Circulatory and Urinary B-Vitamin Responses to Multivitamin Supplement Ingestion Differ between Older and Younger Adults.

Multivitamin and mineral (MVM) supplements are frequently used amongst older populations to improve adequacy of micronutrients, including B-vitamins, but evidence for improved health outcomes are limited and deficiencies remain prevalent. Although this may indicate poor efficacy of supplements, this could also suggest the possibility for altered B-vitamin bioavailability and metabolism in older people. This open-label, single-arm acute parallel study, conducted at the Liggins Institute Clinical Research Unit in Auckland, compared circulatory and urinary B-vitamer responses to MVM supplementation in older (70.1 ± 2.7 y, n = 10 male, n = 10 female) compared to younger (24.2 ± 2.8 y, n = 10 male, n = 10 female) participants for 4 h after the ingestion of a single dose of a commercial MVM supplement and standardized breakfast. Older adults had a lower area under the curve (AUC) of postprandial plasma pyridoxine (p = 0.02) and pyridoxal-5'phosphate (p = 0.03) forms of vitamin B6 but greater 4-pyridoxic acid AUC (p = 0.009). Urinary pyridoxine and pyridoxal excretion were higher in younger females than in older females (time × age × sex interaction, p < 0.05). Older adults had a greater AUC increase in plasma thiamine (p = 0.01), riboflavin (p = 0.009), and pantothenic acid (p = 0.027). In older adults, there was decreased plasma responsiveness of the ingested (pyridoxine) and active (pyridoxal-5'phosphate) forms of vitamin B6, which indicated a previously undescribed alteration in either absorption or subsequent metabolic interconversion. While these findings cannot determine whether acute B6 responsiveness is adequate, this difference may have potential implications for B6 function in older adults. Although this may imply higher B vitamin substrate requirements for older people, further work is required to understand the implications of postprandial differences in availability.

Robotic automation of a UHPLC/MS-MS method profiling one-carbon metabolites, amino acids, and precursors in plasma.

Amino acids (AAs) and one-carbon (1-C) metabolism compounds are involved in a range of key metabolic pathways, and mediate numerous health and disease processes in the human body. Previous assays have quantified a limited selection of these compounds and typically require extensive manual handling. Here, we describe the robotic automation of an analytical method for the simultaneous quantification of 37 1-C metabolites, amino acids, and precursors using reversed-phase ultra-high-pressure liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS-MS). Compound extraction from human plasma was tested manually before being robotically automated. The final automated analytical panel was validated on human plasma samples. Our automated and multiplexed method holds promise for application to large cohort studies.

Sex-Specific Associations of Blood-Based Nutrient Profiling With Body Composition in the Elderly.

The intake of adequate amounts and types of nutrients is key for sustaining health and a good quality of life, particularly in the elderly population. There is considerable evidence suggesting that physiological changes related to age and sex modify nutritional needs, and this may be related to age-associated changes in body composition (BC), specifically in lean and fat body mass. However, there is a clear lack of understanding about the association of nutrients in blood and BC parameters in the elderly. This study investigated the relationships among blood nutrients (amino acids, fatty acids, major elements, trace-elements, and vitamins), BC and nutrient intake in a population of 176 healthy male and female Italian adults between the ages of 65 and 79 years. 89 blood markers, 77 BC parameters and dietary intake were evaluated. Multivariate data analysis was applied to infer relationships between datasets. As expected, the major variability between BC and the blood nutrient profile (BNP) observed was related to sex. Aside from clear sex-specific differences in BC, female subjects had higher BNP levels of copper, copper-to-zinc ratio, phosphorous and holotranscobalamin II and lower concentrations of branched-chain amino acids (BCAAs) and proline. Fat mass, percentage of fat mass, percentage of lean mass and the skeletal muscle index (SMI) correlated the most with BNP in both sexes. Our data showed positive correlations in male subjects among ethanolamine, glycine, albumin, and sulfur with SMI, while palmitoleic acid and oleic acid exhibited negative correlations. This differed in female subjects, where SMI was positively associated with albumin, folic acid and sulfur, while CRP, proline and cis-8,11,14-eicosatrienoic acid were negatively correlated. We investigated the influence of diet on the observed BNP and BC correlations. Intriguingly, most of the components of the BNP, except for folate, did not exhibit a correlation with nutrient intake data. An understanding of the physiological and biochemical processes underpinning the observed sex-specific correlations between BNP and BC could help in identifying nutritional strategies to manage BC-changes in aging. This would contribute to a deeper understanding of aging-associated nutritional needs with the aim of helping the elderly population to maintain metabolic health.

