Analysis of copper in brain by the mass-spectrometric integrated-ioncurrent procedure

Through use of the high-resolution double-focusing mass spectrometer, copper has been identified in various regions of the mouse, rat, guinea pig, rabbit, and human brain. The procedure depends on converting the copper (in ashed tissue) to its chloride salt, followed by derivatization with tetraphenylporphyrin (TPP) to yield a TPP chelate. After chromatographic separation, this chelate is assessed in the mass spectrometer by the integrated-ion-current procedure. Deuterated metal TPP chelates and the rare stable isotope⁶⁵Cu were used as internal standards. Whole brain values obtained were as follows: mouse, 6.67±0.16 (mean±SEM) g/g wet weight of tissue; rat, 1.06±0.05; guinea pig, 5.40±0.63; and rabbit, 7.52±0.76. In the rat, the cerebellum contained the highest concentration (1.25 g/g), and the striatum the lowest (0.70 g/g). In the human brain, the cortex (gray) and the striatum were relatively the highest copper-containing regions, with the cerebellum (white) being the lowest.     

The intracellular distribution and activities of phosphoenolpyruvate carboxykinase isozymes in various tissues of several mammals and birds.

1. The intracellular distribution and/or activities of phosphoenolpyruvate carboxykinase isozymes were determined in liver, kidney, gastrointestinal mucosa, adipose, skeletal muscle, brain, spleen, lung and heart of fed and fasted rabbits, guinea pigs, rats, chickens and pigeons. 2. Liver and kidney of all species contained the highest enzyme activity/g. 3. Carboxykinase activity/g gastrointestinal mucosa of rabbits was quite high compared to the low activity in guinea pig and rat mucosa and essentially undetectable activity in chicken and pigeon mucosa. 4. Activity/g was high in rat brown adipose. 5. Low carboxykinase activity/g was found in skeletal muscle of all species and in white adipose of guinea pig, rabbit and rat although activity was undetectable in white adipose of chicken and pigeon. 6. Carboxykinase activity was essentially undetectable in brain, spleen, lung and heart of all species.     

Light and electron microscopic studies of the paracortical post-capillary high-endothelial venules

Summary The light and electron microscopic structure of the high-endothelial postcapillary venules of lymph nodes were studied in the mouse, rat, guinea pig, and rabbit. The venules were most frequently found in the mouse and rat. In all species, they reached their highest degree of differentiation in the paracortical area. The morphology in the light microscope was identical in all four species. The venules in the rat and mouse were surrounded by a cuff of concentrically-arranged lymphocytes, which was rarely seen in the guinea pig and rabbit.The ultrastructure of the high-endothelial cells in all four species was very complex; a prominent Golgi apparatus was present which often occupied large parts of the cytoplasm. Coated and uncoated vesicles originating in the Golgi apparatus often permeated the cytoplasm. These vesicles emptied their contents into the extracellular space after fusion with the plasma membranes.Numerous lymphocytes traversed vessel walls. During their passage, they were always located between, not inside the high-endothelial cells.     

Developmental Changes of N-Acetyl-L-Aspartic Acid, N-Acetyl-α-Aspartylglutamic Acid and β-Citryl-L-Glutamic Acid in Different Brain Regions and Spinal Cords of Rat and Guinea Pig

Abstract The developmental changes of N -acetylaspartic acid (NA-Asp), N -acetyl-α-aspartylglutamic acid (NA-Asp-Glu), and β -citryl-L-glutamic acid ( β -CG) have been examined in the cerebrum, cerebellum, brain stem and spinal cord of both rat and guinea pig by the gas chromatographic method developed in our studies. A rapid increase in the concentration of NA-Asp was observed postnatally in every region of the rat brain. On the other hand, all regions of guinea pig brain showed the prenatal increases. NA-Asp-Glu showed a different developmental profile, depending on region of the brain, in the two species. The concentration of NA-Asp-Glu remained constantly low during brain maturation in the rostral regions. In the caudal portions it showed a marked increase during maturation and reached a high level in the adult brain. The concentration of β -CG was highest at birth in all regions of rat brain and rapidly decreased by 20 days after birth and remained low thereafter. The rapid decrease occurred in the guinea pig during the foetal period, and β -CG content decreased to an adult level at birth.     

