Preparation Of Hemolysate Research Articles

Metabolic incorporation of H218 O into specific glucose-6-phosphate oxygens by red-blood-cell lysates as observed by 13 C isotope-shifted NMR signals.

Water enriched with oxygen-18 (H218 O) is a potential tracer for evaluating the sources of glucose and glycogen synthesis since it is incorporated into specific sites of glucose-6-phosphate via specific enzyme-mediated exchange/addition mechanisms. Unlike 2 H, 18 O does not experience significant isotope effects for any of these processes. Therefore, H218 O might provide more precise estimates of endogenous carbohydrate synthesis compared with deuterated water provided that positional 18 O enrichments of glucose can be measured. As a proof of concept, H218 O was incorporated into a well characterized hemolysate model of sugar phosphate metabolism and 13 C NMR was applied to quantify positional 18 O enrichment of glucose-6-phosphate oxygens. Human erythrocyte hemolysate preparations were incubated overnight at 37 °C with a buffer containing sugar phosphate precursors and 20% (n = 5) and 80% (n = 1) H218 O. Enrichment of glucose-6-phosphate was analyzed by 13 C NMR analysis of 18 O-shifted versus unshifted signals following derivatization to monoacetone glucose (MAG). 13 C NMR MAG spectra from hemolysate revealed resolved 18 O-shifted signals in Positions 1-5. Mean 18 O enrichments were 16.4 ± 1.6% (Position 1), 13.3 ± 1.3% (Position 2), 4.1 ± 1.1% (Position 3), 12.6 ± 0.8% (Position 4), 10.7 ± 1.4% (Position 5), and no detectable enrichment of Position 6. No 18 O-shifted glucose-6-phosphate signals were detected in preparations containing sugar phosphate precursors only. H218 O is incorporated into Positions 1-5 of glucose-6-phosphate in accordance with spontaneous aldose hydration and specific enzymatic reaction mechanisms. This provides a basis for its deployment as a tracer for glucose and glycogen biosynthesis.

In vitro effects of some metal ions on glutathione reductase in the gills and liver of Capoeta trutta

Many aquatic environmental problems have arisen in consequence of contamination of water by toxic metals and organic pollutants in the present age of technology. Metals play vital roles in enzyme activities and other metabolic events due to their bioaccumulative and nonbiodegradable properties among aquatic pollutants. The aim of this study was to evaluate the inhibitory effects of some metal ions (Ag+, Cu2+, Co2+, Ni2+, Pb2+ and Zn2+) on Capoeta trutta gill and liver glutathione reductase (EC: 1.8.1.7; GR). For this purpose, initially, GR was purified from C. trutta gill and liver. Purification procedure consisted of three steps; preparation of hemolysate, ammonium sulphate precipitation and 2’, 5’-ADP Sepharose 4B affinity chromatography. Using this procedure, C. turtta gill GR, having the specific activity of 19.111 EU/mg proteins, was purified with a yield of 38.8% and 910.05-fold; C. trutta liver GR, having the specific activity of 16.167 EU/mg proteins, was purified with a yield of 21.1% and 734.86-fold. The purity of the enzymes was checked on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and each purified enzyme showed a single band on the gel. In addition, inhibitory effects of some metal ions (Ag+, Cu2+, Co2+, Ni2+, Pb2+ and Zn2+) on GR from gill and liver were investigated in vitro. Ki constants and IC50 values for metal ions which showed inhibition effects were determined by Lineweaver-Burk graps and plotting activity % vs. [I]. In conclusion, IC50 values for fish gill GR were 0.000625, 0.153, 0.220, 0.247 and 0.216 mM and Ki constants for fish gill GR were 0.00045 ± 0.00008, 0.128 ± 0.036, 0.182 ± 0.138, 0.482 ± 0.219 and 0.112 ± 0.047 mM for Ag+, Cu2+, Co2+, Ni2+, Pb2+ and Zn2+, respectively. IC50 values for fish liver GR were 0.000437, 0.217, 0.185, 0.355 and 0.349 mM and Ki constants for fish liver GR were 0.00025 ± 0.00013, 0.532 ± 0.146, 0.123 ± 0.066, 0.093 ± 0.020 and 0.151 ± 0.084 mM for Ag+, Cu2+, Co2+, Ni2+, Pb2+ and Zn2+, respectively. In vitro inhibition rank order was determined as Ag+ > Co2+ > Zn2+ > Ni2+ > Pb2+ for fish gill GR; Ag+ > Cu2+ > Co2+ > Pb2+ > Ni2+ for fish liver GR. From these results, we showed that Ag+ metal ion is the most potent inhibitor of GR enzyme on gill and liver tissues.

