Range of motion exercise of temporo-mandibular joint with hot pack increases occlusal force in patients with Duchenne muscular dystrophy.

The purpose of this study is to evaluate whether the range of motion exercise of the temporo-mandibular joint (jaw ROM exercise) with a hot pack and massage of the masseter muscle improve biting disorder in Duchenne muscular dystrophy (DMD). The subjects were 18 DMD patients (21.3+/- 4.1 years old). The jaw ROM exercise consisted of therapist-assisted training (2 times a week) and self-training (before each meal every day). The therapist-assisted training consisted of the application of a hot pack on the cheek of the masseter muscle region (15 minutes), the massage of the masseter (10 minutes), and jaw ROM exercise (5 minutes). The self-training involved jaw ROM exercise by opening the mouth to the maximum degree, ten times. These trainings continued for six months. Outcomes were evaluated by measuring the greatest occlusal force and the distance at the maximum degree of mouth opening between an incisor of the top and that of the bottom. Six months later, the greatest occlusal force had increased significantly compared with that at the start of jaw ROM exercise (intermediate values: from 73.8N to 97.3N) (p = 0.005) as determined by the Friedman test and Scheffi's nonparametric test. The patients' satisfaction with meals increased. However, the maximum degree of mouth opening did not change after six months of jaw ROM exercise. Jaw ROM exercise in DMD is effective for increasing the greatest occlusal force.

Nonbinding inhibitory antiinsulin receptor antibodies. A new type of autoantibodies in human diabetes.

Sera and their IgG from 10/104 diabetic patients (five with insulin-dependent and five with noninsulin-dependent diabetes, NIDDM), contained antibodies that bound 125I-labeled purified human insulin receptors. 9 of these 10 sera failed to inhibit insulin binding (to rat hepatocytes and human placental membranes), did not stimulate glucose oxidation (by isolated rat adipocytes), and did not bind human placental IGF-1 receptors. Only one serum (and its IgG) modestly inhibited insulin binding and stimulated glucose oxidation. We conclude (a) that sera from 9/104 diabetics (five insulin-dependent and four noninsulin-dependent) contained a newly identified species of IgG antiinsulin receptor autoantibodies (AIRA), which bound to the insulin receptor at a locus different from the insulin binding site and did not inhibit insulin binding; and (b) that only 1/104 diabetic sera contained low-titer "conventional" antiinsulin receptor autoantibodies that bound to the insulin receptor at or near the insulin binding site, inhibited insulin binding and caused a clinical condition, which was difficult to distinguish from typical NIDDM.

Effects of anti-insulin receptor antibodies (AIRA) on downregulation and turnover of insulin receptors on cultured hepatocytes.

We have studied the effects of two polyclonal anti-insulin receptor antibodies (AIRA) on insulin receptor downregulation and turnover in rat hepatocytes in primary culture. Downregulation was determined by measurement of insulin binding after acid washing of cells to remove AIRA. Insulin receptor turnover was estimated by measurement of insulin binding after inhibition of synthesis of functional receptors with tunicamycin (0.5 micrograms/ml). Exposure of hepatocytes to AIRA (both sera were of comparable effectiveness) resulted in progressive, time- and dose-dependent losses of insulin binding (maximal loss was about 55% after 24 h of incubation with AIRA diluted 1:25). Cycloheximide (100 microM) prevented AIRA-mediated downregulation. The t1/2 of disappearance of cell surface insulin binding capacity determined with tunicamycin was 8.0 h. Addition of insulin (1000 ng/ml) or AIRA to tunicamycin reduced the t1/2 to 2.6 h (insulin), 2.2 h (patient B10), and 2.0 h (patient 1). These data suggest that AIRA downregulated insulin receptors on cultured hepatocytes by accelerating their rate of disappearance, inhibition of protein synthesis prevented AIRA-mediated downregulation, and downregulation by AIRA of insulin binding may be partially responsible for the desensitization of target cells to some of the insulin-like actions of these autoantibodies.

Carbohydrate (CHO) oxidation in patients with type B insulin resistance.

