Journal of Clinical Laboratory Analysis 6:123-124 (1992)
Reduced Levels of Free Testosterone in Four Catania-Type Alloalbuminemic Males Giuseppe Banfi, Rita Daverio, and Pierangelo Bonini Laboratorio Analisi, lstituto Scientifico H San Raffaele, via Olgettina 60, 20132 Milano, Italy We studied total testosterone, sex hormone binding globulin, and free testosteronein four males presenting an electrophoretically slowmoving genetic variant of albumin, the alloalbumin Catania. Free testosteronelevels were lower in these cases, found in a year of observation, than those expected for the ages. This Key words:
o 1992Wiley-Liss, Inc.
sex hormone binding globulin, hormone binding, genetic variants
INTRODUCTION Testosterone is a steroid hormone carried in plasma by a specific protein (sex hormone binding globulin, SHBG) and by albumin (1). The physiological role of a hormone is determined by free (unbound) molecules, which are captured by specific receptors on cells. Albumin is crucial in the metabolism of testosterone: this protein presents a lower specificity than SHBG for the hormone but its concentration in biological fluids is very high. Some genetic variants of albumin are present in humans ( 2 ) ;in Italy 16 variants were electrophoretically recognized (alloalbumins) and 12 of them have been sequenced. Alloalbumin Catania presents a modification at the chain end (580 Lys-Leu-Pro-COOH) (3), which provokes a faint cathodic additional band near albumin in electrophoresis at pH 8.6. We studied testosterone metabolism in four males presenting alloalbumin Catania that we found in 1 year of observation in our laboratory.
MATERIALS AND METHODS Alloalbumins Catania were electrophoretically detected (Olympus HITE600, Olympus Optical, Japan) and compared with standards supplied by the Italian Committee for Laboratory Standardization (CISMEL) in order to classify them. Testosterone and free testosterone were measured by radioimmunoassay (RIA; BioRad, Anaheim, California, and Diagnostic Product Corporation, Los Angeles, California, respectively); SHBG was measured by an IFMA method (Delfia, Pharmacia, Bromma, Sweden). The four males were two outpatients and two inpatients. The two outpatients were healthy individuals, whereas the two hospitalized males presented an IgG lambda myeloma and a cardiopathy. Other males, presenting Venezia (572 Pro-Tht-Met-Arg-Ile-Arg-Glu-COOH, 0 1992 Wiley-Liss, Inc.
finding, which is not related to any disease and constantly not recognized in other males with various genetic variants, should induce consideration of a probable difference of the genetic variant in hormone binding.
one case), Milano (unknown mutation, one case), Tagliacozzo (313 Lys-tArg, one case), Verona (570 Glu-Lys, one case), and Roma (321 Glu-rLys, one case) were also analyzed. The values of free testosterone were compared with the reference values of a normal Italian population(4).
RESULTS The values of testosterone, free testosterone, and SHBG of the four males are illustrated in Table 1. Other male carriers of alloalbumins did not present abnormalities of testosterone binding, except one (albumin Venezia, 16 years, SHBG = 133 nmol/liter, free testosterone = 1.4 pgiml, testosterone = 0.6 ng/ml). Electrophoretically quantitated albumin was normal in all the cases.
DISCUSSION The transport of testosterone in plasma is linked to a specific protein, SHBG, and to a nonspecific but highly concentrated protein, albumin. Genetic variants of albumin could be involved in defective or incomplete binding and/or transport of molecules as hormones or drugs (5). Testosterone, reportedly, has some difficulties in binding genetic or pathological (alcohol-dependent diseases, penicillin-induced bisalbuminemia) albumin variants (6). We found low levels of free testosterone in four males presenting alloalbumin Catania, whereas the results were normal in other alloalbuminemic males, except one (an adolescent). It is difficult to explain this finding because testosterone
Received August 30, 1991; accepted December 11, 1991. Address reprint requests to Giuseppe Banfi, Laboratorio Analisi, Istituto Scientifico H San Raffaele, via Olgettina 60, 20132 Milano, Italy.
