Differential Thermal Isomerization: Its Role in the Analysis of Vitamin D3 in Foods.

BACKGROUND: For nutritional purposes, the measurement of vitamin D3 (defined as the sum of vitamin D3 and previtamin D3) is required to obtain an accurate and reliable estimate of its content in foods. An often neglected aspect in the development of methods for the analysis of vitamin D3 is accounting for any potential analytical bias in the results associated with differential thermal isomerization between previtamin D and vitamin D. CONCLUSIONS: For LC-UV methods using a vitamin D2 internal standard, cold saponification, or direct lipid extraction techniques should be avoided, unless chromatographic separation of vitamin D2, vitamin D3, and their previtamin forms is achieved so that UV absorbance corrections can be made. For both LC-UV and LC-MS methods using calciferol internal standards, the simplest solution to avoid analytical bias due to the presence of previtamin D is to utilize heating conditions (typically during saponification) such that previtamin D and vitamin D in the sample and the internal standard reach an equivalent equilibrium state prior to instrumental analysis. Only under such circumstances is the integration of previtamin D unnecessary to obtain accurate results for vitamin D3. HIGHLIGHTS: A detailed discussion of the quantitation of vitamin D3 in food with concise recommendations for avoiding measurement bias as a consequence of differential thermal isomerization.

Comparison of LC-MS/MS and Enzymatic Methods for the Determination of Total Choline and Total Carnitine in Infant Formula and Milk Products.

BACKGROUND: Choline and l-carnitine are classified as pseudo-vitamins because of their conditionally essential status. As they are involved in multiple physiological metabolic pathways in the human body, they are routinely fortified in infant and adult nutritional formulas. OBJECTIVE: The performance of an LC-MS/MS method for the analysis of choline and carnitine, compared with enzymatic methods in routine use for the analysis of total carnitine and total choline, is described. METHOD: Powder samples were reconstituted, with release of carnitine and choline facilitated by both acid and alkaline hydrolysis and the extract analyzed by LC-MS/MS. Quantitation was by internal standard technique using deuterium-labeled carnitine and deuterium-labeled choline. RESULTS: Method range, specificity, sensitivity, precision, recovery, accuracy, and ruggedness were assessed for milk powders, infant formulas, and soy- and milk-based nutritional products. Spike recoveries of 94.0-108.4% were obtained for both total carnitine and choline, and no statistical bias (α = 0.05) between measured results and certified values (choline: P = 0.36; free carnitine: P = 0.67) was found for NIST 1849a certified reference material (NIST1849a). Precision, as repeatability relative standard deviation (RSD), was 2.0% RSDr for total carnitine and 1.7% RSDr for total choline. Equivalent results for total choline and total carnitine were obtained by LC-MS/MS and enzymatic methods (n = 30). CONCLUSIONS: The described LC-MS/MS method is fit for purpose for routine product compliance release testing environments. This validation study has confirmed that alternative enzymatic assays can be used with confidence in laboratories in which LC-MS/MS platforms are unavailable. HIGHLIGHTS: An LC-MS/MS method was evaluated and found to be fit-for-purpose for routine product compliance release testing of infant formula. The LC-MS/MS method was compared with enzymatic methods for the analysis of total carnitine and total choline. Alternative enzymatic assays can be used with confidence in laboratories in which LC-MS/MS platforms are unavailable.

Current Methods for the Analysis of Selected Novel Nutrients in Infant Formulas and Adult Nutritionals.

Infant formula is designed to provide the human infant with a sole source of nutrition and it is intended to imitate breast milk. In recent years, advances in the science of infant nutrition have led to an increasing number of novel ingredients that are supplemented into infant formula. As the list of these nutritionally important nutrients is lengthy, this review summarizes contemporary analytical methods that have been applied to a representative selection (lutein, carnitine, choline, nucleotides, inositol, taurine, sialic acid, gangliosides, triacylglycerides, oligosaccharides, α-lactalbumin, and lactoferrin).

Determination of vitamin A and vitamin E esters in infant formulae and fortified milk powders by HPLC: Use of internal standardisation.

