Sulfated vitamin D metabolites represent prominent roles in serum and in breastmilk of lactating women.

BACKGROUND: Concentrations of vitamin D (VitD) and 25-hydroxyvitamin D (25OHD) in breastmilk are low despite the essential role of VitD for normal infant bone development, yet additional metabolic forms of vitamin D may be present. This study evaluates the contribution of sulfated vitamin D metabolites, vitamin D3-sulfate (VitD3-S) and 25-hydroxyvitamin D3-sulfate (25OHD3-S) for lactating women and assesses the response to high-dose VitD3 supplementation. METHODS: Serum and breastmilk were measured before and after 28 days with 5000 IU/day VitD3 intake in 20 lactating women. Concentrations of VitD3-S and 25OHD3-S in milk, and 25OHD2, 25OHD3, 25OHD3-S, VitD3 and VitD3-S in serum were determined by mass spectrometry. RESULTS: Baseline vitamin D status was categorized as sufficient (mean ± SD serum 25OHD3 69 ± 19 nmol/L), and both serum VitD3 and 25OHD3 increased following supplementation (p < 0.001). 25OHD3-S was 91 ± 19 nmol/L in serum and 0.47 ± 0.09 nmol/L in breastmilk. VitD3-S concentrations were 2.92 ± 0.70 nmol/L in serum and 6.4 ± 3.9 nmol/L in breastmilk. Neither sulfated metabolite significantly changed with supplementation in either serum or breastmilk. CONCLUSIONS: Sulfated vitamin D metabolites have prominent roles for women during lactation with 25OHD3-S highly abundant in serum and VitD3-S distinctly abundant in breastmilk. These data support the notion that 25OHD3-S and VitD3-S may have physiological relevance during lactation and nutritional usage for nursing infants.

Analysis of vitamin D3-sulfate and 25-hydroxyvitamin D3-sulfate in breastmilk by LC-MS/MS.

Sulfated metabolites of vitamin D have been suggested to be in breastmilk, although current methods to measure sulfated vitamin D compounds in breastmilk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) have not adequately accounted for increased aqueous solubility of these sulfated metabolites. The purpose of this study was to generate a method of LC-MS/MS for measuring vitamin D3-3-sulfate (VitD3-S) and 25-hydroxyvitamin D3-3-sulfate (25OHD3-S) specifically in human breastmilk. The resulting method uses methanol to precipitate protein and solid phase extraction to prepare the samples for LC-MS/MS. The limits of quantification for analytes in solvent were 0.23 ng/mL VitD3-S and 0.2 ng/mL 25OHD3-S. Various experiments observed concentrations ranging 0.53 to 1.7 ng/mL VitD3-S and ≤ 0.29 ng/mL 25OHD3-S. Both analytes were present in aqueous skim milk, demonstrating the enhanced aqueous solubility of these vitamin D sulfates. In conclusion, we describe an effective method for measuring VitD3-S and 25OHD3-S in breastmilk by LC-MS/MS.

Rhamnose Is Superior to Mannitol as a Monosaccharide in the Dual Sugar Absorption Test: A Prospective Randomized Study in Children With Treatment-Naïve Celiac Disease.

Background and Aim: We sought to correlate two different measures of gut permeability [lactulose:mannitol (L:M) and lactulose:rhamnose (L:R)] to the severity of duodenal histopathology in children with and without elevated antibodies to tissue transglutaminase (tTG). A secondary objective was to correlate gut permeability with celiac disease (CD) serology and indices of inflammation and bacterial product translocation. Methods: We prospectively randomized children undergoing endoscopy with abnormal (n = 54) and normal (n = 10) concentrations of circulating antibodies to tTG, to either L:M or L:R. Biopsies underwent modified Marsh scoring to measure mucosal injury. Circulating anticore Escherichia coli lipopolysaccharide (LPS) IgG, α-1 acid glycoprotein, LPS-binding protein, and C-reactive protein concentrations were measured by enzyme immunoassays. Results: Of the 54 cases with positive celiac serology, 31 and 69% had modified Marsh 0/1 scores or ≥3a, respectively. Circulating tTG IgA correlated with the modified Marsh score (p = 0.03). L:R, but not L:M or percent L excreted, differed according to modified Marsh scores (p = 0.01). There was no significant association between any systemic marker of inflammation or gut injury, and modified Marsh scores. Concerningly, most participants had evidence of urinary M before the challenge sugar was administered. Conclusions: L:R, but not L:M, is associated with modified Marsh scores in children undergoing small bowel biopsy for suspected CD. Despite increased intestinal permeability, we see scant evidence of systemic exposure to gut microbes in these children. Gut permeability testing with L:R may predict which patients with abnormal celiac serology will have biopsy evidence for celiac disease and reduce the proportion of such patients undergoing endoscopy whose Marsh scores are ≤1. M should not be used as a monosaccharide for permeability testing in children.

