Name: mapping metabolism: monitoring lactate dehydrogenase activity directly in tissue
Uploaded: 2018-06-21T14:00:00.000+00:00
Duration: 6 min 18 s
Description: Watch this Scientific Journal Video about Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue at JoVE.com

HAC was the Milton E. Cassel scholar of the Rita Allen Foundation (http://www.ritaallenfoundation.org). This work was funded by grants to HAC from National Institute of General Medical Sciences R01 GM081686, R01 AR070245, National Institute of General Medical Sciences R01 GM0866465, the Eli &#38; Edythe Broad Center of Regenerative Medicine &#38; Stem Cell Research (Rose Hills and Hal Gaba awards), the Iris Cantor Women&#8217;s Health Center/UCLA CTSI NIH Grant UL1TR000124, the Leukemia Lymphoma Society, Impact awards from the Jonsson Comprehensive Cancer Center to WEL and HAC. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number P50CA092131. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. HAC is a member of the Eli &#38; Edythe Broad Center of Regenerative Medicine &#38; Stem Cell Research, the UCLA Molecular Biology Institute, and the UCLA Bioinformatics Interdepartmental Program.

All experiments described were approved by the Animal Care Committee at the University of California, Los Angeles.
1. Generate Slides of Frozen Mouse Skin
<ol>
	<li>Euthanize mice in accordance with institution policy. 
	Note: Follow institutional policy for protective clothing. All protocols involving animals must be approved by the Institutional Animal Care Committee.</li>
	<li>Remove the mouse&#8217;s hair with an animal hair trimmer.</li>
	<li>Make incisions in the skin using scissors. Using forceps, lift the skin away from the mouse. Using scissors, cut the skin section away.</li>
	<li>Fill the cryomold with freezing reagent compound (see Table of Materials).</li>
	<li>Place skin sections into filled cryomolds using needle-nosed forceps. Orient skin so that tissue slices will generate a cross section of the skin from the epidermis to the dermis, hypodermis and muscle (Figure 2). 
	Note: If possible, mount experimental and control mice together in the same cryomold so they can be sectioned onto the same slide and processed together. Take care not to create bubbles.</li>
	<li>Place cryomold on the flat surface of a block of dry ice in an ice bucket and let freeze. Continue to observe the orientation of the skin. Adjust if necessary. 
	Note: Wear cryogenic gloves when handling dry ice.</li>
	<li>Transfer cryomolds into a -80 &#176;C freezer for storage. Samples can be maintained in the freezer for approximately 3 months without significant loss of enzymatic activity. Do not let frozen sections dry out.</li>
	<li>Using a cryostat at -20 &#176;C, slice tissue to create sections 7 - 10 &#181;m thick for the best skin morphology21.</li>
</ol>
2. Preparing Slides for Staining
<ol>
	<li>Establish the set of slides to be tested. Include a control slide on which NAD will be withheld and another control slide on which the substrate, lactate, will be withheld. Include a positive control slide from a frozen tissue block previously investigated.</li>
	<li>Briefly fix slides containing skin sections with 4% formalin for 5 min either by pipetting 1 mL of 4% formalin onto the slide, or when processing multiple slides, by dipping slides into a container containing 4% formalin. 
	Note: The fixation will ensure the skin does not peel off from the slides during the following procedures and preserve the skin morphology. 
	Caution: Formalin is a carcinogen and hazardous; wear gloves, do not let it touch your skin and perform this step in a chemical fume hood.</li>
	<li>Wash slides with at least 1 mL phosphate buffered saline, pH 7.4, per slide either by pipetting or dipping.</li>
</ol>
3. Preparing Staining Solution
<ol>
	<li>Vortex together reagents for lactate dehydrogenase activity staining (50 mM Tris pH 7.4, 750 &#181;M NADP, 80 &#181;M phenazine methosulfate (PMS), 600 &#181;M nitrotetrazolium blue chloride, and 30 mM lactate) in an appropriately sized tube depending on the amount of staining solution required. 
	Note: 1 mL is sufficient to completely cover one slide.</li>
	<li>Prepare a second solution in which all reagents are present except NAD. Prepare a third solution in which all reagents are present except lactate.</li>
</ol>
4. Incubate Slides in Staining Solution
<ol>
	<li>Gently pipet the staining solution or control solutions onto the prepared slides covering the section in its entirety (1 mL is sufficient for one slide). If multiple slides are being processed together, dip all slides into the staining solution at the same time (make sure the container has enough solution to completely cover the tissue section).</li>
	<li>Incubate the slides in a humidified environment at 37 &#176;C in the dark. If staining solution is on top of the slide, place the slide in a humidified environment to prevent evaporation.</li>
</ol>
5. Monitor the Slides
<ol>
	<li>Monitor the conversion of the staining solution from clear to blue by visual inspection. When the samples have reached the desired level of blueness, stop the reaction by removing the staining solution. 
	Note: For skin, 10 min is sufficient. The time will depend on the organ and the enzymatic activity.</li>
	<li>Rinse slides with phosphate buffered saline by pipetting (1 mL is sufficient for each slide) or dipping. 
	Note: Any samples that will be compared to each other must be maintained in staining solution for the same amount of time.</li>
</ol>
6. Counterstain and mount
<ol>
	<li>Pipet a counterstain onto the slides when the slides have reached an appropriate level of blueness. 
	Note: Counterstains that turn the nuclei red or green will provide good contrast with the blue created by the formazan precipitant.</li>
	<li>Mount with aqueous or non-aqueous mounting medium22. One to three drops (40 - 50 &#181;L) of mounting medium is sufficient depending on the size of the cover slip.</li>
</ol>
7. Image slides and quantify
<ol>
	<li>Image slides under a light microscope. Take photographs of experimental and control samples and all negative controls at 10X, 20X, and 40X magnification.</li>
	<li>Determine the intensity of blue stain in different regions with image analysis software.</li>
</ol>