Dietary salt regulates uroguanylin expression and signaling activity in the kidney, but not in the intestine.

The peptide uroguanylin (Ugn) is expressed at significant levels only in intestine and kidney, and is stored in both tissues primarily (perhaps exclusively) as intact prouroguanylin (proUgn). Intravascular infusion of either Ugn or proUgn evokes well-characterized natriuretic responses in rodents. Furthermore, Ugn knockout mice display hypertension and salt handling deficits, indicating that the Na(+) excretory mechanisms triggered when the peptides are infused into anesthetized animals are likely to operate under normal physiological conditions, and contribute to electrolyte homeostasis in conscious animals. Here, we provide strong corroborative evidence for this hypothesis, by demonstrating that UU gnV (the rate of urinary Ugn excretion) approximately doubled in conscious, unrestrained rats consuming a high-salt diet, and decreased by ~15% after salt restriction. These changes in UU gnV were not associated with altered plasma proUgn levels (shown here to be an accurate index of intestinal proUgn secretion). Furthermore, enteric Ugn mRNA levels were unaffected by salt intake, whereas renal Ugn mRNA levels increased sharply during periods of increased dietary salt consumption. Together, these data suggest that diet-evoked Ugn signals originate within the kidney, rather than the intestine, thus strengthening a growing body of evidence against a widely cited hypothesis that Ugn serves as the mediator of an entero-renal natriuretic signaling axis, while underscoring a likely intrarenal natriuretic role for the peptide. The data further suggest that intrarenal Ugn signaling is preferentially engaged when salt intake is elevated, and plays only a minor role when salt intake is restricted.

The rat kidney contains high levels of prouroguanylin (the uroguanylin precursor) but does not express GC-C (the enteric uroguanylin receptor).

The peptide uroguanylin (Ugn) regulates enteric and renal electrolyte transport. Previous studies have shown that Ugn and its receptor GC-C (a ligand-activated guanylate cyclase) are abundant in the intestine. Less is known about Ugn and GC-C expression in the kidney. Here, we identify a 9.4-kDa polypeptide in rat kidney extracts that appears, based on its biochemical and immunological properties, to be authentic prouroguanylin (proUgn). This propeptide is relatively plentiful in the kidney (~16% of intestinal levels), whereas its mRNA is marginally present (<1% of intestinal levels), and free Ugn peptide levels are below detection limits (<0.4% of renal proUgn levels). The paucity of preproUgn-encoding mRNA and free Ugn peptide raises the possibility that the kidney might absorb intact proUgn from plasma, where the concentration of propeptide greatly exceeds that of Ugn. However, immunocytochemical analysis reveals that renal proUgn is found exclusively in distal tubular segments, sites previously shown not to accumulate radiolabeled proUgn after intravascular infusions. Thus proUgn appears to be synthesized within the kidney, but the factors that determine its abundance (rates of transcription, translation, processing, and secretion) must be balanced quite differently than in the gut. Surprisingly, we also find negligible expression of GC-C in the rat kidney, a result confirmed both by RT-PCR and by functional assays that measure Ugn-activated cGMP synthesis. Taken together, these data provide evidence for an intrarenal Ugn system that differs from the well-described intestinal system in its regulatory mechanisms and in the receptor targeted by the peptide.

Natriuretic and antikaliuretic effects of uroguanylin and prouroguanylin in the rat.

The peptide uroguanylin (Ugn) is stored and released as a propeptide (proUgn) by enterochromaffin cells in the intestine, and converted to Ugn and other metabolites in the renal tubules. Both proUgn and Ugn are natriuretic, although the response to proUgn is thought to depend on its conversion to Ugn within nephrons. To assess the efficiency of intrarenal conversion of proUgn to Ugn, we measured urinary Ugn excretion in rats following intravenous infusions of proUgn or Ugn. Infusion of 2 and 10 nmol proUgn/kg body wt increased plasma proUgn concentration from 2.2 ± 0.3 to 5.6 ± 1.3 pmol/ml and to 37 ± 9.6 pmol/ml, respectively. No proUgn was detected in urine before, during, or after proUgn infusions. These two proUgn infusion doses resulted in total Ugn recovery in urine of 162 ± 64 and 206 ± 39 pmol/kg body wt (9 and 2% of the infused amount, respectively). By contrast, the same molar amounts of Ugn resulted in 1,009 ± 477 and 5,352 ± 2,133 pmol/kg body wt of Ugn in urine (recoveries of ∼50%). Unexpectedly, comparisons of natriuretic dose-response curves for each peptide showed proUgn to be about five times more potent than Ugn, despite the relatively modest amount of Ugn generated from infused proUgn. In addition, both peptides were antikaliuretic at low doses, but in this case Ugn showed greater potency than proUgn. These data do not support Ugn as the primary active principle of proUgn for regulation of renal sodium excretion. Instead, an alternative peptide fragment produced from proUgn may be responsible for natriuretic activity in the kidney, whereas Ugn itself may play an antikaliuretic role.