Detection of Alzheimer's disease amyloid beta 1-42, p-tau, and t-tau assays.

INTRODUCTION: We aimed to provide cut points for the automated Elecsys Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarkers. METHODS: Cut points for Elecsys amyloid beta 42 (Aß42), total tau (t-tau), hyperphosphorylated tau (p-tau), and t-tau/Aß42 and p-tau/Aß42 ratios were evaluated in Mayo Clinic Study of Aging (n = 804) and Mayo Clinic Alzheimer's Disease Research Center (n = 70) participants. RESULTS: The t-tau/Aß42 and p-tau/Aß42 ratios had a higher percent agreement with normal/abnormal amyloid positron emission tomography (PET) than the individual CSF markers. Reciever Operating Characteristic (ROC)-based cut points were 0.26 (0.24-0.27) for t-tau/Aß42 and 0.023 (0.020-0.025) for p-tau/Aß42. Ratio cut points derived from other cohorts performed as well in our cohort as our own did. Individual biomarkers had worse diagnostic properties and more variable results in terms of positive and negative percent agreement (PPA and NPA). CONCLUSION: CSF t-tau/Aß42 and p-tau/Aß42 ratios are very robust indicators of AD. For individual biomarkers, the intended use should determine which cut point is chosen.

Development and Validation of Test for "Leaky Gut" Small Intestinal and Colonic Permeability Using Sugars in Healthy Adults.

BACKGROUND: Oral monosaccharides and disaccharides are used to measure in vivo human gut permeability through urinary excretion. AIMS: The aims were as follows: (1) to obtain normative data on small intestinal and colonic permeability; (2) to assess variance on standard 16 g fiber diet performed twice; (3) to determine whether dietary fiber influences gut permeability measurements; and (4) to present pilot data using 2 selected probes in patients with diarrhea-predominant irritable bowel syndrome (IBS-D). METHODS: Sixty healthy female and male adults, age 18-70 years, participated in 3 randomized studies (2 studies on 16.25 g and 1 study on 32.5 g fiber) in otherwise standardized diets. At each test, the following sugars were ingested: 12C-mannitol, 13C-mannitol, rhamnose (monosaccharides), sucralose, and lactulose (disaccharides). Standardized meals were administered from 24 hours before and during 24 hours post-sugars with 3 urine collections: 0-2, 2-8, and 8-24 hours. Sugars were measured using high-performance liquid chromatography-tandem mass spectrometry. Eighteen patients with IBS-D underwent 24-hour excretion studies after oral 13C-mannitol and lactulose. RESULTS: Baseline sugars (>3-fold above lower limits of quantitation) were identified in the 3 studies: 12C-mannitol in all participants; sucralose in 4-8, and rhamnose in 1-3. Median excretions/24 h (percentage of administered dose) for 13C-mannitol, rhamnose, lactulose, and sucralose were ∼30%, ∼15%, 0.32%, and 2.3%, respectively. 13C-mannitol and rhamnose reflected mainly small intestinal permeability. Intraindividual saccharide excretions were consistent, with minor differences with 16.25 g vs 32.5 g fiber diets. Median interindividual coefficient of variation was 76.5% (10-90 percentile: 34.6-111.0). There were no significant effects of sex, age, or body mass index on permeability measurements in health. 13C-mannitol measurements are feasible in IBS-D. CONCLUSIONS: Baseline 12C-mannitol excretion precludes its use; 13C-mannitol is the preferred probe for small intestinal permeability.

Role of Lactulose Rhamnose Permeability Test in Assessing Small Bowel Mucosal Damage in Children with Celiac Disease.

Increased gut permeability has been suggested in patients with celiac disease (CD). We aimed to compare gut permeability in children using the lactulose/rhamnose permeability test. We prospectively recruited 55 children into 3 groups; 27 in group 1 (children with newly diagnosed celiac disease, 12 in group 2 (siblings of children with celiac disease) and 16 in group 3 (control group). The median age of participants was 11 years 2 months in group 1, 9 years 5 months in group 2 and 10 years 3 months in group 3. Standardized median delta rhamnose was lower in CD group as compared to control group (147.5 vs 3153.1, P = 0.040). The low median rhamnose absorption in children with celiac disease as compared to other groups suggests that this test can differentiate between damaged and healthy mucosa, hence can it potentially can be used as a noninvasive test of mucosal healing in children with celiac disease.

