Calcium-fortified fresh milk ameliorates postmenopausal osteoporosis via regulation of bone metabolism and gut microbiota in ovariectomized rats

The aging of the global population has made postmenopausal osteoporosis prevention essential;however,pharmacological treatments are limited.Herein,we evaluate the effect of calcium-fortified fresh milk(FM)in ameliorating postmenopausal osteoporosis in a rat model established using bilateral ovariectomy.After 3 months of FM(containing vitamin D,and casein phosphopeptides,1000 mg Ca/100 g)or control milk(110 mg Ca/100 g milk)supplementation,bone changes were assessed using dual-energy X-ray absorptiometry,microcomputed tomography,and bone biomechanical testing.The results revealed that FM can regulate bone metabolism and gut microbiota composition,which act on bone metabolism through pathways associated with steroid hormone biosynthesis,relaxin signaling,serotonergic synapse,and unsaturated fatty acid biosynthesis.Furthermore,FM administration significantly increased bone mineral content and density in the lumbar spine and femur,as well as femoral compressive strength,while improving femoral trabecular bone parameters and microarchitecture.Mechanistically,we found that the effects may be due to increased levels of estrogen,bone formation marker osteocalcin,and procollagen typeⅠN-propeptide,and decreased expression of the bone resorption marker C-telopiptide and tartrate-resistant acid phosphatase 5b.Overall,the findings suggest that FM is a potential alternative therapeutic option for ameliorating postmenopausal osteoporosis.

1,25-Dihydroxyvitamin D_3 modulates calcium transport in goat mammary epithelial cells in a dose- and energydependent manner

Background： Calcium is a vital mineral and an indispensable component of milk for ruminants. The regulation of transcellular calcium transport by 1,25-dihydroxyvitamin D3 （1,25-（OH）2D3, the active form of vitamin D） has been confirmed in humans and rodents, and regulators, including vitamin D receptor （VDR）, calcium binding protein Dgk （calbindin-Dgk）, plasma membrane Ca2＋-ATPase ] b （PMCAlb）, PMAC2b and Oral1, are involved in this process. However, it is still unclear whether 1,25-（OH）2D3 could stimulate calcium transport in the ruminant mammary gland. The present trials were conducted to study the effect of 1,25-（OH）2D3 supplementation and energy availability on the expression of genes and proteins related to calcium secretion in goat mammary epithelial cells. Methods： An in vitro culture method for goat secreting mammary epithelial cells was successfully established. The cells were treated with different doses of 1,25-（OH）2D3 （0, 0.1, 1.0, 10.0 and 100.0 nmol/L） for calcium transport research, followed by a 3-bromopyruvate （3-BrPA, an inhibitor of glucose metabolism） treatment to determine its dependence on glucose availability. Cell proliferation ratios, glucose consumption and enzyme activities were measured with commercial kits, and real-time quantitative polymerase chain reaction （RT-qPCR）, and western blots were used to determine the expression of genes and proteins associated with mammary calcium transport in dairy goats, respectively. Results： 1,25-（OH）2D3 promoted cell proliferation and the expression of genes involved in calcium transport in a dose-dependent manner when the concentration did not exceed 10.0 nmol/L. In addition, 100.0 nmol/L 1,25-（OH） 2D3 inhibited cell proliferation and the expression of associated genes compared with the 10.0 nmol/L treatment. The inhibition of hexokinase 2 （HK2）, a rate-limiting enzyme in glucose metabolism, decreased the expression of PMCA1 b and PMCA2b at the mRNA and protein levels as well as the transcription of Oral1, indicating that glucose avaitability was required for goat mammary calcium transport. The optimal concentration of 1,25-（OH）2D3 that facilitated calcium transport in this study was 10.0 nmol/L. Conclusions： Supplementation with 1,25-（OH）2D3 influenced cell proliferation and regulated the expression of calcium transport modulators in a dose- and energy-dependent manner, thereby highlighting the role of 1,25-（OH）2D3 as an efficacious regulatory agent that produces calcium-enriched milk in ruminants when a suitable energy status was guaranteed.

