IgE antibodies in sera from mite-allergic subjects and, when examined in radioallergosorbent test inhibition studies, accounted for up to 30% of the allergenic potency of crude D. farinae extracts [5]. It will now be interesting to isolate the components from crude Dermatophagoides extracts which react with the mouse antiphosphorylcholine and anti-galactan antibodies, characterize the preparations and test them for allergenic activity. Selective approaches using the myeloma antibodies in direct precipitation and affinity chromatograpliy experiments are now in progress. This work was supported by the Princess Margaret Children's MediCal Research Foundation and the Deutsche Forschungsgemeinschaft.
Received August 11, 1977 1. Voorhorst, R., Spieksma-Boezeman, M.I.A., Spieksrna, F.T.M.: Allergie u. Asthma 10, 329 (1964) 2. Pepys, J.,' Chan, M., Hargreave, F.E.: Lancet 1 1968,1270; Assem, E.S.K., MeAllen, M.K. : Brit. Med. J. 2, 504 (1970); Stenius, B., etal.: Clin. Allergy 1, 37 (1971) 3. Woolcock, A.: personal communication; Bronswijk, J.E.M.H. van, Sinha, R.N.: J. Allergy 47, 31 (1971) 4. Baldo, B.A., Turner, K.J., Uhlenbruck, G. : Experientia 32, 641 (1976) 5. Baldo, B.A., Uhlenbruck, G. : Clin. Allergy (in press) 6. Potter, M. : Physiol. Rev. 52, 631 (1972) 7. Baldo, B.A., Fletcher, T.C., Pepys, J. : Immmaology 32, 831 (1977) 8. Capron, A., etal.: Presse retd. 72, 3103 (1964); Longbottom, J.L., Pepys, J. : J. Path. Bact. 88, 141 (1964)
Serum-Ferritin Concentration and Diagnostic 59Fe2+ Absorption in Humans with Iron Deficiency H.C. Heinrich, E.E. Gabbe, and J. Briiggemann Division of Medical Biochemistry, Institute of Physiological Chemistry, University Hospital Eppendorf, D-2000 Hamburg The earliest stage in the development of iron deficiency was called prelatent iron deficiency and is characterized and can be diagnosed by the demonstration of an increased 59Fe absorption from a diagnostic 0.56ms (=10gmole) 59Fe2+ test dose and/or the disappearence of the Berlin blue-reactive diffuse cytoplasmatic nonhaeme storage iron in the bone-marrow macrophages whereas serum iron, total iron-binding capacity, and haemoglobin concentration are still within the normal ranges [1]. Since prelatent iron deficiency is the most prevalent deficiency of mankind both in developing and in industrial countries and precedes latent and manifest iron deficiency, it is of considerable practical interest also to be able to diagnose prolatent iron deficiency without the otherwise necessary whoIe-body-radioactivity detector for measuring the whole-body retention of absorbed 59Fe and/or the bonemarrow biopsy for the Berlin blue staining of the diffuse storage iron. "[he possibility of using a reduced serumferritin concentration as a simple, non-invasive, and cheap substitute for the wholebody-radioactivity measurement or bonemarrow biopsy was investigated by a comparative evaluation of serum-ferritin levels and diagnostic 59Fe2+ absorption in subNaturwissenschaften 64 (1977)
jects with prelatent, latent, and manifest iron deficiency and compared with subjects with normal iron stores. Serum-ferritin concentrations were estimated with an immunoradiolnetric assay using an experimental kit [2] as a modification of the two-site or sandwich solidphase system [3]. 5gFe absorption from a diagnostic 0.56 mg ~9Fe2+ dose was calculated from the whole-body retention of absorbed 59Fe as measured in the 4 ~counting geometry of a whole-body-radioactivity detector with liquid organic scintillator [4]. Sermn-ferritin concentrations with a total range of 28-221 and a geometric mean of Xg=83 ng/ml with a coefficient of standard deviation Cs.n.= 1.7 were estimated in 63 subjects with a normal diagnostic 59FEZ+ absorption of 6-48% and therefore normal iron stores. In 29 subjects with prelatent iron deficiency as indicated by an increased diagnostic 59Fen+ absorption of 52-100% (but normal serum iron, total iron-binding capacity and haemoglobin) the serum-ferritin levels were reduced to a total range of 7.8~64 (X'g= 27; Cs.o. = 1.8) ng/ml without overlap of the 68% ranges between subjects with normal iron stores and subjects with prelatent iron deficiency (Fig. 1). The 95% ranges did, however,
