Effect of autoclaving of a lactose-containing diet on cholesterol and bile acid metabolism of conventional and germ-free rats.

Feeding of lactose in amounts comparable to the adult human intake in developed countries (6% of diet, and in later studies 10%) had no major effect on cholesterol and bile acid metabolism of germ-free and conventional rats. However, when lactose-containing casein-starch diets were sterilized by autoclaving, changes in intestinal and/or fecal bile acids were found. Both germ-free and conventional rats demonstrated some increase in intestinal beta-muricholic acid concentrations ascribable to the mere presence of lactose in the diet. Autoclaving of the diet produced additional changes, especially in the fecal bile acid pattern of conventional rats. Here the ratio between the beta-muricholic-derived secondary bile acids hyodeoxycholic and omega-muricholic acids changed from the usual 5:3 to approximately 1:10, with omega-muricholic acid becoming the major fecal bile acid. These changes point to a notable effect of lactose-derived products, formed during steam-sterilization, on the microbial modification of intestinal bile acids in the lower gut. Similar changes have been observed after oral administration of aureomycin and other, unrelated antibiotics that inhibit growth of gram positive organisms.

'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.

Thyroid function and related hepatic enzymes in the germfree rat.

The adult germfree rat has a low metabolic rate, and the activity of various hepatic enzymes differs substantially from values found in the conventional animal. To promote understanding of the shifts in intermediary metabolism apparent in the germfree rodent, parameters of thyroid function and the activity of three enzyme systems presumably influenced by thyroid function were determined in 40-, 60-, and 100-day-old male germfree and conventional Lobund rats of Wistar origin. Serum thyroxine concentrations generally increased with age. They were below normal levels in 40- and 60-day-old germfree rats but appeared normal in the 100-day-old germfree animal. Serum thyroxine-binding protein saturation levels were significantly reduced in germfree rats of all age groups, but the reduction was least in the 100-day-old group. Mitochondrial succinate oxidase and alpha-glycerophosphate dehydrogenase activities were lower in livers of germfree rats of all age groups, although the significance of the difference in succinic oxidase activity became less in older animals. Monoamine oxidase activity was similar in germfree and conventional animals. Although the data suggest a hypofunction of the thyroid in the immature germfree rat, no definite relation between thyroid function and the characteristic metabolic anomalies of the germfree rat could be established.

Cholesterol metabolism in gnotobiotic gerbils.

Germfree gerbils were associated with a murine-derived hexaflora which produced only minor changes in the primary bile acid pattern of rats. These hexaflora-associated gerbils had relatively small ceca (4% of body weight) and reproduced well. Although serum cholesterol levels of both conventional and hexaflora-associated gerbils increased in response to dietary cholesterol, the hexaflora-associated gerbil showed a greater elevation in serum cholesterol than the conventional gerbil when maintained on a diet containing 0.1% cholesterol. This increase in serum cholesterol manifested itself almost totally in the very low density lipoprotein and low density lipoprotein fractions. The fecal bile acids of the hexaflora-associated gerbil were largely deconjugated, but very little further modification of either cholic or chenodeoxycholic acid had taken place. The data suggest that in the absence of elements of the intestinal microflora that can express a bile acid-modifying potential, and particularly a 7-alpha-dehydroxylating capacity, catabolism of cholesterol to bile acids is reduced, and cholesterol accumulates in the very low density and low density serum lipoprotein fractions.

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.

Oxygen consumption of germfree and conventional mice.

Oxygen consumption was determined in germfree and conventional mice using a respirometer which enabled us to make precise corrections for variations in temperature or barometric pressure. In 20, 3-minute observations on 21 germfree and 21 conventional mice, the germfree animals consumed an average of 2.11 ml of oxygen per gram body weight per hour in comparison to 2.37 ml for the conventional mice. This difference was not accompanied by a difference in rectal temperature.

Exteriorization of the bile duct in germfree rats.

A modified T cannula and a cannula housing receptacle was employed to cannulate the bile duct of germfree rats. Bile was collected without anesthesia or sedation. During collection, the rat was restrained in a plastic holder where it was free to eat.

Fecal neutral steroids and bile acids from germfree rats.