The natriuretic peptide uroguanylin elicits physiologic actions through 2 distinct topoisomers.

The peptide uroguanylin regulates electrolyte transport in the intestine and kidney. Human uroguanylin has 2 conformations that can be stably isolated because of their slow interconversion rate. The A isomer potently activates the guanylate cyclase C receptor found primarily in the intestine. The B isomer, by contrast, is a very weak agonist of this receptor, leading to a widely held assumption that it is physiologically irrelevant. We show here, however, that human uroguanylin B has potent natriuretic activity in the kidney. Interestingly, uroguanylin A and B both induce saluretic responses, but the activity profiles for the 2 peptides differ markedly. The uroguanylin B dose-response curve is sigmoidal with a threshold dose of approximately 10 nmol/kg of body weight, whereas uroguanylin A has a comparable threshold but a bell-shaped dose-response curve. In addition, our study indicates a unique interplay between the A and B isoforms, such that the A form at high concentrations antagonizes the natriuretic action of the B form. These data show that the kidney contains a uroguanylin receptor of which the pharmacological profile does not match that of the well-defined intestinal uroguanylin receptor (guanylate cyclase C), an observation consistent with previous studies showing that the kidney of the guanylate cyclase C knockout mouse remains responsive to uroguanylin. The results presented here also support the unconventional notion that distinct conformations of a single endocrine peptide can elicit different responses in different tissues.

Uroguanylin, an intestinal natriuretic peptide, is delivered to the kidney as an unprocessed propeptide.

Orally delivered salt stimulates renal salt excretion more effectively than does iv delivered salt. Although the mechanisms that underlie this "postprandial natriuresis" are poorly understood, the peptide uroguanylin (UGn) is thought to be a key mediator. However, the lack of selective assays for UGn gene products has hindered rigorous testing of this hypothesis. Using peptide-specific assays, we now report surprisingly little UGn in rat intestine or plasma. In contrast, prouroguanylin (proUGn), the presumed-inactive precursor of UGn, is plentiful (at least 40 times more abundant than UGn) in both intestine and plasma. The intestine is the likely source of the circulating proUGn because: 1) the proUGn portal to systemic ratio is approximately two under normal conditions, and 2) systemic proUGn levels decrease rapidly after intestinal resection. Together, these data suggest that proUGn itself is actively involved in enterorenal signaling. This is strongly supported by our observation that iv infusion of proUGn at a physiological concentration produces a long-lasting renal natriuresis, whereas previously reported natriuretic effects of UGn have required supraphysiological concentrations. Thus, our data point to proUGn as an endocrine (i.e. circulating) mediator of postprandial natriuresis, and suggest that the propeptide is secreted intact from the intestine into the circulation and processed to an active form at an extravascular site.

Circulating prouroguanylin is processed to its active natriuretic form exclusively within the renal tubules.

The intestine and kidney are linked by a mechanism that increases salt excretion in response to salt intake. The peptide uroguanylin (UGn) is thought to mediate this signaling axis. Therefore, it was surprising to find (as reported in a companion publication) that UGn is stored in the intestine and circulates in the plasma almost exclusively in the form of its biologically inactive propeptide precursor, prouroguanylin (proUGn), and, furthermore, that infused proUGn leads to natriuretic activity. Here, we investigate the fate of circulating proUGn. Kinetic studies show rapid renal clearance of radiolabeled propeptide. Radiolabel accumulates at high specific activity in kidney (relative to other organs) and urine (relative to plasma). The principal metabolites found in kidney homogenates are free cysteine and methionine. In contrast, urine contains cysteine, methionine, and three other radioactive peaks, one comigrating with authentic rat UGn15. Interestingly, proUGn is not converted to these or other metabolites in plasma, indicating that circulating proUGn is not processed before entering the kidney. Therefore, our findings suggest that proUGn is the true endocrine agent released in response to salt intake and that the response of the kidney is dependent on conversion of the propeptide to an active form after it reaches the renal tubules. Furthermore, proUGn metabolites (other than small amounts of cysteine and methionine) are not returned to the circulation from the kidney or any other organ. Thus, to respond to proUGn released from the gut, any target organ must use a local mechanism for production of active peptide.

