Measurement of insulin sensitivity indices using 13C-glucose and gas chromatography/combustion/isotope ratio mass spectrometry

Important aspects of glucose metabolism can be quantified by using the minimal model of glucose kinetics to interpret the results of intravenous glucose tolerance tests. The power of this methodology can be greatly increased by the addition of stable isotopically labelled tracer to the glucose bolus dose. This allows the separation of glucose disposal from endogenous glucose production and also increases the precision of the estimates of the physiological parameters measured. Until now the tracer of choice has been deuteriated glucose and the analytical technique has been gas chromatography/mass spectrometry (GC/MS). The consequence of this choice is that nearly 2 g of labelled material are needed and this makes the test expensive. We have investigated the use of (13)C-labelled glucose as the tracer in combination with gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) as the analytical technique. This methodology offers superior analytical precision when compared with the conventional method and so the amount of tracer used, and hence the cost, can be reduced considerably. Healthy non-obese male volunteers were recruited for a standard intravenous glucose tolerance test (IVGTT) protocol but 6,6-(2)H-glucose and 1-(13)C-glucose were administered simultaneously. Tracer/tracee ratios were derived from isotope ratio measurements of plasma glucose using both GC/MS and GC/C/IRMS. The results of these determinations indicated that the two tracers behaved identically under the test protocol. The combination of these results with plasma glucose and insulin concentration data allowed determination of the minimal model parameters S*g and S*i. The parameter relating to insulin-assisted glucose disposal, S*i, was found to be the same in the two techniques, but this was not the case for the non-insulin-dependent parameter S*g.     

Analysis of [U‐13C6]glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry compared with two other mass spectrometry techniques

The use of stable isotope labelled glucose provides insight into glucose metabolism. The ¹³C‐isotopic enrichment of glucose is usually measured by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). However, in both techniques the samples must be derivatized prior to analysis, which makes sample preparation more labour‐intensive and increases the uncertainty of the measured isotopic composition. A novel method for the determination of isotopic enrichment of glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) has been developed. Using this technique, for which hardly any sample preparation is needed, we showed that both the enrichment and the concentration could be measured with very high precision using only 20 µL of plasma. In addition, a comparison with GC/MS and GC/IRMS showed that the best performance was achieved with the LC/IRMS method making it the method of choice for the measurement of ¹³C‐isotopic enrichment in plasma samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of plasma [6,6-2H2]glucose enrichment by a simple and accurate gas chromatographic-mass spectrometric method.

An improved method for the evaluation of glucose turnover rate in humans, using a prime-continuous infusion of [6,6-2H2]glucose, was developed. Deproteinization of plasma and conversion of glucose into the aldononitrile pentaacetate derivative are the only sample manipulations required prior to the gas chromatographic-mass spectrometric analysis. In six normal adults (prime = 5 mg kg-1; continuous infusion = 0.05 mg kg-1 min-1) the hepatic glucose output calculated at steady state by the procedure described here was 2.1 +/- 0.2 mg kg-1 min-1, the isotopic enrichment being determined with a coefficient of variation of ca. 2%. In six additional subjects, with half of the above-mentioned doses of labelled glucose, the mean hepatic glucose output was exactly the same (3.2% coefficient of variation for the isotopic enrichment measurement). The method described allows the hepatic glucose output to be precisely determined with savings both of time and of labelled glucose.     

Determination of 15N enrichment of taurine in cat urine by high resolution fast-atom bombardment mass spectrometry.

A method is described for measuring the stable isotopic enrichment of taurine in cat urine samples by high resolution fast-atom bombardment mass spectrometry, after 15N labelled taurine was given to cats for the purpose of investigating taurine metabolism. The 15N enrichment of taurine was measured after hydrolysis and purification of taurine by anion/cation exchange chromatography. The isotopic ratio of taurine was determined by measuring the [M+H]+ ion peaks in the spectra of the unlabelled and labelled compounds under multiple ion scan conditions. The overall standard deviation of the measurement is better than 4%. This method requires no derivation and uses only 500 microL of urine samples.     

