Red blood cells (RBC) must coordinate their rate of growth and proliferation with the availability of nutrients, such as iron, but the signaling mechanisms that link the nutritional state to RBC growth are incompletely understood. We performed a screen for cell types that have high levels of signaling through mTORC1, a protein kinase that couples nutrient availability to cell growth. This screen revealed that reticulocytes show high levels of phosphorylated ribosomal protein S6, a downstream target of mTORC1. We found that mTORC1 activity in RBCs is regulated by dietary iron and that genetic activation or inhibition of mTORC1 results in macrocytic or microcytic anemia, respectively. Finally, ATP competitive mTOR inhibitors reduced RBC proliferation and were lethal after treatment with phenylhydrazine, an inducer of hemolysis. These results identify the mTORC1 pathway as a critical regulator of RBC growth and proliferation and establish that perturbations in this pathway result in anemia.DOI:http://dx.doi.org/10.7554/eLife.01913.001 To multiply and grow, cells need to create more of the molecules—such as proteins—that make up their structure. This only happens if the cell has a good supply of the nutrients used to build the proteins.Red blood cells are particularly sensitive to the supply of nutrients, especially iron, which is a key component of the hemoglobin molecules that enable the cells to transport oxygen around the body. A lack of iron can lead to a shortage of red blood cells and a condition called anemia. People with mild forms of anemia may feel tired or weak, but more severe forms of anemia can cause heart problems and even death.A protein called mTOR forms part of a protein complex that helps alert the cells of many different organisms to the presence of nutrients. mTOR can add phosphate groups to ribosomes—the molecular machines that translate molecules of mRNA to build proteins. In 2012, researchers developed a technique called Phospho-Trap that can isolate these phosphorylated ribosomes from cells. Cells with an activated mTOR complex express more mTOR protein and in turn have more ribosomes that are modified. Examining the mRNA molecules associated with these ribosomes can reveal which proteins are produced in greater amounts in these cells.Previous experiments using Phospho-Trap found the proteins that make up hemoglobin in unexpectedly high amounts in the mouse brain. Now, Knight et al.—and other researchers involved in the 2012 work—have established that the hemoglobin was not coming from the brain cells but from immature red blood cells circulating within the brain. These immature blood cells were found to have a highly active mTOR complex that promotes the production of hemoglobin and new blood cells.Using genetic techniques in mice, Knight et al. found that the mTOR complex can cause anemia if it is underactive or overactive. Underactive mTOR complexes cause a type of anemia that produces small red blood cells and is usually triggered by a lack of iron. This made sense because mTOR is known to regulate both protein production and cell size. Boosting the activity of the mTOR complex leads to a type of anemia in which the cells are much larger than normal, and which is normally associated with inadequate amounts of folate and B12 vitamins.When Knight et al. gave mice a drug that inhibits the mTOR protein, the mice developed anemia that resolved when the treatment stopped. However, mice that were given the mTOR inhibitor at the same time as a drug that destroys red blood cells, all died within days. Clinical trials are currently testing mTOR inhibitors as a possible cancer treatment; however, a common side effect of chemotherapy is that it stops new red blood cells being produced. Knight et al. suggest that the red blood cells of patients in these clinical trials must be closely monitored before deciding whether to continue the treatment further.DOI:http://dx.doi.org/10.7554/eLife.01913.002 Cells must coordinate their rate of growth and proliferation with the availability of nutrients. mTOR, a serine–threonine kinase, is one the key proteins responsible for nutrient signaling in eukaryotic cells. mTOR is activated by conditions that signal energy abundance, such as the availability of amino acids, growth factors, and intracellular ATP. Activated mTOR then phosphorylates a set of downstream targets that promote anabolic processes, such as protein translation and lipid biosynthesis, while suppressing catabolic processes such as autophagy .mTOR resides in two cellular complexes that have distinct functions and regulation . mTOR coTOR was discovered in yeast, where it functions as a nutrient sensor regulating cell growth and proliferation . This ceIn a previous report, we developed a systematic approach to identify the cell types that show high levels of mTORC1 activity in vivo . This apOur previous report focused on the use of this approach to identify markers for activated neurons in the mouse brain. However, we also noted that genes encoding the protein subunits of hemoglobin were highly enriched in our pS6 immunoprecipitates. In this study, we report that these transcripts are derived from reticulocytes, immature red blood cells (RBCs), that we find have especially high levels of mTORC1 signaling. We further use a combination of pharmacologic, genetic, and nutritional perturbations to delineate a critical role for mTORC1 signaling in RBC development and the pathogenesis of anemia, suggesting that this pathway links the availability of iron to cell growth and hemoglobin synthesis during erythropoiesis.We recently described a method for molecular profiling of activated neurons in the mouse brain . This apHba-a1 and Hbb-b1 were highly enriched transcripts in pS6 immunoprecipitates from the mouse hypothalamus and other brain regions  have high levels of pS6, and VIP mRNA is highly enriched in pS6 immunoprecipitates from the hypothalamus . HoweverHba-a1 and Hbb-b1 are most abundantly expressed in reticulocytes, immature RBCs that circulate in the blood. To test whether the Hba-a1 and Hbb-b1 transcripts originated from the circulating cells, we perfused mice with saline to remove blood from the tissue and then quantified the amount of globin mRNA remaining in hypothalamic extracts. Perfusion removed approximately 95% of Hba-a1 and Hbb-b1 mRNA from hypothalamus but had no effect on transcripts expressed in neurons or glia, such as Actb or Pomc  and RBC hemoglobin content  relative to control animals . To asseTsc1 deletion results in constitutively increased mTORC1 signaling. Previous work has shown that inducible deletion of Tsc1 in adult animals using either Mx1Cre or Gt(ROSA)26SorCre-ERT2 promotes rapid cycling of the hematopoietic stem cells (HSC) and is associated with decreased hematopoiesis due to HSC exhaustion (Mx1 and Gt(ROSA)26Sor are expressed in multiple tissues and do not provide information on the effects of cell autonomous Tsc1 deletion in the RBC lineage. We thus assessed the effect of mTORC1 activation in hematopoietic cells by using Vav1Cre mice to delete a floxed allele of Tsc1  were born at the expected ratio but were smaller than their littermates and had increased mortality in the neonatal period. However, animals that survived to 8 weeks of age had a normal appearance. Western blotting revealed that RBCs from TSC-KO mice had markedly increased levels of pS6 relative to littermate controls, confirming that these cells have constitutive activation of mTORC1 signaling  with more than threefold increase in the size of their spleens  relative to littermate controls . These cRBCs must balance the rate of globin synthesis with the availability of iron, so that free globin peptides do not aggregate in the absence of heme. An excess of free globin decreases red blood cell survival in a number of conditions including thalassemia. Thus, chronic iron deficiency triggers an adaptive response in which hemoglobin synthesis and RBC size are reduced, resulting in a microcytic hypochromic anemia in which the low heme levels are balanced by lower levels of globin synthesis. During iron deficiency the activity of heme-regulated eIF2alpha kinase (HRI), a protein kinase that is selectively expressed in RBCs is increased and acts to reduce hemoglobin synthesis. This kinase is phosphorylated and inhibited by free heme and is disinhibited with iron depletion . HRI knoHowever, since mTORC1 is inhibited by iron deficiency , this raComplete blood counts after 2 months revealed that, similar to controls, TSC-KO mice showed a reduction in the RBC volume and hemoglobin content on the low iron diet. . The iroPten mutation in HSCs using Mx1Cre mice also developed histiocytic sarcomas  offspring (>100 offspring genotyped from >20 litters). The Vav1Cre transgene is expressed beginning at E13, which corresponds to the onset of definitive erythropoiesis in the fetal liver. We therefore dissected and analyzed embryos from these crosses between E13 and E17.5 and found that embryos with the Vav1CreRptorfl/fl genotype were present at the expected ratio and morphologically normal. However, RAP-KO embryos were strikingly pale compared to their littermates  and had less total hemoglobin  . The defCre-mediated deletion of Rptor has led to the conclusion that mTORC1 may be dispensable for the regulation of hematopoiesis in adults  were present at very low levels in the spleen on day 0 but were rapidly mobilized by phenylhydrazine treatment in control animals, reaching more than 30% of total splenocytes by day 6 , black. Taken together, these data reveal a critical role for mTORC1 signaling in mediating the rapid growth and proliferation of erythroid progenitors during stress erythropoiesis. ATP competitive inhibitors block this response much more potently than rapamycin, resulting in a lethal failure to adapt to hemolytic stress. As MLN0128 and other ATP competitive mTOR inhibitors are currently being evaluated in clinical trials, these results suggest that further studies will be necessary to assess the effect of these agents under conditions that trigger stress erythropoiesis, such as hypoxia, blood loss, or drug-induced hemolysis.The mTORC1 pathway coordinates protein translation with nutrient availability in organisms ranging from yeast to humans, and the physiologic functions of mTORC1 signaling in specific mammalian tissues have begun to be elucidated. Within the hematopoietic system, an extensive literature has investigated the role of mTORC1 signaling in leukocyte development and function , includiOur attention was drawn to the potential significance of mTORC1 signaling in RBCs by screening for cell types in the adult brain that have elevated levels of pS6, a ribosomal protein that is a downstream target of mTORC1 activation . In thatWhile few studies have directly examined the role of mTORC1 signaling in RBCs, there is some evidence that dysregulation of protein translation downstream of mTORC1 may play a role in the development of anemia. The hereditary anemias, Diamond-Blackfan and 5q-syndrome, are caused by haploinsufficiency of ribosomal proteins S14 and S19, and recent data show that these anemias can be ameliorated by pharmacologic doses of L-leucine , which aA reduced activity of mTORC1 is known to reduce the size of many cell types. Consistent with this, a reduced level of mTORC activity leads to a microcytic anemia either as a result of iron deficiency or a red cell specific mutation in Raptor. As mentioned, HRI, another iron-dependent kinase, is also known to play a critical role in mediating the response to iron deficiency . FurtherA surprising finding from our study was that S6K signaling in RBCs is rapamycin resistant yet remains sensitive to ATP competitive mTOR inhibitors. Countless studies have shown that rapamycin potently blocks S6 phosphorylation in diverse cellular contexts, and the recent mTOR crystal structure has elucidated the structural origin of this sensitivity . The bioThere is considerable interest in the potential of mTOR inhibitors for the treatment of cancer. While rapamycin analogs have been used clinically for more than a decade, the first ATP competitive mTOR inhibitors are currently in the early-stage clinical trials. A key question for these drugs is how their side-effect