Normal IgG protects against acute graft-versus-host disease by targeting CD4(+)CD134(+) donor alloreactive T cells.

Intravenous immunoglobulin (IVIg) was shown to decrease the severity of acute graft-versus-host disease (aGVHD) in recipients of allogeneic bone marrow transplants. To investigate the mechanisms involved in the protective effect of IVIg, we have used the parent-into-F1 model in which parental lymphocytes are transferred into semi-syngeneic non-irradiated F1 rats. Here we report that IVIg, as well as F(ab')(2) fragments of IVIg, protected (Lewis x Brown-Norway) F1 rats against aGVHD induced by a single injection of Lewis lymphocytes. IVIg was given as five consecutive daily injections, starting on the day preceding that of the transfer of Lewis cells. Protection was associated with a decreased ability of lymphocytes to spontaneously proliferate and to produce NO and IFN-gamma, in the absence of an increased production of IL-10. We further demonstrate that protection was associated with a decrease in CD4(+) T cells bearing the activation marker CD134 in vivo, and with an enhanced apoptosis of activated CD4(+) T cells by IVIg, in vitro. Our observations suggest that the prevention of aGVHD by IVIg in this model is mediated by the induction of apoptosis of activated alloreactive CD4(+)CD134(+) donor T cells. The results further emphasize the role of normal immunoglobulin in modulating alloantigen immune responsiveness.

Neonatal induction of tolerance to T(h)2-mediated autoimmunity in rats.

Brown-Norway (BN) rats are highly susceptible to drug-induced immune dysregulations and when injected with mercuric chloride (HgCl(2)) or sodium aurothiopropanolsulfonate (ATPS), they develop a syndrome characterized by a polyclonal B cell activation depending upon CD4(+) T(h)2 cells that recognize self-MHC class II molecules. Since peripheral tolerance of T(h)2 cells might be crucial in the prevention of immunological manifestations such as allergy, establishing conditions for inducing tolerance to HgCl(2)- or ATPS-mediated immune manifestations appeared to be of large interest. We report here that BN rats neonatally injected with HgCl(2): (i) do not develop the mercury disease, (ii) remain resistant to HgCl(2)-induced autoimmunity at 8 weeks of age and later, provided they are regularly exposed to HgCl(2), (iii) are still susceptible to ATPS-induced immune manifestations, and (iv) exhibit spleen cells that adoptively transfer tolerance to HgCl(2)-induced autoimmunity in naive, slightly irradiated, syngeneic recipients. These findings demonstrate that dominant specific tolerance can be neonatally induced using a chemical otherwise responsible for T(h)2-mediated autoimmunity.

Analysis of NMDA-independent long-term potentiation induced at CA3-CA1 synapses in rat hippocampus in vitro.

1. Excitatory postsynaptic currents (EPSCs) were evoked at synapses formed by Schaffer collaterals/commissural (CA3) axons with CA1 pyramidal cells using the rat hippocampal slice preparation. Long-term potentiation (LTP) was induced at these synapses using a pairing protocol, with 50 microM d,l-APV present in the artificial cerebrospinal fluid (ACSF). 2. Quantal analysis of the amplitudes of the control and conditioned EPSCs showed that the enhancement of synaptic strength was due entirely to an increase in quantal content of the EPSC. No change occurred in the quantal current. 3. These results were compared with those obtained from a previous quantal analysis of LTP induced in normal ACSF, where both quantal current and quantal content increased. The results suggest that calcium entering via NMDA receptors initiates the signalling cascade that results in enhanced AMPA currents because it is adding to cytoplasmic calcium from other sources to reach a threshold for this signalling pathway, or because calcium entering via NMDA receptors specifically activates this signalling pathway.

A model of magnesium metabolism in young sheep. Magnesium absorption and excretion.

A model of Mg metabolism in sheep is proposed. It is based on standard Michaelis-Menten enzyme kinetics to describe the transport of Mg across the rumen wall and passive diffusion to describe the absorption of Mg in the hindgut. Factors known to have an effect on Mg metabolism in farm animals, namely the concentrations of K and Mg in the diet, and the physico-chemical conditions within the rumen as determined by the type of diet, are incorporated into the model. Consideration of the rumen as the only site of Mg absorption provided an inadequate mechanistic description of Mg metabolism in sheep. To ensure compatibility between predicted Mg absorption and recent independent data sets for Mg balances, it was necessary to include in the model aspects of Mg absorption that operate in the hindgut. The results from this model suggest that there is a need for a series of experiments to determine the important aspects of Mg transport in the hindgut of sheep. Mechanisms of homeostasis are discussed.

