Cytomorphometry of developing rat liver and its application to enzymic differentiation.

Quantitative stereological methods have been adapted for the measurement of the volume of liver attributable to parenchymal, hematopoietic, and Kupffer cells and for the measurement of the relative and absolute number (per unit volume) of these cell types and the mean volume of the parenchymal cell. These morphological parameters are the main ones for interpreting the biochemical differentiation of liver. Quantitative changes in these parameters, in rat liver between the 15th day of gestation and adult life, are presented. Despite the large number of hematopoietic cells, the parenchymal cells fill more than half of the liver volume between the 15th and 18th days of gestation and 0.85 of the liver volume at term. The fraction of liver volume occupied by Kupffer cells is never more than 0.02; the number of Kupffer cells per cubic centimeter increases less than twofold between fetal and adult life. The mean volume of individual parenchymal cells undergoes a threefold rise during late fetal life, declines in the neonatal period, and doubles between the 12th and 28th postnatal days. With the morphometric data obtained, it is impossible to convert enzyme concentrations (units per gram, determined in homogenates of whole liver) to enzyme amounts per unit volume of parenchymal or hematopoietic tissue or per individual cell of either type. In late fetal liver, only rises in enzyme concentration less than twofold may be attributed to the enrichment of parenchymal tissue at the expense of hematopoietic elements. The sudden upsurge, by more than twofold, of hepatic enzymes of the late fetal cluster (and also of the neonatal and late suckling cluster) reflects rises per parenchymal mass and per parenchymal cell. Thyroxine and glucagon, the administration of which to fetal rats promotes enzyme differentiation in liver, are without appreciable effect on the cytological parameters studied. Hydrocortisone accelerates the involution of hematopoietic tissue in fetal liver. Enzymes that are diminished by prenatal injection of hydrocortisone may be concentrated in hematopoietic cells.

Subcellular morphometric and biochemical analysis of developing rat hepatocytes.

Livers of rats between the 16th gestational and 100th postnatal day of age were subjected to quantitative biochemical and electron microscope, morphometric analyses. The amount of total mitochondrial protein per gram of liver remained at 34% of the adult level throughout the last 4 days of gestation but this was the period of rapid rise in the levels of cytochrome c oxidase, aspartate aminotransferase, and glutamate dehydrogenase in mitochondria; the nuclear fraction also acquired some glutamate dehydrogenase but lost most of it during postnatal development. During early postnatal life the amount of mitochondrial protein rose in parallel with the levels of cytochrome c oxidase and glutamate dehydrogenase but the upsurges of glutaminase and, later, of ornithine aminotransferase were accompanied by relatively little change in total mitochondrial protein. The surface area of rough endoplasmic reticulum per unit volume of hepatocyte cytoplasm (S(v) (RER)) did not change significantly throughout the period of development studied. From the 16th day of gestation to term the surface area of smooth ER (S(v) (SER)), the volume occupied by mitochondria (V(v) (MT)) and their number (N(v) (MT)) remained at 30, 66, and 45% of their adult values, respectively. V(v) (MT) and N(v) (MT) attained their maximal levels by the 2nd postnatal day and S(v) (SER) between days 2 and 12. Mitochondria of adult liver are thus smaller and contain more protein per unit volume than do those of fetal liver. After the 12th postnatal day, hepatocytes treble their size; they acquire more cytoplasm with additional enzymes but without further change in organelle concentration. The data reveal several distinct phases in the differentiation of hepatocytes. Each phase can be characterized by the extent to which the quantity and composition of various subcellular compartments evolve.

Tryptophan pyrrolase induced in human liver by hydrocortisone: effect on excretion of kynurenine.

Administration of hydrocortisone causes two- to fourfold increase in the level of activity of tryptophan pyrrolase in human liver, as measured in needle-biopsy specimens. Correlation of the higher levels of the enzyme with the amounts of urinary kynurenine suggests that the tryptophan pyrrolase level, which is regulated by adrenocortical hormones, may be the important variable in the increased excretion of tryptophan metabolites that accompanies various diseases.

Alpha-methylphenylalanine, a new inducer of chronic hyperphenylalaninemia in sucling rats.

alpha-Methylphenylalanine reduces the phenylalanine hydroxylase activity of rat liver by 75 percent. Daily injections of this substance (plus phenylalanine) into rats from the 3rd to 15th day of age had no obvious toxic effects, and maintained a plasma concentration of phenylalanine comparable to that of phenylketonuric subjects.

