Variability of low-molecular-weight serum subproteome in healthy humans under the conditions of normal vital activity

To study interindividual variability of the low-molecular-weight serum subproteome in healthy humans, the proteome profiles of blood sera were studied in subjects divided into three age groups: from 20 to 30 years (36 subjects), from 30 to 40 years (11 subjects), and from 40 to 50 years (11 subjects). Serum samples were fractionated by MB WCX magnetic beads using a ClintProt robot prior to the mass spectrometry based profiling. The mass spectra have been obtained using an Autoflex III time-of-flight mass spectrometer (Bruker Daltonics) in the automated mode. The low-molecular-weight serum subproteome in healthy humans was found to be characterized by significant interindividual variability: 21% of all the peaks in the proteome profiles had a coefficient of variation of more than 50%, and 29% of all the peaks had a low variance (CV < 30%). Therefore, the majority of the peaks in the proteome profile had a moderate group variation (the CV was in the interval from 30 to 50%). Fragments of high-molecular-weight kininogen, inter-α-trypsin inhibitor, C3 and C4a complement components, CI apolipoprotein, platelet factor IV, β2-microglobulin, and C cystatin were shown to display a wide variation among the tested groups of healthy humans. The peak area variance of high-molecular-weight kininogen, inter-α-trypsin inhibitor, AII and CIII apolipoproteins increased with age.     

Variability of the healthy human proteome

The aim of this review is to analyze results of studies on characteristics of protein variability and diversity of posttranslational modifications of proteins in healthy humans. Numerous studies have demonstrated that a proteomic profile is characterized by significant intra- and inter-individual variability, and quite often natural (“normal”) variability of some proteins can be comparable to changes observed in pathological processes. Results obtained by our research group have demonstrated high intra-individual variability of serum low-molecular subproteome of healthy volunteers, certified by a special medial committee (the coefficient of variation (CV) of 42.6%). The proteins characterized by high variability under normal conditions (e.g. haptoglobin — 0–40 mg/ml; lysozyme — 0.01–0.1 mg/ml; C-reactive protein — 0.01–0.3 mg/ml) cannot be considered as potential biomarkers of diseases. On the contrary, proteins and peptides characterized by insignificant dispersion in healthy population (such as albumin ( CV = 9%); transferrin-(CV = 14%); C3c complement (CV = 17%), α-1 acid glycoprotein (CV = 21%), α-2-macroglobulin (CV = 20%); transthyretin fragment (CV = 28.3%) and α2-HS-glycoprotein βchain (CV = 29.7%)) can provide valuable information about the state of health. Thus, studies of plasticity in the proteomic profiles of healthy humans will help to correct reference intervals used in clinical proteomics.     

Radioimmunological quantitation of the urinary trypsin inhibitor in normal blood and urine

A radioimmunoassay for measurement of the urinary trypsin inhibitor in human serum and urine is described. Because of the immunological cross-reactivity between the inter-alpha-trypsin inhibitor and the urinary trypsin inhibitor the plasma and serum were treated with perchloric acid to precipitate the inter-alpha-trypsin inhibitor. Gel filtration of serum before and after acid treatment showed identical peaks corresponding to the urinary trypsin inhibitor. The normal level of the urinary trypsin inhibitor in fresh plasma from 30 blood donors was 6.38 +/- 0.33 mg/l (SEM), and in sera from 24 healthy volunteers 7.14 +/- 0.27 mg/l (SEM). In urine from 23 healthy volunteers the normal excretion was 8.17 +/- 1.18 mg/24 h (SEM).     

