Method transfer, partial validation, and cross validation: recommendations for best practices and harmonization from the global bioanalysis consortium harmonization team.

This paper presents the recommendations of the Global Bioanalytical Consortium Harmonization Team on method transfer, partial validation, and cross validation. These aspects of bioanalytical method validation, while important, have received little detailed attention in recent years. The team has attempted to define, separate, and describe these related activities, and present practical guidance in how to apply these techniques.

Insulin-like growth factor-I enhances choleretic action of FK506 in rats.

FK506 is an immunosuppressant for organ transplantation in the same clinical settings as cyclosporine (CsA). In the management of liver transplantation, FK506 has advantages over CsA, in terms of rejection and corticosteroid requirements. Recent clinical findings in liver transplant patients indicate that FK506, but not CsA, stimulates choleresis, suggesting that FK506 treatment may accelerate recovery from cholestatic dysfunction through its choleretic action. Recently, we demonstrated in rats that exogenous treatment with insulin-like growth factor I (IGF-I) results in an increase in bile flow and also that FK506 has the potential to increase hepatic production of IGF-I. However, circulating levels of IGF-I in FK506-treated rats were only 30% higher than in nontreated rats. In this study, we evaluated the combined effect of treatment with both IGF-I and FK506 on bile flow in rats to explore the possibility that combination treatment in liver transplant patients could enhance the choleretic action of FK506, benefiting the transplanted liver. Combination treatment of IGF-I with FK506 resulted in a potent and long-lasting increase in bile flow. Overall, this study demonstrated that IGF-I treatment enhanced the choleretic action of FK506, providing potential clinical utility for combination therapy using these two drugs, in treatment after liver transplantation.

Prediction of functional recovery after coronary bypass surgery using quantitative gated myocardial perfusion SPECT.

Previous studies have demonstrated that myocardial perfusion imaging using 99mTc-tetrofosmin at rest allows viability assessment similar to that obtained with 201Tl imaging and 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET). The simultaneous assessment of perfusion and regional function is now available by quantitative gated myocardial perfusion single-photon emission computed tomography (SPECT). This study was designed to evaluate the utility of quantitative values of wall motion and wall thickening, calculated by quantitative gated myocardial perfusion SPECT, for the prediction of functional recovery after coronary bypass grafting (CABG). Fifty-six patients with coronary artery disease scheduled for CABG were included prospectively. All patients underwent 99mTc-tetrofosmin gated SPECT imaging at rest preoperatively and 3 months after CABG. The myocardium was divided into nine segments and the average quantitative values of regional perfusion (percentage uptake) (%), wall motion (mm) and wall thickening (%) were determined automatically using quantitative gated SPECT (QGS) software. The wall motion score was defined visually using a four-point scale (0, normal; 3, akinesis), and segments with severe asynergy (score of 2 or 3) with patent grafts were assessed. Of 77 segments with severe asynergy, 56 segments showed improved wall motion and 21 segments did not improve after CABG. The area under the receiver operating characteristic curve of wall thickening for the prediction of functional recovery was significantly higher (0.92) than that of the percentage uptake (0.77, P<0.017) or wall motion (0.60, P<0.0001). When each analysis used the optimal threshold, the wall thickening analysis (>or=10%) had a sensitivity of 95% and a specificity of 81%. These values tended to be higher than those of the percentage uptake (sensitivity, 86%; specificity, 67%). The wall motion analysis (>or=1.5 mm) had a significantly lower sensitivity of 75% and specificity of 43% than the wall thickening analysis (P=0.0038 and P=0.011, respectively). The results indicate that wall thickening, calculated by QGS software, may be more useful than regional perfusion or wall motion analysis for the prediction of functional recovery after CABG. The areas of asynergy with relatively preserved wall thickening may have the potential for improved function despite severely decreased perfusion.

Transient left ventricular dysfunction in ischaemic myocardium after stress: comparative study with exercise and pharmacological stress gated myocardial single photon emission computed tomography.

