Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study.

Extracellular vesicles (EVs) have attracted increasing attention because of their potential roles in various biological processes and medical applications. However, isolation of EVs is technically challenging mainly due to their small and heterogeneous size and contaminants that are often co-isolated. We have thus designed a two-step magnetic bead-based (2MBB) method for isolation a subset of EVs as well as their microRNAs from samples of a limited amount. The process involves utilizing magnetic beads coated with capture molecules that recognize EV surface markers, such as CD63. Captured EVs could be eluted from beads or lyzed directly for subsequent analysis. In this study, we used a second set of magnetic beads coated with complementary oligonucleotides to isolate EV-associated microRNAs (EV-miRNAs). The efficiencies of 2MBB processes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) with spiked-in exogenous cel-miR-238 molecules. Experimental results demonstrated the high efficiency in EV enrichment (74 ± 7%, n = 4) and miRNA extraction (91 ± 4%, n = 4). Transmission electron micrographs (TEM) and nanoparticle tracking analysis (NTA) show that captured EVs enriched by 2MBB method could be released and achieved a higher purity than the differential ultracentrifugation (DUC) method (p < 0.001, n = 3). As a pilot study, EV-miR126-3p and total circulating cell-free miR126-3p (cf-miR126-3p) in eight clinical plasma samples were measured and compared with the level of protein markers. Compared to cf-miR126-3p, a significant increase in correlations between EV-miR126-3p and cardiac troponin I (cTnI) and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP) was detected. Furthermore, EV-miR126-3p levels in plasma samples from healthy volunteers (n = 18) and high-risk cardiovascular disease (CVD) patients (n = 10) were significantly different (p = 0.006), suggesting EV-miR126 may be a potential biomarker for cardiovascular diseases. 2MBB technique is easy, versatile, and provides an efficient means for enriching EVs and EV-associated nucleic acid molecules.

An integrated microfluidic system for on-chip enrichment and quantification of circulating extracellular vesicles from whole blood.

Circulating extracellular vesicles (EVs), which can contain a wide variety of molecules such as proteins, messenger ribonucleic acids (mRNAs), micro ribonucleic acids (miRNAs) and deoxyribonucleic acids (DNAs) from cells or tissues of origin, have attracted great interest given their potential to serve as biomarkers that can be harvested in body fluids (i.e., relatively non-invasive). Since enrichment and detection of circulating EVs from whole blood have proven challenging, we report herein a fully integrated microfluidic system combining a membrane-based filtration module (i.e. pneumatically-driven microfluidic devices) and a magnetic-bead based immunoassay capable of automating blood treatment, EV enrichment, and EV quantification directly from human whole blood. Three functional modules were implemented; the first, a stirring-enhanced filtration module for separating plasma from blood cells, was characterized by a plasma recovery rate of 65%, a filtrate flow rate of 22 µL min-1, and a vesicle recovery rate of 94% within only 8 min (using 500 µL of blood). The second module, a magnetic bead-based EV enrichment device for immunocapture of circulating EVs from plasma, was characterized by a capture rate of 45%. The final module performed an on-chip enzyme-linked immunosorbent assay for plasma EV quantification in plasma. Given the automated capacity of this system, it could show promise in circulating EV research and clinical point-of-care applications.

Optimization of an enzyme linked DNA aptamer assay for cardiac troponin I detection: synchronous multiple sample analysis on an integrated microfluidic platform.

In this study, an enzyme linked DNA aptamer based assay was optimized for human cardiac troponin I (cTnI) detection which is a prominent biomarker for acute myocardial infarction (AMI), on an integrated microfluidic platform. This platform allowed for the multiplex detection of six samples (5 µL per sample), and only 30 min were required for detection. First, cTnI-specific aptamers were surface-coated on magnetic beads. Bead-captured proteins were allowed to bind to a primary cTnI antibody and then to a secondary antibody labelled with horseradish peroxidase. Finally, chemiluminescence intensities were detected for quantification of cTnI. Purified proteins, serum from AMI patients and unknown serum samples were used to test the efficacy of the on-chip system. The limit of detection was measured to be only 12 ng L-1, and off-target effects from other proteins were minimal. This sensitive, cTnI-specific aptamer-based assay could consequently be used for reliable diagnosis of AMI.

Simultaneous detection of multiple NT-proBNP clinical samples utilizing an aptamer-based sandwich assay on an integrated microfluidic system.