An HPLC method is described using normal phase conditions with an unbonded silica column to determine concentrations of supplementary vitamin A and vitamin E esters and ß-carotene in infant formulae. The method utilises selective dual-channel fluoresence for vitamins A and E and visible absorbance for ß-carotene. An attribute of the method is the use of retinol propionate, α-tocopheryl propionate and all-E-ß-apo-8'-carotenoic acid ethyl ester internal standards to compensate for analytical variations associated with these labile vitamins. Extraction is performed without saponification, with the aid of protease to remove vitamin encaspsulation and facilitate vitamin partition into hydrocarbon solvent. Figures of merit indicate the method is suitable for its intended purpose in the highly regulated infant formula environment, including liquid formulations. The method is extendable to whole milk powders where total vitamin A content data can be calculated by summing the innate long-chain vitamin A esters with the added esters.

Differential expression of VEGF ligands and receptors in prostate cancer.

BACKGROUND: Prostate cancer disseminates to regional lymph nodes, however the molecular mechanisms responsible for lymph node metastasis are poorly understood. The vascular endothelial growth factor (VEGF) ligand and receptor family have been implicated in the growth and spread of prostate cancer via activation of the blood vasculature and lymphatic systems. The purpose of this study was to comprehensively examine the expression pattern of VEGF ligands and receptors in the glandular epithelium, stroma, lymphatic vasculature and blood vessels in prostate cancer. METHODS: The localization of VEGF-A, VEGF-C, VEGF-D, VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 was examined in cancerous and adjacent benign prostate tissue from 52 subjects representing various grades of prostate cancer. RESULTS: Except for VEGFR-2, extensive staining was observed for all ligands and receptors in the prostate specimens. In epithelial cells, VEGF-A and VEGFR-1 expression was higher in tumor tissue compared to benign tissue. VEGF-D and VEGFR-3 expression was significantly higher in benign tissue compared to tumor in the stroma and the endothelium of lymphatic and blood vessels. In addition, the frequency of lymphatic vessels, but not blood vessels, was lower in tumor tissue compared with benign tissue. CONCLUSIONS: These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer.

Determination of antioxidant activity in foods and beverages by reaction with 2,2'-diphenyl-1-picrylhydrazyl (DPPH): collaborative study First Action 2012.04.

A colorimetric method for the determination of total antioxidant activity in a variety of foods and beverages was validated in both a single-laboratory validation and a collaborative laboratory validation study. The procedure involved extraction of the antioxidants directly into a methanol-water solution containing a known amount of 2,2'-diphenyl-1-picrylhydrazyl (DPPH), thus promoting the rapid reaction of extracted materials with DPPH. The reaction was monitored by spectrophotometric measurement of the absorbance loss at 517 nm. Antioxidant activity was quantified relative to a dilution series of vitamin E analog standards (Trolox), which were analyzed in parallel simultaneously with the food and beverage samples. The antioxidant activities of the samples ranged from 131 to 131 000 micromole Trolox equivalents/100 g. Statistical analysis of the results showed that nine of the 11 matrixes gave acceptable HorRat values, indicating that the method performed well in these cases. The acceptable matrixes include pomegranate juice, blueberry juice, carrot juice, green tea, wine, rosemary spice, ready-to-eat cereal, and yogurt. Two samples failed the HorRat test: the first was an almond milk that had an antioxidant level below the practical LOQ for the method; the second was a sample of canola oil with added omega-3 fatty acid that was immiscible in the reaction medium.

Down-regulation of intra-hepatic T-cell signaling associated with GB virus C in a HCV/HIV co-infected group with reduced liver disease.

