Determination of Total Galactose from Dried Blood Spots—Extensive Assay Evaluation of a CE-Marked Test-Kit

Most newborn screening laboratories use CE-marked or FDA-approved test-kits, like in routine clinical chemistry. National regulations require only minimal evaluation from the customer, if the test-kits are used as specified by the manufacturer. The microtiter-based kit-concept is often based on the perception, that the laboratory always processes whole microtiter plates. However, in the daily routine, this is rather a rare exception, which leads to much higher costs per newborn, compared to the costs per assay in the test-kits. In addition the amount of wasted resources is quite high. Performance of the Neonatal Total Galactose kit from Perkin Elmer was tested. We have determined specificity, limit of detection (LOD), limit of quantitation (LOQ), intra and inter assay variation, recovery, stability of measuring signal and reagents. Results were also compared with the Astoria Pacific Spot Check System. In addition, we had (by chance) the opportunity to test 2 kits, which were already expired for more than 3 years. LOD was 165 - 306 μmol/L and LOQ 475 - 703 μmol/L, depending on the definition of LOD/LOQ. Mean recovery was 112.8%, intra assay CVs were 11.3, 7.3, 4.0, and 3.0, and inter assay CVs 28.7, 15.9, 7.8, and 9.3, at 220, 590, 1200, and 2060 μmol/L respectively. Reconstituted and mixed reagents must be used within some hours, and were unstable even if stored at -20℃. However, if the reconstituted galactose substrate reagent and galactose oxidase reagent were only mixed according to the daily requirements, and the rest stored separately at -20℃, they were stable for at least 12 days. The performance of the expired test-kits did not differ from the others. The performance of the Total Galactose kit is comparable to other tests used for newborn screening. However, we could significantly reduce the costs per newborn and reduce unnecessary production of waste, by thorough validation and modification of the assay procedures.

Antioxidant and Anti-aging Activities of Silybum Marianum Protein Hydrolysate in Mice Treated with D-galactose

Objective In the present study, we investigated the antioxidant and anti‐aging effects of Silybum marianum protein hydrolysate（SMPH） in D‐galactose‐treated mice. Methods D‐galactose（500 mg/kg body weight） was intraperitoneally injected daily for 7 weeks to accelerate aging, and SMPH（400, 800, 1,200 mg/kg body weight, respectively） was simultaneously administered orally. The antioxidant and anti‐aging effects of SMPH in the liver and brain were measured by biochemical assays. Transmission electron microscopy（TEM） was performed to study the ultrastructure of liver mitochondria. Results SMPH decreased triglyceride and cholesterol levels in the D‐galactose‐treated mice. It significantly elevated the activities of superoxide dismutase（SOD） and glutathione peroxidase（GSH‐Px）, and total antioxidant capacity（T‐AOC）, which were suppressed by D‐galactose. Monoamine oxidase（MAO） and malondialdehyde（MDA） levels as well as the concentrations of caspase‐3 and 8‐OHd G in the liver and brain were significantly reduced by SMPH. Moreover, it increased Bcl‐2 levels in the liver and brain. Furthermore, SMPH significantly attenuated D‐galactose‐induced liver mitochondrial dysfunction by improving the activities of Na＋‐K＋‐ATPase and Ca2＋‐Mg2＋‐ATPase as well as mitochondrial membrane potential（ΔΨm） and fluidity. TEM showed that the degree of liver mitochondrial damage was significantly decreased by SMPH. Conclusion The results indicated that SMPH protects against D‐galactose‐induced accelerated aging in mice through its antioxidant and anti‐aging activities.

Multianalyte Biosensors for the Simultaneous Determination of Glucose and Galactose Based on Thin Film Electrodes

A multianalyte biosensor for the simultaneous determination of glucose and galactose was developed by immobilizing glucose oxidase (GOD) and galactose oxidase (GAO) on Nafion-modified thin film platinum disk electrodes. The dual Pt working electrodes with disk shape and the surrounding ring shaped counter electrode were fabricated by thin film technology, which were integrated onto the same microchip. The response of the designed biosensor for glucose and galactose were linear up to 6.0 mmol/L and 3.5 mmol/L with sensitivities of 0.3 mA/mmol/L and 0.12 mA/mmol/L, respectively. No cross-talking effect was observed.

