Evaluation of inosin-5'-monophosphate dehydrogenase activity during maintenance therapy with tacrolimus.

OBJECTIVES: The aim of this study was to identify a target range for inosin-5'-monophosphate dehydrogenase (IMPDH) activity in maintenance therapy with tacrolimus (TCL), and to apply the measurement of IMPDH activity to the therapeutic drug monitoring for mycophenolate mofetil (MMF). METHODS: Eleven patients with renal transplants and 10 healthy volunteers were investigated. All patients were treated with a combination of TCL, steroid and MMF for 2 months after transplantation, and were in stable and good condition. IMPDH activity was determined indirectly by measuring xanthosine 5'-monophophate in cell lysates supplemented with IMP and beta-nicotine adenine dinucleotide using an high-performance liquid chromatography (HPLC) method. RESULTS: The within-run reproducibility of the assay was excellent, with relative standard deviation (RSD) values of 0.41-4.08%. The mean differences between the spiked concentrations of xanthosine 5'-monophophate and their real values (mean relative errors; MREs) were within a range of 2.66-8.89%, showing good accuracy. The interday RSD values were 1.51-6.12% and MREs ranged from 2.10% to 8.89%. Cell lysates showed a 5-6 nmol/L IC(50) mycophenolic acid (MPA) concentration. TCL, cyclosporine and prednisolone did not affect IMPDH activity. The peak MPA concentration was achieved at 1 h after dosing. IMPDH activity decreased to 75% and 67% at 1 and 2 h after dosing respectively. Therefore, the inhibition rates of MPA against IMPDH activity may be adequate at 25-40% in TCL maintenance therapy. CONCLUSION: Inosin-5'-monophosphate dehydrogenase activity in cell lysates could be reliably determined by HPLC. A 25-40% inhibition of IMPDH activity may be an appropriate range for preventing rejection with MPF but this requires further validation using larger studies with harder outcomes such as rejection episodes.

Radical scavenging activity of bisbenzylisoquinoline alkaloids and traditional prophylactics against chemotherapy-induced oral mucositis.

BACKGROUND AND OBJECTIVE: Oral mucositis is a major severe toxic side-effect of systemic chemotherapy and irradiation in patients with cancer. Various free radical scavengers have been shown to prevent chemotherapy-induced skin necrosis. The objective of this study was to determine the antioxidant activity of a bisbenzylisoquinoline alkaloidal compound (BIQAC) and a series of chemicals, including allopurinol, used clinically for the treatment of chemotherapy-induced mucositis. METHODS: Allopurinol, melatonin, camostat mesilate, gabexate mesilate, hydroquinone and BIQAC were tested for their radical scavenging activities on four different radical species: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) cation radical (ABTS(*+)) using standard methods, and superoxide anion radical (O(2) (-)) and hydroxyl radical (OH(*)) using electron spin resonance. RESULTS: Allopurinol had radical scavenging activity against O(2) (-) only. Melatonin had strong radical scavenging activity against ABTS(*+), and weak activity against DPPH radical and OH(*). Camostat mesilate had weak radical scavenging activity against OH(*). Gabexate mesilate had no radical scavenging activity against any of these radicals. Hydroquinone had strong radical scavenging activity against DPPH radical and ABTS(*+), and moderate activity against both O(2) (-) and OH(*). BIQAC had moderate radical scavenging activity against DPPH radical, strong radical scavenging activity against ABTS(*+) and O(2) (-), and weak activity against OH(*). CONCLUSION: The BIQAC had the most braod-spectrum radical scavenging activity, suggesting that it may be effective against chemotherapy-induced mucositis. These findings also suggest that this radical-scavenging activity screening method, against four kinds of radicals, may be useful for the screening of radical scavenging activity of new natural and synthetic chemicals.

Relationship between oral mucositis and high-dose methotrexate therapy in pediatric acute lymphoblastic leukemia.

OBJECTIVE: Oral mucositis is a major toxicity in the high-dose methotrexate (HD-MTX) treatment for children with acute lymphoblastic leukemia (ALL). The first aim of this study was to evaluate the relationship between the MTX serum concentration and occurrence of oral mucositis in pediatric ALL patients. The second aim was to clarify the relationship between MTX exposure and epidermal keratinocyte cell injury using an in vitro study. METHODS: 49 patients were treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL-HR02 protocol. This protocol involves HD-MTX treatment (3 g/m2 for 24-h i.v. infusion). The MTX serum concentrations were measured by a fluorescence polarization immunoassay. The relationship between oral mucositis and MTX serum concentrations 48 and 72 h after administration was determined. The cell toxicity of MTX for human epidermal keratinocytes was analyzed by using a cell viability assay (WST-1 assay). In addition, pharmacokinetic evaluation for clearance, AUC extrapolated from 48 h to infinity (AUC48h-inf) and elimination half-life (t1/2b) were done using the 1-compartmental models. RESULTS: Oral mucositis occurred in 24 patients (49.0%), in whom 20 patients (83.3% in oral mucositis group) showed WHO severity Grade 1 or 2. Only 4 patients (16.7% in oral mucositis group) showed Grade 3 severity. 22 patients (44.9%) had oral mucositis in the group with a concentration under 10-6 M 48 h after MTX administration. There was no significant deference among the cell viabilities in the concentrations of 10-6 M, 10-5 M and 10-4 M 48 h after the MTX exposure. However, the cell viability obtained 24 h after the MTX exposure was significantly different from the respective cell viability 48, 72 and 96 h after the MTX exposure. In the group with oral mucositis, the clearance decreased significantly (p = 0.042), and the t1/2b (p = 0.025) and AUC48h- yen (p = 0.025) increased significantly compared with the non-symptom group. CONCLUSIONS: It seems that there is no significant relationship between the serum MTX concentration and oral mucositis. This in vitro study has demonstrated that the cell injury was related to the duration of MTX exposure rather than a high MTX concentration.

Analysis using fluorescence polarization immunoassay for unbound teicoplanin concentration in serum.

OBJECTIVES: The aim of this study was to develop a simpler and more rapid analytical method for unbound teicoplanin in serum. METHODS: A new analytical method was developed by modifying an existing fluorescence polarization immunoassay (FPIA) method. The validation of the developed FPIA method was compared in the quantification of unbound teicoplanin with that of the high-performance liquid chromatography (HPLC) method reported previously. The developed FPIA method was employed for the measurement of 36 clinical samples collected from patients with methicillin-resistant Staphylococcus aureus (MRSA) infection. RESULTS: The limits of detection and quantification were 0.5 and 0.8 mug/mL, respectively. The recovery rate was 97.5-106.6%. The developed FPIA method showed better accuracy than the HPLC method. The within-run and interday reproducibility of the assay was good, with relative standard deviation values of 4.76-18.75% (within-run) and 5.68-13.95% (interday). Precision and accuracy of this method were within the acceptable limits defined in the US FDA Guidance for bioanalytical method validation. The correlation between the developed FPIA method and the HPLC method was good (r(2) = 0.87). A positive bias with the FPIA method was observed from the result of the Bland-Altman difference plot. CONCLUSION: We firmly believe that the present method is useful for the adjustment of teicoplanin dosages for patients under various conditions.

Estimation of the area under the curve for mycophenolic acid in adult renal transplant patients with concomitant tacrolimus using a limited sampling strategy.

OBJECTIVES: The aim of this study was to develop a limited sampling strategy (LSS) for monitoring the use of mycophenolic acid (MPA) in maintenance therapy with tacrolimus (TCL) in renal transplant patients. METHODS: Eighteen adult patients receiving a first transplant were investigated. All patients were treated with a combination of TCL, steroid and mycophenolate mofetil (MMF). Besides the predose trough concentration (C(0)), whole blood samples were taken for measurement of the MPA concentration at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h for a 14-point 12-h pharmacokinetic (PK) profile. Using stepwise linear regression analysis, an abbreviated area under the concentration time curve (AUC) was calculated using all 14, and any combination of sampling points to give an estimating equation with up to three predictors. RESULTS: The equation derived from C(2), C(7) and C(12,) for AUC estimation: AUC = (2.05 x C(2)) + (8.51 xC(7)) + (2.29 x C(12)) + 4.24. was found to be optimal. Using this formula, there was an excellent correlation between the estimated 3-point AUC and AUC(0-12 h). To assess the agreement between the abbreviated methods and the full PK profile, we plotted the average AUC of the abbreviated estimates and the full PK profile. This Bland-Altman analysis indicated good agreement to within +/-2 SD and a prediction variability of 7.56 microg x h/mL. CONCLUSION: Our proposed three-sampling-point estimate of AUCs is clinically acceptable. However, the sampling times are inconvenient for outpatients, and is recommended only for monitoring MMF treatment of inpatients with suspected toxicity or at high risk of organ rejection.

