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Int.J.Inv.Pharm.Sci.,1(1)2013; 53-58
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INTERNATIONAL JOURNAL OF INVENTIONS IN PHARMACEUTICAL SCIENCES

DETECTION OF PROTEINS IN BODY FLUIDS AND PLASMA PROTEIN BINDING OF VARIOUS DRUGS
RADHIKA KOMMMU*, RAMADEVI BHIMAVARAPU
Sri Siddhartha pharmacy college, Nuzvid, Andhra Pradesh, India

Accepted On: 26thJanuary 2013

For correspondence: radhikakommu38@gmail.com

ABSTRACT: The extent of drug binding to plasma proteins, determined by measuring the free active fraction, has a significant effect on the pharmacokinetics and pharmacodynamics
of a drug. It is therefore highly important to estimate drug-binding ability to these macromolecules in the early stages of drug discovery and in clinical practice. Traditionally, equilibrium
dialysis is used, and is presented as the reference method, but it suffers from many drawbacks. In an attempt to circumvent these, a vast array of different methods has been developed.
This review focuses on the most important approaches used to characterize drugprotein binding and proteins present in various body fluids.
Key Words: Drug, Plasma proteins, Body Fluids.

INTRODUCTION:

PROTEINS PRESENT IN BODY FLUIDS

Protein Binding refers to the binding of a drug to proteins.

Plasma Proteins: Proteins are biochemical chemical compounds

Common proteins involved in binding are Lipoproteins, albumin

comprise of one or more polypeptides alleviating a biological

and glycoproteins. The Bloodstream contains bound form and

function. Blood Proteins are termed as Plasma or blood serum

unbound form of the drug. Normally in blood, many drugs bind

Proteins. There are assorted techniques to determine proteins

to plasma proteins reversibly,

they

[Unbound drug] + Protein [Protein-Drug Complex] 1.

Any drug whether given orally, intravenously, sublingual, sub-

are

microscopy,

protein

immunostaining,

Protein

immunoprecipitation, Immunoelectrophoresis, Immunoblotting,

BCA Protein Assay (to evaluate protein concentrations), Western
blot, Spectrophotometry, Enzyme assay. Protein refinement

cutaneously or intramuscularly, drug is carried by the blood and

could be done in versatile methods such as chromatography

it first enters not only to various cellular components and low

techniques: Ion exchange, Size-exclusion chromatography or Gel

molecular weight compounds but also with the plasma proteins 2.

filtration, Affinity chromatography, Protein Extraction and

The free fraction of the drug diffuses the biological membrane

Solubilization, Protein Concentration determination methods are

and produces therapeutic effect and then eliminates from the

body. The extent of protein binding is expressed as Percentage of
drug bound3. By measuring the free fraction of drug, we can
determine the extent of drug bound to the plasma proteins, which

Bradford protein assay, Concentrating Protein Solutions, Gel

electrophoresis, Gel Elecdsis Under denaturing conditions, Gel
Electrophoresis Under non-denaturing conditions, 2D Gel
Electrophoresis, Electrofocusing. There are diverse plasma

has effect on pharmacokinetics and pharmacodynamics of a

proteins like Albumin(60%), Immunoglobulins, Fibrinogens,

drug4. Drug Protein Binding influences the Bioavailability, Drug

alpha 1- antitrypsin, alpha 2-macroglobulin, Globulins(, , )-

Metabolism, Drug Excretion and Drug Concentration in the

(35%), Lipoproteins, Transferrin, Prothrombin, Regulatory

tissue fluids5,

Proteins like enzymes, zymogens, hormones-(1%). Plasma

. Protein Binding may be intracellular or

extracellular binding. In intracellular binding drug binds to cell

protein, may be a drug receptor and shows pharmacological
response, those receptors are receptors and in extracellular
binding drug binds to an extracellular protein but doesnt shows

protein especially Albumin work comprises to create Osmotic

Pressure which aids in transportation of steroid hormones and
lipids. Globulins function is to carry ions, hormones and lipids
aiding in resistant function. The coagulating of blood line is

pharmacological response and these receptors are termed as

associated with fibrinogen7 -9. The principal site for the

Secondary receptors or Silent receptors.

constitution of fibrinogen, albumin and globulin of the plasma is

Liver and novel plasma proteins may grow from the liver only10.

