BrainResearchBulletin.Vol. 24. pp. 105-111. ~ Pergamon Press plc, 1990. Printed in the U.S.A.
0361-9230/90 $3.00 + .00
Membrane Channel Protein Abnormalities and Autoantibodies in Neurological Disease M A R G U E R I T E M. B. K A Y , * t ~ : 1 J O S E P H G O O D M A N , * ~ : C H R I S T I N E L A W R E N C E § AND GIELJAN BOSMANt
*Department of Medicine and "~Medical Microbiology and Immunology Texas A&M University, College of Medicine, 1901 South First Street STeague Veterans' Center (151), Temple, TX 76504 §Department of Medicine, Bronx Municipal Hospital Center and Albert Einstein College of Medicine, Bronx, NY 10461 R e c e i v e d 18 S e p t e m b e r 1989
KAY, M. M. B., J. GOODMAN, C. LAWRENCE AND G. BOSMAN. Membranechannelproteinabnormalitiesandautoantibodies in neurologicaldisease. BRAIN RES BULL 24(1) 105-111, 1990.--Immunological analogues of band 3, the anion transporter of the human erythrocyte, have been identified in all ceils, including both isolated neurons and neurons of the central nervous system. We hypothesized that the anion channel is altered in neurological disease associated with choreiform movements because ~t-aminobutyric acid (GABA), the major inhibitory neurotransmitter in mammalian brain, binds to its receptor and opens an integral membrane chloride channel. In order to examine this hypothesis, we studied a family with a serious, progressive, genetic neurologic disorder with acanthocytosis (choreoacanthocytosis) that resembles Huntington's chorea. We selected choreoacanthocytosis because erythrocytes, which are readily obtained, are affected in this disease as well as the central nervous system. Biochemical studies of erythrocytes from the proposita, mother, and brother revealed that sulfate transport V,~axwas increased, and glucose effiux was decreased. Erythrocytes exhibited immunological changes indicative of cellular aging/transporter damage. In addition, transporter reactive antibodies were present. This is the first evidence for abnormalities of membrane transport in this neurologic disorder. Anion and glucose transport Alzheimer's disease
Protein band 3
Senescent cell antigen
BAND 3 is a ubiquitous membrane protein (9-11, 18, 27, 33). Like the red blood cell (RBC) proteins spectrin, ankyrin, 4.1, and actin (15,16), it is present in brain and all other tissues examined [(27); for review see (15)]. Band 3 is present not only in cell membranes, but also in nuclear (33), golgi (34), and mitochondria membranes (14,49). Surface immunofluorescence and immunoelectron microscopy studies indicate that the band 3-like proteins in nucleated cells participate in band 3 antibody-induced cell surface patching and capping (33). In red blood cells, band 3 mediates the exchange of anions (e.g., chloride, bicarbonate, phosphate, sulfate, iodide, bromide) across the membrane (7,39), maintains acid-base balance, and is the binding site for the glycolytic enzymes adolase (52), phosphofructokinase (20), and glyceraldehyde-3-P-dehydrogenase (35), and for hemoglobin (47). Water and glucose transport have also been attributed to band 3 (6, 26, 41, 53) although the latter is still open to question (42). Band 3 is the major transmembrane structural protein of the erythrocyte (57). The M r --95,000 band 3 molecule crosses the membrane
Choreoacanthocytosis
GABA
between 3 and 12 times (36, 45, 51). A cytoplasmic segment containing the amino terminus binds to band 2.1 (ankyrin) which attaches to the internal filamentous cytoskeleton (2). Senescent cell antigen, an aging protein that terminates the lifespan of cells, is derived from band 3 (21-32). Appearance of senescent cell antigen initiates IgG binding and cellular removal (1, 5, 21-23, 29). Oxidation generates senescent cell antigen in situ (29). Senescent cell antigen appears to be located on an extracellular portion of band 3 that includes most of the - 3 8 , 0 0 0 Da carboxyl terminal segment and - 3 0 % of the - 17,000 Da anion transport region (24). Although senescent cell antigen was first demonstrated on the surface of senescent human erythrocytes (red blood cells; rbc), it has since been demonstrated on the surface of lymphocytes, polymorphonuclear leukocytes, platelets, embryonic kidney cells, and adult liver cells (23, 25, 33), and isolated neurons and neurons of the central nervous system (27,33). We hypothesized that a defect in the anion channel creates an
IRequests for reprints should be addressed to Marguerite M. B. Kay, Professor of Medicine, Texas A&M University, Building 4, 1901 South First Street, Temple, TX 76504.
