Fasting induces ketoacidosis and hypothermia in PDHK2/PDHK4-double-knockout mice.

The importance of PDHK (pyruvate dehydrogenase kinase) 2 and 4 in regulation of the PDH complex (pyruvate dehydrogenase complex) was assessed in single- and double-knockout mice. PDHK2 deficiency caused higher PDH complex activity and lower blood glucose levels in the fed, but not the fasted, state. PDHK4 deficiency caused similar effects, but only after fasting. Double deficiency intensified these effects in both the fed and fasted states. PDHK2 deficiency had no effect on glucose tolerance, PDHK4 deficiency produced only a modest effect, but double deficiency caused a marked improvement and also induced lower insulin levels and increased insulin sensitivity. In spite of these beneficial effects, the double-knockout mice were more sensitive than wild-type and single-knockout mice to long-term fasting, succumbing to hypoglycaemia, ketoacidosis and hypothermia. Stable isotope flux analysis indicated that hypoglycaemia was due to a reduced rate of gluconeogenesis and that slightly more glucose was converted into ketone bodies in the double-knockout mice. The findings establish that PDHK2 is more important in the fed state, PDHK4 is more important in the fasted state, and survival during long-term fasting depends upon regulation of the PDH complex by both PDHK2 and PDHK4.

Turnover of nonessential fatty acids in cardiolipin from the rat heart.

Cardiolipin (CL) is a unique phospholipid (PL) found in the mitochondria of mammalian cells. CL remodeling is accompanied by turnover of its fatty acid acyl groups. Abnormalities in CL remodeling have been found in Barth's syndrome, diabetes, and obesity. The objective of this study was to determine nonessential fatty acid turnover in CL and phosphatidylethanolamine (PE) in the rat heart in vivo. Sprague-Dawley rats were fed either a regular chow or a high-fat diet for 15 weeks, and consumed 6% deuterium-enriched drinking water as a tracer for 14 days. CL and PE were extracted from cardiac tissue and isolated by TLC. Fatty acids from CL, PE, and plasma were analyzed by GC/MS for deuterium incorporation. Results showed oleate and vaccenate turnover were the highest in CL whereas palmitate and stearate turnover were low. Among the nonessential fatty acids in PE, turnover of stearate and vaccenate were the highest. The high turnover rate in vaccenate was unexpected, because vaccenate previously had no known metabolic or physiologic function. In conclusion, the similarly high turnover rates of both oleate and vaccenate readily suggest that remodeling is an important functional aspect of PL metabolism in CL.

Increased pentose phosphate pathway flux after clinical traumatic brain injury: a [1,2-13C2]glucose labeling study in humans.

Patients with traumatic brain injury (TBI) routinely exhibit cerebral glucose uptake in excess of that expected by the low levels of oxygen consumption and lactate production. This brings into question the metabolic fate of glucose. Prior studies have shown increased flux through the pentose phosphate cycle (PPC) during cellular stress. This study assessed the PPC after TBI in humans. [1,2-(13)C(2)]glucose was infused for 60 mins in six consented, severe-TBI patients (GCS<9) and six control subjects. Arterial and jugular bulb blood sampled during infusion was analyzed for (13)C-labeled isotopomers of lactate by gas chromatography/mass spectroscopy. The product of lactate concentration and fractional abundance of isotopomers was used to determine blood concentration of each isotopomer. The difference of jugular and arterial concentrations determined cerebral contribution. The formula PPC=(m1/m2)/(3+(m1/m2)) was used to calculate PPC flux relative to glycolysis. There was enrichment of [1,2-(13)C(2)]glucose in arterial-venous blood (enrichment averaged 16.6% in TBI subjects and 28.2% in controls) and incorporation of (13)C-label into lactate, showing metabolism of labeled substrate. The PPC was increased in TBI patients relative to controls (19.6 versus 6.9%, respectively; P=0.002) and was excellent for distinguishing the groups (AUC=0.944, P<0.0001). No correlations were found between PPC and other clinical parameters, although PPC was highest in patients studied within 48 h of injury (averaging 33% versus 13% in others; P=0.0006). This elevation in the PPC in the acute period after severe TBI likely represents a shunting of substrate into alternative biochemical pathways that may be critical for preventing secondary injury and initiating recovery.

Lipin deficiency impairs diurnal metabolic fuel switching.