9 by Springer-VerIag 1977
overlap between these two groups so that a serum-ferritin level of 28-64 ng/ml alone is not sufficient for a reliable diagnosis of prelatent iron deficiency. Serum-ferritin levels above 64 ng/ml were only found in subjects with normal iron stores whereas levels below 28 ngjml do indicate depleted iron stores in at least prelatent iron deficiency. Nearly no overlap with the normal serum-ferritin range was observed in 14 subjects with latent #on deficiency (serumferritin range 5.3-32 ng/ml; X'g= 13, CS.D.= 1.6) and 42 subjects with manifest iron deficiency anemia (serum-ferritin range 2.7-12mgjml; X'g=6.1, CS.D.= 1.4). For the most part, the diagnosis of latent and manifest iron deficiency does not depend on a serum-ferritin estimation since the diagnosis and separation from prolatent iron deficiency is based on reduced serum-iron and increased transferrin levels in latent iron deficiency and an additional reduction of haemoglobin in hypochromic microcytic iron-deficiency anemia. Although there is a very close correlation between the reduction of serum-ferritin concentrations and the increase of diagnostic -~9Fe2+ absorption [5], serum ferritin alone is not a reliable indicator of depleted iron stores in the individual subject. Nevertheless it is useful for evaluating the prevalence of depleted iron stores in screening or field studies, especially if Iron deficiency
Normat
300
Fe stores "prelat~ it
Latent
2oo-T
maqifest upper CS.D.
~g
E
*
50 9
@
Cs.B lower
T
i
@ 9
2O c
.'-
I0
E 7
5
7" i
2
tm~6 n- 6327
t mf 79 4 25
tmf 14 4 10
,f 31
Fig. 1. Serum-ferritin concentrations (total range, geometric mean Xg and coefficients of standard deviation CS.D.)in humans with normal iron stores and prelatent, latent, or manifest iron deficiency (t = totals, m = males, f= females, n=number of subjects in each group) 595
no facilities for sensitive and reliable whole-body counting of absorbed 59Fe are available. With the technical assistance of Fatima I~agid and Ellen Pape. Supported in part by a research grant of the Deutsche Forschungsgemeinsehaft.
Received July 15, 1977
1. Heinrich, H.C., Bartels, H.: Ktin. Wschr. 45, 553 (1967); Hausmann, K., et al. : Acta Haemat. 42, 193 (1969) 2. Hoechst Pharmaceutical Research Laboratories, Behring Institute, 1977 3. Miles, L.E.M., etal.: Anal. Biochem. 61, 209 (1974) 4. Heinrich, H.C., et al. : Klin. Wschr. 44, 827 (1966); Heinrich, H.C., Gabbe, E.E., Whang, D.H. : Atompraxis 11, 430, 660 (1965) 5. Heinrich, H.C., Gabbe, E.E., Briiggemann, J. : Z. Naturforsch. (in press)
Transfer of Blood-Group Determinant from Bovine Serum Nonlipids to Erythrocyte Lipids of Various Mammals
I'able 1. Results of transfer of J activity from J-active nonlipids of bovine serum by incubation oferythrocytes in isotonic buffer solution (pH 7.4). The volume (gl) of packed transformed erythrocytes, whose total lipids exert a complete inhibition of immunohemolysis of (jcs) test e~throcytes under standard conditions, is given (+ =J-active, tested qualitatively only) Animal
~tl packed cells
Dog Cat
6.2 +
Rat Rabbit
+ 0.4
Cattle (adult) Calf Pig (miniature) Lama pacos
6.2 + 0.2 +
F. Kr6tlinger and O.W. Thiele
Man
3.1
Physiologisch-Chemisehes Institut der Universit~it, D-3400 G6ttingen
Chicken
0.4
