ABCG2 Mediates Resistance to the Dual EGFR and PI3K Inhibitor MTX-211 in Cancer Cells.

MTX-211 is a first-in-class dual inhibitor of epidermal growth factor receptor (EGFR) and phosphoinositide-3 kinase (PI3K) signaling pathways with a compelling pharmaceutical profile and could enhance the effectiveness of mitogen-activated protein kinase kinase (MEK) inhibitor therapy in colorectal tumors with KRAS mutations. However, the specific mechanisms contributing to the acquired resistance to MTX-211 in human cancers remain elusive. Here, we discovered that the overexpression of the ATP-binding cassette (ABC) drug transporter ABCG2, a prevalent mechanism associated with multidrug resistance (MDR), could diminish the effectiveness of MTX-211 in human cancer cells. We showed that the drug efflux activity of ABCG2 substantially decreased the intracellular accumulation of MTX-211 in cancer cells. As a result, the cytotoxicity and effectiveness of MTX-211 in suppressing the activation of the EGFR and PI3K pathways were significantly attenuated in cancer cells overexpressing ABCG2. Moreover, the enhancement of the MTX-211-stimulated ATPase activity of ABCG2 and the computational molecular docking analysis illustrating the binding of MTX-211 to the substrate-binding sites of ABCG2 offered a further indication for the interaction between MTX-211 and ABCG2. In summary, our findings indicate that MTX-211 acts as a substrate for ABCG2, underscoring the involvement of ABCG2 in the emergence of resistance to MTX-211. This finding carries clinical implications and merits further exploration.

ABCB1 and ABCG2 Overexpression Mediates Resistance to the Phosphatidylinositol 3-Kinase Inhibitor HS-173 in Cancer Cell Lines.

Constitutive activation of the phosphoinositide-3-kinase (PI3K)/Akt signaling pathway is crucial for tumor growth and progression. As such, this pathway has been an enticing target for drug discovery. Although HS-173 is a potent PI3K inhibitor that halts cancer cell proliferation via G2/M cell cycle arrest, the resistance mechanisms to HS-173 have not been investigated. In this study, we investigated the susceptibility of HS-173 to efflux mediated by the multidrug efflux transporters ABCB1 and ABCG2, which are two of the most well-known ATP-binding cassette (ABC) transporters associated with the development of cancer multidrug resistance (MDR). We found that the overexpression of ABCB1 or ABCG2 significantly reduced the efficacy of HS-173 in human cancer cells. Our data show that the intracellular accumulation of HS-173 was substantially reduced by ABCB1 and ABCG2, affecting G2/M arrest and apoptosis induced by HS-173. More importantly, the efficacy of HS-173 in multidrug-resistant cancer cells could be recovered by inhibiting the drug-efflux function of ABCB1 and ABCG2. Taken together, our study has demonstrated that HS-173 is a substrate for both ABCB1 and ABCG2, resulting in decreased intracellular concentration of this drug, which may have implications for its clinical use.

A low-cost, high-throughput microfluidic nano-culture platform for functional metagenomics.

Functional metagenomics is an attractive culture-independent approach for functional screening of diverse microbiomes to identify known and novel genes. Since functional screening can involve sifting through tens of thousands of metagenomic library clones, an easy high-throughput screening approach is desirable. Here, we demonstrate a proof-of-concept application of a low-cost, high-throughput droplet based microfluidic assay to the selection of antibiotic resistance genes from a soil metagenomic library. Metagenomic library members encapsulated in nanoliter volume water-in-oil droplets were printed on glass slides robotically, and cell growth in individual drops in the presence of ampicillin was imaged and quantified to identify ampicillin-resistant clones. From the hits, true positives were confirmed by sequencing and functional validation. The ease of liquid handling, ease of set-up, low cost, and robust workflow makes the droplet-based nano-culture platform a promising candidate for screening and selection assays for functional metagenomic libraries.

P-glycoprotein Mediates Resistance to the Anaplastic Lymphoma Kinase Inhiitor Ensartinib in Cancer Cells.