Vitamin D,calcium homeostasis and aging

Osteoporosis is characterized by low bone mass and microarchitecture deterioration of bone tissue, leading to enhanced bone fragility and consequent increase in fracture risk. Evidence is accumulating for an important role of calcium deficiency as the process of aging is associated with disturbed calcium balance. Vitamin D is the principal factor that maintains calcium homeostasis. Increasing evidence indicates that the reason for disturbed calcium balance with age is inadequate vitamin D levels in the elderly. In this article, an overview of our current understanding of vitamin D, its metabolism, and mechanisms involved in vitamin D-mediated maintenance of calcium homeostasis is presented. In addition, mechanisms involved in age-related dysregulation of 1,25（OH）2D3 action, recommended daily doses of vitamin D and calcium, and the use of vitamin D analogs for the treatment of osteoporosis （which remains controversial） are reviewed. Elucidation of the molecular pathways of vitamin D action and modifications that occur with aging will be an active area of future research that has the potential to reveal new therapeutic strategies to maintain calcium balance.

3D printing of calcium phosphate bioceramic with tailored biodegradation rate for skull bone tissue reconstruction

The bone regenerative scaffold with the tailored degradation rate matching with the growth rate of the new bone is essential for adolescent bone repair.To satisfy these requirement,we proposed bone tissue scaffolds with controlled degradation rate using osteoinductive materials(Ca-P bioceramics),which is expected to present a controllable biodegradation rate for patients who need bone regeneration.Physicochemical properties,porosity,compressive strength and degradation properties of the scaffolds were studied.3D printed Ca-P scaffold(3DS),gas foaming Ca-P scaffold(FS)and autogenous bone(AB)were used in vivo for personalized beagle skull defect repair.Histological results indicated that the 3DS was highly vascularized and well combined with surrounding tissues.FS showed obvious newly formed bone tissues.AB showed the best repair effect,but it was found that AB scaffolds were partially absorbed and degraded.This study indicated that the 3D printed Ca-P bioceramics with tailored biodegradation rate is a promising candidate for personalized skull bone tissue reconstruction.

Vitamin D and calcium are required at the time of denosumab administration during osteoporosis treatment

To evaluate the differences in outcomes of treatment with denosumab alone or denosumab combined with vitamin D and calcium supplementation in patients with primary osteoporosis. Patients were split into a denosumab monotherapy group （18 cases） or a denosumab plus vitamin D supplementation group （combination group; 23 cases）. We measured serum bone alkaline phosphatase （BAP）, tartrate-resistant acid phosphatase （TRACP）-5b and urinary N-terminal telopeptide of type-I collagen （NTX） at baseline, 1 week, as well as at I month and 2, 4, 8 and 12 months. We also measured bone mineral density （BMD） of L1-4 lumbar vertebrae （L）-BMD and bilateral hips （H）-BMD at baseline and at 4, 8 and 12 months. There was no significant difference in patient background. TRACP-5b and urinary NTX were significantly suppressed in both groups from I week to 12 months （except at 12 months for NTX）. In the combination group, TRACP-5b was significantly decreased compared with the denosumab monotherapy group at 2 and 4 months （P 〈 0.05）. BAP was significantly suppressed in both groups at 2-12 months. L-BMD significantly increased at 8 and 12 months （8.9%） in the combination group and at 4, 8 and 12 months （6.0%） in the denosumab monotherapy group, compared with those before treatment. H-BMD was significantly increased in the combination group （3.6%） compared with the denosumab group （1.2%） at 12 months （P 〈 0.05）. Compared with denosumab monotherapy, combination therapy of denosumab with vitamin D and calcium stopped the decrease in calcium caused by denosumab, inhibited bone metabolism to a greater extent, and increased BMD （especially at the hips）.

Phosphate,calcium,and vitamin D signaling,transport,and metabolism in the endometria of cyclic ewes