Sulphasalazine-induced leucopenia in a patient with renal dysfunction.

BACKGROUND: Sulphasalazine is used for the long-term maintenance therapy of ulcerative colitis to prevent the relapse of symptoms. However, its clinical use is often restricted by its serious adverse effects. OBJECTIVE: Leucopenia occurred in a patient with severe renal dysfunction after administration of sulphasalazine. The present study was designed to examine whether the adverse event was associated with a disability in the metabolism of sulphasalazine. SUBJECT: A 29-year-old male patient with ulcerative colitis, who underwent haemodialysis thrice a week because of severe renal dysfunction. The chief complaint was diarrhoea. METHODS: Serum concentrations of three major metabolites of sulphasalazine, (5-aminosalicylic acid, sulphapyridine and N-acetyl-sulphapyridine), were measured. The polymorphism of N-acetyltransferase 2, an enzyme that metabolizes sulphapyridine, was also determined by polymerase chain reaction. RESULTS: The trough levels of 5-aminosalicylic acid, sulphapyridine and N-acetyl-sulphapyridine were 0.77-1.45 microg/mL, 31.20-39.25 microg/mL and 14.19-15.03 microg/mL, respectively. The gene diagnosis of N-acetyltransferase 2 suggested that the type was classified as NAT2*6A/*7B, indicating that the patient was a slow acetylator. CONCLUSION: The patient was a slow acetylator, which might lead to a rise in the serum sulphapyridine concentration. Moreover, the decrease in protein binding of sulphasalazine as a result of severe renal dysfunction might have potentiated the effect because of the extremely high protein binding of this compound. Thus, it is most likely that these two factors contributed to the sulphasalazine-induced leucopenia.

High-performance liquid chromatographic method for mycophenolic acid and its glucuronide in serum and urine.

OBJECTIVE: To develop a simple analytical method for monitoring serum and urine concentrations of mycophenolic acid (MPA), an active metabolic constituent of the immunosuppressive pro-drug mycophenolate mofetil, and its glucuronide. METHODS: Serum samples were prepared by solid-phase extraction (SPE), while urine samples were simply diluted with water. Serum was added to an SPE cartridge, then washed twice with 5% methanol solution. The analytes were eluted with methanol containing benzoic acid as internal standard for mycophenolic acid glucuronide (MPAG). The resultant eluate was directly injected into a high-performance liquid chromatograph (HPLC) to determine MPAG. For the assay of MPA, the remaining eluate was dried under nitrogen and resolved in a mixture of acetonitrile and 20 mM phosphate buffer (pH 3.0). RESULTS: The present methods were reproducible and accurate based on the intra- and inter-assay, and had detection limits of 0.225 microg/mL for MPA and 9.0 microg/mL for MPAG. The present methods enabled us to monitor the time course of changes in the concentrations of MPA and MPAG in serum and urine in a patient with a renal transplant during 12 h after ingestion of mycophenolate mofetil. CONCLUSION: The HPLC method described should be useful for the routine monitoring of serum and urine concentrations of MPA and MPAG during immunosuppressive medication for renal transplantation.

Simple and simultaneous determination of sulphapyridine and acetylsulphapyridine in human serum by column-switching high-performance liquid chromatography.

