Immunoaffinity extraction of morphine, morphine-3-glucuronide and morphine-6-glucuronide from blood of heroin victims for simultaneous high-performance liquid chromatographic determination

The development of an immunoaffinity-based extraction method for the determination of morphine and its glucuronides in human blood is described. For the preparation of an immunoadsorber, specific antisera (polyclonal, host: rabbit) against morphine, morphine-3-glucuronide and morphine-6-glucuronide were coupled to 1,1′-carbonyldiimidazole-activated trisacrylgel and used for immunoaffinity extraction of morphine and its glucuronides from coronary blood. The resulting extracts were analysed by HPLC with native fluorescence detection. The mean recoveries from spiked blood samples were 71%, 76% and 88% for morphine, morphine-3-glucuronide and morphine-6-glucuronide, respectively. The limit of detection was 3 ng/g blood and the limit of quantitation was 10 ng/g blood for all three analytes. The results of the analysis of coronary blood samples from 23 fatalities due to heroin are presented.     

Analgesic responses to intrathecal morphine in relation to CSF concentrations of morphine-3,beta-glucuronide and morphine-6,beta-glucuronide.

This study was performed to determine whether variations in analgesic responses to intrathecal morphine could be explained by cerebrospinal fluid (CSF) concentrations of morphine metabolites. Twenty-four CSF samples were collected at the beginning, middle and end of treatment periods in seven cancer patients with pain of malignant origin. CSF concentrations of morphine-3,beta-glucuronide (M3G) and morphine-6,beta-glucuronide (M6G) metabolites were measured by gas chromatography/mass spectrometry. Analgesic responses to morphine were estimated concurrent with CSF collection using a visual analog scale representing percentages of pain relief. Effective analgesia was defined as > or = 75% pain relief. CSF concentration of M3G and M6G in the 24 samples were 722 +/- 116 ng/ml and 699 +/- 158 ng/ml, respectively. CSF samples were categorized into two groups: (1) those collected during effective analgesia (N=14), and (2) those collected during ineffective analgesia (N=10). M6G levels detected in group 1 samples (effective analgesia) were significantly greater than those found in group 2 samples (ineffective analgesia) (978 +/- 243 ng/ml vs 309 +/- 68 ng/ml, P<0.05). Intergroup differences in CSF M3G concentrations and M3G/M6G ratios were not significant. It is concluded that CSF M6G may be indicative of effectiveness of analgesia in cancer patients subjected to intrathecal morphine.     

Simultaneous determination of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone concentrations in rat serum by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry

An assay using high performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry (ESI-MS-MS) was developed for simultaneously determining concentrations of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone, in 50 microl samples of rat serum. Deuterated (d(3)) analogues of each compound were used as internal standards. Samples were treated with acetonitrile to precipitate plasma proteins; acetonitrile was removed from the supernatant by centrifugal evaporation before analysis. Limits of quantitation (ng/ml) and their between-day accuracy and precision (%deviation and %CV) were--morphine, 3.8 (4.3% and 7.6%); morphine-3-glucuronide, 5.0 (4.5% and 2.9%); oxycodone, 4.5 (0.4% and 9.3%); noroxycodone, 5.0 (8.5% and 4.6%).     

Simultaneous assay of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma using normal-phase liquid chromatography–tandem mass spectrometry with a silica column and an aqueous organic mobile phase

Morphine (MOR) is an opioid analgesic used for the treatment of moderate to severe pain. MOR is extensively metabolized to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). A rapid and sensitive method that was able to reliably detect at least 0.5 ng/ml of MOR and 1.0 ng/ml of M6G was required to define their pharmacokinetic profiles. An LC–MS–MS method was developed in our laboratory to quantify all three analytes with the required sensitivity and a rapid turnaround time. A solid-phase extraction (SPE) was used to isolate MOR, M3G, M6G, and their corresponding deuterated internal standards from heparinized plasma. The extract was injected on a LC tandem mass spectrometer with a turbo ion-spray interface. Baseline chromatographic separation among MOR, M3G, and M6G peaks was achieved on a silica column with an aqueous organic mobile phase consisting of formic acid, water, and acetonitrile. The total chromatographic run time was 3 min per injection, with retention times of 1.5, 1.9 and 2.4 min for MOR, M6G, and M3G, respectively. Chromatographic separation of M3G and M6G from MOR was paramount in establishing the LC–MS–MS method selectivity because of fragmentation of M3G and M6G to MOR at the LC–MS interface. The standard curve range in plasma was 0.5–50 ng/ml for MOR, 1.0–100 ng/ml for M6G, and 10–1000 ng/ml for M3G. The inter-day precision and accuracy of the quality control (QC) samples were <7% relative standard deviation (RSD) and <6% relative error (R.E.) for MOR, <9% RSD and <5% R.E. for M6G, and <3% RSD and <6% R.E. for M3G. Analyte stability during sample processing and storage were established. Method ruggedness was demonstrated by the reproducible performance from multiple analysts using several LC–MS–MS systems to analyze over one thousand samples from clinical trials.     

