Zdeněk Švagera

In situ derivatization-liquid liquid extraction as a sample preparation strategy for the determination of urinary biomarker prolyl-4-hydroxyproline by liquid chromatography-tandem mass spectrometry.

Polar analytes that possess protic functional groups have often been treated with alkyl chloroformates to decrease their polarity and increase their volatility prior to gas chromatography-mass spectrometry analysis. This derivatization reaction has two distinct advantages. It proceeds smoothly in aqueous media, and the desired reaction products are efficiently separated from interfering ionic components by their extraction into a water-immiscible organic phase. In the present work, the derivatization-liquid liquid sample preparation was examined in detail for analysis of a potential urinary dipeptide biomarker L-prolyl-4-L-hydroxyproline (PHP) by downstream liquid chromatography coupled to electrospray mass spectrometry. PHP was treated with a series of alkyl and fluoroalkyl chloroformates in aqueous media, and the detected reaction products were investigated. Smooth conversion of PHP into the N-isobutyloxycarbonyl isobutyl ester was accomplished by the coupled action of isobutanol, isobutyl chloroformate and the pyridine catalyst. This derivative afforded a highest detector response from all the derivatized forms examined, including the nonderivatized PHP. A simple isocratic elution on a common RP-C18 HPLC column coupled with tandem mass spectrometry, and use of the synthesized heptadeuterated analog (D7-PHP) as an internal standard, enabled validation of the method and determination of PHP in human urine in less than 5 min. The in situ derivatization-liquid liquid extraction has thus been demonstrated to be a useful sample preparation strategy for the analysis of polar metabolites by liquid chromatography-tandem mass spectrometry in the complex urine matrix.

Dietary intake and ghrelin and leptin changes after sleeve gastrectomy

Introduction Surgical intervention in obesity is today the most effective treatment method in high level obesity management. Bariatric interventions not only ensure body weight reduction, but may influence dietary habits. Aim To assess changes in adipose hormones and dietary habits in obese patients after sleeve gastrectomy. Material and methods The study set comprised 37 subjects (29 females and 8 males) 24 to 68 years old with body mass index 43.0 ±4.9 kg/m2. Pre-operative examination included baseline measurements of body composition. Dietary habits and intake frequency were monitored by a questionnaire method. Follow-up examinations were carried out in a scope identical to the pre-operative examination, 6 and 12 months after surgery, respectively. Results The average patient weight loss 12 months after surgery was 31.7 kg. Excess weight loss was 55.2 ±20.6%. Patients reported reduced appetite (p < 0.001), increasingly regular food intake (p < 0.001), intake of more meal portions per day (p = 0.003) and a decrease in consuming the largest portions during the afternoon and evening (p = 0.030). Plasma levels of fasting glucose, leptin and ghrelin significantly decreased (p = 0.006; p = 0.0.043); in contrast, the level of adiponectin significantly increased (p < 0.001). Conclusions Sleeve gastrectomy and follow-up nutritional therapy resulted in a significant body weight reduction within 1 year after surgery. An improvement of certain dietary habits in patients was registered. At 12 months after surgery, there were no statistically significant differences in decreases in ghrelin and leptin concentrations between patients without changed appetite and those reporting decreased appetite.

Profiling of urinary amino-carboxylic metabolites by in-situ heptafluorobutyl chloroformate mediated sample preparation and gas chromatography-mass spectrometry.

A novel 1,1,1,2,2,3,3-heptafluorobutyl chloroformate reagent (HFBCF) was examined for in-situ derivatization of amino-carboxylic metabolites in human urine. The arising reaction products exhibit greatly reduced polarity which facilitates combining the derivatization and liquid-liquid microextraction (LLME) from an aqueous urine into an isooctane phase and immediate gas chromatographic-mas spectrometric analysis (GC-MS). The sample preparation protocol is simple, proceeds without an alcohol excess and provides cleaner extracts than other urinary GC-MS based methods. Moreover, thiol metabolites bound in disulfide bonds can be released by reduction with tris(3-hydroxypropyl)phosphine (THP) prior to the developed derivatization and LLME step. In order to evaluate potential of the novel method for GC-MS metabolomics, reaction products of 153 urinary metabolites with HFBCF, particularly those possessing amino and carboxyl groups (56 amino acids and their conjugates, 84 organic acids, 9 biogenic amines, 4 other polar analytes) and two internal standards were investigated in detail by GC-MS and liquid chromatography-mass spectrometry (LC-MS). One hundred and twenty metabolites (78%) yielded a single product, 25 (16%) and 2 metabolites (2-methylcitrate, citrate) generated two and more derivatives. From the examined set, analytically applicable products of 5 metabolites were not detected; the derivatives of 3 metabolites were only suitable for LC-MS analysis. Electron ionization (EI) of the examined analytes contained characteristic, diagnostic ions enabling to distinguish related and isomeric structures. The new method was validated for 132 metabolites using two internal standards in artificial urine and with special attention to potential disease biomarker candidates. The developed sample preparation protocol was finally evaluated by means of a certified organic acid standard mixture in urine and by GC-MS analysis of 100 morning urines obtained from healthy patients (50 males and 50 females), where 112 physiological metabolites were quantified in a 25 μL sample aliquot. The quantification data for the set were satisfactory, most metabolites were found within the range reported in the reference human metabolome (HMDB) database and literature. The reported results suggest that the described method has been a novel promising tool for targeted GC-MS based metabolomic analysis in urine.

