The Internet and the nutritional scientist.

The Internet provides many opportunities to learn, to educate, and to communicate new ideas. This article introduces concepts and terms that will facilitate the use of electronic information media by nutritional scientists. A vast array of sites on the Internet are relevant to the nutritional scientist, including those developed by government, industry, and educational sources, professional societies, and individuals. Using the wide variety of electronic sources that make up the Internet in an efficient and effective manner is an important skill not only for locating specific information, but also for keeping abreast of novel developments and new concepts as they are introduced and discussed. Uncritical acceptance of information appearing in the electronic media, however, is problematic; electronic publishing may occur without the rigorous peer-review process common for publishing in scientific journals. Those intending to publish material electronically must accept responsibility for the veracity of the information, realizing that anyone, from the lay consumer to the professional, may have access to that information. The Internet and its electronic relatives (eg, the World Wide Web and newsgroups) can become invaluable tools for nutritional scientists, extending beyond traditional sources of information (eg, the library) to support research and educational efforts, but use of this new technology must be tempered with knowledge of its limitations as well as potentials.

Scurvy results in decreased collagen synthesis and bone density in the guinea pig animal model.

The effect of severe ascorbic acid deficiency on bone remodeling and collagen synthesis was evaluated in a 21 day experiment, using the scorbutic guinea pig model. Animals (n = 6-7/group) were assigned to one of three groups: scorbutic, pair-fed ascorbic acid-replete, or ad libitum ascorbic acid-replete groups. After 2 weeks, scorbutic animals started voluntarily decreasing food intake and losing weight. By day 19-21, at which time bone and tissue samples were collected and analyzed, scorbutic animals decreased food intake to 46% of usual and lost 9% body weight. Serum 25OHD3, 1,25(OH)2D3, calcium, and albumin were significantly lower (p < 0.05) in the scorbutic animals than in the other groups. Bone mineral density and bone mineral content of the proximal and central femur were significantly lower in the scorbutic group than in the other groups (p < 0.05). Morphometric analysis of tibia indicated significantly lower bone volume, fewer and thinner trabeculae, and a thinner growth plate in the scorbutic group, compared to the pair-fed and ad libitum groups (p < 0.05). Osteoclast surface was about 60% higher in the scorbutic group than in the pair-fed and ad libitum control groups (0.05 < p < 0.10). Mechanical strength of the femur and lumbar vertebral body tended to be lower when bone mass was altered in the same group. Collagen synthesis of articular cartilage and tendons was lower in the scorbutic group than in the pair-fed or ad libitum groups (p < 0.05). In conclusion, scurvy but not food restriction, per se, results in alterations in bone mass and tissue collagen synthesis.

Quantification of plasma cortisol levels in ascorbic acid studies using guinea pigs implanted with ACTH pellets.

A method is described that permits multiple blood collections from unanesthetized, unrestrained guinea pigs with minimal disruption to the animal, as verified by plasma cortisol (PC) levels. Blood (100-200 microliters) was collected from the lateral metatarsal vein using a vacuum-assisted method. Using this method, the effect of subcutaneously-implanted slow-releasing ACTH versus placebo pellets on PC levels was determined. A small, but significant, increase in PC concentration occurred in the control group, reflecting the stress of handling and the implant procedure. In contrast, the peak concentration of PC after implantation of a pellet containing ACTH was 20 times the starting level and occurred within 4 hr. However, the PC level was not correlated with the content of ACTH in the pellet because a similar level was achieved with 0.125 or 8 mg/pellet. A decline in Pc to pre-treatment levels occurred within 24 to 48 hr. The results indicate that administration of ACTH by pellet, although intended to achieve a sustained elevation in PC level, is not suitable for maintaining elevated PC levels.

The effect of bilateral adrenalectomy on metabolism and tissue distribution of ascorbic acid in the rat.

