The Metabolism of (PABA) Para-Amino benzoic Acid
Introduction
PABA or Para amino benzoic acid is a non proteinous amino acid that occurs naturally. The molecular formula of para- amino benzoic acid is C7H7 NO2; the structure of PABA consists of benzene rings substituted with a carboxyl and an amino group. PABA is a white crystalline molecule that is only sparingly soluble in water. PABA is a very essential nutrient for bacteria PABA functions as an intermediate in the synthesis of folic acid in bacteria
The deficiency of PABA does not cause any deficiency diseases in man. Some bacteria in the intestinal tract can synthesise PABA from chorismate. Humans lack the ability of converting PABA to the intermediate folate and therefore need folate from dietary sources like green leafy vegetables
In industry, PABA is used as an ingredient in the manufacture of salts target esters, folic acid, azo dyes and many organic compounds. PABA is also used as UV blocking agent in cosmetics manufacture. It is also used in the preparation of anaesthetics and medical ointments. It is also used as a dietary supplement to counter the effects of fatigue, depression, scleroderma and weeping eczema.
The objective of this practical was to carry out a simple spectrophotometric determination of PABA. The method entailed the diazotisation of PABA followed by the coupling of the product with N – (1- NapthyL Ethylendiamine dihydrochloride to form a very highly coloured dye that is a successful assay of PABA. This practical analysed two samples of urine for the presence of free and acetylated PABA. The PABA present in the sample was measured through diazocoupling PABA to NED (Naphtyl) Ethylenediamine dihydrochloride to produce a pink colouration that was measured using a spectrophotometer to determine the extent of metabolism of PABA in the body.
Procedure
Materials and reagents
Test tubes
Pipettes
Spectrophotometer
Cuvettes
Volumetric flasks
4M HCL
Distilled water
Urine samples treated and untreated
- % w/v Sodium nitrite solution
0.5 % w/v ammonium sulfamate solution
0.1 % w/v NED solution
PABA 10mg l-1
Procedure
For the dilution of urine samples, 25 ml of the control and the treated urine samples were transferred using a pipette each into a 50 ml volumetric flask and 2.5 ml of 4M hydrochloric acid were added to each flask. The volume of each flask was made to 50 ml through additional of distilled water. In The determination of free PABA, Duplicate samples (10 ml each) of the diluted treated and control urine were filled into test tubes and 1.0 ml of sodium nitrite solution added to each tube. The mixture was then mixed well for three minutes.
1.0 ml of ammonium sulfamate solution (0.5% w/w) was added to each duplicate tube to remove excess nitrite. The mixture was then mixed thoroughly and then left for 2 minutes. After mixing the contents of the tubes, 1.ml of NED solution (0.1 % W/V) was then added to each tube and the contents mixed thoroughly. The absorbance values of the contents of each tube were then determined using a spectrophotometer at 510 nm with distilled water used as the blank.
After the absorbance readings for each tube were obtained, Eleven Tubes Were prepared by adding varying concentrations of PABA (10mg L-1) and HCL (0.2 M). Tube 1, 0 ml of PABA and 10.0 ml HCL, tube 2 ,0.5 PABA and 9.5 HCL , Tube 3 0.5 ml of PABA and 9.5 ml of HCL , Tube 4 1.0 ml of PABA and 9.0 ml HCL, tube 5 1.5 ml PABA and 8.5 ml HCL, tube 8 2.0 ml PABA and 8.0 ml Hcl, Tube 9 2.0 ml of PABA and 8.0 ml HCL. Tube 10 2.5 ml PABA and 7.5 Hcl and tube eleven 2.5 ml of PABA and 7.5 ml of Hcl.
The tubes were then each treated with sodium nitrite, ammonium sulfamate as the previous tubes in the earlier steps. The absorbance readings of tube number 2 to tube number eleven were determined at 510 nm using a spectrophotometer with tube number 0 used as the blank
RESULTS
1) Determination of free PABA:
Absorbance:
Control 1: 0.439 treated 1: 0.483
Control 2: 0.449 treated 2: 0.479
2) Preparation of the calibration:
Absorbance:
1/ 0
2/ 0.057
3/ 0.063
4/ 0.101
5/ 0.088
6/ 0.164
7/ 0.169
8/ 0.282
9/ 0.287
10/ 0.395
11/ 0.400
3) Determination of total PABA (Free PABA + N-acetylated PABA)
Absorbance:
Control 1: 0.217 treated 1: 0.252
Control 2: 0.219 treated 2: 0.248
Calibration graph
CALCULATION OF FREE PABA
PABA recovered in complete urine sample:
Control urine:
Control 1 (C1) = 12.5/5 =2.5 mg
Control 2 (C2) = 15.4/5 = 3.08 mg
Treated urine:
Treated 1 (T1) = 12.2/5=2.44 mg
Treated 2 (T2) = 14.4/5=2.88 mg
PABA excreted as a result of taking oral dose:
T1-C1 = 2.44– 2.5 = 0.06 mg
T1-C2 = 2.44 – 3.08= 0.64 mg
T2-C1 = 2.88 – 2.5 = 0.03 mg
T2-C2 = 2.88 – 3.08= 0.02 mg
CALCULATION OF TOTAL PABA (FREE + N-ACETYLATED)
1-PABA recovered in complete urine sample:
Control urine:
Control 1 (C1) = 40.73/2.5=16.29 mg
Control 2 (C2) = 40.65/3.08=13.19 mg
Treated urine:
Treated 1 (T1) = 132.33/2.44 = 54.23 mg
Treated 2 (T2) = 139.52/2.88 = 48.44 mg
Urine contains some small amounts of substances that can react in a similar way to PABA, the amount of PABA present can be determined through subtracting the total mas of the control sample from the mass of pass from the treated sample as indicated below.