Green Electrospun Silk Fibroin/Galactose Chitosan Composite Nanofibrous Scaffolds for Hepatic Tissue Engineering

The electrospun nanofibrous scaffolds made of proteins and polysaccharides were thought to be able to simulate the structure of natural extracellular matrix well.Silk fibroin(SF)and chitosan(CS)are probably the most widely used natural materials in biomedical fields including liver tissue engineering for their good properties and wide variety of sources.The asialoglycoprotein receptors of hepatocyte were reported to specifically recognize and interact with galactose.In this work,a green electrospun SF/galactosylated chitosan(GC)composite nanofibrous scaffold was fabricated and characterized.The data indicated that the addition of GC greatly influenced the spinning effect of SF aqueous solution,and the average diameter of the composite nanofibers was about 520nm.Moreover,the green electrospun SF/GC nanofibrous scaffolds were demonstrated significantly enhancing the adhesion and proliferation of hepatocyte(RH35)according to our data.The present study did a useful exploration on constructing scaffolds for liver regeneration by green electrospinning,and also laid a good foundation for the further applicative research of this green electrospun scaffolds in liver tissue engineering.

The Hainan papaya extract’s effect on anti-aging,learning and memory of aging model mice induced by D-galactose

Objective:To explore the effects of Hainan papaya extract on learning and memory impairment and anti‑aging in D‑galactose‑induced aging mice.Methods:A total of 72 Kunming mice with normal cognitive ability screened by water maze test were randomly divided into negative control group,model group,piracetam group,high,medium and low dose groups of Hainan papaya extract(400 mg/kg,200 mg/kg,100 mg/kg),with 12 mice in each group.Hainan papaya extract and piracetam group were given the above drugs by gavage every day,The negative control and model groups were given the same amount of 0.9%NaCl solution in the same way.Mice in each group were weighed once a week;At the same time,except for the negative control group,mice in each group were intraperitoneally injected with 2%D‑galactose every day,and the negative control group was intraperitoneally injected with normal saline for 7 weeks.After 7 weeks,We observed each group of mice’s capacity of learning and memory by Morris water maze behavioral test;Then,the content of superoxide dismutase(SOD),malondialdehyde(MDA),catalase(CAT),and nitric oxide synthase(NOS)were measured;On the other hand,we observed the hippocampus’histopathological changes by hematoxylin‑eosin staining,and measured the protein expression of nuclear factor‑E2‑related factor(Nrf2)in brain tissue of mice in each group by Western blot.Results:After the intervention of Hainan papaya extract on aging model mice,the high,medium and low dose groups could shorten the swimming time and swimming distance of mice to varying degrees,increase the activities of SOD,CAT and NOS in mouse brain tissue and reduce the content of MDA,The performance of high dose group was better than piracetam group(P<0.01).At the same time,it can improve the histopathological changes of neurons in mouse hippocampus by reducing neuronal nuclear pyknosis,and increase the expression of Nrf2 protein in mouse brain in a dose‑dependent manner.Conclusion:Hainan papaya extract is able to postpone various physical signs of subacute aging mice caused by D‑galactose,and possesses definite anti‑aging and antioxidant effects,which may be related to the regulation of Nrf2 signal pathway.

Differing Roles for Clostridium acetobutylicum’s Galactose Utilization Pathways

There has been a surge of interest in acetone-butanol-ethanol fermentations of Clostridium acetobutylicum due to its capacity to ferment many carbohydrates found in biomass. This metabolic diversity makes it a promising candidate for conversion of inexpensive, heterogeneous carbohydrate feedstocks to biofuels. Galactose is present in many such feedstocks due to its incorporation in plant cell walls. C. acetobutylicum encodes two galactose utilization pathways, the Leloir (LP) and the tagatose-6-P (T6P), and a previous study indicated genes for these pathways was differentially regulated during growth on galactose and lactose. In the current study we utilized quantitative PCR to further investigate gene expression levels and to show both pathways which were subject to carbon catabolite repression. During growth on galactose, mRNA for galactose-6-P isomerase from the T6P was induced to a greater extent than mRNA for glactokinase, the first enzyme in the LP. The galactose-6-P isomerase mRNAs were also more abundant than galactokinase mRNAs during growth on galactose. Analysis of theoretical ATP requirements to generate essential precursor metabolites indicated: 1) the LP is more efficient at generating upper glycolytic intermediates, 2) the T6P is more efficient at forming ATP, lower glycolytic intermediates and TCA cycle intermediates, 3) a combination of the two pathways is most efficient for forming precursor metabolites found in the pentose phosphate pathway. From this it can be suggested that the two pathways have different roles in the organism with the T6P generating most ATP and precursor metabolites and the LP providing upper glycolytic metabolites.