Background Recent evidence suggests important roles for progesterone(P4)and interferon tau in the regulation of calcium,phosphate,and vitamin D signaling in the uteri of pregnant sheep.However,the effects of P4 and estradiol(E2),with respect to the expression of their receptors PGR and ESR1,respectively,in uterine epithelia on mineral signaling during the estrous cycle has not been investigated.Estrous cycles of mature Suffolk ewes were synchronized,prostaglandin F2αwas administered,and ewes were observed for estrus(designated as Day 0)in the presence of vasectomized rams.On Days 1,9,or 14 of the estrous cycle,hysterectomies were performed.Results 25-hydroxyvitamin D was more abundant in plasma from ewes on Day 14 than Day 1(P<0.05).Expression of fibroblast growth factor receptor 2(FGFR2),a disintegrin and metalloprotease 17(ADAM17),and parathyroid hormone-related protein(PTHrP)mRNAs was greater in endometria on Day 9 compared to Days 1 and 14(P<0.01).Similarly,expression of transient receptor potential cation channel subfamily V member 6(TRPV6)mRNA was greater in endometria on Day 9 than Day 1(P<0.05).ATPase plasma membrane Ca^(2+)transporting 4(ATP2B4)and S100 calcium binding protein G(S100G)mRNA expression was greater in endometria on Day 14 than on Days 1 and 9(P<0.01).In contrast,endometrial expression of vitamin D receptor(VDR)mRNA was lower on Days 9 and 14 than Day 1(P<0.01).Expression of klotho(KL)(P<0.05)and cytochrome P450 family 24 subfamily A member 1(CYP24)(P<0.01)mRNAs was lower on Day 14 than Days 1 and 9.ADAM17,FGF23,CYP2R1,CYP27B1,KL,and VDR proteins immunolocalized to the uterine myometrium,blood vessels,and uterine luminal(LE),superficial glandular(sGE),and glandular(GE)epithelia.S100A9 protein was weakly expressed in the uterine myometrium,LE,sGE,and GE.Immunoreactivity of CYP2R1 and KL proteins in uterine LE and sGE was less on Day 1 than on Days 9 and 14.In contrast,S100G protein was expressed exclusively by GE,and immunoreactive S100G protein was less on Day 9.S100A12 protein localized to stromal cells of the uterine stratum spongiosum and blood vessels,but not by uterine epithelial cells.Conclusion Collectively,these results implicate E2,P4,and PGR in the regulation of phosphate,calcium,and vitamin D signaling in cyclic ewes.

Investigation of serum calcium and vitamin D levels in superior semicircular canal dehiscence syndrome: A case control study

Objective: It remains unknown whether calcium metabolism has any effect on the clinical presentation of superior semicircular canal dehiscence(SSCD). Our aim was to analyse the adjusted calcium and vitamin D levels in SSCD patients compared to a control group.Methods: This was a prospective case-control study performed in a tertiary referral center, university teaching hospital in the UK. It included all new patients with SSCD seen in a dedicated skull base clinic over a 5-year period(2015-2019) compared to a gender and age matched control group. The main outcome of the study was adjusted calcium and Vitamin D levels between the two groups.Results: A total of 31 SSCD patients were recruited with a matched number of control patients. The mean Vitamin D level on the SSCD group was 44.8 nmoL/l(SD: 20.8) compared to 47.5 nmoL/l(SD: 27.4) on the control group(p = 0.702). Mean Adjusted calcium level was 2.34 mmoL/l(SD: 0.7) for SSCD compared to2.41 mmoL/l(SD: 0.11) for controls(p = 0.01), being within normal limits for both the SSCD and the control group.Conclusion: Our study did not identify a link between Vitamin D levels and presence of SSCD. Normal adjusted calcium values were found in both groups. Despite that a statistically significant lower calcium level was found in the SSCD group which could indicate that suboptimal levels of calcium may affect the micro-environment of the otic capsule at the SSC region.

Vitamin D and calcium signaling in epidermal stem cells and their regeneration

Epidermal stem cells(SCs)residing in the skin play an essential role for epidermal regeneration during cutaneous wound healing.Upon injury,distinct epidermal SCs residing in the interfollicular epidermis and/or hair follicles are activated to proliferate.Subsequently,SCs and progeny migrate,differentiate and restore the epidermis.We review a role of the vitamin D signaling through its receptor of vitamin D receptor(Vdr)in these processes.Vdr conditional knockout(cKO)mouse skin experiences a delay in wound re-epithelialization under low dietary calcium conditions,stimulating our efforts to examine a cooperative role of Vdr with calcium signaling through the calcium sensing receptor in the epidermis.We review the role of vitamin D and calcium signaling in different processes essential for injury induced epidermal regeneration during cutaneous wound repair.First,we discuss their roles in self-renewal of epidermal SCs through β-catenin signaling.Then,we describe epidermal remodeling,in which SCs and progeny migrate and differentiate to restore the epidermis,events controlled by the Ecadherin mediated adherens junction signaling.Finally,we discuss the potential mechanisms for vitamin D and calcium signaling to regulate injury induced epidermal regeneration mutually and interdependently.

