Interaction of the Gut Microbiome and Immunity in Multiple Sclerosis: Impact of Diet and Immune Therapy.

The bidirectional communication between the gut and central nervous system (CNS) through microbiota is known as the microbiota-gut-brain axis. The brain, through the enteric neural innervation and the vagus nerve, influences the gut physiological activities (motility, mucin, and peptide secretion), as well as the development of the mucosal immune system. Conversely, the gut can influence the CNS via intestinal microbiota, its metabolites, and gut-homing immune cells. Growing evidence suggests that gut immunity is critically involved in gut-brain communication during health and diseases, including multiple sclerosis (MS). The gut microbiota can influence the development and function of gut immunity, and conversely, the innate and adaptive mucosal immunity can influence microbiota composition. Gut and systemic immunity, along with gut microbiota, are perturbed in MS. Diet and disease-modifying therapies (DMTs) can affect the composition of the gut microbial community, leading to changes in gut and peripheral immunity, which ultimately affects MS. A high-fat diet is highly associated with gut dysbiosis-mediated inflammation and intestinal permeability, while a high-fiber diet/short-chain fatty acids (SCFAs) can promote the development of Foxp3 Tregs and improvement in intestinal barrier function, which subsequently suppress CNS autoimmunity in the animal model of MS (experimental autoimmune encephalomyelitis or EAE). This review will address the role of gut immunity and its modulation by diet and DMTs via gut microbiota during MS pathophysiology.

Gut dysbiosis breaks immunological tolerance toward the central nervous system during young adulthood.

Multiple sclerosis (MS) is an autoimmune disease targeting the central nervous system (CNS) mainly in young adults, and a breakage of immune tolerance to CNS self-antigens has been suggested to initiate CNS autoimmunity. Age and microbial infection are well-known factors involved in the development of autoimmune diseases, including MS. Recent studies have suggested that alterations in the gut microbiota, referred to as dysbiosis, are associated with MS. However, it is still largely unknown how gut dysbiosis affects the onset and progression of CNS autoimmunity. In this study, we investigated the effects of age and gut dysbiosis on the development of CNS autoimmunity in humanized transgenic mice expressing the MS-associated MHC class II (MHC-II) gene, HLA-DR2a, and T-cell receptor (TCR) genes specific for MBP87-99/DR2a that were derived from an MS patient. We show here that the induction of gut dysbiosis triggers the development of spontaneous experimental autoimmune encephalomyelitis (EAE) during adolescence and early young adulthood, while an increase in immunological tolerance with aging suppresses disease onset after late young adulthood in mice. Furthermore, gut dysbiosis induces the expression of complement C3 and production of the anaphylatoxin C3a, and down-regulates the expression of the Foxp3 gene and anergy-related E3 ubiquitin ligase genes. Consequently, gut dysbiosis was able to trigger the development of encephalitogenic T cells and promote the induction of EAE during the age window of young adulthood.

[Growth Process of Cystic Lung Cancer Followed with Computed Tomography Findings Over 5 Years].

An estimated 2~16% of primary lung cancers form cavities with cases that form thin-walled cavities being comparatively rare. We treated a patient with squamous cell carcinoma of the lung with a small cystic shadow that showed no changes for 3 years. The cyst then suddenly grew larger, after which the cyst wall thickened over time and a thin-walled cavity was seen. Here we report this important case showing the development process of lung cancer that formed a thin-walled cavity, together with a discussion of the literature.

Advances in the immunopathogenesis of multiple sclerosis.

PURPOSE OF REVIEW: Recent studies indicate a role for immune dysregulation in the pathogenesis of multiple sclerosis, an inflammatory demyelinating and degenerative disease of the central nervous system. This review addresses the current mechanisms of immune dysregulation in the development of multiple sclerosis, including the impact of environmental risk factors on immunity in both multiple sclerosis and its animal models. RECENT FINDINGS: CD4 T-helper (Th) cells have long been implicated as the main drivers of pathogenesis of multiple sclerosis. However, current studies indicate that multiple sclerosis is largely a heterogeneous disease process, which involves both innate and adaptive immune-mediated inflammatory mechanisms that ultimately contribute to demyelination and neurodegeneration. Therefore, B cells, CD8 T cells, and microglia/macrophages can also play an important role in the immunopathogenesis of multiple sclerosis apart from proinflammatory CD4 Th1/Th17 cell subsets. Furthermore, increasing evidence indicates that environmental risk factors, such as Vitamin D deficiency, Epstein-Barr virus, smoking, Western diet, and the commensal microbiota, influence the development of multiple sclerosis through interactions with genetic variants of multiple sclerosis, thus leading to the dysregulation of immune responses. SUMMARY: A better understanding of immune-mediated mechanisms in the pathogenesis of multiple sclerosis and the contribution of environmental risk factors toward the development of multiple sclerosis will help further improve therapeutic approaches to prevent disease progression.

