Detection of N,N-diacetyllactosamine (LacdiNAc) containing free prostate-specific antigen for early stage prostate cancer diagnostics and for identification of castration-resistant prostate cancer patients.

Prostate cancer (PCa) is one of the most common cancer types among men and also acommon cause of death globally. With an increasing incidence, there is aneed for low-cost, reliable biomarkers present in samples, which could be provided non-invasively (without a need to perform prostate biopsy). Glycosylation changes of free-PSA (fPSA) are considered cancer-specific, while the level of different PSA forms can increase under other than cancerous conditions. In the present study, we investigated the role ofN,N-diacetyllactosamine (LacdiNAc) epitope of fPSA (i.e. glycoprofile of fPSA or gPSA) in combination with total-PSA (tPSA), prostate volume, and tPSA density (tPSA level divided by prostate volume i.e. PSAd) as biomarkers for monitoring of PCa development and progression in 105 men. Furthermore, we applied an genetic (evolutionary) algorithm to identify any suspicious individuals in abenign cohort having benign prostatic hyperplasia (BPH). We identified 3 suspicious men originally diagnosed with BPH using gPSA analysis. In thefollow-up we found out that two men should not be considered as BPH patients since multiparametric magnetic resonance imaging (mpMRI) identified one man with clinically significant PCa via Prostate Imaging - Reporting and Data System (PI RADS v2 = 4) and the second man was with High-gradeprostatic intraepithelial neoplasia (HG PIN), commonly described as apre-cancerous stage. Moreover, in the study we described for the first time that changed LacdiNAc on PSA can be applied to identify prostatitis patients and most importantly this is the first study suggesting that changed glycosylation on PSA can be applied to identify castration-resistant prostate cancer (CRPCa) patients.

Simultaneous Quantification of 3'- and 6'-Sialyllactose in Rat Plasma Using Liquid Chromatography-Tandem Mass Spectrometry and Its Application to a Pharmacokinetic Study.

Sialyllactose (SL), an acidic oligosaccharide, has immune-protective effects against pathogens and helps with the development of the immune system and intestinal microorganisms. To elucidate the pharmacokinetic characterization after oral administration to rats, the simultaneous quantification method for 3'-SL and 6'-SL in rat plasma was validated, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an electrospray ionization (ESI) mode. Several types of columns [C18, amide, and hydrophilic interaction liquid chromatography (HILIC) phase] were used to separate the peaks of 3'-SL and 6'-SL, which improved chromatographic selectivity. Ultimately, the HILIC phase column had a good peak shape and quick resolution, with a mobile phase comprising ammonium acetate buffer and acetonitrile obtained by gradient elution. In addition, the simultaneous quantification of 3'-SL and 6'-SL in rat plasma samples were adequately applied to pharmacokinetic study.

Four weeks of probiotic supplementation reduces GI symptoms during a marathon race.

PURPOSE: To evaluate the effects of probiotic supplementation on gastrointestinal (GI) symptoms, circulatory markers of GI permeability, damage, and markers of immune response during a marathon race. METHODS: Twenty-four recreational runners were randomly assigned to either supplement with a probiotic (PRO) capsule [25 billion CFU Lactobacillus acidophilus (CUL60 and CUL21), Bifidobacterium bifidum (CUL20), and Bifidobacterium animalis subs p. Lactis (CUL34)] or placebo (PLC) for 28 days prior to a marathon race. GI symptoms were recorded during the supplement period and during the race. Serum lactulose:rhamnose ratio, and plasma intestinal-fatty acid binding protein, sCD14, and cytokines were measured pre- and post-races. RESULTS: Prevalence of moderate GI symptoms reported were lower during the third and fourth weeks of the supplement period compared to the first and second weeks in PRO (p < 0.05) but not PLC (p > 0.05). During the marathon, GI symptom severity during the final third was significantly lower in PRO compared to PLC (p = 0.010). The lower symptom severity was associated with a significant difference in reduction of average speed from the first to the last third of the race between PLC (- 14.2 ± 5.8%) and PRO (- 7.9 ± 7.5%) (p = 0.04), although there was no difference in finish times between groups (p > 0.05). Circulatory measures increased to a similar extent between PRO and PLC (p > 0.05). CONCLUSION: Probiotics supplementation was associated with a lower incidence and severity of GI symptoms in marathon runners, although the exact mechanisms are yet to be elucidated. Reducing GI symptoms during marathon running may help maintain running pace during the latter stages of racing.