Tissue enzyme changes in parabiotic rats with subcutaneous lymphoma or fibrosarcoma.

A solid lymphoma implanted into normal inbred Kx rats and one partner of parabiotic pairs caused appreciable decreases in hepatic ornithine aminotransferase and arginase about a week earlier (4-6 days after implantation) in single hosts than in parabiotic hosts. By 18-21 days significant decreases in both enzymes were apparent in the host partner also. The hepatic thymidine kinase showed a fivefold elevation in single hosts 4 days after implantation; by 14 days in its levels were about 200 times above normal and had also risen in the parabiotic hosts (20-fold) and host partners (fourfold). Implantation of fibrosarcoma caused qualititatively similar but generally less pronounced changes in these three enzymes in livers of single hosts, parabiotic hosts, and host partners. The splenic thymidine kinase 14 days after implantation was increased from control levels of about 3 U/g to 50.6, 44.8, and 13.5 U/g in single hosts, parabiotic hosts, and host partners, respectively. At later stages, 17-20 days after implantation of the lymphoma, appreciable amounts of thymidine kinase appeared in the plasma: The units of activity per milliliter were 6.2 in single hosts, 0.79 in parabiotic hosts, and 0.55 in host partners (control less than 0.05). Our observations on the changes in hepatic and splenic enzymes in parabiotic rats suggest that effects of neoplasms on distant host tissues are mediated by humoral factors. The less pronounced responses in parabiotic than in single hosts indicate that the tumor-free partner affords some "protection" against these systemic effects.

Enzyme pathology of human mesotheliomas.

In samples of 16 surgically resected mesotheliomas arising from the pleura of the human lung, 6 enzymes from different metabolic pathways, DNA, and mitotic frequency were quantified. The mesotheliomas, irrespective of cell type or grade, showed lower gamma-glutamyl transpeptidase (GGT) concentration than 36 of the 38 pulmonary adenocarcinomas. The mean concentration of this enzyme in the 15 mesotheliomas was an eighth of that in the 56 carcinomas, whereas their DNA content was similar. The quantitative correlation of thymidine kinase (TK), uridine kinase (UK), and phosphoserine phosphatase to mitotic frequency was highly significant for mesotheliomas, as well as for carcinomas. As estimated from their TK [and its recently established quantitative correlation to volume doubling time (DT)], the DT of the 16 mesotheliomas ranged from 50 to over 700 days, with a somewhat longer median than the median for pulmonary carcinomas. Subject survival, though shortest for the 2 sarcomatous mesothelioma cases, varied over an overlapping range for mesotheliomas with epithelial or mixed cell type. The biopsy samples' TK and UK concentrations, however, showed a significant inverse correlation with months of survival after diagnosis. Survival time after the first appearance of symptoms decreased linearly (on log scales) with TK concentration (P less than .001) over the 14 cases. The results of this first quantitative study of a spectrum of biochemical constituents of mesotheliomas identify GGT as an enzyme whose measurement guards against mistaking mesotheliomas and adenocarcinomas for one another and show that the TK concentrations of these mesothelioma samples bear a highly significant, inverse correlation to the postdiagnosis survival time of the individual subjects.

The effect of lymphoma and other neoplasms on hepatic and plasma enzymes of the host rat.

Considerable thymidine kinase and pyrroline-5-carboxylate reductase activities were found in the plasma of rats bearing a transplanted lymphoma; neither activity was detected in plasma of hosts carrying hepatic, renal, mammary, or submaxillary gland tumors. All host livers exhibited signs of biochemical immaturity as indicated by the appropriate increases or decreases in the concentrations of the nine enzymes measured. The extent and time schedule of the changes in host liver varied with the enzyme and with the tumor that caused them. The hepatic concentrations of ornithine aminotransferase, arginase, pyrroline-5-carboxylate reductase, and glucokinase (all diminished), and of peptidyl proline hydroxylase and hexokinase (increased) were sensitive indicators of tumor growth in general. The concentration of ornithine aminotransferase decreased before the tumors became palpable. At more advanced stages, the high hepatic thymidine kinase activity distinguished the presence of hepatoma and lymphoma from those of all other equally fast-growing tumors. However, only in lymphoma-bearing rats did a fivefold elevation of hepatic thymidine kinase occur as early as 4 days after implantation. Additional observations on the lymphoma itself, on blood cells, and on the involuting thymus of normal rats indicate that the striking systemic effects of this tumor cannot be explained by a release of enzymes from the thymus or by the increased number of lymphoma cells present in blood or liver.

