('omp. Biochem. Phlsiol,. 1976, Vol. 53B. pp. 167 to 172. Pergamon Press. Printed in Great Britain
THE PLASMA TRANSPORT PROTEINS OF 25-HYDROXYCHOLECALCIFEROL IN FISH, AMPHIBIANS, REPTILES AND BIRDS A. W. M. HAY AND G. WATSON Nuffield Institute of Comparative Medicine, The Zoological Society of London, Regent's Park, London, NWI, England (Received 17 October 1974)
Abstract--1. The transport proteins of 25-hydroxycholecalciferol were studied in 22 species of fish, 12 species of amphibians, 5 species of reptiles and 19 species of birds. 2. Fish with a cartilaginous skeleton, and amphibia used lipoproteins for 25-hydroxycholecalciferol transport. 3. Bony fish and reptiles had an ~-globulin transport protein. 4. 12 species of birds used fl-globulin transport proteins, 4 species used albumin and 3 species used an ~-globulin.
INTRODUCTION
wider survey is required to incorporate the mode of vitamin D transport observed in random individual species into a more complete phylogenetic study. This paper reports the results for such a study in fish, amphibians, reptiles and birds.
In CHICKS the defective binding of vitamin D 2 to plasma transport proteins may explain the resistance to the action of the vitamin (Edelstein et al., 1973; Belsey et al., 1974). This would result in the lowered efficiency of the hepatic and/or renal hydroxylations of the vitamin and reduced delivery of the dihydroxyMATERIALS AND METHODS metabolites of vitamin D 2 to target tissues (Belsey Sample preparation et al., 1974). The binding of vitamin D and its metabolites to plasma transport proteins may be imporBlood was obtained from fish, amphibians and reptiles tant in the interpretation of its metabolism in mam- by heart puncture, and from birds by venepuncture. The blood was prepared as either serum of plasma (EDTA or mals (Hay & Watson, 1976). As fish are known to have stores of vitamin D citrated). (Bills, 1927; Schmidt-Nielson & Schmidt-Neilson, Isotopes 1930), and its 25-hydroxymetabolite (Fraser, personal [26,27-3H]25-hydroxycholecalciferol (sp. act.: 11 Ci/ communication) but no parathyroid glands (FleischmM) was obtained from Dr. D. E. M. Lawson, Dunn man, 1947)the transport of vitamin D in these verte- Nutrition Laboratory, Cambridge, U.K.). brates may provide a useful phylogenetic interpreElectrophoresis tation of its mode of action (Hay, 1975). Amphibia, the first evolutionary group on land to Polyacrylamide-disc gels with a continuous buffer sysrequire an efficient regulation of calcium absorption tem (Hjerten et al., 1965) were used for the electrophoretic and excretion in order to maintain calcium homeo- analysis. 0.2 ml serum or plasma, was incubated in vitro stasis, use lipoproteins for the transport of cholecalci- with 0.0073 ng [26,27-3H]25-hydroxycholecalciferol at 4°C ferol and its metabolites (Edelstein et at., 1973; Hay, for 16 hr. Two samples of the serum were run simultaneously. At the completion of the run one gel was stained 1975). for protein with Napthalene Black while the other was The two transport proteins in the chick for chole- frozen with solid CO2, cut into 1 mm segments with a calciferol and 25-hydroxycholecalciferol respectively macrotome (Hay & Ray, 1975) and the radioactive mater(Edelstein et al., 1972) migrate with fl-globulin mobi- ial extracted from the gel slices with NCS tissue solubilizer lity, in contrast to the :t-globulin carriers in mammals (Amersham/Searle, High Wycombe, Bucks, U.K.) as de(Chalk & Kodicek, 1961; C h e n & Lane, 1965; scribed by Hellung-Larsen (1971). To locate lipoproteins serum or plasma was incubated Rikkers & DeLuca 1967; Haddad & Chyu, 1971; Edelstein et al., 1973; Nold & Belsey, 1973; Hay & with Oil-Red O before electrophoresis. Duplicate electroWatson, 1976) and the lipoprotein carriers in amphi- phoretic studies were performed on each vertebrate species. bia (Edelstein et al., 1973: Hay 1975). These differ- Measurement of radioactivity ences in the transport of vitamin D highlight the A Packard Tri-carb automatic Liquid Scintillation diversity in transport modes which exist in verte- Spectrometer No. 3003 was used to measure the radioactibrates. As the binding of vitamin D to plasma pro- vity with a counting efficiency of 18-20'~/,,,.The gel slice teins may be important in the regulation of vitamin extracts were counted for radioacti~city in 10 ml of scintilD metabolism (Edelstein et al., 1973; Belsey et al., lant (Hay & Watson, 1976) using the method of Hellung1974; Hay & Watson, 1976) it would appear that a Larsen (1971). 167
i68
A . W . M . HAY AND G. WATSON Table 1. Relative mobility (M) of 25-hydroxycholecalciferol binding globulin (25-OHBG) and albumin (Alb) in fish determined by polyacrylamide disc gel electrophoresis at pH 8.9. Tris/glycine buffer pH 9.5 Orders and species
M-(25-OH-BG)
M-(AIb)
0.02--0.07
u
0.02 0.05
u u
0"02 0"07
u
0.46 0-52 0.51 0.51 0.47 0.47 0.50 0-48 0.47 0.38 0.48 0-47 0.44 0.41 0.40 0.50
u u u 0.70 0'70 u 0.72 0'72 0.71 u u u u u 0.73 u
0.46 0.42
u u
