The Relation of Insulin

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns. 

In keeping with the variability which we have noted in human beings, there is in animals wide interspecies and intraspecies variability in relation to insulin. When the supply of insulin was cut off by removal of the pancreas, the blood sugar levels listed in Table 14 were found in different animals of different species. 17... 

Many chemicals important to genetic research have been produced at high levels, e.g. DNA ligase production in E. coli can be enhanced several hundred fold. Other enzymes can also be produced for industrial use and enhanced degradative ability has been generated for instance, enhanced petroleum degraders can be added to oil spills to accelerate clean-up operations. However, it is in the production of medically related compounds that this technology has been most successfully applied as, for example, in the production of insulin. 

The effects of insulin are modified by various factors. The speed and extent of absorption of insulin depends, for example, on the site of injection (1), the depth of the subcutaneous injection, skin temperature (2), the presence of lipodystrophy, and variation in the extent of inactivation of injected insulin. The disposal of insulin depends on many factors. Exercise and hard work lower the blood glucose and thereby increase the effect of insulin. Infections and obesity reduce its effect. The timing of food intake and the composition of meals are also related to the action of insulin. A thin layer of fat, as sometimes occurs in the upper arm or in the thighs of thin men, can result in intramuscular injection, leading to faster absorption of long-acting insulins. This can reduce the absorption time by half (3). The major factors that affect the fate of injected insulin (and thereby also its risks) are listed in Table 1 (4). 

Disposable syringes can release silicon particles into the insulin vials, reducing the effectiveness of insulin (203). This can happen when insulin is injected back into the vial, during correction for the desired dose, and is specifically seen when low doses are used for long periods. Flocculation of insulin, found before the expiry date, may be related to this problem (SEDA-12, 360). 

In healthy volunteers, there was a dose-related increase in blood insulin concentrations after the administration of insulin detemir (2). However, there was no clear dose-response metabolic effect and individual variability was high. 

Rett, K. 1999. The relation between insulin resistance and cardiovascular complications of the insulin resistance syndrome. Diabetes Obes Metab l(Suppl 1) S8-S16. 

The introduction of "fast HPLC" has proven to be particularly valuable in protein analysis. As stated earlier, assay time in RP-HPLC analysis of proteins is typically long compared to that for smaller organic molecules. We have evaluated the use of 0.6-cm ID x 4-cm columns packed with 3-um particles in the analysis of insulin by RP-HPLC for potency determination, related substances, and in peptide mapping (7). The use of the "fast column" allows considerable savings (40-60%) in analysis time, compared to the regular (0.46-cm ID x 25-cm) columns, without loss in resolving power. 

Another main change is in the insulin sensitivity at different tissues. For example, the effect of insulin on glucose uptake is decreased. Insulin has the same inhibitory effect on lipolysis and on hepatic glucose output, and inside the cells the removal of glucose-6-phosphate is decreased. The different changes in the metabolic traffic result in a spectrum of possible patient variants. Common for them all is an increased plasma glucose concentration and increased meal-related variations. 

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