Benefits of using the ENO-30 for Nitrate and Nitrite Detection

Benefits of using the ENO-30 for Nitrate and Nitrite Detection

Today, I’d like to talk about a system capable of quantitative analysis of nitrate and nitrite from biological samples in minutes, called the ENO-30. The ENO-30 is a fast and highly sensitive system down to 0.1 picomoles.

Let me walk you through how easy it is to use. First, you get your sample ready for analysis. Sample prep is easy. In our manual, we’ve outlined a simple sample preparation protocol for tissue homogenate, blood, cell culture, urine, saliva, and microdialysis samples. Samples are injected into the ENO-30 with a manual injector or an autosampler for laboratory automation.

Once injected, nitrate and nitrite are separated by an HPLC separation column. Once separated, nitrate is reduced to nitrite, forming 2 separated nitrite compounds. Then, the separated nitrites are mixed with a Greiss reagent to form an azo dye, which is then detected by absorbance at 540 nm. In 10 minutes, you will be able to detect both nitrate and nitrite from a single injection.

The ENO-30 combines HPLC with colorimetric analysis. It’s like having a chemist and HPLC expert right on your benchtop. We’ve optimized the conditions to take away any guesswork and made the system straightforward and easy to use.

At Amuza, your success is our priority. It’s important that our products work for you just as well as they do for us here at our facility. When you acquire one of our machines, not only will it include installation and training, you’ll have access to our self-help support center as well as support from our knowledgeable support staff.

Questions?

5-minute Analysis of Dopamine and Serotonin in Brain Microdialysate

5-minute Analysis of Dopamine and Serotonin in Brain Microdialysate

In this video, we’ll show you how to detect and analyze dopamine and serotonin levels in brain microdialysis samples in 5 minutes. For this analysis, we use HPLC-ECD because it is a cost-effective way to get fast and ultra-sensitive data down to the femtomole range.

While HPLC-ECD is a common analysis method, careful consideration must be taken regarding analytical conditions, such as mobile phase preparation, column selection, and other factors. Ideally, we want:

  • Prompt elution to save time
  • Clear separation of the signal peaks from the solvent-front peaks
  • Fewer peaks around the signal peaks
  • A low noise level and a stable baseline

Optimizing these conditions can take time and resources. That’s why many labs require a dedicated person to handle HPLC. Thus, we’ve created an application to take out all the guesswork thereby maximizing the separation conditions for dopamine and serotonin.

For this application, we’re going to use the HTEC, which includes everything you need, integrated into one single unit. Let’s take a look inside:

  • Dual-piston pump with a unique algorithm to reduce noise without any pulse damper.
  • Degasser to remove small air bubbles for better pump performance.
  • Temperature control for consistent results.
  • Separation column uniquely selected for separation of DA and 5-HT.
  • Electrochemical detector cell with a 3 electrode DC potentiostat and an amperometric electrode.

The electrically active analyte is detected as it flows over the surface of a smooth electrode with an applied voltage.

We’ve optimized conditions to make it as easy as possible. Simply inject the sample using a manual injector or autosampler if you’re using laboratory automation. You can monitor signal response in real-time using the dedicated chromatography software. In 5 minutes you will have your results and you’re ready for the next sample. It’s highly sensitive down to 0.16 femtomoles.

Here’s an example of a chromatogram showing dopamine and serotonin.  

For more information on dopamine and serotonin analysis, contact us today!

Questions?

New Loaner Program for Neurotransmitter Detection Systems

New Loaner Program for Neurotransmitter Detection Systems

Are you interested in analyzing neurotransmitters in brain microdialysate or brain tissue?

Perhaps you have a number of samples you’d like to run, but don’t want to fully commit to a new machine. Today, we’re happy to announce a new loaner program for an HTEC system fully ready to use.

The Eicom HTEC is a fully integrated HPLC-ECD for the detection of neurotransmitters from brain microdialysis and/or tissue samples. It is a fast and highly sensitive system that can be placed on a lab benchtop. Sample analysis can range from 5 to 30 minutes per sample, depending on the application. Sample runs can include quantitative analysis neurotransmitters like dopamine, serotonin, norepinephrine, acetylcholine, amino acids like glutamate and GABA. The HTEC is simple and straightforward to use. You don’t have to be an expert on HPLC to run analysis, anyone can be an expert!

