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A method for studying plasma transport of vitamin D applicable to hypervitaminosis D
303
Clinica Chimica Acta, 91 (1979) 303--307 © Elsevier/North-Holland Biomedical Press
CCA 9947
A METHOD FOR STUDYING PLASMA TRANSPORT OF VITAMIN D APPLICABLE TO HYPERVITAMINOSIS D
MENAHEM FAINARU and JUSTIN SILVER *
Department o f Medicine B, tIebrew University-Hadassah Medical School, P.O.B. 499, Jerusalem (Israel) (Received August 14th, 1978}
Summary In man, vitamin D is normally transported on a specific binding globulin (DBP) and on lipoproteins. In addition, binding to albumin occurs in the presence of vitamin D excess. Agarose gel electrophoresis was used to study the binding of radioactive vitamin D to plasma proteins in lipoprotein-free plasma (d > 1.21 g/ml). This method completely separates DBP from albumin and thus enables the quantification of vitamin D bound to these proteins in various clinical and experimental conditions. The same method can be used to study the transport of other vitamin D metabolites.
Introduction Vitamin D and its major metabolite 25-hydroxy vitamin D (25-OH D) are transported in plasma bound to a specific carrier protein, vitamin D binding globulin (DBP) which is identical to the group specific component, Gc [1--3]. In humans, vitamin D but not 25-OH D is also bound in significant amounts to plasma lipoproteins [4,5]. Furthermore, there is also binding to albumin in situations of vitamin D excess either due to clinical toxicity or the in vitro addition of the vitamin [6]. In order to study the transport of vitamin D and its metabolites under such conditions, we have exploited the rapid and simple method of agarose gel electrophoresis to separate and quantify their binding to DBP and other proteins in the lipoprotein free plasma (d > 1.21 g/ml). Materials and methods Plasma (in 0.2% EDTA) was obtained from fasting normolipemic males. Crystalline cholecalciferol (vitamin D-3) was purchased from Sigma (St. Louis, * To whom
correspondence should be addressed.
304
MO), crystalline 25-OH D was a gift of Drs. Guncaga and Donnalek of HoffmanLa Roche (Switzerland). (4-'4C)-Labelled vitamin D-3 (36 mCi/mmol) and 25-hydroxy[26,27-methyl-3H]cholecalciferol (3H-25-OH D) (11.3 Ci/mmol) were obtained from Amersham (Radiochemical Centre, U.K.). Plasma samples (1.5 ml) were incubated with '4C-vitamin D (0.2 nmol in 20 pl ethanol) and 3H-25-OH D (4.4 pmol in 20 p! ethanol) for 16 h at 4 ° C. In some experiments unlabelled vitamin D or 25-OH D (0--150 nmol in 50 pl ethanol) was added to the incubation mixture. The plasma was then brought to a density of 1.21 g/ml with potassium bromide and centrifuged using a 40.3 rotor in a Beckman L-3-50 ultracentrifuge for 48 h at 39 000 rev./min and 4 ° C. The lipoproteins were separated from the lipoprotein-free plasma by tube slicing. The two fractions were dialyzed against 0.9% sodium chloride for 48 h at 4°C (6 changes). 0.1-ml aliquots of incubated plasma and its ultracentrifuged fractions were solubilized in 0.5 ml Soluene 350 (a quaternary ammonium chloride solubilizer from Packard Instrument Company Inc.), for radioassay. The lipoprotein-free plasma ( d > 1.21 g/ml) was electrophoresed in 1% agarose gel (B.D.H., U.K.) in 0.05 M sodium barbital buffer, pH 8 6 , cast on glass plates(12X 20 cm), to a thickness of 2 mm. The samples (100 .ul) were applied in troughs (1 × 15 mm, 5 mm apart) and run with a current of 40 mAmp per plate at 4 ° C. The electrophoresis was stopped when the tracking dye (Bromphenol blue} reached a 55 mm front (5--6 h). The slabs were cut longitudinally and strips 12 mm wide, corresponding to the central portion of each trough, were removed and cut transversely (at 2-mm intervals). The slices were incubated in scintillation vials with 1 ml Soluene 350 for 18 h at 60 ° C. The remainder of the slab was fixed in methanol/acetic acid/water (1.5 : 0.3 : 1.5, v/v) for 1 h and then stained with 0.2% Amidoschwarz 10B (Merck, F.R.G.) for 7 min ar, d destained in the same fixative solution. To the Soluene scflubi!ized samples 10 ml scintillation fluid (containing 0.4% 2,5 diphenyloxazol,~ (PPO) and 0.01% 2,2'-p-phenylenebis(4-methyl-5-phenyloxazole) (POPOP) in toluene) was added. Radioactivity (dpm) was assayed in a Packard scintillation counter (model 3380) with an efficiency of 20% for 3H and of 70% for '4C. In some experiments 4-mm wide longitudinal strips of the agarose slabs were embedded in 1% agarose {0.05 M barbital buffer, pH 8.6) containing 2% of either rabbit anti-human albumin or rabbit anti-Group specific component (Behringwerke, F.R.G.) on 80 × 100 mm glass plates. Crossed electrophoresis was performed according to Ganrot for 5 h with a current of 20 mAmp at 4°C [7]. The plates were washed for 4 days in phosphate buffered saline (8 changes), fixed, stained and destained as described above. Results The recovery of added ['4C]vitamin D and 3H-25-OH D in plasma fractions separated by ultracertrifugation was 82 +_6% (mean + S.E.M., n = 20). The amount of radioactivity recovered in lipoprotein free plasma (d > 1.21 g/ml) depended upon the quar~tity of unlabelled sterols: '4C-vitaminD decreased from 62 + 2 to 38.5 + 2% (mean + S.E.M., n = I0) with increasing amounts of vitamin D (0--26 nmol/ml plasma) and 3H-25-OH D decreased from 98 + 0.4 to
300
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~~ o . , oN
y p o~2 oc, Albumin GLOBULItlS Fig. 1. D i s t r i b u t i o n o f 1 4 C - v i t a m i n D in l i p o p r o t e i n free p l a s m a p r o t e i n s (d ?> 1.21 g / m l ) s e p a r a t e d by e l e c t r o p h o r e s i s in a g a r o s e gel slabs. R a d i o a c t i v i t y was d e t e r m i n e d in 2-ram w i d e s t r i p s o f t h e agarose gel. T h e b r o k e n Hne ( ¢ ) r e p r e s e n t s t h e 1 4 C - v i t a m i n D in n o r m a l p l a s m a a n d t h e solid sline (m) s h o w s t h e d i s t r i b u t i o n o f r a d i o a c t i v i t y w h e n u n l a b e l l e d v i t a m i n D w a s a d d e d ( 1 0 n m o l / m l ) . T h e s t a i n e d strip o f t h e e l e c t r o p h o r e t o g r a m is i n s e r t e d at t h e b o t t o m .
/
/
Fig. 2. T w o - d i m e n s i o n a l e l e c t r o p h o r e s i s o f l i p o p r o t e i n free p l a s m a (d ~> 1.21 g / m l ) . First t h e p l a s m a was r u n h o r i z o n t a l l y as in Fig. 1. Slabs ( 4 - r a m w i d e ) w e r e t h e n i m p r e g n a t e d i n t o 1% a g a r o s e c o n t a i n i n g antib o d i e s a n d r u n v e r t i c a l l y . T h e p i c t u r e is c o m p o s i t e : t h e b o t t o m r e p r e s e n t s t h e e l e c t r o p h o r e s i s in t h e first d i m e n s i o n ( F i g . 1); i m m e d i a t e l y a b o v e it is t h e c r o s s e d e l e c t r o p h o r e s i s i n t o a g a r o s e c o n t a i n i n g 2% r a b b i t a n t i - g r o u p s p e c i f i c ( G c ) a n t i s e r u m ; t h e t o p d e p i c t s t h e c r o s s e d i m m u n o e l e c t r o p h o r e s i s i n t o agarose c o n t a i n i n g 2% r a b b i t a n t i - h u m a n a l b u m i n a n t i s e r u m .
