Plasma Clearance Studies Research Articles

Plasma Clearance of Hemoglobin and Haptoglobin in Mice and Effect of CD163 Gene Targeting Disruption

In humans, plasma haptoglobin (Hp) and the macrophage receptor CD163 promote a fast scavenging of hemoglobin (Hb). In the present study, we have compared the mouse and human CD163-mediated binding and uptake of Hb and HpHb complex in vitro and characterized the CD163-mediated plasma clearance of Hb in CD163 gene knockout mice and controls. Contrary to human Hp, mouse Hp did not promote high-affinity binding to CD163. This difference between mouse and man was evident both by analysis of the binding of purified proteins and by ligand uptake studies in CD163-transfected cells. Plasma clearance studies in mice showed a fast clearance (half-life few minutes) of fluorescently labeled mouse Hb with the highest uptake in the kidney and liver. HPLC analysis of serum showed that the clearance curve exhibited a two-phase decay with a faster clearance of Hb than plasma-formed HpHb. In CD163-deficient mice, the overall clearance of Hb was slightly slower and followed a one-phase decay. In conclusion, mouse Hp does not promote high-affinity binding of mouse Hb to CD163, and noncomplexed mouse Hb has a higher CD163 affinity than human Hb has. Moreover, CD163-mediated uptake in mice seems to only account for a part of the Hb clearance. The new data further underscore the fact that the Hp system in man seems to have a broader and more sophisticated role. This has major implications in the translation of data on Hb metabolism from mouse to man.

Abstract 355: Comparison of biodistribution and accumulation of a thermally responsive polypeptide with and without a cell-penetrating peptide

Abstract Elastin-like Polypeptide (ELP) is a macromolecular carrier for various therapeutic peptides and has the ability to penetrate tumor vasculature. This penetration ability is mediated by a cell-penetrating peptide. The polymer is soluble at normal physiological temperature but aggregates above 40 °C. Peptides without carriers have very short half-lives, so ELP not only allows for longer circulation time, but its thermal properties allow the peptide to accumulate inside the tumor when heated. Bac-ELP-p21 is an ELP conjugate that uses the CPP Bac to penetrate the nucleus and the therapeutic peptide p21 to inhibit the cell cycle at G1/S. ELP-p21 lacks the CPP and therefore the cell penetrating capabilities. Both of these constructs can be actively targeted to the tumor site using local hyperthermia. In this study, tumor accumulation and biodistribution of fluorescently labeled Bac-ELP-p21 and ELP-p21 were monitored in a mouse xenograft model by whole animal imaging using the IVIS® system. Luciferase transfected S2013 pancreatic cells were used to establish the tumor, allowing monitored tumor growth by quantifying luciferase signal over a period of time. The animals were intravenously injected with Alexa750 labeled Bac-ELP-p21 or ELP-p21. Animals were imaged for fluorescent signal at specific times after treatment to observe the level of protein with respect to time. This allowed for the detection and quantification of the protein level throughout the body and compared with uptake inside the tumor. Heated tumors accumulated 3-fold more protein with both Bac-ELP-p21 and ELP-p21. For biodistribution and plasma clearance studies, blood draws were performed on separate groups at 5, 15, 30 min and continuing every 30 min for 4h. At the end of the 4h, organs and tumors were harvested for later dissection. This allowed for comparison of the protein in the organs and monitored the amount of protein in the plasma over time. At 5 minutes, 2 times the amount of ELP-p21 was detectable in blood plasma compared to Bac-ELP-p21. At 2 hours, this ratio increases to 2.5 fold. Protein was still detectable in the tumor at 48h after treatment, indicating that modification of macromolecular carrier with cell penetrating peptide results in its faster plasma clearance due to the rapid accumulation in tumor and/or tissues. In conclusion, fusing a therapeutic peptide to a macromolecular carrier results in its improved pharmacokinetics and accumulation of the peptide, preferentially in the tumor tissue due to passive targeting, which is improved due to externally induced hyperthermia while this active targeting is increased due to the addition of a CPP. This approach will allow us to use lower concentrations of drug systemically and therefore reduce side effects in normal tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 355. doi:1538-7445.AM2012-355