Although cardiovascular diseases such as heart failure (HF) affect 30 million people globally, the early detection of HF has, until recently, been difficult and prone to misdiagnoses. Monitoring the circulatory levels of a relatively new biomarker, the N-terminal prohormone of a B-type natriuretic peptide, could be used for early risk evaluation of HF. Therefore, we developed a pneumatically-driven, automatic integrated microfluidic platform equipped with micromixers, micropumps, and microvalves for the simultaneous detection of NT-proBNP in up to six clinical samples within 25 min by using a novel aptamer-based sandwich assay, and the limit of detection was only 1.53 pg mL-1; given that the chip is 64% more compact than those developed in our prior works and requires only 5 µL of sample input, it may serve as a promising tool for early diagnosis of HF.

Design and Demonstration of Tunable Amplified Sensitivity of AlGaN/GaN High Electron Mobility Transistor (HEMT)-Based Biosensors in Human Serum.

We have developed a swift and simplistic protein immunoassay using aptamer functionalized AlGaN/GaN high electron mobility transistors (HEMTs). The unique design of the sensor facilitates protein detection in a physiological salt environment overcoming charge screening effects, without requiring sample preprocessing. This study reports a tunable and amplified sensitivity of solution-gated electric double layer (EDL) HEMT-based biosensors, which demonstrates significantly enhanced sensitivity by designing a smaller gap between the gate electrode and the detection, and by operating at higher gate voltage. Sensitivity is calculated by quantifying NT-proBNP, a clinical biomarker of heart failure, in buffer and untreated human serum samples. The biosensor depicts elevated sensitivity and high selectivity. Furthermore, detailed investigation of the amplified sensitivity in an increased ionic strength environment is conducted, and it is revealed that a high sensitivity of 80.54 mV/decade protein concentration can be achieved, which is much higher than that of previously reported FET biosensors. This sensor technology demonstrates immense potential in developing surface affinity sensors for clinical diagnostics.

An integrated microfluidic system with field-effect-transistor sensor arrays for detecting multiple cardiovascular biomarkers from clinical samples.

Certain blood-borne biomarkers offer a potent methodology for understanding the risk of cardiovascular diseases (CVDs) with clinicians generally advocating the use of multiple biomarkers for proper risk assessment of CVDs. Herein four such CVDs biomarkers- C-reactive protein (CRP), N-terminal pro b-type natriuretic peptide (NT-proBNP), cardiac troponin I (cTnI), and fibrinogen- were rapidly (5 min) analyzed from clinical samples (~ 4 µL) on an integrated microfluidic platform equipped with 1) immobilized highly specific aptamer probes and 2) field-effect transistor (FET)-based sensor arrays. The calibration curve from the FET sensor arrays showed good agreement in the physiological concentration ranges for CRP (0.1-50 mg/L), NT-proBNP (50-10,000 pg/mL), cTnI (1-10,000 pg/mL), and fibrinogen (0.1-5 mg/mL). The developed prototype of this fully automated portable device requires minimal reagent and sample inputs and consequently shows great promise for next-generation point-of-care devices assaying multiple CVDs biomarkers in clinical samples.

Microfluidic platforms for rapid screening of cancer affinity reagents by using tissue samples.

Cancer is the most serious disease worldwide, and ovarian cancer (OvCa) is the second most common type of gynecological cancer. There is consequently an urgent need for early-stage detection of OvCa, which requires affinity reagent biomarkers for OvCa. Systematic evolution of ligands by exponential enrichment (SELEX) and phage display technology are two powerful technologies for identifying affinity reagent biomarkers. However, the benchtop protocols for both screening technologies are relatively lengthy and require well-trained personnel. We therefore developed a novel, integrated microfluidic system capable of automating SELEX and phage display technology. Instead of using cancer cell lines, it is the first work which used tissue slides as screening targets, which possess more complicated and uncovered information for affinity reagents to recognize. This allowed for the identification of aptamer (nucleic acid) and peptide probes specific to OvCa cells and tissues. Furthermore, this developed system could be readily modified to uncover affinity reagents for diagnostics or even target therapy of other cancer cell types in the future.

An integrated microfluidic platform to perform uninterrupted SELEX cycles to screen affinity reagents specific to cardiovascular biomarkers.

