Cross contamination of intramyocellular lipid signals through loss of bulk magnetic susceptibility effect differences in human muscle using (1)H-MRSI at 4 T.

Cross contamination of intramyocellular lipid (IMCL) signals through loss of bulk magnetic susceptibility (BMS) differences was detected in human muscles using proton magnetic resonance spectroscopic imaging ((1)H-MRSI) at 4 T by varying nominal voxel sizes on healthy subjects. In soleus muscle the IMCL content estimated in 1.00-ml-sized voxels was 15% and 30% higher than that in 0.25-ml voxels for nonobese (P < 0.05) and obese (P < 0.01) subjects, respectively, whereas no effect was observed on IMCL estimation in tibialis posterior (TP) and tibialis anterior (TA) regions for different voxel sizes. The unbiased 0.25-ml voxel size (1)H-MRSI method was applied to measure IMCL content in nonobese sedentary (NOB-Sed), moderately trained (Ath), sedentary obese (OB), and Type 2 diabetic mellitus (DM) subjects. IMCL content in soleus was greatest in OB, with decreasing content in DM, Ath, and NOB-Sed, respectively (12.6 +/- 1.6, 9.7 +/- 1.8, 7.4 +/- 1.0, 4.9 +/- 0.5 mmol/kg wet wt; P < 0.05 by ANOVA; P < 0.05 OB vs. NOB-Sed or Ath). In TA, IMCL was equivalently elevated in DM and OB, which was higher than in Ath or NOB-Sed, respectively (4.2 +/- 0.4 and 4.2 +/- 0.7 vs. 2.7 +/- 0.5 and 1.5 +/- 0.3 mmol/kg wet wt; ANOVA, P < 0.05; P < 0.05 DM or OB vs. NOB-Sed). We conclude that IMCL content is overestimated when voxel size exceeds 0.25 ml despite measurement by optimized high-resolution (1)H-MRSI at high field. When IMCL is measured unbiased by concomitant obesity, we find that it is strongly influenced by muscle type, training status, and the presence of obesity and Type 2 diabetes.

LC/MS quantitative study of glucose by iodine attachment.

We explored the potential of iodine attachment to improve the sensitivity of glucose measurement by LC/MS. After sample preparation, glucose was separated by normal phase chromatography, followed by anionization by I(-)-attachment prior to MS by post-column addition of a methanolic solution of iodoform. Iodine is capable of forming an anionic adduct with neutral monosaccharides in negative ion mode electrospray mass spectrometry. Quasi-molecular ions [M+I]- of glucose, and [6,6-(2)H2]glucose (abbreviated d2-glucose) internal standard were quantitated in selected ion monitoring (SIM) mode. Iodine attachment LC/MS analysis provided high sensitivity, superior to GC/MS. It greatly simplified sample preparation and increased throughput. The advantages of iodine attachment can be realized even on old mass spectrometers. A LOD of 50 pg glucose on column was achieved. Due to iodine's predisposition to sublimate, the iodoform concentration must be minimized, which adds complexity to method development. To optimize reagent concentration we developed an efficient and flexible gradient-based delivery platform. Strategy for method development with iodoform is given.

Calculating glucose fluxes during meal tolerance test: a new computational approach.

A new calculation method is proposed to quantify the endogenous glucose production (EGP), the glucose appearance rate due to meal ingestion (R(a meal)), and the glucose disposal (R(d)) during a three-tracer study design. The method utilizes the maximum likelihood theory combined with a regularization method to achieve a theoretically coherent computational framework. The method uses the two-compartment formulation of the glucose kinetics. Instead of assuming smoothness of unlabeled and labeled glucose concentrations, the method assumes that the EGP, the R(a meal), and the fractional glucose clearance are smooth, increasing plausibility of their individual estimates. The method avoids transformation of the measurement errors, which may skew the estimates of the EGP, R(a meal), and R(d) with the traditional approach. Finally, the sequential nature of the calculations is replaced by calculating the EGP, R(a meal), and R(d) in "one go" to avoid the propagation of the errors from the EGP and R(a meal) into R(d). An example study is shown demonstrating the utility of the approach. A better performance of the new method is demonstrated in a simulation study.

Trace LC/MS quantitative analysis of polypeptide biomarkers: impact of 1-D and 2-D chromatography on matrix effects and sensitivity.

We investigated the impact of one dimension (single reverse phase (RP) column) and two dimension (two different RP columns) chromatographic methods on SIM (MS) and multiple reaction monitoring (MRM; MS/MS) performance from human plasma. We find that MRM analysis is clearly preferable for 1-D applications; however, implementation of SIM detection in conjunction with 2-D separation technique resulted in an over 60-fold increase in analyte peak area and improved S/N compared to MRM for our analyte, human C-peptide. Implementation of a 2-D RP-RP technique with SIM detection is capable of eliminating matrix effects and greatly increases signal response and data quality. For two large peptides in complex biological samples, we found that a 2-D approach performed better than high quality sample preparation together with 1-D chromatography and MRM, even on a high-end mass spectrometer.

Nondigest liberation of biomarkers from plasma: a novel two-stage ultrafiltration approach.

