Challenges in Treating Chlamydia trachomatis, Including Rectal Infections: Is It Time to Go Back to Doxycycline?

OBJECTIVE: To evaluate recent publications on efficacy of single-dose azithromycin and 7-day doxycycline when treating Chlamydia trachomatis. DATA SOURCES: A literature search of MEDLINE, EMBASE, PubMed, and Cochrane library was conducted (1990 to June 13, 2021) using the terms: Chlamydia trachomatis, genital chlamydia, rectal chlamydia, extragenital chlamydia, azithromycin, doxycycline, and treatment guidelines. ClinicalTrials.gov was searched to identify ongoing trials. STUDY SELECTION AND DATA EXTRACTION: English language studies, including controlled studies, retrospective analyses, systematic reviews, meta-analyses, and case reports, reporting microbiological or clinical outcomes in adolescents and adults were considered. DATA SYNTHESIS: Systemic reviews and meta-analyses of randomized trials reported azithromycin efficacy of 96% to 97% in genital chlamydia. However, reports of treatment failure have emerged, especially among symptomatic males, with an increased risk of microbiological failure after azithromycin than doxycycline (relative risk = 2.45; 95% CI = 1.36-4.41). Retrospective analyses and prospective observational cohort studies reported lower efficacy range following azithromycin than doxycycline (74%-87% vs 92%-100%, respectively) in rectal chlamydia. First randomized controlled trial comparing azithromycin and doxycycline reported significantly higher microbiological cure following doxycycline, with absolute difference of 26% (95% CI = 16%-36%; P < 0.001). The proposed 2021 Centers for Disease Control and Prevention treatment guidelines designate doxycycline as the preferred agent for treatment at any site. RELEVANCE TO PATIENT CARE AND CLINICAL PRACTICE: A growing body of evidence for treatment failure following azithromycin, especially in rectal chlamydia supports updating current practice. CONCLUSIONS: Doxycycline continues to achieve high efficacy in genital and rectal chlamydia. Clinicians should consider efficacy with convenience of dosing regimen, medication compliance, and sexual behavior risks when treating chlamydia infections.

Living with advanced cancer and the role of the primary care provider: The missing piece in the survivorship discourse.

The period of survivorship has been identified as a distinct phase of the cancer continuum, and the key role of primary care providers in caring for cancer survivors has been recognized. However, much of the focus to date has been placed on cancer survivors who are treated with curative intent and survive cancer-free. Receiving less attention are those who are living with advanced, non-curative cancer. In this commentary, we review the current evidence about the characteristics of these survivors, their unmet needs and receipt of health care. We offer insights into future research, education and policy initiatives aiming to enhance the care for this population.

Eravacycline, the first four years: health outcomes and tolerability data for 19 hospitals in 5 U.S. regions from 2018 to 2022.

IMPORTANCE: The rise of multidrug-resistant (MDR) pathogens, especially MDR Gram-negatives, poses a significant challenge to clinicians and public health. These resilient bacteria have rendered many traditional antibiotics ineffective, underscoring the urgency for innovative therapeutic solutions. Eravacycline, a broad-spectrum fluorocycline tetracycline antibiotic approved by the FDA in 2018, emerges as a promising candidate, exhibiting potential against a diverse array of MDR bacteria, including Gram-negative, Gram-positive, anaerobic strains, and Mycobacterium. However, comprehensive data on its real-world application remain scarce. This retrospective cohort study, one of the largest of its kind, delves into the utilization of eravacycline across various infectious conditions in the USA during its initial 4 years post-FDA approval. Through assessing clinical, microbiological, and tolerability outcomes, the research offers pivotal insights into eravacycline's efficacy in addressing the pressing global challenge of MDR bacterial infections.

Impact of a pharmacy department-wide transitions-of-care program on inappropriate oral antibiotic prescribing at hospital discharge.

