Native fluorescence spectroscopy of blood plasma in the characterization of oral malignancy

Native fluorescence characteristics of blood plasma were studied in the visible spectral region, at two different excitation wavelengths, 405 and 420 nm, to discriminate patients with different stages of oral malignancy from healthy subjects. The fluorescence spectra of blood plasma of oral malignant subjects exhibit characteristic spectral differences with respect to normal subjects. Different ratios were calculated using the fluorescence intensity values at those emission wavelengths that give characteristic spectral features of each group of experimental subjects studied. These fluorescence intensity ratios were used as input variables for a multiple linear discriminant analysis across different groups. Leave-one out cross-validation was used to check the reliability of each discriminant analysis performed. The discriminant analysis performed across normal and oral cancerous subjects classified 94.7% of the original grouped cases and 93.7% of the cross-validated grouped cases. A classification algorithm was developed on the basis of the score of the discriminant functions (discriminant score) resulted in the analyses. The diagnostic potentiality of the present technique was also estimated in the discrimination of malignant subjects from normal and nonmalignant diseased subjects such as liver diseases. In the discriminant analysis performed across the three groups, normal, oral malignancy (including early and advanced stages) and liver diseases, 99% of the original grouped cases and 95.9% of the cross-validated grouped cases were correctly classified. Similar analysis performed across normal, early stage of oral malignancy, advanced oral malignancy and liver diseases correctly classified 94.9% of the original grouped cases and 91.8% of the cross-validated grouped cases.     

On the search for glycated lipoprotein ApoA-I in the plasma of diabetic and nephropathic patients

The analysis of plasma samples from healthy, diabetic and nephropathic subjects was carried out by 2D gel electrophoresis. This approach shows clear differences among the three classes of subjects. In the case of diabetic and nephropathic patients intense spots appear. Their enzymatic digestion followed by matrix assisted laser desorption ionization/mass spectrometry (MALDI/MS) analysis shows that an overexpression of unglycated and glycated ApoA-I is present in both pathological states. Interestingly, this trend is also observed for the retinol-binding protein (RBP). The data obtained can be relevant to assess possible risks associated either with the glycation level of ApoA-I or with the overexpression of RBP. In fact, in the former case possibly a different functionality of the glycated protein is to be expected, reflecting a different efficiency in cholesterol transport. In the latter case, the increase of RBP level can be related to the overweight of the diabetic subjects under investigation: it is known that obesity leads to RBP overexpression. In the case of nephropathic patients, the RBP level increases in parallel with serum creatinin.     

Sieve-based device for MALDI sample preparation. III. Its power for quantitative measurements

The solid sample inhomogeneity is a weak point of traditional MALDI deposition techniques that reflects negatively on quantitative analysis. The recently developed sieve-based device (SBD) sample deposition method, based on the electrospraying of matrix/analyte solutions through a grounded sieve, allows the homogeneous deposition of microcrystals with dimensions smaller than that of the laser spot. In each microcrystal the matrix/analyte molar ratio can be considered constant. Then, by irradiating different portions of the microcrystal distribution an identical response is obtained. This result suggests the employment of SBD in the development of quantitative procedures. For this aim, mixtures of different proteins of known molarity were analyzed, showing a good relationship between molarity and intensity ratios. This behaviour was also observed in the case of proteins with quite different ionic yields. The power of the developed method for quantitative evaluation was also tested by the measurement of the abundance of IGPP[Oxi]GPP[Oxi]GLMGPP (m/z 1219) present in the collagen-α-5(IV) chain precursor, differently expressed in urines from healthy subjects and diabetic-nephropathic patients, confirming its overexpression in the presence of nephropathy. The data obtained indicate that SBD is a particularly effective method for quantitative analysis also in biological fluids of interest.     

Statistical approach to the rational selection of experimental subjects

Whilst human and animal experimentation receives much attention every year by the general media, the public and the scientific community, little has been done on how to reduce the number of living subjects to be submitted to experimental research. Researchers who design human and animal experiments are conscious that trials on such subjects present a problem, which is not only scientific, but also ethical. The national research and ethics committees regulate the selection and number of patients or mammals to be treated and make the justification of the number of living subjects an aspect of paramount importance in different scientific disciplines. However, a rational methodology to estimate a sensible number that is in accordance with the parameters of the Association of Research Ethics Committees has not been well explored. In this article, a simple statistical approach for the study of the confidence in a model is proposed. The approach can pave the way to a potentially useful method to estimate a sensible number of subjects which is in accordance with the general principles of ethical bodies from all over the world.     