Drug Tissue Distribution of TUDCA From a Biodegradable Suprachoroidal Implant versus Intravitreal or Systemic Delivery in the Pig Model.

Purpose: To determine local ocular tissue levels of the bile acid, tauroursodeoxycholic acid (TUDCA), in the pig model using oral, intravenous (IV), intravitreal injection (IVitI) and low- and high-dose suprachoroidal, sustained-release implants (SCI-L or SCI-H). Methods: Forty-six pigs (92 globes) were included in the study. TUDCA was delivered orally in 5 pigs, IV in 4, IVitI in 6, SCI-L in 17, and SCI-H in 14. Testing timeframes varied from the same day (within minutes) for IV; 1 to 6 days, oral; and 1 to 4 weeks, IVitI and SCI. Enucleated globes were dissected, specimens from specific tissues were separated, and TUDCA was extracted and quantified using mass spectrometry. Results: The highest TUDCA tissue levels occurred after IV delivery in the macula (252 ± 238 nM) and peripheral retina (196 ± 171 nM). Macular choroid and peripheral choroid levels were also high (1032 ± 1269 and 1219 ± 1486 nM, respectively). For IVitI delivery, macular levels at day 6 were low (0.5 ± 0.5 nM), whereas peripheral choroid was higher (15.3 ± 16.7 nM). Neither the SCI-L nor SCI-H implants delivered meaningful macular doses (≤1 nM); however, peripheral retina and choroid levels were significantly higher. Bile acid isoforms were found in the serum specimens. Conclusions: The highest TUDCA tissue levels in the pig model were obtained using IV delivery. Oral delivery was associated with reasonable tissue levels. Local delivery (IVitI and SCI) was able to achieve measurable local ocular tissue levels. Translational Relevance: Diffusional kinetics from the suprachoroidal space follow the choroidal blood flow, away from the macula and toward the periphery.

CSF biomarkers in Olmsted County: Evidence of 2 subclasses and associations with demographics.

OBJECTIVE: We studied interrelationships between CSF biomarkers and associations with APOE ε4 genotype, demographic variables, vascular variables, and clinical diagnosis in Olmsted County, Minnesota. METHODS: We included 774 Mayo Clinic Study of Aging participants (693 cognitively unimpaired [CU]; 71 with mild cognitive impairment [MCI]). CSF ß-amyloid 42 (Aß42), total tau (t-tau), and hyperphosphorylated tau (p-tau) were analyzed using Aß42 CSF, t-tau CSF, and p-tau (181P) CSF electrochemiluminescence immunoassays. Bivariate mixture models were used to evaluate latent classes. We used linear regression models to evaluate independent associations of APOE ε4, demographic factors, cardiovascular risk, and diagnosis with CSF biomarker levels. Results were weighted back to the Olmsted County population. RESULTS: Interrelationships between CSF Aß42 and p-tau/t-tau were consistent with 2 latent classes in the general population. In subgroup 1 (n = 547 [71%]), we found a strong positive correlation between Aß42 and p-tau (ρ = 0.81), while the correlation was much smaller in group 2 (ρ = 0.26, n = 227 [29%]). Group 2 was associated with older age, APOE ε4 genotype, a diagnosis of MCI, and elevated amyloid PET. Overall, APOE ε4 genotype and MCI were associated with Aß42, while age was associated with p-tau/t-tau. There were no associations with sex, education, or vascular risk. CONCLUSION: We hypothesize the population without dementia can be subdivided into participants with and without biological Alzheimer disease (AD) based on the combination of CSF Aß42 and p-tau/t-tau (represented also by the p-tau/t-tau/Aß42 ratio). In those without biological AD, common factors such as CSF dynamics may cause a positive correlation between CSF Aß42 and p-tau/t-tau, while AD leads to dissociation of these proteins.

Serine proteases as luminal mediators of intestinal barrier dysfunction and symptom severity in IBS.

