Histopaque-1077, Trichloroacetic acid (TCA), Thiobarbituric acid (TBA), 5,5′-dithio-bis (2-nitro benzoic acid) (DTNB), acrylamide-bis acrylamide, Tween 20 were purchased from Sigma Aldrich (St. Louis, MO, USA). 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCFDA), pyrogallol, H2O2, ethanol and all other fine chemicals were procured from Merck (Germany). Glutamate ELISA kit was purchased from Abnova (Taiwan). GABA, serotonin and dopamine kits were purchased from QAYEE-BIO (Shanghai, China). Homocysteine ELISA kit was procured from Randox (Antrim, United Kingdom). All antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and other chemicals used were of highest purity grade available.
Ethical approval
The approval from the Institutional Human Ethics Committee, Department of Physiology of University of Calcutta (Ref no. IHEC/SD/P29/13, dated 22.03.2013) was obtained for this work and appropriate measures were taken to follow all ethical norms throughout the study. An information sheet describing the rationale of the study and individual rights was handed to the care givers of the participants. Written informed consents were then obtained from the legally authorized representatives. All experiments were performed in accordance with the approved guidelines and regulations of Human Ethics committee and the Institutional Bio-Safety Committee of University of Calcutta. All the procedures involving the subject handling was done under the supervision of an experienced neuro-physician.
Human Subject Selection
All studies were performed in randomly selected intellectually disabled children [Male, Total number of subjects (N) = 45, Number of subjects in individual groups (n) = 15, age 10 ± 5 years]. The pathophysiology of the disease was diagnosed following the guidelines of Diagnostic and Statistical Manual of Mental Disorders: Fourth Edition-V (DSM-V) (2013), Washington, DC: American Psychiatric Association. Purposive sampling procedure was followed from Government aided NGO-run rehabilitation homes with ID inhabitants. The subjects were then categorized based on their IQ scores using standard psychometric test.
Inclusion criteria
The age ranges of the subjects were selected from 10 ± 5 years. Male subjects were chosen for these experiments.
Exclusion criteria
Individuals with anemia, other blood related diseases, HIV, Hepatitis B and C or already involved in any other clinical trials were not considered for this study.
Psychological Tests
The psychological tests were performed in presence of registered Psychologist and Experienced Practitioner.
Measurement of intelligence quotient (IQ) and mental age by Stanford Binet Intelligence Test
It is a cognitive ability assessment used to measure intelligence (IQ). The Stanford-Binet measures five factors of cognitive ability: Fluid Reasoning, Knowledge, Quantitative Reasoning, Visual-Spatial Processing, and Working Memory. Every factor was tested in two separate domains, verbal and nonverbal32. For this test, IQ levels for mild were considered as 50–70, moderate 35–49 and severe 20–34.
Determination of social age using Vineland Social Maturity Scale (VSMS)
The Vineland Social Maturity Scale is a psychometric assessment instrument that is useful for the assessment of social competence. It is a psychometric questionnaire and measures social maturity or social competence in people from birth to adulthood. It is classified into eight categories of items on the VSMS: self-help general, self-help dressing, self-help eating, communication, self-direction, socialization, locomotion, and occupation33.
Blood collection
Whole blood (10 ml) was collected from the individuals. Serum, peripheral blood mononuclear cells (PBMC) and RBC were isolated from the whole blood by a density gradient technique using Histopaque-107734.
Isolation of PBMC
PBMC were immediately isolated from fresh blood by density gradient centrifugation according to the standard protocol. Briefly, 5 ml blood was layered carefully over an equal volume of Histopaque-1077 and then centrifuged for 30 min at 400 × g. PBMC were collected from the buffy coat formed at the plasma–Histopaque-1077 interface and the pellet was re-suspended in PBS (50 mM, pH 7.4). This process was repeated twice or thrice to remove the platelets35.
Preparation of cell lysate of PBMC
Isolated PBMC (8 × 106) was suspended in 100 μl hypotonic buffer (1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 10 mM HEPES, pH = 7.9) containing protease inhibitor cocktail and sonicated. The suspension containing the lysed cells was centrifuged at 13,000 × g for 10 min at 4 °C. The supernatant (cell homogenate) was then used for antioxidant enzyme assays and western blot analysis35.
Preparation of RBC for antioxidant assay
Blood samples were drawn into glass tubes containing Na-ethylenediaminetetraacetic acid (EDTA). RBCs were separated from plasma by centrifugation at 700 × g at 4 °C for 15 minutes and washed 3 times with 0.9% saline solution with removal of the buffy coat36. Aliquots of the RBCs were taken for determination of GSH and SOD.