Myelin basic protein-specific TCR/HLA-DRB5*01:01 transgenic mice support the etiologic role of DRB5*01:01 in multiple sclerosis.

Genetic susceptibility to multiple sclerosis (MS) has been linked to the HLA-DR15 haplotype consisting of DRB1*15:01(DR2b) and DRB5*01:01(DR2a) alleles. Given almost complete linkage disequilibrium of the two alleles, recent studies suggested differential roles in susceptibility (DR2b) or protection from MS (DR2a). Our objective was to assess the potential contribution of DR2a to disease etiology in MS using a humanized model of autoimmunity. To assess the potential contribution of DR2a to disease etiology, we created DR2a humanized transgenic (Tg) mice and subsequently crossed them to Tg mice expressing TL3A6, an MS patient-derived myelin basic protein 83-99-specific TCR. In TL3A6/DR2a Tg mice, CD4 Tg T cells escape thymic and peripheral deletion and initiate spontaneous experimental autoimmune encephalomyelitis (EAE) at low rates, depending on the level of DR2a expression. The ability to induce active EAE was also increased in animals expressing higher levels of DR2a. Inflammatory infiltrates and neuronal damage were present throughout the spinal cord, consistent with a classical ascending EAE phenotype with minor involvement of the cerebellum, brainstem, and peripheral nerve roots in spontaneous, as well as actively induced, disease. These studies emphasize the pathologic contribution of the DR2a allele to the development of autoimmunity when expressed as the sole MHC class II molecule, as well as strongly argue for DR2a as a contributor to the CNS autoimmunity in MS.

Fecal Lcn-2 level is a sensitive biological indicator for gut dysbiosis and intestinal inflammation in multiple sclerosis.

Multiple Sclerosis (MS) has been reported to be associated with intestinal inflammation and gut dysbiosis. To elucidate the underlying biology of MS-linked gut inflammation, we investigated gut infiltration of immune cells during the development of spontaneous experimental autoimmune encephalomyelitis (EAE) in humanized transgenic (Tg) mice expressing HLA-DR2a and human T cell receptor (TCR) specific for myelin basic protein peptide (MBP87-99)/HLA-DR2a complexes. Strikingly, we noted the simultaneous development of EAE and colitis, suggesting a link between autoimmune diseases of the central nervous system (CNS) and intestinal inflammation. Examination of the colon in these mice revealed the infiltration of MBP-specific Th17 cells as well as recruitment of neutrophils. Furthermore, we observed that fecal Lipocalin-2 (Lcn-2), a biomarker of intestinal inflammation, was significantly elevated and predominantly produced by the gut-infiltrating neutrophils. We then extended our findings to MS patients and demonstrate that their fecal Lcn-2 levels are significantly elevated compared to healthy donors (HDs). The elevation of fecal Lcn-2 levels correlated with reduced bacterial diversity and increased levels of other intestinal inflammation markers including neutrophil elastase and calprotectin. Of interest, bacteria thought to be beneficial for inflammatory bowel disease (IBD) such as Anaerobutyricum, Blautia, and Roseburia, were reduced in fecal Lcn-2-high MS patients. We also observed a decreasing trend in serum acetate (a short-chain fatty acid) levels in MS Lcn-2-high patients compared to HDs. Furthermore, a decrease in the relative abundance of Blautia massiliensis was significantly associated with a reduction of acetate in the serum of MS patients. This study suggests that gut infiltration of Th17 cells and recruitment of neutrophils are associated with the development of gut dysbiosis and intestinal inflammation, and that fecal Lcn-2 level is a sensitive biological indicator for gut dysbiosis in multiple sclerosis.

Effect of switching glatiramer acetate formulation from 20 mg daily to 40 mg three times weekly on immune function in multiple sclerosis.