Cyclostomes Hagfish (M yxine glutinosa) Elasmobranchii Dogfish (Scyliorhinus caniculus) Thornback-ray (Raia clavata) Holocephali Rabbit-fish (Chimaera monstrosa) Actinopteryyii (Bony fish) Salmon (Salmo salar) Pout whiting ( Trisopterus luscus) Whiting (Merlangius merlangus) Common sole (Solea solea) Dragonet (Callionymus lyra) Pipe-fish (Siphostoma typhle) Hooknose (A,qnos cataphractus) Eelpout (Zoarces viviparus) Common eel (Anguilla) Goldfish (Carassius auratus) Plaice (Pleuronectes platessa) Turbot (Psetta maxima) Cichlids (Tilapia leucosticta) Spotted catfish (Anarhichas minor) Blue catfish (Anarhichas denticulatus) Cod (Gadus morhua) Choanichthyes Lungfish (Protopterus) Coelacanth (Latimeria chalumnae) u = Not observed. RESULTS
Using [26,27-3H] 25-hydroxycholecalciferol in an in vitro assay it is possible to locate the single transport protein in mammalian plasma (Edelstein et al., 1973; Hay & Watson, 1976) and the two proteins in chick plasma (Edelstein et al., 1973; Hay, 1975). With this method, in the fish examined (Table 1), the cartilaginous species Hagfish (Myxine glutinosa), Dogfish (Scyliorhinus caniculus), Thornback-Ray (Raia clavata), Rabbit-fish (Chimaera monstrosa) used a lipoprotein for 25-hydroxycholecalciferol transport. The bony fish including the lungfish and coelacanth (Fig. 1) all had a specific or-globulin transport protein. Some binding of 25-hydroxycholecalciferol to lipoproteins was observed in the bony fish but the greater part of the steroid was bound to the a-globulins. Amphibia (Table 2) had no specific 25-hydroxy-D3 transport proteins. All the radioactivity from the gel slices was recovered in a region stained with Oil-Red O. It was reported by Edelstein et al., (1973) that the toad Xenopus laevis used ~-lipoproteins for the transport of cholecalciferol and its 25-hydroxy metabolite. Our results confirm a lipoprotein carrier in X. laevis and also in eleven other amphibia including a mudpuppy (Necturus maculosos) and newt (Salamandra salamandra) (Fig. 2). The reptiles (Table 3) all had specific or-globulin transport proteins. The pattern obtained for the alligator is shown in Fig. 3. In the birds (Table 4) however, considerable diversity in the mode of 25-hydroxycholecalciferol binding to plasma proteins was observed. Like Edelstein et al. (1972), we found 2 proteins in the chicken, (Gallus
gallus), and also in the turkey (Meleaoris gallopauo), goose (Anser anser) and Ring Dove (Columba p. polumbus). Both proteins exhibited fl-globulin mobility. A single protein with fl-globulin mobility was 40--
50
13 .0 ~3
p
20
(D
IO
0
I
20 Gel
40
I
60
I
80
slice
Coelacanth Lat/meria
cholumnae
Fig. 1. Distribution of 3H and protein after polyacrylamide disc gel electrophoresis of Coelacanth plasma incubated with [26,27-aH]25-hydroxycholecalciferol.
25-Hydroxycholecalciferol in fish, amphibians, reptiles and birds
169
Table 2. Relative mobility (M) of 25-hydroxycholecalciferol-binding globulin (25-OH-BG) and albumin (Alb) in amphibians determined by polyacrylamide disc gel electrophoresis at pH 8.9. Tris/ glycine buffer pH 9'5 Orders and species
M-(25-OH-BG)
M-(AIb)
0'03 0-05
0"66 0"72
0"03 0"02-0"12 0"02-0" 11 0'05 0"02 0' 11 0"02 O'O3 0.04 0.03-0.089 0.04
0'71 0'71 0"67 0"69 0'67 0"69 0"70 0-70 0-62 0.69
Caudata Crested newt (Salamandra salamandra) Mudpuppy (Necturus maculosos)
Salientia Marsh frog (Rana ridibunda) Common English frog (Rana temporaria) North American giant bullfrog (Rana catesbiana) Green frog (Rana clamitans) African bullfrog (Pyxicephalus adspera) Colombian bullfrog (Leptodactylus insularium) African green toad (Bufo riridis) Common European toad (Bufo vulgaris) Clawed toad (Xenopus laevis) Surinam toad (Pipa pipa)
responsible for 25-hydroxyeholecalciferol transport in an ostrich (Struthio camelus), rhea (Rhea americana), penguin (Spheniscus demersus), flamingo (Phoenicopterus r. tuber), cockatoo (Cacatua alba), parakeet (Psittacula krameri), rook (Corvus fr~ilegus) and tou-
fractions acted as the transport protein for 25-hydroxy D 3.
can (Ramphastos swainsonii). The pelican (Pelicanus ruJescens), Imperial eagle (Ayuila heliaca) and Tawny owl (Strix aluco), used
In cartilaginous fish, 25-hydroxycholecalciferol is transported in lipoproteins. The hagfish has a skeleton of uncalcified cartilage and no scales or denticles in the skin. The dogfish, rabbit-fish and ray all have a cartilaginous skeleton, which is calcified in the ray but never shows true bone structure. The ray also has dermal denticles corresponding to superficial ornaments, remnants of the bony armour of its placoderm ancestors (Romer, 1966).
a single a-globulin fraction while the kingfisher (Dacelo novaeguineae) and hornbill (Tockus erythrorhynchus) used an albumin fraction for 25-hydroxy D 3 transport. In the plasma of the crane (Grus rubicunda) and ibis (Eudocimus ruber) there was a double albumin fraction and the slower-migrating of these two
DISCUSSION
40--
40-
30--
30
.>_ o "o
,o
o o
20
I0
20--
I0--
I
I 0
20
40
Gel
Crested
$a/amandra
I
60
I
80
0
I\
I
I
20
40
slice
Newt
sa/amandro
Fig. 2. Distribution of 3H and protein after polyacrylamide disc gel electrophoresis of crested newt plasma incubated with [26,27-3H]25-hydroxycholecalciferol.
