Simplified determination of serum cholesterol sulfate by gas-liquid chromatography combined with cyclohexylsilane-bonded phase column purification

Summary A new gas-liquid chromatographic (GLC) determination of cholesterol sulfate (CS) and dehydroepiandrosterone sulfate (DHEAS) for a biochemical diagnosis of recessive X-linked ichthyosis (RXLI) is described. Although the GLC method for determination of CS is known to be more sensitive than the thin layer chromatographic (TLC) method, the former method has not been widely employed because of its complicated pre-purification steps. The present method allows us to measure the serum levels of CS and DHEAS without tedious purification steps such as multiple conventional column chromatography and preparative thin layer chromatography. Sulfated steroids are rapidly purified with a commercially available mini disposable cyclohexylsilane-bonded phase (CH) column, CH BOND ELUT, and the purified steroids after desulfation are converted to water-resistant tert-butyldimethylsilyl ether derivatives for the GLC analysis on dual 2 m glass columns packed with 2% XE-60 on Chromosorb W.By the present method, serum CS concentrations in RXLI patients were shown to be about 10 times higher than those in patients with ichthyosis vulgaris, carriers of RXLI, and healthy subjects. This method is more suitable not only for a biochemical diagnosis of RXLI but also for studies on the metabolism of sulfated steroids than the previous time-consuming GLC methods.     

Ketamine Suppresses Platelet Aggregation Possibly by Suppressed Inositol Triphosphate Formation and Subsequent Suppression of Cytosolic Calcium Increase

Background: Ketamine has been shown to suppress platelet aggregation, but its mechanisms of action have not been defined. The purpose of the current study is to clarify the effects of ketamine on human platelet aggregation and to elucidate the underlying mechanisms of its action.

Methods: Platelet aggregation was measured using an eight-channel aggregometer, and cytosolic free calcium concentration was measured in Fura-2/AM-loaded platelets using a fluorometer. Inositol 1,4,5-triphosphate (IP3) was measured with use of a commercially available IP3 assay kit. To estimate thromboxane A2 (TXA2) receptor binding affinity and expression, Scatchard analysis was performed using [3H]S145, a specific TXA2 receptor antagonist. TXA2 agonist binding assay was also performed. The membrane-bound guanosine 5'-triphosphatase activity was determined using [[gamma]-32P]guanosine triphosphate by liquid scintillation analyzer.

Results: Ketamine (500 [mu]m) suppressed aggregation induced by adenosine diphosphate (0.5 [mu]m), epinephrine (1 [mu]m), (+)-9,11-epithia-11,12-methano-TXA2 (STA2) (0.5 [mu]m), and thrombin (0.02 U/ml) to 39.1 +/- 30.9, 46.3 +/- 4.3, -2.0 +/- 16.8, and 86.6 +/- 1.4% of zero-control, respectively. Ketamine (250 [mu]m-1 mm) also suppressed thrombin- and STA2-induced cytosolic free calcium concentration increase dose dependently. Although ketamine (2 mm) had no effect on TXA2 receptor expression and its binding affinity, it (1 mm) suppressed intracellular peak IP3 concentrations induced by thrombin and STA2 from 6.60 +/- 1.82 and 4.39 +/- 2.41 to 2.41 +/- 0.98 and 1.90 +/- 0.86 pmol/109 platelets, respectively, and it suppressed guanosine triphosphate hydrolysis induced by thrombin (0.02 units/ml) and STA2 (0.5 [mu]m) to 50.3 +/- 3.2 and 67.5 +/- 5.5%versus zero-control, respectively.     

Different postnatal ontogenic profiles of neurons containing beta (beta 1, beta 2 and beta 3) subunit mRNAs of GABAA receptor in the rat thalamus.

The postnatal ontogeny of neurons containing different GABAA receptor beta (beta 1, beta 2 and beta 3) subunit mRNAs were examined in the rat thalamus using in situ hybridization histochemistry. Neurons containing beta 1 or beta 2 subunit mRNA developed remarkably postnatally, while most neurons were already strongly labeled with beta 3 probe at birth. However, beta 3 subunit mRNA decreased rapidly after birth, few cells being labeled with this probe at day 35 and thereafter.     

Morphologic examination of CD3–CD4bright cells in rat liver

Recently, we found CD3-CD4(bright) cells with comparative specificity for normal rat liver. In the current study, we investigated the type and form of both CD3-CD4(bright) cells and CD3-CD4(dull) cells in the rat liver. The surface phenotype of hepatic mononuclear cells in Lewis rats was identified by using monoclonal antibodies including anti-CD4, anti-CD3, and antimacrophage in conjunction with two- or three-color immunofluorescence analysis. CD3-CD4(bright) cells and CD3-CD4(dull) cells were examined morphologically using May-Giemsa staining and scanning electron microscopy. The distribution of CD3-CD4(bright) cells and CD3-CD4(dull) cells 48 hours after intravenous administration of liposome-encapsulated dichloromethylene diphosphate was also investigated. In comparison to CD3-CD4(dull) cells, CD3-CD4(bright) cells were slightly larger macrophages with abundant cytoplasmic granules, being present with comparative specificity for normal rat liver and showing negligible effects by intravenous liposome-encapsulated dichloromethylene diphosphate administration. These data suggest that in normal young rat liver these CD3-CD4(dull) and CD3-CD4(bright) cells may be dendritic cells and Kupffer cells that shift from the liver to the spleen or vice versa. These cells may also be able to locally proliferate in liver or spleen due to changes in the developing liver.     