50 Development of a suitable formulation and robust analytical method for a Chromium-51 - EDTA intravenous solution used in plasma clearance studies for the determination of glomerular filtration rate

Nuclear Medicine, Guy's and St Thomas' NHSFT, London, MSc, PTQA, School of Continuing Professional Development, University of Leeds, Leeds, UK British Nuclear Medicine Society Oral and Poster Abstracts 2007

Folate-Targeted Enzyme Prodrug Cancer Therapy Utilizing Penicillin-V Amidase and a Doxorubicin Prodrug

In antibody-targeted enzyme prodrug therapy, a monoclonal antibody (mAb) covalently linked to an enzyme is commonly exploited to concentrate the enzyme on the tumor cell surface prior to administration of a relatively nontoxic prodrug. The tumor-localized enzyme then converts the prodrug into a cytotoxic agent, which in turn diffuses into the tumor causing localized cell death. In this paper, we have substituted folic acid for the mAb as a mean of delivering an attached enzyme, penicillin-V amidase (PVA), to folate receptor (FR)-positive tumor cells. The enzyme PVA is capable of converting a doxorubicin-N-p-hydroxyphenoxyacetamide prodrug (DPO) into its potent parent drug, doxorubicin. For PVA targeting, each PVA molecule was covalently labeled with three molecules of folic acid via the formation of amide bonds. In vitro binding assays showed that folate-PVA-125I conjugates bind specifically to KB cells (FR-positive tumor cells) but not to A549 cells (FR-negative tumor cells). Moreover, in a series of in vitro cytotoxicity tests, folate-PVA conjugates were found to kill folate receptor positive but not receptor negative cells, and when bound to FR-positive cells, folate-PVA conjugates rendered the DPO prodrug as toxic as free doxorubicin (IC⊂50, 0.6 μM). Finally, preliminary in vivo plasma clearance studies in normal mice revealed that i.v. administered folate-PVA-125I and PVA-125I are both cleared from the blood within a 24 h time period, removing concern that nonspecifically trapped folate-PVA might activate prodrug in nontargeted tissues. In view of the fact that only a small number of folate-PVA molecules are required to mediate killing of target cells in vitro, these data argue that folate-targeted enzyme prodrug therapy should be considered for tumor eradication in vivo.

In Vivo Clearance of Ternary Complexes of Vitronectin-Thrombin-Antithrombin Is Mediated by Hepatic Heparan Sulfate Proteoglycans

Thrombin is inhibited by its cognate plasma inhibitor antithrombin, through the formation of covalent thrombin-antithrombin (TAT) complexes that are found as ternary complexes with vitronectin (VN-TAT). To determine whether the metabolism of VN-TAT ternary complexes is different from that previously reported for binary TAT complexes, plasma clearance studies were done in rabbits using human VN-TAT. 125I-VN-TAT was shown to be cleared rapidly from the circulation (t1/2alpha = 3.8 min) in a biphasic manner mainly by the liver. 125I-TAT had a similar initial clearance (t1/2alpha = 5.3 min) but had a significantly faster beta-phase clearance (t1/2beta = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial alpha-phase of 125I-VN-TAT clearance and reduced its liver-specific association and in vivo degradation. Heparin also reduced the alpha-phase clearance of 125I-TAT and was associated with the appearance of high molecular weight complexes, suggesting enhanced complex formation between VN and TAT. 125I-VN-TAT binding to HepG2 cells was reduced by competition with VN-TAT or heparin but to a much lesser extent in the presence of TAT. The binding of VN-TAT to HepG2 cells was not inhibited by competition with the low density lipoprotein receptor-related protein ligand, methylamine-alpha2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of beta-D-xyloside. Finally, both heparin and chloroquine, but not methylamine-alpha2-macroglobulin, reduced the internalization and degradation of VN-TAT by HepG2 cells. Taken together, these data indicate the importance of VN in TAT metabolism and demonstrate that VN-TAT binds to liver-associated heparan sulfate proteoglycans, which mediate its internalization and subsequent intracellular degradation.

