BIOASSAYS

-Are the techinques, where the estimation of conc/potency

of a drug(s) by measuring its biological responses in living
systems i.e cells, tissues, enzymes etc.
Different types of bio-assays
1. Isolated organ assays(high cost)
2. Tissue based assays
3. Cell based assays
4. Subcellular assays- a enzyme inhibition
b. receptor binding assay
ADVANTAGES OF BIO-ASSAY
Rapid,
Economical
Reproducible
High throughput screening is possible
Requires microgram to milligram
quantities of sample
IMMUNOMODULATORY ASSAYS
Lymphocyte proliferation assay:
Lymphocyte proliferation assay (LPA) is a measure of immune
modulation. It measures the ability of lymphocytes placed in short
term tissue culture to undergo clonal proliferation when exposed to a
foreign substance/mitogen, which is very useful in evaluating the
acute phase effect of test substance on immunity. This assay helps
in the evaluation of the immunostimulatory/immunosuppressive
activity of a substance/herbal test substance.
The assay is performed either alone or with mitogens using
histopaque purified splenocyte population. Cell proliferation is
measured at the end of the incubation period (generally 48 hrs)
using a cell proliferation reagent MTT.


PROCEDURE
Isolation of mouse splenocytes: mouse spleen 40 mesh
Plating splenocytes,
Cell treatment,
Estimation:
Preparation of std curve:

References:
Fernandez-Botran, R. and Vetvicka, V. (2001) Methods in Cellular Immunology, Second Edition by
CRC Press, Jun 26, 2001.
Gooi and Chapel. (1990) Clinical Immunology: A practical approach. 1990 Oxford University Press.
Lamm DL, Riggs DR (2000) The potential application of Allium sativum (garlic) for the treatment of
bladder cancer. Urol Clin North Am, 27: 157-62.
Klein C, Sato T, Meguid MM, Miyata G (2000) From food to nutritional support to specific
nutraceuticals: a journey across time in the treatment of disease. J Gastroenterol, 35: 1-6.
Bauer R, Hoheisel O, Stuhlfauth I, Wolf H (1999) Extract of the Echinacea purpurea herb: an
allopathic phytoimmunostimulant. Wien Med Wochenschr, 149: 185-9.

NO RELEASE ASSAY
Nitric oxide (NO) is an inorganic, free radical that functions as an
intracellular messenger and effector molecule. It is produced during
the conversion of L-arginine to L-citrulline and is catalyzed by the
enzyme nitric oxide synthase (NOS) in the presence of the co-factor
NADPH (Palmer et al., 1988, Nathan, 1992).
Principle:
NO is extremely unstable and undergoes repaid oxidative
degradation to nitrite (NO2-) and nitrate (NO3-), which can be
spectrophotometrically determined. The reduction of nitrate to nitrite
by vanadium (III) chloride, followed by quantification of nitrite by
Griess reaction.
Procedure:
Plating cells
Nitric oxide (NO) quantification
Standard Curve of NaNO
2
Cell proliferation


References:
Palmer, R. M., Ashton, D. S. and Moncada S. (1988) Vascular endothelial cells synthesize nitric
oxide from L-arginine. Nature (Lond) 333: 664-666.
Nathan, C. (1992) Nitric oxide as a secretory product of mammalian cells. FASEB J. 6: 3051-
3064.
Xie, Q. W., Whisnant, R. and Nathan, C. (1993) Promoter of the mouse gene encoding calcium-
independent nitric oxide synthase confers inducibility by interferon gamma and bacterial
lipopolysaccharide. J. Exp. Med. 177: 1779-1784.
Knowles, R. G. and Moncada, S. (1994) Nitric oxide synthases in mammals. Biochem. J. 298(2):
249-258.
MacMicking, J., Xie, Q. W. and Nathan, C. (1997) Nitric oxide and macrophage function. Annu.
Rev. Immunol. 15: 323-350.
Salvemini, D., Doyle, T. M. and Cuzzocrea, S. (2006) Superoxide, peroxynitrite and
oxidative/nitrative stress in inflammation. Biochem. Soc. Trans. 34 (5): 965-970.
Raso, G. M. et. al. (2001) Inhibition of inducible nitric oxide synthase and cyclooxygenase-2
expression by flavonoids in macrophage J774A.1. Life Sci. 68: 921-931.
Attur, M. G. et. al. (2000) Differential anti-inflammatory effects of immunosuppressive drugs:
cyclosporin, rapamycin and FK-506 on inducible nitric oxide synthase, nitric oxide,
cyclooxygenase-2 and PGE
2
production. Inflammation Res. 49 (1):20-26
Holthusen, H. and Arndt, J. O. (1994). Nitric oxide evokes pain in humans on intracutaneous
injection. Neurosci. Lett. 165: 71-74.