Sulphur tracer experiments in laboratory animals using 34S-labelled yeast

We have evaluated the use of ³⁴S-labelled yeast to perform sulphur metabolic tracer experiments in laboratory animals. The proof of principle work included the selection of the culture conditions for the preparation of sulphur labelled yeast, the study of the suitability of this labelled yeast as sulphur source for tracer studies using in vitro gastrointestinal digestion and the administration of the ³⁴S-labelled yeast to laboratory animals to follow the fate and distribution of ³⁴S in the organism. For in vitro gastrointestinal digestion, the combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) showed that labelled methionine, cysteine and other low molecular weight sulphur-containing biomolecules were the major components in the digested extracts of the labelled yeast. Next, in vivo kinetic experiments were performed in healthy Wistar rats after the oral administration of ³⁴S-labelled yeast. The isotopic composition of total sulphur in tissues, urine and faeces was measured by double-focusing inductively coupled plasma mass spectrometry after microwave digestion. It was observed that measurable isotopic enrichments were detected in all samples. Finally, initial investigations on sulphur isotopic composition of serum and urine samples by HPLC-ICP-MS have been carried out. For serum samples, no conclusive data were obtained. Interestingly, chromatographic analysis of urine samples showed differential isotope enrichment for several sulphur-containing biomolecules.     

Quantitative determination of tertatolol in biological fluids by gas chromatography-mass spectrometry

Quantitative determination of tertatolol concentrations in plasma and urine was performed by gas chromatography-mass spectrometry in the chemical-ionization mode with ammonia after successive extractions of the beta-blocking drug in alkaline, acid and final alkaline medium. [2H9]Tertatolol, isotopically stable under the operating conditions employed, was used as an internal standard, thus allowing quantities of 1 ng/ml to be specifically determined. Overall analytical error was less than 10%. Prior to isothermal chromatography at 240 degrees C on a column packed with 3% SE-30, both compounds were silylated with bis(trimethylsilyl)trifluoroacetamide. Detection was performed by monitoring the quasimolecular ions of tertatolol, m/z 368 and m/z 377, for the [2H9]tertatolol in the chemical-ionization mode with ammonia. The calibration curves obtained had linear characteristics for the concentration range 1-1125 ng/ml.     

The role of liquid chromatography and flow injection analyses coupled to isotope ratio mass spectrometry for studying human in vivo glucose metabolism

Under most physiological conditions, glucose, or carbohydrate (CHO), homeostasis is tightly regulated. In order to mechanistically appraise the origin of circulating glucose (e.g. via either gluconeogenesis, glycogenolysis or oral glucose intake), and its regulation and oxidation, the use of stable isotope tracers is now a well-accepted analytical technique. Methodologically, liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) can replace gas chromatography coupled to combustion-isotope ratio mass spectrometry (GC/C/IRMS) for carrying out compound-specific (13)C isotopic analysis. The LC/IRMS approach is well suited for studying glucose metabolism, since the plasma glucose concentration is relatively high and the glucose can readily undergo chromatography in an aqueous mobile phase. Herewith, we report two main methodological approaches in a single instrument: (1) the ability to measure the isotopic enrichment of plasma glucose to assess the efficacy of CHO-based treatment (cocoa-enriched) during cycling exercise with healthy subjects, and (2) the capacity to carry out bulk isotopic analysis of labeled solutions, which is generally performed with an elemental analyzer coupled to IRMS. For plasma samples measured by LC/IRMS the data show a isotopic precision SD(δ(13)C) and SD(APE) of 0.7 ‰ and 0.001, respectively, with δ(13)C and APE values of -25.48 ‰ and 0.06, respectively, being generated before and after tracer administration. For bulk isotopic measurements, the data show that the presence of organic compounds in the blank slightly affects the δ(13)C values. Despite some analytical limitations, we clearly demonstrate the usefulness of the LC/IRMS especially when (13)C-glucose is required during whole-body human nutritional studies.     

Quantitation of cyclosporin A in whole blood by liquid chromatography/stable isotope dilution electrospray ionization mass spectrometry.

Therapy with cyclosporin A (CsA) for immunosuppression after organ transplantation requires monitoring of its levels in blood owing to the narrow therapeutic index of the drug and to the high inter-individual variability of the drug absorption and metabolism. We describe the preparation of CsA labelled with stable isotopes ((13)C and (2)H) with an isotopic enrichment of about 99% using labelled glucose and its use as internal standard for quantification of CsA blood levels by isotope dilution/electrospray ionization mass spectrometry. The method was found to be linear in the tested range (1-1000 ng) with and without the matrix. The accuracy of the bracketting calibration curves prepared using 100 ng ml(-1) labelled CsA was within +/-1.7% (bias). The results confirmed the usefulness of the procedure as a reference method for the external quality assessment of the field methods for the evaluation of CsA blood concentration, the imprecision (relative standard deviation) and accuracy (bias) being <2%.     