A simple procedure for sample preparation of human plasma by two stages of ultrafiltration using one device is described. Our approach is useful for nondigest liberation of biomarkers bound to albumin and other plasma proteins. The analyte contained in the ultrafiltrate can be directly analyzed without additional sample preparation, and quantified by 2-D RP-RP LC/MS.

Direct sensitive quantitative lC/MS analysis of C-peptide from human urine by two dimensional reverse phase/reverse phase high-performance liquid chromatography.

We describe for the first time the direct quantitative analysis of human C-peptide from urine by isotope dilution assay. Implementation of 2-D reverse phase-reverse phase chromatography (2-D RP-RP) with SIM detection resulted in high assay sensitivity (LOQ= 5 pg on column), accuracy, and method ruggedness. Our experiments demonstrate the strong resolving capability of our proposed 2-D RP-RP platform which significantly reduces strong matrix effects and their resulting quantitation error over a wide range of urine concentrations. In contrast, single column methods (both SIM and multiple reaction monitoring) were found acceptable only for strongly diluted urine samples.

Sensitive quantitative analysis of C-peptide in human plasma by 2-dimensional liquid chromatography-mass spectrometry isotope-dilution assay.

BACKGROUND: Isotope-dilution assays (IDAs) are well established for quantification of metabolites or small drug molecules in biological fluids. Because of their increased specificity, IDAs are an alternative to immunoassays for measuring C-peptide. METHODS: We evaluated a 2-dimensional liquid chromatography-mass spectrometry (2D LC/MS) IDA method. Sample preparation was by off-line solid-phase extraction, and C-peptide separation was performed on an Agilent 1100 2D LC system with a purification method based on high-pressure switching between 2 high-resolution reversed-phase columns. Because of the low fragmentation efficiency of C-peptide, multiple-reaction monitoring analysis was omitted and selective-ion monitoring mode was chosen for quantification. Native and isotope-labeled ([M+18] and [M+30]) C-peptides were monitored in the +3 state at m/z 1007.7, 1013.7, and 1017.7. RESULTS: The assay was linear (r(2) = 0.9995), with a detection limit of 300 amole (1 pg) on column. Inter- and intraday CVs for C-peptide were < or =2%. Comparison with an established polyclonal-based RIA showed high correlation (r = 0.964). Plasma concentrations of total C-peptide measured by RIA were consistently higher than by IDA LC/MS, consistent with the higher specificity of IDAs compared with immunoassays. CONCLUSIONS: The 2D LC/MS IDA approach eliminates matrix effects, enhancing assay performance and reliability, and has a detection limit 100-fold lower than any previously reported LC/MS method. Isotope-labeled C-peptide(s) can be clearly differentiated from endogenous C-peptide by the difference in m/z ratio, so that both peptides can be quantified simultaneously. The method is highly precise, robust, and applicable to pharmacokinetic detection of plasma peptides.

Validation in context of bioanalytical research papers.

We have noticed the growing amount of application papers, mainly focused on detailed description of analytical assay validation with limited discussion of method development and optimization in top-ranked chromatographic journals. We analyze the implications of this trend and suggest a re-emphasis on the intellectual component in method development.

Sensitive LC MS quantitative analysis of carbohydrates by Cs+ attachment.

The development of a sensitive assay for the quantitative analysis of carbohydrates from human plasma using LC/MS/MS is described in this paper. After sample preparation, carbohydrates were cationized by Cs(+) after their separation by normal phase liquid chromatography on an amino based column. Cesium is capable of forming a quasi-molecular ion [M + Cs](+) with neutral carbohydrate molecules in the positive ion mode of electrospray ionization mass spectrometry. The mass spectrometer was operated in multiple reaction monitoring mode, and transitions [M + 133] --> 133 were monitored (M, carbohydrate molecular weight). The new method is robust, highly sensitive, rapid, and does not require postcolumn addition or derivatization. It is useful in clinical research for measurement of carbohydrate molecules by isotope dilution assay.

A randomized trial comparing low-fat and low-carbohydrate diets matched for energy and protein.

Several recent studies have found greater weight loss at 6 months among participants on a very-low-carbohydrate (VLC) weight-loss diet compared with a low-fat (LF) weight-loss diet. Because most of these studies were not matched for calories, it is not clear whether these results are caused by decreased energy intake or increased energy expenditure. It is hypothesized that several energy-consuming metabolic pathways are up-regulated during a VLC diet, leading to increased energy expenditure. The focus of this study was to investigate whether, when protein and energy are held constant, there is a significant difference in fat and weight loss when fat and carbohydrate are dramatically varied in the diet. The preliminary results presented in this paper are for the first four of six postmenopausal overweight or obese participants who followed, in random order, both a VLC and an LF diet for 6 weeks. Other outcome measures were serum lipids, glucose, and insulin, as well as dietary compliance and side effects. Our results showed no significant weight loss, lipid, serum insulin, or glucose differences between the two diets. Lipids were dramatically reduced on both diets, with a trend for greater triglyceride reduction on the VLC diet. Glucose levels were also reduced on both diets, with a trend for insulin reduction on the VLC diet. Compliance was excellent with both diets, and side effects were mild, although participants reported more food cravings and bad breath on the VLC diet and more burping and flatulence on the LF diet.