124
Banfi et al.
TABLE 1. Testosterone (T), Free Testosterone (ff)and Reference Values According to Age (Expressed as 2.5-95 percentiles), and Sex Hormone Binding Globulin (SHBG) Values for Four Alloalbuminemic Males' Case
Disease
Age (years)
T (ng/ml)
fr (pg/ml)
SHBG (nmol/liter)
TN CM MM LA
Myeloma Cardiopathy
17 42 52 61
7.97 3.86 1.93 3.75
10.8 ( 12.5-43.2) 12.5 (13-48.8) 1.4 (13-42) 12.9 (12-35)
92 35 33 20
"References values for male testosterone were 1-10 ng/ml, whereas SHBG reference values were 10-70 nmoliliter. We also obtain values lower than expected by dividing the free testosterone concentration for each sample by its total testosterone concentation, and multiplying by 100: respectively, TN 0.31% (the 5th percentile for age group 0-20 years described by manufacturer is 0.38%), CM 0.33% (0.33%), MM 0.15% (0.30%),LA 0.34% (0.31%).
binds albumin in the second domain (7), whereas alloalbumin Catania is modified in the third one, but a steric hindrance andlor variation should be invoked, as in the warfarin defective binding to alloalbumins Parklands and Niigata (7). It is important to remark that the other case that displayed low free testosterone level, although in the presence of high SHBG, presented an albumin variant (Venezia) with a shortened chain mutation and COOH-terminal modification (8). Moreover, it is stated that some genetic variants present an in vitro increase of steroid hormones binding (9). The low levels of free testosterone could not be linked to individual characteristics because two males were apparently healthy and another one presented an underlying disease not involving testosterone production and metabolism. Only in the case of a systemic neoplasia could the disease and its therapy have influenced the hormone cycle; however, the transport proteins presented normal concentrations at the moment of analysis. The anamneses of hospitalized patients and the check-up of the outpatient did not present data of in vivo andrological or, in generic terms, hormonal problems. By contrast, an in vitro artefact could be avoided by the resulting data from other alloalbuminemic males. We should study further cases of alloalbumins Catania to validate our findings or to classify them as accidental data, especially by testing homozygous males subjects or by inves-
tigating in vitro binding of the hormone, using isolated variant protein.
REFERENCES 1. Ismail AAA, Astley P, Burr WA, et al.: The role of testosterone measurement in the investigation of androgen disorders. Ann Clin Biochem 23:113-134, 1986. 2. Tarnoky AL: Genetic and drug-induced variation in serum albumin. Adu ClinChem 1:101-146, 1986. 3. Galliano M, Minchiotti L, Iadarola P, Zapponi MC, Ferri G, Castellani AA: Structural characterization of a chain termination mutant of human serum albumin. JBiol Chem 261:4283-4286, 1986. 4. Ruggeri S, Nunziatini R , Pallotti G: Limiti di riferimento e variazioni del testosterone libero in rapport0 all'eta'. Bioch Clin 15:52-53 (suppl. 1/6), 1991. 5 . Kragh-Hansen U, Brennan SO, Galliano M, Sugita 0: Binding of wartarin, salicylate, and diazepam to genetic variants of human serum albumin with known mutations. Mol Pharmacol37:238-242, 1990. 6. Vermeulen A: Physiology of the testosterone binding globulin in human. Abstracts of the International Congress on Steroid Protein Interactions, Turin, 1987, p41. 7. Kragh-Hansen U: Molecular aspects of ligand binding to serum albumin. PharmacolRev3:17-53, 1981. 8. Minchiotti L, Galliano M, Iadarola P, et al.: The molecular defect in a COOH-terminal-modified and shortened mutant of human serum albumin. JBiol Chem 264:3385-3389, 1989. 9. Kragh-Hansen U, Minchiotti L, Brennan SO, Sugita 0: Hormone binding to natural mutants of human serum albumin. EurJBiochem 193:169171, 1990.
