Measurement of protein degradation is much more limited in terms of the methods available. To measure protein degradation, the protein must be prelabeled. Three methods have been used: (a) removal of the protein from the body, followed by iodination with radioactive iodine, and reinjection into the body to follow the disappearance of the labeled protein; (b) administration of a labeled amino acid to label proteins via incorporation of the tracer during protein synthesis, followed by measurement of labeled amino acid release from degradation of the protein; and (c) use of posttranslational amino acids such as 3-methylhistidine.
The use of iodination limits this methodology to readily removable and reinjectable proteins (i.e., proteins in plasma). Therefore, the applications of this method are limited but it has found use in lipoprotein metabolism (109, 110 and 111). The method is not without problems: proteins that are iodinated do not have the same structure after removal and iodination as they had before removal from the body, and the iodination process may cause untoward effects. However, properly applied, the method can be very specific for measuring the kinetics of select proteins.
Alternatively, proteins may be labeled by long infusions of amino acid tracer. After the tracer infusion is stopped, the tracer enrichment disappears quickly from plasma. At that point, serial sampling of the protein and measuring the decrease in tracer enrichment with time will give its degradation rate. However, another problem occurs: 80% or more of the amino acids released from protein breakdown are reused for synthesis of new proteins. Therefore the amino acid tracer from protein degradation is recycled into new proteins. Because there is generally not a large starting enrichment in the proteins being measured, recycling of low enrichments of tracer greatly complicates interpretation of the labeled protein data obtained by this method.
3-Methylhistidine and Other Posttranslational Amino Acids. In the body, a number of enzymes can modify the structure of proteins after they have been synthesized. The changes are generally modest, occur to specific amino acids, and are either the addition of a hydroxyl group (e.g., conversion of proline to hydroxyproline in collagen ) or methylation of N moieties of amino acid residues such as histidine or lysine. Because t-RNAs do not code for these hydroxylated or methylated amino acids, they are not reused for protein synthesis once the protein containing them is degraded. Of posttranslationally modified amino acids, 3-methylhistidine has found the most extensive application: the measurement of muscle myofibrillar protein breakdown ( 32, 113).
Because of the quantitative importance of muscle to whole-body protein metabolism, measurement of the release of 3-methylhistidine is an important tool for following breakdown of myosin and actin, which are both primary proteins in skeletal muscle and the primary proteins containing 3-methylhistidine ( Fig . 2.15). Analyses of rat carcasses demonstrated that muscle accounts for three-quarters of the 3-methylhistidine pool in body proteins ( 32), and administered 14C-3-methylhistidine has been shown to be quantitatively recovered in the urine of rats (114) and humans (115). There are caveats, however, to the use of 3-methylhistidine excretion for measurement of myofibrillar protein breakdown. Dietary meat will distort urinary 3-methylhistidine collection (116). As much as 5% of the 3-methylhistidine released in the urine may be acetylated in the liver first (a pathway that is much more predominant in the rat), and urinary samples may have to be hydrolyzed before measurement of 3-methylhistidine. Conversion of 3-methylhistidine to balenine (the dipeptide 3-methylhistidine-b-alanine) is of less importance in humans than in other species (117).
Figure 2.15. Schematic depiction of the formation and disposal of 3-methylhistidine in myofibrillar protein in humans. Because 3-methylhistidine is not reused for protein synthesis or oxidized/metabolized, its release into blood represents degradation of myofibrillar protein from tissues containing myosin and actin (muscle, smooth muscle such as gut, skin). In man, 3-methylhistidine is quantitatively excreted into urine; in rat, 3-methylhistidine is also acetylated in the liver before excretion in urine. Dietary intake of meat adds another source of 3-methylhistidine. (Figure drawn from previous descriptions of the metabolism of 3-methylhistidine ( 32) )
Myofibrillar protein and 3-methylhistidine are not specific to skeletal muscle, which means that urinary 3-methylhistidine measurement may not be specific to skeletal muscle protein breakdown (118, 119). The primary argument has been that even though skin and gut may have a small pool of myofibrillar protein compared to the large mass of protein found in skeletal muscle, skin and gut protein turn over rapidly by comparison and, therefore, continually contribute a significant amount of 3-methylhistidine to the urine. More-recent work suggests that skin and gut contributions, while noticeable, can be accommodated in the calculation of human skeletal muscle turnover from urinary 3-methylhistidine excretion (113, 117., 1.20.).
A more specific approach to 3-methylhistidine measurement of skeletal muscle myofibrillar protein breakdown measures release of 3-methylhistidine from skeletal muscle via A-V blood measurements across a muscle bed, such as leg or arm (121). This measurement of protein breakdown from the 3-methylhistidine A-V difference can be combined with the A-V difference measurement of an essential amino acid that is not metabolized in muscle, such as tyrosine. In contrast to 3-methylhistidine, tyrosine released by protein breakdown is reused for protein synthesis. The A-V difference of tyrosine across an arm or leg defines net protein balance (i.e., the difference between protein breakdown and synthesis). Protein synthesis is, therefore, the difference of the 3-methylhistidine and tyrosine A-V difference measurements (56, 1,2,2). The results by this technique should be similar to those obtained by the tracer method of Barrett et al. ( 59).
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