105
106
KAY, GOODMAN, LAWRENCE AND BOSMAN
imbalance between stimulatory and inhibitory impulses in diseases with choreiform movements thus permitting phenotypic expression of the disease. We tested this hypothesis by investigating band 3 structure and function in a patient with familial amyotrophic chorea with acanthocytosis, also called choreoacanthocytosis or neuroacanthocytosis. We selected choreoacanthocytosis because erythrocytes, which are readily obtained, are affected in this disease as well as the central nervous system. This disease is characterized by progressive chorea and erythrocyte acanthocytosis without a-13-1ipoproteinemia (3, 12, 40, 46). This disorder is similar to Huntington's disease in that there is an hereditary, adult onset progressive chorea with decreased lifespan. The age of onset is usually between 20 to 70 years. It differs from Huntington's disease in that acanthocytosis is present, deep tendon reflexes are decreased, and it is inherited in a recessive rather than dominant pattern (3, 8, 46). Choreoacanthocytosis has been reported to have an autosomal recessive mode of inheritance (3,46), but one variety may be inherited as an autosomal dominant (12, 17, 40). Mild to moderate mental deterioration can occur late in the disease. Neuropathology studies have revealed atropy and gliosis of basal ganglia (3). The caudate nucleus and putamen are the most severely affected. Mild diffuse gliosis in the white matter of the cerebral hemispheres, the cerebellum, and spinal cord gray matter is observed (3). Cortical atrophy and denervation atrophy of muscle is also present. Glutamic acid decarboxylase and choline acetyltransferase activity are normal in the cortex, caudate, and putamen (3). The proposita we studied has mild generalized cerebral atrophy and severe atrophy of the caudate nucleus as determined by computerized tomography scan of the brain with contrast. METHOD
Cell Separation Blood was obtained from the 41-year-old affected proposita, and her mother and brother. Her brother, age 43, has a history of seizures. Her mother does not show clinical manifestations of the disease. Whole blood was centrifuged at 1000 rpm for 15 min in a Sorvall RT6000 at 4°C to remove plasma and platelets. Pelleted cells were resuspended to the original volume with phosphate buffered saline without Ca ++ and Mg ÷+ and layered on Ficoll/ Hypaque (1). Mononuclear cells were removed by centrifugation on Ficoll/Hyopaque at 1200 rpm for 15 min in a Sorvall RT6000 at 4°C. Remaining white blood cells were removed with a pipet and red blood cells were separated into populations of different ages on Percoll gradients as previously described (1).
IgG Binding Assay The amount of IgG on cells was quantitated using 12~I-labeled Protein A according to the method of Yam et al. (56).
Sodium Dodecyl Sulfate (NaDodS04) Polyacrylamide Gel Electrophoresis Proteins were analyzed on 6-25% or 12-25% linear NaDodSO4/ polyacrylamide gradient gels using the discontinuous buffer system of Laemmli (36).
Antisera Rabbit antibodies to band 3 and antibody 980, a rabbit antibody to aged band 3 (precursor to senescent cell antigen), were prepared as previously described (30, 32, 33). Control sera consisted of normal, nonimmune rabbit sera, preimmunization sera, and anti-
bodies to band 3 absorbed with band 3.
Immunostaining of the Membrane Proteins Immunoautoradiography was performed by the immunoblotting technique of Towbin et al. (54) with the modifications described previously (30, 32, 33).
Anion Transport Measurements The "self-exchange flux" of sulfate was determined from the sulfate exchange at Donnan equilibrium, essentially following the method of Lepke and Passow (39) and Schnell et al. (48) with the modifications previously described (28-30). Chloride was omitted from the transport media because it is a competitive inhibitor of sulfate influx and stimulates sulfate effiux. Sulfate rather than chloride transport was measured because sulfate transport is slower thus allowing more accurate measurement. Measurements were made at six concentrations of sulfate between two and 30 mM, and at three time-points within the first eight minutes after the start of the assay. Km, the sulfate concentration at which half the maximal velocity, Vmax, is reached, was determined by extrapolation from the linear part of the Lineweaver-Burk plot.
