Endogenous Metabolite; Microbial Metabolite

Tall fescue (Festuca arundinacea Schreb) is a typical cool-season grass that is widely used in turf and pasture. However, high temperature as an abiotic stress seriously affects its utilization. The objective of this study was to explore the effect of spermidine (Spd) on heat stress response of tall fescue. The samples were exposed to 22°C (normal condition) or 44°C (heat stress) for 4 h. The results showed that exogenous Spd partially improved the quality of tall fescue leaves under normal temperature conditions. Nevertheless, after heat stress treatment, exogenous Spd significantly decreased the electrolyte leakage of tall fescue leaves. Spd also profoundly reduced the H2O2 and O2⋅- content and increased antioxidant enzymes activities. In addition, PAs can also regulate antioxidant enzymes activities including SOD, POD, and APX which could help to scavenge ROS. Moreover, application of Spd could also remarkably increase the chlorophyll content and had a positive effect on the chlorophyll α fluorescence transients under high temperature. The Spd reagent enhanced the performance of photosystem II (PSII) as observed by the JIP-test. Under heat stress, the Spd profoundly improved the partial potentials at the steps of energy bifurcations (PIABS and PItotal) and the quantum yields and efficiencies (φP0, δR0, φR0, and γRC). Exogenous Spd could also reduce the specific energy fluxes per QA- reducing PSII reaction center (RC) (TP0/RC and ET0/RC). Additionally, exogenous Spd improved the expression level of psbA and psbB, which encoded the proteins of PSII core reaction center complex. We infer that PAs can stabilize the structure of nucleic acids and protect RNA from the degradation of ribonuclease. In brief, our study indicates that exogenous Spd enhances the heat tolerance of tall fescue by maintaining cell membrane stability, increasing antioxidant enzymes activities, improving PSII, and relevant gene expression.[1]

Crude Enzyme Extraction[1]
For enzyme extracts, a 0.2 g of leaves powder with liquid nitrogen was immersed in 4 mL phosphate buffer (150 mM, pH 7.0) precooled at 4°C homogenized with 0.2 M Na2HPO4 and 0.2 M NaH2PO4. Then, the homogenate was centrifuged at 15,000 × g at 4°C for 30 min. Finally, the supernatant was collected and stored at 4°C to determine enzyme activities.[1]

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Antioxidant Enzyme Activity[1]
For the SOD activity assay, a 0.1 mL enzyme extract was added into 2.9 mL solution plus 50 mM phosphate buffer (pH 7.8), 1.125 mM nitro blue tetrazolium (NBT), 60 μM riboflavin, 195 mM methionine and 3 μM ethylene diamine tetraacetic acid (EDTA). Then, the solution was incubated under 4000 lx irradiance for 30 min. The change of absorbance at 560 nm was recorded with 3 mL of solution without enzyme extract as the control. One unit of SOD activity was defined as the inhibition of NBT reduction by 50%.[1]
The POD activity was measured based on the method described by Fan et al. (2014). In brief, a 50 μL enzyme extract was added into 2.95 mL solution containing 0.075% H2O2, 0.1 M sodium acetate-acetic buffer (pH 5.0), 0.25 mL guaiacol (dissolved in 50% ethanol solution). Then we recorded the absorbance changes at 460 nm per minute for 3 min. One unit POD activity is defined as the increase in absorbance per minute.[1]
The APX activity was measured using Plant APX Elisa Kit.[1]

Evaluation of the Optimum Spd Concentration[1]
To determine the adequate effective spermidine (Spd) concentration for alleviating heat stress, we performed a preliminary experiment by applying different concentration Spd. The concentration of Spd (0, 0.5, 1, and 2 mM) were chosen preliminarily according to the Mostofa experiment on rice (Mostofa et al., 2014). Subsequently, we selected the optimum concentration (0.5 mM) by comparing the fluorescence transients after heat stress 4 h (Figure ​Figure11). Figure ​Figure11 shows the differential changes in chlorophyll fluorescence transients after treatment with different concentration of Spd under heat stress. A 0.5 mM of Spd had the positive impact on photosynthesis by improving FJ, FI, and FP.

Metabolism / Metabolites
Uremic toxins tend to accumulate in the blood either through dietary excess or through poor filtration by the kidneys. Most uremic toxins are metabolic waste products and are normally excreted in the urine or feces.

Toxicity Summary
Uremic toxins such as spermidine are actively transported into the kidneys via organic ion transporters (especially OAT3). Increased levels of uremic toxins can stimulate the production of reactive oxygen species. This seems to be mediated by the direct binding or inhibition by uremic toxins of the enzyme NADPH oxidase (especially NOX4 which is abundant in the kidneys and heart) (A7868). Reactive oxygen species can induce several different DNA methyltransferases (DNMTs) which are involved in the silencing of a protein known as KLOTHO. KLOTHO has been identified as having important roles in anti-aging, mineral metabolism, and vitamin D metabolism. A number of studies have indicated that KLOTHO mRNA and protein levels are reduced during acute or chronic kidney diseases in response to high local levels of reactive oxygen species (A7869).

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Toxicity Summary
Uremic toxins such as spermidine are actively transported into the kidneys via organic ion transporters (especially OAT3). Increased levels of uremic toxins can stimulate the production of reactive oxygen species. This seems to be mediated by the direct binding or inhibition by uremic toxins of the enzyme NADPH oxidase (especially NOX4 which is abundant in the kidneys and heart) (A7868). Reactive oxygen species can induce several different DNA methyltransferases (DNMTs) which are involved in the silencing of a protein known as KLOTHO. KLOTHO has been identified as having important roles in anti-aging, mineral metabolism, and vitamin D metabolism. A number of studies have indicated that KLOTHO mRNA and protein levels are reduced during acute or chronic kidney diseases in response to high local levels of reactive oxygen species (A7869). A7868: Schulz AM, Terne C, Jankowski V, Cohen G, Schaefer M, Boehringer F, Tepel M, Kunkel D, Zidek W, Jankowski J: Modulation of NADPH oxidase activity by known uraemic retention solutes. Eur J Clin Invest. 2014 Aug;44(8):802-11. doi: 10.1111/eci.12297. PMID:25041433

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Health Effects
Chronic exposure to uremic toxins can lead to a number of conditions including renal damage, chronic kidney disease and cardiovascular disease.

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Exposure Routes: Endogenous, Ingestion, Dermal (contact)

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Symptoms
As a uremic toxin, this compound can cause uremic syndrome. Uremic syndrome may affect any part of the body and can cause nausea, vomiting, loss of appetite, and weight loss. It can also cause changes in mental status, such as confusion, reduced awareness, agitation, psychosis, seizures, and coma. Abnormal bleeding, such as bleeding spontaneously or profusely from a very minor injury can also occur. Heart problems, such as an irregular heartbeat, inflammation in the sac that surrounds the heart (pericarditis), and increased pressure on the heart can be seen in patients with uremic syndrome. Shortness of breath from fluid buildup in the space between the lungs and the chest wall (pleural effusion) can also be present.

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Treatment
Kidney dialysis is usually needed to relieve the symptoms of uremic syndrome until normal kidney function can be restored.

[1]. Front Plant Sci. 2017 Oct 12:8:1747.

Spermidine is a triamine that is the 1,5,10-triaza derivative of decane. It has a role as a fundamental metabolite, a geroprotector and an autophagy inducer. It is a triamine and a polyazaalkane. It is a conjugate base of a spermidine(3+).
Spermidine is a polyamine formed from putrescine. It is found in almost all tissues in association with nucleic acids. It is found as a cation at all pH values, and is thought to help stabilize some membranes and nucleic acid structures. It is a precursor of spermine.
Spermidine is a metabolite found in or produced by Escherichia coli (strain K12, MG1655).
Spermidine has been reported in Camellia sinensis, Pseudomonas hydrogenovora, and other organisms with data available.
Spermidine is a polyamine derived from putrescine that is involved in many biological processes, including the regulation of membrane potential, the inhibition of nitric oxide synthase (NOS) and the induction of autophagy.
Spermidine is a uremic toxin. Uremic toxins can be subdivided into three major groups based upon their chemical and physical characteristics: 1) small, water-soluble, non-protein-bound compounds, such as urea; 2) small, lipid-soluble and/or protein-bound compounds, such as the phenols and 3) larger so-called middle-molecules, such as beta2-microglobulin. Chronic exposure of uremic toxins can lead to a number of conditions including renal damage, chronic kidney disease and cardiovascular disease.
Spermidine is a polyamine formed from putrescine. It is found in almost all tissues in association with nucleic acids. It is found as a cation at all pH values, and is thought to help stabilize some membranes and nucleic acid structures. It is a precursor of spermine.
Spermidine is a metabolite found in or produced by Saccharomyces cerevisiae.
A polyamine formed from putrescine. It is found in almost all tissues in association with nucleic acids. It is found as a cation at all pH values, and is thought to help stabilize some membranes and nucleic acid structures. It is a precursor of spermine.
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C7H19N3

145.25

145.157

C, 57.88; H, 13.19; N, 28.93

124-20-9

124-20-9; 334-50-9 (3HCl)

1102

Colorless to light yellow solid if <23°C, and liquid if >25°C

0.9±0.1 g/cm3

246.6±8.0 °C at 760 mmHg

23-25 °C

118.1±22.0 °C

0.0±0.5 mmHg at 25°C

1.475

-0.84

3

3

7

10

56.8

0

NCCCCNCCCN

ATHGHQPFGPMSJY-UHFFFAOYSA-N

InChI=1S/C7H19N3/c8-4-1-2-6-10-7-3-5-9/h10H,1-9H2

spermidine; 1,5,10-Triazadecane; 4-Azaoctamethylenediamine; N1-(3-Aminopropyl)butane-1,4-diamine

spermidine; 124-20-9; 1,5,10-Triazadecane; 4-Azaoctamethylenediamine; N1-(3-Aminopropyl)butane-1,4-diamine; Spermidin; 4-Azaoctane-1,8-diamine; N-(3-aminopropyl)butane-1,4-diamine;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O:~100 mg/mL (688.5 mM)
DMSO: ~16.7 mg/mL (114.8 mM; with ultrasonic and warming as well as adjusting pH to 7 with HCl)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)