The amount and composition of fecal neutral sterols and bile acids excreted by adult male germfree and conventional rats have been determined. The amounts of neutral sterols excreted were 12.8 (germfree) and 19.5 (conventional) mg/kg of body wt per day. The germfree rats excreted cholesterol and lathosterol (methostenol was not assayed); the conventional rats excreted coprostanol and coprostanone in addition. The amounts of bile acids excreted were 11.3 (germfree) and 21.4 (conventional) mg/kg of body wt per day. The bile acids excreted by the rats were tentatively identified as tauro--muricholate, tauro-alpha-muricholate, and tauro-cholate, besides an unidentified component. The conventional rats excreted the corresponding unconjugated acids as well as many other unconjugated bile acids. No significant correlation was found between the amount of coprosterols and the total amount of neutral sterols excreted by the conventional rats. This suggests that bacterial reduction of cholesterol is not an important mechanism of increasing neutral sterol excretion of conventional rats as compared to germfree rats. Evidence is presented that suggests that this difference in neutral sterol excretion is due to changes in intestinal secretion and sloughing between the two types of animal. The factors reponsible for the differences in bile acid excretion have not been identified.

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)

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.

Catabolism and elimination of cholesterol in germfree rats.

Three-month old germfree and conventional male rats were maintained on a complete steam-sterilized, semisynthetic diet. After intravenous injection of cholesterol-26-(14)C the animals were housed in a plastic metabolism chamber for 72 hr. Expired CO(2) was collected throughout the period. The conventional rats released 50% more (14)C as (14)CO(2) than the germfree animals. The total amount of the label recovered as (14)CO(2) during the 72 hr period amounted to 30% and 19% respectively, of the original dose. In both conventional and germfree rats the release of (14)CO(2) accounted for approximately 75% of the (14)C recovered in forms other than the original cholesterol-26-(14)C; 15-20% was found incorporated in water-soluble and fat-soluble fractions other than 3Beta-OH sterol of liver and carcass while the remainder was excreted with feces and urine. After the 72 hr period the specific activities of the cholesterol in plasma and liver were lower in conventional than in germfree animals. The data express the accelerating effect of the intestinal microflora on systemic cholesterol catabolism. They demonstrate that the release of (14)CO(2) from cholesterol-26-(14)C in the intact rat is a suitable and convenient indicator of the oxidative catabolism of cholesterol.

Effect of bile acid deconjugation on the fecal excretion of steroids.

The effect of microbiological deconjugation of bile acids on total bile acid and neutral sterol fecal excretion by adult male rats has been studied. A screening method utilizing mice allowed selection of a Clostridium perfringens type A strain, which accelerated cholesterol catabolism in mice. When this species of bacteria was associated with germfree rats, the fecal bile acids were excreted as free bile acids (deconjugated), however the quantities of bile acids excreted were not increased compared with those of germfree rats. Conventional rats excrete twice as much bile acids (all deconjugated) as do the germfree and C. perfringens-associated rats. It is, therefore, unlikely that the microbiological deconjugation of bile acids is responsible for the increased fecal excretion of bile acids seen in conventional rats. The C. perfringens-associated rats excreted identical kinds and quantities of fecal neutral sterols as did the germfree rats.

Oxygen consumption and thyroid hormones in germfree mice fed glucose-amino acid liquid diet.

Germfree rats and mice fed solid diets have previously been shown to consume less oxygen than their conventional counterparts. Young adult germfree C3H/He mice how serum T3 concentrations comparable to those of conventional mice, but their serum T4 values are 10-15% lower. Fed chemically defined liquid glucose-amino acid diets, germfree C3H mice use 27% more oxygen than comparable germfree mice fed a solid natural ingredient diet, and 12% more than conventional mice fed the solid diet. Like germfree rats, the germfree mice fed natural ingredient diet had smaller hearts and livers than those of conventional mice. However, the chemically defined diet-fed germfree C3H mice showed larger hearts than conventional C3H mice fed solid diets, and livers of comparable size. Serum T3 levels were similar in the two germfree groups at both 3 and 8 months of age, while serum T4 levels were again slightly but significantly higher in the germfree chemically defined diet group at both ages. Both T3 and T4 decline with age in conventional mice, but not in either germfree group, similar to findings in germfree rats. The data suggest that germfree mice fed chemically-defined diet require additional energy expenditure to effectively utilize its major dietary components.

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.