Comparative measurement of spinal CSF microdialysate concentrations and concomitant antinociception of morphine and morphine-6beta-glucuronide in rats

Morphine-6beta-glucuronide (M6G) is well known as a potent active metabolite in humans. To clarify concentration-antinociceptive effect relationships for morphine and M6G, we evaluated comparatively the pharmacokinetics and antinociceptive effects of morphine and M6G. The spinal CSF concentration and antinociception were simultaneously measured by using the combination of a microdialysis method and the formalin test in conscious rats after the s.c. administration of morphine (0.3-3 mg/kg) and M6G (0.1-3 mg/kg). The plasma concentration of M6G after s.c. administration was higher than that of morphine, as shown by the 2.1 times greater value of area under the concentration-time curve (AUC(plasma)). The spinal CSF concentrations of morphine and M6G increased dose-dependently. The AUC(CSF) of M6G was 1.6-1.8 times higher than that of morphine at each dose. Administration of morphine and M6G dose-dependently suppressed the flinching behavior induced by formalin injection. The ED(50) values for M6G were 3 times lower than those of morphine, although the spinal CSF concentration versus antinociceptive effect curves of morphine and M6G were very similar, with similar EC(50) values. These results suggest that the antinociceptive potencies of morphine and M6G, evaluated by simultaneous measurements of spinal CSF drug concentration and antinociception, are equivalent. Simultaneous measurement of spinal CSF concentration and antinociception by using microdialysis should be useful for elucidating the relationship between pharmacokinetics and pharmacodynamics of various opioids.     

Radioimmunoassay of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol 3-glucuronide in serum of patients with cerebrotendinous xanthomatosis.

A rapid, convenient, and specific radioimmunoassay for 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol has been developed. Specific antiserum was obtained from rabbits immunized by the bile alcohol-bovine serum albumin conjugate, which was coupled by an (O-carboxymethyl)oxime bridge at the C-3 position. The assay produces values for serum concentrations of bile alcohol glucuronides in patients with cerebrotendinous xanthomatosis.     

Analysis of serum copper and zinc concentrations in cancer patients

Several studies have shown that plasma copper concentrations are increased in various carcinomas. Zinc acts as a cellular growth protector, including growth of neoplastic cells, and its deficiency was demonstrated to be involved in several stages of malignant transformation. However, the usefulness of the serum zinc and copper determinations in cancer prevention, detection, monitoring treatment, and prognosis requires further investigations. The aim of the present study was to compare the serum copper and zinc levels in patients with cancer of the lung (PC), breast (BC), gastrointestinal tract (GIC), and gynecological (GYNC) malignancy with progress of the disease. The results of the study have shown a significant increase in the mean total serum Cu levels and the serum Cu/Zn ratio in all patient groups with cancer compared to a control group. Increased mean serum concentrations and Cu/Zn ratios were found in the whole group (ALLC), and for the GIC and GYNC groups with local as well as metastasized (Meta) disease in comparison with the control group. The mean serum concentrations of Zn were decreased only in metastasized ALLC and GYNC groups.     

rT3 production in normal man, assessed from variations in serum rT3 during short-term rT3 infusion.

The metabolic clearance rate (MCR) of 3,3'5'-triiodothyronine (reverse T3, rT3) was estimated in normal human subjects by a modified noncompartmental method using the integrated increase in serum rT3 following intravenous infusion of 0.10 nmol/min rT3 for 4 hr. The MCR-rT3 was calculated to be 102.8 +/- 17.01/day and the daily rT3 disposal to be 33.0 +/- 9.5 nmol (mean +/- SD, n = 6). The MCR-rT3 compares well to that of previous studies employing tracer kinetic methods. The disposal rate of rT3 estimated in the present study is considerably lower than found in some previous studies. The discrepancy is due to differences in the measured levels of serum rT3 in normal subjects.     