profile will compare to widely used rapalogs. Some studies have shown that ATP-competitive mTOR inhibitors actually have weaker effects on lymphocytes than rapamycin, suggesting that these drugs may be safer and less immunosuppressive . Our datThe results reported here demonstrate how the transcriptional profiling of activated cells can reveal the cell types that respond to specific stimuli. A basic challenge for biology is to understand how complex physiologic processes emerge from the action of individual cell types, genes, and pathways. Advances in mouse genetics have made it possible to test hypotheses about the function of genes in specific tissues using methods such as Cre-lox recombination. Yet, the existence of thousands of distinct cell types in mammals suggests a need for new approaches to prioritize and guide this effort. In this paper and a companion report , we haveVav-iCre (#008610), hCD2-iCre (#008520) Tsc1fl/fl (#005680), and Rptorfl/fl (#013188) mice were obtained from Jackson laboratory. Rabbit anti-pS6 235/236 , rabbit anti-pS6 240/244 , mouse anti-total S6 , rabbit anti-p4E-BP1 Ser65 , rabbit anti-4E-BP1 rabbit anti-alpha globin , rabbit anti-neuron-specific enolase , and HRP-conjugated rabbit anti-actin  were used for immunoblotting. KU-0063794 was from Selleckchem , MLN0128 was from Active Biochem , phenylhydrazine was from Sigma , and rapamycin was from LC Labs .K562 cells were grown in RPMI supplemented with 10% dialyzed FBS. Cells were treated for 2 hr with either deferoxamine (30 μM), deferasirox (50 μM), or vehicle (0.05% DMSO). Cells were then collected by centrifugation, washed, and lysed in a 1% NP40 buffer.The hypothalamic ribosome profiling experiment that revealed enrichment of hba-a1 and hbb-b1 has previously been described . For perMice were maintained on either a standard chow diet containing 220 ppm iron (Purina 5015) or a low iron diet containing 2–6 ppm iron . Mice were additionally given subcutaneous injections of deferoxamine (150 mg/kg) or vehicle (HBSS) 5 days a week. Reticulocyte lysates for western blotting were generated by collecting blood in EDTA capillaries by cardiac puncture and diluting into HBSS + 20 mM EDTA. This blood was pelleted (3 min at 3000 rpm at 4°C), washed three times with HBSS/EDTA, and then subjected to hypotonic lysis on ice for 20 min with occasional mixing. This was then centrifuged (5 min at 3000 rpm at 4°C), and the supernatant was collected and used for western blotting.6 cells/200 μl. Cells were then stained with PE-Cy-conjugated Ter119 and PE-conjugated CD71 at a concentration of 2.5 μg/ml for 20 min at 4°C in the dark. Cells were washed and resuspended in staining buffer (1 mL) for flow cytometry. Control samples included unstained cells and single-color controls to calculate compensation. Cells were analyzed on an LSRII  flow cytometer and the data were analyzed with FlowJo software.Freshly isolated spleens were mechanically dissociated and strained through a 70-μm strainer in the presence of cold staining buffer (0.2% BSA and 5 mM glucose in PBS). Bone marrow cells were isolated from the femur using cold buffer. Fetal liver cells were extracted from E14.5 embryos, dissociated mechanically by pipetting in cold buffer. All cells were washed twice and resuspended to approximately 1.5 × 10To determine the percentage of reticulocytes in the blood, peripheral blood (5 μl) was collected in EDTA-coated capillary tubes and diluted into 1 mL of BD Retic Count/Thiazole Orange Reagent (BD #349204). Cells were stained in the dark at room temperature for 30 min. Control (unstained) samples were diluted into 1 ml buffer (0.1% BSA and 1 mM EDTA). Samples were analyzed on a LSRII (BD Biosciences) flow cytometer and the data were analyzed with FlowJo software.Complete blood counts were performed on peripheral blood collected using EDTA-coated capillaries. Peripheral blood was diluted 10-fold into cold buffer (HBSS containing 20 mM EDTA) and subjected to automated analysis using a Bayer Advia120 hematology analyzer.Animals were given daily intraperitoneal injections of either rapamycin (10 mg/kg) or twice daily injections of INK-128 (2 mg/kg) or vehicle. All drugs were formulated in a solution of 5% PEG400 + 5% Tween80 in PBS and delivered in a total volume of 200 μl. review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent ; Stuary Orkin and Kun-Liang Guan (peer reviewers).The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.This manuscript demonstrates a clear role for the mTORC1 pathway in erythropoiesis. It has been evident for a long time that a high level of protein synthesis is critical to producing hemoglobin in the quantities required in the red cell, and mTOR, which positively promotes translation, has been shown to be inhibited by iron deficiency in RBCs, suggesting a mechanism of coordination between hemoglobin translation and iron availability. However, the specific link and roles of mTORC1 and mTORC2 have not been previously investigated. The authors' findings demonstrate that abnormally high or low mTORC1 activity induces macrocytic or microcytic anemia, respectively. Furthermore, pharmacological inhibition of mTOR sensitizes mice to hemolytic stress. Moreover, this work yields some surprising findings regarding attenuated effects of rapamycin on S6K signaling  that are of chemotherapeutic relevance. These findings arose from a serendipitous finding of globin RNAs in rpS6 enriched ribosomes. This indicates the potential sensitivity of this method for identifying rare populations with enhanced mTORC1 activity. The experiments are carefully performed, the data of high quality, and the results support the conclusions.1) Does S6 phosphorylation via the mTOR pathway result in altered expression of other erythroid genes in addition to alpha and beta hemoglobin? If available, it would be interesting to see a more complete analysis of mRNAs enriched by pS6 pulldown analyzed with RNAseq. Are all rbc mRNAs similarly selected, or is there a differential effect on a specific subset of mRNAs?2) What is the cause of death in TSC-KO mice on an iron deficient diet? The degree of anemia observed in these animals does not seem to be sufficient to explain this mortality.3) Vav-Cre tissue specific raptor knockouts die perinatally, apparently as a result of a profound defect in erythropoiesis. Though selective knockout of raptor in the lymphoid compartment does not affect survival, is there evidence for a defect in any other hematopoietic compartment (e.g. the megakaryocyte or granulocyte/macrophage lineage)?4) The authors chose Vav-cre mice to reveal the conditional phenotypes, because of variegated effects and incomplete recombination with other erythrocyte or hematopoietic promoters (Results section). However, an erythropoietin receptor (EpoR)-cre line  provides quantitative and exquisite erythroid-specific excision and would have obviated the use of hCD2-Cre mice to exclude potential effects of lymphoid cells. Nonetheless, given the length of time and expense of repeating experiments with this strain, additional experiments of this kind should not be required, given the clear findings in the manuscript.5) 6) 7) Is mTORC1 activated during erythropoietic stress? This can be easily done by Western blotting of pS6 or pS6K1. 1) Does S6 phosphorylation via the mTOR pathway result in altered expression of other erythroid genes in addition to alpha and beta hemoglobin? If available, it would be interesting to see a more complete analysis of mRNAs enriched by pS6 pulldown analyzed with RNAseq. Are all rbc mRNAs similarly selected, or is there a differential effect on a specific subset of mRNAs?We now include a complete list of the genes that were enriched after the polysome precipitation. In response to the reviewers’ question, we do not see enrichment for eyrothoid catalase, carbonic anahydrase II , two cytoplasmic proteins, or sprectrin-a, spectrin-b, and ankyrin, which are membrane proteins.We assume that this is a result of the much greater abundance of hemoglobin compared to these other genes. This is noted in the revised manuscript.2) What is the cause of death in TSC-KO mice on an iron deficient diet? The degree of anemia observed in these animals does not seem to be sufficient to explain this mortality.We have performed autopsies of two of the TSC-KO mice at 6 months of age , one on a chow diet and the other on a low iron diet with an iron chelator. As now mentioned in the revised paper, pathologic analyses showed extensive extra-medullary hematopoiesis and phagocytosis of RBCs, which was greater in the iron deficient animal. At six months of age both animals also showed a widely disseminated histiocytic sarcoma in multiple sites. Consistent with this a prior report showed histiocytic sarcoma with increased Tor activity in hematopoietic cells in animals with a Pten knockout in Mx-1 cre mice. (37) This finding is discussed in the revised manuscript.3) Vav-Cre tissue specific raptor knockouts die perinatally, apparently as a result of a profound defect in erythropoiesis. Though selective knockout of raptor in the lymphoid compartment does not affect survival, is there evidence for a defect in any other hematopoietic compartment (e.g. the megakaryocyte or granulocyte/macrophage lineage)?The only change we saw was an increase in the white blood cell count of the RAP-KO and saw no changes in the basophil, platelets, monocytes or any other hematopoietic cell type in these animals. These data are now included in the revised manuscript.4) The authors chose Vav-cre mice to reveal the conditional phenotypes, because of variegated effects and incomplete recombination with other erythrocyte or hematopoietic promoters (Results section). However, an erythropoietin receptor (EpoR)-cre line  provides quantitative and exquisite erythroid-specific excision and would have obviated the use of hCD2-Cre mice to exclude potential effects of lymphoid cells. Nonetheless, given the length of time and expense of repeating experiments with this strain, additional experiments of this kind should not be required, given the clear findings in the manuscript.We appreciate the reviewers’ consideration.5) We performed these studies albeit with some difficulty owing to our inability to obtain suitable antibodies. The new data in revised 6): Does the ratio of hemoglobin/total protein change? These data should be presented. Such information will show whether mTORC1 is selectively involved in hemoglobin translation.The abundance of hemoglobin represents such a high proportion of the total protein that we were unable to obtain interpretable data addressing this point.7) Is mTORC1 activated during erythropoietic stress? This can be easily done by Western blotting of pS6 or pS6K1.The revised