Quantification of renal haemodynamics with radionuclides

Non-invasive quantification of renal function with radionuclides is an important role of nuclear medicine. With modern commercial preparations of technetium-99m diethylene triamine penta-acetic acid (DTPA), glomerular filtration rate (GFR) can be measured accurately either from the rate of disappearance of the tracer from plasma or from its rate of uptake into the kidneys. Determination of the latter with the gamma camera allows measurement of individual kidney GFR. Renal blood flow (RBF) can be measured from plasma clearance of hippurate or from first-pass kinetics of intravenously injected tracer using the gamma camera. The filtration fraction can be obtained from separate measurement of GFR and RBF, either globally from plasma clearance studies or of each kidney from gamma camera studies. Because they are central to the understanding of plasma clearance studies, the effective distribution volumes of the various tracers used for renal function studies are also discussed in detail.

Clearance of Desialylated Mouse α-Macroglobulin and Murinoglobulin in the Mouse

Mouse alpha-macroglobulin and murinoglobulin were labeled with 125I and utilized for plasma clearance studies performed with mice. Desialylated murinoglobulin was rapidly cleared from the circulation with a half-life of about 5 min. On the other hand, desialylated alpha-macroglobulin showed a biphasic curve: about half was cleared at a rate similar to that of the intact molecule while the remaining half had a shorter half-life of about 20 min which was prolonged by a simultaneous injection of a 200-fold excess of unlabeled asialoorosomucoid. Virtually no cross competition was observed between these asialoglobulins and formaldehyde-treated bovine serum albumin or trypsin-bound alpha-macroglobulin. These results suggest that the intravascular elimination of desialylated alpha-macroglobulin and murinoglobulin is independent of the clearance systems responsible for formaldehyde-modified proteins or proteinase-bound alpha-macroglobulins, and that the structure or spatial arrangement, or both, of oligosaccharide units of alpha-macroglobulin is somewhat different from that of murinoglobulin, resulting in a difference of avidity of interaction with the asialoglycoprotein receptor. The desialylated alpha-macroglobulin and murinoglobulin accumulated principally in the liver.

THE IMPAIRMENT OF TRUE GLOMERULAR FILTRATION RATE IN LONG-TERM CYCLOSPORINE-TREATED PEDIATRIC ALLOGRAFT RECIPIENTS

We performed indium-111-DTPA plasma clearance studies in 61 pediatric kidney and liver recipients treated with cyclosporine to compare true glomerular filtration rate with calculated GFR (cGFR). The mean true GFR of 61.9 +/- 36.6 ml/min/1.73 m2 indicated renal impairment. The mean cGFR of 85.2 +/- 22.4 ml/min/1.73 m2 was significantly higher (P less than 0.001), and overestimated GFR by 38%. cGFR alone did not accurately reflect the degree of renal dysfunction. A group of 48 pediatric orthotopic liver transplant recipients was studied in more detail: 73% of these patients had a true GFR less than 70 ml/min/1.73 m2, while 85% had a true GFR below 90 ml/min/1.73 m2, the lower limit for normal GFR in children. The mean true GFR for patients treated more than 24 months with CsA was lower (P = 0.02) than patients treated with CsA for 12 to 24 months. OLT patients with normal true GFR (greater than 90 ml/min/1.73 m2) had significantly lower plasma CsA levels, and 50% of patients with a true GFR less than or equal to 50 ml/min/1.73 m2 had hypertension. There was no effect on true GFR of age, liver function, azathioprine use, or peritransplant treatment with other nephrotoxic drugs. We conclude that true GFR is significantly impaired in long-term CsA-treated allograft pediatric recipients. Calculations of GFR underestimate the degree of renal dysfunction. As patients treated greater than 24 months had the lowest true GFRs, the fall in GFR may be progressive.

