A sulfur amino acid-free meal increases plasma lipids in humans

The content of sulfur amino acid (SAA) in a meal affects postprandial plasma cysteine concentrations and the redox potential of cysteine/cystine. Because such changes can affect enzyme, transporter, and receptor activities, meal content of SAA could have unrecognized effects on metabolism during the postprandial period. This pilot study used proton NMR ((1)H-NMR) spectroscopy of human plasma to test the hypothesis that dietary SAA content changes macronutrient metabolism. Healthy participants (18-36 y, 5 males and 3 females) were equilibrated for 3 d to adequate SAA, fed chemically defined meals without SAA for 5 d (depletion), and then fed isoenergetic, isonitrogenous meals containing 56 mg·kg(-1)·d(-1) SAA for 4.5 d (repletion). On the first and last day of consuming the chemically defined meals, a morning meal containing 60% of the daily food intake was given and plasma samples were collected over an 8-h postprandial time course for characterization of metabolic changes by (1)H-NMR spectroscopy. SAA-free food increased peak intensity in the plasma (1)H-NMR spectra in the postprandial period. Orthogonal signal correction/partial least squares-discriminant analysis showed changes in signals associated with lipids, some amino acids, and lactate, with notable increases in plasma lipid signals (TG, unsaturated lipid, cholesterol). Conventional lipid analyses confirmed higher plasma TG and showed an increase in plasma concentration of the lipoprotein lipase inhibitor, apoC-III. The results show that plasma (1)H-NMR spectra can provide useful macronutrient profiling following a meal challenge protocol and that a single meal with imbalanced SAA content alters postprandial lipid metabolism.     

NMR-based metabonomic studies reveal changes in the biochemical profile of plasma and urine from pigs fed high-fibre rye bread

This study presents an NMR-based metabonomic approach to elucidate the overall endogenous biochemical effects of a wholegrain diet. Two diets with similar levels of dietary fibre and macronutrients, but with contrasting levels of wholegrain ingredients, were prepared from wholegrain rye (wholegrain diet (WGD)) and non-wholegrain wheat (non-wholegrain diet (NWD)) and fed to four pigs in a crossover design. Plasma samples were collected after 7 d on each diet, and 1H NMR spectra were acquired on these. Partial least squares regression discriminant analysis (PLS-DA) on spectra obtained for plasma samples revealed that the spectral region at 3.25 parts per million dominates the differentiation between the two diets, as the WGD is associated with higher spectral intensity in this region. Spiking experiments and LC-MS analyses of the plasma verified that this spectral difference could be ascribed to a significantly higher content of betaine in WGD plasma samples compared with NWD samples. In an identical study with the same diets, urine samples were collected, and 1H NMR spectra were acquired on these. PLS-DA on spectra obtained for urine samples revealed changes in the intensities of spectral regions, which could be ascribed to differences in the content of betaine and creatine/creatinine between the two diets, and LC-MS analyses verified a significantly lower content of creatinine in WGD urine samples compared with NWD urine samples. In conclusion, using an explorative approach, the present studies disclosed biochemical effects of a wholegrain diet on plasma betaine content and excretion of betaine and creatinine.     

Impaired phenylalanine-tyrosine conversion in patients with iron-deficiency anemia studied by a L-(2H5)phenylalanine-loading test

Ten patients with manifest iron deficiency and without documented relationship to phenylketonuria patients were orally loaded with 25 mg/kg of L-(2H5)phenylalanine. Before loading, the fasting phenylalanine-tyrosine plasma ratio was determined and after loading, the concentrations of labeled and nonlabeled phenylalanine and tyrosine were determined in five consecutive plasma samples. With respect to the fasting phenylalanine-tyrosine ratio and to the post-load ratios of labeled phenylalanine over labeled tyrosine, the iron-deficient patients showed data intermediate between those of normals and heterozygotes for phenylketonuria. Compared to a 100% in vivo activity of phenylalanine hydroxylase in normals and a circa 37% activity in heterozygotes for classic phenylketonuria, iron-deficient patients with an average hemoglobin of 8.6 +/- 1 g/dl showed an activity of circa 56%. After normalization of their iron status, four patients were subjected again to the L-(2H5)phenylalanine-loading test. For three of these individuals, test results shifted into the range of normal.     