It has been shown in a previous paper [1] that the J blood-group activity of cattle can be found in both the lipids and a protein-containing nonlipid fraction of J containing serum. Furthermore, it was demonstrated [2, 3] that the J determinant is transferred from the J-active serum protein onto the erythrocyte membrane by incubation in vitro. Even though the carrier of the J determinant is a lipid-free serum protein (probably a glycoprotein), the transferred J activity is detectable only in the lipid fraction of erythrocytes. Thus, the J determinant (presumably a carbohydrate unit) must have been detached from a s e r u m glycoprotein and transferred to a lipidic receptor at the erythrocyte m e m brane. It was suggested [2, 3] that an enzyme system located at the bovine erythrocyte membrane is responsible for the transfer of J determinant. Furthermore, it has been speculated [2, 3] that a transfer of carbohydrate units from serum glycoproteins to erythrocyte lipids is not confined to the J blood-group-active carbohydrate moiety of cattle. Similar transfers might also take place with carbohydrates other than blood-group-active determinants and might occur in a variety of mammals and other vertebrates and on various tissue cells. In this study, we present evidence on the transfer of bovine J blood-group determinant on erythrocytes of various mammals and of chickens. J-positive bovine serum (jcs serum) was extracted with chloroform-methanol as described earlier [4]. The precipitate (predominantly consisting of proteins) obtained by this procedure served as the lip-
jcs Cattle (naturally acquired J activity)
596
id-free donor of the J determinant. It was incubated at 37 ~ with washed erythrocytes of various animal species either in isotonic saline with added antibiofica for 24 h or in isotonic buffer solution (pH 7.4) for 3 h. The incubation procedure has been described in detail elsewhere [3]. Subsequently, the erythrocytes were washed carefully and extracted with chloroformmethanol [4]. The erythrocytie total lipids thu s obtained were purified [5] and tested for J activity using an immunohemolysisinhibition test in the bovine J system. Controls were run simultaneously with total lipids of unincubated erythrocytes in order to be sure that no cross-reacting lipids were present. All experiments were repeated several times. For a quantitative test, all experiments were done under the same conditions using the same (jcs) test erythroeytes and the same J antiserum. As shown in Table 1, the erythrocytes of all animal species under investigation are able to pick up the J determinant and to incorporate it into a lipid fraction. It is therefore suggested that the erythrocyte membranes of all species tested contain
25 12.5
both an e n ~ m e system catalyzing the transfer of the J determinant and a lipidic receptor which binds to the J determinant. The extent of transferred J activity is higher in all cases tested than that of J-active bovine erythroeytes which have acquired their J activity in vivo. This work was supported by Forschungsmittel des Landes Niedersachsen and by the Fonds der Chemischen Industrie. We wish to thank J. Koch for the supply of anti-J serum, L. Dittrich, J.-N. Meyer, H. W/51fel, C. Niessen, and H. Sander for blood samples of various animals. Received July 5, 1977 1. Schr6ffel,J., etal. : Eur. J. Biochem. 22, 396 (1971) 2. Thiele, O.W., Kr6tlinger, F., Ohl, C. : Naturwissenschaften 62, 586 (1975) 3. Kr6tlinger, F., Thiele, O.W., Ohl, C. : Eur. J. Biochem. 67, 495 (1976) 4. Schr6ffel, J., Thiele, O.W., Koch, J. : ibid. 22, 294 (1971) 5. Wells, M.A, Dittmer, J.C. : Biochemistry 2, 1259 (1963)
