Serum zinc in aging germ-free and conventional rats.

Zinc nutritional status appears to decline with age in humans and rodents. Since germ-free rats outlive their conventional counterparts in better health, serum Zn levels were determined in male germ-free and conventional Lobund Wistar rats in samples originating from the Lobund Aging Study. Starting at 5 months of age, germ-free rats showed significantly higher serum Zn levels than did their conventional counterparts. In conventional rats sacrificed up to 30 months of age in apparently good health, serum Zn levels showed no effect of age, while a slight but significant increase with age was observed in the germ-free rats. In healthy germ-free adults (6-24 months of age), serum Zn concentrations were approximately 25% higher than those in comparable conventional animals. In conventional rats 18-30 months of age (average, 24.5 months), sacrificed because of an obvious moribund condition, serum Zn levels were significantly lower than those in rats of the same age range (average, 24.9 months) that were obviously healthy. Results suggest that the often observed higher absorptive capacity of the germ-free gut might have contributed to higher serum Zn levels, and that a decline in serum Zn concentration with age may be a consequence, rather than a causative factor, of declining health.

Life span, morphology, and pathology of diet-restricted germ-free and conventional Lobund-Wistar rats.

The effect of germ-free life and dietary restriction (DR) on life span and pathology was investigated in isolator housed germ-free (GF) and conventional (CV) Lobund-Wistar rats fed either ad libitum or restricted to 12 grams per day (70% of adult ad libitum intake) of a natural ingredient diet from weaning. The median length of life of ad libitum CV and GF rats was 31.0 and 33.6 months respectively, while DR increased the median length of life of CV and GF rats to 38.6 and 37.8 months respectively. DR reduced the frequency or postponed the occurrence of diseases which eventually lead to death in the Lobund-Wistar rat. This was especially true of prostate adenocarcinoma, prostatitis, and mammary fibroma. The reduced early food intake and smaller body weight of adult GF rats may be the reason ad libitum fed GF rats live slightly longer than their CV counterparts, but GF life was without additional effect on life span when food intake was restricted.

Serum immunoglobulin levels and naturally occurring antibodies against carbohydrate antigens in germ-free BALB/c mice fed chemically defined ultrafiltered diet.

This study investigates the influence of exogenous antigenic stimulation on the serum immunoglobulin levels and the levels of circulating natural antibodies against carbohydrate antigens. Thus, BALB/c mice, raised in a germ-free environment and fed a chemically defined, ultrafiltered diet (GF-CD), were employed. These mice had normal serum IgM levels, but IgG and IgA levels were approximately 5% of conventionally reared littermates. The concentrations of all four IgG isotypes were equally low. The variable part of the heavy chains of naturally occurring BALB/c antibodies against a number of carbohydrate antigens, including 3-fucosyllactosamine (3-FL), levan and dextran, are encoded by VH441, and these antibodies express cross-reactive idiotopes recognized by the monoclonal antibodies 6C4 and 6B1. Antibodies against levan and dextran were lower in GF-CD than in conventional mice, but levels of anti-3FL antibodies, and 6C4 and 6B1 idiotopes, were comparable to those in conventional animals. Peptidoglycan polysaccharide complexes (PPC) are carbohydrate antigens of bacterial origin, like levan and galactan. Naturally occurring antibodies against PPC were found in the serum of conventional mice, but were severely reduced in GF-CD mice. The results indicate that most naturally occurring antibodies against carbohydrate antigens of bacterial origin found in conventional mice are caused by exogenous stimulation.

Role of intestinal microflora in the metabolism of vitamin B-6 and 4'-deoxypyridoxine examined using germfree guinea pigs and rats.