OBJECTIVE: The intestinal lumen contains several proteases. Our aim was to determine the role of faecal proteases in mediating barrier dysfunction and symptoms in IBS. DESIGN: 39 patients with IBS and 25 healthy volunteers completed questionnaires, assessments of in vivo permeability, ex vivo colonic barrier function in Ussing chambers, tight junction (TJ) proteins, ultrastructural morphology and 16 s sequencing of faecal microbiota rRNA. A casein-based assay was used to measure proteolytic activity (PA) in faecal supernatants (FSNs). Colonic barrier function was determined in mice (ex-germ free) humanised with microbial communities associated with different human PA states. RESULTS: Patients with IBS had higher faecal PA than healthy volunteers. 8/20 postinfection IBS (PI-IBS) and 3/19 constipation- predominant IBS had high PA (>95th percentile). High-PA patients had more and looser bowel movements, greater symptom severity and higher in vivo and ex vivo colonic permeability. High-PA FSNs increased paracellular permeability, decreased occludin and increased phosphorylated myosin light chain (pMLC) expression. Serine but not cysteine protease inhibitor significantly blocked high-PA FSN effects on barrier. The effects on barrier were diminished by pharmacological or siRNA inhibition of protease activated receptor-2 (PAR-2). Patients with high-PA IBS had lower occludin expression, wider TJs on biopsies and reduced microbial diversity than patients with low PA. Mice humanised with high-PA IBS microbiota had greater in vivo permeability than those with low-PA microbiota. CONCLUSION: A subset of patients with IBS, especially in PI-IBS, has substantially high faecal PA, greater symptoms, impaired barrier and reduced microbial diversity. Commensal microbiota affects luminal PA that can influence host barrier function.

Sex differences in NSAID-induced perturbation of human intestinal barrier function and microbiota.

Intestinal barrier function and microbiota are integrally related and play critical roles in maintenance of host physiology. Sex is a key biologic variable for several disorders. Our aim was to determine sex-based differences in response to perturbation and subsequent recovery of intestinal barrier function and microbiota in healthy humans. Twenty-three volunteers underwent duodenal biopsies, mucosal impedance, and in vivo permeability measurement. Permeability testing was repeated after administration of indomethacin, then 4 to 6 wk after its discontinuation. Duodenal and fecal microbiota composition was determined using 16S rRNA amplicon sequencing. Healthy women had lower intestinal permeability and higher duodenal and fecal microbial diversity than healthy men. Intestinal permeability increases after indomethacin administration in both sexes. However, only women demonstrated decreased fecal microbial diversity, including an increase in Prevotella abundance, after indomethacin administration. Duodenal microbiota composition did not show sex-specific changes. The increase in permeability and microbiota changes normalized after discontinuation of indomethacin. In summary, women have lower intestinal permeability and higher microbial diversity. Intestinal permeability is sensitive to perturbation but recovers to baseline. Gut microbiota in women is sensitive to perturbation but appears to be more stable in men. Sex-based differences in intestinal barrier function and microbiome should be considered in future studies.-Edogawa, S., Peters, S. A., Jenkins, G. D., Gurunathan, S. V., Sundt, W. J., Johnson, S., Lennon, R. J., Dyer, R. B., Camilleri, M., Kashyap, P. C., Farrugia, G., Chen, J., Singh, R. J., Grover, M. Sex differences in NSAID-induced perturbation of human intestinal barrier function and microbiota.

Measurement of gut permeability using fluorescent tracer agent technology.

The healthy gut restricts macromolecular and bacterial movement across tight junctions, while increased intestinal permeability accompanies many intestinal disorders. Dual sugar absorption tests, which measure intestinal permeability in humans, present challenges. Therefore, we asked if enterally administered fluorescent tracers could ascertain mucosal integrity, because transcutaneous measurement of differentially absorbed molecules could enable specimen-free evaluation of permeability. We induced small bowel injury in rats using high- (15 mg/kg), intermediate- (10 mg/kg), and low- (5 mg/kg) dose indomethacin. Then, we compared urinary ratios of enterally administered fluorescent tracers MB-402 and MB-301 to urinary ratios of sugar tracers lactulose and rhamnose. We also tested the ability of transcutaneous sensors to measure the ratios of absorbed fluorophores. Urinary fluorophore and sugar ratios reflect gut injury in an indomethacin dose dependent manner. The fluorophores generated smooth curvilinear ratio trajectories with wide dynamic ranges. The more chaotic sugar ratios had narrower dynamic ranges. Fluorophore ratios measured through the skin distinguished indomethacin-challenged from same day control rats. Enterally administered fluorophores can identify intestinal injury in a rat model. Fluorophore ratios are measureable through the skin, obviating drawbacks of dual sugar absorption tests. Pending validation, this technology should be considered for human use.

Xenoantigenicity of porcine decellularized valves.