<ol>
	<li>Boonacker, E., Van Noorden, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+cytochemical+techniques+for+metabolic+mapping+in+living+cells,+with+special+reference+to+proteolysis.">Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis.</a> J Histochem Cytochem. 49, 1473-1486 (2001).</li><li>Seidler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tetrazolium-formazan+system:+Design+and+histochemistry.">The tetrazolium-formazan system: Design and histochemistry.</a> Prog Histochem Cytochem. 24, 1-86 (1991).</li><li>Van Noorden, C. J. F., Frederiks, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enzyme Histochemistry: A Laboratory Manual of Current Methods. , (1992).</li><li>Winzer, K., Van Noorden, C. J., Kohler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+cytochemical+analysis+of+glucose-6-phosphate+dehydrogenase+activity+in+living+isolated+hepatocytes+of+European+flounder+for+rapid+analysis+of+xenobiotic+effects.">Quantitative cytochemical analysis of glucose-6-phosphate dehydrogenase activity in living isolated hepatocytes of European flounder for rapid analysis of xenobiotic effects.</a> J Histochem Cytochem. 49, 1025-1032 (2001).</li><li>Negi, D. S., Stephens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+the+histochemical+localization+of+glucose-6-phoshate+dehydrogenase+in+animal+and+plant+tissues.">An improved method for the histochemical localization of glucose-6-phoshate dehydrogenase in animal and plant tissues.</a> J Histochem Cytochem. 25, 149-154 (1977).</li><li>Boren, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+localization+of+transketolase+activity+in+epithelial+cells+of+different+rat+tissues+and+subcellularly+in+liver+parenchymal+cells.">In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells.</a> J Histochem Cytochem. 54, 191-199 (2006).</li><li>Miller, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+metabolic+configurations+of+single+cells+within+complex+tissue+microenvironments.">Exploring metabolic configurations of single cells within complex tissue microenvironments.</a> Cell Metab. , (2017).</li><li>Shen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+value+of+serum+lactate+dehydrogenase+in+renal+cell+carcinoma:+a+systematic+review+and+meta-analysis.">Prognostic value of serum lactate dehydrogenase in renal cell carcinoma: a systematic review and meta-analysis.</a> PLoS One. 11, e0166482 (2016).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+serum+LDH+in+small+cell+lung+cancer:+A+systematic+review+with+meta-analysis.">Prognostic significance of serum LDH in small cell lung cancer: A systematic review with meta-analysis.</a> Cancer Biomark. 16, 415-423 (2016).</li><li>Petrelli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+role+of+lactate+dehydrogenase+in+solid+tumors:+a+systematic+review+and+meta-analysis+of+76+studies.">Prognostic role of lactate dehydrogenase in solid tumors: a systematic review and meta-analysis of 76 studies.</a> Acta Oncol. 54, 961-970 (2015).</li><li>Valvona, C. J., Fillmore, H. L., Nunn, P. B., Pilkington, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+and+function+of+lactate+dehydrogenase+A:+Therapeutic+potential+in+brain+tumor.">The regulation and function of lactate dehydrogenase A: Therapeutic potential in brain tumor.</a> Brain Pathol. 26, 3-17 (2016).