Role of Magnetic Resonance Spectroscopy in Evaluation of Cerebral Metabolic Status Before and After Carotid Endarterectomy/Thromboendarterectomy and Carotid Artery Stenting in Patients with Asymptomatic Critical Internal Carotid Artery Stenosis.

BACKGROUND The relative efficacy of carotid endarterectomy (CEA)/thromboendarterectomy (TEA) and carotid artery stenting (CAS) already has been compared in randomized controlled trials and a meta-analysis, but only limited data exist describing the status of cerebral metabolism before and after these interventions. The aim of the present study was to compare metabolic changes before and after treatment of carotid stenosis and assess their potential clinical implications.   MATERIAL AND METHODS Patients with asymptomatic unilateral critical internal CAS were imaged with proton 3T magnetic resonance spectroscopy (H-MRS) because the technique is more sensitive than regular magnetic resonance imaging for detection of the early signs of ischemic events. Abnormal metabolite ratios detected with H-MRS may precede actual morphological changes associated with hypoperfusion as well as reperfusion changes. Ipsilateral and contralateral middle cerebral artery vascular territories were both evaluated before and after vascular intervention. H-MRS was performed within 24 h before and after surgery. Correlations in the metabolic data from H-MRS for N-acetylaspartic acid (NAA)+N-acetylaspartylglutamate, creatinine (Cr)+phosphocreatinine, and phosphocholine+glycerophosphocholine (Cho) were sought. RESULTS H-MRS voxels from 11 subjects were analyzed. Values for dCho/CrI, dCho/CrC and Cho/Naal (P<0.001) were significantly higher ipsilaterally than contralaterally. Ratios for dNaa/ChoC and Cho/NaaC were significantly higher on the non-operated side (P<0.001). CONCLUSIONS H-MRS may be helpful for assessment of patients with CAS, particularly because unlike other modalities, it reveals postoperative changes in metabolic brain status. Initial results indicate the important role of perioperative neuroprotective treatment.

Ultralong circulating choline phosphate liposomal nanomedicines for cascaded chemo-radiotherapy.

Cancer radiation therapy (RT) is limited by endogenous DNA repair of tumor cells and microenvironmental hypoxia in tumor tissues. Herein, we demonstrated an effective cancer chemo-radiotherapy strategy based on choline phosphate liposomal nanomedicines, which inhibit the intrinsic radioresistance of RT and concomitantly harness the RT-induced hypoxia to produce additional toxicity to overcome post-RT radioresistance. To achieve this strategy, a radiotherapy sensitizer, vorinostat, and a hypoxia-activated banoxantrone dihydrochloride (AQ4N) were simultaneously delivered to a tumor using liposomes composed of an inverted polarity lipid 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe). The DOCPe liposomes exhibited a longer blood circulation time and enhanced tumor accumulation, compared to their zwitterionic phosphocholine counterpart. The RT was sensitized by vorinostat to kill non-tolerant normoxic tumor cells efficiently. The irradiation aggravated hypoxia-activated AQ4N to further potentiate RT treatment. This chemo-radiotherapy combination showed excellent tumor treatment efficacy and is promising for future clinical translation.

A genome-wide association study of IgM antibody against phosphorylcholine: shared genetics and phenotypic relationship to chronic lymphocytic leukemia.

Phosphorylcholine (PC) is an epitope on oxidized low-density lipoprotein (oxLDL), apoptotic cells and several pathogens like Streptococcus pneumoniae. Immunoglobulin M against PC (IgM anti-PC) has the ability to inhibit uptake of oxLDL by macrophages and increase clearance of apoptotic cells. From our genome-wide association studies (GWASs) in four European-ancestry cohorts, six single nucleotide polymorphisms (SNPs) in 11q24.1 were discovered (in 3002 individuals) and replicated (in 646 individuals) to be associated with serum level of IgM anti-PC (the leading SNP rs35923643-G, combined ß = 0.19, 95% confidence interval 0.13-0.24, P = 4.3 × 10-11). The haplotype tagged by rs35923643-G (or its proxy SNP rs735665-A) is also known as the top risk allele for chronic lymphocytic leukemia (CLL), and a main increasing allele for general IgM. By using summary GWAS results of IgM anti-PC and CLL in the polygenic risk score (PRS) analysis, PRS on the basis of IgM anti-PC risk alleles positively associated with CLL risk (explained 0.6% of CLL variance, P = 1.2 × 10-15). Functional prediction suggested that rs35923643-G might impede the binding of Runt-related transcription factor 3, a tumor suppressor playing a central role in the immune regulation of cancers. Contrary to the expectations from the shared genetics between IgM anti-PC and CLL, an inverse relationship at the phenotypic level was found in a nested case-control study (30 CLL cases with 90 age- and sex-matched controls), potentially reflecting reverse causation. The suggested function of the top variant as well as the phenotypic association between IgM anti-PC and CLL risk needs replication and motivates further studies.