As cardiovascular diseases (CVD) are responsible for millions of deaths annually, there is a need for rapid and sensitive diagnosis of CVD at earlier stages. Aptamers generated by systematic evolution of ligands by exponential enrichment (SELEX) processes have been shown to be superior to conventional antibody-based cardiac biomarker detection. However, SELEX is a complicated, lengthy procedure requiring multiple rounds of extraction/amplification and well-trained personnel. To circumvent such issue, we designed an automated, miniaturized SELEX platform for the screening of aptamers towards three protein biomarkers associated with CVDs: N-terminal pro-peptide of B-type natriuretic peptide, human cardiac troponin I, and fibrinogen. The developed microfluidic platform was equipped with microfluidic devices capable of sample transport and mixing along with an on-chip nucleic acid amplification module such that the entire screening process (5 rounds of selection in 8 h.) could be performed consecutively on a single chip while consuming only 35 µL of reagents in each cycle. This system may therefore serve as a promising, sensitive, cost-effective platform for the selection of aptamers specific for CVD biomarkers.

High sensitivity cardiac troponin I detection in physiological environment using AlGaN/GaN High Electron Mobility Transistor (HEMT) Biosensors.

In this study, we report the development of a high sensitivity assay for the detection of cardiac troponin I using electrical double layer gated high field AlGaN/GaN HEMT biosensor. The unique gating mechanism overcomes the drawback of charge screening seen in traditional FET based biosensors, allowing detection of target proteins in physiological solutions without sample processing steps. Troponin I specific antibody and aptamer are used as receptors. The tests carried out using purified protein solution and clinical serum samples depict high sensitivity, specificity and wide dynamic range (0.006-148ng/mL). No additional wash or sample pre-treatment steps are required, which greatly simplifies the biosensor system. The miniaturized HEMT chip is packaged in a polymer substrate and easily integrated with a portable measurement unit, to carry out quantitative troponin I detection in serum samples with < 2µl sample volume in 5min. The integrated prototype biosensor unit demonstrates the potential of the method as a rapid, inexpensive, high sensitivity CVD biomarker assay. The highly simplified protocols and enhanced sensor performance make our biosensor an ideal choice for point of care diagnostics and personal healthcare systems.

Bile acid profiles in neonatal intrahepatic cholestasis caused by citrin deficiency.

BACKGROUND: Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is characterized by conjugated hyperbilirubinemia and increased plasma bile acid concentrations. However, the underlying mechanisms remain unclear. We established a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneously quantifying plasma bile acids and examined bile acid profiles in NICCD infants. METHODS: We measured 15 bile acids within 15min and found a wide linear range for individual bile acids. RESULTS: The within-run and run-to-run CV of all bile acids was 1.2-10.9% and 3.1-10.8%, respectively, with a mean recovery of 90.5-112.6%. Compared to infants with citrullinemia without mutations in SLC25A13 (non-NICCD), NICCD infants showed increased plasma total bile acid concentrations (mean: 201 vs. 42µM, p<0.001), with a distinct bile acid profile characterized by increased conjugated primary bile acid concentrations. The calculated ratios, including primary/secondary bile acid (714 vs. 235, p<0.05) and conjugated/free bile acid (371 vs. 125, p<0.05) ratios, were higher in NICCD infants. The area under receiver operating characteristic curve for conjugated/free bile acid ratio to identify infants with NICCD was 0.871 (95% confidence interval, 0.713-1.0). CONCLUSIONS: Together, our findings indicated plasma bile acid profile as a potential noninvasive diagnostic biomarker for NICCD.

Detection of C-reactive protein on an integrated microfluidic system by utilizing field-effect transistors and aptamers.

Cardiovascular diseases (CVDs) cause more than 17 × 106 deaths worldwide on a yearly basis. Early diagnosis of CVDs is therefore of great need. The C-reactive protein (CRP) is an important biomarker for analyzing the risks of CVDs. In this work, CRP-specific aptamers with high sensitivity and specificity and field-effect-transistor (FET) devices were used to recognize and detect CRP by using an integrated microfluidic system automatically while consuming less volumes of reagents and samples (about 5 µm). In order to package the FET device into the microfluidic chip, a new method to prevent liquid leakage was proposed. Sensitive detection of CRP has been demonstrated on the developed microfluidic system. It is the first time that aptamer-FET assays could be realized on an integrated microfluidic system. Experimental results showed that the aptamer-FET assay was capable of detecting CRP with concentrations ranging from 0.625 mg/l to 10.000 mg/l, which may be promising for early diagnosis of CVDs.

