Scandinavian Journal of Clinical and Laboratory Investigation

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Cobalamin binding proteins in human seminal plasma M. Hansen & E. Nexø To cite this article: M. Hansen & E. Nexø (1992) Cobalamin binding proteins in human seminal plasma, Scandinavian Journal of Clinical and Laboratory Investigation, 52:7, 647-652, DOI: 10.3109/00365519209115508 To link to this article: http://dx.doi.org/10.3109/00365519209115508

Published online: 08 Jul 2009.

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Date: 25 April 2016, At: 13:06

Scand J Clin Lab Invest 1992; 52: 647-652

Cobalamin binding proteins in human seminal plasma M. HANSEN* & E. N E X 0 t 3 $

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*Department of Haematology L , Herlev Hospital, University of Copenhagen, Copenhagen, Denmark, ?Department of Clinical Chemistry, Central Hospital, Hillerad, Denmark, and $Department of Clinical Chemistry, Aarhus University Hospital, Kommune Hospitalet, Aarhus, Denmark

Hansen M, Nex0 E . Cobalamin binding proteins in human seminal plasma. Scand J Clin Lab Invest 1992; 52: 647-652. Transcobalamin (TC) and haptocorrin (HC) are present in normal seminal plasma in substance concentrations ten- to twenty-fold that in blood. The results are given in range and (median). The substance Concentration of seminal plasma TC is 3.6-21.9 (8.4) nmol I-' and of seminal plasma H C 1.4-8.6 (3.0) nmol I-'. Compared to normals the substance concentration of TC is significantly lowered in post-vasectomy seminal plasma 2.2-2.9 (5.3) nmol I-'. The Stokes radius and the isoelectric points of seminal plasma T C are identical to TC in blood.

Key words: cobalamin-binding proteins, haptocorrin, transcobalamin, seminal plasma Ebba N e x l , Department of Clinical Chemistry, KH- University Hospital of Aarhus, 44 NQrrebrogade, DK-8000 Aarhus C, Denmark

The cobalamin binding protein haptocorrin (HC) is a glycoprotein of unknown function, while the cobalamin binding protein transcobalamin (TC) is a peptide that transports cobalamin from its uptake in the intestine to the cell membrane, where it is internalized by specific receptors [ 11. HC is present in plasma [l], and in secretions such as saliva [2], tears [3, 41 and milk [5].The substance concentration of H C varies from 0.2 nmol I-' to 185 nmol I-'. T C is present in plasma [l], in human milk [5], in cerebrospinal fluid [6], in synovial fluid from patients with arthritis [7] and in amniotic fluid [XI. The substance concentration of TC in these fluids varies from 0.2 nmol I-' to 0.6 nmoI I-'. The presence of TC in human semen was first described in 1968 [9, 101. Recently, it has been

reported that seminal plasma contains both T C and H C [ll]. It is the purpose of the present work to study the substance concentration of cobalamin binding proteins in seminal plasma from patients with vasectomy as compared to patients with normal and abnormal fertility as judged from examination of the seminal fluid.

MATERIALS AND METHODS

Patients A total of 62 specimens collected for diagnostic purposes were analysed.

Seminal plasma Semen wascollected into jars by masturbation. Separation of sperms from seminal plasma was 647

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M . Hunsen & E. Nexo

done within 3 h after collection by centrifugation at 600 g for 10 min. The seminal plasma was then centrifuged at 20 000 g for 30 min at 4" C . The supernatant was kept at 20 "C until analysed. Thawed samples were never refrozen.

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Semen

Semen was examined according to the recommendation of WHO [ 121. The sample was collected after two to seven days of abstinence and it was delivered to the laboratory within 2 h . A normal semen specimen fulfilled the conditions in Table I. Three groups were established: normal specimens, abnormal specimens (one o r more of the criteria in Table I were not fulfilled), and vasectomy specimens.

The unsaturated binding capucity of HC und TC The total unsaturated cobalamin binding capacity of the specimens were determined as described by Gottlieb e/ ul. 1131. By adsorption to QUSO G32 (a silica gel, which only adsorb TC, [Bie & Berntsen Ltd, Denmark]), the cobalamin binding capacities of T C and H C were determined as described 114, 151. In brief, 100 pl of the specimen were incubated with cyano15'Co]cobalamin in excess for 15 min at room temperature. Unbound tracer was removed by adsorption to haemoglobin-coated charcoal. After centrifugation (2000 g, 10 min) the supernatant containing the protein-bound tracer was mixed with 100 pl of QUSO G 32 (40 mg ml-I). T h e samples were then centrifugated at 2000 g in 10 min. From the radioactivity of the supernatant and the sediment, the cobalamin binding capacity of H C and TC, respectively was calculated. Gel filtrution

0.9 cm) at 4" C employing 0.1 mol I-' TrisHCI, 1 mol I-' NaCl, pH 8.0 as eluting buffer. Dextran blue (Pharmacia, Uppsala, Denmark) and *'Na (Amersham, Bucks, UK) were used asmarkersofvoid and total volume, respectively. Stokes radius was calculated as described using albumin with a radius o f 3.6 nm as internal standard [ 161.