Total body carbohydrate (CHO) and fat oxidation rates, plasma glucose, free fatty acid, and insulin concentrations were determined in two patients with the type B syndrome of severe insulin resistance and in normal controls in response to insulin infusions (1-100 mU/kg/min) and to a test meal. In addition, insulin was infused at higher rates (10-1000 mU/kg/min) in one of the two patients while plasma glucose concentrations were clamped first at 195 and later at 244 mg/dl. During the postabsorptive state, resting metabolic rates (RMR) were 914 and 979 cal/min/1.73 m2 in the two patients (controls: 1018 +/- 85 cal/min/1.73 m2). Patients met 85% and 83% of their caloric requirements by oxidizing fat (controls: 63 +/- 7%). Protein oxidation accounted for 15% and 13% (controls: 14 +/- 3%) of energy requirements and CHO oxidation for 0% and 0%, respectively, in both patients (controls: 23 +/- 5%). Infusion of insulin at a rate of 10 mU/kg/min raised plasma insulin concentrations from 1400 and 440 microU/ml to 6000 and 2500 microU/ml, respectively, in patients 1 and 2 (controls: from 4 +/- 0.3 to 1288 +/- 50 microU/ml), but had no effects on rates of CHO, fat, or protein oxidation in either patient. By comparison, the rate of CHO oxidation in controls rose about sixfold from 40 +/- 8 to 234 +/- 12 mg/min/1.73 m2. Infusion of 1000 mU/kg/min in combination with an increase in plasma glucose from 195 +/- 1.1 to 244 +/- 1.9 mg/dl in patient 1, however, raised CHO oxidation from 0 to 36 mg/min/1.73 m2 and lowered fat oxidation from 105 to 69 mg/min/1.73 m2.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of anti-insulin receptor antibodies on in vivo insulin metabolism.

The effects of anti-insulin receptor antibodies (AIRA) on receptor binding and insulin metabolism were studied in two patients with the type B, severe insulin resistance syndrome. Insulin binding was determined using rat hepatocytes in primary culture and the patient's own red blood cells. Plasma and urinary insulin concentrations and metabolic clearance rates (MCR) were determined in the two patients and in four normal controls in response to infusions of insulin for 60-120 min at rates ranging from 1 to 925 mU/kg/min. In patient 1, basal insulin concentration was 1400 microU/ml. After infusion of 1, 10, and 925 mU/kg/min of insulin it rose to 3800, 5500, and 225,000 microU/ml, respectively. Respective MCRs were 19, 110, and 186 ml/min. In patient 2, basal insulin concentration was 440 microU/ml. After infusion of 1, 10, and 100 mU/kg/min of insulin it rose to 720, 2500, and 18,800 microU/ml, respectively. Respective MCRs were 193, 262, and 294 ml/min. In controls, basal insulin concentration was 4 +/- 0.3 microU/ml. After infusion of 1 and 10 mU/kg/min of insulin, it rose to 82 +/- 17 and 1288 +/- 50 microU/ml. Respective MCRs were 950 and 630 ml/min. These data showed that, in patients with AIRA: (1) insulin metabolism took place at the same rate but at higher insulin concentrations than in normal controls, and (2) MCR increased with rising insulin concentration but remained subnormal even at the highest insulin concentrations. In contrast, MCR in normal controls decreased with increasing insulin concentrations. The data suggest that prevention of insulin binding prevents insulin metabolism at physiologic insulin concentrations and that supraphysiologically elevated insulin concentrations are needed to activate nonreceptor mechanisms.

Reversal of altered vascular responsiveness in Bartter's syndrome by indomethacin treatment.

The effects of indomethacin treatment on the vascular response to 5-valine-angiotensin II amide, [sarcosine, isoleucine] angiotensin II, and D-3-mercapto-2-methylpropanoyl-L-proline were examined in a patient with Bartter's syndrome. Indomethacin (150 mg/day) was administered for 5 days. PRA decreased from 74.1 to 3.2 ng/ml.h during this period. With this treatment, the normal pressor response to the synthetic angiotensin II was restored, and the hypotensive response to the angiotensin II antagonist and the converting enzyme inhibitor was markedly diminished. These results suggest that altered vascular responsiveness in Bartter's syndrome would be secondary to hormonal derangements.

Fluorescein-isothiocyanated insulin interactions with human platelets.

