A novel method for the analysis of clinical biomarkers to investigate the effect of diet on health in a rat model.

Experiments into the relationship between diet and health have been an area of high interest for a long time. In this study, we investigate the application of multivariate data analysis to differentiate between rat populations fed on two different diets: normal rat diet (control) and Western affluent diet (WAD). Two sets of data were acquired and analysed: one from a biochemical clinical analyser, taking measurements of blood-based biochemical markers; the other from the analysis of the volatile organic compounds (VOCs) emitted from faecal samples from the same animals using selected ion flow tube mass spectrometry (SIFT-MS). Five classes were considered: weanlings, 12 month controls, 12 month WADs, 18 month controls, and 18 month WADs. Data from the biochemical analyser, weanlings and 18 month WAD fed rats showed significant differences from the other measurement classes. This was shown in both the exploratory analysis and through multivariate classification. Classification of control diet versus WAD diets suggested there are differences between classes with 92% accuracy for the 12 month classes and 91% for the 18 month classes. Cholesterol markers, especially as low density lipoprotein-cholesterol (LDL), were the main factor in influencing WAD samples. The data from the SIFT-MS analysis also produced very good classification accuracies. Classification of control diet versus WAD diets using the H3O(+) precursor ion data suggested there are differences between classes with 71% accuracy for the 12 month classes and 100% for the 18 month classes. These findings confirm that total cholesterol and LDL-cholesterol are elevated in the 18 month WAD-fed rats. We therefore suggest that the analysis of VOCs from faecal samples in conjunction with multivariate data analysis may be a useful alternative to blood analysis for the detection of parameters of health.

Changes in lymphokine receptor expression and fatty acid composition of phospholipids and triacylglycerols in rat adipocytes associated with lymph nodes following a transient immune challenge.

Single-photon counting fluorimetry was used to record the time course of the expression of interleukin-10 receptors labelled with fluorescent antibodies on the surface of adipocytes over 24h, following an immune challenge to the rat popliteal lymph node. Homologous perinodal and remote-from-node samples from the stimulated and unstimulated popliteal depots were compared in rats fed on plain chow and chow supplemented with 10% w/w suet, fish or vegetable oils. Receptor expression was maximal 6 h after stimulation, and returned to baseline after 24 h, and was similar in the stimulated and unstimulated depots. Fewer receptors were elicited in tissues from rats fed lipid-supplemented diets compared with the control diet, with fewest of all following the fish oil diet. These data suggest that interleukin-10 is involved in local interactions between perinodal adipocytes and lymph node lymphoid cells. Both triacylglycerols and phospholipids contained more polyunsaturates and fewer saturates in perinodal adipose tissue than in samples from sites not associated with lymphoid tissue. These data are consistent with paracrine interactions between perinodal adipocytes and activated lymphoid cells.

Dietary fatty acids influence the appearance of tumour necrosis factor-alpha receptors on adipocytes following an immune challenge.

Rats were fed from weaning on chow supplemented with suet or sunflower oil (10 % (w/w) each). The appearance of receptors for tumour necrosis factor-alpha on perinodal adipocytes from the popliteal depot following a subcutaneous injection of bacterial lipopolysaccharide was examined. In rats fed on sunflower oil-supplemented chow receptors appeared at a time similar to that described in rats fed unsupplemented chow, but in rats fed on chow supplemented with suet receptor appearance was significantly delayed. The popliteal adipocytes were found to contain different proportions of fatty acids as assessed by GLC. These preliminary results suggest that the fatty acid component of the diet can, by influencing the triacylglycerol-fatty acids within adipocytes, directly alter the time course of an early inflammatory immune response.

Vascularisation in adipose depots surrounding immune-stimulated lymph nodes.

We report a change in the vascularisation of the adipose depots surrounding the popliteal lymph node that has, and the contralateral node that has not, been exposed to a simulated immune challenge. The percentage of the depot that consists of vessels, as measured by image analysis, decreases over a period of 2 d after immune stimulus, then increases in a biphasic manner over the next 2-3 wk. By 1 mo after the stimulus, the vascularisation has returned to baseline values. The adipose tissue surrounding both the stimulated and the unstimulated lymph nodes shows a similar pattern, but the unstimulated depot lags by 3-6 d in reaching its maximum vascularisation. These data support the hypothesis that perinodal adipose tissue is involved in peripheral immune responses.