Alteration of Crystallin Polypeptides in Rat Lenses during the Development of Galactose-induced Cataract

Some striking differences in relative polypeptide abundanceof crystallins were observed in normal and galactose-induced cataractouslenses of rat by means of SDS-PAGE.In the cataractous lenses aprominent band appeared at about 25 kDa and the αA chain increasedmarkedly,whereas the relative amount of the 31 kDa band decreasedsubstantially.These alterations are similar to the changes observed duringthe incubation of young mouse lenses in glucose-free medium.Eye Science1993;9:143-145.

Formulation Containing Phytosomes of Carotenoids from Nyctanthes arbor-tristis and Tagetes patula Protect D-galactose Induced Skin Aging in Mice

Background:Carotenoids play important role in delay of aging process.Orange coloured tubular calyx of flowers of Nyctanthes arbor-tristis contains crocin,an apocarotenoid which forms a major component of stigma of saffron.Due to presence of crocin in orange coloured tubular calyx of Nyctanthes arbor-tristis,it can be used as an economical substitute to saffron for its medicinal and cosmetic utilities.Lutein from flower petals of Tagetes patula L.,is another popular carotenoid which has antioxidant effect and many health benefits.The carotenoids are highly unstable when exposed to atmosphere.One of the ways to improve stability of these phytoconstituents,is their entrapment in phytosomes.Preparation of phytosomes will have dual advantages of improving stability as well as bioavailability of molecule.Objective:In the present study crocin and lutein rich extracts were entrapped into phytosomes to improve stability and efficacy of topical preparation.Methods:The phytosomes of Carotenoid rich extract of tubular calyx of Nyctanthes arbor-tristis L.and the petals of Tagetes patula L.(standardized for crocin and lutein content)were prepared using lipid film hydration technique and these phytosomes were then incorporated into gel base.The gel formulation was evaluated for stability as per ICH guidelines.Efficacy of formulation was evaluated by d-galactose induced aging model.Aging in skin was induced by administration of d-galactose(100 mg/kg bwsc.)to albino mice for 42 days.The gel formulation was applied topically for 42 days.Then the effect of formulation on skin aging was evaluated by estimation of biochemical parameters viz.glutathione and malondialdehyde(MDA)and histopathological studies of treated skin samples.Expression of COL type I and elastin genes in skin samples was also carried out by RT-PCR.Results:Percent entrapment(%w/w)of crocin and lutein in phytosomes were found to be 60.20%and 50.81%,respectively.Accelerated stability studies showed improvement in stability of carotenoids viz.crocin and lutein and the content of crocin and lutein in formulation was found in the range of 99.98%w/w to 99.85%w/w at the end of three months.The formulation containing extract of Phytosomes of carotenoid rich extracts of Nyctanthes arbor-tristis L.and the petals of Tagetes patula L.exhibited potent antiaging activity through significant(P<0.05)increase in dermal and epidermal layers,and increase in GSH levels of skin as compared to the untreated group.The treatment with the gel formulation revealed upregulation of collagen type I and elastin genes.There was significant reduction in lipid peroxidation as revealed through reduction in MDA levels as compared to untreated group.Conclusion:Crocin and lutein have potential to prevent skin aging via upregulation of collagen type I gene and elastin gene.Upregulation of genes resulted into increase in the thickness of epidermal and dermal layer along with reduction in oxidative stress in skin.Entrapment of carotenoid rich extracts of Nyctanthes arbor-tristis and Tagetes patula in phytosomes enhanced the stability and efficacy of the formulation.