Statistical analysis of amplitude fluctuations in EPSCs evoked in rat CA1 pyramidal neurones in vitro.

1. EPSCs were evoked in CA1 pyramidal neurones of young rats in vitro by extracellular stimulation of axons in a restricted stratum radiatum field, and were recorded using the whole-cell technique. 2. Quantal fluctuations in EPSC amplitude could be demonstrated for nineteen of fifty EPSCs analysed. Quantal currents (at the soma) ranged from 2.6 to 9.5 pA (after correction for the access resistance) with a mean of 4.0 +/- 2.0 pA. 3. Quantal variance was negligible for the majority (13/19) of the EPSCs. However, a large quantal variance (with a coefficient of variation > 0.4) is one possible reason why a large number of the EPSCs (29/50) could not be shown to have quantal fluctuations. 4. The statistical pattern of fluctuations in the amplitude of the majority of the quantal EPSCs (18/19) could not be described by conventional models of transmitter release. 5. The time course of the EPSC and a compartmental model of CA1 pyramidal neurones were used to calculate synaptic location. The quantal current (at the soma) was independent of the electrotonic location of the synapse at which it was evoked. The peak quantal conductance generating each quantal current ranged from 0.5 to 6.8 nS (mean 1.3 +/- 1.4 nS), its magnitude increasing with distance from the soma. The mean peak conductance is likely to be generated by the opening of at least 60-160 AMPA channels.

Changes in quantal parameters of EPSCs in rat CA1 neurones in vitro after the induction of long-term potentiation.

1. Long-term potentiation (LTP) was induced in EPSCs evoked in CA1 pyramidal neurones of young rats in vitro by extracellular stimulation of stratum radiatum. Low frequency stimulation was paired with postsynaptic depolarization to induce LTP, using whole-cell recording techniques. 2. Sufficient control and potentiated records were obtained under stable recording conditions to allow a quantal analysis of eleven EPSCs. The fluctuations in amplitude of all eleven EPSCs were quantized before conditioning stimulation, and they remained quantized after LTP induction, usually with an increased quantal variance. 3. Quantal current was increased by conditioning for nine out of eleven EPSCs. The increase in quantal current was correlated with the percentage increase in the EPSC. For only two EPSCs could the entire potentiation be attributed to an increase in quantal current. 4. The amplitude fluctuations of five control EPSCs could be described by binomial statistics, but after conditioning the binomial description held for only one of these EPSCs. For this EPSC, conditioning caused the release probability to increase from 0.39 +/- 0.05 to 0.47 +/- 0.02. 5. Quantal content was increased by conditioning stimulation for ten out of eleven EPSCs. The increase in quantal content was correlated with the percentage increase in the EPSC. However, for only four EPSCs could the entire potentiation be attributed to an increase in quantal content. 6. Most EPSCs were evoked with a high proportion of response failures. The probability of response failures decreased in eight out of eleven EPSCs following the induction of LTP. There was a negative correlation between the change in the probability of response failures and the amount of LTP. 7. The minimal number of sites at which transmission occurred increased for ten out of eleven EPSCs following LTP induction. Increases in the minimal number of active sites following conditioning were associated with decreases in the probability of response failures for seven out of eleven EPSCs. 8. The induction of LTP usually resulted in changes in the time course of the EPSCs. Cable analysis using a passive compartmental model of a CA1 pyramidal cell suggested that these time course changes were associated with shifts in the average electrotonic location of the active sites following LTP induction, rather than being caused by an increased duration of synaptic current. 9. LTP expression involves postsynaptic modifications to enhance the synaptic current at active sites. New sites are recruited, and our data cannot be used to determine if this is a result of a pre- or a postsynaptic change. Evidence for an increase in release probability was found for one EPSC.

Kinetics of lactate and glucose metabolism in the pregnant ewe and conceptus.