Demonstration of glucose-6-phosphate hydrogen 5 enrichment from deuterated water by transaldolase-mediated exchange alone.

Enrichment of glucose position 5 (H5) from deuterated water ((2)H2O) is widely used for quantifying gluconeogenesis. Exchanges of hexose and triose phosphates mediated by transaldolase have been postulated to enrich H5 independently of gluconeogenesis, but to date this mechanism has not been proven. We determined the enrichment of glucose-6-phosphate (G6P), the immediate precursor of endogenously produced glucose, from (2)H2O in erythrocyte hemolysate preparations. Here, transaldolase exchange is active but gluconeogenesis is absent. Hemolysates were prepared from human erythrocytes and incubated with a buffer containing 5% [U-(13)C]G6P, unlabeled fructose 1,6-bisphosphate, and 10% (2)H2O. G6P (2)H-enrichment and (13)C-isotopomer distributions were analyzed by (2)H and (13)C NMR following derivatization to monoacetone glucose. (2)H NMR analysis revealed high (2)H-enrichment of G6P hydrogens 2, 4, and 5; low enrichment of hydrogen 3, and residual enrichments of hydrogens 1, 6R, and 6S. (13)C NMR isotopomer analysis revealed that [U-(13)C]G6P was converted to [1,2,3-(13)C3]G6P, a predicted product of transaldolase-mediated exchange, as well as [1,2-(13)C2]G6P and [3-(13)C]G6P, predicted products of combined transaldolase and transketolase exchanges. Hydrogen 5 of G6P was enriched from (2)H2O through exchanges mediated by transaldolase. These studies prove that G6P can be enriched in hydrogen 5 by (2)H2O independently of gluconeogenesis.

The effect of some antineoplastic agents on glutathione S-transferase from human erythrocytes

Glutathione S-transferase was purified from human erythrocytes and effects of some antineoplastic agents were investigated on the enzyme activity. The purification procedure was composed of Glutathione-Agarose affinity chromatography after preparation of erythrocytes hemolysate. Using this procedure, the enzyme, having the specific activity of 16.00 EU/mg proteins, was purified 1143-fold with a yield of 80%. The purified enzyme showed a single band on the SDS-PAGE. The effects of paclitaxel, cyclophosphamide, and gemcitabine, are antineoplastic agents, were examined on the in vitro enzyme activity of glutathione S-transferase and were determined to be inhibitors for the enzyme. IC50 values were 0.23 mM for paclitaxel, 5.57 mm for cyclophosphamide, and 6.35 mM for gemcitabine. These constants were 0.182 ± 0.028 mM and 0.162 ± 0.062 mM for paclitaxel, 6.97 ± 0.49 mM and 10.50 ± 5.43 mM for cyclophosphamide, and 6.71 mM and 7.93 mM for gemcitabine, with GSH and CDNB substrates, respectively. Inhibition types of all inhibitors were noncompetitive.