A conserved region in .alpha.-macroglobulins participates in binding to the mammalian .alpha.-macroglobulin receptor

Efforts to characterize the receptor recognition domain of alpha-macroglobulins have primarily focused on human alpha 2-macroglobulin (alpha 2M). In the present work, the structure and function of the alpha-macroglobulin receptor recognition site were investigated by amino acid sequence analysis, plasma clearance, and cell binding studies using several nonhuman alpha-macroglobulins: bovine alpha 2M, rat alpha 1-macroglobulin (alpha 1M), rat alpha 1-inhibitor 3 (alpha 1I3), and proteolytic fragments derived from these proteins. Each alpha-macroglobulin bound to the murine peritoneal macrophage alpha-macroglobulin receptor with comparable affinity (Kd approximately 1 nM). A carboxyl-terminal 20-kDa fragment was isolated from each of these proteins, and this fragment bound to alpha-macroglobulin receptors with Kd values ranging from 10 to 125 nM. The amino acid identity between the homologous carboxyl-terminal 20-kDa fragments of human and bovine alpha 2M was approximately 90%, while the overall sequence homology between all carboxyl-terminal fragments studied was 75%. The interchain disulfide bond present in the human alpha 2M carboxyl-terminal 20-kDa fragment was conserved in bovine alpha 2M and rat alpha 1I3, but not in rat alpha 1M. The clearance of each intact alpha-macroglobulin-proteinase complex was significantly retarded following treatment with cis-dichlorodiammineplatinum(II) (cis-DDP). cis-DDP treatment, however, did not affect receptor recognition of purified carboxyl-terminal 20-kDa fragments of these alpha-macroglobulins. A carboxyl-terminal 40-kDa subunit, which can be isolated from rat alpha 1M, bound to the murine alpha-macroglobulin receptor with a Kd of 5 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma clearance in the rat of the LH bioactivity of two human LH standards of differing molecular composition.

The LH biological potency of the International Reference Preparation (IRP) of Human Pituitary LH for Immunoassay (IRP 68/40) relative to that of the 2nd IRP of Human Pituitary FSH and LH for Bioassay (IRP 78/549) is markedly greater when estimated by in-vitro interstitial cell testosterone production (TICT) bioassay than by in-vivo bioassay, and by the 4-h ovarian ascorbate depletion (OAAD) assay than by the 4-day seminal vesicle weight gain assay. Other preparations of human LH which, like IRP 68/40, were highly purified, showed a similar spectrum of bioactivity in these assay systems and also contained a higher proportion of more basic LH isoforms than are found in crude pituitary extracts such as IRP 78/549. In an attempt to explain these differences, a comparison was made of the plasma survival in rats of the LH bioactivity (by TICT assay) of these two preparations. Contrary to expectation, their relative plasma clearance rates over a 4-h period did not account for their differing bioactivities. The plasma half-life of the LH bioactivity (with 95% confidence limits) was estimated to be 42.4 (35.3-49.5) min for IRP 68/40 and 41.3 (31.5-51.0) min for IRP 78/549. Furthermore the time-course of action in vivo of IRP 78/549 did not appear to be more prolonged than that of IRP 68/40. Thus their plasma testosterone responses during the course of these 4-h plasma clearance studies were similar, and estimates of the LH potency of IRP 68/40 relative to that of IRP 78/549 were no greater by 2-h than by 4-h OAAD assay.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the therapeutic effects of free and liposomally encapsulated vincristine in leukemic mice

The neurological toxicity of the Vinca alkaloids restricts their use in cancer chemotherapy. In this study we investigated the potential of liposomal encapsulation of one of these drugs, vincristine, to reduce this toxicity and hence increase the utility of this class of antimitotic agents. Plasma clearance studies for i.v. administration to NMRI mice, indicated that vincristine (VCR) included in liposomes (VCR-Lip) was removed more slowly from the plasma than was free VCR, and that whilst the liposomes were in the circulation most of the VCR remained associated with its liposomal carrier. Early therapeutic trials of vincristine in liposomes, given intravenously to DBA/2 mice previously inoculated with P 388 leukemic cells, demonstrated that considerable toxicity was caused by the lipid composition of the carrier (phosphatidylcholine, cholesterol and stearylamine). This toxicity was confirmed in in vitro screening of empty liposomes of various composition, and from this study a liposome composition of very low toxicity was selected (phosphatidylcholine, cholesterol and phosphatidylserine). Further therapeutic trials with the drug entrapped in ‘non-toxic’ liposomes showed that liposomal encapsulation of Vinca alkaloids did not provide any advantage either in terms of reduced toxicity or in increased therapeutic index over the free drug for the treatment of lymphocytic leukemia.

