[How I do in front of an hemolytic anemia of unknown etiology?] / Que faire devant une anémie hémolytique sans étiologie retrouvée ?

The most frequent causes of hemolytic anemias are immune or infectious diseases, drug induced hemolysis, thrombotic microangiopathies, hereditary spherocytosis, glucose-6-phosphate dehydrogenase or pyruvate kinase deficiencies, thalassemia's and sickle cell disease. Sometimes no cause is found because a rarer etiology is involved. The goal of this review is to remember some unfrequent constitutional or acquired causes and to point out difficulties to avoid wrong interpretations of analysis results.

B Vitamins Can Reduce Body Weight Gain by Increasing Metabolism-related Enzyme Activities in Rats Fed on a High-Fat Diet.

B vitamins are enzyme cofactors that play an important role in energy metabolism. The aim of this study was to elucidate whether B vitamin administration can reduce body weight (BW) gain by improving energy metabolism-related enzyme activities in rats fed on a highfat diet. Fifty rats were randomly assigned to one of the following five groups: control group (C), including rats fed on standard rat chow; four treatment groups (HO, HI, H2, and H3), in which rats were fed on a high-fat diet. Rats in the HI group were treated daily with 100 mg/kg BW thiamine (VB1), 100 mg/kg BW riboflavin (VB2), and 250 mg/kg BW niacin (VPP); rats in the H2 group were treated daily with 100 mg/kg BW pyridoxine (VB6), 100 mg/kg BW cobalamin (VB12), and 5 mg/kg BW folate (FA); and rats in the H3 group were treated daily with all of the B vitamins administered to the HI and H2 groups. After 12 weeks, the BW gains from the initial value were 154.5±58.4 g and 159.1±53.0 g in the HI and C groups, respectively, which were significantly less than the changes in the HO group (285.2±14.8 g, P<0.05). In the HO group, the plasma total cholesterol (CHO) and triglyceride (TG) levels were 1.59±0.30 mmol/L and 1,55±0.40 mmol/L, respectively, which were significantly greater than those in the HI group (1.19±0.18 mmol/L and 0.76±0.34 mmol/L, respectively, P<0.05). The activities of transketolase (TK), glutathione reductase, and Na+/K+ adenosine triphosphatase were significantly increased in the B vitamin-treated groups and were significantly greater than those in the HO group (P<0.05). Furthermore, the glucose-6-phosphate dehydrogenase, pyruvic acid kinase, and succinate dehydrogenase activities also were increased after treatment with B vitamins. Supplementation with B vitamins could effectively reduce BW gain and plasma levels of lipids by improving energy metabolism-related enzyme activities in rats, thus possibly providing potential benefits to humans.

Erythrocyte pyruvate kinase deficiency in an old-order Amish cohort: longitudinal risk and disease management.

Pyruvate kinase deficiency is a chronic illness with age specific consequences. Newborns suffer life-threatening hemolytic crisis and hyperbilirubinemia. Adults are at risk for infections because of asplenia, pregnancy-related morbidity, and may suffer organ damage because of systemic iron overload. We describe 27 Old Order Amish patients (ages 8 months-52 years) homozygous for c.1436G>A mutations in PKLR. Each subject had a predictable neonatal course requiring packed red blood cell transfusions (30 ± 5 mL/kg) to control hemolytic disease and intensive phototherapy to prevent kernicterus. Hemochromatosis affected 29% (n = 4) of adult patients, who had inappropriately normal serum hepcidin (34.5 ± 12.7 ng/mL) and GDF-15 (595 ± 335pg/mL) relative to hyperferritinemia (769 ± 595 mg/dL). A high prevalence of HFE gene mutations exists in this population and may contribute to iron-related morbidity. Based on our observations, we present a strategy for long-term management of pyruvate kinase deficiency.

Tumor M2 pyruvate kinase: a tumor marker and its clinical application in gastrointestinal malignancy.

Proliferating cells, in particular tumor cells, express a dimeric isoenzyme of pyruvate kinase, termed Tumor M2 pyruvate kinase. In the last few years, much attention has been paid to this novel tumor marker that can be determined in EDTA-plasma and in the feces. It has been used in diagnosis and surveillance of a variety of malignant diseases. As compared with the established tumor markers, Tumor M2-PK in EDTA-plasma proves to have at least equal sensitivity in pancreatic, gastric, esophageal, colorectal and cholangiocellular cancer. In combination with established tumor markers, EDTA-plasma M2-PK is a useful tool in diagnosis and surveillance of gastrointestinal tumors. In colorectal cancer, M2-PK in EDTA-plasma even proves superiority as compared with CEA. Fecal Tumor M2-PK testing resembles a good noninvasive screening parameter for colorectal cancer with a reported sensitivity of 68.8-91.0% and a specificity of 71.9-100%. It is superior to fecal occult blood testing in colorectal cancer screening. Since it is effective, easy to handle and bears rather low costs, fecal Tumor M2-PK testing is recommended for large-scale CRC screening.

Antioxidant activity and metabolite profile of quercetin in vitamin-E-depleted rats.

Dietary antioxidants interact in a dynamic fashion, including recycling and sparing one another, to decrease oxidative stress. Limited information is available regarding the interrelationships in vivo between quercetin and vitamin E. We investigated the antioxidant activity and metabolism of quercetin (Q) in 65 F-344 rats (n=13 per group) randomly assigned to the following vitamin E (VE)-replete and -deficient diets: (a) VE replete (30 mg alpha-tocopherol acetate/kg diet) control ad libitum (C-AL), (b) VE replete pair fed (C-PF), (c) VE replete+5.0 g Q/kg diet (R-VE+5Q), (d) VE deplete (<1 mg/kg total tocopherols)+5.0 g Q/kg diet (D-VE+5Q) and (e) D-VE. After 12 weeks, blood and tissue were collected for measurement of plasma vitamin E, quercetin and its metabolites, serum pyruvate kinase (PK), plasma protein carbonyls, malondialdehyde (MDA) and oxygen radical absorbance capacity. D-VE diets decreased serum alpha-tocopherol and increased PK activity in a time-dependent manner. The D-VE diet increased plasma protein carbonyls but did not affect MDA. Dietary quercetin supplementation increased quercetin and its metabolites in plasma and liver but did not affect D-VE-induced changes in plasma alpha-tocopherol, PK or protein carbonyls. Plasma isorhamnetin and its disposition in muscle were enhanced by the D-VE diet, as compared to the R-VE diet. Conversely, tamarixetin disposition in muscle was decreased by the D-VE diet. Thus, quercetin did not slow vitamin E decline in vivo; neither did it provide antioxidant activity in vitamin-E-depleted rats. However, vitamin E status appears to enhance the distribution of isorhamnetin into the circulation and its disposition in muscle.

Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats.

The recent discovery that vitamin E (VE) regulates gene activity at the transcriptional level indicates that VE may exert part of its biological effects by mechanisms which may be independent of its well-recognised antioxidant function. The objective of this study was the identification of hepatic vitamin E-sensitive genes and examination of the effects of VE on their corresponding biological endpoints. Two groups of male rats were randomly assigned to either a VE-sufficient diet or to a control diet deficient in VE for 290 days. High-density oligonucleotide microarrays comprising over 7000 genes were used to assess the transcriptional response of the liver. Differential gene expression was monitored over a period of 9 months, at four different time-points, and rats were individually profiled. This experimental strategy identified several VE-sensitive genes, which were chronically altered by dietary VE. VE supplementation down-regulated scavenger receptor CD36, coagulation factor IX and 5-alpha-steroid reductase type 1 mRNA levels while hepatic gamma glutamyl-cysteinyl synthetase was significantly up-regulated. Measurement of the corresponding biological endpoints such as activated partial thromboplastin time, plasma dihydrotestosterone and hepatic glutathione substantiated the gene chip data which indicated that dietary VE plays an important role in a range of metabolic processes within the liver.

Protection of branched-chain amino acids against ischemic myocardial injury in rats.