Let’s take a look inside:

  •  Dual-piston pump with a unique algorithm to reduce noise without any pulse damper.
  • Degasser to remove small air bubbles for better pump performance
  • Temperature control for consistent results
  • Separation column specific for the application
  • Electrochemical detector cell with a 3 electrode DC potentiostat and an amperometric electrode.

The electrically active analyte is detected as it flows over the surface of a smooth electrode with an applied voltage.

How the loaner program works:

Contact us to discuss the scope of your research or questions you may be asking. Our support specialists will be happy to discuss what kind of analytes you’re interested in as well as the brain region. We’ll get you set up with an application specifically designed for your experiments. We’ll come to your lab and do the installation and training on-site over a 2 day period. From there, you can start running samples. Should you have any questions or need assistance, our support specialists will be available to help along with a self-help support site. At Amuza, it’s important that our instruments work just as well for you as they do in our facility.

We’ll provide shipping materials once your loan of the equipment is completed.

Contact us today for more questions including pricing options.

Price Comparison of 3 Neurotransmitter Detection Methods

Price Comparison of 3 Neurotransmitter Detection Methods

When it comes to neurotransmitter detection such as Dopamine, Serotonin, Norepinephrine, Glutamate, GABA, Acetylcholine, etc., in microdialysis samples, tissue homogenates, blood plasma, and others, there are three (3) main methods; LC-Mass Spectrometry (LC-MS), HPLC-ECD (Electrochemical Detection) & ELISA/Radioimmunoassays.

The first option is LCMS, it provides the highest sensitivity and specificity to the detecting neurotransmitters and also the most expensive option. The price range is between US$200,000 to $600,000.

The second method is HPLC-ECD which is much more cost affordable while offering great sensitivity. The price range is between US$20,000 to $60,000.

The third method is ELISA or Radioimmunoassays which offers less sensitivity. There is also a concern of selectivity due to cross-talk among antibodies.

Therefore, when choosing which method would work best for you, we would recommend HPLC-ECD.

Eicom HPLC-ECD used to study GABA’s role in Diabetes

Eicom HPLC-ECD used to study GABA’s role in Diabetes

The Phelps lab at the University of Florida used the Eicom HTEC-500 HPLC-ECD to determine the time-resolved secretion of GABA, taurine, and other amino acids from pancreatic islet beta cells. This helped them show:

  • GABA in human beta cells is mostly stored in the cytosol, not in vesicles.
  • An ion channel (VRAC) handles GABA secretion from beta cells.
  • Pulsatile release of GABA synchronizes and regulates the secretion of insulin from islets.
  • The depletion of GABA reserves in beta cells correlates with both type 1 and type 2 diabetes in human patients.

Roles of GABA

GABA, short for gamma-aminobutyric acid, has many roles in biology. It is best known as an inhibitory neurotransmitter in the nervous system. In fact, impaired clearance of GABA in the amygdala may contribute to alcoholism. GABA also acts to inhibit immune responses and regulates the secretion of hormones in the pancreas.

How GABA, Insulin, and Glucagon are related to diabetes.

Insulin is secreted in response to high blood sugar levels. It directs cells throughout the body to take up glucose from the blood, thus lowering blood sugar levels. As insulin and blood sugar levels drop, glucagon is secreted. Glucagon directs the liver to convert glycogen into glucose and release it. Bringing blood sugar levels back up and balancing the action of insulin. Beta cells secrete both of these hormones in pancreatic islets. The secretion of both of these hormones is regulated in part by GABA, which is also secreted by beta cells. In type 1 diabetes an autoimmune response attacks and destroys beta cells. But this immune response can be damped by the GABA secreted by beta cells. Administering GABA as a drug not only protects beta cells, it can also cause other pancreatic islet cells (alpha cells) to convert into beta cells. Helping to restore the beta-cell population. Because of this protective role, GABA is already being studied as a treatment for diabetes by the Swedish biotech Diamyd. With clinical trials led by Prof. Kenneth McCormick at the University of Alabama.

How is GABA secreted in the pancreas?

In the brain, secretory vesicles are responsible for storing and then releasing GABA from neurons. This same mechanism was long thought to be at work in the pancreas as well. Yet, the GABA transporter proteins necessary for this mechanism remained elusive in beta cells. Leaving a gap in the understanding of how GABA release occurs. Prof. Edward Phelps previously observed that instead of being held in vesicles, the bulk of GABA is loose within the cytosol of beta cells. This meant that another mechanism could be in play. He and his student Walker Hagan hypothesized that an ion channel, specifically VRAC (Volume Regulatory Anion Channel), could be responsible.