In vitro effects of some drugs on human erythrocyte glutathione reductase

The effects of ketotifen, meloxicam, phenyramidol–HCl and gadopentetic acid on the enzyme activity of GR were studied using human erythrocyte glutathione reductase (GR) enzymes in vitro. The enzyme was purified 209-fold from human erythrocytes in a yield of 19% with 0.31 U/mg. The purification procedure involved the preparation of haemolysate, ammonium sulphate precipitation, 2′′,5′-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies. In the in vitro studies, IC50 values and Ki constants were 0.012 mM and 0.0008 ± 0.00021 mM for ketotifen; 0.029 mM and 0.0061 ± 0.00127 mM for meloxicam; 0.99 mM and 0.4340 ± 0.0890 mM for phenyramidol–HCl; 138 mM and 28.84 ± 4.69 mM for gadopentetic acid, respectively, showing the inhibition effects on the purified enzyme. Phenyramidol–HCl showed competitive inhibition, whereas the others showed non-competitive inhibition.

Evaluation of the free α‐hemoglobin pool in red blood cells: A new test providing a scale of β‐thalassemia severity

β-Thalassemias are characterized by an imbalance of globin chains with an excess of α-chains which precipitates in erythroid precursors and red blood cells (RBCs) leading to inefficient erythropoiesis. The severity of the disease correlates with the amount of unpaired α-chains.Our goal was to develop a simple test for evaluation of the free α-hemoglobin pool present in RBC lysates. Alpha-Hemoglobin Stabilizing Protein (AHSP), the chaperone of α-Hb, was used to trap excess a-Hb. A recombinant GST-AHSP fusion protein was bound to an affinity micro-column and then incubated with hemolysates of patients. After washing, the α-Hb was quantified by spectrophotometry in the elution fraction. This assay was applied to 54 patients: 28 without apparent Hb disorder, 20 β-thalassemic and 6 α-thalassemic. The average value of free α-Hb pool was 93 ± 21 ppm (ng of free α-Hb per mg of Hb subunits)in patients without Hb disorder, while it varies from 119 to 1,756 ppm, in β-thalassemic patients and correlated with genotype. In contrast,the value of the free α-Hb pool was decreased in α-thalassemic patients (65 ± 26 ppm). This assay may help to characterize β-thalassemia phenotypes and to follow the evolution of the globin chain imbalance(α/β+γ ratio) in response to treatment.

In vitro effects of some drugs on human erythrocyte glutathione reductase

The effects of dacarbazine, thiocolchicoside, methotrexate, olanzapine, pantoprazole sodium, and 5-fluorouracil on the enzyme activity of glutathione reductase (GR) were studied using human erythrocyte GR enzymes in in vitro studies, respectively. Materials and methods: The enzyme was purified 3333-fold from human erythrocytes in a yield of 44.44% with 4.0 U/mg. The purification procedure involved the preparation of hemolysate, ammonium sulfate precipitation, 2', 5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. SDS-PAGE showed a single band for enzyme. Purified enzyme was used in the in vitro studies. Results: In the in vitro studies, IC50 values and Ki constants were 0.014 mM and 0.0121 ± 0.0026 mM for dacarbazine, and 0.054 mM and 0.0135 ± 0.0024 mM for thiocolchicoside, 0.31 mM and 0.1213 ± 0.040 mM for methotrexate, 0.35 mM and 0.3173 ± 0.0981 mM for olanzapine, 0.94 mM and 0.5413 ± 0.0728 mM for pantoprazole sodium, and 98.57 mM and 69.611 ± 9.690 mM for 5-fluorouracil, showing the inhibition effects on the purified enzyme. Inhibition types were non-competitive for dacarbazine, methotrexate, olanzapine, and 5-fluorouracil and competitive for thiocolchicoside and pantoprazole sodium. Conclusion: All the drugs indicated the inhibitory effects on the enzyme.

