last year we reported the case of a 58-year - old woman with high concentration of cobalt released from her hip arthroplasty . the woman presented a selective ii and viii cranial nerves impairment and mild distal sensory - motor disturbances , and some months later she became definitely blind , severely deaf , and wheel - chair bounded due to severe limbs motor weakness . during surgery of prosthesis removal of a massive infiltration of periprosthesis tissues by metallic debris ( metallosis ) became evident . the patient has undergone chelation therapy with ethylenediaminetetraacetic acid ( edta ) in order to reduce the cobalt body burden . in the scientific literature there are only five other cases of severe adverse effects on nervous system associated with cobalt released from prosthesis [ 26 ] . moreover , cobalt neurotoxicity for acoustic and optic nerves has been reported following occupational exposures to the metal [ 7 , 8 ] or , few decades ago , after oral intake during therapy of refractory anaemia [ 911 ] . cobalt concentrations in biological fluids have been investigated in subjects with prosthesis [ 1214 ] , but the kinetics of released metal ions is not adequately known , due to the lack of information about amount and rate of release from hip surface , about the solubility of released ions and or about the fraction phagocyted by macrophages and the role of metal deposition - storage mechanisms [ 1517 ] . the distribution of cobalt in the body is also influenced by binding to the plasma proteins . albumin has been indicated to be a physiological carrier of cobalt ions in old and more recent studies [ 18 , 19 ] . the cobalt binding to albumin has been studied because the base of the  albumin cobalt binding test ( acb test ) approved by the food and drug administration for evaluating myocardial ischemia . ischemia modified albumin ( i m a ) is a form of human serum albumin in which the n - terminal amino acids have been altered so as to be unable to bind transition metals . however , other pathologies such as trauma , sclerodermia , diabetes , bacterial or viral infections , end - stage renal disease , liver cirrhosis , brain ischemia , peripheral arterial disease , and cancer are able to influence the binding site of cobalt in albumin [ 2126 ] . in addition there are also evidences of possible genetic variants of human serum albumin , particularly structural changes at the n - terminus abolish transition metals binding [ 27 , 28 ] . the direct measurement of cobalt levels in biological matrices and protein binding of this clinical case may be useful in understanding effects and variability of cobalt neurotoxicity . cobalt in blood , plasma , urine , and cephalorachidian fluid was measured using an inductively coupled - plasma mass spectrometry ( icp - ms , elan drc ii , perkin elmer , waltham , usa ) equipped with dynamic cell reaction . the samples of plasma and blood were diluted with triton x-100 0.05% , while for urine and cephalorachidian fluid was used bidistilled water , tracepur for inorganic trace analysis ( merck kgaa ) . g / l to 1000  g / l cobalt ( in hno3 2% mono elemental standard solution carlo erba reagenti , milano , italy ) . the accuracy of the method was assessed using specific certified materials for different matrices 1a / b ( blood samples ) , 11a / b ( plasma ) 2a / b ( urine ) , all from the german society of occupational and environmental medicine ( erlangen , germany ) . the sample solutions were pumped in the spray chamber by a peristaltic pump detecting mass 59(co ) . the accuracy of methods for the determination of cobalt in blood and urine was 95% , whereas it was 97% for plasma . method precision was 6.1 and 6.9% for blood and plasma determinations , respectively , whereas in the case of urine precision was 4.1% . for each matrix , the lod was 0.02  g / l for co  b and co  p , and 0.05  g / l for co  u . the concentration of i m a can be determined by addition of known amount of cobalt to serum or plasma specimen and measurement of the unbound cobalt by colorimetric assay [ 20 , 23 , 3032 ] . to remove the interference of edta , the plasma was pretreated with calcium chloride ( cacl2 x 2h2o ; sigma - aldrich ) ; the combination of sample and reagent pre - treatment was then centrifuged at 10001200  g for 10 minutes without mixing the two components . plasma was incubated with a cobalt chloride solution ( sigma - aldrich , cocl26h2o ; 1  g / l , 10 minutes ) and dithiothreitol ( sigma - aldrich ; 1.5  g / l , 2 minutes ) before dilution in saline solution ( 0.9% nacl ) and   measurement at 470  nm in a spectrophotometer ( uv - vis spectrophotometer cary 50 , varian , palo alto , usa ) . i m a was calculated from the difference between samples measured with and without dithiothreitol and reported in mean absorbance units ( absu ) . bar - or et al .   