NMR-based Metabonomic Analysis of Serum From Standardbred Yearlings With or
Without Hock Osteochondrosis Dessicans Lesions: Preliminary Study
Sarah Ralston^{1}^, István Pelczer^{2}^, Katherine Shatynski^{1}^, P.F. Spears^{3}^3
^{1}^Rutgers, the State University of New Jersey, New Brunswick, NJ USA
^{2}^Princeton University, Princeton, NJ, USA
^{3}^Hanover Shoe Farms, Hanover, PA, USA
Introduction
Metabonomic analyses use multivariate statistical cluster analyses of Nuclear Magnetic
Resonance (NMR) spectra from biological samples to find metabolic markers associated
with diseases^{1-4^}. The detection of significant differences in large numbers of
metabolites from diseased and normal individuals can lead to the development of novel
diagnostic and therapeutic modalities based on deeper understanding of the metabolic
processes involved^{1-4^}. Osteochondrosis dessicans (OCD) is a developmental
orthopedic disease that has been well documented to have a genetic basis in
Standardbreds and other breeds ^{as quoted in 5}^ but the actual metabolic defects
causing the lesions have not been well defined^{5}^. The presence of OCD lesions has
been correlated with abnormal insulin sensitivity in young horses but never proven to be
a true causal factor^{6, 7}^.
Our overall objective was to determine if metabonomic analyses of NMR spectra of
serum samples of yearling horses would detect consistent metabolic differences between
yearlings that had had hock OCD versus closely related yearling raised in the same
environment that did not have hock lesions.
Materials and Methods
Twenty matched pairs of Standardbred yearlings raised on the same farm under the same
conditions were used for this study. Each pair had the same sire and similarly bred dams.
The only consistent difference between the two members of each pair was that one had
had surgical correction of hock OCD lesions 1 to 6 months prior to sampling (OCD), the
other never had physical or radiographic evidence of lesions (Control).
Blood samples were taken by venipuncture between 1200 and 1500 h on 9/21/07.
Serum samples were subjected to 1H NMR analysis a Varian (Palo Alto, CA, USA)
Unity/Inova 600 MHz spectrometer using excitation sculpting and (ES) water
suppression^{8}^. The spectra were converted to the same scale of total integrated
intensity for comparative purposes (“normalized”) and had peaks alligned to the methyl
doublet of acetate to compensate for the small differences between spectra due to
irrelevant technical variances using HiRes (Columbia University, 2007) ^{9}^ The NMR
metabolic profiles were compared using Principal Component Analysis (PCA) scores
and loading plots (SIMCA-P+11, Umetrics, 2005),comparing horses with and without
OCD. Spectral peaks that had variable importance scores >2.0 were compared between
groups by Students T-Test (Statistix 8, Ocala, FL). Metabolites associated with the peaks
were identified using previously published data^{10}^. Significance was set at P<0.05.
Results
There was distinct clustering of OCD vs Control serum spectra (Figure 1), with three
principle components explaining >15% of the total variation between spectra.
Horses with OCD had higher (P<0.05) peaks in serum choline, tyrosine, threonine,
albumin lysyl and beta-glucose but lower (P<0.05) concentrations of 8 other amino acids,
alpha-glucose, and various low density lipoproteins and lipids. Further analyses are
pending.
Discussion
NMR based metabonomic analysis was able to detect significance differences in protein
and perhaps glucose metabolism in yearling horses that developed OCD lesions versus
half siblings that did not develop lesions, despite having identical nutritional and
environmental histories. Based on these results, it appears that a characteristic metabolic
“profile” may be established for young horses that develop OCD. The profile could be
used to detect foals “at risk” before lesions appear and hopefully identify the defective
metabolic pathways. Once this is accomplished therapeutic modalities may be explored to
prevent lesions in foals at risk. Further studies are needed (and in progress) to verify that
the differences observed in this study are repeatable and also present on foals before
lesions appear.
Key words: metabonomics, Osteochondrosis
Literature Cited
1. Nicholson, JK, Connelly J, Lindon JC, Holmes E. Metabonomics: a platform for
studying drug toxicity and gene function. Nature Rev. Drug Disc, 2002: 1:153161.
2. Hovadance MS, Ralston SL, Pelczer, I. Beyond Blood sugar: The potential of
NMR-Based Metabonomics for Human Diabetes Type 2: the Horse as a
possible model. Analytical Bioanalytical Chem 2007; 387 (2): 533-537.
3. Pelczer I. Structural Biology, ligand binding, Metabonomics-the changing face of high
field, high resolution NMR spectroscopy. J. Mol. Struct. 2003; 667:499-505.
4. Lindon JC, Nicholson JK. Spectroscopic and statistical techniques for information
recovery in metabonomics and metabolomic. Annu.Rev.Anal. Chem. 2008;1:2.1-2.25.
5. Lillich JD, Bertone AL, Malemud CJ, Weisbrode SE, Ruggles AJ, Stevenson S.
Biochemical, histochemical and immunohistochemical characteriztion of distal tibial
osteochondrosis in horses. Am J Vet Res 1997; 58:89-98.
6. Ralston SL. Hyperglycemia/hyperinsulinemia after feeding a meal of grain to young
horses with osteochondritis dissecans (OCD) lesions. Pferdeheilkunde 1996; 12:320322.
7. Pagan JD, Geor RJ, Caddell SE, Pryor PB, Hoekstra KE. The relationship between
glycemic response and the incidence of OCD in Thoroughbred weanlings: a Field
study. Proc Am Assoc Equine Prac 2001; 47:322-325.
8. Hwang TL, Shaka AJ. Water suppression that works:excitation sculpting
using arbitrary waveforms and pulsed field gradients. J Magn Reson, Series A 1995;
112: 275-279.
9. Pelczer I. High-resolution NMR for metabomics. Curr. Op. Drug Res. & Devel 2005;
8:127-133.
10. Nicholson JK, Foxall PJD, Spraul M, Farrant RD, Lindon JC. 750 MHz IH and IHl3C NMR Spectroscopy of Human Blood Plasma. Anal. Chem.1996; 67:793-811.
Acknowledgements: The authors thank Dr. Jablonsky and Skelton for assistance in
collection of samples and Drs. O’Byrne and Williams for assistance with sample
preparation. Deepest gratitude to Hanover Shoe Farm for giving us access to the horses.