The protective effects of Branched-Chain Amino Acids (BCAA) on ischemic myocardium in rats were studied. Thirty Wistar rats (15 female and 15 male) were randomly divided into 3 groups of 10 animals each. Group A: control; group B: isoproternol (ISO); group C: ISO + BCAA. The rats in groups A and B received normal synthetic rat chow while those in group C received BCAA as supplement. After two weeks of dietary treatment, the rats in group A were injected with saline while those of groups B and C were injected with ISO which induced acute ischemic myocardial injury. After 4 days of injections with either saline or ISO, the rats were sacrificed. The activities of lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), alpha-hydroxybutyrate dehydrogenase (alpha-HBDH), creatine kinase (CK), pyruvate kinase (PK) in the serum and in the myocardium, and the concentrations of potassium (K+), magnesium (Mg2+), calcium (Ca2+) in the myocardium were measured. The results showed that the activities of LDH, GOT, alpha-HBDH, CK, PK in the serum and in the myocardium were significantly increased in group B. In addition, the concentrations of Ca2+ in the myocardium were significantly increased. However, the concentrations of Mg2+ in the myocardium were substantially decreased while those of K+ in group B were slightly lowered. In the group C animals both the activities of LDH, alpha-HBDH, PK, CK in the serum and the activities of LDH, GOT, alpha-HBDH, CK in the myocardium were significantly lower than those of the rats in group B, and were not significantly different from those of the control group. More significant was the concentrations of Ca2+ in the myocardium of the rats in group C were comparable to those of the control rats but were significantly lower than those of the rats in group B. It appeared that BCAA was effectively blocking the increase of Ca2+ in the myocardium without raising the level of Mg2+. It was concluded that dietary supplement with BCAA provided some protective effects against ischemic myocardium in rats.

Dietary oxidized frying oil enhances tissue alpha-tocopherol depletion and radioisotope tracer excretion in vitamin E-deficient rats.

Rats fed a diet containing 15% oxidized frying soybean oil (OFO) have been shown to have significantly lower tissue alpha-tocopherol (alpha-T) concentration than rats fed a 15% fresh soybean oil diet. To examine the turnover of alpha-tocopherol, a depletion-repletion experiment and a radioisotope tracer study were conducted. Two groups of male weanling Long-Evans rats were fed vitamin E-deficient diets containing either 15% OFO or 15% vitamin E-stripped fresh soybean oil (control). After 9 wk of depletion, rats fed the OFO diet had significantly higher plasma pyruvate kinase (PK) activity and lower concentrations of alpha-T in RBC, adrenal gland, heart, kidney, liver, spleen, testis and muscle compared with controls (P < 0.05), indicating that the vitamin E-deficient status was aggravated by feeding the OFO diet. After 12 wk, the depleted rats were intraperitoneally injected with a dose of all-rac-alpha-T (2.5 mg/rat, dissolved in Vitamin E-stripped corn oil) every other day. Three doses were administered to each rat during the 1-wk repletion period. Plasma PK activity decreased in both groups (P < 0.05) after repletion but that of the OFO rats was still significantly higher than that of the control group. The repleted OFO gorup also had significantly lower alpha-T concentration in adrenal gland, epididymal fat, liver and spleen than the repleted control group. Two rats from each group that had been vitamin E-depleted for 16 wk were injected intraperitoneally with a single dose of 5-methyl-14C-RRR-alpha-T (740 kBq/kg body weight). During the week after dosing, the radioactivity excreted in urine and feces of the OFO group was 1.3- and 1.7-fold, respectively, that of the control group. Tissue retention of radioactivity was also lower in the OFO rats than in the control rats. The results suggest that more of the alpha-T in the body was catabolized or turned over in rats fed the OFO-containing diet.

Compound heterozygous mutations affecting both hepatic and erythrocyte isozymes of pyruvate kinase.