Total-body-water measurement with (18)O-labeled water in short-bowel patients with an ileostomy

The aim of this study was to evaluate the use of H(2)(18)O for total-body-water (TBW) determination in patients with short bowel because ileostomy losses of labeled water can falsify TBW. Thirteen adult short-bowel patients were studied after an overnight fast. Saliva, blood, urine and ileostomy output were collected before and 30, 60, 90, 120, 240, and 360 min after oral ingestion of 0.88 g/kg of 2.5% H(2)(18)O to measure (18)O abundance by isotope-ratio mass spectrometry. TBW was calculated from the dilution of the isotopic water in saliva and plasma. The quantity of labeled water lost in the ileostomy was calculated from the isotopic enrichment of the ileostomy output from T0 to T360. The values obtained from saliva or plasma with (corrected) and without (uncorrected) considering H(2)(18)O lost in the ileostomy output were compared with a paired t test. Agreement was evaluated using the Bland-Altman method. From T0 to T360, the ileostomy output and the percentage of lost labeled water were 490 +/- 314 mL and 6.38 +/- 8.52%, respectively. TBW calculated from plasma or saliva isotopic enrichment was different, and a significant difference was also observed between corrected and uncorrected TBW values (saliva: uncorrected TBW = 32.35 +/- 7.52 L, corrected TBW = 30.29 +/- 6.09 L; plasma; uncorrected TBW = 30.80 +/- 7.29 L, corrected TBW = 28.79 +/- 5.79 L). The agreement between the values obtained from the two dilution spaces or between the two calculation methods was poor. Because of the large discrepancies between calculation methods, determination of TBW from oral ingestion of (18)O-labeled water in patients with short bowel should be calculated only from plasma (18)O space dilution and should consider ileostomy losses.     

Determination of total body water by deuterium NMR

A new deuterium nuclear magnetic resonance (2H NMR) method is described for determining total body water in humans. The method has been validated against a standard infrared absorption (IR) procedure using a tracer dose of deuterium oxide (2H2O) of approximately 10 g for each human subject. The precision and accuracy for the methods have been compared and found to be very similar. The advantages of the 2H NMR method over other presently available techniques that are based on 2H2O dilution are as follows: it is fast, accurate, needs only a small dose of 2H2O, can be done using any body fluid, and, most importantly, does not require any sample preparation.     

Evaluation of nuclear magnetic resonance spectroscopy for determination of deuterium abundance in body fluids: application to measurement of total-body water in human infants

Nuclear magnetic resonance (NMR) spectroscopy was used to quantitate abundance of 2H in body water of human infants. This method provides precise measurement of total-body water without the extensive sample preparation requirements of previously described methods for determination of 2H content in body fluids. 2H2O (1 g/kg body weight) was administered to infants and saliva and urine were collected for up to 5 h. An internal standard was added directly to the fluid specimen and 2H enrichment in water was measured by NMR spectroscopy. Working range of deuterium abundance was 0.04-0.32 atom %. Coefficients of variation for saliva samples at 0.20 atom % 2H was 1.97%. 2H content in urine and saliva water reached a plateau by 4 h after administration, and amounts in the two fluids were virtually identical. Mean total-body water determination for six infants was 58.3 +/- 5.8% of body weight (range 53-66%).     

Analysis of retinol and dideuterated retinol in rat plasma by gas chromatography combined mass spectrometry

An isotope dilution method for determining the total body stores of vitamin A using a hydrocarbon derivative of rat plasma retinol, anhydroretinol (ANR) or dideuteroanhydroretinol (2H2ANR) has been evaluated by gas chromatography combined mass spectrometry (GC/MS). Rats with negligible vitamin A stores were dosed with retinyl acetate (ROA) or ROA and 11,12dideuteroretinyl acetate (2H2ROA). The 2H2ROA dose was allowed to equilibrate with total body stores for seven days and the deuterium/hydrogen ratio (D/H ratio) of rat plasma retinol fraction was determined. The results indicate that after the equilibration period the plasma D/H ratio is 73 to 108 percent of the loading dose D/H ratio. This study extends earlier reports that exogenous vitamin A storage is lower than the expected fifty percent figure in rats with low initial total body stores of vitamin A [1, 7, 8, 15, 17]. This work supports the concept that the estimation of total body stores of vitamin A by an isotope dilution method is useful for populations with negligible stores (i.e. liver stores) of vitamin A.     

