Relationship between intake and plasma concentrations of vitamin B12 and folate in 873 adults with a physically active lifestyle: a cross-sectional study.

BACKGROUND: Vitamin B12 and folate function as co-factors in pathways used during physical activity. Physical activity may therefore increase vitamin requirements, leading to a risk of deficient plasma concentrations. We aimed to investigate the relationship between intake and plasma concentrations of vitamin B12 and folate in physically active adults, as well as identify other determinants of vitamin B12 and folate plasma concentrations. METHODS: The study population consisted of 873 adults (528 men and 345 women), aged 19-78 years, who participated in a 4-day walking event. The relationship between intake and plasma concentrations of vitamin B12 and folate was assessed using correlation and linear regression analyses. In addition, potential other determinants (sex, age, body mass index, energy intake and physical activity) of vitamin plasma concentrations were investigated. RESULTS: Significant positive correlations were observed between intake and plasma concentrations of vitamin B12 [Pearson's correlation coefficient = 0.15; 95% confidence interval (CI) = 0.08-0.21] and folate (Pearson's correlation coefficient = 0.18; 95% CI = 0.12-0.25). In addition to vitamin intake, sex, age and energy intake were also determinants of both vitamin B12 and folate plasma concentrations in multivariable regression models. CONCLUSIONS: The results suggest a positive association between intake and plasma concentrations for both vitamin B12 and folate in physically active people. By contrast to our hypothesis, physical activity was not a determinant of vitamin B12 and folate plasma concentrations. However, sex, age and energy intake were found to be determinants. Thus, when studying the relationship between intake and plasma concentrations of vitamin B12 or folate, these factors should be taken into account.

Determinants of vitamin D status in physically active elderly in the Netherlands.

PURPOSE: Vitamin D deficiencies are common in elderly, which increases the risk for, e.g., bone fractures. Identification of determinants of vitamin D status may provide leads for specific deficiency prevention strategies. Although determinants of vitamin D status have been studied in various populations, this has not been examined in elderly that have a physically active lifestyle. METHODS: Vitamin D status of 450 physically active elderly who do not use vitamin D supplements was determined and information on possible determinants (demographic, dietary intake and physical activity) was collected around a prolonged four day walking event in July and analyzed in linear regression models. RESULTS: The average summertime serum 25(OH)D concentration was 88.8 ± 22.4 nmol/L. Only 2% of the participants had a 25(OH)D concentration below 50 nmol/L. Dietary intake of vitamin D was 4.0 ± 1.9 µg/day, and the participants spent 12.4 ± 8.6 h/week on outdoor activities. In the multivariate model, lower age (= - 0.48, 95% CI - 0.80 to - 0.16), lower BMI (= - 0.86, 95% CI - 1.62 to - 0.10), being a moderate to high drinker versus a non-drinker (= 7.97, 95% CI 0.43-15.51) and more outdoor physical activity (= 0.25, 95% CI 0.01-0.50) were significantly associated with higher 25(OH)D concentrations. CONCLUSIONS: In physically active elderly, vitamin D status was very high in summertime, with few deficiencies, suggesting that elderly with a physical active lifestyle might not necessarily need supplements during the summer period. Lower age, lower BMI, higher alcohol intake and more outdoor physical activity had a significant association with vitamin D status.

Changes in iron metabolism during prolonged repeated walking exercise in middle-aged men and women.

PURPOSE: The aim of the present study was to assess the effect of prolonged and repeated exercise on iron metabolism in middle-aged adults and to compare differences between sexes. METHODS: 50 male (58.9 ± 9.9 year) and 48 female (50.9 ± 11.2 year) individuals were monitored on 4 consecutive days at which they walked on average 8 h and 44 min per day at a self-determined pace. Blood samples were collected 1 or 2 days prior to the start of the exercise (baseline) and every day immediately post-exercise. Samples were analysed for iron, ferritin, haemoglobin, and haptoglobin concentrations. RESULTS: Plasma iron decreased across days, while ferritin increased across days (both p < 0.001). Haptoglobin showed a decrease (p < 0.001) after the first day and increased over subsequent days (p < 0.001). Haemoglobin did not change after the first day, but increased during subsequent days (p < 0.05). At baseline, 8% of the participants had iron concentrations below minimum reference value (10 µmol/L), this increased to 43% at day 4. There was an interaction between sex and exercise days on iron (p = 0.028), ferritin (p < 0.001) and haemoglobin levels (p = 0.004), but not on haptoglobin levels. CONCLUSION: This study showed decreases in iron, increases in ferritin, a decrease followed by increases in haptoglobin and no change followed by increases in haemoglobin. This is most likely explained by (foot strike) haemolysis, inflammation, and sweat and urine losses. These processes resulted in iron levels below minimum reference value in a large number of our participants.

