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PubMed2005

Changes in tumor cell response due to prolonged dose delivery times in fractionated radiation therapy.

PURPOSE: Dynamic radiation therapy, such as intensity-modulated radiation therapy, delivers more complex treatment fields than conventional techniques. The increased complexity causes longer dose delivery times for each fraction. The cellular damage after a full treatment may depend on the dose rate, because sublethal radiation damage can be repaired more efficiently during prolonged dose delivery. The goal of this study was to investigate the significance of this effect in fractionated radiation therapy. METHODS AND MATERIALS: The lethal/potentially lethal model was used to calculate lesion induction rates for repairable and nonrepairable lesions. Dose rate effects were analyzed for 9 different cell lines (8 human tumor xenografts and a C3H10T1/2 cell line). The effects of single-fraction as well as fractionated irradiation for different dose rates were studied. RESULTS: Significant differences can be seen for dose rates lower than about 0.1 Gy/min for all cell lines considered. For 60 Gy delivered in 30 fractions, the equivalent dose is reduced by between 1.3% and 12% comparing 2 Gy delivery over 30 min per fraction with 2 Gy delivery over 1 min per fraction. The effect is higher for higher doses per fraction. Furthermore, the results show that dose rate effects do not show a simple correlation with the alpha/beta ratio for ratios between 3 Gy and 31 Gy. CONCLUSIONS: If the total dose delivery time for a treatment fraction in radiation therapy increases to about 20 min, a correction for dose rate effects may have to be considered in treatment planning. Adjustments in effective dose may be necessary when comparing intensity-modulated radiation therapy with conventional treatment plans.

Algorithms↗
PubMed2004

Alpha/beta ratio for arteriovenous malformations estimated from obliteration rates after fractionated and single-dose irradiation.

BACKGROUND AND PURPOSE: Results from single-dose radiosurgery have failed to yield reasonable alpha/beta ratios for obliteration rates of arteriovenous malformations (AVMs) in the framework of the linear-quadratic approach. We used outcome data from single-dose and fractionated radiotherapy for AVM to approach this problem. PATIENTS AND METHODS: AVM obliteration rates observed in an updated historical series of fractionated radiotherapy and from six recent series of single-dose stereotactic radiosurgery were analyzed. Reciprocal total doses (1/D) and fraction sizes (d) of isoeffective fractionation schemes were entered into the rearranged form of the linear-quadratic equation: 1/D = (alpha/E) + (beta/E)d, and alpha/beta-ratios were calculated from the parameters of the regression line. RESULTS: Fractionated radiotherapy with 20 Gy/4 Gy fractions, 50 Gy/2 Gy fractions and single-dose radiosurgery of approximately 13 Gy were isoeffective with crude obliteration rates of 13%. The analysis yielded an alpha/beta-ratio of 3.5 Gy. For small-sized AVMs (<3 cm), alpha/beta-ratios of 4.6-6.4 Gy were obtained. CONCLUSION: These results support the view that radiosurgery for AVM can be understood as a typical late tissue effect with a high fractionation sensitivity. Fractionated radiotherapy is ineffective for AVMs and should be evaluated carefully in other benign targets.

Dose Fractionation, Radiation↗
PubMed2004

Treatment margins and treatment fractionation in conformal radiotherapy of muscle-invading urinary bladder cancer.

