Background: Recent advances in telemedicine and mobile applications have enabled greater patient access to medical care. Objectives: This study aims to explore the feasibility of using telemedicine for evaluating dermatomyositis (DM) skin rashes, comparing it to traditional in-clinic assessments. Methods: DM patients, according to the 2017 European League Against Rheumatism/American College of Rheumatology classification criteria, were prospectively enrolled in an observational study called DART, or “Dermatomyositis Assessment of Rash via Telemedicine”. Each patient underwent evaluations by two independent rheumatologists (MD1 and MD2) for both in-clinic and telemedicine visits (2-4 weeks post-clinic evaluation). The Cutaneous Dermatomyositis Disease Area and Severity Index (CDASI) were scored during these assessments. Telemedicine visits were done through a Health Insurance Portability and Accountability Act (HIPAA) -secured platform and aided by patient self-photographs obtained via a mobile application on the patient’s smartphone. In addition, patients were asked to complete a modified CDASI developed for the patient’s self-assessment of the rashes. The patient also completed a Patient-Reported Outcome questionnaire called Skindex to evaluate the patient’s skin quality of life simultaneously. We compared the telemedicine CDASI with in-clinic CDASI. Results: A total of 23 DM patients underwent evaluation, resulting in 30 sets of visits (1-2 sets per patient; 1 set of visits comprised of 1 in-clinic visit and 1 telemedicine visit). Most of the patients were female (82.6%), with a mean age of 48.6 ± 17.4 years and a median (Q1-Q3) disease duration of 38.0 (9.5 - 88.0) months. Their median CDASI activity at baseline visit was 4.5 (2.0 – 17.5). Telemedicine CDASI had strong correlations with in-clinic CDASI, patient CDASI, and Skindex, indicating good convergent validity. The intraclass correlation coefficient (ICC) for inter-rater reliability for telemedicine CDASI was excellent for MD1 and MD2 (ICC= 0.97). The ICC for intra-rater reliability between the same rater in-clinic and telemedicine was 0.97 and 0.96 for MD1 and MD2, respectively. Conclusion: Our results demonstrate favorable validity, inter-rater reliability, and intra-rater reliability of telemedicine CDASI in evaluating DM patients with skin rashes remotely. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: None declared.Table 1The correlation between in-clinic CDASI, telemedicine CDASI, patient CDASI, and SkindexSpearman rank correlation coefficientsClinic MD1 CDASI ActivityClinic MD2 CDASI ActivityTele MD1 CDASI ActivityTele MD2 CDASI ActivityPatient CDASIPatient SkindexClinic MD1 CDASI Activity0.89*0.92*0.91*0.74*0.84*Clinic MD2 CDASI Activity0.89*0.83*0.80*0.68*0.77*Tele MD1 CDASI Activity0.92*0.83*0.89*0.68*0.82*Tele MD2 CDASI Activity0.91*0.80*0.89*0.68*0.71*PatientCDASI0.74*0.68*0.68*0.68*0.66*PatientSkindex0.84*0.77*0.82*0.71*0.66*Abbreviations: CDASI, Cutaneous Dermatomyositis Disease Area and Severity Index; Clinic MD1, in-clinic evaluation by MD1; Clinic MD2, in-clinic evaluation by MD2; Tele MD1, telemedicine evaluation by MD1; Tele MD2, telemedicine evaluation by MD2.*P-value < 0.001