Ionized and Total Magnesium Levels Change during Repeated Exercise in Older Adults.

BACKGROUND: Magnesium is essential for health and performance. Sub-optimal levels have been reported for older persons. In addition, physical exercise is known to temporally decrease magnesium blood concentrations. OBJECTIVE: To investigate these observations in conjunction we assessed total (tMg) and ionized magnesium (iMg) concentrations in plasma and whole blood, respectively, during 4 consecutive days of exercise in very old vital adults. DESIGN: 68 participants (age 83.7±1.9 years) were monitored on 4 consecutive days at which they walked 30-40km (average ~8 hours) per day at a self-determined pace. Blood samples were collected one or two days prior to the start of exercise (baseline) and every walking day immediately post-exercise. Samples were analysed for tMg and iMg levels. RESULTS: Baseline tMg and iMg levels were 0.85±0.07 and 0.47±0.07 mmol/L, respectively. iMg decreased after the first walking day (-0.10±0.09 mmol/L, p<.001), increased after the second (+0.11±0.07 mmol/L, p<.001), was unchanged after the third and decreased on the final walking day, all compared to the previous day. tMg was only higher after the third walking day compared to the second walking day (p=.012). In 88% of the participants, iMg levels reached values considered to be sub-optimal at day 1, in 16% of the participants values were sub-optimal for tMg at day 2. CONCLUSION: Prolonged moderate intensity exercise caused acute effects on iMg levels in a degree comparable to that after a bout of intensive exercise. These effects were not associated with drop-out or health problems. After the second consecutive day of exercise, levels were returned to baseline values, suggesting rapid adaptation/resilience in this population.

Molecular cloning and expression of the relaxin-like factor from the mouse testis.

A complementary DNA encoding the mouse homolog of the testicular relaxin-like factor was cloned from a subtractive complementary DNA library, comprising clones preferentially expressed in the testes of mutant w/wv mice. The predicted amino acid structure conforms with that for other members of the insulin, insulin-like growth factor, and relaxin hormone family, with A, B, and C (connecting) domains. Northern and in situ transcript hybridization showed that the gene is exclusively expressed at a high level in the Leydig cells of both mutant and wild type testes and is up-regulated at puberty. The level of the specific messenger RNA is such as to imply that this novel member of the insulin family represents a major secretory product of Leydig cells, with a potential to be a part of a novel systemic feedback loop to other organs, including the brain.

Ontogeny of oxytocin receptors and oxytocin-induced stimulation of prostaglandin synthesis in prepubertal heifers.