Novel erythrocyte pyruvate kinase gene defects were found in a patient without a family history of consanguinity. The polymerase chain reaction products of the R-type pyruvate kinase cDNA from the propositus contained two point mutations of Ser80 (TCC)-->Pro (CCC) and Arg490 (CGG)-->Trp (TGG). Allele-specific polymerase chain reaction of the genomic DNA revealed that this patient was a compound heterozygote. The mobilities of the patient's L- and R-type pyruvate kinase by thin-layer polyacrylamide gel electrophoresis were abnormal. The results are consistent with the fact that these mutations are within exons common to the hepatic and erythrocyte isozymes.

Functions of vitamin E in reproduction and in prostacyclin and immunoglobulin synthesis in rats.

The bioactivities of RRR-alpha-, beta-, gamma-, and delta-tocopherol (T) and R-alpha-tocotrienol (R-alpha-TT) were determined in rat resorption-gestation tests. The ranking order was RRR-alpha-T greater than RRR-beta-T greater than RRR-gamma-T greater than or equal to R-alpha-TT greater than RRE-delta-T. Accordingly, the biopotency of a palm-oil residue was assessed and expressed as alpha-tocopherol equivalents (alpha-TEs). The release of pyruvate kinase, a variable in the nutrition-linked necrotizing myopathy, into the plasma was dose-dependently inhibited by the RRR-alpha-T standard and the corresponding alpha-TE from this residue. Prostacyclin synthesis from aorta segments induced by thrombin or ionomycin was higher than the spontaneous release. However, there was no difference between the depleted group and groups treated with RRR-alpha-T or alpha-TEs from the palm-oil residue. Quantities of IgG in plasma of vitamin E-depleted rats were the highest. Upon supplementation with RRR-alpha-T or alpha-TEs from the palm-oil residue, reduced IgG concentrations were observed, similar to those of animals on a commercial diet containing adequate amounts of vitamin E.

Effects of vitamin E on toxicity by minute amounts of paraquat fed continuously to rats.

The effects of vitamin E on toxicity by minute amounts of paraquat fed continuously for some period to rats were investigated. Two experiments were carried out as experiments 1 and 2. In both experiments, weaning rats were divided at first into two groups; one group was given a vitamin E-deficient diet, and the other a vitamin E-supplemented control diet (50 mg alpha-tocopherol/kg of diet). They were fed on these diets for 40 days. After that, in both experiments, the rats that had been fed the vitamin E-deficient diet were further divided into two groups, which were either given a paraquat-added diet (+PQ-E) or continuously fed the same vitamin E-deficient diet (-E). The amount of paraquat added was 250 mg of methyl viologen per kg of diet. After the addition of paraquat, these two groups were pair-fed. In experiment 1, paraquat was given to all the rats fed the vitamin E-supplemented control diet (+PQ+E). In experiment 2, rats fed the control diet were divided into paraquat-added (+PQ+E) and non-paraquat-added (+E) groups, similar to those of vitamin E-deficient rats. These two groups were also pair-fed thereafter. In both experiments, about 35 days after paraquat addition, they were sacrificed. Plasma and liver alpha-tocopherol contents were measured by HPLC, and liver peroxidation value was measured by chemiluminescence and the TBA method. And, as parameters of vitamin E deficiency, plasma pyruvate kinase and GOT activities and alpha-cysteine proteinase inhibitor (alpha-CPI) level were measured. When the analyzed values were compared between paraquat-added and the corresponding not-added control groups (+PQ-E vs. -E, +PQ+E vs. +E), the following results were obtained. In experiment 1, the values of plasma and liver alpha-tocopherol levels were significantly lower in the +PQ-E group than those of the -E group; however, liver peroxidation values and values of the three parameters of vitamin E deficiency were not different significantly. In experiment 2, the value of liver alpha-tocopherol level was significantly lower in the +PQ+E group than that of the +E group.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of dietary vitamin E and beta-carotene on oxidation processes in the rat testis.