306 68.5 -~: 3.4% with increasing amounts of 25-OH D (0--25 n m o l / m l plasma). Agarose gel electrophoresis clearly separated the various plasma proteins (Figs. 1 and 2). The resolution between albumin and group specific c o m p o n e n t (DBP) was complete as demonstrated by crossed immunoelectrophoresis (Fig. 2). When no unlabelled sterols were added to the incubation, '4C-vitamin D and 3H-25-OH D were recovered in a single narrow peak with an a, mobility (Fig. 1). Excess vitamin D resulted in a marked displacement of ~4C from the ,-globulin to an additional peak with the same mobility as albumin {Fig. 1). A similar effect was achieved when 25-OH D was added (not shown). The two radioactive peaks corresponded to the rockets produced by crossed immunoelectrophoresis (Fig. 2). The recovery of the applied radioactivity in the agarose strips following electrophoresis was 76 + 5~ and 72 _+ 4.5% (mean + S.E.M., n = 10) for 3H and '4C, respectively. Considering that only 4 of the slab was used for radioassay (see Methods), the true. r(~covery was more than 90%. The a m o u n t of radioactivity in albumin correlated to the quantity of unlabelled sterol added. The intra-assay variation was checked by analyzing two samples in triplicates on different days. The coefficients of variation were 5--7% for both '4C-vitamin D and 3H-25-OH D. Discussion Several methods have been used for studying vitamin D transport on plasma proteins, all of which utilize ~adioactive tracers [1--3,5,8]. Vitamin D binding to lipoproteins may be determined easily by ultracentrifugation [4]. The analysis of binding to other plasma proteins is more complex and consequently many methods have been used. Gel filtration and ion-exchange chromatography are the most accurate but are laborious when a large number of samples have to be processed [ 5,8]. Electrophoreses in various supporting media such as starch gel, agar and polyacrylamide gel, have been reported, but no clear separation of DBP from albumin was demonstrated [2,8--10]. In the present report, agarose gel electrophoresis effectively separated the binding to DBP from albumin. This method cannot be applied to whole plasma because of the normal binding of vitamin D to plasma lipoproteins. High density lipoproteins have an electrophoretic mobility overlapping ~,-globulin and albumin, thus obscuring the clear separation between vitamin D binding to these proteins. Therefore, this method can ,~nly be used on lipoprotein free plasma. In normal lipoprotein free plasma (d > 1.21 g/ml) all vitamin D and 25-OH D resolved in one peak on DBP. Additional binding to albumin as a distinct peak was apparent when excess vitamin D or 25-OH D was present. A similar displacement of vitamin D to albumin was observed in a child with accidental vitamin D toxicity [6]. This indicates that binding to albumin may have an important physiological role. The simple and reproducible methodology described here can be used in the study of the transL)ort of vitam:n D, 25-OH D as well as other vitamin D metabolites among plasma proteins in different physiological and pathological conditions.
307
Acknowledgements We would like to thank Henry Matzner and Tuvia Hadar for their excellent technical assistance. This study was supported in part by grants from the Israeli Ministry of Health and the Joint Research Fund of the Hebrew University and Hadassah. References 1 2 3 4 5 6 7 8 9 10
Bouillon, R., van Baelen, H., R o m b a r t u s , W. and de Moor, P. (1976) Eur. J. B i o c h e m . 6 6 , 2 8 5 - - 2 9 1 Imawari, M.0 Kida,'K. and G o o d m a n , De W.S. (1976) J. Clin. Invest. 5 8 , 5 1 4 - - 5 2 3 Haddad, J.G. and Walgate, J. ( 1 9 7 6 ) J. Biol. Chem. 2510 4 8 0 3 - - 4 8 0 9 Smith, J.E. and G o o d m a n , De W.S. ( 1 9 7 1 ) J. Clin. Invest. 50, 2 1 5 9 - - 2 1 6 6 Edelstein, S. ( 1 9 7 4 ) Biochem. Soc. Spec. Publ. 3, 43--54 Silver, J., Shvil, Y. and Fainard, M. ( 1 9 7 8 ) Br. Med. J. ii, 9 3 - - 9 4 Ganrot, P.O. (1972) Scand. J. Clin. Lab. Invest. 29, Suppl. 124, 39--41 Rosenstreich, S.J., Volwiler, W. a n d Rich, C. (1971) Am. J. Clin. Nutr. 2 4 , 8 9 7 - - 9 0 5 De Crousaz, P., B l a n ~ B. and A n t e n e r , I. (1965) Heir. O d o n t o l . Acta 9 , 1 5 1 - - 1 5 5 Daiger, S.P. and Cavalli-Sforza, L.L. ( 1 9 7 7 ) Am. J. Hum. Genet. 29, 593--604
Clinica Chimica Acta, 91 (1979) 303--307 © Elsevier/North-Holland Biomedical Press
CCA 9947
A METHOD FOR STUDYING PLASMA TRANSPORT OF VITAMIN D APPLICABLE TO HYPERVITAMINOSIS D
MENAHEM FAINARU and JUSTIN SILVER *
Department o f Medicine B, tIebrew University-Hadassah Medical School, P.O.B. 499, Jerusalem (Israel) (Received August 14th, 1978}
Summary In man, vitamin D is normally transported on a specific binding globulin (DBP) and on lipoproteins. In addition, binding to albumin occurs in the presence of vitamin D excess. Agarose gel electrophoresis was used to study the binding of radioactive vitamin D to plasma proteins in lipoprotein-free plasma (d > 1.21 g/ml). This method completely separates DBP from albumin and thus enables the quantification of vitamin D bound to these proteins in various clinical and experimental conditions. The same method can be used to study the transport of other vitamin D metabolites.