BACKGROUND AND PURPOSE: Different treatment margins and fractionation schedules are used in conformal radiotherapy (CRT) of urinary bladder cancer. This study compared intestine and rectum dose-volume histogram (DVH) data and normal tissue complication probability (NTCP) estimates for various clinically applied margins and fractionation schedules in bladder irradiation. PATIENTS AND METHODS: Normal tissue dose distributions in fifteen bladder cancer patients treated with CRT were studied using standard three- and four-field configurations. The impact of margin width on intestine and rectum dose distributions was initially evaluated using DVH data. NTCP modelling with the probit model was used to compare the impact of choice of margin size and fractionation schedule. The analysis included margin combinations of 1.0 cm isotropic (narrow margins) and 1.2-2.0 cm non-isotropic (wide margins) and fractionation schedule alternatives of 52.5Gy/20, 55Gy/20, 57.5Gy/20 and 64Gy/32. RESULTS: Using wide as compared to narrow margins, the volumes of intestine and rectum receiving high doses increased by factors of approximately two and four, respectively. Similar differences between wide and narrow margins were found when calculating intestine and rectum NTCPs. The impact of margin size depended strongly on the volume effect expressed by the NTCP model parameters. With standard parameters, however, the choice of margins and fractionation schedule had a similar impact on intestine NTCPs, while for the rectum, the choice of margin had a greater impact than the choice of fractionation. For a given margin size, the intestine and rectum NTCPs for the 55Gy/20 and the 64Gy/32 schedules were comparable. For clinics using narrow margins and a fractionation of 52.5Gy/20, the NTCP modelling suggested that a change in fractionation schedule (to 55Gy/20 or 64Gy/32) or a change to wide margins would have a similar effect on the intestine NTCP predictions. CONCLUSIONS: This modelling study documented that the choice of margins was as important as the choice of fractionation in terms of intestine and rectum DVH data and NTCP predictions.

Aged↗
PubMed2005

Decreased repopulation as well as increased reoxygenation contribute to the improvement in local control after targeting of the EGFR by C225 during fractionated irradiation.

BACKGROUND AND PURPOSE: Inhibition of repopulation and enhanced reoxygenation has been suggested to contribute to improvement of local tumour control after fractionated irradiation combined with inhibitors of the epidermal growth factor receptor (EGFR). The present study addresses this hypothesis in FaDu human squamous cell carcinoma. For this tumour model marked repopulation and incomplete reoxygenation during fractionated irradiation has previously been demonstrated. Furthermore, the anti-EGFR monoclonal antibody C225 has been shown to significantly improve the results of fractionated irradiation in this tumour. MATERIALS AND METHODS: FaDu tumours in nude mice were irradiated with 18 fractions in 18 days (18f/18d) or 18 fractions in 36 days (18f/36d). Three Gy fractions were given either under ambient or under clamp hypoxic conditions. C225 or carrier was applied four times during the course of treatment. Fractionated irradiations were followed by graded top-up doses to obtain complete dose-response curves for local tumour control. Tumour control dose 50% (TCD50) was determined at day 120 after end of treatment. RESULTS: Significant repopulation and reoxygenation occurred during fractionated irradiation of FaDu tumours (P-values between 0.028 and <0.001). Application of C225 significantly decreased TCD50 for 18f/36d under ambient conditions (P=0.04). Bootstrap analysis revealed decreased repopulation and increased reoxygenation after application of C225 (P=0.06 for the combined effect). This was further corroborated by a significant effect of C225 on the 'repopulated' dose under ambient conditions which is influenced by both, reoxygenation and repopulation (P=0.012). CONCLUSIONS: Our study provides evidence that both decreased repopulation as well as increased reoxygenation contribute to the improvement of local control after targeting of EGFR by C225 during fractionated irradiation of FaDu tumours.

Animals↗
PubMed2006

Phosphorylated histone H2AX in relation to cell survival in tumor cells and xenografts exposed to single and fractionated doses of X-rays.

BACKGROUND AND PURPOSE: Human tumor cell lines grown as monolayers or xenograft tumors were exposed to single or multiple fractions of X-rays and the ability to use residual gammaH2AX to identify radiosensitive cells was assessed. MATERIALS AND METHODS: Twenty-four hour after exposure to single or daily fractions of X-rays, human tumor cells from monolayers or xenografts were analyzed for clonogenic surviving fraction. Cells were also fixed and labeled with anti-gammaH2AX antibodies for analysis by flow and image cytometry. The relative amount of residual gammaH2AX and the percentage of cells with <3 foci were compared with the clonogenic surviving fraction measured for the same population. RESULTS: The fraction of gammaH2AX remaining 24h after X-irradiation relative to peak levels 1h after exposure was correlated with radiosensitivity (SF2) for 18 human tumor cell lines. The fraction of SiHa, C33A and WiDr cells with <3 gammaH2AX foci was predictive of clonogenic surviving fraction for both monolayer cells exposed to either single doses or up to 5 fractions. Similar results were obtained using cells from xenograft tumors of irradiated mice. CONCLUSION: The percentage of tumor cells that retain gammaH2AX foci 24h after single or fractionated doses appears to be a useful measure of cellular radiosensitivity that is potentially applicable in the clinic.

Animals↗
PubMed1998

Characterization of triacylglycerol biosynthesis in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora.

Triacylglycerol (TG) biosynthetic enzymes were characterized in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora. When the membrane or lipid body fraction of this fungus was incubated with [14C]oleoyl-CoA without adding exogenous acyl acceptors, radioactivity was incorporated predominantly into TG, indicating that diacylglycerol acyltransferase (DGAT) used endogenous diacylglycerol to incorporate [14C]oleoyl-CoA into TG. Adding glycerol 3-phosphate or lysophosphatidic acid increased radiolabeled phosphatidic acid (PA) in the membrane fraction, which reflected the presence of glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT). Label accumulation did not occur in lysophosphatidic acid when glycerol 3-phosphate was added, suggesting that GPAT was rate-limiting in sequential acylation. In the lipid body fraction, adding lysophosphatidic acid similarly increased radiolabeled PA, whereas adding glycerol 3-phosphate caused much lower increase in radiolabeled PA. Quantitative assays for GPAT, LPAAT, phosphatidic acid phosphatase (PAP), and DGAT essentially confirmed the results obtained from [1-14C]oleoyl-CoA incorporation; LPAAT had the highest activity in the membrane and lipid body fractions, GPAT was significantly lower in the lipid body fraction, and DGAT was much higher in the lipid body fraction. GPAT and LPAAT in the membrane fraction had a strong preference toward oleoyl-CoA as a substrate over palmitoyl-CoA. Results indicate that TG biosynthetic enzymes had different subcellular distribution with the sequence of enrichment in the lipid body fraction, i.e., GPAT<LPAAT approximately PAP<DGAT. This may reflect a TG biosynthetic process from endoplasmic reticulum membranes to lipid bodies in the fungus.

Acyl Coenzyme A↗
PubMed2001

TiO2 colloidal suspension polydispersity analysed with sedimentation field flow fractionation and electron microscopy.

Sedimentation field flow fractionation (SdFFF) operated at multi gravitational field is used to analyse a highly polydisperse TiO2 colloidal suspension. From the initial sample, time dependent eluted fractions are collected and submitted to electron microscopy (EM) shape and size analysis. To assess the accuracy of FFF in determining the average size of the different fractions, these are re-introduced into the channel by means of two different procedures, the on-channel concentration of the fractions and the direct re-injection of pre-concentrated fractions (DRI). Both methods appear accurate to determine the average size of every fraction, associated to a lower recovery in the case of DRI. The fractogram band spreading characteristics of the re-introduced fractions are correlated to the particle size distribution measured by EM. After density determination of fractionated particles, the fractogram is calibrated in terms of size and size distribution using data obtained from EM for each fraction. Quantitative analyses, based on particle counting showed high recovery (80-90%) of the eluted species. However, this loss limited the possibility to extend signal information to a quantitative one.

Chemical Fractionation↗
PubMed2001

Proton RBE for early intestinal tolerance in mice after fractionated irradiation.

BACKGROUND AND PURPOSE: To determine the influence of the number of fractions (or the dose per fraction) on the proton relative biological effectiveness (RBE). MATERIALS AND METHODS: Intestinal crypt regeneration in mice was used as the biological endpoint. RBE was determined relative to cobalt-60 gamma rays for irradiations in one, three and ten fractions separated by a time interval of 3.5h. Proton irradiations were performed at the middle of a 7-cm Spread Out Bragg Peak (SOBP). RESULTS: Proton RBEs (and corresponding gamma dose per fraction) at the level of 20 regenerated crypts per circumference were found equal to 1.15+/-0.04 (10.0 Gy), 1.15+/-0.05 (4.8 Gy) and 1.14+/-0.07 (1.7 Gy) for irradiations in one, three and ten fractions, respectively. Alpha/beta ratios as derived from direct analysis of the 'quantal radiation response data' were found to be 7.6 Gy for gamma rays and 8.2 Gy for protons. Additional proton irradiations in ten fractions at the end of the SOBP were found to be more effective than at the middle of the SOBP by a factor of 1.14 (1.05-1.23). CONCLUSION: Proton RBE for crypt regeneration was found to be independent of fractionation up to ten fractions. One can expect that it remains unchanged for higher number of fractions as the lethalities for doses smaller than 3 Gy are exclusively due to direct lethal events. As a tendency for increased effectiveness at the end of the SOBP is reported in the majority of the studies, for clinical applications it would be advisable to allow for by arranging a sloping depth dose curve in the deeper part of the target volume. Finally, it must be noticed that most of in vitro and in vivo RBE values for protons are larger than the current clinical RBE (RBE=1.10).

Animals↗
PubMed1998

Audiological assessment before and after fractionated stereotactic irradiation for vestibular schwannoma.

PURPOSE: To find the audiological outcome after LINAC-based fractionated stereotactic irradiation (STI). MATERIALS AND METHODS: Twenty-four patients with vestibular schwannoma treated by fractionated STI between 1991 and 1997 had measurable hearing before STI and were followed audiologically for more than 6 months. The pure tone average (PTA) was measured by averaging the air-conduction threshold for five main frequencies (250-4000 Hz) before and periodically after STI in the 24 patients. Several possible prognostic factors for hearing preservation (defined as a PTA change at the last follow-up of less than 10 dB) were investigated. The median follow-up time was 22 months, ranging from 5 to 69 months. The irradiation schedule was 36 Gy in 20 fractions in 5 weeks to 44 Gy in 22 fractions in 6 weeks followed by 4 Gy/1 fraction boost. RESULTS: The pure tone average before STI was distributed from 7 to 73 dB. Fifty percent of patients showed a change in PTA of less than 10 dB, 79.2% of patients showed a change in PTA of less than 20 dB and 20.8% of patients showed a change in PTA of more than 21 dB at the last follow-up. Only one patient (4%) became deaf. Cases with a sudden loss of hearing were more likely to experience hearing preservation than those with gradual loss of hearing (P<0.05). The mean age was younger in patients whose hearing was preserved (P<0.05). Poor pretreatment PTA appeared to linearly correspond to the changes in PTA (regression coefficient 0.78). The size of the tumor was not related to the change in PTA. No relationship was observed between the maximum or peripheral dose and the PTA change. The real benefit of stereotactic boost after small-field fractionated irradiation was not certain. CONCLUSION: Fractionated STI produced a hearing preservation rate compatible with meticulously collimated multi-spots single fraction irradiation. Further follow-up is required to confirm the long-term benefits of fractionation.

Audiometry↗
PubMed1999

How valid is the assumption of equal effect per fraction?

BACKGROUND AND PURPOSE: The validity of the assumption of equal biological effect with dose per fraction in fractionated radiotherapy has been examined for the acute skin reaction in a rat foot model using a variable number of 2-Gy daily fractions followed by graded top-up doses. MATERIAL AND METHODS: Mature female rats were used. Both hind feet of each rat were irradiated with a range of fractionated and top-up doses of 60Co gamma-rays. The dose-related incidence of moist desquamation was used as an end-point. Quantal data for the incidence of moist desquamation were analysed using probit analysis and ED50 (+/-SE) values were obtained. The results were also compared with predicted values obtained from the application LQ-model. RESULTS: After a single 2-Gy fraction followed by top-up doses 24 h later, the dose effect curve for the top-up doses used was shifted to lower doses as expected and the ED50 for moist desquamation of 19.78 +/- 0.13 Gy was 1.16 Gy less than the ED50 of 20.94 +/- 0.15 Gy for large single dose exposure alone. This implied that only approximately 58% of the initial 2-Gy fraction was effective, and the rest was repaired within a 24-h interval between the 2 Gy and top-up doses. However, after two or three 2-Gy daily fractions the dose effect curves for the subsequent top-up doses moved to the higher doses again and the ED50 for top-up dose increased to 20.33 +/- 0.21 and 20.75 +/- 0.11 Gy, respectively. A further increase in the number of 2-Gy daily fractions shifted the dose effect curves for the top-up doses to lower doses and ED50 values for the top-up doses decreased progressively. CONCLUSIONS: The findings were not in keeping with values predicted based on the assumption of equal effect per fraction and could not be explained by the use of a single alpha/beta ratio in the LQ-model.