Beyond the Debye length in high ionic strength solution: direct protein detection with field-effect transistors (FETs) in human serum.

In this study, a new type of field-effect transistor (FET)-based biosensor is demonstrated to be able to overcome the problem of severe charge-screening effect caused by high ionic strength in solution and detect proteins in physiological environment. Antibody or aptamer-immobilized AlGaN/GaN high electron mobility transistors (HEMTs) are used to directly detect proteins, including HIV-1 RT, CEA, NT-proBNP and CRP, in 1X PBS (with 1%BSA) or human sera. The samples do not need any dilution or washing process to reduce the ionic strength. The sensor shows high sensitivity and the detection takes only 5 minutes. The designs of the sensor, the methodology of the measurement, and the working mechanism of the sensor are discussed and investigated. A theoretical model is proposed based on the finding of the experiments. This sensor is promising for point-of-care, home healthcare, and mobile diagnostic device.

On-chip, aptamer-based sandwich assay for detection of glycated hemoglobins via magnetic beads.

Diabetes can be diagnosed and monitored by measurement of the cutoff ratio between glycated hemoglobins (HbA1c) and total hemoglobin (Hb), which does not require a fasting blood sample and is less influenced by biological variations. In this study, we combined the advantages of the microfluidic system and the selected low-cost, stable and specific aptamers and developed an integrated, aptamer-based microfluidic system for automatic glycated hemoglobin measurements. The detection process of human whole blood can be totally automated in this integrated microfluidic system. According to the experimental results, when compared to conventional bench-top manual assays, reagent consumption was significantly reduced by 75%, and the analysis time was reduced from 3.5h to 30 min. Besides, the novelty in this research also lies in the simultaneously performed two parallel assays for detection of Hb and HbA1c in a single chip. Therefore, this sensitive and low-cost aptamer-based microfluidic system may become a promising tool for point-of -care diagnosis of diabetes.

An integrated microfluidic system for measurement of glycated hemoglobin levels by using an aptamer-antibody assay on magnetic beads.

Blood glycated hemoglobin (HbA1c), reflecting the average blood glucose level in the proceeding 2-3 months, is recommended for screening/diagnosing and patient management of diabetes. However, accurate measurement of the HbA1c level at the point of care is hampered by costly, large-scale instruments (such as high-performance liquid chromatography) or reagent instability of classical immunologic methods, which involve antibody-based immunoturbidimetry. In this work, an integrated microfluidic system using aptamer-based testing to measure HbA1c in blood samples is therefore presented. This measuring system used nucleic-acid aptamers that exhibited high sensitivity and high specificity for hemoglobin and HbA1c to perform a stable and robust testing. The compact microfluidic system consumed less samples and reagents and significantly shortened the detection time. Combining the advantages of microfluidics and aptamers, this integrated microsystem presents a promising tool for accurate and point-of-case HbA1c detection. To demonstrate its clinical utility, whole blood samples with clinically-relevant concentrations of HbA1c and Hb were automatically measured on the integrated microfluidic system. Experimental data showed that the developed aptamer-based microfluidic system is capable of detecting HbA1c and Hb with a good linear response. The entire process was completed within 25 min. The aptamer-antibody on-chip sandwich immunoassay may be further refined to allow diabetes screening and diagnosis at lower cost and earlier phase to minimize the risk of diabetic complications.

Selection of aptamers specific for glycated hemoglobin and total hemoglobin using on-chip SELEX.

Blood glycated hemoglobin (HbA1c) levels reflecting average glucose concentrations over the past three months are fundamental for the diagnosis, monitoring, and risk assessment of diabetes. It has been hypothesized that aptamers, which are single-stranded DNAs or RNAs that demonstrate high affinity to a large variety of molecules ranging from small drugs, metabolites, or proteins, could be used for the measurement of HbA1c. Aptamers are selected through an in vitro process called systematic evolution of ligands by exponential enrichment (SELEX), and they can be chemically synthesized with high reproducibility at relatively low costs. This study therefore aimed to select HbA1c- and hemoglobin (Hb)-specific single-stranded DNA aptamers using an on-chip SELEX protocol. A microfluidic SELEX chip was developed to continuously and automatically carry out multiple rounds of SELEX to screen specific aptamers for HbA1c and Hb. HbA1c and Hb were first coated onto magnetic beads. Following several rounds of selection and enrichment with a randomized 40-mer DNA library, specific oligonucleotides were selected. The binding specificity and affinity were assessed by competitive and binding assays. Using the developed microfluidic system, the incubation and partitioning times were greatly decreased, and the entire process was shortened dramatically. Both HbA1c- and Hb-specific aptamers selected by the microfluidic system showed high specificity and affinity (dissociation constant, Kd = 7.6 ± 3.0 nM and 7.3 ± 2.2 nM for HbA1c and Hb, respectively). With further refinements in the assay, these aptamers may replace the conventional antibodies for in vitro diagnostics applications in the near future.