Introduction Vitamin D and its major metabolite 25-hydroxy vitamin D (25-OH D) are transported in plasma bound to a specific carrier protein, vitamin D binding globulin (DBP) which is identical to the group specific component, Gc [1--3]. In humans, vitamin D but not 25-OH D is also bound in significant amounts to plasma lipoproteins [4,5]. Furthermore, there is also binding to albumin in situations of vitamin D excess either due to clinical toxicity or the in vitro addition of the vitamin [6]. In order to study the transport of vitamin D and its metabolites under such conditions, we have exploited the rapid and simple method of agarose gel electrophoresis to separate and quantify their binding to DBP and other proteins in the lipoprotein free plasma (d > 1.21 g/ml). Materials and methods Plasma (in 0.2% EDTA) was obtained from fasting normolipemic males. Crystalline cholecalciferol (vitamin D-3) was purchased from Sigma (St. Louis, * To whom
correspondence should be addressed.
304
MO), crystalline 25-OH D was a gift of Drs. Guncaga and Donnalek of HoffmanLa Roche (Switzerland). (4-'4C)-Labelled vitamin D-3 (36 mCi/mmol) and 25-hydroxy[26,27-methyl-3H]cholecalciferol (3H-25-OH D) (11.3 Ci/mmol) were obtained from Amersham (Radiochemical Centre, U.K.). Plasma samples (1.5 ml) were incubated with '4C-vitamin D (0.2 nmol in 20 pl ethanol) and 3H-25-OH D (4.4 pmol in 20 p! ethanol) for 16 h at 4 ° C. In some experiments unlabelled vitamin D or 25-OH D (0--150 nmol in 50 pl ethanol) was added to the incubation mixture. The plasma was then brought to a density of 1.21 g/ml with potassium bromide and centrifuged using a 40.3 rotor in a Beckman L-3-50 ultracentrifuge for 48 h at 39 000 rev./min and 4 ° C. The lipoproteins were separated from the lipoprotein-free plasma by tube slicing. The two fractions were dialyzed against 0.9% sodium chloride for 48 h at 4°C (6 changes). 0.1-ml aliquots of incubated plasma and its ultracentrifuged fractions were solubilized in 0.5 ml Soluene 350 (a quaternary ammonium chloride solubilizer from Packard Instrument Company Inc.), for radioassay. The lipoprotein-free plasma ( d > 1.21 g/ml) was electrophoresed in 1% agarose gel (B.D.H., U.K.) in 0.05 M sodium barbital buffer, pH 8 6 , cast on glass plates(12X 20 cm), to a thickness of 2 mm. The samples (100 .ul) were applied in troughs (1 × 15 mm, 5 mm apart) and run with a current of 40 mAmp per plate at 4 ° C. The electrophoresis was stopped when the tracking dye (Bromphenol blue} reached a 55 mm front (5--6 h). The slabs were cut longitudinally and strips 12 mm wide, corresponding to the central portion of each trough, were removed and cut transversely (at 2-mm intervals). The slices were incubated in scintillation vials with 1 ml Soluene 350 for 18 h at 60 ° C. The remainder of the slab was fixed in methanol/acetic acid/water (1.5 : 0.3 : 1.5, v/v) for 1 h and then stained with 0.2% Amidoschwarz 10B (Merck, F.R.G.) for 7 min ar, d destained in the same fixative solution. To the Soluene