpose of this paper is to implement the quality improvement (QI) initiative to improve cystic fibrosis (CF) annual screening rates among patients at one CF center. DESIGN/METHODOLOGY/APPROACH: To improve screening for CFRD at the CF Center, the authors used the Dartmouth Microsystem Improvement Ramp method and formed a collaborative working group. A process map was created to outline the steps and a fishbone analysis was performed to identify barriers and to utilize resources for implementing new interventions. FINDINGS: Prior to these interventions, 21 percent of eligible patients had completed annual screening and after the intervention, it rose to 72 percent. The initial completion rate with the first prescription was only 50 percent, but it improved steadily to 54/75 (72 percent) in response to reminder letters sent six weeks after the initial script was given. PRACTICAL IMPLICATIONS: Close tracking and reminder letters can improve adherence with annual OGTT screening for CFRD among CF patients, with special emphasis on high-risk patients. ORIGINALITY/VALUE: There should be a special emphasis on screening for CFRD in high-risk CF patients (those with low BMI or higher age). This QI initiative brought about several operational changes in the annual OGTT screening process that have now become the standard operating procedure at the center.

Melatonin prevents myeloperoxidase heme destruction and the generation of free iron mediated by self-generated hypochlorous acid.

Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron. Here we show that the presence of melatonin (MLT) can prevent HOCl-mediated MPO heme destruction using a combination of UV-visible photometry, hydrogen peroxide (H2O2)-specific electrode, and ferrozine assay techniques. High performance liquid chromatography (HPLC) analysis showed that MPO heme protection was at the expense of MLT oxidation. The full protection of the MPO heme requires the presence of a 1:2 MLT to H2O2 ratio. Melatonin prevents HOCl-mediated MPO heme destruction through multiple pathways. These include competition with chloride, the natural co-substrate; switching the MPO activity from a two electron oxidation to a one electron pathway causing the buildup of the inactive Compound II, and its subsequent decay to MPO-Fe(III) instead of generating HOCl; binding to MPO above the heme iron, thereby preventing the access of H2O2 to the catalytic site of the enzyme; and direct scavenging of HOCl. Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl. Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation.

Disruption of heme-peptide covalent cross-linking in mammalian peroxidases by hypochlorous acid.

Myeloperoxidase (MPO), lactoperoxidase (LPO) and eosinophil peroxidase (EPO) play a central role in oxidative damage in inflammatory disorders by utilizing hydrogen peroxide and halides/pseudo halides to generate the corresponding hypohalous acid. The catalytic sites of these enzymes contain a covalently modified heme group, which is tethered to the polypeptide chain at two ester linkages via the methyl group (MPO, EPO and LPO) and one sulfonium bond via the vinyl group (MPO only). Covalent cross-linking of the catalytic site heme to the polypeptide chain in peroxidases is thought to play a protective role, since it renders the heme moiety less susceptible to the oxidants generated by these enzymes. Mass-spectrometric analysis revealed the following possible pathways by which hypochlorous acid (HOCl) disrupts the heme-protein cross-linking: (1) the methyl-ester bond is cleaved to form an alcohol; (2) the alcohol group undergoes an oxygen elimination reaction via the formation of an aldehyde intermediate or undergoes a demethylation reaction to lose the terminal CH2 group; and (3) the oxidative cleavage of the vinyl-sulfonium linkage. Once the heme moiety is released it undergoes cleavage at the carbon-methyne bridge either along the δ-ß or a α-γ axis to form different pyrrole derivatives. These results indicate that covalent cross-linking is not enough to protect the enzymes from HOCl mediated heme destruction and free iron release. Thus, the interactions of mammalian peroxidases with HOCl modulates their activity and sets a stage for initiation of the Fenton reaction, further perpetuating oxidative damage at sites of inflammation.

Hepatotoxicity induced by trimethoprim-sulfamethoxazole in a child with cystic fibrosis.

