Transsulfuration, protein synthesis rate and follicle mRNA in the skin of young Merino lambs in response to infusions of methionine and serine.

Methionine (Met) is usually the first limiting amino acid for sheep and supplements of Met may increase production of wool and meat. The wool response may be due to an increased supply of cysteine (Cys) from transsulfuration (TS) of Met. Met is catabolized through homocysteine to form Cys when the S from Met is transferred to serine (Ser). We hypothesized that providing additional Met would create a deficiency of Ser and that by simultaneously providing Met and Ser, TS and wool growth could be increased more than by providing Met alone. The effects of i.v. infusions of Met and Ser to young Merino lambs on TS, fractional synthesis rate (FSR) of protein in skin, follicle mRNA and wool growth were examined. Following 4 d of constant i.v. infusion of 3 g Met/d, or 10 g Ser/d or both, the isotope tracers: L-[3-(13)C]Cys, L-[ring-d5]phenylalanine (Phe) and L-[2,3,3-d3]Ser were infused over 8 h to allow for measurements of irreversible loss rate (ILR), and TS in whole body and skin. Skin biopsies were taken for measurement of FSR. Wool growth rate was measured using autoradiography. An infusion of Met significantly (P < 0.05) improved wool growth rate and increased skin FSR, Cys supply from TS and enhanced levels of follicle mRNA (from the K2.10 intermediate filament gene and three gene families encoding keratin associated proteins KAP1, KAP4 and KAP12). The extra Met lowered Ser ILR. The infusion of Ser doubled Ser ILR in the body and increased skin FSR calculated using the Cys tracer in plasma (P < 0.05). However, there were no significant (P > 0.05) changes in TS, skin FSR calculated using the Phe and Ser tracers, follicle mRNA or wool growth rate as a result of Ser infusion. While there were trends towards increased TS and FSR with Ser infusion, the overall lack of significant changes indicates a high capacity for the de novo synthesis of Ser.

Gas chromatography/mass spectrometry analyses of [2,3,3-d3] serine, [2,3,3-d3] cysteine and [3-13 C] cysteine in plasma and skin protein: measurement of transsulphuration in young sheep.

A new procedure using stable isotope labelled serine (L-[2,3,3-d3] serine) and cysteine (L[13-3-13 C] cysteine) and analysis by gas chromatography/mass spectrometry (GC/MS) has been developed to measure transsulphuration in sheep. The enrichments of the tracers in plasma and skin biopsy samples were measured by GC/electron impact MS analysis of the t-butyldimethylsilyl derivatives. The measured recoveries of the standards enriched with [3-13 C] cysteine from 0.1% to 8%, or with [2,3,3-d3] serine from 0.14% to 14% were greater than 99% of the theoretical values, and the variation coefficients were less than 3% when the enrichment was higher than 0.5%. The use of dithiothreitold (DTT) as a reducing agent before deproteinization of the sample and during the derivatizations successfully increased the cysteine peak area and significantly improved reproducibility in the analysis. The cysteine residues in protein from the skin biopsy were also during the protein hydrolysis with DTT in 6 n HCI. The method was applied to measure transsulphuration of methionine in young sheep. The amount of cysteine derived from transsulphuration accounted for 17% to 21% of the irreversible loss rate of cysteine, depending on the substrate supplies. The results are consistent with other reports. Compared with conventional methods of measuring transsulphuration using radioactive isotopes, the processes of animal experimentation was sample analysis were simple, and there were no radiation hazards. The method should prove useful in studies on the metabolism of methionine and cysteine in human and animals.