Chronic migraines and tension-type headaches are debilitating diseases affecting 1.4 to 2.2% of the population with both quality of life and economic implications. To date, the pain associated with migraine and tension-type headaches has been controlled with a range of medications, with varying levels of success. In addition, the side-effect profile of these medications, as well as their potential for addiction, has been a cause for concern for both patients and physicians.Four women with long histories of migraine or frequent tension-type headache that meet the International Classification of Headache Disorders criteria for Chronic Migraine or Tension-type Headaches were given a systemic treatment(s) of autologous stromal vascular fraction or autologous ‘StroMed’ isolated from lipoaspirate. StroMed is stromal vascular fraction cells prepared by ultrasonic cavitation. Two of the four patients, both of whom are Arab women aged 40 and 36 years, ceased having migraines after 1 month, for a period of 12 to 18 months. The third patient, a Slavic woman aged 43 years, had a significant decrease in the frequency and severity of migraines with only seven migraines over 18 months. The fourth patient, an Asian woman aged 44 years, obtained a temporary decrease for a period of a month and was retreated 18 months later and has been free of migraines to date for 1 month. Pain medication was typically reduced from prescribed opioid analgesia to non-steroidal anti-inflammatory drugs and paracetamol.This case series is the first to provide evidence of the efficacy of autologous StroMed and stromal vascular fraction in the treatment of migraine and tension-type headache. The treatment of this disease by stromal vascular fraction adds a new dimension to its clinical applicability and suggests a relatively simple treatment that may help address the symptoms of the disease. Given what is known about the components of the stromal vascular fraction and how they act, the information presented in this case series may also further our knowledge of the etiology and pathophysiology of migraine and tension-type headaches. This treatment is simple, looks to be extremely effective and has been life changing for these patients. The therapeutic rationale for use of stromal vascular fraction (SVF) cells and the high content of native multi-potent adipose-derived stem/stromal cells (ASCs) within the SVF has gained recent substantial clinical interest. SVF typically has been reported to contain a heterogeneous population of cells including preadipocytes, ASCs, endothelial progenitor cells (EPCs), T-regulatory cells and M2 macrophages . The metCellular populations of SVF such as ASCs have been shown to have significant immunomodulatory capabilities, capable of homing to sites of inflammation or hypoxia and promote recovery in both animal and human models . FurtherSeveral methods for separation of SVF from lipoaspirate are available and include collagenase and ultrasonic cavitation; the SVF product obtained by ultrasonic cavitation has been termed “StroMed”. Although, SVF has not previously been used in the treatment of migraines in animal or human models, we noted that some patients that were treated with SVF for osteoarthritis reported a reduction in migraine frequency and severity. A small case series was performed on patients who had failed all other migraine treatments. Treatment of autologous SVF by a medical practitioner is a procedure permitted by the Therapeutic Goods Administration of Australia (Excluded Goods Order-2011). Informed written consent was obtained from each patient and the study protocol conformed to the principles of the 1975 Declaration of Helsinki.2+ channel blockers [Migraine is a common, episodic neurological disorder. It manifests as episodes of recurrent severe headaches, typically unilateral and throbbing, which may be associated with nausea, vomiting, photophobia or phonophobia. Approximately one-third of patients with migraine also experience ‘aura’ – transient neurological symptoms which are most frequently visual, but may involve speech and/or other senses . Both miblockers . Unfortublockers .While it is generally agreed that migraine is a heterogeneous neurological disorder, the detail of its pathophysiology is as yet unconfirmed with inheritance thought to be as high as 50%. A migraine attack can take place over hours to days and include four overlapping phases: premonitory, aura, headache and postdrome, with the mechanisms leading to an attack largely unknown. The aura and headache phase generally overlap with a large body of indirect evidence suggesting the development of headache relies on the activation and sensitization (A/S) of neurons in the trigeminal nerve. A/S of the trigeminal nociceptors, particularly those that innervate the meninges and large cranial blood vessels, may further propel A/S of second- and third-order central trigeminovascular neurons. As these neurons project to nuclei in the brain stem and forebrain, including the ‘pain matrix’, they are considered to be the underlying cause of both migraine pain and other migrainous symptoms, including loss of appetite and sleepiness . The paiThe origin of the damaging stimuli by which the meningeal receptors become activated is unclear with two central hypotheses put forward: cortical spreading depression (CSD) and neurogenic inflammation. There is increasing evidence that the brief neuronal excitation followed by prolonged inhibition of activity that spreads across the cortex, referred to as CSD, is implicated in the pathophysiology of migraine . The inhThe neurogenic inflammation hypothesis proposes that the release of inflammatory neuropeptides such as calcitonin gene-related peptide, substance P and neurokinin A from trigeminal sensory afferents leads to vasodilatation, mast cell degranulation and ensuing release of cytokines, platelet aggregation and extravasation of plasma proteins. Of interest, most C-type and slow A-delta-type rat dural afferents are activated and sensitized by an inflammatory soup (IF) of potassium, prostaglandins, 5-hydroxtryptamine, bradykinin and histamine , supportRecent theories surrounding TTH also implicate neuronal plasticity in its pathogenesis. As reviewed in Fumal and Schoenen , increasAlthough there exists sufficient evidence to reject the original vascular theory of migraine of dilation of extracranial arteries, vascular dysfunction is still thought to be implicated in migraine. Numerous studies have indicated migraineurs have increased vascular disease, although differences in endothelial function in migraineurs remain controversial. EPCs normally act as an endogenous repair mechanism, counteracting endothelial cell injury and replacing dysfunctional epithelium to maintain endothelial integrity after damage has occurred . Reduced® (lorazepam), 10mg Maxolon® (metoclopramide), 50 to 100mg pethidine and 4mg dexamethasone. The area was cleaned with 0.5% 6-chlorhexidine solution and an incision made to inject the tumescent fluid by a 16-gauge cannula, which is made up of 800mg lignocaine, 1mg adrenaline and 8.4g sodium bicarbonate per liter. The adipose tissue was removed by liposuction using 3mm cannula and collected in a sterile container with a washing solution. This solution was normal 0.9% saline with 8.4g sodium bicarbonate.Patients’ adipose tissue was harvested from subcutaneous locations and they were given 15 to 30mg morphine, 1 to 2mg AtivanThe lipoaspirate was digested with collagenase or by following Cell-Innovations  standard ultrasonic cavitation proprietary protocol under sterile conditions to obtain StroMed. Enzymatic digestion was performed adding 0.05% collagenase with adipose tissue over a period of 45 to 60 minutes. Samples were then centrifuged, and washed three times before being filtered through a 100um filter . The supernatant was removed and the pellet resuspended in phosphate buffered saline or 0.9% saline. A sample was then taken of the SVF yield and the cells counted using a hemocytometer or the Muse Cell Analyzer  with a Count and Viability dye . The total cell number was then determined.Plasma rich in growth factors (PRGF) was prepared by centrifugation of 2 × 10mL of blood with acid-citrate-dextrose formula A (450g × 10 minutes). The plasma was removed and the platelets further concentrated by centrifugation at 2000g for 10 minutes. The platelet-poor plasma was removed and the platelets resuspended in 1.5mL platelet-rich plasma (PRP). The PRP was clotted with the addition of 150uL of calcium gluconate  and after 30 minutes the PRGF was removed.Cells were administered IV by dilution in a 1L saline bag and given intravenously over 1 hour.Case 1 is a 40-year-old Arab woman who is a cigarette smoker with a lifelong history of chronic migraine and TTH, satisfying the International Classification of Headache Disorders-2 (ICHD-2) criteria. She typically experienced two migraines per day with classic aura, as well as frequent episodic TTH. She was given a single IV dose of StroMed (245 million) with PRGF added. At 3 days post-treatment she noted improvement in her migraine symptoms, and had ceased medication for her migraines at 1 month post-treatment. After 3 months post-treatment, she had experienced only one TTH. At 18 months post-treatment she experiences only occasional, minor headaches that are quickly resolved and do not keep her bedridden.Case 2 is a 36-year-old Arab woman who is a cigarette smoker with a long history of migraine, satisfying the ICHD-2 criteria for chronic migraine. She typically experienced more than 20 days of migraine per month, and occasionally required home visits from a doctor for parenteral analgesia. She was given a single IV dose of StroMed (41 million cells) and reported not experiencing any migraines after 1 month post-treatment. At 12 months post-treatment, she stated she had only experienced two strong headaches, which were managed with paracetamol and no migraines.®  to control her symptoms. She was given a single IV dose of autologous adipose tissue-derived SVF (495 million cells), digested by collagenase. After 3 days post-treatment, she noticed a reduction in her migraine symptoms and ceased using transdermal morphine; over the next 5 months she was able to control her symptoms with ibuprofen and paracetamol. After 5 months, minor headaches returned and she was re-treated at 7 months with a further IV dose of autologous adipose tissue-derived SVF (638 million cells) with PRGF. She noticed immediate improvements in her symptoms. After a period of 18 months she had only experienced seven migraines.Case 3 is a 43-year-old Slavic woman who is a non-cigarette smoker with a long history of migraines and headaches, satisfying the ICHD-2 criteria for chronic migraine. She experienced more than 20 days per month of migraine, and was using a combination of transdermal morphine patches, oxycodone hydrochloride, and up to eight tablets a day of Panadeine Forte®  per day for headache-associated pain. She was given a single IV dose of autologous adipose tissue-derived SVF (546 million cells) by enzymatic extraction with added PRGF. In the first month post-administration, she only experienced one headache; it lasted for half an hour and responded to paracetamol. At 1 month, she experienced a relapse of her migraine symptoms. She had a repeat treatment after 18 months and has been free of migraines for at least 1 month afterwards.Case 4 is a 44-year-old Asian woman who is a cigarette smoker satisfying the ICHD-2 criteria for chronic migraine, with a long history of migraines and headaches. She typically had more than 20 days per month of headaches without aura. She experienced mixed tension and migraine headaches, and was taking up to six Panadeine ForteThis case series provides an important novel direction in which StroMed or autologous SVF may be used in treatment of a disease. Given the very heterogeneous nature of headaches and the various proposed pathogeneses, all of which may be true, for the different headaches, we can postulate that there are a number of ways in which the characteristics of MSCs and other cellular components of SVF are able to modulate the frequency, duration and severity of migraine and TTH including cell depletion, immunomodulatory effects and inflammatory pathways. The mechanism (or mechanisms) of action of SVF when unraveled may help to shed further light on the pathophysiology of migraine and TTH.2 and tumor necrosis factor (TNF)-α-induced protein 6 [The inflammatory pathways described in the pathogenesis of migraine may be addressed by many of the SVF constituents including M2 macrophages and ASCs. M2 macrophages are reported to have potent anti-inflammatory and immunoregulatory abilities, in particular, the production of key anti-inflammatory cytokines such as IL-10 and lipid mediators such as lipoxins. The inhibition of proinflammatory molecules by MSCs have also been well documented in number of diseases including acute lung injuries, myocardial infarction and cerebral ischemia [rotein 6 . Indeed rotein 6 . In geneAlthough, as the pain of migraine does not eventuate and is long term this could suggest that the action of stromal cells are sufficient to correct deficiencies that predispose to migraine rather than merely being anti-inflammatory for the neurogenic inflammation. It could be presumed that the therapeutic efficacy of infused SVF relies on either or both the beneficial effects of locally engrafted SVF cells or systemic effects from secreted paracrine factors that diffuse into target tissues. The extravasations of systemically infused SVF and engraftment may also have occurred where they could exhibit both local trophic or paracrine activity in cell depleted areas.SVF is also known to contain a high number