Fatty liver is a common feature of both obesity and lipodystrophy, reflecting compromised adipose tissue function. The lipin-deficient fatty liver dystrophy (fld) mouse is an exception, as there is lipodystrophy without a fatty liver. Using a combination of indirect calorimetry and stable-isotope flux phenotyping, we determined that fld mice exhibit abnormal fuel utilization throughout the diurnal cycle, with increased glucose oxidation near the end of the fasting period and increased fatty acid oxidation during the feeding period. The mechanisms underlying these alterations include a twofold increase compared with wild-type mice in tissue glycogen storage during the fed state, a 40% reduction in hepatic glucose production in the fasted state, and a 27-fold increase in de novo fatty acid synthesis in liver during the fed state. Thus, the inability to store energy in adipose tissue in the fld mouse leads to a compensatory increase in glycogen storage for use during the fasting period and reliance upon hepatic fatty acid synthesis to provide fuel for peripheral tissues during the fed state. The increase in hepatic fatty acid synthesis and peripheral utilization provides a potential mechanism to ameliorate fatty liver in the fld that would otherwise occur as a consequence of adipose tissue dysfunction.

Decreased hepatic futile cycling compensates for increased glucose disposal in the Pten heterodeficient mouse.

Despite altered regulation of insulin signaling, Pten(+/-) heterodeficient standard diet-fed mice, approximately 4 months old, exhibit normal fasting glucose and insulin levels. We report here a stable isotope flux phenotyping study of this "silent" phenotype, in which tissue-specific insulin effects in whole-body Pten(+/-)-deficient mice were dissected in vivo. Flux phenotyping showed gain of function in Pten(+/-) mice, seen as increased peripheral glucose disposal, and compensation by a metabolic feedback mechanism that 1) decreases hepatic glucose recycling via suppression of glucokinase expression in the basal state to preserve hepatic glucose production and 2) increases hepatic responsiveness in the fasted-to-fed transition. In Pten(+/-) mice, hepatic gene expression of glucokinase was 10-fold less than wild-type (Pten(+/+)) mice in the fasted state and reached Pten(+/+) values in the fed state. Glucose-6-phosphatase expression was the same for Pten(+/-) and Pten(+/+) mice in the fasted state, and its expression for Pten(+/-) was 25% of Pten(+/+) in the fed state. This study demonstrates how intra- and interorgan flux compensations can preserve glucose homeostasis (despite a specific gene defect that accelerates glucose disposal) and how flux phenotyping can dissect these tissue-specific flux compensations in mice presenting with a "silent" phenotype.

A novel, view-independent method for strain mapping in myocardial elastography: eliminating angle and centroid dependence.

Robust indices of regional and global cardiac function are a key factor in detection and treatment of heart disease as well as understanding of the fundamental mechanisms of a healthy heart. Myocardial elastography provides a noninvasive method for imaging and measuring displacement and strain of the myocardium for the early detection of cardiovascular disease. However, two-dimensional in-plane axial and lateral strains measured depend on the sonographic view used. This becomes especially critical in a clinical setting and may induce large variations in the measured strains, potentially leading to false diagnoses. A novel method in myocardial elastography is proposed for eliminating this view dependence by deriving the polar, principal and classified principal strains. The performance of the proposed methodology is assessed by employing 3D finite-element left-ventricular models of a control and an ischemic canine heart. Although polar strains are angle-independent, they are sensitive to the selected reference coordinate system, which requires the definition of a centroid of the left ventricle (LV). In contrast, principal strains derived through eigenvalue decomposition exhibit the inherent characteristic of coordinate system independence, offering view (i.e., angle and centroid)-independent strain measurements. Classified principal strains are obtained by assigning the principal components in the physical ventricular coordinate system. An extensive strain analysis illustrates the improvement in interpretation and visualization of the full-field myocardial deformation by using the classified principal strains, clearly depicting the ischemic and non-ischemic regions. Strain maps, independent of sonographic views and imaging planes, that can be used to accurately detect regional contractile dysfunction are demonstrated.

Transforming growth factor beta2 promotes glucose carbon incorporation into nucleic acid ribose through the nonoxidative pentose cycle in lung epithelial carcinoma cells.