BACKGROUND: Many RRMS patients who had been treated for over 20 years with GA 20 mg/ml daily (GA20) switched to 40 mg/ml three times-a-week (GA40) to reduce injection-related adverse events. Although GA40 is as effective as GA20 in reducing annualized relapse rate and MRI activity, it remains unknown how switching to GA40 from GA20 affects the development of pathogenic and regulatory immune cells. OBJECTIVE: To investigate the difference in immunological parameters in response to GA20 and GA40 treatments. METHODS: We analyzed five pro-inflammatory cytokines (IL-1ß, IL-23, IL-12, IL-18, TNF-α), and three anti-inflammatory/regulatory cytokines (IL-10, IL-13, and IL-27) in serum. In addition, we analyzed six cytokines (IFN-γ, IL-17A, GM-CSF, IL-10, IL-6, and IL-27) in cultured PBMC supernatants. The development of Th1, Th17, Foxp3 Tregs, M1-like, and M2-like macrophages were examined by flow cytometry. Samples were analyzed before and 12 months post switching to GA40 or GA20. RESULTS: Pro- and anti-inflammatory cytokines were comparable between the GA40 and GA20 groups. Development of Th1, Th17, M1-like macrophages, M2-like macrophages, and Foxp3 Tregs was also comparable between the two groups. CONCLUSIONS: The immunological parameters measured in RRMS patients treated with GA40 three times weekly are largely comparable to those given daily GA20 treatment.

Dimethyl Fumarate Suppresses Demyelination and Axonal Loss through Reduction in Pro-Inflammatory Macrophage-Induced Reactive Astrocytes and Complement C3 Deposition.

Dimethyl fumarate (DMF) is an oral agent for relapsing-remitting multiple sclerosis (RRMS). In this study, we investigated the therapeutic mechanism of DMF using experimental autoimmune encephalomyelitis (EAE). DMF treatment decreased the proliferation of T cells and the production of IL-17A and GM-CSF. DMF treatment also decreased the development and/or infiltration of macrophages in the central nervous system (CNS), and reduced the ratio of iNOS+ pro-inflammatory macrophage versus Ym1+ immunomodulatory macrophages. Furthermore, DMF treatment suppressed the deposition of complement C3 (C3) and development of reactive C3+ astrocytes. The decrease in iNOS+ macrophages, C3+astrocytes, and C3 deposition in the CNS resulted in the reduction in demyelination and axonal loss. This study suggests that the beneficial effects of DMF involve the suppression of iNOS+ pro-inflammatory macrophages, C3+ astrocytes, and deposition of C3 in the CNS.

Unique clinical and pathological features in HLA-DRB1*0401-restricted MBP 111-129-specific humanized TCR transgenic mice.

Amino acid residues 111-129 represent an immunodominant epitope of myelin basic protein (MBP) in humans with human leukocyte antigen (HLA)-DRB1*0401 allele(s). The MBP 111-129-specific T cell clone MS2-3C8 was repeatedly isolated from a patient with multiple sclerosis (MS), suggesting an involvement of MS2-3C8 T cells in the pathogenesis. To address the pathogenic potential of the MS2-3C8 T cell clone, we generated transgenic (Tg) mice expressing its T cell receptor and restriction element, HLA-DRB1*0401, to examine the pathogenic characteristics of MS2-3C8 Tg T cells by adoptive transfer into HLA-DRB1*0401 Tg mice. In addition to the ascending paralysis typical of experimental autoimmune encephalomyelitis, mice displayed dysphagia due to restriction in jaw and tongue movements and abnormal gait. In accordance with the clinical phenotype, infiltrates of MS2-3C8 Tg T cells and inflammatory lesions were predominantly located in the brainstem and the cranial nerve roots in addition to the spinal cord and spinal nerve roots. Together, these data suggest a pathogenic role of MBP-specific T cells in inflammatory demyelination within the brainstem and cranial nerve roots during the progression of MS. This notion may help to explain the clinical and pathological heterogeneity of MS.

Different development of myelin basic protein agonist- and antagonist-specific human TCR transgenic T cells in the thymus and periphery.