The effect of 17ß-estradiol on lactose in plasma and urine in dairy cows in late lactation.

The aim of this study was to investigate the effect of 17ß-estradiol on mammary tight junctions in cows in late lactation. The experiment included five non-pregnant cows around day 290 in lactation. The cows received injections of 17ß-estradiol for six days. The effect of exogenous 17ß-estradiol on milk yield, milk composition and lactose in plasma and lactose in urine was investigated before, during and after the treatment. Milk yield decreased after 17ß-estradiol injections and lactose in plasma and urine increased, showing an effect on the integrity of the mammary tight junctions. However, there was a delay between hormone injections and the decrease in milk yield and opening of tight junctions, indicating that other factors are involved. A high correlation between lactose in urine and blood plasma was found. More than 30% of the total lactose production was lost in urine after 17ß-estradiol treatment.

Oxytocin Induces Mammary Epithelium Disruption and Could Stimulate Epithelial Cell Exfoliation.

Mammary epithelial cells (MEC) are exfoliated from the epithelium into milk, influencing the number of MEC present in the udder. This process is associated with epithelium integrity. The release of oxytocin (OT) induced by milking causes myoepithelial cell contraction, which, in turn, may stimulate MEC exfoliation through mechanical forces. To investigate the role of OT in MEC exfoliation, we inhibited or induced myoepithelial cell contraction by injecting the OT receptor antagonist atosiban (Ato) or a supraphysiological dose of OT, respectively. Eight cows were assigned to 2 treatments during 2 milkings according to a crossover experimental design: Control+OT (cows were first milked to collect standard milk and then received 5 IU of OT to collect residual milk through a second milking) and Ato + OT (cows were injected with Ato (50 µg/kg of body weight) and milked to collect cisternal milk, then received 5 IU of OT to collect alveolar milk through a second milking). Milk MEC were purified to determine their concentration and number in milk. Mammary epithelium integrity was assessed by measuring the kinetics of plasma lactose concentration. Inhibiting myoepithelial cell contraction by Ato injection decreased the number of exfoliated MEC in milk. In contrast, OT injection increased the concentration of MEC in the residual milk and the number of MEC in the alveolar milk. Ato injection reduced plasma lactose concentration, whereas, in both treatments, OT injections increased it. Our results suggested that myoepithelial cell contraction caused by OT could stimulate MEC exfoliation into milk and was associated with epithelium disruption.

The effect of extended milking intervals (24, 48 and 72 h) on milk yield, milk composition, mammary physiology and welfare traits in dairy ewes.

The majority of the work today regarding the effects of extended milking intervals has focused on dairy cattle and only to a limited extent on dairy goats and sheep. The aim of this study was to investigate the effect of different non-milking intervals on milk yield and composition, mammary physiology and welfare indices in dairy ewes. Thirty-six multiparous ewes in late lactation were allocated to one of four groups of nine and subjected to 24, 48 or 72 h of non-milking or normal milking interval (12 h) (group A, B, C and D, respectively). Data showed that there were no significant differences in milk yield among the experimental groups during the third day after re-milking. Furthermore, no significant differences in milk lactose, protein and fat concentration among the experimental groups were observed after 7, 14 and 21 d of re-milking, respectively. Non-milking for 72 h resulted in a temporary increase of sodium concentration, Na + /K + ratio and enzymatic activities of plasmin (PL), plasminogen (PG) and plasminogen activator (PA) in milk. However, these parameters had similar values among the experimental groups on day 5 after re-milking. The concentration of lactose in blood was also significantly increased as a result of the 72 h non-milking interval and returned to its initial levels the second day after re-milking. These data, taken together, suggest that early involution events that occurred as an effect of non-milking were fully reversible within a short period of time. Finally, no significant signs of welfare impairment were observed in ewes due to extended milking intervals. In conclusion, non-milking up to 72 h had no negative effects on milk yield and composition, mammary physiology and welfare parameters in dairy ewes.

