Characteristics of marrow production and reticulocyte maturation in normal man in response to anemia.

Erythropoiesis in normal man was studied during periods of phlebotomy-induced anemia of varying severity. This study permitted a comparison of marrow production measurements over a wide range of marrow production levels. As long as the serum iron remained above 50 mug/100 ml, measurements of plasma iron turnover provided an excellent index of marrow production at all levels of red cell production. In contrast, the absolute reticulocyte count demonstrated a poor correlation with the other measurements. This was shown to be the result of a prolongation of the time required for circulating reticulocytes to lose their reticulum, which correlated with the severity of the anemia. For the clinical application of the reticulocyte count as a measurement of marrow production, an adjustment must be made for this alteration in the circulating reticulocyte maturation time.

Control of marrow production by the level of iron supply.

The level of erythroid marrow production varies widely in different erythropoietic disorders. In part, this reflects the level of erythropoietin stimulation as determined by the severity of the anemia. However, iron supply plays an equally important role in the control of erythropoiesis. As demonstrated in normal individuals subjected to prolonged periods of phlebotomy-induced anemia, the erythroid marrow will increase production by as little as twice to as much as eight times normal, depending on the iron supply available from different iron pools. Whereas the iron delivered from normal reticuloendothelial stores or orally administered iron is sufficient for a marrow production response of only two to three times normal, the increased iron supply from nonviable red cells, hemolysis, or iron dextran infusions permits marrow production to rise acutely to levels of four to eight times normal.

Effect of alcohol on serum folate level.

Alcohol was given orally and intravenously to normal and chronic alcoholic volunteers to study its effect on folate metabolism. Oral alcohol, given to nine subjects on a low folate diet, caused a greater fall in serum Lactobacillus casei folate levels than that seen in eight subjects on a low folate diet alone. This alcohol-induced fall in serum folate level occurred largely during the 1st day of the protocol. Although brief infusions of intravenous ethanol had no effect on serum folate level, a 13 h infusion caused a striking fall in serum folate level between the 8th and 10th h. When ethanol was stopped, the serum folate level returned rapidly to normal. Two chronic alcoholic subjects with different basal levels of serum folate were studied for several weeks on a low folate diet plus alcohol. The serum folate level fell promptly in each subject, rose when alcohol was temporarily stopped, and fell when alcohol was resumed. Folate-deficient megaloblastic anemia developed in 3 wk in the subject with initially marginal serum folate levels, but failed to develop in almost 7 wk in the subject with normal folate stores, as reflected by initially high serum folate levels. Thus, the alcohol-induced fall in serum folate level was apparently not a result of depletion of folate stores. In vitro experiments ruled out an assay artifact as an explanation for the alcohol-induced fall in serum L. casei folate level. It seems likely that alcohol interferes with the delivery of n-5-methyltetrahydrofolic acid from storage areas.

Kinetics of the normal folate enterohepatic cycle.

Detailed studies were undertaken to better define the role of the liver and the folate enterohepatic cycle in folate homeostasis. Three isotopes of folate were employed in a rat model to study several parameters: (a) intestinal transport; (b) variation in hepatic uptake after different routes of administration; (c) hepatic reduction, methylation, and polyglutamate formation; (d) biliary excretion; (e) transport of folate to tissue and its return to liver for re-entry into the enterohepatic cycle. Folate absorption was not affected by the type of folate administered, but subsequent liver accumulation was greater when PteGlu(1) was given rather than CH(3)H(4)PteGlu(1). After liver uptake, CH(3)H(4)PteGlu(1) is rapidly and quantitatively excreted into bile, whereas nonmethylated folates are either methylated and transported into bile or incorporated into a hepatic polyglutamate pool. Bile folate is then reabsorbed for distribution to both tissue and liver, completing the enterohepatic cycle. The importance of this cycle was demonstrated by long-term bile drainage and by transport studies with two isotopes of CH(3)H(4)PteGlu(1). With bile drainage, serum folate levels fell to 30-40% of normal within 6 h, a much more dramatic drop than that seen with folate-free diets alone. Studies with labeled CH(3)H(4)PteGlu(1) demonstrated that about one-third was taken up by tissue, demethylated, and returned to liver for remethylation and recirculation through the bile and gut. This establishes the enterohepatic cycle as a major factor in folate homeostasis and, for the first time, demonstrates a transport pathway between tissue and liver for nonmethylated folate.

