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Technical Briefs |
1
Hôp. Robert Debré, Lab. de Biochim.-Hormonol., 48 blvd. Sérurier, 75019-Paris, France;
2
Hôp. Saint-Joseph, Lab. de Biochim., 185 rue Raymond Losserand, 75674-Paris Cédex 14, France;
a author for correspondence: fax (33) 1 40 03 47 90
Insulin-degrading enzyme (IDE; EC 3.4.99.45) was first described
40 years ago (1). It is widely distributed in various
tissues, including red blood cells (RBC) (2)(3)(4). IDE may
not play a key role in insulin metabolism, and fundamental questions on
the biological role of IDE remain (2)(3).
Recently, IDE was characterized as a peroxisomal protease
(3). The specificity of IDE is selective: Only insulin and
transforming growth factor-
(Km 
0.1
µmol/L) are good substrates; insulin-like growth factor 1 and
proinsulin are poor substrates (2)(3)(4). On denaturing
polyacrylamide gels, IDE appears as a single polypeptide of 110 kDa
(4), but in nonreducing conditions, IDE has an
Mr of 300 000, suggesting that the enzyme
exists in polymer form (4). The cleavage sites indicate
that IDE recognizes the tertiary structure rather than a particular
amino acid sequence (2). Inhibitors of IDE include
p-hydroxymercuribenzoate (0.1 mmol/L),
p-chloromercuriphenylsulfonic acid (pcMPS, 0.1 mmol/L),
bacitracin (1 g/L), N-ethylmaleimide (1 mmol/L),
1,10-phenanthroline (1 mmol/L), EDTA (5 mmol/L), and diamide (5 mmol/L)
(4)(5)(6). The degradation of insulin by IDE is not inhibited
by lysosomal enzyme inhibitors like aprotinin (500 000 kU/L) or
leupeptin (0.1 g/L) (4)(6).
Although most insulin immunoassay kits indicate that hemolyzed samples should not be analyzed, few extensive studies have been done on the degree of insulin degradation by RBC IDE or how to prevent it (5)(6)(7)(8)(9). To our knowledge, the interference of hemolysis with insulin values has been studied with RIAs (5)(6)(7)(8)(9) but not with specific IRMAs involving monoclonal antibodies. In RIAs, the mechanism of the reduction in insulin concentrations involves IDE-mediated degradation of plasma insulin and I-labeled insulin (used as tracer). Hemolysis is partly dependent on the material used for venipuncture (10) and cannot always be eliminated. We therefore determined the precise influence of hemolysis on human insulin RIA results (Phadeseph Insulin, Pharmacia) using polyclonal antibodies, and those of IRMA (Bi-Insulin IRMA, Sanofi-Pasteur) using monoclonal antibodies without cross-reactivity with intact and des (31,32) proinsulins. We also investigated ways of overcoming the problem.
We studied the effects of hemolysis on insulin degradation by adding lysed RBCs to serum. After centrifugation and removal of serum and white cells by aspiration, RBCs were washed three times in saline and lysed by freezing. Red cell debris was removed by centrifugation and the supernatant was added to serum to obtain hemoglobin concentrations of 0.5, 1, 2, 4, and 6 g/L. Inhibition of IDE was studied either by first adding pcMPS (0.4 mmol/L), diamide (5 mmol/L), 1.10-phenanthroline (1 mmol/L), or EDTA (5 mmol/L) (all products from Sigma) to serum, followed by the RBC hemolysate, or, in another experiment, by maintaining a constant temperature of 4 °C for 1 h after the addition of the RBC hemolysate to serum without inhibitor. To reproduce the usual conditions of blood sampling (temperature, time between sampling and analysis or storage, and the usual degree of hemolysis), we incubated serum for 1 h at 20 °C and 37 °C with hemolyzed RBC.
All insulin samples are measured in duplicate. Mean basal insulin
concentration determined by IRMA was 53 mIU/L (range 17.3–101.2).