Blood lactose after dairy product intake in healthy men.

The absence of a dedicated transport for disaccharides in the intestine implicates that the metabolic use of dietary lactose relies on its prior hydrolysis at the intestinal brush border. Consequently, lactose in blood or urine has mostly been associated with specific cases in which the gastrointestinal barrier is damaged. On the other hand, lactose appears in the blood of lactating women and has been detected in the blood and urine of healthy men, indicating that the presence of lactose in the circulation of healthy subjects is not incompatible with normal physiology. In this cross-over study we have characterised the postprandial kinetics of lactose, and its major constituent, galactose, in the serum of fourteen healthy men who consumed a unique dose of 800 g milk or yogurt. Genetic testing for lactase persistence and microbiota profiling of the subjects were also performed. Data revealed that lactose does appear in serum after dairy intake, although with delayed kinetics compared with galactose. Median serum concentrations of approximately 0·02 mmol/l lactose and approximately 0·2 mmol/l galactose were observed after the ingestion of milk and yogurt respectively. The serum concentrations of lactose were inversely correlated with the concentrations of galactose, and the variability observed between the subjects' responses could not be explained by the presence of the lactase persistence allele. Finally, lactose levels have been associated with the abundance of the Veillonella genus in faecal microbiota. The measurement of systemic lactose following dietary intake could provide information about lactose metabolism and nutrient transport processes under normal or pathological conditions.

Major human milk oligosaccharides are absorbed into the systemic circulation after oral administration in rats.

Human milk oligosaccharides (HMO) are involved in many biological functions influencing infant health. Although HMO act locally at the intestine, recent evidence has demonstrated that HMO are partially incorporated into the systemic circulation of breast-fed infants. In the last few years, a large amount of research has been conducted using preclinical models to uncover new biological functions of HMO. The aim of this study was to evaluate the absorption and urine excretion of HMO in rats. We administered a single oral dose of the following HMO: 2'-fucosyllactose (2'-FL), 6'-sialyllactose and lacto-N-neotetraose at different concentrations to adult rats. The time course of absorption of HMO into the bloodstream and their appearance in urine was studied. Our results showed that rats, similar to human infants, are able to effectively absorb a portion of HMO from the intestine into plasma and to excrete them in urine. On the basis of this, we also conducted a specific kinetic absorption study with 2'-FL, the most predominant HMO in human milk, in 9-11-d-old rat pups. Our results confirmed that a significant amount of 2'-FL was absorbed into the systemic circulation and subsequently excreted in urine during lactation in rats in a dose-depended manner. We also found basal levels of these HMO in plasma and urine of adult rats as well as rat pups as a natural result of nursing. Our data suggest that the rat may be a useful preclinical model that provides new insights into the metabolism and functions of HMO.

Glutamine supplementation reduces markers of intestinal permeability during running in the heat in a dose-dependent manner.

PURPOSE: To examine the dose-response effects of acute glutamine supplementation on markers of gastrointestinal (GI) permeability, damage and, secondary, subjective symptoms of GI discomfort in response to running in the heat. METHODS: Ten recreationally active males completed a total of four exercise trials; a placebo trial and three glutamine trials at 0.25, 0.5 and 0.9 g kg-1 of fat-free mass (FFM) consumed 2 h before exercise. Each exercise trial consisted of a 60-min treadmill run at 70% of [Formula: see text] in an environmental chamber set at 30 °C. GI permeability was measured using ratio of lactulose to rhamnose (L:R) in serum. Plasma glutamine and intestinal fatty acid binding protein (I-FABP) concentrations were determined pre and post exercise. Subjective GI symptoms were assessed 45 min and 24 h post-exercise. RESULTS: Relative to placebo, L:R was likely lower following 0.25 g kg-1 (mean difference: - 0.023; ± 0.021) and 0.5 g kg-1 (- 0.019; ± 0.019) and very likely following 0.9 g kg- 1 (- 0.034; ± 0.024). GI symptoms were typically low and there was no effect of supplementation. DISCUSSION: Acute oral glutamine consumption attenuates GI permeability relative to placebo even at lower doses of 0.25 g kg-1, although larger doses may be more effective. It remains unclear if this will lead to reductions in GI symptoms. Athletes competing in the heat may, therefore, benefit from acute glutamine supplementation prior to exercise in order to maintain gastrointestinal integrity.

Hydroxyurea-Lactose Interaction Study: In Silico and In Vitro Evaluation.

The Maillard reaction between hydroxyurea (a primary amine-containing drug) and lactose (used as an excipient) was explored. The adduct of these compounds was synthesized by heating hydroxyurea with lactose monohydrate at 60 °C in borate buffer (pH 9.2) for 12 h. Synthesis of the adduct was confirmed using UV-visible spectroscopy and Fourier transform infrared, differential scanning calorimetry, high-pressure liquid chromatography, and liquid chromatography-mass spectrometry studies. An in silico investigation of how the adduct formation affected the interactions of hydroxyurea with its biological target oxyhemoglobin, to which it binds to generate nitric oxide and regulates fetal hemoglobin synthesis, was carried out. The in silico evaluations were complemented by an in vitro assay of the anti-sickling activity. Co-incubation of hydroxyurea with deoxygenated blood samples reduced the percentage of sickled cells from 38% to 12 ± 1.6%, whereas the percentage of sickled cells in samples treated with the adduct was 17 ± 1.2%. This indicated loss of anti-sickling activity in the case of the adduct. This study confirmed that hydroxyurea can participate in a Maillard reaction if lactose is used as a diluent. Although an extended study at environmentally feasible temperatures was not carried out in the present investigation, the partial loss of the anti-sickling activity of hydroxyurea was investigated along with the in silico drug-target interactions. The results indicated that the use of lactose in hydroxyurea formulations needs urgent reconsideration and that lactose must be replaced by other diluents that do not form Maillard adducts.

Convergent adaptation of human lactase persistence in Africa and Europe.

A SNP in the gene encoding lactase (LCT) (C/T-13910) is associated with the ability to digest milk as adults (lactase persistence) in Europeans, but the genetic basis of lactase persistence in Africans was previously unknown. We conducted a genotype-phenotype association study in 470 Tanzanians, Kenyans and Sudanese and identified three SNPs (G/C-14010, T/G-13915 and C/G-13907) that are associated with lactase persistence and that have derived alleles that significantly enhance transcription from the LCT promoter in vitro. These SNPs originated on different haplotype backgrounds from the European C/T-13910 SNP and from each other. Genotyping across a 3-Mb region demonstrated haplotype homozygosity extending >2.0 Mb on chromosomes carrying C-14010, consistent with a selective sweep over the past approximately 7,000 years. These data provide a marked example of convergent evolution due to strong selective pressure resulting from shared cultural traits-animal domestication and adult milk consumption.

High-sensitivity immunoassay with surface plasmon field-enhanced fluorescence spectroscopy using a plastic sensor chip: application to quantitative analysis of total prostate-specific antigen and GalNAcß1-4GlcNAc-linked prostate-specific antigen for prostate cancer diagnosis.

A high-sensitivity immunoassay system with surface plasmon field-enhanced fluorescence spectrometry (SPFS) was constructed using a plastic sensor chip and then applied to the detection of total prostate-specific antigen (total PSA) and GalNAcß1-4GlcNAc-linked prostate-specific antigen (LacdiNAc-PSA) in serum, to discriminate between prostate cancer (PC) and benign prostate hyperplasia (BPH). By using this automated SPFS immunoassay, the detection limit for total PSA in serum was as low as 0.04 pg/mL, and the dynamic range was estimated to be at least five digits. A two-step sandwich SPFS immunoassay for LacdiNAc-PSA was constructed using both the anti-PSA IgG antibody to capture PSA and Wisteria floribunda agglutinin (WFA) for the detection of LacdiNAc. The results of the LacdiNAc-PSA immunoassay with SPFS showed that the assay had a sensitivity of 20.0 pg/mL and permitted the specific distinction between PC and BPH within the PSA gray zone. These results suggested that high-sensitivity automated SPFS immunoassay systems might become a powerful tool for the diagnosis of PC and other diseases.