Optimization of N‐methyl‐N‐[tert‐butyldimethylsilyl]trifluoroacetamide as a derivatization agent for determining isotopic enrichment of glycerol in very‐low density lipoproteins

Stable isotope kinetic studies play an important role in the study of very‐low density lipoprotein (VLDL) metabolism, including basic and clinical research. Today, [1,1,2,3,3‐²H₅]glycerol is the most cost‐effective alternative to measure glycerol and triglyceride kinetics. Recycling of glycerol from glycolysis and gluconeogenesis may lead to incompletely labelled tracer molecules. Many existing methods for the measurement of glycerol isotopic enrichment involve the production of glycerol derivatives that result in fragmentation of the glycerol molecule after ionization. It would be favourable to measure the intact tracer molecule since incompletely labelled tracer molecules may be measured as fully labelled. The number of methods available to measure the intact tracer in biological samples is limited. The aim of this project was to develop a gas chromatography/mass spectrometry (GC/MS) method for glycerol enrichment that measures the intact glycerol backbone and is suitable for electron ionization (EI), which is widely available. A previously published method for N‐methyl‐N‐[tert‐butyldimethylsilyl]trifluoroacetamide (MTBSTFA) derivatization was significantly improved; we produced a stable derivative and increased recovery 27‐fold in standards. We used the optimized MTBSTFA method in VLDL‐triglyceride and found that further modification was required to take matrix effects into account. We now have a robust method to measure glycerol isotopic enrichment by GC/EI‐MS that can be used to rule out the known problem of tracer recycling in studies of VLDL kinetics. Copyright © 2010 John Wiley & Sons, Ltd.     

Absolute quantification of superoxide dismutase (SOD) using species-specific isotope dilution analysis

Here we report for the first time the use of species-specific isotope dilution mass spectrometry for the absolute quantification of a metalloprotein using non-denaturing gel electrophoresis laser ablation inductively coupled plasma mass spectrometry (GE-LA-ICP-MS). The concept utilises the intrinsic metals of the metalloprotein for labelling of the isotopically labelled spike (⁶⁵Cu, ⁶⁸Zn SOD). The stability of the metal–protein complex under non-denaturing conditions during 1-D PAGE was confirmed and the performance of the method evaluated. Between 4 and 64 μg, SOD was quantified with a recovery rate between 82% and 110% in a standard. The use of the isotopically enriched SOD was utilised to identify the extent of orthogonal diffusion in 1-D gel electrophoresis. Orthogonal diffusion of natural and isotopically enriched SOD in the gel can interfere with the correct determination of the isotope ratios. The matrix effect of a cytosolic liver extract on the non-covalently bound copper and zinc in SOD was evaluated and no significant metal loss from the SOD spike was observed. This study represents the first step necessary for establishing and evaluating the use of a species-specific isotope dilution approach for the absolute quantification of SOD in real samples based on the combination of gel electrophoresis and LA-ICP-MS.     

The potential of inorganic mass spectrometry in mineral and trace element nutrition research

Over the past two decades, new applications of inorganic mass spectrometry have been made possible by the use of stable isotopes as tracers in studies of mineral and trace element metabolism in man. Stable isotope techniques and radioisotope methods are the only reliable tools available for determination of the absorption, retention, or utilization of a nutrient by the human body. Recent developments in inorganic mass spectrometry might open new perspectives as progress in this field of research depends mainly on improving existing stable isotope techniques and on developing novel concepts. By improving precision in isotope analysis, isotope doses in experiments on man can be reduced to physiologically more meaningful levels. This will also enable reduction of the (often substantial) costs of isotopically labeling a nutrient in a test meal. Improvements in the mass spectrometric sensitivity will enable the development of new tracer techniques that have the potential to provide the information required by: 1. governmental institutions for designing food fortification programs; 2. the food industry for developing nutrient-fortified food products; and 3. public health authorities for establishing reliable dietary recommendations for intake of inorganic nutrients. In this context the current scope and limitations of thermal ionization mass spectrometry, inductively coupled mass spectrometry, accelerator mass spectrometry, and resonance ionization mass spectrometry are evaluated. Iron isotopic variations in the human body are discussed as a possible source of bias that might be a future biological limit to stable isotope-dose reduction in experiments on iron metabolism in man.     