In ischaemic heart disease patients, transient left ventricular dysfunction is observed due to post-exercise stunning. The aim of this study was to determine whether transient left ventricular dysfunction could also be seen after short-acting pharmacological stress (adenosine triphosphate). A 1 day rest/stress gated myocardial single photon emission computed tomography was performed on 362 patients suspected of having ischaemic heart disease by exercise (n=199) or short-acting pharmacological stress (n=163). Left ventricular ejection fraction were estimated both at rest and stress. Based on perfusion findings, patients were subdivided into ischaemia, fixed defect and normal group. For the ischaemia and fixed defect group, left ventricular ejection fraction after stress was significantly decreased compared with the resting value by exercise stress (ischaemia group, 57.5+/-11.0 vs 60.4+/-10.4; fixed defect group, 47.7+/-16.7 vs 49.6+/-16.8; P<0.01), but not by pharmacological stress (ischaemia group, 55.8+/-13.4 vs 57.1+/-13.8; fixed defect group, 50.8+/-13.5 vs 50.6+/-13.1; P=NS). In the normal group, left ventricular ejection fraction after stress was not significantly changed by either exercise (65.7+/-10.4 vs 66.8+/-10.2; P=NS) or pharmacological stress (63.0+/-11.7 vs 64.0+/-12.1; P=NS). It is concluded that a transient decrease in left ventricular ejection fraction after stress was observed following post-exercise, not following a short-acting pharmacological stress in patients showing perfusion abnormalities. Transient left ventricular dysfunction may be the result of post-exercise stunning, not from subendocardial hypoperfusion induced by short-acting pharmacological stress.

Value and limitation of myocardial fluorodeoxyglucose single photon emission computed tomography using ultra-high energy collimators for assessing myocardial viability.

Single photon emission computed tomography (SPECT) using ultra-high energy collimators permits wide clinical application of (18)F-fluorodeoxyglucose (FDG) imaging without the use of expensive positron emission tomography (PET) cameras. This study was designed to evaluate the value of FDG SPECT using ultra-high energy collimators in assessing myocardial viability compared with FDG PET on a regional basis. We prospectively studied 33 patients with ischaemic heart disease. The patients were injected with 555 MBq of FDG under a hyperinsulinaemic glucose clamp, and FDG PET was performed 40 min later. FDG SPECT using ultra-high energy collimators was performed immediately after FDG PET. The images of the left ventricular myocardium were divided into nine segments and the regional defect score was assessed visually using a four-point scale (0=normal to 3=defect). Regional FDG uptake (%uptake) was quantitatively analysed using polar maps. In 297 segments of all the 33 patients, agreement between the defect scores based on FDG SPECT images and those based on FDG PET images was 70%, and agreement within one rank was 96% (kappa value=0.52). The %uptake based on FDG SPECT images significantly correlated with that based on FDG PET images (r=0.77, P<0.01). However, the defect scores in the inferior wall based on FDG SPECT images were higher (1.41+/-1.14) than those based on FDG PET images (1.06+/-1.12, P<0.01). When the viable region is defined as %uptake > or =50% in FDG PET studies, the optimal cut-off level of %uptake based on FDG SPECT images was 60% in the anterior wall, apex, septum and lateral wall (accuracies, 97%, 93%, 96% and 99%, respectively), and 45% in the inferior wall (accuracy, 99%). It is concluded that FDG SPECT using ultra-high energy collimators can be used for the assessment of myocardial viability as accurately as FDG PET. However, a slight difference was observed in the defect scores mainly due to attenuation in the inferior wall. Therefore, a slightly different cut-off level for assessing myocardial viability should be applied to the inferior wall when using FDG SPECT.

Validation of left ventricular function from gated single photon computed emission tomography by using a scintillator-photodiode camera: a dynamic myocardial phantom study.

A scintillator-photodiode camera is able to acquire single photon emission computed tomography (SPECT) images by using a rotating chair system. We validated the left ventricular (LV) parameters of this camera system utilizing a dynamic myocardial phantom. Gated myocardial SPECT of a dynamic myocardial phantom (Hokkaido University type; end diastolic volume (EDV), 143 ml; end systolic volume (ESV), 107 ml; ejection fraction (EF), 25%) was performed with this scintillation camera. LV parameters were calculated using pre-installed software (Mirage Myocardial Perfusion SPECT) (study 1) and the other software (QGS; Cedars-Sinai) (study 2). For comparison, SPECT from a traditional Anger camera were processed by the QGS software (study 3). The estimated volumes were similar among the three studies (EDV, 110+/-8 ml in study 1, 112+/-2 ml in study 2 and 111+/-1 ml in study 3; ESV, 86+/-8 ml in study 1, 93+/-4 ml in study 2 and 91+/-2 ml in study 3). The estimated EFs were 23+/-3%, 17+/-2%, and 18+/-1%, respectively. The calculated volume within each study was underestimated by approximately the same degree. However, each estimated EF value for each study was close to the actual value. The estimated LV function using the scintillator-photodiode camera system may be considered as a suitable alternative to the traditional Anger camera system.

