Deuterium oxide as a contrast medium for real-time MRI-guided endovascular neurointervention.

Rationale: Endovascular intervention plays an important role in the treatment of various diseases, in which MRI-guidance can potentially improve precision. However, the clinical applications of currently available contrast media, including Gadolinium-based contrast agents and superparamagnetic iron oxide particles (SPIO), are hindered by safety concerns. In the present study, we sought to develop D2O as a novel contrast agent for guiding endovascular neurointervention. Methods: Animal studies were approved by institutional ACUC and conducted using an 11.7 T Bruker Biospec system and a 3T Siemens Trio clinical scanner for rodent and canine imaging, respectively. The locally selective blood brain barrier opening (BBBO) in rat brains was obtained by intraarterial (IA) injection of mannitol. The dynamic T2w* EPI MRI sequence was used to study the trans-catheter perfusion territory by IA administered SPIO before mannitol administration, whereas a dynamic T1w FLASH sequence was used to acquire Gd contrast-enhanced MRI for assessing BBBO after injection of mannitol. The contrast generated by D2O assessed by either EPI or FLASH methods was compared with the corresponding results assessed by SPIO or Gd. The utility of D2O MRI was also demonstrated to guide drug delivery to glioma in a mouse model. Finally, the clinical utility of D2O-MRI was demonstrated in a canine model. Results: Our study has shown that the contrast generated by D2O can be used to precisely delineate trans-catheter perfusion territory in both small and large animals. The perfusion territories determined by D2O-MRI show moderate correlation with those by SPIO-MRI (Spearman coefficient r = 0.5234, P < 0.001). Moreover, our results show that the perfusion territory determined by D2O-MRI can successfully predict the areas with BBBO after mannitol treatment similar to that assessed by Gd-MRI (Spearman coefficient r = 0.6923, P < 0.001). Using D2O-MRI as imaging guidance, the optimal infusion rate in the mouse brain was determined to be 150 µL/min to maximize the delivery efficacy to the tumor without serious off-target delivery to the brain parenchyma. The enhanced drug delivery of antibodies to the brain tumor was confirmed by fluorescence imaging. Conclusion: Our study demonstrated that D2O can be used as a negative MRI contrast medium to guide endovascular neurointervention. The established D2O -MRI method is safe and quantitative, without the concern of contrast accumulation. These qualities make it an attempting approach for a variety of endovascular procedures.

Breast milk intake and mother to infant pesticide transfer measured by deuterium oxide dilution in agricultural and urban areas of Mexico.

Vector-borne diseases have increased pesticide use in urban areas (UA) and agricultural areas (AA) in Mexico. Breast milk can be contaminated by pesticide exposure. The objective of the study was to measure breast milk intake by deuterium oxide dilution as well as organochlorine and pyrethroid transfer from mother to infant in AA and UA of Sonora, Mexico. Human milk intake was determined by the 'dose-to-mother' technique using deuterium oxide (D2O) dilution. Mothers' body composition was also assessed by this technique and the intercept method. Pyrethroids (deltamethrin, cypermethrin and cyhalothrin) and organochlorine pesticide residues (p,p'- DDT, p,p'- DDE, p,p'- DDD) in breast milk samples were measured by gas chromatography. Sixty-two lactating women and their infants participated in the study, 32 lived in the UA and 30 lived in the AA. Breast milk intake was approximately 100 mL higher in the AA than in the UA 799 ± 193 and 707 ± 201 mL/day, respectively (p < 0.05). The concentrations of p,p'- DDT and cypermethrin levels in breast milk were higher in the UA than in the AA (p < 0.05 and p = 0.001, respectively). None of the pyrethroids and organochlorine pesticides studied surpassed the Acceptable Daily Intake (ADI) in milk for humans according to EPA and FAO/WHO. In conclusion, breast milk intake was higher in the AA compared to the UA. The p,p'- DDT and cypermethrin levels in breast milk were higher in the UA compared to the AA. Since pesticide levels in human milk did not exceed the ADI, breastfeeding is still a safe practice and should be encouraged.

Determination of (2)H-enrichment of rat brain interstitial fluid and rat plasma by headspace-gas-chromatography - quadrupole-mass-spectrometry.

