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    Issue Date: August 2023

    Milk Oligosaccharides: Every Mammal Has a Different Sugar Mama

    • The creation of a milk oligosaccharide database (MilkOligoDB) from 77 species of mammals allows researchers to easily make direct comparisons between and among mammalian species.
    • MilkOligoDB can be used to understand the biological significance of human milk oligosaccharides and potentially to identify milk sources for infant formula oligosaccharide supplementation.
    • Initial comparisons using MilkOligoDB suggest milk oligosaccharide profiles evolved to meet needs of both mothers and infants, with similarities in oligosaccharide profiles across groups of mammals that have similar reproductive strategies, such as the production of immature offspring or long lactation periods. 

    The scientific literature on mammalian milk oligosaccharides just got a glow up thanks to a team of milk and food scientists from University of California—Davis and the U.S. Department of Agriculture. MilkOligoDB [1] brings together 50 years of research results on milk oligosaccharides from nearly 80 mammal species. The team standardized the available data on milk oligosaccharides and transformed them into a searchable and open access database—no easy feat considering the sheer number of oligosaccharides in mammalian milks and the heterogeneity of data reporting on their identification and concentration. 

    Necessity drives invention, so it shouldn’t be surprising that the team that created MilkOligoDB includes researchers interested in understanding how other mammals’ milks compare with human milk in types and quantities of oligosaccharides. Of particular interest to the team was the identification of nonhuman milks that could be a source of oligosaccharides for supplementing human infant formula. 

    Oligosaccharides are complex carbohydrates made from between three and twenty single sugar molecules. Unlike the milk sugar lactose, which is digested in the small intestine and provides energy for infant growth and development, milk oligosaccharides are generally intact when they reach the large intestine. Once they get there, however, they get to work by providing food for beneficial bacteria that line the infant gut. This well-fed probiotic gut population increases and their consumption of oligosaccharides produces short-chain fatty acids, which have anti-inflammatory properties [1-3]. Some human milk oligosaccharides (HMOs) are also doppelgängers for sugars found on the surface of gut epithelial cells. In an act of biological trickery, they act as decoy receptors for pathogens; pathogens bind to oligosaccharides instead of gut cells and are removed from the gut instead of causing infection [1-4]. HMOs may even support brain development as a source of sialic acid [1-3]

    It is these prebiotic and anti-microbial actions of HMOs that make them highly desirable ingredients for infant formula. But the very thing that makes them so effective in keeping infants healthy—a high degree of structural diversity—makes them difficult to replicate. For reference, researchers have identified over 300 different oligosaccharides across human mothers, each unique structure associated with a potentially unique function [5]. Currently, some formulas do include synthetic oligosaccharides, but they are difficult and expensive to produce and may have functional differences from HMOs [1]. Researchers have also isolated HMOs from human milk for formula supplementation, but this process is also difficult and costly, and only feasible with smaller, less complex oligosaccharides [1]. 

    With these roadblocks, researchers turned to nonhuman mammal milks as potential sources of biologically active carbohydrates. MilkOligoDB was specifically designed to facilitate species comparisons that were not possible with the scattered and heterogenous data from the literature. Pulling from a total of 113 different publications and representing 77 mammal species, the database has 3,193 entries for 783 unique oligosaccharides [1]. But users do not have to go through entry by entry to compare profiles. 

    If a researcher is interested in comparing milk oligosaccharide profiles between two different mammal species (or even a group of species), the database transforms their unique and shared carbohydrates into a concept map that clearly labels and color codes oligosaccharides that are shared, and those that are unique to a species [1]. Did the concept maps from MilkOligoDB reveal any mammal species that produce milks with oligosaccharides that match the structural features in human milk? 

    The short answer is no. But the longer, and infinitely more interesting answer is that all mammals appear to have unique oligosaccharide profiles that reflect species’ adaptations, including their reproductive strategies and pathogen exposure. Because of this, you wouldn’t expect to find a perfect replicate for human milk but rather groups of species with similar oligosaccharide patterns due to similar life history strategies or similar ecological niches. 

    In a perfect example of “one of these things is not like the other,” the team found that milk from chimpanzees, bonobos, and Asian elephants had the best balance of the three key features that typify HMOs [1]. The MilkOligoDB team hypothesized these species converge on this shared suite of features because they all produce offspring with an extended lactation period and have infants that need more immune support due to a slower immune maturation. 