Remote subarachnoid haemorrhage in the posterior fossa following supratentorial surgery

Summary Haemorrhage in regions remote from the site of following intracranial operations is rare, but they do occur. We performed supratentorial craniotomy on 639 patients between the time of introduction of computed tomography (CT) for clinical use in 1983 and June 1992; subarachnoid haemorrhage (SAH) in the posterior fossa occurred postoperatively in six of these cases. These included four patients with tumours in the sellar region, one with an arteriovenous malformation (AVM) and one who underwent superficial temporal artery (STA)-middle cerebral artery (MCA) anastomosis. The ages of the six patients ranged from 17–72 years.Haemorrhage occurred on the day of operation in one case and was detected on CT examination on the day following surgery in the remaining five cases. Of three patients with disturbance of consciousness, two underwent suboccipital craniectomy for reduction of intracranial pressure, while one received barbiturate therapy and later underwent cerebrospinal fluid (CSF) shunt surgery. No special treatment was necessary for the remaining three patients with less serious lesions. Five of the six patients ultimately recovered their pre-operative neurological status apart from the primary diseases.Factors inducing such haemorrhages seem likely to include displacement of the cerebellum by reduced CSF pressure during and after operations, and stretching and tearing of the veins and venules in the sulci of the tentorial surface of the cerebellum. Consideration should therefore be given to the maintenance of an appropriate CSF pressure during operation; this is particularly important in elderly patients and those with an atrophied cerebral cortex.     

An elderly case of systemic lupus erythematosus associated with herpes zoster, anemia, and hemiparesis]

An elderly case of systemic lupus erythematosus (SLE) with suspected hemolytic anemia was experienced. A 70 year-old female was admitted to our hospital on December 31 with complaints of herpetic eruption. She complained of arthralgia since 3 month prior to her admission. The positive findings on examination were skin eruption in the left chest, a systolic heart murmur and a palpable elastic hard liver. Laboratory data showed raised erythrocyte sedimentation rate of 149 mm per hour, decreased Hb (10.1 g/dl), decreased hematocrit (30.0%), increased reticulocytes (33%1000), decreased thrombocytes (73,000/mm3), increased gamma-globulin (33%) and positive rheumatoid factor. During admission, she developed anemia. A stool test for occult blood was negative. The haptoglobin was 38.8 mg/dl and bone marrow aspiration showed increased erythropoiesis, suggesting features of immune hemolytic anemia, except she was negative on Coomb'test. Eye fundi were similar to case of typical bleeding observed in SLE. Concerning immunological findings, the antinuclear factor was x 1280 and the anti-dsDNA antibody was x 80, on which a diagnosis of SLE was based. She experienced numbness of the left arm and developed left hemiparesis 2 days later. Therapy with 15 mg/day prednisone obtained a good response and anemia, abnormal immunological findings and hemiparesis disappeared.     

Species and sex differences of aflatoxin B1-induced glutathione S-transferase placental form in single hepatocytes.

Species and sex differences of aflatoxin B1 (AFB1)-induced glutathione S-transferase placental form (GST-P) positive single hepatocytes have been investigated 48 h after an intraperitoneal injection of AFB1 to young male and female Fischer rats (2 mg AFB1/kg body wt) and male Syrian golden hamsters (6 mg AFB1/kg body wt). The presence of GST-P positive hepatocytes was examined by the immunohistochemical method. Male rats formed three times as many AFB1-induced GST-P positive hepatocytes as females. Pretreatment of both male and female rats with an inhibitor of GSH synthesis, buthionine sulfoximine (BSO) (4 mmol/kg body wt), 2 h and 4 h before AFB1 injection increased AFB1-induced GST-P positive hepatocytes by about 120% above the controls. Male hamsters formed several-fold less AFB1-induced GST-P positive hepatocytes than male rats. Pretreatment with BSO did not increase AFB1-induced GST-P positive hepatocytes in hamsters even though it produced an increase in hepatic necrosis. It appears that GSH and GSH S-transferases play an important role in modulating hepatic AFB1-DNA binding and AFB1-induced GST-P positive hepatocytes in rats and hamsters.     

Suppressive effect of vitamin E on lipid peroxidation in halothane-administered guinea pig liver.

The effect of vitamin E on halothane-induced liver damage was studied in guinea pig halothane hepatitis. Twenty animals were divided into 3 groups, consisting of a control group, a halothane group and a vitamin E + halothane (H) group. The animals in the control group (n = 6) were allowed to inhale air only. The animals in the halothane group (n = 6) and the vitamin E + H group (n = 8) were allowed to inhale 1% halothane with air. Animals in the vitamin E + H group were additionally injected with 30 mg kg-1 of vitamin E 30 minutes prior to inhalation of halothane. Blood was aspirated from the heart immediately after sacrificing to measure the serum activity of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT). A microsomal suspension was prepared from the excised liver. Then the amount of thiobarbituric acid (TBA) reactive products in the microsomes were measured. The amount of tissue TBA-reactive products was increased by inhalation of halothane. The increase in the amount of TBA-reactive product was inhibited by the administration of vitamin E. The serum GPT activity was increased by halothane inhalation. Increased serum GOT and GPT activity were inhibited by the administration of vitamin E. These results demonstrated that vitamin E suppressed halothane-induced liver damage in the guinea pig by inhibiting lipid peroxidation.