Newborn screening for galactosemia:a 30-year single center experience

Background:Galactosemia due to complete or near-complete galactose-l-phosphate uridyltransferase(GALT)deficiency was the first disorder added to the pioneering newborn screening panel besides phenylketonuria.In the last 50 years,many criticisms have been focused on the opportunity of its inclusion.Consequently,long-term single center experiences with this issue are generally lacking.Methods:We reviewed the outcome of newborn screening for hypergalactosemia performed at our department since 1982 and the correspondent long-term clinical outcome.Results:Among 1123909 newborns screened for hypergalactosemia,33 showed abnormal results confirmed at second tier test.Thirteen patients were affected with classic galactosemia,8 partial GALT deficiency,3 severe galactokinase deficiency,7 transient galactosemia,one congenital porto-systemic shunt,and one glucose transporter 2 deficiency.Acute neonatal liver failure in the late first week of life(5.8±1.1 days)unavoidably complicated the clinical course of classic galactosemia,unless in three second-born siblings treated on the basis of presumptive diagnosis immediately after newborn screening sample collection on day 3.Despite early treatment and long­term steadily normal peripheral blood galactose,77%of patients with severe GALT deficiency present mild to severe intellectual disabilities.All patients with partial GALT deficiency showed normal intellectual development on a regular diet,as well as patients with galactokinase deficiency under treatment.Conclusions:Availability of screening results within the fifth day after birth would allow the prevention of acute decompensation in classic galactosemia.A systematic diagnostic work-up in all positive newborns is essential to unravel the etiology of hypergalactosemia.

Erratum on “Galactose functionalized diketopyrrolopyrrole as NIR fluorescent probes for lectin detection and HepG2 cell targeting based on aggregation-induced emission mechanism” [Sci. China Chem., 2018, 61: 898–908]

In a recent publication [1],there is a mistake in Scheme 1,in which a propargyl alpha-mannoside was drawn on the arrow in the synthetic step from compounds 8/9 to 10/11. The correct structure should be a propargyl beta-galactoside. Here we provide corrections to Scheme 1.

Role of Eclipta prostrata extract in improving spatial learning and memory deficits in D-galactose-induced aging in rats

OBJECTIVE: To investigate the role of Eclipta prostrata (E. prostrata) extract in improving spatial learning and memory deficits in D-galactose-induced aging in rats. METHODS: Rats were divided into five groups, with 10 animals in each group. Aging rats were produced by treatment with 100 mg·kg-1·d-1 of D-galactose for 6 weeks. Rats in the E. prostrata treatment groups received an aqueous extract of E. prostrata orally at a concentration of 50, 100, or 200 mg·kg-1· d-1 for 3 weeks. Animals in both the normal and model groups were treated with similar volumes of saline. Spatial memory performance was measured using the Morris water maze. The mRNA levels and enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were analyzed using real- time quantitative PCR and spectrophotometry,respectively. The levels of induced nitric oxide synthase (iNOS), nitric oxide (NO), dopamine (DA), norepinephrine (NE), and serotonin (5-HT) were determined using enzyme-linked immunosorbent assay and spectrophotometry. RESULTS: Compared with the normal group, rats in the D-galactose-treated model group exhibited significant memory loss. There was severe damage to the hippocampal CA1 area, and expression levels of SOD, CAT, GPx, and GR were significantly decreased in the model group compared with the normal group. In the model group, levels of iNOS and NO were significantly increased compared with the normal group. However, treatment with E. prostrata extract reversed the conditions caused by D-galactose- induced aging, especially in the groups with higher treatment concentrations. Compared with the normal group, the levels of DA, NE, and 5-HT were significantly lower in the D-galactose-treated model group. In the E. prostrata extract-treated groups, however, there was a dose-dependent upregulation of DA, NE, and 5-HT expression. CONCLUSION: Our results suggest that administration of E. prostrata extract can result in an improvement in the learning and memory impairments that are induced by D-galactose treatment in rats. This improvement may be the result of enhanced antioxidative ability, decreased iNOS and NO levels, and the induction of DA, NE, and 5-HT expression in the brain.

Study on the dual-frequency oscillations in galactose-BZ system

Three types of dual-frequency oscillations in KBrO3-galactose-acetone-MnSO4-H2SO4 have been observed in a batch reactor depending on the initial concentration of H2SO4 or Br-. Similar phenomena were also observed when galactose was replaced by other aldosugars such as glucose, arabinose, lactose and maltose. However no dual-frequency oscillation has been found when ketosugar like fructose was used instead of galactose as the substrate. The roles of acidity, bromide ion and acetone in dual-frequency oscillations have been analyzed. The mechanism of dual-frequency oscillations has been discussed.