Plasma Metabolites From Choline Pathway and Risk of Cardiovascular Disease in the PREDIMED (Prevention With Mediterranean Diet) Study.

BACKGROUND: The relationship between plasma concentrations of betaine and choline metabolism and major cardiovascular disease (CVD) end points remains unclear. We have evaluated the association between metabolites from the choline pathway and risk of incident CVD and the potential modifying effect of Mediterranean diet interventions. METHODS AND RESULTS: We designed a case-cohort study nested within the PREDIMED (Prevention With Mediterranean Diet) trial, including 229 incident CVD cases and 751 randomly selected participants at baseline, followed up for 4.8 years. We used liquid chromatography-tandem mass spectrometry to measure, at baseline and at 1 year of follow-up, plasma concentrations of 5 metabolites in the choline pathway: trimethylamine N-oxide, betaine, choline, phosphocholine, and α-glycerophosphocholine. We have calculated a choline metabolite score using a weighted sum of these 5 metabolites. We used weighted Cox regression models to estimate CVD risk. The multivariable hazard ratios (95% confidence intervals) per 1-SD increase in choline and α-glycerophosphocholine metabolites were 1.24 (1.05-1.46) and 1.24 (1.03-1.50), respectively. The baseline betaine/choline ratio was inversely associated with CVD. The baseline choline metabolite score was associated with a 2.21-fold higher risk of CVD across extreme quartiles (95% confidence interval, 1.36-3.59; P<0.001 for trend) and a 2.27-fold higher risk of stroke (95% confidence interval, 1.24-4.16; P<0.001 for trend). Participants in the higher quartiles of the score who were randomly assigned to the control group had a higher risk of CVD compared with participants in the lower quartile and assigned to the Mediterranean diet groups (P=0.05 for interaction). No significant associations were observed for 1-year changes in individual plasma metabolites and CVD. CONCLUSIONS: A metabolite score combining plasma metabolites from the choline pathway was associated with an increased risk of CVD in a Mediterranean population at high cardiovascular risk. CLINICAL TRIAL REGISTRATION: URL: http://www.controlled-trials.com. Unique identifier: ISRCTN35739639.

Validation and Clinical Evaluation of a Novel Method To Measure Miltefosine in Leishmaniasis Patients Using Dried Blood Spot Sample Collection.

To facilitate future pharmacokinetic studies of combination treatments against leishmaniasis in remote regions in which the disease is endemic, a simple cheap sampling method is required for miltefosine quantification. The aims of this study were to validate a liquid chromatography-tandem mass spectrometry method to quantify miltefosine in dried blood spot (DBS) samples and to validate its use with Ethiopian patients with visceral leishmaniasis (VL). Since hematocrit (Ht) levels are typically severely decreased in VL patients, returning to normal during treatment, the method was evaluated over a range of clinically relevant Ht values. Miltefosine was extracted from DBS samples using a simple method of pretreatment with methanol, resulting in >97% recovery. The method was validated over a calibration range of 10 to 2,000 ng/ml, and accuracy and precision were within ±11.2% and ≤7.0% (≤19.1% at the lower limit of quantification), respectively. The method was accurate and precise for blood spot volumes between 10 and 30 µl and for Ht levels of 20 to 35%, although a linear effect of Ht levels on miltefosine quantification was observed in the bioanalytical validation. DBS samples were stable for at least 162 days at 37°C. Clinical validation of the method using paired DBS and plasma samples from 16 VL patients showed a median observed DBS/plasma miltefosine concentration ratio of 0.99, with good correlation (Pearson'sr= 0.946). Correcting for patient-specific Ht levels did not further improve the concordance between the sampling methods. This successfully validated method to quantify miltefosine in DBS samples was demonstrated to be a valid and practical alternative to venous blood sampling that can be applied in future miltefosine pharmacokinetic studies with leishmaniasis patients, without Ht correction.