Results are expressed as percentage insulin recovery (Table 1
). Differences were analyzed with the nonparametric Wilcoxon
rank test with StatView 4.1 software (Abacus Concepts). In all analyses
P <0.05 was considered significant.
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Although our study was not performed under the same conditions of hemolysis, time, temperature, and assay, the hemolysis-induced insulin loss determined with IRMA was similar to previous results (5)(7)(8)(9). In particular, slight hemolysis (0.5 g/L) significantly reduced the observed insulin concentration at 20 °C, and massive hemolysis (6 g/L) degraded >90% of insulin after 1 h at 37 °C. Although percentage insulin recovery was lower in IRMA than in RIA, marked insulin loss was also observed with RIA. The difference between the two methods can be explained by the lack of specificity of polyclonal antibodies (which could cross-react with insulin fragments) and (or) degradation of I-labeled insulin used as a tracer in RIA.
Generally, IDE inhibitor activity has been determined in assays measuring insulin degradation by mixing purified IDE and I-labeled insulin at 37 °C, pH 7.4; the reaction is terminated by the addition of trichloroacetic acid (TCA), which precipitates nondegraded insulin (4). With IRMA, the ion chelators EDTA (5 mmol/L) and 1,10-phenanthroline (1 mmol/L) had no influence on insulin degradation (data not shown); in contrast, in the TCA assay they completely inhibit the insulin-degrading activity of IDE (4). As chelators inhibit the activity of IDE but not insulin binding to IDE, the insulin epitopes could be masked, thereby preventing the monoclonal antibodies from binding insulin (11). Conversely, pcMPS (and bacitracin) inhibit insulin binding to IDE, which may explain the difference in activity between the different inhibitors. Although the insulin loss was not completely prevented in the conditions of our study, pcMPS or diamide markedly reduced insulin degradation by IDE. The mean hemolysis-induced insulin loss was <10% when the plasma hemoglobin concentration was <4 g/L. In most situations this insulin loss has little impact on the clinical interpretation of the results.
Our study clearly showed the effect of temperature on IDE
activity (Table 1
). Although IDE activity was reduced by maintaining
hemolyzed samples at 4 °C, the inhibitory effect of low temperature
was less effective than pcMPS or diamide (Table 1
). Moreover,
maintaining a constant low temperature from blood sampling to
plasma/serum freezing is not easy. The effect of diamide on IRMA has
been studied by adding diamide (at a final concentration of 5 mmol/L)
in 32 nonhemolyzed serum (insulin concentration from 1.2 to 155 mIU/L).
Diamide shows no influence on insulin measured by the Bi-Insulin IRMA
kit (P = 0.46).
In summary, our study shows that even slight hemolysis degrades serum insulin immunoreactivity assayed by RIA and IRMA. Ion chelators like EDTA or 1,10-phenanthroline have no effect on insulin degradation; in contrast, when pcMPS (0.4 mmol/L) or diamide (5 mmol/L) are added first, the hemolysis-induced insulin loss is <5% with a serum hemoglobin concentration of 2 g/L and 10% at 4 g/L. Low temperature significantly reduces insulin losses but is less effective than diamide or pcMPS.
References
The following articles in journals at HighWire Press have cited this article:
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  C. B. Ebbeling, M. M. Leidig, H. A. Feldman, M. M. Lovesky, and D. S. Ludwig Effects of a Low-Glycemic Load vs Low-Fat Diet in Obese Young Adults: A Randomized Trial JAMA, May 16, 2007; 297(19): 2092 - 2102. [Abstract] [Full Text] [PDF]
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 R. Sapin, V. Le Galudec, F. Gasser, M. Pinget, and D. Grucker Elecsys Insulin Assay: Free Insulin Determination and the Absence of Cross-Reactivity with Insulin Lispro, Clin. Chem., March 1, 2001; 47(3): 602 - 605. [Full Text] [PDF]
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