5'-Adenosine monophosphate and adenosine metabolism, and adenosine responses in mouse, rat and guinea pig heart.

We examined myocardial 5'-adenosine monophosphate (5'-AMP) catabolism, adenosine salvage and adenosine responses in perfused guinea pig, rat and mouse heart. MVO(2) increased from 71+/-8 microl O(2)/min per g in guinea pig to 138+/-17 and 221+/-15 microl O(2)/min per g in rat and mouse. VO(2)/beat was 0.42+/-0.03, 0.50+/-0.03 and 0.55+/-0.04 microl O(2)/g in guinea pig, rat and mouse, respectively. Resting and peak coronary flows were highest in mouse vs. rat and guinea pig, and peak ventricular pressures and Ca(2+) sensitivity declined as heart mass increased. Net myocardial 5'-AMP dephosphorylation increased significantly as mass declined (3.8+/-0.5, 9.0+/-1.4 and 11.0+/-1.6 nmol/min per g in guinea pig, rat and mouse, respectively). Despite increased 5'-AMP catabolism, coronary venous [adenosine] was similar in guinea pig, rat and mouse (45+/-8, 69+/-10 and 57+/-14 nM, respectively). Comparable venous [adenosine] was achieved by increased salvage vs. deamination: 64%, 41% and 39% of adenosine formed was rephosphorylated while 23%, 46%, and 50% was deaminated in mouse, rat and guinea pig, respectively. Moreover, only 35-45% of inosine and its catabolites derive from 5'-AMP (vs. IMP) dephosphorylation in all species. Although post-ischemic purine loss was low in mouse (due to these adaptations), functional tolerance to ischemia decreased with heart mass. Cardiovascular sensitivity to adenosine also differed between species, with A(1) receptor sensitivity being greatest in mouse while A(2) sensitivity was greatest in guinea pig. In summary: (i) cardiac 5'-AMP dephosphorylation, VO(2), contractility and Ca(2+) sensitivity all increase as heart mass falls; (ii) adaptations in adenosine salvage vs. deamination limit purine loss and yield similar adenosine levels across species; (iii) ischemic tolerance declines with heart mass; and (iv) cardiovascular sensitivity to adenosine varies, with increasing A(2) sensitivity relative to A(1) sensitivity in larger hearts.     

Species Differences in the Brain Regional Distribution of Receptor Binding for Thyrotropin-Releasing Hormone

Abstract: A survey of the regional distribution of binding of 1 nM [³H](3-MeHis²)thyrotropin-releasing hormone ([³H]MeTRH) to TRH receptors in the brains of eight mammalian species revealed major species differences in both the absolute and relative values of TRH receptor binding in different brain regions. Several brain regions exhibited binding equal to or exceeding that in the anterior pituitary gland of the same species, including the amygdaia in the guinea pig and rat, the hypothalamus in the guinea pig, the nucleus accumbens in the rabbit, and all these and other regions in the cat and dog, for which pituitary binding was exceptionally low. Species could be divided into two groups according to which brain region appeared highest in binding: rabbits, sheep, and cattle had highest binding in the nucleus accumbens/septal area, whereas guinea pigs, rats, dogs, cats, and pigs had highest binding in the amygdala/temporal cortex area. The nucleus accumbens consistently exceeded the caudate-putamen in receptor binding. For most brain regions, rabbits, rodents, and sheep tended to be higher than carnivores, cattle, or pigs. Further regions that exhibited appreciable binding in most species included the olfactory bulb and tubercle, hippocampus, and various cortical and brain stem areas. In fact, essentially all brain regions appeared to have detectable levels of TRH receptors in at least some species, but no rat peripheral tissues have yet shown detectable receptor binding. The species differences appeared to reflect largely if not entirely differences in receptor density, although this was not tested in every species.     