Analysis of the Sperm Head Protein Profiles in Fertile Men: Consistency across Time in the Levels of Expression of Heat Shock Proteins and Peroxiredoxins

We investigated the identity and quantitative variations of proteins extracted from human sperm heads using a label-free Gel-MS approach. Sperm samples were obtained from three men with high sperm counts at three different time points. This design allowed us to analyse intra-individual and inter-individual variations of the human sperm head proteome. Each time point was analyzed in triplicate to minimize any background artifactual effects of the methodology on the variation analyses. Intra-individual analysis using the spectral counting method revealed that the expression levels of 90% of the common proteins identified in three samples collected at various time-points, separated by several months, had a coefficient of variation of less than 0.5 for each man. Across individuals, the expression level of more than 80% of the proteins had a CV under 0.7. Interestingly, 83 common proteins were found within the core proteome as defined by the intra- and inter-variation analyses set criteria (CV<0.7). Some of these uniformly expressed proteins were chaperones, peroxiredoxins, isomerases, and cytoskeletal proteins. Although there is a significant level of inter-individual variation in the protein profiles of human sperm heads even in a well-defined group of men with high sperm counts, the consistent expression levels of a wide range of proteins points to their essential role during spermatogenesis.     

Age and contraction type influence motor output variability in rapid discrete tasks.

The purpose of this study was to examine the ability to control knee-extension force during discrete isometric (IC), concentric (CC), and eccentric contractions (EC) in 24 young (mean age +/- SD = 25.3 +/- 2.8 yr) and 24 old (mean age +/- SD = 73.3 +/- 5.5 yr) healthy and active individuals. Subjects were to match a parabola with a time to peak force of 200 ms during IC, CC, and EC at six target levels of force [20, 35, 50, 65, 80, and 90% of the maximum voluntary contraction (MVC)]. ICs were performed at 90 degrees of knee flexion, whereas CCs and ECs ranged from 90 to 80 degrees of knee flexion (0 degrees is full extension) at a slow velocity (25 degrees /s). Results showed that subjects produced similar MVC forces for the three types of contractions. Young subjects produced greater MVC forces than old subjects, and within each age group, men produced greater force than women. The variability (standard deviation) of peak force and impulse in absolute values was greater for young compared with old subjects. When variability was normalized to the force produced [coefficient of variation (CV)], however, old subjects exhibited greater CV than young subjects for peak force and impulse. Both the standard deviation and CV of time to peak force and impulse duration were greater for the old adults. In general, ECs were more variable than ICs and CCs, and old adults exhibited greater CV compared with young adults during rapid, discrete ICs, CCs, and particularly ECs of the quadriceps.     

Variability of urine proteome in healthy humans during a 105-day isolation in a pressurized compartment

The protein composition (proteome) of the body fluids is rather flexible; it can change, responding to various factors of the external environment and changes in the internal environment. In order to study the variability of the proteome profile in healthy humans under the conditions of total control of vital rhythm, physical activity, and diet, urine samples were collected from subjects who had been selected according to special criteria and qualified as healthy by a special physical evaluation board. The subjects took part in an experiment with a 105-day-long isolation in a pressurized compartment, carried out by using an autonomous life-support system at the Institute of Biomedical Problems, Russian Academy of Sciences. The purification and concentration of proteins from the urine samples were carried out using a MB-HIC C8 magnetic bead set (Bruker Daltonics). The mass spectra have been obtained using an Autoflex III time-of-flight mass spectrometer (Bruker Daltonics) in the positive lineal mode. One hundred and seventeen peaks were obtained for each urine sample; technological errors of the method have been studied. The high variability of the urine proteome profile (36 protein MC peaks on average) was shown in healthy humans under the conditions of isolation and controlled vital activity.     

Variability of salivary markers of oxidative stress and antioxidant status in young healthy individuals

Objectives: Salivary advanced glycation end-products (AGEs), advanced oxidation protein products (AOPP), total antioxidant capacity (TAC), and ferric reducing ability of saliva (FRAS) are increased in various diseases. Little data exist for these markers in the healthy population. The aim of this study was to assess the inter-individual and intra-individual variability of AGEs, AOPP, TAC, and FRAS in the saliva of young healthy individuals.

Methods: Unstimulated saliva samples were collected from 16 females and 18 males daily over a period of 30 days. Markers were measured using spectrophotometric and spectrofluorometric microplate-based methods.

