Human Endogenous Metabolite

Owing to the prohibition of cosmetic animal testing, various attempts have recently been made using skin-on-a-chip (SOC) technology as a replacement for animal testing. Previously, we reported the development of a pumpless SOC capable of drug testing with a simple drive using the principle that the medium flows along the channel by gravity when the chip is tilted using a microfluidic channel. In this study, using pumpless SOC, instead of drug testing at the single-cell level, we evaluated the efficacy of α-Lipoic acid (ALA), which is known as an anti-aging substance in skin equivalents, for skin tissue and epidermal structure formation. The expression of proteins and changes in genotyping were compared and evaluated. Hematoxylin and eosin staining for histological analysis showed a difference in the activity of fibroblasts in the dermis layer with respect to the presence or absence of ALA. We observed that the epidermis layer became increasingly prominent as the culture period was extended by treatment with 10 μM ALA. The expression of epidermal structural proteins of filaggrin, involucrin, keratin 10, and collagen IV increased because of the effect of ALA. Changes in the epidermis layer were noticeable after the ALA treatment. As a result of aging, damage to the skin-barrier function and structural integrity is reduced, indicating that ALA has an anti-aging effect. We performed a gene analysis of filaggrin, involucrin, keratin 10, integrin, and collagen I genes in ALA-treated human skin equivalents, which indicated an increase in filaggrin gene expression after ALA treatment. These results indicate that pumpless SOC can be used as an in vitro skin model similar to human skin, protein and gene expression can be analyzed, and it can be used for functional drug tests of cosmetic materials in the future. This technology is expected to contribute to the development of skin disease models. [2]

Lipoic acid (LA) and hyperbaric oxygenation therapy (HBOT) improve chronic wound healing. Objective: We compared the effects of LA or its enantiomer R-(+)-lipoic acid (RLA) on wound healing. Materials and methods: Groups LA + HBOT (L), RLA + HBOT (R) and placebo + HBOT (P). Lesion areas measured before treatment and on 20th and 40th day. The biopsies and plasma were harvested before treatment and on 7th and 14th (measurements of VEGF, vascular endothelial growth factor; EGF, epidermal growth factor, TNF-α and IL-6). Results: Ulcers improved more on RLA. In both L and R groups, EGF and VEFG increased in time. RLA decreased IL-6 on T7 and T14, which did not happen with LA. TNF-α levels decreased on T14 in both LA and RLA. Discussion: The improved wound healing is associated with increased EGF and VEGF and reduced plasma TNF-α and IL-6. Conclusion: RLA may be more effective than LA in improving chronic wound healing in patients undergoing HBO therapy. [1]

Construction of a 3D Skin-Equivalent Model [2]
As shown in Figure 10, rat-tail collagen type I, 10x DMEM, 0.5 N NaOH, primary human fibroblasts suspension (final cell concentration 5.0 × 105 cell/mL), and 1x DMEM were mixed to neutralize the gel. The rat-tail collagen concentration was fixed at 6.12 mg/mL. To fabricate the dermis layer, we seeded the collagen-FBs suspension on the chip to a height of 3 mm and deposited for 40 min at 37 °C in an incubator under 5% CO2. Thereafter, the dermal layer (DL) was cultured in DMEM (with 10% FBS and 1% penicillin/streptomycin) for 5 days, and the medium was changed every day. Next, primary human keratinocytes suspension (final cell concentration 1.0 × 106 cell/mL) were seeded on the dermis layer and cocultured for 2 days. KGM-GoldTM Keratinocyte Growth Medium was supplied only above the DL-KCs, and DMEM was supplied to the channel of the chip. Lipoic acid/ALA supplies E-media that induces the differentiation of KCs for 3 to 7 days and at the same time provides an environment similar to that of real skin by exposing it to air (E-media composition: DMEM/Ham’s F12 (EGF-1 10 ng/mL, hydrocortisone 0.4 μg/mL, insulin 5 μg/mL, transferrin 5 μg/mL, 3,3,5-triiodo-L-thyonine sodium salt 2 × 10−11 M, cholera toxin 10−10 M, 10% (v/v) FBS, 1% penicillin/streptomycin). ALA treatment was performed at the AE stage (1 μM and 10 μM ALA). Human dermal FBs were used at passages 5–7, and human epidermal KCs were used at passages 4–6. All cultures were incubated at 37 °C in an incubator under 5% CO2. The medium was changed every day.