Isolation of RBC membrane
Freshly drawn blood was used for membrane preparation. The RBC membrane was prepared using a standard protocol with minor modification37. Briefly, hypotonic phosphate buffer was added to the suspension of erythrocyte and centrifuged at 25,000 × g for 40 min. Red loosely packed membrane pellet was resuspended in hypotonic phosphate buffer and centrifuged at 20,000 × g for 20 min. This step was repeated five to six times more till a milky-looking membrane pellet was formed and it was re-suspended in PBS (pH 7.4). This was used for determination of LPO and Western blot analysis.
Analysis of clinical parameters
Parameters such as percentage of hemoglobin (Hb), CRP, ESR, glucose (random), urea, creatinine, total lipid profile, liver function tests, mineral content in blood were determined using auto-analyzer in all three ID groups (N = 45) (Purechem Ltd, Ireland).
Protein estimation
Protein content of serum, PBMC and RBC were determined by Bradford method using BSA as a standard38.
Determination of LPO
The lipid peroxidation of RBC and PMBC membrane was estimated using standard protocol of formation of TBARS in the sample. In brief, the cell fractions (PBMC lysate and RBC membrane) were mixed with 15% TCA, 0.375% TBA and 5(N) HCl and incubated for 15 min at 95 °C. After cooling, the mixture was centrifuged at 3000 × g for 10 min at room temperature and the absorbance of supernatant was measured spectrophometrically (Bio-RAD SmartSpecTMPlus Spectrophotometer) at 535 nm against appropriate blank39.
Determination of GSH
Cell fractions (RBC and PBMC in respective cases) were mixed with 0.1 ml of 25% TCA and the mixture was centrifuged at 3,900 × g for 10 min in room temperature. The supernatant was collected in another tube and GSH level of the supernatant was assayed in a total reaction mixture of 3 ml [2 ml of 0.5 mM DTNB prepared in 0.2 M phosphate buffer (pH 8.0), with 1 ml of the supernatant]. The absorbance of resultant yellow complex was measured spectrophotometrically (Bio-RAD SmartSpecTMPlus Spectrophotometer) at 412 nm36.
Determination of SOD activity
The activity of SOD in RBC and PBMC was determined using modified pyrogallol auto-oxidation method40. In brief, 62.5 mM tris-cacodylic acid buffer was mixed with cell fractions (RBC and PBMC in respective cases) followed by addition of 4 mM pyrogallol. The auto-oxidation of pyrogallol was monitored spectrophotometrically (Bio-RAD SmartSpecTM Plus Spectrophotometer) at 420 nm, followed by the estimation of the absorbance of the test samples at specific time intervals.
Determination of serum reactive oxygen species (ROS) level
The serum ROS level was determined by spectrofluorometric method using 2′-7′-dichlorofluorescein-diacetate (H2DCFDA) dye41,42. In brief, 15 µg of serum protein was mixed with 10 µl of 100 µM H2DCFDA, and PBS (pH 7.4) in a total reaction volume of 50 µl and the reaction mixture was incubated for 30 min at 37 °C in a water bath. For blank, 10 µl of 100 µM H2DCFDA was added to the PBS in a total reaction volume of 50 µl and DCF formation was monitored after 30 min incubation at 37 °C in order to subtract background auto-fluorescence values. The reaction was stopped by adding equal volume of ice-cold PBS in the reaction mixture and the fluorescent DCF (oxidized form) was measured in spectrofluorimeter (JASCO, India) using excitation and emission wavelengths at 495 nm and 525 nm, respectively. The entire experiment was performed in a dark room. Serum ROS level was expressed as percentage change of fluoroscence (DCF)/µg of protein.
Determination of serum lactate
The serum lactate level was measured according to the manufacturer’s instructions (Bio-vision Inc., USA)43. Serum containing 50 µg of protein was pipetted into 1.5 ml micro centrifuge tube. The reaction volume of the micro centrifuge tube was adjusted to 256.25 µl (final reaction volume) with PBS (pH 7.4) and 100 µl of color reagent, then the entire solution was mixed well by vortexing. The reaction mixture was incubated for 10 minutes at 37 °C in water bath and the reaction was stopped by adding 244.75 µl of ice cold PBS (pH 7.4) in it. The absorbance was measured in spectrophotometer (UV-1800 UV-VIS, Shimadzu) at 570 nm against a reagent blank prepared from 400 µl of PBS (pH 7.4) and 100 µl of color reagent. The lactate content of the serum was expressed as µg/dl/µg of protein.
Glutamate assay
The level of glutamate was measured according to the manufacturer’s instructions (Abnova, KA1670, Taipei, Taiwan) provided with the ELISA kit12.
