Journal of Hepatology
Volume 36, Issue 1 , Pages 123-125, January 2002

The liver is the main site for metabolism of circulating advanced glycation end products

Department of Biochemistry, Kumamoto University School of Medicine, Honjo, 2-2-1, Kumamoto 860, Japan

See Article, pages 66–71

Article Outline

 

The Maillard reaction is one of post-translational modifications of proteins in which glucose and other reducing sugars, such as ribose and fructose, react with amino residues of proteins to form Schiff base and Amadori products (the most well-known Amadori product in vivo is hemoglobin AIc) [1]. Further incubation converts these early products into irreversible derivatives termed advanced glycation end products (AGEs). AGEs are characterized physicochemically by fluorescence, brown color and cross-linking, and biologically by specific recognition by AGE receptors.

Several AGE structures have been identified such as Nε-(carboxymethyl)lysine (CML) [2], pyrraline [3], pentosidine [4], and imidazolone [5]. Among these products, several in vitro experiments demonstrated that CML is the major AGE structure in vivo [6], [7]. Previous immunological studies using anti-AGE antibodies have demonstrated that in vivo accumulation of AGEs increases with normal aging [8] as well as in several age-enhanced diseases such as diabetic nephropathy [9], atherosclerosis [10], diabetic retinopathy [11], hemodialysis-associated amyloidosis [12], chronic renal failure [13] and Alzheimer's disease [14], [15]. Human plasma proteins are also reported to undergo AGE modification [16], [17]. Therefore, accurate measurement of tissue and plasma AGEs levels is crucial to our understanding of the pathophysiological role of these products in vivo.

Previous studies concluded, but not confirmed, that plasma AGEs are handled mainly by the kidneys because (i) plasma AGE levels increase in patients with chronic renal failure [19] and (ii) because AGE peptides, intermediate degradation products of AGE proteins, are found in urine [20]. In this issue of the Journal, however, Sebekova et al. [18] introduce a new concept regarding the catabolism of AGEs in vivo. They determined plasma CML levels in 51 patients with liver cirrhosis (five of them were followed 36 months after liver transplantation) and 19 healthy controls and clearly showed that the liver plays an important role in the removal of plasma AGE from the circulation. Their major findings were threefold: (i) plasma AGE levels were markedly elevated in patients with liver cirrhosis and correlated with the severity of the disease; (ii) serum CML levels in their patients correlated inversely with the residual liver function represented by serum albumin, plasma bilirubin, and liver enzyme activity (MEGX); and (iii) elevated plasma CML levels in these patients decreased markedly (to about 50% levels) within 3 months after liver transplantation. These data can also be interpreted to mean that plasma AGE levels could be used as a clinical marker for liver function, like serum albumin and plasma bilirubin. This is also a new concept highlighted by the above study because it is generally assumed by physicians and researchers involved in AGE research that plasma AGE levels, or practically plasma CML, could be potentially used as a clinical marker for age-related diseases such as diabetic complications and atherosclerosis.

Three questions arise from the study of Sebekova et al. [18]: (i) what are the mechanism(s) involved in hepatic removal of plasma AGE, (ii) what is the ligand for hepatic AGE receptor, and (iii) what is the most suitable assay system(s) for determination of plasma AGE or CML levels? The main basic question is the mechanism involved in hepatic removal of plasma AGE. The important role of the liver in AGE metabolism has already been emphasized [21], [22], [23]. After intravenous administration of in vitro-prepared AGE-modified bovine serum albumin (AGE-BSA) into rats, more than 90% is eliminated by sinusoidal liver cells [23]. The bulk of the hepatic uptake of AGE-BSA (60–65%) is ascribed to hepatic liver endothelial cells (LECs, 60–65%) and to Kupffer cells (24–28%), whereas the contribution of hepatocytes is low (less than 20%) [23]. In vitro studies using LECs and Kupffer cells isolated after collagenase perfusion demonstrated that AGE-BSA undergoes a receptor-mediated endocytosis by these cells [22], [23]. The receptors that recognize AGE proteins as ligands are collectively categorized as the AGE receptors. The AGE receptors so far reported include a complex of OST-48/80K-H/galectin-3 [24], [25], a receptor for AGE (RAGE) [26], [27] and SR-A (class A scavenger receptor types I and II) [28], [29]. Recent studies further showed that CD36 and SR-BI, members of class B scavenger receptor family, also serve as AGE receptors [30], [31]; the former is known as a receptor for oxidized low-density lipoprotein (Ox-LDL) [32] and the latter is known as the HDL receptor, mediating selective uptake of high-density lipoprotein (HDL) cholesterol by the liver in the reverse cholesterol transport system [33]. Among these AGE receptors, hepatic expression was reported with OST-48/80K-H/galectin-3 [24], SR-A [29] and SR-BI [33].