Adult, male, Sprague-Dawley rats were injected with [1-14C] -ascorbic acid 11 days post bilateral adrenalectomy or sham-adrenalectomy, and immediately placed in metabolism chambers. Excreta (CO2 and urine) were collected for 48 hours. The animals were then sacrificed and 10 tissues were processed and assayed for total ascorbic acid (AA) and radioactivity. There was no significant difference in weight gain between groups post-operation. Adrenalectomized rats excreted significantly less radioactivity, resulting in a significantly longer estimated half-life of AA (173 +/- 7 h) than the sham-adrenalectomized controls (141 +/- 7 h). Weights of tissues were similar between groups. The concentration of AA was significantly higher (43%) in the heart and lower (17%) in the liver in adrenalectomized animals. This group of animals also had significantly higher levels of radioactivity in the heart (51%), kidney, and spleen (19%). The specific activity of AA (% dose/mg AA) was not different between groups for any tissue examined. These results demonstrate that adrenalectomy affects the degradation of AA and its concentration in selected tissues, with a profound effect on the heart.

The influence of anesthesia and surgery on plasma cortisol and tissue ascorbic acid levels in the guinea pig.

The influence of methoxyflurane anesthesia alone compared to anesthesia and laparotomy surgery on plasma cortisol (PC) levels and tissue ascorbic acid (AA) levels was examined in adult male albino guinea pigs (485 +/- 7 g) consuming 500 mg AA/kg diet. Anesthesia resulted in a 60% decrease in food intake on day 1 after treatment, while surgery resulted in a 71% decrease. By day 4, food intake for both groups was equal to that of control animals. Body weight decreased 5-7% on day 1 after both treatments; recovery of body weight to pre-treatment levels was slowest in the animals exposed to surgery. After anesthesia alone, PC levels were twice that of controls by 1 hr and the five-fold higher peak PC level was observed at 9 hr. Surgery induced a more immediate five-fold rise in PC level that plateaued at 1 hr. PC levels decreased to levels similar to the control animals after 72 hr after both treatments. Weight, percent water, and AA levels of tissues examined at seven days after treatment were similar among groups. The results indicate that the adrenal cortical response to surgery is of the same magnitude as for anesthesia alone, although the peak PC level is more quickly achieved. Tissue AA levels are similar seven days after surgery or anesthesia alone even though elevated PC levels sustained for several days.

Comparison of isotope dilution and excretion methods for determining the half-life of ascorbic acid in the guinea pig.

The half-life of ascorbic acid (AA) in guinea pigs was investigated by the isotope dilution and excretion methods. The dilution method measures [1-14C]AA disappearance from the plasma, whereas the excretion method measures the elimination of [1-14C]AA and the metabolites from the body. Two groups of animals underwent both isotope studies in reverse order. Animals were conditioned to the experimental procedures and fed 2.5 mg AA/100 g body weight orally to maintain a daily intake of the vitamin independent of food consumption. The two isotope procedures imposed similar stress on the animals, as determined by plasma cortisol levels and body weight changes. The AA half-life calculations of the rapidly exchangeable pool by the isotope dilution method yielded values of 1.23 and 0.34 hours for the two groups, respectively. The half-life of the slowly exchangeable pool for the two groups was 60.2 and 65.8 hours, respectively. The half-life of AA in the rapidly exchangeable pool, as measured by the excretion studies, was 4.57-8.75 hours. For the slowly exchangeable pool, it was 146-149 hours. The longer half-life of both pools obtained with the excretion method indicates that the isotope is disappearing from the plasma more rapidly than it is being excreted. This suggests that a portion of the [1-14C]AA leaving the plasma is removed to a body pool that is not sampled by the isotope excretion method.

Uptake and release of adrenal ascorbic acid in the guinea pig after injection of ACTH.

The effect of a single injection of ACTH (3 IU/100 g body weight) on the distribution of ascorbic acid (AA) and radiolabeled AA in 20 tissues was studied in adult male guinea pigs consuming 500 mg AA/kg diet. Saline- or ACTH-injected animals were simultaneously injected with [1-14C]AA, and killed at 0.5, 1, 2, 4 and 6 h after injection. There was no significant difference between treatments in the weight of any tissue over the 6-h experimental period. As anticipated, the concentration of AA in the adrenals of animals injected with ACTH was 33% of that of animals injected with saline at 4 h. Unexpectedly, the concentration of radiolabeled AA in the adrenals at 0.5 h after ACTH injection was 172% of that after saline injection. The concentration of radiolabeled AA in the adrenal of the saline-injected animals increased slowly over time to reach a level similar to that of ACTH-injected animals by 6 h. There was no effect of ACTH on the level of AA or uptake in any of the other tissues examined. These results demonstrate that a single dose of ACTH markedly influences the retention of AA in the adrenal gland without similarly altering retention of AA in other tissues. Furthermore, ACTH treatment causes both accelerated uptake and release of AA into the adrenals.