Results: All salivary markers measured were significantly higher in men than in women (P?<?0.05 for AGEs; P?<?0.001 for AOPP, TAC, and FRAS). The inter-individual variability was approximately 60% for AGEs and AOPP and 30–40% for TAC and FRAS in both genders. The inter-individual variability of FRAS was higher in men vs. women (P?<?0.01). Intra-individual variability ranged from 20% for TAC, to 30% for AGES and FRAS and 45% for AOPP.

Discussion: Intra-individual variability of salivary AGEs, AOPP, TAC, and FRAS indicates that their use is currently limited to large cohort studies. Identifying the underlying factors related to the high inter-individual and intra-individual variability is needed. Sex differences should be considered in future studies.     

Unravelling in vitro variables of major importance for the outcome of mass spectrometry-based serum proteomics

The use of mass spectrometry (MS) for analysing low-molecular weight proteins and peptides from biological fluids has a great, yet not fully realized, potential for biomarker discovery. To prune MS-data as much as possible for non-relevant non-biological variation the development of standardized protocols for handling and processing the samples before MS and adjusting data after MS to compensate for method-induced variability are warranted. This calls for knowledge about how different variables contribute to MS-based proteome analyses. In addition, identification of the peptides involved in pre-analytical variation will be helpful in evaluating the clinical significance of predictive models derived from MS data. Using human sera, extraction by weak cation-exchange magnetic beads, and analysis by MALDI-TOF MS we here evaluated pre-analytical variation and identify peptides involved in this. The influences of humidity, temperature, and time for preparation of sera on spectral changes were evaluated. Also, the reproducibility of the methods and the effect of a baseline correction procedure were examined. Low temperatures, short handling times, and a baseline correction procedure minimize the contribution of artifacts to sample variability as observed by MS. The complement split product C3f and fragments thereof appear to be sensitive indicators of sample handling induced modifications. Other peptides that are indicative of such variability are fibrin and kininogen fragments. Using strict experimental guidelines as well as standardized sample collection procedures it is possible to obtain reproducible peak intensities and positions in serum mass profiling using magnetic bead-based fractionation and MALDI-TOF MS.     

Interaction of 0.1-Hz oscillations in heart rate variability and distal blood flow variability

Biophysical features of 0.1-Hz oscillations of heart rate variability (HRV) and distal blood flow (DBF) variability were compared in healthy subjects and patients after acute myocardial infarction (MI). Patients with acute MI (72 men and 53 women; 125 in total) and healthy subjects (23 men and 10 women; 33 in total) aged 30?C83 and 20?C46 years, respectively, participated in the study. The patients were involved in the study for a year after acute MI. The delay in coupling 0.1-Hz oscillations of HRV and DBF variability was estimated. In healthy subjects, the delay in the heart ?? DBF coupling proved to be less than the delay in the DBF ?? heart coupling. Acute MI results mainly in disruption of the heart ?? DBF coupling, which is partially restored by the end of the first year after acute MI, though it remains lower than in healthy subjects. The DBF ?? heart coupling is rapidly restored to the level of healthy subjects within three weeks after acute MI.     

A haplotype constituted of four MMP-2 promoter polymorphisms (-1575G/A, -1306C/T, -790T/G and -735C/T) is associated with coronary triple-vessel disease.

Vascular lesion development is associated with an accumulation of extracellular matrix proteins within the vessel wall. The proteins are degraded by matrix metalloproteinases (MMPs). There is also evidence indicating a participation of the MMPs in the weakening of atherosclerotic plaque that predisposes to lesion disruption. The aim of the study was to test an association among haplotypes of four single nucleotide MMP-2 promoter polymorphisms and the angiographically confirmed coronary triple-vessel disease (TVD). Incidence of haplotypes of four MMP-2 promoter polymorphisms (-1575G/A, -1306C/T, -790T/G and -735C/T) determined by PCR reactions with restriction analyses in 187 patients with coronary TVD (153 men, 34 women, age median 65 years) was compared to 196 control subjects without clinical signs of coronary heart disease (131 men and 65 women, age median 60 years). The incidence of two similar haplotypes was found to be different between patients and healthy subjects. The haplotype GCTC was more frequent in the TVD patients (P=0.01) though the haplotype GCGC was identified only in healthy subjects (P=0.001). Interestingly, the GCTC is the most frequent polymorphic haplotype composed of four promoter SNPs localized in the MMP-2 gene (53% in healthy subjects vs. 66% in patients with TVD) and the haplotype GCGC is the least frequent polymorphic one (4.4% in healthy subjects vs. 0% in patients with TVD). Two different MMP-2 promoter haplotypes differing only in -790T/G allele are significantly more or less frequent in coronary TVD compared to non-ischemic persons. Thus, the -790T/G MMP-2 genotype might be used as a genetic marker representing MMP-2 promoter variability for the TVD with odds ratio for TT and TG genotypes 2.59, 95% confidential interval 1.21-5.55, P=0.009. The analysis of promoter MMP-2 gene variability could help us to understand individual susceptibility to MMP inhibitor treatment of the coronary artery disease.     

Quantitative analysis of the intra- and inter-individual variability of the normal urinary proteome

Urine is a readily and noninvasively obtainable body fluid. Mass spectrometry (MS)-based proteomics has shown that urine contains thousands of proteins. Urine is a potential source of biomarkers for diseases of proximal and distal tissues but it is thought to be more variable than the more commonly used plasma. By LC-MS/MS analysis on an LTQ-Orbitrap without prefractionation we characterized the urinary proteome of seven normal human donors over three consecutive days. Label-free quantification of triplicate single runs covered the urinary proteome to a depth of more than 600 proteins. The median coefficient of variation (cv) of technical replicates was 0.18. Interday variability was markedly higher with a cv of 0.48 and the overall variation of the urinary proteome between individuals was 0.66. Thus technical variability in our data was 7.5%, whereas intrapersonal variability contributed 45.5% and interpersonal variability contributed 47.1% to total variability. Determination of the normal fluctuation of individual urinary proteins should be useful in establishing significance thresholds in biomarker studies. Our data also allowed definition of a common and abundant set of 500 proteins that were readily detectable in all studied individuals. This core urinary proteome has a high proportion of secreted, membrane, and relatively high-molecular weight proteins.     

Variability of urine proteome of healthy persons during 105-daily isolation

Human proteome is very plastic, it changes under the influence of various biological factors. It is of big interest to find out how specific factors of an environment, including a long-term isolation affect on urine proteome. The study was conducted during the experiment with 105-day isolation. In the present investigation we collected urine samples from 6 healthy volunteers (26-41 years old). The physical activity, daily rhythms and diet were controlled. Urine samples were fractionated on magnetic beads MB-HIC C8 (MB - hydrophobic-interaction chromatography) with ClinProt robot (Bruker Daltonics) prior to MALDI-TOF mass spectrometer analysis with Autoflex III TOF/TOF (Bruker Daltonics), working in a positive linear mode. 117 peaks have been obtained in each spectrum of urine. We have shown that even during isolation and under controlled conditions of life a high variability of urine proteome of healthy personas (36 protein MC-peaks in the urine, on average) are revealed.     

The chemical nature of the fluorescing products accumulating in the lipids of the crystalline lenses of mice with hereditary cataract

The content of diene conjugates (lipid hydroperoxides) was shown to be significantly higher in lipids extracted from the lenses of mice with hereditary cataract than in the controls. The same holds true for characteristics of fluorescence of the end-product of lipid peroxidation. Two (low- and high-molecular weight) peaks were detected in chromatographic lipid profile of cataract lenses measured by fluorescence on Sephadex LH-20 column, whereas only one (high-molecular weight) peak was found in lipids from normal lenses. It was established that high-molecular weight fluorescent fractions corresponded to lipid components of lipofuscin-like pigments. NMR and mass spectrometry of low-molecular weight fractions suggested that they contained predominantly products of free radical oxidation of long chain polyunsaturated fatty acids (C22:6).     