I
60
I
80
Gel slice
Alligator
AII/gofor
rn/ss/ssl~Dp/ensz~;
Fig. 3. Distribution of 3H and protein after polyacrylamide disc gel electrophoresis of alligator plasma incubated with [26,27-3H]25-hydroxycholecalciferol.
170
A.W.M. HAy AND G. WATSON Table 3. Relative mobility (M) of 25-hydroxycholecalciferol binding globulin (25-OH-BG) and albumin (Alb) in reptiles determined by polyacrylamide disc gel electrophoresis at pH 8.9. Tris/glycine buffer pH 9.5 Orders and species
M-(25-OH-BG)
M-(AIb)
Squamata Indian python (Python molurus) Gaboon viper (Bitis gabonica) Two-banded monitor (Varanus saluator)
0'42 0-53 0-46
0.64 0.73 0"62
0"49 0.52
0"64 0.60
Crocodylia Alligator (Alligator mississippiensis) Spectacled caymen (Caimen crocodilus)
The appearance of 25-hydroxycholecalciferol in the dogfish at a level of 4.0 ng/ml (Fraser, personal communication) suggests that vitamin D metabolism does indeed occur in this cartilaginous fish, but it is probably no coincidence that these non-bony fish have no specific globulins for vitamin D transport. Among the bony fish, the Actinopterygii and Choanichthyes, marine and fresh-water species without exception all had a specific 25-hydroxy D3 transport protein. The levels of 25-hydroxycholecalciferol in bony fish measured by Fraser (personal communication) were sole (Solea solea) 3"9 ng/ml; goldfish (Carassius auratus) 7.7 ng/rnl and lungfish (Protopterus) 29"7 ng/ml. The level of 25-hydroxycholecalciferol in the lungfish indicates an efficient conversion
of vitamin D to 25-hydroxycholecalciferol in this fish. The trout (Sahno sp.) is reported to metabolise vitamin D3 to 25-hydroxycholecalciferol with an efficiency of metabolism of 30Vo in 6 hr, and to convert 15~/oof 25-hydroxycholecalciferol to 24, 25 dihydroxycholecalciferol in 6 hr (Deftos, personal communication). Oizumi & Monder (1972) reported that goldfish cannot convert vitamin D3 into 25-hydroxycholecalciferol and that 25-hydroxycholecalciferol when administered cannot be held in the tissues. However, the observations made by Fraser in the goldfish suggest that some conversion of vitamin D3 to 25-hydroxycholecalciferol occurs. It may be that metabolism occurs more slowly in the goldfish and that a longer period is necessary for the study of vitamin D meta-
Table 4. Relative mobility (M) of 25-hydroxycholecalciferolbinding globulin (25-OH-BG) and albumin (Alb) in birds determined by polyacrylamide disc gel electrophoresis at pH 8.9. Tris/glycine buffer pH 9.5 Orders and species
M-(25-OH-BG)
M-(AIb)
0-35 0.33
0-73 0-71
0.28
0.69
0,37
0.68
0.38 0.37
0-68 0-68
0.23
0.72
0.28
0.75
0.30 & 0.39
0.77
0-23 & 0.35 0-29 & 0-37
0.67 0.68
0.22 & 038
0.66
0.44
0-67
0-47
0'68
0.42
0"64
0"70 0.66
0"7(1 0"66
0-52 0.51
0.69 0"66
Ratites Ostrich (Struthio camelus) Common rhea (Rhea americana)
lmpennae Blackfooted penguin (Spheniscus demersus~
Ciconi![brmes Greater flamingo (Phoenicopterus ruber ruber)
PsittaciJbrmes Great white cockatoo (Cacatua alba) Ring-necked parakeet (Psittacula krameri)
Passer(formes Rook (Corvus ,fi'ugilegus) Pici[brmes Swainsons toucan (Ramphastos swainsonii) Anseriformes Domestic goose (Anser anser) Galliformes Domestic albino turkey (Meleagris gallopauo) Domestic chicken (Gallus gallus) Columbijormes Ring dove (Columba p. polumbus) Pelican(]brmes Pink-backed pelican (Pelicanus ru[i,scens) FalconiJbrmes Imperial eagle (Aguila heliaca) Strigg([brmes Tawny owl (Strix aluco) M icropod!formes Laughing kingfisher (Dacelo novaeguineae) Red-billed hornbill (Tockus erythrorhynchus) aru(Jbrmes Australian crane (Grus ruhieunda) Scarlet ibis (Eudocimus ruher)
25-Hydroxycholecalciferol in fish, amphibians, reptiles and birds
171
Table 5. The principal Vitamin D transport proteins in vertebrates Lipoproteins
s-Globulin
Cartilaginous (4) Amphibia (12)
Bony fish (18) Reptiles (5) Birds (3) Mammals (65)
fl-Globulin
Albumin
Birds (12)
Birds (4) Mammals (7)