CHOLESTEROL ESTERASE INHIBITION ASSAY
Pancreatic cholesterol esterase is
produced by the exocrine part of
pancreas and released into the intestinal
lumen as part of intestinal juices.
Pancreatic cholesterol esterase hydrolyzes
cholesterol esters into free cholesterol,
further it is required for absorption of free
cholesterol as well.
Esterified cholesterol is rich in non-
vegetarian food.
Thus, inhibition of cholesterol esterase
enzyme leads to a significant reduction in
absorption of dietary cholesterol
PRINCIPLE OF ASSAY

CITRATE SYNTHASE INHIBITION ASSAY
Citrate serves dual functions in the cytoplasm, both as the precursor
of fatty acids and cholesterol and as feed-forward activator of acetyl-
CoA carboxylase (1).

Citrate, the allosteric activator of Acetyl-CoA carboxylase , is
required for both catalysis and polymerization (1).

In obese rats fed on high carbohydrate diet considerable increase in
citrate synthase activity has been observed (2).

MECHANISM OF ACTION

Pyruvate
Oxaloacetate
Pyruvate carboxylase
Citrate
Acetyl-CoA HS-CoA
Citrate synthase
Citrate
Oxaloacetate
Citrate lyase
Acetyl-CoA
ATP
Acetyl CoA
Carboxylase
Malonyl--CoA
FATTY ACID AND CHOLESTEROL BIOSYNTHESIS
+
Malate
Pyruvate
C
Y
T
O
P
L
A
S
M

MITOCHONDRIA
Pyruvate
Principle:
The Citrate synthase assay depends on reduction of chromogenic
reagent 5, 5-dithiobis-(2-nitrobenzoate) (DTNB) with the thiol group
of free CoA as it is produced in the formation of citrate from
oxaloacetate and acetyl-CoA. The reduction of DTNB produces a
yellow colored product which is measured at 412nm.

References:
Watkins, P et al. Proc. Natl. Acad. Sci.74, 1497 1501; 1977.
Dourmashkin, JT. International Journal of Obesity. 111; 2005.
Hassett, P. Analytical Biochemistry. 287, 176179; 2000.


ACYL-COA:MONOACYLGLYCEROL
ACYLTRANSFERASE (MGAT-2) ASSAY
Acyl-CoA: monoacylglycerol acyltransferase (MGAT-2)
catalyzes the synthesis of diacylglycerol, the precursors of
physiologically important lipids such as triacylglycerol and
phospholipids.

In the intestine, MGAT-2 plays a major role in the absorption of
dietary fat because resynthesis of triacylglycerol is required for
the assembly of lipoproteins that transport absorbed fat to
other tissues (1).

Selective inhibition of MGAT-2 enzyme may provide novel
treatment for obesity and its related metabolic complications
associated with excessive fat intake (2, 3).

Principle:
The MGAT-2 assay depends on the detection of the CoA released
due to acylation of monoolein by the enzyme MGAT-2 and
deacylation of palmitoyl-CoA. This assay uses DTNB (5, 5-Dithiobis
(2-Nitrobenzoic acid)) which reacts with the sulfhydryl group of
liberated CoA. This increase in CoA concentration during the
enzyme assay at 412nm.

References:
1.Chi-Liang et al. Proc Natl Acad Sci U S A, 99(13); 8512-8517; 2002.
2.Yuguang Shi and Dong Cheng. Am J Physiol Endocrinol Metab, 2008.
3.Chi-Liang Eric. Nature Medicine, 15; 442-446; 2009.
4.John. B. Rodgers. JR. J Lipid Res, 10; 427-432; 1969.
5.Coleman et al. J. Biol chem, 261(1); 224-228; 1986.
6.Shirkey et al. Anal Biochem, 93; 73-81; 1979.