Glucose Transport Measurements Characteristics of the glucose transport system (zero-trans-exit and infinite-trans-entry)were determined following the methods of Naftalin et al. (43) as previously described (43). Extensively washed erythrocytes were loaded with 50 mM glucose by incubation at 10% hematocrit in 20 mM phosphate/147 mM NaC1/50 mM glucose, pH 7.4 at 37°C for at least two hours, with a change of medium after one hour. For exit experiments, loosely packed cells were incubated with one microCurie D-[U-14C]-glucose (290 mCi/mmol, Amersham) or one microCurie 3-O-methylglucose (290 mCi/mmol, Amersham). Maximal transport velocity, V . . . . and K m of zero-trans-efflux (55), and the Vmax o f infinitetrans-entry was calculated using an integrated rate equation (55).
Isolation of the Brain Anion Transporter The anion transporter was isolated from bovine brain by affinity chromatography with the anion transport inhibitor 4acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) conjugated to Affigel 102 as described (44). Frozen bovine brain was homogenized in "homogenization buffer" (5 mM HEPES, 0.32 M sucrose, 5 mM imidazole, 5 mM benzamidine, 2 mM mercaptoethanol, 3 mM EGTA, 0.5 mM MgSO4, 5 mM glycerophosphate, 5 mM KF, 0.5 mM ZnSO4, 0.1 mg/ml leupeptin, 0.05 mg/ml pepstatin A, 0.1 mg/ml aprotinin, and 1 mM NAN3, pH 8.0). Membranes were obtained by centrifugation at 20,000 rpm in a Ti 65 rotor in a Sorvall OTD 65B centrifuge for 1 hr. Peripheral membrane proteins were removed with 0.1 M NaOH containing 1 mM diisopropylfluorophosphate (DFP), 1 mM EDTA, and 1 mM EGTA. Membranes were pelleted and washed with a low molarity buffer (5 mM sodium phosphate buffer with 1 mM EDTA, 1 mM EGTA, 1 mM DFP, pH 8.0). Membranes were dissolved in 100 mM "Iris HC1 with 1 mM EDTA, 20 mM mercaptoethanol, 1 mM dithiothreitol (DTr), and 2% NaDodSO 4, pH 8.0, diaiysed against column buffer (50 mM Tris acetate with 1 mM EDTA, 1 mM EGTA, 1 mM DFP, 0.03% lithium dodecyl sulfate, and 0.02% azide, pH 8.0) and loaded on an Affigel 102 SITS affinity column. The column was washed with column buffer until the optical density was the same as that of the column buffer as determined with an online spectrophotometer. The column was washed with column buffer containing 150 mM NaC1. The anion transporter
ANION CHANNEL IN NEUROLOGIC DISEASE
C
N
C
107
TABLE 1
N
A N I O N A N D G L U C O S E T R A N S P O R T BY ERYTHROCYTES FROM N O R M A L INDIVIDUALS A N D ERYTHROCYTES FROM C H O R E O A C A N T H O C Y T O S I S F A M I L Y MEMBERS I
Glucose Anion Individual
3
5 m
7
~
Wmax3
Km
Vmax2
Influx
Effiux
Control Proposita
1.1 -- 0.1 1.1 + 0.1
13.1 +_ 0.4 18.1 --_ 0.4?
15.0 -+ 1.2 9.3 + 1.7+
10.0 -- 0.9 2.0 -- 1.Or
Control Proposita
0.7 -- 0.1 0.9 --- 0.1
13.5 -+ 0.7 29.0 --- 1.07
10.1 _+ 0.9 9.8 -+ 1.2
16.5 -- 0.6 8.2 ----- 0 . 3 ? 4
Control Mother
1.2 --- 0.1 1.1 --- 0.2
11.0 - 1.1 16.8 ± 0.4?
9.8 ± 0.9 10.2 ± 0.6
18.2 ± 1.0 7.3 ± 0.4?
Control Brother
1.3 = 0.4 1.3 --- 0.3
11.4 ___ 2.0 15.2 ± 2.2 20.0 ± 2.7 14.3 ± 1.8:~ 13.0 ± 0.1:~ 14.0 ± 0.5?