Reduced Levels of Free Testosterone in Four Catania-Type Alloalbuminemic Males Giuseppe Banfi, Rita Daverio, and Pierangelo Bonini Laboratorio Analisi, lstituto Scientifico H San Raffaele, via Olgettina 60, 20132 Milano, Italy We studied total testosterone, sex hormone binding globulin, and free testosteronein four males presenting an electrophoretically slowmoving genetic variant of albumin, the alloalbumin Catania. Free testosteronelevels were lower in these cases, found in a year of observation, than those expected for the ages. This Key words:
o 1992Wiley-Liss, Inc.
sex hormone binding globulin, hormone binding, genetic variants
INTRODUCTION Testosterone is a steroid hormone carried in plasma by a specific protein (sex hormone binding globulin, SHBG) and by albumin (1). The physiological role of a hormone is determined by free (unbound) molecules, which are captured by specific receptors on cells. Albumin is crucial in the metabolism of testosterone: this protein presents a lower specificity than SHBG for the hormone but its concentration in biological fluids is very high. Some genetic variants of albumin are present in humans ( 2 ) ;in Italy 16 variants were electrophoretically recognized (alloalbumins) and 12 of them have been sequenced. Alloalbumin Catania presents a modification at the chain end (580 Lys-Leu-Pro-COOH) (3), which provokes a faint cathodic additional band near albumin in electrophoresis at pH 8.6. We studied testosterone metabolism in four males presenting alloalbumin Catania that we found in 1 year of observation in our laboratory.
MATERIALS AND METHODS Alloalbumins Catania were electrophoretically detected (Olympus HITE600, Olympus Optical, Japan) and compared with standards supplied by the Italian Committee for Laboratory Standardization (CISMEL) in order to classify them. Testosterone and free testosterone were measured by radioimmunoassay (RIA; BioRad, Anaheim, California, and Diagnostic Product Corporation, Los Angeles, California, respectively); SHBG was measured by an IFMA method (Delfia, Pharmacia, Bromma, Sweden). The four males were two outpatients and two inpatients. The two outpatients were healthy individuals, whereas the two hospitalized males presented an IgG lambda myeloma and a cardiopathy. Other males, presenting Venezia (572 Pro-Tht-Met-Arg-Ile-Arg-Glu-COOH, 0 1992 Wiley-Liss, Inc.
finding, which is not related to any disease and constantly not recognized in other males with various genetic variants, should induce consideration of a probable difference of the genetic variant in hormone binding.
one case), Milano (unknown mutation, one case), Tagliacozzo (313 Lys-tArg, one case), Verona (570 Glu-Lys, one case), and Roma (321 Glu-rLys, one case) were also analyzed. The values of free testosterone were compared with the reference values of a normal Italian population(4).
RESULTS The values of testosterone, free testosterone, and SHBG of the four males are illustrated in Table 1. Other male carriers of alloalbumins did not present abnormalities of testosterone binding, except one (albumin Venezia, 16 years, SHBG = 133 nmol/liter, free testosterone = 1.4 pgiml, testosterone = 0.6 ng/ml). Electrophoretically quantitated albumin was normal in all the cases.
DISCUSSION The transport of testosterone in plasma is linked to a specific protein, SHBG, and to a nonspecific but highly concentrated protein, albumin. Genetic variants of albumin could be involved in defective or incomplete binding and/or transport of molecules as hormones or drugs (5). Testosterone, reportedly, has some difficulties in binding genetic or pathological (alcohol-dependent diseases, penicillin-induced bisalbuminemia) albumin variants (6). We found low levels of free testosterone in four males presenting alloalbumin Catania, whereas the results were normal in other alloalbuminemic males, except one (an adolescent). It is difficult to explain this finding because testosterone
Received August 30, 1991; accepted December 11, 1991. Address reprint requests to Giuseppe Banfi, Laboratorio Analisi, Istituto Scientifico H San Raffaele, via Olgettina 60, 20132 Milano, Italy.