Objective: To evaluate oral antibiotic prescribing for common infections at hospital discharge before and after implementation of a pharmacist-driven transitions-of-care (TOC) program. Design: Single-center before-and-after study. Setting: Acute-care, academic, community hospital in Santa Barbara, California. Patients: Eligible adult patients prescribed oral antibiotics at hospital discharge for community-acquired pneumonia, skin and soft-tissue infections, and urinary tract infections between September 2019 and December 2019 (preimplementation period) and between March 2021 and May 2021 (postimplementation period). Intervention: Antimicrobial stewardship-initiated, department-wide, TOC program requiring all clinical pharmacists to review discharge antibiotic prescriptions in real time. Results: In total, 260 antibiotic prescriptions were assessed for appropriateness: 140 before implementation and 120 after implementation. After implementation, the number of prescriptions considered inappropriate significantly decreased by 18% (52% vs 34%; P = .005). Inappropriate rates decreased in all assessment categories: dosing (15% vs 2%; P < .001), treatment duration (42% vs 31%; P = .08), antibiotic selection based on infection type or microbiology (8% vs 4%; P = .33), and antibiotics not indicated (16% vs 10%; P = .18). Median total antibiotic days decreased by 1 day after implementation (10 days vs 9 days; P = .67), and 30-day readmission rates were similar between both phases. Conclusions: A real-time, pharmacist-driven, TOC program for oral antibiotic prescriptions had a significant impact in reducing inappropriate prescribing of antibiotics at hospital discharge for common infections. Incorporating discharge antibiotic prescription review into pharmacist daily workflow may be a sustainable approach to outpatient antimicrobial stewardship in a setting with limited resources.

Assessing Preceptor and Student Perceptions of Remote Advanced Pharmacy Practice Experience Learning during the COVID-19 Pandemic.

The coronavirus disease 2019 pandemic created a major shift in learning modalities in the Advanced Pharmacy Practice Experience program. This descriptive study aimed to evaluate preceptor and student perceptions of remote learning experiences and student practice readiness upon completion of remote rotations. Preceptors and students who participated in partial to full remote experiential rotations between 17 August 2020 and 26 March 2021 were invited to complete an on-line survey. A cross-sectional survey consisted of closed-ended questions using a 5-point Likert scale assessing perception on adaptability, effectiveness of remote learning in advancing practice knowledge and skills, and confidence in students' practice readiness. A total of 29 preceptors and 43 students completed the survey (response rates of 67% and 57%, respectively). Approximately 70% of the remote rotations were practice-based, with ambulatory care representing the most frequently reported rotation by preceptors (38%) and students (28%). A high level of confidence in preceptor perception of their ability to adapt and provide effective remote experiences (average 4.28) matched with the students' high level of confidence with their preceptors' abilities (86% agree or strongly agree). Upon the completion of remote rotations, both preceptors and students felt confident in student practice readiness based on student ability to design and initiate individualized patient care plans or complete projects using evidence-based resources (79% and 86%, respectively). Most preceptors (69%) reported that students achieved the rotation objectives at the same level as students engaged in-person experiences. The limitations of remote learning included the absence of direct interactions. Overall, both preceptors and students reported achieving practice readiness with remote experiential learning experiences and felt the remote activities should be continued post-pandemic.

Clinical Outcomes of Eravacycline in Patients Treated Predominately for Carbapenem-Resistant Acinetobacter baumannii.

