2-aminoadipic acid is a marker of protein carbonyl oxidation in the aging human skin: effects of diabetes, renal failure and sepsis

Abstract

We hypothesized that the epsilon-amino group of lysine residues in longlived proteins oxidatively deaminates with age forming the carbonyl compound, allysine (alpha-aminoadipic acid-delta-semialdehyde), which can further oxidize into 2-aminoadipic acid. In the present study, we measured both products in insoluble human skin collagen from n=117 individuals of age range 10-90 years, of which n=61 and n=56 were non-diabetic and diabetic respectively, and a total of n=61 individuals had either acute or chronic renal failure. Allysine was reduced by borohydride into 6-hydroxynorleucine and both products were measured in acid hydrolysates by selective ion monitoring gas chromatography (GC)-MS. The results showed that 2-aminoadipic acid (P<0.0001), but not 6-hydroxynorleucine (P=0.14), significantly increased with age reaching levels of 1 and 0.3 mmol/mol lysine at late age respectively. Diabetes in the absence of renal failure significantly (P<0.0001) increased 2-aminoadipic acid up to <3 mmol/mol, but not 6-hydroxynorleucine (levels<0.4 mmol/mol, P=0.18). Renal failure even in the absence of diabetes markedly increased levels reaching up to <0.5 and 8 mmol/mol for 6-hydroxynorleucine and 2-aminoadipic acid respectively. Septicaemia significantly (P<0.0001) elevated 2-aminoadipic acid in non-diabetic, but not diabetic individuals, and mildly correlated with other glycoxidation markers, carboxymethyl-lysine and the methylglyoxal-derived products, carboxyethyl-lysine, argpyrimidine and MODIC (methylglyoxal-derived imidazolium cross-link). These results provide support for the presence of metal-catalysed oxidation (the Suyama pathway) in diabetes and the possible activation of myeloperoxidase during sepsis. We conclude that 2-aminoadipic acid is a more reliable marker for protein oxidation than its precursor, allysine. Its mechanism of formation in each of these conditions needs to be elucidated.

Scheme 1. Oxidation of lysine residues in skin collagen

(A) Formation of 6-hydroxynorleucine and 2-aminoadipic acid as markers for protein oxidation. Insert: fragmentation patterns for trifluoroacetyl methyl ester derivatives of 6-hydroxynorleucine and 2-aminoadipic acid used for quantification by selected ion monitoring GC-MS. (B) Proposed mechanisms for deamination of lysine residues in proteins forming allysine which converts into 2-aminoadipic acid by oxidation: TPQ, 2,4,5-trihydroxyphenylalanine quinone; LTQ, lysine tyrosyl quinone.

Figure 1. Plots of 6-hydroxynorleucine and 2-aminoadipic acid versus chronological age in non-diabetic individuals

Levels determined in relationship to sepsis and acute and chronic renal failure in human insoluble skin collagen. For each graph, regression line and 95% confidence intervals of prediction are shown. Regression line equations where x=age and y=parameter: (A) and (C): 6-hydroxynorleucine, y=5.7×10⁻⁴x+0.124, n=16, r=0.23, P=0.39 (NS); (B) and (D): 2-aminoadipic acid, y=3.1×10⁻⁵x²+0.006x+0.246, n=16, r=0.86, P<0.0001. ○, non-diabetic; ●, non-diabetic+sepsis; ◇, non-diabetic+acute renal failure; ■, non-diabetic+acute renal failure+sepsis; ◇, non-diabetic+chronic renal failure; ◆, non-diabetic+chronic renal failure+sepsis.

Figure 2. Plots of 6-hydroxynorleucine and 2-aminoadipic acid versus chronological age in diabetic individuals

For details see the legend for Figure 1. △, diabetic; ▲, diabetic+sepsis; ▽, diabetic+acute renal failure; ▼, diabetic+acute renal failure+sepsis; , diabetic+chronic renal failure; , diabetic+chronic renal failure+sepsis).

Figure 3. Levels of 2-aminoadipic acid age-adjusted to 50 years and categorized as to the presence of diabetes, sepsis, and acute and chronic renal failure

*The age-adjusted level for each individual is indicated by a black dot. †Three outliers (ages 32, 42 and 82 years) as indicated by crosshair symbols have not been included in the statistical analysis. ‡Statistical probability (P) by the Mann–Whitney Test of a significant difference in the comparison of Gp x versus Gp 1 where x=Gp 2–12. Gp, group.