Reports that the nutrients zinc and taurine interact have prompted interest in the physiological importance and possible mechanisms responsible for this phenomenon (Pasantes-Morales et al. 1987b). Isolated frog rod outer segments exposed to ferrous sulfate display extensive disruption of their structure, characterized by acute swelling and disc membrane disorganization. The addition of zinc or taurine alone to the culture medium is ineffective in protecting against this outer segment damage; however, zinc and taurine, added together, provide protection in a synergistic manner (Pasantes-Morales and Cruz 1984). In vivo experimentation contributes further evidence for this interaction. Plasma taurine concentration is increased in severely zinc-deficient rats (Griffith and Alexander 1972). Griffith and Alexander (1972) found no alteration in the uri nary excretion of taurine in zinc deficiency; other researchers have described an increase (Anthony et al. 1971, Hsu and Anthony 1970). Interest in the interaction between zinc and taurine in neurological disorders has also been reported (Barbeau and Donaldson 1974, Shapcott et al. 1984).

The developing retina may be a sensitive tissue in which to explore the interaction between zinc and taurine. A 50% reduction in retinal taurine in cats results in retinal degeneration characterized by a decrease in a- and b-wave amplitudes of the electroretinogram (Berson et al. 1976) and degeneration of the photoreceptor outer segments (Hayes et al. 1975). With prolonged taurine deficiency, blindness develops. The addition of 200 µmol/L zinc to the drinking water of the taurine-deficient cat can provide partial protection to the a-wave amplitude of the electroretinogram (Pasantes-Morales et al. 1987a). During gestation and early postnatal life, taurine plays an important role in retinal development. Kittens born to taurine-deficient queens have shortened photoreceptors characterized by severely disorganized outer segments (Imaki et al. 1986).

Rats made taurine-deficient using guanidinoethyl sulfonate (GES),⁵ a structural analog of taurine, develop retinal changes similar to those observed in cats. These include reduced a- and b-wave amplitudes of the electroretinogram (Cocker and Lake 1987, Hageman and Schmidt 1987, Lake 1986, Rapp et al. 1987) and degeneration of the photoreceptors (Lake and Malik 1987, Pasantes-Morales et al. 1983). Treatment of rats with 10 g/L GES throughout gestation produces a degeneration of the photoreceptor layer of the retina in pups, similar to that observed in kittens (Bonhaus et al. 1985).

The influence of zinc on the retina is less clear. Examination of fetuses from severely zinc-deficient dams at d 12 and 14 of gestation revealed that invagination of the optic cup was often deficient and that closure of the choroid fissure did not occur. This resulted in retinal folding at the choroid fissure that was visible at term (Rogers and Hurley 1987). When zinc deficiency was instituted at parturition, eye lid opening was delayed but no morphological alterations were noted among photoreceptor cells at weaning (Sinning et al. 1984).

The influence of zinc and taurine on oscillatory potentials (OP) has not been reported. Oscillatory potentials are a series of rhythmic consecutive discharges of retinal neurons found superimposed on the b-wave of the electroretinogram (Speros and Price 1981). It is thought that these oscillations are generated independently from the mechanism producing the primary components of the electroretinogram, the a- and b-waves, at a more proximal location, possibly the inner nuclear layer (el Azazi and Wachtmeister 1990). Our objective was to investigate whether the proposed interaction between zinc and taurine influences the development of retinal structure and function in rats as examined by light microscopy and oscillatory potentials. In addition, we wanted to determine the effect of this interaction on zinc and taurine status of the rats.

MATERIALS AND METHODS

Rats were fed control diet for at least 7 d and bred overnight. On d 0 of gestation, as determined by the presence of sperm in vaginal smears, the females were placed individually in stainless steel cages and randomly assigned to one of five treatments. Treatments consisted of four diets formulated with two levels of zinc (+Zn, 50 µg Zn/g diet; Zn, 15 µg Zn/g diet) and 2 levels of taurine (+Tau, 2 µmol taurine/g diet; Tau, 0 µmol taurine/g diet) in the following combinations: +Zn/+TauAL (control), +Zn/Tau, Zn/+Tau or Zn/Tau. The above groups were provided with free access to modified AIN-93G diets (Reeves et al. 1993) and distilled deionized water. A fifth group of rats (+Zn/+TauPF ) was fed control diet and individually pair-fed to their respective pairs in the Zn/+Tau group. The basal diet presented in Table 1 contains < 0.001 µmol/g taurine as determined by HPLC. GES (10 g/L) was added to the drinking water of the +Zn/Tau and Zn/Tau groups. Zinc concentration of the Zn/+Tau and Zn/Tau diets was further reduced to 7.5 µg/g at parturition.

Table 1. Composition of basal diet1 [View Table]

On d 20 of gestation, dams were transferred to stainless steel cages fixed with plastic mesh bottoms and provided with 30 × 50 cm of absorbent paper (VWR, Edmonton, AB, Canada) as nesting material. Litters were adjusted to 7-10 pups at postnatal d 4. Nesting material was removed within 7 d of birth. At postnatal d 23, male pups (10/treatment) were weaned onto their respective diets. Food intake was recorded daily and weight recorded weekly for the pups.

GES was synthesized and purified (POS Pilot Plant Corporation, Saskatoon, SK, Canada) according to the method of Morrison et al. (1958). Reversed-phase HPLC confirmed that a solution containing 10 g/L GES contained < 0.001 mmol/L taurine; atomic absorption spectrophotometry revealed < 1.5 µmol/L zinc.

The light intensity used was 45 J/cm² , measured at approximate rat eye level, with a Pasco high sensitivity photometer model #OS-8020 (Pasco Scientific, Roseville, CA). Ten 1.2-ms pulses were presented at a rate of 3.6 flashes/min. Oscillatory potential response was recorded using a low-cut filter setting of 30 Hz and a high-cut filter setting of 200 Hz. Data were averaged using a Cadwell 5200A (Cadwell Laboratories) and stored on computer using the Cadwell 5200A Save/Recall Program (Cadwell Laboratories). Amplitude and latency were determined for each oscillatory potential wavelet. OP₁ amplitude was measured from base line; the amplitudes of OP₂ , OP₃ , OP₄ , and OP₅ were measured from the lowest preceding deflection. Oscillatory potential latencies were measured from stimulus onset to the peak of each wavelet.

Table 2. Food intake and body weight of rat pups in response to varied zinc and taurine status throughout gestation and 7.5-8.5 wk of postnatal life1 [View Table]

Table 3. Tissue zinc and taurine concentrations in rats exposed to varying zinc and taurine status throughout gestation and 7.5-8.5 wk of postnatal life1,2 [View Table]

Liver and eye taurine concentrations were determined by HPLC as previously described (Gottschall-Pass et al. 1995). Plasma zinc was determined by flame atomic absorption spectrophotometry (FAAS) after diluting the sample (1:4) with distilled, deionized water. Liver, tibia and eye zinc were determined by FAAS after wet ashing (Clegg et al. 1981). Two eyes were pooled for each analysis. All readings were conducted with the instrument in the absorbance mode. Concentrations were calculated from the absorbance values by use of a linear regression equation, which was obtained with seven standard concentrations made in 0.1 mol/L Ultrex grade HNO₃ (PDI Joldon, Aurora, ON, Canada). National Institute of Standards and Technology (Gaithersburg, MD) bovine serum or liver was included in sample runs as a standard reference material. Recovery for these reference materials was 91.7 and 95.7-102.2%, respectively.

RESULTS

A significant interaction between zinc and taurine was observed for tibial zinc concentration. Zinc deficiency depressed tibial zinc concentrations (+Zn/+TauAL vs. Zn/+Tau); however, this effect was less pronounced when the rats were also taurine deficient (Zn/+Tau vs. Zn/Tau).

Marginal zinc deficiency and taurine deficiency both significantly depressed eye zinc concentrations; however, no interaction between zinc and taurine was detected.

Marginal zinc deficiency significantly depressed liver zinc concentrations, but no interaction between zinc and taurine was observed.

Taurine deficiency significantly depressed eye taurine concentrations, but no interaction between zinc and taurine was observed.

Table 4. Oscillatory potential (OP) amplitudes and latencies in response to a flash of white light provided by Ganzfeld illumination in rats in which zinc and taurine status was varied throughout gestation and 7.5-8.5 wk of postnatal life1,2 [View Table]

Biomicroscopic examination at 7 wk identified the unilateral development of uveitis, a focal keratitis and an anterior cortical cataract in one rat from the +Zn/+TauAL group. These lesions were consistent with incidental trauma and were not included in electroretinographic or histologic assessments.

Examination of retinas from +Zn/Tau rats revealed mild degeneration of the photoreceptors; the outer segments were shorter and contained numerous vesicles, disorientated discs and discontinuous cell membranes (Fig. 4b).

Retinas from the Zn/+Tau and +Zn/+TauAL groups were similar and no abnormalities were noted (Fig. 4c).

DISCUSSION

Oscillatory potentials provide further evidence of an interaction between zinc and taurine; zinc deficiency depressed the amplitude of OP₂ and OP₃ only when the rats were also taurine deficient. The presence of an interaction in only two of the five oscillatory potentials suggests that zinc and taurine may function synergistically in only specific areas of the inner retina. Each of the five oscillatory potentials is believed to originate independently from one another in different regions of the inner nuclear layer of the retina (el Azazi and Wachtmeister 1990). The reversibility of these changes is not known. Interaction was also evident in the depressing effects of taurine deficiency on whole-eye zinc concentration. However, it is difficult to relate these changes to the morphological and functional changes in the retina; whole eye may not be representative of retinal concentrations because the levels of these nutrients in different eye tissues are variable (Eckhert 1983, Heinämäki et al. 1986).

