TY - JOUR
T1 - Method for chronological recording of antigen appearance in human head-hair shafts and its use for monitoring glycation products in diabetes
AU - Nissimov, Joseph
AU - Elchalal, Uriel
AU - Bakala, Hilaire
AU - Brownlee, Michael
AU - Berry, Elliot
AU - Phillip, Moshe
AU - Milner, Yoram
N1 - Funding Information:
The authors gratefully acknowledge Prof. Ehud Ziv from the Diabetes Unit of the Endocrinology Dept, Hadassah Hospital in Jerusalem for suggesting GDM patients as alternative controls for diabetics, Prof. Norman Grover from the Hebrew University Faculty of Medicine, Jerusalem, Israel, for his help with statistical analysis, Prof. Amirav Gordon from the Hebrew University Faculty of Medicine, for his generous donation of anti-EGAs antibodies. Nurses Nancy Novick, Penny Akiva, and Regina Ofan, and Drs. Asher Korkos, Victoria Tarletzki, Hanoch Bar-On, and Arnon Samueloff, were helpful in obtaining the hair samples, and Mrs. Hanna Lan from the Hebrew University Faculty of Dentistry in preparation of the silicone molds used in the production of “dot-blocks”. This study was supported by the Inez and David Myers fund of Cleveland, Ohio (to YM).
PY - 2007/3/30
Y1 - 2007/3/30
N2 - We describe immunochemical assays of non-enzymatic glycation products in human head-hair protein extracts and hair cross sections using Western blots and a novel "dot-block" methodology. In the latter, groups of approximately 15 hair fibers, clipped at about 1 mm proximal to the scalp-skin were aligned, wound around, and attached to 3 mm diameter araldite screw rods. Up to 40 such rods were next embedded lengthwise in additional araldite polymer creating a solid block and the top surface of the block was sectioned off to the half-diameters of the screw rods thus exposing accurately transected hair cross sections at regular (∼ 0.5 cm) intervals. Early- and advanced-glycation products (EGAs and AGEs, respectively) were determined in the exposed cross sections in-situ using specific antibodies and ECL densitometry as in conventional Western blots. Both Western blots and this technique demonstrated 3.1 fold EGAs increases in the proximal 2 cm of hair of diabetics as compared to non-diabetics. Dot-blocks, in addition, were less variable and demonstrated exponential EGAs decreases along fibers distally, with calculated intercepts (at the hair roots) of 4.9 fold increases in diabetics as opposed to non-diabetics and half-lives of 6.0, 5.9 and 9.0 months in hair of non-diabetics, gestational diabetics and diabetic patients, respectively. Correlations in amounts of BG vs. HbA1c, BG vs. EGAs, and HbA1c vs. EGAs, using dot-block and clinical lab data were all significant (p < 0.05). Acute onset T1D patients, defined as previously unsuspected patients diagnosed upon hospitalization due to diabetic complications, exhibited nearly identical EGAs levels in their proximal 0-9 cm hair as did T1D patients with long-established diabetes, thus supporting the notion of long and insidious T1D etiology. Removal of 1-2 μm layers from dot-block surfaces enabled their re-use for multiple assays. Applied anti-AGEs antibodies demonstrated slight decreases or no significant changes in CML and MGI along hair shafts of normal and diabetic subjects. Fluctuations in EGAs and AGEs along hair shafts, indicating alterations in glycemic control were also observed. We conclude that the dot-block method has a potential for early diagnosis and monitoring of diabetes, and more generally, as a long term "biological record" of various chronic medical conditions.
AB - We describe immunochemical assays of non-enzymatic glycation products in human head-hair protein extracts and hair cross sections using Western blots and a novel "dot-block" methodology. In the latter, groups of approximately 15 hair fibers, clipped at about 1 mm proximal to the scalp-skin were aligned, wound around, and attached to 3 mm diameter araldite screw rods. Up to 40 such rods were next embedded lengthwise in additional araldite polymer creating a solid block and the top surface of the block was sectioned off to the half-diameters of the screw rods thus exposing accurately transected hair cross sections at regular (∼ 0.5 cm) intervals. Early- and advanced-glycation products (EGAs and AGEs, respectively) were determined in the exposed cross sections in-situ using specific antibodies and ECL densitometry as in conventional Western blots. Both Western blots and this technique demonstrated 3.1 fold EGAs increases in the proximal 2 cm of hair of diabetics as compared to non-diabetics. Dot-blocks, in addition, were less variable and demonstrated exponential EGAs decreases along fibers distally, with calculated intercepts (at the hair roots) of 4.9 fold increases in diabetics as opposed to non-diabetics and half-lives of 6.0, 5.9 and 9.0 months in hair of non-diabetics, gestational diabetics and diabetic patients, respectively. Correlations in amounts of BG vs. HbA1c, BG vs. EGAs, and HbA1c vs. EGAs, using dot-block and clinical lab data were all significant (p < 0.05). Acute onset T1D patients, defined as previously unsuspected patients diagnosed upon hospitalization due to diabetic complications, exhibited nearly identical EGAs levels in their proximal 0-9 cm hair as did T1D patients with long-established diabetes, thus supporting the notion of long and insidious T1D etiology. Removal of 1-2 μm layers from dot-block surfaces enabled their re-use for multiple assays. Applied anti-AGEs antibodies demonstrated slight decreases or no significant changes in CML and MGI along hair shafts of normal and diabetic subjects. Fluctuations in EGAs and AGEs along hair shafts, indicating alterations in glycemic control were also observed. We conclude that the dot-block method has a potential for early diagnosis and monitoring of diabetes, and more generally, as a long term "biological record" of various chronic medical conditions.
KW - AGEs
KW - Diabetes monitoring
KW - EGAs
KW - Early diagnosis
KW - Hair surrogate tissue analysis
KW - Non-invasive immunodetection
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U2 - 10.1016/j.jim.2006.10.014
DO - 10.1016/j.jim.2006.10.014
M3 - Article
C2 - 17210162
AN - SCOPUS:33847416668
SN - 0022-1759
VL - 320
SP - 1
EP - 17
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -