Quantitative Determination of Collagen Crosslinks - Methods in Molecular Biology: Extracellular Matrix Protocols - page 20

small, e.g., at biopsy, that the limitations of the HPLC procedure alone have to be
accepted. In such a case, reduction of the sample with borohydride and CF1 pre-
treatment can be ignored but it will not then be possible to quantify the interme-
diate crosslinks, deH-HLNL and HLKNL, as these are destroyed by acid
hydrolysis. However, the mature crosslinks hydroxylysyl and lysyl pyridinoline,
and the advanced glycation end-product pentosidine can all be quantified after
hydrolysis without prior reduction.
In summary, amino acid analysis by ion-exchange chromatography using nin-
hydrin detection can be used to assay the following crosslinks:
Hyl-Pyr, Lys-Pyr, HHL, DES, and I-DES.
HLNL and HLKNL can only be assayed after reduction of the sample with
borohydride prior to acid hydrolysis.
Reversed-phase HPLC using fluorescence detection can be used to assay Hyl-
Pyr, Lys-Pyr, and pentosidine. (None of these crosslinks require prior reduction
with borohydride, nor is it essential to use the CF1 clean-up procedure, though its
use will remove other fluorescent compounds resulting in a chromatogram, which
is easier to interpret.)

2. Various methods of homogenization are available and the one chosen should be

appropriate to the tissue under analysis:
Skin or Hide . These should first be cleaned of subcutaneous fat and adher-
ing muscle, and any hair removed with a scalpel or razor blade. The cleaned
skin can then be chopped very finely with a blade or alternatively, homog-
enized in phosphate-buffered saline (PBS) ( see Subheading 3.1. ). We use a
Polytron (Kinematica AG, Lucerne, Switzerland) for this purpose, which
works well for most soft tissues, except tendon , which has a tendency to accu-
mulate at the end of the homogenizer probe. Tendon is best treated by being
chopped finely with a blade.
Bone and cartilage . These are probably best comminuted in a steel “mortar
and pestle” ( see Subheading 3.6. ) at the temperature of liquid nitrogen. The
sample, in a cryothermic container, is frozen in a bath of liquid nitrogen as is
the steel mortar and pestle. This usually takes about 5-10 min. After removal
from the nitrogen bath the sample is placed in the mortar and the pestle, ham-
mered onto the sample, causing it to shatter. With a suitably sized mortar and
pestle, very small samples such as biopsies can be handled in this way with
good recovery of the powdered sample.
Fatty tissues . Lipids are readily removed from tissues with a very high fat
content by overnight extraction in 3:1 chloroform:methanol at 4 C. The chlo-

roform mixture can then be decanted and the tissue rehydrated by several
extractions in PBS.
Muscle . The collagen content of muscle is very low (1-5%), therefore it is
necessary to remove the bulk of the myofibrillar proteins prior to analysis.
This can be achieved by brief ultrasonic homogenization in Hasselbach-
Schneider buffer consisting of 0.6 M potassium chloride, 0.1 M disodium
hydrogen phosphate, 0.01 M sodium pyrophosphate, 0.001 M magnesium

°