Effects of some antibiotics on glutathione reductase activities from human erythrocytes in vitro and from rat erythrocytes in vivo

The effects of streptomycin sulfate, gentamicin sulfate, thiamphenicol, penicillin G, teicoplanin, ampicillin, cefotaxime, and cefodizime on the enzyme activity of glutathione reductase (GR) were studied using human and rat erythrocyte GR enzymes in in vitro and in vivo studies, respectively. The enzyme was purified 5,342-fold from human erythrocytes in a yield of 29% with 50.75 U/mg. The purification procedure involved the preparation of hemolysate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies, and rat erythrocyte hemolysate was used in the in vivo studies. In the in vitro studies, I50 and Ki values were 12.179 mM and 6.5123±4.1139 mM for cefotaxime, and 1.682 mM and 0.7446±0.2216 mM for cefodizime, respectively, showing the inhibition effects on the purified enzyme. Inhibition types were noncompetitive for cefotaxime and competitive for cefodizime. In the in vivo studies, 300 mg/kg cefotaxime and 1000 mg/kg cefodizime when administered to rats inhibited enzyme activity during the first 2 h (p<0.01). Cefotaxime led to increased enzyme activity at 4 h (p<0.05), but neither cefotaxime nor cefodizime had any significant inhibition or activation effects over 6 h (p>0.05).

Effects of some antibiotics on glutathione reductase from bovine erythrocytes

This paper describes effects of some antibiotics on glutathione reductase in vitro. Glutathione reductase was purified from bovine erythrocytes. Purification procedure consisted of four steps; preparation of hemolysate, ammonium sulphate precipitation, 2&amp;rsquo;,5&amp;rsquo;-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtra&amp;shy;tion chromatography. As a result of these four consecutive procedures, the enzyme having the specific activity of 62.5 EU/mg proteins was purified 31 250-fold with a yield of 11.39%. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). The effects of eight different antibiot&amp;shy;ics-streptomycin sulphate, gentamicin sulphate, netilmicin, teicoplanine, thiamphenicol, ampicillin, cefotaxime, and cefodizime- were investigated on the purified enzyme. Six of these antibiotics (streptomycin sulphate, gentamicin sulphate, netilmicin, teicoplanine, thiamphenicol, and ampicillin) increased the activity of glutathione reductase with increase in their concentrations while the two (cefotaxime and cefodizime) inhibited the enzyme activity. I50 values were 7.713 mM and 1.954 mM, and Ki constants were 11.011 mM and 8.956 mM for cefotaxime and cefodizime, respectively. Their inhibition types were competitive.

Inhibitory Effects of Some Antibiotics on Activity of Carbonic Anhydrase from Rainbow Trout Erythrocytes in Vitro and in Vivo

Abstract Carbonic anhydrase (CA) from erythrocytes of rainbow trout Oncorhynchus mykiss was purified by affinity chromatography through a process comprising hemolysate preparation, sepharose- 4B-l-tyrosine-sulfanylamide affinity gel chromatography, and dialysis. Then the effects of three antibiotics—sodium ampicillin, streptomycin, and trimethoprim plus sulfamethoxazole—on this enzyme were investigated in vitro and in vivo. Inhibition effects were determined in vitro by means of the Lineweaver–Burk and activity–antibiotic regression graphs. The dissociation constant of the enzyme inhibitor complex (Ki ) and 50% inhibitory values were 2.16 ± 0.40 and 1.87 mM for sodium ampicillin, 7.40 ± 0.310 and 5.21 mM for streptomycin, and 18.50 ± 0.35 and 9.74 mM for trimethoprim–sulfamethoxazole. The in vivo experiment demonstrated that CA was significantly inhibited by trimethoprim–sulfamethoxazole but not by sodium ampicillin and streptomycin.

