Method for the determination of vitamin K homologues in human plasma using high-performance liquid chromatography-tandem mass spectrometry

We report here the development of a precise and sensitive method for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4), and menaquinone-7 (MK-7) in human plasma using HPLC-tandem mass-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS/MS). The method involves the use of stable isotope (18)O-labeled internal standard compounds, which were synthesized in our laboratory, and the selection of a precursor and product ion with a MS/MS multiple reaction monitoring method. The average intraassay and interassay variation values for PK, MK-4, and MK-7 were <10%. Average spiked recoveries from authentic compounds added to normal human plasma samples for PK, MK-4, and MK-7 were 98-102%. Mean plasma concentrations of PK, MK-4, and MK-7 from healthy subjects (n = 20) were 1.22 +/-0.57, 0.39 +/- 0.46, and 6.37 +/- 7.45 ng/mL, respectively. We conclude that this novel LC-APCI-MS/MS method should be useful for the evaluation of vitamin K status in postmenopausal women and elderly subjects and provides useful information for the treatment and prevention of osteoporosis with vitamin K.     

Determination of 25-hydroxyvitamin D in human plasma using high-performance liquid chromatography--tandem mass spectrometry

We report here the development of a precise and sensitive method to determine 25-hydroxyvitamin D (25-OH-D(2)/ -D(3)) in human plasma using high-performance liquid chromatography-tandem mass-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS/MS). The method involves the use of deuterated 25-OH-D(3) as an internal standard compound for 25-OH-D(2)/-D(3), which was synthesized in our laboratory, and the selection of a precursor and product ion with a MS/MS multiple reaction monitoring method. The average intraassay and interassay variation values (relative standard deviation) were 5.7 and 2.5%, respectively, for 25-OH-D(3) and 4.5 and 5.1%, respectively, for 25-OH-D(2). The average spiked recoveries from authentic compounds added to normal human plasma samples for 25-OH-D(3) and 25-OH-D(2) were 103.8 and 98.8%, respectively. Mean plasma concentrations of 25-OH-D(3) and 25-OH-D(2) in healthy subjects were 20.5 and 0.4 ng/mL, respectively. We conclude that this novel LC-APCI-MS/MS method would be useful for the evaluation of the vitamin D status in postmenopausal women and elderly subjects and provide useful information in the diagnosis of vitamin D insufficiency/deficiency, as well as for the treatment and prevention of osteoporosis with vitamin D.     

<10$$ \bar{1} $$0> Dislocation at a {2$$ \bar{1} $$$$ \bar{1} $$0} low-angle grain boundary in LiNbO3

A LiNbO₃ bicrystal that contains a {2\( \bar{1} \) \( \bar{1} \)0} low-angle grain boundary with both of 2° tilt misorientation and a slight twist misorientation was fabricated, and resulting dislocation structure at the boundary was analyzed by using transmission electron microscopy (TEM) and scanning TEM. The observations revealed that two types of dislocations of b = 1/3 <2\( \bar{1} \) \( \bar{1} \)0> and b = <10\( \bar{1} \)0> are formed at the boundary. A 1/3 <2\( \bar{1} \) \( \bar{1} \)0> dislocation, which dissociates into two partial dislocations with a {2\( \bar{1} \) \( \bar{1} \)0} stacking fault in between, compensates only tilt misorientation of the boundary. On the other hand, it was found that a <10\( \bar{1} \)0> dislocation, which dissociates into three equivalent partial dislocations with b = 1/3 <10\( \bar{1} \)0>, has both edge and screw components in total. That is, the <10\( \bar{1} \)0> dislocations are formed to compensate the twist misorientation of the boundary, in addition to the tilt misorientation. It is interesting that the three partial dislocations from a <10\( \bar{1} \)0> dislocation are arranged in a zigzag pattern with left–right asymmetry. This special configuration is suggested to originate from the presence of stable stacking fault structure on the {2\( \bar{1} \) \( \bar{1} \)3} plane in LiNbO₃.     