(After Hay, 1975, in the Proceedings of the Fifth Parathyroid Conference). bolism in cold blooded vertebrates or, that some other factor is responsible for regulating vitamin D metabolism. The observations made by Deftos jn the trout indicate that there may also be a difference in vitamin D metabolism in different species of fish. It would appear therefore that vitamin D may have some function in fish, although they have no parathyroid glands. The inability offish to respond to mammalian preparations of parathyroid hormone (Simmons 1971; Pang 1973) and the hypercalcemic effect of prolactin (Pang et al., 1973) suggests that certain fish may be useful models for a study of vitamin D metabolism. Although amphibia evolved from the bony fish of the Choanichythes the specific 25-hydroxy D 3 transport protein present in these fish is absent in newts, frogs and toads. Instead lipoproteins are responsible for the transport of the vitamin D metabolite. It may be that the ruling amphibia--an offshoot of the main line of amphibian evolution which led to the reptiles have lost their specific binding protein in the course of their evolution, of alternatively, that they evolved from a species which had no specific vitamin D transport protein. In the reptiles the species studied in the two main groups the Squamata and Crocodylia all had a single
4O
plasma protein with :t-globulin mobility for the transport of 25-hydroxycholecalciferoi. Perhaps the most significant feature of the transport proteins in birds is the use of ~-globulins or albumins by species which are either meat or fish eaters. However a difficulty remains in the interpretation of the two fl-globulin transport proteins in some birds and only one such protein in other species. These results are not entirely satisfactorily explained by the eating and mobility habits of these bird species. Whether the light, often internally strutted, tubular bones of the birds, or their egg-producing habits, place any demands on the transport requirements of vitamin D will have to await considerably more studies on individual species. Similarly, in vivo work will be necessary to confirm many of the avian observations. A comparative study of the 25-hydroxy D 3 transport proteins in mammals has been reported elsewhere (Hay & Watson, 1976), and Table 5 shows that in the course of evolution to the higher vertebrates a more specific mode of vitamin D transport has been developed. Acknowled#ements--We are grateful to Dr. D. E. M. Lawson for the labelled 25-hydroxycholecalciferol, to Dr. C. M. Hawkey, Dr. D. R. Fraser, Dr. H. Vevers, Dr. A. Jamieson, Mr. J. M. Hime (FRCVS), Mr. P. K. C. Austwick, Mr. P. Olney and Mr. A. Howard for supplying plasma samples and to Mr. D. G. Taylor for the photographs.
.4
._> >" 30 f ~3 0 ~- 20
0
REFERENCES
20 40 Gel slice
60
1
8O
Ringdove Columbo p. po/umbus
Fig. 4. Distribution of 3H and protein after polyacrylamide disc gel electrophoresis of Ring Dove plasma incubated with [26,27-3H]25-Hydroxycholecalciferol.
BELSEV R. E., DELuCA H. F. & PoTrs JR. J. T. (1974) Selective binding properties of vitamin D transport protein in chick plasma in vitro. Nature, Lond. 247, 208 209. BILLSC. E. (1927) The distribution of vitamin D with some notes on its possible origin. J. biol. Chem. 72, 751-758. CHALK K. J. I. & KODICEK E. (1961) The association of 14C labelled vitamin D 2 with rat serum proteins. Biochem. J. 79, 1-7. CHEN P. S. & LANE K. (1965) Serum protein binding of vitamin D3. Archs Biochem. Biophys. 112, 7~75. EDELS~IN S., LAWSOND. E. M. & KODlCE~:E. (1972) Separation of binding proteins for cholecalciferol and 25hydroxycholecalciferol from chick serum. Biochim. biophys. Acta 270, 570-574. EDELSTEINS., LAWSOND. E. M. & KODICEKE. (1973) The transporting proteins of cholecalciferol and 25-hydroxycholecalciferol in serum of chicks and other species. Biochem. J. 135, 417-426. FLEISCHMANW. (1947) Comparative physiology of the thyroid gland. Q. Rev. Biol. 22, 119-140. HADDAD J. G. t~ CHYU K. J. (1971) Hydroxycholecalciferol-binding globulin in human plasma. Biochim. biophys. Acta 248, 471 481.
