Biology Reference
In-Depth Information
major, mature crosslinks of bone and cartilage (4) ; tendon, however, contains a
mixture of both mature crosslinks. An additional mature crosslink may be
formed if the keto-imine reacts with a lysine-aldehyde in which case a pyrrole
structure is favored ( see Fig. 1 ). Although the structure of the so-called “pyr-
role” crosslink has not been confirmed there is mounting evidence for its pres-
ence in bone and tendon collagen (2) . An accurate determination of the ratio of
the immature to mature crosslinks provides a valuable indication of the degree
of turnover of a collagenous tissue. It is, therefore, important in any study of
changes to the collagen in a pathological tissue to understand the nature of the
normal, age-related changes that occur in the particular tissue under investigation.
The intermediate crosslinks may be radio-labeled by reduction of the tissue
with tritiated sodium borohydride, thus facilitating their location and identifi-
cation during subsequent chromatography (5) . However, their quantification
requires either ninhydrin, or a similar post-column derivatization technique,
following their separation from the acid hydrolysate of the tissue by ion-
exchange chromatography. Precolumn derivatization of these polyvalent
crosslinks for subsequent analysis by reversed-phase high performance liquid
chromatography (HPLC) can produce multiple derivatives that elute as separate
peaks throughout the subsequent analysis and is therefore not recommended.
The mature crosslinks HHL, Hyl-Pyr, and Lys-Pyr can be simultaneously quan-
tified using the same ion-exchange column (6) . Hyl-Pyr and Lys-Pyr can also
be determined, with greater sensitivity, by HPLC utilizing their natural fluo-
rescence to facilitate their detection and quantification (7) . It has not yet been
possible to analyze the pyrrole crosslink by ion exchange or HPLC chromatog-
raphy and until this is possible a rather nonspecific colorimetric method is
employed (8) .
The other major connective tissue protein, elastin, is also stabilized by
lysine-derived crosslinks based on the same enzymic mechanism, but yields
two tetravalent pyridine compounds, desmosine (DES) and iso-desmosine
(I-DES) (10) . Both of these compounds can be detected by ninhydrin after
elution, under the same conditions, from the same ion-exchange column.
A second crosslinking mechanism occurs when the turnover of collagenous
tissues decreases following maturation and involves the reaction of glucose
with the
-amino group of lysine and subsequent oxidation reactions (9) . Gen-
erally known as glycation, the addition of glucose is nonenzymic, adventitious,
and possibly random. Crosslinks formed by this mechanism, such as
pentosidine, could provide good biomarkers of low metabolism and possible
damage to the functional properties of collagen during aging and in diabetic
subjects. However, to date, none of the glycation crosslinks has been related to
changes in the functional properties of collagen, hence, we have only consid-
ered pentosidine.
ε
Search WWH ::




Custom Search