Forty-six patients were treated with eravacycline (ERV) for Acinetobacter baumannii infections, where 69.5% of isolates were carbapenem resistant (CRAB). Infections were primarily pulmonary (58.3%), and most patients received combination therapy (84.4%). The median (IQR) ERV duration was 6.9 days (5.1 to 11.1). Thirty-day mortality was 23.9% in the cohort and 21.9% in CRAB patients. One patient experienced an ERV-possible adverse event. IMPORTANCE Acinetobacter baumannii, particularly when carbapenem resistant (CRAB), is one of the most challenging pathogens in the health care setting. This is complicated by the fact that there is no consensus guideline regarding management of A. baumannii infections. However, the recent Infectious Diseases Society of America guidelines for treatment of resistant Gram-negative infections provided expert recommendations for CRAB management. The panel suggest using minocycline among tetracycline derivatives rather than eravacycline (ERV) until sufficient clinical data are available. Therefore, we present the largest multicenter real-world cohort in patients treated with ERV for A. baumannii, where the majority of isolates were CRAB (69.5%). Our analysis demonstrate that patients treated with ERV-based regimens achieved a 30-day mortality of 23.9% and had a low incidence of ERV-possible adverse events (2.1%). This study is important as it fills the gap in the literature regarding the use of a novel tetracycline (i.e., ERV) in the treatment of this challenging health care infection.

Effects of a urinary factor from women in early pregnancy on HIV-1, SIV and associated disease.

The effects of clinical grade crude preparations of human chorionic gonadotropin (hCG) on Kaposi's sarcoma, HIV, SIV and hematopoiesis were examined in vitro and in vivo. In contrast to previous studies, we report that the antiviral activity of hCG associated factors is not due to the native hCG heterodimer, including its purified subunits or its major degradation product, the beta-core. Using gel permeation chromatography of the clinical grade hCG and urine concentrates from pregnant women, we demonstrate that an as yet unidentified hCG associated factor (HAF) with anti-HIV, anti-SIV, anti-KS and pro-hematopoietic activities elutes as two peaks corresponding to 15-30 kDa and 2-4 kDa.

Adapting pharmacy experiential education during COVID-19: Innovating remote preceptor resources, tools, and patient care delivery beyond virtual meetings.

PURPOSE: To describe the innovative teaching practices, tools, and resources for remote learning developed by a school of pharmacy with a decentralized experiential program to empower and support preceptors in response to the coronavirus disease 2019 (COVID-19) pandemic. SUMMARY: As the pandemic has continued, there have been significant shifts in pharmacy workflow, staffing, and patient care delivery. Pharmacy students are slowly being reintegrated into these learning environments. Although preceptors are willing and eager to teach, many lack the resources, tools, and support to create remote learning experiences at their facilities. The University of the Pacific Thomas J. Long School of Pharmacy has a decentralized experiential education model in which faculty regional coordinators with clinical practices and diverse expertise are disseminated throughout California. This model allowed us to collaborate and understand preceptor needs from a local level. We created a preceptor COVID-19 guidance document, introduced innovative virtual playbooks to pivot up to 100% remote rotations, and promoted the layered learning model to integrate pharmacy residents into the remote teaching space. Communication and flexibility are key to ensure student and preceptor safety while maintaining high-quality advanced pharmacy practice experiences and preserving patient-student relationships in telehealth. CONCLUSION: Overall, we successfully created innovative solutions and leveraged our decentralized experiential model to meet the teaching and learning demands during an unanticipated crisis. We continue to adapt and plan to assess the effectiveness of the tools by administering surveys of preceptors and pharmacy students.

HIV Viral Rebound Due to a Possible Drug-Drug Interaction between Elvitegravir/Cobicistat/Emtricitabine/Tenofovir Alafenamide and Calcium-Containing Products: Report of 2 Cases.

Elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide (E/C/F/TAF) is a potent fixed-dose, once-daily regimen for HIV-1 treatment and has rare emergence of drug resistance. We report a potential drug-drug interaction in 2 female patients both receiving treatment for HIV and cerebral toxoplasmosis: one case between E/C/F/TAF with calcium carbonate and a second case involving leucovorin as calcium salt. Both cases resulted in rise in HIV RNA levels and emergence of M184 V mutation and resistance to elvitegravir and raltegravir. To the best of our knowledge, these 2 cases are the first reports of rapid emergence of mutation from coadministration of E/C/F/TAF and calcium.

Role of rapid diagnostics for viral respiratory infections in antibiotic prescribing decision in the emergency department.