Mass spectral characterization of peptide transmitters/hormones in the nervous system and neuroendocrine organs of the American lobster Homarus americanus.

The American lobster Homarus americanus is a decapod crustacean with both high economic and scientific importance. To facilitate physiological investigations of peptide transmitter/hormone function in this species, we have used matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-Q-TOF MS/MS) to elucidate the peptidome present in its nervous system and neuroendocrine organs. In total, 84 peptides were identified, including 27 previously known H. americanus peptides (e.g., VYRKPPFNGSIFamide [Val(1)-SIFamide]), 23 peptides characterized previously from other decapods, but new to the American lobster (e.g., pQTFQYSRGWTNamide [Arg(7)-corazonin]), and 34 new peptides de novo sequenced/detected for the first time in this study. Of particular note are a novel B-type allatostatin (TNWNKFQGSWamide) and several novel FMRFamide-related peptides, including an unsulfated analog of sulfakinin (GGGEYDDYGHLRFamide), two myosuppressins (QDLDHVFLRFamide and pQDLDHVFLRFamide), and a collection of short neuropeptide F isoforms (e.g., DTSTPALRLRFamide and FEPSLRLRFamide). Our data also include the first detection of multiple tachykinin-related peptides in a non-brachyuran decapod, as well as the identification of potential individual-specific variants of orcokinin and orcomyotropin-related peptide. Taken collectively, our results not only expand greatly the number of known H. americanus neuropeptides, but also provide a framework for future studies on the physiological roles played by these molecules in this commercially and scientifically important species.

Identification of neuropeptides from the decapod crustacean sinus glands using nanoscale liquid chromatography tandem mass spectrometry.

Neurosecretory systems are known to synthesize and secrete a diverse class of peptide hormones which regulate many physiological processes. The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules--neuropeptides. As an ongoing effort to define the peptidome of the crustacean SG, we determine the neuropeptide complements of the SG of the Jonah crab, Cancer borealis, and the Maine lobster, Homarus americanus, using nanoflow liquid chromatography electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS. Numerous neuropeptides were identified, including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), beta-pigment dispersing hormone (beta-PDH), proctolin and HL/IGSL/IYRamide. Among them, two novel orcokinins were de novo sequenced from the SG of H. americanus. Three CPRPs including a novel isoform were sequenced in H. americanus. Four new CPRPs were sequenced from the SG of C. borealis. Our results show that structural polymorphisms in CPRPs (and thus the CHH precursors) are common in Dendrobranchiata as well as in Pleocyemata. The evolutionary relationship between the CPRPs is also discussed.

Nitric oxide: an inhibitory retrograde modulator in the crustacean heart.

The nervous system innervates most of the organs in the body, and controls and coordinates their activities. Effective coordination depends on accurate feedback from target organs. Recent studies have identified a target-based feedback mechanism that regulates a simple neural circuit, the cardiac ganglion-a network of nine neurons whose rhythmic bursts of action potentials drive the contractions of the crustacean heart. The feedback agent, nitric oxide (NO), is produced by the target organ (the heart), and acts on the neural circuit (the ganglion), thus serving as a retrograde, trans-synaptic signaling molecule. NO decreases the ganglionic burst rate, which has both negative chronotropic and negative inotropic effects on the heartbeat. This article will review the evidence identifying NO as an inhibitory modulator in the crustacean heart, and will present new data showing that these inhibitory effects are not mediated by cGMP, the canonical downstream agent mobilized by NO in many other systems. Rather, our data suggest that in the crustacean heart cGMP may play a secondary role in the process of adaptation that occurs in during prolonged exposures to NO.

Nitric oxide inhibits the rate and strength of cardiac contractions in the lobster Homarus americanus by acting on the cardiac ganglion.