The invasive transformation of A-459 lung epithelial carcinoma cells has been linked to the autocrine regulation of malignant phenotypic changes by transforming growth factor beta (TGF-beta). Here we demonstrate, using stable 13C glucose isotopes, that the transformed phenotype is characterized by decreased CO2 production via direct glucose oxidation but increased nucleic acid ribose synthesis through the nonoxidative reactions of the pentose cycle. Increased nucleic acid synthesis through the nonoxidative pentose cycle imparts the metabolic adaptation of nontransformed cells to the invasive phenotype that potentially explains the fundamental metabolic disturbance in tumor cells: highly increased nucleic acid synthesis despite hypoxia and decreased glucose oxidation.

Oxythiamine and dehydroepiandrosterone inhibit the nonoxidative synthesis of ribose and tumor cell proliferation.

This study investigates the significance of the glucose-6-phosphate dehydrogenase (G6PD) catalyzed oxidative and the transketolase (TK) catalyzed nonoxidative pentose cycle (PC) reactions in the tumor proliferation process by characterizing tumor growth patterns and synthesis of the RNA ribose moiety in the presence of respective inhibitors of G6PD and TK. Mass spectra analysis of 13C-labeled carbons revealed that these PC reactions contribute to over 85% of de novo ribose synthesis in RNA from [1,2-(13)C]glucose in cultured Mia pancreatic adenocarcinoma cells, with the fraction synthesized through the TK pathway predominating (85%). Five days of treatment with the TK inhibitor oxythiamine (OT) and the G6PD inhibitor dehydroepiandrosterone-sulfate (0.5 microM each) exerted a 39 and a 23% maximum inhibitory effect on cell proliferation in culture, which was increased to 60% when the two drugs were administered in combination. In vivo testing of 400 mg/kg OT or dehydroepiandrosterone-sulfate in C57BL/6 mice hosting Ehrlich's ascitic tumor cells revealed a 90.4 and a 46% decrease in the final tumor mass after 3 days of treatment. RNA ribose fractional synthesis through the TK reaction using metabolites directly from glycolysis declined by 9.1 and 23.9% after OT or the combined treatment, respectively. Nonoxidative PC reactions play a central regulating role in the carbon-recruiting process toward de novo nucleic acid ribose synthesis and cell proliferation in vitro and in vivo. Therefore, enzymes or substrates regulating the nonoxidative synthesis of ribose could also be the sites to preferentially target tumor cell proliferation by new anticancer drugs.

Advance in microRNA as a potential biomarker for early detection of pancreatic cancer.

Pancreatic cancer is characterized as a disease with low survival and high mortality because of no effective diagnostic and therapeutic strategies available in clinic. Conventional clinical diagnostic methods including serum markers and radiological imaging (CT, MRI, EUS, etc.) often fail to detect precancerous or early stage lesions. Development of effective biomarkers is unmet for reduction of mortality of pancreatic cancer. MicroRNAs (miRNAs) are a group of small non-protein-coding RNAs playing roles in regulation of cell physiology including tumorigenesis, apoptotic escape, proliferation, invasion, epithelial-mesenchymal transition (EMT), metastasis and chemoresistance. Various altered signaling pathways involving in molecular pathogenesis of pancreatic cancer are mediated by miRNAs as a role of either oncogenes or tumor suppressors. Among biomarkers developed including protein, metabolites, DNA, RNA, epigenetic mutation, miRNAs are superior because of its unique chemical property. Recent study suggests that miRNAs may be promising biomarkers used for early detection of pancreatic cancer. This review will update the progression made in early detection of pancreatic cancer.

Inhibition of Pyruvate Dehydrogenase Kinase 2 Protects Against Hepatic Steatosis Through Modulation of Tricarboxylic Acid Cycle Anaplerosis and Ketogenesis.

Hepatic steatosis is associated with increased insulin resistance and tricarboxylic acid (TCA) cycle flux, but decreased ketogenesis and pyruvate dehydrogenase complex (PDC) flux. This study examined whether hepatic PDC activation by inhibition of pyruvate dehydrogenase kinase 2 (PDK2) ameliorates these metabolic abnormalities. Wild-type mice fed a high-fat diet exhibited hepatic steatosis, insulin resistance, and increased levels of pyruvate, TCA cycle intermediates, and malonyl-CoA but reduced ketogenesis and PDC activity due to PDK2 induction. Hepatic PDC activation by PDK2 inhibition attenuated hepatic steatosis, improved hepatic insulin sensitivity, reduced hepatic glucose production, increased capacity for ß-oxidation and ketogenesis, and decreased the capacity for lipogenesis. These results were attributed to altered enzymatic capacities and a reduction in TCA anaplerosis that limited the availability of oxaloacetate for the TCA cycle, which promoted ketogenesis. The current study reports that increasing hepatic PDC activity by inhibition of PDK2 ameliorates hepatic steatosis and insulin sensitivity by regulating TCA cycle anaplerosis and ketogenesis. The findings suggest PDK2 is a potential therapeutic target for nonalcoholic fatty liver disease.