The undifferentiated enzymic composition of human fetal lung and pulmonary tumors.

The concentrations of 16 to 21 enzymes, representing various metabolic pathways, have been determined in human adult, fetal, and neoplastic lung. At midgestation, 12 enzymes (among them, several that metabolize amino acids) were above their adult values while 3 other enzymes were still at low concentrations. These signs of biochemical immaturity are contrasted and compared with those in fetal human liver and rat lung. The enzymic composition of the 11 human pulmonary tumors studied resembled that of the normal fetal lungs closely; the same 12 enzymes were elevated and the same 2 were decreased (compared to nonneoplastic adult lung) in both. The characteristic abnormality in the overall pattern of enzymes, in the concentrations of individual ones, and in the quality of pyrroline-5-carboxylate reductase was clearly evident in both primary and metastatic tumors. The mean concentrations of 10 enzymes in the tumors were significantly different (higher or lower) from those in the control lungs (p less than 0.001 to less than 0.05). The best markers of neoplasticity were thymidine kinase, peptidyl proline hydroxylase, phosphoserine phosphatase, hexokinase, and pyrroline-5-carboxylate reductase. The results demonstrate that quantification of a small battery of enzymes, none of them tissue specific, can distinguish adult human lung from its neoplasms.

Hormonal and dietary regulation of hepatic enzymes in tumor-bearing rats.

Enzymes in the histologically normal liver of hosts of mammary carcinomas were examined for their responsiveness to endocrine and dietary modulations. Treatments with the developmental stimuli of alanine aminotransferase (glucocorticoids) and of pyruvate kinase (thyroid hormone) which had no effect in control adult rats raised the levels of these enzymes in the tumor-bearing rats. The latter also showed a greater percentage of increase in malic enzyme upon thyroid hormone administration than did control animals. The tumor-induced increase in hexokinase remained unaltered by the various dietary treatments; enzymes at subnormal levels were raised (glucokinase, malic enzymes, and pyruvate kinase) or further decreased (alanine aminotransferase and ornithine aminotransferase) by excessive carbohydrate intake in immature and adult experimental rats. The normal upsurge of glucokinase and malic enzyme upon weaning to the standard solid diet (from the relatively low-carbohydrate-containing milk) was prevented by cancerous growth in the organism. Similarly, the standard diet, which reversed within 2 days the partial loss of these enzymes in normal adult rats fasted for 48 hr, had no restorative effect on the essentially complete loss of the glucokinase and the very low malic enzyme activity in the fasted tumor bearers. The results suggest that failure in the dietary adaptations of hepatic enzymes as well as diminutions of their basal levels contributes to the clinically observed abnormalities in the glucose metabolism of cancer subjects.

Effect of neoplasms on the content and activity of alkaline phosphatase and gamma-glutamyl transpeptidase in uninvolved host tissues.

In rats carrying s.c. or i.p. neoplasms, there were striking (3- to 20-fold) rises in the gamma-glutamyl transpeptidase and alkaline phosphatase activity of several tissues. These included liver, lung, spleen, bone marrow, and circulating granulocytes but not lymphocytes. In response to mammary carcinoma 5A, for example, the gamma-glutamyl transpeptidase activity changed from 0.27 to 1.18 units/g lung and from 0.3 to 4.0 milliunits/million granulocytes; from 2.3 to 17 units and from 2.4 to 46 milliunits were the accompanying increases in alkaline phosphatase. These abnormalities in each host tissue were reversible in that 3 to 7 days after tumor resection the enzymes returned to control levels. Among the secondary factors which might have been responsible for the host tissue changes, it was possible to exclude stimulated adrenocortical secretion, tissue necrosis, and transplantation trauma. Comparisons of the effects of two mammary carcinomas, a fibrosarcoma, and two hepatomas on the gamma-glutamyl transpeptidase and alkaline phosphatase of various "noninvolved" tissues indicate that the faster the growth rate of the tumors, the more striking is this host syndrome.

Uridine kinase, adenylate kinase, and guanase in human lung tumors.

