Goals and Analytical Methodologies for Protein Disposition Studies

This chapter will present an overview of the goals of protein disposition studies and some of the analytical tools used to achieve those goals. The examples cited in the goals portion of this chapter will be briefly presented since these will be explored in greater depth in the ensuing chapters. Analytical issues are of major concern in protein disposition studies since many of the standard protein analysis methods do not individually provide a positive identification of the analyte. The question of which existing methodologies and analytical technologies are most productive will be addressed.

[1]  P. Familletti,et al.  Structural characterization of human interferon γ Heterogeneity of the carboxyl terminus , 1987 .

[2]  R. Kurzrock,et al.  Pharmacokinetics, Single-Dose Tolerance, and Biological Activity of Recombinant γ-lnterferon in Cancer Patients , 1985 .

[3]  J. Villafranca,et al.  Current research in protein chemistry : techniques, structure, and function , 1990 .

[4]  K. Kohnert,et al.  Radioiodination of peptide hormones and immunoglobulin preparations: comparison of the chloramine T and iodogen method. , 2009, Experimental and clinical endocrinology.

[5]  P. Ghezzi,et al.  Recombinant tumor necrosis factor depresses cytochrome P450-dependent microsomal drug metabolism in mice. , 1986, Biochemical and biophysical research communications.

[6]  G. Ooi Insulin-like growth factor-binding proteins (IGFBPs): more than just 1, 2, 3 , 1990, Molecular and Cellular Endocrinology.

[7]  A. Jones,et al.  Hepatic sequestration and biliary secretion of epidermal growth factor: evidence for a high-capacity uptake system. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[8]  B. Desbuquois,et al.  Receptor-Mediated Internalisation of Insulin, Glucagon and Growth Hormone in Intact Rat Liver. A Biochemical Study , 1980 .

[9]  J. Figueiredo,et al.  Renal filtration, transport, and metabolism of low-molecular-weight proteins: a review. , 1979, Kidney international.

[10]  J. Ketelslegers,et al.  Different effects of intermittent and continuous growth hormone (GH) administration on serum somatomedin-C/insulin-like growth factor I and liver GH receptors in hypophysectomized rats. , 1988, Endocrinology.

[11]  E. Rinderknecht,et al.  Natural human interferon-gamma. Complete amino acid sequence and determination of sites of glycosylation. , 1984, The Journal of biological chemistry.

[12]  L. Frohman,et al.  Concomitant association between high plasma levels of growth hormone and low hepatic mixed-function oxidase activity in the young rat. , 1974, Journal of Pharmacology and Experimental Therapeutics.

[13]  T. Iga,et al.  Kinetic analysis of the elimination process of human epidermal growth factor (hEGF) in rats. , 1989, Biochemical pharmacology.

[14]  M. Fukuda,et al.  Survival of recombinant erythropoietin in the circulation: the role of carbohydrates. , 1989 .

[15]  A. B. Jansen Total radioactivity half-lives. , 1979, Drug metabolism and disposition: the biological fate of chemicals.

[16]  E. Arquilla,et al.  Effect of Isologous and Autologous Insulin Antibodies on In Vivo Bioavailability and Metabolic Fate of Immune-Complexed Insulin in Lou/M Rats , 1989, Diabetes.

[17]  Norman A. Mazer,et al.  Pharmacokinetic and pharmacodynamic aspects of polypeptide delivery , 1990 .

[18]  B. Finkle,et al.  The Influence of Recombinant DNA-Derived Human and Murine Gamma Interferons on Mouse Hepatic Drug Metabolism , 1986 .

[19]  F. Komada,et al.  Fate of porcine and human insulin at the subcutaneous injection site. II. In vitro degradation of insulins in the subcutaneous tissue of the rat. , 1985, Journal of pharmacobio-dynamics.

[20]  R. Quirion Bioassays in modern peptide research , 1982, Peptides.

[21]  Gerhard Giebisch,et al.  The Kidney: Physiology and Pathophysiology , 1992 .

[22]  J. Mordenti,et al.  The Role of Pharmacokinetics and Pharmacodynamics in the Development of Therapeutic Proteins , 1991 .