The retinal degeneration observed in combined zinc and taurine deficiency is particularly important given the marginal degree of zinc restriction employed in this study. This model was chosen to represent the degree of zinc deficiency that could occur in the human population. Although zinc deficiency was confirmed by significantly depressed plasma, tibia, liver and whole-eye zinc concentrations, the rats showed no clinical signs. The small decrease in food intake in zinc-deficient rats was not significant although intake was consistently lower than that of control rats at all time points.

The model of taurine deficiency used in this study is similar to that described previously (Bonhaus et al. 1985, Lake 1983, Pasantes-Morales 1983). Rats fed taurine-deficient diets and GES had depressed liver and whole-eye taurine concentrations consistent with other reports (Bonhaus et al. 1985). Depressed retinal taurine has also been described using this model (Cocker and Lake 1989, Lake 1981, Rapp et al. 1987). Other investigators have reported limited data on food intake and weight gain. In the present study, taurine deficiency tended to depress food intake and weight gain in the postweaning period, whereas weight gain at weaning was similar to that of control rats. Previous research that found no intrauterine growth retardation in fetuses of rats deficient in taurine throughout gestation (Gottschall-Pass et al. 1995) and no effect on pup weight during lactation up to postnatal d 17 (Lake 1983) is consistent with this observation.

The biochemical mechanisms responsible for the interaction of zinc with taurine are unknown. Zinc has been proposed to play a physiological role in plasma membranes by its effects on membrane protein conformation and protein-protein interactions (Bettger and O'Dell 1993). Although the precise biochemical roles in the membrane have not been fully elucidated, it has been suggested that the loss of zinc from specific proteins in the plasma membrane alters water and ion channels. Ultimately, intracellular ion and water concentration is changed (Bettger and O'Dell 1993). We hypothesize that taurine, through its role as an intracellular osmoregulator (Huxtable 1992), can respond by regulating water and ion flow across the photoreceptor and inner retinal membrane. The photoreceptor degeneration in combined zinc and taurine deficiency may represent a loss of membrane integrity in the outer retina exacerbated by the loss of an osmoregulatory mechanism. Depressed oscillatory potentials may reflect decreased integrity of neuronal membranes in the inner retinal regions.

In addition to retinal pathology, evidence of focal choroidal atrophy in rats deficient in both zinc and taurine was also observed. This anomaly was initially detected on ophthalmoscopic assessment and confirmed by light microscopy. These data provide additional evidence of an interaction between zinc and taurine in another eye structure. Choroidal atrophy has been reported to occur spontaneously in normal Sprague-Dawley rats (Hubert et al. 1994); the etiological relevance is unknown. We found no evidence of this condition in any other experimental group.

Plasma and tibial zinc concentrations provide evidence that zinc and taurine interact in other tissues. Zinc deficiency depressed plasma and tibial zinc; however, the effect was less pronounced when rats were also taurine deficient, suggesting that taurine influences the zinc status of the animal. Zinc apparently also influences taurine metabolism because liver taurine concentration was elevated by zinc deficiency.

The main treatment effects observed in this study also deserve consideration. Independent effects of taurine deficiency on specific oscillatory potentials were evident with reductions in the amplitudes of OP₁ and OP₄ . Although previous research has identified the importance of taurine in the development of normal photoreceptor morphology and function (Bonhaus et al. 1985, Imaki et al. 1986), our results suggest that taurine also functions in the inner retina. These findings support the work of Lake and Malik (1987) who reported a reduction in inner retinal width on light microscopic examination in rats treated with GES for the last week of gestation and for 8 wk following parturition. We also observed photoreceptor disruption in taurine-deficient rats as has been previously reported (Bonhaus et al. 1985, Lake and Malik 1987, Pasantes-Morales et al. 1983).

The main treatment effect of zinc on certain oscillatory potentials suggests additional independent biochemical functions for zinc in the retina. Marginal zinc deficiency independently depressed the amplitude and increased the latency of OP₁ , and increased the latency of OP₃ . Decreased photoreceptor zinc content has previously been observed by histochemical localization (Hirayama 1990), and morphological changes have been reported in the photoreceptors and retinal pigment epithelium of severely zinc-deficient rats (Leure-duPree and McClain 1982). Little information is available on the influence of marginal zinc deficiency, particularly in regions of the inner retina. Although we did not detect morphological alterations in marginally zinc-deficient rats, future studies using electron microscopy may detect ultrastructural damage that was not apparent on gross histological assessment. The effects of zinc deficiency on retinal electrophysiology and morphology and the mechanism by which zinc exerts these effects deserve further investigation.

In summary, morphological and physiological evidence has been obtained that zinc and taurine interact in the developing rat retina. This has been demonstrated by severe photoreceptor damage, retinal dysplasia and depressed oscillatory potentials in combined zinc and taurine deficiency. Evidence of an interaction in other tissues was noted in plasma zinc, tibial zinc and liver taurine concentrations. Choroidal atrophy was also apparent in the combined deficiency group. The mechanism responsible for this interaction deserves investigation. In addition, independent effects of zinc and taurine on oscillatory potentials were demonstrated. These have not been previously identified, and they suggest additional independent functions for each of these nutrients in the inner retina.

ACKNOWLEDGMENTS