showed that values greater than 0.400 absu indicate lower cobalt binding and values equal or lower than 0.400 absu higher cobalt binding . the distributions , assessed by the one - sample kolmogorov - smirnov test , were normal for plasmatic and hematic cobalt in controls and for i m a in both groups . the trend of cobalt distribution in woman 's plasma and blood and in cob / cop ratio was assessed by ordinary least square ( ols ) . the range of repeated measures of cobalt concentrations , ratio cob / cop and i m a in woman with prosthesis were compared with the arithmetic means of healthy and unexposed subjects . trend of plasma , whole blood , and urine cobalt concentrations are reported in figures 1 and 2 . cobalt basal levels were very high ( 549  g / l in whole blood ; 260  g / l in plasma ) , after the chelation therapy the levels decreased by 70% in whole blood and 61% in plasma . a further reduction occurs after the prosthesis removal ( 94  g / l in whole blood and 64  g / l in plasma ) . the current ( 14 months after removal ) cobalt levels remained higher than reference values ( 33.9 versus 0.052.7  g / l in blood ; 28.5 versus 0.10.6  g / l in plasma ) . in the urine cobalt was rapidly and in great extent eliminated after a chelation therapy , the rate of excretion was 2173 the cobalt in the cephalorachidian fluid was determined before chelation and removal of the prosthesis ( 11.4  g / l and 10.1  g / l ) and two days after ( 5.4 and 2.6  g / l ) ; the normal values ranged between 0.050.15  g / l . in spite of these high levels a clear improvement of clinical evidence for most adverse effects to nervous system , except the visual function , was seen . the investigation of cobalt distribution in blood fractions showed , contrary to the expected , that most of the cobalt was in whole blood than in plasma . normally most of the cobalt is found in the serum and plasma with lower concentrations in the red blood cells ( rbc ) ; the whole blood levels are between those of the rbcs and the plasmatic fraction . the ratio cob / cop is different when considering different kind of metal exposures , in table 1 were reported the ratio of workers exposed to cobalt during hard metals production ( unpublished data ) and a collection of samples from patients after hip arthroplasty . a greater distribution of cobalt in whole blood than in plasma may indicate an alteration in the binding of cobalt to plasma proteins and an increased concentration of nonprotein - bound cobalt . i m a was measured in different samples of patients in study and in healthy unexposed subjects , i m a values significantly higher than controls seem to confirm this hypothesis ( figure 3 ) . according to bar - or   the absorbance units ( absu ) greater than 0.4 indicate a reduced cobalt binding to albumin . i m a 's values and ratio of cob / cop showed a good correlation ( r = 0.6325 ) ( figure 4 ) ; on the contrary , no correlation has been demonstrated between the values of i m a and the concentrations of cobalt in blood or in plasma . cobalt in blood and plasma follows a defined decreasing trend while the ratio of cob / cop does not follow any specific trend . critical points of this hypothesis have been considered ; the possibility that in our conditions the albumin binding site could be only saturated by high amount of cobalt and not altered is disproved by the fact that altered albumin ( i m a 0.92 and 1.17 absu ) corresponds both to high and lower cobalt blood concentrations ( 415 and 38  g / l , resp . ) , excluding a possible interference of cobalt concentration on colorimetric measure . the percentage of blood albumin on total proteins was monitored during the hospitalization period and was 55.68  9.57% ( normal range of laboratory 5569% ) . the alteration of n - terminal binding site of human albumin may be the cause of the reversed cob / cop ratio and the consequent greater amount of cobalt in an available form for higher distribution and interaction into the cells . nonprotein - bound metal can enter cells through a promiscuous metal transporter , divalent metal transporter 1 ( dmt1 ) [ 35 , 36 ] , that is known to actively transport iron , zinc , manganese , cobalt , cadmium , copper , and lead , via a proton - coupled mechanism . dmt1 is predicted to have 12 transmembrane segments and to be expressed in neurons , allowing the incorporation of metals from the extracellular environment and/or from the recycling endosomes . .   