[23]  R. Fernandez-Botran,et al.  A soluble, high-affinity, interleukin-4-binding protein is present in the biological fluids of mice. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[24]  A. Billiau The biology of the interferon system 1984: edited by H. Kirchner and H. Schellekens, Elsevier Science Publishers, 1985. Dfl. 300.00 (xv + 654 pages) ISBN 0 444 80661 X , 1985 .

[25]  W. Ward,et al.  Uptake and intracellular fate of [14C]sucrose-insulin in perfused rat livers. , 1988, The American journal of physiology.

[26]  M. Waters,et al.  Regulation of growth hormone (GH) bioactivity by a recombinant human GH-binding protein. , 1990, Endocrinology.

[27]  B. Beutler,et al.  Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo. , 1985, Journal of immunology.

[28]  B. Finkle,et al.  Effect of murine gamma-interferon on the mouse liver and its drug-metabolizing enzymes: comparison with human hybrid alpha-interferon. , 1985, Journal of interferon research.

[29]  T. Blundell,et al.  Chemistry, structure and function of insulin and related hormones , 1980, FEBS letters.

[30]  J. Parsons,et al.  EVIDENCE THAT PROTEASE INHIBITORS REDUCE THE DEGRADATION OF PARATHYROID HORMONE AND CALCITONIN INJECTED SUBCUTANEOUSLY , 1979, British journal of pharmacology.

[31]  T. Elsasser,et al.  Validation of a heterologous radioimmunoassay for insulin-like growth factor-I in bovine serum. , 1988, The Journal of endocrinology.

[32]  C. Hales,et al.  RADIOIMMUNOASSAY MAY OVERESTIMATE INSULIN IN NON‐INSULIN‐DEPENDENT DIABETICS , 1990, Clinical endocrinology.

[33]  B. Sobel,et al.  Biochemical determinants of clearance of tissue-type plasminogen activator from the circulation. , 1988, Circulation.

[34]  B. Keyt,et al.  Circulatory metabolism of recombinant tissue-type plasminogen activator in monkeys and rabbits , 1988 .

[35]  A. Kerlavage The Use of HPLC in receptor biochemistry , 1989 .

[36]  G. Firestone,et al.  Immunoprecipitation of proteins. , 1990, Methods in enzymology.

[37]  F. Lederer,et al.  Characterization of insulin degradation products generated in liver endosomes: In vivo and in vitro studies , 1990, Molecular and Cellular Endocrinology.

[38]  M. Shaw,et al.  A second, lower affinity growth hormone-binding protein in human plasma. , 1990, The Journal of clinical endocrinology and metabolism.

[39]  J. Henkin,et al.  Enhanced lytic efficacy of multiple bolus injections of tissue plasminogen activator in dogs. , 1990, Thrombosis research.

[40]  L. Creighton,et al.  Investigation by HPLC of the Catabolism of Recombinant Tissue Plasminogen Activator in the Rat , 1988, Thrombosis and Haemostasis.

[41]  M. Huang,et al.  Effects of three recombinant human leukocyte interferons on drug metabolism in mice. , 1982, Drug metabolism and disposition: the biological fate of chemicals.

[42]  R. Wills Clinical Pharmacokinetics of Interferons , 1990, Clinical pharmacokinetics.

[43]  T. Blaschke,et al.  Interferon reduces hepatic drug metabolism in vivo in mice. , 1985, Drug metabolism and disposition: the biological fate of chemicals.

[44]  S. Nielsen,et al.  Luminal uptake of insulin in renal proximal tubules. , 1988, Contributions to nephrology.

[45]  R. D. Wade ELISA and other solid phase immunoassays: Edited by D M Kemeny and S J Challacombe. pp 367. John Wiley, Chichester, UK. 1988. $47.95 ISBN 0-471-90982-3 , 1989 .

[46]  T. Waldmann,et al.  The role of the kidney in the metabolism of plasma proteins. , 1974, Nephron.

[47]  M. Mohler,et al.  Biodisposition and catabolism of tissue-type plasminogen activator in rats and rabbits , 1988 .

[48]  J. Jansson,et al.  Pulsatile intravenous growth hormone (GH) infusion to hypophysectomized rats increases insulin-like growth factor I messenger ribonucleic acid in skeletal tissues more effectively than continuous GH infusion. , 1988, Endocrinology.