New insights into calcium,dairy and colon cancer

This paper is to review recent information about the relationship of calcium and dairy foods to colon cancer. The review focuses on primary prevention, discusses the potential components in dairy foods that might be anti-neoplastic, reviews the epidemiologic information and describes intervention studies demonstrating efficacy of calcium and vitamin D in reducing colorectal polyp recurrence. Since vitamin D is important in cancer prevention, pertinent data is discussed and potential mechanisms of actions presented. Calcium and vitamin D are important agents for the primary prevention of colorectal neoplasia.

Removal and recovery of titanium(Ⅳ) from leach liquor of high-sulfur bauxite using calcium alginate microspheres impregnated with di-(2-ethylhexyl) phosphoric acid

In the leaching solution of high-sulfur bauxite roasted by sulfuric acid,a high concentration of aluminum presented along with titanium and iron.The present work was to remove Ti(IV)from the leach liquor by calcium alginate microsphere sorbent material(CA-P204)based on natural alginate impregnated with di-(2-ethylhexyl)phosphoric acid(D2EHPA)to purify leaching solution.Cation exchange and chelation make major contributions to the adsorption mechanism according to Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy analysis.The results showed that Ti(IV)was successfully removed by the CA-P204 adsorbent from the Ti(IV)-Al(III)-Fe(III)ternary system with a dynamic column experiment.The removal rate of titanium was nearly 95%under optimal conditions and the maximum adsorption capacity was 66.79 mg/g at pH 1.0.Reusability of CA-P204 was evaluated over three consecutive adsorption/desorption cycles.The adsorption process was simple,low-cost,and had no waste discharge,suggesting that the CA-P204 was promising,efficient,and economical for removing Ti(IV)from high-sulfur bauxite leaching solution.

Three-dimensional printing of β-tricalcium phosphate/calcium silicate composite scaffolds for bone tissue engineering

Bioactive scaffolds with interconnected porous structures are essential for guiding cell growth and new bone formation. In this work, we successfully fabricated three-dimensional （3D） porous β-tricalcium phosphate （β-TCP）/calcium silicate （CS） composite scaffolds with different ratios by 3D printing technique and further investigated the physiochemical properties, in vitro apatite mineralization properties and degradability of porous β-TCP/CS scaffolds. Moreover, a series of in vitro cell experiments including the attachment, proliferation and osteogenic differentiation of mouse bone marrow stromal cells were conducted to testify their biological performances. The results showed that 3D printed β-TCP/CS scaffolds possessed of controllable internal porous structures and external shape. Furthermore, the introduction of CS decreased the shrinkage of scaffolds and improved the in vitro apatite formation activity and degradation rate. Meanwhile, compared with pure β- TCP scaffold, the β-TCP/CS composite scaffolds were more conducive to promote cell adhesion, spread and osteogenesis differentiation. However, when the content of CS was increased to 45%, the ions dissolution rate of the composite scaffolds was so high that leaded to the increase in pH value, which inhibited the proliferation of cells. Our results suggested that the introduction of appropriate CS into β-TCP bioceramic is an effective strategy to prepare bioactive 3D printed bioceramic scaffolds for hard tissue regeneration.

Mutant amyloid precursor protein and presenilin-1 genes effect on ischemia vulnerability via calcium homeostasis disturbance