Accuracy and reproducibility of left ventricular function from quantitative, gated, single photon emission computed tomography using dynamic myocardial phantoms: effect of pre-reconstruction filters.

We have investigated the accuracy and reproducibility of left ventricular (LV) functions using quantitative, gated, single photon emission computed tomography (SPECT) software dependent on critical frequencies of pre-reconstruction filters. This study incorporated dynamic myocardial phantoms (myocardial and cone shapes). Gated SPECT of 8-interval sets were pre-filtered with Butterworth filters (critical frequency varying between 0.16 and 1.16 cycles/cm, order 5) and with no filter. Phantoms were repositioned, and SPECT acquisitions were repeated. As the critical frequency increased, the estimated LV volume increased to reach a plateau at the level of the critical frequency, 0.54 cycles/cm. Conversely, the values of ejection fractions, wall motion and wall thickening with different filters which used critical frequency of > or =0.39 cycles/cm remained unchanged. However, LV functions and volumes were underestimated when any pre-reconstruction filter was used. Standard deviations of LV functions after repeated measurements were unaffected by different filters with critical frequencies of > or =0.39 cycles/cm. Standard deviations of LV volume, ejection fraction, wall motion and wall thickening were <2.2 ml, <0.9%, <0.6 mm and <8.7%, respectively. Therefore, with the exception of low critical frequencies, LV functions and volumes were highly reproducible when these routine reconstruction filters were used.

High-speed plasmid isolation using 96-well, size-exclusion filter plates.

The accelerating pace of genomics analysis has necessitated the abbreviation of DNA sample preparation protocols. We have developed a size-exclusion-based system for the rapid isolation ofplasmid DNA in a 96-well microplate format. This high-speed protocol employs a modified alkaline lysis methodfor the preparation of the bacterial lysate, followed by three short vacuum filtration steps. Unlike traditional bind/wash/elute methods, there is no need to use chaotropic salts or ethanol. The samples are recovered from the top side of the MultiScreen96 PLASMID plates. Starting with bacterial cell pellets, the entire prycedure for purifying the plasmid DNA can be performed in 30 min with a multichannel pipettor. The high yields, reproducibility, and quality of the plasmids make this system a good choice for any cloning or DNA sequencing operation.

Processing and mechanical properties of autogenous titanium implant materials.

Pure titanium and some of its alloys are currently considered as the most attractive metallic materials for biomedical applications due to their excellent mechanical properties, corrosion resistance, and biocompatibility. It has been demonstrated that titanium and titanium alloys are well accepted by human tissues as compared to other metals such as SUS316L stainless steel and Co-Cr-Mo type alloy. In the present study, highly porous titanium foams with porosities

Induction of bone loss by bile duct ligation in rats.

Metabolic bone loss is the most common complication of chronic liver disease. However, there is little information available about bone loss in rats with a ligated bile duct, a biliary-type of experimental cirrhosis model. Therefore, in this study, the effect of bile duct ligation (BDL) on bone mineral density (BMD) and plasma insulin-like growth factor-I (IGF-I) levels was examined in rats. Two weeks after BDL operation, the rats with a ligated bile duct showed a pronounced and significant decrease in both trabecular and cortical BMD of the femur. In these rats, decreases in food intake and plasma IGF-I levels plus elevated levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and bilirubin were also observed. When the effect of dietary restriction (30% or 50%) over 2 weeks was then investigated in normal rats, 50% food restriction resulted in a significant decrease in femoral trabecular bone density although BMD was unchanged by 30% dietary restriction. The plasma levels of IGF-I were also decreased in food-restricted rats. Thus, the decrease in femoral bone density in 50% food-restricted rats was less potent compared with BDL rats; nevertheless, the decrease in plasma levels of IGF-I was almost equally potent in both BDL and food-restricted rats. Overall, this study showed that BDL operation resulted in a pronounced bone loss in rats, but also that this bone loss might not be merely due to the decreases in food intake and plasma IGF-I levels. These results suggest that BDL in rats is a useful experimental cirrhosis model for evaluating the bone loss associated with chronic liver disease.