[49]  T. Arakawa,et al.  Role of polycationic C-terminal portion in the structure and activity of recombinant human interferon-gamma. , 1986, The Journal of biological chemistry.

[50]  V. Jörgens,et al.  Absorption Kinetics and Biologic Effects of Subcutaneously Injected Insulin Preparations , 1982, Diabetes Care.

[51]  B. Dunbar,et al.  [51] Protein blotting and immunodetection , 1990 .

[52]  A. Tsuji,et al.  Application of HPLC in Disposition Study of A14-125I-Labeled Insulin in Mice , 1990, Diabetes.

[53]  F. Carone,et al.  Renal tubular processing of small peptide hormones. , 1982, The Journal of laboratory and clinical medicine.

[54]  L. Cecco,et al.  Luteinizing hormone determinations obtained with either a monoclonal and a polyclonal antibody radioimmunoassay and their correlations with clinical findings , 1990, Journal of endocrinological investigation.

[55]  Inhibition of degradation of insulin by ophthalamic acid and by a bovine pancreatic proteinase inhibitor. , 1979, The Biochemical journal.

[56]  T. Buchanan,et al.  In vivo kinetics of a covalent growth hormone-binding protein complex. , 1989, Metabolism: clinical and experimental.

[57]  F. Carone,et al.  Hydrolysis and transport of small peptides by the proximal tubule. , 1980, The American journal of physiology.

[58]  L. Boscato,et al.  Heterophilic antibodies: a problem for all immunoassays. , 1988, Clinical chemistry.

[59]  C. Kluft,et al.  Plasminogen activator inhibitors. , 1987, Blood.

[60]  T. Kamataki,et al.  Effects of hypophysectomy and growth hormone treatment on sex-specific forms of cytochrome P-450 in relation to drug and steroid metabolisms in rat liver microsomes. , 1986, Japanese journal of pharmacology.

[61]  J. Jansson,et al.  Intravenous growth hormone: growth responses to patterned infusions in hypophysectomized rats. , 1985, The Journal of endocrinology.

[62]  D. Drolet,et al.  An enzyme-linked immunosorbent assay to study human relaxin in human pregnancy and in pregnant rhesus monkeys. , 1989, The Journal of endocrinology.

[63]  Sørensen Jt,et al.  Use of a physiologic pharmacokinetic model of glucose homeostasis for assessment of performance requirements for improved insulin therapies. , 1982 .

[64]  M. Stolar,et al.  A specific growth hormone-binding protein in human plasma: initial characterization. , 1986, The Journal of clinical endocrinology and metabolism.

[65]  A. Kossiakoff,et al.  Tertiary structure is a principal determinant to protein deamidation. , 1988, Science.

[66]  L. Häggroth,et al.  Inhibition of the human tissue plasminogen activator in plasma from different species. , 1984, Thrombosis research.

[67]  D. Papachristodoulou,et al.  The binding and degradation of 125I-labelled insulin by rat kidney brush-border membranes , 1983 .

[68]  P. Bugelski,et al.  Internalization of Recombinant Tissue-Type Plasminogen Activator by Isolated Rat Hepatocytes is via Coated Pits , 1990, Thrombosis and Haemostasis.

[69]  K. Tsukamoto,et al.  Effects of Subchronic Treatment with Natural Human Interferons on Antipyrine Clearance and Liver Function in Patients with Chronic Hepatitis , 1990, Journal of clinical pharmacology.

[70]  F. Komada,et al.  Fate of porcine and human insulin at the subcutaneous injection site. I. Degradation and absorption of insulins in the rat. , 1985, Journal of pharmacobio-dynamics.

[71]  T. W. Haeften Clinical significance of insulin antibodies in insulin-treated diabetic patients. , 1989 .

[72]  P. Halban,et al.  The preparation of a semisynthetic tritiated insulin with a specific radioactivity of up to 20 Curies per millimole. , 1975, The Biochemical journal.

[73]  J. Parsons,et al.  Effects of two treatment regimes with synthetic human parathyroid hormone fragment on bone formation and the tissue balance of trabecular bone in greyhounds. , 1983, Endocrinology.