    Human infants are needy in other ways as well. Because of their extended period of brain growth during their first 18 months of life, human infants are considered altricial, or less mature and in need of more parental care. In another fascinating instance of convergent evolution, MilkOligoDB revealed that human milk shares a similar feature in oligosaccharide composition—more fucosylated structures—with other mammals that produce altricial young, including bears, dogs, skunks, and some primates [1]. Fucosylated oligosaccharides, oligosaccharides where a fucose sugar molecule is linked onto the carbohydrate chain, are the largest fraction of HMOs [6]. Fucosylated oligosaccharides may act as decoy receptors and fucose on its own has been shown to have a protective effect on infection and inflammation, features that may be particularly important for immature neonates [1, 6]. 

    Unfortunately, none of these species that humans shared similarities with were reasonable sources (i.e., commercially milked) for milk oligosaccharide isolation [1]. However, some camel and goat breeds did have high concentrations of fucosylated oligosaccharides. In particular, the whey fraction from these milks—the liquid part of milk that separates from the curd during cheese production—could be useful in creating supplements for infants or even as additives to other foods to improve human nutrition [1]. Analyzing milk from domestic species commonly milked outside of the U.S., such as yaks and llamas, for oligosaccharide profiles could reveal additional sources for supplements. Looking for a perfect match for HMOs may be fruitless, but extracting bioactive carbohydrates with known prebiotic and anti-microbial properties from multiple domestic species could provide health benefits to formula-fed infants. 

    MilkOligoDB is far from complete. The concept map format highlights gaps in the literature, indicating a handful of mammalian orders without any corresponding data (anyone have access to flying lemur milk?). And the MilkOligoDB creators are still working hard to improve the functionality of the database by developing a graphical interface to visually see comparisons across species, standardized identifiers for all terms, and even add a programming interface that would allow users to include data from MilkOligoDB in their own databases.

    MilkOligoDB may have been created with a particular research question in mind, but ultimately it will move milk science forward by helping other researchers generate new hypotheses about the evolutionary origins of milk oligosaccharides, why milk oligosaccharides are so structurally variable across species, and even milk’s role in the infant’s immune system.

     

    References

    1. Durham SD, Wei Z, Lemay DG, Lange MC, Barile D. Creation of a milk oligosaccharide database, MilkOligoDB, reveals common structural motifs and extensive diversity across mammals. Scientific Reports. 2023 Jun 26;13(1): 10345.
    2. Bode L. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology. 2012 Sep 1;22(9): 1147-62.
    3. German JB, Freeman SL, Lebrilla CB, Mills DA. Human milk oligosaccharides: evolution, structures and bioselectivity as substrates for intestinal bacteria. Personalized nutrition for the diverse needs of infants and children. Nestle Nutr Workshop Ser Pediatr Program 2008; 62: 205-22.
    4. Morrow AL, Ruiz-Palacios GM, Jiang X, Newburg DS. Human-milk glycans that inhibit pathogen binding protect breast-feeding infants against infectious diarrhea. The Journal of Nutrition. 2005 May 1;135(5): 1304-7.
    5. Ayechu-Muruzabal V, Van Stigt AH, Mank M, Willemsen LE, Stahl B, Garssen J, Van’t Land B. Diversity of human milk oligosaccharides and effects on early life immune development. Frontiers in Pediatrics. 2018 Sep 10; 6: 239.
    6. Orczyk-Pawiłowicz M, Lis-Kuberka J. The impact of dietary fucosylated oligosaccharides and glycoproteins of human milk on infant well-being. Nutrients. 2020 Apr 16;12(4): 1105.

    A Gut Reaction: Lactose Consumption in Lactase-Lacking Adults

    • Genetic variants associated with lactase persistence—the continued production of the enzyme that breaks down the milk sugar lactose into adulthood—may not be the only adaptation humans have to lactose digestion.
    • A new study adds to a growing body of research that demonstrates an association between lactose consumption and gut bacteria populations but only among individuals that do not produce lactase.
    • Continued lactose consumption in the absence of the lactase enzyme is believed to shift gut bacteria populations toward bacteria that ferment lactose into lactic acid or short-chain fatty acids instead of those that produce gas and other symptoms commonly associated with lactose intolerance.