UHPLC/Q-TOFMS-based plasma metabolomics of polycystic ovary syndrome patients with and without insulin resistance.

Polycystic ovary syndrome (PCOS), characterized with menstrual irregularities, hyperandrogenism and ovulatory abnormalities, is usually companied with insulin resistance (IR) and accounts for one of the most prevalent reproductive dysfunction of premenopausal women. Despite accumulating investigations, diagnostic standards of this pathological condition remain obscure. The aim of present study is to characterize the plasma metabolic characteristics of PCOS patients with and without IR, and subsequently identify the potential biomarkers for the diagnosis of PCOS and its IR complication. A total of 59 plasma samples from eligible healthy controls (CON, n=19), PCOS patients without IR (non-IR PCOS, n=19) and PCOS patients with IR (IR PCOS, n=21) were profiled by an ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOFMS) followed by multivariate statistical analysis. Compared to the healthy controls, significant decrease in the levels of phosphocholines (PCs) and lyso PC (18:2), and increase in trilauric glyceride level were observed in the plasma of IR PCOS. Meanwhile, the significant increase in the levels of saturated fatty acids (palmitic acid and stearic acid) and decanoylcarnitine, and decrease in PC (36:2) and PS (36:0) were found in non-IR PCOS patients. Trilauric glyceride and decanoylcarnitine were identified as the potential biomarkers with the highest sensitivity and specificity for the diagnosis of PCOS patients with and without IR, respectively. Furthermore, based on these alterations of metabolites, MetPA network pathway analysis suggested a profound involvement of the abnormalities of glycerophospholipid, glycerolipid and fatty acid metabolisms in the pathogenesis of PCOS and IR complications. Collectively, LC-MS-based metabolomics provides a promising strategy for complementary diagnosis of PCOS and its IR complication and offers a new insight to understand their pathogenesis mechanisms.

Choline Alleviates Parenteral Nutrition-Associated Duodenal Motility Disorder in Infant Rats.

BACKGROUND: Parenteral nutrition (PN) has been found to influence duodenal motility in animals. Choline is an essential nutrient, and its deficiency is related to PN-associated organ diseases. Therefore, this study was aimed to investigate the role of choline supplementation in an infant rat model of PN-associated duodenal motility disorder. MATERIALS AND METHODS: Three-week-old Sprague-Dawley male rats were fed chow and water (controls), PN solution (PN), or PN plus intravenous choline (600 mg/kg) (PN + choline). Rats underwent jugular vein cannulation for infusion of PN solution or 0.9% saline (controls) for 7 days. Duodenal oxidative stress status, concentrations of plasma choline, phosphocholine, and betaine and serum tumor necrosis factor (TNF)-α were assayed. The messenger RNA (mRNA) and protein expression of c-Kit proto-oncogene protein (c-Kit) and membrane-bound stem cell factor (mSCF) together with the electrophysiological features of slow waves in the duodenum were also evaluated. RESULTS: Rats on PN showed increased reactive oxygen species; decreased total antioxidant capacity in the duodenum; reduced plasma choline, phosphocholine, and betaine; and enhanced serum TNF-α concentrations, which were reversed by choline intervention. In addition, PN reduced mRNA and protein expression of mSCF and c-Kit, which were inversed under choline administration. Moreover, choline attenuated depolarized resting membrane potential and declined the frequency and amplitude of slow waves in duodenal smooth muscles of infant rats induced by PN, respectively. CONCLUSION: The addition of choline to PN may alleviate the progression of duodenal motor disorder through protecting smooth muscle cells from injury, promoting mSCF/c-Kit signaling, and attenuating impairment of interstitial cells of Cajal in the duodenum during PN feeding.