[74]  S. Nielsen,et al.  Insulin absorption in renal proximal tubules: a quantitative immunocytochemical study. , 1989, Journal of ultrastructure and molecular structure research.

[75]  N. White,et al.  Diabetes responsive to intravenous but not subcutaneous insulin: effectiveness of aprotinin. , 1981, The New England journal of medicine.

[76]  M. Cronin,et al.  The pharmacokinetics and pharmacodynamics of a human relaxin in the mouse pubic symphysis bioassay. , 1989, Endocrinology.

[77]  V. Bocci Catabolism of therapeutic proteins and peptides with implications for drug delivery , 1989 .

[78]  A C Herington,et al.  Identification and characterization of specific binding proteins for growth hormone in normal human sera. , 1986, The Journal of clinical investigation.

[79]  A. Doble,et al.  Degradation of atrial natriuretic peptides by an enzyme in rat kidney resembling neutral endopeptidase 24.11. , 1988, Biochemical pharmacology.

[80]  S. Nielsen,et al.  Basolateral tubular handling of insulin in the kidney. , 1988, Contributions to nephrology.

[81]  M. Rechler Insulin-like growth factor binding proteins. , 1993, Vitamins and hormones.

[82]  D. Longo,et al.  Resistance to recombinant interferon alfa-2a in hairy-cell leukemia associated with neutralizing anti-interferon antibodies. , 1988, The New England journal of medicine.

[83]  D. Finbloom Internalization and degradation of human recombinant interferon-gamma in the human histiocytic lymphoma cell line, U937: relationship to Fc receptor enhancement and antiproliferation. , 1988, Clinical immunology and immunopathology.

[84]  C. McMartin,et al.  Peptide hormones and their analogues: distribution, clearance from the circulation, and inactivation in vivo. , 1978, Pharmacological reviews.

[85]  B. Clarke,et al.  Influence of insulin antibodies on pharmacokinetics and bioavailability of recombinant human and highly purified beef insulins in insulin dependent diabetics. , 1985, British medical journal.

[86]  A. Bhown Protein/Peptide Sequence Analysis: Current Methodologies , 1987 .

[87]  A. Cerami,et al.  Cachectin (Tumor Necrosis Factor) , 1988 .

[88]  G. Farrell,et al.  Inhibition of antipyrine metabolism by interferon. , 1986, British journal of clinical pharmacology.

[89]  D. Shulman,et al.  Growth Hormone by Daily Injection in Patients Previously Treated for Growth Hormone Deficiency , 1990, Southern medical journal (Birmingham, Ala. Print).

[90]  O. D. de Noord,et al.  Effects of alpha-interferon on theophylline pharmacokinetics and metabolism. , 1989, British journal of clinical pharmacology.

[91]  C. Crowley,et al.  The Influence of Carbohydrate Structure on the Clearance of Recombinant Tissue-Type Plasminogen Activator , 1988, Thrombosis and Haemostasis.

[92]  R. Stach,et al.  Interaction of nerve growth factor with murine α‐macroglobulin , 1989 .

[93]  M. Sporn,et al.  Differential inhibition of transforming growth factor beta 1 and beta 2 activity by alpha 2-macroglobulin. , 1990, The Journal of biological chemistry.

[94]  J. L. Bailey,et al.  Techniques in protein chemistry , 1989 .

[95]  D. Carlo,et al.  Interferon-induced changes in pharmacokinetics and tumor uptake of 111In-labeled antimelanoma antibody 96.5 in melanoma patients. , 1988, Journal of the National Cancer Institute.

[96]  W. Daughaday,et al.  Studies on human growth hormone. II. The physiological disposition and metabolic fate of human growth hormone in man. , 1962, The Journal of clinical investigation.

[97]  G. Pessina,et al.  The role of liver in the catabolism of human alpha- and beta-interferon. , 1982, The Journal of general virology.

[98]  W. Kreutner,et al.  Potentiation of the hypoglycemic effect of insulin by thiorphan, an enkephalinase inhibitor. , 1984, European journal of pharmacology.

[99]  G. Ashwell,et al.  Carbohydrate-specific receptors of the liver. , 1982, Annual review of biochemistry.

[100]  V. Lee Enzymatic barriers to peptide and protein absorption. , 1988, Critical reviews in therapeutic drug carrier systems.

