VANILLYLMANDELIC ACID INTRODUCTION AND ITS ESTIMATION BY VARIOUS METHOD

1. VMA
(Vanillylmandelic Acid)
Raghwendra Sah
Bsc.MLT 4th year(2016 batch)
Gandaki Medical College,
Lekhnath, Kaski

2. Contents
• Introduction
• Methods
• Principle
• Requirements
• Procedure
• Normal Range
• Clinical interpretations

3. Introductions
• Vanillylmandelic acid(3-methoxy,4-hydroxymandelic acid) is the metabolic
end product of catecholamines.
• VMA excreted in urine is formed in the liver, where the presence of alcohol
dehydrogenase leads to oxidation of MHPG derived from portal venous
inflow (60%) or produced locally by O-methlylation of DHPG after removal
from the bloodstream by the liver.
• Catecholamines play important roles as neurotransmitters in the brain or
sympathetic nervous system, or as hormones produced by the adrenal medulla
and other peripheral cellular systems.
• The catecholamines produced naturally in humans—including dopamine,
norepinephrine (noradrenaline), and epinephrine (adrenaline)—are
phenylethylamines with hydroxyl groups on positions three and four of the
benzene ring and an ethylamine sidechain on position one.
• Epinephrine(adrenaline) and norepinephrine(noradrenaline) are the hormones
synthesized by the adrenal medulla.
• These hormones are stored in the adrenal chromaffin cells or
pheochromocytes, which release them in the response to stresses such as
traumatic injury, hypoxia, or hypoglycemia.

4. TYR- Tyrosine
TH-Tyrosine hydoxylase
DOPA- Dihydroxyphenylalanine
DA- Dopamine
NE- Norepinephrine
VL- Vesicular leakage
VS- Vesicular sequestration
DHPG- Dihydroxyphenyl glycol
MAO- Monoamine oxidase
NU- Neuronal reuptake
R- Exocytotic release
EU- Extra neuronal uptake
COMT- Catechol-O-methyltransferase
NMN- Normetanephrine
MHPG- Methoxy, hydroxyphenyl glycol
Metabolism of norepinephrine (NE) in sympathetic
nerve endings in relation to extra neuronal tissue and
the bloodstream

5. TYR- Tyrosine
TH-Tyrosine hydroxylase
DOPA- Dihydroxyphenylalanine
DA- Dopamine
NE- Norepinephrine
VL- Vesicular leakage
VS- Vesicular sequestration
DHPG- Dihydroxyphenyl glycol
MAO- Monoamine oxidase
NU- Neuronal reuptake
R- Exocytotic release
EU- Extra neuronal uptake
COMT- Catechol-O-methyltransferase
NMN- Normetanephrine
MHPG- Methoxy, hydroxyphenyl glycol
EPI-Epinephrine
MN-Metanephrine
Regional pathways of norepinephrine and
epinephrine metabolism

6. Methods
• VMA is estimated by the various methods.
• They are listed as follows:-
1. Spectrophotometric method
2. Colorimetric method
3. Paper chromatographic method--- uses diazotized p-nitroaniline as dye
4. Thin layer chromatographic method
5. High performance liquid chromatography method
6. Enzyme linked immunosorbent assay method
7. Chemiluminescence immunoassay method
Analyze extracted VMA

7. Patient Preparation
There are no fluid restrictions unless by medical direction. Instruct the patient to abstain from smoking
tobacco for 24 hr before testing. Usually, a 24-hr urine collection is ordered. As appropriate, provide
the required urine collection container and specimen collection instructions. Inform the patient of the
following dietary, medication, and activity restrictions in preparation for the test (protocols may vary
among facilities):
• The patient should not consume foods high in amines (bananas, avocados, beer, aged cheese,
chocolate, cocoa, coffee, fava beans, grains, tea, vanilla, walnuts, and red wine) for 48 hr before
testing.
• The patient should not consume foods or fluids high in caffeine (coffee, tea, cocoa, and chocolate)
for 48 hr before testing.
• The patient should not consume any foods or fluids containing vanilla or licorice.
• The patient should avoid self-prescribed medications (especially aspirin) and prescribed
medications (especially pyridoxine, levodopa, amoxicillin, carbidopa, reserpine, and disulfiram) for
2 week before testing and as directed.
• The patient should avoid excessive exercise and stress during the 24-hr collection of urine

8. Spectrophotometric method
• Sample:- 24 hr urine is collected as uninary excretion of VMA is not continuous.
• VMA is extracted by ethyl acetate -- oxidized with metaperiodate --- conversion of VMA to
vanillin -- vanillin extracted with toluene into alkaline solution --- red color --- measured at 360nm
• Low sensitivity and specificity.
Colorimetric method
• The colorimetric method in general, however, suffer from interfering compounds and intensive labor
requiring a number of manual steps.
• Low sensitivity and specificity.