Dual pH and microbial-sensitive galactosylated polymeric nanocargoes for multi-level targeting to combat ulcerative colitis

Ulcerative colitis(UC)is a type of inflammatory bowel disease characterized by inflammation,ulcers and irritation of the mucosal lining.Oral drug delivery in UC encounters challenges because of multifaceted barriers.Dexamethasone-loaded galactosylated-PLGA/Eudragit S100/pullulan nanocargoes(Dexa-GP/ES/Pu NCs)have been developed with a dual stimuli-sensitive coating responsive to both colonic pH and microbiota,and an underneath galactosylated-PLGA core(GP).The galactose ligand of the GP preferentially binds to the macrophage galactose type-lectin-C(MGL-2)surface receptor.Therefore,both stimuli and ligand-mediated targeting facilitate nanocargoes to deliver Dexa specifically to the colon with enhanced macrophage uptake.Modified emulsion method coupled with a solvent evaporation coating technique was employed to prepare Dexa-GP/ES/Pu NCs.The nanocargoes were tested using in vitro,ex vivo techniques and dextran sodium sulfate(DSS)induced UC model.Prepared nanocargoes had desired physicochemical properties,drug release,cell uptake and cellular viability.Investigations using a DSS-colitis model showed high localization and mitigation of colitis with downregulation of NF-ĸB and COX-2,and restoration of clinical,histopathological,biochemical indices,antioxidant balance,microbial alterations,FTIR spectra,and epithelial junctions’integrity.Thus,Dexa-GP/ES/Pu NCs found to be biocompatible nanocargoes capable of delivering drugs to the inflamed colon with unique targeting properties for prolonged duration.

半乳糖修饰人血清白蛋白的质谱分析

研究发现,存在于哺乳动物肝细胞膜上的血浆无唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGP-R)是一种肝结合蛋白(hepatic binding protein,HBP),能专一性识别分子末端带有半乳糖残基的糖蛋白并与之结合,定向转到肝细胞内的溶酶体进行代谢[1],针对ASGP-R作为靶点的肝靶向药物载体半乳糖化人血白蛋白偶合物(galactosyl-neoglycoalbumin,NGA),已引起药物研究工作者的极大重视[2,3].将药物与带有半乳糖残基的蛋白偶合,可提高药物的肝靶向性,提高治疗指数,减少非靶器官的毒副作用.……

Liver-related effects of chronic hepatitis C antiviral treatment

More than five years ago,the treatment of hepatitis C virus infection was revolutionized with the introduction of all-oral direct-acting antiviral(DAA)drugs.They proved highly efficient in curing patients with chronic hepatitis C(CHC),including patients with cirrhosis.The new DAA treatments were alleged to induce significant improvements in clinical outcome and prognosis,but the exact cause of the expected benefit was unclear.Further,little was known about how the underlying liver disease would be affected during and after viral clearance.In this review,we describe and discuss the liver-related effects of the new treatments in regards to both pathophysiological aspects,such as macrophage activation,and the time-dependent effects of therapy,with specific emphasis on inflammation,structural liver changes,and liver function,as these factors are all related to morbidity and mortality in CHC patients.It seems clear that antiviral therapy,especially the achievement of a sustained virologic response has several beneficial effects on liver-related parameters in CHC patients with advanced liver fibrosis or cirrhosis.There seems to be a timedependent effect of DAA therapy with viral clearance and the resolution of liver inflammation followed by more discrete changes in structural liver lesions.These improvements lead to favorable effects on liver function,followed by an improvement in cognitive dysfunction and portal hypertension.Overall,the data provide knowledge on the several beneficial effects of DAA therapy on liverrelated parameters in CHC patients suggesting short-and long-term improvements in the underlying disease with the promise of an improved longterm prognosis.

The Role of Antioxidation in Beneficial Effect of Proper Stress on Mice′sBehavior

The purpose of this study is to elucidate the mechanism of delaying aging process under proper stress. An aging model was established by intraperitoneal injection of D galactose at the dose of 0.12 mg·(g·d) -1 for six weeks, and swimming daily for 15min at 18℃ water was used as proper stressor. Six weeks later, learning and memory ability, the contents of SOD and MDA in hippocampus and heart were measured. The results showed that proper stress could ①retard the decline of learning and memory ability (P<0.001) induced by D galactose;②increase the content of SOD (P<0.001) and reduce the production of MDA (P<0.05). The results indicated that proper stress may delay aging process by improving the antioxidant level and reducing the toxicity of free radicals.