of EPCs, in particular StroMed (unpublished data), which are reduced in both number and function in migraineurs sufferers. One proposed mechanism of recurrent migraine attacks is inflammation, hypoxia, shifts in vascular diameter and blood–brain barrier disruption injury of the vascular endothelium–and that this repeated injury is thought to exhaust the available supply of EPCs . It has et al. in 2011 [Other potential mechanisms for the observed decrease in migraine frequency may be the ability of MSCs to modulate chronic pain. Guo  in 2011  demonstrWhile we have observed a treatment that appears to lead to a relatively effective long-term reduction in migraines and TTH frequency in some patients, we will continue to monitor the patients to observe the length of time the patients will remain relatively migraine free before retreatment is necessary. These original case reports on the treatment of migraines and TTH by StroMed and SVF while encouraging requires further trials and proof of efficacy and understanding of the mechanism of action. This will require significant input from practitioners who are experts in the field.Written informed consent was obtained from the patients for publication of this case report and accompanying images. Copies of the written consents are available for review by the Editor-in-Chief of this journal.Matthew Bright and Shannon Hayne have no competing interests. The use of ultrasonic cavitation to obtain SVF from adipose is a filed Patent Co-operation (PCT) patent by Cell-Innovations. The use of SVF and stem cells for the use of migraines and TTH are filed PCT patents. Ralph Bright and Wayne Thomas are Directors of Cell-Innovations. Pelin Bright is a shareholder of Cell-Innovations.MB, RB, PB and WT performed the procedures, collected the patient data, and assisted with completion of the manuscript. SH was a major contributor in writing the manuscript. All authors read and approved the final manuscript.


G protein-coupled receptors (GPCRs) are flexible integral membrane proteins involved in transmembrane signaling. Their involvement in many physiological processes makes them interesting targets for drug development. Determination of the structure of these receptors will help to design more specific drugs, however, their structural characterization has so far been hampered by the low expression and their inherent instability in detergents which made protein engineering indispensable for structural and biophysical characterization. Several approaches to stabilize the receptors in a particular conformation have led to breakthroughs in GPCR structure determination. These include truncations of the flexible regions, stabilization by antibodies and nanobodies, fusion partners, high affinity and covalently bound ligands as well as conformational stabilization by mutagenesis. In this review we focus on stabilization of GPCRs by insertion of point mutations, which lead to increased conformational and thermal stability as well as improved expression levels. We summarize existing mutagenesis strategies with different coverage of GPCR sequence space and depth of information, design and transferability of mutations and the molecular basis for stabilization. We also discuss whether mutations alter the structure and pharmacological properties of GPCRs. G protein-coupled receptors (GPCRs) are integral membrane proteins that play a central role in signaling pathways being key intermediaries between external stimuli and the intracellular signaling cascades. They consist of a single polypeptide chain with seven transmembrane domains, an extracellular N-terminus and an intracellular C-terminus. They are only found in eukaryotes with over 800 different GPCRs identified in humans so far, 400 of which are non-olfactory receptors  have been used to stabilize GPCRs, reduce flexibility and facilitate their crystallization  , directed evolution is used to screen more than a million mutations, but only selects the most stable and/or best expressing mutants. Directed evolution systems have been previously used to increase functional expression at the cell surface and stability in detergents , usually the 1% highest fluorescent cells are selected and multiple rounds of FACS sorting may be used. The cell sorting allows selection for the highest expression level at the cell surface without selecting for non-functional receptors. An increased diversity can then be achieved by rerandomization or shuffling by staggered extension process (SteP) of the existing library and further rounds of FACS sorting , allows direct selection of detergent-solubilized mutants. A receptor library is expressed in 1b-adrenergic receptor, twofold increase) could not be increased as much as very low initial expression levels  , this strongly suggests that all of these positions are involved in stabilizing interactions and are in structured environments.Structures solved after thermostabilization by alanine scanning or directed evolution are represented in Figure For the majority of crystal structures, modified receptors with little basal activity were used. Most receptors were bound to inverse agonist or antagonist and correspond to receptors in their inactive state  represents an active state of rhodopsin  complex that is, opposite to the native complex, stable in detergents , which can be used as a reference point to test the degree of protein thermostabilization. This method also allows testing of different ligands, ions, detergents on the thermostability of the receptor  using 7-diethylamino-3--4-methylcoumarin (CPM) dye   more conventional biophysical methods can be applied. Since GPCRs have large alpha-helical content, circular dichroism (CD) spectroscopy will be sensitive to protein unfolding, and has been used to characterize olfactory receptor stability . Mutation of this residue to alanine in the NTR1 alters the packing between transmembrane helix 3 (TM3), TM5 and TM6 by reducing the volume of the side chain. As a result, in the structure of the thermostabilized NTR1  that may reduce the energy barrier for its interaction with N5.58, stabilizing an active-like structure.Mutation of residues that are indirectly involved in the activation process. These residues would modulate the conformation of the residues from the first group either through direct interactions or by influencing specific inter-helical packing regions that affect the relative energy between the side chain conformations of the residues in the first group. A representative example is L3107.48M in tβ1AR . Thus, mutation of this residue to alanine in the tβ1AR might allow TM1 to come closer to the transmembrane bundle and improve the packing with TM2 and TM7.Mutation of residues responsible for local structural stability. These residues are located, for instance, in the interface between helices, or in the interface between helices and loops, and their mutation may alter the structural or dynamic properties of the local secondary structure. A representative mutation is I552A receptor using either the agonist 5′-N-ethylcarboxamidoadenosine (NECA) or the antagonist ZM241385 contain 27 and 17 mutations, respectively. Only three mutations were found to stabilize both conformations  domain 3 which contacts both TM4 and TM5. Since position 3.41 is not conserved, introduction of a mutation is less likely to disturb the overall fold of the receptor. The tryptophan at the TM3-TM4-TM5 interface is thought to stabilize the conformationally flexible TM5, and thereby increase thermostability and expression. The effect on β1-adrenergic receptor was similar to thermostabilization by addition of antagonist  and 5-HT2B (M144W) as well as the CXCR4 chemokine receptor (L125W) , W3.41 stabilizes 5-HT1B, 5-HT2B and CXCR4 by its interaction with P5.50 and the carbonyl of 5.46  is 76% and 59% identical to human β1AR (hβ1AR) and β2AR (hβ2AR) respectively , differ by three additional mutations in the JM50 construct  and with the same inverse agonist bound show very similar structures, differences are in flexible parts only which means that the structure is not changed by the stabilizing mutations  show high similarity, the biggest difference is found in the residues contacting the ligand. This is due to ligand differences—additional bulky groups—which push away ECL3 and the extracellular part of TM7 in the 3QAK structure. Additional differences, also caused by the ligand dissimilarities, occur in one turn of TM5 (C1855.46), which is pushed away by 1.6Å in the 3QAK structure. Lack of ECL2 in the 3QAK structure makes this region impossible to compare. The overall RMSD of these two structures is 0.5 Å .Receptors can additionally be stabilized by ligands, which was extensively studied for turkey β1 adrenergic receptor  crystallization, the structures of conformationally thermostabilized or evolved receptors could be determined using crystals obtained in vapor diffusion set-ups. In addition, crystal structures of receptors bound to weak ligands were only obtained with conformationally thermostabilized receptors so far  even when small ligands are used  experiments have been hindered by the limited availability of receptor and the nonspecific partitioning of ligands into detergent micelles. In target-immobilized NMR screening (TINS), thermostabilized receptors solve the problem of availability of active receptor.  of alanine mutations in scanning mutagenesis led to the stabilization of the receptors, suggesting that it is likely that many receptors could be stabilized by this approach.2A receptor, rat neurotensin receptor 1 and turkey β1-adrenergic receptor. The stabilizing mutations can also be combined with a fusion partner approach therefore expanding the repertoire of approaches which can be used to obtain receptor structure. Designed mutations facilitated crystallization of human β2-adrenergic receptor, serotonin receptors 5-HT1B and 5-HT2B as well as chemokine receptor CXCR4 and constitutively active mutations found for many diseases may facilitate structure determination of active-state GPCRs or their G-protein complexes. Addition of mutations is certainly a very powerful technique which can complement all other techniques to stabilize the receptors for structural and biophysical characterization.Stabilization by point mutations in combination with the truncation of the flexible parts of the receptor has led to the successful crystallization and structure determination of several receptors including human adenosine AThe exact molecular reasons for why a particular mutation is stabilizing are not always obvious. In this work, we proposed four categories of stabilizing mutations based on their role in the protein ranging from direct to indirect contribution to the activation mechanism to local stabilization of the structure and facilitate interactions with the lipid or detergent environment. The classification certainly furthers our understanding of the molecular basis for stabilization by mutations and may help in predicting mutations in the future.One might hope that in the future it might be possible to design thermostabilizing mutations, computationally predict them or transfer them from other receptors, or that the development of technology would reduce the effort of finding stabilizing mutations to the point that it would become a widely used approach in GPCR and other unstable dynamic proteins structural and biophysical studies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A cross sectional study was conducted to estimate risk of exposure to lead via tap water ingestion pathway for the population of Seri Kembangan (SK).By using purposive sampling method, 100 respondents who fulfilled the inclusive criteria were selected from different housing areas of SK based on geographical population distribution. Residents with filtration systems installed were excluded from the study. Questionnaires were administered to determine water consumption-related information and demographics. Two water samples (first-flushed and fully-flushed samples) were collected from kitchen tap of each household using HDPE bottles. A total of 200 water samples were collected and lead concentrations were determined using a Graphite Furnace Atomic Absorption Spectrophotometer (GFAAS).