Determination of total body water by Fourier transform infrared analysis

A new technique for determining body water using deuterium isotope dilution and Fourier transform infrared (FTIR) analysis is described. The advantages of the FTIR over conventional dispersion and filter infrared instruments include greater flexibility through computer controlled operations and availability of 'on-line' analytical software. The technique was further improved by the development of a simple procedure for determining D2O concentration in untreated serum samples. A validation study of six normal adults showed that the fat-free-mass determined from the deuterium-space (total body water) correlated well with the results obtained by total body nitrogen (r = 0.997), total body potassium (r = 0.996) and anthropometric (r = 0.995) measurements.     

Application of NMR-based metabonomics suggests a relationship between betaine absorption and elevated creatine plasma concentrations in catheterised sows

The objective of the present explorative study was to determine the absorption dynamics when feeding diets varying in types and levels of dietary fibre in a catheterised animal model. A total of six sows were fed a diet low in fibre (LF), a diet high in soluble fibre and a diet high in insoluble fibre in a repeated 3 × 3 cross-over design. Plasma samples were collected from the mesenteric artery and the portal vein to determine different absorption phases by 1H NMR spectroscopy-based metabonomics. Time profiles were determined for plasma levels of specific metabolites and for the absorption of these metabolites from the small intestine. The LF diet resulted in a higher betaine concentration in the blood than the two high-fibre diets (P=0·008). This leads to higher plasma concentrations of methionine (P=0·0028) and creatine (P=0·020) of endogenous origin. In conclusion, the use of NMR spectroscopy for measuring nutrient uptake in the present study elucidated the relationship between betaine uptake and elevated creatine plasma concentrations.     

Studies on calcium absorption from milk using a double-label stable isotope technique

Calcium absorption was measured in ten male volunteers from skimmed milk, Ca-enriched skimmed milk or watercress (Nasturtium officinale) soup. The foods were labelled extrinsically with 30 mg 44Ca. Shortly after consuming the labelled meal, each subject was given an intravenous injection of 3 mg 42Ca. Fractional absorption from the oral dose was determined from plasma and urine samples collected 24-72 h later, using fast atom bombardment mass spectrometry to measure isotope ratios. The values for urine and plasma were in good agreement. Mean percentage absorption was 45.5 (SEM 1.9)% from the skimmed milk, 35.7 (SEM 4.7)% from the Ca-enriched milk and 27.4 (SEM 1.9)% from the watercress soup. The effect of consuming 568 ml (1 pint) Ca-enriched milk each day for 4 weeks on the efficiency of absorption of Ca was studied. Although there was no statistically significant difference between Ca absorption before and after the supplementation period, the results were considered to be somewhat inconclusive due to the small number of subjects and wide individual variation in Ca absorption.     

Long-term use of an injectable contraceptive: effect of depot-norethisterone oenanthate on carbohydrate metabolism

In order to determine the metabolic effects of long-term use of the injectable contraceptive norethisterone oenanthate, plasma glucose and serum insulin concentrations were studied in two groups of women who had used the method continuously for at least five years. Group 1 comprised 24 subjects, from whom only fasting blood samples were taken. Despite similar plasma glucose concentrations to those of the controls, the subjects had significantly increased serum insulin concentrations (164.5 (39.9) v 120.3 (34.3) pmol/l, p less than 0.01). In addition the insulin:glucose ratios were also significantly increased (34.3 (8.5) v 24.6 (6.7), p less than 0.01), consistent with decreased insulin sensitivity. Group 2 comprised 13 of the original 24 subjects who also had an oral glucose tolerance test. Basal plasma glucose concentrations were similar in the subjects and their controls, whilst the significantly increased insulin:glucose ratios (35.0 (7.7) v 28.7 (5.6), p less than 0.05) were consistent with the results of the larger group. Following oral glucose challenge, plasma glucose concentrations, serum insulin concentrations and insulin:glucose ratios were similar in the subjects and their controls throughout the test. Thus, long-term use of norethisterone oenanthate injections is associated with a decrease in peripheral insulin sensitivity. However, these changes are not associated with any evidence of oral glucose intolerance.     