Determination of in vivo protein binding of homocysteine and its relation to free homocysteine in the liver and other tissues of the rat

Low concentrations (0.5-6 nmol/g) of homocysteine (Hcy) have recently been demonstrated in acid extracts of various tissues of the mouse and rat (Ueland, P.M., Helland, S., Broch, O.-J., and Schanche, J.-S. (1984) J. Biol. Chem. 259, 2360-2364). This is referred to as free Hcy in tissues. This paper describes a method for the determination of protein-bound Hcy, which involves precipitation and washing of tissue protein with ammonium sulfate, release of Hcy from native proteins in the presence of dithioerythritol, and determination of free Hcy by a sensitive radioenzymic assay. Both free and bound Hcy decreased markedly in rat tissues within a few seconds following death of the animal. The amount of protein-bound Hcy was highest in liver, somewhat lower in kidney, brain, heart, lung, and spleen. The ratio between free and bound Hcy was between 1 and 2 in most tissues, except in cerebellum, containing a large excess of free Hcy (free/bound ratio of 18). Free Hcy was almost exclusively localized to the soluble fraction of rat liver, whereas protein-bound Hcy was about equally distributed between this fraction and the microsomes. Isolated rat hepatocytes contained free and protein-bound Hcy in proportions observed in whole liver, but a large amount of Hcy was exported into the extracellular medium. The half-lives, as determined from pulse-chase experiments with [35S] methionine, were 53 s for S-adenosylmethionine, 2 s for S-adenosylhomocysteine and 3 s for Hcy (free and bound regarded as a single pool). Furthermore, isotope equilibrium between these metabolites and between free and bound Hcy throughout the rapid chase period suggests the turnover rates of S-adenosylhomocysteine and Hcy to be production rate limited, and the dissociation rate of the Hcy-protein complex may greatly exceed the turnover rate of Hcy. Thus, the half-lives of Hcy are such that participation of both free and bound Hcy in metabolic regulation is feasible.     

Aldehyde dehydrogenase activity in blood from various vertebrates

1. Aldehyde dehydrogenase activity was determined in whole blood samples from 17 selected vertebrates of 5 classes, using 3,4-dihydroxyphenylacetaldehyde (the aldehyde derived from dopamine) as substrate. 2. Aldehyde dehydrogenase activity in blood was widely but unevenly distributed among the species studied. 3. Mean aldehyde dehydrogenase activities in the range of 40-140 nmol/min.ml blood (measured at 37 degrees C, pH 8.8) were found in blood from man, monkey, rabbit, guinea pig and mouse (C57BL and NMRI strains), with the highest activity in rabbit blood. 4. Much lower aldehyde dehydrogenase activities (0.5-7.5 nmol/min.ml blood) were found in blood from Sprague-Dawley and Wistar rat, dog, cat, horse, pig, chicken, caiman, frog and rainbow trout, whereas the activities in blood from DBA mouse, cow, sheep and crucian carp were close to the detection limit.     

Estrone sulfate sulfohydrolase in the developing brain and pituitary of rat, mouse and guinea pig

The pattern of estrone sulfate sulfohydrolase (estrogen sulfatase) development in the brain of rat, mouse and guinea pig has been established by assaying whole homogenates. Activity was measurable in each species from the fetal state to adulthood. Maximum brain content was reached at about 20 days of age in rat, 14 days in mouse and 15 days in guinea pig. A considerable decrease occurred between 14 days and adulthood in mouse and lesser decreases were seen in rat and guinea pig. The subcellular distribution of enzyme in rat and mouse brain appeared to change from the immature to the adult state. No major differences in enzyme activity occurred between the sexes at any age. Tissue concentration of enzyme in the hypothalamic-preoptic area of rat and mouse was similar to that in the remainder of the brain. In guinea pig the brain concentration was slightly lower than that of the hypothalamic-preoptic region. Sulfatase content of the pituitary was low in all 3 species but the tissue concentration was considerably higher than that of brain, particularly in rat and mouse. Apparent Km values for brain sulfatase were in the range 6-17 microM, with no striking sex difference. Apparent Km's for pituitary sulfatase of immature rat and guinea pig were similar to those for brain in the same animals but that for mouse pituitary (0.9 microM) was much lower. It is unlikely that brain or pituitary sulfatase is by itself, a major factor in making available potentially active estrogen for use during differential sex development in these species.     