~Results are presented as the mean ± one standard deviation; n = 6 - 8 . *Measurement approached limits of detection for this assay. ?p-
0361-9230/90 $3.00 + .00
Membrane Channel Protein Abnormalities and Autoantibodies in Neurological Disease M A R G U E R I T E M. B. K A Y , * t ~ : 1 J O S E P H G O O D M A N , * ~ : C H R I S T I N E L A W R E N C E § AND GIELJAN BOSMANt
*Department of Medicine and "~Medical Microbiology and Immunology Texas A&M University, College of Medicine, 1901 South First Street STeague Veterans' Center (151), Temple, TX 76504 §Department of Medicine, Bronx Municipal Hospital Center and Albert Einstein College of Medicine, Bronx, NY 10461 R e c e i v e d 18 S e p t e m b e r 1989
KAY, M. M. B., J. GOODMAN, C. LAWRENCE AND G. BOSMAN. Membranechannelproteinabnormalitiesandautoantibodies in neurologicaldisease. BRAIN RES BULL 24(1) 105-111, 1990.--Immunological analogues of band 3, the anion transporter of the human erythrocyte, have been identified in all ceils, including both isolated neurons and neurons of the central nervous system. We hypothesized that the anion channel is altered in neurological disease associated with choreiform movements because ~t-aminobutyric acid (GABA), the major inhibitory neurotransmitter in mammalian brain, binds to its receptor and opens an integral membrane chloride channel. In order to examine this hypothesis, we studied a family with a serious, progressive, genetic neurologic disorder with acanthocytosis (choreoacanthocytosis) that resembles Huntington's chorea. We selected choreoacanthocytosis because erythrocytes, which are readily obtained, are affected in this disease as well as the central nervous system. Biochemical studies of erythrocytes from the proposita, mother, and brother revealed that sulfate transport V,~axwas increased, and glucose effiux was decreased. Erythrocytes exhibited immunological changes indicative of cellular aging/transporter damage. In addition, transporter reactive antibodies were present. This is the first evidence for abnormalities of membrane transport in this neurologic disorder. Anion and glucose transport Alzheimer's disease
Protein band 3
Senescent cell antigen
BAND 3 is a ubiquitous membrane protein (9-11, 18, 27, 33). Like the red blood cell (RBC) proteins spectrin, ankyrin, 4.1, and actin (15,16), it is present in brain and all other tissues examined [(27); for review see (15)]. Band 3 is present not only in cell membranes, but also in nuclear (33), golgi (34), and mitochondria membranes (14,49). Surface immunofluorescence and immunoelectron microscopy studies indicate that the band 3-like proteins in nucleated cells participate in band 3 antibody-induced cell surface patching and capping (33). In red blood cells, band 3 mediates the exchange of anions (e.g., chloride, bicarbonate, phosphate, sulfate, iodide, bromide) across the membrane (7,39), maintains acid-base balance, and is the binding site for the glycolytic enzymes adolase (52), phosphofructokinase (20), and glyceraldehyde-3-P-dehydrogenase (35), and for hemoglobin (47). Water and glucose transport have also been attributed to band 3 (6, 26, 41, 53) although the latter is still open to question (42). Band 3 is the major transmembrane structural protein of the erythrocyte (57). The M r --95,000 band 3 molecule crosses the membrane
Choreoacanthocytosis
GABA
between 3 and 12 times (36, 45, 51). A cytoplasmic segment containing the amino terminus binds to band 2.1 (ankyrin) which attaches to the internal filamentous cytoskeleton (2). Senescent cell antigen, an aging protein that terminates the lifespan of cells, is derived from band 3 (21-32). Appearance of senescent cell antigen initiates IgG binding and cellular removal (1, 5, 21-23, 29). Oxidation generates senescent cell antigen in situ (29). Senescent cell antigen appears to be located on an extracellular portion of band 3 that includes most of the - 3 8 , 0 0 0 Da carboxyl terminal segment and - 3 0 % of the - 17,000 Da anion transport region (24). Although senescent cell antigen was first demonstrated on the surface of senescent human erythrocytes (red blood cells; rbc), it has since been demonstrated on the surface of lymphocytes, polymorphonuclear leukocytes, platelets, embryonic kidney cells, and adult liver cells (23, 25, 33), and isolated neurons and neurons of the central nervous system (27,33). We hypothesized that a defect in the anion channel creates an
IRequests for reprints should be addressed to Marguerite M. B. Kay, Professor of Medicine, Texas A&M University, Building 4, 1901 South First Street, Temple, TX 76504.