The interactions of fluorescein-isothiocyanated porcine insulin (FITC-insulin) with human platelets were examined by a fluorescence polarization technique. The degree of polarization of the incubation mixture of FITC-insulin and platelets was changed by the ratio of bound to free insulin. This method enabled us to follow the time course of the reaction in the homogeneous assay system without separating the insulin-receptor complex, and it was shown to be useful in the study of insulin binding and dissociation. Insulin binding to trypsinized platelets was considered to be nonspecific, since this fraction remained relatively constant during the incubation period at 25 C. Specific binding was evaluated by subtracting the nonspecific binding from the binding to untreated platelets; specific binding reached a plateau at 60 min. Scatchard analysis was conducted by adding increasing amounts of FITC-insulin to a platelet suspension. Furthermore, some of the prerequisites in analyzing insulin-receptor interaction were examined by this method.

Effects of antiinsulin receptor antibodies on amino acid uptake by cultured rat hepatocytes.

The effects of two antiinsulin receptor antisera (AIRA) on alpha-amino-2-[14C]isobutyric acid [( 14C]AIB) uptake by and [125I]insulin binding to cultured rat hepatocytes were examined. Diluted 1:100, both antisera inhibited insulin binding by 75-80%, mainly by decreasing available high affinity insulin-binding sites. Diluted 1:500, they decreased insulin binding and high affinity binding sites by 35-40%. Neither antiserum had an effect on basal [14C]AIB uptake, but decreased insulin-stimulated AIB uptake by 83% (dilution, 1:100; 24-h incubation) and 25% (dilution, 1:500; 24-h incubation), respectively. Insulin stimulation of AIB uptake correlated positively (r = 0.96; P less than 0.01) with insulin binding to high affinity receptors on hepatocytes incubated with normal serum or AIRA. We conclude that: 1) insulin stimulation of amino acid uptake by hepatocytes was mediated through insulin receptors, especially high affinity binding sites, and was inhibited by AIRA in parallel with receptor blockade; and 2) AIRA had no insulin-like effect on basal amino acid uptake by hepatocytes.

In vivo and in vitro effects of antiinsulin receptor antibodies.

We studied a patient with Acanthosis nigricans and the type B syndrome of severe insulin resistance. The patient's rates of basal glucose disappearance and appearance were both normal (2.2 and 1.7 mg/kg . min, respectively). FFA, betahydroxybutyrate, and acetoacetate concentrations were stable at 0.8, 1.0, and 0.3 mM, respectively, during a 2-h saline infusion after an overnight fast, indicating continued presence of insulin-like activity (ILA) in her serum. Infusion of insulin at rates of 2.7 and 27 U/h, raising peripheral insulin concentrations from 1400 to 4000 and 6000 microU/ml, respectively, had no effect on glucose disappearance and appearance or plasma concentrations of beta-hydroxybutyrate, acetoacetate, and FFA, suggesting that the observed ILA was not caused by the patient's plasma insulin. To determine the source of the ILA we used the patient's serum containing antiinsulin receptor antibodies (AIRA) to study its acute (2 h) and chronic (24 h) effects on insulin binding and glycogen synthesis in rat hepatocytes in primary culture. Preincubation of hepatocytes with AIRA serum (diluted 1:100) inhibited insulin binding by 84% and 88% after 2 and 24 h, respectively. It increased U-[14C]glucose incorporation into glycogen by 40% and 52% after 2 and 24 h, respectively. These effects were not caused by insulin present in the patient's serum. We conclude that AIRA serum, in addition to causing severe insulin resistance through inhibition of insulin binding, also exerted strong and long lasting insulin-like effects. These findings are compatible with the patient's clinical features of absence of ketoacidosis despite severe insulin resistance, decrease in glucose concentrations during fasting, and postprandial hyperglycemia.

Effect of carbamazepine on the single oral dose pharmacokinetics of alprazolam.

The effect of carbamazepine, an inducer of cytochrome P450 (CYP) 3A4, on the single oral dose pharmacokinetics of alprazolam was examined in a double-blind, randomized crossover study with two phases. Seven healthy male subjects took carbamazepine 300 mg/day or matched placebo orally for 10 days, and on the 8th day they took a single oral 0.8 mg dose of alprazolam. Blood samples were taken and psychomotor function was assessed by the Digit Symbol Substitution Test, Visual Analog Scale, and UKU Side Effect Rating Scale up to 48 h after alprazolam dosing. Carbamazepine significantly (p < .01 to .001) decreased the plasma alprazolam concentrations during the elimination phase. Carbamazepine significantly (p < .001) increased the apparent oral clearance (0.90 +/- 0.21 vs. 2.13 +/- 0.54 ml/min/kg) and shortened the elimination half-life (17.1 +/- 4.9 vs. 7.7 +/- 1.7 h), with no significant effect on the peak plasma concentration (11.7 +/- 1.5 vs. 13.0 +/- 3.5 ng/ml). The majority of psychomotor function parameters during the carbamazepine treatment were not significantly different from those during the placebo treatment, probably because of the sedative effect of carbamazepine itself. The present study suggests that carbamazepine decreases plasma concentration of alprazolam by inducing its metabolism. It also supports the previous studies, suggesting that alprazolam is metabolized predominantly by CYP3A4.