Nonbactericidal secreted phospholipase A2s are potential anti-inflammatory factors in the mammary gland.

The recent burst of duplication and divergence of the bovine PLA2G2D genes is considered typical of immune response genes, and it was recently shown that PLA2G2D is abundantly expressed in mouse leukocytes and acts as an immunosuppressive phospholipase. Analysis of 1,143 Holstein bulls indicated that the four common haplotypes spanning PLA2G2D display copy number variation ranging from 1 to 4 per haploid genome. Association of the fourth haplotype with negative total merit remained significant (P < 0.002) when corrected for population relatedness. We compared the lipase and bactericidal activities of bovine pancreatic PLA2G1B with human PLA2G2A and G2D and bovine PLA2G2D1 and G2D4 proteins, which had been subcloned, expressed, and refolded by us, and the impact of point mutations in the calcium binding site was investigated. All tested phospholipases were ineffective bactericides of Escherichia coli isolated from bovine mastitis. However, in lactating mice treated with E. coli or lipopolysaccharide (LPS), intramammary injection of bovine PLA2G1B relieved visual and histological inflammation and reduced blood levels of infiltrating lactose. Further studies are warranted to determine whether the observed anti-inflammatory effect involves competitive binding of the receptor Pla2r1 which may mimic the LPS resistance effect in Pla2r1-deficient mice.

Lactose in blood plasma and the ability of dairy cows to tolerate once-daily milking in terms of milk loss and milk recovery.

This experiment described the variability among cows with the aim of studying the ability of dairy cows to tolerate once-daily milking (ODM) in terms of milk losses and milk recoveries observed when cows are switched from twice- to once-daily milking and then back to twice-daily milking (TDM). It also aimed to investigate whether or not lactose in blood plasma, which indicates the mammary epithelium permeability, is correlated with milk losses and recoveries and, consequently, a potential candidate assessor of dairy cow tolerance to ODM. The study used 86 crossbred dairy cows (Holstein × Normande) split into 5 groups over 2 yr. The trial consisted of 3 successive periods: a 1-wk control period of TDM, then 3 wk of ODM, followed by 2 wk of TDM. Blood samples were collected 1 to 1.5h before the morning milking and 5 to 6h after milking on d -3, 0, 1, 3, 7, 21, 23, and 28, where d 0 is the last d of the control period. Milk losses measured as kilograms per day were higher in cows with highest control-period milk yields (r=-0.66). When expressed in relative terms (%), milk losses were weakly correlated with the control-period milk yield (r=-0.22). When switched back to TDM, cows recovered only 47% of the milk lost. Milk recovery (kg/d) was weakly correlated with control milk yield (r=0.34) and not correlated with ODM milk yield. Milk recovery was correlated with milk yield losses: the higher the milk yield losses (kg/d or %), the higher the milk recovery (kg/d; r=-0.59 and -0.52, respectively). Rate of milk recovery expressed as the quantity of milk recovered per kilogram of milk lost, was not correlated with milk loss expressed as a percentage. This means that to be considered well-adapted to ODM scheduling, a cow must be well adapted to milk loss, and be well adapted to milk recovery. Blood plasma lactose concentration (log(10)-transformed data) was not correlated with milk production levels in either control, ODM, or back-to-TDM periods. It was poorly correlated with milk loss but was positively correlated with milk recovery (kg/d) and rate of milk recovery. We conclude that blood plasma lactose needs to be coupled with other indicators for evaluating dairy cow tolerance to ODM.

Alteration of the nutrient uptake by the udder over an extended milking interval in dairy cows.