Comparison of human whole blood, plasma, and serum matrices for the determination of perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and other fluorochemicals

Interest in human exposure to perfluorinated acids, including perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), perfluorooctanesulfonate (PFOS), and perfluorooctanoate (PFOA) has led to their measurement in whole blood, plasma and serum. Comparison of measurements in these different blood-based matrices, however, has not been rigorously investigated to allow for across-matrix comparisons. This research evaluated concentrations of PFBS, PFHS, PFOS, and PFOA in whole blood collected in heparin (lithium) and ethylenediamine tetraacetic acid (EDTA), plasma samples collected in heparin and EDTA, and serum (from whole blood allowed to clot). Blood samples were collected from 18 voluntary participants employed at 3M Company. Solid phase extraction methods were used for all analytical sample preparations, and analyses were completed using high-pressure liquid chromatography/tandem mass spectrometry methods. Serum concentrations ranged from: limit of quantitation (LOQ, 5 ng/mL) to 25 ng/mL for PFBS; LOQ (5 ng/mL) to 75 ng/mL for PFHS; LOQ (5 ng/mL) to 880 ng/mL for PFOS; and LOQ (5 or 10 ng/mL) to 7320 ng/mL for PFOA. Values less than the LOQ were not included in the statistical analyses of the mean of the ratios of individual values for the matrices. PFBS was not quantifiable in most samples. Serum to plasma ratios for PFHS, PFOS, and PFOA were 1:1 and this ratio was independent of the level of concentrations measured. Serum or plasma to whole blood ratios, regardless of the anticoagulant used, approximated 2:1. The difference between plasma and serum and whole blood corresponded to volume displacement by red blood cells, suggesting that the fluorochemicals are not found intracellularly or attached to the red blood cells.     

荧光分析法测定血浆和头发中硒含量的比值

采用氧瓶燃烧法对血样和发样进行破坏 ,利用 β-环糊精和十二烷基硫酸钠形成包合物的性质 ,将苯并苤硒脑包合 ,建立了三元包合物荧光分析法 ,不经萃取 ,直接测定血浆和头发中的硒。测血硒的日内精密度 (n=7)为 5.4%～ 9.3% ;日间精密度 (n=7)为 3.5%～ 1 4.5% ;线性范围为 1 0～ 50 0 ng·ml-1;回收率 (n=4)为 91 .0 %～ 97.8%。测发硒的日内精密度 (n=6)为 0 .4%～ 5.9% ;线性范围为 1～ 1 0 0μg· ml-1;回收率为 94.6%～ 1 0 2 .0 %。对 1 8名志愿者的发样和血样测定发现 ,发硒与血硒间存在弱相关 (r=0 .2 0 4 5) ,发硒与血硒比值的平均值为2 .1 7,比值范围在 1 .63～ 2 .88之间     

239,240Pu and inorganic substances in aerosols from the vicinity of a waste isolation pilot plant: the importance of resuspension