A recent report by Matsumoto et al. [34] characterized an interesting aspect of the hepatic AGE receptor. Peritoneal macrophages obtained from SR-A-knockout mice showed a lower endocytic activity for AGE ligands (less than 30–40%) as well as for Ox-LDL (less than 50%), compared with those obtained from wild-type mice. However, LECs isolated from SR-A-knockout mice showed effective endocytic activities for AGE ligands as well as Ox-LDL, which were indistinguishable from the endocytic activities of LECs isolated from wild-type mice [34]. Other studies also showed that the plasma clearance rates of AGE-BSA and Ox-LDL in SR-A-knockout mice were not significantly different from those in wild-type mice [35], [36]. Results of these studies strongly suggest that the hepatic receptor responsible for endocytic uptake of plasma AGE proteins from the blood stream (the hepatic AGE receptor) is distinct from SR-A, but its ligand specificity is closely similar to SR-A since the hepatic AGE receptor seems to recognize both AGE ligand and Ox-LDL as effective ligands. Further studies are needed to determine whether other AGE receptors such as OST-48/80K-H/galectin-3 and SR-BI could also serve as a hepatic AGE receptor, or whether the hepatic uptake of AGEs could be mediated by a novel but not yet identified AGE receptor. If such a receptor is identified, knockout mice exhibiting a high plasma AGE level without liver dysfunction should become available.

The second question is whether plasma CML proteins (probably represented by CML albumin) act as a ligand for the hepatic AGE receptor. To date, it is still not clear whether the interaction of CML proteins with known AGE receptors is specific or not. CML proteins are recognized by RAGE [37], whereas CML-BSA [38] and CML-modified LDL [39] do not show specific binding to SR-A. In our preliminary collaborative study with Smedsrod and his colleagues, the plasma clearance of CML-BSA (in which 14 out of 59 total epsilon amino acids of BSA were converted to CML) in mice and rats did not differ from that of unmodified BSA, suggesting that CML alone does not act as a crucial signal in the hepatic removal system of CML proteins. Rather, the structure formed by CML plus a cluster of negative charges induced by CML modification of epsilon amino acids of lysine residues might be important.

The final question arising from the study of Sebekova et al. [18] concerns the available assay system(s) for determining plasma AGE or CML concentration. Among three available methods, analysis of fluorescent AGEs in plasma samples [40] seems to be no longer valid because (i) the fluorescent properties of AGEs (λex/em=370/440 nm) do not specify their chemical structures, and (ii) fluorescent quenching associated with biological samples prevent accurate measurements. The most accurate method to determine plasma CML is high-performance liquid chromatography analysis after plasma samples are subjected to acid hydrolysis (CML is generated from CML proteins by acid hydrolysis). Since this method is reliable but time-consuming and trained hands are always required, the competitive enzyme-linked immunosorbent assay (ELISA) using a specific antibody against CML is clinically useful. In the study by Sebekova et al. [18], plasma CML levels were measured after proteinase K digestion by competitive ELISA developed by Roche Diagnostics (Penzberg, Germany) using the anti-CML monoclonal antibody 4G9 (Alteon Inc., New York, NY) [41], [42]. One of the critical determinants of ELISA assay for plasma CML level is the specificity of the monoclonal antibody employed in the assay. At present, only a few studies have been reported in which plasma AGE levels were measured by Roche Diagnostics’ ELISA method. The use of this particular ELISA method in many laboratories in the future will not only allow determination of its sensitivity as well as reproducibility for measurement of plasma CML levels, but also whether plasma CML levels are of any clinical diagnostic value.

In summary, the clinical study by Sebekova et al. [18] using normoglycemic patients with liver cirrhosis and liver transplantation has provided the first clinical evidence that liver is the main catabolic site for circulating AGEs, supporting the general notion that accumulation of AGEs is of pathophysiological significance.

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PII: S0168-8278(01)00293-8

Journal of Hepatology
Volume 36, Issue 1 , Pages 123-125, January 2002

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