OBJECTIVE: A high-performance liquid chromatography (HPLC) with an automated on-line column-switching system was used for the simultaneous determination of sulphapyridine and acetylsulphapyridine, two major active metabolites related to the adverse effects of sulphasalazine, in human serum. METHODS: Serum samples were directly injected into the HPLC, with the valve automatically switched on to remove serum proteins and other hydrophilic components remaining in the pre-column after elution of sulphapyridine and acetylsulphapyridine to the analytical column. RESULTS: Serum proteins did not interfere with the analysis of either compound. The recoveries of SLP and Ac-SLP from drug-free human serum were 93.03-99.18% and CV were 2.88-4.34%. The within-run reproducibility of assays was excellent with relative standard deviations (RSD) of 1.01-3.90% (SLP) and 0.77-5.56% (Ac-SLP). The limit of quantification of sulphapyridine and acetylsulphapyridine was 3.13 microg/mL and 0.50 microg/mL, respectively. The serum concentrations in a patient with ulcerative colitis, who took 1.0 g sulphasalazine twice daily, were 31.20 microg/mL for sulphapyridine and 14.64 microg/mL for acetylsulphapyridine at 7 h after ingestion. CONCLUSION: The present simple and reproducible assay was useful for the monitoring of serum sulphapyridine and acetylsulphapyridine.

Simple determination of mycophenolic acid in human serum by column-switching high-performance liquid chromatography.

A column-switching high-performance liquid chromatographic analysis was established to monitor the serum concentration of mycophenolic acid, the active metabolite from mycophenolate mofetil administered for the prophylaxis of acute organ rejection in renal transplantation. The system consisted of two pumps for solvent delivery, a column-switching valve, a precolumn, and a reversed-phase analytical column. The present method enabled us to determine MPA by injecting serum samples directly into HPLC without any pretreatment. The mobile phases with different amounts of organic solvent were delivered to the precolumn and analytical column by separate lines, and samples were applied to the precolumn. The column switching valves were switched automatically following the processes for the elimination of protein and the drug analysis. The peak heights of MPA were linearly related to the concentrations (r=0.999) in the range of 0.1-20 micro g/ml, and the limit of quantification was 0.1 micro g/ml (S/N ratio=3). This method was accurate and reproducible on the basis of the results of recovery (94.0-98.0%) and small coefficient of variations of intra and inter-assay (less than 8.3%).

Application of dynamic laser scattering to the quality control of injectable drugs: polymer formation in ampicillin solution.

OBJECTIVE: To assess the usefulness of dynamic laser scattering for monitoring the stability of ampicillin after reconstitution from commercially available vials with respect to the polymer formation and potency. METHODS: Polymer formation and the remaining potency of the reconstituted ampicillin solution were estimated using dynamic laser scattering and high-performance liquid chromatography. RESULTS: The laser light-scattering submicron particle analyser was sufficiently sensitive for detecting both monomer and polymer aggregates with the average diameter of 1.1 +/- 0.2 and 7.3 +/- 1.7 nm, respectively, in the ampicillin solution. Polymer formation was dependent on both the storage temperature and the storage period, but it was detected, even when no precipitates were visible and when loss of potency was less than 10% of the initial value following storage at 4 or -15 degrees C. CONCLUSION: Submicron particle analysis using scanning electron microscopy, when used in combination with high-performance liquid chromatography, provides a useful method for studying polymer formation in antibiotic solutions and for the quality control of antibiotic injections during storage.

Nasal glucagon delivery using microcrystalline cellulose in healthy volunteers.

We developed an intranasal powder form of glucagon to improve metabolic status and fatty liver in patients with pancreatectomy. Microcrystalline cellulose, which is commonly used in commercial preparations for allergic rhinitis was used as an absorption enhancer. We compared the intranasal powder form with some spray solutions of glucagon with regard to glucagon absorption, concentration of blood glucose, stability and nasal irritation. The absorption of glucagon from the spray solution including 1.5% sodium glycocholate or 1% sodium caprate was 1.3- and 2.6-fold higher than that from the powder form mixed with microcrystalline cellulose at a ratio of 1:69, respectively. The C(max) values of plasma glucose were 2.18, 3.39 and 1.56 mmol l(-1) in the spray solutions including sodium glycocholate and sodium caprate and in the powder form, respectively. However, glucagon in spray solutions was unstable, but that in the powder form was stable at 5 and 25 degrees C for at least 84 days. The spray solution caused strong irritation, but the powder form did not. These results suggested usefulness of the powder form of glucagon for treatment of pancreatectomized patients.

Microanalysis of propofol in human serum by semi-microcolumn high-performance liquid chromatography with UV detection and solid-phase extraction.