Hepatic de novo lipogenesis in stable low-birth-weight infants during exclusive breast milk feedings and during parenteral nutrition.

BACKGROUND: Low-birth-weight (LBW) infants have high energy requirements and are dependent on high fat intake to maintain adequate postnatal growth. Fat energy is transported in plasma as triglycerides, which are either derived from the diet or from de novo lipogenesis (DNL). It is our hypothesis that DNL plays an important physiologic role in adapting to exclusive breast milk (EBM) feeding or to parenteral nutrition (PN). METHODS: We studied hepatic de novo lipogenesis in 14 LBW (<34-week gestation) appropriate for gestational age and receiving either EBM feedings or full PN support. Stable isotope tracer [2-(13)C] acetate was administered for 72 hours to achieve an estimated 10% enrichment of daily fat intake. Fatty acids were extracted from plasma for gas chromatography-mass spectrometry analyses. RESULTS: Percent new synthesis of palmitate was 13.1% +/- 2.5% in the EBM group and 14.9% +/- 0.7% in the PN group (NS), stearate was 11.1% +/- 2.7% in the EBM group and 10.6% +/- 14% in the PN group (NS) and cholesterol was 12.7% +/- 2.1% in the EBM group and 17.4% +/- 4.6% in the PN group (NS) after 72 hours of tracer administration (mean +/- SEM). The plasma lipid fatty acid composition in palmitate, oleate, and stearate with intake of 3.6 +/- 0.6 g/kg/d of IV lipids (ILs) was similar to EBM-feeding infants taking 6.3 +/- 0.13 g/kg/d of fat. CONCLUSIONS: De novo lipogenesis is active in stable LBW infants maintaining standard postnatal growth. Hepatic DNL permits newborn infants to meet the fat energy needs of peripheral tissues for growth and storage and to maintain plasma fatty acid composition in adaptation to different dietary fat intake.

Dynamic profiling of the glucose metabolic network in fasted rat hepatocytes using [1,2-13C2]glucose.

Recent studies in metabolic profiling have underscored the importance of the concept of a metabolic network of pathways with special functional characteristics that differ from those of simple reaction sequences. The characterization of metabolic functions requires the simultaneous measurement of substrate fluxes of interconnecting pathways. Here we present a novel stable isotope method by which the forward and reverse fluxes of the futile cycles of the hepatic glucose metabolic network are simultaneously determined. Unlike previous radio-isotope methods, a single tracer [1,2-13C2]D-glucose and mass isotopomer analysis is used. Changes in fluxes of substrate cycles, in response to several gluconeogenic substrates, in isolated fasted hepatocytes from male Wistar rats were measured simultaneously. Incubation with these substrates resulted in a change in glucose-6-phosphatase/glucokinase and glycolytic/gluconeogenic flux ratios. Different net redistributions of intermediates in the glucose network were observed, resulting in distinct metabolic phenotypes of the fasted hepatocytes in response to each substrate condition. Our experimental observations show that the constraints of concentrations of shared intermediates, and enzyme kinetics of intersecting pathways of the metabolic network determine substrate redistribution throughout the network when it is perturbed. These results support the systems-biology notion that network analysis provides an integrated view of the physiological state. Interaction between metabolic intermediates and glycolytic/gluconeogenic pathways is a basic element of cross-talk in hepatocytes, and may explain some of the difficulties in genotype and phenotype correlation.

The Warburg effect: a balance of flux analysis.