In pulmonary neoplasms, the uridine kinase concentration was higher (2- to 20-fold) than in the noninvolved lung portions of each of the 12 subjects studied. The extent of elevation of uridine kinase in the different tumors showed a significant positive correlation with the rises (1.5- to 30-fold) in thymidine kinase, suggesting that neoplastic transformation in human lung involved coordinated increases in the capacity for the reutilization of different nucleoside phosphates. Adenylate kinase was always at lower levels in neoplasms compared to noninvolved areas of the same lung, and the extent of this loss in the different tumors correlated inversely with the gain in uridine kinase and thymidine kinase. Normal fetal human lung was also deficient in adenylate kinase, while its uridine kinase and thymidine kinase (and also guanase) activities were above the adult levels. The guanase activities of the different neoplasms, unrelated to their uridine kinase or thymidine kinase content, correlated with the activities in the subjects' noninvolved lung. These individual differences were much more striking than those between the neoplastic and control samples. Variations in guanase activity thus appear to be "random," whereas observations on the three other enzymes attest to the orderly nature of biochemical differences among individual tumors and between normal and neoplastic lung.

Peptidyl proline hydroxylase in adult, developing, and neoplastic rat tissues.

A sensitive assay system, optimally supplemented with tritiated protocollagen substrate and cofactors, is described which is suitable for determining the peptidyl proline hydroxylase (PPH) content of a wide spectrum of rat tissues. In most tissues, less than 50 percent of the total activity was soluble; the particulate portion of the activity (concentrated in the mitochondrial and microsomal fractions) was doubled by pretreatment with Triton X-100. Among normal adult tissues, lung had the highest total PPH activity (2.4 times that of liver) and small intestine had the lowest (25 percent that of liver). In brain and lactating mammary gland, the activity was similar to that in skin (60 percent of that in liver). Fetal tissues contained 3 to 8 times more PPH than the corresponding adult tissues, and a much lower portion of the total activity was soluble. In four tissues studied in detail (lung, liver, kidney, and brain), the total PPH declined rapidly during the last few days of gestation; brain attained its low adult value before term, whereas the other three tissues continued to decrease in the course of postnatal development. An injection of cortisol to fetal rats enhanced the decline of PPH in lung, liver, and skull. These experiments suggest that during normal differentiation the decline in collagen synthesis is initiated by fetal glucocorticoid secretion which is maximal on the 19th gestational day. PPH activity appears to be a sensitive indicator of neoplastic growth. In renal, mammary, muscle, and hepatic tumors, the PPH activities were 4 to 10 times higher than in the cognate adult tissue. Even in well-differentiated, slowgrowing tumors, the activity was considerably higher than in any normal, mature, or immature tissue, with the exception of the skull and lung of the 19-day-old fetus.

Responses of bone marrow gamma-glutamyltranspeptidase and alkaline phosphatase in vitro to tumor-elaborated granulocytopoietic factors.

Evidence has been obtained for the humoral mediation of the recently noted tumor-induced rise of the host bone marrow gamma-glutamyltranspeptidase (gamma GT) and alkaline phosphatase (AP) content in vivo: normal rat bone marrow suspensions, if incubated for 18 hr to 3 days with serum from mammary carcinoma hosts, show 2- to 8-fold elevations (per cell) of the same 2 enzymes. The active substance(s) is in the acid-stable, HCI-ethanol-soluble polypeptide fraction of the mammary carcinoma extract, and of the hosts' blood serum. The larger the size of the neoplasm, and the faster its growth rate, the greater the effect of the host serum on the gamma GT and AP of the normal bone marrow cells. In host rats in vivo, this response is followed by increases in the number (as well as the gamma GT and AP content) of circulating granulocytes. Therefore, a positive response on the part of these enzymes in the bone marrow suspension was also sought, and found, upon incubation with preparations which enhance granulocyte colony formation in agar cultures (i.e., colony-stimulating factor and serum from endotoxin-treated rats). The results indicate: (a) that the increase in gamma GT and AP is a necessary prelude to stimulation of granulocyte multiplication by appropriate growth factors; and (b) that measurement of these enzymes in the short-term liquid culture offers a biochemical test for such factors elaborated by cancers or in nonneoplastic conditions.

Enzyme activities and isozyme patterns in human lung tumors.