Comparison of Five Automated Serum and Whole Blood Folate Assays

Serum and whole blood folate measurements are used to establish folate deficiency. Most methods used in clinical laboratories are automated, nonisotopic methods that use folate-binding protein. Linearity, imprecision, and method comparison studies, including serum and whole blood hemolysates, were performed with the Access, Advia Centaur, ARCHITECT i2000, Elecsys 2010, and IMMULITE 2000 methods. The QuantaPhase II radioassay served as the comparison method. (Proprietary information is given in the text.) The Access and IMMULITE 2000 methods had higher systematic errors in linearity studies than the other 3 methods. The imprecision of all methods was acceptable (coefficient of variation, <10%) even at low folate concentrations with the exception of the Elecsys 2010 (coefficient of variation, 16%). Method comparison studies using serum samples revealed calibration differences between the Access and Elecsys 2010 methods and the comparison method. Method comparison studies using whole blood samples showed poorer agreement between each of the automated methods and the comparison method than was seen with serum samples. The ARCHITECT i2000 folate assay demonstrated the best analytic performance. The poor agreement seen with whole blood hemolysates likely is due to calibration differences and differences in hemolysate preparation conditions.

Purification of glucose 6-phosphate dehydrogenase from Buffalo ( Bubalus bubalis) erythrocytes and investigation of some kinetic properties

Glucose 6-phosphate dehydrogenase (G6PD) was purified from buffalo ( Bubalus bubalis) erythrocytes and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: hemolysate preparation and 2 ′,5 ′-ADP–Sepharose 4B affinity gel chromatography. Thanks to the two consecutive procedures, the enzyme, having a specific activity of 69.7 EU/mg proteins, was purified 650-fold with a yield of 31%. Optimal pH, stable pH, optimal temperature, molecular weight, and K M and V max values for NADP + and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. In addition, K i values and the type of inhibition were determined by means of Lineweaver–Burk graphs obtained for such inhibitors as ATP, ADP, NADPH, and NADH.

Effects of Chloramine-T And CuSO4 On Enzyme Activity Of Glucose 6-Phosphate Dehydrogenase From Rainbow Trout (Oncorhynchus Myskiss) Erythrocytes In Vitro An In Vivo

Traditional treatments of parasitic and bacterial diseases are based on chemotherapeutic com- pounds such as chloramine-T and CuSO4. Although many compounds are used in fish treat- ments, their undesirable effects are not known. In this study, glucose 6-phosphate dehydroge- nase (G6PD) was purified from rainbow trout (Oncorhynchus mykiss) erythrocytes by hemolysate preparation, ammonium sulfate precipitation and 2’, 5’-ADP Sepharose 4B affinity gel chromatography in a single day. The enzyme, with a specific activity of 14.51 EU/mg protein, was purified 906.8-fold with a yield of 70.38%. To check the purity of the enzyme, SDS poly- acrylamide gel electrophoresis was performed, which showed a single band. The effects of chlo- ramine-T and copper sulfate (CuSO4) on the G6PD were investigated in vitro. Chloramine-T and CuSO4 had inhibitory effects on the enzyme. I50 values of the chemotherapeutic compounds were determined by plotting percent activity and Ki values, and types of inhibition were deter- mined for each compound by means of Lineweaver-Burk graphs. In vivo studies showed that G6PD in rainbow trout erythrocytes was significantly inhibited by CuSO4 in one hour but not inhibited by chloramine-T.

Unprecedented overmetabolism of a porphyrinogen substrate by coproporphyrinogen oxidase

Harderoporphyrinogen-I is metabolized by avian hemolysate preparations of coproporphyrinogen oxidase to give a trivinylic product; this unprecedented ‘overmetabolism’ of the porphyrinogen substrate provides strong support for a proposed model of the active site of this poorly understood enzyme.

PURIFICATION OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE FROM CHICKEN ERYTHROCYTES. INVESTIGATION OF SOME KINETIC PROPERTIES

ABSTRACT Glucose 6-phosphate dehydrogenase (G6PD) was purified from chicken erythrocytes, and some characteristics of the enzyme were investigated. The purification procedure was composed of three steps: hemolysate preparation, ammonium sulfate precipitation, and 2′,5′-ADP Sepharose 4B affinity gel chromatography. Thanks to the three consecutive procedures, the enzyme, having the specific activity of 20.862 EU/mg proteins, was purified with a yield of 54.68% and 9,150-fold. Optimal pH, stable pH, optimal temperature, molecular weight, and KM and Vmax values for NADP+ and glucose 6- phosphate (G6-P) were also determined for the enzyme. In addition, Ki values and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as ATP, ADP, NADH, and NADPH.