Increased Levels of Sphingosylphosphorylcholine (SPC) in Plasma of Metabolic Syndrome Patients.

Recent developments in lipid mass spectrometry enable extensive lipid class and species analysis in metabolic disorders such as diabesity and metabolic syndrome. The minor plasma lipid class sphingosylphosphorylcholine (SPC) was identified as a ligand for lipid sensitive G-protein coupled receptors playing a key role in cell growth, differentiation, motility, calcium signaling, tissue remodeling, vascular diseases and cancer. However, information about its role in diabesity patients is sparse. In this study, we analyzed plasma lipid species in patients at risk for diabesity and the metabolic syndrome and compared them with healthy controls. Our data show that SPC is significantly increased in plasma samples from metabolic syndrome patients but not in plasma from patients at risk for diabesity. Detailed SPC species analysis showed that the observed increase is due to a significant increase in all detected SPC subspecies. Moreover, a strong positive correlation is observed between total SPC and individual SPC species with both body mass index and the acute phase low grade inflammation marker soluble CD163 (sCD163). Collectively, our study provides new information on SPC plasma levels in metabolic syndrome and suggests new avenues for investigation.

Plasma and cerebrospinal fluid pharmacokinetics of the Akt inhibitor, perifosine, in a non-human primate model.

PURPOSE: Central nervous system tumors are histologically and biologically heterogeneous. Standard treatment for malignant tumors includes surgery, radiation and chemotherapy, yet surgical resection is not always an option and chemotherapeutic agents have limited benefit. Recent investigations have focused on molecularly targeted therapies aimed at critical tumorigenic pathways. Several tumor types, including high-grade gliomas and pediatric pontine gliomas, exhibit Akt activation. Perifosine, an orally bioavailable, synthetic alkylphospholipid and potent Akt inhibitor, has demonstrated activity in some preclinical models, but absent activity in a genetically engineered mouse model of pontine glioma. We evaluated the plasma and cerebrospinal fluid pharmacokinetics of orally administered perifosine in a non-human primate model to evaluate CNS penetration. METHODS: Perifosine was administered orally to three adult rhesus monkeys as a single dose of 7.0 mg/kg perifosine. Serial paired plasma and CSF samples were collected for up to 64 days. Perifosine was quantified with a validated HPLC/tandem mass spectrometry assay. Pharmacokinetic parameters were estimated using non-compartmental methods. CSF penetration was calculated from the areas under the concentration-time curves. RESULTS: Peak plasma concentrations (C max) ranged from 11.7-19.3 µM, and remained >1 µM for >28 days. Time to C max (T max) was 19 h. The median (range) AUCPl was 3148 (2502-4705) µM/h, with a median (range) terminal half-life (t 1/2) of 193 (170-221) h. Plasma clearance was 494 (329-637) mL/h/kg. Peak CSF concentrations were 4.1-10.1 nM (T max 64-235 h). CSF AUCs and t 1/2 were 6358 (2266-7568) nM/h and 277 (146-350) h, respectively. Perifosine concentrations in the CSF remained over  nM for >35 days. The mean CSF penetration was 0.16 %. CONCLUSION: CNS penetration of perifosine after systemic administration is poor. However, levels were measurable in both plasma and CSF for an extended time (>2 months) after a single oral dose.

Interaction of miltefosine with the lipid and protein components of the erythrocyte membrane.

Miltefosine (MT) is an alkylphospholipid that has been approved for the treatment of breast cancer metastasis and visceral leishmaniasis, although its mechanism of action remains poorly understood. Electron paramagnetic resonance spectroscopy of a spin-labeled lipid and a thiol-specific spin label showed that MT causes an increase in the molecular dynamics of erythrocyte ghost membranes and detergent-resistant membranes (DRMs) prepared from erythrocyte ghosts. In the vesicles of lipid raft constituents, it was shown that 20 mol % sphingomyelin could be replaced by 20 mol % MT with no change in the molecular dynamics. The effect of MT in DRMs was more pronounced than in erythrocyte ghosts, supporting the hypothesis that MT is a lipid raft modulator. At the reported MT-plasma concentrations found during the treatment of leishmaniasis (31-90 µg/mL), our measurements in the blood plasma indicated a hemolytic level of 2%-5%. The experiments indicated that MT acts predominantly on the protein component of the membrane. MT aggregates may wrap around the hydrophobic polypeptide chains, forming micelle-like structures that stabilize protein conformations more exposed to the solvent. Proteins with higher hydrophobicity may induce the penetration of the hydrophilic groups of MT into the membrane and cause it to rupture.