Survey of several methods deproteinizing human plasma before and within the chloroformate-mediated treatment of amino/carboxylic acids quantitated by gas chromatography.

Trichloroacetic acid, perchloric acid, phosphotungstic acid, acetonitrile, methanol and some other organic solvents were evaluated for their ability to provide protein and lipid-free plasma supernatants. The residual proteins, total cholesterol and triacylglycerols were assayed in the supernatant on a Beckman Analyzer instrument. The free cholesterol and the neutral lipids were further analyzed by means of high-temperature GC analysis. The conditions for the deproteinizing step were optimized for minimal lipoprotein disruption. A substantial difference regarding contamination by the lipids was found if the plasma supernatant or the whole serum were treated with an alkyl chloroformate reagent. Three plasma sulfur amino acids and the aromatic ones were chosen as model compounds to evaluate compatibility of the precipitation methods with a subsequent methyl chloroformate-mediated derivatization and GC-MS analysis. The results of the total homocysteine assay matched well with that obtained using a commercial immunoassay. Precipitation with trichloroacetic acid has proven to be a method of choice for the analysis of the acido-basic analytes by GC-MS via chloroformates.

Prolyl-hydroxyproline dipeptide in non-hydrolyzed morning urine and its value in postmenopausal osteoporosis.

Abstract Background: Owing to the high correlation between the level of prolyl-4-hydroxyproline dipeptide in non-hydrolyzed urine and that of 4-hydroxyproline in hydrolyzed urine, we examined whether the dipeptide might function as a valuable marker of bone turnover. Methods: Based on densitometric measurements, 68 postmenopausal women were divided into groups of non-osteopathic, osteopenic and osteoporotic subjects. The dipeptide and current urinary resorption markers were assayed in morning urine, the former using liquid chromatography, the others plus serum formation markers by means of immunoassay procedures. Together with the assay of basal levels, diet-related changes and healing effect of yearly alendronate therapy were assessed. Results: Concentration levels in controls and osteoporotic subjects differed significantly; receiver operating characteristics yielded sensitivity of 0.743, specificity of 0.908, area under curve of 0.903, and cut-off of 10.2 μmol/mmol of creatinine. Spearman rank correlation showed the highest pair coefficient between the dipeptide and osteocalcin. Diet-related changes were not found. Following therapy, a significant decline occurred already within a trimester, whilst with the other resorption markers not until 6 months. Conclusions: The ease of the dipeptide assay in non-hydrolyzed urine surpasses that of hydroxyproline, and the results present the compound as a real competition to other commonly assessed markers in osteopathies. Clin Chem Lab Med 2008;46:1391–7.

Effects of sleeve gastrectomy on parameters of lipid and glucose metabolism in obese women – 6 months after operation

Introduction Surgical intervention in obesity is today the most effective treatment method in high level obesity management with long-term clinical results and satisfaction of operated patients. Bariatric interventions not only ensure body weight reduction, but may influence lipid and saccharide metabolism as well. Aim To monitor the dynamics of changes in selected lipid and glucose metabolism parameters after laparoscopic sleeve gastrectomy (LSG) in obese women. Material and methods During the period from September 2010 to June 2011, 35 women, operated on by sleeve gastrectomy, were monitored within a pilot open study. Parameters of lipid and glucose metabolism were measured, and body composition was evaluated, using dual X-ray absorptiometry (DXA). Laboratory parameters were assessed prior to LSG and at 3 and 6 months after the surgery. Results Data of the 35 study subjects are presented. Average age was 41.9 years (27-68 years). Six months after LSG, body weight reduction was achieved from 117.7 ±17.1 kg to 91.2 ±17.2 kg (p < 0.001). The body mass index (BMI) dropped from 42.7 ±4.7 kg/m2 to 33.0 ±4.9 kg/m2 (p < 0.001). The excess weight loss (EWL) was 49.01%. High density lipoprotein (HDL) cholesterol increased from 1.29 mmol/l to 1.39 mmol/l (p < 0.025). Triacylglycerols dropped from 1.97 mmol/l to 1.31 mmol/l (p < 0.001). Glycated hemoglobin dropped from 4.03% to 3.59% (p < 0.001), and C-peptide decreased from 1703 pmol/l to 1209 pmol/l (p < 0.002). The observed changes of low density lipoprotein (LDL) cholesterol, total cholesterol or fasting glucose levels were not significant. Six months after LSG, both weight and BMI significantly decreased. Conclusions Six months after the operation, glucose homeostasis was improved. Despite the rather short-term monitoring period, our study did confirm LSG to influence not only total weight loss and fat tissue reduction but to improve risk factors, mainly glucose homeostasis and dyslipidemia, as well.

Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting.

Objectives We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. Methods We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. Results Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. Conclusions Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.

