Beef insulin

Ultralente Insulin is an almost colorless suspension of a mixture of characteristic crystals, the maximum dimension of which is predominandy 10—40 p.m. It contains, for each 100 USP units of insulin, 0.20—0.25 mg of 2inc (of which 40—65% is in the supernatant Hquid), and not more than 0.70 mg of nitrogen. It also contains on a wt/vol basis 0.15—0.17% sodium acetate, 0.65—0.75% sodium chloride, and 0.09—0.11% methylparaben. The purified form is available as Ultralente Purified Beef Insulin. 

The bioavailability of the insulins is identical when given subcutaneously. The human insulins are slightly less antigenic than pork or beef insulins. Human insulin is the insulin of choice for patients with insulin allergy, insulin resistance, all pregnant patients with diabetes, and any patient who uses insulin intermittently. ... 

Insulin allergy occurs in as many as 3% of patients receiving pork or beef insulin but smaller proportion in those using human insulin. However, the immuno-genicity of insulin is determined more by the purity of its preparations and since the use of monocomponent insulin, insulin allergy has become extremely rare. 

Insulin Preparations. Since diabetes mellitus is a defect of one or more of insulin production, secretion, or action, the administration of insulin replacement as a treatment for diabetes in the 1920s was a landmark discovery. Historically, most commercial insulin came from either bovine or porcine sources. Beef insulin differs from human insulin by three amino acid substitutions pork insulin differs by only one residue. For many years, standard insulin preparations were 70% beef and 30% porcine. However, the biosynthesis of human insulin has now displaced the animal insulins, especially bovine insulin which was more antigenic. Mass production of human insulin by recombinant DNA methods is achieved by inserting the human proinsulin gene into either E. coli or yeast and treating the resulting proinsulin to yield the human insulin molecule. Insulin preparations may be divided into four major types ... 

Insulin is composed of 51 amino acids arranged in two polypeptide chains, designated A and B, which are linked together by two di- sulfide bridges (Figure 23.3A). The insulin molecule also contains an j intramolecular disulfide bridge between amino acid residues of the A chain. Beef insulin differs from human insulin at three amino add positions, whereas pork insulin varies at only one position. 

Figure 25-8 Amino-acid sequence in beef insulin. The A chain of 21 amino-acid residues is linked to the B chain of 30 residues by way of two disulfide bridges.

Historically, commercial insulin in the USA contained beef or pork insulin. Beef insulin differs by three amino acids from human insulin, whereas only a single amino acid distinguishes pork and human insulins (Figure 41-1). The beef hormone is slightly more antigenic than pork insulin in humans. Of the insulins manufactured from animal sources, only purified pork insulin is still available and it requires special ordering. 

The structure of beef insulin was determined by Sanger (see Sec. 36.9) on the basis of the following information. Work out for yourself the sequence of amino acid residues in the protein. 

Beef insulin appears to have a molecular weight of about 6000 and to consist of two polypeptide chains linked by disulfide bridges of cystine residues. The chains can be separated by oxidation, which changes any CyS—SCy or CySH residues to sulfonic acids (CySO H). 

Both pork and human insulin are definitively less immunogenic than beef insulin, producing fewer circulating insulin antibodies, but several studies have indicated no detectable change in antibody concentrations on switching from pork to human insulin or vice versa. Antibodies cause lipoatrophy and are responsible for the substantial insulin resistance seen in some patients, but both events are rare now that purified pork insulin is in common use. Interest has recently been revived in the possible contribution of antibodies in modifying metabolic control. In the short term and under hospital conditions, they are known to prolong the intravenous half-life of injected insulin and to delay the appearance in the circulation of a subcutaneously administered bolus dose. 

The dose of pork insulin for patients with insulin resistance caused by antibodies to beef insulin may be only a fraction of that of beef insulin. Insulin resistance is frequently self-limited after several weeks or months of high dosage, responsiveness may be regained and dosage can be reduced. Insulin resistance is a common feature of insulinoma and can be shown even... 

Historically, insulin came from either beef or pork sources. Beef insulin differs by three amino acids and pork by one amino acid when compared to human insulin. Manufacturers in the United States have discontinued production of beef and pork source insulins as of December 2003, and now exclusively use recombinant DNA technology to manufacture insulin. Eli Lilly and Aventis currently use... 

Fig. 6.13. The primary structure of human insuhn. The substituted amino acids in bovine (beef) and porcine (pork) insulin are shown in blue. Threonine 30 at the carboxy terminal of the B chain is replaced by alanine in both beef and pork insulin. In beef insulin, threonine 8 on the A chain is also replaced with alanine, and isoleucine 10 with vahne. The cysteine residues, which form the disulfide bonds holding the chains together, are invariant. In the bioengineered insulin Humalog (hspro insulin), the position of proline at B28 and lysine at B29 is switched. Insulin is synthesized as a longer precursor molecule, proinsulin, which is one polpeptide chain. Proinsulin is converted to insulin by proteolytic cleavage of certain peptide bonds (squiggly lines in the figure). The cleavage removes a few amino acids and the 31-amino acid C-peptide that connects the A and B chains. The active insulin molecule, thus, has two nonidentical chains.

Di Abietes. Di Abietes treatment was first changed to daily injections j of Humulin instead of beef insulin. Humulin is now mass-produced by recombinant DNA techniques that insert the human DNA sequences for the insulin A and B chains into the Escherichia coli or yeast genome (see Chapter 17). The insulin chains that are produced are then extracted from the media and treated to form the appropriate disulfide bonds between the chains. As costs have fallen for production of the synthetic human insulins, they have replaced pork insulin and the highly antigenic beef insulin. 

How many disulfide linkages are there in each molecule of beef insulin (Figure 20.3)... 

There are three disulfide linkages in beef insulin 10. Guanine and c)Tosine are mutually bonded to each other, as are adenine and thymine. 

Primary structure of beef insulin. The A chain is shown in blue, and the B chain, whose structure determination is described in the text, is shown in pink with black letters. 

Insulin antibodies have been separated by paper electrophoresis from the blood of insulin-treated patients. These antibodies act on insulin labeled with iodine 131, and a cross reaction occurs between the antibodies and insulin obtained from humans, pork, and beef. Insulin antibodies appear in the blood after bovine or foreign insulin administration. The amount of antibody is proportional to the amount of insulin injected. The antibody insulinic complex has little insulinlike activity. Insulin antibody develops in those patients who receive large doses of insulin, and it is therefore impossible to judge whether the insulin resistance results from antibody formation or from the appearance of agents with autoimmune activity. 

The graph in Fig. 2 can be used to address the most fundamental question regarding insulin iontophoresis—is it theoretically possible, under the most favorable conditions, to deliver the required dose Based on the discussion in Section 2.1, the needs are a basal delivery rate of 1-2 units per hour coupled with a bolus of up to 20 units over about a half-hour. Figure 2 indicates that a delivery rate of 40 units per hour could be achieved with a 1 mM solution of insulin, which is equivalent to about 4mg/ml or 100 units/ml. Regular (currently marketed human, pork, or beef) insulin has a water solubility that exceeds this value. Thus, it is theoretically possible to iontophorese insulin at the required rate. However, an idealized model has been used to reach this conclusion. Specifically, it was assumed that insulin exists as an ideal solution (with a MW of 5800), that the mobility of insulin is independent of pH and has a value close to its maximum value, and that the molecule is not degraded on its way through the skin. For regular insulins, these assumptions are not true. In the next section, the physicochemical properties of insulin that impact its deliverability by iontophoresis are described. 

The reaction between insulin and antibody has also been utilized for the bioassay of insulin in body fluids. One unit of nonlabeled crystallin beef insulin was administered to each of 2 normal rabbits, and plasma samples were taken at intervals thereafter. These were assayed for beef insulin concentration by incubation with a minute quantity of labeled beef insulin and with human antisera for which 5[ound]/F[ree] v. 5[ound] relationships had been established with insulin solutions of known concentration. From the B/F ratios observed under these conditions, the concentrations of nonlabeled beef insulin in the rabbit plasmas were determined. ... 

This is sometimes visible in the electron microscope, as is the case for haemocyanin (Fig. 11). In other cases, measurement of physical constants permits the dimensions of the protein molecule to be calculated approximately. Thus, beef insulin (M.W. = 12 x 10 ) is a right prism 44 A long, 26 A wide and 20 A thic whilst tobacco mosaic virus (M.W. = 4 X 10 ) is a rod 2980 A long by 150 A in diameter. 

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