105
106
KAY, GOODMAN, LAWRENCE AND BOSMAN
imbalance between stimulatory and inhibitory impulses in diseases with choreiform movements thus permitting phenotypic expression of the disease. We tested this hypothesis by investigating band 3 structure and function in a patient with familial amyotrophic chorea with acanthocytosis, also called choreoacanthocytosis or neuroacanthocytosis. We selected choreoacanthocytosis because erythrocytes, which are readily obtained, are affected in this disease as well as the central nervous system. This disease is characterized by progressive chorea and erythrocyte acanthocytosis without a-13-1ipoproteinemia (3, 12, 40, 46). This disorder is similar to Huntington's disease in that there is an hereditary, adult onset progressive chorea with decreased lifespan. The age of onset is usually between 20 to 70 years. It differs from Huntington's disease in that acanthocytosis is present, deep tendon reflexes are decreased, and it is inherited in a recessive rather than dominant pattern (3, 8, 46). Choreoacanthocytosis has been reported to have an autosomal recessive mode of inheritance (3,46), but one variety may be inherited as an autosomal dominant (12, 17, 40). Mild to moderate mental deterioration can occur late in the disease. Neuropathology studies have revealed atropy and gliosis of basal ganglia (3). The caudate nucleus and putamen are the most severely affected. Mild diffuse gliosis in the white matter of the cerebral hemispheres, the cerebellum, and spinal cord gray matter is observed (3). Cortical atrophy and denervation atrophy of muscle is also present. Glutamic acid decarboxylase and choline acetyltransferase activity are normal in the cortex, caudate, and putamen (3). The proposita we studied has mild generalized cerebral atrophy and severe atrophy of the caudate nucleus as determined by computerized tomography scan of the brain with contrast. METHOD
Cell Separation Blood was obtained from the 41-year-old affected proposita, and her mother and brother. Her brother, age 43, has a history of seizures. Her mother does not show clinical manifestations of the disease. Whole blood was centrifuged at 1000 rpm for 15 min in a Sorvall RT6000 at 4°C to remove plasma and platelets. Pelleted cells were resuspended to the original volume with phosphate buffered saline without Ca ++ and Mg ÷+ and layered on Ficoll/ Hypaque (1). Mononuclear cells were removed by centrifugation on Ficoll/Hyopaque at 1200 rpm for 15 min in a Sorvall RT6000 at 4°C. Remaining white blood cells were removed with a pipet and red blood cells were separated into populations of different ages on Percoll gradients as previously described (1).
IgG Binding Assay The amount of IgG on cells was quantitated using 12~I-labeled Protein A according to the method of Yam et al. (56).
Sodium Dodecyl Sulfate (NaDodS04) Polyacrylamide Gel Electrophoresis Proteins were analyzed on 6-25% or 12-25% linear NaDodSO4/ polyacrylamide gradient gels using the discontinuous buffer system of Laemmli (36).
Antisera Rabbit antibodies to band 3 and antibody 980, a rabbit antibody to aged band 3 (precursor to senescent cell antigen), were prepared as previously described (30, 32, 33). Control sera consisted of normal, nonimmune rabbit sera, preimmunization sera, and anti-
bodies to band 3 absorbed with band 3.
Immunostaining of the Membrane Proteins Immunoautoradiography was performed by the immunoblotting technique of Towbin et al. (54) with the modifications described previously (30, 32, 33).
Anion Transport Measurements The "self-exchange flux" of sulfate was determined from the sulfate exchange at Donnan equilibrium, essentially following the method of Lepke and Passow (39) and Schnell et al. (48) with the modifications previously described (28-30). Chloride was omitted from the transport media because it is a competitive inhibitor of sulfate influx and stimulates sulfate effiux. Sulfate rather than chloride transport was measured because sulfate transport is slower thus allowing more accurate measurement. Measurements were made at six concentrations of sulfate between two and 30 mM, and at three time-points within the first eight minutes after the start of the assay. Km, the sulfate concentration at which half the maximal velocity, Vmax, is reached, was determined by extrapolation from the linear part of the Lineweaver-Burk plot.