Background: Creatine kinase (CK) is considered an important biomarker for the diagnosis and monitoring of Idiopathic Inflammatory Myopathies (IIM). However, in some cases, CK levels might exhibit only minor elevation or remain within the normal range, even in the setting of active muscle disease. The data regarding normal CK in IIM are limited. Objectives: To characterize IIM patients with normal CK levels, and to investigate the CK level in association with demographic and clinical phenotypes as well as with various aspects of disease activity and outcomes. Methods: Data were collected from the Rituximab in Myositis (RIM) study, a large prospective multicenter, randomized, double-blind, placebo-controlled trial. Enrolled participants included patients with dermatomyositis (DM) or polymyositis (PM) with refractory disease despite the use of glucocorticoids and at least one other immunosuppressive treatment. Data were collected on all core set measures (CSMs), including CK, manual muscle testing (MMT), physician-global disease activity (physician-global) and patient-global disease activity (patient-global), extra-muscular disease activity, and Health Assessment Questionnaire- Disability Index (HAQ-DI). These CSMs were recorded at baseline, weeks 4, 8, 12, 16, 20, 24, 28, 32, 36, and 44. For standardization purposes, CK levels were expressed as a multiple of their lab upper limits of normal [ULN]). "Normal" CK including near normal CK was defined based on a serum level within twice the ULN as per the laboratory reference range, whereas "elevated" CK was determined by levels surpassing this threshold. "Definition of improvement" (DOI) was based on the International Myositis Assessment and Clinical Studies Group (IMACS) preliminary validated response criterion of a ≥ 20% improvement in 3 of any 6 CSMs, with no more than 2 worsening by ≥ 25%. Results: 143 patients were analyzed, including 67 patients with normal/near normal CK and 76 with an elevated CK. The former group had a higher prevalence of Caucasians (84% vs. 59%, p=0.001), females (84% vs. 67%, p=0.023), and DM (64% vs. 37%, p=0.001), while the latter group exhibited a tendency toward increased age, although this trend did not achieve statistical significance (p=0.056). At baseline, the elevated CK group had a worse (lower) MMT score (101± 20 vs. 108±16, p=0.021), more active muscle disease (VAS 5.77± 2.01 vs. 4.28± 1.90, p<0.001), and less extra-muscular global disease activity (VAS 2.38± 2.11 vs. 3.35±1.70, p=0.001). Disease activity (both patient and physician assessed) was higher in the elevated CK group (p=0.024 and p<0.001, respectively). However, the HAQ-DI, patient-global disease damage assessment, and achievement of DOI, were similar between groups (Table 1). At baseline, in the elevated CK group, there was a clear association between CK levels and physician-global assessment (p=0.018) as well as muscle disease activity (p=0.005). Conversely, in patients with normal CK levels, no associations were observed between CK levels and other CSMs at baseline. Employing a linear mixed model with adjustment for age, sex, and race, CK levels exhibited a significant correlation (p<0.001) during the follow-up period with various parameters, including MMT, patient-global, physician-global, muscle disease activity, and extra-muscular disease activity. This correlation was strong regardless of baseline CK status (normal vs. elevated) or myositis sub-type (DM vs. PM) (Figure 1). Conclusion: Normal CK levels are frequently noted in IIM, and do not exclude the presence of active muscle disease. Patients with normal CK levels exhibit distinct demographic and clinical characteristics. CK levels demonstrate a strong longitudinal correlation with other CSMs, irrespective of baseline CK levels or myositis sub-type, highlighting the importance of CK as a biomarker for monitoring disease activity in all IIM patients. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: Shiri Keret: None declared, Lisa Kaly: None declared, Tanya Chandra: None declared, Raisa Lomanto Silva: None declared, Eugenia Gkiaouraki: None declared, Nantakarn Pongtarakulpanit: None declared, Shreya Sriram: None declared, Siamak Moghadam-Kia: None declared, Chester V Oddis: None declared, Rohit Aggarwal 1. Actigraph: Consultant, 2. Alexion: Consultant, 3. ANI Pharmaceuticals: Consultant, 4. Argenx: Consultant, 5. AstraZeneca: Consultant, 6. Boehringer-Ingelheim: Consultant, 7. Bristol Myers-Squibb: Consultant, 8. CabalettaBio: Consultant, 9. Capella Bioscience: Consultant, 10. Corbus: Consultant, 11. CSL Behring: Consultant, 12. EMD Serono: Consultant, 13. Galapagos: Consultant, 14. Horizon Therapeutics: Consultant, 15. I-Cell: Consultant, 16. Janssen: Consultant, 17. Kezar: Consultant, 18. Kyverna: Consultant, 19. Merck: Consultant, 20. Novartis: Consultant, 21. Nuvig Therapeutics: Consultant, 22. Octapharma: Consultant, 23. Pfizer: Consultant, 24. Regeneron: Consultant, 25. Roivant: Consultant, 26. Sanofi: Consultant, 27. Teva: Consultant, 28. Artsome: Consultant, 29. Capstanx: Consultant, 30. Manta: Consultant, 1. Boehringer Ingelheim (BI): 2. Bristol Myers-Squibb, 3. EMD Serono, 4. Janssen, 5. Mallinckrodt, 6. Pfizer, 7. Q32.