Clonal analysis of gliomas.

In malignant gliomas, the characteristically heterogeneous features and frequent diffuse spread within the brain have raised the question of whether malignant gliomas arise monoclonally from a single precursor cell or polyclonally from multiple transformed cells forming confluent clones. Although monoclonality has been shown in surgically resected tissues, these may not include the full spectrum of patterns seen on autopsy material. Little is known about the clonality of low-grade gliomas from which malignant gliomas may sometimes arise. We sought to investigate the clonality of low-grade and malignant gliomas by using and comparing surgical and autopsy material with a Polymerase chain reaction (PCR)-based assay for nonrandom X chromosome inactivation. For that, purpose, archival surgical and autopsy material from 15 female patients (group A) (age 4 to 73 years; median, 45) with malignant gliomas (12 glioblastomas, one gliosarcoma, one anaplastic oligoastrocytoma, one gliomatosis cerebri), surgical material only from 21 female patients (group S) (age 6 to 78 years; median, 60) with low-grade and malignant gliomas (four low-grade astrocytomas, three oligoastrocytomas, two anaplastic astrocytomas, one gemistocytic astrocytoma, four oligodendrogliomas, seven glioblastomas) were analyzed. In group A, representative areas (mean = 5/patient; median = 7) were microdissected from tissue sections and assayed by PCR amplification of a highly polymorphic microsatellite marker locus of the human androgen receptor gene (HUMARA) in the presence of alpha32P with and without predigestion with a methylation-sensitive restriction enzyme (HhaI). Products were resolved by denaturing gel electrophoresis and autoradiographed. In group S, selected tumor areas were used for the assay. Each patient's normal brain tissue was used for control. The band intensity of alleles were measured by densitometric scanning. In group A, 13 of 15 cases were informative (heterozygous). The same pattern of nonrandom X chromosome inactivation was present in all areas of solid dense and moderate tumor infiltration in eight including all components of the gliosarcoma. Two of eight also showed focal loss of heterozygosity (LOH). One of 13 presented global LOH. Two of 13 showed microsatellite instability, one of which in a patient with Turcot syndrome, the other in gliomatosis cerebri. Opposite skewing patterns were seen in distant areas of gliomatosis cerebri consistent with oligoclonal derivation. Clonality remained indeterminate in one glioblastoma and in the anaplastic oligoastrocytoma because of skewed lyonization in the normal control. In group S, 19 of 21 cases were informative. Fifteen of 19 were monoclonal (four low-grade astrocytomas, one anaplastic astrocytoma, one gemistocytic astrocytoma, two oligodendrogliomas, one oligoastrocytoma, six glioblastomas). Four of 19 were indeterminate. We conclude that (1) Low-grade and malignant gliomas are usually monoclonal tumors, and extensively infiltrating tumors must result from migration of tumor cells (2) Gliomatosis cerebri may initiate as an oligoclonal process or result from collision gliomas (3) Biphasic gliomas likely arise from a single precursor cell. (4) LOH at the HUMARA locus is probably related to partial or complete deletion of an X-chromosome, which occurs in malignant gliomas during clonal evolution.

Correlation between p53 immunostaining patterns and gene sequence mutations in breast carcinoma.