Effectiveness of isoascorbate versus ascorbate as an inducer of collagen synthesis in primary avian tendon cells.

In contrast to most biologically active molecules, the isomeric form of ascorbate retains significant biological activity. Moreover, in studies in vitro the isomer was found to be an equally effective cofactor in the enzymatic proline hydroxylation reaction. This raises questions about whether the lower biological activity in vivo results from selective transport into the cell, greater instability of the molecule, or stereospecificity by certain enzyme complexes. Distinguishing these possibilities can be accomplished most directly using a cell culture model. In this study primary avian tendon (PAT) cells were used. With PAT cells isoascorbate was shown to be three- to fivefold less active at inducing procollagen production than ascorbate. Isoascorbate was also internalized by the cell at about one-fifth the ascorbate level. In addition, isoascorbate was degraded in the medium at a slightly higher rate (half-life of 1.6 h) than ascorbate (2.1 h). The data are consistent with a model that postulates that once inside the cell isoascorbate is equally effective at inducing procollagen production but selectivity at the transport step restricts the percentage that is actually internalized. In addition, both ascorbate and isoascorbate were found to degrade very quickly inside the cell in the highly oxygenated environment of cell culture (approximately 2 h half-life). When ascorbate was added to the medium (100 micrograms/mL) the level inside the cell quickly reached a maximum (less than 2 h) and declined rapidly.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma cortisol and adrenal ascorbic acid levels after ACTH treatment with a high intake of ascorbic acid in the guinea pig.

The influence of ascorbic acid (AA) intake on plasma cortisol (PC) and tissue AA levels after ACTH treatment was examined using guinea pigs. ACTH produced an 8- to 10-fold rise in PC levels (p less than 0.0001) over the 4-hour experimental period compared to gel-injected control animals. The magnitude of rise in PC was similar at both normal (0.50 g/kg diet) and high (10 g/kg diet) AA intakes. A 30-100% higher (p less than 0.0001) level of AA in tissues (adrenals, liver, and kidneys) and plasma was observed with the high AA diet. ACTH resulted in a 26-30% lower level of AA in the adrenals (p less than 0.0001), but not in other tissues or plasma. PC and adrenal AA responses were unrelated (r = -0.326). These results suggest that ACTH alters AA only in the adrenals, the PC response to and percent decrease in adrenal AA levels with ACTH are not related to AA status, and the absolute level of AA in the adrenals is not critical for steroidogenesis.

Nutritional factors affecting serum phenylalanine concentration during pregnancy for identical twin mothers with phenylketonuria.

UNLABELLED: The effect of energy, protein, fat, and phenylalanine on serum phenylalanine concentrations during pregnancy for a set of identical twins with phenylketonuria (PKU) was examined. Blood samples were collected one to two times per week. The subjects completed a 3-d food record prior to each blood collection. The effect of the factors on serum phenylalanine levels was evaluated statistically using time-series analysis. Dietary intakes of the nutrients evaluated were similar for the subjects. For one subject, there were highly significant effects of energy, protein, and fat on serum phenylalanine levels. In contrast, these nutrients had no significant effect on serum phenylalanine for the other subject. Dietary phenylalanine had no significant effect on serum phenylalanine for either twin. CONCLUSIONS: There was no effect of phenylalanine intake and no consistent effect of energy, protein, or fat on serum phenylalanine. Other dietary or environmental factors or a combination of factors may impact serum phenylalanine levels of pregnant women with PKU.

Selective reversal of H-2 linked genetic unresponsiveness to lysozymes. I. Non-H-2 gene(s) closely linked to the Ir-2 locus on chromosome 2 permit(s) an antilysozyme response in H-2b mice.

Genes outside of the mouse major histocompatibility complex (H-2) were found to be capable of specifically reversing the previously described nonresponsiveness to hen egg-white lysozyme (HEL) owing to H-2b immune response (Ir) genes. C3H.SW, BALB.B, and C57L, all of the H-2b haplotype, showed responsiveness to HEL, but not to human lysozyme (HUL). Mapping of the reversing gene(s) was attempted by testing H-2b recombinant inbred (RI) strains of mice carrying C3H, BALB, and C57L non-H-2 genes. Analysis of the strain distribution pattern of responsiveness with both CXB and BXH RI strains was consistent with the location of the responsible site within the H-3 region on chromosome 2. The anti-HEL proliferative responsiveness in two H-3 congenic strains of mice, B10.C(28NX)SN and B10.C-H-3cH-3a, that have BALB/c genes within the H-3 region confirmed the mapping, as well as localized the reversing gene(s) near the Ir-2 gene. The data are discussed with regard to the site of expression of the reversing gene(s) and its mechanism of action.