Animals↗
PubMed2003

Impact of prolonged fraction delivery times on tumor control: a note of caution for intensity-modulated radiation therapy (IMRT).

PURPOSE: Intensity-modulated radiation therapy (IMRT) allows greater dose conformity to the tumor target. However, IMRT, especially static delivery, usually requires more time to deliver a dose fraction than conventional external beam radiotherapy (EBRT). The purpose of this work is to explore the potential impact of such prolonged fraction delivery times on treatment outcome. METHODS AND MATERIALS: The generalized linear-quadratic (LQ) model, which accounts for sublethal damage repair and clonogen proliferation, was used to calculate the cell-killing efficiency of various simulated and clinical IMRT plans. LQ parameters derived from compiled clinical data for prostate cancer (alpha = 0.15 Gy(-1), alpha/beta = 3.1 Gy, and a 16-min repair half-time) were used to compute changes in the equivalent uniform dose (EUD) and tumor control probability (TCP) due to prolonged delivery time of IMRT as compared with conventional EBRT. EUD and TCP calculations were also evaluated for a wide range of radiosensitivity parameters. The effects of fraction delivery times ranging from 0 to 45 min on cell killing were studied. RESULTS: Our calculations indicate that fraction delivery times in the range of 15-45 min may significantly decrease cell killing. For a prescription dose of 81 Gy in 1.8 Gy fractions, the EUD for prostate cancer decreases from 78 Gy for a conventional EBRT to 69 Gy for an IMRT with a fraction delivery time of 30 min. The values of EUD are sensitive to the alpha/beta ratio, the repair half-time, and the fraction delivery time. The instantaneous dose-rate, beam-on time, number of leaf shapes (segments), and leaf-sequencing patterns given the same overall fraction delivery time were found to have negligible effect on cell killing. CONCLUSIONS: The total time to deliver a single fraction may have a significant impact on IMRT treatment outcome for tumors with a low alpha/beta ratio and a short repair half-time, such as prostate cancer. These effects, if confirmed by clinical studies, should be considered in designing IMRT treatments.

DNA Damage↗
PubMed1998

Fraction size and dose parameters related to the incidence of pericardial effusions.

PURPOSE: The influence of treatment parameters, such as (a) fraction size and (b) average and maximum dose (as derived from three-dimensional (3D) distributions), on the incidence of pericarditis was analyzed. To understand and predict the dose and volume effect on the pericardium, a normal tissue-complication probability model was tested with these complication data. METHODS AND MATERIALS: Patients (n = 57) entered in 3 consecutive University of Michigan protocols of combined modality for treatment of localized esophageal carcinoma, and having 3D treatment planning for radiation therapy were the subject of this study. Univariate and multivariate analyses were performed to determine the significance of the effect of fraction size and dose parameters on the development of any grade of pericarditis. Dose distributions were corrected for the biological effect of fraction size using the linear-quadratic method. Normal tissue complication probability (NTCP) was calculated with the Lyman model. RESULTS: Nonmalignant pericardial effusions occurred in 5 of the 57 patients; all effusions were in patients who received treatment with 3.5 Gy daily fractions. On multivariate analysis, no dose factor except fraction size predicted pericarditis, until the dose distributions were corrected for the effect of fraction size ("bio"-dose). Then, both "bio-average" and "bio-maximum" dose were significant predictive factors (p = 0.014). NTCPs for the patients with pericarditis range from 62% to 99% for the calculations with the "bio"-dose distributions vs. 0.5% to 27% for the uncorrected distributions. DISCUSSION: A normal tissue complication probability (NTCP) model predicts a trend towards a high incidence of radiation pericarditis for patients who have high complication probabilities. It is important to correct the dose distribution for the effects of fractionation, particularly when the fraction size deviates greatly from standard (2.0 Gy) fractionation.