Myelin basic protein (MBP)-specific T cells are thought to play a role in the development of multiple sclerosis. MBP residues 111-129 compose an immunodominant epitope cluster restricted by HLA-DRB1*0401. The sequence of residues 111-129 of MBP (MBP(111-129)) differs in humans (MBP122:Arg) and mice (MBP122:Lys) at aa 122. We previously found that approximately 50% of human MBP(111-129) (MBP122:Arg)-specific T cell clones, including MS2-3C8 can proliferate in response to mouse MBP(111-129) (MBP122:Lys). However, the other half of T cell clones, including HD4-1C2, cannot proliferate in response to MBP(111-129) (MBP122:Lys). We found that MBP(111-129) (MBP122:Lys) is an antagonist for HD4-1C2 TCR, therefore, MS2-3C8 and HD4-1C2 TCRs are agonist- and antagonist-specific TCRs in mice, respectively. Therefore, we examined the development of HD4-1C2 TCR and MS2-3C8 TCR transgenic (Tg) T cells in the thymus and periphery. We found that dual TCR expression exclusively facilitates the development of MBP(111-129) TCR Tg T cells in the periphery of HD4-1C2 TCR/HLA-DRB1*0401 Tg mice although it is not required for their development in the thymus. We also found that MS2-3C8 TCR Tg CD8(+) T cells develop along with MS2-3C8 TCR Tg CD4(+) T cells, and that dual TCR expression was crucial for the development of MS2-3C8 TCR Tg CD4(+) and CD8(+) T cells in the thymus and periphery, respectively. These results suggest that thymic and peripheral development of MBP-specific T cells are different; however, dual TCR expression can facilitate their development.

Surface Layer Protein A Expressed in Clostridioides difficile DJNS06-36 Possesses an Encephalitogenic Mimotope of Myelin Basic Protein.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Recent studies suggest that migration of Th1 and Th17 cells specific for enteric bacteria from the gut to the CNS may lead to the initiation and/or exacerbation of autoimmune diseases including MS. Human leukocyte antigen (HLA)-DR15 is an MHC class II (MHCII) haplotype highly associated with the development of MS that contains the two HLA-DRB* genes, DRB1*1501 (DR2b) and DRB5*0101 (DR2a). To identify enteric bacteria which harbor antigenic epitopes that activate myelin-specific T cells and drive CNS inflammation, we screened for enteric bacteria which express cross-reactive epitopes ('mimotopes') of an immunodominant myelin basic protein 89-98 (MBP89-98) epitope. Based on known MHCII HLA-DR2a amino acid binding motifs and cultivation with splenic T cells isolated from MBP-T cell receptor (TCR)/DR2a transgenic (Tg) mice, we discovered that a certain variant of surface layer protein A (SLPA), which is expressed by a subtype of Clostridioides difficile, contains an amino acid sequence that activates MBP89-98-reactive T cells. Furthermore, activation of MBP-specific T cells by SLPA upon active immunization induced experimental autoimmune encephalomyelitis (EAE) in MBP-TCR/DR2a Tg mice. This study suggests that a unique strain of C. difficile possesses an encephalitogenic mimotope of MBP that activates autoreactive, myelin-specific T cells.

Sarcopenia is a prognostic factor for TKIs in metastatic thyroid carcinomas.

PURPOSE: Among patients with various cancers receiving anticancer drugs, sarcopenia is associated with poor survival and treatment outcomes. We conducted an observational study using skeletal muscle index (SMI) evaluation to investigate the association between sarcopenia and treatment outcomes of tyrosine kinase inhibitors (TKIs) in metastatic thyroid cancer patients. METHODS: We included 54 patients (19 men, 35 women; age, 66.5 ± 10.9 years) with differentiated thyroid carcinoma (DTC) or medullary thyroid carcinoma (MTC). The records of patients with metastatic DTC and MTC treated with TKIs were retrospectively reviewed. Patients were divided into sarcopenia and non-sarcopenia groups based on SMI. The SMI cutoff values for sarcopenia were 42 and 38 (cm2/m2) for males and females, respectively. Thirty-three patients had sarcopenia before TKI treatment. RESULTS: The sarcopenia group had more females and a lower body mass index. The median progression-free survival (PFS) durations were 13.6 (95% confidence interval (CI): 6.1-29.9) and 41.9 (95% CI: 25.2-not estimable) months in the sarcopenia and non-sarcopenia groups (p= 0.017), respectively. Univariate analysis showed that sarcopenia was significantly associated with PFS (p= 0.037). Sex, age, and performance status did not affect PFS. Multivariate analysis showed that sarcopenia was the only independent prognostic factor for PFS (hazard ratio: 2.488, 95% CI: 1.058-5.846, p= 0.037). CONCLUSIONS: Sarcopenia could be a predictive factor of TKI treatment outcomes in patients with metastatic thyroid cancer as well as intervention target to improve prognosis. Further prospective investigations are needed to confirm these preliminary data.