172
A . W . M . HAY AND G. WATSON
HAY A. W. M. (1975) Comparative aspects of vitamin D. transport. In Proceedings of the Fifth Parathyroid Conference. Excerpta Medica, Amsterdam. (In press). HAy A. W. M. & RAy W. G. (1975) A simple macrotome. J. Lab. Prac. 24, 35. HAY A. W. M. & WATSON G. (1976) The plasma transport proteins of 25-hydroxycholecalciferol in mammals. Comp. Biochem. Physiol. 53B, 163-166. HELLONG-LARSEN P. (1971) Liquid scintillation counting of 3H and a2p RNA in slices of polyacrylamide gels. Analyt. Biochem. 39, 454-461. HJERTEN S., JERS~DT S. & T1SELIUSA. (1965) Some aspects of the use of "'continuous" and "discontinuous" buffer systems in polyacrylamide gel electrophoresis. Analyt. Biochem. 11, 219-223. NOLO J. G. & BELSEV R. E. (1973) Comparative study of rat, human and chick vitamin D binding proteins. Fedn Proc. Fedn Am. Socs exp. Biol. 32, 917a. OIZtJMI K. & MONDER C. (1972) Localisation and metabolism of 1,2-3H-vitamin D3 and 26,27-aH-25-hydroxy-
cholec,,alciferol in goldfish (Carassius auratus h.) Comp. Biochem. Physiol. 42B, 523-532. PANG P. K. T. (1973) Endocrine control of calcium metabolism in teleosts. Am. Zoologist 13, 775-792. PANG P. K. T., SCHREmMANM. P. & GRIFHTH R. W. (1973) Pituitary regulation of serum calcium levels in the killifish (Fundulus heteroclitus) L. Gen. & compar. Endocr. 21, 536-542. RIKKERS H. & DELUCA H. F. (1967) An in vivo study of the carrier proteins of 3H vitamins D3 and D4 in rat serum. Am. J. Physiol. 213, 380-386. ROMER A. S. (1966) Vertebrate Paleontology, p. 37. Univ. of Chicago Press. Chicago. SCHMIDT-NIELSON S. 8,z SCHMIDT-NIELSON S. (1930) Der Gehalt der knorpel fische an antirachitischem Vitamin. Z. phys. Chem. 159, 229-238. SIMMONS D. J. (1971) Calcium and skeletal tissue physiology in teleost fishes. Clin. Orthop. Related Res. 76, 244280.
THE PLASMA TRANSPORT PROTEINS OF 25-HYDROXYCHOLECALCIFEROL IN FISH, AMPHIBIANS, REPTILES AND BIRDS A. W. M. HAY AND G. WATSON Nuffield Institute of Comparative Medicine, The Zoological Society of London, Regent's Park, London, NWI, England (Received 17 October 1974)
Abstract--1. The transport proteins of 25-hydroxycholecalciferol were studied in 22 species of fish, 12 species of amphibians, 5 species of reptiles and 19 species of birds. 2. Fish with a cartilaginous skeleton, and amphibia used lipoproteins for 25-hydroxycholecalciferol transport. 3. Bony fish and reptiles had an ~-globulin transport protein. 4. 12 species of birds used fl-globulin transport proteins, 4 species used albumin and 3 species used an ~-globulin.
INTRODUCTION
wider survey is required to incorporate the mode of vitamin D transport observed in random individual species into a more complete phylogenetic study. This paper reports the results for such a study in fish, amphibians, reptiles and birds.
In CHICKS the defective binding of vitamin D 2 to plasma transport proteins may explain the resistance to the action of the vitamin (Edelstein et al., 1973; Belsey et al., 1974). This would result in the lowered efficiency of the hepatic and/or renal hydroxylations of the vitamin and reduced delivery of the dihydroxyMATERIALS AND METHODS metabolites of vitamin D 2 to target tissues (Belsey Sample preparation et al., 1974). The binding of vitamin D and its metabolites to plasma transport proteins may be imporBlood was obtained from fish, amphibians and reptiles tant in the interpretation of its metabolism in mam- by heart puncture, and from birds by venepuncture. The blood was prepared as either serum of plasma (EDTA or mals (Hay & Watson, 1976). As fish are known to have stores of vitamin D citrated). (Bills, 1927; Schmidt-Nielson & Schmidt-Neilson, Isotopes 1930), and its 25-hydroxymetabolite (Fraser, personal [26,27-3H]25-hydroxycholecalciferol (sp. act.: 11 Ci/ communication) but no parathyroid glands (FleischmM) was obtained from Dr. D. E. M. Lawson, Dunn man, 1947)the transport of vitamin D in these verte- Nutrition Laboratory, Cambridge, U.K.). brates may provide a useful phylogenetic interpreElectrophoresis tation of its mode of action (Hay, 1975). Amphibia, the first evolutionary group on land to Polyacrylamide-disc gels with a continuous buffer sysrequire an efficient regulation of calcium absorption tem (Hjerten et al., 1965) were used for the electrophoretic and excretion in order to maintain calcium homeo- analysis. 0.2 ml serum or plasma, was incubated in vitro stasis, use lipoproteins for the transport of cholecalci- with 0.0073 ng [26,27-3H]25-hydroxycholecalciferol at 4°C ferol and its metabolites (Edelstein et at., 1973; Hay, for 16 hr. Two samples of the serum were run simultaneously. At the completion of the run one gel was stained 1975). for protein with Napthalene Black while the other was The two transport proteins in the chick for chole- frozen with solid CO2, cut into 1 mm segments with a calciferol and 25-hydroxycholecalciferol respectively macrotome (Hay & Ray, 1975) and the radioactive mater(Edelstein et al., 1972) migrate with fl-globulin mobi- ial extracted from the gel slices with NCS tissue solubilizer lity, in contrast to the :t-globulin carriers in mammals (Amersham/Searle, High Wycombe, Bucks, U.K.) as de(Chalk & Kodicek, 1961; C h e n & Lane, 1965; scribed by Hellung-Larsen (1971). To locate lipoproteins serum or plasma was incubated Rikkers & DeLuca 1967; Haddad & Chyu, 1971; Edelstein et al., 