Two monotocous and two ditocous ewes were infused at 124-130 days of gestation with a mixture of L-[U-14C]lactate and D-[6-3H]glucose via a maternal or umbilical catheter, separate days being used for each infusion. Plateaux-specific radioactivities of plasma lactate and glucose were used to derive a four-pool model describing the fluxes between the lactate and glucose pools of the infused conceptus and mother. The average turnover rate of lactate was 23.3 and 13.2 mg carbon min-1 in the conceptus and 32.0 and 44.0 mg carbon min-1 in the mother for monotocous and ditocous ewes, respectively. Glycolysis rates within both conceptus and mother in all sheep were high, accounting for about 80 and 60% of the respective rates of lactate turnover. The synthesis of glucose from lactate accounted for 13 and 31% of the glucose turnover in the mother in monotocous and ditocous ewes, respectively, but was insignificant in the conceptus. Glycolysis within the conceptus used only glucose which had entered and mixed with the conceptus glucose pool; there was no direct transfer of carbon from the maternal glucose pool to the conceptus lactate pool. This finding is an important validation for the use of tracer methods to determine glucose use within the whole conceptus rather than within fetal corporeal tissues alone.

Asymmetric charge movement and calcium currents in ventricular myocytes of neonatal rat.

1. Calcium and sodium currents and non-linear capacitive currents were recorded from isolated ventricular cells from neonatal rats, using the whole-cell patch-clamp technique, usually with a holding potential of -100 mV. 2. When recording with internal and external solutions designed to suppress virtually all ionic currents except the calcium current, careful subtraction of all linear capacitive and ionic currents revealed that depolarizations elicited a small transient outward current which preceded the inward calcium current. This outward current was discernible just below the threshold potential for the calcium current and increased with larger depolarizations to a maximum for potentials of about +30 mV and above. 3. Elimination of the calcium current revealed that at each potential the transient outward current was accompanied by a roughly equal transient inward current upon repolarization. The properties of these currents indicate that they are non-linear capacitive currents. Best-fit Boltzmann curves of the 'on' charge (integral of the transient outward current) gave values for qmax, V and k of 3.9 nC/microF, -29.3 mV and 15.5 mV with internal Cs+. The maximum 'on' charge is similar to that found with calcium currents (4.3 nC/microF). Similar values were obtained with internal TEA+. 4. Boltzmann fits of conductance vs. voltage for the calcium channel gave mean values of -15.5 and 13.3 mV for V and k (with internal Cs+); the corresponding values for the sodium channel were -49.9 and 5.4 mV. 5. Pre-pulses (20 ms) to -60 mV inactivated 77% of the peak sodium current, but only inactivated about 10% of the peak calcium current and reduced the maximum 'on' charge (moved at potentials positive to -60 mV) by 19%. 6. With a holding potential of -100 mV, 10 microM-nifedipine blocked 89% of the calcium current, but had little effect on the amount of 'on' charge. The 'off' charge appeared to be slower in the presence of nifedipine. 7. These results and consideration of the number of calcium channels and high-affinity binding sites for dihydropyridines (DHP), suggest that a large part of the charge movement may be related to DHP binding sites and involved with gating calcium channels. Comparison with skeletal muscle suggests similarities in the mechanisms involved in excitation-contraction coupling.

An assessment of the efficacy and safety of selenium and cobalt included in an anthelmintic for sheep.

A trial was devised to assess whether the administration of selenium and cobalt together with the anthelmintic mebendazole (Ovitelmin S&C) was safe and could improve the supplies of selenium and cobalt for adult sheep fed a whole grain diet, low in both elements, which produced a steady decrease in blood glutathione peroxidase (GSHPx) and plasma vitamin B12 concentrations. Ovitelmin S&C, when given orally in a single dose as a suspension containing 0.34 mg selenium/ml, and 0.44 mg cobalt/ml (to provide 0.11 mg selenium and 0.15 mg cobalt/kg liveweight) significantly increased the GSHPx activity in blood. After a second dose given 28 days later the rate of change increased from 2.5 to 3.5 u/g haemoglobin/day. The responses in GSHPx were similar for a preparation which contained twice the concentration of selenium. Ovitelmin S&C increased the concentration of vitamin B12 in the plasma by about 1000 pg/ml for four to seven days after each dose and the increases were similar to those observed in sheep treated with an Ovitelmin preparation containing 45 times more cobalt (providing 6.7 mg cobalt/kg liveweight). After 63 days, liver vitamin B12 concentrations were 43 per cent higher in the cobalt treated than in the untreated groups (P less than 0.01) with no differences among the groups given cobalt. Neither adverse reactions nor signs of toxicity followed the administration of Ovitelmin S&C or Ovitelmin containing the higher concentrations of selenium and cobalt.