Ensartinib (X-396) is a promising second-generation small-molecule inhibitor of anaplastic lymphoma kinase (ALK) that was developed for the treatment of ALK-positive non-small-cell lung cancer. Preclinical and clinical trial results for ensartinib showed superior efficacy and a favorable safety profile compared to the first-generation ALK inhibitors that have been approved by the U.S. Food and Drug Administration. Although the potential mechanisms of acquired resistance to ensartinib have not been reported, the inevitable emergence of resistance to ensartinib may limit its therapeutic application in cancer. In this work, we investigated the interaction of ensartinib with P-glycoprotein (P-gp) and ABCG2, two ATP-binding cassette (ABC) multidrug efflux transporters that are commonly associated with the development of multidrug resistance in cancer cells. Our results revealed that P-gp overexpression, but not expression of ABCG2, was associated with reduced cancer cell susceptibility to ensartinib. P-gp directly decreased the intracellular accumulation of ensartinib, and consequently reduced apoptosis and cytotoxicity induced by this drug. The cytotoxicity of ensartinib could be significantly reversed by treatment with the P-gp inhibitor tariquidar. In conclusion, we report that ensartinib is a substrate of P-gp, and provide evidence that this transporter plays a role in the development of ensartinib resistance. Further investigation is needed.

Aspartate and Acetate Fuel Gastrointestinal Stromal Tumors Beyond the Warburg Effect.

Metabolic reprogramming is closely linked to the tumorigenesis and drug resistance of gastrointestinal stromal tumors (GISTs). Mapping the metabolic orbit of GISTs is a prerequisite if intervention against the metabolic vulnerability of refractory GISTs is desirable. Methods: A total of 43 patients with treatment-naïve GISTs who had undergone surgical resections were enrolled, on whom a metabolomics profile detected from surgical specimens was constructed based on the 1H-nuclear magnetic resonance (NMR) platform. The mRNA and protein levels of GLUT1, HK2, ACSS2, and FASN were assayed. Dual-tracer 18F-FDG/11C-acetate PET imaging was introduced before surgery in 15 patients. Results: 1H-NMR-based metabolomics revealed that GISTs were characterized by upregulation of glutamate, ascorbate, aspartate and glycine and downregulation of choline, creatine, glucose and glycerol. Bioinformatics analysis showed that the TCA cycle and alanine, aspartate, and glutamate metabolism were the two leading pathways. High- and nonhigh-risk (including intermediate-, low-, and very low-risk) GISTs preferentially displayed upregulation of HK2 and ACSS2, respectively, echoed by in vivo imaging that high- and nonhigh-risk GISTs preferentially exhibited higher uptake of 18F-FDG and 11C-acetate, respectively, while 18F-FDG and 11C-acetate were complementary to each other. Nuclear ACSS2 was exclusively identified in high-risk GISTs. Conclusion: We describe a metabolic landscape of GISTs that read aspartate as a de facto "oncometabolite," which was replenished via the TCA cycle and alanine, aspartate, and glutamate metabolism. Glycolysis and ACSS2-mediated acetate metabolism competed and complemented fatty acid synthesis, although glycolysis remained an aggressive phenotype.

Development of a method for dansylation of metabolites using organic solvent-compatible buffer systems for amine/phenol submetabolome analysis.

Metabolomics, which serves as a readout of biological processes and diseases monitoring, is an informative research area for disease biomarker discovery and systems biology studies. In particular, reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) has become a powerful and popular tool for metabolomics analysis, enabling the detection of most metabolites. Very polar and ionic metabolites, however, are less easily detected because of their poor retention in RP columns. Dansylation of metabolites simplifies the sub-metabolome analysis by reducing its complexity and increasing both hydrophobicity and ionization ability. However, the various metabolite concentrations in clinical samples have a wide dynamic range with highly individual variation in total metabolite amount, such as in saliva. The bicarbonate buffer typically used in dansylation labeling reactions induces solvent stratification, resulting in poor reproducibility, selective sample loss and an increase in false-determined metabolite peaks. In this study, we optimized the dansylation protocol for samples with wide concentration range of metabolites, utilizing diisopropylethylamine (DIPEA) or tri-ethylamine (TEA) in place of bicarbonate buffer, and presented the results of a systemic investigation of the influences of individual processes involved on the overall performance of the protocol. In addition to achieving high reproducibility, substitution of DIPEA or TEA buffer resulted in similar labeling efficiency of most metabolites and more efficient labeling of some metabolites with a higher pKa. With this improvement, compounds that are only present in samples in trace amounts can be detected, and more comprehensive metabolomics profiles can be acquired for biomarker discovery or pathway analysis, making it possible to analyze clinical samples with limited amounts of metabolites.