    Being lactose intolerant and being lactase non-persistent (LNP) were once considered two sides of the same coin. If you are LNP, the gene that carries the instructions for making the lactase enzyme is turned off in the cells of your small intestine. Should you indulge in a banana split, the lactose from the ice cream isn’t digested in the small intestine and instead is fermented by bacteria in the colon. Many of the gut bacteria that commonly dine on lactose produce hydrogen, carbon dioxide, and methane as byproducts as they break the milk sugar down into its single sugars’ glucose and galactose. This results in symptoms of gas and bloating that are commonly associated with lactose intolerance and that can be painful and uncomfortable enough for many LNP individuals to avoid lactose altogether.

    But these unpleasant symptoms may not be inevitable for people that lack lactase. LNP individuals vary in how they experience lactose digestion. The reasons for this variation are not yet clearly understood; however, likely factors include age, malnutrition, alcohol consumption, and overall health [1-3]. In addition, a growing body of research suggests that the variation could also relate to the individual’s lactose consumption [2-5]. An apple a day keeps the doctor away—could a little lactose a day keep the lactose intolerance away?

    It sounds contradictory, but this is precisely what the colonic adaptation hypothesis argues—regular consumption of lactose in LNP individuals is believed to shift the bacterial populations in the colon toward those that metabolize lactose into lactic acid and short-chain fatty acids and away from those that produce hydrogen, carbon dioxide, or methane as byproducts [2-5]. In this way, lactose actually acts as a prebiotic, feeding the population of beneficial bacteria (probiotics) and increasing their numbers. But this hypothesis still requires rigorous testing. Just how frequent the lactose consumption needs to be, how much lactose an individual needs to consume to shift gut microbiota, and which specific types of bacteria are involved in the shift are questions that still need to be addressed.

    A new study [3] from nutritional researchers at the U.S. Department of Agriculture and UC Davis helps to chip away at these pressing questions by studying the interaction between lactose consumption, genetic status for lactase persistence (LP), and gut microbial populations. The study population was an ethnically diverse cohort of 275 healthy adults from Davis, California. All participants had known lactase genotypes for the most common genetic variant associated with LP among individuals of European descent (a single nucleotide polymorphism, or SNP, called rs4988235) [3]. LP individuals express the LCT gene, produce lactase, and can be homozygous dominant (AA genotype) or heterozygous (AG); LNP do not express the LCT gene and are homozygous recessive (GG). Because individuals of African descent could have another genetic variant (conferred by a different SNP) associated with LP, seven LNP study participants (GG genotype) that identified as African American were excluded from analysis. Lactose consumption was calculated from 24-hour recalls from each study participant from two weekdays and one weekend day. To better approximate regular lactose consumption, the researchers also used a food frequency questionnaire (FFQ) to provide data on habitual dairy intake from the previous 12 months. Gut microbiome populations were identified from bacterial DNA analysis on stool samples provided by study participants within 10–14 days from completion of their 24-hour dietary recalls.

    This was an observational study, which means the researchers couldn’t control for lactose intake across participants. However, testing their research questions would only be possible if LNP individuals consumed dairy products with lactose. They found that the average daily lactose consumption was higher in LP compared with LNP participants (12.16 vs. 8.72 grams per day), especially if the participants were men [3]. Luckily there were many LNP individuals who consumed comparable amounts of lactose to LP individuals [3]. Sex-specific differences in lactose consumption were found to correlate to differences in dairy intake; men consumed more fluid milk and women consumed more yogurt, which is lower in lactose than milk [3]. This finding highlights why it is so important to measure lactose intake, not just dairy intake; lactose intake in women would have been overestimated if based only on dairy servings.

    Consistent with predictions from the colonic adaptation hypothesis, variation in lactose consumption across LNP individuals was associated with differences in gut bacteria populations. LNP participants with a higher daily intake of lactose (>12.46 grams) had larger populations of Lactobacillaceae, a family of lactic acid bacteria that ferment lactose into lactic acid, compared with LNP individuals with low daily intakes of lactose (≤ 5.85 grams) [3]. (For reference, an 8-ounce glass of milk has around 12 grams of lactose).