In previous work identification of urinary metabolites of 4'-deoxypyridoxine which had been oxidized in the 5'-position and long-term dilution of labeled urinary metabolites with unlabeled molecules suggested possible microbial contributions. In the current studies germfree guinea pigs were able to convert 4'-deoxypyridoxine to 4'-deoxy-5-pyridoxic acid demonstrating that the ability to oxidize the 5'-position is not restricted to microorganisms. Labelling curves for urinary pyridoxic acid in rats continuously fed [14C]pyridoxine since weaning were similar in conventional and germfree animals indicating that any vitamin B-6 synthesized in the intestinal tract was not readily absorbed and metabolized. Therefore, coprophagy did not make a detectable contribution to vitamin B-6 metabolism in rats receiving a nutritionally complete diet. The difficulty in achieving comparable labeling in adult animals is probably due to very slow turnover of portions of the vitamin B-6 pool and not to microbial production of vitamin B-6. The total pool calculated from the radioactivity in the germ-free rats averaged 16.2 +/- 0.8 nmol vitamin B-6 compounds/g body wt. Only 10% of the ingested label was recovered in the feces. In addition, only about 50% of the label excreted in the urine appeared as 4-pyridoxic acid in rats. These observations suggest that it may be difficult to quantitate the total urinary and fecal excretion of ingested vitamin B-6 without using tracers.

B6 vitamer content of rat tissues measured by isotope tracer and chromatographic methods.

Feeding [14C]pyridoxine to growing rats for 146 days produced uniform labelling of the total vitamin B6 pool, thus permitting the radioactivity to be used as an absolute standard for evaluating the accuracy of vitamin B6 analyses. The results demonstrated that trichloroacetic acid extraction followed by cation exchange chromatography accurately measures the B6 vitamers. It is essential to homogenize tissues in a protein-denaturing agent in order to avoid shifts in the vitamer content, particularly in liver. In rats approximately 80% of the radioactivity was found in carcass and 8-9% each in liver and skin. Pyridoxamine phosphate equalled or exceeded the concentration of pyridoxal phosphate in heart, brain and kidney. The total vitamin B6 pool in weanling and adult rats averaged about 16 nmol/g body wt. Meta-phosphoric acid extraction followed by reverse phase chromatography gave good agreement with the cation exchange method in rat liver but with cat plasma yielded pyridoxal phosphate values below those of the cation exchange or enzymatic methods. The discrepancies encountered between different homogenization techniques and chromatographic methods emphasize the need for constant vigilance and continual verification of results by independent methods.

Serum hormones in diet-restricted gnotobiotic and conventional Lobund-Wistar rats.

In order to clarify the reasons for life extension due to the germ-free state and mild dietary restriction, serum levels of thyroid stimulating hormone, thyroxine, triiodothyronine, prolactin, and testosterone were determined in conventional and gnotobiotic (single microbe contaminations) Lobund-Wistar rats fed either ad libitum or restricted to 12 grams (70% of ad libitum) of diet per day from weaning. Thyroid hormone levels were slightly higher in gnotobiotic rats, declined between 7 and 30 months of age in all rats, and were not affected by dietary restriction. Prolactin showed a significant rise with age within the ad libitum rats only. Testosterone levels declined significantly with age in all rats, but were significantly higher in restricted rats at all ages. These results indicate that mild dietary restriction extends life span without reducing circulating levels of thyroid stimulating hormone, thyroxine, triiodothyronine, or testosterone in mature Lobund-Wistar rats, and prevents the age-associated rise in serum prolactin. Life extension in germ-free rats is not related to differences in endocrine function.

The influence of exogenous antigenic stimulation on the specificity repertoire of background immunoglobulin-secreting cells of different isotypes.

The total number of spontaneously occurring ("background") IgM-, IgG-, and IgA-secreting cells and the frequency of antigen-specific IgM-, IgG-, and IgA-secreting cells were determined in germ-free BALB/c mice fed a chemically defined ultrafiltered diet (GF-CD), in specific pathogen-free BALB/c mice fed an autoclaved natural ingredient diet (SPF-NI), and in conventional BALB/c mice fed nonautoclaved natural ingredients (CV-NI). This was done by means of the ELISA-plaque assay. The results did not show differences among the various groups of mice with regard to the total numbers of IgM-secreting cells in the various lymphoid organs. Also the frequencies of IgM-secreting cells specific for DNP27-BSA and the anti-idiotypic monoclonal antibodies Ac38 and Ac146 did not differ significantly among GF-CD, SPF-NI, and CV-NI mice. GF-CD mice, however, did show substantially decreased numbers of IgG- and IgA-secreting cells in their lymphoid organs. Furthermore, there were striking differences in the frequencies of antigen-specific IgG- and IgA-secreting cells between GF-CD mice and the two other groups of mice. These results indicate that exogenous antigenic stimulation has a great effect on both the total numbers and the specificity repertoires of background IgG- and IgA-secreting cells. Such an influence could not be detected with regard to the background IgM-secreting cells. This suggests two distinct compartments of background Ig-secreting cells: a very stable, endogenously regulated compartment consisting mainly of IgM-secreting cells, and another compartment, consisting mainly of IgG- and IgA-secreting cells, whose numbers and specificity repertoire appeared to be influenced by exogenous antigenic stimulation.

