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Increased complement factor H with decreased factor B determined by proteomic differential displays as a biomarker of tai chi chuan exercise.

Proteomic approaches are increasingly being used for identifying biomarkers that predict common diseases, such as cancer, autoimmune disease, and inflammatory diseases (1,2). Serum biomarkers for tracking the health effects of exercise remain to be identified. Participation in exhaustive physical activity can be associated with short-term immune suppression (3), but moderate exercise such as tai chi chuan (TCC), (5) which combines deep diaphragmatic breathing and relaxation with many fundamental postures (4), has been shown to have beneficial effects on balance and on cardiovascular and respiratory functions (5). The mechanisms responsible for the benefits of moderate exercise remain to be determined, and no potential biomarkers for these beneficial health effects of exercise have yet been identified.

Our previous study had shown that regular TCC exercise for 12 weeks significantly promoted both functional mobility and T-cell functions (6-8); however, which serum biomarkers might be associated with TCC exercise remained unclear. In the present investigation of the differential display of serum proteomes before and after a 12-week TCC exercise program, we have demonstrated and validated a unique proteomic profile of TCC exercise.

In this study, we used a single-group paired pre-and posttest research design, as previously described (6-8). Healthy adults without prior experience in practicing TCC were enrolled in this study. The study protocol was approved by the Institutional Review Board, and informed consent was obtained from all participants. Participants learned to perform 37 standardized movements (tai chi 37 forms), as described previously (6-8). These movements incorporate elements of balance, postural alignment, and concentration (9). Each TCC session was designed to last 60 min with a 10-min warm-up, a 40-min practice, and a 10min cooldown. Sessions were given 3 times a week for 12 weeks. Peripheral blood was collected before and after the TCC exercise program for analyses of complete blood counts, high-sensitivity C-reactive protein, and serum proteomic profiles.

First, we enriched the proteins in serum samples by depleting albumin and IgG with an Albumin and IgG Removal Kit, followed by a 2-D Clean-Up Kit (GE Healthcare). Protein (50 [micro]g) from serum samples obtained from participants before and after TCC exercise were labeled with Cy5 and Cy3 (GE Healthcare), respectively, and were placed on ice in the dark for 30 min. A mixture of equal amounts (25 [micro]g each) of pre-TCC exercise serum protein and post-TCC exercise serum protein was labeled with Cy2 dye as an internal control (10). The Cy5-, Cy3-, and Cy2-labeled protein samples were applied onto each immobilized pH gradient strip (Immobiline DryStrips; GE Healthcare) for isoelectric focusing at 3000 V/h for a total of approximately 30 000 V x h. The Immobiline DryStrips were then loaded with SDS equilibration buffer (50 mmol/L Tris-HCl, pH 8.8, 6 mol/L urea, 300 g/L glycerol, 20 g/L SDS) and subjected to SDS-PAGE on a 100 g/L polyacrylamide gel at 20 mA/gel and 4 [degrees]C. The 2-dimensional fluorescence difference gel electrophoresis (2D-DIGE) gelswere scanned with a Typhoon Trio imaging system (GE Healthcare) and analyzed with the aid of DeCyder 6.0 software (GE Healthcare).

[FIGURE 1 OMITTED]

After imaging analysis, we silver-stained the 2D-DIGE gels and used a pipet tip (Labcon) to excise the spots from the gels that had a noticeably different display before and after the TCC exercise program. The excised gel spots were then chopped into pieces of 1.5-2 mm, washed, destained, and digested with 20 g/L trypsin. The trypsin-digested peptides were extracted twice with 10 g/L trifluoroacetic acid in 100% acetonitrile and spotted onto an AnchorChip (Bruker-Franzen Analytik) that had been prespotted with the matrix (2 g/L [alpha]-cyano-4-hydroxy-irans-cinnamic acid) for MALDI-TOF/TOF analysis of mass spectra (Bruker-Franzen Analytik). The autoproteolysis products of trypsin (m/z 842.51, 1045.56, 2211.10) were used as internal calibrators. The imprecision of molecular weight estimation was <0.5 Da. Identification of peptide and protein matches was performed with Mascot software (Swiss-Prot), showing MOWSE score expressed as -10 log P; the probability (P) of each matched m/z peak is calculated by a training set of protein sequences multiplying all such probability values to compute the composite probability.

Serum samples (30 [micro]g) were subjected to SDS-PAGE for western blotting. The protein blots were transferred to nitrocellulose paper for immunoblotting with antihuman factor H, antihuman factor B, antihuman [alpha]-1B-glycoprotein, antihuman protease C1 inhibitor, or antihuman [beta]-actin antibody (Abcam). The blots were then treated with horseradish peroxidase-conjugated goat antimouse immunoglobulin antibody (Santa Cruz Biotechnology) and visualized with an enhanced-chemiluminescence kit (Pierce) as previously described (11).

In the initial screening study, we used 3 replicate pre- and post-TCC exercise serum samples for the 2D-DIGE analysis. The other 20 pairs of pre- and post-TCC exercise serum samples were used to validate the differentially displayed serum proteins, on the basis of a power of 0.8, an a level of 0.05, and an effect size of 0.45 (45% difference in the protein concentration). The proteins that were differentially displayed in pre- and post-TCC exercise samples and had specific antibodies commercially available were selected for further validation by western blot analysis. SPSS software (version 13.0 for Windows; SPSS) was used for statistical analysis.

Of the 32 healthy adult participants, 23 [11 women and 12 men; mean (SD) age, 52.1 (2.2) years] completed the 12-week TCC exercise program and supplied samples before and after taking part in the program. As shown in Table 1 in the Data Supplement that accompanies the online version of this Brief Communication at http://www.clinchem.org/content/ vol56/issue1, post-TCC exercise measurements of body mass index, high-sensitivity C-reactive protein concentration, hemoglobin concentration, red blood cell count, white blood cell count, and differential counts demonstrated no significant changes from pre-TCC exercise values.

We used the DeCyder program to compare differences in serum protein fluorescence patterns before and after the TCC exercise program (mean ratio >1.2 or a P value in the Student t-test of <0.05 in the 3 replicate experiments). Thirty-nine protein spots comprising 18 proteins with noticeable increases or decreases after the TCC exercise program were identified and subjected to mass spectrometry and peptide sequence matches (see Fig. 1 in the online Data Supplement). Of the 18 differentially displayed proteins, 12 proteins (a-1B-glycoprotein, keratin type II, neurofilament triplet L protein, vitamin D-binding protein precursor, protease C1 inhibitor precursor, keratin type I cytoskeleton, complement factor B, complement C1r subcomponent precursor, transthyretin precursor, zinc finger protein 792, kininogen-1 precursor, and PRAME family member 7) decreased in concentration after TCC exercise; 4 proteins (complement factor H, apolipoprotein C-III precursor, complement C3 precursor, and [[alpha].sub.2]-macroglobulin) increased in concentration after TCC exercise; and 2 proteins (apolipoprotein A-I precursor and serotransferrin precursor) exhibited an increase or a decrease in the concentrations of their different isoforms.

Four of the 18 proteins differentially displayed in pre- and post-TCC exercise serum samples (complement factor H, complement factor B, protease C1 inhibitor, and [alpha]-1B-glycoprotein) have specific antibodies commercially available. The differential protein displays of these 4 proteins were validated by western blotting with 20 paired samples. As is shown in Fig. 2 in the online Data Supplement, western blotting showed that complement factor H was increased after the TCC exercise program and that complement factor B, protease C1 inhibitor, and [alpha]-1B-glycoprotein decreased after the TCC exercise program. Fig. 1 shows that the mean (SD) concentration ratio of complement factor H to [beta]-actin increased from 0.85 (0.07) to 1.48 (0.05) after the TCC exercise program (P = 0.0034). In addition, the mean ratio of the complement factor B concentration to that of [beta]-actin decreased significantly from 1.49 (0.03) to 1.06 (0.04) in the 20 pairs of samples (P = 0.0029). Similarly, the mean ratio of the concentration of protease C1 inhibitor to that of [beta]-actin decreased from 1.41 (0.7) to 0.86 (0.13) (P = 0.0038), and the mean concentration ratio of [alpha]-1Bglycoprotein to [beta]-actin decreased from 1.65 (0.03) to 1.10 (0.02) after the TCC exercise program (P = 0.0005; see Fig. 3 in the online Data Supplement).

Exercise has long been recognized as beneficial to health, but exhaustive exercise can cause immunosuppression (3). Our study, which used 2D-DIGE differential displays of serum proteomes before and after TCC exercise, is the first to demonstrate that an increase in complement factor H associated with a decrease in complement factor B may be a biomarker of TCC exercise.

Complement factor H, a regulator of complement activation, is involved in protection from thrombotic microangiopathies and advanced macular degeneration. Individuals with a congenital deficiency of factor H are susceptible to microangiopathies (12, 13), and those with senile deficiency are susceptible to early development of advanced macular degeneration (14, 15). An increase in complement factor H after TCC exercise may prevent vascular insults and macular degeneration.

In contrast to the increase in complement factor H, we also found that some serum inflammatory markers, such as protease C1 inhibitor, [alpha]-1B-glycoprotein, and complement factor B, decreased after the TCC exercise program. Complement factor B is an enhancer of an alternative pathway of complement activation and is increased and augmented in many inflammatory diseases. Studies with factor B knockout mice have demonstrated that factor B deficiency may limit inflammation of the lung (16) and brain (17). Further studies to investigate whether TCC exercise decreases inflammation in patients with cancer or infection seem to be warranted.

We applied proteomic differential displays, a tool that has been used for biomarker discovery in a variety of diseases (1, 2, 10, 18-20), to identify potential serum proteomic biomarkers of the beneficial effect of TCC exercise. Whether these biomarkers are associated only with TCC exercise or with all kinds of moderate exercise remains to be determined. A limitation of the 2D-DIGE analysis of exercise biomarkers in this study is that such analysis mainly detects proteins with molecular weights >10 kDa. As is shown in Table 1, we detected no protein of < 10 kDa that had a differential display before and after the TCC exercise program.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: Grant NSC95-2314-B-255-001-MY2 from the National Science Council and CMRPG880251 and CMRPF870501 from Chang Gung Memorial Hospital, Taiwan. Expert Testimony: None declared.

