ANALYSIS OF RECOMBINANT BOVINE

GROWTH HORMONE LEVELS IN MILK

Wes Dybedahl

Chem 4101

12/10/10

Background Bovine Somatotropin is a naturally

made hormone found in cow which has been studied and shown to correlate directly with milk production.

In order to help increase milk production, a synthetic version, recombinant bovine growth hormone (rBGH/rbST) was develoved by Monsanto in 1994 under trade name Posilac©, and is now owned by Elanco.(8)

The synthetic version is nearly identical to the natural hormone with the only difference in the 191 amino acid chain being a Methionine on the N-terminus instead of an Alanine.

Image source (2)

Problem• Concerns have been made over the recombinant form as to whether it may cause harm to the cows or humans who consume the milk.

• Results so far have been inconclusive in showing any adverse effects due to the added hormone but studies have shown rBGH supplementation causes an increase in insulin growth factor-1 which has been suggested to increase risk of breast and colon cancer.(1)

• Evidence opposing it’s use suggest that the increased hormone levels cause diseases such as mastitis in cows which forces the farmers to use more antibiotics. The increase in antibiotics has been accused of being present in milk and may be aiding bacterial resistance. (3)

• This evidence has led to the hormone being banned in Canada, Australia, Japan, and the European Union.

Hypothesis

The central hypothesis is to obtain an accurate and efficient method that will be able to distinguish the synthetic form over the natural form and properly measure those levels in milk samples.

HPLC-MS/MS The method that will provide

the best resolution and accuracy for the desired analyte would be HPLC coupled to tandem MS.

Detection of the proteins at low sample volumes possible

Fairly quick analysis time With HPLC, an internal

standard can be used with control samples in order to accurately quantify sample levels.

Instrumentation(1)

HPLC system: Agilent 1200 Column: C18 Interchrom QS Uptisphere 3HDO

150 mm × 2 mm, 3 μm, 100 Å. This column would provide optimum separation for the polarity differences found on the amino acid chains from the proteins found the sample matrix.(4)

Mobile phase: acetonitrile + 0.1% formic acid (A) and water + 0.1% formic acid (B).

The total run time was 20 min The solvent flow rate was 300 μL min−1 and injection volume 20 μL.

MS instrument: API 5000, triple quadrupole, fitted with an electrospray ion source in positive mode.

capillary voltage: 5000 V

nebulizer: 55 psi,

gas flow: 13 L min−1

gas temperature: 300 °C.

(5)

Sample Preparation(1)

To try and remove the desired analyte from the other things found in the matrix for milk several steps need to be made in the preparation.

The sample first must be spiked with 100ng/mL of reST as an internal standard. Then mixed with 10mL of water and 10mL methanol into a solid phase extraction cartridge, followed by two washings, first with 5mL water/0.1% TFA, then 5mL water/acetonitrile (70:30) 0.1%TFA.

The samples are then eluted with 7mL of 20:80 water/acetonitrile mix with 0.1% TFA, followed by an evaporation by nitrogen stream.

The proteins, which contain the hormone, can then be precipitated out by 5mL of cold methanol, and then centrifuged to remove the top layer of un-needed fat. The proteins then need to be digested by 2ug trypsin in 100uL of ammonium bicarbonate, 50mM and 20uL acetonitrile.

Finally, the extracts are evaporated and reconstituted with 40uL of 80:20 mix water/acetonitrile, containing 1ug/mL 13C labelled N-terminal tryptic rbST and 0.1% formic acid for an external standard.

Results

Chromatogram for N-terminal peptides from blank milk samples and those spiked with 25 ng/mL rbST. (1)

•The analytical method gives an accurate quantification of the rbST within the sample, through comparison of the chromatogram with the control samples.•Selectivity is also superb for this technique, with no peak interferences occurring.

Method Specifications(1)

Linearity:Concentration range 20-100 ng/mL

Coefficient of determination R2=0.992

Slope a=0.0106

Intercept b=0.1156

Sensitivity:LOQ 1.24 ng/mL

LOD 1.92 ng/mL

Precision:Relative Standard deviation of repeatability RSD(r)=4.5%

Relative repeatability limit at 95% for duplicate results

r=12.5%

Trueness:RSD(Rec)corrected 7.2%

Measurement of uncertainty:Standard uncertainty u=19.4%

Expanded uncertainty U=38.8%

Alternative methodsMethod Pros Cons

Capillary Electrophoresis(5)

Separation of different protein chains, fast analysis times

Not strong enough to be used alone

Fluorescence Highly sensitive for detecting Trp within the hormone

Sample preparation, matrix interference.

electrochemiluminescent immunoassay(6)

Sensitive and selective method

Difficult to distinguish synthetic from natural hormone

Conclusion

•There is still much debate as to whether or not the use of recombinant bovine growth hormone poses harm to humans.

•With further developments in biological analysis, the hormone will be able to be monitored in the body to determine any possible negative effects that may be occurring.

•The methods previously discussed will provide accurate analysis of the synthetic hormone found in milk samples and, in turn, will be able to help in measuring for determinations of possible adverse effects.

References1) Le Breton, Marie-Hélène; Beck-Henzelin, Andrea; Richoz-Payot, Janique; Rochereau-Roulet,

Sandrine; Pinel, Gaud; Delatour, Thierry; Le Bizec, Bruno. Analytica Chimica Acta, Jul2010, Vol. 672 Issue 1/2, p45-49, 5p; DOI: 10.1016/j.aca.2010.04.030; (AN 51811641)

2) John, Y., and Rodney Pearlman. Stability and characterization of protein and peptide drugs: case histories. 5. New York, NY: Springer Us, 1993. 67-71. Print.

3) “The Issues: rBGH”. Sustainable table. July 2008. December 2010. http://www.sustainabletable.org/issues/rbgh/.

4) http://www.chem.agilent.com/en-US/Products/Instruments/lc/analytical/systems/1290infinitylc/Pages/default.aspx. Dec 5, 2010.

5) Pinel, G.; Buon, R.; Aviat, F.; Larre, C.; Andr-Fontaine, G.; Andr, F.; Le Bizec, B.Recombinant bovine somatotropin misuse in cattle. Evaluation of western blotting and 2D electrophoresis methods on biological samples for the demonstration of its administration Anal. Chim. Acta 2005, 529, 41– 46

6) M.F. McGrath, G. Bogosian, A.C. Fabellar, R. Staub, J.L. Vicini and L.A. Widger, Journal of Agricultural and Food Chemistry 56 (2008), pp. 7044–7048.

7) http://www.chemguide.co.uk/analysis/chromatography/hplc.html. Dec 5, 2010

8) Posilac Information. Elanco. http://www.elanco.us/products/posilac.htm. 2010. Dec 6, 2010