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
Paraoxonase (PON1) is a key enzyme in the metabolism of organophosphates. PON1 can inactivate some organophosphates through hydrolysis. PON1 hydrolyzes the active metabolites in several organophosphates insecticides as well as, nerve agents such as soman, sarin, and VX. The presence of PON1 polymorphisms causes there to be different enzyme levels and catalytic efficiency of this esterase, which in turn suggests that different individuals may be more susceptible to the toxic effect of OP exposure.

Toxicity Summary
(R)-lipoic acid is a cholinesterase or acetylcholinesterase (AChE) inhibitor. A cholinesterase inhibitor (or 'anticholinesterase') suppresses the action of acetylcholinesterase. Because of its essential function, chemicals that interfere with the action of acetylcholinesterase are potent neurotoxins, causing excessive salivation and eye-watering in low doses, followed by muscle spasms and ultimately death. Nerve gases and many substances used in insecticides have been shown to act by binding a serine in the active site of acetylcholine esterase, inhibiting the enzyme completely. Acetylcholine esterase breaks down the neurotransmitter acetylcholine, which is released at nerve and muscle junctions, in order to allow the muscle or organ to relax. The result of acetylcholine esterase inhibition is that acetylcholine builds up and continues to act so that any nerve impulses are continually transmitted and muscle contractions do not stop. Among the most common acetylcholinesterase inhibitors are phosphorus-based compounds, which are designed to bind to the active site of the enzyme. The structural requirements are a phosphorus atom bearing two lipophilic groups, a leaving group (such as a halide or thiocyanate), and a terminal oxygen.

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Health Effects
Acute exposure to cholinesterase inhibitors can cause a cholinergic crisis characterized by severe nausea/vomiting, salivation, sweating, bradycardia, hypotension, collapse, and convulsions. Increasing muscle weakness is a possibility and may result in death if respiratory muscles are involved. Accumulation of ACh at motor nerves causes overstimulation of nicotinic expression at the neuromuscular junction. When this occurs symptoms such as muscle weakness, fatigue, muscle cramps, fasciculation, and paralysis can be seen. When there is an accumulation of ACh at autonomic ganglia this causes overstimulation of nicotinic expression in the sympathetic system. Symptoms associated with this are hypertension, and hypoglycemia. Overstimulation of nicotinic acetylcholine receptors in the central nervous system, due to accumulation of ACh, results in anxiety, headache, convulsions, ataxia, depression of respiration and circulation, tremor, general weakness, and potentially coma. When there is expression of muscarinic overstimulation due to excess acetylcholine at muscarinic acetylcholine receptors symptoms of visual disturbances, tightness in chest, wheezing due to bronchoconstriction, increased bronchial secretions, increased salivation, lacrimation, sweating, peristalsis, and urination can occur. Certain reproductive effects in fertility, growth, and development for males and females have been linked specifically to organophosphate pesticide exposure. Most of the research on reproductive effects has been conducted on farmers working with pesticides and insecticdes in rural areas. In females menstrual cycle disturbances, longer pregnancies, spontaneous abortions, stillbirths, and some developmental effects in offspring have been linked to organophosphate pesticide exposure. Prenatal exposure has been linked to impaired fetal growth and development. Neurotoxic effects have also been linked to poisoning with OP pesticides causing four neurotoxic effects in humans: cholinergic syndrome, intermediate syndrome, organophosphate-induced delayed polyneuropathy (OPIDP), and chronic organophosphate-induced neuropsychiatric disorder (COPIND). These syndromes result after acute and chronic exposure to OP pesticides.

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Symptoms
Symptoms of low dose exposure include excessive salivation and eye-watering. Acute dose symptoms include severe nausea/vomiting, salivation, sweating, bradycardia, hypotension, collapse, and convulsions. Increasing muscle weakness is a possibility and may result in death if respiratory muscles are involved. Hypertension, hypoglycemia, anxiety, headache, tremor and ataxia may also result.

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Treatment
If the compound has been ingested, rapid gastric lavage should be performed using 5% sodium bicarbonate. For skin contact, the skin should be washed with soap and water. If the compound has entered the eyes, they should be washed with large quantities of isotonic saline or water. In serious cases, atropine and/or pralidoxime should be administered. Anti-cholinergic drugs work to counteract the effects of excess acetylcholine and reactivate AChE. Atropine can be used as an antidote in conjunction with pralidoxime or other pyridinium oximes (such as trimedoxime or obidoxime), though the use of '-oximes' has been found to be of no benefit, or possibly harmful, in at least two meta-analyses. Atropine is a muscarinic antagonist, and thus blocks the action of acetylcholine peripherally.