</li><li>Markert, C. L., Shaklee, J. B., Whitt, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+a+gene:+Multiple+genes+for+LDH+isozymes+provide+a+model+of+the+evolution+of+gene+structure,+function+and+regulation.">Evolution of a gene: Multiple genes for LDH isozymes provide a model of the evolution of gene structure, function and regulation.</a> Science. 189, 102-114 (1975).</li><li>Read, J. A., Winter, V. J., Eszes, C. M., Sessions, R. B., Brady, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+altered+activity+of+M-+and+H-isozyme+forms+of+human+lactate+dehydrogenase.">Structural basis for altered activity of M- and H-isozyme forms of human lactate dehydrogenase.</a> Proteins. 43, 175-185 (2001).</li><li>Holness, M. J., Sugden, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pyruvate+dehydrogenase+complex+activity+by+reversible+phosphorylation.">Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation.</a> Biochem Soc Trans. 31, 1143-1151 (2003).</li><li>Yi, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphofructokinase+1+glycosylation+regulates+cell+growth+and+metabolism.">Phosphofructokinase 1 glycosylation regulates cell growth and metabolism.</a> Science. 337, 975-980 (2012).</li><li>Fan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+phosphorylation+of+lactate+dehydrogenase+A+is+important+for+NADH/NAD(+)+redox+homeostasis+in+cancer+cells.">Tyrosine phosphorylation of lactate dehydrogenase A is important for NADH/NAD(+) redox homeostasis in cancer cells.</a> Mol Cell Biol. 31, 4938-4950 (2011).</li><li>Zhao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysine-5+acetylation+negatively+regulates+lactate+dehydrogenase+A+and+is+decreased+in+pancreatic+cancer.">Lysine-5 acetylation negatively regulates lactate dehydrogenase A and is decreased in pancreatic cancer.</a> Cancer Cell. 23, 464-476 (2013).</li><li>Vanderlinde, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+total+lactate+dehydrogenase+activity.">Measurement of total lactate dehydrogenase activity.</a> Ann Clin Lab Sci. 15, 13-31 (1985).</li><li>Nelson, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+imaging+of+patients+with+prostate+cancer+using+hyperpolarized+[1-(1)(3)C]pyruvate.">Metabolic imaging of patients with prostate cancer using hyperpolarized [1-(1)(3)C]pyruvate.</a> Sci Transl Med. 5, 198ra108 (2013).</li><li>Flores, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactate+dehydrogenase+activity+drives+hair+follicle+stem+cell+activation.">Lactate dehydrogenase activity drives hair follicle stem cell activation.</a> Nat Cell Biol. , (2017).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+tissues.">Cryosectioning tissues.</a> CSH Protoc. 2008, (2008).</li><li>Espada, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-aqueous+permanent+mounting+for+immunofluorescence+microscopy.">Non-aqueous permanent mounting for immunofluorescence microscopy.</a> Histochem Cell Biol. 123, 329-334 (2005).</li><li>Hisada, R., Yagi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1-Methoxy-5-methylphenazinium+methyl+sulfate.+A+photochemically+stable+electron+mediator+between+NADH+and+various+electron+acceptors.">1-Methoxy-5-methylphenazinium methyl sulfate. A photochemically stable electron mediator between NADH and various electron acceptors.</a> J Biochem. 82, 1469-1473 (1977).</li></ol>