OBJECTIVE: To describe the frequency of antibiotic prescriptions in patients with known viral respiratory infections (VRIs) diagnosed by polymerase chain reaction (PCR) in 3 emergency departments (EDs) and to identify patient characteristics that influence the prescribing of antibiotics by ED physicians despite PCR confirmation of viral cause. DESIGN: Retrospective, observational analysis of patients with PCR-diagnosed VRI discharged from 3 acute-care hospital EDs within 1 health system. RESULTS: In total, 323 patients were discharged from the ED with a VRI diagnosis, of whom 68 were prescribed antibiotics (21.1%). These patients were older (median, 59.5 vs 43 years; P = .04), experienced symptoms longer (median, 4 vs 2 days; P = .002), were more likely to have received antibiotics in the preceding 7 days (27.9% vs 9.8%; P < .001), and had higher proportions of abnormal chest X-rays (64.5% vs 28.4%; P < .001). Patients were more likely to receive antibiotics for a diagnosis of pneumonia (39.7% vs 1.6%; P < .001) or otitis media (7.4% vs 0.4%; P = .002), and were less likely with diagnosis of upper respiratory infection (2.9% vs 13.7%; P = .02) or influenza (20.6% vs 44.3%; P < .001). CONCLUSIONS: Despite a diagnosis of VRI, one-fifth of ED patients were prescribed antibiotics. Patient characteristics including age, duration of symptoms, abnormal chest X-rays, and specific diagnosis may increase provider concern for concurrent bacterial infections. Opportunities exist for antimicrobial stewardship strategies to incorporate rapid diagnostics in promoting judicious antibiotic usage in the ED.

Amyloid fibril protein in familial amyloidotic polyneuropathy, Portuguese type. Definition of molecular abnormality in transthyretin (prealbumin).

Amyloid fibril protein in patients with familial amyloidotic polyneuropathy is known to be chemically related to transthyretin (TTR), the plasma protein that is usually referred to as prealbumin. A genetically abnormal TTR may be involved in this disease. Studies were conducted on amyloid fibril protein (AFp) isolated from tissues of two Portuguese patients who died with familial amyloidosis, and on TTR isolated from sera of patients with this disease. AFp, purified by affinity chromatography on retinol-binding protein linked to Sepharose, resembled plasma TTR in forming a stable tetrameric structure, and in its binding affinities for both thyroxine and retinol-binding protein. The structural studies included: (a) comparative peptide mappings by reverse-phase high performance liquid chromatography (HPLC) after trypsin digestion; (b) cyanogen bromide cleavage studies; and (c) amino acid microsequence analysis of selected tryptic and CNBr peptides. On the basis of the known amino acid sequence of TTR, comparative tryptic peptide maps showed the presence of a single aberrant tryptic peptide (peptide 4, residues 22-34) in AFp as compared with TTR. This aberrant peptide contained a methionine residue, not present in normal tryptic peptide 4. CNBr cleavage of AFp produced two extra peptide fragments, which were demonstrated, respectively, by HPLC analysis and by sodium dodecyl sulfate-gel electrophoresis. Sequence analyses indicated the presence of a methionine-for-valine substitution at position 30 in AFp as compared with TTR. Thus, the purified amyloid fibril protein comprised a TTR variant with a methionine-forvaline substitution at position 30. A single nucleotide change in a possible codon for valine 30 could explain the substitution. The variant TTR was also present in the TTR isolated from the pooled sera of amyloidoses patients, together with larger (four- to six-fold) amounts of the normal TTR. Thus, in these patients, the variant TTR was circulating in plasma, along with larger amounts of normal TTR. We suggest that the variant TTR represents the specific biochemical cause of the disease, and that this abnormal form of TTR selectively deposits in tissues as the amyloid characteristic of the disease.

Carboxyterminal peptide fragments of the beta subunit are urinary products of the metabolism of desialylated human choriogonadotropin.