The effect of noradrenaline on the ion permeability of isolated mammalian hepatocytes, studied by intracellular recording.

1. The influence of noradrenaline on the membrane potential and conductance of isolated guinea-pig and rabbit hepatocytes in short-term (2-8 h) tissue culture has been studied by intracellular recording. 2. Resting hepatocytes had linear current-voltage relationships, with input resistances of 166 and 216 M omega in guinea-pig and rabbit cells respectively. The recorded membrane potential was -18 mV in each species, though the true resting potential is likely to have been up to 10 mV greater. 3. The hepatocytes sometimes slowly hyperpolarized during intracellular recording, and this was associated with a fall in membrane resistance, and an increase followed by a decrease in membrane potential noise. These effects were abolished by quinine (200 microM) but not by apamin (50 nM), and are attributable to a K+ conductance activated by cell swelling. 4. Noradrenaline (2 microM, in the presence of propranolol at 1 microM) was applied to individual hepatocytes by pressure ejection (puffer pipette technique). After a short latency, the cells hyperpolarized by a mean of 18 mV in both guinea-pig and rabbit preparations. This was associated with a large rise in membrane conductance (50 nS in guinea-pig, 54 nS in rabbit cells). The reversal potential for this action was -38 mV. 5. The experiments were repeated in the presence of apamin (50 nM) to block the Ca2+-dependent K+ permeability which noradrenaline activates in these cells. Noradrenaline still caused some hyperpolarization and a substantial increase (approximately 40 nS) in conductance, with a reversal potential (Er) of -31 mV. This can be attributed to an increase in Cl- conductance. 6. In keeping with this interpretation, noradrenaline applied in the absence of Cl- (replaced by isethionate or gluconate) caused a much greater hyperpolarization (58 mV in guinea-pig, 40 mV in rabbit cells) associated with a smaller rise in conductance (approximately 12 nS). Er for this action was -95 mV (guinea-pig) and -68 mV rabbit), suggesting that the conductance increase was now mainly to K+. 7. The magnitudes of the conductance changes produced by noradrenaline under the various experimental conditions suggest that the increase in the conductance to Cl- (delta GCl) is 3-fold greater than that to K+ (delta GK). 8. The activation of delta GCl occurs either at the same time as delta GK, or (in ca. one cell in ten) a few seconds later.(ABSTRACT TRUNCATED AT 400 WORDS)

Internal perfusion of guinea-pig hepatocytes with buffered Ca2+ or inositol 1,4,5-trisphosphate mimics noradrenaline activation of K+ and Cl- conductances.

External application of noradrenaline to voltage-clamped guinea-pig isolated hepatocytes evoked membrane conductance increases to K+ and Cl-. This effect was reproduced by internal perfusion of the cells with 2 microM buffered Ca2+ and with 20 microM inositol 1,4,5-trisphosphate (IP3). The kinetic properties of the K+ conductance and its selective block by the toxin apamin were the same in each case. Cyclical fluctuations of conductance observed with noradrenaline were reproduced by internal IP3 but not by Ca2+ perfusion, indicating that oscillations of intracellular free Ca2+ may arise from properties of the Ca2+ sequestration mechanism at constant IP3 concentration.

Effect of concurrent infection with Ostertagia circumcincta and Trichostrongylus vitrinus on the performance of lambs.

The interaction between Ostertagia circumcincta and Trichostrongylus vitrinus was investigated in lambs continuously infected over 12 weeks. Four groups of seven lambs were given either zero, 1000 T vitrinus larvae per day, 2500 O circumcincta larvae per day or infected concurrently. Overall liveweight gain was reduced by 17, 20 and 30 per cent in T vitrinus, O circumcincta and concurrent infection, respectively. T vitrinus infection significantly lowered serum phosphorus concentrations by week 4. In the concurrent infection the decline in phosphorus concentration was more gradual and only differed significantly from the controls during the final four weeks of the trial. There were no significant differences between the total numbers of T vitrinus or O circumcincta recovered from single or combined infections. The lack of an additive effect on performance may be due to a delayed establishment of T vitrinus in the presence of O circumcincta.

Effect of phosphorus intake on growth rate, food intake and quality of the skeleton of growing lambs infected with the intestinal nematode Trichostrongylus vitrinus.