Cancer metabolism is characterized by increased macromolecular syntheses through coordinated increases in energy and substrate metabolism. The observation that cancer cells produce lactate in an environment of oxygen sufficiency (aerobic glycolysis) is a central theme of cancer metabolism known as the Warburg effect. Aerobic glycolysis in cancer metabolism is accompanied by increased pentose cycle and anaplerotic activities producing energy and substrates for macromolecular synthesis. How these processes are coordinated is poorly understood. Recent advances have focused on molecular regulation of cancer metabolism by oncogenes and tumor suppressor genes which regulate numerous enzymatic steps of central glucose metabolism. In the past decade, new insights in cancer metabolism have emerged through the application of stable isotopes particularly from 13C carbon tracing. Such studies have provided new evidence for system-wide changes in cancer metabolism in response to chemotherapy. Interestingly, experiments using metabolic inhibitors on individual biochemical pathways all demonstrate similar system-wide effects on cancer metabolism as in targeted therapies. Since biochemical reactions in the Warburg effect place competing demands on available precursors, high energy phosphates and reducing equivalents, the cancer metabolic system must fulfill the condition of balance of flux (homeostasis). In this review, the functions of the pentose cycle and of the tricarboxylic acid (TCA) cycle in cancer metabolism are analyzed from the balance of flux point of view. Anticancer treatments that target molecular signaling pathways or inhibit metabolism alter the invasive or proliferative behavior of the cancer cells by their effects on the balance of flux (homeostasis) of the cancer metabolic phenotype.

NOTCH reprograms mitochondrial metabolism for proinflammatory macrophage activation.

Metabolic reprogramming is implicated in macrophage activation, but the underlying mechanisms are poorly understood. Here, we demonstrate that the NOTCH1 pathway dictates activation of M1 phenotypes in isolated mouse hepatic macrophages (HMacs) and in a murine macrophage cell line by coupling transcriptional upregulation of M1 genes with metabolic upregulation of mitochondrial oxidative phosphorylation and ROS (mtROS) to augment induction of M1 genes. Enhanced mitochondrial glucose oxidation was achieved by increased recruitment of the NOTCH1 intracellular domain (NICD1) to nuclear and mitochondrial genes that encode respiratory chain components and by NOTCH-dependent induction of pyruvate dehydrogenase phosphatase 1 (Pdp1) expression, pyruvate dehydrogenase activity, and glucose flux to the TCA cycle. As such, inhibition of the NOTCH pathway or Pdp1 knockdown abrogated glucose oxidation, mtROS, and M1 gene expression. Conditional NOTCH1 deficiency in the myeloid lineage attenuated HMac M1 activation and inflammation in a murine model of alcoholic steatohepatitis and markedly reduced lethality following endotoxin-mediated fulminant hepatitis in mice. In vivo monocyte tracking further demonstrated the requirement of NOTCH1 for the migration of blood monocytes into the liver and subsequent M1 differentiation. Together, these results reveal that NOTCH1 promotes reprogramming of mitochondrial metabolism for M1 macrophage activation.

Peroxisomal proliferator-activated receptor alpha deficiency diminishes insulin-responsiveness of gluconeogenic/glycolytic/pentose gene expression and substrate cycle flux.

Our previous work led to the hypothesis that peroxisomal proliferator-activated receptor alpha (PPAR alpha) modulates insulin action in a compensatory fashion for hepatic glucose balance vs. peripheral glucose disposal. Therefore, we have examined the expression of insulin-dependent gluconeogenic/glycolytic/pentose cycle enzymes and compared these to insulin responsiveness for peripheral vs. hepatic substrate flux and futile cycling in the PPAR alpha knockout mouse. Hepatic gluconeogenic flux, glucose absorption, clearance and recycling, as well as in vivo glucose disposal were evaluated using new mass isotopomer methods. Insulin-dependent gluconeogenic/glycolytic/pentose cycle enzyme expression and glucose futile cycling were diminished; however, glucose disappearance was increased. This supports the hypothesis of hepatic insulin resistance and increased peripheral glucose uptake as compensatory events secondary to the decrease in fatty acid oxidation characteristic of the PPAR alpha knockout. We conclude that 1) the loss of PPAR alpha results in lower expression levels and diminished response to meal regulation for gluconeogenic/glycolytic enzyme expression; and 2) consequently, substrate/futile cycling of glucose is decreased when PPAR alpha is absent despite increased gluconeogenesis. The compensatory changes in liver and peripheral tissue substrate flux and the resultant adaptation for enzyme expression in the liver to have a diminished insulin dependence reflect the loosely linked correlation between phenotype and genotype in hepatic glucose metabolism.

Inherited abnormal thyroid hormone-binding protein causing selective increase of total serum thyroxine.