Background: Idiopathic inflammatory myopathies (IIM, myositis) are a systemic autoimmune disease leading to debilitating muscle weakness and significant limitations in daily activities. Physical activity monitors (PAM) are validated and recognized measures of the frequency and intensity of physical activities. Given the unmet need for objective, continuous, longitudinal outcome measures of physical activity and function in myositis, we assessed compliance, reliability, and validity of widely used commercial PAM – Fitbit© in evaluating physical activity in myositis patients. Objectives: To investigate the psychometric properties and performance of Fitbit as a wearable activity tracker in an IIM cohort. Methods: “Myositis Patient Centered Tele-Research (MyPACER)” is a multi-center observational prospective study conducted over 6 months on IIM patients with dermatomyositis (DM), necrotizing myopathy (NM) and polymyositis (PM, including anti-synthetase syndrome). The study had two cohorts, 1. Tele-Research Cohort (TRC): remotely enrolled from any location in the United States, and 2. Center-Based Cohort (CBC): a traditional cohort with enrollment from 2 myositis centers. Functional and Patient-Reported Assessments were completed monthly, including a health assessment questionnaire, patient global disease activity, PROMIS-physical function 20, and functional tests Timed-Up-and-Go (TUG) and Sit-To-Stand (STS)). Participants were asked to use their wrist-based Fitbit for (≥10 hours/day) for 7 consecutive 7 days once a month. Average daily steps per minute (Ave step/min) & average daily peak cadence (Ave peak cadence) were evaluated as PAM measures. Results: A total of 120 IIM patients (mean age 55.5±13.43; 75% females; 80.8% White) were enrolled in the study, comprising 82 in the Main TRC group and 38 in the CBC group (Table 1). There were 51.7% DM, 39.2% PM, and 9.1% NM. The TRC and CBC cohorts were similar in demographics and disease subtypes. Age was significantly correlated with the(p=0.01) but not with Ave peak cadence, showing decreased steps with advancing age. Gender, race/ethnicity, or disease subtypes were not associated with PAM measures. On data analysis, 90% of patients completed at least one valid day on their Fitbit devices. The compliance with Fitbit was very high, with participants wearing devices on most days of the week (average = 6.52 days) for the most visits (average 5.73 visits), with similar results for remote or local recruitment. Ave steps/min and Ave peak cadence showed strong test-retest reliability (r=0.89 and r=0.86, p< 0.0001 & p= 0.0001) and were strongly correlated within the patient. On longitudinal analysis, the two groups using a mixed model between the baseline and 6th-month visits had no significant differences in the average steps per minute (p=0.09). However, a difference was noted in the peak cadence (p=0.02). The CBC group had a mean increase of 0.37/visit compared to the TRC group -.04/visit. Conclusion: In a large IIM cohort, Fitbit PAM variables demonstrate favorable compliance and psychometric properties with strong test-retest reliability and a significant difference between baseline and 6th-month visits on average. peak cadence. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: None declared.Figure 1longitudinal analysis measuring the mean change in average steps and peak cadence between the baseline and 6th-month visits