Patients with polycystic ovary syndrome have lower levels of IgM anti-phosphorylcholine antibodies than healthy women.

INTRODUCTION: IgM antibodies against phosphorylcholine (IgM anti-PC) are natural autoantibodies, possibly exerting one of the atheroprotective functions of the immune system. Increased levels of these antibodies reduce the development of atherosclerosis in mice, and low levels of IgM anti-PC have been associated with increased risk for cardiovascular disease (CVD). This study compared levels of IgM anti-PC in women with polycystic ovary syndrome (PCOS, n = 111) and healthy controls (n = 79). METHOD: Levels of IgM anti-PC were measured with ELISA. RESULTS: The median level of IgM anti-PC in patients with PCOS was not significantly different compared to control subjects. However, the proportion of patients with PCOS with low levels of IgM anti-PC, defined as number of individuals below the median level, was significantly higher than among healthy controls, p < 0.05. Patients with PCOS in the oldest age quintile had significantly lower level of IgM anti-PC than control subjects of similar age (p < 0.05) and younger women with PCOS (p < 0.01). CONCLUSION: Our results indicate that women with PCOS more frequently display below-median levels of IgM anti-PC than controls and older women with PCOS have lower median anti-PC levels. Further studies of how this finding translates into actual CVD risk in women with PCOS are needed.

Quantification of CLR1401, a novel alkylphosphocholine anticancer agent, in rat plasma by hydrophilic interaction liquid chromatography-tandem mass spectrometric detection.

A rapid and specific LC-MS/MS based bioanalytical method was developed and validated for the determination of 18-(p-iodophenyl)octadecyl phosphocholine (CLR1401), a novel phosphocholine drug candidate, in rat plasma. The optimal chromatographic behavior of CLR1401 was achieved on a Kromasil silica column (50 mm x 3 mm, 5 microm) under hydrophilic interaction chromatography. The total LC analysis time per injection was 2.8 min with a flow rate of 1.5 mL/min under gradient elution. Liquid-liquid extraction in a 96-well format using ethyl acetate was developed and applied for method validation and sample analysis. The method validation was conducted over the curve range of 2.00-1000 ng/mL using 0.0500 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed < or = 5.9% relative standard deviation (RSD) and -10.8 to -1.4% relative error (RE). The method was successfully applied to determine the toxicokinetics of CLR1401 in rats from three dose groups of 0.4, 4.0, and 10.0 mg/kg/day via intravenous administration.

Probucol therapy overcomes the reproductive defect in CTP: phosphocholine cytidylyltransferase beta2 knockout mice.

The synthesis of phosphatidylcholine (PtdCho), the major phospholipid in mammalian cells, is regulated by the CTP:phosphocholine cytidylyltransferase (CCT). Loss of the CCTbeta2 isoform expression in mice results in gonadal dysfunction. CCTbeta2(-/-) females exhibit ovarian tissue disorganization with progressive loss of follicle formation and oocyte maturation. Ultrastructure revealed a disrupted association between ova and granulosa cells and disorganized Golgi apparati in oocytes of CCTbeta2(-/-) mice. Probucol is a cholesterol-lowering agent that stimulates the uptake and retention of lipids carried by lipoproteins in peripheral tissues. Probucol therapy significantly lowered both serum cholesterol and PtdCho levels. Probucol therapy increased fertility in the CCTbeta2(-/-) females 100%, although it did not completely correct the phenotype, the morphological abnormalities in the knockout ovaries or itself stimulate CCT activity directly. These data indicated that a deficiency in de novo PtdCho synthesis could be complemented by altering the metabolism of serum lipoproteins, an alternative source for cellular phospholipid.

Phase II study of daily oral perifosine in patients with advanced soft tissue sarcoma.

