Muscle protein synthesis by positron-emission tomography with l-[methyl-11C]methionine in adult humans

Existing methods for assessing protein synthetic rates (PSRs) in human skeletal muscle are invasive and do not readily provide information about individual muscle groups. Recent studies in canine skeletal muscle yielded PSRs similar to results of simultaneous stable isotope measurements using l-[1-13C, methyl-2H3]methionine, suggesting that positron-emission tomography (PET) with l-[methyl-11C]methionine could be used along with blood sampling and a kinetic model to provide a less invasive, regional assessment of PSR. We have extended and refined this method in an investigation with healthy volunteers studied in the postabsorptive state. They received ≈25 mCi of l-[methyl-11C]methionine with serial PET imaging of the thighs and arterial blood sampling for a period of 90 min. Tissue and metabolite-corrected arterial blood time activity curves were fitted to a three-compartment model. PSR (nmol methionine⋅min−1⋅g muscle tissue−1) was calculated from the fitted parameter values and the plasma methionine concentrations, assuming equal rates of protein synthesis and degradation. Pooled mean PSR for the anterior and posterior sites was 0.50 ± 0.040. When converted to a fractional synthesis rate for mixed proteins in muscle, assuming a protein-bound methionine content of muscle tissue, the value of 0.125 ± 0.01%⋅h−1 compares well with estimates from direct tracer incorporation studies, which generally range from ≈0.05 to 0.09%⋅h−1. We conclude that PET can be used to estimate skeletal muscle PSR in healthy human subjects and that it holds promise for future in vivo, noninvasive studies of the influences of physiological factors, pharmacological manipulations, and disease states on this important component of muscle protein turnover and balance.

Radioimmunotherapy with a 64Cu-labeled monoclonal antibody: a comparison with 67Cu.

67Cu (t1/2 = 62 h) has demonstrated potential as a radionuclide for radioimmunotherapy, but limited availability severely restricts its widespread use. 64Cu (t1/2 = 12.8 h) has been shown to have comparable effectiveness in vitro and in vivo. The present study was undertaken to examine the therapeutic potential of 64Cu- and 67Cu-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradeca ne-N, N',N",N"'-tetraacetic acid (BAT)-2-iminothiolane (2IT)-1A3 (1A3 is a mouse anti-human colorectal cancer mAb) for treatment of GW39 human colon carcinoma carried in hamster thighs. Hamsters were injected with 64Cu- or 67Cu-BAT-2IT-1A3 or Cu-labeled nonspecific IgG (MOPC) or saline. Hamsters were killed 6-7 months after therapy or when tumors were > or = 10 g. Of the hamsters with small tumors (mean weight 0.43 +/- 0.25 g), 87.5% were disease-free 7 months after treatment with 2 mCi (1 Ci = 37 GBq) of 64Cu-BAT-2IT-1A3 or 0.4 MCi of 67Cu-BAT-2IT-1A3. The mean tumor doses at these activities of 64Cu- and 67Cu-BAT-2IT-1A3 were 586 and 1269 rad (1 rad = 0.01 Gy), respectively. In contrast, 76% of hamsters treated with 2 mCi of 64Cu-BAT-2IT-MOPC or 0.4 mCi of 67Cu-BAT-2IT-MOPC had to be killed before 6 months because of tumor regrowth. When hamsters with larger tumors (mean weight 0.66 +/- 0.11 g) were treated with 64Cu- or 67Cu-BAT-2IT-1A3, survival was extended compared with controls, but only one animal remained tumor-free to 6 months. These results demonstrate that 64Cu- and 67Cu-BAT-2IT-1A3 given in a single administered dose can eradicate small tumors without significant host toxicity, but additional strategies to deliver higher tumor doses will be needed for larger tumors.

Measurement of muscle protein synthesis by positron emission tomography with L-[methyl-11C]methionine.

Positron emission tomography (PET) with L-[methyl-11C]methionine was explored as an in vivo, noninvasive, quantitative method for measuring the protein synthesis rate (PSR) in paraspinal and hind limb muscles of anesthetized dogs. Approximately 25 mCi (1 Ci = 37 GBq) of L-[methyl-11C]methionine was injected intravenously, and serial images and arterial blood samples were acquired over 90 min. Data analysis was performed by fitting tissue- and metabolite-corrected arterial blood time-activity curves to a three-compartment model and assuming insignificant transamination and transmethylation in this tissue. PSR was calculated from fitted parameter values and plasma methionine concentrations. PSRs measured by PET were compared with arterio-venous (A-V) difference measurements across the hind limb during primed constant infusion (5-6 h) of L-[1-13C, methyl-2H3]methionine. Results of PET measurements demonstrated similar PSRs for paraspinal and hind limb muscles: 0.172 +/- 0.062 vs. 0.208 +/- 0.048 nmol-1.min-1.(g of muscle)-1 (P = not significant). PSR determined by the stable isotope technique was 0.27 +/- 0.050 nmol-1.min-1.(g of leg tissue)-1 (P < 0.07 from PET) and indicated that the contribution of transmethylation to total hind limb methionine utilization was approximately 10%. High levels of L-[methyl-11C]methionine utilization by bone marrow were observed. We conclude that muscle PSR can be measured in vivo by PET and that this approach offers promise for application in human metabolic studies.

Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide.

We describe a method to facilitate radioimaging with technetium-99m (99mTc) by genetic incorporation of a 99mTc chelation site in recombinant single-chain Fv (sFv) antibody proteins. This method relies on fusion of the sFv C terminus with a Gly4Cys peptide that specifically coordinates 99mTc. By using analogues of the 26-10 anti-digoxin sFv as our primary model, we find that addition of the chelate peptide, to form 26-10-1 sFv', does not alter the antigen-binding affinity of sFv. We have demonstrated nearly quantitative chelation of 0.5-50 mCi of 99mTc per mg of 26-10-1 sFv' (1 Ci = 37 GBq). These 99mTc-labeled sFv' complexes are highly stable to challenge with saline buffers, plasma, or diethylenetriaminepentaacetic acid. We find that the 99mTc-labeled 741F8-1 sFv', specific for the c-erbB-2 tumor-associated antigen, is effective in imaging human ovarian carcinoma in a scid mouse tumor xenograft model. This fusion chelate methodology should be applicable to diagnostic imaging with 99mTc and radioimmunotherapy with 186Re or 188Re, and its use could extend beyond the sFv' to other engineered antibodies, recombinant proteins, and synthetic peptides.