(2)H2O as nonradioactive, stable marker substance is commonly used in preclinical and clinical studies and the precise determination of (2)H2O concentration in biological samples is crucial. However, aside from isotope ratio mass spectrometry (IRMS), only a very limited number of methods to accurately measure the (2)H2O concentration in biological samples are routinely established until now. In this study, we present a straightforward method to accurately measure (2)H-enrichment of rat brain interstitial fluid (ISF) and rat plasma to determine the relative recovery of a cerebral open flow microperfusion (cOFM) probe, using headspace-gas-chromatography - quadrupole-mass-spectrometry. This method is based on basic-catalyzed hydrogen/deuterium exchange in acetone and detects the (2)H-labelled acetone directly by the headspace GC-MS. Small sample volumes and limited number of preparation steps make this method highly competitive. It has been fully validated. (2)H enriched to 8800 ppm in plasma showed an accuracy of 98.9% and %Relative Standard Deviation (RSD) of 3.1 with n = 18 over three days and with two operators. Similar performance was obtained for cerebral ISF enriched to 1100 ppm (accuracy: 96.5%, %RSD: 3.1). With this highly reproducible method we demonstrated the successful employment of (2)H2O as performance marker for a cOFM probe.

Energy expenditure by multisensor armband in overweight and obese lactating women validated by doubly labeled water.

OBJECTIVE: To validate total energy expenditure (TEE) and activity energy expenditure (AEE) from the portable SenseWear armband (SWA) Pro 2 (TEESWA and AEESWA ; InnerView software versions SWA 5.1 and SWA 6.1) against TEE from doubly labeled water (DLW) and AEE from DLW and indirect calorimetry (TEEDLW and AEEDLW ) in overweight/obese lactating women at 10 weeks postpartum. DESIGN AND METHODS: TEE was measured simultaneously with DLW (14 days) and SWA (first 7 days). Lactating women (n = 62), non-smoking, with a BMI > 25 kg/m(2) and wearing time SWA ≥ 90% were included. RESULTS: Mean TEESWA5.1 was overestimated with 85 kcal/day compared to TEEDLW (P = 0.040), while mean TEESWA6.1 was underestimated with 241 kcal/day compared to TEEDLW (P < 0.001). Mean AEESWA5.1 was similar to mean AEEDLW (P = 0.818), while mean AEESWA6.1 was underestimated with 581 kcal/day compared to AEEDLW (P < 0.001). TEESWA6.1 and AEESWA6.1 were systematically underestimated at higher levels of energy expenditure and BMI while only AEESWA5.1 was systematically overestimated at higher levels of energy expenditure. CONCLUSIONS: TEESWA5.1 and AEESWA5.1 were fairly estimated on a group level while TEESWA6.1 and AEESWA6.1 were significantly and systematically underestimated. Both SWA software versions showed large individual variation in agreement with TEEDLW and AEEDLW , limiting the validity on individual level.

Dynamics of glutathione and ophthalmate traced with 2H-enriched body water in rats and humans.

We developed a LC-MS-MS assay of the (2)H labeling of free glutathione (GSH) and bound glutathione [GSSR; which includes all DTT-reducible forms, primarily glutathione disulfide (GSSG) and mixed disulfides with proteins] and ophthalmate (an index of GSH depletion) labeled from (2)H-enriched body water. In rats whose body water was 2.5% (2)H enriched for up to 31 days, GSH labeling follows a complex pattern because of different rates of labeling of its constitutive amino acids. In rats infused with [(13)C(2),(15)N-glycine]glutathione, the rate of appearance of plasma GSH was 2.1 micromol.min(-1).kg(-1), and the half-life of plasma GSH/GSSR was 6-8 min. In healthy humans whose body fluids were 0.5% (2)H enriched, the (2)H labeling of GSH/GSSR and ophthalmate can be precisely measured after 4 h, with GSH being more rapidly labeled than GSSR. Since plasma GSH/GSSR derives mostly from liver, this technique opens the way to 2) probe noninvasively the labeling pattern and redox status of the liver GSH system in humans and 2) assess the usefulness of ophthalmate as an index of GSH depletion.

Measurement of estrogen effect on bone turnover by 2H2O labeling.