BACKGROUND: The xenoantigenicity of porcine bioprosthetic valves is implicated as an etiology leading to calcification and subsequent valve failure. Decellularization of porcine valves theoretically could erase the antigenicity of the tissue leading to more durable prosthetic valves, but the effectiveness of decellularization protocols in regard to completely removing antigens has yet to be verified. Our hypothesis was that decellularization would remove the more abundant α-gal antigens but not remove all the non α-gal antigens, which could mount a response. METHODS: Porcine aortic valves were decellularized with 1% sodium dodecyl sulfate for 4 days. Decellularized cusps were evaluated for α-gal epitopes by ELISA. To test for non α-gal antigens, valves were implanted into sheep. Serum was obtained from the sheep preoperatively and 1 week, 1 month, and 2 months postoperatively. This serum was utilized for anti-porcine antibody staining and for quantification of anti-pig IgM and IgG antibodies and complement. RESULTS: Decellularized porcine cusps had 2.8 ± 2.0% relative α-gal epitope as compared to fresh porcine aortic valve cusps and was not statistically significantly different (p = 0.4) from the human aortic valve cusp which had a 2.0 ± 0.4% relative concentration. Anti-pig IgM and IgG increased postoperatively from baseline levels. Preoperatively anti-pig IgM was 27.7 ± 1.7 µg/mL and it increased to 71.9 ± 12.1 µg/mL average of all time points postoperatively (p = 0.04). Preoperatively anti-pig IgG in sheep serum was 44.9 ± 1.5 µg/mL and it increased to 72.6 ± 6.0 µg/mL average of all time points postoperatively (p = 0.01). There was a statistically significant difference (p = 0.00007) in the serum C1q concentration before valve implantation (2.5 ± 0.2 IU/mL) and at averaged time points after valve implantation (5.3 ± 0.3 IU/mL). CONCLUSIONS: Decellularization with 1% sodium dodecyl sulfate does not fully eliminate non α-gal antigens; however, significant reduction in α-gal presence on decellularized cusps was observed. Clinical implications of the non α-gal antigenic response are yet to be determined. As such, evaluation of any novel decellularized xenografts must include rigorous antigen testing prior to human trials.

Constipation-Predominant Irritable Bowel Syndrome Females Have Normal Colonic Barrier and Secretory Function.

OBJECTIVES: The objective of this study was to determine whether constipation-predominant irritable bowel syndrome (IBS-C) is associated with changes in intestinal barrier and secretory function. METHODS: A total of 19 IBS-C patients and 18 healthy volunteers (all females) underwent saccharide excretion assay (0.1 g 13C mannitol and 1 g lactulose), measurements of duodenal and colonic mucosal barrier (transmucosal resistance (TMR), macromolecular and Escherichia coli Bio-Particle translocation), mucosal secretion (basal and acetylcholine (Ach)-evoked short-circuit current (Isc)), in vivo duodenal mucosal impedance, circulating endotoxins, and colonic tight junction gene expression. RESULTS: There were no differences in the in vivo measurements of barrier function between IBS-C patients and healthy controls: cumulative excretion of 13C mannitol (0-2 h mean (s.e.m.); IBS-C: 12.1 (0.9) mg vs. healthy: 13.2 (0.8) mg) and lactulose (8-24 h; IBS-C: 0.9 (0.5) mg vs. healthy: 0.5 (0.2) mg); duodenal impedance IBS-C: 729 (65) Ω vs. healthy: 706 (43) Ω; plasma mean endotoxin activity level IBS-C: 0.36 (0.03) vs. healthy: 0.35 (0.02); and in colonic mRNA expression of occludin, zonula occludens (ZO) 1-3, and claudins 1-12 and 14-19. The ex vivo findings were consistent, with no group differences: duodenal TMR (IBS-C: 28.2 (1.9) Ω cm2 vs. healthy: 29.8 (1.9) Ω cm2) and colonic TMR (IBS-C: 19.1 (1.1) Ω cm2 vs. healthy: 17.6 (1.7) Ω cm2); fluorescein isothiocyanate (FITC)-dextran (4 kDa) and E. coli Bio-Particle flux. Colonic basal Isc was similar, but duodenal basal Isc was lower in IBS-C (43.5 (4.5) µA cm-2) vs. healthy (56.9 (4.9) µA cm-2), P=0.05. Ach-evoked ΔIsc was similar. CONCLUSIONS: Females with IBS-C have normal colonic barrier and secretory function. Basal duodenal secretion is decreased in IBS-C.

Pilot study of small bowel mucosal gene expression in patients with irritable bowel syndrome with diarrhea.

