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BioAssay: AID 691

Zebrafish Lipid Metabolism Assay - Dose-Response Confirmation.

The zebrafish processes lipids through its digestive system in a similar way to mammals. Thus it is a useful model organism that provides for in vivo measurement of lipid absorption and processing in a vertebrate organism. Zebrafish larvae are transparent, allowing for observation of lipid metabolism in the whole organism using fluorescent lipid substrates. Zebrafish therefore provide a screening platform to identify compounds that interfere with lipid metabolism and lower the high levels of cholesterol and triglycerides implicated as a major contributor to heart disease in humans. ..more
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 Tested Compounds
 Tested Compounds
All(53)
 
 
Active(12)
 
 
Inactive(38)
 
 
Inconclusive(3)
 
 
 Tested Substances
 Tested Substances
All(53)
 
 
Active(12)
 
 
Inactive(38)
 
 
Inconclusive(3)
 
 
 Related BioAssays
 Related BioAssays
AID: 691
Data Source: PCMD (ZEBRAFISH LIPID METABOLISM-DOSE RESPONSE)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-04-23
Modify Date: 2008-10-07

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 12
Description:
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Dr. Amy Rubenstein, Zygogen LLC, Atlanta, GA
MLSCN Grant: X01-MH077634-01

The zebrafish processes lipids through its digestive system in a similar way to mammals. Thus it is a useful model organism that provides for in vivo measurement of lipid absorption and processing in a vertebrate organism. Zebrafish larvae are transparent, allowing for observation of lipid metabolism in the whole organism using fluorescent lipid substrates. Zebrafish therefore provide a screening platform to identify compounds that interfere with lipid metabolism and lower the high levels of cholesterol and triglycerides implicated as a major contributor to heart disease in humans.

The assay reported here was designed to confirm the activity of compounds previously found to inhibit the fluorescence resulting from cleavage of PED6, a quenched fluorescent lipid substrate for phospholipase A2 [Farber SA, Pack M, H0 S-Y, et al., Science, 292, 1385-1388 (2001)]. Zebrafish larvae (5-7 days old) were fed PED6, which is swallowed, emulsified in the lumen of the intestine, and cleaved by phospholipases. Cleavage of PED6 releases fluorescent fatty acids that are absorbed through the intestinal epithelium, transported to the liver, incorporated into bile, and concentrated in the gall bladder. Fluorescence is observed in the gall bladder and also in the intestine following secretion of fluorescent bile.
Protocol
Materials
Zebrafish were cultivated in the laboratory of Dr. Michael Pack, School of Medicine, University of Pennsylvania. PED6 was purchased from Invitrogen (catalog no. D-23739). Clear flat-bottom and V-bottom polypropylene 96-well microtiter plates were purchased from Corning and Greiner, respectively.
Assay
Zebrafish larvae (5-7 days old) were dispensed into clear flat-bottom microtiter plates to give 5 fish per well in 37.5 uL of E3 media (5 mM sodium chloride, 0.17 mM potassium chloride, 0.33 mM calcium chloride, 0.33 mM magnesium sulfate in deionized water). Test compounds were serially diluted in DMSO from 10 mM to 100 uM. The dilutions in DMSO were transferred into V-bottom polypropylene microtiter plates to give a final range of concentrations of 200 to 1.56 uM in 2% aqueous DMSO. These aqueous compound solutions (37.5 uL) were then transferred by multichannel pipet into the zebrafish plates to give a final range of concentrations of 100 to 0.78 uM. PED6 was added to a final concentration of 100 nM.
After overnight incubation with PED6 and test compounds, zebrafish larvae were viewed under an Olympus IX81 inverted fluorescent microscope. Each well was scored visually based on the fluorescence observed in the gall bladder and intestine of each fish.
Data analysis
Fifty-three compounds previously identified as active at 20 uM in a PED6 screen were retested at concentrations ranging from 100 to 0.78 uM. Each concentration was assigned activity as follows:
ABSENCE _FLUOR_GB&INT
= Complete inhibition of fluorescence in gall bladder and intestine
WEAK_FLUOR_GB&INT
= Partial inhibition of fluorescence in gall bladder and intestine
WEAK _FLUOR_GB_&_ABSENCE_FLUOR_INT
= Partial inhibition of fluorescence in gall bladder and complete inhibition of fluorescence of intestine
TOXIC
= No movement
INACTIVE
= No reduction in fluorescence
A minimum inhibitory concentration (MIC) was assigned to each compound as follows:
MIC = lowest concentration that gave either complete or partial inhibition of fluorescence.
A MIC >100 was reported for compounds that showed no effect on fluorescence at all the concentrations tested. A blank in the MIC #2 column indicates that a given compound was not tested a second time.
Compounds that gave a measurable MIC (i.e. MIC = 100 uM or less) were retested.
Comment
Activity Scoring
Scores were calculated as follows:
Activity score = MIC #1 score + MIC #2 score
Where MIC score = 14 x [-log(MIC in mol/L) -3.5]
Activity Outcome
Active = 12 compounds that gave mean MIC less than or equal to 100 uM.
Inconclusive = 3 compounds that gave an MIC of 25 uM but were toxic at 50 uM
Inactive = 38 compounds that gave no reduction in fluorescence (MIC >100).
Contributors
Zebrafish were cultivated in the laboratory of Dr. Michael Pack, School of Medicine, Unversity of Pennsylvania. Compound plates were prepared by Edinson Lucumi and screening was conducted by David Justin. Data were submitted by Edinson Lucumi and Andrew Napper.
Correspondence
Please direct correspondence to Andrew Napper (napper@seas.upenn.edu).
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierString
2Mean MIC*FloatμM
3Number of MIC determinationsInteger
4QualifierString
5MIC #1FloatμM
6QualifierString
7MIC #2FloatμM
8MIC #1 activity at 0.78 microM (0.78μM**)String
9MIC #1 activity at 1.56 microM (1.56μM**)String
10MIC #1 activity at 3.13 microM (3.13μM**)String
11MIC #1 activity at 6.25 microM (6.25μM**)String
12MIC #1 activity at 12.5 microM (12.5μM**)String
13MIC #1 activity at 25 microM (25μM**)String
14MIC #1 activity at 50 microM (50μM**)String
15MIC #1 activity at 100 microM (100μM**)String
16MIC #2 activity at 0.78 microM (0.78μM**)String
17MIC #2 activity at 1.56 microM (1.56μM**)String
18MIC #2 activity at 3.13 microM (3.13μM**)String
19MIC #2 activity at 6.25 microM (6.25μM**)String
20MIC #2 activity at 12.5 microM (12.5μM**)String
21MIC #2 activity at 25 microM (25μM**)String
22MIC #2 activity at 50 microM (50μM**)String
23MIC #2 activity at 100 microM (100μM**)String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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