Measurement of protein turnover in skeletal muscle strips.

Isolated porcine and bovine muscle strips were incubated in Krebs Ringer bicarbonate buffer to determine in vitro protein synthesis (PS) and protein degradation (PD) rates to validate the in vitro system for use with livestock species. The addition of 5X plasma concentrations of amino acids to the medium stimulated PS 30%. Addition of 3.5 mM leucine to a leucine-deficient buffer supplemented with amino acids decreased PD 37% and stimulated PS 24%. The addition of .1 U/ml insulin reduced PD 28% and increased PS 30%. Protein degradation was elevated in longitudinally split rat soleus and extensor digitorum longus muscles compared to their contralateral intact muscles. Muscle strips must be removed within 15 min of exsanguination because PD rates become greatly elevated thereafter. ATP concentrations declined during incubation, but the addition of ATP or creatine had no effect on either PD or PS. Neither PD nor PS was affected by the addition of transferrin, fetuin, ascorbate, dexamethasone or indomethacin to the incubation medium. However, muscle strips were sensitive to the addition of triiodothyronine (T3), PD was increased up to 75% as T3 concentration was increased, and PS rates doubled compared to controls. Serum from mature barrows or gilts had no effect on protein turnover, but the addition of 10% and 15% serum from boars increased both PD and PS. With fasted pigs a continual decline in PS occurred over 5 d, whereas PD was elevated at 3 d and then declined to rates comparable to the fed state after 5 d. These data suggest that the in vitro system has application for assessing relative changes that occur in vivo following nutritional, physiological and endocrinological manipulation.

Contribution of wood smoke to in vivo formation of N-nitrosothiazolidine-4-carboxylic acid: initial studies.

The effect of administering liquid smoke or smoked food products to rats on in vivo formation of N-nitrosamino acids was investigated. Rats treated by gavage with either cysteine, formaldehyde or nitrite excreted urine containing no detectable levels of N-nitrosothiazolidine-4-carboxylic acid (NTCA). All three precursor compounds were required for NTCA formation. Two liquid smokes, when administered to rats in combination with cysteine and nitrite also produced measurable quantities of NTCA. Ascorbate inhibited in vivo formation of NTCA by approximately 90% when given to rats simultaneously with cysteine, formaldehyde, and nitrite. alpha-Tocopherol was much less effective than ascorbate in blocking NTCA formation. When smoked bacon, smoked Swiss cheese, and chicken barbecued with a sauce containing smoke flavouring were fed to rats along with nitrate, NTCA was again detected in the urine.

Gas chromatographic-thermal energy analysis method for determination of volatile N-nitrosamines in baby bottle rubber nipples: collaborative study.

A collaborative study was conducted on the U.S. Food and Drug Administration (FDA) dichloromethane extraction method for determining volatile N-nitrosamines in baby bottle rubber nipples. Following dichloromethane extraction, N-nitrosamines were determined by gas chromatography-thermal energy analysis. Six pairs of blind duplicate rubber nipple samples representing 6 lots were analyzed by 11 collaborating laboratories. All samples were portions taken from equilibrated composites of cut-up rubber nipples obtained from manufacturers in the United States. Recoveries of the internal standard (N-nitrosodipropylamine) at approximately 20 ppb ranged from 10 to 120%. Reproducibility relative standard deviations (RSDx) were between 35 and 45% for N-nitrosamine levels from 10 to 20 ppb. However, when data from laboratories with recoveries less than 75% were excluded (this is now specified in the method), RSDx values were between 11 and 32% for N-nitrosamine levels from 6 to 26 ppb. Values were consistent with or better than those reported for other analytical techniques designed to quantitate trace contaminants at the low ppb level, e.g., aflatoxin in foods. The method has been adopted official first action for the quantitation of volatile N-nitrosamines in baby bottle rubber nipples.