Interaction of lipopolysaccharides with plasma high-density lipoprotein in rats.

The method of crossed immunoelectrophoresis was used to investigate early changes in plasma proteins of rats treated with lipopolysaccharide (LPS). Intravenous injection of a smooth (S)- and a rough (R)-form preparation led to alterations in the high-density lipoprotein (HDL) precipitation peak. The changes were dose dependent and characteristic for each LPS. The changes were identified as being due to the formation of a complex of LPS with HDL, the complex of the S-form LPS with HDL migrating slower and that of the R-form LPS with HDL migrating faster than free HDL. The fate of the complex was followed in the plasma of injected rats, and it was shown that the R-form LPS complex disappeared after several hours, whereas the S-form LPS complex was still partly present after 2 days. Plasma clearance studies, carried out with 14C-labeled LPS, revealed similar differences in the rate of elimination of the two LPSs. In both cases the time of clearance resembled that of the disappearance of LPS-HDL complex. These results may indicate that HDL represents a transport protein for LPS in plasma to organs of clearance or to other cellular targets.

Clearance of circulating desialylated thyroglobulins in the rat

Canine and rat thyroglobulins were labeled with 125I either in vitro or in vivo, and were utilized for plasma clearance studies performed with rat. The half-life of physiologically radioiodinated asialothyroglobulins was about 6 min, while that of chemically radioiodinated asialothyroglobulin was about 12 min. No marked species difference was observed in this clearance. The label which had disappeared from the blood was recovered mainly in the liver, and this uptake was blocked by the simultaneous injection of desialylated orosomucoid but not by native orosomucoid. Radiolabeled monoiodotyrosine, diiodotyrosine, triiodothyronine and thyroxine were detected in the liver 17 min after intravenous injection of asialothyroglobulin labeled with 125I in vivo, suggesting the possible production of thyroid hormones in extrathyroidal tissues.

125I labeling of fibrinogen by the chloramine-T method without protein denaturation

Purified bovine fibrinogen preparations were radioiodinated with 125I using the Chloramine-T method and examined for degradation by clottability assays and agarose gel filtration. In vivo plasma clearance studies were also conducted using healthy mixed breed calves. The in vitro assays yielded an average clottability of 92.5% and chromatograms comparable to accepted norms for 125I-fibrinogen prepared by ICl and enzymatic methods. The clearance studies yielded an average half life of 5.1 ± 0.4 days for the major exponential component.

Human plasma R-type vitamin B12-binding proteins. II. The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein in the plasma transport of vitamin B12.

The normal human granulocyte vitamin B12-binding protein, transcobalamin I, and transcobalamin III, have been labeled with 125I-labeled N-succinimidyl 3-(4-hydroxyphenyl)propionate and utilized for plasma clearance studies performed with rabbits. Both moieties of 125I-labeled granulocyte vitamin B12-binding protein-[57Co]vitamin B12 were cleared rapidly from the plasma (is less than 90% by 5 min) by the liver. After 30 min, the bulk of the 125I reappeared in the plasma in small molecular weight (less than 1000) form and was rapidly excreted in the urine. After 60 min the bulk of the [57Co]vitamin B12 reappeared in the plasma bound to rabbit transcobalamin II and was subsequently taken up by a variety of tissues. Approximately 15% of the 125I-labeled granulocyte vitamin B12-binding protein-[57Co-a1vitamin B12 was excreted intact into the bile during the period from 10 to 80 min after injection. The hepatic uptake of the protein-vitamin B12 complex was blocked by the prior injection of desialyzed fetuin but not by native fetuin. Similar results were obtained with 125I-labeled transcobalamin III-[57Co]vitamin B12. Approximately 90% of both moieties of 125I-labeled transcobalamin I-[57Co]vitamin B12 had prolonged plasma survivals similar to that of 125I-labeled bovine serum albumin. After treatment with neuraminadase, both moieties of the 125I-labeled transcobalamin I-[57Co]vitamin B12 complex were cleared rapidly from the plasma by the liver in a manner that was indistinguishable from that observed in the case of untreated granulocyte vitamin B12-binding protein and transcobalamin III. These observations indicate that desialyzed transcobalamin I and the native forms of the granulocyte vitamin B12-binding protein and transcobalamin III are cleared from plasma by the mechanism elucidated by Ashwell and Morell (Ashwell, G., and Morell A. G. (1974) Adv. Enzymol. 41, 99-128) that is capable of clearing a wide variety of asialoglycoproteins. These observations have implications concerning the function of the human R-type vitamin B12-binding proteins, the nature of the enterohepatic circulation of vitamin B12, the biological significance of the mechanism described by Ashwell and Morell, and the etiology of the increased plasma concentration of human R-type protein that occurs frequently in chronic myelogenous leukemia and occasionally in hepatocellular carcinoma and other solid tumors.