BACKGROUND & AIMS: Studies have shown that GB virus C (GBV-C) infection leads to reduced liver disease in hepatitis C virus (HCV)/human immunodeficiency virus (HIV) co-infection. Considering that the underlying mechanism(s) are unknown, we aim to identify differential gene and protein expression associated with GBV-C in HCV/HIV co-infection that may be responsible for reduced liver disease. METHODS: Liver, peripheral blood mononuclear cells (PBMCs), and plasma samples were collected from 43 HCV/HIV patients. Plasma was tested for GBV-C RNA by RT-PCR with NS5B gene primers. A microarray was performed on the liver and RT-qPCRs on the liver/PBMC samples. Hepatic protein expression was measured by immunohistochemistry. RESULTS: Sixteen out of 43 patients had GBV-C RNA. GBV-C was associated with reduced hepatic fibrosis (p=0.005) and inflammation (p=0.007). The microarray analysis of the liver samples (n=10) showed down-regulation of genes critical to intra-hepatic T-cell signaling associated with GBV-C. Quantitative RT-PCR of the liver samples (n=13) confirmed the down-regulation of lymphocyte-specific protein tyrosine kinase (LCK) (p=0.02) and docking protein 2 (DOK2) (p=0.04). No differences in the expression levels of these genes were observed in PBMCs (n=22) according to the GBV-C status. The hepatic expression of the LCK protein, measured by immunohistochemistry (n=36), was decreased in CD3-positive T-cells within portal tracts associated with GBV-C (p=0.003). This remained significant in multivariate analysis controlling for hepatic fibrosis and inflammation (p=0.027). No differences were observed in plasma cytokine concentrations (n=25) or ex-vivo peripheral T-cell responses (n=13) versus GBV-C status. CONCLUSIONS: GBV-C infection is associated with down-regulation of critical genes involved in intra-hepatic T-cell signaling in HCV/HIV co-infection. This may be relevant to the pathogenesis of reduced HCV-related liver disease in HIV co-infection.

Virus-specific T-cell immunity correlates with control of GB virus B infection in marmosets.

GB virus B (GBV-B) is a hepatotropic virus that is closely related to hepatitis C virus (HCV). GBV-B causes acute hepatitis in infected marmosets and tamarins and is therefore a useful small-animal model for the study of HCV. We investigated virus-specific T-cell responses in marmosets infected with GBV-B. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay responses in the peripheral blood of two marmosets were assessed throughout the course of GBV-B infection. These T-cell responses were directed against the GBV-B nonstructural proteins 3 (NS3), 4A (NS4A), and 5B (NS5B), and their appearance was temporally associated with clearance of viremia. These marmosets were then rechallenged with GBV-B at least 3 months after clearance of the primary infection to determine if the animals were protected from reinfection. There was no detectable viremia following reinfection, although a sharp increase in T-cell responses against GBV-B proteins was observed. Epitope mapping of T-cell responses to GBV-B was performed with liver and blood samples from both marmosets after rechallenge with GBV-B. Three shared, immunodominant T-cell epitopes within NS3 were identified in animals with multiple common major histocompatibility complex class I alleles. IFN-gamma ELISPOT responses were also detected in the livers of two marmosets that had resolved a primary GBV-B infection. These responses were high in frequency and were directed against epitopes within GBV-B NS3, NS4A, and NS5B proteins. These results indicate that virus-specific T-cell responses are detectable in the liver and blood of GBV-B-infected marmosets and that the clearance of GBV-B is associated with the appearance of these responses.

Defining target antigens for CD25+ FOXP3 + IFN-gamma- regulatory T cells in chronic hepatitis C virus infection.

The mechanism behind the apparent lack of effective antiviral immune responses in chronic hepatitis C virus (HCV) patients is poorly understood. It remains unclear if natural regulatory T cells (Treg) contribute to the induction and maintenance of HCV persistence. We herein report for the first time that CD25(high)IFN-gamma(-)FOXP3(high) Tregs can be rapidly induced by culturing peripheral blood mononuclear cells (PBMCs) of HCV-positive patients with HCV protein-derived peptides. The HCV-specific Tregs, generally CD4(+)CD45RO(+), did not proliferate in response to HCV peptide and failed to produce interferon (IFN)-gamma, in distinct contrast to antiviral effector cells. Stimulation of healthy donor PBMCs with HCV peptides did not result in CD25 and FOXP3 upregulation above non-antigen background. To further investigate the antigen specificity of these potentially disease-associated natural Tregs, CD25(+) cells were isolated from PBMCs, labeled with carboxyfluorescein diacetate succinimidylester and added back to CD25-depleted PBMCs, and the co-cultures were then stimulated with individual peptides derived from the HCV core protein. We found that the actual peptide that can stimulate Treg varied between patients, but within any given subject only a small number of the peptides were able to stimulate Treg, suggesting the existence of dominant Treg epitopes. Although functional experiments for these peptides are ongoing in our laboratory, data presented here suggests that HCV-specific natural Tregs are abundant in infected individuals, in contrast to the extremely low frequency of anti-HCV effector T cells, supporting the view that natural Treg may be implicated in host immune tolerance during HCV infection.

