BIOASSAY OF OXYTOCIN JSK NAGARAJAN by NagarajanKrishnan

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									BIO ASSAY OF OXYTOCIN



J.S.K. NAGARAJAN
ASST. PROF.,
JSS UNIVERSITY,
(OFF CAMPUS: JSS COLLEGE OF PHARMACY,
OOTACAMUND – 643001
Ph: 94431 49945
Email: nagasaki2001@rediffmail.com
                           OXYTOCIN
•   OXY – RAPID      TOCOS- LABOR
•   Synthesized in both sexes, well recognized physiological effects only
    in women.
•   Cyclic Polypeptide hormone - from posterior pituitary gland.
•   Pituitary gland consist posterior labe which produce oxytocin and
    diuretic hormone.
•   Neurosecretary product mainly synthesize in the cell bodies of
    paraventracular nuclear of the hypothalamus.

ROLE OF OXYTOCIN:
• Stimulate the contraction of the uterine smooth muscle & memory
   gland.
• Oestrogen progesterone & prolactin – responsible for production of
   milk by memory gland but milk ejection require oxytocin.
• Facilitates the contraction of uterus.

It is presented as a solid or solution in a solvent containing an
appropriate antimicrobial preservative such as 0.2% w/v of chlorbutol.

Animal species - 90 - 110% stated number of units of oxytocin activity.

Synthetic: Solid - NLT 560 units/mg Calculated with reference to the
peptide content & when liquid NLT 150 units/ml
          Oxytocin – Mechanism of action
 Neuropeptide made in hypothalamus that               stimulates
  contractions that expel the infant from uterus.

 Responsible for milk letdown & triggered by the nipple
  stimulation of suckling

 Called love & bonding hormone. It has a very special affect on
  mothering .

 Psychologically, oxytocin promotes a feeling of well being and
  tranquility.

 It enables the growing sense of love and attachment to the
  infant. The more the infant suck the more oxytocin is produced.

 In mothers it increases their attachment to their infant,
  promoting the feeling of love, and makes her infant more
  valuable to her.

 It also suppresses the fear that would normally cause her to
  back off from threat.
BIOLOGICAL ASSAY OF OXYTOCIN:
PRINCIPLE:
       Potency is determined by comparing its
  activity
   –   Depression of BP
   –   Contraction of Uterus
   –   Milk Ejection Pressure
   –   Vasopressor activity

  with standard preparation of oxytocin

STANDARD PREPARATION:
Consisting free dried synthetic oxytocin peptide
  with human albumin citric acid (12.5 units)
METHOD-A (Depression of the BP in chicken)
Test Animals: Cockerel            (young male chicken),   1.2 - 2.3 Kg, Healthy
            Anaesthesised cock-prolonged & constant high B.P

    Expose gluteus primus muscle(thigh) & remove politeal artery & crural vein.

                 Cannulate the popliteal artery & record B.P response

                        Cannulate the crural or brachial vein.

            Prepare std soln with saline. Inject 0.1 - 0.5ml

            Inject 2 doses of std soln into cannulate vein is record B.P response

            Dose should cause decrease in B.P (reqd. dose between 20-100mUnits)

Interval bween 2 injection, bween 3-10mins depend on rate @ which B.P return normal

      Dil. test preparation with saline so as to get same response as standard

                 The ratio between standard & test should be equal

If animal rapidly becomes insensitive to repeated injection the soln another must used.

Measure all responses are calculated result of the assay by std statistical method.
METHOD—B: (By contraction of the rat uterus):
Test animals: Female rat 120 – 200g
         Inject 100ug of oestradiol benzoate IM into female rat before the assay

 Immediately before assay confirm by vaginal smear that rate in oestrus or pre oestrus.

  Kill rat & suspend one horn of uterus in organ bath containing a solution of following
               Nacl,Kcl,Cacl2, NaHco3, Na2Hpo4, NaH2po4, Mgcl2, Dextrose

                           Maintain the bath at temp at of 32 c

                    Bath liquid required dose between 10-50 units/ml.

  Oxygenate solution with mix of 95% of O2, 5% of CO2 record -contraction of muscle.

   Record contraction produces by addition of two dose of std. ppn (Reqd. Dose 10 &
                             50munits/ml of bath liquid)

   when maximum contraction has been reached replace - bath liquid by fresh solution.

                  Dose should be added at regular interval[3-5minutes]

             Similarly record the contraction of test preparation as standard.

Ratio between two dose of test & two dose of std should be equal. This ratio kept constant
                                 through out the assay.

     Measure all response & calculate result of assay by standard statistical method.
METHOD C: (Milk ejection pressure in Lactating rat)
TEST ANIMALS: Lactating rat, 3-21 day after parturition, 300 g
 Separate from litter & 30-60 minutes later anaesthetise (IP Pentobarbitone Na).

   Tie rat to an operating table, at 37º, by its hind legs leaving front legs free.