Page | 53

Oxidised plasma proteins were watched over by western blotting

methods

to

determine

urinary/CSF

proteins

they

are
23-25

technique with anti-DNP antibody in patient during Alzheimers

turbidimetric,

disease and the oxidised proteins comprised isoforms of

Proteins present in Sub retinal Fluid are Alpha-2 globulin used

fibrinogen Y-chain forerunner and of a-1-antitrypsin precursor11.

by tissue as a source of amino acids, Apoprotein E- maintains

Plasma proteins alike albumin and fractions of gamma globulin,

chylomicron structure, participates in cell recognition or nervous

beta metal blending protein and beta lipoprotein were ascertained

tissue healing26 , Fibronectin involved in cellular adhesion and

to adhere cationic trivalent chromium and the radioactive

migration processes. Plasmin involved in inflammatory and

chromium (cr51) with half life of 26.5 days, a novel biologic

tissue destruction processes 27, 28. Lysozomal enzymes proteinases

decipher was detected to label plasma proteins 12. The protein

capable

CXCL4 exists in plasma of tainted patients, forbids HIV from

Butyrylcholin esterase Acid phosphatase- dephosphorylation-

embarking into healthy human being cells. There are varied

inducing

Fibrous Proteins they are Cytoskeletal proteins such as Actin,

malicodehydrogenase- uses as a marker of cellular destruction,

Arp2/3, Coronin, Dystrophin, FtsZ%, Keratin, Myosin, Spectrin,

Protein kinase- phosphorylation-inducing enzyme, Amines-

Tau (protein), Tubulin, Collagen and Extracellular ground

dopamine is involved in synaptic transmission within the retina,

substance proteins such as Collagen, Elastin, F-spondin,

Retinal S-antigen known for its auto-antigenic properties29,

Pikachurin. There are Globular Proteins such as blood serum

Parathyroid hormone- regulates homeostasis and calcium

Amyloid P Component, blood serum albumin. Plasma in addition

compartmentalisation31,

to comprises Cadherin, Ependymin, Integrin, NCAM, Selectin,

(IRBP) - the predominant protein in photoreceptors. Its function

C-reactive protein, and Hemoproteins. There are Transcription

is to transport retinal from the PE to the retina 33, Opsin- visual

regulative proteins that are in the receptors segment, they are C-

pigment precursor34 , and Interleukin 6 and gamma interferon-

myc, FOXP2, FOXP3, MyoD, and P53. DNA adhering proteins

inflammation

such as Histones are existing in plasma.Proteins in other fluids:

orosomucoid, alpha2-haptoglobin,

Cystatin-D is a protein found in saliva and tears and its function

macroglobulin,

13

electrophoretic,

of

degrading

calorimetric

outer

enzyme,

segments

Lacticode

32

precursors.

methods

and

rhodopsin,

hydrogease

and

30

Interstitial retinal-binding protein

Albumin,

beta-lipoprotein,

alpha1-antitrypsin,

gamma

chains, alpha2-

ceruloplasmin

and

is protease inhibitor . Lactoferrin (Lactotransferrin) is present in

immunoglobulins G, A, and M were present in vaginal fluid35.

milk, saliva, tears and nasal secretions which have antimicrobial

Mostly Albumin, 2u-globulins are present in the urine. Proteins

14

activity . Auto antibodies (a type of proteins) were found in

in saliva are used as Clinical Markers. Plasma CSF Saliva Urine,

urine, semen, tears, perspiration, blood, serum, saliva and body

Serum albumin, Serum albumin, Amylase alpha 1A, Apo

15

tissues . Immunoglobulin A are found in saliva, tears, breast

lipoprotein

milk, sweat and gut fluid16. Thrombomodulin is isolated from

Complement

plasma and urine, which is an endothelial cell membrane protein

microglobulin, Complement C3, Vitronectin are present in

used for rapid activation of plasma protein C (Vitamin k-

saliva. Serum albumin, Uromodulin, Haptoglobin, Cytokeratin,

17

A-IV,
factor

Fibrinogen,
9,

Complement

Immunoglobulin

G,

factor

B,

Alpha-1-

dependent glycoprotein in human plasma) . In bull four seminal

AMBP protein, Alpha-1-microglobulin, Epidermal growth

plasma proteins were present they are 26KDa (PI 6.2) and

factor, Apo lipoprotein D, Complement component C4, Alpha-1-

55KDa (PI 4.5), 16KDa (PI4.1 and 6.7) respectively, and these

acid glycoprotein 1, Protein Z, Prostaglandin D2 synthase,

four were identified by using two-dimensional polyacrylamide

Alpha-2-HS glycoprotein are the proteins present in the urine.

gel electrophoresis and Seminal plasma proteins are used to

There are various proteins that are present in cerebrospinalfluid

18

predict Fertility . In bovine seminal plasma proteins were

(CSF) they are Serum albumin, Transferrin, Cystatin C,

identified they are osteopontin and lipocalin-type prostaglandin-

Immunoglobulin G, Perlecan, A lpha-1-antitrypsin, Complement

D synthase

19, 20

. CSF contains albumin and globular proteins in

which they are lower in ventricular CSF than in lumbar or

factor C4, Prostaglandin D synthase, Alpha-1-acid glycoprotein

1, Fibulin 1 isoform D Actin gamma, Alpha-1-antichymotrypsin.