Circulating insulin-like growth factor binding proteins (IGFBPs) 1 and 2 induced in vitamin C-deficient or fasted guinea pigs inhibit IGF-I action in cultured cells.

Collagen gene expression and proteoglycan synthesis are decreased in vitamin C-deficient guinea pigs losing weight and in fasted guinea pigs receiving ascorbate. Sera from such guinea pigs contain an insulin-like growth factor (IGF)-I-reversible inhibitor of collagen, proteoglycan and DNA synthesis and elevated levels of 29 and 35-kDa IGF binding proteins (IGFBPs). We now have identified the induced proteins as IGFBPs 1 and 2 and investigated their role as inhibitors. Guinea pig sera were treated with antibodies to IGFBPs 1 and 2 and antibody-IGFBP complexes were removed by passage through a Protein A-Sepharose column. Inhibitor content of fasted and scorbutic sera, and Protein A pass-through fractions derived from them, was assessed by their level of stimulation of DNA and collagen synthesis in 3T3 cells, compared to analogously treated normal guinea pig serum. Removal of IGFBP-1 from scorbutic serum reversed inhibition of collagen and DNA synthesis by more than half but removal of IGFBP-2 was less effective. Removal of both IGFBPs reversed inhibition almost completely. Similar results were obtained with fasted guinea pig serum. Conversely, purified rat IGFBPs 1 and 2 inhibited DNA and collagen synthesis in cells cultured in normal guinea pig serum or IGF-I-stimulated DNA synthesis, with IGFBP-1 being more potent. Thus, IGFBP-1 and, to a lesser extent IGFBP-2, cause inhibition of IGF-I action by sera from fasted and scorbutic guinea pigs and may inhibit collagen gene expression in vivo.

Controlled changes in chronic dietary protein intake do not change glomerular filtration rate.

The effect on renal function (creatinine clearance [Ccreat] and inulin clearance [Cinulin]) of changes in chronic dietary protein intake was studied in seven healthy male subjects. Serial 24-hour urine collections were used to determine creatinine excretion (UcreatV) and Ccreat. Subjects were examined after ad libitum (ad lib) food intake and after 2-week periods of high protein diet ([HPD] 1.6 g/kg body weight [BW] per day) and low protein diet ([LPD] 0.5 g/kg BW per day). Inulin clearance (Cinulin) was determined at the end of each 2-week diet period. UcreatV increased from 1,838.8 +/- 97.2 mumol/kg (20.8 +/- 1.1 mg/kg) BW to 2,068.6 +/- 106.1 mumol/kg (23.4 +/- 1.2 mg/kg) BW daily during HPD and decreased significantly to 1,555.9 +/- 167.9 mumol/kg BW per day (17.6 +/- 1.9 mg/kg BW per day) with beginning of LPD. Ccreat rose from 1.54 +/- 0.09 mL/s 1.73 m2 (92.5 +/- 5.5 mL/s.1.73 m2 (104.7 +/- 4.9 mL/min/1.73 m2) during HPD and decreased to 1.23 +/- 0.04 mL/s.1.73 m2 (74.0 +/- 2.2 mL/min/1.73 m2) with initiation of LPD. There was no difference between Cinulin after HPD (1.42 +/- 0.12 mL/s.1.73 m2; 84.9 +/- 7.2 mL/min/1.73 m2) and after LPD (1.36 +/- 0.05 mL/s.1.73 m2; 81.4 +/- 2.9 mL/min/1.73 m2). This study confirms the effect of protein intake on Ccreat and UcreatV, but fails to show an effect of changes in chronic protein intake on glomerular filtration rate (GFR). Ccreat during dietary protein restriction to 0.5 g/kg/d is similar to Cinulin and may be a useful measure of GFR under circumstances where more specific inulin or isotope studies are not available.

Partial overlap of Ir gene-controlled responses to two proteins of limited relatedness: hen egg-white lysozyme and human lysozyme.