Developmental aspects of oxytocin (OT) receptors (OTR) in uterine tissues before puberty are not known. Bovine ovaries secrete some estradiol, but no progesterone, before puberty; the circulating levels of estradiol are between 1 and 3 pg/ml until puberty. Cross-bred Angus-Brahman heifers, in which puberty occurs around 12 months of age, were used to determine the concentrations of OTR from the late fetal stage to adulthood. PGF2alpha release in response to OT was determined in 3-, 6-, and 9-month-old heifers (n = 4 each). Myometrium, endometrium, and cervical mucosa were obtained from 3-week-old, 3-month-old, 6-month-old, and 9-month-old heifers and from adult cows at estrus. Whole uterus and cervix were taken from third trimester fetuses and at birth. [3H]OT binding and specificity, localization of immunoreactive (ir) OTR, OTR messenger RNA, and OT-induced release of PGF2alpha were determined. The uterus from fetuses and the neonate expressed OTR messenger RNA and bound [3H]OT. At 3 weeks of age, OTR concentrations per mg protein were very low, but at 3 months of age they had increased markedly in all three tissues. At 6 and 9 months of age, levels of OTR had risen further and were similar to those in adult cows at estrus. Prepubertal uterus also possessed separate vasopressin VP1 subtype receptors. The ir-OTR was localized in luminal epithelial cells of endometrium and cervical mucosa, most of which were ir positive, whereas in myometrium, clusters of ir-OTR-positive cells were found among large numbers of ir-OTR-negative cells. The PGF2alpha response to OT was insignificant in heifers of all age groups, in contrast to that in cows at estrus. Endometrial cells from 4- to 5-month-old heifers did not respond to OT with PG release in the absence or presence of added arachidonic acid. Tumor promoters, lipopolysaccharide, and interleukin-2 also failed to elicit PG release in vitro, although they induced PG release in similar cell cultures from cyclic cows. In summary, uterine tissues of prepubertal heifers have high levels of OTR, which appear to be developmentally regulated. These receptors are not coupled to PG synthase, or alternatively, the PG synthase gene is not expressed before puberty, possibly because the tissues have had no previous exposure to progesterone.

Relaxin-like factor expression as a marker of differentiation in the mouse testis and ovary.

Expression of the relaxin-like factor (RLF) was studied at the messenger RNA (mRNA) and protein levels in the testes and ovaries of the mouse, as well as through testicular development and differentiation in the mouse testis. In situ hybridization or RT-PCR, and immunohistochemistry using a polyclonal antibody raised against a recombinant protein, provided mutually confirmatory results for a high expression of RLF in the Leydig cells of the adult testis and at a much lower level of expression in the luteal cells of the ovary through the cycle, pregnancy, and in lactation. Analysis of protein and mRNA expression, through postnatal testicular development, indicated moderate RLF expression also in the fetal population of Leydig cells, even in the hpg mutant mouse, lacking an active pituitary-gonadal axis. Prepubertal Leydig cells, however, exhibit only very low-level RLF gene expression, this phenotype persisting in the adult hpg mouse. In summary, fetal Leydig cells express RLF in an LH/human CG-independent fashion, whereas LH/human CG is essential to induce RLF expression in the adult-type Leydig cell. In cultured adult Leydig cells or in the mouse tumor MA-10 cell line, RLF mRNA is expressed in a constitutive fashion. RLF thus seems to be a useful marker of Leydig cell differentiation status.

Progressive inactivation of the haploid expressed gene for the sperm-specific endozepine-like peptide (ELP) through primate evolution.

The endozepine-like peptide (ELP) is a novel intracellular molecule which is expressed in high amounts at both mRNA and protein levels very specifically in late haploid male germ cells. It is closely related to the ubiquitous acyl-CoA binding protein, is highly conserved, shares a similar ability to bind mid-long chain acyl-CoA, and is thus likely to be involved in mature sperm metabolism. While it has been characterized from diverse mammals, it has so far not been possible to identify an equivalent molecule in the primate testis. Using a PCR approach, combined with cDNA cloning and Northern hybridization, testicular transcripts and/or genomic DNA were analysed for different primate species, including human. In the marmoset and cynomolgus macaque normally structured transcripts appear to be expressed, though at a low level. In the human testis, two rare transcripts were characterized, hELP1 and hELP2, the products of independent duplicated genes. Both transcripts were longer than in non-mammalian species, included frame-shift mutations and substantial sequence insertions, preventing the translation of a sensible protein. Genomic PCR analysis of three anthropoid species, chimpanzee, gorilla and orangutan, showed the presence of a similarly mutated hELP1 gene. Only in the gorilla was a hELP2 gene identified, apparently lacking the frame-shift mutation, and thus potentially able to give rise to a functional ELP protein. Taken together, these results show that during primate evolution there has been a progressive inactivation of the ELP gene, initially with a down-regulation in lower primates, and subsequently with inactivating mutations in the open reading frame. At some time during simian evolution prior to these mutations there has been a gene duplication, though this second gene has also become inactivated in humans. In its pattern of evolution the ELP gene shows similarities with the MDC/fertilin family, whose members are also considered essential components of haploid sperm in non-primates, but which are progressively inactivated in anthropoids and humans. We should like to speculate that the established subfertility of the human male may not be a recent event, but the consequence of a longer evolutionary process whereby primates have traded off absolute fertility against social or sexual advantages.