Background: Idiopathic Inflammatory Myopathies (IIM) are a group of rare autoimmune diseases, characterized by heterogeneous manifestations and clinical trajectories. Given its rarity, large IIM clinical trials are scarce. The utilization of tele-research methods has the potential to improve participant recruitment for myositis studies, consequently advancing the understanding of disease characteristics, medication usage patterns, and disease activity. Objectives: To evaluate disease characteristics, including symptoms, diagnosis delay, medication usage, and work absenteeism in IIM patients throughout the U.S., and compare these parameters between patients recruited remotely versus locally. Methods: “Myositis Patient Centered Tele-Research” (My PACER) is a large multi-center prospective 6-month observational study of U.S. IIM subjects, competitively recruited through traditional in-person clinic visits (Center-Based Cohort [CBC]), and remotely using smartphone technology, wearable devices, and telemedicine principles (Tele-Research Cohort [TRC]). Data collection included baseline demographic and clinical parameters, and patient-reported outcomes (PROs) at 6 monthly visits, including myositis core set measures (health assessment questionnaire [HAQ-DI], patient global disease activity, PROMIS physical function), functional tests (six-minute walk, timed up-and-go and sit-to-stand tests), and physical activity monitoring. Clinician Reported Outcomes (ClinRO) including myositis core set measures (manual muscle testing [MMT], physician global disease activity, extra-muscular global disease activity, creatine kinase [CK]) were obtained at baseline and 6 months. Results: 120 IIM patients were enrolled, comprising 82 patients in the TRC and 38 in the CBC. The TRC and CBC were similar in demographics, with a mean age in TRC vs. CBC 54.9 ± 13.8 years vs. 56.9 ± 12.6 years (p=0.44), 62 (76%) vs. 28 (74%) were females (p=1.0), and 68 (83%) vs. 29 (76%) Caucasians (p=0.17), respectively. Overall, 49 dermatomyositis (41%), 32 polymyositis (27%), 28 (23%) anti- synthetase syndrome and 11 necrotizing myositis (9%) patients were enrolled with no significant differences in the prevalence of myositis sub-types observed among the cohorts (p=0.85). Positive myositis-specific antibodies (MSA) or myositis-associated antibodies (MAA) were documented in 63 (53%) and 17 (14%), respectively, with a similar rate among groups (Figure 1a). Diagnostic delay was similar among groups, with a mean duration of 1.08±2.294 years from symptom onset to IIM diagnosis (p=0.70) (Table 1).The groups exhibited comparable employment status, with 68 (57%) patients reporting current active employment (p=0.83).At baseline, all PROs, functional tests, ClinRO, MDAAT, and physical activity measures were similar between the two study groups (Table 1, Figure 1b).The frequency of positive diagnostic tests, such as electromyography (EMG), chest computed tomography (CT) and muscle biopsy consistent with a diagnosis of myositis, showed no significant difference between the groups.Finally, The TRC demonstrated a higher usage of immunosuppressive and immunomodulatory agents, encompassing conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) and biologic treatments (p<0.001 and p=0.013, respectively). When comparing U.S. regions, the prescription of biologic treatments was more prevalent in the Southeast and Southwest regions. Notably, diagnostic delay was similar between the different U.S. regions. Conclusion: Remote recruitment yielded a patient cohort that is demographically and clinically similar to traditionally recruited patients. These findings indicate the viability and effectiveness of remote recruitment for research studies, particularly in rare diseases, offering the potential for robust and diverse patient enrollment across different geographic and ethnic backgrounds. Moreover, these results illustrate the feasibility of remote collection of PROs, conducting functional tests, and measuring physical activity. Acknowledgements: NIL. Disclosure of Interests: Shiri Keret: None declared, Raisa Lomanto Silva: None declared, Tanya Chandra: None declared, Eugenia Gkiaouraki: None declared, Nantakarn Pongtarakulpanit : None declared, Shreya Sriram: None declared, Siamak Moghadam-Kia: None declared, Chester V Oddis: None declared, Rohit Aggarwal: Actigraph: Consultant, Alexion: Consultant, ANI Pharmaceuticals: Consultant, Argenx: Consultant, AstraZeneca: Consultant, Boehringer-Ingelheim: Consultant, Bristol Myers-Squibb: Consultant, CabalettaBio: Consultant, Capella Bioscience: Consultant, Corbus: Consultant, CSL Behring: Consultant, EMD Serono: Consultant, Galapagos: Consultant, Horizon Therapeutics: Consultant, I-Cell: Consultant, Janssen: Consultant, Kezar: Consultant, Kyverna: Consultant, Merck: Consultant, Novartis: Consultant, Nuvig Therapeutics: Consultant, Octapharma: Consultant, Pfizer: Consultant, Regeneron: Consultant, Roivant: Consultant, Sanofi: Consultant, Teva: Consultant, Artsome: Consultant, Capstanx: Consultant, Manta: Consultant, Boehringer Ingelheim (BI): Bristol Myers-Squibb, EMD Serono, Janssen, Mallinckrodt, Pfizer, Q32.

Although corticosteroids remain the mainstay of treatment for the inflammatory myopathies, their use is complicated by many side effects. Other immunosuppressive agents, alone or in combination, are being increasingly used for patients with other severe disease or treatment-related complications. Pulmonary disease remains a serious source of morbidity and mortality in myositis patients. Cyclophosphamide, cyclosporine, and tacrolimus are efficacious in patients with interstitial lung disease. Intravenous immunoglobulin is not only effective for the cutaneous complications of dermatomyositis but has been helpful in other extramuscular manifestations.