The isozyme patterns and activities of six enzymes were determined in surgical biopsy samples of lung tumors and non-neoplastic pulmonary areas. Fetal lungs were also examined. No tissue differences were found in the isozyme pattern of hexokinase or alkaline phosphatase; small differences in pyruvate kinase isozyme proportions were observed. The tumors exhibited significant deviations with respect to the lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) isozyme patterns. Despite the diversity of cell types, the proportions of the M-subunit of LDH in each tumor and that of the mitochondrial isozyme of MDH in all but one tumor were higher than in control samples from the same lung. In contrast, the normal fetal lung showed a higher LDH-H proportion than did adult lung and a mature MDH isozyme pattern. The alpha-glycerophosphate dehydrogenase and adenylate kinase activities of the tumors were about one-tenth and one-fourth, respectively, of those of nonneoplastic adult lung. These lower activities (evident also in normal fetal lung) were accompanied by 3- to 5-fold increases in the LDH, MDH, pyruvate kinase, and hexokinase activities of the tumors; fetal lungs had lesser increases (2- to 3-fold) for the first 3 enzymes. The common features of tumors with different cell types and histological grade identified here point to several enzymes the quantitation or isozyme analysis of which may be of practical use in distinguishing cancerous from nonneoplastic human lung samples. A combination of different indicators, such as opposite changes in LDH and alpha-glycerophosphate dehydrogenase activity, coupled with elevated proportions of LDH-M, may be used to diagnose neoplasia most reliably.

Pulmonary carcinoid tumors: enzymic discriminants, growth rate, and early age of inception.

Analyses of enzymes from various metabolic pathways in pulmonary carcinoid tumors and radiological measurements of their volume increase were compared with those for lung carcinomas of various cell types. The results describe new biochemical features in carcinoid tumors, present the first quantitative evidence for their slow growth rate (i.e., long doubling time) in vivo, and show that measurement of 2 or 3 appropriate enzymes in biopsy samples can guard against instances in which carcinoids and adeno- or oat cell carcinomas are mistaken for one another on histological examination. The uridine kinase to thymidine kinase ratio as well as the beta-galactosidase concentration of carcinoid tumors were 5 times higher than of carcinomas, and their gamma-glutamyl transpeptidase was below that of all 35 adeno- and the 11 squamous cell carcinomas. Thymidine kinase, which bears a quantitative inverse correlation to volume doubling time (irrespective of cell type), had much lower titers in the 9 carcinoids than in the 6 oat cell carcinomas and reflects most clearly their very different (despite common histogenesis) clinical malignancy. Owing to their long doubling time, carcinoid tumors on the average required a much longer period (40.5 years) to attain final volume than did carcinomas (17.8 years). The calculated mean age of the subjects when growth began, -0.5 years (as opposed to 51 years for carcinomas), suggests a prenatal or early childhood inception for pulmonary carcinoid tumors.

Characterization of a mammary carcinoma elaborated factor stimulating gamma-glutamyltranspeptidase expression in bone marrow cells.

The elevation of bone marrow gamma-glutamyltranspeptidase (GGT) and alkaline phosphatase (AP) content in rats carrying mammary carcinoma 5A (MC), reproduced in a short-term (48-h) liquid culture of normal bone marrow cells, was found to be attributable to a blood-borne protein factor with an apparent molecular weight of 60,000. Partial purification, based on the extent of stimulation of GGT expression in this culture, increased the specific activity of the host serum from 1.5 to 40 units and that of MC extracts from 6 to 560 units. Production of the factor by MC in vitro, however, resulted in specific activities of 3000 units in the conditioned medium, and a further 60-fold purification was achieved by DEAE-cellulose, Sephadex G-100, and hydroxylapatite chromatography. The chemical characteristics of the MC-elaborated protein indicate nonidentity to previously isolated colony formation stimulating factors which also induced GGT (and AP) expression by rat bone marrow cells. Most of the AP inducing ability of the MC-serum and MC-conditioned medium copurified with and was still present in preparations with the highest specific activity vis à vis GGT. In mouse (instead of rat) bone marrow cells, however, no AP response accompanied the stimulation of GGT expression by MC (or colony formation stimulating factor) preparations.

Distribution of mitochondrial enzymes between the perikaryal and synaptic fractions of immature and adult rat brain.