The effects of dietary vitamin E and beta-carotene were studied on enzymes involved in arachidonic acid metabolism and other related enzymes in the rat testis. Groups of rats were fed various soybean oil-based semi purified diets. Group 1 was fed a vitamin E-supplemented diet (+E - beta); Group 2 was fed a beta-carotene-supplemented diet (-E + beta); Group 3, the control group (-E - beta) was fed a vitamin E-deficient diet; and Group 4, the standard diet group (S), was fed vitamin E plus beta-carotene-standard diet. Soybean oxidized oil was added to the three diet groups - (+E - beta), (- E + beta) and (- E - beta), whereas the diet of S group contained non-oxidized oil. After 8 weeks rats were killed, blood and testis samples were collected for biochemical determinations. Vitamin E deficiency caused significant increase in testis thiobarbituric acid value and activities of testis NADPH oxidase, testis 15-lipoxygenase and in plasma pyruvate kinase. In contrast, significant decreases were observed in activity of testis prostaglandin synthetase, compared with antioxidant-supplemented diet groups. We also found a significant increase in 15-lipoxygenase activity in (- E + beta) diet group, compared with (- E - beta) diet group. Fatty acid analysis of testis parenchyma indicated decrease in palmitate (16:0) and arachidonate (20:4(n - 6)), and increase in oleate (18:1(n-6)) linoleate (18:2(n - 6)) and linolenate (18:3(n - 3)), when compared (-E - beta) diet group with vitamin E-supplemented diet groups. The results suggest that dietary vitamin E has a role in both enzymatic and non-enzymatic peroxidation of polyunsaturated fatty acids in the testis.

Increased indices of lipid peroxidation in stress-susceptible pigs and effects of vitamin E.

When stress-susceptible and stress-resistant pigs consumed diets containing 10 iu vitamin E kg-1, the stress-susceptible pigs had damaged cell membranes. This was indicated by increased plasma activities of creatine kinase and pyruvate kinase. Plasma concentrations of thiobarbituric acid reactive substances and conjugated dienes were also increased suggesting that the membrane damage was caused by free radicals. Susceptibility to free radicals was further demonstrated by an increased tendency for erythrocytes and tissue homogenates from stress-susceptible pigs to peroxidise when incubated with hydrogen peroxide and iron, respectively. Supplementation of the diets with approximately 20 times normal requirements of vitamin E decreased plasma creatine kinase and pyruvate kinase activities, and inhibited the formation of the indices of peroxidation in vivo and in vitro. It is concluded that the stress-susceptible pig has an abnormality in its antioxidant defence mechanisms. However, this abnormality was not due to a deficiency in selenium-dependent glutathione peroxidase, since activities were significantly increased in the longissimus dorsi of stress-susceptible pigs. Although the nature of the antioxidant defect is unclear, it is suggested that stress-susceptible pigs are under a sustained oxidant stress and that a decreased ability to accommodate even a normal free radical load may contribute to the rapid development of the fatal stress response.

Effect of khat on the metabolism of erythrocytes.

The plant khat "Catha Edulis Forsk" is widely distributed among most East African countries, Yemen and many other areas of the world. Administration of khat extract by the intragastric route in rabbits affected the metabolism of erythrocytes. There is a significant decrease in pyruvate kinase and the level of reduced glutathione (P less than 0.001), and a highly significant increase in both glucose-6-phosphate dehydrogenase and glutathione reductase activities (P less than 0.001) in khat-fed rabbits as compared to controls. On the other hand the activity of uridyl transferase as well as the concentration of 2,3-diphosphoglycerate were not significantly changed in experimental khat-fed rabbits (P greater than 0.5).

Effect of ascorbic acid and vitamin E on biochemical changes associated with vitamin E deficiency in rats.