Apolipoprotein C-III is an amyloid-ß-binding protein and an early marker for Alzheimer's disease.

It has been demonstrated that peripheral injection of anti-amyloid-ß (Aß) antibodies to patients with Alzheimer's disease (AD) and AD transgenic mice facilitate Aß clearance. We hypothesized that peripheral circulating Aß-binding proteins also possess the ability to enhance Aß clearance and the levels of circulating Aß-binding proteins could serve as early AD biomarkers. Circulating Aß-binding proteins were isolated from plasma and identified by LC-MS/MS. Their levels were compared among non-demented individuals without AD family history (ND), with AD family history (ND-FH), and patients with mild AD. The results showed that most of the identified Aß-binding proteins were apolipoproteins, i.e., apoA-I, apoB-100, apoC-III, and apoE. Aß bound preferentially to apoA-I-enriched HDL, followed by apoC-III- and apoE-enriched VLDL, and bound less favorably to apoB-100-enriched LDL. Levels of apoA-I were reduced in AD patients and could be used to discriminate AD from ND groups (AUC: 0.93); whereas levels of apoC-III were reduced in both ND-FH and AD groups and could be used to differentiate ND-FH from ND individuals (AUC: 0.81). Both the levels of apoA-1 and apoC-III positively correlated with CASI and MMSE scores. In conclusion, these results suggest that plasma apoA-I could be a sensitive AD biomarker and individuals with low plasma levels of apoC-III are at risk for AD.

Carbon monoxide may enhance bile secretion by increasing glutathione excretion and Mrp2 expression in rats.

BACKGROUND: Nitric oxide (NO) donors have been reported to induce choleresis via an increased excretion of glutathione. The effects of another gas molecule, carbon monoxide (CO), on bile formation are, however, inconsistent among previous reports. We investigated the sequential changes of bile output and the biliary contents in rats with or without CO supplementation to elucidate the mechanism of CO on bile excretion. METHODS: Dichloromethane (DCM) was gastrically fed to male Sprague-Dawley rats to yield CO by liver biotransformation. The rats were divided into DCM-treated (n = 7), DCM plus L-NAME-treated (n = 6), and corn oil-treated-(n = 8) groups. Bile samples were collected hourly to examine the flow rate and bile content. Serum levels of nitrite and nitrate 4 hours after DCM supplementation with or without NO synthase (NOS) inhibition were measured by capillary electrophoresis. The expression of hepatic inducible NOS was evaluated by Western blotting 6 hours after DCM administration. RESULTS: Levels of carboxyhemoglobin rose to around 10% at 4 hours after DCM supplementation and were maintained until the end of the experiments. Bile flow increased after DCM supplementation and was associated with a concomitant increase of biliary glutathione and higher hepatic multidrug resistance-associated protein 2 (Mrp2) expression. Hepatic inducible NOS expression and serum nitrate/nitrite levels were also increased. Treatment with an NOS inhibitor (L-NAME) abolished the CO-induced glutathione excretion and choleresis, but not Mrp2 expression. CONCLUSION: The present study demonstrated that CO enhanced biliary output in conjunction with NO by increasing the biliary excretion of glutathione. The increment in biliary glutathione was associated with an increased expression of hepatic Mrp2.

The Asian project for collaborative derivation of reference intervals: (1) strategy and major results of standardized analytes.