 Cannulate trachea with a short PE tube of i.d. 2.5 mm in such a manner so as to
        ensure a free airway; apply artificial respiration only if necessary.

 Cannulate an external jugular or femoral vein with a PE tube of i.d. 0.4 mm filled
                         with saline & closed with a pin.

Shave the skin surrounding the inguinal and abdominal teats and excise the tip of
                   one teat, preferably the lower inguinal teat.

  Insert a PE tube of i.d. 0.3 mm & e.d. 0.6 mm, to a depth sufficient to obtain
appropriate measurement of pressure (3-10 mm depth), into the primary teat duct
     which opens onto the cut surface and tie firmly in place with a ligature.

Connect this cannula with a suitable strain gauge transducer (such as that used for
 recording arterial BP in rat) and fill with a 3.8% w/v of Na citrate /saline contain
               50 Units of heparin Na/ml to prevent clotting of milk.

    After cannulation, inject 0.05 - 0.2 ml of this solution into teat duct through
transducer to clear milk from tip of the cannula. (This procedure may be repeated
   during the assay should obstruction arise from milk ejected into the cannula).
 Clamp the strain gauge so that a slight tension is applied to the teat and its natural
alignment is preserved and connect the gauge to a potentiometric recorder adjusted
   to give full-scale deflection for an increase in milk-ejection pressure of 5.3 kPa.

 Inject all solutions through the venous cannula using a 1-ml syringe graduated in
                    0.01 ml and wash them in with 0.2 ml of saline.

  Prepare a solution of Std. & Test Ppn in saline solution so that the volume to be
                          injected is between 0.1 - 0.4 ml.

  Choose two doses of Std Ppn such that the increase in milk-ejection pressure is
           about 1.35 kPa for Lr dose and about 2.7 kPa for Hr dose.

 As an initial approximation, a lower dose of between 0.1 and 0.4 milliUnit and an
                upper dose of 1.5 to 2 times this amount may be tried.

Choose two doses of the Test Ppn with the same inter-dose ratio, matching effects
                 of doses of the Std Ppn as closely as possible.

  Inject four doses (2 doses of Std & 2 doses of Test) at intervals of 3- 5 minutes.

2 doses of Std and 2 doses of test should be given according to randomised block or
         a Latin square design & at least four responses to each -recorded.

  Measure all responses & calculate result of the assay by std statistical methods.

  Potency - 90% - 111%. Fiducial limits of error are 80% - 125%stated potency.
Vasopressor activity:
NMT 0.5 Unit /20 Units of oxytocic activity - by biological assay for vasopressor
activity- comparing activity of Test & Std Ppn of arginine vasopressin
Freeze-dried syn. arginine vasopressin peptide acetate with human albumin & citric acid (supplied
                               in ampoules containing 8.20 Units)

Inject slowly into tail vein of male albino rat weighing 300g -solution of a suitable a-adrenoceptor
        blocking agent, (10 ml/kg body weight of solution prepared by dissolving 5 mg of
 phenoxybenzamine HCl in 0.1 ml of ethanol (95%), adding 0.05 ml of 1 M HCl & dil to 5ml with
                                                saline .

          After 18 hours, anaesthetise rat - that will maintain -prolonged & uniform BP.

        After 45-60 minutes, tie the rat on its back to the operating table by its hind legs.

 Cannulate trachea with short PE of E.D. 2.5 mm & dissect carotid artery ready for cannulation.

                 Then cannulate the femoral vein close to the inguinal ligament.

                 Retract the abdominal muscles to expose the inguinal ligament.

 Retract superficial pudendal vein to one side & dissect femoral vein towards inguinal ligament
                                    from corresponding artery.

   When dissecting, a deep branch reaching femoral vein must be found & tied off to prevent
                                bleeding during cannulation.

 Tie a short PE cannula of E.D. about 1 mm into femoral vein by two ligatures & join by a short
  piece of flexible tubing to a 1-ml burette with an attached thistle funnel containing saline at
                                             about 37º.

 Firmly fix wet absorbent cotton swab to thigh so as to cover incision and cannula. At this stage
  inject through venous cannula 200 Units of heparin, dissolved in saline /100 g of body weight.
 Then tie in a carotid cannula of E.D.about 1 mm & connect by a column of saline
 contain heparin with a pressure measuring device such as Hg manometer of I.D.
                                   about 2-3 mm.

central & peripheral nervous system including both vagus & associated sympathetic
                                nerves is left intact.

                       No artificial respiration is necessary.

No air is injected, inject all solutions through venous cannula by means of a 1-ml
               syringe & wash in with 0.2 ml of saline from burette.

Dil extract of Std & Test Ppn with saline so that volume to be injected is between
                                   0.1 & 0.5 ml.

Choose 2 doses of the Std Ppn such that the elevation of the BP is about 4 kPa for
Lr dose & about 7 kPa but always submaximal for higher, ratio of low to high dose
  being determined by response & usually being 3-5. As an initial approximation
                     doses of 3 and 5 MUnits may be tried.