Gap Junctions in Freeze-fractured Membranes of the Arachnoid Mater F.X. Omlin and A. Bischoff Department of Neurology, University of Berne, Inselspitai, CH-3010 Berne The synonyms for gap junctions [1] are zonulae adherentes [2] and nexus [31. This membrane specialization represents the
morphologic aspect of a functional membrane contact, which provides the electronic coupling from cell to cell by ionic ex-
Naturwissenschaften 64 (1977)
9 by Springer-Verlag 1977
Received August 11, 1977 1. Voorhorst, R., Spieksma-Boezeman, M.I.A., Spieksrna, F.T.M.: Allergie u. Asthma 10, 329 (1964) 2. Pepys, J.,' Chan, M., Hargreave, F.E.: Lancet 1 1968,1270; Assem, E.S.K., MeAllen, M.K. : Brit. Med. J. 2, 504 (1970); Stenius, B., etal.: Clin. Allergy 1, 37 (1971) 3. Woolcock, A.: personal communication; Bronswijk, J.E.M.H. van, Sinha, R.N.: J. Allergy 47, 31 (1971) 4. Baldo, B.A., Turner, K.J., Uhlenbruck, G. : Experientia 32, 641 (1976) 5. Baldo, B.A., Uhlenbruck, G. : Clin. Allergy (in press) 6. Potter, M. : Physiol. Rev. 52, 631 (1972) 7. Baldo, B.A., Fletcher, T.C., Pepys, J. : Immmaology 32, 831 (1977) 8. Capron, A., etal.: Presse retd. 72, 3103 (1964); Longbottom, J.L., Pepys, J. : J. Path. Bact. 88, 141 (1964)
Serum-Ferritin Concentration and Diagnostic 59Fe2+ Absorption in Humans with Iron Deficiency H.C. Heinrich, E.E. Gabbe, and J. Briiggemann Division of Medical Biochemistry, Institute of Physiological Chemistry, University Hospital Eppendorf, D-2000 Hamburg The earliest stage in the development of iron deficiency was called prelatent iron deficiency and is characterized and can be diagnosed by the demonstration of an increased 59Fe absorption from a diagnostic 0.56ms (=10gmole) 59Fe2+ test dose and/or the disappearence of the Berlin blue-reactive diffuse cytoplasmatic nonhaeme storage iron in the bone-marrow macrophages whereas serum iron, total iron-binding capacity, and haemoglobin concentration are still within the normal ranges [1]. Since prelatent iron deficiency is the most prevalent deficiency of mankind both in developing and in industrial countries and precedes latent and manifest iron deficiency, it is of considerable practical interest also to be able to diagnose prolatent iron deficiency without the otherwise necessary whoIe-body-radioactivity detector for measuring the whole-body retention of absorbed 59Fe and/or the bonemarrow biopsy for the Berlin blue staining of the diffuse storage iron. "[he possibility of using a reduced serumferritin concentration as a simple, non-invasive, and cheap substitute for the wholebody-radioactivity measurement or bonemarrow biopsy was investigated by a comparative evaluation of serum-ferritin levels and diagnostic 59Fe2+ absorption in subNaturwissenschaften 64 (1977)
jects with prelatent, latent, and manifest iron deficiency and compared with subjects with normal iron stores. Serum-ferritin concentrations were estimated with an immunoradiolnetric assay using an experimental kit [2] as a modification of the two-site or sandwich solidphase system [3]. 5gFe absorption from a diagnostic 0.56 mg ~9Fe2+ dose was calculated from the whole-body retention of absorbed 59Fe as measured in the 4 ~counting geometry of a whole-body-radioactivity detector with liquid organic scintillator [4]. Sermn-ferritin concentrations with a total range of 28-221 and a geometric mean of Xg=83 ng/ml with a coefficient of standard deviation Cs.n.= 1.7 were estimated in 63 subjects with a normal diagnostic 59FEZ+ absorption of 6-48% and therefore normal iron stores. In 29 subjects with prelatent iron deficiency as indicated by an increased diagnostic 59Fen+ absorption of 52-100% (but normal serum iron, total iron-binding capacity and haemoglobin) the serum-ferritin levels were reduced to a total range of 7.8~64 (X'g= 27; Cs.o. = 1.8) ng/ml without overlap of the 68% ranges between subjects with normal iron stores and subjects with prelatent iron deficiency (Fig. 1). The 95% ranges did, however,