Prior studies in with irritable bowel syndrome with diarrhea (IBS-D) patients showed immune activation, secretion, and barrier dysfunction in jejunal or colorectal mucosa. We measured mRNA expression by RT-PCR of 91 genes reflecting tight junction proteins, chemokines, innate immunity, ion channels, transmitters, housekeeping genes, and controls for DNA contamination and PCR efficiency in small intestinal mucosa from 15 IBS-D and 7 controls (biopsies negative for celiac disease). Fold change was calculated using 2((-ΔΔCT)) formula. Nominal P values (P < 0.05) were interpreted with false detection rate (FDR) correction (q value). Cluster analysis with Lens for Enrichment and Network Studies (LENS) explored connectivity of mechanisms. Upregulated genes (uncorrected P < 0.05) were related to ion transport (INADL, MAGI1, and SONS1), barrier (TJP1, 2, and 3 and CLDN) or immune functions (TLR3, IL15, and MAPKAPK5), or histamine metabolism (HNMT); downregulated genes were related to immune function (IL-1ß, TGF-ß1, and CCL20) or antigen detection (TLR1 and 8). The following genes were significantly upregulated (q < 0.05) in IBS-D: INADL, MAGI1, PPP2R5C, MAPKAPK5, TLR3, and IL-15. Among the 14 nominally upregulated genes, there was clustering of barrier and PDZ domains (TJP1, TJP2, TJP3, CLDN4, INADL, and MAGI1) and clustering of downregulated genes (CCL20, TLR1, IL1B, and TLR8). Protein expression of PPP2R5C in nuclear lysates was greater in patients with IBS-D and controls. There was increase in INADL protein (median 9.4 ng/ml) in patients with IBS-D relative to controls (median 5.8 ng/ml, P > 0.05). In conclusion, altered transcriptome (and to lesser extent protein) expression of ion transport, barrier, immune, and mast cell mechanisms in small bowel may reflect different alterations in function and deserves further study in IBS-D.

Interleukins 6 and 8 and abdominal fat depots are distinct correlates of lipid moieties in healthy pre- and postmenopausal women.

Available data associate lipids concentrations in men with body mass index, anabolic steroids, age, and certain cytokines. Data were less clear in women, especially across the full adult lifespan, and when segmented by premenopausal and postmenopausal status. SUBJECTS: 120 healthy women (60 premenopausal and 60 postmenopausal) in Olmsted County, MN, USA, a stable well studied clinical population. Dependent variables: measurements of 10 h fasting high-density lipoprotein cholesterol, total cholesterol, low-density lipoprotein cholesterol, and triglycerides. INDEPENDENT VARIABLES: testosterone, estrone, estradiol, 5-alpha-dihydrotestosterone, and sex-hormone binding globulin (by mass spectrometry); insulin, glucose, and albumin; abdominal visceral, subcutaneous, and total abdominal fat [abdominal visceral fat, subcutaneous fat, total abdominal fat by computerized tomography scan]; and a panel of cytokines (by enzyme-linked immunosorbent assay). Multivariate forward-selection linear-regression analysis was applied constrained to P < 0.01. Lifetime data: High-density lipoprotein cholesterol was correlated jointly with age (P < 0.0001, positively), abdominal visceral fat (P < 0.0001, negatively), and interleukin-6 (0.0063, negatively), together explaining 28.1 % of its variance (P = 2.3 × 10-8). Total cholesterol was associated positively with multivariate age only (P = 6.9 × 10-4, 9.3 % of variance). Triglycerides correlated weakly with sex-hormone binding globulin (P = 0.0115), and strongly with abdominal visceral fat (P < 0.0001), and interleukin-6 (P = 0.0016) all positively (P = 1.6 × 10-12, 38.9 % of variance). Non high-density lipoprotein cholesterol and low-density lipoprotein cholesterol correlated positively with both total abdominal fat and interleukin-8 (P = 2.0 × 10-5, 16.9 % of variance; and P = 0.0031, 9.4 % of variance, respectively). Premenopausal vs. postmenopausal comparisons identified specific relationships that were stronger in premenopausal than postmenopausal individuals, and vice versa. Age was a stronger correlate of low-density lipoprotein cholesterol; interleukin-6 of triglycerides and high-density lipoprotein; and both sex-hormone binding globulin and total abdominal fat of non high-density lipoprotein cholesterol in premenopausal than postmenopausal women. Conversely, sex-hormone binding globulin, abdominal visceral fat, interleukin-8, adiponectin were stronger correlates of triglycerides; abdominal visceral fat, and testosterone of high-density lipoprotein cholesterol; and age of both non high-density lipoprotein and low-density lipoprotein in postmenopausal than premenopausal women. Our data delineate correlations of total abdominal fat and interleukin-8 (both positively) with non high-density lipoprotein cholesterol and low-density lipoprotein cholesterol in healthy women across the full age range of 21-79 years along with even more specific associations in premenopausal and postmenopausal individuals. Whether some of these outcomes reflect causal relationships would require longitudinal and interventional or genetic studies.

Direct Evidence of Acetaminophen Interference with Subcutaneous Glucose Sensing in Humans: A Pilot Study.