Growth rate of male germfree Wistar rats fed ad libitum or restricted natural ingredient diet.

Germfree life and feed restriction initiated at an early age are known to extend life span. We have examined growth rate and life expectancy in germfree (GF) and conventional (CV) male Lobund Wistar rats, fullfed (F) or restricted (R) to 12 grams/day of natural ingredient diet L-485. GF-F and CV-F rats show comparable growth rates during the first 6 months of life. Thereafter, the GF-F rat falls behind, with its body weight stabilizing at 85% of the CV rat's weight at 2 years of age (510 g vs 435 g). In contrast, GF-R rats become slightly, but significantly, heavier than CV-R rats after an initial 6 months of comparable growth. At 2 years of age GF-R rats weigh 12% more than the CV-R rats (340 g vs 300 g). Physiological parameters were examined in each treatment group in animals that had to be sacrificed because of contamination. These gnotobiotic (GN) rats (see text) and their CV counterparts were grouped as adult (7 to 11 months) and old (18 to 28 months) rats. The most significant findings were: GN-F rats have smaller hearts than CV-F rats, both on an absolute and relative basis; restriction did not affect absolute testes size but elevated serum testosterone levels; serum T4 was reduced by restriction only in CV rats, and declined with age in all groups; and serum T3 was higher in adult GN-F and GN-R rats, but fell to CV levels in old age.(ABSTRACT TRUNCATED AT 250 WORDS)

Early development of Ig-secreting cells in young of germ-free BALB/c mice fed a chemically defined ultrafiltered diet.

The influence of antigenic stimulation on the early development of the "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells has been studied in mice. To evaluate the effect of such exogenous stimulation by an evolving microbial microflora, the young of BALB/c mice that were kept under germ-free conditions and fed a low molecular weight chemically defined synthetic diet (GF-CD) were compared with the young of conventional BALB/c mice fed natural ingredients (CV-NI). The young were first suckling maternal milk and between Days 15 and 18 changed to the same diet as their parents. Background Ig-secreting cells in the spleen were enumerated in the protein A plaque assay. The specificity repertoire of the IgM-secreting cells was determined with plaque assays specific for sheep red blood cells (SRBC) that were haptenized with different concentrations of nitroiodophenyl (NIP), 4-hydroxy-3.5-dinitrophenyl (NNP), and 2,4,6-trinitrophenyl (TNP). The results show that during the first few weeks of life the numbers of background IgM-, IgG-, and IgA-secreting cells in the spleen develop faster in CV-NI mice than in GF-CD mice. At 4 weeks of age equal numbers of IgM- and IgG-secreting cells were found in both groups of mice, but the number of IgA-secreting cells remained reduced in GF-CD mice during the whole period of observation. The frequencies of IgM-secreting cells specific for the differently haptenized SRBC were the same in both groups of mice during the observation period of 10 weeks. This suggests that the ontogenetic appearance of IgM-, IgG-, and IgA-secreting cells in the spleen, and the specificity repertoire of the IgM-secreting cells, as far as was tested in our panel, is independent of exogenous antigenic and/or mitogenic stimulation. However, during neonatal development the rate of development of the background Ig synthesis is enhanced by environmental antigenic stimulation.

'Background' Ig-secreting cells in pregnant germfree mice fed a chemically defined ultrafiltered diet.