Aerosol samples were collected and analyzed to characterize the spatial and temporal variations in the concentrations of plutonium and selected inorganic substances in the atmosphere around the Waste Isolation Pilot Plant (WIPP). High-volume aerosol sampling was conducted at three sites: (1) On Site, (2) Near Field, and (3) Cactus Flats. 239,240Pu was determined by alpha spectrometry following chemical separations; mass loadings were determined gravimetrically. A separate set of low-volume aerosol samples was analyzed for major ions using ion chromatography and for trace elements by inductively-coupled plasma emission spectrometry and mass spectrometry. The average 239,240Pu activity concentrations in total suspended particle (TSP) samples (12 to 16 nBq m(-3)) were consistent with those previously reported, but they varied strongly with season, with the highest values generally in spring. Further, the 239,240Pu activity concentrations were comparable among the three sites, and therefore there was no evidence for elevated 239,240Pu activities due to WIPP operations. The fraction of the 239,240Pu activity concentrations in the PM10, samples (particles less than 10 microm diameter) relative to TSP was lower than the corresponding PM10/TSP ratios of either high-volume mass or several inorganics (sulfate, aluminum or lead), indicating that 239,240Pu tends to be on large particles. Aerosol mass loadings (microg m(-3)) and 239,240Pu activity concentrations were correlated for all sets of samples, but at On Site, the TSP samples showed higher mass to 239,240Pu ratios than the other sites. Thus activities or processes occurring at or near the WIPP site evidently produced aerosols that contributed to the mass loadings but contained less 239,244Pu than ambient aerosols. About 63% of the variability in 239,240Pu activity concentrations was explained by wind travel, sampling location, length of the sampling interval, and aerosol mass. 239,240Pu activity concentrations also were correlated with aluminum (an indicator of mineral dust), further implicating the resuspension of soils as an important determinant of 239,240Pu in aerosols. The 239,240Pu/Al ratios for the aerosols were higher than in soils, and this could be explained by the preferential binding of 239,240Pu to small soil particles that have large surface area to mass ratios and also have higher aluminum contents than larger particles.     

Proton magnetic resonance spectroscopy for assessment of human body composition

BACKGROUND: The usefulness of magnetic resonance spectroscopy (MRS)-based techniques for assessment of human body composition has not been established. OBJECTIVE: We compared a proton MRS-based technique with the total body water (TBW) method to determine the usefulness of the former technique for assessment of human body composition. DESIGN: Proton magnetic resonance spectra of the chest to abdomen, abdomen to pelvis, and pelvis to thigh regions were obtained from 16 volunteers by using single, free induction decay measurement with a clinical magnetic resonance system operating at 1.5 T. The MRS-derived metabolite ratio was determined as the ratio of fat methyl and methylene proton resonance to water proton resonance. The peak areas for the chest to abdomen and the pelvis to thigh regions were normalized to an external reference (approximately 2200 g benzene) and a weighted average of the MRS-derived metabolite ratios for the 2 positions was calculated. TBW for each subject was determined by the deuterium oxide dilution technique. RESULTS: The MRS-derived metabolite ratios were significantly correlated with the ratio of body fat to lean body mass estimated by TBW. The MRS-derived metabolite ratio for the abdomen to pelvis region correlated best with the ratio of body fat to lean body mass on simple regression analyses (r = 0.918). The MRS-derived metabolite ratio for the abdomen to pelvis region and that for the pelvis to thigh region were selected for a multivariate regression model (R = 0.947, adjusted R(2) = 0.881). CONCLUSION: This MRS-based technique is sufficiently accurate for assessment of human body composition.     

Changes in vitamin E concentrations in human plasma and platelets with age.

It has been postulated that vitamin E compounds (tocopherols) can retard the aging process. Therefore, changes in concentrations of tocopherols in human plasma and platelets with aging were studied. Fasting blood samples were obtained from 48 healthy male volunteers aged 24-91 years. Concentrations of the tocopherols in plasma and platelets were determined by a liquid chromatographic method. Alpha and gamma tocopherols were the major forms of vitamin E found in both plasma and platelets. The concentrations of alpha and total tocopherols in plasma did not change significantly with age. However, the plasma gamma tocopherol and the platelet alpha, gamma, and total tocopherol concentrations decreased significantly with age. The platelet to plasma ratios of tocopherol concentrations also decreased with age.     

Changes in vitamin E concentrations in human plasma and platelets with age

It has been postulated that vitamin E compounds (tocopherols) can retard the aging process. Therefore, changes in concentrations of tocopherols in human plasma and platelets with aging were studied. Fasting blood samples were obtained from 48 healthy male volunteers aged 24-91 years. Concentrations of the tocopherols in plasma and platelets were determined by a liquid chromatographic method. Alpha and gamma tocopherols were the major forms of vitamin E found in both plasma and platelets. The concentrations of alpha and total tocopherols in plasma did not change significantly with age. However, the plasma gamma tocopherol and the platelet alpha, gamma, and total tocopherol concentrations decreased significantly with age. The platelet to plasma ratios of tocopherol concentrations also decreased with age.     