A 9-yr-old boy is described in whom increased serum T4 concentration, increased T3 uptake, and increased free T4 index were associated with a euthyroid clinical state with normal total serum T3. T4-binding globulin (TBG), measured by RIA, was decreased. Reverse flow paper electrophoresis of serum proteins after reaction with radioactively labeled T4 demonstrated increased binding of T4 to a protein with electrophoretic mobility corresponding to albumin. Displacement of serum protein-bo-nd [125I]T4 activity by increasing concentrations of T4 revealed the presence of a low affinity, high binding capacity system with an association constant similar to that of T4-binding prealbumin. This low affinity binding protein cochromatographed with TBG on a DEAE-Sephadex column which normally separates TBG from T4-binding prealbumin. At free T4 concentrations equivalent to those present in the plasma of normal individuals, the T4 bound to free ratio is higher in the patient than in normals and the total serum T4 level is increased in the presence of normal free T4 concentrations. The relative affinity of this abnormal T4-binding protein for T3 is low compared to that of TBG. The patient's father had the same abnormal binding protein, which was not found in his mother or fraternal twin brother. These data suggest an autosomal dominant mode of inheritance of an aberration leading to synthesis of a new protein instead of normal TBG. The new protein is different from TBG in electrophoretic mobility, T4 and T3 binding, and antigenic properties.

Oxythiamine and dehydroepiandrosterone induce a G1 phase cycle arrest in Ehrlich's tumor cells through inhibition of the pentose cycle.

Transketolase (TK) reactions play a crucial role in tumor cell nucleic acid ribose synthesis utilizing glucose carbons, yet, current cancer treatments do not target this central pathway. Experimentally, a dramatic decrease in tumor cell proliferation after the administration of the TK inhibitor oxythiamine (OT) was observed in several in vitro and in vivo tumor models. Here, we demonstrate that pentose cycle (PC) inhibitors, OT and dehydroepiandrosterone (DHEA), efficiently regulate the cell cycle and tumor proliferation processes. Increasing doses of OT or DHEA were administered by daily intraperitoneal injections to Ehrlich's ascites tumor hosting mice for 4 days. The tumor cell number and their cycle phase distribution profile were determined by DNA flow histograms. Tumors showed a dose dependent increase in their G0-G1 cell populations after both OT and DHEA treatment and a simultaneous decrease in cells advancing to the S and G2-M cell cycle phases. This effect of PC inhibitors was significant, OT was more effective than DHEA, both drugs acted synergistically in combination and no signs of direct cell or host toxicity were observed. Direct inhibition of PC reactions causes a G1 cell cycle arrest similar to that of 2-deoxyglucose treatment. However, no interference with cell energy production and cell toxicity is observed. PC inhibitors, specifically ones targeting TK, introduce a new target site for the development of future cancer therapies to inhibit glucose utilizing pathways selectively for nucleic acid production.

Value of simultaneous T3, T4, and TSH measurements for management of graves' disease in children.

One hundred simultaneous determinations of serum T4 and T3 were performed during the course of treatment of 23 children and adolescents with Graves' disease. Five patients were previously untreated and six were experiencing relapse after treatment was discontinued. During relapse, increased T3 concentration is frequently present when serum T4 concentration is normal and T3 measurement is therefore more reliable for early detection of relapse. During therapy with thionamides, T4 measurement alone is often misleading in assessing adequacy of control achieved by therapy. Commonly, patients who clinically have hyperthyroidism have serum T4 concentrations within the normal range but continue to have elevated T3 concentrations (T3 toxicosis). Similarly, T4 can be suppressed into the hypothyroid range in the clinically euthyroid patient with either a normal or high T3 concentration. In these patients, determination of serum T3 often prevents premature reduction of thionamide dosage. When T4 and T3 concentrations do not clearly demonstrate presence or absence of hypothyroidism, measurement of serum thyroid stimulating hormone can be of value.

Simultaneous determination of free thyroxine and capacity of thyroxine-binding globulin.

A simple method is described for the simultaneous determination of capacity thyroxine-binding of globulin (TBG) and free thyroxine concentration (FT4). The ratio of bound to free T4 (B/F) is first determined for two total-T4 concentrations using a Sephadex G-25 competitive-binding technique. TBG capacity and FT4 can both the calculated assuming a known value of affinity constant of TBG. The method is linear over a twenty-fold serum dilution. FT4 calculated is identical to that calculated using the method of Irvine. TBG capacity is shown to be linearly correlated to TBG concentration as determined by radioimmunoassay and is consistent with a molecular weight of 69,000 Daltons and one T4 binding site per molecule. FT4 is found to correlate with the free thyroxine index in a complicated way, depending on the degree of TBG saturation.