124
Banfi et al.
TABLE 1. Testosterone (T), Free Testosterone (ff)and Reference Values According to Age (Expressed as 2.5-95 percentiles), and Sex Hormone Binding Globulin (SHBG) Values for Four Alloalbuminemic Males' Case
Disease
Age (years)
T (ng/ml)
fr (pg/ml)
SHBG (nmol/liter)
TN CM MM LA
Myeloma Cardiopathy
17 42 52 61
7.97 3.86 1.93 3.75
10.8 ( 12.5-43.2) 12.5 (13-48.8) 1.4 (13-42) 12.9 (12-35)
92 35 33 20
"References values for male testosterone were 1-10 ng/ml, whereas SHBG reference values were 10-70 nmoliliter. We also obtain values lower than expected by dividing the free testosterone concentration for each sample by its total testosterone concentation, and multiplying by 100: respectively, TN 0.31% (the 5th percentile for age group 0-20 years described by manufacturer is 0.38%), CM 0.33% (0.33%), MM 0.15% (0.30%),LA 0.34% (0.31%).
binds albumin in the second domain (7), whereas alloalbumin Catania is modified in the third one, but a steric hindrance andlor variation should be invoked, as in the warfarin defective binding to alloalbumins Parklands and Niigata (7). It is important to remark that the other case that displayed low free testosterone level, although in the presence of high SHBG, presented an albumin variant (Venezia) with a shortened chain mutation and COOH-terminal modification (8). Moreover, it is stated that some genetic variants present an in vitro increase of steroid hormones binding (9). The low levels of free testosterone could not be linked to individual characteristics because two males were apparently healthy and another one presented an underlying disease not involving testosterone production and metabolism. Only in the case of a systemic neoplasia could the disease and its therapy have influenced the hormone cycle; however, the transport proteins presented normal concentrations at the moment of analysis. The anamneses of hospitalized patients and the check-up of the outpatient did not present data of in vivo andrological or, in generic terms, hormonal problems. By contrast, an in vitro artefact could be avoided by the resulting data from other alloalbuminemic males. We should study further cases of alloalbumins Catania to validate our findings or to classify them as accidental data, especially by testing homozygous males subjects or by inves-
tigating in vitro binding of the hormone, using isolated variant protein.
REFERENCES 1. Ismail AAA, Astley P, Burr WA, et al.: The role of testosterone measurement in the investigation of androgen disorders. Ann Clin Biochem 23:113-134, 1986. 2. Tarnoky AL: Genetic and drug-induced variation in serum albumin. Adu ClinChem 1:101-146, 1986. 3. Galliano M, Minchiotti L, Iadarola P, Zapponi MC, Ferri G, Castellani AA: Structural characterization of a chain termination mutant of human serum albumin. JBiol Chem 261:4283-4286, 1986. 4. Ruggeri S, Nunziatini R , Pallotti G: Limiti di riferimento e variazioni del testosterone libero in rapport0 all'eta'. Bioch Clin 15:52-53 (suppl. 1/6), 1991. 5 . Kragh-Hansen U, Brennan SO, Galliano M, Sugita 0: Binding of wartarin, salicylate, and diazepam to genetic variants of human serum albumin with known mutations. Mol Pharmacol37:238-242, 1990. 6. Vermeulen A: Physiology of the testosterone binding globulin in human. Abstracts of the International Congress on Steroid Protein Interactions, Turin, 1987, p41. 7. Kragh-Hansen U: Molecular aspects of ligand binding to serum albumin. PharmacolRev3:17-53, 1981. 8. Minchiotti L, Galliano M, Iadarola P, et al.: The molecular defect in a COOH-terminal-modified and shortened mutant of human serum albumin. JBiol Chem 264:3385-3389, 1989. 9. Kragh-Hansen U, Minchiotti L, Brennan SO, Sugita 0: Hormone binding to natural mutants of human serum albumin. EurJBiochem 193:169171, 1990.