Regional distribution of the muscarinic cholinoceptor and acetylcholinesterase in guinea pig brain

The muscarinic receptors in membranes prepared from guinea pig brain were studied using a radiolabeled antagonist, [³H]quinuclidinyl benzilate (QNB). The apparent dissociation constant of the QNB-receptor complex (K _d ) was similar in all regions, but the concentration of receptors was highest in the striatum, cerebral cortex, and hippocampus and lowest in the cerebellum. Similar distributions have been reported for other species, although the concentration of receptors in guinea pig brain is higher than in other species. Acetylcholine inhibited QNB binding with a Hill coefficient of 0.4–0.6. The concentration of acetylcholine required to inhibit binding by 50% (I₅₀) was lowest in the brain stem and more than 10 times higher in the hippocampus. Similar results have been reported for mouse brain. The activity of acetylcholinesterase was highest in the striatum, where the concentration of muscarinic receptors is highest, but did not vary greatly in other brain regions.RMD was seconded to the University of Melbourne to undertake this study.     

Regional distribution of the histamine metabolite, tele-methylimidazoleacetic acid, in rat brain: effects of pargyline and probenecid

Abstract: tele -Methylimidazoleacetic acid (t-MIAA), a major brain histamine metabolite, was measured in nine rat brain regions by a gas chromatography-mass spectrometric method that also measures the precursor amine, tele -methylhistamine (t-MH). The t-MIAA concentration of cerebellum, medulla-pons, midbrain, caudate nucleus, hypothalamus, frontal cortex, hippocampus, and thalamus varied 15-fold, hypothalamus showing the highest level (2.21 nmol/g) and cerebellum the lowest (0.15 nmol/ g). The concentrations of t-MIAA and t-MH were significantly correlated in all regions except midbrain, which had relatively more t-MIAA. Probenecid did not alter whole-brain t-MIAA levels. Treatment with pargyline, an inhibitor of monoamine oxidase, lowered the t-MIAA levels in all regions.     

Homocysteine in tissues of the mouse and rat

A method for the determination of L-homocysteine (Hcy) in tissues is described, which involves adsorption of adenosine and S-adenosyl-L-homocysteine (AdoHcy) in the tissue extract to dextran-coated charcoal, while leaving Hcy in solution. Sufficient dilution of the tissue homogenates and the presence of a reducing agent during the adsorption step are required to obtain high recovery of Hcy. Hcy is condensed with radioactive adenosine, and labeled AdoHcy is quantified by high performance liquid chromatography on a 3-micron reversed phase column. The amount of Hcy was determined in several tissues (liver, kidney, brain, heart, lung, and spleen) of mice and rats, and the concentrations of Hcy were in the range 0.5-6 nmol/g, wet weight. Hcy concentration was about 1 microM in mouse plasma. In mice, liver contained the highest amount of Hcy, and kidneys were also rich in Hcy. Similar concentrations were found in rat tissues. S-Adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), the enzyme which is believed to catalyze the only pathway leading to Hcy formation in vertebrates, was nearly completely inactivated in mice injected with the drug combination 9-beta-D-arabinofuranosyladenine plus 2'-deoxycoformycin. This treatment induced a massive accumulation of AdoHcy in all tissues (Helland, S., and Ueland, P. M. (1983) Cancer Res. 43, 1847-1850). The amount of Hcy increased several-fold in kidney, whereas no change was observed in liver, heart, brain, lung, and spleen.     