During syngeneic pregnancy the numbers of 'spontaneously' occurring ('background') Ig-secreting cells were determined in the spleen, bone marrow (BM) and mesenteric lymph nodes (MLN) of BALB/c mice that were kept under germfree conditions and fed a low molecular weight synthetic diet (GF-CD), SPF BALB/c mice fed autoclaved natural ingredient (SPF-NI) and conventional BALB/c mice fed natural ingredient (CV-NI). 'Background' Ig-secreting cells were enumerated in the protein A plaque assay and the specificity repertoire of the IgM-secreting cells was determined with plaque assays specific for differently haptenized sheep red blood cells (SRBC). The numbers of 'background' Ig-secreting cells, especially the IgG- and IgA-secreting cells, are very much reduced in the BM and MLN of GF-CD mice as compared to SPF-NI and CV-NI mice. During pregnancy the total number of Ig-secreting cells increased in all lymphoid organs tested, but the proportional increase was most prominent for the IgG- and IgA-secreting cells in the BM and MLN of the GF-CD mice. This increase could only be due to their pregnant state since all environmental antigenic influences are excluded in GF-CD mice. No changes were found in the background specificity repertoire of the IgM-secreting cells during pregnancy. This suggests a polyclonal activation of the Ig-secreting cells during pregnancy. The reason for this activation remains obscure, but it has to be endogenous. Pregnancy apparently induces a new steady state of the immune system, which can be most properly investigated in GF-CD mice.

Adequacy of chemically defined, water-soluble diet for germfree BALB/c mice through successive generations and litters.

To determine dietary adequacy, germfree BALB/cAnN mice were fed ad libitum an ultrafiltered solution of chemically defined, water-soluble, low-molecular-weight nutrients. They received a measured daily supplement of membrane-filtered, distillation-purified soy oil containing vitamins A, D, E and K. Mice were kept on ash-free filter paper bedding, which they freely consumed. On this regimen, germfree BALB/c mice reproduced through nine generations, and through eight litters in one generation. Average number born per litter was 4.1, compared with 5.1 in control BALB/c mice, which had a conventional microflora and were fed a natural ingredient diet. From 21 to 32 d of age, the experimental mice gained more slowly than controls. After 32 d, experimental mice gained more rapidly than controls; their weights tended to be lower than controls at 45 d and equal to controls at 56 d. Intake-limiting effects of the diet appeared responsible for reduced growth rates and litter size. Experimental females showed a low incidence of deaths from cecal volvulus. Experimental males experienced a high incidence of deaths from colonic impaction of cecally-formed trichobezoars; this site of formation appeared to be unique to BALB/c males on the experimental regimen. These losses were judged to be unrelated to nutritional deficiency. No overt signs of nutritional deficiency developed in female mice which were fed CD diet up to 18 mo of age.

Frequency analysis of functional immunoglobulin C- and V-gene expression by mitogen-reactive B cells in germfree mice fed chemically defined ultra-filtered "antigen-free" diet.

The frequencies of lipopolysaccharide (LPS)-reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of conventional (CV) and "antigen-free" C3H/HeCr mice of various ages. The antigen-free mice were germfree (GF)-raised and were fed an ultrafiltered solution of chemically defined (CD) low m.w. nutrients, and were thus devoid of exogenous antigenic stimulation. Spleen and BM cells were grown in a limiting dilution culture system that allows the growth and development of every newly formed LPS-reactive B cell into a clone of IgM-secreting cells which are capable of switching to other immunoglobulin (Ig) heavy chain isotypes (C-gene expression). The secretion of IgM and IgG1 was determined in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V-gene expression) were detected in plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The absolute frequency of LPS-reactive B cells and their capacity to switch to IgG1-secretion was not significantly different in 8- to 12-wk-old and 52-wk-old GF-CD mice and their age-matched CV controls. Moreover, no differences were observed in the frequencies of antigen-specific B cells within the pool of LPS reactive B cells. These frequencies ranged from 1 in 20 to 1 in 50 for NIP4-SRBC and NNP2-SRBC, from 1 in 100 to 1 in 150 for NIP0.4-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 2000 for SRBC and horse red blood cells. Within the limitations of having determined the switching capacity of IgM to IgG1 only and having assessed only a minor fraction of the total B cell antibody-specificity repertoire, the data indicate that young and old GF-CD mice, although devoid of exogenous antigenic and/or mitogenic stimulation, generate B cells with a similar switching capacity and a similar IgM antibody specificity repertoire as CV mice.