9. • VMA is extracted from acidified urine(pH <1 with HCl) into ethyl acetate and discard aqueous layer.
• Place the drop for chromatogram spotting on 1 foot sq Whatman no.1 paper.
• Standards are spotted for comparison with unknown’.
• Chromatogram are put in covered cylinder tank containing 50 ml mixture of isopropanol : ammonium
hydroxide : water in ratio 40:9:1 for 10-15 hr.
• After air drying they are run in perpendicular direction for 6 hr in tan of benzene : propionic acid :
water (20:15:1).
• After air drying, placed in 250◦c oven for 2 minutes and sprayed with mixture of para- nitroaniline,
sodium nitrite, and potassium carbonate(1:1:2) in that order.
• The spots are compared with different concentration of standard on the basis of size/area/density .
Paper chromatography
Paper chromatography

10.  Advantage:
 Simultaneous study of large number of samples.
 useful during preliminary purification process.
 Disadvantages:
 Takes much time to perform test as compared to TLC.
 Slow as compared to TLC.

11. Thin layer chromatography
• The principle of TLC is the same as described for paper chromatography.
• In place of a paper, an inert substances, such as cellulose, is employed as
supporting material.
• Cellulose is spread as a thin layer on glass or plastic plates.
Advantage:
 Simultaneous study of large number of samples.
 Useful during preliminary purification process.
 Disadvantages:
 Takes much time to perform test as compared to column chromatography.
 Slow.

12. High performance liquid chromatography method
• VMA is measured by solid-phase extraction(SPE) of a 1-mL aliquot of urine.
• A know amount of stable isotope-labeled VMA internal standard(IS) is added to each urine specimen
prior to SPE.
• VMA and IS are eluted from the SPE column with methanol.
• Methanol is evaporated and the VMA and IS are redissolved in liquid chromatography tandem- mass
spectrometry mobile phase.
• A proportion of this prepared extract is injected onto a LC column that separates VMA and IS from the
bulk of any remaining specimen matrix.
• The VMA and IS are measured by mass spectrometry/tandem- mass spectrometry using the selected
reaction monitoring mode.
• VMA is quantified using ratio to IS vs urine calibrators.

13. Methanol (eluant)
+
VMA + IS
Movement of mobile phase
Stationary phase
Where separation occurs
Separated
components
Elution of Separated components and
to detector and fraction collector

14. • Until now, the analysis of catecholamine (CA) metabolites has primarily been based on HPLC with
electrochemical detection (ECD) and fluorescence detection (FLD) because of their inherent high
sensitivity.
• However, the reliability of HPLC–ECD is compromised because of coeluting interference and
electrode fouling and HPLC–FLD generally requires derivatization steps, which is laborious and
time consuming.
• HPLC coupled with mass spectroscopy (HPLC–MS) is a powerful technique that offers excellent
specificity and sensitivity to the target.
• Many studies have been performed using high performance liquid chromatography–tandem mass
spectrometry (HPLC–MS-MS) for the analysis of several compounds in the CA family in varied
biological fluids.

15. ELISA
PRINCIPLE OF THE ASSAY
• VMA ELISA is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the
competition between VMA coated on a microtiter well and that in urine for the monoclonal
antibody. Outlined steps are:
• 1. Sampling and reaction: The samples are incubated in the wells with horseradish peroxidase
conjugated anti-VMA monoclonal antibody.
• 2. Washing: Unbound VMA and the antibody bound to urinary VMA are removed by washing
with 0.9% NaCI solution.
• 3. Enzyme Reaction (Color Development): The amount of bound peroxidase is inversely
proportional to the concentration of the VMA present in the urine sample. Upon addition of the
substrate (TMB), a blue color is developed, and then it is changed to yellow by adding Stopping
Solution. The intensity of this is inversely proportional to the concentration of VMA in the
Calibrator or urine sample.
• 4. Absorbance Detection: After addition of Stopping Solution, absorbance is measured at 450
nm. And the readings are converted into the concentrations from the Calibration curve.

16. MATERIALS PROVIDED IN THE KIT
1. VMA Coated Microwell Plate: 1 VMA coated 96-microwell plate.
2. Anti-VMA-Enzyme Conjugate: Horseradish peroxidase conjugated to an anti-VMA monoclonal
antibody, 10 ml.
3. VMA Color Developing Reagent: Tetramethylbenzidine (TMB) solution, 20 ml.
4. VMA Stop Solution: 2N Sulfuric acid, 20 ml
5. VMA Calibrator Set of 0, 0.0625, 0.125, 0.25, 1, 2, 4 and 8 μg/ml in phosphate buffered saline,
0.01M, pH 7.4 (1.0 ml each).