Detection of erythrocyte membrane structural abnormalities in lecithin: cholesterol acyltransferase deficiency using a spin label approach.

The membrane fluidity of erythrocytes from patients with Lecithin: cholesterol acyltransferase (LCAT) deficiency was studied by means of electron spin resonance. The temperature dependence of the separation of the outer extrema of the spectra of 2-(3-carboxy-propyl)-4,4-dimethyl, 2-tridecyl-3-oxazolidinyloxyl spin probe was monitored for normal, presumed carrier and clinically affected subjects. The temperature profile of controls was significantly different from that of the presumed carriers and the clinically affected individuals. The results show that the compositional abnormalities previously noted in erythrocyte membranes from patients with LCAT deficiency are associated with alterations in the physiocochemical state of the membrane. An investigation of the spectral lineshapes below 10 degrees C allowed a distinction to be made at the membrane level between clinically affected subjects and clinically normal heterozygous carriers. Alterations in the temperature dependence of elec-ron spin resonance parameters may provide a sensitive index of red cell membrane alterations in pathological states of generalized membrane involvement.     

Two-dimensional mapping of three phenotype-associated isoforms of the prion protein in sporadic Creutzfeldt-Jakob disease

Transmissible spongiform encephalopathies (TSE), or prion diseases, are mammalian neurodegenerative disorders characterized by a conformational modification of the host-encoded prion protein (PrP(C)) into an isoform which is detergent-insoluble and partially resistant to protease treatment (PrP(Sc)). Distinct types of PrP(Sc), differing in conformation and variation in the relative amount of their glycoforms, have been associated with different phenotypes of TSE. In sporadic Creutzfeldt-Jakob disease (sCJD), two major types of PrP(Sc), with proteinase K (PK)-resistant fragments of 21 and 19 kDa, have been described. No consensus exists, however, on the molecular classification of PrP(Sc) in sCJD, since further heterogeneity within PrPSc conformers has been reported. We studied 19 subjects with dementia or dementia/ataxia at onset and 12 subjects with ataxia at onset. Following two-dimensional gel electrophoresis, we characterized PrP(C) and PrP(Sc) species in normal and sCJD brains by immunoblotting with antibodies recognizing N-terminal and C-terminal PrP regions. Three types of PrP(Sc) were detected in detergent-insoluble fractions from sCJD brains, mainly consisting of full-length PrP(Sc) in subjects with rapidly progressive dementia, and two different sets of amino-truncated PrP(Sc) glycoforms in subjects with dementia/ataxia and ataxia at onset. Examination of the PrP(Sc) core fragment, following PK treatment and deglycosylation, confirmed the existence of three distinctive patterns. These findings have immediate implications for the molecular classification of sCJD.     

A matrix-assisted laser desorption/ionization mass spectrometry study of the non-enzymatic glycation products of human globins in diabetes

The molecular species present in globins from healthy and diabetic subjects with and without chronic complications have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The technique demonstrated the presence of glycated and glyco-oxidated species of both alpha- and beta-globins. Their abundances show a good linear relationship with respect to HbA1c values and with the mean daily plasma glucose levels over the 6 weeks preceding the investigation. Interestingly, slightly different behaviour is observed in the data from patients with and without chronic complications; the plots of HbA1c vs. the abundance of glycated and glyco-oxidated species show different slopes and different intercepts with the y-axis. To investigate this aspect the mean abundances of glyco-oxidated species from healthy subjects and from diabetic patients with and without complications were calculated. Higher values were found for the two last sets of samples, but no significant difference was found between them. These data could indicate different individual proclivities to oxidation and/or different oxidation kinetics related to behavioural and environmental factors.     