    Gut bacteria were also distinct in a subset of the study population made up of only Caucasian and Hispanic LNP participants [3]. Bacteria from the family Lachnospriaceae were more abundant in high- compared with low-lactose consuming individuals, and the most abundant genera from this family were Blautia, Roseburia, and Coprococcus. This finding is significant because these genera have previously demonstrated the ability to break down lactose into short-chain fatty acids rather than hydrogen, carbon dioxide, and methane, providing additional support for microbial adaptations to lactose consumption across LNP participants [3].

    Importantly, the researchers found no association between lactose intake and gut Lactobacillaceae or Lachnospriaceae populations among LP participants, supporting previous research on lactose consumption and gut microbiomes among LP and LNP individuals. A lactose challenge study on LP and LNP Chinese adults [6] found a significantly greater amount of short-chain fatty acids were produced by fermentation of lactose in stools from LNP compared with LP subjects. And a study on dairy intake among Dutch adults [7] found a higher abundance of gut Bifidobacterium was associated with dairy consumption in LNP individuals but not LP individuals. Taken together, these studies strongly suggest that it was not simply high lactose consumption driving the association with gut bacteria but lactose consumption in the absence of lactase.

    If you lack lactase and are looking to improve the outcomes from lactose digestion by shifting your gut microbiome, take heed: it is still not known how much lactose you need to consume nor how long you need to keep consuming it to call yourself “adapted” from a microbiome perspective. Data like these are provided by intervention studies, which rely on observational studies (like the Davis study) to generate hypotheses about specific bacteria families (or genera or species) and the quantities of lactose needed to observe an effect. With all the nutritional benefits provided by dairy foods and more than half the world’s population possessing the LNP genotype, such studies are clearly important. Although it might not be possible to change your genes to keep the lactase enzyme turned on, it is encouraging that there may be more than one way to digest lactose without pain and discomfort.

    References

    1. del Carmen Tocaa M, Fernándezb A, Orsic M, Tabaccod O, Vinderolae G. Lactose intolerance: myths and facts. An update. Arch Argent Pediatr. 2022 Feb 1;120(1): 59-66.
    2. Brown-Esters O, Mc Namara P, Savaiano D. Dietary and biological factors influencing lactose intolerance. International Dairy Journal. 2012 Feb 1;22(2): 98-103.
    3. Kable ME, Chin EL, Huang L, Stephensen CB, Lemay DG. Association of estimated daily lactose consumption, lactase persistence genotype (rs4988235), and gut microbiota in healthy US adults. The Journal of Nutrition. 2023 Aug ;153(8): 2163-73.
    4. Szilagyi A. Adaptation to lactose in lactase non persistent people: Effects on intolerance and the relationship between dairy food consumption and evaluation of diseases. Nutrients. 2015 Aug 13;7(8): 6751-79.
    5. Forsgård RA. Lactose digestion in humans: intestinal lactase appears to be constitutive whereas the colonic microbiome is adaptable. The American Journal of Clinical Nutrition. 2019 Aug 1; 110(2): 273-9.
    6. He T, Priebe MG, Harmsen HJ, Stellaard F, Sun X, Welling GW, Vonk RJ. Colonic fermentation may play a role in lactose intolerance in humans. The Journal of Nutrition. 2006 Jan 1;136(1): 58-63.
    7. Bonder MJ, Kurilshikov A, Tigchelaar EF, Mujagic Z, Imhann F, Vila AV, Deelen P, Vatanen T, Schirmer M, Smeekens SP, Zhernakova DV. The effect of host genetics on the gut microbiome. Nature Genetics. 2016 Nov;48(11): 1407-12.

    Immune Cells Linked to Lactation Discovered in Mouse and Human Milk

    • Mammary glands in lactating mice contain distinct, short-lived macrophages.
    • Lactation-induced macrophages (LiMACs) appear to play roles in responding to microbial infection.
    • Human milk contains similar immune cells that may have related functions. 
    • LiMACs comprise up to 10 percent of cells in human milk.

    Immune cells known as macrophages in the mammary glands of mice perform important functions such as supporting the formation of mammary ducts during puberty and helping tissues return to the pre-pregnancy state after lactation. 

    But mammary immune cells may also play a part in supporting infants’ immunity. At birth, babies’ immature immune systems rely on additional protection conferred by the antibodies in human milk. But human milk also carries immune cells that increase in number during infections, and less is known about their significance. 