Blood concentrations of interleukin-15 in cancer patients and their variations during interleukin-2 immunotherapy: preliminary considerations

In addition to the better known cytokines IL-2 and IL-12, IL-15, which is mainly produced by macrophages, is a new antitumor cytokine with a mechanism of action similar to that of IL-2. At present, however, there are no data about IL-15 secretion in cancer patients. This study was carried out to evaluate IL-15 blood concentrations in patients with early or advanced cancer and their possible variations in response to IL-2 cancer immunotherapy. The study included 40 patients with solid tumors, 24 of whom had metastatic disease. In addition, IL-15 secretion was evaluated during subcutaneous low-dose IL-2 therapy (6 million IU/day for 6 days/week for 4 weeks) in 14 metastatic renal cell cancer patients by collecting blood samples at weekly intervals. The control group consisted of 40 age-matched healthy subjects. Serum levels of IL-15 were measured by an enzyme immunoassay. No significant difference in mean serum levels of IL-15 was observed between cancer patients and controls. Moreover, the mean serum levels of IL-15 found in metastatic cancer patients were not significantly different from those found in patients with limited disease. Finally, no significant changes in mean levels of IL-15 occurred during IL-2 cancer immunotherapy. This preliminary study would suggest that IL-15 secretion is substantially within the normal range in cancer patients, both in early and advanced disease, and no variation seems to occur in response to IL-2 administration.     

One-year spinal bone change in pre- and perimenopausal Japanese women. A prospective observational study

BACKGROUND: This prospective observational study was designed to determine whether the bone mineral density (BMD) of the lumbar spine decreases before menopause. METHODS: The change in BMD of the second through fourth lumbar vertebrae (delta%L2-4BMD) over the course of 12 months was measured in 197 pre- and perimenopausal Japanese women aged 48.2 +/- 2.3 (mean +/- SD) years. RESULTS: Overall, delta%L2-4BMD decreased significantly, with a greater decrease seen in perimenopausal women. This group also had a significantly higher level of FSH (p < 0.05, t = 7.356), a significantly lower level of estradiol (p < 0.05, t = 4.245), and significantly higher levels of the bone metabolic markers, alkaline phosphatase (p < 0.05, t = 3.841), calcium (p < 0.05, t = 3.939), and osteocalcin (p < 0.05, t = -3.295). Overall, there was a significant positive correlation between osteocalcin and delta%L2-4BMD (r = -0.194, p = 0.0479). CONCLUSION: A subset of perimenopausal women with transient decreases in ovarian function that do not respond to increased FSH may be at increased risk for abnormally low BMD, and may benefit from early management of bone mass.     

Compliance with bladder protocol during concurrent chemoradiation for cancer of the cervix and its impact on enteritis: A prospective observational study

Aim

This prospective study aims to assess the compliance with bladder protocol and the correlation with enteritis during pelvic radiation.

Risk of cancer in relation to serum concentrations of selenium and vitamins A and E: matched case-control analysis of prospective data.

The independent and joint associations of serum selenium and vitamin A (retinol) and E (alpha tocopherol) concentrations with the risk of death from cancer were studied in 51 case-control pairs--that is, 51 patients with cancer, each paired with a control matched for age, sex, and smoking. Case-control pairs came from a random sample of some 12000 people aged 30-64 years resident in two provinces of eastern Finland who were followed up for four years. Patients who died of cancer during the follow up period had a 12% lower mean serum selenium concentration (p = 0.015) than the controls. The difference persisted when deaths from cancer in the first follow up year were excluded. The adjusted risk of fatal cancer was 5.8-fold (95% confidence interval 1.2-29.0) among subjects in the lowest tertile of selenium concentrations compared with those with higher values. Subjects with both low selenium and low alpha tocopherol concentrations in serum had an 11.4-fold adjusted risk. Among smoking men with cancer serum retinol concentrations were 26% lower than in smoking controls (p = 0.002). These data suggest that dietary selenium deficiency is associated with an increased risk of fatal cancer, that low vitamin E intake may enhance this effect, and that decreased vitamin or provitamin A intake contributes to the risk of lung cancer among smoking men with a low selenium intake.     

Oxidase activity of ceruloplasmin and concentrations of copper and zinc in serum of cancer patients.