Choose 2 doses of Test ppn with same inter-dose ratio, matching effects of dose of
                                      Std Ppn.
                   Inject doses at intervals of 10 - 15 minutes.

  2 doses of Std & 2 doses of Test Ppn should given in randomised block / Latin
                square design & 4-5 responses to each recorded.

 Measure all responses & calculate result of the assay by Std statistical methods.
                   METHOD A                              METHOD B                           METHOD C                                 Method D
         Depression of BP                      Contraction of Uterus                Milk ejection Pr.in Lactating rat        Vasopressor Activity

animal   Cockerel, 1.2 to 2.3 Kg               Female rat 120 200g                  Lactating Rat,300g 3-21 days          Male rat 300g
                                                                                    parturitiction.
Organ    Anaesthetised , prolonged             Inject 100ug oestradiol by           anaesthetise(Pentobarbitone           Inject adrenoceptor blocking agent
                                                                                                                          (phenoxybenzamine 5mg) into tail
                                                                                    Na IP). Tie hind legs leaving
         & const high BP                       IM. Confirm rat in oestrus           front        legs       free,@37c.    vein of rat, after 18hrs, anaesthetise
                                               /pre oestrus by vaginal              Cannulate        trachea-respirate
                                                                                                                          rat-prolonged & uniform BP. After 45-
                                                                                                                          60mints, tie rat on its back to
         Gluteus Primus muscle                 smear.                               artificially if reqd. Shave skin      operating table by its hind legs.
                                                                                    around inguinal abdominal
         (thigh) & remove politeal             Kill rat & suspend one horn          teats . Pr. 3-10mm. Connect
                                                                                                                          Cannulate trachea with PE & dissect
                                                                                                                          carotid artery ready for cannulation.
         artery & crural vein.                 of uterus in organ bath              cannula with gauge transducer         Cannulate femoral vein close to
         Cannulate Popliteal artery            contain Na, K, Ca, Mg                &     fill    with    3.8%      Na    inguinal ligament. Retract superficial
                                                                                                                          pudendal vein to one side & dissect
                                                                                    citrate/saline contain 50 Units
         & record BP.                          chrloride NaHCO3,NaHPO4              heparin       Na/ml to prevent        femoral      vein    towards       inguinal
                                               and dextrose- bath 32c               clotting      of     milk.    After
                                                                                                                          ligament from corresponding artery.
                                                                                                                          When dissecting, a deep branch
                                                                                    cannulation, inject 0.05 - 0.2        reaching femoral vein must be found
                                                                                    ml of this solution into teat         & tied off to prevent bleeding. Tie a
                                                                                    duct through transducer to            PE into femoral vein by two ligatures
                                                                                    clear milk from tip of cannula.       & join by a short piece of flexible
                                                                                    Clamp strain gauge so that a          tubing to burette with attached thistle
                                                                                    slight tension is applied to          funnel containing saline-37º.Inject
                                                                                    teat & its natural alignment is       200Units of heparin, dissolved in
                                                                                                                          saline/100g of body weight             thru
                                                                                    preserved & connect gauge to
                                                                                                                          venous cannula. Tie in a carotid
                                                                                    a     potentiometric       recorder   cannula & connect by column of
                                                                                    adjusted to -for increase milk-       saline contain heparin with pressure
                                                                                    ejection pressure of 5.3 kPa.         measuring       device-CNS       &     PNS
                                                                                                                          including both vagus & associated
                                                                                    2 doses of Std Ppn - increase in      sympathetic nerves is left intact. No
                                                                                    milk-ejection pressure is 1.35 kPa    air is injected, inject all solutions thru
                                                                                    for Lr dose & 2.7kPa - Hr dose.       venous cannula by means of syringe
                                                                                                                          & wash with 0.2 ml of saline

Std      With Saline 0.1 -0.5ml                10-50units/ml                        0.1-04 ml                             0.1 & 0.5ml

         Inject 2 doses of std solution into   Oxygenate the soln. with 95% O2,     2 doses of the Test Ppn with          2 doses Std Ppn -elevation BP 4 kPa for Lr &
                                                                                                                          7 kPa higher dose, ratio of low to high dose
         cannulate vein record BP              5%CO2 record the contraction.        the same inter-dose ratio,            being determined using 3-5 munits
                                                                                    matching effects of doses of
         Decrease of BP                        Record the contraction by addition
                                                                                    the Std Ppn as closely as
                                               of 2 doses of STD.
                                                                                    possible.
Interv   3- 10 mins bween 2 injection-         3-5 minutes                          3-5 mints                             Inject doses at intervals of 10
al       depend on rate of BP returns normal                                                                                      - 15 minutes.

Test     Dil. Test ppn with saline get same    As per std.                          2 doses Std & 2 doses test -          2 doses of std & Test
         response                                                                   randomised block/Latin square         randomised block/Latin square
                                                                                    design & at least 4 responses         design & 4-5 response recorded.
                                                                                    to each -recorded.
ratio                                                        Between Standard & Test equal

								
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