干血斑用于HIV-1耐药基因型检测的研究

目的 通过干血斑样本与血浆、全血的HIV-1基因型耐药性检测结果比较,探讨干血斑样本应用于我国患者HIV-1耐药性检测及监测的可行性.方法 选择来自安徽(10例)、云南(13例)、湖南(6例)和新疆维吾尔自治区(10例)4省(自治区)共39例AIDS患者,这些患者感染的HIV-1流行株覆盖中国主要的流行亚型(B、CRF01 AE、CRF07_BC),同时对同一患者的血浆、全血及干血斑3种类型的样本应用实验室自建的套式PCR方法扩增HIV的pol基因区,通过美国斯坦福大学的HIV耐药数据库进行耐药程度判别并比较三者的耐药性结果.结果 干血斑、全血与血浆样本相比总扩增成功率分别为95%(37/39)、92%(36/39)及100%(39/39),各亚型样本基因序列的3种样本的一致性均高于99%,同时干血斑样本的耐药性检测结果与血浆相比一致性为86%(31/36),突变位点不一致的主要原因为混合碱基导致.结论 综合PCR扩增效果及序列分析差异等因素,干血斑样本可以反映出耐药的整体流行趋势,用于我国HIV-1患者的耐药检测及监测值得推广.     

Evaluation of sample preparation methods for nuclear magnetic resonance metabolic profiling studies with Eisenia fetida

The earthworm Eisenia fetida is frequently used in ecotoxicological studies; however, it has not yet been investigated using proton nuclear magnetic resonance ((1)H NMR) metabolic profiling methods. The present study investigates the impact of depuration time, sample homogenization, and different extraction solvents on the quality and reproducibility of the (1)H NMR spectra of E. fetida with the goal of determining whether this species is suitable for future metabonomic studies. A depuration time of 96 h, followed by intact lyophilization before homogenization and extraction into a deuterium oxide (D(2)O)-based phosphate buffer, was found to produce extracts with excellent (1)H NMR reproducibility. The D(2)O buffer extracted the largest quantity of the widest variety of earthworm metabolites, which is consistent with the results from other studies using different earthworm species. Nuclear magnetic resonance assignments of the major metabolites in the D(2)O-based buffer also were performed and found to be similar to those for other earthworm species, such as Eisenia veneta, but also to have characteristic attributes in E. fetida. The major metabolites identified include amino acids (alanine, arginine, glutamic acid, glutamine, glycine, leucine, lysine, phenylalanine, serine, tyrosine, and valine), two sugars (glucose and maltose), the sugar alcohol mannitol, and the polyalcohol inositol. Two other earthworm species (Lumbricus rubellus and Lumbricus terrestris) also were examined using protocols developed for E. fetida, and of the three species, the (1)H NMR spectra of E. fetida had the least variation, indicating this species is well-suited for future metabolomic-based ecotoxicity studies.     

In vivo isotope-fractionation factors and the measurement of deuterium- and oxygen-18-dilution spaces from plasma, urine, saliva, respiratory water vapor, and carbon dioxide

In vivo isotope-fractionation factors were determined for hydrogen and oxygen between plasma water samples and samples of urine, saliva, respiratory water vapor, and carbon dioxide in 20 normal adults. The isotope-fractionation factors ranged from 0.944 to 1.039 for 2H in breath water vapor and for 18O in breath CO2, respectively. When corrected for isotope fractionation, the 2H- and 18O-dilution spaces determined from urine, saliva, respiratory water, and CO2 were within -0.10 +/- 1.09 kg (mean +/- SD, n = 60) and 0.04 +/- 0.68 kg (n = 80), respectively, of the values determined from plasma. In the absence of these corrections, we observed a 6% overestimation of 2H-dilution space and a 1% overestimation of 18O-dilution space from the use of respiratory water values. A 4% underestimation of the 18O-dilution space was observed for breath CO2 without correction for isotope fractionation.