Insight into the mechanism of action of dimethyl fumarate in multiple sclerosis.

Dimethyl fumarate (DMF) is an oral, disease-modifying agent for the treatment of relapsing-remitting multiple sclerosis (RRMS). However, details regarding its mode of action are still emerging. It is believed that the mode of action of DMF involves both nuclear factor erythroid-derived 2-related factor (Nrf2)-dependent and independent pathways, which lead to an anti-inflammatory immune response due to type II myeloid cell and Th2 cell differentiation and neuroprotection. In this review, we will focus on the molecular and signaling effects of DMF that lead to changes in peripheral immune cell composition and function, alteration in CNS cell-specific functions, and effect on the blood-brain barrier.

Germanene Epitaxial Growth by Segregation through Ag(111) Thin Films on Ge(111).

Large-scale two-dimensional sheets of graphene-like germanium, namely, germanene, have been epitaxially prepared on Ag(111) thin films grown on Ge(111), using a segregation method, differing from molecular beam epitaxy used in previous reports. From the scanning tunneling microscopy (STM) images, the surface is completely covered with an atom-thin layer showing a highly ordered long-range superstructure in wide scale. Two types of protrusions, named hexagon and line, form a (7√7 × 7√7) R19.1° supercell with respect to Ag(111), with a very large periodicity of 5.35 nm. Auger electron spectroscopy and high-resolution synchrotron radiation photoemission spectroscopy demonstrate that Ge atoms are segregated on the Ag(111) surface as an overlayer. Low-energy electron diffraction clearly shows incommensurate "(1.35 × 1.35)" R30° spots, corresponding to a lattice constant of 0.39 nm, in perfect accord with close-up STM images, which clearly reveal an internal honeycomb arrangement with corresponding parameter and low buckling within 0.01 nm. As this 0.39 nm value is in good agreement with the theoretical lattice constant of free-standing germanene, conclusively, the segregated Ge atoms with trivalent bonding in honeycomb configuration form a characteristic two-dimensional germanene-like structure.

Fast cleanup method for the analysis of Sudan I-IV and para red in various foods and paprika color (oleoresin) by high-performance liquid chromatography/diode array detection: focus on removal of fat and oil as fatty acid methyl esters prepared by transesterification of acylglycerols.

A fast and effective cleanup method was developed for the analysis of Sudan I, II, III, IV, and Para Red (Sudan dyes) in various foods and paprika color (oleoresin) by high-performance liquid chromatography (LC) with a diode array detector (DAD). Removal of fat or oil in fatty sample was a critical point for reducing the volume of the final sample solution in order to obtain a sufficient level of the analytes. Separation of fat or oil from the dyes with a silica gel solid-phase extraction (SPE) column seemed unfeasible, because elution profiles of oil, fat, and the dyes were similar. Finally, fat and oil were separated from the dyes by elution from the SPE column with n-hexane, not as intact compounds but as fatty acid methyl esters prepared by direct transesterification of acylglycerols in fat and oil, leaving the dyes on the column. The dyes were eluted with n-hexane-diethyl ether (9 + 1). Gradient elution with water and tetrahydrofuran was used for separation on a C18 column by LC. Measurement of spectral of 0.5 microg/g of Sudan dyes in foods and 1 microg/g in paprika color (oleoresin) with the DAD was achieved.

[Properties of commercial licorice extracts used as a food additive].

Properties of eight commercial licorice extracts used as a food additive (sweetener, listed in the List of Existing Food Additives in the Japanese Food Sanitation Law) were surveyed. Residue on ignition ranged from 0.3 to 12.4%, and pH ranged from 4.1 to 6.8, amount of glycyrrhizin, which is the major component in licorice extract, ranged from 10.9 to 77.4%, sodium ranged from 0.1 to 1.2%, potassium ranged from 0.3 to 5.0%, and ammonium nitrogen ranged from 0.03 to 2.5%. These results indicated that various products were distributed on the market. Differences in the manufacturing process may contribute to the variations of product properties.

IL-27: a potential biomarker for responders to glatiramer acetate therapy.