High-performance liquid chromatographic procedure for the determination of di-(2-ethylhexyl)phthalate in human blood specimens. Problems of variable-extraction yield and the use of standard addition for calibration

A high-performance liquid chromatographic procedure was developed for the determination of di-(2-ethylhexyl)phthalate (DEHP) concentrations in human whole blood samples. The solvent extraction of DEHP was found to be highly variable between samples obtained from different subjects (coefficient of variation of 30.4%). The recovery of DEHP following extraction with ethyl acetate was negatively correlated with serum lipid content, as expressed by the sum of serum cholesterol and triglyceride concentrations (r = -0.864). The technique of standard addition of DEHP allowed a single-point calibration of DEHP extractability in individual blood samples, and provided an accurate estimation of DEHP concentration (coefficient of variation of approximately 6% in replicate samples). The potential for intersample variability in the solvent extraction of other highly lipid-soluble compounds should be considered.     

Two-dimensional analysis of tear protein patterns of diabetic patients.

In diabetic patients, dry eye and other ocular surface diseases occur more often than in healthy subjects. The aim of this study was to analyze the tear protein patterns of patients suffering from diabetes mellitus type II (dia) and to compare them to the patterns of healthy volunteers (ctrl). Tear proteins of nonstimulated tears of 20 patients (ctrl n=10, dia n=10) were separated using two-dimensional electrophoretic techniques. The protein patterns of each group were analyzed by a multivariate analysis of discriminance. Furthermore, for all spots of each gel, a 50 x 50 variables pH/Mr (molecular weight) array was generated and subsequently analyzed by a multivariate analysis of discriminance. Additionally, an artificial neural network was trained using the matrix data as input and a sensitivity analysis was performed to figure out, which spots were the most important to differentiate between the tear protein patterns. In both groups a complex staining pattern could be obtained. In diabetic patients significantly more spots were detected compared to the control group (P<0.02). The analysis of discriminance found a highly significant difference between dia and ctrl (P<0.00001). Using the matrix data, the analysis of discriminance showed a significant difference between the two groups, too (P<0.0001). The sensitivity analysis by means of the artificial neural network revealed several spots that were more expressed or more frequently present in the diabetic group. Our findings reveal that the composition of tear proteins of diabetic patients is different from that of healthy subjects. The use of the two-dimensional electrophoretic technique could give more insight into the diabetic-related changes in the tear film composition.     

Gas-liquid chromatography of fecal neutral steriods.

A method is described for the analysis of fecal neutral steriods with a dual-column gas-liquid chromatography (GLC) system. After saponification of the fecal slurry, the neutral steroids were extracted with hexane. The GLC separation of the compounds and quantitation were achieved by simultaneous injection of the derivatized and derivatized aliquots of the extract onto dual colmuns under identical conditions. The neutral steroids of interest were than identified by matching the retention times with those of known standards, and identification was confirmed by use of an interfaced GLC high-resolution mass spectrometry system. The detection limit was 0.003 mg of steroid/g of fecal slurry. The pricision of the method is illustrated by a relative standard diviation of 2-10% and a recovery of neutral steroids from 73-96%. The method was applied to the determination of fecal neutral steroids in a "High protein diet in colon cancer study". A considerably larger level of coprostanone than of coprostanol was observed. Data on neutral steroids in fecal samples from subjects on different diets are the subject of a separate publication.     

Alloxan诱发糖尿病实验大鼠微量元素谱的多元分析

为确认Ａｌｌｏｘａｎ发糖尿病鼠与微量元素的关连，用多元分析研究了糖尿病鼠样品中微量元素，并引出对糖尿病的新见解。用ＩＣＰ－ＡＥＳ测定了Ａｌｌｏｘｚｎ诱发糖尿病鼠和对照鼠的骨、心、肾、脾、肺、肝、血和毛中１９种微量和宏量元素（Ａｌ、Ａｓ、Ｂ、Ｂａ、Ｃｄ、Ｃｏ、Ｃｒ、Ｍｇ、Ｍｎ、Ｎｉ、Ｐｂ、Ｓｅ、Ｚｎ、Ｆｅ、Ｃｕ、Ｓｒ、Ｔｉ、Ｖ） ，结果在糖尿病鼠与对照经组的微量元素间呈现一些显著差异。多元分析找到     

Significant difference in active metabolite levels of ginseng in humans consuming Asian or Western diet: The link with enteric microbiota