The patients were randomly divided into three groups: [1]
Group L: A total of 10 patients (four male and six female with a mean age of 59 (45–83) years old). There were total 13 ulcerated lesions: five caused by arterial impairment, four caused by venous insufficiency, three were diabetic/ischemic origin and one caused by trauma. The average ulcer size was 3.92 cm2 with a mean history of 217 d. The patients were treated with Lipoic acid/LA 600 mg orally 60 min before each session of HBOT.
Group R: A total of 10 patients (five male and five female with a mean age of 71.8 (58–82) years old). There were total 10 ulcerated lesions: five caused by arterial impairment, four were diabetic/ischemic origin and one with diabetic neuropathy. The average ulcer size was 7.45 cm2 with a mean history of 233 d. These patients were treated with RLA, 600 mg orally 60 min before each session of HBOT.
Group P: Seven patients (three male and four female with a mean age of 72.1 (49–88) years old). There were total seven ulcerated lesions: four were diabetic/ischemic origin, two caused by arterial impairment and one from venous insufficiency. The average ulcer size was 3.18 cm2 with a mean history 224 d. These patients were treated with placebo 60 min before each session of HBOT.

Metabolism / Metabolites
Paraoxygenase (PON1) is a key enzyme in organophosphate metabolism. PON1 can inactivate certain organophosphates through hydrolysis. PON1 hydrolyzes active metabolites from various organophosphate pesticides and nerve agents (such as soman, sarin, and VX). The existence of PON1 polymorphism leads to differences in the enzyme activity level and catalytic efficiency of this esterase, which in turn suggests that different individuals may be more susceptible to the toxic effects of organophosphate exposure.

Toxicity Summary
(R)-Lipoic acid is a cholinesterase, or acetylcholinesterase (AChE) inhibitor. Cholinesterase inhibitors (or "anticholinesterases") inhibit the activity of acetylcholinesterase. Because acetylcholinesterase plays a vital physiological role, chemicals that interfere with its activity are potent neurotoxins; even low doses can cause excessive salivation and lacrimation, followed by muscle spasms and ultimately death. Substances used in nerve gases and many pesticides have been shown to exert their effects by binding to serine residues at the active site of acetylcholinesterase, thus completely inhibiting the enzyme's activity. Acetylcholinesterase breaks down the neurotransmitter acetylcholine, which is released at the neuromuscular junction, causing muscle or organ relaxation. Inhibition of acetylcholinesterase results in the accumulation and sustained action of acetylcholine, leading to the continuous transmission of nerve impulses and an inability to stop muscle contractions. The most common acetylcholinesterase inhibitors are phosphorus-containing compounds designed to bind to the enzyme's active site. Its structural requirements include a phosphorus atom with two lipophilic groups, a leaving group (e.g., a halide or thiocyanate), and a terminal oxygen atom.