Role of Sponsor: The funding organizations played a direct role in the design of the study, choice of enrolled patients, review and interpretation of the data, and preparation and final approval of the manuscript.

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Previously published online at DOI: 10.1373/clinchem.2009.126615

Kuender D. Yang, [1] [dagger] Wan-Ching Chang, [1,2] [dagger] Hau Chuang, [1,2] Pei-Wen Wang, [3] Rue-Tsuan Liu, [3] and Shu-Hui Yeh [2,4] *

[1] Proteomic Core Laboratory, Department of Medical Research, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University, Kaohsiung, Taiwan; [2] Chang Gung Institute of Technology, Chiayi, Taiwan; [3] Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University, Kaohsiung, Taiwan; [4] Department of Medical Research, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University, Kaohsiung, Taiwan; [dagger] these authors equally contributed to this study; * Address correspondence to this author at: Department of Medical Research (12F), Chang Gung Memorial Hospital-Kaohsiung Medical Center, #123 Ta-Pei Rd., Niao-Sung, Kaohsiung 833, Taiwan. Fax 886-7-7312867; e-mail yangkd.yeh@msa.hinet.net.

[5] Nonstandard abbreviations: TCC, tai chi chuan; 2D-DIGE, 2-dimensional fluorescence difference gel electrophoresis.
Table 1. Differential displays of serum proteins before and after
the TCC exercise program.

 Proteins identified Accession (a)

Downregulated
proteins

1 PRAME family member 7 PRAM7_HUMAN
2 [alpha]-1B-glycoprotein precursor A1BG_HUMAN
3 Keratin, type II cytoskeletal 1 K2C1_HUMAN
4 Neurofilament triplet L protein NFL_HUMAN
5 Vitamin D-binding protein VTDB_HUMAN
 precursor
6 Protease C1 inhibitor IC1_HUMAN
 precursor
7 Keratin, type I cytoskeletal 10 K1C10_HUMAN
8 Complement factor B CFAB_HUMAN
 precursor
9 Complement C1r C1R_HUMAN
 subcomponent precursor
10 Transthyretin precursor TTHY_HUMAN
11 Zinc finger protein 792 ZN792_HUMAN
12 Kininogen-1 precursor KNG1_HUMAN

Upregulated
proteins

1 Complement factor H CFAH_HUMAN
 precursor
2 Apolipoprotein C-III precursor APOC3_HUMAN
3 [[alpha].sub.2]-Macroglobulin A2MG_HUMAN
 precursor
4 Complement C3 precursor CO3_HUMAN

Mixed up-and
downregulated
proteins

1 Apolipoprotein A-I precursors APOA1_HUMAN
2 Serotransferrin precursors TRFE_HUMAN

 Spots with DIGE index,
 differential mean fluorescence
 display, n ratio (b)

Downregulated
proteins

1 1 -2.31
2 1 -2.04
3 1 -1.65
4 1 -2.74
5 2 -1.5; -5.22

6 2 -2.08; -2.58

7 3 -1.76; -1.93; -3.36
8 2 -1.6; -1.65

9 1 -2.41

10 2 -1.36; -1.79
11 1 -1.68
12 2 -3.25; -3.08

Upregulated
proteins

1 1 5.14

2 1 2.38
3 1 1.48

4 1 1.22

Mixed up-and
downregulated
proteins

1 5 2 up, 3 down
2 12 3 up, 9 down

 MOWSE Protein
 score (c) MW, (d)
 Da

Downregulated
proteins

1 60 53 617
2 124 54 239
3 62 65 847
4 123 61 348
5 58; 76 52 929

6 63; 55 55 119

7 74; 98; 104 59 483
8 86; 72 85 479

9 80 80 122

10 118; 52 15 877
11 58 64 015
12 61; 84 71 900

Upregulated
proteins

1 82 138 979

2 97 10 846
3 91 163 175

4 67 187 030

Mixed up-and
downregulated
proteins

1 66-149 30 759
2 116-263 77 000

(a) UniProt entry name (http://www.uniprot.org).

(b) Some of the identified proteins have 2 or 3 spots with
a noticeable decrease in concentration, as shown by the
mean fluorescence ratio. The DIGE index is a ratio of mean
fluorescence of the protein spot before and after TCC exercise.

(c) Some of the identified proteins have 2 or 3 spots with a
noticeable decrease in mean fluorescence ratio, which is
reflected in the matched MOWSE score.

(d) MW, molecular weight.
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Title Annotation:Brief Communications
Author:Yang, Kuender D.; Chang, Wan-Ching; Wang, Hau Chuang Pei-Wen; Liu, Rue-Tsuan; Yeh, Shu-Hui
Publication:Clinical Chemistry
Date:Jan 1, 2010
Words:3045
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