The subcellular distribution of mitochondrial enzymes was studied in cerebral hemispheres of 15-day-old and adult rats. At both ages the synaptosomal fraction contained very little glutamate dehydrogenase (EC 1.4.1.2) but significant amounts of succinate dehydrogenase (EC 1.3.99.1), glutaminase (EC 3.5.1.2), hexokinase (EC 2.7.1.1), malate NADP dehydrogenase (EC 1.1.1.40) and beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30). In immature brain, in the fraction enriched with free (perikaryal) mitochondria, the concentrations of these enzymes were 9.5, 1.8, 2.0, 0.92, 1.5, and 2.1 times higher, respectively, than in the synaptosomes. The increase with age in succinate dehydrogenase and glutaminase was restricted to free mitochondria while hexokinase and malate NADP dehydrogenase accumulated and beta-hydroxybutyrate dehydrogenase diminished in both fractions. In adult brain, too, where the above ratios became 7.5, 5.2, 3.5, 0.84, 1.4, and 2.0, respectively, the concentrations of enzymes relative to each other distinguished clearly between free and synaptic mitochondria. The results substantiate previously noted signs of mitochondrial heteroeneity in adult brain, and extend them to immature brain. The chemical composition, the quantitative pattern of enzymes, of free and synaptic mitochondria is clearly different, and undergoes separate changes during postnatal differentiation.

gamma-Glutamyltranspeptidase activity in intact leukocytes: flow cytometric analysis and sorting.

A fluorescent method developed for visualizing gamma-glutamyltranspeptidase (GGT) in intact liver cells was adapted to leukocytes and used in a multiparameter flow cytometric study of blood and bone marrow cells from rats with subcutaneous implants of mammary carcinoma 5A. The severe granulocytosis caused by this non-metastatic tumor was preceded by a progressive rise in the percentage of leukocytes with high GGT fluorescence. Both granulocytes and small, immature cells of bone marrow showed increased GGT expression, whereas in blood this increase was attributable entirely to mature granulocytes. At 28 days (but not yet at 14 days) after carcinoma implantation, 20-30% of blood or bone marrow granulocytes constituted a distinct subpopulation in that their GGT fluorescence intensity range was much higher and did not overlap with the range for the rest of the population. The results indicate that fluorescent GGT assay of intact leukocytes provides a useful probe for flow cytometric analysis of population heterogeneity in leukoproliferative disorders.

Cerebral serotonin regulation by phenylalanine analogues and during hyperphenylalaninemia.

Severe hyperphenylalaninemia induced in infant rats by 3 days of treatment with p-chlorophenylalanine (p-cl phe) plus phenylalanine (phe) did not lower the tryptophan concentration of the brain, and the cerebral serotonin (5-HT) deficiency was attributable entirely to the known suppression to tryptophan hydroxylase (TPH) by p-cl phe. The decrease in 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) was thus no more pronounced than in rats which, treated with p-cl phe alone, were devoid of hyperphenylalaninemia. Suppression of TPH was found to also underlie the decrease in cerebral 5-HT caused by treatment with alpha-methylphenylalanine (alpha-mephe) alone: a 22% loss of midbrain TPH activity was detectable 24 hr after an injection only, reverted toward the normal during the next 2 days, and was clearly unrelated to the weak competitive inhibition of the enzyme by alpha-mephe in vitro. However, alpha-mephe (unlike p-cl phe), when administered together with phe, did not suppress TPH, nor did it counterbalance the reduction of cerebral tryptophan uptake by excess phe. Thus the 5-HT diminution in the rat model of phenylketonuria produced by treatment with alpha-mephe plus phe was attributable to hyperphenylalaninemia and the inhibition of tryptophan transport to the brain. Injection of tryptophan was found to restore the cerebral 5-HT level in the face of persistently severe hyperphenylalaninemia.

Lymphocyte thymidine kinase and treatment response in acute lymphocytic leukemia.

The activity of thymidine kinase (TK) and the proportion of its isozymes (TK1/TK2) were studied in peripheral lymphoid cells of 37 children with acute lymphocytic leukemia (ALL). The high TK in 25 untreated subjects (31.5 +/- 8.9) decreased during chemotherapy-induced remission to uniformly low (5.3 +/- 0.4) normal values, and rose again during relapse to a mean of (24.8 +/- 8.1). The proportion of isozyme 1 followed the same pattern but TK was a more sensitive indicator of disease state. The lymphocyte fractions' TK (per mg protein) correlated with the number (per ml blood) of WBCs, blasts and lymphocytes. Although the higher TK of blasts than of apparently normal lymphocytes was confirmed in cases permitting clean physical separation, the lymphocyte fraction of several untreated subjects with minimal blast counts also exhibited elevated TK. Moreover, this elevation was also seen in relapsed cases even if their blood (unlike bone marrow) was devoid of blasts. The results indicate that quantification of TK can reveal a subpopulation of maldifferentiated lymphocytes which are microscopically normal and that it may provide an objective parameter of prognostic differences between ALL subjects with similar hematological characteristics.