Acetate, propionate and butyrate in plasma: determination of the concentration and isotopic enrichment by gas chromatography/mass spectrometry with positive chemical ionization

This study describes a rapid and simple method to determine short-chain fatty acid (SCFA) concentrations and their isotopic enrichments (M(0) + 1 and M(0) + 2) in human plasma. Sample preparation involves SCFA extraction and derivatization with 1-(tert-butyldimethylsilyl)imidazole. Gas chromatography/mass spectrometry was performed using chemical ionization with ammonia as the reagent gas. Outstanding resolution, excellent linearity and good detection limits were obtained. Inter-assay and intra-assay repeatability was below 10% and 3% respectively for SCFA concentration. Inter-assay repeatability was below 5%, 4%, 6%, and 14% for isotopic enrichment determination of [1-(13)C]acetate and [1,2-(13)C(2)]acetate, [1-(13)C]propionate and [1-(13)C]butyrate respectively, with intra-assay being below 6%. Such SCFA concentrations and isotopic enrichments were determined in the plasma of rats infused with a (13)C-labeled SCFA. The turnovers of acetate, propionate and butyrate in rats were 19 micromol kg(-1) min(-1), 2.6 micromol kg(-1) min(-1), 0.3 micromol kg(-1) min(-1) respectively.     

Study of the size-based environmental availability of metals associated to natural organic matter by stable isotope exchange and quadrupole inductively coupled plasma mass spectrometry coupled to asymmetrical flow field flow fractionation

The determination of the isotopically exchangeable fraction of metals in environmental solid samples (soils, composts, sediments, sludges, etc.) is used to know the amount of metal potentially available (E-value). Stable isotopes can be used for determination of E-values through the analysis of the aqueous phases from spiked suspensions. However, the presence of isotopically non-exchangeable metal forms in the aqueous phase led to overestimation of the E-values. In this paper, a method for monitoring the degree of isotopic exchange in function of the molecular mass and/or size of the metal form has been developed based on the direct coupling of asymmetrical flow field flow fractionation (AsFlFFF) with inductively coupled plasma mass spectrometry (ICP-MS) for on-line isotope ratio measurements. ICP-MS data acquisition parameters were stressed to avoid degradation of isotope ratio precision. Two sets of fractionation conditions were selected: a colloids separation, which allowed the separation of substances up to 1 μm, and a macromolecules separation, designed to resolve small size substances up to 50 kDa. The methodology was applied to study the environmental availability of copper and lead in compost samples, where metals are mainly associated to different forms of organic matter. No significant differences on isotopic exchange were observed over the size range studied, validating the E-values determined by direct analysis of the aqueous phases.     

The potential of inorganic mass spectrometry in mineral and trace element nutrition research

Over the past two decades, new applications of inorganic mass spectrometry have been made possible by the use of stable isotopes as tracers in studies of mineral and trace element metabolism in man. Stable isotope techniques and radioisotope methods are the only reliable tools available for determination of the absorption, retention, or utilization of a nutrient by the human body. Recent developments in inorganic mass spectrometry might open new perspectives as progress in this field of research depends mainly on improving existing stable isotope techniques and on developing novel concepts. By improving precision in isotope analysis, isotope doses in experiments on man can be reduced to physiologically more meaningful levels. This will also enable reduction of the (often substantial) costs of isotopically labeling a nutrient in a test meal. Improvements in the mass spectrometric sensitivity will enable the development of new tracer techniques that have the potential to provide the information required by: 1. governmental institutions for designing food fortification programs; 2. the food industry for developing nutrient-fortified food products; and 3. public health authorities for establishing reliable dietary recommendations for intake of inorganic nutrients. In this context the current scope and limitations of thermal ionization mass spectrometry, inductively coupled mass spectrometry, accelerator mass spectrometry, and resonance ionization mass spectrometry are evaluated. Iron isotopic variations in the human body are discussed as a possible source of bias that might be a future biological limit to stable isotope-dose reduction in experiments on iron metabolism in man. Received: 9 February 2001 / Revised: 21 March 2001 / Accepted: 23 March 2001     

Determination on the microscale of plasmatic lactic acid as its t-butyldimethylsilyl derivative by stable-isotope dilution using capillary gas chromatography/mass spectrometry.

A new sensitive and precise method for the determination of lactic acid in plasmatic microsamples (50 microL) has been developed. Lactic acid was directly extracted from plasma by ethyl acetate in acidic conditions, and analysed as its di-t-butyldimethylsilyl derivative by capillary gas chromatography/electron-impact mass spectrometry (GC/MS). The internal standard was a previously synthesized deuterated compound, 3-[2H]-(2R)-lactic acid. The method gives good reproductibility and precision, the overall standard deviation being better than 3%. The GC/MS assay was in good agreement with the enzymatic determination.     