Determination serum GABA, serotonin and dopamine level
The level of GABA, serotonin and dopamine and were measured according to the manufacturer’s instructions (QAYEE-BIO, Shanghai, China) by ELISA kit method44,45,46.
Detection of Homocysteine (Hcy) level
The tissue Hcy level in serum was measured according to the manufacturer’s instructions in auto-analyzer instrument setup (RX Daytona, Randox, Crumlin, Antrim, UK)10.
Scanning Electron microscopy of RBC
RBC morphology was studied on whole blood smears using scanning electron microscope (SEM) following slightly modified the protocol35. Whole blood was diluted 1:1 with phosphate buffered saline (PBS, pH 7.4) and placed it at 37 °C for 10 min. Samples were centrifuged three times at 1,500 rpm for 5 min before they were fixed in 2.5% glutaraldehyde in Dulbecco’s phosphate buffered saline (DPBS) solution with pH of 7.4 for 60 min. The samples were rinsed and washed with phosphate buffer three times for 5 min before being fixed with 1% osmium tetra-oxide (OsO4). This was followed by another three times rinsing with PBS for 5 min each time, followed by a serial dehydration in 30%, 50%, 70%, 90% ethanol and three times with 100% ethanol. A smear of the preparation was prepared on a glass cover slip, dried, mounted and coated with platinum. An EVO-18 special edition SEM system (ZEISS, Germany) was used to study the morphology of erythrocytes.
Immunofluorescence study of RBC
1 ml of each blood sample was centrifuged at 800 × g for 10 minutes at 4 °C and washed twice with PBS for Immunofluorescence study of RBC. The packed cells were smeared onto grease free glass slides and after drying were fixed with 4% paraformaldehyde dissolved in PBS at room temperature for 10 min. It was then permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at 4 °C and washed three times with PBS. Cells were blocked with 1% BSA for 30 min at room temperature and incubated overnight with mouse monoclonal β-actin primary antibody (1:50 dilution in PBS). Following washes in TBS, cells were incubated with specific rabbit anti-mouse FITC tagged secondary antibody (1:100 dilution in PBS) for 1 hr at room temperature. Finally, the fluorescent RBCs were washed followed by covering with mounting solution and visualized using the Olympus confocal microscope (Shinjuku, Tokyo, Japan). The relative fluorescence intensities of β-actin of RBC from three ID groups were estimated from the intensity curves, and each immunofluorescence image was analyzed by ImageJ software9.
Western blot analysis
40 µg of protein (for serum cytokines) and 50 µg of protein for RBC membrane were loaded in each lane on 12.5% SDS-PAGE and transferred it onto a PVDF membrane. After blocking with 5% bovine serum albumin (BSA) in TBST for 1 hr, the membranes were incubated with primary antibody (1:1500 dilution with TBST) for over-night, followed by incubation with secondary antibody (1:2000 dilution with TBST) for 2 hrs at room temperature. Protein bands were visualized using NBT-BCIP solution mixture. The relative protein levels were calculated by normalization to the amount of internal control protein. Polyclonal antibodies were used for TNF-α and IL-6 (Imegenex, San Diego, CA, USA) and monoclonal for β-actin, GAPDH, LDH-A (Cell Signalling Technology, Danvers, MA, USA), BDNF (Abcam, Cambridge, United Kingdom). GAPDH was used as loading control47,48.
Statistical analysis
Statistical analysis of the data was performed using SPSS Statistics, version 23 (IBM Corporation, Armonk, NY). As we found variations in the age range between the three ID groups, we carried out a one-way analysis of covariance (ANCOVA) to study the differences in different blood biomarkers among the ID groups using age as a covariate. Only those biomarkers which exhibited significant difference among the ID groups were reported along with their corresponding by ‘p’ values (Supplementary Tables 1–8).
Linear regression was performed with the biological parameters as dependent variables, and raw/observed IQ scores along with age as independent variables. For each biochemical parameter, a separate regression analysis was performed. Furthermore, we have performed multiple testing adjustments using Bonferroni correction (i.e., ‘p’ values < 0.05/3 were considered as significant after performing ANCOVA or linear regression) for all the analyses (Supplementary Tables 1–8).
Association between different pair-wise combinations of parameters was evaluated using Pearson’s (for parametric biomarkers) or Spearman’s (for non-parametric biomarkers) product moment correlation (r) method. Values of product moment r ranging from 0.8–1.0 were considered as strong association among the parameters (Supplementary Tables 10–16). Densitometric analyses of the western blots, surface plot of RBC and intensity curve of immunofluorescence images were obtained using ImageJ and OriginLab 8.0 softwares.