Purification and Characterization of Glucose 6-phosphate Dehydrogenase from Sheep Erythrocytes and Inhibitory Effects of some Antibiotics on Enzyme Activity

Glucose 6-phosphate dehydrogenase (d -glucose 6-phosphate: NADP + oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2′, 5′-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and K M and V max values for NADP + and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4°C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I 50 values were determined from Activity %-[Drug] graphs and K i values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.

Synthetic substrates for coproporophyrinogen oxidase: mesoporphyrinogen-VI revisited

In contradiction to a previous study, mesoporphyrinogen-VI, a dicarboxylate analog of coproporphyrinogen-III, has been shown to be converted to a monovinylic product by chicken red cell hemolysate preparations of coproporphyrinogen oxidase.

Erythrocytic glucose-6-phosphate dehydrogenase measured by a differential pH technique.

We propose a new quantitative electrochemical method for determining glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity in purified erythrocytes or in whole blood, based on measurement of the pH change caused by oxidation of glucose 6-phosphate to 6-phosphogluconic acid, with simultaneous reduction of NADP+ to NADPH + H+. No sample pretreatment (e.g., preparation of hemolysate) is needed, and the automatic correction for sample blanks obviates interference from 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The method is simple and fast, and the standard curve is linear to at least 2200 U/L at 37 degrees C. The within-day CV was 3.9% for activities in healthy individuals (mean value 1204 U/L), and 10% for deficient ones (classified as belonging to class II, mean value 407 U/L). Results (y) correlated well with those obtained with the WHO-recommended method (x): y = 1.13x + 0.02 (r = 0.971) for purified erythrocytes. Normal reference intervals are reported for the enzyme in purified erythrocytes and in whole blood.

1] Hexokinase from rabbit red blood cells

This chapter describes the purification and the properties of hexokinase found in rabbit reticulocytes. This enzyme shows a marked decrease during cell aging, and two distinct molecular forms (hexokinase Ia and Ib) are found in reticulocytes, but only one of these is consistently present in the mature rabbit red cell. With glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in excess, the rate of glucose phosphorylation is equivalent to the rate of NADP+ reduction, which is measured by the absorbance increase at 340 nm. For each molecule of glucose utilized, two molecules of NADP+ are reduced. Purification procedure for hexokinase from rabbit red blood cells involves preparation of hemolysate, batch treatment with DEAE-Sephadex A-50, ammonium sulfate fractionation, affinity chromatography on Sepharose-N-amino-hexanoylglucosamine column, and preparative polyacrylamide gel electrophoresis. Both hexokinase Ia and Ib can be stabilized by glucose, fructose, glycerol, and sulfhydryl protecting agents, as can hexokinase from mature rabbit red cells.

33] Transketolase from human red blood cells

This chapter describes a method of measuring transketolase activity during purification. Transketolase activity is measured by a coupled assay based on the spectrophotometric measurement of changes in NADH concentration in which triosephosphate isomerase converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate, which is in turn converted to glycerol-3-phosphate by glycerol-3-phosphate dehydrogenase with the concomitant oxidation of NADH. The disappearance of NADH is measured by the decrease of A 340 . Because transketolase activity is stimulated in some hemolysates by thiamine pyrophosphate, this cofactor is included in the assay. The assay involves the preparation of hemolysates and purification of transketolase by chromatography on diethylaminoethyl (DEAE)-cellulose or chromatography on hydroxyapatite. As an alternative purification procedure, the outlet of the DEAE-cellulose column may be coupled directly to the inlet of the hydroxyaparite column with a three-way valve positioned between the two columns for sampling.