A simple test to detect hydrogen/deuterium scrambling during gas-phase peptide fragmentation

Amide hydrogen/deuterium (H/D) exchange coupled with mass spectrometry has become a powerful tool to study protein dynamics. Addition of a proteolysis step between the exchange reaction and mass analysis can be used to localize the positions of deuterium and improve overall resolution. The resolution can be further enhanced by the fragmentation of digested peptides in the gas phase if scrambling of exchangeable hydrogens and deuteriums on the peptides does not occur. Although some laboratories reported successful localization of deuteriums by gas-phase fragmentations, others described total scrambling. Here we propose a simple method to detect the presence or absence of scrambling using a commercially available small peptide, neurotensin (9-13; RPYIL). All exchangeable hydrogens on this pentapeptide are first deuterated by dissolving it in deuterium oxide. The deuterated peptide is loaded onto a reversed-phase column, and then washed with copious amounts of cold acidic aqueous buffer. This washing exchanges all deuteriums on both the terminals and the side chains back to hydrogens. Now only three deuteriums are attached on the pentapeptide, one on each of the amide nitrogens of Y, I, and L. After the partially deuterated peptide is eluted from the column with 95% acidic acetonitrile, collision-induced dissociation (CID) generates a series of b ions, which are analyzed by mass spectrometer. In the absence of scrambling, no deuterium should be observed in the b 2 ion, as neither R nor P have amide hydrogens. On the other hand, in the event of scrambling, b 2 should carry about half of the deuteriums of the parent pentapeptide. In theory, complete scrambling should distribute deuteriums equally among all of the exchangeable hydrogens. The b 2 portion of neurotensin (9-13) has 6 exchangeable hydrogens, whereas the +1 charge state of neurotensin (9-13) has 12 exchangeable hydrogens. We demonstrated that CID caused complete scrambling of hydrogens and deuteriums with an LCQ (a ion trap machine).     

Absorption of ultrashort optical pulses in water

We investigate the linear propagation of 800 and 1530 nm ultrashort optical pulses in water. For all pulse repetition rates studied, we observe pure exponential decay down to a transmission of 2.5 x 10(-5). We further demonstrate that previous observations of nonmonoexponential decay and pulse splitup in broadband pulses are consistent with Beer's law in the purely linear regime.     

Densely arrayed eight-wavelength semiconductor lasers fabricated bymicroarray selective epitaxy

This paper describes a novel epitaxial growth technique, called microarray selective epitaxy (MASE), for fabricating extremely small integrated photonic devices. The MASE technique makes it possible to form densely arrayed (pitch <10 μm) multiple-quantum-well (MQW) waveguides without semiconductor etching as well as to control the bandgap energy of each waveguide. The technique is demonstrated for fabricating an eight-channel 10-μm-spacing microarray MQW structure, and the bandgap wavelength of each channel is successfully controlled by changing the SiO₂ mask pattern over a range of 90 nm. The technique is also applied to the fabrication of densely arrayed, eight-wavelength, Fabry-Perot laser diodes. The laser section is only 70 pm wide and 400 μm long. Eight different lasing wavelengths (each over 80 nm), a uniform threshold current of less than 9 mA, and an output power of over 10 mW are obtained     

Microstructures and luminescent properties of Ce-doped transparent mica glass-ceramics

Transparent mica glass-ceramics were prepared by heating parent glasses that had been doped with 0.5–15 mol% CeO₂. During the melting and heat treatment, Ce⁴⁺ ions in the specimens were reduced to Ce³⁺ ions, and one or both of these ion species were then replaced with Li⁺ ions in the interlayers of the separated mica crystals. However, scanning transmission electron microscope (STEM) and Z-contrast imaging revealed that the mica crystals did not contain the same amount of Ce. On excitation at 254 nm, the parent glasses and glass-ceramics emitted blue light, which originated from the 5d to 4f transition of the Ce³⁺ ions. The emission of the glass-ceramic containing a smaller amount of Ce was attributed to the Ce³⁺ ions in both the glass phase and the mica crystals, whereas that of the glass-ceramics containing a larger amount of Ce was caused mainly by Ce³⁺ ions in the mica crystals. The dependence of the emission band of the parent glasses on the amount of Ce was a unique feature of the Ce-doped transparent mica glass-ceramics and was not observed in previous studies of Eu-doped parent glasses and mica glass-ceramics.     

Who may not benefit from continuous renal replacement therapy in acute kidney injury?

This study aimed to identify factors that may predict early kidney recovery (less than 48 hours) or early death (within 48 hours) after initiating continuous renal replacement therapy (CRRT) in acute kidney injury (AKI) patients. This is a multicenter retrospective observational study of 14 Japanese Intensive care units (ICUs) in 12 tertiary hospitals. Consecutive adult patients with severe AKI requiring CRRT admitted to the participating ICUs in 2010 (n = 343) were included. Patient characteristics, variables at CRRT initiation, settings, and outcomes were collected. Patients were grouped into early kidney recovery group (CRRT discontinuation within 48 hours after initiation, n = 52), early death group (death within 48 hours after CRRT initiation, n = 52), and the rest as the control group (n = 239). The mean duration of CRRT in the early kidney recovery group and early death group was 1.3 and 0.9 days, respectively. In multivariable regression analysis, in comparison with the control group, urine output (mL/h) (odds ratio [OR]: 1.02, 95% confidence interval [CI]: 1.01–1.03), duration between ICU admission to CRRT initiation (days) (OR: 0.65, 95% CI: 0.43–0.87), and the sepsis‐related organ failure assessment score (OR: 0.87, 95% CI; 0.78–0.96) were related to early kidney recovery. Serum lactate (mmol/L) (OR: 1.19, 95% CI: 1.11–1.28), albumin (g/dL) (OR: 0.52, 95% CI: 0.28–0.92), vasopressor use (OR: 3.68, 95% CI: 1.37–12.16), and neurological disease (OR: 9.64, 96% CI: 1.22–92.95) were related to early death. Identifying AKI patients who do not benefit from CRRT and differentiating such patients from the study cohort may allow previous and future studies to effectively evaluate the indication and role of CRRT.     