OBJECTIVE: To develop a simple analytical method for monitoring the low serum levels of propofol found when administered for the sedation of patients in the intensive care unit (ICU). METHODS: A high-performance liquid chromatographic method (HPLC) was used with UV detection. Solid-phase extraction (SPE) cartridges and a semi-microcolumn (TSK gel ODS-80Ts, 2.0 mm i.d. x 25 cm, 5 microm) were used to improve sensitivity. Propofol in the eluate obtained from the SPE cartridge was concentrated to about five times the initial concentration. RESULTS: The sensitivity using the semi-microcolumn was amplified by about three-fold. The assay showed a good linearity with a quantification limit 20 ng/mL. Intra- and inter-assay coefficients of variation were less than 2.2% and 10.0%, respectively. The mean recoveries ranged from 97.6 to 109.5%. CONCLUSION: The HPLC method described should be useful for measuring the low serum propofol levels found when the drug is used for ICU sedation.

Usefulness of forced diuresis for acute boric acid poisoning in an adult.

BACKGROUND: Boric acid is generally not recognized as a poisonous substance. However, boric acid has potentially fatal actions such as hypotension, metabolic acidosis and oliguria. Death may result from circulation collapse and shock. OBJECTIVE: We present a clinical case history of the successful use of forced diuresis with furosemide and intravenous fluid for boric acid poisoning. SUBJECT: A 26-year-old female who attempted suicide by consuming a large quantity of boric acid. She was brought to the hospital in a state of clouded consciousness, fever and erythema 14 h after ingestion. METHOD: 3.25 L of intravenous fluid and 100 mg of furosemide were administered over a period of 4 h in the intensive care unit and the serum and urinary concentrations of boric acid measured. RESULTS: The elimination rate of boric acid obtained with diuresis was similar to that obtained with haemodialysis on a previous occasion when the same patient attempted suicide with boric acid. The patient showed only temporary emesis and diarrhoea along with erythema, and was moved to the general ward 4 h after admission. Although in the general ward the patient's fever persisted and nausea, vomiting and headache often recurred, possibly because of an insufficient dose of furosemide, the patient's condition steadily improved over the 64 h after admission. CONCLUSION: Forced diuresis without haemodialysis is recommended early after admission for boric acid poisoning.

Outcome measurement of problem-based learning.

OBJECTIVE: To determine if there is a significant difference in the national certification examination scores, and in comments received from clinical faculty, between PBL and pre-PBL groups. DESIGN: Retrospective analyses of national certification examination scores and clinical faculty evaluation files. PARTICIPANTS: Medical technology students at the University of Hawaii. MAIN OUTCOME MEASURES: Means of a national certification examination scores of PBL and pre-PBL groups are statistically compared. Types of clinical faculty comments are rank-ordered and compared between the two groups. RESULTS: PBL group did significantly better in the Urinalysis section of the national certification examination. Pre-PBL group received more "Quiet but got better" comments from clinical faculty. CONCLUSION: PBL can affect student outcomes in both national certification exams and perception by clinical faculty.

Surfing the wave of clinical laboratory science evolution in Hawai'i.

OBJECTIVE: To describe the steps taken by the Hawaii Society for Clinical Laboratory Science, an affiliate of the American Society for Clinical Laboratory Science, to inform local laboratory professionals of current trends and to prepare for the future. RESULTS: A Strategic Planning workshop was conducted at the 1997 Hawaii Society for Clinical Laboratory Science Annual Meeting where participants reviewed the essential (but non-traditional) functions of clinical laboratory scientists, and described current realities, identified forces and players affecting the changes, and envisioned the future of our profession. CONCLUSION: As the way health care is provided changes in response to economics and advances in technology, the role of clinical laboratory scientists needs to be redefined. The Hawaii Society for Clinical Laboratory Science continues to provide timely support for members, and plans to work collaboratively with the local chapter of the Clinical Laboratory Managers' Association to advance clinical laboratory science to an appropriate place in the health care community.

Clinical management of boric acid ingestion: pharmacokinetic assessment of efficacy of hemodialysis for treatment of acute boric acid poisoning.