After ingestion of ginseng, the bioavailability of its parent compounds is low and enteric microbiota plays an important role in parent compound biotransformation to their metabolites. Diet type can influence the enteric microbiota profile. When human subjects on different diets ingest ginseng, their different gut microbiota profiles may influence the metabolism of ginseng parent compounds. In this study, the effects of different diet type on gut microbiota metabolism of American ginseng saponins were investigated. We recruited six healthy adults who regularly consumed different diet types. These subjects received 7 days' oral American ginseng, and their biological samples were collected for LC‐Q‐TOF‐MS analysis. We observed significant ginsenoside Rb₁ (a major parent compound) and compound K (a major active metabolite) level differences in the samples from the subjects consuming different diets. Subjects on an Asian diet had much higher Rb₁ levels but much lower compound K levels compared with those on a Western diet. Since compound K possesses much better cancer chemoprevention potential, our data suggested that consumers on a Western diet should obtain better cancer prevention effects with American ginseng intake compared with those on an Asian diet. Ginseng compound levels could be enhanced or reduced via gut microbiota manipulation for clinical utility.     

Determination and identification of amiloride in human urine by high-performance liquid chromatography and gas chromatography-mass spectrometry.

A simple and sensitive high-performance liquid chromatographic method was developed to screen and determine amiloride (I) in human urine. The detection limit of the method is 0.12 micrograms/ml and the recovery of amiloride from urine was 80.4-85.5% at different concentrations. The coefficients of variation were less than 2.8 and 4.4% for intra- and inter-assays, respectively. Total urinary excretion of I in 24 h after oral administration of 5 mg or 15 mg of I ranged from 22.0 to 33.3% of the total dose for three different subjects. I could be detected in urine up to at least 44 h after a 5-mg dose and 72 h after a 15-mg dose. A gas chromatographic-mass spectrometric (GC-MS) confirmatory method was established based on the methanolysis of I to methyl 3,5-diamino-6-chloropyrazine-carboxylate (II). The di-N-trimethylsilyl derivative of II showed very good GC-MS properties and provided reliable structure information for confirmation analysis of I. This is the first time that a reliable GC-MS method has been reported for the detection of urinary I.     

Electrokinetic chromatography.

The important features of electrokinetic chromatography are critically reviewed. Special emphasis is given to systems using micelles as pseudostationary phase. Short and comprehensive overviews are given on the subjects of separation, comparison with capillary electrochromatography, on-line coupling with mass spectrometry, and developments that are expected in the future. A greater coverage on the subject of improvement of detection sensitivity, specifically by on-line concentration was also contributed.     

Dose response for UV-induced immune suppression in people of color: differences based on erythemal reactivity rather than skin pigmentation.

Ultraviolet radiation (UVR) is known to suppress immune responses in human subjects. The purpose of this study was to develop dose responses across a broad range of skin pigmentation in order to facilitate risk assessment. UVR was administered using FS 20 bulbs. Skin pigmentation and UVR sensitivity were evaluated using Fitzpatrick classifications, minimal erythemal dose (MED), slope of the erythemal dose response curve (sED), baseline pigmentation and tanning response. To assess immune responses dinitrochlorobenzene (DNCB) was applied to irradiated buttock skin 72 h after irradiation. Two weeks later DNCB was applied to the inside upper arm. Skin thickness was measured before and after challenge. Dose response was modeled (to obtain a regression line) for the entire group of 185 subjects. With the exception of sED none of the above-mentioned pigmentation indicators contributed significantly to variability around the regression line. Thus, differences in sensitivity for multiple skin types based on Fitzpatrick classification or MED were not observed. However, differences in immune sensitivity to UVR were detected between subjects with steep erythemal dose response curves and those with moderate or flat responses. For subjects with steep erythemal responses the dose calculated to suppress the immune response by 50% was 114 mJ/cm2. This group included individuals with Fitzpatrick skin types I-V, MED for these subjects ranged from 30 to 80 mJ/cm2. The 50% suppression dose for subjects with weak or no erythemal response could not be computed (the dose response was flat). This resistant group included subjects with skin types IV-VI and MED for these subjects ranged from 41 to > 105 mJ/cm2. This study provides a human dose response for UVR suppression of contact sensitivity that will be useful in risk assessment. It is the first study to provide this information using the FS sun lamp and is the first study to include people of color. The sED appears to be a new variable for identifying sensitive subjects at risk of UVR-induced immune suppression.     