Overexpression of Human ABCB1 and ABCG2 Reduces the Susceptibility of Cancer Cells to the Histone Deacetylase 6-Specific Inhibitor Citarinostat.

Citarinostat (ACY-241) is a promising oral histone deacetylase 6 (HDAC6)-selective inhibitor currently in clinical trials for the treatment of multiple myeloma (MM) and non-small-cell lung cancer (NSCLC). However, the inevitable emergence of resistance to citarinostat may reduce its clinical effectiveness in cancer patients and limit its clinical usefulness in the future. In this study, we investigated the potential role of the multidrug efflux transporters ABCB1 and ABCG2, which are two of the most common mechanisms of acquired resistance to anticancer drugs, on the efficacy of citarinostat in human cancer cells. We discovered that the overexpression of ABCB1 or ABCG2 significantly reduced the sensitivity of human cancer cells to citarinostat. We demonstrated that the intracellular accumulation of citarinostat and its activity against HDAC6 were substantially reduced by the drug transport function of ABCB1 and ABCG2, which could be restored by treatment with an established inhibitor of ABCB1 or ABCG2, respectively. In conclusion, our results revealed a novel mechanism by which ABCB1 and ABCG2 actively transport citarinostat away from targeting HDAC6 in cancer cells. Our results suggest that the co-administration of citarinostat with a non-toxic modulator of ABCB1 and ABCG2 may optimize its therapeutic application in the clinic.

Overexpression of ABCB1 and ABCG2 contributes to reduced efficacy of the PI3K/mTOR inhibitor samotolisib (LY3023414) in cancer cell lines.

LY3023414 (samotolisib) is a promising new dual inhibitor of phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR). Currently, multiple clinical trials are underway to evaluate the efficacy of LY3023414 in patients with various types of cancer. However, the potential mechanisms underlying acquired resistance to LY3023414 in human cancer cells still remain elusive. In this study, we investigated whether the overexpression of ATP-binding cassette (ABC) drug transporters such as ABCB1 and ABCG2, one of the most common mechanisms for developing multidrug resistance, may potentially reduce the efficacy of LY3023414 in human cancer cells. We demonstrated that the intracellular accumulation of LY3023414 in cancer cells was significantly reduced by the drug efflux function of ABCB1 and ABCG2. Consequently, the cytotoxicity and efficacy of LY3023414 for inhibiting the activation of the PI3K pathway and induction of G0/G1 cell-cycle arrest were substantially reduced in cancer cells overexpressing ABCB1 or ABCG2, which could be restored using tariquidar or Ko143, respectively. Furthermore, stimulatory effect of LY3023414 on the ATPase activity of ABCB1 and ABCG2, as well as in silico molecular docking analysis of LY3023414 binding to the substrate-binding pockets of these transporters provided additional insight into the manner in which LY3023414 interacts with both transporters. In conclusion, we report that LY3023414 is a substrate for ABCB1 and ABCG2 transporters implicating their role in the development of resistance to LY3023414, which can have substantial clinical implications and should be further investigated.

Glycerophospholipids pathways and chromosomal instability in gastric cancer: Global lipidomics analysis.

BACKGROUND: Based on the breakthrough of genomics analysis, The Cancer Genome Atlas Research Group recently proposed an integrative genomic analysis, dividing gastric cancer (GC) into four subtypes, characterized by the chromosomal instability (CIN) status. However, the CIN status of GC is still vaguely characterized and lacking the valuable easy-to-use CIN markers to diagnosis in molecular and histological detection. AIM: To explore the associations of CIN with downstream lipidomics profiles. METHODS: We collected cancerous and noncancerous tissue samples from 18 patients with GC; the samples were divided into CIN and non-CIN types based on the system of The Cancer Genome Atlas Research Group and 409 sequenced oncogenes and tumor suppressor genes. We identified the lipidomics profiles of the GC samples and samples of their adjacent noncancerous tissues by using liquid chromatography-mass spectrometry. Furthermore, we selected leading metabolites based on variable importance in projection scores of > 1.0 and P < 0.05. RESULTS: Twelve men and six women participated in this study; the participants had a median age of 67.5 years (range, 52-87 years) and were divided into CIN (n = 9) and non-CIN (n = 9) groups. The GC samples exhibited distinct profiles of lysophosphocholine, phosphocholine, phosphatidylethanolamine, phosphatidylinositol, phosphoserine, sphingomyelin, ceramide, and triglycerides compared with their adjacent noncancerous tissues. The glycerophospholipid levels (phosphocholine, phosphatidylethanolamine, and phosphatidylinositol) were 1.4- to 2.3-times higher in the CIN group compared with the non-CIN group (P < 0.05). Alterations in the glycerolipid and glycerophospholipid pathways indicated progression of GC toward CIN. CONCLUSION: The lipidomics profiles of GC samples were distinct from those of their adjacent noncancerous tissues. CIN status of GC is primarily associated with downstream lipidomics in the glycerophospholipid pathway.