Little is known about modifications of the mammary utilization of nutrients circulating in blood plasma when milk yield is strongly decreased by once-daily milking. A trial was carried out to describe the mammary nutritional adjustments linked to the downregulation of milk synthesis as milk accumulated over an extended milking interval in the bovine udder. Three Holstein dairy cows yielding 34.0 kg/d of milk were fitted with an ultrasound flow probe around the left external pudic artery and with catheters inserted into the left carotid and milk vein to estimate mammary blood flow (MBF) and mammary uptake of acetate, ß-hydroxybutyrate, nonesterified fatty acids, glycerol, glucose, O(2), and CO(2) release. The trial was carried out over 2 consecutive weeks, with wk 2 repeating wk 1. Cows were milked twice daily at 12-h milking intervals. On d 3, cows were milked at 0630 h and were not milked for 36 h until d 4 at 1830 h. Over the following days, twice-daily milking was resumed using 12-h milking intervals. Each half-udder was milked separately. Secretion rates of milk and milk proteins decreased 67% during the 12-to-36-h interval of milk accumulation, whereas that of milk fat fell 30%. Timing of changes in MBF and lactose levels in blood plasma was concomitant and significant after 19.5 and 21.5h of milk accumulation in the udder, respectively. The MBF decreased, most likely because the usual increases in MBF no longer occurred when the udder was full of milk. After 24h of milk accumulation, MBF did not increase further when cows lay down, and did not increase as usual 3h after a meal, suggesting a possible physical effect of milk accumulated in the udder on MBF, complementing metabolic regulation. Mammary uptake or release of nutrients was lowered before 24h for glucose, acetate, and ß-hydroxybutyrate and after 24h for total glycerol, O(2), and CO(2), mostly associated with the impaired MBF. However, these decreases ranged from 12 to 17%, and cannot entirely explain the -45 and -20% decreases in milk secretion rates observed during the entire 36 h of milk accumulation, thus confirming the primary role of intramammary metabolic regulation in the downregulation of milk secretion. The larger amount of nutrients taken up by the udder could explain the enhanced milk fat levels, involving a strongly modified metabolic fate of nutrients.

Imidazolium labelling permits the sensitive mass-spectrometric detection of N-glycosides directly from serum.

A novel imidazolium derivative (GITag) shows superior ionisation and consequently allows increased mass spectrometric detection capabilities of oligosaccharides and N-glycans. Here we demonstrate that human serum samples can be directly labelled by GITag on a MALDI target plate, abrogating prevalently required sample pretreatment or clean-up steps.

Maternal waterpipe tobacco smoke exposure during lactation induces hormonal and biochemical changes in rat dams and offspring.

Waterpipe smoking is common among pregnant and breastfeeding women. Herein, the effects of waterpipe tobacco smoke (WTS) exposure during lactation on milk composition, hormonal levels and biochemical profile in dams and pups were investigated. Lactating Wistar rats were randomly assigned to receive either WTS (2 hours per day) or fresh air (control group). Milk was collected on day 21 and analysed for protein, lactose and total fat. Blood, from dams and pups, was analysed for insulin, glucose, lipid profile, leptin, prolactin and corticosterone. WTS exposure during lactation increased the blood level of HDL and corticosterone in dams (P < .05). However, the level of milk lactose and blood glucose was reduced in dams after the exposure to WTS during lactation (P < .05). WTS during lactation significantly increased levels of triglycerides, LDL and leptin (P < .05), and a trend of increase in blood level of nicotine and prolactin in pups. Levels of other parameters were not affected by WTS exposure in dams and pups. In conclusion, WTS exposure during lactation altered the milk composition and altered lipid profile, glucose homeostasis and hormonal levels in dams and pups. It is necessary to adopt strategies to enhance tobacco cessation during breastfeeding.

Human Milk Oligosaccharides in Cord Blood Are Altered in Gestational Diabetes and Stimulate Feto-Placental Angiogenesis In Vitro.

(1) Background: Human milk oligosaccharides (HMOs) are present in maternal serum during pregnancy and their composition is altered in gestational diabetes (GDM). HMOs are also in fetal cord blood and in contact with the feto-placental endothelium, potentially affecting its functions, such as angiogenesis. We hypothesized that cord blood HMOs are changed in GDM and contribute to increased feto-placental angiogenesis, hallmark of GDM. (2) Methods: Using HPLC, we quantified HMOs in cord blood of women with normal glucose tolerance (NGT, n = 25) or GDM (n = 26). We investigated in vitro angiogenesis using primary feto-placental endothelial cells (fpECs) from term placentas after healthy pregnancy (n = 10), in presence or absence of HMOs (100 µg/mL) isolated from human milk, 3'-sialyllactose (3'SL, 30 µg/mL) and lactose (glycan control) and determined network formation (Matrigel assay), proliferation (MTT assays), actin organization (F-actin staining), tube formation (fibrin tube formation assay) and sprouting (spheroid sprouting assay). (3) Results: 3'SL was higher in GDM cord blood. HMOs increased network formation, HMOs and 3'SL increased proliferation and F-actin staining. In fibrin assays, HMOs and 3'SL increased total tube length by 24% and 25% (p < 0.05), in spheroid assays, by 32% (p < 0.05) and 21% (p = 0.056), respectively. Lactose had no effect. (4) Conclusions: Our study suggests a novel role of HMOs in feto-placental angiogenesis and indicates a contribution of HMO composition to altered feto-placental vascularization in GDM.