The authors have no competing interests to disclose.

The method described here can be used to monitor the activity of lactate dehydrogenase or other metabolic enzymes that generate NADH or NADPH, in different cell types within a tissue or within different portions of a tissue over time. Lactate dehydrogenase is an important enzyme for understanding the biology of stem cells and tumors, and the ability to monitor lactate dehydrogenase activity in individual cells is likely to provide important insights into the function of this enzyme.
One import...

Understanding the locations within tissues in which enzymes have high or low activity is essential for understanding development and physiology. Transcript or protein levels are often used as surrogates for enzymatic activity. While such studies can be informative, they do not provide information that can be critical for determining an enzyme&#39;s activity, such as post-translational modifications, the presence of protein complexes, or the enzyme&#39;s subcellular localization. When enzymatic activity is directly measured, it is often monitored in homogenized protein lysates that no longer contain information about individual cells within the mixture or the spatial distribution of the cells with high or low activity within a tissue.
We provide here a detailed protocol for mapping the spatial distribution of enzymatic activity within a tissue sample. The methodology is based on earlier studies demonstrating that tetrazolium salts can be used to localize the activity of dehydrogenases, reductases, and oxidases in frozen tissue1. With these methods, a water-insoluble formazan is formed when protons are transferred to a tetrazolium salt2,3. Glucose-6-phosphate dehydrogenase generates NADPH and a proton, and has been detected with tetrazolium activity. Glucose-6-phosphate dehydrogenase has been monitored in in European flounder hepatocytes4, in alveolar type 2 cells of the lungs5 and nephrons of the kidney5. Tetrazolium salts have also been used to monitor transketolase activity in frozen tissue6. A similar approach was recently&#160;used to monitor the activity of multiple dehydrogenases in the same tissue on adjacent slides7.
We describe here a method to use tetrazolium salts to monitor the spatial distribution of lactate dehydrogenase activity (Figure 1). Lactate dehydrogenase can convert pyruvate generated by glycolysis to lactate, and the reverse reaction. Lactate dehydrogenase activity is consequently an important determinant of pyruvate&#39;s entrance into the tricarboxylic acid cycle versus its secretion as lactate. Lactate levels in the blood are often used to diagnose a range of diseases, including cancer8,9,10, because it can signal that illness or injury has damaged cells and the enzyme has been released.
There are four lactate dehydrogenase genes: LDHA, LDHB, LDHC, and LDHD11. LDHA and LDHB are thought to have arisen from duplication of an early LDHA gene12. LDH is active as a tetramer and LDHA and LDHB can form homotetramers and heterotetramers with each other. LDHA is reported to have higher affinity for pyruvate, while LDHB is reported to have higher affinity for lactate, and to preferentially convert lactate to pyruvate13. The LDHA promoter contains binding sites for the HIF1&#945;, cMYC and FOXM1 transcription factors11. In addition, like many other glycolytic enzymes14,15, LDH can be modified by post-translational modifications. Fibroblast growth factor receptor 1 can phosphorylate LDHA at Y10, which promotes tetramer formation, or Y83, which promotes NADH substrate binding16. LDHA can also be acetylated17. For these reasons, a complete understanding of LDH activity requires monitoring not only LDH protein levels, but the enzyme&#39;s activity as well.
In addition to the method we present here, other approaches have been used to monitor lactate dehydrogenase activity. Lactate dehydrogenase activity can be monitored spectrophotometrically in homogenized protein lysate. The generation of NADH as lactate is converted to pyruvate can be measured based on absorbance at 340 nm, while the disappearance of NADH can be monitored as pyruvate is converted to lactate18. Lactate dehydrogenase activity has also been monitored with magnetic resonance imaging (MRI). 13C- pyruvate can be administered and the conversion of pyruvate to lactate can be monitored as the ratio of [1-13C]lactate/[1-13C]pyruvate. Elevated ratios of [1-13C]lactate/[1-13C]pyruvate have been observed in cancer tissue19. While MRI-based approaches can provide information on lactate dehydrogenase activity in normal and disease tissues, the methodologies do not have the resolution needed to determine the activity level in specific cells. The methodology provided here can provide information on lactate dehydrogenase activity in tissue areas and even in single cells.
Using in situ activity assays, we found that the activity of lactate dehydrogenase is high in the hair follicle stem cells of mouse skin20. We also used the method to monitor the activity of lactate dehydrogenase in cultured embryonic stem cells and found the activity is higher in the stem cells than the feeder layer. Finally, we monitored the activity of lactate dehydrogenase in fresh mouse aorta and observed staining in smooth muscle cells. We describe here a detailed protocol for monitoring lactate dehydrogenase activity in frozen mouse skin.