The constitutional conjugated hyperbilirubinemia

Plasma clearance studies with Indocyanine Green (ICG) and Bromsulfophthalein (BSP) were performed on four patients with chronic conjugated hyperbilirubinemia with or without apparent brown pigments in the liver cells, and free from structural hepatic diseases. An abnormal clearance pattern, characterized by reduced hepatic ICG and BSP clearances, was observed in one case with Rotor’s type of hyperbilirubinemia (R). In the other three cases of Dubin-Johnson syndrome (DJ), an abnormal disappearance curve with a secondary rise of serum BSP 60 and 120 minutes after intravenous administration, was observed, although the hepatic disappearance rates of both ICG and BSP were normal. A two-compartmental analysis revealed that the depressed hepatic dye clearance in R might be due to defects both in the uptake and in the excretion of dye by the liver, and there seems to be an excretory defect of the dye in DJ. The studies on the transport maximum (Tm) and the relative storage capacity (S) of ICG demonstrated the reduction of Tm and S in R, while decreased Tm and normal S in DJ. These results suggest that the current diagnostic criteria for DJ and R would be based upon the dye transport distinction in patients with the same basic hyperbilirubinemia.

Corticosteroid dynamics in Pacific salmon

The tissue distribution of cortisol- 3H and - 14C and cortisone- 3H and - 14C in sexually mature and orchidectomized male sockeye salmon ( Oncorhynchus nerka) was determined at intervals after a single intraarterial injection of cortisol- 3H and cortisone- 14C. Samples of plasma, blood cells, white muscle, red muscle, liver, kidney, head kidney, spleen, gill, gut, brain, heart and, in some cases, bile, were obtained from gonadectomized fish 10, 20, 40, and 80 min after injection and from mature fish 10, 20, 40, 80, and 160 min after injection. Dichloromethane extracts of the tissues were chromatographed on thin-layer silica gel sheets and the quantity of 3H and 14C chromatographically associated with authentic cortisol and cortisone was determined by double isotope scintillation spectrometry. Cortisol and cortisone radioactivity are expressed as dpm/g tissue for a standard 1-kg fish, as a tissue: plasma ratio and as a cortisol- 3H: cortisone- 3H or cortisone- 14C: cortisol- 14C ratio. In most tisues there was a more rapid clearance of cortisol- 3H in the mature than in the gonadectomized fish. Cortisol- 3H tissue:plasma ratios were generally higher in the mature than in the gonadectomized fish. The cortisol- 3H tissue: plasma ratio was especially high in the kidney, liver, and head kidney of the mature fish. The cortisone- 14C tissue: plasma raitos were also higher in the mature than in the gonadectomized salmon but not so high as the cortisol- 3H ratios, suggesting that there are more binding sites for cortisol than for cortisone. These differences in tissue distribution explain the increase in cortisol and cortisone apparent volume of distribution noted during sexual maturation in earlier plasma clearance studies. Tissue cortisol- 3H: cortisone- 3H ratios indicate an apparent rapid conversion of cortisol to cortisone in spleen, heart, and gills in mature fish and in gut and gills in gonadectomized fish. In the liver of the gonadectomized salmon the cortisol- 3H: cortisone- 3H ratio was high and the cortisone- 14C: cortisol- 14C ratio was low, indicating a predominance of 11 keto-reductase activity. This was not the case in the mature salmon.