Immunofluorescent localisation of tumour necrosis factor-alpha receptors on the popliteal lymph node and the surrounding adipose tissue following a simulated immune challenge.

We used immunohistochemical techniques to demonstrate the distribution of receptors for the cytokine tumour necrosis factor-alpha on the popliteal lymph node and the adipose tissue surrounding it for 5 d following a simulated immune challenge to one hind leg in the rat. We found different patterns of expression of receptors on adipocytes surrounding a lymph node to a distance of about 1 mm, and on those more remote from the node. Sites recognised by an antibody to type I tumour necrosis factor receptors appeared on the challenged node and the adipocytes surrounding it within 30 min of an injection of bacterial lipopolysaccharide, but appeared on adipocytes surrounding the unchallenged popliteal node only 24 h later. Adipocytes distant from the node, both within the same depot and in the contralateral depot, showed no response. Sites recognised by an antibody to type II tumour necrosis factor receptors were present at all times on lymph nodes and the adipocytes close to them, but appeared on more distant adipocytes only 24 h after immune challenge, in both challenged and unchallenged legs. These data support the proposal, based on in vitro studies, that the adipose tissue surrounding major lymph nodes is specialised to respond to cytokines derived from lymphoid cells, and participates in the immune responses of the adjacent node.

Fucosylated glycoconjugates in mouse preimplantation embryos.

Preimplantation mouse embryos were metabolically labelled with 3H or 14C-fucose to investigate the synthesis of fucosylated macromolecules. Scintillation counting revealed that there was a progressive increase in both total fucose taken up by the embryo and incorporation of fucose into TCA-precipitable material as embryos developed from the 4-cell to the blastocyst stage. This was reflected in the increasing intensity of bands on autoradiographs of radioactive fucose labelled proteins separated on 10% SDS-PAGs between the 4-cell embryo (at which stage bands were first detectable) and the blastocyst. Minor qualitative changes in fucoproteins were detected at the time of compaction and additional bands appeared at the blastocyst stage. Preliminary analysis of fucolipids in 6- to 8-cell embryos indicated that an approximately equal amount of fucose was incorporated into lipid and protein. Autoradiographs of semi-thin sections of 3H-fucose-labelled embryos showed substantial amounts of radioactive material in the vicinity of the plasma membrane both adjacent to other cells and facing the zona pellucida. These data would support a predominant role for fucoconjugates in cell surface events in the preimplantation embryo from the 8-cell stage.

The fifth cell cycle of the mouse embryo is longer for smaller cells than for larger cells.

Newly-formed pairs of 16-cell blastomeres were collected by periodic observation of isolated 8-cell blastomeres. Any pairs formed by division were recovered and classified as being composed of 1/16 blastomeres that differed in size or were of similar size. All of the latter and some of the former were then cultured for periods of up to 20h. The remaining pairs of different-sized blastomeres were disaggregated to larger or smaller cells. Some of these were reaggregated as smaller: smaller or larger: larger pairs, and these, together with the remaining isolated smaller and larger blastomeres were also cultured for up to 20h. At hourly intervals, cultured cells were sampled and analysed for incidence of division. It was found that larger cells divided on average after 12h whereas smaller cells divided on average after 14h.

Subunits of laminin are differentially synthesized in mouse eggs and early embryos.

Synthesis of laminin by mouse preimplantation embryos was examined by specific immunoprecipitation of [35S]methionine-labeled cell lysates. The three polypeptide subunits of laminin, A, B1, and B2, are not necessarily synchronously expressed during development. Oocytes and eggs synthesize only one immunoprecipitable polypeptide, which comigrates with the intracellular B1 chains made by PYS cells and contains N-linked oligosaccharide residues which are sensitive to endo-beta-N-acetylglucosaminidase H. From the 4- to 8-cell stage, only B1 and B2 polypeptides are synthesized, whereas from the 16-cell stage onwards all three laminin polypeptides are made.

Lethality of radioisotopes in early mouse embryos.

The development of pre-implantation mouse embryos was found to be prevented by exposure of the embryos to [35S]methionine, but not to [3H]methionine. Such embryos have also been shown to be highly sensitive to [3H]thymidine. These observations are discussed with reference to the path lengths and energies of electrons emitted from the different radioisotopes.