Methylamidation for sialoglycomics by MALDI-MS: a facile derivatization strategy for both α2,3- and α2,6-linked sialic acids.

Neutralization of carboxylic acid is an important means to avoid sialic acid dissociation when sialylated glycans are analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In this paper, we describe a simple and rapid method to modify the sialic acids of sialylated glycans in the presence of methylamine and (7-azabenzotriazol-1-yloxy) trispyrrolidinophosphonium hexafluorophosphate (PyAOP). After methylamidation, sialylated glycans can be analyzed by MALDI-MS without loss of the sialic acid moiety. The electrospray ionization mass spectrometry (ESI-MS) and MALDI-MS analysis of both 3'- and 6'-sialyllactose derivatives indicated that the quantitative conversion of sialic acids was achieved, regardless of their linkage types. This derivatization strategy was further validated with the N-glycans released from three standard glycoproteins (fetuin, human acid glycoprotein, and bovine acid glycoprotein) containing different types of complex glycans. Most importantly, this derivatization method enabled the successful characterization of N-glycans of sera from different species (human, mouse, and rat) by MALDI-MS. Because of the mild reaction conditions, the modification in sialic acid residues can be retained. This improvement makes it possible to detect sialylated glycans containing O-acetylated sialic acid moieties using MALDI-MS in positive-ion mode.

Comparable Exogenous Carbohydrate Oxidation from Lactose or Sucrose during Exercise.

PURPOSE: Ingesting readily oxidized carbohydrates (CHO) such as sucrose during exercise can improve endurance performance. Whether lactose can be utilized as a fuel source during exercise is unknown. The purpose of this study was to investigate the metabolic response to lactose ingestion during exercise, compared with sucrose or water. METHODS: Eleven participants (age, 22 ± 4 yr; V[Combining Dot Above]O2peak, 50.9 ± 4.7 mL·min·kg) cycled at 50% Wmax for 150 min on five occasions. Participants ingested CHO beverages (lactose or sucrose; 48 g·h, 0.8 g·min) or water throughout exercise. Total substrate and exogenous CHO oxidation was estimated using indirect calorimetry and stable isotope techniques (naturally high C-abundance CHO ingestion). Naturally low C-abundance CHO trials were conducted to correct background shifts in breath CO2 production. Venous blood samples were taken to determine plasma glucose, lactate, and nonesterified fatty acid concentrations. RESULTS: Mean exogenous CHO oxidation rates were comparable with lactose (0.56 ± 0.19 g·min) and sucrose (0.61 ± 0.10 g·min; P = 0.49) ingestion. Endogenous CHO oxidation contributed less to energy expenditure in lactose (38% ± 14%) versus water (50% ± 11%, P = 0.01) and sucrose (50% ± 7%, P ≤ 0.05). Fat oxidation was higher in lactose (42% ± 8%) than in sucrose (28% ± 6%; P ≤ 0.01); CHO conditions were lower than water (50% ± 11%; P ≤ 0.05). Plasma glucose was higher in lactose and sucrose than in water (P ≤ 0.01); plasma lactate was higher in sucrose than in water (P ≤ 0.01); plasma nonesterified fatty acids were higher in water than in sucrose (P ≤ 0.01). CONCLUSIONS: Lactose and sucrose exhibited similar exogenous CHO oxidation rates during exercise at moderate ingestion rates. Compared with sucrose ingestion, lactose resulted in higher fat and lower endogenous CHO oxidation.