-1kg-1. None of the hazard quotient (HQ) value was found to be greater than 1.Mean lead concentration in first-flushed samples was 3.041± SD 6.967µg/L and 1.064± SD 1.103µg/L for fully-flushed samples. Of the first-flushed samples, four (4) had exceeded the National Drinking Water Quality Standard (NDWQS) lead limit value of 10µg/L while none of the fully-flushed samples had lead concentration exceeded the limit. There was a significant difference between first-flushed samples and fully-flushed samples and flushing had elicited a significant change in lead concentration in the water . It was also found that lead concentration in both first-flushed and fully flushed samples was not significantly different across nine (9) areas of Seri Kembangan (p>0.05). Serdang Jaya was found to have the highest lead concentration in first-flushed water (mean= 10.44± SD 17.83µg/L) while Taman Universiti Indah had the highest lead concentration in fully-flushed water (mean=1.45± SD 1.83µg/L). Exposure assessment found that the mean chronic daily intake (CDI) was 0.028± SD 0.034µgdayThe overall quality of water supply in SK was satisfactory because most of the parameters tested in this study were within the range of permissible limit and only a few samples had exceeded the standard values for lead and pH. Non-carcinogenic risk attributed to ingestion of lead in SK tap water was found to be negligible. Metals could exert effects that are beneficial or harmful to our human body . It can create irreversible intellectual impairment in infants and young children, even at blood lead levels below10 mg/dL .Humans are continuously exposed to lead from natural as well as anthropogenic sources . Since the introduction of unleaded petrol and total phased out of leaded petrol in 1998, the lead level in the atmosphere had declined significantly . In addiDrinking water in the house can be contaminated by corrosion of the plumbing materials used to supply the houses . SignifiIn Malaysia, the water utilities are managed and operated by both state authority and concession companies following the privatization exercise in 1987 . AlthougLead in drinking water is a major public health concern. Risk from exposure to lead contaminated water will vary, depending on the individual, the circumstances, and the amount of water consumed. For example, infants who drink formula prepared with lead-contaminated water may be at a higher risk because of the large volume of water they consume relative to their body size . If therThis cross sectional study was conducted to estimate the risk of exposure to lead via tap water ingestion pathway for the Seri Kembangan (SK) population. SK was selected as the study location because of its large population density with a total population of 66,481 people and there are many housing areas including Taman Sri Serdang, Taman Universiti Indah and Taman Sungai Besi Indah . A studyThe study population was the SK population, and the sampling unit was a resident living in SK who fulfilled the inclusion criteria, which were adult Malaysians (≥ 18 years old) who use the tap water for drinking and cooking. The exclusive criteria were tap water which is not from municipal water supply and personal water filtration systems which are installed at the house.The sample size of this study was 100 respondents. It was calculated based on a formula by Bahasa Malaysia is our national language and the language is understood by all the citizens.For each sampling unit, one person who fulfilled the inclusive criteria was invited to be the respondent. The questionnaire, which inquired about socio-demographics of occupants, health status, water supply and plumbing system in the house, was administered during the visit. The questionnaires were modified from the Baseline, Descriptive and Time-Activity Questionnaires used in NHEXAS-Arizona study  bottles were used for sampling of tap water. HDPE bottles had previously been used in many studies to detect heavy metals in water (Two water samples were collected from each house. The first water sample (first-flush sample) was the very first drops of cold water that came out from the kitchen tap after an overnight of stagnation. This sample determines whether lead accumulated in the water comes from the house's plumbing system. The second water sample (fully-flushed sample) was the water taken from the kitchen tap after flushing for 2 minutes. This sample determines if the main distribution system was the source of lead in the water.Flow diagram of sampling technique is shown in in situ during collection of HDPE bottles from respondents  equipped with the intuitive WinLab32 software, which featured the tools to analyze samples, report and archive data .The analysis of water samples using GF-AAS model Perkin Elmer AAnalyst 600 followed the standard operating procedures (SOPs) as given by the manufacturer. Other instruments such as turbidity meter, pH meter and weighing scale were operated based on the SOPs of the respective equipment as well. Following SOPs could reduce errors due to analysis. All instruments were calibrated before use. For GF-AAS, a calibration curve of approximately 1 was obtained prior to analysis of water samples so that the absorption of the lead atom became more accurate. Pre-testing of questionnaires was conducted on 10% of sample size before data collection to ensure that every question asked in questionnaires could be easily understood and thus answered by respondents. Pre-testing was conducted on 10 households in Kajang area, approximately 10kms from the study area.The following equation was used to calculate the daily exposure for ingestion route .-1 day-1),Where, CDI = Chronic daily intake (µgkg-1),C = Lead concentration in water (mgL-1),DI = Average daily intake rate of water (LDayBW = body weight (kg).Values of these three input variables, unique to each respondent, were used to estimate the respondent's individual chronic daily exposure level. The hazard quotient (HQ) was calculated to estimate non-carcinogenic risk using the following equation:fD value employed in this study was referred to Provisional Weekly Tolerable Intake (PWTI) of 25 μg of lead per kg of body weight  established by JECFA for infants and children but extended to all age groups in 1993 .A HQ value of >1 implied a significant risk level. R-2. In the study, 26% of the respondents were degree holders, 23% of them had completed primary school education level while 4% of them did not receive any formal education. From the questionnaire, it was found that 48% of the respondents had monthly income of less than RM720, 12% of them have monthly income in the range of RM720 to RM1500, 25% of them in the range of RM1501- RM2500 and 15% of them had a higher income of more than RM2500.The socio demographic data of respondents are summarized in The parameters tested in the water samples were lead concentration, temperature, pH, TDS and conductivity. The results are summarized in For other physical properties, none of the parameters had exceeded the NDWSQ and USEPA limit, except for pH value. There were 8 water samples which exceeded the NDWQS limit of pH 9. However, the mean pH value which was 8.19 and median pH value of 8.08 were, in fact, in the range of acceptable value. Conductivity and TDS of water samples were far below the NDWQS limit. According to The study showed that 78 first-flushed water samples had higher lead concentration than fully-flushed water. As shown in -1kg-1, with mean CDI value at 0.028± SD 0.034 µgday-1kg-1 and median at 0.017µgday-1kg-1. Mean HQ value was 0.008± SD 0.0097 while the median was 0.0048. None of the respondents had HQ level of more than 1. HQ values were less than 1 in the whole population indicated that the non-carcinogenic risk associated with exposure to lead in tap water via ingestion pathway was negligible.As summarized in No similar study assessing the risk of exposure to lead in tap water was found in Malaysia. However, a study from A study done by In this study, the mean pH value which was 8.19 and median pH value of 8.08 were in the range of acceptable value. The result was similar with the study by Total dissolve solids (TDS) is the term used to describe the inorganic salts and small amounts of organic matter present in solution in water while conductivity is a measure of the conductance of water to an electric current. The conductivity values indicated the high mineralization of the samples. In this study, mean TDS and conductivity values of the water were far below drinking water standards. Unlike SK, TDS and conductivity value of tap water samples in Haiti were found to be very high and exceeding the limit value . Reliable data on possible health effects associated with the ingestion of TDS in drinking water were not available .For pH, the result was consistent with the study done by In this study, temperature was not correlated with lead in tap water. The study was inconsistent with other studies which found that seasonal variations in temperature between the summer and winter months were correlated with lead concentrations, with the warmer temperatures of the summer months increasing lead concentrations  resulted in excessive scaling in water pipes, and one must consider that lead concentration in tap water was contributed mainly by the corrosion of pipes .The result was consistent with a study done in Sydney, where lead concentrations were significantly different in the three types of samples. Multiple-comparison tests showed that lead levels in first-flushed samples were significantly higher than those in either post first-flushed or fully flushed samples .The finding that first-flushed samples had higher lead concentration than fully-flushed samples was generally in agreement with other literature  and used in many studies (fD of 3.5μg/kg of body weight per day as suggested by JECFA. The HQ values were found ranged from 0 to 0.0647. HQ values were less than 1 in the whole population indicated that the non-carcinogenic risk associated with exposure to lead in tapIn order to estimate risk, hazard quotient, HQ was calculated by dividing CDI by the reference dose, RTaman Bukit Serdang but only 11 water samples were collected from Taman Serdang Jaya. It was because the number of water samples collected was determined by the geographical population distribution where the number of water samples collected was a representative of the population distribution in that area.There were three main limitations in this study. The first limitation was that only the ingestion route was taken into consideration in order to assess exposure associated with lead in drinking water. The second limitation was recall bias as the daily intake rate of water was determined not based on respondent`s water intake rate in 7 consecutive days, but by asking the respondents to recall back the daily water intake rate using standard cup of 200ml. Besides, there was a big variation of number of water samples collected from each housing area that the lead concentration in the water samples from these areas could not be compared statistically. For example, 48 water samples were collected from The overall quality of water supply in SK was found to be satisfactory because most of the parameters tested in this study were within the range of permissible limit and only a few samples had exceeded the standard values for lead concentration and pH values. The study also found that first-flushed samples had higher lead concentration than fully-flushed samples and flushing would significantly reduce the levels of lead. In conclusion, none of the respondents was found to have HQ level more than 1, indicated that the non-carcinogenic risk of exposure to lead in tap water among residents of SK was negligible. Although the risk is negligible, we have to realize that the maximum permissible levels of elements established by the WHO and national legislations are generally based on toxicological studies on animals. Little is known in relation to the long-term human exposure to harmful elements in water. More research and surveillance studies should be conducted to determine exposure on the Malaysian population with regards to trace metals, especially lead in drinking water and to know the long-term effect of these elements to human health.


