Micellar electrokinetic chromatography method for measuring amino acid secretions from islets of Langerhans

Abstract

Islets of Langerhans are responsible for maintaining glucose homeostasis through regulated secretion of hormones and other factors. It is hypothesized that amino acids secreted from islets play a critical role in cell functionality and viability. For example, glutamate and gamma-aminobutyric acid have been proposed to work as paracrine signaling molecules within islets to coordinate the release of hormone secretion; other amino acids, such as glutamine, leucine, alanine, and arginine, have been shown to stimulate or potentiate glucose-stimulated insulin secretion. To characterize the potential roles that these small molecules may play in islet physiology, derivatization of amino acids in high-salt buffers commonly used in islet experiments with naphthalene-2,3-dicarboxaldehyde and MEKC separation conditions were optimized. The optimized conditions used d-norvaline as the internal standard and allowed quantification of 14 amino acids with LODs ranging from 0.2 to 7 nM. The RSDs of the migration times were 0.04-0.54% and the RSDs of the peak areas were 0.2-5.8% for the various amino acids. The effects of glucose and 2,4-dinitrophenol on amino acid secretions from islets were tested and a suppressive effect of glucose on gamma-aminobutyric acid release was observed, likely acting through adenosine triphosphate inactivation of glutamate decarboxylase.

Keywords: 2,4-Dinitrophenol (DNP); CE; Glucose; LIF; Naphthalene-2,3-dicarboxaldehyde (NDA).

Figure 1. Optimization of derivatization conditions

(A) Derivatization times were tested by mixing a solution containing 1 μM each of Ser, Gln, Glu, GABA, Met, Ile, Leu, and D-Nva with 5 mM NDA and 20 mM cyanide and allowing the reaction to proceed for 5, 10, 20, 60, and 120 min. The average peak areas from the resulting peaks in the electropherogram are plotted versus the derivatization time. Each time period was performed in triplicate and the mean value is plotted with error bars corresponding to +/− 1 standard deviation. (B) The effect of the derivatization temperatures on the resulting peak areas is shown using the same reaction conditions as in (A) with a 20 min derivatization time. Each temperature point was performed in triplicate and the average peak area is shown with error bars corresponding to +/− 1 standard deviation.

Figure 2. Optimization of separation conditions

In all electropherograms, a 19 amino acid mixture was separated using 29 kV and the peaks are numbered as the following: 1 = Ser, 2 = Thr, 3 = Asn, 4 = Gln, 5 = Gly, 6 = Ala, 7 = His, 8 = Glu, 9 = Tyr, 10 = Asp, 11 = GABA, 12 = Val, 13 = Met, 14 D-Nva, 15 = Ile, 16 = Leu, 17 = Trp, 18 = Phe, and 19 = Arg. (A) The separation of these 19 amino acids (1 μM each prior to derivatization) with a separation buffer of 15 mM phosphate, 30 mM SDS, at pH 8.0, 8.3, 8.7, and 9.1 are shown. (B) The effect of phosphate concentration was examined by using 15, 20, 25, and 30 mM phosphate. All buffers contained 30 mM SDS at pH 8.3. (C) Using 25 mM phosphate at pH 8.3 with 30 mM SDS, separation temperatures of 15, 20, and 25 °C were examined. All electropherograms are plotted with relative fluorescence units (RFU) on the y-axis and the electropherograms are offset for clarity.

Figure 3. Optimized electropherograms of standard amino acid mixtures

The blue electropherogram (top trace) was obtained after derivatization of a 1 μM amino acid mixture using the optimized conditions. Peak identities are the same as described in Figure 2. The red electropherogram (bottom trace) was obtained when a background BSS solution containing D-Nva was derivatized. In both cases, optimized separation conditions were used, which were a 25 mM phosphate separation buffer, pH 8.3, containing 30 mM SDS with a 29 kV separation voltage and a separation temperature of 25 °C.

Figure 4. Representative electropherograms of islet secretions

Secretions from 25 islets incubated in 3 or 20 mM glucose were derivatized and separated according to the optimized procedures. The blue (bottom) and red (top) traces were the electropherograms obtained when islets were incubated in 3 and 20 mM glucose, respectively. Peak identities are the same as described in Figure 2.

Figure 5. Effect of glucose and DNP on secretion amounts

The secretory amounts of the 14 baseline resolved amino acids are summarized. Amounts measured after 1 hour incubation in 3 mM glucose are shown in black, in 20 mM glucose in red, and in 20 mM glucose with DNP in green. The average of three replicate trials is shown with error bars corresponding to +/− 1 S.E.M. Significance was determined using a one-tailed t-test with: (*) = p < 0.05 compare to 3 mM glucose, or (**) = p < 0.05 compared to 20 mM glucose without DNP. Inset shows a zoomed in view of the amount of GABA detected at the indicated conditions.