Mass-independent isotopic fractionation of tin in chemical exchange reaction using a crown ether

Tin isotopes were fractionated by the liquid-liquid extraction technique with a crown ether, dicyclohexano-18-crown-6. The isotopic ratios of ^mSn/¹²⁰Sn (m: 116, 117, 118, 119, 122 and 124) were measured by multi-collector inductively coupled plasma spectrometry (MC-ICP-MS) on a Nu Plasma 500 with a precision better than 0.05 permil amu⁻¹ on each isotopic ratio. Odd atomic mass isotopes (¹¹⁷Sn and ¹¹⁹Sn) showed depletions compared to the even atomic mass isotopes (¹¹⁶Sn, ¹¹⁸Sn, ¹²²Sn and ¹²⁴Sn). We show that this odd-even staggering property originates from the nuclear field shift effect. The contribution of the nuclear field shift effect to the observed isotope enrichment factor was estimated to be ∼35%.     

Stable isotopes and the international system of units

It is now over 60 years since Nier built the first isotope ratio mass spectrometer. The introduction of continuous-flow techniques heralded a huge expansion in the use of stable isotopes in biomedical and environmental sciences, yet there is no consensus on the appropriate units, especially in the biomedical field. Most isotope ratio mass spectrometry (IRMS) instruments calculate isotopic abundance in terms of delta notation (delta, per thousand, per mil), which is a convention determined by geochemistry, because most of the original IRMS instruments were developed in isotope geochemistry laboratories to measure natural abundance variations. Delta units are not SI units. This paper considers the appropriate units for studies using stable isotopes based on the International System of Units (SI). The SI base unit for concentration is the mol, from which atom fraction and mol fraction are derived. The units of stable isotope abundance, atom % and mol %, are the atom and mol fractions expressed as percentages. Atom % excess and mol % excess are the SI units of enrichment and are to be recommended for use in tracer studies.     

Analysis of tear glucose concentration with electrospray ionization mass spectrometry

We have developed a mass spectrometry-based method that allows one to accurately determine the glucose concentration of tear fluid. We used a 1 microL micro-capillary to collect tear fluid from the tear meniscus with minimal irritation of the eye. We analyzed the 1 muL volume of collected tear fluid with liquid-chromatography electrospray ionization mass spectrometry with the use of D-glucose-6,6-d2 as an internal standard. Repeated measurements and a recovery experiment on pooled, onion-induced tears showed that the analysis of the glucose in tears was precise (4% relative standard deviation) and provided 100% recovery. We found the tear glucose concentration of one fasting nondiabetic subject to be 13 to 51 microM while the onion-induced tear glucose concentration of a different nondiabetic subject to be 211 to 256 microM.     

High yield synthesis and characterization of isotopically enriched monoethylmercury chloride (C2H5201HgCl)

An important but commercially unavailable compound isotopically enriched monoethylmercury chloride (C₂H₅²⁰¹HgCl), has been synthesized from commercially available ²⁰¹HgO (98.11% enriched isotopic purity) and tetraethyltin. The required synthesis time is 1 h at 90 °C, and the product is the single product of monoethylmercury chloride, yielding more than 95% as ²⁰¹Hg in C₂H₅²⁰¹Hg⁺ (98.19 ± 0.22% enriched isotopic purity). The synthesized product was analyzed with high‐performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC‐ICP‐MS) to determine its concentration, isotopic composition and purity. The synthetic isotopically enriched monoethylmercury synthesized can be used in speciated isotope dilution mass spectrometry (SIDMS) and isotope dilution mass spectrometry (IDMS) analyses as a standard. Copyright © 2005 John Wiley & Sons, Ltd.     

Quantitation of plasma 13C-galactose and 13C-glucose during exercise by liquid chromatography/isotope ratio mass spectrometry

The utilisation of carbohydrate sources under exercise conditions is of considerable importance in performance sports. Incorporation of optimal profiles of macronutrients can improve endurance performance in athletes. However, gaining an understanding of the metabolic partitioning under sustained exercise can be problematical and isotope labelling approaches can help quantify substrate utilisation. The utilisation of oral galactose was investigated using ¹³C‐galactose and measurement of plasma galactose and glucose enrichment by liquid chromatography/isotope ratio mass spectrometry (LC/IRMS). As little as 100 μL plasma could readily be analysed with only minimal sample processing. Fucose was used as a chemical and isotopic internal standard for the quantitation of plasma galactose and glucose concentrations, and isotopic enrichment. The close elution of galactose and glucose required a correction routine to be implemented to allow the measurement, and correction, of plasma glucose δ¹³C, even in the presence of very highly enriched galactose. A Bland‐Altman plot of glucose concentration measured by LC/IRMS against glucose measured by an enzymatic method showed good agreement between the methods. Data from seven trained cyclists, undergoing galactose supplementation before exercise, demonstrate that galactose is converted into glucose and is available for subsequent energy metabolism. Copyright © 2011 John Wiley & Sons, Ltd.