BACKGROUND: Recent advances in accuracy and reliability of continuous glucose monitoring (CGM) devices have focused renewed interest on the use of such technology for therapeutic dosing of insulin without the need for independent confirmatory blood glucose meter measurements. An important issue that remains is the susceptibility of CGM devices to erroneous readings in the presence of common pharmacologic interferences. We report on a new method of assessing CGM sensor error to pharmacologic interferences using the example of oral administration of acetaminophen. MATERIALS AND METHODS: We examined the responses of several different Food and Drug Administration-approved and commercially available CGM systems (Dexcom [San Diego, CA] Seven(®) Plus™, Medtronic Diabetes [Northridge, CA] Guardian(®), and Dexcom G4(®) Platinum) to oral acetaminophen in 10 healthy volunteers without diabetes. Microdialysis catheters were placed in the abdominal subcutaneous tissue. Blood and microdialysate samples were collected periodically and analyzed for glucose and acetaminophen concentrations before and after oral ingestion of 1 g of acetaminophen. We compared the response of CGM sensors with the measured acetaminophen concentrations in the blood and interstitial fluid. RESULTS: Although plasma glucose concentrations remained constant at approximately 90 mg/dL (approximately 5 mM) throughout the study, CGM glucose measurements varied between approximately 85 to 400 mg/dL (from approximately 5 to 22 mM) due to interference from the acetaminophen. The temporal profile of CGM interference followed acetaminophen concentrations measured in interstitial fluid (ISF). CONCLUSIONS: This is the first direct measurement of ISF concentrations of putative CGM interferences with simultaneous measurements of CGM performance in the presence of the interferences. The observed interference with glucose measurements in the tested CGM devices coincided temporally with appearance of acetaminophen in the ISF. The method applied here can be used to determine the susceptibility of current and future CGM systems to interference from acetaminophen or other exogenous pharmacologic agents.

Immunologic and mass-spectrometric estimates of SHBG concentrations in healthy women.

OBJECTIVE: Sex-hormone binding globulin (SHBG) concentrations across the adult female lifespan are not well defined. To address this knowledge gap, SHBG was quantified by both immunological and criterion methods, viz, mass spectrometry (MS). SETTING: Center for Translational Science Activities (CTSA). PARTICIPANTS: Healthy nonpregnant women (N=120) ages 21 to 79 years. OUTCOMES: SHBG, testosterone (T), estradiol (E2) and estrone (E1) each determined by MS. Uni- and multivariate regression of SHBG concentrations on age, body mass index (BMI), total and visceral abdominal fat (TAF, AVF), albumin, glucose, insulin, sex steroids, selected cytokines, blood pressure, and lipids. RESULTS: By univariate regression, MS-estimated SHBG correlated negatively with BMI, TAF, AVF, insulin, free T and bioavailable T (bio T) (each P≤10(-4)), but not with blood pressure or lipids. By stepwise multivariate regression analysis, free and total T (both positive) and bio T (negative) were correlated with SHBG in all 4 assays (each P<10(-15), R(2)≥0.481). In addition, TAF and BMI were negatively associated with SHBG (P≤0.0066) in 2 SHBG assays, and estrone and IL-8 with SHBG weakly (P≤0.035) in one SHBG assay each. When nonsignificant cytokines were excluded, SHBG was jointly associated with AVF, total T and HDL (P<10(-9), R(2)=0.358). CONCLUSION: According to MS, three metabolic factors, T, AVF and HDL, together explain more than one-third of the interindividual variation in SHBG levels. We speculate that these measures reflect insulin action.

Immunological and mass spectrometric assays of SHBG: consistent and inconsistent metabolic associations in healthy men.

CONTEXT: SHBG concentrations correlate inconsistently with metabolic parameters. HYPOTHESIS: SHBG assay platforms contribute to nonuniformities according to the literature. DESIGN: The design of the study was a noninterventional quantification of SHBG by two immuno- and two mass spectrometric assays and abdominal visceral fat by computed tomography scan. SETTING: The study was conducted at the Center for Translational Science Activities. PARTICIPANTS: Healthy men (n=120) aged 18-80 years with a body mass index of 20-43 kg/m2 participated I the study. OUTCOMES: Outcomes of the study included a correlation of log SHBG with age, metabolic surrogates [body mass index, albumin, glucose, insulin, abdominal (total and visceral) fat, homeostasis model assessment insulin resistance index], sex steroids (estrone, 17ß-estradiol, T, and dihydrotestosterone by mass spectrometry), and adipocytokines (IL-1ß, IL-6, IL-8, IL-10 and IL-12, TNF-α, and adiponectin). RESULTS: By univariate regression, age (P<10(-4)), dihydrotestosterone (P<10(-4)), T (P≤.00022), and adiponectin (P≤.0084) were positive correlates, and insulin and homeostasis model assessment insulin resistance index were negative correlates (P≤.0060) of SHBG in all four assays. Stepwise multivariate analysis unveiled that age and T together could explain 38.1%-52.5% of the statistical variance in SHBG in all assays (P<10(-11)). Multivariate regression without sex steroids unveiled that age (P<10(-5)) and insulin (P<10(-3)) are jointly associated with SHBG levels in the four assays with overall R2=0.215-0.293 and P<10(-6). In one immunological SHBG assay each, abdominal visceral fat and adiponectin were weak multivariates also. CONCLUSION: Immunological and mass spectrometric SHBG assays yield both consistent and inconsistent correlations with key metabolic variables in healthy men, thereby potentially explaining earlier inconsistencies in the literature.

Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea.

BACKGROUND & AIMS: Variations in genes that regulate bile acid (BA) synthesis are associated with colonic transit in patients with irritable bowel syndrome (IBS). We investigated features of BA synthesis and excretion and genetic features of patients with different types of IBS. METHODS: In 26 healthy volunteers, 26 patients with IBS and constipation (IBS-C), and 26 with IBS and diarrhea (IBS-D), we measured serum levels of 7α-hydroxy-4-cholesten-3-one (C4; a surrogate for BA synthesis) and fibroblast growth factor (FGF) 19 (an ileal hormone that downregulates BA synthesis). For stool samples, we measured concentration of BA, weight, and amount of fat when participants were given high-fat diets. Spearman correlations were used to explore relationships among factors. We analyzed 1 polymorphism in Klotho-ß (KLB) and 3 in fibroblast growth factor receptor-4 (FGFR4) for all members of each group using analysis of covariance. RESULTS: The concentration of BA in stool was associated with group (for a comparison of 3 groups; P = .057); it was higher in patients with IBS-D than IBS-C (P = .017). The serum level of C4 was higher in patients with IBS-D than IBS-C (P = .02) or healthy volunteers (P = .01); 38% of patients with IBS-D had increased serum levels of C4, compared with healthy volunteers. Serum level of C4 correlated with stool concentration of BA (rs = 0.606; P < .001), serum FGF19 (rs = -0.324; P = .007), and stool weight (rs = 0.366; P = .003). Stool concentration of BA correlated with weight (rs = 0.737; P < .001) and level of fat (rs = 0.528; P < .001). Body mass index correlated with serum level of C4 (rs = 0.423, P < .001) and stool concentration of BA (rs = 0.507, P < .001), and was higher in patients with IBS-D compared with other groups (overall P = .036). FGFR4 rs1966265 was associated with stool level of BA (P = .032). CONCLUSIONS: Patients with IBS-D have greater body mass index and synthesize and excrete higher levels of BA than individuals with IBS-C or healthy volunteers. Serum levels of C4 might be used to identify patients with IBS-D who have BA malabsorption; studies are needed to determine if some patients have a genetic predisposition to this disorder.

Candidate serum biomarkers for prostate adenocarcinoma identified by mRNA differences in prostate tissue and verified with protein measurements in tissue and blood.

BACKGROUND: Improved tests are needed for detection and management of prostate cancer. We hypothesized that differential gene expression in prostate tissue could help identify candidate blood biomarkers for prostate cancer and that blood from men with advanced prostate disease could be used to verify the biomarkers presence in circulation. METHODS: We identified candidate markers using mRNA expression patterns from laser-capture microdissected prostate tissue and confirmed tissue expression using immunohistochemistry (IHC) for the subset of candidates having commercial antisera. We analyzed tissue extracts with tandem mass spectrometry (MS/MS) and measured blood concentrations using immunoassays and MS/MS of trypsin-digested, immunoextracted peptides. RESULTS: We selected 35 novel candidate prostate adenocarcinoma biomarkers. For all 13 markers having commercial antisera for IHC, tissue expression was confirmed; 6 showed statistical discrimination between nondiseased and malignant tissue, and only 5 were detected in tissue extracts by MS/MS. Sixteen of the 35 candidate markers were successfully assayed in blood. Four of 8 biomarkers measured by ELISA and 3 of 10 measured by targeted MS showed statistically significant increases in blood concentrations of advanced prostate cancer cases, compared with controls. CONCLUSIONS: Seven novel biomarkers identified by gene expression profiles in prostate tissue were shown to have statistically significant increased concentrations in blood from men with advanced prostate adenocarcinoma compared with controls: apolipoprotein C1, asporin, cartilage oligomeric matrix protein, chemokine (C-X-C motif) ligand 11 (CXCL11), CXCL9, coagulation factor V, and proprotein convertase subtilisin/kexin 6.