Estrogen loss has been known to increase bone turnover through accelerated bone resorption coupled by increased bone formation. In the present study, we measured estrogen effect on bone turnover by incorporation of 2H from 2H2O into amino acids. At 6 weeks of age, rats were either sham-operated (sham) or ovariectomized (ovx). Two weeks after surgery, 17beta-estradiol (est) was implanted subcutaneously to ovx rats. At 9 weeks of age, 2H2O labeling started by administration of 4% 2H2O to rats for 4 or 7 weeks in drinking water after a single intraperitonial bolus injection with 99.9% 2H2O. Body 2H2O enrichments were stable at approximately 3.0% over labeling period. Fractional replacements (f) of the midshaft femur were higher in the sham group (40.36 +/- 4.89% vs 42.47 +/- 11.22%) than the ovx (28.57 +/- 9.67% vs 37.47 +/- 8.34%) and est (26.57 +/- 4.00% vs 30.35 +/- 5.34%) groups 4 and 7 weeks after labeling, respectively. Ovariectomy-induced bone loss was observed in the trabecular bone along with a significantly increased number of osteoclasts, all of which were normalized after estradiol treatment. Taken together, our results indicate that estrogen deficiency significantly reduces the proportion of newly synthesized bone matrix as well as the total amount of bone matrix. The reduced portion of new matrix in ovx rats, presumably caused by activated osteoclastic degradation, was compensated rapidly with time. In addition, estradiol treatment protected the bone matrix by decreasing bone turnover rate.

Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization.

In Arthrobacter aurescens DSM 3747 three enzymes are involved in the complete conversion of slowly racemizing 5'-monosubstituted D,L-hydantoins to L-amino acids, a stereoselective hydantoinase, a stereospecific L-N-carbamoylase and a hydantoin racemase. The gene encoding the hydantoin racemase, designated hyuA, was identified upstream of the previously described L-N-carbamoylase gene in the plasmid pAW16 containing genomic DNA of A. aurescens. The gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Escherichia coli and the recombinant protein purified to homogeneity and further characterized. The optimal condition for racemase activity were pH 8.5 and 55 degrees C with L-5-benzylhydantoin as substrate. The enzyme was completely inhibited by HgCL2 and iodoacetamide and stimulated by addition of dithiothreitol. No effect on enzyme activity was seen with EDTA. The enzyme showed preference for hydantoins with arylalkyl side chains. Kinetic studies revealed substrate inhibition towards the aliphatic substrate L-5-methylthioethylhydantoin. Enzymatic racemization of D-5-indolylmethylenehydantoin in D2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization.

In vivo imaging of extraction fraction of low molecular weight MR contrast agents and perfusion rate in rodent tumors.

Tissue uptake of a fully extractable MR detectable tracer, deuterated water (D2O), was compared with that of a less extractable contrast agent, Gadolinium-DTPA-dimeglumine (Gd-DTPA), in rodent tumor and muscle tissue. This dual tracer method allowed calculation of relative (to muscle) tissue perfusion and extraction fraction of Gd-DTPA in each image pixel in vivo. Solutions of Gd-DTPA and D2O were injected intravenously into Fisher female rats (n = 9) with R3230 mammary adenocarcinomas implanted in the hind limb. Perfusion rate was approximately two times greater (P < 0.005 by paired t test) in tumor than in muscle. Gd-DTPA extraction fraction at the interface between tumor and muscle was 2.0 times the extraction fraction in normal muscle (P < 0.005 by paired t test). Extraction fraction at the tumor center was 1.6 times the extraction fraction in muscle (P < 0.01 by paired t test). High extraction fraction of Gd-DTPA correlated with high capillary permeability determined from Evans Blue staining. Low molecular weight Gd-DTPA derivatives are widely used in clinical practice, and their extraction fractions are crucial determinants of image contrast during the first few passes of the contrast agent bolus. Therefore spatially resolved measurements of contrast agent extraction fractions obtained in vivo have significant clinical utility. The data demonstrate that extraction of low molecular weight tracers is sensitive to increased permeability in tumor vasculature and that this increased permeability can be imaged.

The accuracy and reproducibility of measuring blood flow in murine tumours by the D2O uptake and clearance techniques.

The use of D2O as an NMR visible tracer to monitor murine tumour blood flow (TBF) by both the wash-in and wash-out methods has been investigated. The factors that influence the models used to fit the data and the error on the measurement of the clearance and uptake rates have been assessed. The study concentrates on the uptake method which allows TBF to be measured without the need to use anaesthetic. Also, administering the D2O remotely to the mouse means it can remain undisturbed, in the magnet bore, between control and post-treatment readings. The uptake method in KHT and RIF-1 transplanted murine tumours has been investigated in a series of control experiments and after modifying TBF by hydralazine (5 mg/kg) and photodynamic therapy. These studies showed that four uptake measurements could be made on the same mouse at 20 min intervals without affecting TBF, control values were the same for anaesthetized and unanaesthetized mice and the values obtained for RIF-1 tumours were marginally higher than those obtained for the KHT tumours. The decrease in TBF seen after modification was in good agreement with published data where TBF results were obtained by using D2O clearance, radioactive tracers or laser Doppler flowmetry.