Adenocarcinoma↗
PubMed1999

Fractionated radiosurgery for 9L gliosarcoma in the rat brain.

PURPOSE: Fractionated radiosurgery is being carried out in the clinic to improve the therapeutic ratio of single-dose radiosurgery using various fractionation schemes. Because there is a paucity of experimental radiobiological data in the literature on the tumor response and late-responding normal tissue of critical intracranial structures to radiosurgery, the present animal study was designed to compare the response following a single high dose of radiation with that obtained from calculated fractionated doses of radiosurgery. METHODS AND MATERIALS: Male Fischer rats with 9L gliosarcoma growing in their brains were stereotactically irradiated and assayed for the tumor control rate and brain tissue damage. The radiation dose needed for 50% tumor control (TCD50) was used as the endpoint of the efficacy of radiosurgery. Normal brain damage was measured histologically following a period of time over 270 days. Histological evaluation included hematoxylin-eosin (H & E), Luxol fast blue and periodic acid Schiff (LFB/PAS) for the presence of myelin and glial fibrillary acidic protein (GFAP) for the assessment of astrocytic re-activity. The optical density of optic nerves and chiasms staining with LFB/PAS was quantitatively measured using a computer image analysis to assess the magnitude of demyelination. RESULTS: Radiosurgery (RS) was found to be more effective in curing small tumors than large tumors. The dose required to control 50% of the tumored animals for 120 days was 24, 31, and 40 Gy for 2-, 6-, and 12-day-old tumors, respectively. Using 12-day-old brain tumors, two fractions of 23.5 Gy and three fractions of 18.5 Gy were found to be equivalent to the single dose of 35 Gy for tumor control. For normal brain damages, the visual pathways including optic nerves and chiasm were found to be highly radiosensitive structures. A single dose of 35 Gy produced 100% severe optic neuropathy. The fractionated RS regimens spared substantial optic nerve damage. CONCLUSION: The present data provide a strong radiobiological rationale for the use of fractionated RS in the treatment of tumors located near critical normal structures, including visual pathways. The sparing effect of fractionated RS is greater for late-responding tissues, relative to the rapidly proliferating tumor tissues. This report also characterizes the dose/time tolerance relationship of optic neuropathy after single and fractionated RS.

Animals↗
PubMed2001

Evaluation of hepatic subcellular fractions for Alamar blue and MTT reductase activity.

Alamar blue and MTT are indicators used to measure cytotoxicity of various chemicals in cultured cells. Both Alamar blue and MTT are reduced by mitochondrial enzymes. We observed enhanced fluorescence of Alamar blue when kidney epithelial cells were co-incubated with hepatic post-mitochondrial supernatant (S9) fractions as compared with cells incubated in the absence of S9 fractions. The present studies were carried out to determine whether hepatic cytosolic and/or microsomal enzymes were capable of metabolizing Alamar blue and/or MTT to their reduced products. Livers from female Sprague-Dawley rats were used to prepare S9 fraction, and mitochondrial, microsomal and cytosolic fractions. Fractions containing 1 or 5 mg protein/ml were incubated with Alamar blue or MTT for up to 4 h. Fluorescence (Alamar blue) or absorbance (MTT) were determined and expressed as differences between treated wells and controls. Hepatic fractions (S9, mitochondria, microsomes and cytosol) caused concentration- and time-dependent increases in Alamar blue fluorescence and MTT absorbance. Reduction of Alamar blue and MTT by hepatic S9 fraction was abolished by heating. Reduction of Alamar blue by hepatic mitochondria was approximately equivalent to that catalyzed by hepatic S9 fraction or cytosol. Reduction of MTT by hepatic mitochondria was approximately equivalent to that catalyzed by hepatic S9 fraction or microsomes. These data indicate that mitochondrial, cytosolic and microsomal enzymes reduce Alamar blue and MTT. Therefore, caution should be exercised in ascribing decreases in viability as due solely to mitochondrial damage when using either of these dyes.