<table><tbody><tr><td>Surgical instruments</td><td></td><td></td><td>For collecting skin from euthanized mice</td></tr><tr><td>Tissue-tek cryomold 25 mm x 20 mm x 5 mm</td><td>Fisher Scientific</td><td>NC9511236</td><td>For freezing mouse skin</td></tr><tr><td>Tissue-Tek O.C.T. compound</td><td>Fisher Scientific</td><td>NC9638938</td><td>For mounting cryomolds</td></tr><tr><td>Ice bucket</td><td>Fisher Scientific</td><td>07-210-106</td><td></td></tr><tr><td>Dry Ice</td><td></td><td></td><td></td></tr><tr><td>Polysine Adhesion Slide</td><td>Fisher Scientific</td><td>12-545-78</td><td></td></tr><tr><td>4% formalin</td><td>Fisher Scientific</td><td>23-245-684</td><td>Dilluted in water</td></tr><tr><td>phosphate buffered saline, pH 7.4</td><td></td><td></td><td></td></tr><tr><td>vortex</td><td></td><td></td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>23-245-684</td><td></td></tr><tr><td>NAD</td><td>Sigma-Aldrich</td><td>N7004</td><td></td></tr><tr><td>Phenazine methosulfate</td><td>Sigma-Aldrich</td><td>P9625</td><td></td></tr><tr><td>Nitrotetrazolium blue chloride</td><td>Sigma-Aldrich</td><td>N6876</td><td></td></tr><tr><td>Lithium L-lactate</td><td>Sigma-Aldrich</td><td>L2250</td><td>Substrate</td></tr><tr><td>37&amp;deg;C incubator (or tissue culture incubator)</td><td></td><td></td><td></td></tr><tr><td>Braziliant! Counter stain</td><td>Anatech</td><td>861</td><td>Counter stain</td></tr><tr><td>Mounting medium</td><td>Vector Laboratories</td><td>H-5000&amp;nbsp;</td><td></td></tr><tr><td>Cover slips for slides</td><td>Fisher Scientific</td><td>12-544D</td><td></td></tr><tr><td>Light microscope</td><td></td><td></td><td></td></tr></tbody></table>

mapping metabolism: monitoring lactate dehydrogenase activity directly in tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We have previously reported results for in situ activity assays in mouse skin20. As shown in Figure 3, we observed high levels of lactate dehydrogenase activity in the hair follicle stem cells at the base of the hair follicle when the procedures described above were followed. These findings were corroborated by fluorescence activated cell sorting of skin for hair follicle stem cells and confirming high lactate dehydrogenase ac...

Watch this Scientific Journal Video about Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue at JoVE.com

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

mapping-metabolism-monitoring-lactate-dehydrogenase-activity-directly

Research

JoVE Journal

Biochemistry

11.4K Views.  UCLA. We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Video: Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

Cancer Research

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

Immunology and Infection

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation.
We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase&#39;s activity. The formazan&#39;s absorbance is therefore a direct measure of the dehydrogenase&#39;s activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays.
Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Here, we present a protocol that can be used to microscopically visualize and quantify activity of dehydrogenases in cells and tissues and its kinetics, function and subcellular localization.

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Neuroscience

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, pro-inflammatory (i.e., M1-like) monocytes/macrophages display more reliance on anaerobic glycolysis and less reliance on mitochondrial oxidative phosphorylation, whereas anti-inflammatory (M2-like) macrophages display more reliance on glucose and fatty acid oxidation in the mitochondria. Here, we describe in-depth protocols for extracting macrophages from the two major monocyte/macrophage reservoirs in the body, the spleen and bone marrow, as well as injured tissues such as the heart following myocardial infarction.
Macrophages or monocytes are extracted by immunomagnetic sorting by using antibody-tagged microbeads, which easily bind to cells without compromising their phenotypes. The extracted cells are then cultured in 96-well plates, followed by extracellular flux analysis using a metabolic flux analyzer. Both glycolysis and mitochondrial oxidative phosphorylation can be measured simultaneously in small numbers of cells (as little as 2-3 &#215; 105 cells). This method can easily be performed in 1 day and produces reliable and repeatable results. Ultimately, these methods help to enhance our understanding of metabolic changes during immune and inflammatory responses to injury and disease, which could lead to the development of novel therapeutic targets for immunometabolic pathways.