Separation and Purification of GST-glycerol-3-phosphate Dehydrogenase

In order to investigate the expression of glycerol-3 -phosphate dehydrogenase by GCY1 gene in recombinant Saccharomyces cerevisiae, induction culture of the S. cerevisiaestrain was performed with SD-URA 2% galactose, 3 × YP ＋ 6% glucose, SC-URA 2% galactose, and SC-URA 2% galactose ＋ 5% NaCI glyeerol-3-phosphate dehydregenase, the cultured S. cerevisiaewas comminuted followed by full-automatic high-speed purification, and SDS-PAGE gel electrophoresis was performed for molecular weight of the GST fusion protein. The results showed that after shaking culture of the S. cerevisiae containing GCY1 at 25 ℃, the OD values of its 3 × YP ＋ 6% glucose culture and SC-URA 2% galaetose ＋ 5% NaC1 culture were 8.75 and 7.35, respectively. It was shown by purification with a Profinia low-pressure liquid chromatograph that only the S. cerevisiae cultured in SC-URA 2% galactose ＋ 5% NaC1 medium expressed glycerel-3-phosphate de- hydrogenase, the molecular weight of which was detected as 65 ku by SDS-PAGE gel electrophoresis.

Study on the relationship between Lp-PLA2, D- dimer and Galectin-3 and atherosclerotic vulnerable plaque in patients with coronary heart disease

Objective: To explore the relationship and clinical value of serum phospholipase A2 (Lp-PLA2), d-dimers, and serum galectin-3 (galectin-3) with atherosclerotic vulnerable plaques in coronary artery patients with coronary heart disease. Methods: A total of 248 patients who underwent coronary angiography (CAG) and intravascular ultrasound (IVUS) in our hospital from June 2017 to September 2018 were selected and divided into vulnerable plaque group (89), stable plaque group (89) and control group (70) according to the examination results. The serum levels of Lp-PLA2, d-dimer and galectin-3 in three groups were compared, as well as their correlation with the detection parameters. To evaluate the clinical value of Lp-PLA2, d-dimer and galectin-3 in patients with coronary heart disease (CHD) with atherosclerotic vulnerable plaque. Results: Serum Lp-PLA2, d-dimer and galectin-3 levels were significantly different from the three groups (P<0.05), and the control group < stable plaque group <vulnerable plaque group (P<0.05). Correlation analysis showed that Lp-PLA2, d-dimer and galectin-3 were significantly positively correlated with plaque area, plaque load, necrotic core and calcified tissue (P<0.01), and negatively correlated with fibrous lipid and fibrous tissue (P<0.01). ROC curve showed that Lp-PLA2, d-dimer and galectin-3 had certain predictive value for vulnerable coronary atherosclerotic plaques (AUC=0.939, 0.977, 0.920, P<0.01), and the three combinations (AUC=0.986, P<0.01) had higher predictive value. Conclusion: Serum Lp-PLA2, d-dimer and galectin-3 are significantly correlated with coronary atherosclerotic vulnerable plaques in patients with coronary heart disease, with high sensitivity and specificity, which can be used for the diagnosis and treatment of early atherosclerotic vulnerable plaques.

Hepatocyte selection medium eliminating induced pluripotent stem cells among primary human hepatocytes

Hepatic insufficiency is a fatal liver disease with a significant decrease in functioning hepatocytes. If hepatocytes could be generated from human induced pluripotent stem(hi PS) cells and transplanted into patients with hepatic insufficiency, the disease may become curable. However, a major limitation to this therapeutic strategy is due to the tumorigenicity of hi PS cells and their ability to form cancer. Current methods for eliminating unwanted hi PS cells use genetic manipulation or reagents that are potentially hazardous for hepatocytes; therefore, revised methods are necessary and anticipated. Glucose and arginine are essential cell culture medium ingredients for the survival of most cells, including hi PS cells. However, hepatocytes can produce its own glucose and arginine through galactokinase and ornithine transcarbamylase, respectively. Therefore, it was hypothesized that unwanted hi PS cells could be eliminated in a medium without glucose and arginine, and supplemented with galactose and ornithine instead. This modified medium has been established as hepatocyte selection medium(HSM). So far, attempts to generate a pure colony of mature hepatocytes from hi PS cells have not been successful. After establishment of co-culture in HSM,primary human hepatocytes survive while hi PS cells die within three days. Our latest results regarding a modification of HSM will be introduced in this manuscript.