糖尿病人微量元素谱的多元分析

胜多元分析了糖尿病患者样品中微量元素，了解微量元素与糖尿病的关系，并提出了新见解。用ＩＣＰ－ＡＥＳ测定糖尿病患者血、发中１８种微量和宏量元素 ；结果经多元分析处理，找到血、发共有的相关链：Ｍｎ－Ｎｉ－Ｃｕ－ＳｒＴｉ，它与患者年龄、性别、样品无关，看来它提供了机体的特殊信息。     

Effects of solar radiation on the human immune system.

On UV irradiation of the skin, a complex cascade of immunological changes results, initiated by cutaneous chromophores and ending in suppression of some local and systemic immune responses. In this review, the stages in this process are outlined first, concentrating on the roles of DNA and urocanic acid as photoreceptors. Evidence indicating UV-induced immunomodulation of delayed hypersensitivity and resistance to infectious diseases in human subjects follows. Aspects of genetic susceptibility to the immunosuppressive effects of UV exposure and extrapolation of the data obtained in animal models to the human situation are included. Finally uncertain and unknown factors relating to the impact of UV on the human immune system are discussed.     

Improved high-performance liquid chromatographic method for the determination of coenzyme Q10 in plasma.

Coenzyme (Co) Q10 was dissociated from lipoproteins in plasma by treatment with methanol and extraction with n-hexane. Subsequent clean-up on silica gel and C18 solid-phase extraction cartridges with complete recovery (99 +/- 1.2%) produced a clean extract. High-performance liquid chromatographic (HPLC) separation was performed on a C18 reversed-phase column. Three simple, rapid procedures are presented: HPLC with final UV (275 nm) detection, a microanalysis utilizing a three-electrode electrochemical detector and a microanalysis with column-switching HPLC and electrochemical detection. The methods correlate very well with classical ethanol-n-hexane extraction with UV detection. The identity and purity of the Co Q10 peak were investigated and the resulting methods were concluded to be suitable for total plasma Co Q10 determination. The average level in healthy subjects was 0.80 +/- 0.20 mg/l; the minimum detectable Co Q10 plasma level was 0.05 and 0.005 mg/l for UV and electrochemical detection, respectively. The methods were applied to many samples and the plasma Co Q10 reference values for healthy subjects, athletes, hyperthyroid, hypothyroid and hypercholesterolaemic patients are given.     

Preparation and evaluation of oral dosage form using acylglycerols. II. Effect of food ingestion on dissolution and absorption of aspirin from the granules prepared by acylglycerols in human subjects.

The dissolution behavior of the aspirin enteric granule prepared using acylglycerols, glyceryl monostearate (GMS) and glyceryl trilaurate (GTL), was investigated in vitro and in human subjects in a fasting or non-fasting state. Aspirin was slowly released from the granule in vitro at pH 1.2. No acceleration of the aspirin dissolution rate in the medium without lipase and cholic acid was observed when the pH level of the medium increased to a neutral region (pH 6.4). However, the dissolution of aspirin was significantly increased by increasing the concentrations of lipase and cholic acid in the medium. Lipase appears to play an essential role in the dissolution process of aspirin granules. In human subjects, the average levels of the cumulative amount of total salicylate excreted in a urine-time curve, and the mean residence time (MRT) obtained after oral administration of a granule in the fasting state were markedly delayed in comparison with the results observed using an aqueous solution and a crystalline form of aspirin. In comparing the fasting condition with the non-fasting condition (after food ingestion), no significant difference was recognized in the total amount of salicylate excreted in urine to an infinite time (Ae(infinity)), whether the MRT was obtained by granule, crystalline form or aqueous solution. It can be concluded that aspirin granule prepared by GMS and GTL has a property of pancreatic lipase-sensitive dissolution, and its bioavailability is unaffected by food intake.     

Hair as a bio-indicator: Limitations and complications in the interpretation of results

As part of a larger occupational exposure study in which the concentrations of 18 elements were measured in head hair and toenail collected from steel plant workers, a number of factors associated with interpreting the data obtained were examined. In this paper, some of the limitations and complications associated with hair and nail analysis that were thereby recognised are discussed. Data obtained from the occupational study demonstrated the potential for misinterpreting hair or nail analysis data either through describing results averaged over a group by arithmetic instead of geometric means or through not accounting for the age range of subjects in groups to be compared. Examples that arose from the study indicated that differences between hair from the same subjects grown at different times can both complicate and assist in interpreting hair analysis results. In an investigation into the addition and removal of metallic powders, it was found that both hair and nail can directly incorporate elements through contact with dust.