Glatiramer acetate (GA) is an FDA-approved efficacious drug for the treatment of relapsing-remitting multiple sclerosis (RRMS). However, this treatment is not effective for all RRMS patients. Therefore, it is important to identify reliable biomarkers that can predict a beneficial clinical response to GA therapy. Since an increase in IL-27 has been demonstrated to suppress autoimmune and allergic diseases of inflammatory origin, we examined the effect of GA on the production of IL-27. We observed that IL-27 production in PBMCs cultured with GA was heterogeneous amongst MS patients and healthy donors (HD), and thus, defined these MS patients as either efficient, weak, or non-IL-27 producers. Interestingly, GA could induce the expression of the IL-27p28 subunit more efficiently than the IL-27 EBI3 subunit, and the production of IL-27 depended on MHC class II binding by GA. In addition, we found that GA could augment Toll-like receptor (TLR)-mediated IL-27 production. Importantly, serum production of IL-27 and IL-10 was significantly increased at 6months during GA therapy in clinical responders to GA, but not in GA non-responders. Altogether, our data suggest that GA-induced IL-27 may represent a therapeutic mechanism of GA-mediated immunomodulation and that GA-mediated IL-27 production in PBMCs is worth exploring as a biomarker to screen for GA responders prior to the initiation of GA treatment.

Down-regulation of transcription elogation factor A (SII) like 4 (TCEAL4) in anaplastic thyroid cancer.

BACKGROUND: Anaplastic thyroid cancer (ATC) is one of the most aggressive human malignancies and appears to arise mainly from transformation of pre-existing differentiated thyroid cancer (DTC). However, the carcinogenic mechanism of anaplastic transformation remains unclear. Previously, we investigated specific genes related to ATC based on gene expression profiling using cDNA microarray analysis. One of these genes, transcription elongation factor A (SII)-like 4 (TCEAL4), encodes a member of the transcription elongation factor A (SII)-like gene family. The detailed function of TCEAL4 has not been described nor has any association between this gene and human cancers been reported previously. METHODS: To investigate the role of TCEAL4 in ATC carcinogenesis, we examined expression levels of TCEAL4 in ACLs as well as in other types of thyroid cancers and normal human tissue. RESULTS: Expression of TCEAL4 was down-regulated in all 11 ACLs as compared to either normal thyroid tissues or papillary and follicular thyroid cancerous tissues. TCEAL4 was expressed ubiquitously in all normal human tissues tested. CONCLUSION: To our knowledge, this is the first report of altered TCEAL4 expression in human cancers. We suggest that loss of TCEAL4 expression might be associated with development of ATC from DTC. Further functional studies are required.

Growth-suppressive function of phosphatidylethanolamine-binding protein in anaplastic thyroid cancer.

BACKGROUND: A cDNA microarray analysis of anaplastic thyroid cancer cell lines (ACL) was recently performed and the down-regulation of phosphatidylethanolamine-binding protein (PBP) [RAF kinase inhibitor protein (RKIP)] in ACL compared to normal thyroid tissues was identified. MATERIALS AND METHODS: The expression levels of PBP in primary anaplastic and papillary thyroid cancer, thyroid cancer cell lines (anaplastic, papillary and follicular) and several normal human organs were examined. To examine the function of PBP, cell-growth assays were performed. RESULTS: PBP expression was reduced in anaplastic thyroid cancers, compared to either normal thyroid tissues or differentiated thyroid cancers. PBP was expressed ubiquitously in normal human tissues. Exogenous PBP expression suppressed ACL growth, and suggested a tumor suppressive function of PBP in ACL. CONCLUSION: This is the first report demonstrating that PBP may be a tumor suppressor whose loss is associated with development of anaplastic thyroid cancer from differentiated thyroid cancer.

Determination of magnesium stearate in capsule- or tablet-type supplements.

A simple method for the determination of magnesium stearate in capsule- or tablet-type supplements was developed. Free stearic acid in the sample was removed by extraction with tetrahydrofuran. The remaining stearate was converted to stearic acid by reaction with a cation-exchange resin. The resulting stearic acid was determined by gas chromatography with a polar column. Esters of stearic acid were not converted to stearic acid and would not cause a positive error in the amount of stearate. The amount of magnesium stearate was calculated based on the stearic acid concentration thus obtained. Magnesium stearate levels in 5 out of 25 supplements exceeded 2500 microg/g, which indicated the possible admixture of magnesium stearate.