[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 α-lipoic acid) is able to pass the blood-brain barrier and is putatively used for detoxification of mercury attached to the brain cells. It can mobilise bound mercury into the blood stream as it is a mercaptan (sulfur compound which readily binds to the mercury). In the blood stream, another chelator such as dimercaptosuccinic acid (DMSA) or methylsulfonylmethane (MSM) is used to transfer mercury safely into the urine for excretion. Since DMSA cannot cross the blood-brain barrier, both lipoic acid and DMSA tend to be used together. It is hypothesized that this treatment-along with carnitine, dimethylglycine (DMG), Vitamin B6, folic acid, and magnesium—could be used to treat autism and amalgam poisoning. In this hypothesis, the reason why autism is difficult to treat is that mercury is attached to the brain cells and most medicines and vitamin supplements do not penetrate the blood-brain barrier. However, α-lipoic acid and perhaps vitamin B12 could making it possible for other chelators to remove mercury safely out of the body and could perhaps one day be used as a treatment for autism. Because lipoic acid is related to cellular uptake of glucose and it is both soluble in water and fat, it is being used for treatment in diabetes. It may be helpful for people with Alzheimer's disease or Parkinson's disease.

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Mechanism of Action: Lipoic Acid is generally involved in oxidative decarboxylations of keto acids and is presented as a growth factor for some organisms. Lipoic acid exists as two enantiomers, the R-enantiomer and the S-enantiomer. Normally only the R-enantiomer of an amino acid is biologically active, but for lipoic acid the S-enantiomer assists in the reduction of the R-enantiomer when a racemic mixture is given. Some recent studies have suggested that the S-enantiomer in fact has an inhibiting effect on the R-enantiomer, reducing its biological activity substantially and actually adding to oxidative stress rather than reducing it. Furthermore, the S-enantiomer has been found to reduce the expression of GLUT-4s in cells, responsible for glucose uptake, and hence reduce insulin sensitivity.

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(R)-lipoic acid is the (R)-enantiomer of lipoic acid. A vitamin-like, C8 thia fatty acid with anti-oxidant properties. It has a role as a prosthetic group, a nutraceutical and a cofactor. It is a lipoic acid, a member of dithiolanes, a heterocyclic fatty acid and a thia fatty acid. It is functionally related to an octanoic acid. It is a conjugate acid of a (R)-lipoate. It is an enantiomer of a (S)-lipoic acid.
A vitamin-like antioxidant.
Lipoic acid is a metabolite found in or produced by Escherichia coli (strain K12, MG1655).
(R)-lipoic acid is a metabolite found in or produced by Escherichia coli (strain K12, MG1655).
Lipoic acid has been reported in Solanum lycopersicum with data available.

Lipoic acid is a vitamin-like antioxidant that acts as a free-radical scavenger. Alpha-lipoic acid is also known as thioctic acid. It is a naturally occurring compound that is synthesized by both plants and animals. Lipoic acid contains two thiol groups which may be either oxidized or reduced. The reduced form is known as dihydrolipoic acid (DHLA). Lipoic acid (Delta E= -0.288) is therefore capable of thiol-disulfide exchange, giving it antioxidant activity. Lipoate is a critical cofactor for aerobic metabolism, participating in the transfer of acyl or methylamine groups via the 2-Oxoacid dehydrogenase (2-OADH) or alpha-ketoglutarate dehydrogenase complex. This enzyme catalyzes the conversion of alpha-ketoglutarate to succinyl CoA. This activity results in the catabolism of the branched chain amino acids (leucine, isoleucine and valine). Lipoic acid also participates in the glycine cleavage system(GCV). The glycine cleavage system is a multi-enzyme complex that catalyzes the oxidation of glycine to form 5,10 methylene tetrahydrofolate, an important cofactor in nucleic acid synthesis. Since Lipoic acid is an essential cofactor for many enzyme complexes, it is essential for aerobic life as we know it. This system is used by many organisms and plays a crucial role in the photosynthetic carbon cycle. Lipoic acid was first postulated to be an effective antioxidant when it was found it prevented vitamin C and vitamin E deficiency. It is able to scavenge reactive oxygen species and reduce other metabolites, such as glutathione or vitamins, maintaining a healthy cellular redox state. Lipoic acid has been shown in cell culture experiments to increase cellular uptake of glucose by recruiting the glucose transporter GLUT4 to the cell membrane, suggesting its use in diabetes. Studies of rat aging have suggested that the use of L-carnitine and lipoic acid results in improved memory performance and delayed structural mitochondrial decay. As a result, it may be helpful for people with Alzheimer's disease or Parkinson's disease.

Lipoic acid is a metabolite found in or produced by Saccharomyces cerevisiae.
An octanoic acid bridged with two sulfurs so that it is sometimes also called a pentanoic acid in some naming schemes. It is biosynthesized by cleavage of LINOLEIC ACID and is a coenzyme of oxoglutarate dehydrogenase (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.)