BACKGROUND: A multicenter study conducted in Southeast Asia to derive reference intervals (RIs) for 72 commonly measured analytes (general chemistry, inflammatory markers, hormones, etc.) featured centralized measurement to clearly detect regionality in test results. The results of 31 standardized analytes are reported, with the remaining analytes presented in the next report. METHOD: The study included 63 clinical laboratories from South Korea, China, Vietnam, Malaysia, Indonesia, and seven areas in Japan. A total of 3541 healthy individuals aged 20-65 years (Japan 2082, others 1459) were recruited mostly from hospital workers using a well-defined common protocol. All serum specimens were transported to Tokyo at -80°C and collectively measured using reagents from four manufacturers. Three-level nested ANOVA was used to quantitate variation (SD) of test results due to region, sex, and age. A ratio of SD for a given factor over residual SD (representing net between-individual variations) (SDR) exceeding 0.3 was considered significant. Traceability of RIs was ensured by recalibration using value-assigned reference materials. RIs were derived parametrically. RESULTS: SDRs for sex and age were significant for 19 and 16 analytes, respectively. Regional difference was significant for 11 analytes, including high density lipoprotein (HDL)-cholesterol and inflammatory markers. However, when the data were limited to those from Japan, regionality was not observed in any of the analytes. Accordingly, RIs were derived with or without partition by sex and region. CONCLUSIONS: RIs applicable to a wide area in Asia were established for the majority of analytes with traceability to reference measuring systems, whereas regional partitioning was required for RIs of the other analytes.

Increased aortic stiffness and attenuated lysyl oxidase activity in obesity.

OBJECTIVE: One potential mechanism through which obesity exerts adverse effects on the vascular system is by increasing aortic stiffness, a change known to be predictive of increased cardiovascular mortality. The aim of this study was to investigate the pathophysiology that links obesity to aortic stiffening. APPROACH AND RESULTS: Obese (ob/ob) mice were used to examine physical, morphological, and molecular changes in the aorta in response to obesity. ob/ob mice had increased aortic pulse wave velocity and tissue rigidity. ob/ob aorta exhibited decreases of lysyl oxidase (LOX) activity and cross-linked elastin, and increases of elastin fragmentation and elastolytic activity. The aortas of ob/ob mice were surrounded by a significant amount of proinflammatory and pro-oxidative perivascular adipose tissue. In vitro studies revealed that the conditioned medium from differentiated adipocytes or the perivascular adipose tissue of ob/ob mice attenuated LOX activity. Furthermore, inhibition of LOX in wild-type lean mice caused elastin fragmentation and induced a significant increase in pulse wave velocity. Finally, we found that obese humans had stiffer arteries and lower serum LOX levels than do normal-weight humans. CONCLUSIONS: Our results demonstrated that obesity resulted in aortic stiffening in both humans and mice, and established a causal relationship between LOX downregulation and aortic stiffening in obesity.

Glycine ameliorates liver injury and vitamin D deficiency induced by bile duct ligation.

BACKGROUND: Patients with chronic liver disease had lower serum concentrations 25-hydroxyvitamin D (25OHD). Glycine, a nonessential amino acid, exerts anti-inflammatory, cytoprotective, and immunomodulatory properties. This study aimed to establish a tandem mass spectrometry assay to measure 25OHD in guinea pigs serum and to investigate the effects of glycine against the liver damage induced by bile duct ligation (BDL). METHODS: BDL was performed on male guinea pigs. Glycine, alanine, serine or tyrosine was given by intraperitoneal injection. The animals were sacrificed and examined at 7 and 14 days after BDL. Serum concentrations of total bilirubin and aminotransferase were measured. Serum concentrations of 25OHD2 and 25OHD3 were measured by API 5000 mass spectrometer. In addition, oxidative stress was assessed by serum ischemia-modified albumin (IMA) and hepatic malondialdehyde (MDA), and apoptosis by hepatic caspase 3 activities. RESULTS: Serum 25OHD concentrations were decreased around 50% in the BDL group at days 7 and 14 post ligation, compared to sham (mean 65.3 ng/ml, p<0.005). Glycine but not other amino acid treatment blunted the reduced serum 25OHD (52.6 ng/ml, p<0.05) resulting from BDL. The concentrations of 25OHD were negatively associated with concentrations of IMA (r=-0.305, p<0.05) and caspase 3 (r=-0.562, p<0.0001). At day-14 post ligation, glycine treatment also ameliorated liver damage indicated by serum AST (p<0.005), ALT (p<0.05) and hepatic caspase 3 activities (p<0.05) and oxidative stress. CONCLUSION: Our results indicate that glycine may protect against BDL-induced liver injury through attenuation of oxidative stress, apoptosis and the vitamin D deficiency.