    In a new study, Melanie Greter, an immunologist at the University of Zurich and her colleagues, analyzed the macrophages present in mouse mammary glands and found one distinct subset that was present only during lactation [1]. 

    The team began by comparing the immune cells present in the mammary glands of lactating mice and mice that had never been pregnant. They found that two groups of tissue-resident macrophages previously reported to be in mammary glands were present [2] in lactating and non-lactating mice at constant levels throughout the first three weeks postpartum. But lactating animals also had a third, unique population of macrophages. By the fourth day after giving birth, these immune cells proliferated to become the majority of all myeloid cells and grew tenfold in number within the first twelve days postpartum. The proportion of these cells, named lactation-induced macrophages (liMACs), in mammary tissue decreased three weeks after birth, and they were rarely found in non-lactating animals. 

    LiMACs were largely present only in mammary tissue in areas of milk production. Single-cell RNA sequencing revealed that the genes expressed by these cells did not change significantly over the course of lactation. The cells “seemed to arise uniquely in the period between late pregnancy and early lactation,” the authors wrote in their publication 

    Considering the rapid increase in liMACs, the researchers tested whether they were a result of cells proliferating in the mammary tissue or if they were being formed by monocytes, another kind of immune cell that circulates through the body. The team labeled monocytes in mice and found that liMACs were distinct from the progeny of labeled monocytes. 

    Since immune cells shape the development of mammary ducts during puberty, the authors tested whether liMACs helped remodel the mammary glands during lactation. They found no difference in milk production between mice with these immune cells and those that were treated with an antibody that blocked the formation of liMACs. The researchers also found that liMACs did not alter the antibodies or other proteins present in milk. 

    To understand whether the maternal microbiome had an effect on liMACs, the authors compared conventional mice to germ-free mice that they exposed to four bacterial species. LiMACs were significantly more abundant in conventionally raised mice, suggesting that the microbiome influenced their formation.

    LiMACs expressed several genes related to inflammation and response to a common microbial antigen named LPS, suggesting that they play a role in protecting animals from infections. The researchers also isolated liMACs and exposed them to different microbial proteins, including some from Escherichia coli and Staphylococcus aureus, two bacterial species commonly linked to mastitis. The team found the liMACs were phagocytic and responded to microbial antigens. To elucidate this function, the researchers tested liMACs in a mouse model of mastitis, where they injected animals with LPS to trigger severe inflammation. When mice were treated with a liMAC-depleting antibody prior to the LPS injection, harmful inflammation was significantly reduced, suggesting that these cells play a part in early inflammatory responses. 

    The researchers found cells similar to liMACs in human milk samples collected from a milk bank. Similar to cell proportions in mice, macrophages comprised 1–10% of cells in human milk. Collectively, these data suggest that macrophages in human milk may play a protective role in mastitis and other infections. “These observations indicate that liMacs might participate in immune surveillance to protect the mammary gland from invading pathogens and infection,” the authors wrote in their publication. “These data will open new avenues for investigating the functions of macrophages for mother and infant during the lactation period, in health and disease.”

    References

    [1] Cansever D, Petrova E, Krishnarajah S, Mussak C, Welsh CA, Mildenberger W, Mulder K, Kreiner V, Roussel E, Stifter SA, Andreadou M. Zwicky P, Jurado NP, Rehrauer H, Tan G, Liu Z, Blériot C, Ronchi F, Macpherson AJ, Ginhoux F, Natalucci G, Becher B, Greter M. Lactation-associated macrophages exist in murine mammary tissue and human milk. Nature Immunology. 2023 Jun 19:1-2.

    [2] Gouon-Evans V, Rothenberg ME, Pollard JW. Postnatal mammary gland development requires macrophages and eosinophils. Development. 2000 Jun 1;127(11):2269-82.

    Myo-inositol in Human Milk Spurs Brain Development

    • Samples analyzed in a recent study came from a unique repository collected from mothers in Mexico City, Shanghai, and Cincinnati, OH, each time they fed their babies during their first year.
    • Amounts of myo-inositol are high in human milk for the first few months after birth, and then decrease,  suggesting the molecule is important for infants during that early time.
    • myo-inositol promoted the formation of synapses—connections between neurons—in human and rodent cells grown in a dish and in living mice. 

    It’s no secret that nutrition is a major contributor to health, but scientists know very little about how specific micronutrients affect the brain. Now, new research suggests that a specific carbohydrate in breast milk called myo-inositol enables neurons in the developing brain form connections, called synapses, through which they pass information (1). 