Findings from epidemiological and observational studies have indicated that diets high in omega-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) may reduce the risk of cognitive decline and Alzheimer’s disease (AD). To determine if increasing intake of DHA and EPA through supplementation is beneficial to cognition and mood in individuals with cognitive impairment no dementia (CIND) or Alzheimer’s disease (AD) a four month, randomised, double-blind, placebo controlled study was conducted. Fifty-seven participants with CIND and nineteen with AD were randomised to receive either omega-3 PUFAs (600 mg EPA and 625 mg DHA per day) or placebo (olive oil) over a four month period. Elevating depleted levels of EPA and DHA through supplementation in individuals with CIND or AD was found to have negligible beneficial effect on their cognition or mood. These findings confirm an overall negligible benefit of omega-3 PUFA supplementation for those with cognitive impairment and dementia. More intervention studies need to be undertaken with longer study durations and larger sample sizes. It may prove fruitful to examine effects of different doses as well as effects in other dementia subtypes. There is increasing scientific evidence from epidemiology and animal studies linking omega-3 polyunsaturated fatty acid (PUFA) intake with physical health ,2,3,4,5.Few intervention studies have investigated whether omega-3 PUFAs are beneficial to cognition in either healthy older adults or in older adults with a clinical diagnosis of dementia, specifically Alzheimer’s disease (AD) and, even less explored, cognitive impairment no dementia (CIND) . The few-attendance were illness or hospital admission. Details of the demographic characteristics of the participants receiving omega-3 supplements or placebo are shown in Seventy six individuals (57 CIND and 19 AD) were recruited and there were no dropouts. However, four participants  did not complete all visits. Reasons for nonThere were no differences in age, years of education and pre-morbid (NART) IQ  between Compliance was measured by capsule count and plasma PC fatty acid concentrations. On average both groups consumed 75.6% (SD 0.8) of their capsules.p < 0.00001 vs. baseline, respectively) (Plasma PC EPA and DHA did not change in the placebo group . Howeverctively) . There wctively) . Both EPctively) . The meap > 0.158) These scores were subsequently unaffected by omega-3 PUFAs: there was no significant effect of treatment or significant treatment by month interaction for any of the outcomes  who did not attend their month 4 appointment. In these cases, the missing data were replaced using last observation carried forward. No differences in baseline performance scores for any of the six primary outcome measures between the omega-3 PUFA and placebo groups were found . However, this reflects an absolute increase in EPA content of 2% of fatty acids (from ~1.5% of fatty acids at baseline to ~3.5% after supplementation) and in DHA content of 1.5% of fatty acids (from ~4% of fatty acids at baseline to ~5.5% of fatty acids after supplementation). Nevertheless, EPA seems to be better incorporated than DHA into plasma PC. This conclusion is consistent with previous reports. Provision of 3 g of EPA and 3.54 g of DHA per week for one year in healthy volunteers resulted in a mean 85% relative increase in EPA content of plasma PC from baseline and a mean 44% relative increase in DHA content . This grFrom the results of this study it is possible to surmise that omega-3 fatty acid supplements may have a negligible benefit and thus no clinical usefulness in individuals with AD and may also not be beneficial in primary prevention and thus not useful in people with CIND. However, there are other theoretical reasons why benefits were not seen in these particular populations. Firstly, in an unpublished poster of a research project undertaken in AD mouse models the authors argued that it was possible that a study duration of four months does not give the omega-3 fatty acids enough time to show effects on amyloid accumulation, inflammatory mediators and protein expression . The autIt may also be important to consider that AD is a progressive disorder where cognition is always deteriorating. Therefore, any improvements experienced as a result of intervention may be continuously overcome by the disease progression. A similar view has been argued that omega-3 fatty acids may have a role in primary prevention of AD but not in treatment of the disease once it has manifested . It has Finally, there is a large body of evidence to suggest that the appropriate omega-3 to omega-6 ratio may be the important factor to normal functioning. Even though improvements to the ratio of omega-3 fatty acids to omega-6 fatty acids through supplementation were made in this study , this diIn addition to other theoretical explanations for the present findings there are some limitations evident in this study that need consideration. Firstly, as mentioned above, the sample size was small particularly for the AD group. We acknowledge that this is a limitation. However, we believe that the findings from this study still contribute to the body of evidence  trying to determine the benefits of omega-3 fatty acids in cognition and mood in demented and non-demented individuals. In addition, there were a few measures used in this study that may have lacked the sensitivity necessary for detecting change. This was particularly true for the mood measure (BASDEC) and the BADLS. Both were subject to ceiling affects. In retrospect, the BASDEC may have been inappropriate to use with individuals with AD who lack awareness and the BADLS was inappropriate to ascertain real change in functioning in individuals with CIND. It may have been more appropriate for the study partners of the CIND participants to complete the Alzheimer’s disease Cooperative Study scale for activities of daily living in MCI (ADCS-MCI-ADL) , which aIn summary, findings from this study suggest that even though omega-3 fatty acid supplementation elevates plasma concentrations of DHA and EPA in individuals with CIND and AD, this does not benefit cognition and mood in these populations. In future, studies carried out in this area should use larger sample sizes and longer study durations. Particular attention should also be paid to dose . It is also imperative that cognitive and mood measures are chosen with caution to overcome issues surrounding sensitivity to change and ceiling and floor affects. In conclusion, at the current time it remains unclear whether the treatment of cognitive impairment and AD with omega-3 fatty acid supplements is beneficial and therefore clinically worthwhile.The study was conducted between February 2006 and March 2008. In total, 76 participants  were recruited. The patients with AD were recruited though specialist memory clinics in Bristol, UK. The CIND participants were recruited from the community via local newspaper and radio adverts. All research procedures adhered to the Helsinki Declaration (1975) and good clinical practice. Ethics approval for the study was obtained from Frenchay Research Ethics Committee, North Bristol NHS Trust on 31 October 2005 (05/Q2007/69). Informed consent was obtained from all participants as well as carer assent where necessary. AD: (1) Diagnosis based on the NINCDS-ADRDA (1984) criteria ; (2) HavCIND: (1) Self report or informant-based report of cognitive change; (2) Objective cognitive deficits falling below age-appropriate levels (Z score of −1.5); (3) An absence of dementia; (4) Retain ability to perform activities of daily living; (5) Other possible neurological or psychiatric conditions excluded. The research criteria were based on a combination of criteria for Age-associated Cognitive Decline (AACD) ,33 criteetc.) or neurological conditions ; (3) They had a current drug or alcohol addiction; (4) They had disabilities, disorders or specialist dietary needs that would prevent all of the study’s requirements from being undertaken; (5) They were already taking omega-3 PUFA supplements; (6) They did not have a relative or friend that could accompany them to research visits and who lived with them or visited them at least 2 times a week.Potential participants were excluded if (1) Another dementia was suspected, e.g., Dementia with Lewy Bodies (DLB); (2) They had a history of other psychiatric , schizophrenia, A consecutive sampling design was used to recruit all participants to the two groups (AD and CIND). This allowed the study findings to be representative of the population from which it was drawn.Nineteen patients  were recruited having already had a full medical and neuropsychological examination at one of the memory clinics based in Bristol, UK. In support of their diagnosis based on the NINCDS-ADRDA (1984) criteria , and as Fifty-seven older adults  with a worsening memory or thinking ability were recruited from the general public through a University of Bristol press release, posters in GP surgeries as well as newspaper adverts. All 57 CIND participants were screened for suitability by the researchers using a medical questionnaire developed and based around relevant medical details obtained during diagnostic consultations at the specialist memory clinics. None of the CIND participants had previously been given a diagnosis of dementia.Potential participants or their carer were sent two information sheets about the study, one information sheet for them and the second for their study partner . After one week, they were contacted by phone to determine their interest in taking part. Participants were invited to attend either 3 (AD group) or 4 (CIND group) research appointments. The first was a screening appointment and the second, was a baseline appointment; both appointments lasted approximately one hour. The AD participants were not required to attend a screening appointment as all relevant screening documentation was gathered from their medical records once they had been approached to take part in the study by their treatment team. The last two appointments, which measured performance change from baseline took approximately one and a half to two hours each. The participants took part in the study for 4 months.Participants were randomised to receive two capsules containing a total of 625 mg DHA and 600 mg EPA, or two placebo capsules containing olive oil each day for four months. Each capsule (EPA + DHA and placebo) also contained 10 mg mixed tocopherols. Participants were requested to take the capsules at a convenient time of day for them, preferably with food. This information, as well as their participant ID, visit number and date of visit was also printed on a label attached to the outside of each container. Participants and their carers and the researchers conducting the cognitive assessments and the plasma fatty-acid assays were blind to the identity of the treatments (EPA + DHA or placebo).v/v). PC was isolated by solid phase extraction. Fatty acid methyl esters were prepared by incubation of purified plasma PC with methanol in sulphuric acid and were separated using a Hewlett Packard 6890 gas chromatograph  equipped with a 30 m × 0.25 µm × 0.25 mm BPX-70 fused silica capillary column  and flame ionisation detection. The concentrations of individual fatty acids in plasma PC were determined by measurement of the peak area using ChemStation software (Agilent) and each fatty acid was expressed as a proportion of the total [At baseline, month 1 and month 4 the following examinations were completed: fasting bloods, assessment of functioning in daily living, cognition, mood and visual attention. Blood samples were taken to assess compliance with the intervention. This, the fatty acid profile of plasma phosphatidylcholine (PC) was analysed by gas chromatography. Total lipid was extracted from plasma using chloroform-methanol  method) and per-protocol. Very few differences in results were observed between the two analyses, therefore the data presented here represent the LOCF method unless otherwise stated.Longitudinal changes in the outcome variables were analysed using repeated measures analysis of variance. The data are shown as means, standard deviations, and 95% confidence intervals.This study abided by all the appropriate ethical guidelines outlined by the Declaration of Helsinki and was undertaken according to good clinical practice. Informed consent was obtained from all participants and assent obtained from their study partner. This study was approved by the Local Ethics Committee of North Bristol NHS Trust, Bristol UK and approved by the Research and Development departments of Avon and Wiltshire Mental Health Partnership Trust (AWP) and Bristol Primary Care Trust. The sponsor of the study was the University of Bristol.