Bilirubin Metabolism in Congenital Nonhemolytic Jaundice

Extract: A Negro female with congenital nonhemolytic jaundice (Crigler-Najjar syndrome) was studied over a period of 15 months. During this period the plasma bilirubin concentration averaged 20.6 mg/100 ml. Administration of phenobarbital did not lower the bilirubin concentration. Studies with isotopically labeled bilirubin showed that phenobarbital had not increased the fractional rate of elimination of bilirubin, and the patient's bilirubin production rate while receiving phenobarbital therapy (3.5 mg/kg/24 hr) was not increased over that seen in normal individuals (3.8 \pm 0.6 mg/kg/24 hr, mean \pm sd). Multicompartmental analysis of the patient's labeled bilirubin clearance data, as well as that from two other reported cases of congenital nonhemolytic jaundice, did not furnish new information on pathways of bilirubin catabolism but did demonstrate that the fractional transfer rate of bilirubin from plasma to liver for all three subjects (0.42–0.87 hr-1) was reduced compared with the range seen in normal individuals (0.9–2.0 hr-1). This finding may reflect saturation of either the hepatic uptake process or intracellular binding sites, in the face of a serum bilirubin concentration 20–40 times normal. Menthol conjugation in the patient's parents was normal, unlike reported findings in parents of children with congenital nonhemolytic jaundice unresponsive to phenobarbital therapy. Labeled bilirubin clearances in the parents demonstrated an abnormality in the mother's pattern of clearance, whereas the father's clearance was normal. The patient was found to excrete normal amounts of testosterone conjugated with glucuronic acid, demonstrating that the defect in glucuronide conjugation does not include all physiological compounds. Speculation: Plasma clearance studies with labeled bilirubin can aid in the investigation of modes of inheritance of unconjugated hyperbilirubinemia.

Constitutional hepatic dysfunction (Gilbert's syndrome): A new definition based on kinetic studies with unconjugated radiobilirubin

Plasma clearance studies with unconjugated radiobilirubin were performed in eleven patients who had chronic unconjugated hyperbilirubinemia in the absence of structural liver disease. An abnormal clearance pattern, characterized by increased retention of isotope in the plasma at four hours (24.2±8.4 per cent, mean ± standard deviation; normal=5.0±1.9 per cent) and a reduction in hepatic bilirubin clearance (16±5 ml/minute; normal=47±10 ml/minute), was observed in all eleven cases, despite the fact that the mean red cell survival ranged from normal (112 days) to severely reduced (eleven days). Multicompartmental analysis suggests that the reduced hepatic bilirubin clearance is the result of a defect in both hepatic bilirubin uptake and conjugation. Twenty-one studies in a control group with anemia and/or hemolysis were indistinguishable from normal, indicating that neither of these factors was responsible for the abnormalities noted. These studies suggest that current diagnostic criteria for Gilbert's syndrome, which exclude patients with hemolysis, will result in an artificial distinction among patients with the same basic hepatic abnormality. Plasma clearance studies with unconjugated radiobilirubin were performed in eleven patients who had chronic unconjugated hyperbilirubinemia in the absence of structural liver disease. An abnormal clearance pattern, characterized by increased retention of isotope in the plasma at four hours (24.2±8.4 per cent, mean ± standard deviation; normal=5.0±1.9 per cent) and a reduction in hepatic bilirubin clearance (16±5 ml/minute; normal=47±10 ml/minute), was observed in all eleven cases, despite the fact that the mean red cell survival ranged from normal (112 days) to severely reduced (eleven days). Multicompartmental analysis suggests that the reduced hepatic bilirubin clearance is the result of a defect in both hepatic bilirubin uptake and conjugation. Twenty-one studies in a control group with anemia and/or hemolysis were indistinguishable from normal, indicating that neither of these factors was responsible for the abnormalities noted. These studies suggest that current diagnostic criteria for Gilbert's syndrome, which exclude patients with hemolysis, will result in an artificial distinction among patients with the same basic hepatic abnormality.