We performed p53 immunostaining in 82 invasive breast carcinomas by using two commercially available antibodies, one of which (DO7) was employed in formalin-fixed paraffin-embedded sections. The other antibody (PAb1801) was evaluated in corresponding acetone-fixed cryostat sections. A greater percent of cases were immunostained with DO7 compared to PAb1801 (52% vs 33%); however, the staining was more often heterogeneous (6-50% cells positive) or focal (< or = 5% cells positive) with DO7 (9% vs 31%). To investigate the genetic relevance of p53 immunostaining, single-strand conformational polymorphism (SSCP) analysis and DNA sequencing were performed on exons 2-11 by using archival tissue samples of 18 cases that were selected on the basis of certain immunostaining patterns. Two (33%) of six tumors with negative staining for DO7 had gene sequence mutations; however, one of these mutations was a base-pair deletion that caused a reading-frame shift and the other was a base-pair insertion that resulted in a stop codon. Both of these tumors exhibited immunostaining with PAb1801, although it was weak and cytoplasmic in one case. Conversely, three (30%) of 10 tumors showing immunoreactivity in 6-100% of cells with both reagents lacked a gene sequence mutation. Of the remaining seven tumors that were positive by SSCP, six contained a point mutation resulting in a base-pair substitution. Despite repeat analyses, one of the cases positive by SSCP failed to demonstrate a mutation in the sequenced exons. Four (80%) of five cases with heterogeneous DO7 immunoreactivity (that is, 6-50% of nuclei positive) were positive for gene sequence mutation. Neither of two cases showing focal DO7 nuclear staining in < 5% of tumor cells contained a mutation in the sequenced exons, and neither of these cases was strongly positive with PAb1801. Staining for either antibody was significantly associated with adverse outcome, as determined by disease recurrence at 52 months median follow-up (DO7, p = 0.01; and PAb1801 p = 0.002, chi-squared test). We conclude that a variety of factors may account for discrepancies when immunohistology is used to evaluate p53 status. These include fixation artifacts, differing epitope specificities of monoclonal reagents, presence of immunohistologically "silent" mutations and, possibly, aberrant overexpression of wild-type protein.

A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue.

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.

Auditory effects on the motor responses after magnetic cortical stimulation and on the H-reflexes in patients with Parkinson's disease.

The effects of sound on the responses in teh abductor pollicis brevis muscle after magnetic cortical stimulation and on the H-reflexes in the wrist and finger flexor muscles were examined. Magnetic cortical stimulation and electrical stimulation eliciting H-reflexes were conditioned by sound stimulation. This sound stimulation did not produce the electromyographic response by itself. In the control subjects, sound stimulation produced an increase of the motor responses after cortical stimulation at intervals of 100, 150, 200 and 250 ms. The increase was greater in the patients with Parkinson's disease (PD). In the control subjects, sound stimulation produced an increase of the H-reflexes at intervals of 50, 100, 150 and 200 ms. This H-reflex increase in the PD patients was less than in the normal subjects. The reticular system might play a role in the abnormal motor control system in PD patients.

Anti-insulin receptor antibodies in human diabetes.

Sera and immunoglobulin G from 10/104 diabetic patients (five with insulin-dependent, five with non-insulin-dependent diabetes) were found to contain antibodies that bound 125I-labelled purified human placental insulin receptors. Nine of these sera failed to inhibit insulin binding to cultured rat hepatocytes and did not stimulate glucose oxidation in rat adipocytes. Only one serum modestly inhibited insulin binding and stimulated glucose oxidation. These results suggest that sera from nine of these 104 diabetics contained a new type of anti-insulin receptor antibodies (AIRA) which bound to a locus different from the insulin binding site, and that only one of the 104 diabetic sera contained a low titer of conventional AIRA which could cause a clinical condition not distinguishable from ordinary non-insulin-dependent diabetes.

Carbamazepine toxicity induced by clarithromycin coadministration in psychiatric patients.

Seven psychiatric inpatients receiving carbamazepine 600 mg/day were coadministered clarithromycin 400 mg/day for 5 days to treat atypical pneumonia. Blood samples were taken after clarithromycin coadministration and at 1 and 4 weeks after its discontinuation. Plasma concentrations of carbamazepine and carbamazepine-10,11-epoxide were measured using high-performance liquid chromatography. During clarithromycin coadministration, four out of the seven patients developed moderate-to-severe toxic symptoms of carbamazepine, such as drowsiness, dizziness, and ataxia, which resolved within 5 days after clarithromycin discontinuation. In these four patients, plasma carbamazepine concentrations after clarithromycin coadministration were approximately twice as high as those after its discontinuation. In the seven patients, the mean plasma concentration of carbamazepine, but not of carbamazepine-10,11-epoxide, after clarithromycin coadministration was significantly (p < 0.01) higher than those at 1 and 4 weeks after its discontinuation. The present report suggests that clarithromycin coadministration induces increased plasma carbamazepine concentrations, which may result in carbamazepine toxicity. Therefore, care should be given to prescribing clarithromycin for patients receiving carbamazepine.

