INTRODUCTION

Pantothenic acid, also called vitamin B5 is a water-soluble B vitamin and

therefore an essential nutrient.[7] All animals require pantothenic acid in order to
synthesize coenzyme A (CoA) – essential for fatty acid metabolism – as well as to,
in general, synthesize and metabolize proteins, carbohydrates, and fats.[7][8]

Pantothenic acid is the combination of pantoic acid and β-alanine. Its name derives

from the Greek pantos, meaning "from everywhere", as minimally, at least small
quantities of pantothenic acid are found in nearly every food. [7][9][8] Human
deficiency is very rare.[7][8] As a dietary supplement or animal feed ingredient, the
form commonly used is calcium pantothenate because of chemical stability, and
hence long product shelf life, compared to sodium pantothenate or free pantothenic
acid.

Structure of vitamin B5

Sources of vitamin B5

The best way to make sure you’re getting enough vitamin B5 is to eat a healthy,
balanced diet every day.

Vitamin B5 is an easy vitamin to incorporate into a good diet. It’s found in most
vegetables, including:

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  broccoli

 members of the cabbage family

 white and sweet potatoes

 whole-grain cereals

Other healthy sources of B5 include:

 mushrooms

 nuts

 beans

 peas

 lentils

 meats

 poultry

 dairy products

 egg

DEFICIENCY ASSOCIATED WITH VITAMIN B5

Pantothenic acid deficiency in humans is very rare and has not been thoroughly
studied. In the few cases where deficiency has been seen (prisoners of war during
World War II, victims of starvation, or limited volunteer trials), nearly all
symptoms were reversed with orally administered pantothenic acid. [15]
[10]
 Symptoms of deficiency are similar to other vitamin B deficiencies. There is
impaired energy production, due to low CoA levels, which could cause symptoms

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of irritability, fatigue, and apathy.[15] Acetylcholine synthesis is also impaired;
therefore, neurological symptoms can also appear in deficiency;[31] they include
sensation of numbness in hands and feet, paresthesia and muscle cramps.
Additional symptoms could include restlessness, malaise, sleep disturbances,
nausea, vomiting and abdominal cramps

People take vitamin B5 supplements and derivatives to help with a range of

conditions.These conditions include:

 acne

 ADHD

 alcoholism

 allergies

 asthma

 baldness

 burning feet syndrome

 carpal tunnel syndrome

 celiac disease

 chronic fatigue syndrome

 colitis

 conjunctivitis

 convulsions

 cystitis
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  dandruff

 depression

 diabetic nerve pain

 dizziness

 enlarged prostate

 headaches

 heart failure

DETERMINATION OF VITAMIN B5 IN BLOOD AND URINE

Pantothenic acid, also called vitamin B5 is a water-soluble B vitamin and therefore

an essential nutrient.[7] All animals require pantothenic acid in order to
synthesize coenzyme A (CoA) – essential for fatty acid metabolism – as well as to,
in general, synthesize and metabolize proteins, carbohydrates, and fats.

Pantothenic acid is the combination of pantoic acid and β-alanine. Its name derives

from the Greek pantos, meaning "from everywhere", as minimally, at least small
quantities of pantothenic acid are found in nearly every food. Human deficiency is
very rare. As a dietary supplement or animal feed ingredient, the form commonly
used is calcium pantothenate because of chemical stability, and hence long product
shelf life, compared to sodium pantothenate or free pantothenic acid.

Pantothenic acid is required in the formation of acetyl coenzyme A which holds a

key position in many metabolic pathways. Only the natural dextrorotatory form is
active. Pantothenic acid is found in most foods of plant and animal origin and good

4
sources include liver, kidney, wheat germ, royal jelly, peanuts, spinach, cheese and
peas. There is no quoted RDA, though most diets provide at least 10 mg per day.

Panthenol and pantothenic acid have been identified and quantified in

pharmaceutical preparations by extraction with ethanol or benzyl alcohol and
separated by TLC on silica gel plates developed in propan-2-ol–water (85:15).
Spots are measured by spectrodensitometry. Postaire has applied the over-
pressure derivatization technique following separation of calcium
pantothenate from other hydrophilic vitamins on silica gel HPTLC layers
developed in butan-1-ol–pyridine–water

Food Sources

 Fortified cereals.
 Organ meats (liver, kidney)
 Beef.
 Chicken breast.
 Mushrooms.
 Avocado.
 Nuts, seeds.
 Dairy milk.

EFFECIENCY OF VITAMIN B5

Vitamin B5, also called pantothenic acid, is one of the most important vitamins for
human life. It's necessary for making blood cells, and it helps you convert the
food you eat into energy. Vitamin B5 is one of eight B vitamins. All B vitamins
help you convert the protein, carbohydrates, and fats you eat into energy.

DEFFICIENCY OF VITAMIN B5

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Vitamin B5 deficiency is rare, but may include symptoms such as fatigue,
insomnia, depression, irritability, vomiting, stomach pains, burning feet, and
upper respiratory infections.