Weanling male Sprague Dawley rats were fed a vitamin E and C-free basal diet with or without supplementation of 100 IU vitamin E per kg diet. After 20 weeks, the vitamin E-deficient rats were divided into four groups, six in each group, and received supplemental ascorbic acid and/or vitamin E by tube feeding daily for 7 days: Group I, 30 mg ascorbic acid/100 g body wt.; Group II, 0.03 mg RRR-alpha-tocopheryl acetate/100 g body wt.; Group III, 30 mg ascorbic acid and 0.03 mg RRR-alpha-tocopheryl acetate/100 g body wt.; and Group IV, placebo. The six control rats (Group V) received placebo. The rats were sacrificed, blood and liver samples were collected for biochemical determinations. Vitamin E deficiency significantly increased erythrocyte (RBC) spontaneous hemolysis, liver thiobarbituric acid (TBA) value, activities of glutamateoxaloacetate transaminase (GOT), pyruvate kinase (PK), and creatine phosphokinase (CPK) in plasma, and significantly lowered plasma vitamin E levels and glutathione peroxidase (GPX) activities. Tube-feeding ascorbic acid for 7 days produced partial reversal effect on liver TBA values, activities of plasma PK, GOT, CPK, and plasma vitamin E levels but not on RBC hemolysis and plasma GPX activity. Tube feeding both ascorbic acid and vitamin E showed similar partial reversal effect as feeding vitamin E alone on all the parameters stated above. The results suggest that ascorbic acid may spare the metabolism of vitamin E and partially reverse the changes in some of the biochemical parameters characteristic of vitamin E deficiency.

Dietary vitamin E requirement for optimum immune responses in the rat.

Supplementation of diets with vitamin E has been shown to enhance immune responses in numerous animal models. However, these experiments have not investigated the dietary requirement of vitamin E for optimal T- and B-lymphocyte mitogen responses and compared this directly with the requirement for growth, maintenance of spleen-body weight ratios, platelet count as well as prevention of myopathy and red blood cell lysis. We have found that male weanling rats maintain normal rate of growth and spleen-body weight ratio when fed purified diets containing 7.5 mg/kg vitamin E. A level of 15 mg/kg was adequate to prevent myopathy, and 50 mg/kg was necessary for the prevention of red blood cell hemolysis. The dietary requirement for optimum T- and B-lymphocyte responses to mitogens was greater than 50 mg/kg and was significantly correlated with plasma vitamin E levels over a range of 0.04-18 micrograms/ml. Thus, the requirement for this index of immune system activity was higher than for the other functional parameters of vitamin E adequacy measured. Therefore, the immune system responds to changes in dietary vitamin E well before there are signs of frank vitamin deficiency.

Neocytopheresis: a new approach for the transfusion of patients with thalassaemia major.

At present the treatment of thalassaemia major consists of regular blood transfusions coupled with chelation therapy using deferoxamine. A complementary approach to the problem is the use of blood units enriched with young red cells (neocytes), which reduce the transfusional frequency and thereby diminish the risk of iron overload. Young red cell units were collected from blood from 60 volunteer donors using a cell separator (IBM 2997). Donors' blood was anticoagulated and the young red cell harvesting carried out over 4 h at a constant rotor speed of 500 rpm. Three biological criteria were used to evaluate young red cell quality: the number of reticulocytes, the pyruvate kinase activity and the mean corpuscular volume, all of which show an enrichment of young red cells as compared to standard donor units. The 51Cr young red cell survival in four normal donors and in two splenectomized patients showed an increased red cell half-life compared to the same study performed with standard blood units. Blood consumption was diminished significantly when the two patients were transfused with young red cell units. It must be emphasized that, despite the high cost of this blood product, the efficiency of this transfusion technique, by reducing blood consumption, represents important progress and a hopeful treatment for chronic anaemia.

Some effects of selenium deficiency on glutathione peroxidase (EC 1.11.1.9) activity and tissue pathology in rainbow trout (Salmo gairdneri).

1. Two duplicate groups of rainbow trout (Salmo gairdneri; mean weight 27 g) were given diets of differing selenium content (deficient 0.025 mg Se/kg; supplemented 1.022 mg Se/kg) for 30 weeks. 2. There were no significant differences between treatments in weight gain but packed cell volume, liver vitamin E and liver and plasma Se concentrations were all significantly lower in the Se-deficient trout. 3. Ataxia occurred in about 10% of the Se-deficient trout and histopathologies were evident in nerve cord (damage to axon sheath) and liver (loss of integrity in endoplasmic reticulum and mitochondria with appearance of increased vesiculation). 4. Glutathione peroxidase (EC 1.11.1.9) activity was significantly reduced in liver and plasma of Se-deficient fish but there was no indication, from differential assay, of any non-Se-dependent glutathione peroxidase activity. Glutathione transferase (EC 2.5.1.18) activity was significantly increased in Se-deficient trout.