showed that human neurons in culture were susceptible to metal toxicity in cellular assays involving increasing concentrations of environmental iron   and cobalt . they have identified dmt1 as the key player in the entry of metal ions in the brain . in our case an high concentration of free cobalt in the whole blood could be available to interact with dmt1 and becoming the cause of the neurological effects . the concentration of cobalt in cerebrospinal fluid measured before chelation and before prosthesis removal showed a massive amount of cobalt ( 11.4  g / l ) , that corresponds to a 2.37  cob / cop ratio . an alteration of the bound with the main plasma protein resulting in a greater amount of free cobalt may be the cause of the not frequently neurologic symptoms reported in the scientific literature for cobalt intoxication . some authors suggest that the alteration of binding site of albumin could be the result of acidosis , reduced oxygen tension , and the generation of free radicals [ 39 , 40 ] and this conditions could be caused by cobalt and chromium constantly released from the prosthesis . in addition genetic variants of human serum albumin have been reported , each of these variants has been classified on the basis   of the structural changes at the n - terminus that abolish transition metal binding . many authors reported higher levels of i m a in diabetic patients , and they suggest that the plasmatic albumin of diabetic patients was modified for the chronic hypoxia conditions provoked mainly by hyperglycemia and oxidative stress in diabetes [ 25 , 41 ] . reported that diabetic patients without cardiovascular disease have a higher serum i m a levels compared to healthy controls . diabetes may have been a contributory cause of the alteration of albumin that in the state of cobalt intoxication was not able to correctly bound it   favoring the increase in the haematic fraction . among all the patients with extremely high cobalt concentrations in blood or urine , only few develop a severe neurologic syndrome . the reason is still unclear and , even if it is certain that cobalt can cause potential serious effects on human central nervous system , little is known about the mechanisms through which this metal can cause neurotoxicity to optic and acoustic nerves . we have tried to explain the anomalous reaction to cobalt in different ways and an alteration of plasma protein with a consequent more active fraction seems to be the most plausible hypothesis for the neurological symptoms . a possible explanation for the symptoms may be the altered albumin binding capacity and the consequent increase of the free form of cobalt in the blood . the decreased linkage to albumin for cobalt generates i m a and increases the concentration of biologically active free cobalt . the free cobalt can then interact with specific protein carriers to the cellular targets of the central nervous system . the cause of albumin alteration is not clear , we hypothesize an influence of several factors , which in this specific situation are   the generation of free radicals by cobalt , a genetic predisposition or a possible influence of diabetes . we hope that our study can be the starting point for further studies on the neurological effects related to the toxicity of cobalt .
the distribution and adverse effects , especially to optic and acoustic nerves , of cobalt released from a hip arthroplasty and its association with albumin were studied . 
 the analysis of cobalt was performed in plasma , whole blood , urine , and   cerebrospinal fluid by inductively coupled plasma mass spectrometry ( icp - ms ) . 
 the fraction of albumin binding the metal was determined by colorimetric assay using dithiothreitol ( dtt ) . in all the biological matrices 
 very high levels of cobalt were measured , but contrary to expected , a higher concentration in whole blood than in plasma was observed . 
 the determination of altered albumin confirmed this hypothesis . 
 this evidence might indicate an alteration in the binding of cobalt to albumin and a consequent increase in the concentration of the diffusible ( free ) fraction of the metal . 
 this appears an interesting starting point for further investigations for identifying and better understanding cobalt neurotoxicity , apparently not so frequent in occupational medicine and clinical practice .