A novel endozepine-like peptide (ELP) is exclusively expressed in male germ cells.

A cDNA clone encoding a novel endozepine-like peptide (ELP) was isolated from mouse testes, sequenced, and its mRNA expression characterized by northern and in situ hybridization. ELP mRNA was found exclusively in the late spermatid stages of spermatogenesis in the testes of sexually mature mice and in no other tissue or cell type examined. It was also expressed in rat, bovine, porcine and sheep testes. Mouse ELP-encoding cDNA was used to construct expression vectors for the production of ELP in bacteria, and the purified bacterial protein used to raise polyclonal antibodies in rats. These antibodies identified the predicted endogenous ELP in extracts of mouse testis and epididymis and in no other tissue. Immunohistochemistry confirmed that the ELP antigen was present only in late spermatids and spermatozoa, particularly within the cytoplasmic droplet which is retained by the mature spermatozoa during their transit into the epididymis. We conclude that ELP is an intracytoplasmic peptide exclusively expressed in post-meiotic spermatozoa and which may be involved in the energy metabolism of the mature sperm.

Structure and expression of the bovine oxytocin receptor gene.

The gene for the bovine oxytocin receptor has been sequenced using a combination of clones derived from a bovine endometrial cDNA library from estrus and a bovine genomic DNA library, with confirmation of structure using reverse transcription PCR programmed by term myometrial RNA. The receptor belongs to the seven transmembrane domain family and predicts a protein of 391 amino acids. A comparison of the genomic sequence with the cDNA structure, as well as reverse transcription polymerase chain reaction (RT-PCR) analysis, shows there are two introns, one in the 5'noncoding region that appears to be differentially spliced in the bovine uterus and a conserved intron within the open reading frame between the regions encoding the transmembrane domains VI and VII. Northern blot analysis indicated three major transcripts in myometrium and endometrium in vivo at approximately 6.5 kb, 3.5 kb, and 2.0 kb. In situ hybridization analysis of uterine tissue at term showed highest mRNA concentrations in the endometrial epithelium, particularly in the deep glands, a pattern confirmed also at the immunohistochemical level by monoclonal antibodies raised against a human amino-terminal peptide. Further confirmation of the identity of the receptor was obtained by transient transfection of a reconstituted receptor construct into COS-7 cells. The expressed receptor was shown to have identical pharmacological properties in respect to various oxytocin analogs to the natural bovine endometrial receptor.

Structure and organization of the bovine oxytocin receptor gene.

A DNA probe specific for the V and VI transmembrane domains of the bovine oxytocin receptor was initially prepared by reverse transcription PCR, and its structure and specificity confirmed by DNA sequencing. This probe was then used to screen a bovine genomic DNA library in bacteriophage lambda, and three positive clones were purified, subjected to restriction analysis and relevant fragments sequenced. Parallel to this, a cDNA library prepared using bovine endometrial RNA at the time of ovulation was screened by PCR employing the same primers as above. The longest cDNA clone was also fully sequenced. This clone still lacked, however, a substantial stretch of 5'sequence. The full transcript structure, and hence also the exon-intron organization, was then obtained by RT-PCR using primer oligonucleotides deduced from the cloned genomic sequence. All nucleotide sequence information was derived from a combination of two independent genomic clones, a cDNA clone and several independent RT-PCR reactions programmed by myometrial RNA, all in both strand orientations. The structural organization of the bovine oxytocin gene essentially conforms to that described for the human gene. Unlike the human gene, however, the 5'non-coding region of the primary transcript is interrupted by only a single intron, with a further intron in the coding region separating the sequences encoding the transmembrane domains VI and VII. The difference between this structure and that for the human gene suggests the existence of a differential splicing of 5' non-coding sequences.