1973; Nold & Belsey, 1973; Hay & with Oil-Red O before electrophoresis. Duplicate electroWatson, 1976) and the lipoprotein carriers in amphi- phoretic studies were performed on each vertebrate species. bia (Edelstein et al., 1973: Hay 1975). These differ- Measurement of radioactivity ences in the transport of vitamin D highlight the A Packard Tri-carb automatic Liquid Scintillation diversity in transport modes which exist in verte- Spectrometer No. 3003 was used to measure the radioactibrates. As the binding of vitamin D to plasma pro- vity with a counting efficiency of 18-20'~/,,,.The gel slice teins may be important in the regulation of vitamin extracts were counted for radioacti~city in 10 ml of scintilD metabolism (Edelstein et al., 1973; Belsey et al., lant (Hay & Watson, 1976) using the method of Hellung1974; Hay & Watson, 1976) it would appear that a Larsen (1971). 167
i68
A . W . M . HAY AND G. WATSON Table 1. Relative mobility (M) of 25-hydroxycholecalciferol binding globulin (25-OHBG) and albumin (Alb) in fish determined by polyacrylamide disc gel electrophoresis at pH 8.9. Tris/glycine buffer pH 9.5 Orders and species
M-(25-OH-BG)
M-(AIb)
0.02--0.07
u
0.02 0.05
u u
0"02 0"07
u
0.46 0-52 0.51 0.51 0.47 0.47 0.50 0-48 0.47 0.38 0.48 0-47 0.44 0.41 0.40 0.50
u u u 0.70 0'70 u 0.72 0'72 0.71 u u u u u 0.73 u
0.46 0.42
u u
Cyclostomes Hagfish (M yxine glutinosa) Elasmobranchii Dogfish (Scyliorhinus caniculus) Thornback-ray (Raia clavata) Holocephali Rabbit-fish (Chimaera monstrosa) Actinopteryyii (Bony fish) Salmon (Salmo salar) Pout whiting ( Trisopterus luscus) Whiting (Merlangius merlangus) Common sole (Solea solea) Dragonet (Callionymus lyra) Pipe-fish (Siphostoma typhle) Hooknose (A,qnos cataphractus) Eelpout (Zoarces viviparus) Common eel (Anguilla) Goldfish (Carassius auratus) Plaice (Pleuronectes platessa) Turbot (Psetta maxima) Cichlids (Tilapia leucosticta) Spotted catfish (Anarhichas minor) Blue catfish (Anarhichas denticulatus) Cod (Gadus morhua) Choanichthyes Lungfish (Protopterus) Coelacanth (Latimeria chalumnae) u = Not observed. RESULTS
Using [26,27-3H] 25-hydroxycholecalciferol in an in vitro assay it is possible to locate the single transport protein in mammalian plasma (Edelstein et al., 1973; Hay & Watson, 1976) and the two proteins in chick plasma (Edelstein et al., 1973; Hay, 1975). With this method, in the fish examined (Table 1), the cartilaginous species Hagfish (Myxine glutinosa), Dogfish (Scyliorhinus caniculus), Thornback-Ray (Raia clavata), Rabbit-fish (Chimaera monstrosa) used a lipoprotein for 25-hydroxycholecalciferol transport. The bony fish including the lungfish and coelacanth (Fig. 1) all had a specific or-globulin transport protein. Some binding of 25-hydroxycholecalciferol to lipoproteins was observed in the bony fish but the greater part of the steroid was bound to the a-globulins. Amphibia (Table 2) had no specific 25-hydroxy-D3 transport proteins. All the radioactivity from the gel slices was recovered in a region stained with Oil-Red O. It was reported by Edelstein et al., (1973) that the toad Xenopus laevis used ~-lipoproteins for the transport of cholecalciferol and its 25-hydroxy metabolite. Our results confirm a lipoprotein carrier in X. laevis and also in eleven other amphibia including a mudpuppy (Necturus maculosos) and newt (Salamandra salamandra) (Fig. 2). The reptiles (Table 3) all had specific or-globulin transport proteins. The pattern obtained for the alligator is shown in Fig. 3. In the birds (Table 4) however, considerable diversity in the mode of 25-hydroxycholecalciferol binding to plasma proteins was observed. Like Edelstein et al. (1972), we found 2 proteins in the chicken, (Gallus
gallus), and also in the turkey (Meleaoris gallopauo), goose (Anser anser) and Ring Dove (Columba p. polumbus). Both proteins exhibited fl-globulin mobility. A single protein with fl-globulin mobility was 40--
50
13 .0 ~3
p
20
(D
IO
0
I
20 Gel
40
I
60
I
80
slice
Coelacanth Lat/meria
cholumnae
Fig. 1. Distribution of 3H and protein after polyacrylamide disc gel electrophoresis of Coelacanth plasma incubated with [26,27-aH]25-hydroxycholecalciferol.
25-Hydroxycholecalciferol in fish, amphibians, reptiles and birds
169
Table 2. Relative mobility (M) of 25-hydroxycholecalciferol-binding globulin (25-OH-BG) and albumin (Alb) in amphibians determined by polyacrylamide disc gel electrophoresis at pH 8.9. Tris/ glycine buffer pH 9'5 Orders and species
M-(25-OH-BG)
M-(AIb)
0'03 0-05
0"66 0"72
0"03 0"02-0"12 0"02-0" 11 0'05 0"02 0' 11 0"02 O'O3 0.04 0.03-0.089 0.04
0'71 0'71 0"67 0"69 0'67 0"69 0"70 0-70 0-62 0.69
Caudata Crested newt (Salamandra salamandra) Mudpuppy (Necturus maculosos)
Salientia Marsh frog (Rana ridibunda) Common English frog (Rana temporaria) North American giant bullfrog (Rana catesbiana) Green frog (Rana clamitans) African bullfrog (Pyxicephalus adspera) Colombian bullfrog (Leptodactylus insularium) African green toad (Bufo riridis) Common European toad (Bufo vulgaris) Clawed toad (Xenopus laevis) Surinam toad (Pipa pipa)
responsible for 25-hydroxyeholecalciferol transport in an ostrich (Struthio camelus), rhea (Rhea americana), penguin (Spheniscus demersus), flamingo (Phoenicopterus r. tuber), cockatoo (Cacatua alba), parakeet (Psittacula krameri), rook (Corvus fr~ilegus) and tou-
fractions acted as the transport protein for 25-hydroxy D 3.