Stereoisomers of alpha-tocopheryl acetate. IV. USP units and alpha-tocopherol equivalents of all-rac-, 2-ambo- and RRR-alpha-tocopherol evaluated by simultaneous determination of resorption-gestation, myopathy and liver storage capacity in rats.

Whereas the alpha-tocopheryl acetate (alpha-TA) stereoisomers have frequently been compared in various vitamin E tests, only little studies have been made on the free alpha-tocopherols. For this reason, comparative tests of the alpha-tocopherol (alpha-TOH) stereoisomers were made by means of the resorption-gestation method, not only individually but also against the USP standard all-rac-alpha-TA. In some of these tests, the parameter resorption-gestation was supplemented by determination of pyruvate kinase (PK) activity as well as by measurement of the vitamin E level in the liver. Quantitative statistical evaluation of the results was made by weighted probit analysis or by symmetrical parallel line assays. Comparison of all-rac-alpha-TOH (dl-alpha-TOH) with RRR-alpha-TOH (d-alpha-TOH) in the resorption-gestation test revealed an activity ratio of 1:1.38. Compared with 2-ambo-alpha-TOH, RRR-alpha-TOH was more active by a factor of 1.34 (resorption-gestation) or 1.45 (myopathy). On account of its great importance in practice, the comparison between RRR-alpha-TOH and the USP standard was repeated three times. With the parameters resorption-gestation and myopathy, mean activities on a weight basis of 0.69 and 0.74, respectively, were measured. These results fail to confirm the established activity of 1 mg RRR-alpha-TOH = 1.49 USP units. If vitamin E activity is to be given in alpha-tocopherol equivalents (alpha-TE), the values are referred to RRR-alpha-TOH and the calculation is made with their reciprocals. Depending on the parameter used, the activity of 1 mg all-rac-alpha-TA was equivalent to 1.45 alpha-TE (resorption-gestation) or 1.35 alpha-TE (myopathy) and was thus noticeably higher than the established value of 0.67 alpha-TE. For the first time, three parameters were determined simultaneously in the comparison of all-rac-alpha-TOH with the USP standard. Quantitative evaluation yielded the following activities: Resorption-gestation, 0.52; Myopathy, 0.63; liver storage, 0.68. The currently accepted value of 1.10 USP units for 1 mg all-rac-alpha-TOH was thus not even reached approximately. In the light of our experimental findings as well as of those of other laboratories, the activities of the alpha-tocopherol stereoisomers expressed in USP units and of their esters expressed in alpha-tocopherol equivalents need to be corrected.

Sequential changes in red cell glycolytic enzymes and intermediates and possible control mechanisms in the first two months of postnatal life in lambs.

The sequential changes in several glycolytic enzymes, glucose-6-phosphate dehydrogenase, glycolytic intermediates, and adenosine triphosphate, as well as intracellular pH and plasma inorganic phosphorus were followed simultaneously in eight lambs from birth to 2 months of age. The activities of all glycolytic enzymes and glucose-6-phosphate dehydrogenase were elevated at birth. The 2,3-diphosphoglycerate concentration increased markedly postnatally and was associated with a simultaneous increase in the concentrations of red cell glucose-6-phosphate and total triose phosphate and a decrease in intracellular pH. Inorganic phosphorus also increased and correlated with the 2,3-diphosphoglycerate concentration in the first 10 days of postnatal life. The content of red cell 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and ATP increased slightly. These results suggested increased glycolytic flux through the diphosphoglycerate mutase reaction which resulted in net synthesis of 2,3-diphosphoglycerate. The red cell total triose phosphate peaked and fell initially, followed by glucose-6-phosphate and 2,3-diphosphoglycerate suggesting inhibition of phosphofructokinase activity and a decrease in glycolysis secondary to decreased red cell intracellular pH. After 10 days of postnatal life all glycolytic intermediates fell simultaneously, which correlated with a decrease in activity of the glycolytic enzymes.