BACKGROUND: A multicenter Phase II study was performed to evaluate the clinical activity of an initial loading (150 mg every 6 hours x 4 doses) dose followed by continuous daily oral dosing (100 mg/day) of perifosine in patients with advanced soft tissue sarcomas (STSs). METHODS: Patients with measurable metastatic STS received perifosine as first-, second-, or third-line treatment and underwent disease assessment every 8 weeks until disease progression, excessive toxicity, or patient refusal. RESULTS: Twenty-three patients received 66 cycles (1 cycle = 4 weeks) of perifosine. One partial response of 9 months duration was observed. The overall 3 and 6 month progression-free survival was 22% and 9%. NCI CTC (v2.0) Grade 1 to 2 gastrointestinal toxicity or fatigue were the most common (>50% of subjects) toxicities observed. The steady-state plasma perifosine levels (Css) were similar to prior experience (mean 6 microg/mL). Patients with Css levels >6 microg/mL appeared more likely to remain on study past 2 months than those with levels <6 microg/mL. CONCLUSIONS: Despite not achieving the primary objective of > or =40% 6-month progression-free survival rate, optimism remains for this agent in STS patients. Prolonged responses in heavily pretreated STS patients continue to be observed with perifosine treatment. Continued assessment of perifosine in STS appears warranted, with special attention to specific histologies or tumor characteristics that might identify a more sensitive population and achieving perifosine Css levels >6 microg/mL.

Tumor and normal tissue pharmacokinetics of perifosine, an oral anti-cancer alkylphospholipid.

Clinical use of anti-cancer alkylphospholipids is limited by gastrointestinal toxicity. However, new interest has emerged since it was shown that these drugs enhance the cytotoxic effect of conventional chemotherapy and radiotherapy in preclinical models. The aim of this study was to characterize the pharmacokinetic profile of perifosine, an oral analog of alkylphosphocholine (APC), and to compare in vitro drug uptake with in vivo drug accumulation in three human-derived squamous cell carcinomas (A431, HNXOE and KB). In vitro, KB cells showed a remarkably high uptake and sensitivity for perifosine compared with A431 and HNXOE cells. In vivo, perifosine reached a clinically relevant plasma concentration in mice after a single oral dose of 40 mg/kg. Perifosine was not metabolized and displayed slow elimination, with a terminal half-life of 137 (+/- 20) hours and an apparent volume of distribution of 11.3 l/kg. Comparable tumor accumulation was observed for A431 and HNXOE tumors, whereas perifosine uptake by KB xenografts was substantially higher. Tissue distribution occurred throughout the whole body reaching high perifosine levels in the gastro-intestinal tract, while heart and brain tissue contained relatively low levels. Based on its stability and relatively high tumor uptake in vivo, perifosine is an attractive candidate for further evaluation, e.g. as radiosensitizer.

Quantitative determination of perifosine, a novel alkylphosphocholine anticancer agent, in human plasma by reversed-phase liquid chromatography-electrospray mass spectrometry.

A sensitive and selective reversed-phase LC-ESI-MS method to quantitate perifosine in human plasma was developed and validated. Sample preparation utilized simple acetonitrile precipitation without an evaporation step. With a Develosil UG-30 column (10 x 4 mm I.D.), perifosine and the internal standard hexadecylphosphocholine were baseline separated at retention times of 2.2 and 1.1 min, respectively. The mobile phase consisted of eluent A, 95% 9 mM ammonium formate (pH 8) in acetonitrile-eluent B, 95% acetonitrile in 9 mM ammonium formate (pH 8) (A-B, 40:60, v/v), and the flow-rate was 0.5 ml/min. The detection utilized selected ion monitoring in the positive-mode at m/z 462.4 and 408.4 for the protonated molecular ions of perifosine and the internal standard, respectively. The lower limit of quantitation of perifosine was 4 ng/ml in human plasma, and good linearity was observed in the 4-2,000 ng/ml range fitted by linear regression with 1/x weight. The total LC-MS run time was 5 min. The validated LC-MS assay was applied to measure perifosine plasma concentrations from patients enrolled on a phase I clinical trial for pharmacokinetic/pharmacodynamic analyses.