An interspecies comparison of gangliosides and neutral glycolipids in adrenal glands

Gangliosides and neutral glycolipids of adrenal glands of mouse, rat, guinea pig, rabbit, cat, pig, cow, monkey, and chicken were analyzed by thin layer chromatography (TLC). The major gangliosides common to all species had lactosylceramide in their core structure. GM3 containing N-acetylneuraminic acid (NeuAc) was the major ganglioside in rat, guinea pig, rabbit, and cat, whereas GM3 containing N-glycolylneuraminic acid (NeuGc) was the major one in mouse, cow, and monkey. GD3 was also detected in all species except mouse and GD3(NeuAc)2 was the major in pig adrenal gland. GM4(NeuAc) was detected in the adrenal glands of guinea pig and chicken but not in those of the other species. In the neutral glycolipid fractions, galactosylceramide, glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide were detected and the proportions of these glycolipids varied among the species. Guinea pig and chicken adrenal glands contained large amounts of galactosylceramide, this being consistent with the presence of GM4 in these two species. Globopentaosylceramide was detected in mouse, guinea pig, cat, and chicken by the TLC-immunostaining procedure.     

Interspecies differences in the metabolism of brain norepinephrine to glycol metabolites

Using a highly sensitive and specific gas chromatography-mass spectrometric assay, the glycol metabolites of norepinephrine (NE), 3,4-dihydroxyphenylethyleneglycol (DHPG) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) were determined simultaneously in brain and body fluids of several mammalian species, including humans. Highest molar ratios of DHPG to MHPG were found in rat brain (1.20), a species in which these glycol metabolites were primarily conjugated. In mouse, guinea pig, hamster, monkey, and human brain, DHPG and MHPG were mostly unconjugated, and DHPG concentrations were about 30–60% of the respective MHPG levels. In dog cortex, MHPG occurred predominantly as conjugates, whereas DHPG could only be detected in its unconjugated form. In all species studies, highest DHPG and MHPG concentrations occurred in hypothalamus followed, in general, by midbrain and brainstem whereas cerebral cortex, caudate and cerebellum had the lowest values. These results demonstrate substantial differences in the degree of conjugation and relative abundance of brain DHPG compared to MHPG between the rat and other animal species studied.     

COMPARISON OF THE EFFECTS OF DEPOLARIZING AGENTS AND NEUROTRANSMITTERS ON REGIONAL CNS CYCLIC GMP LEVELS IN VARIOUS''ANIMALS

Abstract— The effects of 121 m m -K⁺, 10 m m -glutamate, 5 m m -GABA, 1 m m -glycine, 0.1 m m -NE, and 1–10 μ m ACh on cyclic GMP levels in tissue slices prepared from cerebral cortex and cerebellum of mouse, rabbit, guinea-pig, cat, and rat were studied. Basal levels of cyclic GMP in the cerebella of mice, guinea-pigs and cats were 4–15 and 70 pmol/mg prot in rat, whereas in the cerebral cortex of the same animals, levels were only 0.6–2 pmol/mg prot. In contrast, basal levels of the cyclic nucleotide were 1–2 pmol/mg prot in both of these regions in rabbit brain. Only 121 m m -K⁺ was capable of increasing cyclic GMP levels in all the tissues studied. Elevations ranged from 30% in rat cerebral cortex to 2800% in mouse cerebellum. Glutamate produced a 30–1000% rise of cyclic GMP levels in all tissues except rabbit cerebellum. NE elevated levels of cyclic nucleotide 2- to 3-fold in slices of cerebellum from all species studied but had no effect in cerebral cortex. GABA and glycine had no effect in any tissue except mouse cerebellum. ACh had no consistent effect on levels of cyclic GMP in any brain region investigated. These results suggest that mechanisms regulating cyclic GMP levels in mammalian CNS vary among brain regions and among animal species.     