2- PABA excreted as a result of taking oral dose:
T1-C1 = 54.23-16.29=36.64 mg
T1-C2 = 54.23-13.79=36 = 40.44 mg
T2-C1 =48.44-16.29= 32.15 mg
T2-C2 = 48.44-13.79 =34.65 mg
Amount of N-acetylated PABA
36.64– 0.06 = 36.58 mg
40.44– 0.64 = 39.8 mg
32.15 – 0.03 = 32.12 mg
34.65 – 0.02 = 34.63 mg
% of PABA dose excreted as free PABA
0.06/200x100 = 0.03%
0.64/200x100 = 0.032%
0.08/200x100 = 0.015%
0.02/200x100 = 0.01%
% of PABA dose excreted as N-acetylated PABA
36.58/200x100 = 18.29%
39.8/200x100 = 19.9%
32.12/200x100 = 16.06%
34.63/200x100 = 17.31%
A Callibratioin Graph Showing The Mass Of PABA Against Absorbance
The absorption spectra of the coloured dye that was formed by the coupling of Diazotised PABA and NED displayed maximum absorbance at 510 in contrast to the blank reagent that showed zero absorption at the same wavelength.
Discussion
The Spectrophotometric determination of PABA entails the diazotisation of PABA followed by the coupling of the corresponding salt with a different coupling agent like Braton Marshall Reagent and the dimethylaminobenzadehyde (Natural standard, 2013). This is called the Griess reagent system that is based on the detection of NO2- ions in a variety of experimental and biological liquid matrixes like serum plasma urine or medium for tissue culture. The chemical reaction utilizes NED 1 napthylethylenediamine dihydrochloride (NED) and sulphanilamide in the presence of acidic conditions (phosphoric acid) to yield a Coloured compound.
The sensitivity of detection depends on the concentration of the Nitrite in the sample. The more the nitrite in the sample , the higher the intensity, The nitrite is usually the rate limiting reactant in the reaction. To ensure an adequate quantification of the nitrite in the sample a reference curve is prepared with a nitrite standard for every assay using the same buffer or the matrix used in the experiment. The NED and sulphamide in this reaction compete for the Nitrite ion in the sample and therefore they are added sequentially. The sulphanilamide is added first and then incubated for about three minutes and then the NED solution is added later (Othman & Mansour, 2005).
The metabolism of Para amino benzoic acid in the body entails glycine and acetylation conjugation to yield hippurated PABA metabolites like PAHA Para aminohippuric acid, Paracaetamidohippuric acid (PAAHA) and P acetamidobenzoic acid (PAABA) (Othman & Mansour, 2005).
The coupling of diazotized P amino benzoic acid in acid medium forms a stable water soluble water Soluble pink dye that portrayed maximum absorbance at 531 nm. According to Salvador et al 2003, Pg, 240), Excess nitrate reacts with PABA to yield a PABA diazonium salt under the presence of acid
The nitrite residue in the experiment is removed through the addition of sulphamic acid
HNO2 +H2N → N2 + H20 + H2SO4
The colored solution is formed through the coupling of the PABA diazolic salt with NED as in the reaction scheme below the diazotization reaction takes place in an acidic medium (Salvador et al, 241).
Conclusion
This experiment was successful and the desired results were obtained. Urine Tests for PABA has found wide application as urine excretion markers for the evaluation of pancreatic function. Measurements of conjugated metabolites of PABA like paracaetamidohippuric acid and (PAHA) paraaminohippuric acid (PAHA) following an oral dose can be used in assessing the function of the liver.
References
Natural standard (2013). Paraminobenozoic acid (PABA). Retrieved from
http://www.naturalstandard.com/index-abstract.asp?create-abstract=paraaminobenzoicacid.asp&title=Para-aminobenzoic%20acid
Othman, N. & Mansour, S. (2005). “Spectrophotometric Determination of P
Amino benzoic acid from procaine drug by using phoroglucinol as coupling agents”. Tikrit J. of Pur. Science, 10, 139.
Salvador A, Choisvert, Rodriquez and Merch J. (2003) “Indirect spectrophotometric
Determination of P. amino benzoic acid in sunscreen formulations by sequential injection analysis. Anal. Chim acta, 493. 233.