The performance of growing lambs dosed daily for 12 weeks with 2500 Trichostrongylus vitrinus larvae (I) or given no larvae (control, C) was compared at two levels of phosphorus intake; 2.75 gP/kg dry matter (normal, NP) and 1.88 pgP/kg dry matter (low, LP). Both low phosphorus and T vitrinus infection reduced dry matter intake and growth rate and the combined effect of low phosphorus and infection were additive. Plasma hypophosphataemia (less than 60 mg per litre) occurred in groups CLP, ILP and INP from week 3, the values gradually returning to normal levels in INP lambs by the end of the trial. T vitrinus infection did not depress plasma phosphorus concentration below that seen in the low phosphorus controls. Both low phosphorus intake and T vitrinus infection reduced the density and mineralisation of bone, the most severely affected bones occurring in groups ILP lambs. T vitrinus infection reduced the content and concentration of phosphorus in the rumen and the effect was greater at the lower intake of phosphorus. Infection also reduced the proportion of phosphorus in the rumen liquid phase. The number of T vitrinus recovered from the intestine was higher and villous atrophy more extensive in lambs from group ILP. It is suggested that development of 'resistance' may be delayed on a low phosphorus intake.

Effects of dietary supplements of thiomolybdates on copper and molybdenum metabolism in sheep.

When 4 groups of 3 lambs were reared on a milk substitute containing 10.5 mg of Cu per kg DM with or without supplements of Cu, 5 mg, and Mo, 3 mg per kg DM in a 2 X 2 factorial design, Mo, given as ammonium tetrathiomolybdate (MoS4) reduced hepatic Cu retention by one-third but had no effect on the amount or distribution of Cu in plasma or on plasma Mo concentrations. When 2 groups of 5 lambs were weaned on to a cereal-based diet containing 3.5 mg Cu per kg DM and one was given MoS4 in the amount used previously, concentrations of Mo and TCA-insoluble Cu in plasma and the rate of depletion of liver Cu were increased. When Cu, 12 mg per kg DM, was finally added to both diets the plasma changes were reversed but the hepatic retention of Cu was still reduced. In a second experiment, 6 groups of 5 hypocupraemic ewes were repleted with a dietary Cu supplement in the presence of one of 3 Mo sources, molybdate (MoO4), dithiomolybdate (MoO2S2) or tetrathiomolybdate (MoS4) providing 3.0 mg Mo per kg DM each in the presence of low or high dietary S. The effects of the S supplement were dominant, causing the MoO4 and MoO2S2 to behave like MoS4 in impairing Cu and Mo absorption and inducing TCA-insoluble Mo and Cu to accumulate in the plasma: S enhanced these responses to MoS4 even further. On the low S diet, the responses to MoO4 and MoO2S2 were similar but small. It is concluded that at high dietary Mo and S concentrations, sufficient MoS4 forms in the rumen and enters complexes with Cu to impair Cu absorption but that the formation of MoO2S2 does not play an important role in the Cu and Mo antagonism.

Absorption and secretion of calcium and phosphorus in the alimentary tract of lambs infected with daily doses of Trichostrongylus colubriformis or Ostertagia circumcincta larvae.

The flows of endogenous and exogenous calcium and phosphorus in the digesta at the abomasum, ileum and in the faeces were measured in growing lambs infected daily with 3000 T. colubriformis larvae (TC), 5000 O. circumcincta larvae or no larvae. The lambs were given 0.70 or 1.0 kg per day of a pelleted diet and the flow rates were measured by reference to soluble and particulate radioactive markers. The fraction of Ca and P in digesta of endogenous origin was measured by the method of isotope dilution involving a single intravenous dose of 45Ca or 32P. Ostertagia circumcincta infection was without effect on the absorption and secretion of Ca and P in the digestive tract, other than producing a small but significant increase in the endogenous faecal Ca excretion. There was, however, a striking increase in the flow of digesta at the abomasum, the DM and Na content of which was consistent with an increased secretion from the mucus-type cells in the abomasal mucosa. The extra digesta was absorbed in the small intestine. Trichostrongylus colubriformis infections reduced the absorption of exogenous P by about 30 per cent and increased losses of endogenous P due to the action of worms on the mucosa of the small intestine. The net effect of this was to induce a P deficiency in TC lambs, leading to a reduced flow of salivary P and a reduction in the plasma concentrations of P. The effect of TC on Ca metabolism was limited to an increase in endogenous faecal excretion.