    “I did not expect that bioactive compounds can so profoundly impact brain development, says Thomas Biederer, a neuroscientist at Yale University School of Medicine and the USDA Human Nutrition Center, who led the work. “I think these effects of micronutrients on the brain are really underappreciated.” 

    What made the study possible was a repository of milk samples assembled and held by Mead Johnson Nutrition/Reckitt, a company that produces pediatric nutrition products. The repository consists of lactation samples from mothers in Mexico City, Shanghai, and Cincinnati, OH, who collected them each time they fed their babies from birth to 1 year of age.

    The company funded the study with the aim of identifying important elements in breast milk that are missing from formula. The researchers analyzed a range of lipids, carbohydrates, and secreted proteins in the samples, but for Biederer, an interesting fact popped out about the level of myo-inositol. “It is high early on, in the first few months after birth, and then it starts to gradually go down,” he says. “That piqued my interest because this is the period when most synapses in the human brain are forming, particularly in brain regions responsible for cognition, in the cerebral cortex.”

    During development, neurons initially form an overabundance of synapses (2). This synapse formation occurs over different time periods across different regions. In humans, sensory regions of the cerebral cortex, which process vision, hearing and touch, go through this burst during infancy. In other areas such as the frontal cortex, which is involved in more complex dimensions of cognition, this process lasts through adolescence.

    To test whether myo-inositol directly affects how neurons develop, the researchers first applied it to human or rat cells grown in a dish. Treating human cells with myo-inositol did not alter the neurons’ length or the degree of complexity in their branching, but it increased the number of synapses in the cells by 29%. The researchers observed the same effect in rat neurons, also noting that the increase in synapses was greater the more myo-inositol they added. 

    Biederer and his colleagues fed myo-inositol to mouse pups from birth to 35 days of age, then examined synapses in their visual cortex. They found that the size of synapses increased by about 50%, which likely  reflects an increase the in the strength of synapses. Finally, they looked at slices of brain tissue that were taken from young mice and grown in a dish until they reached maturity. They determined that the number of synapses increased after they applied myo-inositol. 

    “The conclusion is really robust across all models that myo-inositol promotes how nerve cells can form connections between each other,” Biederer says. “The effects were really, really apparent—when we treated the cultured neurons with myo-inositol they looked healthier than anything we had seen before.”

    Biederer adds that although the current study focused on myo-inositol, breast milk likely has many other functionally important components. “It’s just the first one we had the opportunity to investigate in detail,” he says. 

    The idea that micronutrients can have significant effects on brain function is severely understudied, Biederer notes. There were hints in previous research linking the substance to brain function in adults (3, 4), but the study is the first to flag its potential involvement in the infant brain. He cautions that more research is needed before there’s enough evidence to suggest that people should consume myo-inositol to boost brain function. 

    Biederer’s team is now looking at a slightly later stage of brain development, during which the overproduced connections between neurons get trimmed down so that only the most functional ones remain. “Our interest now is identifying what compounds are supporting the refinement of connectivity,” he says. 

    References

    1. Paquette AF, Carbone BE, Vogel S, Israel E, Maria SD, Patil NP, Sah S, Chowdhury D, Kondratiuk I, Labhart B, Morrow AL, Phillips SC, Kuang C, Hondmann D, Pandey N, Biederer T. The human milk component myo-inositol promotes neuronal connectivity. Proc Natl Acad Sci U S A. 2023:25;120(30):e2221413120.
    2. Rakic P, Bourgeois JP, Eckenhoff MF, Zecevic N, Goldman-Rakic PS. Concurrent overproduction of synapses in diverse regions of the primate cerebral cortex. Science. 1986:232(4747):232-235. 
    3. Vawter MP, Hamzeh AR, Muradyan E, Civelli O, Abbott GW, Alachkar A. Association of myoinositol transporters with schizophrenia and bipolar disorder: Evidence from human and animal studies. Mol Neuropsychiatry. 2019:5(4):200-211.
    4. Levine J, Barak Y, Gonzalves M, Szor H, Elizur A, Kofman O, Belmaker RH. Double-blind, controlled trial of inositol treatment of depression. Am J Psychiatry. 1995:152(5):792-794. 

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