Liquid chromatographic analysis of vitamin B6 in reconstituted infant formula: collaborative study.

A liquid chromatographic (LC) method was validated for the determination of total vitamin B6 in infant formula. Total vitamin B6 was quantified by converting the phosphorylated and free vitamers into pyridoxine. Pyridoxine was determined by ion pair reversed-phase LC with fluorescence detection. The method was subjected to an AOAC collaborative study involving a factory-manufactured, milk- and soy-based infant formula. Each was spiked at 3 concentrations in the range of 0-1 microg/g and sent as blind duplicate to participant laboratories. Nine laboratories returned valid data which were statistically analyzed for outliers and precision parameters. The repeatability relative standard deviation (RSD(r)) ranges were 2.0-4.0 and 3.5-5.9% for fortified milk- and soy-based formulas, respectively. The reproducibility relative standard deviation (RSD(R)) ranges were 8.2-8.4 and 6.7-11.2% for fortified milk- and soy-based formulas, respectively. HORRAT values ranged from 0.42 to 0.53, indicating that the precision of the method is acceptable. The mean RSD(r):RSD(R) values were 0.60 and 0.55 for milk- and soy-based formulas, respectively. As expected, RSDs for the unfortified samples were higher, but their HORRAT values (0.81 and 2.06) helped define a realistic limit of quantitation as 0.05 microg/g. Recovery data were quantitative and varied between 81.4 and 98.0% (mean = 89.8%) for each of 6 spiked materials.

Determination of orotic acid, uric acid, and creatinine in milk by liquid chromatography.

A simultaneous determination of orotic acid, uric acid, and creatinine in milk is described. Following deproteinization, the sample was analyzed by reversed-phase liquid chromatography, using a highly aqueous cationic ion-pair eluent and photodiode array UV detection. In view of their potential dietary significance, the validated method was applied to survey the influence of species, season, and lactation on their contribution to the nonprotein nitrogen pool in milk. Mature bovine milk contained orotic acid, uric acid, and creatinine in the range of 30-70, 9-24, and 6-12 microg/mL, respectively. Although uric acid and creatinine were present in all milks, orotic acid was essentially absent in nonruminant milks. In contrast to urate and creatinine, expression of orotic acid in bovine milk was strongly dependent on stage of early lactation. The co-existence in mammalian milks of related nucleoside and nucleotide components was also determined.

HCV persistence and immune evasion in the absence of memory T cell help.

Spontaneous resolution of hepatitis C virus (HCV) infection in humans usually affords long-term immunity to persistent viremia and associated liver diseases. Here, we report that memory CD4+ Tcells are essential for this protection. Antibody-mediated depletion of CD4+ Tcells before reinfection of two immune chimpanzees resulted in persistent, low-level viremia despite functional intra-hepatic memory CD8+ Tcell responses. Incomplete control of HCV replication by memory CD8+ Tcells in the absence of adequate CD4+ Tcell help was associated with emergence of viral escape mutations in class I major histocompatibility complex-restricted epitopes and failure to resolve HCV infection.

Characterization of HCV-specific Patr class II restricted CD4+ T cell responses in an acutely infected chimpanzee.

Resolution of hepatitis C virus (HCV) infection is associated with strong and sustained virus-specific CD4+ T cell responses. In this study, we investigated the evolution of functional T cell responses during acute infection of a chimpanzee and the longevity of these lymphocytes in blood and liver after resolution of infection. Viremia increased through the first 3 weeks of infection and then remained stable until the onset of T cell responses at weeks 6 and 8 postinfection. CD4+ T cells targeting nonstructural HCV proteins were detected in proliferation assays by week 6 postinfection, but they failed to produce interferon gamma (IFN-gamma). HCV-specific CD4+ and CD8+ T cells with the ability to produce IFN-gamma appeared at week 8 when a rapid 10-fold reduction in plasma viremia was first observed. This cytokine response persisted through to week 24 when infection apparently resolved. T cell lines targeting 3 CD4+ T cell epitopes and 1 CD8+ T cell epitope were derived from liver and their Patr major histocompatibility complex (MHC) restriction elements were identified. In retrospective studies performed on cryopreserved peripheral blood mononuclear cells (PBMCs) collected at various timepoints after infection, the onset of an IFN-gamma response measured against the class II restricted epitopes correlated with viral clearance. In conclusion, the characterization of the HCV epitopes and MHC class II restriction elements described here will facilitate a detailed comparison of CD4+ T cell function in animals with resolved and persistent infections.