Animals↗
PubMed1999

Metabolism of primaquine by liver homogenate fractions. Evidence for monoamine oxidase and cytochrome P450 involvement in the oxidative deamination of primaquine to carboxyprimaquine.

The role of monoamine oxidase (MAO) and cytochrome P450 (P450) in the oxidative deamination of primaquine by rat liver fractions was studied. Rat liver fractions including liver homogenate, mitochondria, microsomes and 100,000 g supematant fractions were prepared from a pool of rat livers and characterised using benzylamine as a probe for MAO activity and N,N-dimethylbenzamide as a probe for P450 N-dealkylation activity. Incubation of all fractions with primaquine yielded carboxyprimaquine as the only metabolite detectable by HPLC. The mitochondrial fraction, which contained MAO activity but not P450 activity, presented the highest Vmax/K(M) value for the formation of carboxyprimaquine (8.5 x 10(-6) dm3mg(-1)h(-1). A substantially lower Vmax/K(M) value (1.3 x 10(-6) dm3mg(-1)h(-1)) was obtained in the microsomal fraction, which contained P450 but not MAO activity. The liver homogenate fraction presented a similar value (1.8 x 10(-6) dm3mg(-1)h(-1), though it contained both enzyme systems. Incubations of all the fractions that presented MAO activity, in presence of the MAO inhibitor pargiline, resulted in a marked inhibition of primaquine oxidation. P450 inhibitor SKF 525-A effectively inhibited primaquine metabolism in the microsomal fraction but inhibition in the liver homogenate was less effective. The results are consistent with an important role for MAO in primaquine biotransformation, though clearly metabolism by P450 has a contribution role.

Animals↗
PubMed1986

Preribosomal ribonucleoprotein particles are a major component of a nucleolar matrix fraction.

Biochemical and morphological studies were performed on Novikoff hepatoma ascites cell nucleolar matrix fractions prepared by deoxyribonuclease I digestion and high-molarity salt extractions essentially according to a published method [Berezney, R., & Buchholz, L. A. (1981) Exp. Cell Res. 20, 4995-5002]. The nucleolar matrix fraction was enriched in polypeptides of molecular mass of 28, 37.5, 40, 70, 72, 110 (protein C23), and 160 kDa, compared to the nuclear fraction in which polypeptides of molecular mass of 31, 33.5, 43.5, 46, 50, 56, and 59 kDa were predominant. About one-fourth of the protein, half of the RNA, and less than 4% of the DNA originally present in the nucleoli remained in the matrix fraction. Addition of single agents such as ethylenediaminetetraacetic acid, ribonuclease A, or mercaptoethanol during preparation had no significant effect on the polypeptide composition of the nucleolar matrix fraction. However, the combination of mercaptoethanol and ribonuclease A caused most of the RNA and protein to be removed, including protein C23 and the 160-kDa polypeptide, with polypeptides in the range of Mr 30 000-50 000 remaining. Electron microscopy of nucleolar matrix fractions revealed the presence of particles similar in size to the granular elements of nucleoli. However, when ribonuclease A and mercaptoethanol were included in the procedure, only amorphous material remained. Many proteins of nucleolar preribosomal RNP particles were also associated with the nucleolar matrix fraction. RNA from the nucleolar matrix fraction was enriched in sequences from 18S and 28S ribosomal RNA. These results indicate that preribosomal RNP particles are major constituents of a nucleolar matrix fraction prepared by the deoxyribonuclease I-high-molarity salt method.