Contents of extracellular DNA in blood of irradiated rats]

Intact rat plasma contains high-molecular DNA which moves as a single fraction in 0.5% agarose electrophoresis. As early as 2-5 hours after gamma-irradiation in a dose of 1-100 Gy there appears low-molecular DNA (about 180 nucleotide pairs), the amount of which directly correlates with the irradiation dose 5 hours after the exposure. Blot-hybridization showed that low-molecular DNA has no common nucleotide sequences with high-molecular DNA, though has sites similar to genome repeat sequences.     

Proteomic analysis of individual variation in normal livers of human beings using difference gel electrophoresis

Normal Chinese Liver Proteome Expression Profile is one of the major parts of Human Liver Proteome Project. Before starting the studies, it is necessary to examine the interindividual variation of normal liver proteome and evaluate the minimal size of samples for proteomic analysis. In this study, normal liver samples from ten individual volunteers were collected and the proteome profiles of these samples were analyzed using 2-D difference gel electrophoresis (DIGE) combined with MALDI-TOF/TOF MS. The individual liver tissue lysates were labeled with Cy3 and Cy5 while the pooled sample was labeled with Cy2 as an internal standard, which minimized gel-to-gel variation. After analysis by the DeCyder software, up to 2056 protein spots were detected on the master gel. The CV of standardized abundance was calculated for the protein spots that were matched across all ten gels. The CV values of these protein spots ranged from 6.4 to 108.5% and the median CV was approximately 19%, which demonstrated that the protein expression of normal liver among different individuals was relatively stable. The eight proteins with CV values over 50% were identified which would be a caveat when considering these proteins as potential disease-related markers. Moreover, the one-way ANOVA feature showed a correlation between sample size and individual variations. The results showed that when the sample size exceeded 7, the individual variations were not significant to the whole pool. Our results are an important basis for liver protein expression profiles and comparative proteomics of liver disease.     

Urine protein profile of IgA nephropathy patients may predict the response to ACE-inhibitor therapy

This study was aimed at the search of urinary biomarkers which might help to predict the clinical response of IgA nephropathy (IgAN) patients to angiotensin converting enzyme inhibitors (ACEi). First, we studied the urinary proteome of 18 IgAN patients (toward 20 healthy controls) who had been chronically treated with ACEi by using 2-D PAGE coupled to nano-HPLC-ESI-MS/MS analysis. We identified 3 proteins, kininogen (p = 0.02), inter-alpha-trypsin-inhibitor heavy chain 4 (35 kDa fragment) (p = 0.02) and transthyretin (p<0.0001), whose urinary excretion was different in IgAN patients' responders when compared to those who had not responded to ACEi. A reduction of daily proteinuria >50% and a stable renal function over time were used to classify patients as responders. Then, we adopted immunoblotting to confirm the predictive power of one of the above proteins, kininogen, in 20 patients with biopsy-proven IgAN, before starting any therapy. Thus, we confirmed that very low levels of kininogen urine excretion were indeed predictive of an inadequate or absent clinical response to ACEi therapy of IgAN patients, after 6-month follow-up. Concluding, the analysis of urine proteome of IgAN patients generated a set of proteins which distinguished subjects responsive to ACEi from those unresponsive to the inhibition of renin-angiotensin system (RAS).     

A practical and reliable method for measuring ethane and pentane in expired air from humans.

We describe a method for the collection of expired air and further document the performance of our analytical technique that is used to measure ethane and pentane simultaneously. Four minutes of breathing hydrocarbon-free air before collection effectively removed high concentrations of residual ambient ethane and pentane from the lungs, with washout times up to 30 min resulting in no further reductions in breath hydrocarbons. Mean (+/-SE) exhalation rates (pmol/kg b.wt./min) in 11 subjects were 2.4 +/- 0.6 for ethane and 1.5 +/- 1.3 for pentane. Total intraindividual variability in exhalation rates (as percent coefficient of variation, %CV), measured from 4 subjects on at least 6 different days, was greater for pentane (44% CV) than for ethane (29% CV). Analytical variability contributed 6% to the total %CV. Advantages of the method are described, and reasons for the large variability in values reported in the literature are discussed.     