SAMPLES

 Blood
 urine

Reagent Preparation

1. Mobile Phase. Ethanol (200 proof) and acetonitrile (HPLC grade) are filtered
separately using 0.45um pore size membranes (Cat. no. HVHP047; Millipore,
Medford, MA). Triethylamine (TEA, ‘Baker’ grade) is added to each prior to
filtering at 32 drops per liter using a glass Pasteur pipette to make 0.1% TEA
solutions. The components are degassed and automated solvent blending takes
place in the Alliance 2695 HPLC system.
2. 10% Ascorbic Acid and 10% NaCl. 0.5 g L-Ascorbic Acid (ACS Certified) and
0.5 g NaCl (Analytical Grade) are dissolved in 5.000 mL deionized water in a
12 x 75 mm test tube. The ascorbic acid is relatively labile but normally can be
used for 2 weeks or until there is a noticeable yellow color to the solution.
Always cap the tube (with a disposable plastic cap) and store at room
temperature in yellow light. If there is significant headspace in the tube, flush
with nitrogen or argon before storage.

Instrumentation

1. Waters HPLC system (Waters Chromatography Division, Milford, MA)

a. Model 2695 Alliance system
b. Model 2996 or 996 photodiode array detector

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 c. Compaq computer with the following specifications: Microsoft Windows
2000 Professional operating system, 1000 MHz, 260 MB RAM, 18 GB
hard drive.
2. Cera column cooler 250 (Cera, Inc. Baldwin Park, CA) or equivalent.
3. Rack-type vortex mixer (American Scientific Products, McGaw Park, IL) or
equivalent.
4. Cary 3E spectrophotometer (Varian Instruments, Palo Alto, CA) or equivalent.
5. Speedvac SC200 and SC210A Systems (Savant Instrument Co., Farmingdale,
NY) or equivalent.
6. Precision Model VP-190 Direct Drive Vacuum Pump (Precision Scientific Inc.,
Chicago, IL) or equivalent.
7. Refrigerated vapor trap, model RVT-4104 (Savant Instrument Co.,
Farmingdale, NY) or equivalent.
8. Magnetic stirrer (American Scientific Products) or equivalent.
9. Digiflex Automatic Diluter/Dispenser, with 200-μL sampling and 2.0mL
dispensing syringes (Titertek Instruments,Inc.,Huntsville, AL) or equivalent.
10.Gilson Microman positive displacement Pipettes (Gilson, Villiers-le, France) or
equivalent. Ranin Pipetman pipette (Ranin, Woburn, MA) or equivalent.

Materials

1. 15 cm x 4.6 mm Phenomenex Ultracarb 3 octadecylsilane, 3 µm particle size

column
2. 15 cm x 4.6 mm Burdick and Jackson OD5 octadecylsilane 5 µm particle
size column
3. 15 cm x 4.6 mm YMC C30 Carotenoid column
4. 25 cm x 10 mm Phenomenx 5 μ micron C18 Luna column
5. Hexane UV

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 6. Acetonitrile HPLC grade
7. Ethanol, absolute (U.S.P.), stored in glass,
8. Chloroform, spectrophotometric grade
9. Triethylamine, ‘Baker’ grade
10.Pantothenic acid
11.Zeaxanthin
12.L-Ascorbic acid, ACS grade
13.Stearic anhydride
14.Pipette tips for SMI Digitron pipette
15.Plastic tuberculin syringes

Sample Preparation

1. Prepare one set of labeled 12 x 75 mm glass tubes and one set of labeled 13 x
100 mm glass tubes for the calibrators, pools and unknowns.
2. Prepare a shallow (2–3’’ deep) ethanol and dry ice bath for freezing samples.
3. With an Eppendorf repeating pipette or equivalent, dispense 10 µL of 10% L-
Ascorbic Acid/10% NaCl solution into the bottom of each 13 x 100 mm glass
tube.
4. Using a micropipette, add 100 µL of serum to each tube containing the 10%
ascorbic acid/10% NaCl solution. Cover the tubes with heavy duty aluminum
foil and vortex for 60 sec.
5. With the Digiflex Dilutor, or equivalent, add 100 µL of the internal standard
solution to each tube.
6. Cover the tubes with foil and vortex the mixture for 60 seconds, being careful
not to allow the liquids to touch the foil during mixing.
7. With the Digiflex Dilutor, add 1.000 mL of hexane. Cover the tubes with foil
and vortex for 60 seconds, being careful not to allow the liquids to touch the