“There is a channel between the interior of the beta cell and the extracellular space, which we thought was worth investigating,” Phelps said. “The volume regulatory anion channel (VRAC) is known for another purpose. It is used to help cells maintain their shape by keeping the osmotic pressure inside and outside the cell in equilibrium. When this balance is disturbed and the cell shape changes, small organic chemicals known as osmolytes are expelled from the cell via the VRAC channel to help the cell regain its shape. When we artificially opened this channel in beta cells using low saline, we found that this channel also transports GABA.”

Changes in cell volume had already been implicated as a trigger for insulin secretion. Further supporting the possibility that VRAC channels could be the principal mechanism for GABA secretion.

Measuring GABA secretion in the pancreas

The Phelps lab used their HTEC-500 HPLC-ECD system to measure GABA and other amino acids in three phases of this project.

First, they confirmed that human pancreatic islets from both type 1 and type 2 diabetic donors are deficient in GABA when compared to those of non-diabetics. Then they measured GABA in perifusate from islets cultured in a Biorep perifusion system. Perifusion washes media though a small group of islets and allows recovery of the perfusate as time-resolved fractions. This way conditions can be varied and resulting changes in the secreted amino acids and hormones can be monitored over time. By varying the media washing through the islets, The Phelps lab was able to confirm that hypotonic media triggers the release of GABA from beta cells. Consistent with VRAC being responsible for secretion. Perifusion also allowed them to study the time-resolved relationship between GABA release and insulin secretion.

Finally, they created a knockout strain of mice unable to express the VRAC channel in beta cells: (βc-LRRC8A−/−). The beta cells in these mice are missing one of the subunits (LRRC8A) of VRAC, and no longer secreted GABA in response to osmotic stress. This lent further support to the role of VRAC in GABA release.

 

Representative HPLC chromatograms from the supernatant of a culture of 200 pancreatic islets. Hypotonic media opens VRAC channels as the cell attempts to relieve osmotic stress. Allowing the release of both GABA and taurine from islets into the supernatant (blue). VRAC channels remain closed in Isotonic media (gray). Resulting in lower concentrations of GABA and taurine in the supernatant. Samples were injected onto an Eicom HTEC-500 equipped with an FA-3ODS column, used for separating GABA, Glu, and other amino acids. An AS-700 autosampler was used to automate both derivatization and subsequent injection of the samples. Data courtesy of the Phelps lab.

 

Levels of GABA and Taurine released into the supernatant of islets suspended in low saline (hypotonic) and isotonic (3G) media. Figure courtesy of the Phelps lab.

Acetylcholine Neurochemical Involvement in Gulf War Illness

Acetylcholine Neurochemical Involvement in Gulf War Illness

For approximately 200,000 US veterans, the 1991 Persian Gulf War marked the beginning of their experience with Gulf War Illness (GWI). GWI encompasses a cluster of chronic symptoms including memory and cognitive problems, fatigue, and fibromyalgia.

GWI has long been associated with a combination of several possible contributory factors: the stress of deployment, altered immune function, and exposure to acetylcholinesterase inhibitors (AChEI), but the exact cause or causes have remained elusive. The AChEI pyridostigmine bromide (PB) was administered to soldiers as a prophylactic against the risk of nerve agent weapons, but many veterans were also exposed to AChEI based pesticides, further complicating the etiology of this illness.

To elucidate the relationship between these factors, Dr. Victoria Macht, her advisor Prof. Lawrence Reagan, and colleagues at the University of South Carolina School of Medicine studied rats exposed to pyridostigmine bromide and repeated restraint stress. The rats were then given either an immune challenge or an acute immobilization stress challenge during in vivo microdialysis. It is the first study to use an in vivo method (microdialysis) to show that PB changes the response of the central cholinergic system to both stress and immune challenges, and does so in a brain region specific manner.

By measuring acetylcholine levels via microdialysis and subsequent HPLC-ECD, they found that cholinergic responses were attenuated in the PFC and hippocampus after immobilization stress. Lipopolysaccharide (LPS) was administered as an immune challenge, after which cholinergic responses were attenuated in the hippocampus but not the PFC. These results indicate that PB and stress interact to shift the cholinergic response to future psychological and immunological stressors, providing a potential mechanism for the persistent and exacerbated cognitive symptoms evidenced in soldiers with GWI.