17. MATERIALS REQUIRED BUT NOT PROVIDED
1. Plate reader with 450 nm filter
2. pH meter of pH paper with the range of 5.0-10.0
3. Pipettor with tips for 10, 50, and 100 μl
4. Pipettor with tips for 50 and 100 μl
5. Volumetric cylinders, 10 and 100 ml
6. Volumetric and serological pipettes, 10 ml
7. Disposable test tubes or vials
8. 5N NaOH solution
9. 5N HCI solution
10. Plate washer (optional)
11. Plate shaker (optional)
12. Sodium Chloride or Saline Solution
13. 0.01M phosphate buffered saline, pH 7.4

18. PREPARATION FOR THE ASSAY
1. Prepare 0.01 M phosphate buffered saline, pH 7.4. This solution is used to dilute
all unknown urine samples prior to analysis.
2. Before beginning the test, bring all urine samples and reagents to room
temperature (15-30°C) and mix well.
3. Set up all reagents and urine samples before running the assay. The entire test
procedure must be performed without any interruption in order to get the most
reliable and consistent results.

19. ASSAY PROCEDURE
Preparation of Washing Solution: Dissolve 9 grams of NaCl in 1 liter of deionized or distilled
water. Commercially available normal saline can also be used.
Preparation of Samples
1. Take 1.0 ml of acidified urine and transfer to a disposable tube in which the pH of urine sample
can be readjusted.
2. Bring pH of all samples within the range of 6 and 9 by stepwise addition of small amounts of 5N
NaOH (e.g. 5 μl) while checking pH either with a pH meter or using pH paper.
3. Dilute pH re-adjusted samples at a 1:10 ratio with phosphate buffered saline. The pH for diluted
samples should be between 7.0 and 8.0.
Standard Procedure for the Assay
1. Make work sheet with Calibrators and sample identification.
2. Sampling:
a. Dispense 50 μl of VMA Calibrators into appropriately designated wells.
b. Dispense 50 μl diluted (1:10) controls or samples to respective wells.
3. Addition of the Anti-VMA-Enzyme Conjugate:
Dispense 50 μl of Anti-VMA-Enzyme Conjugate to each well, using a pipetter.

20. 4. Antigen-Antibody Reaction:
Mix the plate by moving it back and forth slow horizontal movements for a minute. A plate shaker
can be used for this purpose also. Allow the plate to stand at 15-30°C, room temperature for 1 hour.
Calculation of Results
The results obtained above indicate VMA concentration in μg/ml. When the total VMA in 24-hour
urine sample is required;
VMA (μg/ml) X urine volume (ml)/ 1000 = VMA mg/24 hours
or, when VMA/Creatinine value is required;
VMA (μg/ml) ÷ Creatinine (mg/dl) = HVA μg/ml Creatinine (HVA mg/g Creatinine)
HVA- Homovanillic acid

21. CLIA
Principle
The microtiter plate provided in this kit has been pre-coated with an antibody specific to VMA.
Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated
polyclonal antibody preparation specific for and Avidin conjugated to Horseradish Peroxidase (HRP) is
added to each microplate well and incubated.
Then a luminol substrate solution is added to each well.
Only those wells that contain , biotin-conjugated antibody and enzyme-conjugated Avidin will emission
wavelength of radiation 425nm (max) chemiluminescence by HRP catalyzed.
The concentration of in the samples was positively correlated with the light value.
Luminosity is determined by microplate chemiluminescence analyzer (RLU.S-1.PW-1).
The concentration of in the samples is then determined by comparing the light value of the samples to
the standard curve.

22. TABLE :- Reference Intervals for Urinary VMA and
HVA

23. Clinical interpretations
Pheochromocytoma
• Pheochromocytomas are catecholamine-producing neuroendocrine tumors arising from chromaffin
cells of the adrenal medulla or extra-adrenal paraganglia.
• About 85% of pheochromocytomas arise from the adrenal medulla, and about 15% arise from extra-
adrenal chromaffin tissue (paragangliomas).
• Hypertension is the most common sign and can be sustained or paroxysmal.
Symptoms
(1) headache,
(2) palpitations,
(3) diaphoresis,
(4) pallor,
(5) dyspnea,
.
• Neural crest tumor is defined as a tumor originating from neural crest tissue, for example,
neuroblastoma and pheochromocytoma.

24. Neuroblastoma
• Neuroblastomas are malignant neoplasms characterized in most cases by overproduction of
catecholamines and their metabolites.
• Hypertension and other signs of catecholamine excess are uncommon in neuroblastoma.
• Patients commonly present with a tumor mass and clinical signs caused by compression effects on
neighboring structures or by hematologic abnormalities from bone marrow involvement.
(6) nausea,
(7) Attacks of anxiety, and
(8) generalized weakness