The lobster heart is synaptically driven by the cardiac ganglion, a spontaneously bursting neural network residing within the cardiac lumen. Here, we present evidence that nitric oxide (NO) plays an inhibitory role in lobster cardiac physiology. (1) NO decreases heartbeat frequency and amplitude. Decreased frequency is a direct consequence of a decreased ganglionic burst rate. Decreased amplitude is an indirect consequence of decreased burst frequency, attributable to the highly facilitating nature of the synapses between cardiac ganglion neurons and muscle fibers (although, during prolonged exposure to NO, amplitude recovers to the original level by a frequency-independent adaptation mechanism). NO does not alter burst duration, spikes per burst, heart muscle contractility, or amplitudes of synaptic potentials evoked by stimulating postganglionic motor nerves. Thus, NO acts on the ganglion, but not on heart muscle. (2) Two observations suggest that NO is produced within the lobster heart. First, immunoblot analysis shows that nitric oxide synthase (NOS) is strongly expressed in heart muscle relative to other muscles. Second, L-nitroarginine (L-NA), an NOS inhibitor, increases the rate of the heartbeat (opposite to the effects of NO). In contrast, the isolated ganglion is insensitive to L-NA, suggesting that heart muscle (but not the ganglion) produces endogenous NO. Basal heart rate varies from animal to animal, and L-NA has the greatest effect on the slowest hearts, presumably because these hearts are producing the most NO. Thus, because the musculature is a site of NOS expression, whereas the ganglion is the only intracardiac target of NO, we hypothesize that NO serves as an inhibitory retrograde transmitter.

mRNA expression patterns of the cGMP-hydrolyzing phosphodiesterases types 2, 5, and 9 during development of the rat brain.

Recent evidence indicates that cGMP plays an important role in neural development and neurotransmission. Since cGMP levels depend critically on the activities of phosphodiesterase (PDE) enzymes, mRNA expression patterns were examined for several key cGMP-hydrolyzing PDEs (type 2 [PDE2], 5 [PDE5], and 9 [PDE9]) in rat brain at defined developmental stages. Riboprobes were used for nonradioactive in situ hybridization on sections derived from embryonic animals at 15 days gestation (E15) and several postnatal stages (P0, P5, P10, P21) until adulthood (3 months). At all stages PDE9 mRNA was present throughout the whole central nervous system, with highest levels observed in cerebellar Purkinje cells, whereas PDE2 and PDE5 mRNA expression was more restricted. Like PDE9, PDE5 mRNA was abundant in cerebellar Purkinje cells, although it was observed only on and after postnatal day 10 in these cells. In other brain regions, PDE5 mRNA expression was minimal, detected in olfactory bulb, cortical layers, and in hippocampus. PDE2 mRNA was distributed more widely, with highest levels in medial habenula, and abundant expression in olfactory bulb, olfactory tubercle, cortex, amygdala, striatum, and hippocampus. Double immunostaining of PDE2, PDE5, or PDE9 mRNAs with the neuronal marker NeuN and the glial cell marker glial fibrillary acidic protein revealed that these mRNAs were predominantly expressed in neuronal cell bodies. Our data indicate that three cGMP-hydrolyzing PDE families have distinct expression patterns, although specific cell types coexpress mRNAs for all three enzymes. Thus, it appears that differential expression of PDE isoforms may provide a mechanism to match cGMP hydrolysis to the functional demands of individual brain regions.

Expression of nitric oxide synthase and nitric oxide-sensitive guanylate cyclase in the crustacean cardiac ganglion.

The cardiac ganglion is a simple central pattern-generating network that controls the rhythmic contractions of the crustacean heart. Enzyme assays and Western blots show that whole heart homogenates from the crab Cancer productus contain high levels of nitric oxide synthase (NOS), an enzyme that catalyzes the conversion of arginine to citrulline with concomitant production of the transmitter nitric oxide (NO). Crab heart NOS is calcium-dependent and has an apparent molecular weight of 110 kDa. In the cardiac ganglion, antibodies to NOS and citrulline indicate the presence of a NOS-like protein and NOS enzymatic activity in the four small pacemaker neurons and the five large motor neurons of the cardiac network. In addition, all cardiac neurons label positively with an antibody to cyclic guanosine monophosphate (cGMP). The NO donor sodium nitroprusside (SNP, 10 mM) stimulates additional cGMP production in the isolated ganglion. This increase is blocked by [(1)H](1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ, 50 microM), an inhibitor of the NO-sensitive soluble guanylate cyclase (sGC). Taken together, our data indicate that NO- and cGMP-mediated signaling pathways are enriched in the cardiac system relative to other crab tissues and that the cardiac network may be a target for extrinsic and intrinsic neuromodulation via NO produced from the heart musculature and individual cardiac neurons, respectively. The crustacean cardiac ganglion is therefore a promising system for studying cellular and synaptic mechanisms of nitrergic neuromodulation in a simple pattern-generating network.