Quantification of perifosine, an alkylphosphocholine anti-tumour agent, in plasma by pneumatically assisted electrospray tandem mass spectrometry coupled with high-performance liquid chromatography.

An HPLC assay with tandem mass spectrometric detection in the positive-ion Turbo-Ion-Spray (TISP) mode for the fast and sensitive determination of perifosine ((I), D-21266) in human plasma was developed, utilising the structural analogue, miltefosine ((II), D-18506), as internal standard. Automated solid-phase extraction of diluted plasma samples, based on 250-microl plasma aliquots, at pH 6.5, allowed a reliable quantification of perifosine down to 4 ng/ml. Injection of 200 microl of plasma extracts onto a 100x3 mm normal-phase analytical column at a flow-rate of 0.5 ml/min provided retention-times of 2.4 and 2.1 min for perifosine (I) and the internal standard (II), respectively. The standard curves were linear from 4 to 2000 ng/ml using weighted linear regression analysis (1/Y2). The inter-assay and intra-assay accuracies for the calibration standards were within +0.9% and -0.2%, exhibiting precisions (C.V.) of +/-6.5 and +/-7.3%, respectively. Up to 100 unknowns may be analysed each 24 h per analyst.

Choline metabolism in breast cancer; 2H-, 13C- and 31P-NMR studies of cells and tumors.

Choline metabolism in breast cancer cells and tumors has been investigated by multinuclear NMR in order to provide the biochemical basis for the presence of high phosphocholine in breast carcinoma relative to benign breast tumors and normal breast tissue. Choline was found to be transported into MCF7 human breast cancer cells and rapidly phosphorylated to phosphocholine which was then accumulated in the cells to high concentrations. The increased level of phosphocholine did not affect the rate of synthesis of phosphatidylcholine, indicating tight regulation of this pathway. The incorporation of [1,2-13C]choline (100 microM) into phosphocholine and phosphatidylcholine after 24 h was 69.5 and 36% of the total respective pools. Incorporation of 2H9-choline to tumors implanted in nude mice was achieved by infusing the deuterated choline to the blood circulation. The metabolism of deuterated choline was then monitored by 2H localized MRS. The blood level of choline before the infusion was 58.6 +/- 10.3 microM (measured by 1H-NMR of plasma samples) and increased approximately 5-fold during the infusion (measured by 2H-NMR). This increase in the blood level resulted in a gradual increase of a signal at 3.2 ppm due to deuterated choline metabolites. It appears that the increased availability of choline in the blood circulation leads to accumulation of phosphocholine in the tumors by the same mechanism as in the cells.

Distribution of hexadecylphosphocholine and octadecyl-methyl-glycero-3-phosphocholine in rat tissues during steady-state treatment.

The distribution of the alkylphosphocholine hexadecylphosphocholine (He-PC) and the (alkyl)lysophospholipid 1-0-octadecyl-2-0-methyl-rac-glycero-3-phosphocholine (ET18-OCH3) was analyzed in rats. The compounds were given orally at a daily dose of 75 mumol/kg body weight. After 6, 11, and 18 days, three rats in each treatment group were killed and the drug concentration in various tissues and fluids was determined. With the exception of the kidney (He-PC) and brain (He-PC and ET18-OCH3), steady-state levels of the drugs could be achieved in all organs investigated and in serum. Maximal concentrations of He-PC were found in the kidney, adrenal glands, and spleen, whereas the highest concentrations of ET18-OCH3 were detected in the adrenal glands, spleen, and small intestine. The concentrations of He-PC exceeded those of ET18-OCH3 in most tissues by a factor of about 2-25. Since samples of urine and feces did not contain detectable amounts of the compounds, the absorption of both lipid analogues was assumed to be complete. The total amount of He-PC recovered after 6, 11, and 18 days was 15%, 12%, and 6%, respectively, and that of ET18-OCH3 was 1.3%, 0.8%, and 0.3%, respectively. This indicates that the bioavailability of He-PC and ET18-OCH3 is not controlled by differences in the uptake of the two drugs, but by differences in their metabolism. The results could explain the differing efficacy of these two compounds in their antitumor action in animal models.