Previous investigations of patients with gestational trophoblastic neoplasia have shown that their urines often contain carboxyterminal peptide (CTP) fragments of the choriogonadotropin (hCG) beta-subunit as well as forms of hCG deficient in sialic acid. In order to determine whether beta-CTP fragments are among the urinary products of the peripheral degradation of desialylated hCG (as-hCG), using a continuous infusion technique, we gave highly purified as-hCG to humans. Six healthy subjects were given a loading dose of 0.8 mg of as-hCG followed by an infusion of the same preparation. An overall mean infusion rate of 62.9 micrograms/min was maintained for 6 h, and the mean serum concentration of as-hCG achieved during the infusion was 72.1 ng/ml. In all six subjects, beta-CTP fragments were the predominant immunoreactive forms of as-hCG in urine obtained during the infusion. In contrast, the urine of subjects infused with hCG has been shown to contain hCG itself, but nil beta-CTP fragments or as-hCG. After the as-hCG infusion was stopped, the excretion of the beta-CTP fragments in urine declined rapidly. There were no beta-CTP fragments detectable in sera obtained during the infusion or in sera incubated with as-hCG at 37 degrees C. After incubation with as-hCG for 4 h, the urine of normal subjects contained small amounts of beta-CTP fragments; however, the apparent proteolytic activity was too low to account for either the quantity of beta-CTP fragments produced during the infusion or the extremely low levels of as-hCG in the urine. These data demonstrate the existence in humans of a peripheral metabolic pathway that cleaves beta-CTP fragments from as-hCG and allows their excretion in urine. Thus, the frequent presence of beta-CTP fragments in the urines of patients with gestational trophoblastic neoplasia can be accounted for in part by the metabolism of the forms of hCG that bear an altered carbohydrate structure, which are prevalent in this disease.

Characterization of a carboxyterminal peptide fragment of the human choriogonadotropin beta-subunit excreted in the urine of a woman with choriocarcinoma.

We have observed low-molecular weight carboxyterminal fragments of the human choriogonadotropin (hCG) beta-subunit in the urines of several women with choriocarcinoma, and we have characterized one fragment in detail. Its apparent molecular weight by gel chromatography on Sephadex G-100 was 14,200. The fragment was not adsorbed to concanavalin A-Sepharose, indicating that it lacked the asparagine-linked carbohydrate groups of intact hCG beta. It was active in radioimmunoassays (RIA) using antisera either to the hCG beta carboxyterminal peptide (CTP) or to the desialylated hCG beta CTP (hCG beta as-CTP), indicating the presence of not only the hCG beta carboxyterminus but also desialylated O-serine-linked carbohydrate side chains on the fragment. It lacked luteinizing hormone/choriogonadotropin radioreceptor activity and hCG beta conformational immunoreactivity (SB6 RIA). On Sephadex G-100 gel chromatography, the elution profiles of this fragment and the hCG beta as-CTP(115-145) prepared by trypsin digestion of as-hCG were essentially indistinguishable (apparent molecular weights 14,200 and 14,000, respectively). The immunological characteristics of the fragment in both hCG beta CTP and hCG beta as-CTP RIA were indistinguishable from those of the hCG beta as-CTP(115-145) glycopeptide. Carboxyterminal fragments of hCG beta were evident in urine specimens obtained from 10 of 11 patients with choriocarcinoma but not in those obtained from normal subjects who were given an intravenous infusion of highly purified hCG. Of six pregnant women, only the one at term excreted carboxyterminal fragments of hCG beta and then only in trace amounts. We conclude that hCG beta carboxyterminal fragments, including one that is indistinguishable from the tryptic glycopeptide hCG beta as-CTP(115-145), can occur naturally in the urine of patients with choriocarcinoma.

Development of an assay for in vivo human neutrophil elastase activity. Increased elastase activity in patients with alpha 1-proteinase inhibitor deficiency.