[101]  A. Varshavsky,et al.  In vivo half-life of a protein is a function of its amino-terminal residue. , 1986, Science.

[102]  D. Goeddel,et al.  Expression of human immune interferon cDNA in E. coli and monkey cells , 1982, Nature.

[103]  R. Baughman,et al.  Pharmacokinetics and tissue distribution of recombinant human tumor necrosis factor-alpha in mice. , 1988, Drug metabolism and disposition: the biological fate of chemicals.

[104]  L. Goodman,et al.  The Pharmacological Basis of Therapeutics , 1941 .

[105]  B. Beaufrère,et al.  Recycling of an amino acid label with prolonged isotope infusion: implications for kinetic studies. , 1985, The American journal of physiology.

[106]  D. Clemmons The Role of Insulin-Like Growth Factor Binding Proteins in Controlling the Expression of IGF Actions , 1989 .

[107]  C. McClain,et al.  Interleukin-1 (IL-1) depresses cytochrome P450 levels and activities in mice. , 1987, Life sciences.

[108]  S. Rogers,et al.  Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. , 1986, Science.

[109]  D. Chan,et al.  Immunoassay : a practical guide , 1987 .

[110]  R. Clark,et al.  Growth induced by pulsatile infusion of an amidated fragment of human growth hormone releasing factor in normal and GHRF-deficient rats , 1985, Nature.

[111]  G. Singh,et al.  Homogeneous interferon from E. coli depresses hepatic cytochrome P-450 and drug biotransformation. , 1982, Biochemical and biophysical research communications.

[112]  R. Billings,et al.  CELLULAR CATABOLISM OF RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL HIGH AFFINITY UPTAKE SYSTEM ON RAT HEPATOCYTES , 1987, Thrombosis and Haemostasis.

[113]  T. V. van Berkel,et al.  Characterization of the interaction in vivo of tissue-type plasminogen activator with liver cells. , 1988, The Journal of biological chemistry.

[114]  B. Sobel,et al.  Interactions of tissue-type plasminogen activator with plasma inhibitors and their pharmacologic implications. , 1988, Circulation.

[115]  R. Coffey,et al.  Hepatic processing of transforming growth factor beta in the rat. Uptake, metabolism, and biliary excretion. , 1987, The Journal of clinical investigation.

[116]  P. Matsudaira A Practical guide to protein and peptide purification for microsequencing , 1989 .

[117]  A. Kakinuma,et al.  Differential purification by immunoaffinity chromatography of two carboxy-terminal portion-deleted derivatives of recombinant human interferon-gamma from Escherichia coli. , 1987, Journal of interferon research.

[118]  John T. Wilson Alteration of Normal Development of Drug Metabolism by Injection of Growth Hormone , 1970, Nature.

[119]  E. Hersh,et al.  Modification of human leukocyte interferon pharmacology with a monoclonal antibody. , 1985, Cancer research.

[120]  S. Adibi,et al.  Specificity and mechanism of influence of amino acid residues on hepatic clearance of oligopeptides. , 1988, The Journal of biological chemistry.

[121]  L. Benet,et al.  Pharmacokinetics and protein therapeutics , 1990 .

[122]  J. Whicher,et al.  Electrophoresis and densitometry of serum and urine in the investigation and significance of monoclonal immunoglobulins , 1990, Electrophoresis.

[123]  T. Buchanan,et al.  The effect of circulating growth hormone-binding protein on metabolic clearance, distribution, and degradation of human growth hormone. , 1987, The Journal of clinical endocrinology and metabolism.

[124]  J. Merryweather,et al.  Isolation from adult human serum of four insulin-like growth factor (IGF) binding proteins and molecular cloning of one of them that is increased by IGF I administration and in extrapancreatic tumor hypoglycemia. , 1990, The Journal of biological chemistry.

[125]  P. Halban,et al.  Distribution and metabolism of intravenously injected tritiated insulin in rats. , 1979, Metabolism: clinical and experimental.

[126]  J. Koehn,et al.  Degradation of atrial natriuretic factor by kidney cortex membranes. Isolation and characterization of the primary proteolytic product. , 1987, The Journal of biological chemistry.