Purkinje cells in the adult cat cerebellar cortex possess a perineuronal net of proteoglycans.

The Purkinje cells in the adult cat cerebellar cortex were found to possess perineuronal proteoglycans which could be stained with our fine cationic iron colloid and Fujita's highly concentrated aldehyde fuchsin, and digested by chondroitinase ABC/keratanase/ heparitinase and hyaluronidase. The Purkinje cells are surrounded by some collagenous elements which are stained with Gömöri's ammoniacal silver and digested by collagenase. The Purkinje cells also express nerve cell surface glycoproteins which are labeled with lectin Vicia villosa agglutinin and digested by a double treatment with collagenase and endo-alpha-N-acetylgalactosaminidase. Sole digestion by endo-alpha-N-acetylgalactosaminidase never erased the lectin labeling of the nerve cell surface glycoproteins. These findings suggest that the collagenous elements mediate the linkage of the perineuronal proteoglycans to the nerve cell surface glycoproteins. It is presumed that in mice and rats, the perineuronal nets of proteoglycans and nerve cell surface glycoproteins of the Purkinje cells are so thin or coarse that they can not be sufficiently visualized under the light microscope.

Comparison of (18)F-FDG, (131)I-Na, and (201)Tl in diagnosis of recurrent or metastatic thyroid carcinoma.

UNLABELLED: There are several reports about the usefulness of (18)F-FDG PET in thyroid cancer. However, few studies have compared FDG PET with (131)I and (201)Tl scintigraphy. The aim of this study was to evaluate the clinical significance of whole-body FDG PET in differentiated thyroid cancer and to compare the results with those obtained from (131)I and (201)Tl scintigraphy. METHODS: Whole-body FDG PET was performed on 32 patients (10 men, 22 women; age range, 30-77 y; mean age, 54 y) with differentiated thyroid cancer (5 cases of follicular cancer and 27 of papillary cancer) after total thyroidectomy. An overall clinical evaluation was performed, including cytology, thyroglobulin level, sonography, MRI, and CT, to allow a comparison with functional imaging results for each patient. Metastatic regions were divided into five areas: neck, lung, mediastinum, bone, and other. Multiple lesions in one area were defined as one lesion. The tumor-to-background ratio (TBR) was measured for the lesions that were positive for both (201)Tl uptake and FDG PET uptake. RESULTS: The number of lesions totaled 47. Forty-one (87%) were detected by all scintigraphic methods. FDG uptake was concordant with (131)I uptake in only 18 lesions (38%). FDG uptake was concordant with (201)Tl uptake in 44 lesions (94%). Only one lesion was negative for FDG uptake and positive for (201)Tl uptake, and two lesions were positive for FDG uptake and negative for (201)Tl uptake. A significant correlation was seen between the TBR of (201)Tl and that of FDG (r = 0.69; P<0.05). CONCLUSION: These data indicate that for detecting metastatic lesions, FDG PET and (131)I scintigraphy may provide complementary information, whereas FDG PET may provide results similar to those of (201)Tl scintigraphy. Thus, the combination of (131)I scintigraphy and FDG PET (or (201)Tl scintigraphy) is the method of choice for detecting metastatic thyroid cancer after total thyroidectomy.

Induction of choleresis by immunosuppressant FK506 through stimulation of insulin-like growth factor-I production in the liver of rats.

FK506 (Tacrolimus) is an effective immunosuppressant currently used worldwide in organ transplantation. Based on our recent findings that insulin-like growth factor-I (IGF-I) is important for the stimulation of choleresis in vivo, in this study we investigated the effect of FK506 on bile flow and the plasma and hepatic levels of IGF-I in rats. Intravenous treatment of rats with FK506 resulted in a significant increase in bile flow, whereas cyclosporin A induced a significant decrease. A significant increase in plasma levels of IGF-I was observed in rats 30 min after a single intravenous administration of FK506. Oral treatment of rats with FK506 for 1 week also resulted in an increase in both plasma and hepatic levels of IGF-I. Overall, this study showed that FK506 treatment increased bile flow and also induced an increase in the plasma and hepatic levels of IGF-I in rats, suggesting that a stimulation of hepatic IGF-I production by FK506 may contribute to its choleretic profile.