S-Adenosyl-l-homocysteine in brain

Administration of methionine sulfoximine (MSO) to rats and mice significantly decreased cerebral levels ofS-adenosyl-l-homocysteine (AdoHcy). Concurrent administration of methionine prevented this decrease and, when methionine was given alone, significantly elevated AdoHcy levels resulted in both species. Regionally, AdoHcy levels varied from 20 nmol/g in rat cerebellum and spinal cord to about 60 nmol/g in hypothalamus and midbrain. MSO decreased AdoHcy in all regions tested except striatum, midbrain, and spinal cord. AdoMet/AdoHcy ratios (methylation index) varied from 0.48 in hypothalamus to 2.4 in cerebellum, and MSO administration decreased these ratios in all regions except hypothalamus. AdoHcy hydrolase activity was lowest in hypothalamus, highest in brainstem and, generally, varied inversely with regional AdoHcy levels. MSO decreased AdoHcy hydrolase activity in all regions except hypothalamus and spinal cord. Cycloleucine administration resulted in significantly decreased levels of mouse brain AdoHcy, whereas the administration of dihydroxyphenylalanine (DOPA) failed to affect AdoHcy levels. It is concluded that (a) cerebral AdoHcy levels are more tightly regulated than are those of AdoMet after MSO administration, (b) slight fluctuations of AdoHcy levels may be important in regulating AdoHcy hydrolase activity and hence AdoHcy catabolism in vivo, (c) the AdoMet/AdoHcy ratio reflects the absolute AdoMet concentration rather than the transmethylation flux, (d) the decreased AdoMet levels in midbrain, cortex, and striatum after MSO with no corresponding decrease in AdoHcy suggest an enhanced AdoMet utilization, hence an increased transmethylation in the MSO preconvulsant state.Supported by USPHS, NINCDS grant NS-06294.     

Differences in activity of hepatic microsomal triglyceride transfer protein among species

Five sows, five cows, five hens, six guinea pigs, six rabbits, and six rats were used in a study to determine if hepatic microsomal triglyceride transfer protein activity differed among species that varied in site of fatty acid synthesis and rate of hepatic triglyceride export. No differences in plasma nonesterified fatty acids were seen among species. Plasma concentrations of glucose were highest in the hen, intermediate in the rat, guinea pig, and rabbit and lowest in the sow and cow. Liver triglyceride was low in all species with the only significant difference being between the hen and the guinea pig (4.7 and 1.1%, DM basis, respectively). No microsomal triglyceride transfer protein activity was found in muscle. The cow, rat, and guinea pig had the lowest levels and the hen and rabbit the highest levels of duodenal microsomal triglyceride transfer protein activity. Hepatic microsomal triglyceride transfer protein activity was significantly higher in the sow than the other species. Hepatic microsomal triglyceride transfer protein activity was 1.51, 1.63, 2.36, 2.72, 2.95, and 6.70 nmole triolein transferred/h/mg microsomal protein for the guinea pig, rabbit, cow, rat, hen, and sow, respectively. Microsomal triglyceride transfer protein activity in duodenal tissue was 18.0, 18.6, 19.2, 33.4, 113, and 161% of hepatic microsomal triglyceride transfer protein activity for the sow, cow, rat, guinea pig, hen, and rabbit, respectively. Hepatic microsomal triglyceride transfer protein activity scaled to liver weight and metabolic body size was 2.69, 3.36, 4.58, 5.83, 7.49, and 22.3 nmole triolein transferred in the liver/min/kg body weight0.75 for the rabbit, guinea pig, rat, hen, cow, and sow, respectively. There was little relationship between previously published rates for triglyceride export and hepatic microsomal triglyceride transfer protein activity measured in this experiment.     

The interaction of triethyltin with components of animal tissues

1. The distribution of triethyl[(113)Sn]tin chloride in the rat, guinea pig and hamster is not uniform, the highest concentrations being in rat blood and the liver of all three species. 2. Subcellular fractionation of rat liver, brain and kidney shows that triethyltin binds to all fractions to different extents. In the liver of the rat and guinea pig the supernatant fraction contains the largest amount and the highest specific concentration; this triethyltin is bound to a non-diffusible component. 3. Rat haemoglobin is responsible for the binding of triethyltin in rat blood (2 moles of triethyltin/mole of haemoglobin). Haemoglobins from other species have much less affinity for triethyltin. 4. A variety of other proteins do not bind triethyltin.     