Red cell aging in vivo.

Previous studies of red cell structure and metabolism during the aging process have relied upon in vitro techniques of cell separation into various age populations. Probably the most common approach is to isolate the older red cells with the assumption that they are more dense. This may lead to a number of inconsistencies in observations, and may certainly raise questions about possible cell changes secondary to manipulative procedures. For this reason, an experimental system was devised where a normal red cell population could be studied, while aging, in an in vivo environment. The initial red cell mass of a large number of inbred rats was transferred repeatedly into an ever smaller number of animals, making it possible to follow an aging population of red cells up to 48 days while preventing contamination with newly produced cells by suppression of erythropoiesis with transfusion-induced polycythemia. During this period, samples of progressively older red cells could be obtained for measurements of red cell constant. It was noted that the normal rat red cell undergoes both volume reduction and significant hemoglobin content loss with aging. In addition, the hemoglobin concentration within the cell demonstrated an early rise after a return to nearly normal values. These findings are noteworthy in that they help to explain the characteristics of life-spans of cohort labeled red cell populations in small animals, and provide a possible example of a cell's remodeling process within the spleen.

Enterohepatic circulation of methotrexate in rats in vivo.

The pharmacokinetics of the methotrexate enterohepatic cycle were studied in rats in vivo. For plasma levels of methotrexate between 10(-5) and 10(-8) M, biliary levels were directly proportional and concentrated 27-fold. When labeled methotrexate was administered in doses sufficient to achieve plasma levels of 10(-6) M, approximately 50% of methotrexate appeared in the bile in normal animals and up to 80% appeared in anephric animals. In spite of the high percentage of administered methotrexate which appeared in the bile, complete interruption of the enterohepatic cycle in otherwise normal animals did not affect the plasma decay curve of a bolus of methotrexate. The increased biliary excretion which occurred in animals with renal impairment was utilized with possible therapeutic implications. Bile drainage in these animals rapidly decreased plasma methotrexate levels compared to nondrained controls. This suggests that interruption of the methotrexate enterohepatic cycle may provide an alternative for the management of methotrexate toxicity associated with renal insufficiency.

Clinical studies in alcoholic sideroblastosis.

The incidence and characteristics of ring sideroblastic and megaloblastic changes in bone marrow were studied in chronically ill, malnourished alcoholics and well-nourished alcoholics without complicating medical illness. A clear correlation of blood alcohol level with changes in serum pyridoxal-5-phosphate (PLP) values or with the incidence of ring sideroblasts could not be demonstrated. The appearance of ring sideroblasts was associated with dietary restrictions of pyridoxine and noticeable folic-acid-deficient megaloblastosis. The majority of subjects with ring sideroblasts were from the chronic, malnourished alcoholic group; the number of sideroblasts correlated with severity of marrow magaloblastic change. While low serum PLP levels were characteristic of the chronically ill alcoholic with ring sideroblasts, equally low levels were detected in the absence of the marrow abnormality. Thus, a low serum PLP value alone is not a certain indication of the presence of marrow ring sideroblasts.

Use of a physiologic pharmacokinetic model of glucose homeostasis for assessment of performance requirements for improved insulin therapies.

Methods are presented for assessing insulin therapies using a physiologic pharmacokinetic model of glucose homeostasis in man. The model is composed of simultaneous differential equations that represent physiologic compartments and spaces in which glucose and insulin are distributed and undergo metabolic reactions. The model is used to simulate clinical experiments in which blood glucose concentration is controlled by artificial device therapies. Predictions of the theoretical model for responses of normal and diabetic individuals to standard intravenous and oral glucose tolerance tests are compared to clinical data. Reasonable agreement is obtained between predictions of the computer simulations and clinical data for normal individuals. The responses of a diabetic person to oral glucose tolerance tests are simulated by removal of the pancreas from the glucose homeostasis model and introduction of insulin into the model by a prescribed therapy. Model simulations reaffirm expectations concerning the poor blood glucose control attainable by intramuscular insulin injection. Simulations of blood glucose regulation by an artificial pancreas using closed-loop feedback control for controlling insulin delivery rate reveal hyperinsulinemia that results in a net shift in the deposition of a glucose load from liver to peripheral tissues. Simulations of this system in which the time delay for glucose measurement is varied from 1.5 to 30 min show that increases in sensor delay result in progressive loss in glucose regulation, exacerbation of hyperinsulinemia, and increased insulin requirements.

