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A method for analysing small samples of floral pollen for free and protein-bound amino acids.

ABSTRACT: Pollen provides floral visitors with essential nutrients including proteins, lipids, vitamins and minerals. As an important nutrient resource for pollinators, including honeybees and bumblebees, pollen quality is of growing interest in assessing available nutrition to foraging bees. To date, quantifying the protein-bound amino acids in pollen has been difficult and methods rely on large amounts of pollen, typically more than 1 g. More usual is to estimate a crude protein value based on the nitrogen content of pollen, however, such methods provide no information on the distribution of essential and non-essential amino acids constituting the proteins.Here, we describe a method of microwave-assisted acid hydrolysis using low amounts of pollen that allows exploration of amino acid composition, quantified using ultra high performance liquid chromatography (UHPLC), and a back calculation to estimate the crude protein content of pollen.Reliable analysis of protein-bound and free amino acids as well as an estimation of crude protein concentration was obtained from pollen samples as low as 1 mg. Greater variation in both protein-bound and free amino acids was found in pollen sample sizes <1 mg. Due to the variability in recovery of amino acids in smaller sample sizes, we suggest a correction factor to apply to specific sample sizes of pollen in order to estimate total crude protein content.The method described in this paper will allow researchers to explore the composition of amino acids in pollen and will aid research assessing the available nutrition to pollinating animals. This method will be particularly useful in assaying the pollen of wild plants, from which it is difficult to obtain large sample weights.

SUBMITTER: Stabler D

PROVIDER: S-EPMC5856064 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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A method for analysing small samples of floral pollen for free and protein-bound amino acids.

Stabler Daniel D Power Eileen F EF Borland Anne M AM Barnes Jeremy D JD Wright Geraldine A GA

Methods in ecology and evolution 20171016 2

Pollen provides floral visitors with essential nutrients including proteins, lipids, vitamins and minerals. As an important nutrient resource for pollinators, including honeybees and bumblebees, pollen quality is of growing interest in assessing available nutrition to foraging bees. To date, quantifying the protein-bound amino acids in pollen has been difficult and methods rely on large amounts of pollen, typically more than 1 g. More usual is to estimate a crude protein value based on the nitro ...[more]

PMID: 29576862

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Project description:Pigs exposed to heat stress (HS) increase body temperature in which can damage the intestinal epithelia and affect the absorption and availability of amino acids (AA). Protein digestion and metabolism further increase body temperature. An experiment was conducted with six pairs of pigs (of 47.3 ± 1.3 kg initial body weight) exposed to natural HS to assess the effect of substituting dietary protein-bound AA by free AA on morphology and gene expression of intestinal epithelial and serum concentration (SC) of free AA. Treatments were: high protein, 21.9% crude protein (CP) diet (HShp) and low protein, 13.5% CP diet supplemented with crystalline Lys, Thr, Met, Trp, His, Ile, Leu, Phe, and Val (HSaa). The HShp diet met or exceeded all AA requirements. The HSaa diet was formulated on the basis of ideal protein. Pigs were fed the same amount at 0700 and 1900 hours during the 21-d study. Blood samples were collected at 1700 hours (2.0 h before the evening meal), 2030 hours, and 2130 hours (1.5 and 2.5 h after the evening meal). At the end, all pigs were sacrificed to collect intestinal mucosa and a 5-cm section from each segment of the small intestine from each pig. Villi measures, expression of AA transporters (y+L and B0) in mucosa, and SC of AA were analyzed. Ambient temperature fluctuated daily from 24.5 to 42.6 °C. Weight gain and G.F were not affected by dietary treatment. Villi height tended to be larger (P ≤ 0.10) and the villi height:crypt depth ratio was higher in duodenum and jejunum of pigs fed the HSaa diet (P < 0.05). Gene expression of transporter y+L in jejunum tended to be lower (P < 0.10) and transporter B0 in the ileum was lower (P < 0.05) in HSaa pigs. Preprandial (1700 hours) SC of Arg, His, Ile, Leu, Thr, Trp, and Val was higher (P < 0.05), and Phe tended to be higher (P < 0.10) in HShp pigs. At 2030 hours (1.5 h postprandial), serum Lys, Met, and Thr were higher in the HSaa pigs (P < 0.05). At 2130 hours (2.5 h), Arg, His, Ile, Phe, and Trp were lower (P < 0.05); Met was higher (P < 0.05); and Lys tended to be higher (P < 0.10) in HSaa pigs. In conclusion, feeding HS pigs with low protein diets supplemented with free AA reduces the damage of the intestinal epithelia and seems to improve its absorption capacity, in comparison with HS pigs fed diets containing solely protein-bound AA. This information is useful to formulate diets that correct the reduced AA consumption associated with the decreased voluntary feed intake of pigs under HS.

Project description:One-carbon folate metabolites and one-carbon-related amino acids play an important role in human physiology, and their detection in biological samples is essential. However, poor stability as well as low concentrations and occurrence in different species in various biological samples make their quantification very challenging. The aim of this study was to develop a simple, fast, and sensitive ultra-high-performance liquid chromatography MS/MS (UHPLC-MS/MS) method for the simultaneous quantification of various one-carbon folate metabolites (folic acid (FA), tetrahydrofolic acid (THF), p-aminobenzoyl-L-glutamic acid (pABG), 5-formyltetrahydrofolic acid (5-CHOTHF), 5-methyltetrahydrofolic acid (5-CH3THF), 10-formylfolic acid (10-CHOFA), 5,10-methenyl-5,6,7,8-tetrahydrofolic acid (5,10-CH+-THF), and 4-α-hydroxy-5-methyltetrahydrofolate (hmTHF)) and one-carbon-related amino acids (homocysteine (Hcy), methionine (Met), S-ade-L-homocysteine (SAH), and S-ade-L-methionine (SAM)). The method was standardized and validated by determining the selectivity, carryover, limits of detection, limits of quantitation, linearity, precision, accuracy, recovery, and matrix effects. The extraction methods were optimized with respect to several factors: protease-amylase treatment on embryos, deconjugation time, methanol precipitation, and proteins' isoelectric point precipitation on the folate recovery. Ten one-carbon folate metabolites and four one-carbon-related amino acids were detected using the UHPLC-MS/MS technique in various biological samples. The measured values of folate in human plasma, serum, and whole blood (WB) lay within the concentration range for normal donors. The contents of each analyte in mouse plasma were as follows: pABG (864.0 nmol/L), 5-CH3THF (202.2 nmol/L), hmTHF (122.2 nmol/L), Met (8.63 μmol/L), and SAH (0.06 μmol/L). The concentration of each analyte in mouse embryos were as follows: SAM (1.09 μg/g), SAH (0.13 μg/g), Met (16.5 μg/g), 5,10-CH+THF (74.3 ng/g), pABG (20.6 ng/g), and 5-CH3THF (185.4 ng/g). A simple and rapid sample preparation and UHPLC-MS/MS method was developed and validated for the simultaneous determination of the one-carbon-related folate metabolites and one-carbon-related amino acids in different biological samples.

Dataset Information

A method for analysing small samples of floral pollen for free and protein-bound amino acids.

Publications

A method for analysing small samples of floral pollen for free and protein-bound amino acids.

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