Glucose Transport Measurements Characteristics of the glucose transport system (zero-trans-exit and infinite-trans-entry)were determined following the methods of Naftalin et al. (43) as previously described (43). Extensively washed erythrocytes were loaded with 50 mM glucose by incubation at 10% hematocrit in 20 mM phosphate/147 mM NaC1/50 mM glucose, pH 7.4 at 37°C for at least two hours, with a change of medium after one hour. For exit experiments, loosely packed cells were incubated with one microCurie D-[U-14C]-glucose (290 mCi/mmol, Amersham) or one microCurie 3-O-methylglucose (290 mCi/mmol, Amersham). Maximal transport velocity, V . . . . and K m of zero-trans-efflux (55), and the Vmax o f infinitetrans-entry was calculated using an integrated rate equation (55).
Isolation of the Brain Anion Transporter The anion transporter was isolated from bovine brain by affinity chromatography with the anion transport inhibitor 4acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) conjugated to Affigel 102 as described (44). Frozen bovine brain was homogenized in "homogenization buffer" (5 mM HEPES, 0.32 M sucrose, 5 mM imidazole, 5 mM benzamidine, 2 mM mercaptoethanol, 3 mM EGTA, 0.5 mM MgSO4, 5 mM glycerophosphate, 5 mM KF, 0.5 mM ZnSO4, 0.1 mg/ml leupeptin, 0.05 mg/ml pepstatin A, 0.1 mg/ml aprotinin, and 1 mM NAN3, pH 8.0). Membranes were obtained by centrifugation at 20,000 rpm in a Ti 65 rotor in a Sorvall OTD 65B centrifuge for 1 hr. Peripheral membrane proteins were removed with 0.1 M NaOH containing 1 mM diisopropylfluorophosphate (DFP), 1 mM EDTA, and 1 mM EGTA. Membranes were pelleted and washed with a low molarity buffer (5 mM sodium phosphate buffer with 1 mM EDTA, 1 mM EGTA, 1 mM DFP, pH 8.0). Membranes were dissolved in 100 mM "Iris HC1 with 1 mM EDTA, 20 mM mercaptoethanol, 1 mM dithiothreitol (DTr), and 2% NaDodSO 4, pH 8.0, diaiysed against column buffer (50 mM Tris acetate with 1 mM EDTA, 1 mM EGTA, 1 mM DFP, 0.03% lithium dodecyl sulfate, and 0.02% azide, pH 8.0) and loaded on an Affigel 102 SITS affinity column. The column was washed with column buffer until the optical density was the same as that of the column buffer as determined with an online spectrophotometer. The column was washed with column buffer containing 150 mM NaC1. The anion transporter
ANION CHANNEL IN NEUROLOGIC DISEASE
C
N
C
107
TABLE 1
N
A N I O N A N D G L U C O S E T R A N S P O R T BY ERYTHROCYTES FROM N O R M A L INDIVIDUALS A N D ERYTHROCYTES FROM C H O R E O A C A N T H O C Y T O S I S F A M I L Y MEMBERS I
Glucose Anion Individual
3
5 m
7
~
Wmax3
Km
Vmax2
Influx
Effiux
Control Proposita
1.1 -- 0.1 1.1 + 0.1
13.1 +_ 0.4 18.1 --_ 0.4?
15.0 -+ 1.2 9.3 + 1.7+
10.0 -- 0.9 2.0 -- 1.Or
Control Proposita
0.7 -- 0.1 0.9 --- 0.1
13.5 -+ 0.7 29.0 --- 1.07
10.1 _+ 0.9 9.8 -+ 1.2
16.5 -- 0.6 8.2 ----- 0 . 3 ? 4
Control Mother
1.2 --- 0.1 1.1 --- 0.2
11.0 - 1.1 16.8 ± 0.4?
9.8 ± 0.9 10.2 ± 0.6
18.2 ± 1.0 7.3 ± 0.4?
Control Brother
1.3 = 0.4 1.3 --- 0.3
11.4 ___ 2.0 15.2 ± 2.2 20.0 ± 2.7 14.3 ± 1.8:~ 13.0 ± 0.1:~ 14.0 ± 0.5?
~Results are presented as the mean ± one standard deviation; n = 6 - 8 . *Measurement approached limits of detection for this assay. ?p-