Determination of total vitamin C in fruit juices and related products by liquid chromatography: interlaboratory study.

A interlaboratory study was conducted to evaluate a liquid chromatographic (LC) procedure for the determination of total vitamin C in foods at levels of 5-60 mg/100 g. Emphasis was placed on fruit juices, although selected foods were also included in the study. Following dissolution of sample in water, endogenous dehydroascorbic acid was converted to ascorbic acid by precolumn reduction with dithiothreitol at neutral pH. Total ascorbate was determined by C18 reversed-phase LC with a phosphate eluent at pH 2.5, incorporating dithiothreitol to maintain vitamin C in the reduced form, and UV detection at 254 nm. Seven types of fruit juices and foods were tested by 19 collaborators in 7 countries. Three duplicate juices and foods met the criteria for Youden pairs and yielded repeatability relative standard deviation of 5.80-14.66%. Reproducibility relative standard deviation ranged from 6.36 to 35.54% (n = 10) with HORRAT values of 0.82-4.04. The LC method is suitable for routine use in fruit products and foods containing > 5 mg/100 g vitamin C and is recommended for further validation by AOAC INTERNATIONAL and International Fruit Juice Union.

Rapid determination of thiamine, riboflavin, pyridoxine, and niacinamide in infant formulas by liquid chromatography.

A simplified, simultaneous determination of vitamins B1, B2, B3, and B6 in supplemented infant formulas was developed from a single deproteinized sample extract, with analysis by reversed-phase, ion-pair chromatography with an acidified methanol-water mobile phase. The dioctylsulfosuccinate counter-ion facilitates unique retention of the pyridine-based vitamins (niacinamide and pyridoxine) and allows for concurrent measurement of both the pyridoxal and riboflavin 5'-phosphate endogenous components of milk. Other naturally occurring undetected vitamin congeners have minimal analytical significance. UV detection is used for niacinamide, and programmed fluorescence detection is used for riboflavin and the B6 vitamins. Thiamine is routinely determined sequentially under modified elution conditions.

Determination of vitamin K1 isomers in foods by liquid chromatography with C30 bonded-phase column.

Vitamin K1 was determined in a variety of foods by using reversed-phase liquid chromatography with a C30 column followed by post-column reduction to the fluorescent hydroquinone derivatives. Lipids were removed by lipase digestion, followed by single extraction into hydrocarbon, and the protocol was extended to selected natural and processed foods. Biologically active trans- and inactive ci-vitamin K1 isomers were measured individually to evaluate the true nutritional status of the products. Method performance parameters confirmed the validity of the technique. The use of the triacontyl-bonded C30 phase for selective phylloquinone isomer measurement extends previously validated AOAC Method 999.15 for vitamin K1 in milk and infant formula to a wider range of foods important in the human diet. The cis-vitamin K1 isomer contributes up to about 15% of total phylloquinone in certain foods.

Determination of vitamin B12 in milk products and selected foods by optical biosensor protein-binding assay: method comparison.

Biomolecular interaction analysis was evaluated for the automated determination of vitamin B12 in a range of foods. The analytical technique was configured as a biosensor-based, nonlabeled inhibition protein-binding assay using nonintrinsic R-protein. Sample extraction conditions were optimized, and both ligand specificity and nonspecific binding considerations were evaluated. Performance parameters included a quantitation range of 0.08-2.40 ng/mL, recoveries of 89-106%, agreement against assigned reference values for 3 independent certified food reference materials, and a mean between-laboratory reproducibility relative standard deviation of 4.9%. The proposed method was compared with reference microbiological and radioisotope protein-binding methods for a range of food samples. A wide selection of milks, infant formulas, meats, and liver were evaluated for their vitamin B12 content. The influence of season was studied in herd milk, early lactation was followed for a single animal, and the cobalamin content of bovine, caprine, and ovine milks was compared.