The intermediate filament complement of the retina: a comparison between different mammalian species

We compared the intermediate filament expression of the various cell types in the fully differentiated neural retina from rat, mouse, rabbit, guinea pig, cow, pig, and cat. Many cell types had an intermediate filament complement conserved across species boundaries, such as Müller cells and retinal ganglion cells. In some species (rabbit, guinea pig, and cow), however, we were unable to visualize GFA (glial fibrillary acidic)-positive retinal astrocytes, although such profiles were clearly visible in the remainder. Horizontal cell staining proved to be extremely species-variable. In rat and mouse the processes of these cells were identically displayed with antibodies to vimentin and all three neurofilament triplet proteins. In cow they decorated with antibodies to vimentin and antibodies to the two lower molecular weight neurofilament proteins alone, whereas in pig, rabbit and guinea pig all three neurofilament proteins but not vimentin were present. Finally cat horizontal cells stained for all three neurofilament proteins, some finer processes being additionally stainable with vimentin. A further surprise was the visualization of profiles positive only for the two lower molecular weight neurofilament proteins in the inner nuclear layer of both rabbit and guinea pig retina but not the other species. The implications of these results will be discussed.     

A comparative biochemical and ultrastructural study of proteoglycan-collagen interactions in corneal stroma. Functional and metabolic implications.

1. Corneas of mouse, rat, guinea pig, rabbit, sheep, cat, dog, pig and cow were quantitatively analysed for water, hydroxyproline, nucleic acid, total sulphated polyanion, chondroitin sulphate/dermatan sulphate and keratan sulphate, several samples or pools of tissue from each species being used. Ferret cornea was similarly analysed for water and hydroxyproline on one pool of eight corneas. Pooled frog (38) and ferret (eight) corneas and a single sample of human cornea were qualitatively examined for keratan sulphate and chondroitin sulphate/dermatan sulphate by electrophoresis on cellulose acetate membranes. Nine species (mouse, frog, rat, guinea pig, rabbit, sheep, cat, pig and cow) were examined by light microscopy and six (mouse, frog, rat, guinea pig, rabbit and cow) by electron microscopy, with the use of Alcian Blue or Cupromeronic Blue in critical-electrolyte-concentration (CEC) methods to stain proteoglycans. 2. Water (% of wet weight), hydroxyproline (mg/g dry wt.) and chondroitin sulphate (mg/g of hydroxyproline) contents were approximately constant across the species, except for mouse. 3. Keratan sulphate contents (mg/g of hydroxyproline) increased with corneal thickness, whereas dermatan sulphate contents decreased. The oversulphated domain of keratan sulphate was absent from mouse and frog corneas, increasing as percentage of total keratan sulphate with increasing corneal thickness. Sulphation of dermatan sulphate was essentially complete (i.e. one sulphate group per disaccharide unit). 4. Chondroitin sulphate/dermatan sulphate proteoglycans were present at the d bands of the collagen fibrils of all species examined, orthogonally arrayed, with high frequency, and occasionally at the e bands. Keratan sulphate proteoglycans were present at the a and c bands of all species examined, but with far higher frequency in the thicker corneas, where keratan sulphate contents were high. 5. Alcian Blue CEC staining showed much higher sulphation of keratan sulphate in thick corneas, e.g. that of cow, than in thin corneas, e.g. that of mouse, in keeping with biochemical analyses. 6. It is suggested that the constancy of interfibrillar volumes is regulated via the swelling and osmotic pressure of the interfibrillar polyanions, by adjustment of the extent of sulphation in two independent proteoglycan populations, to achieve an 'average sulphation' of the total polyanion similar to that of fully sulphated chondroitin sulphate/dermatan sulphate. 7. The balance of synthesis of the two kinds of proteoglycans may be determined by the O2 supply to the avascular cornea. O2 supply may also determine the conversion of chondroitin sulphate into dermatan sulphate.