The plaque-forming cell (PFC) response to human lysozyme (HUL) is regulated by an Ir gene(s) located within the major histocompatibility complex of the mouse. Mice of H-2a, H-2k, H-2v and H-2r haplotypes respond to HUL, whereas mice with H-2b, H-2d, H-2q, H-2s and H-2u haplotypes fail to generate substantial anti-HUL PFC responses. In contrast, only mice carrying the H-2b and H-2s haplotypes are non-responders to the distantly related hen eggwhite lysozyme (HEL). The major genetic control of the anti-HUL PFC response maps to the I-A subregion of the H-2 complex with perhaps a minor influence by a gene mapping to the right of the I-B subregion. HUL and HEL induce a cross-reactive suppressor cell, directed against a particular determinant found on both lysozymes. Once generated, these antigen-specific suppressor cells can affect the in vitro primary response to either lysozyme conjugated to sheep red blood cells. Despite this overlap, the strain distribution pattern of responsiveness is different for the two lysozymes. In the discussion, this was attributed to the MHC-related failure to process and/or present HEL to the HEL/HUL cross-reactive suppressor T cell in H-2q, d and u strains.

Multiple H-2 and non-H-2 genes controlling the antilysozyme response: alternative gene constellations can lead to responsiveness.

Mice carrying the H-2b and H-2s haplotypes are genetically nonresponsive to hen egg-white lysozyme (HEL). Analysis of the anti-HEL response patterns of F1, F2 and backcross progeny showed that responsiveness was dominant and H-2 linked. From plaque-forming cell and serum assays in intra-H-2 recombinant mice, it was established that two I loci were implicated, the possession of either leading to responsiveness to HEL. One of the I genes maps in I-A, and the second in I-C, S or G. While the nonresponse phenotype was determined by the H-2 haplotype, there were codominant non-H-2 genes which contributed to a severe reduction in the level of antibody produced in responder strains. A model is presented attributing the outcome of an encounter with HEL to the regulatory balance of helper and suppressor T cells, which have been activated by different subregions of the major histocompatibility complex.

Delayed advent of stringent, non-H-2 genetic regulation of the antibody response to a protein antigen.

The role of non-H-2 gene(s) in the control of the antibody response to three lysozymes was investigated. Upon secondary challenge, A/J (H-2a) mice generated at least a 25-fold greater anti-lysozyme plaque-forming cell response than did B10.A (H-2a) mice. Nearly equal, strong peak primary responses, predominantly IgG in nature, were obtained from both A/J and B10.A mice after a single challenge with lysozyme in complete Freund's adjuvant. However, clear differences in responses are seen within 5 days after the peak primary plaque-forming response and by day 28 at the serum antibody level. B10.A mice never equal their primary responses, whereas A/J mice demonstrate positive immune memory. It appears that a non-H-2 gene(s) that regulates the overall antibody level to a protein antigen becomes manifest only after an initial antibody response.

Long-term low ascorbic acid intake reduces bone mass in guinea pigs.

The effect of long-term (1 y) low to excess ascorbic acid (AA) intake on bone mass was evaluated using guinea pigs that were 12-14 d old at the start of the experiment. Dietary AA was low (0.15 g/ kg diet) (n = 7), normal (0.50 g/kg) (n = 8) or excess (10 g/kg) (n = 8). After 12 mo, total body bone mineral density (BMD, mg/cm2) and bone mineral content (BMC, g) were determined by dual energy X-ray absorptiometry. Histomorphometric analysis of the cancellous bone of the proximal tibial metaphysis was completed after in vivo dual fluorochrome labeling. Total body BMD of the low AA group was 4.9% lower (P < 0.05), and total body BMC was 12.4% lower (P < 0.05) than in the normal AA group. Total body BMD and BMC were similar in normal and excess AA groups and in the low and excess AA groups. Histomorphometric analysis indicated significantly greater (P < 0.05) double-labeled bone surface, mineralizing surface, and bone formation rate in the low AA guinea pigs compared with the normal AA animals. Thus, there was greater bone turnover in the low AA group than in the normal AA guinea pigs. No differences in histomorphometric endpoints existed between the normal AA and excess AA groups. Long-term AA deficiency, during the period of rapid growth and slower phases of skeletal maturation, resulted in bone abnormalities in adult guinea pig skeletons. Long-term dietary AA excess caused no such abnormalities.