BACKGROUND： Previous studies have demonstrated that mutant amyloid precursor protein （APP） or presenilin-1 （PS1） genes increase susceptibility to ischemic brain damage induced by middle cerebral artery occlusion. Possible mechanisms include over-production of beta-amyloid peptide （Aβ）. OBJECTIVE： Because Aβ is over-produced in the APP/PS1 double-transgenic mouse, the present study focused on mechanisms of increased ischemic damage due to mutant APP and PS1 genes by measuring oxidative stress, mitochondrial function, and calcium homeostasis. DESIGN, TIME AND SETTING： The non-randomized, controlled, in vivo and in vitro experiments were performed at the Medical Research Center, Second Clinical College, Jinan University between May and October 2008. MATERIALS： Male APP transgenic mice carrying the mutant 695swe gene and female PS1 transgenic mice carrying the mutant Leu235Pro gene were donated from the University of Hong Kong. SHSY5Y human neureblastoma cells were purchased from ATCC （Manassas, VA, USA）, and Aβ1-42 was obtained from Sigma-Aldrich （St. Louis, MO, USA）. METHODS： APP transgenic mice were mated with PS1 transgenic mice to produce APP/PS1 double-transgenic mice and wildtype littermates mice. The photothrombotic stroke model was induced in six APP/PS1 double-transgenic and 6 wildtype littermates mice. SHSY5Y human neuroblastoma cells were cultured in vitro, and were divided into 4 groups： Aβ group, cells were exposed to 5 pmol/L Aβ for 24 hours; oxygen-glucose deprivation （OGD） group, cells were exposed to OGD for 1 hour after treatment with sterile, ultra-pure water for 24 hours; OGD＋Aβ group, cells were exposed to OGD and Aβfor 1 hour after treatment with 5 pmol/L Aβ for 24 hours; sham control group： cells were exposed to sterile, ultra-pure water for 25 hours. OGD was achieved by exposing the cells to glucose-free DMEM and placing the cells in an anaerobic chamber flushed with 5% CO2 and 95% N2 （v/v） at 37 ℃ for 1 hour. MAIN OUTCOME MEASURES： TTC staining was used to measure infarct volume 7 days after photothrombotic stroke. Cell viability was evaluated using the MTT kit. Opening of the mitochondrial permeability transition pore, intracellular concentration of superoxide anion, and calcium after OGD were detected with fluorescence intensity of calcein-AM, hydroethidine, and fluo-3/AM. RESULTS： At 7 days after stroke, total infarct volume and cortical infarct volume were significantly greater in the APP/PS1 transgenic mice compared with the wildtype littermates mice （P 〈 0.01）. Aβ, OGD, and Aβ ＋ OGD significantly decreased cell viability and increased fluorescence intensity of hydroethidine and fluo-3/AM （P 〈 0.01）. Compared with the Aβ or OGD group, Aβ ＋ OGD significantly decreased cell viability （P 〈 0.01） and significantly increased fluorescence intensity of calcein-AM, hydroethidine, and fluo-3/AM （P 〈 0.01 or P 〈 0.05）. CONCLUSION： The APP/PS1 double-transgenic mice were more vulnerable to ischemia. The possible mechanisms included enhanced opening of the mitochondrial permeability transition pore, overproduction of superoxide anion due to pore opening, and disturbed calcium homeostasis induced by excess superoxide anion.

Association of Vitamin D Receptor Gene Polymorphisms with Calcium Oxalate Calculus Disease

To study the relationship between polymorphism of vitamin D receptor (VDR) allele with formation of calcium oxalate calculus and find the predisposing genes of calcium oxalate calculus, we screened out 150 patients who suffered from calcium oxalate calculus. 36 of them had idiopathic hypercalciuria according to analysis of calculus component and assay of urine calcium. The polymorphisms of VDR gene Taq1, Apa1 and Fok1 were detected using PCR-RFLP technique and the correlation were analyzed between the polymorphism and urinary calculus or between the polymorphism and hypercalciuria. The difference in each genotypic frequency of the allele of promoter Fok1 between calculus group and healthy group or between idiopathic hypercalciuria calculus group and health group was significant. The content of 24-h urine calcium of those who had genotype ff was obviously higher than that of those who have other genotypes in the same group. There was no significant difference in the polymorphism of gene Apa1 and Taq1 between each two groups. It is concluded that hypercalciuria and calcium oxalate calculus were related to the polymorphism of VDR gene's promoter Fok1 allele, but it had nothing to do with the polymorphism of gene Apa1 and Taq1. The genotype ff was a candidate heredity marker of calcium calculus disease.

Calcium and vitamin D in the serrated neoplastic pathway: Friends or foes?

Sessile serrated adenoma/polyps(known as SSA/Ps) may play an important role in the development of interval colorectal cancer(CRC). These lesions are more difficult to detect with conventional endoscopy and they may quickly turn into CRC, especially when dysplasia has developed. Therefore, primary or secondary chemoprevention may be an appealing strategy at a population level. Calcium and vitamin D have been shown in epidemiological studies to reduce the risk of CRC and conventional adenomas, but the evidence regarding their effect on SSA/Ps is controversial. In this editorial we comment on the results of a recent randomized controlled trial investigating the effect of calcium and vitamin D on the development of serrated lesions, summarizing the possible antineoplastic mechanisms of calcium and vitamin D, and discussing the differences found with previous observational reports.