Leukocyte extracts contain enzymes that digest fibrinogen and release a fibrinopeptide A-containing fragment. This study was undertaken to identify the responsible proteinase and to characterize the fibrinopeptide A-containing fragment so that it could be used as an index of enzyme activity. Both the fibrinogenolytic activity and the release of the fibrinopeptide A-containing fragment mediated by the leukocyte extracts were shown to be due to human neutrophil elastase (HNE) by the following criteria: activity was completely blocked by a specific HNE inhibitor or by adsorbing HNE from the extracts with a monospecific antibody and reconstitution with purified HNE restored the ability to release the fibrinopeptide A-containing fragment. This fragment was not released by a variety of other proteinases or by HNE-inhibitor complexes indicating that, at least with respect to the enzymes tested, it is a specific product of HNE and its release requires the free enzyme. By separating the products of HNE digestion of fibrinogen using high performance liquid chromatography, identifying the immunoreactive fractions and subjecting them to amino acid analysis, the fragment was identified as A alpha 1-21, indicating an HNE cleavage site at the Val(A alpha 21)-Glu(A alpha 22) bond. The mean plasma A alpha 1-21 level was markedly higher in patients with alpha 1-proteinase inhibitor deficiency as compared to healthy controls (0.2 nM vs. 7.9 nM; P less than 0.0001), consistent with increased in vivo HNE activity in these individuals.

Mycobacterium szulgai in a patient with advanced acquired immunodeficiency syndrome: an unusual pathogen with unusual multidrug resistance.

A 37-year-old Hispanic man with advanced acquired immunodeficiency syndrome developed extensive pulmonary disease with persistent cough, fever, night sweats, worsening dyspnea, and weight loss. Sputum samples showed scant growth of acid-fast bacilli. He failed to respond to the standard tuberculosis regimen of isoniazid, rifampin, ethambutol, and pyrazinamide. Subsequently, Mycobacterium szulgai was identified, and susceptibility tests showed it to be resistant to all four of those agents. Therapy was changed to clarithromycin, doxycycline, ciprofloxacin, and amikacin. Within 2 weeks, the patient's condition improved significantly, and 6 months after treatment, extensive pulmonary infiltrates had nearly resolved. Fewer than 1% of all human isolates of mycobacteria consist of M. szulgai, which is relatively susceptible to standard antimycobacterial agents. To our knowledge, this is the first reported case of M. szulgai with resistance to all primary antituberculosis drugs.

Development of highly sensitive immunoassays to measure human chorionic gonadotropin, its beta-subunit, and beta core fragment in the urine: application to malignancies.

A variety of malignancies have been associated with the presence of human chorionic gonadotropin, hCG, its subunits, and fragments of its beta-subunit in blood and urine. The usefulness of these hCG-related tumor markers in nontrophoblastic malignancies has been inhibited by inadequate assay techniques. In order to achieve the required sensitivity and specificity, concentration steps and other procedures to remove cross-reacting human luteinizing hormone were necessary. In addition, the coexistence of a fragment of the hCG-beta or beta human luteinizing hormone subunit contributes to significant errors of measurement in urine. The importance of the hCG-beta fragment as a potential tumor marker has been recognized previously but no method was available to measure this antigen readily. We report here the development of a series of radioimmunometric, two-site assays which will accurately measure hCG, hCG-beta subunit, and the beta-subunit fragment directly in small volumes of unprocessed urine. These assays are highly specific, extremely sensitive, and not labor intensive since they employ microtiter plate procedures. Application of these assays to urine samples from patients with gynecological malignancies indicated that over 50% of all patients tested excreted the hCG-beta fragment in their urine. Also, this fragment comprised more than 50% of the moles of hCG immunoreactive components present in the specimens that were positive for hCG. This cancer marker is also demonstrable in trophoblastic malignant states such as choriocarcinoma in which the low molecular weight fragment can also be visualized directly by immunoblotting procedures. We conclude that a search for hCG immunoreactivity in the urine of patients with malignancies will be improved by the inclusion of accurate measurements of the prominent quantities of the beta fragment excreted by these individuals.