Deriving a sub-nanomolar affinity peptide from TAP to enable smFRET analysis of RNA polymerase II complexes.

Our capability to visualize protein complexes such as RNA polymerase II (pol II) by single-molecule imaging techniques has largely been hampered by the absence of a simple bio-orthogonal approach for selective labeling with a fluorescent probe. Here, we modify the existing calmodulin-binding peptide (CBP) in the widely used Tandem Affinity Purification (TAP) tag to endow it with a high affinity for calmodulin (CaM) and use dye-CaM to conduct site-specific labeling of pol II. To demonstrate the single molecule applicability of this approach, we labeled the C-terminus of the Rpb9 subunit of pol II with donor-CaM and a site in TFIIF with an acceptor to generate a FRET (fluorescence resonance energy transfer) pair in the pol II-TFIIF complex. We then used total internal reflection fluorescence microscopy (TIRF) with alternating excitation to measure the single molecule FRET (smFRET) efficiency between these two sites in pol II-TFIIF. We found they exhibited a proximity consistent with that observed in the transcription pre-initiation complex by cryo-electron microscopy (cryo-EM). We further compared our non-covalent labeling approach with an enzyme-enabled covalent labeling method. The virtually indistinguishable results validate our smFRET approach and show that the observed proximity between the two sites represents a hallmark of the pol II-TFIIF complex. Taken together, we present a simple and versatile bio-orthogonal method derived from TAP to enable selective labeling of a protein complex. This method is suitable for analyzing dynamic relationships among proteins involved in transcription and it can be readily extended to many other biological processes.

Metabolomic alterations and chromosomal instability status in gastric cancer.

AIM: To explore the correlation of metabolomics profiles of gastric cancer (GC) with its chromosomal instability (CIN) status. METHODS: Nineteen GC patients were classified as CIN and non-CIN type by The Cancer Genome Atlas Research Group system, based on 409 oncogenes and tumor suppressor genes sequenced. The aqueous metabolites of the GC tumor and its surrounding adjacent healthy tissues were identified through liquid chromatography-mass spectrometry. Groups were compared by defining variable importance in projection score of > 1.2, a fold change value or its reciprocal of > 1.2, and a P value of < 0.05 as a significant difference. RESULTS: In total, twelve men and seven women were enrolled, with a median age of 66 years (range, 47-87 years). The numbers of gene alterations in the CIN GC group were significantly higher than those in the non-CIN GC (32-218 vs 2-17; P < 0.0005). Compared with the adjacent healthy tissues, GC tumors demonstrated significantly higher aspartic acid, citicoline, glutamic acid, oxidized glutathione, succinyladenosine, and uridine diphosphate-N-acetylglucosamine levels, but significantly lower butyrylcarnitine, glutathione hydroxyhexanoycarnitine, inosinic acid, isovalerylcarnitine, and threonine levels (all P < 0.05). CIN tumors contained significantly higher phosphocholine and uridine 5'-monophosphate levels but significantly lower beta-citryl-L-glutamic acid levels than did non-CIN tumors (all P < 0.05). CIN GC tumors demonstrated additional altered pathways involving alanine, aspartate, and glutamate metabolism, glyoxylate and dicarboxylate metabolism, histidine metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis. CONCLUSION: Metabolomic profiles of GC tumors and the adjacent healthy tissue are distinct, and the CIN status is associated with downstream metabolic alterations in GC.

Anti-enterovirus 71 activities of Melissa officinalis extract and its biologically active constituent rosmarinic acid.