Foetal and maternal rates of urea production and disposal in well-nourished and undernourished sheep.

1. Five monotocous and two ditocous Scottish Blackface ewes with indwelling catheters in an umbilical artery and vein of one foetus only and in both maternal jugular veins were used. 2. Experiments were conducted over a period of 2 d when [14C]urea was infused intravenously over 9 h into either the mother or foetus, separate days being used for each infusion. Two series of experiments were completed, one in well-nourished and the other in undernourished sheep at 125-141 and 138-143 d of gestation respectively. 3. Plasma urea specific radioactivities of the mother and infused foetus at plateau were used to determine the urea flux-rates within and between mother and foetus. The mean rate of foetal urea production (Ffo) was 1.45 and 1.63 mg/min per kg foetus in well-nourished and undernourished ewes respectively. The corresponding rates of maternal urea production (Fmo) were 0.49 and 0.37 mg/min per kg live weight respectively and there was a close correlation between the rate of maternal urea disposal (Fom) and the dietary nitrogen intake. 4. The values of Ffo were used to calculate the maximum potential for foetal gluconeogenesis from deaminated amino acids. These calculations were compared with published information on the over-all rates of foetal gluconeogenesis in well-nourished ewes. 5. The foetal entry rate of urea expressed on a body-weight basis was high, approximately 8.5 times that of the mother, but it was a threefold overestimate of Ffo. The maternal entry rate was 1.3 times Fmo and the significance of this in relation to assessing differences in urea entry rates in pregnant and non-pregnant sheep is discussed.

Effects of experimental chronic pneumonia on bodyweight, feed intake and carcase composition of lambs.

The growth rate, feed consumption and carcase composition of nine untreated wether lambs (controls) were compared with those of lambs in which a chronic pneumonia had been experimentally induced. Six pneumonic lambs (group 1) were killed with the controls on day 109 and eight (group 2) lambs were killed when they had a similar mean liveweight to the controls (42 kg) on day 172. The mean liveweight gain of infected animals to day 108 was 59 per cent, the mean feed intake 69 per cent and the mean carcase weight of group 1 was 73.5 per cent that of the controls. Group 2 lambs required 25 per cent more feed and nine weeks longer than the controls to reach similar live and carcase weights. This depression of appetite and growth rate was most marked in the first 35 days after inoculation, but growth rates of infected lambs continued to be lower than those of the controls throughout the experiment. At slaughter, all infected lambs had consolidated lesions of pneumonia and a significant correlation was noted between the extent of lung lesions and total liveweight gain in individual lambs. Carcases of group 1 lambs had a proportionately low fat, high lean meat and bone content, indicative of immature development and consistent with a lowered feed intake. Carcases of group 2 lambs showed a similar trend but differences from the controls were not significant.

A model for zinc metabolism in sheep given a diet of hay.

1. Four pairs of Scottish Blackface ewes were given a basal diet of hay, providing 8 mg zinc/d, and one of four levels of supplementary Zn (0,75, 150 or 225 mg/d) continuously by intraruminal infusion. 2. 65Zn (59 microCi) was given intravenously after stabilization for 14 d and the changes in specific radioactivity (SR) of plasma and faeces were monitored for a further 10 and 13 d respectively. The faecal endogenous losses and absorption of Zn were estimated, using the principle of isotope dilution, by two methods using the whole or latter parts of SR:time curves. 3. Faecal endogenous losses were estimated by the part area method to be 4.9, 6.4, 5.1 and 6.3 +/- 0.35 mg/d at 0, 75, 150 and 225 mg supplemental Zn/d i.e. largely unaffected by Zn intake and averaging 0.11 mg/kg live weight (LW). The whole area method gave similar results. 4. Urinary excretion was negligible (probably less than 0.2 mg/d) for all sheep. 5. The amount of Zn absorbed was assumed to equal the irreversible loss of Zn under steady-state conditions and found to remain constant at 7.6 +/- 0.39 and 10.3 +/- 0.6 mg/d when calculated by "whole' and "part area' methods. Zn retention did not increase with Zn intake and homoeostasis was achieved primarily by control of Zn absorption which fell from 0.75 to 0.03 or from 0.96 to 0.05 of intake, depending on method of calculation, as intakes increased.