Lotus pedunculatus.
3.2 METHODOLOGY 1 EXPERIMENTAL DESIGN.
3.2.7 SAMPLE ANALYSIS.
Once it was established that each infusion was at plateau specific radioactivity by 22 hours (Fig. 2), the 26, 28 and 30 hours samples were bulked for detennining the concentration and radioactivity of methionine, cystine and sulphate in plasma.
3.2.7.1 Methionine and Cystine.
Amino acids were separated by ion exchange chromotography on a high performance liquid chromotograph (HPLC; Waters Associates, USA), using a sodium form amino acid analysis column (Waters Associates, USA) maintained at 62oC.
Over the initial 48 min of amino acid separation there was a linear change from 100% 0.2M
sodium citrate/nitric acid buffer (pH 3. 1 ; 7.5x 1O-5% w/v pentachlorophenol) to 100% 0.2M sodium nitratelborate buffer (pH 9.6; 7.5X 1O-3% w/v EDTA). This was followed by a further 27 min of separation by sodium nitratelborate buffer, pH 9.6 and then equilibration of the HPLC with sodium citrate/nitric acid buffer, pH 3 . 1 for 35 min. The combined buffer flow was O.4ml/min, giving a column pressure of 1000 to 1 500 psi (6900-1 0400 lcPa). Amino acid peaks were detected by the fluorescence of ortho-phthaldehyde derivatives of the amino acids, formed by a post-column reaction of the column eluate with
orthophthaldehyde (OPA) using a Waters (USA) 420AC Fluorescence Detector (338nm excitation and 425nm emission wavelength). The OP A solution comprised of 800mg OPA
in 10m! methanol with 400111 mercaptoethanol and 4001l1 30% Brij-35 (polyoxyethylene
lauryl ether) made up to l 000m! with 0.5M borate buffer, pH 10.6 and was pumped at O.4mVmin.
All solutions for use in the HPLC were filtered through a 0.2Jl.m cellulose acetate (aqueous) or polytetrafluoroethylene (non-aqueous) filter (Micro Filtration Systems, USA) and
saturated with oxygen-free nitrogen gas.
The concentration of methionine and cystine were detennined after injection of 40Jl.l of protein-free plasma into the HPLC, by comparison of areas under peaks, calculated by a Waters (USA) 730 Data Module integrator, with an external standard (equimolar (2.5molJl) Amino Acid Hydrolysate (Pierce Chemical Company, USA) with equimolar (2.5molJl) norleucine (BDH) added). The external standard was used for 48 hours before being discarded.
For determining [35S] activity of methionine and cystine, 2ml protein-free plasma was injected into the HPLC and eluate samples ( l ml) containing the relevant peak were collected
into scintillation vials as they flowed from the detector. To this was added 8mls of PCS II
scintillation fluid (Phase Combining System II; Amersham (Australia) Pty Ltd.) and 2m1s
of glacial acetic acid. The acetic acid prevented phase separation and its addition resulted in the formation of a clear, stable gel. Vials were counted in a scintillation counter (Beckman LS 380 1 , USA). Samples were corrected for quenching using Automatic External
Standardization, utilising a spiked quench curve where samples containing known activities of the relevant infusate, were quenched with chloroform.
The recovery by the HPLC of both methionine and [35S] methionine, and cystine and [35S] cysteine, added to protein-free plasma was 0.98±0.03 (SD; methionine) and 0.95±0.04 (SO; cysteine).
3.2.7.2 Inorganic Sulphate.
Inorganic sulphate in protein-free plasma was separated from organic-S with Dowex l -X8 resin (BDH; CI- form, 1 8-52 mesh size, 1 . 33 meq/ml binding capacity), which is a strong base anion exchanger capable of binding inorganic sulphate.
Dowex l -X8 contains benzyltrimethyl ammonium side chains which can degrade to yield free NH3• Ammonia interferes with the colormetric determination of sulphate, so the resin was washed before use by shaking in 1M NaOH for 1 6 hours at room temperature to remove free NH3, followed by shaking in deionised water three times for 4 hours at room temperature and finally regeneration of the resin by shaking in 1 M HCI for 1 6 hours at room temperature.