can (Ramphastos swainsonii). The pelican (Pelicanus ruJescens), Imperial eagle (Ayuila heliaca) and Tawny owl (Strix aluco), used
In cartilaginous fish, 25-hydroxycholecalciferol is transported in lipoproteins. The hagfish has a skeleton of uncalcified cartilage and no scales or denticles in the skin. The dogfish, rabbit-fish and ray all have a cartilaginous skeleton, which is calcified in the ray but never shows true bone structure. The ray also has dermal denticles corresponding to superficial ornaments, remnants of the bony armour of its placoderm ancestors (Romer, 1966).
a single a-globulin fraction while the kingfisher (Dacelo novaeguineae) and hornbill (Tockus erythrorhynchus) used an albumin fraction for 25-hydroxy D 3 transport. In the plasma of the crane (Grus rubicunda) and ibis (Eudocimus ruber) there was a double albumin fraction and the slower-migrating of these two
DISCUSSION
40--
40-
30--
30
.>_ o "o
,o
o o
20
I0
20--
I0--
I
I 0
20
40
Gel
Crested
$a/amandra
I
60
I
80
0
I\
I
I
20
40
slice
Newt
sa/amandro
Fig. 2. Distribution of 3H and protein after polyacrylamide disc gel electrophoresis of crested newt plasma incubated with [26,27-3H]25-hydroxycholecalciferol.
I
60
I
80
Gel slice
Alligator
AII/gofor
rn/ss/ssl~Dp/ensz~;
Fig. 3. Distribution of 3H and protein after polyacrylamide disc gel electrophoresis of alligator plasma incubated with [26,27-3H]25-hydroxycholecalciferol.
170
A.W.M. HAy AND G. WATSON Table 3. Relative mobility (M) of 25-hydroxycholecalciferol binding globulin (25-OH-BG) and albumin (Alb) in reptiles determined by polyacrylamide disc gel electrophoresis at pH 8.9. Tris/glycine buffer pH 9.5 Orders and species
M-(25-OH-BG)
M-(AIb)
Squamata Indian python (Python molurus) Gaboon viper (Bitis gabonica) Two-banded monitor (Varanus saluator)
0'42 0-53 0-46
0.64 0.73 0"62
0"49 0.52
0"64 0.60
Crocodylia Alligator (Alligator mississippiensis) Spectacled caymen (Caimen crocodilus)
The appearance of 25-hydroxycholecalciferol in the dogfish at a level of 4.0 ng/ml (Fraser, personal communication) suggests that vitamin D metabolism does indeed occur in this cartilaginous fish, but it is probably no coincidence that these non-bony fish have no specific globulins for vitamin D transport. Among the bony fish, the Actinopterygii and Choanichthyes, marine and fresh-water species without exception all had a specific 25-hydroxy D3 transport protein. The levels of 25-hydroxycholecalciferol in bony fish measured by Fraser (personal communication) were sole (Solea solea) 3"9 ng/ml; goldfish (Carassius auratus) 7.7 ng/rnl and lungfish (Protopterus) 29"7 ng/ml. The level of 25-hydroxycholecalciferol in the lungfish indicates an efficient conversion
of vitamin D to 25-hydroxycholecalciferol in this fish. The trout (Sahno sp.) is reported to metabolise vitamin D3 to 25-hydroxycholecalciferol with an efficiency of metabolism of 30Vo in 6 hr, and to convert 15~/oof 25-hydroxycholecalciferol to 24, 25 dihydroxycholecalciferol in 6 hr (Deftos, personal communication). Oizumi & Monder (1972) reported that goldfish cannot convert vitamin D3 into 25-hydroxycholecalciferol and that 25-hydroxycholecalciferol when administered cannot be held in the tissues. However, the observations made by Fraser in the goldfish suggest that some conversion of vitamin D3 to 25-hydroxycholecalciferol occurs. It may be that metabolism occurs more slowly in the goldfish and that a longer period is necessary for the study of vitamin D meta-
Table 4. Relative mobility (M) of 25-hydroxycholecalciferolbinding globulin (25-OH-BG) and albumin (Alb) in birds determined by polyacrylamide disc gel electrophoresis at pH 8.9. Tris/glycine buffer pH 9.5 Orders and species
M-(25-OH-BG)
M-(AIb)
0-35 0.33
0-73 0-71
0.28
0.69
0,37
0.68
0.38 0.37
0-68 0-68
0.23
0.72
0.28
0.75
0.30 & 0.39
0.77
0-23 & 0.35 0-29 & 0-37
0.67 0.68
0.22 & 038
0.66
0.44
0-67
0-47
0'68
0.42
0"64
0"70 0.66
0"7(1 0"66
0-52 0.51
0.69 0"66
Ratites Ostrich (Struthio camelus) Common rhea (Rhea americana)
lmpennae Blackfooted penguin (Spheniscus demersus~
Ciconi![brmes Greater flamingo (Phoenicopterus ruber ruber)
PsittaciJbrmes Great white cockatoo (Cacatua alba) Ring-necked parakeet (Psittacula krameri)
Passer(formes Rook (Corvus ,fi'ugilegus) Pici[brmes Swainsons toucan (Ramphastos swainsonii) Anseriformes Domestic goose (Anser anser) Galliformes Domestic albino turkey (Meleagris gallopauo) Domestic chicken (Gallus gallus) Columbijormes Ring dove (Columba p. polumbus) Pelican(]brmes Pink-backed pelican (Pelicanus ru[i,scens) FalconiJbrmes Imperial eagle (Aguila heliaca) Strigg([brmes Tawny owl (Strix aluco) M icropod!formes Laughing kingfisher (Dacelo novaeguineae) Red-billed hornbill (Tockus erythrorhynchus) aru(Jbrmes Australian crane (Grus ruhieunda) Scarlet ibis (Eudocimus ruher)