We have previously proposed that pro-inflammatory cytokines and nitric oxide (NO) contributed to reversible myocardial depression in patients with sepsis and congestive heart failure. Sepsis and heart failure are also associated with refractoriness to beta-adrenoceptor agonists. Therefore, the chronotropic effects of cytokines and the NO synthase inhibitor, N-G-methyl-L-arginine (NMA), on beta-adrenoceptor stimulation of neonatal cardiac myocytes were studied. Tumor necrosis factor alpha, interleukin-1 beta and interleukin-6 but not interleukin-4 or interleukin-5 significantly enhanced spontaneous beating rates compared to untreated myocytes in serum-free media for 48 h (P <0.01; n=12 for each). NMA also significantly enhanced spontaneous beating rates (P <0.01; n=12 for each). Only interleukin-1 beta treatment resulted in significant nitrite production, immunohistochemical staining for inducible nitric oxide synthase and detection of inducible NO synthase messenger RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). However, tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, and NMA each completely blocked the positive chronotropic effects of the beta-adrenoceptor agonist, isoproterenol (P <0.01; n=12 for each). These findings are most consistent with an inducible NO synthase-independent effect of cytokines and NMA on the chronotropic responses of neonatal cardiac myocytes to beta-adrenoceptor stimulation. This effect of cytokines and NMA on adrenergic signaling may involve a myocardial constitutive NO synthase or an NO-independent mechanism.

Cardioprotective effects of dietary fish oil consumption have been attributed to the substitution of eicosapentaenoic acid (EPA) for arachidonic acid (AA) in cellular membranes. This substitution blocks the formation of leukotriene B4 (LTB4) which is elaborated locally in the area of ischemic myocardium. We studied the in vitro inotropic, chronotropic, and radioligand binding characteristics of LTB4 to gain potentially important insights into the mechanisms involved in the cardioprotective effect of EPA. LTB4 was a concentration-dependent and reversible negative inotrope in isolated papillary muscle preparations. LTB4 significantly blocked the positive inotropic effect of Bay K 8644 (BK) (calcium channel regulator) on papillary muscles and the positive chronotropic effect of BK on spontaneously beating neonatal cardiac myocytes. LTB4 had no direct effect on [3H]PN200-110 binding to cardiac sarcolemmal L-type calcium channels or [3H]ryanodine binding to cardiac SR calcium release channels. These studies support a potentially important indirect effect of LTB4 on myocardial E-C coupling.

We have previously reported that interleukin-1 beta (IL-1) alone induced the transcription of inducible nitric oxide synthase (iNOS) mRNA and nitric oxide (NO) production by isolated neonatal rat cardiac myocytes (CM). The present studies were undertaken to explore the signal transduction pathways involved in IL-1-induced NO production by CM. The addition of IL-1 to CM resulted in a peak rise in both adenosine 3',5'-cyclic monophosphate (cAMP) and protein kinase A (PKA) activities by 10 min followed by rapid declines and return to basal levels within 60 min. The PKA inhibitor KT-5720 completely blocked NO-2 production by IL-1-stimulated CM (P < 0.01; n = 12). The protein kinase C (PKC) inhibitor, calphostin C, had no effect on NO2- production by IL-1 stimulated CM [P = not significant (NS); n = 12]. The addition of PKA+cAMP to cytosols derived from IL-1-treated CM did not directly enhance iNOS enzyme activity (P = NS; n = 3). CM treated with IL-1 alone stained positively for iNOS protein by immunohistochemistry. iNOS staining was absent in CM treated with IL-1+KT-5720. KT-5720 resulted in an earlier disappearance of iNOS mRNA from IL-1-treated CM, as detected by semiquantitative reverse transcriptase-polymerase chain reaction. We report for the first time that PKA (but not PKC) activation is required for IL-1-induced NO production by CM.