Protein-free plasma (3ml), washed Dowex 1-X8 ( l g) and deionised water (7m!) were shaken for 16 hours at room temperature. The supernatant, which contains [35S] labelled amino acids was discarded, whilst the resin, which has inorganic sulphate bound to it, was washed three times in 5ml of deionised water to remove all residual protein-free plasma. Inorganic sulphate was eluted off the resin by shaking the resin in 5ml of 1 M HCl for 16
hours at room temperature. The Hel, which contained the inorganic sulphate was decanted
and 1ml was added to 10m! pes IT for scintillation counting as previously described.
Inorganic sulphate concentration was determined by the automated method of Johnson and Nashida (1952), using an Autoanalyser (Technicon Industrial Systems, USA) as follows. Plasma inorganic sulphate was determined colormetrically by reducing inorganic sulphate in the Hel samples to sulphide, which was absorbed in zinc acetate (O.23molll)/sodium acetate (O. 15mol/l) and then reacted with p-aminodimethylaniline to produce methylene blue. All standards used for autoanalysis were made up in 1M Hel which had been treated in an identical fashion to the protein-free plasma samples.
The recovery by Dowex l-X8 resin of both potassium sulphate and (35S] inorganic sulphate, added to protein-free plasma was O.70±O.05 (SD). There was no detectable
rec
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very by Dowex- lX8 resin, of either inert methionine or cystine and [35S] methionine or [35S] cysteine added to protein-free plasma.The [35S] sulphate infusate was treated in an identical fashion to protein-free plasma and had a similar recovery to plasma sulphate; hence for calculation of SA, IRL and TQ no correction for recovery was made, but plasma sulphate concentration was corrected for recovery.
3.2.7.3 Rumen Ammonia.
Rumen NH3 was determined by the method of Williams and Twine (1967) using an auto analyser (Technicon Industrial Systems, USA).
3.2.8 CALCULATION OF DATA AND STATISTICAL ANALYSIS.
Means are presented with the standard error of the difference (SED) or the standard error (SE) as appropriate. Comparison between control and PEG treatments was done by analysis of variance.
3.2.8.1 Specific Radioactivity (SA) of plasma cystine, methionine or sulphate was calculated from the following equation.
SA (dprn/Jlmol) =
Concentration of sampled pool (Jlmol/ml)
3.2.8.2 Irreversible Loss Rate (IRL) measured at plateau SA, is the rate at which plasma cystine, methionine or sulphate, leaves the sampled pool and does not return within the time course of the experiment.
IRL (Jlmol/min) = Infusion into
SA of pool (dpm/Jlmol)
3.2.8.3 Transfer Quotient(TQ) measured at plateau SA is the proportion of radioactive
label detected in a secondary pool (B), which originated from an infusion into a primary
pool (A).
TQ = B
SA of pool A (dpm/Jlmol)
3.2.8.4 Total Flux measured at plateau SA, is the combined total of all outflows
(Jlmol/rnin) from a plasma pool to all processes. When· calculating the mathematical model,
all outflows were assumed to equal all inflows, therefore total flux represents the total flow through a pool.
The results of this experiment are presented as a three pool, compartmentalized model similar to that proposed by Nolan et al. ( 1976) for N transactions in the rumen. To calculate the various flows within the model, methionine IRL and the proportion of cystine and inorganic sulphate derived from methionine (TQ) were calculated from the PSS]
methionine infusion. The procedure was repeated for infusions of (3sS]-labelled cysteine and sulphate. The IRL and TQ were used in mathematical equations proposed by Nolan et al. (1976) to solve the three pool model.
* Sheep into
metabolism crates. DA Y 35: DAY 39:
* Treated for * Rumen sample * Rumen sample
parasites. for ammonia. for ammonia.
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DAY 29: DAY 36:
* Lotus feeding * PEG infusion starts starts.
DAY 42: * Jugular catheters inserted
DAY 45: DAY 47: DAY 50: