Biomedical subjects
R I Henkin
Publications and source records attributed to R I Henkin.
Serum and tissue zinc concentrations in patients with endoscopic esophagitis.
Because zinc is an important metabolic requirement for growth and repair of squamous tissue, we questioned whether changes in serum and esophageal tissue zinc were present in patients with reflux esophagitis. To investigate this question, we prospectively studied 49 patients undergoing upper gastrointestinal endoscopy for symptoms of abdominal pain and discomfort; 19 patients were taking H2 antagonists at the time of the study. Blood was obtained to measure serum zinc concentrations prior to endoscopy and tissue zinc levels were obtained from esophageal biopsies from the distal, middle, and proximal esophagus in patients who were either endoscopically normal or who exhibited endoscopic esophagitis. Serum zinc concentrations were significantly lower in patients with endoscopic esophagitis compared to the endoscopically normal group (77 +/- 3.8 micrograms/dl vs 88 +/- 2.4 micrograms/dl, P less than 0.02). Distal esophageal tissue concentrations were significantly higher in patients with endoscopic esophagitis compared to the endoscopically normal group (200 +/- 30 micrograms/liter vs 135 +/- 15 micrograms/liter, P less than 0.05); whereas there were no differences between values obtained in the proximal or middle esophagus. Serum and tissue zinc concentrations in patients with esophagitis receiving H2 antagonists were more similar to values obtained in patients who were endoscopically normal than to patients with endoscopic esophagitis without treatment. This study suggests that in endoscopic esophagitis: (1) greater amounts of zinc are concentrated in the rapidly proliferating distal esophageal epithelium, (2) the serum zinc pool may serve as a major zinc source, and (3) decreasing esophageal mucosal inflammation with H2 antagonists may decrease zinc loss via the esophageal epithelium.
Decreased NaCl sensitivity in zinc-deprived rats.
NaCl thresholds and ability to discriminate between NaCl and sucrose were assessed in rats using an operant discrimination conditioning procedure before and during moderate and severe zinc deprivation and during zinc supplementation. NaCl thresholds were approximately 1 mM before dietary zinc manipulation. They increased in all zinc-deprived rats tested 10 and 17 days after initiation of deprivation but did not change in pair-fed controls maintained on supplemental zinc. Threshold changes were greater for those rats severely zinc deprived than for those only moderately deprived and were greater as the period of deprivation lengthened. Plasma zinc concentrations decreased significantly in deprived rats from values obtained at baseline, values in severely deprived rats being significantly lower than in those only moderately deprived. Although zinc-deprived rats discriminated NaCl from sucrose, they made more discrimination errors than controls. Following 24 days of zinc supplementation, previously deprived rats exhibited no significant improvement in gustatory performance, although their body weight increased and plasma zinc concentrations increased; but these later changes were not significant. These results demonstrate that zinc deprivation induces decreased gustatory sensitivity and confirm a role for zinc in taste.
Changes in regulation of human zinc metabolism with age.
To assess changes in zinc metabolism with age, kinetic studies were performed in healthy adults (26 men, 21 women) aged 20-84 yr after a single oral or intravenous bolus of 65Zn. Studies covered two consecutive 9-mo periods while subjects were on a basal dietary intake of approximately 10 mg Zn/day and while taking an additional 100 mg Zn/day orally. Zinc metabolism was analyzed by compartmental analysis using data from plasma, red blood cells, urine, feces, liver, thigh, and whole body [M. E. Wastney, R. L. Aamodt, W. F. Rumble, and R. I. Henkin. Am. J. Physiol. 251 (Regulatory Integrative Comp. Physiol. 20): R398-R408, 1986]. Changes in observed and model calculated values of zinc metabolism were assessed on age by regression. During basal state, zinc release from red blood cells decreased with age. During zinc loading, response (defined as change from basal state) of plasma zinc concentration, urinary zinc excretion, and liver zinc increased with age, while response of fraction of zinc taken up by red blood cells decreased with age. In men, response of amount of zinc absorbed increased with age and in women response of fraction of endogenous zinc excreted decreased with age. Four responses that changed with age (urinary excretion, red blood cell exchange, absorption, and endogenous excretion) occurred at previously defined sites of regulation of zinc metabolism. Results show that regulation of zinc metabolism changes with age.