scflubi!ized samples 10 ml scintillation fluid (containing 0.4% 2,5 diphenyloxazol,~ (PPO) and 0.01% 2,2'-p-phenylenebis(4-methyl-5-phenyloxazole) (POPOP) in toluene) was added. Radioactivity (dpm) was assayed in a Packard scintillation counter (model 3380) with an efficiency of 20% for 3H and of 70% for '4C. In some experiments 4-mm wide longitudinal strips of the agarose slabs were embedded in 1% agarose {0.05 M barbital buffer, pH 8.6) containing 2% of either rabbit anti-human albumin or rabbit anti-Group specific component (Behringwerke, F.R.G.) on 80 × 100 mm glass plates. Crossed electrophoresis was performed according to Ganrot for 5 h with a current of 20 mAmp at 4°C [7]. The plates were washed for 4 days in phosphate buffered saline (8 changes), fixed, stained and destained as described above. Results The recovery of added ['4C]vitamin D and 3H-25-OH D in plasma fractions separated by ultracertrifugation was 82 +_6% (mean + S.E.M., n = 20). The amount of radioactivity recovered in lipoprotein free plasma (d > 1.21 g/ml) depended upon the quar~tity of unlabelled sterols: '4C-vitaminD decreased from 62 + 2 to 38.5 + 2% (mean + S.E.M., n = I0) with increasing amounts of vitamin D (0--26 nmol/ml plasma) and 3H-25-OH D decreased from 98 + 0.4 to
300
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y p o~2 oc, Albumin GLOBULItlS Fig. 1. D i s t r i b u t i o n o f 1 4 C - v i t a m i n D in l i p o p r o t e i n free p l a s m a p r o t e i n s (d ?> 1.21 g / m l ) s e p a r a t e d by e l e c t r o p h o r e s i s in a g a r o s e gel slabs. R a d i o a c t i v i t y was d e t e r m i n e d in 2-ram w i d e s t r i p s o f t h e agarose gel. T h e b r o k e n Hne ( ¢ ) r e p r e s e n t s t h e 1 4 C - v i t a m i n D in n o r m a l p l a s m a a n d t h e solid sline (m) s h o w s t h e d i s t r i b u t i o n o f r a d i o a c t i v i t y w h e n u n l a b e l l e d v i t a m i n D w a s a d d e d ( 1 0 n m o l / m l ) . T h e s t a i n e d strip o f t h e e l e c t r o p h o r e t o g r a m is i n s e r t e d at t h e b o t t o m .
/
/
Fig. 2. T w o - d i m e n s i o n a l e l e c t r o p h o r e s i s o f l i p o p r o t e i n free p l a s m a (d ~> 1.21 g / m l ) . First t h e p l a s m a was r u n h o r i z o n t a l l y as in Fig. 1. Slabs ( 4 - r a m w i d e ) w e r e t h e n i m p r e g n a t e d i n t o 1% a g a r o s e c o n t a i n i n g antib o d i e s a n d r u n v e r t i c a l l y . T h e p i c t u r e is c o m p o s i t e : t h e b o t t o m r e p r e s e n t s t h e e l e c t r o p h o r e s i s in t h e first d i m e n s i o n ( F i g . 1); i m m e d i a t e l y a b o v e it is t h e c r o s s e d e l e c t r o p h o r e s i s i n t o a g a r o s e c o n t a i n i n g 2% r a b b i t a n t i - g r o u p s p e c i f i c ( G c ) a n t i s e r u m ; t h e t o p d e p i c t s t h e c r o s s e d i m m u n o e l e c t r o p h o r e s i s i n t o agarose c o n t a i n i n g 2% r a b b i t a n t i - h u m a n a l b u m i n a n t i s e r u m .