Animals↗
PubMed1997

Analysis of drug/plasma protein interactions by means of asymmetrical flow field-flow fractionation.

PURPOSE: The applicability of Asymmetrical Flow Field-Flow Fractionation (Asymmetrical Flow FFF) as an alternative tool to examine the distribution of a lipophilic drug (N-Benzoyl-staurosporine) within human plasma protein fractions was investigated with respect to high separation speed and loss of material on surfaces due to adsorption. METHODS: Field-Flow Fractionation is defined as a group of pseudochromatographic separation methods, where compounds are separated under the influence of an externally applied force based on differences in their physicochemical properties. This method was used to separate human plasma in its protein fractions. The drug distribution in the fractions was investigated by monitoring the fractionated eluate for drug content by fluorescence spectroscopy. RESULTS: Human plasma was separated into human serum albumin (HSA), high density lipoprotein (HDL), alpha 2-macroglobulin and low density lipoprotein (LDL) fractions in less than ten minutes. Calibration of the system and identification of the individual fractions was performed using commercially available protein reference standards. The influence of membrane type and carrier solution composition on the absolute recovery of N-Benzoyl-staurosporine and fluorescein-isothiocyanate-albumin (FITC-albumin) was found to be quite significant. Both factors were optimized during the course of the investigations. N-Benzoyl-staurosporine was found to be enriched in the fraction containing HSA. CONCLUSIONS: If experimental conditions are thoroughly selected and controlled to suppress drug and plasma protein adsorption at the separation membrane, Asymmetrical Flow FFF shows high recoveries and fast separation of human plasma proteins, and can be a reliable tool to characterize drug/plasma protein interactions. For analytical purposes it has the potential to rival established technologies like ultracentrifugation in terms of ease-of-use, precision, and separation time.

Adsorption↗
PubMed1973

Heterogeneous distribution of glucose 6-phosphatase in rat liver microsomal fractions as shown by adaptation of a cytochemical technique.

1. A novel technique for the subfractionation of rat liver smooth and rough microsomal fractions according to their content of glucose 6-phosphatase is described. This technique, based on the Gomori lead histochemical procedure, involves incubation of smooth and rough microsomal fractions with low concentrations of Pb(NO(3))(2) and glucose 6-phosphate. Control experiments, in which enzyme was assayed in the presence of various amounts of Pb(NO(3))(2) or in which microsomal fractions were reisolated after incubation with low concentrations of Pb(NO(3))(2) and glucose 6-phosphate, showed that lead does not interfere with glucose 6-phosphatase activity. 2. Discontinuous sucrose-density-gradient centrifugation of microsomal fractions which had previously been incubated with various amounts of Pb(NO(3))(2) and glucose 6-phosphate showed that it is possible to subfractionate both smooth- and rough-microsomal fractions into several bands, owing to a differential modification of the density of the microsomal vesicles by the trapping of lead phosphate within them. 3. When the material in the bands obtained by density-gradient centrifugation of incubated microsomal fractions was assayed for glucose 6-phosphatase activity, it was found that the modification of the density of the microsomal fractions was directly related to their relative enrichment in glucose 6-phosphatase activity. Control experiments, in which microsomal fractions were incubated with Pb(NO(3))(2) and glucose 6-phosphate and then treated with EDTA, showed that the subfractionation was not due to aggregation of microsomal vesicles, lead and glucose 6-phosphate. Thus the resolution of microsomal preparations into subfractions with different glucose 6-phosphatase activities is interpreted as indicating heterogeneity of glucose 6-phosphatase distribution in the microsomal vesicles. 4. Electron micrographs of both smooth- and rough-microsomal subfractions show deposits of lead phosphate within the microsomal vesicles. The frequency and extent of these deposits correlate with the different amounts of glucose 6-phosphatase activity measured biochemically. 5. The nature of the heterogeneous distribution of glucose 6-phosphatase is discussed and the more general applicability of the technique for studying membrane fractions containing a heterogeneous distribution of phosphatases is indicated.

Animals↗