Kinetic analysis of zinc metabolism in humans after simultaneous administration of 65Zn and 70Zn.
Zinc kinetics were studied and compared after oral simultaneous administration of two tracers, radioactive (65Zn) and stable (70Zn) isotope, to four normal human volunteers. Both tracers and zinc concentration were measured in plasma, red blood cells (RBC), urine, and feces for up to 78 days. Radioactive zinc was also measured by external counting over whole body, liver, and thigh. Data from each individual were analyzed using a compartmental model for zinc metabolism. Values calculated for absorption, fractional zinc excretion in urine, exchange with RBC, and secretion into gut using 70Zn data did not differ from values calculated using 65Zn data. Results show that human zinc metabolism can be investigated using stable isotopes as tracers to determine parameters of whole body zinc metabolism, including zinc absorption, excretion, and secretion.
Hyperosmia and depression following exposure to toxic vapors.
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Radiochemical neutron activation analysis of zinc isotopes in human blood, urine, and feces for in vivo tracer experiments.
Enriched stable isotopes are being increasingly used for study of trace element nutrition in humans who cannot be studied by use of in vivo radioactive tracers (e.g., subjects under age 18 and pregnant women). Zinc metabolism in these subjects can be evaluated by administration of Zn enriched to 65% in the minor isotope, 70Zn (0.6% natural abundance). The enhanced 70Zn is detected later in red blood cells, plasma, urine, and feces by measuring 70Zn/64Zn or 70Zn/68Zn ratios. Stable isotope concentrations are measured by neutron activation of the samples and observation of their products: 244-day 65Zn, 14-h 69mZn, and 4-h 71mZn. Zinc-65 can be observed in these samples without chemical separations 3 weeks after irradiations, but large amounts of 24Na and other short-lived species preclude direct observation of the short-lived Zn activities. Preirradiation chemistry was developed to remove most interferences, the major steps being to place the sample on Chelex resin, elute alkali metals and alkaline earths from it, and irradiate the resin containing the Zn. gamma-Rays of 69mZn can be observed on the irradiated resin, but additional precipitation and solvent extraction steps are needed to remove 56Mn and 64Cu for clear observation of 71mZn and 65Zn within hours after irradiation. Yields for pre- and postirradiation separations are typically 85% and 70%, respectively. The stable isotope tracer method was validated by simultaneous in vivo tracing with radioactive 65Zn in four subjects.
Calculation of zinc absorption in humans using tracers by fecal monitoring and a compartmental approach.
Zinc absorption is often determined following oral administration of tracer by fecal monitoring as dose minus tracer excreted in feces. The value obtained for absorption with this method is influenced by excretion of absorbed tracer into feces during the fecal collection period and by any incomplete elimination of unabsorbed tracer. In the present study a published, physiologically based compartmental model of zinc metabolism has been used to calculate, after oral tracer administration, the fecal appearance of unabsorbed and absorbed tracer and the appearance when fractional rates of endogenous excretion and elimination were varied. Absorption was determined by fecal monitoring and compared to the value determined using parameter values of the compartmental model. The value calculated for absorption using fecal monitoring varied with length of fecal collection, rate of excretion of absorbed tracer (secretion) and rate of elimination of unabsorbed tracer. Absorption was determined correctly by fecal monitoring only when the amount of absorbed tracer excreted was equivalent to the amount of unabsorbed tracer remaining in the gut. Fecal monitoring determines a parameter representing the combined processes of absorption, endogenous excretion (or secretion) and fecal elimination.
Zinc deficiency decreases the activity of calmodulin regulated cyclic nucleotide phosphodiesterases in vivo in selected rat tissues.
The effect of zinc deficiency on calmodulin function was investigated by assessing the in vivo activity of two calmodulin regulated enzymes, adenosine 3',5'-monophosphate (c-AMP) and guanosine 3',5'-monophosphate (c-GMP) phosphodiesterase (PDE) in several rat tissues. Enzymatic activities in brain, heart, and testis of rats fed a zinc deficient diet were compared with activities in these tissues from pair fed, zinc supplemented rats. In testis, a tissue in which zinc concentration decreased with zinc deficient diet, enzyme activities were significantly decreased over those in rats who were pair fed zinc supplemented diets. In brain and heart, tissues in which zinc concentrations did not change with either diet, enzymatic activities between the groups were not different. These results indicate that zinc deficiency influences the activity of calmodulin-regulated phosphodiesterases in vivo supporting the hypothesis that zinc plays a role in calmodulin function in vivo in zinc sensitive tissues.
Development and application of a model for zinc metabolism in humans.
A model has been developed to describe zinc metabolism in humans. The model was developed from a series of studies in normal volunteers and patients with various clinical disorders. Zinc metabolism was studied with radiotracers for 9-12 mo before and after a daily oral zinc load of 100 mg. Data were analysed by compartmental analysis and a model was developed that consists of compartments for zinc in the gut, plasma, red blood cells, liver, muscle, bone and other tissues with excretion via urine and feces. Using the model parameters of zinc metabolism including absorption, tissue exchange, secretion and excretion have been determined, together with mass of zinc in tissues and in the whole body. The model has been used to identify five sites of long-term regulation of zinc metabolism in humans. These sites are absorption, excretion, exchange with red blood cells, release of zinc from muscle and secretion of zinc into gut. The model is currently being applied to several areas of nutrition including the effects of dietary fiber on zinc stores, the effects of aging on zinc metabolism and the zinc requirements of neonates.
In vivo effects of zinc deficiency on calmodulin concentrations in selected rat tissues.
Two groups of male Sprague-Dawley rats, one fed zinc-deficient diet, ad libitum, the other, pair-fed with the same diet, but given supplemental zinc in the drinking water (8 mg Zn++/ml) were studied. After ten weeks of diet, rats were exsanguinated and zinc and calmodulin concentrations in brain and testis were measured. Mean zinc concentration in testis was significantly decreased in rats fed zinc-deficient diet without supplemental Zn++, but mean zinc concentration in brain was not different. Similarly, mean calmodulin concentration in testis was decreased in rats fed zinc-deficient diet without supplemental Zn++ whereas mean calmodulin concentration in brain was not different. Distribution studies of zinc and calmodulin showed that both zinc and calmodulin were released more freely into soluble fractions of testis in rats fed zinc-deficient diet without supplemental Zn++. These results indicate, for the first time in in vivo studies, that zinc influences the calmodulin content of testis.
Metal binding characteristics of human salivary and porcine pancreatic amylase.
With the exception of calcium very little is known about metal binding characteristics of either human salivary or porcine pancreatic amylase. In order to learn more about these protein-metal binding interactions, calcium-free human salivary and porcine pancreatic amylase [P(protein)] were obtained by carboxymethylcellulose chromatography of the partially purified proteins. Because these proteins acquired small amounts of calcium after further preparatory studies, they were dialyzed against 1 mM EDTA, pH 7.4, at 22 degrees C, which removed essentially all acquired calcium. The calcium-free amylases were then subjected to equilibrium dialysis against copper or zinc solutions with or without added glycine. The experimental data were fitted to appropriate mathematical equations, and binding constants of the metal complexes were calculated. Both human salivary and porcine pancreatic amylase were found to have two metal ion binding sites, only one of which was selective for calcium. Copper or zinc appeared to bind to the second site forming the species CuCaLP (or ZnCaP), where L, a ligand, is the glycine anion. Neither copper nor zinc displaced calcium from human salivary amylase, although copper bound to both binding sites in human salivary apoamylase to form the species Cu2L2P in which the amylase molecule appeared to form a bridge between the two copper atoms. In the case of the zinc-human salivary apoamylase system, the experimental data could not be analyzed quantitatively since the protein formed an insoluble complex species. Copper displaced calcium from porcine pancreatic amylase and formed a mixed ligand species similar to that formed with human salivary apoamylase. Zinc bound to both metal binding sites of porcine pancreatic apoamylase, forming species ZnP and Zn2P, although it did not displace calcium from the protein. While calcium in amylase is known to be critical for its amylolytic activity, little is known about the function of either zinc or copper in amylase albeit both of these metals are important in biological systems.
Human salivary gustin is a potent activator of calmodulin-dependent brain phosphodiesterase.
Human salivary gustin stimulated activity of brain calmodulin-dependent cyclic nucleotide phosphodiesterase (cAMP PDEase; 3',5'-cyclic-nucleotide phosphodiesterase, EC 3.1.4.17) in a dose-dependent manner in the absence of calmodulin. At physiological levels found in human saliva, gustin activated cAMP PDEase 5- to 6-fold. Activation of PDEase occurred with as little as 500 ng of gustin. Comparative sensitivity of activation of PDEase by gustin was intermediate between calmodulin and lysophosphatidylcholine with maximal activation and half-maximal activation (indicated in parentheses) at 3 X 10(-8) M (4.3 X 10(-9) M), 3.4 X 10(-6) M (3.4 X 10(-7) M), and 2.5 X 10(-3) M (4.0 X 10(-5) M) for calmodulin, gustin, and lysophosphatidylcholine, respectively. No other major salivary protein activated PDEase. Anticalmodulin antibody completely inhibited calmodulin-activated cAMP PDEase activity, but the antibody had no effect on gustin-activated cAMP PDEase activity. A sensitive calmodulin RIA indicated that no calmodulin was detected in any gustin preparation that activated cAMP PDEase. Both gustin and calmodulin rendered cAMP PDEase thermally labile to a similar extent and increased Vmax without affecting the apparent Km for the substrate cAMP. Activation by gustin and calmodulin was unaffected by lubrol-PX, trypsin inhibitor, pepstatin A, or leupeptin. In the presence of 1 mM EGTA, gustin activated cAMP PDE 5- to 6-fold, but the activating ability was completely lost after gustin was heated at 100 degrees C for 5 min. In contrast, calmodulin lost all activating ability in the presence of 1 mM EGTA, whereas heating calmodulin at 100 degrees C for 5 min did not affect its activation of cAMP PDEase. Lysophosphatidylcholine-activation of cAMP PDEase, like gustin activation, was unaffected by EGTA, but lysophosphatidylcholine-activation of cAMP PDEase, like calmodulin activation, was unaffected by heating at 100 degrees C for 5 min.
Low parotid saliva calmodulin in patients with taste and smell dysfunction.
Parotid saliva calmodulin was found both in 32 normal volunteers and in 60 patients with taste and smell dysfunction; salivary calmodulin concentration was significantly lower in the patients than in the volunteers. There were no differences in salivary calmodulin concentration with respect to age, sex, or salivary flow rate in either normal volunteers or patients. When patients were categorized by diagnosis, calmodulin concentration was found to be decreased in all patient groups. The concentration of calmodulin in saliva was about 10 times that found in serum, suggesting that the parotid gland is a major source of this protein.
Kinetic analysis of zinc metabolism and its regulation in normal humans.
Zinc metabolism was studied in 32 normal volunteers after oral (n = 25) or intravenous (n = 7) administration of 65Zn. Data were collected from the blood, urine, feces, whole body, and over the liver and thigh regions for 9 mo while the subjects consumed their regular diets (containing 10 mg Zn ion/day) and for an additional 9 mo while the subjects received an exogenous oral supplement of 100 mg Zn ion/day. Data from each subject were fitted by a compartmental model for zinc metabolism that was developed previously for patients with taste and smell dysfunction. These data from normal subjects were used to determine the absorption, distribution, and excretion of zinc and the mass of zinc in erythrocytes, liver, thigh, and whole body. By use of additional data obtained from the present study, the model was refined further such that a large compartment, which was previously determined to contain 90% of the body zinc, was subdivided into two compartments to represent zinc in muscle and bone. When oral zinc intake was increased 11-fold three new sites of regulation of zinc metabolism were identified in addition to the two sites previously defined in patients with taste and smell dysfunction (absorption of zinc from gut and excretion of zinc in urine). The three new sites are exchange of zinc with erythrocytes, release of zinc by muscle, and secretion of zinc into gut. Regulation at these five sites appears to maintain some tissue concentrations of zinc when dietary zinc increases.