Comparison of arrhythmogenicity of atrial pacing at several right atrial pacing sites: evaluation of canine atrial electrograms during atrial pacing and arrhythmogenicity for atrial fibrillation

The changes in the duration of atrial electrograms and the appearance of AF during atrial pacing were compared among five atrial pacing sites in dogs to clarify the arrhythmogenicity of atrial pacing at different atrial pacing sites. In seven mongrel dogs (15-20 kg), the right atrial surface was exposed by right thoracotomy. Atrial electrograms were recorded via bipolar electrodes with an interelectrode distance of 1.2 mm at four right atrial sites: (1) the high right atrium (HRA), (2) the mid-right atrium (MRA), (3) the low right atrium (LRA), and (4) the center of the pectinate muscle (PM). The duration of the atrial electrograms at these four recording sites were measured during atrial pacing with fixed cycle lengths of 200, 150, and 120 ms delivered at five atrial sites: (1) the HRA, (2) the inferior vena cava (IVC), (3) the right atrial appendage (RAA), (4) Bachman's bundle (BB), and (5) the atrial septum (AS). In each dog, the atrial pacing with the 120-ms cycle length was performed five times at each pacing site to evaluate the inducibility of AF. When AF was induced, the atrial recording site which first showed a fragmented atrial electrogram was considered the initiation site of the AF. AF was induced during 9 of 35 episodes of atrial pacing at the HRA site, 11 of 35 at the IVC site, 5 of 35 at the RAA site, 3 of 35 at the BB site, and none at the AS site. The initiation site of AF was in the HRA site in 11 of 28 episodes of induced AF, in the MRA site in 9 of 28, and in the LRA site in 8 of 28. At each recording site, the shorter the paced cycle length, the longer the duration of the atrial electrogram regardless of the pacing site. During the atrial pacing with the 200-ms cycle length, the HRA pacing resulted in the shortest duration of the atrial electrogram at each recording site in comparison with the other pacing sites. However, during atrial pacing at the two shorter paced cycle lengths, the duration of the atrial electrogram was shorter during the pacing at the BB or AS sites in comparison with the other three pacing sites, i.e., the HRA, IVC, and RAA sites. These results were the same for all atrial recording sites, but the prolongation of the atrial electrogram was most prominent at the HRA and MRA recording sites, which are most likely initiation sites of the induced AF. In the canine atria, (1) the initiation sites of AF were likely to be the HRA, MRA, or LRA sites in comparison with the PM site; and (2) the atrial pacing at the BB or AS sites was considered less arrhythmogenic for AF than the pacing at the HRA, LRA, or RAA sites.     

Identification of pharmacological chaperones for Gaucher disease and characterization of their effects on beta-glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry

Point mutations in beta-glucocerebrosidase (GCase) can result in a deficiency of both GCase activity and protein in lysosomes thereby causing Gaucher Disease (GD). Enzyme inhibitors such as isofagomine, acting as pharmacological chaperones (PCs), increase these levels by binding and stabilizing the native form of the enzyme in the endoplasmic reticulum (ER), and allow increased lysosomal transport of the enzyme. A high-throughput screen of the 50,000-compound Maybridge library identified two, non-carbohydrate-based inhibitory molecules, a 2,4-diamino-5-substituted quinazoline (IC(50) 5 microM) and a 5-substituted pyridinyl-2-furamide (IC(50) 8 microM). They raised the levels of functional GCase 1.5-2.5-fold in N370S or F213I GD fibroblasts. Immunofluorescence confirmed that treated GD fibroblasts had decreased levels of GCase in their ER and increased levels in lysosomes. Changes in protein dynamics, monitored by hydrogen/deuterium-exchange mass spectrometry, identified a domain III active-site loop (residues 243-249) as being significantly stabilized upon binding of isofagomine or either of these two new compounds; this suggests a common mechanism for PC enhancement of intracellular transport.