Problem Louis C

Louis was born in 1967, at term, after an uneventful pregnancy. He was a sickly infant and did not grow well. On a number of occasions his mother noted that he appeared drowsy, or even comatose, and said that there was a 'chemical, alcohol-like' smell on his breath and in his urine. The GP suspected diabetes mellitus, and sent him to the Middlesex Hospital for a glucose tolerance test, which showed clearly that he was diabetic (see Figure 10.14).

Blood samples were also taken for measurement of insulin at zero time and 1 hour after the glucose load. At this time a new method of measuring insulin was being developed, radioimmunoassay, and therefore both this and the conventional biological assay were used. The biological method of measuring insulin is to determine the uptake and metabolism of glucose in rat muscle in vitro. This can be performed relatively simply by measuring the radioactivity in the 14CO2 produced after incubating samples of rat muscle with [14C]glucose in the presence and absence of the patient's blood. The then newly developed method of measuring insulin involves measuring the ability of insulin to bind to anti-insulin antibodies, in competition with radioactively labelled insulin — this is radioimmunoassay, and it is generally preferred because it is possible to assay a large number of blood samples at the same time. The antibody recognizes, and binds to, the surface of the tertiary structure of the protein. The results are shown in Table 10.1.

As a part of their studies of the new radioimmunoassay for insulin, the team at the Middlesex Hospital performed gel exclusion chromatography of a pooled sample of normal serum and determined insulin in the fractions eluted from the columns both by radioimmunoassay (graph A in Figure 10.16) and by stimulation of glucose oxidation (graph B). Gel exclusion chromatography separates compounds by their molecular mass, so that larger molecules flow through the column faster, and are eluted earlier, than smaller molecules. Three molecular mass markers were used; they eluted as follows: 9000 in fraction 10, 6000 in fraction 23 and 4500 in fraction 27.

The investigators also measured insulin in the fractions eluted from the chromatography column after treatment of each fraction with trypsin. The results are shown in graph C.

After seeing the results of these studies, they subjected the same pooled serum to brief treatment with trypsin, and performed gel exclusion chromatography on the resulting sample. Again they measured insulin by radioimmunoassay (graph D) and biological assay (graph E).

What conclusions can you draw from these results?

More recently, the gene for human insulin has been cloned. Although insulin consists of two peptide chains, 21 and 30 amino acids long, these are coded for by a single gene, which has a total of 330 base pairs between the initiator and stop codons. As you would expect for a secreted protein, there is a signal sequence coding for 24 amino acids at the 5' end of the gene.

Table 10.1 Serum insulin by biological assay and radioimmunoassay

Fasting

One hour after glucose

Louis C

Control subjects

Louis C Control subjects

Biological assay

0.8

6 ± 2

5 40 ± 11

Radioimmunoassay

10

6 ± 2

biological assay

id Si

o"

biological assay radio-immunoassay

What does this information suggest about the processes that occur in the synthesis of insulin?

What is likely to be the underlying biochemical basis of Louis' problem?

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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