To derive and validate a clinical prediction model to estimate the risk of liver disease diagnosis following liver function tests (LFTs) and to convert the model to a simplified scoring tool for use in primary care.Population-based observational cohort study of patients in Tayside Scotland identified as having their LFTs performed in primary care and followed for 2 years. Biochemistry data were linked to secondary care, prescriptions and mortality data to ascertain baseline characteristics of the derivation cohort. A separate validation cohort was obtained from 19 general practices across the rest of Scotland to externally validate the final model.Primary care, Tayside, Scotland.Derivation cohort: LFT results from 310 511 patients. After exclusions , the derivation cohort contained 95 977 patients with no clinically apparent liver condition. Validation cohort: after exclusions, this cohort contained 11 653 patients.Diagnosis of a liver condition within 2 years.From the derivation cohort (n=95 977), 481 (0.5%) were diagnosed with a liver disease. The model showed good discrimination (C-statistic=0.78). Given the low prevalence of liver disease, the negative predictive values were high. Positive predictive values were low but rose to 20–30% for high-risk patients.This study successfully developed and validated a clinical prediction model and subsequent scoring tool, the Algorithm for Liver Function Investigations (ALFI), which can predict liver disease risk in patients with no clinically obvious liver disease who had their initial LFTs taken in primary care. ALFI can help general practitioners focus referral on a small subset of patients with higher predicted risk while continuing to address modifiable liver disease risk factors in those at lower risk. Our study has successfully developed and validated the first risk prediction model and subsequent user-friendly scoring tool, the Algorithm for Liver Function Investigations, for liver condition diagnosis in patients with no obvious liver condition at the time of incident liver function testing in primary care.This model can be used to facilitate general practitioner decision-making about whom to refer to secondary care.γ-Glutamyltransferase (GGT) was found to be a powerful predictor of liver disease, over and above alkaline phosphatase and transaminase. Since some laboratories do not routinely test for GGT, its use should be re-evaluated.The observational data lacked some potential predictors of liver disease, for example, alcohol intake and body mass index. However, other available predictors such as liver function tests and deprivation may act as surrogate markers for such factors.General practitioners (GPs) commonly request liver function tests (LFTs), but the results rarely identify cases of liver disease.The primary care management of these types of patients might be facilitated through the use of a clinical prediction model which estimates the risk of a specific outcome adjusted for patient characteristics.11To derive a clinical prediction model using a cohort of patients from Tayside that would estimate the risk of a liver disease over the 2 years following LFTs taken in primary care;To validate this model geographically and temporally using a different cohort of patients from 19 general practices in Scotland;To convert this model into a user-friendly clinical scoring tool that will allow the GP to easily calculate the risk of liver disease diagnosis at 6 months and 2 years.This population-based historical cohort study followed up two separate cohorts of patients living in Scotland with no clinically recognised liver disease who initially had LFTs undertaken in primary care.Separate populations were used to develop the prognostic model (derivation cohort) and then validate it .The study population was initially derived from a laboratory database which contains all electronically available LFT results from patients within Tayside during the 15-year period from January 1989 to December 2003 linked to hospital admission and GP prescribing data.Bilirubin greater than 35 µmol/L at baseline, suggesting jaundice.Diagnosis of ascites, encephalopathy, varices or portal hypertension within 6 weeks of their first LFTs.History of any liver disease before baseline.Patients aged 16 and above with no obvious or reported clinical signs of a liver disease on presentation to their GP, and with at least two different LFTs requested from the index appointment between 1989 and 2003, were eligible for inclusion. The following exclusion criteria ensured that the study population of patients had no clinically recognised liver disease at presentation in primary care:Box 1Death registry from the General Register Office for Scotland including date and causes of death.Deprivation score was assessed by the Carstairs method.Tayside Community Health Index (CHI) files: Contains CHI number,Regional biochemistry database: All liver function tests (LFTs) came from the largest hospital laboratory in the Tayside region, Ninewells. Two smaller hospital laboratories contributed electronic LFT results later, one from 1998 and the other from 2003.Scottish Morbidity Records 1 (SMR01) database: Patient-specific morbidity data are routinely collected in Scotland, and collectively these are known as SMR. SMR01 contains hospital admissions and procedures for all hospitals in Scotland and is one of the oldest and most complete national health datasets in the world.16SMR04 database: Holds all inpatient and day case episodes for mental health specialties, which identified patients admitted/discharged with diagnoses of alcohol dependency or drug misuse.Pharmacist dispensed prescription database: A community-based database that holds encashed prescriptions in Tayside,Epidemiology of Liver Disease In Tayside (ELDIT) database: Contains all Tayside patients with liver diseases who have been ‘electronically’ diagnosed using record linkage of biomedical datasets, including virology and immunology.The databases relevant to this study are described briefly in Written informed consent from patients was waived by the Tayside Committee for Medical Research Ethics because the databases were anonymised so that no patient identifiable information was accessible. The databases used in this study see  covered As well as the five analysed LFTs, baseline characteristics included age, gender, deprivation,The primary outcome for this study was liver disease diagnosis during the 2 years following the initial analysis of LFTs in primary care. Liver diseases were identified from the Epidemiology of Liver Disease in Tayside (ELDIT) database , describSurvival analysis was conducted using parametric regression models to estimate the risk of liver disease within 2 years. The starting point was taken as the date of the initial LFT test and the endpoint for each patient was whichever one of the following events came first: end of follow-up , death, end of study period , date of emigration or liver disease diagnosed. All patients whose endpoint was not liver disease diagnosis were censored in the model.The Weibull regression model was used for model building using potential predictors at baseline. A manual stepwise technique was used to arrive at a model that contained only significant predictive characteristics. The functional form of continuous characteristics  was assessed by plotting each against the Martingale residuals and, subsequently, appropriate transformations were carried out where necessary. Clinically important two-way predictor interactions were also investigated and were included in the final model if they were statistically significant. The significant predictors from the Weibull model were then refitted to different parametric model distributions including the generalised γ, log-logistic, log-normal and exponential distributions to find the one that fitted best.The problem of missing data occurs in almost all retrospective studies using routine health databases. The simplest way of dealing with this is to use only cases with complete data in the analysis. However, this leads to the loss of potentially valuable information from the incomplete cases (and hence loss of power), and introduces bias, especially if there are systematic differences between the complete and incomplete cases. Therefore, the findings from an analysis using only the complete cases may not be a true reflection on what would be found if all the cases were analysed. To analyse only the complete data assumes that the missing data are missing completely at random (MCAR), which is unlikely.The integrated discrimination index (IDI) was used to measure the improvement in the final model for each individual covariate.23A separate cohort of patients was obtained from the Primary Care Clinical Informatics Unit (PCCIU), University of Aberdeen,The parameter estimates of the final model from the derivation cohort were applied to the validation cohort to assess its predictive ability. The C-statistic was used as a measure of discrimination.Decision curve analysis was used to determine a range of threshold predicted probabilities of liver disease where the primary care decision to refer a patient to secondary care would be better than assuming all patients are disease free  and assuming that all patients have liver disease .A point-based scoring tool was created from a simpler version of the final model for potential use in primary care.Model development and validation were performed using SAS (V.9.3)  and the decision curve analysis was performed using the stdca function in R V.3.0.1.30–32Before applying the exclusion criteria, LFTs were extracted for 310 511 patients. After excluding patients under 16 years of age, non-Tayside