21

cisternal fluid . In CSF there are proteins like Amyloid beta

(Abeta); total (t-tau) and phorphorylated tau (p-tau) are present
and they are used to differentiate Alzheimer diseased patients
from normal patients i.e. In AD patients there will be increased
tau levels and decreased Abeta42 levels 22. There are many

Page | 54

PLASMA PROTEIN BINDING OF VARIOUS DRUGS: In

plasma the amount of drug bound is depend on the protein
concentration, the number of sites on the protein, the affinity of
binding site for the drug, and the total drug concentration36. The

Protein Binding of drugs shows effects on Hepatic Metabolism

1975), antimicrobial agents (Craig and Wagnild, 1974),

Rate, Renal Excretion, Biomembrane Permeation Rate, Steady

Digitoxin (Shoeman and Azarnoff, 1972), Digoxin (Stor stein,

State Distribution Volume and Pharmacodynamics. Drugs that

1976), Phenylbutazone and thiopentone (Andreason, 1973) in

bind to plasma proteins have shorter half-life. Binding of drugs

uraemic patients. In plasma proteins, total proteins can be

involves weak bonds. Drugs will have small apparent volume of

measured by Auto Analyzer Technique and Bromocresol green

distribution when bind to plasma proteins. There are various

technique is used to analyse albumin (Nisbet et al., 1973).

factors that affect the plasma-drug binding they are Drug related

Binding

of

drug

to

plasma

proteins

determines

the

49

factors, Protein related factors, Drug Interactions and Patient

pharmacological response of that drug . By Erythrocyte

related factors. Protein Binding can be determined by Direct and

Partitioning Technique, the binding of etretinate and acitertin to

Indirect techniques. Indirect techniques were applied in

plasma proteins at a percentage of >99 was investigated50. By

biological samples like serum, blood, plasma. Various indirect

using Liquid Scintillation spectroscopy and equilibrium dialysis,

analytical methods

we

include dynamic dialysis, equilibrium

can

determine

plasma

protein

binding

of

51

dialysis, ultrafiltration, diafilteration, ultracentrifugation, gel

diphenylhydantoin . Salicylate (Sturman and Smith, 1967),

filteration.

spectroscopy,

Warfarin (OReilly, 1967), Thiopental (Dayton and Perel, 1971),

fluorimetry and ion-selective electrodes. The order of binding of

Diphenylhydantoin, in these drugs protein binding is inversely

drugs to plasma proteins is:

proportional to concentration of the drug, but some drugs are

Direct

techniques

include

UV

concentration independent (Eg: Amphetamine (Baggot and Other

Albumin> 1-Acid Glycoprotein> Lipoproteins> Globulins.

1972), desmethylimiprimanine (Borga, Azarnoff, plym Forshell

Many drugs bind to Human Serum Albumin. Drugs like

and Sjoqvist, 1969), Morphine (Baggot and Davis, 1973) and

Imipramine, Lidocaine, and Quinidine which are basic mainly

Methadone (Olsen, 1972). The plasma protein binding of

bind to 1-Acid Glycoprotein. Chlorpromazine which is basic

anticoagulants- Phenprocoumon, warfarin, acenocoumarol was

and lipophilic drug mainly bind to Lipoproteins. Steroid like

measured by equilibrium dialysis, flourimetry52 , Scintillation

ions and some vitamins A, D, E, and K bind to 2Globulin and

ultrafiltration with uv detection . The P lasma Protein Binding of

Heamoglobulin.