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Biology

Monitoring Brain Metabolite Transport in Drosophila Using FRET Sensors

Visualizing Monocarboxylates and Other Relevant Metabolites in the Ex Vivo Drosophila Larval Brain Using Genetically Encoded Sensors

The high energy requirements of brains due to electrical activity are one of their most distinguishing features. These requirements are met by the production of ATP from glucose and its metabolites, such as the monocarboxylates lactate and pyruvate. It is still unclear how this process is regulated or who the key players are, particularly in Drosophila.
Using genetically encoded F&#246;rster resonance energy transfer-based sensors, we present a simple method for measuring the transport of monocarboxylates and glucose in glial cells and neurons in an ex-vivo Drosophila larval brain preparation. The protocol describes how to dissect and adhere a larval brain expressing one of the sensors to a glass coverslip. 
We present the results of an entire experiment in which lactate transport was measured in larval brains by knocking down previously identified monocarboxylate transporters in glial cells. Furthermore, we demonstrate how to rapidly increase neuronal activity and track metabolite changes in the active brain. The described method, which provides all necessary information, can be used to analyze other Drosophila living tissues.

Author Spotlight: Advances in Brain Energy Metabolism Research Using the Drosophila Model

Here we present a protocol to visualize the transport of monocarboxylates, glucose, and ATP in glial cells and neurons using genetically encoded F&#246;rster resonance energy transfer-based sensors in an ex-vivo Drosophila larval brain preparation.

The high energy requirements of brains due to electrical activity are one of their most distinguishing features. These requirements are met by the ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

An Introduction to Cell Metabolism

In cells, critical molecules are either built by joining together individual units like amino acids or nucleotides, or broken down into smaller components. Respectively, the reactions responsible for this are referred to as anabolic and catabolic. These reactions require or produce energy typically in the form of a &#8220;high-energy&#8221; molecule called ATP. Together, these processes make up &#8220;Cell Metabolism,&#8221; and are hallmarks of healthy, living cells.JoVE&#8217;s introduction to cell metabolism briefly reviews the rich history of this field, ranging from early studies on photosynthesis to more recent discoveries pertaining to energy production in all cells. This is followed by a discussion of some key questions asked by scientists studying metabolism, and common methods that they apply to answer these questions. Finally, we&#8217;ll explore how current researchers are studying alterations in metabolism that accompany metabolic disorders, or that occur following exposure to environmental stressors.

Cell Metabolism, Metabolic Pathways and Assays

In cells, critical molecules are either built by joining together individual units like amino acids or nucleotides, or broken down into smaller ...

Education

JoVE Science Education

Advanced Biology

Cell Biology

Colorimetric Assay for Quantification of Lactate in Test Samples

Source: Yanase, S., et al. <a href="https://app.jove.com/v/57807">Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans.</a> J. Vis. Exp. (2018).
This video describes a colorimetric assay for lactate measurement in cell lysates by using the lactate dehydrogenase enzyme. This enzyme converts lactate into pyruvate, along with the conversion of NAD to NADH that reacts with tetrazolium dyes and gives a colored product that can be measured spectrophotometrically to quantitate the lactate.

Source: Yanase, S., et al. Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans. J. Vis. Exp. ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Summary

Explore More Videos

Chapters in this video

Hyperpolarized ¹³C Metabolic Magnetic Resonance Spectroscopy and Imaging

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Visualizing Monocarboxylates and Other Relevant Metabolites in the Ex Vivo Drosophila Larval Brain Using Genetically Encoded Sensors

The Lactate Dehydrogenase Sequestration Assay — A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

An Introduction to Cell Metabolism

Colorimetric Assay for Quantification of Lactate in Test Samples