Origin and occurrence of human chorionic gonadotropin beta-subunit core fragment.

Human chorionic gonadotropin (hCG) beta-subunit core fragment (beta-fragment) is present in the urine of pregnant individuals as well as those with trophoblast disease and certain other cancers at concentrations 0.8 (early pregnancy) to 7 (second trimester pregnancy)-fold greater than that of hCG. The core fragment may be directly secreted by trophoblast tissue into the circulation or possibly originates from peripheral degradation of circulating hormone by the kidney. We examined the former hypothesis. We examined 24-h organ cultures of trophoblast tissue from first, second, and third trimester pregnancy. The media from this tissue contained hCG, free beta-subunit, and beta-fragment. The amount of beta-fragment present exceeded that of hCG, as was observed in second and third trimester pregnancy urine. The beta-fragment immunoreactive material produced by trophoblast tissue was compared to a standard preparation of urinary beta-fragment. The material in medium was identical to the standard beta-fragment in its elution pattern from a gel filtration column, from a reverse-phase HPLC column, from an ion-exchange gel, and from an immobilized lectin affinity column, and also by electrophoresis and immunoblotting with fragment-reactive monoclonal antibodies. We conclude that beta-fragment can also originate directly from trophoblast tissue, and could be the principal hCG beta-immunoreactive molecule secreted.

The heterogeneity of human chorionic gonadotropin (hCG). II. Characteristics and origins of nicks in hCG reference standards.

hCG, the hormone produced by the trophoblast throughout pregnancy, has peptide bond cleavages, or nicks, in the beta-subunit. We sought to compare the nature of these nicks in standard reference preparations of hCG, to determine the enzymes that may be responsible for generating the peptide bond cleavages, and to devise means of separating nicked from intact hormone. The standard reference preparations of hCG, which are purified from a commercial product made from large pools of pregnancy urine, were found to have varying concentrations of nicked hormone. The preceding report showed that 11 of 13 hCG preparations isolated from individual pregnancy urine samples were nicked at the beta 47-48 bond, with 2 of 13 having a second nick at beta 44-45. As shown here, all of the hCG reference standards are nicked to similar extents at both the beta 47-48 bond and the beta 44-45 bond. The percentage of peptide bond nicking in the various hCG standard preparations ranged from 10-20% and appeared higher in the more recent preparations. We showed that human leukocyte elastase is capable of specifically cleaving the beta 44-45 bond, and in extended digests it can also cleave the beta 48-49 and beta 51-52 peptide bonds. Thus, human leukocyte elastase may be the origin of some of these cleavages in the individual samples and the reference standards. Furthermore, we report that a monoclonal antibody directed to hCG alpha-beta dimer binds preferentially to nonnicked hCG and much less to nicked hCG.

High resolution high performance liquid chromatography fingerprinting of purified human chorionic gonadotropin demonstrates that oxidation is a cause of hormone heterogeneity.

To characterize the structures of the various forms of hCG and its immunologically related fragments measured in blood, urine, and tissue culture media, we developed a highly sensitive HPLC tryptic fingerprinting system that can characterize as little as 15 micrograms of material. Standard preparations of the subunits of hCG purified from pregnancy urine were found to exhibit heterogeneity on reverse phase HPLC chromatography, complicating efforts to characterize such forms of hCG. The same type of chromatographic heterogeneity was observed when the hormone was reduced and S-carboxymethylated (RCM) and its RCM subunits separated on HPLC. One major and one minor peak were observed in the alpha- and beta-subunits from both native and RCM hormone, with a ratio of between 5:1 and 10:1. Development and application of a high resolution, semimicrobore HPLC fingerprint technique used together with an examination of selected peptides by amino acid sequencing and mass spectrometry defined the differences between these forms as oxidation. Using each of the isolated RCM subunit's major and minor HPLC peaks, the oxidation was shown to be reversible and probably due to interconversion of methionine with its sulfoxide form.