Oxytocin and male reproductive function.

In the male mammal, the small peptide hormone oxytocin is produced in similar quantities within the hypothalamo-pituitary magnocellular system as in the female, yet for the male little is known about the physiology associated with this hormone. The present review summarizes what is known about the function of oxytocin in the male mammal and tries to take account of both central and systemic effects, and those linked with a local production of oxytocin within the male reproductive organs. In several species a pulse of systemic oxytocin, presumably of hypothalamic origin, appears to be associated with ejaculation. The systemic hormone could act peripherally stimulating smooth muscle cells of the male reproductive tract, but could also reflect central effects in the brain modulating sexual behaviour. In addition to systemic oxytocin, the peptide is also made locally within the testis, and possibly also the epididymis and prostate. In the former tissue it appears to have an autocrine/paracrine role modulating steroid metabolism, but may in addition be involved in contractility of the seminiferous tubules. However, the latter function may involve the mediacy of Sertoli cells which under some circumstances can also exhibit the components of a local oxytocin system. In the prostate of the rat and the dog oxytocin is linked again to steroid metabolism and may also act as a growth regulator. Finally, oxytocin in seminal fluid is discussed and its possible role in respect to the fate of the semen following ejaculation.

The evolution of the endozepine-like peptide (ELP) in the mammalian testis.

The endozepine-like peptide (ELP) is a testis-specific isoform of the acyl-CoA binding protein (ACBP) and shares the latter's peptide motif for binding mid-long chain acyl-CoA groups. ELP is expressed both as mRNA and protein at high levels in the testes of a wide range of mammals, including rodents, carnivores and ruminants. However, the ELP gene is progressively inactivated through primate evolution, with no protein detectable in a range of primates studied, including human. In nonprimate species, ELP is expressed in very late postmeiotic germ cell stages only, such that its function in these species is probably associated with the metabolism of the mature spermatozoon. Current research is looking at both the function of the ELP protein and the haploid regulation of the gene.

The evolution of the endozepine-like peptide (ELP) in the mammalian testis.

The endozepine-like peptide (ELP) is a testis-specific isoform of the acyl-CoA binding protein (ACBP) and shares the latter's peptide motif for binding mid-long chain acyl-CoA groups. ELP is expressed both as mRNA and protein at high levels in the testes of a wide range of mammals, including rodents, carnivores and ruminants. However, the ELP gene is progressively inactivated through primate evolution, with no protein detectable in a range of primates studied, including human. In nonprimate species, ELP is expressed in very late postmeiotic germ cell stages only, such that its function in these species is probably associated with the metabolism of the mature spermatozoon. Current research is looking at both the function of the ELP protein and the haploid regulation of the gene.

Molecular cloning and in vivo expression of the bovine steroidogenic acute regulatory protein.

Differential screening was used to select clones from a bovine luteal cDNA library, which were specifically expressed in late luteal stages. One of these clones encoded the bovine homologue of the Steroidogenic Acute Regulatory protein (StAR). The bovine StAR gene is transcribed as 3kb and 1.8kb transcripts, which differ in their sites of 3' polyadenylation. Both transcripts are highly expressed in corpora lutea of mid to late cycle and through pregnancy, but only at low levels in the early cycle. Northern analysis showed expression only in the corpus luteum and in the adrenal gland, but not in any other tissue examined. Within the protein coding region of the bovine StAR gene, there is a marked 124 base homology to the 5' non-coding region of another luteal transcript, TIMP-1, suggesting a possible common regulatory function for this sequence.

Relaxin-like factor gene is highly expressed in the bovine ovary of the cycle and pregnancy: sequence and messenger ribonucleic acid analysis.

Relaxin-like factor (RLF) is a new member of the insulin/insulin-like growth factor family of hormones and growth factors, which appears to be predominantly expressed in the Leydig cells of the testis. An analysis of male and female bovine tissues indicated that in the cow the RLF gene is also highly expressed in the female, mainly in the follicular theca interna and in the corpus luteum, with a pattern of gene expression very similar to that for the related peptide relaxin in other species. Sequence analysis of bovine testicular and luteal RLF and cDNA shows that the same gene is expressed in both male and female gonads. Because the bovine RLF sequence, like those from pigs, humans, and mice, retains, the putative receptor binding motif described for relaxin, it seems plausible that RLF might functionally substitute for relaxin in the cow, in which the latter peptide appears not to be significantly expressed.