Bone mineral density and body composition after laparoscopic sleeve gastrectomy in men: A short-term longitudinal study.

INTRODUCTION Longitudinal changes in bone and body composition occurring in obese men after laparoscopic sleeve gastrectomy (LSG) has been evaluated. METHODS In short-term longitudinal study, 25 obese men in mean baseline age 44.8 ± 10.9 years and mean body mass index (BMI) 43.3 ± 4.4 kg/m(2)were assessed after undergoing LSG for obesity. Bone mineral density (BMD) (spine, femoral neck [FN], total hip [TH], and total body [TB]) and body composition (TB bone mineral content [BMC], fat, % of fat, lean, lean BMC, total mass) were assessed at baseline, and after three and six months. RESULTS Mean body measurements, including weight, BMI, waist and hips, decreased significantly over the study period (p < 0.0001). FN BMD (p < 0.01) and TH BMD (p < 0.001) decreased, and spine BMD increased significantly (p < 0.001). TB BMD did not change. Weight decreased by 21.3 ± 7.3%, BMI by 21.2 ± 7.3%, FN BMD by 3.32 ± 6.35%, TH BMD by 3.51 ± 3.95% whereas spine BMD increased by 2.89 ± 5.1%. TB BMC increased by 2.4 ± 4.62%; all other variables relating to body composition decreased: fat by 33.0 ± 9.6%, lean mass by 12.8 ± 6.1%, lean BMC by 12.3 ± 5.9%, total mass by 20.1 ± 6.4%, and % fat by 15.8 ± 7.2%. CONCLUSIONS After LSG, body size and variables related to body composition (except for TB BMC) decreased with an accompanying decrease in FN BMD in the men in this study. Spine BMD increased, and TB BMD did not change.

Body Size, Bone Mineral Density, and Body Composition in Obese Women After Laparoscopic Sleeve Gastrectomy: A 1-Year Longitudinal Study

Changes in body weight, waist and hip circumferences, body composition, and skeletal status in women after bariatric surgery were evaluated. Thirty-six women [mean age 41.2 ± (SD) 9.5 years, weight 115.7±18.0 kg, and BMI 42.1±5.3 kg/m(2)] underwent laparoscopic sleeve gastrectomy. Bone mineral density (BMD) at lumbar spine, femoral neck, total hip, and total body, and body composition were evaluated at baseline and 3, 6, and 12 months after surgery.Weight, BMI, waist and hip circumferences decreased significantly. Total body bone mineral content (TBBMC) increased by 2.5±3.5%, and fat, lean body mass, total mass and fat-% decreased significantly by 38.9±12.0%, 15.4±5.9%, 26.5±8.1%, and 17.6±8.9%, respectively. Slight decreases in total body (0.6±2.2%) and spine (1.2±7.1%) BMD were not significant, whereas total hip and femoral neck BMD decreased significantly by 5.3±8.2%, and 6.2±7.0% (p<0.001). Change (Δ) in TBBMC correlated only with Δ in weight (r=0.38, p<0.05) whereas Δ in all other body composition parameters correlated significantly with Δ in body weight and circumferences (r=0.46-0.98). The Δ in BMD (except total body BMD) correlated significantly with Δ in body composition parameters (r=0.34-0.59). Baseline fat and lean content besides changes in body fat and lean mass accounted for bone changes. In conclusion, bone loss after bariatric surgery is related to post-operative changes in body composition, as well as to weight loss and decrease in waist and hip circumferences.

Laparoscopic gastric plication and its effect on saccharide and lipid metabolism: a 12-month prospective study

Introduction Laparoscopic greater curvature plication (LGCP) is a novel restrictive technique that reduces gastric volume by plication of the greater curvature. The advantage of LGCP is its reversibility in comparison to laparoscopic sleeve gastrectomy. Nowadays, the long-term LGCP efficacy, safety and metabolic effect are being investigated. Aim To assess body composition, clinical complications and metabolic changes in obese patients 6 and 12 months after laparoscopic greater curvature plication. Material and methods A total of 70 subjects underwent LGCP; 52 of them (33 women and 19 men) completed 1-year follow-up study. Anthropometry and biochemical parameters (glucose, glycated haemoglobin, lipids, ghrelin, leptin, adiponectin and fibroblast growth factor 21 [FGF-21]) were assessed before and 3, 6, and 12 months after surgery. Results All study participants exhibited statistically significant weight loss at both 6 and 12 months following the LGCP compared to baseline, with significant reductions in body composition – body weight, body mass index, percentage excess weight loss (%EWL), and percentage excess BMI loss (%EBL) (p ≤ 0.001). Moreover, significant lowering of glucose and glycated haemoglobin, triacylglycerols and leptin was observed 12 months after LGCP. On the other hand, plasma concentrations of ghrelin, adiponectin and LDL cholesterol increased significantly. Total cholesterol, LDL cholesterol and FGF-21 levels did not change significantly. Conclusions Laparoscopic greater curvature plication appears to be a procedure with good restriction results, which might be mediated through alteration in incretin metabolism. Technical aspects and standardization of the procedure still remain to be worked out.