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Mike Churchill: What story do the different responses to the immune challenge and the immobilization challenge tell?

Victoria Macht: By using two different types of challenges, we were able to test both the diversity and consistency of effects of PB and stress on the cholinergic system. LPS is a novel challenge which specifically elicits a response from the innate immune system. The immobilization challenge is more of a psychological stressor, and as it shares some similar qualities with the prior restraint stress, this allowed us to test if rats with PB and restraint stress had impaired neurochemical adaptations to recurrent stressors.

MC: How might these results relate to changes in fear memory and cognitive function?

VM: ACh is an important regulator for a variety of factors in fear memory including coordination of local circuits to help with sensory and cortical processing of stimuli as well as the consolidation process. Interestingly, regional differences in the cholinergic response of the PFC and hippocampus to immobilization stress suggested that PB impairs cortical processing of novel stressful stimuli and impairs the neurochemical adaptation to recurrent stressful stimuli. In our fear conditioning studies, we similarly found impairments in the way PB and stress interacted to impair context and cue related retrieval. This suggested to us that impairments in the function of the cholinergic systems impacts a variety of psychological stressful stimuli, indicating that this is a global deficit in cognitive function rather than a specific deficit to only one type of stressor.

MC: How do the microdialysis results relate to the tests for inflammation you ran?

VM: ACh is really fascinating because while it is not only central in learning and memory, it is also an important negative regulator for the inflammatory response via α7 nicotinic ACh receptors. We found that PB blunted the central cholinergic response to an innate immune challenge, which could suggest an exacerbated chronic inflammatory response in the brain. Interestingly, these microdialysis results for acetylcholine parallel some of our findings with peripheral inflammatory markers. Peripheral levels of c-reactive protein were elevated after the LPS challenge in rats which had received PB, suggesting a dysregulated inflammatory response. While we need to confirm these results with cytokine levels in the brain, our results suggest that impaired cholinergic feedback to inflammatory stimuli could underlie some of the changes in the sensitivity of the immune system which are evident in clinical populations with GWI.

MC: Does PB have to cross the BBB to cause these effects?

VM: It does not. There has been a big debate on this topic. One suggestion was that stress caused a leaky barrier, allowing PB to get through. However, tests on this have been inconsistent on this. What our studies demonstrate is that PB changes the function of the central cholinergic system regardless of whether it is able to get through the BBB.

MC: What will be the next steps for this project?

VM: Prof. Reagan will continue the project: measuring cytokine responses in the brain to see if they match peripheral cytokine responses. There is also an opportunity to see if aging exacerbates the decline of the cholinergic responses and cognitive deficits in our model of GWI. The goal would be to see if animal models of GWI can predict further changes in veterans as they age, and plan treatment accordingly. We have a unique opportunity with this population for the preclinical research on treatments to get ahead of the patient population as they age.

MC: How did you like using the Eicom HTEC HPLC-ECDs in Prof. Jim Fadel’s lab?

VM: It is amazing! I can’t imagine having done these projects without it, and I miss using it.  We used the system daily for two years to measure acetylcholine without any real problems. It made my dissertation a much more pleasant experience!

MC: Had you used HPLCs before using the HTEC?

VM: We used a different system before but it was not reliable, so when it was working people felt they had to immediately run all of their samples before it went down again, and watch it all of the time when it was running.

MC: How many samples do you think you ran over the course of this project?

VM: That makes my head spin! We looked at both ACh and glutamate, in two brain regions, each rat underwent microdialysis 2 separate days, there were approximately 8 animals per group, and 4 groups. So at least 3500 samples – plus the pilot study! Plus there were other studies going on during this time which were also using the HTEC.

MC: Where is your career taking you next?

VM: I am now doing a postdoc at UNC Chapel Hill, working with Prof. Fulton Crews, studying the long term effects of binge drinking in adolescents. Interestingly, while this is a different clinical population, changes in the cholinergic system and innate immune system are also common features here.

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The article appears in the April issue of Brain, Behavior, and Immunity:

Pyridostigmine bromide and stress interact to impact immune function, cholinergic neurochemistry and behavior in a rat model of Gulf War Illness
V.A. Macht, J.L. Woodruff, E.S. Maissy, C.A. Grillo, M.A. Wilson, J.R. Fadel, L.P. Reagan
doi: 10.1016/j.bbi.2019.04.015