Measurement of haloperidol in human breast milk by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the assay of haloperidol in human breast milk. The method involves rapid extraction of haloperidol and chromatography with a reversed-phase C18 column and a mobile phase of phosphate buffer (pH 4.0):acetonitrile (70:30, v/v). Moperone was used as the internal standard. Haloperidol and moperone were detected with ultraviolet detection at 254 nm. The sensitivity was 5 ng/mL of milk, and the standard curve was linear in the concentration range 5-250 ng/mL. The average interassay coefficient of variation for samples with drug in the concentration range 25-125 ng/mL was less than 8.8%. The absolute recovery of haloperidol by the method was greater than 94.4%. No interference with endogenous substances in human milk was observed. The method was used to determine haloperidol levels in breast milk from patients undergoing chronic haloperidol treatment.

Monitoring of carbamazepine and carbamazepine 10,11-epoxide in breast milk and plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the measurement of carbamazepine (CBZ) and carbamazepine 10,11-epoxide (CBZE) in human breast milk and plasma. The method involves rapid C18 solid-phase extraction of CBZ and CBZE. Chromatographic separation was achieved with a reversed-phase C8 column using a mobile phase of potassium dihydrogenphosphate (pH 2.5) and acetonitrile (67:33 v/v), with ultraviolet detection at 254 nm. 2-Methyl CBZ was used as the internal standard. Determination of both CBZ and CBZE was possible in the range of 0.01-6.0 mg/L and 0.02-6.0 mg/L in milk and plasma, respectively. The recoveries of CBZ and CBZE added to the milk and plasma were 90.6-98.0% and 88.9-104.0%, respectively, with coefficients of variation less than 8.3% and 10.5%, respectively. The method has been used for drug level monitoring in milk and plasma samples obtained from CBZ-treated patients. The mean (SD) levels for CBZ in milk and plasma samples were 3.50 (0.4) mg/L and 6.18 (2.9) mg/L, and for CBZE were 1.28 (0.3) mg/L and 1.85 (1.0) mg/L, respectively. The mean (SD) milk/plasma ratios of CBZ and CBZE were 0.64 (0.2) and 0.79 (0.3), respectively. The milk/plasma ratio of CBZE was slightly higher than that of CBZ.

Monitoring of phenytoin in human breast milk, maternal plasma and cord blood plasma by solid-phase extraction and liquid chromatography.

A rapid liquid chromatographic method for the quantitation of phenytoin in human breast milk, maternal plasma and cord blood plasma was developed using a Develosil C85 micron reverse phase column and a potassium dihydrogen phosphate buffer/acetonitrile mobile phase. Phenytoin and mephenytoin as an internal standard were detected by ultraviolet absorbance at 240 nm. The sample preparation method involves a rapid and simple procedure based on solid-phase extraction using a C18-bonded phase. Phenytoin could be determined in the concentration range of 0.05-3 micrograms ml-1. The recovery of phenytoin added to human breast milk and plasma were 91.6-94.7 and 91.6-96.0%, respectively, with coefficient of variation less than 4.2 and 8.7%. The method has been used for drug level monitoring in the human breast milk, maternal plasma and cord blood plasma samples that were taken from patients treated with phenytoin. The average ratio between the breast milk concentrations versus the plasma concentration was 0.28 +/- 0.1, with a rather poor correlation (r = 0.3033).

Does edema formation occur in the rabbit brain exposed to head-down tilt?

Earlier studies showed that exposure to microgravity caused cephalad fluid shift, increased capillary pressure in the head, and produced facial edema and nasal congestion. In the present study, edema formation in the brain was investigated in rabbits exposed to simulated microgravity, head-down tilt (HDT), by measuring water content and histological examinations. Water content in the brain tissues of rabbits exposed to 2 and 8 days of HDT did not increase significantly compared with that of control animals. Neither vital staining using Evans blue nor immunohistochemical examination demonstrated extravasation of plasma constituents in the brain tissues of the HDT rabbits. Although marked congestion was noted in the brain, hematoxylin and eosin staining did not show edematous changes, such as distension of the perivascular and pericellular spaces and vacuolar appearance, in the tissues obtained from HDT rabbits. Transmission electron microscopy revealed that tight junctions of the capillary endothelium were intact in the HDT rabbits. These results suggest that either HDT up to 8 days does not cause brain edema in rabbits or it induces only a slight brain edema which is hard to be demonstrated by measurement of water content or histological examinations.