Patients with cystic fibrosis (CF) have chronic and progressive lung infections with various bacterial organisms that require treatment with oral and intravenous antibiotics on a regular basis. Trimethoprim-sulfamethoxazole (TMP-SMX) is one of the medications used to treat acute pulmonary infectious exacerbations in patients with CF. Hepatic toxicity secondary to TMP-SMX was previously described in normal subjects but has not been reported in children with CF. Here we describe a 14-year-old female child with CF who was given oral TMP-SMX for an acute pulmonary infectious exacerbation. She developed a rash, severe constitutional symptoms, and significant elevation of liver enzyme concentrations secondary to immunity-mediated reaction to TMP-SMX. Discontinuation of TMP-SMX and supportive treatment led to resolution of her symptoms and normalization of liver enzyme concentrations.

Why does adenotonsillectomy not correct enuresis in all children with sleep disordered breathing?

PURPOSE: We analyzed the outcome of nocturnal enuresis after adenotonsillectomy in children with sleep disordered breathing. We also evaluated differences in demographic, clinical, laboratory and polysomnography parameters between responders and nonresponders after adenotonsillectomy. MATERIALS AND METHODS: We prospectively evaluated children 5 to 18 years old diagnosed with sleep disordered breathing (snoring or obstructive sleep apnea syndrome) on polysomnography and monosymptomatic primary nocturnal enuresis requiring adenotonsillectomy to release upper airway obstruction. Plasma antidiuretic hormone and brain natriuretic peptide were measured preoperatively and 1 month postoperatively. RESULTS: Sleep studies were done in 46 children and 32 also underwent blood testing preoperatively and postoperatively. Mean ± SD patient age was 8.79 ± 2.41 years and the mean number of wet nights weekly was 6.39 ± 1.26. Polysomnography revealed obstructive sleep apnea syndrome in 71.7% of patients and snoring in 28.3%. After adenotonsillectomy 43.5% of patients became dry. Preoperative polysomnography findings indicated that responders, who were dry, had significantly more arousals and obstructive apnea episodes but fewer awakenings than nonresponders, who were wet. Significant increases in plasma antidiuretic hormone and significant decreases in plasma brain natriuretic peptide were seen in all children with no difference between responders and nonresponders. No difference between the groups was noted in age, gender, race, body mass index, constipation, preoperative number of wet nights weekly or type of sleep disordered breathing. CONCLUSIONS: Nocturnal enuresis resolved after adenotonsillectomy in almost half of the children with sleep disordered breathing. Those who became dry had more frequent arousal episodes caused by apnea events than those who remained wet.

Improving the Rate of Sufficient Sweat Collected in Infants Referred for Sweat Testing in Michigan.

Objective. Sweat collected for testing should have quantity not sufficient (QNS) rate of ≤10% in babies ≤3 months of age. Michigan (MI) cystic fibrosis (CF) centers' QNS rates were 12% to 25% in 2009. This project was initiated to reduce sweat QNS rates in MI. Methods/Steps. (a) Each center's sweat testing procedures were reviewed by a consultant. (b) Each center received a report with recommendations to improve QNS rates. (c) Technicians visited other participating centers to observe their procedures. Results. A total of 778 infants were identified as positive via CF newborn screening over a 2-year period. The mean age at time of sweat test was 23.2 days (SD ± 13.0 days). The overall QNS percent decreased from 14.4% to 9.5% (P = .04) during the study. Conclusion. This project and teamwork approach led to a decrease of sweat test QNS rates, opportunities to solve a common problem, and improved quality of care.

Sleep architecture parameters that predict postoperative resolution of nocturnal enuresis in children with obstructive sleep apnea.