9 by Springer-VerIag 1977
overlap between these two groups so that a serum-ferritin level of 28-64 ng/ml alone is not sufficient for a reliable diagnosis of prelatent iron deficiency. Serum-ferritin levels above 64 ng/ml were only found in subjects with normal iron stores whereas levels below 28 ngjml do indicate depleted iron stores in at least prelatent iron deficiency. Nearly no overlap with the normal serum-ferritin range was observed in 14 subjects with latent #on deficiency (serumferritin range 5.3-32 ng/ml; X'g= 13, CS.D.= 1.6) and 42 subjects with manifest iron deficiency anemia (serum-ferritin range 2.7-12mgjml; X'g=6.1, CS.D.= 1.4). For the most part, the diagnosis of latent and manifest iron deficiency does not depend on a serum-ferritin estimation since the diagnosis and separation from prolatent iron deficiency is based on reduced serum-iron and increased transferrin levels in latent iron deficiency and an additional reduction of haemoglobin in hypochromic microcytic iron-deficiency anemia. Although there is a very close correlation between the reduction of serum-ferritin concentrations and the increase of diagnostic -~9Fe2+ absorption [5], serum ferritin alone is not a reliable indicator of depleted iron stores in the individual subject. Nevertheless it is useful for evaluating the prevalence of depleted iron stores in screening or field studies, especially if Iron deficiency
Normat
300
Fe stores "prelat~ it
Latent
2oo-T
maqifest upper CS.D.
~g
E
*
50 9
@
Cs.B lower
T
i
@ 9
2O c
.'-
I0
E 7
5
7" i
2
tm~6 n- 6327
t mf 79 4 25
tmf 14 4 10
,f 31
Fig. 1. Serum-ferritin concentrations (total range, geometric mean Xg and coefficients of standard deviation CS.D.)in humans with normal iron stores and prelatent, latent, or manifest iron deficiency (t = totals, m = males, f= females, n=number of subjects in each group) 595
no facilities for sensitive and reliable whole-body counting of absorbed 59Fe are available. With the technical assistance of Fatima I~agid and Ellen Pape. Supported in part by a research grant of the Deutsche Forschungsgemeinsehaft.
Received July 15, 1977
1. Heinrich, H.C., Bartels, H.: Ktin. Wschr. 45, 553 (1967); Hausmann, K., et al. : Acta Haemat. 42, 193 (1969) 2. Hoechst Pharmaceutical Research Laboratories, Behring Institute, 1977 3. Miles, L.E.M., etal.: Anal. Biochem. 61, 209 (1974) 4. Heinrich, H.C., et al. : Klin. Wschr. 44, 827 (1966); Heinrich, H.C., Gabbe, E.E., Whang, D.H. : Atompraxis 11, 430, 660 (1965) 5. Heinrich, H.C., Gabbe, E.E., Briiggemann, J. : Z. Naturforsch. (in press)
Transfer of Blood-Group Determinant from Bovine Serum Nonlipids to Erythrocyte Lipids of Various Mammals
I'able 1. Results of transfer of J activity from J-active nonlipids of bovine serum by incubation oferythrocytes in isotonic buffer solution (pH 7.4). The volume (gl) of packed transformed erythrocytes, whose total lipids exert a complete inhibition of immunohemolysis of (jcs) test e~throcytes under standard conditions, is given (+ =J-active, tested qualitatively only) Animal
~tl packed cells
Dog Cat
6.2 +
Rat Rabbit
+ 0.4
Cattle (adult) Calf Pig (miniature) Lama pacos
6.2 + 0.2 +
F. Kr6tlinger and O.W. Thiele
Man
3.1
Physiologisch-Chemisehes Institut der Universit~it, D-3400 G6ttingen
Chicken
0.4