Trimethoprim,

Sulphadiazine

and

Sulphamethoxazole- plasma-protein binding was studied and

confirmed that trimethoprim was 50% bound, sulphadiazine was

H-labelled

phenprocoumon53 ,

counting

Drugs like Phenytoin, pentobarbital and phenothiazines bind to

of

corticosterone, thyroxine and vit B12 bind to 1Globulin. Cupric

continuous

52

antideprassants was studied by ultrafiltration technique using

labeled compounds54. The protein binding of the antibiotics
moxifloxacin, gatifloxacin, linezolid and telithromycin to human
serum albumin (HSA) and Bovine serum albumin (BSA) and the

56.2% and sulphamethoxazole was 76.9% bound . The presence

protein

of increased concentrations of salicylic acid like drugs

propylxanthine (enprofylline), were determined by automated

simultaneously malnutrition, kidney and liver diseases may

continuous ultrafiltration method55,

37

influence the protein binding38. Plasma albumin concentration is

reduced due to aging39. Acidic compounds mostly bind to plasma

binding

of

l-methyl-3-propylxanthine,
56

and

3-

. A racemic drug protein

binding is potentially different between enantiomers, which show

the difference in their pharmacokinetic character 57,

58

. High

albumin and basic compounds bind to 1-acidglycoprotein and

Performance Frontal Analysis is useful for plasma protein

lipoproteins40,

binding study of hydrophobic drugs and this method is rapid and

41

. Generally many phenothiazines bind to 1-

acidglycoprotein42,

43

. In pathological conditions such as

inflammation, infection, myocardial infarction and cancer, 1acidglycoprotein shows increased concentration
equilibrium

dialysis,

we

can

study

the

44-47

. By using

binding

of

reversible59.Clozapine binds to Human Serum Albumin (bound

fraction -9.1%) and other plasma proteins, average bound
fraction in plasma was found to be 84% 60. Tolbutamide, a
hypoglycemic agent highly bound to plasma protein- albumin61,
62

chlorpromazine, trifluoperazine, perphenazine, desipramine,

propranolol and salicylic acid to human plasma proteins 48. The
plasma protein binding is decreased in some drugs like
pentobarbital, diphenyl hydantoin (Ehrnebo and odar- Cederlof,

. Semotiadil and levosemotiadil bind to human serum albumin

upto 99% and is determined by sensitive analytical method63.

Using highperformance frontal analysis (HPFA), P lasma protein
binding of oxybutynin (OXY) was investigated quantitatively
and enantioselectively64.

Page | 55

SOME DISEASES WHICH ALTER THE PROTEIN

increased which was shown by Piafsky et al. (1978) 73. Finally it

BINDING

is concluded that an increased plasma protein binding of Glycoprotein in diseased states decreases apparent volume of

CARDIAC DISEASES: An increased concentration of Glycoprotein occurs after myocardial infarction65. studied that
there are changes in serum protein when anti-arrhythmic drugs

distribution and total plasma concentration of drugs.

REFERENCES:

like flecainide and disopyramide binds after myocardial

infarction, Flecainide binds to serum at a percentage of 61% to
53% as it is weakly bound drug where as disopyramide which is
a strongly bound drugs binds to serum proteins at a percentage of
80% to 87%. In severe chronic cardiac failure drugs like
diazepam, digitoxin, warfarin and Phenytoin binds to serum
albumin and drugs like propranolol and imipramine binds to Glycoprotein.
KIDNEY DISEASES: Major organ for elimination and
detoxification is Kidney; due to poor kidney function protein
binding of drugs in plasma varies by endogenous waste
.zomepirac a non-narcotic analgesic shows decreased plasma
protein binding in uraemia condition upto 4% than in normal
condition it shows 98% plasma protein binding66. plasma protein
binding of prednisolone varies in nephrotic syndrome, In renal
disease, the drug Tolfenamic acid % free is increased to 0.17
than in normal stage as this drug binds upto 99.9% to plasma

proteins.67
HEPATIC DISEASES: Drugs like phenylbutazone, salicylate
and sulphadiazine binding is reduced during liver diseases like
cirrhosis and hepatitis. Verapamil binding was also decreased
during liver disease 68. but there are some conflicts regarding this
statement that is, Echizen and Eichelbaum (1986)

69

reported that

there is no changes in binding of verapamil during liver diseases.

DIABETES MELLITUS: As we know that patients with
diabetic mellitus have high glucose levels causing non-enzymatic
glycosylation of various proteins which was shown by RuizCabello and Erill (1984)70. Valproic acid binding capacity to
serum is decreased for Insulin Depended Diabetes Mellit us
patients, which was showed by (Grainger-Rousseau, 1988)71.
OTHER

DISEASES:

Patients

with chronic

obstructive

pulmonary disease (COPD) in which theophylline protein

binding may change which was reported by Zarowitz et al.
(1985)72. In cystic fibrosis (CF), Antibiotics like flucloxacillin,
penicillin will alter the protein binding .Due to the disease
induced levels of -Glycoprotein, the plasma protein binding of
some basic drugs like propranolol and chlorpromazine is

Page | 56

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