residents and those whose initial LFTs were measured in secondary care, 99 165 patients remained. After excluding those with clinically recognised liver disease at baseline, the derivation cohort contained 95 977 patients with incident initial LFTs taken in primary care and with no obvious liver disease. There were more female patients (57.9%) than male patients (42.1%), and the median (IQR) age was 54.6 (39.2–68.8) years . The mosOnly 8388 (8.7%) patients had all five LFTs. The percentage of complete data for each LFT was as follows: ALP (99.2%), albumin (99.2%), bilirubin (93.6%), transaminases (76.5%) and GGT (10.9%). There were more male patients with complete data  than female patients (54.6% vs 45.4%) compared with the incomplete data group (40.9% vs 59.1%). The group with complete data was also more deprived and contained more alcohol-dependent patients than the incomplete data group (see online supplementary appendix 2). When those without GGT measurements were compared with those with GGT measurements, the results were similar to the above since those without GGT measurements comprised the majority of the incomplete data group. The group of patients without transaminase measurements contained a higher proportion of female patients (63.5% vs 36.5%) than the group with transaminase measurements (56.2% vs 43.8%) and had a higher median ALP result (86 vs 73 U/L).A total of 481 patients (0.5%) were diagnosed with a liver disease during the 2-year follow-up period. Of these, 339 (70.5%) had at least one abnormal LFT (out of 22 673). Cirrhosis was the most frequently diagnosed condition , and in 19 of these patients the cause was recorded as alcohol-related liver disease. Primary biliary cirrhosis was diagnosed in 73 patients but only as definite in 13 . The next most frequent diagnoses were hepatitis C (n=68), alcoholic-related liver disease (n=68), hepatitis B (n=40), hepatocellular carcinoma (n=34), autoimmune hepatitis (n=29) and fatty liver disease (n=28).The log-normal regression model had the lowest AIC and therefore was proven to have the best fit to the survival distribution. The following baseline characteristics were significant predictors of liver disease diagnosis: increasing GGT, decreasing albumin, alcohol dependency, being female, increasing ALP, living in a deprived area and younger age . There wAfter exclusions, the external cohort contained 11 653 patients with incident initial LFTs taken in primary care. A total of 57 patients (0.5%) were diagnosed with a liver disease within 2 years. The proportion of male and female patients was reasonably similar to the derivation cohort , but the external cohort was older, more deprived and had a greater proportion of comorbidities and medications . HoweverThe C-statistic for the final model parameter estimates applied to the external cohort was 0.78 (95% CI 0.72 to 0.84). The calibration plot (see online supplementary appendix 3) showed some visible evidence of overfitting for the highest risk group; however, the calibration slope showed no significant deviation from one (slope=0.871 (95% CI 0.696 to 1.047); p=0.15).The sensitivity, specificity, PPV and NPV for different cut-offs of predicted risk of liver disease diagnosis, along with the receiver operating characteristic curves for the final model applied to the derivation and external validation cohorts, are displayed in online supplementary appendix 4. A cut-off greater than or equal to the 75th centile of the risk score  had sensitivity and specificity of approximately 75% each. The NPV was very high at almost 100% for all cut-offs. Although PPV was very low, it rose to over 10% for predicted risks over 5% and reached 20–30% for higher risks (over 20%). These performance measures were similar for derivation and validation cohorts. From the decision curve in The final model and how to calculate the risk of liver disease directly from it is presented in online supplementary appendix 5. A user-friendly paper-based scoring tool was developed from a slightly simpler version of the final model with the albumin and transaminase interaction term removed . The C-sThe correlation between the predicted probabilities from the scoring tool probabilities and the predicted probabilities from the final model when applied to the validation cohort was 0.91. Online supplementary appendix 6 shows the scatterplot of the relationship between the two.A user-friendly clinical scoring tool has been developed that can estimate the risk of liver disease diagnosis at 6 months and 2 years in patients who had their initial LFTs measured in primary care. The tool is based on a clinical prediction model which was derived and validated using large population databases. This model adds morbidity and a longer duration to a previous algorithm that predicts 1-year mortality as part of our Algorithm for Liver Function Investigations (ALFI).33This is the first large-scale population-based analysis of LFTs with complete determination of liver disease outcome. ALFI has been derived from unselected ‘real-world’ observations in a geographically defined population: an approach recommended by the National Institutes of Health.The aim of the study was to combine known significant predictors of liver disease into one source of prognostic information that could provide the GP with an individualised estimate of the predicted risk of liver disease at the point where the patient has had their initial LFTs measured. If, at that point, the patient has a high risk of liver disease based on this model, then the GP can also use other contextual information and their own clinical judgement to make a decision as to whether to monitor or refer the patient to a liver clinic according to national or local protocols.The predicted probabilities from the scoring tool correlated well with those from the model. However, it is evident from the scatterplot in online supplementary appendix 6 that for high predicted probabilities from the final model the tool underestimates the predicted probability. This is because many of these higher probabilities come from patients who have very highly abnormal LFTs. The tool assigns such patients the points based on the highest cut-off in the tool. For example, if a patient has an ALP measure of 825, the tool assigns the same points as someone with an ALP of 181  since ≥180 U/L is the highest cut-off in the tool. It would be inefficient to expand the number of groups for the LFTs to include such large values as the score card would be huge. In such circumstances, the GP would automatically refer without the need for a scoring tool. Also, it is advisable to reduce the influence of extreme values when creating such a scoring tool by using the 99th centile value of measurements as the largest possible value when creating the cut-offs.29The validation cohort came from 19 general practices across nine different regions of Scotland during a different time period than the derivation cohort. Good discrimination and calibration demonstrated that ALFI is transportable to different geographical and temporal populations. The C-statistic for discriminatory ability was 0.78, which is comparable to that found in the validation of the Framingham equation on different cohorts ranging from 0.63 to 0.83.GGT and, to a lesser extent, transaminase were either missing or not tested for a large proportion of patients in the derivation cohort, but much less so in the validation cohort. From the database, it was not possible to determine the exact reason for the non-presence of these two LFTs. However, in Tayside, the laboratories do not routinely include GGT with the other four LFT results unless specifically requested by the primary care physician. The demographics of the patients with complete data  suggested that some primary care physicians may have requested GGT where they suspected substance abuse.36At a cut-off of 0.6% (the 75th centile) for liver disease risk, sensitivity and specificity were similar with values of 73.7% and 75.3% in the validation cohort. However, the PPV for low cut-offs such as this was poor. The specificity at a cut-off of 1.2% risk is greater than 90% and rises to 100% at the 10% cut-off. The NPV of the model is very high even for reasonably small cut-offs of risk, meaning that the model is good at ruling out risk of liver disease within 2 years. However, this is not surprising, given the low prevalence of liver disease in the sample of 0.5%.The decision curve analysis showed that the model was of value for threshold probabilities between 0.5% and 7.5%. To determine whether the model is of clinical value, we should consider the possible range of threshold probabilities of liver disease at which GPs would decide to refer the patient to secondary care. The decision to refer is not a particularly risky intervention for the patient. Initially, it will lead to further blood tests and possible ultrasound in secondary care. The outcome of these results will determine whether the patient requires a liver biopsy which has some risk attached to it. However, if the patient gets to that stage, then the clinician must have a high suspicion of liver disease, meaning that the decision to refer was correct. If the GP does not refer and the patient does have or develop liver disease, then the risk to the patient  depends on the type and stage of the disease. Therefore, the GP may think that the intervention of referral is much less risky than not referring and decide that a probability threshold of, say, 5% is enough to refer. For thresholds between 5% and 7.5%, the model is also reasonably accurate. Therefore, ALFI could help GPs focus referral on a small subset of patients with higher predicted risk and reasonable PPVs while taking clinical factors that were not included in the model (but which may have improved prediction) into account. However, where the risk is lower, the long time frame of the development of many liver diseases means that GPs can continue to address modifiable liver disease risk factors .The next step is to evaluate ALFI as a complex intervention while taking into account the cost associated with referral of false positives.38GGT should be considered just as important as other LFTs in the prediction of liver disease since only albumin explained more variation in the model. Proper use of ALFI in practice will depend on GGT being a routine part of the LFT panel. Local health economies will have to decide whether the additional cost of an extra test in the panel is worth it in terms of GGT's value in improving the ability to predict those patients who are at high (or low) risk and, subsequently, change referral practice. Before such decisions are made, further research is necessary involving cost-effectiveness analysis.In summary, this study has developed and externally validated the ALFI model for prediction of liver disease diagnosis in patients with no clinically obvious liver disease having their LFTs taken within primary care. From this model, a simple scoring tool was developed to facilitate GP decision-making with regard to retesting or referring their patients. GP decisions regarding referral of patients to secondary care should be based on probability thresholds where benefits outweigh harm.