A balance between oxidant carcinogens and endogenous antioxidant defence is of particular relevance to the carcinogenesis. Ceruloplasmin (Cp) carries up to 90% of Cu in plasma and performs ferroxidase, antioxidant and amine oxidase activity. Cu and Zn, as trace elements, have been recognized to play an important role as cofactors of SOD. The study presents the relationship of the Cp oxidase activity and concentrations of Cu and Zn in serum of 62 patients with breast (BCA), lung (LCA), gastrointestinal (GICA) and gynecological (GYNCA) cancer. The Cp oxidase activity was determined in serum with o-dianisidine as a substrate. Cu and Zn concentrations in serum were measured by using atomic absorption spectrometry. The results of the study have shown significant increase in the mean serum Cp oxidase activity and total Cu concentrations in all patient groups compared with the control one. The total mean serum Zn concentration was found to be decreased only in LCA group as compared with the control. The effect of the cancer progress on the Cp oxidase activity and concentrations of Cu and Zn was observed within the group of all cancer patients (ALLCA) and within the GICA group. The only significant difference in Cu concentrations among various stages of the disease was observed in GICA between local and distant one. Significant positive correlation coefficients were caLculated for the Cp activity and Cu concentrations in the control group and all patients groups, also according to the cancer progress. Future research is needed to evaLuate the consequences of the elevation of the serum Cp oxidase activity and concentration of Cp, Cu and Zn for the host antioxidant-oxidant balance.     

A prospective observational study of community-acquired bacterial bloodstream infections in Metro Manila,the Philippines

Community-acquired bacterial bloodstream infections are caused by diverse pathogens with changing antimicrobial-resistance patterns. In low-middle income countries in Southeast Asia, where dengue fever is endemic and a leading cause of fever, limited information is available about bacterial bloodstream infections due to challenges of implementing a blood culture service. This study describes bacterial bloodstream pathogens and antimicrobial-resistance patterns in Metro Manila, the Philippines. We aimed to identify the proportion of patients with a positive blood culture, the bacteria isolated and their antimicrobial resistance patterns, and the clinical characteristics of these patients, in this dengue endemic area. We conducted a prospective observational study in a single hospital enrolling febrile patients clinically suspected of having a community-acquired bacterial bloodstream infection between 1^st July 2015 and 30^th June 2019. Each patient had a blood culture and additional diagnostic tests according to their clinical presentation. We enrolled 1315 patients and a significant positive blood culture was found in 77 (5.9%) including Staphylococcus aureus (n = 20), Salmonella enterica Typhi (n = 18), Escherichia coli (n = 16), Streptococcus pneumoniae (n = 3) and Burkholderia pseudomallei (n = 2). Thirty-four patients had meningococcal disease diagnosed by culture (n = 8) or blood PCR (n = 26). Additional confirmed diagnoses included leptospirosis (n = 177), dengue virus infection (n = 159) and respiratory diphtheria (n = 50). There were 79 (6.0%, 95%CI 4.8%−7.4%) patients who died within 28 days of enrollment. Patients with a positive blood culture were significantly more likely to die than patients with negative culture (15.2% vs 4.4%, P<0.01). Among S. aureus isolates, 11/20 (55%) were methicillin-resistant (MRSA) and ST30: USA1100 was dominant sequence type (88.9%). Antimicrobial-susceptibility was well preserved in S. enterica Typhi. Among hospitalized patients with clinically suspected community-acquired bacterial bloodstream infection in Metro Manila, the Philippines, 5.9% had a blood culture confirmed infection of whom 15.6% died. S. aureus, including a significant number of MRSA (USA1100 clones), S. enterica Typhi, E.coli and Neisseria meningitidis were frequently identified pathogens.     

Levels of serum pentraxin 3, IL-6, fetuin A and insulin in patients with rheumatoid arthritis

ObjectiveThe aim of this study was to investigate the relationship between disease severity and biochemical parameters such as pentraxin-3, fetuin-A, IL-6, insulin and HOMA-IR levels in patients with rheumatoid arthritis.MethodsThis study included 60 patients with RA and 20 healthy controls. Serum pentraxin-3, fetuin-A, IL-6 and insulin concentrations were measured. Also, HOMA-IR values were calculated. Disease activity was assessed with Disease Activity Score (DAS28). To evaluate quality of life, the Health Assessment Questionnaire disability index was applied.ResultsThe serum values for ESR, CRP, pentraxin-3 and fetuin-A in patients with RA were found to be higher than control subjects (p values = 0.001, 0.001, 0.000, 0.000, 0.01, 0.02, respectively). A positive correlation was evident between the DAS 28 score and IL6 levels (r = 0.263, p = 0.045). We found no correlation between the DAS28 score and HOMA-IR, the levels of pentraxin 3, fetuin A, insulin (p < 0.05). Fetuin A levels were positively correlated with cumulative steroid dose (r = 0.382, p = 0.035). A statistically significant correlation was evident between presence of cardiovascular disease and HOMA-IR values in RA patients (r = 0.437, p = 0.032).ConclusionElevated levels of pentraxin-3, fetuin-A, CRP, ESR might play a role in the pathogenesis of RA. Levels of fetuin-A, insulin HOMA-IR, pentraxin-3, CRP and ESR were not associated with clinical severity of the RA.