Familiarization and reliability of multiple sprint running performance indices

The aims of this study were to evaluate the time-course of the familiarization process associated with a test of multiple sprint running performance and to determine the reliability of various performance indices once familiarization had been established. Eleven physically active men (mean age: 21 +/- 2 years) completed 4 multiple sprint running trials (12 x 30 m; repeated at 35-s intervals) with 7 days between trials. All testing was conducted indoors, and times were recorded by twin-beam photocells. Results revealed no apparent learning effects as evidenced by no significant (p > 0.05) between-trial differences in measures of fastest or mean 30-m sprint time. Within-subject test-retest reliability determined over 4 trials by coefficient of variation (CV) and intraclass correlation coefficient (ICC) showed excellent reliability for measures of fastest and mean sprint times (CV range: 1.34-2.24%; ICC range: 0.79-0.94). Pre- and posttrial blood lactate concentrations showed good reliability when judged in context with typical values (CV range: 12.08-18.21%; ICC range: 0.72-0.78). In contrast, and in line with previous research, fatigue data showed much greater variability (CV: 26.43%; ICC: 0.66). The results of this study suggest that high degrees of test-retest reliability can be obtained in many multiple sprint running indices without the need for prior familiarization.     

Evidence of association of the CYP2E1 genetic polymorphism with micronuclei frequency in human peripheral blood

Micronuclei (MN) are used as one of the cytogenetic biomarkers, and intra- and inter-individual variations in this frequency have been reported in human blood lymphocytes. Polymorphisms in a few metabolic enzyme genes seem to account for a proportion of this variability, but the impacts of specific genetic variants on the MN frequency have not yet been clarified. Here, we investigated the relationship between the MN frequency and several gene polymorphisms in 90 healthy Japanese men. The subjects with the CYP2E1(*)3 variant allele had a statistically lower mean MN frequency than subjects with the CYP2E1(*)1/(*)1 wild type. Furthermore, the adjusted odds ratio (OR) of the CYP2E1(*)3 variant with higher MN frequency levels was also significantly lower and calculated to be 0.25 (95% CI 0.07-0.83), when the OR for the subjects with the CYP2E1(*)1/(*)1 wild type was defined as 1.00. These data suggest that the CYP2E1(*)3 polymorphism may have the potential to influence the baseline frequency of MN.     

Impact of controlling shear rate on flow-mediated dilation responses in the brachial artery of humans.

The reactive hyperemia test (RHtest) evokes a transient increase in shear stress as a stimulus for endothelial-dependent flow-mediated vasodilation (EDFMD). We developed a noninvasive method to create controlled elevations in brachial artery (BA) shear rate (SR, estimate of shear stress), controlled hyperemia test (CHtest), and assessed the impact of this vs. the RHtest approach on EDFMD. Eight healthy subjects participated in two trials of each test on 3 separate days. For the CHtest, SR was step increased from 8 to 50 s(-1), created by controlled release of BA compression during forearm heating. For the RHtest, transient increases in SR were achieved after 5 min of forearm occlusion. BA diameter and blood flow velocity (ultrasound) were measured upstream of compression and occlusion sites. Both tests elicited significant dilation (RHtest: 6.33 +/- 3.12%; CHtest: 3.00 +/- 1.05%). The CHtest resulted in 1) reduced between-subject SR and EDFMD variability vs. the RHtest [SR coefficient of variation (CV): 4.9% vs. 36.6%; EDFMD CV: 36.16% vs. 51.80%] and 2) virtual elimination of the impact of BA diameter on the peak EDFMD response (peak EDFMD vs. baseline diameter for RHtest, r(2) = 0.64, P < 0.01, vs. CHtest, r(2) = 0.14, P < 0.01). Normalization of the RHtest EDFMD response to the magnitude of the SR stimulus eliminated test differences in between-subject response variability. Reductions in trial-to-trial and day-to-day SR variability with the CHtest did not reduce EDFMD variability. Between-subject SR variability contributes to EDFMD variability with the RHtest. SR controls with the CHtest or RHtest response normalization are essential for examining EDFMD between groups differing in baseline arterial diameter.