Isoelectric focusing In brief, partly purification of T C and H C was done by applying the sample (100 pl) to a S-200 Sephacryl column ( 4 7 ~ 0 . 9cm) using the same eluting buffer as described above. Human albumin (Behringwerke, Marburg, Germany) was added t o the tubes collecting T C (final concentration 0.02%) in order to avoid adhesion of the peptide t o the tubes. The fractions containing T C or H C were pooled and dialysed against distilled water for 48 h at 4" C. The dialysates were lyophilized and kept frozen until used and redissolved in distilled water. The redissolved H C (250 pl) was mixed with 250 pl acetate buffer, 0.15 mol I-' NaCI, 3 mmol I-' CaCI2, p H 5.0 and treated with neuraminidase (Behringwerke, Marburg ORKO 0405, Germany) in a final concentration of 0.7 iu I-' for 30 min at 37" C . The samples were dialysed overnight at room temperature against 2x10 ml0.01 mol I-' NH4HC03,p H 8.9 and lyophilized. Isoelectric focusing of TC was done by applying $11 of the redissolved sample (20 pl) 1171. HC was focused by applying 15 pl of the sample redissolved in 25 pl 1 % glycine "1. After focusing the pH gradient was immediately measured and autoradiography done. The individual isopeptides was identified due to the isoelectric point.

Seminal plasma was labelled with cyano[s7Co] Stafi,stics cobalamin in amounts corresponding to 1.5 The Mann-Whitney U-test was used to test times the unsaturated cobalamin binding the null hypothesis postulating that the samples capacity. The sample (100 pl) was applied to in the normal group and in the abnormal group the G-200 Sephadex column (column size 9 5 ~ or the group of vasectomy were from the same T A B I . I~. : ?'he criteria used f o r a normal semen population. The level of significance employed specimen. was 0.05. 2-6 ml Qualitative motility Active forward progression Quantitative motility > 60'% Spcrm concentration > 40x10'' I-' Morphology > 5O'X normal spermatozoa Leukocytes None

Sample volume

RESULTS Seminal plasma contains both H C and TC as judged from gel filtration of seminal plasma labelled with c y a n ~ [ ~ ~ C o ] c o b a l a m(Fig. i n 1).

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Cobaiamin binding pruteins in human seminal plasma This was in accord with previous observations Ill]. The substance concentration of T C was very high (Table 11). In normals the median was 8.4 nmol I-'. In the abnormal group the median was 7.0 nmol I-'. The value was not significantly different from the normal group of semen (p=0.36). In the vasectomy group, the median of T C was 5.3 nmol I-'. This was significantly different from the values obtained for the normal group (p=0.01). As seen in Table 11, there was a high substance concentration of H C in semen. The substance concentration of HC in the normal group (median of 3.0 nmol I-') did not differ from the substance concentration of HC in the abnormal group (median 4.0 nmol I-'; p=0.75) or in the vasectomy group (median 4.0 nmol I-'; p =0.66). In order to study possible degradation of T C or HC in seminal plasma, centrifugation was cpm x

lo3

649

done immediately after arrival. The determination of HC and TC was then done immediately after centrifugation, after 3 h at room temperature and after 20 h at room temperature. All samples were frozen at -20" C immediately after analysis, thawed and analysed the following day. No difference was found between the substance concentration of TC and H C in the three groups (data not shown). T C in seminal plasma was further characterized with regard to size and isoelectric point (PI). The Stokes radius was 2.7 nm (Fig. 1). The pl was determined in 6 samples. In three samples the TC isopeptide pattern MX (PI 6.6 and 7.0) and in the remaining three samples the pattern M (PI 6.6) was identified. Isoelectric focusing of T C without partly purification showed a fuzzy band around pH 6 (Fig. 2). HC in seminal plasma was also characterized. The Stokes radius was 5.5 nm (Fig. 1). Isoelectric focusing showed the seminal HC t o be more acidic than H C present in saliva. The samples were treated with neuraminidase by a technique previously shown t o efficiently remove sialic acid [ 181. After treatment with neuraminidase the isopeptide pattern in seminal plasma was comparable to the pattern of HC in saliva (Fig. 3).

