alpco

Background

Adiponectin Background

Adiponectin circulates in several oligomeric isoforms with varying degrees of biological activity. Current research emphasizes a role for high-molecular weight (HMW) adiponectin, and the ratio between total and HMW adiponectin (annotated as HMWR or S_A) in studies examining the etiology of various metabolic disorders.

The HMWR has been shown to be a better predictor of insulin sensitivity than total adiponectin levels ^{(1, 2)}. Additionally, literature also shows that a high HMWR is related to a decreased risk for type 2 diabetes independent of total adiponectin ⁽³⁾, and that HMW adiponectin and the HMWR may be a better predictor of coronary atherosclerosis than total adiponectin ⁽⁴⁾. HMW adiponectin levels provide predictive value relative to the development of various metabolic syndrome cluster traits (including insulin resistance, glucose tolerance, lipoprotein distribution and the progression to a type II diabetic phenotype)⁽⁵⁾.

Because of the novel findings in regards to the importance of specifically characterizing levels of HMW in humans, many researchers are utilizing pre-clinical models to assess HMW levels in different metabolic phenotypes. The mouse has, and continues to be, a widely used model in the field of metabolic research. Until recently, researchers have relied on a number of tedious methods for separation of different adiponectin complexes, including partially denaturing, non-reducing gel electrophoresis, velocity sedimentation coupled to western blot and gel filtration chromatography analysis. Although these are viable methods for independent isoform quantification, there is room for improvement. Some of the reported issues with current methods are⁽⁶⁾.

• Susceptibility to slight changes in temperature and denaturing state
• Labor intensiveness
• Failure to resolve "bundled" complexes

As with our widely published human multimeric ELISA, ALPCO's new Mouse Total and HMW Adiponectin ELISA addresses these issues through utilization of a simple protease pretreatment step which selectively digests isomers that are not of interest. This pretreatment allows for specific, accurate measurement of both total and HMW adiponectin on the same plate.⁽⁷⁾

1. 1. Pajvani, et al. 2004. J Biol Chem 279 (13): 12152-62.
2. 2. Hara, et al. 2006. Diabetes Care 29: 1357-62.
3. 3. Heidemann, et al. 2008. Annals of Internal Medicine 149 (5): 307-316.
4. 4. von Eynatten, et al. 2008. European Heart Journal 29: 1307-1315.
5. 5. Lara-Castro, et al. 2006. Diabetes 55: 249-259.
6. 6. Schraw, et al. 2008. Endocrinology 149 (5): 2270-2282
7. 7. Ebinuma H, et al. Clin Chim Acta 2009; 401:181-3.

Method Comparison:

Young (<6 weeks at inclusion), male db/db mice were fed a normal chow diet and treated with rosiglitazone (n=7) or vehicle (n=8) for 29 days. Serum was collected and stored at -80 °C until analysis. Adiponectin complex distribution and HMWR was assessed in aliquots by three methods:

1. ALPCO/Sekisui Mouse HMW and Total Adiponectin ELISA
2. Gel filtration chromatography followed by Western Blot
3. Alternative commercially available ELISA for Total Adiponectin

Method Summary: ALPCO/Sekisui Mouse HMW and Total Adiponectin ELISA

Aliquots from each sample were assayed in duplicate according to the manufacturer's instructions to determine both HMW and total adiponectin. The performance of the assay met the manufacturer's specifications according to the characteristics included in the package insert. The HMWR was assessed by dividing the calculated concentration for the HMW fraction by the calculated concentration for total adiponectin and multiplying by 100.

Method Summary - Gel filtration chromatography/western blot

Gel filtration chromatography and Western Blot were conducted according to the method described by Schraw, et al⁽¹⁾. Briefly: Adiponectin complexes in the samples were separated by gel filtration chromatography and eluted. Fractions were subjected to SDS-PAGE to insure reduction of all adiponectin complexes into monomeric form. Western blot analysis was performed using rabbit polyclonal anti-adiponectin antibodies followed by goat anti-rabbit secondary antibodies coupled to IRDye 800.

The intensity of the fluorescence corresponding to the band in the 30 kDa position was quantified by densitometry. Each eluent fraction was plotted on an x-y graph as follows: the relative intensity of each eluent fraction was plotted on the y-axis, while the retention volume of that fraction was plotted on the x-axis. The resulting densitometric graph (Figure 2) depicts the adiponectin complexes as they eluted from the column and each peak (HMW, LMW, trimer) represents the relative amount of adiponectin in each fraction.

The area under the curve (AUC) for each peak was determined, as was the total AUC for all fractions. The percent HMWR adiponectin is calculated by dividing the area associated with the HMW peak by the total AUC and multiplying by 100. The same method is used to establish LMW and trimeric ratios.

Method Summary: Alternative Commercially Available ELISA for Total Adiponectin

Concentrations for total adiponectin were assigned to the gel filtration/Western blot data based on values generated using an alternative commercial ELISA (Millipore, St. Charles, MO). Relative concentrations for each of the complexed forms (HMW, MMW and trimeric) were extrapolated from the total concentration in the ELISA based on their respective peak's AUC.

Results:

The values for HMW, total adiponectin, and HMWR, were compared across methods.

The values calculated using the ALPCO/Sekisui ELISA correlated with the values generated using the Milipore ELISA for total adiponectin (r² = 0.90).

The HMW adiponectin values calculated using the ALPCO/Sekisui ELISA correlated with the values extrapolated from the gel filtration/western blot method (r² = 0.97).

The slope differences for both total and HMW adiponectin can most likely be attributed to a lack of standardization between methods, which is inherent in the industry.

The HMWR generated by both methods were also compared (Figure 3), and although the correlation was less robust (r² = 0.8), the differences between the rosiglitazone-treated and vehicle treated groups was significant whether measured by the ALPCO/Sekisui ELISA kit (p=0.001) or the Western Blot method (p=0.00004).

Conclusion:

The ALPCO/Sekisui ELISA for mouse HMW and total adiponectin ELISA accurately measures complex distribution of adiponectin in mouse samples, demonstrating significant differences between treatment groups:

1. 1. Schraw, et al. 2008. Endocrinology 149 (5): 2270-2282