Acidimetric assay for phospholipase A using egg yolk suspension as substrate

Tan, Nget Hong; Tan, Chon-Seng

doi:10.1016/0003-2697(88)90632-x

Cited by 81 publications

(31 citation statements)

References 11 publications

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“…Mice used in this study were of albino Institute of Cancer Research (ICR) strain (20)(21)(22)(23)(24)(25) 18 pipette tips were purchased from Merck Millipore (Billerica, MA). Resource S (1 mL) column was purchased from GE Healthcare (Stockholm, Sweden).…”

Section: Methodsmentioning

confidence: 99%

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Venom and Purified Toxins of the Spectacled Cobra (Naja naja) from Pakistan: Insights into Toxicity and Antivenom Neutralization

Wong¹,

Tan²,

Tan³

2016

The American Society of Tropical Medicine and Hygiene

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Abstract. Geographical variations of snake venoms can result in suboptimal effectiveness of Indian antivenoms that are currently used in most South Asian countries. This study investigated the toxicity and neutralization profile of the venom and toxins from Pakistani spectacled cobra, Naja naja, using VINS polyvalent antivenom (VPAV, India), Naja kaouthia monovalent antivenom (NKMAV, Thailand), and neuro bivalent antivenom (NBAV, Taiwan). Cationexchange and reverse-phase high-performance liquid chromatography fractionations followed by toxin identification through liquid chromatography-mass spectrometry (MS)/MS indicated that the venom comprised mainly of postsynaptic neurotoxins (NTXs) (long neurotoxins [LNTXs], 28.3%; short neurotoxins [SNTXs], 8%), cytotoxins (CTXs) (31.2%), and acidic phospholipases A 2 (12.3%). NKMAV is the most effective in neutralizing the lethal effect of the venom (potency = 1.1 mg venom/mL) and its LNTX (potency = 0.5 mg toxin/mL), consistent with the high content of LNTX in N. kaouthia venom. VPAV was effective in neutralizing the CTX (potency = 0.4 mg toxin/mL), in agreement with the higher CTX abundance in Indian cobra venom. All the three antivenoms were weak in neutralizing the SNTX (potency = 0.03-0.04 mg toxin/mL), including NBAV that was raised from the SNTX-rich Taiwanese cobra venom. In a challenge-rescue experiment, envenomed mice were prevented from death by a maximal dose of VPAV (intravenous 200 μL) but the recovery from paralysis was slow, indicating the need for higher or repeated doses of VPAV. Our results suggest that optimal neutralization for Pakistani N. naja venom may be achieved by improving the formulation of antivenom production to enhance antivenom immunoreactivity against long and SNTXs.

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Section: Methodsmentioning

confidence: 99%

“…The enzymatic activity of phospholipases (PLA 2 ) was tested on egg yolk substrate using an acidimetric method described by Tan and Tan. 22 The activity of the enzyme was expressed in micromoles of fatty acid released/min/mg enzyme.…”

Section: Methodsmentioning

confidence: 99%

Venom and Purified Toxins of the Spectacled Cobra (Naja naja) from Pakistan: Insights into Toxicity and Antivenom Neutralization

Wong¹,

Tan²,

Tan³

2016

The American Society of Tropical Medicine and Hygiene

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“…Phospholipase A 2 activity was determined by the acidimetric method (27). The egg yolk substrate suspension was prepared by mixing one part chicken egg yolk, one part 18 mM calcium (II) chloride, and one part 8.1 mM sodium deoxycholate.…”

Section: Phospholipase a 2 Assaymentioning

confidence: 99%

Enzymatic and toxinological activities of Hypnale hypnale (hump-nosed pit viper) venom and its fractionation by ion exchange high performance liquid chromatography

Tan¹,

Sim²,

Gnanathasan³

et al. 2011

J. Venom. Anim. Toxins incl. Trop. Dis

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Abstract:Hypnale hypnale (hump-nosed pit viper) has been recently identified as one of the medically important venomous snakes in Sri Lanka and on the southwestern coast of India. The characterization of its venom is essential for understanding the pathophysiology of envenomation and for optimizing its management. In the present study, the biological properties of Hypnale hypnale venom and venom fractions obtained using Resource Q ion exchange chromatography were determined. The venom exhibited toxic activities typical of pit viper venom, comparable to that of its sister taxon, the Malayan pit viper (Calloselasma rhodostoma). Particularly noteworthy were its high activities of thrombin-like enzyme, proteases, phospholipase A 2 , L-amino acid oxidase and hyaluronidase. The thrombin-like enzyme was mainly acidic and distributed over several chromatography fractions, indicating its existence in multiple isoforms. The hemorrhagic and necrotic activities of the venom were likely associated with the proteolytic enzyme found mainly in the basic fraction. Phospholipase A 2 and phosphomonoesterase exist in both acidic and basic isoforms, while L-amino acid oxidase and hyaluronidase are highly acidic. The venom clotting activity on fibrinogens showed distinct species specificity in the following increasing order for clotting time: bovine < rabbit < goat < human < horse < < dog, and was comparable to that of C. rhodostoma venom. Its clot formation on human fibrinogen is gradual and prolonged, a phenomenon suggestive of consumptive coagulopathy as a complication observed clinically. At an intramuscular sublethal dose, the venom did not cause acute kidney injury in a rodent model, contrary to the positive control group treated with Daboia russelii venom. Nephrotoxicity may result from higher venom doses in the context of coagulopathy, as a complication provoked by venom hematoxicity.

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“…Phospholipase A2 assay was determined according to the acidimetric method with little modification (Tan and Tan, 1988). Briefly, egg yolk suspension was prepared by mixing 1% lecithin, 18 mM calcium chloride and 8.1 mM sodium deoxycholate.…”

Section: Neutralization Of Phospholipase A2 Assaymentioning

confidence: 99%

In vitro neutralization of Eachis carinatus and Naja naja venom by Canthium parviflorum and its GC-MS analysis

Shrikanth

Janardhan

2016

Bangladesh J Pharmacol

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Canthium parviflorum is used in the traditional therapy to treat snakebite victims. In the present study, partially purified methanol root extract of C. parviflorum was used for the neutralization of Eachis carinatus and Naja naja venom. The extract inhibited in vitro phosphomonoesterase, phosphodiesterase, acetylcholinesterase, hyaluronidase, protease, phospholipase A2 and 5’nucleotidase activities of both venoms. One of the promising band exhibited neutralization of all the in vitro enzyme activities and was further subjected to GC-MS analysis which revealed the presence of eight active phytocompounds. These phytochemicals might be responsible for in vitro enzyme neutralization. Methanol extract possesses potent active phytochemicals against the site specific toxins and hydrolytic enzymes analyzed.Video Clip of Methodology:<a href="https://youtube.com/v/oSLdFcfaJxU">Phospholipase A2 assay</a>: 1 min 36 sec

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Acidimetric assay for phospholipase A using egg yolk suspension as substrate

Cited by 81 publications

References 11 publications

Venom and Purified Toxins of the Spectacled Cobra (Naja naja) from Pakistan: Insights into Toxicity and Antivenom Neutralization

Venom and Purified Toxins of the Spectacled Cobra (Naja naja) from Pakistan: Insights into Toxicity and Antivenom Neutralization

Enzymatic and toxinological activities of Hypnale hypnale (hump-nosed pit viper) venom and its fractionation by ion exchange high performance liquid chromatography

<i>In vitro</i> neutralization of <i>Eachis carinatus</i> and <i>Naja naja</i> venom by <i>Canthium parviflorum</i> and its GC-MS analysis

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