Enterovirus 71 (EV71) infection is endemic in the Asia-Pacific region. No specific antiviral drug has been available to treat EV71 infection. Melissa officinalis (MO) is a medicinal plant with long history of usage in the European and Middle East. We investigated whether an aqueous solution of concentrated methanolic extract (MOM) possesses antiviral activity. MOM inhibited plaque formation, cytopathic effect, and viral protein synthesis in EV71-infected cells. Using spectral techniques, we identified rosmarinic acid (RA) as a biologically active constituent of MOM. RA reduced viral attachment and entry; cleavage of eukaryotic translation initiation factor 4 G (eIF4G); reactive oxygen species (ROS) generation; and translocation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) from nucleus to cytoplasm. It alleviated EV71-induced hyperphosphorylation of p38 kinase and EPS15. RA is likely to suppress ROS-mediated p38 kinase activation, and such downstream molecular events as hnRNP A1 translocation and EPS15-regulated membrane trafficking in EV71-infected cells. These findings suggest that MO and its constituent RA possess anti-EV71 activities, and may serve as a candidate drug for therapeutic and prophylactic uses against EV71 infection.

Impaired embryonic development in glucose-6-phosphate dehydrogenase-deficient Caenorhabditis elegans due to abnormal redox homeostasis induced activation of calcium-independent phospholipase and alteration of glycerophospholipid metabolism.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a commonly pervasive inherited disease in many parts of the world. The complete lack of G6PD activity in a mouse model causes embryonic lethality. The G6PD-deficient Caenorhabditis elegans model also shows embryonic death as indicated by a severe hatching defect. Although increased oxidative stress has been implicated in both cases as the underlying cause, the exact mechanism has not been clearly delineated. In this study with C. elegans, membrane-associated defects, including enhanced permeability, defective polarity and cytokinesis, were found in G6PD-deficient embryos. The membrane-associated abnormalities were accompanied by impaired eggshell structure as evidenced by a transmission electron microscopic study. Such loss of membrane structural integrity was associated with abnormal lipid composition as lipidomic analysis revealed that lysoglycerophospholipids were significantly increased in G6PD-deficient embryos. Abnormal glycerophospholipid metabolism leading to defective embryonic development could be attributed to the increased activity of calcium-independent phospholipase A2 (iPLA) in G6PD-deficient embryos. This notion is further supported by the fact that the suppression of multiple iPLAs by genetic manipulation partially rescued the embryonic defects in G6PD-deficient embryos. In addition, G6PD deficiency induced disruption of redox balance as manifested by diminished NADPH and elevated lipid peroxidation in embryos. Taken together, disrupted lipid metabolism due to abnormal redox homeostasis is a major factor contributing to abnormal embryonic development in G6PD-deficient C. elegans.

Lipidomic Analysis of Caenorhabditis elegans Embryos.

Metabolomic is an emerging field of system biology. Lipidomic, a branch of metabolomic, aims to characterize lipophilic metabolites in biological systems. Caenorhabditis elegans (C. elegans) is a genetically tractable and versatile animal model for novel discovery of lipid metabolism. In addition, C. elegans embryo is simple and homogeneous. Here, we demonstrate detailed procedures of C. elegans culture, embryo isolation, lipid extraction and metabolomic data analysis.

Docosapentaenoic acid and docosahexaenoic acid are positively associated with insulin sensitivity in rats fed high-fat and high-fructose diets.

BACKGROUND: The aim of the present study was to compare insulin resistance and metabolic changes using a global lipidomic approach. METHODS: Rats were fed a high-fat diet (HFD) or a high-fructose diet (HFrD) for 12 weeks to induce insulin resistance (IR) syndrome. After 12 weeks feeding, physiological and biochemical parameters were examined. Insulin sensitivity and plasma metabolites were evaluated using a euglycemic-hyperinsulinemic clamp and mass spectrometry, respectively. Pearson's correlation coefficient was used to investigate the strength of correlations. RESULTS: Rats on both diets developed IR syndrome, characterized by hypertension, hyperlipidemia, hyperinsulinemia, impaired fasting glucose, and IR. Compared with HFrD-fed rats, non-esterified fatty acids were lower and body weight and plasma insulin levels were markedly higher in HFD-fed rats. Adiposity and plasma leptin levels were increased in both groups. However, the size of adipocytes was greater in HFD- than HFrD-fed rats. Notably, the lipidomic heat map revealed metabolites exhibiting greater differences in HFD- and HFrD-fed rats compared with controls. Plasma adrenic acid levels were higher in HFD- than HFrD-fed rats. Nevertheless, linoleic and arachidonic acid levels decreased in HFrD-fed rats compared with controls. Plasma concentrations of docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) were significantly reduced after feeding of both diets, particularly the HFrD. There was a strong positive correlation between these two fatty acids and the insulin sensitivity index. CONCLUSIONS: The systemic lipidomic analysis indicated that a reduction in DHA and DPA was strongly correlated with IR in rats under long-term overnutrition. These results provide a potential therapeutic target for IR and metabolic syndrome.