306 68.5 -~: 3.4% with increasing amounts of 25-OH D (0--25 n m o l / m l plasma). Agarose gel electrophoresis clearly separated the various plasma proteins (Figs. 1 and 2). The resolution between albumin and group specific c o m p o n e n t (DBP) was complete as demonstrated by crossed immunoelectrophoresis (Fig. 2). When no unlabelled sterols were added to the incubation, '4C-vitamin D and 3H-25-OH D were recovered in a single narrow peak with an a, mobility (Fig. 1). Excess vitamin D resulted in a marked displacement of ~4C from the ,-globulin to an additional peak with the same mobility as albumin {Fig. 1). A similar effect was achieved when 25-OH D was added (not shown). The two radioactive peaks corresponded to the rockets produced by crossed immunoelectrophoresis (Fig. 2). The recovery of the applied radioactivity in the agarose strips following electrophoresis was 76 + 5~ and 72 _+ 4.5% (mean + S.E.M., n = 10) for 3H and '4C, respectively. Considering that only 4 of the slab was used for radioassay (see Methods), the true. r(~covery was more than 90%. The a m o u n t of radioactivity in albumin correlated to the quantity of unlabelled sterol added. The intra-assay variation was checked by analyzing two samples in triplicates on different days. The coefficients of variation were 5--7% for both '4C-vitamin D and 3H-25-OH D. Discussion Several methods have been used for studying vitamin D transport on plasma proteins, all of which utilize ~adioactive tracers [1--3,5,8]. Vitamin D binding to lipoproteins may be determined easily by ultracentrifugation [4]. The analysis of binding to other plasma proteins is more complex and consequently many methods have been used. Gel filtration and ion-exchange chromatography are the most accurate but are laborious when a large number of samples have to be processed [ 5,8]. Electrophoreses in various supporting media such as starch gel, agar and polyacrylamide gel, have been reported, but no clear separation of DBP from albumin was demonstrated [2,8--10]. In the present report, agarose gel electrophoresis effectively separated the binding to DBP from albumin. This method cannot be applied to whole plasma because of the normal binding of vitamin D to plasma lipoproteins. High density lipoproteins have an electrophoretic mobility overlapping ~,-globulin and albumin, thus obscuring the clear separation between vitamin D binding to these proteins. Therefore, this method can ,~nly be used on lipoprotein free plasma. In normal lipoprotein free plasma (d > 1.21 g/ml) all vitamin D and 25-OH D resolved in one peak on DBP. Additional binding to albumin as a distinct peak was apparent when excess vitamin D or 25-OH D was present. A similar displacement of vitamin D to albumin was observed in a child with accidental vitamin D toxicity [6]. This indicates that binding to albumin may have an important physiological role. The simple and reproducible methodology described here can be used in the study of the transL)ort of vitam:n D, 25-OH D as well as other vitamin D metabolites among plasma proteins in different physiological and pathological conditions.
307
Acknowledgements We would like to thank Henry Matzner and Tuvia Hadar for their excellent technical assistance. This study was supported in part by grants from the Israeli Ministry of Health and the Joint Research Fund of the Hebrew University and Hadassah. References 1 2 3 4 5 6 7 8 9 10
Bouillon, R., van Baelen, H., R o m b a r t u s , W. and de Moor, P. (1976) Eur. J. B i o c h e m . 6 6 , 2 8 5 - - 2 9 1 Imawari, M.0 Kida,'K. and G o o d m a n , De W.S. (1976) J. Clin. Invest. 5 8 , 5 1 4 - - 5 2 3 Haddad, J.G. and Walgate, J. ( 1 9 7 6 ) J. Biol. Chem. 2510 4 8 0 3 - - 4 8 0 9 Smith, J.E. and G o o d m a n , De W.S. ( 1 9 7 1 ) J. Clin. Invest. 50, 2 1 5 9 - - 2 1 6 6 Edelstein, S. ( 1 9 7 4 ) Biochem. Soc. Spec. Publ. 3, 43--54 Silver, J., Shvil, Y. and Fainard, M. ( 1 9 7 8 ) Br. Med. J. ii, 9 3 - - 9 4 Ganrot, P.O. (1972) Scand. J. Clin. Lab. Invest. 29, Suppl. 124, 39--41 Rosenstreich, S.J., Volwiler, W. a n d Rich, C. (1971) Am. J. Clin. Nutr. 2 4 , 8 9 7 - - 9 0 5 De Crousaz, P., B l a n ~ B. and A n t e n e r , I. (1965) Heir. O d o n t o l . Acta 9 , 1 5 1 - - 1 5 5 Daiger, S.P. and Cavalli-Sforza, L.L. ( 1 9 7 7 ) Am. J. Hum. Genet. 29, 593--604