Stimulation of choleresis by insulin-like growth factor-I in rats.

Insulin-like growth factor-I (IGF-I) is an endogenous growth factor which is mainly produced in the liver. The functions of IGF-I can be summarized as growth-promoting and insulin-like metabolic actions. In the present study, the effect of IGF-I on bile flow and bile acid secretion was investigated in rats. In normal rats bile flow was significantly increased by single exogenous administration of IGF-I, and by 1 week treatment of IGF-I, both bile flow and bile acid secretion were significantly increased. Moreover, to further understand the relationship between IGF-I and bile acid secretion, hypophysectomized rats were next used. We found that the decreases in bile flow and bile acid secretion observed in rats after hypophysectomy, as well as the decrease in the endogenous level of IGF-I in the blood, were partially reversed by 1 week exogenous IGF-I treatment. Overall, this study showed that IGF-I stimulates choleresis associated with an elevation of bile acid secretion in both normal and hypophysectomized rats when exogenously administered, suggesting the importance of IGF-I in the stimulation of choleresis in vivo.

Lesser reduction in bone mineral density by the immunosuppressant, FK506, compared with cyclosporine in rats.

BACKGROUND: Posttransplantation osteopenia leading to osteoporosis induced commonly by treatment with immunosuppressants including cyclosporine (CsA) is a severe complication and results in lowering the quality of life in patients receiving organ transplantation. FK506 is a newly developed immunosuppressant and is currently being used for the prevention of rejection after organ transplantation. In this study, to investigate whether FK506 as well as CsA would cause osteopenia or not, we evaluated the effect of FK506 on bone mineral density and several parameters relevant to bone metabolism in comparison with that of CsA using normal rats. METHODS: Ten-week-old male Sprague-Dawley rats were treated with FK506 (vehicle, 1 mg/kg, and 3.2 mg/kg) or CsA (vehicle, 10 mg/kg, and 32 mg/kg) by daily oral gavage for 28 days. Bone mineral density of the femur, plasma insulin-like growth factor I (IGF-I), and urinary deoxypyridinoline were determined by peripheral quantitative computerized tomography, radioimmunoassay, and enzyme-linked immunosorbent assay, respectively. RESULTS: The reduction in bone mineral density of the femur was observed in both FK506- and CsA-treated rats. The reduction in CsA-treated rats, however, was statistically significant and strikingly severe, whereas that in FK506-treated rats was much less severe than CsA. Plasma IGF-I levels were significantly elevated in FK506-treated rats but not in CsAtreated rats. Urinary deoxypyridinoline levels were unchanged in FK506-treated rats but elevated in CsA-treated rats. CONCLUSIONS: Compared with CsA, FK506 does not appear to induce severe osteopenia by high-turnover bone metabolism in the rat by mediating via IGF-I induction in part. The results suggest that FK506 may exert favorable effects on bone metabolism in patients with organ transplantation compared with CsA. To assess this idea, further clinical investigations focused on bone metabolism will be required.

Determination of imidapril and imidaprilat in human plasma by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

A sensitive and specific assay of imidapril and its active metabolite, imidaprilat, in human plasma has been developed. This method is based on rapid isolation and high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS-MS). Imidapril and imidaprilat were isolated from human plasma using OASIS HLB (solid-phase extraction cartridge), after deproteinization. The eluent from the cartridge was evaporated to dryness, and the residue was reconstituted in mobile phase and injected into the HPLC-ESI-MS-MS system. Each compound was separated on a semi-micro ODS column in acetonitrile-0.05% (v/v) formic acid (1:3, v/v). The selected ion monitoring using precursor-->product ion combinations of m/z 406-->234 and 378-->206, was used for determination of imidapril and imidaprilat, respectively. The linearity was confirmed in the concentration range of 0.2 to 50 ng/ml in human plasma, and the precision of this assay, expressed as a relative standard deviation, was less than 13.2% over the entire concentration range with adequate assay accuracy. The HPLC-ESI-MS-MS method correlates well with the radioimmunoassay method, therefore, it is useful for the determination of imidapril and imidaprilat with sufficient sensitivity and specificity in clinical studies.

Immobilon-Ny+ nucleic acid blotting membrane: an advanced nylon membrane optimized for superior fixation and reprobing.