The rat endozepine-like peptide gene is highly expressed in late haploid stages of male germ cell development.

The structure of the endozepine-like peptide (ELP) gene is closely related to the intracellular acyl-CoA binding protein (ACBP), but unlike the generalized distribution of the latter, it is restricted to the male germ cells of the testis. In the present study, a combination of nonradioactive in situ mRNA hybridization and immunohistochemistry was used to precisely determine the cellular expression patterns of ELP mRNA and protein in control and methoxyacetic acid (MAA)-treated rat testes. ELP transcripts are first detectable in late stages (step 6) of round spermatids, with transcription increasing through late-elongating steps. Translation of the ELP mRNA is delayed, with first immunohistochemical staining occurring in elongated spermatids at step 16, and protein accumulating through step 19. ELP immunoreactivity proves to be an excellent marker for late spermatid stages and highlights the presumably clonal recovery of spermatids following MAA treatment.

Structure and expression of the mouse gene encoding the endozepine-like peptide from haploid male germ cells.

The endozepine-like peptide (ELP) represents a testis-specific isoform of the ubiquitous acyl-CoA binding protein (ACBP) and is highly expressed in late haploid stages of male germ cell development. The genomic sequence of the functional ELP gene as well as that of a pseudogene were analysed from independent bacteriophage clones of a 129sv mouse genomic library. Unlike the ACBP gene, which comprises four exons, the ELP gene has only a single intron within the region of the 5' untranslated region, suggesting that, like some other haploid expressed genes, the ELP gene might have evolved by retroposon-mediated gene duplication. Primer extension analysis was used to define the start site for transcription and hence the 5' promoter region. Electrophoretic mobility shift analysis was carried out on this region comparing nuclear extracts from adult mouse testis with those from mouse liver. Several testis-specific DNA-protein complexes were observed throughout 700 bp upstream of the transcription start site. One of these could be identified as corresponding to a steroidogenic factor-1 (SF-1) binding element. Further analysis using pure transcription factors showed that this element at position -340 was able to bind specifically to both SF-1 and to the germ cell nuclear factor (GCNF). Immunohistochemical analysis using an ELP-specific antibody showed that expression was very restricted within the testis to the postmeiotic germ cells, and in the ovary to interstitial/luteal cells, cell-types known to express GCNF and SF-1, respectively. Testes of CREM-tau knockout mice, lacking all spermatogenic stages later than round spermatids, were devoid of ELP immunoreactivity, whereas in RAD6 knockout mice the few remaining elongated spermatids were clearly defined by this excellent late haploid marker product. The ELP gene and its product thus offer an ideal system with which to investigate the differentiation of late haploid stages of spermatogenesis.

Relaxin-like factor: a highly specific and constitutive new marker for Leydig cells in the human testis.

The complete protein-coding region of the human relaxin-like factor (RLF; formerly Ley-I-L) was cloned by reverse transcription-polymerase chain reaction from human testis and subcloned into a bacterial expression plasmid for the production of recombinant human RLF in Escherichia coli. Polyclonal antibodies were raised against the recombinant RLF, as well as against a peptide epitope from the B-domain of the RLF polypeptide. Antibodies were used for immunohistochemistry of Bouin-fixed, paraffin-embedded samples of human testis tissues. Specific immunoreactivity was located exclusively in the Leydig cells with a consistent high intensity of staining, showing similar spatial distribution to other Leydig cell markers, such as the luteinizing hormone (LH) receptor and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and to the pattern of RLF mRNA shown by in-situ transcript hybridization. In biopsy samples from patients with severe disturbances of spermatogenesis, RLF staining intensity was consistently high in all cases, unlike staining for 3 beta-HSD which varied considerably between patients. Immunostaining for RLF would thus appear to offer an interesting new marker for Leydig cells in human testis samples.