Seven hours after suicidal ingestion of about 21 g of boric acid, a 26-year-old female admitted to our hospital in a state of slightly impaired consciousness, with frequent vomiting, shivering, fever and skin flush. Immediately, gastric lavage, followed by administration of activated charcoal and laxative (MgSO4), was performed. In order to ensure her urination, fluid infusion therapy was conducted with the aid of diuretics (furosemide). Since the serum concentrations of boric acid was very high, hemodialysis was carried out twice during the first 39 h. She responded well to the above mentioned treatment and was discharged 12 d post-admission without any sequelae. The concentrations of boric acid in serum and urine were measured in appropriate intervals with our modified Miyamoto's method, and the pharmacokinetics of boric acid were analyzed. The concentration of boric acid in serum and urine at the beginning of treatment was 465 micrograms/ml and 3.40 mg/ml, respectively. The half-life of boric acid in serum was 13.46 h, whereas it was shortened to 3.76 h during hemodialysis. The total body clearance was 0.99 l/h, while it increased to 3.53 l/h by hemodialysis. The additional removal of boric acid by hemodialysis was estimated to be about 5 g. It was concluded that the hemodialysis was very useful in the treatment of boric acid poisoning, because it accelerated the elimination of boric acid about four times faster than with conventional treatment.

Concomitant carriage of hepatitis B virus and human T-lymphotropic virus type I among blood donors in Kitakyushu, Japan.

The seroprevalence of hepatitis B surface antigen (HBsAg) by gender and age and of human T-lymphotropic virus type I (HTLV-I), and their concomitant carriage was examined among blood donors at the Kitakyushu Red Cross Blood Centre in the fiscal year of 1988. The positive rates of HBsAg among males were consistently higher than those of females; the peaks were detected in male donors aged 30-39 years and in females aged 40-49 years. Declining seropositive rates in individuals aged 50 years or over were observed for both genders. Self-selection due to chronic HBV infection may partly account for such tendencies. On the other hand, the prevalence rates of anti-HTLV-I antibodies among males were uniformly lower than those of females, which must be attributable to male-to-female transmission of HTLV-I via sexual contact. Elevated positive rates in proportion to age were noted for both genders, which may be explained in part by birth cohort effect. The seropositive rates of HBsAg among HTLV-1 carriers were not statistically different from those of non-HTLV-I carriers. Conversely, the prevalence of antibodies to HTLV-I was unrelated to the status of seropositivity of HBsAg.

Follow-up of asymptomatic HTLV-I carriers among blood donors in Kyushu, Japan.

We examined mortality from adult T-cell leukemia/lymphoma (ATL/ATLL) and other diseases alleged to be associated with human T-lymphotropic virus type I (HTLV-I) among anti-HTLV-I antibody-positive blood donors in Kyushu, Japan. During 1984-87, a total of 3,991 blood donors aged 40 years or over were followed from the date of donation to the date of death or the end of the study. Crude mortality rates from ATL (with 95 percent confidence intervals) were 68 per 100,000 (13-202) for males and 36 per 100,000 (3-132) for females. The rates were underestimated by approximately 50 percent because of self-selection and short observation periods. Neither death rates from other cancers nor death rates from all cancers were elevated.

Efficacy of emetic and United State pharmacopoeia ipecac syrup in prevention of drug absorption.

The efficacy of both the emetic syrup prepared in the previous report and the United States Pharmacopoeia (USP) ipecac syrup concerning the prevention of drug absorption was investigated in 4 beagle dogs using a randomized and cross-over design. In order to control the intragastric pH of the beagle dogs, the administration of pentagastrin or hydrochloric acid (HCl)-glycine buffer (pH 1.5) was tested. The intragastric pH changed from 7.2 to 1.8 with the intramuscular administration of pentagastrin, but the primary emesis occurred more slowly. On the other hand, the HCl-glycine buffer (pH 1.5) gave the appropriate emesis. Therefore, the HCl-glycine buffer (pH 1.5) was used to control the intragastric pH of the beagle dogs. Acetaminophen (AcA), salicylic acid (SA) and kanamycin (KM) as markers were administered orally after conditioning the intragastric pH at 1.5. The emetic syrup or the USP ipecac syrup was then administered. The recovery rate of AcA and KM from vomit was 42-65%. The emetic syrup and the USP ipecac syrup significantly reduced the absorption of AcA from the calculation of pharmacokinetic parameters compared to the control syrup. It was observed that the absorption of cephaeline (CP) in the emetic syrup was less than that of CP in the USP ipecac syrup.