Regulation of vitamin B6 metabolism in human red cells.

The effects of pyridoxine and pyridoxal 5-phosphate (PLP) administration on pyridoxine kinase (PnK) and asparate aminotransferase (EGOT), a PLP-dependent enzyme, were studied in human red cells separated into young and old populations by density centrifugation. After a delay of 48 hr, both pyridoxine and PLP increase EGOT activity in mature red cells by activating preformed GOT apoenzyme. In addition, in young erythroid cells, pyridoxine therapy induces synthesis of PnK, while both pyridoxine and PLP induce synthesis of GOT apoprotein. Thus, PLP stimulates EGOT induction without a change in PnK activity, suggesting that PLP enters erythroid precursor cells without prior dephosphorylation. However, with both pyridoxine and PLP, the full induction of enzyme activities reflect the gradual replacement of circulating red cells by newly formed cells with higher enzyme levels. Therefore, the use of EGOT as a measure of vitamin B6 nutritional status requires recognition of the complexities of intracellular enzyme regulation.

The significance of platelets with increased RNA content (reticulated platelets). A measure of the rate of thrombopoiesis.

Direct flow cytometric measurement of nucleic acid content in individual platelets is possible using the fluorescent dye Thiazole Orange (Becton-Dickinson, San Jose, CA). When applied to studies of thrombocytopenic patients, platelets with elevated nucleic acid content ("reticulated platelets") can be identified and quantitated. Labeling of these platelets is saturable and is abolished by treatment with RNAse. It has been suggested that, similar to the erythrocyte reticulocyte response to anemia, the number of these platelets appearing in the circulation may provide an estimate of the rate of thrombopoiesis. The authors studied 229 thrombocytopenic patients, measuring both reticulated platelets and platelet-associated immunoglobulin. The results show that for the subset of patients with normal levels of platelet-associated immunoglobulin, the average absolute number of reticulated platelets is independent of platelet count and remains in the normal range. For those with elevated levels of platelet-associated immunoglobulin, the absolute number of reticulated platelets increases in patients who are moderately thrombocytopenic (60 to 100 x 10(9)/L) but decreases to normal or subnormal levels as thrombocytopenia worsens. The latter finding has been duplicated in studies of mice made thrombocytopenic by injection of anti-platelet antiserum. These results are consistent with the hypothesis that reticulated platelets are subject to peripheral destruction at the same rate as mature platelets, and that in the severely thrombocytopenic patient their level may decrease despite an appropriate marrow thrombopoietic response.

Quantitative prediction of transdermal iontophoretic delivery of arbutamine in humans with the in vitro isolated perfused porcine skin flap.

The use of the isolated perfused porcine skin flap (IPPSF), an alternative in vitro animal model, to predict the profile of the concentration of arbutamine in plasma samples from humans after transdermal iontophoretic administration of this novel catecholamine is described. The strategy involved administering the drug in the IPPSF (n = 8) and assaying concentrations of drug in the venous efflux versus time (IPPSF venous efflux profile). Intravenous infusion (n = 7) and transdermal studies (n = 32) were also conducted in humans. The IPPSF profile was then used as an input into an intravenous pharmacokinetic model obtained from the human experiments to predict the profile of concentration of drug in plasma versus time (plasma concentration-time profile) seen after iontophoretic administration. The IPPSF profiles were denormalized according to the parameters used in the human studies (i.e., multiplied by in vivo concentration, electrode area, current, and dosing time). For two different sets of iontophoretic dosing conditions, the concentration-time profiles that were predicted on the basis of the IPPSF study were compared with those seen after delivery to humans.

Blood-loss anemia.

Effective management of blood-loss anemia depends on an understanding of the physiologic response to volume loss, the pattern of marrow response to acute depletion of red cell mass, and the controlling influence of iron supply. Each of these elements must be evaluated and incorporated into the plan of management.

Adverse hematologic effects of alcohol.

Alcohol can interfere with the production of new blood cells--RBCs, granulocytes, and platelets; the ability of the host to resist infection; and normal hemostasis. Appreciation of the mechanisms behind this interference and the relative incidence and clinical patterns of each abnormality can be important in the management of affected patients. A limited history, physical examination, and a few screening tests should allow identification of persons who may have difficulty with anesthesia, infection, bleeding, and wound healing because of alcohol's hematologic toxicity.