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 foil during mixing. Repeat this mixing cycle (60 sec) six times for a total of 6
min of extraction with hexane. For good recovery, it is important to mix as
vigorously as possible without touching the foil (liquid should reach up to
twothirds of the tubes while shaking).
8. Allow the samples to rest for 10 min in order for aqueous and organic phases to
separate.
9. Carefully transfer the rack of tubes to the dry ice/ethanol bath. Allow the
aqueous phase to freeze (≥ 25 min). Pour the hexane (upper) layers into the
second set of labeled 12 x 75 mm tubes and evaporate the hexane in the
Speedvac (without heat) according to the number of tubes (9 minutes for 40–60
tubes,
10.10 minutes for 60–100 tubes, and 12 minutes for >100 tubes). Each Speedvac
has slight differences in dry times which the analyst has documented. Make
sure not to over-dry the extracts. 10. Add 50 µL of ethanol to the tubes
containing the dried extracts.
11.Vortex the tubes for 2 cycles of 60 seconds each.
12.Add 50 µL of acetonitrile to each dissolved extract and vortex the tubes for 2
cycles of 60 seconds each.
13.Draw each extract into a 1.0 mL tuberculin syringe taking care to leave an air
space between the plunger tip and the solution, place a 0.45 µ pore-size syringe
filter on the loaded syringe, and filter the extract into an autosampler vial.
14.Cap the vials and place them in the Alliance carousels at 20°C.

CALCULATION

Both the Packard COBRA and Titertek Apex 10/600 Plus counters have full data
reduction capabilities. Linear B/B0 vs log10 concentration with a cubic or auto
spline curve fit is used in both counters where:

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B/B0 = CPM of Standard or Unknown Sample - CPM of NSB X 100

CPM of 0 Standard - CPM of NSB

and B = corrected counts/min (blank subtracted) for each tube, and Bo = corrected
counts/min of 0 standard (blank subtracted).

Results

The method described here is designed to detect serum B5 values from 5 to 100
ng/mL, which is beyond the normal range of values expected to be observed for
human serum B5 concentration. Values 70 ng/mL are verified by reassay,
including re-extraction. For re-extraction, dilute elevated specimens (>100 ng/mL)
with 0 standard prior to extraction. Any samples with CVs >10% are also
reassayed.

The sensitivity of this assay, when defined as the lowest quantity differentiated
from zero at 2 standard deviations below the mean cpms of the zero standard, has
been shown to be at or below 1.5 ng/mL. Any values less than the lowest standard,
5.0 ng/mL, are reported as "3.0." These levels could occur physiologically and
would indicate severe B5 deficiency. Values greater than 70 ng/mL indicate
prolonged exposure to ultraviolet radiation or excessive supplementation.

ADVANTAGE OF VITAMIN D DETERMINATION

 Pantothenic acid deficiency. Taking 5-10 mg of pantothenic acid

by mouth daily prevents and treats pantothenic acid deficiency.

DISADVANTAGES OF VITAMIN D DETERMINATION

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 Skin damage caused by radiation therapy (radiation dermatitis). Applying
dexpanthenol, a chemical similar to pantothenic acid, to irritated skin does not
seem to reduce skin damage caused by radiation therapy.

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References

"Scientific Opinion on the safety and efficacy of pantothenic acid (calcium D-

pantothenate and D-panthenol) as a feed additive for all animal species based
on a dossier submitted by Lohmann Animal Health". EFSA Journal. Parma,
Italy: European Food Safety Authority. 9 (11): 2409.
2011. doi:10.2903/j.efsa.2011.2409.
Lide, David R., ed. (2009). CRC Handbook of Chemistry and
Physics (90th ed.). Boca Raton, Florida: CRC Press. ISBN 978-1-4200-9084-
0.
"Calcium D-pantothenate". CHEMICALLAND21, AroKor Holdings Inc.
Retrieved 5 September 2014.
"MSDS of D-pantothenic acid" (PDF). Human Metabolome Database. Retrieved 5
September 2014.
Leenheer AP, Lambert WE, Bocxlaer JF, eds. (2000). Modern Chromatographic
Analysis of Vitamins: Revised And Expanded. Chromatographic Science.
Vol. 84 (3rd ed.). Marcel Dekker. p. 533. ISBN 978-0-203-90962-1.
"DL-Pantothenic acid calcium salt". Retrieved 5 September 2014.
"Pantothenic acid: Fact Sheet for Health Professionals". Office of Dietary
Supplements, US National Institutes of Health. 3 June 2020. Retrieved 27
November 2020.
"Pantothenic acid". Linus Pauling Institute at Oregon State University.
Micronutrient Information Center. 1 July 2015. Retrieved 27
November 2020.
"Pantothenic acid ordered by nutrient content per 100 g". US Department of
Agriculture Agricultural Research Service, Food Data Central. February
2020. Retrieved 3 June 2020.
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Miller, JW; Rucker, RB (2020). "Pantothenic Acid". In BP Marriott; DF Birt; VA
Stallings; AA Yates (eds.). Present Knowledge in Nutrition, Eleventh
Edition. London, United Kingdom: Academic Press (Elsevier). pp. 273–
88. ISBN 978-0-323-66162-1.

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