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Health Effects
Acute exposure to cholinesterase inhibitors can cause cholinergic crisis, characterized by severe nausea/vomiting, salivation, sweating, bradycardia, hypotension, collapse, and convulsions. Increased muscle weakness is also possible, and if respiratory muscles are involved, it can lead to death. The accumulation of acetylcholine in motor nerves can lead to overexpression of nicotine receptors at the neuromuscular junction. When this occurs, symptoms such as muscle weakness, fatigue, muscle spasms, fasciculations, and paralysis may appear. When acetylcholine (ACh) accumulates in autonomic ganglia, it leads to overexcitation of nicotine receptors in the sympathetic nervous system. Related symptoms include hypertension and hypoglycemia. Overexcitation of nicotine acetylcholine receptors in the central nervous system due to acetylcholine accumulation can lead to anxiety, headache, convulsions, ataxia, respiratory and circulatory depression, tremors, general weakness, and even coma. When excessive acetylcholine on muscarinic acetylcholine receptors leads to over-excitation of these receptors, symptoms such as visual disturbances, chest tightness, wheezing due to bronchoconstriction, increased bronchial secretions, increased salivation, lacrimation, sweating, intestinal peristalsis, and urination may occur. Certain reproductive effects, including male and female fertility and growth and development, have been shown to be closely related to organophosphate pesticide exposure. Most studies on reproductive effects have focused on farmers using pesticides and insecticides in rural areas. In women, menstrual cycle disorders, prolonged pregnancy, spontaneous abortion, stillbirth, and some developmental problems in offspring are associated with organophosphate pesticide exposure. Prenatal exposure is associated with impaired fetal growth and development. Neurotoxicity is also associated with organophosphate pesticide poisoning, leading to four types of neurotoxic symptoms in humans: cholinergic syndrome, intermediate syndrome, organophosphate-induced delayed polyneuropathy (OPIDP), and chronic organophosphate-induced neuropsychiatric disorder (COPIND). These syndromes are caused by acute or chronic exposure to organophosphate pesticides. Symptoms
Symptoms of low-dose exposure include excessive salivation and lacrimation. Acute poisoning symptoms include severe nausea/vomiting, salivation, sweating, bradycardia, hypotension, collapse, and convulsions. Worsening muscle weakness may occur, potentially leading to death if respiratory muscles are involved. Other possible side effects include hypertension, hypoglycemia, anxiety, headache, tremor, and ataxia.
Treatment
If the compound is ingested, immediate gastric lavage with a 5% sodium bicarbonate solution should be performed. Skin contact should be washed immediately with soap and water. If the compound gets into the eyes, flush immediately with copious amounts of physiological saline or water. In severe cases, atropine and/or pralidoxime should be used. Anticholinergic drugs can antagonize the effects of excessive acetylcholine and reactivate acetylcholinesterase (AChE). Atropine can be used as an antidote in combination with pralidoxime or other pyridine oximes (such as trimeoxime or obizzime), but at least two meta-analyses have shown that the use of "oximes" is not beneficial and may even be harmful. Atropine is a muscarinic receptor antagonist, thus blocking the peripheral effects of acetylcholine.

[1]. Effects of alpha lipoic acid and its R+ enantiomer supplemented to hyperbaric oxygen therapy on interleukin-6, TNF-α and EGF production in chronic leg wound healing. J Enzyme Inhib Med Chem. 2014 Apr;29(2):297-302.

[2]. Effect of α-Lipoic Acid on the Development of Human Skin Equivalents Using a Pumpless Skin-on-a-Chip Model. Int J Mol Sci. 2021 Feb 22;22(4):2160.

Pharmacodynamics
Lipoic acid (or alpha-lipoic acid) can cross the blood-brain barrier and is presumably used to remove mercury attached to brain cells. Because it is a thiol (a sulfur-containing compound that readily binds to mercury), it can release the bound mercury into the bloodstream. In the blood, another chelating agent, such as dimercaptosuccinic acid (DMSA) or methanesulfonylmethane (MSM), is needed to safely transfer the mercury to the urine for excretion. Since DMSA cannot cross the blood-brain barrier, lipoic acid and DMSA are often used together. This therapy—along with carnitine, dimethylglycine (DMG), vitamin B6, folic acid, and magnesium—is presumably used to treat autism and amalgam poisoning. According to this hypothesis, autism is difficult to treat because mercury binds to brain cells, and most medications and vitamin supplements cannot cross the blood-brain barrier. However, alpha-lipoic acid, and perhaps vitamin B12, may help other chelating agents safely remove mercury from the body and holds promise for future use in treating autism. Because lipoic acid is involved in cellular glucose uptake and is soluble in both water and fat, it is used to treat diabetes. It may also be helpful for patients with Alzheimer's or Parkinson's disease. Mechanism of action: Lipoic acid is commonly involved in the oxidative decarboxylation of keto acids and is considered a growth factor in some organisms. Lipoic acid exists in two enantiomers: the R-enantiomer and the S-enantiomer. Normally, only the R-enantiomer of the amino acid is biologically active, but for lipoic acid, the S-enantiomer helps reduce the R-enantiomer when using a racemic mixture. Some recent studies have shown that the S-enantiomer actually inhibits the R-enantiomer, significantly reducing its biological activity and actually increasing rather than decreasing oxidative stress. Furthermore, studies have found that the S-enantiomer reduces the expression of GLUT-4, which is responsible for glucose uptake in cells, thereby reducing insulin sensitivity. (R)-Lipoic acid is the (R)-enantiomer of lipoic acid. It is a vitamin C8 thiocarboxylic acid with antioxidant properties. It can be used as a cofactor, nutritional supplement, and cofactor. It is an alpha-lipoic acid, belonging to the dithioheterocyclic pentane class of compounds, and is a heterocyclic fatty acid and a thiocarboxylic acid. Functionally, it is related to octanoic acid. It is the conjugate acid of (R)-alpha-lipoic acid and also the enantiomer of (S)-alpha-lipoic acid. It is a vitamin-like antioxidant. Alpha-lipoic acid is a metabolite found in or produced by Escherichia coli (K12 strain, MG1655 strain). (R)-Alpha-lipoic acid is a metabolite found in or produced by Escherichia coli (K12 strain, MG1655 strain). Tomatoes have also been reported to contain alpha-lipoic acid, and there is relevant data. Alpha-lipoic acid is a vitamin-like antioxidant that can scavenge free radicals. α-Alpha-lipoic acid, also known as thioctic acid, is a naturally occurring compound that can be synthesized by plants and animals. Lipoic acid contains two sulfhydryl groups and can be oxidized or reduced. The reduced state is called dihydrolipoic acid (DHLA). Lipoic acid (ΔE = -0.288) can therefore undergo thiol-disulfide bond exchange, thus exhibiting antioxidant activity. Lipoic acid is a key cofactor in aerobic metabolism, participating in the transfer of acyl or methylamine groups via 2-ketoate dehydrogenase (2-OADH) or the α-ketoglutarate dehydrogenase complex. This enzyme catalyzes the conversion of α-ketoglutarate to succinyl-CoA. This activity leads to the catabolism of branched-chain amino acids (leucine, isoleucine, and valine). Lipoic acid also participates in the glycine cleavage system (GCV). The GCV is a multi-enzyme complex that catalyzes the oxidation of glycine to 5,10-methylenetetrahydrofolate, a crucial cofactor in nucleic acid synthesis. Because lipoic acid is an essential cofactor for many enzyme complexes, it is vital for aerobic life as we know it. Many organisms utilize this system, which plays a key role in the photosynthetic carbon cycle. Lipoic acid was initially considered a potent antioxidant because it was found to prevent vitamin C and vitamin E deficiencies. It scavenge reactive oxygen species and reduce other metabolites, such as glutathione or vitamins, thus maintaining a healthy cellular redox state. Cell culture experiments have shown that lipoic acid can increase cellular glucose uptake by recruiting the glucose transporter GLUT4 to the cell membrane, suggesting its potential use in treating diabetes. Studies on rat aging have shown that the use of L-carnitine and lipoic acid can improve memory and slow mitochondrial structural decline. Therefore, it may be beneficial for patients with Alzheimer's or Parkinson's disease. Lipoic acid is a metabolite found or produced in Saccharomyces cerevisiae. It is an octanoic acid, bridged by two sulfur atoms, and is therefore also called valerate in some nomenclature schemes. It is biosynthesized from the cleavage of linoleic acid and is a coenzyme of α-ketoglutarate dehydrogenase (the ketoglutarate dehydrogenase complex). It is used in dietary supplements.

C8H14O2S2

206.3256

206.043

C, 46.57; H, 6.84; O, 15.51; S, 31.08

1200-22-2

Lipoic acid; 1200-22-2; 1077-28-7 (racemate)

6112

Light yellow to dark brown solid powder

1.2±0.1 g/cm3

362.5±11.0 °C at 760 mmHg

46-49ºC

173.0±19.3 °C

0.0±1.7 mmHg at 25°C

1.562

2.16

1

4

5

12

150

1

S1[C@@]([H])(C([H])([H])C([H])([H])S1)C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(=O)O[H]

AGBQKNBQESQNJD-SSDOTTSWSA-N

InChI=1S/C8H14O2S2/c9-8(10)4-2-1-3-7-5-6-11-12-7/h7H,1-6H2,(H,9,10)/t7-/m1/s1

5-[(3R)-dithiolan-3-yl]pentanoic acid

Thiogamma oral; R-(+)-alpha-Lipoic acid; (+)-alpha-Lipoic acid; Lipoate;Verla Lipon; Verla-Lipon; VerlaLipon; Lipoic Acid; Thioctacide T; Thioctic Acid; (R)-5-(1,2-Dithiolan-3-yl)pentanoic acid; Thiogamma Injekt; thioctic acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO: ≥ 100 mg/mL (~484.7 mM)
H2O: ~1 mg/mL (~4.9 mM)

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.)