The use of charged nylon membranes in nucleic acid blotting applications has become an important factor in the success of hybridization-based assays. Retention of nucleic acids on these membranes is promoted by baking at 80 degrees C under vacuum or by exposure to short wavelength UV light, with the latter method preferred. Immobilon-Ny+ is an advanced, positively charged nylon membrane that has been optimized to show superior retention of target DNA and RNA under hybridization conditions. Higher signal levels are obtained in these assays compared to competitive membranes, even after 13 cycles of probing. This report illustrates the superior performance of Immobilon-Ny+ in 32P and chemiluminescent hybridization assays on blotted DNA and RNA.

[Visualization of normal organs in whole-body FDG-PET imaging].

It is important to know FDG accumulation in the normal distributions for interpreting whole-body PET imaging for tumor detection. Twenty-eight normal subjects were studied with whole-body PET imaging and were examined the intensity of FDG uptake in major organs and the factors which caused it's variety. Emission images were acquired and images were reconstructed without attenuation correction. The intensity of FDG uptake was classified into 4 grades visually. No accumulation was found in the thyroid, the esophagus and the spleen. The oral cavity, the liver, the stomach, and the colon were visualized in all subjects. The laryngeal muscle, the cervical muscle, and the heart accumulated FDG with various grade from 1 to 4 grades. No association was found between the intensity of uptake in the organs and volunteer's age. The fasting time was shorter in volunteers whose heart showed "high" grade than those showed less accumulation (p < 0.05). Serum concentration of free fatty acid was significantly lower in them, too (p < 0.05). Various FDG uptake was observed in many organs, especially the laryngeal muscle, the cervical muscle, and the heart. In our study, there was no facter which caused FDG uptake in organs except for the fasting time and the value of free fatty acid in the heart. Such analysis of whole-body FDG distributions in the normal subjects is valuable for tumor detection with FDG-PET.

Coexistence of rare arteries in the human celiaco-mesenteric system.

Some rare anomalies of the celiaco-mesenteric system were observed postmortem in a Japanese adult male: a) The left gastric, common hepatic, splenic and superior mesenteric arteries arose independently from the abdominal aorta. b) The anterior inferior pancreaticoduodenal artery of the superior mesenteric artery issued a hepatic artery which ascended along the anterior surface of the pancreas and gave off the right gastroepiploic, right gastric and cystic arteries. c) The common hepatic artery gave off an anastomosing branch to the superior mesenteric artery. d) The left gastric artery gave off the left accessory hepatic artery. e) The splenic artery issued the accessory middle colic artery. f) The left inferior phrenic artery gave off the esophageal branch. These anomalies are discussed in light of a typological system which we proposed in a previous paper for the celiaco-mesenteric system.

Effects of phenobarbital on aniline metabolism in primary liver cell culture of rats with ethionine-induced liver disorder.

In experiment 1, the amount of aniline (AN) metabolites in the primary cell culture medium of the liver cells obtained from ethionine (ET)-treated rats was compared with that of the control (normal) rats. Although the metabolites detected in both groups were p-aminophenol (p-AP), N-acetyl-p-AP (AAP), acetoanilide (AAN), AAP-glucuronide (AAPG), phenylhydroxylamine sulfate (PHAS) and p-AP-glucuronide (p-APG), the amount of AAP was lower and that of p-APG was markedly higher in the ET-treated rats than in the control rats. In experiment 2, phenobarbital (PB) was orally administered to the ET-treated and control rats at a dose of 100 mg/kg. The time course changes in AN metabolites in the primary cell culture medium of liver cells obtained at 2 or 48 hr after PB treatment were compared with those without PB treatment. In the ET-treated rats, the amount of PHAS was slightly higher at 2 hr after PB treatment, and that of AAP was lower and that of p-APG was higher at 48 hr after PB treatment as compared with those without PB treatment. In the control rats, the amounts of AAP, AAN, p-AP and p-APG at 2 hr after PB treatment remained lower than those without PB treatment, and that of AAP was markedly lower and that of p-APG was higher at 48 hr after PB treatment as compared with those without PB treatment. These findings indicated greater detoxication in the primary liver cell culture in the ET-treated rats than in the control rats. Furthermore, detoxication was greater in the primary cell culture of liver cell obtained from the ET-treated rats after PB treatment than from those without PB treatment, because the production of acetylates (AAP) decreased and p-APG increased (induction of conjugated enzyme) in the PB treatment group.