25-Hydroxycholecalciferol in fish, amphibians, reptiles and birds
171
Table 5. The principal Vitamin D transport proteins in vertebrates Lipoproteins
s-Globulin
Cartilaginous (4) Amphibia (12)
Bony fish (18) Reptiles (5) Birds (3) Mammals (65)
fl-Globulin
Albumin
Birds (12)
Birds (4) Mammals (7)
(After Hay, 1975, in the Proceedings of the Fifth Parathyroid Conference). bolism in cold blooded vertebrates or, that some other factor is responsible for regulating vitamin D metabolism. The observations made by Deftos jn the trout indicate that there may also be a difference in vitamin D metabolism in different species of fish. It would appear therefore that vitamin D may have some function in fish, although they have no parathyroid glands. The inability offish to respond to mammalian preparations of parathyroid hormone (Simmons 1971; Pang 1973) and the hypercalcemic effect of prolactin (Pang et al., 1973) suggests that certain fish may be useful models for a study of vitamin D metabolism. Although amphibia evolved from the bony fish of the Choanichythes the specific 25-hydroxy D 3 transport protein present in these fish is absent in newts, frogs and toads. Instead lipoproteins are responsible for the transport of the vitamin D metabolite. It may be that the ruling amphibia--an offshoot of the main line of amphibian evolution which led to the reptiles have lost their specific binding protein in the course of their evolution, of alternatively, that they evolved from a species which had no specific vitamin D transport protein. In the reptiles the species studied in the two main groups the Squamata and Crocodylia all had a single
4O
plasma protein with :t-globulin mobility for the transport of 25-hydroxycholecalciferoi. Perhaps the most significant feature of the transport proteins in birds is the use of ~-globulins or albumins by species which are either meat or fish eaters. However a difficulty remains in the interpretation of the two fl-globulin transport proteins in some birds and only one such protein in other species. These results are not entirely satisfactorily explained by the eating and mobility habits of these bird species. Whether the light, often internally strutted, tubular bones of the birds, or their egg-producing habits, place any demands on the transport requirements of vitamin D will have to await considerably more studies on individual species. Similarly, in vivo work will be necessary to confirm many of the avian observations. A comparative study of the 25-hydroxy D 3 transport proteins in mammals has been reported elsewhere (Hay & Watson, 1976), and Table 5 shows that in the course of evolution to the higher vertebrates a more specific mode of vitamin D transport has been developed. Acknowled#ements--We are grateful to Dr. D. E. M. Lawson for the labelled 25-hydroxycholecalciferol, to Dr. C. M. Hawkey, Dr. D. R. Fraser, Dr. H. Vevers, Dr. A. Jamieson, Mr. J. M. Hime (FRCVS), Mr. P. K. C. Austwick, Mr. P. Olney and Mr. A. Howard for supplying plasma samples and to Mr. D. G. Taylor for the photographs.
.4
._> >" 30 f ~3 0 ~- 20
0
REFERENCES
20 40 Gel slice
60
1
8O
Ringdove Columbo p. po/umbus
Fig. 4. Distribution of 3H and protein after polyacrylamide disc gel electrophoresis of Ring Dove plasma incubated with [26,27-3H]25-Hydroxycholecalciferol.
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172
A . W . M . HAY AND G. WATSON
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cholec,,alciferol in goldfish (Carassius auratus h.) Comp. Biochem. Physiol. 42B, 523-532. PANG P. K. T. (1973) Endocrine control of calcium metabolism in teleosts. Am. Zoologist 13, 775-792. PANG P. K. T., SCHREmMANM. P. & GRIFHTH R. W. (1973) Pituitary regulation of serum calcium levels in the killifish (Fundulus heteroclitus) L. Gen. & compar. Endocr. 21, 536-542. RIKKERS H. & DELUCA H. F. (1967) An in vivo study of the carrier proteins of 3H vitamins D3 and D4 in rat serum. Am. J. Physiol. 213, 380-386. ROMER A. S. (1966) Vertebrate Paleontology, p. 37. Univ. of Chicago Press. Chicago. SCHMIDT-NIELSON S. 8,z SCHMIDT-NIELSON S. (1930) Der Gehalt der knorpel fische an antirachitischem Vitamin. Z. phys. Chem. 159, 229-238. SIMMONS D. J. (1971) Calcium and skeletal tissue physiology in teleost fishes. Clin. Orthop. Related Res. 76, 244280.