Fatty acids and small organic compounds bind to mineralo-organic nanoparticles derived from human body fluids as revealed by metabolomic analysis.

Nanoparticles entering the human body instantly become coated with a "protein corona" that influences the effects and distribution of the particles in vivo. Yet, whether nanoparticles may bind to other organic compounds remains unclear. Here we use an untargeted metabolomic approach based on ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry to identify the organic compounds that bind to mineral nanoparticles formed in human body fluids (serum, plasma, saliva, and urine). A wide range of organic compounds is identified, including fatty acids, glycerophospholipids, amino acids, sugars, and amides. Our results reveal that, in addition to the proteins identified previously, nanoparticles harbor an "organic corona" containing several fatty acids which may affect particle-cell interactions in vivo. This study provides a platform to study the organic corona of biological and synthetic nanoparticles found in the human body.

Footprints of natural and artificial selection for photoperiod pathway genes in Oryza.

Asian rice, Oryza sativa, consists of two major subspecies, indica and japonica, which are physiologically differentiated and adapted to different latitudes. Genes for photoperiod sensitivity are likely targets of selection along latitude. We examined the footprints of natural and artificial selections for four major genes of the photoperiod pathway, namely PHYTOCHROME B (PhyB), HEADING DATE 1 (Hd1), HEADING DATE 3a (Hd3a), and EARLY HEADING DATE 1 (Ehd1), by investigation of the patterns of nucleotide polymorphisms in cultivated and wild rice. Geographical subdivision between tropical and subtropical O. rufipogon was found for all of the photoperiod genes in plants divided by the Tropic of Cancer (TOC). All of these genes, except for PhyB, were characterized by the existence of clades that split a long time ago and that corresponded to latitudinal subdivisions, and revealed a likely diversifying selection. Ssp. indica showed close affinity to tropical O. rufipogon for all genes, while ssp. japonica, which has a much wider range of distribution, displayed complex patterns of differentiation from O. rufipogon, which reflected various agricultural needs in relation to crop yield. In japonica, all genes, except Hd3a, were genetically differentiated at the TOC, while geographical subdivision occurred at 31°N in Hd3a, probably the result of varying photoperiods. Many other features of the photoperiod genes revealed domestication signatures, which included high linkage disequilibrium (LD) within genes, the occurrence of frequent and recurrent non-functional Hd1 mutants in cultivated rice, crossovers between subtropical and tropical alleles of Hd1, and significant LD between Hd1 and Hd3a in japonica and indica.

[Study on the lowest acceptable intake of cow's milk for healthy adults].

OBJECTIVE: [corrected] To find out the lowest acceptable intake of Cow's Milk in subjects with lactose intolerance(LI) and then observe intolerance symptoms in 280 healthy adults after they drank the milk. METHODS: Eight LI subjects with watery diarrhea who were selected from 38 subjects by hydrogen breath test(HBT) were divided into group A, group B to go on drinking cow's milk test. The subjects in group A ingested 25g milk powder, the subjects in group B ingested 38.2g milk powder( contain 6.25g, 9.55g lactose respectively) according to literature and single factor optimization method (0.618 golden mean) to analyze their hydrogen excretion(ppm), symptom score and stool lactose. The 100 younger, the 80 middle-age and the 100 elder subjects were selected to observe symptoms after they ingested 25g milk powder. RESULTS: hydrogen excretion(ppm) and symptom score in the high dose group (group B)are a little higher than those in low dose group(group A). No case with diarrhea occurred in these two group but mild symptoms. Most of healthy adults (80.3%) have no intolerance symptoms after they drank the milk. CONCLUSION: The study demonstrates that most of adults, even with LI, can tolerance 25g cow's milk powder (6.25g lactose). It is suggested 20g cow's milk powder (5g lactose, make 160 ml of milk solution) is the lowest acceptable intake of healthy adults in China.