OBJECTIVES: We performed a prospective cohort study in a pediatric tertiary care center to determine whether preoperative sleep architecture is associated with complete resolution of nocturnal enuresis (NE) after adenotonsillectomy. METHODS: Thirty-seven pediatric patients with primary NE who underwent adenotonsillectomy for obstructive sleep apnea (OSA) were evaluated. Preoperative polysomnograms, as well as preoperative and postoperative reports of NE, were recorded. We performed chi2 analysis, Fisher's exact test (for p values), and t-tests to evaluate the impact of multiple demographic characteristics on sleep architecture, comparing children with resolved NE to those with unresolved NE after adenotonsillectomy. RESULTS: The patients' mean age was 8.0 years (SD, 2.32 years). All children had presurgical primary NE. No age or gender differences were identified between children with resolved NE and those with unresolved NE. After surgery, more than half of the participants had resolution of NE. A higher percentage of boys had unresolved NE (chi2 = 3.63; p = 0.06). Improvement of NE was identified in children with a higher obstructive apnea-hypopnea index and more desaturation events. Eleven of the 12 children with prolonged stage 2 sleep reported resolution of NE (p = 0.001). Children with an obstructive apnea-hypopnea index of greater than 10 had a significantly greater rate of resolution of NE (p = 0.01). Logistic regression demonstrated that an elevated body mass index and the interaction of severe OSA and prolonged stage 2 sleep predicted resolution of NE. All 10 children with severe OSA and an abnormal total time spent in stage 2 sleep had resolution of NE. CONCLUSIONS: Adenotonsillectomy is a treatment option for children with OSA and NE. Postoperative resolution of NE was seen in 51.4% of patients who underwent adenotonsillectomy. The children with both severe OSA and prolonged stage 2 sleep were 3.4 times as likely to have postoperative resolution of NE. These results suggest that there are significant differences in preoperative sleep architecture between children whose NE resolves after adenotonsillectomy and those whose NE does not resolve.

Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway.

Myeloperoxidase (MPO) is a heme-containing enzyme that generates hypochlorous acid (HOCl) from chloride (Cl(-)) and hydrogen peroxide (H2O2). It is implicated in the pathology of several chronic inflammatory conditions such as cardiovascular and pulmonary diseases and cancer. Recently we have shown that HOCl can destroy the heme prosthetic group of hemoproteins. Here, we investigated whether the HOCl formed during steady-state catalysis is able to destroy the MPO heme moiety and thereby function as a major source of free iron. UV-visible spectra and H2O2-specific electrode measurements recorded during steady-state HOCl synthesis by MPO showed that the degree of MPO heme destruction increased after multiple additions of H2O2 (10 µM), precluding the enzyme from functioning at maximum activity (80-90% inhibition). MPO heme destruction occurred only in the presence of Cl(-). Stopped-flow measurements revealed that the HOCl-mediated MPO heme destruction was complex and occurred through transient ferric species whose formation and decay kinetics indicated it participates in heme destruction along with subsequent free iron release. MPO heme depletion was confirmed by the buildup of free iron utilizing the ferrozine assay. Hypochlorous acid, once generated, first equilibrates in the solution as a whole before binding to the heme iron and initiating heme destruction. Eliminating HOCl from the MPO milieu by scavenging HOCl, destabilizing the MPO-Compound I-Cl complex that could be formed during catalysis, and/or inhibiting MPO catalytic activity partially or completely protects MPO from HOCl insults. Collectively, this study elucidates the bidirectional relationship between MPO and HOCl, which highlights the potential role of MPO as a source of free iron.

Evaluation of genetic counseling among cystic fibrosis carriers, Michigan Newborn Screening.