Need of Calcium and Vitamin D in Patients after a Recent Fracture

Adequate calcium and vitamin D intake is advocated in guidelines of osteoporosis. However, the dosage needed to achieve an optimal calcium intake and vitamin D status is still a point of debate. Of 902 consecutive patients older than 50 years presenting at the time of fracture, 502 were evaluable for measurement of calcium intake and serum 25(OH)D concentration. We calculated the percentage of patients who needed calcium supplements to achieve intake of ?1000 mg/d and who needed cholecalciferol supplementation to achieve serum levels of ?50 nmol/l. Calcium intake ranged between 250 and 2050 mg/d and serum 25(OH)D between <10 and 130 nmol/l. A combination of calcium intake of ≥1000 mg/d and serum 25(OH)D concentration of ?50 nmol/l was present in 11% of patients. To achieve 1000 mg/d of calcium, 57% of patients needed supplementation of 500 mg/d and 12% needed 1000 mg/d. Systematic calcium supplements of 500 mg/d would achieve an intake of 1000 mg/d in 88%. To achieve serum 25(OH)D concentrations of 50 nmol/l, 41% of patients needed a supplement of 800 IU D3/d and 25% needed higher doses. Systematic supplementation of 800 IU/d would achieve 50 nmol/l in 75% of patients. Calcium intake and vitamin D status vary considerably between fracture patients. Conclusion: calcium supplements need to be titrated individually to achieve desirable levels. Most patients achieved 50 nmol/l of 25(OH)D with 800 IU D3/d. Prospective studies are needed to study how to achieve and maintain optimal serum vitamin D levels and adequate calcium intake.

Validation of a Food Frequency Questionnaire for Vitamin D and Calcium Intake in Healthy Female College Students

Objective: The objective of this study was to examine the reproducibility and validity of a Food Frequency Questionnaire (FFQ) and assess calcium and vitamin D intake in health female college students. Methods: Thirty-five healthy female students were conveniently selected to participate in the study. None of the subjects were taking any supplements. The FFQ was validated against intakes from a three-day diet food record report (FR). Results: Positive correlations were observed of daily vitamin D (r = 0.82, p –8, 9, p < 0.676 and 43 mg/d (95% CI: 20, 65, p < 0.01). Conclusions: The FFQ used in this study shows promising validation evidence to be used in the future for assessing vitamin D and calcium intakes in female students.

The Deleterious Effect of Inappropriate Calcium and Vitamin D Supplementation during Pregnancy in Women Predisposed to Calcium Oxalate Urolithiasis

Background: Gestational formation of new urolithiasis is rare yet the impact of inappropriate gestational calcium and vitamin D supplementation (Ca/DS) is underestimated. Patients and Methods: we retrospectively evaluated 75 pregnant women with history of UL, yet were stable for >2 years on dietary restrictions, for new UL after Ca/DS. Results: During the past 5 years 21 (48%) of those who had received Ca/DS had developed UL and all had high Vitamin D with hypercalcemia while the remaining 31 patients, without Ca/DS, did not have UL and maintained normal vitamin D urinary calcium without need for supplementation. Overt UL was evident by 30th weeks of gestations and most were diagnosed by ultrasonography and managed by medical expulsive therapy. Conclusion: in patients with history of UL, prudent use of Ca/DS is indicated to avoid new UL.

A sequential continuous flow synthesis and purification process of calcium dibutyryladenosine cyclophosphate

Calcium dibutyryladenosine cyclophosphate is a widely used cardiovascular drug.The traditional batch synthesis process suffers from long reaction times,tedious operations,and unstable yields.Herein,a sequential continuous flow synthesis combined with a multistage in-line purification process of calcium dibutyryladenosine cyclophosphate was developed.The acylation reaction was completed in a continuous coil reactor at 160℃ in 20 min.And the high toxic solvent pyridine was replaced by acetonitrile.Furthermore,the multistage in-line purification process was integrated into the homemade 3D circular cyclone-type micromixer chip.Combining with the membrane phase separators,the residence time of the purification step was 30 s.The isolated yield of this sequential continuous process was 92%with 99%purity.