Fractlon No

FIG. 1. G-200 Sephadex gel filtration of seminal plasma. V,, void volume, V, elution of albumin, V.,. total volume. V,,c and V.,.,. indicate the elution volumen of HC and T C from serum. Seminal plasma contained one fraction (Stokes radius 5.5 nm) identical to HC and one fraction (Stokes radius 2.7 nm) identical to TC.

- 6.0 - 6.6 - 7.0

TABLE 11. The unsaturated cobalamin binding capacity of TC and HC in human seminal plasma

Semen

n

Normal

15

Abnormal

26

Vasectomy

21

Transcobalamin range (median) nmoI I-'

Haptocorrin range (median) nmol I-'

3.6-21.9 (8.4) 3.0- 19.3 (7.0) 2.2-9.9 (5.3)

1.4-8.6 (3.0)

1.5-26.6 (4.0) 1.2-19.5 (4.0)

A

B

PH

FIG.2. Autoradiogram of transcobalamin in seminal plasma separated by isoelectric focusing. Lane A: unfractionated transcobalamin and lane B: partly purified transcobalamin (B). Transcobalamin phenotype MX. The pH is indicated t o the right.

6.50

M . Hunsen & E Nex@

- 4.3

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-5.0

A

6

C

D

pH

FIG. 3 . Autoradiogram of haptocorrin from seminal plasma and saliva separated by isoelectric focusing. Lane A and B: Haptocorrin from scminal plasma (A) and saliva (B). Lane C and D: Desialated haptocorrin from seminal plasma (C) and saliva (D). The pH is indicated to the right.

DISCUSSION Both T C and HC are present in all of the seminal fluids studied. We found that normal seminal plasma contains around 8 nmol I-' of TC, that is around 10 fold more than in plasma. TC is essential for the transport of cobalamin into the cells 111 and because of that the very high substance concentration of T C in seminal plasma is interesting. It is well-known that cobalamin is essential to spermatogenesis, that cobalamin deficiency reduces male fertility and that treatment with cobalamininjectionsrestore the fertility (19-211. Similar observations have been made in females [22,231. Recently, it has been reported that T C receptors are present in rabbit and rat germ cells and that TC-cobalamin is internalized and dissociated from the receptor in acidic endosomes 1241. The endocytosis of TC is highest in spermatocytes and 'round' spermatids and lowest in elongated spermatids [2.51. suggesting that the uptake of cobalamin is essential to the early development of the germ cells. The origin of T C in seminal plasma is unknown, but it has been reported that two patients with congenital absence of seminal

vesicles had markedly diminished transcobalamin levels in seminal plasma. This suggests that the seminal vesicles are the chief source of TC [ 111. However, in our study no difference is found between the substance concentration of TC in the normal and abnormal groups, whereas the TC level in vasectomized patients was significantly reduced, Part of the transcobalamin may originate either from the testicular tissues or from the epididymis. TC in seminal plasma has the same size and pI as TC found in plasma 117, 261. Seminal plasma TC react with anti-TC antisera [ l l ] . Partly purification of TC was necessary prior to isoelectric focusing, which may suggest that seminal plasma contains, substances that form complexes with TC, as does plasma (27. 281. The nature of such substances is unknown. H C is present in a number of exocrine secretions most often in concentrations that are higher than the 3 nmol I-' found in seminal plasma. In human milk the binding capacity is around 15 nmol I-' [ 5 ] , in saliva around SO nmol I-' [2], in tears around 185 nmol I-' 131 and in nasal secretions around 190 nmol I-' [ 181. There is no difference between the substance concentration of HC in the different fertility groups including the vasectomized patients. Carmel et al. reported a significant increase in H C levels after vasectomy 1111. The reason for the difference between the two studies is unknown. The H C in seminal plasma was studied by isoelectric focusing and compared with HC in saliva. While the pattern for the native proteins differed, no difference was observed for the desialated proteins. This supports the idea that the desialated H C d o not show tissue specificity, while sialated H C show tissue specific changes due to different degrees of sialation [291. Similar observations have been reported by others 1301. In conclusion, seminal plasma contains TC as its major cobalamin binding protein in addition to HC. The substance concentration of TC is significantly reduced in vasectomized patients, while the substance concentration of H C is unchanged.

ACKNOWLEDGMENT The authors acknowledge the skillful technical

Cobalamin binding proteins in human seminal plasma assistance of Marianne Rye Hansen and Helle Holm. The present study was in part supported by grants from the Danish Cancer Society (J.No OW - 82).

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in human seminal plasma. The First International Congress of “Vitamins and Biofactors in Life Science”, Kobe September 16-20 1991; 98. (Abstract) Received 25 March 1992 Accepted 3 April 1992

Cobalamin binding proteins in human seminal plasma.

Transcobalamin (TC) and haptocorrin (HC) are present in normal seminal plasma in substance concentrations ten- to twenty-fold that in blood. The resul...
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