Cytokines are soluble mediators that possess both intra- and intercellular signaling properties. Their function has been investigated most thoroughly in the context of immune reactions. Cytokines, however, form an important component of the inflammatory response to trauma and are found in elevated concentrations following cardiopulmonary bypass (CPB). We were, therefore, interested in studying mRNA expression for the cytokine tumor necrosis factor alpha (TNF-alpha) in patients undergoing cardiac surgery. White cells isolated from the circulation of seven patients before and after CPB were assayed for the expression of mRNA with TNF-alpha specific primers using the polymerase chain reaction. Message for TNF-alpha was found in all patients, with the highest values demonstrated in patients whose CPB times exceeded 1 1/2 hours. Enhanced message expression was seen as early as 1 hour after the start of CPB. Genes responsible for cytokine (TNF-alpha) expression and production are activated during cardiac surgery.

We and others have provided indirect evidence for the presence of a constitutive nitric oxide synthase (cNOS) in the mammalian heart. We now provide more direct evidence for the regulation of a myocardial cNOS in the hearts of patients undergoing elective cardiopulmonary bypass (CPB). cNOS enzyme activity was demonstrable in both cytosolic (8.3 +/- 0.02 pmol/min/mg) and membrane (11.1 +/- 0.4 pmol/min/mg) preparations derived from human atrial pectinate muscles obtained at the time of CPB (n = 6). Plasma nitrite (NO2-) + plasma nitrate (NO3-) levels from the beating hearts of patients before bypass were reduced from 146 +/- 33 to 5.1 +/- 50 pmol/min/g after cardiac arrest during CPB (n = 23; p < 0.002 by Student's t test). Thus, the human myocardium constitutively produces nitric oxide that is regulated by the contractile state of the heart.

We provide evidence for an immediate effect of NG-monomethyl-L-arginine (L-NMMA) on the force-frequency relationship in isolated hamster papillary muscles. L-NMMA (competitive inhibitor of nitric oxide synthase) reversed the force-frequency relationship (staircase effect) in isolated hamster papillary muscles from negative to positive (P < .01; ANOVA; n = 6). The addition of L-arginine (substrate for nitric oxide synthase) blocked the L-NMMA effect (P < .01; ANOVA; n = 6). The addition of the nitric oxide (NO) donor, sodium nitroprusside (NTP), significantly increased the level of cGMP in the tissue bath (P < .01; test; n = 6) and reversed the positive inotropic effect of L-NMMA on staircase (P < .01; ANOVA; n = 6). The addition of 8-Br-cGMP to the bath resulted in a concentration-dependent decrease in tension generated by the papillary muscles (n = 6). Methylene blue (known inhibitor of cGMP) mimicked the effect of L-NMMA on staircase (P < .01; ANOVA; n = 6). L-NMMA also significantly blunted the negative inotropic effect of ryanodine (SR calcium release channel regulator) (P < .01; ANOVA; n = 6). The positive inotropic effect of Bay K 8644 (sarcolemmal, L-type calcium channel regulator) was not affected by L-NMMA (P = NS; ANOVA; n = 6). L-NMMA had no effect on either [3H]ryanodine or [3H]PN200-110 (sarcolemmal, L-type calcium channel regulator) binding to cardiac membranes. These findings support a cGMP-dependent role for endogenous NO in myocardial E-C coupling.

Platinum electrodes (PLE) have been used recently to study the physiologic effects of the enzymatic conversion of L-arginine (L-arg) to nitric oxide (NO) by nitric oxide synthase (NOS). We sought to determine if PLE alone could catalytically convert L-arg to NO without NOS. Accordingly, NO2- + NO3- levels (NOx) were measured by HPLC from a physiologic Tyrode's buffer in the presence and absence of electrical stimulation with PLE or silver chloride electrodes (SCE) for 10 min (n = 6 for each condition). PLE stimulated at 1Hz produced 24 +/- 0.7 and 15.4 +/- 2.8 microM NOx from D-arg and L-arg (100mM) respectively. PLE + L-arg stimulated from 0 to 5 Hz produced increasing NOx (2.9 +/- 0.27;15.4 +/- 2.8;19.7 +/- 4;32 +/- 5;37 +/- 6.2;43 +/- 7 microM). SCE+L-arg showed no increase in NOx above background. The addition of 1% BSA to the Tyrode's buffer further increased NOx levels. NO2- levels were undetectable, indicating all NOx levels were NO3-. We conclude that PLE can catalytically convert L-Arg to NO3- via electron transfer without NOS.