OBJECTIVE: A quality improvement (QI) strategy to improve the rate of genetic counseling (GC) services was initiated in cystic fibrosis (CF) care Center E in 2010. This statewide study was conducted to determine: (1) GC rates before and after implementation of the QI strategy at Center E; (2) characteristics associated with not receiving GC; and (3) topic areas addressed during GC. METHODS: The retrospective study included 1,097 CF carriers born from 2008 to 2011 identified through Michigan's Newborn Screening Program. Rate of GC services was determined for Center E and the other four CF centers before and after the QI change. Bivariate and multivariable logistic regression was used to determine associations between select characteristics and not receiving GC. Topic areas discussed during GC sessions were assessed using frequency tables. RESULTS: Rate of GC services in Center E increased from 23% in 2008-2010 to 91% in 2011, while at the other centers approximately 92% received GC services across those years. In 2008-2010, being seen at Center E and black race were significantly associated with increased likelihood of not receiving GC services in adjusted analyses. In 2011, neither characteristic was associated with receipt of GC. Of 16 target topic areas, all were discussed in 85% of GC sessions. CONCLUSIONS: Implementing a QI strategy of providing sweat test results at the GC appointment within Center E resulted in more CF carriers receiving comprehensive GC services. Center-specific procedure differences should be assessed to increase rate of GC services following a positive CF newborn screen.

Desensitization to inhaled aztreonam lysine in an allergic patient with cystic fibrosis using a novel approach.

OBJECTIVE: To report the successful desensitization of a highly allergic patient with cystic fibrosis (CF) to inhaled aztreonam lysine using the novel approach of intravenous desensitization followed by full-dose inhaled therapy without any adverse reactions. CASE SUMMARY: A 19-year-old woman with CF had persistent Pseudomonas aeruginosa-positive cultures and a history of type I hypersensitivity reactions to multiple medications, including aztreonam and tobramycin (intravenous and inhaled). To start therapy with an inhaled antipseudomonal antibiotic on a chronic basis, she underwent rapid desensitization to intravenous aztreonam followed by initiation of inhaled aztreonam lysine. Following intravenous desensitization with aztreonam, there was no adverse reaction or decline in lung function noted with inhaled aztreonam lysine and the chronic therapy was continued at home, with a modified regimen to maintain desensitization. DISCUSSION: Aztreonam lysine has been used for treatment of patients with CF with chronic P. aeruginosa colonization. Previous allergic reaction to intravenous aztreonam is considered a contraindication for use of aztreonam lysine. Our patient had a history of hives and facial swelling following administration of intravenous aztreonam (type I hypersensitivity reaction) as well as hypersensitivity to tobramycin. Rapid desensitization can be done for drugs that mediate a type I hypersensitivity reaction, with mast cells and basophils being the cellular targets. There are a few case reports of desensitization to inhaled antibiotics such as tobramycin and colistin, but desensitization to aztreonam lysine has not previously been reported. CONCLUSIONS: Desensitization of a patient with CF who is allergic to intravenous aztreonam was successfully accomplished with the novel approach of rapid intravenous desensitization followed by inhaled therapy. As inhaled antibiotics are being increasingly used for patients with CF, this novel strategy can be used for desensitizing allergic patients with CF to ensure that they can continue to receive these medications safely.

Melatonin attenuates hypochlorous acid-mediated heme destruction, free iron release, and protein aggregation in hemoglobin.

In inflammatory diseases, where hypochlorous acid (HOCl) is elevated, iron homeostasis is disturbed, resulting in accumulation of free iron. Free iron is toxic by virtue of its ability to generate free radicals through the Fenton reaction. HOCl is generated by myeloperoxidase, (MPO) using chloride and hydrogen peroxide as substrates. Recent studies demonstrate that HOCl binds to the heme moiety of hemoglobin (Hb), which generates a transient ferric species whose formation and decay kinetics indicate it participates in protein aggregation, heme destruction, and free iron release. Here, we show that melatonin prevents HOCl-mediated Hb heme destruction and protein aggregation, using a combination of UV-vis spectrophotometry, ferrozine colorimetric assay, and in-gel heme staining. We also show that melatonin treatment prevents HOCl-mediated loss of red blood cell (RBC) viability, indicating biologic relevance of this finding. The mechanism by which melatonin prevents HOCl-mediated Hb heme destruction is by direct scavenging of HOCl and/or through the destabilization of the higher Hb oxidative states intermediates, ferryl porphyrin radical cation Hb-Fe(IV)=O(+π•) and Hb-Fe(IV)=O, which are formed through the reaction of HOCl with Hb. Our work establishes a direct mechanistic link between melatonin and its protective effect in chronic inflammatory diseases. Collectively, in addition to acting as an antioxidant and as a MPO inhibitor, melatonin can also exert its protective effect by inhibiting HOCl-mediated heme destruction of hemoproteins and subsequent free iron release.