It has been shown in a previous paper [1] that the J blood-group activity of cattle can be found in both the lipids and a protein-containing nonlipid fraction of J containing serum. Furthermore, it was demonstrated [2, 3] that the J determinant is transferred from the J-active serum protein onto the erythrocyte membrane by incubation in vitro. Even though the carrier of the J determinant is a lipid-free serum protein (probably a glycoprotein), the transferred J activity is detectable only in the lipid fraction of erythrocytes. Thus, the J determinant (presumably a carbohydrate unit) must have been detached from a s e r u m glycoprotein and transferred to a lipidic receptor at the erythrocyte m e m brane. It was suggested [2, 3] that an enzyme system located at the bovine erythrocyte membrane is responsible for the transfer of J determinant. Furthermore, it has been speculated [2, 3] that a transfer of carbohydrate units from serum glycoproteins to erythrocyte lipids is not confined to the J blood-group-active carbohydrate moiety of cattle. Similar transfers might also take place with carbohydrates other than blood-group-active determinants and might occur in a variety of mammals and other vertebrates and on various tissue cells. In this study, we present evidence on the transfer of bovine J blood-group determinant on erythrocytes of various mammals and of chickens. J-positive bovine serum (jcs serum) was extracted with chloroform-methanol as described earlier [4]. The precipitate (predominantly consisting of proteins) obtained by this procedure served as the lip-
jcs Cattle (naturally acquired J activity)
596
id-free donor of the J determinant. It was incubated at 37 ~ with washed erythrocytes of various animal species either in isotonic saline with added antibiofica for 24 h or in isotonic buffer solution (pH 7.4) for 3 h. The incubation procedure has been described in detail elsewhere [3]. Subsequently, the erythrocytes were washed carefully and extracted with chloroformmethanol [4]. The erythrocytie total lipids thu s obtained were purified [5] and tested for J activity using an immunohemolysisinhibition test in the bovine J system. Controls were run simultaneously with total lipids of unincubated erythrocytes in order to be sure that no cross-reacting lipids were present. All experiments were repeated several times. For a quantitative test, all experiments were done under the same conditions using the same (jcs) test erythroeytes and the same J antiserum. As shown in Table 1, the erythrocytes of all animal species under investigation are able to pick up the J determinant and to incorporate it into a lipid fraction. It is therefore suggested that the erythrocyte membranes of all species tested contain
25 12.5
both an e n ~ m e system catalyzing the transfer of the J determinant and a lipidic receptor which binds to the J determinant. The extent of transferred J activity is higher in all cases tested than that of J-active bovine erythroeytes which have acquired their J activity in vivo. This work was supported by Forschungsmittel des Landes Niedersachsen and by the Fonds der Chemischen Industrie. We wish to thank J. Koch for the supply of anti-J serum, L. Dittrich, J.-N. Meyer, H. W/51fel, C. Niessen, and H. Sander for blood samples of various animals. Received July 5, 1977 1. Schr6ffel,J., etal. : Eur. J. Biochem. 22, 396 (1971) 2. Thiele, O.W., Kr6tlinger, F., Ohl, C. : Naturwissenschaften 62, 586 (1975) 3. Kr6tlinger, F., Thiele, O.W., Ohl, C. : Eur. J. Biochem. 67, 495 (1976) 4. Schr6ffel, J., Thiele, O.W., Koch, J. : ibid. 22, 294 (1971) 5. Wells, M.A, Dittmer, J.C. : Biochemistry 2, 1259 (1963)
Gap Junctions in Freeze-fractured Membranes of the Arachnoid Mater F.X. Omlin and A. Bischoff Department of Neurology, University of Berne, Inselspitai, CH-3010 Berne The synonyms for gap junctions [1] are zonulae adherentes [2] and nexus [31. This membrane specialization represents the
morphologic aspect of a functional membrane contact, which provides the electronic coupling from cell to cell by ionic ex-
Naturwissenschaften 64 (1977)
9 by Springer-Verlag 1977