[127]  T. Ulich,et al.  Effect of IgG Subclasses on In Vivo Bioavailability and Metabolic Fate of Immune-Complexed Insulin in Lewis Rats , 1987, Diabetes.

[128]  D. Bier Intrinsically difficult problems: the kinetics of body proteins and amino acids in man. , 1989, Diabetes/metabolism reviews.

[129]  Charles Ward Parker,et al.  Radioimmunoassay of biologically active compounds , 1976 .

[130]  J. Gauldie,et al.  Alpha 2-macroglobulin and serum preferentially counteract the mitoinhibitory effect of transforming growth factor-beta 2 in rat hepatocytes. , 1990, Laboratory investigation; a journal of technical methods and pathology.

[131]  M. Binoux,et al.  Analysis of serum insulin-like growth factor binding proteins using western blotting: use of the method for titration of the binding proteins and competitive binding studies. , 1986, Analytical biochemistry.

[132]  T. Tuvemo What is the Best Mode of Growth Hormone Administration? , 1989, Acta paediatrica Scandinavica. Supplement.

[133]  F. Komada,et al.  Pharmaceutical approach to subcutaneous dosage forms of insulin. , 1983, Journal of pharmaceutical sciences.

[134]  U. Lewis,et al.  Enhancement of the growth promoting activity of human growth hormone. , 1975, Biochemical and biophysical research communications.

[135]  B. Howard,et al.  Effects of intraperitoneal versus subcutaneous insulin administration on lipoprotein metabolism in type I diabetes. , 1990, Metabolism: clinical and experimental.

[136]  P. Halban,et al.  The Effect of Aprotinin on the Absorption of Subcutaneously Injected Regular Insulin in Normal Subjects , 1980, Diabetes.

[137]  J. Moore,et al.  Metabolism, Endocrinology and ImmunologyInfluence of Various Purified Interferons on Effects of Drugs in Mice , 1983 .

[138]  V. Bocci Metabolism of protein anticancer agents. , 1987, Pharmacology & therapeutics.

[139]  E. Góth,et al.  [Studies on human growth hormone]. , 1963, Orvosi hetilap.

[140]  N. LaRusso Proteins in bile: how they get there and what they do. , 1984, The American journal of physiology.

[141]  G. Bolli The pharmacokinetic basis of insulin therapy in diabetes mellitus. , 1989, Diabetes research and clinical practice.

[142]  Deborah L. Higgins,et al.  Tissue plasminogen activator: the biochemistry and pharmacology of variants produced by mutagenesis. , 1990, Annual review of pharmacology and toxicology.

[143]  F. Bidlingmaier,et al.  Circulating antibodies to mouse monoclonal immunoglobulins caused false-positive results in a two-site assay for alpha-fetoprotein. , 1989, Clinical chemistry.

[144]  M. Muscettola,et al.  The lymphatic route--III. Pharmacokinetics of human natural interferon-beta injected with albumin as a retarder in rabbits. , 1986, General pharmacology.

[145]  K. Hall,et al.  Growth factors : from genes to clinical application , 1990 .

[146]  Ajit K. Thakur,et al.  New Trends in Pharmacokinetics , 1991, NATO ASI Series.

[147]  W. Hagopian,et al.  Hepatic glucagon metabolism. Correlation of hormone processing by isolated canine hepatocytes with glucagon metabolism in man and in the dog. , 1987, The Journal of clinical investigation.

[148]  J. Gosling A decade of development in immunoassay methodology. , 1990, Clinical chemistry.

[149]  J. Heersche,et al.  Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: differential effects of intermittent and continuous administration. , 1982, Endocrinology.

[150]  R N Bergman,et al.  Assessment of insulin sensitivity in vivo. , 1985, Endocrine reviews.

[151]  A. Katz,et al.  Metabolism of polypeptide hormones by the normal kidney and in uremia. , 1978, Nephron.

[152]  E. Goldwasser,et al.  Purification of human erythropoietin. , 1977, The Journal of biological chemistry.

[153]  J. Playfair,et al.  Serum IL-2 inhibitor in mice. I. Increase during infection. , 1985, Immunology.

[154]  H. Burger,et al.  Metabolic clearance rate of radioiodinated human growth hormone in man. , 1969, The Journal of clinical investigation.