Maternal smoking during pregnancy, polymorphic CYP1A1 and GSTM1, and lung-function measures in urban family children.

PURPOSE: Understanding the interplay between genes and in-utero tobacco exposure in affecting child lung development is of great significance. In this study, we tested the hypothesis that tobacco-related lung-function reduction in children differs by maternal polymorphic genes Cytochrome P450 1A1 (CYP1A1) and Glutathione S-transferase Mu 1 (GSTM1). MATERIALS AND METHODS: Data were collected among 370 children (6-10 years old, 81.6% African-Americans) and their biological mothers visiting a large children's hospital. Study hypotheses were tested using multiple regression method. RESULTS: Among the study sample, 143 mothers smoked throughout pregnancy and 72 smoked on a daily basis. Spirometric measures (mean±SD) included were: forced vital capacity (FVC)=1635±431 mL, forced expiratory volume in the first 1s (FEV1)=1440 ±360 mL, percent FEV1/FVC ratio=89±12, and forced expiratory flow between the 25% and 75% of FVC (FEF25-75)=1745±603 mL. In addition to a tobacco effect on FVC (-131 mL, 95% CI: -245, -17) and FEV1/FVC ratio (42, 95% CI: 1, 83), regression analysis controlling for covariates indicated that for the subsample of children whose mothers were CYP1A1⁎2A homozygous, maternal daily smoking was associated with -734 mL (95% CI: -1206, -262) reductions in FEV1 and -825 mL (95% CI: -909, -795) reductions in FVC; reduced smoking was still associated with -590 mL (95% CI: -629, -551) reductions in FVC. For children of mothers with GSTM1 deletion, persistent daily smoking was associated with -176 mL (95% CI: -305, -47) reductions in FVC. DISCUSSION AND CONCLUSIONS: Maternal smoking during pregnancy was significantly associated with lung-function reduction in children, particularly for those whose mothers possessed the polymorphic CYP1A1*2A and GSTM1 deletion.

Reaction of hemoglobin with HOCl: mechanism of heme destruction and free iron release.

Hypochlorous acid (HOCl) is generated by myeloperoxidase using chloride and hydrogen peroxide as substrates. HOCl and its conjugate base (OCl(-)) bind to the heme moiety of hemoglobin (Hb) and generate a transient ferric species whose formation and decay kinetics indicate it can participate in protein aggregation and heme destruction along with subsequent free iron release. The oxidation of the Hb heme moiety by OCl(-) was accompanied by marked heme destruction as judged by the decrease in and subsequent flattening of the Soret absorbance peak at 405 nm. HOCl-mediated Hb heme depletion was confirmed by HPLC analysis and in-gel heme staining. Exposure of Hb to increasing concentrations of HOCl produced a number of porphyrin degradation products resulting from oxidative cleavage of one or more of the carbon-methene bridges of the tetrapyrrole ring, as identified by their characteristic HPLC fluorescence and LC-MS. A nonreducing denaturing SDS-PAGE showed several degrees of protein aggregation. Similarly, porphyrin degradation products were identified after exposure of red blood cells to increasing concentrations of HOCl, indicating biological relevance of this finding. This work provides a direct link between Hb heme destruction and subsequent free iron accumulation, as occurs under inflammatory conditions where HOCl is formed in substantial amounts.